Poster Sessions 2018-03-28T12:40:16+00:00

12th SCT

April 18-21, 2018. Belgrade, Serbia

At least one author of an abstract must be registered for the Congress in order to be included in the abstract book. One author can present only ONE abstract.

Poster Sessions



Air Pollution in Novi Sad (Serbia) – Long-term Level of Benzene (2011-2017)

Ljilja Torović 1,2, Stanka Bobić 2, Milan Jovanović 2, Maja Ćirković 2, Nataša Dragić 1,2, Emil Živadinović 2, Sanja Bijelović 1,2

1 Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia, 2Institute of Public Health of Vojvodina, Novi Sad, Serbia


The scope of the study was monitoring of volatile aromatic hydrocarbons included in BTEX group in ambient air of the City of Novi Sad. In the context of human health risk, benzene as group 1 carcinogen is of special interest.

Urban traffic (A) and industrial (B) site were monitored during seven years (2011-2017). Samples were collected on charchoal sorbent tubes (24 hours), with annual time coverage 27-34% (except 2011 – A 16%, 2014 – B 22%). Analytes of interest were determined by GC-MS, as described by reference method EN 14662-2. Temporal distribution in terms of mean monthly concentrations over calendar year, and between years during monitoring period, as well as spatial distribution between monitoring sites was considered.

The highest concentration of benzene recorded on site A ranged from 4.1 in 2011 to 37.1 mg/m3 in 2015, whereas on site B the range was 7.1-27.3 mg/m3 (2012-2016). The month with the highest mean benzene level was December or January, except in 2011 and 2012. On site A, annual mean benzene levels were 0.5, 1.2, 1.2, 3.9, 1.9, 2.1, and 2.5 mg/m3, during consecutive years, with the overall mean at 1.9 mg/m3. Site B was characterized with higher annual mean benzene levels than site A (except in 2012): 1.0, 0.9, 1.7, 5.0, 3.2, 3.0, and 3.2 mg/m3 (overall mean 2.6 mg/m3). The highest annual mean was recorded in 2014, on both monitoring sites.

Importantly, regulatory annual mean benzene limit value of 5 mg/m3 was not exceeded, although it was reached in 2014.

Keywords: ambient air, benzene, GC-MS

Comparison of Methods for Determination of Sulphur Dioxide Concentrations in Ambient Air


Nenad Petrović,


Air pollution is one of the major environmental issues and one of the main pollutant of concern in ambient air is sulfur dioxide (SO2).  One of the most frequently used method for determining concentration of sulfur dioxide in ambient air is tetrachloromercurate (TCM)/pararosaniline method in which tetrachloromercurate is present in absorption solution. The objective of the present study was to compare tetrachloromercurate/pararosaniline method with methods that don’t contain mercury in the absorption solution and determine if they can be used as suitable replacement for TCM/pararosaniline method. Two methods were compared with TCM/pararosaniline method: spectrophotometric thorin/barium perchlorate method and nepheleometric barium chloride method.  Samples were prepared using certified reference gas cylinder of sulfur dioxide and gas calibrator MCZ-CMK 5. All samples were prepared by transferring diluted gas through absorption bottle filled with a 20 ml of absorption solution with constant flow of 0,5 l/min. The three methods in the order tetrachloromercurate/pararosaniline method, spectrophotometric  thorin/barium perchlorate method and nepheleometric barium chloride method gave the following results: limit  of quantification(LOQ),   3,6 μg/m3, 22,2 μg/m3  and 18,3 μg/m3; measurement range up to 106,7 μg/m3, 222,2 μg/m3 and 275,1 μg/m3; sensitivity 0,72 μg/m3, 1,94 μg/m3 and 1,97 μg/m3; precision expressed as relative standard deviation(RSD%) 3,3 %, 6,2 %, and 5,6 %; accuracy  98,3-103,9 %, 92,6-102,9 % and 94,7-108,4 %. Although it has been shown that tetrachloromercurate/pararosaniline method has more advantages concerning sensitivity, (LOQ), accuracy and precision, conclusion is that both spectrophotometric and nepheleometric method can be used as suitable replacement for tetrachloromercurate/pararosaniline method.

Keywords: tetrachloromercurate, pararosaniline, thorin, nepheleometry, SO2

Inflammatory Response Modulation of Human Airway 3D-model Depending on the PM’s Chemical Composition


Khaled Boukerma, Emeline Seurat, Sophie Achard

Université Paris Descartes, Faculté de Pharmacie de Paris, EA 4064 “Impact des pollutions sur la santé”, Paris, France


Many epidemiological studies describe the existence of an association between air pollution and some respiratory disorders like asthma and allergic rhinitis. Within the major pollutants, the particulate matter (PM) is a complex mixture depending on the emitting sources.

To provide elements of biological plausibility to the epidemiological observations, the present study evaluates the impact of fine particles (PM0.3-2.5) coming from two different places: Dunkerque (France) for PMD and Cotonou (Benin) for PMC ; with similar sizes but different chemical compositions.
An innovative and sensitive in vitro 3D-model of human reconstituted airway epithelium (MucilAir, Epithelix®), cultured in an air-liquid interface was used.

The epithelia were exposed to PM at 30 or 60µg once or twice a week for three consecutive weeks. Forty hours after each exposure, the culture medium on basal side was collected and inflammatory response was assessed. At the end of each week the membrane integrity was evaluated using the TEER (transepithelial electrical resistance) test.

Our results showed that IL-8 production was time-dependent, whatever the PM sample and the quantity tested: a significant increase of the inflammatory response was observed from 6h to 96h after exposure. In addition, the IL-8 production was the highest during the first week of exposure, certainly due to the adaptation capacity of our 3D-model to its environment. Lastly, measured inflammatory response was higher after PMC exposure compared to PMD exposure, probably in relation with it chemical composition. No loss of tissue integrity is observed during the experiment.

Keywords: airborne particulate matter, human respiratory reconstituted epithelium, repeated air-liquid exposures, inflammatory response

Carcinogenic and Non-Carcinogenic Risk Assessment of BTEX Compounds in Ambient Air of Tehran, Iran

Masud Yunesian1,2, Mansour Shamsipour2, Hassan Amini3, Mahammad Sadegh Hassanvand4, Maryam Zare-jeddi4, Vahi Hosseini5, Sasan Faridi1, Farzad Younesian6

1Department of Environmental Health Engineering, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, 2Department of Research Methodology and Data Analysis, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran, 3Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute (Swiss TPH), University of Basel, Basel, Switzerland, 4Center for Air Pollution Research (CAPR), Institute for Environmental Research (IER), Tehran University of Medical Sciences, Tehran, Iran, 5 Department of Mechanical Engineering, Sharif University of Technology, Tehran, Iran, 6Department of Civil and Environmental Engineering, Shahrood Uinversity of Thechnology, Shahrood, Iran


We used ambient concentrations of benzene, toluene, ethylbenzene, different species of xylene (BTEX) measured in Tehran Study of Exposure Prediction for Environmental Health Research (Tehran SEPEHR). Samples were collected over three 2-week periods in summer, winter, and spring at 174 randomly selected sites in Tehran, capital of Iran using passive BTEX samplers (Passam Co. Männedorf, Switzerland). After cleaning and correcting data (by subtracting mean value of negative control from all measured samples), carcinogenic and non-carcinogenic risk of exposure to these compounds were assessed using reference dose and cancer slope factors developed by US Integrated Risk Information system (IRIS) for age and sex specific  groups of inhabitants in Tehran. The annual median for benzene was 7.8 μg/m3.  This value exceeded the national air quality standard level (5 μg/m3) in about 90% of the samples, and concentrations of almost all compounds were peaked  in the summer period. Results of performed risk assessment indicated that Excess Lifetime Cancer Risk (ELCR) for women (7.79E-5) was greater compared to that of for men (6.0E-5), both more than World Health Organization’s recommended negligible cancer risk (1E-5). Hazard quotient (HQ) for each compound and hazard index (HI) for total BTEX compounds were far less than unity. Our data suggested that younger children (age group 0-4) are most susceptible to the carcinogenic effects.

 Keywords: BTEX, carcinogenic, non-carcinogenic, kriging

The Use of a 0.20µm Particulate Matter Filter Decreases Cytotoxicity in Lung Epithelial Cells Following Air-liquid Interface Exposure to Motorcycle Exhaust


Tao Yu1 , Bin Li1, Ping Bin1

1National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, 29 Nanwei Road Beijing,100050


Our study was designed to establish and apply a dynamic in vitro model for direct exposure of human cells to gaseous contaminants to investigate the cellular responses to airborne chemical exposures and investigate whether the use of a 0.20 µm particulate matter (PM) filter reduced the cytotoxicity induced by motorcycle exhaust (ME), a mixture of gases and particles, in lung epithelial cells cultured in air-liquid interface (ALI) inserts. The concentrations of PM, carbon monoxide, carbon dioxide, total hydrocarbons (THC), total volatile organic compounds, and nitrogen oxides were measured. Lung epithelial cells were exposed to clean air, fME, or non-fME in the ALI chamber. Cell relative viabilities (CRV) and the reactive oxygen species (ROS) generation were determined. Our results revealed that PM and THC levels were significantly reduced, as compared with non-fME. When compared with the clean air exposed group, the CRV in both fME and non-fME-exposed group was significantly reduced (p < 0.001), while their ROS generation were markedly increased (p < 0.001). When compared with non-fME-exposed group, the CRV and ROS generation were significantly improved following fME exposure (p < 0.05). As a result, PM and THC concentrations were decreased approximately 90% and 22.71%, respectively, the CRV was improved from 40.4% (non-fME) to 55.7% (fME), and the increased ROS generation by non-fME was decreased about 51.6%. Our results provided evidence that levels of PM and THC in ME were significantly reduced, and oxidative stress was significantly improved after filtration as compared with non-fME.


Keywords: motorcycle exhausts, air-liquid interface, cytotoxicity, lung epithelial cells


Fine Particulate Matter (PM2.5) and Heavy Metals in the Atmosphere of Dokki Urban Area, Egypt


Salwa K. Hassan,, Waleed H. Shetaya, Asmaa El-Mekawy, Elham F. Mohamed, Atef M.F. Mohammed

Air Pollution Department, National Research Centre, 33 El-Bohouth St., Dokki, Giza 12622, Egypt



A total of 90 PM2.5 samples (24 h) were collected using a high-volume sampler for a year (2017-2018) in one of the most populated urban districts of Cairo (Dokki).  The annual average concentration of PM2.5 was 33.5 g m-3 which is almost triple the WHO annual limit of 10 g m-3.  The elevated PM2.5 levels were not exclusive to one particular season and recorded 49.7, 41.8, 30.5 and 21.8 g m-3 in winter, spring, summer and autumn, respectively.  This indicates that the residents of urban Cairo are exposed to dangerously high levels of PM2.5 and may suffer from associated long-term health problems. In addition, in 56 % of the measured days the PM2.5 concentrations exceeded the WHO 24 h limit of 25 g m-3; and seasonally, 78, 76, 53 and 33 % of the days also exceeded the WHO limit.  This highlights a year-round high possibility of short-term health consequences for the urban Cairo local residents especially in winter and spring when conditions are either very calm (winter) leading to minimal dispersion of the locally emitted particulates, or stormy (spring) and bringing long-range transported aerosols.  All samples were acid extracted and assayed by ICP-MS for trace metals.  The concentrations of 13 elements including Al, V, Mn, Fe, Co, Ni, Cu, Zn, As, Cd, Mg, Hg and Pb in PM10 were determined.

Keywords: heavy metals particles, air, Cairo, ICP-MS

The Impact of Ambient Air Pollution on Suicide Mortality in Albania


Enkelejda Shkurti1, Diamant Shtiza2, Esmeralda Thoma3

1,3University of Medicine, Tirana, Albania, 2University Hospital Centre : “Mother Theresa”, Tirana, Albania


While the outcomes of social and individual risk features of suicide have been extensively examined, proof of environmental results of exposure to air pollution is insufficient. We explored the consequences of ambient air pollution on suicide mortality in Tirana,Albania during 2010-2016.

A provisional logistic regression analysis with a transversal design was carried out to evaluate the outcomes of daily exposure to three standard air contaminants, counting particulate matter less than 10 μm (PM10), sulphur dioxide (SO2) and nitrogen dioxide (NO2), on suicide mortality, after altering for the confounding outcomes of daily mean temperature, relative humidity, atmospheric pressure and sunshine extent. Additional analyses were stratified by season, sex, age group, educational accomplishment and suicide category.

Between 2010 and 2016, 370 suicide deaths were recorded in Tirana. A considerable increase in suicide risk was related to interquartile-range increases in the concentration of air contaminants, with an odds ratio of 1.06 (95 % confidence interval (CI): 1.00, 1.18) and 1.13 (95 % CI: 1.02, 1.14) for PM10 and NO2 at lag 01, and 1.09 (95 % CI: 1.01, 1.17) for SO2 at lag 01, correspondingly. The suicide hazards linked to air pollution for males and people with high education rank were higher than for females and those with low education level, correspondingly. Considerable air pollution consequences were established on aggressive suicide mortality and in cool season but not on non-violent suicide mortality or in warm season.

These results would offer significant information for the health impact appraisal of air pollution and for the expansion of valuable strategies and involvements for the prevention of suicide.

Keywords: air pollution, Tirana, mortality, suicide





Development of an Alternative Method to Evaluate In Vitro Skin Sensitisation Potency of Chemicals


  1. Buzzella1,2, R. Vicini2, G. Mazzini1,3, C. Angelinetta2, O. Pastoris1.

 1Department of Biology and Biotechnology ‘L. Spallanzani’, University of Pavia, Pavia (Italy),  2Bio Basic Europe S.r.l., Pavia, (Italy),  3Institute of Molecular Genetics (IGM-CNR), Pavia (Italy)


Allergic contact dermatitis, a common condition for human health, is an inflammatory reaction caused by repeated skin exposure to specific chemicals. In order to comply with the requirements of European legislation, concerning the risk assessment of potential skin sensitizers, considerable progress has been made in developing alternative methods, such as human cell line activation test (h-CLAT). H-CLAT is based on measurement, obtained by cytometric analysis, of fluorescence variations emitted by anti-CD54 and anti-CD86 antibodies in THP-1 cells. Following this method, a range of substances have been analysed; the emitted fluorescence, generally at low intensity, has caused problems concerning the interpretation of results, preventing the discrimination between sensitizers and non-sensitizers.

For this reason, the aim of the study was to find an alternative cytometric parameter for evaluating the sensitising potential of chemicals. Cells have been analyzed with flow cytometry after treatment with sensitising compounds administered at non-cytotoxic concentrations. A set of cells have not been exposed to any product (negative control).

Sensitizers, once in contact with cells, were able to induce alterations in cell morphology to a more “condensed” one allowing the identification of cells under microscope as a “sensitized” subpopulation. These variations cause similar modifications in “scattering” parameters, making thus cells easily monitorable by flow cytometry. No changes have been observed in cells treated with non-sensitizers or in untreated cells. In conclusion, this method based on the analysis of forward scatter and side scatter parameters, can be used as a valid alternative method for identifying sensitisation potential of chemical compounds.

Keywords: in vitro method, skin sensitisation, flow cytometry, morphology



An Innovative Approach for Evaluating the Safety of Cosmetic Products Through a Combination of in vitro and in vivo Methods


Riccardo Vicini1, Alice Buzzella1,2, Giuliano Mazzini3, Claudio Angelinetta1, Eliana Regola1, Fernando Bianchi1, Ornella Pastoris2

1Bio Basic Europe S.r.l., Milan, Italy; 2 Dpt. of Biology and Biotechnologies, University  of Pavia, Italy; 3Institute of Molecular Genetics, CNR, Pavia, Italy



European Regulation no. 1223/2009 prohibits animal testing for cosmetic products and it requires their safety for human health. Hence the need to use alternative validated methods based on in vitro techniques on cell cultures, which are easy to manage and rapid, but on the other hand they provide forecasting models not always suitable for in vivo system. Our aim was to develop a multi-step test to evaluate the high tolerability of cosmetic products that involves the sequential execution of in vitro and in vivo tests.

We performed a pro-sensitising test on human monocytes on 193 cosmetic products, evaluating by flow cytofluorimetry the variation of cell morphology and CD86/CD54 expression. The products were then subjected to in vivo patch test on 25 volunteers, and to a subsequent clinical investigation on 20 volunteers for 28 days, to check the long-term skin tolerability.

182 products (94.3%) did not show any in vitro pro-sensitising activity. On these products also patch test (absence of erythema/oedema), and skin tolerability test (absence of undesirable effects) were negative. 11 products (5.7%) showed in vitro pro-sensitizing effect, but were negative to patch test and skin tolerability test.

We can conclude that in vitro tests on cell cultures can predict with good approximation the in vivo safe use of cosmetic products. Nevertheless, the in vitro presence of an undesirable effect seems not to cause the appearance of the same reaction in vivo. In these cases it is therefore appropriate to deepen the analysis with specific in vivo tests.

Keywords: alternative methods, cosmetic products, skin sensitization, integrated test strategy

Development of a Full-thickness 3D Autologous Skin Equivalent Model to Determine Immunogenicity of Therapeutics


Asif S. Tulah, Shaheda S. Ahmed, Anne M. Dickinson

Alcyomics Ltd, Newcastle upon Tyne, United Kingdom


There is growing demand for human-based assays to test compounds/therapeutics for adverse immune reactions prior to entering clinical trials. Human skin equivalent models are useful in vitro testing platforms. We describe a unique full-thickness 3D autologous skin equivalent model made from primary human tissue which is representative of normal human skin and an autologous platform for testing therapeutics under development.

Primary human fibroblasts and keratinocytes were grown from healthy volunteer tissue. The full-thickness skin equivalent model was generated by first forming a dermal equivalent by culturing fibroblasts on a scaffold before adding donor-matched keratinocytes and culturing at the air-liquid interface. Histology and immunofluorescence for protein markers (involucrin, cytokeratin 14 and collagen 3) was completed and compared to normal skin. We co-cultured the model with activated peripheral blood mononuclear cells (PBMCs) and observed similar immune damage to that in our skin explant assay. Immunofluorescence staining for heat shock protein 70 (HSP70) as a marker of apoptosis was used to confirm these findings.

Our model was representative of normal human skin, showing similar structure observed by haematoxylin and eosin staining and positive immunofluorescence staining  for protein markers of epidermal differentiation and skin structure (involucrin, cytokeratin 14 and collagen 3). Our data also suggest this model could be used to detect immune damage as shown by positive HSP70 staining.

We have generated an autologous 3D skin equivalent model, representative of normal human skin. Preliminary data has shown this to be a useful additional platform for testing drugs under development.

Keywords: 3D skin model, immunogenicity testing, safety testing, non-animal




Ecotoxicity Assessment of Waste Hazard Using Clutches of the Mollusk Lymnaea stagnalis

Boris Olga, Ilyukova Irina, Shevtsova Svetlana

Republican unitary enterprise «Scientific and practical centre of hygiene», Minsk, Belarus



The method for studying embryotoxicity of waste in the test-model Lymnaea stagnalis for the application to determine the hazard class of industrial waste on hazard characteristic «ecotoxicity». A study of more than 30 production waste identified toxic effects from exposure to waste and set evaluation criteria harmful effects, scientifically substantiated rational setting conditions of the experiment, the methodological aspects as well as the objective criteria for evaluating the ecotoxicity of waste and test validity criteria.

Such estimation criteria as EC50 (the average effective concentration), EC15 (the threshold concentration) and the acute zone have been developed. The use of these indicators will provide further investigations of waste ecotoxicological profile. The threshold concentration is useful for making out waste concentrations that should be considered conditionally safe in case of contact with the environment.

The clutches in the gastrula stage of snails are used in this test-system. The total number of hatchlings as well as the total number of dead embryos are counted per each experimental group. The experiment with Lymnaea stagnalis test-system is aimed to estimate the inhibition of hatching efficacy.

The development allows reducing the negative impact of waste production on the environment well-being and preventing negative consequences for the human health.

Keywords: the waste products, Lymnaea stagnalis, biotesting, ecotoxicity.



Ultrastructural and Histological Changes in the Intestines of  the Earthworm (Eudrilus eugeniae) Exposed to Heterocyclic Amines


Balogun WG1, Mail MH1, Mat Hassan H2, Seeni A1,3

1Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bandar Putra Bertam, 13200 Kepala Batas, Penang, Malaysia, 2School of Biological Sciences, Universiti Sains Malaysia, 11800 Gelugor, Penang, Malaysia, 3Malaysian Institute of Pharmaceuticals and Nutraceuticals, National Institutes of Biotechnology Malaysia, Bukit Gambir, 11700 Gelugor, Pulau Pinang, Malaysia


Haze formation and bush burning are two major environmental problems facing the South-East Asia countries; these environmental pollution results into the release of heterocyclic amines, known potent carcinogenic compounds into the environment.

This study investigates the effects of heterocyclic amines on the earthworm, a major inhabitant of the soil.

Earthworms and vermicast were gotten from the School of Biological Sciences of USM and divided into 8 groups. Group A and B were treated with distilled water and DMSO, group C, D and E were treated with different concentration of PhIP while group F, G and H were treated with different concentration of MeIQx. Another set of earthworms were used for the contact test for preliminary investigation of the toxic effects of the heterocyclic amine. The contact test was performed for 24 hours and during that time signs of toxicity and mortality were observed. The soil exposure test was carried out for 28 days and after that the tissues were fixed in formalin and karnovsky fixative for further analysis.

Results from the contact test showed that heterocyclic amines induced signs of toxicity within the first 6th hour and mortality from the 12th hour. There was no death from the soil exposure test, but light and electron microscopy observations of the body wall revealed damage and disruption of the cuticle, circular and longitudinal muscles. There was also damage to the intestine, an organ involved in detoxification and metabolism of the earthworm.

This work has demonstrated that presence of the heterocyclic amines in the environmental media could be deleterious to the organisms inhabiting it.

Keywords: heterocyclic amines, toxicity, earthworm, environment, apoptosis





Analytical Method Development of Methylisothiazolinone, a Preservative, in Rat Plasma Using LC-MS/MS


Hyang Yeon Kim, Yong Jae Lee, Kyu-Bong Kim

College of Pharmacy, Dankook University, 119 Dandae-ro, Dongnam-gu, Cheonan, Chungnam 31116, Republic of Korea.


Methylisothiazolinone (MI) is a preservative in personal care products to control bacteria and fungi. It might be one of the toxic sensitizer and irritant on the skin, and initiator of lung diseases, so it is important to understand the characterization and toxic mechanism of MI in the body. In this study, we developed the HPLC-MS/MS analytical method in rat plasma using multiple reaction monitoring (MRM) technique, which is following the fragments of target metabolite. MRM transition of MI was m/z 116 à 101 and lower limit of quantification (LLOQ) was set at 10 ng/mL. Including the concentration of LLOQ, seven-point calibration curve have good linearity (R2= 0.9998) and its intra- and inter-day accuracy and precision values were within 15% of the relative error (RE%) and standard deviation (RSD%) along with FDA guideline. In the pharmacokinetic study using rats, presented analysis method was useful for detecting the profile of MI in the plasma. We determined half-life, clearance and distribution of MI by WinNonlin software and these might be useful information for the study on the toxicokinetics in body.

Keywords: methylisothiazolinone, HPLC-MS/MS, MRM, pharmacokinetics

Determination of Methadone in Blood by Solid-phase Extraction and Liquid Chromatography-Mass Spectrometry

Tatjana Ćebović1, Vesna Kilibarda2, Snežana Đorđević2

1Faculty of Medicine Novi Sad, Clinical centre of Vojvodina, Novi Sad, Serbia; 2Military Medical Academy, Poison Control Centre, Belgrade, Serbia


Methadone, synthetic opiate analgesic, belongs to a class of compounds referred to as diphenylpropylamine derivatives. In spite of the introduction of new agents such as L-LAAM and buprenorphine, methadone continues to be the drug of choice for the treatment of heroin addiction. Due to the wide variation in half-life among individuals (reported values 13-58 hours), there is a significant ovedose risk. Together with the requirement for effective monitoring of replacement therapy, it emphasizes the need for development of fast, precise and reliable analytical method for quantification of methadone in biological samples.

In this research, liquid chromatography-mass spectrometry (LC-MS) method for determination of methadone in blood was developed, using solid-phase (SPE) extraction for the preparation of samples.

Methadone was determined by LC-MS method in a single ion monitoring mode (SIM) at m/z 310. The chromatographic separation was performed on XTerra MS C18 column, using a mixture of formic buffer (pH 3,5) – acetonitrile (60 : 40 V/V) as mobile phase, with the flow of 0.5 mL/min.

Linearity was achieved in the range from 0.05 to 2 mg/L. Retention time of methadone was 5.1 min. Limit of detection and limit of quantification were 1.0 ng/mL and 3.2 ng/mL, respectively. The intra-assay precisions (RSD) were about 0.07-1.01%. The inter-assay precisions (RSD) were 0.18-2.11%. The accuracy varied from 92.0 to 101.1% for intra-assay and 99.7 to 100.9% for inter-assay.

Described LC-MS method for analysis of methadone in blood is precise, accurate, reproducible and reliable, with significantly shortened time of analysis (run time 10 min).

Keywords: methadone, overdose, therapeutic laboratory monitoring, validation

Determination of Carbamazepine Concentration in Serum – Comparison of Methods


Milanka Ljubenovic1, Vladan Cosic1, Jasna Lalic1, Svetlana Stojiljkovic1,

Maja Vujovic2, Biljana Milosavljevic2, Bojan Ljubenovic3

1Centre of medical biochemistry, Clinical Centre Nis, Serbia, 2Laboratory of toxicology, Institute of Forensic Medicine, Nis, Serbia, 3Faculty of medicine, University of  Nis, Serbia


Carbamazepine is antiepileptic widely used for the treatment of epilepsy. Due to low therapeutic index of carbamazepine, there is a need for routine measuring its concentrations in serum. Monitoring of the drug level helps to adjust the dose and achieve optimal therapeutic effects while avoiding subtherapeutic and toxic level. The aim of this study was to determine serum concentration of carbamazepine in patients with epilepsy who are on monotherapy, as well as to show the correlation of different methods on different systems for determination of the drug in serum (spectrophotometric and chromatographic method).

For the study were used serums of the patients with epilepsy, 35 serums total. The samples were analyzed with homogen immuno-enzyme test on the Beckman Coulter Analyzer System and high performance liquid chromatography with diode array detector (HPLC/PDA) on Waters system.

Concentration of carbamazepine in serum, in the interval of reference values had 71.5% of patients, the lower values had 28.5% patients, and there were no increased values. The average value of carbamazepine concentration in serum determined photometric on Beckman Coulter Analyzer System is = 5,44±2,06. The correlation coefficient obtained by comparing the spectrophotometric and chromatographic methods is,  r = 0,896, p > 0,05.                   .

Our results show that the most of patients on carbamazepine therapy have the correct dosage regimen. The statistical difference between different systems and methods is not significant. There is a strong correlation and connection, so the photometric method is reliable and applicable for routine operation.

Keywords: carbamazepine, analytics, immuno-enzyme test, HPLC/PDA


The Analysis on Physicochemical Parameters to Evaluate the Water Quality of Wellspring at Mountain Kukavica Exploited for Drinking


Tomislav Tosti 1, Bojan Nikolic 2, Radoslav Daljevic 3, Katarina Karljikovic Rajic 2

1 University of Belgrade Faculty of Chemistry-Department of Analytical Chemistry P.O. Box 51, 2University of Belgrade Faculty of Pharmacy-Department of Analytical Chemistry P.O. Box 146, 3Institute of general and physical chemistry, P.O. Box 45, Belgrade, Republic of Serbia


At mountain Kukavice, in the southeast part of Serbia, several wellsprings are adapted and exploited for drinking for any person walking or working in this region, but without any notice on safe usage. The objectives of this study were to evaluate water quality of wellspring based on physicochemical parameters: pH, TDS (total dissolved solid), TSS (total suspended solids), specific conductivity, total water hardness (ppm CaCO3) determined by classical complexometric titrations and compared with ICP-OES analysis, as well as ICP/MS for trace analysis of micro elements.

The results of volumetric analysis of total water hardness (27.5 ppm CaCO3) with the ratio Ca2+/Mg2+=2.57, classified investigated water sample in the type of very soft (<75 ppm CaCO3) and were in accordance with ICP-OES analysis: 28.0 ppm CaCO3 with slightly higher ratio Ca2+/Mg2+=3.06. The satisfactory accuracy of volumetric analysis was confirmed by Ca2+ content of 9.74 ppm in commercial drinking water “Rosa” (labeled 9.6 ppm Ca). For filtered sample the obtained TDS revealed 77 ppm (180 °C) correlated with results for unfiltered sample (TDS 126 ppm and TSS 47.8 ppm at 105 °C). The pH value and specific conductivity were 6.91 and 81.5 mS/cm (18.3 °C), respectively. The calculated TDS from specific conductivity was 61.2 ppm. The ion chromatography (IC) analysis for anions provided adjacent chloride and nitrate content 0.87 and 0.82 ppm, respectively and 12.57 ppm of sulphate. The results of micro elements analysis obtained by ICP/MS provided concentrations (As; Cd; Pb) lower than maximum allowable limits, confirming safe water usage of investigated adapted wellspring.

Keywords: adapted wellspring, water quality, total water hardness, TDS and TSS, anions’ analysis

Tools for Identification of Unknown Compounds After Gas Chromatographic-mass Spectrometric (GC-MS) Analysis of Urinary Volatile Organic Metabolites (VOMs)


Tanja Živković Semren,  Irena Brčić Karačonji, Andreja Jurič, Blanka Tariba Lovaković, Nataša Brajenović, Alica Pizent

Analytical Toxicology and Mineral Metabolism Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia


Volatile organic metabolites (VOMs) in human urine can be used in non-targeted metabolomics research to identify specific metabolites that may be useful as new diagnostic, predictive, and prognostic disease biomarkers. Determination of urinary metabolite profile provides novel information on phenotypic characteristics of an organism that cannot be obtained from target measurement. The headspace-solid phase microextraction (HS-SPME) technique coupled with gas chromatography-mass spectrometry (GC-MS) proved to be the most successful in VOMs analysis. After data processing of raw GC-MS data crucial step is to identify compounds of interest. Due to the complexity of the matrix, wide chemical diversity of the metabolites and their wide concentration range, metabolite identification is intrinsically difficult. In our laboratory, we use automated mass spectral deconvolution and identification system (AMDIS) for identification of unknown VOMs. AMDIS first deconvolutes the raw GC-MS data file to find all components, and then compare mass spectral data against a library of target compound (e.g. National Institute of Standards (NIST) mass spectral library). To reduce possible solutions of identification offered by NIST Kovats retention index (RI) is used. A Kovats retention index system uses a series of standards, homologous series of n-alkanes applied as reference peak. Despite some limitations, presented methods could be very useful in VOMs identification. To confirm identification of unknown VOMs unequivocally, analysis of available analytical standards using the same GC-MS conditions is recommended.

Keywords: metabolomics, HS-SPME, AMDIS, Kovats retention index, NIST

Identification of Synthetic Cannabinoid MMB CHMICA in ‘‘Spice-like’’ Herbal Mixture: Update of the Serbian Situation for the October 2017


Vera Lukić1, Ružica Micić2*, Tatjana Verbić3, Anja Jokić2

1Institute of Forensic Medicine, Faculty of Medicine, University of Belgrade, 2Faculty of Science and Mathematics, University of Priština, Kosovska Mitrovica, 3Faculty of Chemistry, University of Belgrade, Belgrade


Synthetic cannabinoids, which were synthesized to improve the therapeutic effects of cannabis, have become a major issue when they are abused. They have different chemical structures from tetrahydrocannabinol (THC) but similar effects on endocannabinoid receptors. “Spice” named products have more serious side effects than cannabis and can even cause death. These mixtures are prepared by spraying chemicals onto small pieces of herbs and are being dishonestly sold as ‘‘natural’’ and ‘‘legal’’ products over the internet. Their popularity is continuously increasing. Although, various products are labeled with warnings “not for human consumption”, they are intended to mimic psychoactive effects of illicit drugs of abuse. This paper shows the application of accurate-mass quadrupole time-of-flight (Q-TOF) LC/MS method and GC-EI-MS for identification of MMB CHMICA (methyl 2-{[1-(cyclohexylmethyl)-1H-indol-3-yl] formamido}-3-methylbutanoate) synthetic cannabinoid in plant material. By reviewing the UNODC Early Warning Advisory on the New Psychoactive Substances, the first time this substance was reported by Slovenia in 2015. In 2017, the emergence of this cannabinoid was reported by two countries, the USA and Cyprus. The plant material was analyzed in October 2017 upon user’s personal request-According to the information we received, it was purchased in one of the smart shops in Belgrade, Serbia, in the original package “Milf”. It is declared as a freshener with a warning that it is not for human use. A sample of plant material was prepared by extraction with methanol in the ultrasonic bath for 30 min. The analysis was firstly performed by GC-EI-MS. The resulting mass spectrum did not give an appropriate match with the base of the spectrum used at that moment in the laboratory. Sample extract was further analyzed by LC-QTOF MS and we got accurate-mass 370,2256 Da, Diff (ppm) -0,76 for our compound and proposed formula C22H30N2O3. On the basis of the obtained GC-EI-MS spectra, the exact mass and the recommended formula, MMB CHMICA was identified in the plant material.


Ion Chromatography for the Determination of Chlorites in Drinking Water


Vesna Milutinović, Sežana Vukčević

Institute of Public Health, Belgrade, Bulevar despota Stefana 54a, 11000 Belgrade, Serbia


Chlorine dioxide is used and in water-treatment facilities to make water safe for drinking. Its byproduct, chlorite and chlorate ions may be present at low levels in tap water. Chlorite ion is very reactive chemical. The EPA has set the maximum concentration in the drinking water at 1,0 mg/L for chlorite ion and domestic regulation at 0,2 mg/L chlorite.

Available human and animal data indicate that oral exposure to relatively large amounts of chlorite may result in irritation of the digestive tract and increased levels of methemoglobin in the blood, which reduces the ability of oxygen to bind with hemoglobin.

The samples that were analyzed were collected twice a week from the „Vinca“ plant, starting from 2014. Chlorite concentrations were determined by ion chromatography with suppressor, sodium carbonate as eluent, conductometric detector (Metrohm 930 IC flex) and EPA 300.1 method. Approximately 5 mL of the sample was injected with pre-nitrogen degassing and with a sample-loop of 50 μL. The quantification limit is 0,005 mg/L chlorite. Accuracy and precision were determined via recovery of the spiked samples into nine repetitions. The values obtained were in the range of 75-115%. During 2014, 2015 and 2016, concentrations above the limit of quantification were determined in the samples. About 2% of the samples from the total analyzed had values above the maximum allowed concentration according to domestic regulations and ranged to 0.35 mg/L. During 2017, the treatment of water with chloridoxide has been temporarily suspended, but it is scheduled for 2018 and next years.

Keywords: byproduct, regulation, exposure, ion chromatography

Identification of Gamma-butyrolactone (GBL) by Liquid Chromatography Mass Spectrometric Method

Gordana Brajković1, Snežana Đorđević1,2,  Jasmina Jović-Stošić1,2, Vesna Kilibarda1,2, ZoricaBrajković3, SnežanaBojović1, SlavicaVučinić1,2

1National Poison Control Centre, Military Medical Academy, Belgrade, Serbia, 2Medical Faculty, Military Medical Academy, Ministry of Defence, 3School of Medicine University of Belgrade, Belgrade, Serbia



Recreational use of gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL) as its precursor is very popular in recent years. They are clear, odourless, oily liquids. GHB is used as an anesthetic andrelaxant, and also haseuphoric effects. GBL is its precursor. It has legitimate use as a chemical for cleaning. GBL represents a serious threat to public safety because in the body it converts to GHB which is on the list of forbidden drugs. This is the reason why the identification of unknown sample is very important. The aim of this work is to present analytical method for identification of GBL in liquid sample.

Laboratoriy received a liquid sample suspected of GHB or GBL. The sample was qualitatively analyzed by using a liquid chromatography-electrospray ionization-mass spectrometer (LC-ESI-MS), on XTerra MS RP18 column under following conditions: source temperature 125° C, desolvation temperature 430° C, flow gas: desolvatacion 400 L/h, cone 50 L/h. The mobile phase: 5 mM ammonium formate (pH 3.5) : acetonitril with 0,1 %  formic acid. MS data were recorded in the full scan mode (m/z 40–150). For qualitative analysis of GBL the protonated molecular peaks ([M+H]+) of these compounds were monitored in the full – scan. The monitoring ions for GBL were m/z 87, 44, 42, and peak od GBL was detected at 3.48 min.

Presented LC-MS method is a method of choice for reliable identification of unknown sample that can make a difference between GHB and GBL.

Keywords: gamma-hydroxybutyrate (GHB); gamma-butyrolactone (GBL), LC-MS.



Distribution of Environmental Lead in Brown Bear Body Compartments


Maja Lazarus1, Tatjana Orct1, Slaven Reljić2, Jasna Jurasović1, Đuro Huber2

1Analytical Toxicology and Mineral Metabolism Unit, Institute for Medical Research and Occupational Health, 2Department of Biology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia


Lead (Pb) is a very stable inorganic environmental pollutant exchanged between blood, mineralized and soft tissue compartments upon absorption. Comprehensive distribution of Pb in the body of brown bears (Ursus arctos) has not been previously investigated. The need for such a study arose upon earlier reports suggesting that 1-5% of the bear population might be at risk of adverse effects caused by hepatic and renal Pb levels over threshold levels established for terrestrial mammals. Therefore, the aim of this study was to explore the bone as the principal deposition compartment and marker of long-term exposure to Pb, and soft tissues (heart, kidney, liver, lungs, muscle and spleen) that reflect recent exposure, but also endogenous Pb sources. Lead was quantified in tissues of 40 brown bears hunted in 2015 according to the Brown Bear Management Plan in Croatia. The highest Pb levels were found in the kidney cortex (median, 4398 µg/kg dry mass), followed by the femoral bone (2502-3980), liver (1761), lungs (303), spleen (209), heart (22.7) and muscle (11.1). Microdistribution inside the bone revealed higher Pb in the compact (3980 µg/kg) than trabecular bone (2502 µg/kg) at the same location (femoral metaphysis). Also, the Pb level differed depending on the location on the bone (epiphysis vs. metaphysis and metaphysis vs. diaphysis). Age was a significant factor in prediction of bone Pb, which was higher in adults compared to young animals. Unlike soft tissues, the femoral bone of brown bears was confirmed as a good biomarker of Pb, known for its accumulative nature.

Keywords: Ursus arctos, femoral bone, soft tissues, kidney, liver


Ten Years of Human Biomonitoring of Environmental Chemicals in Canada


Julie Yome, Annie St-Amand

Healthy Environments and Consumer Safety Branch, Health Canada. 269 Laurier Ave. W, Ottawa, Ontario, Canada, K1A 0K9.


The Chemicals Management Plan is part of the Government of Canada’s comprehensive environmental agenda to ensure the safe management of chemicals. Human biomonitoring of chemical exposure is a key element of this program and is used along with interpretation tools to quantify exposure and provide information for setting priorities and taking action to protect public health. The biomonitoring component of the Canadian Health Measures Survey (CHMS) is the cornerstone of the national biomonitoring program, collecting nationally-representative data on environmental chemical exposure in Canadians aged 3-79 years in ongoing two-year cycles. Over the past decade over 250 chemicals were measured in over 29,000 Canadians at 81 sites across the country. In the first three cycles, half of the chemicals measured were above the limit of detection in 60% of individual blood and urine samples. Indoor air and tap water samples were also collected in selected cycles. Tools have also been developed to interpret and communicate biomonitoring data in a public health context. Reference values (RV95) are statistical tools that allow the identification of individuals with increased exposure. RV95s derived from the CHMS are proposed for heavy metals, and persistent and non-persistent chemicals. Biomonitoring equivalents (BEs) are risk-based tools to prioritize vulnerable populations potentially at risk. Experience gained over the past decade has made Canada a world leader in human biomonitoring. This effort has established population baseline concentrations that have been used to inform regulatory risk assessment and improve evidence-based decision making in public policies to reduce exposure to toxic chemicals in Canada.

Keywords: biomonitoring, exposure

A Novel, Rapid, Sensitive Bioassay System for Dioxins Determination in Various Types of Samples


Songyan Zhang, Heidi Qunhui Xie, Li Xu, Yangsheng Chen, Bin Zhao

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China


The pollution caused by persistent organic pollutants (POPs) is a worldwide problem. Among these POPs, dioxin and those structurally dioxin-like chemical compounds with highly toxic are present in environmental media such as ambient air, soil, waste water at the national scale in China. The toxic of dioxins are mediated through binding to and activating aryl hydrocarbon receptor (AhR) and then producing the adverse outcomes. Thus several bioassay systems based on AhR-mediated reporter gene assays have been developed and used as supplemental tools for large scale pre-screening of dioxins instead of high resolution gas chromatography-high resolution mass spectrometry (HRGC-HRMS) analysis. Here, we developed a new and sensitive recombinant mouse hepatoma cell line, CBG2.8D, in which a novel luciferase-reporter plasmid containing 2 copies of a newly designed dioxin-responsive domain and an endogenous promoter was integrated. The minimal detection limit for 2,3,7,8-tetrachlorodibenzo-p-dioxin was 0.1 pM. We have employed the CBG2.8D bioassay to determine the dioxin levels in several environmental samples. The measured bioanalytical equivalent values (BEQs) were closely correlated with the toxic equivalent values (TEQs) obtained from HRGC-HRMS analysis, for example in air samples r2 = 0.97 and similar correlations between the BEQs and TEQs were achieved for soil and fly ash samples. Based on this suitable pre-screening character in diverse environmental media, we would apply this bioassay to monitor the total AhR activity in food samples or human blood samples and try to provide reference data for human and environmental risk assessment.

Keywords: persistent organic pollutants, aryl hydrocarbon receptor, environmental media


Mercury in Hares (Lepus europaeus Pallas) Collected in the Vicinity of the Natural Gas Treatment Plant in Northern Croatia During the Last Ten Years

Andreja Prevendar Crnić and Emil Srebočan

Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia

correspondence to:

In this study hares organs were analyzed for total mercury concentration by ICP-MS method as a part of a comprehensive monitoring of the eco-system in the vicinity of the natural gas production and treatment plant Molve in the north of Croatia. During the last ten years the range of the median mercury concentration values (wet weight) in hares organs were 0.00002-0.00436, 0.00417-0.01942, 0.0234-0.11729 and 0.00007-0.00102 μg/g for muscle, liver, kidney and brain, respectively. The results of mercury measurements in muscle and brain demonstrate mostly uniform values with a small increase in concentration values of mercury in muscle. Median Hg concentrations in liver and kidney differ from year to year but generally show consistent pattern in fluctuations, meaning that increased concentration of Hg in liver is followed by the same increase in kidney. Comparing our results with results published in available data on mercury concentration in hare’s tissue it can be concluded that investigated area near Molve belongs to low mercury contaminated region. Results also point to the fact that closed mercury removal system installed in 1993 is effective in mercury removal from natural gas, and that research and production of natural gas contaminated by mercury doesn’t threaten human health and the environment. Nevertheless, further eco-monitoring and mercury measurements in various hares organs are valuable and necessary and will be continued.

Keywords: hares, total mercury, natural gas, environmental monitoring


Can Measurement of Arsenic Concentration be Comparable in Human Buccal Cells, Hair Samples and Urine Samples and Correlate with DNA Damage Assessment with Micronucleus Buccal Cytome Assay?


Simone Brauer1, Walter Goessler1, Mirta Milić2, Vatroslav Šerić3,4, Marija Milić3,4, Ivan Pavičić5, Ana Marija Marjanović Čermak5, Stefano Bonassi6,7, Višnja Oreščanin8, Ivana Vinković Vrček9

1 Institute for Chemistry, University of Graz, Graz, Austria, 2 Mutagenesis Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia, 3 Department of Clinical Laboratory Diagnostics, Osijek University Hospital, Osijek, Croatia, 4 Faculty of Medicine, University of Osijek, Osijek, Croatia, 5 Radiation Dosimetry and Radiobiology Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia, 6 Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Rome, Italy, 7 Department of Human Sciences and Quality of Life Promotion, San Raffaele University, Rome, Italy, 8 ORESCANIN Ltd., Zagreb, Croatia,  9 Analytical Toxicology and Mineral Metabolism Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia


Although chronic arsenic (As) exposure is associated with toxic effects, both malignant and non-malignant, the dose response for As toxicity is still not clearly determined for human population, and therefore there are still not reliable biomarkers of the exposure and the effect for chronic As exposure that would helpful in assessment of DNA damage and prediction of cancer risk development. Sampling of urine, hair and buccal cells is part of noninvasive methods, but the potential of those three sampling types is still not fully investigated. With no literature data on arsenic measured in buccal cells and its correlation with DNA damage in buccal cells and arsenic concentrations in hair and urine samples, the aim of this study was to evaluate the buccal cells measurement with two other sampling materials in order to give an insight whether these methods can be used together. As and other elements were measured in more than 100 samples of the individuals from the Eastern Croatia chronically exposed to As through ground water, drinking water (As levels above 10 µg) and water used for crops. Liquid chromatography coupled to inductively coupled plasma mass spectrometry-ICPMS was used and the results were compared with the DNA damage assessed in buccal cells.   Results demonstrated that As concentrations determined in buccal cells were not in the correlation with those determined in hair and urine samples.

The study was financially supported by the Grant of Ministry of Science and Education of the Republic of Croatia and Austrian Federal Ministry of Science, Research and Economy 05/2016 Possible early noninvasive biomarkers of chronic exposure to arsenic.


Keywords: arsenic, urine, buccal cells, hair, DNA damage



Defining the Pesticide Exposome: Characterizing Longitudinal Seasonal and Occupational Trends of Pesticides in House Dust


Breana Bennett1,2, Tomomi Workman1,2, Marissa Smith1,2, William C. Griffith1,2, Beti Thompson3, Elaine M. Faustman1,2

1 Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Seattle, Washington, USA, 2 Institute for Risk Analysis and Risk Communication, University of Washington, Seattle, Washington, USA, 3 Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA


Agriculture often relies on chemical inputs such as pesticides, many of which have known adverse health effects. Children of farmworkers are especially vulnerable, and early childhood pesticide exposure may have lasting health effects. Because children of farmworkers are exposed to a variety of pesticides, we propose using an exposome framework to explore child pesticide exposure.

Using household dust samples from a children’s agricultural cohort, we examined the pesticide exposome for the 2005 and 2011 agricultural seasons. In particular, we analyzed how the pesticide exposome changes over time, and investigated the differences in the pesticide exposome between farmworker and non-farmworker households.

Dust samples were collected from floor areas where children played and analyzed by liquid chromatography-mass spectrometry (LC-MS). Individual pesticides were grouped into pesticide classes using the US EPA pesticide chemical classifications, and trends in concentrations were analyzed at the class level.

Across the entire cohort, the levels of organophosphates, pyridazinones, and phenols significantly decreased between 2005 and 2011, whereas levels of anilides, 2,6-dinitroanilines, chlorophenols, and guanidines significantly increased. Among farmworkers alone, there were significantly lower levels of N-methyl carbamates and neonicotinoids in 2011. For several common agricultural pesticides, changes in use were reflected in the dust of farmworker households.

We have defined the pesticide exposome in a children’s agricultural cohort and examined how that exposome changed between two agricultural seasons six years apart. This study exemplifies the utility of an exposome approach in evaluating longitudinal data on the changes in the pesticide exposome for pesticides.



Keywords: biomonitoring, epidemiology, exposure, children




The First National Biomonitoring Study of Environmental Metals


Anita Cvetkovska1, Biljana Manevska2, Fljamure Zekiri-Keka3, Irena Bojadzieva4, Elisaveta Stikova5

1,2,3,4Public Health Institute of the Republic of Macedonia 5Public Health Institute of the Republic of Macedonia, Medical Faculty, University Sts. Cyril and Methodius


Biomonitoring is a key tool used as an indicator and quantitative measure of exposure to chemicals in the environment. Biomonitoring is recognized as a standard for assessing people’s exposure to toxic substances and for responding to serious environmental public health issues. Public Health Institute of the Republic of Macedonia in 2014 and 2015 perform many activities, aiming to develop and establish the national human biomonitoring study with emphasises on heavy metals.

The main purpose of this study was to determine the baseline levels of Pb, Hg, Cd, Mn and Zn in blood in the representative sample of the adult population living in the 5 hot spots in the country and one control non-contaminated area, with intention to compare the differences of the concentration of these metals among different populations groups by age, sex, occupations and lifestyle (nutrition, smoking and living area). In this paper are shown the results of the first two years of the study (2014-15). The concentration of heavy metals was measured with electrothermal atomic absorption spectrophotometry and apparatus Analyst 600-Perkin Elmer.

The results showed that average concentration of the BLL was 14,5µg/l (2,1-19,7µg/l) ; Hg was 2,5µg/l (0,3-3,8µg/l); Cd was 0,7µg/l (0,1-2,0µg/l); Mn was 7,3µg/l (1,4-14,0µg/l) and Zn was 4,1µg/l (2,7-5,1 µg/l). Except the average concentration of the Mn in one and Cd in two hot spots, all other concentration are below the recommended levels by WHO, but with statistical significant differences in accordance with the age and living area i.e. environmental exposition, which are shown in the paper.

Keywords: lead, mercury, cadmium, manganese, zinc, blood levels, Macedonia


Assessment of Genotoxicity from Exposure to Low Dose Ionizing Radiation Among Workers in Radiology Department, Kuala Lumpur, Malaysia


Mazrura Sahani1, Razinah Sharif1, Mastura Hamzah1, Mohamed Ariff Jaafar Sidek2, Selvarajah Elavaras2,  Hamzaini Abdul Hamid2 and Hanizah Mohd Yusoff2

1Faculty of Health Sciences, University Kebangsaan Malaysia, 2Faculty of Medicine, University Kebangsaan Malaysia


This study was conducted to assess the relationship between chromosome damage and exposure to ionizing radiation among workers in Radiology Department, in one of the hospital in Kuala Lumpur, Malaysia. A cross-sectional study that involved 96 workers was conducted from April 2017 to May 2017. Methodology for this study includes reviewing RPL cumulative dose data, questionnaire survey, random blood sampling of 60 workers that representative of working unit and exposure group, alkaline comet assay and Cytokinesis Block Micronucleus Assay. Two of the workers have cumulative dose exceeded effective dose of 20 mSv as stated in Atomic Energy Licensing (Basic Safety Radiation Protection) Regulations 2010 and both of them are from Angiography Unit. From the questionnaire survey, 42 of the workers are from high exposure group and 54 personnel from the low exposure group. All workers compliant to the use of personal protective equipment and RPL dosimeter. Respondents from high exposed group were shown to have higher DNA damage with the tail moment values than respondent from low exposed group. Statistical comparison showed frequency of MN formation is significantly higher in elder individuals above 35 years of age (p=0.002). There is a significant weak correlation (p=0.033; r=0.21) between cumulative radiation dose and MN frequency. In conclusion, this study found that there is a relationship between occupational exposure to ionizing radiation and MN frequency. The management workers’ safety and health should be optimized to minimized toxicity from radiation exposure in Radiology Department.

Keywords: genotoxicity, ionizing radiation, radiology department


Urinary 8-hydroxyl-2′-deoxyguanosine (8-OHdG) Levels of Electronic Cigarette (e-cigarette) Users

Göksel Koç Morgil 1, Awat Abdullah Ali 2, İsmet Çok2, Onur Kenan Ulutaş2, Edibe Nurzen Bozkurt1, Yıldırım Cesaretli1

1 Minister of Health, General Directorate of Public Health, Department of Consumer Safety and Public Health Laboratories, Toxicology Laboratory, Sıhhıye, Ankara,Turkey, 2 Gazi University, Faculty of Pharmacy, Department of Toxicology, 06330, Hipodrom, Ankara, Turkey


The usage of e-cigarettes has increased dramatically during the last decade because e-cigarettes have been widely advertised in many countries, mostly through the Internet. Despite the recent popularity of e-cigarettes, to date only limited data is available on their safety and effectiveness  for users. Recent chemical  analysis  of  e-cigarettes  has  showed  that variety toxicants  and  carcinogens  were  detected in the refill solutions and aerosols of e-cigarettes such as nitrosamines, formaldehyde, acetaldehyde, acrolein, metals, and PAHs. The health risks of these compounds are of major concern to health care authorities. One of the most important of risks posed by these chemicals are creating alterations in DNA. We need to identify these alterations that could occur in DNA molecule due to the use of e-cigarettes. 8-hydroxyl-2′-deoxyguanosine (8-OHdG)  has been presented as a sensitive biomarker for determination of  alterations in DNA especially oxidative DNA damage and genotoxicity. In this study we aimed to investigate the possible oxidative stress by measuring 8-OHdG  in urine samples of subjects who use e-cigarette and compare the results with those obtained from classic cigarette and passive smokers.

Between May 2016 and September 2017, spot urinary samples were collected from e-cigarette users (n:28), cigarette smokers (n:25), passive smokers (n:25) and healthy non smokers (n:25). 8-OHdG levels were determined using Liquid chromatography tandem-mass spectrometry (LC-MS/MS) and expressed as ng/g creatinine. 8-OHdG levels in e-cigarette users (16.59±12.56) were significantly higher (p>0.0023) than those of healthy controls group (7.36±7.39).

Keywords: e-cigarettes, health risk, biomarker, DNA damage

Urinary Excretion of Aflatoxin M1: A Survey


Raul Ortiz-Martinez1, Ma. Carolina de Luna-Lopez1, Arturo G. Valdivia-Flores2, Teodulo Quezada-Tristán2

1 Department of Animal Science. Agricultural Science Center. UAA, 2 Department of Veterinary Clinic. Agricultural Science Cente, UAA


Aflatoxin M1 (AFM1) is one of the less toxic forms of aflatoxin B1 (AFB1). When the presence of AFM1 is detected in urine, means in an indubitable way, that people consume food contaminated whit AFB1. Precisely, the determination of the AFM1 concentration in urine correlates with the amount of ingested AFB1 so it is a linear indicator associated with its consumption.

A biomarker is a variation in cellular or biochemical components or processes, structures, or functions that is measurable in a biological system or sample; these changes are induced by xenobiotics. Based in this fact, the presence of AFM1 in organic fluids, has been consider an excellent exposure biomarker of AFB1.

The objective of the study is to explore the exposure to AFB1, through measuring AFM1 concentration in urine of volunteer adults.

A convenient sampling (non-probabilistic sampling), was performed in Aguascalientes, Mexico. In the study both female and male gender was included. Informed consent was signed by the participants in the research.

The samples were analyzed according to directions of an ELISA commercial kit (Detection range: 0-4.0 ng/mL).

At the present, 223 volunteer adults, were sampled in order to determinate AFM1 in urine. The study has had 46% male and 54% female volunteers. The average age of the sampled participants was 20 year-old. The 60% of samples had detectable levels of AFM1. The average concentration of AFM1 in urine samples was 0.40 ng/mL.

These results implicate the importance of biomarkers as predictive tools to detect early potential alterations and eventually, improve public health.

Keywords: aflatoxin B1, aflatoxin M1, biomarkers, urine




 Antigenotoxic Effects of Cinnamic Acid in Diabetic Rats


Hatice Gül Anlar1, Merve Bacanlı2, Tuğbagül Çal1,3, Sevtap Aydın2, Nuray Arı4, Ülkü Ündeğer Bucurgat2, Arif Ahmet Başaran5, Ayşe Nurşen Başaran2

1Bülent Ecevit University Faculty of Pharmacy Department of Pharmaceutical Toxicology 67600 Zonguldak, Turkey 2Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Toxicology 06100 Ankara, Turkey 3Karadeniz Technical University Faculty of Pharmacy Department of Pharmaceutical Toxicology 61080 Trabzon, Turkey 4Ankara University Faculty of Pharmacy Department of Pharmacology 06100 Ankara, Turkey 5Hacettepe University Faculty of Pharmacy Department of Pharmacognosy 06100 Ankara, Turkey


Diabetes mellitus (DM) is a major health problem worldwide. Cinnamic acid (CA) and its derivatives are synthesized in the plants and increasing attention has been received in the recent years due to the high number of beneficial health properties attributed to their consumption. The aim of this study was to investigate the antigenotoxic effects of CA on streptozotocin (STZ)-induced diabetes in Wistar albino rats. The rats were divided into four groups: Group 1: Sham group (n=6), group 2: diabetic group (n=6), group 3: CA treated group (n=6), group 4: CA treated diabetic group (n=6). CA dose (50 mg/kg b.w. per oral) was selected according to our unpublished studies. At the end of the experimental period, all animals were decapitated under the anesthesia. Whole blood samples were obtained via the intracardiac method. Liver and kidneys were removed. DNA damage was evaluated in the blood, liver and kidney cells of rats by the alkaline comet assay. 8-hydroxy-2’-deoxyguanosine (8-OHdG) levels, a critical biomarker of oxidative stress, were also evaluated by in the plasma samples by spectrophotometrically using kit following the manufacturer’s instruction at 535 nm. As a result of this study, DM caused genotoxic and oxidative damage. CA treatment ameliorated these effects and it seems that CA might have a role in the prevention of the complication of diabetes.

Keywords: diabetes mellitus, DNA damage, oxidative stress, 8-hydroxy-2’-deoxyguanosine, comet assay

Estrogen Receptor Antagonısts as a Target for Treatment of Estrogen-Induced Carcınogenesıs


Elif Ince1 , Alev Tascioglu1, Ozlem Oztürk-Ceylan2, Sibel Suzen2, Hande Gurer-Orhan1

1Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Ege University, Izmir, Turkey,2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey


Breast cancer is the leading cause of death among female cancers and prolonged exposure to elevated levels of estrogens is suggested to play an important role in the process. Melatonin (MLT) is reported to exert oncostatic effect which is partly mediated by its selective ER modulator and aromatase inhibitor activities. Although MLT has low toxicity, its short half life limits its therapeutic use. This drawback can be conquered by synthesizing MLT analogues with longer half life. Our group has already been working on investigating antioxidant and aromatase inhibiting activity of indole-based MLT analogues. The aim of the present study is to evaluate potential antiestrogenic activity of analogues which were found to have antioxidant and aromatase inhibitor activity. Their antiestrogenic effect is investigated in a two-stepped, modified miniaturized E-Screen assay, which is based on estrogen dependent MCF-7 cells to proliferate only in the presence of estrogenic compounds. In the first step various concentrations of the compounds are added to the medium containing fixed dose of estradiol. Some of the compounds were found to inhibit estrogen action in a dose-dependent manner. Those analogues were further tested in step-2, in order to evaluate whether this effect was receptor dependent. Increasing concentrations of estradiol are added onto fixed dose of the compound and fortunately three of the compounds are found to be promising ER antagonists. As a conclusion some of our indole-based MLT analogues seem to be promising candidates for breast cancer therapy via their triple benefits; as antioxidant, aromatase inhibitor and ER antagonist.

This work was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK) Grant 117S065 and 109S099. Analyses are performed at Ege University Faculty of Pharmacy, Pharmaceutical Sciences Research Center (FABAL).


Keywords: breast cancer, estrogen receptor antagonist, melatonin analogues

Oxıdatıve Stress and Dna Damage Caused by Helıcobacter Pylorı in Human Gastrıc Adenocarcınoma Cells


Didem Orala, Gizem Ozkemahlıa,b, Unzile Sura,c, Belma Kocer-Gumusela, Pınar Erkekoglua,*

aHacettepe University, Faculty of Pharmacy, Department of Toxicology, 06100 Ankara, Turkey< bErzincan University, Faculty of Pharmacy, Department of Toxicology, Erzincan, Turkey, cAtatürk University, Faculty of Pharmacy, Department of Toxicology, Erzurum, Turkey


The association between chronic inflammation and the development of cancer has been asserted for a long time. Understanding the role of chronic bacterial inflammation in oncogenesis has been gaining importance for the past 20 years. Certain bacteria can also promote carcinogenesis due to their direct effects on cell transformation. Helicobacter pylori is helix-shaped gram-negative anaerobic bacterium that leads to gastritis and peptic ulcer, and later to gastric cancer. The aim of this study was to investigate whether oxidative stress is one of the mechanisms underlying the toxicity of Helicobacter pylori. Human gastric adenocarcinoma cells were exposed to different multiplicities of infection (MOIs) with the bacterium. The intracellular reactive oxygen species (ROS) production and oxidative stress parameters were elucidated. After incubation of the adenocarcinoma cells with different MOIs (25, 50, 75, 100, 200 and 400) of Helicobacter pylori, we observed that this bacterium causes MOI-dependent cytotoxicity. The median inhibitory concentration 50 (IC50) was found to be 218 bacterium/ live cell and IC30 was 138 bacterium/ live cell. We observed that at IC30 MOI, this bacterium caused significant increases in ROS and DNA damage. Moreover, there were significant changes in intracellular glutathione and lipid peroxidation after human gastric adenocarcinoma cells were exposed to Helicobacter pylori. We can suggest that one of the mechanisms underlying the toxicity of Helicobacter pylori is oxidative stress. More mechanistic studies are needed to prove the toxicity mechanisms of Helicobacter pylori.

Keywords: chronic inflammation, oncogenesis, adenocarcinoma, Helicobacter pylori



Role of Circadian Regulated Gene Expression in Mutagenesis and Carcinogenesis


Helmut Zarbl, Howard Kipen and Mingzhu Fang.

Environmental and Occupational Health Sciences Institute. Rutgers University. Piscataway, New Jersey, U.S.A.  08854


Methylselenocysteine (MSC) reduces chemically-induced mammary carcinogenesis in all susceptible rat strains. MSC mediates its effects by restoring circadian regulated expression of hormone receptor and DNA repair genes disrupted by carcinogen exposure. Expression of core circadian genes is regulated by oscillation in the acetylation of histones on specific E-box enhancer elements in their promoters. Acetylation is increased by the intrinsic histone acetylation activity of the Clock protein, while deacetylation is mediated by Sirtuin 1, a NAD+-dependent protein deacetylase enzyme. The cellular clocks in an organism are synchronized by entrainment with environmental cues that include light, temperature and metabolism, and can be disrupted by a wide variety of environmental toxicants and stressors. Mechanistic studies showed that carcinogen exposures ablate circadian rhythm by depleting intracellular levels of NAD+/NADH, which in turn inhibits the activity of the Sirtuin 1 histone deacetylase enzyme. Increased sensitivity to circadian disruption by chemicals or shifting light-dark cycles (chronotype) contributes to the differential genetic susceptibility of rodents the mammary carcinogenesis.

Disruption of circadian gene expression by exposure to light-at-night through shift work or jet lag is also mediated by depletion of intracelluar NAD+/NADH. Animal studies showed that MSC restores circadian gene expression and reduces carcinogenesis in animals exposed to jetlag protocols. These studies were translated into a placebo-controlled MSC intervention trial in shift workers who experience circadian disruption.

Keywords: circadian gene expression, carcinogenesis, MSC  






Determination of Serum Manganese in Women with Polycystic Ovaries Syndrome and in the Ukraine Population


Serhiy Gunkov, Yana Kolianchuk,  Oleksiy Makarov

  1. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health, Ukraine


Polycystic ovarian syndrome (PCOS) belongs to polyethiologic diseases. Manganese may provoke the occurrence of endocrine pathology. The purpose of this study was to determine the serum manganese in women with polycystic ovaries.

The method ICP-MS was used for determination the manganese in the blood serum of 52 women with PCOS. The control group was consisted of 38 reproductive age women. Manganese indicator in 61 specimens of the donor’s blood of both sexes was determined with goal to assess the exposure level of the population. Data of manganese exposure indicators on Ukraine’s population, published in domestic journals from different regions and scientific institutions of Ukraine was taken into account.

Our results indicate that the median index equal 0.0035 mg/l, 95th-percentile – 0.014 mg/l in the control group of women. In women with PCOS, the rates were significantly higher (p 0.0012), median – 0.0095 mg/l, 95th-percentile – 0.13 mg/l. Results showed that women with PCOS have significantly higher levels of manganese exposure. As manganese applies to endocrine disruptors, this gives grounds to assume that high concentrations in the serum may contribute to cause of PCOS. The results showed that in the donors blood, the median was 0.011 mg/l, 95th-percentile – 0.018 mg/l.

Part of the Ukraine population is highly exposed to manganese. Women with PCOS revealed high concentrations of manganese in serum. We can conclude, the occurrence of PCOS may be associated with high levels of manganese exposure.


Keywords: polycystic ovarian syndrome, manganese, exposure.

Intoxications by Heavy Metals in Patients with Hip Arthroplasties


Fernandez-Ibanez Ana, Ugalde-Herra Rodrigo, Rodriguez-Getino Jose Angel, Perez-Casas Juan Bautista, Diaz-Suarez Jorge Cipriano1

Department of Medicine – University of Oviedo (Asturias) Spain


The appearance of trace elements in routine serum analysis can be an indicator of the beginning of the wear of a prosthesis.

The aim of this research was to check, in patients who have inflammatory joint symptoms, if heavy metals in the toxicity range appear in their serum.

19 patients aged between 36-78 years who presented with pain or infection in their hip prosthesis were classified according to the prosthesis’ composition as cobalt, chromium and titanium. These elements were determined corresponding patients. The samples were centrifuged at 10,000 rpm for 10 minutes, separated and frozen at -80ºC until processing. Elements were all analysed using induced coupling plasma mass spectrometry (ICP-MS).

3 groups were differentiated according to the trace element analysed. Each element has its own reference limit for toxicity. For Titanium (n=6), one patient had a level of 18μg/L which is indicative of prosthetic deterioration and confirmed the clinical suspicion. For Cobalt (n=5), a patient with concentrations of 27μg/L was operated urgently for a prosthetic replacement and followed up with routine analytical controls. For Chromium (n=15), several patients had prostheses that needed checking because the concentrations found were between 1.2μg/L-4.4μg/L.

Heavy metals’ concentrations should not be used in isolation, but must be interpreted in the context of the complete clinical problem. The determination of trace elements is difficult because the detection technique has a high cost and is not always available in the clinical environment, which sometimes implies a delay in the diagnosis of the patient.


Keywords: cobalt, trace element, titanium, chromium, arthroplasties



Severe Benzodiazepine Poisoning in Elderly – A Case Report


Natasa Perkovic Vukcevic 1, Gordana Vukovic Ercegovic1, Vesna Mijatovic2, Olivera Potrebic1, Snezana R Jankovic3, Zivanovic Dragan1, Jasmina Jovic Stosic1

1 National Poison Control Centre, Military Medical Academy, Belgrade, Serbia, 2 Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Novi Sad, Serbia, 3 Institute for Scientific Information, Military Medical Academy, Belgrade, Serbia


A case of an elderly woman with a prolonged decreased level of consciousness and a decreased rate of bromazepam elimination in acute-bromazepam-overdose settings is described. Previously healthy 67-year-old woman was found unconscious with Glasgow Coma Score of 3, normotensive, normofrequent and euglycemic. MSCT scan was normal. After the flumazenil application, a positive response was observed. The patient reported the ingestion of 90 mg of bromazepam. She was referred to the intensive care unit in coma, while vital and laboratory parameters were within a normal range. Toxicological analysis of the blood identified bromazepam in concentration of 2.73mg/l. Flumazenil treatment was continued (both bolus and infusion). EEG was consistent with the findings in benzodiazepines overdoses. On the fifth day of hospitalization, the patient became febrile. Chest radiography revealed a pneumonic infiltration. Leukocytosis and elevated CRP were detected and an antibiotic treatment was introduced. Rhabdomyolysis (CPK level up to 7900i.u.) was also developed as a complication. Due to very slow recovery of consciousness, the blood bromazepam concentrations were determined repeatedly. At day 7, the bromazepam blood level was 2.32mg/l. The patient was hypotensive. The 20% Intravenous Fat Emulsion was applied. After its administration, the bromazepam blood level was 1.2mg/l and 0.6mg/l. Flumazenil treatment was continued up to the 14th day of hospitalization when the patient was conscious, respiratory and cardiocirculatory stable, with bromazepam blood concentration of 0.1mg/l.

Altered metabolism and slower elimination of benzodiazepines in elderly could be responsible for a more severe clinical course of acute benzodiazepines poisoning.

Keywords: bromazepam, overdose, flumazenil




Society Transition and Trends of Acute Poisonings in Republic of Macedonia


Niko Bekjarovski, Daniela Chaparoska, Zhanina Perevska, Natasha Simonovska, Irena Jurukov, Aleksandra Babulovska

University clinic for toxicology, University campus “Mother Theresa”Skopje, Macedonia


Last three decades big transition happened in all former socialist countries. New economic system developed new habits and illness among citizens in these countries.

The aim of the study is to present the changes and new trends in acute poisonings in last three decades in Republic of Macedonia.We analyzed patients treated in University Clinic for toxicology in Skopje, Macedonia after acute poisoning in the period from 1987 till 2017.The total number of acute poisonings has a trend of slow increase, especially in the last 5 years. The trend of acute poisonings with pesticides dramatically dropped down after 1992. Poisonings with barbiturates are almost totally replaced with acute poisoning with benzodiazepines. Benzodiazepines are the most frequent drugs of abuse, and have a trend of continues rising. The numbers of acute corrosive poisonings in last thirty years is almost tripled and are the second reason for hospitalization in our Clinic.

Poisonings with benzodiazepines and corrosive substances are the new challenges in our everyday clinical practice. Poisonings with pesticides and barbiturate, typical for the 80”s of last century are very rare in now days practice.

Keywords: intoxications, pesticides, corrosive, drugs






A Systems Biology Approach to Discovery the Mechanism of Regulation of Repetitive Prophylaxis of Stable Iodide on Sodium/Iodide Symporter (NIS)


David P.A. Cohen1, Dalila Lebsir1, Karine Tack1, Marc Benderitter2, Maâmar Souidi1

1 Institut de Radioprotection et de Sûreté Nucléaire (IRSN), PSE-Santé/SESANE/LRTOX, 92262 Fontenay-aux-Roses, France, 2 Institut de Radioprotection et de Sûreté Nucléaire (IRSN), PSE-Santé/SERAMED, 92262 Fontenay-aux-Roses, France


A single dose of potassium iodide (KI) against prolonged exposure to repeated radioactivity might not be effective enough to protect the thyroid. Our group showed that repetitive dose of KI for eight days offers efficient protection without toxic effects on rats. However, we also have shown that the expression of the genes involved in the Wolff-Chaikoff effect changes during this period. This effect may result in a decrease in thyroid hormones and hypothyroidism. Notably, a decrease in the sodium/iodide symporter (NIS) gene expression has been observed. NIS is responsible for the uptake of KI and plays an important role in the Wolff-Chaikoff effect. The mechanism of a single dose of KI on the toxicity of the thyroid is well known in contrast to repetitive administration of KI for eight days.

In the present study, we try to understand the Wolff-Chaikoff regulation and its molecular constituents during repetitive administration of KI. For this we have constructed a biochemical reaction network that is visualised as a “geographical” map of the thyrocyte depicting iodide and thyroid hormone metabolism. Subsequently, Path analysis of the network has been performed to investigate if a path exists from the node iodide going to the node representing “nis transcription”.

This map reviews the most updated information about iodide and thyroid hormone metabolism. Besides as a source of information, it can help to elucidate the mode of action of KI on gene transcription after repetitive KI administration. We have found two mechanisms that might explain the inhibition of nis.


Keywords: network, map, mechanism, Wolff-Chaikoff, thyroid


In Silico Methods to Predict the Toxicity of Mixtures: Current Status and Future Directions


Mark Cronin, Steve Enoch, James Firman, Judith Madden, Samuel Belfield

School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, England



In silico models provide invaluable tools to fill gaps where toxicological (and other) information is missing. However, approaches such as the use of (Quantitative) Structure-Activity Relationships ((Q)SARs) and read-across have usually been targeted towards the effects of single chemical substances. This study has reviewed the use of (Q)SARs and read-across for the prediction of the toxicity of mixtures with a view to providing recommendations for their future implementation. The term “mixture” covers many types of chemistries ranging from commercial formulations and products; unknown or variable composition, complex reaction products or biological materials (UVCBs); botanicals; nanomaterials and other substances such as raw products and surfactants of variable chain length. Development of predictive models for mixtures requires that its composition is fully understood to enable mixture toxicity effects such as concentration addition, synergy and antagonism to be captured. Modelling is often based on the identification of the hazard of individual components with a “worst-case scenario” enacted. This is simplistic and in some cases effective, however, it lacks appreciation of relative exposure which can be increased or decreased by the other components in a mixture. A better mechanistic understanding and knowledge of “complex” Molecular Initiating Events within the Adverse Outcome Pathway paradigm will be the cornerstone of the development of better models – especially if the theory can be supported by relevant mechanistic data. The challenges remain the need to obtain such data, the anchoring to in vivo results and extrapolation to human exposure.


Keywords: computational, toxicology, QSAR

Phthalates (diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP)) and Obesity: a Toxicogenomics Approach


Katarina Baralić1, Dragica Jorgovanović1, Danyel Jennen2 Danijela Đukić-Ćosić1

1Department of Toxicology “Academic Danilo Soldatović”, University of Belgrade – Faculty of Pharmacy, Serbia 2Department of Toxicogenomics, Maastricht University, The Netherlands


The etiology of many chronic diseases, including obesity, involves interactions between environmental contaminants and genes that modulate physiological processes. Diethylstilbestrol, bisphenol A, phytoestrogens, and phthalates are considered the most well-known obesogens. People are most often simultaneously exposed to multiple phthalates as a result of their use as plasticizers and additives in a variety of consumer products.

The aim of this study was to explore the relationship between the two most commonly used phthalates (diethylhexyl-phthalate (DEHP) and dibutyl phthalate (DBP)) and obesity, using the Comparative Toxicogenomics Database (CTD).

A set of genes of interest was constructed, consisting of 850 curated genes affected by both phthalates. Obtain results indicate that DEHP and DBP correlate with the development of obesity – DEHP by interacting with 55 genes involved in 63 molecular pathways, and DBP by interacting with 79 genes involved in 47 molecular pathways. Both phthalates affect the activity of 41 genes associated with the development of obesity, involved in 38 common molecular pathways. Among these genes, PPARA, the gene with the majority of curated interactions for DEHP (519),  PPARG and ATP citrate lyase gene (ACLY) stand out as key factors in lipid metabolism. Additionally, both phthalates interact with insulin 1 gene (INS1), which is important having in mind that obesity is linked to an increased risk of developing insulin resistance and type 2 diabetes.

These results provide a basis for further in vitro and in vivo studies in order to better understanding the molecular mechanisms of phthalate obesogenic properties.

Keywords: plasticizers, obesogens, toxicogenomics, CTD, genes






Effect of Low Oral Cadmium Exposure During Pregnancy on Steroid Hormones in Mother Rats and Female Offsprings


Anja Mikolić, Martina Piasek, Tatjana Orct, Antonija Sulimanec Grgec, Ljerka Prester, Jasna Jurasović

Institute for Medical Research and Occupational Health, Zagreb, Croatia


Cadmium is a toxic metal and environmental pollutant with the potential to act as an endocrine disrupting chemical of reproduction and reproductive development. The main cadmium exposure source is food. Its gastrointestinal absorption in laboratory rats is up to 1% and increases 2-3 times during gestation. We evaluated the effects of low cadmium exposure (5 mg Cd/L as CdCl2 in demineralised water) in Wistar rats exposed from gestation day (GD) 1 to 20 on steroid hormones in the placenta and maternal serum on GD20 and in sera of 14-day-old female pups (at weaning) and at the onset of puberty. On GD20, part of the mother rats were euthanized under anaesthesia, blood was taken from the heart and the liver, kidney, placentas and foetuses were sampled. Blood, liver, kidney and brain were sampled in 14-day-old and pubertal female offspring. Tissue cadmium was analysed by ICP-MS and progesterone in placenta and serum by ELISA. In the exposed rats, cadmium increased in all of the sampled maternal tissues, placenta, foetuses and 14-day-old pups’ liver. Placental progesterone did not change, whereas progesterone levels decreased in maternal serum on GD20 and in serum of 14-day-old female pups. Onset of puberty was between postnatal days 47-54, a week later in female than in male offspring, irrespective of cadmium exposure. The results show that low cadmium exposure during gestation may disrupt serum progesterone levels in both mother rats and female pups before puberty, which may have impact on foetal viability in utero and/or female reproductive development.

Keywords: cadmium, gestation, steroid hormone disruption, reproductive development, rat



Effects of BPA on Global DNA Methylation and Global Histone 3 Lysine Modifications in SH-SY5Y Cells: An Epigenetic Mechanism Linking the Regulation of Chromatin Modifiying Genes


Mine Senyildiz1, Ecem Fatma Karaman1, Serap Sancar Baş2, Pelin Arda Pirincci2, Sibel Ozden1

1 Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, 34116-Beyazit, Istanbul, Turkey, 2 Department of Biology, Faculty of Science, Istanbul University, 34134-Vezneciler, Istanbul, Turkey.


Bisphenol A (BPA), an estrogenic endocrine disruptor, is widely used in the production of polycarbonate plastic and epoxy resins, resulting in high risk on human health. In present study we aimed to investigate the effects of BPA on global and gene specific DNA methylation, global histone modifications and regulation of chromatin modifiying enzymes in human neuroblastoma cells (SH-SY5Y). Cells were treated with BPA at 0.1, 1 and 10 µM concentrations for 48 and 96 h. IC50 value of BPA was determined as 183 and 129 µM in SH-SY5Y cells after 24 h by MTT and NRU tests, respectively. We observed significant alterations on the 5-mC% levels (1.3 fold) and 5-hmC% levels (1.67 fold) after 10 µM of BPA for 96 h. Significant decrease was identified in H3K9me3 and H3K9ac after 10 µM of BPA for 96 h while decrease was observed in H3K4me3 at 10 µM of BPA for 48 h. Alterations were observed in chromatin modifiying genes including G9a, EZH2, SETD8, SETD1A, HAT1, SIRT1, DNMT1, RIZ1 and Suv39h1 after 96 h of BPA exposure. We suggest that especially dramatic increase of SIRT1 regulation accordance with the decrease on H3K9ac might be useful target for the BPA inducing toxicity. In addition, Suv39h1 regulation might involve to the mechanisms of the BPA toxicity. Our study might provide a new perspective for epigenetic alterations of BPA in SH-SY5Y cells due to the fact that limited studies are available on the epigenetic effects of BPA toxicity in neuroblastoma cells.

Keywords: bisphenol A, DNA methylation, histone modifications, chromatin modifiying genes, neuroblastoma cells

Determination of Phthalates in Toys


Marija Stanković1, Ana Stanisavljev1, Anka Cvetković1, Nenad Vuković1

1Institute for Public Health, Bulevar Despota Stefana 54a, Belgrade



Phthalates are used as plasticisers, primarily in PVC, and can be also used in paints and adhesives. Phthalates can disrupt our hormones and may cause fertility problems. Furthermore they are connected with childhood,  obesityasthmaneurological problems obesityasthmaneurological problems, cardiovascular issues and even cancer. In our laboratory we analyze toys before they reach the market. Six phthalates determined for that purpose are: bis (2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), benzyl butyl phthalate (BBP)  di-isononyl phthalate (DINP)  and  di-isodecyl phthalate (DIDP)  and  di-n-octyl phthalate (DNOP) . Phthalates are extracted from samples with tetrahydrofuran  for 30 minutes on rotating mixer or ultrasonic bath. After that hexane is added to release PVC polymer. Samples are left for 15 minutes in fridge, for better precipitation of PVC polymer. After that supernatant is filtrated and content of phthalates is determined by GC-MS.

In our laboratory, 111 toys were analyzed for the content of phthalates in the last year. Content of phthalates in nine samples was above maximum allowed concentration defined by National Regulation, that is less than 10 percent of all samples.

Keywords: plasticizers, GC-MS



Do Biflavonoid Constituents of St. John’s Wort Have Endocrine Modulating Effets?


Alev Tascioglu1, Senem Ozcan Sezer1, Duysal Uslu1, Elif Ince1, Hande Gurer-Orhan1

1 Ege University, Faculty of Pharmacy, Pharmaceutical Toxicology Department, Izmir/Turkey


St. John’s Wort (SJW) is used as a dietary supplement in depression mostly in postmenopausal women. In addition, an active constituent of SJW, hyperforin, is shown to modulate endogenous estrogen levels. SJW has more active compounds such as naphthodianthrones, flavonoids (flavonol glycosides and biflavonoids) which aren’t evaluated for their potential hormone-related effects. In the present study, SJW extract and two of its biflavonoid constituents, amentoflavone and biapigenin, were investigated for their endocrine modulating effects via two mechanisms: estrogen receptor modulating effect was evaluated by E-Screen assay and estrogen synthesis modulating effect was evaluated by aromatase inhibition assay since aromatase is responsible for local estrogen synthesis. E-screen results showed that, SJW extract has agonistic effects on estrogen receptor alpha (ERα) with 0,01 mg/ml EC50 value while its biflavonoids didn’t show any effects on ERα. In aromatase inhibition assay, SJW extract inhibited aromatase as well as amentoflavone which showed aromatase inhibition with a 93,6µM IC50 value. The extract of St. John’s Wort is a herbal dietary supplement intended to treat symptoms of depression. Possible endocrine modulating effects of SJW found in the present study suggests that the consumption of SJW extract by vulnerable populations, such as pregnant women and children, might cause adverse effects. Furthermore amentoflavone, one of the biflavonoid constituents of SJW, is found to be involved in aromatase inhibitory effect of the extract.

This work was supported by TUBITAK grants 108S202 & 112S375 and Ege University Research Fund grant 13ECZ008. Analyses were performed at Ege University Pharmacy Faculty Research Laboratories, FABAL.


Keywords: St. John’s Wort, amentoflavone, biapigenin, E-Screen, aromatase inhibition



Current Status of Public Awareness About Endocrine Disrupting Chemicals in Slovenia


Lucija Kolar ,1,2 Igor Muršec 2,

1 Complementarium, Institute for Environmental Technologies and Research of Nature (CMP Lopata), Lopata 60, SI-3000 Celje, Slovenia. 2 Environmental Protection College, Trg mladosti 7, SI-3320 Velenje, Slovenia.


Our research shows current situation regarding public recognition of the term endocrine disrupting chemical (EDC) and public awareness about pollution with them at three different sites in Slovenia. EDCs may interfere with the body’s endocrine system and produce adverse developmental, reproductive, neurological, and immune effects in humans and wildlife. Chemicals commonly detected include DDT, polychlorinated biphenyls (PCB’s), bisphenol A (BPA), polybrominated diphenyl ethers (PBDE’s), and variety of phthalates.

267 respondents answered the questionnaire we used in the research. There were several important findings carried out. Our focus was public opinion and awareness about which EDCs are most widely recognized and what the most important routes of body entering are.

Respondents mainly include plant protection products, pharmaceutical agents, phytoestrogens and metals in the EDC group. Slovenians know that EDCs are introduced to the body primarily through food, medicines and cosmetics. They are aware that EDCs have an impact on the organism already in small quantities and that the most endangered are children. We also investigated how many Slovenians know whether there are EDC polluted areas (Mežica Valley, the Krupa River, north-eastern part of Slovenia). Most of the respondents knew about the problems of the Mežica valley but not many were aware about the other two areas. Respondents also gave an opinion on whether substances containing EDCs should be properly labeled or even withdrawn, and we received an affirmative majority answer to both questions.

With this research we set the basics to further raise the awareness and spread the knowledge about EDCs.

Keywords: endocrine disrupting chemicals, recognition, public awareness, national opinion


Teratogenic and Embryotoxic Effects of Diisononyl Phthalate


Hrynchak Vitali, Sychik Sergei, Il’yukova Irina

Republican unitary enterprise «Scientific practical centre of hygiene», Academic 8, Minsk, Belarus


A toxicological study was conducted to investigate the effect of diisononyl phthalate on the reproductive system of white rats. The embryotrophic and teratogenic effects were registered taking into account the dynamics of development of offspring in the postnatal period. The presence of anomalies in the development of the internal organs of embryos was determined by the sagittal section method according to W. Wilson. It was found that intragastric administration of the studied compound to females during the pregnancy period at doses of 100, 1000 and 10,000 mg/kg initiated external and internal malformations of embryos (absence of interventricular septum, intestinal and/or liver ejection, microphthalmia, anencephaly, hydrocephalus, acronia, encephalocele, micrognathia and hypoplasia of the lower lobe of the lung). The exposure level of 10000 mg/kg is characterized by an increase in total postimplantation and embryonic mortality, the presence of multiple embryonic developmental defects. Observations of the process of postnatal development of the rats were carried out starting from the day of birth of the rats up to 60 days of age. It was established that postnatal mortality at a level of exposure to diisononyl phthalate of 10000 mg/kg significantly increased by 22.7%. According to the parameters of physical development (weight and body length), the female rats of the experimental groups receiving phthalate did not differ from the control groups. Also, there were no reliable deviations in the estimated coefficients of testes and appendages, functional spermatozoa.

Keywords: diisononyl phthalate, embryotoxicity, teratogenicity, toxicity



In vitro Estimation of Bisphenol S Toxicity

Maja Milanović1, Dragana Četojević-Simin2, Nataša Milošević1, Milica Medić Stojanoska3, Nataša Milić1

1University of Novi Sad, Faculty of Medicine, Department of Pharmacy, Novi Sad, Serbia, 2University of Novi Sad, Faculty of Medicine, Experimental Oncology Department, Oncology Institute of Vojvodina, Sremska Kamenica, Serbia, 3University of Novi Sad, Faculty of Medicine, Clinic for Endocrinology, Diabetes and Metabolic Diseases, Clinical Center of Vojvodina, Novi Sad, Serbia


The association of bisphenol A (BPA) with a range of health disorders such as fertility problems, diabetes, obesity, cardiovascular diseases and increased carcinogenic risk was confirmed in the considerable number of studies. Due to the extensive exposure to BPA through the variety of everyday products, the major issue is to find the safe replacement of this compound. Nowadays, bisphenol S (BPS) is used as BPA alternative without restriction. Toxicological data on BPS are very scarce. However, similar physico-chemical properties could lead to the potent toxicological profile. Therefore, in vitro toxicity after acute BPS exposure was estimated in the panel of mammalian cell lines. Cell growth effects of BPS were evaluated in human cervix carcinoma (HeLa), breast adenocarcinoma (MCF7), colon adenocarcinoma (HT-29) fetal lung (MRC-5) and rat hepatoma (H-4-II-E) cell lines using colorimetric sulforhodamine B assay. EC50 values were calculated from the concentration response curves following 48 h exposure time. BPS significantly impaired the growth of all investigated cell lines, i.e. the EC50 values were reached in the range from 35.94 to 103.15 mg/mL. When compared to BPA, BPS was from 3 to 15-fold less toxic. Narrow span of EC50 values that were obtained at low concentrations indicate general toxic mode of action of BPS and raises awareness of the health risks associated with its ubiquitous presence in the environment.

Acknowledgment: This research has been financially supported by the Provincial Secretariat for Science and Technological Development, AP Vojvodina, Republic of Serbia, Grant No 114-451-2216/2016.


Keywords: endocrine disruptors, bisphenol S, toxicity, cell lines



Levels of Bisphenol-A in Thermal Paper Receipts from Serbia and Greece


Milan Milenković 1,2, Tatjana Nedeljković 1, Zorica Blagojević 1

1 Institute of Public Health of Serbia “Dr Milan Jovanović Batut”, 2 University of Belgrade-Faculty of Pharmacy


Bisphenol A (BPA) is a chemical substance which is globally produced in great quantities, and widely used as a color developer in thermal paper. Thermal paper is every day present in daily life, in form of cash register receipts. BPA used for this purpose is in unbound, free form, therefore risk of human exposure is present. BPA is one of the most common endocrine disrupting chemicals, and is also linked to certain developmental disorders, cardiovascular disease, and diabetes. Aim of this study was to determine concentration levels of BPA in thermal paper, in two countries. For this study, 20 thermal paper receipts, 10 from each country, were collected from various market places, randomly selected in Serbia and Greece, and analyzed. BPA was extracted and its analysis was performed using liquid chromatography/fluorescence technique. LOQ of method was 0.001% (0.01 mg BPA/g paper). BPA was detected in 19 samples. Samples concentrations were found in range of 0.59 – 1.32% (5.9 and 13.2 mg BPA/g paper), and 0.02 – 1.61% (0.2 and 16.1 mg BPA/g paper), in Serbia and Greece, respectively. Only one sample from Serbia had concentration below LOQ. The BPA concentrations measured in thermal paper receipts were comparable to those reported in similar studies.


Keywords: BPA, thermal paper receipts, endocrine disrupting chemicals, HPLC-FLD

Changes in the Oocyte Integrity and Bone Marrow Induced by 3-Methylcholanthrene and Prevented by α-Naphthoflavone


Rhon-Calderón EA1, Galarza RA1,2, Zurita S1, Faletti AG1,2.

1Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Centros de Estudios Farmacológicos y Botánicos (CEFYBO), Facultad de Medicina, Buenos Aires-Argentina, 2Universidad de Buenos Aires, Facultad de Medicina, Dto. de Toxicología y Farmacología, Buenos Aires-Argentina.


3-Methylcholanthrene (3MC) is an environmental pollutant that causes reproductive toxicity and genotoxicity. Previous studies showed that 3MC alters the ovarian function by affecting the follicle integrity, and causes DNA damage in peripheral blood, bone marrow and oocytes. Now, we studied the effect of daily exposure to 3MC on the oocyte integrity, induction of chromosome aberrations in bone marrow and the ability of α-naphthoflavone (αNF) to prevent this action. Immature rats were daily injected with a combination of 3MC (0.1 and 1 mg/kg) and αNF (80 mg/kg) for 20 days. Oocyte integrity, by morphological parameters; oocyte viability, by fluorescent dyes; and chromosome aberrations (CA) in bone marrow were evaluated. Compared with controls (C), both doses of 3MC increased the area (C 12.4±0.2 μm2; 3MC 14.5±0.7 μm2; P<0.001), perimeter (C 39±1 μm; 3MC 48±2 μm; P<0.001), and perivitelline space (C 1.6±0.1 μm; 3MC 2.3±0.1 μm; P<0.001); and decreased the thickness of the zona pellucida (C 1.7±0.1 μm; 3MC 1.1±0.1 μm; P<0.001) and the number of viable oocytes (C 86±3%; 3MC 60±3%; P<0.001). 3MC-treated rats exhibited a higher number of CA (C 7±2%; 3MC 20±4%; P<0.001), especially metaphases with dicentric chromosomes (C 2.4±0.5%; 3MC 8±2%; P<0.001) and fragments of chromosomes (C 1.0±0.5%; 3MC 3.6±0.4%; P<0.001). All these changes were prevented by daily treatment with αNF. In conclusion: i) daily exposure to 3MC alters the oocyte integrity; ii) low doses of 3MC are enough to induce CA in bone marrow; and iii) αNF prevents both the systemic and oocyte toxic effect of 3MC.

Key words: 3-methylcholanthrene, α-naphthoflavone, oocyte, bone marrow.








































































Resveratrol Inhibits Ovary Cells Proliferation Induced by Low Doses of Polychlorinated Biphenyls


Marina Miletić, Teuta Murati, Sanja Marđetko, Ivana Kmetič

Laboratory for Toxicology, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierotti St 6, 10000 Zagreb, Croatia


Toxic potential of PCBs is linked to various deleterious effects on human health and, in 2016 IARC has upgraded the classification of PCBs to Group 1 “Carcinogenic to humans”. Increased ROS levels by PCBs are able to promote tumor growth and malignant progression. This study was focused on low doses effects of PCB 77 congener at ovarian cellular level with the intention of reducing PCB induced proliferation by plant polyphenol-resveratrol. Resveratrol was selected because of protective effects on reproductive tissues and antioxidative activity. Initially, ovary cells (CHO-K1 cell line) were exposed sole to resveratrol in order to determine non-toxic doses to further experiments. Dose dependent cell growth inhibition after resveratrol exposure (2.5-150 μM) was confirmed with Trypan Blue method with significant effects in concentrations ≥ 5 μM (p<0.025-p<0.001). Neutral Red method, unequal to results obtained with Trypan Blue method, showed statistically significant cell growth inhibition in doses above 50 μM (p<0.001), while lower doses have shown beneficial effects on cell viability. Cytofluorimetric analysis confirmed elevated late apoptotic/death cells fraction for resveratrol ≥50 μM and therefore concentrations lower than 50 μM were further used in experiments. Cells were pre-treated with resveratrol, then treated with 1 μM PCB 77 (most effective dose in stimulation of cell proliferation) and after 24h cell viability was determined.

Presence of resveratrol efficiently suppressed PCB elevated cellular proliferation–statistically significant in doses 15-50 μM (p<0.05-p<0.01) confirmed with Trypan Blue method. These findings suggest that resveratrol alters cellular progression and may be effective in suppression of malignant progression.

Keywords: PCB 77, protection, plant polyphenol, cell viability, cancer



Biochemical Distrubances in Testes of Albino Rats with Metabolic Syndrome Induced in Prepubertal Age and Metformin Treatment


Oleksandr Tkachenko, Ganna Shayakhmetova, Alla Voronina, Valentina Kovalenko

SI “Institute of Pharmacology & Toxicology of NAMS of Ukraine”, Kyiv



Adolescence is decisive with regard to the formation of reproductive function, so almost in half of cases childhood and adolescence diseases are causes of male infertility. To date no emphasis has been placed on examination of effect of childhood MS on testes parameters in puberty. The aim of present study was an estimation of biochemical indices in rat testes following MS induced in childhood and metformin treatment. Wistar albino male rats of 21 days age were divided into 3 groups (10 animals in each): (1) control, (2) MS, (2) MS+metformin (266 mg/kg, 1 month). MS was induced by full replacement of drinking water by 10% fructose solution. Development of MS in childhood greatly affected testicular pro- and antioxidant systems: we recorded lipid peroxidation processes intensification 2,2 times, increase of catalase activity 22% and decrease of glutathione content 12% as compared with control. In group with metformin administration a tendency for inhibition of lipid peroxidation processes was detected. At the same time metformin forwarded full restoration of testicular reduced glutathione content and catalase activity. Thus, induction of MS in childhood provoked remote disturbances in testicular cells at biochemical level. These and other pathological events in testicular tissues need to be investigated profoundly as processes affecting spermatogenesis and probably underlying male infertility in adults. It should be highlighted that in very high-risk insulin-resistant children, pharmacotherapy with metformin may be indicated and this medicine use could also protect male reproductive parameters, probably due to attenuation of insulin-mediated oxidative stress.

Key words: metformin, male gonads, lipid peroxidation, glutathione, catalase


Multi-element Profile of Wines from Fruska Gora (Vojvodina)

Danijela Lukić 1, Milan Jovanović 1, Ivana Beara 2, Ljilja Torović 1,3

1Institute of Public Health of Vojvodina, Novi Sad, Serbia, 2 Department of Chemistry, Biochemistry and Environmental Protection, University of Novi Sad Faculty of Sciences, Novi Sad, Serbia, 3 Department of Pharmacy, University of Novi Sad Faculty of Medicine, Novi Sad, Serbia


The aim of this study was to provide elemental profile of wines from Fruska Gora, a vineyard in Vojvodina vine growing region, by reporting analysis of 23 elements (Be, B, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Sr, Mo, Cd, Sn, Sb, Te, Ba, Hg, Tl, Pb) in 91 bottled wines from 24 wineries, originating from 2009-2015. Analysis was carried out using ICP-MS, on diluted samples with internal standard addition.

Levels of elements that could pose a health risk (Pb, Cd, Sn, As, Fe, Cu, Zn) in all analysed wines were far below the current regulatory limits. It is of interest to note the content of As, due to the widely known problem of As contaminated ground water in Vojvodina: mean levels in red and white wines were 3.84 and 4.43 mg/kg, respectively, with maximum concentration (8.92 mg/kg) measured in a white wine. Generally, there were no substantial differences in mean levels of analysed elements between red, rosé and white wines, with the exception of Be, Al and Ni. Al concentration varied from 137 to 4671 mg/kg, with mean at 861 and 1536 mg/kg, in red and white wines, respectively.

Elemental composition of wine is influenced by endogenous sources and winemakers interventions. Certain elements can affect sensory properties of wine (Zn, Fe), some present health risk; furthermore, multi-element profile could contribute to differentiation of wines in terms of geographical origin. Results of this study should contribute to the recognition and positioning of wines from Fruska Gora.

Keywords: metals, wine, ICP-MS

Antibacterial Activity of Some Lamiaceae Essential Oils Against Some

Foodborne Pathogens


Danijela Pecarski1 ,Dragana Dragaš Milovanović 2, Jasmina Stanišić Bašić3

1,2,3  College of Professional Health Studies in Belgrade, Cara Dušana 254, Belgrade



The aim of this study was to evaluate and compare antimicrobial activity of 4 essential oils that belong Lamiaceae family (sage, oregano, thyme, ) and eucalyptus oil, against pathogenic and food spoilage bacteria and to choose the best one that can be potentially used as a food preservative. Gas chromatography with mass spectrometry (GC-MS) revealed that the highest percent of essential oils 98,93% include three classes of compounds – monoterpene hydrocarbons, aromatic hydrocarbons and oxidized monoterpenes. The main components are oxidized monoterpenes: carvacrol (59,03%); thymol (36,12%), eucalyptol (20.66%), hydrocarbon monoterpenes: limonene (30.96%) i a-pinene (12,21%) and aromatic monoterpene, p-cymene (22,25%) All essential oils showed great potential of antimicrobial activity against several bacteria and yeast C.albicans, using the agar diffusion method with wells. Minimum inhibitory concentration (MIC) for essential oils has been determined by the broth dilution method and valued in range of 1 µL/ml – 5 µL/ml., depends of essential oil and bacteria tested , and up to 100 µL/ml for C.albicans. Essential oils of Lamiaceae family provided the strong antibacterial activity for tested microorganisms, while the essential oil of thyme was especially recognized according to results of chemical and microbiological analysis. Considering its origin and numerous biochemical and pharmacological examinations, the probability for this essential oil to be toxic during pharmacological tests is very small, that is very important for its potential application as food preservative.

Keywords: essential oils, Lamiaceae, food bacteria, antibacterial activity, food preservative

Investigation of Toxic Metals, Nitrate and Nitrite in the Commercial Packed Drinking Water in Mashhad, Northeastern Iran


Seyed Reza mousavi 1, Mahdi Balali Mood 1, Sam Elmi 1, Mahmood Sadeghi 1, Monavar Afzalaghaee1,Bamdad Riahi Zanjani 1

 1Medical Toxicology Research Centre, Mashhad University of Medical Sciences, Mashhad, Iran.


Due to the increasing consumption of  bottled water in recent years, we planned to determinate the concentration of 6 toxic metals, nitrate and nitrite components in the bottled water which commonly sold in Mashhad, Iran.

The 11 best-selling brand of packaged drinking water in Mashhad’s market were identified. Eight bottles from each brand were randomly prepared and stored in a fridge at -4 oC. The concentrations of metals (lead, mercury, arsenic, chromium, aluminum, cadmium) in the samples were measured with atomic absorption spectrometric methods. Nitrate and nitrite were determined by a spectrophotometeric method. The results were analyzed by SPSS software and compared with the WHO and Australian guidelines. Also, the discrepancy between measured components and the depicted labels’ values were compared by means of one sample T-test, by assessing the P value of which being less than 0.05 would be assumed as significant difference.

The mean and SD of concentrations of the toxic metals in 11 brands were as below: lead 1.62±0.86 (µg/L), chromium 1.03±0.84 (µg/L), cadmium 0.17±0.07 (µg/L), mercury 3.86±1.57 (µg/L), arsenic 0.89±0.46 (µg/L), aluminum 6.56±4.54 (µg/L). The mean and SD measured quantities of nitrate, nitrite and pH were 9.96±5.95 (mg/L), 0.01±0.03 (mg/L) and 7.92±5.95, respectively. There were significant difference between the labels values and quantified levels of constituent totally (p-values between 0.000 and 0.003 ).

The toxic metals, nitrate and nitrite concentrations in all samples were within the national and WHO and Australian ranges, except for mercury in 9 samples that exceeded the Australian standard.


Keywords: nitrate, nitrite, toxic metals, drinking water, bottled water

Food Additives in Food Intended for Children in the Republic of Srpska Market


Ljubica Bojanić 1,2, Miodrag Marjanović 1 , Mirjana Đermanović1,2, Zorica Jusupović 1, Janja Bojanić 1,2

1Public Health Institute of Republic of Srpska, 2Medical Faculty, University of Banja Luka 


 Food additives are added to food in order to improve the technological and sensory properties, but may also have adverse health effects. Children, especially in pre-school age, are particularly vulnerable. This age is characterized by intense growth and development of the organism and intense metabolism, which leads to increased energy and nutrients requirements, and consequently increased food intake per kg of body weight compared to adults.According to legislation in the Republic of Srpska, it is allowed to use food additives for children older than three years, including preservatives, sweeteners and colours.The aim of this paper is to present the potential exposure of children to additives by controlling the declarations of products labeled as intended for children.

Declarations of 25 randomly taken samples were examined, of which 11 soft beverages, 9 candies, 3 lollipops and 2 chewing gums. Of these, Na-benzoate and K-sorbate preservatives were declared on 8 beverages, 5 samples contain artificial sweeteners acesulfame K and aspartame (3 beverages and 2 chewing gums), while the presence of colors was found in all tested samples, including natural E100, E120, E141, E 150, E153, E160a, E160e, E162, E163, E171 and E172, and artificial E102, E110, E122, E124, E131, E132, E133 and E151, depending on the sample.

The obtained results give a general insight into the quality of products intended for children in our market and show that a significant number of these products contain additives, which indicates the need for further and more detailed research on the assessment of the exposure and impact of additives on children’s health in the Republic of Srpska.

Keywords: food additives, children, exposure, Republic of Srpska

Dietary Exposure to Mycotoxins Through Ready-to-eat Food Consumption


Guillermina Font 1 Dionisia Carballo2, Emilia Ferrer 1, Houda Berrada 1

1 Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Spain, 2 Faculty of Agricultural Science, National University of Asunción, Paraguay



Knowledge of human exposure to mycotoxins through processed diet is an important component of food safety strategies. The present study investigates the evaluation of mycotoxins in ready-to-eat meals as a reliable tool for risk assessment. For this objective,  the presence of twenty-seven mycotoxins including AFB1, AFB2, AFG1, AFG2, EN A, EN A1, EN B, EN B1, BEA, FB1, FB2, STG, DON, 3-ADON, 15-ADON, NIV, NEO, DAS, FUS-X, ZEA, αZAL, β-ZAL, αZOL, β-zol, T2 and HT-2 toxin, was assessed in 25 ready-to-eat food samples using QuEChERS extraction and determination by chromatographic methods coupled to mass spectrometry in tandem.

Results showed presence of deoxynivalenol at 36% of samples (2.61-21.59 µg/kg), enniatin B at 20% of samples (9.83-86.32 µg/kg), HT-2 toxin at 16% of samples (9.06-34.43 µg/kg) and aflatoxin G2 at 4% of samples (2.84 µg/kg). Mycotoxins were detected mainly in ready-to-eat food samples prepared with cereals, vegetables and legumes at levels below those often obtained from raw food. The obtained results indicated that mycotoxins are present in different ready-to-eat food samples which point out the need to perform continuous surveys to insure consumer health. The dietary exposure of the adult population was estimated using the deterministic approach, through the evaluation of the consumption and data of foods mycotoxin contamination to assess the estimated daily intake (EDI) and values obtained resulted below the tolerable daily intake for the selected mycotoxins.

This research was supported by the Spanish Ministry of Economy and Competitiveness (AGL2016-77610-R) and Government Scholarship program “Carlos Antonio López – Paraguay.

Keywords: Mycotoxins, ready-to-eat food, mass spectrometry, dietary exposure



Investigation of the Relative Hepatotoxic and Genotoxic Potency of Selected Pyrrolizidine Alkaloids


Lan Gao, Lukas Rutz, Karl-Heinz Merz, Dieter Schrenk

Food Chemistry and Toxicology, University of Kaiserslautern, 67663 Kaiserslautern, Germany


Pyrrolizidine alkaloids (PAs) having a 1,2-unsaturated pyrrolizidine core are known to be cytotoxic, genotoxic and carcinogenic occurring naturally in many plant species. They exert toxic effects through metabolic activation which form the corresponding dehydropyrrolizidine derivatives, primarily in the liver, catalyzed by cytochrome P450 monooxygenases. The presence of PAs as contaminants in food and feed, depending on the dose, has become a relevant safety issue in recent years. Although hints for a structure dependent toxicity exist, no sufficient conclusions on the relative toxicities of individual PAs can be drawn due to the limited and not comparable data.

In order to investigate further the connection between structure and toxicity, a series of PAs congeners were tested at doses ranging from 1 to 300 µM for mutagenicity in the Ames fluctuation assay with Salmonella typhimurium in the strains TA98 and TA100. None of the selected PAs showed mutagenicity despite metabolic activation with S9-mix. Cytotoxicity was assessed by the Alamar blue assay in HepG2 cells and primary rat hepatocytes. In HepG2 cells, possibly due to the lack of CYPs, none of the selected PAs showed cytotoxic effects. On the contrary, the probable structure dependent cytotoxicity was demonstrated with rat hepatocytes in primary culture. Lasiocarpine, an open-chained di-ester with 7S-structure, proved to be the most cytotoxic followed by the other di-esters echimidine, retrorsine, seneciphylline and senecionine. The mono-esters heliotrine, indicine, europine and lycopsamine were much less cytotoxic.


Keywords: food contaminants, Ames fluctuation assay, Alamar blue assay, metabolic activation, structure-dependent toxicity



Human Intervention Trial with Strawberry Tree (Arbutus unedo L.) Honey: Impact on DNA Stability and Haematological Parameters


Andreja Jurič 1, Marin Mladinić 2, Davor Želježić 1, Marija Pezer 3, Mirjana Turkalj 4, Karlo Jurica 5, Nevenka Kopjar 1, Irena Brčić Karačonji 1

1 Institute for Medical Research and Occupational Health, Zagreb, Croatia 2 Xellia Ltd, Zagreb, Croatia 3 Genos Ltd, Zagreb, Croatia 4 Srebrnjak Children’s Hospital, Zagreb, Croatia 5 Ministry of the Interior, Zagreb, Croatia



Due to its high phenol content, strawberry tree honey possesses many beneficial health effects. The aim of this study was to evaluate potential DNA and cytoprotective effects of A. unedo honey after short-term supplementation. Participants (six healthy men, aged 26-38) consumed 70 g of honey (from Pelješac peninsula, Croatia) dissolved in 200 mL of water daily for 14 days. Their blood samples were collected before honey supplementation and one day after the last consumption (day 15). We monitored changes in leukocyte DNA integrity and haematological parameters. To establish how the supplementation modified the response of DNA to external stimuli with a genotoxic chemical, we performed a challenge test with hydrogen peroxide (H2O2) by measuring the levels of spontaneous and H2O2-induced DNA damage using the alkaline comet assay. Results showed significant lowering of comet tail intensity compared to the baseline value (0.21% vs. 0.88% DNA in comet tail). We found significant DNA-protective effects in leukocytes of almost all participants after an ex vivo challenge with H2O2. Comet tail intensity recorded after the H2O2 challenge was significantly lower after honey supplementation (2.42% DNA at day 0 vs. 0.38% DNA at day 15). Additionally, a significant increase in neutrophil count was observed after honey supplementation. Although limited by a small number of participants, the results of this intervention trial point to strawberry tree honey having promising DNA- and cytoprotective effects, which should be further clarified in forthcoming studies using other complementary methods on a much larger population exposed for longer periods.

Keywords: alkaline comet assay, challenge test, supplementation



Organochlorine Pesticide Residues in Farmed Fish in Machakos and Kiambu Counties, Kenya


Isaac Omwenga1. Laetitia Kanja1.Joseph Nguta1. James Mbaria1.Patrick Irungu2

1Department of public health, pharmacology and toxicology ,Faculty of Veterinary Medicine, University of Nairobi, Box 30197,00100 Nairobi, Kenya.

2 Department of Agricultural Economics, Faculty of Agriculture, University of Nairobi .P.O.

Box 30197, 00100 Nairobi, Kenya.



This study was conducted to evaluate occurrence of organochlorine pesticide residues in Oreochromis niloticus fish reared by aquaculture in Kiambu and Machakos Counties. A total of 213 fish organ samples were analyzed for organochlorine pesticide residues from Kiambu and Machakos Counties in Kenya using gas liquid chromatography. Overall, betahexachlorocyclohexane (BHC) group, the cyclodiens, aldrin, heptachlor, dieldrin, endrin and the dichlodiphenyltrichloroethane (DDT) group of compounds were detected in fish samples. p,p’ – DDT and its metabolite p,p’-DDD were detected in the muscle, liver and gonad and their concentration ranged between not detectable (ND) to 0.916 μg Kg-1 for p,p’-DDT and ND to 1.684 μg Kg-1 for p,p’-DDD. Brain had the highest concentration of DDT metabolites o, p’ -DDT ( 2.098±4.097 μg Kg-1) and p’ p-DDD (1.684±3.666 μg Kg-1 ). Organochlorine pesticide residues were generally higher in Kiambu County compared to Machakos County but there was no significant difference. The residue levels of organochlorines in all fish samples analyzed were lower than the benchmark levels set by FAO, FDA (2001) and NAS/NAE (1972).

Keywords: organochlorines, residues, farmed fish, gas liquid chromatography

Monitoring of Metals and Metalloids in Samples of Plastic Food Packaging During 2013-2017


Ž. Ljubičić1,  B. Antonijević2, N.Zec Petković1

Institute of Public Health Sremska Mitrovica, Sremska Mitrovica, Serbia

2Department of Toxicology “ Akademik Danilo Soldatovic” , Faculty of Pharmacy, University of Belgrade, Serbia

Health safety of packaging materials represents the basic prerequisite of their safe use in food and beverage packaging. In contact with food, chemicals can migrate from the packaging to the food content and contaminate it. Therefore, according to the official rules, food contact materials (FCM) have to be investigated in order to ensure that the substances migrating from the material into the food do not endanger human health or change the food itself.

The aim of this study was to present the results of arsenic, copper, barium, zinc, iron, chromium, cadmium, cobalt, lithium, manganese, lead and mercury content in FCM during the last five-year period.

Measurement of the elements in the migration solutions (3% acetic acid, 50% ethanol) was carried out by atomic absorption spectrophotometry FLAAS, with exception of arsenic which was determined by HGAAS. Sample preparation was carried out according to the requirements of EU Commission Regulation No 10/2011 for plastic materials and articles intended to come into contact with food and the Role book on the conditions regarding the health safety of consumer products that may be placed on the market, Official Gazette SFRJ No. 26/1983, 61/1984, 56/1986, 50/1989 and 18/1991 and standards EN 1886-1, EN 1886-5, EN 1886-9, EN 1886-13 and EN 1886-14. All measurements were done using accredited methods.

The measurements were carried out during the period 2013-2017 in different samples of FCM obtained from several manufacturers on the territory of Srem, Serbia. A total of 745 samples was analyzed. All the concentrations of potential contaminants in migration solutions were below the maximum allowable levels, established by the regulation in the EU and the national regulation, which indicate adequate health safety of FCM covered by this monitoring.


Keywords: food contact materials, contaminants, migration


Safety Assessment of the Extracts of Overground Part of Hedychium Coronarium Koenig in SD rats


Ling-Shan Tse 1, Po-Lin Liao 1, Jiunn-Wang Liao3, Jaw-Jou Kang2, Yu-Wen Cheng1 *

1 School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan, R.O.C, 2 School of Pharmaceutical Science, National Tang Ming University, Taipei, Taiwan, R.O.C, 3 Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taiwan, R.O.C


Hedychium Coronarium (HC) has a long history using in food and folk medicine from different countries, however, the potential toxicity of HC was remained unknown. In this study, groups of male and female SD rats were orally administered 0, 1.5, 3.0 and 5.0 g/kg/day water extracts of HC leaf for 90 days (N=15), ten rats from each group were randomly scarified at 91 days, other 5 rats from each group were stopped gavaging and maintained observation for the following 14 days resting phase as followed up recovery study. There were no toxicologically relevant findings in female groups. In male groups, after treatment with HC 3.0 and 5.0g/kg/day for 90 days, urine volume was increased, imbalanced electrolyte and aldosterone was decreased, those indicating the side effect of HC. Hence, the no-observed adverse- effect level of water extracted HC leaf is suggested as 1.5g/kg/day for male.


Keywords: subchronic toxicity, folk medicine, herb, aldosterone, diuretics








































































Carbamate Derivatives of Short-acting Bronchodilator Albuterol Inhibits Human Acetylcholinesterase and Butyrylcholinesterase


Anita Bosak 1, Anamarija Knežević 2, Katarina Zlatić 2, Robert Kerep 1, Zrinka Kovarik 1

1 Institute for Medical Research and Occupational Health, Zagreb, 2 Ruđer Bošković Institute, Zagreb


Novel synthesised biscarbamate and monocarbamate derivatives of the short-acting bronchodilator albuterol (biscalb) resemble the structure of biscarbamate bambuterol, whose bioconversion to the bronchodilator terbutaline in an organism is enabled through the inhibition of butyrylcholinesterase (BChE). Bambuterol’s high therapeutic index is associated with an extremely selective BChE inhibition compared to acetylcholinesterase (AChE). We determined the inhibition potency of biscalb and monocalb toward human BChE and AChE to evaluate their selectivity. Both carbamates proved to be potent inhibitors of both cholinesterases with ki constants within 103-105 M-1min-1. Biscalb and monocalb inhibited AChE only 10 times slower than they did BChE, meaning that the selectivity of both carbamates was very poor compared to the 20,000 times faster inhibition of BChE determined for bambuterol. Therefore, the novel compounds lack potential for prodrug development in analogy with bambuterol. However, due to their inhibition potency, these novel carbamates could be considered structural and functional lead compounds for the design of new compounds, whose primary action would be inhibition of cholinesterases, like pesticides or neurodegenerative disease drugs.

Supported by CSF Grant no. 4307.

Keywords: cholinesterase, selectivity, monocarbamate, biscarbamate, prodrug


Exposure to Exhaust Gas and Changes in Liver Functions, Biomarkers

of Oxidative Stress and Heavy Metals in Automobile Workers


Augusta Nsonwu-Anyanwu1*, Sunday Offor1, Edmund Egbe1, Chinyere Usoro1.

1Department of Medical Laboratory Science, University of Calabar, Cross River State, Nigeria.


Biomarkers of oxidative stress (malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO), total antioxidant capacity (TAC), total plasma peroxides (TPP), oxidative stress index (OSI)), liver function enzymes (Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), Gamma glutamyl transferase (GGT)) cadmium (Cd) and lead (Pb) were assessed in sera of automobile workers.

One hundred and twenty (120) men (18-60 years) comprising of 40 automobile mechanics (AMM), 40 automobile spray painters (ASP) and 40 non-automobile workers were studied. The MDA, GSH, NO, TAC, TPP, ALT, ALP, GGT were estimated colorimetrically, Cd and Pb by AAS. Systolic and diastolic blood pressure (SPB & DBP) was obtained. Data were analyzed using ANOVA and correlation at p<0.05.

Non automobile workers had lower SBP, TPP, OSI, NO, ALP, GGT, Pb and Cd and higher TAC and GSH compared to AMM and ASP; and lower DBP, MDA and ALT compared to ASP only (p<0.05).  ASP had higher DBP, MDA, ALT, ALP, GGT, Pb and Cd compared to AMM (p<0.05). Duration of exposure correlated negatively with NO (r = -0.230, p= 0.040) and positively with TPP (r = 0.830, p = 0.000), OSI (r = 0.801,p = 0.000), Pb (r = 0.776, p = 0.000) and Cd (r = 0.674, p = 0.000), ALT (r=0.274, p=0.014), ALP (r = 0.473, p=0.00) and GGT (r = 0.535, p=0.000) in both ASP and AMM.

Chronic exposure to automobile exhaust gas is associated with increased liver enzymes activities, toxic elements, lipid peroxidation and depletion of antioxidants in automobile workers studied.


Keywords: exhaust gas, liver enzymes, antioxidants, lipid peroxidation, toxic metals

Mutagenicity Study of Generic Insecticides Lambda Cyhalothrin in the Mammalian In Vivo Micronucleus Test

Tetiana Tkachuk, Mykola Prodanchuk, Nadiiya Nedopytanska, Oleksandr Kravchuk, Volodymyr Bubalo, Oleksander Tkachuk, Olena Zubko, Olena Kostik

  1. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety MH, Kiev, Ukraine


Experimental mutagenicity data is a mandatory part of toxicological assessment of the justifying their safety usage in environment. One of the methods applied in our Laboratory is studying mutagenicity in the mammalian in vivo micronucleus test. The purpose of the micronucleus test is to identify test-substances that cause cytogenetic damage which results in the formation of micronuclei containing either lagging chromosome fragments or whole chromosomes. The test evaluates micronucleus formation in polychromatophilic erythrocytes (PCE) of the mice bone marrow. Following OECD 474 guidelines we modified test for rapid screening of generic pesticides for mutagenicity. The tests are conducted following the SOP, in compliance with GLP requirements.

Two samples of technical pesticide lambda cyhalothrin obtained from different manufacturers and content different purity percentage were studied. The mutagenic activity was studied on CD1 healthy young mice, male. Acclimatization of the animals took 5 days before dosing. The test substances were administered as an aqueous emulsion, orally.

Each sample was investigated in the following doses: 5.0; 1.0; 0.2 mg/kg/bw and was accompanied with positive and negative control. Exposure time – 24 hours.

As a result of analyzing lambda cyhalothrin samples in high concentrations were observed significant (р≤0.05) increase in the frequency of PCE micronuclei in compared to the negative control and historical control data.

All two samples of generic insecticide lambda cyhalothrin showed mutagenic effect in highest doses (5.0 mg/kg/bw) in the micronucleus test in the mice bone marrow in vivo.


Keywords: mutagenicity, micronucleus test, pesticides, lambda cyhalothrin.

Isolatıon and Characterızatıon, Antıcarsınogenıc and Apoptotıc Effects of Humıc Acid


Ayse Demir Aktas1, Sultan Mehtap Buyuker, Derya Ozsavci1, Ozlem Bingol Ozakpinar1

1Department of Biochemistry, School of Pharmacy, Marmara University, Istanbul, Turkey, 2Department of Pharmacy Services, Vocational School of Health Service, Uskudar University, Turkey



Humic acid (HA), which are part of the organic structure of soils, are the resultant compounds of the biomolecules that undergo physical, chemical and microbiological transformation. Chemical and infrared spectroscopic analyses have revealed the presence of aromatic rings, and phenolic hydroxyl, ketonyl, quinone carbonyl, carboxyl, and alkoxyl groups in HA. Therefore, HA extracts are the most powerful antioxidants in the world. Because of the anti-inflammatory, anti-allergic, anti-apoptotic, anti-irritant, heavy metal binding and antiviral properties of HA, it is increasingly being used in industrial, agricultural, environmental and biochemical applications. The goals of this study were twofold. First, it aimed to isolation and characterization of HA from soils. Second, it aimed to investigation of in vitro anti-carsinogenic and anti-apoptotic effects of synthetic and isolated HA on human servical cancer cell line. For this purpose, we isolated HA from soil samples collected from Antalya, Turkey by the California Department of Food and Agriculture (CDFA) method. Then, isolated HA was characterized by HPLC and compared to the standard HA. HeLa cells were grown in petri dishes in a humidified atmosphere at 37°C. Five different concentrations were prepared using standart and isolated HA. The cell  proliferation was measured by MTT method.  On the other hand, the apoptotic mechanisms induced by HA in cancer cells were investigated by measuring caspase-3, 8 and 9 enzyme activities.

According to our results, HA has an growth inhibitory and apoptotic effects on HeLa cells. However, further studies are required to determine the exact mechanism.

Keywords: cancer, CDFA method, humic acid, isolation, soil

























































Effect of Myrtenal on Social Behavior and Memory of Rats


Stela Dragomanova1,2, Radoslav Klisurov1,3, Marieta Georgieva2, Maria Lazarova1, Christophor Dishovsky1, Reni Kalfin1, Lyubka Tancheva1,*

1 – Institute of Neurobiology, Bulgarian Academy of Sciences, Acad. Georgi Bonchev Str., Block 23, Sofia 1113, Bulgaria; 2 – Medical University of Varna, 55 Marin Drinov Str., Varna 9002, Bulgaria; 3 – Medical University of Sofia, 2 Zdrave str, Sofia 1000, Bulgaria; * – Weston Visiting Professor of Weizmann Institute of Science, Israel


Plant monotherpen Myrtenal (M) presents in many essential oils and has rich specter of pharmacological activities. Our previous studies established significant effects of M on behavior of mice.

Aim of this study was to investigate effect of M on the cognition and social behavior of rats.

Male Wistar rats were daily injected Myrtenal (40 mg/kg, i. p.) for 9 days. 24 hours after the last treatment the changes in behavior and cognition were tested via Open field test, Step-through test and Mouse-killing test. Brain levels of some neurotransmitters (dopamine – DA, serotonin – 5-HT, acetylcholine – ACh, adrenaline – Adr and noradrenaline – NA) were also measured. Significant improved cognitive performance in M-treated group was established. Learning and memory in this group was better than in the control group at the end of the experiment. Elevated brain neurotransmitter levels of DA, 5-HT and Ach confirmed behavioral data. Interesting changes in social behavior of M-treated animals were recorded, as well. After 3rd day of M-administration increased incidences of dominant and aggressive behavior were established accompanied by vocalization and fighting in the group. We assume that it is due to interaction of M odor with rats pheromones related to animal hierarchy in the group. Brain levels of Adr and NA in M-group were not significantly elevated.  At the same time incidences of inter-species aggression were observed according to Mouse-killing test.

Present study indicates significant effects of M on CNS accompanied by changes in brain mediation systems and deserve further studies.

Keywords: cognition, aggression, mediators, monoterpene

Gentiana Lutea Radix Extract Exerts In Vitro Dose- and Time-dependent Response in Peripheral Blood Mononuclear Cells

Ana Valenta Šobot, Jelena Filipović Tričković, Dunja Drakulić

Vinča Institute of Nuclear Sciences, University of Belgrade, Mike Petrovica Alasa 12-14, 11001 Belgrade, Serbia


Although Gentiana lutea radix extract (GRE) is believed to strengthen organism by stimulating circulation and the activity of many organs, it has certain active components that can cause oxidative and genotoxic stress. The aim of the current study was to estimate which concentration (0.5, 1 and 2 mg/ml) of GRE initiates DNA damage and lipid peroxidation, disrupts oxidative balance following 48h lasting treatment, and whether those parameters modulate survival of human peripheral blood mononuclear cells in vitro after 48 and 72h. DNA damage was assessed using alkaline Comet assay, oxidative status by PAB assay, lipid peroxidation by determining MDA levels and cell count by tripan blue dye exclusion test. Obtained results indicate that the lowest tested concentration increases lipid peroxidation along with DNA damage and has a mild cytotoxic effect. Higher concentrations provoke only significant DNA damage, probably due to the other mechanisms involved in initiation of cell death rather than elevated oxidative stress. Albeit cell death after 48h of treatment at the highest tested concentration is not significant, DNA fragmentation is more than doubled compared to control. The impact of detected fragmentation is seen as reduction of cellular survival observed after 72 hours of treatment. According to presented findings DNA fragmentation could be a predictive tool for the cytotoxic effects estimation of GRE treatment since increased fragmentation is observed 24h prior to significant increase in cell death. Parallel monitoring of oxidative status and DNA damage might be valuable parameter in determining sublethal concentrations of GRE in other experimental setups.

Keywords: DNA damage, Comet assay, lipid peroxidation, viability

Betulinic Acid Reversed High-Fructose Diet-Induced Hyperglycemia, Dyslipidemia, Inflammation and Oxidative Stress in Rats


1Rafiat A. Ajala-Lawal, 1Najeeb O. Aliyu, 2Oluwayemisi B. Ibitoye, and 1Taofeek O. Ajiboye.

1Nile University of Nigeria, Abuja, Nigeria; 2Al-Hikmah University, Ilorin, Nigeria


Betulinic acid, a pentacyclic triterpene, is known for its widely reported pharmacological activities. In this study, we investigated the protective effect of betulinic acid in high-fructose diet-induced metabolic syndrome. Metabolic syndrome was induced in rats using high-fructose diet (60%). Metabolic syndrome rats were treated 5, 10 and 20 mg/kg bodyweight betulinic acid or metformin. Oral administration of betulinic acid significantly reversed high-fructose diet-mediated increase in body mass ratio, abdominal circumference and blood glucose. Furthermore, betulinic acid restored HFD-mediated alterations in metabolic hormones (insulin, HOMA-IR, leptin and adiponectin). Similarly, elevated TNF-a, IL-6 and IL-8 were significantly lowered. In addition, betulinic acid restored HFD-mediated increase in the levels of lipid profile parameters and indices of atherosclerosis, cardiac and cardiovascular diseases. High-fructose diet-mediated decrease in antioxidant enzymes (superoxide dismutase and catalase) and increase in oxidative stress biomarkers (reduced glutathione, lipid peroxidation products, protein oxidation and fragmented DNA) were significantly restored by betulinic acid. The data generated from this study revealed that betulinic acid protects against high-fructose diet-induced hyperglycemia, inculin resistance, dyslipidemia and oxidative stress in rats

Keywords: triterpenes, metabolic syndrome, antioxidant enzymes, inflammatory cytokines, lipid peroxidation

Lophirones B and C Protect against Colistin Sulphate-Induced Neurotoxicity by Upregulating Nrf-2 through Akt and Protein Kinase C Pathway


Ajiboye, T. O.

Department of Medical Biochemistry, Nile University of Nigeria, Abuja



Chalcones, including lophirones B and C, are versatile plant phytochemicals with electrophilic capability. Lophirones B and C are chalcone dimers in Lophira alata stem bark. In this study, the protective influence of lophirones B and C on colistin sulphate-induced neurotoxicity in rats was investigated. Lophirones B and C (20 mg/kg body weight) lowered colistin sulphate (15 mg/kg body weight)-mediated increase in activities of acetylcholinesterase, butyrylcholinesterase, monoamine oxidases A and B, ENTPDase and Ecto-5’ nucleotidase. Colistin sulphate-mediated decrease in the activities of superoxide dismutase, catalase, glutathione S-tranferase and reduced glutathione activities was significantly reversed by lophirones B and C. Conversely, elevated levels of products of lipid peroxidation (malondialdehyde) and protein oxidation (protein carbonyl) were lowered by lophirones B and C. Furthermore, Lophirones B and C raised the level of Nrf-2, Akt1 and PKC proteins while lowering keap1 protein. Conversely, there was no significant difference in the level of MAPK and ERK1 The results of this study show that lophirones B and C protects against colistin sulphate-induced neurotoxicity by lowering the activities of cholinergic, monoaminergic and purinergic enzymes, enhancing the antioxidant enzymes through Akt1 and PKC pathways.

Keywords: chalcone dimmers, neurodegeneration, cholinergic enzymes, purinergic enzymes, transcription factors

Effects of Commercially Available Dietary Supplement Based on Soybean Extract (Glycine max. L.) on Hepatic and Renal Function and Clinically Relevant Interactions with Conventional Drugs


Jelena Hogervorst1, Aleksandar Rašković2, Milan Ubavić3, Ana Tomas2, Bojana Gaćeša1, Vladan Borčić2, Nebojša Stilinović2

1Department of Pharmacy, Faculty of Medicine, University of Novi Sad, 2Department of Pharmacology, toxicology and clinical pharmacology, Faculty of Medicine, University of Novi Sad, 3Faculty of Pharmacy, European University, Novi Sad


Dietary supplements based on soybean extracts (DSU) are widely used by general public, but there is very limited scientific data to support their efficacy, safety and potential interactions with conventional therapy. The aim of this study was to examine the effect of commercially available DSU on liver and renal function, oxidative status and interactions with several conventional drugs in animal models. Standard pharmacodynamics tests were performed on Swiss albino mice treated with DSU in addition to several central nervous system acting drugs. Parameters of liver and renal function, and oxidative status in liver homogenates were determined in healthy and Wistar rats subjected to oxidative stress with CCl4 after treatment with dietary supplement. DSU administration weakened the analgesic activity of codeine, significantly potentiated diazepam induced motor coordination impairment in 10th and 30th minute after the administration of diazepam and had the opposite effect on alprazolam effect. DSU pretreated group also exhibited significantly shorter pentobarbital sleep induction and sleeping time. These findings go in favor of DSU interference with metabolic pathways of tested drugs and subsequent altered pharmacodynamics. DSUs supplementation did not affect the liver and renal function, and ameliorated the liver injury and oxidative stress caused by CCl4. Despite exhibiting no negative effects on liver and renal function, and demonstrated antioxidant in vivo potential, the safety of DSU in combination with conventional drugs is questionable. The results of our study implicate the potential of DSU for serious herb-drug interactions.

Keywords: phytoestrogens, soybean extract, interactions, Cytochrome P-450, conventional drugs

Protective Effect of Ellagic Acid on 6-Hydroxydopamine Hemistriatal Intoxication


Andrey Popatanasov1, Lyubka Tancheva1, Maria Lazarova1 , Stela Dragomanova1,2, Albena Aleksandrova1, Elina Tsvetanova1, Almira Georgieva1, Reni Kalfin1

1Institute of Neurobiology, Bulgarian Academy of Sciences, 2Faculty of Pharmacy, Department of Pharmacology, Toxicology and Pharmacotherapy, Medical University of Varna, 2


Ellagic Acid (EA) is used in human diet as ingredient of many fruits and vegetables as pomegranate, grapes, etc. EA is known to have also chemopreventive action on metabolite disturbances, liver intoxications, cancer, dementia etc.

Scope of the study was to evaluate EA effects on hemistriatal intoxication with the neuronal and mitochondrial toxin 6-hydroxydopamine (6-OHDA) known to induce Parkinson’s disease (PD) symptoms in rodents.

The intoxication was produced via right striatal 6-OHDA (2 µg in 2 µl saline) injection of male Wistar rats. PD-symptoms were verified by apomorphine rotations test on the 2nd and 3rd week after the operation and were compared to sham operated animals. EA preventive effect (400 mg/kg i.p. 5 days) was evaluated via behavioral and biochemical tests- learning and memory (Step-through test), motor coordination (Rot-a-rod-test) and antioxidant status in brain (lipid peroxidation-LPO, glutathione, SOD, CAT, GPx).

Significant improving effect of EA on learning and memory processes and motor coordination in treated animals was established (both on 2nd and 3rd week) in compare to PD-controls. At the same time significant antioxidant effects of EA in brains were established -LPO is 45% lower and CAT 336% higher in EA group and some changes in glutathione and SOD, CAT, GPx were established. Interhemispheric differences were also observed (10 to 30%). EA probably “shields” lipid bilayers and thus reducing lipid peroxidation. As chelator and radical scavenger EA probably reduces free radical levels and in this way minimizes the mitochondrial and neuronal damage in 6-OHDA intoxicated animals.


Keywords: Parkinson’s disease, antioxidant, chemoprotection, polyphenols



Effect of Crocin Against NicotineInduced Damages on Liver Parameters of Male Mice


Cyrus Jalili1, Faramarz Jalili 2

1 Department of Anatomy and Cell Biology, Kermanshah University of medical Sciences, Kermanshah, Iran, 2 Students Research Committee, Kermanshah University of medical Sciences, Kermanshah, Iran,


Nicotine is a major pharmacologically active substance in cigarette smoke. It is mainly metabolized in liver and causes devastating effects. Crocin is the chemical ingredient primarily responsible for the color of saffron. It has different pharmacological effects such as antioxidant and anticancer. This study was designed to evaluate the protective role of crocin against nicotine on the liver of mice.

Forty‑eight mice were equally divided into 8 groups; control (normal saline), nicotine (2.5 mg/kg), crocin (12.5, 25 and 50 mg/kg) and crocin plus nicotine treated groups. Saline, crocin, nicotine and crocin/nicotine (once a day) were intraperitoneally injected for 4 weeks. The liver weight and histology, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and serum nitric oxide levels have been studied.

The results indicated that nicotine administration significantly decreased liver weight (48.37%) and increased the mean diameter of hepatocyte (239%), central hepatic vein (28.45%), liver enzymes level (ALP 29.43%, AST 21.81%, ALT 21.55%), and blood serum nitric oxide level (57.18%) compared to saline group (P < 0.05). However, crocin and crocin plus nicotine administration significantly boosted liver weight (49.54%) and decreased the mean diameter of hepatocyte (40.48%), central hepatic vein (15.44%), liver enzymes (ALP 22.02%, AST 19.05%, ALT 23.11%), and nitric oxide levels (35.80%) in all groups compared to nicotine group (percentages represent the maximum dose) (P < 0.05).

Crocin showed its partly protective effect against nicotine‑induced liver toxicity.


Keywords: crocin, liver, mice, nicotine

The Effect of Hydro-alcoholic Extract of  Falcaria Vulgaris on Liver Damages  Induced by Ethanol in Male Rats


Faramarz Jalili1, Cyrus Jalili 2

1 Students Research Committee, Kermanshah University of medical Sciences, Kermanshah, Iran, 2 Department of Anatomy and Cell Biology, Kermanshah University of medical Sciences, Kermanshah, Iran


In this study, 68 male rats were divided in to 8 groups:control, ethanol-treated group (20%); hydro-alcoholic extract of falcaria vulgaris-treated groups (50, 100, 150 mg/kg./day); and ethanol and hydro-alcoholic extract of falcaria vulgaris treated group were  administration orally for 28 consequent days. These mice were randomly assigned to 8 groups(n=8). After 24 hours animal were killed, the liver was sampled: tissue sections were prepared and examined by light microscope. weight of liver  and number of hepatocyte and diameter and serum levels of nitric oxide, ALT,AST,ALP,TG and TAC (Total antioxidant capacity) were analyzed (one-way ANOVA). Data with P<0.05 was considered significant

The results indicate that ethanol administration significantly increased ALT,AST,ALP,TG and nitric oxide levels compared to saline group(P<0/05). Histopatology of the liver confirmed the changes induced by ethanol and the hepatoprotection effect of hydro-alcoholic extract falcaria vulgaris. Oral administration of hydro-alcoholic extract falcaria vulgaris resulted in significant reduction in the levels ALT,AST,ALP,TG and nitric oxide (p<0.05). Ethanol administration significantly decreased TAC levels compared to saline group(P<0/05).

The present study suggest that hydro-alcoholic extract falcaria vulgaris offers protection against oxidative stress and this ability might be attributed to its antioxidant potential. It seems that hydro-alcoholic extract falcaria vulgaris administration could improve liver changes and prevente ethanol-induced adverse effects on serum levels of nitric oxide, ALT,AST, ALP, TG and Total antioxidant capacity. Hydro-alcoholic extract Falcaria vulgaris may improve liver dysfunction in ethanol-induced in rats by modulation of detoxification enzymes and its antioxidant effects.


Keywords: Falcaria vulgaris, ethanol, liver, rats


Oral Acute Toxicity of Candlenut Seeds (Aleurites moluccana) in Rats


Dennis Olivares1, Nicolás Cáceres1, María Fernanda Cavieres1

1 Facultad de Farmacia, Universidad de Valparaíso, Valparaíso, CHILE.


Ingestion of candlenut seeds (Aleurites moluccana) induces severe abdominal pain, vomiting and diarrhea. However, these seeds are intentionally and extensively used as a “safe” and “effective” option for weight loss. Here we evaluate and describe the acute toxicity of seeds bought by contacting a provider on the internet. The protocol was approved by the Bioethics Committee of the Faculty of Pharmacy, Universidad de Valparaíso.  8 weeks old, female, Sprague-Dawley rats (n=3 per treatment), weighing  230 ± 20 g were dosed with an extract of 0,  1/2 or 1 seed in 2 ml of cold or boiling water. Animals were observed for up to 72 h, after which they were euthanized for necropsy and histopathology study. All animals dosed with candlenut seed extracts showed toxicity symptoms including gastrointestinal (diarrhea, constipation), neurotoxicity (central and autonomic). There were no differences in blood biochemistry or hemogram values, except for an increase in white blood cells. Necropsy showed abdominal distension, neurotoxicity and an increase of lymphoid tissue associated to the intestinal mucosa. Brain histopathology showed an increase of leucocytes while gastrointestinal tissues showed necrosis, edema, congestion and leucocytic infiltration. Effects may have a dose-dependency and did not vary between the cold or the boiling water extracts.  We show that candlenut seeds induce in rats a gastroenterocolitic syndrome with necrosis of the first and second third of the microvilli which agrees with acute manifestations observed in humans. We also observed neurotoxicity not reported in humans, which may be due to different sensibility of rodents.


Keywords: Candlenut seeds, Aleurites moluccana, acute toxicity

Does Ursolic Acid Protect The Kidneys of Diabetic Rats from the Oxidative Stress?


Merve Bacanlı1, Sevtap Aydın1, Hatice Gül Anlar1, Tuğbagül Çal1,2, Nuray Arı3, Ülkü Ündeğer Bucurgat1, A. Ahmet Başaran4, Nurşen Başaran1

1 Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Toxicology 06100 Ankara, Turkey 2 Karadeniz Technical University Faculty of Pharmacy Department of Pharmaceutical Toxicology 06100 Trabzon, Turkey 3 Ankara University Faculty of Pharmacy Department of Pharmacology 06100 Ankara, Turkey 4 Hacettepe University Faculty of Pharmacy Department of Pharmacognosy 06100 Ankara, Turkey


Diabetes, a heteregenous metabolic and chronic disease, is a growing health problem in most countries. It has claimed that diabetes is associated with the increased formation of free radicals and decreased antioxidant potential. Ursolic acid is a well-known pentacylic triterpene which is commonly used in traditional Chinese medicine due to its health beneficial effects. The aim of this study was to investigate the effects of ursolic acid in the kidneys of Wistar albino rats with streptozotocin (STZ)-induced diabetes. For this purpose, the oxidative stress parameters such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx) enzyme activities and total glutathione (GSH) and malondialdehyde (MDA) levels were evaluated. It is observed that diabetes caused increases in GR enzyme activities and MDA levels, as well as decreases in GSH levels and CAT, SOD, and GPx enzyme activities. Ursolic acid treatment was found to significantly decrease GR enzyme activities and MDA levels and significantly increase GSH levels and CAT, SOD, and GPx enzyme activities in diabetic rats. According to our results, it seems that ursolic acid might be beneficial against diabetes induced renal damage.

This study was funded by a grant from The Scientific and Technological Research Council of Turkey (Project number: 114S919).

Keywords: ursolic acid, oxidative stress, kidney
















































































Preview of Deontological Beginnings of Toxicology in Serbia


Bojana Petrović1, Vesna Matović2, Predrag Vukomanović1, Veljko Todorović3

1Medical Sanitary School of Applied Sciences“Visan”, Belgrade, Serbia, 2Faculty of Pharmacy, University of Belgrade, 3Military Medical Academy


The health situation in Serbia began to change significantly in 1830 when Serbia was recognized for constitutional status of the principality, political autonomy, approved military, police, judiciary, health, education, institution formation. After the opening of the first pharmacies, the development of Serbian pharmacy and toxicology was on the rise. However, the situation in these areas was unsettled, as general stores, in addition to other goods, made and sold drugs and poisons. Legislation was necessary, and in 1865 Prince Mihailo Obrenovic passed the “Law on Pharmacies and Pharmacists, Holding, Selling Drugs and Poisons”, which contributes to regulation of the pharmaceutical practice. On the basis of this law, the Minister of Internal Affairs issued “Pharmaceutical tax for Citizens, Public and Private Pharmacies” and “Short Serbian Pharmacopoeia”. The law defines the rules and ethical standards for the keeping and distribution of drugs and toxins. In addition pharmacies were obliged to make toxicological and chemical analyzes for the needs of state authorities. The law prescribed a list of allowed substances in pharmacies, among which there were extremely poisons: cantaridine, colchicine, strychnine, compounds of arsenic, lead, mercury.  In Pharmacopoeia there were recipes for the preparation of the extractive compositions of distinct toxins and opiates, teriyaki, colchicine wine and tincture, cantharidin tincture and patches, extracts of helleborus, hemlock, aconite, opium. Besides the fact that it has laid the foundation of modern toxicological deontology, it testifies to the level of health culture, and provides a complete picture of Serbia in the second half of the XIX century.

Keywords: poisons, history, XIX century, laws


The Influence of Alchemy on the Development of Toxicology


Aleksandar Vidaković1, Bojana Petrović2, Predrag Vukomanović2, Milica Nikolić3, Slavoljub Jović3, Veljko Todorović4

1Serbian Institute of Occupational Health “Dr Dragomir Karajović”, 2Medical Sanitary School of Applied Sciences“Visan”, Belgrade, Serbia, 3Faculty of Veterinary Medicine University of Belgrade, Serbia, 4Military Medical Academy


Alchemy covers a period of nineteen centuries (300 B.C.-1600 A.D.), from the Greek philosophers to the chemists in the 17th century. It was developed on the territory of ancient Egypt, the Middle East, India, China, ancient Greece and Rome. Decline and stagnation of medieval European medicine and pharmacy, and the strengthening of mysticism and superstition, alchemy begins to flourish. Alchemy had two basic approaches, practical skill of possible transformation of non-precious metals a precious, and spiritualistic, soul-purification and the attainment of immortality. Despite the fact that both approaches to alchemy were unsuccessful, the importance of alchemy in the development of toxicology and science is extremely important. The toxicological contribution is reflected in the identification of many toxic substances, and their application. Thanks to the continuous work of alchemists in the search for the Philosopher’s stone, the elixir of life, gold, they found new chemical substances and compounds, the Royal water (a mixture of nitric and hydrochloric acid), ethanol, nitrate, mercury, iron, copper, antimony, arsenic, bismuth, phosphorus, etc. In the late Middle Ages, alchemists used different poisons for therapeutic purposes, opiates in analgesia, iron in the treatment of anemia, mercury in venereal diseases, arsenic in skin diseases, having regard to the influence of the dose on toxicity. In the treatment of alcoholism, the emetic effect of antimony tartarate was used, obtained in reaction of antimony with wine. It is especially interesting to point out that the foundations of modern toxicology and science are the result of numerous accidental discoveries by alchemists.

Keywords: alchemy, poisons, metals, Philosopher’s stone

Paracelsus – a Man behind a Myth


Vesna Matović1, Bojana Petrović2, Predrag Vukomanović2

1Faculty of Pharmacy, University of Belgrade, 2Medical Sanitary School of Applied Sciences,“Visan”, Belgrade, Serbia.


Philippus Aureolus Theophrastus Bombastus von Hohenheim-Paracelsus (1493–1541) was one of the most famous alchemists, a Swiss physician, chemist, the “Father of toxicology”. The most accurate definition of drugs: “All substances are poisons; there is none which is not a poison. The right dose differentiates a poison from a remedy”, by Paracelsus is the basic toxicology principle. In spite of criticism of colleagues and toxicity of metals he advocated their usage, antimony for treatment of digestive problems and mercury for syphilis.  However, mercury was tacitly used due to the rapid spread of syphilis epidemic. Paracelsus first connected drinking water minerals with goiter. He studied miners diseases in Carinthia (present-day Austria), caused by inhalation of mercury and arsenic fumes. Despite the undisputed contribution to medicine and toxicology, Paracelsus was a controversial figure, confirmed by the fact that he chose pseudonym Paracelsus (“beyond” Roman physician Celsus) to express contempt for the doctors and scientists of that time. During his tenure as professor of medicine at the University of Basel, he publicly burned the most authoritative medical textbook The Canon of Medicine of Avicenna, and declared Galenus’s teaching obsolete. His hard temper led to conflicts with leading scientist of that time, which resulted in frequent moving. Named “Luther of medicine” because his papers and lectures were in German and not in Latin that was common practice. In spite of turbulent career, he introduced the more rational approach to diagnosis and therapy, and the usage of chemical instead of herbal drugs.

Key words: alchemy, Paracelsus biography, poisons, doses

Poisons Information Service of South Africa


Kate Balm2, Linda Curling2, Catharina du Plessis1, Carine Marks1, Farahnaz Mohamed2, Cindy Stephen2, Cherylynn Wium1

1 TPIC, Division Clinical Pharmacology, Stellenbosch University, Cape Town, South Africa 2RXHPIC, Department of Paediatrics and Child Health, Red Cross War Memorial Children’s Hospital, Cape Town, South Africa


South Africa initiated a Poisons Information service over 45 years ago, which was strengthened by the establishment of the Tygerberg Poison Information Centre (TPIC) and Red Cross Children’s Hospital Poisons Information Centre (RXHPIC). In 2015, the two centres combined their telephone service to form the Poisons Information Helpline of the Western Cape (PIHWC).  The PIHWC provides a 24/7 consultant based telephone service to medical professionals and the public.  Consultants, known as specialists in poisons information (SPIs), assist with the prevention, diagnosis and management of poisoning. Seven of the fifteen SPIs are medical doctors, one of whom is always available to provide clinical support to non-medical SPIs. Data from poisoning calls are entered, real-time, onto a server-hosted database, the AfriTox TeleLog. The locally-compiled AfriTox poisons information database underpins call data entry, providing for both substance definitions and treatment advice given. The PIHWC provides a system of toxicovigilance, which contributes epidemiological data on poisoning, describes new hazards, provides information on national trends, and identifies potentially vulnerable populations. Where necessary, the WHO is informed about any potentially serious or unusual public health impacts.  The PIHWC is actively involved in education and research. It offers regular toxicology workshops and formal lectures to under- and post-graduate students. Public education and information programmes on prevention and immediate management of acute poisoning are essential activities.  Research findings are regularly published in peer-reviewed journals and staff members actively participate in national and international conferences, as well as international WHO workshops.

Keywords: Poison information helpline, poisonings data, toxicovigilance

Almost Four Decades of History of Spanish Association of Toxicology (AETOX)


Font Guillermina1, Cameán Ana María2

1Laboratory of Toxicology and Food Chemistry, Faculty of Pharmacy, University of Valencia, Valencia, Spain, 2Area of Toxicology, Faculty of Pharmacy, University of Seville. Seville Spain



The Spanish Association of Toxicology (AETOX) was founded in 1980. It is an entity with more than 300 researchers, professionals and professors of Toxicology of Spain and other countries covering all the fields of Toxicology.

The main objective of AETOX is to promote the relationship and cooperation among its members and with other international societies of Toxicology to coordinate their efforts, in order to contribute to the development and dissemination of scientific knowledge in the different areas of the Toxicology.

The Society edits the “Revista de Toxicología” since 1983 maintaining homogeneity in its editorial line and the purposes of the publication. It is an open access journal, which is designed to facilitate the dissemination of current research in the field of Toxicology.

AETOX has the Spanish Register of Toxicologists to include all those professionals specialized in applied or basic Toxicology who apply for registration. Recognizing and accrediting the abilities and toxicological activities of those people registered, and disseminate this information who may be interested is the objective.

AETOX regularly organizes congresses every two years. The last one was celebrated in Valencia in 2017 being the XXII Spanish Congress of Toxicology and VI Iberomerican. In 2019, the congress will be celebrated in Seville. In addition, sessions, meetings and workshops, on topics of interest, are organized by the different sections.

AETOX primary goals consists of improving health conditions of living beings and the protection of the environment motivating young researchers in the field of Toxicology.

Keywords: toxicology, society, Spain

Theriaca – Poison or Antidote?


Dragan Joksović1, Vesna Matović2, Bojana Petrović3, Predrag Vukomanović3, Slavoljub Jović4,  Milica Nikolić4, Saša Ivanović4


1Military Madical Academy, 2Faculty of Pharmacy, University of Belgrade, 3Medical Sanitary School of Applied Sciences“Visan”, Belgrade, Serbia, 4Faculty of Veterinary Medicine University of Belgrade, Serbia.


Mithridates VI Eupator – toxicologist and king of Pontus (120-63 B.C.), has made Theriaca (described in the files “Antidotum Mithridaticum“), a sticky mixture made from 54 substances, like opium, flesh of poisonous snakes and blood of ducks feed with toxic substances. Great ruler and sworn enemy of the Roman empire, it is believed that he poisoned 80000 Romans. In constant fear of poisoning, he increasingly took small doses of known poisons, until he was able to withstand lethal doses (a process known as mithridatism). His alleged immunity to poisons is evidenced by the fact of Mithridates unsuccessful suicide attempt with arsenic trioxide after defeat from Roman commander Pompeius. Theriaca was important drug and universal antidote used over 1000 years. After Mithridates death, Theriaca recipe was brought to Rome, where it was perfected and supplemented to 64 toxic substances, by the Nero’s personal physician Andromachus. Despite a wide range of application, it was primarily used for treatment of plague and bites of poisonous animals. The making of Theriaca was shrouded in mystery, made on a particular day in large quantities and was kept in fine containers. Only some doctors had the honor of making it, and before making the Theriaca they had to check every ingredient. For years, it was one of the most expensive drugs. Up to the middle of 20th century and Pharmacopoeia Jugoslavica editio secunda (1951), Theriaca was in every official pharmaceutical textbook.

Keywords: Mithridatism, Pharmacopoeia, Roman Empire, universal antidote

An Overview of the Accomplishments of the German MAK Commission


Kyriakoula Ziegler-Skylakakis

Scientific Secretariat of the Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, Freising, Weihenstephan, Germany


The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, the MAK-Commission, is a Permanent Senate Commission of the German Research Foundation, Deutsche Forschungsgemeinschaft (DFG) and was established in the year 1955. Its task cording to DFG regulations is to provide scientific policy advice. Its innovative work has helped improve occupational safety and health in Germany.

In its 62 years of existence, the MAK Commission has evaluated over 1000 chemicals. It has established MAK-values and BAT-Values, as well as having developed approaches to strengthen scientific knowledge about potential hazardous substances in the work area. Further tasks include establishing and examining analytical methods to detect hazardous chemicals in the air or in biological materials.

In the poster, the milestones of the work accomplished by the MAK Commission during the past six decades will be highlighted. In addition the methods used for the derivation of the MAK-values, and for the designation of chemicals as to their carcinogenic potential, their sensitizing potential, their germ cell mutagenic potential and their contribution to systemic toxicity after percutaneous absorption will be presented. The cooperation taking place with different national and international Institutions will be also pointed out.

Keywords: MAK-values, BAT-values, health at the work area



About the Toxicological Risk Assessment Center in Serbia

Biljana Antonijević, Danijela Đukić-Ćosić, Marijana Ćurčić, Evica Antonijević

Department of Toxicology “Academic Danilo Soldatović”, University of Belgrade – Faculty of Pharmacy


Toxicological Risk Assessment Center (TRAC) at the Faculty of Pharmacy, University of Belgrade was established in 2013 and represents a unique institution of its kind in the Republic of Serbia. In 2017, the Center became a member of the World Health Organization Chemical Risk Assessment Network. Two principal goals of the center are training and research through networking and coordinating activities between academia and other professionals dealing with chemicals safety. The research interests primarily focus on mixture toxicology with particular emphasis on understanding the links between the environment and health risks, the mechanistic aspects of toxicity and how emerging knowledge could be translated into prevention strategies. The mission of the TRAC is to promote public/chemical safety and regulatory initiatives, and to train professionals on human health risk assessment by organizing seminars, workshops and consultancy. Short-term trainings and info days regarding hazard assessment of chemicals, REACH implementation, safety assessment of plant protection products, biocides and cosmetics have been designed to increase the national capacity to manage environmental, toxicological and health issues. Being a member of a global network, TRAC is open for true collaboration across scientific community, and with regulatory and industry players, all united in the vision to improve and promote the science of toxicological risk assessment.

Keywords: risk assessment, center, mission, vision


History of Undergraduate Teaching in Toxicology at the University of Belgrade, Faculty of Pharmacy


Danijela Đukić-Ćosić, Katarina Baralić, Marijana Ćurčić, Evica Antonijević, Aleksandra Buha Đorđević, Zorica Bulat, Dragana Vujanović, Mirjana Đukić, Biljana Antonijević, Vesna Matović, Mirjana Nedeljković

Department of Toxicology “Academic Danilo Soldatović”, University of Belgrade – Faculty of Pharmacy, Serbia



Undergraduate teaching in Toxicology has been carried out as an independent subject at the University of Belgrade for almost 80 years. It was first introduced under the name Toxicological Chemistry with the establishment of Department of Pharmacy at the Medical Faculty in 1939. The first professor of Toxicological Chemistry and chief of the Institute of Toxicological Chemistry was Dr. Momčilo Mokranjac (a student of Gabriel Bertrand and later a member of L′Académie de Pharmacie de Paris), and in 1964, he was succeeded by Dr. Danilo Soldatović. Apart from Pharmacy study program, another study program, Pharmacy-Medical Biochemistry, was introduced at the University of Belgrade, Faculty of Pharmacy in 1984. In 1987, the second mandatory subject, Clinical Analyses in Toxicology, was added to the curriculum of the new study program. With the start of Bologna Process in 2008, the latest curriculum of the Faculty of Pharmacy defined the mandatory course Toxicology with Analytics for both study programs and Clinical Analyses in Toxicology for Pharmacy-Medical Biochemistry. These courses are carried out through lectures, case studies analysis, and practical training. This enables students to acquire the necessary knowledge in general and special toxicology, as well as the skills relevant for laboratory work, with emphasis given to the sample preparation, detection, and determination of the most important poisons. In addition, numerous elective courses are available for both study programs: Acute Drug Poisoning with Analytics, Human Health Risk Assessment, Substances of Abuse with Analytics, Free Radicals and Antioxidants-laboratory analyses, Ecotoxicology, Toxicology in Practice and Chemical Carcinogens.

Keywords: Toxicological Chemistry, Pharmacy study program, Toxicology with Analytics, elective courses, Belgrade University

Paracelsus – the Founder of Toxicology Science


Danijela Đukić-Ćosić1 and Steven Gilbert2

1Department of Toxicology “Academic Danilo Soldatović”, University of Belgrade-Faculty of Pharmacy, 2Institute of Neurotoxicology & Neurological Disorders, Washington


Paracelsus (Philippus Theophrastus Aureolus Bombastus von Hohenheim, 1493-1541), a contemporary of Leonardo da Vinci, Martin Luther, and Nicholas Copernicus, was a Swiss-German physician, chemist, botanist, astrologer, alchemist, and general occultist. As headstrong, stubborn and independent young man, he decided to be a physician/chemist like his father. He studied at a number of universities in Europe, receiving his baccalaureate in medicine in 1510 and doctorate in 1516 from the University of Ferrara (Italy). Paracelsus stated that knowledge plus experience makes an expert, and therefore traveled throughout Europe, Africa, and Asia Minor to broaden his knowledge and gain experience. His influence on toxicology was enormous. He very clearly expounded the dose-response concept, suggesting that “the dose makes the poison”. He used this saying to defend the use of inorganic substances in medicine because his critics claimed that they were too toxic to be applied as therapeutic agents. Apart from the basic concept in toxicology, Paracelsus was the creator of the idea of target organ toxicity. His belief that diseases locate in a specific organ was extended to include target organ toxicity, which means that a chemical has a specific site within the body where it exerts its greatest effect. Paracelsus also encouraged the use of experimental animals to study the beneficial, as well as toxic effects of chemicals. In addition, he wrote the paper On the Miners’ Sickness and Other Diseases of Miners documenting the occupational hazards of metalworking, including treatment and prevention strategies. While clearly a pioneer in toxicology, his concept of “dose makes the poison” needs to be considered in the light of our new knowledge of low dose effects and vulnerable populations.

Keywords: father of toxicology, physician/chemist, alchemist, dose response concept



The Secret of Paracelsus’ Sword


Danijela Đukić-Ćosić1 and Philip Wexler2  ,

1Department of Toxicology “Academic Danilo Soldatović”, University of Belgrade-Faculty of Pharmacy, 2National Library of Medicine, Washington Correspondence:

Philippus Theophrastus Aureolus Bombastus von Hohenheim, better known as Paracelsus (1493-1541), was a German-Swiss physician who introduced the dose-response concept to toxicology. The influence of this famous doctor on other sciences, including chemistry, medicine, physiology, and philosophy was enormous as well. Paracelsus’ portrait dating from 1540, depicts him holding his sword, gripping its spherical pommel. Posthumous portraits of Paracelsus and his statues in Beratzhausen, Bavaria, where he prepared Paragranum, his main work on medical philosophy, show him in the same pose, his hand on the sword’s pommel. He came in possession of this famous longsword during the wars when he served as an army physician. The word “Azoth” was inscribed on the handle of the sword. According to some broadsheets, Azoth was the elixir vitae (mercury), a secret medicine Paracelsus had discovered and kept hidden in the handle of his sword. Paracelsus theorized that materials which are poisonous in large doses may be curative when used in small doses; he demonstrated this with the examples of magnetism and static electricity, wherein a small magnet can attract much larger metals. He was acquainted with the therapeutic powers of the magnet and used it in the treatment of various diseases. His doctrines in regard to effects of magnetism on humans have been confirmed to a great extent since the time of his death. If his sword was forged from magnetite, an iron oxide and the primary component of naturally occurring iron ore, it might suggest that it was used as magnetic therapy.

Key words: portraits of Paracelsus, long sword, magnet therapy

Meet prof. Momčilo Mokranjac – the Father of Serbian Toxicology


Danijela Đukić-Ćosić, Vesna Matović

Department of Toxicology “Academic Danilo Soldatović”, University of Belgrade – Faculty of Pharmacy, Serbia


Prof. Momčilo Mokranjac (1899-1967) was a respected professor at the University of Belgrade and the founder of education in the field of toxicology in our country. He was born in Belgrade in the family of the most famous Serbian composer, Stevan Mokranjac, and Marija Mokranjac, born Predić, a relative of Uroš Predić (one of the most important Serbian painters). Nevertheless, his education was aimed at the natural sciences, far from the music and art. He graduated from the Faculté des Sciences in Paris in 1920, and two years later defended the doctoral thesis (Recherches sur la présence du zinc, du nickel et du cobalt dans les terres arables) at Sorbonne under the supervision of Gabriel Bertrand, “father of microelements”. Upon returning to Belgrade, Dr. Mokranjac worked as a chemist in several institutions. In 1946, he was elected the first professor of Toxicological Chemistry at the Faculty of Pharmacy, University of Belgrade. He was the vice-dean (1947-1949), and then dean (1949-1952) of the Faculty, and spread the importance of pharmaceutical education in our country. Prof. Mokranjac published a book in Serbian Toxicological Chemistry and over 60 important scientific papers. Therefore, he was elected a correspondent member of the Paris Academy of Pharmacy in 1954, and the following year awarded Lavoazie’s medal of the French National Academy of Pharmacy. Prof. Mokranjac was a member of numerous associations as well as the first chief of the Institute of Chemistry of the Serbian Academy of Sciences. This first professor of toxicology in Serbia was a professional, humanist, and “man of science”.


Keywords: professor, toxicological chemistry, Faculty of Pharmacy, University of Belgrade


Founding of the Society of Toxicology


On Saturday, March 4, 1961, a small group met in Washington, DC, to talk about the need for providing a forum where toxicologists could meet and share their research findings. By the end of their day-long meeting the Founders had concluded that the advantages of forming a society outweighed the disadvantages. They had even suggested a name, “The Society of Toxicology” (SOT) and it was to be an international learned society drawing together persons trained in the various disciplines related to toxicology. The follow-up work from this organizational meeting required preparation of a draft constitution, bylaws, scheduling presentations at upcoming scientific meetings, and notifying key people in the field of their plans. Dr. Lehman accepted the role of Honorary President. Dr. Hodge was elected President; Dr. DuBois, Vice President; Dr. Deichmann, Treasurer; Dr. Hays, Secretary; Drs. Coulston, Drill, Larson and Shaffer, members of the Council. To finance all this, each attendee at the first meeting contributed $5 to the treasury. Accordingly, the Society of Toxicology was launched with assets of $35. In 2018, SOT remains a professional and scholarly organization of scientists from academic institutions, government, and industry representing the great variety of scientists who practice toxicology in the US and abroad. The membership has grown to 8,200+ members with 18 Regional Chapters, 6 Special Interest Groups, and 29 Specialty Sections. The SOT Annual Meeting provides a forum where scientists can share their research findings and learn about cutting-edge science and the impact of toxicology research on human and environmental health. The Society’s mission is to create a safer and healthier world by advancing the science and increasing the impact of toxicology. Recordings of the history of the Society of Toxicology are available via the website [].



History of Olimpic Doping Control Laboratory at Faculty of Pharmacy in Sarajevo


Aleksandra Marjanović, Elma Omeragić, Jasmina Đeđibegović, Miroslav Šober 

Faculty of Pharmacy, University of Sarajevo, Zmaja od Bosne 8, Sarajevo, BiH


The most outstanding period in history and development of Faculty of Pharmacy, University of Sarajevo was during XIV Winter Olympic Games (ZOI’84) when the Doping control laboratory was established. This laboratory was organized within the Department for Quality Control of Drugs and Department for Toxicological Chemistry and originally financed by International Olympic Committee and Organizing Committee of ZOI’84. Laboratory was equipped with state-of-the-art chromatographic and spectroscopic equipment that existed in Bosnia and Herzegovina at that time. Laboratory was organized in six operational departments: Department for Collecting and distribution of samples; Department for Extraction and derivatization of samples; Department for Thin Layer Chromatography; Department for Gas Chromatography and High Performance Liquid Chromatography; Department for hyphenated technique GC-MS and Department for Radioimmunologic Analysis. The multidisciplinary team was made up of 60 experts from different professions: pharmacists, doctors, chemists and computer scientists.

During January 1984, the Doping Control Laboratory was accredited by the International Olympic Committee and at that point there were eleven such laboratories in the world. At that time Laboratory at Faculty of Pharmacy together with the laboratory at Los Angeles University of California (UCLA), participated in the wider project of the International Olympic Committee, which aimed to establish reliable evidence for the abuse of exogenous testosterone in sports. After the Olympics, the Laboratory was re-accredited for the control of doping at international competitions in 1987 by the International Athletic Amateur Federation (IAAF).

Keywords: ZOI’84, doping control, Faculty of Pharmacy Sarajevo

Former Yugoslav and Latter Serbian Society of Toxicology


Vesna Matović1, Dragan Joksović2, Veljko Todorović2, Aleksandar Vidaković3

1Department of Toxicology Akademik Danilo Soldatović“, University of Belgrade-Faculty of Pharmacy, 2Military Medical Academy, 3Serbian Institute of Occupational Health “Dr Dragomir Karajović”,



Serbian Society of Toxicology is a national union of toxicologists, dealing with different fields of toxicology: clinical toxicology, environmental toxicology, occupational toxicology, etc. The Society was registered by Bussiness Registry Agency of Republic of Serbia on 4th of March 2010 with the purpose to gather the persons who are actively engaged or interested in research, teaching and application of toxicology. The main goal of the society is to foster the science and education of toxicology, promote the safety of humans, animals and the environment, and protect global health.

However, Yugoslav Society of Toxicology was established back in 1969 and the first Congress of Toxicology was organized in Herceg Novi, Montenegro in October, 1974. This Congress was followed by Congresses in Portorož, Slovenia (October, 1979), in Struga, FYR Macedonia (October, 1982), in Belgrade, Serbia (September, 1985) and in Brioni, Croatia (1990). During nineties, right after the Yugoslavia separation, Society continues to operate through the Section for toxicological chemistry of Pharmaceutical Society of Serbia and newly formed Section for toxicology of Serbian Medical Society. In this period, two congresses were held: VI Congress of Toxicologists on Tara in 1994 and VII Congress of Toxicologists in Igalo (1998) and the journal “Archives of Toxicology, Kinetics and Xenobiotic Metabolism“ with international review was published quarterly. VIII and IX Congresses of Toxicology of Yugoslavia were organized on Tara, in 2002 and 2006,  respectively.

Serbian Society of Toxicology organized X Congress of Toxicology of Serbia with international participation held in Palić in 2010 while XI Congress of Toxicology accredited as International Congress was held in Sremski Karlovci in 2014. Both congresses gathered eminent international scientists.

Serbian Society of Toxicology has been member of EUROTOX and IUTOX ever since 2010.

The joint10th Congress of Toxicology in Developing Countries and 12th Serbian Congress of Toxicology is going to be held in April 2018, 18-21, Belgrade, Serbia.

Keywords: toxicology, society, Yugoslavia, Serbia, congresses, mission

About the Milestones in Toxicology and Toxipedia


Dragica Jorgovanović, Katarina Baralić, Danijela Đukić-Ćosić

Department of Toxicology “Academic Danilo Soldatović”, University of Belgrade – Faculty of Pharmacy


Toxipedia is a free toxicology encyclopedia initiated by Steven G. Gilbert on June 16, 2006. It provides a way to share the knowledge about Toxicology for the benefit of the common good by offering articles and resources about toxic chemicals, health conditions, ethical considerations, history of toxicology, laws and regulation, etc. Toxipedia is a project of the Institute of Neurotoxicology and Neurological Disorders, a nonprofit organization that distributes scientific information about the health and environmental impacts of toxic chemicals, empowering the public and policymakers to make choices that create healthy communities and environments.

Through the ages, Toxicology shaped civilization and taught us very important life lessons. Significant milestones and discoveries in Toxicology can be seen in the poster presentation, made by Steven G. Gilbert and Antoinette Hayes in 2006, available as an interactive version at This poster presentation has been translated into 16 different languages- Amharic, Arabic, Chinese, French, German, Italian, Japanese, Korean, Persian, Portuguese, Russian, Serbian, Slovenian, Spanish, Turkish and Vietnamese. Beside the beginnings of Toxicology, the examples in this poster address further milestones that occurred from antiquity to the postmodern era. The poster also explains how toxicology, which is now a separate science whit multidisciplinary approach, interacted with other disciplines in the past, including religion and politics.

In 2016, 10 years after the founding of Toxipedia, Steven G. Gilbert was recognized by the Society of Toxicology with Public Communications Award for broadening the public’s understanding of toxicology.

Keywords: free toxicology encyclopedia, Steve Gilbert, Milestones of Toxicology

The Slovenian Society of Toxicology


Lucija Perharič, Jernej Kužner, Lucija Peterlin Mašič, Žiga Jakopin, Marjan Vračko

Gerbičeva 60, 1000 Ljubljana, Slovenia



Founded in March 2000, the Slovenian Society of Toxicology (SST), a member of EUROTOX and IUTOX, is an independent, voluntary, non-profit organization of professionals working in toxicology. SST aims to foster and promote toxicology in Slovenia and to facilitate information exchange amongst various fields of toxicology ( The SST is also a recognized stakeholder within the Slovenian chemical safety network ( The SST’s official journal is the Archives of Industrial Hygiene and Toxicology (, co-edited with the Croatian Society of Toxicology. In January 2018, the SST counted 52 members from various professional backgrounds including biochemistry, biology, chemistry, food science, medicine, microbiology, pharmacy, sanitary engineering and veterinary medicine, working in a range of institutions. Four SST members are European Registered Toxicologists.

Within its mission, the SST organized a number of lectures and public debates, a symposium Toxicology (2001), a workshop Environmental Biomarkers in Bioindicators, supported by IUTOX (2002), and national Congresses with international participation (2004, 2015, 2017). The latest congress was devoted to pharmaceutical pollutants in the environment, risk communication and contradictions concerning glyphosate. To encourage education in toxicology the SST organized teaching courses in 2004 and 2005/06. The EUROTOX Basic Toxicology course held in Ljubljana in 2004, was the first of its kind. In co-operation with the University of Ljubljana, the SST prepared an MSc programme in toxicology, unfortunately not accredited due to financial restrains.

In 2018, the SST plans to organize a specialized EUROTOX course in Regulatory Toxicology and continue with the preparations for hosting the EUROTOX2021.

Keywords: Slovenia, Society of toxicology, mission, activities

Postgraduate Studies in the Field of Toxicology – Faculty of Pharmacy, University of Belgrade


Vesna Matović, Dragana Javorac , Evica Antonijevć, Aleksandra Buha Đorđević, Marijana Ćurčić, Danijela Đukić-Ćosić, Zorica Bulat,  Dragana Vujanović, Mirjana Đukić, Biljana Antonijević, Mirjana Nedeljković

University of Belgrade – Faculty of Pharmacy, Department of Toxicology “Akademik Danilo Soldatović”


Since the founding of the Department of toxicology in 1939 postgraduate studies have been established for the students to advance towards the Master of Sciences degree which, from 1966 on, became a precondition for defending a doctoral thesis. In the 1960s, specialist studies began to be organized with the aim of educating experts in the field of toxicology for the needs of healthcare. In addition to these studies, since 2013, academic specialist studies have also been conducted.

Among the first doctoral dissertations at the Faculty of Pharmacy were dissertations in the field of toxicology: Contribution to the question of the application of polarography in toxicological and chemical analyses, by candidate Dušan Jovanović and Contribution to cobalt as a microelement, by candidate Zagorka Pavković-Filipović, under the supervision of professor Momčilo Mokranjac in 1956. A few years later, in 1963, among the first three master thesis at the Faculty of Pharmacy was Matej Maksimović’s thesis in the field of toxicology, also under mentorship of professor Momčilo Mokranjac. In 1970 Knežević Mileva became the first specialist of Toxicological chemistry. Since the founding of the Department of Toxicology until today, 27 doctoral theses, 20 master theses and over 60 specialist papers were defended.

Nowadays, the Department of Toxicology “Akademik Danilo Soldatović” organizes postgraduate studies: PhD studies in Toxicology, specialist studies in Toxicological chemistry and specialist academic studies in Toxicological risk assessment, with the aim of acquiring and improving knowledge and skills in the field of toxicology.

Keywords: doctoral dissertations, master theses, specialist studies, Momčilo Mokranjac


Toxicological Laboratory of Institute of Forensic Medicine in Belgrade –

from State Chemistry to Reference


  1. Lukic, K. Denic, B. Zdrale, D. Stojkov, D. Cvetkovic, V. Zivkovic1, S. Nikolic

Institute of Forensic Medicine, School of Medicine, Belgrade University



In October 1859, Prince Milos Obrenovic signed the decree on the foundation of the chemistry laboratory within the Sanitary Department of the Ministry of Interior, where samples for the State would be analysed. Its first warden was Pavle Ilic. The first building of this, State Chemistry Laboratory was built in 1882 by Ferdinand Sems, the chemist and laboratory warden since 1873, in Kralja Milutina Street – where it still stands. In this laboratory, chemical analyses for forensic, medical and technological purposes were performed for the needs of all Ministries. From 1893 until 1920, laboratory’s third warden was Marko Leko. From 1929 until 1941, this laboratory was part of the Central Hygienic Institute. In 1947, its Toxicology Department became part of the Faculty of Medicine, specifically Toxicology Sector of the Institute of Forensic Medicine. After World War II, laboratory executives were Dusan Gregovic and Bisenija Hristic. The laboratory was still located in 25 Kralja Milutina Street and a small improvised laboratory was established at the Institute of Forensic Medicine for alcohol detection in biological samples. In 1980, this laboratory was officially moved to a new building of the Institute of Forensic Medicine, Faculty of Medicine. Around 150 years after its foundation, this laboratory became the Reference Laboratory for Identification and Determination of Psychoactive Controlled Substances.

Keywords: State Chemistry Laboratory, Toxicology Sector of the Institute of Forensic Medicine

The Toxicology Society of South Africa (TOXSA)


Werner Cordier, Mary Gulumian, Carine Marks, Vanessa Steenkamp, Wilna van Rijssen, Robyn van Zyl, Melissa Vetten, Cherylynn Wium

Toxicology Society of South Africa



TOXSA was established in 2001 with the aim of promoting and advancing the study and application of toxicology in all its aspects in South Africa. Our first national conference was held in 2002 and the Society was admitted to IUTOX in 2004. In 2012, following extensive negotiations through TOXSA, South African Council for Natural Scientific Professions (SACNASP) accepted Toxicological Sciences as a separate, recognized discipline. TOXSA has hosted a number of international and local conferences, including the 7th Congress of Toxicology in Developing Countries (7CDTC) in 2009, the 7th International Symposium on Nanotechnology, Occupational and Environmental Health (NanOEH) in 2015, and participated in the 17th World Congress of Basic & Clinical Pharmacology (WCP2014) in 2014. TOXSA has provided training in the form of risk assessment and hazard identification workshops and, recently, courses on basic, advanced, and clinical toxicology. Later this year, TOXSA is teaming up with various local societies to co-host the First Conference of Biomedical and Natural Sciences and Therapeutics (CoBNeST 2018) to be held in Stellenbosch, South Africa. In addition, TOXSA is proud to present a session at CTDC10 entitled “Arachnids: Fallacies, Clinical Manifestations, Differential Diagnosis and Management of Spider Bite and Scorpion Sting”, and looks forward to fruitful discussions and possible collaborations.


Keywords: TOXSA, South Africa, Africa, societies

A Brief History of the Department of Toxicology “Academic Danilo Soldatović” at the Faculty of Pharmacy, University of Belgrade


Danijela Đukić-Ćosić, Evica Antonijević, Aleksandra Buha Đorđević, Marijana Ćurčić, Zorica Bulat, Dragana Vujanović, Mirjana Đukić, Biljana Antonijević, Vesna Matović, Mirjana Nedeljković

Department of Toxicology “Academic Danilo Soldatović”, University of Belgrade – Faculty of Pharmacy, Serbia


The current Department of Toxicology “Academic Danilo Soldatović” was established as the Institute of Toxicological Chemistry at the Faculty of Pharmacy, University of Belgrade in 1946. The first chief of the Institute and professor of Toxicological Chemistry was prof. Momčilo Mokranjac. At the time, the Institute consisted only of a small room at the University Children’s Clinic, where practical work with students and scientific research were performed. After three years, thanks to the efforts of prof. Mokranjac, the Institute developed and expanded. The Institute of Toxicological Chemistry had separate laboratories for students, professors and assistants within the Institute of Pathology building, as well as a physical chemistry laboratory. After 45 years of establishment, it was transferred to the dedicated premises of the new building of the Faculty of Pharmacy, where teaching and a part of the scientific research are carried out today. In 2006, the Institute of Toxicological Chemistry changed the name into the Department of Toxicology “Academic Danilo Soldatović”. Even today, this Department provides the widest education in the field of toxicology, not only in Serbia, but throughout the former Yugoslavia. Apart from education in undergraduate and postgraduate studies, its academic staff participates in numerous national and international projects, and works with the expert group of ministries and agencies in Serbia responsible in the field of chemicals, biocides, plant protection products, genotoxic impurities, psychoactive substances, cosmetics. Also, the Department cooperates with scientific and professional institutions in the country and abroad in order to improve the knowledge about toxic substances.

Key words: Institute, education of Toxicology, scientific research, Serbia


Danilo Soldatović-Great Toxicologist, Pharmacist and Man


Uroš Čakar and Vesna Matović

Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia


History of toxicology in Serbia does not remember many experts who were known in the world too. Professor Danilo Soldatović is one of them, and he left a significant mark in the world toxicology. He gained university education from the Faculty of Pharmacy, University of Belgrade in 1952, and PhD degree in toxicology on the same institution in 1959 where he continued professional career. He was also exceptional volleyball player and member of National team. During the years he brought to homeland new achievements from the field of toxicology, which he acquired from famous professors in France, Belgium and Switzerland. Besides his activity in academia and science, he left a great mark in the public health, especially regarding the professional poisoning and ecotoxicology. His most significant research was regarding the ability of magnesium as an antagonist of toxic metals. His exceptional scientific findings from toxicology were recognized by French National Academy of Medicine. In 2006, Danilo Soldatović was first pharmacists from Serbia who became a member of it respectful institution. The achievements of professor Soldatović point out that he was important person for the Serbian toxicology, pharmacy and ambassador of our science in the world.


Keywords: toxicology, pharmacy, magnesium



The History of the National Poison Control Centre in Serbia


Slavica Vucinic, Jasmina Jović-Stosic, Vesna Kilibarda, Nataša Perkovic-Vukcevic, Snezana Djordjevic, Gordana Babic, Dragana Djordjevic, Olivera Potrebic, Gordana Vukovic-Ercegovic, Tomislav Rezic, Gordana Brajkovic

National Poison Control Centre, Military Medical Academy, Belgrade, Serbia


The National Poison Control Centre (NPCC) is a national referential institution in which the medical services of prevention and therapy of poisoning by chemical agents, detection of chemical substances in biological and other materials, education and scientific research work in the field of toxicology and pharmacology are carried out.

The forerunner of the present Centre is the Department of Toxicology of the Military Medical Academy (MMA), established in 1961, with the main task to treat acute poisonings and study the poisoning that are of particulate interest to the medical corps service. The first Head of the Department was col. dr Mirko Kramer. In 1984 the Department became a Clinic for toxicology.

The toxicology we practice today is a complex scientific discipline, developed in accordance with the growing risks for human health and the environment. With this regard, we can speak about the serbian analytical, experimental and clinical toxicology that have functioned separately in the Army for decades. According to the guidelines of the WHO, IPCS and the EAPCCT, the Ministry of Health of the former Federal Republic of Yugoslavia, established the NPCC in 1997. Since its foundation up to now the NPCC has grown into the most prestigious institution of its kind in South-Eastern Europe. The Heads of the NPCC were dr Dusan Jovanovic (1998-1999; 2007-2010) and col. prof. dr Dragan Joksovic (1999-2007). Since 2010 to date Prof. dr Slavica Vucinic is the Head of the NPCC.

In terms of medical prevention and rational treatment of human poison exposures in Serbia, the current organization of NPCC has so far proven to be highly effective, which was recognized by the serbian government that has declared the NPCC as the institution of national importance for the defense of the country.

Keywords: poison control centre, toxicology, poisonings









































Positive Results From a Four-year Educational Campaign on Proper Disposal of Pharmaceutical Waste


Predrag Vukomanović1 , Bojana Petrović 1

1 Medical Sanitary School of Applied Sciences,“Visan”, Belgrade, Serbia.


Pharmaceutical waste includes medicines, primary packaging, and accessories for the use, which have become unusable due to expiration date, defects in quality, contamination, or unusable for other reasons. Incorrectly disposed pharmaceutical waste can cause toxic, corrosive, teratogenic, infectious, carcinogenic and other harmful effects on living beings and the environment.

The educational campaign was conducted with the aim of raising public awareness about the importance of proper disposal of expired medicines.

It was conducted in the period from 01/01/2014-31/12/2017. at the Health Center “Lekovita”, Mladenovac, which has 12 pharmacies. In each drug store, it was clearly pointed out that the expired drugs can be brought by citizens and other pharmacies.  The container was prominently located (a red-black hard plastic waste bucket, with a good breathing lid). Education was carried out in pharmacies via flyers and personal contact with pharmacists. The results of the action were followed by measuring the amount of waste material annually.

During the first three years of the campaign, the amount of waste material was collected on average over 20kg, in 2014, 20.5kg, 2015, maximum quantity 26 kg, and 2016, 24 kg. During 2017, lowest quantity of waste was recorded 16 kg, with a greater response of citizens with small amount of expired drugs which indicates that the citizens reacted positively to the campaign and deposited waste in the previous years, so they only disposed of current. Perennial constant work with patients gave positive results in terms of increasing awareness about the necessity of the proper disposal of drugs.

Keywords: management of pharmaceutical waste, environment, compliance, hazardous waste

Exposure Assessment of Female Population to Dibutyl Phthalate (DBP) via Two Cosmetic Products: Application of ConsExpo Tool


Stefan Simeunovic1, Marijana Curcic1, Evica Antonijevic1, Katarina Baralic1, Gonca Cakmak2, Ksenija Durgo3, Biljana Antonijevic1, Danijela Djukic-Cosic1

1University of Belgrade, Faculty of Pharmacy, Department of Toxicology “Akademik Danilo Soldatović”, Serbia, 2Gazi University, Faculty of Pharmacy, Department of Toxicology, Turkey, 3University of Zagreb, Faculty of Food Technology and Biotechnology, Department of Biochemical Engineering, Laboratory for Biology and Microbial Genetics, Hrvatska


Exposure assessment is an important step in determining risks to chemicals in consumer products, including cosmetics, cleaning and pest control products. Exposure models can be used to estimate exposures to chemicals in the absence of biomonitoring data and as tools in chemical risk screening. We have applied a modelling tool ConsExpo (Consumer Exposure) version 5.0 (RIVM, NL) to assess exposure to dibutyl phthalate (DBP), a chemical present in a variety of consumer products, raising health concerns regarding developmental and/or reproductive toxicity.

As a case study, we quantified exposure of female population to dibutyl phthalate (DBP) via two cosmetic products, hair spray and nail polish, containing DBP as plasticizing agent.

Input data on female´s physiology and product usage habits relevant for calculations were selected from ConsExpo default database. The data on DBP weight fraction in products were obtained from available literature. Inhalation and dermal routes were considered relevant for aggregative exposure scenario. Estimated potential chronic dermal exposure (external dose) to DBP was 0.36 mg/kg/day (0.013 and 0.36 mg/kg/day for the hair spray and nail polish, respectively) while the calculated chronic systemic dose (internal dose) was 0.012 mg/kg/day (0.012 and 6,1×10-6 mg/kg/day for the hair spray and nail polish, respectively). The calculated exposures by inhalation route were negligible. Thus, dermal exposure route was shown to be dominant for both products. Potential dermal exposure was shown to be higher to DBP in nail polish compared to hair spray. However, systemic dose was result of almost only dermal exposure to DBP in hair spray.

Keywords: systemic dose, aggregative, dermal, hair spray, nail polish

National Inventory of Mercury Releases in the Republic of Macedonia


Elisaveta Stikova1, Aleksandar Mickovski2, Marjan Mihajlov3, Trajče Stafilov4

1Institute of Public health, Medical Faculty, Sts. Cyril and Methodius University, 2POPs Unit, MOEPP Macedonia, 3MANEKO Solutions, 4Institute of Chemistry, Faculty of Science, Sts. Cyril and Methodius University


The intentional use of mercury in products and processes is a large anthropogenic source of Hg to the global environment. The purpose of this paper is to show the sources of the mercury released in the country, to estimate and quantify of  those releases and to distribute the same in the corresponding output pathways.

The identification was made by inventory of the known sources with subsequent quantification in main 10 source categories. The distribution in the corresponding output pathways was made by performing analysis of the input factors and output fractions or by using the default factors provided in the UNEP’s toolkit. The inventory was developed in December 2017 and referent base year is 2013.

The results showed that the total mercury release in the country is 3.282,77 kg/Hg//y. The most significant category is waste deposition/landfilling and waste water treatment, contributing with 1,476.8 kg/Hg/y of the total mercury releases. The second most significant source is the waste incineration and burning with total release of 382,90 kg/Hg/y (12%). The leading output of the released mercury is in the air, with total amount of the 1,225.34 kg/Hg/y (37,3%), following by land with 977.79 kg/Hg/y (29.8%). The individual mercury release sub-categories contributing with the highest inputs of new mercury are: production of recycled ferrous metals, coal combustion, thermometers with mercury, dental mercury amalgam and the cement production.

The national inventory on mercury releases shall present a basis for establishing a national inventory of emissions from relevant sources.

Keywords: inventory, mercury release, output pathways

Integrated Approaches to Testing and Assessment (IATA) for Risk Assessment: Scoping of Available Guidance with an Emphasis on Uncertainty Assessment


Andrea-Nicole Richarz, Stephanie K Bopp, Raffaella Corvi, Andrew P Worth

European Commission Joint Research Centre, Directorate for Health, Consumers and Reference Materials, Ispra (VA), Italy


Integrated Approaches to Testing and Assessment (IATA) constitute a framework for considering and integrating multiple data sources in a weight of evidence (WoE) to conclude on the hazard/risk of chemicals in view of informing regulatory decision-making. IATA are composed of different, ideally mechanistically derived, building blocks such as data from Test Guideline and non-standard studies, high throughput in vitro assays, omics data, and in silico predictions. They can also be combined in Defined Approaches. Uncertainty evaluation is recognised as a crucial part of risk assessment. All IATA components are associated with uncertainty at different levels, including overarching issues of data and methodological quality, adding uncertainties of the integration and WoE. It is important to analyse and transparently document all uncertainties, in order to allow for informed regulatory decisions.

A scoping exercise was undertaken to identify guidance related to the IATA components and overarching issues, from official regulatory sources to the peer-reviewed literature. Particular emphasis was placed on identifying practical elements such as user-friendly templates, and guidance on characterisation and reporting of uncertainties. It was concluded that guidance is available at different levels, however more addressing the “technical level” than the integration and weight of information. Many initiatives support uncertainty reduction via standardisation and good practice. However, the guidance is fragmented and sometimes duplicated across sectors, scientific areas, countries and legislations. Some overall integration and harmonisation will be beneficial, allowing to increase confidence in risk assessment results as well as to support the aim of mutual acceptance.

Keywords: weight of evidence, data quality, reliability, relevance, alternative methods

Environmental Risk Assessment of Effluent Discharges into the Ogun River from Kara Cow Market, Ogun State, Nigeria


Esther Olaniran 1, Temitope Sogbanmu 2

1,2 Ecotoxicology and Conservation Unit, Department of Zoology, Faculty of Science, University of Lagos, Lagos, Nigeria.


Effluent discharges into freshwater ecosystems are sources of pollutants which can have adverse effects on aquatic organisms. The study aim was to assess the environmental risk posed by the discharge of effluents from Kara cow market, Ogun state, Nigeria into the Ogun river. The studies which were conducted following standard methods include: administration of structured questionnaires to market stakeholders, fish species diversity, physico-chemical analysis, acute and sublethal toxicity studies (biochemical and histological indices) of the effluent using Clarias gariepinus (African Catfish) over a period of 28 days. The questionnaire survey showed that effluents were not treated and stakeholders were ignorant of the environmental risks. Some effluent physico-chemical parameters were higher than set limits. Total PAHs level in the effluent and sediment was 6.73 mg/L and 8.07 mg/kg respectively. Tetracyline levels in the effluent and surface water were 0.23 µg/mL and 0.85µg/mL respectively. Fish species diversity was lower at the test site (Margalef index – 0.45) compared the control (Margalef index – 0.71). The 96 hLC50 of the effluent was 126 mL/L. Neither histological alterations nor significant biochemical changes were observed in the gills and liver of C. gariepinus exposed to sublethal concentrations of the effluent. The results demonstrate the adverse impacts of the effluent constituents on the physico-chemistry of the river and fish diversity though these were not evidenced in the biomarkers assessed. Consequently, it is recommended that more holistic evaluations of the effluent should be conducted in order to provide evidence-based risk communication to the public and stakeholders.


Keywords: abattoir wastewater, biomonitoring, Clarias gariepinus, freshwater ecosystems, persistent organic pollutants

Organochlorine Pesticides in Placenta and Birth Outcomes


  1. Anand and A. Taneja

1Department of Chemistry, Dr. B.R. Ambedkar University, Khandari Campus, Agra-282002, India


Among the agricultural chemicals, pesticides have been the most intensively considered for their association with preterm birth. Dichloro di phenyl trichloro ethane (DDT) is the most notorious pesticides because of its persistent nature, poorly excreted, its property to mimic estrogen hormone and biomagnified in the food chain, thereby increasing potential for human exposure. Organochlorine pesticides cross the placenta; this prenatal exposure has been associated with adverse pregnancy outcomes. An attempt was made to evaluate the relationship between placental organochlorine pesticides (DDT & HCH) and birthweight. Total of 90 mother-child pairs were selected from Agra city. Organochlorine pesticides were measured in the placental tissue by Gas Chromatography equipped with electron capture detector. Regression analyses were performed between birth weight and organochlorine pesticide concentrations. There was a significant decrease in birthweight with increasing organochlorine pesticides such as with every unit increase in alpha-HCH, beta-HCH, gamma-HCH, delta-HCH, total-HCH, p,p-DDE and p,p-DDT the birthweight decreases by 5.81 g, 1.94 g, 4.72 g, 2.09 g, 2.65 g, 6.85 g, and 10.8 g respectively. Preliminary results indicate that exposure to organochlorine pesticides may be related to a lower birthweight. Further robust multicentric studies are needed to conclude the link.

Keywords: placenta, organochlorine pesticides, gas chromatography, birth outcomes

Slovenian Experience in Setting up and Developing Plant Protection Product Risk Asssessment for Human Health


Lucija Perharič1,Tanja Fatur1, Mateja Bolčič Tavčar1, Sanja Vranac2, Jernej Drofenik2

1National Institute of Public Health

2Administration of the republic of Slovenia for food safety, veterinary and plant protection


The administration of the republic of Slovenia for food safety, veterinary and plant protection (ARSFSVPP) is the responsible authority for national registrations of plant protection products (PPP) and risk assessment (RA). It also coordinates national activities concerning zonal registrations, mutual recognitions, and European Union active substance (a.s.) RA.

In 2003, ARSFSVPP established a multidisciplinary group of professionals with expertise in chemistry, efficacy, toxicology, ecotoxicology, environmental fate and behaviour and residues. The members were initially trained at the British Pesticide Safety Directorate and supported to continuously develop their expertise. The group currently consists of 15 professionals. The National Institute of Public Health (NIPH) was commissioned to carry out work related to human health. Three NIPH professionals assess risk according to the Regulation 1107/2009 and relevant international technical guidelines.

Since 2003, NIPH assessed 464 PPPs, acted as rapporteur or co-rapporteur for five a.s.: Azimsulfuron, Metsulfuron—metyl, Imazosulfuron, Rimsulfuron and Tritosulfuron. We actively participated in the PPP RA group at the European Food Safety Authority and the Expert Advisory Group for the endocrine disruptor (EDs) identification criteria. We surveyed plant protection first aid, decontamination and treatment instructions of PPP labels in order to bring them in line with the current guidelines. We regularly communicate to the professional and lay public, and participate in capacity building on national and international level.

In 2018, we plan to embark on identification of EDs in the context of Regulation 1107/2009, prepare RA monographs for GiberellicAcid and Gibberellins, to continue the routine and ad-hoc RAs and training a ctivities.


Keywords: pesticides, human risk assessment, capacity building

One-year Study on Exposure Assessment to Marine Biotoxins via Consumption of Shellfish from the Black Sea, Bulgaria


Stanislava Georgieva 1, Zlatina Peteva 1, Bernd Krock 2, Anelia Gerasimova-Peneva 1, Mona Stancheva 1

1Medical University – Varna, Department of Chemistry, Marin Drinov 55, 9002 Varna, Bulgaria, 2 Alfred Wegener Institute, Helmholz Zentrum für Polar und Meeresforshung, Chemische Ökologie, am Handelshafen 12, 27570 Bremerhaven, Germany  


Human poisonings after consumption of shellfish contaminated with microalgae toxins (phycotoxins) have been recorded in Europe for several decades. In this regard EFSA has

set limits of some marine biotoxins present in seafood. Although in Bulgaria no outbreaks have ever been reported, some of the regulated marine toxins – domoic acid (DA), pectenotoxin-2 (PTX-2) and yessotoxin (YTX) have been detected in mussel samples.

The aim of this study was the human acute and chronic exposure to these toxins through shellfish consumption to be evaluated. For this purpose, data of toxins concentrations in samples collected in the period spring – summer 2017 has been linked consumption data obtained from the National Bulgarian Fish and Shellfish Consumption Survey including 4891 adults.

The spring toxin profile represents 61% DA, 22% YTX and 17% PTX-2 as in summer only YTX was detected. It was calculated that the acute exposure to DA, PTX-2, YTX in spring and YTX in summer is respectively 0,62; 0,00055; 0,00054 and 0,003 μg phycotoxin /kg bodyweight (bw) that are not exceeding the acute reference doses (ARfDs). Chronic exposure only for DA was calculated as for PTX-2 and YTX no data on their chronic effects in animals were available, so tolerable daily intake (TDI) is not established. With 0,0076 μg DA/kg bw/day the chronic exposure was established to be beneath the tolerable daily intake of 0,075 μg/kg bw per day.

The present study showed that Bulgarian population is posed to no risk through consumption of studied shellfish.


Keywords: shellfish poisoning, domoic acid, pectenotoxin-2, yessotoxin, mussels
























































































Predicting Adverse Immune Reactions Using a Novel Human In-Vitro Skin Test


Shaheda Ahmed2 , Louis Bibby1, Anne Dickinson1, 2
1Institute of Cellular Medicine, Newcastle University, Framlington Place, Newcastle upon Tyne, United Kingdom NE2 4HH
2Alcyomics LTD, Bulman House, Regent Centre, Gosforth, Newcastle upon Tyne, United Kingdom, NE3 3LSC


There are currently no preclinical human in vitro tests to determine the potential of hypersensitivity related to adverse drug responses. Here we describe human in-vitro skin tests to assess the safety, efficacy and dose responses to pharmaceuticals from a hypersensitivity perspective. Responses also correlate to T cell proliferation and IFN-ƴ assays which give a predictive read out for adverse immune reactions. The used tests are human blood and skin biopsies from healthy donors which are co-cultured with the test compound. Skin is assessed for histopathological damage following fixation of skin in formalin for 24 hours and staining for haematoxylin and eosin (H&E). Sections are graded according to the Lerner classification system (grade I negative or grade II-IV positive) which correlates to immune damage. A number of therapeutic biologics and small molecule drugs have been tested in our human in vitro test model. Results show a statistically strong positive correlation with histopathological grading in our skin tests to expected clinical response for biologics (r=0.96, p<0.0001) and small molecules (r=0.72, p<0.01). Additionally, an analogue of TGN (TGN1412) was tested and showed a strong positive response (grade III) in our assay, indicating that this assay could have predicted the serious life-threatening cytokine storm observed in the 2006 Northwick park trial disaster. The novel human in vitro skin tests can be used to determine safety, efficacy and dose responses to biopharmaceuticals including monoclonal antibodies and small molecule drugs as well as providing information on adverse immune reactions from a hypersensitivity perspective.

Keywords: hypersensitivity, in vitro test, biopharmaceuticals


Safety Assessment of Vaccine Adjuvants: Effect of MPLA and QS-21 on In Vitro Co-culture Model

1,2 Carolina Campos Estrada, 1 Benjamin Riquelme, 1,2 María Fernanda Cavieres.

1 Escuela de Química y Farmacia, Facultad de Farmacia, Universidad de Valparaíso, Valparaíso, Chile. 2 Centro de Investigación Farmacopea Chilena, Universidad de Valparaíso, Valparaíso, Chile.


Currently the vaccines are oriented not only to prevention but also the treatment of various pathologies. In this scenario, must be co-administered with an adjuvant to elicit a potent immune response immunological. Thus, there is a major need for developing powerful adjuvants or adjuvant systems for use in these vaccines. Numerous adjuvants are currently subject to studies and many of these formulations include QS21 and MPL. The main drawbacks in the development and approval of new adjuvants are unacceptable side-effects, lack of understanding of their mechanism, unknown safety in special populations, and use of preclinical models with limitations in their prediction of toxic effects. We standardize a co-culture model between HUVECs and PBMCs from healthy donors to evaluate both vaccine adjuvants to obtain physiologically relevant data that deliver genetically-diverse and meaningful pathophysiological evidence for the country. The viability was evaluated by MTT and PI incorporation. The release of 13-Cytokine was determined with multiplex assay. The induction of immune response was evaluated by ECAMs expression, leucocyte adhesion and NFκB activation. We found that both adjuvants were able generate an increase IL-1, IL-6, IFN-β, TNF-α, and TGF β. However, QS-21 but not MPLA was able to increase the IL-17 release. Both adjuvants increased ECAMs expression, cell-cell adhesion indicating endothelial activation and leukocyte recruitment. Interestingly, we found an optimum MPLA/QS21 combination, in order to establish the balance between efficacy and safety. Finally, this model could be an alternative to the use of experimental animals, evaluating more precisely the immune response triggered by the adjuvanted vaccines.

Keywords: cytokines, ECAMs, cell-cell adhesion, adjuvants, NFκB


MicroRNA Regulation of TNF sSgnaling; Changing the Balance Between Pro-death and Pro-survival Effects

Samira Ghorbani1,2, Farideh Talebi1, Farshid Noorbakhsh1

1 Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

2 Shefa Neuroscience Research Center, Khatam Al-Anbia Hospital, Tehran, Iran


MicroRNAs are small RNA species which are known to regulate various aspects of cell biology, including cell survival, proliferation and apoptosis. Tumor necrosis factor alpha (TNF), is an innate immune cytokine which is involved in regulating inflammation as well as cell survival. Interestingly, both pro-survival and pro-death effects have been reported for TNF in various physiological and/or pathological settings. Whether binding of TNF to its receptors leads to pro-survival or pro-death effects is mostly attributed to the type of adaptor molecules which are recruited to the cytoplasmic domain of the receptors. Two such adaptor molecules are TRADD (TNF receptor associated death domain) and MADD (MAP kinase activating death domain). In this study, we investigated the potential effects of miRNAs on the expression of MADD and downstream TNF signaling. Initial bioinformatic analyses revealed miR-181a and miR-181b as potential regulators of MADD expression. Molecular analyses using luciferase vectors which contained a luciferase coding sequence upstream of MADD’s 3’UTR sequence confirmed direct interaction between miR-181a and miR-18b and MADD transcripts. Overexpression of miR-181a/b mimicking sequences in L929 fibroblast cells suppressed MADD’s transcript and protein levels, while downregulation of endogenous miR-181a/b levels by antagomirs enhanced MADD’s expression. miR-181b mimic sequences enhanced the occurrence of apoptosis in L929 cells following TNF treatment, a phenomenon that was associated with decreased mitochondrial membrane potential in miR-181b overexpressing cells. Overall, our results point to a potential role for miRNAs in regulating TNF pro-death versus pro-survival signaling.

Keywords: MADD, TNF signaling, microRNA, apoptosis






































Genotoxic Effects of Metformin in Human Cervix Adenocarcinoma Cells Using Comet Assay


Tuba Sarıaydın1, Tuğba Gül Çal1, Sevtap Aydin1, Ülkü Ündeğer Bucurgat1, Nurşen Başaran1,

1Department of Pharmaceutical Toxicology, Faculty of Pharmacy, University of Hacettepe, 06100, Ankara, Turkey


Metformin is commonly used in the therapy of type 2 diabetes. Antioxidant effect of metformin has been suggested to be responsible for protecting oxidative stress-related DNA damage. However there are contradictory results in the literature.

This study was focused on the cytotoxic and genotoxic effects of metformin in human cervix adenocarcinoma (HeLa) cells.

The cytotoxicity of metformin (0.5-64 μM) was determined by Thiazolyl Blue Tetrazolium Blue (MTT) assay and its genotoxicity (5-1000 μM) was determined by alkaline comet assay in HeLa cells treated with/without H2O2. DNA damage was expressed as DNA tail intensity.

The cell viability significantly decreased at the doses of 32 μM and 64 μM. The IC50 dose of metformin was 76.97 μM for 24h in HeLa cells. Metformin alone did not induce DNA damage at all studied concentrations (5-1000 μM) for 24h. However, at the concentration ranges between 5 μM and 1000 μM, it significantly increased H2O2-induced DNA strand breakage damage (p < 0.05).

In conclusion, our results suggest that metformin has a potential role in increasing the oxidative stress-related genotoxicity. In order to get more pronounced results, more mechanistic studies should be performed.

Keywords: metformin, cytotoxicity, genotoxicity, comet assay, human cervix adenocarcinoma cells

Interactions of Pycnogenol with Cisplatin on V79 Cell Viability


Sevtap Aydin1, Merve Becit1, Arif Ahmet Basaran2, Nurşen Başaran1,

1Department of Pharmaceutical Toxicology, Faculty of Pharmacy, University of Hacettepe, 06100, Ankara, Turkey

2Department of Pharmacognosy, Faculty of Pharmacy, University of Hacettepe, 06100, Ankara, Turkey



Cisplatin is commonly used in the therapy of many solid tumors. In the treatment of cancer, it has been intended to increase anticancer effect and decrease cytotoxicity using various plant-derived phenolic compounds with chemotherapeutic drugs. Pycnogenol (PYC) obtained from Pinus pinaster is commonly consumed as a dietary food supplement because of its strong antioxidant effect and has been the subject of many studies. The mechanisms of the interactions of PYC with cisplatin needs to be clarified, therefore we aimed to determine the effects of PYC on cisplatin cytotoxicity in Chinese lung fibroblast cell lines (V79). The cell viability was determined by MTT assay for 24h and 48h. The IC50 values of PYC were 670 μM and 119 μM for 24h and 48h, respectively.  PYC significantly increased the cytotoxicity of cisplatin at concentrations of 500 μM and1000 μM (1.75 fold and 4.04, respectively, vs. IC50 doses of cisplatin) for 24h and 250-500 μM (2.92 fold, 6.96 fold and 12.43 fold, respectively, vs. IC50 doses of cisplatin) for 48h in a dose dependent manner. In conclusion, our findings show that PYC may interact with cisplatin and play a important role in the chemotherapy; however, further studies are required to confirm their interactions with cisplatin.

Keywords: Pycnogenol, cisplatin, cytotoxicity, V79 cells

Interaction of Zebrafish (Danio rerio) Organic Anion Transporter 2d (Oat2d) with Xeno- and Endobiotics


Jelena Dragojević1, Ivan Mihaljević1, Tvrtko Smital1*

1 Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, Zagreb, Croatia,,

Organic anion transporters (OATs/Oats) are polyspecific membrane uptake transporters that mediate entrance of compounds into the cell. Consequently, they are key determinants of toxicological response to various xenobiotics. Despite their physiological importance and role in cellular detoxification, the knowledge about uptake transporters in non-mammalian species is scarce. Zebrafish (Danio rerio) has seven OAT orthologs: Oat1, Oat2a-e and Oat3. Mammalian OAT2 was characterized in human, rat and mouse, and was shown to play an important role in uptake and distribution of physiological compounds, as well as anionic toxins and drugs. As OAT2 orthologs have been poorly studied in non-mammalian species, the goal of our study was to determine phylogenetic relationships, tissue distribution and substrate specificity of zebrafish Oat2d. Phylogenetic analysis of OAT/Oat genes confirmed similarities among mammalian and zebrafish Oat transporters. Zebrafish Oat2d highly expressed in intestine and moderately expressed in testes and brain. Western blot analysis revealed protein band of 60 kDa, and immunocytochemistry showed its correct localization in the plasma membrane. Functional studies using HEK293T cells overexpressing zebrafish Oat2d revealed two model fluorescent substrates of Oat2d: lucifer yellow (LY, Km = 56.36 µM) and 6-carboxyfluorescein (Km = 210.1 µM). The initial screening with various endo- and xenobiotics showed significant inhibition of Oat2d mediated uptake of LY by endogenous compounds (α-ketoglutarate, fumarate, bilirubin and deoxycholic acid) and exogenous compounds (p-aminohippurate, perfluorooctanesulfonic acid, perfluorooctanoic acid, furosemid, chlorpyrifos, tetracycline and diclofenac). Selected potent inhibitors, fumarate and indomethacin, showed dose dependent inhibition of Oat2d transport (IC50 = 68.24 μM and 20.41 μM, respectively).

Keywords: membrane proteins, phylogeny, tissue expression, cell localization, functional analysis


Biological Responses to Hybrid Fe-Si Nanoparticles in Caco2 Cells


Mihaela Balas 1, Florian Dumitrache 2, Madalina Andreea Badea 1, Andreea Luminita Radulescu 1, Claudiu Fleaca2, Claudiu Locovei2, Eugenia Vasile2,3, Anca Dinischiotu 1

1 Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 9195 Splaiul Independenţei, 050095 Bucharest 5, Romania, 2 National Institute for Lasers, Plasma and Radiation Physics (NILPRP), Atomistilor 409, 077125 Magurele, Romania,3 Politehnica University of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of Oxide Materials and Nanomaterials, Gh. Polizu 1-7, Bucharest, Romania


Our study aimed to assess the biological responses of hybrid Fe-Si nanoparticles in cancer cells.

Hybrid Fe-Si nanoparticles synthetized by laser pyrolysis using Fe(CO)5 vapors and SiH4 as Fe and Si precursors were coated with L-3,4-dihydroxyphenylalanine (L-DOPA) and sodium carboxymethyl cellulose (CMC-Na). Doses ranging from 0 – 200 µg/mL nanoparticles were exposed to colorectal adenocarcinoma cells (Caco2 cell line) for 24 and 72 hours. Cancer cell viability and integrity were evaluated by the level of mitochondrial enzymes (MTT assay) and lactate dehydrogenase (LDH) activities. Biological responses to nanoparticles were evaluated by analyzing reactive oxygen species (ROS) production, reduced glutathione (GSH) content and Nrf-2 protein expression. Cellular morphology and internalization of nanoparticles were also assessed.

The results showed no changes of viability and integrity of Caco2 cells exposed to a dose below 100 μg/mL L-DOPA-coated Fe-Si nanoparticles but over a dose of 50 μg/mL CMC-Na-coated nanoparticles cell viability significantly decreased in a time-dependent manner. Cellular morphology and organization of F-actin filaments were not altered after exposure to 25 and 50 μg/mL from both L-DOPA and CMC-Na-coated Fe-Si nanoparticles. After 72 hours, internalization of both types of nanoparticles were observed in cancer cells. However, the ROS production increased starting with 24 hours exposure to 25 and 50 μg/mL of both types Fe-Si nanoparticles inducing the increase of GSH intracellular level and activation of Nrf-2.

Our findings suggest that Caco2 cells managed to survive after exposure to both L-DOPA and CMC-Na-coated Fe-Si nanoparticles by triggering antioxidant defense mechanisms in response to oxidative stress.

Keywords: Fe-Si nanoparticles, laser pyrolysis, colorectal adenocarcinoma cells, cytotoxicity, oxidative stress

Saccharomyces cerevisiae – a Simple Unicellular Eukaryotic Model Organism for In Vitro Toxicity Screening of Nanoparticles


Kaja Kasemets1, Sandra Käosaar1, Anne Kahru1

1 Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia


The yeast Saccharomyces cerevisiae is a promising unicellular eukaryotic model organism for the NPs toxicity screening as its cellular structure and function has many similarities (e.g., oxidative stress response) with the higher-level organisms. Moreover, yeast has a short generation time and can be easily cultivated. In addition, the genome of S. cerevisiae has been sequenced and there are lots of mutated strains available for mechanistic studies. Yeast cells have also endocytosis, being a simple particle-uptake model. Differently from the higher eukaryotic cells yeast has rigid cell wall consisting of chitin and glucan – main specific antifungal molecular targets.

In this study we profiled the toxicity mechanism of copper oxide (CuO) and silver (Ag) NPs using S. cerevisiae BY4741 wild-type (wt) and its single gene-deletion mutants (EUROSCARF) defective in oxidative stress response (OS), cell wall/membrane integrity and endocytosis. The growth inhibition assay in organic-rich growth medium (YPD) and cell-viability assay in deionized water (DI) was applied.

Results showed that the studied CuO and AgNPs were ~30–100 more toxic in DI than in YPD. Comparison of the toxicity pattern of wt and mutant strains revealed that the toxicity of CuO and Ag NPs was not caused by the OS or cell wall/membrane disturbance. Confocal microscopy showed that wt but not the endocytosis-deficient mutant internalized AgNPs. The role of dissolved ions in the studied NPs toxicity will be discussed.

This work was supported by the IUT 23-5.


Keywords: CuO and Ag NPs, dissolution, oxidative stress, cell wall/membrane integrity, endocytosis

Investigation into Metabolic Profiles Following Diethylstilbestrol on p53+/- Heterozygous Mice Using 1H-NMR Spectroscopy


Mohd Nazil Salleh1, Henkie Isahwan Ahmad Mulyadi Lai1 andTaufiq Yap Yun Hin2

1Department of Health Sciences, Faculty of Engineering and life Sciences, Universiti Selangor, Shah Alam City Campus, 40000 Shah Alam Selangor Malaysia. 2Catalysis Science and Technology Research Centre, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Malaysia.


Metabonomic approach based on 1H-NMR Spectroscopy and chemometric analysis has been applied to investigate endogenous metabolite profiles of biochemical effects of clastogenic carcinogen, diethylstilbestrol (DES) in the urine of female wild-type mice after severed changes of genes profiles compared to p53+/- heterozygous mice. Mice (n=80) were housed individually in metabolism cages in a well-ventilated room, at a temperature of 21 ± °C and a relative humidity of 50 ± 10%, with a 12-h light/darkness cycle. Each animal received a single dose (i.p) injection of 500 umole/kg b.w of DES, once daily for four days and animals were sacrificed. Urine samples were collected at various time-points: pre-dose (control), 8, 24, 32, 48, 56, 72, 80 and 96 hours. Chemometric analysis of 1H-NMR data have shown that there are endogenous metabolite changes between vehicle and DES-treated mice between wild-type and p53+/- heterozygous mice. Major biochemical changes included elevated urinary levels of creatine, lactate, taurine, hippurate and reduced tricarboxylic acid cycle (TCA) especially 2-oxoglutrate and trimethylamine-n-oxidase levels in DES-treated compared to vehicle mice. The elevated taurine and creatine levels in the urine of treated mice in response to hepatocyte necrosis has been shown in the histological evaluation. The reduction of 2-oxoglutrate indicates a general increase in energy metabolism. Therefore, the application of 1H-NMR spectroscopy, coupled with chemometric methods analysis, could be of importance for generating new diagnostic tools in the early detection of toxicity.

Keywords: metabonomic, diethylstilbestrol, p53+/- heterozygous, 1H-NMR Spectroscopy

Toxic Mechanisms of Cyanobacterial Secondary Metabolites from Oscillatoria Strain Revealed by the Effects-directed Analyses (EDA)


Petra Marić1, Marijan Ahel2, Olivera Babić3, Dajana Kovač3, Jelica Simeunović3, Tvrtko Smital1

1Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, 10 000 Zagreb, Croatia, 2Laboratory for Analytical Chemistry and Biogeochemistry of Organic Compounds, Division for Marine and Environmental Research, Ruđer Bošković Institute, 10 000 Zagreb, Croatia, 3Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia


Cyanobacterial strains are producers of various toxic secondary metabolites referred as cyanotoxins that represent a significant threat to the environment and human health, especially during periods of intensive blooms. Increased occurrence of cyanobacterial blooms, as well as human and animal poisonings has led to numerous studies aimed to reveal mechanisms of cyanobacterial toxicity on cellular defense systems. Yet, the knowledge on accumulation-detoxification molecular pathways directly affected by cyanotoxins produced by specific strains of cyanobacteria, as well as a deeper insight on their ecotoxicological effects is still missing. The concept of the effects-directed analyses (EDA) is the most advanced methodology available in environmental science for addressing complex environmental toxicity issues, able to determine the identity of the toxic compounds, mechanisms of toxicity and their effects. Accordingly, the main goal of this study was to identify molecular mechanisms of cyanobacterial toxicity in the extract of aquatic cyanobacterial strain Oscillatoria K3 found in toxic cyanobacterial blooms in Vojvodina region (Serbia) using EDA approach. Ecotoxicological endpoints that were determined included interaction with toxicologically relevant zebrafish uptake membrane transporters DrOatp1d1 and DrOct1 (phase 0 of cellular detoxification), and CYP1A1 enzymes (phase I). Extracts of Oscillatoria K3 obtained by non-selective and non-target preparation techniques demonstrated the strongest interaction with DrOatp1d1 transporter as well as CYP1A1 enzyme in the first, second and third tier of our EDA study. In conclusion, our data showed that toxic secondary metabolites from Oscillatoria K3 include polar cationic and anionic DrOatp1d1/DrOct1 inhibitors and lipophilic CYP1A1 inducers of the cellular detoxification mechanism.

Keywords: cyanobacteria, secondary metabolites, uptake membrane transporters, CYP1A1 enzyme, effects-directed analyses (EDA)

Oxidative Stress and Antioxidative Defense Parameters in Female Workers  Exposed to Volatile Organic Compounds


Nina Umicevic1, Jelena Kotur-Stevuljevic2, Evica Antonijevic3, Danijela Djukic-Cosic3 and Biljana Antonijevic3

1University of Banja Luka, Faculty of Medicine, Department of Toxicology , 2University of Belgrade, Faculty of Pharmacy, Department of Medical Biochemistry, 3University of Belgrade, Faculty of Pharmacy, Department of Toxicology “Akademik Danilo Soldatović”

Workers in the shoe manufactures are constantly exposed to commonly used volatile organic compounds (VOCs), including xylene, propane, hexane, cyclohexane, butene, toluene, acetaldehyde, ethanol, acetone, dimethyl sulfide, dimethyl disulfide etc. The aim of this study was to investigate  relationship between inhalation exposure to volatile organic compounds (VOCs) and VOCs’ capacity to induce oxidative stress in women employed in shoe production facility. The study included 55 female workers and 25 healthy unexposed female controls. The superoxide anion (O2–), advanced oxidation protein products (AOPP), total oxidative status (TOS), prooxidative-antioxidative balance (PAB), oxidative stress index (OSI) and antioxidative defence parameters (superoxide dismutase (SOD) enzyme activities, values of SH groups, paraoxonase-1 (PON1) activities and total antioxidant status (TAS) were determined in plasma in all subjects. The obtained results have shown that all investigated oxidative stress parameters (O2–, AOPP, PAB, TOS and OSI) as well as antioxidative defence parameters were significantly higher (p<0.001) in exposed workers compared to controls. Moreover, paraoxonase-1 (PON1) activities (p<0.001) and total antioxidant status (TAS) (p<0.001) significantly decreased in the workers exposed to VOCs. These results suggest that occupational exposure to even permissible levels of VOCs in the shoe manufactures may induce oxidative stress in the plasma of workers proving that oxidative stress is among the mechanisms, which positively contribute to the development of adverse effects caused by VOCs.

Keywords: volatile organic compounds, occupational exposure, oxidative stress,  antioxidative defense

Influence of Hypothyroidism on Testicular Mitochondrial Oxidative Stress by Activating the p38MAPK and JNK Signaling Pathways in Rats


X-R Chang1, Y-L Yao1, D Wang1, H-T Ma1, P-H Gou2, C-Y Li1, J-L Wang1

1Department of Toxicology, School of Public Health, Lanzhou University, Lanzhou, Gansu Province 730000, China

2INSERM UMR-S 1131, Institut Universitaire d’Hématologie, Université Paris Diderot, Paris 75475, France

Thyroid hormone (THs) deficiency can impair testicular function. However, knowledge of the effects of MAPK pathways on testicular mitochondrial oxidative damage induced by hypothyroidism is still rudimentary. This study aims to explore the possible mechanisms of testicular mitochondrial oxidative damage in hypothyroidism rats. Wistar male rats were randomly divided into control (C), low- (L) and high-hypothyroidism (H) groups [0, 0.1 and 10 mg propylthiouracil (PTU)/kg respectively] by intragastric gavage for 60 days. Blood samples were collected to measure the levels of serum triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH). Testicular mitochondrial homogenates were to measure the activities of superoxide dismutase (SOD), catalase (CAT) and Ca2+-ATPase as well as protein and mRNA expression of androgen receptor (AR), p38 mitogen-activated protein kinase (p38MAPK) and c-Jun NH2-terminal kinase (JNK). The results showed that the body weight, testis weight, and levels of T3 and T4 were all significantly decreased and the relative testes weights and level of TSH were significantly increased in the H group. There were significant decreases in SOD activity in the H group as well as decreases in CAT and Ca2+-ATPase activities in the L and H groups. Additionally, protein expression of AR decreased significantly and protein expression of phosphorylated p38MAPK and JNK increased significantly in the H group. Therefore, the study suggests that hypothyroidism could affect male reproductive function by disturbing expression of AR, inducing oxidative stress and changing the activity of Ca2+-ATPase, leading to activation of p38MAPK and JNK signaling in the testicular mitochondria.

Keywords: hypothyroidism, testicular mitochondria, oxidative stress, p38MAPK, JNK

















































Influence of Chelation Therapy on Oxidative Stress Parameters in Occupationally Lead Exposed Workers


Asli Karacan Dincer, Tugce Cetin, Suna Sabuncuoglu, Gozde Girgin, Turkan Nadir Ozis 

Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey.


Lead (Pb), which has many toxic effects in human organ systems, is a well-known environmental and occupational toxic heavy metal. The mechanism of Pb induced oxidative stress involves structural and functional changes in tissues and cellular components leading to inactivation of enzymes, lipid peroxidation and DNA damage. The present study was designed to investigate the possible influence of chelation therapy on oxidative stress induced by occupational Pb exposure. For this purpose, heparinized blood samples were collected from occupationally Pb exposed workers (aged 25 to 48, n=39) before and after chelation therapy. 8-hydroxy-deoxyguanosine (8-OHDG), malondialdehyde (MDA), protein carbonyl (CO), glutathione peroxidase (GPx) and aminolevulinate delta dehydratase (ALAD) enzyme, which is an important target of Pb, levels were measured in plasma by using ELISA kits. Besides that, to evaluate possible cholesterol oxidation caused by Pb exposure, 7-ketocholesterol (7-KC) and cholestane-3β,5α,6β-triol (triol) levels were also measured using HPLC-MS/MS. The levels of 8-OHDG, MDA, 7-KC and triol significantly decreased when compared its values before chelation therapy (p<0.05). ALAD enzyme activity significantly increased following chelation therapy. Nevertheless, CO and GPx levels were not significantly affected. Our results showed that chelation therapy decreases Pb induced oxidative stress in occupationally Pb exposed workers. Furthermore, cholesterol oxidation is also significantly increased by Pb exposure and decreased after chelation therapy. The data indicate that chelation therapy, which is mainly used to decrease blood Pb level, is beneficial against Pb induced oxidative stress. Further studies are needed to clarify the role of chelation therapy on Pb exposure induced oxidative stress.


Keywords: lead, chelation therapy, oxidative stress, cholesterol oxidation


Predicting Phytoavailability of Metals in Vegetables UsingSoil Characteristics, Melbourne Australia


Slavica Kandic 1, Dr. Susanne Tepe 2, Dr. Ewan Blanch 3, Maddumage Don Erandika Abeywardane 4, Dr. Suzie Reichman5

1 School of Engineering, RMIT University, Melbourne 3001, Australia, 2 School of Engineering, RMIT University, Melbourne 3001, Australia, 3School of Applied Chemistry and environmental Science, RMIT University, Melbourne 3001, 4 School of Engineering, RMIT University, Melbourne 3001, Australia,


The objectives of present study were to investigate acumulation of metals in nine vegetable species and to  model the uptake of metals (Pb, Cd, Ni and Cr) in vegetables from garden soil with different levels of historical pollution. Five vegetable groups were cultivated in home gardens and categorized in five groups. Among the vegetable groups, concentrations of metals in edible parts were in descending order:

herbs>leafy>legumes>solanaceous>cucurbitae. Metal concentrations in vegetables decreased in the order of Ni> Pb> Cr>Cd.  The highest ability for metal uptake was in leafy vegetables, while the Cucurbitae had the lowest concentrations accumulated in plant tissue. This suggest that low metal accumulators such as Solanaceae and Cucuritae are suitable for cultivation in contaminated soil. By including soil characteristics, pH, electrical conductivity, soil organic matter, cation exchange capacity and vegetable garden soil metals, 20 empirical models  were developed using stepwise multiple regression model to predict metal concentrations in vegetables. We assessed human health risk from metals due to vegetable consumption using Target Hazard Quotient (THQ). The total diet THQ values for  adults and children were below 1.0 indicating that there was minimal risk to adverse health due to oral exposure to vegetables in this study.

Keywords: phytoavailability, vegetables, soil characteristics, metals

Mercury Levels in the Food and İts Biological Derivatives in Turkey

                                        A Meta-Analysis Study

Elif Aslihan CAYIR 1, Sinan KARACABEY 1,2, Yasser BATSH 1, Ahmet AYDIN 1

1 Yeditepe University, Faculty of Pharmacy, Department of Toxicology, Istanbul, Turkey,

2 Marmara University, School of Medicine, Department of Emergency, Istanbul, Turkey.


Mercury is a toxic heavy metal and the accumulation of it inside food and its biological derivatives is a common and serious issue. Food and Drug Administration (FDA) estimates that most people are exposed, on average, to about 50 ng of mercury per kilogram of body weight per day (50 ng/kg/day) in the food they eat..  A large part of this mercury comes from eating fish. However,. In addition to fish, many kinds of contaminated food and its derivatives may be a source of toxicity with mercury. In this study, a meta-analysis was done to many kinds of food and biological derivatives which were consumed in Turkey assessing published articles dealt Separately with the level of mercury in these products. PubMed and Medline databases were searched with mercury, food, biological derivatives, and Turkey keywords. We found about 60 articles, the foods in these articles were classifieds according to specific foodstuffs like fish, breast milk, mushrooms, olives, baby food, cheese, honey, cow sheep and chicken liver. Mercury levels in general was below the maximum accepted limit but wide ranges of the values have been seen for example mercury level in many kinds of fishes was between 0,003 to 0,6 ppm. As a conclusion, many factors affected the level of mercury, like the distance from the surroundings industry areas, which has had high effect to the results, in addition to the time of study, and the method of analysis used to determine it.


Keywords: heavy metal, toxic metal, fish, food contamination, bioaccumulation



Lead Levels in the Food and its Bıologıcal Derıvatıves in Turkey

A Meta- Analysis Study


Ezgi TALO 1, Neslihan ŞAHİN 1,  Sinan KARACABEY 1,2, Ahmet AYDIN 1

1 Yeditepe University, Faculty of Pharmacy, Department of Toxicology, Istanbul, Turkey,

2 Marmara University, School of Medicine, Department of Emergency, Istanbul, Turkey.


The lead is a bluish gray metal with the symbol Pb and a toxic substance present in our environment in small amounts. People may be exposed to lead from drinking water or food. In 1993, the FDA established a maximum daily intake level (6 µg/day), based on 10 micrograms of lead per deciliter of blood (µg/dL). Large part of lead comes from by contaminated food. In this study, a meta-analysis was done to many kinds of food and biological derivatives which were consumed in Turkey assessing published articles dealt with the level of lead in these products. PubMed and Medline databases were searched with lead, food contamination, heavy metal, and Turkey keywords. We found about 163 different articles. The analysed material in these articles were both food products and biological samples. Most of the articles have been made on various fish species. The lead levels in these studies were in concentrations that would not pose a threat to human health (0.405±0.025 µg/g), but some suggestions were made that some fish species would not be suitable for human consumption. As a conclusion, many factors affected the level of lead, like the distance from the highways, which has had high effect to the results, in addition to the time of study, and the method of analysis used to determine it.


Keywords: heavy metals, lead, food contamination, Turkey



Selected Effects of in ovo Aluminium Exposure on Developing Nile Crocodiles


  1. Christoff Truter1, Johannes H van Wyk2, Natalia Garcia-Reyero Vinas3, Jan G Myburgh4, Anna-Maria Botha1

1 Department of Genetics, Stellenbosch University, South Africa; 2 Department of Botany and Zoology, Stellenbosch University, South Africa; 3 Environmental Laboratory, US Army Engineer Research and Development Center, US Army Corps of Engineers, USA; 4 Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, South Africa


High concentrations of aluminium (Al) have been detected in the surface water and sediments of waterbodies impacted by mining and other anthropogenic activities. Nile crocodiles, Crocodylus niloticus nest in damp sediment, and developing young may be at risk of Al exposure. Our objectives were to determine whether Al can (1) enter C. niloticus eggs and be absorbed by embryos during development, (2) affect hatching success and the general health of hatchlings, (3) result in abnormalities in gonad development and liver morphology, and (4) alter global DNA methylation. C. niloticus eggs obtained from a commercial farm were exposed in ovo to 3.3 g/L Al (pH 4), a pH control (pH 4) and negative control (pH 7). The animals were sacrificed three months after hatching. Al concentrations were higher in the exposed egg shells than previously shown for C. niloticus eggs collected from the wild. A significantly lower proportion of Al exposed hatchlings were able to hatch without assistance relative to the controls, suggesting that Al exposure may compromise survival in the wild. Corresponding growth rates were observed among treatments after three months, indicating that the Al exposed animals did recover. Although Al concentrations in liver, blood and kidney tissues did not vary significantly among treatments, the femurs of Al treated animals contained significantly lower Al levels than the controls, indicating effective detoxification. No apparent abnormalities were observed in ovary and liver tissues. Global 5mC-methylation was significantly higher in the livers of Al exposed individuals, suggesting epigenetic modification in response to Al treatment.

Keywords: metal pollution, reptile ecotoxicology, bioaccumulation, DNA methylation.

The Dental Amalgam Toxicity Fear: A Myth or Actuality


DelfinLovelina Francis

Department of Public Health Dentistry, Dr MGR Medical University,Chennai, Tamil Nadu, India.



Amalgam has been used in dentistry since about 150 years and is still being used due to its low cost, ease of application, strength, durability, and bacteriostatic effect. When aesthetics is not a concern it can be used in individuals of all ages, in stress bearing areas, foundation for cast-metal and ceramic restorations and poor oral hygiene conditions. Besides all, it has other advantages like if placed under ideal conditions, it is more durable and long lasting and least technique sensitive of all restorative materials, but, concern has been raised that amalgam causes mercury toxicity. Mercury is found in the earth’s crust and is ubiquitous in the environment, so even without amalgam restorations everyone is exposed to small but measurable amount of mercury in blood and urine. Dental amalgam restorations may raise these levels slightly, but this has no practical or clinical significance. The main exposure to mercury from dental amalgam occurs during placement or removal of restoration in the tooth. Once the reaction is complete less amount of mercury is released, and that is far below the current health standard. Though amalgam is capable of producing delayed hypersensitivity reactions in some individuals, if the recommended mercury hygiene procedures are followed the risks of adverse health effects could be minimized. For this review the electronic databases and PubMed were used as data sources and have been evaluated to produce the facts regarding amalgam’s safety and toxicity.

Keywords: amalgam, mercury, myth, restoration, safety, tooth, toxicity

Evidence of Immunomodulatory Properties of Cadmium;  is Oxidative Stress Involved


Milena Anđelković1,2, Dragana Javorac2, Katarina Baralić2, Evica Antonijević2, Aleksandra Buha Đorđević2, Vesna Matović2, Zorica Bulat2

1Health Center Kosovska Mitrovica, 2University of Belgrade – Faculty of Pharmacy, Department of Toxicology “Akademik Danilo Soldatović”


Cadmium (Cd) is industrial and environmental pollutant with major concern on human health. Among many mechanisms of toxicity, oxidative stress is tightly connected with Cd exposure disease risk. The aim of the study was to find relation between oxidative stress and potential modulation of immune responses after exposure to single doses of Cd.

The study was carried out on 20 male Wistar rats, randomly divided in experimental groups: control, Group 1-received 15 mg Cd/kg body weight (bw), and Group 2-received 30 mg Cd/kg bw, acute by oral gavage. After rats were sacrificed, collected blood was used for total antioxidative status (TAS) assessment, measurement of white blood cells count (WBC) with leukocyte differential count and Cd levels monitoring, while thymus was used for Cd estimation. For statistical analyses parametric and nonparametric ANOVA followed by appropriate post hoc test was used.

TAS were significantly lower in treated groups compared to controls (p<0.05). Also, inside treated groups significantly differences were noticed (p<0.05). Significantly lower relative lymphocytes counts were observed in both treated groups if compared with control (p<0.01) while WBC significantly decreased only in Group 2, for almost 40% (p<0.01), compared with control. Blood Cd levels were significantly higher in both treated groups (p<0.05) as well as in the thymus of Group 2 (p<0.05) compared to control.

These changes in relative lymphocytes counts are in agreement with increasing Cd levels in blood and thymus and decreasing TAS in treated group. Correlation analysis showed strong negative correlations for Cd levels in blood and TAS as well as relative lymphocyte count which is undoubtedly evidence of the involvement of oxidative stress in Cd-induced immune response modulation.

Keywords: total antioxidative status, leukocyte differential count, blood, thymus

Heavy Metals Blood Level in Tobacco Smokers

Aleksandra Repić1,2, Zorica Bulat2, Vesna Matović2

1Serbian Institute for Occupational Health “Dr Dragomir Karajović”, Belgrade, Serbia, 2University of Belgrade – Faculty of Pharmacy, Department of Toxicology “Akademik Danilo Soldatović”, Belgrade, Serbia


According to World Health Organisation, in 2015, over 1.1 billion people smoked tobacco. Serbian population rank among the highest in Europe: 43.6 % males and 39.7% females aged 15 and older smoke on regular basis. Tobacco is a plant known to accumulate metals, especially cadmium and to a less extent lead, chromium and nickel. Smoking of 20 cigarettes a day could result in the inhalation of 2–4 μg of Cd and 1–5 μg of Pb.

We determined cadmium (Cd-B) and lead (Pb-B) concentration in the blood of 81 smokers and 30 nonsmokers using graphite furnace atomic absorption spectrometry. Smokers were divided into groups according to the number of daily smoked cigarettes and cigarette type. We used ANOVA for statistical analysis.

Cd-B was 3.5 times and Pb-B 1.5 times higher ( 21.4 nmol/L and 0,163 µmol/L, respectively) in smokers compared with nonsmokers (6.0 nmol/L; 0.108 µmol/L, respectively). Smokers who smoked less than 10 cigarettes per day had significantly lower Cd-B (11.4 ± 3.9 nmol/L ) and Pb-B (0.124 ± 0.024 µmol/L) than participants who smoked 11-20 cigarettes.(23.7 ± 5.0 nmol/L and 0.168 ± 0.026 µmol/L respectively) and more than 21 cigarettes (30.5 ± 15.8 nmol/L and 0.237 ± 0.102 µmol/L, respectively). Smoking of manually prepared cigarettes made of tobacco bought at public market, caused significantly higher Cd-B (30,6±9,9) and Pb-B (0,229 ± 0,070) levels than legal cigarettes (19.5 ± 4.4 nmol/L and 0,151 ± 0,022 µmol/L, respectively).

Keywords: lead, cadmium, cigarettes



Reduction of PARK2 Expression among Smelting Workers Exposed to Manganese (Mn)


Wei Zheng 1, Ximin Fan 2, Wendy Jiang 1, and Qiyuan Fan 2,3

1 School of Health Sciences, Purdue University, West Lafayette, IN, United States; 2 School of Public Health, Zunyi Medical College, Zunyi, Guizhou, China; 3 Department of Health Management, Zunyi Medical and Pharmaceutical College, Zunyi, Guizhou, China


Modern industry demands the high quantity of ferroalloys. Smelting workers engaged in production of Mn-containing ferroalloys are frequently exposed to airborne Mn in workshop. Occupational exposure to Mn is known to cause clinical syndromes similar, but not identical to, Parkinson’s disease. Loss-of-function mutations in PARK2 have been associated with certain forms of PD. In this human cohort study, we tested the hypothesis that Mn-exposed smelters had an altered PARK2 expression, leading to Mn-induced neurotoxicity. Mn-exposed smelters (n=26) and non-Mn-exposed control smelters (n=20) were recruited from a Mn-iron (Fe) alloy smelting factory and an Fe smelting factory, respectively, from Zunyi City in China. Subjects were matched with socioeconomic status and background for environmental factors. Analyses by atomic absorption spectrophotometry (AAS) of Mn concentrations indicated that Mn concentrations in plasma, red blood cell (RBC) and saliva, and the cumulative Mn-exposure were about 2.2, 2.0, 1.7 and 3.0 fold higher, respectively, in Mn-exposed smelters than those in control subjects (p<0.01). Total RNA from the blood samples was isolated and analyzed by RT-PCR to quantify PARK2. The data showed that the expression of PARK2 in Mn-exposed smelters was significantly decreased by 42% as compared to controls (p<0.01). Linear regression analysis further established that the expression of PARK2 mRNA was inversely correlated with Mn levels in plasma, RBC and saliva, as well as the cumulative Mn exposure (p<0.01). Taken together, our data suggest that Mn exposure among smelters may lead to a reduced expression of PARK2, which may partly explain the Mn-induced Parkinsonian disorder.

Keywords: manganese, PARK2, worker, smelter, biomarker



Accumulation of Hg in Prawns of Areas Near Open Dumps in the Amazon


Brenda Costa1,2, Barbara Silva1,2, Maria Tavares1,2, Helena Almeida1,2, Lucas Gallat1, Sarita Loureiro1, Thaís Queiroz2, Marcelo Lima2

1Federal University of Pará, 2Evandro Chagas Institute


Waste disposal in open dumps in the Amazon provides liquid effluents (slurry) for the rivers that are in their surroundings. The slurry contains toxic metals which can cause damage to aquatic organisms. In this region it is common for communities to live on the banks of rivers and they may be exposed to mercury (Hg) from these open dumps. This study evaluated the concentration of total mercury (THg) in the shrimp muscles (Macrobrachium amazonicum) captured in the river Aurá, located about 1 km from the open dump of the Metropolitan Region of Belém (BMR). In the months of November of 2015 and February, May and August of 2016, 3 sampling points were selected, P1, P2 and P3, which are 2, 1.5 and 1 km distant from open dump, respectively. THg quantification was performed in 15 pools of muscle and the analysis was by ICP-MS. In all months it was observed that the highest concentrations of THg were recorded in P1. This, although the most distant, it is a meeting point of the waters of two stretches (P2 and P3) of the Aurá river that surrounds the open pit and can be a point of convergence of the flow of slurry from this anthropic source. Higher concentrations of THg were observed in the dry months. In spite of the low rainfall rates in the region, Hg tends to increase its concentration in surface waters. The shrimp of the Aurá river are consumed by the riverside community that resides in BMR.

Keywords: ICP-MS, Macrobrachium Amazonicum, river, pollution, slurry


Measurement of Arsenic, Arsenic Species and Other Llements in Urine, Drinking Water and Hair Samples- Screening of the Situation in Eastern Croatia


Walter Goessler1, Simone Brauer1, Mirta Milić2, Vatroslav Šerić3,4, Marija Milić3,4, Ivan Pavičić5, Ana Marija Marjanović Čermak5, Stefano Bonassi6,7, Višnja Oreščanin8, Ivana Vinković Vrček9

1 Institute for Chemistry, University of Graz, Graz, Austria, 2 Mutagenesis Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia, 3 Department of Clinical Laboratory Diagnostics, Osijek University Hospital, Osijek, Croatia, 4 Faculty of Medicine, University of Osijek, Osijek, Croatia, 5 Radiation Dosimetry and Radiobiology Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia, 6 Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Rome, Italy, 7 Department of Human Sciences and Quality of Life Promotion, San Raffaele University, Rome, Italy, 8 ORESCANIN Ltd., Zagreb, Croatia,  9 Analytical Toxicology and Mineral Metabolism Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia


Chronic exposure to arsenic (As) both in organic but also in inorganic (more toxic) forms, is still a major public health problem worldwide. It is estimated that hundreds of millions of persons are affected with As problem exposure not limited only to developing countries, but developed countries as well, primarily from natural geological sources (ground water, water used for food preparation or crop irrigation and drinking water) or from anthropogenic sources (e.g. industrial activities). In Eastern part of Croatia, naturally occurring inorganic As in ground water can be present in concentrations less than 1 µg/L up to 610 µg/L. Although public actions were taken to provide affected population with safe drinking water (≤10 µg As/L under the EU Drinking Water Directive), progress is gradual and variable and the concentrations are still above the limit. The aim of this study was make a screening of the situation in Eastern Croatia. We have examined As concentrations in drinking water samples and urine As-concentrations and As-species (organic: dimethylarsinic acid-DMA, monomethylarsonic acid-MA; inorganic; and the sum of cationic arsenic species) in more than 100 exposed individuals from Eastern Croatia using liquid chromatography coupled to inductively coupled plasma mass spectrometry-ICPMS and correlated the results of As-concentrations and other elements measured in hair samples in order to get a better insight in the amount of arsenic that could stay in the human body from the chronic exposure to arsenic and to make assessment of the toxic effect of such exposure.

The study was financially supported by the Grant of Ministry of Science and Education of the Republic of Croatia 05/2016 Possible early noninvasive biomarkers of chronic exposure to arsenic.


Keywords: ICPMS, arsenic, urine, human health


Heavy Metals and Minerals Use in Traditional Medicines and Potential Human Health Outcomes


Renée A. Street1,2, Gaëtan M. Kabera3 and Catherine Connolly4

1Environment & Health Research Unit, South African Medical Research Council, South Africa,2Discipline of Occupational and Environmental Health, University of KwaZulu-Natal, Durban, South Africa 3Department of Statistics, University of South Africa, South Africa, 4Biostatistics Unit, South African Medical Research Council, South Africa


The World Health Organization (WHO) recognize the incorporation of plant, animal and/or mineral based products in traditional medicines. As a consequence of the legal acknowledgement of traditional health practitioners (THPs) in South Africa, traditional medicine products must now also be brought under regulatory measures. The aim of this study was to determine and describe the prevalence and practices of five metals and minerals in traditional medicine by South African THPs. Traditional health practitioners (n=201) were interviewed in the local language. Mercury (Hg), potassium permanganate (KMnO4) and copper sulfate (CuSO4) was used by 78 (39%), 158 (79%) and 145 (72%) THPs, respectively. Ammonium chloride (NH4Cl) and calcium sulfate (CaSO4) was used by 152 (76%) and 114 (57%) THPs, respectively. Modes of use depended on the metal/mineral but included oral use, via enema or subcutaneous implantations. Certain metals/minerals were given to both children and adults. Socio-cultural use of metals is a potential source of metal exposure. This study has identified traditional medicine users at risk of poisoning due to socio-cultural practices involving metal and minerals.

Keywords: poisoning, metal exposure, mercury, South Africa

The Influence of pH on the Removal of As3+ from Aqueous Solutions with Acid-activated Clay Modified with Sodium Carboxymethyl Cellulose


Vojkan Miljković 1, Milan Jokanović 1, Maja Vujović 1, Maja Stanković 2, Aleksandra Pavlović 2

1 Faculty of Medicine, Department of Pharmacy, University of Niš, Serbia, 2 Faculty of Science and Mathematics, Department of Chemistry, University of Niš, Serbia


In this study, the suitability of the use of acid-activated clay modified with NaCMC to remove As3+ from aqueous solutions was investigated.

Since ancient times, arsenic has been known as a toxic element and as such has often been the cause of many deliberate or accidental poisoning. The origin of arsenic in the human environment is mainly industrial. Most arsenic compounds are rapidly absorbed from the digestive tract (about 80% of the amount ingested), and well absorbed through the skin. Chemical compounds in which arsenic is present as As3+ are more toxic than compounds with As5+, while inorganic compounds are considered more toxic than organic ones. According to the IARC classification, arsenic belongs to the group 1 of human carcinogens. According to the WHO experts, long-term exposure to arsenic through drinking water containing 0.2 mg arsenic/l can cause skin cancer in 5% of the population.

The sorbent characterization was performed by SEM-EDS and FTIR spectral analysis. Adsorption of arsenic (50 ml of As3+ solution, concentration 100 mg/l) on modified clay (1 g) was tested at different pH values – 1, 3, 5, 7 and 9.

It has been found that adsorption is variable with a pH change. Asorption of As3+ is very successful at pH=1, or when adsorption is carried out in a highly acidic medium. Adsorption is also very successful in the base environment, at pH=9, for all three samples of modified clay (GI, GII and GIII).

This study was financed by the Serbian Ministry of Education, Science andTechnological Development through the Grant No. TR 34012.

Keywords: clay, NaCMC, SEM-EDS, FTIR, As3+








































Oleic Acid Double Coated Iron Oxide Nanoparticles as New Relevant Biocompatible Nanoparticles with aParticular Mechanism of Activity


Elena-Alina Moacă1, Dorina Coricovac1, Cristina Dehelean1, Cornelia Păcurariu2

1“Victor Babeş” University of Medicine and Pharmacy of Timişoara, Faculty of Pharmacy, Department of Toxicology, Timişoara, Romania; 2Politehnica University of Timişoara, Faculty of Industrial Chemistry and Environmental Engineering, Timişoara, Romania 


Iron oxide nanoparticles have gained an increased interest in recent years due to their unique features, like: superparamagnetism, biocompatibility and stability in aqueous solutions. However, a lack of knowledge concerning the toxicity associated to their administration confines their use. This study was aimed to offer relevant information about the cytotoxicity induced by oleic acid double coated magnetic iron oxide nanoparticles (MIONPs) to a panel of healthy (keratinocytes, fibroblasts) and tumor (human and murine melanoma, lung carcinoma and breast carcinoma) cell lines. The MIONPs were obtained by combustion method followed by coating with a double layer of oleic acid. The physico-chemical properties of the biocompatible colloidal suspension were evaluated by means of suitable techniques, such as: optic microscopy (TEM and SEM), magnetic measurements (VSM) and dynamic light scattering (DLS). The cytotoxicity was detected with colorimetric cell-viability bioassays like MTT and Alamar blue. The dimensions of the coated MIONPs were in the range of 30 nm, size considered non-toxic for in vivo administration. The nanoparticles exerted a significant cytotoxic effect on all the tumor cell lines even at low concentrations (10 µM), whereas in the case of healthy cells the viability was affected only at the highest concentration tested (50 µM). The MIONPs induced a different kind of cell death, a particular enucleation process that was not described for other types of nanoparticles. These results show that MIONPs displayed a high stability in aqueous solutions (mandatory criteria for in vivo administration), and anticancer properties, making them suitable as nanoplatforms for chemotherapeutic agents.

This work was supported by a grant of Minister of Research and Innovation, CNCS – UEFISCDI, project number PN-III-P4-ID-PCE-2016-0765, within PNCDI III.


Keywords: magnetic iron oxide nanoparticles, oleic acid, cytotoxicity, melanoma, breast carcinoma


Electrochemical Polymerization of Polyaniline and Polypyrrole Modified Carbon Cloth Anode for High Performance of Microbial Fuel Cells


Praveena Mishra

Schools of Studies in Chemistry, Jiwaji University, Gwalior 474011 INDIA


The development of highly efficient anode materials is critical for enhancing the current output of microbial fuel cells. In this study, an approach of improving power production and biocompatibility of microbial fuel cells (MFCs) by using conducting polymers modified carbon cloth (CC) anode is reported. The modification of CC anode is accomplished by electrochemical deposition of aniline and pyrrole with multiwalled carbon nanotubes (MWCNTs). CC offers conducting porous surface for the development of the macroporous polymer composite network. Biocompatibility of modified electrodes has been evaluated and compared with unmodified carbon cloth (CC) electrode in a microbial fuel cell setup as anodes. A mediatorless dual-chamber MFC reactor filled with sewage waste water as substrate has been used in the comparative study of current and power production using different anodes. During MFC experiment with the polyaniline/polypyrrole/MWCNTs-MnO2 anode achieved a maximum power density of 2768.37 mW m-2, which is higher than the corresponding values of the MFC with other anodes. The SEM images taken after 45 days of the experiment confirm biocompatibility of modified anodes. Evidence from the electrochemical tests confirmed that the electrochemical activity of the modified anode is enhanced. Stability and charge transfer is facilitated by polyaniline (PANI), Polypyrrole (PPy) and MWCNT modification. All the results illustrated that the PANI and PPy modification of the carbon cloth surface is an efficient approach for enhancement of the performance of MFCs, while the chemical composition, size and shape of the nanocomposite determine the extent of the enhancement.

Keywords: microbial fuel cells, conducting polymers, electrochemistry, metal oxides

Antioxidant Potential and Toxicity Study of Pullulan Mediated Cerium Oxide Nanoparticles Synthesized in Rats


Hamid Reza Sadeghnia1-4, Majid Darroudi 1,5, Mohammad Bagher Khorrami1,6, Bamdad Riahi-Zanjani6, Alireza Pasdar1,7, Majid Ghayour-Mobarhan1,8, Fatemeh Forouzanfar1,3, Azar Hossani2, Zohreh Mehrdadpour1,10, Mohammad Zare6

1Department of Modern Sciences and Technologies, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, 2Pharmacological Research Center of Medicinal plants, Mashhad University of Medical Sciences, Mashhad, Iran, 3Division of Neurocognitive Sciences, Psychiatry and Behavioral Sciences Research Center, Mashhad University of Medical Sciences, Mashhad, Iran, 4Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, 5Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad, Iran 6Social Security Organization, 17th Shahrivar Hospital, Mashhad, Iran, 7Medical Toxicology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran, 8Division of Applied Medicine, Medical School, University of Aberdeen, Foresterhill, Aberdeen, UK, 9Biochemistry of Nutrition Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. 10Department of Medical Parasitology and Mycology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran



The purpose of this study was to investigate the acute toxic potential of cerium oxide nanoparticles synthesized with pullulan (CNPs) in adult male Wistar rats. Thirty male Wistar rats were randomly divided into five experimental groups of six animals each. The animals received 50, 100 and 400 mg/kg CNPs for 14 consecutive days. At the end of the experiment, the rats were euthanized and histopathological evaluation of the liver and renal tissues, as well ass, the markers of serum oxidative stress including thiobarbituric acid reactive substances (TBARS), total sulfhydryl (SH) content, and antioxidant capacity (using FRAP assay) were assessed. Hematological parameters and the activity of liver function enzymes were also measured. The results of this study showed that CNPs caused no significant changes in the activity of liver enzymes, hepatic and renal histopathology, hematological parameters, while improved serum redox status. In conclusion, acute administration of pullulan mediated CNPs is safe and possess high strength of antioxidant activity.

Keywords: cerium oxide, nanoparticles, acute toxicity, oxidative stress, kidney damage, liver injury


Effects of Silver Nanoparticles on Neurodevelopment Using C57BL/6 and A/J Primary 3D Organotypic Mouse Midbrain Cultures


Brittany A. Weldon1,2 and Julie Juyoung Park1,2, Sungwoo Hong1,2, Tomomi Workman1,2, Russell Dills2, Ji Hyun Lee 2, William C. Griffith1,2, Terrance J. Kavanagh2, and Elaine M. Faustman1,2*

1Institute for Risk Analysis and Risk Communication, University of Washington, Seattle, WA; 2Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA, USA


Many consumer, commercial, and medical products have been increasingly using silver nanoparticles (AgNPs) for their antimicrobial properties. Observations of silver in adult and fetal brain following in vivo AgNPs exposures have also led to concerns about the potential of AgNPs as neurotoxicants. In this study, we investigated effects of gold-cored AgNPs of differing sizes and coatings (20nm AgCitrate, 110nm AgCitrate, and 110nm AgPVP) on neurodevelopment across two mouse strains using our 3D organotypic embryonic midbrain micromass cultures. Primary cells from gestational day (GD) 11 C57BL/6 or GD 12 A/J mouse embryos were used. After 24-hour AgNP exposures at three different time points of development (days in vitro (DIV) 7, 15, and 22), cytotoxicity was assessed by both nominal and dosimetric dose. Dosimetry of silver and gold was evaluated in cultures, where gold acted as a tracer for uptake of intact gold-cored AgNPs and silver as a tracer for dissolved particles. Results by nominal and dosimetric dose demonstrated significantly increased cell death in a dose-dependent manner at DIV 15 and 22, which represents differentiation stages of neurodevelopment in both strains. When assessed by dosimetric dose, cultures were more sensitive to smaller particles in both strains despite less uptake of Ag. The extent of AgNP dissolution in the micromass cultures across two mouse strains was different, suggesting potential genetic differences in AgNP uptake mechanisms. Future research is needed to elucidate uptake mechanisms for AgNPs with various sizes and coatings and to validate the effects of genetic background on AgNP uptake mechanisms.

Keywords: developmental neurotoxicity, in vitro nanotoxicology, dosimetry, genes x environment


Impact of Sample Preparation of MWCNT for Developmental Toxicity by Intratracheal Instillation


Akihiko Hirose 1, Motoko Hojo 2, Norihiro Kobayashi 1

1 National Institute of Health Sciences, 2 Tokyo Metropolitan Institute of Public Health


Some studies reported that the intratracheal or intraperitoneal administration with multi-walled carbon nanotube (MWCNT) to pregnant mice caused reproductive and developmental toxicity. We have recently suggested that the reproductive and developmental toxicity could depend on the fiber length of MWCNTs, by comparison of the intraperitoneal administration with various typed MWCNTs. Recently, we preliminary found that the strength of developmental toxicity was varied depending on the preparation methods of the dispersed solution of MWCNT. We postulated that the inflammation caused by the MWCNT administration may be related with preparation methods as well as the fiber length of MWCNT. In order to evaluate the reproductive and developmental toxicity based on various sample preparations of MWCNT, we conducted repeated intratracheal instillation studies in pregnant mice. Three types of MWCNT dispersions (bulk, heat-treatment, single dispersion by Taquann method) were administered to pregnant mice on gestational days 6, 9, and 12 at dosages of 4.0 mg/kg/day. The pregnant mice were dissected on the gestational day 15, and then reproductive and developmental parameters were evaluated. Body weights of the heat-treatment MWCNT exposed mice significantly decreased. Body weights of fetuses were significantly decreased in the bulk MWCNT exposed groups and decreased in the heat-treatment MWCNT exposed groups, although the change was not statistically significant. The increased numbers of neutrophil and eosinophil cells and the increased LDH activities in lavage of dam’s lung were correlated with the body weight effects. These suggested that the developmental toxicity of MWCNT could depend on inflammation in dams.


Keywords: nanomaterials, MWCNT, intratracheal instillation, inflammation


Demonstration of the Uptake of Gold Nanoparticles Using CytoViva Technology and Transmission Electron Microscopy


Melissa Vetten1,2, Mary Gulumian1,2

1National Institute for Occupational Health, South Africa, 2University of the Witwatersrand, South Africa


Gold nanoparticles (AuNPs) have a wide range of potential applications which necessitates the need for toxicity studies prior to their commercialization. The uptake and intracellular fate of nanoparticles will influence their ability to cause toxicity and therefore needs to be determined. In this study, 14 nm AuNPs were found to be non-toxic to the human bronchial epithelial cell line BEAS-2B for up to 24 hours incubation. The uptake of these particles was then assessed using two approaches, namely through the CytoViva HSI system and Transmission Electron Microscopy (TEM). The CytoViva HSI system allowed for the acquisition of dark field microscopy images and the confirmation of the presence of AuNPs using hyperspectral imaging and spectral angle mapping. In addition, their 3D imaging technology confirmed the uptake and identified nanoparticles within the cells without any fluorescent labelling. This newly developed technology can locate non-labelled nanoparticles within a three dimensional space relative to their surroundings; however, staining with fluorescent markers is necessary to locate nanoparticles relative to subcellular organelles. Since AuNPs are known to interfere with some fluorescent dyes, the use of dyes must be validated prior to use in nanoparticle studies. TEM imaging was used to confirm the uptake and the presence of AuNPs in vesicles/vacuoles and in the cytoplasm. Both of these techniques require the availability of specialized equipment, however the sample preparation for TEM can be somewhat tedious. On the other hand, the sample preparation for CytoViva is quick and relatively easy once validated.


Keywords: gold nanoparticles, uptake, CytoViva, TEM, nanotoxicology

Interference of Gold Nanoparticles (AuNPs) in Molecular Biology Assay Systems

Natasha Sanabria1, Mary Gulumian1,2

1National institute for Occupational Health, 2University of the Witwatersrand, Johannesburg South Africa.


It has now been established that intracellular nanomaterials interfere with different toxicity and genotoxicity assay systems. There is, however, a lack of validation when conducting routine tests for nucleic acid isolation, quantification, integrity and purity analyses, as well as, in the verification of qPCR-related gene expression analyses. Investigations were, therefore, conducted to assess the interference of gold nanoparticles (AuNPs) in these assay systems.

Results have indicated that the introduction of AuNPs to BEAS-2B cells produced absorbance peak shifts, which indicated changes in the quality of the isolated RNA. Although the RNA isolated from the 24 h AuNP-treated samples was considered to be suitable for RNA-based techniques when using the traditional methods, additional screening identified changes that are associated with structural alterations of functional groups. The wavelength shift observed was most probably due to these AuNPs interacting with the amines found in nitrogenous bases of the nucleic acid. Results have also indicated that AuNPs have the potential to interfere with the amplification and detection within the RT-qPCR assay mechanism, which relies heavily on the quantification of stably expressed reference genes.

In conclusion, caution is advised when only assessing DNA/RNA quantity, since structurally altered or damaged nucleic acids could be falsely interpreted as simply a low yield and, subsequently, produces false genetic expression data. Moreover, AuNPs have the potential to interfere with the assay mechanism of RT-qPCR, thus, assay verification is required for AuNP-related gene expression studies used to evaluate toxicity.

Keywords: qPCR, assay-interference, engineered nano-materials, gold nano-particles (AuNPs)


























































Antitumoral Activity of MTX-II, a Basic Myotoxic Phospholipases A2, Isolated from Bothrops asper Snake Venom from Panama.


Aristides Quintero1, 2, Sulamita S. Setúbal 3,4, Leonardo A. Calderón3, 4, Rodrigo G. Stábeli3 ,4, Juliana P. Zuliani3 ,4, Andreimar M. Soares3 ,4

1Center for Information and Toxicological Research and Applied Chemistry   (CEIITOXQUIA), UNACHI, Chiriqui, Panama, 2Department of Chemistry of the Autonomous University of Chiriquí (UNACHI), Chiriqui, Panama, 3Centre for Studies of Biomolecules Applied to the Health (CEBio), Rondônia, Brazil, 4Oswaldo Cruz Foundation (Fiocruz Rondônia), Rondônia, Brazil


Cancer is one of the leading causes of morbidity and mortality worldwide. The major drawback of the current methods of cancer treatment is that patients often do not respond or eventually develop resistance after initial treatment. This has led to the increased use of anticancer drugs developed from natural resources. Phospholipases A2 (svPLA2) are abundant components of snake venoms that have been extensively studied due to their pharmacological and pathophysiological effects on living organisms. This study aimed to assess the antitumor potential of MTX-II, a basic myotoxic PLA2 isolated from crude Bothrops asper snake venom by a single-step chromatography using a CM Sepharose ion-exchange column (1.5 × 15 cm). The 2D SDS-PAGE analysis revealed that the protein has a single chain and molecular mass next to 14.2 kDa, confirmed by MALDI-TOF mass spectrometry. The isoelectric focusing revealed that the protein has pI value approximately to 8.2. Analysis of the N-terminal sequence demonstrated that MTX-II belongs to the enzymatically inactive Lys49 PLA2-like subclass. It was observed that MTX-II does not affect the viability in vitro of J774.1 macrophages and induces your activation to start phagocytic activity and superoxide production. The MTX-II showed cytotoxic activity tumor cell lines of JURKAT cells (T cell leukemia) and SK-BR-3 (human breast adenocarcinoma) of 70%and 40%, respectively. The results obtained revealed that in vitro antitumor activity is independent of the enzymatic activity. However, further studies must be carried out to ensure the safety and efficacy of MTX-II for the development of anticancer drugs.

Keywords: snake venom, toxins, anticancer agents, apoptosis inducer, cancer

Develompent and Application of Method for Analysis of Ochratoxin A in Grapes

Bojana Špirović Trifunović 1, Ljilja Torović3,  Vojislava Bursić4 Dragica Brkić 1, Sanja Lazić4, Gorica Vuković 1,2

1 Faculty of Agriculture, University of Belgrade, Belgrade, Serbia; 2Institute of Public Health Belgrade, Belgrade, Serbia; 3Institute of Public Health of Vojvodina, Novi Sad, Serbia;    4Faculty of Agriculture, University of Novi Sad, Novi Sad, Serbia

Corresponcence e-mail address

The aim of the study was development of a method for determination of ochratoxin A, a mycotoxin produced by Aspergillus and Penicillium fungi, in grapes, and its application for analysis of grapes harvested in Serbia in 2016.

QuEChERS procedure was applied for extraction of ochratoxin A from grape in several variations: acetonitrile or acidified acetonitrile extraction, without further treatment or followed by dispersive solid phase extraction clean-up on different sorbents used for pesticide residue analysis. LC-MS/MS analysis confirmed straightforward acetonitrile extraction as the most efficient. Optimization of LC-MS/MS conditions, performed using Mass Hunter Optimizer Software, based on quantification/qualification ion 239/221, resulted with fragmentation and collision energy of 105/120 and 20/33 V, respectively. Linearity of detector response was investigated based on solvent (acetonitrile), matrix match and standard addition calibrations, for black and white grape matrices. Black grape exhibited substantial matrix effect (29%), as opposed to white (-11%), demanding matrix calibration. Limit of detection of ochratoxin A was determined using Qualitative Mass Hunter program, and 1 mg/kg was established as limit of determination. Accuracy of the method was studied through recovery assay on several concentration levels, using black (81-104%) and white grape (94-117%). Precision, in terms of RSD, was 2.7% for black, and 2.6% for white grape. Following successful fulfilment of the relevant criteria of SANTE/11945/2015, optimised method was applied for the analysis of 250 grape samples (black and white 1:1), including 10 black and 25 white grapes from organic production. Fortunately, presence of ochratoxin A in analysed samples was not detected.

Keywords: ochratoxin A, grape, LC-MS/MS, method development

The First Report on Ochratoxin A Concentrations in the Kidneys of the European Brown Bear (Ursus arctos L.)


Dubravka Rašić1, Maja Lazarus2, Đuro Huber3, Slaven Reljić3 and Maja Peraica1

1Toxicology Unit, 2Analytical Toxicology and Mineral Metabolism Unit, Institute for Medical Research and Occupational Health, 3Department of Biology, Veterinary Faculty of the University of Zagreb, Zagreb, Croatia


Ochratoxin A (OTA) is a nephrotoxic and carcinogen mycotoxin that contaminates different food commodities. Its toxicokinetics and accumulation in plasma, kidneys and liver are species-specific. The European brown bear (Ursus arctos L.) is the largest terrestrial mammal in Croatia and most its diet consists of plant food. Maize from supplemental feeding sites makes up an important part of the bears’ diet, especially in periods and years of low natural food resources. This supplemental maize is often of poor quality to begin with and is further adulterated by weather conditions, which enhance the production of OTA. We hypothesized that the OTA level in kidneys would reflect the intake of OTA-contaminated maize. A total of 56 kidney samples were collected (19 samples in 2013 and 37 samples in 2015) from bears hunted according to the Brown Bear Management Plan in Croatia. After liquid-liquid extraction, OTA concentration was measured using HPLC with a florescent detector. Regression analysis revealed that OTA concentration was significantly lower (13.82 ± 12.55 ng g-1 tissue; median 12.25) in samples collected during 2013 than in samples collected during 2015 (142.30 ± 237.53 ng g-1 tissue, median 53.31), while controlling for age, sex and season of collection (b=0.60, p<0.001, R2=0.46). These results are the first report on OTA concentrations in the kidney of the European brown bear. OTA in bear kidneys depends on the specific diet habits, bears’ microlocation, quality of supplemental food and the conditions of food storage.

Keywords: brown bear, HPLC, kidneys, maize, ochratoxin A

Correlation Between Na+/K+ ATPase Isoforms and the In Vitro Cells Sensitivity to the Algal Toxin Palytoxin


Marco Pelin, Valentina Brovedani, Chiara Florio, Silvio Sosa, Aurelia Tubaro

Dept. of Life Sciences, University of Trieste, 34127 Trieste, Italy


Palytoxins (PLTXs) are highly toxic compounds identified in marine Palythoa zoanthids, Ostreopsis dinoflagellates and Thricodesmium cyanobacteria, and involved in adverse effects in humans after different exposure routes. Epidemiological and molecular evidences suggest a variable inter-individual sensitivity to PLTXs, possibly related to genetic-dependent differences in the Na+/K+-ATPase expression, the molecular target of these toxins.

To identify the specific Na+/K+-ATPase isoforms correlated with the in vitro cells sensitivity to PLTX, 9 cell lines (from skin, liver, breast, intestine and pancreas) were used evaluating PLTX cytotoxicity (EC50, concentration reducing cell viability by 50 %; MTT assay) and its cells binding (Kd, binding affinity, and Bmax, maximum PLTX binding; cell-based ELISA). The results were then correlated with the Na+/K+-ATPase protein expression (flow cytometry) and the gene expression for the isoforms of the α (α1-4) and β (β1-3) subunits (real time PCR).

Among the 9 cell lines, a significant variability of the sensitivity parameters was recorded (median EC50=5.7×10-10 M; interquartile range=1.5×10-10-1.9×10-9 M; median Kd=8.1×10-10 M; interquartile range=2.2×10-10-2.4×10-9 M; median Bmax=0.015; interquartile range=0.0095–0.02738). Even though cell sensitivity to PLTX was not related with Na+/K+-ATPase protein expression, a significant correlation was observed with gene expression of specific Na+/K+-ATPase α and β isoforms: a significant positive correlation was found between Kd values and β2 gene expression (r=0.8052, p value=0.0159; Pearson correlation) and between Kd values and the ratio of α1/α2 gene expressions (r=0.7225, p value=0.0279; Pearson correlation). These data, for the first time, suggest a significant role of these isoforms in PLTX binding to Na+/K+-ATPase.


Keywords: palytoxin, Na+/K+-ATPase, genetic variants, Ostreopsis


Devil’s Trumpet: Beautiful danger


Marko Antunović, Jelena Džudovć1, Zorica Bulat2, Snežana Đorđević1, Vesna Matović2, Vesna Kilibarda1

1 National Poison Control Centre, Military Medical Academy, Belgrade, 2Faculty of Pharmacy, University of Belgrade


Datura stramonium (DS), also known as Tatula, Angel’s Trumpet and Devil’s Trumpet, is a widespread annual plant growing in rural and urban areas. Plant is toxic because of high content of tropane alkaloids (atropine, hyosciamine and scopolamine), causing anticholinergic syndrome as a result of central and peripheral muscarinic neurotransmission. Typical signs and symptoms of poisoning are: mydriasis, blurred vision, dry mouth, tachycardia, nausea and vomiting, hyperthermia, hallucinations, loss of consciousness and coma. Severity of symptoms depends on quantity and part of the consumed plant. Differential diagnosis of Datura stramonium intoxication can be difficult.

Datura stramonium poisoning is mostly intentional because of abuse, especially among teenage population due to its hallucinogenic and euphoric effects. In 2017, six patients were admitted to National Poison Control Center of Serbia due to DS poisoning, followed by mild signs of poisoning. Cause for concern is that two teenage girls (less than 18 years old) were admitted because of intentional ingestion of DS. They used internet to find out how to consume DS.

Atropine was laboratory confirmed in all patients. Management was mainly supportive and involved nonspecific detoxification therapy and sedation with benzodiazepines. All patients were treated without complications and discharged after short-term hospitalization. DS intoxication should be suspected in patients with unexplained anticholinergic symptoms, especially among teenagers. It is also important to consider influence of social media on youth in a manner of poisonings.

Keywords: Datura stramonium, anticholinergic syndrome, substance abuse, social media, teenagers

Early Diagnosis Associated with Pharmacological and Hyperbaric Therapy in a Certain Case of Loxosceles Bite

Franca  Davanzo1, Anna Celentano1, Valeria Dimasi1, Fabio Garuti2, Giovanni Sesana2, Rossella Giacomello2, Gianluca Basso3

1.Milan Poison Control Center, ASST Grande Ospedale Metropolitano “Niguarda” Milano Italy; 2. Center of Hyperbaric Medicine, ASST Grande Ospedale Metropolitano “Niguarda” Milano Italy; 3.Plastic Surgery, ASST Grande Ospedale Metropolitano “Niguarda” Milano Italy.



Loxosceles rufescens (LR) is indigenous to the Mediterranean Europe. It is described a severe case of skin necrosis, caused by this spider’s bite which was treated with pharmacological and hyperbaric oxygen therapy (HBOT) and treatment of the difficult wound.

Case report: a 38 years old male, arrived at the emergency department four days after the spider bite. The patient had found the spider at home and killed it. The spider was identified by the entomologist as LR. The bite has not been felt and has affected the distal fly part of the right arm. After a few hours a small lesion appeared, which progressively enlarged, complicated by pain, itching, and edema that spreads both proximally and distally of the arm and caused functional impairment. Fever (over 39 ° C) and erythema appeared. The subject was visited and treated with oral antibiotic, antihistaminic and topical cortisone. The local lesion, that was approximately 11×5 cm, worsened rapidly with ecchymosis, edema, hemorrhage and dermonecrosis at the bite site. Leukocytosis and an increase of aminotransferase with hyperglycemia. Twenty cycles of HBOT was performed due to the worsening of skin lesions with rapid pain, edema decrease, and injury reduction. After 80 days, a surgical toilette was performed with total recovery.

Regardless of the score, the HBOT results in a decrease in pain and a faster demarcation of the eschar. The timely antibiotic and antihistamine therapy, in addiction to the HBOT, has enabled rapid healing without any local damage for this patient.

Keywords: spider bite, case report


Fluoride a Double Edged Sword – Fluoride Exposure and Its Effect on the Intelligence of School Children in Tamil Nadu, India


DelfinLovelina Francis

Department of Public Health Dentistry, Dr MGR Medical University,Chennai, Tamil Nadu, India.


The objective of this study was to assess the relationship between drinking water fluoride levels exposure and children’s intelligence in Tamil Nadu, India.

This cross-sectional study was conducted among 12-year-old school children of Tamil Nadu, India. 100 children were selected from low (< 1.5 parts per million) and 100 children from high (≥1.5 parts per million) fluoride areas. A pretested questionnaire was used to collect information on the children’s personal characteristics, residential history, medical history, educational level of the head of the family, and socioeconomic status of the family. The children’s intelligence was measured using a psychological test and mental age calculator. Data analysis was done using the chi-square. P value <0.05 was considered statistically significant. Differences in participant’s sociodemographic characteristics were statistically not significant (P>0.05). Reduction in intelligence was observed with increased water fluoride levels. It can be concluded that children in endemic areas of fluorosis are at risk for impaired development of intelligence.

Keywords: child, India, fluoride poisoning, intelligence, water

The Retrospective Investigation of the Impacts of Alien and Native Taxa on Human Health: A Case Study of Two Poison Information Centers.


Moleseng C. Moshobane

Directorate of Biological Invasions, South African National Biodiversity Institute, Pretoria National Botanical garden, Silverton, South Africa.



Alien species cause considerable negative impacts on biodiversity, economy and public health. Impacts of alien species on public health have received a degree of attention worldwide, largely in developed countries, but scarce in developing countries. Here, we provide a review of human exposures and poisonings cases from native and alien plant species reported to poison information centers. A retrospective review of the Tygerberg Poison Information Centre (TPIC) and Poisons Information Centre (PIC) at Red Cross War Memorial Children’s Hospital (RCWMCH) was conducted over approximately 2-year period (1 June 2015 through to 06 March 2017). Results: Combined, TPIC and PIC handled 626 cases during the 2-year period. Toxicity cases were more abundant in Gauteng (47.1%), followed by Western Cape (29.4%). The primary mechanism of injury was ingestion (96.7%) and all cases were predominantly accidental. Most reported cases involved infants (20.6%), with few fully-grown adults related cases (5.8%). Adults presented minor to moderate toxicity, while infants none to minor toxicity. We conclude that reported toxicity cases on human health is biased towards few alien species, and that several cases relates to unknown species of mushrooms.  Public awareness is essential to reducing the poisoning incidences.

Keywords: Alien species, invasive species, natural toxins, poisoning, public health



Necrotizing Fasciitis Due to Loxosceles rufescens: A Case Report


Anna Celentano1, Franca  Davanzo1, Angelo Travaglia1, Gianluca Basso2, Marco Bragagliotti3, Umberto Valentinotti4, Fabio Garuti5, Rossella Giacomello5

1.Milan Poison Control Center, Niguarda Great Metropolitan Hospital, Milan, Italy; 2.Plastic Surgery, Niguarda Great Metropolitan Hospital, Milan, Italy; 3.IRCCS Polyclinic Foundation San Matteo, Pavia; 4. Orthopedics and Traumatology, Niguarda Great Metropolitan Hospital, Milan, Italy; 5.Hyperbaric Medicine Center, Niguarda Great Metropolitan Hospital, Milan, Italy.



Loxosceles rufescens is indigenous to the Mediterranean Europe. One severe case of skin necrosis, caused by this spider’s bite which was treated with pharmacological and hyperbaric oxygen therapy (HBOT) and rehabilitation is described.

Case report: A 55 years old lumberjack was bitten by a spider that had entered inside his gloves. After a 7-8 hours asymptomatic period, a burning pain  to his left thumb irradiated to his whole hand appeared.  A tendonitis was diagnosed and thus, after the hand was immobilized, a therapy of NSAID and pain relievers was instituted. During the next 48 hours, the clinical picture worsened dramatically with an unbereable pain and swelling sensation from the hand up to the forearm. An orthopaedic visit produced a diagnosis of necrotizing fasciitis. The patient underwent surgery and, after other 24 hours, began HBOT. At the same time antibiotics and pain relievers were administered. During recovery a total of 36 HBOT sessions were administered along with 3 surgeries. Finally, autologous skin transplant was performed. A regenerative skin substitute was used along with a silk membrane to favour a better wound evolution.

Conclusion: the symptoms of most cases of loxoscelism tend to appear late and thus make an immediate diagnosis. The correct diagnosis was largely delayed, HBOT was instrumental in the recovery and prevented the loss of the affected limb.

It is important to keep in mind that the venom injected by these spiders contains anaerobic bacteria and, therefore, it is imperative to establish a broad spectrum antibiotic therapy.

Keywords: spider’s bite, skin necrosis, therapy





























































CAR-mediated Expression of CYP2B1 in Primary Rat Hepatocytes After Isolation by Means of EDTA Perfusion


Marc Wollenweber 1, Dunja Dimitrijevic1, Bennard van Ravenzwaay2, Dieter Schrenk1

1 University of Kaiserslautern, Food Chemistry and Toxicology, Kaiserslautern, Germany, 2 BASF SE, Experimental Toxicology and Ecology, Ludwigshafen am Rhein, Germany


The constitutive androstane receptor (CAR) is an orphan nuclear receptor. In rodent liver, CAR is involved in the phenobarbital-mediated tumor promotion. However, due to the constitutive activity of CAR in several immortalized cell lines, it is difficult to measure in vitro CAR activation above baseline. In this study, we compare two different perfusion methods for the isolation of primary rat hepatocytes (pRHs) to establish an alternative and sensitive method for measuring CAR activation in vitro.

For the isolation of pRHs we used two perfusion methods, a two-step collagenase perfusion and an EDTA-based perfusion. CAR activation was determined by RT-qPCR of CYP2B1 mRNA after exposure to phenobarbital (PB), phenytoin (PHY) or TCPOBOP (TC).

PB, PHY and TC significantly increased CYP2B1 expression in pRHs (preliminary data). In collagenase isolated pRHs expression levels peaked at 120-fold (PB, 0.5 mM), 85-fold (PHY, 50 mM) and 75-fold (TC, 10 µM) relative to the solvent control. In comparison, EDTA isolated pRHs showed similar expression patterns but with up to 7 times higher CYP2B1 induction compared to collagenase-treated pRHs.

In this study, we showed that pRHs isolated by EDTA perfusion showed significantly higher CYP2B1 induction than pRHs obtained by collagenase perfusion after exposure to CAR activators. As a possible reason it has been suggested that the collagenase perfusion may also digest structures on the cell surface of pRHs and thus disrupts cell signaling. Our findings provide a basis for further refinement of an in vitro assay for CAR activation.

Keywords: constitutive androstane receptor, primary rat hepatocytes, EDTA perfusion, CYP2B1, phenobarbital



Lessons Learned: Altertox Academy Hands-On Trainings in Non Animal Testing


Ilija Prachkovski  and Francois Busquet

Altertox Academy


Over the last thirty years, dozen of validated alternative test methods exist in the EU and even more thanks to ICATM collaboration. Nevertheless, when one looks at the number of testing proposals submitted to REACH it is clear these methods are not being put to sufficient use. While ad-hoc events, tailor-made training, webinars, and scientific meetings regularly provide training in these new methods, more efforts should be invested into “after-sales” services to disseminate the emerging technologies and reach new audiences. The European Commission and the member states are actively filling the gaps in training via EU research programs such as Horizon2020, and the innovative medicines initiatives.

Altertox Academy has a mission to increase the use of validated alternative methods among researchers and toxicologists in Europe. Altertox Academy two-day training sessions are made up of 20% lectures and 80% hands-on-training in the lab. This study proves that there is an added and unique value of Altetox Academy training format to address its mission. Since its creation in 2012, twenty hands-on trainings took place and gathered in total approx. 200 participants in the lab and via webinars. This presentation intends to share feedback and lessons learned on reaching out the participants and how to perform successful trainings. Last, the sustainability of such initiatives will be described and the objectives of the medium-term listed.


Keywords: education, training, 3Rs, in vitro, in silico


New In Vitro Toxicity Pathway-based Bioassays for Toxicity Screening of Chemicals


A’edah Abu-Bakar1,3, Hao Hu2, Ting Yu3, and Matti A Lang4

1Faculty of Pharmacy, Universiti Teknologi MARA Selangor, 42300 Puncak Alam, Malaysia; 2Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA; 3The University of Queensland, Queensland Alliance for Environmental Health Science (QAEHS), 4072 Brisbane, Queensland, Australia, 4Alaric Consultants, Centre for Molecular Genetics, Friisilantie 9b, 02240, Espoo, Finland.


With the implementation of 3Rs principle (Replacement, Reduction, Refinement) in animal use for toxicity testing of xenobiotics, the demand for in vitro toxicity bioassays is in the rise, especially those that are based on toxicity pathways. Existing commercial toxicity pathway-based bioassays were developed on the principle of one toxicity pathway working in isolation. In reality, various transcription factors are activated in response to toxic stimuli to express various toxicity pathways in defence. Thus, there is dire need for more accurate in vitro bioassays that can measure various toxicity pathways. One strategy is to develop recombinant human cells that express gene reporter plasmid carrying specific binding sites for the various stress activated transcription factors. We have successfully constructed luc gene promoter-based bioassays that are driven by an entire gene promoter containing cis-elements for various transcription factors, including AHR/Arnt dependent-XRE and Nrf2-dependent StRE. Three bioassays were constructed by stably transfected human breast cancer cell line (MCF-7) with: (i) wildtype bioassay containing the full length (2kb) promoter region of the Cyp2a5 gene; (ii) XRE-MUT bioassay with the AHR/Arnt cis-element mutated; and (c) StRE-MUT bioassay with the Nrf2 cis-element mutated. The bioassays were able to detect responses of various environmental contaminants at concentration range below LC50. Additionally, concurrent application of the three bioassays would determine the predominant toxicity pathway of a given compound. The bioassays could potentially analyse and predict toxicity of environmental contaminants, which will benefit regulators and researchers in the field of risk assessment, as well as mining and remediation industries.

Keywords: in-vitro toxicity testing, toxicity pathway-based bioassay, chemical risk assessment, alternatives to animal testing, luc gene promoter-based bioassay

Effect of Metformın on Doxorubıcın Induced Cytotoxıcıty in Hep2 and Hepg2 Cells


  1. Zeynep Ünal1, Suna Sabuncuoglu1, Kaya FA2
    1Hacettepe University, Faculty of Pharmacy, Department of Toxicology, Ankara, Turkey

2Hacettepe University, Faculty of Medicine, Department of Pediatrics, Division of Hematology-Bone Marrow Transplantation Unit, Ankara, Turkey


Doxorubicin (DOX) is a medication in a class of anthracyclines used in cancer chemotherapy including leukemia, breast and liver cancer. DOX is considered as cell-cycle specific and acts during multiple phases of the cell cycle. Metformin, a commonly used oral anti-hyperglycemic agent of the biguanide family, also activates AMPK. In the present study, the effect of MET on cytotoxicity of DOX  have been evaluated in vitro. In order to do present study, drugs were applied on HepG2 and Hep2 cell lines for 24, 48 and 72 hours in different concentrations. In cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release assays were performed for analysis of cell viability. Apoptotic and necrotic cell ratios were determined by flow cytometry and real-time cell motions were evaluated at XCELLigence. Before the main study, ideal doses were determined (in the Hep2 cell line doxorubicin 100-0.8 μM, metformin 1-10 mM; in the HepG2 cell line doxorubicin 0.08-3.2×10-3 μM, metformin 10-0.04 mM).  According to the results, MET synergise the cytotoxic effect of DOX when the drugs were used together at different doses in 24, 48 and 72 hours. At the high concentration of MET and DOX combinations, cell viability ratio decreased and DOX induced apoptotic cell counts increased. Furthermore, XCELLigence analysis (120 hours) results showed that 1-10 mM MET combinations increased the cytotoxic potential of DOX. In conclusion, cell properties and assay principals may affect the response for the interactions between DOX and MET. Cancer types and administered drug doses have to be considered for interactions.

Keywords: doxorubicin, metformin, cytotoxicity, interaction























In Vivo Reactivating Efficacy of Oximes K203 and K027 Against a Direct Acetylcholinesterase Inhibitor: Dose-response Modeling


Evica Antonijevic1, Kamil Musilek2, Kamil Kuca3, Danijela Djukic-Cosic1, Marijana Curcic1, Zorica Bulat1, Biljana Antonijevic1

1University of Belgrade, Faculty of Pharmacy, Department of Toxicology “Akademik Danilo Soldatović”, Serbia, 2University of Hradec Kralove, Faculty of Science, Department of Chemistry, Czech Republic, 3University Hospital in Hradec Kralove, Biomedical Research Center, Czech Republic, 4National Poison Control Center, Military Medical Academy, Serbia


Reactivation of organophosphate(OP)-inhibited acethylcholinesterase (AChE) as specific endpoint is used in efficacy testing of experimental oximes, antidotes in OPs poisoning. According to our best knowledge, the majority of in vivo studies tested only one or two oxime doses resulting in qualitative oxime efficacy evaluation. However, quantitative analysis of in vivo dose-response data would improve identification and quantification of the effect as well as rigorous comparison of different oximes efficacies. Thus, we have evaluated in vivo dose-response relationship for two promising experimental oximes, K203 and K027, concerning reactivation of AChE inhibited by dichlorvos (DDVP). To compare the oximes effects, benchmark (BMD) covariate approach was used to estimate oxime dose (with 90% confidence intervals) that elicits a pre-specified effect size of 100% (2-fold increase in AChE activity compared to DDVP-treated group). Wistar rats (5/group) were treated with oxime (1.25%, 2.5%, 5%, 25% and 50% LD50 im) immediately after DDVP challenge (75% LD50 sc). Activity of AChE was measured in erythrocytes by Ellman´s method 60 min after the treatment. Dose-response and BMD modeling was done in PROAST software (version 64.13, RIVM, Nederlands). Exponential model m5-b (y=a[c-(c-1)exp(-bxd)]) was selected as best estimate with parametes: a=0.8019, bK203=0.0015, bK027=0.003355, c=2.662 and d=1.218. Derived BMD100 were K203=194 (153, 243) and K027=100 (81, 125) µmol/kg bw, indicating that oxime K027 induces the same effect size with 2-times lower dose compared to oxime K203. Moreover, obtained confidence intervals of BMDs did not overlap allowing the conclusion that more potent dose-response relationship belongs to experimental oxime K027.


Keywords: benchmark dose, effect size, potency, erythrocytes, rat

Hematological and Cytochemical Parameters of Peripheral Blood after Acute Exposure to Epoxiconazole


Tetiana Usenko, Valentyna Shulyak, Volodymyr Bubalo

Laboratory of Pathology, L.I.Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety Ministry of Health, Ukraine, Kiev


Epoxiconazole is one of the most frequently used substances from triazole fungicides. The aim of the study was to assess hematotoxic effects of generic epoxiconazole, 95% on hematological parameters of peripheral blood and cytochemical status of leukocytes. 10 healthy males of Wistar Han rats were equally divided into control (0 mg/kg/bw) and experimental groups. Dose 1580 mg/kg/bw of epoxiconazole (1/2 LD50) was administrated once orally by gavage to 5 experimental rats. Peripheral blood was studied at 0 and 1, 3, 7, 14 day after exposure (DAE). RBC, HGB, HCT, erytrocyte indices MCV, MCH, MCHC, WBC and PLT were, hemogram and morphological disturbances of cells were studied. Cytochemical investigations of succinate dehydrogenase (SDH), acid phosphatase (AP) and specific naphtol-AS-D-chloracetate esterase (NCAE) in leukocytes were studied. As a result: significant RBC increase and MCHC decrease, polychromasia of erythrocytes and trend to decrease of HGB, MCV up to 14 DAE showed anemic effect. 1 DAE the amount of segmented neutrophiles increased: that describes the activation of blood system compensatory mechanisms in rats. WBC elevated on 3 DAE: monocytes, plasmocytes, lymphocytes were significantly increased. Young cells and leukocytes with degenerative changes in nucleus and cytoplasm were observed. At 3 DAE AP and at 7 – SDH and NCAE significantly raised. As a conclusion: epoxiconazole had hematotoxic action, altered hematological and cytochemical parameters of rats peripheral blood.

Keywords: pesticides, hematotoxicity, anemia, leukocytes, cytochemistry

The Effect of Thiamine on Activity of Enzymes (with a special emphasis on MAPK) in the Brain of Japanese Quails Treated with Chlorpyrifos


Dejana Ćupić Miladinović1, Sunčica Borozan2, Sanja Peković3, Sanja Dacić4, Danijela Đukić-Ćosić5, Vitomir Ćupić1, Saša Ivanović1

1 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Belgrade, Serbia, 2Department of Chemistry, Faculty of Veterinary Medicine, University of Belgrade, Serbia, 3Department of Neurobiology, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Serbia, 4Department for Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Serbia, 5 Department of Toxicology “Akademik Danilo Soldatović“, Faculty of Pharmacy, University of Belgrade, Serbia


The aim of this study was to investigate the influence of vitamin B1 (thiamine) on biochemical changes in the brain tissue of Japanese quail (Coturnix japanica) treated with chlorpyrifos. The following parameters were examined: cholinesterase activity (acetylcholinesterase – AChE and butyrylcholinesterase – BChE), nitrite concentration–NO2 (parameter of oxidative/nitrosative stress), activity of inducible nitric oxide synthase –iNOS, arylesterase –ARE, cyclooxygenase –COX and extracellular signal–regulated kinase –ERK (MAPK).

The study was conducted on eighty male Japanese quails (2 controls and 6 experimental groups, n= 10), 3-4 weeks old. One control group was treated only with vitamin B1, while the second one received pure corn oil. CPF dissolved in corn oil was administered to three groups of quails by gavage for 7 consecutive days at doses of 1.5 mg/kg BW, 3 mg/kg BW and 6 mg/kg BW. Another three groups were treated with 10 mg/kg BW of vitamin B1 i.m. 30 min after CPF administration (in above mentioned doses) for 7 consecutive days. Our studies have shown that CPF significantly inhibited both cholinesterases and ARE in brain, while vitamin B1 increased activity of enzymes in a dose dependent way. Also CPF has led to increase in the concentration of NO2, activity of iNOS and COX, but after thiamine treatment there has been a decrease of these parameters. There has been a decrease of ERK expression after CPF treatment that demonstrates an increase of apoptotic vulnerability of cells exposed to CPF.

Overall these results confirm that CPF causes oxidative/nitrosative stress and apoptosis, but also support the hypothesis that thiamine belongs to the group of “antistress vitamins”.

Keywords: acetylcholinesterase, butyrylcholinesterase, oxidative/nitrosative stress, arylesterase, apoptosis

Evaluation of Biocidal Products Enquiries to the Austrian Poisons Information Centre 2015


Angelika Holzer, Tara Arif, Kinga Bartecka-Mino, Helmut Schiel, Dieter Genser

Poisons Information Centre, Austria


A biocidal product is any substance or mixture intending to destroy, deter, render harmless, prevent the action of, or exert a controlling effect on any harmful organism by any means other than mere physical or mechanical action. Biocidal products are divided into 4 main groups: disinfectants, preservatives, pest control and other biocidal products.

On behalf of and funded by the Austrian Federal Ministry of Agriculture, Forestry, Environment and Water Management the local Poison Information Centre (PIC) evaluated retrospectively enquiries regarding exposures to biocidal products in 2015.

PIC Austria received in total 25718 telephone enquiries in 2015. Regarding biocidal product exposure the PIC was contacted in 643 cases: 341 (53%) under the age of 15, 302 (47%) persons over 15 years of age. In 542 cases a poisoning could be excluded due to minor exposure. In 54 cases the risk of intoxication could not be estimated due to lack of sufficient information at the time of consultation. In 37 cases intoxication was suspected and medical observation was recommended. In only 10 patients an intoxication could be verified due to the severity of the symptoms. The causative substances were disinfectants (industrial n=5, household n=3) and chlorine gas (n=2).

In relation to the total number of calls, enquiries regarding biocidal products are relatively rare and the number of human intoxications seems to be small. Only 10 cases with severe symptoms, which had to be treated medically, were recorded. No deaths were recorded in the local PIC.

Keywords: biocide, consultation, intoxication

Comparative Analysis of Freshwater Species Sensitivity Distributions and Ecotoxicity for Priority Pesticides: Implication for Water Quality Criteria

Huanqi He1, Zhenguang Ya1n*, Xin Zheng1, Jia He1, Yizhe Wang1, Li He1, Zhengtao Liu1

Chinese Research Academy of Environmental Sciences, Beijing 100012, China



Organochlorine pesticides (OCPs) and organophosphate pesticides (OPPs) posed severe threats to the aquatic environment in China. The toxicity data of 21 priority OCPs and OPPs for American and Chinese species were collected, and their species sensitivity distributions (SSDs) and ecotoxicity were compared. Mode-of-action (MOA) and physicochemical characteristics were used to analyze the difference in the pesticide toxicity. Results suggested no significant difference between pesticide SSDs for Chinese and American species, yet HC5 values could differ dramatically. American HC5 values of half of the pesticides were beyond the 2-fold difference of Chinese HC5 values. Applications of American thresholds of methyl parathion, parathion, dipterex, dimethoate, dichlorvos, chlorpyrifos, heptachlor, alpha-1,2,3,4,5,6-hexachloro cyclohexane (666), and chlorfenvinphos led to an over-protection for Chinese species, while American thresholds of atrazine, simazine, aldrin, chlordane, and diuron led to an under-protection. This result demonstrated the urgency of deriving native water quality criteria based on Chinese species and unique regional characteristics. In general, OCPs with the MOA of neurotoxicity were more toxic towards the ecological community. The regression analysis indicated a decrease in HC5 values with the increase in molecular weight (MW) and Octanol-Water Partition Coefficient (Kow), suggesting the potency of these two physicochemical characteristics to predict the pesticide toxicity.

Keywords: priority pesticides, species sensitivity distribution, water quality criteria, China, America

Behaviour of Vegetable Farmers in Respect of the Employment of a Panoply of Phytosanitary Products Case of Ain Naga And Doucen (Biskra -Algeria)

Soudani Nafissa; Belhamra Mohammed

Laboratory of diversity of ecosystems and dynamic systems of agricultural production in arid zones. Mohamed Khider university – Biskra (Algeria).


Around the world, intensifying agricultural production to limit the needs of the growing population, calls for the use of chemical inputs (pesticides and fertilizers) as a legitimate reality to ensure protection against pests and diseases that are increasingly intense, and that weigh strongly on agricultural productivity.

This study aims to verify the level of behavior of vegetable farmers (greenhouse and open fields) as regards the use of agricultural pesticides intended for plant protection. For this, a survey conducted from October 2016 to September 2017, among 90 producers and 20 phytosanitary product seller, randomly selected in two zones poles of the region of Biskra (Ain Naga and Doucen).

The results show that 80% of vegetable farmers in both areas are unaware that the misuse uncontrolled spread of a diversity of active substances coupled with a lack of solid knowledge can cause heavy impact on the natural environment, on their health and the health of consumers, thus threatening the development of agriculture and its sustainability in these arid regions. A series of factors justify well this alarming percentage are: the heterogeneity of farmers in terms of age, level of education and experience, Agricultural extension, the application of good phytosanitary practices and protective equipment, the respect of the recommended doses, the DAR and the management of empty packaging of pesticides.

Keywords: active ingredients, comportment, health, environment, Algeria

Place of Oximes in the Management of Acute Poisoning with Cholinesterase Inhibitors: Experience of the Pharmacology Toxicology Department of University Hospital of Oran


Haciba Rezk-kallah 1,2,3, Bilel Chefirat 1,2,3, Sameh Benzerga 1,2, Anissa Zergui 1,2

1 Department of Pharmacology Toxicology, University Hospital of Oran, Algeria, 2 Department of Pharmacy, Faculty of Medicine, University of Oran 1, Algeria, 3 Environmental Health Research Laboratory, University of Oran 1, Algeria


Acute poisoning with pesticides is a serious public health problem. The indication of oximes in the treatment of poisoning by anticholinesterase pesticides is still controversial in practice, although it seems theoretically interesting because of the reversibility of the toxic cholinesterase binding and the atropine-like effect. The aim of this work is to instigate, through our experience, a rational and scientific approach of the use of oximes in the treatment of acute anticholinesterase poisoning. This is a descriptive study of cases of acute pesticide poisoning received at Oran University Hospital during the last twelve years. Data was collected prospectively, using a pre-established information sheet, accompanying the samples. Diagnosis and monitoring were performed by the determination of cholinesterase activity. The interest of the oximes will be discussed through the analysis of some observations and a review of the literature. A total of 944 cases of acute pesticide poisoning were recorded, representing 10% of total acute intoxications. They occupy second place after drug poisoning. Among our patients, the indication of oximes, when available, has been justified in organophosphate poisoning, life-threatening carbamate poisoning, and in the presence of a severe cholinergic table where the pesticide is not identified but cholinesterase activity is collapsed.

Oximes keep their place in the treatment of acute intoxication with cholinesterase inhibitors. Their indication should be discussed for each case by taking into account the anamnesis, clinical picture and rate of cholinesterase activity.

Keywords: pesticides, pralidoxime, organophosphorate, carbamate, cholinesterase activity

S-metolachlor: Acute and Subacute Effects on Common Carp

(Cyprinus carpio L.)


Božidar Rašković1, Vesna Poleksić1, Gorica Vuković2, Dejana Ćupić-Miladinović3, Gavrilo Božić1, Zoran Marković 1, Dragica Brkić1

1Faculty of Agriculture, University of Belgrade, Belgrade, Serbia, 2Institute of Public Health Belgrade, Belgrade, Serbia, 3Faculty of Veterinary Medicine, University of Belgrade, Belgrade, Serbia


Toxicological assessment of S-metolachlor, synthetic organic herbicide frequently used for efficient weeds control, was the subject of the present study. Two exposure experiments (acute – 4 day and subacute – 28 days) were conducted on five months old common carp originated from the same family from the selective breeding program. 96h LC50 was calculated to be 16.31 mg L-1, which is generally high value, since environmental concentrations of S-metolachlor are usually determined to be below 1 mg L-1. Concerning this, three concentrations were set for a subacute test, carried out in triplicates: C0 – 0.0 mg L-1, C1 – 0.5 mg L-1, C2 – 1.4 mg L-1, C3 – 4.1 mg L-1. Biomarkers used for the assessment of fish health status were: histopathology of gills and liver, as well as nuclear abnormalities on erythrocytes. Histological assessment found a number of alterations in both sampled tissues: hyperaemia, presence of eosinophilic granular cells, epithelial lifting, hyperplasia of epithelial cells and focal necrosis in gills; leukocyte infiltration, hyperaemia, fibrosis of blood vessels and focal necrosis in liver. Only two of all mentioned alterations (epithelial lifting and gills hyperemia) had higher levels in the control, while majority were absent from control group. Different morphological abnormalities were noticed on the erythrocyte nuclei: micronucleus, nuclear buds, fragmented-apoptotic, and bi-nucleated cells. Almost all alterations, either histopathological or nuclear, were higher compared to control, but due to the high variation in between groups, statistical significance was not established. This study confirms low toxicity of S-metolachlor to common carp.


Keywords: herbicide, histopathology, gills, LC50, erythrocytes


Post-exposure Treatment with the Oxime RS194B Rapidly Reactivates Brain Acetylcholinesterase Activity in Mice Exposed to Sarin and VX


Nikolina Maček Hrvat1, Carol Green2, Suzana Žunec1, Zoran Radić3, Palmer Taylor3, Zrinka Kovarik1

1Institute for Medical Research and Occupational Health, Zagreb, Croatia; 2SRI International, Palo Alto, CA, USA; 3Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA, USA


Acetylcholinesterase (AChE) has a vital function in cholinergic neurotransmission but is irreversibly disrupted after exposure to organophosphate (OP) nerve agents, resulting in onset of toxicity symptoms which may lead to death. Currently used therapy consists of quaternary pyridinium aldoximes as reactivators of inhibited AChE, given along with atropine. The permanent cation precludes these reactivators rapidly crossing the BBB in appreciable concentrations to reactivate synaptic AChE, thereby restricting their activity to the periphery. Alternatives encompass oximes lacking a permanent cationic charge or presenting a tertiary amine as found in the zwitterionic hydroxyiminoacetamido alkylamines (RS194B). We have shown RS194B to be an effective in vitro reactivator of human AChE inhibited by VX, sarin, other methylphosphonates and various alkylphosphorates. Here we examine the pharmacokinetic properties, oral bioavailability and antidotal efficacy of RS194B against OP exposure in mice. The results show that 2 h sequential administrations out to 10 h result in steady-state plasma and brain levels of the oxime. Moreover, within the 40 min period brain concentrations of RS194B exceed the plasma concentrations prior to the next administration. Also, RS194B substantially protected mice when administered by gastric lavage prior to OP exposure, whereas 2-PAM exhibited no protection when similarly administered. Furthermore, the observed recovery of the mice brain activity after administering RS194B after exposure to both, VX and sarin is consistent with its rapid tissue disposition and BBB penetration. Those results, along with low toxicity of RS194B in mice, make this oxime a lead candidate for analyzing efficacy, tissue disposition and pharmacokinetics in other animal species.

Acknowledgements: This work was supported by the Croatian Science Foundation (4307) and National Institutes of Health (NS UO1-058046)

Keywords: CNS-active antidote, nerve agents, pharmacokinetics, tissue disposition

Genotoxicity Evaluation of Chlorpyrifos, Imidacloprid and α-cypermethrin in Low Concentrations on Human Peripheral Blood Lymphocytes by Cytokinesis-block Micronucleus Assay


Vedran Mužinić, Davor Želježić

Institute for Medical Research and Occupational Helath, Mutagenesis Unit, Zagreb, Croatia


The use of insecticides is increasing in modern society, but their toxic effects in low concentrations relevant for real scenario exposure have not received large attention. We have treated whole peripheral blood in vitro individually with three active insecticidal substances chlorpyrifos (a conventionally used organophosphate), imidacloprid (a novel neonicotinoid) and α-cypermethrin (a pyrethroid). Applied concentrations were estimate equivalents of acceptable daily intake (ADI), residual exposure level (REL), occupational exposure level (OEL) and a fourth common concentration (3 μg/mL). After treatment according to cytokinesis-block micronucleus assay, binucleated lymphocytes were analyzed for presence of micronuclei (MN), nuclear buds (NB) and nucleoplasmic bridges (NPB), and ratios of mono-, bi-, tri- and tetranuclear cells were calculated for estimation of cytokinesis-block proliferation index (CBPI).  The results have shown that none of the tested substances exhibited significant induction of any markers of secondary DNA damage or effect on cell proliferation rates. We conclude that chlorpyrifos, imidacloprid and α-cypermethrin induce no significant secondary DNA damage nor affect proliferation across the tested concentrations.

This work has been supported by Croatian Science Foundation under the project 8366.


Keywords: chlorpyrifos, imidacloprid, α-cypermethrin, micronucleus assay, genotoxicity

Neurotoxic Disorders Caused by Organophosphorus Insecticides: An Overview

Milan Jokanović

Faculty of Medicine, University of Nish, Nish, Serbia


In this presentation the neurotoxic disorders appearing in patients poisoned with organophosphorus pesticides and known mechanisms involved are reviewed. Organophosphorus compounds cause four main neurotoxic effects in humans: the cholinergic syndrome, the intermediate syndrome, organophosphate-induced delayed polyneuropathy and chronic organophosphate-induced neuropsychiatric disorder. Compared to the cholinergic syndrome, that causes millions of cases of poisoning with fatality of more than 15% each year, other disorders involve much smaller number of patients. Possible association of exposure to organophosphorus pesticides with neurodegenerative diseases, Parkinson’s disease and dementia will also be reviewed. This presentation is focused on neurotoxic disorders appearing after acute and chronic exposure to organophosphates with emphasis on molecular mechanisms, clinical presentation, pathogenesis, and possibilities for prevention/medical treatment.

Keywords: organophosphorus; neurotoxicity; cholinergic syndrome; acetylcholinesterase; intermediate syndrome; organophosphate-induced delayed polyneuropathy

Effect of Bifenthrin on TNF α and Interleukin 1β in Mice Kidneys


Barbara Nieradko-Iwanick, Andrzej Borzęcki

Chair and Department of Hygiene, Medical University of Lublin

Correspondence:  bnieradkoiwanicka

Pyrethroids, including bifenthrin, are used as insecticides of neurotoxic properties. Their use in pest control  increases. There is data that pyrethroids can also induce organ toxicity, immunotoxicity and inflammation.

The aim of the study was to find out if 28-day exposure to bifenthrin  affects  TNF α and interleukin 1β levels in mice kidneys.

32 female mice were divided into 4 groups of 8. The experiment was accepted by Local Ethical Committee. Group 0 were controls. Groups 1, 2 and 3 received bifenthrin intraperitoneally at the dose of 1.61 mg/kg, 4.025 or 8.05 for 28 days. On day 29 they were anesthetized and kidneys were collected. TNF α and interleukin 1β were measured with use of ELISA kits (Cloud-Clone Corp.USA).

Mean TNF α level in the kidneys of mice from group 0 was 6 pg/ml, in group 1=5,4 pg/ml, in group 2=6,3pg/ml and in group 3=9.5 pg/ml. Statistical significance was obtained for Group 3 vs 0 (p<0.05). Bifenthrin  increased the level of interleukin 1 β in a single dose proportionate manner in comparison to the control group. Mean interleukin 1 β values were: in group 0 3.9 pg/ml, group 1 6.8, group 2=9.8, and group 3 11. There was a statistically significant difference between Group 3 and 0 (p<0.05).

Bifenthrin increases the level of interleukin 1β  and TNF α in mice kidneys in a single dose  proportionate manner which might disrupt cytokine regulatory processes and lead to disease.

Keywords: pyrehroid bifenthrin, TNF α, interleukin 1β, kidney, female mice

Porsulan, an Ancient Medicinal Herb, Could Have Protective Effects on Paraquat-induced Cytotoxicity on Isolated Mice Splenocytes


Seyed Hadi Mousavi1,2, Vafa Baradaran Rahimi2, Vahid Reza Askari2

1Medical Toxicology Reserch Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

2Department of Pharmacology and Pharmacological Research Center of  Medicinal Plant, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran



Paraquat is an herbicide that has a high toxicity to various organs of the body, especially lungs and kidneys. Because of its structural similarity to the active metabolite MPTP, it is thought to be the cause of neurodegenerative diseases, including Parkinson’s. The main mechanism of this combination is the induction of oxidative stress and apoptosis in various tissues and cells. Induced oxidative stress and apoptosis have been observed by paraquat in various tissues and cells including peripheral blood lymphocyte cells. It has also been shown that Porsulan has antioxidant and anti-apoptotic effects.

In this study, the protective effect of aqueous extract of Porsulan on cytotoxicity and oxidative stress by paraquat in mice splenocytes were investigated. Cell viability was assessed using MTT dye at 24 and 48 h after incubation with paraquat as well as MDA, ROS and NO, and TNF-a measured as oxidative and inflammatory markers, respectively.

Results indicated that all tested concentrations of the extract (20-160 µg/mL) significantly increased cell viability in concentration and time dependent manner (p<0.05 to 0.001). Also, inflammatory marker (TNF-a) and oxidative stress, especially MDA were significantly decreased after both 24 and 48 of pretreatment with the extract (p<0.05 to 0.001).

Our study could suggest that this plant is useful for reduction of cytotoxicity and inflammation induced by paraquat. Moreover, complementary therapy with this extract may have protective effect on farmers to avoid or delay the neurodegenerative diseases, such as Parkinson’s.

Keywords: paraquat, porsulan, Portulaca oleracea L, cytotoxicity

Primary Toxicological Assessment of the Fungicide Formulation Based on Azoxystrobin

Popel Alina, Vasilyeva Marina, Yurkevich Elena

Republican Unitary Enterprise «Scientific-Practical Centre of Hygiene»

The study of the toxic properties of plant protection products is conducted with a view to preventing or minimizing potential adverse effects on the human body and the environment. Therefore, it is now advisable to offer manufacturers of agrochemicals a wide range of promising plant protection products that will minimize the negative impact on public health, the environment and the associated economic damage.

The purpose of this work is to carry out a primary toxicological evaluation of a fungicide based on azoxystrobin.

In the course of the experiment it was found that LD50 formulation of fungicide with intragastric intake of more than 5000 mg / kg body weight. At a single exposure to the skin of LD50 more than 2000 mg / kg of body weight. When determining the parameters of acute inhalation toxicity, no cases of death of animals were recorded during the whole observation period. Doesn’t irritate the skin of rabbits, has a weak irrigation effect on the mucous membranes of rabbit eyes. Doesn’t have a sensitizing effect when applied to the test in guinea pigs. The cumulative coefficient is greater than 5.

When assessing the means of plant protection data from scientific literature, international databases, as well as scientific and technical documentation, guidance and methodological materials are used.

The results of the work done will allow increasing the range of applied plant protection products, use the drugs that are less hazardous to health and the environment in the agro-industrial complex.

Keywords: fungicide, primary toxicological evaluation, acute toxicity, plant protection, azoxystrobin.
























































New Psychoactive Substances (NPS): The Results of Toxicology Laboratory, Serbia, Nis in UNODC International Quality Assurance Programme (IQAP) and International Collaborative Exercises (ICE) in the Period 2015-2017


Maja Vujovic1, 2, Biljana Milosavljevic2, Jovana Simic2

1University of Niš, Faculty of  Medicine, Department of Pharmacy, Toxicology, Serbia, 2Institute of Forensic Medicine Niš, Toxicology Laboratory, Serbia


Illegal production and use of new NPS represent a worldwide health risk problem. The synthetic NPS are often labeled as “legal” and sold on the drug markets and darkened as substitutes for international controlled drugs. To struggle against illicit drugs, UN, through Drug Control Programme (UNDCP) and Office on Drug and Crime (UNODC), collaborate with many countries, governments, and institutions, continuously monitor and research global illicit drug markets. To participate in solving the drug problem, the Toxicology Laboratory at the Institute of Forensic Medicine in Nis, joined the UNODC International Quality Assurance Programme (IQAP) and International Collaborative Exercises (ICE) in 2015. In this assay, we present our results covering a three -year period between 2015 and 2017. Methods: GC/MS, UHPLC-MS/MS, HPLC/PDA and immunoassay tests. In 2015, we identified the following drugs in BS1: Ketamine, Norketamine, Buprenorphine, Norbuprenorphine; BS2: Blank, MDA; BS3: MDA, Methadone; BS4: 6-MAM, Morphine, Nordazepam, SM-1: Caffeine, Cocaine, Lidocaine, Procaine; SM2: Amphetamine, Caffeine; SM3: Blank; SM4: Heroine, Morphine. In 2016, BS1: Morphine; BS2: Benzoylecgonine, Methylecgonine, Morphine; BS3: 3,4 MDMA; BS4: 2CB; SM1: Cocaine; SM2: JWH-073; SM3: Ketamine; SM4: Heroin. In 2017, BS1: Blank; BS2:  Amphetamine, Nordazepam, Oxazepam, Temazepam; BS-3: GHB; BS4: Morphine; SM1: MDPV; SM2: Cocaine; SM3: 3,4 MDMA; SM4: Blank. In each of the two rounds per year the laboratory receives test samples that contain controlled drugs and their metabolites in urine (BS) and/or drugs in seized materials (SM). In all samples, the test drugs were successfully identified with acceptable Z-score values.


Keywords: drugs, biological specimens, seized materials, LC-MS/MS, GC-MS


Use of Benzodiazepines in Methadone Treatment Patients


Daniela Chaparoska, Niko Bekjarovski, Danil Petrovski, Aleksandra Babulovska, Irena Jurukov,Dushan Petkovski and Tanja Petrushevska.

Universiry Clinic  of Toxicology, Medical Faculty, University ‘Sts Cyril and Methodius’, Skopje, Macedonia


Introduction:  The aim of the study is to explore the use/abuse of benzodiazepines by patients on methadone treatment and indicate needs and recommend intervention.

Material and Methods: The research is quantitative analytical study which was implemented from December 2016 to July 2017.A randomized anonymous survey, in terms of the use/abuse of benzodiazepines, was conducted on 458 addicts who are on  methadone substitution treatment Results: The prevalence of abuse of BZD in patients on methadone treatment is 86.66%. The average age in the first try of BZD is 21.9+/_ 6.1 years. For 24.1% BZD were prescribed by a doctor the first time when it was used. BZD without prescription were use by 85.8% of the respondents. At the moment treatment with BZD prescription receive 22.4 %, usually 38.7% use more than the prescribed dose of BZD, use prescribed dose 17.3%, use less than the prescribed 6,8% and never had prescription for BZD 37.2% vs 50%. BZD use before entering the methadone program 70.7% of respondents in the experimental group. BZD has increased/started after the methadone program 60.1%. In the life always used only BZD which were prescribed by a doctor said 7.8% of respondents in the experimental group.In the total sample of patients of methadone treatment as the most common health problem is selected  heroin at 42% followed by hepatitis C in 41.6% and anxiety/ stress at 33.5%. Prescribed treatment for psychological/ emotional problems (including depression or anxiety), receiving 54.3% of the participants in the experimental group.

Keywords: benzodiazepine use, methadone program, substances abuse, epidemiology

Detection of Designer Drug – 4-bromo-2,5-dimethoxyphenyletanamine (2CB) in Urine Using GC-MS Method


Lazar Grahovac¹, Danijela-Đukić Ćosić2, Vera Lukić3, Snežana Đorđević4, Biljana Antonijević2, Marijana Ćurčić2

¹University of Belgrade-Faculty of Pharmacy, PhD student, modul Toxicology,Belgrade, Serbia, 2University of Belgrade-Faculty of Pharmacy, Department of toxicology “Akademik Danilo Soldatović”, Belgrade, Serbia, 3University of Belgrade, Institute of Forensic Medicine “Milovan Milovanovic”, Belgrade, Serbia, 4National Poison Control Center, Military Medical Academy, Belgrade, Serbia


4-bromo-2,5-dimethoxyphenyletanamine (2CB) is psychoactive substance from the family of phenylethylamines. 2CB is a popular and recreational psychostimulant phenylethylamine, known among other street names as ‘nexus’ or ‘cyber’. It can act as partial agonist or antagonist of serotonin 5-HT2 receptors in central nervous system. Increase in recreative use of 2CB to achieve entactogenic effect indicates need for proposing adequate method for its detection in humans.

The objective of this analysis was to develop method for urine sample preparation for 2CB   detection by GC-MS method.Urine samples spiked with the 2CB were prepared by liquid-liquid extraction (LLE) or solid phase extraction (SPE). Liquid-liquid extraction was done by using ethyl acetate and sodium hydroxide, while SPE was done by elution with the mixture of 2-propanole/methylene chloride/ammonia (74:24:2).  Derivatization of 2CB in extracts was done with the 2,2,2-trifluoro-N-methyl-N-(2,2,2-trifluoroacetyl)acetamide (MBTFA) during 20 minutes at 80 °C. Prepared samples were analyzed by GC-MS method.Obtained retention times for derivatized 2CB were 11 min while principal ions (m/z) for its identification after GC-MS analysis were 262 and 240. Chromatography peak areas imply equal efficacy of the both used sample preparation methods. Set conditions for urine sample preparation and derivatization show that developed GC-MS method is adequate for 2CB detection.

Keywords: 2CB, liquid-liquid extraction, solid-phase extraction, derivatization, GC-MS

Can Passive Inhalation of Cannabis Smoke Affect Someone’s Driving Abilities?


Ljubiša Božić1, Saša Bovan1

1Department of theory, sociology and philosophy of law, Faculty of Law, University of Belgrade


Cannabis is one of the most frequently encountered illicit drugs in Serbia; therefore it poses a significant risk factor for traffic accidents. Serbia is one of European countries where legal limit, for presence of drugs in body fluids, is set at zero. Still, defense lawyers sometimes argue that the presence, of cannabinoid metabolites in the defendant biological specimen, is result of passive unintentional inhalation of environmental cannabis smoke.

Several authors have studied passive exposure to cannabis smoke under extreme conditions (small, unventilated room, high smoke exposure, etc.) in order to demonstrate that passive inhalation affects blood or urine drug testing. Concentrations of THC (Δ9-tetrahydrocannabinol) and its metabolites, gained in such unrealistic circumstances, are in most cases lower than legal cannabinoids thresholds for drivers in some European states. Also, majority of measured cannabinoids concentration are below cutoff concentration of various immunoassay techniques, which implies possibility of staying undetected in routine drug screening. Nevertheless a real-life conditions experiment was conducted, where non-smoker participants spent three hours in well-attended coffee shop in Netherlands. Absorbed concentration of THC were measured and obtained result indicate that passive exposure to cannabis smoke in such circumstances may only lead to trace amounts of THC in serum.

All results given, obtaining such concentrations of cannabinoids, which would lead to impairment of driver, is possible only at extreme smoke exposure, for long period of time in confined space, and making it impossible for a person to be unaware of his/hers environment and potential consequences.


Keywords: Cannabis, impaired, driving, traffic, accidents

Review of Analyzed Confiscated Illicit Substances. Uncommon Combination of Drugs of Abuse


Dragana Stojkov, Branislava Zdrale, Kristina Denic, Vera Lukic

Institute of Forensic Medicine, Faculty of Medicine, University of Belgrade


During 2017 substances of abuse, earlier confiscated on the territory of Republic of Serbia, were submitted to Reference Laboratory for identification of controlled psychoactive substances of Institute of Forensic Medicine. Gas chromatography with mass and liquid chromatography with tandem mass spectrometry were used for qualitative and quantitative determination of all samples (123). In marijuana samples (60), tetrahydrocannabinol, cannabidiol and cannabinol were detected and quantificated. According to the amount of tetrahydrocannabinol (0.32 – 11.67 %) all marijuana samples were defined as substances of abuse. In 43 brown powder samples was detected heroin with its concomitant fillers like acetaminophen, caffeine, thebaol, meconin. Average heroin content in samples was 16.01 % (0.25–75.70 %). Lidocaine, levamisole, caffeine and cocaine as main component were identified in 11 analyzed samples. Cocaine content was from 0.95 % up to 67.80 %. Beside these, only 5 from all of analyzed samples were positive on amphetamine and 4 on MDMA. One of MDMA positive samples was with extremely specific content. It was capsule fulfilled with white and red small crystals. After analysis, it was proved that analyzed crystals contained mixture of MDMA (35.00%), amphetamine (0.75 %) and cocaine (0.40 %). This is rare and unusual composition among the most common ”ecstasy” form. This combination has pronounced cardiotoxicity which makes it extraordinary dangerous.

According to these results , marijuana and heroin are probably the most present “street“ drugs. However new mixtures of known drugs may be extremely dangerous and of special concern.

Keywords: illicit substances, drugs of abuse, MDMA, cocaine

Use of Mephedrone During Pregnancy Induces Neurotoxicity in Offspring and Increases the Risk of Stillbirth

Ahmad Ghorbani1, Gholamreza Naseri2, Alireza Fazel2, Mohammad Jafar Golalipour3, Hossein Haghir2,4, Hamid Sadeghian5, Majid Mojarrad4, Mahmoud Hosseini6, Shokouh Shahrokhi Sabzevar4, Farimah Beheshti7

1Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran; 2Department of Anatomy and Cellular Biology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; 3Gorgan Congenital Malformations Research Center, Golestan University of Medical Sciences, Gorgan, Iran; 4Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; 5Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran; 6Division of Neurocognitive Sciences, Psychiatry and Behavioral Sciences Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; 7Department of Basic Science and Neuroscience Research Center, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran


Mephedrone is a synthetic derivative of cathinone, the natural psychostimulant alkaloid present in the Khat plant. Recently, regular and recreational use of mephedrone has increased among young adults in several countries. No experimental study has yet evaluated the toxicity of mephedrone in the gestational stage. This study was aimed to evaluate the effects of mephedrone exposure in pregnancy on newborn outcomes, focusing on hippocampal damage. The pregnant mice received mephedrone (50 mg/kg, sc) as regular schedule (once daily, day 5 to 18 of gestation) or repeated schedule (thrice daily at 5th, 6th, 11th, 12th, 17th, 18th day of gestation) to simulate regular or recreational uses of mephedrone, respectively. In both regular and repeated mephedrone groups, the percent of weight gain in pregnant mice was significantly lower than control group. Mephedrone significantly decreased the weight and number of delivered pups and increased the rate of stillbirth. Results of immunohistochemistry and TUNEL assays showed an inhibition of cell proliferation and an increase of apoptosis in the hippocampus of delivered pups of repeated mephedrone group. This apoptotic effect was associated with increased expression of the proapoptotic gene Bax and reduced the expression of antiapoptotic gene Bcl-2. Data of Morris water maze showed an impairment of the spatial learning and reference memory in offspring (60-day-old) born from mephedrone treated mothers. In conclusion, this study demonstrated that regular and repeated exposure to mephedrone during pregnancy increased the risks of low birth weight, stillbirth, and neurotoxicity in offspring.

Keywords: apoptosis, hippocampus, memory, mephedrone, pregnancy

Mephedrone – “White magic”. Qualitative and Quantitative Analyse in Urine by LC-MS/MS


Branislava Zdrale, Dragana Stojkov, Kristina Denic, Vera Lukic

Institute of Forensic Medicine, Faculty of Medicine, University of Belgrade


Mephedrone, also known as “white magic” is a designer stimulative drug from cathinone class, present in Catha edulis plant leaves. It is usually in the form of white powder, tablets or capsules. The users can take it by mouth, inject, smoke, snort or use rectally. Onset of the effects and their duration depends on the way mephedrone is used. Mephedrone causes effects similar to the effects of cocaine, amphetamine and MDMA such as stimulation, euphoria, elevated mood, improved mental function. Because of its structure similar to biogenic amines mephedron influences cardiovascular and neurological system provoking thus side effect.

In Laboratory of toxicology, Institute of Forensic Medicine,urine sample from ICE program organized by scientific department of UNODC was analyzed. Qualitative and quantitative analyze of mephedrone was performed in urine by LC-MS/MS. Its identification was also confirmed by GC-MS.

Solid phase prepared sample using Strata-XC column was derivatized with MBTFA and analyzed by GC/MS system. It was identified on 8.25 min with target ions 119,154 and 91.

Alkaline liquid-liquid extracted sample was analyzed by LC-MS/MS system with a C18 column (Kinetex 100Å, 2.6 µm, 75×4.6 mm). Assay was performed using 10mM ammonium formate (A) and methanol (B) as a mobile phase with a flow rate 0.8 ml/min. Target ions were 178, 160, 145. Mephedrone retention time was 0.98 min.

For quantitative analyze mephedrone standard (purity ≥ 99.8 %) was used. In submitted urine sample mephedrone is detected in concentration of 966 ng/mL.

LC-MS/MS is fast and precise method for reliable mephedrone identification and quantification in biological sample.

Keywords: mephedrone, white magic, mephedrone detection, synthetic cathinone, LC-MS/MS















































Testing of Tobacco and Tobacco Products in Croatia Before Transposing the 2014/40 / EU Tobacco Directive in May 2017

Ivona Vidić Štrac, Nino Dimitrov, Buga Kovačić, Rina Oliver Grbavec, Bernarda Damianić
Croatian Institute of Public Health


In December 2016, cigarettes and related products were bought at random. The tests were carried out in accordance with the requirements of the Ordinance on Health Safety of Consumer Items (OG 125/2009), REACH requirements for lead, cadmium and nickel, and the requirements for nicotine in e-liquids prescribed by the new tobacco directive.

Molds were detected in four samples of cigarettes or 20%.  In 10% of cigarette samples, the carbon monoxide content in the smoke condensate is higher than maximum emission levels, while in one sample the tar content is higher than the stated value. Organochlorinated pesticides examination was compliyed with requirements.  In fine- cut tobacco, molds were found in 50% of samples.  Rolling paper complies also with the requirements prescribed in the Ordinance. The total content of lead in the mouthpiece was more than 0,05 % by weight, as prescribed by the requirements for products Commission Regulation (EU) 2015/628 of 22 April 2015 coming into contact with skin.  The nicotine content was found to be greater than the maximum permissible value of 20mg/mL in 50% e-liquid samples.

There is few references on tobacco testing from the market. Values for nicotine in e-liquids declared and measured content are in accordance with the literature data that indicates the results deviations. The results for mouthpiece samples are of particular concern since no research has been done so far and there is no legislation on the matter, instead analogue regulations are applied.

Keywords: tobacco and related products, market, legislation

Application of Physiologically-Based Pharmacokinetic Modelling to Pesticides in South Africa:  Requirements, Feasibility, and Challenges

Turgay Celik1, Wells Utembe2, Mary Gulumian2,3

1 School of Computer Science and Applied Mathematics, University of the Witwatersrand, Johannesburg, south Africa, 2 Department of Toxicology at the South African National Institute for Occupational Health, South Africa, 3 School of Pathology, University of the Witwatersrand

South Africa


Physiologically-based pharmacokinetic (PBPK) models use physiologic properties of organisms and biophysical properties of substances to describe the absorption, distribution, metabolism and elimination (ADME) of substances.  This information is very important for, inter alia, prediction of internal dose at target organs as well as interspecies dose and route-to-route extrapolation necessary for the risk assessment process in the registration of pesticides.   PBPK modelling is especially invaluable in cases where specific target organ toxicity (STOT) is anticipated, and also for the determination of toxicokinetically and toxicodynamically derived chemical-specific adjustment factors (CSAFs) that are important in the derivation of parameters such as the acceptable daily intake (ADI) and reference dose (RfD).

Publicly available generic PBPK models, such as the USEPA’s Exposure Related Dose Estimating Model (ERDEM), as well as the Canadian Centre for Environmental Modelling and Chemistry’s generalized PBPK model make it possible for their applications by developed and developing countries. However, despite their availability, PBPK modelling is conspicuously underutilized in South Africa where numerous challenges may have hampered this possibility. Steps are therefore taken to urgently remedy the situation in South Africa by establishing a unit specializing in much needed computational toxicology in collaboration between the Department of Toxicology at the South African National Institute for Occupational Health (NIOH) and the School of Computer Science and Applied Mathematics at the University of the Witwatersrand. This in turn, will be able to deliver more adequately on the risk assessment requirements for the registration of pesticides in South Africa.

Keywords:  PBPK modelling, dose

























Abnormal Development of Reproductive Organs was Caused by Exposure to Cigarette Smoke via Respiratory System in Female Diabetic Rats


Soo-Min Kim and Kyung-Chul Choi

Laboratory of Biochemistry and Immunology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea


Cigarette smoke (CS) is harmful to human fertility, reproduction, and development. Moreover, CS has been shown to have a greater impact on patients suffering from metabolic diseases such as diabetes than on healthy individuals. This study was conducted to investigate the relationship between CS exposure and histological alterations of reproductive organs in diabetic rats. We evaluated the histology of uteruses and ovaries obtained from female rats exposed to smoke from standard cigarettes for four weeks (28 h a week). After CS exposure, we manufactured tissue slides from uterine and ovarian samples and examined them after H&E staining. Immunohistochemistry (IHC) was used for detection of matrix metallopeptidase 9 (MMP- 9), C-X-C chemokine receptor type 4 (CXCR4) and estrogen receptor (ER) α in the uterus and ovary. In the uterus, expression of MMP9, CXCR4, ERα and the number of endometrial glands were increased by CS exposure, while in the ovary, expression of MMP9, CXCR4, ERα, PCNA and the number of corpus lutea (CL) or cyst follicles (CF) was increased by CS exposure. This study indicates that exposure to CS via the respiratory system exerted a harmful impact on the uterus and ovary in female rats under induction diabetes.

This research was supported by a grant (14182MFDS977) from the Ministry of Food and Drug Safety in 2017.


Keywords: cigarette smoke exposure, ovarian follicle, uterus, diabetic rats



New Insight into the Toxicity of Polychlorinated Biphenyls: Study on Animal Model


Aleksandra Buha 1, Vesna Milovanovic 2, Sasa Jankovic 3, Zorica Bulat 1, Biljana Antonijevic1, Vesna Matovic1

1 Department of Toxicology “Akademik Danilo Soldatović”, University of Belgrade-Faculty of Pharmacy, Serbia, 2 University Children’s Hospital, Belgrade, Serbia, 3 Institute of Meat Hygiene and Technology, Belgrade, Serbia


Polychlorinated biphenyls (PCBs) are synthetic chemicals widely spread in the environment. Due to PCBs chemical stability and lipophilic nature, i.e. ability to accumulate and biomagnify in the food chain, humans interact with these chemicals on a daily bases. This study was aimed to investigate the effects of exposure to different doses of PCBs on body weight gain, hematological parameters, liver, kidney and thyroid function in animal model.

Six groups of male albino Wistar rats (7 animals/group) were receiving commercial PCBs mixture Aroclor 1254 dissolved in corn oil in six different doses (chosen to reflect the environmental exposure) by oral gavage during 28 days while controls were receiving corn oil only.

PCBs produced profound effects on body weight gain suggesting possible developmental toxicity. Critical effects of PCBs in blood was the diminishing effect on white blood cells count while platelets number was lowered only in group receiving the highest dose of PCBs. The study demonstrated both hepatotoxic and nephrotoxic effects of PCBs with oxidative stress as an important mechanism of their toxicity. Namely, PCBs were shown to affect enzymatic and non-enzymatic components of antioxidant defence system and to cause both lipid and protein degradation in liver and kidneys. The obtained results also gave the confirmation of the thyroid disrupting effects of PCBs with profound effects on T4 hormone levels presumably as the result of PCBs direct effect on thyroid gland.

It can be concluded that PCBs impose significant adverse effects in even in relatively low doses that correspond to environmental ones.

Keywords: environmental exposure, blood, liver, kidney, thyroid gland


Cadmium Levels in Rats Blood and Testes after Acute Oral Treatment with Two Doses


Milena Anđelković1,2, Simona Tatović2, Danijela Đukić-Ćosić2, Aleksandra Buha Đorđević2, Vesna Matović2, Zorica Bulat2

1Health Center Kosovska Mitrovica, 2University of Belgrade – Faculty of Pharmacy, Department of Toxicology “Akademik Danilo Soldatović”


Cadmium (Cd) affects reproductive organs by still unclear mechanisms of toxicity. Disruption of the blood-testis barrier, followed by permeability changes, is one of them. The aim of the study was to investigate Cd level in the blood and testes after low and moderate acute oral administration.

Male Wistar rats were randomly assigned to non-treated (control) and treated (Cd15 and Cd30) groups,administered by oral gavage 15 and 30 mgCd/kg body weight, respectively. After 24 hours, animals were sacrificed and blood and testes were used for Cd analyses. Mineralisation was carried out by microwave digestion system and Cd levels were measured by atomic absorption spectrophotometer with graphite tube. Nonparametric ANOVA, post hoc Mann Whitney test and Spearman’s rho correlation coefficient have been used for statistical analyses.

Blood Cd levels were 14.47 μgCd/L (controls), 51.60 μgCd/L (Cd15) and 52.03 μgCd/L (Cd30), indicating significantly higher Cd in treated groups compared with controls, but with no  difference between the treated groups. In testes, Cd levels were 0.0024 μgCd/g, 0.0354 μgCd/g and 0.0798 μgCd/g in control, Cd15, and Cd30 groups, respectively. Treated groups have significantly higher Cd level than control one, and in contrast to blood Cd levels, higher Cd dose induced significantly higher Cd concentration in testes, compared with lower one (p<0.05). Furthermore, correlation analysis has showed significant correlation between levels of Cd in blood and testes (p< 0.01).

This study demonstrated that oral treatment with low and moderate Cd doses induced significant increase in both blood and testes, although the dose dependence was observed for testes only.

Keywords: reproductive organs, blood-testis barrier, correlation

Stability of Mitochondrial Intactness for Further Toxicological Studies


Ege Arzuk, Hilmi Orhan

Ege University, Faculty of Pharmacy, Pharmaceutical Toxicology Department, 35100 Izmir, Turkey


Mitochondria are critical subcellular organelles as they provide more than 95% of the energy for biochemical and physiological functions, as well as they play a critical role in lipid metabolism, steroidogenesis and programmed cell death. Recently we have shown their metabolic capacity for various drugs as well. Nevertheless, mitochondria from different species, as well as from different organs in the same organism exhibit diverse properties, which entails special attention in working on structures and functions of this organelle. Intactness of mitochondria as the whole organelle must be provided and maintained along in vitro studies, since these varieties may significantly influence both toxic pathways and toxic end-points in in vitro studies. In the current study, therefore, we have explored mitochondrial stability in isolated organelle and in frozen tissue by mitochondrial pore transition (MPT). Mitochondria were isolated from fresh tissue and either mitochondria or whole tissue were kept in various temperatures (room temperature, +4 °C and -86°C) for a day, for a week, for 3 months, respectively. MPT pore opening was assessed as mitochondrial swelling by a rapid decrease in absorbance at 540 nm. It has been found that mitochondria and tissue maintain functionality for limited time due to the ambient temperature. However, mitochondria keep stability much longer when frozen.

This study is supported by The Scientific and Technical Research Council of Turkey (TUBITAK, SBAG-114S310).


Keywords: subcellular isolation, integrity, mitochondrial pore transition

Exposure of Cigarette Smoke Extract May Induce Apoptosis and Autophagy in Human Placenta Choriocarcinoma JEG-3 Cells


Hae-Miru Lee and Kyung-Chul Choi

Laboratory of Biochemistry and Immunology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea


In this study, the effects of cigarette smoke (CS) on the induction of apoptosis via reactive oxygen species (ROS) production and endoplasmic reticulum stress (ER-stress) of JEG-3 human choriocarcinoma cells were examined to confirm the relationship between CS and placenta development. Upon TUNEL assay, CS extract (3R4F; 0.3 μM and 2.1 μM) increased JEG-3 apoptosis. Western blot assay revealed that the protein expressions of p53, Bax and CCAAT-enhancer-binding protein homologous protein (CHOP) increased, while the levels of Bcl-2 were reduced following CS extract treatment. Moreover, DCFH-DA assay revealed increased ROS production. Upon MTT assay, isoprene (IP), one of ingredients of CS, deceased JEG-3 cell viability (10-11 M to 10-6 M). After based on the MTT assay, two IP concentrations of 10-11 M and 10-8 M were selected and the protein expressions of cyclin D1, cyclin E1, p21 and p27 decreased in response to IP. Furthermore, IP showed the greatest increase in autophagy at 24 h and further induction of cell death at 72 h upon MDC and TUNEL assay. Western blot analysis confirmed the increase in autophagy markers, LC3β and p62, as well as the increase or decrease of apoptosis markers p53, Bax, CHOP and Bcl-2 in response to its treatments. In addition to confirming increases in ROS through DCFH-DA, we also confirmed the expression of Nrf2, an antioxidant marker, and the expression of Kelch-like ECH-associated protein 1 (KEAP1), which specifically degrades Nrf2, by Western blot. Taken together, these results indicate that CS may inhibit the development of placenta via activation of ROS by inducing apoptosis and autophagy by affecting the expression of KEAP1, which regulates Nrf2 expression. [This research was supported by a grant (14182MFDS977) from the Ministry of Food and Drug Safety in 2017.]

Keywords: cigarette smoke, isoprene, placenta choriocarcinoma, cell cycle, EMT


































































Harmful Effects of the Misuse of Diclofenac in the Geriatric Population


Bojana Petrović2, Predrag Vukomanović2, Saša Ivanović3

1Medical Sanitary School of Applied Sciences“Visan”, Belgrade, Serbia, 3Faculty of Veterinary Medicine University of Belgrade, Serbia.


The geriatric population is affected by multiple physiological deficiencies, polymorbidity, poliotherapy and polypragmasy. Based on a detailed overview of relevant literature, theoretical and pharmacoepidemiological studies, diclofenac is one of the most frequently prescribed medications for patients over 65 years for painful conditions and rheumatic ailments in the Republic of Serbia. In addition, elderly very often takes diclofenac at the recommendation of an incompetent person or at his own discretion. This fact is particularly alarming, due to the known pharmacovigilance of diclofenac, especially in uncontrolled usage and overdosage. Diclofenac achieves analgesic, antinflammatory and antipyretic activity by non-selective inhibition of cyclooxygenase: physiological COX1 (which contributes to harmful effects) and pathological COX2. The most common harmful effects of diclofenac are gastrointestinal haemorrhage and ulcer perforation, which are caused by various pathogenetic mechanisms, such as: weakening and disorders in the regeneration of the mucous barrier of the stomach, decreased bicarbonate and mucus secretion, disinhibition of HCl secretion, reduced circulation in the mucous membrane. Diclofenac can lead to nephrotoxicity, cardiotoxicity, hepatotoxicity, anaphylactic reactions. Furthermore, aging leads to changes in pharmacodynamics and pharmacokinetics, which affects the choice, dosage and frequency of the use of many medicines, and therefore diclofenac. Diclofenac can lead to an increase in the serum concentration of cardiac glycosides, and lowering effect of β-blockers, ACE inhibitors and thiazide diuretics, drugs that are often used in the therapy of elderly. Based on the above facts, it can be concluded that diclofenac is widely used in any pain symptom and is very commonly misused.

Keywords: chronic pain, elderly, diclofenac, pharmacovigilance.


Bile Acid Potentiates Apoptosis of Human Breast Adenocarcinoma Cells Induced by Doxorubicin


Bojan Stanimirov1, Karmen Stankov1, Nebojša Pavlović1, Maja Đanić1, G. Bogdanović2, Vesna Kojić2, Momir Mikov1

1 Faculty of Medicine, University of Novi Sad, Novi Sad, Vojvodina, Serbia

2 Oncology Institute of Vojvodina, Sremska Kamenica, Vojvodina, Serbia


The use of doxorubicin (Dox) is limited by cumulative, dose-dependent toxicity. Ursodeoxycholic acid (Udca), as a bile acid with amphipathic structure, has propensity to modulate transport of xenobiotics across biological membranes but also to modulate cell metabolism, proliferation and cell death. The aim of this study is to evaluate whether Udca potentiates apoptosis-inducing effects of DOX in MCF-7 cell line by measuring expression of apoptosis-regulating genes.

MCF-7 cells were treated either with 250 nM of Dox or with combined treatment by adding 0.05 mM Udca. Cell viability was determined by a colorimetric assay using MTT whereas gene expression was analysed using qRT-PCR.

The co-incubation of MCF-7 cells with Dox and UDCA resulted in significant inhibition of cell growth (p<0.05) compared to Dox. Relative expression of gene of pro-apoptotic Bax was increased in both Dox-treated cells (p=0.001) and co-treated cells (p=0.007), compared to untreated control. Relative quantitation of Bcl-2 mRNA revealed that expression of this anti-apoptotic marker was significantly reduced in cells co-treated with Udca (p=0.0002) compared to Dox-treated cells. The values of Bax to Bcl-2 ratio indicated that co-treatment with Udca induced apoptosis in higher level than Dox alone compared to control ( p=0.004, p=0.008)

Udca potentiates mitochondrial pathway of Dox-induced apoptosis in MCF-7 cells, suggesting that Udca may be further investigated as a novel agent with primary aim to improve therapeutic index of DOX.

Supported by HORIZON2020 MEDLEM project No.690876, Project for Scientific and Technological Development of Vojvodina No.114-451-2072-/2016-02.


Keywords: chemotherapy, toxicity, mitochondria

Effects of Subtle Chemical Changes on Drug-induced Liver Toxicity: A Structure-toxicity Relationship Study on Non-steroidal Anti-inflammatory Drugs


Yi Yun Pang , Han Kiat Ho

Department of Pharmacy, National University of Singapore.


Diclofenac and lumiracoxib are non-steroidal anti-inflammatory drugs (NSAIDs) carrying the 2-phenylaminophenyl acetic acid scaffold. They exemplify an interesting case study of a pair of compounds that exhibit subtle chemical differences, and yet producing highly contrasting hepatotoxicity. To elucidate the role of these substituents to the toxicophore of the compounds, their chemical differences were extrapolated by synthesizing a series of 24 analogues, differing only in the size of their aliphatic and halogen side chains. Their pharmacological activities as well as in vitro toxicity were profiled and analyzed. Among which, the most toxic in the series was found to be the most lipophilic and brominated compound. A clear trend could be observed between toxicity with metabolic stability, as well as with electrophilic reactivity. Bioactivation by Phase I metabolism is a prerequisite for toxicity whereby the propensity to generate electrophilic intermediate as measured by glutathione depletion correlated well with the extent of toxicity. Further LC-MS/MS analysis of the glutathione-trapped intermediates facilitated the identification of putative structures of such reactive metabolites. Pooling this effort together with COX-1 and COX-2 evaluations of the compounds unravels a divergent role of the same substituents to its pharmacology, thereby providing an approach to optimize the safety and efficacy of NSAIDs.

Keywords: hepatotoxicity, structure-toxicity relationship, reactive metabolites, non-steroidal anti-inflammatory drugs, diclofenac.

Association of GSTP1 Genes Polymorphisms with Frequency of Hemorrhagic Cystitis in Patients Treated with Cyclophosphamide


Samin Sabzevari1, Neda Setayesh2, Vahideh Montazeri1, Mohammad Vaezi3, Mahsa Amoli3, Omid Sabzevari1,4

1Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran-1417614411, Iran, 2Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran-1417614411, Iran

3Shariati Hospital, Tehran University of Medical Sciences, Tehran-1417614411, Iran

4Toxicology and Poisoning Research Centre, Tehran University of Medical Sciences, Tehran-1417614411, Iran

Glutathione S-transferases (GSTs) are phase P superfamily enzymes with important role in catalyzing many drugs such as cyclophosphamide (CP). CP is a commonly used alkylating drug in bone marrow transplantation due to immunosuppressive activity in AML and ALL patients The active metabolite, 4-hydroxy-cyclophosphamide is responsible for cyclophosphamide’s alkylating potency. Functional SNPs in GST are important in the activity of enzymes. Polymorphisms of GSTM1 (GSTM1 null allele) deletion and one transition point mutation of A1578>G that creates the GSTP1 Ile105Val polymorphism with reduced activity are important in the activation of CP. Hemorrhagic cystitis (HC) is the main adverse effect in patients treated with high dose of CP for transplantation. We investigated association of GSTM1 & P1 polymorphisms with hemorrhagic cystitis in AML and ALL patients treated with high dose of cyclophosphamide.

Blood samples of 200 patients diagnosed with ALL or AML in which 17 experienced HC were collected. For the control group, 100 healthy subjects were randomly chosen. DNA were extracted with Yekta Tajhiz extraction kit and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for GSTP1 (M24485.1, GeneBank) genotyping. GSTM1 null genotype was determined using polymerase chain reaction (PCR).

Our study showed that GSTP1 mutant allele increased risk of ALL and Hemorrhagic Cystitis (the adverse effect of Cyclophosphamide) while GSTM1 polymorphism showed no association with Acute Leukemia Type and hemorrhagic Cystitis.

Keywords: ALL, GSTM1, PCR, AML, blood

Lamivudine, Tenofovir Disoproxil Fumarate and Efavirenz Induced Somatic and Germ Cell Mutation in Mice


Adekunle Bakare, Bayonle Dauda, Kehinde Akinseye, Francis Ofoegbu, Anifowoshe Saheed,  Stork Abruda, Opeoluwa Fadoju, Olusegun Ogunsuyi and Chibuisi Alimba.

Cell Biology and Genetics Unit, Department of Zoology, University of Ibadan, Ibadan, Nigeria


The lifelong dependence of HIV-infected patients on a combination of antiretroviral (ARV) drugs may increase the risk of various side effects, including DNA damage. This study was designed to investigate the genotoxicity of Tenofovir disoproxil fumarate (TDF), Lamivudine, Efavirenz and their combination (1:1:1) using the bone marrow micronucleus (MN) and sperm morphology assays. All experiments were carried out at concentrations corresponding to the x0.125, x0.25, x0.5, x1, and x2 of the individual and combination of the ARV drugs. Male Swiss albino mice (6-8 weeks for MN assay; 11-15 weeks for sperm morphology assay) were intraperitoneally exposed to the individual and combination of the drugs for 5 consecutive days. For the sperm morphology assay, mice were sacrificed after the first (35 days) and second spermatogenic (70 days) cycles. Another set of mice (11-15 weeks) were orally exposed to the ARV combination for 35 consecutive days. A significant (p < 0.05) increase in micronucleated polychromatic erythrocytes (MNPCE) was observed in the individual and ARV combination groups compared to the negative control group. At the 35-day exposure period, a significant (p < 0.05) increase in sperm abnormality was observed in groups exposed to TDF, Efavirenz and the ARV combination irrespective of the exposure route. A significant (p < 0.001) reduction of abnormal sperm cells was observed between the 35- and 70-day exposure periods for the intraperitoneally administered mice. Data demonstrate that the individual and combination of ARV drugs have the potential of inducing DNA damage to the somatic and germ cells in mice.

Keywords: antiretroviral drugs, micronucleus, sperm abnormality, DNA damage



Сurrent Issues of Reglamentation of Air Pollution in the Production of  Cytotoxic Drugs Vasilkevich Vadzim , Pavel LiapioshkaRepublican unitary enterprise “Scientific Practical Centre of Hygiene”Correspondence: For the treatment of patients with tumors in the Republic of Belarus are widely used cytotoxic drugs. Actual hygiene issue is the prevention of air pollution of the working area and the environment of products of the synthesis of cytotoxic drugs. This task is solved through the establishment