ANTIMICROBIAL COATINGS IN HEALTHCARE SETTINGS: EFFICIENCY VERSUS SAFETY
Chair: Anne Kahru1,2, email@example.com, Co-Chair: Angela Ivask 1, firstname.lastname@example.org
1 National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia, 2 Estonian Academy of Sciences, Kohtu 6, 10130 Tallinn, Estonia
Infections and infectious diseases are considered a major challenge to human health in healthcare units worldwide. As antimicrobial materials (such as silver and copper) are inherently toxic, the application of (nano-enabled) antimicrobial surface coatings (AMCs) in healthcare settings may cause harm in addition to benefits. Indeed, in parallel to cutting down the spread of potentially infectious microbes, AMCs may induce (eco) toxicological hazard and antimicrobial resistance. This session will cover briefly all these aspects: potential of AMCs in the development and spread of antimicrobial resistance (A. Ivask), introduction of the adverse environmental effects (A. Kahru), the need for balancing the efficiency and safety of AMCs by ‘safe-by-design’ (I. Vinković Vrček), aspects of industrial up-scaling of safe AMCs (P. Mantecca) and importance of internationally coordinated research and networks (F. Crijns). The session was suggested by Estonian Society of Toxicology and the speakers cooperate in the COST Action AMiCI (AntiMicrobial Coating Innovations, CA15114), supported by the European Cooperation in Science and Technology (COST). More information on this COST network is available in the joint paper of the authors: http://www.mdpi.com/1660-4601/14/4/366.
Keywords: nanomaterials, safety, healthcare associated infections, antimicrobial resistance, risk-benefit analysis
Antimicrobial Coatings in Healthcare: Possible Benefits and Need for Internationally Coordinated Research
Francy Crijns 1, Rinaldo van Meel 1, Olaf Brouwers 1, Jim Odekerken 1, Patrick van de Poel 2, Minna Keinänen-Toivola 3, Gabrielle Tuijthof 1
1 Zuyd University of Applied Sciences, Faculty of Bèta Sciences and Technology, P.O. Box 550, 6400 AN Heerlen, The Netherlands; 2 Zuyderland Hospital, P.O. Box 5500, 6130 MB Sittard-Geleen, The Netherlands, 3 Faculty of Technology, Satakunta University of Applied Sciences, P.O. Box 211, FI-26101 Rauma, Finland
Infections are a continuous threat to human health. According to the European Centre for Disease prevention and Control (ECDC), each year over 4 million people are estimated to acquire a HealthCare Associated Infection (HCAI). A potential and promising weapon against bacterial growth and possibly the development of multi-drug resistant bacteria has been found in AntiMicrobial (nano)-Coatings (AMC). In coatings fortified with an active ingredient, the ingredient is responsible for the elimination of the microorganisms.
Nowadays, a lot of these coatings are commercially available and establish a high reduction of bacterial loads on surfaces, when studied in standardized laboratory conditions. Field studies to test the efficacy of AMC in healthcare settings are scarce. This is one of the main reasons for so far limited use of AMCs in hospitals. Zuyd University is currently performing field studies in Zuyderland Hospital, to assess the efficacy of 2 different antimicrobial TiO2 coatings in patient rooms. Both coatings have proven efficacy in controlled lab conditions using the intimate contact test ISO-19622. The aim of the current study is to explore the efficacy of these coatings in a living lab situation by assessing bacterial load and diversity. Moreover, experience built up in these living lab studies is valuable to extend field studies through the AMiCI network.
The COST Action AMiCI (AntiMicrobial Coating Innovations, CA15114), supported by the European Cooperation in Science and Technology (COST), brings together universities, research institutes and companies to evaluate the impact of (introducing) antimicrobial coatings in healthcare on spreading of infections.
Keywords: healthcare associated infections, nanocoatings, COST Action AMiCI, living lab, performance assessment
Design and Evaluation of Efficient and Safe Antimicrobial Coatings: Connections with the Industry
Paride Mantecca1, Kaja Kasemets1,2, Ehud Banin3, Ilana Perelsthein4, Aharon Gedanken4
1Department of Earth and Environmental Sciences, Research Centre POLARIS, University of Milano-Bicocca, 1 Piazza della Scienza, Milan, Italy; 2Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia; 3The Mina and Everard Goodman Faculty of Life Sciences, Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan, Israel; 4Department of Chemistry and Nanomaterials, Bar-Ilan University Center for Advanced Materials and Nanotechnology, Ramat-Gan, Israel
The increase of infectious diseases is a global scale concern with extremely high social and economic costs. Special concern is spreading of the nosocomial infections typically derived from increased exposure to pathogenic bacterial in hospitals or healthcare units. The development and use of antimicrobial coatings (AMCs) is a promising industrially up-scalable research field to mitigate spreading of infectious diseases and antimicrobial resistance (AMR). The primary goal is to obtain more effective AMCs and currently the devices/materials impregnated with antibiotics or silver are most widely used. As the scientific community has questioned the human and environmental safety of nanosilver, the identification of safer AMCs is urgently needed applying safe-by-design approach. Textiles are among the materials with the highest demand for antimicrobial functional coating, due to their ubiquitous use in healthcare settings (e.g. bandages, pajamas, bed-sheets). The growing need for antibacterial textiles has resulted in revolutionary progress in the textile industry, leading to new technologies and products able to improve the antibacterial efficacy and to concomitantly reduce the environmental and health hazard, which finally has the potential to open new market and business opportunities for the companies.
The EU project PROTECT has developed novel nano-enabled AMCs (CuO, ZnO and Zn-doped CuO) to coat textiles to achieve enhanced antibacterial activity (also against resistant bacterial strains), in concomitance with reduced health hazards. Pilot-scaled industrial plants were developed and the refinement of the technologies is ongoing to match the market demand.
EU-H2020 project PROTECT (n. 720851) and EU COST Action network CA15114 AMiCI are acknowledged.
Keywords: COST Action CA15114 AMiCI, antibacterial textiles, nano-coatings, metal oxides, safety
Can Antimicrobial Coatings Promote Development of Resistant Microorganisms?
Angela Ivask1, Siiri Kõljalg2
1 Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia; 2 Institute of Biomedicine and Translational Medicine, Faculty of Medicine, University of Tartu, Ravila 19, Tartu 50411, Tartu, Estonia
Microbe-caused infectious diseases and development of antimicrobial resistance (AMR) are increasing global concerns associated with significant medical costs. Antimicrobial coatings (AMCs) have been suggested as a promising solution against the spread of microbes and related infections in public and healthcare sectors. Although certain AMCs affect microbial cells via topology or anti-fouling properties, most often AMCs are based on antimicrobial active substances – traditional antibiotics, antimicrobial peptides, enzymes, quaternary ammonium compounds (QACs), or nanoparticles e.g., of silver, copper, zinc – that kill microbes either after release from the surface or by direct contact. Due to the release of these chemicals from AMCs and rapid genetic exchange and mutations in microbes there are concerns that increasing use of AMCs may induce the appearance of new or spread of already existing AMR microbes. The fact that traditional antibiotics, even if released from AMCs only at low concentrations may cause the spreading of AMR microbes via horizontal gene transfer, is well-recognised. However, microbes may develop resistance also against antimicrobial peptides and QACs e.g., via mutations for modified membrane composition, expression of stress-response, repair system, or efflux pump genes. Also various genes granting microbial resistance to metals have been discovered and there is growing evidence of co-resistance between e.g., QACs or metal-ions and traditional antibiotics. Thus, by aiming to control microbial infections by AMCs one has to take care not to open the door for resistant microbes instead. Financial support from COST Action CA15114 AMiCI and Estonian Research Council grant PUT748 is acknowledged.
Keywords: antimicrobial surfaces, co-resistance, horizontal gene transfer, mutations, membrane composition, COST Action CA15114 AmiCI
Can Antimicrobial Coatings Pose Risk to the Environment?
Anne Kahru1,2, Kaja Kasemets1, Merja Ahonen3
1 Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia;2 Estonian Academy of Sciences, Kohtu 6, Tallinn 10130, Estonia; 3Faculty of Technology, Satakunta University of Applied Sciences, P.O. Box 211, FI-26101 Rauma, Finland
Bacteria-caused (infectious) diseases are currently among the most burdensome to patients and society in terms of distress, therapy needs and financial burden to healthcare budgets. The problem expands as microbial resistance to antibiotics increases and can be alleviated by application of antimicrobial coatings (AMCs) in health-care settings. To this end, nanoparticles (NPs), e.g., silver, copper, and zinc NPs are increasingly used in AMCs (bandages, face masks, privacy curtains, bed-sheets) due to their antimicrobial efficiency. Unfortunately, most AMCs are also toxic to humans, animals and to the environment at large. For example, Ag+, Cu2+, Zn2+-ions that are the drivers of the antimicrobial effects of respective AMCs are highly toxic to algae and crustaceans – important members of the aquatic food-webs, although at low concentrations copper and zinc are essential micronutrients. Therefore, AMCs can be a double-edge sward: in addition to inhibition/killing of noxious microbes adverse effects to environmental organisms can occur via various waste-streams originating e.g., from the production, application, wear and tear, cleaning and/or disposal of AMCs. Thus, the risk assessment of AMCs over the entire life cycle into different environmental compartments is needed for the sustainable application of AMCs. Currently there are some data available for hazard evaluation of AgNPs – the antimicrobial nanomaterial that has been most efficiently studied – but not yet enough to conduct a detailed risk-benefit assessment. The data gaps are even more severe for other (nano)antimicrobials that have remarkably less available information.
Estonian Research Council project IUT23-5 and EU COST Action CA15114 AMiCI are acknowledged.
Keywords: silver, copper, aquatic food-web, risk-benefit assessment, COST Action CA15114 AMiCI
Safe-by-design Approach for Development and Use of Antimicrobial Coatings
Ivana Vinković Vrček1, Angela Ivask2
1 Institute for Medical Research and Occupational Health, Ksaverska cesta 2, Zagreb, Croatia; 2Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia.
A state-of-the-art innovation to combat HealthCare Associated Infections (HCAIs) is the creation of self-disinfecting surfaces through the application of antimicrobial coatings (AMCs). Nanotechnology, one of the key enabling technologies, allows for significant improvements in the AMC development providing better healthcare but also enabling the design and clinical use of innovative solutions for HCAIs. However, nano-specific behavior should be taken into account during manufacturing, application and final disposal of nano-enabled AMCs at the Safe-by-Design level. This is especially relevant for exposure, absorption, distribution, accumulation, and toxic effects. The Safe-by-Design is well accepted approach to ensure safety for three different, but interrelated communities – the workplace, consumers and environment.
There are following key issues: identification and characterisation of nano-based biocidal agent; transformation of nanomaterials; dose metrics; fate and kinetics. It is crucial to identify difficulties related to experience and knowledge about safety assessment of AMCs. Opinions are needed to provide response, challenges, needs and recommendation for Safe-by-Design approach in addressing the application of AMCs in healthcare setting.
Financial support from EU COST Action CA15114 AMiCI is acknowledged.
Keywords: nanotechnology, healthcare associated infections, safety, COST Action CA15114 AMiCI
EXPOSURE AND RISK ASSESSMENT OF PESTICIDE USE IN AGRICULTURE: APPROACHES, TOOLS, AND ADVANCES
Chair: Claudio Colosio 1, email@example.com, Co-Chair: Aristidis Tsatsakis 2, firstname.lastname@example.org
1 Department of Health Sciences of the University of Milan and International Centre for Rural Health of the S. Paolo Hospital, Italy, 2 Medical School, Division Morphology, University of Crete, Greece
Pesticide exposure remains an outstanding problem for public health, and the attention of the scientific community on the topic is growing. Plant Protection Products are used worldwide, their presence is ubiquitous in the living and working environments and doubts persist on the real health risks they pose, especially for continuous, long term, low-dose environmental and occupational exposures. In assessing pesticide risk, the main challenge is quantifying and qualifying exposure levels. This activity often leads to biases due to under and overestimation of exposure, impairing, risk assessment and management. Researchers face the problem of choosing adequate analytical methods and approaches for their study, including both pre-analytical and analytical phases, and, in any case, any analytical approach is hampered by the difficulties created by the instability of the climatic conditions and the variation of the exposure timing and patterns. In the meanwhile, the continuous development of new technologies and computational approaches creates the possibility to perform risk assessment without doing measures, using models and simulations. This Symposium is aimed at offering data regarding the sources of difficulty in doing biological and environmental monitoring, and at overviewing the different methods available for pesticide exposure and risk assessment, from the regulatory field to real-life exposure studies, A particular attention will be addressed at the new tools which may be developed using modern technologies. Each Speaker will first offer a short theoretical background on each method presented, followed by the practical approach and experience. Each presentation will discuss also prospects for development of these activities.
Keywords: pesticides, exposure assessment, risk assessment, biological monitoring, modeling
From Regulation to Risk Assessment: Outlining the Process of Regulatory Exposure and Risk Assessment and the Practical Use of Limit Values
Martin F. Wilks
Swiss Centre for Applied Human Toxicology, University of Basel, Switzerland
The process for human health risk assessment of pesticides involves 4 steps: identification of toxicological hazards; characterization of dose-response relationships; assessment of human exposure; and comparison of exposure estimates to a limit value that is defined as the dose which will not harm humans. Limit values are normally set using threshold levels at which no adverse effects are seen in animal studies and applying a number of uncertainty or modifying factors to account e.g. for interspecies and interindividual differences, or sensitive subpopulations. Different limit values are set for different regulatory purposes: the Reference Dose (RfD) is the amount of a chemical that a person can be exposed to on a daily basis that is not anticipated to cause adverse health effects over the entire lifetime. For pesticide handlers, the Acceptable Operator Exposure Level (AOEL) is the maximum amount of active substance to which the operator may be exposed without adverse health effects. Both RfD and AOEL can also be calculated for acute exposures (typically 24 h or less). An alternative approach to the setting of limit values involves calculating the margin between an observed (adverse) effect and the measured or estimated exposure (MoE). Although current risk assessment methodologies have been successfully used for many years, they need to be developed to address existing and future challenges, e.g. how to integrate novel data (in vitro, omics, biomarkers) into risk assessments; how to deal with chemicals for which no threshold can be established; or how to account for exposure to chemical mixtures.
Keywords: hazard, threshold, reference dose, acceptable operator exposure level, margin of exposure
Introduction to Pesticide Exposure Monitoring, Practical Guidance, and Perspectives
Claudio Colosio, Stefan Mandić-Rajčević, Federico Maria Rubino
Department of Health Sciences of the University of Milan and International Centre for Rural Health of the S. Paolo Hospital, Italy
The use of pesticides has continued to grow since their introduction to modern agriculture, and more than 2 billion kilograms of these substances are used per year worldwide. Their main characteristic, that they impact living organisms, makes them a chemical hazard, and their use results in potential health risk, especially in agricultural workers. In agriculture, there is a notable instability of working conditions, disregard of good agricultural practices, and misuse of personal protective devices. Estimating the exposure and absorbed dose in this scenario is, therefore, extremely challenging. Pesticide field studies still represent the main way to collect real-life exposure data, to perform absorbed dose and risk assessment, and to verify the presence or absence of health effects from pesticide use. Here we present the main methods for pesticide exposure monitoring in real-life field conditions, with their advantages, disadvantages, and ways to improve them. Since most of the exposure in open field conditions comes from dermal exposure, OECD “patch” and “whole-body” methodologies are the two most widely used methods for exposure assessment. The main advantages of the “patch” methodology is that it preserves the real-life working conditions and allows the combined use of personal exposure and biological monitoring. The use of biological monitoring is limited in because of the lack of health-based occupational biological exposure limits. Ideally, a method to produce biological exposure limits for pesticide use in agriculture, similar to the ACGIH BEIs, could be developed taking into account skin as the main route of exposure in this setting.
Keywords: methodology, patch, whole-body, biomonitoring, biological exposure limit
The Need for Biological Monitoring in Agriculture and New Promising Methods
Ioanna Katsikantami, Athanasios Alegakis, Manolis Tzatzarakis, Elena Vakonaki, Aristidis M. Tsatsakis
Center of Toxicology Science and Research, Medical School, University of Crete, GR-71003, Heraklion, Crete, Greece.
Biological monitoring (biomonitoring) in occupational health is an essential tool for the exposure assessment of workers to pollutants by measuring their chemical or biochemical markers in biological matrices and tissues. Biomonitoring provide information about the total uptake of the pollutant from all routes of exposure and its distribution in human body, metabolism, absorption and excretion. Different biomarkers among biological samples indicate current, recent or past exposures. The importance of biomonitoring lies in the correlation of systemic and chronic exposure with clinical symptoms and signs of diseases appeared in workers. However, environmental sources of exposure often contribute to occupational exposure and together with the existence of nonspecific biomarkers, occupational risk assessment becomes unclear and complicated.
Strict regulation on pesticides has resulted in lower occupational exposure and there is a strong demand for analytical tools and protocols which allow trace analysis of chemicals. Chromatographic and mass spectrometric techniques offer high sensitivity, multi component and target analysis as well as identification of unknown samples. Besides urine and blood, noninvasive matrices such as saliva and exhaled air have been employed for human biomonitoring. Except for metabolites, promising biomarkers have been developed like protein and DNA adducts. Information derived from genomics, proteomics and metabolomics techniques is combined with biomonitoring data and give explanations to observed differences in health impact, pharmacokinetics and metabolism between individuals, possibly due to population discrepancies.
Keywords: pesticides, biomonitoring, biomarkers, analytical tools
Duration of Skin Exposure: a Neglected Variable in Absorbed Dose Assessment
Stefan Mandić-Rajčević, Federico Maria Rubino, Claudio Colosio
Department of Health Sciences of the University of Milan and International Centre for Rural Health of the S. Paolo Hospital, Italy
The use of pesticides has become unavoidable in agriculture as it ensures the massive production of food crops and their global trade, as well as solves public health problems by eradicating vectors of human diseases such as malaria. Besides risk assessment done in the pre-marketing phase, field studies allow for the re-evaluation of exposure and risk in real-life working conditions, opening new possibilities for risk assessment and modeling. In agriculture, special attention must be given to the skin as the main route of exposure, but the fixed fractional approach to dermal absorption might not represent the perfect solution to absorbed dose assessment. Here we present a practical method for integrating the information on the duration of exposure into the absorbed dose assessment, using a group of mancozeb applicators as a case study. Assumption of an 8-hour exposure resulted in a gross overestimation of absorbed dose from hands’ exposure. Absorbed dose from body exposure was overestimated in those workers working less than 8 hours, but somewhat underestimated in those working more than 8 hours, which is common in agriculture. In total, an 80% reduction of the absorbed dose estimate resulted from the introduction of the duration of exposure as a factor. This reduction did not influence risk assessment significantly for substances with low toxicity such as mancozeb, but implications for modeling might be much more important.
Keywords: methodology, patch, biological monitoring, absorbed dose assessment, modeling
Integrating Epidemiology Along with other Lines of Scientific Evidence into Pesticide Risk Assessment
Antonio F. Hernández
Department of Legal Medicine and Toxicology, University of Granada, School of Medicine, Avenida de la Investigación, 11, 18016- Granada, Spain.
Regulatory agencies currently conduct a formal human risk assessment for pesticide active substances based on mandated regulatory toxicology studies. Although this process is mainly based on experimental studies according to specific study protocols, human observational epidemiological studies could add relevant information to the risk assessment process. However, a better use of epidemiology data is needed to improve the understanding and characterization of risks from pesticide exposures. To this end, individual studies addressing the association between pesticide exposure and human health should be subject to a quality assessment of methodology and reporting to meet quality standards. Then, studies should be combined and summarized using systematic reviews and meta-analysis. The impact of evidence synthesis on risk assessment will be particularly useful for problem formulation and hazard identification, though if quantitative data are available dose-response modelling of pooled data could be used for hazard characterization. Therefore, the new paradigm of pesticide risk assessment should be based on all available lines of evidence, from animal regulatory studies to independent peer-reviewed studies, including human data. In addition, novel tools for identifying biological pathways and mechanisms of toxicity (in vitro/mechanistic studies) could provide biological support to the findings observed in animal or human studies. All these lines of evidence can form part of the overall weight of evidence using modified Bradford Hill criteria as an organizational tool to increase the likelihood of underlying causal relationships. This integrative process would allow for a more realistic human risk assessment of pesticides, though a harmonised framework will be required.
Keywords: epidemiology, risk assessment, evidence synthesis, weight of evidence, integration of evidence
Oxime Efficacy in Acute Organophosphate Poisoning: Challenges and Perspectives
University of Belgrade-Faculty of Pharmacy, Department of Toxicology “Akademik Danilo Soldatovic”, Serbia
Organophoshorus (OP) compounds are used as pesticides causing every year thousands of fatalities especially in developing countries, mainly resulting from suicidal or accidental poisonings. Recent homicidal use of chemical warfare nerve agent sarin confirmed that OPs still represent permanent threat on the global scale. High toxicity of OPs is based on inhibition of acetylcholinesterase (AChE), which leads to excessive accumulation of acetylcholine in the synaptic cleft and subsequent overstimulation of cholinergic receptors. Current standard treatment of acute OP poisoning includes a combined administration of causal antidotes (oximes), antimuscarinic drug (atropine) and anticonvulsive drug (diazepam).
The principal mechanism of action of oximes is reactivation of OP-inhibited AChE. Since the synthesis of the first pyridinium oxime in 1955, a number of oximes have been invented and tested in order to identify structures sufficiently potent to restore inhibited AChE and to enable therapeutic effectiveness. Despite numerous in vitro and in vivo studies none of the known oximes fulfills the crucial requirement, i.e. ability to recover AChE inhibited by a broad range of structurally different OPs. Aimed to overcome present drawbacks and limitations of the oximes, recent research efforts are focused on the development of broad spectrum, less toxic oximes, the synthesis of mono-charged or uncharged reactivators able to penetrate blood-brain barrier, to restore ”aged” AChE, and to reactivate OP-inhibited bioscavengers. Designing an oxime with universal antidotal properties is of utmost interest but potentially a never-ending goal. Therefore, alternatively improvement of oximes therapeutic value could be achieved by combination of oximes with complementary spectrum towards critical OPs.
Keywords: therapeutic effectiveness, reactivation, drawbacks and limitations, novel strategies
ADVANCES IN MOLECULAR METAL TOXICOLOGY
Chair: Yoshito Kumagai, email, email@example.com
Faculty of Medicine, University of Tsukuba, Japan
Dr. Naranmandula will especially focus on the anticancer effects of arsenic trioxide and its active metabolites on PML-RARA fusion protein degradation in acute promyelocytic leukemia (APL). He will introduce that the actual arsenic metabolic pathway would be beneficial in elucidating the probable role of different arsenic species in relation to toxicities along with the uses of arsenic as a therapeutic drug. Dr. Hara will discuss recent advances in understanding the change in lipid metabolism in methylmercury (MeHg) and other metals-induced toxicity, focusing on the LC-MS/MS-based lipidomics approach. He will also show the role of some lipid metabolizing enzymes in detoxification of metal-toxicity by using these enzymes gene-engineered mice. Dr. Aschner will discuss recent studies that tested whether the redox sensitive cofactor nicotinamide adenine dinucleotide (NAD(+)) is affected by MeHg and whether supplementation of NAD(+) prevents MeHg-induced toxicities. He will show studies in worms treated with MeHg where depletion in cellular NAD(+) levels was prevented by NAD(+) supplementation. Dr. Kumagai will introduce that electrophilic metals such as MeHg and cadmium are able to activate cellular redox signal transduction pathways (e.g., Keap1/Nrf2 pathway, HSP90/HSF1 signaling and PTEN/Akt signaling) involved in adaptive response, quality control of cellular proteins and cell survival at lower concentrations. In addition, he will also introduce that persulfide/polysulfides capture the environmental metals, leading to formation of their sulfur adducts, thereby regulating the activation of redox signaling and cytotoxicity.
Keywords: arsenicals, methylmercury, cadmium, molecular toxicology
Metabolism, Toxicity and Anticancer Activities of Arsenicals
School of Medicine and Public Health, Zhejiang University, China
A variety of studies indicated that inorganic arsenic and its methylated metabolites have paradoxical effects, namely, carcinogenic and anticancer effects. Especially, arsenic trioxide (As2O3) is successfully used in the treatment of refractory or relapsed acute promyelocytic leukemia (APL), but its exact antileukemic mechanism in APL is still under investigation. The probable explanation for As2O3-induced cell differentiation is the direct targeting of PML-RARα oncoprotein by As2O3, which results in initiation of PML-RARα degradation. However, after injection, As2O3 is able toy methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), therefore, it remains unknown that which arsenic specie is actually responsible for the therapeutic effects against APL. Here we have shown the role of iAsIII and its intermediate metabolites (i.e., MMAIII/DMAIII) in NB4 cells. Inorganic iAsIII predominantly showed induction of cell differentiation, while MMAIII and DMAIII specifically showed to induce apoptosis. Additionally, our results also found that the binding of arsenicals to PML proteins is not associated with the degradation of PML-RARα fusion protein.
Keywords: arsenic trioxide, acute promyelocytic leukemia, PML-RARα, cell differentiation
Metal Toxicity and Toxicolipidomics
Division of Health Chemistry, Department of Healthcare and Regulatory Sciences, School of Pharmacy, Showa University, Tokyo, Japan
Some metals such as methylmercury cause their toxicity by the induction of oxidative stress in the target organ. Oxidative stress is more likely to enhance lipid peroxidation of membrane phospholipids and formation of bioactive lipid mediators leading to the tissue injury. In order to clarify novel mechanisms of metal toxicity, we focused on lipid molecules as targets of metal toxicity and performed toxicolipidomics, in which we comprehensively investigated alterations in lipid profiles including membrane phospholipid composition and production of bioactive lipids. As the results, we found that mouse cerebellum tissue and cerebellar granule cells, both of which are sensitive to methylmercury, contain high levels of polyunsaturated fatty acids in their membrane phospholipids. Methylmercury treatment affected their membrane phospholipid composition but did not enhance the production of eicosanoids. It was also found that some inhibitors of calcium-independent phospholipase A2s (iPLA2s), which have an ability to eliminate lipid peroxides from membrane phospholipids, enhanced methylmercury-induced cell death of cerebellar granule cells. These results suggested that in cerebellum tissues, polyunsaturated fatty acids in membrane phospholipids might be targets of metal toxicity and that iPLA2s might function as detoxifying enzymes. Furthermore, we have established several lipid-metabolizing enzymes gene-engineered mice and found that iPLA2g (one of iPLA2 isozymes) or ACSL4 (one of long-chain acyl-CoA synthase isozymes) gene knockout changed membrane phospholipid compositions in mouse tissues/cells. The effects of these gene knockouts on metal toxicity will be also discussed.
Keywords: methylmercury, cerebellum, lipidomics, polyunsaturated fatty acids,
NAD+ Supplementation Attenuates Methylmercury Dopaminergic and Mitochondrial Toxicity in Caenorhabditis elegans
Albert Einstein College of Medicine, Bronx, NY 10461
Correspondence email: firstname.lastname@example.org
We have previously shown that MeHg causes both morphological and behavioral changes in the Caenorhabditis elegans dopaminergic (DAergic) neurons that are associated with oxidative stress. We were therefore interested in whether the redox sensitive cofactor nicotinamide adenine dinucleotide (NAD(+) may be affected by MeHg and whether supplementation of NAD( + )may prevent MeHg-induced toxicities. Worms treated with MeHg showed depletion in cellular NAD( + )levels, which was prevented by NAD( + )supplementation prior to MeHg treatment. NAD( + ) supplementation also prevented DAergic neurodegeneration and deficits in DAergic-dependent behavior upon MeHg exposure. In a mutant worm line that cannot synthesize NAD( + ) from nicotinamide, MeHg lethality and DAergic behavioral deficits were more sensitive to MeHg than wildtype worms, demonstrating the importance of NAD( + ) in MeHg toxicity. In wildtype worms, NAD( + )supplementation provided protection from MeHg-induced oxidative stress and mitochondrial dysfunction. These data show the importance of NAD( + ) levels in the response to MeHg exposure. NAD( + ) supplementation may be beneficial for MeHg-induced toxicities and preventing cellular damage involved in Parkinson’s disease.
Keywords: methylmercury, dopamine, nicotinamide adenine dinucleotide (NAD). C. elegans
Activation of Redox Signal Transduction Pathways Mediated by Electrophilic Metals
Environmental Biology laboratory, Faculty of Medicine, University of Tsukuba, Japan.
Methylmercury (MeHg) accumulated in fish such as tuna and cadmium (Cd) contaminated in rice are electrophilic metals that covalently modify protein thiols, resulting in formation of protein adducts. While it is believed that such protein modifications are associated with cell damage and tissue injury due to nonselective and excess modification of cellular protein thiols at higher concentrations, accumulated studies indicate that MeHg and Cd modulate cellular signaling as well. From these observations, we hypothesized that MeHg and Cd would activate redox signaling consisting of sensor proteins and effector molecules through covalent modification of the sensor proteins at lower concentrations because sensor proteins with reactive thiols showing low pKa values should be selectively modified by these metals.
We found that 1) MeHg and Cd modify sensor protein Keap1 and thus repress its activity to hold transcription factor Nrf2, thereby activating Nrf2 and up-regulating proteins responsible for detoxification and excretion of these metals. 2) MeHg causes S-mercuration of phosphatase PTEN, leading to inhibition of its enzyme activity, thereby activating kinase Akt and its downstream transcription factor CREB; as a result, anti-apoptotic protein Bcl-2 involved in cell survival was up-regulated. However, MeHg at higher concentrations disrupted PTEN/Akt/CREB signaling through S-mercuration of CREB. 3) Cd modifies sensor protein HSP90, leading to blockage of its activity to interact with HSF1, thereby substantially activating HSF1 and up-regulating HSP70 associated with quality control of cellular proteins. 4) Persulfides and polysulfides capture MeHg and Cd, resulting in formation of their sulfur adducts with little toxicity.
Keywords: methylmercury, cadmium, covalent modification of proteins, adaptive response, reactive sulfur species
EMERGING AND KNOWN NATURAL TOXINS: ENVIRONMENTAL FATE AND HUMAN RISK
Chair: Aurelia Tubaro1, email@example.com, Co-Chair: Cesare Montecucco2, firstname.lastname@example.org
1Department of Life Sciences, University of Trieste, Via Alfonso Valerio 6, Trieste, Italy, 2Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, Padova, Italy
Natural toxins are secondary metabolites produced by bacteria, cyanobacteria, fungi, algae, plants and animals. Usually, they are produced in proximity of essential resources for humans and animals, such as food, feed and drinking water. However, their entrance in the food web or their effects after different exposure routes does not represent the only sanitary problem since their impact on environment may affect human welfare.
Although some toxins represent a challenge in toxicological studies and in their management due to the limited availability and not always commercially available, several studies allowed a correct risk assessment and management for some toxins. However, there are no analytical methods for many toxins, and no monitoring data and/or regulations notwithstanding their well-documented toxicities. Examples are represented by “emerging natural toxins”, which, in view of climate changes, may open new scenarios from a toxicological point of view. However, the concept “emerging toxins” is quite subjective, being used for new toxins, known toxins appearing in new geographical areas and non-regulated known toxins requiring additional toxicological evidence before establishing regulations. Hence, an update of the state-of-art of emerging natural toxins and their impacts on the environment and humans is necessary to understand and profile the future goals and challenges to be addressed by the scientific community.
This session will focus on multiple aspects of natural toxins, from the novel findings on the biochemical, molecular and clinical effects, to the new methods for their detection, with special emphasis on the emerging species representing the urgency in the near future.
Keywords: human risk, environmental toxicology, natural toxins, human toxicity, monitoring methods
Many Novel Botulinum Neurotoxins
Marco Pirazzini, Ornella Rossetto, Cesare Montecucco
Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, Padova, Italy
Botulinum neurotoxins, produced by anaerobic bacteria of the Clostridium genus, are the most potent toxins known so far, with a MLD50 as low as 10 picograms/Kg. Traditionally seven serotypes were known, with Botulinum Neurotoxin type A known for therapeutic and cosmetic uses. Many novel botulinum neurotoxins are being discovered by NGS, bioinformatics and metagenomics studies. These neurotoxins will be discussed also with respect of the possible identification of novel human therapeutics. Recently, the Weissella oryzae SG25T genome was sequenced and a botulinum neurotoxin (BoNT) like gene was identified by bioinformatics methods. It showed the typical three-domains organization of BoNTs with a N-terminal metalloprotease domain, a translocation and a cell binding domains. The BoNT family is rapidly growing, but this was the first indication of the possible BoNT toxin expression outside the Clostridium genus. Molecular modeling and dynamics simulations show that the 50 kDa N-terminal domain folds very similarly to the metalloprotease domain of BoNT/B, the binding part being different. However, neither the recombinant metalloprotease nor the binding domains showed cross-reactivity with the standard antisera defining the seven BoNTs serotypes. We found that the purified Weissella metalloprotease cleaves VAMP at a single site untouched by the other VAMP-specific BoNTs. This is a unique Trp-Trp peptide bond located within the juxtamembrane segment of VAMP, essential for neurotransmitter release. This study identifies the first non-Clostridial BoNT-like metalloprotease that cleaves VAMP at a novel and relevant site. Further studies are in progress to identify the possible role of this Weissella putative toxin.
Keywords: neuromuscular junction, neurotoxins, peripheral neuroparalysis, metalloprotease
Control of Marine Biotoxins in the European Union: Regulatory Levels and Analytical Methods
Ana Gago Martínez
University of Vigo, Dpt. of Analytical and Food Chemistry and EU Reference Laboratory for Marine Biotoxins (EURLMB), Campus Universitario de Vigo, 36310-Vigo, Spain
Marine biotoxins are natural contaminants with important socioeconomic impact in geographical areas with relevant bivalve molluscs production. Animal assays have been used as reference methods to detect these compounds, but changes in the EU Legislation have been made to replace them by analytical methods. The methodological transition is being challenging due to the lack of experience on these methods as well as of standards/reference materials for methods setting up and their validation. The lack of reference materials has been also responsible for the limited toxicological studies and the total toxicity evaluation using Toxicity Equivalent Factors (TEF), needed to ensure public health. Nevertheless, the application of new reference methods has been generally improved while new advanced ones are being developed for the sensitive detection/quantitation of biotoxins and even for confirmation through High Resolution Mass Spectrometry. Moreover, sensitive alternative methods are being developed for screening purposes. These advances allow us to conclude that we are definitively on a different era in the control of marine biotoxins: new and emerging toxins are appearing in areas where they had not been detected before, and modern analytical techniques are being developed to identify these compounds. From the perspective of an EU Reference Laboratory, these advances are extremely important and show the successful work carried out in this field, but a proper work to reach a harmonized and multidisciplinary response has to be ensured: Analysts and Toxicologists must work close together to ensure an adequate control of marine biotoxins for human health and economic resources protection.
Keywords: marine toxins, new reference methods, screening, control
Cyclic Imines Phycotoxins: Pharmacological Characterization, Biodistribution, Musculo-skeletal Effect and Detection of these Emergent Families of Neurotoxic Agents
Denis Servent 1, Sophie Creuzet 2, Carole Malgorn 1, Vincent Dive 1, Armen Zakarian 3, Romulo Aràoz 1,2, Jordi Molgó 1,2
1 Service d’Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay, F-91191 Gif sur Yvette, France, 2 Institut des Neurosciences Paris-Saclay, UMR 9197, CNRS/Université Paris-Sud, 91191 Gif sur Yvette, France, 3 Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, USA
Cyclic imines produced by various species of marine dinoflagellate microorganisms, constitute a widely distributed group of phycotoxins with increasing prevalence in oceanic environment due to recent climate change. During active dinoflagellate blooms, phycotoxins may accumulate in shellfish tissues and can be transferred into fish, marine mammals and ultimately to humans. Among these phycotoxins, pinnatoxins (PnTx-A to H), produced by the dinoflagellate Vulcanodinium rugosum, were recently identified and shown to exhibit the highest oral acute mouse toxicity among cyclic imine toxins. We will describe binding and electrophysiological experiments highlighting the exceptional ability of these toxins to interact with various subtypes of nicotinic receptors (nAChRs), especially the muscle and neuronal (α7) subtypes. Biodistribution analyses by digital radioimaging in rats, after oral or i.v administration of tritiated PnTx-G, revealed the presence of the toxin in various peripheral organs, as well as in the central nervous system, highlighting its property to cross both the intestinal and the blood-brain barrier. Moreover, using the chick embryo as a first model, we show that during embryonic development, PnTx-A exposure reduces embryo motility by decreasing the embryo spontaneous movements, which affected the maturation of the musculoskeletal system. Finally, in order to be able to detect cyclic imine phycotoxin families in the environment, original receptor-based assays were developed in microplate or lateral flow test formats.
Keywords: pinnatoxins, nicotinic receptors, cyclic imines, V. rugosum
Toxic Effects of Co-exposure to Mycotoxins
Institute for Medical Research and Occupational Health, 10000 Zagreb, Ksaverska c.2, Croatia
Humans and animals are constantly exposed to mycotoxins mostly through the ingestion of contaminated food and feed, although other ways of exposure are possible. Food is usually contaminated with several mycotoxins due to co-contamination by various molds but also because some mold strains may produce various mycotoxins under modified conditions of growth. There are about 400 known mycotoxins that, using very sensitive methods, can be found in food and feed, but only some have been found to have a toxic effect in mammals. Unfortunately, even fewer have known toxicokinetics and mechanisms of toxicity. The mechanisms of toxicity of mycotoxins are rarely studied on experimental animals, while the results of in vitro studies on cell cultures are often contradictory. In the available literature on the effect of co-exposure to mycotoxins either in experimental animals or in in vitro studies, many different end-points were measured which creates difficulties in results interpretation. In animals these were changes in body weight, parameters of kidney, liver and immune system lesions, diuresis, activity of plasma, organs and urine enzymes, teratogenicity and mortality rate. In various cell cultures, cell viability, reactive oxygen species production, apoptosis and parameters of oxidative stress were measured. It was found that the same combinations of mycotoxins may have an antagonistic, additive or synergistic effect, depending on the dose of mycotoxins. Although some mycotoxins in combination with others have always had a clear antagonistic effect at any tested dose, the best protection against them is the prevention of their overall appearance.
Keywords: additive effect, antagonism, mycotoxins, synergism, toxicity
Human Health Risks Associated to Cyanotoxins Exposure
Istituto Superiore di Sanità – Environment and Health Department, Rome, Italy
Since 3.5 billion years ago, when cyanobacteria were already present on the earth, they have colonized almost all terrestrial and aquatic ecosystems, mainly but not exclusively freshwaters, where they can grow up to very high densities, forming blooms and scums. Cyanobacteria produce a high number of bioactive molecules, among which cyanotoxins, such as microcystins (the most studied group), nodularins, cylindrospermopsin and neurotoxins. Increasing occurrence of blooms, in terms of extension and frequency, associated with excess of nutrients due to anthropogenic activities and climate changes, has given rise to some concern for human health and animal life exposed to cyanotoxins. Numerous cases of lethal poisonings have been associated with cyanotoxins ingestion in wild animal and livestock. In humans few episodes of lethal or severe human poisonings have been recorded after acute or short-term exposure, but the repeated/chronic exposure to low cyanotoxin levels remains a critical issue. Indeed, the cyanotoxins known and studied so far have a large spectrum of toxic effects from hepato- and nephro-toxicity, to neurotoxic effects and tumor promotion, depending on the toxin involved and the exposure scenarios. Despite this, data on the kinetic behavior, toxicological profile and exposure levels are still scant and often limited to few variants within each cyanotoxin group. Nevertheless the WHO organization has derived health based values and considerations on the risks for human health can be drawn to protect the potentially exposed populations.
Keywords: cyanobacteria, cyanotoxins, toxicological profile, exposure, risk assessment
MODIFIED MYCOTOXINS – AN EMERGING RISK IN FOOD SAFETY
Chair: Angela Mally1, email@example.com
1Department of Toxicology, University of Würzburg, Germany
Modified (or “masked”) mycotoxins are metabolites of the parent mycotoxin formed in the fungus, in plants or in animals. Modified forms of certain mycotoxins, which are not captured by routine analysis, may therefore be present in food and contribute to the overall mycotoxin exposure. Preliminary exposure estimates for modified Fusarium mycotoxins indicate that human exposure to certain masked mycotoxins may be as high as exposure to the parent mycotoxin. This suggests that current risk assessment and legal limits, which are based on parent compounds only, may underestimate human health risk resulting from dietary intake of mycotoxins. The European Food Safety Authority (EFSA) therefore identified modified mycotoxins as an emerging risk in food safety. Focusing on fusarium mycotoxins and their modified forms as exemplary mycotoxins of key concern, the session will summarize the current knowledge and highlight data gaps regarding the occurrence, exposure to and toxicity of modified mycotoxins. Recent approaches to risk assessment of modified forms of mycotoxins in food and feed will be presented.
Keywords: modified mycotoxins, mycotoxin exposure, food safety, risk assessment
Application of Human Biomonitoring to Assess Human Exposure to Mycotoxins and their Modified Forms
Michele Solfrizzo and Lucia Gambacorta
Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), Via Amendola 122/O, 70126 Bari, Italy
Total mycotoxins exposure in humans could be estimated by the combined measures of urinary free mycotoxins, phase I metabolites and their glucuronides and/or sulphates derivatives. The predigestion of urine with β-glucuronidase/sulfatase enzymes deconjugate the glucuronides and sulphates derivatives to form free mycotoxins and phase I metabolites, thus reducing the number of analytes (mycotoxin biomarkers) to be measured. It has been demonstrated that foods can be frequently contaminated by mixtures of mycotoxins and their modified forms. These last can be hydrolysed in the gut to form the parent mycotoxins. Therefore the analysis of urine for multi-biomarker is a powerful approach to identify the type and the number of mycotoxins ingested by each individual. Moreover, the urinary concentrations of mycotoxin biomarkers can be used to estimate the probable daily intake of those mycotoxins for which human excretion rate is available. We have developed an highly sensitive multi-biomarker LC-MS/MS method based on predigestion of urine with β-glucuronidase/sulfatase enzymes and SPE/immunoaffinity concentration and cleanup for the determination of urinary biomarkers of deoxynivalenol (DON), zearalenone (ZEA), aflatoxin B1 (AFB1), fumonisin B1 (FB1), and ochratoxin A (OTA). These are the main mycotoxins frequently co-occurring in food/beverage worldwide and are mainly produced by Fusarium graminearum (DON, ZEA), Aspergillus flavus and A. parasiticus (AFB1), F. verticillioides and F. proliferatum (FB1), Penicillium verrucosum and A. carbonarius (OTA). Our method was used to analyse 356 human urines collected in Italy, South Africa and Sweden and allowed the identification of mycotoxins mostly ingested in each country as well as the probable daily intake of each mycotoxin.
Keywords: mycotoxin, food, urine, biomarker, assessment
In Vitro and in Vivo Toxicity of Modified Fusarium Mycotoxins: Current Status and Knowledge Gaps
Department of Toxicology, University of Würzburg, Germany
Based on current exposure estimates, it appears that application of the threshold of toxicological concern (TTC) concept, which may be used to assess known structures of unknown toxicity present at very low levels in the diet, may not be applicable to modified mycotoxins. Therefore, in order to reliably assess the contribution of individual modified mycotoxins to health risks related to the presence of modified mycotoxins in food, it is essential to understand both their toxic potential and the extent and form in which modified mycotoxins become bioavailable. Due to the lack of commercially available reference standards, however, only limited data on modified forms of fusarium mycotoxins are available so far. The examples of conjugated forms of zearalenone and deoxynivalenol, and of fumonisin bound to components of the matrix demonstrate that conjugated and matrix-associated mycotoxins may be released in gastrointestinal tract and significantly add to the systemic exposure to the free mycotoxin. While in vitro data indicate that conjugates frequently show lower toxic potential compared to the free mycotoxin, the few data available on in vivo toxicity of selected conjugated forms suggest that conjugates may exhibit comparable in vivo toxicity to that of the parent compound, consistent with their cleavage in the gastrointestinal tract. Importantly, a reductive metabolite of zearalenone, (a-zearalenol), was shown to be 60-times more estrogenic than its parent mycotoxin, indicating a potentially large contribution of some modified forms to the overall health risk.
Keywords: modified mycotoxins, fusarium mycotoxins, toxicity, food safety
Current Approaches to Health Risk Assessment of Modified Mycotoxins in Food and Feed
Nicole Lorenz 1, Sven Dänicke 2, Lutz Edler 3, Christoph Gottschalk 4, Eva Lassek 5, Doris Marko 6, Michael Rychlik 7, Angela Mally 8
1Federal Institute for Risk Assessment (BfR), Max-Dohrn-Str. 8-10, D-10589 Berlin, Germany
2Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, Bundesallee 50, D-38116 Braunschweig, Germany
3German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
4Chair of Food Safety, Ludwig-Maximilians-Universität München, Schönleutnerstr. 8, D‑85764 Oberschleißheim, Germany
5Bavarian Health and Food Safety Authority, Luitpoldstr. 1, D-97082 Würzburg, Germany
6Institute of Food Chemistry and Toxicology, University of Vienna, Währingerstr. 38, A-1090 Vienna, Austria
7Chair of Analytical Food Chemistry, Technische Universität München, Alte Akademie 10, D‑85354 Freising, Germany
8Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany
A systematic definition consisting of four hierarchic levels introducing the term “modified mycotoxins” for all mycotoxin compounds that are altered in their chemical structure compared to the corresponding parent compound was proposed in order to encompass all possible forms in which mycotoxins and their modifications can occur. Potential exposure to modified mycotoxins due to their presence in food and feed has raised concern that modified mycotoxins may pose a substantial additional risk to human and animal health.
Considering the range of modified forms of zearalenone (ZEN) detected in food and feed, the endocrine activity of several of these metabolites and the fact that estimated dietary exposure to free ZEN alone is already close to the tolerable daily intake, ZEN is used as an exemplary mycotoxin of key concern. Recently published scientific opinions of the European Food Safety Authority (EFSA) are taken as starting point for the evaluation of the current situation and identification of data gaps for a comprehensive health risk assessment of ZEN and its modified forms.
The development of sufficiently sensitive analytical methods for the detection of modified mycotoxins, the application of these methods to generate reliable occurrence data, the reduction of uncertainties in the toxicology of modified mycotoxins, the derivation of appropriate health-based guidance values, the utilization of biomonitoring data to reduce uncertainties in the exposure assessment as well as the development of strategies for health risk assessment of chemical mixtures were identified as key challenges which will be presented along with recommendations for further research.
Keywords: zearalenone, oestrogenicity, dietary exposure, biomonitoring, chemical mixtures
APPLICABILITY AND LIMITATION OF NON ANIMAL TESTING IN SAFETY ASSESSMENT
Chair: Emanuela Corsini1, email: firstname.lastname@example.org
Co-Chair: Emanuela Testai 2, email: email@example.com
1Università degli Studi di Milano, Milan, Italy 2 Istituto Superiore di Sanità, Rome, Italy
A fundamental change occurring in toxicity testing to improve efficiency and human relevance in chemical safety assessments is the use of testing strategies, integrating human cell-based in vitro assays, including advanced models such as the microfluidic systems, and new predictive computational tools. This allows to move away from animal-based risk assessment strategies and default factors used for extrapolating to humans and to meet many pieces of regulation in Europe, asking for reduction in animal testing.
The session will present consolidated and emerging methodologies and how industry, regulatory agencies and academy are facing the use of non animal testing methods, highlighting the successful cases in which their applicability has been already accepted, the limitation(s) of their use in other cases and the challenges for the future regarding the increase in their predictivity of in vivo effects in response to chemical exposures.
Keywords: safety assessment, in vitro methods, in silico methods, regulatory acceptance
Think-exposure-first! Industry Perspective on Predictive Tools for Exposure-based Safety Assessment
‘Dow Agrosciences, UK’
In the global agrochemical regulatory testing programme, risk assessment is driven by “external doses” administered to 4 animal species used (humans clinical testing is not currently possible). Despite not strictly required for registration, translational evaluation of internal exposure alongside toxicodynamic characterisation has been supported by numerous publications and regulations. Introductory traditional examples from animal programs will be given for species sensitivity/human relevance characterisation and dose selection, using more relevant kinetically-derived maximum doses (not MTDs).
A toolbox of non-animal ADME models has been developed for decades in the pharmaceuticals regulatory packages and is further developing with the changes in the cosmetic industry regulation. These tools provide unique opportunity to characterise internal exposure and inter-species toxicokinetic differences. In addition, some of the more complex predictive tools are also being characterised by regulatory agencies for predictivity of target-organ toxicodynamic, with a variety of complex endpoints such as high content or toxicogenomic techniques. The usefulness of these tools for the characterisation of the human relevance of toxicology findings and factors influencing the regulatory uptake will be discussed.
Current and future applications include the use to design more sustainable “green chemistry” in the R&D agrochemical process and the potential for a shift towards (internal) exposure-based safety assessment, already in place in other sectors of regulated industries.
Keywords: non-animal ADME models, toxicogenomic techniques, regulatory acceptance
Regulatory Use of Non Animal Models: the Present and the Future Challenges
Istituto Superiore di Sanità, Rome, Italy
The currently hazard-driven approach in toxicological risk assessment has strong reliance on animal testing. However, as a consequence of advances in scientific knowledge, modelling and measuring techniques, the emphasis of the scientific community is moving towards integrating in vitro human cell-based assays, imaging and highthrouput methodologies (including ‘-omics’ and microfluidic organ-on-a-chip) with computational modeling, with a shift to an exposure-driven, more mechanism-based paradigm, with identification of AOP.
Since regulators demand validated and internationally accepted non-animal methods, during the last two decades, a number of in vitro methods were submitted to international validation bodies. This exercise showed that most of the available in vitro and in silico methods still require considerable improvements in design, robustness and reliability to generate data sets useful to support regulatory decisions, the major issues being the establishment of in vitro preparations preserving in vivo original properties for prolonged periods of time, the biokinetics measurement and the establishment of in vitro biomarkers of adversity. EURL ECVAM is currently coordinating the issuing of a guidance on Good In Vitro Method Practices (GIVIMP). ECHA is working on a ‘Report on the regulatory applicability of alternative and non-animal approaches’.
Some animal-free tests have been already adopted as Guidelines and accepted by regulators in different areas (e.g. some non-testing methods such as TTC or read across, and some genotoxicity tests, dermal absorption, skin sensitization testing) and others are included in the data requirements by regulatory authorities (e.g. the in vitro comparative metabolic profile of pesticide active substance asked by EFSA).
Keywords: non animal testing, alternative methods, regulatory acceptance
Applicability of Non-animal Based Tools for Food and Feed Safety Assessment
Application of non-animal based tools in food and safety is of critical importance both to reduce animal testing and to apply predictive tools dealing with data poor chemicals. In 2017, EFSA published OpenFoodTox: its open source database which provides summary hazard data for more than 5000 chemicals in the human, animal and ecological risk assessment (HRA, ARA, ERA). From openfoodtox and other relevant databases (US-EPA terrestrial database, Fraunhofer RepDose), a number of predictive QSAR models have been developed. For HRA, these include continuous QSAR models predicting NOAEL values in rats for general sub-chronic toxicity and sub-chronic liver toxicity. For ARA and ERA, models include a continuous one for acute contact toxicity in trout and a classification model predicting acute toxicity in honey bees were.
Finally, generic toxicokinetic (TK) models were developed for HRA, ARA and ERA using physiological, metabolism data and their use has been illustrated for a number of single and multiple chemicals. These TK tools provide a means to further integrate exposure, TK processes and toxicity through quantitative modelling of inter-species differences and inter-individual differences.
The future of open source mechanistic tools as alternatives to animal testing is discussed including in silico and in vitro tools and tiered weight of evidence approaches tailored to support risk assessment. International cooperation between national and international scientific advisory bodies and academic institutions concludes as the corner stone for the translation of 21st century toxicological research into harmonised methodologies and tools and for the training of the next generation of risk assessors.
Keywords: food, feed, in silico methods, harmonised methodologies
History of a Success: Contact Hypersensitivity
ESP, Università degli Studi di Milano, Italy
Over the last two decades, an incredible progress has been made in the field of in vitro immunotoxicology, in particular, in the area of contact hypersensitivity. Several in vitro methods to support the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in combination with other complementary information (i.e. in the context of an Integrated Approach for Testing and Assessment, OECD guidance document No. 256) have been developed. These methods have been formally validated and OECD guidelines are available. In addition to the already validated tests, several other methods will be soon validated for skin sensitization. Currently validated methods are useful for hazard identification, classification and labeling. With no doubt, contact hypersensitivity represents a real success in the field of alternative methods to the use of the animals: from the description of the first Adverse Outcome Pathway to the validation of several in vitro methods, this field of research is a history of a success. However, it is important to remember that to achieve a complete replacement of animals in skin sensitization assessment, dose-response information and evaluation of relative skin sensitizing potency to support effective risk assessment are necessary. In this presentation, the state-of-the-art in the field of in vitro assessment of skin sensitization and the in vitro identification of the no induction sensitization level for contact sensitizers will be discussed.
Keywords: skin sensitization, integrated testing strategy, effective risk assessment
Future of Non Animal models: 3D Models and Human on a Chip
CAAT, Baltimore, USA
The increasing doubt into the value of animal models given their ethical and pecuniary costs and the concomitant emergence of predictive non-animal models have brought us to a tipping point, where new approaches are more and more dominating life science research. Objective assessments of the reproducibility of animal studies even under quality-assurance by Good Laboratory Practice are showing tremendous problems contributing to the reproducibility crisis perceived in science. In recent years, for example, pharmaceutical companies have dramatically reduced animal usage while shifting to novel tools. Four trends contributing to this paradigm change will be discussed: (1) the favoring of mechanistic assays, (2) the generation and mining of big data, (3) the creation of more relevant organotypic cell cultures (microphysiological systems) and (4) the integration of different information sources in Integrated Testing Strategies, Systems Biology approaches and Systematic Reviews (evidence-based approaches).
Keywords: organ on-a-chip, adverse outcome pathway, big data, systematic review
ENVIRONMENTAL POLLUTION AND TOXIC OUTCOMES: DOSES, MOLECULAR BIOMARKERS, AND ASSOCIATIONS
1Ege University, Faculty of Pharmacy, İzmir, Turkey
2Retired, Gazi University, Faculty of Pharmacy, Ankara, Turkey
Environmental pollution is a growing problem all over the world. Various chemicals at different emission rates enter one or more environmental compartments by industrial, agricultural and/or household usage, as well as by road traffic. These chemicals directly and/or indirectly affect wildlife, and eventually reach humans by atmosphere, water, and food chain. There have been epidemiological, as well as experimental findings that they may trigger several pathophysiological mechanisms and cause various diseases. The possible link between persistent organic pollutants, metals, therapeutic agents and diseases and proposed hypotheses will be thoroughly discussed and recent data on this field will be presented in this symposium.
Key words: Environmental pollutants, exposure, disease, human, biomarker
Kidney, Breast and Stomach Cancers in Relation to Internal Concentrations of Persistent Organic Pollutants, DNA Damage Markers and Related Polymorphisms
Hilmi Orhan 1, Sinan Süzen2, Rasih Kocagöz1, İlgen Onat1, Merve Demirbügen2, Burak Turna3, Koray Atilla4, Levent Yeniay3, Banu Sarsık3, Osman Zekioğlu3, Murat Özdemir3
1Ege University, Faculty of Pharmacy, 2Ankara University, Faculty of Pharmacy, 3Ege University, Faculty of Medicine, 4Dokuz Eylül University Faculty of Medicine, İzmir, TURKEY
Environmental pollution has been linking to various diseases including cancers since soon after the industrial revolution. Among the numerous synthetic pollutants, organochlorine pesticides, polychlorinated biphenyls and polybrominated diphenyl ethers are the most concerned ones because of their chemical stability and lipophilicity, and called “persistent organic pollutants; POPs”. In majority of the human studies on the link between POPs and cancers, exposure data have been based on self-declaration questionnaire, which potentially diminish the accuracy and reliability of quantitative assessment. In order to accurately assess whether environmental exposure to POPs increase risk of kidney, breast and stomach cancers, tumour tissues as well as related specimen were collected from surgically operated patients. Cellular DNA and protein oxidative damage markers (8-OHdG and dityrosine, respectively), have also been analysed in the patients and healthy control group, and assessed whether there are changes in these parameters because of the disease. The preliminary data suggest that tissue and blood POP concentrations were, although weakly, associated with cancers in patients. DNA and protein damage was found to be higher in patients compared to healthy volunteers, although inter-individual variability and sample size analysed so far prevented statistical significances. Current findings confirmed that glutathione S-transferase theta null-polymorphism is a risk factor for kidney tumours. Data suggest that environmental exposure to POPs may induce tumour initiation and/or progression in humans.
The study is supported by The Scientific and Technical Research Council of Turkey (TUBITAK, grant SBAG-114S310).
Key words: Persistent organic pollutants, cancer, human, DNA damage, biomarker
Drugs and their Metabolites as Environmental Pollutants
Momir Mikov1,3, Svetlana Golocorbin-Kon2
1 School of Pharmacy, Faculty of Medicine, University of Banja Luka, Bosnia and Herzegovina, 2Faculty of Medicine, Department of Pharmacy, University of Novi Sad, Serbia
3Faculty of Medicine, Department of Pharmacology and Toxicology, University of Novi Sad, Serbia
Drugs enter into the environment through various routes. The general perception is that drugs in the environment are the result of inappropriate disposal of expired and/or unused drugs. The reality is that the most drugs detected in the environment are the result of their therapeutic use in humans and animals. When drugs finish their therapeutic role they take another role as environmental pollutants.
Ingested or injected drugs are excreted unmetabolized via the urine and feces or as active or inactive metabolites. Domestic sewage is a dominant and hospital sewage is a secondary source of environmental pollution with drugs and their metabolites. Drugs and their metabolites can escape degradation in standard sewage treatment facilities. The metabolites, especially their conjugates can be transformed to the parent drug. Many questions regarding environmental pollution are unanswered like: what is the impact of the short term and long term of low levels of drugs, what is the impact of the exposure to the mixtures of drugs, their metabolites and chemicals, what populations are most vulnerable like elderly, young, pregnant women, disease, what is the significance for the animals and plants which are used as food?
Treatment of patients (human and animal) with drugs is beneficial to them, but its role as environmental pollutant contributor is underestimated. The evaluation of the risk of drugs and their metabolites should be a part of their drug evaluation process before drug registration. The implementation of the system of ecopharmacovigilance is crucial for the monitoring and intervention regarding their impact on the environment.
Keywords: drugs as contaminants, ecopharmacovigilance
Neurodegenerative and Neurodevelopment Disorders Linked to Chemicals: What are the Underlying Mechanisms?
Antonio F. Hernández 1, Fernando Gil1, Beatriz González-Alzaga2,3, Clemente Aguilar-Garduño3, Marina Lacasaña2,3,4
1 University of Granada School of Medicine, 2Andalusian School of Public Health, 3ibs.GRANADA, Spain. 4CIBERESP, Spain;
The developing brain is much more susceptible to toxic injury than the adult brain. The developmental origin of health and disease hypothesizes that the environment during fetal and childhood development affects the risk for many chronic diseases in later stages of life, including neurodevelopmental disorders. Expose to environmental factors, such as neurotoxicants, mental stress and malnutrition during fetal and neonatal periods can induce epigenetic alterations of the offspring. Alternatively, these exposures can disrupt the normal function of neurotransmitter receptors during developmental periods, affecting the mechanisms driving neural progenitor cell proliferation, migration, differentiation, neurite outgrowth, dendritic maturation, synaptogenesis, and apoptosis, eventually leading to neurodevelopmental disorders. On the other hand, epidemiological studies have identified exposure to neurotoxic substances as a risk factor for various neurodegenerative diseases, particularly in genetically vulnerable people as a result of gene–environment interactions. The underlying pathogenic mechanisms include oxidative stress, mitochondrial dysfunction, epigenetic modifications, impairment of the ubiquitin proteasome system (UPS) and deregulated autophagy. Many environmental factors can produce highly reactive molecules in nerve tissue or affect antioxidant defense mechanisms, leading to oxidative protein modifications, oxidative modification in the mitochondrial DNA, mitochondrial dysfunction and neuronal death. Insufficient cellular repair mechanisms may contribute to premature aging and neuronal apoptosis. Moreover, epigenetic modifications play a pivotal role in the epigenetic regulation of gene expression and many other cellular events, including growth, differentiation, development, learning and memory, and apoptosis. Defects in the UPS and autophagy lead to the accumulation and aggregation of toxic proteins, which may eventually result in neurodegeneration.
Keywords: neurodegeneration, neurodevelopment, mode of action, mechanisms of action, toxicodynamic
Cadmium Modulation of Immune Defense and Susceptibility to Inflammatory Diseases: Insight from Animal Models
Milena Kataranovski 1,2
1Department of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
2 Department of Ecology, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Despota Stefana Boulevard 142, 10000 Belgrade, Serbia
Cadmium (Cd) is important food and water contaminant, which allocates gastrointestinal tract as target for its toxicity. The effect of 30-day oral intake of 5 ppm and 50 ppm of Cd to gut immune reactivity in DA and AO rats (which accumulate similar levels of Cd in the intestine) was assessed. More pronounced intestine damage, inflammation, along with reduction of commensal bacteria of Lactobacillus strain, were noted in DA rats only. Intestinal damage and inflammation was seen in cadmium-exposed AO rats, but immune priming of major gut-associated (mesenteric) lymph nodes (MLN) was absent. Immune priming of MLN [(increase of cell proliferation, inflammatory cytokine (IFN-γ and IL-17) production] in DA rats, is devoted to protect vulnerable intestine from bacterial overgrowth. However, activity in this otherwise tolerogenic lymphoid microenvironment might be an introduction to perturbation of immune-mediated homeostasis in the gut, making MLN a player in the disease pathogenesis. Systemic immune-modulatory effects of oral cadmium intake in rats were also seen in the skin, barrier tissue responsible both for immune defense and immune tolerance to commensal microorganisms. Neutrophil infiltration, mast cells hyper-granulation and change in inflammatory (IL-6, TNF and IL-1) epidermal cell cytokine production and their responsiveness to commensal Staphylococcus epidermidis were observed in the skin of DA rats which consumed cadmium. This depicted skin not only as a place of cadmium accumulation, but also indicate impact on tissue immune homeostasis. Both local and systemic immune-toxic effects should be taken into account when assessing dietary cadmium as health risk factor.
Keywords: oral cadmium, gut inflammation, gut-associated lymphoid tissue, skin immunity
Environmental lead exposure in children: a problem of developing countries?
Petar Bulat 1, 2, Stefan Mandić-Rajčević 3, Zorica Bulat 4, Vesna Matović 4
1 University of Belgrade, Faculty of Medicine, Belgrade, Serbia, 2 Serbian Institute for Occupational Health “Dr Dragomir Karajović” Department of Toxicology, Belgrade, Serbia, 3 Department of Health Sciences of the University of Milan and International Centre for Rural Health of the San Paolo Hospital, Via San Vigilio 43, 20142 Milan, Italy, 4 Department of Toxicology “Akademik Danilo Soldatović,” University of Belgrade – Faculty of Pharmacy, Serbia
Environmental exposure to lead, although not an important cause of mortality, represents one of the main causes of morbidity among children and adolescents. In general, rural communities are expected to have significantly lower blood lead levels (BLLs) than urban communities. However, this is not the case in populations living in the vicinity of lead mines and smelting facilities, where higher BLLs may occur, particularly among young children. Around 50% of Global lead production can be traced back to car battery recycling.
The aim of this study was to quantify blood lead levels (BLLs) of children living near a car battery smelting facility in Serbia, compare the levels with developed and developing countries’ standards, and identify the main determinants of lead exposure in this population. BLLs were quantified in 75 children from Zajača, a village where a car battery smelting factory is located, and 52 children from Paskovac, village 5 kilometers away from Zajača. The median BLL for both groups were 12 μg/dl, 7.60 μg/dl in children from Paskovac, and 17.5 μg/dl in children from Zajača. Even 87% of children from Zajača had the BLL above 10 μg/dl, which is comparable to urban schoolchildren in South Africa and Bangladesh. Although a European country, a candidate country for the European Union, BLLs of Serbian children were comparable to that of children living in developing countries and higher than those expected in children living near lead smelting facilities in developed countries.
Keywords: combined exposure, blood lead levels, mixed-effects model
EVALUATION OF SAFETY PROFILE OF HERBAL PRODUCTS
Chair: Ahmet Aydın¹, firstname.lastname@example.org; Co-Chair: Nan Mei², email@example.com
¹Department of Toxicology, Faculty of Pharmacy, Yeditepe University, Turkey; 2Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration (FDA), United States.
Herbal supplements and plants have a long history in many cultures for use in heath improvement and treatment of various illness and ailments. Their long term use is a guaranty of their safety according to the international regulatory documents, although there are some concerns on their safety profile. The complex chemical nature of herbal products makes it difficult to evaluate their efficacy and safety. Published international regulatory issues about herbal products were discussed from safety concern. The toxicological data on some herbal products have been reviewed. Interaction is one of the important topics about herbal use. Possible interactions and endpoints have been investigated during co-administration of herbal product and other therapeutic drugs. Among the toxicological endpoints, genotoxicity attracts great attention because of direct relationship between genotoxicity and carcinogenic development. Evaluation of mutagenic and genotoxic effects of herbal formulations in multiple test systems will be discussed in this session. Threshold for toxicological concern (TTC) is an efficient regulatory tool for risk assessment. This TTC approach, along with innovative applications, will be discussed in order to conduct risk assessment of the genotoxic compounds found in herbal drugs. As an example for genotoxicity evaluation of herbal products, goldenseal, which is available in a wide array of herbal products on the international market and has been used for the treatment of a wide variety of ailments, has been studied for their carcinogenicity potential and mechanism of action. Recent studies suggest that topoisomerase II inhibitory effect might contribute to its carcinogenic development.
Keywords: regulatory issues, threshold for toxicological concern, genotoxicity, interaction, goldenseal
Safety Evaluation of Herbal Products from the View Point of Regulations
Department of Toxicology, Faculty of Pharmacy,Yeditepe University, Turkey, Correspondence: firstname.lastname@example.org
Herbal products have been used for years in the world for their different health benefits. However their long term use is a guarantie of their safety according to the regulatory documents there are some missing information on their safety profile. Many of the responsible authorities indicates that in case of enough data animal use for same type of toxicity test should be restricted. According to the European Medicine Agency (EMA), if a herbal product has been used in the world for 30 years and at least 15 years in the Europe there is no need to carry out general toxicity tests except for developmental and reproductive toxicity, genotoxicity, carcinogenicity and toxicokinetic data. Especialy genotoxicity test data should be available for many of the herbal products due to the direct relationship between genotoxicity and carcinogenesis development. Even if they are used in the world for a long time there is generaly a gap at these type of data. The published regulatory documents, mainly EMA documents, were reviewed and present situation was discussed. Missing information on herbal products should be complated for the need for further evaluation of herbal products.
Keywords: regulatory issues, long term use, genotoxicity
Toxicological Endpoints of Herbal Product-Drug Interactions
Nurşen Başaran1, Merve Bacanlı1, A. Ahmet Başaran2
1Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Toxicology 06100, Ankara, Turkey, 2Hacettepe University Faculty of Pharmacy Department of Pharmacognosy 06100, Ankara, Turkey
In recent years, because of the belief of herbal medicines are safe in the treatment or prevention of diseases and the protection of the overall health status, their usage has been increasing. However, as the contents of herbal medicines have many bioactive components, the lack of sufficient study on their efficacy and toxicity, inadequate controls of their availability reduce their safety. Many medicinal herbs and pharmaceutical drugs can be therapeutic at one dose and toxic at another. Interactions between herbs and drugs may increase or decrease the pharmacological or toxicological effects of the active component. In addition, patients with chronic diseases who also use herbal medicines must consider the adverse effects and interactions of these substances. The herbal medicines contain substances which can induce or inhibit enzymes that can take part in drug metabolism. Particularly, drugs with narrow therapeutic index (warfarin, digoxin, etc.), and many plants which affect drug metabolizing enzymes (Hypericum perforatum, Panax ginseng, Ginkgo biloba etc.) when they used together, may lead to undesirable results. The concurrent use of these drugs wih some medicinal plants can cause serious adverse effects and also the decrease of the efficacy of the therapy. In order to prevent unwanted medicine-herbal drug interactions, it is important to have more information about this area and patients using herbal medicines must consult with their physician and pharmacist.
Keywords: herb, drug, interaction, toxicity
Evaluation of Mutagenic and Genotoxic Effects of Different Herbal Formulation in Multiple Test Systems
Ashok K. Giri
Molecular Genetics Division, Indian Institute of Chemical Biology, Kolkata-700032, India
Different herbal formulation including herbal tea, herbal health drink and black tea are more popular throughout the world. More than 100 varieties of herbal tea presently consumed by human being in the different parts of the world. As per WHO, OECD or the individual countries guide lines, it is mandatory that all herbal products must be evaluated for their toxic and mutagenic effects before it can be used for human consumption. It is also important to evaluate its potential for animutagenic effects so that it may evantually help us to find out the best suitable herbal formulation with antimutagenic effects which can be used as anticancer drug in near future. We have been working on the antimutagenic and anticancer effects of Indian black tea and its polyphenols and Turkish black tea in multiple test systems. A significant decrease in the mutagenic effects were observed in the Salmonella assay when black tea and its polyphenols were used against known mutagenic compounds. Similarly a significant decrease in the mutagenic effects of Turkish black tea were observed both in vivo and in vitro against known positive mutagens. These results clearly indicates the antimutagenic effects of these tea in both in vivo and in vitro. Black tea and its polyphenols also showed significant anticancer activities in vitro. Details of these results will be discusseed during the lecture. Thus the overall results indicates that both black tea and its polyphenols and Turkish tea showed significant antimutagenic and anticancer activities both in vivo and in vitro.
Keywords: herbal tea, black tea, Turkish tea, antimutagenic and anticancer effects
Mechanistic Study of Goldenseal-Associated Genotoxicity
Nan Mei¹, Si Chen², Lei Guo²
¹Division of Genetic and Molecular Toxicology, ²Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States.
Goldenseal (Hydrastis canadensis) is available in a wide array of herbal products on the international market, and it has been used for the treatment of a wide variety of ailments. The five major alkaloid constituents in goldenseal are berberine, palmatine, hydrastine, hydrastinine, and canadine. When goldenseal was evaluated by the National Toxicology Program (NTP) in the standard 2-year bioassay, goldenseal induced an increase in liver tumors in rats and mice. It is of great interest to investigate the potential mechanisms that may contribute to its carcinogenicity. In this study, the toxicity of the five goldenseal alkaloid constituents was characterized, and their toxic potencies were compared. As measured by the Comet assay and the expression of γ-H2A.X, berberine, followed by palmatine, appeared to be the most potent DNA damage inducer in human hepatoma HepG2 cells. Berberine and palmatine suppressed the activities of both topoisomerase (Topo) I and II. In berberine-treated cells, DNA damage was shown to be directly associated with the inhibitory effect of Topo II, but not Topo I by silencing gene of Topo I or Topo II. In addition, DNA damage was also observed when cells were treated with commercially available goldenseal extracts and the extent of DNA damage was positively correlated to the berberine content. Our findings suggest that the Topo II inhibitory effect may contribute to berberine- and goldenseal-induced genotoxicity and tumorigenicity. The use of goldenseal or berberine products may be not without risk due to this “off-target” toxicity.
Keywords: goldenseal, berberine, DNA damage, Comet assay, γ-H2A.X
FROM ASSESSMENT OF INTERNAL EXPOSURE TO CHEMICALS TO ACTION TO PREVENT ADVERSE HEALTH IMPACTS: THE ROLE OF HUMAN BIOMONITORING
Chairs: Dorota Jarosinska, WHO Regional Office for Europe, WHO European Centre for Environment and Health, email@example.com; Ashok Kumar Giri, Molecular Genetics Division, Indian Institute of Chemical Biology, Kolkata-700032, India, firstname.lastname@example.org
Human biomonitoring (HBM) studies are an important means for assessing exposure to chemicals of public health concern and linking this to disease outcomes. The development of harmonized approaches to HBM will enlarge and strengthen the evidence base on exposures, and expand our capacities to investigate the role of chemicals in the origin of disease and developmental disorders. The use of HBM in birth cohort studies is providing valuable data on the link between early life exposures to certain chemicals and the development of disease later in life. The symposium will discuss progress in the development of harmonized approaches for HBM studies, the opportunities that this presents and also the challenges and limitations.
Keywords: human biomonitoring harmonized approach; early life exposure; diseases
Harmonization of Approaches to Human Biomonitoring: Benefits and Challenges
Irina Zastenskaya, Dorota Jarosinska, Elizabet Paunovic
WHO Regional Office for Europe, WHO European Centre for Environment and Health
Human biomonitoring (HBM) is a reliable tool for assessment of human exposure to hazardous chemicals. HBM provides information on cumulative exposure to chemicals from different sources, opens opportunities for investigating links between exposure and health effects, allows assessing geographical and temporal trends of exposure, and provides the basis for cost-effectiveness and social-economic impact analysis. But HBM isn’t applicable for all chemicals of interest and for monitoring of sources of exposure. Ideally, HBM and environmental monitoring should complement each other. Harmonization of approaches to HBM allows getting comparable data, which enable monitoring of geographical distribution of exposure, collecting knowledge on sources of chemicals to humans, prioritizing chemicals of public health concern, and identifying the effective risk reduction measures. In addition, the WHO recommendations on HBM create opportunities for setting national biomonitoring programs, especially in developing countries.
The first experience of applying a harmonized approach to HBM comes from a WHO/UNEP survey of POPs in breast milk. Conducted in the framework of the Stockholm Convention, the survey provides valuable data on distribution of POPs globally, effectiveness of preventing measures, and allows identifying hotspots, also unexpected.
Another successful experience is the development of a harmonized approach to assessment of prenatal exposure to mercury. Lessons learned from both initiatives show challenges in harmonizing all components of HBM and the need to adapt the WHO recommendations to specificities and capacities in countries. In certain areas, such as methods for analysis of chemicals in biological matrices, harmonization is challenging due to differences in analytical capacities.
Keywords: human biomonitoring, exposure, hazardous chemicals, harmonization, mercury
Towards Expanding the HBM in Europe – the European Human Biomonitoring Initiative HBM4EU
Argelia Castaño 1, on behalf of the HBM4EU Initiative 2
1 National Center for Environmental Health (CNSA). Institute of Health Carlos III (ISCIII). Majadahonda. Madrid, 2 https://www.hbm4eu.eu/
Humans are exposed to a wide range of chemicals in their daily life at the workplace. Exposure to some of those chemicals can seriously damage human health. In order to effectively tackle chemical risks, policies must be grounded in a robust scientific understanding of the chemical exposure and the potential impacts on human health.
HBM measures chemicals or their metabolites in the human body. It provides an integrated measure of the level of exposure to chemicals independently of their source and considering individual susceptibility. As such, HBM is an important tool for assessing exposures of the human population to chemicals, and estimating potential health risks linked to the exposure. Analysed over time, HBM data allow evaluation of trends in exposure and can be used to assess the efficiency of implemented policies.
HBM4EU is a joint effort of 28 countries, the European Environment Agency and the European Commission, co-funded under Horizon 2020. The main aim of the initiative is to coordinate and advance human biomonitoring in Europe. HBM4EU will provide better evidence of the actual exposure of citizens to chemicals and the possible health effects to support policy making. HBM4EU will run for five years, from 2017 to 2021, building on previous activities undertaken at EU and national levels. The HBM4EU initiative represents a novel collaboration between scientists and chemical risk assessors and risk managers, including several Commission services, EU agencies and national representatives. The project will build bridges between the research and policy worlds and deliver benefits to society in terms of enhanced chemical safety.
Application of a harmonized approach to HBM: insights from a multi-country project on mercury
Irina Ilchenko1, Tatiana Boyarskaya1, Kamila Timoshenko1, Sergey Lyapunov2, Olga Okina2
1I.M. Sechenov Moscow State Medical University (Sechenov University), 2Institute of Geology Russian Academy of Sciences
Mounting evidence confirms that mercury affects the developing brain at early life. This human biomonitoring (HBM) survey in Russia, aiming at assessment of prenatal exposure to mercury in Republic of Karelia (RK) and its determinants, was conducted using the WHO methodology including the survey protocol and standard operating procedures for sampling of biological matrices and mercury analysis. Some amendments were required to apply the WHO harmonized procedures in the country.
In cross-sectional survey, 252 women from RK were examined. Epidemiological information was obtained using the WHO questionnaire. Mercury level in hair and cord blood was assessed using AAS with cold vapor. One-way ANOVA was used to compare mercury levels with SES trajectories in the region. Determinants of exposure, social gradients in exposure were examined with multiple and logistic regression.
The geometric mean for mercury was 0.534 µg/g in maternal hair, 2.29 µg/L in cord blood. Among the most important determinants of exposure to mercury were municipality and urban vs rural residency as well as maternal education level. There was a significant effect of the education level on concentrations of mercury in hair of women (F=4.30; p<0.01), on mercury in cord blood (F=4.74; p<0.01; urban/rural residence for mercury in hair (F=6.68; p<0.01).
The survey demonstrated applicability of the WHO harmonized approach. The findings provide comparable exposure data, which might be useful for long-term mercury HBM. Results also illustrate the magnitude and distribution of inequality in mercury exposure within Karelia and help to identify target groups and prioritize areas for intersectoral action.
Keywords: mercury, human biomonitoring, prenatal exposure, Republic of Karelia
Role of HBM in Managing Contaminated Sites: Exposure to Lead Close to Antimony and Lead Mining and Metal-processing Complex in Serbia
Branislava Matic1, Igor Dragičević2
1Institute of Public Health of Serbia, Belgrade, Serbia 2Institute of Public Health of Šabac, Šabac, Serbia
Objective of this cross-sectional study was assessing exposure to lead and its dynamics through examining blood lead levels (BLL) in children, living in the vicinity of the Zajača antimony and lead mining and metal-processing complex, as compared to partially exposed (Paskovac), and non-exposed (Gornja Borina) population.
Two cross-sectional studies were conducted in 2012 and 2013. Atomic absorption spectrometry was used to determine lead concentration in blood. To assess the statistical significance of mentioned comparisons we used univariate methods and the non-parametric tests for the attributable variables: Hi-square test, proportion test. To test significance of the difference between non-parametric variables, we used: Kolmogorov-Smirnov Z test for testing distribution’s normality, ANOVA for the normal distribution, and Kruskal Wallis test in cases without normal distribution.
142 participants were tested on lead in blood: exposed (Zajača, 58 children, 18 adults), partially exposed (Paskovac, 38 children), and non-exposed (Gornja Borina, 30 children). Mean BLLs in children living less than 1 km from the smelter was 18.98 µg/dl (SD 6.06), at the first test series (year 2012), and 12.21 µg/dl (SD 6.41) in the second round (year 2013). For those living further than 3 km mean BLLs at first series was 8.30 µg/dl (SD 1.51), and at the second series it was 5.85 µg/dl (4.39).
Blood lead levels in children from Zajača are high and risk reduction measures are necessary to minimize the exposure. Due to the complex pattern of multi-source exposure to lead, cadmium and arsenic, abatement and further monitoring are needed at the site to assess the effectiveness of risk reduction measures, primarily, continuous HBM.
Key words: human biomonitoring; blood lead levels; exposure; children, lead smelter
THE SIGNIFICANCE OF DRUG/XENOBIOTIC METABOLIZING ENZYME POLYMORPHISMS IN CANCER/DISEASES
Chairs: Mumtaz Iscan1 email: email@example.com, Ann K. Daly2 email: firstname.lastname@example.org
1 Department of Toxicology, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey,2 Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK
Cancer is the worldwide leading cause of mortality. In addition, neurodegenerative diseases, like Parkinson’s disease (PD), are also known to be very important health problem. Besides inter-individual differences that are important in susceptibility to these diseases, great inter-individual differences in response to therapy treatment also occur among these patients. The biotransformation enzyme systems inactivate environmental carcinogens and certain drugs used in therapy depending on their genetic polymorphisms. Therefore, it is important to identify individual genetic factors that modify such diseases prognosis in order to develop preventive and therapeutic strategies.
- Simic will focus on whether common GST polymorphisms confer the risk and the progression of malignant (clear renal cell carcinoma, ccRCC) and non-malignant (end stage renal disease, ESRD) renal diseases.
A.O. Ada will present their recent findings with respect to the association between non-small cell lung cancer (NSCLC) risk and polymorphisms of CYP2E1 and GST genes encoding these xenobiotic metabolizing enzymes and TP53.
A.K. Daly will report the associations of CYP polymorphisms with risk of various cancers including lung, liver, colon and nasopharyngeal based on recent results of both meta analyses and genome-wide association studies.
I.Novakovic will present their recent data on the association between polymorphisms in genes encoding for brain derived neurotrophic factor (BDNF), apolipoprotein E (APOE) and catechol-O–methyltransferase (COMT) and response to therapy and adverse outcomes in PD patients.
M.Iscan will provide their recent findings with respect to the role of CYP and GST gene polymorphisms in response to chemotherapy and survival in NSCLC patients.
Keywords: risk, prognosis, polymorphisms, CYPs and GSTs, COMT
Role of GST Polymorphisms in Malignant and Non-malignant Diseases of the Kidney
Institute of Medical and Clinical Biochemistry, Faculty of Medicine, University of Belgrade, Belgrade, Serbia
Almost all members of the mammalian cytosolic glutathione transferases (GSTs) exhibit genetic polymorphism, resulting in inter-individual differences in GST isoenzyme profile that might affect both biotransformation and antioxidant capacity. Therefore, we aimed to discern whether common GST polymorphisms confer the risk and the progression of oxidative stress associated malignant (clear renal cell carcinoma, ccRCC) and non-malignant (end stage renal disease, ESRD) renal diseases. GST genotypes were determined by various PCR methos whereas oxidative stress byproducts and soluble adhesion molecules were measured using ELISA method. GSTP1*Val (variant) and GSTM1-null genotypes showed a significant individual association with ccRCC risk. On the other hand, GSTM1-null genotype was associated with better survival in ccRCC. Regarding ESRD patients, the results of this study showed that variant forms of genes encoding antioxidant enzymes GSTA1, GSTM1, GSTP1, GSTT1 and GSTO2 were more frequent in ESRD patients than in corresponding healthy subjects. Besides, the presence of variant genotypes was associated with the higher levels of oxidative stress byproducts and soluble adhesion molecules in these patients. Moreover, GSTM1-null genotype conferred higher risk and worse prognosis in terms of overall and cardiovascular survival in patients with ESRD. Taken together, the results presented in this study suggest that GST genotypes might serve as a valuable indicator in both risk assessment and prognosis stratification.
Keywords: GST polymorphism, risk, prognosis, RCC, ESRD
CYP2E1, TP53 and GST Gene Polymorphisms and Lung Cancer Risk
Ahmet Oguz Ada Department of Toxicology, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey Correspondence: email@example.com
Lung cancer is an increasing worldwide public health problem and the main cause of deaths arising from cancers in most countries. Majority of lung carcinomas have non-small cell lung carcinoma (NSCLC) histology. Although smoking and environmental carcinogens are well known risk factors, genetic factors indicate individual susceptibility in the development of lung cancer. Possible cancer susceptibility genes have been sought among tumor suppressor genes and genes encoding phase I and phase II xenobiotic metabolizing enzymes. Cytochrome P450 2E1 (CYP2E1), a main member of CYP450 superfamily, has been shown to metabolically activate various carcinogens including nitrosamines in tobacco smoke. Polymorphisms of CYP2E1 have been shown to alter the activity of the corresponding enzyme that leads to individual variations in microsomal oxidation capacity. The well-known tumour suppressor gene TP53, is an essential regulator of cell-cycle arrest, DNA repair and apoptosis. The variant allele of TP53 has been shown to cause decrease in the activity of the corresponding protein that is crucial for the apoptotic functions of P53. Glutathione S-transferase (GST) enzymes occupy a key position in biological detoxification processes. Polymorphisms of genes encoding GST enzymes alter their corresponding enzyme activities. Therefore individuals with reduced or loss of GST enzymatic activity may be at a greater risk for cancer due to decreased detoxification of carcinogenic and mutagenic compounds. As lung cancer is an important health problem, our recent findings with respect to association between NSCLC risk and polymorphisms of genes encoding these xenobiotic metabolizing enzymes and TP53 will be evaluated herein.
Keywords: non-small cell lung cancer, environmental carcinogens, risk, polymorphism
Relevance of CYP Polymorphisms to Cancer Susceptibility
Ann K. Daly
Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK
Associations of cytochrome P450 polymorphisms with risk of cancer have been reported widely. Studies on lung cancer susceptibility have been particularly common. There are a large number of published studies on polymorphisms in CYP1A1, CYP2D6 and CYP2A6as lung cancer risk factors. However, recently both meta analyses and genome-wide association studies suggest that only the CYP2A6 association, where genotypes associated with low activity decrease susceptibility, appears significant. This may be due to the role of CYP2A6 in nicotine metabolism. Associations with lung cancer susceptibility have also been reported for CYP1A2, CYP1B1 and CYP2E1 polymorphisms but these, though biologically plausible, have not been well replicated. For cancers where exposure to non-tobacco related xenobiotics affects risk, cytochrome P450 polymorphisms may also be relevant. Examples include CYP1A2 for colon cancer associated with heterocyclic arylamine exposure, CYP3A for hepatocellular carcinoma due to aflatoxin exposure and CYP2E1 for nitrosamine-related nasopharyngeal cancer. Data supporting each of these associations will be considered. In general, cytochrome P450 polymorphisms are relevant to risk for some cancers but the importance of this risk may have been overstated in the past.
Keywords: lung cancer, tobacco smoke, nicotine, cytochrome P450, polymorphism
Pharmacogenetics of Drug Response in Parkinson’s Disease
Ivana Novakovic, Eleonora Dzoljic, Milena Jankovic, Ana Marjanovic, Marija Brankovic, Natasa Dragasevic, Vladimir Kostic
Clinic of Neurology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia
Parkinson’s disease (PD) is the most common movement disorder affecting 1% of population at the age of 60 years. Individual therapy optimization is one of the important issues for medical doctors, pharmacists and health insurance in whole. There are recent evidences that variable efficacy of treatment and risk of motor and mental complications could have genetic basis. Pharmacogenetic and pharmacogenomic studies of PD plays leading role in that research. Variability in genes coding for drug-metabolizing enzymes, drug receptors and proteins involved in drug pathway signaling is an important factor determining inter-individual variability in drug responses. In this study we have analyzed polymorphisms in genes encoding for brain derived neurotrophic factor (BDNF), apolipoprotein E (APOE) and catechol -O -methyltransferase(COMT) in a group of PD patients on standard antiparkinsonian drug treatment. Molecular genetic analysis was performed using real time PCR method and pre-designed TaqMan genotyping assays. Interpersonal differences in drug responses have been clearly documented although individualized treatment of PD was not widely applied. Treatment with antiparkinsonian drugs was associated with the development of complications, such as L-DOPA-induced dyskinesia (LID), hallucinations and excessive daytime sleepiness. Carriers of specific genetic polymorphisms showed particular susceptility to development of some of these drug adverse effects. Our results confirm that many genetic variations and polymorphisms in different proteins can influence individual response to anti-PD drugs.
Keywords: antiparkinsonian drugs, individual response, gene polymorphisms
Association of Drug/Xenobiotic Metabolizing Enzyme Polymorphisms with Treatment Outcome of Advanced Non-small Cell Lung Cancer Patients with Platinum-based Chemotherapy
Department of Toxicology, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey
Lung cancer is an increasing worldwide public health problem. Most of the lung cancer patients have non-small cell lung cancer (NSCLC). These patients are mainly treated with standard platinum based chemotherapy. The poor response and great inter-individual variety in response to this chemotherapy occur among these patients. There are accumulating evidences to support the hypothesis that genetic polymorphisms alter the drug response and survival. The cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes metabolize xenobiotics including antineoplastic drugs and involve in drug resistance. The polymorphic CYPs and GSTs have altered enzyme activities and thus they may have influences on the response to chemotherapy and survival in lung cancer patients. Herein, our recent findings with respect to the role of CYP and GST gene polymorphisms in response to chemotherapy and survival in NSCLC patients will be evaluated which could be useful for the clinicians in the prognosis of these patients treated mainly with platinum based chemotherapy.
Keywords: CYPs, GSTs, polymorphisms, response to chemotherapy, survival
SUBSTANCES OF ABUSE: GLOBAL TRENDS, PREVENTION AND MANAGEMENT
Chairs: Prof. dr Slavica Vučinić, Head of the National Poison Control Centre MMA,Medical faculty University of Defense, Belgrade, Serbia, firstname.lastname@example.org;
Prof. dr Hans Maurer, University of Saarland, Homburg, Germany, advisor to the U.S. Federal Bureau of Investigation, email@example.com
Over the past 20 years, the spectrum of substances available on the global drug market has widened considerably, paralell with the significant increase in the number of drug users. In 2015, about 250 million people used drugs at least once, of these, around 29.5 million suffered from drug use disorders. Opioids were the most harmful drug type in health terms, with almost 12 million years of „healthy“ life (disability-adjusted life years – DALYs) lost worldwide. Overall, hepatitis C has caused greatest harm among drug users. Albeit the opioid market is becoming more diversified and the cocaine market is expanding, marijuana is top drug in every country by the proportion of users. The World Drug Report 2017 of UNODC reinforced the importance of united action to address drug challenges and the need for science and rights-based drug use prevention and treatment.
The world is witnessing an alarming problem of an unprecedented increase in the number, type and availability of new psychoactive substances (NPS) in Europe with over 620 being monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). The EMCDDA’s main responsibilities in this field are to operate the EU Early Warning System (EWS), with its partner Europol and to undertake risk assessments of NPS when necessary. The EU EWS works by collecting information on the appearance of NPS from the 28 EU Member States, Turkey and Norway, allowing the EU to respond rapidly to emerging threats. Serbia has promoted the National EWS in 2016, with the National Poison Control Centre as one of key subjects.
The strong EWS can play a critical role in ensuring a timely response in order to protect public health.
Keywords: designer drugs, new psychoactive substances, synthetic cannabinoids, early warning system, liquid chromatography
Analytical Strategy for Effective Clinical Toxicology Services
Hans H. Maurer
Department of Experimental and Clinical Toxicology, Saarland University, D-66421 Homburg (Saar), Germany
Various analytical tools allow today a broad range of analysis in clinical toxicology, particularly in big centers. Of course, long distances may limit the usefulness. Current strategies for efficient analytical diagnostics in clinical toxicology are presented. The tasks for such diagnostics, different analytical strategies and various methods are reviewed. They cover procedures mainly using low and high-resolution mass spectrometry coupled to gas or liquid chromatography (GC-MS or LC-(HR)MS) for target or comprehensive screening for drugs (of abuse), poisons, and their metabolites as well as for quantification in blood. Quality control aspects are discussed as well as strategies for competent interpretation of the analytical result in correlation with the clinical signs presented by the patient. However, the service must be available around the clock and reliable results must be provided in a short time frame relevant for treatment and at reasonable costs.
Keywords: clinical toxicology, analytical strategy, mass spectrometry, toxicological interpretation, quality control
The Role of the National Poison Control Centre in the Early Warning System on New Psychoactive Substances
National Poison Control Centre, MMA, Medical Faculty, University of Defense, Crnotravska 17, Belgrade, Serbia
The drug market in Serbia is characterized with the avaliability of all types of drugs, including new psychoactive substances (NPS), the steadily rising number of patients treated for overdose, and great difficulties that come with NPS analysis and determination of substance-specific effects. From January 2013 to December 2016, 58 patients, aged 14 to 25, with the effects of synthetic cannabinoides, were treated in the NPCC. Products “Biljni tamjan”, “Beli slez”, “Rainbow Special”etc, were obtained from tabacco shops or online as natural herbal products and air fresheners, and consumed by smoking or inhalation of smoke. Liquid chromatography-electrospray ionization-mass spectrometry with Xterra column, liquid chromatography with PDA detector and GC-MS (ion trap detector) were used for analysis. The clinical picture included: tachycardia in 54 (93.1), mydriasis in 31 (53.4%), somnolence, nausea, vomiting, agitation in 16 (27.6%), dizziness in 10 (17.2%), disorientation in 9 (15.5%), chest pain, dispnea in 5 (8.6%), loss of consciousness, pallor, paresthesia, muscle twitches and short-term memory impairment in 2 (3.4%) patients. After receiving treatment, all patients had fully recovered within up to 8 hours in the emergency ward and were discharged shortly afterwards. AB-PINACA AB-FUBINACA, JWH 18, JWH-122, JWH-210, 5F-AKB48, MDMB-CHMICA and AB-CHMINACA were detected in urine samples and/or herbal products.
The efficient and systematic collection of the relevant data on synthetic cannabionoids and other NPS, adverse effects, changes of purity and composition of controlled PS and NPS-based products, along with raising the public awareness on the NPS will pave the way to the national EWS.
Keywords: synthetic cannabinoids, clinical picture, analytics, poison control centre
The EU Early Warning System – 20 Years of Monitoring New Psychoactive Substances in Europe
EMCDDA, Lisbon, Portugal
Over the past 20 years, the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) has played a central role in Europe’s response to new psychoactive substances (NPS). Its main responsibilities in this field are to operate the EU Early Warning System (EWS), with its partner Europol and to undertake risk assessments of new substances when necessary. The EU EWS works by collecting information on the appearance of new substances from the 28 EU Member States, Turkey and Norway, and then monitoring them for signals of harm, allowing the EU to respond rapidly to emerging threats.
Over the last two decades there has been an unprecedented increase in the number, type and availability of NPS in Europe. The EMCDDA is currently monitoring in excess of 620 new psychoactive substances, which are not covered by international drug controls. The rapid increase in growth in this market is evidenced by the fact that approximately 70% of these new substances were detected in the EU in the past 5 years alone.
The growth in the market is also responsible for the increase in serious harms reported to the EMCDDA in recent years. Most of these concern non-fatal intoxications and deaths, but also include broader social harms. During 2016, serious harms requiring urgent attention led the EMCDDA to undertake investigations into 3 new substances – the synthetic cannabinoid receptor agonist MDMB-CHMICA, and two fentanyl derivatives: acryloylfentanyl and furanylfentanyl.
It is likely that the growth of the market in NPS will continue to pose a range of challenges for public health and drug policy over the next few years i.e. the speed at which they appear, their open sale, and the little or no information provided on their effects and harms. It is here that strong early warning systems can play a critical role in ensuring a timely response in order to protect public health.
Keywords: EMCDDA, international drug controls, synthetic cannabinoinds, fentanyl derivatives
Alternative Biological Samples for Determination of Psychoactive Substances
National Poison Control Center, Military Medical Academy, Medical Faculty, University of Defence Belgrade, Serbia
In recent years unconventional biological samples such as saliva and hair are used for determination of psychoactive substances (PAS), although the drug levels are often lower than the corresponding ones in urine or blood.
The aim of this study was to describe the possibility of determination of PAS in saliva and hair samples.
Saliva samples were collected from patients after abuse of alcohol, benzodiazepines and heroin, and hair samples were taken from patients who abused PAS.
Alcohol in saliva was determined by gas chromatography with flame ionization detection, and presence of benzodiazepines and heroin metabolites was confirmed after alkaline chlorophorm extraction, by liquid chromatography with mass spectrometry. After decontamination, pulverization and solubilization, hair samples were prepared by alkaline liquid-liquid extraction with chlorophorm for benzodiazepines and heroin and acidic extraction with n-hexane:ethylacetate (9:1) for THC-COOH. The chromatographic separation was performed on XTerra®RP18 column, using a gradient of acetonitrile/formic acid 1% and formic buffer pH 3.5 as the mobile phase.
Our result showed good correlation of alcohol and benzodiazepines concentration between blood and saliva, as well as heroin metabolites in urine and saliva. We also proved presence of benzodiazepines, heroin and THC metabolites in hair samples of chronic abusers.
Saliva and hair samples could be used as an alternative to blood or urine. The detection of PAS in saliva is a sign of recent drug use and it may be used for their detection. Hair analysis may provide useful information about abusing PAS and the history of usage after their intake.
Keywords: saliva, hair, liquid chromatography with mass spectrometry
Drugs of Abuse: Trends in Croatia
Irena Brčić Karačonji, Andreja Jurič, Nataša Brajenović
Analytical Toxicology and Mineral Metabolism Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia
According to data provided by the European Monitoring Centre for Drugs and Drug Addiction, approximately one quarter of Europeans have used an illicit drug at some point in their lives. A study on illicit drug use in Croatia carried out in 2015 on 4,992 respondents showed that 20.3% of the population aged 15-64 years used illicit drug at least once during their lifetime. Cannabis was the most frequently used illicit drug in Croatia with a lifetime prevalence of 19.4%, followed by amphetamines (3.5%), ecstasy (3%), cocaine (2.7%), LSD (1.7%), and heroin (0.3%). The largest concern is the high lifetime prevalence (6.9%) of taking new psychoactive substances, mainly synthetic cannabinoids, in the 15-24 age group.
Advances in the sensitivity of analytical techniques have enabled analyses of substances of abuse in alternative biological matrices such as hair. Since 1999, over 1700 hair samples were analysed by gas chromatography-mass spectrometry at the Institute for Medical Research and Occupational Health, Croatia for the presence of methadone, cocaine, heroin, codeine, morphine, and amphetamine-type stimulants. A total of 22% of the tested samples were positive for one or more drugs of abuse. Ecstasy was the most frequently detected substance (in 10% of samples).
Croatia’s National Strategy on Combating Drug Abuse was adopted in 2012, with an aim to reduce both the demand and the supply of drugs of abuse. Its implementation should significantly contribute to protecting the health of individuals and the general community through an integrated approach to the problem of drug abuse.
Keywords: substances of abuse, new psychoactive substances, hair analysis, statistical data
BIOMARKERS IN CHRONIC DEGENERATIVE DISEASES AND RISK ASSESSMENT
Department of Pharmacy and Biotechnology, University of Bologna, Italy
The identification of biomarkers is an important stage in the development of strategies for prevention, prognosis and treatment of any disease. Indeed, understanding the relationship between measurable biological process and disease development is vital to expand our arsenal of biomarkers risk assessment and prevention, and of treatments for all disease. Besides these, biomarkers might play a critical role in deepening our understanding of normal, healthy physiology.
Keywords: biological process, disease development, prevention, treatment
COPD Risk Evaluation Through the Exposome, Genetic Traits and Enzymatic Activities
Sabrina Angelini1, Francesca Maffei2, Silvana Hrelia2, Patrizia Hrelia1
1 Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy; 2 Department for Life Quality Studies, University of Bologna, Rimini, Italy.
Chronic obstructive pulmonary disease (COPD) is a multifactorial disease with cigarette smoke the main risk factor. Only a small proportion of smokers develop symptomatic disease, suggesting the existence of other risk factors. We perform a multidisciplinary project, Breath Bologna, with the aim to uncover socio-economic features, biomarkers of genetic damage and susceptibility, and enzymatic activities as risk factors in COPD. 229 subjects were recruited among outpatients undertaking respiratory function tests at the Pneumology Unit of the Sant’Orsola-Malpighi Hospital, Bologna. Comprehensive socio-demographic, lifestyle and clinical data, revealed that fragility is associated with COPD stage and that comorbidities and the low body mass index are predictors of mortality or hospitalization. Regarding biomarkers of genetic damage, we evaluated micronuclei frequency in peripheral blood lymphocytes. Data showed no significantly different micronuclei frequencies associated with the disease stage. Interestingly, comparing micronuclei frequency in COPD patients and healthy subjects, we observed a trend for a higher micronuclei frequency in patients. With regard to biomarkers of susceptibility, we investigate whether polymorphisms of mEH and other oxidative stress genes (CAT, GR, GPX, SOD) influenced individual susceptibility to COPD onset and severity. Currently statistical analysis of association with the analyzed polymorphisms is ongoing. We also aimed to define biochemical biomarkers of oxidative stress, predicting the risk of COPD, determining by spectrophotometric test the activity of GR, CAT, GPX and SOD. At present, we are evaluating the association of these enzymatic activities with COPD risk and severity. Furthermore, we are assessing the influence of polymorphisms in the observed enzymatic activities.
Keywords: COPD, biomarkers, micronuclei, polymorphisms, antioxidant activity
hCOMET Network: Results of the First Statistical Analysis and New Findings in the Buccal Micronucleus Assay, as Biomarkers in Human Biomonitoring and Early Disease Detection
Mirta Milić1, Stefano Bonassi2,3, Emilio Rojas4, Claudia Bolognesi5, hCOMET Consortium5
1 Institute for Medical Research and Occupational Health, Mutagenesis Unit, Croatia; 2 Scientific Institute for Research, Hospitalization and Health Care San Raffaele Pisana; 3IRCCS San Raffaele Pisana, Italy; 4 National Autonomous University of Mexico, Mexico; 5 Environmental Carcinogenesis Unit, Ospedale Policlinico San Martino, Genova, Italy;
The alkaline comet assay is a test for measuring single strand DNA breaks in eukaryotic cells. Method exists for more than 30 years and is used in genotoxic research. Since 2014 (revised 2016) it has become the official method for assessing genomic damage in vivo after exposure to both physical and/or chemical agents (Organization for Economic Co-operation and Development- OECD Test No. 489). With its additional variants it is also used in the biomonitoring of exposed populations, but also as an auxiliary method for disease diagnosis and in therapies for patients’ health improvement, but for such an official application, the method should be improved and validated. That task has been addressed by the newly formed group hCOMET that has gathered results on human biomonitoring studies from 41 laboratories, including 90 methods, 108 studies and more than 19 000 individuals. We will discuss the efforts and the results of the group, and will also talk about a relatively new method of micronucleus buccal test (buccal mucosa) that has a potential to replace or to be added value to the classical in vitro micronucleus method on mammalian cells. Much shorter assay than the classical micronucleus method in vitro, the assay has already shown possible application in the study of exposure to asbestos and the occurrence of Alzheimer’s disease. We will discuss the efforts already done in this area and further steps.
Keywords: comet assay, human biomonitoring, buccal micronucleus assay, human biomarkers, early disease detection
Genotoxicity Biomarkers in the Clinical and Environmental Molecular Epidemiology Studies for Children
Gazi University, Faculty of Pharmacy, Department of Toxicology, Ankara, Turkey
Children are among the most susceptible group of the population regarding environmental exposures, diseases and medical treatments. There is an increasing attempt to determine the sensitivity of children to environmental agents and clinical outcomes, especially to predict likely cancer development. Genotoxicity, as the intermediate step in cancer can be evaluated by use of early effect biomarkers. In order to protect children’s health there is crucial need for genotoxicity data derived directly from children studies. The information gained from those studies could be crucial in the hazard identification step of risk assessments to drive regulatory actions and in developing efficient and safe treatment strategies. Additionally, to maintain conscious future generations, increasing the awareness of children on indoor and outdoor chemicals could be complementary. Herewith the focus point is to overview the molecular epidemiology studies on children for environmental exposures (air pollution) and for clinical outcomes (children with thalassemia minor, chronic kidney disease, etc.), in which genotoxicity was the main endpoint.
Keywords: genotoxicity, children, clinical biomonitoring, environmental biomonitoring, awareness
Role of Oxidants and Antioxidants in Degenerative Diseases Development: in vitro Models
Ksenija Durgo, Ana Huđek, Ana Belsčak-Cvitanović, Arijana Bušić, Draženka Komes, Višnja Bačun-Družina
Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia
Cells of our organism are permanently exposed to different endogenous and exogenous oxidants, originated from basal metabolism, immune reactions, xenobiotics, detoxyfing processes and intermediates formed during biotransformations. At cellular level, oxidants cause DNA damage, oxidation of the proteins and destruction of lipids by lipid peroxidation chain reactions. On the other hand, numerous chemicals (of exogenous or endogenous origin) easily interacts with reactive oxidative species, preventing macromolecules destruction and consequently, damages of the cells and tissues. Significant role in this oxidative/antioxidative balance play bioactive compounds from plants, fruits or vegetables. Under certain circumstances, these compounds can decrease level of oxidative damage, but at the same time, under some other circumstances they can enhance pro-oxidative response. There are different assays for measurement of the antioxidant status and oxidative damage. The simplest ones are chemical in vitro reactions and tests in cell cultures. They can yield useful information about mechanisms of action, but extrapolation to effects of dietary antioxidants in vivo is not simple and often it may be incorrect, because uptake from the gastrointestinal tract, influence of microflora and metabolism are not considered. In this presentation, some of in vitro models for detection of pro-oxidative/antioxidative effects will be presented, including the most common biomarkers that are measured in order to determine changes of cellular macromolecules that are the first step in the development of degenerative diseases.
Keywords: oxidative damage, antioxidants, degenerative diseases, biomarkers, phytochemicals
INFLUENCE OF ENDOCRINE-DISRUPTING CHEMICALS (EDCS) ON DEVELOPMENT AND REPRODUCTION
1Clinic of Endocronology, Diabetes and Metabolic Diseases, Faculty of Medicine, University of Belgrade, Belgrade, Serbia, 2Endocrine Unit, ARETAIEION hospital, Athens Faculty of Medicine, National and Kapodistrian University of Athens, Athens, Greece
Endocrine-disrupting chemicals (EDCs) are ubiquitous chemicals that affect all endocrine systems. Male and female reproduction is a supreme physiological process that is under the control of signalls from central nervous system and gonadal steroid. Reproduction could be under the influence of various disruptors among which chemicals are playing still unresolved role. EDCs may influence gonadal system on the level of hypothalamus, pituitary, testicles, ovary, prostate and uterus, and resulting in subfertility, infertility, improper hormone production, estrous and menstrual cycle abnormalities, anovulation, and early reproductive senescence. These substance could interfere with developmental processess and mediate carcinogenesis as well. First lecture of the symposium will provide an introduction to the complex issue of EDCs. Second lecture of the symposium will demonstrate how the xenograft system could be used for the demonstration important species differences in the effects of exposure to chemicals such as di-n-butyl phthalate, diethylstilboestrol and that paracetamol could interfere with testosterone production and germ cell development in the human fetal testis. Third lecture will emphasize the effects of EDCs on reproductive system in males, namely semen quality, infertility, urogenital tract abnormalities, and testicular germ cell cancer. Fourth lecture will provide clinical data on the effects of phthalates and bisphenol A in polycystic ovary syndrome as common female reproductive disorder. Fifth lecture will provide preclinical data on the endocrine disrupting potential of herbal dietary supplements on estrogen receptor function, and estrogenic activity of mixtures of persistent organic pollutants in breast cancer patients.
Keywords: endocrine-disrupting chemicals, testosterone, infertility, polycystic ovary syndrome, estrogen receptor
EDCs: an Introduction
Clinic of Endocronology, Diabetes and Metabolic Diseases, Faculty of Medicine, University of Belgrade, Belgrade, Serbia
Endocrine disrupting chemicals (EDCs) are substances present in the environment, food, and consumer products that could interfere with hormone biosynthesis, metabolism, or action. A variety of synthetic chemicals could affect human development, as well as fertility at different periods of humans life, endorine-related cancers as breast and prostate ones, neuroendocrine pathways, thyroid function, obesity and metabolism. Among EDCs we are aware on the influence of pesticides (organochlorines, organophosphates, carbamates, pyrethroids, triazines), heavy metals (arsenic, lead, mercury), plasticizers, different pharmaceutical compounds and others. The mechanisms of EDCs involve different receptors and enzymes including estrogenic, androgenic, thyroid, peroxisome proliferator-activated receptors, retinoid, other nuclear receptors, steroidogenic enzymes, neurotransmitters. Moreover, we are concerned on the non-monotonous effects of even small concentrations of the specific chemical or their mixtures, latency to the occurence of clinical manifestation of the derangement or diseasea as well as their transgenerational effect. Results obtained from studies on animal models, in vitro analyses and epidemiological data on humans led us to the conclusion for the existence of EDCs on human development and general health, and epidemiological studies converge to implicate EDCs as a significant concern to public health.
Keywords: endocrine disrupting chemicals, hormones, receptors, neurotransmitters, reproduction
EDCs: Assessing the Risks of Exposure to Environmental Chemicals and Pharmaceuticals
1MRC Centre for Reproductive Health, Queens Medical Research Institute, 2Department of Diabetes and Endocrinology, Royal Hospital for Sick Children, Edinburgh, UK
Many industrial chemicals and pharmaceutical products have been proposed to result in endocrine disruption in humans. This includes potential effects on reproductive development in males and females. Chemicals that have been proposed to impact on male reproductive development include plasticizers, synthetic oestrogens and analgesics. Much of the data on the effect of exposure to these agents are based on studies conducted in rodent models; however, confirmation of such findings in human model systems at human-relevant exposure levels are lacking.
We have developed model systems to determine the effects of exposure to a variety of proposed ‘EDCs’ on human fetal testis development and function. Using a xenograft system designed to reproduce normal fetal testis development and in-utero hormonal environment, our results demonstrate important species differences in the effects of exposure to chemicals such as di-n-butyl phthalate (DBP) and diethylstilboestrol (DES) in terms of testosterone production. We have also demonstrated that exposure to analgesics, such as paracetamol, result in a significant reduction in testosterone production and also impact on germ cell development in the human fetal testis. Importantly, these effects are apparent at therapeutic levels of exposure using a standard therapeutic regimen.
Our work, in addition to that of several other groups, highlight the importance of choosing an appropriate model species, experimental system and relevant exposure regimen in order to determine the potential impact of EDC exposure in humans. Findings from rodent studies should, where possible, be confirmed using human tissue models in order to determine the relevance to human health.
Keywords: testis development, testosterone, phthalate, paracetamol, diethylstilboestrol
Endocrine Disruption and Male Gonadal Function
Endocrine Unit, ARETAIEION hospital, Athens Faculty of Medicine, National and Kapodistrian University of Athens, Athens, Greece
Humans are exposed to dozens of endocrine-disrupting substances (EDCs). Some act as antiandrogens while androgenic EDCs have been identified. The latency between exposure to EDCs and occurrence of clinical disorders can be long or transgenerational. Three male reproductive health endpoints are affected by EDCs: disrupted reproductive function (semen quality, infertility); urogenital tract abnormalities, (hypospadias, cryptorchidism); and testicular germ cell cancer (TGCC). These endpoints have been connected to EDCs and genetic factors. Cryptorchidism, hypospadias, oligospermia, and testicular cancer have been associated in the so-called testicular dysgenesis syndrome (TDS). TDS was reproduced in rodents with phthalates and PCBs. Reduced anogenital distance was observed in the rat and in epidemiological studies in human male newborns. Inverse relationship between maternal phthalate levels and human offspring’s anogenital index has been described. Prostate hyperplasia has been described after exposure to BPA. Regarding PCBs, pesticides, and phthalates, limited evidence supports a relationship between adult exposure and reduced semen quality; timing of exposure to dioxins has been associated to semen quality. The dramatic recent upward trend in the incidence rate of TGCC indicates that apart genetic factors, environmental and lifestyle factors might be involved. Blood organochlorine levels measured in mothers, decades after their sons’ birth, were predictive of increased TGCC risk in the latter. Because the current scientific evidence on associations of EDCs with male reproductive health endpoints remains limited there is need for further research.
Keywords: endocrine-disrupting chemicals, male reproduction, infertility, prostate, testicular cancer
Influence of Phthalates and Bisphenol A on Fertility in Woman
Milica Medic Stojanoska
University of Novi Sad, Faculty of Medicne, Clinical Center of Vojvodina, Novi Sad, Serbia
Endocrine disrupting chemicals (EDCs) as bisphenol A (BPA) diphenylmethane derivate and phthalaes, often called plasticizers (diestars of 1,2-benzendicarbonic acid), are the most produced chemicals worldwide. Numerous studies indicate that they can adversely affect ovaries, uterus, anterior pituitary and/or steroid hormone production, gonadotropins, GnRH pulsatility and signaling. This can lead to reproductive disorders, such as early puberty, infertility, premature ovarian failure, endometriosis and adverse pregnancy outcomes. It is known that fetuses, infants and /or young adolescents are more susceptible groups. Exposure to them may have trans-generation effects on female fertility. Recently published studies suggest association between BPA and some phthalates (diethyl-hexyl phthalates – DEHP and others) with polycystic ovary syndrome (PCOS). Our previously performed examination confirmed association of BPA and DEHP with obesity and other parameters of metabolic syndrome, which can exacerbate PCOS phenotype and infertility. Now we found increased level metabolites of DEHP and diethyl phthalate (DEP) and BPA in some groups of patients with PCOS. Little information is available on the effects of phthalates and BPA on the other causes of infertility in humans. Mechanisms by which BPA and phthalates disrupt folliculogenesis and steroidogenesis are not studied enough and they include estrogenic, anti-estrogenic, oxidative stress response or PPAR activation. The evaluation in vitro of acute BPA exposition in the panel of mammalian cell lines was confirmed to be toxic, which may also suggest its toxic model of action. Conclusion: BPA and some kinds of phthalates have a proven negative influence on the female fertility by insufficiently studied mechanisms.
Keywords: EDCs, BPA, phthalates, infertility, PCOS
Exploring EDCs and the mechanisms by which they adversely affect reproduction
Department of Toxicology, Faculty of Pharmacy, Ege University, Izmir, Turkey
Exposure to various endocrine disrupting chemicals (EDCs) is suggested to play an important role in hormone-dependent cancers and reproductive disorders. A recent comprehensive meta-regression analysis reported a significant decline in sperm counts among men from developed countries suggesting an urgent need for research on the causes of this continuing decline. OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters suggests a battery of in vitro assays providing data about selected endocrine mechanism(s)/pathways(s) at Level 2. Our group has been evaluated endocrine disrupting potential of active molecules from widely used herbal dietary supplements. Their estrogen receptor (ER) binding affinities, ER agonist/antagonistic effects in MCF-7 proliferation assay and aromatase inhibitory potential have been investigated. Some of our findings were confirmed by molecular modelling (OECD Level 1) and estrogenic activity observed in vitro was further investigated by in vivo uterotrophic assay (OECD Level 3). Recently we are working on investigating possible utilization of total estrogenic activity of adipose tissues from breast cancer patients as biomarker for exposure to mixtures of persistent organic pollutants (POP). For this reason we improved an existing method to separate POPs in the tissue samples from endogenous hormones by HPLC, and the estrogenic potential of the eluates containing POPs was evaluated in the E-Screen bioassay. The amount and type of POPs in the samples were further analysed by LC-tandem MS. Results of this assay make it possible to evaluate the total estrogenic potential of the mixtures of POPs at various combinations and concentrations.
Keywords: endocrine disruptors, screening, mechanism of action, phytoestrogens, persistent organic pollutants
DEVELOPMENT IN METHODOLOGIES TO ADDRESS MIXTURE RISK ASSESSMENT
Chair: Alan Boobis, Imperial College London, UK;
Deriving methodologies for human-relevant risk assessment for mixtures is leading to a number of approaches driven by exposure. These approaches need to be scientifically-based, feasible within and across chemical sectors, consistent globally and provide relevant assessments to protect public health using the most current tools such as read-across, in silico or in vitro approaches and AOPs. Problem formulation and exposure mapping will aid in first focussing the drivers for risk assessments to address relevant population concerns.
This symposium will describe approaches which are being developed for combined risk assessment of multiple chemicals.
Keywords: mixtures, human-relevant risk assessment
Human Health Protection from Combined Exposure to Multiple Chemicals in the Global Context
Alan R Boobis
Department of Medicine, Imperial College London, London W12 0NN, UK
Exposure to chemicals never occurs in isolation; rather there is co-exposure to multiple chemicals, both natural and synthetic. It is not possible to eliminate all such exposure, and indeed it may not be desirable as some chemicals are beneficial to health. Hence, some means of assessing which combinations are of concern, that is both efficient and effective in protecting human health, is needed. As it is not feasible, or indeed necessary, to test all potential combinations, a tiered approach to determining which chemicals should be considered in combination has been proposed. An assessment should start with problem formulation, which includes explicit statement of the purpose of the assessment and the risk management goals. Given the breadth of chemical space involved, it is essential to consider which chemicals should even be considered within the scope of the assessment. The chemicals are then triaged using tiered exposure and hazard assessments, higher tiers resulting in less uncertainty but involving greater resources. The outcome is identification of a set of chemicals that should be assessed together, a cumulative assessment group. The combined toxicity of the group is estimated using an appropriate model, usually dose addition, but if supported by the data, response addition might be more appropriate. If the margin of exposure is such that there is potential concern for effects on public health, dialogue with the risk manager is necessary to determine whether there is time for further refinement or given the magnitude of potential concern, immediate action is necessary.
Keywords: cumulative assessment groups, dose addition, problem formulation, tiered assessment
Opportunities for Grouping/Read-across in the Risk Assessment of Combined Exposures to Multiple Chemicals
Andrea-Nicole Richarz 1, Andrew Worth 1, Stephanie Bopp 1
1 European Commission Joint Research Centre, Directorate for Health, Consumers and Reference Materials, Ispra (VA), Italy
Grouping approaches are considering similar chemicals together, defining similarity based not only on chemical structure, but also biological/toxicological similarity. The definition and justification of the similarity are crucial for the assessment approaches built upon the grouping. One widely discussed use of grouping for mixture assessment is the grouping into assessment groups. It is assumed that based on the similarity of mode of action of the chemicals, concentration addition methods can be used for combined risk assessment. Read-across is inferring unknown properties, such as toxic effects, for the target substance(s) based on available data from the similar (source) substance(s). It is useful for predicting toxicity of large groups of chemicals in absence of actual testing. Again, the crucial step is to find the relevant similar analogues. In view of applying read-across to combined exposure assessment, ideally analogues for the whole mixture should be considered. For mixtures in the sense of the REACH (IUCLID) substance definition, databases such as AMBIT can be searched. For non-defined mixtures and general combined exposure the read-across approach has to be further explored, starting with reading across toxicity for single substances or specific chemical groups within the mixture. In that case, however, possible synergistic effects are not taken into account and have to be considered additionally. The adverse outcome pathway (AOP) framework can support this approach by identifying modes of action and thus chemicals acting towards the same adverse effects, even if relating to different molecular initiating events, or influencing each other through the AOP network.
Keywords: mixtures, safety assessment, mode of action, adverse outcome pathways
Chemical Mixtures in Food – What Tools and Test Strategies will the EUROMIX Project Deliver?
Jacob van Klaveren
RIVM National Institute for Public Health and the Environment
The main deliverable of EuroMIx is a mechanism-based test strategy, aiming to reduce the use of animals in testing, for refining risk assessment of multiple chemicals from multiple exposure routes. EuroMix developed Adverse Outcome Pathway (AOP) networks and practical tests for measuring the mixture effect at several key-events. The EuroMix consortium tested QSARs, molecular docking and exposure tools for pesticides, additives and contaminants aiming to set test priorities. EuroMix will provide in vitro tests and standard operating procedures describing how these tests can be used. The in vitro testing will be validated against in vivo testing, which is required in current risk evaluation. The test results will refine uncertainties embedded in current risk assessment of mixtures such as worst-case assumptions on in- or exclusion of chemicals into cumulative assessment groups, the dose addition and the potency of each separate chemical in a mixture. The results will be used in deterministic and probabilistic exposure and hazard modelling approaches. The EuroMix will develop a web-based model and data toolbox available for stakeholders involved in the chemical safety evaluation. Case studies addressing several chemical classes will illustrate how the web-based toolbox can be used overarching regulatory sectors such as pesticides, additive and contaminant legislation. Practical guidance will be written and training will be given on how to use the EuroMix toolbox. Countries outside Europe participating in the Codex Alimentarius will try to reproduce these case studies using consumption data of their countries, which will simulate international implementation and discussions on harmonizing approaches.
Keywords: mixtures, AOP, risk, probabilistic, harmonisation
Utility of AOPs for Mixture Safety Assessments
Department of Biomedical and Clinical Sciences, Università degli Studi di Milano, and ICPS at ASST-Fatebenefratelli-Sacco, Milano, Italy
Adverse Outcome pathways (AOPs) consist of a sequence of KE (Key Events) connecting an MIE (Molecular Initiating Event) to an AO (Adverse Outcome). For compounds that share the same MIE (and AOP) (also identified as compounds with similar MOA) the approach is rather straightforward. However, when compounds have different MIEs, that, further down the chain of events, lead to the same KE(s) and AO (dissimilar MOA), the approach becomes more complex. Quantitative and temporal definition of dose-response relationships with KE(s) are key to determine whether the additive model for combined exposures needs to be applied or whether it entails excessive conservatism, particularly at environmentally relevant doses that are expected to cause sub-threshold KEs.
Keywords: pathways, key events, molecular initiating events, adverse outcomes
Exposure-Driven Cumulative Risk Assessment in Practice
Toxicology and Risk Assessment Leader, Human Health Assessment, Dow AgroSciences, UK
Deriving methodologies for human-relevant risk assessment for mixtures is leading to a number of approaches driven by exposure. The problem formulation applied to cumulative risk assessment of agrochemicals needs to be explored further. An exercise in mapping and integrating scenarios enables the risk assessor to identify and clarify the cumulative exposure for which a risk assessment is needed. The aim of this presentation is to present a case example of how to map such an exposure scenario and then apply the relevant tools to addressing the risk. The types and extent of data that would be required will differ according to the defined protection goals. A primary outcome is to identify the risk drivers and thus focus the assessment to the relevant populations or exposure scenario of concern, and define individual substances leading to that concern. This will then enable appropriate risk mitigation or risk reduction measures to be applied, thus leading to relevant decision-making for achieving the risk management goals.
Key words: cumulative assessment groups, problem formulation, exposure scenario, exposure data, tiered assessment
TOXICITY OF RESPIRABLE PARTICULATE MATTER IN AMBIENT AIR
Chair: M. Jovašević-Stojanovć1, Vinča Institute of Nuclear Sciences, University of Belgrade, Serbia, firstname.lastname@example.org
Air pollution is the single largest environmental health risk in Europe. Recently, a new WHO air quality model confirmed that 92% of the world’s population lives in places where air quality levels exceed WHO limits. Numerous studies underlined contribution of exposure to toxic spices contained in ambient airborne respirable particulate matter (RPM) for development different cardiopulmonary diseases and lung cancer. In addition, air pollutants and its most toxic past that is RPM associated with increase in incidence of numerous additional diseases. IACR designated outdoor air pollution as a Group 1 carcinogenic substance. In addition RPM mixture, that contain variable amount of organic (e.g. PAHs, OC) and inorganic (e.g. metals and metalloids) species was evaluated separately and also classified in the Group 1. Current research on airborne particle-induced health effects investigates the critical characteristics of RMP that determine their biological effects. It need to be take in account that the size of the airborne particles and their surface area determine the potential to elicit inﬂammatory injury, oxidative damage, and other biological effects. Associations between chemical compositions and particle toxicity tend to be stronger for the ﬁne and ultraﬁne particulate matter size fractions. Extractable organic compounds contribute to various mechanisms of cytotoxicity, in addition, the water-soluble faction (mainly transition metals with redox potential) play an important role in the initiation of oxidative DNA damage and membrane lipid peroxidation.
Keywords:respirable particulate matter (RPM), particle size, particle composition, toxicity
Comparative Toxicity of Airborne Fine and Ultrafine Particles from Different Regions and Emission Sources
- Mantecca1, S.K. Hassan2, A. A. El-Abssawy2, W. H. Shetaya2, A. El-Mekawy2, E. F. Mohamed2, A. M. F. Mohammed2, R. Bengalli1, S. Marchetti1, A. Zerboni1, E. Longhin1, M. Camatini1
1 University of Milano-Bicocca, Department of Earth and Environmental Sciences, Research Center POLARIS, Milan, Italy; 2 Air Pollution Department, Environmental Sciences Division, National
Research Centre, Dokki, Giza, Egypt
Air pollution, mainly with airborne particulate matter (PM), is one of the leading global risks to human health. The toxicity of airborne PM is linked to particles’ dimension and chemical composition and is greatly affected by regional and seasonal variations, as well as by the local emission sources. Fine (FP) and ultrafine (UFP) particles are nowadays considered the most deleterious fractions, because they penetrate in the profound lung, where can hit the respiratory barrier and translocate to secondary organs.
In this presentation, the cytotoxic and genotoxic effects of PM2.5 collected in Milan (Italy) and Giza (Egypt) on in vitro lung cells will be displayed. The variations in PM2.5 chemical composition, especially polycyclic aromatic hydrocarbons (PAHs) and heavy metals, will also be discussed in relation to the geographical and seasonal variations.
Since diesel and biomass combustion-derived ultrafine particles (UFPs) largely contribute to the PM2.5 pollution, additional toxicity studies have been performed by testing combustion particles from different diesel and biomass sources. The results clearly indicate the variability in the cytotoxic effects exerted by UFPs derived from different sources and burning conditions.
The chemical and toxicity data presented will improve our knowledge on the health hazards associated with PM2.5 and UFP exposure and may be considered in the planning of future mitigation strategies.
Acknowledgments: Italian Ministry of Foreign Affairs and International Cooperation (proj. ID PGR00786); Egyptian Science and Technology Development Fund (STDF) (proj. ID 26001); Fondazione Cariplo (proj. ID 2013-1038)
Keywords: PM2.5, ultrafine particles, cytotoxicity, genotoxicity, emission sources
The Influence of Metal Components on the Health Effects of Ambient Particulate Matter
Jasmina Jović-Stošić1, Dragan Alavantić2, Milena Jovašević-Stojanović21National Poison Control Centre, Military Medical Academy, Belgrade, 2Vinča Institute of Nuclear Sciences, Belgrade
In order to assess the contribution of metal fraction to particulate matter (PM) toxicity, we made a review of the relevant literature, comparing results of epidemiological studies, experimental research and professional exposure.
Except for heavy metals, well known for their toxicity, epidemiological studies tended to be internally consistent in identifying some transition metals (iron, nickel, vanadium, zinc) more often than others (arsenic, copper, manganese, selenium) as potentially affecting health. The limitations of these studies are primarily related to characterization of exposure and the presence of co-pollutants possibly contributing to the same effects. Occupational studies may not be representative for general population exposure, but provide valuable information on metals toxicity. Metal fume fever as acute illness, and pulmonary fibrosis, nasal and lung tumours after chronic exposure, are examples of metals toxicity. In such cases, concentrations of metals are much higher than those measured in polluted air. These observations are consistent with the experiments demonstrating that metals from ambient PM can produce acute inflammatory responses only in doses which are much greater than in ambient air.
Epidemiologic studies have found statistically significant association between certain metals in ambient PM and health effects (respiratory and cardiovascular morbidity and mortality). However, experimental research and experience from professional exposure indicate that toxic effects occur at doses many orders of magnitude greater than in airborne PM. From the medical point of view, some metals are normal constituents of the body and we may suppose that relatively low ambient air concentration may be controlled by body homeostasis.
Keywords: air pollution, PM, metal, toxicity
(Bio)monitoring of Polycyclic Aromatic Hydrocarbons
REQUIMTE-LAQV, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, R. Dr. António Bernardino de Almeida 431, 4200-072 Porto, Portugal.
Polycyclic aromatic hydrocarbons (PAHs) are a large group of ubiquitous pollutants. Many PAHs present cytotoxic and mutagenic properties being some of them considered carcinogenic or probable/possible carcinogenic to humans. PAHs monitoring is complicated by their partitioning between air particulate and vapour phases. Main exposure routes to these chemicals are inhalation, food ingestion, and dermal contact, which make biomonitoring the most appropriated way to assess total exposure to PAHs. Thus urinary metabolites of PAHs, monohydroxyl-PAHs (OH-PAHs), constitute valuable biomarkers.
Children are a sensitive group because their systems (respiratory, central nervous, etc.) are not fully developed. Firefighters are an occupational group that are regularly exposed to a high number of pollutants released during fires, including PAHs. Thus ongoing research aims to attain a comprehensive characterization of preschool children and firefighters exposure to PAHs based on biomonitoring and environmental monitoring.
Airborne PAHs with 2–3 rings were the most abundant compounds, being followed by 4-6 ring PAHs. A similar distribution profile was observed between airborne PAHs and urinary OH-PAHs, with 1-hydroxynaphthalene and 1-hydroxyacenaphthene contributing the most for ∑OH-PAHs, followed by 2-hydroxyfluorene, 1-hydroxypyrene, and 1-hydroxyphenanthrene. Levels of 1-hydroxypyrene were lower than the proposed benchmark level; urinary 3-hydroxybenzo[a]pyrene, the PAH biomarker of carcinogenicity, was not detected. Regardless the group considered, strong to moderate Spearman correlations were observed between the levels of ∑PAHs and ∑OH-PAHs, suggesting air as the one of the major exposure source of PAHs.
This work was supported by EU (FEDER/COMPETE) and National Funds (Fundação para a Ciência e Tecnologia) through project UID/QUI/50006/2013.
Keywords: air pollution, polycyclic aromatic hydrocarbons, total exposure, biomarkers of exposure, urine.
Toxicity of Indoor and Outdoor Respirable Particulate Matter in Schools/Kindergartens
- Jovašević-Stojanović1, A. Filipović2, M. Živković1, M. Jovanović1, I. Lazović1, M. Davidović1, R. Kovačević3
1 Vinča Institute of Nuclear Sciences, University of Belgrade, Serbia 2 Public Health Institute of Begrade, Serbia, 3 Mining and Metallurgy Institute Bor, Serbia
Kindergartens and later schools are the microenvironments where adolescents spend significant period of time. Outdoor ambient particulate matter (PM) originate mainly from soil, traffic and industrial sources, while indoor PM in schools represent mixture of both, infiltrated and indoor-generated particle. In many studies there are notified high concentrations of PM in classrooms comparatively with typical home indoor environment. In addition level and content of indoor PM in schools are under influence: air exchange rate; cleaning practice; resuspension; age and physical activity of children in relation to room dimension, due to higher inhalation rates and levels of physical activity children may be more exposed than adults.
PM in ambient air shows differences in size, physical and chemical properties and thus in the toxicological, mutagenic and/or carcinogenic effects. Hazardous pollutants such as Polycyclic Aromatic Hydrocarbons-PAHs and heavy metals are strongly associated with finer particles (< PM2.5). PAHs generated primarily emitted to atmosphere during the incomplete combustion of organic materials such as coal, oil, petrol and wood. Level and characteristics of PAHs in school indoor environment depends of different factors including building location (traffic/residential area), season (heating/nonheatong), heating (local/district), etc.… Metal-enrichment in PM is of particular importance for schools located in industrialized regions since anthropogenic metal emissions to the ambient outdoor atmosphere can transport indoors and negatively impact children’s health.
For data collected in last decade risk of exposure to PM, bonded PAHs and metals in selected school in Serbia will be presented and compared with similar studies performed in other countries.
Acknowledgments: Ministry of Education, Science and Technological Development of Serbia: III 42008, (2011-2017) and TR 21009 (2007-2010) ; SANCO/2009/C4/04, contract SI2.570742, SINPHONIE project (2010-2012)
Keywords: indoor air pollution, Respirable particulate matter, schools, toxicity
Development of an Assay to Assess Genotoxicity by Particulate Matter Extract
Alexandros Priftis1, Konstantinos Papikinos1, Marina Koukoulanaki1, Efthalia Kerasioti1, Dimitrios Stagos1, Konstantinos Konstantinopoulos2, Demetrios Spandidos3, Marianthi Kermenidou4, Spyros Karakitsios4, Dimosthenis Sarigiannis4,5, Arisitidis Tsatsakis6 and Demetrios Kouretas1
1Department of Biochemistry and Biotechnology, University of Thessaly, Larissa 41500, Greece, 2Coffee Island S.A., Patras 26334, Greece, 3 Laboratory of Clinical Virology, University of Crete, Medical School, Heraklion 71409, Greece, 4 Aristotle University of Thessaloniki, Department of Chemical Engineering, Environmental Engineering Laboratory, 54124 Thessaloniki, Greece, 5 Institute of Advanced Study (IUSS), Environmental Health Engineering, Piazza della Vittoria 15, 27100 Pavia, Italy, 6 School of Medicine, University of Crete, Greece
The current study describes a method for assessing the oxidative potential of common environmental stressors (ambient air particulate matter), using a plasmid relaxation assay where the extract caused single strand breaks, easily visualized through electrophoresis. This assay utilizes a tiny amount (11μg) of particulate matter (PM) extract compared to other, cell-based methods (~3000μg). The negative impact of air pollution on human health has been extensively recognised. Among air pollutants, PM holds an eminent role reflected in the broad scientific and regulatory interest. PM toxicity highly depend on its composition (metals and organic compounds), which in turn has been linked to multiple health effects (such as cardiorespiratory diseases and cancer) through multiple toxicity mechanisms; oxidative stress induction is consider one major mechanism among them. In this study the PM levels, the oxidative potential, the cytotoxicity and the genotoxicity of PM in the region of Larissa were examined using the plasmid relaxation assay. Finally, coffee extracts from different varieties, derived from both green and roasted seeds, were examined for their ability to inhibit particulate matter induced DNA damage. These extracts also displayed an inhibitory activity towards xanthine oxidase and catalase, while having no effect against superoxide dismutase. Overall, the study highlighted the importance of assays for assessing the oxidative potential of widespread environmental stressors (PM), as well as the antioxidant capacity of beverages and food items, with the highlight being the development of a plasmid relaxation assay to assess the genotoxicity caused by PM using only a tiny amount.
Keywords: particulate matter, genotoxicity, antioxidant activity, coffee, polyphenols
INCORPORATING INFORMATION ON CHEMICAL MIXTURES INTO CHEMICAL RISK ASSESSMENTS
Chair: Richard Brown1, email@example.com, Co-Chair: Irina Zastenskaya2, firstname.lastname@example.org
1World Health Organization HQ, Switzerland, 2WHO Regional Office for Europe, WHO European Centre for Environment and Health, Germany
Combined exposures to multiple chemicals is a concern in many environmental matrices, including chemicals in food, in drinking water and in air. Many tools have been developed for risk assessment of combined exposures of chemicals, including by the World Health Organization (WHO).
This symposium will describe an electronic tool for prioritization of chemicals for risk assessment using monitoring data of chemical mixtures in indoor air, and a published framework for assessing mixtures of chemicals in drinking water using tiered approaches for hazard and exposure assessment. Use of chemical registers to characterize chemical mixtures in various sectors, and development of “in silico” methods to identify chemicals of concern will also be described. The web-based platform set up by the European Commission for searching for and gaining access to chemical monitoring programme data from multiple sources will be demonstrated. A case study of assessing interactions between chemicals in mixtures (cadmium and PCBs) will also be presented.
Keywords: chemical mixtures, combined exposures, tools, cocktail effect, prioritization
Tools Available and Developing for the Risk Assessment of Mixtures of Chemicals in Indoor Air
Irina Zastenskaya, Dorota Jarosinska, Elizabet Paunovic
WHO Regional Office for Europe, WHO European Centre for Environment and Health
Indoor air is a source of exposure to a number of chemicals. Around 1000 chemical substances have been identified in indoor air to date. They can interact, and synergistic and cumulative effects of exposure on human health are of particular concern.
To assess health risks due to exposure to multiple chemicals, WHO, supported by leading experts, has developed a framework for a tiered exposure and hazards assessment, which allows also assessing risks having limited data on exposure. The framework can be applied for assessment of health risks of chemicals in indoor air.
A number of tools for cumulative risk assessment (CRA) exist to enforce the EU legislation on chemicals. They were applied to assess risks of indoor pollution in several projects. Risks of irritative and respiratory health effects were assessed in relation to acute and long-term exposure to indoor air pollutants emitted during household use of selected consumer products.
To assist countries in implementing CRA of exposure to indoor chemicals, WHO is working on developing an electronic tool to facilitate assessment of risks using existing monitoring data. Given the complexity of the task, a pragmatic approach is taken for the development of a tool for assessment of risk of chemical mixtures in indoor air.
Development of the tool will contribute to the implementation of the Ostrava Declaration on Environment and Health in priority areas such as improvement of quality of the indoor environment and assessment of risks of chemical mixtures.
WHO Europe acknowledges the government of Germany for financial support of the project.
Keywords: chemical mixtures, indoor air, exposure, risk assessment
Assessment and Management of Chemical Mixtures in Source and Drinking Water
M.E. (Bette) Meek
McLaughlin Centre for Risk Science, University of Ottawa, Canada
A recently released WHO Publication on Chemical Mixtures in Source and Drinking Water provides an overview of available tools for assessment of combined exposures. It also includes practical recommendations to support the prioritization of mixtures in drinking and source water for risk assessment and management. Illustrative case studies address pharmaceuticals, non-steroidal anti-inflammatory drugs, statins, microcystins, carbamate insecticides and natural and synthetic oestrogens. Prioritization of pesticides which co-occur in drinking water on the basis of risk ranking and detection frequency is also addressed in an additional case study.
The pragmatic recommendations in the publication are based on experience in the drinking water sector in application of the World Health Organization (WHO) International Programme on Chemical Safety (IPCS) Framework on combined exposures to multiple chemicals. This framework, which was developed drawing from assessments internationally, includes formal problem formulation followed by stepwise consideration of both exposure and hazard in several tiers of increasingly data-informed analyses.
Implications of this additional experience are considered. This includes the critical role and impact of the early consideration of exposure in setting priorities and additional aspects related to treatment and analysis. Priorities for further work to facilitate more systematic consideration of combined exposures in drinking and source water including the potential application of bioanalytical tools are also identified.
Keywords: drinking-water, problem formulation, prioritization, risk assessment
Sharing Data on Chemicals in Different Environmental Matrices and Humans – How Data from Chemical Monitoring Programmes Can Be Easily Discovered, Accessed and Retrieved (the European Commission’s IPCHEM Platform)
Stylianos Kephalopoulos1 & Silvia Dalla Costa1
1European Commission, DG Joint Research Centre
Directly following-up to the European Commission’s Communication “Combined Effects of Chemicals – Chemical mixtures” (EC, 2012), DG JRC has developed the EC’s reference Information Platform on Chemical Monitoring data (IPCHEM).
IPCHEM supports a more coordinated approach for searching, accessing, retrieving, assessing and sharing data related to the occurrence of chemicals and chemical mixtures across various media (e.g. environment, humans, food & feed, indoor air and consumer products) (https://ipchem.jrc.ec.europa.eu/).
IPCHEM was designed and implemented as a distributed infrastructure, providing where feasible, remote access to existing relevant information systems and data providers.
European Commission services (DGs ENV, SANTE, JRC, RTD), European Agencies (EFSA, EEA, ECHA) and EU MS are actively involved in IPCHEM’s development, promoting its use and engaging new data providers.
IPCHEM’s primary objectives are focused on: (1) Assisting policy makers and scientists to discover and access chemical monitoring data on existing, new, emerging and less-investigated chemicals covering a range of matrices and media; (2) Hosting data currently not readily accessible (e.g. outcomes of research projects, off-line stored monitoring data, etc.) that will be searchable and accessible through the platform; (3) Providing chemical monitoring data and information of defined quality in terms of spatial, temporal, methodological and metrological traceability; (4) Facilitating exposure and risk assessment practices in support of EU policies to adequately address the risks from exposure to multiple chemicals from different sources and pathways.
IPCHEM contains already a wealth of data thanks to the contributions of several EU bodies, government agencies and research consortia: more than 33 million of chemical concentration measurements data are accessible, retrievable and downloadable today via IPCHEM.
Keywords: information platform, chemical monitoring data, chemical mixtures
Development of National Chemical Registers and Using Information on Chemical Mixtures to Prioritize Chemicals for Risk Assessment. Use of In Silico and Testing Methods
Mykola Prodanchuk, Serhii Kolesnyk, Oleksandr Kravchuk, Petro Zhminko, Olena Ryabuha
L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health of Ukraine
There are several chemical registers in Ukraine: registers of Hazardous Factors, Food Additives, Flavorings, Pesticides and Agrochemicals, Disinfectants. According to the legislation, chemical and biological substances are permitted for use only being certified through the state registration procedure. Most information required for classification, labelling and for safety datasheets is included in the certificate: product identification; physico-chemical properties; use and storage; risk of ignition and burning; environmental and human toxicity and biological effects; safety standards; hazard class; analytical methods; first aid for acute poisoning; ecological safety. Thus, existing registers include some information necessary for risks assessment of mixtures (RAM).
To pull out data necessary for RAM quickly and effectively, the existent chemical registers should be digitalized and unified. Changes in regulations are also needed to advance work on RAM.
Given high attention to endocrine disrupting properties of pesticides and biocides, algorithms for using “in silico” methods for identification of substances with high affinity for the same nuclear receptors are being developed in our center for further identification of groups for toxicity assessment and RAM.
We are conducting a research of effects of combined exposure to mixtures of pesticides in the pesticide formulations. Acute toxicity studies were performed and showed that combined effects of two or more active substances contained in the same product often lead to potentiation of toxicity. Further studies of repeated dose toxicity are planned. In addition, a study of combined action of pesticides that are a part of single-component plant protection products but used in one agrotechnology is also interesting.
Keywords: chemical mixtures; pesticides, in-silico methods, toxicity, risk assessment
Assessing Possible Interactions in Chemical Mixtures: Case Study on Cadmium and Polychlorinated Biphenyls
Aleksandra Buha 1 and Vesna Matovic1
1 Department of Toxicology “Akademik Danilo Sodatović“, University of Belgrade-Faculty of Pharmacy, Serbia
Humans are concurrently exposed to a great number of chemicals from different sources, thus toxicological evaluation should be focused on their “cocktail effects“. Up to date, several types of models for assessing the mixture toxicity have been proposed; all having certain limitations and/or being difficult to interpret.
The aim of the study was to assess possible interactions between chemicals in mixtures using the concept that analyzes the differences in the slope of dose–response curves for single chemicals and their mixtures. A mixture of cadmium (Cd) and polychlorinated biphenyls (PCBs) was chosen as a representative one.
The experimental design consisted of twenty-three groups of rats orally treated during 28 days with different doses of Cd (six groups), different doses of PCBs (six groups), different dose combinations of Cd and PCBs (nine groups), and vehicles (two controls). Investigated end points were effects on blood, liver, kidney, and thyroid function.
The study revealed certain important limitations of the applied method. Namely, for some investigated parameters, data on the assessed interactions between chemicals in mixtures were dependent on the chemical chosen as a comparative one. Furthermore, if the effect was not dose dependent it was not possible to assess whether chemicals acted independently or produced joint actions.
It can be concluded that defining an adequate model for the mixture toxicity assessment i.e. the model that will consider interactions between chemicals in mixtures, is of paramount importance in predicting the entire range of mixture toxicity.
Keywords: cocktail effects, slopes, dose-response curves, limitations