9^th Euro-Global Summit on Toxicology and Applied Pharmacology June 22-24, 2017 Paris, France

Anaïs Wakx has graduated in Toxicology from the Paris Descartes University, works on the impact of micropollutants, particularly endocrine disruptors and carcinogenic, mutagenic and reprotoxic substances on placenta. She is the recipient of the OPAL Prize awarded by the National Academy of Pharmacy for her work on alternative methods for animal experimentation. Her current work is supported by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES).

Statement of the Problem: Benzo(a)pyrene (BaP) is a persistent environmental pollutant produced by organic combustion from various sources, and is currently classified as highly carcinogenic, mutagenic and reprotoxic substance. Human placenta-an interface between the fetus and the mother plays a critical role as a barrier against many xenobiotics. However, pollutants such as BaP could reach and cross this barrier and thus modify its physiological functions, with significant implications on pregnancy outcome. Within the cell, the main target of BaP is the aryl hydrocarbon receptor (AhR), a cytosolic ligand-activated transcription factor which relocates and triggers the expression of genes involved in xenobiotic metabolism. However, recent data suggest that the AhR is not limited to this process and may assure functions other than detoxification. Our aim was to evaluate the expression, localization and activation of the AhR in several placental models, as well as the impact of BaP exposure toward these parameters. Methodology & Theoretical Orientation: we characterized, for the first time, the expression profile of the AhR and its activity toward its main target genes in human placental chorionic villi during different periods of pregnancy. Using different methods such as cell fractionation and fluorescence microscopy, we unexpectedly demonstrated a nuclear localization of the AhR in 1) freshly harvested chorionic villi throughout pregnancy, 2) purified term placental primary cells during differentiation into syncytiotrophoblast and 3) BeWo cell line in the absence of any exogenous activator, suggesting an intrinsic activation of the AhR. Furthermore, we studied the impact of BaP exposure toward the localization and activity of this receptor in human trophoblast cells. The expression of AhR, its partner ARNT and its repressor AhRR were unaffected. However, we demonstrated a role of the AhR upon BaP exposure (using RT-qPCR) on the expression of various critical genes involved in different processes such as detoxification, placental function, oxidative stress, inflammation, and epithelial to mesenchymal transition. Conclusion & Significance: To our knowledge, this is the first study of a global functional cartography of the AhR in human placenta. Recent Publications 1.Wakx A, Regazzetti A, Dargère D, Auzeil N, Gil S, Evain-Brion D, Laprévote O, Rat P (2016) New in vitro biomarkers to detect toxicity in human placental cells: The example of benzo[A]pyrene. Toxicol. In Vitro 32: 76-85. 2.Stejskalova L, Pavek P (2011) The function of cytochrome P450 1A1 enzyme (CYP1A1) and aryl hydrocarbon receptor (AhR) in the placenta. Curr. Pharm. Biotechnol. 12(5): 715-30.

Anja Mikolić has graduated from the Faculty of Food Technology and Biotechnology, University of Zagreb in 2005. She acquired her PhD in Biomedicine and Health Sciences from the Faculty of Pharmacy and Biochemistry, University of Zagreb in 2015. She is employed in Analytical Toxicology and Mineral Metabolism Unit at Institute for Medical Research and Occupational Health (IMROH), Zagreb, Croatia since 2007. Her professional fields are designing and performing in vivo studies on experimental animals (rats); studying biomarkers of oxidative stress; assessment of human exposure to essential and toxic metals by bio-monitoring, interactions of metals and reproductive toxicity; development and application of analytical methods regarding hormone and antioxidant enzyme analysis. She has published 6 original scientific papers in the international peer-reviewed journals, participated in few national and international scientific conferences and is a member of few scientific associations.

Terbuthylazine is a chlorotriazine herbicide that acts primarily as an inhibitor of photosynthesis. It is used mainly in the production of maize but is also applied in the cultivation of various fruits, and vegetables, as well as in forestry. We evaluated the in vivo effects of terbuthylazine following a 28-day oral administration in adult male Wistar rats at 2.300 mg/kg b.w./day, 0.400 mg/kg b.w./day, and 0.004 mg/kg b.w./day. After treatment, oxidative stress responses were measured in the blood of both the terbuthylazine-treated and the control rats using biomarkers of lipid peroxidation, activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase. To establish DNA damaging effects, we applied an alkaline comet assay on the leukocytes of the exposed and control animals. Exposure to terbuthylazine slightly disturbed lipid peroxidation, which was not dose-dependent. Treatment also led to increases of GSH-Px activity in blood, which was statistically significant at 0.400 mg/kg b.w./day. The observed increase of SOD activity in erythrocytes was most prominent at the highest dose tested. On the other hand, plasma SOD activity dropped significantly at two lower doses. Catalase activity in plasma decreased, which was statistically significant at the highest dose. Terbuthylazine produced measurable effects on leukocyte rat DNA. Based on the values measured for comet tail length and tail intensity, which were both lower at the two higher concentrations, it can be concluded that one of possible mechanisms of terbuthylazine’s effect on the DNA molecule includes intercalation. This may in turn slow down the migration of DNA during electrophoresis. Taken together, our findings suggest that the 28-day exposure of rats to very low doses of terbuthylazine, which were within toxicology reference values, resulted in oxidative stress responses and low-level DNA instability. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios.

Chungsik Min has been working for many years at the MFDS (Ministry of Food and Drug Safety) of Korea. He has a professional experience in risk assessments for food safety management and reviewing data for drug authorization. Nowadays, he works at cosmetic research team under NIFDS (National Institute of Food and Drug Safety Evaluation, under MFDS), to manage the safety of cosmetics. He has a lot of interest in not only the safety of preservative and sunscreen in their effect but also the management of pollutant in cosmetics.

Nickel is present naturally in the earth. Chronic exposure of nickel may cause skin allergic reaction, skin rash, etc. Also, IARC classified nickel compounds in Group 1 (carcinogenic to humans) and metallic nickel in Group 2B (possibly carcinogenic to humans). For these reasons, the cosmetic regulation of nickel provides that nickel use in cosmetic products should be prohibited in Korea and EU. However, most cosmetic products in the markets can contain technically unavoidable traces of metals. First, we analyzed the content of nickel in 123 commercialized cosmetics for monitoring of degree of nickel contamination by using ICP-Mass. As the results, nickel is contained as up to 0.0039% in eye products (not detected 9 products), 0.0029% in make-up products (not detected 7 products) and 0.0003% in all other cosmetic products (not detected 30 products), respectively. Using the monitoring results and HT25 of nickel which is 0.188 mg/kg/day, life time cancer risk was evaluated for nickel in cosmetics to be 3.0x10-6. So, nickel has a risk of carcinogenesis of less than 10-5. Also, using the monitoring results and EC3 2.5% of nickel, the skin sensitization (AEL/CEL) was evaluated for nickel in cosmetics to be 1.1, which is greater than 1.

Maistro E L has completed his PhD from Universidade Estadual Paulista (UNESP), São Paulo, Brazil and Post-doctoral studies from Universidade Estadual de Londrina (UEL), Brazil. He works as a Professor and Researcher in the Speech and Hearing Science department. He has published more than 75 papers in reputed journals. His studies focus on genotoxic and anti-genotoxic assessment of natural products, medicinal plants and its major constituents.

Statement of the Problem: Crataegus oxyacantha L. berries, leaves and flowers and their extracts have been used as astringent, antispasmodic, cardiotonic, diuretic, hypotensive and antiatherosclerotic agents. Popularly known as hawthorn, is particularly used in the treatment of various heart problems. In traditional Chinese medicine, hawthorn fruits are used to treat stomach diseases, diarrhea, abdominal pain, as well as amenorrhea, hypertension, and hyperlipidemia. Its fruits are also consumed as foodstuff (canned fruit, jam, jelly, drink, and wine). Despite of the pharmacological potential of C. oxyacantha, our literature review showed absence of genetic toxicity studies specifically on its fruits. Therefore, the present study was carried out to evaluate the genotoxic potential of this fruits extract. Methodology: In vitro analysis was performed using leukocytes and liver (HepG2) human cultured cells. In the in vivo protocol, mice were treated by oral gavage (50, 100, and 200 mg/Kg body weight doses) during seven consecutive days at 24 hours interval. SCGE (comet) and micronucleus assays were the cytogenetic tests used in the genotoxic assessment. Results & Conclusion: The in vitro results showed that the fruits extract presented DNA and chromosome damage at concentrations of 5, 10, 50, and 100 µg/ml in both human cell types. The in vivo findings showed that extract did not induce significant DNA damage in leukocytes and bone marrow cells by the comet assay; however, was observed a significant increase in micro-nucleated polychromatic erythrocytes at the three tested doses. Our experimental conditions allowed us to conclude that C. oxyacantha fruits extract present genotoxic effects in cultured human cells, which were confirmed in the in vivo assay on mice bone marrow cells. These results suggest caution on its use by humans and indicate the necessity of complementary genotoxic studies in different formulations containing extracts of this plant.

Maria João Silva has graduated in Pharmaceutical Sciences and PhD in Human Genetics and Toxicology. She is the Principal Investigator of the Group of Genetic Toxicology, Department of Human Genetics, National Institute of Health, Portugal. She is also a member of the Scientific Coordination of the Center of Toxicogenomics and Human Health, NOVA Medical School, Lisbon. Her research interests include environmental genotoxicity (including nanotoxicology, radiobiology, and mixtures toxicology), DNA repair and molecular epidemiology. She has authored and co-authored over 30 scientific publications.

Statement of the Problem: Our previous work has shown the presence of the hazardous chemicals within a complex mixture of contaminants (e.g., metals, pesticides and polycyclic aromatic hydrocarbons) trapped in sediments of a Portuguese estuary. In that case-study, an epidemiological survey confirmed the exposure of the local population mainly through food chain, suggesting the need of a biomonitoring study that includes the quantification of contaminants in biological fluids as well as biomarkers of early biological effects (e.g., biochemical, genetic and omics-based endpoints) in the target population. Recognizing the knowledge gap between exposure to hazardous substances and health outcomes, not only in Portuguese population, but also throughout Europe, the project European Human Biomonitoring Initiative (HBM4EU) has just started, with the overarching goal of generating knowledge towards the safe management of chemicals. Methodology & Theoretical Orientation: Human biomonitoring will be used to understand the human exposure to chemicals and resulting health impacts. The first steps rely on harmonizing procedures for human biomonitoring across countries, to provide policy makers with comparable data on human internal exposure to chemicals and mixtures of chemicals at EU level. Then, linking data on internal exposure with the hazardous chemicals, will allow to aggregate external exposure and identifying exposure pathways and upstream sources. Conclusion & Significance: By generating scientific evidence on the causal links between human exposure to chemicals and negative health outcomes, an evidence-base will be established to allow the use of human biomonitoring in chemical risk assessment methodologies to data. The risk management and communication with stakeholders and policy makers will ensure that results are applied in the design of new regulations for chemicals and for supporting public health protection policies. Recent Publications 1.Pinto M, Louro H, Costa P, Caeiro S, Silva M J (2015) Exploring the potential interference of estuarine sediment contaminants with the DNA repair capacity of human hepatoma cells. Journal of Toxicology and Environmental Health, Part A: Current Issues 78(9): 559-570. 2.Costa P M, Pinto M, Vicente A M, Gonçalves C, Rodrigo A P, Louro H, Costa M H, Caeiro S, Silva M J (2014) An integrative assessment to determine the genotoxic hazard of estuarine sediments: combining cell and whole-organism responses. Front. Genet. 5: 437. 3.Pinto M, Costa P M, Louro H, Costa MH, Lavinha J, Caeiro S, Silva M J (2014) Determining oxidative and non-oxidative genotoxic effects driven by estuarine sediment contaminants on a human hepatoma cell line. Science of the Total Environment 478: 25-35.

Mariana Pinhão has worked mainly in genetic toxicology, focusing on the impact of several nanomaterials on the human genome, such as titanium, silicon, silver and carbon nanoparticles, among others. She has also studied the influence of mycotoxins on the DNA stability, as well as the antioxidant potential of natural extracts, using human digestive system in vitro cell models. Currently, she is enrolled in a multidisciplinary PhD program related to biological systems, where she is working with bioinformatic tools applied to human genomics.

Statement of the Problem: The emergence of newly developed nanomaterials (NMs), with physicochemical features useful for innovative applications in consumer products and medicine, generated the need of fast- and cost-effective toxicology screening strategies for safety assessment. Overall toxicity can be evaluated using survival or cell death as an endpoint. However, in view of the NMs’ specificities, many methods are vulnerable to interference of the NMs with the detection process. We analyzed the cytotoxic effects of a panel of 10 benchmark NMs in respiratory cells (A549) using plating efficiency (PE) assay, and compared with a spectrophotometric method. Methodology: The NMs were used for PE assay in cells exposed for 7 days to silicon dioxide (NM-200 and NM-203), titanium dioxide(NM-100, NM-101, NM-103), silver (NM-300 k, NM-302), cerium oxide (NM-212), barium sulphate (NM-220), zinc oxide (NM-110) nanomaterials or carbon nanotubes (NM-401). For a core group of NMs, NM-212, NM-100, NM-101, NM-103 and NM-220, the MTT assay was also performed in 24 h exposed cells. In addition, the batch dispersions and their dilutions in the cell medium were analyzed by dynamic light scattering (DLS). Findings: The size distribution of the dispersions showed that in general the NMs showed peaks around 100 nm, but a multimodal size-distribution was observed for NM-103, NM-200, NM-302 and NM-401. Considering the PE assay, NM-212, NM-101, NM-110, silicon dioxides and both silver NMs were cytotoxic. On the contrary, NM-100, NM-103 and NM-220 were not cytotoxic. In the MTT assay following 24h- exposure to the core NMs, no significant cytotoxicity was observed. Conclusion & Significance: Overall, cytotoxicity determined in PE assay yielded positive results in the majority of the NMs, suggesting that this methodology may be more sensitive than MTT to NMs’ induced toxicity. Furthermore, long-term exposure to NMs is possibly more relevant to address health impact of NMs.

W D V Karunaratne has done her BSc degree in Chemistry from the University of Colombo and the MSc degree from the University of Peradeniya, Sri Lanka. She joined the Toxicology Laboratory of the Government Analyst’s Department of Sri Lanka as an Assistant Analyst in the year 1996. She has completed her training in Analytical Skills Development course and Laboratory Quality Management course in VERIFIN at the University of Helsinki, Finland. Since 2006, she serves as the Head of the Toxicology Laboratory, where she is responsible for the analysis of alcohol, other volatiles, carbon monoxide, drugs and poisons such as cyanide, phosphene, metallic poisons and pesticides in biological samples for forensic toxicology casework island wide and issue reports to the judicial medical officers and to the courts of law. She also serves as the Technical Manager for the ISO/IEC 17025 accreditation of the Toxicology Laboratory. She has also published several pier reviewed scientific papers. She is a member of International Association of Forensic Toxicology (TIAFT), Medico-legal Society of Sri Lanka, Institute of Chemistry Sri Lanka and the Sri Lanka Association for the Advancement of Science.

Globally there is a scarcity of information on the magnitude of intentional poisoning, as well as the relative importance of different pesticides. Community based data on the pesticides responsible for fatal self-harm are not available for most of the rural Asia. The variety of pesticides available in communities for intentional poisoning is large, reflecting the pesticides used in local agriculture. This paper discuss the particular types of pesticides identified in 59 fatal self-poisoning cases analyzed in the toxicology laboratory of the Government Analyst’s Department of Sri Lanka during the year 2016. Out of 59 cases 36 (61.0%) were carbamate poisoning cases and 19 (32.2%) were organophosphate poisoning cases. In one case (1.7%) both organophosphate and carbamate were identified. Tartan was identified in 3 (5.0%) cases. Particular types of carbamate and organophosphate pesticides identified the sex and age patterns and their occupation and residing areas will be discussed in the paper.

Airton da Cunha Martins Jr. is a PhD student from School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo. He has experience in toxicology, with emphasis in toxicology of metals.

Statement of the Problem: Arsenic (As) is a toxic element that is widely distributed in the Earth’s crust and may be found in a variety of chemical forms, including organic and inorganic species. Rice (Oryza sativa L.) is global staple food that can be accumulated more than other commodities. Studies have demonstrated the toxic effects of As due to exposure and accumulation of this element through rice consumption. The purpose of this study was to evaluate the As species found in a Brazilian cultivar and As distribution in animals after consumption of rice containing As. Methodology: The BR IRGA 409 rice cultivar was planted in soil contaminated with As at 10 ppm concentration; A control rice cultivar was planted without addition of As in soil. After analyzing the As concentration in rice and speciation, Swiss mice were divided into 2 groups and chronically treated with As through the diet. The tissue concentration was realized by ICP-MS. Findings: The mean of As rice concentration in control cultivar was 55±0.01 ng g-1 of As, while the concentration of this element in exposed cultivar was 426 ng g-1. The chemical species present in the highest concentration were As3+ and DMA. We observed higher concentrations of these species in exposed cultivar (172 and 218 ng g-1) compared to control cultivar (40.4 and 17.7 ng g-1). The highest concentration of As in the group treated with rice contending As was found in testicles followed by skin and hair. Conclusion & Significance: The preliminary results of this study indicate that after rice consumption, testicles were the organ with highest concentration of As. Future studies on the speciation of As in this tissue will be performed. Recent Publications 1.Segura F R, de Oliveira Souza J M, De Paula E S, da Cunha Martins A, Paulelli A C C, Barbosa F, Batista B L (2016) Arsenic speciation in Brazilian rice grains organically and traditionally cultivated: Is there any difference in arsenic content? Food Research International 89: 169-176.