Environmental Risk of carcinogenic toxicants and emerging pollutants: Testing their interactions in vivo and in vitro

Event Abstract Back to Event Environmental Risk of carcinogenic toxicants and emerging pollutants: Testing their interactions in vivo and in vitro Carla Martins1, 2*, Virginia Cunha3, Kristian Dreij3 and Pedro M. Costa1 1 Unidade de Ciências Biomoleculares Aplicadas, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Portugal 2 Centro de Ciências do Mar e do Ambiente (MARE), Portugal 3 Unit of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Sweden Environmental pollution and loss of biodiversity in aquatic ecosystems has long been awakening the society to the real problems generated by human activities. However, the levels of both ‘traditional’ pollutants, like metals and polycyclic aromatic compounds (PAHs), and ‘emerging’ substances, such as pharmaceuticals and derivatives, are still increasing in the aquatic milieu (with emphasis in estuaries, which act as ‘sinks’), part due to inefficient removal at wastewater treatment plants (EEA, 2010; Escher et al, 2011; Verlicchi et al, 2012). Nonetheless, for both types of pollutants, environmental quality guidelines are drawn for individual compounds and neglect that, in the environment, toxicants almost invariably occur as mixtures. The concerns over the complex mixtures of toxicant being formed are now far from restricted to an ecological matter but also as a human health problem. To address the problem of toxicant mixtures, we selected the genotoxicant and carcinogen benzo[a]pyrene (B[a]P) among traditional pollutants, whose sources can be, for example, from combustion of fuels or cigarette smoke. To illustrate the emerging pollutants Diclofenac was chosen, a Non-Steroid Anti-Inflammatory Drug (NSAID), whose production and discharge to the environment increases already caught authorities’ attention, including the European Environmental Agency (EEA, 2010). This drug is considered one of the most toxic NSAIDs for wildlife (Oaks, 2004). In fact, the interesting about these two pollutants is their possible intersection in toxicopathological pathways. Whereas B[a]P is a potential carcinogen (Grivennikov, 2010), Diclofenac is judged to have an anti-tumour effect (Kaur and Sanyal,2011). Consequently, the objective of this work, more than understanding individual effects, is to study the effects of the interaction between emerging (Diclofenac) and traditional aquatic pollutants (Benzo[a]Pyrene) under ecologically-realistic circumstances. In order to evaluate the contaminants’ effects, we applied both zebrafish embryos and HepG2 cells (human) as biological models in for testing. In brief, zebrafish adults (males and females) were placed in individual spawning tanks during the night and at the morning the eggs are collected to a Petri dish with reconstituted water, according to the OECD guidelines for the fish embryo test (FET). The viable eggs were dechorionated with the aim of improve the action of the contaminants and placed in glass Petri dishes. In each dish were placed ten eggs in 10 mL of embryo medium. At 4 h post-fertilization (sphere stage) the eggs were exposed to two individual concentrations (‘low’ and ‘high’) with environmental relevance for both Diclofenac (3.14E-4 µM and 3.14E-3 µM) and B[a]P (1.98E-4 µM and 1.98E-3 µM) and the correspondent mixtures of them, as well as control (with solvent DMSO and water) and blank (embryo medium only), in duplicates. This assay was repeated four times to assure replicability and cope with natural variability. The assays had a duration of 96 h and both development observations and mortality were recorded every 24 h. Medium was changed daily. The live embryos were recovered after 96 h and fixed in glutaraldehyde 2.5%, 0.1 M (pH7.4) and archived for future evaluations. HepG2 cells were cultured in medium with 10% fetal bovine serum and appropriate supplements and maintained at 37 °C. Prior to exposure cells were seeded at 2 X 104 cells/ml in 96-well plates and cultured for 24 h. Cells were exposed to solvent control (0.1% DMSO), B[a]P, Diclofenac and mixtures for up to 48 h. We tested B[a]P and Diclofenac between 1.28E-4 µM and 50 µM and 2.56E-4 µM and 500 µM, respectively. In both models, the low concentrations for Diclofenac and B[a]P were selected based on European limits and maximum quantities found in European waters. In order to evaluate the viability of the cells exposed to contaminants and establish the EC50 the Alamar Blue test was performed. The zebrafish embryo assays did not reveal significant alterations to the normal embryonic development (Figure 1) from eggs to larval stage (96 h). Additionally, mortality was not significantly modulated by exposure. In fact, even though there was a mortality increase on the first 48 h (Figure 2), no differences were found between controls, individual toxicants and mixtures. Therefore, this increase is related to the viability of the eggs and dechorionation process. The low concentration of individual toxicants (deemed to be environmental relevant) was probably the reason for the absence of gross toxicopathological alterations. However, chronic internal damage to developing organs is still under assessment and such alterations fall better within the class of alterations that would be expected for either toxicant to fish, as they possess high EC50 in vivo albeit interfering with neoplasia and inflammation. At this point concentration are to be increased to levels around to (1/5) EC50 in order to simulate a bad pollution scenario. Nevertheless, these findings assisted in developing the best assay to address mixtures and non-lethal effects. In parallel, HepG2 cells permitted checking a much wider range of concentrations and showed that they provide an expeditious and consistent alternative to develop tests for pollutant mixtures. The results so far indicate important evidence for potential chronic, rather than acute, effects of the toxicants and their mixture. Indeed, the findings confirm that cell death may not necessarily correlate to hazard when organic toxicants known to interfere with specific toxicological pathways are involved, especially those involving CYP-mediated bio-activation of compounds into more hazardous metabolites. The combination of the two tests is seemingly able to reliably test complex mixtures of toxicants and provide vital clues to their-mode-of action that can be used to steer future research and eventually contribute to re-evaluate guidelines and safety thresholds for all surface waters, freshwater, brackish and marine, which are invariably the ultimate fate of pollutants. Figure 1 Figure 2 Acknowledgements Portuguese Foundation for Science and Technology (FCT) for the funding of the research project GreenTech (PTDC/MAR-BIO/0113/2014) and to UCIBIO, financed nationally through the strategic programme (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). The grants SFRH/BD/120030/2016 to C.M. and IF/00265/2015 to P.M.C. (from FCT). This study was supported by the Swedish Cancer and Allergy foundation and the ÅForsk foundation (to K.D.) References EEA (2010). Pharmaceuticals in the environment. Technical Report nº 1/2010 Escher B.I., Baumgartner, R., Koller, M., Treyer, K., Lienert, J., McArdell, C.S. (2011). Environmental toxicology and risk assessment of pharmaceuticals from hospital wastewater. Water Res. 45, 75-92. doi: 10.1016/j.watres.2010.08.019 Grivennikov, S.I., Greten, F.R., Karin, M. (2010). Immunity, Inflammation and cancer. Cell 140, 883-899. doi: 10.1016/j.cell.2010.01.025 Kaur, J., Sanyal, S.N. (2011). Diclofenac, a selective COX-2 inhibitor, inhibits DMH-induced colon tumorigenesis through suppression of MCP-1, MIP-1alpha and VEGF. Mol. Carcinog. 50:707-718 doi: 10.1002/mc.20736 Oaks, J.L., Gilbert, M., Virani, M., Watson, R.T., Meteyer, C.U., Rideout, B.A., Shivaprasad, H.L., Ahmed, S., Chaudry, M.J.I., Khan, A.A. (2004). Diclofenac residues as the cause of vulture population decline in Pakistan. Nature 427, 630-633. doi:10.1038/nature02317 Verlicchi, P., Al Aukidy, M., Zambello, E. (2012). Occurrence of pharmaceutical compounds in urban wastewater: removal, mass load and environmental risk after a secondary treatment-a review. Sci. Total Environ. 429, 123-155. doi: 10.1016/j.scitotenv.2012.04.028 Keywords: aquatic pollution, HepG2, Non-steroid anti-inflammatory drugs, polycyclic aromatic hydrocarbons, Zebrafish Conference: IMMR'18 | International Meeting on Marine Research 2018, Peniche, Portugal, 5 Jul - 6 Jul, 2018. Presentation Type: Oral Presentation Topic: Biodiversity, Conservation and Coastal Management Citation: Martins C, Cunha V, Dreij K and Costa PM (2019). Environmental Risk of carcinogenic toxicants and emerging pollutants: Testing their interactions in vivo and in vitro. Front. Mar. Sci. Conference Abstract: IMMR'18 | International Meeting on Marine Research 2018. doi: 10.3389/conf.FMARS.2018.06.00035 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Apr 2018; Published Online: 07 Jan 2019. * Correspondence: MD. Carla Martins, Unidade de Ciências Biomoleculares Aplicadas, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Caparica, Portugal, c.martins@campus.fct.unl.pt Login Required This action requires you to be registered with Frontiers and logged in. 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