|GENOPES - Mechanisms of DNA damage induced by pesticides in fish and interference of co-exposure to other contaminants|
Programme - PTDC/AAC-AMB/114123/2009
Execution dates - 2011-04-01 - 2014-03-31 (36 Months)
Funding Entity - Fundação para a Ciência e Tecnologia (FCT)
Funding for CESAM - 79250 €
Total Funding - 90050 €
Proponent Institution - Universidade de Aveiro
Universidade de Trás-os-Montes e Alto Douro (UTAD)
Genotoxicity stands for the most adverse impact of chemicals on wild organisms. Keeping in view pesticides as one of the most ubiquitous and hazardous group of contaminants, the leitmotiv for the present project was the recognition of a gap on the literature concerning the genotoxic potential of these chemicals to fish. The well documented genotoxicity to bacterial and mammalian systems [1-3], allied to the positive results found in the few available studies on fish [4-7], constitute the rationale to redirect scientific efforts on this direction. Though mammal studies demonstrated that DNA damage and oxidative stress are mechanistically linked in exposures to pesticides [8-9], in relation to fish the mechanisms involved remain unidentified.
Therefore, the fundamental research question of the project is the assessment of the genotoxicity of pesticides to fish and the development of a biologically based knowledge on DNA damage mechanisms. The strategy includes the evaluation of genotoxic endpoints (i) coupled with crucial processes on determining the extent and type of damage, viz. (ii) activation/detoxification enzymes, (iii) defense mechanisms and (iv) DNA repair/cellular turnover.
As genotoxic endpoints (i) the comet assay in the standard version and in combination with specific DNA repair enzymes (FPG, endonuclease III) will be adopted to determine DNA strand breaks and oxidized bases, respectively. In addition, the micronucleus (MN) and erythrocytic nuclear abnormalities (ENA) assays will be adopted as markers of clastogenic/aneugenic effects. As indicators of bioactivation and detoxification (ii), P450 mixed-function oxidases and conjugative enzymes will be evaluated. Antioxidant status and stress proteins will be estimated as defense mechanisms (iii). To evaluate the (hypothetical) transient nature of the DNA lesions, as a result of DNA repair/cellular turnover (iv), the previous genotoxic endpoints will be followed along a post-exposure period.
In order to assess tissue-specific vulnerability, all the parameters will be determined in blood, gill, kidney, liver and brain, with the exception of MN and ENA assays that will be applied only in blood.
The European eel (Anguilla anguilla) will be adopted as test organism since it fulfils most requirements as bioindicator species.
In agricultural practice, multiple pesticide applications are a rule and not an exception. In addition, the real situation is even more complex as agrochemicals appear in the aquatic environment as part of a cocktail of different classes of contaminants. Hence, the co-exposure to pesticides and metals or nutrients is a challenging, but likely, scenario, namely in euthrophic riverine and lacustrine habitats. Accordingly, to assess pesticide genotoxicity under ecologically relevant conditions, the project will be developed in three consecutive and complementary steps:
1 – Laboratory exposures (hours-days) to pesticides representative from the main classes, viz. endosulfan (organochlorine), glyphosate (organophosphate) and carbofuran (carbamate), individually and in binary mixtures;
2 – Laboratory exposures (hours-days) to mixtures pesticide-metal (Cr or Cu) and pesticide-nutrient (nitrate or nitrite);
3 – Field caging experiments (hours-days) in a freshwater lagoon - Pateira de Fermentelos (central region of Portugal), adopted as a prototype of multi-contaminated system, as it receives inputs of domestic sewage, pesticides and industrial effluents containing metals. In situ exposures will be carried out seasonally and the assessed biomarkers will be complemented with quantification of pesticides and metals in water, sediment and fish tissues.
The research team has vast expertise in the toxicity (biochemical, physiological, cytogenetic and histological changes) of various contaminants in fish [10,11], and also holds young researchers keeping in view the training of scientists. The team has been demonstrating the perception of the importance of interactions in multi-contamination conditions (in field  and laboratory studies ), as well as its commitment to search mechanistic linkages between oxidative stress/biotransformation and DNA damage [14,15], which is a determinant background to achieve the proposed goals. Furthermore, the team has an excellent knowledge of the experimental model (A. anguilla), supported in two decades of investigations .
The proposed strategy is completely new in the context of fish, on one hand, due to its integrative character substantiated in the broad spectrum of processes addressed playing a modulatory role on DNA damage, and on the other, by the application of the comet assay with lesion-specific endonucleases that can offer valuable data on the mechanisms of breaks generation. Finally, the expected results will provide useful recommendations for policy-making, fundamental to (re)formulate regulatory processes for protecting the environment health.