Study of genotoxic effects on exocrine pancreas after chronic dietary exposure to cocktail of pesticides
Résumé
Pancreatic ductal adenocarcinoma (PDAC) is projected to become the third leading cause of cancer-related death by 2030. One reason could be the environmental exposures, including pesticides, which could induce or alter cellular mechanisms such as genotoxicity, microenvironment remodelling and autophagy. The aim of our work was first to study the genotoxic effects of pesticide exposure on exocrine pancreas by comet assay. To avoid the DNA damage caused by the tissue autodigestion and/or isolation method, we optimized the protocol of acinar cells isolation to reach a very low basal DNA damage (<5% of tail DNA). Pancreatic tissue was grinded mechanically using dounce in HBSS-EDTA 0.02M buffer supplemented with STI (Soybean Trypsin Inhibitor). Acinar cell isolation was confirmed by flow cytometry before performing alkaline comet assay, analysed with Instem Comet IV software. We studied thereafter the effects of five month exposure to a cocktail of pesticides (Imazalil, Thiabendazole, Boscalid, Lambda-Cyhalothrine) called ITBC on exocrine pancreas of mice fed with those components at low doses (acceptable daily intake (ADI) or 10 ADI of the pesticide cocktail). The levels of DNA damage were assessed in both control chow diet (CD) and obesogenic western diet (WD). These effects will be illustrated by statistical graphs, some based on a logit transformation of the tail intensity, and evaluated through statistical tests. To complete this study, we performed histological analysis on pancreas with hematoxylin/eosin colouration. Autophagy markers were also studied such as autophagy inducer Vps34, expressed mostly in pancreatic acinar granules. Regeneration and remodelling of the tissue were studied by analysis of proliferation markers, fibroblast activation and fibrillar collagen composition. In conclusion, we developed a suitable method for isolating fresh acinar pancreatic cells without damaging their DNA for comet assay analysis. We applied this novel methodology to analyse the genotoxic effects of chronic dietary exposure to pesticides.