Effects of p,p-DDE on the oxidative stress in rat tissues and in cell cultures. Study of the mitochondrial dynamics variations in vitro.

Exposure to environmental contaminants is one of the most associated risk factors for health damage. The most well-known and harmful synthetic molecules introduced in the environment are the pesticides. Acts to reduce the infectious risk on plants, animals and humans, the pesticides can cause a variety of toxic effects due to ingestion, contact direct with skin or by their entry into the body through the respiratory tract. The toxicity of these substances also depends by their ability to persist in the environment. In fact, the persistent substances are subject to bioaccumulation and biomagnification phenomena in the food chain. For example, Dichlorodiphenyltrichloroethane (DDT) and its metabolites go against to these processes. The purpose of this work was done to evaluate the effects of the Dichlorodiphenyldichloroethylene (p,p-DDE), the first metabolite of p,p-DDT, in male Wistar rats for 4 weeks of treatment at a dose of 10 mg / Kg b.w. Since p,p-DDE is a lipophilic organic pesticide, its effects were also monitored in association with a diet rich in saturated fatty acids. Four experimental groups were studied: N (Normal Diet); D (High-Fat Diet, 45% saturated); N + DDE (Normal Diet + p,p-DDE 10 mg / Kg b.w.); D + DDE (High-Fat Diet, 45% saturated, + p,p-DDE 10 mg / Kg b.w). The analyzes were conducted in vivo on three different organs (testis, kidney and liver) and in vitro on the experimental model of human hepatocarcinoma cell (Huh7). The results obtained in vivo indicate for Testis, morphological alteration in some tubules, in their distal part, in D group; release of non-differentiated cells in both groups in presence of p,p-DDE; positives spermatogonia to cleaved caspase 3 in group D were found; spermatogonia, spermatocytes and spermatidis positives to cleaved caspase 3 in presence of the pesticide were observed. Probably, the activation of the apoptotic pathway is started by mitochondria. This hypothesis was formulated because in both DDE-treated groups there is an increase of Bak and BAX proteins (regulated by p53 incremented levels) when there is a need to activate cell death. Down-regulation of MTs, particularly significant in the N + DDE group. Activation of the antioxidant enzymes (SOD1 / 2, GPX1) especially in the N + DDE group, indicating a major effect of DDE on the oxidative stress generation in absence of saturated fats. Increased cell proliferation in all groups, most significant in the N + DDE group, probably to mitigate the negative effects on testis due to the apoptotic stimuli. In the Kidney, morphological alteration on the tubule-proximal component in presence of saturated fats accumulated in the cells; epithelial-tubular erosion in N + DDE with some damage on the glomerular component; peritubular collagen deposition in presence of p, p-DDE and recruitment of macrophages; activation of the antioxidant system in the enzyme component analyzed (SOD1 / 2 and GPX1) and up-regulation of reticular chaperones (Grp78 and Grp75); Up-regulation of MT and nuclear translocation particularly in the presence of pesticides to protect DNA from oxidative stress and ensure the transport of useful metals during the transcriptional processes. Finally, in the liver, we showed fatty acids accumulation in Group D and cellular vacuolization in the presence of pesticide were observed; mitochondrial superoxide anion production in all experimental groups vs. N; accumulation of lipid peroxides in presence of p,p-DDE (D + DDE, N + DDE); mitochondrial decoupling and up-regulation of the antioxidant system (SOD1 / 2, GPX1); increase of mitochondrial BAX and release of cytochrome c from mitochondria in the p,p-DDE treated groups; activation of caspase 3, bulging of the endoplasmic reticulum cisterns and increased levels of Grp78; MT down-regulation associated to their nuclear translocation in all experimental groups vs N; recruitment of macrophages in the tissue to eliminate cells death. Moreover, in vitro studies were performed on the hepatocarcinoma line cell Huh7. Cells treated with DDE exhibit, after 24h of exposition, increased levels of mitochondrial superoxide anion and hydrogen peroxide. Decrease of mitochondrial membrane potential and ATP synthesis were observed, indicating that p,p-DDE play a role in the mitochondrial disease generation. Additionally, mitochondria vary in their dynamics by fragmenting and assuming donut morphology. Marker of apoptosis were measured by western blotting analyses indicating activation of mitochondrial apoptotic pathway. Conclusions: Our results indicate that p, p-DDE generates both in vivo and in vitro oxidative stress. In vivo, at the dose and time used, buffer systems are activated to control tissue physiology; In vitro, mitochondrial damage activates cellular apoptosis (similarly to what was observed in the liver of rat model)