Evaluación de los efectos toxicogenómicos y genotóxicos de la Vinclozolina en invertebrados acuáticos

Abstract

The general objective of this work was to evaluate the toxicogenomic and genotoxic effects of the pesticide Vinclozolin (Vz) in aquatic invertebrates. It has been shown that Vz acts as an endocrine disruptor with anti-androgenic activity in mammals. Its effect on the reproduction of aquatic organisms (fish and invertebrates) has also been described, although via an unknown mechanism. In this doctoral thesis, I worked with two species of invertebrates (embryo and larva of the dipteran insect Chironomus riparius and the adult phase of the freshwater mollusk Physa acuta), with the objective of improving our knowledge of the mechanism of action of Vz in invertebrates. The toxicogenomic effects of Vz were determined following acute exposure by analysis of the expression of genes related to different physiological processes: endocrine system (EcR, E74, Kr-h1, DIS, Cyp18a1, FOXO, MAPR , INR, MET, ER); cellular stress response (Hsp16.6, Hsp17, Hsp20.4, Hsp24, Hsp40, Hsp60, Hsp70, Gp93, Cu/Zn SOD); xenobiotic metabolism (Cyp4G, Cyp2U1, Cyp3A7, Cyp4F22, GSTt2, GSTo1, GstD3, MRP1-ABC); and DNA repair mechanisms (ATM, ATR, CASP3, DECAY, NLK, RAD-1, XRCC-1, XRCC-2 and XRCC-4). To study the impact of Vz on DNA repair mechanisms, the comet assay was used to determine the DNA damage induced by double-strand breakage. Finally, the effects of Vz on reproduction were evaluated in C. riparius through the chronic exposure of larvae until emergence of the adult phase in water-sediment systems. Although Vz did not affect the survival of larvae of C. riparius or adults of P. acuta, it does affect the development of C. riparius embryos, and more severely when the parental generation was previously exposed. However, no effect was observed in the reproduction test of C. riparius except for a non-significant tendency to increase fecundity as the concentration of Vz increased. Gene expression results in both embryos and larvae of C. riparius demonstrated endocrine system effects at the level of hormonal regulation, by altering the expression of EcR, E74, MET, MAPR, FOXO and at the same time modifying the metabolism of ecdysone as reflected by DIS and Cyp18a1. However, the only hormone receptor analyzed in P. acuta (ER) did not experience changes in its expression after exposure to Vz. On the other hand, the results observed for the response to cellular stress in both embryos and larvae of C. riparius showed an activation of Hsp40, Hsp24 and Hsp70, and additionally the activation of detoxification mechanisms (Cyp4G). Again, P. acuta showed a different behavior, with inhibition of Hsp20.4 and a lack of activation of any of the genes related to detoxification processes. Comet assays demonstrated the ability of Vz to induce DNA damage in both test species, although a significant dose-response relationship for all analyzed parameters (percentage of DNA in tail, length of tail, tail moment and olive moment) was only found in the case of C. riparius. Finally, the upregulation of genes involved in DNA repair mechanisms (ATM and XRCC-1) observed in C. riparius demonstrates that this organism is capable of activating said mechanisms, while in the case of P. acuta, an inhibition was observed for the analogous genes ATM and XRCC-2. In this doctoral thesis, the genotoxic effects of Vz on aquatic insects and mollusks were verified, for the first time linking them to the transcriptional activity of genes involved in DNA repair mechanisms. The results allow us to conclude that Vz can alter key genes in the ecdysone-mediated signaling pathway, suggesting a direct interaction of this contaminant with the endocrine system of insects. In the same way, activation of cell stress targets and detoxification pathways in this group of invertebrates was confirmed that Vz can alter key genes in the ecdysone-mediated signaling pathway of C. riparius. The lack of change in endocrine pathway genes, cellular stress and detoxification observed in P. acuta brings to light the need to continue exploring other molecular targets that may play a role in the mechanism of action of endocrine disruptors in mollusks. Finally, these results highlight the necessity of using different molecular targets in various representative species from different invertebrate taxa during such studies.