RESUMO
Chlorpyrifos (CPF) is an organophosphate pesticide, commonly detected in water and food. Despite CPF toxicity on aquatic species has been extensively studied, few studies analyze the effects of CPF on fish transcriptional pathways. The Pregnane X receptor (PXR) is a nuclear receptor that is activated by binding to a wide variety of ligands and regulates the transcription of enzymes involved in the metabolism and transport of many endogenous and exogenous compounds. We evaluated the mRNA expression of PXR-regulated-genes (PXR, CYP3A27, CYP2K1, ABCB1, UGT, and ABCC2) in intestine and liver of the rainbow trout, Oncorhynchus mykiss, exposed in vivo to an environmentally relevant CPF concentration. Our results demonstrate that the expression of PXR and PXR-regulated genes is increased in O. mykiss liver and intestine upon exposure to CPF. Additionally, we evaluated the impact of CPF on other cellular pathway involved in xenobiotic metabolism, the Aryl Hydrocarbon Receptor (AhR) pathway, and on the expression and activity of different biotransformation enzymes (CYP2M1, GST, FMO1, or cholinesterases (ChEs)). In contrast to PXR, the expression of AhR, and its target gene CYP1A, are reduced upon CPF exposure. Furthermore, ChE and CYP1A activities are significantly inhibited by CPF, in both the intestine and the liver. CPF activates the PXR pathway in O. mykiss in the intestine and liver, with a more profound effect in the intestine. Likewise, our results support regulatory crosstalk between PXR and AhR pathways, where the induction of PXR coincides with the downregulation of AhR-mediated CYP1A mRNA expression and activity in the intestine.
Assuntos
Clorpirifos , Inseticidas , Oncorhynchus mykiss , Animais , Clorpirifos/toxicidade , Inseticidas/toxicidade , Fígado , Oncorhynchus mykiss/genética , Receptor de Pregnano X/genética , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
The development of oil and gas production together with the fruit production in nearby areas of North Patagonia, Argentina, suggests aquatic pollution scenarios which include permanent oil pollution combined with short events of pesticides application. It has been reported that oil hydrocarbons activate the aryl hydrocarbon receptor (AhR) pathway in the rainbow trout, Oncorhynchus mykiss, and that the insecticide Chlorpyrifos (CPF) interacts with these effects. Thus, it is interesting to investigate whether hydrocarbons and insecticides, applied by separate or combined, can affect fish health and reproductive signaling by acting on different nuclear receptors' regulatory pathways. To study this kind of interactions, we exposed juvenile rainbow trout to water accommodated fraction (WAF) of crude oil (62 µg L-1 TPH) for 48 h and subsequently exposed the livers ex vivo to the insecticide Chlorpyrifos (CPF) (20 µg L-1) for 1 h. We analyzed the mRNA expression of nuclear receptors and proteins involved in detoxifying, antioxidant, immune and apoptosis responses by qRT-PCR. We also performed histopathological analysis. WAF induced the expression of the androgen (AR) and the Liver X receptor (LXR) by 8- and 3-fold, respectively. AR induction was reversed by subsequent exposure to CPF. The progesterone receptor (PR) and glucocorticoid receptor (GR) were increased 2-fold and 3-fold by WAF respectively, while estrogen and mineralocorticoid receptors were not affected. GR was also induced by CPF with an additive effect in the WAF-CPF treatment. The antioxidant genes, gamma glutamyl transferase (GGT), superoxide dismutase (SOD1) were induced by WAF (2-3-fold). WAF upregulated the ATP Binding Cassette Subfamily C Member 2 (ABCC2, MRP2) (4-fold) and downregulated alkaline phosphatase. WAF also induced the inflammatory interleukins (IL) IL-8, and IL-6 and the anti-inflammatory IL-10, while CPF induced the inflammatory tumor necrosis factor (-α) and IL-6, and activated the intrinsic apoptotic pathway through the induction of caspases 3 and 9. Both, WAF and CPF downregulated the expression of the extrinsic apoptosis initiator caspase 8 and the inflammatory caspase 1. In conclusion, WAF hydrocarbons alter O. mykiss endocrine regulation by inducing AR, PR and GR. The subsequent exposure to CPF reverses AR, suggesting a complex interaction of different pollutants in contaminated environments, WAF hydrocarbons alter liver metabolism by inducing the expression of LXR, GR, antioxidant and detoxifying enzymes, and both inflammatory and anti-inflammatory cytokines, and causing mild hepatic steatosis. CPF activates inflammatory and stress responses associated with the induction of inflammatory cytokines together with apoptosis initiator and executioner caspases.