Effects Of Diclofenac And Simvastatin On Nrf2 And PXR Key Detoxication Signaling Pathways In Gambusia Affinis

Recently,pharmaceuticals and personal care products(PPCPs)are widely detected in aquatic environment,which may pose potential threats to non-target aquatic organisms(i.e.fish).However,studies on the effects of PPCPs and the detoxification processes of PPCPs in fish are relatively limited.In the present study,diclofenac(a non-steroidal anti-inflammatory drug,DCF)and simvastatin(one type of lipid-regulating drug,SV),were chosen as typical representative PPCPs and a kind of widespread small fresh water fish—mosquitofish(Gambusia affinis),was employed as the experimental model organism to explore the toxic effects of diclofenac and simvastatin.The sequences of transcriptional factors Nrf2 and PXR and their downstream detoxification-related genes were cloned firstly,and the qPCR methods were established based on the cloned sequences.Moreover,the primary antibodies of Nrf2 and PXR were also sythesized on the basis of cloned genes.The expressions of Nrf2 and its downstream target genes(GCLC,NQO1,UGT,MRP2,CAT and SOD2)and PXR and its downstream target genes(P-gp,CYP3 and GSTA)in the mosquitofish under the exposures of environmentally relevant concentration DCF/SV were measured by the qPCR methods.The activities of ERND,GST,SOD and CAT and the levels of GSH and MDA were determined.In addition,the relative protein expressions of Nrf2 and PXR were analyzed by Western Blotting(WB).Moreover,the histopathological changes in liver tissues of the mosquito fish were observed after HE stain,immunohistochemical stain of Nrf2 protein(IHC),and transmission electron microscope(TEM)under SV exposure,and the relative protein expressions of ERK,JNK and p38 belonged to the MAPK pathway were also measured.Results showed that both DCF and SV had significant effects on transcriptional expression of Nrf2 and its downstream genes(GCLC,GSTA,NQO1,CAT and SOD2),enzyme activities(SOD,CAT and GST),the contents of GSH and MDA,and the protein expression of Nrf2.Transcriptional changes of PXR and its pathway genes(P-gp,CYP3 A,UGT and MRP2),ERND enzyme activity,the protein expression of PXR under the same exposures also showed significant changes.Both genes transcription and enzymes activity changes showed time dependent or concentration dependent patterns to some extent,especially Nrf2 mRNA rose with the extended exposure time,which was higher at 168 h than 24 h and 72 h.The transcriptional expressions of PXR and CYP3 A under DCF and SV exposure first increased and then decreased with increasing concentrations.Up-regulation of Nrf2,more disorganized cells distribution,larger mitochondria under SV exposure were observed.ERK,JNK and p38 protein expressions exhibitted significant changes too,suggestting there might exist the regulation relationships between MAPK pathway and Nrf2/PXR detoxification pathways.Overall,this study confirmed DCF and SV have detrimental effects to the detoxification system in the mosquitofish at different levels,and revealed the potential risk of PPCPs like diclofenac and simvastatin to non-target organisms like fish.