Responses Of The Nrf2/Keap1 Signaling Pathway In Mugilogobius Abei(M.abei) Exposed To Environmentally Relevant Concentration Aspirin And Atorvastatin

The extensive use of pharmaceuticals and personal care products(PPCPs)and possible ecological effects caused by their environmental residues have drawn worldwide attention.PPCPs in environments may cause negative effects on organisms due to their pseudo-persistence and accumulation.Aspirin and atorvastatin are two typical NSAIDs and antilipemic agents respectively,which are both characterized by wide application and large dosage.Their high detection frequency in the environments and potential adverse effects on organisms deserve more attention.In this study,Mugilogobius abei,a small marine fish distribute widely in aquatic ecosystems in southern China,was employed as the testing organism and the acute and subchronic toxicity of aspirin and atorvastatin were investigated,focusing on the responses of the Nrf2/Keap1 oxidative stress signaling pathway.The transcription expression of the key gene(Nrf2,Keap1,GCLC,GPx,GST,SOD,CAT,Trx2,Trx R,HMG-Co AR and PGC-1?)as well as the changes of the related enzymatic activities(GPx,GST,SOD,CAT and Trx R)and GSH,MDA content were measured.These responses and the results of histological examination were combined to reveal the antioxidant regulation mechanisms of M.abei under the exposure of the two typical drugs.The genes of Keap1,Trx2 and Trx R in M.abei were cloned,with the length of 209 bp,229 bp and 391 bp respectively,encoding 68,74 and 119 amino acids.The sequences had high homology with Boleophthalmus pectinirostris and Paralichthys olivaceus.Suitable primers were filtered and a stable real-time quantitative PCR method was established.Under the exposure of ASA,the transcriptional expression of Nrf2 and Keap1 showed a negative correlation to some extent,and the related genes downstream the Nrf2/Keap1 signaling pathway showed an inhibition at 24 h and induction trend at 72 h,168 h.The activity of antioxidant enzymes increased in different degrees during the ASA exposure.MDA content was significantly increased along with the rapid consumption of GSH at 24 h and 72 h after exposure,but MDA content decreased at 168 h with the recovery of the GSH content.The exposure to ASA at the environmentally relevant concentrations did not cause alterations on the liver of M.abei.Under the ATV exposure,the Nrf2/Keap1 signaling pathway in M.abei was activated with the up-regulation of Nrf2 and downstream antioxidant genes.Antioxidant enzymatic activities were inhibited at 24 h and 72 h after exposure but induced/recovered at 168 h.The responses of MDA and GSH were negatively correlated.The expression of HMG-Co AR was down-regulated at 24 h but up-regulated at 168 h.PGC-1? expression was up-regulated at 24 h but down-regulated at 72 h and 168 h.Histopathological examination showed that the volume of the lipid vacuoles decreased with the increasing concentration and exposure time.In summary,two typical PPCPs were able to activate the Nrf2/Keap1 signaling pathways.ASA exposure can activate the antioxidant system by increasing reductive small molecules and improving the enzymatic activity.ATV brought about oxidative stress to M.abei at the initial stage of exposure,but with the prolonged exposure time,the enhanced antioxidant capacity reversed this situation.ATV also affected the lipid metabolism of M.abei by lowering the cholesterol content and accelerating lipid decomposition.This study explored the effects of ASA and ATV on the Nrf2/Keap1 antioxidant detoxification system in fish combined with gene expressions,enzymatic activities and histopathological changes,which provided scientific basis for risk assessment of NSAIDs and antilipemic agents in aquatic ecosystems.This study could help better understand the antioxidant mechanism of aspirin and atorvastatin in aquatic organisms.