Effects Of Atorvastatin And Aspirin On Nrf2/Keap1 Signaling Pathway And DNA Methylation In Daphnia Magna

Pharmaceuticals and personal care products(PPCPs)are a new type of pollutant that has attracted attention.They are widely present in water environments around the world.Due to their "pseudo-persistence" and bioaccumulation,they may pose potential ecological risks to non-targeted aquatic organism.Atorvastatin(ATV)and Aspirin(ASA)are typical representatives of lipid-lowering drugs and non-steroidal anti-inflammatory drugs,respectively,and are often detected in various water environments.However,there are currently few studies on the potential effects of Atorvastatin and Aspirin on aquatic organisms,especially on aquatic invertebrates.In this study,the model organism Daphnia magna was used as the test organism to study the responses of Nrf2/Keap1 signaling pathway and DNA methylation under acute exposure to atorvastatin and aspirin,and the changes in physiological indicators of Daphnia magna under chronic exposure.In this study,we firstly established the RT-PCR method for DNA methylation-related genes(DNMT3,TET2)and target gene of atorvastatin(HMG-Co AR).At the molecular level,we detected the transcription expression of the key genes(Nrf2,Keap1,HO-1,GCLC,GPx,TRX,Trx R and Prx1)in Nrf2/Keap1 signaling pathway,DNA methylation-related genes(DNMT1,DNMT3 and TET2)and HMG-Co AR gene in Daphnia magna exposed to ATV or ASA for 24 h,48 h and 96 h.At the biochemical level,we measured the activity of antioxidant enzymes(SOD,CAT,GST,GPx and Trx R)and the content of GSH and MDA under a single exposure of ATV and ASA for 48 h.At the individual level,we investigated the toxic effects of ATV and ASA on the growth and reproduction of Daphnia magna through the 21-day chronic toxicity test.The results showed that both Atorvastatin and Aspirin have a significant effect on the transcriptional expression of antioxidant associated genes in the Nrf2/Keap1 signaling pathway with dose/time-effect relationship to different degrees.They also affect the antioxidant enzymatic activity,GSH and MDA content.Under ATV exposure,the expression of Nrf2 and Keap1 showed generally a negative correlation,Nrf2 and its downstream antioxidant genes were induced at 24 h and 48 h,however most of them were inhibited at 96 h.DNA methylation-related genes(DNMT1,DNMT3)showed a trend of first down-regulation and then up-regulation with prolonged exposure time.HMG-Co AR was induced by high-dose ATV at 24 h and 96 h.The increase in antioxidant enzymes(SOD,GST,GPx and Trx R)activity,consumption of GSH and accumulation of MDA indicated that Daphnia magna was under oxidative stress when exposed to ATV for 48 h.In the 21-day chronic toxicity test,ATV caused adverse effects on the growth,reproduction of Daphnia magna(e.g.decreased number of molting,decreased number of lavae born in the first pregnancy and total litter size,shortened body length at 21 days,etc.).The highest concentration of ATV showed lethal toxicity.Under ASA exposure,the expression of Nrf2 was up-regulated at 48 h and down-regulated at 96 h.Antioxidant genes were induced to varying degrees.The thioredoxin system genes(Trx R,TRX and Prx1)were mainly up-regulated during the later period of exposure(96 h)and play an role in anti-oxidant process.DNA methylation-related genes(DNMT1,DNMT3)were first up-regulated and then down-regulated as the exposure time increases.When Daphnia magna was exposed in ASA for 48 hours,the activity of antioxidant enzymes(SOD,GST and GPx)increased,GSH content decreased but MDA content increased in a dose-dependent manner,indicating that Daphnia magna was subjected to oxidative stress.In the 21-day chronic toxicity test,ASA also showed inhibition effects on the growth and reproductive ability of Daphnia magna,for example,the delayed first time to pregnant eggs,the shortened body length,and the slower heartbeat frequency.In summary,when exposed to both ATV and ASA pollutants,Daphnia magna can regulate redox-regulation-related gene expression and enzyme activity through the Nrf2/Keap1 signaling pathway,which plays a coordinated role against oxidative stress.To a certain extent,the growth and reproduction ability of Daphnia magna was inhibited under the chronic exposure to ATV or ASA.The responses of DNA methylation-related gene expression suggested that epigenetic regulation may be involved in the process of cellular antioxidant responses,but further research is needed.In this article,we explored the effects of ATV and ASA on the Nrf2/Keap1 signaling pathway and antioxidant detoxification metabolic in the aquatic invertebrate organisms at the molecular level and enzymatic activity level,and addressed the physiological effects of ATV and ASA on the growth,development and reproduction of the aquatic invertebrates.We evaluated the potential risks of ATV and ASA exposure to Daphnia magna,which lay a foundation for further research on the toxicity mechanism and ecological risk of typical PPCPs such as statins and non-steroidal anti-inflammatory drugs in the aquatic environment to aquatic non-target organisms.