Different Toxicity Response Mechanisms Of Benzo[a]pyrene And Cadmium In Earthworms

Soil is a major sink for organic and inorganic pollutants due to the industrial development.Benzo[a]pyrene(BaP)is the PAH with the highest toxicity equivalent factor(TEF)and cadmium(Cd)more frequently exceeded permissible limits in soil samples than any other toxic chemical and had the highest single pollution index(SPI).Although these toxic chemicals pose serious environmental risks,their ecotoxicological risks are unclear.Earthworms(Annelida,Oligochaeta),which promote a healthy soil structure and play a key role in terrestrial food chains,are commonly used as bioindicators in standard bioaccumulation tests and ecotoxicological studies.Besides,some studies have been looking at differences in contaminant accumulation between earthworm ecotypes,and this difference is species-dependent.Meanwhile,the pollutants distribution in earthworm organs or tissues could help to clarify earthworm detoxification and pollutant tolerance mechanisms.Besides,the different types of soil also could change the toxicities of pollutant.Furthermore,Inorganic pollutants(such as nonessential heavy metals)and organic pollutants(such as polycyclic aromatic hydrocarbon)could induce different responses in earthworms and,due to differences in their chemical properties,the toxic chemicals exhibit different patterns of accumulation and distribution,andenzymatic responses,gene expressions once absorbed.Here,our study mainly focused on the different accumulations and distributions in different ecotype earthworms,and the toxic responses on BaPor Cdexposed to the different type soils.Besides,we analysed the protein expression and the metabolic pathway under BaP or Cd stress in earthworm Eisenia fetida by using the proteomic and metabolomics technologies,respectively.Finally,we investigated the oxidation-related indexes by using the model hormesis when earthworm exposed to the Inorganic salt solution.The results showed below:1,The accumulations and distributions of BaP or Cd were relied on the different ecotypes on earthworm.We found that the three earthworm ecotypes showed different sensitivities toBaP and Cd,and accumulated different levels of these pollutants.E.fetida(epigeic)hadhigher sensitivity to BaPthanCd,while A.carnosus(endogeic)and M.guillelmi(anecic)were more sensitive to Cd than to BaP.E.fetida and M.guillelmiaccumulated more BaP than Cd,predicted maximum internal concentrations of BaP were 1946.27±306.29 mg/kg and 2046.61±90.64 mg/kg,respectively.While the opposite was true for A.carnosus,and predicted maximum internal concentrations was 927.10 mg/kg.The accumulation patterns at the organ level were similar between the different earthworms;BaP accumulation was highest in the reproductive organs,while Cd mainly accumulated in the digestive organs.The higher sensitivity of A.carnosus and M.guillelmimakes them potential bioindicators for environmental risk assessment.2,The availability and toxic responses of BaP or Cd were depended on the soil types.Under BaPtreatment,the increasing available BaP(5.76±0.36mg/kg)were found in fluvo-aquic soil.While,the the increasing available Cd were found in red soil(3.43±0.15mg/kg).BaPcould induce much ROS(reactive oxygen species)level than Cdin earthworm,and ROS levels were higher under both BaP and Cd treatments in red soil than in fluvo-aquic soil.3,BaP and Cd induced unique protein expressions in E.fetida.There were more protein spots in the 2-DE by using TCA-Acetone method.we found that earthworms respond to different pollutants via unique complex mechanisms.Proteomics analysis revealed that BaP exposure markedly induced oxidation/reduction proteins,which are involved in stress-related responses,whereas Cd exposure mainly induced proteins involved in transcription and translation.Some proteins exhibited opposite responses following exposure to BaP or Cd;for instance,CAT(catalase)and Hsp70(heat shock protein 70)were up-regulated under BaP treatment and down-regulated under Cd treatment.Furthermore,a significant increase in ROS levels and CAT activity demonstrated that BaP treatment induced oxidative stress.qRT-PCR analysis showed that the expression of MT(metallothionein)expression was induced by Cd treatment,and that expression of mRNA did not correlate well with the protein expression.4,Metabolic pathway was relied on the different types of soils and pollutants.Under the BaP treatment,BaP could induce the glutathione metabolism,glyoxylate and dicarboxylate metabolism and sulfur metabolism in red soil,while the pathways of alanine,aspartate and glutamate metabolism and pantothenate and CoA biosynthesis were induced in fluvo-aquic soil.For Cd exposure,Cd could induce lanine,aspartate and glutamate metabolism and ABC transporters in red soil,while aminoacyl-tRNA biosynthesis,ABC transporters and cyanoamino acid metabolism were induced in fluvo-aquic soil.p38-MAPK was activated by both BaP and Cd.Earthworm could accumulation higher BaP level in fluvo-aquic soil,while more Cdaccumulation was found in red soil.BaP could induce higher level of BPDE-DNA adduct in fluvo-aquic soil than in red soil.5,BaP or Cd could induce the hormesis effect in earthworm E.fetida after 24h,48h and 72h exposures under semi-static solution.ROS could be stimulated at shorter time than other biomarkers under both BaP and Cd treatments,and BaP induced stronger ROS level than Cd.Under BaP treatments,higher hormesis factors(as described by P value,AUCH/AUCZEP)were found in CAT than SOD(superoxidative dismutase).while the opposite results were found in Cd treatment after 24h and 48h exposures.CAT might impact on the GSH(glutathione)to remove the ROS by using structural equation models.Furthermore,the results of hormetic effect showed that the CAT played a key role in hormetic effect.Therefore,our results implied that CAT might act as a switch in response to the ROS in E.fetida.