Studies On The Reproductive Endocrine-disrupting Effects Of Tetrabromobisphenol A(TBBPA) And Decabromodiphenyl Ethane(DBDPE) In Mussel Mytilus Galloprovincialis

Tetrabromobisphenol A(TBBPA)and decabromodiphenyl ethane(DBDPE)are typical brominated flame retardants,which have been frequently detected in marine environment and the concentration and frequency of their detection yearly increased.TBBPA and DBDPE have posed potential threats on marine ecosystem.Previous studies showed that TBBPA and DBDPE could significantly affect the reproductive endocrine system of vertebrates,but there are few studies on the reproductive endocrine disrupting effect and its mechanism in invertebrates.The representative invertebrate mussel Mytilus galloprovincialis was selected as the experimental animal in this study.Transcriptomics together with traditional ecotoxicological methods were used to analyze the reproductive endocrine-disrupting effects and mechanisms of TBBPA and DBDPE in M.galloprovincialis.This study may provide references for risk assessment of TBBPA and DBDPE pollution in marine environment.The results are as follows:1.TBBPA-induced reproductive endocrine disruption and its mechanism in M.galloprovincialisProliferating mussels were exposed to 0,0.6,3,15,75 and 375 μg/L TBBPA for 30 days.The accumulation of TBBPA in the whole soft tissue of mussels was determined by liquid chromatography-mass spectrometry(HPLC-MS).Significant TBBPA accumulation presented an exponential increase with TBBPA concentration increasing from 0.6 to 375 μg/L(P < 0.01),ranging from 62.32 to 11176.81 ng/g dry weight.Real-time reverse transcription PCR(q RT-PCR)was used to detect the expression levels of sex-specific genes(vitelline envelope receptor for lysin(VERL)and vitelline coat lysin(VCL)).The relative expression of VERL and VCL reflected the sex of mussels and the development stage of gametes.U-shaped curves exhibiting relative expression profiles of VERL and VCL occurred in females and males with TBBPA exposure dose,respectively.The results indicated that TBBPA exposure markedly promoted the gametogenesis in both sexes and were associated with the post- spawning stage of gonads.The development stages of mussel gametes and gonadal index(GI)were determined based on histological observation.The GI showed a gross increase trend with increasing TBBPA dose,and a large proportion of individuals in TBBPA-treated groups were at the mature,spawning or resting stages.Especially,high concentrations(75 and 375 μg/L)of TBBPA significantly promoted the development of gametes in both sexes(P < 0.05).The vertebrate sex hormone levels in gonads of mussels were determined by HPLC-MS.The results showed that the decreased sex hormone levels were found in 75 and 375 μg/L TBBPA-treated groups,and closely associated with the post-spawning stage of gonads.These findings indicated that TBBPA promoted the gonad development in both sexes,and interfered with reproductive endocrine of mussels without gender-specific manner.Transcriptomic profiling revealed that the number of differentially expressed genes(DEGs)induced by TBBPA in male mussels was higher than that in female mussels.The significantly enriched GO terms and KEGG pathways included sulfuric ester hydrolase activity,steroid biosynthesis pathway,unsaturated fatty acid biosynthesis pathway and arachidonic acid metabolism which were commonly altered in both female and male mussels.Further analysis on the mechanisms of TBBPA-induced reproductive endocrine-disrupting effects in mussels suggested that TBBPA biotransformation was implemented by not only step-wise metabolism of phase I/Ⅱ/Ⅲ(cytochrome P450s(CYPs),glutathione-S-transferase(GST)and P-glycoprotein(Pg P)),but also direct sulfation of TBBPA mediated by sulfotransferases 1B1(SULT1B1)and SULT1C1.The up-regulation of SULT1E1 was largely attributed to the sharp increase in SULT1B1 and SULT1C1.TBBPA-sulfate catalyzed by SULT1 activated the expression of steroid sulfatase(STS).The effects of TBBPA on the enzyme activities of SULT1E1 and STS were in a sex-specific manner,375 μg/L TBBPA significantly inhibited the activity of SULT1E1 in females and STS in males,respectively.In summary,TBBPA disrupted the steroid hormone biosynthesis pathway in mussels through promoting steroids sulfonation and hydrolysis of sulfate steroids and therefore induced reproductive endocrine disruption in M.galloprovincialis.In addition,TBBPA promoted fatty acid β-oxidation and arachidonic acid metabolism supported by up-regulation of acyl Co A oxidase(ACOX1),ACOX3 and CYP2U1.TBBPA interfered with lipid metabolism and might induced toxic effects on reproductive endocrine system.2.DBDPE-induced reproductive endocrine disruption and its mechanism in M.galloprovincialisDeveloping mussels were exposed to 0,1,10,50,200 and 500 μg/L DBDPE for 30 days.The accumulation of DBDPE in the whole soft tissue of mussels was determined by gas chromatography-mass spectrometry(GC-MS).DBDPE accumulation presented an exponential increase with exposure DBDPE concentration increasing from 1 to 500 μg/L,ranging from 121.46 to 577.93 ng/g dry weight.Significant DBDPE accumulation was observed in 50,200 and 500 μg/L DBDPEtreated groups compared to control group(P < 0.05).q RT-PCR was used to detect the expression levels of sex-specific genes(VERL and VCL).The relative expression of VERL and VCL reflected the sex of mussels and the development stage of gametes.Linear increase and inverted U-shaped curves of VERL and VCL expression occurred in females and males with DBDPE exposure dose,respectively.The development stages of mussel gametes,GI and histological morphometric parameters of gonads were determined based on histological observation.The effects of DBDPE on the gonad development of mussels were in a gender-specific manner.The GI and relative expression of VERL showed a gross increase trend with increasing DBDPE dose.Especially,the highest concentration(500 μg/L)of DBDPE significantly promoted the development of gametes in female mussels(P < 0.05).The results of histological morphometric parameters in females were consistent with the development stages of gametes.With respect to female mussels,the GI and relative expression of VCL in male mussels showed a gross increase trend and inverted U-shaped curves with increasing DBDPE dose,respectively.All individuals in 50,200 and 500 μg/L DBDPE-treated groups were at the spawning stage.The significantly decreased area of VC cells indicated abnormal gonad development and the abnormality of VC cells in male mussels,which might be one of the adverse effects of DBDPE.These results indicated that DBDPE promoted the gonad development in both sexes and induced genderspecific reproductive endocrine-disrupting effects in mussels.Transcriptomic profiling showed that the number of DEGs induced by DBDPE in male mussels was higher than that in female mussels.No enriched GO terms and KEGG pathways were commonly altered in both female and male mussels.The mechanisms of reproductive endocrinedisrupting effects induced by DBDPE were sex-specific.DBDPE suppressed the processes of cholesterol homeostasis in both sexes via low-density lipoprotein receptor related proteins(LRPs)-induced pathways.The down-regulated ABC transporter family(ABCs)indicated that DBDPE inhibited cholesterol transport.DBDPE resulted in cholesterol transport disruption and directly inhibited the resources for steroid hormone synthesis.DBDPE also activated nongenomic signaling pathway including signaling cascades G protein-coupled receptor 157-inositol 1,4,5-triphate receptor-Ca2+(GPR157-IP3-Ca2+)and secondary messengers(cyclic guanosine monophosphate,c GMP),subsequently altering the expression of genes(vitelline membrane outer layer 1 VMO1),barrier-to-autointegration factor(Banf1),zonadhesin(ZAN)and protein hook homolog 1(Hook1))which were associated with reproduction.In addition,the effects of DBDPE on apoptosis and energy metabolism might interfere with reproductive endocrine system of mussels.DBDPE induced apoptosis via Caspase and eukaryotic translation initiation factor 2-alpha kinase 2(EIF2AK2)mediated pathways in the female and male mussels,respectively.The concentrations of 50,200 and 500 μg/L DBDPE also resulted in the increase in energy demand and insufficient energy supply of male mussels,but did not cause the same toxic effects with females.3.Comparison of reproductive endocrine-disrupting effects and mechanisms between TBBPA and DBDPETBBPA induced reproductive endocrine-disrupting effects in mussels without gender-specific manner,while DBDPE induced gender-specific reproductive endocrine-disrupting effects in mussels.Both TBBPA and DBDPE induced reproductive endocrine-disrupting effects in mussels of both sexes,which was supported by the promotion of gametogenesis.However,the mechanisms of reproductive endocrine disruption caused by TBBPA and DBDPE in M.galloprovincialis were different:(1)Different mechanisms of interfering with steroid hormone biosynthesis pathways.TBBPA disrupted the steroid hormone biosynthesis in mussels through promoting steroids sulfonation and hydrolysis of sulfate steroids by up-regulated SULT1E1/STS and inhibited SULT1E1/STS activities.DBDPE disturbed the steroid hormone biosynthesis by affecting the transport of steroid hormone synthesis substrates(cholesterol)via LRPs-induced pathways.DBDPE also inhibited cholesterol transport through the down-regulation of ABCs.(2)Different activation of non-genomic signaling pathway.DBDPE activated non-genomic signaling pathways in M.galloprovincialis through GPR157-IP3-Ca2+ pathway in male mussels and c GMP signaling molecules in female mussels,while TBBPA did not activate cytoplasmic or plasma membrane receptor-mediated non-genomic pathways.(3)Other possible mechanisms.TBBPA promoted fatty acid β-oxidation and arachidonic acid metabolism,and interfered with lipid metabolism in mussels.There were no significant changes in DEGs related to lipid metabolism in DBDPE-treated groups.DBDPE induced apoptosis in digestive glands of mussels,which might affect the normal function of digestive glands.DBDPE also increased the energy demand and inhibited the energy supply of male mussels.The reduction of availability of reproductive energy in male mussels might indirectly affect the reproductive function of mussels.DEGs related to apoptosis and energy metabolism were not significantly altered in TBBPA-treated groups.