Toxicity Effects And Different Gene Selected In Chlamys Farreri In Response To Exposure To Tetrabromobisphenol A

Brominated flame retardants （BFRs） have been widely added to or applied as atreatment to polymeric materials （plastics, textiles, electronic equipment） for thepurpose of fire prevention. The widely used BFRs contain polybrominated diphenylesters （PBDEs）, hexabromocyclodecane （HBCD） and the phenolic compoundtetrabisphenol A （TBBPA）. It has become increasingly in the environment andpresents a potential health risk to organisms exposed to these emerging classes ofenvironmental contaminants. Among brominated flame retardantsTetrabromobisphenol A （TBBPA） has the largest production volume in the world（over120,000tons annually or30%of all BFRs）. TBBPA as well as most of the otherbrominated aromatic flame retardants show a high persistency in the environmentbecause of its persistency brominated aromatic compounds which can bioaccumulateand are globally traceable in environmental samples. It is reported that TBBPA and itsderivatives have been detected in the various of environmental medium, such ashuman blood samples, eggs from predatory bird species, moreover sediments, soils,marine and fresh water animals, which suggested that this compound could affectaquatic organisms. TBBPA has high acute toxicity to algae, molluscs, crustaceans andfish. However, little information on the molecular biomarkers and gene expressionprofiles involved is available. Suppression subtractive hybridization （SSH） can beemployed to construct differentially expressed gene library between normalizationand subtraction in a single procedure based primarily on suppression polymerasechain reaction （PCR）. Many researches showed that is a highly useful technology inaquatic vertebrates where no genomic data are available. This usefulnessmethodology in aquatic invertebrates was assessed using SSH and Real-time PCR toinvestigate altered gene expression in gill of Chlamys farreri in response to exposure to Tetrabromobisphenol A （TBBPA）. Studies on the changes in gene expressionprofile may help in identifying the genes which play an important part in bivalvemollusks detoxification metabolic system. Also, the resulting data may show moregenomic information of the bivalve and molecular biomarkers for TBBPA stress.Molecular biomarkers may be selected to monitor the TBBPA pollution in aquaticenvironment.1The primary study of TBBPA in chlamys farreri in response toexposure to tetrabromobisphenol AThis study was conducted to determine single acute toxicity of TBBPA on C.farreri and the effect of BDE-47on the aryl hydrocarbon hydroxylase （AHH）,Glutathione S-transferase （GST）, superoxide dismutase （SOD） activities, Glutathionecontent and DNA single strand breaks of the gills and digestive gland of C. farreri.The result showed that24h、48h、72h、96h LC₅₀of young and adult C. farreri are5.924、4.169、3.095、2.614mg/L and2.458、1.657、1.502、1.342mg/L; All the indexesshowed no significant change in the control during the experimental time. The AHHand SOD activities were restrained all the time and exhibit a peak value; GST werechanged significantly while in the higher dose-group（100、200μg/L） were restrained.DNA single strand breaks levels were induced by all the dose-groups, and the damagewas permanent. And there were good linear correlations and dose-time effectrelationship between all the indexes and TBBPA concentrations. AHH、GST、SODactivities in the gills and digestive gland of Chlamys farreri were selected as thebiomakers to evaluate the pollution level of TBBPA.2Cloning, sequence analysis and tissue distribution of ARNT gene inchlamys farreriThe degenerate primers were designed from ARNT protein multiple sequencealignments from related species. The ARNT cDNA sequence was obtained throughthe technology of RT-PCR and RACE. The results show that the length cDNA ofARNT was1268bp, and an open reading frame （ORF） of1020bp encoding a protein of340amino acid residues. The comparison of the deduced amino acid sequenceswith ARNT from other species showed that ARNT in C. farreri in highly conserved.Specific amplifications of the ARNT mRNA were performed on the gill, digestivegland, adductor muscle, mantle and foot. The results revealed that the ARNT in gillsare highly expressed, while that in mantle is the lowest.3Construction of gene expression profiles in chlamys farreri inresponse to exposure to tetrabromobisphenol ATBBPA-induced genes were identified using suppression subtractivehybridisation （SSH） from Chlamys farreri. A total of203and44clones from SSHforward and reverse library were respectively obtained including cellular process,immune system process, response to stimulus, metabolic process and signaling etc.Differential gene expressions were compared between scallops from control andTBBPA treatment groups （400μg/L,15d） using quantitative real time RT-PCR. Forfurther research, eight significant genes expression from scallops exposed to TBBPA（0;100;200;400μg/L） sampling at0,1,3,6,15d, were utilized for Q-RT-PCR. Theresults revealed that the expression level of most selected cDNAs was dominantlyup-regulated or down-regulated in the TBBPA-induced scallops. These findingsprovide basic genomic information of the bivalve and the selected genes may be thepotential molecular biomarkers for TBBPA pollution in aquatic environment.