Effects Of Antioxidant Enzymes And AHR-related Gene Expression By Pyrene Exposure On Pinctada Martensii

Polycyclic aromatic hydrocarbons (PAHs) as a class of persistent organic pollutants are existing in marine environment, the majority of PAHs are teratogenic, carcinogenic, mutagenic toxicity, which are harmful to environmental organisms and even humans. With the increasing pollution of the marine PAHs, carrying toxic studies on marine organisms has become a hot issue. Pyrene as a typical tetracyclic polycyclic aromatic hydrocarbons, has higher levels of organisms in the marine environment which shows genotoxicity and carcinogenicity. Up to now, research on marine organisms by pyrene is still limited, particularly at the molecular gene level. In present study, the related genes of aryl hydrocarbon receptor, including ahr, cyp4and hsp90were cloned and the potential toxic effect of pyrene on Pinctada martensii (the antioxidant defense system, gene expression) was studied in order to select the useful molecular biomarkers for monitoring PAHs pollution. The resulting data may provide early warning of PAHs and scientific basis for ecological risk assessment. Following research conclusions were reached:1、Pyrene pollution had an obvious impact on antioxidant in different tissues of P.martensii:the sensitivity of enzyme activitis varies during time-depentent changes and different tissues. Three enzyme activities (SOD、POD、GPx) in gills were mainly induced by pyrene exposure, meanwhile, The activities of SOD GPx showed relative dose-response relationship during treatment, In7d, POD activity had dose-response relationship, however, in10d, there was no change at POD activity; In hepatopancreas, the activities changed from inhibition to induction with exposure time extanding, and in10d, all the enzyme activities showed clear dose-response relationship.2、The full-lengthes of ahr-related genes(ahr、cyp4、hsp90) were obtained by RACE method for the first time, and genes showed differnet transcriptions in tissues:the full-length ahr cDNA of pearl oyster was4253bp, encoding an open reading frame of2547bp with848amino acids. There were two nuclear translocation sequences founded in ahr sequence. ClustalW analysis showed the deduced amino acid sequence of ahr had a high identity with other ahrs. The pearl oyster ahr transcription was detected in mantle, gill, hepatopancreas, adductor muscle, gonad and pleopod, the highest level was showed in gill.The cyp4cDNA full-length was1855bp, encoding an ORF of498amino acids, cyp4cysteine heme-iron ligand signature pattern (F438SAGPRNCIG447) was found in the sequence and the heme-iron ligand cys445was highly conservative. Blast showed that cyp4had a high identity from other mollusks with highest similarity with clam C. farreri (50.2%). RT-PCR showed highest transcription was in gill of pearl oyster cyp4, followed in mantle and hepatopancreas and also detected in other tissues.hsp90cDNA length is2584bp, coding an open reading frame (ORF) of725aa. the deduced amino acid sequence has a high identity with other species, five conserved hsp90family regions are detected in P.martensii hsp90and the C-terminal amino acid sequence (MEEVD) was also found in hsp90. The highest expression of hsp90was detected in gonad.3、Pyrene exposure had an effect on the expression of ahr、cyp4、hsp90in gills of pearl oyster, different gene expression levels had time difference in response to pyrene:for3d exposure, the expression levels of three genes showed the same trend, in the high concentration group32μgL-1there were significant changes between ahr and cyp4genes levels compared with control group(p<0.01); in5d, comparing with the control group, the expression levels of ahr was significantly induced (p<0.01), cyp4and hsp90mRNA expression had the same trend, low concentration groups had obvious expression levels; after7d treatment, for hsp90,8and16μ.g-L-1concentration groups were significantly induced (p＜0.01), while ahr and cyp4mRNA expression levels showed a downward trend with prolonged exposure time.4、By comparing LOEC, we found that in antioxidant enzymes indicators, POD and GPx can be used as sensitive biomarkers, in expression of key genes of ahr-mediated detoxification metabolism of pearl oyster exposed to pyrene, ahr and cyp4genes are more sensitive as molecular biomarkers for pyrene contamination indicating and providing early warning of marine PAHs pollution.