The Primary Study On Gene Expressions And Toxic Effects Of BDE-47 On Venerupis Philippinarum

Polybrominated diphenyl ethers (PBDEs) are a new type of brominated flame retardants which are widely used in electronic products, coatings and various textiles. It was reported that the concentrations of PBDEs are increasing exponentially in biota and abiota in recent years. Because of their refractory, environmental stability, high-fat-soluble and bio-amplification can be transferred through the food chain, so that at a high trophic level of biological contamination and ultimately lead to the body's nervous, endocrine, reproductive system harm. In recent years, the environment and increasing concentrations of PBDEs have been prevalent throughout the world, and also because the chemical structure of PBDEs and Polychorinated biphenyls (PCB) and 2,2-bis (4-Chlorophenyl)-1,1,1-trichloroethane (DDT) are very similar, the toxicity of PBDEs in the environment attracts more and more the concern of scholars. Manila clam, Venerupis philippinarum is a kind of burrowing bivalve in Veneridae with wide distribution, and often considered as an indicator organism of marine sediment environmental pollution. In this paper, we chose the V. philippinarumis as the research object, and try to reveal of the BDE-47 toxic effects of V. philippinarum is from the level of molecular biology and enzymology respectively.1 Cloning, sequence analysis and tissue distribution of CYP4 gene inⅤ. philippinarumThe degenerate primers were designed from CYP4 protein multiple sequence alignments from related species. The complete CYP4 cDNA sequence was obtained through the technology of RT-PCR and RACE. The results show that the full length cDNA of CYP4 was 2107 bp, with a 5'untranslated region (UTR) of 461 bp, a 3' UTR of 318 bp, and an open reading frame (ORF) of 1329 bp encoding a protein of 442 amino acid residues. The comparison of the deduced amino acid sequences with CYP4 from other species showed that CYP4 in V. philippinarum in highly conserved. Specific amplifications of the CYP4 mRNA were performed on the gill, digestive gland, adductor muscle, mantle and foot. The results revealed that the GST-pi was only expressed in gill, digestive gland and adductor muscle and not found in mantle and foot.2 Cloning, sequence analysis and tissue distribution of Glutathione S-transferase (GST-pi) gene in V. philippinarumIn this study, we used degenerated primers designed in the highly conserved regions of GST to amplify the corresponding mRNA in the gill of V. philippinarum. Full-length coding sequences were obtained by rapid amplification of cDNA ends (RACE) for the first time. The full length cDNA of GST-pi was 1142 bp, with a 5' untranslated region (UTR) of 461 bp, a 3'UTR of 438 bp, and an open reading frame (ORF) of 618 bp encoding a protein of 205 amino acid residues with an estimated molecular mass of 23.9 kDa and an predicted isoelectric point (pI) of 7.90. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzymes belong to the pi-class and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) were highly conserved. Specific amplifications of the GST-pi mRNA were performed on the gill, digestive gland, adductor muscle, mantle and foot. The results revealed that the GST-pi expression level was observed higher in gill, adductor muscle, mantle and foot while extremely lower in digestive gland.3 Effects of BDE-47on the metabolic genes mRNA expression in the tissues ofⅤ. philippinarumClams V. philippinarum were exposed to different concentrations of BDE-47 (0, 0.25μg/L,6.25μg/L) for 21 days, and sampled on 0 d,1 d,3 d,6 d,10 d,15 d and 21 d. The technology of RT-PCR was used to detect AhR, CYP4, GST-pi and PgP mRNA expression. The results show that all the indexes showed no significant change in the control during the experimental time. AhR gene of gill tissue exposed to different concentrations of BDE-47 was induced a peak change, while in digestive gland AhR gene was significantly induced higher under conditions of 0.25μg/L and directly inhibited by high concentration. This was the similar trends with GST-pi in organizations of V. philippinarum.0.25μg/L BDE-47 treatment group PgP gene of gill tissue in the 1d was significantly induced higher and maintain stability, and 6.25μg/ L treatment group showed significant peak changes. In digestive gland the PgP gene in lowe concentration treatment group was the state continues to rise, while in high concentration was significant decreased keep lower than the control group level, but the difference was not significant (P> 0.05).4 Effects of BDE-47on the metabolic enzymes and DNA damages in the tissues ofⅤ. philippinarumThis study was conducted to determine the effect of BDE-47 on the aryl hydrocarbon hydroxylase (AHH), Glutathione S-transferase (GST), superoxide dismutase (SOD) activities, Glutathione content and DNA single strand breaks of the gills and digestive gland of V. philippinarum. The result showed that all the indexes showed no significant change in the control during the experimental time. The AHH activities exhibit a distinct dose-time response relationship except dealt with lower dose (0.01μg/L) and keep stability after 15 d. The GST, SOD activities and GSH contents changed significantly (P<0.05) among the treatments, and a peak value appeared in the lower dose-groups (0.01,0.05,0.25μg/L) during the experimental time. However, those in higher dose-groups (1.25,6.25μg/L) were restrained all the time, as the duration of the treatments and enhancement of the concentrations, the restraining effects were more heavier. DNA single strand breaks levels were induced by all the dose-groups except 0.01μg/L, and the damage was permanent. And there were good linear correlations and dose-time effect relationship between all the indexes and BDE-47 concentrations. SOD activities and damages of DNA in the gills and digestive gland of Venerupis philippinarum were selected as the biomakers to evaluate the pollution level of BDE-47.