Molecular Mechanism Behind The Toxic Effects Of Benzo(a)pyrene On Scallop Chlamys Farreri

Based on the contaminated situation of polycyclic aromatic hydrocarbon at the coastal area of China, the toxic mechanism of benzo(a)pyrene (BaP) effect on scallop Chlamys farreri was studied. Aryl hydrocarbon receptor, CYP450, Glutathione S-transferase and P-glycoprotein were cloned. The relationship between BaP accumulation, gene expression and DNA damage were studied in order to illustrate the behind mechanism of the toxic effect of BaP, and provide a theoretic support for the biomarkers selection in PAHs biomonitoring.The scallop ChfAhR cDNA length is 2891 bp, coding an open reading frame with 821 amino acids. Three nuclear translocation sequences are found in the ChfAhR sequence. BLAST analysis showed that ChfAhR gene has a high identity with AhR genes from other species. The ChfAhR protein structure model is conservative. There is a basic Helix-Loop-Helix domain and a PAS domain in the sequence which indicated that the ChfAhR belongs to the bHLH-PAS transcript factor family. The ChfAhR bHLH-PAS domain has a high identity with other invertebrate AhR and a relative low identity with vertebrate AhR. ChfAhR transcript was detected in gill, digestive gland, mature female gonad, mantle and adductor, and showed the highest level in digestive gland.ChfCYP4 cDNA length is 1927 bp, coding an open reading frame of 438 amino acids. There is a endoplasmic reticulum location sequence in ChfCYP4. ChfCYP4 has a high identity with CYP4 genes from other species. The cytochrome P450 cysteine heme-iron ligand signature pattern F461SAGPRNCIG470 was found in the sequence and the heme iron ligand Cys468 is conservative. The highest transcript of ChfCYP4 was found in mature female gonad, and also detected in gill, digestive gland, mantle and adductor.ChfGSTpi cDNA length is 692 bp, coding an open reading frame of 205 amino acids. ChfGSTpi showed a high identity with GSTpi genes from other species and a conservative protein structure. The soluble GST N-terminal and C-terminal domain profile were found in the 205 amino acid sequence, including the GSH binding site (G-site) and a non-specific xenobiotic binding site (H-site).The consevative amino acid Tyr109 was found in H-site which is important for ligand binding. Highest ChfGSTpi transcript level was found in mantle, and also detected in gill, digestive gland, gonad and adductor.ChfPgp cDNA partial length is 1345 bp, coding a peptide of 210 amino acids. The ChfPgp gene shows a high identity with Pgp genes from other species, and a conservative protein structure. The ATP binding domain and ABC transport site L107SGGQKQRVA116 are found in the ChfPgp. Semi-quantitative PCR result indicated that ChfPgp has a lowest expression in mantle and higher expression in gill, digestive gland, mature female gonad and adductor.A BaP (0.05,0.2,0.8μg/L) exposure(10 d)and depuration (5 d) experiment was conducted and the result showed that BaP burden in digestive gland is about 11.3 fold higher than in whole soft tissue when data showed by wet weight, and about 5.7 fold higher than in whole soft tissue when data showed by dry weight. The BaP exposure induced gene expression of ChfAhR and ChfGSTpi, and showed a depress effect on gene expression of ChfCYP4. GSTpi expression and BaP burden in digestive gland has a positive relation and a Hyperbola equation (single RectangularⅡ,3 parameters) is established. DNA adduct and 8OHdG (8-oxodeoxyguanosine) have positive relation with BaP burden in digestive gland and exponential equations (single,3 parameters) are established.DNA adduct and 8OHdG content in cells of digestive gland from C.farreri exposed to BaP showed that CYP450 does not have a significant effect on DNA adduct and 8OHdG formation. GSTpi has an important effect on reduce the DNA adduct and 8OHdG content. SOD showed a significant effect on reducing the 8OHdG formation. Pgp showed a potential promotive effect on DNA adduct and 8OHdG formation.