| In this study, we assayed the activity of enzymes CYP1A and CYP3A, mRNA expression and protein expression in Tanichthys albonubes. Effects of heavy metals (Cu2+and Cd2+) on the enzymes of P450in two cytochromes were studied at three levels including enzymes activity, gene transcription and translation. The relationship between Cu2+and Cd2+stress and the above indicators were discussed. The results are as follows:1. Effect of Cu2+and Cd2+exposure on activity of enzymes CYP1A and CYP3AEffects of different concentration of Cu2+and Cd2+, under single exposure and joint exposure, on activity of enzymes CYP1A and CYP3A in Tanichthys albonubes liver weere determined. The results showed that CYP1A enzyme activity under low dose of Cu2+exposure increased significantly at72h (p<0.05). It continued to increase in96h and had significant difference with the control group (p<0.05). In high dose group, the CYP1A enzyme activity increased significantly at48h (p<0.05) and kept increasing; it had significant difference with the control group (p<0.05) at72h and96h. CYP1A enzyme activity under low dose of Cd2+exposure had no difference with the control group (p<0.05) at each time point. In high dose group, the CYP1A enzyme activity had no difference with the control group within48h (p<0.05), but then increased significantly at72h and96h (p<0.05). Joint exposure of Cu2+and Cd2+on CYP1A enzyme activity showed synergetic effects at24h,48h and72h, respectively and antagonistic effect at96h. CYP3A enzyme activity under low dose of Cu2+exposure increased significantly at72h (p<0.05) and continue to increase at96h and had significant difference with the control group (p<0.05). In high dose group, the CYP3A enzyme activity under low dose of Cu2+exposure increased significantly at48h (p<0.05), decreased slightly at72h and continue increased at96h, significant difference with the control group (p<0.05). CYP3A enzyme activity under low dose and high dose of Cd2+exposure had no significant difference with the control group at24h,48h and72h, CYP3A enzyme activity increased significantly at96h (p<0.05). Joint exposure on CYP3A enzyme activity showed synergetic effect at48h and72h, and independent effect at96h.2. Effect of Cu2+and Cd2+exposure on the relative expression of CYP1A and CYP3A mRNATanichthys albonubes CYP3A gene cDNAs431bp were obtained using homology cloning (Genbank number:KC844034). The Effect of Cu2+and Cd2+single exposure on the transcription of CYP1A and CYP3A mRNA was determined using fluorescence quantitative PCR. The results showed that, CYP3A mRNA gene expression level under low dose of Cu2+exposure increased significantly (p<0.05) at48h, while decreased at72h and increased in96h (p<0.05). CYP3A mRNA gene expression level with low dose of Cd2+exposure was the highest at48h (p<0.05), it decreased at72h but increased at96h (p<0.05). In high dose group, the CYP3A mRNA gene expression level raised significantly at72h and96h (p<0.05). Joint exposure on CYP3A mRNA gene expression level showed synergetic effect. CYP1A mRNA gene expression level under low dose of Cu2+exposure increased significantly at48h and96h (p<0.05), significantly decreased at72h (p<0.05) under high dose of Cu2+exposure;CYP1A mRNA gene expression level under low dose of Cd2+exposure increased significantly at48h (p<0.05), and gradually decreased after48h, and decreased significantly under high dose of Cd2+exposure at48h and72h (p<0.05), Joint exposure on CYP1A mRNA expression level showed antagonistic effect at24h, and synergetic effect at72h and96h.3. Effect of Cu2+and Cd2+exposure on the translation of CYP1A proteinEffect of different concentration of Cu2+and Cd2+single exposure and joint exposure on the translation of CYP1A protein in Tanichthys albonubes liver was determined using protein western blot hybridization. The results showed that CYP1A protein expression level with low dose of Cu2+exposure raised significantly (p<0.05) at48h, then it reached the maximum at72h (p<0.05), but the difference decreased at96h. In high dose group, the CYP1A protein expression level had significant difference with control group (p<0.05) at24h, and decreased at48h and72h, but increased at96h (p<0.05). CYP1A protein expression level with low dose of Cd2+exposure raised significantly at24h (p<0.05), and decreased slightly at48h. In high dose group, the CYP1A protein expression level had significant deference with control group at24h (p<0.05), but it decreased in48h,72h and96h. In low dose group of joint exposure, CYP1A protein expression level had significant different at each time point (p<0.05). In high dose group, the CYP1A protein expression level was high at24h and72h, and decreased at48h and96h.This study showed that, Cu2+and Cd2+exposure on the different level indicators in Tanichthys albonubes presented measurability and regularity, which can serve as biomarkers to monitor and evaluate the pollution level of heavy metal in water environment. |
PDF Full Text Request