The Toxical Effect Of Main Biomarkers On Tilapia Exposed To Benzo(A)Pyrene

Benzo(a)pyrene(BaP) is a pentacyclic polycyclic aromatic hydrocarbon, which is carcinogenic and teratogenic. Benzo(a)pyrene has stable physical and chemical properties, so it is hard to degradate. It can enter the water environment through a variety of ways, including rainfall, industrial wastewater discharge, surface runoff and so on. In recent years, with the rapid development of industrialization and urbanization, the BaP pollution in water is getting more and more serious, which has attracted people’s attention.Biological monitoring of pollutants is important in BaP risk assessment. The experiments used tilapia as trial animal to study the effects of different concentrations of benzo(a)pyrene on bile BaP metabolites, liver CYP1A1and GST activity, DNA damage and liver vitellogenin gene expression, which provides a scientific basis for the analysis and evaluation of ecotoxicity and genotoxicity of BaP, as well as a feasible method for BaP monitoring in the freshwater environment.The BaP metabolites in bile were detected using fixed wavelength fluorescence (FF). First, the FF method for tilapia was developed. Bile of tilapia was diluted to1:1000,1:2000,1:4000and1:8000, and the excitation/emission wavelength of spectrofluorophotometer was set to380/430nm. Results showed that the dilution about2000-fold was reasonable. Combining with other studies, the1600-fold dilution was used in the follwing test. The results of BaP exposure test showed that the BaP metabolism in0.1μg/L concentration group did not have obvious fluctuation during the exposure time and significant differences compared with the control group(P>0.05); the other three dose groups were significantly different with control group from2h(P<0.05, P<0.01). The fluorescence intensity in the concentrations of1μg/L,10μg/L,50μg/L showed a trend of increased at first and then decreased. It began to decline in50ug/L group after48h. There were no obvious peaks in1μg/L and10μg/L groups, but the fluorescence intensity also decreased after7d in1μg/L and4d in10μg/L. The bile BaP metabolites increased following the exposure concentration, showing a significant dose-response relationship.The activities of CYP1A1and GST in liver were measured in the exposure durations of2h,6h,12h,48h,4d,7d,14d. The results showed that the CYP1A1activity in the concentrations of0.1μg/L,1μg/L,10μg/L had no significant difference compared with control group (P>0.05), and it was significantly induced in6h,7d in50μg/L dose(P<0.01). The liver GST activity in the groups of0.1μg/L,1μg/L had no significant difference compared with the control group(P>0.05), and had significant differences in groups of10μg/L and50μg/L in14d(P<0.05). Similar change trend was found between CYP1A1activity with GST activity, especially in the1μg/L and50μg/L groups, suggesting that there might exist certain correspondence between the two enzymes.The liver cell DNA damage was detected using single cell gel electrophoresis(SCGE). The SCGE was conducted immediately after sampling. Four indicators, tail DNA content (TDNA%), tail length (TL) and tail moment (TM), Olive tail moment (OTM) were used to evaluate DNA damage. Results showed that BaP exposure could cause liver cell DNA damage in tilapia. Among the concentration of0.1μg/L,1μg/L,10μg/L, DNA damage increased following the BaP exposed concentrations, but decreased in50μg/L group. In addition to the tail DNA content of1μg/L dose group, tail DNA and Olive tail moment of10μg/L group, other parameters decreased in7d and then increased, especially in the0.1μg/L dose group.Vitellogenin (Vtg) is an important biomarker for monitoring environmental estrogens (EEs) in aquatic environment. Vtg mRNA in male fish is a new kind of biomarker. In order to research whether BaP has environmental estrogen effect, the Vtg mRNA was also studied in our paper. Total liver RNA was extracted from the liver by Trizol kit. The RT-PCR was conducted with vitellogenin special primers designed by Esterhuyse and by ourselves. The isolated422bp and687bp tilapia vitellogenin cDNAs were obtained. The effect of BaP on tilapia liver Vtg mRNA expression was detected with the help of real-time PCR. Results showed that there were no significant differences of Vtg gene expression among control group and the exposure concentrations of10μg/L and50μg/L in day4and17(P>0.05), in day10, the Vtg mRNA level increased by113.18%and121.79%respectively in10μg/L group and50μg/L group which were significantly higher than that in control group(P<0.01).