| Pharmaceuticals and personal care products(PPCPs) have the characteristics with strong persistence, biological activity, bioaccumulation and slow biodegradation in ecosystem. It has been widely reported that these compounds present potential risk to ecological environment as well as human health due to their specific physicochemical properties and increasing usage. When PPCPs enter organisms, it would induce physiological defensing and metabolizing procedure, the response would be firstly displayed on the molecular and cellular levels. CYP 1A, CYP 3A, GST and P-gp are the enzymes and proteins which can metabolize pollutants including PPCPs in the organism. They could be used as biomarkers for monitoring environmental pollutants and warning of their potential risk. In this paper, the effects of Norfloxacin(NFLX) and Triclosan(TCS) on swordtail fish (Xiphophorus helleri) were measured by considering a number of parameters, including Phase I, Phase II metabolic enzyme activities and the expression of mRNA. Therefore, the potential hazard of NFLX and TCS on aquatic ecosystem were evaluated.The major contents of this research are listed as follows:In the first part, CYP 3A cDNA was cloned from the liver of swordtail fish using RT-PCR. Sequence analysis revealed that the cloned CYP 3A cDNA fragment was 582 bp in length, encoding 194 amino acids. Search results of the BLAST protein database revealed that the deduced amino acid sequence was highly homologous with Fundulus heteroclitus, shared 90%. And the homologous with other fishes was greater than 65%, with mammals was nearly 60%. The results showed that the CYP 3A gene of swordtail fish was successfully cloned.In the second part, five pairs of primers were designed according to the sequences ofβ-actin, GST, CYP 1A, CYP 3A and P-gp of swordtail fish respectively, a real-time quantitative PCR method was developed for detection of the five genes using the SYBR Green I. The amplification efficiencies of this assay were between 95% and 105%, and correlation coefficients were between 0.9971 and 0.9999. The coefficients of variation for both intra-experimental reproducibility and inter-experimental reproducibility were less than 2%.The results showed that the developed SYBR Green I Real-time quantitative PCR was simple, sensitive, efficient and stable, which could be used as a quantitative method for analysis of biological samples. In the third part, the effect of NFLX on swordtail fish was analyzed with the SYBR Green I Real-time quantitative PCR methods, the measured parameters included the expression of GST, CYP 1A, CYP 3A and P-gp mRNA. At the same time, the activities of GST, EROD and ERND were determined. Results showed that the activities of Phase I, Phase II metabolic enzyme and the relative expression of mRNA of swordtail fish were either induced or inhibited when exposed to NFLX, genes were more sensitive than enzymes, and differences were observed when compared females and males. The activity of EROD and the relative expression of CYP 1A mRNA sensitively responsed to NFLX exposure, which implied they could act as candidate biomarkers of NFLX. The results showed that the EROD and CYP 1A mRNA could be used as sensitive biomarkers for monitoring pollutants in aquatic ecosystem.In the fourth part, the toxic effect of TCS on swordtail fish was studied with in series of concentrations. The results showed that TCS was high-toxic to swordtail fish, the 96h LC50 was 1.47 mg-L-1. The activities of GST, EROD, ERND and the relative expression of mRNA of swordtail fish had a certain response to TCS exposure, while a gender difference was also abserved. GST activity and GST mRNA were more sensitive compared with other parameters, therefore, they may be used as potential biomarkers for TCS exposure. |
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