Characters Of Vitellogenin MRNA In Zebrafish (Danio Rerio) And Gobbid Fish (Odontobutis Potamophila) And The Application For Biomonitoring On Environmental Estrogens

In recent years, vitellogenin (VTG) has been an important biomarker in the research of detecting environmental estrogens. Hepatic vitellogenin mRNA in the male individual can be induced sensitively and steadily by estrogens, so it can be a new biomarker in the detecting of environmental estrogens.In the present study, both the standard animal zebrafish (Danio rerio) and gobbid fish (Odontobutis potamophila) which exist distinguishingly in the middle and backward reaches of Yangtze River have been used to obtain their vitellogenin expressing genes and as a biomarker to detect the responses to some potential estrogen treatments, such as E₂, NP and BPA, We will evaluate the two kinds of fish molecular models in development of a biomonitoring system for environmental estrogens.Total RNA was abstracted from livers with Trizol reagent, and its ratio of A₂₆₀/A₂₈₀ was between 1.8 and 2.0. Using RNA as a template, we conduct the RT-PCR with vitellogenin special primers designed by ourselves and by Yan Tong. The isolated 569bp and 464bp zebrafish vitellogenin cDNAs are obtained according to the full zebrafish vitellogenin l(zvtgl) which is 3644bp long. After compared vitellogenin cDNAs of several fishes, we cloned a small segment of gobbid fish vitellogenin cDNA according the conservative vitellogenin sequences. The gobbid fish vitellogenin cDNA we obtained is 780bp long and it was about 84% identical to the Japanese common goby sequence (Vg-530) and 62. 4% identical to zebrafish (zvtgl). The protein sequence of 260 amino acids was deduced from the gobbid fish cDNA clone and it was most closed to the amino acid sequence of Japanese common goby. The results proved that the cDNAs we have got are the vitellogenin expressing genes. The vitellogenin cDNAs were cloned from the lives of female zebrafish and gobbid fish, whose sizes were about 569bp and 388bp. In contrast, no vitellogenin cDNA was found in other tissues, for example, gill, ovary, pellicle, muscle, intestine, and eye. There was also no vitellogenin mRNA expressing in livers of male zebrafish and gobbid fish.The semi-quantified reserve transcriptase polymerase chain reaction (RT-PCR)assay was developed for quantification of vitellogenin mRNA level in both control male fish and estrogen treatment. In male zebrafish, the lowest-observed-effect concentrations of E₂, NP and BPA for the induction of vitellogenin mRNA was respectively observed at 20ng/L, 20μg/L, and 50μg/L in a 14-day exposure experiment. The expressions diversely increased with the exposed concentrations of the three estrogens. Further time courses following 250ng/LE₂, 250μg/LNP and 500μg/LBPA of zebrafish VTG mRNA expressions were detected. The results indicated that mRNA could be induced in 2 days by NP, while in 4 days by E₂ and BPA; which OD values are respectively 147.97±0.01> 29.57±0.06 and 113.91±0.10. The OD values increased straightly till the last exposure day. It was able to respond much faster to NP treatment than E₂ and BPA, while the E₂ exposure could cause much higher expressing level of VTG mRNA. After stopping exposure, the VTG mRNA expressing level inducted by E₂ was reduced much more quickly than that inducted by NP and BPA. There was no VTG mRNA expressing induced by E₂ 4 days later, and the expressing induced by NP and BPA was completely disappeared 6 days later. In addition, an enzyme-linked immnuosorbent assay (ELISA) for measuring plasmatic vitellogenin in zebrafish was developed and validated. It was testified that the plasmatic vitellogenin appeared a bit later than the. VTG mRNA. 250ng/LE₂ and 500μg/LBPA treatments result in the increasing of vitellogenin level in plasma after 8 days; the 250μg/LNP treatment needed 4 days, synchronously a little earlier. Instead of the depression of the plasmatic vitellogenin, occurred a continuous appearance or an unconspicuous reduce after stopping estrogen exposure. Finally, we proved that in gobbid fish it needed much higher exposure concentrations of E₂ and NP to induce VTG mRNA expression compared with zebrafish. The mR...