Purification And Determination Of Vitellogenin For Application To The Joint Toxicity Of Diethylstilbestrol And 17α-Ethynylestradiol

Vitellogenin(Vtg) is the egg yolk protein precursor in the oviparous animals.It is usually produced in sexually active female individuals.However,Vtg can also be detected in the male and the juvenile exposed to estrogen or estrogen mimic chemicals.Therefore,Vtg in male serum or plasma is generally considered to be a candidate of protein biomarker for environmental estrogens,thus widely used for the study of estrogenic effect of chemicals.Currently,most approaches for Vtg purification are the combination method of ion exchange chromatography and gel permeation chromatography,which suffer from low recovery,time-consuming and easy degradation for protein.Therefore,it is necessary to develop a new purification protocol that is simple,rapid and has high recovery.Specific assay methods termed immunoassays have been widely used for analysis of Vtg.It appears that immunological techniques applied to Vtg proteins have a great potential for developing into specific,sensitive quantitative methods.However, these techniques are cumbersome and require a long time for sample preparation.Therefore,the prerequisite for the study of environmental estrogens is to establish a rapid,reliable and valid method for the determination of Vtg in some species.Conventional ecotoxicity study of chemicals for their estrogenic effects has predominantly employed systems designed mostly for single estrogen,but few for the combinations.With the economical development along the coastal areas in China,a lot of discarded chemicals deteriorate the water quality of the coastal and estuary region.It is essential to investigate the joint effects of the persistent and toxic environmental estrogens on marine organisms.In this study,a two-step method had been developed for purification of Vtg with ultrafiltration and weak anion exchange liquid chromatography.And Vtg was determinated by the method of intrinsic fluorescence or UV.The method was sensitive, simple and with a wide linear range.Based on the established methods of the purification and the determination,the joint toxic effect of diethylstilbestrol(DES) and 17α-ethynylestradiol(EE₂) was studied using Vtg as the main monitoring index.The detail information,divided into three sections,is as follows.1.Two-step purification and primary characterization of VtgVtg from sea bream(Pagrosomus major) was induced by 17β-estradiol(E₂) with the application of intraperitoneal injection.With weak anion exchange liquid chromatography and ultrafiltration,a two-step method had been developed for rapid and reliable purification of Vtg from the serum of sea bream.It was found that most impurities and salt could be removed during the first step by weak anion exchange liquid chromatography and further clean-up in the second step through ultrafiltration. The concentrated Vtg purified with the two-step method had the same characteristics of chromatography,PAGE electrophoresis and protein molecular as those reported by other researchers.The purified Vtg could be used as the standard sample inner laboratory.The results showed that the two-step purification was simple and effective.2.Determination of Vtg in fish serum by HPLC with UV and intrinsic fluorescence detection in tandemA sensitive and simple determination method for Vtg in fish serum with a wide linear range was developed.The Vtg separated by high-resolution anion exchange chromatography was detected by intrinsic fluorescence and UV detection in tandem. The linearity curve ranged from 24.1 to 96.4μg/mL with a detection limit 8.0μg/mL (S/N=5) for UV detection and from 1.9 to 30μ/mL with a detection limit 1.0μg/mL (S/N=5) for fluorescence detection.The relative standard deviations(RSD) for UV detection and for fluorescence detection were 2.4%(n=5) and 3.4%(n=5), respectively.The method was simple,rapid and replicable.The proposed method was successfully applied to determine Vtg in fish serum from red drum(Sciaenops ocellatus).The validation results showed that the Vtg of red drum and sea bream had the same chromatographic behaviors.It indicated that the developed method could be applied to evaluate Vtg in various fishes. 3.Study of Vtg as a biomarker for environmental estrogensThe concentration-effect and exposed time-effect of DES,EE₂ and their mixture on red drum were studied.Moreover,with Vtg as the main biomarker,the joint estrogen effect of DES and EE₂ on red drum was investigated.The results are as follows.(1) DES and EE₂ were toxic to juvenile red drum.They could lead the individuals to abnormality,and even death.(2) The condition factor(CF),hepatopancreas somatic index(HSI) and renal somatic index(RSI) increased with the increasing expose time and concentrations of DES,EE₂ and their mixture.The effect of DES,EE₂ and their mixture in the experiment of concentration series was most significant for HIS and lest for CF.The significant effect in the experiment ot:time series was found at the exposed time of 30 days for CF,18 days for RSI and 9 days for HIS,respectively.(3) The concentrations of total protein and Vtg in serum enhanced with the increasing concentrations of DES,EE₂ and their mixture at relatively low concentrations(less than 320,160 and 120 ng/L for DES,EE₂ and their mixture,respectively).However,the concentrations of total protein and Vtg reduced with further increase of the concentrations.It demonstrated that the effect of DES,EE₂ and their mixture on total protein and Vtg accorded with Hormesis effect.The concentrations of total protein and Vtg were elevated with the prolonging expose time in the experiment of time series.Compared to other physiological indicators and the total serum protein,Vtg was the most significant and rapid one.It could well reflect the estrogenic effect of DES and EE₂.It was thus known that Vtg was a good candidate of protein biomarker for environmental estrogen.(4) From the relationships between Vtg and the other indicators,it was found that there was no significant correlation between Vtg and CF,but positive highly significant correlation(P＜0.001) between Vtg and total serum protein.The relationship between Vtg and HIS was similar to that between Vtg and RSI. There were significant correlations(P＜0.05) between Vtg and either HSI or RSI when the concentration of Vtg was in the range of 0-90 mg/mL.And there were highly significant correlations(P is less than or close to 0.01) between Vtg and the two when the concentration of Vtg was in the range of 0-25 mg/mL.However,there were no correlations(P＞0.05) between Vtg and the two when the concentration of Vtg was in the range of 25-90 mg/mL.(5) The relationships of concentration-effect for juvenile red drum exposed to DES and EE₂ coincided with the curve of typical s-shape.It could be fitted with the Logistic equation.Moreover,using the models of concentration addition and independent action it had shown that the joint effect of DES and EE₂ could be predicted from the activity of the individual chemicals. Compared to the model of independent action,the joint effect of the binary mixture could be well predicted by the model of concentration addition.