Analysis of plasma protein in newborn rats treated with diethylstilbestrol by using rapid micro two-dimensional electrophoresis

J. Suzuki, Shogo Ujiie, Ryosuke Kira, K. Sakaguchi, Yoichi Kakuno, M. Honda
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Abstract

We examined the effect of diethylstilbestrol(DES) administration on newborn male rats by rapid micro two-dimensional polyacrylamide gel electrophoresis (RM2D-PAGE) under non-denaturing/denaturing conditions and a 10-17% second-dimensional gel. We began by visually analyzing the changes at each week over time (weeks 1-9) in plasma protein spots from the same individual DES-administered male rats. We found the tendency for mercaptoalbumin (reduced form) spots to increase in week 1 in DES-administered rats. We also analyzed the quantitative alterations in α2u-globulin (AUG), a biomarker of endocrine disruptors, and found a two-week delay in expression in DES-administered rats in comparison to those not given DES (control rats). We then investigated the reproducibility of these results by using samples taken from multiple male rats in weeks 5, 7 and 9 to perform electrophoretic analysis three times for each individual, and observed the changes in AUG. AUG production was not seen in week 5 in the control rats, but was present at weeks 7 and 9. In DES-administered rats, expression was not observed at week 5 or 7, but was present at week 9. In addition, we identified the spots of AUG by Western blotting after electrophoresis. We consequently confirmed that AUG production delayed by two weeks in DES-administered rats, in comparison with control rats, which suggests that the effect of either a decline in AUG synthesis function or its depletion results in stunting of the reproductive organs, which was confirmed through autopsy.
二乙烯雌酚处理新生大鼠血浆蛋白的快速微双向电泳分析
本研究采用微二维快速聚丙烯酰胺凝胶电泳(RM2D-PAGE)技术,在非变性/变性条件下和10-17%的二维凝胶中,检测了己烯雌酚(DES)给药对新生雄性大鼠的影响。我们首先从视觉上分析同一只雄性大鼠的血浆蛋白斑的每周变化(第1-9周)。我们发现在给药的大鼠中,巯基白蛋白(还原形式)斑点在第1周增加的趋势。我们还分析了α - u-球蛋白(AUG)(内分泌干扰物的生物标志物)的定量变化,发现与未给予DES的大鼠(对照大鼠)相比,给予DES的大鼠的表达延迟了两周。然后,我们通过在第5、7和9周取多只雄性大鼠的样本,对每个个体进行三次电泳分析,研究了这些结果的可重复性,并观察了AUG的变化。对照大鼠在第5周没有观察到AUG的产生,但在第7和9周存在。在给药des的大鼠中,在第5周或第7周未观察到表达,但在第9周出现。此外,电泳后用Western blotting鉴定AUG的斑点。因此,我们证实,与对照大鼠相比,使用des的大鼠的AUG生成延迟了两周,这表明AUG合成功能下降或耗竭的影响导致生殖器官发育迟缓,这一点通过尸检证实。
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