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

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.