Synergistic effects of parabens on the induction of calbindin-D9k gene expression act via a progesterone receptor-mediated pathway in GH3 cells

Abstract

Although the endocrine-disrupting bioactivity of parabens is weakly estrogenic (parabens are xenoestrogens), their combined synergistic effect is unknown. The aim of this study was to investigate the effects of methyl paraben (MP), ethyl paraben (EP), propyl paraben (PP), isopropyl paraben (IPP), butyl paraben (BP), and isobutyl paraben (IBP), either alone or in combination (MP + EP + PP + BP; PP + IPP; and BP + IBP) on the induction of the estrogenic biomarker gene, calbindin-D9k( CaBP-9k), in rat pituitary lactosomatotrophic GH3 cells. The expression of CaBP-9k mRNA and protein was analyzed using real-time PCR and Western blot analysis, respectively. After 24 h of treatment, a significant increase in CaBP-9k expression was observed. This was dependent upon the length of the paraben alkyl chains (shortest in MP and longest in IBP). Interestingly, the synergistic effects of these paraben combinations were observed at a dose (10−5 M) of these parabens, which induced the highest expression of CaBP-9k mRNA and protein. To investigate the involvement of estrogen receptors (ERs) and progesterone receptors (PRs), through which parabens exert their effects, the expression levels of ERα and PR-B were also examined. The expression of ERα mRNA and protein fluctuated after paraben treatment in GH3 cells, which was not significant. However, the expression level of ERα gene was induced when cotreated with 17β-estradiol (E2) and ICI 182, 780 (estrogen receptor antagonist). The different combinations of parabens induced the expression of the PR-B gene, which was abolished by cotreatment with ICI 182,780. The expression patterns of CaBP-9k and PR-B genes appeared to be similar in response to paraben treatments. This implied that CaBP-9k expression in GH3 cells may be induced by parabens via a PR-mediated pathway. Taken together, these results suggest that exposure to multiple parabens at low concentrations may increase their synergistic estrogenic activities in GH3 cells through a PR-mediated pathway.