No abstract

The alkyl esters of p-hydroxybenzoic acid (parabens) are used widely as preservatives in foods, pharmaceuticals and cosmetics to which the human population is exposed. Recent studies have reported that methylparaben, ethylparaben, n-propylparaben and n-butylparaben all possess oestrogenic activity in several in vitro assays and in animal models in vivo. This study reports on the oestrogenic activity of isobutylparaben in a panel of assays in vitro and in vivo. Isobutylparaben was able to displace [(3)H]oestradiol from cytosolic oestrogen receptor alpha of MCF7 human breast cancer cells by 81% at 100 000-fold molar excess. Using a clonal line of MCF7 cells containing a stably transfected oestrogen-responsive ERE-CAT reporter gene, CAT gene expression could be increased by isobutylparaben such that the magnitude of the response was the same at 10(-5) M isobutylparaben as with 10(-8) M 17beta-oestradiol. Isobutylparaben could also increase expression of the endogenous oestrogen-responsive pS2 gene in MCF7 cells and maximal expression at 10(-5) M isobutylparaben could be inhibited with the anti-oestrogen ICI 182 780. The proliferation of two oestrogen-dependent human breast cancer cell lines MCF7 and ZR-75-1 could be increased with isobutylparaben such that at concentrations of 10(-5) M the proliferation response was of the same magnitude as with 10(-8) M 17beta-oestradiol. Evidence for oestrogen receptor mediation of proliferation effects was provided by the inability of isobutylparaben to influence the growth of oestrogen-unresponsive MDA-MB-231 human breast cancer cells and by the ability of the anti-oestrogen ICI 182 780 to inhibit the isobutylparaben effects on MCF7 cell growth. The proliferation response to 10(-10) M 17beta-oestradiol was not antagonized with isobutylparaben at any concentration from 10(-9) M to 10(-4) M in either MCF7 or ZR-75-1 cells. Finally, subcutaneous administration of isobutylparaben was able to increase the uterine weight in the immature mouse after three daily doses of 1.2 or 12.0 mg per mouse. Previous work using linear-alkyl-chain parabens has shown that oestrogenic activity increases with alkyl chain length from methylparaben to n-butylparaben. The results here show that branching of the alkyl chain to isobutylparaben increases oestrogenic activity beyond that of the equivalent length linear alkyl chain in n-butylparaben.

Previous work has demonstrated that the alkyl esters of p-hydroxybenzoic acid (parabens) possess oestrogenic activity, which increases with length of alkyl chain from methylparaben to n-butylparaben and with branching in the alkyl chain from n-butylparaben to isobutylparaben. This study reports on the oestrogenic activity of benzylparaben in a variety of assays in vitro and in vivo. Benzylparaben was able to displace [(3)H]oestradiol from cytosolic oestrogen receptor (ER) of MCF7 human breast cancer cells by 22% at 1000-fold molar excess, by 40% at 10,000-fold molar excess, by 57% at 100 000-fold molar excess and by 100% at 1,000,000-fold molar excess. It was able to increase expression of a stably transfected oestrogen responsive reporter gene (ERE-CAT) in MCF7 cells after 24 h at 10(-5)M/10(-4)M and after 7 days at 10(-6)M/10(-5)M/10(-4)M. Proliferation of MCF7 cells could be increased by 10(-6)M/10(-5)M benzylparaben and this could be inhibited by 10(-7)M pure anti-oestrogen ICI 182,780, indicating that growth effects were ER mediated. Further evidence for ER-mediation was provided from the ability of benzylparaben to increase the growth of a second oestrogen-dependent human breast cancer cell line ZR-75-1, but not the oestrogen-insensitive MDA-MB-231 cell line. When tested in the presence of 10(-10)M 17beta-oestradiol, benzylparaben gave no antagonist response on the growth of either MCF7 or ZR-75-1 cells. Finally, benzylparaben could increase uterine weight in the immature mouse following topical application of three daily doses of 33 mg to dorsal skin. These results demonstrate that the oestrogenicity of methylparaben can be increased by the addition of an aryl group as well as by lengthening or branching the alkyl grouping.