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Catherine A. Harris, ¹ Pirkko Henttu, ² Malcolm G. Parker, ² and John P. Sumpter ¹

¹ Department of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex, United Kingdom
² Molecular Endocrinology Laboratory, Imperial Cancer Research Fund, London, United Kingdom

Abstract

A large number of phthalate esters were screened for estrogenic activity using a recombinant yeast screen. A selection of these was also tested for mitogenic effect on estrogen-responsive human breast cancer cells. A small number of the commercially available phthalates tested showed extremely weak estrogenic activity. The relative potencies of these descended in the order butyl benzyl phthalate (BBP) >dibutyl phthalate (DBP) >diisobutyl phthalate (DIBP) >diethyl phthalate (DEP) >diisononyl phthalate (DINP) . Potencies ranged from approximately 1 10 ⁶ to 5 10 ⁷ times less than 17ß-estradiol. The phthalates that were estrogenic in the yeast screen were also mitogenic on the human breast cancer cells. Di(2-ethylhexyl) phthalate (DEHP) showed no estrogenic activity in these in vitro assays. A number of metabolites were tested, including mono-butyl phthalate, mono-benzyl phthalate, mono-ethylhexyl phthalate, mono- n -octyl phthalate ; all were found to be inactive. One of the phthalates, ditridecyl phthalate (DTDP) , produced inconsistent results ; one sample was weakly estrogenic, whereas another, obtained from a different source, was inactive. Analysis by gel chromatography-mass spectrometry showed that the preparation exhibiting estrogenic activity contained 0.5% of the ortho -isomer of bisphenol A. It is likely that the presence of this antioxidant in the phthalate standard was responsible for the generation of a dose-response curve--which was not observed with an alternative sample that had not been supplemented with o , p ´-bisphenol A--in the yeast screen ; hence, DTDP is probably not weakly estrogenic. The activities of simple mixtures of BBP, DBP, and 17ß-estradiol were assessed in the yeast screen. No synergism was observed, although the activities of the mixtures were approximately additive. In summary, a small number of phthalates are weakly estrogenic in vitro . No data has yet been published on whether these are also estrogenic in vivo ; this will require tests using different classes of vertebrates and different routes of exposure. Key words : contaminated standards, estrogenicity, MCF-7, metabolites, phthalates, recombinant yeast screen, ZR-75. Environ Health Perspect 105:802-811 (1997) .

Address correspondence to C.A. Harris, Department of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex, UB8 3PH, U.K.