In recent studies, both diesel exhaust (DE) and diesel exhaust particles (DEP) have been reported to exert toxic effects on both the male and female reproductive systems. These compounds suppress spermatogenesis in mice 7) and rats 8) ; in one study, serum testosterone levels and the weights of accessory sex glands increased significantly in F344 male rats exposed to DE for 8 months.9) In addition, pregnant C57BL mice injected with diesel exhaust particulate extract (DEPE) showed a significant increase in the rate of abortions, 10) and DEPE increases the uterine weight and myometrial contractility in this strain of mice.11) These results suggest that chemical substances present in diesel exhaust emission cause disturbance of endocrine systems. Meek 12) reported that dichloromethane extracts of DEP could act as activating ligands for estrogen receptors, and Taneda et al. 13,14) reported that crude DEP, as well as successively extracted hexane, benzene, dichloromethane, and methanol fractions, showed estrogenic activity on hER-yeast screen assay. Mori et al. 15) reported that 4,6-and 2,8-dimethyldibenzothiophenes isolated from DEP had estrogenic activity in hER-yeast assay. However, the specific compound that is responsible for this phenomenon remains unclear.We recently isolated the nitrophenol derivatives 4-nitrophenol, 2-methyl-4-nitrophenol, 3-methyl-4-nitrophenol, and 4-nitro-3-phenylphenol from DEP and showed that they had vasodilatory activity. 16,17) These compounds captured our attention because it has long been known that alkylphenols are associated with estrogenic activity.2,4) In receptor-binding studies, alkylphenols have been shown to interact directly with the estrogen receptors of rainbow trout and to act in an identical way to 17b-estradiol (E 2 ) in stimulating receptor transcription.2) Furthermore, some estrogenic compounds are known to possess anti-androgenic activity.18) Thus the alkylphenols of DEP are likely to be associated with both estrogenicity and anti-androgenicity.We threrfore evaluated the estrogenic and anti-androgenic activity of the nitrophenols with alkylphenolic structures that we had previously isolated from DEP.
MATERIALS AND METHODS
ReagentsWe purchased 4-nitrophenol (PNP), 2-methyl-4-nitrophenol (4-nitro-o-cresol, PNOC), and 3-methyl-4-nitrophenol (4-nitro-m-cresol, PNMC) from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan), and we synthesized the 4-nitro-3-phenylphenol (PNMPP) by the method described previously.
17)All other reagents used were of the purest grade commercially available.Human Estrogen Receptor and Human Androgen Receptor Yeasts Human estrogen receptor (hER)-yeast and human androgen receptor (hAR)-yeast were kindly provided by Prof. John P. Sumpter of Brunel University, Uxbridge, U.K. These strains were developed in the Genetics Department at Glaxo Wellcome, plc. (Stevenage, Herts, U.K.).The DNA sequences of either hERa or hAR were integrated stably into the genome of a strain of the yeast Saccharomyces cerevisiae. The yeast also contained expression plasmids ca...
