ESTROGENIC ACTIVITY OF BISPHENOL-A AND OTHER BISPHENOLS ASSESSED BY YEAST TWO-HYBRID ASSAY Masanori Fujita, Michihiko Ike* and Min-Yu Chen Department of Environmental Engineering, Graduate School of Engineering, Osaka University, 2-1, Yamada-oka, Suita, Osaka 565-0871, Japan Tel: (81 6) 6879 7672 Fax: (81 6) 6879 7675 Email: email@example.com ABSTRACT Bisphenol-A (BPA: 2,2-bis(4-hydroxyphenyl)propane) is an industrially important compound which is used in production of polycarbonates, epoxy rasins, lacquer coatings on food cans and food storage vessels etc. Total annual production of BPA in the world reached 930 million pounds in the beginning of 1990s, and it has become a most ubiquitous contaminant in all kinds of aquatic environment. It has been detected at especially high concentrations in the leachate from solid waste disposal sites. A group of chemicals structurally similar to BPA is also abundantly and widely utilized in the manufacture of resin and plastics. These chemicals, which consist of two phenolic rings joined together through a bridging carbon or other chemical structures, are named BPA-related compounds or bisphenols (BPs). Some of these compounds are considered to be able to partly replace BPA in the industrial applications. Especially, the production of bisphenol-S (BPS: 4,4-dihydroxyphenylsulfone) which has an excellent stability against high temperature and resistance to sunlight tends to increase year by year. Resultantly, the discharge of BPs into the environment will increase, and BPs can be widespread environmental pollutants in future as well as BPA. Recently there has been increasing public concern that various chemicals in the environment are adversely affecting wildlife and human health by disrupting normal endocrine function through interaction with steroid hormone receptors, and BPA was identified as a weakly estrogenic chemical (Colborn et al., 1996). Several researchers have confirmed the estrogenicity of BPA using various in vivo and in vitro assays (e.g., Brotons et al., 1993; Rehmann et al., 1998). However, much less attention was paid to the estrogenic activity of other BPs, though they have structures similar to BPA. It can be said that the estrogenic activity of other BPs has not been fully studied, and it is required to assess their possible hazardous effects. Therefore, we have investigated the estrogenic activity of a variety of BPs in comparison with BPA in this study using a simple yeast screening system. Structures of BPs used in this study are shown in Fig. 1. To assess the estrogenic activity of BPs, a yeast two-hybrid system developed by Nishikawa et al. (1999). This system is based on the ligand-dependent interaction of two proteins, an estrogen receptor ER-alfa and a coactivator TIF2, and the estrogenic activity is detected by beta-galactosidase (b-gal) activity. BPs were dissolved in dimethyl sulfoxide and the yeast screen was exposed to them for 4 hours. The 17beta-estradiol (E2) dose-response curve was used as the positive control for evaluating the estrogenic activity of BPs. The b-gal activity was induced by over 0.1-1.0 mg/l of BPA in the yeast screen, and it was confirmed that BPA is estrogenic. BPA was 4-5 orders of magnitude less potent than E2. All the BPs used in this study showed the concentration-dependent increase of b-gal activity, i.e. estrogenic activity to a certain degree. Among the tested diphenylalkanes (BPs of which two phenolic rings are joined with alkanes) 2,2-bis(4-hydroxyphenyl)butane (BPB) showed the highest estrogenic activity, about 1 order of magnitude higher than BPA. The estrogenic activity of 4,4-ethylidenebisphenol (BPE) was comparable to that of BPA, while bis(4-hydroxyphenyl)methane (BPF) was a slightly less potent than BPA. A trend that higher estrogenic activity of diphenylalkanes was obtained from longer alkyl substituent at the center bridging carbon was previously reported also (Perez et al., 1998). Compared with BPA. 2,2-bis(4-hydroxy-3-methyl)phenylpropane (BPP) and 4,4-thiodiphenyl (TDP) showed considerably higher and comparable estrogenic activity, respectively. On the other hand, 4,4'-hydroxyphenylbenzophenone (HBP) and BPS were much less potent. Especially the estrogenic activity of BPS was estimated at about 2 orders of magnitude lower level of BPA. BPs including BPA seem to possess estrogenic activity as a common property. Some of them such as BPS are considered to have lower estrogenicity, and the use of such BPs instead of BPA may reduce the possible risk of the plastic industry. However, their environmental fate including the biodegradability should be taken into consideration to totally assess the impact of BPs on the ecosystem and human health. m BPP: 2,2-bis(4-hydroxy-3- ethyl)- BPA: 2,2-bis(4-hydroxyphenyl)propane phynylpropane CH 3 H3 C CH 3 CH 3 ++ HO C OH ++ HO C OH CH 3 CH 3 BPF: bis (4-hydroxyphenyl)methane - HBP: 4,4'-hydroxyphenyl benzophenone ++ HO CH 2 OH + HO CO OH BPE: 4,4-ethylidenebis phenol BPS: 4,4'-dihydroxyphenylsulfone CH 3 ++ HO CH OH + HO SO2 OH BPB: 2,2-bis(4-hydroxyphenyl)butane TDP: 4,4-thiodiphenyl CH 3 +++ HO C OH +++ HO S OH CH 2 CH 3 +++, Estrogenic activity was significantly higher than BPA; ++, Estrogenic activity was comparable to BPA; +, Estrogenic activity was significantly lower than BPA. Fig. 1. BPs used in this study and their estrogenic activity. REFERENCES Brotons, J.A., O-Serrano, M.F., Villalobos, M., Pedraza, V. and Olea, N. 1995. Xenoestrogens released from lacquer coatings in food cans. Environ. Health Perspect 104: 608-612. Colborn, T., Dumanoski, D. and Myers, J.P. 1996. Our stolen future. New York. Nishikawa, J., Saito, K., Goto, J., Dakeyama, F., Matsuo, M. and Nishihara, T. 1999. New screening methods for chemicals with hormonal activities using interaction of nuclear hormone receptor with coactivator. Toxicol. Appl. Pharmacol. 154: 76-83. Perez, P., Pulgar, R., Olea, S.F., Villalobos, M., Rivas, A., Metzler, M., Pedraza, V. and Olea, N. 1998. The estrogenicity of bisphenol A-related diphenylalkanes with various substituents at the center carbon and the hydroxy groups. Environ. Health Perspect 106: 167-174. Rehmann, K., Schramm, K.W. and Kettrup, A.A. 1999. Applicability of a yeast oestrogen screen for the detection of oestrogen-like activities in environmental samples. Chemosphere 38: 3303-3312.
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