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:


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

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.

                                                   BPP : 2,2-bis(4-hydroxy-3-methyl)-
    BPA: 2,2-bis(4-hydroxyphenyl)propane           phynylpropane
                         CH3                                 H3 C        CH3    CH3
      ++    HO           C          OH               ++     HO           C       OH
                         CH3                                             CH3

    BPF: bis(4-hydroxyphenyl)methane              HBP: 4,4'-hydroxyphenyl-

      ++    HO          CH2          OH               +     HO          CO       OH

    BPE: 4,4-ethylidenebisphenol                   BPS: 4,4'-dihydroxyphenylsulfone
      ++    HO           CH          OH               +     HO          SO2       OH

   BPB: 2,2-bis(4-hydroxyphenyl)butane             TDP: 4,4-thiodiphenyl
     +++    HO           C          OH               +++    HO          S       OH
+++, 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.

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:

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:

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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