* Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands, and BioDetection Systems B.V., Badhuisweg 3, 1031 CM, Amsterdam, The Netherlands
Received August 20, 2004; accepted November 8, 2004
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ABSTRACT |
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Key Words: polycyclic musks; UV filters; estrogen receptor; androgen receptor; progesterone receptor.
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INTRODUCTION |
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Recently, parabens, which are used as preservatives in cosmetics, have been found in human breast tumors. It has been suggested that these chemicals might contribute to the rising incidence of breast cancer (Darbre, 2003). In several laboratory experiments, parabens have shown hormone-disrupting properties, including weak estrogenic activity in vitro and estrogenic activity in vivo (Byford et al., 2002
; Darbre et al., 2002
, 2003
; Lemini et al., 1997
; Routledge et al., 1998
). In recent years, UV filters and polycyclic musks have been analyzed for possible hormone-related activity. Several UV filters have been found to be estrogenic in in vitro systems (Miller et al., 2001
; Mueller et al., 2003
; Nakagawa and Suzuki, 2002
; Schlumpf et al., 2001
; Schreurs et al., 2002a
) and also in vivo (Ashby et al., 2001
; Holbech et al., 2002
; Schlumpf et al., 2001
; Tinwell et al., 2002
). The polycyclic musks AHTN and HHCB were found to be only weakly estrogenic, but more potent anti-estrogens in an in vitro reporter gene assay (Schreurs et al., 2002b
; Seinen et al., 1999
). In an in vivo transgenic zebrafish assay, anti-estrogenic effects were observed for both compounds (Schreurs et al., 2004
). Interaction with androgen receptors has been investigated for UV filters (Ashby et al., 2001
, Ma et al., 2003
), some of which some exerted anti-androgenic effects (Ma et al., 2003
). As far as we know, interaction of polycyclic musks and UV filters with the progesterone receptor has never been reported.
In this paper we assess seven UV filters: benzophenone-3 (Bp-3), octyl-methoxycinnamate, 4-methylbenzylidene camphor (4-MBC), butyl-methoxydibenzoylmethane (B-MDM), homosalate (HMS), octyl-dimethyl-p-aminobenzoic acid (OD-PABA), 3-benzylidene camphor (3-BC), and five polycyclic musk fragrances (Tonalide®, Galaxolide®, Celestolide®, Versalide®, Phantolide®) for their (anti)androgenic and (anti)progestagenic activity in newly developed and sensitive in vitro reporter gene assays.
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MATERIALS AND METHODS |
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AR and PR CALUX® bioassays. The generation of stable hAR and hPR transfectants of U2-OS cells is described elsewhere (Sonneveld et al., in press; Sonneveld et al., in press). In short, these cells contain a pSG5-neo-hAR or pSG5-neo-hPR expression vector in combination with a 3 x ARE-TATA-Luc-reporter construct, respectively. AR CALUX® cells were trypsinized and suspended in phenol-red-free DF medium, supplemented with 5% dextran charcoal stripped FCS. Cells were plated in 96-well tissue culture plates (Nunc, Roskilde, Denmark) (6000 cells/well) at a volume of 200 µl per well. After 48 h the medium was changed, and the compounds to be tested (dissolved in ethanol) were added directly to the medium in a 1:1000 dilution. After 24 h, the cells were lysed in 30 µl Triton-lysis buffer (1% Triton X-100, 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA and 1 mM DTT). A 25-µl portion of cell lysate was transferred to a black 96-well plate to which 25 µl luciferine substrate (LucLite reporter gene assay kit, Packard Instruments, Meriden, CT) was added. Luciferase activity was measured in a luminometer (Lucy2; Anthos Labtec Instruments, Wals, Austria) for 0.1 min per well. The PR CALUX® bioassay is performed in exactly the same way as the AR CALUX® bioassay.
Gene expression assay in stable ER and ERß reporter cell lines. The generation of stable hER
and hERß transfectants of HEK293 cells is described previously (Lemmen et al., 2002
). The performance of the assay is similar to the AR and PR CALUX® assays.
Data analysis. Luciferase activity per well was measured as light units. In every experiment, each concentration was analyzed in triplicate. From these values, fold induction was calculated by dividing the mean value of light units in exposed and nonexposed wells. ARE-luc activity as a percentage of maximal dihydrotestosterone induction was calculated by setting the highest fold induction of dihydrotestosterone at 100%. The same was done for the ER- and PR-reporter cell lines, using estradiol and ORG2058 as reference compounds, respectively.
Dose-response curves were fitted using the sigmoidal function: y = y0 + a/[1 = exp((x x0)/b)] in SigmaPlot 2002 for Windows version 8.02 (SPSS Inc., Chicago, IL, USA). Using this fit, the values of EC50 and IC50 were calculated. The figures were drawn using GraphPad Prism 4.00 (GraphPad Software, Inc.).
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RESULTS |
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DISCUSSION |
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In the literature, several chemical compounds have been assessed for their interaction with the androgen receptor. Only a few environmental xeno-androgens have been found, namely triphenyltin (Schulte-Oehlmann et al., 2000), and unidentified compounds in pulp-mill effluent (Parks et al., 2001
). Many more environmental anti-androgens have been found, for example the pesticides vinclozolin, p,p'-DDE, DDT, procymidone, and linuron (Gray et al., 1999
, 2001
; Kelce et al., 1997
). The phthalates diethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP) have been found to be nonclassical anti-androgens, in that they inhibit enzymes concerned with hormone metabolism, rather than competing with endogenous androgens for receptor binding (Parks et al., 2000
). The same phenomenon can be observed for the progesterone receptor. As far as we know, the flavonoid apigenin is the only environmental PR agonist found (Willemsen et al., 2004
). Several compounds have been reported to act like PR antagonists, for example DDT and its metabolites, 4-tert-octylphenol, 4-nonylphenol, lindane, and endosulfan (Jin et al., 1997
; Klotz et al., 1997
; Tran et al., 1996
). These studies were undertaken using a yeast strain hPR-PRE with ß-galactosidase as the read-out signal. The most potent compounds were 4-nonylphenol (IC50 0.50 µM) and the DDT-metabolite DDOH (IC50 0.35 µM). In this research, we also find only antagonists for the AR and PR, rather than agonists.
It is striking that such high concentrations of the clinically used anti-androgen flutamide are needed to exert an effect. It may be that flutamide is not metabolized into the more potent anti-androgen hydroxyflutamide, as occurs in vivo. The UV filter HMS is only a factor of two less potent than flutamide, and the polycyclic musk HHCB is roughly a factor of 5 less potent. Although some of the test compounds were found to be nearly as potent anti-androgens as flutamide, the effect concentrations are quite high and have not been found in the environment.
The most variable range of IC50 values were found in the PR CALUX bioassay. Apart from ADBI, the polycyclic musks were found to be the most potent PR antagonists. The IC50 values of AHTN and AHMI are even lower than those found for 4-nonylphenol and the DDT-metabolite DDOH, while HHCB and AETT have similar IC50 values. When comparing the IC50 values of these four polycyclic musks on the different receptors, those on the PR are roughly a factor of 10100 lower. This means that the tested polycyclic musks are more potent antagonists on the PR than on the ER or AR, when effect concentrations are taken into account.
Some discrepancies with respect to the UV filters 3-benzylidene camphor and 4-methyl benzylidene camphor were found when comparing our results with other studies. The UV filter 3-BC has recently been investigated and compared with the UV filter 4-MBC by Schlumpf et al. (2004). In an E-SCREEN assay, both compounds induced MCF-7 cell proliferation, with EC50 values of 0.69 µM 3-BC and 3.9 µM 4-MBC. In an in vivo uterotrophic assay, 3-BC was found to be more potent than 4-MBC. In our study, such difference between 3-BC and 4-MBC was not observed. In an ER-binding assay, Schlumpf et al. showed that both compounds displaced 16
-125I-estradiol from human ERß, but not from human ER
. This would seem to indicate that 3-BC and 4-MBC are unable to directly bind ER
, and that their agonistic effects in this study could be caused by metabolites. The fact that we do not find different potencies between 3-BC and 4-MBC may be due to differences in metabolism between experimental systems.
Another discrepancy concerns observed anti-androgenic effects. Ma et al. (2003) only found anti-androgenic effects for the UV filters Bp-3 and HMS, while we also found anti-androgenic effects with 3-BC and 4-MBC. These differences may be explained by the fact that Ma et al. used an MDA-kb2 cell line containing low endogenous AR and GR levels. In this study we used a U2-OS cell line overexpressing AR which is probably more selective and sensitive for measuring AR interaction.
In this study we found interaction of cosmetic ingredients with estrogen, androgen, and progesterone receptors. Four polycyclic musks were found to be antagonists toward ERß, AR, and PR. The UV filters were found to be mainly ER-agonists, and antagonists toward AR and PR. For a detailed risk assessment of cosmetic ingredients concerning endocrine disruption in humans, more data is needed, especially in vivo data. Recently, parabens, which are used in deodorants, were found to be present in human breast tumors (Darbre et al., 2004
). Several in vitro studies have shown that parabens are estrogenic (Byford et al., 2002
; Routledge et al., 1998
). The same effect has also been shown in in vivo studies (Darbre et al., 2003
; Routledge et al., 1998
). Due to the apparent estrogenic activity of parabens and their presence in breast tumors, it has been speculated that parabens might contribute to the rising incidence of breast cancer (Darbre et al., 2004
). However, parabens are not the only xenoestrogens that can be found in breast tissue. Polychlorinated biphenyls (PCBs), DDT, DDE, and dieldrin have been found in breast tissue too. Further, it cannot be ruled out that the tested estrogenic UV filters, which have been found in mother's milk, can also be found in breast tissue or breast tumors. The polycyclic musks AHTN and HHCB have also been found in human breast milk as well, but these compounds act as ER antagonists. The consequences of exposure to an anti-estrogenic effect induced by xenobiotics for human health so far remain unclear. In literature, anti-estrogenic effects in wildlife have been rarely found. Several estrogenic chemicals have been found to also be anti-androgenic (Sohoni and Sumpter, 1998
). In this paper some UV filters were also found to have these properties. A small number of chemical-compound-induced anti-androgenic effects in animals have been reported, for example by vinclozolin and DDE (reviewed in Kelce and Wilson, 1997
). As far as we know, disruption of the PR-mediated pathway has not been investigated. As these latter effects in our research were found to be relatively strong, future studies should focus on in vivo endocrine disruptive effects produced by anti-progestins.
This research shows that a single compound can exert different effects on several hormone receptors. However, it is not possible to predict the overall endocrine disruptive effect from in vitro studies. Therefore, in vivo studies are needed to find out whether a compound can exert endocrine-related adverse health effects. Screening chemicals in suitable in vitro assays and finding out whether a compound is an agonist and/or an antagonist toward a given hormone receptor is a useful and potentially important starting point in the search for environmental endocrine disruptive chemicals. Using in vitro assays can provide a mechanistically based, early focus on compounds of interest and a potentially significantly reduction in the number of in vivo studies with the associated ethical issues and costs involved. The bioassays used in this paper are sensitive, straightforward and fast to use, which makes them a suitable tool for the screening of suspected endocrine active compounds.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Institute for Risk Assessment Sciences, Utrecht University, PO Box 80176, 3508 TD, Utrecht, The Netherlands. Fax. +31 30 2535077. E-mail: w.seinen{at}iras.uu.nl
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