Institute of Pharmacology and Toxicology, University of Zurich,CH-8057, Zurich, Switzerland
Received November 23, 2003; accepted March 24, 2003
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ABSTRACT |
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Key Words: MDA-kb2 cells; androgen receptor; androgen; antiandrogen; endocrine disruptor; pesticide; UV filter.
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INTRODUCTION |
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In this study, we validated the human AR expressing cell line MDA-kb2 with known androgens and antiandrogens and used the system to test UV filters for androgenic or antiandrogenic activity in vitro. Eight of the most frequently used compounds were chosen among the total of 30 UV filters admitted for use in Switzerland. The human breast carcinoma cell line, MDA-kb2, expresses high levels of functional endogenous androgen receptor (AR) and also glucocorticoid receptor (GR), while estrogen receptor alpha and progesterone receptor are not detectable at the RNA level, and estrogen receptor beta is expressed only at low levels (Hall et al., 1992). The cells are stably transfected with a luciferase transporter plasmid driven by the mouse mammary tumor virus promoter (MMTV) that can be activated through both AR and GR (Wilson et al., 2002
). Compounds acting through AR or GR can therefore induce luciferase expression. It was shown earlier that known AR antagonists like hydroxyflutamide, the vinclozolin metabolites M1 and M2, p,p'DDE, and linuron-inhibited dihydrotestosterone (DHT)-induced gene expression in this cell line (Wilson et al., 2002
).
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MATERIALS AND METHODS |
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Stock solutions of the compounds were prepared in absolute ethanol at a concentration of 10-2 M, stored at -20°C, and diluted to desired concentrations in L-15 (LEIBOVITZ) medium (Gibco, Cat No. 11415049, Lot No. 3041839). The final ethanol concentrations in the medium did not exceed 1% (v/v). This concentration did not affect cell proliferation (Schlumpf et al., 2001).
Cell line and cell culture conditions.
The MDA-kb2 cell line was kindly provided by K. Bobseine and L. E. Gray (Endocrinology Branch, U.S. EPA, Research Triangle Park, NC). For routine maintenance, cells were grown in 25-cm2 canted neck tissue culture plastic flasks (Falcon, Oxnard, CA) in Leibovitzs L-15 medium at 37°C in a humidified incubator under regular atmospheric conditions (no CO2). The medium was supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS, Lot No. 1077868, Cat No. 16000044, Life & Technologies, GIBCo, Grand Island, NY), and 1% (v/v) (final concentration) antibiotic-antimycotic agent (Gibcobrl, Cat No. 15240062, Lot No. 1078238).
AR-mediated gene-reporter activation assay in MDA-kb2 cells.
Tests were carried out according to the protocol of Wilson et al.(2002) with several modifications. MDA-kb2 cells were trypsinized and seeded into 96-well plates (Costar NY, USA) at a density of about 1 x 104 cells/well with 100 µl media/well using a multichannel pipettor. After the cells had attached, medium was removed and replaced by dosing medium. For every experiment, wells in column no. 6 were filled with medium as negative control, wells in column no. 7 with 1% ethanol in medium as solvent control, wells in column no. 12 with 10 nM DHT as a positive control (0.1 or 0.5 nM for testing AR antagonists), and wells in column no. 1 with 1µM flutamide or bicalutamide alone (for comparison with the test chemical alone). Wells in columns no. 25 and 811 were filled with different concentrations of the test chemical, rows 14 together with DHT and rows 58 without DHT. Cells were incubated overnight at 37°C.
For measuring luciferase activity, medium was removed. Cells were washed gently two times with Dulbeccos phosphate-buffered saline (PBS) at room temperature. Lysis Buffer (Promega, Cat No. E1351, Lot No. 119684) was added (25 µl/well), and cells were left at room temperature for 30 min. The contents of the wells (25 µl/well) were transferred onto a white Dynatech microtiter plate (DYNEX Technologies, Inc., Chantilly, VA). The plate was read in a luminometer ML 1000 [Dynatech Laboratories, Chantilly, VA], with injection set to deliver 25 µl 1 mM D-luciferin and 25 µl reaction buffer (25 mM glycylglycine, 15 mM MgCl2, 5 mM ATP, 0.1 mg/ml BSA, pH 7.8) using a flash-detection program.
MTT reduction assay.
Cytotoxicity was estimated with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye reduction assay as described by Mosmann (1983), with some modifications. Briefly, cells were seeded at 10,000 cells/100 µl into 96-well flat-bottom culture plates, grown for 24 h, and then treated with the chemicals at the indicated concentrations and time intervals. After incubation with chemicals, 0.1 mg (20 µl/well of 5 mg/ml in PBS) MTT was added. Plates were further incubated for 4 h at 37°C. The medium was then discarded, 100 µl of DMSO was added to each well to dissolve the formazan crystals formed, and the plate was agitated for 1 min. Absorption at 540 nm (reference filter 620 nm) was quantified with a microplate reader (Anthos labtec reader). MTT-reduction for each treatment was expressed as a percentage of control values.
Data analysis.
Results were expressed as mean fold induction compared to negative control (medium) ± SE of the mean (SEM). Luciferase activities of negative control and solvent control were identical. Data were analysed by two-way ANOVA followed by Bonferroni pairwise comparisons (SYSTAT 5.01 software). Differences between groups were considered statistically significant at p < 0.05.
Nonlinear regression and calculation of EC50 and IC50 values were performed with GraphPad Prism 2.01 (GraphPad Software, Inc., San Diego, CA 92121). Mean EC50 or IC50 values of compounds were calculated from the EC50 or IC50 values of individual experiments.
EC50 is the concentration of the agonist producing 50% maximal induction of luciferase activity in MDA-kb2 cells, whereas, IC50 is the concentration of the antagonist producing 50% inhibition of agonist-induced luciferase activity in the cells.
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RESULTS |
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Screening of UV Filters for Androgenic or Antiandrogenic Activity
In order to assess possible agonistic or antagonistic actions of UV filters on AR in MDA-kb2 cells, the cells were exposed to eight UV filters in the absence or presence of 0.1 or 0.5 nM DHT. The list of test chemicals included 4-MBC, 3-BC, OD-PABA, B-MDM, OMC, Bp-3, Bp-4, and HMS. None of the compounds displayed agonistic activity (Fig. 5). However, two UV-filters, Bp-3 and HMS, reduced DHT-induced AR activation in MDA-kb2 cells in a concentration-dependent manner (Figs. 5A
and 5B
; Table 2
). As indicated by the MTT reduction assay, this effect was not due to cytotoxicity, which was seen only at and above 200µM (Fig. 6
). 4-MBC, 3-BC, OD-PABA, B-MDM, Bp-4, and OMC did not inhibit AR activation by DHT across a wide concentration range (Figs. 5C
and 5D
). Bp-3 and HMS were also tested with respect to possible interactions with GR activation (Fig. 7
). HMS did not change the effect of 50 nM dexamethasone on luciferase activity. High concentrations of Bp-3 induced a small increase in luciferase activity beyond the level produced by dexamethasone alone. This effect was not prevented either by the estrogen antagonist ICI 182,780 nor by hydroxyflutamide (Fig. 7B,C
).
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DISCUSSION |
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The assay likewise proved to be suitable and sensitive for the detection of AR antagonistic effects of nonsteroidal antiandrogens such as hydroxyflutamide, flutamide, bicalutamide, and vincolozolin (Bauer et al., 1998; Gray et al., 1994
; Kelce et al., 1994
; Waller et al., 1996
; Wong et al., 1995
). Vinclozolin is converted in vivo into two metabolites, M1 and M2, with higher antiandrogenic activity than the parent compound (Kelce et al., 1994
). The IC50 value of the parent compound observed in the present study is in the range of the activity reported by Wilson and coworkers (2002)
.
A complex situation was encountered with cyproterone actetate (CPA). CPA was characterized by marked agonistic activity across a wide concentration range when given alone, a slight, nonsignificant tendency of DHT antagonism at low CPA concentrations (0.1µM), and an identical dose-response relationship at high CPA concentrations in the presence or absence of DHT. CPA is known to possess significant partial AR agonist activity (Labrie et al., 1987). However, the failure by hydroxyflutamide to reduce its agonistic effect speaks against a major role of AR for the agonistic CPA action in the present cell system. The effect might result from an action on the GR expressed by MDA-kb2 cells; CPA has been reported to exhibit glucocorticoid activity (Lamberts et al., 1988
; Poulin et al., 1991
). A predominance of agonistic effects of CPA was also observed in MCF7-AR1 cells expressing the human AR (A-SCREEN assay) (Ma, 2002
). Thus, antiandrogens with mixed activities may present problems for analysis in both assay systems.
As mentioned in the introduction, the use of UV filters has greatly increased over the last decades. Since they are lipophilic, they may bioaccumulate in the food chain. Evidence for this has been presented for fish (perch and roach in the Meerfelder Maar lake, Germany; Nagtegaal et al., 1997), and for human milk (Hany and Nagel, 1997
). The presence in human milk has recently been confirmed for one UV filter in this laboratory (unpublished observations). When analyzed for potential endocrine activity, 4-MBC, OD-PABA, OMC, Bp-3, and HMS exhibited estrogenic activity in vitro on MCF-7 cells (increased proliferation, pS2 protein induction). 4-MBC, OMC, and Bp-3 also increased uterine weight in immature rats (Schlumpf et al., 2001
). Two of these compounds, benzophenone- 3 (Bp-3) and homosalate (HMS), also showed significant androgen (DHT) antagonism in vitro in MDA-kb2 cells. Both chemicals were completely devoid of agonistic actions when tested alone. The IC50 values of the two UV filters were in the low micromolar range, which is the effective range of other environmental antiandrogenic chemicals such as vinclozolin and linuron (Vinggaard et al., 1999
; Wilson et al., 2002
). While in this cell line, HMS appeared only to antagonize androgen actions and did not interfere with glucocorticoid effects, high concentrations of Bp-3 elicited an unexpected increase of luciferase activity in the presence of dexamethasone, beyond the level induced by the glucocorticoid. Since hydroxyflutamide and the estrogen antagonist ICI 182,780 were unable to block this effect, its nature remains uncertain.
In conclusion, our investigation identified two out of eight UV filters tested, Bp-3 and HMS, as antiandrogens in the in vitro MDA-kb2 cell transcription assay. Both compounds also act as estrogen agonists on MCF-7 cells in vitro, and Bp-3 is estrogenic in the in vivo uterotrophic assay (Schlumpf et al., 2001). In vitro estrogenic potencies (ED50 3.73 µM for Bp-3 and 1.56 µM for HMS) and antiandrogenic potencies (Table 2
) are in a similar range, but such comparisons should be done with caution, considering the different endpoints of the two assays (proliferation versus reporter gene). Our data support the notion that some endocrine disrupting chemicals may interact with endocrine regulation by more than one mechanism (Sohoni et al., 1998
). Whether Bp-3 and HMS are also AR antagonists in vivo remains to be clarified.
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ACKNOWLEDGMENTS |
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NOTES |
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