* EcoScreen R & D Section, Endocrine Disrupting Chemical Analysis Center, Otsuka Life Science Initiative, Otsuka Pharmaceutical Co., Ltd. 224-18 Ebisuno Hiraishi, Kawauchi-cho, Tokushima, 771-0195, Japan; and Department of Medicine and Bioregulatory Science (Third Department of Internal Medicine), Graduate School of Medicine Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
Received July 30, 2004; accepted September 7, 2004
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ABSTRACT |
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Key Words: aromatase; endocrine disrupter; screening assay; KGN cell line; benomyl.
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INTRODUCTION |
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Aromatase is a key enzyme in the conversion of androgens to estrogens and has an important role in maintaining a homeostatic balance between them. Some flavonoid chemicals, for example, -naphthoflavone, apigenin, and chrysin, are known to inhibit aromatase activity in vitro (Campbell and Kurzer, 1993
; Jeong et al., 1999
; Kellis and Vickery, 1984
; Le Bail et al., 1998
; Pelissero et al., 1996
). In addition, the herbicide atrazine has recently been shown to induce aromatase activity, and various imidazole-like fungicides have been shown to be aromatase inhibitors (Sanderson et al., 2002
). Also, the biocides triphenyltin (TPT) and tributyltin (TBT), which are used in antifouling paints and wood preservatives, are suspected to inhibit aromatase activity and cause imposex in gastropods (Heidrich et al., 2001
; Saitoh et al., 2001
).
Two kinds of in vitro assay have been developed to measure aromatase activity: a cell-free assay using human placental microsomes (Njar et al., 1995; Vinggaard et al., 2000
) or human recombinant aromatase protein; and a cell-based assay using mammalian cell lines, such as the human JEG-3 (Drenth et al., 1998
; Yue and Brodie, 1997
) and JAr (Brueggemeier et al., 1997
) cell lines. In either case, aromatase activity is determined by measuring the amount of 3H-water released upon enzymatic conversion of radiolabeled androstenedione. However, these assays require the use of radioactive materials and specialized equipment for radiometric measurement. An alternative fluorescent cell-free method has recently been developed using human recombinant aromatase protein (Stresser et al., 2000
), but this method cannot detect aromatase induction because it utilizes a cell-free system.
In the current study, we developed a novel nonradioactive cell-based assay that can detect both inhibition and induction of aromatase. This assay was performed using KGN cells, which are a steroidogenic human ovarian granulosa-like tumor cell line that was established from a patient with invasive granulosa cell carcinoma (Nishi et al., 2001). This cell line possesses normal properties of granulosa-like cells, including relatively high aromatase activity that is stimulated by follicle stimulating hormone or cAMP. In addition, the KGN cells cannot synthesize androgen or estrogen by themselves due to the absence or low level of 17
-hydroxylase. Therefore, the aromatase activity can be evaluated simply by culturing the cells with androstenedione and measuring the estrone level in the culture medium with a specific enzyme-linked immunosorbent assay (ELISA). This novel assay should be useful for the high-throughput screening of chemicals for aromatase inhibition or induction.
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MATERIALS AND METHODS |
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Chemical preparations. Test chemicals (Table 1) and androstenedione (Sigma, St. Louis, MO) were dissolved in DMSO at a concentration of 10 mM and stored at 20°C. The maximum concentration of each chemical used in the experiments was 10 µM, 1 µM, or 100 nM, depending on its solubility in the cell culture medium. Then the test chemicals and the substrate were diluted with serum-free DMEM/F-12 medium by 50 and 100 folds, respectively. And there were six dose points (by 10 folds diluted from maximum concentrations) tested in this study. The final concentration of DMSO in the cell growth medium was 0.15% (v/v), and we confirmed that 0.15% DMSO had no statistically significant effect on the estrone production in KGN cells by Student's t-test (p = 0.65, n = 8). All treatments were tested in triplicate, except for the controls, which were performed in sextuplicate.
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Estrone ELISA. The estrone ELISA was carried out as described in the manufacturer's instructions, except that 3,3',5,5'-tetramethylbenzidine solution and Stop Buffer (Scytech, Logan, UT) was used instead of the o-phenylenediamine and the stop buffer provided in the kit. The absorbance in each well was measured at 450 nm using an ARVOsx 1420 multilabel counter (Wallac, Turku, Finland), and the estrone concentration in each well was calculated based on a standard curve and using SOFTmax Pro 4.0 (Molecular Devices, Sunnyvale, CA).
Measurement of cell viability. The cytotoxicity of the various test chemicals was assessed using AlamarBlue assay (Ahmed et al., 1994). An 8 µl volume of the AlamarBlue reagent (Serotec Ltd., Oxford, UK) was added to the wells, and cells were incubated for 3 to 4 h at 37°C according to the manufacture's protocol. The fluorescence was measured at 590 nm with excitation at 544 nm using the ARVOsx 1420 microplate reader. The cytotoxicity was determined by comparing the fluorescence in each well with the fluorescence of solvent control wells (0.15% DMSO).
Data and statistical analysis. Effects on aromatase activity and cell toxicity were expressed as a relative ratio of estrone concentrations at each dosing point divided by the estrone concentration in solvent controls. If there was more than 50% inhibition of aromatase activity, IC50 values were calculated by GraphPad PRISM ver 4.0 (GraphPad, San Diego, CA) using sigmoidal dose-response curve fitting (variable slope). Data were analyzed by one-way factorial ANOVA using SatView for Windows (SAS Institute Inc., Cary, NC).
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RESULTS |
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Effects of Test Chemicals on Aromatase and Cell Viability
Using the optimized assay, we tested the effect of 23 chemicals that had previously been reported to affect aromatase activity. Most of these were selected from a draft of a detailed review paper on aromatase by the U. S. Environmental Protection Agency (2002). Included in the 23 chemicals were flavonoids, a pesticide, pharmaceutical compounds, and organotin compounds. We first tested the eight flavonoid chemicals for effects on aromatase activity and cell viability (Fig. 3). All flavonoids were found to inhibit aromatase activity at relatively high concentrations, and flavone and flavanone were very weak inhibitors. These chemicals did not have cell toxicity on KGN cells, excepting for apigenin, which had some cell toxicity at maximum concentration. Next, we investigated the effects of 10 pesticides, pharmaceutical compounds, and organotins (Fig. 4). With the exception of o,p-DDT (o,p-dichloro-1, 1-diphenyl-2,2,2-trichloroethane), all of the chemicals inhibited the aromatase activity. The organotins also decreased aromatase activities, but they were very cytotoxic. These inhibitory effects on aromatase activity by organotins seemed to be affected by these cell toxicities. Finally, we tested five chemicals that are suspected to induce aromatase activity (Fig. 5). We found that benomyl and forskolin induced aromatase activity, but the other three, atrazine, diclobutrazole, and vinclozolin, had no inducible effects. A summary of these results and the related chemical information is shown in Table 1. The table also shows the mean IC50 values (n = 3) calculated for compounds that inhibited aromatase activity as well as any previously reported IC50 values. Overall, the IC50 values increased the following order: 4-hydroxyandrostenedione (4-OHA) < imazalil <<
-naphthoflavone < propiconazole < 7-methoxyflavone < chrysin < fenarimol < aminoglutethimide < naringenin < apigenin < triadimefon << 7-hydroxyflavone < p,p'-DDD (p,p'-dichlorodiphenyldichloroethane).
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DISCUSSION |
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Our testing of potential endocrine disrupters showed that the eight flavonoids, which are weak inhibitors of aromatase in vitro (Ibrahim and Abul-Hajj, 1990; Kao et al., 1998
; Kellis et al., 1984
; Le Bail et al., 2001
; Saarinen et al., 2001
), also inhibited aromatase activity in our cell-based assay. In addition, flavone and flavanone, which are weaker aromatase inhibitors than other flavonoids, could be detected using our assay, indicating that the method has high sensitivity. However, IC50 values were not calculated for these two compounds because the highest concentrations tested produced less than 50% inhibition.
Among agricultural chemicals, pesticides, and fungicides suspected to be inhibitors of aromatase, fenarimol, triadimefon, imazalil, propiconazole, and p,p'-DDD had inhibitory effects in our assay, whereas o,p'-DDT had little or no effect at the concentration range we tested (100 pM10 µM). On the contrary, o,p'-DDT was reported to inhibit aromatase activity at 10 µM in H295R cells (Sanderson et al., 2002). Although the reason for this discrepancy is unclear, it may be due to the difference in cell type or origin. We also found that the pharmaceutical compounds 4-OHA (also called formestane) and imazalil strongly inhibited the aromatase activity. In fact, the IC50 of imazalil was lower in our assay than in previous reports (Sanderson et al., 2002
). Sanderson et al. (2002)
investigated imazalil at concentration from 0.1 µM to 1 mM, and their IC50 value of imazalil was 0.1 µM in H295R cell. Our estimated IC50 value of imazalil was about 20-folds lower than that of H295R results (Table 1).
Organotin compounds were very toxic to the KGN cells. In fact, both TBT (tributyltin chloride) and TPT (triphenyltin chloride) had previously been reported to have strong cell toxicity (Saitoh et al., 2001). They showed TBT and TPT had inhibitory effect on aromatase activity in KGN cells with no cell toxicity at concentration lower than 20 ng/ml. In this study, decrease of the cell viability was also observed in TBT and TPT (Fig. 4). In TBT, the reduction of aromatase activity was also observed, however the cell viability was described in parallel. Therefore we considered reduction of aromatase activity in TBT mostly depended on its cell toxicity. TPT also had cell toxicity at concentration of 1000 ng/ml; however aromatase activity at 200 ng/ml was clearly decreased without cytotoxic effect. TPT could inhibit aromatase activity, but that range was very limited. We have used 24 h for preincubation of chemicals with KGN cells and an additional 24-h incubation for aromatase reaction with 96-well plate; meanwhile Saitoh's have 48 h and 6 h incubation with petri dish, respectively. Cell culture methods and exposure time with KGN cells and/or enzyme reaction time may have contributed to the difference about organotin compound between two studies. Although Bettin et al. (1996)
concluded that the induction of imposex in gastropods by organotin compounds may be due to inhibition of aromatase (Bettin et al., 1996
), our results as well as those of Sanderson et al. (2000) and Morcillo and Porte (1999)
suggest that the induction of imposex in gastropods by TBT or TPT occurs via mechanisms other than inhibition of aromatase activity.
In our investigation of aromatase inducers, we found that forskolin, a known inducer of cAMP, strongly enhanced aromatase activity. This is not surprising, because cAMP promotes aromatase gene expression by binding to a cAMP response element upstream of the aromatase gene. At 10 mM, benomyl, one of fungicides, induces aromatase activity twofold, which is consistent with previously reported results in KGN cells (Morinaga et al., 2004). They further reported that benomyl and its metabolite carbendazim induce aromatase activity through stimulation of CYP19 (aromatase) expression. The mechanism was unclear, although it was confirmed to not be through elevation of intracellular cAMP. This suggests that long-term exposure of wildlife and humans to chemicals like benomyl might lead to estrogen-mediated pathologies, such as tumor promotion, inappropriate sexual differentiation, and inappropriate feminization. Therefore, it is urgent to further investigate the physiological effects of this and similar compounds.
In contrast to forskolin and benomyl, there was no enhancement of aromatase by atrazine, o,p'-DDT, diclobutrazole, or vinclozolin, even though they have been reported to increase aromatase activity in H295R cells (Sanderson et al., 2002). Particularly, atrazine has been reported to cause over twofold induction on aromatase activity at 30 µM in H295R cells (Heneweer et al., 2004
; Sanderson et al., 2002
). However we could not observe such inducible effects at 5 µM or 50 µM in KGN cells. Because the H295R cell line is derived from a human adrenocortical carcinoma cell, the observed discrepancy may be due to the different origins of the cells. This could also be due to alternative transcriptional mechanisms; for example, the promoter sites in the aromatase (CYP19) gene are different in various tissues and are regulated by cellular conditions or signaling pathways (Bulun et al., 2004
; Simpson, 2004
). In fact, one study reported that atrazine induces aromatase activity in H295R but not rat R2C (Heneweer et al., 2004
). Furthermore, another report using KGN cells and a radioisotope-based aromatase assay showed no enhancement of activity by 10 µM of atrazine (Morinaga et al., 2004
). Also, although atrazine has been reported to enhance cAMP levels in H295R cells (Sanderson et al., 2002
), it had no effect on aromatase in KGN cells despite the fact that forskolin promoted aromatase in our assay. Although the reason for this is unclear, again, it could be due to alternative promoter site usage. Considering these results, if the objective is to screen endocrine disruptors, it would be better to use cells from the reproductive tissue origin.
In summary, in the current studies we introduced a novel method for measuring aromatase activity. Whether KGN cells are the most suitable for aromatase screening remains to be determined, but we expect that this assay will be very useful for the screening of large numbers of samples because the assay is simple, nonradioactive, and adaptable to high-throughput screening. Therefore, it should be possible to perform large-scale screening for identification of chemicals that can affect aromatase activity and thereby lead to abnormal steroidogenesis.
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SUPPLEMENTARY DATA |
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ACKNOWLEDGMENTS |
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The KGN cells are available from the Riken Cell Bank as the stock number RCB No.1154 (http://www.brc.riken.go.jp/).
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NOTES |
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1 To whom correspondence should be addressed. Fax: 81-88-665-3613. E-mail: iidam{at}hq.otsuka.co.jp.
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