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Endocrinology, doi:10.1210/en.2003-1182
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Endocrinology Vol. 145, No. 4 1860-1869
Copyright © 2004 by The Endocrine Society

A Benzimidazole Fungicide, Benomyl, and Its Metabolite, Carbendazim, Induce Aromatase Activity in a Human Ovarian Granulose-Like Tumor Cell Line (KGN)

Hidetaka Morinaga, Toshihiko Yanase, Masatoshi Nomura, Taijiro Okabe, Kiminobu Goto, Nobuhiro Harada and Hajime Nawata

Department of Medicine and Bioregulatory Science (Third Department of Internal Medicine), Graduate School of Medical Sciences, Kyushu University (H.M., T.Y., M.N., T.O., K.G., H.N.), Higashi-ku, Fukuoka 812-8582, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Corp. (H.M., T.Y., M.N., T.O., K.G., H.N.), Kawaguchi, Saitama 332-0012, Japan; and Department of Biochemistry, Fujita Health University School of Medicine (N.H.), Toyoake, Aichi 470-1192, Japan

Address all correspondence and requests for reprints to: Toshihiko Yanase, M.D., Department of Medicine and Bioregulatory Science (Third Department of Internal Medicine), Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: yanase{at}intmed3.med.kyushu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endocrine disruptor chemicals are known to cause a range of abnormalities in sexual differentiation and reproduction. One mechanism underlying such effects may be via alteration of aromatase activity, which is responsible for estrogen production. A good screening system for identifying endocrine disruptors has long been desired. We have recently established a human ovarian granulosa-like tumor cell line, KGN, which possesses a relatively high level of aromatase expression and is considered a useful mammalian model for investigating the in vitro effects of various chemicals on aromatase activity. In this study we screened 55 different candidate chemicals for endocrine disruptors by assaying aromatase activity. Only benomyl, known as both a benzimidazole fungicide and a microtubule-interfering agent, was found to induce aromatase activity in association with increased levels of aromatase mRNA in KGN cells. The effect of benomyl was presumed to be mediated by its metabolite carbendazim, because it produced an effect equivalent to that of benomyl. The mechanism underlying the benomyl-induced increase in aromatase activity appears independent of the cAMP-protein kinase A pathway. Treatment with taxol, another class of microtubule-interfering agents, also caused induction of aromatase in KGN cells. Both benomyl and taxol changed KGN cell morphology, including the development of cell roundness and a disorganized network of microtubules. These results indicate that benomyl is a potential endocrine disruptor that provides a novel estrogenicity and operates through a microtubule-interfering mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE ARE SERIOUS concerns that certain environmental contaminants and commercial products have the potential to disturb endocrine function in humans and wildlife, lead to impaired reproductive capacity, and have other toxic effects on sexual differentiation, growth, and development (1, 2). These chemicals are called endocrine disruptors and act as estrogenic or antiestrogenic agents by affecting both the synthesis and the action of estrogen. Many chemicals exert their effects by blocking or activating steroid hormone receptors and/or affecting the levels of sex hormones. These effects can disrupt the development or differentiation of both the male and the female reproductive system. These facts emphasize the importance of screening candidate chemicals for a wide range of hormone-mimicking effects.

One important enzyme in the steroid synthesis pathway is cytochrome P450 aromatase (aromatase), which is a product of the CYP19 gene and catalyzes the rate-limiting step in the conversion of androgens into estrogens. Aromatase is responsible for maintaining the homeostatic balance between androgens and estrogens in both sexes. This enzyme is expressed by many human tissues, including the ovary, testis, placenta, brain, liver, adipose, muscle, and breast tissue. This pattern of aromatase expression is regulated by a tissue-specific promoter derived from alternative splicing of the CYP19 gene (3, 4).

For several years the activating or inhibiting effects of some endocrine disruptors on aromatase activity have been reported. Aromatase activity can be induced by 2-chloro-s-triazine herbicide (atrazine, simazine, and propazine) in vitro (5) and by p,p'-dichlorodiphenyldichloroethane (p,p'-DDE) in vitro and in vivo (6). Recently, we demonstrated that tributyltin and triphenyltin, commonly used as biocides in antifouling paint and wood preservatives, inhibited aromatase activity in vitro (7). Also, several chemicals, including pesticides such as prochloraz, fenarimol, triadimefon, triadimenol, and mono-(2-ethylhexyl) phthalate, suppress aromatase activity in vitro (8, 9, 10, 11, 12).

We have recently established a steroidogenic human ovarian granulosa-like tumor cell line, KGN, from a patient with invasive granulosa cell carcinoma (13). The cell line possesses properties similar to those of normal granulosa cells, including relatively high aromatase activity. The cell line is thus considered to be a useful model for investigating the effects of some endocrine disruptors on aromatase activity in vitro (7). Using this system, we screened 55 candidate chemicals for endocrine disruption in assays of aromatase activity and found that only benomyl [methyl 1-(butylcarbamoyl)-2- benzimidazolecarbamate], a benzimidazole fungicide commonly used on a variety of food crops and ornamental plants, induces aromatase activity in KGN cells. Benomyl is also able to interfere with the assembly of fungal microtubules (14), can rapidly degrade into carbendazim in aqueous (15) or organic (16) solutions, or as a result of hydroxylation in the microsomal monooxygenase system (17). In this study we investigated some of the mechanisms involved in benomyl-induced aromatase activity in association with the properties stated above.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and supplies
Chemicals were obtained from Wako Pure Chemical Co. (Osaka, Japan) and AccuStandard, Inc. (New Haven, CT). Carbendazim was obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). [1ß-3H]Androstenedione was obtained from NEN Life Science Products (Boston, MA). Wortmannin, U0126, PD98059, genistein, AG490, and AG1478 were obtained from Sigma-Aldrich Corp. (St. Louis, MO).

Cell culture
KGN cells were maintained in a DMEM/Ham’s F-12 medium (Invitrogen, Grand Island, NY) supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml) as previously described (13).

Human ovarian granulosa cells were obtained from four women who underwent in vitro fertilization. Written informed consent was obtained from each subject before the study. The brief protocol of in vitro fertilization was described previously (18). Granulosa cells were plated on a 24-well plate (Nulge, Nunc International, Naperville, IL) and cultured as previously described (18).

Aromatase assay
Aromatase activity was determined by measuring the amount of [3H]H2O released with the conversion of [1ß-3H]androstenedione into estrone as described previously (13). Briefly, the cells were cultured in a 24-well dish in DMEM/Ham’s F-12 with 10% FCS in the presence or absence of various chemicals and incubated for 24 h. The cells were then incubated with [1ß-3H]androstenedione for an additional 6 h. The medium was extracted with chloroform and centrifuged. The aqueous phase was then mixed with 5% charcoal/0.5% dextran and incubated for 30 min. The mixture was subsequently centrifuged, and the supernatant was added to 5 ml scintillation fluid and assayed for radioactivity. The amount of radioactivity in [3H]H2O was standardized from the protein concentration determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Measurement of cAMP and progesterone
The cAMP content in the cell culture medium, incubated with various chemicals, was measured by RIA using a commercially available kit (Yamasa Syoyu Co. Ltd., Chiba, Japan) (19). The content of progesterone in the medium was also measured by a specific RIA (SRL, Tokyo, Japan).

Luciferase assay and transfection
KGN cells were transfected using SuperFect transfection reagent (Qiagen, Hilden, Germany) with the pGL3-basic luciferase reporter plasmid (Promega Corp., Madison, WI) that contains 4 kb CYP19 promoter II (20) or cAMP-responsive element (CRE) plasmid-luciferase (Clontech Laboratories, Palo Alto, CA) plasmid, which contains two copies of the CRE consensus sequence (TGACGTCA). The Renilla luciferase control reporter plasmid phRG-TK was used as an internal control for transfection efficiency. Luciferase assays were performed in the cell lysate by treating the luciferase lysis buffer with a dual luciferase reporter assay system (Promega Corp.). Luminescence activity was measured with a LUMAT LB9507 luminometer (Berthold Technologies GmbH & Co., Bad Wildbad, Germany).

RNA extraction, semiquantitative RT-PCR, and quantitative real-time PCR
KGN cells were cultured in the presence or absence of various chemicals. After 24 h of culture, total RNA was extracted with ISOGEN (Wako Pure Chemical Co.) and stored at -80 C for later analysis. The extracted RNA was subjected to a reverse transcriptase reaction using Superscript II (Invitrogen). The oligonucleotides used for PCR were custom-ordered from Hokkaido System Science Co. Ltd. (Sapporo, Japan). First-strand cDNA was synthesized using 5 µg total RNA as a template. PCR carried out with T-Gradient Thermoblock (Biometra Biomedizinische Analytik GmbH, Gottingen, Germany). The sense/antisense primers used were: aromatase, 5'-GGTCACAGTCTGTGCTGAATCC-3'/5'-ACTCGAGTCTGTGCATCCTTAA-3'; cytochrome P450 side-chain cleavage (P450scc), 5'-GAGATCCCCTCTCCTGGTGA-3'/5'-TGGCGCTCCCCAAAAATGA-3'; steroidogenic acute regulatory protein (StAR), 5'-GCAGCAGCAGCGGCGGCA-3'/5'-TGTGTCCATGCCAGCCAGC-3'; and ß-actin, 5'-TCGTGCGTGACATTAAGGAG-3'/5'-GATGTCCACGTCACACTTCA-3'. Based on preliminary experiments, to achieve linear amplification, the PCRs for aromatase, P450scc, StAR, and cDNA were performed in 35, 30, and 30 cycles, respectively, whereas that for ß-actin cDNA was performed in 28 cycles. Each PCR amplification for aromatase, P450scc, StAR, and ß-actin was started by an initial denaturing reaction at 94 C for 5 min, followed by 35, 30, 30, and 28 cycles of denaturing (30 sec, 94 C) and elongation (1 min, 72 C) reactions, respectively. Annealing was performed for 30 sec at 60 C for aromatase, 50 C for P450scc, 48 C for StAR, and 60 C for ß-actin, respectively. The PCR products were visualized by electrophoresis on a 1.5–2.0% agarose gel containing 0.5 mg/ml ethidium bromide and luminescence was measured by NIH image. We finally verified the nucleotide sequence of each PCR product by direct sequencing using the appropriate primers.

To reconfirm the relatively small increase in aromatase mRNA by benomyl obtained by RT-PCR analysis, we also performed a quantitative real-time PCR. First-strand cDNA was synthesized using 5 µg total RNA as a template. The sense/antisense primers for real-time PCR were the same as those used in RT-PCR, except for aromatase (5'-ACGCA-GGATTTCCACAGAAGAG-3'/5'-CTTCTAAGGCTTTGCGCATGAC-3') and glyceraldehyde-3-phosphate dehydrogenase (5'-CCACCCATGGCAAATTCCATGGCA-3'/5'-TCTAGACGGCAGGTCAGGTCCACC-3'). PCRwas carried out with a LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, 1 µl cDNA (or H2O control) was placed into a 20-µl reaction volume containing 1 µl of each primer and 2 µl LightCycler-FastStart DNA Master SYBR Green I (Roche, Mannheim, Germany). Nucleotides, Taq DNA polymerase, and buffer were included in the LightCycler-FastStart DNA Master SYBR Green I. The thermal cycling conditions comprised an initial denaturation step at 95 C (10 min), followed by 40 cycles of 95 C (0 sec), 63 C (15 sec), and 72 C (40 sec). Threshold values were obtained where fluorescent intensity was in the geometric phase of amplification, as determined via LightCycler software version 3.5. Products were verified on a 2% agarose gel.

Western blotting
Cell lysates were prepared by treating cells with CelLytic-M (Sigma-Aldrich Corp.) containing protease inhibitor. Lysate was sonicated for 20 sec on ice and centrifuged at 10,000 x g for 10 min to separate the particulate materials. SDS-PAGE was performed under reducing conditions on 10% polyacrylamide gels. The resolved proteins were transferred onto a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ) and then incubated with rabbit antiserum raised against human aromatase (1:1000) (21). After several washes with PBS, the membrane was incubated with the secondary antibody to IgG conjugated with horseradish peroxidase (1:2000). The blots were then probed with the ECL+ Western blot detection system (Amersham Pharmacia Biotech) in accordance with the manufacturer’s instructions.

Immunofluorescence microscopy
KGN cells were seeded in the chambers of a two-well glass slide (Nulge, Nunc International). After drug treatments and incubation, the growth medium was removed and then immediately fixed with 4% paraformaldehyde for 30 min. Cells were permeabilized using 0.2% Triton X-100 in PBS for 10 min at room temperature and then incubated with blocking buffer (Block Ace, Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) for 1 h. Anti-{alpha}-tubulin (Molecular Probes, Eugene, OR) diluted 1:200 with 10% Block Ace in PBS was added to the samples for 1 h at room temperature. Samples were then washed three times with PBS, and Alexa Fluor 488 goat antimouse (Molecular Probes) antibodies diluted 1:1000 with 10% Block Ace in PBS was applied to the samples for 1 h at room temperature. Samples were mounted in ProLong Antifade Mounting Media (Molecular Probes) for preservation and observation. Images were collected using a fluorescent microscope (Olympus Optical Co. Ltd., Tokyo, Japan) and a High Sensitive CCD camera (Keyence Corp., Osaka, Japan).

Statistics
All experiments were carried out at least three times, with triplicate plates per point. All values represent the mean ± SD. A one-way ANOVA was used for statistical evaluation. P < 0.05 indicated statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Screening of endocrine disruptor chemicals for aromatase activity in KGN cells
We obtained 55 chemicals that may act as endocrine disruptor chemicals and examined the effects of these chemicals, at a concentration of 10-5 M (except for 10-7 M tributyltin and 10-7 M triphenyltin), on aromatase activity in KGN cells. Only benomyl was found to induce aromatase activity (Fig. 1AGo). At a concentration of 10-5 M, benomyl induced a remarkable 3-fold increase in aromatase activity compared with the dimethylsulfoxide (DMSO) control. Benzo(a)pyrene and heptachlor also caused a slight, but significant, increase in aromatase activity, whereas heptachlor, p,p'-DDD, p,p'-DDE, cypermethrin, metribuzine, kelthane, pentachlorophenol, dicyclohexyl phthalate, diethylhexyl phthalate, and bisphenol A produced a significant decrease in activity (P < 0.01). Moreover, in agreement with a previous study of ours (7), tributyltin inhibited aromatase activity in this cell line. As the effect of benomyl was most remarkable, in this study we focused on the mechanism underlying benomyl stimulation of aromatase activity. Importantly, 10-5 M benomyl also caused a 2-fold increase in aromatase activity in cultured human granulosa cells obtained from a woman who underwent in vitro fertilization (Fig. 1BGo), indicating that the effect of benomyl on aromatase activity is not a cell line-specific effect. The stimulatory effect of benomyl on aromatase activity exhibited a dose-dependent relationship for concentrations ranging from 10-6–10-5 M (Fig. 2AGo). The effect of 10-5 M benomyl also exhibited a time dependence for at least the 80-h incubation tested here (Fig. 2BGo).



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  		FIG. 1. Screening of 55 endocrine disruptor chemicals for aromatase activity in cultured KGN cells. A, KGN cells were cultured and 10-5 M of each compound (except for 10-7 M tributylin and 10-7 M triphenyltin) or DMSO (control) was added to the medium, incubation was performed for 24 h, and aromatase activity was assayed as described in Materials and Methods. B, Normal granulosa cells were cultured in the presence or absence of 10-5 M benomyl, and aromatase activity was assayed as described in Materials and Methods. Each value indicates the mean ± SD of three experiments, with triplicate plates per point. *, P < 0.05; **, P < 0.01 (vs. control).

 


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  		FIG. 2. Effect of benomyl on aromatase activity in cultured KGN cells. A, KGN cells were cultured in the presence or absence of 10-6–10-5 M benomyl for 24 h (dose dependency). B, KGN cells were cultured in the presence of 10-5 M benomyl for 24–96 h (time dependency). Aromatase activity was assayed as described in Materials and Methods. Each value indicates the mean ± SD of three experiments, with triplicate plates per point. *, P < 0.05; **, P < 0.01 (vs. control).

 
Effect of benomyl on transcription of CYP19
To investigate whether these effects of benomyl were regulated at the level of transcription, we determined the levels of aromatase mRNA expression by both RT-PCR and quantitative real-time PCR. The KGN cells were incubated with 10-7 M forskolin, 500 mIU/ml human menopausal gonadotropin (hMG), 10-5 M benomyl, or DMSO (control) and total RNA was extracted. We amplified both CYP19 and ß-actin mRNA (used as an internal control to normalize the levels of CYP19 expression). Both forskolin and hMG, which are known to activate the cAMP-protein kinase A (PKA) pathway, stimulated the expression of aromatase mRNA. Similarly, benomyl increased aromatase mRNA levels relative to ß-actin (Fig. 3AGo). This was further confirmed using real-time PCR methods. Namely, 10-5 M benomyl caused a 3.47-fold increase in aromatase mRNA compared with the treatment with DMSO (control; Table 1Go). We next analyzed the effects of these compounds on transcription of the CYP19 promoter. KGN cells were transfected with the pGL3-basic luciferase reporter plasmid that contains 4 kb of aromatase promoter II. Forskolin, benomyl, or DMSO (control) were then added and incubated for 24 h. As shown in Fig. 3BGo, benomyl significantly stimulated luciferase activity. All these data clearly indicate that benomyl predominantly up-regulates aromatase activity through the transcription of CYP19.



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  		FIG. 3. Effect of benomyl on the expression of aromatase mRNA in cultured KGN cells. A, KGN cells were cultured in the presence or absence of 10-7 M forskolin, 500 mIU/ml hMG, or 10-5 M benomyl for 24 h. Total RNA was then extracted, and semiquantitative RT-PCR was performed as described in Materials and Methods. Images of agarose gel electrophoresis of RT-PCR products are indicated. The relative amount of cytochrome P450 aromatase to ß-actin mRNA was determined from the intensity of the ethidium bromide. B, KGN cells were cultured in the presence or absence of 10-7 M forskolin, 500 mIU/ml hMG, or 10-6–10-5 M benomyl for 24 h, andthe relative luciferase activity of the CYP19 gene was measured as described in Materials and Methods. Each value indicates the mean ± SD of three experiments, with triplicate plates per point. *, P < 0.05; **, P < 0.01 (vs. control).

 

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  		TABLE 1. Relative quantitation of mRNA expressions of aromatase, StAR, and P450scc determined by quantitative real-time PCR

 
Effects of benomyl on cAMP production and CRE-mediated transcription
It is well known that the expression of CYP19 in ovarian granulosa cells is mainly regulated by the cAMP-PKA pathway (3, 4, 22, 23, 24), resulting in an adenylate cyclase-mediated rise in cAMP levels and activation of PKA. We thus examined whether the stimulatory effect of benomyl on aromatase activity is mediated by this system. For this analysis, KGN cells were treated with 10-7 M forskolin, 500 mIU/ml hMG, 10-5 M benomyl, or DMSO (control). To prevent the metabolic effect of phosphodiesterase on cAMP, 0.5 mM 3-isobutyl-1-methylxanthine (a specific inhibitor of phosphodiesterase activity) was added to the culture medium. The concentration of cAMP in the culture medium was measured after 48 h. As shown in Fig. 4AGo, forskolin and hMG increased cAMP levels, whereas benomyl did not. We further analyzed the effect of benomyl on CRE-mediated transcription using a reporter plasmid containing two copies of the CRE sequence. As shown in Fig. 4BGo, both forskolin, a known activator of adenylate cyclase, and hMG stimulated CRE-mediated activity, whereas benomyl had no effect. These results strongly suggest that the cAMP-PKA pathway is unlikely to be the main mechanism that mediates the benomyl-mediated increase in aromatase activity.



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  		FIG. 4. Effect of benomyl on cAMP production and CRE-mediated transcription in cultured KGN cells. A, KGN cells were cultured in the presence or absence of 10-7 M forskolin, 500 mIU/ml hMG, or 10-5 M benomyl for 48 h. The cAMP concentration in the medium was then measured. B, KGN cells were cultured in the presence or absence of 10-7 M forskolin, 500 mIU/ml hMG, or 10-5 M benomyl for 24 h, and CRE-mediated transcription was measured by the relative luciferase activity of the pGL3-Basic luciferase reporter plasmid containing two copies of CRE. Each value indicates the mean ± SD of three experiments, with triplicate plates per point. *, P < 0.05; **, P < 0.01 (vs. control).

 
Several kinases other than PKA are activated by FSH receptors, including protein kinase B (PKB)/Akt, serum and glucocorticoid-induced kinase (Sgk), ERK, and p38 MAPK, protein tyrosine kinase also stimulates FSH signal (25, 26, 27, 28). Using a variety of kinase inhibitors, the effect of benomyl on kinases other than PKA was examined. The increased aromatase activity by benomyl was not significantly blocked by kinase inhibitors for PKB/Akt and Sgk (0.1–10 µM wortmannin), ERK (10 µM U0126 or 10 µM PD98059), protein tyrosine kinase (10 µM genistein, 50 µM AG490, and 100 nM AG1478), which suggests that these kinases do not mediate the effects of benomyl.

Effect of benomyl on the production of progesterone, P450scc mRNA, and StAR mRNA
We have previously shown that KGN cells mainly express StAR and P450scc, rather than aromatase (13). We measured the levels of progesterone after incubation with 10-7 M forskolin, 500 mIU/ml hMG, 10-5 M benomyl, or DMSO (control). Progesterone levels in the medium were significantly increased by forskolin and hMG (Fig. 5AGo). Benomyl, however, did not alter basal progesterone production. Additionally, we determined whether benomyl affected the expression of mRNA for these steroidogenesis-related proteins and enzymes. The KGN cells were incubated with 10-7 M forskolin, 500 mIU/ml hMG, 10-5 M benomyl, or DMSO (control), and the total RNA was extracted. We then determined the levels of P450scc and StAR mRNA by both RT-PCR and quantitative real-time PCR. As shown in Fig. 5BGo and Table 1Go, benomyl did not affect the mRNA expression levels of P450scc and StAR relative to ß-actin or glyceraldehyde-3-phosphate dehydrogenase. Together these results indicate the effect of benomyl on steroidogenesis in KGN cells is specific to aromatase.



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  		FIG. 5. Effect of benomyl on the production of progesterone (A) and expressions of P450scc and StAR mRNA in cultured KGN cells (B). A, KGN cells were cultured in the presence or absence of 10-7 M forskolin, 500 mIU/ml hMG, or 10-5 M benomyl for 24 h. The progesterone concentration in the medium was then measured by specific RIA. B, KGN cells were cultured in the presence or absence of 10-7 M forskolin, 500 mIU/ml hMG, or 10-5 M benomyl for 24 h, and the expressions of P450scc, StAR, and ß-actin mRNA were determined by RT-PCR. Each value indicates the mean ± SD of three experiments, with triplicate plates per point. *, P < 0.05; **, P < 0.01 (vs. control).

 
Effect of the benomyl metabolite, carbendazim, on aromatase expression
It is well established that in either aqueous solutions or soil, benomyl rapidly degrades to its breakdown product carbendazim (Fig. 6AGo) (15, 16). We therefore investigated the effect of carbendazim on aromatase expression. As expected, like benomyl, 10-5 M carbendazim significantly induced transcription of CYP19 (Fig. 6BGo). We also determined aromatase expression levels by Western blotting. Benomyl and carbendazim increased the expression of aromatase almost equally, although the effect was weaker than that of 10-7 M forskolin or 500 mIU/ml hMG (Fig. 6CGo), which is consistent with the results of the luciferase assay (Fig. 6AGo). Carbendazim (10-5 M) increased aromatase activity to a level similar to that observed with 10-5 M benomyl. These results suggest that the effect of benomyl on aromatase is mediated by carbendazim.



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  		FIG. 6. Effect of the benomyl and its metabolite, carbendazim, on transcription of the CYP19 promoter (B) and aromatase protein expression (C). A, Chemical structure of benomyl and its metabolite, carbendazim. B, Effect of benomyl or carbendazim on the transcription of CYP19 promoter (promoter II) in cultured KGN cells. KGN cells were cultured in the presence or absence of 10-7 M forskolin, 500 mIU/ml hMG, 10-5 M benomyl, or 10-5 M carbendazim for 24 h, and the relative luciferase activity was measured as described in Materials and Methods. C, Effect of benomyl or carbendazim on aromatase expression in cultured KGN cells. KGN cells were cultured in the presence or absence of 10-7 M forskolin, 500 mIU/ml hMG, 10-5 M benomyl, or 10-5 M carbendazim for 24 h, and the expression of aromatase was examined by Western blot analysis using antibodies for aromatase. Thirty micrograms of protein were loaded into each lane. Each value indicates the mean ± SD of three experiments, with triplicate plates per point. *, P < 0.05; **, P < 0.01 (vs. control).

 
Effect of taxol on benomyl-stimulated aromatase activity
Microtubules are ubiquitous components of cytoskeletons that contribute to the translocation of cellular organelles and secretory materials. A dynamic equilibrium exists between dimeric tubulin and microtubules, with net assembly and disassembly occurring at opposite ends of each complex, and is thought to be closely associated with the function of the cytoskeleton. Benomyl is known to interfere with the assembly of microtubules (14), whereas taxol is a potent promoter of microtubule assembly and is thought to stabilize the polymerized form of tubulin in vitro (29, 30). We thus compared the effect of benomyl on aromatase activity with that of taxol. As shown in Fig. 7AGo, treatment with 10-5 M benomyl or 10-9 M taxol caused clear changes in KGN cell morphology. Namely, KGN cells treated with benomyl or taxol became rounder than those incubated with DMSO (control) or 10-7 M forskolin. In addition, immunofluorescence staining with anti-{alpha}-tubulin revealed that taxol caused dense staining of bundles in the perinuclear region. These responses by the cells treated with taxol have been previously reported in various cell types, including ovarian granulosa cells (30, 31). Benomyl caused a similar change in cell shape and in the subcellular bundle pattern. Although we could not refer to fine structural changes in the bundles, it was evident that both treatments clearly produced a different pattern compared with the control or forskolin. Additionally, we investigated the effect of taxol on aromatase promoter II. Taxol increased aromatase-luciferase activity to the same extent as benomyl (Fig. 7BGo). Taxol also increased aromatase activity to similar levels as benomyl (data not shown). These results suggest that disruption of the dynamic equilibrium between dimeric tubulin and microtubules by microtubule-interfering agents (MIAs) may lead to increased aromatase activity.



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  		FIG. 7. Effects of MIAs on KGN cells. A, Anti-{alpha}-tubulin staining of control cells (a) and cells treated with 10-7 M forskolin (b), 10-5 M benomyl (c), and 10-9 M taxol (d), respectively. Immunofluorescence staining was performed as described in Materials and Methods. B, KGN cells were cultured in the presence or absence of 10-5 M benomyl or 10-9 M taxol for 24 h, and the relative luciferase activity of the CYP19 gene was measured. Each value indicates the mean ± SD of three experiments, with triplicate plates per point. *, P < 0.05; **, P < 0.01 (vs. control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study the screening of 55 potential chemicals as endocrine disruptors found that only benomyl stimulated aromatase activity in cultured human granulosa cell line or KGN cells. The stimulatory effect of benomyl on aromatase activity was associated with comparable changes in aromatase mRNA levels. In addition, the luciferase activity of aromatase promoter II (4 kb) in transfected KGN cells was increased with the addition of benomyl. Based on these findings, the benomyl-induced increase in aromatase activity appears to be mainly regulated by the transcription of CYP19. Importantly, the rapidly produced metabolite of benomyl, carbendazim (15, 16, 17), was equally effective in stimulating aromatase activity. Thus, the effect of benomyl could be mediated by carbendazim, rather than benomyl itself, as suggested in benomyl-induced testicular toxicity (32).

The stimulatory effect of the benzimidazole fungicide, benomyl, on aromatase activity is somewhat surprising because some imidazoles, such as fadrozole and vordozole, suppress aromatase activity and act as potent aromatase inhibitors through the formation of tightly associated aromatase-inhibitor complexes that disturb heme binding and substrate binding of aromatase (33). Thus, benomyl is considered to be a unique type of imidazole because it stimulates aromatase activity.

FSH is the major physiological stimulator of human CYP19 expression, by increasing cAMP levels. This mechanism is mediated by the CRE, which binds the trans-acting factors of the CRE-binding protein/activating transcription factor family (22, 34). However, it is unlikely that this cAMP-PKA pathway mediates the stimulatory effect of benomyl on CYP19 expression, because benomyl did not increase either cAMP levels or CRE-mediated transcriptional activity. In addition, several other kinases, including PKB/Akt, Sgk, ERK, and protein tyrosine kinase, which are reported to be activated by FSH signal (25, 26, 27, 28), seemed to be unrelated to this mechanism, based on the effect of inhibitors for these kinases.

Cellular cytoskeletons consist of an integrated network of microtubules, microfilaments, and intermediate filaments, in which changes in the distribution of one component can profoundly influence that of another. It can thus be imagined that steroidogenesis involves controlled organization of microtubules, which may facilitate the movement of substrates into organelles such as mitochondria or microsomes, possibly by bringing these cellular inclusions closer together. This role of microtubules in regulating ovarian granulosa cell steroidogenesis has been previously demonstrated (35, 36, 37, 38, 39, 40). For example, colchicine and nocodazole, two agents that depolymerize microtubules, significantly stimulate progesterone production in antral stage granulosa cells of Sprague-Dawley rats, whereas taxol, a stabilizing agent of microtubules, markedly reduced FSH-stimulated production of progesterone in both preantral and antral cells (35). In our study, both benomyl, a depolymerizing agent of microtubules, and taxol, a stabilizing agent of microtubules, had similar effects on the stimulation of aromatase activity in KGN cells. The effect of benomyl or taxol was accompanied by characteristic changes in cell shape and subcellular reorganization of microtubules. Interestingly, in agreement with our results, Cameron et al. (40) reported that nanogram per milliliter concentrations of taxol increase estrogen production in three ovarian cancer cell lines and in JEG-3 choriocarcinoma cells as well as immunostaining for aromatase in cancer cells. These results strongly suggest that deregulation of the dynamic equilibrium of microtubule assembly/disassembly caused by MIA may play an important role in increasing aromatase activity in KGN cells.

It is also noteworthy that the effect of benomyl in steroidogenesis was specifically observed in the microsomal enzyme, aromatase, but was not evident in the mitochondrial protein, StAR, or in the enzyme P450scc. When it reorganizes the intracellular distribution and spacing toward microsomes and mitochondria, benomyl may influence the cross-bridges linking microtubules and organelles. These differences may be simply dependent on the MIA type used or the cell type and conditions. Although little is known about the molecular mechanism of how MIA works, the partial involvement of the c-Jun N-terminal kinase signal, one subgroup of MAPK (41), has been implicated in the effects mediated by taxol. It has been reported that microtubules can anchor the transcription factors, Smads, in an inactive state in cytoplasm. Activation by a ligand or MIA results in dissociation of Smads from the microtubule network (42). Likewise, MIA may activate some transcription factors, which are essential for CYP19 gene transcription. Further investigation is needed to determine the mechanism of MIA and benomyl-mediated stimulation of CYP19 expression.

A logical concern based on our results would be that long-term or excessive exposure of wildlife and humans to benomyl, carbendazim, or taxol might contribute to serious estrogen-mediated pathologies, such as tumor promotion and inappropriate sexual differentiation. During critical development periods, such as embryonic perinatal and pubertal development, benomyl-mediated induction of aromatase could result in inappropriate feminization. In human females, the local production of estrogens due to the increased levels of aromatase expression in breast adipose or stromal tissue has been associated with an increased risk of breast cancer (3, 43). Interestingly, taxol inhibits TNF-{alpha}-stimulated aromatase activity in stromal fibroblasts derived from normal or malignant breast tissues, suggesting that taxol may have a therapeutic potential in the treatment of breast cancer (44). However, they observed no such effect by another class of MIA, indicating that the effect of each MIA is tissue specific and should be carefully considered in its evaluation as an endocrine disruptor chemical.

Benomyl is especially known to be toxic to the male reproductive system of rodents and dogs, producing hypospermatogenesis and multinucleated germ cells. Because benomyl or its metabolite carbendazim inhibits microtubule formation in fungi, it has been proposed by many investigators that severe testicular dysfunction may be caused by the impediment of cell division and other microtubule-dependent processes of spermatogenesis (45, 46, 47, 48, 49). However, it is not clear whether this occurs in human males. In addition, because the same promoter usage of CYP19 has been identified in the ovary and testis (23, 24), it might be intriguing to examine the possibility that benomyl contributes to male feminization by affecting testicular aromatase activity.

In summary, the human granulosa cancer cell line, KGN, cell system that we have developed has proven useful in identifying a potentially essential target for endocrine disruptors in aromatase assays. Using this system, we have demonstrated for the first time that benomyl and its metabolite, carbendazim, stimulate aromatase activity independently of cAMP activation and several other kinase-related pathways. The effect of benomyl or carbendazim was regulated mainly at the level of transcription and seems to be closely related to the unbalanced assembly/disassembly organization of microtubules.


   Footnotes
 
This work was supported in part by a grant from the Ministry of Health, Labor, and Welfare of Japan.

Abbreviations: CRE, cAMP-responsive element; DMSO, dimethylsulfoxide; hMG, human menopausal gonadotropin; MIA, microtubule-interfering agent; PI3 kinase, phosphatidylinositol-3OH-kinase; PKA, protein kinase A; PKB, protein kinase B; p,p'-DDD, p,p'-dichlorodiphenyldichloroethane; P450scc, cytochrome P450 side-chain cleavage; Sgk, serum and glucocorticoid-induced kinase; StAR, steroidogenic acute regulatory protein.

Received September 8, 2003.

Accepted for publication December 15, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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