Alterations of FSH-stimulated progesterone production and calcium homeostasis in primarily cultured human luteinizing-granulosa cells induced by fenvalerate

Toxicology 203 (2004) 61–68

Alterations of FSH-stimulated progesterone production and calcium homeostasis in primarily cultured human luteinizing-granulosa cells induced by fenvalerate Jun He, Jianfeng Chen, Ru Liu, Shoulin Wang, Lin Song, Hebron C. Chang, Xinru Wang∗ Jiangsu Key Laboratory of Applied Toxicology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China Received 10 April 2004; received in revised form 22 May 2004; accepted 24 May 2004 Available online 1 July 2004

Abstract Fenvalerate, a synthetic pyrethroid, is widely used in agriculture and other domestic applications in China. Recently, Fenvalerate has been suspected to be one of the endocrine-disrupting chemicals (EDC). In this study, we investigated the effects of fenvalerate on follicle-stimulating hormone (FSH)-stimulated progesterone (P4) production by human ovarian luteinizing-granulosa cells (hGLCs). After 24 h incubation, fenvalerate inhibited FSH-stimulated P4 production. At the same time, FSH-stimulated cAMP also decreased. Due to calcium and Ca2+ –calmodulin (CaM) system involving gonadotropin-stimulated steroidogenesis by granulosa cells, we then evaluated the effects of fenvalerate on trifluoperazine (TFP)- and verapamil-driven FSH-stimulated P4 production. The results showed that calcium or calmodulin might play a role in fenvalerate-induced alterations in FSH-stimulated P4 biosysthesis. Then, the effects of fenvalerate on calcium homeostasis in hGLCs were studied. The result showed that 5 ␮M fenvalerate induced a slow increase in [Ca2+ ]i in hGLCs by using a fluorescent Ca2+ indicator fluo-3/AM. The changes in total concentration of CaM in hGLCs induced by fenvalerate were evaluated by a method of immunofluorescence. There is a significant increase in all treated groups. In summary, fenvalerate could inhibit FSH-stimulated P4 production. Also, fenvalerate interferes with calcium homeostasis in hGLCs. The effects of fenvalerate on FSH-stimulated ovarian steroidogenesis may be mediated partly through calcium signal. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Endocrine disruptor; Pyrethroids; Granulosa cell; Progesterone; Calcium homeostasis

1. Introduction Fenvalerate(4-chloro-␣-(1-methylethyl)benzeneaceticacid cyano(3-phenoxyphenyl) methyl ester), a ∗ Corresponding author. Tel.: +86 25 86862939; fax: +86 25 86527613. E-mail address: [email protected] (X. Wang).

member of the family of synthetic pyrethroid, type II, is widely used in agricultural and other domestic applications in China due to its high insecticidal activity and a low hazard potential to humans. Although it is generally considered that fenvalerate is relatively immobile and readily degraded in soil, they are moderately persistent in soils because of low water solubility, high octanol–water partition

0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.05.014

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coefficients (Adelsbach and Tjeerdema, 2003). Also, fenvalerate has been identified having the potential to accumulate in aquatic sediments and biota. Therefore, with increased application, it is essential to assess its possible toxic effects comprehensively. Fenvalerate has been found to cause severe neurotoxic effects. However, some recent works indicate that fenvalerate is capable of disrupting reproductive and endocrine function. Fenvalerate has been enlisted as one kind of endocrine-disrupting chemical (EDC) and has aroused worldwide concern. Fenvalerate induced significant decrease of testis weight, epididymal sperm counts, sperm motility and marker testicular enzymes for testosterone biosynthesis (Mani et al., 2002). Also, Garey and Wolff (1998) showed certain pyrethroid compounds including fenvalerate had estrogenic potential by using the Ishikawa Variant-I human endometrial carcinoma cell line. The same results were obtained from other independent studies on MCF-7 human breast carcinoma cell lines (Go et al., 1999; Chen et al., 2002). However, Kunimatsu et al. (2002) reported that three pyrethroids (esfenvalerate, fenvalerate, and permethrin) were lack of (anti-) androgenic or estrogenic effects by using Hershberger’s and uterotrophic assays. Because study on endocrine disruption is an emerging field, researches are needed to give deep perception in endocrine disrupters. Endocrine-disrupting chemicals are known to act at multiple sites through multiple modes of action, but the mechanisms of action including putative exposure–response relationships are poorly understood (Damstra, 2003). It is necessary to explore the mechanisms of action from multiple points of view. Intracellular Ca2+ homeostasis and the messenger role of the Ca2+ ion in the regulation and control of cell functions have been a very active and productive areas of basic biological research. The chemical–physical events mediated by calcium are various such as the release of neurotransmitter, steroidogenesis, fertilization, and synthesis of DNA, etc. Based on the central role of the Ca2+ –messenger system in these aspects of cell functions, it is logical to examine possible disturbance in Ca2+ homeostasis and Ca2+ -mediated functions as underlying mechanisms of toxicant action (Pounds, 1990). Several studies suggest that some toxic symptoms induced by pyrethroids have been found to be associated with calcium homeostasis. The changes induced by fenvalerate in circulatory

T3 and T4 are accompanied by increased levels of total calcium as well as protein-bound calcium in whole brain and hypothalamus (Kaul et al., 1996). Deltamethrin, another type II pyrethroid, causes an increase in neurotransmitter release and intrasynaptosomal free Ca2+ levels and protein phosphorylation activities at the same time (Enan and Matsumura, 1993). In addition, pyrethroids have been documented to bind to the Ca2+ , Mg2+ -ATPase adopting a folded conformation with both the acid and alcohol moieties in contact with hydrophobic regions of the ATPase (Michelangeli et al., 1990). To our knowledge, the effects of pyrethroid on ovarian cell calcium homeostasis has not been elucidated up-to-date. If fenvalerate directly or indirectly, or secondarily alter Ca2+ homeostasis in ovarian cells, it may impair the reproductive endocrine functions. Furthermore, accumulating evidences indicate calcium ions modulate gonadotropin-stimulated steroidogenesis by granulosa cells (Veldhuis et al., 1984; Jayes et al., 2000). The calcium–calmodulin (CaM) system participates in the regulation of steroidogenesis at different stages of granulosa cell differentiation (Carnegie and Tsang, 1984). In the study, we used primarily cultured human ovarian granulosa-luteinizing cells (hGLCs) model. Human ovarian granulosa-luteinizing cells are responsible for ovary functions including steroidogenesis, oocyte development along with theca cells. Firstly, we selected various concentration of fenvalerate (0, 1, 5, 25, 125 ␮M) to study the effects of this pesticide on follicle-stimulating harmone (FSH)-stimulated progesterone (P4) production. Then, we investigated the effects of fenvalerate on calcium homeostasis in hGLCs. We also explored the relationship between Ca2+ –calmodulin system and the alterations of steroidogenesis induced by fenvalerate.

2. Materials and methods 2.1. Human luteinizing-granulosa cell primary culture Human ovarian luteinizing-granulosa cells were obtained by follicular aspiration from women taking part in in vitro fertilization programs. The study was approved by People’s Hospital of Jiangsu Province

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(China) and the informed written consents were given by subjects. After removal of the cumulus–oocyte complex, the cells from all follicles of each woman were pooled, pelleted and centrifuged at 500 × g for 10 min. Then, the supernatants was trypsinated for 10 min at 37 ◦ C with a ethylenediamine tetraacetate (EDTA) at a final concentration of 0.25% trypsin and 0.02% EDTA, respectively. Blood cells were separated from granulosa cells by centrifugation in 50% percoll (Sigma, MO) for 5 min at 700 × g. The cells were then washed and plated in DMEM (GIBCO BRL, NY) supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamine (Life Technologies, Paisley, Scotland), 100 IU/mL penicillin, and 100 ␮g/ml streptomycin sulfate at 37 ◦ C in a 5% CO2 –95% air atmosphere. 2.2. RIA for P4 and cAMP Human ovarian granulosa-luteinizing cells were seeded in 24-well plates (2 × 105 cells per well). Because the women were undertaken continuous stimulation by FSH before follicular puncture, cells were cultured for 4 days in medium containing FCS before initiating the treatments to allow the cells to recover the responsiveness to gonadotrophins (Schipper et al., 1993). After that, cells were transferred to serum free medium containing 0.1% BSA. At the same time, fenvalerate (99.9% purity, Sigma) at doses of 0, 1, 5, 25, 125 ␮M and 200 ng/ml rhFSH (Sigma, MO) was added to the corresponding wells. Final concentration of ethanol used as chemical solvent was 0.1% included in controls. At the end of the 24 h incubation, the medium was collected and stored at −20 ◦ C until measurement. The P4 production was measured by using commercial RIA kits (Beijing North Institute of Biological Technology, China). The assay detection limit was 0.2 ng/ml. Inter- and intra-assay coefficients of variation were <10 and <15%, respectively. The generation of cAMP was tested in cell culture for 24 h with fenvalerate (0, 1, 5, 25, 125 ␮M) as described previously (Crellin et al., 1999). In brief, at the end of incubation, phosphodiesterase inhibitor isobutyl methylxanthine ( IBMX, 125 ␮M) was added in the culture medium for 30 min. Medium was then discarded and cAMP was extracted with absolute alcohol. The extract was dried and resuspended in 0.01M acetate buffer, pH 6.2, and was stored at −20 ◦ C. The

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cAMP level was determined by RIA kit provided by Shanghai University of Traditional Chinese Medicine. The detection limit of cAMP was 0.08 pM. Sample protein was quantitated by the Bradford method. All treatments were performed in triplicate or quadruplex within an experiment and with three replicates. 2.3. Cytosolic calcium concentration ([Ca2+ ]i ) Relative change in [Ca2+ ]i was measured with indicator fluo-3/AM (Molecular Probe, USA). Cells were grown on the 24 mm × 24 mm glass coverslips and washed twice with Hanks’ HEPES buffer, pH 7.4 (Machelon et al., 1998). Then, cells were incubated for 30 min at 37 ◦ C in the same buffer including 6 ␮M fluo-3/AM in the dark. After loading, cells were washed and then placed on the heat stage of a confocal laser scanning microcope (LSM510, Zeiss, Germany). Measurements were performed on 8–10 cells in one field of vision. Fenvalerate was dissolved in ethanol. The final concentration of ethanol was 0.1% in Hanks HEPES buffer and was used as vehicle control. Fenvalerate was added to the cells from the beginning of the scan. Fluorescence images were collected at the excitation wavelength of 488 nm. The intracellular calcium concentration was calculated using the following equation: [Ca2+ ]i =

Kd (F − Fmin ) (Fmax − F)

The Ca2+ affinity of fluo-3 (Kd ) is 400 nM. Fmax was obtained by addition of 0.1% Triton X-100, Fmin by addition of 10 mM EGTA. 2.4. Immunofluorescence for calmodulin Cells were grown on the glass coverslips in the presence of fenvalerate for 24 h. We found that there was no significant difference induced by fenvalerate at the doses of between 1 and 5 ␮M in our previous study. Therefore, we used 0, 5, 25, and 125 ␮M for the study. Cells were fixed in methanol for 10 min at 4 ◦ C and were allowed air dried. Then the cells were subsequently subjected to immunofluorescence labeling. After being washed with phosphate-buffered saline containing 0.5% Tween (PBST), the cells were incubated with the goat serum diluted to 1:100 as blocking

J. He et al. / Toxicology 203 (2004) 61–68

2.5. Statistical analysis Values in the tables and figures are expressed as mean ± S.D. Comparison between two means was performed by Student’s t-test. Differences were considered significant at a value of P < 0.05.

3. Results 3.1. The effects of fenvalerate on FSH-stimulated P4 production Follicus stimulating hormone (FSH)-stimulated P4 production was significantly higher than the basal P4 production. That shows the cells have recovered the responsiveness to gonadotrophins through 4 days culture in serum contained medium. Fenvalerate had shown the inhibitory effect on the FSH-stimulated P4 production. P4 concentrations decreased to 59, 55, and 43 of control (FSH) in hGLCs incubated with 5, 25 and 125 ␮M fenvalerate, respectively for 24 h (Fig. 1). The involvement of the calcium–calmodulin system in the gonadotropic regulation of granulosa cell steroidogenesis during follicular development had been studied previously (Carnegie and Tsang, 1984), we confirmed this result by investigating the influence of various agents that had been known to alter calcium metabolism or calmodulin activity on basal and FSH-stimulated production of progesterone by hGLCs. (Ethylene-bis (oxyethylene-nitrilo))

0. 8 0. 7 0. 6 0. 5 *

0. 4

*

**

0. 3 0. 2 0. 1

fenvalerate (µM)

FS H +2 5 FS H +1 25

FS H +5

FS H +1

FS H

0 ba sa l

solution for 30 min. The rabbit calmodulin polyclonal antibody (Santa Cruz., CA) as primary antibodies were diluted to 1:300. The incubation with the primary antibody was carried out 2 h at 37 ◦ C. This was followed by three washes. Then, cells were incubated with sencondary goat anti-rabbit fluorescence isothiocyanate (FITC) conjugated antibodies diluted to 1:200 for 1 h at 37 ◦ C. After rinsing, the slides were mounted with Vectashield in order to prevent bleaching. As a negative control, the primary antibody was replaced by PBST. The FITC-mediated emission was detected using confocal laser scanning microscopy. The fluorescence intensity was measured in a total of approximately 100 cells at different field of visions at each sample. Each treatment was repeated for three times.

progesterone (ng/µg protein)

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Fig. 1. The effect of fenvalerate on FSH-stimulated progesterone production by hGLCs. Cells were treated with increasing concentrations of fenvalerate for 24 h in the presence of FSH (200 ng/ml). Values are presented as mean ± S.D. from four wells in a single experiment which was repeated for three times. ∗ Statistically different from control (FSH) using t-test, P < 0.05.

tetraacetic acid (EGTA, 0.1 mM) markly reduced FSH-stimulated P4 production. Verapamil (Sigma), an inhibitor of calcium uptake and trifluoperazine (TFP, Sigma), a kind of calmodulin inhibitor, both attenuated the FSH-stimulated P4 production with the concentration of 10 and 40 ␮M, respectively. No significant effect was observed on basal P4 production with these agents (Table 1). To explore the possible role of calcium in fenvalerate induced alteration in steroidogenesis, TFP and verapamil were used as CaM inhibitor and calcium uptake inhibitor, respectively. We selected 5 and 25 ␮M as representative

Table 1 The effects of various agents on basal and FSH-stimulated progesterone production by hGLCs Progesterone production (ng/␮g protein) Basal Control EGTA (0.1mM) Verapamil (10 ␮M) TFP (40 ␮M)

0.17 0.22 0.21 0.21

± ± ± ±

With FSH (200 ng/ml) 0.03 0.07 0.02 0.06

0.58 0.37 0.30 0.15

± ± ± ±

0.15 0.05a 0.04a 0.05a

Values are presented as mean ± S.D. from four wells in a single experiment which was repeated for three times. a Statistically different from control using t-test, P < 0.05.

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Table 2 The effects of fenvalerate on TFP- and verapamil-driven FSH-stimulated progesterone production Fenvalerate (␮M)

FSH (200 ng/ml)

Progesterone production (ng/␮g protein) 0 0.58 ± 0.15 5 0.34 ± 0.04a 25 0.32 ± 0.03a

FSH (200 ng/ml) +TFP(40 ␮M)

FSH (200 ng/ml) + verapamil(10 ␮M)

0.15 ± 0.05 0.21 ± 0.02 0.41 ± 0.05a

0.30 ± 0.04 0.40 ± 0.06 0.51 ± 0.08a

Values are presented as mean ± S.D. from four wells in a single experiment which was repeated for three times. a Statistically different from corresponding control using t-test, P < 0.05.

3.4. The effects of fenvalerate on FSH-stimulated cAMP production in hGLCs The effects of fenvalerate on FSH-stimulated cAMP production were variable (Fig. 4). The results showed a

500 400 300 200

0

10 50 82 139 220 300 421 542 662 793 953 1114 1275 1434

100

(a)

Time (seconds)

600 500 400 300 200 100 0

(b)

939

778

618

457

297

156

vehicle control

107

Calmodulin, the primary intracellular Ca2+ receptor, is required as an obligatory intermediate in many Ca2+ -dependent processes. We semi-quantitatively evaluated the total CaM in hGLCs by the method of immuofluorescence. The fluorescence intensities reflecting the total concentrations of CaM were approximately 1.3-, 2.2- and 4.3-fold higher than that of the control (Fig. 3).

EGTA 10 mM

fenvalerate 5µM

58

3.3. The effects of fenvalerate on CaM

600

10

The effects of fenvalerate on changes in [Ca2+ ]i in hGLCs was investigated using confocal laser scanning microscopy in individual cells. Fenvalerate was dissolved in ethanol. The final concentration of ethanol never exceeded 0.1% in Hanks HEPES buffer and was used for vehicle control. As shown in Fig. 2a, fenvalerate (5 ␮M) induced a slow increase in intracellular calcium concentration in hGLCs. The concentrations were 82.58 ± 11.28, 236.66 ± 50.69, 519.22 ± 172.92, 592.24 ± 189.2 nM at 1, 5, 9 and 13 min, respectively (n = 8). The vehicle control did not show any effect (Fig. 2b).

[Ca2+]i(nM)

3.2. The effects of fenvalerate on [Ca2+ ]i

kind of biphascic effect. At the concentration of 1 ␮M, fenvalerate augmented FSH-stimulated cAMP production compared with FSH alone. Then, as the dosage increased, fenvalerate showed the inhibitory effects in a dose dependent manner. At 125 ␮M, FSH-stimulated cAMP concentrations were declined to 50% of control (FSH alone).

[Ca2+]i(nM)

doses, and observed that fenvalerate activated both TFP-driven and verapamil-driven FSH-stimulated P4 production. The results show that TFP and verapamil could reverse the inhibition of FSH-stimulated P4 biosynthesis (Table 2).

Time (seconds)

Fig. 2. [Ca2+ ]i oscillations in individual fenvalerate-stimulated hGLC. The [Ca2+ ]i levels were calibrated using the equation [Ca2+ ]i = 400nM × (F − Fmin )/(Fmax − F). Fenvalerate (5 ␮M) induced a slow increase in free cellular Ca2+ . The final addition of 10 mM EGTA induced a rapid decrease in [Ca2+ ]i (a). The ethanol was 0.1% in Hanks HEPES buffer (vehicle control) had no effect on the intracellular calcium concentration (b). The scanning frequency was one image every 4 s at the beginning and then every 10 s. These traces were representative of 8–10 cells in a single experiment and were successfully repeated in three preparations.

J. He et al. / Toxicology 203 (2004) 61–68

fluoresence intensity

66

**

200 150 **

100 **

50 0

0

5

25

125

fenvalerate (µM)

cAMP(nM/µg protein)

Fig. 3. The effects of fenvalerate on total concentration of CaM in hGLCs. Cells were treated with increasing concentrations of fenvalerate for 24 h. The concentration of CaM was quantified by the method of immunofluorescence and was expressed by fluorescence intensity. Values are presented as mean ± S.D. from 100 individual cells. ∗∗ Statistically different from control using t-test, P < 0.01. 0. 02 0. 016 0. 012 **

0. 008 0. 004

5 H +1 25 FS

SH

+2

+5 FS H

+1 FS H

FS H

ba

sa l

0

fenvalerate (µM)

Fig. 4. The effects of fenvalerate on FSH-stimulated cAMP production in hGLCs. Cells were treated with increasing concentrations of fenvalerate for 24 h in the presence of FSH (200 ng/ml). The production of cAMP was measured by RIA. Values are presented as mean ± S.D. from four wells in a single experiment which was repeated for three times. ∗∗ Statistically different from control (FSH) using t-test, P < 0.01.

4. Discussion Fenvalerate has recently been linked to endocrine disruption. The endocrine disrupting chemical has been defined as exogenous agent that interferes with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body (Kavlock et al., 1996). Our results showed that FSH-stimulated P4 by hGLCs was declined at the presence of fenvalerate for 24 h. The finding is consistent with the experiments performed in cultured rat granulosa cells carried out in our laboratory (unpublished data). It has been documented that fenvalerate is likely to be a

kind of estrogenic mimic compound (Go et al., 1999; Chen et al., 2002). Studies on effects of 17␤-estradiol on serum hormone concentration in famale rats indicate serum P4 concentrations were decreased at each stage of estrous cycle (Biegel et al., 1998). Therefore, the effects of fenvalerate might be explained as an estrogen mimic. Intracellular free calcium concentration is kept within the physiological limits by maintaining a delicate balance between influx and enflux of calcium. Disruption of cell calcium homeostasis is often an early event for impairment of Ca2+ -mediated cell functions. Our results suggest that fenvalerate interrupts calcium homeostasis in ovarian cells. Fenvalerate induced an slow increase in [Ca2+ ]i in hGLCs. It is reported pyrethroids can largely bind to biological membranes with non-specific binding site (Lombet et al., 1988). Also, Sarkar et al. (1993) reported fenvalerate could interact with biological membrane by localizing itself near the hydrophobic tail region of the lipid acyl chain and perturbs its ordered structure to make the membrane more fluid. That means fenvalerate is likely to affect the activities of membrane proteins including various calcium channels or Ca2+ -ATPase that are responsible for calcium transport. Certainly, further exploration for the exact mechanisms for interruption of calcium homeostasis in hGLCs is necessary in the future studies. Calmodulin has been shown to be the primary mediator of Ca2+ mediator Ca2+ -dependent signaling in eukaryotic non-muscle and smooth muscle cells by serving as high affinity intracellular receptor (Means et al., 1991). The increased Ca2+ binds to CaM, and the binding induces the latter a conformational change exposing hydrophobic patches that are involved in interaction with and activation of target enzymes (Lu and Means, 1993). CaM is known to be expressed in all eukaryotic cells especially rich in brain, testis and ovaries (Moriya et al., 1993). In the present studies, we found fenvalerate could increase the total concentration of CaM in hGLCs. There is substantial evidence to support that the actions of FSH are mediated by the adenylyl cyclase-cAMP-PKA pathway in ovarian granulosa cells (Richards, 1994). However, not all the actions of FSH are completely reproduced by cAMP analogues or other activators of adenylyl cyclase (Flores et al., 1990). It is well studied that calcium

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and calcium–calmodulin system involves in the gonadotropic regulation of granulosa cell steroidogenesis during follicular development. As confirmed in Table 1, using intracellular calcium inhibitors (verapamil and EGTA) or the calmodulin inhibitor trifluoperazine, FSH-stimulated P4 biosynthesis was attenuated. It has been proposed that this calcium dependency should be located at least two distinct sites in the stimulatory pathway, one is at the level of FSH-induced cyclic AMP production, and another at an unidentified biochemical sites subsequent to the formation of this cyclic nucleotide (Carnegie and Tsang, 1984). In addition, the interaction of these two intracellular messengers in gonadotropin promoted steroidogenesis also existed. Jamaluddin et al. (1992) studied the influence of Ca2+ on cAMP production in hen granulosa cells by using two inophores, A23187 and ionomycin. They found higher [Ca2+ ]i would inhibit LH-promoted cAMP generation while lower [Ca2+ ]i would result in a stimulatory effect on cAMP generation. We therefore speculate if a toxicant perturbed the calcium homeostasis or calmodulin in ovarian granulosa cells, gonadotropic promoted steroidogenesis would be finally affected. In the present study, the production of P4 was inhibited in the absence of TFP or verapamil. On the contrary, the production of P4 was activated in the presence of 40 ␮M TFP or 10 ␮M verapamil (Table 2). We concluded that calcium or CaM might play a role in alterations in FSH-stimulated steroidogenesis induced by fenvalerate. Since fenvalerate could induce the elevation of intracellular calcium concentration and last quite a long time, the possible relationship between increased [Ca2+ ]i and decreased cAMP level induced by fenvalerate is deserved to be explored. In addition, it is well known that CaM acts as an activator of cyclic phosphodiesterase (PDE), which are responsible for degrading the cyclic AMP in cells. Therefore, the cyclic AMP in hGLCs decreased in the present study partly due to increased CaM, which finally lead to the inhibitory effect on P4 biosynthesis. This is consistent with the results obtained in the presence of TFP(Table 2). However, the discrepancy between previous reports and our results existed. Rashatwar and Matsumura (1985) reported that cypermethrin, permethrin inhibited CaM using abovine heart phosphodiesterase–calmodulin system. However Fakata et al. (1998) suggested that the pyrethroids did

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not interfere with calmodulin-dependent signaling. The contradict might be due to differences in experimental methodology or species or organs differences. The present studies are explored to explain decreased FSH-stimulated P4 production induced by fenvalerate partly through calcium signal pathway. The results indicate that calcium or Ca2+ –calmodulin system may be involved in fenvalerate-induced alterations in steroidogenesis. Obviously, further and detailed studies may be necessary to elucidate the exact mechanisms. Establishing cell calcium as a key target for the action of a toxicant is a complex and a challenging process. In summary, we confirmed that fenvalerate is able to disrupt endocrine function by inhibit FSH-stimulated P4 biosynthesis. The studies also showed that fenvalerate interrupted calcium homeostasis in hGLCs. And, FSH-stimulated P4 biosynthesis is inhibited by fenvalerate partly through calcium signal pathway. The mechanisms and the downstream events are deserved to be developed in future studies.

Acknowledgements This work was funded by grants from Nature Science Foundation of China (No. C03010501), the Preliminary Study of an Important Project in the National Basic Research (200150) and the Greatest Project in the National Basic Research (2002CB512908).

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