Sex-specific induction of apoptosis by cyproterone acetate in primary rat hepatocytes

P. Kasper1 and L. Mueller

Federal Institute for Drugs and Medical Devices, Seestrasse 10, D-13353 Berlin, Germany


    Abstract
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 Abstract
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The synthetic steroid cyproterone acetate (CPA) has been reported to be hepatogenotoxic in female rats depending on sex-specific expression of a hydroxysteroid sulfotransferase (HST) which is involved in the bioactivation of CPA to reactive metabolites. In the present study the ability of CPA to initiate apoptosis in rat hepatocytes in vitro was investigated with respect to sex-specific effects and dependency on HST activity. Incubation of primary hepatocytes of female rats with CPA (0.1–30 µM) caused a strong increase in percent of cells undergoing apoptosis. The lowest concentration leading to apoptosis was 0.3 µM. In contrast, hepatocytes isolated from male rats showed a very weak response at high exposure to CPA (30 µM) only. Treatment with transforming growth factor-ß1 induced high levels of apoptotis in hepatocytes of both genders. Megestrol acetate and chlormadinone acetate, two structural analogues of CPA with a much lower genotoxic potency, did not induce apoptosis. Pre-addition of 10 or 50 µM dehydroepiandrosterone (DHEA), a known inhibitor of hepatic HST, almost completely inhibited CPA-induced apoptosis in hepatocytes of female rats. Using similar test concentrations, DHEA also reduced CPA-induced DNA excision repair as measured in the unscheduled DNA synthesis test. The results suggest that apoptosis induction is directly related to DNA damage induced by HST-dependent CPA metabolites.

Abbreviations: 2-AAF, 2-acetylaminofluorene; CMA, chlormadinone acetate; CPA, cyproterone acetate; DAPI, 4',6-diamidino-2-phenylindole; DHEA, dehydroepiandrosterone; DMSO, dimethylsulfoxide; HBSS, Hanks' balanced salt solution; HST, hydroxysteroid sulfotransferase; MGA, megestrol acetate; TGF-ß1, transforming growth factor-ß1; UDS, unscheduled DNA synthesis; WEM, Williams' E medium.


    Introduction
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 Abstract
 Introduction
 References
 
Cyproterone acetate (CPA) is a synthetic anti-androgen and progestin widely used in anti-androgenic drugs. In early rodent long-term studies CPA as well as other steroidal sex hormones were found to induce liver tumors (1). These findings were related to a promoting effect of the steroids based on their ability to stimulate liver cell growth (2,3). More recent findings have given clear evidence of a sex-specific DNA damaging potential of CPA in the liver of female rats (4). A single dose of CPA efficiently causes DNA adducts which persist for several weeks (4,5) and was also shown to induce gene mutations in the liver of female Big Blue transgenic rats (6). Involvement of a hydroxysteroid sulfotransferase (HST) which is highly and sex specifically expressed in female rats (7), was found to be responsible for the sex-specific genotoxicity of CPA (8,9). Another mechanism which may contribute to the hepatocarcinogenicity of CPA was proposed by Roberts et al. (10), who found that administration of CPA to male rats significantly reduced the basal level of apoptosis. Such an effect would prevent the removal of spontaneously DNA-damaged, potentially initiated cells (10). Unfortunately, female rats were not investigated in this study and thus it is unclear whether even at elevated levels of DNA damage suppression of apoptosis by CPA occurs. In the present study investigations with primary rat hepatocytes of both sexes were done and the following aspects were experimentally addressed: (i) comparative measurement of the apoptotic activities of CPA in hepatocytes of male and female rats; (ii) investigations of the effects of the HST inhibitor dehydroepiandrosterone (DHEA) on CPA-induced apoptosis and CPA-induced genotoxicity; (iii) comparison of the potency of CPA in inducing apoptosis with that of chlormadinone acetate (CMA) and megestrol acetate (MGA), two structural analogues of CPA.

Rat liver cells were obtained from 8–10-week-old male and female Wistar rats (150–200 g body wt). Isolation of hepatocytes was done according to the two step collagenase perfusion technique of Seglen (11). Briefly, the rats were anaesthetized with an i.p. injection of pentobarbital sodium (Nembutal; WDT, Hannover, Germany) and their livers were perfused in situ for 10 min with Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) supplemented with 10 mM HEPES and 0.5 mM EGTA, followed by collagenase digestion solution (HBSS supplemented with 10 mM HEPES, 5 mM CaCl2 and 0.05% collagenase Type IV; Sigma, Deisenhofen, Germany) for 10 min. After the collagenase digestion the hepatocytes were dispersed in ice-cold William's E medium (WEM) and filtered through a sterile gauze. The cell suspension was washed three times with cold WEM (centrifugation at 50 g for 2 min) to remove cell debris and non-parenchymal liver cells. The final cell pellet was resuspended in complete WEM (William's E medium, 10% fetal calf serum, 2 mM L-glutamine, 0.1 mg streptomycin/ml and 100 U penicillin/ml). The viability was >75% as determined by Trypan Blue exclusion.

For the analysis of apoptotic cells, hepatocytes were plated at a density of 8x104 cells/cm2 onto rat tail collagen- coated 6 mm dishes in 5 ml complete WEM supplemented with 10–7 M insulin. After 1.5 h the medium was changed to remove unattached cells. Twenty-four hours after initiating the hepatocyte cultures fresh medium with the test compound was added. As a positive control the growth inhibitory cytokine transforming growth factor-ß1 (TGF-ß1), a well-known inducer of apoptosis in rat hepatocytes, was used (12). A further 24 h later all cultures were changed into serum-free conditions with the test compounds and exposure continued for 24 or 48 h. It has been reported that serum-free conditions increase the sensitivity of hepatocytes to the effects of apoptosis-inducing agents (13). In experiments where the influence of DHEA on CPA-induced apoptosis was investigated, DHEA was added 30 min before treatment with CPA started and co-exposure continued until the end of treatment as described above. After treatment the monolayers were fixed in ice-cold methanol/acetic acid (4:1) for 10 min and dried overnight. The fixed cells were stained at 4°C for 1 h with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) (0.5 µg/ml). In each experiment 1000 cells/dish from two parallel dishes were analysed for each condition using a fluorescent microscope with a UV filter. Under these conditions apoptotic cells were easily recognizable and were identified as those with brightly fluorescent, condensed chromatin or chromatin pieces (fragmented nuclei).

Measurement of unscheduled DNA synthesis (UDS) was basically done as described (14). In brief, ~2x105 viable hepatocytes in 2 ml complete WEM were seeded onto collagen-coated 25 mm Thermanox coverslips (Nunc, Wiesbaden, Germany) in 35 mm 6-well dishes. After an attachment period of 1.5 h the medium was replaced by WEM containing 5 µCi/ml [3H]TdR (sp. act. 25 Ci/mmol; Amersham Buchler, Braunschweig, Germany) and DHEA. CPA or 2-acetylaminofluorene (2-AAF), both dissolved in dimethylsulfoxide (DMSO), was added 30 min later. After an exposure period of 18 h the hepatocytes were fixed and prepared for autoradiography. Net grains per nucleus were determined by subtracting the plasma grains from the nuclear grains (14).

The induction of apoptosis in primary hepatocytes of male and female rats after exposure to CPA in DAPI-stained cultures is shown in Figure 1Go. In control cultures treated with the solvent DMSO (1%) the incidence of cells with apoptotic nuclei was low and varied in the different experiments between 0.05 and 0.30% without differences between both genders. Exposure to 2 ng/ml TGF-ß1 caused a clear increase in apoptosis in hepatocytes of both male and female rats with ~4–5% cells with apoptotic morphology. Treatment with CPA over a concentration range from 0.1 to 30 µM did not induce cell death in hepatocytes of male rats after a 72 h treatment period. A very slight effect with a doubling of the control values was observed after 48 h treatment at the highest concentration only. In contrast, in hepatocytes from female rats exposure to CPA resulted in a marked and concentration-dependent increase in the percentage of cells undergoing apoptosis. The lowest concentration of CPA found to induce apoptosis was 0.3 µM after 72 h exposure. Maximum levels of apoptotic cells for both exposure times were observed at 30 µM CPA with ~5% apoptotic cells, which was in the range of effects found for the positive control TGF-ß1. The sex-specific pattern of the apoptotic potential of CPA in rat liver cells is similar to that found for the genotoxic activity of CPA, such as DNA adduct formation or induction of DNA repair (4,15). To investigate whether HST-mediated metabolites which have been reported to be responsible for the genotoxicity of CPA (8) are also involved in the induction of apoptosis, experiments with the selective HST inhibitor DHEA (16) were performed. As shown in Figure 2Go, co-treatment with DHEA significantly reduced the amount of CPA-induced apoptosis at both DHEA concentrations tested. At the higher concentration of 50 µM DHEA the apoptotic effects of CPA were completely inhibited. DHEA alone had no effect on the spontaneous level of apoptosis. The influence of DHEA on CPA-induced genotoxicity was investigated in the UDS test (Figure 3Go). As was already demonstrated earlier (15,17,18), CPA induced a considerable UDS effect in hepatocytes of female rats with mean net grain counts of ~10 and 15 at 10 and 30 µM, respectively. Pre-addition of DHEA led to a concentration-dependent decrease in CPA-induced UDS with very drastic inhibitory effects at 50 µM DHEA, which agrees with data from 32P-post-labelling studies, where DHEA in the concentration range 20–100 µM strongly inhibited the formation of CPA–DNA adducts in rat hepatocytes of female rats (8). Exposure of hepatocytes to DHEA alone had no effect on the spontaneous level of UDS. Induction of UDS by another mutagen, 2-AAF (1 µg/ml), was not reduced when cells were co-treated with DHEA, indicating that DHEA has no inhibitory effects on enzymes involved in excision repair. Both, the inhibitory effect of DHEA and the marked sex difference suggest that CPA-induced apoptosis depends on HST-mediated bioactivation of CPA and is most likely related to the formation of DNA damage by CPA metabolites. DNA damage is widely recognized as a primary signal for the induction of apoptosis. Genotoxic compounds such as etoposide, hydroxyurea or 2-AAF and also UV irradiation have been shown to be inducers of apoptosis in rat hepatocytes (19,20). In contrast, CMA and MGA, two structural analogues of CPA with a qualitatively comparable genotoxic activity, i.e. sex-specific induction of DNA adducts and DNA repair in rat liver cells of females (21,22), were negative in the apoptosis assay when tested in the same concentration range as CPA (Table IGo). However, both steroids possess markedly lower genotoxic potencies, as compared with CPA (21,22). In a 32P-post-labelling study with hepatocytes of female rats using similar exposure levels as in the present study (i.e. 3, 10 and 30 µM) CMA and MGA formed 10, 29, 53 and 6, 16, 29 adducts/109 nucleotides, respectively, in comparison with 699, 1170 and 1670 adducts/109 nucleotides formed by CPA (22). It may be concluded from this comparison that DNA damage has to exceed a certain threshold to initiate the apoptotic pathway.



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Fig. 1. Induction of apoptotic cells in primary hepatocyte cultures of male and female rats treated with CPA or TGF-ß1. Twenty-four hours after initiation of primary hepatocyte cultures cells were exposed to the test compounds for 48 or 72 h. The percentage of cells with an apoptotic morphology was determined in DAPI-stained monolayers using a fluorescence microscope. In each experiment 1000 cells/dish from two parallel dishes were scored for each condition. Data points are the mean of two independent experiments.

 


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Fig. 2. Effects of DHEA on CPA-induced apoptosis in primary hepatocytes of female rats. Twenty-four hours after initiation of primary hepatocyte cultures cells were exposed to CPA with or without DHEA for 48 h. Determination of apoptotic cells was as described in Figure 1Go. Data points are the means ± SD of two independent experiments.

 


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Fig. 3. Effects of DHEA on UDS induction by CPA and 2-AAF in primary hepatocytes of female rats. Hepatocytes were exposed to CPA or 2-AAF (1 µg/ml) with or without DHEA in the presence of tritiated thymidine for 18 h. UDS was measured by autoradiography. Three slides in which 50 cells were counted per slide were analysed for each condition in each experiment. Data represent the means ± SD from two independent experiments.

 

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Table I. Induction of apoptotic cells in primary hepatocyte cultures of female rats treated with CPA, CMA and MGA
 
Reports on apoptotic activity of CPA in cultured rat hepatocytes are limited so far to studies with cells from male animals only and are in agreement with our findings from experiments with male rats. Oberhammer and Qin (12) found a slightly increased number of apoptotic cells at the maximum test concentration of 10 µM CPA only. In another study CPA at a concentration of 12 µM had no effect on the spontaneous level of apoptotic cells in primary hepatocytes of male rats (23). Both studies showed in addition a co-apoptogenic potential of CPA, i.e. CPA increased the apoptotic activity of TGF-ß1. It was suggested that CPA, due to its mitogenic activity, shifts the cells to a point before S phase entry where they become more vulnerable to the apoptosis-inducing activity of TGF-ß1 (12,23). It may be speculated that in CPA-exposed hepatocytes from female rats the additional mitogenic stimulus also renders the cells more sensitive to apoptotic signals induced by DNA damage, thus explaining the strong induction of apoptosis in the present study. In in vivo studies using female rats, only a very slight increase in the level of liver cells with apoptotic morphology was observed 24 h after treatment of animals with 100 mg/kg CPA for 3 days followed by 130 mg/kg for 4 days (24). Under similar dosing conditions CPA was reported to induce high adduct levels as well as cell proliferation in female rat liver (2,4). The reason for the obviously different apoptogenic activity of CPA in vivo and in vitro is unclear, but may reflect the more sensitive detection of apoptotic cells under in vitro conditions in hepatocyte cultures.

In summary, the present findings show that CPA is a potent apoptogen in primary hepatocytes of female rats but not of males. The induction of apoptosis can be related to the sex-specific, HST-dependent genotoxicity of CPA in rat liver. In addition, these studies demonstrate that cultivated hepatoctes represent a promising tool to investigate the correlation of apoptosis, genotoxicity and metabolism under defined experimental conditions.


    Acknowledgments
 
The authors are grateful to Mrs Karin Gindler for skilful technical help.


    Notes
 
1 To whom correspondence should be addressed Email: p.kasper{at}bfarm.de Back


    References
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 Abstract
 Introduction
 References
 

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Received March 15, 1999; revised July 16, 1999; accepted July 23, 1999.