Gender Differences in Microsomal Metabolic Activation of Hepatotoxic

The gender differences in the in vitro microsomal metabolic activation of hepatotoxic ... expressed in the male rat, might play a significant role in ...
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Chem. Res. Toxicol. 2003, 16, 768-774

Gender Differences in Microsomal Metabolic Activation of Hepatotoxic Clivorine in Rat Ge Lin,* Yan-Yan Cui, and Xiao-Quan Liu† Department of Pharmacology, The Chinese University of Hong Kong, Shatin, Hong Kong, Special Administrative Region Received February 18, 2003

The gender differences in the in vitro microsomal metabolic activation of hepatotoxic clivorine, a representative naturally occurring hepatotoxic otonecine type pyrrolizidine alkaloid, in Sprague-Dawley rats and their relation to the gender differences in susceptibility to clivorine intoxication were reported in the present study. Clivorine-induced liver damage in the male rat via metabolic activation to form the reactive pyrrolic ester followed by covalent binding to liver tissue constituents has been reported previously by our research group. The present study demonstrated, for the first time, that cytochromes P450 3A1 and 3A2, which are constitutively expressed in the male rat, might play a significant role in the metabolic activation of clivorine in the rat. Thus, in the male rat, the metabolic activation by liver microsomes to form the reactive pyrrolic ester was found as the only direct metabolic pathway of clivorine followed by subsequent formation of the toxic tissue-bound pyrroles leading to hepatotoxicity. In the case of the female rat, a less significant metabolic activation was observed, whereas the formations of two novel nonpyrrolic metabolites were determined as the predominant biotransformations. None of the four cDNA-expressed rat enzymes (cytochrome P450 2C12, 2E1, 3A1, 3A2) tested could catalyze the formation of these two new metabolites. Furthermore, the female rat (LD50 ) 114 ( 9 mg/kg, ip) was found to be significantly less susceptible to clivorine intoxication than the male rat (LD50 ) 91 ( 3 mg/kg, ip). Therefore, the results suggested that a significantly lower metabolic activation due to the lack of cytochrome P450 3A1 and P450 3A2 activities mainly accounted for the smaller susceptibility of the female rat to clivorine intoxication.

Introduction PA1

poisoning has been reported in both human and animals, and the majority of naturally occurring PAs is found to be hepatotoxic and/or carcinogenic (1-4). PAinduced liver damage in man has been reported to be mainly due to the consumption of PA-containing plants, herbal teas, and herbal medicines and PA-contaminated food products (5-8). Clivorine is an otonecine type PA found in various Ligularia species including the traditional Chinese medicinal herb Ligularia hodgsonii Hook (9, 10). Clivorine is also known to be hepatotoxic and carcinogenic in ACI rats and mutagenic in Salmonella typhimuriun in the presence of a mammalian microsomal enzyme system, suggesting that metabolic activation is critical in the development of its toxicity (11-13). The mechanisms of hepatotoxicity induced by different types of PAs have been extensively investigated. It has been well-established that the hepatic metabolic activation of different types of PAs to form the corresponding chemically reactive pyrrolic esters, which then rapidly generate covalent adducts with cellular macromolecules in the liver, is the key mechanism responsible for hepatotoxicity * To whom correspondence should be addressed. Tel: (852)26096824. Fax: (852)2603-5139. E-mail: [email protected]. † Current address: Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, People’s Republic of China. 1 Abbreviations: PA, pyrrolizidine alkaloid; DHR, dehydroretronecine; 7-GSH-DHR, 7-glutathionyldehydroretronecine; 7,9-diGSH-DHR, 7,9-diglutathionyldehydroretronecine; PDA, photodiode array; SD, Sprague-Dawley.

induced by all different types of hepatotoxic PAs (1-3, 14-17). Our research group has been mainly focused on the study of otonecine type PAs. Our previous studies on the microsomal metabolism of clivorine in the male SD rat demonstrated that metabolic activation of clivorine to form the reactive pyrrolic ester, dehydroclivorine, followed by covalent binding of this unstable intermediate with liver cellular macromolecules was the primary mechanism responsible for the induction of hepatotoxicity (18-20). However, in the present study, significant gender differences in microsomal metabolism of clivorine and susceptibilities to clivorine intoxication in SD rats were found. In this paper, the gender differences in hepatic metabolic activation of clivorine in rat and their association with the different susceptibilities to clivorine intoxication are reported.

Materials and Methods Materials. The reduced form of GSH, monocrotaline, retrorsine, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, ketoconazole, phenobarbitone, and dexamethasone were purchased from Sigma Chemical Co. The Supersomes samples, which are cDNA-expressed rat cytochrome P450 enzymes, including cytochrome P450 2C12, 2E1, 3A1, and 3A2, were purchased from Gentest Corporation (Woburn, MA) and stored at -80 °C till use. Clivorine was isolated from L. hodgsonii Hook by a standard alkaloid extraction procedure for PAs (9, 10). The identity of the isolated clivorine was confirmed by UV, IR, NMR, and MS analyses, and its purity was determined to be higher

10.1021/tx0340302 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/08/2003

Gender Differences in Metabolism of Clivorine in Rat than 99% by TLC, HPLC, and NMR spectroscopy. The reference samples of putative metabolites were either synthesized using monocrotaline as the starting material for DHR, 7-GSH-DHR, and 7,9-diGSH-DHR or isolated from a scaled-up male rat microsomal incubation with clivorine for clivoric acid as described previously (19). Animals and Preparation of Liver Microsomes. Adult SD rats of both sexes (body weight 220-250 g) supplied by the Laboratory Animal Services Centre at the Chinese University of Hong Kong were utilized. Liver microsomes were prepared by a standard procedure (18-20) from the normal rats and rats pretreated with different enzyme inducers as described below. Protein content was determined using a modification of the Lowry method (21). Cytochrome P450 content was determined by a published standard procedure (22), and cytochrome P450 3A activity referring to the selective mediation of erythromycin N-demethylation was measured using a reported assay (23). The prepared microsomes were divided into aliquots and stored at -80 °C until use. Microsomal Incubation and Treatment of Incubated Samples. Our previously reported procedures were used (1820). Briefly, a typical 1 mL microsomal incubation mixture included potassium phosphate buffer (10 mM, pH 7.4) containing either liver microsomes (2 mg protein/mL) or cDNAexpressed rat enzyme (128 pmol P450 2C12/mL, 128 pmol P450 2E1/mL, 135 pmol P450 3A1/mL, 110 pmol P450 3A2/mL), 250 µM clivorine, 2.0 mM GSH, and an NADPH-generating system (5 mM MgCl2, 1 mM NADP, 10 mM glucose 6-phosphate, 1 unit/ mL glucose 6-phosphate dehydrogenase). As described previously, the addition of GSH into the incubation system enabled this nucleophilic agent to trap the reactive dehydroclivorine forming the corresponding GSH conjugates (19, 20). The biotransformation was initiated by addition of the NADPH-generating system. After it was incubated at 37 °C for 60 min, the reaction was terminated by chilling in an ice bath. Incubation for each individual condition was conducted at least in triplicate. The resultant incubates were centrifuged at 105 000g at 2 °C for 20 min. Aliquots (200 µL) of the supernatant were directly subjected to HPLC/MS for qualitative studies and HPLC/PDA for quantitative determinations. In addition, the resultant pellets obtained from all different incubates were utilized for the determination of total DHR-derived tissue-bound pyrroles as previously reported (19, 24). Briefly, the pellets obtained after centrifugation of incubates were homogenized in ethanol and centrifuged. The residue obtained was washed with ethanol and reconstituted into ethanolic silver nitrate. The resultant mixture was shaken for 30 min and then centrifuged. The residue obtained was extracted with ethanol and reacted with Ehrlich reagent at 55 °C for 10 min. Absorbance of the sample was measured at both 562 and 625 nm, respectively. Adjusted absorbance (A) was determined from A ) 1.1(A562 A625) accordingly (24). Enzyme Induction and Inhibition Studies. Two hepatic enzyme inducers, namely, phenobarbitone and dexamethasone, were utilized for the study of effects of enzyme induction on the metabolism of clivorine. SD rats were randomly divided into four groups with two groups for male and the other two for female rats. Rats in two groups with different sexes were administered with sodium phenobarbitone (80 mg/kg/day, ip) for three consecutive days, while rats in the other two groups were given with dexamethasone (50 mg/kg/day, ip) for four consecutive days. Liver microsome of each pretreated rat was prepared similarly for the subsequent microsomal incubation of clivorine under the identical condition as described above. In addition, a selective cytochrome P450 3A inhibitor ketoconazole was studied for its effects on the microsomal metabolism of clivorine in rats of both sexes. Ketoconazole (5 µM) was preincubated for 10 min prior to the microsomal incubation of clivorine. Furthermore, because the activities of metabolizing enzymes may be altered by preincubation, the controls were also pretreated identically to enable a direct comparison with the results obtained from the corresponding enzyme inhibition experiment.

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Figure 1. Representative chromatograms of HPLC/PDA analysis of the male rat microsomal incubate (A) and the female rat microsomal incubate (B). Peak 1, clivorine; peak 2, DHR; peak 3, 7-GSH-DHR; peak 4, 7,9-diGSH-DHR; peak 5, clivoric acid; IS, internal standard retrorsine. The UV absorbance was recorded at 230 nm. Analysis of Metabolites. The supernatants of incubates were directly subjected to HPLC/MS analysis for the identification of metabolites. All analytical conditions were identical to those previously reported (20). Full scan mass spectra were obtained over the mass range of m/z 150-850 in a negative ion mode for the identification of 7-GSH-DHR, 7,9-diGSH-DHR, and clivoric acid or in a positive ion mode for the determination of DHR and the intact clivorine. A direct comparison of the retention time and mass spectrometric data of each metabolite with that for the authentic sample was also conducted for the confirmation of the identity of each metabolite found. The quantitative study of each individual incubate was conducted by using our previously developed HPLC/PDA assay using a specific two column setup with a Hamilton PRP-1 analytical column (250 mm × 4.6 mm, 5 µm) and Hamilton PRP-1 guard column (50 mm × 4.1 mm, 5 µm) (25). The incubated blank samples spiked with standards were also analyzed under identical conditions, and a direct comparison of the retention time and UV spectrum of each analyte with that of the corresponding standard was also performed. Samples for the construction of calibration curves for the intact clivorine and all known metabolites were prepared as previously described (25). Calibration curves for all analytes within the testing concentration ranges were linear. The quantity of each analyte present in different incubates was determined from the corresponding calibration curve. Data Analysis. ANOVA with the Bonferroni test was used to compare results in different groups. A probability (p) value less than 0.05 was considered statistically significant.

Results Metabolic Pathways. With a direct comparison of the retention time, mass, and UV spectrometric data of the corresponding authentic samples, four and three known metabolites generated from male and female rat microsomal incubations were unequivocally identified, respectively, by using both HPLC/MS and HPLC/PDA analyses. As shown in the HPLC/PDA chromatogram in Figure 1A, four known metabolites of clivorine, namely, DHR,

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Figure 2. In vitro microsomal metabolic pathways of clivorine in the male (M) and female (F) rat. NuS, nucleophilic biological macromolecules; GS, glutathionyl. The unstable intermediate is shown in the large bracket. The forward arrow indicates the major metabolic pathway of clivorine in the female rat.

7-GSH-DHR, 7,9-diGSH-DHR, and clivoric acid, were found in the male rat microsomal incubations, which are in a good agreement with our previous in vitro studies (19, 20). Similarly, a racemic mixture with a 1:1 ratio of 7(S) and 7(R) enantiomers was found for both GSH conjugates. In the case of the female rat microsomal metabolism of clivorine, except 7,9-diGSH-DHR, the same three metabolites were identified. Moreover, two additional peaks with retention times of 28.3 (M1) and 14.5 min (M2) were observed (Figure 1B), which were not found in the control incubates. In addition, similar to our previous in vitro studies (19, 20), DHR-derived tissuebound pyrroles were also found in the microsomal incubates of rats of both sexes. Figure 2 illustrated a comparison of metabolic pathways of clivorine in the male and female rats. For the two new metabolites found in the female rat microsomal metabolism of clivorine, their MS spectra analyzed by HPLC/MS suggested that they are both novel and nonpyrrolic metabolites. However, their molecular structures have not been unequivocally identified and studies are currently under progress. Our preliminary studies (data not shown) indicated that M2 was a product of further metabolism of M1. Moreover, the formations of these two metabolites were not inhibited by the cytochrome P450 inhibitor SKF 525-A, suggesting that they were cytochrome P450-independent. The detailed studies of these two novel nonpyrrolic metabolites of clivorine will be reported separately. Metabolic and Metabolite Formation Rates. As shown in Figure 3A,B, the formation rates for 7-GSHDHR, clivoric acid, and the tissue-bound pyrroles were significantly lower in the female rat. However, the metabolic rates of clivorine (Figure 3C) in both sexes were the same with approximately 49% of clivorine metabolized in 1 h. Apparently, formations of M1 and M2 accounted for the biotransformations of clivorine in the female rat to a significant extent. On the basis of the apparent peak areas in both HPLC/PDA (Figure 1B) and

Figure 3. Quantification of the in vitro metabolites of clivorine and the intact clivorine in the male and female rat. *** p < 0.001 as compared with male rat. Table 1. Total Cytochrome P450 Content and Cytochrome P450 3A Activity in Rat Liver Microsomesa

microsome male control phenobarbitone induction dexamethasone induction female control phenobarbitone induction dexamethasone induction

P450 3A erythromycin P450 (nmol/ N-demethylation mg protein) (pmol/mg protein/min) 0.54 ( 0.06 1.0 ( 0.05* 0.89 ( 0.09* 0.45 ( 0.06 0.63 ( 0.04* 0.83 ( 0.04*

2900 ( 52 5090 ( 110* 7700 ( 130* 1860 ( 100 2920 ( 79* 7520 ( 79*

a Data were expressed as mean ( SD (n ) 3). * p < 0.001 as compared with the corresponding control.

HPLC/MS (data not shown) chromatograms, M2 could be considered as the predominant metabolite, suggesting that the formation of this metabolite was the major metabolic pathway, while the oxidative N-demethylation was the minor pathway in the female rat (Figure 2). Enzyme Induction and Inhibition Studies. As summarized in Table 1, a total cytochrome P450 content and activity of cytochrome P450 3A in both male and female rat microsomes were significantly induced by two cytochrome P450 inducers tested. The degree of the induction of P450 3A activity was more severe in dexamethasone pretreated than in phenobarbitone pretreated in both sexes, since dexamethasone is a selective P450 3A inducer (26, 27) whereas phenobarbitone is a nonselective cytochrome P450 inducer and induces activities

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Table 2. Effect of Enzyme Inducers and Inhibitors on the In Vitro Metabolism of Clivorine in Rats of Both Sexes formation rate of metabolite (nmol/mg protein/1 h) 7-GSH-DHR 7,9-diGSH-DHR clivoric acid

sample

DHR

control phenobarbitone dexamethasone controla ketoconazole

8.9 ( 0.6 15.5 ( 0.4** 16.7 ( 0.2** 4.8 ( 0.1

7.4 ( 0.2 17.4 ( 0.8** 11.6 ( 0.2** 3.8 ( 0.2

control phenobarbitone dexamethasone controla ketoconazole

8.8 ( 0.8 11.1 ( 0.4** 12.6 ( 0.6 5.8 ( 0.1

2.4 ( 0.1 8.3 ( 0.2** 5.3 ( 0.3** 3.6 ( 0.2

male 1.8 ( 0.1 3.0 ( 0.1* 2.3 ( 0.1* 2.3 ( 0.1 female

intact clivorine (nmol/mL)

total tissue bound pyrroles (nmol)

52.1 ( 0.6 108 ( 0.9** 124 ( 0.3** 21.9 ( 0.2

129 ( 0.9 32.1 ( 1.1**

1.3 ( 0.2 2.0 ( 0.1** 2.1 ( 0.1** 0.7 ( 0.1

8.7 ( 0.4 65.5 ( 0.8** 123 ( 2** 4.8 ( 0.1

129 ( 2 20.4 ( 0.1**

153 ( 5 204 ( 10**

178 ( 21 181 ( 9

0.4 ( 0.1 0.9 ( 0.1** 1.9 ( 0.2** 0.2 ( 0.1

a Preincubated for 10 min prior to the addition of the NADPH-generating system. Data were expressed as mean ( SD (n ) 3). * p < 0.01, ** p < 0.001 as compared with the corresponding control. Blank entries, not detected.

Table 3. In Vitro Metabolism of Clivorine by CDNA-Expressed Rat Cytochrome P450 Enzymes formation rate of metabolite (pmol/mg protein/min) sample P450 2C12 P450 2E1 P450 3A1 P450 3A2 a

DHR

99 ( 5 109 ( 13

7-GSH-DHR

107 ( 2 134 ( 6

clivoric acid

intact clivorine (nmol/mL)

total tissue bound pyrroles (nmol)a

364 ( 2 312 ( 21

231 ( 17 246 ( 8 135 ( 10 147 ( 3

0.88 0.59

The total quantity in combined three incubated samples. Data were expressed as mean ( SD (n ) 3). Blank entries, not detected.

of various P450 enzymes in rats (28). As compared with the control microsomal incubations, the metabolic rates of clivorine in rats of both sexes were significantly increased by phenobarbitone (Table 2). After 1 h of incubation, about 13 (male) and 8% (female) of the intact clivorine was found, respectively, and the formation rates of all pyrrolic related metabolites were significantly elevated. In the case of dexamethasone induction, more severe induction of metabolic rate of clivorine was observed in both sexes. Clivorine was completely metabolized within 1 h, and the formation rates of all pyrrolic related metabolites significantly increased with a similar pattern in both sexes (Table 2). Furthermore, incubation with ketoconazole (29, 30), a selective cytochrome P450 3A inhibitor, was conducted. The control incubations were also pretreated under the same conditions as for the inhibitory incubations, since metabolic rates for control samples were markedly lower with 10 min preincubation as compared with that without such preincubation (Table 2). The results showed that ketoconazole completely inhibited the formations of all metabolites generated from the oxidative N-demethylation pathway of clivorine in both male and female rats (Table 2). However, the metabolic rate of clivorine and formation rates for two novel metabolites M1 and M2 (data not shown) were not significantly affected in the female rat. The results indicated that the P450 3A subfamily was the primary enzyme catalyzing the oxidative N-demethylation of clivorine in rats of both sexes (Figure 2). Metabolism of Clivorine by cDNA-Expressed Cytochrome P450 Enzymes. Further studies were conducted by using four cDNA-expressed rat cytochrome P450 enzymes to confirm the primary enzyme(s) catalyzing the metabolism of clivorine in rats. The results (Table 3) demonstrated that both cytochrome P450 3A1 and 3A2 mediated the oxidative N-demethylation of clivorine with the similar metabolic rates to form DHR,

7-GSH-DHR, clivoric acid, and tissue-bound pyrroles. However, none of the enzymes examined were involved in the formation of two novel M1 and M2 metabolites.

Discussion The male rat has been previously reported to be more susceptible to retronecine type PA intoxication than the female rats of different strains (2, 5, 7, 8, 31). Our current study on clivorine, a representative otonecine type PA, in SD rats also found the similar gender difference. The male rat was significantly more susceptible to clivorine toxicity (LD50 ) 91 ( 3 mg/kg, ip) than the female rat (LD50 ) 114 ( 9 mg/kg, ip, p < 0.05). Furthermore, our previous studies in the male rat have demonstrated that clivorine-induced hepatotoxicity was primarily due to its hepatic metabolic activation (20); thus, gender differences in the microsomal metabolism of clivorine and the susceptibilities to clivorine intoxication in SD rats and whether there is a relation between these differences were investigated in the present study. As previously reported (19, 20), the metabolic activation of clivorine is the oxidative N-demethylation pathway to form a reactive pyrrolic ester, dehydroclivorine, which is similar to the reactive pyrrolic esters generated from various retronecine type PAs (1-3, 14-17). Once formed, it may rapidly bind nucleophilic macromolecules to generate tissue-bound pyrroles leading to hepatotoxicity (Figure 2). Thus, the tissue-bound pyrroles are considered as the toxic pyrrolic metabolites directly related to PA intoxication (1-5, 11, 14, 32). On the other hand, the reactive pyrrolic ester may also undergo further biotransformations to produce pyrrolic related metabolites including DHR and two GSH conjugates with a concurrent generation of clivoric acid (Figure 2). The results obtained from the present study in the male rat are in good agreement with our previous findings (19, 20). The metabolic activation to form the reactive pyrrolic

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ester was identified to be the only direct metabolic pathway of clivorine in the male rat. On the other hand, in the female rat, in addition to the same metabolic activation, one more direct metabolic pathway of clivorine to generate nonpyrrolic metabolites M1 and M2 was observed (Figure 2). Although identities of these two novel metabolites have not been unequivocally determined, whether they are toxic metabolites is unknown; as the nonpyrrolic metabolites, they are highly unlikely to directly relate to the formation of the tissue-bound adducts leading to hepatotoxicity. Further studies are needed to clarify this hypothesis. Although the reactive pyrrolic ester cannot be determined directly, two important metabolites, the tissuebound pyrroles, which directly relate to the hepatotoxicity, and clivoric acid, which can be accounted as the sum of all pyrrolic related metabolites formed from further metabolisms of the reactive pyrrolic ester (19, 20), were quantified. The quantitative studies further confirmed that as compared with the male rat the metabolic activation of clivorine in the female rat was a minor pathway. As shown in Figure 3, although about half of clivorine was metabolized by both sexes after 1 h of incubation, 42% of clivorine was converted to the pyrrolic related metabolites via metabolic activation in the male rat, while only about 7% of such conversion was determined in the female rat. The results indicated that in the female rat the metabolic activation was a minor pathway whereas the other available pathways to generate nonpyrrolic metabolites M1 and M2 might be predominant (Figure 2). Furthermore, the amount of total tissue-bound pyrroles generated, which directly relate to the liver damage, was significantly lower in the female rat (0.4 ( 0.1 nmol) than in the male rat (1.3 ( 0.2 nmol). Therefore, the overall results suggested that the significant lower metabolic activation of clivorine to generate the tissue-bound pyrroles in the female rat correlated with less susceptibility of the female rat to clivorineinduced hepatotoxicity. It has been reported that both cytochrome P450 3A and P450 2B subfamilies are responsible for the hepatic metabolism of retronecine type PAs in rats (16, 23, 28, 31, 32). Our previous study also demonstrated that the P450 3A subfamily is the primary enzyme mediating microsomal metabolism of clivorine, an otonecine type PA, in the male SD rat (20). It is well-known that P450 3A1 and P450 3A2 are constitutively expressed in the male rat but their levels are significantly lower in the female rat, and both enzymes are inducible in both sexes (33-36). The present study demonstrated, for the first time, that P450 3A1 and P450 3A2 were the key enzymes responsible for the microsomal metabolic activation of clivorine in rats. As illustrated in Table 3, study with the selected cDNA-expressed rat enzymes confirmed that among four cytochrome P450 enzymes tested P450 3A1 and 3A2 were responsible for the metabolic activation of clivorine to generate the reactive pyrrolic ester and tissue-bound pyrroles. However, M1 and M2 metabolites were not formed by any of these enzymes studied. It is well-known that the female adult rat lacks cytochrome P450 3A1 and P450 3A2 enzymes (36). When the P450 3A activities were significantly induced by dexamethasone and became the same in rats of both sexes, the metabolic profiles of clivorine appeared to be very similar (Table 1) and clivorine was completely metabolized (Table 2) in both sexes. The conversion of

Lin et al.

clivorine to the pyrrolic-related metabolites via the metabolic activation accounted for about 99 (male) and 98% (female) of the clivorine metabolized, respectively, and the formation of total tissue-bound pyrroles was also significantly induced (2.0 ( 0.1 nmol in male and 1.9 ( 0.2 nmol in female). Furthermore, M1 and M2 were not produced in the dexamethasone-induced female rat microsomal incubation. These results indicated that when P450 3A activity in the female rat was induced to the same level as in the male rat, the metabolic activation pathway predominated and thus significant amounts of toxic metabolites were formed in the female rats. Furthermore, ketoconazole, a selective P450 3A inhibitor (29, 30), abolished the metabolic activation pathway of clivorine in both sexes (Table 2); thus, no metabolites were determined in the male rat. However, in the case of the female rat, although there were no metabolites generated from the metabolic activation, the metabolic rate of clivorine was not significantly affected because the other cytochrome P450-independent pathways to form M1 and M2 were not inhibited by ketoconazole. All of the above results demonstrated that the significantly lower metabolic activation of clivorine in the female rat was due to its lack of P450 3A1 and P450 3A2 activities. It was reported that eight of 12 ACI rats of both sexes developed malignant liver tumors after given drinking water containing 0.005% clivorine for 340 days (12). However, this reported study did not describe gender differences in clivorine-induced tumorigenicity in ACI rats. Whether there are strain differences is unknown. Further investigations are warranted for the confirmation of whether there are strain differences in metabolic activation and correlation of the metabolic differences with clivorine-induced carcinogenicity. Moreover, the pyrrolic alcohol DHR has been reported to be genotoxic, since it can also alkylate nucleophiles and generate DHRderived DNA adducts but in a slower and probably more selective manner than the reactive pyrrolic ester (1, 2, 37-39). However, DHR is more stable and hydrophilic than the reactive pyrrolic ester and may widely distribute throughout the body; therefore, it is generally considered as less hepatotoxic but may induce extrahepatic toxicity (40-42). In the present study, similar formation rates of DHR (about 9 nmol/mg protein/h) were found in both male and female rat liver microsomal metabolism of clivorine. However, whether such amounts of DHR formed in the SD rats of both sexes may induce genotoxicity needs further investigation. In conclusion, the results suggested, for the first time, that hepatic cytochrome P450 3A1 and P450 3A2 might be the major enzymes responsible for the metabolic activation of clivorine in SD rats. The male rat is more susceptible to clivorine intoxication, because clivorine is predominantly metabolized via the metabolic activation pathway mediated by highly expressed cytochrome P450 3A1 and P450 3A2 enzymes, leading to hepatotoxicity. In the case of the female rat, the cytochrome P450independent pathways to generate nonpyrrolic related metabolites predominated, whereas metabolic activation was the minor pathway due to the significantly lower expressed levels of cytochrome P450 3A1 and P450 3A2 enzymes. Therefore, the lack of P450 3A1 and P450 3A2 activities leading to a significantly lower metabolic activation is the main reason responsible for the less susceptibility of the female rat to clivorine-induced hepatotoxicity. Therefore, the male SD rat but not the

Gender Differences in Metabolism of Clivorine in Rat

female SD rat should be a suitable animal model for the studies of the mechanism(s) of PAs, especially the otonecine type PAs, induced hepatotoxicity.

Acknowledgment. The financial support from the Research Grant Council of Hong Kong (Earmarked Research Grant CUHK 415/95M) for this research project was greatly acknowledged. We also thank Mr. Tin-Yan Cheng (Department of Pharmacology, the Chinese University of Hong Kong) for the quantification of the tissue bound pyrroles.

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