Sex-Dependent Toxicity of a Novel Acyl-CoA:Cholesterol Acyltransferase Inhibitor, YIC-C8-434, in Relation to Sex-Specific Forms of Cytochrome P450 in Rats

Kimiyuki Kaneko,1, Kazumi Uchida, Toshihide Kobayashi, Kouzou Miura, Keiko Tanokura, Kayoko Hoshino, Ikuo Kato, Masaharu Onoue and Teruo Yokokura

Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186-8650, Japan

Received June 26, 2001; accepted September 10, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
YIC-C8-434 is a novel inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT). To clarify the toxicity of YIC-C8-434, the compound was given orally to Sprague-Dawley rats for 28 days at 0, 4, 20, 100, or 500 mg/kg/day. The toxicity of the drug differed significantly between male and female rats. In female rats treated at 500 mg/kg, many symptoms including moribund condition, suppression of weight gain and food consumption, abnormal blood chemistry, and decreases in organ weights (thymus, ovaries, and uterus) were observed. In male rats by contrast, no significant toxicity was observed at any dose. After a single administration of YIC-C8-434 at 500 mg/kg, female rats had a higher blood concentration of the compound than male rats. Little elimination of YIC-C8-434 was observed in female rats on analysis of drug-elimination kinetics. Furthermore, the metabolism of YIC-C8-434 was analyzed using rat hepatic microsomal preparations from both sexes. Consistent with the observations in vivo, hepatic microsomes from male rats better metabolized YIC-C8-434 than those from females. In addition, the metabolism of YIC-C8-434 by hepatic microsomes from male rats was blocked by SKF525A, a P450 inhibitor. Inhibition experiments using anti-rat CYP1A1, CYP1A2, CYP2B1, CYP2C11, CYP2E1, CYP3A2, and CYP4A1 antisera indicated that CYP3A2 played the predominant role in the metabolism of YIC-C8-434 in rats. Since there is less CYP3A2 in the liver of female than male rats, the involvement of CYP3A2 in YIC-C8-434 metabolism has implications for the sex-related metabolic activity and toxicity of YIC-C8-434.

Key Words: rats; ACAT inhibitor; toxicity; sex differences; hepatic CYP enzymes; CYP3A2.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acyl CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) is the enzyme responsible for the intracellular esterification of cholesterol (Suckling et al., 1985Go). Although ACAT activity has been demonstrated in most mammalian tissues, ACAT has a variety of roles in each tissue. In the liver, ACAT-derived cholesteryl esters are secreted as a component of very low-density lipoprotein (VLDL) (Drevon et al., 1980Go; Suckling et al., 1985Go). In the gastrointestinal tract, ACAT-mediated cholesterol esterification is suggested to be the rate-limiting step in the absorption of food- and bile-derived cholesterol (Clark et al., 1984Go; Suckling at al., 1985; Sviridov et al., 1993Go). In macrophages, ACAT may participate in foam-cell formation and thereby contribute to atherosclerotic lesions (Brown et al., 1983Go; Matsuda, 1994Go; Suckling at al., 1985). Thus, numerous ACAT inhibitors have been developed during the past 20 years (reviewed in Krause et al., 1993Go; Sliskovic et al., 1991Go; Vaccaro et al., 1996Go), several of which have been reported to have adrenal toxicity (Dominick et al., 1993aGo,bGo; Matsuo et al., 1996Go; Reindel et al., 1994Go; Vernetti et al., 1993Go; Wolfgang et al., 1995Go). These toxic effects are not unrelated to the ACAT isozyme and its localization. Recently, 2 ACAT genes have been identified in mammals. ACAT-1 is expressed ubiquitously and ACAT-2 is expressed in the liver and small intestine (Chang et al., 2000Go; Meiner et al., 1997Go; Uelmen et al., 1995Go). ACAT-1 is highly expressed, not only in macrophage-derived foam cells in atherosclerotic lesions (Miyazaki et al., 1998Go) but also in steroid hormone-producing cells and other human tissues (Sakashita et al., 2000Go). In steroidogenic organs such as the adrenal gland and ovaries, cholesteryl esters formed by ACAT are stored as substrates for use in the synthesis of steroid hormones, and ACAT activity is regulated reciprocally with steroid hormone synthesis (Civen et al., 1984Go; Schuler et al., 1981Go; Suckling et al., 1985Go). This implies that the toxicity in cells due to the buildup of cellular free cholesterol can be malignant (Warner et al., 1995Go). A recent study shows that selective inhibition of ACAT-1 in lesion macrophages in the setting of hyperlipidemia can lead to the accumulation of free cholesterol in the artery wall, and may be detrimental, rather than therapeutic for atherosclerosis (Fazio et al., 2001Go). Therefore, unanticipated toxic effects may result from a selective and/or potent inhibition of ACAT-1. This would be desirable to explore in that selective inhibition of ACAT-2 is beneficial for treating hypercholesterolemia.

YIC-C8-434, chemically designated as N-(3,5-dimethoxy-4-n-octyloxycinnamoyl)-N`-(3,4-dimethylphenyl)piperazine, is a potent ACAT inhibitor that acts in rat liver microsomes, in hepatocyte-like HPG2 cells, and in intestinal enterocyte-like Caco-2 cells (Ohishi et al., 2001Go). Furthermore, YIC-C8-434 showed anti-hypercholesterolemic activity in cholesterol-fed rats at 2.4 mg/kg/day (Ohishi et al., 2001Go). Considering the predominant expression of ACAT-1 and ACAT-2 in HPG2 cells and Caco-2 cells, respectively (Chang et al., 2000Go), it is assumed that YIC-C8-434 might have sufficient ability to inhibit both ACAT-1 and ACAT-2. However, we are investigating the possibility that YIC-C8-434 strongly inhibits ACAT-2 rather than ACAT-1, since YIC-C8-434 did not inhibit ACAT in THP-1 macrophages (unpublished communication) in which ACAT-1 is expressed.

We demonstrated that the toxicity of YIC-C8-434 was restricted to female rats. To date, there have been no reports on sex-related differences in the toxicity of ACAT inhibitors. The purpose of this study is to show that the sex-related toxicity is caused not by the inhibition of ACAT in steroidogenic tissues, especially in ovaries, but by sex-related differences in YIC-C8-434 metabolism in rats.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
YIC-C8-434 (N-(3,5-dimethoxy-4-n-octyloxycinnamoyl)-N`-(3,4-dimethylphenyl)piperazine) was synthesized at Yakult Central Institute for Microbiological Research (Tokyo, Japan). The chemical structure of YIC-C8-434 is shown in Figure 1Go. The purity of the material was 99.8% as analyzed by HPLC. Rat liver microsomes were prepared freshly in our laboratory. Anti-rat CYP antibodies were obtained from Daiichi Pure Chemicals Co., LTD. (Tokyo, Japan). All other reagents used in this study were of analytical grade.



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FIG. 1. Chemical structure of YIC-C8-434.

 
Animals.
Male and female Sprague-Dawley rats (Crj:CD[GS]), 6 weeks of age, were purchased from Charles River Japan, Inc. (Kanagawa, Japan) and used after 7 days acclimation. Animals were housed individually in cages with a wire mesh floor and were freely given water and commercial laboratory chow (F2: Funabashi Farm Co., Ltd., Chiba, Japan). Room temperature, relative humidity, and the light-dark cycle were adjusted to 23 ± 3°C, 50 ± 10%, and 12 h (8:30–20:30), respectively. Rats were randomly assigned to test groups.

Twenty-eight-day repeated oral toxicity study.
YIC-C8-434 was suspended in a 0.5% carboxymethylcellulose solution at concentrations of 0.4, 2, 10, and 50 mg/ml. The dosing volume was 10 ml/kg. Each group consisted of 10 rats, which were administered the drug by gavage once daily for 28 days. Control rats were given the same volume of 0.5% carboxymethylcellulose as received by drug-treated rats. Body weight was measured weekly during the test period. Food weight was measured twice a week, and we calculated food consumption per day. After the final administration of YIC-C8-434, rats were fasted overnight. The next day, they were sacrificed under pentobarbital anesthesia. Blood was collected from the abdominal vena cava. Blood for hematological analysis was collected in tubes containing potassium EDTA. Hematological determinations, including erythrocyte, leukocyte, platelet, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and leukocyte differential (lymphocyte, neutrophil, eosinophil, basophil, monocyte) were performed using an automated cell counter (Bayer H • 1E, Bayer Co., Tarrytown, NY). Blood was collected in tubes with heparin for plasma chemistry and without anticoagulant for serum protein analyses. In blood chemical examinations, we analyzed the following items; transaminase (GOT, GPT), alkaline phosphatase, glucose, total cholesterol, triglyceride, phospholipid, total bilirubin, urea nitrogen, creatinine, calcium, sodium, potassium, chloride, and total protein using an Automatic Clinical Analyzer (7170, HITACHI Ltd., Tokyo, Japan). The weights of organs (brain, pituitary, thymus, heart, liver, spleen, kidney, adrenal, testis, prostate, uterus, and ovary) were determined at necropsy of the animals. Tissues were fixed in 10% neutral buffered formalin and processed for paraffin sectioning followed by hematoxylin and eosin (H&E) staining for microscopic observation.

The following tissues from the 500-mg/kg dose group and control group were examined microscopically: brain, pituitary, thymus, heart, liver, spleen, kidney, adrenal, femur with marrow, skin, mammary gland, harderian gland, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), pancreas, mesenteric lymph nodes, thyroid gland, parathyroid gland, muscle, testis, epididymis, seminal vesicle, prostate, uterus, and ovary. Adrenal, ovary and bone marrow were examined in the 100-mg/kg dose group, for female rats.

In vivo pharmacokinetic study.
Three rats of each sex received a single oral administration of YIC-C8-434 at 500 mg/kg. Blood was collected from the cervical vein at 1, 4, 10, 24, and 48 h after the administration. Plasma was obtained after immediate centrifugation and the concentration of YIC-C8-434 in plasma was assayed by HPLC. The area under the blood drug concentration-time curve (AUC) was calculated from the drug-concentration for 48 h after a single administration by the linear trapezoidal method.

Absorption and elimination kinetics was investigated by a residual method. It is assumed that YIC-C8-434 is absorbed into the body and is eliminated from the systemic circulation by a first-order process. Based on this assumption, kinetics of absorption and elimination were compared between male and female rats. Utilizing the data from a single administration test, separate linear regression analyses were performed with plots for 4–48 h (elimination term) and residual plot-time profiles based on the slope of the residual plot (1–4 h for absorption term) were constructed. The constants of absorption rate (Ka) and elimination rate (Ke) were calculated from the slope.

In vitro metabolism studies.
Hepatic microsomes were prepared freshly from livers of male and female rats. Microsomes were washed, and the pellets were resuspended in 0.1 M phosphate buffer (pH 7.4). The metabolism of YIC-C8-434 by hepatic microsomes was assayed in a reaction mixture consisting of an NADPH-generating system (20 mM glucose-6-phosphate, 1.2 IU glucose-6-phosphate dehydrogenase, 20 mM MgCl2), hepatic microsomes (0.5 mg protein/ml) and YIC-C8-434 (0.67, 1.33, 2.66, 6.67, 13.3, 26.6, or 53.3 µM). After pre-incubation for 5 min at 37°C, the reaction was initiated by adding NADPH (1 mM). The final volume of the reaction mixture was 1.5 ml. After incubation for 10 min, the reaction was terminated by the addition of 2 ml of ice-cold acetonitrile. Since metabolites of YIC-C8-434 were unknown, the metabolism was evaluated as the difference in the amount of residual YIC-C8-434 between the start (0 min) and the end of the reaction (10 min).

For inhibition by SKF525A, hepatic microsomes were mixed with various amounts of SKF525A (0, 2, 10, 50, or 250 µM) and pre-incubated at 37°C for 5 min. An aliquot of this microsome solution was transferred to a reaction mixture containing the NADPH-generating system and YIC-C8-434 (2.66 µM). After pre-incubation at 37°C for 5 min, the reaction was initiated by adding NADPH (1 mM) and terminated after a 10-min incubation (final volume of 1.5 ml).

Immunoinhibition of the YIC-C8-434 metabolism with microsomes prepared from the liver of male rats was performed in the presence of 50 µl of anti-P450 antisera (0.4 mg protein/ml) against CYP1A1, CYP1A2, CYP2B1, CYP2C11, CYP2E1, CYP3A2, or CYP4A1. Microsomes were premixed with each antibody for 30 min and then were added to a reaction mixture containing the NADPH-generating system and YIC-C8-434 (15 µM). After incubation for 10 min, the amount of residual YIC-C8-434 with/without anti-P450 antiserum was measured and the inhibition rates were calculated.

Measurement of YIC-C8-434 in plasma and the reaction mixture.
The concentration of YIC-C8-434 was measured by HPLC. Acetonitrile (2 ml) was added to 0.4 ml of plasma or 1.5 ml of reaction mixture. After shaking and ultrasonic treatment for 5 min, the sample was centrifuged (3000 rpm, 15 min). The upper layer was transferred to a clean glass tube, dried under N2 gas, and reconstituted with 100 µl of acetonitrile/water (9/1). Twenty µl of the solution was injected onto a Symmetry C18 analytical column (3.5 µm, 4.6 x 75 mm. Water Co., Milford, MA). The temperature of the column was adjusted to 40°C. The flow rate of the mobile phase, acetonitrile/water/acetic acid/triethylamine (500:475:25:1), was adjusted according to the retention time of YIC-C8-434 to be about 12 min. Column effluent was monitored by UV absorption at 310 nm, and the limit of detection was 15 ng/ml.

Statistical analysis.
Data were represented as the mean ± SD. The homogeneity of variance was tested by Bartlett's method. If homogeneous, the data were analyzed by one-way ANOVA, and if not, by the Kruskal Wallis test. In significant cases, Dunnett's test for multiple group comparisons was performed. Significance was accepted at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Twenty-Eight-Day Repeated Oral Toxicity Study
There was no death and no remarkable toxicity at any dose in male rats treated for 28 days with YIC-C8-434. In contrast, 3 female rats in the 500-mg/kg dose group were sacrificed in a moribund condition on the 19th day. Before the sacrifice, some abnormal symptoms such as piloerection, crouching, emaciation, and soilure from periurinary area to abdomen were observed. Additionally, the remaining rats in this group showed marked leanness.

In Figure 2Go, the changes in body weight during the 28 days are shown. In male rats, the body weight gains were normal. In contrast, weight gain was markedly suppressed in the high-dose group (500 mg/kg) of female rats compared with the control and other groups. The mean body weight of this group was significantly lower than that of the control group during the test period.



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FIG. 2. Effects of repeated administration of YIC-C8-434 on body weight. Rats were orally treated with 4, 20, 100 (dashed lines), or 500 mg/kg/day (solid line and triangles) of YIC-C8-434 or solvent (0.5% carboxymethylcellulose, solid line and circles) for 28 days. Symbols and bars represent the mean ± SD, respectively; **p < 0.01 significantly different from the control.

 
The graph in Figure 3Go illustrates the changes in food intake during the 28 days. Although a significant decrease in food consumption in the 500 mg/kg group was observed in the second week in male rats, there were no significant differences compared with the control group at other times. In contrast to male rats, the food consumption of females of the 500-mg/kg dose group was significantly suppressed at all weeks. The rats in this group took in only about 40% of the amount digested by control rats throughout the test period.



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FIG. 3. Effects of YIC-C8-434 on food intake in rats. Rats were orally treated with 4, 20, 100, (dashed lines) or 500 mg/kg/day (solid line and triangles) of YIC-C8-434 or solvent (0.5% carboxymethylcellulose, solid line and circles) for 28 days. Symbols and bars represent the mean ± SD, respectively; *p < 0.05, ** p < 0.01 significantly different from the control.

 
The results of hematology and blood chemistry are summarized in Table 1Go. Changes were observed in females of the YIC-C8-434 group (500 mg/kg). In marked contrast to males, females in this group exhibited significant increases of plasma lipids (total cholesterol, phospholipid, triglyceride) and total bilirubin. The total amount of cholesterol in plasma was about 3 times higher in this group than the control group. Furthermore, decreases in MCV, MCH, and leukocytes, including lymphocytes and eosinophils, were observed.


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TABLE 1 Hematology and Blood Chemistry of Male and Female SD Rats Administered YIC-C8-434 by Gavage for 28 Days
 
The effects of YIC-C8-434 on body and organ weights of females are summarized in Table 2Go. Relative weights of brain, heart, liver, and kidneys were increased in female rats treated with YIC-C8-434 at the 500-mg/kg dose, due to the low body weight. However, the absolute weights and relative weights of thymus, ovaries, and uterus in this group were lower than the control values. No significant differences in organ weight were observed in male rats at any dose (data not shown).


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TABLE 2 Body and Organ Weights of Female SD Rats Administered YIC-C8-434 by Gavage for 28 Days
 
Microscopically, YIC-C8-434 induced histopathologic changes in bone marrow, ovaries, and adrenal glands in females (Table 3Go). In bone marrow, the number of fatty cells and immature hematopoietic cells increased slightly on treatment with YIC-C8-434 at 500 mg/kg (Fig. 4Go). Necrosis in follicular cells was observed in the ovaries (Fig. 5Go). Apparent changes were not seen in the cells of the adrenal medulla or zona glomerula of the adrenal cortex in either sex of any group, although cortical cells containing little lipid in the zona fasciculata were seen in 3 females given the 500-g/kg dose (Fig. 6Go).


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TABLE 3 Incidence and Severity of Non-neoplastic Lesions in Female SD Rats in the 28-Day Gavage Study with YIC-C8-434
 


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FIG. 4. Micrograph of bone marrow from a female rat treated with solvent (0.5% carboxymethylcellulose (a) or 500 mg/kg YIC-C8-434 (b) for 28 days. (a) No remarkable changes; (b) there is reduction of hematopoietic tissue with replacement by fat. H&E stain, x130.

 


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FIG. 5. Micrograph of ovary from a female rat treated with solvent (0.5% carboxymethylcellulose (a), or 500 mg/kg YIC-C8-434 (b) for 28 days. (a) No remarkable changes; (b) there is necrosis of follicular cells (arrowheads). H&E stain, x520.

 


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FIG. 6. Micrograph of adrenal from a female rat treated with solvent (0.5% carboxymethylcellulose (a), or 500 mg/kg YIC-C8-434 (b) for 28 days. (a) No remarkable changes; (b) there is moderate decrease of vacuoles of cortex cells in the fascicular zone. H&E stain, x260.

 
Pharmacokinetics of YIC-C8-434 after a Single Administration
Marked sex-related differences in the concentration of YIC-C8-434 in blood were observed (data not shown). As shown in Table 4Go, when YIC-C8-434 (500 mg/kg) was given orally, AUC and t1/2 were substantially greater in female than in male rats. Ke values (min-1) were 0.009 for female rats and 0.164 for male rats. However, a sex-related difference in Ka values was not observed.


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TABLE 4 Effect of Gender on the Kinetics of YIC-C8-434 after a Single Administration of 500 mg/kg by Gavage
 
In Vitro Metabolism by Liver Microsomes
Figure 7Go shows the effects of substrate concentration on the rate of YIC-C8-434 metabolism by liver microsomes from male and female rats. In the presence of NADPH, the metabolism of YIC-C8-434 by liver microsomes from male rats depended on the concentration of substrate, and conformed to Michaelis-Menten kinetics. The Lineweaver-Burk (double reciprocal) plot for metabolism, using microsomes from male rats, was linear (Fig. 7Go). This result suggested that the metabolism of YIC-C8-434 in male rats is catalyzed predominantly by a single P450 enzyme, with substrate concentrations of ~53.3 µM. Values of Km and Vmax for metabolism, using microsomes from male liver, were calculated as 12.22 µM and 0.66 µmol/mg/min, respectively. Contrary to the behavior in male rat hepatic microsomes, a plot of V (velocity) vs. [S] (reciprocal of substrate concentration) for the metabolism of YIC-C8-434 in female rat hepatic microsomes did not display a normal shape.



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FIG. 7. Effect of substrate concentration on the metabolism of YIC-C8-434 by rat liver microsomes. YIC-C8-434 (0.67–53.3 µM) was incubated with rat liver microsomes (0.5 mg/ml) for 10 min. The upper graph is direct plots of reaction rates versus substrate concentration. The lower graph is a Lineweaver-Burk plot of YIC-C8-434 metabolism. Symbols represent the average of duplicate incubations.

 
In subsequent experiments using a P450 inhibitor, the concentration of substrate was 2.66 µM, between 0.67 and 6.67 µM, because YIC-C8-434 metabolism by female liver microsomes reached a plateau in this lower concentration range of substrate (Fig. 7Go). As shown in Figure 8Go, the effects of a P450 inhibitor (SKF525A) on YIC-C8-434 metabolism by liver microsomes were determined. While metabolism by the liver microsomes from male rats was inhibited, that by microsomes from female rats was not. These results pointed to P450 enzymes being responsible for the metabolism of YIC-C8-434 in male rats, and the expression of a specific P450 enzyme that contributed to metabolism being diminished in female rats. The effects of various anti-P450 antisera on the metabolism of YIC-C8-434 are shown in Figure 9Go. In the reaction mixture containing microsomes from male rats, anti-rat CYP3A2 antiserum inhibited the metabolism of YIC-C8-434 by 50%. In contrast, other antisera had little effect on this activity.



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FIG. 8. Effect of P450 inhibitor, SKF525A, on YIC-C8-434 metabolism by rat hepatic cytochrome P450 systems. YIC-C8-434 (2.66 µM) was incubated with rat liver microsomes (0.5 mg/ml) and SKF525A (0–250 µM) for 10 min. Symbols represent the average of duplicate incubations. Solid lines represent approximate lines.

 


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FIG. 9. Effect of anti-CYP antisera on YIC-C8-434 metabolism in male rat liver microsomes. Each bar represents average of triplicate incubations.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, toxicological profiles of YIC-C8-434, a novel ACAT inhibitor, were assessed after oral administration to rats for 28 days, and the principal finding was a marked sex-related toxicity at the highest dose (500 mg/kg). Although toxicity of YIC-C8-434 in male rats was not observed at all, the toxicity was remarkable in female rats. Namely, body weight and food consumption were decreased, and rats showed a marked reduction in body weigh gain in the highest-dose group during the treatment. Furthermore, lipids (total cholesterol, triglyceride, phospholipids) and total bilirubin in plasma significantly increased in the females, and the circulating leukocyte count decreased on repeated administrations of YIC-C8-434. Additionally, weights of thymus, ovary, and uterus were decreased, and fatty marrow was observed in female rats.

Studies have shown that the adrenal gland is very susceptible to the toxicity of ACAT inhibitors (Dominick et al., 1993aGo,bGo; Matsuo et al., 1996Go; Vernetti et al., 1993Go; Wolfgang et al., 1995Go). However, unlike other ACAT inhibitors, YIC-C8-434 had little effect on adrenal glands compared with thymus and ovaries. The inhibition of ACAT by YIC-C8-434 is potentially toxic to the ovary and thymus since the ACAT enzyme is expressed in these organs, as well as the adrenal gland (Matsuda et al., 1998Go; Meiner et al., 1997Go; Uelmen et al., 1995Go). Thus, if toxic effects on the thymus and ovary resulted from inhibition of ACAT that was expressed in these organs, we may point out that the thymus and ovary are the target organs of toxicity induced by YIC-C8-434.

However, the increase in plasma bilirubin, and the atrophies of thymus, ovary, and fatty marrow are arguably not irrelevant to the decrease in food consumption. Our findings indicated that YIC-C8-434 induced anorexia in female rats. Food consumption was reduced to about 40% of control in female rats during the treatment period at the highest dose (500 mg/kg) and weight gain was suppressed. Generally, in food-restriction studies, decreases of tissue weight (thymus and ovary) and increase of fat in the marrow are exhibited in rats (Bronson, 1986Go; Levin et al., 1993Go; Seki et al., 1997Go). The restricted rats also exhibit a decrease of hematopoiesis and increase of total bilirubin (Levin et al., 1993Go; Seki et al., 1997Go). We observed similar changes caused by the toxicity of YIC-C8-434. It seems entirely possible that the loss of bone marrow is reflected in the appearance of fatty marrow and a decrease in the number of circulating leukocytes in rats. As for the suppression of food consumption occurring from the first week of treatment in female rats, it is conceivable that YIC-C8-434 has an ability to induce hypophagia.

Meiner et al. (1996) found that ACAT knockout mice had increased plasma cholesterol levels. Our finding of an increase in lipids among female rats is similar to this. Although strong inhibition of the ACAT enzyme could be a causal factor in plasma lipid disorders, the increase of plasma lipids might also be explained by anorexia. Stone (1994) reported that anorexia could be correlated with hyperlipidemia. However, we cannot explain why anorexia should be induced in female rats during treatment with YIC-C8-434. Further study into the influence of YIC-C8-434 on the hypophagic effect is required.

The major characteristic of the toxicity induced by YIC-C8-434 was that it was sex-related. We suggested that the finding was attributable to a higher concentration of YIC-C8-434 in the blood of female than of male rats. As shown in Table 4Go, greater values of the concentration of YIC-C8-434 in blood and AUC in female rats were interpreted to reflect a difference in the rate of metabolism or absorption of YIC-C8-434 from the intestinal tract in males and females. To investigate the mechanism behind the higher concentration of YIC-C8-434 in blood in female rats, in vivo and in vitro studies were performed. From the result of the in vivo study with a single oral administration, sex-related differences in the metabolism of YIC-C8-434 were predicted reasonably well. Kato and Kamataki (1982) have reported sex differences in the toxicological and pharmacological potency of certain drugs. There is evidence that sexual dimorphism in drug metabolism in rats results from a sex-related differential expression of hepatic P450s (Kobliakov et al., 1991Go). Therefore, we performed additional experiments on the metabolism of YIC-C8-434 by hepatic microsomes of the rat. As shown in Figure 8Go, these in vitro experiments indicated that the microsomal cytochrome P450 from male rats could metabolize YIC-C8-434. However, consistent with the observations in vivo, hepatic microsomes from female rats poorly metabolized YIC-C8-434. Furthermore, inhibition of the metabolism in microsomes from female rats was not observed with the P450 inhibitor, SKF525A. This result indicated that cytochrome P450 did not play a role in YIC-C8-434 metabolism in female rats. It is known that the sexual dimorphism of drug metabolism in the rat is due to the differential expression of certain cytochromes P450. For example, CYP2A2, CYP3A2, and CYP2C11 are male-predominant or expressed only in male rats, whereas CYP2A1, CYP2C7, and CYP2C12 are female-predominant or limited to female rats (Bandiera, 1990Go; Kobliakov et al., 1991Go; Legraverend et al., 1992Go; Waxman et al., 1985Go, 1990Go). In the examination with anti-rat P450 (CYP) antisera, we tried to identify the CYP isoform for metabolism. Anti-rat CYP3A2 antiserum showed the most potent inhibition of YIC-C8-434 metabolism: 50%. Because the CYP3A2 isoform is responsible for the metabolism of YIC-C8-434 in rats, and because there is more CYP3A2 isoform in the liver of males than females, these results strongly suggest that the sexual dimorphism in the metabolism of YIC-C8-434 in rats results from the sex-related differential expression of the CYP3A2 isoform.

In summary, the results of the present study implicate the sex-related differential expression of CYP3A2 in the toxicological findings relevant to reduced metabolism and the accumulation of YIC-C8-434 in female rats. It would be reasonable to conclude that the metabolite of YIC-C8-434 had no effect on the toxicity, since toxic findings were not observed in male rats in which YIC-C8-434 was well metabolized. The existence of sex differences in drug metabolism is not unique to rats. Such differences have been shown in humans (Hunt et al., 1992Go). However, the magnitude of the sex differences in humans is invariably far subtler than that found in rats. Further study will be required to ascertain whether YIC-C8-434 shows a sex-related difference in clearance in humans.


    NOTES
 
1 To whom correspondence should be addressed. Fax: 042–5773020. E-mail: kimiyuki-kaneko{at}yakult.co.jp. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bandiera, S. (1990). Expression and catalysis of sex-specific cytochrome P450 isozymes in rat liver. Can. J. Physiol. Pharmacol. 68, 762–768.[ISI][Medline]

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