Introduction

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Diabetes has been found to potentiate hepatotoxicity of certain compounds, such as thioacetamide (TA), chloroform, 1,1,2-trichloroethane, CCl₄, and bromobenzene (Hanasono et al., 1975; El-Hawari and Plaa, 1983; Watkins et al., 1988). Preliminary studies in our laboratory (Wang et al., 2000) have shown that a nonlethal dose of TA (300 mg/kg, i.p.) in normal rats caused 90% lethality in the streptozotocin (STZ)-induced diabetic rats. Plasma alanine aminotransferase (ALT), sorbital dehydrogenase, and histopathological studies showed that TA-induced liver injury was highly exaggerated in diabetic rats (Wang et al., 2000).

TA was first used to control the decay of oranges and then as a fungicide (Childs and Siegler, 1945). Now it is being used in leather, textile, and paper industries as an accelerator in the vulcanization of buna rubber and as a stabilizer for motor fuels (Sittig, 1985). It is well understood that, in the liver, TA (CH₃-C(S)NH₂) is S-oxidized at the thioamide group to TA sulfoxide (CH₃-C(SO)NH₂) and subsequently to di-S-oxide (CH₃-C(SO₂)NH₂). Reactive intermediate(s) in this pathway covalently bind to hepatic macromolecules and eventually cause liver injury (Hunter et al., 1977; Porter and Neal, 1978). Specific enzymes responsible for the bioactivation of TA have not been identified. Neal and his coworkers (Hunter et al., 1977; Porter and Neal, 1978) have shown that pretreatment with a variety of cytochrome P450 inhibitors, such as pyrazole, SKF-525A, cobalt chloride, metyrapone, and an antibody to cytochrome P450 protected against TA-induced liver necrosis. De Ferreyra et al. (1983) reported that prior administration of substrates of flavin-containing monooxygenase (FMO), such as chlorpromazine, imipramine, mercaptoethylamine, 1-(1-naphthyl)-2-thiourea, or phenyl-thiocarbamide, also prevented TA-induced liver necrosis. Thiobenzamide (C₆H₅-C(S)NH₂), a structural analog of TA, has been shown to be metabolized to thiobenzamide sulfoxide by both cytochrome P450 (35%) and FMO (65%) (Tynes and Hodgson, 1983) in rat liver microsomal incubations.

Diabetes is known to modify a plethora of metabolizing enzymes in liver, the most prominent effect being a 6- to 8-fold induction of CYP2E1 (Favreau and Schenkman, 1988). CYP2E1 is responsible for the bioactivation of an extensive array of drugs, solvents, and environmental carcinogens (Koop, 1992). In addition to diabetes, CYP2E1 is also induced by diet restriction, fasting, as well as exposure to ethanol (Johansson et al., 1988; Leakey et al., 1991; Hu et al., 1995). All these conditions are known to potentiate hepatotoxicity of TA (Maling et al., 1975; Strubelt et al., 1981; Ramaiah et al., 1998). These data collectively suggest that CYP2E1 might mediate TA hepatotoxicity, but no studies have been done to test this hypothesis. FMO is another family of monooxygenases involved in the oxidation of many sulfur-containing compounds and other soft nucleophiles. FMO1 is the predominent form of FMO in rat liver and intestine. Unlike cytochrome P450s, FMOs appear not to be significantly inducible by xenobiotics. Significant changes in FMO expression have been observed following endocrine manipulations such as gonadectomy (Dannan et al., 1986) and also in special physiological states such as pregnancy and starvation (Osimitz and Kulkarni, 1982; Dixit and Roche 1984). Rouer et al. (1988) reported that FMO activity increased 2-fold in STZ-induced diabetic rats and mice. Few reports exist on dietary modulation of FMO (Ziegler, 1993; Larsen-Su and Williams, 1996). However, Larsen-Su and Williams (1996) have demonstrated that dietary indole-3-carbinol (I3C), a constituent derived from cruciferous vegetables, inhibits hepatic FMO1. FMOs and cytochrome P450s often catalyze many of the same substrates.

The objective of this study was to investigate the relative roles of CYP2E1 and FMO1, if any, in the bioactivation of TA. We report here that metabolism via CYP2E1 appears to be the primary mechanism underlying TA-induced liver injury and that increased expression of CYP2E1 underlies the potentiated TA hepatotoxicity observed in the diabetic rat. FMO1 is unlikely to play a significant role in bioactivation-based liver injury by TA in the diabetic rat.

	Materials and Methods

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Animals

Animal maintenance and research were conducted in accordance with the National Institutes of Health Guide for Animal Welfare Act. Male Sprague-Dawley rats (250 to 300 g) were obtained from our central animal facility. Rats were housed individually in wire-bottom cages in air-conditioned quarters (21 ± 1°C) with a 12-h photoperiod. All rats had free access to water and commercial chow (Rat Chow 7001, Harlan Teklad, Madison, WI) unless otherwise stated.

Chemicals

STZ, TA, CCl₄, and isoniazid (INH) were purchased from Sigma Chemical Co. (St. Louis, MO). Diallyl sulfide (DAS), I3C, and 5,5'-dithiobis(nitrobenzoic acid) were obtained from Aldrich Chemical Co. (Milwaukee, WI). p-Nitrophenol (PNP) was purchased from Fisher Scientific Co. (Baton Rouge, LA).

Treatments

Induction of Diabetes and Effects of Diabetes on TA Hepatotoxicity and Hepatic CYP2E1. On day 0, male Sprague-Dawley rats were treated with either a single dose of STZ (60 mg/kg, i.p.) to induce diabetes or citrate buffer (0.01 M, pH 4.3, 1 ml/kg, i.p.) as the nondiabetic control. Tail vein blood was collected to measure plasma glucose daily after STZ injection. Animals were considered diabetic if plasma glucose was >200 mg/dl, as measured using Sigma kit 315-100. On day 10, both diabetic and nondiabetic rats were treated with TA (300 mg/kg, i.p.). At 0, 12, and 24 h after TA treatment, blood was collected from the dorsal aorta under diethyl ether anesthesia for plasma ALT measurement. Liver samples were collected before TA administration (0 h after TA treatment) for CYP2E1 and FMO1 studies (n = 3 per group per time point).

CYP2E1 Induction Study. Rats were pretreated either with INH (250 mg/kg, i.p.) to induce CYP2E1 (Park et al., 1993) or 0.9% NaCl (saline, 1 ml/kg, i.p.). TA (300 mg/kg, i.p.) was administered to both groups 18 h later. Liver samples were collected before TA administration (0 h after TA treatment), and blood samples were collected at 0, 12, and 24 h after TA administration (n = 3 per group per time point).

CYP2E1 Inhibition Study. DAS is a competitive inhibitor and inactivator of CYP2E1 (Chen et al., 1994). STZ-induced diabetic rats were pretreated with either two doses of DAS (200 mg/4 ml of corn oil/kg, p.o.) or an equal volume of corn oil (4 ml/kg, p.o.). The time interval between the two doses was 12 h. Four h after the second dose, TA (300 mg/kg, i.p.) was given to all the rats. Similarly, two groups of nondiabetic rats (one was treated with DAS, the other given corn oil) received TA 4 h after the last treatment. Liver samples were collected before TA administration (0 h after TA treatment), and blood samples were collected at 0, 12, and 24 h after TA treatment (n = 3 per group per time point).

FMO1 Inhibition Study. To investigate the involvement of FMO1 in TA-induced liver injury, an FMO1 inhibition study was conducted with both nondiabetic and diabetic rats. I3C, an in vivo inhibitor of FMO (Larsen-Su and Williams, 1996) was mixed in powdered rat chow at a concentration of 0.25% (w/w). One nondiabetic group was given regular chow powder, the other group was given chow containing 0.25% I3C. For diabetic rats, on day 0 all rats were treated with STZ (60 mg/kg, i.p.) to induce diabetes. On this day, one diabetic group was maintained on the I3C diet, the other group was maintained on regular chow powder. All rats were maintained on their respective diet regimen till the end of the study. On day 10, all rats were treated with TA (300 mg/kg, i.p.). Liver samples were collected before TA administration (0 h after TA treatment), and blood samples were collected at 0, 12, and 24 h after TA treatment (n = 3 per group per time point).

Hepatotoxicity

Plasma ALT activity was measured as a biomarker of liver damage. After the blood samples were collected, plasma was separated by heparinization and centrifugation. Plasma ALT (EC 2.6.1.2) was measured using Sigma kit 59 UV.

Preparation of Microsomes

Liver microsomes were prepared by differential ultracentrifugation using the method of Chipman et al. (1979). Protein concentration of the microsomes was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL) with crystalline bovine serum albumin as a standard. Microsomes were stored at 80°C until needed.

Western Immunoblot Analysis

Microsomal protein (20 µg) from each rat was separated by SDS-polyacrylamide gel electrophoresis and transferred to membranes. For detecting CYP2E1 protein, nitrocellulose membranes (Bio-Rad, Hercules, CA) were incubated with a primary rabbit polyclonal antibody raised against rat CYP2E1 (a generous gift from Dr. Magnus Ingelman-Sundberg, Karolinska Institute, Stockholm, Sweden). Then the blots were further incubated with a secondary ¹²⁵I-labeled donkey anti-rabbit antibody purchased from Amersham Pharmacia Biotech (Piscataway, NJ). For detecting FMO1 protein, polyvinylidene difluoride membranes (Amersham Pharmacia Biotech) were incubated with a primary rabbit antibody raised against hog hepatic FMO1 (a generous gift from Dr. David Williams, Oregon State University). The blots were then probed with a donkey anti-rabbit secondary antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech) and visualized using a chemiluminescence kit obtained from Amersham Pharmacia Biotech. Immunoquantitation was carried out using a GS-700 imaging densitometer (Bio-Rad).

Enzyme Activity Assays

Microsomal CYP2E1-dependent hydroxylation of PNP to p-nitrocatechol was used as a standard assay for quantifying CYP2E1 activity as described by Koop (1986). FMO1 catalyzes oxidation of methimazole to formamidine sulfenic acid, which oxidizes thiocholine to thiocholine disulfide. The loss of thiocholine was measured to quantify FMO1 activity as described by Guo et al. (1992).

Statistical Analysis

Data were expressed as means ± S.E. Comparison between two groups was made by Student's paired t test; comparison among values was made by analysis of variance followed by the Bonferroni t procedure.

	Results

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Effect of Diabetes on TA-Induced Liver Injury and CYP2E1. Diabetes was induced in male Sprague-Dawley rats after a single dose of STZ (60 mg/kg, i.p.) as shown in Table 1. Plasma ALT activity in diabetic rats measured at 12 and 24 h after TA administration was 7- to 8-fold higher than that of nondiabetic rats.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effect of diabetes on hepatic CYP2E1 expression and enzyme activity. On day 0, rats were treated with either STZ (60 mg/kg, i.p.) to induce diabetes or 0.01 M citrate buffer (1 ml/kg, i.p.) as controls. On day 10, liver samples were collected under diethyl ether anesthesia and prepared for the microsomes (n = 3). A, microsomal CYP2E1 expression was detected by Western blot and quantitated by densitometry. The mean value obtained from the nondiabetic group was taken to be 100%, and the value obtained from the diabetic group was expressed as a percentage of control. A representative CYP2E1 blot is shown in the upper left corner. B, CYP2E1 activity was measured by PNP hydroxylase activity. *, significantly different from the nondiabetic group. P  .05.

CYP2E1 Induction Study. INH was used as a specific inducer of CYP2E1 to investigate if CYP2E1 is involved in TA hepatotoxicity. Plasma ALT showed that TA-induced liver injury was significantly increased in INH-pretreated rats. At the 24-h time point, the elevated ALT activity of INH-exposed rats was approximately 2.5-fold as much as that of control (Fig. 2). Western blot analysis showed (Fig. 3A) that CYP2E1 expression was increased 2.2-fold in the INH-pretreated rat. Correspondingly, PNP hydroxylation (Fig. 3B) was induced 2.5-fold.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of INH pretreatment on TA-induced liver injury. Rats were treated with INH (250 mg/kg, i.p.) or 0.9% NaCl (saline, 1 ml/kg, i.p.). 18 h later, both groups received TA (300 mg/kg, i.p.). Plasma ALT was measured at 0, 12, and 24 h after TA administration (n = 3). *, significantly different from the respective 0-time control; #, significantly different from the control group at the same time point. P  .05.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effect of INH on the hepatic CYP2E1 expression and enzyme activity. Experimental detail is as in Fig. 2. Liver samples were collected before TA administration (n = 3). A, microsomal CYP2E1 protein was detected by Western blot and quantitated by densitometry. The mean value obtained from the control was taken to be 100%, and the value obtained from the INH-treated group was expressed as a percentage of control. A representative CYP2E1 blot is shown in the upper left corner. B, CYP2E1 activity was measured by PNP hydroxylase activity. *, significantly different from the control group. P  .05.

CYP2E1 Inhibition Study. If CYP2E1 is involved in the bioactivation of TA, inhibition of CYP2E1 should lead to decreased liver injury by TA. As shown in Fig. 4, pretreatment with the inhibitor of CYP2E1, DAS, decreased liver injury in both nondiabetic and diabetic rats. In nondiabetic rats, DAS pretreatment decreased plasma ALT activity approximately 60% at 12 and 24 h after TA administration. In diabetic rats, pretreatment with DAS yielded a maximum of 75% inhibition in TA-induced liver injury. DAS pretreatment reduced microsomal PNP hydroxylation (Fig. 5) by 90% in nondiabetic rats and suppressed the induced CYP2E1 by 75% in diabetic rats.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effect of DAS on TA-induced liver injury. Two groups of nondiabetic and two groups of diabetic rats were treated 12 h apart intragastrically either with two doses of DAS (200 mg/4 ml of corn oil/kg, gavage) or corn oil (4 ml/kg, gavage). TA (300 mg/kg, i.p.) was administered 4 h after the second dose. At 0, 12, and 24 h after TA administration plasma ALT was measured (n = 3). *, significantly different from the respective 0-time control; !, significantly different from the nondiabetic group without DAS pretreatment at the same time point. #, significantly different from the diabetic group without DAS pretreatment at the same time point. P  .05.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of DAS on hepatic CYP2E1 activity. Experimental detail is as in Fig. 4. Liver samples were collected before TA administration (n = 3). Microsomal CYP2E1 activity was measured by PNP hydroxylase activity. *, significantly different from rats without DAS pretreatment. P  .05.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Effect of DAS on CCl4-induced liver injury. Normal rats were challenged 12 h apart intragastrically with two doses of DAS (200 mg/kg in 4 ml of corn oil/kg, gavage) or corn oil (4 ml/kg, gavage). CCl4 (0.1 ml/kg, i.p. in 1 ml of corn oil/kg, gavage) was given 4 h after the second dose. At 0, 12, and 24 h after CCl4 administration, plasma ALT was measured (n = 3). *, significantly different from the respective 0-time control; #, significantly different from the control group at the same time point. P  .05.

FMO Inhibition Study. If FMO1 is involved in the bioactivation of TA, in vivo inhibition of FMO1 should result in decreased TA-induced liver injury. Hepatic FMO1 protein expression (Fig. 7) and activity (Fig. 8) were induced 2.5- and 1.8-fold in the diabetic rat compared with the nondiabetic rat. Dietary administration of I3C for 10 days decreased both FMO1 protein level and activity (Figs. 7 and 8). In nondiabetic rats, hepatic FMO1 expression was reduced by 37% (Fig. 7) and FMO1 activity was inhibited by 32% (Fig. 8). In diabetic rats, hepatic FMO1 protein was reduced by 67% (Fig. 7) and FMO1 activity was inhibited by 55% (Fig. 8). However, TA-induced liver injury was substantially increased in I3C-exposed rats (Fig. 9). In nondiabetic rats exposed to I3C, the plasma ALT activity increased to 220 and 190% of the nondiabetic + TA groups at 12 and 24 h after TA administration, respectively. In diabetic rats fed with I3C diet, there were increases of plasma ALT levels to 220 and 170% of values obtained for the diabetic + TA groups observed at 12 and 24 h after TA treatment, respectively, suggesting that FMO1 is unlikely to mediate TA-associated liver injury.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Hepatic FMO1 expression. Nondiabetic rats were given either regular chow or chow containing 0.25% I3C for 10 days. For diabetic rats, on day 0, rats were treated with STZ (60 mg/kg. i.p.), one group maintained on the I3C diet, the other group remained on regular chow. On day 10, liver samples were collected under diethyl ether anesthesia and used to prepare for microsomes (n = 3). Hepatic FMO1 protein was detected by Western blot and quantitated by densitometry. The mean value obtained from the nondiabetic control was taken to be 100%, and values obtained from all other experimental groups were expressed as a percentage of control. A representative FMO1 blot is shown in the upper right corner. *, significantly different from the nondiabetic group; #, significantly different from the diabetic group without I3C exposure; !, significantly different from the nondiabetic group without I3C exposure. P  .05.

View larger version (12K): [in this window] [in a new window]   Fig. 8.   Hepatic FMO1 activity. Experimental detail is as in Fig. 7. Microsomal FMO1 activity was measured by methimazole-dependent oxidation of thiocholine. *, significantly different from the nondiabetic group; #, significantly different from the diabetic group without I3C exposure; !, significantly different from the nondiabetic group without I3C exposure. P  .05.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Effect of I3C on TA-induced liver injury. Nondiabetic rats were given regular chow or chow containing 0.25% I3C for 10 days. For diabetic rats, on day 0, rats were treated with STZ (60 mg/kg. i.p.), one group maintained on the I3C diet, the other group remained on regular diet. On day 10, all the rats were treated with TA (300 mg/kg, i.p.). At 0, 12, and 24 h after TA administration, blood samples were collected to measure plasma ALT (n = 3). *, significantly different from the respective 0-time control; !, significantly different from the nondiabetic group without I3C pretreatment; #, significantly different from the diabetic group without I3C pretreatment. P  .05.

	Discussion

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TA hepatotoxicity is potentiated in STZ-induced diabetic rats (El-Hawari and Plaa, 1983; Wang et al., 2000). This study was undertaken to investigate the underlying mechanisms. These studies suggest that diabetes-induced CYP2E1 is primarily responsible for the potentiated TA hepatotoxicity in the diabetic rat. Furthermore, FMO1 is unlikely to mediate TA-induced liver injury.

We used in vivo enzyme induction and inhibition approaches to characterize the roles of CYP2E1 and FMO1 in TA-induced liver injury. Pretreatment with INH (250 mg/kg, i.p.) increased CYP2E1 protein concentration and activity 2.2- and 2.5-fold, respectively. Attendant with this induction of CYP2E1 was an approximate 2.5-fold increase in TA liver injury. When CYP2E1 is induced by diabetic state as well as by prior exposure of normal rats to INH, potentiated TA liver injury was observed, suggesting the involvement of this enzyme in the bioactivation of TA. To further confirm the role of hepatic CYP2E1 in TA-associated liver injury, CYP2E1 was inhibited by DAS (Chen et al., 1994) before TA administration. In in vivo experiments with nondiabetic and diabetic rats, DAS pretreatment inhibited TA liver injury by 60% in nondiabetic and by 75% in diabetic rats. Hepatic microsomal PNP hydroxylation and in vivo CCl₄ hepatotoxicity studies confirmed that CYP2E1 activity was greatly inhibited by DAS pretreatment. Therefore, CYP2E1 inhibition studies lend further support to the participation of CYP2E1 in bioactivation of TA. Data presented in Fig. 10 were derived from a number of widely different experiments of this study in which hepatic CYP2E1 levels and activities were manipulated. The high degree of correlation between CYP2E1 levels (Fig. 10A) and activity (Fig. 10B) with TA liver injury offers very strong support for a predominant role of CYP2E1 in TA hepatotoxicity.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   A, correlation (r = 0.959, p < .001) between relative CYP2E1 protein expression and plasma ALT activity; B, correlation (r = 0.999, P < .001) between PNP hydroxylation and plasma ALT activity in rats with different treatments. The solid line represents the line of best fit. Values of hepatic CYP2E1 expression and activity (PNP hydroxylation) taken from various experiments are plotted against the corresponding TA liver injury (plasma ALT). Nondiabetic (NonDB); diabetic (DB); normal rats with INH (INH) or saline pretreatment (saline); nondiabetic and diabetic rats with DAS pretreatment (NonDB + DAS; DB + DAS).

Supporting evidence exists in the literature for a cytochrome P450-mediated TA bioactivation. Neal and his coworkers (Hunter et al., 1977; Porter and Neal, 1978) showed that S-oxidation of TA was inhibited by cytochrome P450 inhibitors, like carbon monoxide, SKF-525A, and an antibody to rat cytochrome P450. Furthermore, Chieli et al. (1990) have shown that CYP2E1 mediates biotransformation of thiobenzamide, which is a structural analog of TA and shares the same metabolic pathway with TA (Tynes and Hodgson, 1983). In contrast, Castro et al. (1974) reported pretreatment of rats with the cytochrome P450 inhibitors, SKF-525A and Sch 576, did not decrease TA hepatotoxicity. The apparently conflicting observations of Hunter et al. (1977) and Castro et al. (1974) probably arose from the different dosing regimens of TA and inhibitors used by these investigators. Moreover, Castro et al. (1974) reported that TA hepatotoxicity was partially prevented by pretreatment with disulfiram, which has since been shown to be a potent inhibitor of CYP2E1 (Brady et al., 1991).

Diabetes up-regulates several other cytochrome P450 enzymes, and the possibility of their involvement in potentiating TA hepatotoxicity should be considered. For example, CYP2B1 is induced in diabetes (Yamazoe et al., 1989). However, Castro et al. (1974) and El-Hawari and Plaa (1983) did not observe any potentiation of TA toxicity after pretreatment with phenobarbital, ruling out the involvement of CYP2B1 in TA bioactivation. CYP1A is another cytochrome P450 subfamily induced in male diabetic rats (Yamazoe et al., 1989). However, pretreatment with 3-methylcholanthrene and 3,4-benzypyrene did not cause increased TA liver injury (El-Hawari and Plaa, 1983). CYP3A2 is not increased in male diabetic rats but increased in the female diabetic rats (Schenkman, 1991). These studies were conducted using male Sprague-Dawley rats. These considerations suggest that several major hepatic cytochrome P450 enzymes (CYP1A, CYP2B, and CYP3A) are unlikely to mediate TA-induced liver injury.

DAS pretreatment decreased 60% of TA liver injury in normal rats. The remnant liver injury might be due to remaining CYP2E1 activity or could be due to FMO1, because it has been shown that in vitro FMO catalyzes TA S-oxidation (Venkatesh et al., 1991). Western blot and methimazole-dependent oxidation of thiocholine studies showed that hepatic FMO1 expression and activity were induced 2.5- and 1.8-fold in STZ-induced diabetic rats. Dietary exposure to I3C for 10 days inhibited hepatic FMO1 protein concentration and activity in both nondiabetic and diabetic rats. However, TA-induced liver injury was not decreased, in fact it was substantially increased in these I3C-exposed rats. The enhanced TA hepatotoxicity following FMO1 inhibition suggests that FMO1 is unlikely to mediate TA bioactivation and liver injury in vivo.

The mechanisms of markedly increased TA liver injury during FMO1 inhibition by I3C are unclear. Four plausible mechanisms might be considered. These in the order of likelihood are: 1) The role of FMO1 is to detoxify TA in contrast to the proposed role in bioactivation. 2) By blocking FMO1 pathway, additional TA becomes available for bioactivation via CYP2E1. 3) I3C induces other cytochrome P450 enzymes. 4) I3C induces CYP2E1. First, in vivo FMO1 might catalyze conversion of TA to TA sulfoxide, but further metabolism by FMO1 might occur to detoxify TA to one or more nontoxic metabolites instead of forming TA sulfone. Alternatively, FMO1 may metabolize TA via mechanisms that do not involve the formation of reactive intermediates. Inhibition of FMO1 would mean that a detoxification pathway is blocked, thereby explaining the increased TA-induced liver injury. Chiba et al. (1988) have shown that FMO acts as a detoxifying enzymatic pathway to detoxify 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Therefore, a possible role of FMO1 in detoxication of TA in the liver and the ensuing metabolic pathway (Fig. 11) are of considerable interest. Second, blocking the FMO1 pathway of TA metabolism might result in higher concentration of TA for bioactivation via CYP2E1 pathway, leading to increased liver injury in rats treated with I3C. Our data are more consistent with a combination of the above two possibilities. A third possibility is that I3C induction of cytochrome P450 enzymes contributes to additional TA liver injury. It has been shown that I3C induces a number of different cytochrome P450 enzymes, including CYP1A, CYP2B, and CYP3A (Larsen-Su and Williams, 1996; Renwick et al., 1999) along with the inhibition of FMO1. However, neither pre-exposure to 3-methylcholanthrene nor to phenobarbital, which induce CYP1As (El-Hawari and Plaa, 1983) and CYP2B1 + CYP3A1 (Corcos, 1992), respectively, result in increased TA hepatotoxicity, indicating that CYP1As, CYP2B1, or CYP3A1 are not likely to mediate TA bioactivation. Therefore, induction of cytochrome P450s by I3C is unlikely to be responsible for the enhanced TA hepatotoxicity in I3C-treated rats. Regarding the fourth possibility, Jellinck et al. (1994) have shown that I3C does not induce CYP2E1. Therefore, induction of CYP2E1 by I3C is not a likely mechanism of substantially increased TA liver injury in I3C-treated rats. Collectively, the first two of the above four possibilities are the most likely.

View larger version (13K): [in this window] [in a new window]   Fig. 11.   Proposed role of CYP2E1 and FMO1 in TA-induced liver injury. Normally, hepatic microsomal CYP2E1 mediates TA bioactivation and liver injury. In the STZ-induced diabetic rats, CYP2E1 is induced 6- to 8-fold leading to higher liver injury of TA. FMO1 is likely to detoxify TA. FMO1 is induced 1.8-fold in the diabetic rats and may help to temper down the consequences of heightened bioactivation-mediated injury via the CYP2E1 pathway.

In conclusion, our studies suggest a major involvement of CYP2E1 in TA hepatotoxicity (Fig. 11). Diabetes-induced CYP2E1 appears to be predominantly responsible for the potentiated TA liver injury in diabetic rats. Although FMO1 is induced in diabetic rats, it is unlikely to be a contributor to the potentiated TA hepatotoxicity. These findings are more consistent with a role for FMO1 in TA detoxication in normal and diabetic rats (Fig. 11).