Introduction

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The cytochrome P450-dependent microsomal monooxygenase system consists of a flavoprotein, cytochrome P450 reductase, and a multigene superfamily of hemeproteins (the cytochrome P450s). This system plays a major role in the metabolism of a wide variety of drugs, xenobiotics as well as endogenous compounds such as fatty acids, vitamins, and steroids (Guengerich, 1995). Cytochrome P450s belonging to the CYP3A family account for 25 to 35% of the total cytochrome P450 present in adult human or rat liver (Guengerich, 1995) and represent the majority of P450s present in human small intestine (Watkins et al., 1987). CYP3A enzymes catalyze 6-hydroxylation of steroids such as cortisol, testosterone, and estradiol and are responsible for metabolism of numerous drugs, including psychotropic, cardiac, analgesic, hormonal, immunosuppressant, antineoplastic, and antihistaminic agents (Guengerich, 1995).

In the rat, four CYP3A cDNAs have been characterized: CYP3A1 (also referred to as CYP3A23), CYP3A2, CYP3A9, and CYP3A18 (Gonzalez et al., 1985, 1986; Kirita and Matsubara, 1993; Komori and Oda, 1994; Strotkamp et al., 1995; Wang et al., 1996; Mahnke et al., 1997; Robertson et al., 1998). The expression of CYP3A genes is age- and gender-dependent and is regulated by a number of agents such as phenobarbital, dexamethasone, and clotrimazole (Lehmann et al., 1998). CYP3A9 is the most recently cloned member of the family and its mRNA is detected only in adult rats, with a higher expression in females (Mahnke et al., 1997). The regulation of CYP3A9 appears different from the other CYP3A isoenzymes because phenobarbital is a more effective inducer than dexamethasone or clotrimazole, which are strong inducers of the other CYP3A family members (Mahnke et al., 1997). Diet also affects the expression of CYP3A because there was a significantly higher induction by dexamethasone of CYP3A2 and CYP3A activity in rats fed soy protein isolate compared with casein-fed controls (Ronis et al., 1999). Moreover, there is a diet and alcohol suppression of CYP3A expression and activity in the rat small intestine (Hakkak et al., 1993).

We have previously reported that dietary carbohydrate to fat ratios (CHO/fat) significantly impact ethanol hepatic toxicity because focal necrosis was seen only in animals infused ethanol in a low-CHO diet compared with rats fed ethanol in a high-CHO diet (Korourian et al., 1999). In these studies we also reported that ethanol induced approximately a 4-fold greater increase in hepatic CYP2E1 apoprotein levels in the low-CHO-fed animals compared with high-CHO-fed animals, indicating the CHO/fat ratios impact CYP2E1 regulation. In the current study we examined the effects of CHO/fat and chronic ethanol on activities and expression of hepatic CYP3As in male rats and provide evidence that CYP3A9 has a similar substrate specificity to that of CYP2E1.

	Materials and Methods

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Chemicals and Reagents NADPH and testosterone were purchased from Sigma Chemical Co. (St. Louis, MO). 6-Hydroxytestosterone standard was purchased from Steraloids Inc. (Wilton, NH). [¹⁴C]Testosterone (50-60 mCi/mmol) was purchased from DuPont NEN (Boston MA). Rabbit polyclonal antibodies against rat CYP3A1 (CYP3A23) were the kind gift of Dr. Magnus Ingelman-Sundberg (Karolinska Institute, Stockholm, Sweden). ¹²⁵I-Goat anti-rabbit IgG and ¹²⁵I-goat anti-mouse IgG were purchased from ICN Biomedicals Inc. (Costa Mesa, CA). Linear-k high-performance normal phase silica thin layer chromatography plates were purchased from Whatman International Ltd. (Maidstone, Kent, England).

Animal Treatments. The experiments received prior approval from the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences. All animals were housed in an American Association for the Accreditation of Laboratory Animal Care-approved animal facility at Arkansas Children's Hospital Research Institute and all animal housing and husbandry conformed to United States Department of Agriculture guidelines. Virus-free adult male Sprague-Dawley rats (c. 320 g) were purchased from Harlan Industries (Indianapolis, IN). Rats were kept in a 12-h light cycle and constant humidity. Each rat was conditioned by handling for 7 days before surgical implantation of a single intragastric cannula and allowed to recover for 14 days before infusion with total enteral nutrition test diets (Korourian et al., 1999).

Experiments. For ease in presenting the data, high-CHO/low-fat diets are referred to as high-CHO and low-CHO/high-fat diets are referred to as low-CHO. Rats were randomly assigned (n = 4-6) to high-CHO (experiment 1) or low-CHO (experiment 2) diets with ethanol or without ethanol (control). Total enteral nutrition diets (Table 1) were formulated and infused as described previously (Korourian et al., 1999). Hepatic monooxygenase activities, cytochrome P450 enzyme apoprotein expression, and mRNA levels were examined after 55 days for experiment 1 (high-CHO) or 42 days for experiment 2 (low-CHO) of continuous diet infusion.

Ethanol. Ethanol was introduced at 8 to 10 g/kg/day on day 1, progressively increased to 13 g/kg/day (experiment 1) or 12.5 g/kg/day (experiment 2) over approximately 10 days, and remaining at that level thereafter. The ethanol dose was carefully titrated to produce only mild signs of intoxication at peak blood ethanol concentrations.

CYP3A Activity Assay. Testosterone 6-hydroxylase was assayed by incubating hepatic microsomal fractions with [¹⁴C]testosterone and the resulting metabolites separated by high-performance thin layer chromatography as described previously (Ronis et al., 1991). 6-Hydroxylated products were identified by comigration with pure standards and quantified by phosphorimaging using a Bio-Rad GS525 molecular imager (Richmond, CA).

Source and Properties of the Recombinant Purified CYP3A9. The source of recombinant P450 3A9 and a detailed description of its preparation and properties were presented previously (Wang and Strobel, 1997). Briefly, the enzyme was obtained by overexpression in Escherechia coli and purification. The enzyme was stored in 100 mM potassium phosphate buffer (pH 7.25) containing 20% glycerol. The P450 content was 16.4 nmol/ml and the specific content was 8.7 nmol/mg of protein.

Chlorzoxazone Hydroxylation Assay The chlorzoxazone hydroxylation assays were performed using the conditions described previously with a modification (Lucas et al., 1996; Wang and Strobel, 1997). Enzyme activity measurements were performed in a final volume of 0.5 ml with 100 mM potassium phosphate buffer (pH 7.25) containing 50 pmol of P450 3A9, saturating NADPH-cytochrome P450 reductase (0.5 U), 10 µl of lipid (1:1:1 mixture of L--dilauroyl phosphatidylcholine, phosphatidylserine, and dioleoyl phosphatidylcholine), 3.0 mM reduced glutathione, and 100 µg of sodium cholate (pH 7.25). The reaction mixtures were preincubated at 37°C for 3 min and the reaction was initiated by addition of 0.5 mM NADPH. NADPH-cytochrome P450 reductase was purified from rat liver microsomes in our laboratory. Cytochrome b₅ was not added because it was shown to be unnecessary to achieve maximal catalytic activity. Chlorzoxazone 6-hydroxylation was performed using the reconstitution conditions described above with a substrate concentration of 400 µM. The reactions were terminated by the addition of 25 µl of 43% H₃PO₄. The reaction mixture was extracted with 2.0 ml of chloroform/isopropanol (85:15, v/v) in Teflon-capped tubes by vortexing for 20 min and centrifuged at 3000g for 10 min. After removing the aqueous phase, the organic phase was dried under the constant flow of N₂ at 37°C and reconstituted in 200 µl of mobile phase. The products of chlorzoxazone hydroxylation were separated by HPLC using the Nucleosil CIS column and HPLC system used for steroid hydroxylations. Isocratic mobile phase consisting of 0.5% (v/v) acetic acid in water/acetonitrile (70:30, v/v) was used to elute the substrate and product. UV detection was performed at 287 nm.

Western Immunoblot Analysis. Western blotting was conducted on liver microsomes (2.5 µg of protein/well) or recombinant purified CYP3A9 (1 and 10 pmol of protein/well) using a rabbit anti-rat polyclonal antibody directed against CYP3A1 (CYP3A23) at a dilution of 1:1000 as described previously (Ronis et al., 1994). Immunoquantitation was obtained by densitometric scanning of the resulting autoradiographs using a Bio-Rad GS525 molecular imager.

Northern Analysis of CYP3A mRNA Levels CYP3A1, CYP3A2, and CYP3A18 antisense oligonucleotides and CYP3A9 cDNA were used to measure mRNA from total liver RNA as previously described (Ronis et al., 1999). Bands were quantitated by densitometry of the autoradiograph or ethidium bromide-stained gel image (18S rRNA) and the ratio of CYP3A message to 18S rRNA in the same sample was determined and expressed as relative RNA units or as percentage of that for the control.

Statistical Analysis. The high-CHO (experiment 1) and low-CHO (experiment 2) were run at different times, thus the studies were not compared with each other and only the effects of ethanol within the same diet study were determined. Data were analyzed using Student's t test with P < .05 considered statistically significant. Data are presented as mean ± S.E.

	Results

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Effect of Ethanol and CHO/Fat on CYP3A Levels and Activities. The mean CYP3A-dependent testosterone 6-hydroxylase activities in hepatic microsomes from rats fed either high- or low-CHO plus ethanol were 77% lower than that in microsomes from high- or low-CHO-fed control rats (P < .05), respectively (Table 2). This ethanol-dependent decrease in testosterone 6-hydroxylase activity was mirrored by similar decreases in CYP3A apoprotein expression of 62 or 74% (P < .05) in rats fed high- or low-CHO diets plus ethanol, respectively (Table 2; Fig. 1).

View this table: [in this window] [in a new window]   TABLE 2 CYP3A-dependent testosterone 6-hydroxylase activity and CYP3A apoprotein level expression from male rats fed low- or high-CHO diets plus or minus ethanol Data are presented as mean ± S.E. (n = 4-6).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Autoradiographs of Western immunoblots of hepatic CYP3A apoprotein expression from male rats fed low- or high-CHO diets plus or minus ethanol. Westerns were performed as described under Materials and Methods.

Effect of Ethanol and CHO/Fat on CYP3A1, CYP3A2, CYP3A9, and CYP3A18 mRNA Steady-State Levels. To further study the mechanism of ethanol-induced inhibition of CYP3A, we used specific antisense oligonucleotides against CYP3A1, CYP3A2, and CYP3A18 and a CYP3A9 cDNA to measure CYP3A mRNA's steady-state levels using Northern blots (Fig. 2; Table 3). There were detectable mRNAs for all four of the hepatic CYP3A genes with no differences in mRNA steady-state levels expressed for CYP3A18 in rats fed ethanol compared with control rats. In contrast, CYP3A2 mRNA levels decreased (P < .05) by 73 and 85% in the rats fed low- or high-CHO plus ethanol versus control rats, respectively. Surprisingly, in the livers of these same rats, ethanol in the low- or high-CHO diets induced a 2.6- and 11.3-fold (P < .05) increase in CYP3A9 mRNA levels, respectively. There was a small ethanol-induced increase (1.6-fold; P < .05) in CYP3A1 mRNA in the high-CHO-fed rats with no effect in the low-CHO-fed rats (Fig. 2; Table 3).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Autoradiographs of Northern blots of hepatic CYP3A1, CYP3A2, CYP3A9, and CYP3A18 steady-state mRNA levels from male rats fed low- or high-CHO diets plus or minus ethanol. Northerns were performed as described under Materials and Methods.

Chlorzoxazone Metabolism by CYP3A9. The ethanol induction of CYP3A9 resembles that of CYP2E1, the major ethanol-inducible P450. Chlorzoxazone 6-hydroxylation has been used both in vivo and in vitro as a probe for the activities of CYP2E1 (Lucas et al., 1996). Therefore, chlorzoxazone was chosen as a substrate to determine whether CYP3A9 has a similar chlorzoxazone 6-hydroxylase activity. The time course of recombinant purified CYP3A9-catalyzed chlorzoxazone turnover is shown in Fig. 3. As can be seen, CYP3A9 catalyzed the transformation of chlorzoxazone to 6-hydroxychlorzoxazone as a single product. Lineweaver-Burk plot of the substrate saturation curve (data not shown) revealed a K_m value of 310 µM and a V_max value of 1.3 nmol/min/nmol of P450.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Time course of 6-hydroxychlorzoxazone formation by P450 3A9. The reconstitution assays and HPLC analysis of the product were performed as described under Materials and Methods using a chlorzoxazone concentration of 400 µM.

	Discussion

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Demasculinization of hepatic cytochrome P450-dependent testosterone metabolism occurred in male rats infused with ethanol-containing diets, irrespective of the CHO/fat ratio. In these rats, decreases in CYP3A2-dependent, male-specific testosterone 6-hydroxylase activity and CYP3A apoprotein levels were measured and are most likely due to the ethanol-induced decrease in CYP3A2 mRNA levels. In contrast, ethanol in both the low- or high-CHO diets induced a 2.6- or 11.3-fold increase in CYP3A9 mRNA levels, respectively.

The CYP3A antibody used cross-reacted with the recombinant CYP3A9 apoprotein (data not shown). However, the increase in CYP3A9 mRNA did not lead to increases in CYP3A9 apoprotein levels sufficient to compensate for the ethanol-induced decrease in CYP3A2 apoprotein because there was an overall decrease in the total immunoreactive CYP3A measured in the Western blot (Fig. 1). In addition, because recombinant CYP3A9 does not have testosterone 6-hydroxylase activity (Jager et al., 1999) any increased protein expression would not be reflected in the activity assay used here.

The effects of low- and high-CHO diets were studied in two separate experiments lasting 42 and 55 days, respectively. Because they were conducted at different times and there was a difference in the duration of these experiments it would be statistically inappropriate to compare the two. However, ethanol differentially affected the mRNA expression of CYP3A gene family members in these two experiments and it is tempting to speculate that this effect is modulated by dietary CHO/fat ratios. For CYP3A2, ethanol inhibited its expression and this was independent of the CHO/fat ratios, whereas significant ethanol induction of CYP3A1 and CYP3A9 steady-state mRNA levels was measured only in rats fed high-CHO diets. Alternatively, the lack of ethanol-induced CYP3A1/CYP3A9 mRNA expression in the low-CHO-fed rats may be due to signal transduction and gene expression changes that lead to the greater hepatic total pathology score measured in the these animals, compared with the high-CHO ethanol group (Korourian et al., 1999). We are currently investigating the impact of changes in the CHO/fat ratio on ethanol-regulated genes.

It is also possible that the longer ethanol exposure time in the high-CHO experiment might be the underlying cause for the increases in CYP3A1 and CYP3A9 mRNA expression measured. Analysis of reports of ethanol regulation of CYP3A indicates a relationship between CYP3A expression and the duration of alcohol exposure (Table 4). Using cultured human and rat hepatocytes or rat hepatoma cells, short-term ethanol treatment increased CYP3A (Sinclair et al., 1991; de Waziers et al., 1992; Kostrubsky et al., 1995). Similarly, in rats exposed to ethanol in their diets for 7 to 14 days, there was a significant increase in immunoreactive CYP3A protein (Ronis et al., 1991; Roberts et al., 1995; Kostrubsky et al., 1997) and CYP3A2 mRNA steady-state levels (Louis et al., 1994). In contrast, in rats chronically fed ethanol diets for 38 days, there was a significant decrease in CYP3A2-dependent hepatic testosterone 6-hydroxylase activity (Badger et al., 1993) that is similar to the results presented here where the rats were fed ethanol diets for 42 to 55 days. Taken together, these results show that ethanol is a CYP3A inducer in cells and rats fed ethanol for short periods of time, but in chronically fed rats that develop liver pathology, the end result of ethanol feeding is a remarkable increase in CYP3A9 with an inverse decrease in CYP3A2 activity, protein levels, and mRNA expression. These opposing ethanol effects on CYP3A expression indicate that there are at least two different mechanisms of ethanol action. One mechanism is a primary effect of acute ethanol exposure that leads to CYP3A2 induction. However, chronic ethanol exposure causes CYP3A2 inhibition and CYP3A9 induction and this mechanism is most likely a secondary effect of ethanol related to the growth hormone (GH) secretion changes associated with chronic ethanol exposure.

Expression of CYP3A enzymes is regulated by numerous endocrine systems, including growth hormone, insulin, and gonadal steroids (Waxman et al., 1990; Hussain et al., 1995; Wang and Strobel, 1997; Woodcroft and Novak, 1997; Robertson et al., 1998). Gender-specific expression of CYP3As has been reported with a male-predominant expression for CYP3A2 and a female predominance for CYP3A9 (Wang and Strobel, 1997; Mahnke et al., 1997). Expression of gender-dependent rat hepatic cytochrome P450s is regulated mainly by the gender-specific pattern of GH secretion and is subject to androgen imprinting (Waxman and Chang, 1995). CYP3A2 is suppressed in adult female rats by their continuous pituitary GH secretion profile, but its expression occurs with the male pattern of GH secretion (Waxman et al., 1990). In addition, exposure of male rats to the female pattern of GH secretion increased hepatic CYP3A9 mRNA (Robertson et al., 1998). Chronic ethanol exposure to male rats resulted in a demasculinization of the GH pulse pattern with a corresponding decrease in the male-specific expression of CYP2C11 (Badger et al., 1993). Similarly, we observed CYP2C11 decreases in rats from the present study (Badger et al., 1998), indicating a demasculinization. Collectively, these results support the hypothesis that chronic ethanol treatment causes a demasculinization of the male rats with a shift from the male-predominant expression of CYP3A2 to the female-predominant CYP3A9.

The biological significance of these differential CYP3A effects and their relationship to alcohol-induced liver injury are not clear. Ethanol was metabolized by heterologous expression of human CYP3A4 in HepG2 cells and ethanol metabolism was measured in human liver microsomes and this CYP3A-dependent ethanol metabolism was significantly reduced by a specific CYP3A4 inhibitor (Salmela et al., 1998). Additionally, in human liver microsomes and in dexamethasone-treated rats, specific CYP3A-dependent hepatic p-nitrophenol catalytic activities were reported (Zerilli et al., 1997). Other cytochrome P450s such as CYP2E1 have both ethanol oxidation and p-nitrophenol hydroxylase activities (Ingelman-Sundberg and Johansson, 1984; Koop, 1986). The 6-hydroxylation of chlorzoxazone was also reported to be catalyzed primarily by CYP2E1 in human liver microsomes and had been used both in vivo and in vitro as a specific probe for CYP2E1 activity (Lucas et al., 1996; Amato et al., 1998). Our data show that CYP3A9 is active in the 6-hydroxylation of chlorzoxazone with a turnover number 1.3 nmol/min/nmol of P450. This was consistent with the recent results from the study using human liver microsomes, which showed that rabbit anti-human CYP3A antibodies reduce the formation of 6-hydroxychlorzoxazone formation by 47% (Gorski et al., 1997). Similar results were obtained using the recombinant P450 proteins: recombinant CYP2E1 and CYP3A4 showed comparable chlorzoxazone 6-hydroxylase activities (Gorski et al., 1997). Data presented in this article support the role of CYP3A enzymes in the metabolism of chlorzoxazone. However, the affinity of CYP3A9 for chlorzoxazone is much lower than that for CYP2E1 with K_m values of 310 and 39 µM, respectively (Lucas et al., 1996). Thus, it would be expected that in vivo, CYP3A9-dependent chlorzoxazone hydroxylase would contribute only a minor portion of the total hydroxylase activity with the major activity derived from CYP2E1. It remains to be determined whether CYP3A9 metabolizes ethanol or other CYP2E1 substrates such as lipids and whether there is a potential role for CYP3A9 in the microsomal ethanol-oxidizing system (Lieber, 1999). Other CYP3A9 substrates include imipramine, erythromycin, benzphetamine, ethylmorphine, and 17-estradiol (Wang and Strobel, 1997).

The results from this study demonstrate that chronic ethanol treatment of male rats causes a significant demasculinization of the rats exhibited by a change in the expression of hepatic CYP3A from male-predominant CYP3A2 to the female predominant CYP3A9. In addition, these results show that ethanol regulation of the CYP3A2 is complex, involving an initial inducing effect as reported by others and resulting in inhibition after prolonged ethanol exposure. Thus, the effects of alcohol on drug metabolism may differ in acute and chronic ethanol exposure. In addition, diet may also be an important factor in the regulation of the CYP3A family mRNA expression. These differences need to be taken into account when attempting to determine the ethanol effect on P450-dependent drug metabolism, especially because studies have reported that alcohol affects drug-metabolizing systems with acceleration of blood clearance rates of numerous substances, including meprobamate, pentobarbital, warfarin, diphenylhydantoin, tolbutamide, propranolol, and rifampicin (Lieber and DeCarli, 1989).