ToxSci Advance Access originally published online on March 31, 2004
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Toxicological Sciences 79, 233-241 (2004)
Toxicological Sciences vol. 79 no. 2 © Society of Toxicology; all rights reserved.
Regulation of CYP2E1 by Ethanol and Palmitic Acid and CYP4A11 by Clofibrate in Primary Cultures of Human Hepatocytes
* California Toxicology Research Institute, 1989 Palomar Oaks Way, Suite B, Carlsbad, California 92009, and Institute for Biomedical Research, Hackensack University Medical Center, Hackensack, New Jersey 07601
Received January 12, 2004; accepted March 1, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CYP2E1 and CYP4A11 are cytochrome P450 enzymes that are regulated by physiological conditions including diabetes and fasting. In addition, the xenochemical clofibrate has been reported to induce both rodent CYP2E1 and CYP4A. These findings suggest similar modes of regulation. Also in common to both enzymes is the ability to metabolize fatty acids such as laurate and arachidonic acid. Here, we used primary cultures of human hepatocytes to determine if certain xenochemicals could regulate CYP2E1 and CYP4A11. Ethanol significantly (p < 0.05) increased expression of CYP2E1 mRNA by 216 ± 32% of control, but did not alter CYP4A11 mRNA accumulation (145 ± 22% of control). In contrast, hepatocytes exposed to ethanol exhibited only a slight elevation in CYP2E1 protein (122 ± 13% of control) and a negligible effect on CYP4A11 protein. Clofibrate significantly (p < 0.05) enhanced both CYP4A11 mRNA and protein by 239 ± 30% and 154 ± 10% of control, respectively, but did not increase CYP2E1. Because rodent CYP4A is reportedly regulated by fatty acids through peroxisome proliferator activated receptor
(PPAR) and CYP2E1 is induced by high fat diets, we examined the effects of a medium chain fatty acid, palmitate on CYP2E1 mRNA content. Palmitic acid significantly (p < 0.05) increased CYP2E1 mRNA to 326 ± 57% of control. Collectively, results presented here identify agents that enhance CYP2E1 and CYP4A11 at the transcription level and suggest that fatty acids may represent a similar mode of regulation for these P450 enzymes. The lack of induction of CYP2E1 protein by ethanol in human hepatocytes indicates that for certain P450 enzymes, isolated hepatocytes may not be an adequate tool for predicting in vivo responses. Key Words: human hepatocytes; CYP4A11; CYP2E1; ethanol; palmitate; clofibrate.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytochrome P450 2E1 and P450 4A11 are constitutively expressed in human liver and kidney and both enzymes are involved in highly conserved catalytic functions, including the oxidation of fatty acids. Among the fatty acids metabolized by these P450 enzymes are arachidonic acid (Capdevila et al., 1992
; Laethem et al., 1993) and medium chain fatty acids such as laurate (Amet et al., 1994; Roman et al., 1993). An additional similarity between these P450 enzymes is that their expression is governed by physiological states. Common factors underlying the expression of CYP2E1 and CYP4A have been identified in rodents and include streptozotocin-induced diabetes (Funae et al., 1988; Imaoka et al., 1988) and fasting (Savas et al., 2003; Tu and Yang, 1983). CYP2E1 exhibits additional physiological functions including involvement in gluconeogenesis through its ability to metabolize acetone to acetol (reviewed in Lieber, 1999). Despite the role of CYP2E1 in fatty acid homeostasis and gluconeogenesis, this enzyme does not appear to be essential for normal mammalian development or physiology. The CYP2E1-null mouse does not exhibit any obvious phenotype or pathological abnormalities (Lee et al., 1996). CYP4A11 exhibits a more pronounced physiological role in homeostasis than does CYP2E1. Its role in the formation of arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE), implicates this enzyme as important in regulating systemic blood pressure, controlling cellular proliferation and inflammation, and in producing antipyretic effects (reviewed in Kroetz and Zeldin, 2002). Because of the physiological functions associated with EETs, DHETs, and 20-HETE generated by CYP4A11, alterations in expression can significantly affect the ability of this enzyme to control such functions.
Regulation of CYP2E1 and CYP4A can also occur by exposure to xenochemicals. Compounds known to induce CYP4A in rodents and rabbits include peroxisome proliferators, such as the hypolipidemic drug, clofibrate (Johnson et al., 1996). Conversely, CYP2E1 expression is regulated by agents such as ethanol (Kim et al., 1988; Lasker et al., 1987; Lieber and DeCarli, 1970), isoniazid, and pyridine (reviewed in Lieber, 1999 and Raucy et al., 1993). CYP4A11 is elevated by fatty acids (Tollet et al., 1994) and CYP2E1 by high fat diets (Lieber et al., 1988), suggesting that fatty acids are an additional mode of regulation that may be common to both enzymes. In animals, enhanced expression of CYP4As by fatty acids and peroxisome proliferators including the hypolipidemic drugs, involves the orphan nuclear receptor, PPAR. There is also evidence to suggest that human hepatic CYP4As, CYP4A11, and CYP4A22, are regulated by PPAR (Savas et al., 2003). PPAR is a ligand-activated transcription factor that binds to peroxisome proliferator-responsive elements (PPREs) as a heterodimer with the retinoid X receptor (RXR). PPREs have been identified in the promoter sequences of rabbit CYP4A6 (Muerhoff et al., 1992) and rat CYP4A1 (Bardot et al., 1993). In contrast, the regulation of human CYP4A genes is poorly understood because a PPRE has not been identified. The regulation of CYP2E1, at least in animals, does not appear to involve a nuclear receptor or transcriptional activation. Indeed, the primary mechanism governing altered expression of CYP2E1 by xenochemicals is protein stabilization (reviewed in Raucy et al., 1993). Moreover, studies to date have identified few transcriptional activators of the human enzyme.
While CYP4A11 primarily metabolizes endobiotics, CYP2E1 plays an important role in the metabolism of xenobiotics including alcohol. Additional pharmaceutical agents metabolized by CYP2E1 include the NSAID, acetaminophen (Raucy et al., 1989), the anesthetic, halothane (Gruenke et al., 1988); and the muscle relaxant, chlorzoxazone (Peter et al., 1990). Metabolic transformation of a majority of CYP2E1 substrates results in the production of toxic metabolites or oxygen radicals and underlies hepatotoxicity associated with such agents. For example, alcoholic liver disease (ALD) stems from CYP2E1-mediated ethanol metabolism and generation of free oxygen radicals (reviewed in Lieber, 1999). Progression of the disease is most likely due to an increase in the production of these radicals caused by ethanol-mediated induction of CYP2E1. Metabolism of arachidonic acid by CYP2E1 also generates oxygen radicals and enhanced levels of this P450, produced by exposure to sodium salicylate or acetylsalicylic acid, causes cellular toxicity in HepG2 cells engineered to express CYP2E1 (Wu and Cederbaum, 2001). In addition, acetaminophen mediated hepatotoxicity is more pronounced in individuals such as alcohol abusers that exhibit elevated CYP2E1 enzyme levels (Takahashi et al., 1993).
In the present study, we employed primary cultures of human hepatocytes from 23 individuals to identify xenochemicals that alter CYP2E1 and CYP4A11 mRNA and/or protein expression. We hypothesized that given the similarities in their regulation by physiological factors including fasting and diabetes, CYP2E1 and CYP4A11 would exhibit common mechanisms of regulation by xenobiotics and fatty acids. Xenobiotics that alter expression of both enzymes include ethanol and the fibrates. Indeed, Zangar et al. (1996) demonstrated that the peroxisomal proliferator, ciprofibrate, induced both CYP2E1 and CYP4A1 in primary cultures of rat hepatocytes. Furthermore, previous studies in isolated human fetal hepatocytes exhibited an elevation in CYP4A11 protein by ethanol (unpublished observations). Despite the large variability in response among the samples examined here, we found that ethanol and the fatty acid, palmitic acid, enhanced CYP2E1 at the level of transcription and that clofibrate increased CYP4A11 mRNA levels in human hepatocytes. That clofibrate induced CYP4A11 mRNA suggests that PPAR may be involved in the regulation of this P450. Because fatty acids are ligands for PPAR and have been shown to induce CYP4A1 in rat hepatocytes (Tollet et al., 1994), this suggests that fatty acids would also induce the human gene. Thus, fatty acids may be common regulators of CYP2E1 and CYP4A11.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Primary human hepatocyte cultures.
Human hepatocytes plated on rat-tail collagen coated-flasks (T-25) in serum-supplemented media were obtained from the Liver Tissue Procurement and Distribution System (LTPADS, University of Minnesota, Minneapolis, MN). Characteristics of the hepatocyte donors are given in Table 1. Upon arrival of plated hepatocytes, media was replaced with serum-free HMM (hepatocyte maintenance medium, Clonetics, San Diego, CA) containing 10–7 M dexamethasone plus 10–6 M insulin, and the cells were maintained in an atmosphere of 95% air and 5% CO2 at 37°C for 24 h as previously described (Raucy, 2003
; Raucy et al., 2002). Initial experiments were performed in medium lacking insulin. However, following the outcome of these experiments, all subsequent cultures contained 10–6 M insulin in the medium. After the 24 h equilibration period, hepatocytes were treated with 10 µM rifampicin (RIF), or 1 mM clofibrate for 48 h. This exposure time was selected based on previous studies with human hepatocytes and other inducers (Raucy, 2003; Raucy et al., 2002). These compounds were dissolved in dimethylsulfoxide (DMSO), which was added to the serum-free culture media at a final concentration of 0.1% (13.4 mM).
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Treatment of hepatocytes with 50 mM ethanol was performed by adding 31 µl of a 50% ethanol solution (v/v) in water to 5 ml of culture media. Control cells were left untreated. The plates were sealed with Teflon tape and placed in the incubator. Cells were treated for various periods of time including 3, 6, 12, 24, and 48 h. For hepatocytes exposed to ethanol for 48 h, the media containing 50 mM ethanol was replaced at 24 h. The fatty acid, palmitate, was prepared in a bovine serum albumin (BSA) solution by dissolving 500 mg BSA in 25 ml of 10 mM potassium phosphate buffer (KPO4), pH 7.4 (2% solution). To 10 ml of the 2% BSA, 600 µl of 15 mM palmitic (6 mg of fatty acid in 1 ml isopropanol) was added slowly dropwise while mixing. Once the fatty acid was added, the solution was stirred for an additional 30 min at room temperature and then aliquoted into microfuge tubes. The tubes were speed vacuum-dried for 6 h to produce a powdery material. This material was then resolubilized by adding 1 ml of HMM media and vortexed to produce a 900 µM solution of palmitate. One ml of redissolved fatty acid was transferred to an additional 3.5 ml of culture medium and then added directly to the hepatocyte cultures for a final concentration of 200 µM of palmitic acid. Fatty acid treatment of cells was for 48 h, based on previous observations with other compounds, and the media was removed daily and replaced with fresh media containing either palmitate dissolved in BSA or BSA alone. After drug, fatty acid, or ethanol treatment, hepatocytes were harvested and homogenates and/or total RNA were prepared (Carpenter et al., 1996
; Raucy, 2003; Raucy et al., 1997, 2002).
RNA isolation and Northern blot analysis.
Hepatocyte RNA was prepared using RNeasy kits, and quantitated by the absorbance at 260 nm; purity was assessed from the 260/280 nm absorbance ratio and by integrity of the 28s and 18s bands on agarose gels. Total RNA (10 µg) was subjected to electrophoresis on 1% agarose-2.2 M formaldehyde gels, followed by transfer to nylon membranes. RNA was bound to the membranes using a Stratalinker UV crosslinker (Stratagene, La Jolla, CA), after which the membranes were hybridized with random-primed 32P-labelled cDNA probes encoding human CYP2E1 or CYP4A11 and probed a second time with 18s rRNA. The CYP2E1 probe (297 bp) complemented the region 501 bp to 799 bp and the CYP4A11 probe (129 bp) spanned the region between 1520 to 1649 bp. Hybridization conditions have been described elsewhere (Raucy, 2003; Raucy, et al., 2002). DNA-RNA hybridization signals were measured on autoradiograms with a ScanMaker II flat bed scanner (Microtek, Redondo Beach, CA), and the signal intensities digitized using Un-Scan-It software (Silk Scientific, Orem, Utah). Hybridization signals obtained with a human 18s rRNA probe were used to normalize the amounts of RNA loaded onto the gels.
Protein blot analysis.
Hepatocytes were harvested after chemical exposure by scraping cells in ice-cold 2 ml of homogenization buffer (0.1 M Tris, 0.1 M KCl, 1 mM ethylenediaminetetraacetic acid [EDTA]) containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The cells were pelleted by centrifugation at 1000 rpm for 5 min, the supernatants discarded and pellets overlaid with 0.2 ml of homogenization buffer containing 0.5 mM PMSF and frozen at –80°C until use. Prior to immunoblot analysis, cells were thawed and sonicated (3 x 15 s bursts; 30 s cooling on ice between bursts). The cell lysates were transferred to 1.5 ml Eppendorf tubes and microfuged (12,000 rpm) for 15 min in the cold room. The supernatants were aspirated and protein concentrations determined with the bicinchoninic acid (BCA) procedure using BSA as the standard. Western blotting of hepatocyte lysate proteins (20 µg) to nitrocellulose, and subsequent immunochemical staining with 200 µg of anti CYP2E1, CYP4A11, or CYP3A4 IgG was performed as described elsewhere (Carpenter et al., 1996; Raucy et al., 1989). The properties of the polyclonal antibodies to CYP2E1, CYP4A11, and CYP3A4 used for these studies have been reported earlier (Jin et al., 1998; Raucy et al., 1989, 2002). Hepatocyte CYP2E1, CYP3A4, and CYP4A11 enzyme levels were quantified by scanning the blots with the ScanMaker II scanner, and then integrating immunostaining intensities with Un-Scan-It software.
Ethanol content in tissue culture plates.
To determine ethanol evaporation in hepatocyte cultures, T-25 culture plates (without hepatocytes) were filled with 2.5 ml of HMM containing DEX, insulin, and 25 or 50 mM ethanol (added as a 50% solution). After removing a small aliquot of medium, the plates were sealed with Teflon tape, and incubated at 37°C in an atmosphere containing 5% carbon dioxide. Additional aliquots of media were removed after 12 and 24 h of incubation. Ethanol content in the medium aliquots was then assessed using a commercial diagnostic kit (Sigma Chemical Co., St. Louis, MO).
Data analysis.
Results are presented as the mean ± standard deviation for three or more samples. Statistical significance was determined by the Student's paired t-test.
Materials.
Human 18s rRNA was from Ambion (Austin, TX). RIF, palmitic acid, BSA, and clofibrate were from Sigma Chemical (St. Louis, MO). The bicinchoninic acid was from Pierce Chemical Co. (Rockford, IL). Nitrocellulose and SDS-PAGE reagents were from BioRad Laboratories (Richmond, CA). RNeasy kits were obtained from Qiagen (Valencia, CA) and nylon membranes were purchased from Molecular Simulations, Inc. (Westboro, MA). All other reagents used have been described elsewhere or were of the highest quality available.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Primary cultures of human hepatocytes were obtained from 23 subjects, three of whom were female, ranging in age from 2 to 78 years (Table 1). Hepatocytes were used to assess the inductive or repressive effect of clofibrate, rifampicin, ethanol and the fatty acid, palmitate, on human CYP2E1 and CYP4A11. In certain hepatocyte samples, CYP3A4 content was measured for comparison. In initial experiments, the stability of CYP2E1 and CYP4A11 in culture under a defined medium (HMM, 10–6 M insulin and 10–7 M DEX) were determined in hepatocytes obtained from subjects HH988, HH984, and HH993. Both enzymes were stable under the culture conditions employed here for at least the first 48 h in culture. At this time, there was 97 ± 18% and 92 ± 19% of the CYP2E1 and CYP4A11, respectively, content found in zero-time cells (data not shown). At 72 h in culture, protein levels of CYP2E1 and CYP4A11 were 76 ± 36% and 74 ± 26% of those at zero time, respectively. However, at 96 h in culture CYP2E1 and CYP4A11 enzyme levels were 56 ± 40% and 46 ± 17%, respectively, of those at zero time. The stability of CYP3A4 in cultured cells was described elsewhere (Raucy et al., 2002
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Hepatocytes were initially cultured in an insulin free medium because of a previous report that suggested that insulin down-regulated CYP2E1 expression in rat hepatocytes (Woodcroft and Novak, 1999). Thus, we determined if a similar effect occurred in human hepatocytes. The addition of 10–6 M insulin to cultures from subjects HH943, HH832, and HH833 did not significantly alter constitutive levels of CYP2E1 (Table 2). Furthermore, induction of CYP2E1 by ethanol was unaffected by insulin in the media. All subsequent experiments were performed in cells cultured in medium containing insulin. In addition, it should be noted that cell quantity and availability governed the selection of hepatocytes used for the following induction studies.
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To assess the ability of ethanol to increase expression of CYP2E1 or CYP4A11 in primary cultures of human hepatocytes, we initially determined the solubility and volatility of ethanol in the culture media. T-25 culture plates without hepatocytes containing 5 ml of HMM medium were exposed to either 25 or 50 mM ethanol. At various time periods including 0, 12, and 24 h, aliquots of media were removed and ethanol concentrations determined. At both concentrations, ethanol content changed little in the culture medium over 24 h. Moreover, in the presence of hepatocytes (HH930), medium ethanol concentrations decreased only slightly (34%) over the 24 h incubation time (data not shown). In subsequent experiments where hepatocytes were exposed to ethanol for 48 h, we replaced medium at 24 h with fresh HMM containing ethanol. To determine the concentration of ethanol required to produce optimal induction of CYP2E1 protein, various concentrations of ethanol including 25, 50, and 75 mM were examined in hepatocytes from HH893 (data not shown). The greatest increase in CYP2E1 protein levels occurred at 50 mM and was 130% of control. This concentration was utilized in all subsequent experiments.
To evaluate the effect of ethanol exposure on transcriptional activation of CYP2E1 and CYP4A11, human hepatocytes from various subjects were exposed to 50 mM ethanol for selected time periods and Northern blot analyses performed on the isolated RNA. A representative Northern blot, used to determine mRNA levels in hepatocytes from subject HH966, is shown in Figure 1A. Hepatocytes from this subject were exposed to ethanol for 3, 6, 12, 24, and 48 h, RNA isolated, and subjected to Northern analysis. The blot, hybridized with CYP2E1 and CYP4A11 cDNA probes, was exposed to film and the developed film quantified (Fig. 1B). Northern blots that contained RNA from hepatocytes of additional subjects exposed to 50 mM ethanol for the same time periods were used to quantify mRNA levels of CYP2E1 and CYP4A11 (Fig. 2). Results are the mean of CYP2E1 mRNA assessed in six samples (HH955, HH966, HH973, HH1029, HH1034, and HH1040) and of CYP4A11 mRNA determined in the same six hepatocyte samples. Northern blot analysis revealed that at 48 h of ethanol exposure, the mean CYP2E1 mRNA levels were significantly elevated (p < 0.05) (216 ± 32%) above untreated cells while CYP4A11 mRNA content in these same hepatocytes was not increased (138 ± 32%; Fig. 2). At 3, 6, and 12 h, CYP4A11 mRNA was not significantly altered by ethanol treatment of hepatocytes (Fig. 2). The greatest increase in CYP4A11 mRNA occurred at 24 h (145 ± 22% above control), but this increase was not significantly different from control levels. At 3, 6, 12, and 24 h of exposure to ethanol, CYP2E1 mRNA content was elevated (128 ± 21%, 107 ± 9%, 141 ± 29%, and 164 ± 17% of control content, respectively), and was significantly higher (p < 0.05) than that in control cells at 24 h.
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Because ethanol-mediated induction of CYP2E1 is known to occur by protein stabilization, we used immunoblot analyses to assess protein levels. CYP4A11 protein content in hepatocytes treated with ethanol, RIF, and clofibrate was also examined. In addition, CYP3A4 levels were determined and included as a control for integrity and inducer responsiveness as this P450 enzyme consistently exhibits optimal enhanced expression in hepatocyte cultures upon exposure to 10 µM RIF (LeCluyse et al., 2000
; Luo et al., 2002; Raucy, 2003; Raucy et al., 2002). A representative immunoblot containing hepatocyte homogenates from subject HH994 was developed with antibodies to CYP2E1, CYP4A11, and CYP3A4 (Fig. 3A). Hepatocytes from this subject were treated for 48 h with 50 mM ethanol, 1 mM clofibrate, or 10 µM RIF prior to immunochemical analysis. The selected concentration of clofibrate was based on previous studies that demonstrate optimal induction at this dose of the fibrate (Ozaki et al., submitted manuscript). Hepatocytes from three additional subjects were also exposed to ethanol, clofibrate, and rifampicin and immunoblot analyses performed. Figure 3B represents the mean values obtained by scanning and digitizing immunoblots from all four subjects (HH994, HH998, HH1032, and HH1000). Following exposure to ethanol, CYP2E1 protein from these subjects was elevated to 122 ± 13% of that expressed in untreated cells and CYP4A11 protein was slightly repressed to 87 ± 35% of control in the same four subjects. Conversely, clofibrate treatment of hepatocytes produced significantly enhanced (p < 0.001) expression of CYP4A11 (154 ± 10% of control) in these same hepatocyte cultures (Fig. 3B). Clofibrate negligibly enhanced CYP2E1 protein levels in hepatocytes from subjects HH994, HH998, and HH1000 to 112 ± 2% of control. With regards to CYP3A4 expression, only RIF significantly elevated protein levels of this P450 in cultured hepatocytes (Figs. 3A and 3B). RIF treatment produced a decline in CYP2E1 protein to 56 ± 30% of control, albeit not significantly, in hepatocytes from HH994, HH1014, and HH1029 (Figs. 3A and 3B) and caused a dramatic decrease in CYP4A11 protein to 33 ± 15% (p < 0.02) of control in the same hepatocytes (Fig. 3B).
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Examination of CYP4A11 mRNA accumulation revealed a significant (p < 0.05) elevation to 239 ± 30% of control from subjects HH955, HH956, HH1029, HH1034, and HH1040 by clofibrate (Fig. 3C). However, this agent did not increase CYP2E1 mRNA levels (123 ± 20% of control) in these same hepatocyte samples. Conversely, ethanol significantly increased CYP2E1 mRNA accumulation but had no effect on CYP4A11 mRNA levels in hepatocytes from these same subjects (Fig. 3C). Rifampicin produced a dramatic decline in CYP2E1 mRNA levels (Fig. 3C). At rifampicin concentrations used to elevate CYP3A4 in cultured cells (10 µM), CYP2E1 mRNA was significantly decreased (p < 0.05) to 34 ± 6% of control levels. However, this antibiotic did not significantly decrease CYP4A11 mRNA content in the five hepatocyte samples examined here to the same extent (75 ± 12% of control).
Because high fat diets regulate CYP2E1 and fatty acids induce CYP4A, we wanted to determine if fatty acids could also enhance expression of CYP2E1 and thereby, identify a common regulator of CYP2E1 and CYP4A11. Thus, we examined the ability of the fatty acid, palmitate to transcriptionally regulate CYP2E1. In previous observations, we found that doses above 200 µM palmitate were toxic to cells and 50 and 100 µM enhanced CYP2E1 mRNA accumulation in a dose dependent fashion (data not shown). In hepatocytes from HH1092, HH1091, HH1087, palmitic acid (200 µM) exposure for 48 h significantly enhanced (p < 0.05) CYP2E1 mRNA levels to 326 ± 57% of control content (Fig. 4). This is the first report to demonstrate that palmitic acid regulates human CYP2E1 at the transcriptional level.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study we examined the effects of various agents on the expression of CYP2E1 and CYP4A11 mRNA and protein in multiple samples of human hepatocytes. Unexpectedly, we found that ethanol significantly enhanced expression of CYP2E1 mRNA (216 ± 32% of control). These results were unanticipated because animal studies showed that ethanol-mediated expression of CYP2E1 occurs by protein stabilization rather than transcriptional activation (reviewed in Raucy et al., 1993
; Koop and Tierney, 1990; Lieber, 1999). An additional surprise was that ethanol slightly enhanced CYP2E1 protein (122 ± 13% of control) in primary cultures. The extent of induction found here was less than that observed in human tissue samples obtained from alcohol abusers where hepatic CYP2E1 protein ranged from 2 to 4-fold greater than those samples from nonabusers (Mishin et al., 1998; Tsutsumi et al., 1989). The extent of induction observed here in hepatocytes was also less than that determined in vivo utilizing chlorzoxazone pharmacokinetic parameters to assess CYP2E1 induction (Girre et al., 1994; Raucy et al., 1997). Rates of chlorzoxazone metabolism were at least 1.9 to 4-fold higher in alcoholics than in nonalcoholics. Furthermore, metabolism of chlorzoxazone correlated well with either lymphocyte or liver CYP2E1 protein levels. That there was less induction by ethanol of CYP2E1 protein in primary cultures in the present investigation suggests that human hepatocytes may not be the best mechanism for assessing induction of certain P450 enzymes.
One factor that may influence the inducibility of this P450 in hepatocytes may be varied culture conditions such as hormones. Despite reports demonstrating that insulin suppressed CYP2E1 induction by ethanol in rodent hepatocyte cultures (Woodcroft and Novak, 1999; Yang et al., 2001), this hormone did not alter the inducibility of human CYP2E1 mRNA or protein by ethanol in human hepatocytes. However, there may be other factors that affect in vivo regulation of this enzyme by ethanol that are lacking in the culture media or are absent in isolated hepatocytes. Another factor that may influence the inducibility of CYP2E1 in human hepatocytes is the variability observed in response to ethanol exposure. In the present study, we observed a 216% increase in CYP2E1 mRNA in five separate samples, but the range was 102 to 285% of control. This large variation in range suggests that hepatocytes from certain subjects respond well to inducers while others do not. Because only three samples were utilized to assess CYP2E1 protein levels in the present investigation, there may not have been an adequate number to reflect induction of the protein. However, the variability seen here was in good agreement to that of Donato et al. (1995) where ethanol enhanced CYP2E1-mediated p-nitrophenol hydroxylation ranged from 1 to 3.5 fold above that in untreated human hepatocytes. Thus, one of the major complicating factors in quantitatively predicting enzyme induction in human hepatocytes is the large interindividual variability in response to inducers such as that observed here.
Among the agents examined in this investigation, we found that the fatty acid, palmitate, caused significant increases in CYP2E1 mRNA in human hepatocyte cultures. Obesity and high fat diets have been shown to induce CYP2E1 in animals and humans (O'Shea et al., 1994; Raucy et al., 1991; Tsukamoto et al., 1986; Wan et al., 2001; Yun et al., 1992). Thus the increase in expression of CYP2E1 mRNA by the fatty acid examined here was not surprising. That the increase occurred at the transcriptional level was unanticipated. Very few agents that induce CYP2E1 at the level of transcription have been identified. Most agents have been shown to increase expression of this enzyme by protein stabilization. Due to the small quantity of cells available, we were unable to perform immunoblot analyses on hepatocytes treated with palmitate and therefore did not measure CYP2E1 protein. Whether the elevations in CYP2E1 mRNA levels by palmitate would reflect higher concentrations of the corresponding protein leading to a significant alteration in metabolism is unclear and needs to be determined. Also, we did not determine the mechanism by which palmitate increased CYP2E1 mRNA levels. In contrast, we demonstrated that CYP4A11 mRNA and protein were enhanced by clofibrate, suggesting that PPAR may be involved in the induction of this gene. Fatty acids, their metabolites, eicosanoids, and fibrates have all been identified as ligands of human PPAR (Murakami et al., 1999). Therefore involvement of this receptor in clofibrate induction of CYP4A11, suggests that PPAR activates CYP4A11 by other ligands, such as fatty acids, in human hepatocytes. Although not shown here, palmitate and other medium chain fatty acids induce CYP4A1 (Tollet et al., 1994), suggesting a similar effect on CYP4A11. Whether PPAR plays a role in palmitate mediated induction of CYP2E1 remains to be determined. Regardless of the mechanism, we were able to identify an inducer of CYP2E1 that in all likelihood, increases expression of CYP4A11, namely palmitic acid.
In summary, we found that ethanol and palmitate significantly enhanced CYP2E1 mRNA levels greater than 200% of control in primary cultures of human hepatocytes. This is the first report to identify these agents as transcriptional activators of hepatocyte CYP2E1. Moreover, clofibrate produced a 239% increase above untreated cells in CYP4A11 mRNA content. To the best of our knowledge, this is also the first report demonstrating induction of CYP4A11 by PPAR ligands in primary cultures of human hepatocytes. Clofibrate-mediated induction of CYP4A11 mRNA and protein agrees with results reported elsewhere (Ozaki et al., submitted manuscript). We also found that despite the significant increase in CYP2E1 mRNA by ethanol, protein levels of this enzyme from hepatocytes treated with ethanol were not significantly higher than control cells. Alcohol abusers exhibit enhanced hepatic and lymphocytic concentrations of CYP2E1 protein and chlorzoxazone metabolism that is two-fold or greater than that in nonalcohol abusers. (Chen and Yang, 1996; Girre et al., 1994; Lucas et al., 1996; O'Shea et al., 1994; Raucy et al., 1997). Thus, it is unclear why ethanol did not mediate a significant increase in hepatocyte CYP2E1 protein. Only one additional study measured CYP2E1 by immunoblot analysis in human hepatocytes treated with ethanol but that investigation did not include quantified results (Kostrubsky et al., 1995). Thus, results presented here cannot be compared to previous observations. Despite the small increases in CYP2E1 protein that occurred from ethanol exposure in vitro, CYP2E1 mRNA and in vivo metabolism by this P450 are clearly altered accounting for hepatotoxicity associated with alcoholic liver disease, high doses of acetaminophen, or carcinogenesis from nitrosamines and various halogenated hydrocarbons.
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ACKNOWLEDGMENTS |
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This research was supported by NIH grant AA08990 (J.L.R.), AA07842 (J.M.L.), and by the Liver Transplant, Procurement, and Distribution System (#N01-DK-9-2310).
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NOTES |
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1 To whom correspondence should be addressed. Fax: (760) 929-9834. E-mail: jraucy{at}ctri-np.org
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