* 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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Key Words: human hepatocytes; CYP4A11; CYP2E1; ethanol; palmitate; clofibrate.
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INTRODUCTION |
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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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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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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 106 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 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 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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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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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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