MONTSERRAT MARí AND ARTHUR I. CEDERBAUM

Transcription

1 Induction of Catalase, Alpha, and Microsomal Glutathione S-Transferase in CYP2E1 Overexpressing HepG2 Cells and Protection Against Short-Term Oxidative Stress MONTSERRAT MARí AND ARTHUR I. CEDERBAUM Induction of cytochrome P450 2E1 (CYP2E1) and the formation of reactive oxygen species (ROS) appear to be one of the mechanisms by which ethanol is hepatotoxic. Glutathione peroxidase and catalase detoxify H 2 O 2. Glutathione S- transferases (GST) provide protection from membrane lipid peroxidation, have GSH peroxidase activity, and reduce lipid hydroperoxides. Previous studies showed an up-regulation of GSH synthesis in CYP2E1 expressing HepG2 cells; this finding prompted an evaluation of the levels of other antioxidant exzymes. In CYP2E1 expressing cells, the alpha and microsomal GST messenger RNA (mrna) are increased by 4- and 2-fold, respectively, and catalase protein and mrna is increased by 2-fold. The increase in alpha and microsomal GST mrna correlates with increased total enzymatic activity and is caused by increased transcription as evidenced by run-on transcription assays. In HepG2 cells transfected to express a different cytochrome P450, CYP3A4, there was an increase in alpha GST. However, in contrast to the CYP2E1 expressing cells, neither microsomal GST nor catalase were induced, suggesting some specificity for CYP2E1. In agreement with an increased antioxidant defense system, the sensitivity to added prooxidants such as menadione, antimycin A, H 2 O 2, and 4-hydroxynonenal was lower in the CYP2E1 expressing cells as compared with control cells. In conclusion, overexpression of CYP2E1 in HepG2 cells, besides elevating total GSH levels, also induces expression of catalase and alpha and microsomal GST. This induction confers resistance to the cells against several prooxidants and is suggested to reflect an adaptive response by the cells against CYP2E1-mediated oxidative stress. (HEPATOLOGY 2001;33: ) The glutathione S-transferases (GST; EC ) are a family of enzymes that catalyze the conjugation of glutathione Abbreviations: GST, glutathione S-transferase; GSH, glutathione; 4-HNE, 4-hydroxy- 2,3,-nonenal; ROS, reactive oxygen species; CYP2E1, cytochrome P450 2E1; cdna, complementary DNA; AMT, 3-amino-1,2,4-triazol; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; SSC, sodium saline citrate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CYP3A4, cytochrome P450 3A4; mrna, messenger RNA. From the Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine of New York University, One Gustave L. Levy Place, New York, NY Received August 7, 2000; accepted December 18, Supported by USPHS Grant AA03312 from the National Institute on Alcohol Abuse and Alcoholism. Address reprints request to: Arthur I. Cederbaum, Ph.D., Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, NY Arthur.Cederbaum@mssm.edu; fax: Copyright 2001 by the American Association for the Study of Liver Diseases /01/ $35.00/0 doi: /jhep (GSH) to a wide variety of hydrophobic molecules of both exogenous and endogenous origin. 1-3 The cytosolic GSTs are dimeric proteins of subunits having a molecular mass in the 22- to 28-kd range. On the basis of their amino acid sequence, 5 classes have been described, namely alpha, mu, pi, theta, and sigma. 4-6 In humans the alpha class, subunits A1 and A2, is the predominant GST accounting for about 85% of the total GST protein. The major GST subunits expressed in rat liver are the alpha class, subunits A1 and A2, and the mu class, subunits 3 and 4, 7,8 whereas most of the cytosolic GST in HepG2 cells is the alpha isozyme. 9,, unsaturated aldehydes, such as 4-hydroxy-2,3-nonenal (4-HNE) and other endogenous or exogenous aldehydes, are generated in response to a variety of oxidative stimuli such as free intracellular iron, 10 chronic ethanol consumption, 11,12 and treatment with anticancer drugs. 13 GST-mediated conjugation of 4-HNE to GSH is the major pathway for 4-HNE metabolism. 14,15 Among the numerous cytosolic GST isozymes, the alpha-class displays the highest activity toward lipid hydroperoxides and has been implicated in the protection against oxidative stress The microsomal GST, different from the cytosolic family, comprise a family of small (16- to 18-kd), membrane-associated proteins that have been shown to be active as a trimer of identical subunits ; its activity can be increased by treatment with N-ethylmaleimide and other sulphydryl reagents, heat, oxidative stress, and protein dimerization. 23,24 Microsomal GST is strongly expressed in liver and has a wide specificity for lipophilic and electrophilic substrates. 25 The fact that the microsomal GST also exhibits glutathione peroxidase activity, 25 an activity also found in the alpha-class cytosolic GST, suggests that this enzyme might be of particular importance as a defense mechanism against lipid peroxidation. Its localization bound to the membrane of the endoplasmic reticulum, where potential substrates are produced by the action of the cytochrome P450 system and where hydrophobic xenobiotics and/or their metabolites are expected to accumulate, suggests a central role for this enzyme in drug metabolism and opens the possibility that hydrophobic substrates, such as lipid hydroperoxides, may be more efficiently eliminated by microsomal GSTs than by cytosolic GSTs. Endogenous hydrogen peroxide can be removed either by GSH in the presence of glutathione peroxidase or by catalase in the peroxisomes. In high doses, reactive oxygen species (ROS) are noxious to the cell leading to impaired metabolic functions, growth inhibition, and ultimately cell death. 26 Cells, therefore, employ several antioxidant enzyme systems to maintain low levels of ROS.

2 HEPATOLOGY Vol. 33, No. 3, 2001 MARÍ AND CEDERBAUM 653 Induction of cytochrome P450 2E1 (CYP2E1) and the formation of reactive oxygen intermediates, including ROS and lipid peroxidation derivatives, appear to be one of the mechanisms by which ethanol is hepatotoxic. 27,28 It has been shown that CYP2E1 when reduced by NADPH-cytochrome P450 reductase, is a loosely coupled enzyme that displays high NADPH oxidase activity. 29,30 Formation of reactive oxygen intermediates can occur even in the absence of added substrate, e.g., formation of superoxide and H 2 O 2 by microsomes from CYP2E1-expressing cells was not altered by the addition of substrates and ligands of CYP2E1. 31 To directly demonstrate that ROS generated by CYP2E1 can promote hepatotoxicity, a HepG2 cell line with enhanced expression of CYP2E1 was established. 32 The CYP2E1-expressing cells, E47 cells, have a slower growth rate than the empty vector transfected control C34 cells when GSH levels are maintained but displayed a loss of viability and an increase in lipid peroxidation when GSH synthesis is inhibited by treatment with l-buthionine sulfoximine (BSO). 32 However, despite a 40% to 50% increase in intracellular H 2 O 2, E47 cells showed an up-regulation of the levels of reduced GSH due to transcriptional activation of -glutamylcysteine synthetase, 33 the gene coding for the rate-limiting enzyme in GSH synthesis. This may reflect an adaptation to cope with the CYP2E1- derived oxidative stress. This up-regulation of GSH synthesis prompted us to evaluate the levels of other antioxidant enzymes important for the removal of ROS, and to determine whether any potential changes indeed allowed the CYP2E1- expressing cells to cope more effectively with oxidants and reactive intermediates. MATERIALS AND METHODS In Vitro Model and Cell Culture Conditions. Two human hepatoma HepG2 sub lines, which were established previously, 32 were used as a model in this study. E47 cells contain the human CYP2E1 complementary DNA (cdna) (kindly provided by Dr. F. Gonzalez, NCI/ NIH) inserted into the Eco RI restriction site of the pci-neo expression vector (Promega, Madison, WI) in the sense orientation. C34 cells contain the pci-neo vector alone. The HepG2 transduced C34 and E47 clones were cultured in minimum essential medium, supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 g/ml streptomycin, and 2 mmol/l glutamine in a humidified atmosphere in 5% CO 2 at 37 C. Cells were maintained in the presence of 0.5 mg/ml of geneticin. Most reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The protein content of cell lysates was determined using the DC-20 Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Specific reagents are described below. CYP2E1 levels were routinely monitored by assaying the oxidation of p-nitrophenol to p-nitrocatechol. Animal Treatment. Male Sprague-Dawley rats ( g) were used. All animals received humane care according to the National Institutes of Health guidelines. Rats were given 1% acetone (vol/vol) in the drinking water for 10 days and after overnight starvation, the liver was removed after perfusion in situ with ice-cold saline. The liver was homogenized with 50 ml of 210 mmol/l mannitol, 60 mmol/l sucrose, 10 mmol/l KCl, 0.25 mmol/l DTT, 0.1 mmol/l EGTA, and centrifuged at 700g for 10 minutes. The supernatant was centrifuged at 9,000g for 15 minutes and the resulting supernatant was centrifuged at 105,000g for 60 minutes. The supernatant was considered as the cytosol fraction. The microsomal pellet was resuspended in 0.05 mol/l potassium phosphate buffer (ph 7.4) containing 0.3 mmol/l EDTA and 0.25 mol/l sucrose. Cytosolic and microsomal fractions were used immediately or within a week after storage at 80 C. MTT Assay. Cytotoxicity of menadione, H 2 O 2, Antimycin A, or 4-hydroxy-2-nonenal to C34 and E47 cells was determined by the MTT assay. A total of cells/ml/well were plated onto a 24-well plate and incubated in 5% CO 2 at 37 C. Test reagents were added to the culture medium for a designated incubation time, typically 18 hours. The MTT assay was performed using the Cell Titer 96 Non-Radioactive Proliferation Assay Kit (Promega, Madison, WI). Briefly, 15% volume of dye solution was added to each well after the appropriate incubation time. After 4 hours of incubation at 37 C, an equal volume of solubilization/stop solution was added to each well for an additional hour of incubation. The absorbance of the reaction solution at 570 nm was recorded. The absorbance at 630 nm was used as a reference. The net A 570 -A 630 was taken as the index of cell viability. The reading taken from the wells of untreated cultured cells was used as the 100% viability value. The percent viability was calculated by the formula (A 570 A 630 ) sample /(A 570 A 630 ) control 100. GST Activity. GST activity was determined according to the method of Habig et al. 34 with some modifications. The reaction was carried out in 0.1 mol/l potassium phosphate, ph 6.5, 1 mmol/l GSH for cytosolic GST or 5 mmol/l GSH for microsomal GST, and 1 mmol/l 1-chloro-2, 4-dinitrobenzene in the presence of 50 L of cell lysate or isolated microsomal fraction (about 0.3 to 1.0 mg protein), and the increase in absorbance was monitored at 340 nm and 25 C over a 4-minute time period. Results are expressed as units of specific activity defined as the amount of the enzyme that produces 1 mol of conjugated product per minute per milligram of protein. Catalase Activity. Fresh sonicated extracts from cells were used. Catalase activity was determined at 25 C according to Claiborne and Fridovich. 35 The decomposition of hydrogen peroxide by catalase was followed by ultraviolet spectroscopy at 240 nm. The reaction was performed using a solution of 20 mmol/l hydrogen peroxide in 50 mmol/l KH 2 PO 4 containing 20 g of total cellular protein in a final volume of 1 ml. Specific activity of catalase was calculated from the equation: specific activity (units/mg protein/min) A 240 nm (1 min) 1,000/43.6 mg protein. Glutathione Reductase, Superoxide Dismutase, and Glutathione Peroxidase Activities. Glutathione reductase, superoxide dismutase, and glutathione peroxidase were assayed following the methods of Carlberg and Mannervik, 36 Paoletti and Mocali, 37 and Flohé and Günzler, 38 respectively, as described previously. 33 H 2 O 2 Removal. In these experiments H 2 O 2 levels were measured by the method of Jiang et al. 39 The colorimetric substrate was a mixture of 100 mol/l xylenol orange, 250 mol/l ammonium ferrous sulfate, and 100 mmol/l sorbitol dissolved in 25 mmol/l sulfuric acid. A total of 100 L of diluted culture medium sample (1:20) was combined with 100 L of substrate at room temperature for 45 minutes. Absorbance was read at 570 nm. A standard curve was generated by including in the assay known amounts of H 2 O 2 (from 0-5 mol/l, linear range). For the measurement of the H 2 O 2 removal rate, a solution of 300 L of minimum essential medium plus 10% fetal bovine serum (without phenol red) plus 100 mol/l H 2 O 2 was added to each well of a 24-well plate containing C34 or E47 cells. Some C34 and E47 cells were also preincubated with 3-amino-1,2,4-triazole (AMT) overnight. At times 0, 1, 2, 3, 5, 7.5, and 10 minutes, 100- L portions of medium were withdrawn, diluted 20 times, and subjected to H 2 O 2 determination as described above. Western Blot. Cells isolated from 80% confluent 75 cm 2 culture flasks were washed twice with 1 phosphate-buffered saline and harvested by scraping and subsequent sonication for 30 seconds. Total cell extract or microsomes were prepared by differential centrifugation and were resuspended in 0.1 mol/l potassium phosphate buffer (ph 7.4). After protein determination, 10 g of either denatured microsomes, cell extract, or rat liver homogenate from control or acetone-treated rats were resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro blotted onto nitrocellulose membranes (Bio-Rad). Membranes with transferred proteins were incubated with rabbit antihuman CYP2E1 polyclonal antibody (1:1,000) (kindly provided by Dr. Jerome Lasker, Mount Sinai School of Medicine, New York, NY), alpha GST

3 654 MARÍ AND CEDERBAUM HEPATOLOGY March 2001 A1-1 polyclonal antibody (1:1,000) (Oxford Biomedical Research), microsomal GST antibody (1:200) (kindly provided by Dr. Ralf Morgenstern, Karolinska Institutet, Stockholm, Sweden), or rabbit anti-human catalase antibody (1:1,000) (Calbiochem) as primary antibody, followed by incubation with horseradish peroxidase conjugated to goat anti-rabbit IgG (1:10,000) (Sigma) as the secondary antibody. Chemiluminiscence reaction using the ECL kit (Amersham, UK) was carried out for 1 minute followed by exposure to Kodak X-Omat radiograph film (Eastman Kodak, Rochester, NY). Northern Blot. Total RNA was isolated using the Trizol reagent (Gibco/BRL). Ten micrograms of RNA were electrophoresed under denaturing conditions in 0.9% agarose/formaldehyde gels and transferred onto Gene-screen nylon membranes (Dupont, Boston, MA). RNA was UV-cross linked at 1,200 Jules 100 for 30 seconds and prehybridized in a hybridization oven for 2 hours at 42 C in 5 sodium saline citrate (SSC), 50% formamide, 50 mmol/l potassium phosphate, ph 6.5, and 1 Denhardt s solution containing 0.1 mg/ml of denatured salmon sperm DNA. A 447-bp probe corresponding to nucleotides 69 to 515 of the human alpha GST (A1 and A2, 95% homologous) and a 563-bp probe corresponding to nucleotides 82 to 645 of the human microsomal GST1 were generated from HepG2 cells RNA by polymerase chain reaction using synthetic oligonucleotides. Probes for rat microsomal and alpha or Ya GSTs were also generated from a rat liver RNA corresponding to nucleotides 290 to 612 and 82 to 445, respectively. A human cdna for catalase was generated after digesting the plasmid pzeosv2-cat (kindly provided by Dr. J. A. Melendez, Albany Medical College, New York, NY) with NotI and HindIII. cdna probes were radiolabeled with 32 P-dCTP (specific activity 3,000 Ci/mmol, Amersham) using a random priming DNA labeling kit (Amersham). Immobilized RNA was hybridized in 0.1% SDS, 5 SSC, 50% formamide, 50 mmol/l potassium phosphate, ph 6.5, 1 Denhardt s solution, and 12.5 dextran sulfate with 10 6 cpm/ml of labeled cdna and 0.1 mg/ml of denatured salmon sperm DNA for 16 hours at 42 C. Blots were washed 4 times for 15 minutes in 2 SSC and 0.5% SDS at 65 C, exposed to a phosphor storage screen for a fixed period of time according to the hybridized cdna, and scanned in a Molecular Dynamics Phosphorimager (Molecular Dynamics, Sunnyvale, CA). Values were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) messenger RNA (mrna) using the ImageQuant software. Nuclear In Vitro Run-on Transcription Assays. Cultured cells were washed twice with sterile 1 phosphate-buffered saline, scraped off, and transferred into 15-mL Falcon tubes for centrifugation at 500g at 4 C for 5 minutes. Pellets were loosened and resuspended in 4 ml of Buffer A (10 mmol/l Tris, ph 7.4, 10 mmol/l NaCl, and 3 mmol/l MgCl 2 ). After addition of 0.5% (vol/vol) NP-40, samples were vortexed for 10 seconds and kept on ice for 10 minutes. After a second centrifugation, supernatants containing cytoplasmic RNA were discarded, and 4 ml of Buffer A was added. Samples were kept on ice for 10 minutes and spun down. Nuclei were collected in 100 L of storage buffer (50 mmol/l Tris, ph 8.3, 40% glycerol, 5 mmol/l MgCl 2, and 0.1 mmol/l EDTA) and immediately stored at 70 C until analyzed. Transcription was assayed in vitro with 100 L nuclei (isolated from about cells), 100 Ci of 32 P-UTP, and 100 L of assay buffer (2.5 mmol/l MnCl 2, 125 mmol/l Tris, ph 7.4, 12.5 mmol/l MgCl 2, 2.5 mmol/l DTT, 2.5 mmol/l ATP, 1.25 mmol/l GTP, 1.25 mmol/l CTP, and 375 mmol/l KCl). The reaction was carried out at 25 C for 45 minutes and stopped by adding DNase I (RNase free) at a final concentration of 20 g/ml. After incubating at 30 C for 5 minutes, RNA was released from the nuclei by treating with 1% SDS, 5 mmol/l EDTA, 10 mmol/l HEPES, ph 7.5, and 200 g/ml of proteinase K at 42 C for 30 minutes. Newly transcribed RNA was isolated using the Trizol reagent. Eight micrograms of each cdna of interest were denatured in 0.25 N NaOH/0.5 mol/l NaCl for 10 minutes at room temperature, and diluted in 0.1 SSC/0.125N NaOH. Samples were blotted onto Gene-screen membranes, neutralized in 0.5 N NaCl/0.5 M Tris, ph 7.5, and UV-crosslinked. Prehybridization was carried out for 6 hours at 42 C, and cpm/ml of purified denatured riboprobes were used for hybridization for 72 hours at 42 C. The composition of the hybridization buffer was the same as described for the Northern Blot assay, using 0.1 mg/ml of bakers yeast trna instead of the salmon sperm DNA. After 4 washes of 15 minutes each in 2 SSC/ 0.5% SDS at 65 C, membranes were exposed to a phosphor storage screen and the signals were quantified using the GAPDH mrna signal as a control. Transient Transfection Experiments. Transient transfection experiments were carried out with pci-neo vector or pci-neo vector containing the CYP2E1 cdna in the sense orientation. Transfection of HepG2 cells was carried out with 10 g of the appropriate plasmid, 3 g of pl7-lac (kindly provided by Dr. Robert Krauss, Mount Sinai School of Medicine, New York), and 30 L of Fugene 6 (Roche Molecular Biochemicals). After 48 hours, the efficiency of the transfection was determined by X-gal staining; and at 48 and 96 hours, total RNA was isolated to study alpha-gst, mic-gst, catalase, and GAPDH mrna expression by Northern blotting. Statistics. Results refer to mean SD and are average values from 3 to 6 values per experiment; experiments were repeated at least twice. Statistical evaluation among groups was carried out using the Student s t test and P.05 was considered significant. RESULTS Antioxidant Enzyme Activities. In a recent publication 33 we described the up-regulation of GSH synthesis in CYP2E1 overexpressing cells. To maintain the intracellular GSH levels, GSSG is reduced back to GSH by glutathione reductase or de novo synthesis of GSH by -glutamylcysteine synthetase occurs. Organic hydroperoxides can be reduced either by glutathione peroxidase or GSTs. Superoxide is converted to H 2 O 2 by superoxide dismutase; H 2 O 2 can be removed either by reduction by GSH in the cytosol, as catalyzed by glutathione peroxidase, or by catalase in the peroxisomes. As shown in Table 1 there was no difference in the activity of glutathione reductase or superoxide dismutase between C34 and E47 cells. There was a 30% decrease in glutathione peroxidase activity in CYP2E1-expressing cells. However, there was a 2-fold increase in the activities of total GST and catalase in E47 cells compared with C34 cells. An increase in GST activity in microsomal fraction isolated from the E47 cells was also found and the percent increase in GST activity was similar in both cytosolic and microsomal fractions of CYP2E1-expressing cells. Microsomal GST can be activated by the covalent modification of the sufhydryl group 23 ; treatment of microsomes with 10 mmol/l DTT for 10 minutes at 37 C did not affect the microsomal GST activity suggesting that enzyme TABLE 1. Antioxidant Enzyme Activities in C34 and E47 Cells Antioxidant Enzyme (Activity) C34 E47 Glutathione reductase (mu/mg) Superoxide dismutase (U/mg) Glutathione peroxidase (mu/mg) * Catalase (U/mg) * Glutathione S-transferase (mu/mg) * Glutathione S-transferase (mu/mg), microsomal fraction * NOTE. The activities of glutathione reductase, superoxide dismutase, glutathione peroxidase, glutathione S-transferase, and catalase were determined as described under Materials and Methods and refer to mean SD, n 6. *P.05 when compared with C34. The microsomal fraction contains microsomal and cytosolic glutathione S-transferases.

4 HEPATOLOGY Vol. 33, No. 3, 2001 MARÍ AND CEDERBAUM 655 FIG. 2. Representative Western blot of microsomal GST. To determine the amount of microsomal GST protein Western blots were performed in control and CYP2E1-expressing cells. Data represent 1 (carried out in triplicate) of 3 different experiments. thiol oxidation did not play a significant role in the enhanced activity of the microsomal fraction isolated from the E47 cells. Expression of the Microsomal-GST mrna and Alpha-GST mrna in C34 and E47 Cells. Since among the cytosolic GSTs, the alpha class GST (GST A1 and GST A2) is the predominant form present in human liver and HepG2 cells, a 447-bp cdna was synthesized by reverse transcription-polymerase chain reaction and used as a specific probe complementary to GST A1 and GST A2, which are 95% homologous. Northern blots of alpha-gst and GAPDH (used as a loading control) mrna are shown in Fig. 1A. The mrna for the alpha GST in control C34 cells is almost undetectable, but was present in the CYP2E1 expressing cells. Levels of GAPDH mrna were similar between the 2 cell lines. Northern Blots for the microsomal GST (Fig. 1B) revealed the presence of 2 transcripts with size around 1 kb. There was a 2-fold increase (P.05) in the microsomal GST mrna in the CYP2E1-expressing cells compared with the control cells. After the observation that activity and mrna for the alpha and microsomal GST were increased in the E47 cells, we assayed for protein content of these enzymes by Western Blot analysis. Alpha-GST protein could not be detected in C34 and E47 cells. The antibody used is specific for the alpha forms of GST in human and rat, and the alpha-gst protein could be detected in rat (data not shown). We assume that in these transduced clones of HepG2 cells, the amount of alpha-gst protein is probably below the detection limit of the assay. The lower levels of expression of alpha-gst mrna and protein in HepG2 cells, compared with human liver cells, is consistent with a 10-fold lower catalytic activity of GST in the HepG2 cells compared with human liver. 9 The content of microsomal GST protein in E47 cells, as shown in Fig. 2, was 2- to 3-fold higher (P.05) than in C34 cells, consistent with the increase in mrna levels. An increase in alpha or microsomal GST mrna may reflect either an increased transcription rate of the gene or a stabilization of the mrna. It was impossible to compare the mrna decay in the presence of Actinomycin D in the case of the alpha GST because the mrna transcript in the control C34 cells was undetectable. For the microsomal GST, there was no decay for the first 24 hours after Actinomycin D addition suggesting a relatively stable mrna. Although we cannot rule out the possibility of mrna stabilization in E47 cells, nuclear run-on experiments were carried out to evaluate transcriptional activation of the alpha and/or microsomal GST genes. Indeed, the nuclear run-on revealed an increased capacity of E47 cells to transcribe both the alpha and the microsomal GST gene (Fig. 3). The GST/GAPDH ratio of the newly synthesized mrna was elevated from a value of 1 in the C34 cells to a value of 2 in the E47 cells for the alpha GST, and from 1 to a value of 1.8 for the microsomal GST. This increase in transcription under steady state conditions is similar to the increase found in microsomal GST mrna levels as detected by Northern blot. These experiments were replicated in another CYP2E1-expressing clone (E43 cells) to avoid a cloning artifact. Similar results were observed as described above for the E47 cells (data not shown). Effect of Catalase and Vitamin E. To further characterize the nature of CYP2E1-induced transcriptional activation of alpha and microsomal GST, extracellular catalase (500 U/mL) and vitamin E (50 mol/l) were added to the culture medium for 7 days (because of the long half-life of mrna and protein), and the effect on alpha GST and microsomal GST mrna levels were determined by Northern blot. As shown in Fig. 4, both catalase and vitamin E partially prevented (40% decline, P.05, in the alpha-gst/gapdh mrna ratio) the increase in alpha GST mrna in E47 cells. However, there was no significant decrease found for the microsomal GST mrna by these compounds (data not shown). The effectiveness of catalase and vitamin E suggested the possibility that H 2 O 2 and FIG. 1. Representative Northern blots of alpha and microsomal GST. (A) Northern blot of alpha GST in control and CYP2E1 expressing cells. (B) Northern blot of microsomal GST in control and CYP2E1 expressing cells. The same blots were sequentially probed with cdna for alpha (A) or microsomal GST (B) and GAPDH. Data represent 1 (carried out in triplicate) of at least 6 different experiments. FIG. 3. Transcriptional regulation of alpha and microsomal GST genes. Intact nuclei of control (C34) and CYP2E1 expressing (E47) cells were isolated and the transcription in vitro of the alpha and microsomal GST genes was determined as described under Materials and Methods. Data represent 1 of 2 different experiments.

5 656 MARÍ AND CEDERBAUM HEPATOLOGY March 2001 FIG. 4. Effect of catalase and vitamin E on alpha-gst mrna levels in control and CYP2E1-expressing cells. Control and CYP2E1- expressing cells were treated with 500 U/mL catalase or 50 mol/l vitamin E. After 7 days of incubation, RNA was isolated and Northern blots for alpha GST and GAPDH were performed. Data represent 1 (carried out in duplicate) of 3 different experiments. lipid peroxidation derivatives may play a role in the induction of alpha GST. Comparison Between CYP2E1 and CYP3A4 on Alpha and Microsomal GST Expression. The C34 control cells, like the parental cell line, do not contain or express very low levels of P450 enzymes. To ascertain if the induction of GSTs in the E47 cells could be due to the expression of any P450, we evaluated the ability of cytochrome P450 3A4 to increase alpha and microsomal GST mrna and protein. A HepG2 cell line has been recently developed, by methods similar to those used for establishing the E47 and E43 cells, that constitutively and stably expresses CYP3A4 (Feierman D.E., Department of Biochemistry and Anesthesiology, Mount Sinai School of Medicine, unpublished observations). These CYP3A4-expressing HepG2 cells were previously used for comparative purposes with the E47 cells with respect to induction of -glutamylcysteine synthetase expression. 33 As shown in Fig. 5, the mrna of the alpha GST in the CYP3A4-expressing cells was detectable and similar to the level found in E47 cells. In contrast, the mrna level of the microsomal GST in CYP3A4-expressing cells was similar to control C34 cells and lower than that found in the E47 cells. The amount of microsomal GST protein (not shown) correlated with the mrna showing no increase in the CYP3A4 cells as compared with control C34 cells but a 2- to 3-fold increase in E47 and E43 cells. This result suggests that the increase in alpha GST is not CYP2E1 dependent but P450 dependent because at least 2 different P450s (2E1 and 3A4) showed induction of alpha-gst mrna; however, there may be some selectivity in the induction of microsomal GST by CYP2E1- expressing cells, at least compared to one other P450 enzyme. Effect of the Induction of CYP2E1 In Vivo. Since microsomal GST was increased in CYP2E1 but not in CYP3A4-expressing HepG2 cells, the possible in vivo induction of this enzyme was evaluated after treatment of rats with acetone, a known CYP2E1 inducer. After 10 days of treatment with a 1% acetone in the drinking water, rats were killed and livers were assayed for the content of CYP2E1, alpha, and microsomal GST by Western blot. As shown in Fig. 6, CYP2E1 protein (Fig. 6A) was increased by 2-fold and there was a 50% increase in the content of microsomal GST protein (Fig. 6B); however, there was no change in the content of the alpha GST isoform (data not shown). The total GST activity of the liver extract was increased by 30% after the acetone treatment. Expression of Catalase mrna and Protein in CYP2E1-Expressing HepG2 Cells. After the observation that catalase activity was increased in CYP2E1-expressing cells, Northern and Western blots were performed to determine the nature of the induction. As shown in Fig. 7A, Northern blots for catalase display a single transcript of 2.7 kb. There was a 2-fold increase in catalase mrna in CYP2E1-expressing E47 cells compared with control C34 cells and CYP3A4-expressing cells. This increase in catalase mrna correlates with an increased cata- FIG. 5. Comparison of induction of alpha and microsomal GST in 2 different P450-containing HepG2 cell lines. Northern blotting of alpha (A) or microsomal (B) GST and GAPDH mrna were performed in control, CYP3A4-, and CYP2E1-expressing HepG2 cells. Data represent 1 (carried out in triplicate) of 3 different experiments. FIG. 6. Expression of rat microsomal GST after CYP2E1 induction in vivo. After 10 days of treatment with 1% acetone in the drinking water, rat liver microsomes were assayed for protein content of CYP2E1 (A) and microsomal GST (B) by Western blot. Data represent samples from 4 different control and acetone-treated rats.

6 HEPATOLOGY Vol. 33, No. 3, 2001 MARÍ AND CEDERBAUM 657 FIG. 7. Representative Northern blots and Western blots of catalase. Northern blot (A) and Western blot (B) of catalase mrna and protein were performed in control, CYP3A4-, and CYP2E1-expressing cells. Data represent 1 (carried out in triplicate) of 2 different experiments. the assay completely inhibited catalase in both cell lines and also inhibited CYP2E1 in E47 cells (by 90%). As shown in Fig. 8, CYP2E1-expressing cells remove H 2 O 2 more efficiently than control cells and this more rapid removal is completely prevented by preincubating with AMT. The AMT treatment also decreased H 2 O 2 removal by the control C34 cells. This latter result indicates that catalase plays an important role in the capacity to remove H 2 O 2 in these cell lines. Effect of the Induction of CYP2E1 on Toxicity of Prooxidants. Alpha and microsomal GST can protect against oxidative stress and these isoforms also function as selenium-independent glutathione peroxidases. The sensitivity to the cytotoxic effects of H 2 O 2 was compared for the control, CYP3A4-, and CYP2E1-expressing cells using an MTT cytotoxicity assay. As shown in Fig. 9A, CYP3A4- and CYP2E1-expressing cells were more resistant to H 2 O 2 than control cells. After inhibition of catalase by pretreatment with AMT there was a decrease in the IC 50 for H 2 O 2 for all cell lines, but the CYP2E1- lase protein content in the CYP2E1-expressing cells (Fig. 7B). The fact that glutathione peroxidase activity was decreased whereas catalase was induced in the CYP2E1-expressing cells, suggests the possibility that the catalase induction helps the cell to cope with H 2 O 2 derived from CYP2E1 expression. Removal of Extracellular H 2 O 2 by Control and CYP2E1-Expressing Cells. The observation that CYP2E1-expressing cells have a 2-fold increase in catalase activity suggests that this cell line is better equipped than control cells to efficiently remove H 2 O 2. Using a xylenol orange assay, the kinetics of H 2 O 2 removal were determined in control and CYP2E1-expressing cells in the presence and absence of AMT, a catalase inhibitor. Pretreatment of the cells with 6 mmol/l AMT for 18 hours before FIG. 8. Removal of extracellular H 2 O 2 by control and CYP2E1-expressing cells. The kinetics of H 2 O 2 removal were compared between control and CYP2E1-expressing cells, and in control and CYP2E1-expressing cells preincubated with 6 mmol/l AMT, a catalase inhibitor, for 18 hours. Cells were challenged with 100 mol/l H 2 O 2 and at different time points the remaining H 2 O 2 concentration was measured. Data represent 1 (carried out in triplicate) of 3 experiments. *P.05 vs. control. The inhibition by AMT was significant for both cell lines. FIG. 9. Cytotoxicity of extracellular H 2 O 2 in control, CYP3A4-, and CYP2E1-expressing cells. (A) Cells were challenged with different concentrations of H 2 O 2 for 18 hours. (B) Cells were preincubated with 6 mmol/l AMT for 8 hours and then challenged with H 2 O 2 for 18 hours. The viability was measured by an MTT assay. Data represent 1 (carried out in triplicate) of at least 3 different experiments. *P.05 vs. control.

7 658 MARÍ AND CEDERBAUM HEPATOLOGY March 2001 and CYP3A4-expressing cells were still more resistant than control cells to H 2 O 2 cytotoxicity. This result suggests that even though catalase is important for cytoprotection against H 2 O 2, alpha GST also confers resistance to H 2 O 2 because the two cell lines that overexpress alpha GST, CYP2E1, and CYP3A4 are more resistant to H 2 O 2 than control cells, even after inhibition of catalase. It is important to note that because AMT is also a CYP2E1 inhibitor, the CYP2E1-associated induction of other genes would be abolished; due to the long half-life of alpha and microsomal GST mrna and protein, the inhibition of CYP2E1 would not affect appreciably the activities of alpha and microsomal GST in this time frame but effects on other antioxidant systems cannot be ruled out. We also cannot rule out a role for microsomal GST in the protection against H 2 O 2, but since CYP3A4-expressing cells display similar sensitivity to H 2 O 2 as CYP2E1-expressing cells, the contribution of microsomal GST towards protection against H 2 O 2 seems negligible. The above experiments involved cytotoxicity of an external-added bolus of H 2 O 2. Sensitivity to toxicity of intracellularly generated ROS was evaluated by studying the effects of addition of menadione or of Antimycin A. Consistent with increased catalase and possibly microsomal GST in CYP2E1- expressing cells, the E47 cells were more resistant to toxicity than CYP3A4 and control cells after being challenged with 20 mol/l menadione or 15 mol/l Antimycin A for 8 hours (Fig. 10). Interestingly, the CYP3A4 cells were more resistant than control cells to these agents, perhaps a reflection of the enhanced alpha GST activity. Effect of the Induction of CYP2E1 on Toxicity of 4-HNE., unsaturated aldehydes, e.g., 4-HNE, are generated in response to a variety of oxidative stimuli. The conjugation of 4-HNE with GSH, mediated by GST, is the major pathway for 4-HNE detoxification. To evaluate if increased microsomal and/or alpha GST expression confers resistance to 4-HNE, control, CYP3A4-, and CYP2E1-expressing cells were challenged with different doses of 4-HNE, and toxicity was determined after FIG. 11. Cytotoxicity of 4-HNE in control, CYP3A4-, and CYP2E1-expressing cells. Cells were challenged with different concentrations of 4-HNE for 18 hours and viability was measured by an MTT assay. Data represent 1 (carried out in triplicate) of at least 3 different experiments. *P.05 vs. control, P.05 vs. CYP3A4. 18 hours by the MTT assay. As shown in Fig. 11 the protection against 4-HNE followed the order CYP2E1 CYP3A4 control cells, the same order as increase in number of different GSTs isoforms. Taken together, these results suggest that the expression of both alpha and especially microsomal GST is important in protecting against the toxicity of lipid peroxidation derivatives. Effect of Challenge With Prooxidants and Transient Expression of CYP2E1 in HepG2 Cells. To rule out an effect on the selection of the cell line due to the possible generation of mutants as a result of an oxidative stress rather than a physiologic response, transient transfection experiments with CYP2E1 were performed in the parental HepG2 cell line. As shown in Fig. 12, 96 hours after transfection with CYP2E1 there was an increase in the content of catalase, mic-gst, and alpha-gst mrna, as compared with transfection with the empty plasmid. The increases observed (about 30%), normalized by GAPDH mrna, was in range with the efficiency of the trans- FIG. 10. Cytotoxicity of menadione and Antimycin A in control, CYP3A4-, and CYP2E1-expressing cells. Cells were incubated with 15 mol/l menadione or 15 mol/l Antimycin A for 8 hours. The viability was measured by an MTT assay. Data represent 1 (carried out in triplicate) of at least 3 different experiments. *P.05 vs. control, P.05 vs. CYP3A4. FIG. 12. Transient transfection of CYP2E1 in HepG2 cells. HepG2 cells were transiently transfected with the empty or CYP2E1 containing vector and the expression of catalase, alpha, and microsomal GST mrna was determined after 96 hours and normalized by GAPDH mrna. Data represent 1 (carried out in triplicate) of 2 different sets of transfection.

8 HEPATOLOGY Vol. 33, No. 3, 2001 MARÍ AND CEDERBAUM 659 fection obtained. Thus, transient transfection with CYP2E1 produced an increase in the same mrna species found to be increased in the HepG2 cell lines constitutively expressing CYP2E1. These results confirmed that the effects observed in the E47 cells are not due to a mutation or artifact on the selection of the CYP2E1-expressing cell line. Moreover, challenge of parental HepG2 cells with prooxidants such as 5 mol/l menadione or U/mL glucose oxidase induced an increase in catalase and alpha-gst mrna (data not shown), validating that the HepG2 cells up-regulate these mrnas in response to an oxidative challenge. DISCUSSION The liver has relatively high metabolic activity and is the major organ involved in the metabolism of drugs/xenobiotics. These activities put this organ at an increased risk for exposure to oxidative stress as the result of formation of ROS. Glutathione peroxidase and catalase are able to detoxify H 2 O 2. Hepatic GSTs are able to not only catalyze the conjugation of endogenous and exogenous electrophiles to GSH, 1-3 but also provide protection from membrane lipid peroxidation. In human liver the alpha GST, the cytosolic predominant form, and the microsomal GST have GSH peroxidase activity and are able to reduce lipid hydroperoxides ,25,40 The cytochrome P450 family of proteins is known to be critical in the oxidative, peroxidative, and reductive metabolism of numerous xenobiotics Chronic alcohol consumption leads to an increase in the content of CYP2E1 in the liver and enhances its catalytic activity in the microsomal fraction. Induction of CYP2E1 in alcohol-fed animals has been shown to increase the lipid peroxidation of hepatic microsomes. 41,42 A previous study 33 reported on the up-regulation of -glutamylcysteine synthetase and of increased GSH levels in CYP2E1-expressing HepG2 cells. In the present study, overexpression of CYP2E1 in HepG2 cells was found to induce expression of catalase and alpha and microsomal GST, and this induction confers at least some resistance to the cells against several prooxidants. These results suggest the possibility that the induction of GSH synthesis and the activity of GST and catalase may play a role as an adaptive mechanism to cope with CYP2E1-derived oxidative stress and lipid peroxidation. Indeed, when GSH levels are lowered upon inhibition of -glutamylcysteine synthetase with l-buthionine sulfoximine, the E47 cells lose their viability as a result of increased lipid peroxidation. 32 The increase in alpha and microsomal GST mrna in E47 (and E43) cells correlates with increased GST protein and activity. The alpha and microsomal GST mrna are increased by 4- and 2-fold, respectively, in the CYP2E1-expressing cells and this is due, at least in part, to increased transcription of the gene as evidenced by run-on transcription assays. Increased GST activity can protect cells against oxidative stress and membrane lipid peroxidation by removing reactive metabolites. In liver, the peroxidation of microsomal membranes yields n-alkanals, 2-alkenals, 4-hydroxyalkenals, and a variety of other aldehydes with diverse chemical structures. Whereas most of these aldehydic products are produced in very low concentration, 2 - -unsaturated aldehydes, malondialdehyde, and 4-hydroxy-2,3-nonenal (4-HNE) are produced in relatively large quantities and therefore are classified as major products of lipid peroxidation HNE elimination by conjugation with GSH, catalyzed by GST, represents 50% to 60% of the total 4-HNE removal by hepatocytes; other pathways responsible for 4-HNE metabolism are alcohol/aldehyde dehydrogenases. 14,15 Both, alpha and microsomal 25 GST are able to metabolize 4-HNE. Results of the present study show that induction of alpha GST alone, by expression of probably either CYP2E1 or CYP3A4, provided protection against the toxicity elicited by 4-HNE; and that this protection was even greater when there was an additional induction of microsomal GST, as a result of the expression of CYP2E1. Even though the specific activity of purified microsomal GST, with 4-HNE as a substrate, is lower than that for the alpha GST family, 25 our data suggest that the role of microsomal GST in protecting and detoxifying lipid peroxidation derivatives is important. The fact that microsomal GST is a membrane-bound protein and 4-HNE can activate microsomal GST posttranslationally raises the possibility that this compound could increase its own metabolism by reacting with the enzyme, presumably through reaction with the cysteine sulphydryl group and, as a consequence, rendering the protein more efficient in handling these hydrophobic substrates. The K m value of catalase for hydrogen peroxide is much higher than that of GSH peroxidase. Hence, GSH peroxidase has been postulated to degrade low levels of H 2 O 2 physiologically, while catalase might function when cellular levels of H 2 O 2 are increased. In CYP2E1-expressing cells the 2 enzymes appear to compensate for each other in scavenging H 2 O 2 in intracellular and extracellular compartments, because GSH peroxidase is decreased by 30% but catalase is increased 2-fold. GSH peroxidase is known to be sensitive to oxidative stress. As a consequence, the CYP2E1-expressing cells are more efficient in handling high levels of H 2 O 2 added extracellularly or generated intracellularly, as evidenced by the toxicity assays with H 2 O 2, menadione, and Antimycin A. The fact that CYP2E1 cells, which overexpress catalase and microsomal plus alpha GST, are more resistant than CYP3A4 cells, which only overexpress alpha GST, whereas the CYP3A4 expressing cells, in turn, display less toxicity than control cells towards these prooxidants suggest that catalase and microsomal and alpha GST are important in attenuating the toxicity generated either by H 2 O 2 or the intracellular production of radicals by menadione or Antimycin A. Huan and Koop, 43 using a tetracycline-controlled expression system, showed that menadione was less toxic to Hela cells after induction of CYP2E1 when compared with noninduced cells, suggesting that the expression of CYP2E1, before menadione exposure, primed the cells to be less susceptible to oxidative stress. Indeed, previous studies in our laboratory showed that CYP2E1 overexpression induced GSH synthesis, which is important for maintaining the viability of these cells as increased ROS generation and lipid peroxidation occurs under conditions of GSH depletion. 32 In the present study we observed that while induction of alpha GST occurs with at least 2 different P450 enzymes, the induction of microsomal GST and catalase may be more specific (as was the induction of -glutamylcysteine synthetase 33 ) and related to CYP2E1 but not CYP3A4 expression in this tissue culture model. The effects of other P450s would be of interest to evaluate. The observation that the increase in alpha-gst mrna could be partially blocked by prolonged incubation of the cells in the presence of catalase and vitamin E suggest that H 2 O 2 and lipid peroxidation derivatives play an important role in the induction of this cytosolic enzyme. The failure of these

9 660 MARÍ AND CEDERBAUM HEPATOLOGY March 2001 antioxidants in blocking the microsomal GST induction in CYP2E1-expressing cells suggests that other mechanisms or other reactive oxidants may be involved in the induction of this gene. The stability of the microsomal GST mrna is another complication in efforts to assay down-regulation of the gene by various treatments. Nonetheless, the fact the acetonetreated rats also displayed induction of microsomal GST, along with CYP2E1, suggest that the 2 phenomena may be related. Future studies are needed to understand the nature of this induction. We had previously shown that ethanol caused a loss of viability and produced apoptosis in a HepG2 cell model that expresses lower levels of CYP2E1 than the E47 cells (E9 cells 44,45 ). In fact, ethanol produced little toxicity in the E47 cells despite the approximately 4-fold higher content of CYP2E1. It appears that the up-regulation of antioxidants such as GSH and the antioxidant enzymes such as catalase, microsomal, and alpha GST in the E47 cells, may explain the resistance of the E47 cells to ethanol analogous to their resistance to H 2 O 2, menadione, antimycin A, or 4-HNE. More powerful prooxidants, however, such as the polyunsaturated fatty acid arachidonic acid or an iron-nitrilotriacetatec complex were more toxic to the E47 cells than control cells 46,47 even though the former have increased antioxidant defense. Most likely, the balance between oxidative stress and antioxidant defense is shifted with time and with powerful prooxidants towards the former, with a resulting loss in cellular viability. In summary, CYP2E1 overexpression in HepG2 cells upregulates catalase, alpha, and microsomal GST expression, and this may serve as a mechanism to attenuate CYP2E1- derived lipid peroxidation and oxidative stress. 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