Increased toxicity by transforming growth factor-beta 1 in liver cells overexpressing CYP2E1

Free Radical Biology & Medicine 41 (2006) 1100 – 1112 www.elsevier.com/locate/freeradbiomed

Original Contribution

Increased toxicity by transforming growth factor-beta 1 in liver cells overexpressing CYP2E1 Jian Zhuge, Arthur I. Cederbaum ⁎ Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, One Gustave L. Place, New York, NY 10029, USA Received 17 February 2006; revised 21 June 2006; accepted 22 June 2006 Available online 4 July 2006

Abstract Ethanol treatment causes an increase in expression of TGF-β1 and CYP2E1 in the centrilobular area. Alcoholic liver disease is usually initiated in the centrilobular region of the liver. We hypothesized that the combination of TGF-β1 and CYP2E1 produces increased oxidative stress and liver cell toxicity. To test this possibility, we studied the effects of TGF-β1 on the viability of HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells, which do not express CYP2E1. E47 cells underwent greater growth inhibition and enhanced apoptosis after TGF-β1 treatment, as compared to the C34 cells. There was an enhanced production of reactive oxygen species (ROS) and a decline in reduced glutathione (GSH) levels in the TGF-β1-treated E47 cells and the enhanced cell death could be prevented by antioxidants. The CYP2E1 inhibitor diallyl sulfide prevented the potentiated cell death in E47 cells validating the role of CYP2E1. Mitochondrial membrane potential declined in the TGF-β1-treated E47 cells, prior to developing toxicity, and cell death could be prevented by trifluoperazine, an inhibitor of the mitochondrial membrane permeability transition. TGF-β1 also produced a loss of cell viability in hepatocytes from pyrazole-treated rats with elevated levels of CYP2E1, compared to control hepatocytes. In conclusion, increased toxic interactions by TGF-β1 plus CYP2E1 can occur by a mechanism involving increased production of intracellular ROS and depletion of GSH, resulting in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of TGF-β1-induced cell death by CYP2E1 may contribute to mechanisms of alcohol-induced liver disease. © 2006 Elsevier Inc. All rights reserved. Keywords: CYP2E1; TGF-β1; Apoptosis; Reactive oxygen species; Reduced glutathione; Mitochondrial membrane potential; HepG2 cells; Rat hepatocytes

Introduction It has become increasingly evident that endotoxemia, alcohol metabolism, oxidative stress, and cytokines play a key role in alcohol-mediated liver injury [1]. Transforming growth factorbeta (TGF-β) is a multifunctional cytokine, whose numerous cell and tissue activities include cell cycle control, the regulation Abbreviations: CYP2E1, cytochrome P450 2E1; DAS, diallyl sulfide; DCFDA, 2′,7′-dichlorofluorescein diacetate; FBS, fetal bovine serum; GSH, reduced glutathione LDH, lactate dehydrogenase; MEM, minimal essential medium; Δψm, mitochondrial membrane potential; MnTMPyP, Mn(III)tetrakis(L-methyl4-pyridyl)porphyrin pentachloride; MTT, 3(4,5-dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide; O2 −, superoxide anion; PI, propidium iodide; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TFP, trifluoperazine; TGF-β, transforming growth factor-beta; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. ⁎ Corresponding author. Fax: +1 212 996 7214. E-mail address: [email protected] (A.I. Cederbaum).

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0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.06.017

of early development, differentiation, extracellular matrix formation, hematopoiesis, angiogenesis, chemotaxis, immune functions, and the induction of apoptosis [2]. Three TGF-β isoforms are expressed in mammals, i.e., TGF-β1, TGF-β2, and TGF-β3. TGF-β1 is the most abundant and universally expressed isoform [3]. In the liver, TGF-β1 is expressed in Kupffer cells, sinusoidal endothelial cells, and fat-storing perisinusoidal cells [4]. In a rat model of alcohol liver disease, TGF-β1 mRNA and protein levels were increased in Kupffer cells [5]. The proapoptotic effects of TGF-β1 in normal adult/ fetal rat/mouse hepatocytes have been shown [2]. TGF-β1 also causes apoptosis in human hepatoma cell lines, such as Hep3B or HuH7 cells [4]. The induction of apoptosis by TGF-β1 to fetal rat hepatocytes [6–8] or rat hepatocytes [9] involves enhanced production of reactive oxygen species (ROS), loss of glutathione, suppression of antioxidant gene expression [10], and decreases in mitochondrial membrane potential (Δψm) [11,12].

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Cytochrome P450 2E1 (CYP2E1), the ethanol-inducible form, metabolizes and activates many toxicologically important substrates including ethanol, carbon tetrachloride, acetaminophen, and N-nitrosodimethylamine to more toxic products [13]. Induction of CYP2E1 by ethanol is one of the central pathways by which ethanol generates a state of oxidative stress in hepatocytes. CYP2E1 is predominantly expressed in the perivenous or centrilobular region both under control conditions and subsequent to ethanol treatment, in both rats and human [14–16]. Alcoholic liver disease is usually initiated in the centrilobular region of rat liver [17,18]. The majority of phagocytic Kupffer cells are normally located in the periportal region, whereas Kupffer cells active in cytokine production are to a greater extent localized in the centrilobular region [19]. Ethanol treatment was shown to cause a very pronounced increase in expression of TGF-β1 mRNA and proprotein selectively in the perivenous area [19]. Since ethanol increases CYP2E1 and TGF-β, especially in the perivenous area of the liver acinus, we hypothesize that the combination of CYP2E1 plus TGF-β1 may promote an enhanced toxicity to liver cells and this may be an important mechanism of alcoholic liver disease. To test this hypothesis, we used a HepG2 cell line that constitutively expresses CYP2E1 (E47 cells) and a control HepG2 cell line transfected with the empty vector (C34 cells) [20]. CYP2E1-dependent toxicity in the presence of ethanol, arachidonic acid, and arachidonic acid plus iron has been characterized in these cell lines; these agents did not produce significant toxicity in C34 cells, whereas their addition to E47 cells decreased cell viability and caused either necrosis or apoptosis [21]. Though HepG2 cells express type I, II, and III TGF-β1 receptors [22] and display a primary TGF-β response [23], HepG2 cells are normally resistant to TGF-βmediated apoptosis and cell cycle arrest [24]. The high expression of Bcl-2 [25] or enhanced c-Src activity [26] may protect HepG2 cells from TGF-β1-induced apoptosis. Buenemann et al. [23] suggested that HepG2 cells may have yet unknown defects in pathways downstream of Smad and TIEG1 proteins that are involved in regulation of cell growth and apoptosis. In this study, we show that the E47 cells are more sensitive to TGF-β1-induced cell death than the control C34 cells. Production of ROS, reduced glutathione (GSH) levels, ATP levels, and mitochondrial membrane potential were evaluated as factors which may sensitize the HepG2 cells to the enhanced toxicity of TGF-β1 in CYP2E1-expressing liver cells. Materials and methods Reagents All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO), unless otherwise stated. Minimal essential medium (MEM) without phenol red, G418, penicillin, streptomycin and L-glutamine were from Invitrogen Corp. (Carlsbad, CA). MEM and trypsin-EDTA were from Mediatech, Inc. (Hemdon, VA). Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) was from EMD Biosciences, Inc. (San Diego, CA). All the plastic wares were from Becton Dickinson and Co. (Frank-

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lin Lakes, NJ). The protein concentration was determined using a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). Cell culture and rat hepatocyte preparation and treatments HepG2 cells, stably expressing the P450 enzyme CYP2E1 (E47) or vector controls (C34), were cultured as previously described [20]. Before treatments, cells were seeded on dishes or multiwell plates. After overnight culture in MEM containing 10% fetal bovine serum (FBS), the cells were washed twice with serum-free MEM or PBS and the medium was replaced with MEM containing 2% FBS and different concentrations of TGF-β1. Human recombinant TGF-β1 was reconstituted in distilled water and further diluted with PBS containing 2 mg/ml of bovine serum albumin as indicated by the manufacturer. Rats received humane care, and studies were carried out according to the criteria outline in the Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Committee approval. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), 200–250 g body weight, were injected intraperitoneally with pyrazole, 200 mg/kg body weight, once a day for 2 days to induce CYP2E1. After overnight fasting, pyrazole-treated and control rat hepatocytes were isolated by a two-step collagenase perfusion method [27]. Hepatocytes were seeded onto 24-well plates, which were coated with Matrigel basement membrane matrix (BD Biosciences, San Jose, CA) and cultured in HeptoZYME-SFM medium (Invitrogen Corp.) containing 2 mM glutamine and 100 units/ml of penicillin and 100 μg/ml streptomycin. Two to 4 hours after seeding, unattached cells were washed out, and TGF-β1 was added. Cell viability change and cytotoxicity assay Cell viability and proliferation was measured by the 3(4,5dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described [28]. Cell cytotoxicity was determined by a lactate dehydrogenase (LDH) leakage assay using the cytotoxicity detection kit (Roche Diagnostics GmbH, Penzberg, Germany). The cytotoxicity was expressed as percentage LDH release: 100% × LDHout/(LDHout + LDHin). Microscopic examination of nuclei C34 and E47 cells (1 × 104) were seeded on 24-well plates. After incubation for 72 h with 2 ng/ml of TGF-β1 in MEM containing 2% FBS, the cells were fixed in ice-cold 80% methanol solution for 30 min. The nuclei were stained with 50 μg/ml propidium iodide (PI) and the cells were subjected to inspection under a fluorescent microscope (Nikon Eclipse, TE2000). Dead cells were identified by condensed or broken nuclei [29]. Assessment of cell death by flow cytometry Cells (5 × 104) were seeded onto 6-well culture plates and treated with 2 ng/ml TGF-β1 for 72 h. Cells were trypsinized and pooled with floating cells and washed once with binding buffer (10 mM Hepes, 140 mM NaCl, 25 mM CaCl2) and incubated

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with annexin V-FITC (Molecular Probes, Inc., Eugene, OR) for 15 min at room temperature. Then 20 μg/ml of PI was added and fluorescence analysis was performed using a FACS Calibur flow cytometry apparatus (BD Biosciences) and the CELL QUEST analysis software. Annexin V-FITC generated signals were detected with the FL1 detector and PI signals were detected with the FL2 detector. The DNA content analysis for detection of apoptotic cells was carried out as described in [30]. Briefly, cells (5 × 104) were seeded onto 6-well culture plates and treated with or without TGF-β1 for 72 h. The cells were trypsinized and pooled with detached cells, washed with PBS, and centrifuged at 200g for 10 min. The pellet of cells was resuspended in 1 ml of PBS and mixed with 10 ml of 80% ethanol and stored in fixative at −20°C. Cells were centrifuged 5 min at 200g and DNA extraction buffer (0.2 M phosphate citrate buffer, pH 7.8) was added. After incubation at 37°C for 30 min and followed by centrifugation, the cells were resuspended in 0.5 ml freshly prepared staining solution containing 20 μg/ml PI, 200 μg/ml RNase A, 0.1% (v/v) Triton X-100 in PBS. Flow cytometry was carried out after 30 min incubation at room temperature. CYP2E1 activity and western blot analysis The CYP2E1 enzymatic activity was assessed with 7methoxy-4-trifluoromethylcomarin as substrate [31,32]. Cells (5 × 104) were seeded onto 6-well cell culture plates and incubated with 2 ng/ml of TGF-β1 for varying times. 7-Methoxy-4trifluoromethylcomarin in acetonitrile (final concentration of 100 μM) was added and the cells were incubated in serum and phenol red-free MEM for 2 h at 37°C. Cells were scraped into 2 ml of medium and the intensity of fluorescence was immediately read in a fluorescence spectrophotometer (Perkin-Elmer 650-10S) at 409 nm for excitation and at 530 nm for emission. Background readings from cells in which 7-methoxy-4-trifluoromethylcomarin was added just before the measurement of fluorescence were subtracted. To assay for CYP2E1 protein expression levels, 3 × 105 cells were plated in 10-cm dishes. After treatment with TGF-β1 for different times, the cells were washed twice with PBS, harvested by scraping, and centrifuged and the cell pellet was dispersed in PBS with a protease inhibitor cocktail mix (Roche Applied Science, Indianapolis, IN) and subsequently sonicated for 10 s on ice. Fifty micrograms of denatured protein was resolved on 10% SDS-PAGE and electroblotted onto nitrocellulose membranes (Osmonics Inc., Westboro, MA). The membrane was incubated with rabbit antihuman CYP2E1 polyclonal antibody (1:10,000, provided by Dr. J.M. Lasker, Hackensack Biomedical Research Institute, NJ), followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1:10,000). Detection by the chemiluminescence reaction was carried out for 1 min using the ECL Western blotting substrate (Pierce Biotechnology, Rockford, IL), followed by exposure to CL-XPosure film (Pierce Biotechnology).

diacetate (DCF-DA) as the probe [33]. C34 and E47 cells (3 × 105) were seeded in 10-cm dishes and incubated without or with 2 ng/ml of TGF-β1 for the indicated times, followed by incubation with 5 μM DCF-DA in MEM for 30 min at 37°C in the dark. The cells were washed in PBS, trypsinized, and resuspended in 3 ml of PBS, and the intensity of fluorescence was immediately read in a fluorescence spectrophotometer at 485 nm (slit width = 5 nm) for excitation and at 530 nm (slit width = 5 nm) for emission. Background readings from cells incubated without DCF-DA were subtracted (10.6 ± 1.1 AU/mg protein, n = 4). The oxidation-dependent fluorescent dye dihydroethidium (Molecular Probes, Eugene, OR) was used to S evaluate in situ production of superoxide anion (O2 − ) [28]. C34 4 or E47 cells (1 × 10 ) were seeded in 24-well plates and incubated with TGF-β1 for 72 h, followed by the addition of 40 μM dihydroethidium and 1 mM NADPH in serum and phenol red-free MEM to the surface of the cells. The cells were incubated for 30 min at 37°C in the dark and visualized with a S fluorescent microscope. To quantify the production of O2 − [34], 5 E47 cells (3 × 10 ) were seeded in 10-cm dishes and incubated with TGF-β1 for 72 h, followed by the addition of 10 μM dihydroethidium in MEM. The cells were incubated for 30 min at 37°C in the dark. The cells were washed in PBS, trypsinized, and resuspended in 2 ml of PBS, and the intensity of fluorescence was immediately read in a fluorescence spectrophotometer at 475 nm for excitation (slit width = 3 nm) and at 610 nm for emission (slit width = 20 nm). Background fluorescence readings from cells incubated without dihydroethidium were subtracted. Intracellular GSH measurement GSH levels were determined using the fluorometric substrate o-phthalaldehyde (MP Biomedicals, LLC, Aurora, OH). C34 and E47 cells (5 × 104) were cultured in 6-well plates and treated with or without 2 ng/ml of TGF-β1 for various times. Cells were washed twice with PBS. The amount of 0.4 ml of 5% (W/V) of trichloroacetic acid was added to each well and the samples were left at room temperature for 10 min to extract the GSH. The cells were scraped and centrifuged at 10,000g for 5 min at 4°C, the precipitated cellular protein pellets were resuspended in 5% (W/V) SDS in 0.1 M NaOH for protein content assay, and the supernatants were diluted 1:10 (V/V) with 0.1 M phosphate-EDTA buffer, pH 8.0. The final assay mixture contained 0.1 ml diluted supernatant, 1.8 ml of phosphate-EDTA buffer, and 0.1 ml of the o-phthalaldehyde solution containing 0.1 mg of o-phthalaldehyde. After thorough mixing and incubation at room temperature for 15 min, the solution was transferred to a cuvette. Fluorescence was determined at 420 nm for emission and 350 nm for excitation. Standard curves with known concentrations of GSH were carried out.

Intracellular production of ROS

ATP assay

Fluorescence spectrophotometry was used to measure intracellular production of ROS with 2′,7′-dichlorofluorescein

Intracellular ATP was assayed using the ENLITEN ATP assay system bioluminescence detection kit (Promega, Madison,

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Fig. 1. Cell proliferation/viability and cytotoxicity assay after TGF-β1 treatment. (A) Cell proliferation/viability after treatment with different amounts of TGF-β1 was assayed with MTT. Ten thousand of C34 and E47 cells were incubated in MEM with 2% FBS containing 1, 2, and 4 ng/ml of TGF-β1 for 72 h. The A570–630 nm of C34 and E47 cells treated with 0 ng/ml of TGF-β1 was regarded as 100%. One-way ANOVA, * P < 0.001, $ P < 0.05, $$P < 0.005 vs 0 ng/ml TGF-β1-treated cells. (B) Cell proliferation/viability after treatment for different times with 2 ng/ml TGF-β1 was assayed with MTT. The A570–630 nm of C34 and E47 cells treated with 0 ng/ml of TGF-β1 at each time point was regarded as 100%. One-way ANOVA, * P < 0.001 vs 0 h E47 cells, $P < 0.05 vs 0 h C34 cells. (C) Cytotoxicity after treatment with 2 ng/ml TGF-β1 was assayed by a LDH leakage method. Ten thousand of C34 and E47 cells were incubated in MEM without phenol red containing 2% FBS with 2 ng/ml of TGF-β1 for the indicated times. The cytotoxicity was expressed as percentage LDH release: 100% × LDHout/(LDHout + LDHin). One-way ANOVA, *P < 0.001, **P < 0.01 vs E47 cells at 0 h, respectively. $P < 0.001 vs C34 cells at 0 h.

WI) according to the kit instructions. Briefly, 5 × 104 cells were seeded in 6-well plates and treated with 2 ng/ml TGF-β1 for 24, 48, and 72 h. Four hundred microliters of 2.5% (V/V) of trichloroacetic acid was applied to extract ATP from the cells and the suspension was neutralized by adding pH 7.75 Tris-acetate buffer (1:10, V/V). Ten microliters of buffered sample was added to 100 μl of recombinant luciferase/luciferin reagent and luminescence was read in a LKB-Wallac 1251 luminescence photometer (Turku, Finland). Standard curves with known concentrations of ATP were carried out. Flow cytometry analysis of the Δψm Changes in Δψm were examined by monitoring the cells after double staining with rhodamine 123 and PI [28]. C34 and E47 cells (5 × 104) were seeded in 6-well cell culture plates and treated with or without 2 ng/ml of TGF-β1 for the

indicated times. At the end of the treatment, cells were incubated with medium containing 5 μg/ml rhodamine 123 for 1 h and harvested by trypsinization and resuspended in 0.5 ml of PBS containing 20 μg/ml of PI and analyzed by flow cytometry. Statistical analysis Results are expressed as mean ± standard deviation of at least three independent experiments carried out in duplicate or triplicate. Statistical differences were analyzed by one- or twoway ANOVA followed by multiple comparisons performed with post hoc Bonferroni test (SPSS version 10.0) with P < 0.05 as the level of significance. In the case of two-sample comparisons, a Student’s t test assuming unequal variances was used to determine whether there was a significant difference between two sample means.

1104 J. Zhuge, A.I. Cederbaum / Free Radical Biology & Medicine 41 (2006) 1100–1112 Fig. 2. TGF-β1-induced cell death in C34 and E47 cells. (A) Morphology of C34 and E47 cells after 72 h treatment with 2 ng/ml of TGF-β1 (magnification, ×300). C34 and E47 cells were fixed in ice-cold 80% methanol solution and stained with PI (50 μg/ml). The E47 cells treated with TGF-β1 displayed condensation of nuclear chromatin (arrows). (B) Assessment of cell death by flow cytometry was carried out as described under Materials and methods. Early apoptosis cells, which are the annexin V-FITC-positive/PI-negative population of cells, are reported in the lower right-hand quadrant. Necrosis or late apoptotic cells, which are the annexin V-FITC-positive/PI-positive population of cells are reported in the upper right-hand quadrant. The figure shows one typical dot plot for C34 cells and E47 cells treated with or without TGF-β1. * P < 0.01 vs control E47 cells or C34 cells treated with TGF-β1. #P < 0.01 vs C34 cells treated with TGF-β1 and P < 0.05 vs control E47 cells. (C) The DNA content analysis for detection of apoptotic cells was carried out as described under Materials and methods. The apoptotic cells are represented by the sub-G1 (M1) populations. * P < 0.01 vs C34 cells treated with TGF-β1 or control E47 cells.

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Results Cell viability change and cytotoxicity caused by TGF-β1 treatment C34 and E47 cells were treated with 1, 2, and 4 ng/ml of TGFβ1 and cell proliferation and viability were evaluated by a MTT assay. HepG2 cells are relatively insensitive to TGF-β1 treatment as reviewed in [2] and reported in [26,35] and this was confirmed for the C34 HepG2 cells. However, E47 cells were sensitive to TGF-β1 as they displayed growth inhibition compared with that of TGF-β1-treated C34 cells or control E47 cells (Fig. 1A). After 72 h of TGF-β1 treatment, C34 cells showed some growth inhibition by TGF-β1 treatment; however, this was less than the inhibition found in TGF-β1-treated E47 cells (Fig. 1B). Cytotoxicity caused by TGF-β1 was evaluated by a LDH leakage assay. E47 cells were much more sensitive than C34 cells to TGF-β1 treatment, as 10 and 31% of LDH were released to the medium after 48 and 72 h of treatment, whereas, only 3 and 7% LDH leakage was found in C34 cells (Fig. 1C). Morphological changes and apoptosis caused by TGF-β1 treatment After 72 h of TGF-β1 treatment, cell nuclear morphology was assessed by PI staining. C34 cells exhibited a normal distribution of chromosomal DNA, whereas E47 cells showed condensed chromatin, suggesting an apoptosis model of cell death (Fig. 2A). Cell death was also assessed with flow cytometry after double staining with annexin V and PI. The annexin

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V-FITC-positive/PI-negative population of cells (apoptotic cells; lower right quadrant) was not increased in C34 cells after 72 h of TGF-β1 treatment compared with control C34 cells (Fig. 2B). With the E47 cells, the apoptotic cell population increased after TGF-β1 treatment compared with that of C34 cells or control E47 cells (Fig. 2B). The annexin V-FITCpositive/PI-positive population of cells (necrosis and late apoptotic cells; upper right quadrant) also increased in E47 cells after TGF-β1 treatment compared with control E47 cells and that of C34 cells (Fig. 2B). The DNA content analysis for detection of apoptotic cells by flow cytometry showed that the percentage of pre-G1 E47 cells (17.7%) was higher than that of control E47 cells (4.8%) or C34 cells (1.6%) after TGF-β1 treatment for 72 h (Fig. 2C). These results suggest that the increased toxicity produced by the combination of TGF-β1 plus CYP2E1 was apoptotic in nature. CYP2E1 activity and expression during TGF-β1 treatment The increased toxicity found with the E47 cells compared to the C34 cells after TGF-β1 treatment is likely due to the expression of CYP2E1 in the E47 cells, and the lack of CYP2E1 expression in the C34 cells. CYP2E1 activity was assessed with 7-methoxy-4-(trifluoromethyl)comarin, an effective substrate for metabolism by CYP2E1 in intact cells [31]. E47 cells displayed stable CYP2E1 activity after 24, 48, and 72 h of culture and there was no effect on this activity by TGF-β1 treatment. C34 cells, as expected showed very low activity in the absence or presence of treatment with TGF-β1 (Fig. 3A). Western blot assay of CYP2E1 protein expression showed that the treatment

Fig. 3. CYP2E1 activity and expression in C34 and E47 cells after TGF-β1 treatment. (A) Oxidation of 7-methoxy-4-trifluoromethylcomarin was carried out as described under Materials and methods. The CYP2E1 activities of C34 and E47 cells treated with or without 2 ng/ml of TGF-β1 for 24, 48, and 72 h are shown. (B) Western blot analysis of CYP2E1 expression. C34 cells and E47 cells treated with or without 2 ng/ml of TGF-β1 for 24, 48, and 72 h were harvested and sonicated. Fifty micrograms of denatured protein was resolved on 10% SDS-PAGE and immunoblotted with rabbit anti-CYP2E1 antibody. The nonspecific immunoreactive bands were used as loading control. One representative experiment of three is shown.

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with TGF-β1 did not alter CYP2E1 protein levels in the E47 cells nor induce CYP2E1 production in the C34 cells (Fig. 3B). Protection against CYP2E1 plus TGF-β1-induced cell death To evaluate mechanisms which contribute to the TGF-β1 toxicity in CYP2E1-expressing cells, Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble vitamin E derivative antioxidant, trifluoperazine (TFP), a mitochondrial permeability transition inhibitor [36], catalase, which removes hydrogen superoxide, MnTMPyP, a cell-permeable

superoxide dismutase mimetic, which can interact with and S remove O2 − [37], and diallyl sulfide (DAS), an inhibitor of CYP2E1 [38] were added to the cell incubation during the TGFβ1 treatment, followed by assays of cell viability and LDH leakage. None of these compounds had any effect on C34 or E47 cell viability in the absence of TGF-β1. The solvent control of dimethyl sulfoxide (≤0.2%, V/V) or ethanol (0.1%, V/V) had no effects on cell viability and cytotoxicity in this model (data not shown). The MTT assay showed that the viability of E47 cells was decreased about 50% after 72 h of TGF-β1 treatment and was significantly lower compared with that of C34 cells (Fig.

Fig. 4. Protection against loss of cell viability produced by TGF-β1 treatment. C34 and E47 cells (1 × 104 cells) were seeded onto 24-well plates, and treated with 2 ng/ ml of TGF-β1 in MEM with 2% FBS (for MTT assay) or MEM without phenol red with 2% FBS (for LDH leakage assay). Some cells were also treated with either 100 μM Trolox, 2 μM TFP, 50 μM MnTMPyP, 1 KU/ml catalase, or 0.2 mM DAS and incubated for 72 h. (A) MTT assay, relative viability refers to the 100% × A570-630 nm of cells with treatment/A570–630 nm of cells without any treatment (Control). *P < 0.005, t test vs C34 cells treated with TGF-β1. #P < 0.005, ## P < 0.001, one-way ANOVA, vs TGF-β1-treated E47 cells. (B) LDH leakage assay, *P < 0.001, vs C34 cells treated with TGF-β1. #P < 0.001, one-way ANOVA, vs E47 cells treated with TGF-β1. @P < 0.05, t test vs control C34 cells. (C) Percentage of pre-G1 cells after TGF-β1 treatment. C34 and E47 cells (5 × 104) were seeded in 6-well cell culture plates and treated with or without 2 ng/ml of TGF-β1 for the indicated times. Some E47 cells incubated with TGF-β1 for 72 h were also treated with 100 μM Trolox, 2 μM TFP, or 0.2 mM DAS. At the end of the treatment, cells were treated and analyzed as described under Materials and methods. One-way ANOVA, *P < 0.05, **P < 0.001 vs control E47 cells. #P < 0.005, ##P < 0.001, vs E47 cells treated with TGF-β1 for 72 h.

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4A). Trolox, TFP, catalase, MnTMPyP, and DAS increased the viability of the E47 cells subjected to TGF-β1 treatment (Fig. 4A). Similarly the LDH leakage of E47 cells induced by TGFβ1 treatment for 72 h was largely prevented by Trolox, TFP, MnTMPyP, or DAS (Fig. 4B). LDH leakage in C34 cells was increased by TFP and TGF-β1 treatment compared with that of control C34 cells without treatment (P < 0.05, Fig. 4B) for unknown reasons; this is similar to previous results with cyclosporine A, which increased LDH leakage in serumdeprived C34 cells [28]. The percentage of pre-G1 E47 cells increased after TGF-β1 treatment for 48 and 72 h compared with control E47 cells or that of TGF-β1-treated C34 cells (Fig. 4C). This increase of pre-G1 cells could be arrested by treatment with Trolox, TFP, or DAS (Fig. 4C). These findings suggest that increased production of ROS (protection by Trolox, MnTMPyP, and catalase) and decline in Δψm (protection by TFP) may contribute to TGF-β1 and CYP2E1 potentiated toxicity. The protection by DAS further validates

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the role of CYP2E1 in the E47 cells in the enhanced toxicity with TGF-β1. Intracellular ROS production is increased after TGF-β1 treatment DCF-DA is a widely used fluorescence probe, frequently being used to detect the redox state of a cell. Although DCF-DA is used to measure the concentration of hydrogen peroxide in cells, superoxide, nitric oxide, and lipid peroxide generation are also capable of oxidizing 2′-7′-dichlorodihydrofluorescein [39]. DCF fluorescence in nontreated E47 cells was slightly higher compared with C34 cells in 10% FBS (0 h), or after incubation with 2% FBS for 24, 48, and 72 h (Fig. 5A). DCF fluorescence in E47 cells was increased after 48 and 72 h of TGF-β1 treatment compared to control E47 cells or C34 cells treated with TGF-β1 (Fig. 5A). E47 cells treated with Trolox, catalase, and DAS showed less DCF fluorescence than that of the nontreated E47

Fig. 5. Effect of TGF-β1 on intracellular ROS production. (A) Intracellular ROS level after TGF-β1 treatment was assayed using DCF-DA as described under Materials and methods. The results are expressed as arbitrary units of the fluorescence intensity per milligram of protein. *P < 0.05, t test vs control C34 cells. #P < 0.01 vs the same time point control E47 cells. (B) Effect of treatment with 100 μM Trolox, 2 μM TFP, 1 KU/ml catalase, or 0.2 mM DAS on intracellular ROS levels in E47 cells. One-way ANOVA, *P < 0.005, vs control, #P < 0.001, @P < 0.01 vs E47 cells treated with TGF-β1. (C) Production of O2 − after 72 h of TGF-β1 treatment was assayed using dihydroethidium as described under Materials and methods. Images were visualized with a fluorescent microscope (Magnification, ×300). Arrows indicate strong red fluorescence in the nuclei. (D) Effect of treatment with 100 μM Trolox, 2 μM TFP, 50 μM MnTMPyP, or 0.2 mM DAS on O2 − production by E47 cells. #P < 0.05, ##P < 0.001, one-way ANOVA, vs E47 cells treated with TGF-β1.

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cells after 72 h of TGF-β1 treatment (Fig. 5B). TFP did not decrease the elevated DCF fluorescence level of the TGF-β1treated E47 cells (Fig. 5B). The decrease in DCF fluorescence by catalase indicates that hydrogen peroxide is one of the ROS responsible for oxidation of DCF-DA. The oxidation-dependent fluorescent dye dihydroethidium S was used to evaluate in situ production of O2 −. E47 cells showed a very strong red fluorescence in the nuclei after 72 h of TGF-β1 treatment, whereas C34 cells showed weak fluorescence (Fig. S 5C). Quantification of O2 − in E47 cells after 72 h of TGF-β1 S treatment is shown in Fig. 5D. Trolox and DAS decreased O2 − S − production by E47 cells treated with TGF-β1. The O2 scavenger MnTMPyP strongly decreased fluorescence validating S the role of O2 − as the ROS interacting with dihydroethidium. S TFP did not decrease the elevated intracellular level of O2 − in TGF-β1-treated E47 cells. These results suggest that CYP2E1 is upstream of ROS production, whereas the decline in Δψm is downstream of ROS production. Intracellular GSH and ATP levels GSH is among the most important intracellular antioxidants. The intracellular GSH levels of E47 cells were higher than C34

cells when cultured in MEM with 2% FBS (two-way ANOVA, P < 0.01). After TGF-β1 treatment, the GSH levels of C34 cells were relatively stable and did not change (Fig. 6A). However, the GSH levels of E47 cells declined after TGF-β1 treatment compared with E47 cells cultured alone in 2% FBS or C34 cells treated with TGF-β1 (two-way ANOVA, P < 0.001 and P < 0.05, respectively, Fig. 6A). Oxidized glutathione levels were not elevated in the E47 cells treated with TGF-β1 (data not shown). Levels of ATP were lower in E47 cells than C34 cells without treatment as reported in [20] (Fig. 6B). Levels of ATP were stable in the E47 and C34 cells cultured in 2% FBS MEM for up to 72 h (Fig. 6B). TGF-β1 has no effect on ATP levels in the C34 cells, but produced a small decrease in the E47 cells after 72 h of treatment (Fig. 6B). Δψm is lowered by TGF-β1 treatment Δψm was assessed by flow cytometry after double staining with rhodamine 123 and PI. Rhodamine 123, a cationic dye, is selectively taken up by mitochondria to an extent that is directly proportional to Δψm. PI enters cells whose plasma membrane integrity is lost. The Δψm in C34 cells was relatively insensitive to TGF-β1 treatment as only a few percent of cells appeared on

Fig. 6. Effect of TGF-β1 on intracellular levels of reduced glutathione and ATP. (A) GSH levels were determined as described under Materials and methods after incubating with or without TGF-β1 for the indicated times. *P < 0.05, t test vs control C34 cells. (B) ATP levels were assayed using the ENLITEN ATP assay system bioluminescence detection kit. *P < 0.05, t test vs C34 cells at the same time points. #P < 0.005, t test vs 72 h untreated E47 cells.

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Fig. 7. Flow cytometry analysis of the mitochondrial membrane potential (Δψm). (A) The percentage of cell populations with decreased Δψm (low rhodamine 123 staining) but still viable (PI negative) is shown. *P < 0.01, t test, vs control E47 cells (48 h). ** P < 0.05, vs control E47 cells (72 h). (B) Protection against the decrease in Δψm. The percentage of E47 cells with low Δψm and PI negative which are not treated (2% FBS control) or treated with 2 ng/ml TGF-β1 in the absence or presence of 100 μM Trolox, 2 μM TFP, or 0.2 mM DAS for 72 h is shown. Results for E47 cells cultured in 10% FBS are also shown in the first bar. One-way ANOVA, *P < 0.001, vs control E47 cells cultured in MEM with 2% or 10% FBS. #P < 0.001, vs E47 cells treated with TGF-β1.

the rhodamine 123-negative, PI-negative field compared with control cells (Fig. 7A). After 48 or 72 h of TGF-β1 treatment, E47 cells show a significantly decreased Δψm compared with C34 cells (Fig. 7A). Importantly, this decline in Δψm occurs in PI-negative cells, i.e., cells that are still viable. Fig. 7B shows the percentage of E47 cells with lower Δψm and PI negative was significantly decreased by treatments with Trolox, TFP, and DAS (one-way ANOVA, P < 0.001, Fig. 7B). This suggests that CYP2E1 and ROS production are upstream of the decrease in Δψm. TGF-β1 decreases viability of rat hepatocytes with high levels of CYP2E1 To extend the results found with HepG2 cells to primary hepatocytes, the effect of TGF-β1 on viability of hepatocytes from pyrazole-treated rats with elevated levels of CYP2E1 was compared to that of hepatocytes from control rats. Relatively small toxicity was observed with the control hepatocytes after 3 days of treatment of TGF-β1 (Fig. 8). However, a decline in viability was found with the hepatocytes from the pyrazoletreated rats after TGF-β1 treatment for 3 days (P < 0.05, Fig. 8).

Discussion CYP2E1 plays an important role in the toxicity of various hepatotoxins, in ethanol toxicity and in elevating oxidant stress and promoting the development of nonalcoholic steatohepatitis [13]. TGF-β1 treatment induces oxidative stress and causes cell toxicity and apoptosis in a variety of models [6,9,40]. The current study evaluated whether the combination of CYP2E1 and TGF-β1 can promote oxidant injury and cellular toxicity. HepG2 cells are resistant to TGF-β1 treatment and indeed the C34 control HepG2 cells showed only a small decrease in cell growth in response to TGF-β1 treatment (MTT assay), and cell viability was minimally affected (low LDH leakage, various indices of apoptosis, and normal morphology). E47 cells that express CYP2E1 were considerably more sensitive to TGF-β1. E47 cells exhibited significant growth inhibition and cell death compared with control E47 cells incubated with MEM containing 2% FBS and TGF-β1-treated C34 cells (Fig. 1). Cell nuclear morphology, flow cytometry assays with annexin V/PI double staining, and DNA content assays all indicated that TGF-β1 treatment induced apoptosis in E47 cells to a much greater extent than in C34 cells. Cell morphology, LDH leakage, and flow

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Fig. 8. TGF-β1 toxicity in rat hepatocytes. Hepatocytes (3 × 104 cells) from pyrazole-treated and control rats were seeded onto 24-well plates and incubated in HepatoZYME with 2 ng/ml of TGF-β1 for 24, 48, and 72 h. Cell viability was assayed by MTT. The A570–630 nm of hepatocytes treated with 0 ng/ml of TGF-β1 was regarded as 100%. *P < 0.05, t test vs 72 h control hepatocytes.

cytometry assays indicated that limited necrosis may also be occurring and contributing to E47 cell death. TGF-β1 treatment caused a loss of cell viability in pyrazole-treated hepatocytes compared to control hepatocytes, extending the results with the HepG2 cells to primary liver cells. Future studies are planned to characterize the hepatocyte TGF-β1 interaction, especially with respect to the role of CYP2E1. A likely explanation as to why TGF-β1 toxicity is increased in the CYP2E1-expressing cells is an elevated oxidant stress, as ROS is being generated via TGF-β1 (likely mitochondrial) and by CYP2E1 in the endoplasmic reticulum. Indeed, DCF S fluorescence and O2 − production were elevated in E47 cells subjected to TGF-β1 as compared to C34 cells. The antioxidants Trolox, catalase, and MnTMPyP could rescue E47 cells from TGF-β1-induced cell death. They also decreased the elevated S intracellular O2 − and ROS, produced by TGF-β1 in the E47 cells, indicating that enhanced ROS production appears to be central to the mechanism leading to E47 cell death. DAS also prevented E47 cell death and decreased the elevation in S intracellular O2 − and ROS production by TGF-β1, which validates that CYP2E1 plays a critical role in the TGF-β1induced E47 cell death. In addition to an increased production of ROS, a decrease of the intracellular GSH level has been found to be an important mechanism for TGF-β1-induced apoptosis in Hep3B cells [41], the TAMH murine hepatocyte cell line [42], and fetal rat hepatocytes [6]. The GSH levels of E47 cells but not C34 cells declined gradually after 24 h of TGF-β1 treatment. The lower intracellular GSH levels likely sensitize the E47 cells to the increase in ROS caused by the combination of CYP2E1 plus TGF-β1 treatment. TFP had been used to inhibit mitochondrial membrane permeability transition and subsequently the loss of Δψm [36]. TFP effectively protected E47 cells from toxicity and the decrease of Δψm induced by TGF-β1 treatment, which suggests that a decrease in Δψm contributes to the potentiation of cell death caused by TGF-β1 treatment in the CYP2E1-expressing cells. Δψm was decreased to a greater extent in the E47 cells

than the C34 cells after TGF-β1 treatment. Importantly the decline in Δψm occurred in PI-negative cells, indicating that the decline was not a consequence of the developing toxicity but rather preceded the toxicity. The decrease of Δψm was significantly prevented by treatment with either Trolox or DAS. This indicates that ROS and CYP2E1 are upstream of the decline in Δψm. Fig. 9 shows a scheme of the proposed mechanism of TGFβ1 plus CYP2E1-induced cell death. Expression of CYP2E1 or TGF-β1 treatment each increases intracellular ROS production, which coupled to the decline of the intracellular GSH levels causes elevated oxidant stress. The increase in oxidant stress results in mitochondrial damage with loss of Δψm. Ultimately, the decline in Δψm leads to apoptosis and necrosis. Sites within this scheme where DAS, catalase, MnTMPyP, Trolox, or TFP react are shown to clarify how these additions protect against the TGF-β1 plus CYP2E1 enhanced toxicity. Chronic ethanol treatment causes liver damage largely in the pericentral zone of the liver lobule [17,18]. This area contains

Fig. 9. Summary of a proposed mechanism for the enhancement of cell death produced by TGF-β1 treatment of E47 cells. Expression of CYP2E1 and TGFβ1 treatment each increases intracellular production of ROS, which coupled to the decline of the intracellular GSH levels, results in mitochondrial damage with loss of Δψm. This ultimately leads to apoptosis and necrosis.

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the highest levels of CYP2E1. Ethanol treatment caused a very pronounced increase in expression of TGF-β1 mRNA and proprotein selectively in the perivenous area [19]. In the presence of elevated CYP2E1 expression, TGF-β1 may subject liver cells to elevated oxidant stress and potentiate cellular toxicity, analogous to the TGF-β1 treatment model with E47 cells. In summary, TGF-β1 treatment and CYP2E1 expression combine to lead to an increased oxidative stress and decline of GSH and Δψm in E47 cells, which plays a crucial role in execution of the downstream events of apoptosis and necrosis.

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This work was supported by USPHS Grant AA-06610 from the National Institute on Alcohol Abuse and Alcoholism. [21]

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