Volume 128, Issue 1, Pages 96-107 (January 2005) Hepatitis C virus core protein, cytochrome P450 2E1, and alcohol produce combined mitochondrial injury and cytotoxicity in hepatoma cells  Kazuhiro Otani, Masaaki Korenaga, Michael R. Beard, Kui Li, Ting Qian, Lori A. Showalter, Aman K. Singh, Ting Wang, Steven A. Weinman  Gastroenterology  Volume 128, Issue 1, Pages 96-107 (January 2005) DOI: 10.1053/j.gastro.2004.10.045 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Characterization of core/CYP2E1-expressing cells. (A) Time course of HCV core expression in L14 (+CYP2E1) and I10 (−CYP2E1) Huh-7 subclones. The cells were cultured in the presence (off) or absence (on) of tetracycline 2 μg/mL and harvested at the indicated times. Cell lysates were subjected to Western blot analysis with anti-HCV core monoclonal antibody. No difference was seen in the time course or expression levels of HCV core protein between L14 and I10 cells. (B) Immunofluorescence staining of HCV core protein in L14 and I10 cells. The cells were cultured in the presence or absence of tetracycline 2 μg/mL for 72 hours. HCV core protein (green) was detected by indirect immunofluorescence with anti-HCV core antibody, and nuclei were counterstained blue with 4′,6-diamidino-2-phenylindole. (C) Western blot analysis of CYP2E1 in L14 and I10 cells. HCV core induction did not affect the CYP2E1 expression. Gastroenterology 2005 128, 96-107DOI: (10.1053/j.gastro.2004.10.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Effect of HCV core protein and CYP2E1 on ROS production. Cellular ROS production was measured by oxidation of DCFDA in L14 and I10 cells with or without core expression. Cells were measured under control conditions (untreated) or after a 5-hour incubation with tBOOH 0.1 μmol/L. *P < .05 compared with untreated control; **P < .01 compared with untreated control. Gastroenterology 2005 128, 96-107DOI: (10.1053/j.gastro.2004.10.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Confocal images of ROS generation. L14 cells expressing core protein were treated with tBOOH 0.1 μmol/L and incubated with the mitochondrially localized dye TMRM (200 nmol/L). They were subsequently exposed to DCFDA (500 nmol/L) and imaged at various times. (A) TMRM fluorescence. (B–F) Oxidized DCF fluorescence at 0 minutes (B), 3 minutes (C), 6 minutes (D), 10 minutes (E), and 14 minutes (F). (G–I) Magnified details of TMRM (G), DCF (H), and overlay image (I) at 6 minutes after exposure to DCFDA. Gastroenterology 2005 128, 96-107DOI: (10.1053/j.gastro.2004.10.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Effect of HCV core protein, CYP2E1, and ethanol on oxidative cell death. (A) Cells were treated with ethanol 25 mmol/L for 24 hours, and cytotoxicity was determined by the methylthiazoletetrazolium assay. In the absence of exogenous peroxide, there was no cytotoxicity of core/CYP2E1 expression. (B) Cells were exposed to tBOOH 0.1 μmol/L with or without ethanol 25 mmol/L for 24 hours. Cytotoxicity was assessed by trypan blue exclusion. In some cases, cells were pretreated with NAC 20 mmol/L overnight and subsequently exposed to tBOOH 0.1 μmol/L, ethanol 25 mmol/L, or both in the presence of NAC 20 mmol/L for 24 hours. Expression of core and CYP2E1 synergistically enhanced the cytotoxicity induced by tBOOH (P < .05). Ethanol increased cytotoxicity in the CYP2E1-expressing cell types (P < .05), and NAC completely blocked the cytotoxicity in all cases. Gastroenterology 2005 128, 96-107DOI: (10.1053/j.gastro.2004.10.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Effect of ethanol on mitochondrial membrane potential in CYP2E1/core-expressing cells. Mitochondrial membrane potential was analyzed by flow cytometry by using the JC-1 dye. (A) Examples of ΔΨ distribution. The intensity of green (x-axis) and red (y-axis) fluorescence of each cell was plotted. Cells with a low ΔΨ are found in the right upper and right lower quadrants. L14 cells were measured after no treatment, ethanol (25 mmol/L), tBOOH (0.1 μmol/L for 48 hours), tBOOH and ethanol, or tBOOH and ethanol after preincubation with NAC 20 mmol/L. Valinomycin (0.5 μmol/L), a strong inducer of mitochondrial depolarization, was used in otherwise untreated cells for a positive control. (B) The percentage of cells with depolarized mitochondria in each condition is presented (n = 3 separate experiments). In the presence of tBOOH, core protein expression significantly increased depolarization (P < .05). The effect of ethanol on core-expressing cells was also significant (P < .05). Gastroenterology 2005 128, 96-107DOI: (10.1053/j.gastro.2004.10.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Characteristics of cell death induced by tBOOH, ethanol, and TNF. (A) Cell viability of L14 cells expressing core protein was assessed by the propidium iodide assay with or without exposure of cells to tBOOH 0.1 mmol/L, ethanol 25 mmol/L, and cyclosporin A 1 μmol/L. The percentage of dead cells at 3 hours is presented. (B) Effect of the caspase inhibitor zVAD-fmk (25 μmol/L) on oxidant-induced cell death in L14 cells expressing core and CYP2E1. Cells were exposed to tBOOH/ethanol as in (A), and cell death was monitored until approximately 60% killing occurred (4–8 hours). Cell death was compared with that in identically treated cell cultures containing zVAD-fmk. *P < .001 compared with tBOOH/ethanol without zVAD-fmk. (C) Time course of cell killing of CYP2E1-expressing L14 cells with or without core protein expression. TNF (30 ng/mL) and actinomycin D were added as indicated at time 0. *P < .005. Gastroenterology 2005 128, 96-107DOI: (10.1053/j.gastro.2004.10.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Effects of ethanol on mitochondrial antioxidant capacity and ROS production. (A) Cells were cultured in the presence or absence of ethanol for 24 hours and subsequently harvested for determination of whole-cell and mitochondrial glutathione. Core/CYP2E1 decreased mitochondrial GSH (P < .001), and ethanol produced a further decrease (P = .016). (B) ROS production was measured by confocal microscopy performed 6 minutes after the addition of 1 μmol/L DCFDA to L14 cells expressing core protein treated with either tBOOH 0.1 μmol/L or tBOOH and ethanol 25 mmol/L. Data presented are the average intensity of the perinuclear mitochondria-rich region of the cells (n = 4; *P < .05). (C) Effect of N-acetylcysteine incubation on mitochondrial GSH content in L14 cells expressing core protein. *P < .05. Gastroenterology 2005 128, 96-107DOI: (10.1053/j.gastro.2004.10.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Mitochondrial association of CYP2E1 and core protein. Mitochondria were isolated from L14 cells expressing CYP2E1 and core protein and subjected to digestion with proteinase K at the indicated concentrations for 10 minutes at 4°C, followed by immunoblotting. Markers were used to determine successful digestion of associated ER proteins (protein disulfide isomerase), mitochondrial outer membrane proteins (voltage-dependent anion channel), and intermembrane space proteins (cytochrome c). Gastroenterology 2005 128, 96-107DOI: (10.1053/j.gastro.2004.10.045) Copyright © 2005 American Gastroenterological Association Terms and Conditions