**6. Some new findings with effects of ethanol-induced oxidative stress on apoptosis in SK-Hep1 cells**

Overconsumption of alcohol is associated with deaths of about 2 million people per year throughout the world. In its early stage, fatty liver is induced, which may progress to liver disorders accompanied with hepatocyte death, inflammation, and fibrosis, further to cirrhosis and hepatocarcinoma [56, 57]. Rate of incidences of acute alcohol intoxication and heavy alcohol drinking indifferent to one's health is globally increasing and acute liver injury caused by alcohol is attracting attention [58]. Suggested molecular mechanisms for liver disorders induced by alcohol include increase of ROS and changes in various signaling pathways, but the molecular mechanism for hepatocyte death was not clarified yet. In experimental studies using model animals of ethanol-induced liver injury, it was emphasized that apoptosis is playing an important role in the pathogenesis of alcoholic

**Figure 3. The effect of GCDCA on BAP31 in HepG2 cells.**HepG2 cells treated with GCDCA (300 µM for 24 hours) were double-labeled for BAP31 with rat anti-BAP31 antibody (A, C, E, G) and for ER with Alexa Fluor 488 (B, D, F, H). Original magnification × 200. (untreated hepatocytes (A, B), hepatocytes treated with 300 µM GCDCA (C, D), 300 µM GCDCA + Z-IETD-FMK (E, F), recombinant active caspase-8 (G, H)

Some Findings on Apoptosis in Hepatocytes 213

212 Apoptosis and Medicine

be interrelated to each other.

**apoptosis in SK-Hep1 cells** 

stress-mediated pathways via death receptor, but mitochondria and ER themselves seem to

**6. Some new findings with effects of ethanol-induced oxidative stress on** 

Overconsumption of alcohol is associated with deaths of about 2 million people per year throughout the world. In its early stage, fatty liver is induced, which may progress to liver disorders accompanied with hepatocyte death, inflammation, and fibrosis, further to cirrhosis and hepatocarcinoma [56, 57]. Rate of incidences of acute alcohol intoxication and heavy alcohol drinking indifferent to one's health is globally increasing and acute liver injury caused by alcohol is attracting attention [58]. Suggested molecular mechanisms for liver disorders induced by alcohol include increase of ROS and changes in various signaling pathways, but the molecular mechanism for hepatocyte death was not clarified yet. In experimental studies using model animals of ethanol-induced liver injury, it was emphasized that apoptosis is playing an important role in the pathogenesis of alcoholic

**Figure 3. The effect of GCDCA on BAP31 in HepG2 cells.**HepG2 cells treated with GCDCA (300 µM for 24 hours) were double-labeled for BAP31 with rat anti-BAP31 antibody (A, C, E, G) and for ER with Alexa Fluor 488 (B, D, F, H). Original magnification × 200. (untreated hepatocytes (A, B), hepatocytes treated with 300 µM GCDCA (C, D), 300 µM GCDCA + Z-IETD-FMK (E, F), recombinant active caspase-8 (G, H)

**Figure 4. The effect of GCDCA on the CHOP mRNA expression in isolated heaptocytes.** (A) CHOP mRNA expression was determined by semiquantitative PCR. Line graphs representation of the observed fold induction normalized to β-actin for each time point. (B) RT-PCR analysis of CHOP and βactin expression at each incubation time after treatment with GCDCA. (C) Reaction cycles-PCR product yield curves of each reaction mixture were plotted. The intensity of fluorescence was fitted to the data in the linear portion of curves. The resulting CHOP mRNA / β-actin mRNA ratio is represented as the mean+SEM of 8-15 samples. \*p<0.05 and \*\*p<0.01: significant difference from untreated hepatocytes (control).

hepatitis or alcoholic liver cirrhosis [3, 59]. Clinical studies suggested similar findings. Various factors including cytotoxicity by alcohol and its metabolites, changes in metabolizing enzymes, invasion of inflammatory cells, reactive oxygen species, cytokines, hepatic microcirculation, nutritional factors, etc. are involved in the onset of apoptosis. Among others, correlation between ROS or oxidative stress and hepatocyte apoptosis is attracting attention. Orally fed ethanol is absorbed in the upper digestive organs, mainly small intestine, and 90% of it is metabolized in the liver. Upon metabolization, ROS is generated in the liver by alcohol dehydrogenase, microsome-ethanol oxydizing system (MEOS) via cytochrome P450, and NADPH oxidase (NOX) in the cell membrane, which is a center for ROS generation (Fig. 5). In general, oxidative stress inflicts cytotoxicity when excessive ROS is generated in the cells. Antioxidant was shown to reduce hepatocyte apoptosis in acute ethanol-addicted rats [60]. On the other hand, several death receptors and their ligands (especially Fas/FasL) are over-expressed in the liver cells of alcoholic hepatitis patients compared with those in healthy subjects. The levels of Fas and FasL are increased in serious alcoholic hepatitis patients, but there are still many uncertainties about the details of their mediators and biological importance [61]. The increase in FasL might be mediated by ROS or increase in NF-кB which increases the transcription of Fas and FasL genes based on TNF-α inducing activity [62]. In fact, serum TNF-α level increases also in alcoholic hepatitis patients, and plays an important role in inflicting hepatotoxicity [63]. Chronic ethanol administration increases expressions of TNF-R in hepatocytes [64], and during the exposure to ethanol, hepatocytes underwent apoptosis induced by TNF-α. TNF-α/ TNF-R1 system seems to be required in the cell death mediated by Fas. In fact, recent studies showed that in TNF-R1/TNF-R2 double knockout mice, apoptosis mediated by TNF-α did not occur, and the mice showed resistance against induced fulminant hepatic failure [65]. The activation of TNF-α/TNF-R1 complex may be co-working with the signaling conveyed by Fas for inducing hepatocyte apoptosis.

Some Findings on Apoptosis in Hepatocytes 215

**Figure 6. The effect of ethanol on NOX-4 and p22phox mRNA in SK-HEP-1 cells.** (A): The expression of NOX-4 mRNA in SK-HEP1 treated with ethanol (200 mM) and NAC (10 mM). (B): The expression of p22phox mRNA in SK-HEP1 treated with ethanol and NAC. The resulting NOX-4 or p22phox mRNA/βactin mRNA ratio is represented as the mean ± S.E.M. of 8-12 samples. (C): RT-PCR analysis of NOX-4 or p22phox and β-actin expression after treatment with ethanol or ethanol+NAC. Lane1, 2: untreated SK-HEP1 cells, Lane3, 4: 200 mM ethanol, Lane5, 6: ethanol+NAC, Lane7, 8: 10 mM NAC. \*p < 0.05:

significant difference from untreated cells, # p < 0.05: significant difference from 200 mM ethanol-treated

In a recent study on the actions of ethanol on hepatocytes, the importance of mitogenactivated protein kinases (MAPKs) (mainly, ERK1/2, p38 and JNK1/2) and histone modification (acetylation, methylation, or phosphorylation) is emphasized. MAPK pathway is correlated with many signaling pathways including tyrosine, serine/threonine kinase, G protein and calcium signals [69]. MAPKs are a family of protein kinases of which main members are ERK1 and ERK2, p38MAPK, and c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK). MAPKs regulate various biological processes including cell growth, proliferation, movement, inflammation, fatty degeneration, necrosis and apoptosis [70]. In a primary culture of rat hepatocytes, ethanol showed modest activation of ERK1/2 and notable activation of JNK [71]. In rat cultured hepatocytes, when ERK1/2 phosphorylation was inhibited by U-0126 (a MEK1/2 inhibitor), phosphorylation of JNK by ethanol was increased [72]. In previous studies on neurons, ethanol was found to activate

MAPK cascade and increase ROS generation via p38MAPK pathway [73, 74].

cells.

**Figure 5. The effect of ethanol on ROS generation in SK-HEP1 cells.** SK-HEP1 cells (1×106 cells/ well) were treated with ethanol (25-300 mM) for 5 hours at 37C. The generation of ROS in SK-HEP1 treated with ethanol was analyzed spectrofluorometrically. Each value represents the mean ± S.E.M. of 6-12 samples. \*:p<0.05: significant difference from untreated SK-HEP-1 cells.

Histologically, nick-end label positive apoptotic cells were abundantly found around Mallory body in biopsied tissue from patients with alcoholic liver injury [66]. In in vivo animal studies, hepatocyte apoptosis is increased in animals fed with alcohol-containing feed, and long-term alcohol administration is suggested to induce hepatocyte death [67]. Apoptotic cells are observed even in normal cells in some part around the central vein, but in rats fed with alcohol for a long term, many apoptotic cells were observed in various parts around the central vein.

We ourselves reported the ethanol-induced apoptosis in cultured hepatocytes, and showed that apoptosis is induced in vitro in the presence of alcohol. We also found that at ethanol concentrations lower than those that induce apoptosis, significant increase in ROS generation was observed. It was suggested that in the process of apoptosis induced by ethanol, ROS generation by NOX was important as inducer of apoptosis [68]. As shown in Fig. 6, in the presence of 200 µM ethanol, the mRNA expression of p22phox, which is a protein constitutively bound to NOX to enhance the action of NOX4, was found significantly increased and the increase of the expression was suppressed by the pretreatment with Nacetyl-L-cysteine (NAC), a precursor of glutathione and an antioxidant.

inducing hepatocyte apoptosis.

around the central vein.

TNF-α inducing activity [62]. In fact, serum TNF-α level increases also in alcoholic hepatitis patients, and plays an important role in inflicting hepatotoxicity [63]. Chronic ethanol administration increases expressions of TNF-R in hepatocytes [64], and during the exposure to ethanol, hepatocytes underwent apoptosis induced by TNF-α. TNF-α/ TNF-R1 system seems to be required in the cell death mediated by Fas. In fact, recent studies showed that in TNF-R1/TNF-R2 double knockout mice, apoptosis mediated by TNF-α did not occur, and the mice showed resistance against induced fulminant hepatic failure [65]. The activation of TNF-α/TNF-R1 complex may be co-working with the signaling conveyed by Fas for

**Figure 5. The effect of ethanol on ROS generation in SK-HEP1 cells.** SK-HEP1 cells (1×106 cells/ well) were treated with ethanol (25-300 mM) for 5 hours at 37C. The generation of ROS in SK-HEP1 treated with ethanol was analyzed spectrofluorometrically. Each value represents the mean ± S.E.M. of 6-12

Histologically, nick-end label positive apoptotic cells were abundantly found around Mallory body in biopsied tissue from patients with alcoholic liver injury [66]. In in vivo animal studies, hepatocyte apoptosis is increased in animals fed with alcohol-containing feed, and long-term alcohol administration is suggested to induce hepatocyte death [67]. Apoptotic cells are observed even in normal cells in some part around the central vein, but in rats fed with alcohol for a long term, many apoptotic cells were observed in various parts

We ourselves reported the ethanol-induced apoptosis in cultured hepatocytes, and showed that apoptosis is induced in vitro in the presence of alcohol. We also found that at ethanol concentrations lower than those that induce apoptosis, significant increase in ROS generation was observed. It was suggested that in the process of apoptosis induced by ethanol, ROS generation by NOX was important as inducer of apoptosis [68]. As shown in Fig. 6, in the presence of 200 µM ethanol, the mRNA expression of p22phox, which is a protein constitutively bound to NOX to enhance the action of NOX4, was found significantly increased and the increase of the expression was suppressed by the pretreatment with N-

samples. \*:p<0.05: significant difference from untreated SK-HEP-1 cells.

acetyl-L-cysteine (NAC), a precursor of glutathione and an antioxidant.

**Figure 6. The effect of ethanol on NOX-4 and p22phox mRNA in SK-HEP-1 cells.** (A): The expression of NOX-4 mRNA in SK-HEP1 treated with ethanol (200 mM) and NAC (10 mM). (B): The expression of p22phox mRNA in SK-HEP1 treated with ethanol and NAC. The resulting NOX-4 or p22phox mRNA/βactin mRNA ratio is represented as the mean ± S.E.M. of 8-12 samples. (C): RT-PCR analysis of NOX-4 or p22phox and β-actin expression after treatment with ethanol or ethanol+NAC. Lane1, 2: untreated SK-HEP1 cells, Lane3, 4: 200 mM ethanol, Lane5, 6: ethanol+NAC, Lane7, 8: 10 mM NAC. \*p < 0.05: significant difference from untreated cells, # p < 0.05: significant difference from 200 mM ethanol-treated cells.

In a recent study on the actions of ethanol on hepatocytes, the importance of mitogenactivated protein kinases (MAPKs) (mainly, ERK1/2, p38 and JNK1/2) and histone modification (acetylation, methylation, or phosphorylation) is emphasized. MAPK pathway is correlated with many signaling pathways including tyrosine, serine/threonine kinase, G protein and calcium signals [69]. MAPKs are a family of protein kinases of which main members are ERK1 and ERK2, p38MAPK, and c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK). MAPKs regulate various biological processes including cell growth, proliferation, movement, inflammation, fatty degeneration, necrosis and apoptosis [70]. In a primary culture of rat hepatocytes, ethanol showed modest activation of ERK1/2 and notable activation of JNK [71]. In rat cultured hepatocytes, when ERK1/2 phosphorylation was inhibited by U-0126 (a MEK1/2 inhibitor), phosphorylation of JNK by ethanol was increased [72]. In previous studies on neurons, ethanol was found to activate MAPK cascade and increase ROS generation via p38MAPK pathway [73, 74].

We found that exposure of cultured hepatocytes to ethanol increase generation of ROS and MAPK (p38MAPK and JNK) phosphorylation activity. However, ROS generation was not significantly affected when hepatocytes were pretreated with MAPK inhibitors (SB202190 for p38MAPK, and SP600125 for JNK) [75]. These results suggest that ROS may be generated by the upstream effector of p38 MAPK (Fig. 7).

Some Findings on Apoptosis in Hepatocytes 217

found to be correlated with the increase in mRNAs of CYP2E1, GRP78, SREBP-1c and also increase in protein levels of CYP2E1, GRP78, nuclear SREBP-1c and caspase-12 activity [78].

We found that when human hepatocarcinoma cell line, SK-Hep1, were exposed to ethanol, expressions of mRNAs of BIP, CHOP, and sXBP-1 were increased. The ethanol-induced increase in expressions were suppressed by NAC, an antioxidant, and we speculated the increase to be associated with oxidative stress induced by ethanol. We also observed a transient increase in intracellular Ca2+ and calpain activity, but they were not suppressed by NAC, and we believe these were independent from ethanol-induced oxidative stress. It was suggested, therefore, there are two pathways of ethanol-induced apoptosis mediated by ER stress, that is, ethanol-induced oxidative stress-dependent and independent pathways [79]. Possible approaches for the treatment of ER stress induced by ethanol include decreasing homocysteine and increase in SAM by betain or folic acid [80-82], improvement of protein folding using chemical chaperone, PBA(sodium phenylbutyrate) and TUDCA [81, 83, 84], inhibition of dephosphorylation of eukaryotic initiation factor-2α (eIF2α) using its inhibitor salubrinal [85], and reducing oxidative stress by decreasing ROS generation from oxidized protein using antioxidants. However, the results of clinical study cannot be obtained. Mechanism of ethanol-induced ER stress is too complex, and the approaches for treatment in human may not be so simple or universal. It may be necessary to employ properly

**7. Proposed conceptual diagram of apoptosis process in hepatocytes** 

Apoptosis is an indispensable process in line with cell proliferation for maintenance of tissue homeostasis and health by removal of injured and/or aged cells. This is especially important in the liver where cells are exposed to toxins and viruses [1]. Any loss of balance between cell death and proliferation due to excessive or insufficient apoptosis always leads to pathologic conditions due to unstable state. In the liver, massive hepatocyte apoptosis is observed in acute hepatic failure, and persistent hepatocyte apoptosis is associated with fibrilization, chronic dysfunction and cancerous transformation of the liver [86]. Apoptosis is induced by various intracellular and extracellular stimuli. In all types of hepatocytes, death receptors (especially Fas) are universally expressed [87], and hepatocyte apoptosis is usually transmitted by external pathways. Especially, activations of Fas and TNF-R1 are correlated with hepatocyte apoptosis in various liver diseases including viral hepatitis, fulminant hepatitis, cholestatic liver disease, alcoholic hepatitis, non-alcoholic fatty-liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), Wilson's disease, and ischemiareperfusion injury [88]. In the study of cholestasis, because no liver injury was observed after ligation of the common bile duct (a model of extrahepatic cholestasis) in Fas-knockout mice, high levels of toxic intracellular bile salts were speculated to increase Fas in the cell membrane resulting in the activation of the receptor [89, 10]. Furthermore, apoptosis is induced via ER stress. Caspase-8 activated via death receptor may regulate ER stress mediated by BAP31 on ER [54]. In alcoholic liver injury, ROS generation is enhanced via NOX, and the excessive ROS interact with Fas death receptor [8] to induce mitochondria-

combined therapy of every known beneficial medication.

Furthermore, as described above, overconsumption of alcohol induces various pathological stress responses and a part of them is endoplasmic reticulum (ER) stress response. ER stress is associated with alcoholic injury in such organs as the liver, pancreas, heart and brain. The possible mechanism for triggering alcoholic ER stress response is directly or indirectly correlated with alcohol metabolism, which, in turn, is correlated with toxic acetaldehyde and homocysteine, oxidative stress, upset of calcium or iron homeostasis, decrease in the ratio of S-adenosylmethionine/S-adenosyl- homocysteine and abnormal epigenetic modifications. Inhibition of triggering process of ER stress could hopefully be beneficial in the treatment of alcoholic diseases.

In the study of genetic expression in ethanol-fed mice, remarkable increase in caspase-12 mRNA and BIP, a ER chaperone, and CHOP mRNA [76]. When the protein levels were examined, BIP, CHOP and caspase-12 were increased. When CHOP null mice was fed with ethanol, apoptosis of hepatocyte was found to be dependent on CHOP [77], showing that ethanol-induced hepatic injury was associated with hepatocyte apoptosis mediated by CHOP. Furthermore, when micropigs were fed with ethanol, fatty liver and apoptosis are found to be correlated with the increase in mRNAs of CYP2E1, GRP78, SREBP-1c and also increase in protein levels of CYP2E1, GRP78, nuclear SREBP-1c and caspase-12 activity [78].

216 Apoptosis and Medicine

treated cells.

the treatment of alcoholic diseases.

by the upstream effector of p38 MAPK (Fig. 7).

We found that exposure of cultured hepatocytes to ethanol increase generation of ROS and MAPK (p38MAPK and JNK) phosphorylation activity. However, ROS generation was not significantly affected when hepatocytes were pretreated with MAPK inhibitors (SB202190 for p38MAPK, and SP600125 for JNK) [75]. These results suggest that ROS may be generated

**Figure 7. The effect of MAPK inhibitors on ROS generation in ethanol-induced apoptotic SK-Hep1 cells.** Generation of ROS in SK-Hep1 cells treated with 200 mM ethanol or MAPK inhibitors (SB202190: p38 inhibitor, SP600125: JNK inhibitor) + 200 mM ethanol was analyzed spectrofluorometrically. \*p < 0.05: significant difference from untreated cells, # p < 0.05: significant difference from 200 mM ethanol-

Furthermore, as described above, overconsumption of alcohol induces various pathological stress responses and a part of them is endoplasmic reticulum (ER) stress response. ER stress is associated with alcoholic injury in such organs as the liver, pancreas, heart and brain. The possible mechanism for triggering alcoholic ER stress response is directly or indirectly correlated with alcohol metabolism, which, in turn, is correlated with toxic acetaldehyde and homocysteine, oxidative stress, upset of calcium or iron homeostasis, decrease in the ratio of S-adenosylmethionine/S-adenosyl- homocysteine and abnormal epigenetic modifications. Inhibition of triggering process of ER stress could hopefully be beneficial in

In the study of genetic expression in ethanol-fed mice, remarkable increase in caspase-12 mRNA and BIP, a ER chaperone, and CHOP mRNA [76]. When the protein levels were examined, BIP, CHOP and caspase-12 were increased. When CHOP null mice was fed with ethanol, apoptosis of hepatocyte was found to be dependent on CHOP [77], showing that ethanol-induced hepatic injury was associated with hepatocyte apoptosis mediated by CHOP. Furthermore, when micropigs were fed with ethanol, fatty liver and apoptosis are We found that when human hepatocarcinoma cell line, SK-Hep1, were exposed to ethanol, expressions of mRNAs of BIP, CHOP, and sXBP-1 were increased. The ethanol-induced increase in expressions were suppressed by NAC, an antioxidant, and we speculated the increase to be associated with oxidative stress induced by ethanol. We also observed a transient increase in intracellular Ca2+ and calpain activity, but they were not suppressed by NAC, and we believe these were independent from ethanol-induced oxidative stress. It was suggested, therefore, there are two pathways of ethanol-induced apoptosis mediated by ER stress, that is, ethanol-induced oxidative stress-dependent and independent pathways [79].

Possible approaches for the treatment of ER stress induced by ethanol include decreasing homocysteine and increase in SAM by betain or folic acid [80-82], improvement of protein folding using chemical chaperone, PBA(sodium phenylbutyrate) and TUDCA [81, 83, 84], inhibition of dephosphorylation of eukaryotic initiation factor-2α (eIF2α) using its inhibitor salubrinal [85], and reducing oxidative stress by decreasing ROS generation from oxidized protein using antioxidants. However, the results of clinical study cannot be obtained. Mechanism of ethanol-induced ER stress is too complex, and the approaches for treatment in human may not be so simple or universal. It may be necessary to employ properly combined therapy of every known beneficial medication.
