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Heme oxygenase 1 protects ethanol-administered liver tissue in Aldh2 knockout mice
Alcohol 52 (2016) 49e54
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Heme oxygenase 1 protects ethanol-administered liver tissue in Aldh2 knockout mice Akiko Matsumoto a, *, David Thompson b, Ying Chen c,1, Vasilis Vasiliou c,1, Toshihiro Kawamoto d, Masayoshi Ichiba a a
Department of Social Medicine, Saga University School of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan Department of Clinical Pharmacy, University of Colorado School of Pharmacy, 12850 E. Montview Blvd., Aurora, CO 80045, USA Department of Pharmaceutical Sciences, University of Colorado School of Pharmacy, Aurora, CO 80045, USA d Department of Environmental Health, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi, Kitakyushu, Fukuoka 807-0804, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 August 2015 Received in revised form 14 December 2015 Accepted 18 February 2016
A genetic polymorphism of the aldehyde dehydrogenase 2 (ALDH2) gene, ALDH2*2, encodes an enzymatically defective ALDH2 protein. Recent epidemiological studies suggest that possessing ALDH2*2 is a protective factor for liver tissue in healthy individuals, although these studies lack a mechanistic explanation. Our animal studies have shown the same trend: levels of serum alanine transaminase (ALT), hepatic malondialdehyde (MDA), and hepatic tumor necrosis factor alpha (TNF-a) were lower in Aldh2 knockout (Aldh2/) mice than in wild-type (Aldh2þ/þ) mice after ethanol administration. To propose a mechanistic hypothesis, residual liver specimens from the previous experiment were analyzed. An antioxidative protein, heme oxygenase 1 (HO-1), and an oxidative stress-producing protein, cytochrome P450 2E1 (CYP2E1), were detected at higher levels in Aldh2/ mice than in Aldh2þ/þ mice, regardless of ethanol treatment. Other oxidative stress-related proteins and inflammatory cytokines did not show such a significant difference. To conclude, we propose a protective role of HO-1 in individuals with A LDH2*2. Our continued studies support the epidemiological finding that possession of ALDH2*2 is a protective factor in the liver of the healthy individual. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: ALDH2 CYP2E1 HO-1 Oxidative stress Polymorphism
Introduction Generally, alcohol is recognized to have an adverse effect on liver tissue. Excessive drinking causes alcoholic liver disease or more rapid progression of chronic liver disease (Jamal, Saadi, & Morgan, 2005; Liangpunsakul, 2011; Parés et al., 1990). However, according to epidemiological studies, a drinking habit in healthy people (with normal BMI and triglyceride) does not elevate serum ALT, AST, and GGT levels (Murata et al., 2003; Takeshita, Yang, & Morimoto, 2000). This trend is also observed in experimental animals. For example, our previous in vivo study showed that ethanol administration itself did not induce serum ALT elevation, inflammation, or necrotic change (Matsumoto et al., 2008). To induce pathological changes in liver tissue, a ‘stimulus’ or ‘promoter’ such as a high-fat diet or lipopolysaccharide treatment needs to be applied (Bertola, Mathews, Ki, Wang, & Gao, 2013;
Conflicts of interest: none. * Corresponding author. Tel.: þ81 952 34 2289; fax: þ81 952 34 2065. E-mail address: [email protected] (A. Matsumoto). 1 Present address. Department of Environmental Health Sciences, Yale School of Public Health, 60 College St., New Haven, CT 06520, USA. http://dx.doi.org/10.1016/j.alcohol.2016.02.004 0741-8329/Ó 2016 Elsevier Inc. All rights reserved.
McMullen et al., 2005; Minagawa, Deng, Liu, Tsukamoto, & Dennert, 2004; Thakur, Pritchard, McMullen, & Nagy, 2006). A genetic polymorphism of aldehyde dehydrogenase 2 (ALDH2) has been studied as a candidate ‘promoter’ of alcoholic liver injury (Yokoyama et al., 2013). ALDH2 plays a primary role in the metabolism of acetaldehyde, an oxidized metabolite of ethanol (Isse, Matsuno, Oyama, Kitagawa, & Kawamoto, 2005; Peng, Chen, Tsao, Wang, & Yin, 2007). Individuals possessing a defective ALDH2 allele, ALDH2*2, are highly sensitive to the adverse effects of ethanol because their severe deficiency in ALDH2 enzymatic activity results in an accumulation of acetaldehyde (Peng et al., 2007). The ALDH2*2 allele is common in East Asia, with some studies estimating that approximately half of the populations of Japan and China carry it (Chen et al., 2006; Takeshita, Morimoto, Mao, Hashimoto, & Furuyama, 1993). Surprisingly, the presence of ALDH2*2 is a protective factor in certain disease scenarios (Kato et al., 2011; Nagasawa et al., 2007; Suzuki et al., 2004; Wang et al., 2013; Yoshimasu et al., 2015), although it is a well known risk factor for cancerous diseases (Chen et al., 2006; Hira et al., 2013). In liver tissue, the lack of ALDH2 has been suggested to have adverse effects such as fibrogenesis (Chen, 2002; Novitskiy, Potter, Rennie-Tankersley, & Mezey, 2004) and tumorigenesis (Matsuda et al., 2007; Sakamoto et al., 2006).
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However, epidemiological studies reported that alcohol-related liver disease and chronic hepatitis C were not exacerbated by the presence of the ALDH2*2 allele (Yang, Takeshita, Hirata, Sato, & Morimoto, 1999; Zintzaras, Stefanidis, Santos, & Vidal, 2006). Moreover, Takeshita and colleagues showed that in subjects consuming moderate to high levels of alcohol, serum ALT, AST, and GGT were lower in those with ALDH2*2 (Takeshita et al., 2000). Murata and colleagues demonstrated that ALDH2*2 is an independent protector against an elevated serum ALT level, using logistic regression analysis with multiple variates including BMI and drinking habits (Murata et al., 2003). This is an important observation, although is not yet recognized in the field, possibly because a persuasive explanation of the mechanism is still lacking. Currently, serum ALT, AST, and GGT levels are used for markers of alcohol ingestion. However, these are of no use in people with ALDH2*2, as they could become even lower than baseline levels after alcohol intake (Matsumoto et al., 2008; Matsumoto, Vasiliou, Kawamoto, Tanaka, & Ichiba, 2014; Murata et al., 2003; Takeshita et al., 2000). As a result, the opportunity to warn people with ALDH2*2 of the potential for alcohol overdose could be lost. Furthermore, individuals with ALDH2*2 may need to refrain from drinking as they show a higher incidence of cancerous disease (Matsuda et al., 2007; Yokoyama, Mizukami, & Yokoyama, 2015). Our previous studies using Aldh2/ mice have shown a consistent trend, in which lower levels of serum ALT (Kwon et al., 2014; Matsumoto et al., 2008, 2014), tumor necrosis factor alpha (TNF-a) (Matsumoto et al., 2008), and a marker of oxidative stress, malondialdehyde (MDA) (Matsumoto, Ichiba, et al., 2007; Matsumoto, Kawamoto, et al., 2007), in liver tissue were observed in Aldh2/ mice compared with those in Aldh2þ/þ mice. This evidence suggests the existence of a liver-protective mechanism that is upregulated, or a deteriorating mechanism that is downregulated, by carrying the ALDH2*2 allele. Heme oxygenase (HO) metabolizes heme, a molecule that is highly toxic in large concentrations, to biliverdin. HO-1 is the inducible isoform of HO, being expressed in certain organs including liver tissue (Tenhunen, Marver, & Schmid, 1968; Wegiel, Hauser, & Otterbein, 2015). Subsequently, biliverdin is reduced to bilirubin by biliverdin reductase. The bilirubin-biliverdin cycle is an essential defense mechanism against cellular oxidative stress (Sedlak et al., 2009). Similarly, glutathione (GSH) is a well-known and effective cytoprotective molecule (Sedlak et al., 2009). It was previously reported that mice that overexpressed ALDH2*2 showed a remodeled metabolism with an enhanced GSH-cytoprotective system (Endo et al., 2009). In our previous study, higher GSH levels were observed in Aldh2/ mice than in wild-type mice after a single dose of ethanol-administered by gavage (Matsumoto, Ichiba, et al., 2007). Additionally, cytochrome P450 2E1 (CYP2E1) is a well-known oxidant producer (Caro & Cederbaum, 2004), and its mRNA level was lower in Aldh2/ mice than in wild-type mice in our previous report (Matsumoto, Kawamoto, et al., 2007). Continuing from our previous report, the present study explores why the expected adverse effects of ethanol could be counteracted by the possession of the ALDH2*2 gene. As possible protecting factors, HO-1, glutamate-cysteine ligase, catalytic subunit (GCLC), and thioredoxin reductase 1 (TXNRD1) were analyzed in mouse liver tissue, as well as a possible deteriorating factor, CYP2E1.
mice (10e11 weeks old) were provided with one bottle of ethanol solution and standard hard feed for 5 weeks. Mean ethanol intake was approximately 14 g/day/kg body weight for the ethanol treatment groups of both genotypes. A summary of basic information on the subjects is shown in the supplementary material (Table S1). Saga University approved all the animal experiments. Sample analysis Western blot Hepatic tissue was homogenized in 0.25 M sucrose. Protein concentration was determined using a Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Samples (30 mg of protein) were subjected to 4e12% bis-tris gel (Invitrogen, Carlsbad, CA) electrophoresis. After transfer to a polyvinylidene fluoride membrane, proteins were analyzed by Western blotting using commercial antibodies of ALT, CYP2E1, cytochrome P450 reductase (CPR), GAPDH, GCLC, HO-1, and TXNRD1 (Table S2). CPR, distributed over the endoplasmic reticulum (ER), supplies electrons to ER-resident enzymes including CYP2E1 and HO-1 (Huber et al., 2009; Strittmatter et al., 1974). Therefore it was chosen as an internal control for CYP2E1 and HO-1. Secondary antibodies were detected by enhanced chemiluminescence using an LAS-3000 fluoro-image analyzer (Fuji Film, Tokyo, Japan). Signal intensities for ALT, GCLC, and TXNRD1 were normalized to GAPDH, and CYP2E1 and HO-1 were normalized to CPR. Immunohistochemistry Immunohistochemistry for CYP2E1 was performed on deparaffinized and dehydrated liver sections (4 mm). Slides were treated with 3% H2O2 in 8% skim milk to prevent nonspecific reactions. Tissue sections were incubated for 1 h at 4 C with the primary antibody, rabbit polyclonal CYP2E1 antibody (BIOMOL International LP, Plymouth Meeting, PA). Color development required the reaction of biotinylated secondary antibody and chromogen (DAB) (Sigma Chemical, St. Louis, MO). Sections were further stained with hematoxylin to detect cell nuclei. Multiplex immunoassay Pro-inflammatory cytokines, interleukin (IL)-1b, IL-2, interferon-g (IFNg) and granulocyte-macrophage colony-stimulating factor (GM-CSF), and an anti-inflammatory cytokine, IL-10, in liver tissue were analyzed with a Bio-Plex system (Bio-Rad, Hercules, CA) using a Mouse Cytokine 8-Plex Panel (Bio-Rad). Liver protein was extracted using a Bio-Plex Cell Lysis Kit (Bio-Rad). Statistical analyses The ManneWhitney U test was performed for comparison between genotypes or between control and ethanol groups. Spearman’s rank correlation analysis was performed to detect the association between two parameters. p < 0.05 was considered to indicate significance. StatView for Windows Version 5.0 (SAS Institute Inc., Cary, NC) was used for statistical analysis.
Materials and methods
Results
Animals and experimental protocols
Low serum ALT level does not reflect reduced ALT content in liver cells
All experiments described herein used a part of the residual female mouse liver from a previously performed experiment (Matsumoto et al., 2008) that had been stored at 80 C. Female
To confirm that low levels of serum ALT were not the consequence of reduced ALT in the hepatocytes, the residual liver tissue
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Fig. 1. Hepatic ALT was unaffected by Aldh2 genotype and ethanol administration. ALT expression levels in liver tissue were assessed by Western blotting. Female mice (10e11 weeks old) were fed with one bottle of ethanol solution and standard hard feed for 5 weeks (mean ethanol intake was approximately 14 g/kg/day body weight).Quantitative data are shown as the mean SEM (n ¼ 4e8).
was analyzed (Fig. 1). There was no significant difference in hepatic ALT levels either between genotypes (p ¼ 0.17) or with ethanol treatment (p ¼ 0.65). The correlation analysis between the hepatic ALT level and serum ALT level (Table S1) showed no significant association (r ¼ 0.07). Oxidative stress-related proteins The level of HO-1 in Aldh2/ mice was almost twice that of the wild-type mice, regardless of ethanol treatment, while there were no significant differences in GCLC and TXNRD1 levels (Fig. 2). The level of CYP2E1, a radical-producing protein, was also higher in Aldh2/ mice than in wild-type mice, regardless of ethanol treatment (Fig. 3). Ethanol treatment caused a further increase of CYP2E1 in Aldh2/ mice (Fig. 3). CYP2E1 was detected in the centrilobular area without a distributive difference between genotypes (Fig. 3). CYP2E1 and HO-1 levels were significantly correlated in both genotypes (r ¼ 0.8, p < 0.05 for both genotypes). No such correlation was found for GCLC and TXNRD1 levels. Inflammatory cytokines Further investigation of the biochemical mechanism underlying these observations was attempted by analyzing five types of cytokine. In our previous report, 10 days of ethanol feeding with a highfat/low-carbohydrate diet was followed by binge ethanol feeding. We found Aldh2/ mice exhibited greater inflammatory responses than wild-type mice did after the ethanol administration, even though Aldh2/ mice showed lower levels of serum ALT than wildtype mice (Kwon et al., 2014). In the present study, inflammatory cytokine levels did not change, even in Aldh2/ mice (Fig. 4). Discussion HO-1 is a potential protector of Aldh2
/
hepatic MDA and TNF-a in Aldh2/ mice than in wild-type mice (Matsumoto et al., 2008). Because hepatic ALT expression in liver tissue was not reduced (Fig. 1), lowering serum ALT may reflect reduced injury of hepatic cells. The present study suggests that HO-1 is a potential protective factor that ameliorates hepatocyte injury because of its antioxidant role (Kim et al., 2011; Wegiel et al., 2015). One limitation of this study, however, is that the actual activity of HO-1 was not assessed because the protein level and its activity do not correspond completely. It has been reported that HO-1 enzyme activity can be affected by some environmental factors, such as hydrogen peroxide concentration, without any change in the protein concentration (Huber et al., 2009). Induction of an anti-stress system may compensate for genetic disadvantage Because HO-1 was induced in untreated Aldh2/ mice (Fig. 2), its induction occurred in stages earlier than 15-weeks old. This is a reasonable alteration because individuals lacking ALDH2 may have been exposed to relatively high levels of endogenous aldehydes from birth, such as 4-hydroxy-2-nonenal and MDA (Brichac et al., 2007; Yoval-Sánchez & RodríguezZavala, 2012), and an appropriate defense response is required. A similar stress-response mechanism is also indicated in cardiac tissue. Induction of a series of glutathione-producing enzymes in ALDH2*2-overexpressing mice was shown by Endo and colleagues (Endo et al., 2009), and a further caseecontrol study proved their hypothesis (Zhang, Gong, Zhang, Li, & Hu, 2012). A more recent study suggested another mechanism for the amelioration of hepatocyte injury in Aldh2/ mice; IL-6-induced STAT3 relieves hepatic steatosis and lowers serum ALT levels (Kwon et al., 2014). Upregulation of CYP2E1 in Aldh2/ mouse liver
mouse liver tissue
Our previous study showed decreased levels of serum ALT after ethanol intake in Aldh2/ mice (Table S1) and lower levels of
As well as HO-1, CYP2E1 is also shown to be upregulated in Aldh2/ mouse liver (Fig. 3). It is thought that CYP2E1 is induced in individuals lacking ALDH2 to compensate for the loss of metabolic
Fig. 2. Modulation of anti-oxidative proteins in liver tissue by Aldh2 genotype and ethanol administration. Anti-oxidative protein levels were assessed by Western blot. Female mice (10e11 weeks old) were fed with one bottle of ethanol solution and standard hard feed for 5 weeks (mean ethanol intake was approximately 14 g/kg/day body weight). Quantitative data are shown as the mean SEM (n ¼ 4e8). ap < 0.05 vs. Aldh2þ/þ.
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Fig. 3. Modulation of hepatic CYP2E1 by Aldh2 genotype and ethanol administration. Evaluation of CYP2E1 level was performed by Western blot (A, B) and immunohistochemistry (C) (positive staining is shown in brown). CYP2E1 and HO-1 showed a positive correlation (D) (r ¼ 0.84, p < 0.0001). Female mice (10e11 weeks old) were fed with one bottle of ethanol solution and standard hard feed for 5 weeks (average ethanol intake was approximately 14 g/day/kg body weight). Quantitative data are shown as the mean SEM (n ¼ 4e8). **p < 0.01 vs. control group. aap < 0.01 vs. Aldh2þ/þ. CV, central vein.
capacity, because CYP2E1 metabolizes various kinds of chemicals, including acetaldehyde (Kunitoh et al., 1997; Terelius, NorstenHöög, Cronholm, & Ingelman-Sundberg, 1991). This finding was seemingly inconsistent with our previous study, which reported that CYP2E1 mRNA levels in Aldh2/ mouse liver decreased 12 h after 5 g/kg ethanol by gavage (Matsumoto, Kawamoto, et al., 2007). However, a subsequent study revealed that CYP2E1 protein production was induced 12 h after an intraperitoneal injection of the same ethanol dosage (Matsumoto et al., in preparation). As such, CYP2E1 protein stabilization is often reported to be induced by its substrates through a post-translational mechanism (Gonzalez, 2007). A possible role of CYP2E1 to upregulate anti-stress systems It is well known that CYP2E1 generates oxidative stress (Caro & Cederbaum, 2004). However, a series of studies by Cederbaum and colleagues suggested CYP2E1 has a role in inducing anti-oxidative proteins. They showed that the CYP2E1-overexpressing HepG2 cell line had higher HO-1, and identified Nrf2 as the regulator of antioxidant genes (Cederbaum, 2006). This proposed mechanism, the CYP2E1-Nrf2-HO-1 pathway, is also supported by in vivo studies recently performed by others (Ahmad et al., 2014; Lu, Zhang, & Cederbaum, 2012). The present study also showed a
significant correlation between HO-1 and CYP2E1 levels in the tissue samples (Fig. 3D). However, it failed to show the correlative induction of GCLC and TXNRD1, which is a downstream factor of Nrf2 as well as HO-1 (Hayes & Dinkova-Kostova, 2014; Mulcahy, Wartman, Bailey, & Gipp, 1997). This indicates that further investigation is required to consider other proposed regulatory mechanisms of HO-1 (Wegiel et al., 2015) or factors interfering in the expression of GCLC and TXNRD1. Our current series of animal studies supported the epidemiological finding that possessing the ALDH2*2 allele is a hepatoprotective factor in healthy individuals (Murata et al., 2003; Takeshita et al., 2000; Takeuchi et al., 2011), and this has important consequences, especially in the field of preventive medicine. Unfortunately it has been neglected in both the clinical and laboratory settings, possibly because of the lack of understanding and knowledge of the overall cellular response. We herein suggested a hypothetical mechanism based on induction of a compensatory anti-stress system, and the subsequent upregulation of HO-1 by CYP2E1. Further studies on other stress-related factors and signaling pathways are required to elucidate the entire cytoprotective mechanism of Aldh2/ mice. Further verification of HO-1 using other ethanol administration models, including an alcoholic fatty liver-inducing model, are needed to help confirm the hypothesis discussed in this study.
Fig. 4. Hepatic levels of inflammatory cytokines in females. Pro-inflammatory cytokines, interleukin (IL)-1b, IL-2, interferon-g (IFNg) and granulocyte-macrophage colonystimulating factor (GM-CSF), and an anti-inflammatory cytokine, IL-10, in liver tissue were analyzed by multiplex immunoassays. Female mice (10e11 weeks old) were fed with one bottle of ethanol solution and standard hard feed for 5 weeks (average ethanol intake was approximately 14 g/kg/day body weight). Data are shown as the mean SEM (n ¼ 4e8).
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Conclusion The antioxidant protein HO-1 represents a potential factor in providing a compensatory protective mechanism for Aldh2/ mice. While induced CYP2E1 in Aldh2/ mice would produce adverse effects in the cells, it may play a role in upregulating the expression of antioxidant proteins such as HO-1. Our serial studies provide strong support for the important epidemiological finding that possession of ALDH2*2 is a protective factor against overexposure to alcohol in healthy individuals. Acknowledgments This work was supported by JSPS Grant-in-Aid for Young Scientists (B) Grant Number 25870515. The authors are grateful to Tatsuya Takahashi, Akane Matsumoto, Akiko Inoue Kisaki, and Nicole Payton for their technical assistance. Appendix. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.alcohol.2016.02.004. References Ahmad, I., Shukla, S., Singh, D., Chauhan, A. K., Kumar, V., Singh, B. K., et al. (2014). CYP2E1-mediated oxidative stress regulates HO-1 and GST expression in maneb- and paraquat-treated rat polymorphonuclear leukocytes. Molecular and Cellular Biochemistry, 393, 209e222. Bertola, A., Mathews, S., Ki, S. H., Wang, H., & Gao, B. (2013). Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nature Protocols, 8, 627e637. Brichac, J., Ho, K. K., Honzatko, A., Wang, R., Lu, X., Weiner, H., et al. (2007). Enantioselective oxidation of trans-4-hydroxy-2-nonenal is aldehyde dehydrogenase isozyme and Mg2þ dependent. Chemical Research in Toxicology, 20, 887e895. Caro, A. A., & Cederbaum, A. I. (2004). Oxidative stress, toxicology, and pharmacology of CYP2E1. Annual Review of Pharmacology and Toxicology, 44, 27e42. Cederbaum, A. I. (2006). Cytochrome P450 2E1-dependent oxidant stress and upregulation of anti-oxidant defense in liver cells. Journal of Gastroenterology and Hepatology, 21(Suppl. 3), S22eS25. Chen, A. (2002). Acetaldehyde stimulates the activation of latent transforming growth factor-beta1 and induces expression of the type II receptor of the cytokine in rat cultured hepatic stellate cells. The Biochemical Journal, 368, 683e693. Chen, Y. J., Chen, C., Wu, D. C., Lee, C. H., Wu, C. I., Lee, J. M., et al. (2006). Interactive effects of lifetime alcohol consumption and alcohol and aldehyde dehydrogenase polymorphisms on esophageal cancer risks. International Journal of Cancer, 119, 2827e2831. Endo, J., Sano, M., Katayama, T., Hishiki, T., Shinmura, K., Morizane, S., et al. (2009). Metabolic remodeling induced by mitochondrial aldehyde stress stimulates tolerance to oxidative stress in the heart. Circulation Research, 105, 1118e1127. Gonzalez, F. J. (2007). The 2006 Bernard B. Brodie Award Lecture. Cyp2e1. Drug Metabolism and Disposition, 35, 1e8. Hayes, J. D., & Dinkova-Kostova, A. T. (2014). The Nrf2 regulatory network provides an interface between redox and intermediary metabolism. Trends in Biochemical Sciences, 39, 199e218. Hira, A., Yabe, H., Yoshida, K., Okuno, Y., Shiraishi, Y., Chiba, K., et al. (2013). Variant ALDH2 is associated with accelerated progression of bone marrow failure in Japanese Fanconi anemia patients. Blood, 122, 3206e3209. Huber, W. J., 3rd, Marohnic, C. C., Peters, M., Alam, J., Reed, J. R., Masters, B. S., et al. (2009). Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system. Drug Metabolism and Disposition, 37, 857e864. Isse, T., Matsuno, K., Oyama, T., Kitagawa, K., & Kawamoto, T. (2005). Aldehyde dehydrogenase 2 gene targeting mouse lacking enzyme activity shows high acetaldehyde level in blood, brain, and liver after ethanol gavages. Alcoholism: Clinical and Experimental Research, 29, 1959e1964. Jamal, M. M., Saadi, Z., & Morgan, T. R. (2005). Alcohol and hepatitis C. Digestive Diseases (Basel, Switzerland), 23, 285e296. Kato, N., Takeuchi, F., Tabara, Y., Kelly, T. N., Go, M. J., Sim, X., et al. (2011). Meta-analysis of genome-wide association studies identifies common variants associated with blood pressure variation in East Asians. Nature Genetics, 43, 531e538. Kim, H. P., Pae, H. O., Back, S. H., Chung, S. W., Woo, J. M., Son, Y., et al. (2011). Heme oxygenase-1 comes back to endoplasmic reticulum. Biochemical and Biophysical Research Communications, 404, 1e5.
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Heme oxygenase 1 protects ethanol-administered liver tissue in Aldh2 knockout mice Akiko Matsumoto a, *, David Thompson b, Ying Chen c,1, Vasilis Vasiliou c,1, Toshihiro Kawamoto d, Masayoshi Ichiba a a
Department of Social Medicine, Saga University School of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan Department of Clinical Pharmacy, University of Colorado School of Pharmacy, 12850 E. Montview Blvd., Aurora, CO 80045, USA Department of Pharmaceutical Sciences, University of Colorado School of Pharmacy, Aurora, CO 80045, USA d Department of Environmental Health, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi, Kitakyushu, Fukuoka 807-0804, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 August 2015 Received in revised form 14 December 2015 Accepted 18 February 2016
A genetic polymorphism of the aldehyde dehydrogenase 2 (ALDH2) gene, ALDH2*2, encodes an enzymatically defective ALDH2 protein. Recent epidemiological studies suggest that possessing ALDH2*2 is a protective factor for liver tissue in healthy individuals, although these studies lack a mechanistic explanation. Our animal studies have shown the same trend: levels of serum alanine transaminase (ALT), hepatic malondialdehyde (MDA), and hepatic tumor necrosis factor alpha (TNF-a) were lower in Aldh2 knockout (Aldh2/) mice than in wild-type (Aldh2þ/þ) mice after ethanol administration. To propose a mechanistic hypothesis, residual liver specimens from the previous experiment were analyzed. An antioxidative protein, heme oxygenase 1 (HO-1), and an oxidative stress-producing protein, cytochrome P450 2E1 (CYP2E1), were detected at higher levels in Aldh2/ mice than in Aldh2þ/þ mice, regardless of ethanol treatment. Other oxidative stress-related proteins and inflammatory cytokines did not show such a significant difference. To conclude, we propose a protective role of HO-1 in individuals with A LDH2*2. Our continued studies support the epidemiological finding that possession of ALDH2*2 is a protective factor in the liver of the healthy individual. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: ALDH2 CYP2E1 HO-1 Oxidative stress Polymorphism
Introduction Generally, alcohol is recognized to have an adverse effect on liver tissue. Excessive drinking causes alcoholic liver disease or more rapid progression of chronic liver disease (Jamal, Saadi, & Morgan, 2005; Liangpunsakul, 2011; Parés et al., 1990). However, according to epidemiological studies, a drinking habit in healthy people (with normal BMI and triglyceride) does not elevate serum ALT, AST, and GGT levels (Murata et al., 2003; Takeshita, Yang, & Morimoto, 2000). This trend is also observed in experimental animals. For example, our previous in vivo study showed that ethanol administration itself did not induce serum ALT elevation, inflammation, or necrotic change (Matsumoto et al., 2008). To induce pathological changes in liver tissue, a ‘stimulus’ or ‘promoter’ such as a high-fat diet or lipopolysaccharide treatment needs to be applied (Bertola, Mathews, Ki, Wang, & Gao, 2013;
Conflicts of interest: none. * Corresponding author. Tel.: þ81 952 34 2289; fax: þ81 952 34 2065. E-mail address: [email protected] (A. Matsumoto). 1 Present address. Department of Environmental Health Sciences, Yale School of Public Health, 60 College St., New Haven, CT 06520, USA. http://dx.doi.org/10.1016/j.alcohol.2016.02.004 0741-8329/Ó 2016 Elsevier Inc. All rights reserved.
McMullen et al., 2005; Minagawa, Deng, Liu, Tsukamoto, & Dennert, 2004; Thakur, Pritchard, McMullen, & Nagy, 2006). A genetic polymorphism of aldehyde dehydrogenase 2 (ALDH2) has been studied as a candidate ‘promoter’ of alcoholic liver injury (Yokoyama et al., 2013). ALDH2 plays a primary role in the metabolism of acetaldehyde, an oxidized metabolite of ethanol (Isse, Matsuno, Oyama, Kitagawa, & Kawamoto, 2005; Peng, Chen, Tsao, Wang, & Yin, 2007). Individuals possessing a defective ALDH2 allele, ALDH2*2, are highly sensitive to the adverse effects of ethanol because their severe deficiency in ALDH2 enzymatic activity results in an accumulation of acetaldehyde (Peng et al., 2007). The ALDH2*2 allele is common in East Asia, with some studies estimating that approximately half of the populations of Japan and China carry it (Chen et al., 2006; Takeshita, Morimoto, Mao, Hashimoto, & Furuyama, 1993). Surprisingly, the presence of ALDH2*2 is a protective factor in certain disease scenarios (Kato et al., 2011; Nagasawa et al., 2007; Suzuki et al., 2004; Wang et al., 2013; Yoshimasu et al., 2015), although it is a well known risk factor for cancerous diseases (Chen et al., 2006; Hira et al., 2013). In liver tissue, the lack of ALDH2 has been suggested to have adverse effects such as fibrogenesis (Chen, 2002; Novitskiy, Potter, Rennie-Tankersley, & Mezey, 2004) and tumorigenesis (Matsuda et al., 2007; Sakamoto et al., 2006).
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However, epidemiological studies reported that alcohol-related liver disease and chronic hepatitis C were not exacerbated by the presence of the ALDH2*2 allele (Yang, Takeshita, Hirata, Sato, & Morimoto, 1999; Zintzaras, Stefanidis, Santos, & Vidal, 2006). Moreover, Takeshita and colleagues showed that in subjects consuming moderate to high levels of alcohol, serum ALT, AST, and GGT were lower in those with ALDH2*2 (Takeshita et al., 2000). Murata and colleagues demonstrated that ALDH2*2 is an independent protector against an elevated serum ALT level, using logistic regression analysis with multiple variates including BMI and drinking habits (Murata et al., 2003). This is an important observation, although is not yet recognized in the field, possibly because a persuasive explanation of the mechanism is still lacking. Currently, serum ALT, AST, and GGT levels are used for markers of alcohol ingestion. However, these are of no use in people with ALDH2*2, as they could become even lower than baseline levels after alcohol intake (Matsumoto et al., 2008; Matsumoto, Vasiliou, Kawamoto, Tanaka, & Ichiba, 2014; Murata et al., 2003; Takeshita et al., 2000). As a result, the opportunity to warn people with ALDH2*2 of the potential for alcohol overdose could be lost. Furthermore, individuals with ALDH2*2 may need to refrain from drinking as they show a higher incidence of cancerous disease (Matsuda et al., 2007; Yokoyama, Mizukami, & Yokoyama, 2015). Our previous studies using Aldh2/ mice have shown a consistent trend, in which lower levels of serum ALT (Kwon et al., 2014; Matsumoto et al., 2008, 2014), tumor necrosis factor alpha (TNF-a) (Matsumoto et al., 2008), and a marker of oxidative stress, malondialdehyde (MDA) (Matsumoto, Ichiba, et al., 2007; Matsumoto, Kawamoto, et al., 2007), in liver tissue were observed in Aldh2/ mice compared with those in Aldh2þ/þ mice. This evidence suggests the existence of a liver-protective mechanism that is upregulated, or a deteriorating mechanism that is downregulated, by carrying the ALDH2*2 allele. Heme oxygenase (HO) metabolizes heme, a molecule that is highly toxic in large concentrations, to biliverdin. HO-1 is the inducible isoform of HO, being expressed in certain organs including liver tissue (Tenhunen, Marver, & Schmid, 1968; Wegiel, Hauser, & Otterbein, 2015). Subsequently, biliverdin is reduced to bilirubin by biliverdin reductase. The bilirubin-biliverdin cycle is an essential defense mechanism against cellular oxidative stress (Sedlak et al., 2009). Similarly, glutathione (GSH) is a well-known and effective cytoprotective molecule (Sedlak et al., 2009). It was previously reported that mice that overexpressed ALDH2*2 showed a remodeled metabolism with an enhanced GSH-cytoprotective system (Endo et al., 2009). In our previous study, higher GSH levels were observed in Aldh2/ mice than in wild-type mice after a single dose of ethanol-administered by gavage (Matsumoto, Ichiba, et al., 2007). Additionally, cytochrome P450 2E1 (CYP2E1) is a well-known oxidant producer (Caro & Cederbaum, 2004), and its mRNA level was lower in Aldh2/ mice than in wild-type mice in our previous report (Matsumoto, Kawamoto, et al., 2007). Continuing from our previous report, the present study explores why the expected adverse effects of ethanol could be counteracted by the possession of the ALDH2*2 gene. As possible protecting factors, HO-1, glutamate-cysteine ligase, catalytic subunit (GCLC), and thioredoxin reductase 1 (TXNRD1) were analyzed in mouse liver tissue, as well as a possible deteriorating factor, CYP2E1.
mice (10e11 weeks old) were provided with one bottle of ethanol solution and standard hard feed for 5 weeks. Mean ethanol intake was approximately 14 g/day/kg body weight for the ethanol treatment groups of both genotypes. A summary of basic information on the subjects is shown in the supplementary material (Table S1). Saga University approved all the animal experiments. Sample analysis Western blot Hepatic tissue was homogenized in 0.25 M sucrose. Protein concentration was determined using a Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Samples (30 mg of protein) were subjected to 4e12% bis-tris gel (Invitrogen, Carlsbad, CA) electrophoresis. After transfer to a polyvinylidene fluoride membrane, proteins were analyzed by Western blotting using commercial antibodies of ALT, CYP2E1, cytochrome P450 reductase (CPR), GAPDH, GCLC, HO-1, and TXNRD1 (Table S2). CPR, distributed over the endoplasmic reticulum (ER), supplies electrons to ER-resident enzymes including CYP2E1 and HO-1 (Huber et al., 2009; Strittmatter et al., 1974). Therefore it was chosen as an internal control for CYP2E1 and HO-1. Secondary antibodies were detected by enhanced chemiluminescence using an LAS-3000 fluoro-image analyzer (Fuji Film, Tokyo, Japan). Signal intensities for ALT, GCLC, and TXNRD1 were normalized to GAPDH, and CYP2E1 and HO-1 were normalized to CPR. Immunohistochemistry Immunohistochemistry for CYP2E1 was performed on deparaffinized and dehydrated liver sections (4 mm). Slides were treated with 3% H2O2 in 8% skim milk to prevent nonspecific reactions. Tissue sections were incubated for 1 h at 4 C with the primary antibody, rabbit polyclonal CYP2E1 antibody (BIOMOL International LP, Plymouth Meeting, PA). Color development required the reaction of biotinylated secondary antibody and chromogen (DAB) (Sigma Chemical, St. Louis, MO). Sections were further stained with hematoxylin to detect cell nuclei. Multiplex immunoassay Pro-inflammatory cytokines, interleukin (IL)-1b, IL-2, interferon-g (IFNg) and granulocyte-macrophage colony-stimulating factor (GM-CSF), and an anti-inflammatory cytokine, IL-10, in liver tissue were analyzed with a Bio-Plex system (Bio-Rad, Hercules, CA) using a Mouse Cytokine 8-Plex Panel (Bio-Rad). Liver protein was extracted using a Bio-Plex Cell Lysis Kit (Bio-Rad). Statistical analyses The ManneWhitney U test was performed for comparison between genotypes or between control and ethanol groups. Spearman’s rank correlation analysis was performed to detect the association between two parameters. p < 0.05 was considered to indicate significance. StatView for Windows Version 5.0 (SAS Institute Inc., Cary, NC) was used for statistical analysis.
Materials and methods
Results
Animals and experimental protocols
Low serum ALT level does not reflect reduced ALT content in liver cells
All experiments described herein used a part of the residual female mouse liver from a previously performed experiment (Matsumoto et al., 2008) that had been stored at 80 C. Female
To confirm that low levels of serum ALT were not the consequence of reduced ALT in the hepatocytes, the residual liver tissue
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Fig. 1. Hepatic ALT was unaffected by Aldh2 genotype and ethanol administration. ALT expression levels in liver tissue were assessed by Western blotting. Female mice (10e11 weeks old) were fed with one bottle of ethanol solution and standard hard feed for 5 weeks (mean ethanol intake was approximately 14 g/kg/day body weight).Quantitative data are shown as the mean SEM (n ¼ 4e8).
was analyzed (Fig. 1). There was no significant difference in hepatic ALT levels either between genotypes (p ¼ 0.17) or with ethanol treatment (p ¼ 0.65). The correlation analysis between the hepatic ALT level and serum ALT level (Table S1) showed no significant association (r ¼ 0.07). Oxidative stress-related proteins The level of HO-1 in Aldh2/ mice was almost twice that of the wild-type mice, regardless of ethanol treatment, while there were no significant differences in GCLC and TXNRD1 levels (Fig. 2). The level of CYP2E1, a radical-producing protein, was also higher in Aldh2/ mice than in wild-type mice, regardless of ethanol treatment (Fig. 3). Ethanol treatment caused a further increase of CYP2E1 in Aldh2/ mice (Fig. 3). CYP2E1 was detected in the centrilobular area without a distributive difference between genotypes (Fig. 3). CYP2E1 and HO-1 levels were significantly correlated in both genotypes (r ¼ 0.8, p < 0.05 for both genotypes). No such correlation was found for GCLC and TXNRD1 levels. Inflammatory cytokines Further investigation of the biochemical mechanism underlying these observations was attempted by analyzing five types of cytokine. In our previous report, 10 days of ethanol feeding with a highfat/low-carbohydrate diet was followed by binge ethanol feeding. We found Aldh2/ mice exhibited greater inflammatory responses than wild-type mice did after the ethanol administration, even though Aldh2/ mice showed lower levels of serum ALT than wildtype mice (Kwon et al., 2014). In the present study, inflammatory cytokine levels did not change, even in Aldh2/ mice (Fig. 4). Discussion HO-1 is a potential protector of Aldh2
/
hepatic MDA and TNF-a in Aldh2/ mice than in wild-type mice (Matsumoto et al., 2008). Because hepatic ALT expression in liver tissue was not reduced (Fig. 1), lowering serum ALT may reflect reduced injury of hepatic cells. The present study suggests that HO-1 is a potential protective factor that ameliorates hepatocyte injury because of its antioxidant role (Kim et al., 2011; Wegiel et al., 2015). One limitation of this study, however, is that the actual activity of HO-1 was not assessed because the protein level and its activity do not correspond completely. It has been reported that HO-1 enzyme activity can be affected by some environmental factors, such as hydrogen peroxide concentration, without any change in the protein concentration (Huber et al., 2009). Induction of an anti-stress system may compensate for genetic disadvantage Because HO-1 was induced in untreated Aldh2/ mice (Fig. 2), its induction occurred in stages earlier than 15-weeks old. This is a reasonable alteration because individuals lacking ALDH2 may have been exposed to relatively high levels of endogenous aldehydes from birth, such as 4-hydroxy-2-nonenal and MDA (Brichac et al., 2007; Yoval-Sánchez & RodríguezZavala, 2012), and an appropriate defense response is required. A similar stress-response mechanism is also indicated in cardiac tissue. Induction of a series of glutathione-producing enzymes in ALDH2*2-overexpressing mice was shown by Endo and colleagues (Endo et al., 2009), and a further caseecontrol study proved their hypothesis (Zhang, Gong, Zhang, Li, & Hu, 2012). A more recent study suggested another mechanism for the amelioration of hepatocyte injury in Aldh2/ mice; IL-6-induced STAT3 relieves hepatic steatosis and lowers serum ALT levels (Kwon et al., 2014). Upregulation of CYP2E1 in Aldh2/ mouse liver
mouse liver tissue
Our previous study showed decreased levels of serum ALT after ethanol intake in Aldh2/ mice (Table S1) and lower levels of
As well as HO-1, CYP2E1 is also shown to be upregulated in Aldh2/ mouse liver (Fig. 3). It is thought that CYP2E1 is induced in individuals lacking ALDH2 to compensate for the loss of metabolic
Fig. 2. Modulation of anti-oxidative proteins in liver tissue by Aldh2 genotype and ethanol administration. Anti-oxidative protein levels were assessed by Western blot. Female mice (10e11 weeks old) were fed with one bottle of ethanol solution and standard hard feed for 5 weeks (mean ethanol intake was approximately 14 g/kg/day body weight). Quantitative data are shown as the mean SEM (n ¼ 4e8). ap < 0.05 vs. Aldh2þ/þ.
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Fig. 3. Modulation of hepatic CYP2E1 by Aldh2 genotype and ethanol administration. Evaluation of CYP2E1 level was performed by Western blot (A, B) and immunohistochemistry (C) (positive staining is shown in brown). CYP2E1 and HO-1 showed a positive correlation (D) (r ¼ 0.84, p < 0.0001). Female mice (10e11 weeks old) were fed with one bottle of ethanol solution and standard hard feed for 5 weeks (average ethanol intake was approximately 14 g/day/kg body weight). Quantitative data are shown as the mean SEM (n ¼ 4e8). **p < 0.01 vs. control group. aap < 0.01 vs. Aldh2þ/þ. CV, central vein.
capacity, because CYP2E1 metabolizes various kinds of chemicals, including acetaldehyde (Kunitoh et al., 1997; Terelius, NorstenHöög, Cronholm, & Ingelman-Sundberg, 1991). This finding was seemingly inconsistent with our previous study, which reported that CYP2E1 mRNA levels in Aldh2/ mouse liver decreased 12 h after 5 g/kg ethanol by gavage (Matsumoto, Kawamoto, et al., 2007). However, a subsequent study revealed that CYP2E1 protein production was induced 12 h after an intraperitoneal injection of the same ethanol dosage (Matsumoto et al., in preparation). As such, CYP2E1 protein stabilization is often reported to be induced by its substrates through a post-translational mechanism (Gonzalez, 2007). A possible role of CYP2E1 to upregulate anti-stress systems It is well known that CYP2E1 generates oxidative stress (Caro & Cederbaum, 2004). However, a series of studies by Cederbaum and colleagues suggested CYP2E1 has a role in inducing anti-oxidative proteins. They showed that the CYP2E1-overexpressing HepG2 cell line had higher HO-1, and identified Nrf2 as the regulator of antioxidant genes (Cederbaum, 2006). This proposed mechanism, the CYP2E1-Nrf2-HO-1 pathway, is also supported by in vivo studies recently performed by others (Ahmad et al., 2014; Lu, Zhang, & Cederbaum, 2012). The present study also showed a
significant correlation between HO-1 and CYP2E1 levels in the tissue samples (Fig. 3D). However, it failed to show the correlative induction of GCLC and TXNRD1, which is a downstream factor of Nrf2 as well as HO-1 (Hayes & Dinkova-Kostova, 2014; Mulcahy, Wartman, Bailey, & Gipp, 1997). This indicates that further investigation is required to consider other proposed regulatory mechanisms of HO-1 (Wegiel et al., 2015) or factors interfering in the expression of GCLC and TXNRD1. Our current series of animal studies supported the epidemiological finding that possessing the ALDH2*2 allele is a hepatoprotective factor in healthy individuals (Murata et al., 2003; Takeshita et al., 2000; Takeuchi et al., 2011), and this has important consequences, especially in the field of preventive medicine. Unfortunately it has been neglected in both the clinical and laboratory settings, possibly because of the lack of understanding and knowledge of the overall cellular response. We herein suggested a hypothetical mechanism based on induction of a compensatory anti-stress system, and the subsequent upregulation of HO-1 by CYP2E1. Further studies on other stress-related factors and signaling pathways are required to elucidate the entire cytoprotective mechanism of Aldh2/ mice. Further verification of HO-1 using other ethanol administration models, including an alcoholic fatty liver-inducing model, are needed to help confirm the hypothesis discussed in this study.
Fig. 4. Hepatic levels of inflammatory cytokines in females. Pro-inflammatory cytokines, interleukin (IL)-1b, IL-2, interferon-g (IFNg) and granulocyte-macrophage colonystimulating factor (GM-CSF), and an anti-inflammatory cytokine, IL-10, in liver tissue were analyzed by multiplex immunoassays. Female mice (10e11 weeks old) were fed with one bottle of ethanol solution and standard hard feed for 5 weeks (average ethanol intake was approximately 14 g/kg/day body weight). Data are shown as the mean SEM (n ¼ 4e8).
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