Methylenetetrahydrofolate reductase gene polymorphisms in patients with nonalcoholic steatohepatitis (NASH)

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1 cell biochemistry and function Cell Biochem Funct 2008; 26: Published online 11 June 2007 in Wiley InterScience ( Methylenetetrahydrofolate reductase gene polymorphisms in patients with nonalcoholic steatohepatitis (NASH) Ali Sazci 1 *, Emel Ergul 1, Cem Aygun 2, Gurler Akpinar 1, Omer Senturk 2 and Sadettin Hulagu 2 1 Faculty of Medicine, Department of Medical Biology, University of Kocaeli, Umuttepe, Kocaeli, Turkey 2 Faculty of Medicine, Department of Gastroenterology, University of Kocaeli, Umuttepe, Kocaeli, Turkey Nonalcoholic fatty liver disease (NAFLD) is the most common cause of abnormal hepatic steatosis in the absence of a history of alcohol use. Nonalcoholic steatohepatitis (NASH) is the progressive form of NAFLD. Hyperhomocysteinemia causes steatosis, and the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms result in hyperhomocysteinemia. To examine whether the C677T and A1298C polymorphisms of the MTHFR gene were associated with NASH, we analysed the allele and genotype distribution of the MTHFR C677T and A1298C polymorphisms in 57 well-diagnosed NASH patients, 324 healthy controls in a case-control study of Turkish subjects of Caucasian origin. The diagnosis of the NASH patients was based on liver biopsy. The method used in the analysis of genotypes was PCR-RFLP. The MTHFR A1298C polymorphism was significantly associated with NASH (x 2 ¼ 8.439; p ¼ 0.015) in the total NASH patients compared with healthy controls. The MTHFR 1298C allele (odds ratio (OR) ¼ 2.480; 95%CI ¼ ; x 2 ¼ 7.703; df ¼ 1; p ¼ 0.006) was significantly associated with NASH in the total NASH patients. The MTHFR C677C/A1298C compound genotype (OR ¼ 2.218; 95%CI ¼ ; x 2 ¼ 3.998; df ¼ 1; p ¼ 0.046) in men patients was also significantly associated with NASH. Likewise the MTHFR C1298C genotype was significantly associated with NASH in women patients with NASH (OR ¼ 2.979; 95%CI ¼ ; x 2 ¼ 4.343; df ¼ 1; p ¼ 0.037). In conclusion, the MTHFR 1298C allele in all NASH patients, C1298C genotype, C677C/C1298C compound genotype in women NASH patients and C677C/A1298C compound genotype in men NASH patients were genetic risk factors for NASH. Copyright # 2007 John Wiley & Sons, Ltd. key words MTHFR; polymorphism; gender association; nonalcoholic steatohepatitis (NASH); Turkey INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is the most common metabolic syndrome in liver disease ranging from simple fatty liver to steatohepatitis, cirrhosis and hepatocellular carcinoma. 1 NAFLD is known as liver steatosis in patients whose alcohol consumption is not enough to cause hepatic injury. NAFLD is also known to be associated with some drugs, genetic defects, obesity, insulin resistance and type 2 diabetes. Nonalcoholic steatohepatitis (NASH) is the most progressive form of NAFLD. 2 NASH has been shown to have the * Correspondence to: Dr A. Sazci, PhD, Professor of Human Genetics, Department of Medical Biology, University of Kocaeli, Umuttepe, Kocaeli, Turkey.Tel: Fax: alisazci@gmail.com potential to progress to cirrhosis and hepatocellular carcinoma. 3,4 NASH may be a leading cause of cryptogenic cirrhosis in which aetiologically pathological features can no longer be differentiated. 5 NAFLD is also recognized as a significant form of liver disease in paediatric populations, that in some, it may progress to cirrhosis in adulthood. 6,7 The enzyme methylenetetrahydrofolate reductase (MTHFR; EC ) catalyses the conversion of the 5,10-methylenetetrahydrofolate into 5-methytetrahydofolate which is then used for remethyletion of homocysteine to methionine. 8 DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity during cellular differentiation, is catalysed by methyltransferases that utilize the universal methyl donor S-adenosyl-Lmethionine. 9 The genomic DNA methylation directly Copyright # 2007 John Wiley & Sons, Ltd. Received 19 February 2007 Revised 28 February 2007 Accepted 12 April 2007

2 292 a. sazci ET AL. correlates with folate status and inversely with plasma homocysteine levels. The MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status. 10 DNA methylation status is a fundamental mechanism in the epigenetic modulation of gene expression and maintenance of genomic integrity. 9,11 DNA methylation, a characteristic feature of many eukaryotic genomes, necessitates the addition of a methyl group at the carbon 5 0 position of cytosine within the cytosine guanine (CpG) dinucleotide. 12 DNA methylation typically occurs in CpG dinucleotide-rich regions, which are highly represented in gene promoters. 13 Aberrant genomic DNA methylation is widely recognized to be associated with an expanding spectrum of diseases including cancer. 12,14 The MTHFR C677T and A1298C polymorphisms have been recently shown to be associated with higher levels of homocysteine, under the conditions of low folate plasma levels, 15,16 neurovascular disease, 17 psychiatric disorders, 18,19 neurodegenerative diseases, 20 ischemic and hemorrhagic stroke 21 and breast cancer. 22 In the present study, we examined whether the MTHFR C677T and A1298C polymorphisms were associated with NASH in a case-control study of Turkish subjects of Caucasian origin. MATERIALS AND METHODS Subjects The subjects were 57 patients with NASH (31 men (54.39%) and 26 women (45.61%); age range years) and 324 controls (171 men (52.78%) and 153 women (47.22%); age range years) matched for age, sex, ethnicity and geographical area. These subjects were recruited from Kocaeli University Hospital Gastroenterology Department from May 2002 to May Mean age and standard deviation (SD) were years in patients and years in controls, respectively. The histological findings of NASH were interpreted and scored according to the classification as previously proposed. 23,24 All the patients and controls were unrelated Turkish Caucasians. Patients with histologically confirmed NASH who had elevated liver aminotransferases and negative serologic markers of the autoimmune hepatitis with no use of alcohol were studied. Healthy controls did not have any known disease or symptoms. All subjects were from the city of Kocaeli, Turkey. Informed consent was obtained from each individual included in the study. The institutional review board approved the study. Histological evaluation Liver biopsy specimens were fixed in Bouin s and embedded in paraffin and the sections were stained with hematoxylin eosin and Masson s trichrome. The diagnosis of NASH was established, if abnormal liver enzymes were associated with greater than 10% steatosis in the presence of lobular and/or portal inflammation, with or without Mallory bodies, ballooning degeneration or fibrosis and the exclusion of other viral, metabolic or immunologic liver diseases for more than 3 months. 25 The histological classification of NASH was made according to the Brunt classification. 23 Genotyping Genomic DNA was isolated from peripheral blood samples of NASH patients as well as healthy controls. PCR-RFLP conditions were as previously described. 26 Statistical analysis Differences between groups were examined by the x 2 test and Students s t-test and Mann Whitney U test. Whenever appropriate, relative risk as odds ratio (OR) analysis was carried out with 2 2 cross-tabulation and a binary logistic regression model for the age and sex. All statistics were performed with the SPSS v12.0 statistical package (SPSS, Chicago, IL, USA). A value of p 0.05 was taken as significant. RESULTS This is the first study to show that the MTHFR C677T and A1298C polymorphisms are associated with NASH. Table 1 shows the allele and genotype distribution of MTHFR C677T and A1298C polymorphisms in 57 patients with NASH and 324 healthy controls. The MTHFR A1298C polymorphism (x 2 ¼ 8.439; p ¼ 0.015) in the total NASH patients (x 2 ¼ 5.960; p ¼ 0.051) and in women patients with NASH was significantly associated with increased risk for NASH (Tables 1, 2, 5). The MTHFR 1298C allele was also significantly associated with NASH in all NASH patients (OR ¼ 2.480; 95%CI ¼ ; x 2 ¼ 7.703; df ¼ 1; p ¼ 0.006). The MRHFR C677C/A1298C compound genotype (OR ¼ 2.218; 95%CI ¼ ; x 2 ¼ 3.998; df ¼ 1; p ¼ 0.046) in men patients with NASH, MTHFR C1298C genotype (OR ¼ 2.979; 95%CI ¼ ; x 2 ¼ 4.343; df ¼ 1; p ¼ 0.037) and C677C/ C1298C compound genotype in women patients with

3 Table 1. Allele and genotype distributions of the methylenettetrahydrofolate reductase C677T and A1298C polymorphisms in NASH in overall patients and controls Genotype Cases N ¼ 57 Controls N ¼ 324 mthfr polymorphism in nash 293 Cases (%) T of 677 Allele frequency: C of 1298 OR; 95%CI; x 2 ; df; p MTHFR (%) (%) x 2 ¼ 0.832; df ¼ 2; p ¼ C677C 32 (56.1) 161 (49.7) ( ); x 2 ¼ 0.807; df ¼ 1; p ¼ C677T 21 (36.8) 139 (42.9) ( ); x 2 ¼ 0.731; df ¼ 1; p ¼ T677T 4 (7.0) 24 (7.4) ( ); x 2 ¼ 0.011; df ¼ 1; p ¼ MTHFR (%) (%) x 2 ¼ 8.439; df ¼ 2; p ¼ A1298A 13 (22.8) 137 (42.3) ( ); x 2 ¼ 7.703; df ¼ 1; p ¼ A1298C 34 (59.6) 154 (47.5) ( ); x 2 ¼ 2.848; df ¼ 1; p ¼ C1298C 10 (17.5) 33 (10.2) ( ); x 2 ¼ 2.622; df ¼ 1; p ¼ NASH were significantly associated with NASH (Tables 4 6). In contrast, the MTHFR A1298A genotype (OR ¼ 0.403; 95%CI ¼ ; x 2 ¼ 7.703; df ¼ 1; p ¼ 0.006) in the total patients (OR ¼ 0.392; 95%CI ¼ ; x 2 ¼ 4.437; df ¼ 1; p ¼ 0.035), in men patients with NASH and C677C/A1298A (OR ¼ 0.856; 95%CI ¼ ; x 2 ¼ 4.262; df ¼ 1; p ¼ 0.039) compound genotypes in women patients with NASH had protection towards NASH (Tables 1, 3, 6). DISCUSSION In our study of a cohort of the Turkish population, we report that the MTHFR 1298C allele was significantly associated with NASH. Although our population was limited in size, the MTHFR 1298C allele in the total, C1298C genotype, and C677C/C1298C compound genotype in women NASH patients and C677C/ A1298C compound genotype in men NASH patients were significantly associated with NASH. In contrast, we did not find any association between the MTHFR 677T allele and NASH. To our knowledge, this is the first time that the MTHFR 1298C allele has been shown to be significantly associated with NASH. The MTHFR A1298A genotype in all NASH patients and men NASH patients and C677C/A1298A in women NASH patients had protection towards NASH. It is still unclear what causes progression from steatosis to steatohepatitis and from steatohepatitis to bridging fibrosis and cirrhosis. Alcohol consumption, hyperhomocysteinemia, 27 hepatic iron deposition, 28 drugs, endotoxin 29,30 and polymorphism in cytochrome p450 enzymes 31 have been proposed as putative mechanisms. However, the definitive mechanism is yet to be determined. More recently, Wanless and Shiota 32 proposed a four-step model in patients with NAFLD, including steatosis facilitated by insulin resistance (first step), necrosis induced by lipid peroxidation (second step), release of lipid from hepatocytes into the interstitium leading to direct and inflammatory injury to small hepatic veins (third step) and the venous obstruction with secondary collapse and ultimately fibrosis (fourth step). The involvement of various genetic and acquired thrombotic risk factors in the pathogenesis of vein thrombosis is well established but their role in NAFLD progression, to our knowledge, has not yet been investigated. Table 2. Comparison between the MTHFR C677T and A1298C polymorphisms in NASH in overall NASH patients and controls MTHFR677 MTHFR1298 Cases Controls OR; 95%CI; x 2 ; df; p CC AA 3 (5.3) 44 (13.6) ( ); x 2 ¼ 3.101; df ¼ 1; p ¼ CC AC 20 (35.1) 86 (26.5) ( ); x 2 ¼ 1.762; df ¼ 1; p ¼ CC CC 9 (15.8) 31 (9.6) ( ); x 2 ¼ 1.997; df ¼ 1; p ¼ CT AA 6 (10.5) 69 (21.3) ( ); x 2 ¼ 3.556; df ¼ 1; p ¼ CT AC 14 (24.6) 68 (21.0) ( ); x 2 ¼ 0.367; df ¼ 1; p ¼ CT CC 1 (1.8) 2 (0.6) ( ); x 2 ¼ 0.802; df ¼ 1; p ¼ TT AA 4 (7.0) 24 (7.4) ( ); x 2 ¼ 0.011; df ¼ 1; p ¼ TT AC 0 0 TT CC 0 0

4 294 a. sazci ET AL. Table 3. Allele and genotype distributions of the MTHFR C677T and A1298C polymorphisms in NASH in men patients and men controls Genotype Cases (%) N ¼ 31 N ¼ 171 Allele frequency: Cases (%) T of 677 C of 1298 OR; 95%CI; x 2 ; df; p MTHFR (%) (%) x 2 ¼ 2.035; df ¼ 2; p ¼ C677C 19 (61.3) 81 (47.4) ( ); x 2 ¼ 2.035; df ¼ 2; p ¼ C677T 10 (32.3) 75 (43.9) ( ); x 2 ¼ 1.449; df ¼ 1; p ¼ T677T 2 (6.5) 15 (8.8) ( ); x 2 ¼ 0.183; df ¼ 1; p ¼ MTHFR (%) (%) x 2 ¼ 4.550; df ¼ 2; p ¼ A1298A 7 (22.6) 73 (42.7) ( ); x 2 ¼ 4.437; df ¼ 1; p ¼ A1298C 20 (64.5) 79 (46.2) ( ); x 2 ¼ 3.523; df ¼ 1; p ¼ C1298C 4 (12.9) 19 (11.1) ( ); x 2 ¼ 0.084; df ¼ 1; p ¼ More recently, Ji and Kaplowitz 33 have shown that hyperhomocysteinemia is implicated in the pathogenesis of liver disease through a mechanism involving endoplasmic reticulum (ER) stress. Hyperhomocystenemia is associated with increased expression of ER stress response genes. 33 Homocysteine-induced ER stress induces lipid biosynthesis and uptake through activation of the sterol regulatory element binding protein 1 (SREBP-1) 34 thus leading to severe hepatic steatosis in transgenic mice. 35 In conclusion, hyperhomocysteinemia-induced ER stress response in the liver leads to fatty liver and inflammation and/or fibrosis in NASH. 36 Insulin resistance has been shown to be associated with elevated homocysteine levels in healthy nonobese subjects. 37,38 A recent study also suggested that plasma homocysteine levels were significantly higher in patients with NASH than in patients with simple Table 4. Comparison between the MTHFR C677T and A1298C polymorphisms in NASH in men patients and men controls MTHFR677 MTHFR1298 Cases (%) N ¼ 31 N ¼ 171 OR; 95%CI; x 2 ; df; p CC AA 3 (9.7) 22 (12.9) ( ); x 2 ¼ 0.246; df ¼ 1; p ¼ CC AC 13 (41.9) 42 (24.6) ( ); x 2 ¼ 3.998; df ¼ 1; p ¼ CC CC 3 (9.7) 17 (9.9) ( ); x 2 ¼ 0.002; df ¼ 1; p ¼ CT AA 2 (6.5) 36 (21.1) ( ); x 2 ¼ 3.663; df ¼ 1; p ¼ CT AC 7 (22.6) 37 (21.6) ( ); x 2 ¼ 0.014; df ¼ 1; p ¼ CT CC 1 (3.2) 2 (1.2) ( ); x 2 ¼ 0.758; df ¼ 1; p ¼ TT AA 2 (6.5) 15 (8.8) ( ); x 2 ¼ 0.183; df ¼ 1; p ¼ TT AC 0 0 TT CC 0 0 Table 5. controls Allele and genotype distributions of the MTHFR C677T and A1298C polymorphisms in NASH in women patients and women Genotype Cases N ¼ 26 Controls N ¼ 153 Allele frequency: Cases (%) T of 677 C of 1298 OR; 95%CI; x 2 ; df; p MTHFR (%) (%) x 2 ¼ 0.142; df ¼ 2; p ¼ C677C 13 (50.0) 80 (52.3) ( ); x 2 ¼ 0.047; df ¼ 1; p ¼ C677T 11 (42.3) 64 (41.8) ( ); x 2 ¼ 0.002; df ¼ 1; p ¼ T677T 2 (7.7) 9 (5.9) ( ); x 2 ¼ 0.126; df ¼ 1; p ¼ MTHFR (%) (%) x 2 ¼ 5.960; df ¼ 2; p ¼ A1298A 6 (23.1) 64 (41.8) ( ); x 2 ¼ 3.282; df ¼ 1; p ¼ A1298C 14 (53.9) 75 (49.0) ( ); x 2 ¼ 0.207; df ¼ 1; p ¼ C1298C 6 (23.1) 14 (9.2) ( ); x 2 ¼ 4.343; df ¼ 1; p ¼ 0.037

5 mthfr polymorphism in nash 295 Table 6. Comparison between the MTHFR C677T and A1298C polymorphisms in NASH in women patients and women controls MTHFR677 MTHFR1298 Cases (%) N ¼ 26 N ¼ 153 OR; 95%CI; x 2 ; df; p CC AA 0 22 (14.4) ( ); x 2 ¼ 4.262; df ¼ 1; p ¼ CC AC 7 (26.9) 44 (28.8) ( ); x 2 ¼ 0.037; df ¼ 1; p ¼ CC CC 6 (23.1) 14 (9.2) ( ); x 2 ¼ 4.343; df ¼ 1; p ¼ CT AA 4 (15.4) 33 (21.6) ( ); x 2 ¼ 0.518; df ¼ 1; p ¼ CT AC 7 (26.9) 31 (20.3) ( ); x 2 ¼ 0.590; df ¼ 1; p ¼ CT CC 0 0 TT AA 2 (7.7) 9 (5.9) ( ); x 2 ¼ 0.126; df ¼ 1; p ¼ TT AC 0 0 TT CC 0 0 steatosis. 27 They also reported that plasma homocysteine was a statistically significant predictor of severity of necroinflammatory activity in NASH. Elevated plasma levels of homocysteine are associated with hepatic fat accumulation, thus altering intracellular lipid metabolism. 35 A recent study also reported that hyperhomocysteinemia in CHC patients was associated with steatosis and liver fibrosis and MTHFR polymorphism. 39 The MTHFR polymorphism was not associated with the HCV genotype and hepatic necroinflammatory activity. HCV patients having the C677T genotype of the MTHFR gene have an increased risk of developing high levels of steatosis that is sixfold greater than that observed for patients with the C677C genotype. Whereas patients having the T677T genotype for MTHFR have an increased risk that is 20-fold greater than that observed in patients with C677C genotype and 1.9-fold greater than that observed in patients with the C677T genotype. 39 Individuals with elevated homocysteine plasma levels display increased risk for fatty liver. 39 The data are consistent with results of Schwahn et al., 40,41 who, using MTHFR knockout mice as a model for moderate and severe hyperhomocysteinemia, demonstrated that such mice spontaneously develop marked steatosis. Huang et al., 42 reported that folate depletion and elevated homocysteine promote oxidative stress in rat livers. In conclusion, the MTHFR 1298C allele in all patients, C1298C genotype, C677C/C1298C compound genotype in female NASH patients and C677C/ A1298C compound genotype in male NASH patients were genetic risk factors for NASH. REFERENCES 1. Tolman KG, Fonseca V, Tan MH, Dalpiaz AN. A narrative review: hepatobiliary disease in type 2 diabetes mellitus. Ann Intern Med 2004; 141: Ludwig J, Viggiano TR, McGill DB, Oh BJ. Nonalcoholic steatohepatitis: mayo clinic experiences with a hitherto unnamed disease. Mayo Clin Proc 1980; 55: Sanyal AJ. American Gastroenterological Association. AGA technical review on nonalcoholic fatty liver disease. Gastroenterology 2002; 123: McCullough AJ. Update on nonalcoholic fatty liver disease. J Clin Gastroenterol 2002; 34: Caldwell SH, Swerdlow RH, Khan EM, et al. Mitochondrial abnormalities in non-alcoholic steatohepatitis. J Hepatol 1999; 31: Schwimmer JB, Deutsch R, Rauch JB, Behling C, Newbury R, Lavine JE. Obesity, insulin resistance, and other clinicopathological correlates of pediatric nonalcoholic fatty liver disease. J Pediatr 2003; 143: Roberts EA. Steatohepatitis in children. Best Pract Res Clin Gastroenterol 2002; 16: Rozen R. Genetic predisposition to hyperhomocysteinemia: deficiency of methylenetetrahydrofolate reductase (MTHFR). Thromb Haemost 1997; 78: Jones PA, Gonzalgo ML. Altered DNA methylation and genome instability: a new pathway to cancer? Proc Natl Acad Sci USA 1997; 94: Friso S, Choi SW, Girelli D, et al. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status. Proc Natl Acad Sci USA 2002; 99: Wolffe AP, Matzke MA. Epigenetics: regulation through repression. Science 1999; 286: Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet 1999; 21: Razin A, Riggs AD. DNA methylation and gene function. Science 1980; 210: Robertson KD, Wolffe AP. DNA methylation in health and disease. Nat Rev Genet 2000; 1: Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983; 132: Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10: Kara I, Sazci A, Ergul E, Kaya G, Kilic G. Association of the C677T and A1298C polymorphisms in the 5,10 methylenetetrahydrofolate reductase gene in patients with migraine risk. Mol Brain Res 2003; 111: Sazci A, Ergul E, Guzelhan Y, Kaya G, Kara I. Methylenetetrahydrofolate reductase gene polymorphisms in patients with schizophrenia. Mol Brain Res 2003; 117:

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