Fibrosis in Chronic Hepatitis C Correlates Significantly With Body Mass Index and Steatosis

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1 Fibrosis in Chronic Hepatitis C Correlates Significantly With Body Mass Index and Steatosis LUKE F. H OURIGAN, 1 GRAEME A. MACDONALD, 2 DAVID PURDIE, 3 VICKI H. WHITEHALL, 4 CLAUDIA SHORTHOUSE, 1 ANDREW CLOUSTON, 5 AND ELIZABETH E. POWELL 1 SEE EDITORIAL ON PAGE 1328 Steatosis is a frequent histological finding in chronic hepatitis C infection; however, the pathophysiology of steatosis and its role in disease progression have not been established. We studied 148 consecutive patients with untreated chronic hepatitis C to assess the effect of body mass index, diabetes mellitus, alcohol consumption, hepatic iron content, and viral load on steatosis and hepatic fibrosis. Ninety-one patients (61%) had steatosis: grade 1 (F30% hepatocytes involved) in 61 (41%), grade 2 (30%- 70% hepatocytes involved) in 17 (11%), and grade 3 (G70% hepatocytes involved) in 13 (9%). After adjusting for potential confounding variables, a highly significant relationship was found primarily between steatosis and body mass index (P F.0001). The mean ( SD) body mass index of patients with no steatosis was kg/m 2, whereas for grade 1 steatosis it was kg/m 2, and for grade 2 and 3 steatosis combined the body mass index was kg/m 2. Hepatic fibrosis was significantly associated with age (P.002). After adjusting for potential confounding variables, including age, hepatic fibrosis was also significantly associated with steatosis (P F.03). There was no significant association between hepatic iron content, alcohol intake, gender, and viral load and steatosis or fibrosis. These findings suggest that increasing body mass index has a role in the pathogenesis of steatosis in chronic hepatitis C and that steatosis may contribute to fibrosis. The association between body mass index and steatosis and fibrosis has important prognostic and therapeutic implications in the management of patients with chronic hepatitis C virus. (HEPATOLOGY 1999;29: ) Abbreviations: HCV, hepatitis C virus; NASH, nonalcoholic steatohepatitis; BMI, body mass index; HIC, hepatic iron content. From the Departments of 1 Gastroenterology and Hepatology and 5 Pathology, The Princess Alexandra Hospital, the Departments of 2 Medicine and 3 Social and Preventive Medicine, The University of Queensland, the 2 Clinical Sciences Unit, Queensland Institute of Medical Research, and the 4 Conjoint Gastroenterology Laboratory, Royal Brisbane Hospital Research Foundation, Brisbane, Australia. Received August 21, 1998; accepted November 24, Address reprint requests to: Elizabeth E. Powell, M.D., Director of Clinical Training and Gastroenterologist, Princess Alexandra Hospital, Ipswich Rd, Woolloongabba, Brisbane, Queensland, 4102 Australia. powelle@health.qld.gov.au; fax: (61) Copyright 1999 by the American Association for the Study of Liver Diseases /99/ $3.00/0 Steatosis is a frequent histological finding in chronic hepatitis C virus (HCV) infection; however, the mechanisms underlying its development remain uncertain. Although most of the experimental and clinical data suggest that the liver injury in chronic HCV is immunologically mediated, it has been suggested that fatty change within hepatocytes may reflect a direct cytopathic effect of the virus. In support of this, a transgenic mouse model carrying the HCV core gene developed progressive hepatic steatosis. 1 An alternate possibility is that steatosis in hepatitis C is unrelated to the viral infection or host response to the virus. Hepatic steatosis occurs frequently in obese individuals, non-insulin-dependent diabetics, and after heavy alcohol ingestion, and the influence of these variables on the development or severity of steatosis in association with chronic HCV infection has not been established. The natural history and long-term prognosis of chronic HCV is difficult to define. Although up to 30% of patients with this chronic liver disease may develop cirrhosis, in any individual the factors that determine increasing fibrosis and progressive disease remain unknown. In epidemiological studies of chronic hepatitis C infection, age, duration of infection, alcohol consumption, and male gender are independent factors related to histological severity, 2 but the role of steatosis in disease progression has not been studied. Steatosis alone, as in nonalcoholic steatohepatitis (NASH) can be associated with increased hepatic fibrosis. However, only a minority of patients with NASH ultimately develop cirrhosis. The factors associated with the progression of fibrosis in this setting are not clear. It has been shown previously that mildly increased iron stores in patients with NASH are associated with increased hepatic fibrosis and that fibrosis was associated with increasing grades of steatosis. 3 There is also evidence that liver iron levels are mildly increased in patients with chronic viral hepatitis and suggestions that this iron may potentiate hepatocyte injury. 4 The mechanism for the development of fibrosis associated with iron and steatosis probably involves lipid peroxidation with subsequent activation of hepatic stellate cells, the principal collagen-producing cells within the liver. We hypothesized that the hepatic steatosis seen in patients with hepatitis C is multifactorial, and significantly related to the same factors that cause steatosis in patients without hepatitis C infection. These factors include increased body weight, diabetes mellitus, and alcohol consumption. Our second hypothesis was that in patients with chronic hepatitis C, steatosis and increased hepatic iron stores are associated with increased fibrosis. To investigate the first of these 1215

2 1216 HOURIGAN ET AL. HEPATOLOGY April 1999 hypotheses, we studied 148 consecutive patients with untreated chronic HCV to assess the effects of body mass index (BMI), diabetes mellitus, alcohol consumption, hepatic iron content (HIC), and viral load on steatosis. To investigate our second hypothesis we examined the association between hepatic iron stores, steatosis, and hepatic fibrosis. PATIENTS AND METHODS Patients. The study involved 148 consecutive patients with chronic HCV who had undergone liver biopsy before treatment with interferon at the Princess Alexandra Hospital, Brisbane, Australia, between February 1995 and December Informed consent in writing was obtained from each patient and the study protocol was approved by the Princess Alexandra Hospital Research Ethics Committee. Diagnosis of chronic HCV was based on standard serological assays and abnormal serum aminotransferase levels (greater than or equal to 1.5 times the upper limit of normal) for at least 6 months. All patients were positive for HCV antibody by the second-generation enzyme-linked immunosorbent assay (Abbott Laboratories, North Chicago, IL) with infection confirmed by detection of circulating HCV RNA by polymerase chain reaction using the Amplicor HCV assay (Roche, Branchburg, NJ) and were negative for hepatitis B surface antigen or antibodies to human immunodeficiency virus. Serum HCV RNA was quantitated in 73 patients using the Roche Amplicor HCV Monitor assay (Roche) and viral genotyping was performed in 42 patients using the Inno-Lipa HCV II assay (Innogenetics, Zwijnaarde, Belgium). Patients with other forms of chronic liver disease or previous treatment with interferon were excluded from the analysis. Details about weight and height and average alcohol intake (g/d) during the preceding 12 months were obtained from all patients at the time of liver biopsy. Information regarding average alcohol intake (g/d) before the last 12 months was also obtained. Alcohol consumption was assessed retrospectively by interview on at least three occasions. The number and types of alcoholic drinks consumed each day were recorded and the alcohol content of each drink was calculated. The alcohol intake over a weekly period was averaged and recorded in grams per day. Non-insulin-dependent diabetes mellitus was present in four patients at presentation. Random blood glucose levels were normal in the remaining patients. No patient was taking steatosis-inducing drugs. Histopathological Evaluation and Measurement of HIC. Liver biopsy specimens were fixed in buffered formalin and embedded in paraffin. The degree of inflammation and fibrosis was assessed and graded according to the method of Scheuer. 5 Steatosis was graded as follows: 0, 1 ( 30% of hepatocytes affected), 2 (30%-70% of hepatocytes affected), or 3 ( 70% of hepatocytes affected). Perls stain was available in 137 patients and was graded 0 to 4. All grading was performed by a single pathologist (A.C.) without knowledge of the patient s clinical or laboratory data. HIC was measured by atomic absorption spectrophotometry as previously described. 6 Detection of the Hemochromatosis Gene Mutation. Sixty-five of the patients in this study were tested for the HFE Cys282Tyr mutation by the polymerase chain reaction as described previously using DNA extracted from peripheral blood mononuclear cells. 7 Statistical Analysis. Clinical and histopathological characteristics of the patients were compared across levels of steatosis and fibrosis. These data are presented in terms of percentages for categorical or ordinal variables (gender and levels of steatosis and fibrosis), means and standard deviations for approximately normally distributed variables (age, HIC, and BMI), and medians for skewed variables (past and current alcohol consumption and viral load). For more detailed investigation, current alcohol consumption was grouped based on average daily intake into either less than or equal to 10 g (1 standard drink) per day or greater than 10 g; past alcohol consumption was also classified as either less than or equal to 50 g/d or greater than 50 g/d. The influence of gender, the categories of alcohol consumption, and HIC on steatosis and fibrosis were tested using the Pearson s 2 statistic, and the Mantle-Haenszel 2 test for linear association. Mean differences in age and BMI across levels of steatosis and fibrosis were assessed using one-way ANOVA; median alcohol consumptions, HIC, and viral load were compared using the Kruskal-Wallis nonparametric analysis. The independent effects of age, gender, BMI, and alcohol consumption on steatosis and the effect of all these and HIC on fibrosis were assessed using ordinal logistic regression. RESULTS Histological Evaluation. Steatosis was present in 91 of 148 (61%) patients with chronic HCV, 61 with grade 1 (41%), 17 with grade 2 (11%), and 13 with grade 3 (9%). In view of the relatively small number of patients with grades 2 and 3 steatosis, patients with these grades of steatosis were combined for statistical analyses. General Characteristics of the Patients Studied. The age of the patients (73% male) ranged from 22 to 66 years, with a mean age of 37 years (SD, 6.9). The possible source of hepatitis was previous intravenous drug use in 100 (67.6%), posttransfusion in 19 (12.8%), and other risk factors or unknown in 29 (19.6%). BMI ranged from 16.9 to kg/m 2, with a mean of kg/m 2 (SD, 5.0). Alcohol intake in the past and in the 12 months preceding the liver biopsy ranged from none to very heavy ( 540 g/d). Circulating HCV RNA levels before liver biopsy ranged from to One patient was homozygous for the hemochromatosis mutation Cys282Tyr, 14 patients were heterozygous, and 50 patients were homozygous normal. The HIC ranged from 0 to 41 µmol/g dry weight and did not differ between patients heterozygous for the Cys282Tyr mutation and homozygous normal patients. The Perls stain was 0 in 115 patients, 1 in 21 patients, and 2 in 2 patients. The 51-year-old patient homozygous for the hemochromatosis mutation Cys282Tyr had an HIC of 37.7 µmol/g dry weight and a Perls stain of 0. Factors Associated With the Grade of Steatosis. The characteristics and laboratory features of the patients within each grade of steatosis are summarized in Table 1. In this series of patients with chronic HCV, a highly statistically significant association (P.001) was found between increasing BMI and grade of steatosis. The mean BMI ( SD) of patients with no steatosis was 23.9 kg/m 2 4.3, which is within the acceptable weight range for men and women. The mean BMI of patients with grade 1 steatosis and patients with the combined grades 2 and 3 steatosis were within the overweight range, 26.5 kg/m and 28.4 kg/m 2 4.9, respectively. Of the 64 patients with an acceptable weight (BMI up to 25.5), 4 had grade 3 steatosis and 3 had grade 2 steatosis. Of the 23 obese patients (BMI 30), only 3 patients had no steatosis and 10 had grade 1 steatosis. After adjusting for age, sex, and alcohol consumption using multiple ordinal logistic regression, the effect of BMI on grade of steatosis remained significant (P.0002). Diabetes was an infrequent finding in this group of patients (n 4), and therefore its impact could not be evaluated. However, two of these patients had grade 1 steatosis and the other two had grade 2/3 steatosis. Crudely, there appeared to be higher grades of steatosis in men compared with women (P.08); however, after

3 HEPATOLOGY Vol. 29, No. 4, 1999 HOURIGAN ET AL TABLE 1. Characteristics and Laboratory Features of Patients Within Each Grade of Steatosis 0 (n 57) Grade of Steatosis 1 (n 61) 2/3 (n 30) Crude P Value Mean age (SD) 36.5 (6.6) 37.9 (7.0) 37.2 (7.6).56 Mean BMI (SD) 23.9 (4.3) 26.5 (5.1) 28.4 (4.9).001 Male (n 109) 33.9% 42.2% 23.9%.08 Female (n 39) 51.3% 38.5% 10.3% Alcohol current: Median * 10 g/d (n 98) 42.9% 35.7% 21.4% g/d (n 46) 28.3% 52.2% 19.6% Alcohol past: Median g/d (n 72) 44.4% 36.1% 19.4% g/d (n 72) 33.3% 44.4% 22.2% Mean HIC (µmol/g dry wt) (SD) 14.0 (9.5) 17.2 (10.1) 13.5 (8.5).23 Viral genotype: 1a or 1b (n 23) 39.1% 43.5% 17.4%.10 3a (n 17) 17.6% 35.3% 47.1% Median viral load ( 10 6 /ml) NOTE. All percentages are row percentages. *Kruskal-Wallis statistic P value. 2 Statistic P value. adjustment for BMI this effect was no longer observed (P.26). Similarly, higher grades of steatosis were seen in patients with viral genotype 3a compared with genotypes 1a or 1b. However, the number of patients for whom genotyping had been performed was small and the association was therefore not statistically significant. There was no statistically significant relationship, either crudely or adjusted, between grade of steatosis and alcohol intake, viral load, hepatic iron stores, or age (Table 1). Factors Associated With the Severity of Fibrosis. The effects of age, gender, viral load, alcohol intake, and HIC on the severity of fibrosis were also assessed (Table 2). A statistically significant association was found between age and severity of fibrosis, with older people having more severe levels of fibrosis (P.001), which remained significant after adjusting for the other factors (P.003). There were significant effects of both steatosis and portal inflammation on severity of fibrosis (Table 2). Higher grades of steatosis were predictive of more severe levels of fibrosis (P.01), and a portal inflammation of 2 to 3 compared with 0 to 1 was also predictive of much more severe fibrosis (P.001). After adjustment for age, gender, BMI, and levels of alcohol intake, increasing steatosis and portal inflammation remained associated with more severe fibrosis (P.05 and.0001, respectively). No significant associations were found between hepatic iron stores, gender, or alcohol intake and level of fibrosis. Crudely, BMI appeared to be related to severity of fibrosis; however, this association was confounded by the association with steatosis (discussed later). After adjusting the effect of BMI for grade of steatosis, BMI was no longer significantly associated with severity of fibrosis (P.52). This implies that BMI is associated with fibrosis through its effect on steatosis. DISCUSSION In our series of 148 consecutive patients with chronic HCV, steatosis was present in 91 (61%) of these. Steatosis was grade 1 ( 30% hepatocytes involved) in 61 (41%), grade 2 (30%-70% hepatocytes involved) in 17 (11%), and grade 3 ( 70% hepatocytes involved) in 13 (9%). This is similar to other histopathological studies of chronic HCV, which have reported microvesicular and macrovesicular fatty change in 31% to 72% of patients Steatosis has been cited as a characteristic feature of chronic HCV, but it remains uncertain whether it is directly related to the virus or secondary to host factors. Our findings indicate that obesity has an important role in the pathophysiology of steatosis in chronic HCV. Similar results were noted in an Italian study evaluating steatosis in chronic liver disease with and without serum antibodies to HCV. 11 Steatosis was found in 60% of HCV-positive and 52% of HCV-negative patients and was strongly associated with obesity. Using simple logistic regression and multivariate analysis the association between HCV and steatosis was not significant. A recent study from the Mayo Clinic also showed that patients with chronic HCV and hepatic steatosis were more commonly overweight than their counterparts without these histological changes. 12 However, the number of patients studied with mild or moderate steatosis was relatively small and the authors were unable to show a relationship between the degree of steatosis and BMI. It has been suggested that steatosis may represent a direct cytopathic effect of HCV. In support of this, a transgenic mouse model expressing the HCV core gene developed progressive hepatic steatosis. 1,13 The HCV core protein can be detected in the liver of patients with chronic HCV infection, and the level of expression appears to be similar to that seen TABLE 2. Characteristics and Laboratory Features of Patients Within Each Level of Fibrosis 0 (n 35) Level of Fibrosis 1 (n 46) 2 (n 30) 3/4 (n 36) Crude P Value Mean age (SD) 34.2 (4.8) 36.0 (7.0) 38.6 (5.4) 40.3 (8.5).001 Mean BMI (SD) 24.5 (3.7) 25.0 (4.7) 26.2 (6.4) 28.5 (4.4).009 Male (n 109) 18.3% 32.1% 21.1% 28.4%.43 Female (n 39) 39.5% 28.9% 18.4% 13.2% Alcohol current: Median g/d (n 98) 22.7% 34.0% 19.6% 23.7% g/d (n 46) 23.9% 23.9% 23.9% 28.3% Alcohol past: Median g/d (n 72) 23.9% 31.0% 22.5% 22.5% g/d (n 72) 22.2% 30.6% 19.4% 27.8% Median HIC (µmol/g dry wt) Median viral load ( 10 6 /ml) Grade of steatosis: 0(n 57) 35.7% 35.7% 16.1% 12.5%.01 1(n 61) 18.0% 32.8% 18.0% 31.1% 2/3 (n 30) 13.8% 17.2% 34.5% 34.5% Portal inflammation: 0/1 (n 79) 38.8% 38.8% 16.3% 6.3%.001 2/3 (n 60) 6.7% 25.0% 26.7% 41.7% NOTE. All percentages are row percentages.

4 1218 HOURIGAN ET AL. HEPATOLOGY April 1999 in the transgenic mouse liver. 13 The mechanism by which overexpression of the core protein may promote accumulation of triglycerides in mouse hepatocytes remains unclear. Expression of the core protein in mitochondria disrupts the normal double membrane structure of this organelle and may produce steatosis through impairment of fatty acid oxidation. 13 Other viruses such as the influenza B virus may cause hepatic steatosis by altering mitochondrial enzyme activity. 14 In this study we found no statistically significant relationship between viral load and genotype and fatty liver, suggesting that host factors may be more important than viral factors in the pathogenesis of steatosis. An important finding in this study was the significant relationship between hepatic fibrosis and steatosis, suggesting that in chronic HCV infection steatosis may play a role in disease progression. After adjustment for age and other potential confounding variables, the relationship between fibrosis and steatosis remained significant. Statistical modelling of interactions between hepatic fibrosis and other histological features of chronic HCV has also shown a strong relationship between fibrosis and steatosis. 15 Czaja et al. 12 also found that patients with chronic HCV and moderate steatosis had higher Knodell scores for inflammatory activity and fibrosis than patients with mild steatosis. Unfortunately no information was provided about potential confounding variables, such as the association between age and fibrosis. Steatosis of any cause can be associated with the development of inflammatory changes and fibrosis in the setting of oxidative stress. 16 Sources of oxidative stress include mitochondrial dysfunction induced by drugs or viruses and an increase in the intrahepatic concentration of iron. These factors may result in the formation of reactive oxygen species, which may initiate lipid peroxidation leading to cell death. Animal studies have shown that lipid peroxidation occurs in both acute and chronic steatosis induced by a variety of agents. 17,18 Lipid peroxidation is associated with stellate cell activation and the synthesis of collagen type 1, the major collagen in fibrotic liver tissue The association between lipid peroxidation and hepatic fibrosis appears to be present in other liver diseases including hemochromatosis and alcoholinduced liver injury In chronic hepatitis C infection, steatosis correlates with lipid peroxidation, 25 with evidence of lipid peroxidation found in areas of active fibrogenesis. 26 However, the link between steatosis, lipid peroxidation, and fibrosis has not been examined, and we hypothesize that in hepatitis C infection, steatosis increases lipid peroxidation, which in turn increases stellate cell activation and collagen synthesis. Several recent reports have suggested that hepatic iron may be important as a cofactor in producing fibrosis in chronic HCV. Although HIC falls within the normal range, the levels are increased by comparison with hepatitis B virus-related chronic liver disease and are associated with increased lipid peroxidation and glutathione turnover. 25 In patients with NASH, mildly increased hepatic iron stores are usually caused by heterozygosity for the hemochromatosis Cys282Tyr mutation and are associated with an increased risk of fibrosis. 3 Similar findings have recently been reported by Smith et al. 27 for individuals with chronic HCV who are heterozygous for hemochromatosis. In our series of patients with chronic HCV, hepatic iron levels were generally very low, and there was no inter-relation between HIC and the HFE mutation and steatosis and fibrosis. This indicates that iron does not appear to play an important role in fibrogenesis in our patients with hepatitis C. However, we cannot draw any conclusions about the effect of more significant hepatic iron stores detected by some other groups. These findings suggest that increasing BMI has a role in the pathogenesis of steatosis in chronic HCV and that this steatosis may contribute to fibrosis. These findings may have important prognostic and therapeutic implications in the management of patients with chronic HCV. It may be important for future treatment trials to direct some efforts towards restoring a lean BMI (as an adjunct to other therapies) in these patients. REFERENCES 1. Moriya K, Yotsuyanagi H, Shintani Y, Fujie H, Ishibashi K, Matsuura Y, Miyamura T, et al. Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. J Gen Virol 1997;78: Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. 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