Non-alcoholic Fatty Liver Disease Damage Research Articles

Role of adipokines in the pathogenesis of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome. The increased prevalence of obesity, diabetes, hypertension, hypertriglyceridaemia and hypercholesterolemia are considered to be the potential causative factors for NAFLD. NAFLD is emerging as a major clinical problem worldwide. Recently much attention has been focused in India as the prevalence of obesity and diabetes is rising. NAFLD is responsible for unexplained raise in transaminases, and an important cause of cryptogenic cirrhosis and cryptogenic hepatocellular carcinoma in India. NAFLD is a spectrum of disease ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), potentially leading to fibrosis and cirrhosis. Studies have suggested that the adipokines are involved in the pathogenesis of NAFLD and its progression to NASH, through their metabolic and pro- or anti-inflammatory activity. Adipokines in particular tumor necrosis factor-α and interleukin-6 are believed to mediate the shift in pathology from steatosis to steatohepatitis. In addition, other adipokines such as adiponectin, leptin and resistin also play a crucial role in the development and progression of NAFLD through their metabolic and pro-or anti-inflammatory activity. This suggests that imbalance between pro-inflammatory and anti-inflammatory cytokines may have a role in the development of liver damage in NAFLD. Understanding the relationship between adipokines and NAFLD may play an important role in the early identification/diagnosis, treatment and also help in preventing disease progression.

Serum ferritin and liver inflammation: which come first? Chicken or egg?

Beaton MD, et al. For the clinician the serum elevation of ferritin in chronic liver diseases (CLD), with the exception of Emochromatosis, is always assumed to be a non-specific marker of hepatic inflammation and not of iron overload.1 Nonalcoholic fatty liver disease (NAFLD) is now the most common cause of CLD.2 The spectrum of NAFLD is ranging from simple steatosis through nonalcoholic steatohepatitis (NASH).3 Iron is regarded as a putative element that interacts with oxygen radicals, and high rates of hyperferritinemia together with increased hepatic iron stores have been demonstrated in NASH.4 The role of hepatic iron in the progression and in the pathogenesis of NASH remains controversial. We know that phlebotomy or iron-restricted diet reduce hepatic damage as well as insulin resistance (IR) in patients with NAFLD/NASH.5 However, exact mechanisms involved in iron accumulation in NASH remain to be clarified. Genetic factors linked to IR, dysregulation of iron-regulatory molecules, erythrophagocytosis by Kupffer cells may be responsible for hepatic iron overload in NASH.6,7 Chakraberti and Adams, in this Annals of Hepatology issue8 reported a simple clinical study of phlebotomy treatment performed in a series of 56 patients with histology-proven NAFLD. Liver biopsy and liver iron concentration (LIC) was performed at entry and 6 months after phlebotomy. They did not found any significant correlation between LIC and the level of serum ferritin, hepatic inflammation as measured by NAS score, and other generic markers of inflammation, such as ESR and CRP. The authors concluded that elevated serum ferritin is linked to hepatic iron accumulation, but that liver inflammation is not the cause of increased serum ferritin in patients with NAFLD. Other authors7 recently demonstrated that elevated ferritin level reflects iron stores, and not hepatic inflammation, being a predictors of vascular damage in NAFLD. The mechanisms involved in iron accumulation in NAFLD, and in inducing IR, metabolic, hepatic, and vascular damage by iron accumulation are not yet well know and should be further investigated. They may involve up-regulation of hepcidin by increased iron stores in patients not carrying HFE mutations, and iron compartmentalization into macrophages, or the hepatic expressions of the ironexport protein ferroportin-1, and of the iron-sensing molecule hemojuvelin, which have been found to be significantly lower in NAFLD patients.7,9 Whatever the mechanisms should be the only certainty is that an increased serum ferritin level in NAFLD is not a marker or a cause of inflammation, but a consequence of iron accumulation inside the hepatocyte.

Association of Vitamin D Deficiency to Liver Enzyme Status in Nonalcoholic Fatty Liver Disease in Veterans: A Retrospective Analysis

Purpose: Vitamin D is a lipophilic molecule with anti-inflammatory, antifibrotic, immunomodulatory effects. Prior studies indicate a high prevalence of vitamin D deficiency in patients with nonalcoholic fatty liver disease (NAFLD). NAFLD is now considered a hepatic component of the metabolic syndrome and increases the risk of cardiovascular disease. The prevalence of NAFLD is observed in 20-30% of general population.In this study, we explore the relationship and extent of vitamin D deficiency to normal and abnormal liver enzymes among veterans with NAFLD and assess improvement in liver enzyme status with vitamin D supplementation. Methods: A retrospective review of electronic medical records of patients with a diagnosis of NAFLD at Veterans Affairs Medical facilities in the Southeastern United States was conducted. Those with one serum 25(OH) vitamin D level drawn between 2001-2008 were included and monitoring of 25(OH)D level over three subsequent years was noted. Patient demographic, initial and follow-up vitamin D values along with liver function were recorded. Vitamin D deficiency was defined as a value less than 20 ng/ml. The normal values for AST and ALT were less than 40 units and alkaline phosphatase 44-147 units/L. Vitamin D supplementation with ergocalciferol or cholecalciferol was also noted. Results: There were 376 veterans with NAFLD and an available 25(OH) vitamin D level. 44% had abnormal liver enzymes. Patients with abnormal liver enzymes were 53% more likely to be vitamin D deficient. Liver enzymes status did predict compliance vitamin D monitoring among study patients. Supplementation with any type of vitamin D was not associated with improvement in liver enzymes.Table: Table. Liver enzyme status and vitamin D variablesTableConclusion: Our data suggest that veterans with abnormal liver enzymes are more likely to be vitamin D deficient. Vitamin D deficiency could be due to impaired liver 25-hydroxylation that is a manifestation of disease severity. Vitamin D is implicated in insulin resistance that contributes to NAFLD. Vitamin D supplementation did not improve liver enzymes which could be due to need for robust replacement with vitamin D, patient non compliance for follow up. Increasing hepatocyte damage in NAFLD leads to decreased expression of VDR for any replaced vitamin D level to provide positive effect on liver enzyme values.Vitamin D3 appears to be approximately 87% more potent in raising and maintaining a sustained serum 25(OH)D level than D2. In our study, ergocalciferol was used to a little more extent than cholecalciferol.The lack of improvement in liver enzymes could also be explained by inability of ergocalciferol to maintain an adequate serum vitamin D level.

The inhibitory effect of black soybean on hepatic cholesterol accumulation in high cholesterol and high fat diet-induced non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is defined as excess of fat in the liver. We investigated the effects of black soybean on the cholesterol metabolism and insulin resistance of mice fed high cholesterol/fat diets. Mice were randomly allocated into four groups that were fed different diets: the normal cholesterol/fat diet; high cholesterol/fat diets (HCD); and HCD with 1%, and 4% black soybean powder (1B-HCD, and 4B-HCD). Liver total cholesterol and triglyceride concentrations were significantly lower in the black soybean-supplemented groups than that in the HCD group. PCR revealed significantly lower hepatic SREBP2 and HMG-CoA reductase mRNA levels of black soybean-supplemented mice. Real-time PCR revealed significantly higher hepatic ABCA1 mRNA level of black soybean-supplemented mice, which may increase cholesterol efflux. Liver bile acids concentration was significantly high in the 4B-HCD group. Black soybean stimulated secretion of adiponectin, activation of pAMPK, and eliminated free fatty acids in the liver. Black soybean supplementation decreased MDA and nitrate level. The activities of SOD, catalase, and GPx were restored by black soybean supplementation. Our data strongly indicate that black soybean influences the balance between oxidative and antioxidative stress. We suggest that black soybean improves cholesterol metabolism, insulin resistance, and alleviates oxidative damage in NAFLD.

Hedgehog pathway activation parallels histologic severity of injury and fibrosis in human nonalcoholic fatty liver disease

The Hedgehog (HH)-signaling pathway mediates several processes that are deregulated in patients with metabolic syndrome (e.g., fat mass regulation, vascular/endothelial remodeling, liver injury and repair, and carcinogenesis). The severity of nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome generally correlate. Therefore, we hypothesized that the level of HH-pathway activation would increase in parallel with the severity of liver damage in NAFLD. To assess potential correlations between known histologic and clinical predictors of advanced liver disease and HH-pathway activation, immunohistochemistry was performed on liver biopsies from a large, well-characterized cohort of NAFLD patients (n = 90) enrolled in the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) Database 1 study. Increased HH activity (evidenced by accumulation of HH-ligand-producing cells and HH-responsive target cells) strongly correlated with portal inflammation, ballooning, and fibrosis stage (each P < 0.0001), supporting a relationship between HH-pathway activation and liver damage. Pathway activity also correlated significantly with markers of liver repair, including numbers of hepatic progenitors and myofibroblastic cells (both P < 0.03). In addition, various clinical parameters that have been linked to histologically advanced NAFLD, including increased patient age (P < 0.005), body mass index (P < 0.002), waist circumference (P < 0.0007), homeostatic model assessment of insulin resistance (P < 0.0001), and hypertension (P < 0.02), correlated with hepatic HH activity. In NAFLD patients, the level of hepatic HH-pathway activity is highly correlated with the severity of liver damage and with metabolic syndrome parameters that are known to be predictive of advanced liver disease. Hence, deregulation of the HH-signaling network may contribute to the pathogenesis and sequelae of liver damage that develops with metabolic syndrome.

Hepatoprotective Activities of Black Soybean in High Cholesterol Diet‐induced Non‐alcoholic fatty liver disease

Non‐alcoholic Fatty Liver Disease(NAFLD) is characterized by an increase in intra‐hepatocellular triglycerides, which is not due to alcohol. This study investigated the effect of black soybean supplementation on high cholesterol diet induced NAFLD. Mice were divided into five groups: normal control(NCD), high cholesterol control(HCD), and the high cholesterol diet supplemented with black soybean powder(1B‐HCD:1%, 2BHCD: 2%, 4B‐HCD:4%). Blood glucose, insulin, lipid profiles, adiponectin, and malondialdehyde(MDA) were determined. Blood glucose and insulin in HCD(163.2mg/dl, 3.07μIU/ml) were higher than in NCD(100.0mg/dl, 1.62μIU/ml), reducing in all experimental groups(129.2mg/dl, 1.76μIU/ml; 98.6mg/dl, 2.30μIU/ml; 113.7mg/dl, 2.29μIU/ml). Total cholesterol and triglyceride were only decreased in 4B‐HCD compared to HCD. Adiponectin was significantly decreased in HCD(6.68ng/ml), while increased in experimental groups(8.13ng/ml; 8.89ng/ml; 9.12ng/ml). MDA was dropped in all experimental groups(20.04μM; 33.89μM; 15.89μM), compared to HCD(35.96μM). The results indicate that supplementation of black soybean may improve lipid profiles and oxidative damage in NAFLD. This work was supported by SRC and BK 21.Grant Funding Source: Brain Korea 21

The epidemiology, pathogenesis and histopathology of fatty liver disease

Fatty liver disease includes non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD), each of which is increasing in prevalence. Each represents a histological spectrum that extends from isolated steatosis to steatohepatitis and cirrhosis. NAFLD is associated with obesity, diabetes, and insulin resistance, and is considered to be the liver manifestation of the metabolic syndrome. The pathogenesis of NAFLD and ALD involves cytokines, adipokines, oxidative stress, and apoptosis. Histopathology is the gold standard for assessing the severity of liver damage in NAFLD and ALD. We have reviewed the literature, and described and compared the epidemiology, natural disease history, pathogenesis and histopathology of NAFLD and ALD.

Diagnostic and therapeutic implications of the association between ferritin level and severity of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD), defined by excessive liver fat deposition related to the metabolic syndrome, is a leading cause of progressive liver disease, for which accurate non-invasive staging systems and effective treatments are still lacking. Evidence has shown that increased ferritin levels are associated with the metabolic insulin resistance syndrome, and higher hepatic iron and fat content. Hyperferritinemia and iron stores have been associated with the severity of liver damage in NAFLD, and iron depletion reduced insulin resistance and liver enzymes. Recently, Kowdley et al demonstrated in a multicenter study in 628 adult patients with NAFLD from the NAFLD-clinical research network database with central re-evaluation of liver histology and iron staining that the increased serum ferritin level is an independent predictor of liver damage in patients with NAFLD, and is useful to identify NAFLD patients at risk of non-alcoholic steatohepatitis and advanced fibrosis. These data indicate that incorporation of serum ferritin level may improve the performance of noninvasive scoring of liver damage in patients with NAFLD, and that iron depletion still represents an attractive therapeutic target to prevent the progression of liver damage in these patients.

Pathogen- or damage-associated molecular patterns during nonalcoholic fatty liver disease development

Pathogen- or damage-associated molecular patterns during nonalcoholic fatty liver disease development

Lack of association between peroxisome proliferator-activated receptors alpha and gamma2 polymorphisms and progressive liver damage in patients with non-alcoholic fatty liver disease: a case control study

BackgroundPeroxisome proliferator-activated receptors (PPARs) play key roles in the pathogenesis of nonalcoholic fatty liver disease (NAFLD).Aimto assess the effect of functional single nucleotide polymorphisms (SNPs) of PPARα and PPARγ2, previously associated with insulin resistance and dyslipidemia, on liver damage in NAFLD, whose progression is influenced by metabolic abnormalities and inherited factors.MethodsThe Leu162Val PPARα and Pro12Ala PPARγ2 SNPs were evaluated by restriction analysis. We considered 202 Italian patients with biopsy-proven NAFLD.ResultsThe frequency of the evaluated SNPs did not differ between patients and 346 healthy controls. The presence of the PPARα 162Val allele (prevalence 57%), but not of the PPARγ2 12Ala allele (prevalence 18%), was associated with higher insulin resistance (HOMA-IR index 4.71 ± 3.8 vs. 3.58 ± 2.7, p = 0.026), but not with hyperglycemia. The PPARα 162Val and PPARγ2 12Ala alleles were not associated with the severity of steatosis, necroinflammation, or fibrosis.ConclusionsThe presence of the PPARα 162Val allele was associated with insulin resistance, but not with liver damage in NAFLD. Because of the limited power of the present sample, larger studies are needed to exclude a minor effect of the PPARγ2 12Ala allele on necroinflammation/fibrosis in NAFLD.

Serum ferritin levels are associated with vascular damage in patients with nonalcoholic fatty liver disease

Background and aims Increased ferritin and body iron stores are frequently observed in nonalcoholic fatty liver disease (NAFLD), associated with heightened susceptibility to vascular damage. Conflicting data have been reported on the role of iron in atherosclerosis, with recent data suggesting that excess iron induces vascular damage by increasing levels of the hormone hepcidin, which would determine iron trapping into macrophages, oxidative stress, and promotion of transformation into foam cells. Aim of this study was to investigate the relationship between iron status and cardiovascular damage in NAFLD. Methods and results Vascular damage was evaluated by common carotid arteries intima-media thickness (CC-IMT) measurement and plaque detection by ecocolor-doppler ultrasonography in 506 patients with clinical and ultrasonographic diagnosis of NAFLD, hemochromatosis gene (HFE) mutations by restriction analysis in 342 patients. Serum hepcidin-25 was measured by time-of-flight mass spectrometry in 143 patients. At multivariate analysis CC-IMT was associated with systolic blood pressure, glucose, LDL cholesterol, abdominal circumference, age, and ferritin ( p = 0.048). Carotid plaques were independently associated with age, ferritin, glucose, and hypertension. Ferritin reflected iron stores and metabolic syndrome components, but not inflammation or liver damage. Hyperferritinemia was associated with increased vascular damage only in patients with HFE genotypes associated with hepcidin upregulation by iron stores ( p < 0.0001), and serum hepcidin-25 was independently associated with carotid plaques ( p = 0.05). Conclusion Ferritin levels, reflecting iron stores, are independent predictors of vascular damage in NAFLD. The mechanism may involve upregulation of hepcidin by increased iron stores in patients not carrying HFE mutations, and iron compartmentalization into macrophages.

Genetic variants regulating insulin receptor signalling are associated with the severity of liver damage in patients with non-alcoholic fatty liver disease

Background/aimsThe aim of this study was to assess the effect of functional ENPP1(ectoenzyme nucleotide pyrophosphate phosphodiesterase 1)/PC-1 (plasma cell antigen-1) and IRS-1 (insulin receptor substrate-1) polymorphisms influencing insulin receptor activity...

Nonalcoholic fatty liver disease andHFEgene mutations: A Polish study

To describe a Polish population with nonalcoholic fatty liver disease (NAFLD) with regard to HFE gene mutations, as well as analyzing demographic and clinical data. Sixty-two consecutive patients with biopsy-proven NAFLD were included in the study. Demographic, clinical, and laboratory data were summarized in a database. C282Y and H63D mutations of the HFE gene were analyzed using polymerase chain reaction-restriction fragment lenght polymorphism. The analyzed cohort consisted of 62 homogeneic Caucasian participants, 66.1% men and 33.9% women, with a median age of 48 years. The median body mass index was 29.05 kg/m(2). Hypercholesterolemia was observed in 74.2% of patients and hypertriglyceridemia in 32.2%; 16.1% had type 2 diabetes mellitus (DMt2). On liver biopsy, 22.6% of NAFLD patients were found to have severe fibrosis. There were no differences between frequencies of HFE gene mutations in subgroups of NAFLD patients with less and more severe liver fibrosis. Obesity, older age, female gender and DMt2 were associated with more advanced fibrosis in this Polish cohort, as well as higher glucose level, serum iron and transaminase aspartate aminotransferase/alanine aminotransferase ratio. HFE mutations conferred no additional hepatic fibrosis risk in NAFLD, but higher serum iron was a risk factor for severe liver damage in NAFLD, regardless of HFE mutations.

Serum Ferritin Levels Are Associated with Vascular Damage in Patients with Nonalcoholic Fatty Liver Disease.

Abstract 5098 Background and AimsIncreased ferritin and body iron stores are frequently observed in nonalcoholic fatty liver disease (NAFLD), associated with heightened susceptibility to vascular damage. Conflicting data have been reported on the role of iron in atherosclerosis, with recent data suggesting that excess iron induces vascular damage by increasing levels of the hormone hepcidin, which would determine iron trapping into macrophages, oxidative stress, and promotion of transformation into foam cells. Aim of this study was to investigate the relationship between iron status and cardiovascular damage in NAFLD. Methods and ResultsVascular damage was evaluated by common carotid arteries intima-media thickness (CC-IMT) measurement and plaque detection by ecocolor-doppler ultrasonography in 506 patients with clinical and ultrasonographic diagnosis of NAFLD, HFE mutations by restriction analysis in 342 patients. Serum hepcidin-25 was measured by time-of-flight mass spectrometry in 143 patients. At multivariate analysis CC-IMT was associated with systolic blood pressure, glucose, LDL cholesterol, abdominal circumference, age, and ferritin (p=0.048). Carotid plaques were independently associated with age, ferritin (p=0.0004), glucose, and hypertension. Ferritin reflected iron stores and metabolic syndrome components, but not inflammation or liver damage. Hyperferritinemia was associated with increased vascular damage only in patients with HFE genotypes associated with hepcidin upregulation by iron stores (p<0.0001), and serum hepcidin-25 was independently associated with carotid plaques (p=0.05). ConclusionFerritin levels, reflecting iron stores, are independent predictors of vascular damage in NAFLD. The mechanism may involve upregulation of hepcidin levels by increased iron stores in patients not carrying HFE mutations, and iron compartmentalization into macrophages. DisclosuresNo relevant conflicts of interest to declare.

Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis

In the early stages of nonalcoholic fatty liver disease (NAFLD), triglycerides accumulate in hepatocytes. Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in hepatocyte triglyceride biosynthesis. DGAT2 antisense oligonucleotide (ASO) treatment improved hepatic steatosis dramatically in a previous study of obese mice. According to the 2-hit hypothesis for progression of NAFLD, hepatic steatosis is a risk factor for nonalcoholic steatohepatitis (NASH) and fibrosis. To evaluate this hypothesis, we inhibited DGAT2 in a mouse model of NASH induced by a diet deficient in methionine and choline (MCD). Six-week-old genetically obese and diabetic male db/db mice were fed either the control or the MCD diet for 4 or 8 weeks. The MCD diet group was treated with either 25 mg/kg DGAT2 ASO or saline intraperitoneally twice weekly. Hepatic steatosis, injury, fibrosis, markers of lipid peroxidation/oxidant stress, and systemic insulin sensitivity were evaluated. Hepatic steatosis, necroinflammation, and fibrosis were increased in saline-treated MCD diet-fed mice compared to controls. Treating MCD diet-fed mice with DGAT2 ASO for 4 and 8 weeks decreased hepatic steatosis, but increased hepatic free fatty acids, cytochrome P4502E1, markers of lipid peroxidation/oxidant stress, lobular necroinflammation, and fibrosis. Progression of liver damage occurred despite reduced hepatic expression of tumor necrosis factor alpha, increased serum adiponectin, and striking improvement in systemic insulin sensitivity. Results from this mouse model would suggest accumulation of triglycerides may be a protective mechanism to prevent progressive liver damage in NAFLD.

α1-Antitrypsin mutations in NAFLD: High prevalence and association with altered iron metabolism but not with liver damage

Hyperferritinemia, a common feature of nonalcoholic fatty liver disease (NAFLD), has been associated with steatohepatitis and fibrosis. Heterozygosity for alpha 1-antitrypsin (AAT) mutations is a cofactor of liver damage, and AAT influences inflammation and iron metabolism. This study evaluated the prevalence of the common AAT PiS/PiZ mutants in 353 patients with NAFLD, 195 of whom had hyperferritinemia, versus 114 matched controls and their influence on iron metabolism and the severity of liver damage in the 212 patients submitted to biopsy. PiS and PiZ alleles were searched for by restriction analysis. Thirty-eight patients (10.8%) carried non-MM genotypes versus 4/114 (3.5%) controls (P = .02). Patients carrying AAT mutations had higher ferritin (573 [454-966] vs. 348 [201-648]; P = .001) with similar transferrin saturation. The difference was more evident in males (P < .0001) and significant in patients not carrying HFE genotypes associated with iron overload (P = .015). The prevalence of non-MM genotypes was higher in patients with hyperferritinemia than in those without (28/195, 14% vs. 10/158, 6%, P = .016), and AAT mutations were associated with higher prevalence of sinusoidal siderosis (17/27, 63% vs. 70/180, 39%; P = .02), and sinusoidal/total iron score (46.3 +/- 38% vs. 25.1 +/- 35%, P = .01). Although ferritin was independently associated with fibrosis (P = .047), AAT mutations favoring sinusoidal iron deposition did not affect liver damage. In conclusion, AAT mutations are associated with hyperferritinemia and sinusoidal iron accumulation, but not with more severe liver damage in NAFLD.

In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases

Background/Aims : Although oxidative stress is an important candidate in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), the localization and pathological significance of oxidative stress-induced cellular damage in NAFLD remains unclear. Methods : Hepatic expression of 4-hydroxy-2 ′-nonenal (HNE) and 8-hydroxydeoxyguanosine (8-OHdG), as reliable markers of lipid peroxidation and oxidative DNA damage, respectively, was immunohistochemically investigated in NAFLD and the results were compared with histological findings. Results : While no HNE adducts were observed in control livers, they were frequently detected in NAFLD. In NASH, the localization of the adducts was in the cytoplasm of sinusoidal cells and hepatocytes with a predominance in zone 3. The grade of necro-inflammation as well as the stage of fibrosis significantly correlated with the HNE index. Regarding 8-OHdG, although no 8-OHdG expression was observed in normal liver and only a few in fatty liver, 11 of 17 cases (64.7%) with NASH exhibited nuclear expression of 8-OHdG in hepatocytes and sinusoidal cells in areas of active inflammation. The 8-OHdG index significantly correlated with the grade of necro-inflammation. Conclusions : Oxidative cellular damage occurs frequently in livers with NAFLD and may be associated with some clinico-pathological features of NAFLD including liver fibrosis and possibly, hepatocarcinogenesis.