Excessive production of collagen type I is a major contributor to hepatic fibrosis. Activated (myofibroblastic), but not quiescent, hepatic stellate cells (lipocytes) have a high level of collagen type I and a-smooth muscle actin expression. Therefore, stellate cell activation is a critical step in hepatic fibrosis. Here we show that quiescent stellate cells were activated by the generation of free radicals with ascorbate/ FeSO4 and by malondialdehyde, a product of lipid peroxidation. In addition, stellate cell activation by collagen type I matrix and TGFa was blocked by antioxidants, such as da-tocopherol and butylated hydroxytoluene. Moreover, oxidative stress, TGFa and collagen type I markedly stimulated stellate cell entry into S-phase, NFkB activity and c-myb expression, which were prevented by antioxidants. c-myb antisense oligonucleotide blocked the activation and proliferation of stellate cells induced by TGFa. Nuclear extracts from activated, but not from quiescent, stellate cells formed a complex with the critical promoter E box of the a-smooth muscle actin gene, which was disrupted by c-myb and NFkB65 antibodies, and competed by c-myb and NFkB cognate DNA. c-Myb expression was also stimulated in activated stellate cells in carbon tetrachloride-induced hepatic injury and fibrogenesis. This study indicates that oxidative stress plays an essential role, through the induction of c-myb and NFkB, on stellate cell activation. (J. Clin. Invest. 1995. 96:2461-2468

In hepatic iron overload, iron-catalyzed lipid peroxidation has been implicated in the mechanisms of hepatocellular injury. Lipid peroxidation may produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), which may form aldehyde-protein adducts. We investipted whether lipid peroxidation occurred in rats fed a diet containing 3% carbonyl iron for 5-13 wk, and if this resulted in the formation of MDA-and 4-HNE-protein adducts. Chronic iron feeding resulted in hepatic iron overload (> 10-fold) and concomitantly induced a 2-fold increase in hepatic lipid peroxidation. Using an antiserum specific for MDA-lysine protein adducts, we demonstrated by immunohistochemistry the presence of aldehyde-protein adducts in the cytosol of periportal hepatocytes, which co-localized with iron. In addition, MDAand 4-HNE-lysine adducts were found in plasma proteins of animals with iron overload. Only MDA adducts were detected in albumin, while other plasma proteins including a 120-kD protein had both MDA and 4-HNE adducts. In this animal model of hepatic iron overload, injury occurs primarily in periportal hepatocytes, where MDA-lysine protein adducts and excess iron co-localized. (J.

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Background and Aims: Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non‐alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade. Methods: Liver biopsies from 39 patients with varying degrees of hepatic steatosis were assessed for evidence of lipid peroxidation (malondialdehyde adducts), hepatic iron, inflammation, fibrosis, hepatic stellate cell activation (α‐smooth muscle actin and TGF‐β expression) and collagen type I synthesis (procollagen α1 (I) mRNA). Results: Lipid peroxidation occurred in and adjacent to fat‐laden hepatocytes and was maximal in acinar zone 3. Fibrosis was associated with steatosis (P < 0.04), lipid peroxidation (P < 0.05) and hepatic iron stores (P < 0.02). Multivariate logistic regression analysis confirmed the association between steatosis and lipid peroxidation within zone 3 hepatocytes (P < 0.05), while for hepatic iron, lipid peroxidation was seen within sinusoidal cells (P < 0.05), particularly in zone 1 (P < 0.02). Steatosis was also associated with acinar inflammation (P < 0.005). α‐Smooth muscle actin expression was present in association with both lipid peroxidation and fibrosis. Although the effects of steatosis and iron on lipid peroxidation and fibrosis were additive, there was no evidence of a specific synergistic interaction between them. Conclusions: These observations support a model where steatosis exerts an effect on fibrosis through lipid peroxidation, particularly in zone 3 hepatocytes.

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