Arthur I. Cederbaum

Alcohol and oxidative liver injury

Acute and chronic ethanol treatment has been shown to increase the production of reactive oxygen species, lower cellular antioxidant levels, and enhance oxidative stress in many tissues, especially the liver. Ethanol‐induced oxidative stress plays a major role in the mechanisms by which ethanol produces liver injury. Many pathways play a key role in how ethanol induces oxidative stress. This review summarizes some of the leading pathways and discusses the evidence for their contribution to alcohol‐induced liver injury. Many of the seminal reports in this topic have been published in Hepatology , and it is fitting to review this research area for the 25th Anniversary Issue of the Journal. (Hepatology 2006;43:S63–S74.)

Role of oxidative stress in alcohol-induced liver injury

Reactive oxygen species (ROS) are highly reactive molecules that are naturally generated in small amounts during the body’s metabolic reactions and can react with and damage complex cellular molecules such as lipids, proteins, or DNA. Acute and chronic ethanol treatments increase the production of ROS, lower cellular antioxidant levels, and enhance oxidative stress in many tissues, especially the liver. Ethanol-induced oxidative stress plays a major role in the mechanisms by which ethanol produces liver injury. Many pathways play a key role in how ethanol induces oxidative stress. This review summarizes some of the leading pathways and discusses the evidence for their contribution to alcohol-induced liver injury. Special emphasis is placed on CYP2E1, which is induced by alcohol and is reactive in metabolizing and activating many hepatotoxins, including ethanol, to reactive products, and in generating ROS.

Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury.

HepG2 cells were transfected with vectors containing human catalase cDNA and catalase cDNA with a mitochondrial leader sequence to allow comparison of the effectiveness of catalase overexpressed in the cytosolic or mitochondrial compartments to protect against oxidant-induced injury. Overexpression of catalase in cytosol and in mitochondria was confirmed by Western blot, and activity measurement and stable cell lines were established. The intracellular level of H2O2 induced by exogenously added H2O2 or antimycin A was lower in C33 cell lines overexpressing catalase in the cytosol and mC5 cell lines overexpressing catalase in the mitochondria as compared with Hp cell lines transfected with empty vector. Cell death caused by H2O2, antimycin A, and menadione was considerably suppressed in both the mC5 and C33 cell lines. C33 and mC5 cells were also more resistant to apoptosis induced by H2O2 and to the loss of mitochondrial membrane potential induced by H2O2 and antimycin A. In view of the comparable protection by catalase overexpressed in the cytosol versus the mitochondria, catalase produced in both cellular compartments might act as a sink to decompose H2O2 and move diffusable H2O2 down its concentration gradient. The present study suggests that catalase in cytosol and catalase in mitochondria are capable of protecting HepG2 cells against cytotoxicity or apoptosis induced by oxidative stress.

Lipid peroxidation and oxidant stress regulate hepatic apolipoprotein B degradation and VLDL production

How omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) lower plasma lipid levels is incompletely understood. We previously showed that marine omega-3 PUFAs (docosahexaenoic acid [DHA] and eicosapentaenoic acid) stimulate a novel pathway, post-ER presecretory proteolysis (PERPP), that degrades apolipoprotein B100 (ApoB100), thereby reducing lipoprotein secretion from liver cells. To identify signals stimulating PERPP, we examined known actions of omega-3 PUFA. In rat hepatoma or primary rodent hepatocytes incubated with omega-3 PUFA, cotreatment with the iron chelator desferrioxamine, an inhibitor of iron-dependent lipid peroxidation, or vitamin E, a lipid antioxidant, suppressed increases in thiobarbituric acid-reactive substances (TBARSs; a measure of lipid peroxidation products) and restored ApoB100 recovery and VLDL secretion. Moreover, omega-6 and nonmarine omega-3 PUFA, also prone to peroxidation, increased ApoB100 degradation via intracellular induction of TBARSs. Even without added fatty acids, degradation of ApoB100 in primary hepatocytes was blocked by desferrioxamine or antioxidant cotreatment. To extend these results in vivo, mice were infused with DHA, which increased hepatic TBARSs and reduced VLDL-ApoB100 secretion. These results establish a novel link between lipid peroxidation and oxidant stress with ApoB100 degradation via PERPP, and may be relevant to the hypolipidemic actions of dietary PUFAs, the basal regulation of ApoB100 secretion, and hyperlipidemias arising from ApoB100 overproduction.

A high-fat diet leads to the progression of non-alcoholic fatty liver disease in obese rats

Fatty livers of obese fa/fa rats are vulnerable to injury when challenged by insults such as endotoxin, ischemia‐reperfusion or acute ethanol treatment. The objective of this study was to evaluate whether a high‐fat diet can act as a “second hit” and cause progression to liver injury in obese fa/fa rats compared with lean Fa/? rats. Accordingly, obese fa/fa rats and their lean littermates were fed a diet low in fat (12% of total calories) or a diet with 60% calories as lard for 8 weeks. Hyperglycemia and steatohepatitis occurred in the fa/fa rats fed the high‐fat diet. This was accompanied by liver injury as assessed by alanine aminotransferase, hematoxilin and eosin staining, increased TNFα and stellate cell‐derived TGFβ, collagen deposition, and up‐regulation of α‐smooth muscle actin. Active MMP13 decreased in fa/fa rats independently of the diet, and TIMP1 expression increased with the high‐fat diet, especially in fa/fa rats. Although UCP2 expression was higher in fa/fa rats regardless of the diet, minor changes in ATP levels were observed. Oxidative stress occurred in the fa/fa rats fed the high‐fat diet as lipid peroxidation and protein carbonyls were elevated, while glutathione and antioxidant enzymes were very low. Expression and activity of cytochrome P450 2E1 and xanthine oxidase activity were down‐ regulated in fa/fa compared with Fa/? rats, and no effect was seen by the high‐fat diet. However, NADPH oxidase activity increased 2.5‐fold in fa/fa rats fed with the high‐fat diet. In summary, a high‐fat diet induces liver injury in fa/fa rats leading to periportal fibrosis. A role for oxidative stress is suggested via increased NADPH oxidase activity, lipid peroxidation, protein carbonyl formation, and low antioxidant defense.

Production of Reactive Oxygen Species by Microsomes Enriched in Specific Human Cytochrome P450 Enzymes

Few studies have evaluated the production of reactive oxygen intermediates by human microsomes, especially the influence of the specific form of cytochrome P450. Experiments were carried out to evaluate the ability of CYP1A1, 1A2, 2B6, and 3A4 to consume NADPH, reduce iron, and catalyze production of reactive oxygen species. Microsomes enriched in each of these CYPs were obtained from commercial +/- lymphoblast cells that had been transfected with cDNA encoding the specific human CYP. On a per nanomole cytochrome P450 basis, CYP3A4 was the most active P450 evaluated in catalyzing NADPH oxidation, production of superoxide anion radical, NADPH-dependent chemiluminescence, oxidation of dichlorofluorescein diacetate, and reduction of either ferric-EDTA or ferric-citrate. CYP1A1 was the next most reactive CYP, whereas CYP1A2 and 2B6 displayed a comparable, lower activity. Nitric oxide, which reacts with and inactivates hemoproteins, inhibited superoxide production by all the CYPs to a similar extent. Because CYP3A4 is present in high amounts in human liver microsomes and is active in catalyzing the formation of reactive oxygen species, this CYP may make an important contribution in the overall ability of human liver microsomes to generate active oxygen species.

Oxidative stress and alcoholic liver disease.

Reactive oxygen species (ROS) are highly reactive molecules that are naturally generated in small amounts during the bodys metabolic reactions and can react with and damage complex cellular molecules such as lipids, proteins, or DNA. This review describes pathways involved in ROS formation, why ROS are toxic to cells, and how the liver protects itself against ROS. Acute and chronic ethanol treatment increases the production of ROS, lowers cellular antioxidant levels, and enhances oxidative stress in many tissues, especially the liver. Ethanol-induced oxidative stress plays a major role in the mechanisms by which ethanol produces liver injury. Many pathways play a key role in how ethanol induces oxidative stress. This review summarizes some of the leading pathways and discusses the evidence for their contribution to alcohol-induced liver injury.

CYP2E1-dependent toxicity and oxidative stress in HepG2 cells.

Induction of CYP2E1 by ethanol is one of the central pathways by which ethanol generates a state of oxidative stress in hepatocytes. To study the biochemical and toxicological actions of CYP2E1, our laboratory established HepG2 cell lines which constitutively overexpress CYP2E1 and characterized these cells with respect to ethanol toxicity. Addition of ethanol or an unsaturated fatty acid such as arachidonic acid or iron was toxic to the CYP2E1-expressing cells but not control cells. This toxicity was associated with elevated lipid peroxidation and could be prevented by antioxidants and inhibitors of CYP2E1. Apoptosis occurred in the CYP2E1-expressing cells exposed to ethanol, arachidonic acid, or iron. Removal of GSH caused a loss of viability in the CYP2E1-expressing cells even in the absence of added toxin or pro-oxidant. This was associated with mitochondrial damage and decreased mitochondrial membrane potential. Surprisingly, CYP2E1-expressing cells had elevated GSH levels, due to transcriptional activation of gamma glutamyl cysteine synthetase. Similarly, levels of catalase, alpha-, and microsomal glutathione transferase were also increased, suggesting that upregulation of these antioxidant genes may reflect an adaptive mechanism to remove CYP2E1-derived oxidants. While it is likely that several mechanisms contribute to alcohol-induced liver injury, the linkage between CYP2E1-dependent oxidative stress, mitochondrial injury, and GSH homeostasis may contribute to the toxic action of ethanol on the liver. HepG2 cell lines overexpressing CYP2E1 may be a valuable model to characterize the biochemical and toxicological properties of CYP2E1.

Cytochrome P450 2E1 contributes to ethanol‐induced fatty liver in mice

Cytochrome P450 2E1 (CYP2E1) is suggested to play a role in alcoholic liver disease, which includes alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. In this study, we investigated whether CYP2E1 plays a role in experimental alcoholic fatty liver in an oral ethanol‐feeding model. After 4 weeks of ethanol feeding, macrovesicular fat accumulation and accumulation of triglyceride in liver were observed in wild‐type mice but not in CYP2E1‐knockout mice. In contrast, free fatty acids (FFAs) were increased in CYP2E1‐knockout mice but not in wild‐type mice. CYP2E1 was induced by ethanol in wild‐type mice, and oxidative stress induced by ethanol was higher in wild‐type mice than in CYP2E1‐knockout mice. Peroxisome proliferator‐activated receptor α (PPARα), a regulator of fatty acid oxidation, was up‐regulated in CYP2E1‐knockout mice fed ethanol but not in wild‐type mice. A PPARα target gene, acyl CoA oxidase, was decreased by ethanol in wild‐type but not in CYP2E1‐knockout mice. Chlormethiazole, an inhibitor of CYP2E1, lowered macrovesicular fat accumulation, inhibited oxidative stress, and up‐regulated PPARα protein level in wild‐type mice fed ethanol. The introduction of CYP2E1 to CYP2E1‐knockout mice via an adenovirus restored macrovesicular fat accumulation. These results indicate that CYP2E1 contributes to experimental alcoholic fatty liver in this model and suggest that CYP2E1‐derived oxidative stress may inhibit oxidation of fatty acids by preventing up‐regulation of PPARα by ethanol, resulting in fatty liver. (HEPATOLOGY 2008.)

Effects of chronic ethanol treatment on mitochondrial functions damage to coupling site I

Abstract Chronic ethanol feeding to rats produces changes in hepatic mitochondria which persist in the absence of ethanol metabolism. The integrity of isolated mitochondria is well preserved, as evidenced by unchanged activities of latent, Mg 2+ - and dinitrophenol-stimulated ATPase activity, and unaltered permeability to NADH. With succinate or ascorbate as substrates, oxygen uptake by mitochondria from ethanol-fed rats was decreased compared to pair-fed controls. The decrease was comparable under state 4 or state 3 conditions, or in the presence of an uncoupler. However, with the NAD + -dependent substrates, ADP-stimulated oxygen consumption (state 3) was decreased to a greater extent than state 4 or uncoupler-stimulated oxygen consumption in mitochondria from ethanol-fed rats. This suggests that the decrease in energy-dependent oxygen consumption at site I may be superimposed upon damage to the respiratory chain. Using NAD + -dependent substrates (glutamate, α-ketoglutarate or β-hydroxybutyrate) the respiratory control ratio and the P O ratio of oxidative phosphorylation were significantly decreased in mitochondria isolated from the livers of rats fed ethanol. By contrast, when succinate or ascorbate served as the electron donor these functions were unchanged. The rate of phosphorylation is decreased 70% with the NAD + -dependent substrates because of a decreased flux of electrons, as well as a lower efficiency of oxidative phosphorylation. With succinate and ascorbate as substrates, the rate of phosphorylation is decreased 20–30%, owing to a decreased flux of electrons. These data suggest the possibility that, in addition to effects on the respiratory chain, energy-coupling site I may be damaged by ethanol feeding. Energy-dependent Ca 2+ uptake, supported by either substrate oxidation or ATP hydrolysis, was inhibited by chronic ethanol feeding. Concentrations of acetaldehyde (1–3 m m ) which inhibited phosphorylation associated with the oxidation of NAD + -dependent substrates had no effect on that of succinate or ascorbate. Many of the effects of chronic ethanol feeding on mitochondrial functions are similar to those produced by acetaldehyde in vitro .