Arthur I. Cederbaum - US grants

Pyrazole and 4-methylpyrazole are potent inhibitors of alcohol dehydrogenase, and have been used to block the metabolism of ethanol and for treatment of methanol or ethylene glycol poisoning. Pyrazole, but not 4-methylpyrazole, has been shown to be hepatotoxic. The reasons for these differences are not known. 4-Hydroxypyrazole and 4-hydroxymethylpyrazole are major metabolites found in the urine after in vivo administration of pyrazole or 4-methylpyrazole. The enzyme system(s) responsible for the metabolism of these compounds are not known. Preliminary results show that microsomes from rats treated with pyrazole or 4-methylpyrazole display several properties which are very similar to properties found with microsomes from chronic ethanol-fed rats, suggesting the possibility that pyrazole and 4-methylpyrazole may induce a cytochrome P-450 isozyme which is similar to the ethanol-inducible cytochrome P-450. The overall objective of this application is to study the interaction of pyrazole and 4-methylpyrazole with microsomes and to compare the properties of microsomes from pyrazole- and 4-methylpyrazole-treated rats to those found with microsomes from ethanol-treated rats. Properties to be studied include substrate and alcohol metabolism and specificity, kinetic experimens, substrate binding spectra, content of microsomal enzymes, effect of inhibitors, generation of oxygen radicals and promotion of lipid peroxidation. The metabolism of pyrazole and 4-methylpyrazole by microsomes from control and induced rats will be determined and the metabolites characterized. Attempts to purify the pyrazole- and 4-methylpyrazole-inducible cytochrome P-450 and reconstitution experiments with purified enzymes will be carried out. Some experiments on metabolic consequences of pyrazole and 4-methylpyrazole treatment, and the effects of their metabolites, on hepatocyte function will be performed to evaluate why pyrazole, but not 4-methylpyrazole, is hepatotoxic. It is hoped that these studies will provide important information on the metabolism and on biochemical and pharmacological properties of these important compounds, which are widely used in the alcohol field.

Pyrazole and 4-methylpyrazole are potent inhibitors of alcohol dehydrogenase, and have been used to block the metabolism of ethanol and for treatment of methanol or ethylene glycol poisoning. Pyrazole, but not 4-methylpyrazole, has been shown to be hepatotoxic. The reasons for these differences are not known. 4-Hydroxypyrazole and 4-hydroxymethylpyrazole are major metabolites found in the urine after in vivo administration of pyrazole or 4-methylpyrazole. The enzyme system(s) responsible for the metabolism of these compounds are not known. Preliminary results show that microsomes from rats treated with pyrazole or 4-methylpyrazole display several properties which are very similar to properties found with microsomes from chronic ethanol-fed rats, suggesting the possibility that pyrazole and 4-methylpyrazole may induce a cytochrome P-450 isozyme which is similar to the ethanol-inducible cytochrome P-450. The overall objective of this application is to study the interaction of pyrazole and 4-methylpyrazole with microsomes and to compare the properties of microsomes from pyrazole- and 4-methylpyrazole-treated rats to those found with microsomes from ethanol-treated rats. Properties to be studied include substrate and alcohol metabolism and specificity, kinetic experimens, substrate binding spectra, content of microsomal enzymes, effect of inhibitors, generation of oxygen radicals and promotion of lipid peroxidation. The metabolism of pyrazole and 4-methylpyrazole by microsomes from control and induced rats will be determined and the metabolites characterized. Attempts to purify the pyrazole- and 4-methylpyrazole-inducible cytochrome P-450 and reconstitution experiments with purified enzymes will be carried out. Some experiments on metabolic consequences of pyrazole and 4-methylpyrazole treatment, and the effects of their metabolites, on hepatocyte function will be performed to evaluate why pyrazole, but not 4-methylpyrazole, is hepatotoxic. It is hoped that these studies will provide important information on the metabolism and on biochemical and pharmacological properties of these important compounds, which are widely used in the alcohol field.

Oxygen can be toxic to cells when it is transformed into reactive intermediates called oxygen radicals. Many agents damage cells by promoting the production of oxygen radicals and lipid peroxidation. The mechanism(s) by which alcohol damages the liver is not understood. Based upon recent results, it appears that alcohol can promote the production of oxygen radicals and induce lipid peroxidation. The specific objective is to evaluate the interaction between alcohol and oxygen radicals in promoting liver cell damage and in increasing the susceptibility of the alcoholic to oxidative stress and to the toxicity of other agents. This information may be of value in preventing or ameliorating some of the toxic effects associated with alcohol abuse. Experiments will be conducted to extend previous observations that production of hydroxyl radicals (.OH) by microsomes from chronic ethanol-fed rats is increased. Rates of superoxide, hydrogen peroxide, .OH production and lipid peroxidation by microsomes from ethanol-treated rats and controls will be determined. Sensitivity to superoxide dismutase, catalase, iron chelators and the role of different iron chelates will be assessed as will the microsomal enzymes which participate in generating oxy-radicals. The activation of compounds which become toxic because of increased generation of oxy-radicals and promotion of lipid peroxidation will be studied. The generation of .OH and promotion of lipid peroxidation by intact hepatocytes will also be evaluated. The overall objective will be to extend studies with microsomal systems to intact cells. Special emphasis will be placed on use of dimethylsulfoxide, tert butyl alcohol and benzoate, as new chemical probes for the detection of .OH-like species. Viability will be monitored to determine if the alcohol-derived cells are especially sensitive to damage by the generated oxy-radicals. Some in vivo experiments to detect .OH and lipid peroxidation after ethanol treatment will be carried out. The regulation of drug metabolism by intact liver cells, the effects produced by acute and chronic ethanol on hepatic drug metabolism, and the role of acetaldehyde in the actions of ethanol will be studied. NADPH availability may regulate oxygen radical generation, and alcohol-drug interactions may explain why alcoholics show different sensitivities to drugs, and are more susceptible to damage by external toxins.

Pyrazole and 4-methylpyrazole are potent inhibitors of alcohol dehydrogenase, and have been used to block the metabolism of ethanol and for treatment of methanol or ethylene glycol poisoning. Pyrazole, but not 4-methylpyrazole, has been shown to be hepatotoxic. The reasons for these differences are not known. 4-Hydroxypyrazole and 4-hydroxymethylpyrazole are major metabolites found in the urine after in vivo administration of pyrazole or 4-methylpyrazole. The enzyme system(s) responsible for the metabolism of these compounds are not known. Preliminary results show that microsomes from rats treated with pyrazole or 4-methylpyrazole display several properties which are very similar to properties found with microsomes from chronic ethanol-fed rats, suggesting the possibility that pyrazole and 4-methylpyrazole may induce a cytochrome P-450 isozyme which is similar to the ethanol-inducible cytochrome P-450. The overall objective of this application is to study the interaction of pyrazole and 4-methylpyrazole with microsomes and to compare the properties of microsomes from pyrazole- and 4-methylpyrazole-treated rats to those found with microsomes from ethanol-treated rats. Properties to be studied include substrate and alcohol metabolism and specificity, kinetic experimens, substrate binding spectra, content of microsomal enzymes, effect of inhibitors, generation of oxygen radicals and promotion of lipid peroxidation. The metabolism of pyrazole and 4-methylpyrazole by microsomes from control and induced rats will be determined and the metabolites characterized. Attempts to purify the pyrazole- and 4-methylpyrazole-inducible cytochrome P-450 and reconstitution experiments with purified enzymes will be carried out. Some experiments on metabolic consequences of pyrazole and 4-methylpyrazole treatment, and the effects of their metabolites, on hepatocyte function will be performed to evaluate why pyrazole, but not 4-methylpyrazole, is hepatotoxic. It is hoped that these studies will provide important information on the metabolism and on biochemical and pharmacological properties of these important compounds, which are widely used in the alcohol field.

Pyrazole and 4-methylprazole (4-MP) are potent inhibitors of alcohol dehydrogenase and block the metabolic actions of ethanol. These compounds have been used in the treatment of methanol and ethylene glycol poisoning and to ameliorate the disulfiram-ethanol reaction and acetaldehyde toxicity in "flushers". Biochemical and pharmacological properties of pyrazole and 4-MP and enzymatic loci for their metabolism, have not been investigated in detail. Pyrazole and 4-MP were found to induce the same isozyme of cytochrome P-450 as does ethanol, P-450 IIE.1. The pyrazole- induced P-450 has been purified, characterized, and a polyclonal antibody produced Pyrazole and 4-MP produce Type II binding spectra with microsomes and cytochrome P-450, and inhibit the oxidation of drugs, including ethanol, by microsome. Pyrazole was shown to be a good substrate for P-450 IIE.1 and was oxidized to 4-hydroxy-pyrazole. Experiments are planned to continue to characterize the interactions of pyrazole and 4-MP with the microsomal mixed-function oxidase system. We have developed a new method to detect 4-hydroxymethylpyrazole. The ability of microsomes, P-450, hepatocytes and oxygen radicals to oxidize 4-MP to this metabolite, and the effect of inducers of P-450 IIE.1 will be evaluated. Studies to understand at the molecular level mechanisms responsible for the induction of P-450 IIE.1 by pyrazole, 4-MP and ethanol will involve effect of inhibitors of transcription and translation, detailed time and dose response curves comparing changes in P-450 IIE.1 mRNA levels and protein levels and measures of the turnover of P-450 IIE.1 in the absence and presence of the inducer. The acinar localization and induction of P-450 IIE.1. and activation of hepatotoxins in periportal and perivenous hepatocytes will be determined. The ability of glycerol (a possible physiological relevant inducer) to induce P-450 IIE.1 and to interact with and be metabolized by microsomes and P-450 will be investigated. Additional studies will employ intact hepatocytes and human livers, characterize metabolic actions of the metabolites 4- hydroxypyrazole and 4-hydroxymethylpyrazole, and extend the substrate preference of P-450 IIE.1 to toxicologically important agents such as nitriles, e.g., cyanamide and glycols, e.g., anti-freeze. It is hoped that these studies will provide important information and continue to characterize the metabolic, biochemical and pharmacological properties of these valuable compounds.

This award supports cooperative research in cellular biochemistry to be performed by Arthur I. Cederbaum of Mt. Sinai Hospital in New York and Susana Puntarulo and Alberto Boveris, both in the Department of Biochemistry at the University of Buenos Aires in Argentina. Metals, especially ferric complexes are required to promote the formation of oxidizing species responsible for DNA and cellular damage. This work will study the interaction of various ferric complexes, including ferritin, with organelles such as isolated nucleii and their ability to catalyze the generation of oxygen-active radicals in rat liver nucleii. This collaborative effort will bring together the expertise of two laboratories concerned with the role of transition metals in catalyzing the production of active oxygen radicals by biological systems. The work will provide new insights into oxygen radical formation and lipid peroxidation in such systems.

There is increasing evidence that ethanol toxicity may be associated with elevated production of reactive oxygen intermediates. Among the mechanisms suggested by which ethanol produces oxidative stress, ethanol induction of the microsomal mixed-function oxidase system and cytochrome P450 2E1, and ethanol-derived NADH are of special interest. The specific objective of this application is to employ electron spin resonance (ESR) spectroscopy to determine production of radicals such as superoxide, hydroxyl and carbon center radicals such as the hydroxyethyl radical by liver cell organelles, particularly microsomes, and assess the influence of ethanol treatment on this production of reactive radical intermediates. The major advantages of ESR are that it provides unambiguous, direct determination of radicals, is highly sensitive, and is the only method to detect reactive intermediates such as HER. Experiments will be carried out which compare the effectiveness of NADH with that of NADPH in promoting radical generation and in interacting with a variety of iron complexes to catalyze radical generation by liver cell organelles such as microsomes, mitochondria, nuclei and plasma membranes. The role of P450 2E1 will be assessed using substrates, inhibitors and antibodies. The effects of anti-oxidants and redox cycling agents, and comparisons of results obtained by ESR and chemical detection, will be made. The effect of chronic ethanol treatment on ESR- detectable rates of oxygen and hydroxy radicals and HER production will be evaluated. Since ethanol toxicity originates in the perivenous zone, microsomes and other organelles will be isolated from periportal hepatocytes prepared from control and ethanol-treated rats, and production of reactive intermediates determined by ESR. To identify microsomal enzymes which play a role in the NADH-and NADPH-dependent generation of reactive radical intermediates, experiments with purified NADH-b5 reductase, NADPH-P450 reductase, b5 and P450 (especially P450 2E1) will be carried out, as will selected experiments with human liver microsomes and human liver P450 2E1. A final aim will be to use ESR to evaluate production of glycerol and other polyhydroxylated alcohol radicals. It is anticipated that direct and specific ESR studies will provide new information on the generation of, and the role of, reactive radical intermediates in alcohol toxicity.

Cedarbaum 9224797 This U.S.-Argentina Cooperative Science Program award supports travel expenses of A. Cederbaum, Mount Sinai School of Medicine, New York to Argentina. Dr. Cederbaum will carry out research in biochemistry, in collaboration with S. Puntarulo and A. Boveris, of the Univ. of Buenos Aires. The award also covers disposable supplies needed for the research and some publication costs. The research id directed towards understanding the role of transition metals in catalyzing the production of active oxygen radicals by biological systems, in particular the ferritin system. The physiological role of ferritin is important, and the approach taken by the investigators is likely to solve some long-standing problems involving the action of ferritin.

Pyrazole and 4-methylprazole (4-MP) are potent inhibitors of alcohol dehydrogenase and block the metabolic actions of ethanol. These compounds have been used in the treatment of methanol and ethylene glycol poisoning and to ameliorate the disulfiram-ethanol reaction and acetaldehyde toxicity in "flushers". Biochemical and pharmacological properties of pyrazole and 4-MP and enzymatic loci for their metabolism, have not been investigated in detail. Pyrazole and 4-MP were found to induce the same isozyme of cytochrome P-450 as does ethanol, P-450 IIE.1. The pyrazole- induced P-450 has been purified, characterized, and a polyclonal antibody produced Pyrazole and 4-MP produce Type II binding spectra with microsomes and cytochrome P-450, and inhibit the oxidation of drugs, including ethanol, by microsome. Pyrazole was shown to be a good substrate for P-450 IIE.1 and was oxidized to 4-hydroxy-pyrazole. Experiments are planned to continue to characterize the interactions of pyrazole and 4-MP with the microsomal mixed-function oxidase system. We have developed a new method to detect 4-hydroxymethylpyrazole. The ability of microsomes, P-450, hepatocytes and oxygen radicals to oxidize 4-MP to this metabolite, and the effect of inducers of P-450 IIE.1 will be evaluated. Studies to understand at the molecular level mechanisms responsible for the induction of P-450 IIE.1 by pyrazole, 4-MP and ethanol will involve effect of inhibitors of transcription and translation, detailed time and dose response curves comparing changes in P-450 IIE.1 mRNA levels and protein levels and measures of the turnover of P-450 IIE.1 in the absence and presence of the inducer. The acinar localization and induction of P-450 IIE.1. and activation of hepatotoxins in periportal and perivenous hepatocytes will be determined. The ability of glycerol (a possible physiological relevant inducer) to induce P-450 IIE.1 and to interact with and be metabolized by microsomes and P-450 will be investigated. Additional studies will employ intact hepatocytes and human livers, characterize metabolic actions of the metabolites 4- hydroxypyrazole and 4-hydroxymethylpyrazole, and extend the substrate preference of P-450 IIE.1 to toxicologically important agents such as nitriles, e.g., cyanamide and glycols, e.g., anti-freeze. It is hoped that these studies will provide important information and continue to characterize the metabolic, biochemical and pharmacological properties of these valuable compounds.

The overall goal is to evaluate the role of reactive oxygen intermediates (ROl) in the molecular mechanisms by which ethanol is toxic. Major focus will be on the role of P4502E1, interaction with iron, and reactivity of NADH and NADPH as cofactors. Specific Aim 1 - Our laboratory developed a HepG2 cell line which stably expresses human P4502E1. Ethanol was toxic to these cells, but not to control cells. These cells appear to represent a novel model to assess the role of P4502E1 or ROl generated during the metabolism of ethanol by P4502E1 in the hepatotoxic actions of ethanol. Studies will be carried out to characterize ethanol toxicity, effect of P4502E1 inhibitors, antioxidants, iron chelators, enrichment of cell membranes with PUFA and the ability of ethanol, acting via P4502E1,to produce a state of oxidative stress: Lipid, protein, and DNA targets for ROl produced by ethanol will be studied. Acetaldehyde and lipid adducts will be immunochemically detected. A final study will be to assess whether ethanol toxicity involves an apoptotic mechanism; if so, protection by BCl-2 will be studied. Specific Aim 2 - The role of NADH-cyt b5 reductase, cyt b5, and cyt P450 in the NADH-dependent production of ROl by microsomes will be studied employing reconstituted systems containing the purified enzymes, and inhibitors and antibodies against these enzymes. Specific Aim 3 - The interaction of NADPH-P450 reductase and P4502E1 with various ferric complexes to produce ROl will be investigated to test the hypothesis that the ability of the reductase versus P450 to interact with iron is not only dependent upon the chelated form of the iron, but also the concentration of iron. Specific Aim 4 - The production of ROl by microsomes and mitochondria isolated from periportal and pericentral hepatocytes of control and ethanol-treated rats will be determined since P4502E1 is present at highest levels in the PC zone and ethanol injury originates here. Specific Aim 5 - Studies will be carried out to compare the production of ROl by human P4502E1 with other human P450 isozymes. Ethanol toxicity and the role of 2E1 and ROl will be studied in immortalized normal human liver cells. These studies will help define the role of P4502E1 and ROl in the mechanism by which ethanol is hepatotoxic, help to design therapeutic interventions to prevent or ameliorate this toxicity, and provide basic mechanistic information of value to the oxygen radical and P450 areas of research.

APPLICANT'S ABSTRACT: There is much current interest in the role of oxidative stress and ethanol generation of reactive radical species in the mechanism(s) by which ethanol is toxic. It has been difficult to establish direct linkage between CYP2El, oxidative stress, and ethanol toxicity. Our laboratory has established a HepG2 cell line which constitutively expresses the human CYP2El. Ethanol or a polyunsaturated fatty acid (PUFA) was toxic to the E9 cells which express CYP2El, but not to control cells. Toxicity by ethanol and PUFA was prevented by antioxidants. These cells appear to be a valuable model to assess the role of CYP2El-dependent formation of reactive species such as the 1-hydroxyethyl radical (HER) in the hepatotoxic actions of ethanol. SPECIFIC Aim I is designed to study the role of HER, lipid, and other free radicals in the toxic actions of ethanol and PUFA to cells expressing CYP2El. The major analytical technique to be used is ESR spectroscopy. Studies are planned to study the mechanism of HER formation by CYP2El, and the role of HER and lipid radicals in the toxicity exerted by ethanol and PUFA; the interaction of HER with cellular constituents including adduct formation with CYP2El will be evaluated; activation of the transcription factor, NF-kB, by ethanol or CYP2El derived HER and other radical species will be determined. These studies should identify production of HER and lipid radicals in cells expressing CYP2El and assess the role of these radicals in the toxicity by ethanol and PUFA. Aim II will characterize NADPH- and NADH-dependent formation of HER, 02-, OH, and other reactive intermediates by microsomes from cell lines which express only one human P450 isoform. Toxicity by ethanol or PUFA to these cells will be evaluated, to indicate if human CYP2El is uniquely reactive in activating ethanol to HER, in formation of free radicals, and in promoting ethanol toxicity via a free radical, oxidative stress type of mechanism. The effect of NO on CYP2El catalytic activity and generation of HER and other free radicals will be evaluated in Aim Ill. If NO inhibits CYP2El, NO may prove to be useful as a protectant against the toxicity of ethanol and other toxins which are activated by CYP2El; this will be directly determined with the E9 cells. These studies will provide new information on the ability of NO to modulate CYP2El catalytic activity, generation of reactive intermediates, and ethanol and PUFA toxicity.

DESCRIPTION (provided by applicant): There is interest in the role of oxidative stress and generation of reactive radical species in the mechanism(s) by which ethanol is toxic. Induction of CYP2E1 by ethanol is one central pathway by which ethanol generates oxidative stress. Understanding the biochemical and toxicological properties of CYP2E1 will be important in providing mechanistic information on the actions of CYP2E1 in intact cells. A1M1 will evaluate the role of mitogen activated protein kinases (MAPK) in CYP2E1-dependent toxicity in HepG2 cells which express CYP2E1 and in pyrazole hepatocytes with high levels of CYP2E1. The cells will be treated with ethanol, arachidonic acid, iron or depleted of GSH and activation of MAPK-ERK, JNK, P38 evaluated. The ability of specific inhibitors of different MAPK to prevent CYP2E1 toxicity and oxidative stress will be determined. Experiments will be carried out to evaluate if NF-kappaB is activated in CYP2E1 expressing cells exposed to the above toxic agents and whether this activation protects or increases sensitivity to the toxin. AIM2 will characterize the role of calcium in CYP2E1-dependent toxicity. Using the above models, toxicity will be evaluated in Ca2+ -containing and Ca2+-free medium, and in the presence of intracellular Ca2+ chelating agents or inhibitors of Ca2+ import into the cell. Intracellular Ca2+ levels will be determined, as will possible linkage between changes in Ca2+ homeostasis, oxidative stress, cell viability and mitochondrial function. Downstream possible mediators such as the Ca2+-activated hydrolases calpain and phospholipase A2 will be studied for their role in CYP2E1-dependent toxicity. AIM3-Since oxidative stress plays an important role in alcoholic liver injury, we will evaluate whether such injury occurs in mice deficient in critical antioxidant enzymes such as copper-zinc SOD-1, manganese SOD-2 and GPX-1 knockout mice. These mice will be fed an ethanol diet for varying times and liver injury evaluated. Oxidative stress parameters will be measured, as will mitochondrial function, CYP2E1 and TNFalpha. Toxicity that is associated with binge alcohol treatment or after induction of CYP2E1 in these knockouts and controls will be evaluated. It is hoped that such studies in hepatocyte and HepG2 cell culture models and in-vivo models, will help to define further the biochemical and toxicological actions of CYP2E1 and its role in alcohol-induced liver injury.

NAD(H) phosphate; free radical oxygen; liver function; alcoholism /alcohol abuse; ethanol; acetaldehyde; oxidative stress; xanthine oxidase; chelating agents; hepatotoxin; cytochrome P450; isozymes; microsomes; mitochondria; adduct; unsaturated fatty acids; iron; liver metabolism; metal complex; antioxidants; nicotinamide adenine dinucleotide; tissue /cell culture; laboratory rat;

DESCRIPTION: (Adapted from the Applicant's Abstract): There is much interest in the role of oxidative stress as contributing to mechanisms by which ethanol is hepatotoxic. Induction of CYP2E1 by ethanol appears to be a major pathway by which ethanol produces a state of oxidative stress. Besides ethanol, CYP2E1 oxidizes many other important compounds and is induced under a variety of pathophysiological conditions. A major level of regulation of CYP2E1 is at the posttranscriptional stage as several ligands for CYP2E1, including ethanol protect the enzyme against degradation by uncharacterized intracellular proteolytic pathways. The goal of this application is Specific Aim 1 will evaluate the role of the proteasome complex in CYP2EI degradation in human hepatocytes. Turnover of CYP2E1 in cultured human hepatocytes will be studied. The major proteolytic pathways, and whether ubiquitination is required for CYP2E1 degradation will be determined, as will the effects of ethanol on this process. Specific Aim 2 will evaluate the role of reactive oxygen species in triggering CYP2E1 degradation by the proteasome complex. The ability of a variety of antioxidants to alter CYP2E1 levels and turnover in intact cells and reconstituted systems will be evaluated, and evidence for CYP2E1 oxidative modification will be provided. Specific Aim 3 will evaluate the effect of molecular chaperones on steady state levels and turnover of CYP2E1. Experiments will involve studying the effects of inhibitors of heat shock proteins or antibodies or of irnmunodepletion followed by readdition of the modulator in the reconstituted system, or adding inhibitors or addition of plasmids expressing the putative modulator to intact cell models. Does ethanol and other CYP2E1 ligands block the effect of the modulator? These experiments will provide new information on the role of the proteasome and ubiquitination in CYP2E 1 turnover in human hepatocytes, how ethanol protects CYP2E1 against degradation, and identify factors which may trigger rapid turnover of this enzyme. Modulators of any of these newly identified steps may have therapeutic implications for individuals with high levels of CYP2E1.

DESCRIPTION (provided by applicant): There is interest in the role of oxidative stress and generation of reactive radical species in the mechanism(s) by which ethanol is toxic. Induction of CYP2E1 is one pathway by which ethanol generates oxidative stress. S-Adenosyl- L-Methionine (SAM) is a regulator of cellular growth, differentiation and function. Impairment of SAM synthesis plays an important role in hepatic injury induced by various agents, including alcohol. CYP2E1 levels were increased in the MAT1A knockout mouse suggesting SAM could regulate or modulate CYP2E1. The goal of this application is to study possible interactions/modulation between CYP2E1 and SAM and to investigate the effects of SAM on CYP2E1- dependent toxicity and generation of reactive oxygen species. Aim 1 will evaluate the effect of SAM, and the SAM metabolite 5-methylthioadenosine (MTA) on CYP2E 1-dependent toxicity in cultured hepatocytes from pyrazole-treated rats and HepG2 cells overexpressing CYP2E1 (E47 cells). Aim 2 will study the effect of SAM and MTA on hepatic stellate cell activation by CYP2El-derived diffusible mediators in co-cultures of primary hepatic stellate cells with pyrazole hepatocytes or E47 cells. Aim 3 will assess the effect of SAM and MTA on CYP2El-dependent activation of antioxidants genes which reflect an adaptive response to CYP2El-dependent oxidative stress. The ability of SAM or MTA to prevent activation of P38 MAP kinasc or other stress kinascs by CYP2E1 will be determined, since such actions may be important in mechanisms by which SAM or MTA prevent CYP2E1 toxicity. Aim 4 will study in-vivo effects of SAM and MTA on CYP2E1 expression, content and actions. Control rats or rats induced by pyrazole, ethanol, starvation with high levels of CYP2E 1 will be treated with SAM or MTA in-vivo and the effect on basal or induced CYP2E1 protein, activity, mRNA level on up regulation of GSH and antioxidants and on CYP2El-dependent toxicity in several in-vivo models determined. The effect of CYP2E1 induction on expression of the MAT1A and MAT2A genes or enzyme activities responsible for the synthesis of SAM will be determined. Aim 5 will evaluate the ability of SAM or MTA, in-vitro, to inhibit CYP2El-dependent generation of reactive oxygen species. It is hoped that this study utilizing hepatocyte cell culture models, in-vivo models and mechanistic studies will help to define the effects of SAM on CYP2E 1-dependent toxicity and may prove valuable in understanding the hepatoprotective actions of SAM in many models of liver injury, including alcohol-induced liver injury.

[unreadable] DESCRIPTION (provided by applicant): CYP2E1 metabolizes and activates many toxicologic important compounds. Toxicity of these agents is enhanced by ethanol, due to induction of CYP2E1. Moreover, CYP2E1 is induced under a variety of physiological and pathophysiological conditions such as diabetes, obesity and non-alcoholic steatohepatitis. Further understanding the biochemical and toxicological properties of CYP2E1 will be important in providing mechanistic information on the actions of CYP2E1 in intact cells and this is the major goal of this application. AIM 1- Adaptation to oxidant stimuli is critical for survival of cells exposed to oxidative stress. We found an up- regulation of important antioxidant genes by CYP2E1. Nrf2 is a key transcription factor regulating these antioxidant genes. We will evaluate Nrf2 levels and actions in vivo and in vitro after treatment of mice and rats with ethanol or inducers of CYP2E1 such as pyrazole and acetone and in our HepG2 cell models which express human CYP2E1. CYP2E1 null mice will be used to evaluate whether CYP2E1 plays a role in regulating Nrf2 levels and induction of antioxidant genes by ethanol. SiRNA-Nrf2 will be used to down-regulate Nrf2 and the consequences of this on CYP2E1- and ethanol-induced oxidant stress and toxicity determined. Nrf2 null mice will be treated with ethanol acutely and chronically to attempt to develop an oral alcohol model of liver injury. AIM 2- Damage to mitochondria is an early event in the loss of cellular viability when cells with high levels of CYP2E1 are exposed to ethanol and other prooxidants. We propose to further characterize the effects of CYP2E1 and the role of CYP2E1 in the effects of ethanol on mitochondrial function and intactness. We will evaluate the onset of the mitochondrial permeability transition (MPT) in HepG2 cells expressing CYP2E1 or in hepatocytes from pyrazole and ethanol-treated rats exposed to prooxidants. The specific role of CYP2E1 in onset of the MPT by ethanol and prooxidants will be determined using CYP2E1 null mice and inhibitors. CYP2E1 has recently been shown to be present in the mitochondria. We have developed a HepG2 cell line which expressed CYP2E1 only in the mitochondria. We propose to study the functional consequences associated with mitochondrial CYP2E1 when such cells are exposed to ethanol and to prooxidants. AIM 3- Possible interactions between LPS and CYP2E1 or between CYP2E1 and the Fas system have not been evaluated yet these are all independent risk factors involved in alcoholic liver disease. We will explore the susceptibility and possible synergistic effect of CYP2E1 overexpression to LPS or to Fas antibody hepatotoxicity and assess the involvement of CYP2E1 in the increase of hepatotoxicity of LPS or Fas antibody-induced liver injury following pyrazole or ethanol pretreatment in mice. These studies may provide an experimental model to better understand mechanisms of ethanol-induced liver damage. The role of oxidative and nitrosative stress and of Kupffer cells in the CYP2E1 potentiation of toxicity will be evaluated to provide mechanistic insights of the toxicity process. It is hoped that such studies in hepatocyte and HepG2 cell culture models, and in vivo control and knockout mouse models will help to define further the biochemical and toxicological actions of CYP2E1 and its role in alcohol- induced liver injury. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The mechanism(s) by which alcohol causes cell injury are still not clear. A major mechanism that is a focus of considerable research is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a key role on how ethanol induces "oxidative stress". We hypothesize that increased oxidative stress from CYP2E1 induction in vivo sensitizes hepatocytes to chronic ethanol induced hepatotoxicity and steatosis. We propose that oxidants such as peroxynitrite, activation of MAP kinases such as JNK and/or p38 MAPK, a decline in activation of NF-:B and synthesis of survival factors, and mitochondrial dysfunction are downstream mediators of the CYP2E1 potentiated hepatotoxicity. We propose that such downstream mediators from CYP2E1 in association with ethanol-induced elevation of TNF1, homocysteine and ER stress activate lipogenic transcription factors and enzymes while decreasing activation of lipolytic transcription factors and enzymes, thereby resulting in fatty liver. Preliminary data in support of these hypotheses are the findings that ethanol-induced fatty liver is blunted in CYP2E1 knockout mice and restored in humanized CYP2E1 knockin mice. Hepatotoxicity and elevated oxidative/nitrosative stress in found in the CYP2E1 knockin mice with high levels of human CYP2E1 after chronic ethanol feeding. The ethanol-fed CYP2E1 knockin mice appear to be an effective ORAL model of alcohol-induced hepatotoxicity. Wild type, CYP2E1 knockout and CYP2E1 knockin mice will be fed the high fat Lieber-DeCarli diet for varying times, e.g. 1 to 6 weeks. Controls will be pair-fed with dextrose. AIM1 is designed to evaluate mechanisms by which elevated expression of CYP2E1 causes hepatotoxicity in the ethanol-fed CYP2E1 knockin mice and will include assays of oxidative/nitrosative stress, mitochondrial dysfunction, CYP2E1, iNOS, TNF1 , MAPK and NF:B. AIM 2 is designed to evaluate pathways by which CYP2E1 contributes to alcohol-induced fatty liver, including assays of levels of transcription factors such as SREBP-1c and PPAR1, content of key and rate- limiting lipogenic and lipolytic enzymes;rates of fatty acid oxidation will be determined in the wild type, CYP2E1 knockout and CYP2E1 knockin mice fed ethanol or dextrose. The time course of changes in the downstream CYP2E1 effectors will be determined relative to that for ethanol-induced steatosis and hepatotoxicity. The effect of inhibitors of CYP2E1, iNOS, oxidative/nitrosative stress, MAPK, mitochondrial dysfunction, TNF1 production on ethanol-induced steatosis and liver injury in wild type and CYP2E1 knockin mice will be determined. We believe these experiments will provide molecular mechanisms by which CYP2E1 promotes ethanol-induced liver injury and fatty liver and may further the development of new therapeutic designs to treat or minimize progression of ethanol-induced liver injury, a stated goal of RFA-AA-006. PUBLIC HEALTH RELEVANCE: Mechanisms responsible for alcohol-induced hepatotoxicity and fatty liver are not fully understood. With the novel use of CYP2E1 knockout mice and humanized CYP2E1 knockin mice, a role for CYP2E1 in these toxic actions of alcohol has been defined. Identification of downstream effectors of alcohol/CYP2E1 actions would be very informative in developing therapeutic interventions against alcohol-induced liver injury and fatty liver.

DESCRIPTION (provided by applicant): The mechanism(s) by which alcohol causes cell injury are still not clear. A major mechanism that is a focus of considerable research is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a key role on how ethanol induces "oxidative stress", including mitochondrial dysfunction, activation of MAP kinases, ethanol-induced increases in cytokine formation, especially TNF1 and induction of CYP2E1. These pathways are not exclusive of each other, however, associations and interactions between them, especially under in vivo conditions, remain to be evaluated and clarified. We hypothesize that increased oxidative stress from CYP2E1 induction in vivo sensitizes hepatocytes to TNF1-induced hepatotoxicity. We propose that oxidants such as peroxynitrite, activation of MAP kinases such as JNK and/or P38 MAPK, inactivation of NF-kB and mitochondrial dysfunction are downstream mediators of the CYP2E1- TNF1 potentiated hepatotoxicity. We also hypothesize that induction of CYP2E1 plays a central role in mechanisms by which in vivo treatment with alcohol potentiates TNF1 hepatotoxicity. Since CYP2E1 and TNF1 are considered key risk factors in the development of alcoholic liver injury, possible interactions and potentiation of their actions in vivo is important to evaluate. AIM 1 will evaluate the potentiation of TNF1-induced liver injury by induction of CYP2E1. TNF1 or saline will be administrated to either saline-treated mice with basal levels of CYP2E1, or pyrazole-treated mice with high levels of CYP2E1, and to CYP2E1-knockout mice with no CYP2E1. Various concentrations of TNF1 will be given and mice killed at various times after TNF1 challenge. We will assay for the following: Hepatotoxicity, oxidative/nitrosative stress, activation of NF-kB, activation of MAP kinases, mitochondrial dysfunction. AIM 2 will evaluate the role of CYP2E1 in the potentiation of TNF1-induced liver injury by ethanol and to identify downstream targets for the CYP2E1-ethanol- TNF1 interactions. A binge model of alcohol intake followed by TNF1 or saline challenge will be the acute ethanol model evaluated. For chronic ethanol feeding, mice will be fed a control or an ethanol Lieber-DeCarli diet for 2, 4 or 6 weeks, followed by challenge with TNF1 (or saline). Assays similar to the above will be carried out. To prove that CYP2E1 is responsible for the potentiated injury in the pyrazole and ethanol models, CYP2E1 knockout mice and humanized CYP2E1 knockin mice will be used. PUBLIC HEALTH RELEVANCE: We believe that these experiments will help to clarify how two independent risk factors believed to be important for alcohol-induced liver injury, CYP2E1 and TNF1 interact to promote this liver injury, will provide clear evidence for a role for CYP2E1 in acute and chronic alcohol/ TNF1 potentiated liver injury, and help to identify downstream mediators, signaling pathways and targets of the CYP2E1-ethanol- TNF1 interactions. The latter may have potential therapeutic value. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page

DESCRIPTION (provided by applicant): Alcohol-induced liver injury is a significant global health problem and a leading cause of death. The mechanisms by which ethanol treatment causes cell death are not clear. CYP2E1 is induced by ethanol, is an active producer of reactive oxygen species and plays a role in ethanol-induced liver injury. Autophagy is a lysosomal-mediated pathway for removal and recycling of long-lived proteins, cellular organelles and lipid droplets. The goal of this R21 application is to evaluate whether autophagy can modulate CYP2E1-dependent ethanol toxicity in vitro and in vivo after acute and chronic ethanol treatment. The rationale is that CYP2E1 plays a role in ethanol-induced oxidant stress, fatty liver and liver injury. Autophagy, in some settings is protective against cell injury, while in other settings autophagy can promote cell toxicity. If autophagy is protective against ethanol/CYP2E1 toxicity, attempts to stimulate autophagy may prove to be helpful in lowering ethanol-induced liver injury. If autophagy promotes ethanol/CYP2E1 toxicity, inhibitors of autophagy may help to ameliorate ethanol hepatotoxicity. We will treat HepG2 cells which express CYP2E1 (E47 cells) or do not (C34 cells) with ethanol (0-100mM, 1-10 days) in the absence and presence of inhibitors of autophagy or activators of autophagy and assay the following: cell viability, apoptosis, oxidant stress, levels and activity of CYP2E1, mitochondrial dysfunction, steatosis, activation of mitogen activated protein kinases, hepatoprotective defense, autophagy and autophagy regulators such as Bcl-2, AMPK, mTOR. For in- vivo studies, wild type SV129 mice, SV129 CYP2E1 knockout (KO) mice, and SV129 CYP2E1 knockin (KI) mice in which human CYP2E1 has been knocked in will be treated acutely with ethanol (3g/kg, body wt. twice a day for 1, 2 and 4 days) or saline or be fed the high fat Lieber-DeCarli diet containing ethanol or isocaloric dextrose for 2 to 8 weeks. Some mice will also be treated with the autophagy inhibitor 3-methyladenine or the autophagy activator rapamycin. The effects of acute and chronic ethanol in the absence and presence of modifiers of autophagy in WT mouse with normal levels of mouse CYP2E1, in KO mice without CYP2E1 and in KI mice with elevated levels of human CYP2E1 on reactions described above will be evaluated. Time course experiments are designed to help define the sequence of events from the interactions between ethanol and CYP2E1 when autophagy is inhibited or activated. Time course experiments may also be informative as to whether the acute or chronic ethanol feeding may initially activate autophagy as an adaptive response to ethanol, which subsequently is not sustained with more prolonged acute ethanol treatment or chronic ethanol feeding. PUBLIC HEALTH RELEVANCE: CYP2E1 plays a major role in ethanol-induced liver injury. Autophagy can protect or promote cell toxicity. We will evaluate whether autophagy can modulate CYP2E1-dependent ethanol toxicity after acute and chronic ethanol treatment.