The public health issues, economic impact and human suffering associated with alcohol abuse are staggering. Although alcohol-related research has been on-going for decades, there are many unanswered questions concerning mechanisms of alcohol-induced tissue injury. Elevated blood homocysteine (hyperhomocysteinemia) is strongly associated with chronic alcoholism. However, the molecular mechanism of elevated homocysteine in alcohol abusers is largely unknown. Of greater importance is the pathological impact and clinical consequences of hyperhomocysteinemia in these individuals. This proposal will provide novel information on the role of methionine synthase (MS) in alcohol-associated hyperhomocysteinemia, homocysteine-induced oxidative and endoplasmic reticulum (ER) stress in models of alcoholic liver disease (ALD), and changes in the one-carbon proteome and metabolome of alcohol-treated cells and liver. The central hypotheses that drive this project are: 1) alcohol-induced oxidative stress in the liver will lead to the inactivation of B12-dependent MS;2) impaired remethylation of homocysteine results in elevated intracellular homocysteine and hyperhomocysteinemia;3) molecular targeting of specific intracellular proteins by homocysteine causes enhanced production of reactive oxygen species (ROS), ER stress, and apoptosis;and, 4) the one-carbon proteome and metabolome of the liver is substantially altered in ALD. The specific aims of this project are: 1. to determine the mechanism of inactivation of B12-dependent MS in alcohol-treated cells in culture and in livers from ethanol-fed rats;2. to establish the role that molecular targeting of specific proteins by L-homocysteine has in the generation of oxidative stress and in the induction of ER stress in alcohol-treated cells and rats;and, 3. to determine whether the decreased SAM/SAH ratio that accompanies ALD is (a) a direct result of alcohol or its metabolites on the levels and/or activities of the methionine cycle enzymes, or (b) an indirect result of the hyperhomocysteinemia that accompanies increased alcohol consumption. These hypotheses will be tested at the cellular level using cultured alcohol-treated or untreated HepG2 E47 and C34 cells and in vivo using livers from rats on the Lieber-DeCarli ethanol feeding model and pair-fed controls. The mechanistic insight gained from this proposal may be useful for establishing novel therapeutic interventions in the treatment of alcoholic liver disease. PUBLIC HEALTH RELEVANCE: The public health issues, economic impact and human suffering associated with alcohol abuse are staggering. Elevated blood homocysteine (hyperhomocysteinemia) is strongly associated with chronic alcoholism. However, the molecular cause of elevated homocysteine and its role in mediating tissue injury in alcoholism is largely unknown. This proposal will provide mechanistic insight on the cause of hyperhomocysteinemia and its deleterious effects to tissues in chronic alcohol abuse.