Epidemiological data suggest that excessive alcohol consumption may cost the US economy $2 billion annually in skeletal pathology. Patients with alcoholic bone disease display marked impairment in bone formation. Alcohol may alter bone metabolism indirectly by elevating the secretion of cytokines shown to be critical factors in postmenopausal osteoporosis. It has been demonstrated that cytokines mediate bone loss due to estrogen deficiency by increasing bone resorption and decreasing bone formation. Furthermore, clinical studies show that cytokines are over-produced in alcohol-induced liver disease. Distraction osteogenesis (DO) is a clinical and experimental procedure for lengthening bones that has been used to isolate and study the effects of chronic ethanol exposure on intramembranous (direct osteoblastogenesis) bone formation, which is well organized and spatially distinct from resorptive processes. Previous work demonstrates that chronic ethanol exposure in the rat (enteral nutrition model) decreases tibial bending strength, inhibits bone formation during DO, and increases the expression of Tumor Necrosis Factor alpha (TNF) in the liver and DO gap. Further, treatment with TNF antagonists during DO prevents osteoinhibiton by ethanol, while treatment of control rats with TNF during DO inhibits bone formation. Preliminary results, combining a novel murine DO model with liquid ethanol delivery, demonstrate that chronic ethanol exposure decreases tibial strength and inhibits bone formation during DO. In this murine model, analogous to the rat, TNF antagonist treatment during DO protects bone formation and exogenous TNF inhibits bone formation. The Specific Aims of this project are to determine 1) the downstream mediators of TNF actions, 2) the roles of cytochrome p450 2E1 (CYP2E1) and 3) the role of reactive oxygen species (ROS) in ethanol's induction of TNF. The working hypotheses are that ethanol induction of CYP2E1 increases ROS, inducing TNF, which binds to a specific receptor and triggers signal transduction pathways that inhibit bone formation. The long term goals of this research are to support future pharmacological and/or nutritional interventions in orthopaedic procedures in patients with ethanol or cytokine excess as risk factors.