EXCEED THE SPACE PROVIDED. There is interest in the role of oxidative stress and generation of reactive radical species in the mechanism(s) by which ethanol is toxic to the liver. Inductionof CYP2E1 is one pathway by which ethanol is believed to generate a state of oxidative stress. CYP2E1 can be also induced by a variety of pathophysiological conditions and chemicals. The goal of this renewal is to evaluate the biochemical and toxicological effects of CYP2E1 in intact cells and to assess the impact of CYP2E1-derived reactive oxygen species (ROS) on several cellular metabolic reactions which play a role in the actions of ethanol on the liver.
Aim 1 will evaluate the modulation of stress associated proteins by CYP2E1 or CYP2E1-derived ROS. HepG2 cells expressing CYP2E1 and their controls will be incubated in the absence or presence of ethanol, arachidonic acid or iron, and the activity, content and mRNA levels of enzymes such as y-glutamyl cysteine synthase, glutathione transferase alpha, microsomal glutathione transferase, heat shock proteins such as Hsp90 and Hsp70 and heme oxygenase 1 determined. Sensitivity of any change to inhibitors of CYP2E1 and antioxidants will be studied. Transcriptional activation will be assessed by nuclear run-on experiments and reporter assays.
Aim 2 will evaluate whether CYP2E1 or CYP2E1-derived diffusable mediators, produced in the absence or presence of ethanol, PUFA or iron can activate collagen gene transcription by stellate cells. Models will include co-cultures of HepG2 cells or rat hepatocytes withwild type or primary stellate cells. The role of ROS and TGF0 in the CYP2E1 effects will be evaluated, as will possible increased binding activity of oxidant-sensitive transcription factors.
Aim 3 will evaluate the role of mitochondria in the toxicity produced by ethanol, PUFA or iron in CYP2E1-expressing cells. Mitochondrial antioxidant defense will be enhanced by novel transfection methods and effects on cellular viability evaluated. Loss of membrane potential or development of membrane transition pores will be compared to the kinetics of loss in viability.Reconstituted systems with mitochondria plus microsomes will be developed to mimic events occurring in the CYP2E1-expressing cells. Rats will be treated in vivo with pyrazole or acetone to induce CYP2E1 and effects on targeted genes, collagen expression and mitochondria determined. It is anticipated that new information will be gained on the ability of CYP2E1-derived ROS to up- or down-regulate specific genes important for ethanol metabolic effects on the liver, on promoting collagen production by HSC, a key event in hepatic fibrosis and on modulating mitochondrial membrane potential and function thereby contributing to the developing toxicity and apoptosis produced by ethanol, PUFA or iron in CYP2E1-expressing cells. PERFORMANCE SITE ========================================Section End===========================================
