The main research efforts are to evaluate the production of an increased state of oxidative stress by ethanol and the role of reactive oxygen species in the hepatotoxicity produced by ethanol. We are focusing on the ability of ethanol to increase the levels of a cytochrome P450 isoform called CYP2E1, which has been shown to be a powerful producer of superoxide radical and H2O2. We have developed stable HepG2 lines that overexpress CYP2E1 and have shown that ethanol produces cytotoxicity in cells expressing CYP2E1, but not cells lacking CYP2E1. The cytotoxicity is apoptotic in nature and can be prevented by a variety of antioxidants, inhibitors of CYP2E1, caspase 3 inhibitors and transfection with a plasmid that expresses bcl-2. Ethanol toxicity is enhanced by administration of polyunsaturated fatty acids or by iron, which promote lipid peroxidation. Impairment of mitochondrial function is an early step in the CYP2E1 plus ethanol toxicity. We are currently co-incubating our CYP2E1 cell lines with stellate or Kupffer cells to evaluate possible activation of collagen or cytokine production. Reactive oxygen species are detected by ESR spectroscopy, but new HPLC spin-trapping methods have been developed which are more sensitive and we are characterizing the utility of these new approaches. A major mechanism by which ethanol enhances the level of CYP2E1 is by stabilizing the enzyme against degradation. We are characterizing this degradation process and the role of the proteasome-ubiquitin system and molecular chaperones. Anti-oxidant defense systems are up-regulated in the CYP2EI-overexpressing cells, most likely an adaptation to the CYP2EI-derived oxidant stress. The lab is studying the mechanisms responsible for this increased transcriptional response and the signaling pathways and factors involved. In-vivo models of alcohol toxicity in knockout mice deficient in antioxidant defense or other hepatoprotective factors are being developed.
Arthur Cederbaum, PhD
Pharmacology and Systems Therapeutics
1468 Madison Avenue
Annenberg Building Room 19-10
New York, NY 10029