Ethanol synergizes with hydrogen peroxide, peroxyl radical, and trypsin to kill epithelial cells in culture

Monkey kidney epithelial cells, labeled with chromium and grown in culture,were killed in a synergistic manner when subtoxic amounts of ethanol were combined either with subtoxic amounts of glucose oxidase-generated hydrogen peroxide, or with mixtures of peroxide and with 2,2′-Azobis (2-amidinopropane)HCl (AAPH)-generated peroxyl radical. A further enhancement of cytotoxicity occurred when subtoxic amounts of trypsin were added to mixtures of all three agents. While ethanol alone caused shrinkage of the monolayers and cell rounding, no visible cytotoxic changes were observed. Hydrogen peroxide at the concentrations used (about 1 mM), caused only some cell rouding. On the other hand, cells exposed simultaneously to ethanol and to H₂O₂ developed extensive membrane damage characterized by the formation of large polar blebs, which is compatible with altered membrane permeability. The presence of trypsin markedly enhanced cellular cytotoxicity induced by mixtures of peroxide, peroxyl radical, and ethanol. This could markedly be depressed by catalase and by dimethylthiourea. The tissue culture model described might serve to further investigate the role played by synergy among oxidants and a variety of membrane-damaing agents, and by xenobiotics in tissue damage induced by inflammatory processes.