During the normal aging process, cellular enzymes accumulate as catalytically inactive or less active forms. The mechanism(s) by which these enzymes became altered, as yet unknown, may involve the oxidative modification of critical amino acid residues. In order to define the mechanism(s) responsible for these modifications, we have developed an in vitro model system to investigate the relationship between enzyme modification by mixed-function oxidase (MFO) systems and the accumulation of altered enzymes during aging. To this end, the level of carbonyl groups, known to be generated in MFO systems, have been determined in crude extracts of freshly isolated hepatocyte cultures derived from rats of different ages. Preliminary studies have shown an increase in carbonyl content in hepatocytes isolated from old rats and in hepatocytes from rats of all ages cultured in the presence of an acute oxidative stress. The oxidative modification of proteins will also be investigated by determining carbonyl content in hepatocyte cultures derived from rats exposed to vigorous exercise, or dietary regimens known to effect longevity. Additionally, effects of factors which activate or inhibit MFO-protein oxidation in these cells will be examined.