The effects of low, moderate and severe oxidative stress on the steady-state levels of the metabolites involved in the transmethylation/transsulfuration pathway were studied in lung epithelial (A549) cells. When cells were exposed to low (0.1 mM) or moderate (1.0 mM) concentrations of hydrogen peroxide (H(2)O(2)) or tert-butylhydroperoxide (t-butOOH), intracellular levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were significantly decreased, while the SAM/SAH ratio remained the same or elevated. Likewise, extracellular levels of SAM and SAH metabolites remained steady or elevated. Both intracellular and extracellular levels of homocyst(e)ine and cyst(e)ine were decreased. Cell contents of serine, cystathionine and methionine were also decreased. Total intracellular glutathione content was decreased only by moderate t-butOOH exposure. When cells were exposed to high concentrations (10mM) of either of the peroxides, extracellular levels of methionine, cystathionine, and total cyst(e)ine were depleted, mostly due to direct oxidation of sulfur amino acids by peroxides, as indicated by oxidative treatment of culture media alone. Similar to low and moderate oxidative conditions, the levels of SAM, SAH, and sulfur amino acids were decreased, while cell SAM/SAH ratio increased. Paradoxically, under high peroxide exposure, extracellular concentrations of SAM, SAH, and cyst(e)ine were increased, indicating cellular release, despite the severe methionine depletion. Intracellular total glutathione was also decreased. The results indicate that lung epithelial cells release high levels of SAM, probably as an adaptive response to increased oxidative stress, even when the substrate for SAM formation, methionine, is critically depleted.