Accumulating data support the view that sepsis is associated with an acquired intrinsic derangement in the ability of cells to consume O₂, a phenomenon that has been termed "cytopathic hypoxia." We sought to use an in vitro "reductionist" model system using cultured cells stimulated with proinflammatory cytokines to test the hypothesis that cytopathic hypoxia is mediated, at least in part, by depletion of intracellular levels of NAD⁺/NADH secondary to activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We measured O₂ consumption by Caco-2 enterocytes growing on microcarrier beads after cells were incubated for 24 h under control conditions or with cytomix, a mixture of tumor necrosis factor-, interleukin-1, and interferon-. Immunostimulated cells consumed O₂ at about one-half the rate of control cells, but this effect was largely prevented if any one of the following pharmacological agents was present during the period of incubation with cytomix: 4,5-dihydroxy-1,3-benzene disulfonic acid, a superoxide radical anion scavenger; 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger; 5,10,15,20- tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III], a peroxynitrite (ONOO) decomposition catalyst; urate, an ONOO scavenger; 3-aminobenzamide, a PARP inhibitor; or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide HCl, a chemically dissimilar and more potent PARP inhibitor. The decrease in O₂ uptake induced by cytomix was associated with decreased cellular levels of NAD⁺/NADH. The decrease in cellular NAD⁺/NADH content and the decrease in O₂ uptake induced by cytomix were completely abrogated if liposome-encapsulated NAD⁺ was added to the cultures during immunostimulation. Empty liposomes also increased O₂ uptake by immunostimulated Caco-2 cells, but much less effectively than liposomes containing NAD⁺. These data are consistent with the view that enterocytes exposed to proinflammatory cytokines consume less O₂ due to NAD⁺/NADH depletion secondary to activation of PARP by ONOO or other oxidants.