CO decreases nitrite production and iNOS protein from hepatocytes transfected with adenoviral vector carrying a human iNOS gene. Modified adenoviral vectors (MOI of 3) carrying a human iNOS gene or β-galactosidase were prepared. (A). The hepatocytes were incubated with CO (250 ppm) for 4h prior to infection of adenoviral vector. The cells were transfected with adenoviral vector in a serum-free medium for 6h, and recovered for indicated time (8, 16, 24h) in fresh medium with 5 % serum in the absence or presence of CO. Monolayers were washed with ice-cold PBS, and lysed by three cycles of freeze and thaw. Western blot was performed from cytosolic fractions collected by centrifugation at 12,000×g for 20min. (B). Accumulated nitrite was measured in the culture medium using Griess reagent. Data represent the mean ± S.D. of two independent experiments.