Galactose treatment downregulates nitric oxide production and the mitochondrial anti-oxidant SOD2 without altering oxidative homeostasis. (A) Cell glutathione content was not altered by 1 or 5 days of galactose culture (n = 4). (B) glutathione redox homeostasis was maintained after 5 days of galactose treatment (n = 4). (C) Five days of galactose culture significantly reduced nitrate and nitrite (NOx) release into the culture media (n = 13). (D) Out of a panel of 12 cytosolic and mitochondrial anti-oxidant proteins, 5 days of galactose culture selectively reduced the abundance of the mitochondrial SOD isoform, SOD2, as determined by selected reaction monitoring mass spectrometry (n = 3). (E) Western blot analysis further verified the reduction in SOD2 abundance (n = 5). (F) Real-time polymerase chain reaction analysis showed a significant reduction in gene expression of SOD2 (n = 5) but not the cytosolic SOD isoform, SOD1 (n = 4), after 5 days of galactose treatment. (G) Five days of galactose treatment reduced the total activity of superoxide dismutase (tSOD) (n = 7). The reduction in tSOD activity after 5 days in galactose culture was due primarily to a reduction in the activity of the mitochondrial SOD isoform, SOD2 (n = 7), (H) which paralleled the reduction in SOD2 protein expression. Bars represent mean ± standard error of the mean. *P <0.05, **P <0.01, and ***P <0.001 between glucose and galactose. Gal, galactose; Glu, glucose; GSH:GSSG, ratio of reduced to oxidized glutathione; SOD, superoxide dismutase.