AIM: To understand the role of mitochondrial-produced superoxide(O 2 ?) in the regulation of iron-regulatory hormone, hepcidin by alcohol in the liver. METHODS: For alcohol experiments, manganese superoxide dismutase knockout mice heterozygous for Sod2 gene expression(Sod2 +/) and age-matched littermate control mice(LMC), expressing Sod2 gene on both alleles, were exposed to either 10%(w/v) ethanol in the drinking water or plain water(control) for 7 d. Total cellular O 2 ? levels in hepatocytes isolated from the livers of mice were measured by electron paramagnetic resonance spectroscopy. The mitochondrial-targeted, O 2 ?-sensitive fluorogenic probe, MitoSOX Red and flow cytometry were utilized to measure O 2 ? in mitochondria. Gene and protein expression were determined by Taqman Real-time quantitative PCR and Western blotting, respectively. RESULTS: Sod2 +/- mice expressed 40% less MnSOD protein(SOD2) in hepatocytes compared to LMC mice. The deletion of Sod2 allele did not alter the basal expression level of hepcidin in the liver. 10% ethanol exposure for 1 wk inhibited hepatic hepcidin mRNA expression three-fold both in Sod2 +/ and LMC mice. O 2 ? levels in hepatocytes of untreated Sod2 +/ mice were three-fold higher than in untreated LMC mice, as observed by electron paramagnetic resonance spectroscopy. O 2 ? levels in mitochondria of Sod2 +/ mice were four-fold higher than in mitochondria of untreated LMC mice, as measured by MitoSOX Red fluorescence and flow cytometry. Alcohol induced a two-fold higher increase in O 2 ? levels in hepatocytes of LMC mice than in Sod2 +/ mice compared to respective untreated counterparts. In contrast, 1 wk alcohol exposure did not alter mitochondrial O 2 ? levels in both Sod2 +/- and control mice. CONCLUSION: Mitochondrial O2 ? is not involved in the inhibition of liver hepcidin transcription and thereby regulation of iron metabolism by alcohol. These findings also suggest that short-term alcohol consumption significantly elevates O 2 ? levels in hepatocytes, which appears not to originate from mitochondria. METHODS: For alcohol experiments, manganese superoxide dismutase knockout mice heterozygous for Sod2 gene expression (Sod2 + /) and age-matched littermate control mice (LMC), expressing Sod2 gene on both alleles, were exposed to either 10% (w / v) ethanol in the drinking water or plain water (control) for 7 d. 2? Levels in hepatocytes isolated from the livers of mice were measured by electron paramagnetic resonance spectroscopy. The mitochondrial-targeted, 02? -sensitive fluorogenic probe, MitoSOX Red and flow cytometry were utilized to measure O2? In mitochondria. Gene and protein expression were determined by Taqman Real-time quantitative PCR and Western blotting, respectively. RESULTS: Sod2 +/- mice expressed 40% less MnSOD protein (SOD2) in hepatocytes compared to LMC mice. The deletion of Sod2 allele did not alter the basal expression level of hepcidin in the liver. 10% ethanol exposure for 1 wk inhibited hepatic hepcidin mRNA expression three-fold both in Sod2 + / and LMC mice. O 2? levels in hepatocytes of untreated Sod2 + / mice were three-fold higher than in untreated LMC mice, as observed by electron paramagnetic resonance spectroscopy. O 2? levels in mitochondria of Sod2 + / mice were four-fold higher than in mitochondria of untreated LMC mice, as measured by MitoSOX Red fluorescence and flow cytometry. a two-fold higher increase in O 2? levels in hepatocytes of LMC mice than in Sod2 + / mice compared to respective untreated counterparts. In contrast, 1 wk alcohol exposure did not alter mitochondrial O 2? levels in both Sod2 +/- and control mice. CONCLUSION: Mitochondrial O2? is not involved in the inhibition of liver hepcidin transcription and thereby regulation of iron metabolism by alcohol. These findings also suggest that short-term alcohol consumption significantly elevates O 2? levels in hepatocytes, which appears not to originate from mitochondria.