title: Effect of alcohol exposure on hepatic superoxide generation and hepcidin expression.
abstract: OBJECTIVE
  To understand the role of mitochondrial-produced superoxide (O2 (•-)) 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 O2 (•-) levels in hepatocytes isolated from the livers of mice were measured by electron paramagnetic resonance spectroscopy. The mitochondrial-targeted, O2 (•-)-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. O2 (•-) levels in hepatocytes of untreated Sod2 (+/-) mice were three-fold higher than in untreated LMC mice, as observed by electron paramagnetic resonance spectroscopy. O2 (•-) 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 O2 (•-) 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 O2 (•-) levels in both Sod2 (+/-) and control mice.
  
  
  CONCLUSIONS
  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 O2 (•-) levels in hepatocytes, which appears not to originate from mitochondria.
authors: Duygu Dee Harrison-Findik, Sizhao Lu, Emily M Zmijewski, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198, United States.
authors: Duygu Dee
authors: DD
authors: Harrison-Findik
authors: Sizhao
authors: S
authors: Lu
authors: Emily M
authors: EM
authors: Zmijewski
authors: Jocelyn
authors: J
authors: Jones
authors: Matthew C
authors: MC
authors: Zimmerman
keywords: Alcohol; Electron paramagnetic resonance; Hepcidin; Iron; Liver; Manganese superoxide dismutase; Mitochondria; Superoxide; Superoxide dismutase