NADPH oxidase–derived free radicals are key oxidants in alcohol-induced liver disease
Hiroshi Kono, Ivan Rusyn, Ming Yin, Erwin Gäbele, Shunhei Yamashina, Anna Dikalova, Maria B. Kadiiska, Henry D. Connor, Ronald P. Mason, Brahm H. Segal, Blair U. Bradford, Steven M. Holland, Ronald G. Thurman
Hiroshi Kono, Ivan Rusyn, Ming Yin, Erwin Gäbele, Shunhei Yamashina, Anna Dikalova, Maria B. Kadiiska, Henry D. Connor, Ronald P. Mason, Brahm H. Segal, Blair U. Bradford, Steven M. Holland, Ronald G. Thurman
View: Text | PDF
Article

NADPH oxidase–derived free radicals are key oxidants in alcohol-induced liver disease

Abstract

In North America, liver disease due to alcohol consumption is an important cause of death in adults, although its pathogenesis remains obscure. Despite the fact that resident hepatic macrophages are known to contribute to early alcohol-induced liver injury via oxidative stress, the exact source of free radicals has remained a mystery. To test the hypothesis that NADPH oxidase is the major source of oxidants due to ethanol, we used p47phox knockout mice, which lack a critical subunit of this major source of reactive oxygen species in activated phagocytes. Mice were treated with ethanol chronically, using a Tsukamoto-French protocol, for 4 weeks. In wild-type mice, ethanol caused severe liver injury via a mechanism involving gut-derived endotoxin, CD14 receptor, production of electron spin resonance–detectable free radicals, activation of the transcription factor NF-κB, and release of cytotoxic TNF-α from activated Kupffer cells. In NADPH oxidase–deficient mice, neither an increase in free radical production, activation of NF-κB, an increase in TNF-α mRNA, nor liver pathology was observed. These data strongly support the hypothesis that free radicals from NADPH oxidase in hepatic Kupffer cells play a predominant role in the pathogenesis of early alcohol-induced hepatitis by activating NF-κB, which activates production of cytotoxic TNF-α.

Authors

Hiroshi Kono, Ivan Rusyn, Ming Yin, Erwin Gäbele, Shunhei Yamashina, Anna Dikalova, Maria B. Kadiiska, Henry D. Connor, Ronald P. Mason, Brahm H. Segal, Blair U. Bradford, Steven M. Holland, Ronald G. Thurman

×

Figure 3

Options: View larger image (or click on image) Download as PowerPoint
Ethanol administration activates the transcription factor NF-κB and incr...
Ethanol administration activates the transcription factor NF-κB and increases expression of mRNA for the inflammatory cytokines TNF-α and IL-6 in wild-type but not NADPH oxidase–deficient mice. (a) NF-κB DNA binding activity in the liver was assessed by electrophoretic mobility shift assay using whole liver nuclear extracts from wild-type mice fed high-fat control (lane 1) or ethanol-containing (lane 2) diet, or p47phox–/– mice fed high-fat control (lane 3) or ethanol-containing (lane 4) diet. No binding was detected with no nuclear extract added (lane 5). Nuclear extracts from wild-type mice fed ethanol-containing diet (same as in lane 2) were used for competition experiments (200-fold excess of the unlabeled oligonucleotide, lane 6) and supershift experiments (p50 or p65 anti-serum, lanes 7 and 8, respectively). Representative data from four separate experiments are shown. (b) Data shown are results of densitometric analysis of the NF-κB/DNA complex images. Density of the NF-κB/DNA complex image in livers from wild-type mice fed high-fat control diet was set to 100%. Data represent mean ± SEM (n = 4). CON, high-fat control diet; ETH, high-fat ethanol-containing diet. AP < 0.05 compared with wild-type mice fed high-fat control diet, BP < 0.05 compared with wild-type mice fed ethanol-containing diet by two-way ANOVA with Bonferroni’s post-hoc test. (c) Total mRNA was prepared from livers of wild-type or p47phox–/– mice administered high-fat (CON) or ethanol-containing (ETH) diets for 4 weeks, and RNase protection assays were performed. Representative data from four separate experiments are shown.