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Title: Increased 4-hydroxynonenal protein adducts in male GSTA4–4/PPAR-alpha double knockout mice enhance injury during early stages of alcoholic liver disease

Author
item RONIS, MARTIN - Arkansas Children'S Nutrition Research Center (ACNC)
item MERCER, KELLY - Arkansas Children'S Nutrition Research Center (ACNC)
item GANNON, BRENDA - University Arkansas For Medical Sciences (UAMS)
item ENGI, BRIDGETTE - University Arkansas For Medical Sciences (UAMS)
item ZIMNIAK, PIOTR - University Arkansas For Medical Sciences (UAMS)
item SHEARN, COLIN - University Of Colorado
item ORLICKY, DAVID - University Of Colorado
item ALBANO, EMANUELE - Universita Del Piemonte Orientale "amedeo Avogadro"
item Badger, Thomas - Arkansas Children'S Nutrition Research Center (ACNC)
item PETERSEN, DENNIS - University Of Colorado

Submitted to: American Journal of Physiology - Gastrointestinal and Liver Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2014
Publication Date: 3/1/2015
Citation: Ronis, M.J., Mercer, K.E., Gannon, B.M., Engi, B., Zimniak, P., Shearn, C., Orlicky, D., Albano, E., Badger, T.M., Petersen, D.R. 2015. Increased 4-hydroxynonenal protein adducts in male GSTA4–4/PPAR-alpha double knockout mice enhance injury during early stages of alcoholic liver disease. American Journal of Physiology - Gastrointestinal and Liver Physiology. 308(5):G403-G415.

Interpretive Summary: Alcohol-associated liver damage progresses from simple appearance of fat in the liver to inflammation (hepatitis), fibrosis, and ultimately cirrhosis. However, how this process happens and why this type of progression only happens in a relatively small proportion of alcoholics remains unclear. One possible mechanism involves oxidative stress generated during alcohol breakdown. Oxidation of fat in the liver results in formation of a number of small molecules called short chain aldehydes including hydroxynonenal (4HNE) which are highly reactive and which can bind to proteins to form adducts. These adducts may prevent proteins from functioning normally and may make them targets for the immune system. In the current study, we investigated the relative importance of HNE in progression of alcohol-induced liver damage by using genetically manipulated mice. We fed alcohol-containing liquid diets to normal mice, mice in which breakdown of HNE is blocked by knockout of the enzyme (GSTA4-4) that is responsible for this, mice in which fat breakdown is blocked as a result of knockout of a factor that controls this process (PPAR alpha), or double knockout mice in which both GSTA4-4 and PPAR alpha were knocked out. We observed almost no liver injury after 40 days of alcohol feeding to normal mice. In contrast, the knockout mice all had fatty liver and inflammation and the double knockout mice had the most injury, including fibrosis. These results suggest that HNE is important in progression of alcoholic liver damage and that the process of fat oxidation may be a target for new drugs to block alcoholic liver injury.

Technical Abstract: To test the significance of lipid peroxidation in the development of alcoholic liver injury, an ethanol (EtOH) liquid diet was fed to male wild type 129/SvJ mice, and glutathione S-transferase A4-4 null (GSTA4-/-) mice for 40 d. GSTA4-/- mice were also crossed with peroxisome proliferator-activated receptor-alpha null mice (PPAR alpha-/-), and the effects of EtOH in the resulting dKO mice were compared to the other strains. EtOH increased lipid peroxidation in all except WT mice (P< 0.05). Increased steatosis, mRNA expression of inflammatory markers: CXCL2 and tumor necrosis factor alpha (TNF) and of smooth muscle actin ( alpha SMA) were observed in EtOH GSTA4-/- compared to EtOH WT mice (P<0.05). EtOH PPAR alpha -/- mice had increased steatosis, serum alanine aminotransferase (ALT) and elevated mRNA encoding CD14, CXCL2, TNF, IL-6, tumor growth factor beta, platelet derived growth factor receptor beta (PDGFR), matrix metalloproteinase (MMP) 9 and 13, SMA and collagen type 1 compared to EtOH WT mice. dKO mice displayed elevation of centrilobular hepatic 4-HNE adducts and serum antibodies against malondialdehyde-adducts compared to EtOH-feeding of GSTA4-/-, PPAR alpha -/- and WT mice (P<0.05). ALT was higher in EtOH dKO mice compared to all other groups (P<0.001). EtOH dKO mice also had elevated mRNAs for TNF and CD14, histological evidence of fibrosis, and increased PDGFR, MMP 9 and 13 mRNAs, compared to either the EtOH GSTA4-/- or EtOH PPAR alpha -/- genotype (P<0.05). These findings demonstrate the central role lipid peroxidation plays in mediating progression of alcohol-induced necroinflammatory liver injury, stellate cell activation, matrix remodeling and fibrosis.