Knockout of the Gsta4 Gene in Male Mice Leads to an Altered Pattern of Hepatic Protein Carbonylation and Enhanced Inflammation Following Chronic Consumption of an Ethanol Diet.
BACKGROUND
Glutathione S-transferase A4-4 (GSTA4) is a key enzyme for removal of toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE). In this study, we examined the potential role of GSTA4 on protein carbonylation and progression of alcoholic liver disease by examining the development of liver injury in male wild-type (WT) SV/J mice and SV/J mice lacking functional GSTA4 (GSTA4-/- mice).


METHODS
Adult male WT and GSTA4-/- mice were fed chow (N = 10 to 12) or high-fat Lieber-DeCarli liquid diets containing up to 28% calories as ethanol (EtOH) (N = 18 to 20) for 116 days. At the end of the study, half of the EtOH-fed mice were acutely challenged with an EtOH binge (3 g/kg given intragastrically) 12 hours before sacrifice. Carbonylation of liver proteins was assessed by immunohistochemical staining for 4-HNE adduction and by comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) of purified carbonylated proteins.


RESULTS
Chronic EtOH intake significantly increased hepatic 4-HNE adduction and protein carbonylation, including carbonylation of ribosomal proteins. EtOH intake also resulted in steatosis and increased serum alanine aminotransferase. Hepatic infiltration with B cells, T cells, and neutrophils and mRNA expression of pro-inflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)γ was modest in WT mice. However, an EtOH binge increased hepatic necrosis, hepatic cell proliferation, and expression of TNFα mRNA (p < 0.05). EtOH treatment of GSTA4-/- mice increased B-cell infiltration and increased mRNA expression of TNFα and IFNγ and of matrix remodeling markers MMP9, MMP13, and Col1A1 (p < 0.05). GSTA4-/- mice exhibited panlobular rather than periportal distribution of 4-HNE-adducted proteins and increased overall 4-HNE staining after EtOH binge. Comprehensive LC-MS of carbonylated proteins identified 1,022 proteins of which 189 were unique to the GSTA4-/- group.


CONCLUSIONS
These data suggest long-term adaptation to EtOH in WT mice does not occur in GSTA4-/- mice. Products of lipid peroxidation appear to play a role in inflammatory responses due to EtOH. And EtOH effects on B-cell infiltration and autoimmune responses may be secondary to formation of carbonyl adducts.
['Shearn|Colin T|CT|0000-0002-9618-9885', 'Pulliam|Casey F|CF|', 'Pedersen|Kim|K|', 'Meredith|Kyle|K|', 'Mercer|Kelly E|KE|', 'Saba|Laura M|LM|', 'Orlicky|David J|DJ|', 'Ronis|Martin J|MJ|', 'Petersen|Dennis R|DR|']
D000818:Animals D000431:Ethanol D005982:Glutathione Transferase D007249:Inflammation D008099:Liver D008108:Liver Diseases, Alcoholic D008297:Male D051379:Mice D057507:Mice, 129 Strain D018345:Mice, Knockout D050050:Protein Carbonylation D017433:Protein Structure, Secondary
Protein Carbonylation Ethanol Liver Detoxification Oxidative Stress