Volume 120, Issue 5, Pages 1241-1250 (April 2001) Delivery of the Cu/Zn–Superoxide dismutase gene with adenovirus reduces early alcohol-induced liver injury in rats  Michael D. Wheeler, Hiroshi Kono, Ming Yin, Ivan Rusyn, Matthias Froh, Henry D. Connor, Ronald P. Mason, R.J. Samulski, Ronald G. Thurman  Gastroenterology  Volume 120, Issue 5, Pages 1241-1250 (April 2001) DOI: 10.1053/gast.2001.23253 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Representative photomicrographs of immunohistochemical staining for adenovirus transgene 3 weeks after Ad.lacZ infection. Animals were injected with (A) saline or (B) adenovirus (1 × 109 pfu) containing the β-galactosidase reporter gene (Ad.lacZ). Formalin-fixed sections (20 μm) were immunostained by using an antibody against β-galactosidase followed by light hematoxylin stain as described in the Materials and Methods section. Original magnification 200×. Gastroenterology 2001 120, 1241-1250DOI: (10.1053/gast.2001.23253) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Expression of transgenes in hepatocytes and Kupffer cells. Rats were infected with either Ad-lacZ or Ad-SOD1 (1 × 109 pfu). Three weeks after the injection, hepatocytes and Kupffer cells were isolated as described in the Materials and Methods, and cytosolic extract was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by immunoblotting with a monoclonal antibody against (A) β-galactosidase or (B) Cu/Zn-SOD. Lysates from human embryonic kidney (HEK.293) cells infected with Ad.lacZ and Ad-SOD1 were used as positive controls. Gastroenterology 2001 120, 1241-1250DOI: (10.1053/gast.2001.23253) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Increases in serum transaminases caused by ethanol are blunted by Ad.SOD1. Serum alanine transaminase levels were measured by standard serum enzyme kits (Sigma, St. Louis, MO). Data are expressed as mean ± SEM of 5 individual experiments in each group. aP < 0.05 compared with high-fat control; bP < 0.05 compared with Ad.lacZ, ethanol-treated samples by 2-way ANOVA followed by the Tukey post hoc analysis. Con, high-fat control diet; EtOH, ethanol-containing diet. Gastroenterology 2001 120, 1241-1250DOI: (10.1053/gast.2001.23253) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Representative photomicrograph of livers after 3 weeks of ethanol treatment. Livers were harvested from animals infected with either (A and C) Ad.lacZ or (B and D) Ad.SOD1 and fed either (A and B) high-fat control diet or (C and D) diet containing ethanol for 3 weeks. Tissues were fixed with 10% neutral-buffered formalin and stained with H&E. Original magnification 200×. Data are representative of 5 animals in each group. Gastroenterology 2001 120, 1241-1250DOI: (10.1053/gast.2001.23253) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 ESR spectra of bile from animals after 3 weeks of enteral ethanol. Rats given (A and B) Ad.lacZ or (C and D) Ad.SOD1 were fed (A and C) high-fat control diet or (B and D) diet containing ethanol for 3 weeks. After POBN (1 g/kg, intravenously) injection, bile was collected and ESR analysis was performed as described in the Materials and Methods. Representative ESR spectra are shown. Gastroenterology 2001 120, 1241-1250DOI: (10.1053/gast.2001.23253) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 NFκB activation is blunted by Cu/Zn-SOD overexpression. EMSAs were performed as described in the Materials and Methods. (A) Nuclear extracts (40 μg) from Ad.lacZ-treated animals that received ethanol were incubated with radiolabeled probe (lane 2) in the presence of p50 antibody (lane 3), p65 antibody (lane 4), or excess unlabeled probe (lane 5). Lane 1 is radiolabeled probe alone. (B) EMSA was performed on nuclear extracts from animals infected with Ad.lacZ (lanes 1 and 2) or Ad.SOD1 (lanes 3 and 4) and fed high-fat control diet (lanes 1 and 3) or diet containing ethanol (lanes 2 and 4) for 3 weeks. Data are representative of 5 individual experiments. (C) Image analysis performed on 5 individual experiments in each group. aP < 0.05 compared with high-fat control; bP < 0.05 compared with Ad.lacZ, ethanol-treated samples by 2-way ANOVA followed by the Tukey post hoc analysis. Gastroenterology 2001 120, 1241-1250DOI: (10.1053/gast.2001.23253) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Cytokine mRNA analysis by RNase protection assay. RNase protection assay was performed as described in the Materials and Methods section. Lanes: (1) undigested probe; (2) yeast RNA; (3) Ad.lacZ-treated, high-fat control diet; (4) Ad.lacZ-treated ethanol diet; (5) Ad.SOD1-treated, high-fat control diet; and (6) Ad.SOD1-treated ethanol diet. Data are representative of 5 individual experiments. Gastroenterology 2001 120, 1241-1250DOI: (10.1053/gast.2001.23253) Copyright © 2001 American Gastroenterological Association Terms and Conditions