Volume 121, Issue 6, Pages 1451-1459 (December 2001) The superoxide dismutase mimetic MnTBAP prevents Fas-induced acute liver failure in the mouse  Benoît Malassagne, Pierre–Jacques Ferret, Renaud Hammoud, Micheline Tulliez, Sassia Bedda, Hélène Trébéden, Patrick Jaffray, Yvon Calmus, Bernard Weill, Frédéric Batteux  Gastroenterology  Volume 121, Issue 6, Pages 1451-1459 (December 2001) DOI: 10.1053/gast.2001.29590 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Survival rates and histologic data. (A) Survival rates in mice injected with a lethal (0.25 μg/g) dose of agonist anti-Fas mAb (●; n = 27); in mice injected with MnTBAP (10 mg/kg) 2 hours before anti-Fas mAb (♦; n = 15) (P < 0.001 vs. untreated mice) or 2 hours after anti-Fas mAb (■; n = 12) (P < 0.001 vs. untreated mice), in control mice treated with PBS (○; n = 16), or with MnTBAP alone (▵; n = 16). Curves are the compiled results of 3 experiments. Survival rates in the groups treated preventively and curatively were still significantly increased at 24 hours (P < 0.001 vs. untreated mice). (B) Microscopic views of livers 12 hours after injection of a sublethal (0.15 μg/g) dose of anti-Fas mAb. H&E staining (250-fold magnification), left quadrants; TUNEL staining (400-fold magnification), right quadrants; anti-Fas mAb alone, upper quadrants; anti-Fas mAb and preventive injection of MnTBAP, middle quadrants; and anti-Fas mAb and curative injection of MnTBAP, lower quadrants. Gastroenterology 2001 121, 1451-1459DOI: (10.1053/gast.2001.29590) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Flow cytofluorometry analysis of isolated hepatocytes after anti-Fas challenge (gray lines) and in controls without anti-Fas mAb (black lines), after preincubation or not with MnTBAP. The various parameters were plotted as fluorescence intensity on the abscissa and cell counts on the ordinate. All determinations were done on hepatocytes at an early stage of apoptosis (annexin V–positive, PI, or YO-PRO-1–negative cells). ROS production was assessed by (A) CM-DCFDA, (B) O2°− production by DHE, and (C) anthracen staining. (D) Variations in ΔΨm were evaluated using the cationic dye CMXRos, and (E) alterations in cardiolipin content were evaluated by NAO incorporation. Data from 1 of 4 representative experiments with similar results are presented. Gastroenterology 2001 121, 1451-1459DOI: (10.1053/gast.2001.29590) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Flow cytometry analysis of isolated hepatocytes submitted to 1 μg/mL of agonist anti-Fas mAb after preincubation or not with 1 mg/mL MnTBAP. (A) ROS production as assessed by inhibition of anthracen staining. (B) Early apoptosis as assessed by annexin V. (C) Cell death as assessed by YO-PRO-1. Data are presented as means ± SEM of 6 independent experiments. Anti-Fas + MnTBAP vs. anti-Fas alone: *P < 0.05; **P < 0.02; ***P < 0.01; and ****P < 0.001. Gastroenterology 2001 121, 1451-1459DOI: (10.1053/gast.2001.29590) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Preventive (P) or curative (C) MnTBAP prevents cytochrome c release from mitochondria and preserves mitochondrial Bcl-2 in livers from mice with Fas-induced ALF. After lethal (0.25 μg/g) doses of anti-Fas mAb, determinations were performed at the time of death or 24 hours after anti-Fas mAb injection in surviving mice treated with MnTBAP and in controls injected with MnTBAP alone. After sublethal (0.15 μg/g) doses of anti-Fas, all determinations were performed 12 hours after antibody injection. (A) Western blot analysis of cytochrome c in liver mitochondrial (HM) and cytosolic (S100) fractions from control mice injected with PBS or MnTBAP alone, and from mice injected with anti-Fas mAb, treated or not with MnTBAP10. (B) Mitochondrial expression of Bcl-2. Western blot analysis of Bcl-2 was performed in enriched mitochondrial fractions of livers. Gastroenterology 2001 121, 1451-1459DOI: (10.1053/gast.2001.29590) Copyright © 2001 American Gastroenterological Association Terms and Conditions