Volume 131, Issue 1, Pages 165-178 (July 2006) c-Jun N-Terminal Kinase Plays a Major Role in Murine Acetaminophen Hepatotoxicity  Basuki K. Gunawan, Zhang–Xu Liu, Derick Han, Naoko Hanawa, William A. Gaarde, Neil Kaplowitz  Gastroenterology  Volume 131, Issue 1, Pages 165-178 (July 2006) DOI: 10.1053/j.gastro.2006.03.045 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Effect of JNK inhibitor (SP600125) on APAP induced GSH depletion and covalent binding in cultured mouse hepatocytes. (A) Total glutathione (GSH). Total GSH was measured at 3 hours after addition of APAP. (B) Covalent binding. [14C]APAP covalent binding was measured 1 hour after APAP treatment. See Materials and Methods for details. Gastroenterology 2006 131, 165-178DOI: (10.1053/j.gastro.2006.03.045) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Effect of JNK inhibitor (SP600125) on APAP and BSO induced necrosis. Primary mouse hepatocytes were treated with 2.5 mmol/L APAP or 1 mmol/L BSO or combination of 2.5 mmol/L APAP and 1 mmol/L of BSO with and without 20 μmol/L JNK inhibitor pretreatment. Cell death was assessed at 15 hours. Results are mean ± SD for 3 experiments. *P < .05 vs APAP + BSO group. Gastroenterology 2006 131, 165-178DOI: (10.1053/j.gastro.2006.03.045) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Effect of JNK inhibitor (SP600125) on APAP induced JNK activity and toxicity in vivo in C57BL/6 mice. (A) Western blot of phospho-c-Jun. Phospho-c-Jun was measured at 4 hours after APAP treatment in the absence and presence of JNK inhibitor pretreatment. (B) Phospho-c-Jun time course. Phospho-c-Jun levels were measured at different time points after APAP treatment with densitometric comparison for 3 different experiments shown in C. *P < .05 vs other groups. (D) Serum ALT. Mice received 800 mg/kg APAP alone (n = 22, 23% mortality) or were pretreated 1 hour before APAP treatment with 10 mg/kg JNK inhibitor (n = 10, 0% mortality), 20 mg/kg p38 inhibitor (n = 6, 33.3% mortality), or 20 mg/kg Erk inhibitor (n = 6, 16.7% mortality), respectively. The surviving mice were then killed at 24 hours after APAP treatment, and serum ALT was determined. Results are mean ± SD. *P < .05 vs all other groups. (E) Survival. Using 1000 mg/kg APAP, survival was determined at 24 and 48 hours after APAP treatment in the presence or absence of pretreatment with JNK inhibitor. Each group had 6 mice. (F) Liver histology. Representative H&E-stained sections of liver of mice that received 800 mg/kg APAP. The mice were killed at 24 hours after APAP treatment either pretreated 1 hour with vehicle or SP600125. Gastroenterology 2006 131, 165-178DOI: (10.1053/j.gastro.2006.03.045) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 Effect of JNK inhibitor on APAP induced glutathione (GSH) depletion in mice. (A) Total GSH. Total liver GSH (percentage of control) was measured at 1 (n = 27), 3 (n = 6), and 6 (n = 16) hours after 800 mg/kg APAP injection in C57BL/6 mice in the absence or presence of 1 hour JNK inhibitor pretreatment. Mean total liver GSH for control was 7.12 ± 2.49 μmol/g liver. Results are mean ± SD. (B) Mitochondrial GSH. Mitochondrial GSH (percentage of control) was measured at 1 and 3 hours after 800 mg/kg APAP injection in the absence or presence of 1 hour JNK inhibitor pretreatment. Mean mitochondrial GSH for control was 0.24 ± 0.4 μmol/g liver. Results are mean ± SD. Each group had 3 mice for each time point. Gastroenterology 2006 131, 165-178DOI: (10.1053/j.gastro.2006.03.045) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Effect of timing of JNK inhibitor administration on APAP hepatotoxicity. C57BL/6 mice received 800 mg/kg APAP at time 0 and JNK inhibitor (10 mg/kg) from −6 to +8 hours. For each time point, 3 or more mice were used, and serum ALT was measured at 24 hours after APAP injection. Results are mean ± SD. At right, ALT values for APAP + vehicle at 8 hours are depicted (n = 12). Mortality was only seen in the 8-hour delayed treatment group (42% with JNK inhibitor and 50% with vehicle). Gastroenterology 2006 131, 165-178DOI: (10.1053/j.gastro.2006.03.045) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Effect of APAP on JNK1 k/o and JNK2 k/o mice. (A) Serum ALT. All mice received 800 mg/kg APAP (wild-type/C57BL/6: 8 mice; JNK1 k/o: 6 mice; JNK2 k/o: 6 mice). Mice were killed at 24 hours, and serum ALT was measured. *P < .05 vs wild type. (B and C) Histology in JNK k/o mice. Representative H&E-stained sections of liver of JNK1 k/o mouse (B) and JNK2 k/o mouse (C) 24 hours after 800 mg/kg APAP. Gastroenterology 2006 131, 165-178DOI: (10.1053/j.gastro.2006.03.045) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Effect of JNK1 and JNK2 antisense oligonucleotides (ASO) on APAP induced toxicity. (A and B). Western blots of JNK. C57BL/6 mice received either control oligonucleotide or both JNK1 and JNK2 ASO for 2 weeks prior to liver harvesting. Using antisera to both JNK1 and 2, representative Western blot is shown in A and densitometric comparison for 4 individual mice per group (each normalized to actin) in B. *P < .05. (C) Serum ALT. After 2 weeks of treatment with different ASO, mice were injected with 800 mg/kg APAP (control antisense: 7 mice, JNK1 antisense: 4 mice, JNK2 antisense: 4 mice, JNK1 + 2 antisense: 8 mice). The mice were then killed at 24 hours after APAP treatment, and serum ALT was measured. *P < .05 vs all other groups. #P < .05 vs control antisense and JNK1 antisense groups. Results are mean ± SD. (D) Survival. Using 1000 mg/kg APAP, none of 3 mice pretreated with control antisense survived at 24 hours after APAP, whereas all 3 mice pretreated with JNK1 + 2 antisense survived. (E) Glutathione (GSH) depletion. Total GSH was measured at 3 hours after 800 mg/kg APAP injection in mice that received control oligonucleotides or both JNK1 and JNK 2 ASO. Nearly identical (>86%) GSH depletion was observed. Each group had 3 mice. (F) Liver histology. After 2 weeks of treatment with different ASO, mice were given 800 mg/kg APAP and then killed at 24 hours. Representative H&E-stained liver sections are shown from mice pretreated with control oligonucleotides, JNK1 ASO, JNK2 ASO, and both JNK1 and JNK2 ASO. Gastroenterology 2006 131, 165-178DOI: (10.1053/j.gastro.2006.03.045) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Effect of JNK1 and JNK2 antisense oligonucleotides (ASO) on APAP induced toxicity. (A and B). Western blots of JNK. C57BL/6 mice received either control oligonucleotide or both JNK1 and JNK2 ASO for 2 weeks prior to liver harvesting. Using antisera to both JNK1 and 2, representative Western blot is shown in A and densitometric comparison for 4 individual mice per group (each normalized to actin) in B. *P < .05. (C) Serum ALT. After 2 weeks of treatment with different ASO, mice were injected with 800 mg/kg APAP (control antisense: 7 mice, JNK1 antisense: 4 mice, JNK2 antisense: 4 mice, JNK1 + 2 antisense: 8 mice). The mice were then killed at 24 hours after APAP treatment, and serum ALT was measured. *P < .05 vs all other groups. #P < .05 vs control antisense and JNK1 antisense groups. Results are mean ± SD. (D) Survival. Using 1000 mg/kg APAP, none of 3 mice pretreated with control antisense survived at 24 hours after APAP, whereas all 3 mice pretreated with JNK1 + 2 antisense survived. (E) Glutathione (GSH) depletion. Total GSH was measured at 3 hours after 800 mg/kg APAP injection in mice that received control oligonucleotides or both JNK1 and JNK 2 ASO. Nearly identical (>86%) GSH depletion was observed. Each group had 3 mice. (F) Liver histology. After 2 weeks of treatment with different ASO, mice were given 800 mg/kg APAP and then killed at 24 hours. Representative H&E-stained liver sections are shown from mice pretreated with control oligonucleotides, JNK1 ASO, JNK2 ASO, and both JNK1 and JNK2 ASO. Gastroenterology 2006 131, 165-178DOI: (10.1053/j.gastro.2006.03.045) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 8 Effect of JNK inhibitor (SP600125) on Bax translocation to mitochondria. C57BL/6 mice received 800 mg/kg APAP with or without 1-hour JNK inhibitor pretreatment and were killed at 6 hours after APAP treatment. Mitochondrial fraction was obtained (see Materials and Methods section). Using antisera to Bax and cytochrome oxidase IV (COX IV) as loading control, representative Western blot is shown in A and densitometric comparison for 3 individual mice per group (each normalized to COX IV) in B. *P < .05. Gastroenterology 2006 131, 165-178DOI: (10.1053/j.gastro.2006.03.045) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 9 Effect of JNK inhibitor (SP600125) on CCl4 hepatotoxicity in mice. C57BL/6 mice received CCl4 IP with or without 1-hour JNK inhibitor pretreatment. Each group had 3 or 4 mice. The mice were then killed at 24 hours after CCl4 treatment, and serum ALT was measured. Gastroenterology 2006 131, 165-178DOI: (10.1053/j.gastro.2006.03.045) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 10 Effect of JNK inhibitor (SP600125) on Con A hepatotoxicity in mice. C57BL/6 mice received 15 mg/kg Con A IV with or without 1-hour JNK inhibitor pretreatment. Each group had 6 mice. Serum ALT was measured at 6 hours and 24 hours after Con A treatment. Gastroenterology 2006 131, 165-178DOI: (10.1053/j.gastro.2006.03.045) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions