The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation.
Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4-6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions.
Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA.
Kuo
K
Du
C David
CD
Williams
Mitchell R
MR
McGill
Yuchao
Y
Xie
Anwar
A
Farhood
Mathieu
M
Vinken
Hartmut
H
Jaeschke
D000082:Acetaminophen / Q000008:administration & dosage / Q000009:adverse effects*; D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology*; D056486:Chemical and Drug Induced Liver Injury / Q000188:drug therapy* / Q000517:prevention & control; D017630:Connexins / Q000037:antagonists & inhibitors / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D015398:Signal Transduction; D020122:tert-Butylhydroperoxide / Q000008:administration & dosage / Q000009:adverse effects; D000097002:Gap Junction beta-1 Protein
D000082:Acetaminophen; D001896:Boron Compounds; D017630:Connexins; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System; D004121:Dimethyl Sulfoxide; D048031:JNK Mitogen-Activated Protein Kinases; D020122:tert-Butylhydroperoxide; D000097002:Gap Junction beta-1 Protein; C109986:2-aminoethoxydiphenyl borate
2-APB; 2-Aminoethoxy-diphenyl-borate; 2-aminoethoxy-diphenyl-borate; 7-EFC; 7-ethoxy-4-trifluoromethylcoumarin; ALT; APAP; Acetaminophen hepatotoxicity; Connexin32; Cx32; DMSO; GSH; Gap junctions; HPLC-ECD; JNK; LDH; N-acetyl-p-benzoquinone imine; N-acetylcysteine; NAC; NAPQI; Oxidative stress; PH; ROS; TAA; acetaminophen; alanine aminotransferase; c-jun-N-terminal kinase; connexin32; dimethyl sulfoxide; glutathione; high-pressure liquid chromatography with electrochemical detection; lactate dehydrogenase; p-JNK; phorone; phospho-JNK; reactive oxygen species; t-BHP; tert-butylhydroperoxide; thioacetamide