No evidence for caspase-dependent apoptosis in acetaminophen hepatotoxicity
Authors: Hartmut Jaeschke, C. David Williams, Anwar Farhood
Journal: Hepatology (2010)

In a recent article, Hu and Colletti suggest that acetaminophen (N-acetyl-p-aminophenol [APAP]) hepatotoxicity in mice is caused by caspase-dependent apoptosis.1 However, we have considerable concerns regarding experimental design, data interpretation, and the conclusions. First, the authors do not show apoptotic cellular morphology in sections stained with hematoxylin and eosin, which is considered the gold standard for apoptosis.2, 3 Instead, they rely mainly on the terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL) assay. However, this assay indicates DNA strand breaks, which are not specific for apoptosis.2 If one compares TUNEL-positive cells caused by APAP overdose with apoptotic cells induced by galactosamine/endotoxin, there is a difference in cellular staining patterns (Fig. 1). However, there is no difference in DNA ladder formation between APAP-induced necrosis and galactosamine/endotoxin-induced apoptosis, suggesting that endonucleases are involved in both cases.4 Whereas pancaspase inhibitors eliminate DNA fragmentation after galactosamine/endotoxin-induced apoptosis,2, 3, 5 they have no effect on APAP-induced DNA fragmentation.3, 5 In contrast, scavenging of reactive oxygen and peroxynitrite in mitochondria prevents APAP hepatotoxicity.4 DNA damage after APAP overdose is associated with mitochondrial dysfunction and nuclear translocation of intermembrane proteins (endonuclease G, apoptosis-inducing factor).6 DNA damage during apoptosis is caused by caspase-activated deoxyribonuclease2; however, there is no relevant caspase activation after APAP overdose.3-5 A transient appearance of caspase fragments shown in overexposed gels1 is insufficient evidence for caspase activation that could be responsible for 30% of hepatocytes undergoing apoptosis.3 Fasted C57BL/6 mice were treated with APAP (300 mg/kg body weight) in saline for 6 hours (A,C) or galactosamine/endotoxin (700 mg/kg, 100 μg/kg, respectively) for 5 hours (B,D). Tissue sections were stained with (A,B) hematoxylin and eosin or (C,D) with TUNEL. Hematoxylin and eosin–stained sections (×200) and insets (×400) show necrotic cells with vacuolization, cell swelling and nuclear disintegration after APAP overdose (black arrows). Sections of galactosamine/endotoxin-treated mice show nuclear condensation, cell shrinkage, and formation of apoptotic bodies which are all consistent with apoptosis (white arrows). (C) TUNEL staining shows APAP-induced release of nuclear DNA into the cytoplasm and eventually extracellular space as indicated by diffuse staining in the area of necrosis whereas (D) galactosamine/endotoxin-induced apoptosis causes only distinct nuclear staining. Second, the authors showed that the caspase inhibitor quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh), which is only soluble in dimethyl sulfoxide (DMSO), eliminated APAP hepatotoxicity.1 Based on the dose, we estimate that 2-10 mL/kg of DMSO was injected. We have demonstrated that pretreatment with as little as 0.25 mL/kg DMSO with or without caspase inhibitor eliminates APAP toxicity because DMSO is a potent inhibitor of cytochrome P450.5 Treatment after APAP metabolism but before injury with DMSO-soluble or water-soluble caspase inhibitors is ineffective.3, 5 Thus, the results by Hu and Colletti can only be interpreted one way: First, the control group did not receive the solvent; Second, the protective effect of Q-VD-OPh was caused by the solvent DMSO, not the caspase inhibitor. Taken together, there is no credible evidence presented in this article that APAP hepatotoxicity is caused by caspase-dependent apoptosis. Hartmut Jaeschke Ph.D.*, C. David Williams B.Sc*, Anwar Farhood M.D , * Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, Department of Pathology, Brackenridge Hospital, Austin, TX.