Necrosis versus apoptosis in acetaminophen-induced hepatotoxicity
Authors: Klaus Schulze- Osthoff, Heike Bantel
Journal: Hepatology (2010)

We read with interest the article by Hu and Colletti investigating the mechanisms of acetaminophen (N-acetyl-para-aminophenol [APAP])-induced liver injury.1 The authors suggest that APAP hepatotoxicity is caused by the mitochondrial apoptosis pathway and facilitated by chemokine (C-X-C motif) receptor 2 (CXCR2) receptor signaling. We would like to bring to the readers' attention that, due to the increasing knowledge of nonapoptotic cell death and the development of novel biomarkers, recent evidence indicates that acute liver failure (ALF) following an APAP overdose is mainly mediated by necrosis rather than by apoptosis.2, 3 Moreover, we have reported that not only in experimental models, but even in critically ill patients with ALF, necrosis is the predominant cause of APAP hepatotoxicity.4 A distinction between both cell death mechanisms is important, because there are now increasing possibilities for therapeutic interventions with these distinct cell death forms. Results from our and other groups further suggest that determination of the mode of cell death might be of predictive value for the disease outcome of patients with ALF.4-6 The mechanism of APAP-induced liver injury involves the generation of the toxic metabolite N-acetyl-p-benzoquinoneimine by the cytochrome P450 system, which causes glutathione depletion, oxidative stress, alterations of calcium homeostasis, and finally results in mitochondrial damage and adenosine triphosphate (ATP) depletion. However, even though mild forms of APAP intoxication might cause signs of apoptosis, there is now a general agreement that apoptosis is strictly ATP-dependent and therefore inhibited under conditions of ATP depletion.7 Instead, necrosis as a result of mitochondrial dysfunction is consistent with high lactate levels that are especially observed in patients with ALF with a poor outcome.8 Unfortunately, the methods used by Hu and Colletti preclude the evaluation of the relevant cell death pathways, but might have led to the misinterpretation of apoptosis as the principal mechanism of APAP hepatotoxicity. First, the authors show that a caspase inhibitor, which was solubilized in dimethylsulfoxide (DMSO), prevented terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining in hepatocytes. TUNEL is an unspecific marker that cannot distinguish between apoptotic and necrotic DNA fragmentation. Moreover, a DMSO control was not presented but is mandatory, because DMSO is a radical scavenger and by itself exerts cytoprotective effects.9 Second, although the authors show that APAP-induced DNA fragmentation was prevented, it remains unclear whether caspase inhibition indeed improves the survival of mice. There are many cases known in which caspase inhibitors prevent apoptotic alterations but do not affect cell survival. Finally, on the basis of the assessment of proteolytic caspase fragments, the authors suggest that caspase-9 is activated by APAP. However, they do not present data on the enzymatic caspase activity. Indeed, caspase-9 does not require cleavage to be activated. Moreover, calpains that are activated by APAP can induce proteolytic cleavage of caspase-9.10 These cleavages generate fragments of similar size but occur at sites that render the caspase-9 proteolytically inactive. Hence, the mere cleavage of caspase-9 cannot be taken as sufficient evidence for its activation. Altogether, we have serious concerns regarding the interpretation of the results by Hu and Colletti. Apoptosis is certainly of major importance in many chronic liver diseases. APAP-induced ALF is, however, one of the few examples where necrosis but not apoptosis predominates. An understanding of the cell death processes will be essential for effective interventions in ALF and other liver diseases. Klaus Schulze- Osthoff M.D*, Heike Bantel M.D , * Interfaculty Institute for Biochemistry, University of Tübingen, Tübingen, Germany, Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany.