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. Author manuscript; available in PMC: 2012 Feb 1.
Published in final edited form as: Hepatology. 2010 Dec 17;53(2):718–719. doi: 10.1002/hep.23940

NO EVIDENCE FOR CASPASE-DEPENDENT APOPTOSIS IN ACETAMINOPHEN HEPATOTOXICITY

Hartmut Jaeschke 1, C David Williams 1, Anwar Farhood 2
PMCID: PMC3058812  NIHMSID: NIHMS231231  PMID: 21274895

To the Editor

In a recent paper, Hu and Colletti suggest that acetaminophen (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 H&E sections, which is considered the gold standard for apoptosis.2,3 Instead, they rely mainly on the TUNEL assay. However, this assay indicates DNA strandbreaks, 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 (Figure 1). However, there is no difference in DNA ladder formation between APAP-induced necrosis and Gal/ET-induced apoptosis suggesting that in both cases endonucleases are involved.4 Whereas pancaspase inhibitors eliminate DNA fragmentation after Gal/ET-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 DNase.2 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

Figure 1.

Figure 1

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Fasted C57BL/6 mice were treated with 300 mg/kg APAP in saline for 6 h (A,C) or 700 mg/kg galactosamine/100μg/kg endotoxin for 5 h (B,D). Tissue sections were stained with hematoxylin and eosin (H&E) (A,B) or with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (C,D). H&E sections (x200) and inserts (x400) show necrotic cells with vacuolization, cell swelling and nuclear disintegration after APAP overdose (black arrows). Sections of Gal/ET-treated mice show nuclear condensation, cell shrinkage and formation of apoptotic bodies all consistent with apoptosis (white arrows). 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 (C) while Gal/ET-induced apoptosis causes only distinct nuclear staining (D).

Second, the authors showed that the caspase inhibitor Q-VD-OPh, which is only soluble in dimethylsulfoxide (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 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 manuscript that APAP hepatotoxicity is caused by caspase-dependent apoptosis.

References

  • 1.Hu B, Colletti LM. CXC receptor-2 knockout genotype increases X-linked inhibitor of apoptosis protein and protects mice from acetaminophen hepatotoxicity. Hepatology. 2010;52:691–702. doi: 10.1002/hep.23715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Jaeschke H, Lemasters JJ. Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology. 2003;125:1246–1257. doi: 10.1016/s0016-5085(03)01209-5. [DOI] [PubMed] [Google Scholar]
  • 3.Gujral JS, Knight TR, Farhood A, Bajt ML, Jaeschke H. Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis? Toxicol Sci. 2002;67:322–328. doi: 10.1093/toxsci/67.2.322. [DOI] [PubMed] [Google Scholar]
  • 4.Cover C, Mansouri A, Knight TR, Bajt ML, Lemasters JJ, Pessayre D, Jaeschke H. Peroxynitrite-induced mitochondrial and endonuclease-mediated nuclear DNA damage in acetaminophen hepatotoxicity. J Pharmacol Exp Ther. 2005;315:879–887. doi: 10.1124/jpet.105.088898. [DOI] [PubMed] [Google Scholar]
  • 5.Jaeschke H, Cover C, Bajt ML. Role of caspases in acetaminophen-induced liver injury. Life Sci. 2006;78:1670–1676. doi: 10.1016/j.lfs.2005.07.003. [DOI] [PubMed] [Google Scholar]
  • 6.Bajt ML, Cover C, Lemasters JJ, Jaeschke H. Nuclear translocation of endonuclease G and apoptosis-inducing factor during acetaminophen-induced liver cell injury. Toxicol Sci. 2006;94:217–225. doi: 10.1093/toxsci/kfl077. [DOI] [PubMed] [Google Scholar]

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