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LETTERS TO THE EDITOR

Comments on "Glycogen Synthase Kinase-3 Mediates Acetaminophen-Induced Apoptosis in Human Hepatoma Cells"

Liver Research Institute, University of Arizona, Tucson, Arizona

Received May 18, 2005 ; accepted June 9, 2005.

After more than 30 years of studying AAP-induced hepatotoxicity, there is no disagreement among investigators that AAP-induced liver cell death is initiated by the formation of a reactive metabolite, which is first detoxified by glutathione. After cellular glutathione levels are exhausted, the reactive metabolite covalently binds to cellular proteins (Nelson, 1990; Jaeschke et al., 2003). These initiating events occur in livers of animals and humans after AAP overdose in vivo as well as in all primary cultured hepatocytes. It is therefore difficult to comprehend why mechanisms triggered in cancer cell lines, which are not able to generate this reactive metabolite, cannot deplete glutathione, and will not cause any covalent binding, may have any relevance for events occurring early after AAP exposure in primary hepatocytes. In addition, immortalized cell lines have a dramatically different gene expression profile than primary hepatocytes (Boess et al., 2003). Since the authors specifically wanted to investigate early signaling events, it is particularly puzzling that they chose a hepatoma cell line as an experimental model when not only critical initiating events for cell toxicity are absent but the outcome (mode of cell death) is completely different between cancer cell lines and primary hepatocytes. Although Boulares et al. (2002) outright state that SK-Hep1 cells cannot metabolically activate AAP, Macanas-Pirard et al. (2005) suggest that HuH7 cells may be able to form a reactive metabolite. However, the experimental evidence for this conclusion is a 40 to 50% depletion of glutathione at 30 to 48 h, which correlates with the loss of cell viability of 30 to 68% at that time. In contrast, liver glutathione is depleted within 30 min after AAP overdose in mice (Knight et al., 2001) and within 3 h in primary cultured mouse hepatocytes (Bajt et al., 2004). Since the earliest loss of cell viability occurs between 2 to 3 h in vivo and 3 to 6 h in vitro, glutathione depletion precedes cell death by several hours. In addition to these fundamental differences in the mechanism of AAP-induced hepatotoxicity during the initiation of cell death between primary hepatocytes and cancer cell lines, later events also seem to be completely different. For example, it was recently demonstrated that AAP-induced necrosis depends on the mitochondrial permeability transition pore opening and collapse of the mitochondrial membrane potential in vitro (Kon et al., 2004) and in vivo (Masubuchi et al., 2005). On the other hand, AAP-induced caspase-dependent apoptosis in HuH7 cells is independent of the mitochondrial permeability transition (Macanas-Pirard et al., 2005). Thus, the mechanisms of AAP-induced cell death in HuH7 hepatoma cells reproduce neither the initiating events nor later mechanisms of injury that occur in primary hepatocytes after AAP overdose.

I also take issue with the statement by the authors that the initial form of AAP-induced cell damage is through apoptosis. Apoptosis is defined by morphological characteristics such as cell shrinkage, chromatin condensation, formation of apoptotic bodies with predominantly intact cell organelles, absence of cell content release, and no significant inflammation (Jaeschke and Lemasters, 2003). Even if many cells are affected, apoptosis remains a single cell event. On the other hand, AAP-induced liver injury in vivo is characterized by early mitochondrial swelling and dysfunction, karyolysis, cell swelling, loss of cell contents, and substantial inflammation (Gujral et al., 2002). AAP-induced cell injury affects large numbers of contiguous cells in centrilobular areas. These morphological features, which are completely opposite to the classic definition of apoptosis, are observed with different doses of AAP in overnight-fasted and in fed animals (Gujral et al., 2002). Thus, AAP-induced cell death in primary hepatocytes in vitro and in vivo never shows morphological features of apoptosis but is an unambiguous oncotic process. Since almost all biochemical parameters of apoptosis are not specific for this cell death process, certain similarities between apoptosis and AAP-induced cell death (e.g., DNA ladders, mitochondrial Bax translocation, etc.) do not prove AAP-induced apoptosis in vivo. The argument that AAP-induced apoptosis rapidly deteriorates to necrosis is not justified based on experimental data. Any secondary necrotic process is always preceded by apoptosis with its clearly identifiable morphological and biochemical features (Jaeschke and Lemasters, 2003; Jaeschke et al., 2004). In addition, during secondary necrosis, the cells may swell and release cell contents but still maintain many characteristics of the preceding apoptosis, e.g., massive caspase activation (Jaeschke et al., 2004). None of these features of apoptosis or secondary necrosis applies to hepatocytes exposed to AAP overdose.

In summary, significant advances in our understanding of intracellular signaling events in response to AAP have been made in recent years. These efforts need to be continued. However, it is important to use appropriate experimental tools, which provide answers relevant for the in vivo hepatotoxicity in animals and humans. Cancer cell lines, which cannot metabolically activate AAP and have a dramatically different gene expression profile compared with primary hepatocytes, are certainly not suitable models to study mechanisms of AAP-induced liver injury.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.089821.

ABBREVIATIONS: AAP, acetaminophen.

	References

Top References

 

Bajt ML, Knight TR, Lemasters JJ, and Jaeschke H (2004) Acetaminophen-induced oxidant stress and cell injury in cultured mouse hepatocytes: protection by N-acetyl cysteine. Toxicol Sci 80: 343-349.[Abstract/Free Full Text]
Boess F, Kamber M, Romer S, Gasser R, Muller D, Albertini S, and Suter L (2003) Gene expression in two hepatic cell lines, cultured primary hepatocytes and liver slices compared to the in vivo liver gene expression in rats: possible implications for toxicogenomics use of in vitro systems. Toxicol Sci 73: 386-402.[Abstract/Free Full Text]
Boulares AH, Zoltoski AJ, Stoica BA, Cuvillier O, and Smulson ME (2002) Acetaminophen induces caspase-dependent and Bcl-x(L) sensitive apoptosis in human hepatoma cells and lymphocytes. Pharmacol Toxicol 90: 38-50.[CrossRef][Medline]
Gujral JS, Knight TR, Farhood A, Bajt ML, and Jaeschke H (2002) Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis? Toxicol Sci 67: 322-328.[Abstract/Free Full Text]
Jaeschke H, Gujral JS, and Bajt ML (2004) Apoptosis and necrosis in liver disease. Liver Int 24: 85-89.[CrossRef][Medline]
Jaeschke H, Knight TR, and Bajt ML (2003) The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity. Toxicol Lett 144: 279-288.[CrossRef][Medline]
Jaeschke H and Lemasters JJ (2003) Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology 125: 1246-1257.[CrossRef][Medline]
Knight TR, Kurtz A, Bajt ML, Hinson JA, and Jaeschke H (2001) Vascular and hepatocellular peroxynitrite formation during acetaminophen-induced liver injury: role of mitochondrial oxidant stress. Toxicol Sci 62: 212-220.[Abstract/Free Full Text]
Kon K, Kim JS, Jaeschke H, and Lemasters JJ (2004) Mitochondrial permeability transition in acetaminophen-induced necrotic and apoptotic cell death to cultured mouse hepatocytes. Hepatology 40: 1170-1179.[CrossRef][Medline]
Macanas-Pirard P, Yaacob NS, Lee PC, Holder JC, Hinton RH, and Kass GEN (2005) Glycogen synthase kinase-3 mediates acetaminophen-induced apoptosis in human hepatoma cells. J Pharmacol Exp Ther 313: 780-789.[Abstract/Free Full Text]
Masubuchi Y, Suda C, and Horie T (2005) Involvement of mitochondrial permeability transition in acetaminophen-induced liver injury in mice. J Hepatol 42: 110-116.[Medline]
Nelson SD (1990) Molecular mechanisms of the hepatotoxicity caused by acetaminophen. Semin Liver Dis 10: 267-278.[Medline]

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