The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and nuclear DNA fragmentation (original) (raw)

The objective of this investigation was to gain further insight into the intracellular mechanisms of APAP hepatotoxicity in humans and to assess the mode of cell death. Because there is no diagnostic benefit that would justify the risk of a liver biopsy during the early injury phase after APAP overdose, our approach was to use plasma biomarkers, validated in a mouse model, to obtain reliable mechanistic information. We studied 3 groups of patients. In addition to an age- and sex-matched control group, APAP-overdose patients were divided into 2 groups: one with evidence of liver injury (peak plasma ALT activities ≥ 1,000 U/l and peak PT ≥ 18 s) and one with no or very limited liver injury (PT < 18 s and ALT < 1,000 U/l; highest: 17.5 s and 158 U/l, respectively). The low injury in this group was likely due, in part, to early admission and treatment prior to development of toxicity. This would be consistent with the higher plasma APAP levels on admission in the group with normal LTs. Additionally, some of these patients may have taken lower overdoses.

Mitochondrial dysfunction and APAP hepatotoxicity. Mitochondrial dysfunction after toxic doses of APAP has been recognized in rodents since the 1980s, when inhibition of mitochondrial respiration and depletion of ATP were first described (15, 16, 44). More recent studies have shown the development of oxidative and nitrosative stress within mitochondria and occurrence of the mitochondrial MPT after APAP treatment (18–21). Using a human cell line with high expression levels of CYP (HepaRG), mitochondrial dysfunction has also been shown to occur in cultured human hepatocytes (25). We reasoned that, if mitochondrial membrane integrity is compromised and mitochondrial contents leak into the cytosol in patients, these mitochondrial components must be released into the circulation when the hepatocytes become necrotic. Our data showing the presence of the specific mitochondrial components GDH and mtDNA in plasma of patients with highly elevated ALT levels provide evidence that mitochondrial dysfunction occurs in humans after APAP overdose. The high activity of GDH in the plasma from these patients is consistent with the higher GDH expression in zone 3 of the liver (34), where APAP causes the greatest tissue injury.

The assumption behind these mechanistic conclusions is that GDH and mtDNA release occurs only when mitochondrial damage is involved, not just cell injury. This hypothesis was confirmed by demonstrating that liver cell damage caused by furosemide, a hepatotoxicant not believed to affect mitochondria (43), resulted in ALT release, but not a statistically significant increase in either mtDNA or GDH in mouse plasma. In contrast, APAP overdose triggers release of both ALT and large amounts of GDH and mtDNA into the plasma of mice. Thus, it is justified to conclude that APAP-induced liver injury in humans involves mitochondrial damage. The time course of the release of GDH and mtDNA correlated well with the appearance of ALT in plasma of all patients, suggesting that mitochondrial damage may be closely related to cell necrosis. In experimental animals, interventions that restored the scavenging capacity for ROS and peroxynitrite in mitochondria (30, 45–47) or prevented the MPT (19–21) protected against APAP-induced liver injury. In addition, selective impairment of mitochondrial antioxidant defenses (partial MnSOD deficiency) aggravated APAP hepatotoxicity (48). Moreover, mitochondrial dysfunction and oxidant stress preceded cell necrosis by several hours in murine hepatocytes and in human HepaRG cells (25, 49). Thus, mitochondrial damage is central to APAP-induced cell death in murine models and in a human hepatocyte cell line (19, 21, 25, 49, 50). Based on the close correlation between release of biomarkers of mitochondrial damage and cell necrosis, it is likely that mitochondrial dysfunction is a main determinant of liver cell damage in APAP-overdose patients. However, despite these close correlations, our data do not establish that mitochondrial damage is the cause of cell death in humans.

Nuclear DNA damage and APAP hepatotoxicity. In addition to the biomarkers of mitochondrial damage, nuclear DNA fragments were detectable in plasma of patients with severe APAP-induced liver injury. The assay for DNA fragments is based on detection of nuclear histones, which are not present in mtDNA (39). Thus, the antihistone ELISA measures specifically nuclear DNA fragments. No plasma DNA fragments were detectable in healthy volunteers or in patients without severe liver injury. In contrast, the time course of plasma DNA fragment levels in patients with liver injury closely followed the release of ALT, i.e., cell necrosis. A comparison of plasma DNA fragments and nuclear DNA damage in mouse liver after APAP overdose revealed that both parameters correlate with ALT release, suggesting that nuclear DNA damage occurs along with cell death.

Previous studies in rodents documented that DNA fragments after APAP are indistinguishable from apoptotic DNA fragments (40, 51). In addition, no nitrotyrosine residues were detectable in the nucleus (51). This indicated that DNA damage was not caused by oxidant stress or peroxynitrite formation, but involved endonucleases. Because of the lack of relevant caspase activation during APAP hepatotoxicity (23, 52), the traditional caspase-activated DNase can be excluded. Instead, EndoG and AIF are released from the mitochondrial intermembrane space and translocate to the nucleus during APAP-induced cell death (22). Protection against mitochondrial oxidant stress or prevention of the MPT eliminated nuclear DNA fragmentation (21, 49, 51). In a later study, the Bcl-2 family member Bax, which forms pores in the outer mitochondrial membrane during the early phase after APAP exposure, was found to facilitate release of EndoG and AIF from mitochondria and nuclear translocation, which triggered the initial DNA damage (53). Partial AIF deficiency reduced the mitochondrial oxidant stress, nuclear translocation of AIF, and DNA fragmentation (54). There is also evidence that a general endonuclease inhibitor attenuated APAP-induced cell death (55). Together, these findings indicate that nuclear DNA damage is dependent on mitochondrial dysfunction and the release of endonucleases from the intermembrane space. Thus, nuclear DNA damage is closely related and even contributes to liver cell death in the murine model. Given the similar appearance of nuclear DNA in patients with APAP-induced liver injury, it is likely that the same mechanisms of DNA damage apply in human liver.

DNA as DAMP. Our data indicate that nuclear DNA fragments and mtDNA are released into the plasma of patients and in mice after APAP overdose. These molecules can act as DAMPs through activation of TLRs, especially TLR9, to induce cytokine formation after APAP (56). In the mouse model, there is extensive formation of proinflammatory mediators and recruitment of neutrophils into the liver in response to APAP overdose (57). However, the preponderance of the experimental evidence argues against a relevant contribution of neutrophils to the injury process (58). Consistent with these findings, neutrophils isolated from the injured liver or from the blood are not activated during the main injury phase of APAP hepatotoxicity in mice (59). Preliminary data for neutrophil activation in APAP-overdose patients appear to confirm the lack of neutrophil activation during the injury phase, but show a progressive activation at later time points (C.D. Williams and H. Jaeschke, unpublished observations). Thus, it is likely that in patients, the release of DAMPs, such as nuclear DNA fragments and mtDNA during the injury phase, contributes to activation of innate immune cells, which are involved in the removal of necrotic cell debris and thus contribute to the recovery as observed in mice (58).

Mode of APAP-induced cell death in patients. It is generally accepted that the mode of APAP-induced liver cell death in mice is oncotic necrosis. This is based on morphological evidence (cell swelling, vacuolization, karyorrhexis, and karyolysis), the massive release of cell contents (ALT), and the resulting inflammation (23). However, there is limited evidence of apoptosis. Hallmarks of apoptotic cell death include several morphological features, such as cell shrinkage, chromatin condensation, and formation of apoptotic bodies as well as extensive caspase activation (60). In general, only very few apoptotic cells are detectable after APAP overdose (23), and there is no relevant caspase activation in mouse livers (23, 52, 61). Furthermore, pancaspase inhibitor did not protect against APAP hepatotoxicity (23–25, 52, 62, 63). However, most of these experiments were done with overnight-fasted animals. Recently, it was suggested that in fed mice with higher cellular ATP content, APAP causes limited caspase activation and some apoptotic cell death (62). This conclusion was also based on the detection of a K18 cleavage product, which is thought to be specific for caspase-3 activity (62). The use of this assay has resulted in conflicting data in the clinical literature. A case report showed no significant increase of the cleavage product over the time course of injury and recovery of an APAP-overdose patient (64). In contrast, 2 studies found significant increases of this caspase cleavage product in APAP-overdose patients (32, 33). However, as noted by Volkmann et al. (33), necrotic full-length K18 was the dominant form, suggesting that apoptosis plays a relatively minor part in the mechanism of cell death after APAP overdose in humans. Our data directly analyzing caspase-3 activity or the active fragment of caspase-3 by Western blotting did not reveal any evidence for caspase-3 activation in these patients. However, in the overdose patients with extensive liver injury, pro–caspase-3 protein was present in plasma, reflecting the release of cell contents of necrotic cells. These clinical data are similar to our observations in APAP-treated mice, which had an increase in plasma pro–caspase-3 levels, but no active fragments and no increase in enzyme activity. In contrast, in the G/E model, which is a positive control for caspase-dependent apoptosis in hepatocytes (42), extensive caspase-3 activity and the active fragment of caspase-3 were clearly detectable. In a similar experiment, caspase-3 activity was readily detectable in plasma after galactosamine-induced apoptosis in rat liver despite the fact that only 5%–6% of hepatocytes were apoptotic (65). Based on these experiments, it can be concluded that the absence of active caspase-3 fragments and of detectable caspase-3 activity in plasma of APAP-overdose patients with severe liver injury suggests that apoptotic cell death is not relevant for the overall pathophysiology. The discrepancy between the detection of minor levels of caspase-dependent K18 cleavage product in some studies (32, 33) and our lack of direct plasma caspase-3 activity measurement in APAP patients requires further study. This could be related to differences in assay sensitivity or potentially even specificity, i.e., K18 could be cleaved by other proteases. However, there is principal agreement among all studies that necrotic cell death is dominant in these patients.

Currently, much emphasis is placed upon the development of novel biomarkers to aid in both the diagnosis and prognosis of various liver diseases. It is possible that mitochondrial markers such as GDH and mtDNA can also predict outcome. In this study, only one patient did not survive, and none received a liver transplant. With these numbers, we are unable to assess the prognostic value of these biomarkers. Larger cohorts will be needed to determine whether or not these biomarkers are predictive of patient outcome.

In summary, our data demonstrated the release of biomarkers reflecting mitochondrial damage and nuclear DNA fragmentation in patients with severe APAP-induced liver injury. These events led to predominantly necrotic cell death. The use of these biomarkers and the mechanistic conclusions were extensively validated by parallel studies in mice in which tissue injury and the release of these markers were measured. Our findings provide strong support for the hypothesis that mitochondrial dysfunction and DNA damage are critical events in the mechanism of cell necrosis after APAP overdose in patients. In addition, these data confirm that the in vivo mouse model, primary murine hepatocytes, and human HepaRG cells are appropriate experimental systems to study cell death mechanisms that appear to be relevant for APAP-overdose patients.