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

Abstract

Acetaminophen (APAP) overdose is the predominant cause of acute liver failure in the United States. Toxicity begins with a reactive metabolite that binds to proteins. In rodents, this leads to mitochondrial dysfunction and nuclear DNA fragmentation, resulting in necrotic cell death. While APAP metabolism is similar in humans, the later events resulting in toxicity have not been investigated in patients. In this study, levels of biomarkers of mitochondrial damage (glutamate dehydrogenase [GDH] and mitochondrial DNA [mtDNA]) and nuclear DNA fragments were measured in plasma from APAP-overdose patients. Overdose patients with no or minimal hepatic injury who had normal liver function tests (LTs) (referred to herein as the normal LT group) and healthy volunteers served as controls. Peak GDH activity and mtDNA concentration were increased in plasma from patients with abnormal LT. Peak nuclear DNA fragmentation in the abnormal LT cohort was also increased over that of controls. Parallel studies in mice revealed that these plasma biomarkers correlated well with tissue injury. Caspase-3 activity and cleaved caspase-3 were not detectable in plasma from overdose patients or mice, but were elevated after TNF-induced apoptosis, indicating that APAP overdose does not cause apoptosis. Thus, our results suggest that mitochondrial damage and nuclear DNA fragmentation are likely to be critical events in APAP hepatotoxicity in humans, resulting in necrotic cell death.

Figure 1. GDH activity in plasma from APAP-overdose patients.

GDH activity was measured in the plasma from APAP-overdose patients throughout their time in the hospital. (A–C) Time course data from 3 representative patients showing both GDH and ALT activity. (D) Plasma GDH activity at the time of peak ALT in healthy volunteers (V), normal LT group (Norm LT), and abnormal LT group (Abnorm LT). (E) Average plasma GDH activity over time for the patients with abnormal LT results. (F) Average plasma ALT activity over time for patients with abnormal LT results. Data are expressed as mean ± SEM for n = 20. *P < 0.05 compared with healthy volunteers.

Figure 2. mtDNA in plasma from APAP-overdose patients.

The concentration of mtDNA was determined in the plasma of patients after APAP overdose. Concentrations were measured by absolute quantification real-time PCR using primers for subunits of complex I (NADH deh) and complex IV (Cyt c ox) of the electron transport chain exclusively encoded in mtDNA. (A and B) Time course data from 2 representative patients (Pt) showing both mtDNA concentration (Cyt c ox) and ALT activity in plasma. (C) Scatterplot of peak plasma ALT activity against peak mtDNA (Cyt c ox) concentration in plasma from patients of the abnormal LT group. Pearson’s correlation coefficient is shown. (D) Average mtDNA concentrations in plasma from healthy volunteers (n = 6), normal LT group (n = 20), and abnormal LT group (n = 20), expressed as mean ± SEM. *P < 0.05 compared with healthy volunteers.

Figure 3. Nuclear DNA fragments in plasma from APAP-overdose patients.

Nuclear DNA fragments were measured by ELISA in plasma from patients after APAP overdose as an indirect marker of mitochondrial injury. (A–C) Time course data from 3 representative patients showing both DNA fragments (expressed as percentage of the average change in OD/min with plasma from healthy volunteers) and ALT. (D) Average DNA fragments, expressed as percentage of the average change in OD/min with plasma from healthy volunteers, for healthy volunteers (n = 6), for the normal LT group (n = 20), and the abnormal LT group (n = 20). Data are expressed as mean ± SEM. *P < 0.05 compared with healthy volunteers.

Figure 4. Caspase-3 activation is not detectable in plasma from APAP-overdose patients.

(A) Caspase-3 processing was assessed by immunoblot in plasma from patients or from mice treated with either APAP or G/E or from control mice. The human and mouse samples were run on different gels but probed with the same antibody. Full-length (32 kDa) pro–caspase-3 was detectable in samples from the abnormal LT patient group, as well as from mice treated with APAP or G/E. Cleaved active caspase-3 fragment (17 kDa) was detectable only in plasma from the positive control G/E-treated mice. (B) Average caspase-3 activity in plasma from G/E-treated mice (n = 3), healthy volunteers (n = 6), normal LT group (n = 20), and abnormal LT group (n = 20) based on cleavage of the caspase-3 substrate Ac-DEVD-AMC. Expressed as mean ± SEM. *P < 0.05 compared with healthy volunteers.

Figure 5. Release of GDH and mtDNA into blood is not a secondary effect of necrosis.

Mice were treated with APAP or furosemide (FS) to cause liver necrosis, and mitochondrial markers were measured in plasma from these animals near the peak of injury for each model (24 hours for FS and 12 hours for APAP). (A) Average GDH and ALT activity. (B) Average mtDNA concentration (Cyt c ox). Data are expressed as mean ± SEM for n = 3–6. *P < 0.05 versus control.

Figure 6. APAP and furosemide both cause centrilobular necrosis.

Mice were treated with APAP or furosemide to cause liver necrosis, and liver sections were stained with H&E near the peak of injury for each model (24 hours for FS and 12 hours for APAP). (A and B) Representative images of livers from untreated control mice or (C and D) 12 hours after 300 mg/kg APAP or (E and F) 24 hours after 400 mg/kg FS. Original magnification, ×50 (A, C, and E); ×100 (B, D, and F).

Figure 7. ALT activity and DNA fragments in plasma correlate with tissue injury.

Mice were treated with 300 mg/kg APAP. At various time points, plasma was collected for measurement of ALT and DNA fragments and tissues were collected for histology. (A) Time course of ALT activity in plasma from APAP-treated mice. (B) H&E sections of liver from APAP-treated mice. Necrosis is evident as pale eosinophilic staining and loss of hepatocyte nuclei. (C) Time course of DNA fragments in plasma from APAP-treated mice (closed circles) and of TUNEL staining in tissue (expressed as TUNEL-positive cells per high power field [HPF]). (D) Representative sections from TUNEL-stained livers over time. Original magnification, ×50. Data are expressed as mean ± SEM; n = 6 per time point.