Apoptosis-inducing factor modulates mitochondrial oxidant stress in acetaminophen hepatotoxicity

Abstract

Acetaminophen (APAP) overdose causes liver injury in humans and mice. DNA fragmentation is a hallmark of APAP-induced cell death, and nuclear translocation of apoptosis-inducing factor (AIF) correlates with DNA fragmentation after APAP overdose. To test the hypothesis that AIF may be a critical mediator of APAP-induced cell death, fasted male AIF-deficient Harlequin (Hq) mice and respective wild-type (WT) animals were treated with 200 mg/kg APAP. At 6 h after APAP, WT animals developed severe liver injury as indicated by the increase in plasma alanine aminotransferase (ALT) activities (8600 ± 1870 U/l) and 61 ± 8% necrosis. This injury was accompanied by massive DNA strand breaks in centrilobular hepatocytes (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling [TUNEL] assay) and release of DNA fragments into the cytosol (anti-histone ELISA). In addition, there was formation of reactive oxygen (increase in liver glutathione disulfide (GSSG) levels and mitochondrial protein carbonyls) and peroxynitrite (nitrotyrosine [NT] staining) together with mitochondrial translocation of activated c-jun-N-terminal kinase (P-JNK) and release of AIF from the mitochondria. In contrast, Hq mice had significantly less liver injury (ALT: 330 ± 130 U/l; necrosis: 4 ± 2%), minimal nuclear DNA damage, and drastically reduced oxidant stress (based on all parameters) at 6 h. WT and Hq mice had the same baseline levels of cyp2E1 and of glutathione. The initial depletion of glutathione (20 min after APAP) was the same in both groups suggesting that there was no relevant difference in metabolic activation of APAP. Thus, AIF has a critical function in APAP hepatotoxicity by facilitating generation of reactive oxygen in mitochondria and, after nuclear translocation, AIF can be involved in DNA fragmentation.

FIG. 1.

Hepatic expression of AIF and cytochrome P450 2E1 in WT and AIF-deficient (Hq) mice. AIF (A) and cyp2e1 (B) levels were determined by western blot analysis. Densitometric analysis was performed in three animals per group. In addition, animals were either untreated or injected with 200 mg/kg APAP, liver samples were obtained between 0 and 60 min and the glutathione levels were analyzed (C). All data are expressed as mean ± SE of four animals per group or time point. *p < 0.05 (compared with WT or controls).

FIG. 2.

APAP-induced liver injury in WT and AIF-deficient (Hq) mice. (A) Representative H&E-stained sections are shown (×200). (B) Plasma ALT activities and the area of necrosis were measured as indicators for liver injury in untreated controls (C) or 6 h after administration of 200 mg/kg APAP. Data represent means ± SE of n = 6 animals per group. *p < 0.05 (compared with controls), **p < 0.05 (compared with WT/APAP).

FIG. 3.

Effect of APAP on hepatic glutathione content in WT and AIF-deficient (Hq) mice. Glutathione (GSH + GSSG) (Panel A) and GSSG (B) were measured in untreated controls (C) and 6 h after administration of 200 mg/kg APAP. The GSSG:GSH ratio was calculated for each sample (C). Data represent means ± SE of six animals per group. *p < 0.05 (compared with controls), **p < 0.05 (compared with WT/APAP).

FIG. 4.

APAP-induced oxidant and nitrosative stress in WT and AIF-deficient (Hq) mice. Protein carbonyls (A) were measured in hepatic mitochondria and liver sections were stained for NT protein adducts (B) at 6 h after 200 mg/kg APAP. For protein carbonyls data represent means ± SE of three animals per group. For NT staining, representative sections are shown (×100). *p < 0.05 (compared with controls), **p < 0.05 (compared with WT/APAP).

FIG. 5.

Translocation of cytosolic proteins to mitochondria, release of mitochondrial intermembrane proteins, and nuclear DNA fragmentation in WT and AIF-deficient (Hq) mice. (A) Western blot analysis of P-JNK and Bax in the mitochondria and AIF, endonuclease G, Smac, and cytochrome c in the cytosol. DNA strandbreaks (TUNEL Assay) (B), and DNA fragmentation (antihistone ELISA) (C) were determined. Samples were obtained from WT and AIF-deficient (Hq) mice either untreated (C) or 6 h after treatment with 200 mg/kg APAP. Representative animals (n = 3 per group) are shown for the western blots and representative tissue sections are shown for the TUNEL assay. DNA fragmentation data represent means ± SE of six animals per group. *p < 0.05 (compared with controls), **p < 0.05 (compared with WT/APAP).

FIG. 6.

APAP-induced liver injury in WT and AIF-deficient (Hq) mice. DNA strand breaks (TUNEL assay) (A) and plasma ALT activities and the area of necrosis (B) were determined as indicators of APAP-induced liver injury in untreated controls (C) or 24 h after administration of 200 mg/kg AAP. Data represent means ± SE of five animals per group. *p < 0.05 (compared with controls), **p < 0.05 (compared with APAP/WT).

Fig. 7.

APAP-induced PCNA expression in WT and AIF-deficient (Hq) mice. (A) Western blot analysis of PCNA levels in liver homogenates from WT and Hq mice 24 h after treatment with 200 mg/kg APAP. (B) Densitometric analysis of the PCNA western blot. Data represents mean ± SE of four animals per group.