Robust protein nitration contributes to acetaminophen-induced mitochondrial dysfunction and acute liver injury

Abstract

Acetaminophen (APAP), a widely used analgesic/antipyretic agent, can cause liver injury through increased nitrative stress, leading to protein nitration. However, the identities of nitrated proteins and their roles in hepatotoxicity are poorly understood. Thus, we aimed at studying the mechanism of APAP-induced hepatotoxicity by systematic identification and characterization of nitrated proteins in the absence or presence of an antioxidant, N-acetylcysteine (NAC). The levels of nitrated proteins markedly increased at 2h in mice exposed to a single APAP dose (350mg/kg ip), which caused severe liver necrosis at 24h. Protein nitration and liver necrosis were minimal in mice exposed to nontoxic 3-hydroxyacetanilide or animals co-treated with APAP and NAC. Mass-spectral analysis of the affinity-purified nitrated proteins identified numerous mitochondrial and cytosolic proteins, including mitochondrial aldehyde dehydrogenase, Mn-superoxide dismutase, glutathione peroxidase, ATP synthase, and 3-ketoacyl-CoA thiolase, involved in antioxidant defense, energy supply, or fatty acid metabolism. Immunoprecipitation followed by immunoblot with anti-3-nitrotyrosine antibody confirmed that the aforementioned proteins were nitrated in APAP-exposed mice but not in NAC-cotreated mice. Consistently, NAC cotreatment significantly restored the suppressed activity of these enzymes. Thus, we demonstrate a new mechanism by which many nitrated proteins with concomitantly suppressed activity promotes APAP-induced mitochondrial dysfunction and hepatotoxicity.

Fig. 1

Differential effects of APAP or AMAP on liver histology and ALT levels. Hematoxylin and eosin (H&E) stained liver slides for mice treated for 2 h are presented for (A) vehicle control (SHAM), (B) APAP, and (C) AMAP (magnification 100 ×). (D) Serum ALT levels for different groups are presented. *, significantly different (*p<0.05) from the control. A few necrotic regions in APAP-exposed mice are marked with broken lines (B) (n=4-6/group).

Fig. 2

Differential effects of APAP or AMAP on protein nitration. Hepatic mitochondrial (A) and cytosolic (B) proteins from mice exposed to the indicated agents for 2 h were subjected to immunoblot analysis with the specific anti-3-NT (upper panels), while specific anti-ATP synthase and actin were used to detect equal protein loading respectively (lower panels). Silver stained images of the affinity-purified nitrated proteins from mitochondria (C) and cytosol (D) are shown (n=4-6/group). Similar results to those shown in the Figures (A-D) were observed by at least two separate experiments.

Fig. 3

Summary of nitrated mitochondrial proteins in APAP-exposed mice. Some newly-identified nitrated mitochondrial proteins in APAP-exposed mouse liver are summarized with respect to the function of each protein identified by mass spectrometry. The five proteins written in bold and italic characters were further characterized for reversible nitration and enzyme activity changes following APAP exposure in the absence or presence of NAC co-treatment.

Fig. 4

Protective effects of NAC on APAP-mediated protein nitration and liver injury. The levels of nitrated proteins in mitochondria (A and B) and cytosol (C and D) from mice exposed to APAP ± NAC for 2 or 24 h, as indicated, were evaluated with the specific anti-3-NT (upper panels). Anti-ATP synthase and actin were used for equal loading for mitochondria and cytosolic fractions, respectively (lower panels). (E) Typical H&E-stained liver slides are presented for APAP-exposed mice with or without NAC co-treatment, as indicated (magnification 100 ×). (F) Serum ALT levels for the indicated groups are presented. *, significantly different (*p<0.05) from other groups (n=4-6/group). Severe necrotic regions of APAP-exposed samples are marked with broken lines (E).

Fig. 5

Reversible changes of nitration and activity of ALDH2 or thiolase. The levels of mitochondrial ALDH2 (A) or thiolase (D) in mice exposed to APAP ± NAC for 2 or 24 h were determined by immunoblot analysis (top panels) while the level of ATP synthase was used to demonstrate similar protein loading (lower panels). Numbers above the panels A and D represent densitometric values compared to those of sham group, which was set at 1. (B) Mitochondrial proteins from the indicated groups were immunoprecipitated with anti-ALDH2 (B) or anti-thiolase (E) antibody. The immunoprecipitated proteins were subjected to immunoblot analysis with anti-3-NT antibody (upper panels in B and E) and anti-ALDH2 (B) or anti-thiolase (E) antibody (bottom). ALDH2 (C) or thiolase (F) activity in mitochondrial extracts was determined for the indicated samples. *, significantly different from sham group; ^&significantly different from corresponding APAP+NAC; ^#significantly different from other groups (n=4-6/group), (*p<0.05).

Fig. 6

Reversible changes of nitration and activity of ATP synthase. (A) The levels of mitochondrial ATP synthase in APAP-exposed mice ± NAC for 2 or 24 h were determined by immunoblot analysis. (B) Mitochondrial proteins from the indicated groups were immunoprecipitated using the specific anti-ATP synthase antibody. The immunoprecipitated proteins were then subjected to immunoblot analysis with anti-3-NT antibody (upper panel) or anti-ATP synthase antibody (bottom). (C and D) Mitochondrial ATP synthase activity and levels were determined for the indicated samples, respectively. ^#, significantly different (*p<0.05) from other groups (n=4-6/group).

Fig. 7

Reversible changes of nitration and activity of SOD2 or Gpx. The levels of mitochondrial SOD2 (A) or Gpx (D) in APAP-exposed mice ± NAC for 2 or 24 h were determined by immunoblot analysis (top panels). ATP synthase levels were used to show similar protein loading (lower panels). Numbers above the panels A and D respresent densitometric values compared to those of sham group which was set at 1. (B) Mitochondrial proteins from the indicated groups were immunoprecipitated with anti-SOD2 (B) or anti-Gpx (E) antibody. The immunoprecipitated proteins were then subjected to immunoblot analysis with anti-3-NT antibody (upper panels in B and E) and anti-SOD2 (B) or anti-Gpx (E) antibody (bottom). SOD2 (C) or Gpx (F) activity in mitochondrial extracts was determined for the indicated samples. *, significantly different from sham group; ^&significantly different from corresponding APAP+NAC; ^# significantly different from other groups (n=4-6/group), (*p<0.05).