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

Abstract

Acetaminophen (APAP), a widely-used analgesic 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 anti-oxidant N-acetylcysteine (NAC). The levels of nitrated proteins markedly increased at 2 h in mice exposed to a single APAP dose (350 mg/kg ip), which caused severe liver necrosis at 24 h. 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 anti-oxidant defense, energy supply, and fatty acid metabolism, respectively. Immunoprecipitation followed by immunoblot with anti-3-NT antibody confirmed that the aforementioned proteins were nitrated in APAP-exposed mice but not in NAC-co-treated mice. Consistently, NAC co-treatment significantly restored the suppressed activities of these enzymes. Thus, we demonstrate a new mechanism by which many nitrated proteins with concomitantly suppressed activities promotes APAP-induced mitochondrial dysfunction and hepatotoxicity.

Binge alcohol promotes hypoxic liver injury through a CYP2E1–HIF-1α-dependent apoptosis pathway in mice and humans

“…Particularly, significant relationships among BAC, CYP2E1, HIF-1α andiNOS provide evidence about the involvement of HIF-1α in binge alcohol-induced iNOS induction and subsequent events such as protein nitration under hypoxia, at least partially, in binge alcohol-exposed humans and mice. Although not conducted in this study, it is likely that increased protein nitration, that is likely mediated by the increased CYP2E1 and iNOS protein levels, in different sub-cellular fractions might be also involved in inactivation of their functions, contributing to mitochondrial dysfunction, endoplasmic reticulum stress, and apoptosis in alcoholexposure, as recently demonstrated [17,52,53]. …”

Functional Roles of Protein Nitration in Acute and Chronic Liver Diseases

“…Using this method of immunoaffinity purification, we were able to identify more than 30 and 70 nitrated cytosolic and mitochondrial proteins, respectively, in APAP-treated mouse liver. Detection and functional consequences of nitration in five selected mitochondrial proteins (Figure 2 and Table 1) were confirmed via immunoprecipitation followed by immunoblot analysis with anti-3-NT antibody and activity measurements in the absence or presence of an antioxidant, which fully prevented APAP-mediated protein nitration and hepatic damage [35]. Tyr-nitrated peptides of many proteins including SOD2 in APAP-exposed mice could not be identified by the MS analysis.…”

Nitric oxide in liver diseases

“…Acetaminophen was also reported to inhibit the activity of various mitochondrial enzymes through nitration, including aldehyde dehydrogenase, Mn-SOD (SOD2), glutathione peroxidase, ATP synthase, and 3-ketoacyl-CoA thiolase, which are involved in antioxidant defense, energy supply, or fatty acid metabolism [136]. …”