Jack A. Hinson

High doses of acetaminophen (APAP) result in hepatotoxicity that involves metabolic activation of the parent compound, covalent binding of the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI) to liver proteins, and depletion of hepatic glutathione. Impaired fatty acid β-oxidation has been implicated in previous studies of APAP-induced hepatotoxicity. To better understand relationships between toxicity and fatty acid β-oxidation in the liver in APAP toxicity, metabolomic assays for long chain acylcarnitines were examined in relationship to established markers of liver toxicity, oxidative metabolism, and liver regeneration in a time course study in mice. Male B6C3F1 mice were treated with APAP (200 mg/kg IP) or saline and sacrificed at 1, 2, 4, 8, 24 or 48 h after APAP. At 1 h, hepatic glutathione was depleted and APAP protein adducts were markedly increased. Alanine aminotransferase (ALT) levels were elevated at 4 and 8 h, while proliferating cell nuclear antigen (PCNA) expression, indicative of hepatocyte regeneration, was apparent at 24 h and 48 h. Elevations of palmitoyl, oleoyl and myristoyl carnitine were apparent by 2–4 h, concurrent with the onset of Oil Red O staining in liver sections. By 8 h, acylcarnitine levels were below baseline levels and remained low at 24 and 48 h. A partial least squares (PLS) model suggested a direct association of acylcarnitine accumulation in serum to APAP protein adduct and hepatic glutathione levels in mice. Overall, the kinetics of serum acylcarnitines in APAP toxicity in mice followed a biphasic pattern involving early elevation after the metabolism phases of toxicity and later depletion of acylcarnitines.

In overdose acetaminophen (APAP) is hepatotoxic. Toxicity occurs by metabolism to N-acetyl-p-benzoquinone imine, which depletes GSH and covalently binds to proteins followed by protein nitration. Nitration can occur via the strong oxidant and nitrating agent peroxynitrite, formed from superoxide and nitric oxide (NO). In hepatocyte suspensions we reported that an inhibitor of neuronal nitric-oxide synthase (nNOS; NOS1), which has been reported to be in mitochondria, inhibited toxicity and protein nitration. We recently showed that manganese superoxide dismutase (MnSOD; SOD2) was nitrated and inactivated in APAP-treated mice. To understand the role of nNOS in APAP toxicity and MnSOD nitration, nNOS knockout (KO) and wild-type (WT) mice were administered APAP (300 mg/kg). In WT mice serum alanine aminotransferase (ALT) significantly increased at 6 and 8 h, and serum aspartate aminotransferase (AST) significantly increased at 4, 6 and 8 h; however, in KO mice neither ALT nor AST significantly increased until 8 h. There were no significant differences in hepatic GSH depletion, APAP protein binding, hydroxynonenal covalent binding, or histopathological assessment of toxicity. The activity of hepatic MnSOD was significantly lower at 1 to 2 h in WT mice and subsequently increased at 8 h. MnSOD activity was not altered at 0 to 6 h in KO mice but was significantly decreased at 8 h. There were significant increases in MnSOD nitration at 1 to 8 h in WT mice and 6 to 8 h in KO mice. Significantly more nitration occurred at 1 to 6 h in WT than in KO mice. MnSOD was the only observed nitrated protein after APAP treatment. These data indicate a role for nNOS with inactivation of MnSOD and ALT release during APAP toxicity.

In overdose the analgesic/antipyretic acetaminophen (APAP) is hepatotoxic. Toxicity is mediated by initial hepatic metabolism to N-acetyl-p-benzoquinone imine (NAPQI). After low doses NAPQI is efficiently detoxified by GSH. However, in overdose GSH is depleted, NAPQI covalently binds to proteins as APAP adducts, and oxygen/nitrogen stress occurs. Toxicity is believed to occur by mitochondrial dysfunction. Manganese superoxide dismutase (MnSOD) inactivation by protein nitration has been reported to occur during other oxidant stress-mediated diseases. MnSOD is a critical mitochondrial antioxidant enzyme that prevents peroxynitrite formation within the mitochondria. To examine the role of MnSOD in APAP toxicity, mice were treated with 300 mg/kg APAP. GSH was significantly reduced by 65% at 0.5 h and remained reduced from 1 to 4 h. Serum alanine aminotransferase did not significantly increase until 4 h and was 2290 IU/liter at 6 h. MnSOD activity was significantly reduced by 50% at 1 and 2 h. At 1 h, GSH was significantly depleted by 62 and 80% at nontoxic doses of 50 and 100 mg/kg, respectively. No further GSH depletion occurred with hepatotoxic doses of 200 and 300 mg/kg APAP. A dose response decrease in MnSOD activity was observed for APAP at 100, 200, and 300 mg/kg. Immunoprecipitation of MnSOD from livers of APAP-treated mice followed by Western blot analysis revealed nitrated MnSOD. APAP-MnSOD adducts were not detected. Treatment of recombinant MnSOD with NAPQI did not produce APAP protein adducts. The data indicate that MnSOD inactivation by nitration is an early event in APAP-induced hepatic toxicity.

Acetaminophen (APAP) toxicity in primary mouse hepatocytes occurs in two phases. The initial phase (0−2 h) occurs with metabolism to N-acetyl-p-benzoquinoneimine which depletes glutathione, and covalently binds to proteins, but little toxicity is observed. Subsequent washing of hepatocytes to remove APAP and reincubating in media alone (2−5 h) results in toxicity. We previously reported that the reincubation phase occurs with mitochondrial permeability transition (MPT) and increased oxidative stress (dichlorodihydrofluorescein fluorescence) (DCFH₂). Since DCFH₂ may be oxidized by multiple oxidative mechanisms, we investigated the role of reactive nitrogen species (RNS) leading to 3-nitrotyrosine in proteins by ELISA and by immunoblots. Incubation of APAP with hepatocytes for 2 h did not result in toxicity or protein nitration; however, washing hepatocytes and reincubating in media alone (2−5 h) resulted in protein nitration which correlated with toxicity. Inclusion of the MPT inhibitor, cyclosporine A, in the reincubation media eliminated toxicity and protein nitration. The general nitric oxide synthase (NOS) inhibitor L-NMMA and the neuronal NOS (NOS1) inhibitor, 7-nitroindazole, added in the reincubation media decreased toxicity and protein nitration; however, neither the inducible NOS (NOS2) inhibitors L-NIL (N6-(1-iminoethyl)-l-lysine) nor SAIT (S-(2-aminoethyl)isothiourea) decreased protein nitration or toxicity. The RNS scavengers, N-acetylcysteine, and high concentrations of APAP, added in the reincubation phase decreased toxicity and protein nitration. 7-Nitroindazole and cyclosporine A inhibited the APAP-induced loss of mitochondrial membrane potential when added in the reincubation phase. The data indicate a role for RNS in APAP induced toxicity.

Although considered safe at therapeutic doses, at higher doses, acetaminophen produces a centrilobular hepatic necrosis that can be fatal. Acetaminophen poisoning accounts for approximately one-half of all cases of acute liver failure in the United States and Great Britain today. The mechanism occurs by a complex sequence of events. These events include: (1) CYP metabolism to a reactive metabolite which depletes glutathione and covalently binds to proteins; (2) loss of glutathione with an increased formation of reactive oxygen and nitrogen species in hepatocytes undergoing necrotic changes; (3) increased oxidative stress, associated with alterations in calcium homeostasis and initiation of signal transduction responses, causing mitochondrial permeability transition; (4) mitochondrial permeability transition occurring with additional oxidative stress, loss of mitochondrial membrane potential, and loss of the ability of the mitochondria to synthesize ATP; and (5) loss of ATP which leads to necrosis. Associated with these essential events there appear to be a number of inflammatory mediators such as certain cytokines and chemokines that can modify the toxicity. Some have been shown to alter oxidative stress, but the relationship of these modulators to other critical mechanistic events has not been well delineated. In addition, existing data support the involvement of cytokines, chemokines, and growth factors in the initiation of regenerative processes leading to the reestablishment of hepatic structure and function.

The role of mitochondrial permeability transition (MPT) and oxidative stress in chloroform toxicity was determined in freshly isolated female B6C3F1 mouse hepatocytes. Incubation of chloroform (12 mM) with hepatocytes resulted in cell death (alanine aminotransferase release and propidium iodide fluorescence). Chloroform had volatilized from the incubation and glutathione was depleted by 1 h; however, toxicity was not significantly different between control and chloroform-incubated cells. Hepatocytes were washed and reincubated in fresh media at 1 h. Subsequent reincubation of chloroform-treated hepatocytes resulted in significant toxicity at 3–5 h. Inclusion of the MPT inhibitor cyclosporine A or the antioxidant N-acetylcysteine (NAC) in the reincubation media at 1 h prevented toxicity. Confocal microscopy studies with the dye calcein AM indicated MPT that was blocked by cyclosporine A or NAC. Fluorescence microscopy studies utilizing JC-1 indicated loss of mitochondrial membrane potential, which was also blocked by cyclosporine A or NAC. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress, and the increase was blocked by cyclosporine A. Since oxidative stress may occur by peroxynitrite, its role in toxicity was examined. Either of the nitric oxide synthase inhibitors N ^G-methyl-L-arginine (L-NMMA) and 7-nitroindazole (7-NI) at 1 h blocked toxicity. Western blot analysis of hepatocytes for 3-nitrotyrosine in proteins, a biomarker of peroxynitrite, indicated one major nitrated protein at 81 kD. Nitration of this protein was inhibited by cyclosporine A, L-NMMA, 7-NI, or NAC. The data indicate that chloroform-induced cell death occurs in two phases: a metabolic phase characterized by glutathione depletion, and an oxidative phase characterized by MPT and protein nitration.