Acetaminophen-treated Mice Research Articles

Mechanism of acetaminophen-induced hepatotoxicity: covalent binding versus oxidative stress.

The hepatotoxicity of acetaminophen is believed to be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine; however, the mechanism by which this metabolite produces the toxicity is unknown. The metabolite, which is both an electrophile and an oxidizing agent, may covalently bind to critical proteins, or it may initiate oxidative damage. We have previously developed a Western blot assay for detection of acetaminophen covalently bound to protein and have reported the relationship between covalent binding and the development of hepatotoxicity. Recently, we developed a Western blot assay for protein aldehyde formation, which may occur via the reactive oxygen species, the hydroxyl radical. In this paper, we have compared covalent binding to protein aldehyde formation. Toxic doses of acetaminophen (400 mg/kg) were administered to mice, and the mice were subsequently killed at 0, 1, 2, 4, and 6 h. Since the oxidizing agent FeSO4 has been reported to potentiate lipid peroxidation when administered with acetaminophen, other mice received FeSO4 (100 mg/kg) plus acetaminophen. Compared to saline-treated control mice, acetaminophen treatment significantly increased serum alanine aminotransferase levels, an index of hepatotoxicity, at 4 and 6 h, but not at 1 or 2 h. Acetaminophen plus FeSO4 treatment of mice significantly increased serum alanine aminotransferase levels at 2, 4, and 6 h compared to controls. Levels of alanine aminotransferase in serum of acetaminophen plus ferrous sulfate-treated mice were higher at 4 and 6 h than those of acetaminophen-treated mice, but not significantly different. FeSO4 alone did not increase alanine aminotransferase levels. Western blot assays revealed that acetaminophen did not cause an increase in protein aldehydes over control at any time, nor did acetaminophen plus FeSO4; however, FeSO4 alone increased the intensity of staining of the immunoblot for protein aldehydes over control at all times after 0 time. Acetaminophen-protein adducts were detected in acetaminophen- and acetaminophen plus FeSO4-treated mice. In vitro experiments indicated that FeSO4 plus tert-butyl hydroperoxide in the presence of bovine serum albumin increased protein aldehyde formation. Inclusion of acetaminophen in the incubation mixture inhibited protein oxidation of bovine serum albumin in a concentration dependent manner. The data indicate that acetaminophen quenches protein oxidation, presumably by reacting with the hydroxyl radical. These data are consistent with the theory that acetaminophen covalent binding is the primary mechanism of toxicity and argue against a role for protein oxidation in acetaminophen hepatotoxicity.

Immunoblot analysis of protein containing 3-(cystein- S-yl)acetaminophen adducts in serum and subcellular liver fractions from acetaminophen-treated mice

The hepatotoxicity of acetaminophen is believed to be mediated by the metabolic activation of acetaminophen to N-acetyl- p-benzoquinone imine which covalently binds to cysteinyl residues on proteins as 3-(cystein- S-yl)acetaminophen adducts. The formation of these adducts in hepatic protein correlates with the hepatotoxicity. In this study the formation of 3-(cystein- S-yl)acetaminophen adducts in specific cellular proteins was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected using affinity-purified antisera specific for 3-(cystein- S-yl)acetaminophen adducts on immunoblots. These techniques were used to investigate the liver 10,000 g supernatant and serum from B6C3F1 mice that received hepatotoxic doses of acetaminophen. More than 15 proteins containing 3-(cystein- S-yl)acetaminophen adducts were detected in the liver 10,000 g supernatant. The most prominent protein containing 3-(cystein- S-yl)acetaminophen adducts in the hepatic 10,000 g supernatant had a relative molecular mass of 55 kDa. Serum proteins containing 3-(cystein- S-yl)acetaminophen adducts had molecular masses similar to those found in the liver 10,000 g supernatant (55, 87, and approximately 102 kDa). These data, combined with our previous findings describing the temporal relationship between the appearance of 3-(cystein- S-yl)acetaminophen adducts in protein in the serum and the decrease in the levels of 3-(cystein- S-yl)acetaminophen adducts in protein in the liver, suggested that liver adducts were released into the serum following lysis of hepatocytes. The temporal relationship between the formation of specific adducts and hepatotoxicity in mice following a hepatotoxic dose of acetaminophen was examined using immunoblots of mitochondria, microsomes, cytosol, and plasma membranes. Hepatotoxicity indicated by serum alanine aminotransferase levels was increased at 2 and 4 hr after dosing. The cytosolic fraction contained numerous proteins with 3-(cystein- S-yl)acetaminophen adducts, the most intensely stained of which was a 55-kDa protein. 3-(Cystein- S-yl)acetaminophen adducts were detected in the 55-kDa liver protein 30 min after dosing and prior to the development of significant toxicity. Examination of gels suggested that maximal levels of immunochemically detectable adducts in the 55-kDa protein occurred at 1–2 hr, with a decrease in intensity 4 hr after dosing. The presence of 3-(cystein- S-yl)acetaminophen adducts in proteins prior to hepatotoxicity suggests a threshold for adduct formation in the development of toxicity. Protein in microsomes which contained 3-(cystein- S-yl)acetaminophen adducts ranged in molecular weight from 38 to approximately 106 kDa. The major proteins containing 3-(cystein- S-yl)acetaminophen adducts in the mitochondria had molecular masses of 39, 50, 68, and 79 kDa. The plasma membranes consisted of four major proteins containing 3-(cystein- S-yl)acetaminophen adducts with approximate molecular masses of 72, 82, 115, and 118 kDa. The presence of electrophoretically distinct acetaminophen adducts may be important to the mechanism of acetaminophen hepatotoxicity.

Acetaminophen-induced alterations in pancreatic β cells and serum insulin concentrations in B6C3F1 mice

Administration of acetaminophen (500 mg/kg) to male B6C3F1 mice resulted in alterations of pancreatic β cell ultrastructure. These alterations were characterized by pronounced intercellular spaces, cytoplasmic vacuolization, damaged membranes of cytoplasm, secretory granules, and other organelles, and pyknotic nuclei with disrupted membranes. Concomitant with these changes, acetaminophen also caused increases in serum insulin concentrations from 24 μU/ml at 0 time to 160 μU/ml at 8 hr and increases in serum alanine aminotransferase (ALT) concentrations from 42 to 13,279 U/liter, which indicated hepatic damage. Quantitation of 3-(cystein- S-yl)acetaminophen adducts in hepatic 10,000 g supernatant protein using a particle concentration fluorescence immunochemical assay indicated a positive correlation between binding and the occurrence of the hepatotoxicity consistent with that has been previously reported; however, 3-(cystein- S-yl)acetaminophen protein adducts were not detected in pancreatic 10,000 g supernatant. Immunohistochemical analysis of the liver and pancreas from acetaminophen-treated mice revealed acetaminophen-protein adducts in the centrilobular regions of the liver but not in the pancreatic islets. Doses of 100 and 200 mg/kg produced no evidence of hepatotoxicity and no increase in serum insulin; 300 mg/kg and higher doses produced both hepatotoxicity and increased serum insulin concentrations. A comparison of the time course for the increase in serum levels of ALT and insulin following a toxic dose of acetaminophen indicated that the increase in ALT preceded the increase in insulin. Thus the hepatotoxicity of acetaminophen correlates with the formation of 3-(cystein- S-yl)acetaminophen protein adducts in liver, which supports the concept that this toxicity is mediated by the reactive metabolite N-acetyl- p-benzoquinone imine; however, the toxicity of acetaminophen to β cells in the pancreas is apparently not mediated by this mechanism.

Direct protection against acetaminophen hepatotoxicity by propylthiouracil. In vivo and in vitro studies in rats and mice.

Hepatotoxicity caused by acetaminophen can be prevented by enzyme-catalyzed conjugation of its reactive metabolite with glutathione (GSH). Since we have shown in previous studies that 6-N-propyl-2-thiouracil (PTU) can substitute for GSH as a substrate for the GSH S-transferases, we examined the possibility that PTU might also protect against acetaminophen hepatotoxicity by direct chemical interaction with the reactive metabolite of acetaminophen. In an in vitro system consisting of [(3)H]acetaminophen, liver microsomes from phenobarbital-pretreated rats, and an NADPH-generating system, we found that PTU had a dose-dependent additive effect with GSH on inhibition of acetaminophen covalent binding. PTU administration also resulted in a dose-dependent decrease in both GSH depletion and covalent binding in vivo in acetaminophen-treated mice. To examine the possible mechanisms by which PTU exerts its protective effect, we studied the action of PTU on both acetaminophen conjugation and metabolic activation. PTU had no effect upon acetaminophen pharmacokinetics in phenobarbital-pretreated rats, as examined by measuring acetaminophen concentration in bile, urine, and blood after an intraperitoneal dose, nor did it alter the total amount of polar conjugates formed. Microsomes from PTU-treated rats were unaltered in cytochrome P-450 concentrations and p-nitroanisole-O-demethylase, benzo-alpha-pyrene hydroxylase, and cytochrome c-reductase activities. Furthermore PTU did not decrease acetaminophen-GSH adduct formation in vitro, suggesting that there was no reduction in drug activation. However, in bile from [(35)S]PTU and [(3)H]acetaminophen treated rats, as well as in incubates of the two drugs with liver microsomes, a new (35)S- and (3)H-containing product could be identified. By both thin layer chromatography and high pressure liquid chromatography this new product, which co-eluted with [(3)H]acetaminophen, was separated from unreacted [(35)S]PTU. The formation of this product in vitro was a function of PTU concentration and reached a maximum of 0.06 mumol/min per mg protein at 0.5 mM PTU. In vivo, the total biliary excretion of this product over 4 h (116 nmol) equaled the net reduction in acetaminophen metabolite covalent binding in the liver of phenobarbital-pretreated rats (108 nmol). We conclude that PTU, independent of its antithyroid effect, diminishes hepatic macromolecular covalent binding of acetaminophen reactive metabolite both in vivo and in vitro, and it does so by detoxifying the reactive metabolite through direct chemical interaction in a manner similar to GSH. These observations may define the mechanism by which PTU is protective against liver injury caused by acetaminophen.

Hepatic glutathione content and aryl hydrocarbon hydroxylase activity of acetaminophen-treated mice as a function of age.

Glutathione (GSH) plays an important role in cells as an antioxidant, and participates in the detoxification of drugs and foreign chemicals. The effect of age on hepatic glutathione (GSH) levels and aryl hydrocarbon hydroxylase (AHH) activity and the ability of these mice to respond to the acute administration of acetaminophen (AAP) has been studied. Female Swiss Webster mice at ages 3, 9, and 18 mo were treated i.p. with 500 mg/kg AAP, and hepatic GSH content and AHH activity were determined at 0, 0.5, 2, 4, 6, 8, 10, 12, and 24 hrs. The hepatic GSH levels and the AHH activity in 18 mo old mice were 43% and 36% lower, respectively, as compared to the 3 mo old animals. Maximum AAP-induced depletion of GSH occurred 2-4 hrs after treatment. Only in 18 mo animals did GSH fail to return to or exceed control levels 24 hr after AAP administration. Maximum decrease in hepatic AHH activity occurred in all age groups at 10 hr post-AAP administration, and had not returned to control values 24 hr after treatment. The results support the hypothesis that a decrease in GSH and mixed function oxidases may contribute to changes associated with aging as well as to the increased susceptibility to disease and drugs which occur with advanced age.