Inhibition of carbamyl phosphate synthetase-I and glutamine synthetase by hepatotoxic doses of acetaminophen in mice

Abstract

The primary mechanisms proposed for acetaminophen-induced hepatic necrosis should deplete protein thiols, either by covalent binding and thioether formation or by oxidative reactions such as S-thiolations. However, in previous studies we did not detect significant losses of protein thiol contents in response to administration of hepatotoxic doses of acetaminophen in vivo. In the present study we employed derivatization with the thiol-specific agent monobromobimane and separation of proteins by SDS-PAGE to investigate the possible loss of specific protein thiols during the course of acetaminophen-induced hepatic necrosis. Fasted adult male mice were given acetaminophen, and protein thiol status was examined subsequently in subcellular fractions isolated by differential centrifugation. No decreases in protein thiol contents were indicated, with the exception of a marked decrease in the fluorescent intensity, but not of protein content, as indicated by staining with Coomassie blue, of a single band of approximately 130 kDa in the mitochondrial fractions of acetaminophen-treated mice. This protein was identified by isolation and N-terminal sequence analysis as carbamyl phosphate synthetase-I (CPS-I) (EC 6.3.4.16). Hepatic CPS-I activities were decreased in mice given hepatotoxic doses of acetaminophen. In addition, hepatic glutamine synthetase activities were lower, and plasma ammonia levels were elevated in mice given hepatotoxic doses of acetaminophen. The observed hyperammonemia may contribute to the adverse effects of toxic doses of acetaminophen, and elucidation of the specific mechanisms responsible for the hyperammonemia may prove to be useful clinically. However, the preferential depletion of protein thiol content of a mitochondrial protein by chemically reactive metabolites generated in the endoplasmic reticulum presents a challenging and potentially informative mechanistic question.

FIG. 1

Hepatic protein thiols in subcellular fractions 2 hr (A) or 6 hr (B) after acetaminophen. Subcellular fractions were isolated by differential centrifugation (nuclear, NUC; mitochondrial, MITO; microsomal, MICRO; soluble, SOL) after intraperitoneal administration of 400 mg/kg of acetaminophen (AP) or equal volumes of saline to controls (CO). The fractions were treated with mBBr and the proteins were separated by one-dimensional SDS–PAGE as described under Materials and Methods. The fluorescent bands reflect derivatization of the proteins with the thiol-selective reagent. The electrophoretic mobility of the molecular mass standards is indicated on the left. The arrow denotes the 130-kDa protein band.

FIG. 2

Hepatic protein thiols in mitochondrial fractions 2 hr (lanes 1–4) and 6 hr (lanes 5–8) after acetaminophen. Subcellular fractions were isolated by differential centrifugation after intraperitoneal administration of 400 mg/kg of acetaminophen (AP) or equal volumes of saline to controls (CO). The fractions were treated with mBBr and the proteins were separated by one-dimensional SDS–PAGE as described under Materials and Methods. The fluorescent bands reflect derivatization of the proteins with the thiol-selective reagent. The electrophoretic mobility of the molecular mass standards is indicated on the left. The arrow denotes the 130-kDa protein band.

FIG. 3

Ponceau-S-stained gel pattern of the mitochondrial fractions 2 hr (lanes 1–4) and 6 hr (lanes 5–8) after acetaminophen. Mitochondrial fractions shown in Fig. 2 were separated on one-dimensional SDS–PAGE, electroblotted onto PVDF membranes, and stained with Ponceau-S, as described under Materials and Methods. No differences are evident in the intensities of the protein bands at 130 kDa (arrow). Similarly, examination of gels from these fractions stained with Coomassie blue showed no difference in intensities of this protein (not shown). The differences in band intensity shown in Fig. 2 are therefore less likely to be due to loss of this protein than to loss of thiol content by the protein.

FIG. 4

Coomassie blue-stained gel of the 130-kDa protein isolated for characterization. The protein band at 130 kDa was isolated from a gel after one-dimensional SDS–PAGE of a mitochondrial fraction as described under Materials and Methods. The eluted protein was subjected to a second one-dimensional SDS–PAGE separation and stained with Coomassie blue. Molecular weight standards are on the left.

FIG. 5

Dose-dependent inhibition of hepatic CPS-I by acetaminophen in mice. Livers were collected from mice treated with saline or acetaminophen and homogenized, and CPS-I activities were determined as described under Materials and Methods. Data are means ± SEM, n = 7–10 per group. *CPS-I activities in mice given 200 mg/kg or more of acetaminophen were different from activities in the control group by one-way ANOVA, with Student–Newman–Keuls, p < 0.05. In addition, the activities in the animals treated with 200, 300, or 400 mg/kg were different from each other.

FIG. 6

Dose-dependent inhibition of hepatic glutamine synthetase by acetaminophen in mice. Livers were collected from the mice described in the legend to Fig. 5 and homogenized, and glutamine synthetase activities were determined as described under Materials and Methods. Data are means ± SEM, n = 7–10 per group. *Activities in mice given 300 or 400 mg/kg of acetaminophen were different from activities in the control group by one-way ANOVA, with Student–Newman–Keuls, p < 0.05, but were not different from each other.

FIG. 7

Dose-dependent hyperammonemia caused by acetaminophen in mice. Ammonia concentrations of plasma samples collected from the mice described in the legend to Fig. 5 were measured as described under Materials and Methods. Data are means ± SEM, n = 7–10 per group. *Ammonia concentrations in mice given 300 or 400 mg/kg of acetaminophen were different from levels in the other groups by one-way ANOVA, with Student–Newman–Keuls, p < 0.05, but were not different from each other.

FIG. 8

Dose-dependent hepatic injury by acetaminophen in mice. Plasma was collected from anesthetized animals 6 hr after dosing, and ALT activities were determined as described under Materials and Methods. Data are means ± SEM, n = 7–10 per group. *Different from respective control group by one way ANOVA, with Student–Newman–Keuls, p < 0.05.

FIG. 9

Time course of acetaminophen-induced inhibition of hepatic CPS-I. Male ICR mice were fasted 18 hr prior to intraperitoneal administration of 400 mg/kg of acetaminophen in saline (AP) or equal volumes of saline (CONTROL). Livers were collected from anesthetized animals 2, 4, or 6 hr after dosing and homogenized, and mitochondrial fractions were separated by differential centrifugation. CPS-I activities were determined as described under Materials and Methods. Data are means ± SEM, n = 6–10 per group. *Different from respective control group by ANOVA Newman–Keuls, p < 0.05.

FIG. 10

Time course of acetaminophen-induced inhibition of hepatic glutamine synthetase. Glutamine synthetase (GS) activities in livers obtained from the acetaminophen or saline-treated animals described in the legend to Fig. 9 were measured as described under Materials and Methods. Data are means ± SEM, n = 6–10 per group. *Different from respective control group by ANOVA Newman–Keuls, p < 0.05.

FIG. 11

Time course of acetaminophen-induced hyperammonemia in mice. Plasma ammonia levels were measured in mice treated with acetaminophen or saline, as described in Fig. 9. Data are means ± SEM, n = 6–10 per group. *Different from respective control group by ANOVA Newman–Keuls, p < 0.05.

FIG. 12

Time course of acetaminophen-induced hepatic injury. Plasma was obtained from the animals treated with acetaminophen or saline as described in the legend to Fig. 9, and ALT activities were determined. Data are means ± SEM, n = 6–10 per group. *Different from respective control group by ANOVA Newman–Keuls, p < 0.05.