Liver cytosol catalyzed conjugation of reduced glutathione with a reactive metabolite of acetaminophen

doi:10.1016/0041-008X(79)90328-4

Toxicology and Applied Pharmacology

Volume 47, Issue 2, February 1979, Pages 331-339

https://doi.org/10.1016/0041-008X(79)90328-4 Get rights and content

Abstract

The effect of liver cytosol enzymes on the rate of formation of a glutathione (GSH) conjugate and on the rate of covalent binding of acetaminophen to microsomal proteins was studied in vitro. Mouse liver microsomes were incubated with $[^{14} C] acetaminophen$ , GSH, a NADPH-generating system, and Sephadex G-25-treated mouse liver supernatant. The addition of liver cytosol to the microsomal incubation increased the rate of conjugate production, as measured by high pressure liquid chromatography and decreased the rate of covalent binding of the reactive intermediate of acetaminophen to microsomal proteins at all concentrations of GSH and acetaminophen studied. The effect of the liver supernatant enzymes was most pronounced at low GSH concentrations. Although cysteine and N-acetyl-l-cysteine form conjugates with acetaminophen, the cytosol preparations did not facilitate conjugate formation with these nucleophiles. Cytosol enzymes may be important in the detoxification of acetaminophen particularly when the liver concentration of GSH is low and may contribute to the marked “threshold” effect seen with acetaminophen toxicity.

References (20)

A.R. Buckpitt et al.
Quantitative determination of the glutathione, cysteine and N-acetyl cysteine conjugates of acetaminophen by high pressure liquid chromatography
Anal. Biochem.
(1977)
D.C. Davis et al.
Species differences in hepatic glutathione depletion, covalent binding and hepatic necrosis after acetaminophen
Life Sci.
(1974)
G.L. Ellman
Tissue sulfhydryl groups
Arch. Biochem. Biophys.
(1959)
W.H. Habig et al.
Glutathione S-transferases. The first enzymatic step in mercapturic acid formation
J. Biol. Chem.
(1974)
O. Lowry et al.
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
(1951)
E. Piperno et al.
Reversal of experimental paracetamol toxicosis with N-acetyl cysteine
Lancet
(1976)
L.F. Prescott et al.
Plasma paracetamol half-life and hepatic necrosis in patients with paracetamol overdosage
Lancet
(1971)
H. Borner et al.
The enzymatic conjugation of GSH with the reactive metabolite of acetaminophen
Pharmacologist
(1976)
M.R. Boyd et al.
In vivo studies on the relationship between target organ alkylation and the pulmonary toxicity of a chemically reactive metabolite of 4-ipomeanol
J. Pharmacol. Exp. Ther
(1978)
M. Dixon et al.

There are more references available in the full text version of this article.

Cited by (48)

Nilotinib alleviated acetaminophen-induced acute hepatic injury in mice through inhibiting HIF-1alpha/VEGF-signaling pathway
2022, International Immunopharmacology
Citation Excerpt :
Sixty to Ninty percent of acetaminophen is metabolized into glucuronide and sulfate conjugate via conjugation process, while 5–10 % is oxidized into N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of APAP formed by drug-metabolizing enzymes cytochrome P-450, (CYP2E1, CYP1A2, CYP3A4, and CYP2D6). NAPQI is quenched via conjugation with glutathione (GSH), which depletes GSH resources after a toxic dose [2]. Hypoxia-inducible factor −1 induction may be enhanced through oxidative stress [3,4] by inactivating prolyl hydroxylase domain-containing proteins (PHD) enzymatic activity.
The current study inspects the impact of nilotinib (Nil) on liver damage caused by acetaminophen (APAP). Adult male mice were pre-treated with nilotinib (Nil,5 and 10 mg/kg) orally once daily for 7 days followed by a single intraperitoneal administration of acetaminophen (APAP, 200 mg/kg) on the 7th day.
The results indicated that nilotinib significantly decreased APAP-induced elevation of biochemical markers (ALT, AST, ALP, LDH, ɤ GT, and total bilirubin). Additionally, nilotinib significantly increased hepatic GSH and SOD content, while decreased MDA content. Nil significantly suppressed the expression of HIF-1α and VEGF. Histopathological examination of hepatic tissue from Nil-treated mice revealed that Nil reduced acetaminophen-induced necrosis.
Contribution of acetaminophen-cysteine to acetaminophen nephrotoxicity in CD-1 mice: I. Enhancement of acetaminophen nephrotoxicity by acetaminophen-cysteine
2005, Toxicology and Applied Pharmacology
Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Recent studies suggest a contributory role for glutathione (GSH)-derived conjugates of APAP in the development of nephrotoxicity. Inhibitors of either γ-glutamyl transpeptidase (γ-GT) or the probenecid-sensitive organic anion transporter ameliorate APAP-induced nephrotoxicity but not hepatotoxicity in mice and inhibition of γ-GT similarly protected rats from APAP nephrotoxicity. Protection against APAP nephrotoxicity by disruption of these GSH conjugate transport and metabolism pathways suggests that GSH conjugates are involved. APAP-induced renal injury may involve the acetaminophen-glutathione (APAP-GSH) conjugate or a metabolite derived from APAP-GSH. Acetaminophen-cysteine (APAP-CYS) is a likely candidate for involvement in APAP nephrotoxicity because it is both a product of the γ-GT pathway and a probable substrate for the organic anion transporter. The present experiments demonstrated that APAP-CYS treatment alone depleted renal but not hepatic glutathione (GSH) in a dose-responsive manner. This depletion of renal GSH may predispose the kidney to APAP nephrotoxicity by diminishing GSH-mediated detoxification mechanisms. Indeed, pretreatment of male CD-1 mice with APAP-CYS before challenge with a threshold toxic dose of APAP resulted in significant enhancement of APAP-induced nephrotoxicity. This was evidenced by histopathology and plasma blood urea nitrogen (BUN) levels at 24 h after APAP challenge. APAP alone was minimally nephrotoxic and APAP-CYS alone produced no detectable injury. By contrast, APAP-CYS pretreatment did not alter the liver injury induced by APAP challenge. These data are consistent with there being a selective, contributory role for APAP-GSH-derived metabolites in APAP-induced renal injury that may involve renal-selective GSH depletion.
Cell Death via Interactions of Agents with DNA
1997, Advances in Molecular and Cell Biology
This chapter reviews the evidence that DNA damage may be involved in chemical-induced cell death. The importance of DNA damage for radiation-induced cell killing has been demonstrated in experiments where the cell nucleus and the cytoplasm are selectively irradiated using suitably constructed beams of ionizing particles. It is shown that when the cytoplasm is the target, the dose required to kill a cell is larger by orders of magnitude than the dose required in the nucleus. A general problem shared by most genotoxic chemicals is that they also damage non-DNA targets, such as RNA, proteins and lipids, and cellular processes in the nucleus or in other organelles including mitochondria, endoplasmic reticulum, plasma membrane, and cytoskeleton. When evaluating the importance of chemical-induced DNA damage in organ toxicity, extranuclear injury has to be taken into consideration.
Characterization of cannabidiol-mediated cytochrome P450 inactivation
1993, Biochemical Pharmacology
Cannibidiol (CBD) has been shown to impair hepatic drug metabolism in several animal species and to markedly inhibit mouse hepatic microsomal Δ¹-tetrahydrocannabinol (THC) metabolism by inactivating specific cytochrome P450s (P450) belonging to the 2C and 3A subfamilies. Elucidation of the mechanism of CBD-mediated P450 inhibition would be clinically very important for predicting its effect on metabolism of THC and the many other clinically important drugs known to be metabolized by P450s 2C and 3A. CBD-mediated inactivation of mouse hepatic microsomal P450s did not decrease hepatic microsomal heme content. However, [⁴C]CBD was found covalently bound to microsomal protein in an approximately equimolar ratio to P450 loss. Immunoprecipitation of microsomal protein with antibodies raised against either P450 2C or 3A revealed that approximately equal amounts of [¹⁴C]-CBD were bound to each of these P450s after CBD-mediated inactivation. Furthermore, this specific P450 binding was equivalent to P450 loss and accounted for nearly all of the microsomal [¹⁴C]CBD irreversible binding. Although >80% of the enzyme activities attributed to P450s 2C and 3A were inactivated by CBD at the anticonvulsant dose of 120 mg/kg, P450 2C was approximately 3-fold more sensitive than P450 3A at the lower CBD doses tested. CBD analogs were synthesized in order to elucidate the chemical pathways for CBD-mediated P450 inactivation in vivo. Oxidations at allylic carbon positions or saturation of either the exocyclic double bond or both double bonds of the terpene moiety did not markedly affect the inhibitory properties of the analogs. Methylation of both phenolic groups of the resorcinol moiety completely blocked the P450-inhibitory properties of this analog, revealing the involvement of a free hydroxyl group in the inactivation process. Rotation of the resorcinol moiety in abnormal-CBD did not impair the inhibitory properties of the analog, suggesting that the position of the hydroxyl group relative to the terpene ring is unimportant. Further studies are required to fully understand the chemical basis of CBD-mediated P450 inactivation.
Paracetamol (acetaminophen) poisoning
1993, Pharmacology and Therapeutics
<sup>32</sup>P-Postlabelling analysis of DNA adducts of benzo[a]pyrene formed in complex metabolic activation systems in vitro
1989, Cancer Letters
The influence of cytochrome P-450-linked monooxygenase, epoxide hydrolase, UDP-glucuronyltransferase and glutathione S-transferases on the metabolic activation of benzo[a]pyrene (BP) was studied by incubating BP with preparations of rat-liver microsomal and cytosolic fractions in the presence of exogenous DNA. ³²P-Postlabelling analysis of the DNA revealed the presence of covalently bound adducts formed by BP, which were visualised as radioactive spots on autoradiographs of thin-layer chromatograms. The effects on the adduct profile of adding different combinations of 1,2-epoxy-3,3,3-trichloropropane (TCPO), UDP-glucoronic acid (UDPGA) and glutathione (GSH) to the incubation mixture were determined. As many as 14 different DNA adducts were resolved and quantitated on the chromatograms, the numbers and quantities of which varied within a large range depending on the incubation conditions. The influence of the enzyme inhibitor and cofactors on the adduct patterns reflects the complex effects of simultaneous enzyme interactions on the metabolic activation of BP.

View all citing articles on Scopus

¹: Staff Fellow, Pharmacology Research Associate Program, National Institute of General Medical Sciences.

²: Supported by a National Heart, Lung and Blood Institute Fellowship 1F32-HLO5236.

View full text

Article preview

Toxicology and Applied Pharmacology

Abstract

References (20)

Quantitative determination of the glutathione, cysteine and N-acetyl cysteine conjugates of acetaminophen by high pressure liquid chromatography

Anal. Biochem.

Species differences in hepatic glutathione depletion, covalent binding and hepatic necrosis after acetaminophen

Life Sci.

Tissue sulfhydryl groups

Arch. Biochem. Biophys.

Glutathione S-transferases. The first enzymatic step in mercapturic acid formation

J. Biol. Chem.

Protein measurement with the Folin phenol reagent

J. Biol. Chem.

Reversal of experimental paracetamol toxicosis with N-acetyl cysteine

Lancet

Plasma paracetamol half-life and hepatic necrosis in patients with paracetamol overdosage

Lancet

The enzymatic conjugation of GSH with the reactive metabolite of acetaminophen

Pharmacologist

In vivo studies on the relationship between target organ alkylation and the pulmonary toxicity of a chemically reactive metabolite of 4-ipomeanol

J. Pharmacol. Exp. Ther

Cited by (48)

Nilotinib alleviated acetaminophen-induced acute hepatic injury in mice through inhibiting HIF-1alpha/VEGF-signaling pathway

Contribution of acetaminophen-cysteine to acetaminophen nephrotoxicity in CD-1 mice: I. Enhancement of acetaminophen nephrotoxicity by acetaminophen-cysteine

Cell Death via Interactions of Agents with DNA

Characterization of cannabidiol-mediated cytochrome P450 inactivation

Paracetamol (acetaminophen) poisoning

<sup>32</sup>P-Postlabelling analysis of DNA adducts of benzo[a]pyrene formed in complex metabolic activation systems in vitro