Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures

doi:10.1016/0041-008X(85)90356-4

Toxicology and Applied Pharmacology

Volume 79, Issue 2, 30 June 1985, Pages 342-347

https://doi.org/10.1016/0041-008X(85)90356-4 Get rights and content

Abstract

Acetaminophen (APAP) metabolism, cytotoxicity, and genotoxicity were measured in primary cultures of rat hepatocytes. Although 3 mm APAP caused a slight increase in cellular release of lactate dehydrogenase into the culture medium, cellular glutathione concentration (an index of APAP metabolism) was reduced by 50%. APAP at 7 mm was significantly more toxic to these hepatocytes and had a similar but more marked effect on glutathione concentrations. In spite of its cytotoxicity, neither dose of APAP stimulated DNA repair synthesis when monitored by the rate of incorporation of [³H]thymidine into DNA following exposure to APAP. Thus, although APAP has been shown to be both hepato- and nephrotoxic in several in vivo and in vitro systems, the reactive toxic metabolite of APAP is not genotoxic in rat primary hepatocyte cultures.

References (35)

D. Acosta et al.
Cytotoxicity of acetaminophen and papaverine in primary cultures of rat hepatocytes
Toxicol. Appl. Pharmacol.
(1980)
G.M. Decad et al.
Maintenance of cytochrome P-450 and metabolism of aflatoxin B₁ in primary hepatocyte cultures
Biochem. Biophys. Res. Commun.
(1977)
G.M. Decad et al.
Metabolism of aflatoxin B₁ in cultured mouse hepatocytes: Comparison with rat and effects of cyclohexene oxide and diethyl maleate
Toxicol. Appl. Pharmacol.
(1979)
C.E. Green et al.
Metabolism and cytotoxicity of acetaminophen in hepatocytes isolated from resistant and susceptible species
Toxicol. Appl. Pharmacol.
(1984)
P.J. Hissin et al.
A fluorometric method for determination of oxidized and reduced glutathione in tissues
Anal. Biochem.
(1976)
J. Mohandas et al.
Metabolic oxidation of acetaminophen (paracetamol) mediated by cytochrome P-450 mixed-function oxidase and prostaglandin endoperoxide synthetase in rabbit kidney
Toxicol. Appl. Pharmacol.
(1981)
C.B. Salocks et al.
Effects of butylated hydroxytoluene pretreatment on the metabolism and genotoxicity of aflatoxin B₁ in primary cultures of adult rat hepatocytes: Selective reduction of nucleic acid binding
Toxicol. Appl. Pharmacol.
(1984)
C.B. Salocks et al.
Butylated hydroxytoluene pretreatment protects against cytotoxicity and reduces covalent binding of aflatoxin B₁ in primary hepatocyte cultures
Toxicol. Appl. Pharmacol.
(1981)
A. Sibatani
Precipitation and counting of minute quantities of labeled nucleic acids as cetyltrimethylammonium salt
Anal. Biochem.
(1970)
R.S. Andrews et al.
Isolation and identification of paracetamol metabolites
J. Int. Med. Res.
(1976)

W.J. Bodell et al.

Enzyme digestion of intermediates of excision repair in human cells irradiated with ultraviolet light

Biochemistry

(1982)

H.M. Carpenter et al.

Acetaminophen nephrotoxicity: Studies on renal acetylation and deacetylation

J. Pharmacol. Exp. Ther.

(1981)

R. Dulbecco et al.

Plaque formation and isolation of pure lines with poliomyelitis viruses

J. Exp. Med.

(1954)

L.J. Fischer et al.

Levels of acetaminophen and its metabolites in mouse tissues after toxic dose

J. Pharmacol. Exp. Ther.

(1981)

A. Flaks et al.

Induction of liver cell tumours in IF mice by paracetamol

Carcinogenesis

(1983)

M.W. Gemborys et al.

Formation and disposition of the minor metabolites of acetaminophen in the hamster

Drug Metab. Disp.

(1981)

J.W. Grisham

Use of hepatic cell cultures to detect and evaluate the mechanisms of action of toxic chemicals

Int. Rev. Exp. Pathol.

(1979)

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Organ-on-chip technology is a promising in vitro approach recapitulating human physiology for the study of responses to drug exposure. Organ-on-chip cell cultures have paved new grounds for testing and understanding metabolic dose-responses when evaluating pharmaceutical and environmental toxicity. Here, we present a metabolomic investigation of a coculture of liver sinusoidal endothelial cells (LSECs, SK-HEP-1) with hepatocytes (HepG2/C3a) using advanced organ-on-chip technology. To reproduce the physiology of the sinusoidal barrier, LSECs were separated from hepatocytes by a membrane (culture insert integrated organ-on-chip platform). The tissues were exposed to acetaminophen (APAP), an analgesic drug widely used as a xenobiotic model in liver and HepG2/C3a studies. The differences between the SK-HEP-1, HepG2/C3a monocultures and SK-HEP-1/HepG2/C3a cocultures, treated or not with APAP, were identified from metabolomic profiles using supervised multivariate analysis. The pathway enrichment coupled with metabolite analysis of the corresponding metabolic fingerprints contributed to extracting the specificity of each type of culture and condition. In addition, we analysed the responses to APAP treatment by mapping the signatures with significant modulation of the biological processes of the SK-HEP-1 APAP, HepG2/C3a APAP and SK-HEP-1/HepG2/C3a APAP conditions. Furthermore, our model shows how the presence of the LSECs barrier and APAP first pass can modify the metabolism of HepG2/C3a. Altogether, this study demonstrates the potential of a “metabolomic-on-chip” strategy for pharmaco-metabolomic applications predicting individual response to drugs.
A comprehensive weight of evidence assessment of published acetaminophen genotoxicity data: Implications for its carcinogenic hazard potential
2021, Regulatory Toxicology and Pharmacology
In 2019, the California Office of Environmental Health Hazard Assessment initiated a review of the carcinogenic hazard potential of acetaminophen, including an assessment of its genotoxicity. The objective of this analysis was to inform this review process with a weight-of-evidence assessment of more than 65 acetaminophen genetic toxicology studies that are of widely varying quality and conformance to accepted standards and relevance to humans. In these studies, acetaminophen showed no evidence of induction of point or gene mutations in bacterial and mammalian cell systems or in in vivo studies. In reliable, well-controlled test systems, clastogenic effects were only observed in unstable, p53-deficient cell systems or at toxic and/or excessively high concentrations that adversely affect cellular processes (e.g., mitochondrial respiration) and cause cytotoxicity. Across the studies, there was no clear evidence that acetaminophen causes DNA damage in the absence of toxicity. In well-controlled clinical studies, there was no meaningful evidence of chromosomal damage. Based on this weight-of-evidence assessment, acetaminophen overwhelmingly produces negative results (i.e., is not a genotoxic hazard) in reliable, robust high-weight studies. Its mode of action produces cytotoxic effects before it can induce the stable, genetic damage that would be indicative of a genotoxic or carcinogenic hazard.
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.
The genotoxicity and carcinogenicity of paracetamol: A regulatory (re)view
1996, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
The publication of several studies reporting genotoxic effects of paracetamol, one of the world's most popular over-the-counter drugs, has raised the question of regulatory action. Paracetamol does not cause gene mutations, either in bacteria or in mammalian cells. There are, however, published data giving clear evidence that paracetamol causes chromosomal damage in vitro in mammalian cells at high concentrations and indicating that similar effects occur in vivo at high dosages. Available data point to three possible mechanisms of paracetamol-induced genotoxicity: (1) inhibition of ribonucleotide reductase; (2) increase in cytosolic and intranuclear Ca²⁺ levels; (3) DNA damage caused by NAPQI after glutathione depletion. All mechanisms involve dose thresholds. Studies of the relationship between genotoxicity and toxic effects in the rat (induction of micronulei in rat bone marrow including dose-response relationship, biotransformation of paracetamol at different dosages, concomitant toxicity and biochemical markers) have recently been completed. These studies, which employed doses ranging from the dose resulting in human therapeutic peak plasma levels to highly toxic doses, give convincing evidence that genotoxic effects of paracetamol appear only at dosages inducing pronounced liver and bone marrow toxicity and that the threshold level for genotoxicity is not reached at therapeutic dosage. Reliable studies on the ability of paracetamol to affect germ cell DNA are not available. However, based on the amount of drug likely to reach germ cells and the evidence of thresholds, paracetamol is not expected to cause heritable damage in man. Various old and poorly designed long-term studies of paracetamol in the mouse and rat have given equivocal results. A few of these studies showed increased incidence of liver and bladder tumours at hepatotoxic doses. National Toxicology Program (U.S.A.) feeding studies have shown that paracetamol is non-carcinogenic when given at non-hepatotoxic doses up to 300 mg/kg/d to the rat and up to 1000 mg/kg/d to the mouse. Taking into account the knowledge of the hepatotoxicity and metabolism of paracetamol and the existence of thresholds for its genotoxicity, the animal studies do not indicate a carcinogenic potential at non-hepatotoxic dose levels. Based on this updated assessment of the genotoxicity and carcinogenicity of paracetamol, it is concluded that there is no need for regulatory action.
Evaluation studies on the in vitro rat hepatocyte micronucleus assay
1995, Mutation Research/Environmental Mutagenesis and Related Subjects
Based on a previous study with 8 chemicals (Müller et al., 1993) the applicability of the in vitro rat hepatocyte micronucleus assay was evaluated by testing a further 21 compounds of different chemical classes. The obtained results are in good agreement with the known genotoxic profiles of about 90% of the in total tested compounds. Several known mutagens and carcinogens, i.e., alkylating agents, aromatic amines, nitrosamines, nitro compounds, cross-linking agents, and pyrrolizidine alkaloids gave clear positive results in this assay, whereas all of the tested non-carcinogens were negative. The hepatocyte micronucleus assay was shown to distinguish between carcinogenic/non-carcinogenic isomers, such as 2- and 4-acetylaminofluorene (AAF) and 2- and 1-nitropropane (NP). Furthermore, the non-genotoxic nature of several hepatocarcinogens, i.e., the peroxisome proliferating agents fenofibrate, nafenopin, Wy-14,643, diethyl(hexyl)phthalate (DEHP), and the sedative phenobarbital, could be confirmed in this assay. The hepatocarcinogen coumarin exerted mitogenic but no mutagenic properties in the rat hepatocyte micronucleus assay. This compound may act as a liver tumor promoter. benzo[a]pyrene (B[a]P) and 7,12-dimethylbenzanthacene (DMBA), both beloning to the group of known carcinogenic and mutagenic polycyclic aromatic hydrocarbons, failed to induce micronucleus formation in rat hepatocytes. The high susceptibility of in vitro proliferating hepatocytes to mitotic inhibition, exerted by the strong cytotoxic actions of these compounds, seems to be responsible for these negative results. A strongly reduced mitotic activity can prevent the formation of micronuclei, even when clastogenic effects may have occurred. In the present stage, the in vitro rat hepatocyte micronucleus assay cannot be recommended for screening genotoxicity testing. It should rather be used for special purposes, e.g., when liver-specific mutagenic effects are expected.
An evaluation of the genetic toxicity of paracetamol
1995, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
During the last years, several reports have indicated genotoxic effects of paracetamol, a widely used non-prescription analgesic and antipyretic drug. Thus, a careful evaluation of a possible genotoxic effect related to paracetamol use is warranted. Studies in vitro and in vivo indicate that the reactive metabolite of paracetamol can bind irreversibly to DNA and cause DNA strand breaks. Paracetamol inhibits both replicative DNA synthesis and DNA repair synthesis in vitro and in experimental animals. Paracetamol does not cause gene mutations, either in bacteria or in mammalian cells. On the other hand, a co-mutagenic effect of paracetamol has been reported. Furthermore, paracetamol increases the frequency of chromosomal damage in mammalian cell lines, isolated human lymphocytes and experimental animals. Two independent studies have shown an increase in chromosomal damage in lymphocytes of human volunteers after intake of therapeutic doses of paracetamol, whereas a third study was negative. Paracetamol-induced chromosomal damage appears to be caused by an inhibition of ribonucleotide reductase. This indicates that a threshold level for the paracetamol-induced chromosomal damage may exist. Genotoxic effects of paracetamol have, however, been demonstrated both in vitro and in vivo at or near therapeutic concentrations. The data indicate that the use of paracetamol may contribute to an increase in the total burden of genotoxic damage in man. Thus, there may be a need to evaluate the therapeutic benefit of paracetamol, taking into consideration not only its potential to induce acute and chronic organ damage, but also genotoxic effects.

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Short communicationAcetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures

Abstract

Toxicol. Appl. Pharmacol.

Biochem. Biophys. Res. Commun.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Anal. Biochem.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Anal. Biochem.

Isolation and identification of paracetamol metabolites

J. Int. Med. Res.

Enzyme digestion of intermediates of excision repair in human cells irradiated with ultraviolet light

Biochemistry

Acetaminophen nephrotoxicity: Studies on renal acetylation and deacetylation

J. Pharmacol. Exp. Ther.

Plaque formation and isolation of pure lines with poliomyelitis viruses

J. Exp. Med.

Levels of acetaminophen and its metabolites in mouse tissues after toxic dose

J. Pharmacol. Exp. Ther.

Induction of liver cell tumours in IF mice by paracetamol

Carcinogenesis

Formation and disposition of the minor metabolites of acetaminophen in the hamster

Drug Metab. Disp.

Use of hepatic cell cultures to detect and evaluate the mechanisms of action of toxic chemicals

Int. Rev. Exp. Pathol.

Short communication
Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures