Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: Effects of Ca2+-endonuclease, DNA repair, and glutathione depletion inhibitors on DNA fragmentation and cell death

doi:10.1016/0041-008X(92)90276-X

Toxicology and Applied Pharmacology

Volume 112, Issue 1, January 1992, Pages 32-40

https://doi.org/10.1016/0041-008X(92)90276-X Get rights and content

Abstract

Hepatotoxic alkylation of mouse liver cells by acetaminophen is characterized by an early loss of ion regulation, accumulation of Ca²⁺ in the nucleus, and fragmentation of DNA in vitro and in vivo. Acetaminophen-induced DNA cleavage is accompanied by the formation of a “ladder” of DNA fragments characteristic of Ca²⁺-mediated endonuclease activation. These events unfold well in advance of cytotoxicity and the development of necrosis. The present study utilized cultured mouse hepatocytes and mechanistic probes to test whether DNA fragmentation and cell death might be related in a “cause-and-effect” manner. Cells were isolated by collagenase perfusion, cultured in Williams' E medium for 22–26 hr, and exposed to acetaminophen. Aurintricarboxylic acid, a general Ca²⁺-endonuclease inhibitor, and EGTA, a chelator of Ca²⁺ required for endonuclease activation, significantly decreased DNA fragmentation at 6 and 12 hr and virtually abolished cytotoxicity. N-Acetylcysteine also eliminated DNA fragmentation and cytotoxicity. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase-stimulated DNA repair, failed to alter the amount of DNA fragmentation at 6 hr but substantially increased acetaminophen cytotoxicity in hepatocytes at 12 hr. With the exception of when DNA repair was inhibited by 3-aminobenzamide, Ca²⁺ accumulation in the nucleus, DNA fragmentation, and hepatocyte death varied consistently and predictably with one another. Collectively, these findings suggest that unrepaired damage to DNA contributes to acetaminophen-induced cell death in vivo and may play a role in necrosis in vivo.

References (62)

D. Beales et al.
Lipid peroxidation, protein synthesis, and protection by calcium EDTA in paracetamol injury to isolated hepatocytes
Biochem. Pharmacol.
(1985)
G.B. Corcoran et al.
Immediate rise in intracellular calcium and glycogen phosphorylase a activities during acetaminophen covalent binding leading to hepatotoxicity in mice
Toxicology
(1988)
E. Dybing et al.
Genotoxicity studies with paracetamol
Mutat. Res.
(1984)
J.C. Gaal et al.
Cellular euthanasia mediated by a nuclear enzyme: A central role for nuclear ADP-ribosylation in cellular metabolism
Trends Biochem. Sci. April
(1987)
P. Garberg et al.
Studies on the role of DNA fragmentation in selenium toxicity
Biochem. Pharmacol.
(1988)
M.A. Hayes et al.
Comparative influences of different PB-type and 3-MC type polychlorinated biphenyl-induced phenotype on cytocidal hepatotoxicity of bromobenzene and acetaminophen
Toxicol. Appl. Pharmacol.
(1984)
D.R. Hewish et al.
The calcium dependent endonuclease activity of isolated nuclear preparations. Relationships between its occurrence and the occurrence of other classes of enzymes found in nuclear preparations
Biochem. Biophys. Res. Commun.
(1973)
J.K. Hongslo et al.
Genotoxic effects of paracetamol in V79 Chinese hamster cells
Mutat. Res.
(1988)
D.P. Jones et al.
Calcium-activated DNA fragmentation in rat liver nuclei
J. Biol. Chem.
(1989)
E.J. Landon et al.
Effects of calcium channel blocking agents on calcium and centrilobular necrosis in the liver of rats treated with hepatotoxic agents
Biochem. Pharmacol.
(1986)

O. Lowry et al.

Protein measurement with the folin phenol reagent

J. Biol. Chem.

(1951)

J.R. MacDonald et al.

Structural requirements for cytoprotective agents in galactosamine-induced hepatic necrosis

Toxicol. Appl. Pharmacol.

(1985)

N. Masaki et al.

tert-Butyl hydroperoxide kills cultured hepatocytes by peroxidizing membrane lipids

Arch. Biochem. Biophys.

(1989)

D.J. McConkey et al.

Stimulation of endogenous endonuclease activity in hepatocytes exposed to oxidative stress

Toxicol. Lett.

(1988)

E.G. Miller

Stimulation of nuclear poly(adenosine diphosphateribose) polymerase activity from HeLa cells by endonucleases

Biochim. Biophys. Acta

(1975)

M. Moore et al.

The toxicity of acetaminophen and N-acetyl-p-benzoquinone imine in isolated hepatocytes is associated with thiol depletion and increased cytosolic calcium

J. Biol. Chem.

(1985)

S. Orrenius et al.

Role of calcium in toxic cell killing

Trends Pharmacol. Sci.

(1989)

S.D. Ray et al.

Early loss of large genomic DNA in vivo with accumulation of calcium in the nucleus during acetaminophen-induced liver injury

Toxicol. Appl. Pharmacol.

(1990)

M.H. Sarma et al.

RNA synthesis in isolated HeLa cell nuclei

Biochim. Biophys. Acta

(1976)

W. Shen et al.

Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes. I. Correlation of nuclear Ca²⁺ accumulation and early DNA fragmentation with cell death

Toxicol. Appl. Pharmacol.

(1991)

P.E. Starke et al.

Calcium-dependent and calcium-independent mechanisms of irreversible cell injury in cultured hepatocytes

J. Biol. Chem.

(1986)

R.E. Thiers et al.

The effect of carbon tetrachloride poisoning on subcellular metal distribution in rat liver

Biochem. Pharmacol.

(1960)

J.N. Vanderbilt et al.

Endogenous nuclease. Properties and effects on transcribed genes in chromatin

J. Biol. Chem.

(1982)

H. Weissbach et al.

Inhibition of transfer factor Ts by aurintricarboxylic acid

Biochem. Biophys. Res. Commun.

(1970)

K. Yoshihara et al.

Inhibition of rat liver Ca²⁺, Mg²⁺-dependent endonuclease activity by nicotinamide adenine dinucleotide and poly(ADP-ribose) synthase

Biochem. Biophys. Res. Commun.

(1974)

M.J. Arends et al.

Apoptosis. The role of the endonuclease

Am. J. Pathol.

(1990)

E.S. Baginski et al.

Direct microdetermination of serum calcium

Clin. Chim. Acta

(1973)

N.A. Berger

Symposium: Cellular response to DNA damage; the role of poly(ADP-ribose). Poly(ADP-ribose) in the cellular response to DNA damage

N.A. Berger et al.

Association of poly(adenosine diphosphoribose) synthesis with DNA damage and repair in normal human lymphocytes

J. Clin. Invest.

(1979)

M.K. Bruno et al.

Antidotal effectiveness of N-acetylcysteine in reversing acetaminophen-induced hepatotoxicity. Enhancement of the proteolysis of arylated proteins

Biochem. Pharmacol.

(1988)

K. Burton

A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid

Biochem. J.

(1956)

Cited by (124)

A comprehensive weight of evidence assessment of published acetaminophen genotoxicity data: Implications for its carcinogenic hazard potential
2021, Regulatory Toxicology and Pharmacology
Citation Excerpt :
Elevated cytosolic Ca2+ concentrations can contribute to mitochondrial dysfunction and ATP depletion (Jaeschke, 1990), and after accumulation in the nucleus (Ray et al., 1990) can promote endonuclease activation and DNA fragmentation (Collins and Rivas, 1993; McConkey and Orrenius, 1994). This is supported by data showing that Ca2+ antagonists such as chlorpromazine, verapamil and diltiazem protect against acetaminophen-induced hepatotoxicity (Ray et al., 1993; Satorres et al., 1995; Shen et al., 1991, 1992). Increased Ca2+ levels have also been shown to lead to mitochondrial dysfunction, increased levels of reaction oxygen species, and cytotoxicity via necrosis (Miao et al., 2019; Orrenius et al., 2010, 2015).
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.
Detection of biomarkers to differentiate endocrine disruption from hepatotoxicity in zebrafish (Danio rerio) using proteomics
2020, Chemosphere
Citation Excerpt :
Similarly, apoptosis of liver cells triggered by DNA fragmentation, and induced oxidative stress resulting in excessive mitochondria perturbation in the hepatocytes of female zebrafish (Fig. S1 C, D) were significantly affected upon APAP exposure. These results further support the premise that apoptosis-induced DNA fragmentation and induced oxidative stress is central to APAP-induced liver injury (Shen et al., 1991, 1992; Lee, 2007; Bajt et al., 2011; Li et al., 2015; Lee et al., 2018; Ramachandran and Jaeschke, 2018). In addition, a significant change in estrogen-dependent gene expression (Fig. S1 C) (Gadd et al., 2002) suggests that the observed APAP-induced liver injury may trigger the perturbation of VTG synthesis (Fig. 1 D) (Anderson et al., 1996; Ruepp et al., 2002).
Measurement of specific biomarkers identified by proteomics provides a potential alternative method for risk assessment, which is required to discriminate between hepatotoxicity and endocrine disruption. In this study, adult zebrafish (Danio rerio) were exposed to the hepatotoxic substance acetaminophen (APAP) for 21 days, in a fish short-term reproduction assay (FSTRA). The molecular changes induced by APAP exposure were studied in liver and gonads by applying a previously developed combined FSTRA and proteomics approach. We observed a significant decrease in egg numbers, an increase in plasma hyaluronic acid, and the presence of single cell necrosis in liver tissue. Furthermore, nine common biomarkers (atp5f1b, etfa, uqcrc2a, cahz, c3a.1, rab11ba, mettl7a, khdrbs1a and si:dkey-108k21.24) for assessing hepatotoxicity were detected in both male and female liver, indicating hepatic damage. In comparison with exposure to fadrozole, an endocrine disrupting chemical (EDC), three potential biomarkers for liver injury, i.e. cahz, c3a.1 and atp5f1b, were differentially expressed. The zebrafish proteome response to fadrozole exposure indicated a significant regulation in estrogen synthesis and perturbed binding of sperm to zona pellucida in the ovary. This study demonstrates that biomarkers identified and quantified by proteomics can serve as additional weight-of-evidence for the discrimination of hepatotoxicity and endocrine disruption, which is necessary for hazard identification in EU legislation and to decide upon the option for risk assessment.
Trifluoperazine inhibits acetaminophen-induced hepatotoxicity and hepatic reactive nitrogen formation in mice and in freshly isolated hepatocytes
2017, Toxicology Reports
Citation Excerpt :
Moreover, they found that an increase in cytosolic calcium correlated with development of toxicity [48]. Corcoran et al. found a toxic dose APAP to mice caused an increase in nuclear calcium levels using cultured hepatocytes [49] and that EGTA, a calcium ion chelator, inhibited hepatocyte death; a finding that suggested toxicity was mediated by extracellular calcium since EGTA is ionized and does not enter the cells. In freshly isolated hepatocytes we previously found that EGTA, as well as Quin-2, inhibited APAP hepatotoxicity [50].
The hepatotoxicity of acetaminophen (APAP) occurs by initial metabolism to N-acetyl-p-benzoquinone imine which depletes GSH and forms APAP-protein adducts. Subsequently, the reactive nitrogen species peroxynitrite is formed from nitric oxide (NO) and superoxide leading to 3-nitrotyrosine in proteins. Toxicity occurs with inhibited mitochondrial function. We previously reported that in hepatocytes the nNOS (NOS1) inhibitor NANT inhibited APAP toxicity, reactive nitrogen and oxygen species formation, and mitochondrial dysfunction. In this work we examined the effect of trifluoperazine (TFP), a calmodulin antagonist that inhibits calcium induced nNOS activation, on APAP hepatotoxicity and reactive nitrogen formation in murine hepatocytes and in vivo. In freshly isolated hepatocytes TFP inhibited APAP induced toxicity, reactive nitrogen formation (NO, GSNO, and 3-nitrotyrosine in protein), reactive oxygen formation (superoxide), loss of mitochondrial membrane potential, decreased ATP production, decreased oxygen consumption rate, and increased NADH accumulation. TFP did not alter APAP induced GSH depletion in the hepatocytes or the formation of APAP protein adducts which indicated that reactive metabolite formation was not inhibited. Since we previously reported that TFP inhibits the hepatotoxicity of APAP in mice without altering hepatic APAP-protein adduct formation, we examined the APAP treated mouse livers for evidence of reactive nitrogen formation. 3-Nitrotyrosine in hepatic proteins and GSNO were significantly increased in APAP treated mouse livers and decreased in the livers of mice treated with APAP plus TFP. These data are consistent with a hypothesis that APAP hepatotoxicity occurs with altered calcium metabolism, activation of nNOS leading to increased reactive nitrogen formation, and mitochondrial dysfunction.
Acetaminophen from liver to brain: New insights into drug pharmacological action and toxicity
2016, Pharmacological Research
Acetaminophen (APAP) is a well-known analgesic and antipyretic drug. It is considered to be safe when administered within its therapeutic range, but in cases of acute intoxication, hepatotoxicity can occur. APAP overdose is the leading cause of acute liver failure in the northern hemisphere. Historically, studies on APAP toxicity have been focused on liver, with alterations in brain function attributed to secondary effects of acute liver failure. However, in the last decade the pharmacological mechanism of APAP as a cannabinoid system modulator has been documented and some articles have reported “in situ” toxicity by APAP in brain tissue at high doses. Paradoxically, low doses of APAP have been reported to produce the opposite, neuroprotective effects. In this paper we present a comprehensive, up-to-date overview of hepatic toxicity as well as a thorough review of both toxic and beneficial effects of APAP in brain.
Experimental models of hepatotoxicity related to acute liver failure
2016, Toxicology and Applied Pharmacology
Citation Excerpt :
This model is often selected due to the epidemiological relevance in humans. Furthermore, the pathophysiology in mice reflects very closely what is observed in humans, including reactive metabolite formation (McGill and Jaeschke, 2013), mitochondrial damage with oxidative stress (Adamson and Harman, 1993; Cover et al., 2005; Jaeschke, 1990; Kon et al., 2004, 2007; Reid et al., 2005; Saito et al., 2010a, 2010b), followed by DNA fragmentation (Bajt et al., 2008; Cover et al., 2005; Shen et al., 1992) and necrosis (Bajt et al., 2008; Gujral et al., 2002; Jaeschke et al., 2012a; Williams et al., 2011). As an APAP overdose mainly causes oncotic necrosis, alternative models are needed to study apoptosis and secondary necrosis in liver injury.
Acute liver failure can be the consequence of various etiologies, with most cases arising from drug-induced hepatotoxicity in Western countries. Despite advances in this field, the management of acute liver failure continues to be one of the most challenging problems in clinical medicine. The availability of adequate experimental models is of crucial importance to provide a better understanding of this condition and to allow identification of novel drug targets, testing the efficacy of new therapeutic interventions and acting as models for assessing mechanisms of toxicity. Experimental models of hepatotoxicity related to acute liver failure rely on surgical procedures, chemical exposure or viral infection. Each of these models has a number of strengths and weaknesses. This paper specifically reviews commonly used chemical in vivo and in vitro models of hepatotoxicity associated with acute liver failure.
Standardized extract of Vitex doniana Sweet stalls protein oxidation, lipid peroxidation and DNA fragmention in acetaminophen-induced hepatotoxicity
2015, Journal of Ethnopharmacology
Vitex doniana fruits are locally used in Nigeria as a remedy in the treatment of jaundice and liver related disease. The effect of methanolic extract of Vitex doniana fruits on acetaminophen induced protein oxidation, lipid peroxidation and DNA fragmentation was investigated in mice.
Antioxidant activity of the extract (0.2–1.0 mg/mL) was investigated in vitro using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide ion, hydrogen peroxide, hydroxyl radical and ferric ion reducing system. Vitex doniana extract at 1.0 mg/mL scavenged DPPH, superoxide ion, hydrogen peroxide, and hydroxyl radical by 86%, 78%, 80% and 72% respectively, it also reduced ferric ion significantly. Hepatoprotective effect of Vitex doniana fruits was monitored in acetaminophen-induced hepatotoxicity in mice.
Acetaminophen-mediated alterations in serum alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, albumin and total bilirubin levels in mice were significantly (P<0.05) attenuated by the extract. Similarly, acetaminophen-mediated decrease in activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase was significantly (P<0.05) attenuated in the liver of mice. Increased levels of conjugated dienes, lipid hydroperoxides, malondialdehyde, protein carbonyl and fragmented DNA were significantly (P<0.05) lowered by methanolic extract of Vitex doniana fruits.
Overall, the results of this study show that Vitex doniana fruits possess antioxidant properties and halted acetaminophen-mediated oxidative rout on cellular proteins, lipids and DNA, made possible by β-sitosterol, platycodin D, apigenin, saikosaponin, chrysin and ellagitanin in the extract.

View all citing articles on Scopus

^☆: This work was supported in part by Grant GM 41564 from the National Institute for General Medical Sciences, National Institutes of Health. Portions of this work were presented at the Annual Meeting of the Federation of American Societies for Experimental Biology held in Atlanta, GA April 21–25, 1991, and published in abstract form (FASEB J. 5, A1563).

View full text

Regular articleAcetaminophen-induced cytotoxicity in cultured mouse hepatocytes: Effects of Ca2+-endonuclease, DNA repair, and glutathione depletion inhibitors on DNA fragmentation and cell death☆

Abstract

Biochem. Pharmacol.

Toxicology

Mutat. Res.

Trends Biochem. Sci. April

Biochem. Pharmacol.

Toxicol. Appl. Pharmacol.

Biochem. Biophys. Res. Commun.

Mutat. Res.

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

Toxicol. Appl. Pharmacol.

Arch. Biochem. Biophys.

Toxicol. Lett.

Biochim. Biophys. Acta

J. Biol. Chem.

Trends Pharmacol. Sci.

Toxicol. Appl. Pharmacol.

Biochim. Biophys. Acta

Toxicol. Appl. Pharmacol.

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Apoptosis. The role of the endonuclease

Am. J. Pathol.

Direct microdetermination of serum calcium

Clin. Chim. Acta

Symposium: Cellular response to DNA damage; the role of poly(ADP-ribose). Poly(ADP-ribose) in the cellular response to DNA damage

Association of poly(adenosine diphosphoribose) synthesis with DNA damage and repair in normal human lymphocytes

J. Clin. Invest.

Antidotal effectiveness of N-acetylcysteine in reversing acetaminophen-induced hepatotoxicity. Enhancement of the proteolysis of arylated proteins

Biochem. Pharmacol.

A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid

Biochem. J.

Regular article
Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: Effects of Ca²⁺-endonuclease, DNA repair, and glutathione depletion inhibitors on DNA fragmentation and cell death☆