Special Contribution

Yonago Acta medica 2004;47:17–28

Acetaminophen-Induced Hepatotoxicity: Still an Important Issue
Isao Sumioka*†, Tatsuya Matsura† and Kazuo Yamada†*Healthcare Research Institute, Wakunaga Pharmaceutical Co., Ltd., Akitakata 739-1195 and†Division of Medical Biochemistry, Department of Pathophysiological and Therapeutic Science,
School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503 Japan
Acetaminophen (APAP), the most commonly sold over-the-counter antipyretic analgesic,
is generally considered harmless at therapeutic doses. However, APAP overdose causes
severe and sometimes fatal hepatic damage in humans and experimental animals. In the
United States and Europe, the incidence of liver injury due to APAP overdose, either withsuicidal intent or by accident, is increasing. Recently, even in Japan, APAP has become
more commonly used alone, especially in children with a view to prevention of Reye's
syndrome. Thus, understanding the hepatotoxicity induced by APAP overdose is very important. To date, the mechanisms underlying APAP toxicity are considered to be associated with i) covalent binding to cellular macromolecules of a reactive intermediate metaboliteof APAP produced by cytochrome P450 (CYP), N-acetyl-p-benzoquinoneimine, and ii)
oxidative stress. APAP-induced hepatotoxicity is modified by associated risk factors suchas alcohol abuse, fasting and concomitant drugs. Even therapeutic doses of APAP sometimes
induce hepatic damage in the presence of these risk factors. N-Acetylcysteine (NAC), oneof the cysteine prodrugs, is the most widely used for treating APAP-induced liver injury.
In addition to NAC, CYP2E1 inhibitors and antioxidants may also serve as mechanismbased antidotes for APAP overdose. Moreover, inducers of heat shock protein (HSP) (inparticular HSP25 and HSP70i) without any side effects might be new-type antidotes.
key words: acetaminophen; N-acetyl-p-benzoquinoneimine; cytochrome P450 2E1; hepatotoxicity;oxidative stresscauses hepatotoxicity (Davidson and Eastham, 1966;
Mitchell, 1988). In addition, susceptibility to
APAP-induced hepatotoxicity is modified by various factors such as alcohol abuse, fasting and concomitant drug use. In Japan, APAP has usually been
consumed as combination tablets or granules andrarely used alone except as a suppository. However,since recent reports that the use of salicylic acid in
combination cold remedies is associated withAcetaminophen (N-acetyl-p-aminophenol, APAP),
also referred to as paracetamol, is widely used as ananalgesic and antipyretic drug throughout theworld. In the United Kingdom, about 3.2 × 109 tablets of APAP are consumed every year, which isequivalent to an average of 55 tablets/person (Jones,
1998). As an over-the-counter drug, it can be readily obtained without prescription. Although APAP
is generally harmless at therapeutic doses, overdose

Abbreviations: ALT, alanine aminotransferase; APAP, acetaminophen; CoQ10, oxidized coenzyme Q10; CoQ9H2,
reduced coenzyme Q9; CoQ10H2, reduced coenzyme Q10; CYP, cytochrome P450; GSH, reduced glutathione; GSSG,
oxidized glutathione; HSP, heat shock protein; MEOS, microsomal ethanol oxidizing system; NAC, N-acetylcysteine;
NAPQI, N-acetyl-p-benzoquinoneimine; ROS, reactive oxygen species; SAMC, S-allylmercaptocysteine; TBARS,
thiobarbituric acid-reactive substance; α-Toc, α-tocopherol

17

I. Sumioka et al.

Reye’s syndrome in children with influenza or
varicella infection (Belay et al., 1999), APAP has
become more likely to be used alone in Japan and inother countries. Therefore, it is important that we
review APAP toxicity, even though APAP-induced
hepatic injury is not a newly emerging problem.
APAP-induced hepatotoxicity has been demonstrated in experimental animals as well as clinicalcases. Mice and hamsters have been shown to bevery sensitive to the hepatotoxic effects of APAP,
developing a fulminant centrilobular necrosis similar to that observed in humans (Mitchell et al.,
1973; Davis et al., 1974; Potter et al., 1974). Rats,
rabbits and guinea pigs are, however, relatively
resistant to APAP insult (Boyd and Bereczky, 1966;
Madhu et al, 1992).
This review focuses on the mechanisms underlying APAP-induced hepatotoxicity, risk factorsthat increase susceptibility to the hepatotoxic effects of APAP, and protection against the toxicity.

to cellular macromolecules (Jollow et al., 1973;
Potter and Hinson, 1986). This is thought to lead toa loss of protein thiol groups (Moore et al., 1985;
Kyle et al., 1990) and ultimately to cell death. In
humans, CYP2E1, CYP1A2 and CYP3A4 have beenthought to contribute to the metabolism of APAP toform NAPQI (Raucy et al., 1989; Patten et al., 1993;
Thummel et al., 1993; Nelson, 1995). However,recent pharmacokinetics studies in human volunteers have demonstrated that involvement of
CYP1A2 and CYP3A4 in NAPQI formation in vivo
is much less than that of CYP2E1, as omeprazole(CYP1A2 inducer) and rifampicin (CYP3A4 inducer) treatment have no effect on the formation of
NAPQI from APAP, whereas disulfiram (CYP2E1
inhibitor) treatment decreases NAPQI formation(Sarich et al., 1997; Manyike et al., 2000). Studies
using CYP2E1 and CYP1A2 knockout mice haveshown that the former, but not the latter, are moreresistant to APAP-induced hepatotoxicity thanwild-type animals (Lee et al., 1996; Tonge et al.,
1998; Zaher et al., 1998), indicating that CYP2E1 isa primary contributor to APAP biotransformation
among CYPs. Concerning the other CYP enzymes
responsible for bioactivation of APAP, Hazai et al.
(2002) recently reported that selective inhibition of
CYP2A6 as well as CYP2E1 significantly decreased NAPQI formation in human liver microsomes,
whereas CYP1A2 and CYP3A4 inhibition did notaffect NAPQI production, suggesting that CYP2A6
may also be responsible for APAP bioactivation.
However, the role of CYP2A6 in APAP metabolism in vivo remains to be established. Overall, it isfeasible that the principal CYP responsible for
APAP toxicity is CYP2E1.

Mechanisms of APAP-Induced
Hepatotoxicity

Reactive intermediate metabolite of APAP
APAP is biotransformed and eliminated as nontoxic
glucuronic acid and sulfate conjugates. Glucuronide is provided by UDP-glucuronic acid, and sulfation is dependent on phosphoadenylsulfate(Clements et al., 1984). Furthermore, the mixedfunction oxidase system cytochrome P450 (CYP)
participates in metabolizing a small proportion of
APAP at therapeutic doses. The metabolism of
APAP by CYP leads to the formation of N-acetyl-pbenzoquinoneimine (NAPQI), a highly reactive intermediate metabolite (Dahlin et al., 1984), which isnormally detoxified by conjugation with reduced
glutathione (GSH).
After high doses of APAP, the capacity for its
removal by hepatic conjugation with glucuronide
and sulfate is exceeded, and more of the reactive
metabolite NAPQI is formed. Consequently, more
NAPQI is conjugated with GSH, and when hepatic
GSH is depleted, more NAPQI will bind covalently

Oxidative stressOxidative stress is also considered to be involved in
the induction of hepatotoxicity by APAP. The oneelectron oxidation of APAP by CYPs may generate
reactive oxygen species (ROS). Hydrogen peroxide and superoxide are produced during metabolic
activation of APAP in the mixed function oxidase
system (Nordblom and Coon, 1977; Kuthan et al.,
1978; de Vries, 1981). It has been reported that18

Acetaminophen-induced hepatotoxicity

APAP overdose causes decreases in antioxidant
enzyme activities such as catalase and glutathione
peroxidase (Lores Arnaiz et al., 1995; Chen andLin, 1997), levels of endogenous antioxidants suchas α-tocopherol (α-Toc) and reduced forms of coenzyme Q9 (CoQ9H2) and coenzyme Q10 (CoQ10H2)(Amimoto et al., 1995; Sumioka et al., 1998), and
the GSH/oxidized glutathione (GSSG) ratio(Jaeschke, 1990; Lores Arnaiz et al., 1995; Amimoto
et al., 1995) in animal livers. These endogenous
antioxidants, especially α-Toc, CoQ 9 H 2 and
CoQ10H2 scavenge lipid peroxyl radicals and consequently their own levels decrease (Matsura et al.,1992a, 1992b). Although there are arguments that
lipid peroxidation may be a consequence rather
than the cause of APAP-induced cell damage(Mitchell et al., 1981, 1984), our time course dataobtained by quantitative analysis of biomarkers inmice revealed that the reduction in hepatic CoQ9H2and CoQ10H2 levels preceded increases in the thiobarbituric acid-reactive substance (TBARS) content as an index of lipid peroxidation and plasma
alanine aminotransferase (ALT) activity following
APAP treatment (Amimoto et al, 1995). Moreover,pretreatment with oxidized coenzyme Q10 (CoQ10)
resulted in an increase in hepatic CoQ10H2 and amarked reduction in hepatic TBARS content andplasma ALT activity without affecting hepatic GSH
after APAP injection (Amimoto et al., 1995).
Pretreatment with α-Toc also suppressed the increase in the hepatic TBARS content and plasma
ALT activity without affecting the hepatic GSH
level (Amimoto et al., 1995). These results strongly
suggest that lipid peroxidation is involved in themechanism of APAP-induced hepatic injury.
Recently, it has been reported that the formation of reactive nitrogen species such as peroxynitrite followed by extensive protein nitration is
also associated with APAP-induced hepatotoxicity(Hinson et al., 1998; Knight et al., 2001).

which increase susceptibility to the hepatotoxic
effects of APAP.

AlcoholInteraction between alcohol and APAP has beenrecognized since the late 1970s (McClain et al.,1980). However, the APAP-alcohol interaction isvery complicated, because acute and chronic alcohol intakes have opposite effects. Chronic alcohol
intake increases hepatic CYP2E1 activity (Perrot etal., 1989) and decreases the GSH level (Lauterburg
and Velez, 1988), especially the liver mitochondrial
GSH level (Hirano et al., 1992; Zhao et al., 2002;
Zhao and Slattery 2002). These changes lead to anincrease in NAPQI formation and a decrease in
NAPQI detoxification, resulting in accumulation of
NAPQI. Consequently, the severity of APAP-induced
hepatotoxicity is enhanced by chronic alcohol intake (Sato et al., 1981a; Zimmerman and Maddrey,
1995; Schmidt et al., 2002), and even a therapeutic
dose of APAP (generally considered to be nontoxic
in nonalcoholics) may lead to hepatotoxicity. On
the other hand, acute alcohol intake reduces theseverity of APAP-induced hepatotoxicity (Rumack
et al., 1981; Banda and Quart 1982). In rats, thiseffect may be due to direct inhibition by alcohol ofthe biotransformation of APAP to NAPQI (Sato etal., 1981b, 1981c). Because alcohol itself is metabolized not only by alcohol dehydrogenase but also
by CYP2E1 (microsomal ethanol oxidizing system;
MEOS) and MEOS plays a major role when blood
ethanol levels are high (Lieber, 1990), alcohol may
competitively inhibit biotransformation of APAP to
NAPQI. In these studies, APAP was administeredwhen ethanol was still present in the blood circulation. Because ethanol is both a substrate and inhibitor of CYP2E1 (Lieber, 1997), the relative timing of APAP and ethanol ingestion is critical whenattempting to elucidate the effect of short-term
ethanol exposure on APAP metabolism. Recently,
Thummel et al. (2000) reported, from studies inhealthy adult volunteers, that the increase in
NAPQI formation caused by ethanol occurred only
when APAP was given after ethanol had cleared
from the body.

Risk Factors
Even therapeutic doses of APAP sometimes induce
hepatotoxicity in the presence of risk factors (suchas alcohol abuse, fasting and drug interaction),
19

I. Sumioka et al.

Table 1. Protective effect of cysteine prodrugs on APAP-induced hepatotoxicity
CompoundN-Acetylcysteine

L-2-Oxothiazolidine-4-carboxylate
L-2-Methylthiazolidine-4-carboxylate

2-(Polyhydroxyalkyl)thiazolidine-4(R)carboxylic acids
Cystathionine

Experimental model
Mouse hepatocytes(in vitro)
Rats (in vivo)
Mice (in vivo)
Mice (in vivo)
Mice (in vivo)

Massey and Racz

1981

Lauterburg et al.
Corcoran et al.
Hjelle et al.
Corcoran and Wong

1983
1985
1986
1986

Mice (in vivo)

Hazelton et al.

1986

Mice (in vivo)

Roberts et al.

1987

Kitamura et al.
Wlodek and Rommelspacher
Wlodek and Rommelspacher
Roberts et al.
Lucas et al.
Srinivasan et al.
Crankshaw et al.

1989
1997a
1997b
1992
2000
2001
2002

Mice (in vivo)
HepG2 cells (in vitro)
2-Methyl-thiazolidine-2,4-dicarboxylic acidMice (in vivo)
Mice (in vivo)
Ribose cysteine
Mice (in vivo)
2(R,S)-n-Propylthiazolidine-4(R)-carboxylic acid Mice (in vivo)
Mice (in vivo)
N,S-bis-Acetyl-L-cysteine

References

Minton et al., 1988; Crippin, 1993; Li et al., 2002). In
contrast, susceptibility to APAP liver injury is decreased by pretreatment with CYP inhibitors suchas piperonyl butoxide and 4-methylpyrazone (Brady
et al., 1991; Brennan et al., 1994; Kucukardali et al.,2002, Hazai et al., 2002).

Fasting
Fasting has also been considered a risk factor for
APAP-induced hepatotoxicity. Whitcomb andBlock (1994) reported a clinical study which foundthat APAP-induced hepatotoxicity after an overdose is more likely to be associated with recent fasting than recent alcohol use. In rats, fasting causes
severe malnutrition, and the capacity of hepaticconjugation of APAP with glucuronide and sulfate
decreases (Price et al., 1986, 1987). Consequently,
more APAP is metabolized by CYPs, resulting in
more NAPQI production. Although NAPQI is detoxified by conjugation with GSH, fasting also decreases hepatic GSH (Langley and Kelly, 1992; Vogt
and Richie, 1993). Therefore, fasting may potentiate
APAP-induced hepatotoxicity.

Protection against APAP-Induced
Hepatotoxicity

Cysteine prodrugs
GSH plays an important role in protecting the liver
against APAP-induced hepatotoxicity, because
NAPQI is detoxified by conjugation with GSH.
Therefore, stimulation of hepatic GSH synthesis isfeasible for prevention of APAP-induced hepatic
injury. GSH, a tripeptide comprising glutamate,cysteine and glycine, is synthesized by a two-stepreaction. The first step is catalyzed by γ-glutamylcysteine synthase to form γ-glutamylcysteine. In
the second step, GSH synthase catalyzes the reaction between glycine and γ-glutamylcysteine toform GSH (Wang and Ballatori, 1998). The firststep catalyzed by γ-glutamylcysteine synthase

Drug interactions
Finally, long-term treatment with drugs that induce
CYPs may increase the risk of APAP-induced liver
damage. Carbamazepine, phenytoine, isoniazid
and troglitazone are considered to induce CYPs,
and therefore long-term use of these drugs enhances
APAP-induced hepatotoxicity (Smith et al., 1986;20

Acetaminophen-induced hepatotoxicity(rate-limiting enzyme) is regulated by feedback
inhibition of GSH (Richman and Meister, 1975).
When GSH is consumed and the regulation of feedback inhibition is lost, the availability of cysteine asa precursor can become the limiting factor for GSH
synthesis (Wang and Ballatori, 1998).
Various cysteine prodrugs have been reported
to protect the liver from APAP-induced hepatotoxicity in experiments using animals or cultured cells(Table 1). The mechanism responsible for this protection may be metabolism of these prodrugs to Lcysteine, which is incorporated into hepatic GSH(Lauterburg et al., 1983; Corcoran and Wong, 1986;
Hazelton et al., 1986; Roberts et al., 1987).
Among these cysteine prodrugs, N-acetylcysteine (NAC) is the most widely used antidote for
APAP overdose in clinical practice. Intravenous
administration of NAC to patients with APAP poisoning is effective for preventing APAP-induced
hepatotoxicity (Prescott, 1981; Smilkstein et al.,1991). Prescott (1981) reported that NAC given
intravenously 8 to 10 h after APAP ingestion was

effective for preventing liver damage, hepatic failure, renal damage and death. Oral administrationof NAC to patients with APAP poisoning was also
shown to be effective for preventing liver damage(Rumack et al., 1981; Smilkstein et al., 1988).
Analysis of the national multicenter study (1976 to
1985) in the United States reported by Smilkstein etal. (1988) demonstrated that oral treatment with
NAC was protective regardless of the initial plasma
APAP concentration when given within 8 h of
APAP ingestion. They concluded that NAC treatment should be started within 8 h of APAP ingestion, but that treatment is still indicated at least as
late as 24 h after ingestion. As shown in Fig. 1, wedemonstrated that oral treatment of mice with NAC
1 h after APAP administration significantly suppressed the increase in plasma ALT activity as anindex of liver injury. The most appropriate routeand dose regimen of NAC for APAP-induced hepatotoxicity is still controversial. However, retrospective and prospective studies suggest that NAC
treatment may be effective for fulminant liver failure after APAP overdose (Harrison et al., 1990;
Keays et al., 1991).

(IU/L)

Plasma ALT activity

4000

CYP2E1 inhibitors

3000

The metabolism of APAP by CYP leads to the formation of NAPQI as discussed in the “Reactive intermediate metabolite of APAP” section. Therefore, CYP inhibitors may be effective for APAPinduced hepatotoxicity. Of the CYP enzymes,
CYP2E1 plays the most important role in metabolizing APAP. Studies of CYP2E1 knockout mice
have revealed that CYP2E1 is the most importantfactor in APAP biotransformation (Lee et al., 1996;
Zaher et al., 1998).
We have demonstrated that S-allylmercaptocysteine (SAMC), an organosulfur compound inaged garlic extract, protects mice from APAPinduced liver injury. Oral treatment with SAMC
(100–200 mg/kg) suppressed the increase in plasma
ALT activity (Fig. 1) and the hepatic necrosis after
APAP overdose, and reduced APAP-induced mortality. The mechanism underlying this protection
involved the suppression of CYP2E1 activity

2000

1000

0

*
**
Intact Vehicle SAMC NAC+ APAP

Fig. 1. Effect of SAMC or NAC treatment on APAPinduced liver injury in male ddY mice. APAP (500 mg/kg) was orally administered to mice, and then SAMC
(100 mg/kg) or NAC (80 mg/kg) was given orally 1 h
after APAP administration. The mice were anesthetizedwith diethyl ether and blood samples were taken fromthe right ventricle with a heparinized syringe 6 h after
APAP. Plasma ALT activity was determined spectrophotometrically with a commercially available kit. The
data are expressed as means ± SE for 4 to 5 animals.
Plasma ALT activity in intact mice was 8 ± 1 IU/L (Note:these values are too small to visualize). *,** Significantly
different from the APAP + vehicle group (P < 0.05 and
0.01, respectively). Unpublished data, Sumioka et al.

21

I. Sumioka et al.

(Nakagawa et al., 1989; Sumioka et al., 1998, 2001).
Diallyl sulfide and phenethyl isothiocyanate, compounds derived from garlic and cruciferous plants,
respectively, have also been shown to inhibit
CYP2E1 activity, and thereby to exhibit a protective effect against APAP-induced hepatotoxicity(Hu et al., 1996; Li et al., 1997). A synthetic compound, 2-(allylthio)pyradine, inhibited CYP2E1mRNA and protein expression, resulting in prevention of APAP-induced liver damage (Kim et al.,1997). SAMC or diallyl sulfide treatment, not only
before but also after APAP overdose, can protect
animals from APAP-induced hepatotoxicity (Hu etal., 1996; Sumioka et al., 2001), suggesting thatCYP2E1 inhibitor could serve as an antidote for
APAP overdose.
CYP2E1 is also known to be a key bioactivator of various carcinogens, such as azoxymethane
and N-nitrosodimethylamine (Yoo et al., 1990; Sohn
et al., 2001). In this context, a CYP2E1 inhibitor
could be a potential inhibitor of carcinogenesis byenvironmental carcinogens (Smith et al., 1995), andits clinical application could be extended fromtreatment of APAP-induced hepatotoxicity to treatment of all CYP2E1-related disorders.

Antioxidants
Oxidative stress followed by lipid peroxidation is
also thought to contribute to the initiation or progression of APAP-induced hepatotoxicity (Wendel
et al., 1979; Albano et al., 1983, 1985), as describedin the “Oxidative stress” section.
We have demonstrated that intravenous pretreatment with the lipid-soluble antioxidants, CoQ10(5 mg/kg) and α-Toc (20 mg/kg), can limit hepatic
injury produced by APAP overdose (Amimoto et al.,1995). CoQ10 is converted to CoQ10H2 in liver cells,
and thereafter acts as an antioxidant (Matsura et al.,1992a, 1992b). These lipid-soluble antioxidants
suppressed the increase in plasma ALT activity(Fig. 2) and hepatic TBARS level without affecting
the hepatic GSH level. L-Ascorbic acid and its esters(Raghuram et al., 1978; Lake et al., 1981; Mitra et al.,1991) and β-carotene (Baranowitz and Maderson,1995) also exert protective action against APAPinduced hepatotoxicity. In addition, antioxidant enzymes such as superoxide dismutase and catalase
protect the liver from APAP-induced injury (Kyleet al., 1987; Nakae et al., 1990). Iron is well known
to play a major role in lipid peroxidation (Halliwell
and Gutteridge, 1990). Sakaida et al. (1995) reported that deferoxamine, an iron chelator, can protect
against APAP-induced hepatotoxicity.
Given that these antioxidants are effective
when administered before APAP, taking APAP
with them might be helpful for protecting against
toxicity by misadventure, especially as antioxidants
in nature, such as CoQ10, α-Toc, ascorbic acid andβ-carotene, have few side effects.

Plasma ALT activity(IU/L)
1500
1200
900
600
300
0

*

*

Intact Vehicle CoQ10 Vehicle α-Toc+ APAP+ APAP

Fig. 2. Effect of CoQ 10 or α-Toc pretreatment on
APAP-induced liver injury in male ICR mice. CoQ10 (5mg/kg), α-Toc (20 mg/kg) or vehicle was intravenously
injected into mice 12 h before intraperitoneal administration of APAP (400 mg/kg). The mice were anesthetized with diethyl ether and blood samples were takenfrom the right ventricle with a heparinized syringe 3 hafter APAP. Plasma ALT activity was determined spectrophotometrically with a commercially available kit.
The data are expressed as means ± SE for at least 5 animals. * Significantly different from the APAP + vehicle
group (P < 0.05). From Amimoto et al., 1995 with somemodifications.

Heat shock protein (HSP) inducers
HSP is ubiquitous in nature, and expressed in bothprokaryotic and eukaryotic cells in response to avariety of stresses (Hendrick and Hartl, 1993). Initially, HSP was identified as a cellular response
protein to hyperthermia, but HSP induction has also
been observed after treatment of cells with a num22

Acetaminophen-induced hepatotoxicity

HNCOCH3

HNCOCH3

Sulfateconjugation

COOH

UDP-glucuronyltransferase

Sulfotransferase

NHCOCH3

OO
OH
HO

OSO3H

OH

OH

APAP

Glucuronide
conjugation

CYPs (2E1) inhibitors
CYPs (2E1)
OH-N-COCH3NHCOCH3

O2–,H2O2
Cysteine prodrugs
O•OH

GSH synthesis
HNCOCH3

Antioxidants
NCOCH3

GSH

Lipid peroxidation
SG
OH

O

Glutathione
conjugation

NAPQI
Covalent
binding

HNCOCH3

Detoxication

S
OH

Hepatic
protein

HSP25
HSP70iLiver injury

Repair of damaged
hepatic protein

HSP inducers

Fig. 3. The metabolic pathways of APAP, and the mechanisms of, and protection against, APAP-induced hepatotoxicity. +, stimulation; –, inhibition.

ber of chemical toxicants. APAP-induced hepatotoxicity has been shown to increase the expressionof HSP25 and HSP70i (Salminen et al., 1997a). It is
generally accepted that the stimulus for increased
HSP synthesis in response to stresses is the presence of non-native proteins (Ananthan et al., 1986;
Goff and Goldberg, 1985). In the case of APAPinduced hepatotoxicity, covalent binding of NAPQI
to hepatic macromolecules is thought to play apivotal role in triggering HSP induction (Salminen
et al., 1998). HSP70i together with HSP25 mayfunction as a cytoprotective HSP to repair damaged
protein in the necrotic lesion. The patterns of hepatic HSP25 and HSP70i induction are clearly different. The level of HSP70i increases initially after
APAP overdose, and thereafter the level of HSP25
increases. The induction time after APAP overdose
may differ with the severity of hepatotoxicity. For
23

I. Sumioka et al.

imine cytotoxicity. Mol Pharmacol 1985;28:306–311.
3 Amimoto T, Matsura T, Koyama S, Nakanishi T,
Yamada K, Kajiyama G. Acetaminophen-induced
hepatic injury in mice: the role of lipid peroxidationand effects of pretreatment with coenzyme Q10 and αtocopherol. Free Radic Biol Med 1995;19:169–176.
4 Ananthan J, Goldberg AL, Voellmy R. Abnormal
proteins serve as eukaryotic stress signals and trigger
the activation of heat shock genes. Science 1986;232:
522–524.
5 Banda PW, Quart BD. The effect of mild alcohol consumption on the metabolism of acetaminophen in man.
Res Commun Chem Pathol Pharmacol 1982;38:57–70.
6 Baranowitz SA, Maderson PF. Acetaminophen toxicity is substantially reduced by beta-carotene in mice.
Int J Vitam Nutr Res 1995;65:175–180.
7 Belay ED, Bresee JS, Holman RC, Khan AS, Shahriari
A, Schonberger LB. Reye’s syndrome in the UnitedStates from 1981 through 1997. N Engl J Med 1999;
340:1377–1382.
8 Boyd EM, Bereczky GM. Liver necrosis from paracetamol. Br J Pharmacol 1966;26:606–614.
9 Brady JT, Birge RB, Khairallah EA, Cohen SD. Posttreatment protection with piperonyl butoxide against acetaminophen hepatotoxicity is associated with changes inselective but not total covalent binding. Adv Exp Med
Biol 1991;283:689–692.
10 Brennan RJ, Mankes RF, Lefevre R, Raccio-Robak N,
Baevsky RH, DelVecchio JA, et al. 4-Methylpyrazole
blocks acetaminophen hepatotoxicity in the rat. Ann
Emerg Med 1994;23:487–494.
11 Chen LH, Lin SM. Modulation of acetaminopheninduced alterations of antioxidant defense enzymes byantioxidants or glutathione precursors in cultured
hepatocytes. Biochem Arch 1997;13:113–125.
12 Clements JA, Critchley JA, Prescott LF. The role ofsulphate conjugation in the metabolism and dispositionof oral and intravenous paracetamol in man. Br J Clin
Pharmacol 1984;18:481–485.
13 Corcoran GB, Racz WJ, Smith CV, Mitchell JR.
Effects of N-acetylcysteine on acetaminophen covalentbinding and hepatic necrosis in mice. J Pharmacol Exp
Ther 1985;232:864–872.
14 Corcoran GB, Wong BK. Role of glutathione in prevention of acetaminophen-induced hepatotoxicity by
N-acetyl-L-cysteine in vivo: studies with N-acetyl-Dcysteine in mice. J Pharmacol Exp Ther 1986;238:54–
61.
15 Crankshaw DL, Berkeley LI, Cohen JF, Shirota FN,
Nagasawa HT. Double-prodrugs of L-cysteine: differential protection against acetaminophen-induced hepatotoxicity in mice. J Biochem Mol Toxicol 2002;16:
235–244.
16 Crippin JS. Acetaminophen hepatotoxicity: potentiation by isoniazid. Am J Gastroenterol 1993;88:590–
592.
17 Dahlin DC, Miwa GT, Lu AYH, Nelson SD. N-Acetylp-benzoquinone imine: a cytochrome P-450-mediatedexample, at a lethal dose, HSP25 could be detected
24 h after APAP treatment (Sumioka et al., 2004),
whereas, at a necrotic but not lethal dose, it was detectable 6 h after APAP treatment (Salminen et al.,1997a). Salminen et al. (1997b) reported that intraperitoneal pretreatment with amphetamine (15 mg/kg) in mice causes an acute rise in core body temperature to 40˚C for at least 1 h, increases the levelsof HSP25 and HSP70i, and protects the liver from
APAP-induced hepatotoxicity. We have reported
that the level of expression of hepatic HSP25 may
be a crucial determinant of the fate of mice exposed
to APAP insult, because mortality following APAP
overdose declined rapidly after the appearance ofhepatic HSP25 (Sumioka et al., 2004). These findings suggest that HSP (HSP25 and 70i) inducer
might be a new type of antidote for APAP overdose.

Conclusion
APAP overdose causes hepatotoxicity, which issometimes fatal. This hepatotoxicity is initiated by
the metabolic activation of APAP to NAPQI, areactive metabolite. ROS generation and subsequent lipid peroxidation during APAP biotransformation is likely to exacerbate APAP insult.
Even at therapeutic doses, APAP hepatotoxicity
can occur when there are risk factors such as alcohol abuse, fasting and concomitant drug use. NAC,
one of the cysteine prodrugs, has been traditionallyused to prevent the development of hepatotoxicity
following a significant overdose of APAP. In addition to NAC, HSP inducers as well as CYP2E1 inhibitors and antioxidants may also be antidotes for
APAP overdose, on the basis of the mechanisms
underlying APAP toxicity (Fig. 3).

References
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Paracetamol-stimulated lipid peroxidation in isolated
rat and mouse hepatocytes. Chem Biol Interact 1983;
47:249–263.
2 Albano E, Rundgren M, Harvison PJ, Nelson SD,
Moldeus P. Mechanisms of N-acetyl-p-benzoquinone

24

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oxidation product of acetaminophen. Proc Natl Acad
Sci USA 1984;81:1327–1331.
18 Davidson DG, Eastham WN. Acute liver necrosis following overdose of paracetamol. Br Med J 1966;
5512:497–499.
19 Davis DC, Potter WZ, Jollow DJ, Mitchell JR. Species
differences in hepatic glutathione depletion, covalentbinding and hepatic necrosis after acetaminophen. LifeSci 1974;14:2099–2109.
20 de Vries J. Hepatotoxic metabolic activation of paracetamol and its derivatives phenacetin and benorilate:oxygenation or electron transfer? Biochem Pharmacol
1981;30:399–402.
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Received and accepted March 30, 2004
Corresponding author: Tatsuya Matsura, MD, PhD

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