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Prednisolone stimulates hepatic glutathione synthesis in mice.
Protection by prednisolone against acetaminophen hepatotoxicity in
Speck RF; Schranz C; Lauterburg BH
Department of Clinical Pharmacology' University of Bern' Switzerland.
J Hepatol, 18(1):62-7 1993 Apr
Mediators of inflammation modulate the extent of hepatocellular
necrosis following the administration of hepatotoxins. Since
corticosteroids interfere with the generation of some of these
mediators they might thus protect against the hepatotoxicity of drugs
such as acetaminophen. To test this hypothesis mice were pretreated
with two doses of prednisolone (10 and 20 mg/kg i.p.' 17 and 2 h'
respectively) prior to a hepatotoxic dose of 375 mg/kg acetaminophen
and the metabolism and toxicity of acetaminophen were assessed.
Twenty-four hours after acetaminophen the activity of ALT in plasma
(737 vs. 6775 U/l) and the extent of hepatocellular necrosis (4 vs. 45%
necrotic hepatocytes) were significantly lower in
prednisolone-pretreated mice. Prednisolone pretreatment resulted in
decreased covalent binding of the toxic metabolite in vivo and an
increased urinary excretion of glutathione-derived conJugates of
acetaminophen' indicating an enhanced detoxification of the reactive
metabolite by glutathione. Nevertheless' hepatic glutathione was less
depleted by acetaminophen in the prednisolone group' indicating an
increased capacity to resynthesize glutathione. This was confirmed in
experiments with diethyl maleate which depletes hepatic glutathione
without causing cell inJury. Following the administration of diethyl
maleate to fed and fasted mice' hepatic glutathione was depleted to the
same extent after 45 min' but was significantly higher after 2.5 h in
prednisolone-pretreated mice. The present results indicate that
prednisolone increases the capacity to replete depleted hepatic
glutathione stores in mice.

Efficacy of oral versus intravenous N-acetylcysteine in acetaminophen
overdose: results of an open-label, clinical trial [see comments
Perry HE; Shannon MW
Division of Emergency Medicine, Children's Hospital, Boston,
Massachusetts, USA.
J Pediatr, 132(1):149-52 1998 Jan
We compared the clinical course of pediatric patients (n = 25) with
acetaminophen poisoning treated with an investigational intravenous
preparation of N-acetylcysteine (IV-NAC) with that of historical
control subjects (n = 29) treated with conventional oral NAC (O-NAC)
therapy. Patients received IV-NAC for 52 hours; historical control
subjects received O-NAC (72 hours). There were no significant
intergroup differences between treatment groups in age (15.5 vs 15.9
years), gender (88% vs 90% female) or distribution of risk categories
(probable risk, 12 vs 15; high risk; 13 vs 14). The peak prothrombin
time was significantly higher in the IV-NAC group (14.2 vs 13.6
seconds; p = 0.048). Mean treatment delay was significantly longer in
the IV-NAC group (14.4 vs 10.4 hours; p = 0.001). Hepatoxicity was
noted in two (8.0%) patients in the IV-NAC treatment group and two
(6.9%) patients in the O-NAC group. All patients recovered. Our results
indicate that 52 hours of intravenous NAC is as effective as 72 hours
of oral NAC.

Selective protein arylation and acetaminophen-induced hepatotoxicity.
Cohen SD; Khairallah EA
Department of Pharmaceutical Sciences' University of Connecticut'
Storrs 06269' USA.
Drug Metab Rev, 29(1-2):59-77 1997 Feb-May
More than 20 years have passed since the early reports of acute
hepatotoxicity with APAP overdose. During that period investigative
research to discover the "mechanism" underlying the toxicity has been
conducted in many species and strains of intact animals as well as in a
variety of in vitro and culture systems. Such work has clarified the
primary role of biotransformation and the protective role of GSH.
Understanding the former provides explanations for the toxic
interactions which may occur with alcohol or other xenobiotics' while
understanding of the latter led to the development of antidotes for the
treatment of acute poisoning. acetaminophen (APAP)-induced
hepatotoxicity: roles for protein arylation. Initiating events in
toxicity require biotransformation of APAP to NAPQI followed by
arylation of several important proteins with subsequent alteration of
protein structure and function. The immediate consequence of the
alterations is detectable in several organelles and these may represent
multiple initiating events which are depicted as acting in concert to
cause cell inJury (large arrowheads). Arylation of cytosolic 58-ABP
with subsequent translocation to the nucleus is depicted as a possible
signaling mechanism for determining outcome at the cell or organ level
(within dotted boundary). For simplicity NAPQI`s potentials for
oxidizing protein sulfhydryls and direct binding to DNA have been
omitted. Significant light has also been shed on the biochemical and
cellular events which accompany APAP-induced hepatotoxicity. However'
such studies have not identified a unique mechanism of toxicity that is
universally accepted. The recent identification of several protein
targets which become arylated during toxicity--along with the findings
that arylation of some of those target proteins results in loss of
protein function--demonstrates that covalent binding does' indeed' have
biological consequences and is not merely an indicator of the fleeting
presence of reactive electrophiles. These observations further suggest
that multiple independent insults to the cell may be involved in
toxicity. it is now apparent that the concept of a multistage process
that involves both initiation and progression events is appropriate for
APAP toxicity' and it is unlikely that a unique initiating event will
ever be identified. In light of recent findings it is more likely that
a number of such cellular events occur very early after toxic
overdosage' and that they collectively set in motion and perpetuate the
biochemical' cellular' and molecular processes which will determine
outcome. The importance of 58-ABP arylation with early' apparently
selective' translocation to the nucleus remains to be elucidated. To
date there is nothing to suggest that this represents an initiating
event in toxicity. rather it is plausible that the translocation may
play a role in signaling electrophile presence and in calling for
cellular defense against electrophile insult. This is reflected in the
hypothetical model presented in Fig. 3. Critical experimental testing
of this model will advance our understanding of the cellular and
molecular responses to toxic electrophile insult.

Zonation of acetaminophen metabolism and cytochrome P450 2E1-mediated
toxicity studied in isolated periportal and perivenous hepatocytes.
Anundi I; L ahteenm aki T; Rundgren M; Moldeus P; Lindros KO
Biomedical Research Center' ALKO Ltd.' Helsinki' Finland.
Biochem Pharmacol, 45(6):1251-9 1993 Mar 24
To study the mechanism of centrilobular damage developing in the
centrilobular region after high doses of acetaminophen (APAP)' its
metabolism and toxicity were compared in periportal and perivenous
hepatocytes isolated by digitonin/collagenase perfusion. Contrary to
earlier reports' based on perfusions' no evidence for a periportal
dominance of APAP sulfation could be observed. Glucuronidation' the
dominant pathway of conJugation at high (5 mM) APAP concentration' was
faster in perivenous cells. During primary culture' prolonged exposure
(> or = 24 hr) to 5 mM APAP damaged perivenous cells' with a higher
P450 2E1 level than periportal cells. When cells were isolated from
ethanol-pretreated rats' to induce P450 2E1 levels specifically in the
perivenous region' perivenous hepatocytes exhibited enhanced APAP
vulnerability and extensive glutathione depletion. In contrast'
corresponding periportal cells retained good viability. Isoniazid' an
inhibitor of cytochrome P450 2E1' protected cells against APAP toxicity
and prevented glutathione depletion. Induction of P450 2E1 also caused
a 3-fold increase in the covalent binding of reactive intermediates
from [14C APAP' and this increase was mainly confined to perivenous
cells. These results indicate that in rat liver there is only slight
perivenous zonation of APAP conJugation and suggest that zone-specific
APAP activation' mediated by the regional expression of
ethanol-inducible cytochrome P450 2E1' is responsible for the
characteristic centrilobular liver damage elicited by APAP.

In vivo murine studies on the biochemical mechanism of acetaminophen
Wells PG; Wilson B; Winn LM; Lubek BM
Faculty of Pharmacy' University of Toronto' ON' Canada.
Can J Physiol Pharmacol, 73(8):1123-9 1995 Aug
C57BL/6 and DBA/2 mice are' respectively' susceptible and resistant
both to the induction of aryl hydrocarbon hydroxylase (cytochrome P450
1A1' or CYP1A1) and to the cataractogenicity of acetaminophen' which
may involve its bioactivation to a toxic reactive intermediate'
catalysed by P450 and (or) prostaglandin H synthase (PHS). Following
induction of P450 using beta-naphthoflavone' the cataractogenicity of
acetaminophen (400 mg/kg ip) in C57BL/6 mice was reduced by
pretreatment with the P450 inhibitors SKF 525A and metyrapone' the
glutathione precursor N-acetylcysteine' the antioxidant vitamin E' and
the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (p <
0.05). acetaminophen (200 mg/kg) cataractogenicity was enhanced by
pretreatment with the glutathione depletor diethyl maleate (DEM) and
the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine
(BSO) (p < 0.05). No significant effect on acetaminophen
cataractogenicity was observed using the PHS cyclooxygenase inhibitors
aspirin or naproxen' or the glutathione reductase inhibitor
1'3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Accordingly' acetaminophen
cataractogenicity in C57BL/6 mice does not appear to be dependent upon
bioactivation by PHS. In DBA/2 mice treated with beta-naphthoflavone' a
high dose of acetaminophen (750 mg/kg ip) was not cataractogenic' even
after pretreatment with DEM' BSO' or BCNU. The resistance of DBA/2 mice
to acetaminophen cataractogenesis' despite concomitant pretreatments
with an inducer of P450 and several agents that interfere with
glutathione-dependent detoxifying pathways' suggests differences in
this strain involving cytoprotective pathways subsequent to
acetaminophen bioactivation and detoxification of the cataractogenic
reactive intermediate. These results indicate that acetaminophen
cataractogenicity in C57BL/6 mice results from P450-catalysed
bioactivation of acetaminophen to a reactive intermediate' possibly a
benzoquinone imine and (or) a free radical' the toxicity of which is
reduced by glutathione-dependent reactions.

Tylenol Extended Relief overdose.
Cetaruk EW; Dart RC; Hurlbut KM; Horowitz RS; Shih R
Rocky Mountain Poison Center' Denver' CO' USA.
Ann Emerg Med, 30(1):104-8 1997 Jul
In this report we describe the toxicokinetics of the Tylenol Extended
Relief (TER) preparation of acetaminophen in human overdose. We
collected 41 cases of TER overdose from five regional poison centers.
Patients who met the following criteria were studied: a single
ingestion of TER alone; confirmed time of ingestion; at least four
acetaminophen determinations; and normal concentrations of liver
function enzymes. With the exception of standard decontamination
measures' treatment with N-acetylcysteine (NAC) if any acetaminophen
level was above the treatment line of the Rumack-Matthew nomogram' and
additional acetaminophen determinations' no interventions were
recommended. Our study group comprised 13 patients' 12 female and 1
male' with single overdoses of 10.4 to 65 g TER. The acetaminophen
elimination half-life was 3.1 +/- .8 hours (mean +/- SD; range' 1.3 to
4.0 hours; n = 12). The elimination phase for patients 2' 3' 4' 6' 8'
9' 11' 13 was delayed until 8.0 +/- 2.8 hours (range' 5 to 14 hours)
after ingestion. Patients 3' 8' and 11--who had initial acetaminophen
levels below the "possible toxicity" line of the Rumack-Matthew
nomogram--later had acetaminophen levels above this line. No patient
demonstrated a late or second acetaminophen peak. We conclude that the
elimination half-life of TER acetaminophen is similar to that reported
in overdose of immediate-release acetaminophen overdose. In a subgroup
of patients' drug absorption continued beyond the 2 to 4 hours
previously reported in immediate-release acetaminophen overdose. On the
basis of our data' the use of a single 4-hour acetaminophen
determination may lead to failure to recognize patients with
potentially toxic TER ingestion. Until more toxicokinetic data are
available' a reasonable approach would be to obtain at least one
additional acetaminophen determination at least 4 to 6 hours after the
first' if the first is obtained 4 to 8 hours after ingestion. NAC
treatment should be initiated if either level is above the nomogram
line but not if both levels fall below the nomogram line.

acetaminophen-induced hepatotoxicity. Analysis of total covalent
binding vs. specific binding to cysteine.
Matthews AM; Roberts DW; Hinson JA; Pumford NR
Division of Toxicology' University of Arkansas for Medical Sciences'
Little Rock 72205' USA.
Drug Metab Dispos, 24(11):1192-6 1996 Nov
acetaminophen-induced hepatotoxicity is believed to be mediated by
covalent binding of the reactive metabolite N-acetyl-p-benzoquinone
imine to essential proteins in liver. It has been shown that the
primary reaction of this metabolite with hepatic proteins is the
formation of 3-(cysteine-S-yl)-acetaminophen adducts. The importance of
covalent binding to other amino acids that may be formed by reaction of
N-acetyl-p-benzoquinone imine with protein is unclear. Previously' we
developed immunochemical assays for the acetaminophen cysteine adducts
by immunizing animals with the conJugate
3-(N-acetylcystein-S-yl)acetaminophen-keyhole limpet hemocyanin'
wherein the carboxyl group of the N-acetyl-cysteine moiety was coupled
to amino groups on the protein. A very sensitive and specific
immunochemical assay was developed for acetaminophen specifically bound
to cysteine groups on protein [3-(cystein-S-yl)acetaminophen protein
adducts . Analysis of protein adducts indicated that after toxic doses'
acetaminophen covalently bound at high levels to cysteine residues on a
relatively small number of hepatic proteins. In the present work' a new
antiacetaminophen antiserum was prepared by immunizing mice with
4-acetamidobenzoic acid coupled to keyhole limpet hemocyanin.
Competitive ELISA data indicate that the resulting antiserum has
excellent recognition of acetaminophen and related arylacetamide
derivatives. Using this new antiserum' Western blot analyses of liver
proteins from acetaminophen-intoxicated mouse livers were performed and
compared with similar assays using the
anti-3-(cystein-S-yl)acetaminophen antiserum. Visual and densitometric
analyses of the Western blots indicate that the two antisera detect the
same primary acetaminophen protein adducts; however' minor differences
in the intensity of certain bands were observed. These differences may
represent either differences in antibody accessibility to
3-(cystein-S-yl)acetaminophen adducts or differences in the proportion
of acetaminophen bound to cysteine vs. binding to other amino acids.

Acute renal failure due to acetaminophen ingestion: a case report and
review of the literature.
Blakely P; McDonald BR
Division of Nephrology and Hypertension' UCSD Medical Center' La Jolla
92093-0623' USA.
J Am Soc Nephrol, 6(1):48-53 1995 Jul
acetaminophen is the most commonly reported drug overdose in the United
States. Acute renal failure occurs in less than 2% of all acetaminophen
poisonings and 10% of severely poisoned patients. At the therapeutic
dosages' acetaminophen can be toxic to the kidneys in patients who are
glutathione depleted (chronic alcohol ingestion' starvation' or
fasting) or who take drugs that stimulate the P-450 microsomal oxidase
enzymes (anticonvulsants). Acute renal failure due to acetaminophen
manifests as acute tubular necrosis (ATN). ATN can occur alone or in
combination with hepatic necrosis. The azotemia of acetaminophen
toxicity is typically reversible' although it may worsen over 7 to 10
days before the recovery of renal function occurs. In severe overdoses'
renal failure coincides with hepatic encephalopathy and dialysis may be
required. Recognition of acetaminophen nephropathy requires the
following: (1) a thorough drug history' including over-the-counter
medications such as Tylenol or Nyquil; (2) knowledge of the risk
factors that lessen its margin of safety at therapeutic ingestions'
i.e.' alcoholism; and (3) consideration of acetaminophen in the
differential diagnosis of patients who present with combined hepatic
dysfunction and ATN.

The killing of cultured hepatocytes by N-acetyl-p-benzoquinone imine
(NAPQI) as a model of the cytotoxicity of acetaminophen.
Harman AW; Kyle ME; Serroni A; Farber JL
Department of Pathology' Thomas Jefferson University' Philadelphia' PA
Biochem Pharmacol, 41(8):1111-7 1991 Apr 15
The killing of isolated hepatocytes by N-acetyl-p-benzoquinone imine
(NAPQI)' the maJor metabolite of the oxidation of the hepatotoxin
acetaminophen' has been studied previously as a model of liver cell
inJury by the parent compound. Such studies assume that the toxicity of
acetaminophen is mediated by NAPQI and that treatment with exogenous
NAPQI reproduces the action of the endogenously produced product. The
present study tested these assumptions by comparing under identical
conditions the toxicity of acetaminophen and NAPQI. The killing of
hepatocytes by acetaminophen was mediated by oxidative inJury. Thus' it
depended on a cellular Source of ferric iron; was potentiated by
1'3-bis(2-chloroethyl)-1-nitrosourea (BCNU)' an inhibitor of
glutathione reductase; and was sensitive to antioxidants. By contrast'
the cytotoxicity of NAPQI was not prevented by chelation of ferric
iron; was unaffected by BCNU; and was insensitive to antioxidants.
Thus' the killing of cultured hepatocytes by NAPQI occurs by a
mechanism different from that of acetaminophen. The killing by NAPQI
was preceded by a collapse of the mitochondrial membrane potential and
a depletion of ATP. Monensin potentiated the cell killing' and
extracellular acidosis prevented it. These manipulations are
characteristic of the toxicity of mitochondrial poisons' and are
without effect on the depletion of ATP and the loss of mitochondrial
energization. Thus' mitochondrial de-energization by a mechanism
unrelated to oxidative stress is a likely basis of the cell killing by
NAPQI. It is concluded that treatment of cultured hepatocytes with
NAPQI does not model the cytotoxicity of acetaminophen in these cells.


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