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acetaminophen toxicity to cultured rat embryos.
Weeks BS; Gamache P; Klein NW; Hinson JA; Bruno M; Khairallah E
Center for Environmental Health' Department of Animal Science'
University of Connecticut' Storrs.
Teratog Carcinog Mutagen, 10(5):361-71 1990
We tested the effects of acetaminophen on cultured rat embryo
development. When added directly to culture media at 300 microM' a
concentration approximately twice the human therapeutic blood level'
acetaminophen caused abnormalities in the cultured embryos. Sera from
both rats and monkeys following gavage with acetaminophen were also
toxic to cultured embryos. The sera toxicities were related to
acetaminophen concentrations' and the toxicity could be removed by
serum dialysis. With regard to the metabolism of acetaminophen'
glutathione levels in the yolk sac decreased in a concentration related
fashion with addition of the drug. Also' buthionine sulfoximine' an
inhibitor of glutathione synthesis' appeared to enhance acetaminophen
embryo toxicity' and N-acetylcysteine' a glutathione precursor'
appeared to protect embryos from acetaminophen toxicity. These results
suggested that acetaminophen embryo toxicity resulted from direct
exposure of embryos to acetaminophen and not a maternal metabolite.

Predictive value of liver slices for metabolism and toxicity in vivo:
use of acetaminophen as a model hepatotoxicant.
Miller MG; Beyer J; Hall GL; deGraffenried LA; Adams PE
Department of Environmental Toxicology' University of California' Davis
Toxicol Appl Pharmacol, 122(1):108-16 1993 Sep
To establish the usefulness of liver slices as predictive models for in
vivo metabolism and toxicity' acetaminophen was used as a model
hepatotoxicant for which the role of metabolism in toxicity is well
documented. acetaminophen was incubated with liver slices prepared from
the rat and hamster since these species differ in susceptibility to
acetaminophen-induced hepatotoxicity. Formation of acetaminophen
metabolites (sulfate' glucuronide' and glutathione conJugate)' slice
glutathione levels' and slice histopathology were assessed.
acetaminophen (0.5' 1' and 2 mM) induced a dose-dependent depletion of
glutathione in hamster slices (sensitive species) but not rat slices
(insensitive species). Formation of the acetaminophen toxic metabolite'
as measured by glutathione conJugate formation' was much lower in rat
slices compared with hamster slices. Rat slices also showed greater
activity of the nontoxic sulfation and glucuronidation pathways. These
data predicted an 11-fold greater susceptibility to toxicity in the
hamster based on the proportion of the dose metabolized to the reactive
species. In vivo data' using hepatic glutathione depletion as an
indicator of toxic metabolite formation' would predict a similar
difference (13.3-fold) between the hamster and the rat. To ascertain if
the characteristic centrilobular lesion induced by acetaminophen in
vivo could be reproduced in vitro' liver slice histopathology was
assessed 6 and 12 hr after a 2-hr treatment with acetaminophen (2 mM).
Discrete damage to the centrilobular regions of the liver were noted in
hamster but not rat slices. Overall' based on metabolite formation'
glutathione depletion' and slice histopathology' the liver slices were
excellent predictors of acetaminophen hepatotoxicity in vivo.

Ca2+ antagonists inhibit DNA fragmentation and toxic cell death induced
by acetaminophen.
Ray SD; Kamendulis LM; Gurule MW; Yorkin RD; Corcoran GB
Toxicology Program' College of Pharmacy' University of New Mexico'
Albuquerque 87131-1066.
FASEB J, 7(5):453-63 1993 Mar
Ca2+ accumulates in the nucleus and DNA undergoes enzymatic cleavage
into internucleosome-length fragments before acetaminophen and
dimethylnitrosamine produce hepatic necrosis in vivo and toxic cell
death in vitro. However' Ca(2+)-endonuclease fragmentation of DNA is
characteristic of apoptosis' a type of cell death considered
biochemically and functionally distinct from toxic cell death. The
present studies investigate DNA fragmentation as a critical event in
toxic cell death by testing whether the Ca(2+)-calmodulin antagonist
chlorpromazine and the Ca2+ channel blocker verapamil prevent
acetaminophen-induced hepatic necrosis by inhibiting Ca2+ deregulation
and DNA damage. acetaminophen overdose in mice produced accumulation of
Ca2+ in the nucleus (358% of control) and fragmentation of DNA (250% of
control) by 6 h' with peak release of ALT occurring at 12-24 h (38'000
U/l). Pretreatment with chlorpromazine prevented increases in nuclear
Ca2+ and DNA fragmentation and nearly abolished biochemical evidence of
toxic cell death. Verapamil pretreatment also decreased Ca2+
accumulation and DNA damage while attenuating liver inJury. The Ca2+
antagonists did not protect against toxic cell death through
hypothermia because neither produced the delay in toxicity that is
customarily associated with hypothermia. Nor did chlorpromazine or
verapamil protect through inhibiting acetaminophen bioactivation.
Chlorpromazine failed to diminish glutathione depletion in whole liver
and isolated nuclei. Verapamil (250 microM) also failed to alter
glutathione depletion in whole liver and had no effect on
acetaminophen-glutathione adduct formation by mouse liver microsomes
and by cultured mouse hepatocytes. Collectively' these results support
the hypothesis that Ca(2+)-induced DNA fragmentation plays a
significant role in cell necrosis produced by acetaminophen and may
contribute to toxic cell death caused by other alkylating hepatotoxins.

Aniline and its metabolites generate free radicals in yeast.
Brennan RJ; Schiestl RH
Department of Molecular and Cellular Toxicology' Harvard School of
Public Health' Boston' MA 02115' USA.
Mutagenesis, 12(4):215-20 1997 Jul
The carcinogen aniline is negative in the Ames Salmonella mutagenicity
assay. Aniline does' however' induce intrachromosomal recombination
between repeated sequences in Saccharomyces cerevisiae' resulting in
deletion (DEL) of intervening sequences. We have investigated whether
the generation of oxidative free radical species by aniline and/ or its
metabolites may be responsible for its recombinagenic activity in
yeast. The toxicity and recombinagenicity of aniline in yeast were
greatly reduced in the presence of the free radical scavenger N-acetyl
cysteine. Aniline cytotoxicity was many-fold increased in strains of
S.cerevisiae lacking the antioxidant enzyme superoxide dismutase.
Aniline also induced oxidation of the intracellular free
radical-sensitive reporter compound 2'4-dichlorofluorescin diacetate to
its fluorescent derivative 2'4-dichlorofluorescein in vivo in
S.cerevisiae. The aniline metabolites 4-aminophenol and 2-aminophenol
were significantly more potent inducers of DEL recombination in yeast
than aniline. In contrast' the secondary metabolite 4-acetamidophenol
(acetaminophen) was non-toxic and non-recombinagenic in yeast.
4-Aminophenol and 2-aminophenol were also significantly more toxic than
aniline in a superoxide dismutase deficient yeast strain. 4-aminophenol
was a significantly more potent oxidizer of 2'4-dichlorofluorescin
diacetate than aniline. The Escherichia coli soxS promoter' which is
induced in the presence of redox cycling agents like paraquat' was
induced weakly by aniline at toxic doses. The soxS promoter was
strongly induced by 4-aminophenol and 2-aminophenol. The results
indicate a role for oxidative stress' mediated by generation of
superoxide radical' in the toxicity and recombinagenicity of aniline.
The increased activity of 4-aminophenol and 2-aminophenol suggests that
ring hydroxylation may be an important activating step in this process.

Restoration of cerebrovascular responsiveness to hyperventilation by
the oxygen radical scavenger n-acetylcysteine following experimental
traumatic brain inJury.
Ellis EF; Dodson LY; Police RJ
Department of Pharmacology and Toxicology' Medical College of Virginia'
Virginia Commonwealth University' Richmond.
J Neurosurg, 75(5):774-9 1991 Nov
Previous experiments have shown that' following experimental
fluid-percussion brain inJury' cyclo-oxygenase-dependent formation of
oxygen radicals prevents arteriolar vasoconstriction in response to
hyperventilation. The oxygen radical scavengers superoxide dismutase
and catalase restore normal reactivity; however' they are not routinely
available for clinical use. The present study tested whether
n-acetylcysteine (Mucomyst)' an agent currently available for
acetaminophen toxicity' could be used as a radical scavenger to restore
reactivity after brain inJury. N-acetylcysteine (163 mg/kg) was given
intraperitoneally prior to or 30 minutes after fluid-percussion brain
inJury (2.6 atm) in cats' and reactivity to hyperventilation was tested
1 hour after inJury. The authors found either that pre- or postinJury
administration led to normal reactivity. Additional experiments
supported the hypothesis that n-acetylcysteine is an oxygen radical
scavenger' since it reduced or prevented the free radical-dependent
cerebral arteriolar dilation normally induced by the topical
application of arachidonic acid or bradykinin. The mechanism by which
n-acetylcysteine is effective in trauma may involve direct scavenging
of radicals or stimulation of glutathione peroxidase activity. The
results suggest that n-acetylcysteine may be useful for treatment of
oxygen free radical-mediated brain inJury.

Influence of rifampicin on the toxicity and the analgesic effect of
Dimova S; Stoytchev T
Department of Drug Toxicology' Bulgarian Academy of Sciences' Sofia.
Eur J Drug Metab Pharmacokinet, 19(4):311-7 1994 Oct-Dec
The influence of rifampicin on the toxicity' analgesic effect and
pharmacokinetics of acetaminophen was studied in male albino mice.
Repeated administration of rifampicin (50 mg/kg i.p. daily for 6 days)
shortened hexobarbital sleeping time and increased liver weight'
microsomal cytochrome P-450 and heme contents' NADPH-cytochrome c
reductase and ethylmorphine-N-demethylase activities. Aniline
hydroxylase activity was decreased and glucuronidation of p-nitrophenol
was unaffected. Rifampicin pretreatment changed neither the LD50 of
acetaminophen nor the hepatic glutathione level nor the glutathione
depletion provoked by the toxic dose of acetaminophen (737 mg/kg p.o.).
This suggests that rifampicin has no influence on the amount of
acetaminophen toxic metabolites formed in the liver. Rifampicin
decreased the acetaminophen analgesic effect in mice. Rifampicin
decreased the Cmax' the half-time' the MRT and the AUC of acetaminophen
and accelerated its clearance. The plasma concentration of
acetaminophen glucuronide and acetaminophen sulfate was increased. It
is assumed that the most probable mechanism by which rifampicin
decreases acetaminophen analgesia is the accelerated acetaminophen

Phenylpropanolamine potentiation of acetaminophen-induced
hepatotoxicity: evidence for a glutathione-dependent mechanism.
James RC; Harbison RD; Roberts SM
Center for Environmental and Human Toxicology' University of Florida'
Gainesville 32615.
Toxicol Appl Pharmacol, 118(2):159-68 1993 Feb
Pretreatment of male ICR mice with the adrenergic agonist
phenylpropanolamine (200 mg/kg' ip) resulted in a marked potentiation
of hepatotoxicity produced by acetaminophen (400 mg/kg' ip). Enhanced
liver necrosis with phenylpropanolamine pretreatment was evident both
by measurement of serum aminotransferase activity and by
histopathologic examination. Several lines of experimental evidence
suggest this interaction is a result of the hepatic glutathione
depression produced by alpha-adrenergic compounds' which adds to the
glutathione depression caused by toxic' or nearly toxic' doses of
acetaminophen. First' the potentiation of acetaminophen hepatotoxicity
was time-dependent' being observed only when phenylpropanolamine was
administered as a 3-hr pretreatment and not when given 1 hr before'
with' or 3 hr after acetaminophen. The 3-hr interval between
phenylpropanolamine and acetaminophen doses corresponds to the
characteristic lag period required for alpha-adrenergic agents
(including phenylpropanolamine) to produce significant and maximal
effects on hepatic glutathione content. Second' dose-response
relationships for phenylpropanolamine and acetaminophen were such that
increased toxicity was observed only when the interaction was
sufficient to lower hepatic glutathione concentrations below a level
regarded as critical in preventing acetaminophen-induced
hepatotoxicity. Third' when animals were pretreated with two
nonadrenergic depletors of hepatic glutathione' diethylmaleate (125
mg/kg' ip) or the glutathione synthesis inhibitor buthionine
sulfoximine (222 mg/kg' ip)' at doses producing glutathione depletion
approximating that observed with the adrenergic agents' acetaminophen
hepatotoxicity was potentiated to the same extent. From these
observations it is postulated that a variety of adrenergic compounds
known to deplete hepatic glutathione by a moderate 30-50% may
potentiate the hepatotoxicity of acetaminophen and possibly other
hepatotoxic compounds for which glutathione conJugation is an important
detoxification pathway.

Effects of ethanol and inhibitors on the binding and metabolism of
acetaminophen and N-acetyl-p-benzoquinone imine by hepatic microsomes
from control and ethanol-treated rats.
Prasad JS; Chen NQ; Liu YX; Goon DJ; Holtzman JL
Department of Pharmacology' University of Minnesota' Minneapolis 55455.
Biochem Pharmacol, 40(9):1989-95 1990 Nov 1
acetaminophen is metabolized by cytochrome P450 to
N-acetyl-p-benzoquinone imine (NABQI). This metabolite reacts with
critical cellular macromolecules to give toxicity. The administration
of 10% ethanol in the drinking water to 100 g male rats for 6 weeks
markedly increases the toxicity of acetaminophen. This increase was
associated with a 71% increase in microsomal protein binding of
acetaminophen [4.8 pmol/min/mg protein in control microsomes versus 8.2
pmol/min/mg protein in ethanol microsomes (P less than 0.01) and a 131%
increase in aniline hydroxylase [0.52 nmol/min/mg protein in control
microsomes versus 1.20 nmol/min/mg protein in ethanol microsomes (P
less than 0.001) . On the other hand' cysteine conJugation of
acetaminophen showed an increase of only 12% [2.8 nmol/min/mg protein
in control microsomes versus 3.1 nmol/min/mg protein in ethanol
microsomes (P less than 0.05) . Ethylmorphine- and benzphetamine
N-demethylases did not increase. In microsomes from both control and
ethanol animals' imidazole (1 mM) inhibited the two N-demethylases'
aniline hydroxylation and acetaminophen binding by 85-95% but inhibited
the cysteine conJugation by only 50%. For control and ethanol animals'
both 80% CO/20% O2 and SKF-525A (1 mM) totally inhibited cysteine
conJugation but only inhibited the other activities by about 36-60%.
KCN (1 mM) had no effect on any of the activities except protein
binding (60-67% inhibition). Scavengers of reactive oxygen [mannitol (1
mM)' dimethyl sulfoxide (1 mM)' superoxide dismutase (15 micrograms/mL)
and catalase (65 micrograms/mL) had no effect on any of the reactions.
Of all these treatments only CO/O2 decreased the protein binding and
cysteine conJugation of NABQI in the presence of either NADP+ or NADPH.
The data from the inhibitor studies and the effect of ethanol on
acetaminophen and NABQI metabolism would suggest that protein binding
and cysteine conJugation are catalyzed by different isozymes of
cytochrome P450. Finally' the current results indicate that the
increased toxicity of acetaminophen observed with ethanol more closely
parallels the increase in protein binding activity rather than cysteine

Metabolism and toxicity of 4-hydroxyphenylacetone in rat liver slices:
comparison with acetaminophen.
Thompson DC; Perera K; London R
Department of Medical Pharmacology and Toxicology' College of Medicine'
Texas A&M University Health Science Center' College Station 77843-1114'
Drug Metab Dispos, 24(8):866-71 1996 Aug
acetaminophen is oxidized by cytochrome P450 to a reactive quinone
imine' N-acetyl-p-benzoquinone imine' which is thought to be
responsible for its hepatotoxic effects. 4-Hydroxyphenylacetone (4-HPA)
is a structural analog of acetaminophen in which the amine group is
replaced by a methylene group. Following a similar metabolic pathway'
4-HPA would be oxidized to form a reactive quinone methide
intermediate. We compared the metabolism and toxicity of 4-HPA and
acetaminophen in liver microsomes and precision-cut liver slices from
male Sprague-Dawley rats. Both 4-HPA and acetaminophen formed
glutathione conJugates in microsomal incubations. 4-HPA formed
diastereomeric glutathione conJugates' which is consistent with the
formation of an intermediate quinone methide. The rate of conJugate
formation with 4-HPA was 8.5-fold greater than that with acetaminophen.
In rat liver slices a concentration of 5 mM 4-HPA killed approximately
50% of hepatocytes after 6 hr of incubation' whereas acetaminophen was
not toxic at concentrations up to 50 mM. N-Acetylcysteine protected
slices from 4-HPA-induced toxicity' whereas phenobarbital enhanced
metabolism and toxicity. In summary' 4-HPA is more hepatotoxic than
acetaminophen' and this may be the result of differences in the
metabolic rate and/or the type of reactive intermediate formed.

Acute hepatic and renal toxicity from low doses of acetaminophen in the
absence of alcohol abuse or malnutrition: evidence for increased
susceptibility to drug toxicity due to cardiopulmonary and renal
Bonkovsky HL; Kane RE; Jones DP; Galinsky RE; Banner B
Department of Medicine' University of Massachusetts Medical School'
Worcester 01655.
Hepatology, 19(5):1141-8 1994 May
A 67-yr-old man with chronic cardiopulmonary disease exhibited severe
hepatic and moderately severe renal inJury after short-term ingestion
of therapeutic doses of acetaminophen (1 to 3 gm/day for 3 days). Drug
metabolism and other studies' performed 5 mo after recovery from the
acute insult' indicated that the patient had decreased rates of hepatic
metabolism of acetaminophen to its primary' nontoxic metabolites and
decreased kidney function that was compromised further by acetaminophen
ingestion. He also had abnormally low concentrations of hepatic and
plasma reduced glutathione. Alcohol abuse and malnutrition could not be
implicated in the pathogenesis of inJury; rather it appeared that
advancing age with chronic renal' cardiac and pulmonary insufficiency
contributed to acetaminophen toxicity in this patient.


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