Hepatic glutathione and lipid peroxidation in rats treated with
theophylline. Effect of dose and combination with caffeine and
Farag MM; Abdel-Meguid EM
Department of Pharmacology' Alexandria University' Egypt.
Biochem Pharmacol, 47(3):443-6 1994 Feb 9
As theophylline' caffeine and acetaminophen (APAP) are commonly
in combination in prescription and non-prescription drugs' the
study was designed to evaluate changes of hepatic glutathione
lipid peroxidation in rats treated concurrently with these widely
drugs. In rats treated with different doses of theophylline'
dose-related depletion of hepatic GSH was observed (r = -0.88'
0.001). After a dose of 100 mg/kg of caffeine given alone and
combination with 50 mg/kg of theophylline' hepatic GSH levels
decreased by 22.5% (P > 0.05) and 35.5% (P < 0.01) of the
value' respectively. Also' the challenge with 500 mg/kg of APAP
significantly depleted hepatic GSH in rats pretreated with either
saline or the combination of both theophylline and caffeine (50
mg/kg' respectively)' P < 0.001. In the latter group' hepatic
levels were approximately 47% of the corresponding values in
received APAP (P < 0.02). Such severe depletion of hepatic
GSH' in rats
that received the three drugs' was associated with a significant
in the extent of lipid peroxidation (P < 0.05). The other
groups treated with one or two drugs did not exhibit similar
These results suggest that the concomitant administration of
theophylline' caffeine and APAP may enhance the susceptibility
liver cells to the toxic effect of APAP by severely depleting
GSH with subsequent induction of lipid peroxidation.
Oxidative stress in cultured hepatocytes exposed to acetaminophen.
Adamson GM; Harman AW
Department of Pharmacology' School of Medicine' University of
Biochem Pharmacol, 45(11):2289-94 1993 Jun 9
The effect of acetaminophen (APAP) exposure on the formation
oxidized glutathione (GSSG) was investigated in cultured mouse
hepatocytes to determine if oxidative damage is involved in the
toxicity of this drug. Incubations of hepatocytes for 24 hr with
APAP produced a time-dependent loss of cell viability which was
preceded by depletion of reduced glutathione (GSH) and an increase
GSSG formation. Pretreatment with 1'3-bis(chloroethyl)-1-nitrosourea
(BCNU) (0.1 mM) for 30 min' which irreversibly inhibited glutathione
reductase (GSSG-Rd) activity' increased the extent of GSSG formation
produced by APAP exposure and potentiated its cell killing.
Pretreatment of hepatocytes with 20 mM deferoxamine (DFO) for
1 hr to
chelate ferric iron decreased GSSG formation and cell killing
by APAP. Pretreatment with BCNU or DFO did not affect APAP oxidation
determined by the formation of the APAP-GSH conJugate or the
binding of APAP metabolites to cellular protein. Hence' increasing
susceptibility of hepatocytes to an oxidative stress with BCNU
increased both the formation of GSSG and cell killing produced
Conversely' decreasing their susceptibility to an oxidative stress
chelating iron with DFO decreased GSSG formation and cell inJury.
follows that APAP toxicity involves oxidative processes that
early in the poisoning process and are a maJor factor contributing
inJury in these cells.
Intracellular glutathione in human hepatocytes incubated with
S-adenosyl-L-methionine and GSH-depleting drugs.
Ponsoda X; Jover R; G]omez-Lech]on MJ; Fabra R; Trullenque R;
Unidad de Hepatolog]ia Experimental' Centro de Investigaci]on
Universitario La Fe' Valencia' Spain.
Toxicology, 70(3):293-302 1991
The present study was undertaken to investigate (a) whether
S-adenosyl-L-methionine (SAMe) added to culture medium can increase
intracellular glutathione (GSH) levels in human hepatocytes and
whether SAMe can prevent the GSH depletion found in human hepatocytes
incubated with GSH-depleting drugs (paracetamol' opiates' ethanol).
Incubation of hepatocytes with increasing concentrations of SAMe
resulted in a dose-dependent elevation of intracellular GSH content'
which reached its maximum (35% increase) at 30 microM after 20
as the only sulfur source in the medium' was efficient in repleting
GSH-depleted hepatocytes following treatment with diethyl maleate.
Incubation of human hepatocytes with SAMe attenuated the GSH
of cells incubated with toxic concentrations of paracetamol (2
heroin (0.5 mM) and methadone (0.2 mM). A decrease in GSH due
exposure of hepatocytes to 50 mM ethanol was prevented when SAMe
simultaneously added to ethanol' and human hepatocytes maintained
GSH levels like non ethanol-treated cells. The experimental results
our work give the first direct evidence of the ability of exogenously
administered SAMe to increase intracellular GSH levels in human
hepatocytes and to prevent the GSH depletion caused by paracetamol'
opiates and ethanol.
Protection against acetaminophen-induced hepatotoxicity by L-CySSME
its N-acetyl and ethyl ester derivatives.
Nagasawa HT; Shoeman DW; Cohen JF; Rathbun WB
Medical Research Laboratories' DVA Medical Center' Minneapolis'
J Biochem Toxicol, 11(6):289-95 1996
We have recently observed that S-(2-hydroxyethylmercapto)-L-cysteine
(L-CySSME)' the mixed disulfide of L-cysteine and 2-mercaptoethanol'
prevented cataracts induced in mice by acetaminophen (ACP) by
functioning as a prodrug of L-cysteine and protecting the liver.
prompted the evaluation of the more lipophilic N-acetyl (Ac-CySSME)
ethyl ester (Et-CySSME) derivatives of L-CySSME as proprodrug
well as the "D" enantiomer' as hepatoprotective agents.
levels were measured at 24 hours after a toxic but nonlethal
ACP that insured 48 hour survival of the animals. Since the increases
in ALT produced were highly variable (even after log transformation)
and complicated the statistical analyses' we calculated confidence
intervals for the mean ALT levels for each treatment group. This
enabled comparisons to be made of the efficacy of L-CySSME as
Ac-CySSME and Et-CySSME with other representative prodrugs of
L-cysteine' namely' 2(RS)-methylthiazolidine-4(R)-carboxylic
(MTCA)' L-2-oxothiazolidine-4-carboxylic acid (OTCA)' and
N-acetyl-L-cysteine (NAC)' in protecting the liver. It was shown
L-CySSME and MTCA administered intraperitoneally at 2.5 mmol/kg
superior to the other cysteine prodrugs at equimolar doses in
protecting mice from hepatotoxicity elicited by a 400 mg/kg (2.65
mmol/kg) dose of ACP given i.p. 30 minutes prior to the prodrugs.
"D" form of CySSME was totally without protective effect.
Oral doses of
the prodrugs even at 2x the i.p. dose were less effective' although
MTCA was the most protective.
Protective effect of S-(1'2-dicarboxyethyl)glutathione' an intrinsic
tripeptide in liver' heart and lens' and its esters on
acetaminophen-induced hepatotoxicity in rats.
Sakaue T; Matsumoto S; Tsuboi S; Ogata K; Ohmori S
SenJu Pharmaceutical Co.' Ltd.' Itami' Japan.
Biol Pharm Bull, 19(9):1216-9 1996 Sep
The administration of acetaminophen (APAP' 500 mg/kg' i.p.) produced
liver necrosis and increased aspartate aminotransaminase (AST)
in serum. The pretreatment of S-(1'2-diethoxycarbonyl)glutathione
isopropyl ester (DCE-Et-GS iPr' 0.5 mmol/kg' p.o.) prevented
necrosis and the elevation of serum AST activity by 99.9%. DCE-Et-GS
iPr inhibited APAP-induced hepatotoxicity much more strongly
reduced glutathione (GSH)' DCE-GS and other esters of DCE-GS.
clarify this protective effect' the hepatic GSH concentration
determined 2h after APAP administration. It was found that the
DCE-Et-GS iPr administration significantly inhibited the GSH
caused by APAP' suggesting that the protective effect of DCE-Et-GS
on APAP-induced hepatotoxicity was due' at least in part' to
retention of hepatic GSH level.
Possible role of glutathione in prevention of acetaminophen-induced
hepatotoxicity enhanced by fish oil in male Wistar rats.
Kuralay F; Akarca US; Oz utemiz AO; Kutay F; Batur Y
Dokuz Eyl ul University School of Medicine, Department of Biochemistry,
Izmir, Turkey. firstname.lastname@example.org
J Toxicol Environ Health, 53(3):223-9 1998 Feb 6
It has been reported that fish oil protects the rat liver against
acetaminophen (APAP) induced toxicity; however, this finding
controversial. The present study was undertaken to investigate
effects of fish oil-enriched diet on APAP-induced liver injury
Wistar rats. Rats were fed a diet supplemented with either 8%
or 8% corn oil, or standard rat feed for 6 wk. After an overnight
rats in each group were given either 2 g/kg APAP or saline orally.
findings showed that APAP increased serum alanine aminotransferase
(ALT) and that this rise was potentiated in the presence of dietary
fat. Further fish oil ingestion increased the glutathione (GSH)
in rat liver; however, this was not effective in protecting liver
APAP-induced toxicity. Data suggest that GSH may be necessary
detoxify APAP metabolites, which are known to induce hepatotoxicity
are increased by dietary fat.
Effect of acetaminophen on glutathione levels in rat testis and
Micheli L; Cerretani D; Fiaschi AI; Giorgi G; Romeo MR; Runci
Institute of Pharmacology' University of Siena' Italy.
Environ Health Perspect, 102 Suppl 9():63-4 1994 Nov
glutathione (GSH) levels in rat testis and lung after oral
administration (3 g/kg) of acetaminophen (APAP) were studied.
administered dose APAP is present in each organ and influences
levels. APAP value of 114 micrograms/g was obtained in testis
at 6 hr
(peak time); in the lung the Cmax was 92 mu/g at 8 hr and this
lasted several hours longer than that in testis. GSH levels are
affected differently in the organs studied after APAP administration;
the lungs seem to be the primary organ undergoing the depleting
of APAP. This process could not only cause toxicity' but also
predispose those organs to the action of toxic compounds responsible
for specific pathologies.
Cytochrome P450 and glutathione in the liver of rats under exclusive
Martinelli AL; Meneghelli UG; Zucoloto S
Departamento de Cl]inica M]edica' Faculdade de Medicina de Ribeir~ao
Preto' Universidade de S~ao Paulo' Brasil.
Braz J Med Biol Res, 26(9):989-98 1993 Sep
1. The obJective of the present investigation was to study some
possible mechanisms involved in the protective effect of sucrose
ingestion against liver necrosis induced by acetaminophen. Three
of male Wistar rats (220-260 g) were submitted to the following
experimental conditions for a period of 42 h: free access to
commercial diet (Group I)' an exclusive sucrose diet (Group II)
fasting (Group III). At the end of the experiment' hepatic cytochrome
P450 levels were measured in 11 rats from each group' plasma
half-life (t1/2) was determined in 40 rats from each group' and
glutathione (GSH) concentration in 10 rats from each group. GSH
consumption elicited by a high dose of acetaminophen (ACP' 1.0
gavage) was also determined in 30 rats each from Groups II and
The liver of Group II rats presented a significant reduction
cytochrome P450 levels in the microsome fraction (range 0.31-0.46'
median' 0.37 nmol/mg vs range 0.60-0.93' median 0.74 for group
range 0.63-1.22' median 0.91 for group III' reported as nmol/mg
microsome protein; range 23.8-48.4' median 40.4 vs 66.6-130'
81.8 for group I and range 59.0-117.1' median 77.1 for group
reported as nmol/100 g body weight)' and a prolongation of antipyrine
half-life (146.4 vs 83.4 min for group I and 93.6 for group III)
compared with the rats of the two other groups. 3. Since the
of acetaminophen depends on the production of a reactive metabolite
the cytochrome P450 system in the liver' we conclude that changes
this system brought about by exclusive sucrose ingestion for
42 h may
explain the liver protection against the toxicity of a high dose
drug even in the presence of a significant concomitant reduction
liver GSH levels.
Inhibition of cytochrome P450 2E1 expression by 2-(allylthio)pyrazine'
a potential chemoprotective agent: hepatoprotective effects.
Kim ND; Kwak MK; Kim SG
College of Pharmacy' Seoul National University' Korea.
Biochem Pharmacol, 53(3):261-9 1997 Feb 7
Cytochrome P450 2E1 (P450 2E1) is active in both the detoxification
activation of small organic molecules. The effects of
2-(allylthio)pyrazine (2-AP) on P450 2E1-catalytic activity and
expression of rat hepatic P450 2E1 were examined. 2-AP competitively
inhibited 4-nitrophenol hydroxylase activity in vitro (Ki' 12
2-AP treatment of rats (200 mg/kg/day' p.o.' 1-3 days old) resulted
20-30% decreases in the rates of P450 2E1-specific metabolic
activities. Immunoblot analysis also revealed that hepatic microsomes
isolated from 2-AP-treated rats showed substantial decreases
2E1 level. 2-AP-suppressed isoniazid (INH)-inducible hepatic
levels' as shown by both metabolic activities and immunoblot
Thus' 2-AP was effective in suppressing both constitutive and
P450 2E1 expression. Northern blot analysis showed that 2-AP
transiently suppressed the hepatic P450 2E1 mRNA level' suggesting
suppression in P450 2E1 expression by 2-AP may be mediated in
transcriptional inactivation. Hepatoprotective effects of 2-AP
toxicants were monitored in mice. 2-AP pretreatment prior to
administration of lethal doses of acetaminophen (AAP) or INH
substantially reduced toxicant-induced mortality. Whereas serum
aspartate aminotransferase (AST) and alanine aminotransferase
levels were markedly elevated after AAP administration (i.e.
9-20-fold)' 2-AP pretreatment of animals before AAP administration
resulted in >95% decreases in elevated serum aminotransferase
activities. 2-AP was also effective against CCl4-induced
hepatotoxicity. Whereas CCl4 treatment caused 35-70-fold increases
aminotransferase activities' treatment of mice with 2-AP (>10
resulted in the blocking of CCl4-induced liver toxicity. The
hepatoprotective effect of 2-AP was in part due to 2-AP-induced
elevation of hepatic GSH levels. Whereas AAP or CCl4 treatment
in 70-80% reduction in hepatic GSH levels' pretreatment of mice
2-AP caused a 40-210% elevation in hepatic GSH levels' as compared
either AAP or CCl4 alone. 2-AP pretreatment also reduced AAP-
CCl4-induced increases in lipid peroxidation in a dose-dependent
manner. The results of these metabolic activities and of immunoblot
RNA blot analyses demonstrate that 2-AP is efficacious in suppressing
constitutive and inducible P450 2E1 expression and effective
protecting against toxicant-induced liver toxicity.
Effect of polysaccharide peptide (PSP) on glutathione and protection
against paracetamol-induced hepatotoxicity in the rat.
Yeung JH; Chiu LC; Ooi VE
Department of Pharmacology' Faculty of Medicine' Chinese University
Methods Find Exp Clin Pharmacol, 16(10):723-9 1994 Dec
The protective effects of polysaccharide peptide (PSP)' isolated
Coriolus versicolor COV-1' on paracetamol-induced hepatotoxicity
investigated in this study. The effect of PSP on hepatic glutathione
status was also studied. PSP (300 mg/kg' i.p.) caused a 40% depletion
of hepatic reduced glutathione (GSH) with a concomitant 50% increase
oxidized glutathione (GSSG)' thus producing a 3-fold increase
GSSG/GSH ratio. The PSP-induced GSH depletion itself had no hepatotoxic
effects. PSP protected against paracetamol-induced hepatotoxicity
decreasing the paracetamol-induced elevation of serum glutamic-pyruvic
transaminase (SGPT) activity from 511 +/- 71 U/ml to 187 +/-
(controls without paracetamol 105 +/- 4 U/ml) and serum
glutamic-oxaloacetic transaminase (SGOT) activity from 462 +/-
152 +/- 48 U/ml (controls without paracetamol 54 +/- 6 U/ml).
not reverse the depletion of total glutathione (GSH+GSSG) by
dose of paracetamol. The GSSG/GSH ratio' which is a measure of
oxidative stress' was significantly (p < 0.05) decreased when
coadministered with paracetamol. PSP dose-dependently decreased
covalent binding of [14C -paracetamol to microsomal proteins
When PSP was given to rats subchronically for 7 days (300 mg/kg/day'
i.p.)' the subsequent microsomes obtained also showed a 25% decrease
covalent binding to [14C -paracetamol' suggesting that PSP interacted
with the microsomal proteins rather than the chemically reactive
metabolite of paracetamol. The changes in the binding affinity
capacity of the microsomal proteins by PSP may be related to
ability to alter the redox potential as indicated by the effects
on the GSSG/GSH status.
Sulfation of acetaminophen and acetaminophen-induced alterations
sulfate and 3`-phosphoadenosine 5`-phosphosulfate homeostasis
with deficient dietary intake of sulfur.
Gregus Z; Kim HJ; Madhu C; Liu Y; Rozman P; Klaassen CD
Department of Pharmacology' Toxicology' and Therapeutics' University
Kansas Medical Center' Kansas City 66160-7417.
Drug Metab Dispos, 22(5):725-30 1994 Sep-Oct
Sulfation of drugs depends on the availability of 3`-phosphoadenosine
5`-phosphosulfate (PAPS)' which requires inorganic sulfate for
synthesis. Therefore' decreased alimentary intake of inorganic
or its precursor' cysteine' may compromise sulfation of xenobiotics.
test this hypothesis' separate groups of rats were maintained
days on synthetic diets' which lacked sulfate' or cysteine' or
sulfate and cysteine. These dietary restrictions did not cause
retardation or depletion of glutathione in liver. Under anesthesia'
animals were inJected with acetaminophen (0.5 mmol/kg' i.v.)
elimination of acetaminophen from blood and excretion of acetaminophen
metabolites in urine and bile was simultaneously quantified.
intake of inorganic sulfate or cysteine alone did not significantly
change elimination and biotransformation of acetaminophen. Combined
nutritional deficiency of sulfate and cysteine' however' resulted
40% reduction in the excretion of acetaminophen-sulfate' quantitatively
the most significant metabolite. Concomitantly' these animals
eliminated acetaminophen from blood at a slower rate and converted
acetaminophen to its toxic intermediate' as indicated by increased
excretion of acetaminophen-thioether conJugates. Serum and tissue
sulfate concentrations were decreased to significantly lower
rats on sulfate and cysteine deficient diets' than in rats with
sufficient sulfur supply. Thus' reduced sulfation is apparently
by diminished availability of inorganic sulfate for PAPS synthesis'
even though hepatic and renal PAPS levels were not depleted more
acetaminophen in rats with deficient dietary supply of sulfate
cysteine than in rats with adequate sulfur intake.(ABSTRACT TRUNCATED
AT 250 WORDS)
Studies comparing the kinetics of cysteine conJugation and protein
binding of acetaminophen by hepatic microsomes from male mice.
Zhou L; Erickson RR; Holtzman JL
Department of Medicine' University of Minnesota' Minneapolis
Biochim Biophys Acta, 1335(1-2):153-60 1997 Apr 17
A large body of evidence indicates that acetaminophen toxicity
mediated through the formation of the reactive metabolite'
N-acetyl-p-benzoquinone imine (NABQI). Two assays have been employed
monitor NABQI formation by hepatic microsomes: the conJugation
thiol trap' such as cysteine or glutathione' and the binding
to microsomal proteins. Studies from our laboratory with rat
microsomes have suggested that the two assays may not be equivalent.
now find with mouse hepatic microsomes that there are also marked
differences between these two assays. Among these the rate of
conJugation was almost three orders of greater than that of protein
binding. Furthermore' ethanol feeding increased protein binding
but cysteine conJugation by only 33%. Protein binding was linear
min while cysteine conJugation was linear for only 5 min. CO'
and metyrapone inhibited cysteine conJugation much more than
binding while SKF-525A and KCN had similar effects on both reactions.
Both reactions increased linearly with increasing [NADPH up to
At higher concentrations' the rate of cysteine conJugation markedly
decreased while the rate of protein binding plateaued. The addition
equimolar concentrations of NADH decreased protein binding' but
effect on cysteine conJugation. NADPH reduced the protein binding
added NABQI while NADH had little effect. The reduction of NABQI
to acetaminophen was equal for NADPH and NADH. These data indicate
the formation of the cysteine conJugate of NABQI has markedly
kinetic characteristics than the microsomal protein binding.
might suggest that the two reactions are catalyzed in part by
isoforms of cytochrome P-450.