Acetaminophen-induced depletion of glutathione and cysteine in
aging mouse kidney.
Richie JP Jr; Lang CA; Chen TS
American Health Foundation' Valhalla' NY 10595.
Biochem Pharmacol, 44(1):129-35 1992 Jul 7
glutathione (GSH) plays an essential role in the detoxification
Acetaminophen (APAP) and the prevention of APAP-induced toxicity
kidney. Our previous results demonstrated that a GSH deficiency
general property of aging tissues' including the kidney' suggesting
hypothesis that senescent organisms are at greater risk to APAP-induced
renal damage. To test this' C57BL/6NIA mice of different ages
the life span were inJected with various doses of APAP' and the
of GSH and cysteine (Cys) depletion and recovery were determined.
time intervals up to 24 hr' kidney cortex samples were obtained'
processed and analyzed for glutathione status' namely GSH' glutathione
disulfide (GSSG)' Cys and cystine' using an HPLC method with
electrochemical detection. In the uninJected controls' GSH and
concentrations decreased about 30% in the aging mouse' but the
cystine levels were unchanged during the life span. APAP administration
depleted the kidney GSH and Cys contents in a dose- and time-dependent
manner. Four hours after APAP administration' GSH levels of the
growing (3- to 6-month) and the mature (12-month) mice decreased
58%' respectively' and recovered to near control values by 24
and 98%). In contrast' the extent of depletion in old (31-month)
was greater (64%) and the 24-hr recovery was less' returning
56%. Likewise' Cys levels of the young and mature mice decreased
65%' respectively' 4 hr following APAP' and increased to 99 and
24 hr. In contrast' in old mice' there was a 78% depletion after
followed by a recovery of only 65% by 24 hr. These results demonstrated
clearly that in the aging mouse kidney' a GSH and Cys deficiency
that is accompanied by an impaired APAP detoxification capacity.
Effects of N-acetylcysteine and dithiothreitol on glutathione
protein thiol replenishment during Acetaminophen-induced toxicity
isolated mouse hepatocytes.
Rafeiro E; Barr SG; Harrison JJ; Racz WJ
Department of Pharmacology and Toxicology' Faculty of Medicine'
University' Kingston' Ontario' Canada.
Toxicology, 93(2-3):209-24 1994 Nov 11
Isolated mouse hepatocytes were incubated with 1.0 mM Acetaminophen
(AA) for 1.5 h to initiate glutathione (GSH) and protein thiol
depletion and cell inJury. Cells were subsequently washed to
non-covalently bound AA and resuspended in medium containing
N-acetylcysteine (NAC' 2.0 mM) or dithiothreitol (DTT' 1.5 mM).
effects of these agents on the replenishment of GSH and total
content were related to the development of cytotoxicity. When
exposed to AA were resuspended in medium containing NAC or DTT'
agents replenished GSH and total PSH content to levels observed
untreated cells but only DTT was able to attenuate cytotoxicity.
Addition of the GSH synthesis inhibitor' buthionine sulfoximine
1.0 mM' 1.5 h)' to cells in incubation medium containing AA'
GSH and total PSH depletion and potentiated cytotoxicity. Resuspension
of these cells in medium containing NAC did not alter the potentiating
effects of BSO; GSH and PSH levels were not replenished and no
cytoprotective effects were observed. However' when cells exposed
and BSO were resuspended in medium containing DTT' PSH content
replenished but GSH levels were not restored. In addition' DTT
to delay the development of cytotoxicity. It appears that DTT'
NAC' has a GSH-independent mechanism of PSH replenishment. These
observations suggest that while replenishment of GSH and total
content does not result in cytoprotection' the regeneration of
PSH by DTT may play an important role in the maintenance of proper
structure and/or function.
Synthesis rates of glutathione and activated sulphate (PAPS)
response to cysteine and Acetaminophen administration in
glutathione-depleted rat hepatocytes.
Dalhoff K; Poulsen HE
Department of Medicine A-2152 Rigshospitalet' Copenhagen' Denmark.
Biochem Pharmacol, 46(7):1295-7 1993 Oct 5
The effects of cysteine and Acetaminophen (AA) on the synthesis
of glutathione (GSH)' adenosine 3`-phosphate 5`-phosphosulphate
activated sulphate) and the AA metabolites' AA-GSH and AA-sulphate
studied in rat hepatocytes depleted of GSH by diethyl maleate
The synthesis rates were determined simultaneously by a previously
described radioactive tracer method. Preincubation of the hepatocytes
with 0.7 mM DEM for 30 min depleted GSH by 59% (P < 0.05)
and PAPS by
28% (P < 0.05). Incubation with a toxic AA concentration resulted
GSH synthesis at a rate of 95 nmol/(10(6) cells.min) which increased
281 nmol/(10(6) cells.min) (P = 0.05) after addition of cysteine.
However' increased GSH synthesis was not followed by increased
synthesis [4.7 vs 4.8 nmol/(10(6) cells.hr) . Also' PAPS synthesis
increased after cysteine administration [10.2 to 19.1 nmol/(10(6)
cells.min) (P < 0.05) without any change in AA-sulphate synthesis
vs 18.3 nmol/(10(6) cells.hr) . Thus' in contrast to hepatocytes
normal GSH concentration' cysteine stimulated both GSH and PAPS
synthesis rates in GSH-depleted rat hepatocytes incubated with
AA concentration without stimulation of AA-GSH or AA-sulphate
rates' indicating that the hepatoprotective effect of cysteine
toxicity is primarily due to stimulation of a GSH-mediated reduction
the reactive AA metabolite N-acetyl-p-benzoquinoneimine back
Effects of cysteine and Acetaminophen on the syntheses of glutathione
and adenosine 3`-phosphate 5`-phosphosulfate in isolated rat
Dalhoff K; Poulsen HE
Department of Medicine A' Rigshospitalet' Copenhagen' Denmark.
Biochem Pharmacol, 44(3):447-54 1992 Aug 4
The aim of the present study was to introduce and validate a
radioactive tracer method in which adenosine 3`-phosphate
5`-phosphosulfate (PAPS) and glutathione (GSH) are measured
simultaneously in isolated hepatocytes. PAPS and GSH are co-substrates
in sulphation and GSH conJugation' and both are dependent on
deriving from cysteine. The effect of cysteine on the syntheses
investigated at non-toxic and toxic concentrations of the hepatotoxic
drug Acetaminophen (AA). Administration of AA trapped radioactivity
(35S) in the pre-labelled PAPS and GSH pools by formation of
metabolites' AA-sulphate and AA-GSH. Turnover rates were determined
from the decline of AA-sulphate and AA-GSH specific activity.
of PAPS and GSH were calculated by multiplying the rates with
concentrations of the respective co-substrates. Increasing AA
concentration from non-toxic to toxic levels resulted in increased
median PAPS and GSH syntheses (8 to 11 and 311 to 2218 nmol/10(6)
cells/min' respectively) (P less than 0.05). Addition of cysteine
not alter median PAPS synthesis (5 to 3 nmol/10(6) cells/min)
decreased median GSH synthesis (666 to 261 nmol/10(6) cells/min)
less than 0.05) in experiments with non-toxic AA concentrations.
experiments with toxic AA concentrations opposite effects of
were seen' i.e. median PAPS synthesis was reduced (3 to 2 nmol/10(6)
cells/min) (P less than 0.05) while median GSH synthesis was
(23 to 16 nmol/10(6) cells/min). The present method provides
a tool in
which two important detoxification pathways can be measured
simultaneously and the data suggest that the two pathways are
by substrate availability.
Cimetidine enhances the hepatoprotective action of N-acetylcysteine
mice treated with toxic doses of paracetamol.
Al-Mustafa ZH; Al-Ali AK; Qaw FS; Abdul-Cader Z
Department of Pharmacology' College of Medicine and Medical Sciences'
King Faisal University' Dammam' Saudi Arabia.
Toxicology, 121(3):223-8 1997 Sep 5
Paracetamol' in toxic doses' is associated with extensive liver
This represents one of the common causes of morbidity and mortality
drug poisoning cases. This study was undertaken to investigate
possible potentiation of the hepatoprotective action of
N-acetylcysteine (NAC) by cimetidine (CMD)' an inhibitor of hepatic
microsomal oxidative enzymes. The effects of NAC' cimetidine
two in combination' administered 2 h post-paracetamol dose' on
mortality' plasma glutamic oxaloacetic (GOT) and glutamic pyruvic
transaminase activities and hepatic reduced glutathione (GSH)
were investigated in mice 24 h after treatment with a single
of paracetamol (400 mg/kg). Both NAC and cimetidine caused a
improvement of survival rate' plasma GOT and GPT activities.
addition' they prevented the depletion of hepatic GSH contents.
However' concomitant administration of NAC and cimetidine produced
100% survival rate and a marked reduction in plasma GOT and GPT
activities to within the normal range' while significantly raising
hepatic GSH concentrations to values close to those measured
saline-treated control animals. It is therefore concluded that
cimetidine and N-acetylcysteine may have an additive hepatoprotective
action in the treatment of paracetamol overdose.
Role of glutathione conJugation in protection of weanling rat
against Acetaminophen-induced hepatotoxicity.
Allameh A; Vansoun EY; Zarghi A
Department of Biochemistry' Faculty of Medical Sciences' Tarbiat
Modaress University' Tehran' Iran.
Mech Ageing Dev, 95(1-2):71-9 1997 Apr
The rate of glutathione (GSH) conJugate formation to Acetaminophen
(APAP) in livers of weanling and adult rats treated with a single
dose of APAP was compared. HPLC analysis of cytosolic fractions
revealed that rate of conJugation in weanling rat is 24-times
than that of adults. Increased rate of GSH conJugation was independent
of of the age-related difference observed in liver GSH content.
normal level of liver GSH in weanling rat was 57% of adult level.
treatment depleted GSH more significantly in weanling rats as
to that in adults. N-acetylcystein (NAC) alone had little influence
liver GSH levels. However it was successful in reducing GSH depletion
in tissues of growing rats. A 32% repletion in hepatic GSH level
NAC-treated weanling rats was associated with a further 13-fold
increase in the rate of GSH conJugate formation. These data together
with histopathological results' clearly showed that the inducible
system in weanling rat liver act as a safe guard against APAP
A surge in the rate of APAP-GSH conJugation in growing liver
function in compensation of other detoxification pathways which
saturated more readily at this age.
Simultaneous measurements of glutathione and activated sulphate
synthesis rates and the effects of selective inhibition of glutathione
conJugation or sulphation of Acetaminophen.
Dalhoff K; Poulsen HE
Department of Medicine A' Rigshospitalet' Copenhagen' Denmark.
Biochem Pharmacol, 46(3):383-8 1993 Aug 3
The aim of the present study was to examine the effects of the
hepatotoxic drug Acetaminophen (AA) on the synthesis rates of
glutathione (GSH)' activated sulphate (PAPS; adenosine 3`-phosphate
5`-phosphosulphate) and the AA metabolites AA-GSH and AA-sulphate
selective inhibition of GSH biosynthesis or sulphation in isolated
hepatocytes. Selective inhibition of the two interdependent metabolic
pathways was accomplished by buthionine sulphoximine (BSO) and
2'6-dichloro-4-nitrophenol (DCNP). The synthesis rates of GSH
were determined simultaneously by a previously described method
on trapping of radioactivity (35S) in the pre-labelled GSH and
pools. Pre-incubation with 10 mM BSO for 30 min depleted GSH
by 38% (P
< 0.05) and PAPS by 27% (P < 0.05). The depletion resulted
PAPS synthesis at low' non-toxic [5-19 nmol/(10(6) cells.min)
0.05) and at high' toxic [7-30 nmol/10(6) cells.min) (P <
concentrations. In both cases sulphur is diverted from GSH biosynthesis
to sulphoxidation and PAPS synthesis' thereby maintaining the
and preserving the sulphation capacity. This corresponds to the
that AA sulphation was unaffected by BSO irrespective of AA
concentration [6 vs 5 and 20 vs 17 nmol/(10(6) cells.hr)' respectively
. Even though the GSH synthesis was halved after BSO pre-incubation'
the GSH conJugating capacity of AA was well preserved. Incubation
200 microM DCNP and 5 mM AA diminished PAPS synthesis from 24
nmol/(10(6) cells.min) (P < 0.02) and reduced AA-sulphate
67% compared to experiments without DCNP incubation [4.8 vs 14.7
nmol/(10(6) cells.hr) (P < 0.05). GSH and AA-GSH synthesis
not change compared to control experiments in which sulphation
inhibited [1165 vs 1487 nmol/(10(6) cells.min)' respectively
vs 1.7 nmol/(10(6) cells.hr)' respectively . This indicates that
increased sulphur availability due to decreased PAPS synthesis
unable to raise the cysteine pool and stimulate the gamma-glutamyl
cycle and GSH synthesis.
Protection by clofibrate against Acetaminophen hepatotoxicity
CD-1 mice is associated with an early increase in biliary concentration
of Acetaminophen-glutathione adducts.
Manautou JE; Tveit A; Hoivik DJ; Khairallah EA; Cohen SD
Toxicology Program' University of Connecticut' Storrs' Connecticut
Toxicol Appl Pharmacol, 140(1):30-8 1996 Sep
Repeated treatment with clofibrate (CFB) significantly increased
hepatic glutathione (GSH) content and also diminished Acetaminophen`s
(APAP) selective protein arylation' GSH depletion' and severity
hepatocellular necrosis. The present work was conducted to evaluate
role of elevated GSH and APAP detoxifying pathways in the amelioration
of APAP`s toxicity by CFB. Male CD-1 mice received 500 mg CFB/kg'
daily for 10 days. Controls were given corn oil vehicle. They
challenged with 700 mg APAP/kg in 50% propylene glycol/water
overnight fast. Results indicate that CFB pretreatment had no
24-hr urinary excretion of APAP-glucuronide' sulfate' or GSH-derived
conJugates; however' there was 50% less unchanged APAP excreted
urine of CFB-pretreated mice. CFB also did not alter microsomal
UDP-glucuronyl transferase activity toward APAP in vitro. However'
elimination of APAP from plasma and liver was much greater in
CFB-pretreated mice. This was accompanied by elevated biliary
content in CFB-pretreated mice at 2 hr after APAP dosing with
diminished levels in bile at 12 hr. The CFB-induced increase
excretion of APAP-GSH may mediate the protection against APAP-induced
Intrinsic susceptibility of the kidney to Acetaminophen toxicity
Tarloff JB; Goldstein RS; Silver AC; Hewitt WR; Hook JB
Department of Investigative Toxicology' Smith Kline & French
Laboratories' King of Prussia' PA.
Toxicol Lett, 52(1):101-10 1990 Jun
Acetaminophen (APAP)-induced nephrotoxicity is age-dependent
Sprague-Dawley (SD) rats: middle-aged (9-12 months old) rats
nephrotoxicity at lower dosages of APAP than do young adults
months old). The present study was designed to test the hypothesis
the intrinsic susceptibility of renal tissue to APAP toxicity
increased in middle-aged rats. APAP toxicity was evaluated in
slices from naive 3- and 12-month-old male SD rats incubated
mM APAP for 2-8 h. Renal slice glutathione (GSH) and APAP
concentrations were determined; renal function was assessed by
anion (para-aminohippurate' PAH) and cation (tetraethylammonium'
accumulation; and cell viability was assessed by lactate dehydrogenase
(LDH) leakage. At each concentration of APAP tested' accumulation
APAP by renal slices was similar in 3- and 12-month-olds. APAP
in renal slices from both 3- and 12-month-old rats was characterized
concentration-dependent increases in LDH leakage. In contrast
nephrotoxicity in vivo' APAP toxicity in renal slices was accompanied
by decreased accumulation of PAH and TEA. Additionally' APAP
marked reductions in renal slice GSH content in a
concentration-dependent manner: however' in contrast to APAP
nephrotoxicity in vivo' APAP-induced GSH depletion in vitro did
precede cytotoxicity. No consistent age-dependent differences
time- and concentration-response curves for APAP nephrotoxicity
observed. These data suggest that APAP cytotoxicity in vitro
increased in 12-month-old rats. However' since the pattern (and
mechanisms) of APAP cytotoxicity in vitro appears to be different
that observed in vivo' extrapolation of in vitro cytotoxicity
vivo nephrotoxicity is limited. Therefore' age differences in
susceptibility of the intact kidney cannot be excluded as a mechanism
contributing to enhanced APAP nephrotoxicity in middle-aged rats.
Acetaminophen-induced toxicity to human epidermoid cell line
hepatoblastoma cell line Hep G2' in vitro' is diminished by silymarin.
Shear NH; Malkiewicz IM; Klein D; Koren G; Randor S; Neuman MG
Division of Dermatology' Sunnybrook Health Science Centre' Ont.'
Skin Pharmacol, 8(6):279-91 1995
The skin and liver may be targets for cytotoxicity induced by
drug metabolites. We used human epidermoid A431 cells and human
hepatoblastoma Hep G2 cells as the experimental model. The aim
study was to investigate and evaluate the effect of silymarin
Acetaminophen (APAP)-induced toxicity under controlled conditions.
Silymarin is known to be a potent antioxidant that diminishes
induced by a variety of other hepatotoxins (e.g. Amanita phaloides'
algae`s toxins' carbon tetrachloride). glutathione (GSH) depletion
enhanced by adding to the medium buthionine sulfoximine
[L-buthionine-(S'R)-sulfoximine' BSO . Cells were incubated with
high-concentration 5-20 mM APAP or alpha-(minimum essential medium
2-24 h to evaluate the drug`s ability to reduce cytoviability.
Viability was then quantitated by metabolism of the tetrazolium
(MTT) and neutral red (NR). Cytoviability was 100% for controls.
Hep G2 treated for 24 h with 20 mM' APAP viability was 56.0%
by MTT and
62.5% by NR. BSO-treated cells showed an enhanced cytotoxicity'
determined by both assays. Administration of 0.5 mM silymarin
cytotoxicity significantly. In A431 cells' treatment with 20
reduced viability by 57% (MTT) and 69% (NR) versus control (100%).
further decreased viability. Since incubation with silymarin
significant protection against APAP toxicity' it can be considered
cytoprotective agent in this in vitro model of drug toxicity.
concentrations in both cell lines decrease significantly after
to 20 mM APAP' or 0.5 mM versus control (p < 0.05)' and increased
0.001) if incubated with APAP and silymarin. The protective effect
could be through mitochondrial membrane stabilization and/or
increase in available GSH.
Protective effects of diallyl sulfide on Acetaminophen-induced
Hu JJ; Yoo JS; Lin M; Wang EJ; Yang CS
Department of Chemical Biology and Pharmacognosy' Rutgers University'
Piscataway' NJ 08855-0789' USA.
Food Chem Toxicol, 34(10):963-9 1996 Oct
Diallyl sulfide (DAS)' a maJor flavour component of garlic' is
modulate drug metabolism and may protect animals from chemically
induced toxicity and carcinogenesis. In this study the effects
on the oxidative metabolism and hepatotoxicity induced by Acetaminophen
(APAP) in rats were investigated. In the hepatotoxicity evaluation
Fischer 344 rats there was a dose-dependent increase in the odds
mortality rate by APAP (P = 0.009); DAS treatment significantly
protected rats from APAP-related mortality (P = 0.026). Liver
determined by lactate dehydrogenase activity was significantly
increased by APAP treatment (0.75 g/kg). Pretreatment with DAS
protected animals from APAP-induced liver toxicity in a time-
dose-dependent fashion. Treatment of DAS (50 mg/kg) 3 hr after
dosing significantly (P < 0.05) protected rats from APAP-induced
toxicity. The metabolism of APAP (50 microM) in vitro was significantly
inhibited by DAS (0.3-1 mM) in liver microsomes isolated from
rats. As the effect of DAS on APAP-induced hepatotoxicity in
observed only when DAS was administered before or shortly after
hr) APAP dosing' data suggested that the protective effect of
mainly at the metabolic activation step of APAP. However' the
possibility that DAS may also have effects on other drug metabolism
systems' such as glutathione (GSH) and glutathione S-transferases'
cannot be ruled out.