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 Acetaminophen

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Title
Acetaminophen-induced depletion of glutathione and cysteine in the
aging mouse kidney.
Author
Richie JP Jr; Lang CA; Chen TS
Address
American Health Foundation' Valhalla' NY 10595.
Source
Biochem Pharmacol, 44(1):129-35 1992 Jul 7
Abstract
glutathione (GSH) plays an essential role in the detoxification of
Acetaminophen (APAP) and the prevention of APAP-induced toxicity in the
kidney. Our previous results demonstrated that a GSH deficiency is a
general property of aging tissues' including the kidney' suggesting a
hypothesis that senescent organisms are at greater risk to APAP-induced
renal damage. To test this' C57BL/6NIA mice of different ages through
the life span were inJected with various doses of APAP' and the extent
of GSH and cysteine (Cys) depletion and recovery were determined. At
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 dual
electrochemical detection. In the uninJected controls' GSH and Cys
concentrations decreased about 30% in the aging mouse' but the GSSG and
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 young'
growing (3- to 6-month) and the mature (12-month) mice decreased 34 and
58%' respectively' and recovered to near control values by 24 hr (95
and 98%). In contrast' the extent of depletion in old (31-month) mice
was greater (64%) and the 24-hr recovery was less' returning only to
56%. Likewise' Cys levels of the young and mature mice decreased 49 and
65%' respectively' 4 hr following APAP' and increased to 99 and 85% by
24 hr. In contrast' in old mice' there was a 78% depletion after 4 hr
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 occurs
that is accompanied by an impaired APAP detoxification capacity.

Title
Effects of N-acetylcysteine and dithiothreitol on glutathione and
protein thiol replenishment during Acetaminophen-induced toxicity in
isolated mouse hepatocytes.
Author
Rafeiro E; Barr SG; Harrison JJ; Racz WJ
Address
Department of Pharmacology and Toxicology' Faculty of Medicine' Queen`s
University' Kingston' Ontario' Canada.
Source
Toxicology, 93(2-3):209-24 1994 Nov 11
Abstract
Isolated mouse hepatocytes were incubated with 1.0 mM Acetaminophen
(AA) for 1.5 h to initiate glutathione (GSH) and protein thiol (PSH)
depletion and cell inJury. Cells were subsequently washed to remove
non-covalently bound AA and resuspended in medium containing
N-acetylcysteine (NAC' 2.0 mM) or dithiothreitol (DTT' 1.5 mM). The
effects of these agents on the replenishment of GSH and total PSH
content were related to the development of cytotoxicity. When cells
exposed to AA were resuspended in medium containing NAC or DTT' both
agents replenished GSH and total PSH content to levels observed in
untreated cells but only DTT was able to attenuate cytotoxicity.
Addition of the GSH synthesis inhibitor' buthionine sulfoximine (BSO'
1.0 mM' 1.5 h)' to cells in incubation medium containing AA' enhanced
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 to AA
and BSO were resuspended in medium containing DTT' PSH content was
replenished but GSH levels were not restored. In addition' DTT was able
to delay the development of cytotoxicity. It appears that DTT' unlike
NAC' has a GSH-independent mechanism of PSH replenishment. These
observations suggest that while replenishment of GSH and total PSH
content does not result in cytoprotection' the regeneration of critical
PSH by DTT may play an important role in the maintenance of proper cell
structure and/or function.

Title
Synthesis rates of glutathione and activated sulphate (PAPS) and
response to cysteine and Acetaminophen administration in
glutathione-depleted rat hepatocytes.
Author
Dalhoff K; Poulsen HE
Address
Department of Medicine A-2152 Rigshospitalet' Copenhagen' Denmark.
Source
Biochem Pharmacol, 46(7):1295-7 1993 Oct 5
Abstract
The effects of cysteine and Acetaminophen (AA) on the synthesis rates
of glutathione (GSH)' adenosine 3`-phosphate 5`-phosphosulphate (PAPS'
activated sulphate) and the AA metabolites' AA-GSH and AA-sulphate were
studied in rat hepatocytes depleted of GSH by diethyl maleate (DEM).
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 in
GSH synthesis at a rate of 95 nmol/(10(6) cells.min) which increased to
281 nmol/(10(6) cells.min) (P = 0.05) after addition of cysteine.
However' increased GSH synthesis was not followed by increased AA-GSH
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 18.5
vs 18.3 nmol/(10(6) cells.hr) . Thus' in contrast to hepatocytes with
normal GSH concentration' cysteine stimulated both GSH and PAPS
synthesis rates in GSH-depleted rat hepatocytes incubated with a toxic
AA concentration without stimulation of AA-GSH or AA-sulphate synthesis
rates' indicating that the hepatoprotective effect of cysteine on AA
toxicity is primarily due to stimulation of a GSH-mediated reduction of
the reactive AA metabolite N-acetyl-p-benzoquinoneimine back to AA.

Title
Effects of cysteine and Acetaminophen on the syntheses of glutathione
and adenosine 3`-phosphate 5`-phosphosulfate in isolated rat
hepatocytes.
Author
Dalhoff K; Poulsen HE
Address
Department of Medicine A' Rigshospitalet' Copenhagen' Denmark.
Source
Biochem Pharmacol, 44(3):447-54 1992 Aug 4
Abstract
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 sulphur
deriving from cysteine. The effect of cysteine on the syntheses was
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 the
metabolites' AA-sulphate and AA-GSH. Turnover rates were determined
from the decline of AA-sulphate and AA-GSH specific activity. Syntheses
of PAPS and GSH were calculated by multiplying the rates with the
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 did
not alter median PAPS synthesis (5 to 3 nmol/10(6) cells/min) but
decreased median GSH synthesis (666 to 261 nmol/10(6) cells/min) (P
less than 0.05) in experiments with non-toxic AA concentrations. In
experiments with toxic AA concentrations opposite effects of cysteine
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 unchanged
(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 regulated
by substrate availability.

Title
Cimetidine enhances the hepatoprotective action of N-acetylcysteine in
mice treated with toxic doses of paracetamol.
Author
Al-Mustafa ZH; Al-Ali AK; Qaw FS; Abdul-Cader Z
Address
Department of Pharmacology' College of Medicine and Medical Sciences'
King Faisal University' Dammam' Saudi Arabia.
Source
Toxicology, 121(3):223-8 1997 Sep 5
Abstract
Paracetamol' in toxic doses' is associated with extensive liver damage.
This represents one of the common causes of morbidity and mortality in
drug poisoning cases. This study was undertaken to investigate the
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 and the
two in combination' administered 2 h post-paracetamol dose' on
mortality' plasma glutamic oxaloacetic (GOT) and glutamic pyruvic (GPT)
transaminase activities and hepatic reduced glutathione (GSH) levels
were investigated in mice 24 h after treatment with a single oral dose
of paracetamol (400 mg/kg). Both NAC and cimetidine caused a partial
improvement of survival rate' plasma GOT and GPT activities. In
addition' they prevented the depletion of hepatic GSH contents.
However' concomitant administration of NAC and cimetidine produced a
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 in
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.

Title
Role of glutathione conJugation in protection of weanling rat liver
against Acetaminophen-induced hepatotoxicity.
Author
Allameh A; Vansoun EY; Zarghi A
Address
Department of Biochemistry' Faculty of Medical Sciences' Tarbiat
Modaress University' Tehran' Iran.
Source
Mech Ageing Dev, 95(1-2):71-9 1997 Apr
Abstract
The rate of glutathione (GSH) conJugate formation to Acetaminophen
(APAP) in livers of weanling and adult rats treated with a single i.p.
dose of APAP was compared. HPLC analysis of cytosolic fractions
revealed that rate of conJugation in weanling rat is 24-times greater
than that of adults. Increased rate of GSH conJugation was independent
of of the age-related difference observed in liver GSH content. The
normal level of liver GSH in weanling rat was 57% of adult level. APAP
treatment depleted GSH more significantly in weanling rats as compared
to that in adults. N-acetylcystein (NAC) alone had little influence on
liver GSH levels. However it was successful in reducing GSH depletion
in tissues of growing rats. A 32% repletion in hepatic GSH level in
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 GSH
system in weanling rat liver act as a safe guard against APAP toxicity.
A surge in the rate of APAP-GSH conJugation in growing liver may
function in compensation of other detoxification pathways which are
saturated more readily at this age.

Title
Simultaneous measurements of glutathione and activated sulphate (PAPS)
synthesis rates and the effects of selective inhibition of glutathione
conJugation or sulphation of Acetaminophen.
Author
Dalhoff K; Poulsen HE
Address
Department of Medicine A' Rigshospitalet' Copenhagen' Denmark.
Source
Biochem Pharmacol, 46(3):383-8 1993 Aug 3
Abstract
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 after
selective inhibition of GSH biosynthesis or sulphation in isolated rat
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 and PAPS
were determined simultaneously by a previously described method based
on trapping of radioactivity (35S) in the pre-labelled GSH and PAPS
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 in increased
PAPS synthesis at low' non-toxic [5-19 nmol/(10(6) cells.min) (P <
0.05) and at high' toxic [7-30 nmol/10(6) cells.min) (P < 0.05) AA
concentrations. In both cases sulphur is diverted from GSH biosynthesis
to sulphoxidation and PAPS synthesis' thereby maintaining the PAPS pool
and preserving the sulphation capacity. This corresponds to the finding
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 with
200 microM DCNP and 5 mM AA diminished PAPS synthesis from 24 to 10
nmol/(10(6) cells.min) (P < 0.02) and reduced AA-sulphate synthesis by
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 rates did
not change compared to control experiments in which sulphation was not
inhibited [1165 vs 1487 nmol/(10(6) cells.min)' respectively and [1.7
vs 1.7 nmol/(10(6) cells.hr)' respectively . This indicates that
increased sulphur availability due to decreased PAPS synthesis is
unable to raise the cysteine pool and stimulate the gamma-glutamyl
cycle and GSH synthesis.

Title
Protection by clofibrate against Acetaminophen hepatotoxicity in male
CD-1 mice is associated with an early increase in biliary concentration
of Acetaminophen-glutathione adducts.
Author
Manautou JE; Tveit A; Hoivik DJ; Khairallah EA; Cohen SD
Address
Toxicology Program' University of Connecticut' Storrs' Connecticut
06269-2092' USA.
Source
Toxicol Appl Pharmacol, 140(1):30-8 1996 Sep
Abstract
Repeated treatment with clofibrate (CFB) significantly increased
hepatic glutathione (GSH) content and also diminished Acetaminophen`s
(APAP) selective protein arylation' GSH depletion' and severity of
hepatocellular necrosis. The present work was conducted to evaluate the
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' i.p.'
daily for 10 days. Controls were given corn oil vehicle. They were
challenged with 700 mg APAP/kg in 50% propylene glycol/water after an
overnight fast. Results indicate that CFB pretreatment had no effect on
24-hr urinary excretion of APAP-glucuronide' sulfate' or GSH-derived
conJugates; however' there was 50% less unchanged APAP excreted in
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 APAP-GSH
content in CFB-pretreated mice at 2 hr after APAP dosing with
diminished levels in bile at 12 hr. The CFB-induced increase in biliary
excretion of APAP-GSH may mediate the protection against APAP-induced
hepatotoxicity.

Title
Intrinsic susceptibility of the kidney to Acetaminophen toxicity in
middle-aged rats.
Author
Tarloff JB; Goldstein RS; Silver AC; Hewitt WR; Hook JB
Address
Department of Investigative Toxicology' Smith Kline & French
Laboratories' King of Prussia' PA.
Source
Toxicol Lett, 52(1):101-10 1990 Jun
Abstract
Acetaminophen (APAP)-induced nephrotoxicity is age-dependent in male
Sprague-Dawley (SD) rats: middle-aged (9-12 months old) rats exhibit
nephrotoxicity at lower dosages of APAP than do young adults (2-3
months old). The present study was designed to test the hypothesis that
the intrinsic susceptibility of renal tissue to APAP toxicity is
increased in middle-aged rats. APAP toxicity was evaluated in renal
slices from naive 3- and 12-month-old male SD rats incubated with 0-50
mM APAP for 2-8 h. Renal slice glutathione (GSH) and APAP
concentrations were determined; renal function was assessed by organic
anion (para-aminohippurate' PAH) and cation (tetraethylammonium' TEA)
accumulation; and cell viability was assessed by lactate dehydrogenase
(LDH) leakage. At each concentration of APAP tested' accumulation of
APAP by renal slices was similar in 3- and 12-month-olds. APAP toxicity
in renal slices from both 3- and 12-month-old rats was characterized by
concentration-dependent increases in LDH leakage. In contrast to APAP
nephrotoxicity in vivo' APAP toxicity in renal slices was accompanied
by decreased accumulation of PAH and TEA. Additionally' APAP produced
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 not
precede cytotoxicity. No consistent age-dependent differences in the
time- and concentration-response curves for APAP nephrotoxicity were
observed. These data suggest that APAP cytotoxicity in vitro is not
increased in 12-month-old rats. However' since the pattern (and
mechanisms) of APAP cytotoxicity in vitro appears to be different from
that observed in vivo' extrapolation of in vitro cytotoxicity to in
vivo nephrotoxicity is limited. Therefore' age differences in intrinsic
susceptibility of the intact kidney cannot be excluded as a mechanism
contributing to enhanced APAP nephrotoxicity in middle-aged rats.

Title
Acetaminophen-induced toxicity to human epidermoid cell line A431 and
hepatoblastoma cell line Hep G2' in vitro' is diminished by silymarin.
Author
Shear NH; Malkiewicz IM; Klein D; Koren G; Randor S; Neuman MG
Address
Division of Dermatology' Sunnybrook Health Science Centre' Ont.'
Canada.
Source
Skin Pharmacol, 8(6):279-91 1995
Abstract
The skin and liver may be targets for cytotoxicity induced by oxidative
drug metabolites. We used human epidermoid A431 cells and human
hepatoblastoma Hep G2 cells as the experimental model. The aim of the
study was to investigate and evaluate the effect of silymarin on
Acetaminophen (APAP)-induced toxicity under controlled conditions.
Silymarin is known to be a potent antioxidant that diminishes toxicity
induced by a variety of other hepatotoxins (e.g. Amanita phaloides'
algae`s toxins' carbon tetrachloride). glutathione (GSH) depletion was
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 for
2-24 h to evaluate the drug`s ability to reduce cytoviability.
Viability was then quantitated by metabolism of the tetrazolium dyes
(MTT) and neutral red (NR). Cytoviability was 100% for controls. For
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 reduced
cytotoxicity significantly. In A431 cells' treatment with 20 mM APAP
reduced viability by 57% (MTT) and 69% (NR) versus control (100%). BSO
further decreased viability. Since incubation with silymarin showed
significant protection against APAP toxicity' it can be considered a
cytoprotective agent in this in vitro model of drug toxicity. GSH
concentrations in both cell lines decrease significantly after exposure
to 20 mM APAP' or 0.5 mM versus control (p < 0.05)' and increased (p <
0.001) if incubated with APAP and silymarin. The protective effect
could be through mitochondrial membrane stabilization and/or an
increase in available GSH.

Title
Protective effects of diallyl sulfide on Acetaminophen-induced
toxicities.
Author
Hu JJ; Yoo JS; Lin M; Wang EJ; Yang CS
Address
Department of Chemical Biology and Pharmacognosy' Rutgers University'
Piscataway' NJ 08855-0789' USA.
Source
Food Chem Toxicol, 34(10):963-9 1996 Oct
Abstract
Diallyl sulfide (DAS)' a maJor flavour component of garlic' is known to
modulate drug metabolism and may protect animals from chemically
induced toxicity and carcinogenesis. In this study the effects of DAS
on the oxidative metabolism and hepatotoxicity induced by Acetaminophen
(APAP) in rats were investigated. In the hepatotoxicity evaluation of
Fischer 344 rats there was a dose-dependent increase in the odds of
mortality rate by APAP (P = 0.009); DAS treatment significantly
protected rats from APAP-related mortality (P = 0.026). Liver toxicity
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- and
dose-dependent fashion. Treatment of DAS (50 mg/kg) 3 hr after APAP
dosing significantly (P < 0.05) protected rats from APAP-induced liver
toxicity. The metabolism of APAP (50 microM) in vitro was significantly
inhibited by DAS (0.3-1 mM) in liver microsomes isolated from F344
rats. As the effect of DAS on APAP-induced hepatotoxicity in vivo was
observed only when DAS was administered before or shortly after (< 3
hr) APAP dosing' data suggested that the protective effect of DAS is
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.

 

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