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Effect of ascorbic acid esters on hepatic glutathione levels in mice
treated with a hepatotoxic dose of acetaminophen.
Mitra A; Kulkarni AP; Ravikumar VC; Bourcier DR
Division of Pharmacology and Toxicology' School of Pharmacy' Northeast
Louisiana University' Monroe 71209.
J Biochem Toxicol, 6(2):93-100 1991 Summer
acetaminophen (APAP) with or without ascorbyl stearate (AS) or ascorbyl
palmitate (AP) was administered by gavage to male Swiss-Webster mice at
a dose of 600 mg/kg for each chemical. The biochemical markers of
hepatotoxicity' serum transaminases (serum glutamate pyruvate
transaminase [SGPT ' serum glutamate oxaloacetic transaminase [SGOT )
and serum isocitrate dehydrogenase (SICD) activities were monitored
after APAP and APAP + AP or AS dosing. There were significant
reductions in serum transaminase and SICD activities in the APAP- +
ascorbate ester-treated animals as compared to APAP-positive controls.
Oral coadministration of APAP with AP or AS did not prevent the initial
hepatic GSH depletion (15 min-4 hr postdosing). However' hepatic GSH
content began to rise in the APAP + AS or AP-treated animals at 4 hr
and reached control values within 12 hr postdosing. Urinary
mercapturate conJugates were also significantly higher in the APAP + AP
or AS-treated animals as compared to APAP alone when measured over a
60-min postdosing period. Plasma sulfobromophthalein (BSP) retention
was approximately eight times higher in APAP-treated animals as
compared to the APAP + ascorbate ester treatments indicating
maintenance of hepatic excretory functions in presence of AP or AS.
Prior depletion of hepatic GSH by diethyl maleate (DEM) did not alter
hepatoprotective effects of AP or AS in the presence of APAP. Hepatic
ascorbate levels also peaked at 4 hours after APAP + AP or AS
treatments. The possible role of L-ascorbic acid esters in GSH
regeneration following co-administration of a hepatotoxic dose and APAP
is discussed.

Comparison of covalent binding of acetaminophen and the regioisomer
3`-hydroxyacetanilide to mouse liver protein.
Matthews AM; Hinson JA; Roberts DW; Pumford NR
Division of Toxicology' University of Arkansas for Medical Sciences'
Little Rock 72205' USA.
Toxicol Lett, 90(1):77-82 1997 Jan 15
The hepatotoxicity of the analgesic acetaminophen has been previously
attributed to metabolic activation by cytochrome P450 to the reactive
intermediate N-acetyl-p-benzoquinone imine. At therapeutic doses this
species is detoxified by reaction with glutathione; however' following
a hepatotoxic dose' liver glutathione levels are depleted and the
metabolite covalently binds primarily to cysteine groups on proteins as
3-(cystein-S-yl)acetaminophen adducts. Altered function of critical
proteins has been postulated to be the mechanism of hepatotoxicity.
Covalent binding has been studied by both radiochemical methods and
immunochemical methods. Utilizing Western blot analysis with an
antiserum which recognizes acetaminophen we have previously shown that
covalent binding occurs on a number of proteins in various hepatic
fractions. In an effort to better understand the role of covalent
binding in the toxicity' others have studied the non-hepatotoxic isomer
3`-hydroxyacetanilide. Administration of large doses of radiolabeled
acetaminophen or 3`-hydroxyacetanilide resulted in similar levels of
covalent binding to proteins. To better understand the role of covalent
binding in toxicity we have administered mice 3`-hydroxyacetanilide and
acetaminophen' and analyzed liver fractions for protein adducts using
anti-3-(cystein-S-yl)acetaminophen and anti-arylacetamide antisera in
Western blot assays. Analysis of liver fractions from
acetaminophen-treated mice' with both antisera showed' as has been
previously reported' that acetaminophen covalently binds to a number of
hepatic proteins. In liver from 3`-hydroxyacetanilide-treated mice'
covalent adducts were detected with an anti-arylacetamide antiserum
only. A maJor 3`-hydroxyacetanilide protein adduct was observed in
microsomes at 50 kDa. Minor adducts were observed at 47 kDa in
microsomes and 56 kDa in cytosol. 3`-Hydroxyacetanilide protein adducts
were not observed in the 10'000 x g pellet. Densitometric analysis of a
time course of 3`-hydroxyacetanilide protein adducts indicated that
peak levels of the 50 kDa microsomal protein adduct occurred at 1 h and
subsequently decreased.

Effect of pregnenolone-16 alpha-carbonitrile and dexamethasone on
acetaminophen-induced hepatotoxicity in mice.
Madhu C; Maziasz T; Klaassen CD
Department of Pharmacology' Toxicology and Therapeutics' University of
Kansas Medical Center' Kansas City 66103.
Toxicol Appl Pharmacol, 115(2):191-8 1992 Aug
Recently' we demonstrated that a microsomal enzyme inducer with a
steroidal structure' pregnenolone-16 alpha-carbonitrile (PCN)' markedly
decreased the hepatotoxicity of acetaminophen (AA) in hamsters.
Therefore' it was of interest to determine if PCN' as well as another
steroid microsomal enzyme inducer' dexamethasone (DEX)' would decrease
the toxicity of AA in mice' another species sensitive to AA
hepatotoxicity. Mice were pretreated with PCN or DEX (100 and 75 mg/kg'
ip' for 4 days' respectively) and were given AA (300-500 mg/kg' ip).
Twenty-four hours after AA administration' liver inJury was assessed by
measuring serum activities of sorbitol dehydrogenase and alanine
aminotransferase and by histopathological examination. Neither PCN nor
DEX protected markedly against AA hepatotoxicity in mice; PCN tended to
decrease AA-induced hepatotoxicity' whereas DEX was found to enhance
AA-induced hepatotoxicity and it produced some hepatotoxicity itself.
DEX decreased the glutathione concentration (36%) in liver and
increased the biliary excretion of AA-GSH' which reflects the
activation of AA' whereas PCN produced neither effect. Thus' whereas
PCN has been shown to markedly decrease the hepatotoxicity of AA in
hamsters' apparently by decreasing the isoform of P450 responsible for
activating AA to N-acetyl-p-benzoquinoneimine' this does not occur in
mice after induction with either PCN or DEX. In contrast' DEX enhances
AA hepatotoxicity apparently by decreasing liver GSH levels and
increasing the activation of AA to a cytotoxic metabolite.

Studies of paracetamol/phenacetin toxicity: isolation and
characterization of p-aminophenol-glutathione conJugate.
Eyanagi R; Hisanari Y; Shigematsu H
Daiichi College of Pharmaceutical Sciences' Fukuoka-shi' Japan.
Xenobiotica, 21(6):793-803 1991 Jun
1. p-Aminophenol' a minor metabolite of phenacetin' is a potent
nephrotoxic agent. 2. We have examined the binding of p-aminophenol to
glutathione (GSH)' a model amino acid' in the presence of horseradish
peroxidase' which catalyses one electron oxidation. 3. The reaction
product was purified by preparative h.p.l.c.' and its structure was
determined by FAB mass spectrometry and 1H-n.m.r. to be a
p-aminophenol-GSH conJugate. The conJugate was formed between the ortho
carbon of the amino group of p-aminophenol and the SH group of GSH. 4.
It was confirmed by h.p.l.c. and 1H-n.m.r. that formation of the
conJugate was catalysed in vitro by rat liver microsomes and cumene

Protective effect of pregnenolone-16 alpha-carbonitrile on
acetaminophen-induced hepatotoxicity in hamsters.
Madhu C; Klaassen CD
Department of Pharmacology' Toxicology and Therapeutics' University of
Kansas Medical Center' Kansas City 66103.
Toxicol Appl Pharmacol, 109(2):305-13 1991 Jun 15
Overdosage of acetaminophen (AA) is known to produce acute liver
toxicity in both humans and laboratory animals. Hamsters are especially
sensitive to the hepatotoxic effect of AA. In the present study'
hamsters pretreated with pregnenolone-16 alpha-carbonitrile (PCN; 75
mg/kg' ip' daily for 4 days) were given a single dose of AA (350-1200
mg/kg' ip) and liver function was determined 24 hr later. Serum
activities of alanine aminotransferase (ALT) and sorbitol dehydrogenase
(SDH) as well as histopathology were used as indices of hepatotoxicity.
PCN pretreatment decreased AA-induced mortality. PCN dramatically
decreased ALT (93-97%) and SDH (63-98%) activities relative to control
values from hamsters treated with AA alone' and remarkably decreased
hepatic centrilobular necrosis produced by AA. To investigate the
mechanism of this protective effect' the biliary and urinary excretion
of AA metabolites were measured for 1 hr after administration of AA
(150 mg/kg' iv) in bile-duct-cannulated hamsters. PCN pretreatment
resulted in increased urinary and biliary excretion of AA-glucuronide
and decreased biliary excretion of AA-glutathione. Microsomes from
PCN-pretreated hamsters produced less benzoquinoneimine intermediate
than controls' as determined by the formation of AA-glutathione. In
addition' hepatic UDP-glucuronic acid and UDP-glucuronosyltransferase
were significantly increased in PCN-pretreated hamsters. In conclusion'
PCN pretreatment protected against AA-induced hepatotoxicity. The
mechanism of this protection appears to be due to decreased formation
of the reactive metabolite by the cytochrome P450 pathway' and an
increased detoxication by enhanced glucuronidation of AA.

[Influence of caffeine on toxicity and pharmacokinetics of paracetamol
Rai]nska-Giezek T
Zak adu Farmakologii KliniczneJ Instytutu Farmakologii i Toksykologii
PomorskieJ Akademii MedyczneJ w Szczecinie.
Ann Acad Med Stetin, 41():69-85 1995
The aim of this study was an experimental assessment of the influence
of caffeine on the symptoms of the toxic action of paracentamol in mice
as well as a detailed analysis if paracetamol pharmacokinetics in men
receiving caffeine at the same time. The toxicologic investigations
were performed in 620 Swiss mice. The LD50 and LD100 were determined
after an administration of paracetamol intraperitoneally. The effects
of two doses of caffeine on the survival time and number of animal
deaths were investigated. The degree of hepatic damage was assessed on
the basis of biochemical serum criteria' i.e. alanine aminotransferase'
aspartate aminotransferase' alkaline phosphatase and concentration of
bilirubin in serum' as well as on the basis of biochemical
investigations of liver homogenates' estimating the concentration of
reduced glutathione and P-450 cytochrome in the liver. The
anatomicopathologic liver evaluation was also performed' including
histological and histopathological examinations (glycogen' lipids). The
pharmacological investigations were performed in 9 healthy volunteers
in two randomized subgroups with the use of a cross-over method twice
at one week intervals. The blood paracetamol level was determined
according to the method of Thoma et al. The course of changes of
paracetamol plasma levels was described with a one-compartment model
for extravascular administration of the drug. The biexponential
equation' describing the assumed model' was solved with the method of
the smaller squares' using non-linear approximation. (Tab 1-6' Fig.
1-3). The experimental studies demonstrated a decrease in both the
acute toxicity and hepatotoxic action of paracetamol administered in
combination with caffeine' which was indicated by a significant
decrease in aminotransferase and alkaline phosphatase activity and in
concentration of bilirubin as well as by an increase in the
concentration of P-450 cytochrome and GSH in the liver which decreased
after administration of paracetamol alone and also by limitation or
lack of hepatic necrosis. The pharmacokinetic calculations in men
demonstrated an interaction between paracetamol and caffeine which was
indicated by a decrease in plasma paracetamol levels' by a smaller
surface under the curve of changes of paracetamol levels indicating
faster elimination of the drug after simultaneous administration with
caffeine. Therefore' paracetamol preparations with caffeine may be less
toxic than paracetamol alone.

Effect of captopril on glutathione level in the liver and
paracetamol-induced liver damage in rats
Habior A
Kliniki Gastroenterologii i Przemiany Materii Centrum Medycznego Kszta
cenia' Podyplomowego' Warszawie.
Pol Arch Med Wewn, 87(6):332-40 1992 Jun
Captopril' an inhibitor of angiotensin converting enzyme is widely used
in the treatment of hypertension and congestive heart failure. It
contains active sulfhydryl group and shares other structural feature
with cysteine' the main substrate of glutathione. Experiments were
undertaken to examine the effect of captopril on concentration of
endogenous glutathione in the liver and to examine the ability of
captopril to protect against paracetamol-induced hepatotoxicity. Single
doses of captopril (30 mg/kg) given to male Sprague-Dawley rats
produced a significant time dependent depletion of hepatic glutathione:
at 3 h--16% (controls--10% as the effect of fasting; p less than 0.02)'
at 5 h--25% (controls--17%; p less than 0.02). Pretreatment of rats
with single doses of captopril (30 mg/kg) 2 hours prior to
administration of toxic doses of paracetamol (2500 mg/kg) produced a
significant depletion of hepatic glutathione level as compared with
animals without pretreatment with captopril (median: 2.95 mumol/g liver
and 3.50 mumol/g liver' respectively; p less than 0.01). This was not
accompanied by a difference in the hepatotoxic effect of paracetamol as
assessed by histological staging of necrosis. Studies on covalent
binding of paracetamol showed that neither captopril at the doses 30
mg/kg' nor penicillamine (20 mg/kg) affected covalent binding of
paracetamol metabolites to cell protein. The results suggest that
captopril despite its structural similarity to cysteine depletes
hepatic glutathione level and does not protect against paracetamol

N-acetylcysteine in experimental and clinical acute lung injury.
Bernard GR
Department of Medicine' Vanderbilt University' Nashville' Tennessee
Am J Med, 91(3C):54S-59S 1991 Sep 30
Clinically' lung inJury is characterized by one or more of the
following: altered gas exchange' dyspnea' decreased static compliance'
and nonhydrostatic pulmonary edema. Although many antioxidants have
been investigated in in vitro systems and in animal models' only some
are at the developmental stage' or safe for clinical trials.
Considerable evidence has recently accumulated supporting the
hypothesis that leukocyte activation involves release of large
quantities of highly reactive oxygen radicals' and hydrogen peroxide is
partially responsible for diffuse microvascular and tissue inJury in
septic patients. Granulocyte depletion in animal models reduces the
degree of fall in dynamic lung compliance and the increase in airflow
resistance' lymph flow' and hypoxemia secondary to endotoxin
administration. We hypothesized that the partial benefit derived from
granulocyte depletion was due to the effective removal of a maJor
source of oxygen radicals. Among the list of free radical scavengers'
N-acetylcysteine stands out' because of its established usefulness in
at least one human disease thought to be secondary to free radical
organ damage (acetaminophen or paracetamol overdose). It is an
extremely safe agent with a wide toxic-therapeutic window. An
increasing number of animal studies indicate efficacy for this agent in
the prevention and therapy of lung inJury involving toxic oxygen
species. We developed a randomized' double-blind protocol for the study
of intravenous N-acetylcysteine in patients with established adult
respiratory distress syndrome (ADRS). Results of this trial are
preliminary. Nevertheless' they indicate that plasma and red cell
glutathione levels are decreased in ADRS patients' and that
N-acetylcysteine increases plasma cysteine as well as plasma and red
cell glutathione. There are also indications that cardiopulmonary
physiology is favorably affected by such therapy including improvements
in chest radiograph edema scores' pulmonary vascular resistance' static
compliance' oxygen delivery' and oxygen consumption.

Hepatoprotective mechanism of silymarin: no evidence for involvement of
cytochrome P450 2E1.
Miguez MP; Anundi I; Sainz-Pardo LA; Lindros KO
Biomedical Research Center' ALKO Ltd' Helsinki' Finland.
Chem Biol Interact, 91(1):51-63 1994 Apr
The involvement of the alcohol-inducible cytochrome P450 2E1 in the
hepatoprotective mechanism of the plant flavonoid extract silymarin'
and its main active component silybin' was investigated in isolated
hepatocytes. Allyl alcohol toxicity' associated lipid peroxidation and
GSH depletion was efficiently counteracted by silymarin (0.01-0.5 mM)'
and at higher concentrations by silybin. Cell damage by t-butyl
hydroperoxide was also prevented by silymarin and silybin' but less
efficiently. However' the covalent binding of the acetaminophen
intermediate' formed via P450 2E1' was unaffected by addition of the
flavonoids. Silybin did not influence microsomal 2E1-catalyzed
demethylation of N-nitrosodimethylamine. Neither did demethylation of
N-nitrosodimethylamine or aminopyrine by isolated microsomes affect the
in vivo administration of silybin. Addition of silymarin or silybin to
primary cultures of isolated hepatocytes did not prevent cell damage
induced by exposure to the P450 2E1 substrate CCl4. In contrast' the
mere presence of low concentrations (25-50 microM) of these compounds
was found to inhibit cell attachment to the matrix and eventually
resulted in cell damage. We conclude that contrary to earlier reports
we found no evidence for an interaction of silymarin or silybin with
cytochrome P450 2E1. This suggests that the antioxidant and free
radical scavenging properties may account for most of the therapeutic
effect of these compounds. The untoward effect of silymarin on cultured
cells may have consequences when considering long-term prescription of
this therapeutic agent.

Molecular mechanism for prevention of N-acetyl-p-benzoquinoneimine
cytotoxicity by the permeable thiol drugs diethyldithiocarbamate and
Lauriault VV; O`Brien PJ
Faculty of Pharmacy' University of Toronto' Ontario' Canada.
Mol Pharmacol, 40(1):125-34 1991 Jul
The present study was carried out to elucidate the mechanism by which
the permeable thiol drug diethyldithiocarbamate (DEDC) exhibited an
antidotal effect against acetaminophen-induced hepatotoxicity in vivo.
DEDC was found to act as an antidote against acetaminophen-induced
cytotoxicity in hepatocytes isolated from a pyrazole-pretreated rat
without affecting cytochrome P-450 levels. The mechanism of protection
exhibited against reactive intermediate N-acetyl-p-benzoquinoneimine
(NAPQI)-induced cytotoxicity by DEDC was then investigated and compared
with that exhibited by the permeable thiol-reductant dithiothreitol
(DTT). Cytotoxicity induced by the dimethylated analogue
2'6-dimethyl-N-acetyl-p-benzoquinoneimine (2'6-diMeNAPQI) was prevented
if the hepatocytes were preincubated with DEDC for 5 min and removed
before addition of 2'6-diMeNAPQI. Both DEDC and DTT were also found to
act as antidotes against NAPQI- and 2'6-diMeNAPQI-induced cytotoxicity
in isolated rat hepatocytes if added within 2 min of the addition of
the quinoneimines. However' the addition of DEDC or DTT 10 min after
either quinoneimine did not prevent subsequent cytotoxicity or restore
GSH levels' indicating that the alkylation of GSH and of protein thiols
was irreversible at that time. Fast atom bombardment mass spectrometry
was used to show that DEDC formed conJugates with both NAPQI and
2'6-diMeNAPQI. Furthermore' these conJugates were found to be nontoxic.
This suggests that DEDC acts as a trap for the toxic quinoneimines'
thus preventing alkylation of essential macromolecules. In contrast'
DTT reduced the quinoneimines to their respective nontoxic parent
compounds and presumably also reduced mixed-protein disulfides and
GSSG' thereby regenerating protein thiols and GSH. Therefore' this
study suggests that DEDC and DTT act as antidotes by two different

Protection against acetaminophen hepatotoxicity by ribose-cysteine
Roberts JC; Charyulu RL; Zera RT; Nagasawa HT
Department of Medicinal Chemistry' University of Utah' Salt Lake City
Pharmacol Toxicol, 70(4):281-5 1992 Apr
oxylic acid (Ribose-Cysteine' RibCys)' a latent form of L-cysteine'
releases the sulfhydryl amino acid in vivo by non-enzymatic ring
opening and solvolysis. The liberated L-cysteine then stimulates
hepatic glutathione biosynthesis. In the present studies' the efficacy
of hepatoprotection by RibCys was evaluated to explore its potential
utility as an acetaminophen (APAP) antidote. Protection was evaluated
in the Swiss-Webster mouse model both by survival data as well as by
quantitative histological criteria of hepatic damage. A dose-response
study showed increased protection with increased intraperitoneal doses
of RibCys ranging from 0.5 to 8.0 mmol/kg. RibCys administration 30
min. prior to and up to four hours after the APAP dose showed varying
degrees of protection; however' the best protection was seen when
RibCys was given shortly after APAP administration. A single RibCys
dose given by the intraperitoneal or intravenous route gave better
protection than when administered orally; however' RibCys given in
three doses' one hour apart' regardless of the mode of administration'
offered the best protection after an LD90 dose of APAP. Overall' RibCys
continues to exhibit promising protective capabilities against APAP
hepatotoxicity' which may be capitalized upon in clinical overdose

Effects of astragalus (ASI' SK) on experimental liver inJury
Zhang YD; Shen JP; Zhu SH; Huang DK; Ding Y; Zhang XL
Department of Pharmacology' NanJing Medical College.
Yao Hsueh Hsueh Pao, 27(6):401-6 1992
The saponins (ASI' SK) used in this study was extracted from the root
of Astragalus membranaceous Bge and Astragalus sieversianus Pull. ASI
and SK were found to protect liver from chemical inJury induced by
CCl4' D-galactosamine and acetaminophen in mice. The two saponins were
shown to impede the elevation of SGPT level' decrease the MDA content
and increase the GSH concentration in mouse liver. Obvious improvement
of histological changes were also observed. The protective action of
ASI and SK against the hepatotoxicity was also shown in experiments
using primary cultured rat hepatocytes. The average value of GPT in the
medium treated with different concentration of ASI and SK (0.00075-0.18
mmol/L) was lower than that in control. Analyzing through multiple
linear correlation' we showed that the lowering of SGPT was negatively
related to the increase of GSH' positively related to the decrease of
MDA in mice given CCl4 or acetaminophen in combination with ASI or SK.
These results indicate that the hepato-protective effects of ASI and SK
may be due to their anti-oxidation activities' since the content of
liver protein in mice given ASI or SK was more than that in the
controls. Moreover' the level of hepatic microsomal cytochrome P-450 in
all mice given the two saponins were significantly increased' the liver
metabolism and immunoregulating action produced by ASI and SK may be
also involved in their hepato-protective effects.


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