Effect of ascorbic acid esters on hepatic glutathione levels
treated with a hepatotoxic dose of acetaminophen.
Mitra A; Kulkarni AP; Ravikumar VC; Bourcier DR
Division of Pharmacology and Toxicology' School of Pharmacy'
Louisiana University' Monroe 71209.
J Biochem Toxicol, 6(2):93-100 1991 Summer
acetaminophen (APAP) with or without ascorbyl stearate (AS) or
palmitate (AP) was administered by gavage to male Swiss-Webster
a dose of 600 mg/kg for each chemical. The biochemical markers
hepatotoxicity' serum transaminases (serum glutamate pyruvate
transaminase [SGPT ' serum glutamate oxaloacetic transaminase
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
Oral coadministration of APAP with AP or AS did not prevent the
hepatic GSH depletion (15 min-4 hr postdosing). However' hepatic
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
60-min postdosing period. Plasma sulfobromophthalein (BSP) retention
was approximately eight times higher in APAP-treated animals
compared to the APAP + ascorbate ester treatments indicating
maintenance of hepatic excretory functions in presence of AP
Prior depletion of hepatic GSH by diethyl maleate (DEM) did not
hepatoprotective effects of AP or AS in the presence of APAP.
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
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
intermediate N-acetyl-p-benzoquinone imine. At therapeutic doses
species is detoxified by reaction with glutathione; however'
a hepatotoxic dose' liver glutathione levels are depleted and
metabolite covalently binds primarily to cysteine groups on proteins
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
immunochemical methods. Utilizing Western blot analysis with
antiserum which recognizes acetaminophen we have previously shown
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
3`-hydroxyacetanilide. Administration of large doses of radiolabeled
acetaminophen or 3`-hydroxyacetanilide resulted in similar levels
covalent binding to proteins. To better understand the role of
binding in toxicity we have administered mice 3`-hydroxyacetanilide
acetaminophen' and analyzed liver fractions for protein adducts
anti-3-(cystein-S-yl)acetaminophen and anti-arylacetamide antisera
Western blot assays. Analysis of liver fractions from
acetaminophen-treated mice' with both antisera showed' as has
previously reported' that acetaminophen covalently binds to a
hepatic proteins. In liver from 3`-hydroxyacetanilide-treated
covalent adducts were detected with an anti-arylacetamide antiserum
only. A maJor 3`-hydroxyacetanilide protein adduct was observed
microsomes at 50 kDa. Minor adducts were observed at 47 kDa in
microsomes and 56 kDa in cytosol. 3`-Hydroxyacetanilide protein
were not observed in the 10'000 x g pellet. Densitometric analysis
time course of 3`-hydroxyacetanilide protein adducts indicated
peak levels of the 50 kDa microsomal protein adduct occurred
at 1 h and
Effect of pregnenolone-16 alpha-carbonitrile and dexamethasone
acetaminophen-induced hepatotoxicity in mice.
Madhu C; Maziasz T; Klaassen CD
Department of Pharmacology' Toxicology and Therapeutics' University
Kansas Medical Center' Kansas City 66103.
Toxicol Appl Pharmacol, 115(2):191-8 1992 Aug
Recently' we demonstrated that a microsomal enzyme inducer with
steroidal structure' pregnenolone-16 alpha-carbonitrile (PCN)'
decreased the hepatotoxicity of acetaminophen (AA) in hamsters.
Therefore' it was of interest to determine if PCN' as well as
steroid microsomal enzyme inducer' dexamethasone (DEX)' would
the toxicity of AA in mice' another species sensitive to AA
hepatotoxicity. Mice were pretreated with PCN or DEX (100 and
ip' for 4 days' respectively) and were given AA (300-500 mg/kg'
Twenty-four hours after AA administration' liver inJury was assessed
measuring serum activities of sorbitol dehydrogenase and alanine
aminotransferase and by histopathological examination. Neither
DEX protected markedly against AA hepatotoxicity in mice; PCN
decrease AA-induced hepatotoxicity' whereas DEX was found to
AA-induced hepatotoxicity and it produced some hepatotoxicity
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'
PCN has been shown to markedly decrease the hepatotoxicity of
hamsters' apparently by decreasing the isoform of P450 responsible
activating AA to N-acetyl-p-benzoquinoneimine' this does not
mice after induction with either PCN or DEX. In contrast' DEX
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
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
determined by FAB mass spectrometry and 1H-n.m.r. to be a
p-aminophenol-GSH conJugate. The conJugate was formed between
carbon of the amino group of p-aminophenol and the SH group of
It was confirmed by h.p.l.c. and 1H-n.m.r. that formation of
conJugate was catalysed in vitro by rat liver microsomes and
Protective effect of pregnenolone-16 alpha-carbonitrile on
acetaminophen-induced hepatotoxicity in hamsters.
Madhu C; Klaassen CD
Department of Pharmacology' Toxicology and Therapeutics' University
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
sensitive to the hepatotoxic effect of AA. In the present study'
hamsters pretreated with pregnenolone-16 alpha-carbonitrile (PCN;
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
values from hamsters treated with AA alone' and remarkably decreased
hepatic centrilobular necrosis produced by AA. To investigate
mechanism of this protective effect' the biliary and urinary
of AA metabolites were measured for 1 hr after administration
(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
PCN-pretreated hamsters produced less benzoquinoneimine intermediate
than controls' as determined by the formation of AA-glutathione.
addition' hepatic UDP-glucuronic acid and UDP-glucuronosyltransferase
were significantly increased in PCN-pretreated hamsters. In conclusion'
PCN pretreatment protected against AA-induced hepatotoxicity.
mechanism of this protection appears to be due to decreased formation
of the reactive metabolite by the cytochrome P450 pathway' and
increased detoxication by enhanced glucuronidation of AA.
[Influence of caffeine on toxicity and pharmacokinetics of paracetamol
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
as well as a detailed analysis if paracetamol pharmacokinetics
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
of two doses of caffeine on the survival time and number of animal
deaths were investigated. The degree of hepatic damage was assessed
the basis of biochemical serum criteria' i.e. alanine aminotransferase'
aspartate aminotransferase' alkaline phosphatase and concentration
bilirubin in serum' as well as on the basis of biochemical
investigations of liver homogenates' estimating the concentration
reduced glutathione and P-450 cytochrome in the liver. The
anatomicopathologic liver evaluation was also performed' including
histological and histopathological examinations (glycogen' lipids).
pharmacological investigations were performed in 9 healthy volunteers
in two randomized subgroups with the use of a cross-over method
at one week intervals. The blood paracetamol level was determined
according to the method of Thoma et al. The course of changes
paracetamol plasma levels was described with a one-compartment
for extravascular administration of the drug. The biexponential
equation' describing the assumed model' was solved with the method
the smaller squares' using non-linear approximation. (Tab 1-6'
1-3). The experimental studies demonstrated a decrease in both
acute toxicity and hepatotoxic action of paracetamol administered
combination with caffeine' which was indicated by a significant
decrease in aminotransferase and alkaline phosphatase activity
concentration of bilirubin as well as by an increase in the
concentration of P-450 cytochrome and GSH in the liver which
after administration of paracetamol alone and also by limitation
lack of hepatic necrosis. The pharmacokinetic calculations in
demonstrated an interaction between paracetamol and caffeine
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
caffeine. Therefore' paracetamol preparations with caffeine may
toxic than paracetamol alone.
Effect of captopril on glutathione level in the liver and
paracetamol-induced liver damage in rats
Kliniki Gastroenterologii i Przemiany Materii Centrum Medycznego
cenia' Podyplomowego' Warszawie.
Pol Arch Med Wewn, 87(6):332-40 1992 Jun
Captopril' an inhibitor of angiotensin converting enzyme is widely
in the treatment of hypertension and congestive heart failure.
contains active sulfhydryl group and shares other structural
with cysteine' the main substrate of glutathione. Experiments
undertaken to examine the effect of captopril on concentration
endogenous glutathione in the liver and to examine the ability
captopril to protect against paracetamol-induced hepatotoxicity.
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
at 5 h--25% (controls--17%; p less than 0.02). Pretreatment of
with single doses of captopril (30 mg/kg) 2 hours prior to
administration of toxic doses of paracetamol (2500 mg/kg) produced
significant depletion of hepatic glutathione level as compared
animals without pretreatment with captopril (median: 2.95 mumol/g
and 3.50 mumol/g liver' respectively; p less than 0.01). This
accompanied by a difference in the hepatotoxic effect of paracetamol
assessed by histological staging of necrosis. Studies on covalent
binding of paracetamol showed that neither captopril at the doses
mg/kg' nor penicillamine (20 mg/kg) affected covalent binding
paracetamol metabolites to cell protein. The results suggest
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.
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
been investigated in in vitro systems and in animal models' only
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
partially responsible for diffuse microvascular and tissue inJury
septic patients. Granulocyte depletion in animal models reduces
degree of fall in dynamic lung compliance and the increase in
resistance' lymph flow' and hypoxemia secondary to endotoxin
administration. We hypothesized that the partial benefit derived
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
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
the prevention and therapy of lung inJury involving toxic oxygen
species. We developed a randomized' double-blind protocol for
of intravenous N-acetylcysteine in patients with established
respiratory distress syndrome (ADRS). Results of this trial are
preliminary. Nevertheless' they indicate that plasma and red
glutathione levels are decreased in ADRS patients' and that
N-acetylcysteine increases plasma cysteine as well as plasma
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'
compliance' oxygen delivery' and oxygen consumption.
Hepatoprotective mechanism of silymarin: no evidence for involvement
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
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
GSH depletion was efficiently counteracted by silymarin (0.01-0.5
and at higher concentrations by silybin. Cell damage by t-butyl
hydroperoxide was also prevented by silymarin and silybin' but
efficiently. However' the covalent binding of the acetaminophen
intermediate' formed via P450 2E1' was unaffected by addition
flavonoids. Silybin did not influence microsomal 2E1-catalyzed
demethylation of N-nitrosodimethylamine. Neither did demethylation
N-nitrosodimethylamine or aminopyrine by isolated microsomes
in vivo administration of silybin. Addition of silymarin or silybin
primary cultures of isolated hepatocytes did not prevent cell
induced by exposure to the P450 2E1 substrate CCl4. In contrast'
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
we found no evidence for an interaction of silymarin or silybin
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
cells may have consequences when considering long-term prescription
this therapeutic agent.
Molecular mechanism for prevention of N-acetyl-p-benzoquinoneimine
cytotoxicity by the permeable thiol drugs diethyldithiocarbamate
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
the permeable thiol drug diethyldithiocarbamate (DEDC) exhibited
antidotal effect against acetaminophen-induced hepatotoxicity
DEDC was found to act as an antidote against acetaminophen-induced
cytotoxicity in hepatocytes isolated from a pyrazole-pretreated
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
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
if the hepatocytes were preincubated with DEDC for 5 min and
before addition of 2'6-diMeNAPQI. Both DEDC and DTT were also
act as antidotes against NAPQI- and 2'6-diMeNAPQI-induced cytotoxicity
in isolated rat hepatocytes if added within 2 min of the addition
the quinoneimines. However' the addition of DEDC or DTT 10 min
either quinoneimine did not prevent subsequent cytotoxicity or
GSH levels' indicating that the alkylation of GSH and of protein
was irreversible at that time. Fast atom bombardment mass spectrometry
was used to show that DEDC formed conJugates with both NAPQI
2'6-diMeNAPQI. Furthermore' these conJugates were found to be
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
GSSG' thereby regenerating protein thiols and GSH. Therefore'
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
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
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
quantitative histological criteria of hepatic damage. A dose-response
study showed increased protection with increased intraperitoneal
of RibCys ranging from 0.5 to 8.0 mmol/kg. RibCys administration
min. prior to and up to four hours after the APAP dose showed
degrees of protection; however' the best protection was seen
RibCys was given shortly after APAP administration. A single
dose given by the intraperitoneal or intravenous route gave better
protection than when administered orally; however' RibCys given
three doses' one hour apart' regardless of the mode of administration'
offered the best protection after an LD90 dose of APAP. Overall'
continues to exhibit promising protective capabilities against
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
of Astragalus membranaceous Bge and Astragalus sieversianus Pull.
and SK were found to protect liver from chemical inJury induced
CCl4' D-galactosamine and acetaminophen in mice. The two saponins
shown to impede the elevation of SGPT level' decrease the MDA
and increase the GSH concentration in mouse liver. Obvious improvement
of histological changes were also observed. The protective action
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
MDA in mice given CCl4 or acetaminophen in combination with ASI
These results indicate that the hepato-protective effects of
ASI and SK
may be due to their anti-oxidation activities' since the content
liver protein in mice given ASI or SK was more than that in the
controls. Moreover' the level of hepatic microsomal cytochrome
all mice given the two saponins were significantly increased'
metabolism and immunoregulating action produced by ASI and SK
also involved in their hepato-protective effects.