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Title
Specificity and directionality of thiol effects on sinusoidal
glutathione transport in rat liver.
Author
Lu SC; Kuhlenkamp J; Ge JL; Sun WM; Kaplowitz N
Address
Department of Medicine' University of Southern California School
of
Medicine' Los Angeles 90033.
Source
Mol Pharmacol, 46(3):578-85 1994 Sep
Abstract
In rats the sinusoidal glutathione (GSH) carrier transports GSH
bidirectionally' and its activity is influenced by the thiol-disulfide
status; the Vmax of sinusoidal GSH efflux was increased by
dithiothreitol (DTT) and decreased by cystine. In the present
work we
examined the specificity and directionality of the thiol effect.
Using
in situ perfused livers' we found that 1 mM DTT and other dithiols'
including 1'2-ethanedithiol' 1'3-propanedithiol' and 1'4-butanedithiol'
stimulated sinusoidal GSH efflux by 200-500% but dihydrothioctic
acid'
which is negatively charged' had no effect. Uncharged or positively
charged monothiols (2 mM)' such as dimercaprol' monothioglycerol'
2-mercaptoethanol' 3-mercapto-2-butanol' 1-mercapto-2-propanol'
and
cysteamine' also exerted a stimulatory effect on sinusoidal GSH
efflux.
In contrast' monothiols containing a negatively charged substituent'
such as penicillamine' captopril' N-acetylcysteine'
mercaptopropionylglycine' mercaptoethanesulfonic acid' mercaptoacetic
acid' and mercaptopropionic acid' had no effect. The thiol moiety
was
essential for activity' inasmuch as ethanol' propanol' propanediol'
and
glycerol had no effect on sinusoidal GSH efflux. The effect of
DTT or
cystine pretreatment (2 mM or 0.5 mM' respectively' for 30 min)
on GSH
uptake was then examined using cultured rat hepatocytes. The
linear
rate of [35S GSH uptake and the concentration dependence were
measured
after cells were pretreated with acivicin (0.5 mM' for 15 min)
and
buthionine sulfoximine (10 mM' 15 min)' to prevent breakdown
and
resynthesis of GSH from precursors' respectively. Uptake buffer
also
contained 20 mM alpha-(methylamino)isobutyric acid and 20 mM
threonine
(inhibitors of amino acid transport systems A and ASC' respectively)'
to prevent uptake of cysteine. Pretreatment with DTT decreased
the Vmax
of GSH uptake by approximately 50% (control Vmax value' 24 nmol/10(6)
cells/30 min)' whereas the Km remained unaffected (approximately
8 mM).
Cystine pretreatment had no influence on GSH uptake but inhibited
efflux. In conclusion' the presence of at least one thiol group
and the
absence of negative charge are required to stimulate sinusoidal
GSH
efflux. The direction of GSH transport is modulated by the
thiol-disulfide status' so that thiol reduction changes the GSH
transporter from a bidirectional GSH transporter into a preferentially
unidirectional (outward) transporter by inhibiting uptake while
stimulating efflux and thiol oxidation favors inward transport
by
inhibiting only efflux.
Title
Comparative study of the damage produced by acute ethanol and
acetaldehyde treatment in a human fetal hepatic cell line.
Author
Olivares IP; Bucio L; Souza V; C]arabez A; Guti]errez-Ruiz MC
Address
Departamento de Ciencias de la Salud' Universidad Aut]onoma
Metropolitana-I' Unidad Iztapalapa' Mexico' D.F.
Source
Toxicology, 120(2):133-44 1997 Jun 27
Abstract
The effects of acute ethanol and acetaldehyde treatment on cell
proliferation' cell adhesion capacity' neutral red incorporation
into
lysosomes' glutathione content' protein sulfhydryl compounds'
lipid
peroxidation' inner mitochondrial membrane integrity (MTT test)'
lactate dehydrogenase activity (LDH) and ultrastructural alterations
were investigated in a human fetal hepatic cell line (WRL-68
cells).
WRL-68 cells were used' due to the fact that' although this cell
line
expresses some hepatic characteristics' it does not express alcohol
dehydrogenase or cytochrome P450 activity' so it could be a good
model
to study the effect of the toxic agents per se. Cells were exposed
during 120 min with 200 mM ethanol or 10 mM acetaldehyde. Under
these
conditions' cells presented 100% viability and no morphological
alteration was observed by light microscopy. Acetaldehyde-treated
cells
reduced their proliferative capacity drastically while the
ethanol-treated ones presented no difference with control cells.
Cell
adhesion to substrate' measured as time required to adhere to
the
substrate and time required to detach from the substrate' was
diminished in acetaldehyde WRL-68-treated cells. Cytotoxicity
measures
as neutral red and MTT test showed that acetaldehyde-treated
cells
presented more damage than ethanol-treated ones. Cellular respiratory
capacity was compromised by acetaldehyde treatment due to 40%
less
oxygen consumption than control cells. Lipid peroxidation values'
measured as malondialdehyde production' were higher in ethanol-treated
WRL-68 cells (127%) than in acetaldehyde-treated ones (60%) to
control
cell values. Lactate dehydrogenase activity (LDH) in extracellular
media of ethanol-treated cells presented the highest values.
GSH
content was reduced 95% and thiol protein content was diminished
severely in acetaldehyde-treated cells. Transmission electron
microscopy showed more ultrastructural alterations in cells treated
with acetaldehyde. The results indicate that acetaldehyde' like
ethanol' produced damage at cellular level' although more damage
could
be observed in acetaldehyde WRL-68-treated cells.
Title
Evidence of toxic metabolite stress in black South Africans with
chronic pancreatitis.
Author
Gut A; Chaloner C; Schofield D; Sandle LR; Purmasir M; Segal
I;
Braganza JM
Address
Gastrointestinal Unit' Baragwanath Hospital' Johannesburg' South
Africa.
Source
Clin Chim Acta, 236(2):145-53 1995 May 15
Abstract
We report the results of a further study to test our hypothesis
that
toxic metabolite stress is germane to heightened free radical
activity
and hence to the genesis of chronic pancreatitis. Consecutive
black
South African patients with clinically quiescent chronic pancreatitis
were studied' provided that the diagnosis had been made within
the
previous 2 years and that they did not have overt liver disease.
All of
them had been advised to stop drinking alcohol. Analysis of an
early
morning sample of urine showed a lower ratio of inorganic to
ester
sulphate (P < 0.001) and a higher ratio of D-glucaric acid
to
creatinine (P < 0.02) in the group of 14 patients than in
15 local
controls' while plasma analysis showed a lower concentration
of
glutathione (GSH) in the patients (P < 0.001). This evidence
of
increased utilisation of phase II conJugative pathways of xenobiotic
disposal was in keeping with on-going toxic metabolite stress
from
heightened phase I oxidative metabolism in the group of patients.
Parallel studies of theophylline pharmacokinetics showed heightened
drug clearance compatible with induced cytochrome P-4501A2 in
two
patients' whereas increased activity of gamma-glutamyl transferase
in
serum suggested persisting induction of P-4502E1' as by ethanol'
in
several others. The contemporaneous increases in free radical
activity
and utilisation of xenobiotic disposal pathways in Sowetan Africans
with chronic pancreatitis is in line with the toxic metabolite
concept
of disease pathogenesis.
Title
GSH transporters: molecular characterization and role in GSH
homeostasis.
Author
Kaplowitz N; Fern]andez-Checa JC; Kannan R; Garcia-Ruiz C; Ookhtens
M;
Yi JR
Address
USC Center for Liver Diseases' USC School of Medicine' Los Angeles'
90033-4581' USA.
Source
Biol Chem Hoppe Seyler, 377(5):267-73 1996 May
Abstract
Considerable progress has been made in the last few years in
the
molecular identification and characterization of hepatic GSH
transporter-associated polypeptides. We are now poised to determine
their precise mechanisms of action and regulation at the
transcriptional and post-translational level. It is also anticipated
that molecular characterization of the mitochondrial GSH transporter
and sodium GSH co-transporters will be accomplished in the near
future.
With this information' a more complete understanding of GSH/cysteine
homeostasis can be achieved which can be applied to furthering
the
prevention and treatment of the diseases of oxidative stress'
such as
aging' HIV' cataract' atherosclerosis' cancer and alcoholic liver
disease.
Title
glutathione homeostasis in rats chronically treated with ethanol.
Evidence for an increased hepatic GSH export in vivo.
Author
Kretzschmar M; Reinhardt D; Schlechtweg J; Machnik G; Klinger
W;
Schirrmeister W
Address
Clinic of Anaesthesiology and Intensive Care' Friedrich Schiller
University Jena' Germany.
Source
Exp Toxicol Pathol, 44(6):344-8 1992 Oct
Abstract
The influence of chronic ethanol feeding to rats on the hepatic
glutathione (GSH and GSSG) system (synthesis' catabolism' export)
and
on the GSH and GSSG concentrations in extrahepatic tissues was
investigated. Histological examination of livers from ethanol
pretreated rats revealed a minor dilatation of the hepatic sinusoids.
After ethanol administration the distribution pattern of
gamma-glutamyltranspeptidase (enzymehistochemistry) was nearly
unchanged' but the hepatic activity of this enzyme was increased.
The
ethanol pretreatment led to a decrease in hepatic GSH content.
The
hepatic activity of the GSSG-reductase were increased after ethanol
treatment whereas the activities of the GSH synthesizing enzymes
(gamma-glutamyl-cysteinyl-synthetase and GSH-synthetase) were
not
affected. A strong increase in sinusoidal GSH export was found
in the
ethanol-pretreated rats. The GSH- and GSSG concentrations of
brain'
lung' kidney and skeletal muscle were unchanged. It can be concluded
that the ethanol-induced alteration of the hepatic GSH metabolism
is
caused mainly by changes of the sinusoidal membrane of the hepatocytes
(direct effect of ethanol on the sinusoidal GSH carrier) leading
to an
increased GSH export into plasma. This effect should not due
to an
increased extrahepatic requirement for GSH.
Title
Protective role of intracellular glutathione against ethanol-induced
damage in cultured rat gastric mucosal cells.
Author
Mutoh H; Hiraishi H; Ota S; Yoshida H; Ivey KJ; Terano A; Sugimoto
T
Address
Second Department of Internal Medicine' Faculty of Medicine'
University
of Tokyo' Japan.
Source
Gastroenterology, 98(6):1452-9 1990 Jun
Abstract
This study investigated whether intracellular glutathione is
cytoprotective against ethanol-induced inJury to cultured rat
gastric
mucosal cells in vitro. Secondly' it investigated whether reduced
glutathione or oxidized glutathione is responsible for this
cytoprotection. Cytolysis was quantified by measuring 51Cr release
from
prelabeled cells. Concentrations of ethanol greater than 12%
caused
cell damage and increased 51Cr release in a dose-dependent and
time-related fashion. When a substrate for glutathione synthesis'
N-acetyl-L-cysteine' was provided to cultured cells for 4 h before
challenge with ethanol' cytolysis was significantly decreased
corresponding with an increase in cellular glutathione content.
Pretreatment with diethyl maleate' which depletes reduced glutathione
without forming oxidized glutathione' potentiated ethanol-induced
cell
damage in a dose-dependent manner with the decrease of cellular
glutathione content. The administration of tert-butyl hydroperoxide
(which is specifically reduced by glutathione peroxidase to generate
oxidized glutathione from reduced glutathione) or diamide (which
nonenzymatically oxidizes reduced glutathione to oxidized glutathione)
enhanced ethanol inJury. We conclude that in cultured gastric
mucosal
cells' (a) intracellular glutathione maintains integrity of gastric
mucosal cells against ethanol in vitro; and (b) reduced glutathione
rather than oxidized glutathione is responsible for this
cytoprotection. We postulate that the presence of reduced glutathione
is essential to allow glutathione peroxidase to catalyze the
ethanol-generated toxic oxygen radical' hydrogen peroxide.
Title
Combined effects of ethanol and cigarette smoke on hepatic and
pulmonary xenobiotic metabolizing enzymes in rats.
Author
Eke BC; Vural N; I,scan M
Address
Department of Toxicology' Faculty of Pharmacy' Ankara University'
Turkey.
Source
Chem Biol Interact, 102(3):155-67 1996 Dec 20
Abstract
The combined effects of ethanol (EtOH) and cigarette smoke (CS)
on
hepatic and pulmonary monooxygenase (MO) activities (aniline
4-hydroxylase (AH)' aminopyrine N-demethylase (AMND)' 7-ethoxyresorufin
O-deethylase (EROD)' p-nitroanisole O-demethylase (p-NAOD))'
lipid
peroxidation (LP) and reduced glutathione (GSH) levels and glutathione
S-transferase (GST) activities toward several substrates
(l-chloro-2'4-dinitrobenzene (CDNB)' 1'2-dichloro-4-nitrobenzene
(DCNB)' ethacrynic acid (EAA)' 1'2-epoxy-3-(p-nitrophenoxy)-propane
(ENPP)) were determined and compared with those of EtOH or CS
alone in
rats. When the male adult rats (225-275 g) were treated with
10% EtOH
(v/v) in their drinking for 21 days AH' AMND and EROD activities
and LP
and GSH levels increased significantly whereas GST activity for
EAA
decreased significantly in liver as compared to controls. EtOH
did not
change the hepatic p-NAOD and GST activities toward CDNB' DCNB
and
ENPP. In lung' EtOH increased GST activities toward CDNB and
ENPP and
LP level but decreased GST activity toward DCNB' significantly.
No
alterations were noted in pulmonary MO activities and GST activity
toward EAA and GSH level by EtOH treatment. When the animals
were
exposed to CS five times a day' with 1 h intervals' for 3 days
in a
chamber where smoke and fresh air lead alternatively' AMND' EROD
and
p-NAOD activities' GST activity toward EAA and GSH level increased
but
LP level and GST activity for ENPP decreased significantly in
liver. CS
did not alter the hepatic AH and GST activities toward CDNB and
DCNB.
In lung' CS increased AH' EROD and p-NAOD activities and LP and
GSH
levels and decreased all the GST activities studied significantly.
CS
had no influence on pulmonary AMND activity. For the combined
treatment' the animals were treated with 10% EtOH (v/v) in their
drinking water for 21 days and during the last 3 days they were
exposed
to CS five times a day' with 1 h intervals' in a chamber where
smoke
and fresh air lead alternatively. In these animals' augmentation
of
elevations were noted in AH and p-NAOD activities and LP and
GSH levels
but not in EROD and AMND activities in liver. Combined treatment
significantly decreased GST activity toward CDNB' ameliorated
the
alteration caused by either EtOH or CS treatment alone on GST
activity
toward EAA and potentiated the depression of GST activity toward
ENPP
to a greater degree. No change was observed in GST activity toward
DCNB. In lung' combined treatment potentiated the elevations
of AMND
and p-NAOD activities and LP level and not those of AH and EROD
activities. GST activities toward CDNB' DCNB and ENPP were highly
elevated by the combined treatment. No changes were observed
in
pulmonary GSH level and GST activity for EAA by the combined
treatment.
These results reveal that the regulations of the hepatic and
pulmonary
MO and GST are differentially influenced by EtOH' CS and the
combined
treatment.
Title
Effect of abstinence from alcohol on the depression of glutathione
peroxidase activity and selenium and vitamin E levels in chronic
alcoholic patients.
Author
Girre C; Hispard E; Therond P; GuedJ S; Bourdon R; Dally S
Address
Clinique Toxicologique Hopital Fernand Widal' Paris' France.
Source
alcohol Clin Exp Res, 14(6):909-12 1990 Dec
Abstract
glutathione peroxidase activity and selenium and vitamin E levels
were
measured in the plasma and erythrocytes of 25 chronic alcoholic
patients without liver cirrhosis before and after 14 days of
abstinence
from alcohol' and compared with the levels in 25 sex- and age-matched
healthy controls. Before abstinence' all three levels were shown
significantly depressed in the alcoholic patients compared with
the
controls' in both plasma (80' 71' and 89% of control values)
and
erythrocytes (68' 70' and 83% of control values). After a 14-day
abstinence period with no dietary supplementation' a trend towards
normalization was noted in erythrocyte (vitamin E and glutathione
peroxidase 74 and 91% of control values respectively)' in whole
blood
selenium (82%) and plasma in vitamin E (74%). However' plasma
selenium
and glutathione peroxidase values were lower than pre-abstinence
values
(76% and 86% of control values respectively). Our results point
to a
deficiency in the antioxidant defense system of chronic alcoholics
before the occurrence of severe liver disease. This lack of protection
against lipoperoxides is all the more important in circumstances
like
chronic alcohol consumption' in which lipid peroxidation is known
to
increase. However' the present study also demonstrated that during
14
days of a normal diet free of ethanol' a rapid trend occurred
towards
the normalization of the factors.
Title
Chronic ethanol ingestion-induced changes in open-field behavior
and
oxidative stress in the rat.
Author
Harkany T; Sasvari M; Nyakas C
Address
Central Research Division of Clinical and Experimental Laboratory
Medicine' Haynal Imre University of Health Sciences' Budapest'
Hungary.
harkany@lib.hiete.hu
Source
Pharmacol Biochem Behav, 58(1):195-201 1997 Sep
Abstract
The effects of chronic ethanol intoxication on the open-field
behavior'
on antioxidant enzyme activities' and the degree of lipid peroxidation
were investigated. Rats consuming a liquid diet containing 7%
ethanol
for 4' 7' 14' or 21 days exhibited a significantly decreased
ambulation
activity' accompanied by a reduced frequency and duration of
explorative rearing in an open-field task 4' 7' and 14 days after
chronic ethanol ingestion' whereas presumed adaptation to the
neurologic effects of ethanol was observed on day 21. Changes
in the
activities of glutathione peroxidase (GSH-Px): glutathione reductase
(GSH-R)' and catalase' and in the content of reduced glutathione
(GSH)
in blood samples were determined by means of biochemical methods.
The
degree of lipid peroxidation was measured via thiobarbituric
acid
assays. Chronic ethanol ingestion elicited a significant increase
in
GSH-Px activity (by a maximum of approximately 32% on day 14)'
whereas
opposite alterations in GSH-R and catalase activities were recorded
(49% of the control value on day 4 and 17% on day 21' respectively).
Highly elevated contents of thiobarbituric acid reactive substances
reflected extensive lipid peroxidation processes throughout the
experiment. These changes indicate that ethanol toxicity induces
profound changes in explorative behavior' mediated' at least
partly' by
changes in the free radical metabolism. |
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