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Title
Characterization of benzo[a pyrene quinone-induced toxicity to
primary
cultured bone marrow stromal cells from DBA/2 mice: potential
role of
mitochondrial dysfunction.
Author
Zhu H; Li Y; Trush MA
Address
Department of Environmental Health Sciences' Johns Hopkins University'
School of Hygiene and Public Health' Baltimore' Maryland 21205.
Source
Toxicol Appl Pharmacol, 130(1):108-20 1995 Jan
Abstract
Oral exposure of DBA/2 mice to benzo[a pyrene (BP) has been shown
to
result in hematotoxicity which is manifested as aplastic anemia
and
leukemia. Since normal hematopoiesis is regulated by bone marrow
stromal cells' in this study we have characterized the bone marrow
stromal toxicity induced by BP and BP-derived metabolites' particularly
quinones. Incubation of stromal cells with various concentrations
of
BP-1'6-' 3'6-' 6'12-' or 7'8-quinone for 24 hr resulted in a
significant decrease of cell survival in a concentration-dependent
manner' while cells treated with BP or BP-7'8-dihydrodiol did
not
exhibit any significant loss of cell survival. Among the BP quinones
examined' BP-1'6-quinone was the most cytotoxic to stromal cells.
The
cytotoxicity induced by BP-1'6-quinone also exhibited a time-dependent
relationship. Pretreatment of stromal cells with 1'2-dithiole-3-thione
(D3T) resulted in a significant induction of both cellular reduced
glutathione (GSH) content and quinone reductase (QR) activity
in a
concentration-dependent manner. However' D3T pretreatment did
not offer
any protection against BP-1'6-quinone-induced toxicity. Furthermore'
dicumarol' a potent inhibitor of QR' or buthionine sulfoximine'
a
specific inhibitor of GSH biosynthesis' did not potentiate
BP-1'6-quinone-induced cytotoxicity was not altered. However'
incubation of stromal cells with BP-1'6-quinone resulted in a
significant depletion of cellular ATP content and mitochondrial
morphological changes' which preceded the loss of cell survival.
In
addition to BP-1'6-quinone' other cytotoxic BP quinones also
exhibited
a capacity to deplete cellular ATP level in stromal cells' while
BP'
which was not cytotoxic to stromal cells' did not elicit any
significant decrease in cellular ATP level. These observations
suggest
that mitochondria may be a potential target of BP quinones. Overall'
the above results indicate that neither cellular GSH and QR nor
reactive oxygen species appear to be involved in BP quinone-induced
stromal cell inJury and that BP quinones may elicit cytotoxicity
to
stromal cells through directly disrupting mitochondrial energy
metabolism.
Title
Intracellular high energy metabolite depletion and cell membrane
inJury
with antioxidant enzymes during oxidant exposure in vitro.
Author
Aalto K; Raivio KO; Pietarinen P; Kinnula VL
Address
University of Helsinki' Department of Pediatrics' Finland.
Source
Toxicol Lett, 85(2):93-9 1996 May
Abstract
We compared oxidant-induced intracellular adenine nucleotide
catabolism
and cell membrane inJury in 4 different human cell types. Responses
to
oxidant exposure were correlated with endogenous antioxidant
enzyme
activities in these cells. Blood monocytes' amniotic fibroblasts'
umbilical vein endothelial cells in primary culture' and transformed
bronchial epithelial cells (BEAS 2B) were exposed to 0.1-5 mM
hydrogen
peroxide (H2O2) for 4 h. Some experiments were conducted in cells
pretreated with 3-amino 1:2'4-triazole (ATZ) to inactivate catalase
or
with 1'3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inactivate
glutathione (GSH) reductase. Depletion of adenine nucleotides
and
accumulation of their catabolic products (hypoxanthine' xanthine
and
uric acid) occurred to varying extent' monocytes being the most
resistant. There was a mutual relationship between catalase and
GSH
reductase activities and maintenance of cellular adenine nucleotide
levels during H2O2 exposure. GSH reductase inhibition rendered
BEAS 2B
cells susceptible to lytic inJury by H2O2' assessed by release
of
lactate dehydrogenase and intact nucleotides into the medium'
there was
no correlation between these markers of such inJury and endogenous
antioxidant enzymes. We conclude that adenine nucleotide depletion
and
nucleotide catabolite accumulation relate closely with the antioxidant
enzyme activities' whereas the lack of a similar correlation
between
the enzyme levels and markers of lytic cell inJury suggest that
intracellular antioxidant enzymes do not protect cells from membrane
damage due to extracellular oxidants.
Title
Comparison of erythrocyte antioxidant enzyme activities and embryologic
level of neural tube defects.
Author
Graf WD; Oleinik OE; Pippenger CE; Eder DN; Glauser TA; Shurtleff
DB
Address
Department of Pediatrics' University of Washington School of
Medicine'
Seattle' USA.
Source
Eur J Pediatr Surg, 5 Suppl 1():8-11 1995 Dec
Abstract
Increased exposure to oxidant-derived free radicals or inadequate
systems for antioxidant defense could alter cellular response
at
critical points in development. We measured 5 antioxidant enzymes'
glutathione peroxidase (GSH-Px)' glutathione reductase'
glutathione-S-transferase' catalase and superoxide dismutase
in
erythrocytes and their plasma cofactor trace elements (Se' Zn'
Cu) in
37 children with myelomeningocele and in 37 age-matched controls.
We
placed the patients into 3 groups according to motor level of
the
lesion at birth. We found significantly lower GSH-Px activities
(p =
0.007) in children with myelomeningocele. For paired comparisons
among
the 3 patient groups and controls' there were significant differences
(p < 0.05) between controls and both high (thoracic) and raid
(lumbar)
level embryologic lesions. The finding of antioxidant enzyme
variations
in our patients with myelomeningocele may indicate a role for
abnormal
oxidative metabolism in the development of this defect. The
contribution of oxidative stress to human birth defects warrants
investigation. We discuss potential relationships between oxidative
stress and energy metabolism during primary neurulation.
Title
Thiol depletion induces lethal cell inJury in cultured cardiomyocytes.
Author
Dhanbhoora CM; Babson JR
Address
Department of Pharmacology and Toxicology' College of Pharmacy'
University of Rhode Island' Kingston 02881.
Source
Arch Biochem Biophys, 293(1):130-9 1992 Feb 14
Abstract
Treatment of cultured neonatal cardiomyocytes with ethacrynic
acid (EA)
induced a rapid depletion of glutathione (GSH) that preceded
a gradual
elevation of cytosolic Ca2+ (monitored by phosphorylase a activation)'
a loss of protein thiols' and a marked inactivation of the
thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase
(G3PD).
A subsequent decline of mitochondrial transmembrane potential
(delta
psi) and ATP occurred prior to the onset of lipid peroxidation
which
closely paralleled a loss of cardiomyocyte viability. The antioxidant
N'N`-diphenyl-p-phenylenediamine prevented lipid peroxidation
and cell
death but had no effect on elevated cytosolic Ca2+' delta psi
loss' GSH
depletion' or G3PD inactivation. Pretreatment with the iron chelator'
deferoxamine' decreased both lipid peroxidation and cell death.
EA-induced lipid peroxidation and cell damage were also diminished
by
preincubation with acetoxymethyl esters of the Ca2+ chelators
Quin-2
and ethylene glycol bis(beta-aminoethyl ether) N'N`-tetraacetic
acid'
even though cytosolic Ca2+ remained elevated. The extent of GSH
depletion was unaltered by either chelator; however' Quin-2 did
protect
G3PD from inactivation by EA. An inhibitor of the mitochondrial
respiratory chain' antimycin A' decreased EA-induced lipid peroxidation
and cell death but had no effect on thiol depletion or elevated
cytosolic Ca2+. These data suggest that cardiomyocyte thiol status
may
be linked to intracellular Ca2+ homeostasis and that peroxidative
damage originating in the mitochondria is a maJor event in the
onset of
cell death in this cardiomyocyte model of thiol depletion.
Title
Neurotrophin regulation of energy homeostasis in the central
nervous
system.
Author
Jackson GR; Werrbach-Perez K; Pan Z; Sampath D; Perez-Polo JR
Address
Department of Human Biological Chemistry and Genetics' University
of
Texas Medical Branch at Galveston 77555-0652' USA.
Source
Dev Neurosci, 16(5-6):285-90 1994
Abstract
Our hypothesis is that one cause of neuronal cell death and shrinkage
in the aged central nervous system is an inability of neurons
to
maintain oxidant homeostasis in the face of increased levels
of
reactive oxygen species' decreased endogenous antioxidants' and
impaired energy metabolism associated with physiological senescence'
Alzheimer`s' and Parkinson`s diseases. Since treatment with nerve
growth factor (NGF) reverses behavioral impairments in aged rats
and
stimulates cholinergic activity in the basal forebrain' while
brain-derived neurotrophic factor appears to play a similar role
in the
striatum' we propose that neurotrophin-mediated cell-sparing
reflects
effects on oxidant homeostasis. Neurotrophins may play a similar
cell-sparing role in hypoxic/ischemic inJury to the nervous system'
which also is mediated in part by reactive oxygen species. The
degradation of one such species' H2O2' is catalyzed by catalase
and
glutathione peroxidase (GSH Px). The activity of the latter enzyme
is
dependent on glutathione reductase and the availability of NADPH
for
regeneration of reduced GSH. The GSH redox cycle is also regulated
by
enzymes of the hexose monophosphate shunt. NGF protects PC12
cells from
H2O2 inJury by stimulating the synthesis of antioxidant enzymes
including catalase' GSH Px' glucose-6-phosphate dehydrogenase'
and
gamma-glutamylcysteine synthetase' the rate-limiting enzyme for
glutathione synthesis. NGF also enhances recovery from the NAD+
losses
occurring as a consequence of H2O2 treatment.
Title
Respective roles of free radicals and energy supply in hypoxic
rat
liver inJury after reoxygenation.
Author
Videla LA
Address
Departamento de Ciencias Biol]ogicas' Facultad de Medicina' Universidad
de Chile' Santiago.
Source
Free Radic Res Commun, 14(3):209-15 1991
Abstract
Livers from fasted rats subJected to 60 min of hypoxia followed
by 25
min of reflow exhibited a significant release of lactate dehydrogenase
(LDH) and protein into the perfusate together with high rates
of oxygen
consumption' depletion of hepatic glutathione (GSH) and accumulation
of
thiobarbituric acid reactants (TBAR) in the liver. These changes
were
observed in the presence and absence of added xanthine (25 microM)
and
were significantly diminished when experiments were carried out
in the
presence of either 1 mM allopurinol or 100 microM Trolox. Allopurinol
inhibited by 79% the production of uric acid by the liver' which
was
not altered by Trolox. Hypoxia-reflow studies performed in the
presence
of 25 microM 2'4-dinitrophenol (DNP) showed a drastic enhancement
in
LDH (244%) and protein (104%) efflux from the liver' compared
with the
effects found in its absence' with a moderate increase (35%)
in tissue
TBAR levels. Liver perfusion in the presence of both allopurinol
and
DNP exhibited a normalization of the tissue content of GSH and
TBAR'
while the net increase in LDH and protein release elicited by
DNP alone
was inhibited by only 20 and 25%' respectively. Similar results
were
obtained in experiments in which allopurinol was replaced by
Trolox.
These studies indicate that production of oxygen free-radicals
are
involved in hypoxic liver inJury upon reflow' but its relative
importance is significantly diminished when energy stores are
severely
diminished.
Title
Endothelium-derived oxidative stress may contribute to exercise-induced
muscle damage.
Author
Duarte JA; Appell HJ; Carvalho F; Bastos ML; Soares JM
Address
Department of Sport Biology' Faculty of Sport Sciences' University
of
Porto' Portugal.
Source
Int J Sports Med, 14(8):440-3 1993 Nov
Abstract
In exercise-induced muscle damage' oxidative stress derived from
the
liberation of reactive oxygen species (ROS) is assumed to be
of
etiological importance. Xanthine oxidase (XO) located in capillary
endothelium is one of the possible sources for ROS' mainly investigated
so far under conditions of ischemia/reperfusion. XO can be inhibited
by
allopurinol. To investigate the contribution of XO for the oxidative
stress-induced development of muscle damage' mice were subJected
to a
single bout of exhaustive running exercise. Another exercised
group
received allopurinol. The reduced form of glutathione (GSH) was
measured to estimate the amount of oxidative stress in soleus
muscle'
and the same muscle was examined in the light and electron microscope
at different periods of time (0' 48' 96 h) after exercise. While
exercise alone resulted in a marked reduction of GSH indicative
for
oxidative stress' which only recovered at 96 h' the administration
of
allopurinal to exercised animals induced a complete recovery
already at
48 h after exercise. Muscle damage was more pronounced in the
exercised
animals which had not been treated with allopurinol. It is concluded
that endothelium-derived ROS contribute reasonably to oxidative
stress
to exercised muscle and to fiber and capillary damage.
Title
Mitochondrial glutathione redox and energy producing function
during
liver ischemia and reperfusion.
Author
Kurokawa T; Kobayashi H; Nonami T; Harada A; Nakao A; Takagi
H
Address
Department of Surgery II' Nagoya University School of Medicine'
Tsurumai 65' Showa-ku' Nagoya' 466' Japan.
Source
J Surg Res, 66(1):1-5 1996 Nov
Abstract
The glutathione in mitochondria is thought to play a crucial
role in
protecting mitochondria against ischemia-reperfusion-induced
inJury.
However' little is known about the mitochondrial redox system.
This
study was conducted to clarify changes of mitochondrial glutathione
redox during liver ischemia and reperfusion and its role on energy
producing function. Rats were divided into three groups each
of which
were treated respectively with saline (Control)' buthionine sulfoximine
(BSO)' which induces a rapid decrease in tissue glutathione
concentrations' and gamma-glutamylcysteine ethyl ester (GCE)'
which
conversely induces a rapid increase in tissue glutathione
concentrations before induction of ischemia. Liver ischemia was
induced
for 120 min' and blood reflow was subsequently restored for 60
min.
Total and mitochondrial glutathione concentrations' mitochondrial
respiratory function' and tissue adenine nucleotide were determined
after both the ischemic and the reperfusion periods. In all groups'
concentrations of the reduced form of glutathione (GSH) gradually
decreased during ischemia and reperfusion. On the other hand'
significant increases in mitochondrial GSH were apparent after
reperfusion despite significant decreases during ischemia in
the
control and GCE groups. Total and mitochondrial GSH in the BSO
and GCE
groups were significantly lower and higher' respectively' compared
with
the control throughout the experiment. Recovery of the mitochondrial
energy producing function and cellular adenine nucleotide after
reperfusion were dependent on GSH concentrations. We conclude
that
mitochondrial GSH concentrations dramatically change in a different
manner from cytosolic concentrations after reperfusion' and that
recovery of the mitochondrial energy-producing function might
be
closely associated with mitochondrial GSH concentrations.
Title
Adaptation of chondrocytes to low oxygen tension: relationship
between
hypoxia and cellular metabolism.
Author
RaJpurohit R; Koch CJ; Tao Z; Teixeira CM; Shapiro IM
Address
Department of Biochemistry' School of Dental Medicine' University
of
Pennsylvania' Philadelphia 19104-6003' USA.
Source
J Cell Physiol, 168(2):424-32 1996 Aug
Abstract
In endochondral bone' the growth cartilage is the site of rapid
growth.
Since the vascular supply to the cartilage is limited' it is
widely
assumed that cells of the cartilage are hypoxic and that limitations
in
the oxygen supply regulate the energetic state of the maturing
cells.
In this report' we evaluate the effects of oxygen tension on
chondrocyte energy metabolism' thiol status' and expression of
transcription elements' HIF and AP-1. Imposition of an hypoxic
environment on cultured chondrocytes caused a proportional increase
in
glucose utilization and elevated levels of lactate synthesis.
Although
we observed a statistical increase in the activities of
phosphofructokinase' pyruvate kinase' lactate dehydrogenase'
and
creatine kinase after exposure to lowered oxygen concentrations'
the
effect was small. The cultured cells exhibited a decreased utilization
of glutamine' possibly due to down regulation of mitochondrial
function
and inhibition of oxidative deamination. With respect to total
energy
generation' we noted that these cells are quite capable of maintaining
the energy charge of the cell at low oxygen tensions. Indeed'
no
changes in the absolute quantity of adenine nucleotides or the
energy
charge ratio was observed. Hypoxia caused a decrease in the glutathione
content of cultured chondrocytes and a concomitant rise in cell
and
medium cysteine levels. It is likely that the fall in cell glutathione
level is due to decreased synthesis of the tripeptide under reduced
oxygen stress and the limited supply of glutamate. The observed
rise in
cellular and medium cysteine levels probably reflects an increase
in
the rate of degradation of glutathione and a decrease in synthesis
of
the peptide. To explore how cells transduce these metabolic effects'
gel retardation assays were used to study chondrocyte HIF and
AP-1
binding activities. Chondrocyte nuclear preparations bound an
HIF-oligonucleotide; however' at low oxygen tensions' no increase
in
HIF binding was observed. In addition' we found that AP-1 binding
activities in chondrocytes exposed to low oxygen tensions was
elevated'
although the response was lower than that exhibited by fibroblasts
exposed to the same range of oxygen concentrations. We compared
these
results to HIF and AP-1 binding by cells in the growth plate.
There was
strong HIF and AP-1 binding throughout the plate' but no evidence
of
selective binding to any one zone. The results of the study lend
strong
support to the view that chondrocytes are very well adapted to
low
oxygen tensions; thus' under hypoxic conditions' there is a high
level
of expression of both HIF and AP-1' and energy conservation appears
to
be near-maximum.
Title
Cellular responses of cultured cerebellar astrocytes to ethacrynic
acid-induced perturbation of subcellular glutathione homeostasis.
Author
Huang J; Philbert MA
Address
Department of Pharmacology and Toxicology' Rutgers College of
Pharmacy'
Piscataway' NJ 08854' USA.
Source
Brain Res, 711(1-2):184-92 1996 Mar 4
Abstract
glutathione (GSH) and glutathione-related enzyme systems in astrocytes
play an important role in cellular defense against oxidative
stress in
the nervous system. The present study was designed to characterize
the
cellular responses of cultured astrocytes to chemically-induced
perturbations of mitochondrial and cytosolic GSH homeostasis.
Treatment
of astrocytes in culture with ethacrynic acid (EA)' a
mitochondrion-penetrating thiol reagent' induced rapid and extensive
depletion of both cytosolic and mitochondrial pools of GSH. Concomitant
with the effects of EA on cellular GSH were significant and
concentration-dependent increases in intracellular generation
of
reactive oxygen species (ROS) as indicated by the oxidation of
preloaded 2`'7`-dichlorofluorescein diacetate. Significant elevation
of
intracellular ROS occurred by 15 min following exposure to 100
microM
EA and reached peak levels by 30 min which were approximately
7-fold
higher than corresponding control levels. Ethacrynic acid-induced
GSH
depletion and intracellular ROS elevation was followed by marked
decreases in glutathione reductase (GR) activity in mitochondria'
and
to a lesser extent' in cytosolic fractions of cultured astrocytes.
This
inhibitory effect was time- and concentration-dependent' and
other
GSH-related enzymes' glutathione peroxidase and glutathione
S-transferase' were not or only slightly affected. Kinetic studies
showed that EA markedly diminished V(max) values of both mitochondrial
and cytosolic GR without affecting K(m)' suggesting noncompetitive
inhibition of this thiol-dependent enzyme. Another thiol-dependent
enzyme glyceraldehyde-3-phosphate dehydrogenase was also markedly
inhibited by EA in a time-dependent fashion. Subsequent decline
of
mitochondrial transmembrane potential (rhodamine 123 uptake)
and
cellular ATP production following EA treatment occurred prior
to the
onset of loss of cell viability as indicated by lactate dehydrogenase
leakage. These results suggest that the loss of mitochondrial
GSH may
render the astrocytes unable to combat the pathological sequelae
of
endogenous oxidative stress' leading to perturbations of
thiol-dependent enzyme activities' mitochondrial function and
energy
metabolism.
Title
Cadmium-induced changes of antioxidant and metabolic status in
red
blood cells of rats: in vivo effects.
Author
Kosti]c MM; OgnJanovi]c B; DimitriJevi]c S; Ziki]c RV; StaJn
A; Rosi]c
GL; Zivkovi]c RV
Address
Institute of Physiology' Faculty of Medicine' University of KraguJevac'
Yugoslavia.
Source
Eur J Haematol, 51(2):86-92 1993 Aug
Abstract
Chronic exposure of adult rats to dietary intake of cadmium (15
mg
CdCl2/day/kg for 30 days) leads to development of anemia and
thrombocytosis. Anemia is characterized by significant reticulocytosis
(13.1 +/- 1.0%)' anysocytosis' poikilocytosis' iron deficiency
and
marked alterations of antioxidant and metabolic status of red
blood
cells. Activities of SOD' catalase' GPx and GR were significantly
increased in red blood cells of cadmium-treated rats. In treated
animals cadmium induced an increase of red cell reduced and oxidized
glutathione with no changes of GSSG/GSH ratio. However' significant
reduction of lipid peroxidation was found. Plasma levels of tocopherol
and ascorbate' as well as activity of glutathione-S-transferase'
were
all significantly increased in cadmium-treated rats. The energy
metabolism of red blood cells was deeply altered in cadmium-treated
rats. The levels of ATP' ADP' AMP and TAN were significantly
increased
while ATP/ADP ratio and adenylate energy charge (AEC) were
significantly reduced. The level of 2'3-BPG was somewhat lower'
but
2'3-BPG/Hb ratio was considerably higher' in red blood cells
of
cadmium-treated rats.
Title
glutathione metabolism in skeletal muscle derived cells of the
L6 line.
Author
Sen CK; Rahkila P; H anninen O
Address
Department of Physiology' University of Kuopio' Finland.
Source
Acta Physiol Scand, 148(1):21-6 1993 May
Abstract
Skeletal muscle derived L6 myoblasts possess a considerably high
resting total glutathione (TGSH) pool. Exposure to L-buthionine-[S'R
-sulphoximine resulted in a 90% depletion of the intracellular
TGSH
pool. All the key enzymes of glutathione metabolism' especially
glutathione S-transferase' were observed to be considerably active
in
the undifferentiated cells. Se-dependent glutathione peroxidase
activity appeared to account for most of the total GSH peroxidase
activity of the cells. A significant contribution of gamma-glutamyl
transpeptidase-independent (5 mM acivicin insensitive) mechanism
to the
extracellular GSH uptake capacity of the muscle cells was evident.
Efflux of oxidized glutathione from the cells exposed to t-butyl
hydroperoxide was rapid and appeared to be energy linked.
Title
Role of glutathione and nitric oxide in the energy metabolism
of rat
liver mitochondria.
Author
Nishikawa M; Sato EF; Kuroki T; Inoue M
Address
Department of Biochemistry, Osaka City University Medical School,
Japan.
Source
FEBS Lett, 415(3):341-5 1997 Oct 6
Abstract
Previous studies have suggested that nitric oxide (NO) inhibited
mitochondrial respiration. NO and/or its intermediate(s) react
with
various molecules, such as hemeproteins and free SH groups. The
inhibitory effect of NO on mitochondrial respiration was decreased
by
exogenously added glutathione (GSH). However, a decrease of
intramitochondrial GSH by pretreating animals with L-buthionine
sulfoximine had no appreciable effect on the inhibitory effect
of
isolated mitochondria. Furthermore, the effect of NO was not
affected
by depleting free SH residues in mitochondria by N-ethylmaleimide.
These results suggest that cytosolic but not intramitochondrial
GSH
might be an important factor that determines the NO-dependent
regulation of mitochondrial energy metabolism. |
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