ANTIOXIDANTS,

NUTRITION &c
HEALTH

OXYGEN 2000 S27

r-=-l I 54 I

ONOIDS ON DNA DAMAGE IN Pyruvate protects cerulein induced acute pamreatitis.

CELLS
NORA M. O’BRIEN,
Department of Food Science
College Cork, Ireland
The effects of the flavonoids, quercetin and rutin, were
with butylated hydroxytoluene (BID’), a synthetic
t, and desferrioxamine (DFO), a specific iron
terr-butylhydroperoxide (rerr-BOOH)- and
ed DNA damage in HepG2 and Caco-2 cells.
and menadione induced DNA damage in a
r, as assessed using the alkaline comet
uercetin and rutin protected against
damage by way of their metal ion
quercetin decreased menadione
ng possibly as both a metal ion
scavenger. In HepG2 cells the flavonoids
not protect against terr-BOOH-induced DNA damage.
rcetin and the metal ion chelator, DFO, protected to some
nt against DNA damage evoked by menadione in HepG2
s. Greater protection by the flavonoids was seen in the
o-2 cell model. Metabolism of the flavonoids may have
urred in the HepG2 cell model thus resulting in their
ted effectiveness.
J A tosiewia, R. A. Olek, J. Popinigis. Jednej Sniadecki University
S&i1 of Physical Education, Department of Bioenergetics, Wiejska 1,
XI-336 Gdansk, Poland
and Ceruleln induced acute pancreatitis (AP) has been related to increased free
radical formation. It has been shown that different antioxidants can inhibit
this kind of stress. The aim of this work was to lind out, that pyrwate
can protect cerulein induced AP. Despite of being the substrate in many en-
zymatic reactions pymvate can also nonenzymatically react with hydrogen
= ~~
oemxide. Thus it can be consider as an antioxidant. Twelve male rats have
been used in this study. Rata were divided into three groups, one received
food with 2% of sodium pyruvate. AP was induced by two intraperitoneal in-
jection of cemlein (40 pg/kg body wt) at hourly intervals. In cemlein treated
rats typical symptoms of pancreatitis, hyperamylasemia (1969 vs. 7569 U/dl)
and pancreas edema (pancreas weight 0.28 vs. 0.493 g) were observed. In
pyrovate group the amylase activity and pancreas weight were almost the
same as in control group and were 2567 U/dl and 0.363 g respectively. Pm-
tein mdfhydryl groups have been observed to decrease more after cemlein
treatment in control than in pyruvate group. The obtained data suggest
that pyruvate protects pancreas from cerulein induced AP. This may imply
that metabolic changes, which lead to decrease intracellular pyrwate con-
centration, may predispose pancreas into pancreatitis. The role of pyruvate
as a substance, which can din&d& oxidatlve stress and NADH level inside
the cell, is also discussed. This work was supported from KBN grant 4P05A
125 19.

I 55 I 1
Adenovirus-mediated Overexpression of CataIase in CytosoIic or
Mitochondrial Compartment Protects Against Cytochrome P460
2El-dependent Toxicity in HepG2 Cella
JingximgBai & Arthur I Cederbaum. Department of Biochemistry and
Molecular Biology, Mount Sinai School of Medicine, New York, NY 10029
Cytochrome P450 2El (CYPZEl) is an effective producer of ROS, which
may contribute to the development of alcohol liver dii. In order to invee-
tigate the protective role ofcatalase against CYP2El dependent cytotoxicity,
Et7 cells, a transfected HepG2 cell line overexpressing CYP2E1, were infected
with adenoviral vectors containing human catalase cDNA(AdCat) and cata-
lase cDNA with a mitochondrial leader sequence(AdmCat). 46 hours after
infection with 100 MO1 AdCat or AdmCat, intracellular cat&se protein was
increased more than two fold as compared to uninfected B47 cells and E47
cells infected with empty adenoviral vector(AdNull) as detexmined by West-
ern blot and cat&se activity measurements. Overexpression of cat&se in
cytosol (AdCat) and in mitochondria (AdmCat) was cm&rmed by confocal
microscopy. Cell death caused by arachidonic acid plus iron was considerably
suppressed in both AdCat and AdmCat infected E47 cells ea determined by
ways of MTT, LDH release, and morphology changes. AdCat and AdmCat
infected oells were also more resistant to the loss of mitochondrial membrane
potential and to the bxree.se in lipid peroxidation induced by AA and iron.
The present study indicates that cat&se in cytwol and mitochondria are
capable of protecting HepG2 cells expressing CYPZEI against cytotoxicity
induced by oxidants which promote lipid pemxidation, and suggest the pee-
sibility that such agents may be useful in protecting against the development
of alcohol liver injury.
Production of extracelhdar phenolic a&oxidants provides a
buffer against low-level buildup of ROS in plants
C. Jacyn Baker, Nichole O’NeilI and Kenneth De&l. USDA, ARB, Plant
Science Institute, Beltsville, MD
We have been investigating the role of ROS in the early events of
plant/pathogen interactions. Potato and tobacco suspension cells were first
washed and suspended in assay buffer, then placed on a shaking water
bath and finally inoculated with virulent and avirulent bacterial pathogens.
Within the first few hours there is a two-phased ROS burst. The first phase,
which begins immediately after inoculation, occurs in both susceptible and
resistant interactims. The second phase begins about 2 hr. after inoculation
and only occurs in resistant interactions. The scavenging capability of these
cells was monitorad during this period using a technique that measures the
rate of decrease of exogenously added hydrogen peroxide. This lead to the
observation in control cells that antioxidant levels were building up and neu-
tralizing the emgenously added hydrogen peroxide within 1 to 2 sec. The
antioxldants appear to be small molecular weight phenolic compounds. Based
on the spectrum from 210-350 nm there are several different compoonds in-
volved depending on the age and type of plant cell. These compounds are
currently being isolated and identified. It appears they do not accumulate
stressad cells because they react with the endogenous hydrogen peroxide as
it is produced. Therefore due to the production of these antioxidants, the
levels of ROS in plant cell interactions is likely underestimated.

S28 OXYGEN 2000

I 57 I
CHLORINATION OF THE RED WINE POLYPHENOL
QUERCETIN BY HOCI-PRODUCING NEUTROPHILS:
DISPARATE EFFECTS OF THE FREE AND CONJUGATED
FORMS.
R. Binsack, R. Pate& M. Kirk, H. Kim, V. Da&y-Usmar,
J. E&rich and D.A. Parks. Depts. of Anesthesiology, Pathology,
Pharmacology and UAB Center for Free Radical Research, Univ. of
Alabama at Birmineham. Birmineham. AL 35233. USA.
Epidemiological stidies’indicate-that moderate red wine consumption low-
ers the incidence of cardiovascular disease, with the beneficial effect being
partly attributed to the red wine polyphenol quercetin. The precise mech-
anism of the cardioprotection remains unclear, and is complicated by the
fact that quercetin exists in plasma in ““conjugated and conjugated forms.
Moreover, recent data indicate that (poly)phenolic compounds can be chlo-
rinated in reactions with hypochlorous acid (HOCl), a proinflammatory oxi-
dant produced by activated neutrophils. To investigate potential differences
of quercetin and its conjugated forms as well as related chlorinated derivates
in more detail, stimulated neutrophils were mixed with quercetin or one of
its conjugated forms, quercetin-rutinoside and quercetin-glucuronoside. Only
the conjugated forms of quercetin reacted with HOC1 to stable mow_+ and
dichloroderivates. However, since the stimulated neutrophils also released
a-glucuronidase, nonconjugated forms were also detected. Although conju-
gated quercetin did not activate the estrogen receptors ERa and ERP in
transfected COS cells, quercetin and, to a lesser extent, the chloroderivates
did. In contrast, the chloroderivates of quercetin exhibited a 2-fold greater
antioxidant potential than the unmodified form. In conclusion, quercetin may
result in cardioprotection via several pathways depending on the pathological
state.
A NOVEL MECHANISM FOR ANTIOXIDANT
ACTMTY OF PHYTOESTROGENS: EVIDENCE FOR
POLYPHENOGMEDIATED PEROXIDATION
Brenda J. Boersma, Rakesh P&l, Marion Kirk, Stephen Barnes,
and Victor Darley-Usmar, Depta. of Pharmacology and
Toxicology and Pathology, University of Alabama at
Birmingham, Birmingham, AL 35294
Oxidation of low density lipoprotein (LDL) results in the accumulation
of macrophage foam cells in atherosclerotic plaques. Genistein, a soy
isoflavone, has been shown to inhibit the oxidation of LDL in vitro and
inhibits the development of atherosclerosis in animal models. It is
unclear whether genistein is potent enough to act as an anti-
atherosclerotic agent via a classical antioxidant mechanism. Using
liposomes and LDL oxidized by ABAP (2. 2’- azobis- amidino-
propane hydrochloride), genistein inhibited LDL oxidation but
paradoxically was notconsumed. However, when genistein and ABAP
were reacted without liposomes, genistein consumption was detected.
Based on these findings, we propose that genistein is capable of
exhibiting polyphenol-:ncdiated peroxidation, analogous to tocopherol-
mediated peroxidation. This would both explain the lack of
consumption during lipid peroxidation and a profound synergy in
antioxidant potency we have observed when ascorbate is added to the
oxidation system. These data suggests that polyphenols with
intrinsically low antioxidant ability in vitro may be greatly enhanced in
viva The criteria for assessing the antioxidant properties of genistein
and polyphenols as mediators ofperoxidation will be discussed.
OXYGEN
Effects of a cyst&e precursor on neural damage in sulfur amino
acid (SAA) deficient rats after global hemispheric
hypoxia-ischemia (GHHI).
P.J. Bobyn*, J.L. Franklin*, CM. Wall*, B.H.J. Juurlink#,
J.A. Thornhill**, P.G. Paterson * *College of Pharmacy and Nutrition,
#Dept. of Anatomy and Cell Biology, ‘*Dept. of Physiology, University of
Saskatchewan, Saskatoon, SK., Canada S7N 5C9
The SAA cyst&e is essential for synthesis of glutathione (GSH), a tripep-
tide critical in antioxidation of reactive oxygen species in conditions such as
stroke. In a rat model of GHHI, in which the right, common carotid artery
was ligated and severed followed by exposure to 35 minutes of hypoxia, our
laboratory has previously shown that dietary SAA deficiency decreases brain
GSH and increases neural damage. A cysteine precursor, CZ-oxothiazolidine-
4-carboxylic acid (OTC), or carrier, was injected subcutaneously 15 minutes
after GHHI and every 12 hours for 3 days in both SAA deficient (-SAA) and
SAA sufficient (+SAA) rats. Neural damage was assessed histologically in
2 brain sections from the hemisphere ipsilateral to the ligated artery 7 days
after GHHI. Semiquantitative scoring (maximum score 8) of damage to the
neocortex, hippocampus, striatum and thalamus suggests OTC may be pro-
tective in +SAA (p=O.O5) but not -SAA (p=O.62) rats (Mann-Whitney test).
Our laboratory also developed a more quantitative scoring system restricted
to the hippocampus. Preliminary data suggest OTC does not protect total
hippocampus in -SAA or +SAA rats, but a trend towards protection is seen
in the CA4 hippocampal region of +SAA animals (p=O.O8, l-tail t-test).
Neuroprotective effects of OTC in stroke may vary with nutritional status.
finded by the Heart and Stroke Foundation of Saskatchewan
I 60 I
Phytochemicals in Tomatoes Increase Survival of Rats With
NMU-Testosterone-Induced Prostate Cancer
T. Boileau. S. Clinton I-9Z. Liao -9
M. Monaco S. Donovan, & J. Erdman, Jr.
University of Illinois, Urbana, IL & The Ohio State University, Columbus,
OH.
Consumption of tomatoes has been suggested to decrease prostate cancer
risk. We tested the effect of dietary lycopene (the red pigment in toma-
toes) in different forms on survival of rats with chemicallv-induced or&ate
can&r. Five-week old male Wistar-Unilever rats were randomly &signed
to one of 3 AIN-baaed experimental diets (control, lycopene beadlets, or
tomato powder) and fed for one week prior to the initiation of tumors by
sequential anti-androgen, androgen, carcinogen treatment, followed by pro-
motion of tumor formation with subcutaneous testosterone implants. Since
calorie restriction has also been shown to decrease cancer risk, each group
was further randomized to 20% diet restriction or ad libitum 3 days after
carcinogen treatment. Increased survival was noted for tomato powder-
fed (39% increase, R&0.613; P=O.O056) and lycopene beadlet-fed (17%
increase, RR=O.637; P=O.162) rats as compared to controls. Diet restric-
tion also significantly increased overall survival (23% increase, RR=0.770;
PzO.0429) as compared to ad libitum feeding. We concluded that the con-
sumption of lycopene from beadlets or tomato powder enhances survival in
this chemically-induced prostate cancer model. Furthermore, tomato pow-
der may contain substaneea, in addition to lycopene, which may enhance host
antioxidant defenses and favorably modulate prostate carcinogenesis.
2000 s29
L!?!
Activation of Nuclear Factor kappa B (NFkH) Causes Cell Death
induced by Diabetogenic Compounds in Cell Culture
Thomas W.-M. Boileau,Yu-HwaiTsai, and Tammy M. Bray . The Ohio
State University, Columbus, OH
Our laboratory has previously demonstrated that in viva activation of the
nuclear factor kappa B (NFkB) is a critical early step in pancreatic b cell
death in experimental models of diabetes. Antioxidants that inhibit NFkB
activation reduced hyperglycemia induced by diibetogenic compounds. The
objective of this experiment is to determine if NFkB activation is cell type-
specific. NFkB activation in b c&r (RINm5F) or a liver cell line (HepG2)
following allmtan and streptoaotocin (STZ) exposure was determined by elec-
trophoretic mobility shift assay (EMSA). BIN 5F cells or HepG2 cells (105 -
107) were treated with various levels of alloxan or STZ. At diierent time in-
tervals, cells were harvested and nuclear extractions from cells were prepared.
Results of this study demonstrated the tie course and mode of cell death
(apoptosis vs necrosis) induced by various diabetogenic compounds. We also
demonstrated that various classes of antioxidants have different capacity in
inhibiting NFkB activation in a cell type specific manner.
1 1
Formation of reactive oxygen species during exercise is reduced by
adudrdtration of pyruvate as antioxidant or by exercise training
-9
B. Fii 0. Sommerand E. Bassenge. Inst. of Applied Physiology,
University Freiburg, Germany
Heavy physical exercise increases oxygen consumption and potentially ini-
tiates enhanced formation of reactive oxygen species @OS). This in turn
leads to oxidative stress and cellular damage if not appropriately counter-
acted. The objective of this study was to quantify in viva formation of ROS
and hemodynamic parameters at rest, as well as during incmasing levels of
treadmih exercise before and after a training period of 8 weeks, or under
administration of pymvate. Dogs prepared with art&a carotis loops (blood
sampling and hemodynamic measurements) were exercised at 5, 10 and 15
km/h (5% grade). In viva generated ROS were scavenged using IV infusion
of 1-hydroxy-3earboxy-pyrrolidine and quantified by electron spin resonance.
Plasma lactate concentrations were measured spectrophotometrically. As
depicted in the table, art&al systolic pressure (APsys). heart rate (HR),
and ROS formation increased signiscantly with increasing levels of exercise
before and after training (see table, * p < 0.05 vs. resting values). rest
15 km/h 15 km/h+pyr. 15 km/h+training APsys (mmfig) 143f5 2Olf8*
190&3*’ 195f8.0*+ HR (beats/min) 113flO 222f12* 217flO* 198f7*+
ROS (mM) 2.85f0.45 5.13&0.66* 4.42&0.31* 2.75&0.05*+ Exercise train-
ing or a continuous infusion of pyrnvate (30 mg/min/kg) as antioxidant to
maintain appropriate redox potentials significantly reduced enhanced ROS
production. These in viva analyses demonstrate that exercise causes an en-
hanced formation of ROS, which is decreased by training and/or pyruvate.
s30 OXYGEN
El62
Release of NO and Enhanced formation of reactive oxygen species
in hypercholesterolemia is modulated by various statins
B. Fink and E. Bassenge. Inst. of Applied Physiology, University Fteiburg,
Germany
Hypercholesterolemia is associated with enhanced formation of reactive
oxygen species (ROS). We analyzed the effects of superimposed atorvas-
tatin/simvastatin given over a period of 8 weeks with an atherogenic choles-
terol diet (0.5%) on ROS production in hyperchol~terolemic guinea pigs.
The cholesterol diet was given without or with atorvastatin or simvastatin
(10 mg/kg/day). BGS production was analyzed both in the perfusate of Lan-
gendorff hearts and in blood samples. We analysed concentrations of reduced
and oxidized SBgroups in plasma and nitrates bioconversion using ESR tech-
nique. ROS production in blood samples and perrirsates of guinea pig hearts
treated with cholesterol increased from 1.73f0.06 to 2.73~0.09pM/min and
from 0.237fO.O3fiM/min to 0.364fO.O24~M/min respectively. This exces-
sive production was significantly reduced by atorvastatin approaching control
values both in en viva blood samples (1.36fO.O2/&/min) and in perfirsates
(0.31f0.92 /~MM/min)(simvastatin caused a similar but lesser reduction). Sur-
prisingly, concentrations of redwed SIX-groups were signi6cantly increased
from 205i9.1 to 254f18pM after 8 weeks cholesterol diet, and were signifi-
cantly augmented when atorvastatin (236f14pM) or simvastatin (264zk9pM)
was added to the cholesterol diet. Both concentration of reduced S&groups
productibn of ROS during experimental hypercholesterolemia are effec-
tively reduced by appropriate doses of ‘atorvastatin and less by simvastatin
administration.
I
64 1
I
Synergistic toxicity of iron and arachidonic acid in HepG2 cells
overex~ressimz CYP2El
AndresCam a112 Arthur Cederbaum. Mount Sinai School of Medicine,
Department of Biochemistrv and Mokscuhar Bioloav
Priming of the liver for ethanol-induced injury, b~nutrients such as polyun-
saturated fat and iron, has been suggested to play a key role in the develop-
ment of alcoholic liver disease. The objective of thii work was to evaluate
the effect of the combination of Fe-NTA and arachidonic acid on the viabil-
ity of a HepG2 cell line (E47) transduced to express the ethanol-inducible
CYPSEl. Cells were plated, preloaded with aracbidonic acid (AA, up to 10
uM), washed, and exposed to Fe-NTA (up to 25 uM) for variable periods.
Fe-NTA (10 uM) or AA (5 uM) alone showed low toxicity to E47 cells (19%
and 16% respectively at 36 h), while the combination produced synergis-
tic injury (75% toxicity at 36 h). Exposure of control cells not expressing
any cytochrome P450, or HepGZC3A4 cells (expressing human CYP3A4) to
Fe+AA did not produce significant toxicity (~20%). Toxicity in E47 cells was
prevented by antioxidants, which also decreased lipid permcidation. Damage
to mitochondria plays a role in the CYP2El-dependent toxicity of Fe+AA,
as the mitochondrial transmembrane potential decreased early in the process,
and cyclosporine A prevented the toxicity. Tmdcity in E47 cells exposed to
Fe+AA is mainly necrotic in nature, based on morphology, early alteration
of plasma membrane integrity, depletion of ATP levels, and non significant
DNA degradation at early periods. The results p-ted suggest that low
concentrations of Fe and AA (that are not cytotoxic by themselves), can act
as priming or sensitizing factors for CYP2El-induced iqjury in HepG2 cells.
2000
1 1
ANTIOXIDANT ACTIVITY OF ORGANIC PALM OIL
FRACTIONS
Milvia Chicca Marilena Lais, Monica Borghl, Damiano Mucchi,
Arianna Co&*, France De Paniilis’, Siivano Pmamonti. Department of
Biology, University of Ferrara, Ferrara, Italy *OrganicSur, via A. Costa
112/2, Rastignano (Bologna), Italy
Fractions of organic virgin palm oil, extracted from drupae of cultivars
of E&is gumeensis Jacq. (Magnoliophyta Liliopsida Arecaceae), currently
employed for alimentary purposes, were tested for antioxidant activity. The
palm oil fractions were collected from drupae at di&rent ripening stages
and showed diierent degrees of oxidative stability when evaluated by the
Bancimat test (induction time of volatile acid production under oxldii
conditions). The antioxidant activity was evaluated by measuring the ability
of each fraction to reduce oxidized cytochrome c solutions and to quench DNA
damage induced by oxygen radicals during fluorometric analysis of DNA
unwinding. The results showed that palm oil fractions had different degrees of
reducing activity upon cytochrome c and protective effects against oxidative
DNA damage, in good correlation with their ripening stage and &dative
stability. The antioxidant properties of organic raw palm oil fractions are
probably related to a higher content in carotenoids, tocopherols and other
minor antioxidant components when compared to reilned and bleached palm
oil fractions.
I 67 I
I I
Increased sensltyvity of G(IPD-deficient skin 5broblast cells to
hydrogen peroxid&nduced senescence
Ho, Cheng ML, Stern A. Chiu DTY. Graduate Institute of Basic
Medical %iences and School of MedicalTechnology, Chang Gung Univ.,
Taoyuan, Taiwan; Detp. of Pharm., NYU, New York, USA
Glucose-6-phosphate dehydrogensse(Gf3PD) is involved in the generation
of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and the
maintenance of the cellular redox balance. Increased susceptibility of GGPD-
deficient red cells(BBC) to oxidative stress rseulting in accelerated IlBC de
struction has been well documented. However, very little is known about
the susceptibility of G6PDde&ient nucleated cells to coddative stress. In
this study, we compared the susceptibility of GBPDdeticient skin fibrob
last cells(SFC) and normal SFC to hydrogen peroxide-induced senescence.
Both normal SFC and G6PDdefmient SFC were obtained by circumcision
of normal and G6PD-deilcient child, respectively. SFC were cultured and
subjected to various treatments with hydrogen p&de at di&ent pop&
tion doubling level(PDL). Cellular senescence wss determii by morphology
and beta-galwmeidsse stain. Our results show that cells with lower GGPD
activity and NADPH/NADP ratio were particularly susceptible to hydrogen-
elicited senescence. Overexpression of GGPD greatly increased the cellular
resistance to the efkxt of hydrogen peroxide. This study clearly demonstat-
eds that GGPD-deficient nucleated cells are also more susceptible to or&dative
stress than their normal counter parts. In addition, this study lends support
to the notion that oxidative stress is relatede to the onset of seneecence.
OXYGEN
Vitamin C and NAC supplementation increases oxldetive stress
in humans after eccentric muscle iury.
Chiids, A., Jacobc C., Kaminski T., *Halliwell B., and C. Leeuwenburgh.
University of Florida; *National University of Singapore
There is sign&ant debate on whether antioxidants could act as pro-oxidants
in viva, namely in the presence of increased levels of redox-active metal ions.
Most studies show that vitamin C is a protective antioxidant with or with-
out the presence of redax-active metals. However, we have investigated an
inflammatory model that strongly suggests vitamin C and NAC supplemen-
tation increases oxidative stress. We studied an acute muscle injury, induced
by eccentric exercise in humans, character&d by massive inflammation and
signiilcant increases in levels of bleomycin-detectable-imn. Following the in-
jurious bout, subjects consumed a placebo or vitamin C (12.5 mg/kg body
weight) and n-acetyl cysteine (NAC;10 n&kg body weight) combiition
for 7 days. Levels of MPO, II&, LDH, CK, and myuglobm were sign&
cantly elevated 2, 3, and 4 days post-injury, but returned to baseline after
7 days. Lipid hydroperoxidea, MDA, aud &iiprostanes were significautly
elevated post-iqjury. Despite an increase in total antioxidant status with
supplementation, levels of lipid hydroperoxides in the supplemented group
were signiilcantly elevated above the placebo group. SOD and GPX activity
in the serum was significautly increased in subjects receiving antioxidanta
This study has two major findings 1) the recruitment of leukocy& and their
production of reactive intermediates plays a sign&ant role in the develop
ment of acute muscle injury 2) vitamin C and NAC can act as prooxidants,
manifested by acute increases in redox-active metal ions.
I
1
68 I
I
MICE WITH COMBINED DISRUPTION OF GPXl AND
GPX2 GENES HAVE GROWTH RETARDATION,
HYPOTHEHMIA, AND COLITIS
Fang- Fang Chu*, Richard Arg&,= -1 &cott W. Binder**,
Martin G. Martin#, &mes II. Domshow*, and Il. Steven Eeworthy*.
*Department of Medical Oncology, City of Hope National Medical Center,
Duarte, CA 91010, =Division of Digestive Dies, Department of
Medicine, #Division of Gestrceuterology and Nutrition, Department of
Pediatrics, University of California School of Medicine, Los Angeles, CA
99919, **Department of Pathology, Division of Medical Genetics,
CedawSmsi Medical Center, Los Angeles, CA 90048, USA
Mice deilcient in single Gpxl or Gpx2 gene appear normal under regular
housing condition. GPX-1 and GPX-GI, encoded by Gpxl and Gpx2, sre the
two major GPX activity p-t in the eplthellum of gsstrointestbral tract.
Mice with combined disruntion of GnuI and Gnu2 series. double-KO. have
cache&. These double=KO-mice -&s hypo&&lc, especially under stress.
They also have a high incidence of diarrhea, anal mucous d&charge, and
perianal ulceration. Hiiologlcal study shows severe colitis and some lleitis
starting before weaning. Thus, we have provided the first direct evidence to
show GPX plays an lmportaut role in maintaming animal health.
2000 s31
VASORELAXANT AND ANTIOXIDANT PROPERTIES OF
BRAZILIAN WINES
1. ZCkless, K.; 3Cru2,C.D; BSchuldt, M.C.; SSchuldt,
E.Z..; BRibeBe, R.M. 1Depto. de Bioquhnica - UFSC,
Florian6polis/SC. ZCurso de FarmLia - UNISUL, Tuba&$X. 3Depto. de
Farm8cologia - UFSC, Florian6polii/SC. Brash. ckless@mboxl.ufsc.br
The scientific data confirm the popular belief that wine consumption is se-
sociated with a reduced risk of coronary heart disesse (CAD). These effects
could be attributed to the presence of phenolic compounds, which possess
a strong antic&idant activity, in wine. Recently, it has been shown that ex-
tracts of medicinal plants, grape and wine can induce vsxnelaxation through
endotheiium-dependent nitric oxide/cGMP. The aim of this study was to
evaluate the vasorelaxant properties in rings of thoracic aorta and antioxi-
dant capacity of red and white Brazilian wines. Alcohol-free wines were used
in all assays. The antioxidant properties of the wines were analyzed by de-
oxyribose degradation assay, nitro blue tetraaolium (NBT) reduction test and
lipoperoxidation assay. Through the analysis of deaxyribose degradation and
superoxide scavenging activity, as well as, lipoperoxidation assay it was found
that red wines possessed greater antioxidant capacity than white wines. This
effect was more evident in the deoxyribcee assay. In the endothelium intact
rings contracted with phenylephrine (10-100 nM), cumulative additions of
increasing concentrations of red wine (1 to 3.000 pg n&l) c8used graded
relaxations. This effect wss not observed when the white wine w8.s tested
in the same conditions. The v8sorelaxant properties of red wine could be
related to the strong antioxidant capacity showed by this wine, as well as its
phenoiic contents. Supported by: CNPq
I 71
EVALUATION OF THE DNA DAMAGE EFFECT OF RED
BRAZILIAN WINE BY THE SINGLE CELL GEL
ELECTROPHORESIS (COMET ASSAY)
1.2Ckless, K.; BRibeirodoValle, I&&& 1Departament.o de Bioqufmica -
CCB - UFSC, Floriaw$olis/SC. 2Cureo de Farmkia - UNISUL,
‘Brbarti/SC. 3Departamento de Farmacologia - CCB - UFSC,
Florian6polii/SC. Bmsil. ckies&mbmtl.ufsc.br
It has been widely reported that diets rich in fruit and vegetables are protec-
tive against certain diseases. These protective e&cts have been attributed, in
large part, to the antioxklant activity of plant phenolic compounds. In con-
trast to the beneikriai effects, some phenolic compounds have also been found
to be prooxidant and genotcoic in vitro. The ahn of this study was to inves-
tigate the genotoxic and/or antigenotoxic effects in hunnm blood cells (HBC)
using Comet ass8y. Whole human blood was incubated with alcohol-free red
wine for 4h at 37 OC in the presence or absence of Ii202. The results demon-
strated that the red wine ems not able to protect against the genotzxic effect
of II202 in the HBC. Furthermore, at higher concentration (I:10 dilution) red
wine increased DNA damage. This genotcuic effect w-8s also observed when
red wine was incub8ted at the same concentration without H202. These
results suggest that red wine poswses genotacic potentialin vitro. This
effect could be r&ted to complex formation of phenoiic compounds and iron
in the hemoglobii. Such complex iron-phenolic compounds could increase
free radicai generation and consequently increase DNA danmge. This effect
corroborates data reported in the literature, that demonstrated a germtoxic
e&t in vitro for some phenolic acids in the presence of transition metals.
Financi8i support: CNPq
S32 OXYGEN
ANTIOXIDANT PROPERTIES OF NATURAL AND
PROCESSED ORANGE JUICES
1, PCkless, K.;, 4Franke, S.I.H.; BRibeiro-do_Vaile, R.M.; 3Henriquea,
J.A.P. IDepto. de Bioqufmic8CCBUFSC, Florian6poiis/SC; 2Curso de
Farmkia-UNISUL, Tubar&o/SC; 3Centro de Biotecnologia-UFRGS, Porto
Alegre/BS; 4Curso de Nutri&UNISC, Sta. Cruz do Sul/RS; 5Depto. de
Farm8cologiaUFSC, Floriamipolis/SC. Brssil. ckless@mboxl.ufsc.br
In some studies, intake of fruits has been associated with the prevention
of certain diseases. These omtective effects have been attributed. mainlv. to
phenolic compounds. Since phenolic compounds are present in~severrr~fruit
juices and the preparation and/or processing of these juices is an important
factor in the retention of their antioxidant properties, the aim of this study
WBSto investigate the antioxidant properties of natural and processed, frozen
and fresh orange juices. The processed juices were purdmsed at the super-
market and fresh orange juice was prepared prior to the experiments. A
sample of esch juice was kept froeen for several days and later, submitted to
the assays. The juices were diluted to 0.01; 0.1; 1.0 and 10%. The antioxi-
dant properties of the juices were analyzed by deoxyribcee degradation assay
and nitro blue tetra8olium fNBT) reduction test. All tested oranee iuiws
inhibited deoxyribose degradation’in a similar profile. These juices were able
to inhibit deoxyribose degradation by around 60% at 1% concentrations and
by around 75% at 10% concentrations. In the NBT reduction test, one of
the proosssed juices had no effect. The others showed 02-. scavenger ca-
pacity only at the highest concentration tested. The scavenger properties
of these juices probably are related to the presence of phenolic compounds.
Supported by: CNPq
El72
Thioredoxin, a Singlet Oxygen Quencher and Hydroxyl Radical
Scavenger: Radon Independent Functions
Kumuda C. Dss and Chandan K. Das. University of Texas Health Center
at Tyler
Thioredoxin is a ubiquitous small protein known to protect ceils and tis-
sues against oxidative stress. However, its ex8ct antioxidsnt nature has not
been elucidated. In this report, we present evidence that human thioredoxin
is a powsrtid singlet oxygen quencher 8nd hydroxyl radical scavenger. Hu-
man thioredoxin at 3 PM caused 50% inhibition of TEMP-102 (TEMPO)
adduct formation in a photolysis EPR study. In contrast, E. Coli thiore-
doxin c8used 50% inhibition of TEMPO formation at SO pM. Both E.coli
thioredoxin 8nd human thioredoxin inhibited .OH dependent DMPG-OH
formation 8s demonstrated by EPR spectrornetry. The quenching of 102
or scavenging of .OH w8s not dependent upon the redon state of thioredoxin.
Using a human thioredcodn in which the structural cysteines were mutated
to alanine, Ttx-C3A, we show that structural cysteines that do not take part
in the catalytic functions of the protein are also imports& for its reactive
oxygen sc8wnging properties. In addition, using a quadruple mutant Trx-
C4A, where one of the catalytic cystsines, C35 wan mutated to 8hanine in
addition to the mutated structural cysteines, we demonstrated that catalytic
cysteines are also required for the sc8wnging action of thioredoxin. Iden-
tification of thionxkxin as a 102 quencher and .OH scavenger may be of
signMeant importance in explaining various redmc-related antioxidant func-
tions of thioredoxin. Supported by AHA and ACS grants.
2 0 0 0
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Non-transferrin-bound serum iron after therapeutical iron dosage EFFECT OF CATECHINS ON UV-C MEDIATED DNA
B.Dresow P.Nielsen, M. Diirken* and R.Fischer. Med.
-, OXIDATION.
Biochemie/Molekularhiologie, Kinderklinik*, UKE - University Hamburg,
!&ar G. Frm, Javier I. Ottaviani. Fernando Carrasquedo, Carl L.
Germany.
Non-transferrin bound iron (NTBI) leads to an enhanced generation of re- Keen’. and Harold H. Schmitz’. PRALIB (CONICET)Physical
active oxygen species and to oxidative stress mediating cell and tissue dam- Chemistry. School of Pharmacy and Biochemistry, U. of Buenos Aires.
age. NTBI has been reported in most forms of iron overload, in patients I I I3-Buenos Aires, Argentina: ‘Department of Nu~rmon. I iniversity 3f
with leukemia under conventional chemotherapy and in patients after bone
California, Davis CA 956161: ‘MARS Inc., Hacketlstown, NJ 07840.
marrow or stem cell transplantation.Very recently NTBI was found in ane-
mic patients on hemodialysis after intravenous iron application. The present Catechins are a group of polyphenols (PP) that can exert
study demonstrates for the first time the appearance of NTBI after oral iron
antioxidanr activity in biological systems. However, a recent report
intake. Serum iron (SI), transferrin saturation (Tfs) and NTBI were mea-
sured in patients with mild iron deficiency before and up to 8 hours after indicated the possiblicy of a pmoxidanr effect of PP. specially when
intake of a single dose of 100 mg Fe2+ as a pharmaceutical iron preparation. present 81 high concentrations. Here we evaluated the effects of catechil
Before intake, SI and Tfs was low in all patients, NTBI was not detectable. (0. epicatechin (EC), quercetin, and EC oligomers at concentritions ’
After ingestion there was an increase in SI and Tfs with maxima at about
ranging from 0.1 m 50 pM. on UV-C mediated DNA oxidation. DNA
3 to 4 hours. In parallel, max. NTBI leveler between 3.5 - 11.5 pmol/L
oxidation was evaluared by the formation of 8.oxo-2’-desoxyguanosine
(normally found in severe iron overload) was measured in the serum of all
patients. Even 6 - 8 hours after oral iron intake NTBI was still measurable. (oxo8dG). In the absence of added PP, the oxo8dG levels ranged from
This study demonstrates for the first time the appearance of NTBI, widely I.3tO.ito5.9kO.l and 14.0+00.9 oxo8dGllO’dG (p<O.O5) after I5 and
discussed to be responsible for in viva lipid peroxidation, after a pharmaceu- 30 min of irradiation. respectively. oxo8dG formation was reduced
tical dose of oral iron. The physiological relevance of this finding has to be
further elucidated, also in view of the widely administration of iron prepara- significantly in the presence of concentrations as low as 0.1 NM of EC

tions in one of the most common deficiencies in humans. One may also think trtrilmrr or EC hexamer and I pM of C. EC or EC dimer. PP
of a coadministration of antioxidants, especially, vitamin E together with the concentrations as high as 50 pM did not provide better protection and
iron preparation. &d not generate higher oxo8dG Irvels. Catechins and related oligomers
protected DNA from oxidation 81 concentrations that can be obkked HI
physmlogical conditiow Higher PP concentrations, which are unlikely
IO occurr following the ingestion of foods. did not lead IO further UV-C
medioled DNA oxidation (supporrrd by UBA and MARS 1~).

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ANTIOXIDANT PROPERTIES OF HUMAN MILK & INFANT
FORMULA
JK F&l, WL Andrew, M Langdon. Memorial Univ
Milk from mothers of pre-term (PT) and full-term (FT)infants may have
antioxidant ability not seen in humanized formulas. Mothers may respond
to early gestation by increasing milk (HM) antioxidant roperties as seen for
other milk components. We assessed the ability of HM at weeks 1, 2 and
12 of lactation (17 PT,28 FT) to resist oxidative stress. HX/XO generated
the radicals and oxygen consumption, CAT activity and MDA levels were
assessed. Normal & high-iron formulas were tested with/without CAT, SOD
and GHSPX. There was no difference (p<O.OS)between PT & FT groups for
oxygenronsumption (“moles 02/min). Means f SEM for the Pr/FT groups
following stress,were: wk 1: 7.97 f 0.56; 6.68 f 0.88;wk 2 4.62 i 0.42, 5.17
f 0.36, wk 12 4.08 f 0.39, 4.80 f 0.49. MDA levels & CAT activity did
not differ between groups. PT milk is as good ss but not better than FT
milk. R2=.443 between 02 consumption and MDA and R2=-0.30 between
02 consumed & CAT for all weeks, suggesting a protective role of CAT in
HM. 02 consumption differed over time between HM (44.75 f 1.66)& normal
(98.88 f 3.11)& high-iron (168.94 f 9.47) formulas. Adding CAT, SOD &
GHSPX increased antioxidant capacity by decreasing 02 consumption to
66.11 f 6.48 and 104.81 f 8.65 respectively. No CAT activity was seen
naturally in formula. Pasteurization of HM did not compromise antioxidant
capability suggesting that enzymes play a role but are not solely responsible
for protection observed in HM. Funded by the MRC and Janeway Research
Foundation.
ELEVATED IRON INTAKES AND ANTIOXIANT STATUS IN
PREMATURE INFANTS
Friel JK, Andrew WL, Azis K. Issa T, Kwa PJ. Biochemistry and
Pediatrics. Memorial Universitv of Newfoundland. Canada.
Prernatire infants are often”given erythropoieid (EPO) and iron, (2.12
mg/kg/d) for periods of up to 4 weeks soon after birth. EPO is to stimulate
RBC production and Fe is to prevent anemia. We hypothesized that high
Fe doses may cause oxidative stress. We studied 11 infants (1032 f 216 g
birthweight) at 3 times: Tie 1 (baseline, no FE, no EPO); Time 2 (2 wks
later, EPO and 2-4 mg/kg/d Fe; Time 3 (2 wks later again, EPO and 46
mg/kg/d Fe). We measured plasma MDA, RBC CAT, ZnCuSOD and total
antioxidant potential (TRAP). No differences over time occurred except for a
trend (p=O.O7) in CAT. Time 1,66f17; Time 2, 77*19; Tie 3,86f16. For
40 control infants CAT did not rise over time. We hypothesize CAT increase
in response to elevated Fe intakes either due to oxidant stress or incorporation
of newly formed heme. Further, human milk fed infants had higher TRAP
values (629f281 vs 176f178) at Time 2 and higher CAT (95f7 vs 68f15) at
Time 3 suggesting that method of feed afTects antioxidant status. Supported
by the Medical Research Council and the Janeway Research Foundation.

OXYGEN 2 0 0 0 S33
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OPTIMIZATION OF AN IMMUNOASSAY FOR Deuwsad MnSOD Expression May Be Involved In Neointimal

ISFZt-ISOPROSTANE THAT DOES NOT REQUIRE SOLID
PHASE EXTRACTION.
S. Gupta#, D. Szwaki#, McGoweo#, J. Morrow+,
L.J. Roberta-. #OxfordBiicalBeseamh,
P.O. Box 522. Oxford MI. USA ; +Dept. Pharmacology, vaoderbilt
U., Nashville, TN, USA 37232. *Dept. Chemistry, Oaklaadv., I&heater
MI, USA 48309
Isoprcetaaes arc the biomarkers of choice for the meaauem eat of oxidant
stress. A competitive ELISA for 15~Isopnmtane developed by us n-s.9vai-
idated by GC/MS. The standard procedure rquirea biological sampled be
subjected to solid phase extraction to remove interking substaacen prior
to F&ISA. These manipulations are tedious and eapensive aad caa introduce
errors. We therefore sought to eliminate the extraction step without comprw
misiig the accuracy or sensitivity ofisoprostane ELISAs. Urine samplea (n =
8,21), for which 15F2t&oprostaoe levels bad been determined by GC/MS,
were diluted ia buffer systems wataining vary& quantities of surfactants
and/or blocking agents to minimize n&c biding aad matrix effecta.
The diluted/non-extracted samples were then subjacted to ELISA. The 4
parameter logistic curw Bt was us& io compare the seasitivity (50% B/BO<
5 n&l) and the usable eoncentratioo ranga for theds ELISAs. Fbllowiag op
timization, agood correlation (r2 0.85) and statistically signSkant dmkity
(ANOVA; p < 0.05) was observed between the ELlSA valuea obtained for
non-extracted urine samplea and GC/MS. The results iadicate that 15F2t-
Isoprostaae in urine samples can be analyzed without solid-phase extmcti~n,
thereby reducing time aad effort, and eliminating errors due to extraction
or incomplete solvent removal. [Supported in part by NIEHS contract #
N44-ES-054481.
Smooth Muscle Cell Proliferation
Tile, PIL Wein
aad LW Ober,;ak~ ek=L:2z2k Jr”
Folk&g vascuku iajury, medial smooth muscle cells (SMCs) of the ‘ep-
ithelioid’ pkmotyps migrate and cloaally proliferation to form the neointima.
We recently reported that lewls of superoxide were increased in neointimal
as compared with medisl SMCa, and superoxide has been reported by other
ioveatigators to inducs SMC proliferation. The purpose of this study was
the&&e to dewmine the role of aatioxidaat emzymea in the neointiial SMC
superoxide accumula&a aad proliferatioa. Neointimal and medial SMCs,
and epithelioid and spindle-&aped clones were enzymaticaUy isolated from
rat aorta two weeks after balloon injury or sham injury. Under identical
culture conditions, neointimd SMCS proliferated more rapidly than medial
SMCs. We&era blottiag showed that the ratio of MaSOD, CuZaSOD, aad
catslass in neoiatimai SMCs versus msdial SMCs was 0.732 f 0.048,1.172 f
0.088, 1.858 f 0.387, mspectively. Siily, epithelioid clones, whether de-
rived from 04 msdia or naointima, had lower MaSOD aad hi&r catalase
proteia lev& as compared with spiadk-shaped clones derived from the same
-. Sii patterns of dtexatioas of the enzyme activities were also
ohwrwd. Northern Blotting iadicatad that epitkelioid clones had deereased
hfaSOD mRh’A &on compared to spindle-shaped clones under ether
serum-frsa or lO%FCS culture eonditkms. These data suggsst that decreased
MnSOD exptwsioa might contribute to the accumulation of superoxide and
the increwsd proliferation of SMCs in rat aortic neoiatima s&r balloon in-
jury.

Effects of Vitamin E and Pulmonary Hypertension Syndrome on Ethanol-induced he~atotoxicity and waventiva effect of betaine
Lunu Mitochondrial Fbttv Ad& d Ant-. Gii&r KANBAK, &&&. &man&i University,The Medical
rbtlc&KK~~ “tje. Dept. of Poultry S&ace, 00. DeDartmedofBioQmiatrV
P&c& e&c& of betaine in ethanol hepatotoxicity were investigated.
As mitochondrisl’dy&mcti has been obaerwd in puhmmary bypatension 24 bale w&tar albino rata were divided into three group:control,ethaad
syndrmne (PBS) ia broilers, the objective of this study was to determine the and ethaaol+bstaine groupRats were fed ethanol 8 g/kg/day for 2
effects of PHS and vitamin E (VE) on loag chaia fatty dds and aatiax- months.Ethanol+b group were fed ethanol plus betaiae (0.5% W/Y).
idantsialungmitochoadria. Luugmitodn&iavcreiaokadfrombirds Reduosd glutathiooe, malondiaidehyde and vitamin A were determined in
raised under cold coaditions (to induce PHS) aad fed dieta coata&@ 15 or ths liver tissue. Alaaiae ease wers also measured intracardiac
100 IU dl-a4ocopherol/kg (Coatrol aad VE, respectively). Oxidatiw stress blood samples. GSH levels in the ethanol group were si@fxantly lower than
WMobservedinP~mitoehondriathatineludedlorerlsvebda-~dT the control grmtp (p<O.OOl). GSH elevated in betain group as comparsd
~~her;~ys aad an skated GSSG/GSE. PHS lung mitndmmdria to the e&awl group (p<O.OOl). MDA io ethanol group were significantly
. . . pew&ageofC10:0andafowetpercanta8edC18:1 higher thao the control group (~~0.05). MDA were decxewsd in betaine
comparsdtobiiwitboutPES.IBghdistaryVEelevatedhmg*twopbad group as c0mpam.d to the ethanol group (~~0.05). Vitamin A ia the ethanol
inbothvEsndvEPHsgaupsmanpsndtocmtmb,butdidwtprcvasrta group were signifkcz&ly lower thanin the control group (p<O.Ol). In the
decreaseinGSIiinVEPH!3mitochoadriaBtipaovidadsu~VE ethuml+betaine group wwe high as compared to the ethaool group (p<O.Ol).
exhibited ekated w ofC18zOandC2OOaadlowersd~ ALT in the ethanol gmup were higher thaa the control group (pc0.05). Ox-
of Cl&l, C1&2,18:3, and C2Oz3 in lung mitodkoadria compared to bkls fed idative stress mighthave a majorrole in the ethanol-mediated hepatotoxicity.
theckmtroldiet. Thus,highdietaryVEwas modatedwithabweringof Betaiaa may protects liver iqjury and it may prexents vitamin A depletion.
the~~otunsaturationnlativetolvturstionof~~aeidsinl~ ThemfoE, it may u&id nutritional ageat ia the prevention of clinical prob
mim rqardkm of ths presence or absence of PHS. (Supported by 1em.sdepends on ethsaol-induced vitamin A depletion ia liver.
USDA-NIU #OS42123 to W. Bottje.)

s34 OXYGEN 2000

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Antioxidative properties of carvedilol, an antihypertensive agent THIOREDOXIN-LINKED LIPID HYDROPEROXIDE

Karl Oettl, Joachim Greilberger, Klaus Zan er, Ernst Haslinger
Gilbert Reibnegger and Guenther +titute
Juergens. for Medic&
Chemistry and Pregl Laboratory and Institute of Pharmaceutical
Chemistry, Karl-fianzens-University Graz, Harrachgasse 21/H, A-8010
Graz, Austria
Carvedilol is an antihypertensive agent and in clinical use since years. In
addition to its function as a ~-blocker carve&lo1 was shown to act aa an an-
tioxdant. However, there is some controversy about how carvedilol achieves
its antioxidative ability: by radical scavenging or ion chelation? We therefore
used a method for radical generation independent of metal ions to investi-
gate the antioxidative properties of carvedilol. We show that carvedilol de-
creases LDL oxidation induced by peroxyl radicals generated by 2,2/-ar,obis(2-
amidinopropane) hydrochloride (AAPH). Formation of thiobarhituric acid
reactive substances, lipid peroxides and epitopes against oxidised LDL were
used for monitoring LDL oxidation. We further show that carwdilol reacts
with peroxyl radicals being consumed during reaction. However, carvedilol
showed no reaction with nitrogen centered radicals which are often used in
assays determining antioxidative properties. On the other hand we found
that carvedilol acts &s a chelator of ferric ions. Using mass spectrometry and
NMR spectrometry we show complex formation with free and acetylacete
nate complexed ferric ions. The binding constant with Fe3+ is in the range
of 105 l/mol. Ram our data we conclude that arvedilol acts as both, as a
metal chelator and a radical scavenger. However, it is selective in reacting
with different radicals.
PEROXIDASE ACTIVITY OF HUMAN SERUM ALBUMIN IN
THE PRESENCE OF PALMITOYL COENZYME A
IL-HAN KIM National Creative Research Initiative Center for
Antioxidant Proteins, Department of Biochemistry, Paichai University,
Taejon 302735, Republic of Korea
Human Serum Albumin (HSA) showed a weak, but significant peroxidase
activity with the use of a thiol reducing equivalent such as dithiothreitol
(DTT). Carboxyl group-modified HSA (CM-HSA) showing lo-fold strong
peroxidase activity (1.6 nmol/min/mg) as that of HSA (0.17 nmol/min/mg),
exerted the substrate selectivity toward lipid hydroperoxide (such as linoleic
acid hydroperaxide). Replacement of DTT with thioredoxin (‘I&) as an in
viva thiol electron donor resulted in an increase in the lipid peroxidase activ-
ity of CM-HSA. In contrast to CM-HSA, HSA did not reduce lipid peroxide
with the use of ‘I&. In the presence of pahnitoyl coenzyme A, however,
HSA utilized nx &s an electron donor to the reduction of lipid hydroper-
oxide. The ‘&x-linked percaidase activity of HSA sharply increased with
elongation in the carbon chain of the acyl moiety from a&y1 CoA (2:0)
up to palmitoyl-CoA (16:0), and beyond pahnitcyl-CoA the activity steeply
decreased. Fluorescence study indicates the conformational changes of HSA
induced by palmitoyl-CoA. Together, these data suggest that pahnitoyl-CoA-
bound HSA has a capability to remove lipid peroxide with the use of electron
given by Trx system. Key words: human serum albumin, peraxidase activity,
palmitoyl coenzyme A, thioredoxin.

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RELATIONSHIP BETWEEN ANTIOXIDANTS IN THE DIET Ascorbic acid in respiratory secretions and its modulation by
AND PREGNANCY INDUCED HYPERTENSION oral supplementation
Korraa’. S.S., El-w* E M &deek* ,
M.A., Khater*** y-7
M.F. and N. S.Hu&n*. *National Center for Radiation Research and Sun e Kwack John M. Koostra Cartoll E. Cross, Robert
Technology **Prof. Of Biochemistry -Institute for Environmental Studies Ha&man, Eri~Gershwin, and Albert van der Vliet
and Flesearch- Ain Shams University - Cairo, Egypt. ***Prof. of Obstetrics ents of Internal Medicine and Nutrition, University of
and Gynecology - Faculty of Medicine - Aii Shams University. * ProF. Of %%%a, Davis, CA 95616, USA.
Nutritional Biwhemistry - Nutritional Institute
The relation between the oxidative stress outcome in terms of plasma malon- There is much interest in the role of oxidant stress in
dialdehyde (MDA) and antioxidant status in terms of superoxide dismutase
enzyme (SOD) blood level together with serum and dietary intake of vitamin
E was investigated among thirty five strictly defined late pregnancy hyper-
tensive women of age range from 20-28 years old matched with 30 controls of
pregnant non- hypertensive women. Rem&s showed significant high levels of
plasma MDA among hypertensive casea compared with controls (13.22+2.47
via 8.73+2&l nmol/ml). The latter was positively correlated with both sys-
tolic and diastolic blood preasure.Increased consumption of dietary vitamin E
(59.77+27&l vis 46.86+23.17 &day) was observed among late pregnancy
hypertensive women. It was observed that patients exhibitedhigher serum
vitamin E with respect to controls (1306+774.3via 1633.04+692.99). Blood
SOD was tignilicantly decreased in patients with respect to controls (169.51
vis 194.31 units/ml). There was a paitive correlation between the severity of
the disease and plasma levels of MDA and a negative correlation between the
severity of the dii and blood SOD. This study given support to previous
studies that oxidative stress plays a digni6cant role in the pathophysiology
of preeclamsia.

OXYGEN 2000 s35

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Vitamin E dllrimlnation in a-tocopherol transfer
protein-deficient mice
Leonard
-I SW
_, i
-, Terasawa Y 3 ’ Fareae
-1 _I -,_?- RV4 6 ltaber MG’ r ‘Linus
Pauling Institute & ‘Dept of Nutrition and Food Man&G. 6SU.
Corvalhs, OR, 3Dept of-Nutrition, UC, Berkeley, CA, ‘&&tone Institute
of Cardiovascular Disease, 5Cardiovascular FLeaezucbInstitute & BDept of
Medicine, UCSF, San Francisco, CA
A genetic model of vitamin E deilciency was developed by disrupting the
mouse a-tocopherol transfer protein (TTP)gene (7’tpe by using homologous
recombination in embryonic stem cells (Terasawa et al. PNAS 97:in press
2000). This mouse model was used to teat TTP’s role in vitamin E transport.
A diet containing RRR-a-[5,7-(C’&)]- and elf ma-a-[5-(C*H&]-tocopheryl
acetates (30 mg each/kg diet, 1:l RRR/d-me, naturabsynthetic) wa8 fed
to Ttpe+l+, T&&l-, and Ttpa-I- mice (n=5/group) for 3 months. Mice
were fasted 4 h, sacrificed, plasma and tissues h-ted and analyzed by
LC/MS/MS. The RRR/oll-mc ratios for plasma and 16 tissues (excluding
liver) from Ttpa+l+, l&m+‘-, and Ttpa-/- mice were 1.6 f 0.2, 1.9 f 0.2,
and 1.1 f 0.1 (mean f SD, p<O.ooOl for -/- vs. +/+ or +/-) respectively,
showing that TTP is required for discrimination between natural and syn-
thetic a-tocopherol. Total (labeled + unlabeled) vitamin E in meet Ttp-I-
tissues ranged from 220% of Ttpa+‘+tissues, demonstrating that TTP is
required for maintenance of vitamin E concentrations. Labeled represented
8990% of total vitamin E in all genot ea. Notable exceptions were nervous
tissue, which were 3555% for Ttpa+F+ and Ttpa+~-; adipose, eye and skin
tissue which were 55-75% labeled. Thus, TTP is required for maintenance
of plasma and tissue vitamin E as well as discrimination between different
vitamin E forms.
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Carotenoids inhibit c&dative DNA damage and liver necrosis in
rats treated with ferric nitrilotriacetate.
(1)Humberto R. Mates, (2)Vera Capelozzi, (1)Osmar F. Gomes,
(1)Paolo Di Mascio and (1)Marisa II. G. Medeiros. (1)Departamento de
Bioqufmica, Institute de Qufmica, Universidade de Sfio Paulo, CP 26077,
CEP 05513?970, S&o Paulo, SP, Brazil. (2)Departamento de Patolcgia,
Faculdade de Madicina, Universidade de Sgo Paula, Sgo Paulo, SP, Brazil.
A hi incidence of cancer has been correlated with chronic iron over-
load, and camtenoids are of interest as possible anticarcinogens. We have
investigated the e&t of lycopene and beta-carotene on lipid peroxidation,
Box~7,8dihydro-2’-d~guanosine (6-oxodGuo) formation and liver hiatol-
ogy in animals subjected to ferric nitrllitriacetate (Fe-NTA) administration.
Male Wiitar rats received daily i.p. injections of lycopene or beta-carotene 10
mg/kg body welgth for five consecutive days. At the 6fth day of carotenoid
treatment, animala were given 10 mg/kg body wigth F&lTA, or saline, i.p.,
forming four experimental groups: (a) control, (b) Fe-NTA, (c) lycopene
FeNTA and (d) beta-carotene-Fe-NTA. Each group contahmd 10 animals.
Animalsweresacrifieedsfter3and24h~NTAiqjectionandthelivetwere
immediately removed. Compared with control rats, liver of Fe-NTA treated
rata showed a significant incream in the &oxodGuo level, malodialdehyde
(MDA) accumulation concomitantly with liver necro& Both lycopene and
be&carotene were wry e&dent in reducing MDA and &xodGuo levela and
protect the liver against the observed histological alterations. These results
indicate that lywpene and be&carotene play a role against EBNTA induced
tissue damage. Financial support: CNPq, FAPESP, USP/COFECUB and
PRONEX
S36 OXYGEN
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Asseesment of &dative stress in athletes during endurance
exercise using deuterium-labeled vitamin E
Mastaloudis A’*‘, Leonard SW’, Leklem JEr, Traber MG’*r. ‘Linus
Pauling Institute, & ‘Dept of Nutrition & Food Mgt, OSU, Corvallis, OR
To determine whether extreme endurance exercise induces lipid peroxi-
dation, we studied 14 athletes (5 F/ 9 M) during a 50 km ultramarathon
(trial 1) and a sedentary day (trial 2) 1 month later. The night before the
race (or sedentary trial), subjects consumed 75 mg each ds-RRR and de-o11
moo-tocopheryl acetates. Blood samples were taken at baseline, 30 min
pre-race, mid-race, post-race, 1 h post-race, 24 h pcet-race, and at corre-
spondmg times during trial 2. All subjects completed the race; average run
time was 390 i 67 mm, at a rate of 13 f 2 nun/mile. Plasma labeled and
unlabeled a-tocopherols, Fr-isoprostanea (Fr-I), and ascorbic acid (AA) were
measured. Deuterated da a-tocopherol (d3 a-Tot) disappearance rate9 were
faster (2.8x10-” f 0.5x10-‘) during the race compared to the sedentary trial
(2.3~10~’ f 0.6x10-‘; p<O.O3). Moreover, Fr-I nearly doubled during the
race (pmrace vs post-race, 76 f 24 vs 130 f 54 pg/ml, p<O.ooOl), then
returned to baseline by 24 h post-race, but were unchanged during trial 2.
Plasma AA increased from 75 f 11 at pre-race to 156 f 36 pM at race end
(p<O.OOfR) then decreased to below baseline concentrations at 24 h post-
race (40 f 5 &f p<0.0001). AA levels also increased during trial 2, largely
as a result of antioxidant-fortified foods. Thus, lipid peroxidation increased
during the ultramarathon BSdocumented by increased vitamin E disappear-
ance rates and Fr-I concentrations. Increased plasma AA maybe a protective
mechanism invoked in response to thii oxidative stress.
I 88 I
DIFFERENTIAL RESPONSE BETWEEN YOUNG AND OLD
TO ECCENTRIC EXERCISE-INDUCED OXIDATIVE STRESS
I$ul~~ury, Jennifer Sache&, BRoubenoff. and Jeffrey Blumberg.
“man Nutrition Flesearcb Center on Aging at Tufts University,
Boston, MA 02111
Exentric exercise can be utilii to initiate an acutepham response and
provide a model wherein respiratory oxidative metabolism or immune re-
sponse induced oxldative stress may be studied in healthy hulividuah. Young
(2335 yr) and elderly (66-78 yr) men performed eccentric exercise by running
down an incllled tmadmill at 70% of VOOmax for 45 min. Plasma markers
of immune response and biomarkem of oxidative streamstatus (BOSS) were
assayed at baseline, 0,6,24 and 72 hr po&exerciee. Elderly men exhibit ele-
rated (p<O.O07) baseline F2 ieoprostanen (2.635 f 0.64 &ml) compared to
young men (1.717 f 0.634 rig/ml). Baseline malondialdehyde (MDA) did not
differ slgniicantly betwen groups. Baseline MDA and F2 kmprcetanea did
not correlate. Baseline elderly ORAC values tended to be lower compared to
young men (p=O.OsS). OBAC values decmaaed in both groups postexercise
with those obeerwd ln the young decreasing 10% by 24 hr and the elderly
reaching 10% decrease by 72 hr. MDA hmreamd signiiicantly hi young men
hnmedlately poaexereise (p<O.O4), returning to baseline within 6 hr. MDA
did not change in elderly men. By six hr postexerdse plasma IL6 was ele-
rated (p<O.O5) in both age groups. F2 isoprostanea were elevated by 50% at
24hrinboth. At72hrF2isoprostaneswereelevated~and13046inel-
derly and young men respectively. These data indicate di&rential expression
of BOSS between young and elderly men in response to eccentric exercise.
MDA and F2 isoprostane kinetica differ within the age groups.
2000
l-=-l
Enzyme inhibition and protein-binding action of the
procyanldin-rich Rench maritime pine bark extract,
PvcnozenolfR): Effect on xantidne oxidase.
H&ii Miini, &ohg Guo and Lester P&r. Department of Molecular and
Cell Biolozx
-. 251 Lie Sciences Addition, Universitv of California at
Berkeley, Berkeley, CA 94720-3200, USA _ Objectiv: Obstructive sleep apnea may have significant effects on incidence
Pycnogenolr, an extract from French maritime pine bark (PBE), is an ef-
fective scavenger of reactive oxygen species, and its main constituents are
procyanidins of various drain lengths. In order to find out the biochemical
basis of action of PBE on enzyme activity, involvement of its redox activity
and/or direct binding to the enzyme in its subsequent action on enzyme ac-
tivity have been investigated. PBE dosedependently inhibited the activities
of xanthiie oxidase and xantbiie dehydrogenase but it did not affect the
activities of glucose cmidase and ascorbate oxidase. Under non-denaturing
condition, PBE changed the electrophoretic mobility of xanthiie oxidase but
not of glucose oxidsse. It was found that hydrophobic bondii might be
the dominant mode of interaction between PBE and xanthine axidase. The
importance of the biidii in the effect of PBE on enzyme activity was sup
ported by the observation that PBE bids to and inhibits cat&se, but not
superoxide dismutase. However, no correlation was found between superox-
ide/hydroxyl radical scavenging activity and the inhibitory e&t on xantbine
oxidase activity of PBE, various purified flavonoids, or other complex mix-
tures of bioflavonoids. The results indicate that PBE selectively inhibits
xanthiie oxidase through binding to the enzyme rather than by the redox
activity.
I 9’ I I
Characterhmtion of the Diierentiated ProlUe of Human Saliva
Antioxidant Systems
Nagler Klein D&i&s and Ileznick Hambam MedicsJ Center and Faculty
of Medicine, Technion - Israel Institute of Technology, Haifa
Saliva has various defense mechanisms, including immunological and en-
zymatic systems aimed at pathological microorganisms, and the abiity to
protect mucosa against mechanical insults and when required to promote
healing by agents such as the epidermal growth factor. However, another
defense mechanism exists, the antioxidant system, which seems to be of
paramount importance. This study found much higher concentrations of
the various salivary molecular and enzymatic antioxidant parametera (SOD,
pertidase, uric acid and TAS) in the parotid saliva as compared to the
submandibular/sublingual saliva, and also a distiiction between the sali-
vary antioxidant system and the immunological and enzymatic pmtective
systems as represented reapsctively by the salivary concentrations of score
tory IgA and lysozyme which were similar in parotid and submandibular/
sublingual saliva. These iindll suggest that the profound antioxidant c&
pacity of saliva secreted from parotid glands as compared to that secreted
from submandibular elands is related either to the d&rent ohvsiolozical
. . de-
mands related to eatinn (oamtid oredo lninancel. oral blt&itv maintenance
;edox active trahsitio& n&l ions in This signifies that our
oral cavity environment is only pa&sJIy protected against caddative stress
during most of the day and night.
OXYGEN
Igo I
I I
GENIOGLOSSAL ADVANCEMENT - A SIMPLE SURGICAL
PROCEDURE FOR SLEEP APNEA
B.&e1 M. Nagler DMD, MSc, PhD, and Dov Laufer DMD. Rambarn
Medical Center and Facultv of Medicine. Tech&m - Israel Institute of
Technolozv. Haifa
of mortality if left untreated. The use of a standard nasal continuous oositive
airway pressure device is often efIective but is neither curative nor universally
well-t&rated. Surgical intervention to correct either soft or bony tissues is
often inevitable. In severe cases of abnormal sagittal morphology of the skull,
an involved maxill+mandibular advancement is often indicated to enlarge the
pharyngeal airway. However, such a surgical procedure is relatively radical
and may have significant attendant complications. Patient/Method. We de-
scribe a simple, conservative surgical procedure performed on a patient with
profound mandibular retrognathia accompanied by sleep apnea. The sleep
apnea was partly overcome by using thii simple modification of the standard
genioplasty procedure, based on extensive advancement with upper tilting
and rigid internal fixation of a bony symphysial segment following limited
anterior stripping of its periceteum. Result. The procedure resulted in a
significant pharyngeal &way enlargement of 53% and 87% at the mandible
angle and hyoid bone levels, respectively, increased oxygen saturation, re
duction in the respiratory disturbance index by 50X, and improved sleep
quality.
I 92 I
I I
Decreased Antioxidant Capacity of Saliva Related to Age and
The Effect of Cigarette Smoking
i$t$=JJ=J;~i Q&z&,K~~.&R~~~~, Hebrew
The use of cyclic voltsmmetry has shown that donor age significantly de-
cresses the reducing ability of human saliva. We recently initiated a study of
the eifect of cigarette smoke (CS) on salivary enzyme activities , s&vary an-
tioxidant defense systems, and the ability of several exogenous antimddants
to reduce the CSassociated damages. In vitro exposure of saliva to nine puffs
of CS showed a 4OG%increase in salivary protein carbonyls. Ascorbate and
desferri wunine (DES) had little e&t on protein carbonyl accumulation,
while glutathione (GSH) and N-acetylcysteine (NAC) considerably inhibited
this accumulation. After exposure to CS, a @n&ant reduction was found
in the activities of several salivary enzymes D amylase (83%), lactic dehy-
drogenase (LDH) (57%), and phcephatase (ACP) (77%). The addition of 1
mM of GSH and NAC considerably protected LDH and amylase activities,
suggesting that iiSII groups are al&ted in LDH and amylase. On the other
hand, the addition of 1 n&l -bate caused further km of LDH and amylase
activities. which could be partially p-ted by the addition of DES, impli-
cating metal-catalyzed oxidation promaw. However, loss of ACP activity
was completely w&&ted by any of the above antioxidants. It is concluded
that the loss of salivary ensyme activities may be due to various agents in
the CS which a&t the enzyme activities by means ofdifferentmeehanisms.
2000 s37
I 93 I
Inhibition of ceramide-induced apoptosis by ca!Feic acid in U937
cells. Commuison with other antioxidants and protein kinases
inhibitors.
M. Nardini (1) I_,F. Leonardi C. Scaccini and F. Virgili. (1) Division of
Pubnonarv Medicine. UCDavis School of Medicine. Davis. CA; Ftee Radical
Research Group, National Institute for Food and &it& Research,
Rome, Italy.
&amide acts as second messenger in the signal transduction triggered by
a variety of stress stimuli and extracellular agents. Stress response through
ceramide is involved in the development of many human diseases. Dietary
phenolic compounds have been reported to exhert a beneficial effect on the
onset and development of most of them. However the mechanism underlying
this beneficial effect are mostly not understood at the present. We investi-
gated the effect of ca&ic acid, a widespread phenolic acid largely present in
human diet, in the modulation of ceramid+induced apoptosis in U937 cells,
in comparison with other established antioxidants of nutritional interest (N-
a&y1 cyst&x?, d-a-tocopherol acetate and ascorbic acid). Our results show
that cafTeic acid efficiently inhibits ceramide-induced apoptc&. Other an-
tioxidants tested are totally in&e&w. Moreover, several tyrosine kinase
inhibitors also display an inhibitory effect. Preliminary results indicate that
the inhibition of tyrceine kinase activity is involved in the mechanism of
action of cal%ic acid. Our results underline the contribute of dietary phe-
nolic compounds in the modulation of cellular functions. The identifteation
of inhibitors of nutritional interest of ceramide-induced apoptosis may help
in providing novel strategies for therapeutic intervention and prevention of
many human disease.
r95 I
I I
The Potential Therapeuticii@Role of Lecithinized Superoxide
Dismutase in Cardiovascular Field
Minoru Ohno, Miiako Hangaishi, Hiroyoshi Nakajima, Nobukazau Ishiiaka,
Ryozo Nag& Cardiovascular Medicine, UniversityiiOofTokyo
Althoueh%avennine of suoeroxide was exoected as theraoeutic strateev for
cardiovascular field, many of basic and clinical study failed to show the t&a-
peutic efficiency of SOD, for example, to reperfusion injury. Lecithinized hu-
man recombinant Cu,Zn-SOD&SOD) has a much longer half-lie and much
higher affinity to the cell membrane than unmodiied (UM)-SOD. The re-
markable protective effect of lecithin&d SOD was demonstrated on several
kinds of cmdiovaacular disease models in our studies. In rat myocardial
reperfusionii&nodel( 45min coronary ligation and 12Omin reperfusion), in-
travenous administration of GSOD just before reperfusion strikingly inhib-
ited infarct size compared with UM-SOD, although bemodynamics were not
d&rent between the groups. ELISA assay revealed that vary high amount of
L-SOD was successfully delivered to ischemic myocardium. L-SOD also pro-
tected myocardium from prolonged ischemia (24 hours) without reperfusion
in viva. Furthermore, the number of tunnel positive cells and DNA fragmen-
tation was also significantly reduced in L-SODtreated group. GSOD was
found to affiliate to survive prolonged hypoxia up to 72 hours in rat cultured
cardiomyocyte. GSOD was e9lciently uptaken by cultured cardiomyocyte
and delivered into mitochondria ii@ Hypoxia-induced apoptosis, indicated
with DNA laddering and Hoechst 33342, was also significantly reduced in L-
SOD-treated cells, not in U-SOD-treated cells. GSOD also preserved BCL-2
under prolonged hypoxia. Thus,lecithinization of SOD has therapeutic po-
tential in the cardiovascular disease.
S38 OXYGEN
I 94
I
I
I
OXIDATIVE STRESS IN ALPHA-TOCOPHEROL TRANSFER
PROTEIN KNOCKOUT MOUSE
Noriko Nogucbi, Hongliao Shi, Hiroyuki Arai(*) and Etsuo Niki. RCAST
and Faculty of Pharm. Sci., University of Tokyo
Alpha-tocopherol transfer protein (alpha-TTP) specifically binds alpha-
tocopherol, by which it keeps alpha-tocopherol level high in plasma and tis-
sues and eliminates other tocopherol isomers. The mutant mice generated
with a targeted disruption of the alpha-TTP gene have very low levels of vita-
min E. Vitamin E has been well known as one of the ma% abundant lipophilic
antioxidant in viva. It has been also reported that alpha-tocopherol acts as
a prooxidant under certain circumstances. In the present study, we inves-
tigated whether more extensive c&dative stress is observed in alpha-TTP
knockout mice than wild mice. Alpha-TTP knockout mice and wild mice
were fed with normal diet. After overnight fast&on, AAPH (40 &kg) was
administered intra-peritoneally. PBS was administered as a control for both
types of mice. Mice were sacrificed by decapitation and blood was removed
by heart punctuation. The concentration of cholestryl ester (CEOOH) in
plasma of alpha-TTP knockout mice was significantly higher than that of
wild mice even without administration of AAPH. TBARS in liver, kidney,
and brain were measured. TBARS in liver of alpha-TTP knockout mice were
significantly higher than wild mice, while the prominent effects of AAPH
adminiitration were not observed in any of tissues of alpha-TTP knockout
mice. These results suggest that cuidative stress is increased in alpha-TTP
knockout mice spontaneously.
1 96 1
I I
The Peroxiredoxin Gene Family in Drosophila melanogaster
William C. Orr, Svetlana N. Radyuk,
Vladimir I. Klichko and Benedetta Spin&. Southern Methodist University
The peroxiredoxins comprise a family of antioxidant genes represented in
a wide variety of organisms. The proteins encoded by these genes function
as peroxidases when coupled to a sulfhydryl reducing system. In Drosophila
mehnogluter, wehave identified by sequence analysis five distinct perox-
iredadns, three of which have not been previously described. The coding
domains of all 5 were cloned and expressed in E. co&. Recombinant proteins
were purified and all five were shown to have peroxidase activity in the pres-
ence of dithiotbreltol. In addition, three of the family members were active
in the presence of thioredoxin and, consequently fall into the thioredaxin
peroxidase group. E&h of the five family members has a distinct expression
pattern based on developmental Northems and sub-cellular localization of
proteins expressed in tissue culture.
2000
I 97 I
I I
New amay systems to estimate the effects of food extracts on the
expression of antioxidant enzymes
Monet&a Ozekil, Tomoyuki Tan&al, Yamjim Morimitsu2,
Enji Nakatanit, yasue Kiiozaki3, Hibi Hii&, and Sbinya Toyokunil. 1
Department ofPathology and Biology of Dii, Graduate School of
Medicine, Kyoto University. 2 Department of Nutrition and Food Science,
Faculty of Human life and Environmental Science, Ochanomizu University.
3 Department of Food and Nutrition, Faculty of Human Life Science, Osaka
City University.
Oxidative stress has a great impact on tbe living organisms. A variety of
diseases have been associated with c&dative stress. Food is ao important
factor for modulating oxidative stress in that molecules with antioxidative
effects are reportedly contained. We have developed an efficient system by
the we of luciferrae reporter assay for screeoing molecules from food extracts
for modulating expression of antioxidant enzymes. DNA fragment containing
promoter region (-2425 to +44) of manganese superaide dismutase, one of
the antioxidant enzymes, was prepared from rat liver genome by polymerase
chain reaction. Tbia was integrated iota pGL3B luciferase reporter assay vec-
tor (QIAGEN). pGL3B-MoSOD and pRL-TK, internal control vector, were
introduced by transient transfection into COS7 or RL34 cells and incubated
for 24 hr at 37 OC with 5% COa. Then food extracts were added to celh and
incubated for 24 hr. Luciferase activity of cell lysates were evaluated using
Dual-luciferase reporter assay system (Promega). Evaluation of dietary plant
extracts are now in progress.
I 99 I
Moderate ethanol consumption and the red wine polyphenol
quercetin increase MnSOD protein in the vasculature.
,“&:::k??!?&t$$tk!!!$& Pathology and UAB
Cker for l+ee Radical Biolonv.-. Univ. of Alabama h Bin&n.I
Birmingham, AL
Considerable epidemiological data indicate that moderate consumption
of red wine (l-2 glasses/day) decreases the incidence of cardiovascular dia-
ease. The precise me&a&m of cardiovsxular protection remains unclear,
althoogb there is accunmlatiig evidence that alcohol and red wine polyphe
nols mediate thii cardioprotection through increased biivailabiity of nitric
aide (NO). The increased NO could result from (1) induction of NO syn-
thawa (NOS); (2) decreasing the interaction of NO with super&de through
induction of superoxide dismutases (SOD); or (3) a combiition. We previ-
ously demonstrated that alcohol and the red wine polyphenol quercetin in-
crease eNOS protein in the vasculature. The current studies tested whether
consumption of moderate alcobol or quercetin could induce the mitocbondrii
form of the supwxidwaxwenging enzyme, MnSOD protein in the vwcula-
tore. Rats consumed alcohol in the dri&ng water (7.5 percent) for 8 wks
or were gavaged daily with quercetin (0.033 mg/kg) for 3 nks. The expres-
sion of MnSOD protein in the thoracic aorta was aseaed by Western blot
analysis. Moderate alcohol and quercetin increased MnSOD protein 5.5 and
a&fold, respectively. These data indicate that the induction of MnSOD and
a diminiied reaction of NO with superoxide may increase NO bioavailability
and play a role in alcohol-induced cardiovascular protection.
OXYGEN
I 98 I
I I
Rapid kinetic analysin for determining structure-antioxidant
activity relationships of &won&la
Anantb SekherPamala, CatherineA. Rice-Evans.Centre for Age Related
Diseases, GKT School of Biomedical Sciences, King’s College London,
London-SE1 SILT, UK
Scavenging of the stable ABTS radical cation using fast reaction kinetics has
been applied to screen for the antioxidant activity oftlavonoids. The reaction
is carried out in a stop&w kinetic equipment coupled to a diodearray spec-
trophotometer. Antioxidant activity is expressed in tenus of stoichiometric
value c&olated on the basis of extent of ABTS radical cation scavenged.
Scavenging of ABTS radical cation by phenolic compoon& follows two dis-
tinct modes of reaction. Monophenolicn follow a rapid reaction complete
witbin 0.1 sec. The mechanism of action for these compounds is possibly via
the donation of a single electron to ABTS radical cation resulting in tbe for-
mation of phenoxyl radical. Compounds containing a c&echo1 group follow
a fast initial phase in 0.1 set with a slow secondary phase in the sulxequent
2.9 sec. The mechaism of actionfor catechola is possibly via the donation
of two electrons resulting in the formation of a quinone. The hierarchy of
antioxidant activity is trihydraxy compounds > catecbola > hindered phenols
(dimethoxy compounds) > hindered phenols (monomethoxy compounds) >
monophenolics. Among the dierent classes of the a&oxidants tested it
was found that the position and number of hydroxyl groupa play a key role
in the antioxidant activity of the compounds and the extent of scavenging
during the rapid initial phase of the reaction is a sensitive indicator of the
antioxidant activity.
I 100 I
A new role for oxidized fatty acids: bile acid mimic?
Meera Penma Nadya Khan and Sampath Parthasarathy. Emory
university ’
F&cent studies haveshownthat dietary axidized fatty acids (Ox-FFA) may
contribute to atherosclerosis, by enhancbxg the oxidation of lipoproteins such
as LDL and VLDL. We pmpoee an additional role for Ox-FFA in the patho-
genesis of atherceclerosis. We tented the hypothesisthat oxidizedlinoleic
acid (0x-18:2) increaea the micek&ation/uptake of cbole~terolcompared
to unoxidizedlinoleicacid (Unox-l&2) by mimickingbile acids due to its
ampiphilicity.In thisstudywe showthat Ox-l&2 promotedthe formationof
[4”cjcho1estercdmicelleaby 7 fold comparedto Unarm-l&2.Fkrthermore,
the uptakeof [414cJcholesterol was 50% gwter in Caco-2 c& and everted
rat intestinalsacsin the presenceof 0x-18:2 comparedto Unax-l&2. These
resultsshowthat 0x-18:2, by enhancingmieellarization of cholesterd might
increase dietarycholesterol absorption. In concltin, Ox-FFA couldenhance
athemsclerosia not only by increaing the oxidationpotentialof lipoproteins
but also by inaeadng dmleaterolabsorption.
2000 s39
GLUTATHIONE AND GLUTATHIONE ETHYL ESTER
SUPPLEMENTATION ATTENUATES GLUTATHIONE
LOSSES IN HEART, LUNG AND DIAPHRAGM AND
PROLONGS ENDURANCE TIME
Tracey Phillips, Li Li Ji*, and Christiaan Leeuwenburgh. Aging
Biochemistry Laboratory, Center for Exercise Science, Univ. of Florida,
*Departm& of Kinesioiogy Univ. of Wisconsin.
The effects of glutathione (GSH) and GSH ethyl ester (GSHE) supplemen-
tation on GSH states in the lung, heart, and costal diaphragm muscles were
investigated at test and after prolonged swim exercise. Male Swiss-Webster
mice (2 mo) were fasted for 24 h and then divided into 3 groups and injected
with either saline, GSH, or GSHE (6 mmol/kg body wt) 1 hour before ex-
ercise. Half of each group swam to exhaustion prior to kill; the other half
was timematched and killed at rest. Glutatbione levels of the control, GSH,
and GSHE supplemented animals were very similar in the lung, heart, and
diaphragm at rest. After the exhaustive exercise GSH content of the lung,
heart and diaphragm decreased significantly in all groups to similar levels.
In spite of this, GSH and GSHE supplemented animals swam almost 2 hours
longer than the control animals. Taking into consideration the longer swim-
ming time, GSH or GSHE supplementation attenuated the losses of GSH
in the diaphragm, heart, and lung. The significantly enhanced endurance
performance with GSH and GSHE may be due to au increased GSH antioxi-
dant reserve or sparing of liver and muscle glycogen by increasing amino acid
availability.
HAIR SILICON IN DEGENERATIVE ARTHRITIS
H.Povoa, C.C.Andrade, C.Santos and L.G.Povoa CLINICE - Centro de
Medicina Avanada
After 40 years it is very common to find degenerative arthritis (osteoarthri-
tie.) in human body. Recent papers demonstrate that silicon is a mineral that
is involved in collagen metabolism, 88 well as in synthesis of glycceaminogly-
cana (mainly of heparan sulfate). Due to this fact and also considering lack
of data in world scientiic literature on this subject, we thought of interest to
study hair silicon in patients suffering from osteoarthritis. They were divided
into 2 (two) groups: controls (39 cases) and patients suffering from thii con-
dition (43 cases). These two groups were in a similar range of age (between
40 and 60 years). The results were the following: Controls (39): 45,96f9,34
(hair tilicon=ppm)* ; 4,5194,3 (range). Patients (43): 19,43*3,3 (hair sil-
icon=ppm)* ; 3,2-106,O (range). *Mean f Standard Deviation of mean In
patients, hair silicon has a stat&ally signi6caut decrease compared with con-
trols: t=2,7 ; p< 0,Ol.
S40 OXYGEN
FREE RADICALS IN HUMAN FAECES (BRIEF NOTE)
H.Povoa, L.Ayer, C.C.Andrade, C.Santos, J.S.Roque and A.F.Carneiro.
CLINICEmro de Medicina Avanada
Babbs found enormous concentrations of l+ee Radicals (FR) in farces of
rats. We could previously confirm the same finding in human faeces. As
vegetarians ingest a greater amount of antioxidants and a smaller amount of
fat, this may cause a lower level of iron in intestine, decreasing the amount
of Hydroxyl Radical (HO*) in farces. Due to this fact, we thought of interest
to study effect of a similar diet during 7 days in FR content in faeces of
adults. 15 individuals were submitted to a diet without animal proteins
and a large amount of raw fiber (fruit and vegetable). They were interned
during one week and did not ingest also milk derivatives. The diet was
hypocaloric and the fat was only vegetal and monounsaturated. FR were
determined according to the technique of Babbs and tehe results were the
following: Before diet (15 cases): FR= 16,4f5,4 nM/g faxes (range= 4,9
- 24,7). After diet (15 cases): FR= 12,9 f4,2 nM/g faeces (range= 5,4 -
20,7). According to a non parametric test (Wilcoxon), we found a statistically
significant decrease: p<O,O22.
HAIR CHROMIUM AND VANADIUM IN DIABETIC
PATIENTS
H.Pwoa, C.C.Andrade, C.Sa0tc.s and L.G.Pwoa. CLINICE - Centro de
Medicina Avanada
Many minerals are involved in carbohydrate metabolism: zinc, manganese,
selenium and magnesium have relevant functions in glucose metabolism. Re-
cently, chromium and vanadium have been considered to he fundamental in
metaboliim of insulin, specially in cases of diabetes mellitus type II. They
have a powerful insulinomlmetic action. Due to these facts, we thought of
interest to study content of cbromhun and vanadium in hair collected from
scalp. 146 persons (men or women) Were studied: 73 controls and 73 dia-
betic patients (type II). The ages were between 30 and 55 years. The results
were the following: Diabetic (73 cases): Chromium* (ppm)= 0,264 f 0,043;
Vanadium* (ppm)= 0,121 f 0,011. Non diabetic (73 cases): Chromium*
(ppm)= 0,428 f 0,024; Vauadium* (ppm)= 0,208 f 0,0144. * Mean& Stan-
dard Deviation of mean. Deaeasen of these minerals were statistically sig-
nificant: Chromium: t-3,41 ; pcO,Ol and Vanadium: t=4,9 ; p<O,Ol. It
was also observed a statistically positive correlation between hair chromium
and vanadium. Chromium/Vanadium: diabetic => r=+ 0,289; ~~0.05 Non
diabetic=> r=+ 0,591; p<O,Ol.
2 0 0 0
 I105 1 t 106 1
ACTIVATION OF COX-2 AND iNOS IS MEDlATED BY Biomarkers for bioavailability and metabolism of c&&c acid
derivatives in humans
NF-KB DUBING SELENIUM DEFICIENCY. C. C. Reddy,
Andreas R. Rechner, Jeremy P. E. Spencer, Catherine A. Rice Evans.
F. Zamamiri-Davis, Y. Lu, J. Thompson. S.K. Prabhu, J. Stewart Centre of Age-Related Diseases, GKT School of Biomedical Sciences, Kings
and L.M. Sordillo, Center for Molecular Toxicology and College London, London SE1 9RT
Carcinogenesis. Department of Veterinary Science, The The purpose of this investigation was to obtain biomarkers for the bioavail-
Pennsylvania State University, University Park, PA 16802. ability and metabolism of hydroxycinnamates. Coffee consists mainly of
chlorogeoic acids and was used as a source rich in cai%ic acid derivatives.
Following the pharmacokinetics of urinary excretion no caffeic acid conju-
Cellular oxidant-antioxidant imbalance is a potential gates were detected but ferulic, isoferulic and vanillic acid were identified in
consequence of inadequate selenium (Se) nutrition. The present urine after beta-glucuronidase treatment. These are specific biomarkers for
study is based on the premise that, in RAW264.7 cells, oxidant the bioavailability and metabolism of the caf%ic acid derivatives. lsoferulic
acid is the more specific marker for caffeic acid derivatives then ferulic acid
stress resulting from Se deficiency mediates its effects on
since it is not a dietary component. Both showed a highly significant increase
prointlammatory genes through redox-sensitive transcription in the excreted amounts post-supplementation relative to the pre-study day
factor kappa B (NF-KB). The levels of intracellular reactive and a strong correlation between the excreted amount in the single eliiina-
oxygen species (ROS) were markedly elevated during Se tion samples and the time points of consumption of the coffee which indicates
relative fast absorption and metabolism. As a non-specific biomarker for gen-
deficiency and it was further exacerbated by LPS stimulation,
eral intake of phenolic and benzoic compounds hippuric acid was identified
validating our oxidant stress model. Compared to Se- while 3-hydroxyhippuric acid was found as a more specific biomarker for
supplemented cells, the expression of COX-2 and iNOS in Se- bioavailability and metabolism of thydroxylated phenolic compounds. The
deficient cells was increased significantly during LPS origin of the precursors of both compounds is most likely the large intesti-
stimulation as judged by RT-PCR and protein immunoblot nal microllora. Glycination to hippuric acids may occur in the liver. The
amounts of both hippuric acids increased significantly for every volunteer on
analysis as well as enzymatic activities. This increased COX-2
the study day with excretion maxima appearing more than 12 hours after
and iNOS expression in Se-deficient cells paralleled with an the first coffee consumption.
increase in the translocation of NF-tcB into nucleus during LPS
stimulation. We propose that the transcriptional activation of
these two proinflammatory genes is mediated by the way of NF-
KB activation during Se deficiency. Molecular aspects
underlying the interplay of various complex kinase pathways
involved in NF-xB activation during Se deficiency will be
presented. (Supported by PHS grants DK 49122 and HL 06347)

I 107 I 11081
I 1

Stability of Antimicrobial Activity against Streptococcus mutans
of Cashew Nut Shell Liquid (CNSL) during Industrialised
Heatine Process
IRico. < 1Gaitao. SY. 2Medina. R, 1% ral R and 3Bra TM
1Deoartment of Chemistrv and 20donto -?-$ ow ac
+A&tv. National
de dolombia, and 3Depa&ent of Humao”Nutrition,‘The Ohio State
University.
The liquid in the cashew nut shell (CNSL) has been reported to have aoti-
tumor, antimicrobial, antiacne and molluscacidal effects. Thii study assessed
if temperatures used to process cashew nuts at&t the antibacterial activity
of CNSL (Criollo Llaoero variety) against S. mutans, one of the bacteria re-
sponsible for tooth decay. The crude CNSL was fractionated by HPLC. The
antimicrobial activity (MIC and MBC) of individual fraction was aaseaed by
the serial dilution method and compared to that of the crude CNSL. Four
most active compounds were identified in the major fractions. The &xt of
3 heating temperatures, i.e. 96C (10 min), 15OC (5 tin) sod 2ODC (2 min)
of crude CNSL on antibacterial activity was asses&. Heating at 96C did
not a&ct the antibacterial activity against S. mutans (MIC and MBC are at
B.OOug/mL), and 15OC diminished the bactericide capacity but did not affea
the bacteriostatic capacity (MIC S.OOug/mL, MBC lO.OOug/mL). Heating at
2OOC,the temperature meet frequently wed in industrialized nut processing,
dimiied both MIC and MBC (> lOug/mL and >lOug/mL). The results
showed that industrial process of cashew nuts (fryii nuts at ZOOC)destroys
the antibacterial activity of CNSL. We rscommsnd that the cashew nuts
should be processed around 96C in order to pwerve the antibacterial ac-
tivity. Other biological activities of compounds in CNSL, byproduct of food
industry, should be measured.
CAT’S CLAW (Uncaria tomentosa) INHIBITS TNF alpha
PRODUCTION AND SCAVENGES FREE RADICALS: ROLE
IN CYTOPROTECTION
Manuel Saodoval, Randi M. Charbonnet, Natly N. Okuhama,
Jarod Roberts, Zdenka Krenova, Anne Marie Trentacosti*,
and Mark J. S. Miller. Albany Medical College, center for cardiovascular
Sciences, Albaoy, NY 12208, ‘Rainforest Phytoceuticals, Delmar, NY
12054.
Cat’s claw is a medicinal plant from the Amazon that is widely used for
inflammatory disorders and previously described as an inhibitor of NF-LB.
Cat’s claw (CC) wva8 prepared as a deco&on of micropulverized (CCmp)
bark with and without concentration by freeze-drying (CCfd). Murine
macrophagen (RAW 264.7) were used in cytotoxicity sssays in response to
the free radical l,ldiphenyl-2picrilhydrazyl (DPPH, 0.3 PM) and ultraviolet
liiht (UV). TNFa production was induced by lipopolysacdmride (LPS 0.5
pg/ml). Cat’s claw was an effective scavenger of DPPH; the EC50 value for
CCfd was leas than CCmp (18 vs. 150 pg/ml, P < 0.05). CCfd (10 fig/ml)
was fully protective against DPPH and UV irradiation induced cytotoxicity.
LPS increased TNFa levels from 3 to 97 ng/ml. Cat’s claw suppressed TNF
Q production by approxima tiy 6585% (P < 0.01) but at concentrations
considerably lower than its aotioxidant activity: CCfd EC50 = 1.2 ng/nd,
CCmp EC50 = 28 r&d. In con&sion, CC is an effective a&oxidant and a
potent inhibitor of TNFa production. The primary mechanism for cat’s claw
anti-in6ammatory actions appears to be immunomodulation via suppression
of TNFa synthesis.

OXYGEN 2 0 0 0
I 109 I
I I
Red Wine Vs Gram Juice: Antioxidant or Prooxidant
N. Santanam, S. Rick, A.A. Murphy and S. Parthasarathy. Dept.
Gvnecoloev & Obstetrics, Emorv Univemitv. Atlanta. GA
A cup z red wine a day is suggested to be beneficial for coronary artery
dkease (CAD). The polyphenolic (“antioxidant”) component of red wine is
responsible for its beneficial action. However, red wine also contains alcohol.
Both polyphenols and alcohol can affect lipoprotein metabolism. Grape juice
has similar or increased content of polyphenols but no alcohol content. In
this study, we asked the question if increased polyphenol content of the grape
juice is beneficial over lower polyphenol and alcohol content in red wine.
Baseline and final fasting blood was drawn from healthy volunteem (n=20)
who consumed either 150 ml of red wine or grape juice per day for 36 days.
There was no change in the lipid profile between the baseline and final blood
draw in both the groups. However, there was an increase in the oxidative
stress markers (1.5 fold higher #CAM, 2 fold higher MPO and 2 fold higher
auto antibodies to oxidatively modified proteins) after one month of grape
juice consumption compared to its initial baseline values. No such change
was observed after one month of red wine consumption. The results were
consistent with our recent observation that i) phenolic compounds can be
converted to prooxidants by peroxidaees and ii) increamd oxidative stress in
the plasma may be beneficial because it increases the antioxidant defense in
the artery, thereby preventing CAD. We conclude that i) increased oxidatiw
stress olxwrved after grape juice (hi polyphenol content) consumption is
beneficial and ii) the alcohol content of red wine may counteract the beneficial
effect of increased polyphenols.
I 111 I
Bioavailable forms of flavanols protect flbroblaats tkoomoxidative
stress-induced apoptoeis
Jeremy P. E. Spencer, If. Schroeter, B. Shenoy, G. Kuhnle, ES. Debnam,
and C. Riee_hrans C-e&e for Age-Related Dii,
Cal Sciences, ~&g’s College London, London, SE1
9RT. Royal Fkee and Uniwraity College Medical School, London, NW3
2PF. University of Leipzig, 94103, Germany
There is considerable interest in the bioavailabifity of polyphenoln and
their biiivity in viva. We have studied the abwrption and metabolism of
catechin and epicatechin in the small intestine and the ability of the result-
ing metabolitm to protect c& against ROS-induced apoptc&. Pemtsion
of Isolatedrat small intestinewith the flavanoh~ resulted in extensive glu-
curonidation and O-methyiation of the compounds during transfer - the
entemcytes of the jejunum but to a lamer extent acmm the ileum. O-Methyl
and 0-mathylghr~ catechiw were the predominate met&olitea
detected in the ti fluid suggwting these speciea LYI
the most bioavailable
forms. The total percentage of &wan01 transfer acrom the jejunum wae lower
than - the ikum which was primarily due to a pea&r transfer of un-
metabohsed Savanol. Pm-treatment of human dermal fibroblasm with 3’-O-
methyl- and 4’-O-mathyl-epicatecMn resulted in prokction against apoptotic
cell death induced by hydrogan per&de. However, the level of inhibition
evoked by the O-methylated metabohtea vm9 not significantly d&rent from
that emlmd by epicateddn itself. Them data mrggeat that although the mea-
sured Hdonatmg potential of them compounda are very difkrent (TEAC),
the ability to protect cells against caddative stress in viva is not dependent
on the redox potential alone.
s42 OXYGEN
I 110 I
Antioxidants and protein carboqyls in bronchoalveolar Image
lkdd of children without wheesez normal data
PS Fit&$, R Taylora,
menta of 1CIimc’ al Biochemistry and
2Child Health, Queen’s University of Belfast, UK
Antioxidant-oxidant imbalan& in bronchoalveolar lavage Buid (BAL) are
thought to contribute to oxidative stress in respiratory disease. However,
normal reference ranges for BAL antioxidanta and oxidized proteins in chil-
dren are not known. In thii study, we recruited 124 children (age 1.6612.6
y) attending for elective surgery for a non-inflammatory condition, 83 were
non-asthmatic, non-atopic (N) and 41 were non-asthmatic, atopic (NA). A
non-bronchoacopic lavage was performed and -bate, urate, a-tocopherol
and protein carbonyl concentrations were determined in BAL fluid. The 95%
reference range was 0.11261.897 pmol/l Ior ascorbate, 0.14962.163 pmol/l for
urate, 0.69360966 qol/l for a-tocopherol and 0.28964.529 nmol/mg for pro
kin carbonyls. Age, gender and exposure to tobacco smoke did not affect
concentrations of antioxidants or protein carbonyls. However, in multiple lin-
ear regression analyses, the type of home heating (glass fronted &es) reduced
concentrations of ascorbate and urate in BAL fluid (&coefficient -bate:
0.445, p=O.631, uratez 0.114, p=O.691). Them wan no signilicant difkence
between both groupa in BAL fluid concentrations of -bate, urate or pro
tein carbonyls. The a-tocopherol concentration ~88 significantly increased in
the NA group (p=O.637). Only -bate concentrations of .wum and BAL
fluid were significantly correlated (r=0.297, p=O.OlB, n=63), which may of-
fer an explanation why supplementing the diet with vitamin C can improve
asthma symptoms.
Contrasting action of ascorbic acid against oxklative stress
Bru Take&& Kanji, Kohji Aoyama, Baohui Xq,
Kazufumi Ha&igu&i, wKomatsu, NaoM K&&,
Kunitomo Watanabe*. Kanehisa Morimoto+. Department of hygiene,
Kagwhima University Faculty of Medicine 6-35-l Sakuragaoka, Kagoshima
899-8529, Japan *Institute of Anaerobic Bacteriology, Gii University
School of Medicine, Gii 5698795, Japan +Department of Social and
Environmental Medicine, Course of Social Medicine, O&a University
Graduate School of Medicine, 22 Yamadwka, Suita, O&a 5656871, Japan
We have investigated the effects of -bit acid on coddative damage in-
duced by 02 exposure in P. mdaninogenica, a strict anamobe. We deter-
mined cell viabiity and E-hydroxydecayguancaine(8OHdG), typical of &da
tiw DNA damage. A brief exposure to 02 w~ylhighly toxic to the bacterium
and it decreased the cell viabiity to 0.05%. Ascorbic acid at acidic pH, but
not at neutral pH, markedly attenuated the toxicity and increased the cell
viabiity after 02 exposure to 59%. 02 exposure increased 80HdG in P.
mehminogenica. Aworbic acid at acidic pH enhanced the 80HdG increase,
whileit had no &ecta at neutralpfi. Acidic pH by itself, adjusted with HCl,
slightly decrezwd both mortality and 8OHdG levela induced by 02 exposure.
Catalme also decreamd both the mortality and the t3OHdG induction. These
6ndingn indicate that the effecta of -bit acid are pIi dependent, and that
-bit acid acts a,e both antioxidant and prooxidant. The findings also
suggest that P. malaninogenica is useful to evabmte the action of chemicals
against o&dative stress.
2 0 0 0
113
El
Rationale of Antioxidant-based Composite Therapy of Brain
Oxidative Stress by naditional Chinese Herb Medicine
Tetsuya Konishi, Haruyo Ichikawa, Wang. Niigata College of
Pharmacy
Traditional medicines such as Chinese herbal medicines have been used for
treating many lifestyle- and/or age-related diseases such as diabetes militias
and cancer, and obtained certain successes. Although the precise mechanism
of their action is not yet fully understand, the mixed formula plays synergis-
tic role in the action. It is also known that otidative stress is implicated in
pathogenesis of these complex disorders. Thus, the antioxidant-based com-
posite therapy must be a promised approach for the prevention and repair
of oxidative stress related disorders, and the antioxidative Chinese herbal
medicines would be a good model to examine the propriety of the strat-
egy. We studied here the antioxidant potential of two traditional formulas
of Chinese herbal medicines, Shengmai San and Qizhu Tang, in prevent-
ing cerebral oxidative damage after ischemia-reperfusion in rats and revealed
that both formulas effectively prevented TBARS formation and loss of Glu-
tathione peroxidase activity in the brain when were adiminstered before is-
chemic treatment. Further analysis of antioxidant potential of component
herbs and the mixed decoctions revealed the rationale of composite formula
of Chinese herbal medicine in terms of antioxidant potential. The limitation
will be shown for the use of in vitro antioxidant indications to evaluate the in
viva effectiveness of herbal composite in preventing oxidative stress-induced
brain injury.
I I
REDOX STATUS AND MODULATION OF STRESS PROTEIN
SYNTHESIS
Kenneth Watson, Graham Jones and Jun Peng. University of New
England, School of Biological Sciences, Human Biology, Armidale 2351,
Australia
There is a need to develop biomarkers that may act as monitors of cellular
deface aa influenced bv free radical attack and redox status of the cell.
In human lymphocytes,” oxidative stress imposed by AAPH [2,2’az&ii-(2-
amidinopropane)dihydrochloride]induced the synthesis of heme oxygenase 1
(HO-l), transcription factor NF-kappaB and heat shock proteins(hsp)h@O
and hsp27. Antioxidants such as N-acetylcysteine(NAC)modnlated protein
synthesis by down regulation of HO-l and NF-kappaB and up regulation of
hsp. In lymphocytes from individuals receiving antioxidant supplements and
subjected to a heat shock in the presence of AAPH, enhanced synthesis of
hsp105, hsp90, hsp70 and hsp40 by contrast with decreased synthesis of HO-
1 were noted. UVB stress imposed on HaCaT cells, a transformed human
keratinocyte cell line, induced the synthesis of NF-kappaB, hsp70 and HO-
1. The syntheses of these proteins were downregulated in the presence of
NAC. DNA microarrays were used to con&m these observations and allowed
a global analysis of the redox regulation of gene expression. It was concluded
that the synthesis of stress proteins was modulated by cellular redox status.
OXYGEN
I, 114 I I
Ascorbate Protects against Myeloperoxidase-Mediated Lipid and
Protein Oxidation in Low-Density Lipoprotein
Terry Tijerina, Anitra Carr and Balz tiei. Linus Pauling Institute, Oregon
State University, Corvallis OR 97331
Myeloperoxidase (MPO)-modified low-density lipoprotein (LDL) is impli-
cated in the pathogen&s of atherosclerosis. MPO generates a number of
oxidants, including hypochlorous acid (HOCL) from chloride ions (Cl-)and
nitrogen dioxide radicals from (NO*-). MPO-derived HOC1 oxidizes the prw
tein protion (apo B) of the LDL particle forming primarily N-chloramines,
which can subsequently break down to form N-centered radicals; the latter
may initiate lipid peroxidation in LDL. Nitrogen dioxide can nitrate ape B
tyrosine residues and directly initiate lipid peroxidation. Ascorbate may pro-
tect against LDL modification by scavenging MPO-derived oxidants and by
recycling vitamin E from its radical. An in-uitmsystem of LDL/MPO/H20z
containing physiological concentrations of either Cl-or NOZ-was found to
moderately increase levels of cholesteryl ester hydroperoxides in LDL with
a concomitant decrease in LDGassociated a-tocopherol, indicative of lipid
peroxidation. Apo B damage, as indicated by an up to 1.8-fold increase in
the relative ekxtrophoretic mobility of LDL, was also observed in the pres-
ence of Cl-, but not NO*-. Ascorbate (12.5 - 2OOpM) added to either of
the two systems (Cl- or NOz-) protected against lipid and protein oxidative
damage in LDL. Our data show that physiological concentrations of ascor-
bate are able to prevent MPO-mediated modiication of LDL by HOC1 or
reactive nitrogen species, thereby potentially decreasing macrophage up&e
and lowering the risk and severity of atherosclerosis.
OXIDATIVE DAMAGES IN DIGOXIN-TREATED HUMAN
CELLS
J. S. Wei. C. Y. Kao, Y. S. Chen, Y.T. Chow and. School of
Medical Technology and Graduate Institute of Basic Medicine, Chang Gung
University, Kweisan, Taoyuan, Taiwan.
We had previously demonstrated that digoxin caused apoptceis in exper-
imental cells. In thii studv. the oossible roles of oxidative darnaxe in cvto-
toxicity during digoxin tre&nent’ in several human hepatoma cell-l&s were
studied. Time course studies revealed significant increases in the production
of nitric oxide at 8 hours and hydrogen peroxide at 18 hours in digoxin treated
SK-Hep-1 cells. The addition of NOS inhibiton such aa GNAME (10T5M)
and aminoguaoidiie (IO-‘M) but not ‘I-nitroindazole (up to 10w3M), recov-
ered partial cell viability of digoxin-treated SK-Hep-1 cells. Also, alterations
in gene expression of eNOS isoform, but not iNOS, in SK-H-1 cells in
response to dioxin treatment were coniirrned by RT-PCR. Indeed, upon
digoxin addition, an increase of fluorescent intensity from cells loaded with
non-fluorescent dichloroRuorwcein diacetate allowed us to visualize “oxida-
tive stress” in living cells. The inactivation of endogenous cat&w in the
SK-Hepl(but not Hep G2cells), as well in membrane-bound alkaline phos-
phatase in 55 cells, were observed. Antioxidanta ameliorate the cytotoxicity
of digcxin in decreasing order of effectiveness as follows: ascorhate, rnela-
tonin, toeopherd, and &arotene. Furthermore, the addition of exogenous
cat&se in the growth medium increased the cell survival rate from digoxin
induced apoptosis. Taken together, we co&m the involvement of nitric ox-
ide and hydrogen per&de in digoxin-induced cytotcuicity and antioxidants
prevent its effect. (Supported by grant NSC 89-231~B-182-080 to JSW).
2 0 0 0 s43
Ascorbate Does not Act an a Prwoxidant in Iron-Overloaded
Human Plasma, even in the Presence of H202
Ben-Zhau Zhu, Jung Sub and Balz Ftei. Linus Pauliig Institute, Oregon
State University, Corvallis, OR 97331
Ascorbate (Asc) acts as an anticxidaut or a pro-oxidaut in vitro in the
absence or presence, respectively, of redox-active metal ions. We have pre-
viously shown that Cu-induced oxidation of human LDL and Fe-mediated
lipid peroxidation in human plasma are strongly inhibited, not enhanced, by
Asc. Nevertheless, it is not dear whether the same holds true when H202 is
simultaneously present (complete Udenfrieud system - H202/Asc/Fe), and
whether oxidative damage to biological macromolecules other than lipids, e.g.
proteins, is also inhibited. Therefore, in this study we used Fe-overloaded
human plasma to investigate whether Asc acts as an antioxidant or a pro-
oxidant in the presence of H202. Protein carhonyl and lipid hydropemxide
formation were used as two sensitive markers of protein aud lipid oxida-
tion, respectively. We found that iron (0.1-l mM) induced time and dose-
dependent protein carhonyl formation in human plasma, which was further
enhanced by addition of H202 (0.2 mM). However, protein carbonyl fonna-
tion induced hy iron (0.1 mM) or by Fe/H202 (0.2 u&f) was not enhanced by
Asc (0.2 mM). H202 also markedly enhanced lipid peroxidation induced hy
iron (0.05 - 0.1 mM). Depletion of endogenoua Asc by Asc oxidaae further en-
hanced Fe/H202-induced lipid hydroperccdde formation. Furthermore, ad-
dition of exogenouli Asc (0.2 mM) did not increase, but rather decreased
Fe/H2OZinduced lipid peroxidation. Our results demonstrate that physic+
logical concentrations of Asc do not act as a pro-oxidant in Feoverloaded
human plasma, even in the presence of H202.

s44 OXYGEN 2000