comprehensive list of all LPI scientific papers published in 2002 and
2003 can be found here. Summaries of some representative
papers appear below. LPI scientists are identified in boldface.
JS. Nitric oxide, peroxynitrite and ageing. In: Critical
Reviews of Oxidative Stress and Aging (Cutler RG and Rodriguez H,
eds), vol. 1, pp. 54-83, 2002.
In this book chapter,
Dr. Beckman discusses the biological role of peroxynitrite, which is a
relatively stable and very damaging oxidant formed from the reaction between
nitric oxide and the superoxide radical. Its formation can be attenuated
by the endogenous anti oxidant enzyme superoxide dismutase. In fruit flies,
the deletion of superoxide dismutase results in a 60% reduction in life
span, which is corrected when super-oxide dismutase production is increased.
Peroxynitrite serves an important function in the immune system, where
it is generated by inflammatory cells as an antimicrobial defense. Peroxynitrite
can damage proteins by nitrating the amino acid tyrosine, and nitration
of structural proteins abundant in cells can result in loss of normal
function. Since there are only about one million motor neurons in a human
and they are not regenerated, damage to proteins in these neurons over
time may be critical and result in some of the manifestations of aging.
For instance, mutations to superoxide dismutase, the enzyme that inhibits
the formation of peroxynitrite, are found in amyotrophic lateral sclerosis
(ALS) patients. The abnormal superoxide dismutase loses its affinity for
zinc and helps to generate, rather than suppress, the formation of peroxynitrite,
which then damages the motor neurons. Peroxynitrite is a double-edged
sword—small amounts generated by immune cells are beneficial, but
residual or inappropriately generated peroxynitrite can damage biological
molecules and vulnerable motor neurons, contributing to age-related decline.
TM, MOREAU R, SUH JH, and VISIOLI F. Mitochondrial decay in the
aging rat heart: evidence for improvement by dietary supplementation with
acetyl-L-carnitine and/or lipoic acid. Ann. N.Y. Acad. Sci.
959: 491-507, 2002.
This paper summarizes
a number of experiments with rats supplemented with acetyl-L-carnitine,
a non-protein amino acid, or R-alpha lipoic acid. Heart muscle
cells from old rats show both decreased oxygen consumption and increased
production of damaging oxidants, or free radicals. Supplementation of
old rats with acetyl-L-carnitine for one month had no effect
on vitamin C levels or oxidant levels in cardiac cells, but reversed mito
chondrial decay. Lipoic acid supplementation for two weeks increased vitamin
C levels in cardiac cells in both young and old rats and restored the
vitamin C status in cells from old rats to that of young, unsupplemented
rats. These results suggest that the combined use of acetyl-L-carnitine
and lipoic acid may help maintain myocardial function in aging mammals.
J, HEAD E, GHARIB AM, YUAN W, INGERSOLL RT, HAGEN TM,
COTMAN CW, and AMES BN. Memory loss in old rats is associated with brain
mitochondrial decay and RNA/DNA oxidation: partial reversal by feeding
acetyl-L-carnitine and/or R-alpha-lipoic acid. Proc.
Natl. Acad. Sci. USA 99: 2356-2361, 2002.
In this study, the
authors examined the effect of supplemental acetyl-L-carnitine
and/or R-alpha lipoic acid on memory function in old rats. They
found increased oxidative damage in the hippocampus, a region of the brain
associated with memory, in old rats, which may explain the decline in
memory function with increasing age. Using two tests of memory performance—the
Morris water maze and a time discrimination procedure—the investigators
observed improved memory function in old rats supplemented with either
acetyl-L-carnitine or lipoic acid. The greatest benefit was found
when both supplements were given together, which also resulted in the
reduction of oxidative damage in the hippocampus to levels seen in young
AJ, JOISHER N, and HAGEN TM. Age-related decline
of sodium-dependent ascorbic acid transport in isolated rat hepatocytes.
Arch. Biochem. Biophys. 410: 112-120, 2003.
The vitamin C concentration
in liver cells from old rats is about
70% lower than that in young rats. Two forms of a sodium-dependent vitamin
C transporter (SVCT) are found in cells—SVCT1 and SVCT2—that
allow the uptake of vitamin C from extracellular fluids into cells. The
authors found that the amount of SVCT1 in liver cells from old rats was
only 55% of the amount in young rats, whereas the amount of SVCT2 remained
unchanged with age. These results may help explain the decline in vitamin
C status associated with aging, as has been observed in elderly humans,
and suggest that higher plasma concentrations of vitamin C may be needed
to offset age-related changes in SVCT1 in the elderly in order to maintain
optimal vitamin C status.
and oxidative stress
M, BLOCK G, HUDES M, MORROW JD, NORKUS EP, TRABER MG,
CROSS CE, and PACKER L. Antioxidant supplementation decreases lipid peroxidation
biomarker F(2)-isoprostanes in plasma of smokers. Cancer Epidemiol.
Biomarkers Prevent. 11: 7-13, 2002.
It is well established
that smokers exhibit high levels of oxidative stress. The experiments
in this paper were designed to evaluate whether antioxidants can lower
oxidative stress in smokers. One hundred and twenty-eight male and female
smokers from 20 to 78 years old were enrolled in the study and given daily
either a placebo; 500 mg of vitamin C; or a combination of 500 mg of vitamin
C, about 800 mg of total vitamin E as mixed tocopherols and tocotrienols,
and 95 mg of lipoic acid. Plasma levels of F2-isoprostanes, markers of
oxidative damage to lipids, were measured at baseline and after two months.
In smokers with a body mass index (BMI) above the median, vitamin C decreased
levels of F2-isoprostanes, but, surprisingly, the antioxidant combination
did not. F2-isoprostanes were strongly associated with increasing BMI.
No effect of antioxidants was observed in smokers with a BMI less than
the median, suggesting that vitamin C can reduce oxidative stress only
in smokers who are overweight.
TM, LIU J, LYKKESFELDT J, WEHR CM, INGERSOLL RT, VINARSKY V,
BARTHOLOMEW JC, and AMES BN. Feeding acetyl-L-carnitine and lipoic
acid to old rats significantly improves metabolic function while decreasing
oxidative stress. Proc. Natl. Acad. Sci. USA 99:
The authors fed acetyl-L-carnitine
and/or R-alpha lipoic acid to young and old rats and measured
several parameters of metabolic function and oxidative stress. Young rats
and old rats, which showed a three-fold decline in their activity level,
were fed acetyl-L-carnitine and lipoic acid for one month. Both
populations exhibited increased activity, although the increase was greatest
in old rats. Oxygen consumption in liver cells from old supplemented rats
also increased, reflecting improved metabolic function. Acetyl-L-carnitine
plus lipoic acid also increased vitamin C levels in liver cells from both
young and old rats. While acetyl-L-carnitine alone increased
malondialdehyde (MDA), a marker of lipid oxidative damage, in liver cells
from young and old rats, lipoic acid alone, as well as the combination
of acetyl-L-carnitine and lipoic acid, lowered MDA levels in
the livers of young and old rats.
SW, TERASAWA Y, FARESE RV Jr, and TRABER MG.
Incorporation of deuterated RRR- or all-rac-alpha-tocopherol
in plasma and tissues of alpha-tocopherol transfer protein-null mice.
Am. J. Clin. Nutr. 75: 555-560, 2002.
The vitamin E family
has eight members—four tocopherols and four tocotrienols. The recommended
dietary allowance (RDA) is based only on RRR-alpha-tocopherol,
also known as natural or d-alpha-tocopherol, because this is the only
form that the body retains. A protein synthesized in the liver, the alpha-tocopherol
transfer protein (alpha-TTP), preferentially recognizes RRR-alpha-tocopherol,
which it transfers into lipoproteins that circulate in the plasma. In
the experiments reported in this paper, the investigators found that mice
without the gene that codes for alpha-TTP accumulate alpha-tocopherol
in the liver, resulting in the depletion of alpha-tocopherol in peripheral
tissues. The results also demonstrate that the bioavailability of natural
alpha-tocopherol is twice that of synthetic vitamin E, or d,l-alpha-tocopherol.
RA, HUMMER KE, FINN CE, FREI B, and WROLSTAD RE. Anthocyanins,
phenolics, and antioxidant capacity in diverse small fruits: Vaccinium,
Rubus, and Ribes. J. Agric. Food Chem. 50:
Many fruits contain
a variety of antioxidants like vitamin C and other substances called phytochemicals,
some of which, known as flavonoids, also function as antioxidants. In
this investigation, the authors measured the antioxidant capacity and
anthocyanin (a dark pigment) content of various cultivars of blueberries,
blackberries, and black currants. The antioxidant capacity was found to
be related more to total flavonoid content than to anthocyanin content.
Blueberries exhibited the highest anthocyanin content, followed by black
currants and blackberries. While one test of antioxidant capacity found
blueberries to have the highest value, a different test found higher values
in blackberries, although there was a lot of variability among the specific
cultivars. These results show that these fruits are an important source
of antioxidants and that selective breeding of particular cultivars may
enhance these properties.
S and TRABER M. Gamma-Tocopherol, the new vitamin E?
Am. J. Clin. Nutr. 77: 530-531, 2003.
In this editorial,
the authors call for urgent clinical studies to determine the respective
protective functions of the different tocopherols in platelet aggregation,
inflammation, and heart disease. Although the body preferentially retains
the d-alpha-tocopherol form of vitamin E, gamma-tocopherol has been found
in some experiments to exhibit greater anti-inflammatory and antioxidant
properties. Unlike alpha-tocopherol, gamma-tocopherol scavenges reactive
nitrogen species, such as peroxynitrite. Alpha-tocopherol has a more pronounced
effect than gamma-tocopherol on the antiatherogenic behavior of vascular
smooth muscle cells. Dose and duration of use, as well as monitoring oxidative
stress, inflammation, plasma levels of vitamin E, and relevant clinical
endpoints, are important factors to consider when designing effective
clinical studies to assess the relative merits and functions of the different
J, ZHU B-Z, and FREI B. Ascorbate does not act
as a pro-oxidant towards lipids and proteins in human plasma exposed to
redox-active transition metal ions and hydrogen peroxide. Free Radic.
Biol. Med. 34: 1306-1314, 2003.
The potential pro-oxidant
function of vitamin C in vivo has been controversial. The experiments
reported in this paper demonstrate that vitamin C functions as an antioxidant
even in circumstances in which it might be expected to be pro-oxidant.
The authors depleted human plasma of vitamin C or added vitamin C to other
samples. Iron or copper salts were then added to the plasma samples. In
classical chemical experiments, vitamin C becomes pro-oxidant when treated
with these transition metal ions in solution. However, in this study,
vitamin C strongly protected against lipid oxidation in plasma, as measured
by hydro-peroxide and malondialdehyde formation. Lipid oxidation was promoted
only in the plasma that had been depleted of vitamin C. Vitamin C also
did not increase protein oxidation, as measured by protein carbonyl formation.
AA, CUSTER LJ, COONEY RV, TANAKA Y, XU M, and DASHWOOD
RH. Inhibition of colonic aberrant crypt formation by the dietary
flavonoids (+)-catechin and hesperidin. Adv. Exp. Med. Biol.
505: 123-133, 2002.
are mutagens formed in protein-rich food, such as meat, by high-temperature
cooking. When rats are fed these cooked-meat mutagens, they develop precancerous
lesions in the intestine called aberrant crypts, which are presumed to
be reliable biomarkers for human colon cancer. In this study, the investigators
tested the effects of hesperidin, which is a flavonoid from citrus fruit,
and catechin, a flavonoid from tea, on the inhibition of colonic aberrant
crypts in rats treated with cooked-meat mutagens. Catechin and hesperidin
inhibited the development of aberrant crypts, suggesting that they may
be effective chemopreventive substances against colon cancer.
SK, WILLIAMS DE, YUEH M-F, MARTIN SR, HINES RN, RAUCY
JL, DOLPHIN CT, SHEPHARD EA, and PHILLIPS IR. Genetic polymorphisms of
flavin-containing monooxygenase (FMO). Drug Metab. Rev. 34:
monooxygenase (FMO) system comprises a number of genes that code for enzymes
that metabolize drugs and other foreign compounds (xenobiotics) in mammals.
Mutations in these genes are associated with various diseases, including
trimethylaminuria, in which the affected patient excretes trimethylamine,
a substance with a potent fishy odor, in urine and sweat. Specific forms
of FMO are found in different tissues, and the form found in the lung
is called FMO2. FMOs exist in different populations in a number of closely
related but different forms called polymorphisms. This study reports the
finding that one variant form, or polymorphism, of FMO2 is present in
a significant percentage of African Americans and Hispanics, which may
affect drug metabolism or xenobiotic toxicity in the lung and has implications
for determining dosage in drug therapy.
G, ORNER GA, XU M, IZQUIERDO-PULIDO M, and DASHWOOD RH.
Inhibition by white tea of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced
colonic aberrant crypts in the F344 rat. Nutr. Cancer 41:
This paper reports
experiments showing that white tea—the least processed type of tea—strongly
inhibited the formation of colonic aberrant crypts in rats treated with
a cooked-meat mutagen known as PhIP. White tea is prepared by steaming
and drying the buds and young leaves but without withering. It has higher
levels of polyphenols called catechins than green or black tea. Tea was
prepared as it is for humans and given to rats for 8 weeks, along with
five exposures to PhIP, resulting in a 75% decrease in the number of aberrant
crypts compared to rats given water instead of tea. Further studies revealed
that white tea altered cytochrome P450 enzymes that metabolize drugs and
xenobiotics, leading to enhanced metabolism of PhIP and increased excretion
of its metabolites in the urine. Additionally, in another test of mutagenicity,
nine substances extracted from white tea and then combined were less effective
than whole tea in inhibiting mutagenicity.
CA, XU M, ORNER GA, DÍAZ GD, LI Q, DASHWOOD W-M, BAILEY GS,
and DASHWOOD RH. Promotion versus suppression of rat
colon carcinogenesis by chlorophyllin and chlorophyll: modulation of apoptosis,
cell proliferation, and beta-catenin/Tcf signaling. Mutat. Res.
523-524: 217-223, 2003.
This paper reports
that chlorophyll and chlorophyllin, a derivative of chlorophyll, had differential
effects on the development of aberrant crypts in the rat colon induced
by carcinogens, including the cooked-meat mutagen IQ and the chemical
carcinogen 1,2-dimethylhydrazine (DMH). Chlorophyll or chlorophyllin were
fed to rats in their diet or given in drinking water after the rats had
been exposed to, or initiated with, either of the carcinogens. Post-initiation
treatment with low-dose chlorophyllin promoted the formation of aberrant
crypts by DMH only, whereas high-dose treatment was inhibitory against
IQ-induced aberrant crypts. In another experiment, an intermediate dose
of natural chlorophyll suppressed aberrant crypts induced by exposure
to IQ or a metabolite of DMH. Further experiments are planned to understand
the mechanisms responsible for these effects more thoroughly.
RH and XU M. The disposition and metabolism
of 2-amino-3-methylimidazo[4,5-f]quinoline in the F344 rat at
high versus low doses of indole-3-carbinol. Food Chem. Toxicol.
41: 1185-1192, 2003.
The authors examined
the effect of a range of doses of indole-3-carbinol (I3C), a substance
found in cruciferous vegetables that has anticancer properties, on the
metabolism of a cooked-meat mutagen known as IQ in rats. At very low doses
of I3C, the metabolism of IQ by liver enzymes was unfavorably altered,
whereas larger doses induced the enzymes that detoxify IQ, leading to
decreased DNA damage. However, the specific liver enzyme induced by I3C
is also known to activate certain carcinogens, such as polycyclic aromatic
hydrocarbons that are found in tobacco smoke and grilled meats. Therefore,
I3C may not only inhibit cancer, but depending on the dosage and timing,
may also enhance certain types of cancer.
WILLIAMS DE, BAILEY GS, REDDY
A, HENDRICKS JD, OGANESIAN A, ORNER GA, PEREIRA CB, and
SWENBERG JA. The rainbow trout (Oncorhynchus mykiss) tumor model:
recent applications in low-dose exposures to tumor initiators and promoters.
Toxicol. Pathol. 31 (Suppl): 58-61, 2003.
The authors of this
study have developed trout as a model for human carcinogenesis. The use
of trout has a number of advantages: a vast number of small, relatively
inexpensive animals can be used for high statistical power, trout
are sensitive to human carcinogens and metabolize them similarly to humans,
and they have a low incidence of spontaneous tumors. These attributes
allow dose-response studies in trout that reliably evaluate low doses
of potential carcinogens. In one experiment, trout were exposed to the liver carcinogen
aflatoxin B1, produced by molds on grains, and
then fed diets containing increasing amounts of indole-3-carbinol (I3C),
an anticancer substance in cruciferous vegetables. I3C significantly increased
tumor incidence and multiplicity (number of tumors per animal) at levels
in the diet of 500 ppm and higher, corresponding to a daily intake of
14-28 mg I3C/kg of body weight. Therefore, the use of some commercial
I3C supplements that achieve this range of intake in humans may pose risks.
The second experiment found that the relationship between the amount of
the carcinogen dibenzo[a,l]pyrene (DBP), a polycyclic aromatic hydrocarbon
found in charred meat and tobacco smoke, administered to trout and the
resultant tumor incidence was not linear at low doses, i.e., very low
doses of DBP may not be as carcinogenic as previously thought.
KJ, FAN S, ROSEN EM, GOODWIN L, CHANDRASKAREN A, WILLIAMS DE,
CHEN D, and CARTER TH. Indole-3-carbinol is a negative regulator of estrogen.
J. Nutr. 133: 2470S-2475S, 2003.
suggest that the consumption of cruciferous vegetables decreases the risk
of breast cancer, and animal studies support the role of indole-3-carbinol
(I3C), an anticancer substance in these vegetables, in preventing estrogen-enhanced
cancer, such as cancers of the breast, cervix, and uterus. This study
investigated the effect of diindolylmethane (DIM), which is an I3C metabolite,
and genistein, which is the major flavonoid in soy, on the growth of estrogen-sensitive
human breast cancer cells in culture. At physiologically relevant concentrations,
DIM and genistein had slight effects on cell death, but when combined,
they dramatically increased cell death by increasing the amount of certain
proteins that stimulate programmed cell death, increasing the expression
of a tumor suppressor gene, and interfering with estrogen signaling.
JC, KINLAY S, BELTRAME J, HIKITI H, WAINSTEIN M, BEHRENDT D, SUH
J, FREI B, MUDGE GH, SELWYN AP, and GANZ P. Effect of vitamins
C and E on progression of transplant-associated arteriosclerosis: a randomised
trial. Lancet 359: 1108-1113, 2002.
This clinical study
reported that daily supplementation with 1,000 mg of vitamin C and 800
IU of vitamin E for one year after cardiac transplantation significantly
retarded the progression of arteriosclerosis in the coronary arteries.
In the placebo group of 21 subjects, the intimal index—a measure
of atherosclerotic plaque—increased about 8%, but did not increase
at all in the 19 patients treated with vitamins C and E. The first few
years following transplantation is the period in which thickening of the
arterial wall, which predicts clinical outcome, proceeds most rapidly.
The authors speculated that the benefit of vitamins C and E observed in
this study may be applicable to patients undergoing other organ transplants.
F, SMITH A, ZHANG W, KEANEY JF Jr, HAGEN T,
and FREI B. Lipoic acid and vitamin C potentiate nitric
oxide synthesis in human aortic endothelial cells independently of cellular
glutathione status. Redox Rep. 7: 223-227, 2002.
or stiffness of the arteries is associated with hypertension and heart
disease. A number of clinical studies have found that vitamin C promotes
relaxation of the arteries (vasodilation), probably through its effect
on the generation of nitric oxide, a signaling molecule that induces the
relaxation of vascular smooth muscle cells and also inhibits platelet
aggregation. In this study, the authors reported that lipoic acid and
vitamin C enhanced nitric oxide production in cultured vascular endothelial
cells, but had no effect on the levels of another antioxidant, glutathione,
suggesting that depletion of glutathione is not involved in vascular dysfunction.
Vitamin C enhances nitric oxide synthesis by stabilizing and increasing
the amount of a molecule critical to the synthesis of nitric oxide, tetrahydrobiopterin,
but the mechanism by which lipoic acid stimulates the production of nitric
oxide is unknown.
B. To C or not to C, that is the question! J. Am. Coll. Cardiol.
42: 253-255, 2003.
In this editorial,
Dr. Frei briefly reviewed the evidence on the role of vitamin C in the
prevention of heart disease. Specifically, he discussed a recent paper
that showed an impressive 28% reduction in the risk of heart disease in
women taking vitamin C supplements. Unlike a number of other studies,
dietary vitamin C intake, mainly from fruits and vegetables, was not associated
with protection against heart disease, suggesting that vitamin C itself
is beneficial. The dietary range of intake in the study was 61-209 mg/day
of vitamin C, while the supplement users had a median daily intake of
672 mg. Recent pharmacological data indicate that a daily intake of 400
mg is necessary to fully saturate circulating cells in women, which is
compatible with these results. Dr. Frei also noted that the study did
not reveal any increased risk of heart disease attributable to vitamin
C. The mechanism by which vitamin C affords protection against heart disease
is still under investigation and may be related to improved endothelial
cell function and antioxidant defense.
P, PELUFFO H, PEHAR M, MARTINEZ-PALMA L, RESSIA A, BECKMAN JS,
ESTEVEZ AG, and BARBEITO L. Peroxynitrite triggers a phenotypic transformation
in spinal cord astrocytes that induces motor neuron apoptosis. J.
Neurosci. Res. 67: 21-29, 2002.
Astrocytes are star-shaped
cells that nurture neurons, the specialized cells in the nervous system
that conduct nerve impulses. In the cell culture experiments reported
in this paper, spinal cord astrocytes became morphologically altered after
exposure to peroxynitrite, a powerful oxidant formed in the body from
reactions between nitric oxide and the superoxide radical. Additionally,
the altered astrocytes became toxic to motor neurons and induced apoptosis
(programmed cell death). Peroxynitrite scavengers protected motor neurons
R-J, YE Y-Z, PARKS DA, and BECKMAN JS. Urate produced
during hypoxia protects heart proteins from peroxynitrite-mediated protein
nitration. Free Radic. Biol. Med. 33: 1243-1249,
caused by the highly reactive peroxynitrite is associated with many pathologies.
Peroxynitrite and its metabolites damage proteins by a process known as
nitration. In Parkinson’s disease, tyrosines in certain enzymes
are nitrated, resulting in loss of function. In amyotrophic lateral sclerosis
(ALS), neurofilaments are damaged by nitration. In this study, the investigators
examined the effect of various substances, including urate, vitamin C,
and sulfur-containing (thiol) antioxidants, on the nitration of proteins
in rat heart and brain. Vitamin C and thiols had little effect on nitration
by peroxynitrite in either the heart or brain, but urate, which accumulated
in the heart to levels higher than those in the brain, protected against
protein nitration. Urate is formed naturally in the body as a by-product
of DNA metabolism and can also be formed from dietary inosine.
H and BECKMAN JS. Oxidative stress and nitration in neurodegeneration:
cause, effect, or association? J. Clin. Invest. 111:
In this perspective
paper, the authors offered the view that oxidative and nitrative processes
that generate reactive oxygen radicals and reactive nitrogen radicals
are central to the death of motor neurons in neurodegenerative diseases
like Parkinson’s and ALS. Oxidants like peroxynitrite, formed from
the reaction between nitric oxide and the superoxide radical, as mentioned
above, damage proteins, resulting in abnormal protein aggregation, compromised
integrity of the blood-brain barrier, and, indirectly, the necrosis or
apoptosis (programmed cell death) of motor neurons. In models of Parkinson’s
disease, certain environmental toxins generate reactive species that produce
cellular dysfunction and death. In ALS, mutations in an important endogenous
antioxidant enzyme, superoxide dismutase, have been linked to muscle degeneration.
While maintaining or improving antioxidant status may prove important
in preventing or treating these diseases, other metabolic dysfunctions,
such as failures in energy production, changes in the regulation of metals
like iron, copper, and zinc, and the inability to repair damaged proteins,
also contribute to the pathologies.
updated November, 2003