From the Departments of Ophthalmology and
¶ Chemistry, Columbia University, New York, New York 10028
Received for publication, January 15, 2003, and in revised form, March 12, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The autofluorescent pigments that accumulate in
retinal pigment epithelial cells with aging and in some retinal
disorders have been implicated in the etiology of macular degeneration. The major constituent is the fluorophore A2E, a pyridinium bisretinoid. Light-exposed A2E-laden retinal pigment epithelium exhibits a propensity for apoptosis with light in the blue region of the spectrum
being most damaging. Efforts to understand the events precipitating the
death of the cells have revealed that during irradiation (430 nm), A2E
self-generates singlet oxygen with the singlet oxygen in turn
reacting with A2E to generate epoxides at carbon-carbon double bonds.
Here we demonstrate that A2E-epoxides, independent of singlet oxygen,
exhibit reactivity toward DNA with oxidative base changes being at
least one of these lesions. Mass spectrometry revealed that the
antioxidants vitamins E and C, butylated hydroxytoluene, resveratrol, a
trolox analogue (PNU-83836-E), and bilberry extract reduce
A2E-epoxidation, whereas single cell gel electrophoresis and cell
viability studies revealed a corresponding reduction in the incidence
of DNA damage and cell death. Vitamin E, a lipophilic antioxidant,
produced a more pronounced decrease in A2E-epoxidation than vitamin C,
and treatment with both vitamins simultaneously did not confer
additional benefit. Studies in which singlet oxygen was generated by
endoperoxide in the presence of A2E revealed that vitamin E, butylated
hydroxytoluene, resveratrol, the trolox analogue, and bilberry reduced
A2E-epoxidation by quenching singlet oxygen. Conversely, vitamin C and
ginkgolide B were not efficient quenchers of singlet oxygen under these conditions.
The di-retinal conjugate A2E forms as a consequence of
light related vitamin A cycling in the retina. This orange-emitting fluorophore is formed synthetically as the condensation product of
all-trans-retinal and ethanolamine (1-3). NMR and
corroborative total chemical synthesis revealed A2E to be a pyridinium
bisretinoid consisting of an unprecedented pyridinium polar head group
and two hydrophobic retinoid tails (4, 5). A2E, its slightly less polar
photoisomer, iso-A2E, and other minor cis-isomers together constitute the most prominent age-related hydrophobic pigments (lipofuscin) in retinal pigment epithelial
(RPE)1 cell extracts assayed
by reverse phase HPLC (3, 6). In vivo, A2E is generated by
phosphate hydrolysis of the fluorophore phosphatidylpyridinium
bisretinoid, the latter precursor forming from reactions between
all-trans-retinal and phosphatidylethanolamine in the
photoreceptor outer segment membrane (3, 7, 8).
Although certain levels of A2E are clearly tolerated by RPE cells,
adverse effects of its accumulation have also been reported. Thus, not
only can A2E mediate detergent-like effects on cell membranes (9), its
accumulation can also lead to the alkalinization of lysosomes (10) and
to the detachment of proapoptotic proteins from mitochondria (11). A2E
also bestows a sensitivity to blue light damage (11-13) that is
proportional to the A2E content of the cells that is not exhibited by
cells devoid of A2E and that exhibits a wavelength dependence that
reflects the excitation spectrum of A2E (12). Evidence indicates that
the generation of oxygen reactive species upon photoexcitation of A2E
is integral to the death of the cells. For instance, an enhancer
(D2O) and quenchers (histidine, DABCO, and azide) of
singlet oxygen modulate the incidence of nonviable A2E-laden RPE
following blue light illumination (14). Importantly, A2E itself
undergoes photoxidation during irradiation with blue light to produce a
series of epoxide rings along the retinoid side arms of the molecule
(A2E-epoxides) (14, 15). The extent of epoxidation is dependent on the
intensity and duration of illumination with irradiated A2E forming a
mixture of compounds exhibiting epoxides of varying numbers, including an unprecedented nonaoxirane. The involvement of singlet oxygen in the
photoxidation of A2E is indicated by the phosphorescence detection of
singlet oxygen upon 430-nm irradiation of A2E by deuterium solvent
potentiation, by inhibitory effects of singlet oxygen quenchers, and by
experiments demonstrating that endoperoxide-derived singlet oxygen can
substitute for blue light irradiation (14, 15).
We recently demonstrated that DNA is one of the subcellular targets of
the photodynamic events initiated by the interaction of blue light and
A2E (16). Although the exact mechanism by which the DNA damage occurs
is not known, chemical reactions between DNA and the epoxides of
photooxidized A2E may be a critical step. Because of the large ring
strain and electrophilicity of these carbon- and oxygen- (2:1)
containing three-membered epoxide rings, nucleophilic macromolecules
such as proteins and DNA tend to react with them spontaneously (17,
18). In fact, several classes of carcinogens that induce structural
changes in DNA are known to react with guanosine and to a lesser extent
adenosine and cytosine through epoxide moieties (19-26). In some
cases, these reactions generate bulky adducts. Intrastrand and
interstrand cross-links can also form when two or more epoxides are
available for reaction (23, 27). Moreover, modifications associated
with skeletal aromatic nitrogens of DNA bases can be destabilizing.
Consequently, secondary transformations such as facile depurination or
imidazole ring opening at N-7-alkyl-deoxyguanosines (17) can occur.
Here we report that A2E-epoxides can generate DNA lesions independent
of direct damage by singlet oxygen. By mass spectrometry and
quantitative HPLC, we have also screened some antioxidants for their
ability to inhibit A2E-epoxide formation.
Reagents--
Resveratrol and PNU-83836-E were obtained from
Pharmacia. (L)-Ascorbic acid (vitamin C), BHT,
m-chloroperoxybenzoic acid, ethanolamine, and trifluoroacetic
acid were purchased from Aldrich. HEPES, Cell Culture and Illumination--
Human adult RPE cells
(ARPE-19, American Type Culture Collection, Manassas, VA) lacking
endogenous A2E (9) were grown as described previously (9, 12). To
generate A2E-laden RPE, nonconfluent cultures were allowed to
accumulate A2E from a 20 µM concentration in
medium (9). For some experiments, cultures were treated prior to
blue light illumination with A2E-epoxides--
A2E-epoxides were generated by illuminating
(430 nm, 10-min exposure, 0.36 milliwatt/mm2) 200 µM A2E (3) in DPBS with calcium, magnesium, and glucose. Under these conditions, >50% A2E in the sample undergoes epoxidation with 1-7 epoxides (out of a possible maximum of nine) forming on the
parent A2E molecule (14). A2E-bisepoxide, a compound containing a
single epoxide on each of the two side arms of A2E (positions 7,8 and
7',8'), was also generated by oxidation with m-chloroperoxybenzoic acid
as described previously (15). To determine whether DNA damage can be
induced by epoxidized A2E, A2E-epoxides and A2E-bisepoxide were
subsequently incubated with ARPE-19 cells (specifically, A2E-free
cells). To ensure that epoxidized A2E could permeate the cell membrane
to access the nuclear material in these experiments, two modifications
of the protocol were used. First, the cells were permeabilized on ice
(for 1 h) before incubation with A2E-epoxides (for 3 h) or A2E-bisepoxide (for 5 h). As a second approach, a modified
comet assay was performed in which the agarose-embedded cells were
incubated with A2E-epoxides or A2E-bisepoxide (at 1 h) before
lysis (29). The results were similar with these two approaches, and the
data were pooled.
For analysis by FAB-MS, preparations of A2E (200 µM) in
DPBS with and without the antioxidants (100 µM) vitamin
E, vitamin C, BHT, ginkgolide B, bilberry, resveratrol, or PNU-83836E
were exposed to 430-nm light (0.36 milliwatt/mm2). In other
experiments, A2E (500 µM) was epoxidized by incubation with a singlet oxygen generator, the endoperoxide of
1,4-dimethylnaphthalene (20 mM in 75 µl of methanol).
Stock solutions of the antioxidants vitamin E, vitamin C, BHT,
ginkgolide B, bilberry, or resveratrol were prepared in
Me2SO and added (25 µl) to a final concentration of 40 mM. The mixture was stirred for 14-15 h in the dark
at room temperature, and afterward, the sample was stored at
Single Cell Gel Electrophoresis--
DNA damage was probed using
the comet assay (Trevigen, Gaithersburg, MD). After treatment, cells
were released from the well by brief trypsinization, and after being
added to low melting point agarose (1 × 105 cells/ml,
1/10 dilution, 37 °C), 75-µl aliquots of the mixture were pipetted
onto comet slides and allowed to gel. After incubating in lysis
solution at 4 °C for 90 min followed by washing and denaturation in
an alkali buffer for 45 min (room temperature and darkness), the slides
were transferred to a horizontal chamber for electrophoresis (1 V/cm,
30 min) in alkali solution (0.3 M NaOH, 1 mM
EDTA, pH >13) on ice. Finally, the slides were immersed in ethanol (5 min), air-dried, and stained with SYBR Green. Because comet tail
moment departs from normality (30), the means were compared using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (Prism, GraphPad Software, San Diego, CA). The level of
significance was 0.05.
Immunostaining for 8-Oxodeoxyguanosine--
Detection of
8-oxodeoxyguanosine (8-oxo-dG), the oxidized derivative of the parent
nucleoside deoxyguanosine, was performed by immunostaining with
monoclonal antibody to 8-oxo-dG (Trevigen Inc.). After the cells were
fixed in 70% ethanol ( HPLC--
A Waters 600E HPLC equipped with Waters 996 photodiode
array detector was used with a reverse phase C18 column (250 × 4.6 mm, Cosmosil 5C18, Nacalai Tesque, Osaka, Japan). A2E was
eluted with the following gradient of acetonitrile in water (containing 0.1% trifluoroacetic acid): 84-96% (10 min), 96% (5 min), 96-100% (2 min), and 100% (3 min) and a flow rate of 1 ml/min with monitoring at 430 nm. For A2E quantitation, an external standard of A2E was used.
Mass Spectrometry--
FAB-MS was performed on a JEOL
JMS-HX110A/110A tandem MS (Akishima, Tokyo, Japan) using 10-kV
acceleration voltage and fitted with a Xe beam FAB gun (6 kV) on the
MS-1 ion source. 3-Nitrobenzyl alcohol was used as matrix.
Assay of Cell Viability--
The nuclei of nonviable cells were
labeled with the membrane-impermeant dye Dead Red (Molecular Probes,
Eugene OR), and the nuclei of all of the cells were labeled with DAPI
(4',6-diamidino-2-phenylindole) as described previously (14).
Counting was performed from digital images, nonviable cells were
expressed as a proportion of the total number of cells in an
illuminated field, and means were compared by ANOVA followed by the
Student's-Newman Keul multiple comparison test. The level of
significance was 0.05.
A2E-epoxides Can Induce Cellular Damage--
To test the
reactivity of A2E-epoxides with cellular macromolecules, A2E-epoxides
(1-7 epoxides on each A2E molecule) were incubated with permeabilized
ARPE-19 cells. To detect direct strand breaks and alkali labile sites
(31, 32), single cell gel electrophoresis was performed under alkaline
conditions that allow DNA to be drawn out into a comet tail when
subjected to an electrical field. As shown in Fig.
1, the presence of strand breaks was
visualized by the emergence of comet tails from the nuclei of
A2E-epoxide-treated cells. The nuclei of A2E- and vehicle-treated cells
remained spherical. The presence of comet tails in A2E-epoxides-treated
cells corresponded to significantly elevated (p < 0.01) measures of tail moment, a parameter whose magnitude reflects the
frequency of DNA strand breaks per nucleus (Fig.
2A) (30, 33, 34). Moreover,
83% nuclei were associated with values of mean tail moment that were 2 S.D. greater than the mean of vehicle-treated cells (range 48-98%, five experiments). Conversely, the values for mean tail moment obtained
for the A2E-treated and vehicle-treated cells were consistent with that
of intact nuclei. In the experiments described above, the cells were
permeabilized on ice for 1 h before incubation with A2E-epoxides.
Nevertheless, less pronounced (60% of that observed with permeabilized
cells) measures of tail moment were also observed when nonpermeabilized
cells were incubated with A2E-epoxides. Elevated mean tail moment,
indicative of DNA damage, was also observed when A2E-bisepoxide was
incubated with ice-permeabilized cells or with nonlysed cells embedded
in agarose (Fig. 2B). The detection of comets after cells
are incubated with A2E-epoxides indicates that the latter can injure
DNA independent of singlet oxygen. Under the current experimental
design, singlet oxygen that was generated by blue light irradiation of
A2E (15) but which has a short life time would have been unavailable to
react directly with DNA.
A2E-epoxides Induce the Formation of 8-Oxo-dG--
The epoxide
moiety of aflatoxin B1 (35, 36) among several other agents (37) has
been shown to oxidize the C8 position of guanine to form 8-oxo-dG.
Therefore, we sought to determine whether cells treated with
A2E-epoxides exhibited the same base modification. Immunocytochemical
labeling with monoclonal antibody to 8-oxo-dG revealed specific nuclear
staining in cells incubated with A2E-epoxides, whereas staining was
absent in control cells incubated with vehicle (DPBS) (Fig.
3).
Vitamins E and C Protect against Blue Light-mediated DNA Damage to
A2E-laden RPE--
We have previously established that singlet oxygen
is involved in the epoxidation of A2E (15). Accordingly, because
vitamins E and C are naturally occurring compounds with antioxidant
capability, we tested their efficacy in preventing DNA damage when
A2E-laden RPE are blue light-irradiated. When analyzed by alkaline
comet assay with the measurement of tail moment, pretreatment with
vitamin E resulted in a dose-dependent decrease in the
formation of single strand breaks with significant decreases
(p < 0.01) being obtained at 1, 10, and 100 µM concentrations (Fig. 4).
Preincubation with
The ability of vitamins E and C to confer a resistance to blue light
damage in the context of intracellular A2E was further exemplified in
experiments in which the viability of A2E-containing RPE was assayed
after blue light illumination (Fig. 4). Again, pre-incubation with
vitamin E yielded a more pronounced protective effect than that of
vitamin C with the additive effects of vitamins E and C being small and
not significantly different.
Vitamins E and C Reduce Blue Light-induced A2E Epoxidation--
To
establish whether the protective effects of vitamins E and C were
mediated, at least in part, by inhibiting light-induced A2E-epoxidation, A2E in aqueous medium was exposed to 430-nm
illumination followed by FAB-MS analysis (Fig.
5). The FAB-MS spectra of blue light-irradiated A2E disclosed a molecular ion peak at
m/z 592, corresponding to the mass of A2E,
and an additional series of higher molecular mass peaks
representing the sequential insertion of oxygens at carbon-carbon
double bonds. We have previously shown that these additional peaks
correspond to A2E-epoxides (15). When A2E was irradiated in the
presence of vitamin E or C, the higher mass peaks arising from the
epoxides were suppressed as evidenced by the overall reduction in
epoxide peak intensity and by the loss of the peaks at
m/z 688 and 704. Moreover, vitamin E appeared to
be more effective than vitamin C at reducing the generation of
molecular ion peaks corresponding to A2E-epoxides. Not only were the
molecular ion peaks of lower intensity, only three epoxides were
detectable (m/z 608, 624, and 640) in the presence of vitamin E as compared with six with vitamin C.
Vitamin E but Not Vitamin C Protects against A2E-epoxidation by
Quenching Singlet Oxygen--
To determine whether vitamins E and C
inhibit A2E-epoxidation by quenching singlet oxygen, we generated
singlet oxygen from the decomposition of the endoperoxide of
1,4-dimethylnaphthalene, which decomposes with a half-life of ~5 h at
25 °C into 1,4 dimethylnapthalene and singlet oxygen. We previously
showed that for the generation of A2E-epoxides, the endoperoxide can
substitute for blue light irradiation (15). Methanol solutions of A2E
(500 µM), the endoperoxide of 1,4-dimethylnaphthalene (20 mM), and antioxidant (40 mM) were incubated for
14-15 h at room temperature. The extent of A2E-epoxidation in the
presence and absence of vitamin E or vitamin C was monitored by
quantifying the consumption of A2E by HPLC (14). As shown in Fig.
6, the incubation of A2E in the presence
of the singlet oxygen generator but without antioxidant resulted in
approximately a 50% decrease in measurable levels of A2E. The addition
of vitamin E ( Antioxidants Vary in Their Ability to Inhibit A2E-epoxidation by
Quenching Singlet Oxygen--
In addition to vitamins E and C, we
compared several other antioxidants in terms of their ability to
inhibit the epoxidation of A2E. From the FAB-MS spectra obtained after
samples of A2E were irradiated at 430 nm in the absence of antioxidant,
it was apparent that as many as seven epoxides
(m/z 608, 624, 640, 656, 672, 688, and 704)
formed along the retinal-derived side arms of A2E (Fig.
7). However, only the monoepoxides and
bisepoxides (m/z 608 and 624) formed in the
presence of BHT, a well known antioxidant. Similarly, only the
monoepoxides, bisepoxides, and triepoxides (m/z
608, 624, and 640) formed in the presence of resveratrol, an
antioxidant derived from grapes (38) or PNU-83836E, a water-soluble
trolox analogue and chroman derivative, which is related to vitamin E
and which acts to restore endogenous vitamin E (39). Up to four
epoxides (monoepoxides through tetraepoxides; m/z
608, 624, 640, and 656) were generated when irradiation was performed
in the presence of bilberry (Vaccinium myrtillus), a widely
used nutritional supplement exhibiting antioxidant activity (40, 41).
On the other hand, ginkgolide B, a diterpenoid trilatone (from Ginkgo
biloba), which has demonstrated antioxidant effects under other
conditions (42), showed only a small effect in reducing A2E-epoxidation
when evaluated by FAB-MS (Fig. 7).
To establish whether the effect of these antioxidants on
A2E-epoxidation occurred through the quenching of singlet oxygen, the
latter was generated from the decomposition of endoperoxide and the A2E
consumption that accompanies epoxidation was quantified by HPLC. In the
presence of BHT, the loss of A2E was reduced from 50 to 4%, such that
the amount of A2E in the sample was not significantly different from
that in the absence of the endoperoxide (Fig. 6). Similar protection
was mediated by bilberry and resveratrol with bilberry, reducing the
loss of A2E from 50 to 3% and resveratrol diminishing the loss to 5%.
Just as with vitamin C, however, ginkgolide B did not protect against
A2E-epoxidation and consumption of A2E when the latter was exposed to
the singlet oxygen generated from endoperoxide. Thus, at least under
these conditions, ginkgolide B appears not to quench singlet oxygen.
We previously observed that the double bond structure of A2E
predisposes it to reaction with the singlet oxygen that is
autogenerated by irradiation with blue light, the oxidation of the
double bonds leading to epoxide formation (14, 15). Thus, at the outset of this work, we reasoned that as alkylating agents, these epoxides would exhibit reactivity toward cellular macromolecules. Accordingly, we have shown that A2E-epoxides, both a mixture with varying numbers of
epoxides on the A2E side arms and bisepoxide, can induce DNA lesions
within cultured RPE cells. Moreover, by immunoperoxidase labeling with
monoclonal antibodies that recognize 8-oxo-dG, we demonstrated in
individual cells that at least one of these lesions is an oxidatively
modified guanine base. Although cells, including ARPE-19 cells (16),
have developed mechanisms for repairing oxidative lesions to DNA bases,
a decline in repair activity with age has been recognized (43, 44).
Indeed, for cells that do not turnover as is the case for RPE, there is
a greater accumulation of such lesions (45). The persistence of DNA
damage can result in altered gene expression with the transcription of
some genes being arrested, while the transcription of other genes is
induced (46). DNA is not the only macromolecule in RPE cells
toward which A2E-epoxides are likely to react because epoxides can
react with a large range of nucleophiles, the latter attacking the
electrophilic carbons of the epoxide, causing it to open. Examples of
common cellular nucleophiles include the sulfhydryl (SH) groups
of proteins, the electron-rich nitrogen atoms of the amino groups
(NH2) of proteins and DNA, and the oxygen atom in a
hydroxyl ion (OH).
It is well established that RPE cells that have accumulated A2E are
subject to blue light-mediated injury (12-14, 16), and we showed that
at least one of the cellular macromolecules damaged is DNA. Although we
report here that A2E-epoxides can damage DNA and probably other
cellular constituents, the singlet oxygen generated by the
photosensitization of A2E is an additional potentially important
cytotoxic agent. Within polar solvents, A2E generates singlet oxygen
with modest efficiency (0.03) (15, 47, 48), although the
photosensitizing ability of a hydrophobic compound such as A2E is
probably greater in nonpolar surroundings such as that can be found in
the membrane environment of a cell (49, 50). Nevertheless, it is clear
that much of the singlet oxygen generated under these conditions is
quenched by A2E (15, 51), and in the process, A2E-epoxides are
generated (15). Moreover, the ability of singlet oxygen to diffuse only
a short distance (~10-20 nm) (52) within the cell may limit its
ability to generate DNA damage. Thus, the potential for A2E-mediated
photodamage in RPE cells in the eye may be very much dependent on the
formation of A2E-epoxides. In keeping with this observation, it is
important to note that some photosensitizers with low singlet oxygen
quantum yields (0.005) exert a phototoxicity that is by orders of
magnitude more potent than expected on the basis of their photophysical characteristics. The pronounced ability of these compounds to mediate
light-induced cellular damage is explained by a conjugated double bond
structure that serves to directly quench the singlet oxygen produced to
form damaging oxidized intermediates (53-55). The proximity of the
nucleus to the site of epoxide generation is likely to influence the
ability of these compounds to interact with DNA. Thus, it is important
to note that A2E accumulates intracellularly in lysosomal storage
bodies that assume a perinuclear distribution (9). The access to the
nucleus may be further facilitated if the A2E-epoxide fragment into
readily diffusible compounds and/or if redistribution of the epoxides
occurs because of lysosomal photodamage, the latter being the case for
some other photosensitizers (56).
Based on analogy with other DNA alkylation reactions and the secondary
chemical transformations they undergo, it is possible to conceive
mechanisms by which A2E-epoxide can generate a carbonyl product at the
C8 position of guanine. For instance, the dietary carcinogen
aflatoxin-B1 undergoes cytochrome P450-mediated oxidation to an aflatoxin-epoxide that can then form DNA adducts through covalent
binding to the ring nitrogen at position 7 of guanine (57).
Aflatoxin-N(7)-guanine adducts, however, are very unstable and
can spontaneously yield 8-oxo-dG (35, 36, 57). Other alkyl epoxides
have also been shown to prefer the N(7) position of guanine,
principally because it has high nucleophilicity and is sterically
accessible (17) and some of these other N(7)-substituted guanines can
also undergo hydrolytic rearrangement to form 8-oxo-dG (58). Thus, we
propose that an epoxy-A2E may react at the position 7 of guanine to
form an iminium salt. Reaction of the iminium salt with
H2O hydroxide ion would form a DNA adduct, which could subsequently eliminate its A2E moiety to give 8-oxo-dG. The foregoing mechanism is currently under investigation as is the potential for DNA
cross-linking. The latter is feasible because A2E can undergo
epoxidation at multiple double bonds and thus may be capable of
reacting at two sites on DNA, leading to cross-link formation.
In addition to transforming to 8-oxo-dG, N(7)-guanine adducts derived
from epoxides can also proceed to depurination (59). The resulting base
loss can transpire under physiological conditions and occurs because
alkylation at guanine N(7) introduces a positive charge on the nitrogen
atom (quaternary nitrogen) that leads to cleavage of the glycosyl bond
to regain ring stability (17). These abasic sites created by
spontaneous fission of the base-sugar linkage can subsequently be
transformed into DNA strand breaks by the alkaline unwinding solution
of the comet assay (31, 32). Such alkali-labile abasic sites probably
account, at least in part, for the single strand breaks we detected by
comet assay. Some of the DNA strand breaks may have also resulted from
excision of damaged bases by specific DNA glycosylases in the process
of DNA repair (44).
We observed that vitamin E, vitamin C, and a combination of both
vitamins reduced the number of A2E molecules undergoing epoxidation, diminished the number of epoxides formed on a given A2E-epoxide adduct,
and decreased the incidence of associated DNA injury and cell death.
However, a clear synergistic effect of these two vitamins was not
observed. Vitamin E is considered to be a major lipophilic antioxidant,
and in some (60) but not all (61) cases, its oxygen-scavenging
efficiency can be increased by combined treatment with vitamin C. Our
mass spectrometry and HPLC data indicate that vitamin E served to
scavenge singlet oxygen after its generation by photosensitized A2E but
before it was inserted into the hydrophobic side arms of A2E to form
epoxides. The notion that the effect of vitamin E was to intercept
singlet oxygen-induced A2E epoxidation rather than to block reactivity
between A2E-epoxides and cellular targets is consistent with reports
that treatment with vitamin E does not inhibit covalent binding of
aflatoxin B1-epoxide to DNA (36). A spectroscopic study
(9) of A2E supports the assertion that the hydrophobic domains
of this amphiphilic molecule are associated with the nonpolar lipid
portions of intracellular membranes. With A2E positioned as such,
vitamin E may function by neutralizing singlet oxygen at the site of
its formation within the lipid bilayer of the membrane. This scenario
may also account for the far more pronounced effect observed with
vitamin E as compared with aqueous-soluble vitamin C and for the
absence of additional benefit from combined treatment. It has been
reported (62) that in addition to direct scavenging of reactive oxygen
species, vitamin C can exert an antioxidant effect by maintaining
vitamin E in its reduced functional form. Because under our
experimental conditions significant quenching of singlet oxygen by
vitamin C was not observed, we suggest that the protection afforded by
vitamin C was related to its ability to recycle tocopheroxyl radicals.
The significance of our findings with respect to vitamins C and E and
their suppression of A2E-epoxidation is underscored by studies
suggesting that individuals with low plasma levels of the antioxidants
vitamins C and E are at increased risk for AMD (63, 64). Moreover, the
Age-related Eye Disease Study, a 9-year multicenter clinical trial,
recently reported that supplementation with zinc, vitamins E and C, and
Of the other presumed antioxidants we surveyed, bilberry and
resveratrol are both plant-derived dietary constituents. Bilberry is
widely used in the U. S. as a nutritional supplement to improve vision, although the mechanism by which this may occur is not clear
(40, 41) and little work has been done in the U. S. to elucidate
indications for its use. The ability of bilberry to inhibit
A2E-epoxidation in our studies may be related to its content of
anthocyanosides (70, 71), the latter being reputed to have antioxidant
activity (40). Resveratrol is a natural compound found in grapes (38).
It is reported to have antioxidant properties, and its presence in wine
in a soluble form that results in improved bioavailability is thought
to be responsible for the cardioprotective effects of red wine. Both
resveratrol and bilberry extract can inhibit A2E-epoxidation by
quenching singlet oxygen. This appears not to be the case for
ginkgolide B.
The oxidative damage to which all of the cells are subjected is
considered to be a significant cause of an age-related decline in cell
function (72-74). Nevertheless, as a nonreplicating cell that is
exposed to wavelengths of light in the visible spectrum and accumulates
a naturally occurring photoreactive chromophore with age, the RPE cell
may be anomalous. The damage that A2E epoxidation bestows on the RPE
cell may be the element linking the associations between RPE atrophy
and lipofuscin accumulation (75-78) on the one hand and AMD and light
exposure (79-82) on the other. Photoxidative mechanisms involving A2E
may also underlie the known susceptibility of RPE to blue light damage
in vivo (83-85).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tocopherol acetate,
-tocopherol, and dehydroascorbic acid were obtained from Sigma.
Bilberry extract was from Nature's Resource Premium Herb (Mission
Hills, CA). Acetonitrile was purchased from Fisher, and Dulbecco's
phosphate-buffered saline (DPBS) was from Invitrogen. Glass-backed TLC
were purchased from Merck (Darmstadt, Germany). All of the other
chemicals were from Sigma. A2E was synthesized as described previously
(3).
-tocopherol (100 µM) or
-tocopherol acetate (100 µM) prepared as a 1/1000 dilution of stock solution in ethanol for 24 h. For the
accumulation of ascorbic acid, cells were pretreated for 30 min with
dehydroascorbic acid (100 µM), the latter being reduced
intracellularly to ascorbate (28). Dehydroascorbic acid was diluted in
medium from a 10 mM stock solution in DPBS. Cells
were subsequently exposed to 430-nm illumination (0.36 milliwatt/mm2) as described previously (12-14). To assess
cell viability, cultures were exposed to a 430-nm light line (0.8-mm
wide, 0.34 milliwatt/mm2).
80 °C. The samples either underwent HPLC quantitation (3, 9, 12) using A2E as external standard or were analyzed by FAB-MS.
20 °C, 10 min) and DNA was denatured (4 N
HCl, 7 min), the cells were incubated in blocking serum (10% fetal
bovine serum in 10 mM Tris-HCl, pH 7.5, for 1 h at
37 °C) followed by anti-8-oxo-dG diluted 1/300 in blocking serum
(4 °C, overnight). Visualization was done by an alkaline phosphatase
detection system (ABC-AP, Vector Laboratories, Burlingame, CA) with
Vector Red (Vector Laboratories) as substrate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (58K):
[in a new window]
Fig. 1.
A2E-epoxides induce DNA damage. Cells
were processed by alkaline comet assay. RPE cells were incubated with
A2E after it was irradiated at 430 nm (A2E-epoxides) or with
nonirradiated A2E (A2E) and PBS (control). Comets
induced with A2E-epoxides are indicative of DNA damage.
View larger version (14K):
[in a new window]
Fig. 2.
Quantitation of DNA damage induced by
epoxidized A2E. A, cells were incubated with
A2E-epoxides generated by irradiation at 430 nm, nonirradiated A2E
(A2E), or PBS (vehicle). B, incubation
with A2E-bisepoxide produced by oxidation with m-chloroperoxybenzoic
acid. Comet assay performed with quantification by tail moment.
Mean ± S.E. of 2-5 experiments. *, p < 0.01 as
compared with A2E and vehicle. Insets in A and
B illustrate structures of A2E-nonaepoxide (the species
within the mixture of A2E-epoxides that has the maximum number of
epoxides) and A2E-bisepoxide (two epoxides), respectively.
View larger version (80K):
[in a new window]
Fig. 3.
A2E-epoxides mediate base-specific DNA
damage. Immunocytochemical staining with antibody to 8-oxo-dG is
shown. Cells were incubated with A2E that had been previously
irradiated at 430 nm (A2E-epoxides) (A) or with PBS
(C). B and D, phase-contrast images of
fields shown in A and C, respectively.
Arrows, stained (A) and unstained (C)
nuclei. Scale bar, 10 µm.
-tocopherol (100 µM) and
-tocopherol acetate (100 µM), a hydrophilic analog of
-tocopherol, attenuated mean tail moment to a similar extent (44-58
and 52-66%, respectively), and thus, the data were pooled.
Pretreatment with dehydroascorbic acid, the oxidized and readily
accumulated form of ascorbic acid (vitamin C) also provided protection
against DNA damage in blue light-illuminated A2E-laden RPE, although
vitamin E was more effective than vitamin C at lower doses (1 and 10 µM). Specifically, only vitamin E generated statistically significant differences (p < 0.01) at the dose of 1 µM. Although the data at the 10 µM
concentration were statistically significant for both vitamins E and C
(p < 0.01 and p < 0.05, vitamins E
and C, respectively), the effect was greater with vitamin E (47 versus 19% reduction, vitamins E and C, respectively). When
treatment with vitamins E and C was combined, the difference in tail
moment as compared with vitamin E alone was not statistically
significant (Fig. 4). Conversely, combined treatment produced a 40%
decrease over that with vitamin C alone, a decrease that was
statistically significant (p < 0.01). Taken together,
these findings indicate that vitamin E is more effective than vitamin C
in resisting blue light-mediated DNA damage to A2E-laden RPE.
View larger version (17K):
[in a new window]
Fig. 4.
Modulation of DNA damage and cell death in
the presence of vitamin E, vitamin C, and the latter antioxidants in
combination. DNA damage was detected by single cell gel
electrophoresis (comet assay) and was quantified by tail moment (50 nuclei/experiment). Percentage of nonviable cells was determined by
labeling all of the nuclei with DAPI and nuclei of nonviable cells with
a membrane-impermeable dye. Concentrations of 100 µM were
used for combined treatment and cell death assay. Values are the
mean ± S.E. of 2-5 experiments. *, p < 0.05;
**, p < 0.01 as compared with conditions of A2E and
blue light (A2E BL).
View larger version (13K):
[in a new window]
Fig. 5.
Vitamins E and C reduce the formation of
A2E-epoxides. FAB-MS of nonirradiated A2E (A2E
control), A2E exposed to blue light (A2E + Blue Light),
and A2E irradiated with blue light in the presence of vitamin E (200 µM) or vitamin C (200 µM) is shown. The
molecular ion peak at m/z 592 corresponds to the
molecular mass of A2E. The formation of A2E-epoxides by illumination is
indicated by the presence of additional peaks that differ by mass 16. Illumination in the presence of vitamin E or C reduces the formation of
these epoxides. The peak at 613 is a matrix peak.
-tocopherol, 40 mM) conserved A2E at
levels comparable to control samples not exposed to the endoperoxide
(p < 0.01). The same concentration of vitamin C
(L-ascorbic acid), however, did not attenuate the loss of
A2E (p > 0.05). This finding was consistent with our
results, indicating that vitamin E was more effective than vitamin C in
suppressing A2E-epoxidation. Moreover, it appears that whereas vitamin
E reduces A2E-epoxidation by singlet oxygen quenching, the protective
effect of vitamin C depends, to at least some extent, on mechanisms
other than singlet oxygen quenching.
View larger version (21K):
[in a new window]
Fig. 6.
Antioxidants vary in their ability to inhibit
A2E-epoxidation by quenching singlet oxygen. The consumption of
A2E, which accompanies A2E-epoxidation, was quantified by HPLC after
A2E was exposed to singlet oxygen generated from the endoperoxide of
1,4-dimethylnaphthalene (20 mM) with and without the
addition of various antioxidants (40 mM). The symbols (+)
and ( ) indicate the presence or absence, respectively, of a compound.
*, values not different from levels of A2E present in the absence of
singlet oxygen generation; ANOVA, p > 0.05. The
absence of a difference is indicative of antioxidant protection.
View larger version (29K):
[in a new window]
Fig. 7.
Effect of various antioxidants on A2E
epoxidation. FAB-MS of A2E (200 µM in PBS) exposed
to blue light (A2E blue light) and A2E exposed to blue light
in the presence of the antioxidants (100 µM) BHT,
ginkgolide B, bilberry, resveratrol, and PNU-83836E. The molecular ion
peak at m/z 592 correlates with the molecular
mass of A2E. The higher molecular weight peaks correspond to
A2E-epoxides with the exception of 613, which is a matrix peak.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carotene can reduce the risk of progression to advanced AMD (65).
Although the Age-related Eye Disease Study did not attribute this
finding to particular mechanisms, investigators have speculated for
some time that antioxidant vitamins provide a shield against oxidative
injury originating, at least partially, from light (64). Because the
death of RPE is central to the etiology of atrophic AMD (66-69),
defense against the damaging effects of light-induced A2E-epoxidation
may be one means by which these vitamins afford protection.
![]() |
FOOTNOTES |
---|
* This work was supported by the National Institutes of Health Grants EY 12951 (to J. R. S.) and GM 34509 (to K. N.), Macula Vision Research Foundation (to J. R. S.), and unrestricted funds from Research to Prevent Blindness.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Research to Prevent Blindness Lew R. Wasserman Merit Award. To whom correspondence may be addressed: Dept. of Ophthalmology, Columbia University, New York, NY 10032. Tel.: 212-305-9944; Fax: 212-305-9638; E-mail: jrs88@columbia.edu.
Supported by a National Eye Institute Vision Sciences training
grant to Columbia University.
** To whom correspondence may be addressed: Dept. of Chemistry, Columbia University, New York, NY 10027. Tel.: 212-854-2169; Fax: 212-932-8273; E-mail: kn5@columbia.edu.
Published, JBC Papers in Press, March 19, 2003, DOI 10.1074/jbc.M300457200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RPE, retinal pigment epithelial cells; 8-oxo-dG, 8-oxo-deoxyguanosine; A2E, pyridinium bisretinoid; AMD, age-related macular degeneration; BHT, 2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene); D2O, deuterium oxide; DABCO, 1,4-diazabicyclooctane; DPBS, Dulbecco's phosphate buffered saline; FAB-MS, fast atom bombardment ionization mass spectrometry; HPLC, high performance liquid chromatography; ANOVA, analysis of variance.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Eldred, G. E., and Lasky, M. R. (1993) Nature 361, 724-726[CrossRef][Medline] [Order article via Infotrieve] |
2. | Eldred, G. E., and Katz, M. L. (1988) Exp. Eye Res. 47, 71-86[Medline] [Order article via Infotrieve] |
3. |
Parish, C. A.,
Hashimoto, M.,
Nakanishi, K.,
Dillon, J.,
and Sparrow, J. R.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14609-14613 |
4. | Sakai, N., Decatur, J., Nakanishi, K., and Eldred, G. E. (1996) J. Am. Chem. Soc. 118, 1559-1560[CrossRef] |
5. | Ren, R. F., Sakai, N., and Nakanishi, K. (1997) J. Am. Chem. Soc. 119, 3619-3620[CrossRef] |
6. |
Ben-Shabat, S.,
Parish, C. A.,
Vollmer, H. R.,
Itagaki, Y.,
Fishkin, N.,
Nakanishi, K.,
and Sparrow, J. R.
(2002)
J. Biol. Chem.
277,
7183-7190 |
7. |
Liu, J.,
Itagaki, Y.,
Ben-Shabat, S.,
Nakanishi, K.,
and Sparrow, J. R.
(2000)
J. Biol. Chem.
275,
29354-29360 |
8. |
Mata, N. L.,
Weng, J.,
and Travis, G. H.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7154-7159 |
9. |
Sparrow, J. R.,
Parish, C. A.,
Hashimoto, M.,
and Nakanishi, K.
(1999)
Invest. Ophthalmol. Visual Sci.
40,
2988-2995 |
10. | Holz, F. G., Schutt, F., Kopitz, J., Eldred, G. E., Kruse, F. E., Volcker, H. E., and Cantz, M. (1999) Invest. Ophthalmol. Visual Sci. 40, 737-743[Abstract] |
11. |
Suter, M.,
Reme, C. E.,
Grimm, C.,
Wenzel, A.,
Jaattela, M.,
Esser, P.,
Kociok, N.,
Leist, M.,
and Richter, C.
(2000)
J. Biol. Chem.
275,
39625-39630 |
12. |
Sparrow, J. R.,
Nakanishi, K.,
and Parish, C. A.
(2000)
Invest. Ophthalmol. Visual Sci.
41,
1981-1989 |
13. |
Sparrow, J. R.,
and Cai, B.
(2001)
Invest. Ophthalmol. Visual Sci.
42,
1356-1362 |
14. |
Sparrow, J. R.,
Zhou, J.,
Ben-Shabat, S.,
Vollmer, H.,
Itagaki, Y.,
and Nakanishi, K.
(2002)
Invest. Ophthalmol. Visual Sci.
43,
1222-1227 |
15. | Ben-Shabat, S., Itagaki, Y., Jockusch, S., Sparrow, J. R., Turro, N. J., and Nakanishi, K. (2002) Angew. Chem. Int. Ed. Engl. 41, 814-817[CrossRef][Medline] [Order article via Infotrieve] |
16. | Sparrow, J. R., Zhou, J., and Cai, B. (2003) Invest. Ophthalmol. Visual Sci., in press |
17. | Koskinen, M., and Plna, K. (2000) Chem. Biol. Interact. 129, 209-229[CrossRef][Medline] [Order article via Infotrieve] |
18. | Henderson, R. F. (2001) Chem. Biol. Interact. 135-136, 53-64[CrossRef] |
19. | Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C., Autrup, H., Kasai, H., and Nakanishi, K. (1976) Science 193, 592-595[Medline] [Order article via Infotrieve] |
20. | Jeffrey, A. M., Blobstein, S. H., Weinstein, I. B., Beland, F. A., Harvey, R. G., Kasai, H., and Nakanishi, K. (1976) Proc. Natl. Acad. Sci., U. S. A. 73, 2311-2314[Abstract] |
21. | Jeffrey, A. M., Weinstein, I. B., Jennette, K. W., Grzeskowiak, K., Nakanishi, K., Harvey, R. G., Autrup, H., and Harris, C. (1977) Nature 269, 348-350[Medline] [Order article via Infotrieve] |
22. | Jeffrey, A. M., Grzeskowiak, K., Weinstein, I. B., Nakanishi, K., Roller, P., and Harvey, R. G. (1979) Science 206, 1309-1311[Medline] [Order article via Infotrieve] |
23. |
Koivisto, P.,
Kilpelainen, I.,
Rasanen, I.,
Adler, I. D.,
Pacchierotti, F.,
and Peltonen, K.
(1999)
Carcinogenesis
20,
1253-1259 |
24. |
Selzer, R. R.,
and Elfarra, A. A.
(1999)
Carcinogenesis
20,
285-292 |
25. | Sun, D., Hansen, M., Clement, J. J., and Hurley, L. H. (1993) Biochemistry 32, 8068-8074[Medline] [Order article via Infotrieve] |
26. | Giri, I., Jenkins, M. D., Schnetz-Boutaud, N. C., and Stone, M. P. (2002) Chem. Res. Toxicol. 15, 638-647[CrossRef][Medline] [Order article via Infotrieve] |
27. | Carnelley, T. J., Barker, S., Wang, H., Tan, W. G., Weinfeld, M., and Le, X. C. (2001) Chem. Res. Toxicol. 14, 1513-1522[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Welch, R. W.,
Wang, Y.,
Crossman, A.,
Park, J. B.,
Kirk, K. L.,
and Levine, M.
(1995)
J. Biol. Chem.
270,
12584-12592 |
29. | Szeto, Y. T., Collins, A. R., and Benzie, I. F. F. (2002) Mutat. Res 500, 31-38[Medline] [Order article via Infotrieve] |
30. | Green, M. H. L., Lowe, J. E., Delaney, C. A., and Green, I. C. (1996) Methods Enzymol. 269, 243-265[Medline] [Order article via Infotrieve] |
31. | Rojas, E., Lopez, M. C., and Valverde, M. (1999) J. Chromatogr. 722, 225-254 |
32. | Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L. (1988) Exp. Cell Res. 175, 184-191[Medline] [Order article via Infotrieve] |
33. | Fairbairn, D. W., Olive, P. L., and O'Neill, K. L. (1995) Mutat. Res 339, 37-59[Medline] [Order article via Infotrieve] |
34. | McCarthy, P. J., Sweetman, S. F. S., Mckenna, P. G., and McKelvey-Martin, V. J. (1997) Mutagenesis 12, 209-214[Abstract] |
35. | Yarborough, A., Zhang, Y.-J., Hsu, T.-M., and Santella, R. M. (1996) Cancer Res. 56, 683-688[Abstract] |
36. | Shen, H.-M., Ong, C.-N., Lee, B. L., and Shi, C.-Y. (1995) Carcinogenesis 16, 419-422[Abstract] |
37. | Kasai, H. (1997) Mutat. Res. 387, 147-163[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Bhat, K. P. L.,
and Pezzuto, J. M.
(2002)
Ann. N. Y. Acad. Sci.
957,
210-229 |
39. | Campo, G., Squadrito, F., Campo, S., Altavilla, D., Avenoso, A., Ferlito, M., Squadrito, G., and Caputo, A. P. (1997) Free Radical Res. 27, 577-590[Medline] [Order article via Infotrieve] |
40. | Muth, E. R., Laurent, J. M., and Jasper, P. (2000) Altern. Med. Rev. 5, 164-173[Medline] [Order article via Infotrieve] |
41. | Zadok, D., Levy, Y., and Glovinsky, Y. (1999) Eye 13, 734-736[Medline] [Order article via Infotrieve] |
42. | Van Beek, T. A. (2000) Ginkgo Biloba, Medical and Aromatic Plants-Industrial Profiles , Harwood Academic Publishers, Amsterdam |
43. | Cabelof, D. C., Raffoul, J. J., Yanamadala, S., Ganir, C., Guo, Z. M., and Heydari, A. R. (2002) Mutat. Res. 500, 135-145[Medline] [Order article via Infotrieve] |
44. | Bohr, V. A. (2002) Free Radical Biol. Med. 32, 804-812[CrossRef][Medline] [Order article via Infotrieve] |
45. | Sohal, R. S., Agarwal, S., Candas, M., Forster, M. J., and Lal, H. (1994) Mech. Ageing Dev. 76, 215-224[CrossRef][Medline] [Order article via Infotrieve] |
46. | Bohr, V. A. (2001) in Advances in Cell Aging and Gerontology (Gilchrest, B. A. , and Bohr, V. A., eds) , pp. 191-205, Elsevier Science Publishers B.V., Amsterdam |
47. | Lamb, L. E., Ye, T., Haralampus-Grynaviski, N. M., Williams, T. R., Pawlak, A., Sarna, T., and Simon, J. D. (2001) J. Phys. Chem. B. 105, 11507-11512[CrossRef] |
48. | Gaillard, E. R., Atherton, S. J., Eldred, G., and Dillon, J. (1995) Photochem. Photobiol. 61, 448-453[Medline] [Order article via Infotrieve] |
49. | Aveline, B. M., Hasan, T., and Redmond, R. W. (1995) J. Photochem. Photobiol. B Biol. 30, 161-169[CrossRef][Medline] [Order article via Infotrieve] |
50. | Krieg, M., Srichai, M. B., and Redmond, R. W. (1993) Biochim. Biophys. Acta 1151, 168-174[Medline] [Order article via Infotrieve] |
51. | Roberts, J. E., Kukielczak, B. M., Hu, D. N., Miller, D. S., Bilski, P., Sik, R. H., Motten, A. G., and Chignell, C. F. (2002) Photochem. Photobiol. 75, 184-190[Medline] [Order article via Infotrieve] |
52. | Moan, J., and Berg, K. (1991) Photochem. Photobiol. 53, 549-553[Medline] [Order article via Infotrieve] |
53. | Bunting, J. R. (1992) Photochem. Photobiol. 55, 81-87[Medline] [Order article via Infotrieve] |
54. | Krieg, M., and Bilitz, J. M. (1996) Biochem. Pharmacol. 51, 1461-1467[CrossRef][Medline] [Order article via Infotrieve] |
55. | Delaey, E., van Laar, F., De Vos, D., Kamuhabwa, A., Jacobs, P., and de Witte, P. (2000) J. Photochem. Photobiol. B Biol. 55, 27-36[CrossRef][Medline] [Order article via Infotrieve] |
56. | Selbo, P. K., Sivam, G., Fodstad, O., Sandvig, K., and Berg, K. (2001) Int. J. Cancer 92, 761-766[CrossRef][Medline] [Order article via Infotrieve] |
57. |
Egner, P. A.,
Wang, J. B.,
Zhu, Y. R.,
Zhang, B. C.,
Wu, Y.,
Zhang, Q. N.,
Qian, G. S.,
Kuang, S. Y.,
Gange, S. J.,
Jacobson, L. P.,
Helzlsouer, K. J.,
Bailey, G. S.,
Groopman, J. D.,
and Kensler, T. W.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
14601-14606 |
58. | Kohda, K., Tada, M., Kasai, H., Nishimura, S., and Kawazoe, Y. (1986) Biochem. Biophys. Res. Commun. 139, 626-632[Medline] [Order article via Infotrieve] |
59. |
Smela, M. E.,
Hamm, M. L.,
Henderson, P. T.,
Harris, C. M.,
Harris, T. M.,
and Essigmann, J. M.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
6655-6660 |
60. | Kontush, A., Mann, U., Arlt, S., Ujeyl, A., Luhrs, C., Muller-Thomsen, T., and Beisiegel, U. (2001) Free Radical Biol. Med. 31, 345-354[CrossRef][Medline] [Order article via Infotrieve] |
61. |
Huang, H. Y.,
Appel, L. J.,
Croft, K. D.,
Miller, E. R.,
Mori, T. A.,
and Puddey, I. B.
(2002)
Am. J. Clin. Nutr.
76,
549-555 |
62. | Stocker, R. (1994) Curr. Opin. Lipidol. 5, 422-433[Medline] [Order article via Infotrieve] |
63. | Beatty, S., Koh, H.-H., Henson, D., and Boulton, M. (2000) Surv. Ophthalmol. 45, 115-134[CrossRef][Medline] [Order article via Infotrieve] |
64. | Snodderly, D. M. (1995) Am. J. Clin. Nutr. 62, (suppl.), 1448-1461 |
65. |
The Age-related Eye Disease Study Research Group.
(2001)
Arch. Ophthalmol.
119,
1417-1436 |
66. | Curcio, C. A., Madeiros, N. E., and Millican, C. L. (1996) Invest. Ophthalmol. Visual Sci. 37, 1236-1249[Abstract] |
67. | Green, W. R., McDonnell, P. J., and Yeo, J. H. (1985) Ophthalmology 92, 615-627[Medline] [Order article via Infotrieve] |
68. | Sarks, J. P., Sarks, S. H., and Killingsworth, M. C. (1988) Eye 2, 552-577[Medline] [Order article via Infotrieve] |
69. | Young, R. W. (1988) Surv. Ophthalmol. 32, 252-269[Medline] [Order article via Infotrieve] |
70. | Casoli, U., Cultrera, R., and Dall'Aglio, G. (1967) Ind. Conserve (Parma) 42, 11-16 |
71. | Nyman, N. A., and Kumpulainen, J. T. (2001) J. Agric. Food Chem. 49, 4183-4187[CrossRef][Medline] [Order article via Infotrieve] |
72. | Burkle, A. (2001) Eye 15, 371-375[Medline] [Order article via Infotrieve] |
73. |
Baynes, J. W.
(2002)
Ann. N. Y. Acad. Sci.
959,
360-367 |
74. | Olshansky, S. J., Hayflick, L., and Carnes, B. A. (2002) Sci. Am. 286, 92-95 |
75. | Dorey, C. K., Wu, G., Ebenstein, D., Garsd, A., and Weiter, J. J. (1989) Invest. Ophthalmol Visual Sci. 30, 1691-1699[Abstract] |
76. | Feeney-Burns, L., Hilderbrand, E. S., and Eldridge, S. (1984) Invest. Ophthalmol. Visual Sci. 25, 195-200[Abstract] |
77. | Holz, F. G., Bellmann, C., Margaritidis, M., Schutt, F., Otto, T. P., and Volcker, H. E. (1999) Graefe's Arch. Clin. Exp. Ophthalmol. 237, 145-152[CrossRef][Medline] [Order article via Infotrieve] |
78. |
Holz, F. G.,
Bellman, C.,
Staudt, S.,
Schutt, F.,
and Volcker, H. E.
(2001)
Invest. Ophthalmol. Visual Sci.
42,
1051-1056 |
79. | Liu, I. Y., White, L., and LaCroix, A. Z. (1989) Am. J. Public Health 79, 765-769[Abstract] |
80. | Pollack, A., Bukelman, A., Zalish, M., Leiba, H., and Oliver, M. (1998) Ophthalmic Surg. Lasers 29, 286-294[Medline] [Order article via Infotrieve] |
81. | Pollack, A., Marcovich, A., Bukelman, A., and Oliver, M. (1996) Ophthalmology 103, 1546-1554[Medline] [Order article via Infotrieve] |
82. | Taylor, H. R., West, S., Munoz, B., Rosenthal, F. S., Bressler, S. B., and Bressler, N. M. (1992) Arch. Ophthalmol. 110, 99-104[Abstract] |
83. | Ham, W. T. J., Mueller, H. A., Ruffolo, J. J. J., Millen, J. E., Cleary, S. F., Guerry, R. K., and Guerry, D. I. (1984) Curr. Eye Res. 3, 165-174[Medline] [Order article via Infotrieve]J. E.S. F. |
84. | Ham, W. T. J., Ruffolo, J. J. J., Mueller, H. A., Clarke, A. M., and Moon, M. E. (1978) Invest. Ophthalmol. Visual Sci. 17, 1029-1035[Abstract] |
85. | Busch, E. M., Gorgels, T. G. M. F., and van Norren, D. (1999) Vision Res. 39, 1233-1247[CrossRef][Medline] [Order article via Infotrieve] |