(Received for publication, April 4, 1995; and in revised form, May 25, 1995)
From the
Exposure of the eye to intense light, particularly blue light, can cause irreversible, oxygen-dependent damage to the retina. However, no key chromophores that trigger such photooxidative processes have been identified yet. We have found that illumination of human retinal pigment epithelium (RPE) cells with light induces significant uptake of oxygen that is both wavelength- and age-dependent. Analysis of photoreactivity of RPE cells and their age pigment lipofuscin indicates that the observed photoreactivity in RPE cells is primarily due to the presence of lipofuscin, which, under aerobic conditions, generates several oxygen-reactive species including singlet oxygen, superoxide anion, and hydrogen peroxide. We have also found that lipofuscin-photosensitized aerobic reactions lead to enhanced lipid peroxidation as measured by accumulation of lipid hydroperoxides and malondialdehyde in illuminated pigment granules. Hydrogen peroxide is only a minor product of aerobic photoexcitation of lipofuscin. We postulate that lipofuscin is a potential photosensitizer that may increase the risk of retinal photodamage and contribute to the development of age-related maculopathy.
Although the anterior eye tissues, the cornea and lens, filter
out the most damaging components of solar radiation(1) , the
retina can be subjected to intense illumination from focal light that
includes the relatively energetic photons from the blue part of the
solar spectrum (2) . As a result, photic retinopathy may
develop(3) , and this process will be enhanced by the presence
of oxygen(4) . Solar radiation has also been implicated in the
development of ARM()(5) , a degenerative disease
that is recognized as the predominant cause of blindness in people over
60 in many developed Western countries(6, 7) . The
primary lesion associated with photoreceptor degeneration and loss of
vision in ARM is believed to be located in the RPE(8) .
However, no key chromophores that trigger primary events in either
light-induced phenomena have been identified yet(9) . Among
several potential photosensitizing molecules that are normally present
in human RPE, melanin and lipofuscin deserve special attention. These
two pigments, present in the cell exclusively in the particle form,
have broad optical absorption bands(10) . Their biogenesis is
very different; whereas RPE melanin appears very early in the fetal
development(11) , RPE lipofuscin becomes apparent only during
the second decade of life and accumulates with age(12) . Even
though melanin can photogenerate superoxide anion and hydrogen
peroxide(13) , it is believed that under typical in vivo conditions, melanin acts as an efficient antioxidant(10) .
In this regard little is known about the photosensitizing abilities of
lipofuscin, except that this age pigment stimulates photoreduction of
cytochrome c under aerobic conditions, which suggests possible
photoformation of superoxide anion(14) .
To determine whether the human RPE exhibits any substantial photoreactivity that may lead to retinal phototoxicity, we measured action spectra of photoinduced oxygen uptake and photoformation of hydrogen peroxide for RPE cells. In a search for the major retinal chromophores responsible for the observed RPE photoreactivity, we studied aerobic photoreactions in suspensions of purified retinal lipofuscin and melanin granules and identified some products of the interaction of photoexcited lipofuscin with oxygen.
Rates of photo-dependent oxygen uptake were obtained by
measuring kinetics of oxygen concentration changes in irradiated
samples. In these measurements ESR oximetry was employed using
10M CTPO or mHCTPO as the nitroxide spin
probes(19, 20) . Samples in flat quartz cells (0.25
mm) were illuminated in situ, in a resonant cavity, at ambient
temperature. Instrument settings were: microwave power, 1 mW;
modulation amplitude, 0.1 G (for mHCTPO) or 0.05 G (for CTPO).
Rates
of HO
formation were determined at 4 °C in
order to minimize decomposition of H
O
. Samples
were illuminated in a thermostated optical chamber of a miniature
oxygen monitor (Instech Laboratories, Horsham, PA). Aliquots of granule
suspension were taken during sample illumination, and the concentration
of hydrogen peroxide was measured using an oxidase electrode (Yellow
Spring Instruments Co. (YSI), Yellow Springs, OH) as described
previously(21) .
MDA was analyzed using
the thiobarbituric acid assay followed by HPLC separation and
fluorometric determination ( = 523 nm,
= 550 nm) of MDA-thiobarbituric acid complex,
as described elsewhere(25) .
Figure 1:
Action spectra for photo-dependent
oxygen uptake in suspensions of isolated RPE cells from human donors
(60-70-year-olds, ), and bovine (the non-pigmented tapetal
fundus (
) and the pigmented non-tapetal fundus (
). Samples
were equilibrated with a mixture of 50% N
and 50% air.
Rates of photo-dependent oxygen uptake were obtained by ESR oximetry
using CTPO as a spin probe. Data were normalized to equal protein
content (mg/ml) and fluxes of incident photons
(einstein/s/m
). Values are the means ± S.D. of
triplicate experiments. Dark consumption of oxygen, under the
conditions employed, was negligible. Inset, ESR spectra of
melanin radicals in human (top) and bovine non-pigmented (middle) and pigmented (bottom) RPE cells. Instrument
settings: microwave power, 20 µW; modulation amplitude, 3
G.
Analysis of blue light photoreactivity observed in human RPE cells, isolated from individuals 16-97 years of age, demonstrated a marked increase in the rate of photo-dependent oxygen uptake as the donor age increased (Fig.2A), and only a modest change in the corresponding rate of hydrogen peroxide photoproduction (Fig.2B). The data also indicate that hydrogen peroxide is only a minor product of aerobic photoactivation of human RPE cells, accounting for no more than 6% of the oxygen consumed.
Figure 2:
The
effect of donor age on the consumption of oxygen (A) and the
formation of hydrogen peroxide (B) in irradiated samples
(408-495 nm, 220 mW/cm) of isolated human RPE cells.
Data were normalized to 1 mg of protein/ml of phosphate-buffered
saline. Solidline represents the best linear
approximation of the experimental data; dottedlines,
95% confidence interval.
Figure 3:
Action spectra for photo-dependent oxygen
uptake (A) and photoformation of hydrogen peroxide (B) in air-equilibrated suspensions of lipofuscin () and
melanosomes (
) isolated from human RPE cells. Rates of
photo-dependent oxygen uptake were obtained by ESR oximetry using
mHCTPO as a spin probe. Data were normalized to equal concentration of
granules (10
granules/ml) and fluxes of incident photons
(einstein/s/m
). Values are mean ± S.D. of at least
four experiments. Dark consumption of oxygen, under the conditions
employed, was negligible.
Figure 4:
Chromatographic separation of cholesterol
hydroperoxides from photooxidized liposomes fused with lipofuscin
granules: no illumination (a) and after illumination with blue
light for 18 min (b), 65 min (c) or 65 min in the
absence of lipofuscin (d). Peak identities are as follows: 1, hydroperoxide of cis-11-eicosenoic acid methyl ester as
internal standard; 2, 7-OOH; 3, 7
-OOH; 4, 5
-OOH; 5, 6
-OOH; 6, 6
-OOH; 7, not identified lipofuscin derived solute. Samples were
illuminated with blue light, as described under ``Materials and
Methods.''
Figure 5:
ESR signals of DMPO spin adducts in
illuminated aqueous suspension of human RPE lipofuscin (A),
and simulated spectra of DMPO-OH, DMPO-OOH, and their superposition
(1:0.7 ratio of the corresponding species) (B). In A,
the first (top) signal was recorded in dark (0 min
illumination time), while the consecutive signals were recorded after
indicated illumination time (in minutes). Lipofuscin was isolated from
human donors 60-70 years of age and suspended in 50 mM phosphate buffer with 200 mM DMPO. ESR samples were
irradiated with blue light (408-480 nm, irradiance, 38
mW/cm). Instrumental settings: time constant, 0.128 s;
sweep time, 120 s; microwave power, 20 mW; modulation amplitude, 1.25
G.
Figure 6: The effect of inclusion of superoxide dismutase (0.10 mg/ml) (A), both superoxide dismutase and catalase (0.15 mg/ml, about 1900 units/ml) (B) and oxygen removal (C) on formation of DMPO spin adduct during illumination of ESR sample containing lipofuscin suspension in the presence of DMPO. Experimental conditions and instrumental settings the same as in Fig.5.
The mechanism of DMPO-OH generation during
aerobic illumination of lipofuscin is unknown at present.
Interestingly, the spin adduct appears to be
HO
-independent, since it is also formed in the
presence of catalase (Fig.6B).
Figure 7:
Formation of hydroperoxides (A)
and malondialdehyde (B) during aerobic illumination with blue
light (408-495 nm, 220 mW/cm) of human lipofuscin
isolated from 60-70-year-old donors. Values are means ±
S.D. of triplicate experiments.
The observed photoreactivity of human RPE cells is, to a
significant extent, determined by their lipofuscin content. This is
evident from the comparison of action spectra of photo-dependent oxygen
uptake in RPE cells and in purified lipofuscin granules ( Fig.1and Fig. 3). Both action spectra exhibit
significant similarities in that the efficiency of illuminating light,
to induce oxygen uptake and generation of HO
,
falls steeply with the wavelength between 300 and 600 nm. Thus, blue
light seems to be the most efficient radiation under physiologically
relevant conditions. This is because ultraviolet radiation is
completely filtered out by the cornea and lens of the adult human eye
and, as a result, virtually no light below 400 nm is transmitted to the
retina(1) .
Retinal melanin does not seem to be the major contributor to photo-dependent oxygen uptake in either human RPE cells or bovine pigment epithelium, which may prove that lipofuscin is a major retinal chromophore that photoexcitation leads to consumption of oxygen. Such a conclusion about the dominant role of lipofuscin in aerobic photoreactions of human RPE is further supported by the distinct changes of the RPE photoreactivity with age of the donors (Fig.2). The increase in the rate of photo-dependent oxygen uptake between the second and ninth decade of life, observed in this work, is consistent with the reported accumulation of retinal lipofuscin with age(12) . The presence of lipofuscin, or its precursors, may also account for photo-dependent oxygen uptake, albeit reduced, in bovine RPE cells. It must be stressed that the molecular nature of chromophores of human RPE lipofuscin responsible for its photoreactivity remains unknown.
Melanolipofuscin granules exhibit aerobic photoreactivity which is intermediate between that of lipofuscin and melanin. However, the contribution of melanolipofuscin to photo-induced consumption of oxygen, observed in RPE cells, is difficult to evaluate. One has to consider that in human RPE a significant number of these complex granules become apparent in individuals 60-100 years of age, when lipofuscin granules are more numerous than any other pigment granules (31) .
Since
superoxide anion has been detected in irradiated lipofuscin samples (Fig.5), it is conceivable that O may be a major intermediate
in lipofuscin-photosensitized generation of HO
.
However, our data clearly show that in isolated pigment granules
photo-induced consumption of oxygen is not accompanied by
stoichiometric production of H
O
(Fig.3). Since similar results have been observed for RPE
cells, we may conclude that an identical or closely related mechanism
of photo-dependent oxygen uptake occurs in isolated pigment granules
and in RPE cells.
Molecular oxygen consumed during illumination of RPE cells may in part be utilized in lipid peroxidation processes. This can be inferred from the detection of hydroperoxides that were induced by blue light in purified lipofuscin granules (Fig.4), even though the yield of photoformation of lipofuscin-derived hydroperoxides, as well as that of the accompanying accumulation of MDA, has not been determined in this work.
An important question is
the identity of reactive species that initiate lipofuscin-sensitized
photoperoxidation, and lead to the observable photoconsumption of
O. Two major mechanisms of lipid peroxidation should be
considered: the free radical and singlet oxygen pathways(27) .
Our ESR spin-trapping data and the observed formation of both
7
-OOH and 7
-OOH epimers, seem to indicate that the free
radical mechanism operates. Although superoxide anion is rather a
nonreactive species, and in aqueous media acts predominantly as a
moderate reductant(32) , its protonated form, the hydroperoxyl
radical, can attack double-allylic hydrogen atoms in a number of
unsaturated fatty acids(33) . However, powerful antioxidants,
such as butylated hydroxytoluene and
-tocopherol, only partially
inhibited the photo-dependent uptake of O
. It may suggest
that peroxidation, induced in lipofuscin granules by light, is mostly a
non-radical process or a mixed-type process, in which singlet oxygen is
involved. In such processes oxygen is consumed during the primary
formation of lipid hydroperoxides, while the secondary
chain-propagating reactions are less efficient(34) . However,
at this point it cannot be ruled out that the relatively small
inhibitory effect of tested antioxidants on photo-dependent uptake of
oxygen, is also due to their inefficient penetration of the lipofuscin
granule.
Due to the particle character of lipofuscin and the
molecular heterogeneity of its main constituents, quantitative
determination of reactive species, which may be generated by lipofuscin
granules, is rather difficult. One has to realize that any short-lived
species, generated within the lipofuscin granule, are likely to be
scavenged or quenched by several reactive lipofuscin components, before
having chance to diffuse out of the granule. Therefore, the yield of
photoformation of reactive species, such as singlet oxygen, determined
by its interaction with a selective acceptor, homogeneously distributed
in the entire sample volume, would be severely underestimated. The
situation can be improved if a suitable acceptor of
O(
) is preferentially located
in the granule or in its close proximity. Indeed, singlet oxygen
photoformation has unambiguously been detected by the chemical method
using cholesterol as an exogenous singlet oxygen acceptor (Fig.4). However, at this stage we cannot determine quantum
yield of the formation of O
(
)
by lipofuscin.
In conclusion, human RPE lipofuscin exhibits substantial photoreactivity and, under aerobic conditions, is able to form several potentially cytotoxic species. It is important to stress that these are typical conditions for a normally functioning eye; the RPE in vivo is constantly exposed to high oxygen tension and must endure substantial fluxes of light that reach the posterior eye segments(35) . The extent to which the long term effects of lipofuscin photoreactivity affect the structural integrity and function of the RPE-retinal complex in vivo has yet to be elucidated, as has its role in ARM.