Eye lens color: formation and function.

Aromatic amino acids are photooxidized by near-ultraviolet light to colored products that are bound very tightly to protein amino groups. The resulting colored proteins absorb near-ultraviolet light more strongly and are rendered more hydrophobic than the untreated compounds, and they fluoresce at 440 nanometers when excited at 360 nanometers. Coloration in the lenses of diurnally active animals (including man) may be caused by this reaction, and senile cataracts may result. Such changes in many other proteins (as in the skin and retina) could lead to more serious consequences.

Inhibition of cell division and growth by a redox series of cyanine dyes.

A series of cyanine dyes used in photography, with reduction potentials from -1.35 to -0.20 volts, were tested for their ability to inhibit mitosis and cell growth in fertilized sea urchin eggs. Low concentrations of dyes with reduction potentials more negative than -1.0 volt generally inhibited mitosis and growth, whereas those with more positive reduction potentials did not. The active dyes penetrated the cell, entered all subcellular compartments, were bound to numerous macromolecules, and inhibited synthesis of macromolecules. Thus mitosis and growth may be retarded with substances that can alter electrochemical activity in cells.

Black light induction of skin tumors in mice.

Albino inbred mice (A/J strain exposed to 40-w black light fluorescent lamps (BLB) for 12 hr a day for up to a year developed inflammatory and hyperplastic responses on hairless ear and tail skin, but not on back skin covered by hair. After 1 year of such exposure, many individuals developed papillomas, carcinomas, and sarcomas on their ears and tails. It may be concluded that black light is a skin carcinogen for A/J mice.

Sunlight and human cataracts.

Studies of the cataractous lenses of humans in three different geographic locations indicate that where the ultraviolet components of sunlight are more intense, dark brown cataracts result at a higher frequency than in locations where they are weak. Individuals exposed to sunlight regularly by virtue of their outdoor occupations seemed to develop this dark brown type of cataract much more frequently than those who work indoors. The biochemical characteristics of all brunescent cataracts were found to be very similar, whether derived from individuals exposed to high or low levels of sunlight. These findings support the idea that exposure to sunlight specifically enhances brunescent cataract development in humans.

Biochemical features of the grey squirrel lens.

The ocular lens of the grey squirrel (Sciurus carolinensis) is an excellent model for studies of eye-light interactions that apply to the human system. In this diurnal animal, lens size, shape, yellow pigmentation, and light absorption properties have important similarities to those of young children. This article describes the observations of soluble to insoluble protein conversion with chronological aging, and the loss of heavier lens crystallins in the internal as compared to the external layers of the lens. Such changes are related to aging, as the older lens material is present in the nuclear core, while the younger lens material is superficial. It describes the subunit peptides of the soluble crystallins and of the extrinsic and instrinsic proteins associated with fiber cell membranes. Squirrel lens fiber membranes release most of their extrinsic peptides in 8 M urea, as do those of other young animals. Due to the presence of near-UV absorbing species of low molecular weight, the squirrel lens has great potential for studies of the effects of near-UV radiation on the lens.

Temporal sequence of changes to the gray squirrel retina after near-UV exposure.

Monocularly aphakic gray squirrels (Sciurus carolinensis) were exposed for 10 min to monochromatic near-ultraviolet radiation (lambda = 366 nm, radiant exposure = 4.3 J/cm2) to determine if their yellow pigmented lens protected retinal tissue from photochemical damage. Eyes were examined from 1 to 30 days after exposure to determine the temporal sequence of retinal damage and the extent of recovery from such exposures. Light microscopy of exposed aphakic retinas revealed irreversible lesions to the photoreceptors. Swelling of inner segments, accumulation of heavy pigment deposits in the PE, presence of macrophages in the subretinal space, and pyknosis of photoreceptor nuclei were observed in the exposed region of the aphakic eye. Eyes exposed to ultraviolet radiation with their lenses intact were devoid of significant retinal lesions. This study represents a model system for studying the potential damaging effects of near-UV radiation to the aphakic eyes of humans.

Effect of chronic near-ultraviolet radiation on the gray squirrel lens in vivo.

The effects of ambient exposure to near-ultraviolet (near-UV) radiation (300-400 nm) on the ocular lens of the diurnal squirrel (Sciurus carolinensis) are reported. Gray squirrels lived in cages illuminated for 12 hr a day with near-UV light (6 mW/cm2, 365 nm) for 1 yr. The non-UV-exposed controls were housed separately. In the lenses of UV-exposed animals, anterior pole changes occurred. Central epithelial cells swelled, disappeared, or underwent proliferation. A band of disoriented degenerating fiber cells was seen in the midcortex, with a degree of liquefaction. When lens protein compartments were separated by centrifugation, water-insoluble but urea-soluble fractions were enhanced in the outer and inner cortex and the nucleus. Both high-performance liquid chromatography and polyacrylamide gel electrophoresis revealed that proteins mainly in the midcortex and nucleus were altered considerably. Evidence of a loss of sulfhydryl compounds (by chemical and Raman spectroscopic analyses) and an increase of protein-thiol mixed disulfides (chemically) was also observed. These data prove that repetitive ambient exposure of diurnal animals to near-UV radiation at subsolar levels damages the lens by interfering with the maintenance of epithelial cells and altering the structural proteins; some of this may be due to the conversion of sulfhydryls to mixed disulfides.

Tinting of intraocular lens implants.

Intraocular lens (IOL) implants of polymethyl methacrylate (PMMA) lack an important yellow pigment useful as a filter in the visual process and in the protection of the retina from short-wavelength radiant energy. The ability to produce a yellow pigment in the PMMA used in IOL implants by exposure to near-ultraviolet (UV) light was tested. It was found that the highly cross-linked material in Copeland lens blanks was tinted slightly because of this exposure. The absorptive properties of lens blanks treated with near-UV light in this way approached that of the absorptive properties of human lenses. This finding shows that it is possible to alter IOL implants simply so as to induce a pale-yellow pigment in them to improve the visual process and to protect the retinas of IOL users.

Lack of photosensitization of ocular tissues by allopurinol.

Based on careful double-blind studies using albino mouse ocular tissues, we conclude that allopurinol does not act as a photosensitizer for ocular tissue damage in mice relative to exposure to environmental near ultraviolet (UV) light. Damage to lens epithelial cells and retinal photoreceptors was equivalent in UV light-exposed mice that were either fed or not fed a dose of allopurinol equivalent to that used by humans in the treatment of gout. There was also no direct in vitro photosensitizing interaction between allopurinol and protein or nucleic acids. We conclude that patients in whom cataracts developed after using allopurinol and exposure to high irradiances of near UV light were most likely affected by the UV light itself.

The role of sunlight in human cataract formation.

This review summarizes and integrates new findings concerning the role of near-ultraviolet radiation, as is present in sunlight and common artificial light sources, in stimulating human and animal cataract formation. Epidemiological and basic research studies are summarized and critical statements concerning them are offered. Although certain questions still remain unanswered, the evidence that near-UV radiation does stimulate cataract formation is very strong. Avoidance of excessive exposure to near-ultraviolet light and the use of protective lenses that filter it out are suggested to prevent the enhancement of human cataract formation by near-UV light.

Structural and functional changes in catalase induced by near-UV radiation.

Part one of this study shows that exposure of purified beef liver catalase in buffered solutions to BL lamps that provide a mixture of 99% UVA and 1% UVB (to be labeled UVA) alters its chemistry and enzymatic activity. Thus, its spectral absorbance lost detail, it aggregated and exhibited a lower isoelectric point and its enzymatic activity was substantially reduced. These photochemically induced changes were increased by irradiation in phosphate buffer or in physiological medium (minimal essential medium) containing riboflavin and tryptophan. Neither alpha-tocopherol nor deferoxamine were protective against these UVA-induced changes in pure catalase. We further investigated the effect of UVA radiation on the activity of catalase in cultured lens epithelial cells and the protective effects of antioxidants. Cultured lens epithelial cells of rabbits and squirrels were exposed to near-UV radiation with representation in the UVA region of 99% and 1% UVB. Catalase assays were done on homogenate supernatants of cells kept dark or UV exposed. In some instances, cells were cultured in medium containing alpha-tocopherol or deferoxamine prior to UV radiation. Comparisons were made between UV-exposed lens cell catalase activity when exposure was done with or without the antioxidants. The UVA radiation was strongly inhibitory to both rabbit and squirrel lens epithelial cell catalase activities. The range of fluxes of near UV radiation was compatible with that which could reach the lens from the sunlit environment. Catalase inactivation was lessened in cells preincubated with alpha-tocopherol and deferoxamine. This suggests that both singlet oxygen and hydroxyl radical formation may be involved in near-UV damage to lens epithelial cell catalase. Such inhibition of catalase by near-UV would enhance H2O2 toxicity and stimulate SH oxidation so as to damage the lens.

Fluorescence studies of lens epithelial cells and their constituents.

Steady-state and time-resolved fluorescence measurements have been made of human and rabbit lens epithelial cells and their total soluble protein. Excitation at 350 nm results in broad fluorescence spectra peaking at 450 nm and stretching into the visible past 650 nm. The fluorescence excitation spectra peak around 350 nm. We assign the species responsible for this absorption and fluorescence as NADPH. Because the absorption of near-UV light (300-400 nm) is responsible for cell damage and death, we postulate that excited states of NADPH are implicated in the mechanisms of cell damage. Preirradiation with 355 nm light leads to a loss of NADPH fluorescence but with no change in decay kinetics. Possible mechanisms for cell damage are explored.

The effect of near-UV light on Na-K-ATPase of the rat lens.

The influence of in vitro near-UV radiation exposure on the physical state of the rat lens and on its membrane-bound Na-K-ATPase activity was investigated. Lens swelling was correlated to the appearance of opacities and the inactivation of the enzyme. The results show a significant decrease in the Na-K-ATPase activity which may be an early change leading to osmotic type cataracts. The dose-effect curves obtained for cortical and epithelial enzymes were different. Since the data do not follow a monoexponential function, the existence of two forms of Na-K-ATPase in the lens is discussed.

DNA damage and repair in rabbit lens epithelial cells following UVA radiation.

Since ultraviolet light may be a contributing factor to cataractogenesis, we investigated the response of the lens epithelium, a potential target for UV insult, to UVA radiation. Cell survival and the induction and repair of DNA single-strand breaks (SSBs) were measured in cultured rabbit lens epithelial cells following UVA exposure. The light was passed through a filter which eliminated wavelengths below 335 nm in order to ensure that the cells were exposed only to UVA. In order to study the effect of various fluences of UVA on cell survival, 2 x 10(6) cells suspended in Tyrode's buffer were exposed to UVA. During all irradiations the cells were maintained at 0.5 degrees C in order to minimize DNA repair. Following UVA treatment, 200 cells were cultured in minimal essential medium containing 10% rabbit serum, and a colony forming assay was used to quantify cell survival. UVA induced cell death in a dose-dependent manner. In additional experiments, confluent epithelial cells on glass slides immersed in Tyrode's buffer were irradiated and SSBs were quantified using the alkaline elution technique. A 30 min exposure to UVA (180 KJ/m2) induced measurable SSBs. An increase in UVA fluence brought about an increase in the number of DNA SSBs. Rejoining of SSBs was measured after the cells were irradiated in Tyrode's for 2 hrs and allowed to repair in the dark for 4 hrs at 36 degrees C in MEM containing 10% serum. Eighty percent of the DNA SSBs were repaired within 4 hrs as determined by analysis of the alkaline elution profile. The repair kinetics were biphasic with an initial fast and subsequently slower component. The results indicate that UVA can induce SSBs in lens epithelial cells, that the cells can repair most UVA-induced SSBs, and that UVA treatment can be toxic to the epithelium.

Effects of near-UV radiation on the protein of the grey squirrel lens.

In vivo exposure of grey squirrels to 40W BLB illumination resulted in alterations in the state of the lens crystallins, mainly in the outer layer of the lens. HPLC revealed an increase of the void volume or crosslinked crystallins and an increase in peptides with molecular weights lower than 20,000 d. In vitro exposure of squirrel lens aqueous extracts to Woods lamp radiation (predominantly 365 nm) led to similar but more exaggerated changes as viewed by high performance liquid chromatography. When viewed by polyacrylamide gel electrophoresis (PAGE), soluble protein crosslinking was also observed. The near-UV absorbing chromophores of low molecular weight present in the lens served as photosensitizers that enhanced the protein changes. Sodium azide inhibited the changes, indicating a role for singlet oxygen in the crosslinking.

Comparative response to UV irradiation of cytoskeletal elements in rabbit and skate lens epithelial cells.

PURPOSE: This work reports a differential effect of ultraviolet A (UVA) irradiation on the three major cytoskeletal structures, actin and vimentin filaments and microtubules of lens cells in primary culture. The effect on cells from lens of the skate (a bottom-dwelling marine elasmobranch) was compared with that on rabbit lens, in order to assess UVA sensitivity as a function of exposure to these wavelengths in the native habitat. METHODS: Exposure intervals of irradiation time up to 6 hours were selected, at fluences from 13.5 to 54.4 J/cm2 and at 365 +/- 45 nm wavelength, to represent mild to moderate physiological levels. Cultures were fixed and processed with anti-alpha-tubulin-FITC and rhodamine phalloidin, or with anti-vimentin FITC and rhodamine phalloidin conjugates. RESULTS: With epifluorescence microscopy, it was found that microtubules were most sensitive to UVA irradiation (in depolymerizing), followed by actin, with vimentin hardly at all affected. Irradiation for 6 hours followed by incubation for 3 days in fresh medium showed no recovery of actin but good recovery of microtubule organizing centers, followed by mitosis in many (rabbit) cells. Skate cells were more sensitive and showed no recovery. CONCLUSIONS: In view of the role of cytoskeletal elements in intracellular structure, cell division and transport, their disruption supports the hypothesis that UVA may damage lens epithelial cells in vivo so as to contribute to cataract formation. In addition, the data suggest that the lenses of animals exposed to sunlight require effective cytoskeletal repair mechanisms to avoid loss of function.

Effects of intermittent UVA exposure on cultured lens epithelial cells.

PURPOSE: This paper addresses the question of whether a single non-lethal dose of UVA radiation or the same dose divided three daily one-third doses have like or unlike effects on the growth and the catalase activity of cultured rabbit epithelial (RLE) and immortalized human epithelial (HLE) cells. METHODS: Non-confluent cultured RLE and HLE cells were used to study the effects of UVA radiation on their growth. The effects of three doses of 3 J/cm(2) given one day apart on cell growth (i.e. live cell number) over a three day period were compared with those of a single 9 J/cm(2 ) exposure. Estimation of live cell numbers was done 24 hrs after each exposure by counting trypan blue exclusive cells using a hemocytometer. Confluent cultures of RLE cells were used to study the effects of UVA radiation on their catalase activity. The effects of three doses of 1.25 J/cm(2) each given one day apart on catalase activity (breakdown of H( 2) O( 2) measured spectro-photometrically at 240 nm) were compared with that of a single 3.75 J/cm(2) exposure. RESULTS: A single 9 J/cm(2) dose of UVA reduced the cell growth to only 10% of controls after 24 hrs. By three days after a single 9 J/cm( 2) exposure, the number of live cells was only 70% of controls. Three intermittent exposures of 3 J/cm(2) each for three consecutive days reduced the cell number to 76% of controls. Similar results were obtained for HLE cells. Little if any recovery of cell numbers occurred after three intermittent exposures. A single dose of 3.7 J/cm(2) reduced RLE catalase activity to 20% of controls by one day. By three days after exposure, catalase activity returned to 90% of controls. After three intermittent exposures to 1.25 J/cm(2) each, over three days, RLE catalase activity was reduced to 75% of the controls. There appeared to be no recovery within two additional days. CONCLUSIONS: While single below lethal doses of UVA allow no recovery of RLE or HLE cell growth, partial recovery does occur after several daily intermittent exposures. Recovery of the catalase activity by RLE cells does occur after single sub lethal exposures, but intermittent exposures allow no recovery. The recovery of lens epithelial cell growth and catalase activity from UVA damage varies with the exposure regimen.

Lipids of human lens fiber cell membranes.

A study of the membranes of human lens fiber cells revealed a very high protein to lipid ratio, which tended to increase with aging and in brunescent cataract. Phospholipids were more abundant than cholesterol, cholesterol esters, and other neutral lipids. With aging and cataract formation, a marked decrease in membranes phospholipid content occurred. Sphingomyelin was present in highest amount. Phosphatidylethanolamine, phosphatidylcholine, lysophosphatidyl-ethanolamine, phosphatidylserine, and phosphatidylglycerol were also present. Cholesterol represented approximately 40% of the total lipids. Saturated and unsaturated fatty acids having 16 to 24 carbons were present. The lipid composition varied according to the portion of the lens examined and the state of the lens. The results do not support a conclusion that lipid peroxidation represents a major mechanism of membrane damage that contributes to cataract formation, since there is no decrease in unsaturated fatty acids with age or in cataractous lenses. We suggest that the aggregation of soluble proteins, and their association with lens membranes, and altered membrane function due to the loss of phospholipids are important processes leading to loss of transparency.

Measurement of oxygen production by in vitro human and animal lenses with an oxygen electrode.

PURPOSE: This paper describes an advantageous method of measuring the activity of the enzyme catalase, which has an important antioxidative role in the lens. This method allows the measurement of catalase in whole lenses. METHODS: Exposure to UVA (99% UV-A) radiation was used to stress animal and human (Eye Bank) lenses in vitro. The ability of lens catalase to convert H2O2 into O2 was measured directly, using an oxygen electrode and meter. This method is very specific, as catalase is the only enzyme that converts H2O2 to O2. RESULTS: Catalase in the lenses of humans, rabbits, and squirrels catalyzed the production of O2 from H2O2 very efficiently. The anterior equatorial regions of these lenses were the most active O2 producing areas. More than 95% of lens catalase activity was found in the capsule-epithelium layer. Exposure to UVA radiation, up to approximately 100 J/cm2 in 18 h, strongly inhibited O2 production from 0.77 mM H2O2 by the lenses. Catalase activity decreased with increasing age. Mixed cataractous human lenses produced O2 from H2O2 at only 60% of the rate of normal lenses of similar ages. Nuclear cataracts produced O2 at only 75% of the rate of normal lenses. Alpha-tocopherol (10(-5) M) protected lens catalase activity strongly. Alpha-tocopherol is known to accumulate in and protect against cell membrane peroxidation, and against singlet oxygen formation. These oxidative mechanisms appear to contribute to catalase photoinactivation. CONCLUSIONS: The method described indicated that catalase is a crucial antioxidative enzyme in the normal lens. Its inactivation could upset the oxidation-reduction balance in the lens and stimulate lens opacification.

Sub-threshold near-UV radiation effects on aphakic guinea pig retinas.

Monocularly aphakic guinea pigs (Cavia porcellis), prepared by removing the lens by phacoemulsification, were maintained under near-UV lighting conditions for several months. Exposure to near-UV energy was at much lower irradiance levels than that of sunlight, and was at lower than the threshold level for near-UV damage to the aphakic monkey retina as reported by Ham, et al (1). In some aphakic eyes, regenerated lens-like structures formed which scattered light appreciably. After increasing light exposure periods, the eyes of control and irradiated animals were studied histologically. Other animals were periodically examined by electroretinographic (ERG) techniques. While there was no observable histopathological damage, aphakic-UV irradiated eyes with little or no lens regrowth exhibited depressed b-waves, late time constants and altered wave forms when compared with control eyes. The results demonstrate that ambient near-UV light exposure can adversely influence retinal electrical activity in aphakic eyes at irradiance levels below threshold for morphological damage. The protective function of the lens is also supported by these findings.