Lens UVA photobiology.

Although UVA radiation was considered to be benign as a factor that could contribute to cataract formation, the studies briefly summarized herein show that UVA may provide the more damaging radiant energy involved in the formation of human cataract. Of the UVA impinging onto the eye from sunlight, a sufficient dose does reach into the lens to enhance cataractous changes. The adverse physiological and biochemical effects on lens epithelial cells and their ability to differentiate into clear adult lenses include cell growth inhibition, cell membrane and cytoskeletal anomalies, and enzyme inactivation. Many of the damaging events are related to the excitation of sensitive UVA absorbing chromophores which generate singlet oxygen and free radicals. This process enhances lens oxidative stress. Some protection against this damage is provided by vitamin E and tea polyphenols. Thus, an imbalance between photooxidative stress, antioxidant protection, and repair processes would increase the potential to produce cataracts.

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.

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.

Replication protein A stimulates long patch DNA base excision repair.

Two pathways for completion of DNA base excision repair (BER) have recently emerged. In one, called short patch BER, only the damaged nucleotide is replaced, whereas in the second, known as long patch BER, the monobasic lesion is removed along with additional downstream nucleotides. Flap endonuclease 1, which preferentially cleaves unannealed 5'-flap structures in DNA, has been shown to play a crucial role in the long patch mode of repair. This nuclease will efficiently release 5'-terminal abasic lesions as part of an intact oligonucleotide when cleavage is combined with strand displacement synthesis. Further gap filling and ligation complete repair. We reconstituted the final steps of long patch base excision repair in vitro using calf DNA polymerase epsilon to provide strand displacement synthesis, human flap endonuclease 1, and human DNA ligase I. Replication protein A is an important constituent of the DNA replication machinery. It also has been shown to interact with an early component of base excision repair: uracil glycosylase. Here we show that human replication protein A greatly stimulates long patch base excision repair.

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.

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.

Human RAD2 homolog 1 5'- to 3'-exo/endonuclease can efficiently excise a displaced DNA fragment containing a 5'-terminal abasic lesion by endonuclease activity.

Repair of abasic lesions, one of the most common types of damage found in DNA, is crucial to an organism's well-being. Studies in vitro indicate that after apurinic-apyrimidinic endonuclease cleaves immediately upstream of a baseless site, removal of the 5'-terminal sugar-phosphate residue is achieved by deoxyribophosphodiesterase activity, an enzyme-mediated beta-elimination reaction, or by endonucleolytic cleavage downstream of the baseless sugar. Synthesis and ligation complete repair. Eukaryotic RAD2 homolog 1 (RTH1) nuclease, by genetic and biochemical evidence, is involved in repair of modified DNA. Efficient endonucleolytic cleavage by RTH1 nuclease has been demonstrated for annealed primers that have unannealed 5'-tails. In vivo, such substrate structures could result from repair-related strand displacement synthesis. Using 5'-tailed substrates, we examined the ability of human RTH1 nuclease to efficiently remove 5'-terminal abasic residues. A series of upstream primers were used to increasingly displace an otherwise annealed downstream primer containing a 5'-terminal deoxyribose-5-phosphate. Until displacement of the first annealed nucleotide, substrates resisted cleavage. With further displacement, efficient cleavage occurred at the 3'-end of the tail. Therefore, in combination with strand displacement activity, RTH1 nucleases may serve as an important alternative to other pathways in repair of abasic sites in DNA.

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.

Environmental near-UV radiation and cataracts.

This report compares sunlight UV-A and UV-B fluxes in the Northeastern United States that reach the crystalline lens with thresholds that cause lens damage. The fluxes of UV-A and UV-B radiation that reach earth to penetrate the cornea and to reach the lens were calculated. Ten hours of continuous UV-A exposure or 23 min of UV-B would exceed the rabbit cornea threshold for photokeratitis. The lens threshold would be reached by 26 h of UV-A or 245 h of UV-B continuous exposure. The sequence of UV-induced damage follows: (1) UV-B photokeratitis; (2) UV-A photokeratitis; (3) UV-A lens damage; and (4) UV-B lens damage.

Damage to cultured lens epithelial cells of squirrels and rabbits by UV-A (99.9%) plus UV-B (0.1%) radiation and alpha tocopherol protection.

The purpose of this research is to observe the near-UV radiation induced damage to cultured rabbit and squirrel lens epithelial cells as related to destruction and alterations of specific biochemical targets in the cells and to determine protective effects on the cells and targets that are provided by alpha-tocopherol. Confluent monolayers of cultured rabbit and squirrel lens epithelial cells were exposed to black light (BL) lamps, which emit predominantly UV-A radiation. These cells received a mixture 3 J/cm2 of UV-A and 4 mJ/cm2 of UV-B per h. This mixture is termed near UVA (i.e.: predominantly UV-A). Cells were exposed in Tyrode's or in MEM without or with alpha-tocopherol added at 2.5-10 micrograms/ml. Analyses of cell viability and survival, the physical state of cytoskeletal actin, and the activities of Na-K-ATPase and catalase were made. Exposure to near UVA damaged these cells as measured by vital staining and colony forming ability. Pretreatment with alpha-tocopherol decreased the magnitude of near UVA cytotoxicity. Near UVA exposure in MEM always produced more damage to the cells and biochemical targets than in Tyrode's. Cytoskeletal actin was degraded and the activities of Na-K-ATPase and catalase were markedly inhibited by UV-exposure. All of these targets were at least partially protected by alpha-tocopherol in the medium. Without alpha-tocopherol added to the media, the viability and survival of the cells did not recover even after 25 h of incubation. Cell viability was better protected from near UVA by alpha-tocopherol than was the ability to grow into colonies. This indicates that alpha-tocopherol protects actin, catalase, and Na-K-ATPase from near UVA damage.

Effects of near UV radiation and antioxidants on the response of dogfish (Mustelus canis) lens to elevated H2O2.

In vitro exposure of dogfish (Mustelus canis) lenses to near-UV energy not incompatible with that of the environment, causes an opalescence that is not present in unirradiated lenses or those irradiated after soaking in alpha-tocopherol or deferoxamine. The ability of whole lenses to destroy H2O2, as shown by their ability to produce O2 bubbles in H2O2 containing media, is markedly diminished by UV exposure without these antioxidant/free radical scavengers added. The lens capsule epithelium is the major site of catalase activity. Bubble formation was prevented by presoaking the lenses in 3-amino-triazole (3-AT), a potent catalase inhibitor. Analytical measurements confirmed the above observations. Near-UV inhibition of catalase may allow H2O2 in the aqueous humor to damage the lens by exerting oxidative stress.

Classification and protein distribution in a series of intracapsular cataracts.

The crystallin profiles of the cortices and nuclei of intracapsular cataractous lenses were studied by high pressure liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), and dot blotting. The complete personal and medical history of 381 patients and the Cooperative Cataract Research Group (CCRG) classification of each were obtained. Few statistically significant associations between patient personal history and cataract types were found. Protein profiles of selected cataracts which had specifically located opacities (i.e., nuclear only, cortical only, etc.) were studied in detail. Sodium dodecyl sulphate-PAGE revealed few differences in lens-soluble proteins between cataractous and normal cortices or nuclei. By HPLC, the proteins of cataractous cortices and their nuclei differed very little from age-matched controls. The cortical proteins of nuclear cataracts appeared normal. However, two major alterations of proteins were observed in the nuclei of dense nuclear cataracts. Increased high molecular weight protein and increased components with molecular weights < 20,000 Da were found in cataractous nuclei as compared with normal age-matched control lens nuclei. Dot blot (immunological) analyses identified the crystallins of normal lenses that eluted from the HPLC column more efficiently than those of cataractous lenses. Cortical protein HPLC samples had the most specificity. Nuclear protein HPLC samples of older normal and cataractous lenses had little if any alpha crystallin specificity in the void volume peak. A relation between the presence of opacities and changes in molecular weight distribution of crystallins was found in the opaque nuclei but not in the opaque cortices.(ABSTRACT TRUNCATED AT 250 WORDS)