Photoreactions of human lens monomeric crystallins.

Human lens beta s- and gamma A-crystallins exhibit very similar tryptophan fluorescence emission maxima (329 nm). gamma A isolated from infant human lenses is photo-oxidized by 300 nm irradiation and forms water-insoluble aggregates; beta s or gamma A from young human lenses form a small amount of water-soluble crosslinked species. At least part of the mechanism of photodamage by 300 nm irradiation is photogeneration of the oxidant H2O2 via the generation of O2- radical, this reaction occurs via photosensitization by the tryptophan photo-oxidation product N-formylkynurenine (N-FK) or related species. These results indicate that even though the tryptophan residues of beta s- and gamma A-crystallins are in hydrophobic (buried) microenvironments as compared to those of the alpha- and beta-crystallins, the photogeneration of N-FK is sufficient to produce O2- and H2O2.

Spectroscopic studies on human lens crystallins.

Human lens crystallins were studied by absorption, circular dichroism and fluorescence spectroscopy. The absorption spectra in the near-ultraviolet region show some differences in intensity, but spectral features are similar, except for the alpha-crystallin, which gives a fine structure due to phenylalanine between 250 and 270 nm. Tryptophan fluorescence and near-ultraviolet circular dichroism indicate that tryptophan residues are more exposed in alpha-crystallin than in either beta- or gamma-crystallin, and that the degree of exposure decreases in the order of alpha less than beta 1 greater than beta 2 greater than beta 3 greater than gamma. The far ultraviolet CD suggests that these proteins exist mainly in a beta-sheet conformation and that the amount does not vary much among them. The greater exposure of the tryptophan residues in the high-molecular-weight crystallins may reflect greater unfolding in their protein domains. Spectroscopic measurements are thus useful in predicting protein tertiary structure in the absence of the complete sequence and X-ray data. The fact that the high-molecular-weight proteins exist in a more unfolded state may render them more vulnerable to exogeneous insults, and these effects may be studied by spectroscopic measurements.

Generation of oxidants in the near-UV photooxidation of human lens alpha-crystallin.

In this study we report on the generation of superoxide anion (O2-) and hydrogen peroxide in the near-UV irradiation of human lens alpha-crystallin by monochromatic light at 300 nm. Photolysis of human lens alpha-crystallin at 300 nm, at irradiances similar to those encountered in sunlight causes an alteration of protein tertiary structure, a loss of tryptophan fluorescence and increase of nontryptophan fluorescence. The nontryptophan fluorescence is likely to be due to the photooxidation of tryptophan to N-formylkynurenine (N-FK or related species), which is a good photodynamic sensitizer, has significant absorption at 300 nm, and can thus react via its triplet state with O2 to generate 1O2 or with reducing substrates (amino acids of the protein) to generate free radicals. The latter, in the presence of O2 can lead to the generation of O2- and H2O2. These species have been directly assayed in this study in photolyzed solutions of fetal, young and old human lens alpha-crystallin. The addition of superoxide dismutase (SOD) to the protein solution prior to photolysis increased the amount of H2O2 generated by 3- to 4-fold. This observation not only provides definitive evidence for the photogeneration of O2-, but also indicates that only a fraction of this species is transformed into H2O2 in the absence of SOD. Significant amounts of O2- and H2O2 were formed by 340 nm irradiation of old human lens alpha-crystallin, in which the basal level of N-FK is high. The role of 1O2 in these photoreactions has been studied by investigating the quenching effect of azide and the enhancing effects of D2O on the rate of loss of tryptophan fluorescence yield and the effect of azide on the rate of H2O2 generation.

Expression of growth control and differentiation genes in human lens epithelial cells with extended life span.

PURPOSE: Peptide growth factors including hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) are mitogens for many cell types and may act as regulators of lens epithelial cell growth and differentiation. The present study was undertaken to investigate the expression of growth factor receptors and crystallin genes in the human lens epithelial cell line HLE B-3, created by infection with Adeno12-simian virus 40 (Ad12-SV40) hybrid virus. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers was used to detect transcripts, and Southern and western blot analyses were used for identification of gene products. Functional analysis of PDGF receptor was performed by measuring the effect of PDGF-BB on Ca2+ release, cell growth, and western blot analysis, by using an antiphosphotyrosine antibody. RESULTS: Human lens epithelial B-3 cells expressed the growth factor receptors HGF-R, EGF-R, and PDGF-Rbeta, but not PDGF-Ralpha, and also expressed the oncogenes H-ras and raf and the growth inhibitor transforming growth factor-beta1. Stimulation of PDGF-Rbeta with PDGF-BB in HLE B-3 cells increased phosphorylation of the receptor, was associated with an increase in intracellular Ca2+ levels, and produced a small increase in cell growth. In addition, HLE B-3 cells expressed transcripts for alphaA-, alphaB-, and betaB2-crystallins, and expressed the corresponding proteins. The transcripts for alphaA-crystallin decreased markedly at higher passages. CONCLUSIONS: The above findings suggest that the increased growth potential of human lens epithelial cells by Ad12-SV40 infection maintained certain lens-specific properties and response to PDGF.

Regulation of GSH in alphaA-expressing human lens epithelial cell lines and in alphaA knockout mouse lenses.

PURPOSE: To study the mechanism of regulation of GSH in HLE-B3 cells expressing alphaA-crystallin (alphaA) and in alphaA knockout mouse lenses. METHODS: GSH levels and maximal rates of GSH synthesis were measured in immortalized, alphaA-transfected HLE-B3 cells containing varying amounts of alphaA. The mRNA and protein for the rate-limiting enzyme for GSH synthesis, gamma-glutamylcysteine synthetase (GCS), were also determined in alphaA- and mock-transfected cells by Northern blot analysis and Western blot analysis of heavy (GCS-HS) and light (GCS-LS) subunits. The effect of absence of alphaA and alphaB on lens GSH concentrations was evaluated in whole lenses of alphaA knockout and alphaB knockout mice as a function of age. GCS-HS mRNA and protein were determined in young, precataractous and cataractous alphaA knockout lenses. RESULTS: GSH levels were significantly higher in HLE-B3 cells expressing alphaA- compared with mock-transfected cells and were correlated positively with alphaA content. Mean rate of GSH synthesis was also higher in alphaA-expressing cells than in mock controls (0.84 vs. 0.61 nmol. min(-1) per mg protein, respectively). GCS-HS mRNA and GCS-LS mRNA were approximately twofold higher in alphaA-expressing cells, whereas the heavy and light GCS subunit proteins increased by 80% to 100%. In alphaA(-/-) mouse lenses, GSH level was not different from that of wild type up to 2 months from birth, after which it dropped to approximately 50% of controls. On the other hand, GCS-HS and GCS-LS proteins showed a significant decrease before cataract formation as early as 15 days after birth. GSH level in cataract-free alphaB(-/-) lenses was similar to that of wild type for up to 14 months. CONCLUSIONS: Expression of alphaA caused an increase in cellular GSH, in part, because of an increase in mRNA and protein of both GCS subunits. GSH levels decreased with increasing age in cataractous alphaA(-/-) lenses but not in the noncataractous alphaB(-/-) lenses. It is suggested that neonatal precataractous lenses (with normal GSH and decreased GCS) may maintain their GSH level by other compensatory mechanisms such as increased GSH transport.

The role of prostaglandins E2 and F2 alpha in ultraviolet radiation-induced cortical cataracts in vivo.

PURPOSE: Previous work has shown that exposure of lens epithelial cells or rabbit eyes in vivo to ultraviolet B (UVB) radiation enhanced prostaglandin (PG)E2 synthesis. Such enhanced PGE2 synthesis was related to the increased DNA synthesis that followed UVB exposure. The current study examined the relationship between enhanced prostaglandin synthesis and UVB-induced cataract formation. METHODS: Seventy albino (New Zealand white) rabbit eyes were exposed to UVB radiation in vivo. Fluence of radiation at the cornea was 2.8 J/cm2, 5.6 J/cm2, or 11.2 J/cm2. Eyes were examined 24 hours after UVB exposure and for as long as 10 days by slit lamp biomicroscopy. Mass spectrometry was used to measure PGE2, PGF2 alpha, and 6-keto-PGF1 alpha content of the lens and iris-ciliary body using authentic standards. To determine the effect of inhibition of prostaglandin synthesis on UVB-induced cataract formation, animals were given indomethacin intraperitoneally. Other pharmacologic agents, such as PGE2, PGF2 alpha, and misoprostol, were applied topically to the eye. The effect of UVB on K+ pump was determined by incubating isolated lenses with [86Rb+]. RESULTS: Twenty-four hours after UVB exposure, PGE2 and PGF2 alpha concentrations in aqueous humor were increased by 100- and 30-fold, respectively. Lens PGE2 and PGF2 alpha increased by 6- and 4-fold, respectively, after UVB radiation exposure. Pretreatment of animals with indomethacin prevented the rise in lens and aqueous humor PGE2 and PGF2 alpha levels. Furthermore, indomethacin was partially protective against UVB cataract formation and lowered cataract severity from stage 3 to stage 1, but it did not prevent UVB-induced lens changes completely. Topical application of PGE2 before UVB exposure completely prevented cataract formation in the UVB-exposed eye. In contrast, topical administration of PGF2 alpha increased cataract severity. UVB-induced cataract formation preceded changes in [86Rb]+ uptake in lenses subsequently incubated in K(+)-free Tyrode's. CONCLUSIONS: Enhanced synthesis of cyclooxygenase products of arachidonic acid metabolism in the lens is associated with UVB-induced cataract formation in albino rabbit eyes, and inhibition of cyclooxygenase by indomethacin decreased the severity of cataracts. PGE2, the principal arachidonic acid metabolite, appears to have a protective role because pretreatment of the eye with topical PGE2 completely prevented UVB-induced cataract formation, whereas PGF2 alpha increased the severity of the cataract. The evidence presented for a role of PGF2 alpha in the development of cataract suggests that caution be exercised in the use of PGF2 alpha derivatives in the therapy of glaucoma.

Glutathione transport in immortalized HLE cells and expression of transport in HLE cell poly(A)+ RNA-injected Xenopus laevis oocytes.

PURPOSE: To determine reduced glutathione (GSH) transport in cultured human lens epithelial cells (HLE-B3) and plasma membrane vesicles and to study the expression of GSH transport in Xenopus laevis oocytes injected with poly(A)+ RNA from HLE-B3 cells. METHODS: Confluent HLE-B3 cells pretreated with 10 mM DL-buthionine sulfoximine and 0.5 mM acivicin were used in GSH uptake studies. The uptake of 35S-GSH was performed for 30 minutes in either NaCl medium (Na+-containing) or choline chloride medium (Na+-free) at 37 degrees C and 4 degrees C. The molecular form of 35S uptake was determined by high-performance liquid chromatography. GSH uptake kinetics were studied in acivicin and buthionine sulfoximine-treated HLE-B3 cells in NaCl medium in the concentration range 0.01 microM to 50 mM. The transport of GSH and the effect of Na+ on uptake also were determined in mixed plasma membrane vesicles from HLE-B3 cells. In oocyte expression studies, HLE-B3 poly(A)+ RNA was injected into X. laevis oocytes and GSH uptake experiments were performed 3 days after injection. The uptake of 35S-GSH and GSH efflux rates were determined in HLE-B3 poly(A)+ RNA-injected oocytes. RESULTS: No significant difference was found in the uptake of 1 mM GSH+/-acivicin (17.7+/-4.3 versus 15.7+/-1.4 picomoles/min(-1) per 10(6) cells). However, GSH uptake was significantly lower in Na+-free medium compared with Na+-containing medium (10.3+/-0.7 versus 16.8+/-0.9 picomoles/min(-1) per 10(6) cells; P < 0.01). GSH uptake in NaCl medium was carrier mediated. GSH uptake showed partial sodium dependency from 5 microM to 5 mM GSH in mixed plasma membrane vesicles from HLE-B3 cells. Oocytes injected with HLE-B3 poly(A) RNA expressed uptake and efflux of GSH. Uptake showed partial Na+ dependency at various GSH concentrations. The efflux rates were approximately 30-fold higher than those in water-injected oocytes (0.48+/-0.03 versus 0.016+/-0.005 (nanomoles per hour(-1) per oocyte, respectively). The molecular form of uptake in cultured cells and in oocyte studies was predominantly as intact GSH. CONCLUSIONS: HLE-B3 cells and plasma membrane vesicles transported GSH by a carrier-mediated process. HLE-B3 poly(A)+ RNA injected X laevis oocytes expressed GSH transport. GSH uptake was partially Na+ dependent in all systems. HLE-B3 cells offer a useful model for characterizing GSH transport and for studying its regulatory role in the etiology of cataracts.

Deficiency in light-dependent opsin phosphorylation in Irish setters with rod-cone dysplasia.

A deficiency in light-dependent opsin phosphorylation and a slight reduction in opsin synthesis were observed during photoreceptor cell development (22-26 days) preceding photoreceptor cell loss in Irish setters with rod-cone dysplasia. In addition to opsin, two other phosphoprotein bands were found associated with the photoreceptor cell layer; synthesis and phosphorylation of one of these (band 3; 44-48 Kd) appeared reduced, while synthesis and phosphorylation of the other (band 1; 29-31 Kd) was within the normal range in 25-day-old affected setters. The deficiency in light-dependent opsin phosphorylation in affected setters was not due to a deficiency in opsin kinase, since soluble proteins from affected or normal outer segments catalyzed equally well opsin phosphorylation in partially kinase-depleted outer segment membranes from normal, while both kinase preparations failed to promote light-dependent opsin phosphorylation in those from affected setters. A deficiency in light-dependent opsin phosphorylation was also observed in rd/rd mice at all ages studied. In contrast, in Royal College of Surgeons (RCS) rats, light-dependent opsin phosphorylation was within the normal range prior to photoreceptor loss, and became nondetectable only after 50% or more of the photoreceptors had degenerated.

Action spectrum for cytotoxicity in the UVA- and UVB-wavelength region in cultured lens epithelial cells.

PURPOSE: This study was done to quantitate the biologic effects of different wavelengths of radiation in the UVA- and UVB-wavelength region on cultured rabbit lens epithelial cells. METHODS: An action spectrum for UV-induced cytotoxicity as measured by colony-forming ability was determined using six different monochromatic wavelengths from 297 to 405 nm in rabbit lens epithelial cell line N/N1003A. Cell survival was determined by clonogenic assay. Fluence rates were monitored with a calibrated radiometer. RESULTS: Survival curves show that cell killing was most efficient at 297 nm. After quantum correction, the efficiency of 297-nm radiation in cell killing was 7 times greater than was 302-nm radiation. Radiation at 297 nm was more than 170, 340, 560, and 2000 times as effective in cell killing as 313-, 325-, 334-, and 365-nm radiation, respectively. The action spectrum had a shape similar to the DNA absorption spectrum in the UVB region, suggesting that DNA may be one of the critical targets for damage to the cells. At wavelengths longer than 313 nm, the shape of the action spectrum deviated from the DNA absorption spectrum. CONCLUSIONS: Cytotoxicity of UV radiation in cultured lens epithelial cells varies greatly with wavelength within the UVA and UVB regions. Different mechanisms may predominate in the two wavelength regions. Cultured cells may provide a suitable system for investigating the mechanisms by which UV radiation damages lens epithelial cells and leads to cataract formation.

Modulation of lens epithelial cell proliferation by enhanced prostaglandin synthesis after UVB exposure.

PURPOSE: The goals of this investigation were to examine the synthesis of prostaglandins after UVB exposure of lens epithelial cells and to investigate their role in cell proliferation. METHODS: Cultured rabbit lens epithelial cells (cell line N/N1003A) were exposed to low levels of UV radiation. Prostaglandins were assayed by radioimmunoassay; products of arachidonic acid metabolism were analyzed by thin-layer chromatography and mass spectroscopy. Cell proliferation was measured by [3H]thymidine incorporation and proliferative autoradiography. RESULTS: Cultured lens epithelial cells exposed to UVB radiation showed a dose-dependent increase in basal prostaglandin synthesis measured 24 hours after UV exposure. At an optimal dose (250 J/m2) of UVB, prostaglandin E2 (PGE2) synthesis was enhanced tenfold. Product identity was confirmed using thin-layer chromatography and mass spectroscopy with authentic standards. Incubation of irradiated cells with exogenous arachidonic acid followed by extraction and thin-layer chromatography revealed that the cultures produced PGE2, prostaglandin I2 (measured as 6-keto-prostaglandin F1 alpha), prostaglandin F2 alpha, and hydroxyeicosatetraenoic acid. The synthesis of all of these products was enhanced threefold in cells exposed to 250-J/m2 UVB. Indomethacin pretreatment eliminated the synthesis of prostaglandins, further confirming their identity. To discover the relationship between PGE2 synthesis and irradiation-induced cell proliferation, [3H]thymidine incorporation into DNA was determined 24 or 48 hours after exposure. These experiments revealed a fivefold increase in incorporation induced by UVB exposure. UVB-enhanced incorporation of thymidine was eliminated by pretreatment of cultures with indomethacin to eliminate PG synthesis. However, when 100 nM PGE2 was added to the indomethacin-treated irradiated cultures, incorporation of the label was restored toward the level detected in the UVB-stimulated cells. Addition of other prostaglandins to the cultures was ineffective. CONCLUSIONS: The results indicate that the synthesis of PGE2 is enhanced by exposure of lens epithelial cells to UVB radiation. PGE2 seems to play a specific role in cell proliferation after UV exposure. This increase in PGE2 synthesis may be important in posterior subcapsular cataract formation in humans and in animals exposed to UVB radiation in vivo.

Expression of prostaglandin receptors EP4 and FP in human lens epithelial cells.

PURPOSE: To examine the expression of prostaglandin (PG) receptors EP2, EP4, and FP in a human lens epithelial cell line (HLE-B3) at molecular and pharmacologic levels. METHODS: Reverse transcription-polymerase chain reactions (RT-PCR) were performed with total RNA preparations from HLE-B3 cells using sense and antisense primers for each of the three prostaglandin receptors. The PCR products were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. At the pharmacologic level, the expression of EP4 receptors was determined by measuring intracellular cyclic adenosine monophosphate (cAMP) formation in response to PGE2 (EP1, EP2, EP3, and EP4 agonist) and the EP4 receptor-selective antagonist AH23848. The expression of FP receptors in HLE-B3 cells was explored by measuring intracellular [Ca2+]i mobilization. RESULTS: RT-PCR generated DNA products of predicted sizes corresponding to the EP2, EP4, and FP receptors. Hybridization of the PCR products with specific 32P-labeled probes and restriction digestion of the PCR products further confirmed that they were generated from the respective EP2, EP4, and FP mRNAs. The EP receptor agonist PGE2 significantly increased the cAMP level in HLE-B3 cells. The formation of cAMP by PGE2 was concentration-dependently inhibited by the EP4 receptor-selective antagonist AH23848. Stimulation of HLE-B3 cells by the FP receptor agonist fluprostenol increased [Ca2+]i in a time-dependent manner. CONCLUSIONS: The results of the molecular biologic and pharmacologic experiments showed conclusively the presence of EP4 and FP receptor messenger RNAs and proteins, respectively, in HLE-B3 cells.

Propagation and immortalization of human lens epithelial cells in culture.

PURPOSE: To establish primary and immortalized cell cultures of human lens epithelial cells for a model system investigating human lens epithelial physiology and cataract. METHODS: Human lens epithelial cells in culture were grown by isolating epithelium fragments from infant human lenses from patients who underwent treatment for retinopathy of prematurity and by allowing epithelial cells to grow from explants. To immortalize cells, the cultures were infected with an adenovirus 12-SV40 virus (Ad12-SV40). RESULTS: The primary cells from infant eyes proliferated for three passages before senescence was observed. However, the immortalized cells remained proliferative and retained the morphology of the primary cells. Immunohistochemical analysis demonstrated that these immortalized cells were SV40 large T antigen-positive and ceased to produce infectious virus after a few passages. Immortalized cells passaged to population doubling levels of 76 continued to form confluent cultures within 7 days of subculture. Analysis of proteins by SDS-PAGE and immunoblotting showed that immortalized cells produce a protein with molecular weight of about 25 kD, which reacted with an antibody to beta H-crystallin. CONCLUSIONS: This report constitutes the first successful immortalization of human lens epithelial cells. Currently, two cell lines have been created (B-3 and B-4) and passaged to population doubling levels of 76 and 52, respectively. These cells may provide an important human cell line specific to in vivo human lens epithelial cell physiology and would be of interest in establishing a human model to study lens cell differentiation and the etiology of cataract. These cells may also provide a constant and reproducible source of lens epithelial cells for eye-related toxicology studies and to assay inhibitory drugs for the prevention of cataracts and posterior capsular opacification observed after cataract extraction.

Enhanced prostaglandin synthesis after ultraviolet-B exposure modulates DNA synthesis of lens epithelial cells and lowers intraocular pressure in vivo.

PURPOSE: To study the functional significance of prostaglandin synthesis after ultraviolet-B (UVB) exposure of cultured human lens epithelial cells and rabbit eyes in vivo. METHODS: Prostaglandin E2 (PGE2) was assayed using a radioimmunoassay (RIA) and mass spectroscopy. An immortalized human lens epithelial cell line (HLE-B3) was exposed to UV irradiation, and the synthesis of PGE2 was compared with the rabbit lens epithelial cell line N/N1003A. Intact human lenses were exposed to UVB in organ culture. [3H]Thymidine incorporation was measured in cultured lens epithelial cells by incubation with the radiolabel. The effects of isobutyl methyl xanthine (IBMX), an inhibitor of phosphodiesterase and of dibutyryl cyclic adenosine monophosphate (cAMP), an analog of cAMP, on PGE2 synthesis and DNA synthesis, were determined. Rabbit eyes were exposed to UVB radiation in vivo. Intraocular pressure was measured at specific times after exposure. Aqueous humor was remove from rabbit eyes, and its PGE2 content was measured by RIA. RESULTS: Cultured human lens epithelial cells (HLE), like rabbit lens epithelial cells (RLE), showed a dose-dependent increase in basal PGE2 synthesis 24 hours after UVB exposure. However, the amount of PGE2 synthesis was 2000-fold higher in the rabbit cells. Ultraviolet-B radiation enhanced the incorporation of [3H]thymidine in lens epithelial cells. Pretreatment of cells with indomethacin reduce PGE2 synthesis and [3H]thymidine incorporation. The human and rabbit cells responded in a similar manner to changes in DNA synthesis after UVB exposure. The addition of IBMX or dbcAMP to indomethacin-treated, UVB-exposed cells restored DNA synthesis toward the levels observed in the UVB-exposed cells. An increase in the concentration of cAMP was observed in lens epithelial cells exposed to exogenous PGE2. PGE2 synthesis in intact human lenses also increased twofold 24 hours after UVB exposure. Exposure of the rabbit eye in vivo to an optimal dose of UVB produced an increase in the PGE2 levels of the lens and the aqueous humor. Measurements of the intraocular pressure (IOP) of the animals showed a decrease in IOP by 2.21 +/- 0.66 and 6.45 +/- 0.79 mm Hg (mean +/- SEM, P = 0.004, t-test) at 6 and 24 hours after UVB exposure, respectively. The decrease in IOP was prevented by pretreatment with indomethacin. Exposure of the rabbit lens to UVB radiation in vivo enhanced [3H]thymidine incorporation twofold into the lens. Pretreatment of rabbits with indomethacin before exposure reduced this response. CONCLUSIONS: Results indicate that UVB exposure enhances PGE2 synthesis in HLE cultures as well as in rabbit lenses irradiated in vivo. This increased PGE2 synthesis is related to the increase in DNA synthesis observed after UVB treatment. The modulation of DNA synthesis in cultured lens epithelial cells after UVB exposure may be mediated by a cAMP-dependent mechanism.

Ultraviolet action spectra for photobiological effects in cultured human lens epithelial cells.

The action spectrum for cell killing by UV radiation in human lens epithelial (HLE) cells is not known. Here we report the action spectrum in the 297-365 nm region in cultured HLE cells with an extended lifespan (HLE B-3 cells) and define their usefulness as a model system for photobiological studies. Cells were irradiated with monochromatic radiation at 297, 302, 313, 325, 334 and 365 nm. Cell survival was determined using a clonogenic assay. Analysis of survival curves showed that radiation at 297 nm was six times more effective in cell killing than 302 nm radiation; 297 nm radiation was more than 260, 590, 1400 and 3000 times as effective in cell killing as 313, 325, 334 and 365 nm radiation, respectively. The action spectrum was similar in shape to that for other human epithelial cell lines and rabbit lens epithelial cells. The effect of UV radiation on crystallin synthesis was also determined at different wavelengths. To determine whether exposure to UV radiation affects the synthesis of beta-crystallin, cells were exposed to sublethal fluences of UV radiation at 302 and 313 nm, labeled with [35S]methionine and the newly synthesized beta-crystallin was analyzed by immunoprecipitation and western blotting using an antibody to beta-crystallin. The results show a decrease in crystallin synthesis in HLE cells irradiated at 302 and 313 nm at fluences causing low cytotoxicity. The effect of radiation on membrane perturbation was determined by measuring enhancement of synthesis of prostaglandin E2 (PGE2). Synthesis of PGE2 occurs at all UV wavelengths tested in the 297-365 nm region.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of near-UV radiation on human lens beta-crystallins: protein structural changes and the production of O2- and H2O2.

beta-Crystallins (beta 1-, beta 2- and beta 3-crystallin) comprise nearly half the protein of the human lens. The effect of near-UV radiation, which is one of the possible risk factors in cataract formation, on the beta-crystallins is investigated in this study. Protein intersubunit crosslinking, change in charge of the protein subunits to more acidic species and changes in protein tertiary structure (conformation) by 300 nm irradiation are reported. The fluorescence yield of protein tryptophan residues decreases by 300 nm irradiation. There is an increase in nontryptophan fluorescence (lambda cx 340 nm, lambda cm 400-600 nm), and in protein absorption at 340 nm, due to the formation of tryptophan photooxidation products. Both tryptophan and its oxidation products can be photoexcited by 300 nm irradiation and the latter are known to be good photosensitizers. The results provide evidence for the generation of H2O2 in the irradiated human beta-crystallin solutions by the Type I photosensitizing action of the chromophores absorbing at 300 nm. The H2O2 is generated via the intermediate production of O2 anion; the latter spontaneously dismutates to H2O2, presumably via O2- protein interactions. The amount of H2O2 generated per absorbed photon is compared for various solutions of beta 1-, beta 2- and beta 3-crystallins from human lenses of different age.

Action spectrum for photocross-linking of human lens proteins.

The action spectrum for photocross-linking was measured for human lens beta gamma-crystallins from young adult noncataractous lenses at wavelengths of 297, 302, 313, 334 and 365 nm. The action spectrum had a maximal effectiveness at 297 nm that sharply decreased in effectiveness up to 313 nm, then remained flat until 334 nm and decreased markedly as wavelength increased to 365 nm. Radiation at 297 nm was 36 times more effective in producing cross-linking than 302 nm radiation. The 297 nm radiation was 220, 195 and 1300 times more effective than 313, 334 and 365 nm radiation, respectively. The action spectrum had a shape similar to the absorption spectrum of the lens proteins but the response was lower than expected from the absorption data, suggesting that some of the absorptions are not effective at cross-linking. Because most animal experimentation and epidemiological studies include broadband radiation, these studies would be useful in predicting the biological response of the lens to environmental UV stress.

Spectroscopic studies on the photooxidation of calf-lens gamma-crystallin.

The photooxidation of calf gamma-crystallin has been investigated in (a) riboflavin-sensitized reaction and (b) direct photolysis of the tryptophan residues of the protein at 300 nm. Partial insolubilization of the protein is evident by the increase in turbidity of the photolyzed protein solution. The turbidity is diminished when anaerobic conditions are used for irradiation. Spectroscopic studies on the soluble phase of the photolyzed protein reveal significant changes in the near-UV circular dichroism spectrum, suggesting that changes in the tertiary structure of the protein precede insolubilization. Isoelectric focusing analyses reveal that the gamma-crystallin polypeptides have more acidic pI's after photooxidation under both conditions. The insoluble protein is highly crosslinked into dimers and higher oligomers via covalent, non-disulfide crosslinks, but inter-subunit crosslinking is negligible in the soluble phase of the photolyzed protein. The mechanism of photooxidation involves the generation of H2O2 in the riboflavin-sensitized reaction. In the direct photolysis of tryptophan residues by 300 nm irradiation, there is a rapid disruption of the protein structure, apparently by the oxidation of the hydrophobic tryptophan residues.

DNA repair and survival in human lens epithelial cells with extended lifespan.

PURPOSE: Ultraviolet-B radiation (290-320 nm) produces cataracts in animals and has been associated with human cataract formation in several epidemiological studies. UVB radiation decreases the long-term cell survival and changes the pattern of protein synthesis in cultured lens epithelial cells. However, the relationship between DNA photoproduct formation and long term cell survival in human lens epithelial cells is not known. In the present work, we used human lens epithelial cells with extended lifespan (HLE B-3 cells) to examine the kinetics of DNA repair and cell survival after UVB exposure. METHODS: Cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts were analyzed by radioimmunoassay. Long-term survival of the cells was determined by measuring their ability form colonies when plated at low density. RESULTS: HLE B-3 cells were repair competent after UVB (302 nm) exposure. Excision repair of the (6-4) photoproduct was more efficient than that of the cyclobutane dimer. Ninety five percent of the (6-4) photoproducts were repaired 24 h after 400 J/m2 UVB exposure, whereas 50% of the cyclobutane dimers were repaired during this time. When cells were split for the clonogenic assay immediately after irradiation, only 10% of the cells formed colonies following 7 days of culture in the serum-containing medium. When cells were split for the clonogenic assay after a 48 hour incubation in serum-containing medium, the colony-forming ability of the irradiated cells increased to 60% following culture in a serum-containing medium. CONCLUSIONS: These results indicate a close correlation between the repair of cyclobutane dimers and the increase in the long-term survival of the cells as measured by their colony-forming ability. The extended lifespan human lens epithelial cells HLE B-3 may be a useful model to investigate the mechanism and regulation of UVB-induced DNA repair in human lens cells.

Lens hexokinase deactivation by near-UV irradiation.

Photodamage to lens hexokinase has been investigated by exposing the lenses of rat, rabbit and calf eyes to 300 nm irradiation. Hexokinase activity was diminished by 15.9% +/- 5.4 and 23.4% +/- 5.0 upon irradiation of the isolated rat lens for 1 and 2 hours respectively. Irradiation of the whole eye for 2 hours resulted in hexokinase deactivation of 13.6% +/- 5.8 and 19.2% +/- 6.2 for rat and rabbit lens homogenates and 55% +/- 7 for calf lens capsule plus epithelium. Enzyme deactivation was prevented when the isolated lens was irradiated with the vitreous attached. Glucose, catalase or ascorbate added to the medium prior to irradiation, each had a protective effect on hexokinase deactivation. The results are consistent with a mechanism in which photochemical generation of active species of oxygen, via the photosensitizing action of tryptophan photoproducts, plays a significant role in enzyme deactivation.

Fluorescence studies on the age related changes in bovine and human lens membrane structure.

Bovine and human lens fiber cell plasma membranes were isolated as the urea-insoluble fraction for a study of age related changes. The changes in fluorescence intensity, both intrinsic (tryptophan) and extrinsic probes ANS (1-anilinonaphthalene-8-sulfonic acid) and DPH (1,6-diphenyl 1,3,5-hexatriene), as well as DPH anisotropy and lifetime were measured. The results of tryptophan fluorescence indicate that tryptophan residues in membrane proteins are in a very hydrophobic environment and do not show a change with aging. ANS reacts with surface protein in the polar-apolar interface, while DPH penetrates into the interior of membranes. Both probes show a decrease in fluorescence intensity in the old membranes. The decrease in ANS fluorescence may result from the conversion of MP26 to MP22, while the decrease of DPH fluorescence intensity may indicate a decrease in accessibility of lipid to DPH. To further delineate the change, fluorescence anisotropy and lifetime data of the lipid probe DPH were obtained. While lifetimes do not change with age, anisotropy shows a definite age-dependent increase. Anisotropy is related to the degree of lipid structural order. Greater anisotropy values were found for older membrane samples, indicating an increased rigidity with age, which may be partially caused by the increased cholesterol/phospholipid (C/P) ratio as reported in the literature.