ULTRAVIOLET ACTION SPECTRA FOR PHOTOBIOLOGICAL EFFECTS IN CULTURED HUMAN LENS EPITHELIAL CELLS

Abstract— 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 β-crystallin, cells were exposed to sublethal fluences of UV radiation at 302 and 313 nm, labeled with [³⁵S]methionine and the newly synthesized βY-crystallin was analyzed by immunoprecipitation and western blotting using an antibody to β-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. The slope of the PGE₂ response curves was higher than that of cell killing curves in cultured HLE cells. These data show that cultured HLE cells with extended lifespan are a suitable system for investigating photobiological responses of cells to UV radiation.     

CONFORMATIONAL CHANGES OF BOVINE LENS CRYSTALLINS IN A PHOTODYNAMIC SYSTEM

Abstract— Conformational changes of bovine lens crystallins in a photodynamic system generating singlet oxygen, have been investigated. The formation of intersubunit crosslinks was observed in all three classes (α-, β and γ-) of crystallins by irradiation in the presence of the photosensitizer methylene blue. Near-UV circular dichroism (CD) spectra of the crystallins were significantly altered by irradiation under these conditions, indicating changes in tertiary structure but the far-UV CD remained unchanged suggesting that the secondary structure ((β-sheet conformation) remains unchanged. Significant changes in the absorption and fluorescence spectra were also observed. Measurement of total sulfhydryl content showed a decrease of 27%, 50% and 37% for α-, β- and γ-crystallins respectively, after irradiation. Fluorescence lifetime measurements of N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenediamine-labeled crystallins showed a significant decrease of the lifetime of the major decay components of the label bound to sulfhydryl groups of α- and γ-crystallins, but showed no change in the microenvironment of the sulfhydryl groups of β-crystallin. The results are consistent with the microenvironments of the tryptophan and sulfhydryl groups predicted from sequence studies.     

THE EFFECTS OF NEAR-UV RADIATION ON HUMAN LENS β-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.     

Phototoxicity in human lens epithelial cells promoted by St. John's Wort

St. John's Wort (SJW), an over-the-counter antidepressant, contains hypericin, which absorbs light in the UV and visible ranges and is phototoxic to skin. To determine if it also could be phototoxic to the eye, we exposed human lens epithelial cells to 0.1-10 microM hypericin and irradiated them with 4 J/cm2 UV-A or 0.9 J/cm2 visible light. Neither hypericin exposure alone nor light exposure alone reduced cell viability. In contrast, cells exposed to hypericin in combination with UV-A or visible light underwent necrosis and apoptosis. The ocular antioxidants lutein and N-acetyl cysteine did not prevent damage. Thus, ingested SJW is potentially phototoxic to the eye and could contribute to early cataractogenesis. Precautions should be taken to protect the eye from intense sunlight while taking SJW.     

CHANGES IN TERTIARY STRUCTURE OF CALF-LENS α-CRYSTALLIN BY NEAR-UV IRRADIATION: ROLE OF HYDROGEN PEROXIDE

The effect of 300 nm irradiation on the three lens crystallins, α-, β-, and γ-, was studied by using fluorescence and circular dichroism techniques. α-Crystallin showed a pronounced change in tertiary structure as manifested in fluorescence and circular dichroism measurements. This finding is in agreement with our earlier findings that the tryptophan residues of α-crystallin are more exposed than those of the other two crystallins. The results of studies using inhibitors specific for the different active species of oxygen suggest that H₂O₂-mediated damage is involved in the change of tertiary structure of the proteins. Analyses of circular dichroism spectra indicate that, upon irradiation, the secondary structure of α-crystallin remains virtually unaltered, and that the change in tertiary structure results primarily from photoinduced damage to the tryptophan residues.     

CHANGE IN SULFHYDRYL GROUP MICROENVIRONMENT OF CALF LENS α-CRYSTALLIN BY 300 nm LIGHT

Abstract— The effect of 300 nm irradiation on the sulfhydryl groups of calf lens a-crystallin has been investigated by using specific, covalently bound fluorescent sulfhydryl probes 4–(N-iodoacetoxy)ethyl-N-methylamino-7-n-itrobenz-2-o-xa-1,3-d-iazole (IANBD), N-iodoacetyl-N'-(5-s-ulfo-l-naphthyl) ethylene-diamine (1,5 IAEDANS) and 5-i-odoacetamidofluorescein (IAF). The decrease in tryptophan fluorescence with time of irradiation of a-crystallin, is accompanied by a decrease in the fluorescence of the hydrophobic sulfhydryl label IANBD. In addition, the fluorescence of the surface-sulfhydryl label IAF increased in the irradiated a-crystallin. These results indicate that the sulfhydryl groups are in a more exposed (hydrophilic) environment in the irradiated protein than in the control, possibly because of partial unfolding of the protein. This result is confirmed by fluorescence lifetime measurements with IAEDANS. The decay curve of IAEDANS-α-crystallin has a major lifetime of 15.7 ns and a minor one of 24.6 ns. Upon irradiation, the lifetime of the major component decreases to 10.2 ns and that of the minor component to 21.7 ns. Denatured IAEDANS-α-crystallin has a single lifetime of 10.4 ns. These results show that the photoinduced damage to the tryptophan residues of α-crystallin alters the environment of the sulfhydryl groups and induces a change in the tertiary structure of the protein. Proximity of the cysteine residues to tryptophan in the tertiary structure of the protein may be an important determinant of their susceptibility to photoinduced change.     

THE EFFECT OF TREATMENT OF CHLOROPLAST MEMBRANES WITH GUANIDINE HC1 AND AQUEOUS ACETONE ON THE FLUORESCENCE OF BOUND ANS AND CHLOROPHYLL-a†

Abstract The fluorescence intensity of the extrinsic chromophore 1-anilino-naphthalene-8-sulfonate (ANS) bound to pea chloroplast fragments shows a sigmoidal rise as the pH of the suspending medium is decreased by the addition of HC1. The abrupt increase occurs at pH – 4.5. A 70% decrease in the maximal fluorescence intensity (pH range 3.5-4.5) of bound ANS was observed when soluble chloroplast proteins were removed by washing with water. Extraction of chloroplast membranes with 6 M guanidine-HC1 abolishes the acid–induced enhancement of ANS fluorescence. However, the subsequent removal of lipids (by 80% acetone extraction) from the guanidine-HC1-extracted naked membranes restores the acid-induced fluorescence increase. These results suggest that ANS binds mainly to the surface of the chloroplast membrane and the fluorescence changes of ANS by acidification mainly reflect the changes in the associated proteins. The lack of enhancement of the fluorescence of ANS by acidification of the guanidine-HCl treated membranes and the recovery of the acid-induced fluorescence rise after extraction of the lipids from the guanidine-HCl treated membranes suggest that the boundary lipids somehow prevent the entry of the ANS molecules into the hydrophobic interior of the naked membrane. The lipid-depleted, guanidine-HCl extracted naked membrane fragments do not show any shift in the position of the peak of emission of ANS (λ= 470 nm) upon acidification as the lipid-depleted preparations without guanidine-HCl treatment do (shift from 460 to 470 nm). Divalent cations (Mn²⁺, Ca²⁺, Mg²⁺) also increased ANS fluorescence intensities when added to both types of lipid-depleted chloroplast preparations. A comparative analysis of ANS fluorescence bound to the lipid-depleted and guanidine-HCl treated chloroplast fragments with that of just lipid-depleted fragments shows that the acidification of the latter brings about a greater change in the value of the relative binding sites (n) and the dissociation constant k_d of ANS than the protonation of the former. The role of chloroplast protein and lipid components in the structural changes of the thylakoid membrane imposed by external perturbations is discussed.     

Immunological Detection of N‐formylkynurenine in Porphyrin‐Mediated Photooxided Lens α‐crystallin

Crystallin proteins are responsible for maintaining lens transparency and allowing the lens to focus light undistorted onto the retina. The α‐crystallins are the major lens crystallins, and function as both structural proteins and chaperones to protect all lens proteins from damage leading to lens deterioration. Because lens crystallin proteins do not turn over, the damage they accumulate can lead to cataracts, the world’s leading cause of blindness. Photosensitizing porphyrins can accumulate in the eye through either endogenous metabolism or through therapeutic or diagnostic procedures. Porphyrin buildup exacerbates lens aging through increased levels of singlet oxygen, resulting in protein polymerization and amino acid residue alteration. Tryptophans oxidize to kynurenine and N‐formylkynurenine (NFK) causing irreversible changes in the refractive index of the normally transparent lens, leading to development of cataracts. Additionally, NFK is itself a photosensitizer, and its presence exacerbates lens deterioration. This work uses anti‐NFK antiserum to study porphyrin‐facilitated photooxidation of α‐crystallin tryptophan residues. In vitro experiments show that four biologically interesting porphyrins mediate α‐crystallin polymerization and accumulation of both protein radicals and NFK. Confocal microscopy of cultured human lens epithelial cells indicates that while all four porphyrins photosensitize cellular proteins, not all oxidize the tryptophans of cellular α‐crystallin to NFK.