NEAR UV LIGHT AND CATARACTS*

Abstract. Sunlight and many types of artificial lighting contain near-UV light (300–400 nm). These wavelengths can enter the eye and are maximally absorbed in the lens due to its chemical composition. The lenses of certain animals develop cataracts from exposure to this light, and changes similar to those that occur in human lenses with certain types of cataracts and with aging are inducible in isolated human lenses. These changes seem to be associated with chemical alterations in the essential amino acid tryptophan either as a part of proteins or in free form. Such changes in tryptophan would result in lens cell toxicity, in increased pigmentation of the lens, and in large aggregates of proteins. The latter two changes would result in losses in the ability of the lens to transmit visible light needed for vision, and the abnormal state called cataract would result. Much more work is needed to prove that near-UV light can accelerate cataractous changes in the lenses of living humans. Studies at the basic chemical level are needed, but population studies would be most essential for the final proof. Many preventive measures could become available, including the use of special types of spectacles and dietary additives.     

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.     

DIFFERENTIAL EFFECTS OF TRYPTOPHAN PHOTOPRODUCTS AND H2O2 ON THE GROWTH OF MOUSE EMBRYONIC FIBROBLASTS*

Abstract. Exposure of dilute solutions of L-tryptophan in balanced salt mixtures to near-ultraviolet light (ca. 320–390nm) yield several photoproducts capable of inhibiting the growth of cultured mouse embryonic fibroblasts. Recent literature suggested a need to test the possibility that the major toxic photoproduct is hydrogen peroxide, and therefore several lines of experimentation were pursued. On the basis of chemical and morphological studies and an analysis of the uptake of ³H-thymidine by these cells and its incorporation into their DNA, it is concluded that sufficient hydrogen peroxide could not be generated without the addition of high concentrations of oxygen to influence the growth or DNA synthesis of mouse embryonic fibroblasts.     

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 UV_A) 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 α-tocopherol nor de-feroxamine were protective against these UV_A-induced changes in pure catalase. We further investigated the effect of UV_A 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 ho-mogenate supernatants of cells kept dark or UV exposed. In some instances, cells were cultured in medium containing a-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 UV_A 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 a-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.     

ISOLATION AND IDENTIFICATION OF TRYPTOPHAN PHOTOPRODUCTS FROM AQUEOUS SOLUTIONS OF TRYPTOPHAN EXPOSED TO NEAR-UV LIGHT*

Abstract—One of the previously unidentified photoproducts isolated from the photolysate of aqueous tryptophan solutions was identified as 2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo(2,3b)-indole by direct comparison with the authentic reference compound synthesized using the established procedure. This pyrroloindole alcohol has been shown to be the reduction product of the 3a-hydroperoxy intermediate (structure 4 in Fig. 1) by Nakagawa et al . (1977). The isolation and identification of this derivative and the detection of the peroxy intermediate 3a-hydroperoxypyrrolidinoindole ( 4 ). from irradiated aqueous tryptophan solutions suggests that the direct photooxidation of l -tryptophan to fromylkynurenine may follow a pathway via a tricyclic intermediate instead of the energetically unfavorable dioxotane intermediate. This scheme is similar to the mechanistic model proposed by Nakagawa et al . (1977) for the rose bengal sensitized photooxidation of tryptophan.     

Enhancement of the non-tryptophan fluorescence of human lens proteins after near-UV light exposure

Abstract. In this work, the non-tryptophan fluorescence (360 nm excited; 440 nm emitted) of human lens proteins was found to be intensified by exposing whole lens homogenates to near-UV light in the presence of tryptophan photoproducts. The induced fluorescence accumulates mainly in the soluble phase proteins, whereas in aging and brown cataractous lenses, the major fluorescence is found in the insoluble proteins. Using SDS-polyacrylamide gel electrophoresis with densitometric and fluorescence scanning techniques, the polypeptide chains of the three major protein fractions were analyzed for their specific non-tryptophan fluorescences. The same chains were found in all fractions. Two chains (11,000 and 45,000 daltons) were found to accumulate most of the induced fluorescence. These also contained the greatest intrinsic fluorescence initially. The data indicates that specific polypeptide chains in the lens proteins are most sensitive to modifications due to their exposure to near-UV light in the presence of tryptophan photoproducts.