UVA light-excited kynurenines oxidize ascorbate and modify lens proteins through the formation of advanced glycation end products: implications for human lens aging and cataract formation.

Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320-400 nm, 100 milliwatts/cm(2), 45 min to 2 h), young human lenses (20-36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans.

Photoactivity of kynurenine-derived UV filters.

Quantum yields of photodecomposition and triplet state formation under aerobic and anaerobic conditions are determined for kynurenine (KN), 3-hydroxykynurenine (3OHKN), xanthurenic acid (XAN), and kynurenine adducts of glutathione (GSH-KN), cysteine (Cys-KN), histidine (His-KN), and lysine (Lys-KN) in aqueous solutions. The highest yields of anaerobic photodecomposition were obtained for GSH-KN and His-KN adducts, which correlates with the highest triplet yields for these compounds. In aerobic conditions, the photodecomposition yields for all compounds under study increase; the highest decomposition rates were observed for His-KN and 3OHKN. The fast decomposition of the latter is attributed to the dark autoxidation of the starting compound.

Photochemical and thermal reactivity of kynurenine.

The thermal and photochemical reactivity of kynurenine (KN), a tryptophan metabolite found in human lenses, has been studied in aqueous solution. The decarboxylation reaction of KN, resulting in the formation of 4-hydroxyquinoline, is reported for the first time. Rate constants for KN deamination and decarboxylation were determined in the temperature range 50-90 degrees C. The quantum yields for KN photodecomposition under argon were measured to be Phi Ar=(2.0+/-0.2) x 10(-5) and under oxygen Phi O2=(1.1+/-0.1) x 10(-4).

Impact of UVR-A on whole human lenses, supernatants of buffered human lens homogenates, and purified argpyrimidine and 3-OH-kynurenine.

PURPOSE: Yellow chromophores and fluorescent compounds accumulate in the lens with age. Some of these compounds are photochemically active. The present study aimed to examine the photochemical effect of ultraviolet radiation-A (UVR-A) on the human lens. METHODS: Intact human lenses and supernatants of buffered lens homogenates were exposed to UVR-A. The effect of UVR-A was evaluated by time-resolved and steady-state fluorescence spectroscopy, visual evaluation of colour and protein gel electrophoresis. RESULTS: Intact lenses exposed to UVR-A showed no changes in time-resolved or steady-state fluorescence properties but the yellow coloration was visibly attenuated. The supernatants of buffered lens homogenates exposed to UVR-A demonstrated a reduction in time-resolved and steady-state fluorescent properties and protein cross-linking. CONCLUSIONS: Exposure of the intact lens to UVR-A causes chromophore bleaching without affecting fluorescence, indicating that non-fluorescent chromophores have been destroyed. After homogenization, both chromophores and fluorophores from the lens suffer damage and proteins aggregate. This indicates that powerful mechanisms of protection against UVR-A found in the intact lens are disturbed by homogenization of the lens, suggesting that isolated lens proteins cannot be used as a model system for studying cataractogenesis. Hypothetically, the protective mechanism could be related to the rigidly packed three-dimensional structure of the lens proteins or to the abundance of antioxidative and free radical scavenging defence systems.

Photochemical properties of kynurenine pathway metabolites and indoleamines.

Photochemical damages to the biological system may occur through photodynamic action in the presence of photosensitive molecules. Photodynamic action contains the following processes; 1) photosensitisation and/or 2) electron transfer, in which singlet oxygen and superoxide radical production for each in the presence of oxygen molecules. We have studied those processes after the absorption of light by kynurenine pathway metabolites and indoleamine derivatives. We found that kynurenine and 3-hydroxykynurenine generate superoxide radical after electron transfer from their excited state molecules to oxygen molecules, and superoxide makes reduction reaction. On the other hand, it was found that kynurenic acid, melatonin, 5-methoxytryptamine and 5-methoxytryptophol work as photosensitisers with the detection of singlet oxygen production by using the N, N-dimethyl-4-nitrosoaniline bleaching method, while xanthurenic acid, serotonin and N-acetylserotonin generate no detectable amount of singlet oxygen. We have determined the photochemical quantum yields of singlet oxygen production for those photosensitisers, in which quantum yields are not so high except kynurenic acid (f3 = 0.101). In view of the multiple roles played by their metabolites in various systems, these results are relevant to taking into consideration of their photoeffect in the presence of light.

Photochemically modified alpha-crystallin: a model system for aging in the primate lens.

The purpose of this study was to quantitatively study the changes that occur upon irradiation of 3-hydroxykynurenine (3-HK) in the presence of alpha-crystallin under conditions similar to those in the lens. The samples were prepared in 10 mM phosphate buffer at pH 7.4, bubbled with O2 or Ar and irradiated with 300-400 nm light. The amount of light absorbed by the samples (Iabs) was measured using azobenzene as an actinometer. Modifications to alpha-crystallin were monitored by ultraviolet-visible and fluorescence spectroscopy. Aerobic samples had increased absorption around 320 nm and above 400 nm while the 3-HK maximum at 368 nm decreased. The isolated modified protein showed that there was increased absorption throughout the spectrum. Changes in the anaerobic samples were similar to those of the aerobic but occurred more slowly. As irradiation time increased fluorescence emission of the isolated protein red shifted and quantum yields of fluorescence (phi f) were calculated at different irradiation time intervals by comparison to 3-HK. By comparing OD320/OD365 for the model system to values from primate lenses, Iabs can be correlated with age and transmission of the sample in the blue region of the spectrum and thus allows lenticular aging to be quantitated.

Photooxidized products of recombinant alpha A-crystallin and W9F mutant.

Human lenses contain many photosensitizers that absorb light at wavelengths above 300 nm, most notably UVA light (320-400 nm). Kynurenine (Kyn) and 3-hydroxykynurenine (HK), two of the best-known photosensitizers in the human lens, may play a significant role in photooxidation-related changes in lens proteins, such as conformational change and aggregation. In vitro irradiation experiments with proteins indicate that the Trp residue (with maximal absorption at 295 nm) is more susceptible to photooxidation by UVB light (280-320 nm) than by UVA light, but most UVB light below 300 nm is screened by the cornea and little reaches the lens, especially the nuclear region where nuclear color develops. Therefore, if photooxidation is an important contributor to nuclear color or nuclear cataract, it must arise from a photosensitized reaction. In the present study, we use recombinant alpha A- and its Trp-deficient mutant W9F as models to study the effects of UVA irradiation in the presence of HK or Kyn and of UVB (300 nm) irradiation on alpha-crystallins. alpha A-crystallin showed a large decrease in Trp fluorescence and a large increase in non-Trp (blue) fluorescence after the HK-sensitized or 300 nm photooxidation. For the W9F mutant, a smaller decrease in protein fluorescence (lambda ex at 280 nm) and a smaller increase in blue fluorescence than for the wild-type alpha A-crystallin were observed. A decrease in the near-UV CD was also observed for both photooxidized alpha A and the W9F mutant. The effect of Kyn sensitization is smaller than that of HK sensitization. A study of chaperone-like activity indicated that only 300 nm photooxidized alpha A and the W9F mutant increased the ability to protect insulin from dithiothreitol-induced aggregation. Thus, sensitized photooxidation can occur in amino acids other than Trp by UVA in the presence of HK or Kyn with effects similar to, albeit smaller than, those of direct UVB (300 nm) photooxidation.

Model studies on the photochemical production of lenticular fluorophores.

With aging, human lens proteins accumulate fluorophores having blue and green emissions. Model studies were undertaken to determine the role of 3-hydroxykynurenine (3-HK) and its glucoside (3-HKG) in the photochemical production of those fluorophores. Experiments were carried out using 10(-3) M 3-HK solutions in the presence or absence of glycine (1 M), which was used to mimic the environment of the lens. The solutions were photolyzed (transmission above 295 nm) for various periods of time while the loss of starting material and the formation of fluorescent photoproducts (blue emission at 470 nm, and green emission at 520 nm) were monitored using fluorescence and UV-visible spectroscopy and thin-layer and high-pressure liquid chromatography analysis. Several parameters were varied such as oxygen tension and the addition of the free radical scavenger, penicillamine. The photolytic loss of 3-HK in the absence of glycine occurred approximately 5-10 times faster than in its presence. Conversely, blue and green fluorophores formed in irradiated solutions containing glycine but not with the photolysis of 3-HK alone. The blue fluorophore was formed first and appeared then to be photochemically converted to the green one, with the rate of formation of the latter increasing with an increase in UV dosage or oxidizing conditions. The addition of penicillamine drastically reduced the photochemical formation of both fluorophores. Both the blue and green fluorophores appear to result from the photochemically induced covalent attachment of 3-HK to glycine.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of coloration of human lenses induced by near-ultraviolet-photo-oxidized 3-hydroxykynurenine.

Tha absorption spectra of human cataractous lenses, classified into four groups on the basis of coloration, were measured by a spectrophotometer equipped with an integrating sphere that is able to detect diffuse reflectance of solid materials. The results revealed that pale-yellow cataractous lenses showed a characteristic absorption spectrum with a peak near 365 nm, coincident with that of 3-hydroxykynurenine, and that brunescent cataractous lenses with high pigmentation showed an absorption spectrum with a peak over 400 nm, coincident with that of xanthommatin, a dimerized compound of 3-hydroxykynurenine. Depending on the coloration of the lens, the position of maximum absorption shifted from 365 to 410 nm and the optical density increased at visible regions. The changes in absorption spectra of pale-yellow lens homogenates were studied in the presence or absence of 3-hydroxykynurenine and by near-ultraviolet (UV) irradiation. Brown pigments were formed in the insoluble proteins of lens homogenates by 48 h of exposition of the samples with 3-hydroxykynurenine to UV irradiation. The absorption spectra of the brown pellets detected by an integrating sphere corresponded well to those of pellets which were obtained from the homogenates of intact human brunescent cataractous lenses. These results strongly suggest that 3-hydroxykynurenine and UV irradiation are involved in the brown pigmentation of human cataractous lenses by formation of xanthommatin.

Photochemistry of proteins: a review.

Proteins are an important target of photochemical damage to the eye. The wavelength of irradiation is a major determinant of the initial mechanisms. The effects of ultraviolet radiation are initiated by absorption in the aromatic amino acid residues and cystine. Photoionization of tyrosine and tryptophan residues leads to aromatic free radicals. The ejected electrons are stabilized in the aqueous medium as hydrated electrons and may be temporarily trapped by cystyl bridges. N-formylkynurenine is an important tryptophan photoproduct, which can act as an endogenous photosensitizer of near-ultraviolet radiation by generating singlet oxygen and superoxide. Singlet oxygen is produced also by exogenous sensitizers absorbing in the visible and near ultraviolet regions. The mechanisms are illustrated by porphyrins and furocoumarins, in which dark binding interactions influence the photosensitization pathways.