Vitamin C mediates chemical aging of lens crystallins by the Maillard reaction in a humanized mouse model

Abstract

Senile cataracts are associated with progressive oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. We hypothesized that the Maillard reaction, which leads browning and aroma development during the baking of foods, would occur between the lens proteins and the highly reactive oxidation products of vitamin C. To test this hypothesis, we engineered a mouse that selectively overexpresses the human vitamin C transporter SVCT2 in the lens. Consequently, lenticular levels of vitamin C and its oxidation products were 5- to 15-fold elevated, resulting in a highly compressed aging process and accelerated formation of several protein-bound advanced Maillard reaction products identical with those of aging human lens proteins. These data strongly implicate vitamin C in lens crystallin aging and may serve as a model for protein aging in other tissues particularly rich in vitamin C, such as the hippocampal neurons and the adrenal gland. The hSVCT2 mouse is expected to facilitate the search for drugs that inhibit damage by vitamin C oxidation products.

Fig. 1.

Selected chemical pathways linking vitamin C oxidation with the formation of advanced Maillard reaction products assayed in this study.

Fig. 2.

Effects of hSVCT2 overexpression in mouse lens epithelial and fiber cells on uptake and lenticular levels of vitamin C and DHA. Immunohistochemical staining of a typical control (a Left) and transgenic (a Right) lens with an antibody against the C-terminal portion of hSVCT2 reveals a diffuse presence of hSVCT2 along the cell membrane in cortical layers of the lens. (b) 6-fluoro-6-deoxy ASA uptake into cultured lenses after incubation with 100 μM for 120 min (P < 0.001). (c) ASA concentration in 16 lenses from hSVCT2 mice, 13 controls from the same litter, and six human lenses. (d) DHA concentration in 10 lenses from hSVCT2 and control mice.

Fig. 3.

Effects of hSVCT2 overexpression on lens protein-bound fluorescence and advanced ascorbylation product. Student's t test was used for all comparisons. (a) Fluorescence at 335/385 nm excitation/emission in enzymatic protein digest was increased at all ages in hSVCT2 compared with wild type (P < 0.05 to P < 0.0001). (b) Fluorescence at 370/440 nm was similarly increased in transgenic mice (P value from nonsignificant at 6 months to P < 0.0001 for 12 vs. 9 months. (c) Cross-link K2P content in transgenic vs. wild type was higher at all ages (P < 0.0001). (d) Pentosidine levels were significantly higher in transgenics at all ages (P < 0.001 to P < 0.0001). (e) Vesperlysine A was detectable only in transgenic mice. (f) The CML level was significantly higher in transgenic mice (P < 0.0001 for 6 months; P < 0.003 for 9 months; P < 0.001 for 12 months). (g) CEL was elevated in transgenic mice (P < 0.05) but did not increase with age. (h) Fructosyl-lysine measured as furosine was unchanged in transgenic mice (P > 0.05).

Fig. 4.

Macroscopic appearance of the hSVCT2^+/+ lenses at 12 months. The transgenic lenses are colored yellow. All mouse lenses were photographed in the same dish to allow accurate comparison between wild-type and transgenic lenses. A fresh 67-year-old normal human lens obtained at autopsy is shown for comparison.