UV-B-induced DNA damage and repair in the mouse lens

Abstract

Purpose: Epidemiologic studies have linked UV-B exposure to development of cortical cataracts, but the underlying molecular mechanism(s) is unresolved. Here, we used a mouse model to examine the nature and distribution of DNA photolesions produced by ocular UV-B irradiation.

Methods: Anesthetized mice, eye globes, or isolated lenses were exposed to UV-B. Antibodies specific for 6-4 photoproducts (6-4 PPs) or cyclobutane pyrimidine dimers (CPDs) were used to visualize DNA adducts.

Results: Illumination of intact globes with UV-B-induced 6-4 PP and CPD formation in cells of the cornea, anterior iris, and central lens epithelium. Photolesions were not detected in retina or lens cells situated in the shadow of the iris. Photolesions in lens epithelial cells were produced with radiant exposures significantly below the minimal erythemal dose. Lens epithelial cells rapidly repaired 6-4 PPs, but CPD levels did not markedly diminish, even over extended postirradiation recovery periods in vitro or in vivo. The repair of 6-4 PPs did not depend on the proliferative activity of the epithelial cells, since the repair rate in the mitotically-active germinative zone (GZ) was indistinguishable from that of quiescent cells in the central epithelium.

Conclusions: Even relatively modest exposures to UV-B produced 6-4 PP and CPD photolesions in lens epithelial cells. Cyclobutane pyrimidine dimer lesions were particularly prevalent and were repaired slowly if at all. Studies on sun-exposed skin have established a causal connection between photolesions and so-called UV-signature mutations. If similar mechanisms apply in the lens, it suggests that somatic mutations in lens epithelial cells may contribute to the development of cortical cataracts.

Keywords: DNA damage; UV-B; cortical cataract.

Figure 1

Distribution of photolesions following UV-B irradiation of intact globes. Mid-Sagittal paraffin sections of eyes from 2-month-old mice following irradiation with 500 mJ/cm² UV-B. Lesions were visualized with antibodies against CPDs or 6-4 PPs (green). Nuclei were counterstained with DRAQ5 (red). Strong CPD (A) and 6-4 PP (C) signals are detected in cornea, iris, and central lens epithelium. Boxed regions in (A, C) are shown at higher magnification in (B, D). Note the presence of CPD- and 6-4 PP-positive nuclei throughout the full thickness of the cornea. Nuclei at the anterior surface of the iris (arrows) and lens epithelial cells located in the pupillary space (asterisk) are labeled strongly. RT, retina; Le, lens; I, iris; Co, cornea. Scale bars: 0.5 mm (A, C); 100 μm (B, D).

Figure 2

Analysis of photoproduct formation in the lens epithelium of irradiated eyes. Intact globes from 2-month-old mice were irradiated with UV-B at (A) 5 mJ/cm², (B) 250 mJ/cm², and (C) 50 mJ/cm². Lens epithelia were dissected, fixed and immunostained (green) with anti-CPD (A, B) and anti–6-4 PP (C). Nuclei were counterstained with DRAQ 5 (red). Both kind of photolesion are restricted to an elliptical region (arrowed in [B]) in the center of the epithelium. The size and shape of the elliptical region match the dimensions of the pupil. Scale bar: 0.5 mm.

Figure 3

Cyclobutane pyrimidine dimer production in lens epithelia irradiated in situ or in isolation. Cyclobutane pyrimidine dimers were visualized by immunofluorescence and quantified using image analysis. The level of UV-B–induced CPDs in lens epithelial cell nuclei is significantly reduced when lenses are irradiated in situ (A) compared with irradiation in isolation (B). Quantitative analysis (C) suggests that lens CPD levels are reduced by 70% to 80% when intact globes rather than isolated lenses are irradiated. The shielding afforded by an intact globe is similar in 1-month-old and 4-month-old animals. Data represent the mean ± SEM of three to five independent experiments in each case. Scale bar: 25 μm.

Figure 4

Differential repair of CPDs and 6-4 PPs in the lens epithelium of UVB-irradiated eyes. Intact globes from 1-month-old mice were irradiated with 5 mJ/cm² (A) or 50 mJ/cm² (B) of UV-B to induce formation of CPDs (A) or 6-4 PPs (B), respectively (green). Nuclear DNA (red) was visualized with DRAQ5 (red). Following irradiation, lenses were cultured for the periods shown, during which the levels of photolesions were quantified (C). Levels of 6-4 PPs return to baseline values after 24 hours in culture. In contrast, there is no reduction in CPDs over the culture period. Data represent the mean ± SEM of four to seven independent experiments for each time point. Scale bar: 25 μm.

Figure 5

Ultraviolet-B–induced CPDs are not repaired over prolonged periods in vivo. Levels of CPDs (green) were quantified one week (A) after irradiation of 2-month-old mice with low level (3mJ/cm²) UV-B and compared to values measured immediately after irradiation (B). There is a small (<30%) reduction in CPD levels one week after irradiation (C). Deoxyribonucleic acid was counterstained with DRAQ5 (red). Scale bar: 0.1 mm.

Figure 6

Regional distribution of proliferative activity in the lens epithelium. Mitotic cells (arrows) are visualized with PCNA staining in the lens epithelium of a 2-month-old mouse. Proliferating cells are localized to the GZ near the equatorial margin of the epithelium. Proliferating cells were rarely detected in the CZ of the epithelium. Scale bar: 0.1 mm.

Figure 7

Deoxyribonucleic acid repair capacity is not related to proliferative activity. Lenses were exposed to UV-B (5 mJ/cm²) to induce 6-4 PP formation and then placed in culture to assess the capacity of cells in the CZ and GZ to repair UV-B–induced DNA damage. (A) Photolesions were visualized with anti–6-4 PPs (green). The distribution of mitotic cells was determined by PCNA immunofluorescence (red) and nuclei were counterstained with DRAQ5 (blue). 6-4 PPs levels are initially comparable in the two regions (B) and subsequently decline at similar rates (C). After 6 hours the levels had fallen to approximately 70% of the value at t = 0. Scale bar: 50 μm.