Aggregation of Guinea Pig Lens Cortical and Nuclear Crystallins Induced in vitro by UVA Light

Abstract

Purpose:: A possible role for UVA light in the formation of maturity-onset nuclear cataract is not well understood. Here we have compared the ability of UVA light to induce increased light scattering and crystallin aggregation in guinea pig lens cortical and nuclear supernatants (guinea pig lens contains large amounts of the UVA chromophore NADPH bound to ζ-crystallin).

Methods:: Guinea pig lens nuclear and cortical water soluble proteins (1mg/ml) were exposed to UVA light (338-400nm, 200mW/cm²) for 4h. Preliminary studies described here were conducted in room air; future studies will be conducted at lower levels of oxygen to better mimic physiological conditions. H₂O₂ production was measured by ferrous oxidation in xylenol orange (FOX-1 assay), light scattering by increase of absorbance at 360nm, and formation of high molecular weight (HMW) proteins by gel electrophoresis with and without reduction by dithiothreitol. All results were compared to dark controls. Western blotting was conducted using antibodies to αA-, αB-, ß-, γ- and ζ-crystallins.

Results:: After 4 hours of UVA-irradiation, H₂O₂ concentrations were 12.4µM in nuclear supernatant (NS) and 5.6µM in cortical supernatant (CS), with no detectable H₂O₂ in dark controls (H₂O₂ production was confirmed separately by inclusion of catalase with the FOX-1 reagents). UVA-induced light scattering in NS was nearly 5-fold higher than NS dark controls, and 10-fold higher than UVA-irradiated CS. The concentration of GSH in CS (0.5mM) was 10x higher than NS (0.05mM). Formation of high molecular weight (HMW) crystallin aggregates >120 kDa, by both disulfide and non-disulfide crosslinking, was detected in UVA-treated NS, but not in UVA-treated CS, nor in dark controls. The aggregates were found to include both γ- and ζ-crystallins. When either dithiothreitol (12.5mM) or GSH (5.0mM) was added to NS before UVA-irradiation, light scattering was reduced by 50-65%.

Conclusions:: Guinea pig lens nuclear proteins were more susceptible to UVA-induced increases in light scattering and aggregation than cortical proteins, possibly because of lower levels of GSH present in the nucleus. UVA-induced aggregates consisted mainly of γ- and ζ-crystallins. The results may have relevance to the formation of maturity-onset nuclear cataract.

Keywords: crystallins • oxidation/oxidative or free radical damage • cataract