Focal Length Variability and Protein Leakage as Tools for Measuring Photooxidative Damage to the Lens¶

Abstract

Hypericin is the ingredient used to standardize the popular over‐the‐counter antidepressant medication St. John's Wort. Because hypericin readily produces singlet oxygen and other excited state intermediates, it is a very efficient phototoxic agent in the eye that can potentially induce the development of the cataract photooxidative mechanism. Hypericin absorbs in the UV and visible ranges, binds to the lens crystallins (α, β and γ) and damages these proteins through a photooxidative mechanism. Effects were measured previously using fluorescence, UV and mass spectrometry. We report here two additional methods to monitor lens damage: (1) measuring focal length variability using a ScanTox™ instrument and (2) measuring protein leakage from the damaged lens. Because nonenzymic glycation results in free radical production, we chose to use elevated glucose concentrations as a convenient model for studying oxidative stress. To compare and contrast photooxidative damage against oxidative damage to the lens, we also measured the focal length variability and protein leakage induced by the presence of elevated glucose concentrations. We found that the total accumulated protein leakage was positively correlated (r= 0.9) with variability in focal length. Lenses treated with hypericin and irradiated with UVB had an increase in focal length variability as compared with the lenses that were only UVB‐irradiated. Lenses without UVB irradiation had much lower focal length variability than irradiated lenses. For non–hypericin‐treated lenses, UVB‐irradiated lenses had a larger variability (4.58 mm) than the unirradiated lenses (1.78 mm). The lenses incubated in elevated glucose concentrations had a focal length variability (3.23 mm) equivalent to that of the unirradiated hypericin‐treated lenses (3.54 mm). We conclude that photooxidative damage by hypericin results in changes in the optical properties of the lens, protein leakage and finally cataract formation. In contrast to this, high concentrations of glucose induced protein leakage but not changes in optical properties or the opacity associated with a cataract. This work provides further evidence that people should protect their eyes from intense sunlight when taking St. John's Wort.

Long-term Lens Organ Culture System with a Method for Monitoring Lens Optical Quality¶

“…The system is also used to measure ocular irritancy of contact lens solutions (39) and oxidative damage (40). Using the lens-culture system, it was found that the lens can recover in culture conditions from damage induced by different insulting factors (21,27,32,39,41).…”

UV‐Mediated DNA Strand Breaks in Corneal Epithelial Cells Assessed Using the Comet Assay Procedure¶

“…Fortunately, UVC radiation is completely absorbed by the ozone layer (2, 3). However UVA and some UVB radiation pass through the ozone layer, and it has been suggested that UVA and UVB irradiation mediate agerelated changes in the eye and can cause ocular disease (1, 4–10). This is of particularly importance to the cornea because it is the first layer of the eye exposed to sunlight (4–6).…”

Phototoxicity in Human Lens Epithelial Cells Promoted by St. John's Wort¶

“…In vitro studies have determined that hypericin inhibits the growth of retinal pigment epithelial cells ( 17 ). Our previous studies have shown that hypericin is taken up by the intact lens ( 18 ), photooxidizes lens proteins ( 19 ) and distorts the transmission of light through the lens ( 20 ). Therefore, hypericin fulfills the criterion of a potential ocular phototoxic agent.…”