Surfactant and UV-B-induced damage of the cultured bovine lens.

PURPOSE: To evaluate in vitro methods for testing the toxicity of the surfactants, sodium dodecyl sulfate (SDS) and benzalkonium chloride (BAK), and Ultraviolet (UV)-B radiation to the bovine lens. METHODS: Lenses were dissected from bovine eyes--obtained from a local abattoir--and incubated in M199 culture medium at 37 degrees C, with 4% CO(2) and 96% air atmosphere. For the SDS and BAK experiments, the lenses (n = 153) were exposed directly to 0.001%, 0.01%, 0.1%, and 1.0% solutions for 15 min. These lenses were then rinsed five times each with saline and medium. Another group of lenses (n = 36) was irradiated with broadband UV-B at energy levels of 1.0 and 2.0 J/cm(2) (0.445 and 0.89 J/cm(2) in the biologically effective energy levels). For all of the above experiments, lens optical quality and cellular viability of lens epithelial cells were evaluated. RESULTS: The analysis of optical quality, using a scanning laser in vitro assay system, of exposed lenses treated with SDS and BAK at concentrations of 0.01%, 0.1%, and 1.0%, and with UV-B at energy levels of 0.445 and 0.89 J/cm(2) showed a dose- and time-dependent increase in back vertex distance (BVD) variability, indicating loss of sharp focus in comparison with control lenses. Both 0.001% SDS and 0.001% BAK-treated lenses did not show any optical damage until 8-days after exposure. Lenses treated with 0.01% SDS showed recovery from optical damage 6-days later after exposure. Optical damage was not shown immediately for UV-B-exposed lenses. The Alamar Blue assay data for SDS, BAK and UV-B-exposed lenses, except the 0.001% SDS treated lens group, showed also dose- and time-dependent decreases in cellular viability in comparison with the control lenses, and there was no cellular recovery during the entire culture period. Lenses treated with 0.001% SDS did not show biological damage until 8-days after exposure. It appears that cellular changes appeared earlier than optical changes. CONCLUSIONS: The findings suggest that cultured bovine lenses can be evaluated by assays that probe optical properties and cellular function after exposure to surfactants and UV-B irradiation, and that the optical and biological assay methods are valuable for in vitro mild ocular toxicity research.

Effect of hyperbaric oxygen on guinea pig lens optical quality and on the refractive state of the eye.

The main objective of this study was to investigate the effect of in vivo hyperbaric oxygen (HBO) treatment of albino guinea pigs on ocular refractive state and optical properties of the lens in vitro, as well as on the integrity of the mitochondria of the lens. The animals were treated 30-35 times (2.5-3 months) or 70 times (6 months) with HBO. An increased level of lens nuclear light scattering was evident by slit-lamp at 30 treatments, and this increased at 70 treatments. After 30-35 HBO treatments a myopic shift in refractive state of the eye was seen in two separate studies with two different refractionists. Also, the average back vertex distance of the lens was significantly shorter after 35 HBO treatments while spherical aberration (focal variability) increased after 70 treatments. No difference in refractive state was noted after 70 HBO treatments (a reversal of the initial myopic effect). The mitochondrial distribution and morphology of the lens epithelium and the superficial cortical fibre cells were normal after both 35 and 70 HBO treatments, highlighting that HBO treatment does not affect the superficial cortex of the lens. The results of the in vitro lens optical analysis carried out in this study correlate with the myopia observed after 30-35 HBO in vivo treatments. A similar reversible myopia and increase in lens nuclear light scattering is known to occur in humans treated with HBO for extended periods and the results suggest that the myopia was caused by a change in the refractive index of the lens. The significant loss of sharp focus after 70 HBO treatments can be correlated with previous reports of biochemical and morphological changes associated with HBO-induced loss of lens nuclear transparency in mature guinea pigs. The guinea pig HBO model may be a useful approach for the study of lens development and refractive error.

AlamarBlue bioassay for cellular investigation of UV-induced crystalline lens damage.

PURPOSE: The use of the alamarBlue fluorescence dye for cellular study of UV-induced photodamage in cultured ocular lenses was examined by comparing the results from the fluorometric assay to lens optical quality using a scanning laser system to measure the focal lengths of the lenses following UVB treatment. METHODS: Excised porcine lenses were cultured in M199 supplemented with 1% antibiotics and 4% porcine serum. After 1 week of pre-incubation at 37 degrees C, baseline measurements were taken. Treated lenses were irradiated with a range of UVB radiant exposures from 0.019 to 0.076 J cm-2. The lenses were maintained for a further 4 weeks, with measurements carried out every 48 h in the first 9 days post-UVB treatment and then once each week. At each measurement session, treated and control lenses were transferred into a 24-well plate, one lens per well containing the assay. The lenses were incubated for 50 min, after which fluorescence readings were taken with a plate reader. RESULTS: Analyses showed significant (p < 0.05) inhibition of lens metabolic activity and optical function in the 0.038 and 0.076 J cm-2 UVB treated lenses. Lenses treated with 0.019 J cm-2 UVB did not exhibit any photodamage. CONCLUSIONS: These results suggest that the alamarBlue assay is useful for the in vitro study of UV-induced lens damage. The decrease in the capacity of treated lenses to reduce alamarBlue over time confirms that UVB photo-oxidation can cause diminution of viable lens epithelial and fibre cells. The results also suggest that the energy threshold for broadband UVB induced cataractogenesis in vitro ranges between 0.019 and 0.038 J cm-2.

Evaluation of a porcine lens and fluorescence assay approach for in vitro ocular toxicological investigations.

Cell biology, as monitored with the fluorescent indicator dyes Alamar Blue and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), and lens optical quality, as measured with an in vitro scanning laser system, have been used to evaluate in vitro the condition of porcine lenses after being placed in a culture medium. The measurements, beginning from week one of culture, were compared statistically. Optical quality and cellular viability, as measured with either dye, were unchanged in lenses that had been maintained for 6 weeks in modified M199 medium. Some lenses were treated with 0.152J/cm(2) UVB radiation, and a decline was observed after 48 hours in both optical and metabolic capabilities, as indicated by a decreased capacity of the lenses to reduce Alamar Blue. The measurements with CFDA-AM did not show complete concordance with the other indicators of lens health after UV treatment, making this dye less reliable as applied currently to lens cultures. Overall, the findings suggest that porcine lenses can be maintained for weeks in culture, and that their condition can be evaluated quantitatively by assays that probe cellular functions and optical properties. Such a system should prove valuable for in vitro ocular pharmacotoxicological research.