High lenticular tolerance to ultraviolet radiation-B by pigmented guinea-pig; application of a safety limit strategy for UVR-induced cataract.

PURPOSE: The purpose of this study was to determine a threshold measure, maximum tolerable dose (MTD), for avoidance of UVR-B-induced cataract in the pigmented guinea-pig. METHODS: Thirty pupil-dilated anesthetized young female guinea-pigs, divided into five equal groups, received between 0 and 84.9 kJ/m(2) unilateral UVR-B. Lens extraction and in vitro lens photography occurred 24 hr after exposure. Measurement of intensity of lens light scattering served as quantifying tool for the degree of cataract. Data analysis included regression, using a second order polynomial model. The applied MTD concept was based on the UVR-B dose-response curve obtained for the pigmented guinea-pig. A smaller number of pigmented guinea-pigs, pigmented rats and albino rats underwent morphometric analysis of the anterior segment geometry. RESULTS: All eyes exposed to UVR-B developed cataract in the anterior subcapsular region. MTD for avoidance of UVR-B-induced cataract was 69.0 kJ/m(2) in the pigmented guinea-pig. Iris was considerably thicker in the guinea-pig than in the rats. Lens blockage by the dilated iris was lowest in the guinea-pig. CONCLUSIONS: Maximum tolerable dose for avoidance of UVR-B-induced cataract in the pigmented guinea-pig was 69.0 kJ/m(2), over 10-fold higher than the threshold 5 kJ/m(2) obtained by Pitts et al. in the pigmented rabbit. Maximum tolerable dose is an appropriate method for estimation of toxicity for UVR-B-induced cataract in the guinea-pig. The pigmented guinea-pig is significantly less sensitive to UVR-B exposure than the pigmented rabbit and pigmented rat.

Drinking water supplementation with ascorbate is not protective against UVR-B-induced cataract in the guinea pig.

PURPOSE: To study if ascorbate supplementation decreases ultraviolet radiation (UVR)-induced cataract development in the guinea pig. METHODS: Sixty 6-9-week-old pigmented guinea pigs received drinking water supplemented with or without 5.5 mm l-ascorbate for 4 weeks. After supplementation, 40 animals were exposed unilaterally in vivo under anaesthesia to 80 kJ/m(2) UVR-B. One day later, the animals were killed and lenses were extracted. Degree of cataract was quantified by measurement of intensity of forward lens light scattering. Lens ascorbate concentration was determined with high-performance liquid chromatography (HPLC) with UVR detection at 254 nm. Twenty animals were used as non-exposed control. RESULTS: Supplementation increased lens ascorbate concentration significantly. In UVR-exposed animals, mean 95% confidence intervals (CIs) for animal-averaged lens ascorbate concentration (micromol/g wet weight lens) were 0.54 +/- 0.07 (no ascorbate) and 0.83 +/- 0.05 (5.5 mm ascorbate). In non-exposed control animals, mean 95% CIs for animal-averaged lens ascorbate concentration (micromol/g wet weight lens) were 0.72 +/- 0.12 (0 mm ascorbate) and 0.90 +/- 0.15 (5.5 mm ascorbate). All non-exposed lenses were devoid of cataract. Superficial anterior cataract developed in all UVR-exposed lenses. The lens light scattering was 39.2 +/- 14.1 milli transformed equivalent diazepam concentration (m(tEDC)) without and 35.9 +/- 14.0 m(tEDC) with ascorbate supplementation. CONCLUSION: Superficial anterior cataract develops in lenses exposed to UVR-B. Ascorbate supplementation is non-toxic to both UVR-B-exposed lenses and non-exposed control lenses. Ascorbate supplementation does not reduce in vivo lens forward light scattering secondary to UVR-B exposure in the guinea pig.

Ultraviolet radiation-B-induced cataract in albino rats: maximum tolerable dose and ascorbate consumption.

PURPOSE: To investigate the maximum tolerable dose (MTD) for cataract induced by ultraviolet radiation-B (UVB) in 7-week-old albino rats and to study the effect of UVB eye exposure on lens ascorbate content. METHODS: Fifty 7-week-old albino Sprague Dawley rats were unilaterally exposed in vivo to 300-nm UVB under anaesthesia, receiving 0, 0.25, 3.5, 4.3 and 4.9 kJ/m(2). The MTD was estimated based on lens forward light scattering measurements. Lens ascorbate content was determined in the processed lens using high performance liquid chromatography with UVR detection. RESULTS: Animals exposed to UVB doses >or=3.5 kJ/m(2) developed cortical cataracts. The MTD for avoidance of UVB-induced cataract was estimated to 3.01 kJ/m(2). UVB exposure decreased lens ascorbate concentration in the exposed lens in line with UVB dose, H(e), according to the models: C = C(NonCo) + C(Co)e(-kH(e) ) for exposed lenses; C = C(NonCo) + C(Co) for non-exposed lenses, and C(d) = C(Co)(e(-kH(e) ) - 1). Parameters for consumable and non-consumable ascorbate were estimated to C(NonCo) = 0.04 and C(Co) = 0.11 micromol/g wet weight of lens. For lens ascorbate difference, tau = 1/k = 0.86 kJ/m(2). A total of 63% of UVB consumable ascorbate has been consumed after only tau = 0.86 kJ/m(2), while MTD(2.3 : 16) = 3.01 kJ/m(2), indicating that ascorbate decrease is in the order of 3.5 times more sensitive to detecting UVR damage in the lens than forward light scattering. CONCLUSIONS: The MTD for avoidance of UVB-induced cataract in the 7-week-old albino Sprague Dawley rat was estimated to be 3.01 kJ/m(2). In vivo UVB exposure of the rat eye decreases lens ascorbate content following an exponential decline, and suprathreshold doses cause greater effect than subthreshold doses.

Time dependency of metabolic changes in rat lens after in vivo UVB irradiation analysed by HR-MAS 1H NMR spectroscopy.

The lens ability to protect against, and repair ultraviolet radiation (UVR) induced damages, is of crucial importance to avoid cataract development. The influence of UVR-induced damage and repair processes on the lens metabolites are not fully understood. Observation of short- and long-term changes in light scattering and the metabolic profile of pigmented rat lenses after threshold UVR exposure might serve to better understand the protective mechanisms in the lens. By using high resolution magic angle spinning (HR-MAS) 1H NMR spectroscopy it was possible to investigate the metabolites of intact rat lenses. Brown-Norway rats were exposed to 15 kJm(-2) UVB irradiation. One eye was exposed and the contralateral served as control. The rats were sacrificed 5, 25, 125, and 625 hr post-exposure and the lenses were removed. The degree of cataract was quantified by measurement of lens forward light scattering. Thereafter, proton NMR spectra from intact lenses were obtained and relative changes in metabolite concentrations were determined. The light scattering in the lens peaked at 25 hr post-exposure and decreased thereafter. The lowest level of light scattering was measured 625 hr after exposure. No significant changes in concentration were observed for the metabolites 5 and 25 hr post-exposure except the total amount of adenosine tri- and diphosphate (ATP/ADP) that showed a significant decrease already 5 hr after exposure. At 125 hr the lens concentrations of lactate, succinate, phospho-choline, taurine, betaine, myo-inositol, and ATP/ADP showed a significant decrease (p<0.05). Phenylalanine was the only metabolite that revealed a significant increase 125 hr post-exposure. At 625 hr most of the metabolic changes seemed to normalise back to control levels. However, the concentration of betaine and phospho-choline were still showing a significant decrease 625 hr after UVB irradiation. The impact of UVB irradiation on the metabolic profile did not follow the same time dependency as the development of cataract. While the light scattering peaked at 25 hr post-exposure, significant changes in the endogenous metabolites were observed after 125 hr. Both the metabolic changes and the light scattering seemed to average back to normal within a month after exposure. Significant decrease in osmolytes like taurine, myo-inositol and betaine indicated osmotic stress and loss of homeostasis. This study also demonstrated that HR-MAS 1H NMR spectroscopy provides high quality spectra of intact lenses. These spectra contain a variety of information that might contribute to a better understanding of the metabolic response to drugs or endogenous stimuli like UVB irradiation.

Ascorbate in the rat lens: dependence on dietary intake.

PURPOSE: To establish a method for sample preparation to measure ascorbate in whole lenses and to investigate whether lens ascorbate concentration is dependent on dietary ascorbate intake. METHODS: Four groups of 3 young Sprague-Dawley rats each were fed chow containing L-ascorbate, either 0.0, 5.7, 57.0 or 114.0 mmol/kg for a duration of 4 weeks. Thereafter, each rat was sacrificed. The lens was extracted, photographed, and lens wet weight was measured. The lens was homogenized in 1.0 ml of 0.25% metaphosphoric acid, the homogenate was centrifuged and the supernatant ultrafiltered. The filtrate was injected into an ion exchange, reversed-phase Polypore H HPLC column equipped with a 254-nm ultraviolet detector. Samples were calibrated against an L-ascorbate standard. Polynomial regression analysis was performed on the data. RESULTS: All lenses were devoid of cataract. A 95% confidence interval for baseline content of ascorbate without any dietary intake was estimated to be 0.16+/-0.01 micromol/g wet weight of lens. The lens ascorbate concentration increased linearly with dietary ascorbate intake with an increased rate, estimated as a 95% confidence interval of 0.33+/-0.18 (micromol ascorbate) (g lens)-1)(mol ascorbate)-1 (kg chow) with r2=0.62. CONCLUSION: Lens ascorbate concentration linearly increases with dietary ascorbate intake without cataract development in the rat. The currently presented method for sample preparation to measure the whole-lens content of ascorbate is applicable.

Ascorbate in the guinea pig lens: dependence on drinking water supplementation.

PURPOSE: To investigate whether lens ascorbate concentration can be elevated with drinking water supplementation. METHODS: Pigmented guinea pigs received drinking water supplemented with L-ascorbate, concentration 0.00, 2.84, 5.68 or 8.52 mm for a duration of 4 weeks. In addition, the chow fed to all animals contained 125 mmol L-ascorbate per kg of chow. At the end of the supplementation period, the guinea pigs were killed. Each lens was extracted. The lens was processed and ascorbate concentration was measured using high performance liquid chromatography (HPLC) with 254 nm ultraviolet radiation detection. The data were analysed with regression. RESULTS: At the end of the test period, all lenses were devoid of cataract as observed by slit-lamp examination. All lenses contained a detectable concentration of ascorbate. Estimated 95% confidence intervals for mean animal-averaged lens ascorbate concentrations (micromol/g wet weight of whole lens) per group were 0.51 +/- 0.04 (0.00 mm; n = 6), 0.70 +/- 0.18 (2.84 mm; n = 6), 0.71 +/- 0.11 (5.68 mm; n = 5), and 0.71 +/- 0.06 (8.52 mm; n = 6). Animal-averaged lens ascorbate concentration [Asc(lens)] (micromol/g wet weight lens) increased with ascorbate supplementation in drinking water [Asc(water)] (M), in agreement with the model: [Asc(lens)] = A - Be(-kAsc(water)]. CONCLUSION: Lens ascorbate concentration increases with drinking water supplementation in the guinea pig without cataract development. The currently presented method for measurement of whole lens ascorbate content is suitable.