Physiological Effects of UVB Irradiation on Cultured Rabbit Lens

Transcription

1 Investigative Ophthalmology & Visual Science, Vol. 33, No. 5, April 1992 Copyright Association for Research in Vision and Ophthalmology Physiological Effects of UVB Irradiation on Cultured Rabbit Lens Kenneth Hightower and Janet McCready The purpose of this study was to investigate the physiological effects of irradiation in the spectral range nm in cultured rabbit lenses. Ultraviolet B cataract was produced in lenses exposed to low levels of irradiation, 1-2 mw/cm 2. Opacification was assessed by laser transmittance measurements. The changes observed during lens culture after a 1 hr dose (4 J/cm 2 ) include a gradual increase in hydration, sodium concentration, and calcium levels. Loss in membrane voltage and a rise in ^Cl accumulation indicate that membrane permeability was increased. The cation pump was impaired within 20 hr of irradiation, as concluded by an observed fall in 22 Na efflux. Availability of glucose for cation transport was diminished based on the reduced rate of uptake of tritiated 3-o-methylglucose in irradiated lenses, but this reduced accumulation was observed much later than was sodium elevation. Ionic imbalances and opacification required less than 1 d of culture for 4-12-wk-old lenses and required nearly 7 d of culture for 100-wk-old lenses. Invest Ophthalmol Vis Sci 33: , 1992 Ultraviolet (UV) cataracts in animals may serve as useful models for human cataracts. However, detailed mechanisms related to low-level UV irradiation in the nm region remain unclear. Potential targets include tryptophan, which may produce byproducts that can subsequently bind to membrane components or crystallins Much of our information on UV cataract comes from Zigman's studies describing effects of UVA irradiation, ie, UV light in the 360 nm region. 4 Cataracts were produced in the squirrel and in the rat using a wavelength spectrum between 340 and 380 nm, a range found to correspond to the absorption spectra of tryptophan photo-oxidation products such as kynurenine. 5 In one study, the loss of tryptophan after UVA (360nm) irradiation was correlated with an inhibition of Na/K-ATPase in homogenates of rat lens. 6 Dillon 5 has shown that photo-oxidation products of tryptophan can be produced by 290 nm irradiation of lens proteins. One such product is 3-hydroxy-kynurenine glucoside, which also can absorb UV at 360 nm. Thus, it and other related compounds appear to be sensitizers or targets of UVA. Whether such products directly inhibit Na/K-ATPase activity in irradiated rat lenses is unknown. Except for a number of studies involving high energy UV laser irradiation, 7 ' 8 ' 9 few studies have been From the Eye Research Institute, Oakland University, Rochester, Michigan. Supported by National Eye Institute grant No. EY Submitted for publication: May 15, 1991; accepted November 1, Reprint requests: Kenneth Hightower, the Eye Research Institute, Oakland University, Rochester, MI done to explain low-level UVB cataract such as that produced by Pitts 10 and Jose. 11 Importantly, the results demonstrated that lower wavelengths had a lower threshold for opacification than the wavelength region of UVA. The biochemical and physiological processes altered in UVB cataract have received little investigation. The present study offers an in vitro model for UVB cataract in cultured rabbit lenses and attempts to characterize the ionic changes and identify possible targets. Particular emphasis is placed on using brief exposure times rather than chronic exposure, using wavelengths confined to nm, with a peak at 311 nm. The conclusion is that the resulting opacification and the ionic imbalances observed are a result of irreversible alterations in membrane transport and permeability properties. Methods Lenses were irradiated with a Spectroline lamp/system (Model EB-180C, Markson Scientific, Phoenix, AZ) through glass vials, at a dose of approximately 1-2 mw/cm 2 at the surface of the lens. Thus, a 1 hr dose delivers 4 J/cm 2. As shown in Figure 1, the wavelength spectrum reaching the lens had a maximum of 311 nm and a range of nm, with 90% of the energy between 300 and 330 nm. Power levels were monitored with an YSI-Kettering Radiometer (Model 65, Yellow Springs Instrument Co., Yellow Springs, OH), and relative intensities were measured with a model 902 GBC Atomic Absorption Spectrophotometer (Applied Research Laboratories, Dearborn, MI.) 1783

2 1784 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / April *- Larrp/filter lamp/filter + glass Wavelength (nm) Fig. 1. Graph of relative intensity of UVB Spectroline black lamp with its filter in place (dashed line) and through glass as used to culture lenses (solid line). Peak wavelength occurs at 311 nm at a maximum power of 1 mw/cm2 as measured with YSI-Kettering Radiometer. New Zealand White albino rabbits were killed with injection of t-61 euthanasia solution (Hoechst-Roussel Agri-Vet Co., Somerville, NJ). Immediately upon dissection, lenses were placed anterior down in glass vials with tissue culture medium 199 (Gibco Laboratories, Life Technologies, Inc., Grand Island, NY). Vials were corked and placed on the UV lamp, which maintained a temperature of 30 C during the irradiation. Control lenses were similarly placed, posterior side up and on the lamp with the light blocked. Lenses then were transferred to petri dishes containing medium with gentamicin (Gibco) for further culture in a tissue culture chamber with 5% C0 2 and 20% 0 2. Efflux experiments were carried out by loading control and 1 hr irradiated lenses with 22Na sodium chloride (50,000 cpm/0.1 ml) in medium 199 for 20 hr immediately following irradiation. The efflux, unless stated otherwise, occurred over a 3 hr period. Details of this procedure were published previously.'2 In experiments measuring 36C1 accumulation, lenses were transferred to medium 199 containing 36 C1 sodium chloride (25,000 cpm/0.1 ml). The amount of 36C1 taken up by the lens in 20 hr was measured. Data are expressed as the ratio of cpm in the lens to cpm in the medium. For experiments that measured glucose accumulation, lenses were exposed to UV for 1 hr and were transferred to medium 199 containing 5,000 cpm/ 0.10 ml 3H 3-o-methylglucose. Lenses in this medium were incubated at 35 C. After 24 hr, culture lenses were weighed, homogenized in 10% trichloroacetic acid and spun down on a table top centrifuge (Multifuge C1387-1, American Scientific Products, McGaw Park, IL). One hundred microliters of supernatant was counted (see below). Glucose uptake values are Vol. 33 expressed as the ratio of cpm in the lens water to the cpm in the medium. Radioisotopes (3H 3-O-methylglucose, 22Na sodium chloride, 36C1 sodium chloride) used for the above procedures were obtained from the New England Nuclear division of the Du Pont Co. (Wilmington, DE). Isotopes were counted in Ecolume scintillation cocktail (ICN Biomedicals, Inc., Irvine, CA) on a Beckman series LS 5801 scintillation counter (Beckman Instruments, Inc., Arlington Heights, IL). Measurements of ion concentrations and lens voltage were routinely performed as described in detail previously.13 These investigations conformed to the AR VO Resolution on the Use of Animals in Research. Results Initial experiments were aimed at establishing a correlation between the UVB dose and the onset and extent of opacification. Exposure times from 1-5 hr caused opacification within 20 hr of subsequent culture, as shown in Figure 2. As measured by laser transmittance, the loss of transparency ranged from 8095% (n = 8). Lenses also were observed to swell, gaining an average of 13 mg (± 2 mg, n = 5) within 6 hr of Fig. 2. Photograph of rabbit lenses following 1-hr UVB irradiation and subsequent culture for 0,24, and 48 hr, the latter lens being the most opaque against the black background.

3 No. 5 IN VITRO UVD CATARACT / Highrower ond McCready 1785 culture, and 26 mg (± 3 mg, n = 9) after 20 hr of culture. Because lenses were clearly opaque and swollen after overnight culture, membrane leakiness was studied by measuring the uptake of the passively distributed chloride ion and the extent of depolarization of the lens voltage. Figure 3 shows values of Cl 36 uptake ratios (lens/media) following a 20 hr uptake period. Irradiated lenses accumulated significantly more label than controls by a factor of approximately three. Another indication of membrane damage was the observed depolarization (Fig. 4) in lens voltage from an average of-67 ± 3 mv to -55 ± 5 mv within 1-2 hr of irradiation. Additional culture for 20 hr resulted in a final lens voltage of only -25 ± 8 mv. When the dose was only l h hr at 1 mw the lens voltage recovered to the normal 67 mv. These results suggest that a 1 hr dose would be appropriate for use in subsequent experiments to assess the extent of ionic imbalances. The results in Figure 5 illustrate the rapid increase in sodium and calcium levels during culture following a 1 hr exposure to UVB. The greatest rate of increase in sodium levels occurred within the first 6 hr after irradiation, with the mean concentration rising from 15 to 43 mm. At this point, lenses were still transparent, but equatorial vacuoles were evident upon microscopic examination. Calcium levels rose from 0.2 mm to nearly 0.5 mm within the same 6 hr period. At 20 hr, calcium and sodium levels had increased to approximately 1.4 and 87 mm, respectively. Notable is that further culture beyond 20 hr under optimum conditions did not reverse ion concentrations or recovery of lens transparency. The changes described indicate a loss in the integrity of lens membranes leading to alterations in passive ionfluxes. To assess the possibility that ion trans control Irradiated (1-5 hr) Paired lenses (20 hr culture) Fig. 3. Chloride 36 accumulation in control and experimental lenses irradiated for 1-5 hr. Data expressed as the ratio of activity in the lens to the medium, following 20-hr uptake period started after irradiation. > <-» 46 O > + controls A full dose subthresh Time (hr) Fig. 4. Lens voltage measured with 1 Meg. ohm glass microelectrodes after irradiation. Pluses denote control lenses. Triangles represent lenses which received full 1-hr dose, whereas circles denote lenses receiving ^-hr dose of UVB. port also might be impaired, active export of sodium was measured using Na 22. As evident in Figure 6, compared to control efflux (bottom line), the 3 hr efflux from UVB-exposed lenses (upper trace) was reduced by 45%. Because membrane transport apparently was affected by UVB irradiation, the question of glucose availability for operation of the cation pump was investigated. The rate of glucose accumulation was assessed by measuring the uptake of labelled 3-o-methylglucose. The results in Figure 7 show a plot of the ratio of concentration of cpm in lens to medium. The results show that compared to control lenses, uptake was 30% slower and plateaued at a ratio of approximately 1 in the UVB exposed lenses, suggesting equilibration between the lens interior and the bathing medium. Whether transport of glucose into the lens or leak out is involved, the results show that the ability of the lens to accumulate the glucose analog was significantly compromised in UVB-exposed lenses. To determine whether susceptibility to irradiation damage at low light levels was age dependent, older lenses were exposed to similar conditions. A number of lenses approximately 100 wk old were irradiated for 1-4 hr and cultured for various periods of time. Seven days of culture after 1 hr of irradiation were required to make an older lens appear opaque and gain as much sodium (68 mm, n = 5) as a 4-wk-old lens cultured for only 1 d. Old lenses were still clear after 1 d. Finally, experiments were done to determine whether tryptophan might be a target of UVB irradiation. Aqueous solutions of tryptophan (0.1 mm) were irradiated under the same conditions used for lens experiments. The colorless solution turned slightly

4 1786 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1992 Vol o Fig. 5. Calcium (triangles) and sodium (circles) concentrations in control lenses (lower curves) and irradiated lenses (upper curves) following 1-hr UVB irradiation Time (hrs) yellow within 1 hr. Because hydrogen peroxide can be formed in such instances, its presence was monitored and was found to reach levels averaging approximately 10 IJM within 1 hr. Discussion The results of this study demonstrate that, similar to thefindingsof Torriglia and Zigman on rat lenses, 6 cataracts can be produced in rabbit lenses in vitro following low-level UV irradiation. One important difference, however, is that cataracts produced in this study were caused by UVB in the wavelength region between 295 and 340 nm, considerably below the UVA spectrum used in the rat lens investigation (360 ± 20 nm). While lens physiology was not the focus in the rat lens study, it was demonstrated that Na/K-ATPase in lens homogenates was markedly impaired in a dose dependent manner. In the present study, the effect of UVB radiation on the Na/K transport system was measured directly in the intact lens by assessing active export of 22 Na from irradiated lenses. Approximately half of the active efflux of sodium was abolished. Considering the inhibition evident for rat lens ATPase, it is probable that cation pump inhibition observed in UVB cataract may also have been due to enzyme modification. An alternative explanation for diminished Na 22 efflux is that elevated intracellular calcium inhibits Na/ K-ATPase. Earlier studies in this laboratory have demonstrated the susceptibility of the rabbit lens Na/K pump to calcium elevation. 14 The likely cause of calcium accumulation may be a breakdown in the permeability characteristics of the lens membranes, particu- Irradlated ( 1 hr ) control irradiated ( 1 hr ) CO c «_1, c o»1o mm eg E « Time (min) Fig. 6. Efflux of 22 Na from control (lower line) and irradiated lenses (upper line) plotted as the fraction (%) of remaining counts per minute in the lens at time zero (based on 20-hr load-up of tracer). Time (hrs) Fig. 7. Tritiated 3-o-methylglucose accumulation in control (upper curve) and irradiated lenses (lower curve) following 1-hr dose, plotted as the ratio of concentration of counts per minute in the lens to the medium.

5 No. 5 VITRO UVD CATARACT / Highrower ond McCreody 1787 larly on the anterior surface directly exposed to UVB light. That there is an early, steady decline in the lens voltage within 1-2 hr of irradiation suggests that membranes already have been damaged before visible changes such as opacification or lens swelling occur. Torriglia and Zigman 6 also reported that there was a 4 hr lag period following rat lens irradiation before swelling or any degree of opacification was observed. Membrane damage also is indicated by the observed increase in the accumulation of chloride, an ion that is passively distributed according to concentration gradients and lens voltage rather than a pump. Interestingly, doses beyond 1 hr do little to further increase the leak of an already leaky lens. An increase in membrane permeability of nonspecific channels would permit increased influx of calcium, which in turn could bind to the sodium pump before it could be exported by the Ca pump. The inhibition of the cation pumps cannot be explained by the reduced availability of glucose. As shown in Figure 7, sodium and calcium levels were already elevated by 6 hr post irradiation. At that point, however, glucose accumulation still was normal. In fact, glucose levels, as assessed by 3-o-methylglucose, were not depressed until nearly 20 hr later, at a time when membrane leakiness was maximal. Thus, glucose would be expected to leak out of the lens, but at a time long after cation imbalances are observed. While slightly diminished levels of glucose probably do not cause impaired Na extrusion, glucose metabolism still might be a contributing factor. Tung et al reported that near UV irradiation inhibited hexokinase activity in rabbit and rat lenses. 15 While the mechanism of UVB cataract was not the focus of the present investigation, it is important to note that UVA cataract by comparison appears to involve tryptophan and its photo-oxidation product, kynurenine. 3 ' 4-5 Clearly, in UVA cataract studied by Zigman, 6 UV-produced products also can absorb the UV energy in the 360 nm region and also may involve subsequent free radical and peroxide production as described by Borkman et al 7 or suggested by Tung et al. 15 In the experiment in which tryptophan solutions (1 mm) were irradiated for 1 hr and the peroxide measured periodically, we detected peroxide levels of approximately 10 /xmol/1 within 1 hr. If this also were occurring in the intact lens, catalase or peroxidase probably could detoxify, unless these enzymes were damaged. Until these enzymes are investigated, the role of hydrogen peroxide in UVB cataract remains unknown. In conclusion, whatever the photochemical basis of UVB cataract, the initial effect appears to be on lens membranes because permeability and transport functions are impaired. Whether membrane physiology is compromised by the loss of tryptophan or the appearance of bound photoproducts, initial ionic imbalances and hydration suggest an osmotic-type cataract, with opacification correlated with elevated calcium at a later time. Further studies are underway to help characterize targets directly or indirectly affected by UVB radiation. Key words: calcium, cataract, lens, membrane, UVB References 1. Van Heyningen R: Photo-oxidation of lens proteins by sunlight in the presence offluorescentderivatives of kynurenine, isolated from the human lens. Exp Eye Res 17:137, Zigler JS, Sidbury JB, Yamanashi BS, and Wolbarsht M: Studies on brunescent cataracts. Ophthalmic Res 8:379, Borkman RF: Ultraviolet action spectrum for tryptophan destruction in aqueous solution. Photochem Photobiol 25:163, Zigman S and Vaughan T: Near-ultraviolet light effects on the lenses and retinas of mice. Invest Ophthalmol 13:462, Dillon J: Photolytic changes in lens proteins. Curr Eye Res 3:145, Torriglia A and Zigman S: The effect of near-uv light on Na- K-ATPase of the rat lens. Curr Eye Res 7:539, Borkman RF, Dalrymple A, and Lerman S: Ultraviolet action spectrum for fluorogen production in the ocular lens. Photochem Photobiol 25:129, Zuclich JA and Connolly JS: Ocular damage induced by nearultraviolet laser radiation. Invest Ophthalmol 15:762, MacKeen D, Fine S, and Fine BS: Production of cataracts in rabbits with the ultraviolet laser. Ophthalmic Res 5:317, Pitts DG: The ocular effects of ultraviolet radiation. Am J OptomPhysiol Opt 55:19, Jose JG and Pitts DG: Wavelength dependency of cataracts in albino mice following chronic exposure. Exp Eye Res 41:545, Hightower KH and McCready JP: Effects of selenium on ion homeostasis and transparency in cultured lenses. Invest Ophthalmol Vis Sci 30:171, Hightower KH, Harrison SE, Unakar NJ, and Tsui J: Effects of intracellular calcium on lens membrane permeability. Curr Eye Res 4:693, Hightower KH and Hind D: Cytotoxic effects of calcium on sodium-potassium transport in the mammalian lens. Curr Eye Res 2:239, Tung WH, Chylack LT, and Andley UP: Lens hexokinase deactivation by near-uv irradiation. Curr Eye Res 7:257, 1988.