Exp. Eye Res. (1992) 54, 621-626

Lens GSH Depletion and Electrolyte Changes Preceding Cataracts Induced by Buthionine Sulfoximine in Suckling Mice HAROLD

l.CALVIN”*,

STANLEY

VON HAGENb, JOHN L. HESS”, SUBHASH S.-C. JOSEPH FW

A. PATEL”

AND

Departments of aOphthalmology, Biochemistry and Molecular Biology, and bPharmacology and Toxicology, UMD-NJ Medical School, Newark, NJ 07703 and cDepartment of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A. (Received Bethesda 18 March 1991 and accepted in revised form 27 June 7991) Cataractswere inducedin sucklingmice by multiple injectionsof L-buthionine-S,R-sulfoximine (BSO),a specificinhibitor of GSHbiosynthesis,starting on post-natalday 7. The earliestvisible lensaberrations beganapproximately 2 days after t,, following 99% depletionof lensGSH.Cataract developmentthen proceededthrough four stageswithin lessthan 24 hr. ElevatedNa+and Ca+and decreasedK+were first detectedin pre-cataractous(stage0) lenses.During stage0, lensNa+and K+levelsdisplayeda significant inversecorrelation: by contrast, Ca2+levelswere poorly correlatedwith thoseof Na+.The initial increase in Na+exceededthe decreasein K’. This suggested the presenceof osmotic stress prior to cataract stage 1 (developingfloriform). Increasedlenshydration wasfirst apparentin stage1, coincidentwith a marked elevationof Ca2+,further increasein Na+and decreasein K+.Thesetrendspersistedin the stage2 cataract (completedfloriform). Subsequentchangesin lenshydration and cation content during cataract stages 3 (degeneratefloriform) and 4 (amorphoustranslucent)suggestedsubstantialinflux of extracellular fluid into the affectedlenses.The BSOcataract may representa usefulin vivo modelto study the functions of GSHin maintaining normal lens cation balanceand transparency. Key words: mouse; lens; sodium: calcium; potassium: buthionine sulfoximine: GSH depletion: hydration ; cataracts.

1. Introduction The tripeptide, reduced glutathione (GSH), is present in high concentrations in the mammalian lens, particularly in the epithelium, and is believed to be of importance in preventing harmful oxidations of lens proteins and lipids, thereby potentially reducing the severity or incidence of cataracts (Reddy, 1990 ; Giblin et al., 1990). In most cataracts where GSH levels have been compared with those in normal lenses,depletion of GSH either accompanies or precedes opacification (Rathbun. 1989). Production of lens cation abnormalities in vitro by agents which deplete GSH has been repeatedly demonstrated. The observed effects include decreaseduptake and/or increased efflux of s6Rband suggest compromise of mechanisms that maintain normal intracellular concentrations of Na+ and K+ (Epstein and Kinoshita, 19 70 ; Giblin, Chakrapani and Reddy, 1976; Cheng et al., 1984; Reddy et al., 1988). Upset of Na+/K+ balance in the GSH-depleted lens tends to be associated with increased Ca2+ levels (Duncan, Gandolfi and Maraini, 1988), perhaps partially as a result of impaired Na+/Ca+ exchange (Tomlinson et al., 1991). The effects of GSH-depleting agents upon electrolyte balance are exacerbated by oxidative stress (Reddy et al., 1988), without which * For correspondence at: Department of Biochemistry and Molecular Biology, UMD-NJ Medical School, 18 5 South Orange Ave. Newark, NJ 07103, U.S.A. 00144835/92/040621+06 41

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opacification generally does not occur (Srivastava, Ansari and Awasthi, 1984). They may bear some relationship to perturbations induced by blocking of membrane SH groups (Kern et al., 1984; Hightower, 1986 ; Walsh and Patterson, 1991) or treatment with H,O, (Delamere, Paterson and Cotton, 1983 ; Walsh and Patterson, 1991). In vivo production of dense cataracts by an agent which specifically affects GSH levels was lirst reported in suckling mice administered repeated injections of Lbuthionine-S,R-sulfoximine (BSO), a specific inhibitor of GSH biosynthesis (Griffith, 1982), shortly after 1 week of post-natal age (Calvin, Medvedovsky and Worgui, 1986). The initiation of opacification was correlated with nearly undetectable levels of GSH and did not occur in animals treated similarly at 2 weeks of age, despite 95% depletion of total glutathione (GSH+GSSG) (Calvin et al., 1986). More recently, it was demonstrated that the lensesof neonatal rats and mice are even more sensitive to BSO than those of week-old animals, and develop cataracts when lens glutathione is reduced to 0.2 nmol mg-’ lens (approximately 3 % of normal levels) (Martensson et al., 1989). The younger lenses developed cataracts when lens glutathione was reduced to 0.2 nmol mg-’ lens (approximately 3 % of normal levels), but remained clear when the level was maintained above 0.7 nmol mg-’ lens by concomitant administration of GSH ester (Martensson et al., 1989). Examination of the histological changes induced by 0 1992 Academic Press Limited EER54

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BSO in week-old mice revealed a very rapid degeneration of lens fibers within a 24-hr period, beginning approximately 48 hr after initiation of treatment (Calvin et al., 1991). Observations on whole lenses have enabled division of the initial 24-hr progression of the cataract into four stages: ( 1) developing floriform ; (2) completed floriform ; (3) degenerate floriform : and (4) amorphous translucent. Modifications of lens crystallin polypeptides are first detectable in late stage 1 cataracts and progress rapidly during succeeding stages (Calvin et al., 1992). The present communication presents evidence that elevation of lens Na+ and non-compensatory decrease in K’ occur before stage 1 of BSO cataract progression, suggesting analogy to other osmotic cortical cataracts (Kinoshita, 1974). Increased Ca2+ was also detected prior to stage 1. Thus, significant abnormalities in lens electrolyte composition appear to precede the reported fiber cell deterioration and modifications of lens crystallin polypeptides induced by BSO.

2. Materials

and Methods

Generation of Cataracts Adult male and female mice originally derived from a random-bred Swiss-Webster strain (Dominion Laboratories, Dublin, VA) were bred continuously in the Research Animal Facilities at UMDNJ to generate litters. L-buthionine-S.R-sulfoximine (BSO) was purchased from Schweizerhall (So. Plainfield, NJ) and was dissolved at a concentration of 0.2 M in 0.1 M NaCl. Cataracts were generated by repeated subcutaneous injections of BSO (4 pmol g-’ body weight). A previously described protocol of four injections daily on post-natal days 8 and 9 (Calvin et al., 199 1) was employed for survey of lens glutathione depletion and recovery during BSO-induced cataract development (protocol 1, Fig. 1). For detailed analysis of early cation changes induced by BSO (Figs 2 and 3), the injection protocol was modified slightly by administering three injections at 2.5-3 hr intervals after noon on day 7, followed by four injections on day 8 (protocol 2), in order to produce a high yield of stage 0 and stage 1 lenses on the third experimental day (i.e. post-natal day 9). Processing of Lenses After decapitation of the mice, their lenses were released from the enucleated eye globes by a posterior approach, suspended in 0.02 M Hepes-0.13 M NaCl (HBS), and dissected free of all other eye structures except minute quantities of zonule material. The lenses were observed at ambient temperature under a dissecting microscope for staging of cataract development (Calvin et al., 1992). Those lenses scheduled for assay of cations were rolled briefly on filter paper soaked in HBS-OS mM EGTA. Each lens was then

ET AL.

suspended in a platinum wire loop, the excess medium was carefully entrained with filter paper and the lens was transferred to a 600-~1 capped microfuge tube for weighing (two to four lenses per tube, pooled according to stage). Samples were stored at - 80°C until analysis. Analytical Procedures Lens total glutathione. Frozen lenses were homogenized directly within the microfuge tubes in 12 or 2 5 volumes of ice-cold 0.9 M HClO,-0.05 M H,PO, (homogenizing medium). The lower volume was employed for those samples expected to possess less than 5% of normal glutathione levels. The samples were neutralized with one-tenth volume of 6 M K,CO, and analysed for total glutathione (GSH+GSSG) by the glutathione reductase-catalysed cycling procedure of Tietze (1969). The total assay volume of 180 /sl contained 10 ~1 neutralized sample. Samples of glutathione content beyond the working range of the assay were diluted with 1: 10 6 M K&O,: homogenizing medium prior to analysis. Reagent blanks (equivalent to approximately 8 pmol of GSH) were minimized, as suggested by Brehe and Burch (19 76), by limiting the concentration of glutathione reductase in the assay reagent. Lens cations. The stored lenses were dried overnight at 110°C and pooled in groups of nine to 11 (stages O-2) or six to seven (stages 3 and 4) to obtain dry weight. At least four dried samples were analysed for each determination. They were transferred to borosilicate tubes (Pyrex 9820) for two cycles of digestion to dryness with concentrated HNO, (Ultrex grade, Baker Chemicals, Phillipsburg, NJ), followed by 30% H,O, (certified reagent grade, Fisher Scientific, Springfield, NJ) (Bunce, Hess and Batra, 1984). The tubes in which the digestions were performed were pretreated with concentrated HNO,. Simultaneous analyses for Ca, K and Na were carried out by inductively coupled plasma spectrometry in the Soil Testing and Plant Analysis Laboratory at Virginia Polytechnic Institute and State University. Statistical Analyses Data has been expressed as means+ S.E. Medians and their interquartile ranges (IQR) have, in some instances, been furnished for skewed sets of values. Significance of differences between means was established by Student’s unpaired t-test when the data was normally distributed. Otherwise, statistical difference between groups was established non-parametrically by the Mann-Whitney U-test. Evaluation and Photography of Lenses Prior to storage of lenses for chemical analyses, their morphological status was carefully evaluated at ambient temperature (24+ 1°C) with a dissecting

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1. Depletion and recovery of lens total glutathione induced by BSO administration according to protocol 1 (see Materials and Methods). Determinations of glutathione in untreated control mice at selected ages are also shown (post-natal day 8 = experimental day 0). Data points shown as means+S.E. (X) Controls: (0) precataractous: (+) cataractous. FIG.

microscope (Model AO-570, AO-Reichert, Buffalo, NY), under transillumination with diffusely reflected light. During the initial 24-hr period of cataract development, the lenses were scored as stage 0 (no observable abnormalities) or stages 14, using criteria described elsewhere (Calvin et al., 1992). Stages O-3 were obtained at 44-54 hr after t,, stage 4 at approximately 72 hr after t,.

3. Results Glutathione Levels The pattern of total glutathione (GSH+GSSG) changes during cataract development induced by BSO (injection protocol 1) is summarized in Fig. 1. Also depicted are the lens glutathione levels in untreated mice at various post-natal ages. Following a steep decline to essentially undetectable levels during the first 3 days of cataract induction, glutathione concentrations remained very low until 5 days after t,, and then increased sharply. By 7 days after t,, mean glutathione was > 10% of normal levels and continued to rise gradually thereafter. However, the level remained lower than controls at 35 days after t,. The data summarized in Fig. 1 include a low mean level of lens glutathione in precataractous (stage 0) lenses at 2 days after t,:0.14 f0.03 ,umol g-l wet weight. The median value of glutathione determined for this group of 31 pairs of stage 0 lenses was even lower: 0.03 pmol g-l (IQR 0.11) and suggested that the typical precataractous lens at this point retained less than 1% of the normal glutathione concentration of 6.0 pmol g-l. Determinations of lens glutathione at 2 days after t,, in stage 0 samples (16) obtained after the alternative injection procedure (protocol 2). confirmed nearly complete glutathione depletion shortly before visible lens changes : median concentration, 0.04 ,umol g-l (IQR 0.08). The level of lens glutathione was still lower in stage 1 samples (8) : median value of

0.02 pmol g-’ (IQR 0.02). Glutathione then became undetectable (< 0.01 pmol g-l) as the cataracts progressed further (stages 24). Electrolyte Changes The alterations in lens cation composition and hydration induced as result of BSO treatment (protocol 2) are summarized in Figs 2 and 3. As lens pathology progressed from the normal untreated state used as the control (C), the following general trends could be seen for the concentrations of Na+, K+, and Ca2+, expressed in pmol g-’ lens wet weight [Fig. 2(A)]: (1) steadily increasing Na+ during stages O-2, little further change thereafter; (2) steadily decreasing K+ during stages O-3, then no further change ; and (3) increasing Ca2+ during stages O-2, followed by a constant level during stages 24. From this, it appears that the cationic composition of lens water had attained approximate stability by stage 3 of the cataract. However, additional increases in Na+ and Ca2+ concentration were indicated during stages 3 and 4, when calculated on the basis of pmol g-l dry weight [Fig. 2(B)] or kmol per lens (data not shown). These changes coincided with an increase in lens wet weight from 2.0f0.08 mg per lens in stage 2 to 2.8 20.03 mg per lens in stage 4 [Fig. 2(A)] and increased lens wet weight/dry weight from 4.3 + 0.07 in stage 2 to 6.1 L-O.16 in stage 4 [Fig. 2(B)]. These coincidental increases in Na+, Ca2+ and lens water during stages 3 and 4 suggest substantial influx of extracellular fluid into the lens. The wet weight/dry weight ratio of stage 0 samples (3.8 f 0.0 7) was indistinguishable from that of controls (3.7kO.05). However, by stage 1, the wet weight/dry weight ratio (4.0 f 003) was significantly elevated [Fig. 2(B)]. Thus, increased lens hydration began shortly after modifications in lens cation content detected in stage 0 lenses and coincided approximately with the initiation of visible changes in lens refractility (i.e. stage 1). 41-2

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Stage FIG. 2. Mean stage-dependent lens cation concentrations, expressed in ,umol g-’ wet weight (A) or dry weight (B), following BSO administrations to mice according to protocol 2. Stage C represents untreated age-matched controls. Also shown are wet weight per lens pair in (A) and wet weight/dry weight ratio in (B). The ratio of wet weight to dry weight (R) is related to lens hydration (H) by the equation: H = (R-l/R) x 100. Error bars indicate S.E. Number of determinations for each mean is indicated in parentheses. (m) Na; (a) K; (a) Cax 10: (m) wet weight per lens pair x 10; (0) wet weight/dry weight ratio x 100.

Those lenseswhich appeared to be normal (stage 0) were of special interest during these studies. A scatter plot of the individual values of Na+ vs. those of K+, calculated on the basis of sample dry weight, is presented in Fig. 3(A). Each data point is categorized according to lens stage. It is quite evident that there was an inverse relationship between the concentrations of these two ions in stage 0 lenses and that this relationship persisted as the cataract progressed through stages 1 and 2. However, at the level of approximately 500 pmol g-l Na+, K’. concentration reached a minimum of about 40 pmol g-’ dry weight and remained there, as Nat continued to increase in stage 3 and stage 4 lenses. Figure 3(A) contains an interesting stage 0 data point (circled) with slightly high Na+ and a level of K’ that was typical of control values. This behaviour was in fact consistent with the indication that Na+ elevation at first exceedscompensatory lossof K’. This is revealed in an additional plot [Fig. 3(A), inset] demonstrating that all values of Na++K+ in stage 0 sampleswere > the maximum concentration seen in controls. A second scatter plot compares Na+ and Ca2+ (pmol g-’ dry weight) in individual samples.Although

all of the Na+ concentrations in stage 0 sampleswere higher than any of control values, the behaviour of lens Ca2+in this group was considerably more variable, ranging from a minimum level (0.83) approximately equal to the lowest of the controls (0.77) to a maximum value (6.6) which was five times as high as the mean for controls (1.26 f0.19). The levels of Ca2+ were not well-correlated with those of Na+ during stage 0. The group of four samples of lowest Ca2+ content (circled) displayed an especially poor correlation between Na+ and Ca”+ concentration. The remaining stage 0 data point with high Ca”+ and relatively high Na+ represents a sample for which wet weight ratio was slightly elevated (4.0). suggesting early fiber swelling. It is likely that this sample included lenseswith refractile aberrations incorrectly scored as stage 0. The concentration of Ca”+ became consistently and substantially elevated in all stage 1 samples,attaining a mean dry weight concentration which was nearly ten-fold that found in controls. Remarkable discontinuities were seenbetween Ca2+levels in stage 0, stage 1 and stage 2 samples,respectively. By contrast, the Na+ measurementsin successivestagesoverlapped considerably.

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FIG. 3. Relationship between cation concentrations of individual samples, expressed in pmol g-l dry weight. Stage of pooled dried lenses used for each determination is indicated by symbols. A, Na+ vs. K+ in controls and stages 04, showing inverse relationship during early cataract development. Inset, (Na’ + K’) vs. K’ in stages C, 0 and 1. All values of (Na’ +K+) in stage 0 samples are > those in controls. The single sample with normal K’ in both plots is indicated by a circled data point and displays a value of (Na’ + K’) well above that seen in controls. B, Na+ vs. Ca 2+.A subset of data from lenses scored as stage 0 is encircled. This group displayed poor correlation between Na+ and Ca2+ concentrations. The remaining stage 0 sample displayed a profile of cation concentration and hydration more typical of stage 1 (see text).

4. Discussion Histological studies have confirmed that fiber swelling is one of the very early events in the development of the mouse BSO cataract. Such changes begin along the suture boundaries in the anterior cortex and spread very rapidly to the rest of the lens (Calvin et al., 1991). These observations have suggested that the cataract begins as an osmotic cortical cataract, in which dramatic changes in lens hydration occur within the space of hours. According to the mechanism proposed for the hereditary Nakano mouse cataract (Kinoshita. 19 74), such increased hydration

would be caused by influx of Na’ which was not compensated by loss of K+. The data in Fig. 3 (A) (inset) indicate that the BSO cataract obeyed this pattern during progression from stage 0 to stage 1. The sharp rise of lens Ca2+ manifested in stages 1 and 2 may then have played a key role in subsequent cataract progression. Indeed, an accelerated increase in hydration during stages 3 and 4 followed these substantial increases in Ca2+ content (Fig. 2). It is wellestablished that elevation of lens calcium is closely correlated with opacification (Hightower, 1986; Marcantonio, Duncan and Rink, 1986 ; Shearer, David and Anderson, 1987). An important factor in this

H. I. CALVIN

process is Ca2+-activated proteolysis, catalysed by the enzyme, calpain II (Shearer et al., 1987). The timing of crystallin polypeptide modifications in the BSO cataract (Calvin et al., 1992), is consistent with the interpretation that they follow prior increases in lens Ca2+ during stage 1 and are largely dependent upon activation of calpain. The high levels of Ca2+ observed in stage 1 may largely reflect rapid increases in lens extracellular space, as fiber cell membranes deteriorated and lysed. The events that preceded this in late precataractous (stage 0) lensesare more relevant to the initiation of the cataract and deserve special attention. As shown in Fig. 3(B), most of the stage 0 samples possessed considerably lower levels of Ca”’ than those scored as stage 1 and elevated Na+ was not consistently associated with elevated Ca2+.This suggeststhat the sequence of changes in cation content of precataractous lenses induced by BSO may be further dissectable in future experiments. In vitro experiments have provided considerable data concerning the effects of GSH depletion, loss of membrane SH groups and oxidative stress upon lens cation active transport and diffusion. These three categories of insult are, of course, not mutually exclusive, and may result in altered lenscation balance by common mechanisms. The mouse BSO cataract may be a valuable in vivo system to investigate the relationships between lens GSH depletion, oxidative changes in proteins and lipids, and membrane dysfunction leading to electrolyte disturbances and cataracts.

Acknowledgements This work was supportedin part by NIH researchgrant EY-07355 (H.C.), NIH biomedicalresearchsupport grant RR-05393 to UMD-NJMedical Schooland by a grant from Researchto Prevent Blindness,Inc. to the Department of Ophthalmology.UMD-NJMedical School.The authors wish to thank Benjamin Knopp for technical assistanceand Nancy Phillips for performing inductively coupledplasma spectrometry.

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Calvin, H. I., Patel. S.A., Zhang, J. P.. Li. M. Y. and Fu. S.C. J. (1992). Progressivemodificationsof mouselens crystallins in cataracts induced by buthionine sulfoximine. Exp. Eye Res. 54, 611-9. Cheng. H.-M., Saltza, I., Gonzalez, R. G., Ansari, N. H. and Srivastava, S. K. (1984). Effect of glutathione deprivation on lens metabolism. Exp. Eye Res. 39. 355-64. Delamere. N. A., Paterson, C. A. and Cotton, T. R. (1983). Lens cation transport and permeability changes following exposure to hydrogen peroxide. Exp. Eye RPS. 37,45-53.

Duncan, G.. Gandolfi, S. A. and Maraini. G. ( 1988). Diamide alters membrane Na+ and K+ conductances and increased internal resistance in the isolated rat lens. Exp. Eye Res. 47, 807-18. Epstein. D. L. and Kinoshita, J. H. (lV70). The effect of diamide on lens glutathione and lens membrane function. Invest. Ophthalmol. Vis. Sci. 9. 629-38. Giblin, F. J.. Chakrapani, B. and Reddy, V. N. ( 1976). Glutathione and lens epithelial function. Invest. Ophthab moi. Iris. Sci. 15, 381-93. Giblin. F. J., Reddan. J. R.. Schrimscher. L.. Dziedzic, D. C. and Reddy. V. (1990). The relative roles of the glutathione redox cycle and catalase in the detoxification of H,O, by cultured rabbit lens epithelial cells. Exp. Eye Res. 50, 795-804. Griffith, 0. W. (1982). Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis. 1. Biol. Chem. 257, 13704-12. Hightower, K. R. (1986). Superficial membrane -SH groups inaccessible by intracellular GSH. Cum. lhge Res. 5. 421-7.

Kern, H. L.. Ingalls. L. K., Weiner. B. C. and Zolot, S. (1984). Patterns in effects of sullhydryl reagents on transport in bovine lens. Cur. Eye Res. 3. 1373-82. Kinoshita. J. H. (19 74). Mechanisms initiating cataract formation. Invest. Ophthalmol. 13, 713-24. Marcantonio, J. M.. Duncan, G. and Rink, H. (1986). Calcium-induced opacification and loss of protein in the organ-cultured bovine lens. Exp. Eye Res. 42, 617-30. Martensson. J., Steinherz, R., Jain. A. and Meister. A. (1989). Glutathione ester prevents buthionine sulfoximineinduced cataracts and lens epithelial cell damage. Proc. Natl. Acad. Sci. U.S.A. 86, 8727-31. Rathbun, W. B. (1989). Glutathione in ocular tissues. In Glutathione (Eds Dolphin, D., Poulson, R. and Avramovie. 0.) Pp. 467-510. John Wiley: New York. Reddy, V. (1990). Glutathione and its function in the lensan overview. Exp. Eye Res. 50. 771-8. Reddy, V. N.. Garadi, R.. Chakrapani, B. and Giblin. F. J.

(1988). Effect of glutathione depletion on cation transportand metabolismin the rabbit lens.Ophthalmic References Brehe. J. E. and Burch. H. B. (1976). Enzymatic assayfor glutathione. Awl. Biochem. 74, 189-V 7. Bunce. G. E., Hess,J. L. and Batra, R. (1984). Lenscalcium andselenite-induced cataract. Curr. Eye Res. 3, 3 15-20. Calvin, H. I., Medvedovsky,C., David,J. C., Broglio, T., Hess, J. L., Fu, S.C. J. and Worgul, B. V. (1991). Rapid deteriorationof lensfibersin GSH-depleted mousepups. Invest. Ophthalmol. Vis. Sci. 32, 1916-24. Calvin, H. I., Medvedovsky, C. and Worgul. B. V. (1986). Near-total glutathione depletion and age-specific cataractsinducedby buthionine sulfoximinein mice.Science 233,

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Res. 20, 191-9. Shearer, T. R., David, L. L. and Anderson, R. S. ( 1987). Selenite cataract: a review. Curr. Eye Res. 6, 289-300. Srivastava. S. K.. Ansari. N. H. and Awasthi. Y. C. (1984). Lens glutathione depletion of 1-chloro-2,4-dinitrobenzene and oxidative stress. Curr. Eye Res. 3. 117-V. Tietze, F. (1969). Fazymic method for quantitative determination and total and oxidized glutathione : applications to blood and other tissues. Anal. Biochem. 27. 502-22.

Tomlinson. J., Bannister, S. C.. Croghan. P. C. and Duncan, G. (1991). Analysis of rat lens 4sCa2* fluxes: evidence for Na+-Ca2+ exchange. Exp. Eye Res. 52, 619-27. Walsh, S. and Patterson. J. W. (1991). Effects of oxidants on lens transport. Invest. Ophthalmol. Vis. Sci. 32, 1648-58.