Cataract Development in γ-Glutamyl Transpeptidase-deficient Mice

Cataract Development in γ-Glutamyl Transpeptidase-deficient Mice

Exp. Eye Res. (2000) 71, 575±582 doi:10.1006/exer.2000.0913, available online at http://www.idealibrary.com on Cataract Development in g-Glutamyl Tra...

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Exp. Eye Res. (2000) 71, 575±582 doi:10.1006/exer.2000.0913, available online at http://www.idealibrary.com on

Cataract Development in g-Glutamyl Transpeptidase-de®cient Mice PAT R I C I A C H EÂ V E Z - B A R R I O S ab, AM Y L . W I S E M A N a, E M IL IO RO J AS c, CH I N G - N A N O U a A N D M I C H A EL W. LI E B E R M A N ad* a

Department of Pathology, Baylor College of Medicine, Houston, TX 77030, U.S.A., bDepartment of Ophthalmology, Baylor College of Medicine, Houston, TX 77030, U.S.A., cInstituto de Investigaciones Biomedicas, UNAM, Coyoacan, Mexico and dDepartment of Cell Biology, Baylor College of Medicine, Houston, TX 77030, U.S.A. (Received Rochester 27 March 2000, accepted in revised form 16 August 2000 and published electronically 9 October 2000) The present study was undertaken to analyse the relationship of lens glutathione (GSH) and light to cataract development in mice de®cient in g-glutamyl transpeptidase (GGT). These mice have reduced levels of cysteine and GSH in the eye and develop cataracts. GGT-de®cient mice raised under normal vivarium conditions, showed no cataractous changes at birth, but by 1 week they had developed nuclear opacities. By 3 weeks more severe cataracts develop, and lens GSH levels are approximately 6±7 % of wild type levels. By 6±11 weeks cataracts show nuclear and cortical involvement, liquefaction and calci®cation. Single cell DNA electrophoresis (comet assay) demonstrated mild DNA damage in the lens epithelium. GGT-de®cient mice raised in the dark beginning the day after conception all developed cataracts, but these were less severe than those in GGT-de®cient mice raised with normal vivarium lighting. Administration of N-acetyl cysteine (NAC) raises lens GSH and almost completely prevents cataract development. Our data indicate that cataract development in GGT-de®cient mice is multifactorial and results from exogenous damage (exposure to light), reduced lens GSH levels, and # 2000 Academic Press nutritional effects secondary to low cysteine levels. Key words: g-glutamyl transpeptidase; cataract models; cataractogenesis; N-acetylcysteine; DNAdamage; glutathione; light damage; cataract; transgenic mice.

1. Introduction Throughout the world, cataracts are the largest single cause of blindness and are responsible for visual impairment in 30±40 million people (Steinberg et al., 1993; Thylefors et al., 1995; Thylefors, 1998). With these lesions becoming more common with increasing age, they are often referred to as `senile cataracts' and are thought to result from progressive accumulation of damage to the lens leading to opaci®cation of the lenticular nucleus and cortex. Many studies have linked oxidative damage resulting from exposure to light as a cause of cataract formation; such damage may occur via direct photochemical reaction or secondary via photosensitization (Andley, 1994). However, the reason why some individuals develop cataracts while others do not remains largely unknown. All lens cells formed throughout life are retained as anuclear lens ®bers. These ®bers possess refractivity because of the way in which the cells and their proteins are arrayed and the low water content. Any damage to these ®bers, the proteins that comprise them (crystallins) or the anterior layer of metabolically active, dividing epithelial cells will result in * Address correspondence to: Michael W. Lieberman, Department of Pathology, Baylor College of Medicine, Houston, TX 77030, U.S.A. E-mail: [email protected]

0014-4835/00/120575‡08 $35.00/0

opaci®cation and cataract formation. Mechanisms related to senile cataract formation have been grouped as either `oxidative' or `degenerative' or a combination of the two. Although the mechanism by which cataracts form is not well understood, many investigators have suggested that GSH is a major factor in protecting the lens against such damage (Spector, 1995). There is also a large literature demonstrating the importance of growth factors in crystallin synthesis and lens formation and maintenance, but the role of such factors has not been extensively evaluated in cataract formation (Alemany et al., 1989; Klok et al., 1998). In addition, nutrition is believed to play a role in cataract development (Bunce, Hess and Davis, 1984; Waddell, 1998). It has been dif®cult to evaluate the role of GSH in the prevention of cataracts in experimental animal models because, until recently, the only way to lower GSH levels was to administer reagents which themselves might have toxic effects or interact with the other experimental variables (Reddy et al., 1988). Further, long-term studies have been dif®cult because of the need for continued administration of reagents and uncertainty about dose level as a function of time after administration (Calvin et al., 1992). Using a targeted deletion strategy, we developed mice de®cient in g-glutamyl-transpeptidase (GGT), the enzyme that # 2000 Academic Press

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initiates the catalysis of degradation of GSH by cleaving the g-glutamyl-cysteine bond (Lieberman et al., 1996). An interesting ®nding in these experiments was that these mice developed cataracts in the ®rst few weeks of life. In the eye, as in many other organs of these mice, GSH levels were paradoxically low. We determined that these reductions in GSH were secondary to a cysteine de®ciency caused by the excretion of large amounts of cysteine (as GSH) in the urine. Administration of N-acetylcysteine (NAC) corrected this de®ciency and many of the sequelae of cysteine de®ciency. Thus GGT-de®ciency provides a unique model in which to undertake a more comprehensive study of cataract formation. The present communication presents our analysis of the development of these lesions and the role of NAC in preventing them.

2. Materials and Methods Generation of GGT-de®cient Mice GGT-de®cient mice were generated on C57BL/ 6X129SvEv hybrid background by homologous recombination as detailed in a previous report. Mice were maintained as heterozygotes and bred as needed to produce wild type mice, heterozygous and homozygous mice. Homozygous (GGT-de®cient) mice were identi®ed by Southern blotting (Lieberman et al., 1996). Mice received a standard lab chow diet (Purina Rodent Diet 5001) and water ad libitum. One set of mice were exposed to vivarium lighting on a standard 12 hr light/dark cycle while a second set was maintained in complete darkness from the day after conception until the time of observation. Cages were changed once a week under a red darkroom light. Various groups of mice were supplemented with NAC (Sigma Ultragrade, Sigma) starting on day 3 of life (0.5 mg g ÿ1 body weight injected subcutaneously twice a day) and/or on day 21 [at weaning; 10 mg ml ÿ1 NAC dissolved in the drinking water (Lieberman et al., 1996)].

Morphological Analysis Necropsies were performed on newborn, 1, 2, 3, 6, 11 and 14 week old mice, and the eyes were harvested by immediate enucleation. Intact lenses were extracted under a dissecting microscope using a variation of intracapsular extraction through a large corneal incision and through the pupil after removal of the zonules and iris. Eyes for morphologic examination were ®xed in 10 % formalin. Conventional processing of formalin-®xed tissues was performed for paraf®n embedding, and 5 m sections were stained with hematoxylin and eosin (H and E) or periodic acid Schiff (PAS).

Biochemical Analyses GSH was measured in whole-homogenized lenses by the method of Tietze by using a COBAS-BIO centrifugal analyser (Tietze, 1969; Lieberman et al., 1996). Brie¯y, the freshly extracted lenses were immediately homogenized in a PCA/BPDS solution of 5 % perchloric acid (EM Science, Cherry Hill, New Jersey, U.S.A.) containing 1 mM BPDS (bathophenanthroline disulfonic acid) (Sigma Co., St. Louis, MO, U.S.A.) and frozen overnight. To prevent GGT from breaking GSH down, 5 mM AT-125 (Sigma Co., St. Louis, MO, U.S.A.) was added to the PCA/BPDS solution. Then they were centrifuged to pellet down precipitated proteins and the supernatant was assayed for GSH analysis. DNA damage evaluation was assessed by the single cell gel electrophoresis (SCGE, `Comet' assay). The alkaline SCGE assay was performed as described with minor modi®cations (Tice, Strauss and Peters, 1992; Rojas et al., 2000). The lenticular epithelial cells were removed from the anterior and equatorial capsule and suspended in ice cold normal saline (Fig. 1). A small volume (20 ml) of cells was mixed with 75 ml of 0.5 % of low melting agarose maintained at 378C, and 75 ml from this mixture was pipetted onto a slide with 180 ml of standard agarose and immediately covered with a coverslip to make a microgel on the slide. Slides were placed on an ice-cold steel tray on ice for 1 min to allow the agarose to gel. The coverslip was removed, and the slide was overlayered with 75 ml of agarose as before. Slides were immersed in an icecold lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris±Base, pH 10). After lysis at 48C for 1 hr, slides were placed on a horizontal electrophoresis unit. The DNA was allowed to unwind for 20 min in electrophoresis running buffer solution (300 mM NaOH and 1 mM Na2EDTA, pH 13). Electrophoresis was conducted for 20 min at 25 V and 300 mA. All technical steps were conducted using very dim indirect light. After electrophoresis, the slides were gently removed, and the alkaline pH was neutralized with 0.4 M Tris, pH 7.5. They were then dehydrated in two steps with absolute ethanol for 10 min each. Ethidium bromide (75 ml of a 20 mg ml ÿ1 solution) was added to each slide and a coverslip was placed on the gel. DNA migration was analysed with a Nikon microscope ®tted with ¯uorescence equipment, (excitation ®lter 515±560 nm and a barrier ®lter of 590 nm). The extent of migration was measured with a scaled ocular as the tail length of the comet. One hundred cells per mouse were scored. 3. Results We have previously described phenotypic and biological ®ndings in gGGT (Lieberman et al., 1996). Brie¯y, GGT de®cient mice were half of the size of wild-types at 6 weeks. The mice begin to die at about

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F IG . 1. Preparation of lens epithelial cells for `Comet' electrophoresis. (A) Opening of the posterior capsule and extraction of nuclear and cortical material. (B) Under a drop of saline-balanced salt solution, the epithelial cells are removed from the capsular attachments with gentle mechanical force. (C) Cells are retrieved with a pipette and placed in cold saline solution (D.1) and then placed on a glass slide coated with agarose (D.2).

F IG . 2. Analysis of progression of lens damage in GGT-de®cient mice. (A) One day old GGT-de®cient mouse with no cataractous changes of the lens (L, lens). (B) One week old GGT-de®cient mouse with mild nuclear opacity (N, nucleus). (C) Three week old GGT-de®cient mouse with nuclear densities and cortical vacuoles (N, nucleus; C, cortex). Hematoxylin and Eosin, original magni®cation 10.

12 weeks. Administration of NAC in the drinking water beginning on day 21 resulted in normal growth and effectively prevented early death. Cataract Development in GGT-de®cient Mice At birth no cataractous changes were observed in GGT-de®cient mice (0/10) [Fig. 2(A)]. In mutant mice

raised under normal vivarium lighting we observed nuclear opacities by week 1; these were identi®ed by external observation (data not shown) and microscopic observation in 4/4 mice [Fig. 2(B) and Table I]. By week 2±3 12/12 GGT-de®cient mice showed more severe cataractous changes [Fig. 2(C) and Table I]. All had nuclear opacities, 10/12 had cortical vacuolization and 3/12 had anterior subcapsular plaques.

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F IG . 3. Modulation of cataract development in GGT-de®cient mice treated with NAC. (A) Lens from a 6 week old GGTde®cient mouse after 3 weeks of NAC in drinking water shows mild nuclear density and mild cortical changes (N, nucleus; C, cortex). (B) In contrast, lens from an untreated GGT-de®cient mouse at 6 weeks showing vacuolated nuclear degeneration (*), cortical changes (C) and dense subcapsular anterior plaque (arrow). (C) Lens from a 6 week old GGT-de®cient mouse after NAC treatment beginning on day 3 (see Materials and Methods); note the mild nuclear opacity (N) and the absence of cortical and epithelial changes. (D) Lens from a 6 week old wild (control) type mouse showing no cataractous changes. Hematoxylin and Eosin, original magni®cations 10.

Beginning at week 6 and progressing to weeks 11±14, 14/14 mutant mice had cataracts involving the nucleus and the cortex with most of the lenses showing liquefaction of nuclear material and calci®cations [Fig. 3(B) and Table II]. Eight of 14 mice had anterior subcapsular plaques. No cataractous changes were observed in wild type mice.

TABLE I Comparison of cataract development in GGT-de®cient mice raised in the dark versus those raised in normal vivarium lighting GGT-de®cient mice raised in the dark

Cataract Development in GGT-de®cient Mice Raised in Darkness Eight litters were raised in darkness from the day after conception. Four GGT-de®cient mice from this cohort were killed at 1 week; three showed only minor nuclear cataracts. Of seven `dark-raised' mutant mice killed at 3 weeks, six showed some degree of cataractous change including total cataract in 3/6 mice (Fig. 4 and Table I). However, these changes were less severe than those seen in GGT-de®cient mice raised under standard vivarium conditions (Fig. 4 and

Lenticular changes No change Mild cortical Marked cortical Nuclear Anterior subcapsular plaque Calci®cation Liquefaction

GGT-de®cient mice raised in normal lighting

1 week 3 week 1 week 3 week old old old old 3/4 1/4

4/7 2/7 4/7 2/7

4/4 4/4

10/12 12/12 3/12

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F IG . 4. Analysis of cataract development in GGT-de®cient mice raised in the dark. (A) Mild cataractous changes in the cortex of a lens from a 3 week old GGT-de®cient mouse raised in the dark. (B) More severe changes in the lens of a GGT-de®cient mice raised under vivarium light. Hematoxylin and Eosin, original magni®cation 10.

TABLE II Cataract development in 11±14 week old GGT-de®cient mice treated with N-acetyl cysteine (NAC) or maintained on a standard diet Lenticular changes No changes Mild cortical Marked cortical Nuclear Anterior subcapsular plaque Calci®cation Liquefaction

Standard diet

NAC in drinking water, post weaning

14/14 14/14 7/14 10/14 11/14

4/5 3/5

NAC injected from day 3, followed by NAC in drinking water, post weaning 7/12 1/12 4/12

NAC was adminstered to a GGT-de®cient mice as described in Materials and Methods.

Table I). These ®ndings demonstrate that GGTde®ciency results in cataract formation even in the complete absence of light and that light exacerbates these changes. Wild type mice (n ˆ 4) and heterozygous mice (n ˆ 21) had no cataractous changes. The Role of N-acetyl Cysteine in Cataract Prevention We examined the role of NAC in cataract formation using two different protocols. In both protocols, control lenses from wild type mice show no morphological changes compared with control lenses untreated. In the ®rst we fed NAC (10 mg ml ÿ1 in the drinking water) to GGT-de®cient mice beginning at age 3 weeks (post weaning). Compared to untreated GGT-de®cient mice, the fed mice showed less consistent cataract development, and cortical changes were less than those in the untreated mutant mice (Fig. 3 and Table II). By week 11±14 most of the mutant mice fed NAC (3/5) developed nuclear cataracts. In a second experiment we began NAC treatment of GGT-de®cient mice on day 3 with subcutaneous injections twice a day (see Materials

and Methods) and at day 21 switched to supplementation in drinking water. This regimen was more effective than drinking water supplementation alone. Seven of 12 mice were completely protected from cataract development (Table II). Four of 12 GGTde®cient mice had only minor nuclear opacity, and one showed a nuclear cataract and rare small vacuoles of the cortex. None had calci®cation or anterior subepithelial plaques (Table II). These data demonstrate that NAC provides substantial protection for GGT-de®cient mice from cataract development. GSH Levels To assess the role of GSH in cataract formation we measured levels of this tripeptide in the lenses of untreated wild type mice, GGT-de®cient mice, and GGT-de®cient mice fed NAC for 3 weeks beginning on day 21. In 6 week old GGT-de®cient mice, lens GSH levels were approximately 5±6 % of wild type values (Table III). NAC treatment of GGT-de®cient mice resulted in an `over shooting' of GSH values so that

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TABLE III Comparison of lens GSH levels in wild type and GGT-de®cient mice

Wild type (control) Heterozygous mice GGT-de®cient GGT-de®cient, NAC fed

GSH levels (mmol g ÿ1)

Percentage of control ( %)

5.82 + 0.39 6.01 + 0.30 0.32 + 0.05 6.10 + 0.6

100.0 103.2 5.5 104.8

Beginning on day 21 mice were fed a normal lab chow diet. NACtreated mice received NAC in the drinking water (10 mg ml ÿ1). Mice were killed at 6 weeks of age and GSH was determined in individual lenses (n ˆ 4 from two mice for the GGT-de®cient untreated and NAC fed; n ˆ 8 from four mice for control animals and n ˆ 3 for each group from three mice for heterozygous animals) as decribed in Materials and Methods.

levels in lens from treated GGT-de®cient mice were higher than wild type values (6.1 mmol g ÿ1 vs 5.82 mmol g ÿ1; Table III). Thus restoration of lens GSH values by NAC is correlated with its protection against cataract development. DNA Damage in Epithelial Cells of GGT-de®cient Mice Light and oxidative injury are known to cause DNA damage, therefore it was reasoned that this process might be a precursor of cataract formation. To assess this hypothesis we analysed accumulated DNA damage in individual lens epithelial cells as a function of age in wild type mice and GGT-de®cient mice. The approach consisted of harvesting epithelial cells from the lenses of individual mice and using alkaline treatment of cells to separate DNA strands with strand breaks and to lyse alkali-labile sites followed by electrophoresis (see Materials and Methods). Following electrophoresis, cells with undamaged DNA have circular images while those with damage have a tail (`Comet' structures) from the rapid migration of damaged (smaller) DNA. The migration distance is a measure of extent of DNA damage. We scored 100 cells for all data points and found no differences between new born wild type and GGTde®cient mice by this assay (Fig. 5). By 6 weeks there was a slight, but detectable increase in the migration distance (tail length) in cells from GGT-de®cient mice. This increase is indicative of only mild DNA damage and is unlikely to be a major contributing cause of cataract formation in these mice. 4. Discussion Our results demonstrate that cataract formation in GGT-de®cient mice is progressive. At birth these mice have normal lenses, but by 3 months all have severe cataracts including calci®cation and liquefaction

F IG . 5. DNA damage in lens cells from normal and GGTde®cient mice. Electrophoretic migration distance of DNA from individual lens cells is plotted for (A) normal mice and (B) GGT-de®cient mice. One hundered cells were analysed for each group. (A) New born (nb), 4 week old (WT4) and 6 week old mice (WT6) mice. (B) GGT-de®cient mice; new born (NbGGT), 4 week (GGT4), and 6 week (GGT6).

(Tables I and II). Progression of cataract development can be arrested and modi®ed in GGT-de®cient mice by administration of NAC beginning on day 21, and cataract formation can be largely prevented by administration of this agent beginning shortly after birth (Table II). Raising GGT-de®cient mice in complete darkness also ameliorates cataract formation (Table I). This ®nding indicates that light is a source of lenticular damage in this model; however, even in complete darkness, GGT-de®cient mice develop cataracts. The fact that there are only mild changes in the DNA damage pro®le of GGT-de®cient mice (raised under normal vivarium lighting) indicates that DNA damage is not a major contributor to lens epithelial damage and cataract formation in this model (Fig. 5). This conclusion is supported by the observation that

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cataractous changes appear well before changes in the DNA damage pro®le (Fig. 5). Reduced levels of ocular GSH and to a lesser extent altered cysteine metabolism are the major contributors to cataract formation in GGT-de®cient mice. GGTde®ciency results in a paradoxical drop in tissue GSH levels resulting from loss of cysteine (as GSH) in the urine and failure to synthesize GSH in the absence of this amino acid (Lieberman et al., 1996). In most organs of GGT-de®cient mice, including the entire eye, GSH levels range from 25 to 80 % of normal (Lieberman et al., 1996). The fall of lens GSH in these mice to 5±6 % of normal values is the most dramatic change we have seen to date in GSH levels. In shortterm cell culture studies using inhibitors of GSH synthesis, levels of GSH below 10 % of control values have been found to sensitize cells to damage. Thus low GSH levels are likely to sensitize the lens in these mice to exogenous insults such as light and endogenous insults as well (Xu, Zigler and Lou, 1992; Dickerson, Lou and Gracy, 1995; Shamsi et al., 2000). Studies of cataractous human lenses have revealed increases in intramolecular disul®de bond formation in b-B2 and a-A crystallin (Takemoto, 1996, 1997). In another study of cataractous lens, up to 100 % of cysteine groups were oxidized to form disul®de groups (Patterson and Delamere, 1992). Formation of disul®de bonds resulting from cysteine oxidation induces high molecular weight aggregates and turbidity in human cataract, and experimental data support the idea that crystallin fragmentation and oxidation may be causally linked to turbidity (Nakamura et al., 1999). Although cysteine represents only a small fraction of the amino acids that comprise lens crystallins, it is apparently located at strategic sites that determine protein conformation, and even a small disturbance in the structure of these proteins might result in ®ber distortion and/or admission of water into the structure of the lens (Pal and Ghosh, 1998). Low GSH levels would be expected to promote these reactions (Mossner et al., 1999). The formation of GGT-de®cient cataract in the mouse may be similar to selenete-induced cataract in the young rat. Selenite cataracts are believed to be caused by oxidative damage to the lens epithelium, causing an increase in the level of lens calcium and initiating the activation of calpain in the lens mucleus, the precipitation of proteins and a swollen cataract (Shearer et al., 1992). Low cysteine levels in GGT-de®cient mice produces growth retardation and failure of sexual maturation in both male and female in addition to cataract formation (Lieberman et al., 1996). All of these defects are corrected by NAC. Without NAC supplementation, plasma cysteine and tissue cysteine levels are approximately 10 % of wild type levels (Lieberman et al., 1996). These data suggest that cataract formation in GGT-de®cient mice has a `nutritional' component and might result from a general failure of protein synthesis secondary to cysteine de®ciency in

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many tissues including the lens. Several authors have suggested the importance of nutritional status including low dietary cysteine levels as a risk factor in cataract development, and recently cataract development has been found to be related to low socioeconomic and nutritional status (Bunce et al., 1984; Leske, Chylack and Wu, 1991; Waddell, 1998). A component of this nutritional block might also be humeral. We have found that GGT-de®cient mice have unmeasurable levels of circulation IGF 1 (submitted for publication). IGF 1 is a key growth hormone that has also been shown to have speci®c effects on lens development and crystallin synthesis (Alemany et al., 1989; Alemany, Borras and de Pablo, 1990; Klok et al., 1998). It is unlikely that IGF 1 de®ciency alone would be suf®cient to produce cataracts since this defect has not been reported in IGF 1-de®cient mice; however, IGF 1 de®ciency or other humeral de®ciencies might participate as cofactors in cataract formation. In summary, cataract formation in GGT-de®cient mice is a multifactorial phenomenon involving spontaneous damage, low GSH levels and nutritional effects. By taking advantage a targeted deletion in GGT, we have been able to demonstrate the importance of low GSH in cataract formation in the absence of reagents that might have toxic or unexpected effects independent of their inhibition of GSH synthesis (Reddy et al., 1988; Calvin et al., 1992; Martensson and Meister, 1991; Meister, 1991). Our ®ndings have direct implications for cataract formation in humans, especially senile type cataracts and those related to cysteine and GSH de®ciencies. They substantiate previous ideas and experimental work on the central importance of GSH and cysteine for maintaining a clear crystalline lens. Our data also support the possible use of NAC in patients prone to cataractogenesis such as those with end-stage renal disease undergoing dialysis (GSH losses) (Patterson and Delamere, 1992). Other metabolic diseases, such as diabetes, in which cataracts develop might also be evaluated for the use of NAC therapy (Mitton et al., 1997; Ozmen et al., 1997). In conclusion, our ®ndings underscore the complexity of the interplay of environmental and nutritional factors in cataract development. Acknowledgements We would like to thank Subhendu Chakraborty for excellent technical assistance. This work was supported by NIH grant ES-07827.

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