Cataract Induction Research Articles

Temporal and Spatial Growth Patterns in the Normal and Cataractous Human Lens

This study presents a computational model of the growth of the normal human lens and the induction of spoke-like cortical cataract in the aging lens. The anterior lens is modelled as a 2-D disk with a circumferential germinative zone. Lens cortical fibre cells in the same generation cover the surface in three identical 120deg growth wedge-shaped sectors, with centre cardinal fibres at the 90, 210 and 330deg meridians. In the foetal lens all primary fibre cells begin to elongate simultaneously. Anterior migration is spatially asynchronous, where the centre fibre begins to move towards the anterior pole first. The fibres at the end of the sector move last in the anterior direction. Fibre elongation advanced at constant speed until the boundary of the sector is reached. Spatio-temporal asynchrony and random fluctuations were increased for the adult lens. The model foetal lens evolved Y-shaped sutures anteriorly, and an inverted Y-shaped posteriorly. Fibre length varied periodically with meridional angle. The adult lens displayed irregular growth. If clusters of germinative cells are caused to opacify the resultant opacities are predominantly spoke-shaped. The model mimics crystalline lens fibre growth to the extent of successfully evolving lens sutures. Fluctuations in lens mass are consistent with an ordered pattern of growth. Lens senescence includes a progressive loss of spatio-temporal synchrony in fibre migration from the germinative zone. Peripheral light focusing by the anterior eye is a possible explanation for the nasal predilection and cuneiform shape of age-related cortical cataract.

ζ-Crystallin catalyzes the reductive activation of 2,4,6-trinitrotoluene to generate reactive oxygen species: a proposed mechanism for the induction of cataracts

Exposure to 2,4,6-trinitrotoluene (TNT) has been shown to cause induction of cataract in which oxidative stress plays a critical role. From bovine lens we purified to homogeneity and identified an enzyme that catalyzes the reduction of TNT, resulting in the production of reactive oxygen species. The final preparation of TNT reductase showed a single band with a subunit molecular weight of 38 kDa on SDS–PAGE. Sequence data from peptides obtained by digestion with lysylendopeptidase Achromobacter protease I (API) revealed that TNT reductase is identical to ζ-crystallin. Superoxide anions were formed during reduction of TNT by ζ-crystallin, though negligible enzyme activity or protein content for superoxide dismutase, a superoxide scavenging enzyme, was found in the lens. Thus, the present results suggest that the induction of cataracts by TNT may be associated with increased oxidative stress, as a result of reductive activation of TNT generating superoxide anions, there being minimal antioxidant enzyme activity for defense against reactive oxygen species exogenously produced in the lens.

Photodynamic cross-linking of proteins: V. Nature of the tyrosine–tyrosine bonds formed in the FMN-sensitized intermolecular cross-linking of N-acetyl- l-tyrosine

Some types of proteins are intermolecularly cross-linked via the formation of Tyr–Tyr (dityrosine) bonds on illumination in the presence of certain kinds of photosensitizers. The study of reactions of this type is of importance since they appear to be involved in several processes of biomedical importance such as the photosensitized (photodynamic) treatment of tumors, pathological effects of sunlight (skin photoaging, induction of cataracts), etc. The mechanisms involved in the photosensitized formation of dityrosine cross-links are not clear, and the chemical nature of the cross-link(s) is not known. In the present work, these problems have been studied using N-acetyltyrosine as a model substrate. This compound was illuminated under aerobic conditions at pH 6.0 with FMN as the photosensitizer. The Tyr–Tyr cross-linked products were isolated and purified by a preparative silica gel 60 column and by preparative reverse phase HPLC. Three main cross-linked products, designated as 1 (C6, C6′-linked di-( N-acetyltyrosine)), 2 (C6, O7′-linked di-( N-acetyltyrosine)), and 3 (C6, C4′-linked di-( N-acetyltyrosine)), were identified, and their chemical structures were determined by MS, two-dimensional NMR spectral methods, and other standard techniques. Detailed reaction pathways for the formation of the carbon–carbon ( 1 and 3) and carbon–oxygen ( 2) coupled products were proposed.

Induction of cataracts of varying degrees of hardness in human eyes obtained postmortem for cataract surgeon training

PURPOSE: To report a method of inducing nuclear cataracts of varying degree of hardness using intralenticular injection of the Karnovsky solution. METHODS: Twelve postmortem human eyes were prepared according to the Miyake–Apple posterior video technique. After performing capsulorhexis and hydrodissection/delineation, 0.2 ml of the Karnovsky solution was injected into the lens nucleus of the eyes. Manual extracapular cataract extraction techniques and various two-handed phacoemulsification maneuvers were then practiced at various time intervals after the injection. RESULTS: Uniform nuclear cataracts with varying degrees of hardness were successfully induced in all globes. The optimum dose of the Karnovsky solution and time for induction of nuclear hardness of grade 3 (ideal for practicing two-handed phacoemulsification maneuvers) were 0.2 ml and 15 minutes, respectively. CONCLUSION: The Karnovsky solution can be successfully used for the induction of hard, uniform nuclear cataracts for learning/practicing extracapular cataract extraction and phacoemulsification.

Differential Gene Expression in Male and Female Rat Lenses Undergoing Cataract Induction by Transforming Growth Factor-β (TGF-β)

Epidemiologic studies in humans as well as immunohistologic studies in animals have demonstrated significant sex differences in the propensity to develop cataract. Several studies suggest that estrogen may play a protective role against cataractogenesis. Indeed, male and ovariectomized female rat lenses have a greater susceptibility to cataract induced by transforming growth factor-beta (TGF-β) than do normal female lenses. However, in spite of the current evidence that estrogen may play a pivotal role in cataractogenesis, the molecular mechanisms behind this phenomenon are largely undetermined. Our study utilized the differential display procedure to examine gene up- and down-regulation in male, normal female and ovariectomized female rat lenses exposed to TGF-β. Male and normal female rat lenses were cultured with or without 0.15 ng ml−1TGF-β . Lenses were then harvested, and total RNA was isolated for analysis by reverse-transcriptase differential display. Differentially expressed mRNAs were subcloned, sequenced and identified through GenBank database searches. The original experiment was repeated with the addition of ovariectomized female TGF-β(+/-) conditions, and all differential patterns of gene expression were verified using Northern blot and RT-PCR analysis. Screening of approximately 12% of the mRNA population led to the identification of 27 differentially expressed cDNAs. Notably, strong gender differences were found in expression levels of γB-crystallin. In addition, proteasome Z subunit was up-regulated in TGF-β-treated male and ovariectomized female lenses, but was down-regulated in TGF-β-treated normal female lenses. This pattern of expression is consistent with the increased susceptibility of male and ovariectomized lenses to TGF-β-induced cataract. We conclude that differential display is a useful and expedient method for analysing changes in gene expression in the lens. Structural and functional studies of the genes identified in this study may further elucidate mechanisms underlying the TGF-β-induced cataract formation and differential rates of cataractogenesis in males vs females. In particular, our data suggest that the role of proteasome Z subunit in TGF-β-induced anterior subcapsular cataract warrants further investigation.

Cataract induction after total body irradiation and bone marrow transplantation: Dose-effect relationship

Cataract induction after total body irradiation and bone marrow transplantation: Dose-effect relationship

Photochemical studies on xanthurenic acid .

The tryptophan metabolite xanthurenic acid (Xan) has been isolated from aged human cataractous lenses. The photophysical properties of Xan were examined to determine if it is a potential chromophore for age-related cataractogenesis. We found that Xan produces singlet oxygen (psi delta = 0.17 in CD3OD) with the same efficiency as the lenticular chromophore N-formyl kynurenine and quenches singlet oxygen at a rate similar (2.1 x 10(7); CD3OD) to other tryptophan metabolites found in the eye. As the mechanisms of induction of cataracts may also involve redox reactions, the interactions of hydrated electrons (e(aq)-), the azide radical (N3*) and hydroxyl radical (OH*) with Xan were studied using the technique of pulse radiolysis. The reaction rate constants of e(aq)-, N3* and OH* with Xan were found to be of the same order of magnitude as other tryptophan metabolites. The rate constant for reaction of Xan with e(aq)- solvated electrons was found to be diffusion controlled (k = 1.43 x 10(10) M(-1) s(-1); the reaction with N3* was very fast (k = 4.0 x 10(9) M(-1) s(-1)); and with OH* was also near diffusion controlled (k = 1.0 x 10(10) M(-1) s(-1)). Superoxide O2*- production by irradiated Xan in methanol was detected by electron paramagnetic resonance and substantiated by determining that the enhanced rate of oxygen consumption of Xan irradiated in the presence of furfuryl alcohol was lowered by superoxide dismutase.

Late ophthalmological complications after total body irradiation in non-human primates

Purpose : To investigate the long-term eff ects of total body irradiation (TBI) on the incidence and time course of ocular complications. Materials and methods : Rhesus monkeys treated with TBI photon doses up to 8.5Gy and proton doses up to 7.5Gy were studied at intervals up to 25 years post-irradiation. They were compared with control groups with a similar age distribution. Cataract formation and ocular fundus lesions were scored according to a standardized protocol. Fluorescein angiography and histopathology was performed in selected animals. Results : Cataract formation occurred after a latent period of 3-5 years. Significant cataract induction was observed for photondoses of 8 and 8.5Gy and beyond 20 years after proton irradiation. The severity of the lesions represents significant impairment of vision and would require cataract surgery if similar results occurred in human bone marrow transplant patients. Fluorescein angiography demonstrated a normal pattern of retinal vessels in 13 out of 14 animals (93%) from the irradiated group and in eight out of nine animals (89%) from the control group. No additional lesions apart from age-related degenerative changes could be demonstrated. Histological evaluation revealed no radiation-associated vasculopathy. Conclusions : Radiation alone for doses up to 8.5Gy of photons does not carry a potential risk for fundus pathology, whereas clinically important cataract induction should be anticipated within 5 years after photon doses of 8.0 and 8.5Gy and proton doses in excess of 2.5Gy.

Overlapping effects of different members of the FGF family on lens fiber differentiation in transgenic mice.

Fibroblast growth factors (FGFs), such as FGF-1, have been shown to induce differentiation of lens epithelial cells both in tissue culture and in transgenic mice. In the present study, using the alpha A-crystallin promoter, we generated transgenic mice that express different FGFs (FGF-4, FGF-7, FGF-8, FGF-9) specifically in the lens. All four FGFs induced changes in ocular development. Microphthalmic eyes were evident in transgenic mice expressing FGF-8, FGF-9 and some lines expressing FGF-4. A developmental study of the microphthalmic eyes revealed that, by embryonic day 15, expression of these FGFs induced lens epithelial cells to undergo premature fiber differentiation. In less severely affected lines expressing FGF-4 or FGF-7, the lens epithelial cells exhibited a premature exit from the cell cycle and underwent a fiber differentiation response later in development, leading to cataract formation. The responsiveness of lens cells to different FGFs indicates that these proteins stimulate the same or overlapping downstream signalling pathway(s). These overlapping effects of different FGFs on a common cell type indicate that the normal developmental roles for these genes are determined by the temporal and spatial regulation of their expression patterns. The fact that any of these FGFs can induce ocular defects and loss of lens transparency implies that it is essential for the normal eye to maintain very specific spatial control over FGF expression in order to prevent cataract induction.

Antioxidants and cataract: (cataract induction in space environment and application to terrestrial aging cataract).

The effect of several antioxidants and cysteine-elevating precursor drugs (prodrugs) was tested on lens damage occurring after in vitro exposure to low levels of 60Co-gamma-irradiation, to simulate in vitro the exposure to radiation in vivo of (1) astronauts (2) jet crews (3) military radiation accident personnel. Tocopherol (100 microM), ascorbic acid (1 mM), R-alpha-lipoic acid (1 mM), and taurine (0.5 mM) protected against radiation-associated protein leakage. MTCA and ribocysteine protected lenses against opacification, LDH and protein leakage, indicating that antioxidants and prodrugs of cysteine appear to offer protection against lens damage caused by low level radiation.

Progression of Mouse Buthionine Sulfoximine Cataracts in vitro is Inhibited by Thiols or Ascorbate

Mouse lens cultures were employed to study the progression of cataracts initiated by injection of buthionine sulfoximine, an inhibitor of glutathione (GSH) biosynthesis. Culture of lenses removed from untreated mice on postnatal day 7, for 48 hr in the presence of 4 mmBSO, resulted in only limited cataractous changes. To enable substantial progression of cataracts in vitro, it was therefore necessary to pretreat the mice with BSO prior to lens culture. A single injection of BSO (4 nmol mg−1lens), administered on day 7, resulted in >90% depletion of lens GSH within 3 days, but no visible cataractous changes. The clear lenses were incubated for 29±1 hr at 37°C in Medium HL-1, supplemented with EGF, insulin and Ca2+, in the presence or absence of BSO, and were scored for cataract development by previously described criteria. In the absence of BSO, only 4 of 10 lenses developed large opacities. However, in the presence of 4 mmBSO, 40 out of 45 experimental lenses developed opacities affecting at least 50% of the lens visual field and were scored as stages 1C-4, depending upon the extent and density of the cataracts. In addition, three lenses had opacities involving 20–50% of the field (stage 1B). By contrast, less than 10% of lenses from untreated mice incubated in the absence of BSO developed opacities. The cataracts developed in 4 mmBSO were accompanied by reduction of lens glutathione levels to <0·010 nmol mg−1lens. They were almost completely prevented by 1 mmascorbate, 2 mmGSH, 2 mmGSH monoethyl ester and 2 mmcysteamine. GSH and GSH ester maintained lens glutathione content between 0·1 and 0·2 nmol mg−1in the presence of BSO, whereas ascorbate did not prevent near-total GSH depletion. The prevention of cataracts by thiols and ascorbate was confirmed by lens Na/K ratios not significantly different from those in control lenses. The above combination of GSH depletion in vivo by a single injection of BSO, followed 3 days later with lens culture in the presence of BSO, may yield a useful system to elucidate and control the biochemical mechanisms involved in oxidative cataract induction by this GSH-depleting agent.

Modifications in Lens Protein Biosynthesis Signal the Initiation of Cataracts Induced by Buthionine Sulfoximine in Mice

Cataract induction in preweanling mice by l-buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis, was correlated with perturbations in vitro protein biosynthesis. These were detected by incubation of late precataract and early cataract lenses with 35S-labeled amino acids, followed by dissection of lenses into capsule-epithelium and decapsulated fiber fractions, further processing of the fibers into water-soluble, urea-soluble and urea-insoluble fractions, and analysis by 2D electrophoresis and fluorography. Most of the protein labeling in control lenses was in the water-soluble fiber and capsule-epithelium fractions (80% and 14% of total cpm, respectively). Labeling in all three fiber fractions was decreased by cataract induction. The urea-insoluble fraction displayed a transient increase in labeled high molecular weight basic protein, as labeling of polypeptide monomers decreased. Densitometric analysis of fluorograms from the water-soluble and urea-soluble fiber fractions revealed a sharp decrease in fiber γ-crystallin polypeptide labeling preceding and accompanying early cataract development, a delayed decrease in labeling of αA-crystallin and increased relative percentage of several labeled β-crystallin polypeptides, especially in the urea-soluble fraction. By contrast with diminished labeling of the fiber fractions during cataract initiation, protein labeling of the corresponding capsule-epithelium fraction was stimulated dramatically and persisted at reduced levels during early opacification (stage 3), when nearly all of the protein labeling in the lens was found in capsule-epithelium. Capsule-epithelium polypeptides showing increased labeling during cataract initiation included αA-crystallin, several acidic polypeptides of M r=40–50 kDa and a group of neutral to mildly acidic polypeptides of M r=20–28 kDa. This transient activation, which was relatively non-specific, may relate to previously reported observations of polyribosome accumulation in lens epithelium during initial development of BSO cataracts. The labeled capsule-epithelium preparations are known to include newly differentiating fibers near the lens equator as well as epithelial cells. Both of these cell populations survive in mature BSO cataracts. It is suggested that modifications of the normal pattern of gene expression in the lens may be involved in initiation of the mouse BSO cataract and its subsequent pattern of development.

Maintenance of hepatic glutathione homeostasis and prevention of acetaminophen-induced cataract in mice by L-cysteine prodrugs

Administration of acetaminophen (ACP, 3.0 mmol/kg, i.p.) to β-naphthoflavone-induced C57 BL/6 mice led to the formation of bilateral cataracts within 8 hr with a 71% incidence. The hepatic glutathione (GSH) levels were reduced 99% and lenticular GSH levels reduced 42% in cataractous mice. Cataract formation was completely prevented by the co-administration of the L-cysteine prodrugs 2( R, S)-methylthiazolidine-4( R)-carboxylic acid (MTCA) and 2( R, S)- n-propylthiazolidine-4( R)-carboxylic acid (PTCA) in two divided i.p. doses totaling 4.5 mmol/kg. 2-Oxo-L-thiazolidine-4-carboxylic acid (OTCA) was nearly equipotent, yielding only one cataract in 16 mice, but D-ribose-L-cysteine (RibCys, 5 16 ) and N-acetyl-L-cysteine (NAC, 9 14 ) were much less effective. Hepatic and lenticular GSH were maintained at near normal levels by MTCA, PTCA and OTCA. These results suggest that maintenance of adequate cellular GSH levels in the presence of ACP protects against cataract induction.

Quantitative comparisons of continuous and pulsed low dose rate regimens in a model late-effect system

There is increasing interest and usage of pulsed low dose rate (PDR) brachytherapy, in which a single source is shuttled through the catheters of an implant, typically for about 10 min each hour. This study was designed to compare the late effects produced in various PDR regimens with those from the corresponding continuous low dose rate (CLDR) regimens. A model late-responding system was used, namely, cataract induction in the rat lens. This system has the advantage of being highly quantifiable. The rats eyes were exposed to a total dose of 15 Gy either continuously over 24 h, or with three different PDR regimens, all with the same total dose and overall time. We addressed three questions: (a) are late effects increased when a CLDR regimen is replaced with 10-min pulses repeated every hour? (b) Are late effects increased if hourly 10-min pulses are replaced with 10-min pulses repeated every 4 h? (c) Are late effects increased if 10-min pulses are replaced with 100-s pulses? We found that the four regimens under test, continuous, 10-min pulses each hour, 10-min pulses every 4 h, and 100-s pulses every hour, showed no significant differences in cataractogenic potential, as estimated with the Wilcoxon-Gehan test. Power tests indicated that the experimental design was adequate to detect relatively small differences in cataractogenicity between regimens. The equality of late effects from CLDR and PDR in these experiments must imply that sublethal damage repair is quite slow in this model late-responding system, in agreement with trends observed in the clinic for sublethal damage repair of late sequelae. Such trends would suggest that PDR is unlikely to produce significantly worse late effects than the corresponding CLDR regimen, which is in agreement with early clinical data using PDR. Caution, however, is strongly recommended.

Cataractogenesis in Rats Induced by in Utero Exposure to RG 12915, a 5-HT 3 Antagonist

Cataractogenesis in Rats Induced by in Utero Exposure to RG 12915, a 5-HT 3 Antagonist. Lerman, S. A., Delongeas, J. L., Plard, J. P., Veneziale, R. W., Clark, R. L., Rubin, L., and Sanders, J. E. (1995). Fundam. Appl. Toxicol. 27, 270-276. RG 12915, a selective 5-HT 3 antagonist developed for the treatment of emesis and nausea associated with cancer chemotherapy, was administered by gavage to four groups of pregnant rats from Gestation Day 6 to 17 at doses of 0, 1, 10, and 100 mg/kg/day, as part of a Segment II (developmental toxicity) study. The 100 mg/kg/day dose was maternally toxic as indicated by decreased body weight gain and food consumption during the treatment period. A portion of the rats were allowed to deliver and rear their litters and three pups from two litters in the 100 mg/kg/day group were observed to have lens opacities (visible to the naked eye) at weaning. At a later examination, when the offspring were approximately 4 months old, four additional animals from the same two litters had cataracts. A slight growth retardation was also observed postweaning in the offspring of the 100 mg/kg/day group. To confirm the lens findings and more precisely define the no-effect dose, another study was conducted in which pregnant rats were administered daily RG 12915 doses of 0, 10, 30, 60, or 100 mg/kg/day from Gestation Day 6 to 17. There was a dose-related decrement in maternal body weight gain during the treatment period in the 30, 60, and 100 mg/kg/day groups (12, 28, and 47%, respectively) compared to the control group. A treatment-related incidence of nuclear cataract was observed in the offspring of the 60 and 100 mg/kg/day groups (litter incidence 6 and 45%, respectively). Cataracts were subsequently observed in a Segment III (peri- and postnatal toxicity) study in which RG 12915 was administered to four groups of pregnant rats from Gestation Day 15 through Postpartum Day 21 at doses of 5, 20, and 80 mg/kg/day. A high incidence of treatment-related lens opacities was observed in the offspring of the 80 mg/kg/day group (95% of the litters were affected). Since the common treatment period between the Segment II and III studies is Gestational Days 15-17 and the major organogenesis of the lens nucleus occurs during Gestation Days 11-15 in the rat, it is suggested that the sensitive period for cataract induction by RG 12915 may be close to Gestational Day 15. In summary, maternally toxic doses of this serotonergic agent during gestation produced cataracts in the offspring.

A review of the evidence supporting melatonin's role as an antioxidant

This survey summarizes the findings, accumulated within the last 2 years, concerning melatonin's role in defending against toxic free radicals. Free radicals are chemical constituents that have an unpaired electron in their outer orbital and, because of this feature, are highly reactive. Inspired oxygen, which sustains life, also is harmful because up to 5% of the oxygen (O2) taken in is converted to oxygen-free radicals. The addition of a single electron to O2 produces the superoxide anion radical (O2-.); O2-. is catalytic-reduced by superoxide dismutase, to hydrogen peroxide (H2O2). Although H2O2 is not itself a free radical, it can be toxic at high concentrations and, more importantly, it can be reduced to the hydroxyl radical (.OH). The .OH is the most toxic of the oxygen-based radicals and it wreaks havoc within cells, particularly with macromolecules. In recent in vitro studies, melatonin was shown to be a very efficient neutralizer of the .OH; indeed, in the system used to test its free radical scavenging ability it was found to be significantly more effective than the well known antioxidant, glutathione (GSH), in doing so. Likewise, melatonin has been shown to stimulate glutathione peroxidase (GSH-Px) activity in neural tissue; GSH-PX metabolizes reduced glutathione to its oxidized form and in doing so it converts H2O2 to H2O, thereby reducing generation of the .OH by eliminating its precursor. More recent studies have shown that melatonin is also a more efficient scavenger of the peroxyl radical than is vitamin E. The peroxyl radical is generated during lipid peroxidation and propagates the chain reaction that leads to massive lipid destruction in cell membranes. In vivo studies have demonstrated that melatonin is remarkably potent in protecting against free radical damage induced by a variety of means. Thus, DNA damage resulting from either the exposure of animals to the chemical carcinogen safrole or to ionizing radiation is markedly reduced when melatonin is co-administered. Likewise, the induction of cataracts, generally accepted as being a consequence of free radical attack on lenticular macromolecules, in newborn rats injected with a GSH-depleting drug are prevented when the animals are given daily melatonin injections. Also, paraquat-induced lipid peroxidation in the lungs of rats is overcome when they also receive melatonin during the exposure period. Paraquat is a highly toxic herbicide that inflicts at least part of its damage by generating free radicals.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucocorticoid-Induced Cataract of Developing Chick Embryo as a Screening Model for Anticataract Agents

In order to develop an effective screening model for anticataract agents, we examined the age dependence of cataract induction by glucocorticoid in developing chick embryos. Hydrocortisone sodium succinate (0.25 mumol) was administered to chick embryos on day 15 (15-day-old) and cataract formation was examined 48 hr later. Administration earlier than on day 13 or later than on day 15 was a little or ineffective. These results indicate that the formation of glucocorticoid-induced cataract in developing chick embryos depends on developing stages. The embryos treated with hydrocortisone sodium succinate on day 15 decreased GSH amount in the lens, approximately 50% of the control in 48hr. However, the embryos treated at other ages, in which cataract was not induced, showed little or no decrease of GSH. The cataract formation in chick embryos appeared to depend on structure of steroid and was due to biological activities of glucocorticoids. Since cataract is easily produced in a reproducible manner with high incidence by glucocorticoid, our chick embryo model will be a valuable model system for screening anticataract agents.

The influence of dose, dose-rate and particle fragmentation on cataract induction by energetic iron ions

Because activities in space necessarily involve chronic exposure to a heterogeneous charged particle radiation field it is important to assess the influence of dose-rate and the possible modulating role of heavy particle fragmentation on biological systems. Using the well-studied cataract model, mice were exposed to plateau 600 MeV/amu 56Fe ions either as acute or fractionated exposures at total doses of 5 – 504 cGy. Additional groups of mice received 20, 360 and 504 cGy behind 50 mm of polyethylene, which simulates body shielding. The reference radiation consisted of 60Co γ radiation. The animals were examined by slit lamp biomicroscopy over their three year life spans. In accordance with our previous observations with heavy particles, the cataractogenic potential of the 600 MeV/amu 56Fe ions was greater than for low-LET radiation and increased with decreasing dose relative to γ-rays. Fractionation of a given dose of 56Fe ions did not reduce the cataractogenicity of the radiation compared to the acute regimen. Fragmentation of the beam in the polyethylene did not alter the cataractotoxicity of the ions, either when administered singly or in fractions.

The Effect and Recovery of Long-term H 2O 2 Exposure on Lens Morphology and Biochemistry

Oxidative stress has long been speculated to play an important role in cataractogenesis. In the H 2O 2-induced cataract model, rat lens showed extensive biochemical damage but very mild morphological changes after being exposed to H 2O 2 (0·5 mM) for 24 hr in culture. This damage included reduced glutathione (GSH) depletion, protein-GSH mixed disulfide (PSSG) elevation but not protein-protein disulfide (PSSP) formation. In order to understand the role of protein-thiol mixed disulfide formation in relation to the sequence of events during cataract induction, we conducted a long term H 2O 2 exposure study for up to 96 hr to monitor the dynamic changes in GSH and PSSG levels, the formation of PSSP aggregate, protein solubility, and the progression in lens opacity. Rat lenses were cultured in 0·5 mM H 2O 2 and harvested at intervals of 24, 48, 72 and 96 hr for the examination of morphological and biochemical changes. Contralateral lenses cultured in H 2O 2-free media were used as controls. It was found that the lenses had only patchy opacity at the equator after 24 hr, but became hydrated suddenly at 48 hr (31% heavier than the control), with an opacity which involved the entire outer cortical region. By 72 hr incubation, the nucleus was opacified. Lens GSH progressively decreased with time of H 2O 2 exposure, 40% was lost by 24 hr and over 95% by 48 hr. There was a concomitant elevation of PSSG, 16-fold over the controls by 24 hr and 45-fold by 48 hr followed by a decline to 34-fold after 72 hr. In addition, the level of protein-cysteine mixed disulfide (PSSC) was elevated after 48 hr incubation in H 2O 2. At this time point, PSSP aggregates began to appear both in water soluble (WS) and urea soluble (US) fractions along with a drastic reduction in protein solubility. Western blot analysis of the protein fractions identified β and γ, but not α-crystallin in the disulfide-containing aggregates. The lens clarity and biochemical changes partially recovered if the oxidant was removed within 24 hr, indicating a potential therapeutic role for antioxidants. The complete normalization of PSSG level under this recovery condition signifies that cells may have a natural defense system for controlling PSSG elevation.

Comparison of cell-permeable calpain inhibitors and E64 in reduction of cataract in cultured rat lenses

E64, an inhibitor of calpain (EC 3.4.22.17) and other cysteine proteases, slows the rate of formation of cataract in cultured rat lenses. The purpose of this study was to determine (1) why E64, a charged compound with little cell permeability, was effective in reducing cataract in cultured lens and (2) whether uncharged more permeable protease inhibitors are more effective than E64 in preventing cataract. Results showed that E64 entered the lens, but only after the lens was treated with the calcium ionophore, A23187, or sodium selenite, both of which cause cataracts. Therefore, the uptake and subsequent effectiveness of E64 may be related to a generalized increase in membrane permeability during induction of cataract in culture. Three protease inhibitors, reported to have improved cell permeability, were compared with E64 for their ability to prevent cataracts in cultured lenses. cBz-ValPheH, calpain inhibitors I and II, are uncharged-aldehyde inhibitors of calpain. Calpain inhibitors I and II even at high concentrations were not effective at reducing lens opacity caused by calcium ionophore and were toxic to the lens. cBz-ValPheH, which is slightly toxic to the lens, was able to significantly reduce lens opacity induced by calcium ionophore. The presented data suggest that while E64 decreases cataract formation in cultured lens, the more cell permeable inhibitor, cBz-ValPheH, may have greater efficacy as an anticataract drug in vivo.