Lenticular Glutathione Research Articles

Prevention of age-related truncation of γ-glutamylcysteine ligase catalytic subunit (GCLC) delays cataract formation.

A sharp drop in lenticular glutathione (GSH) plays a pivotal role in age-related cataract (ARC) formation. Despite recognizing GSH's importance in lens defense for decades, its decline with age remains puzzling. Our recent study revealed an age-related truncation affecting the essential GSH biosynthesis enzyme, the γ-glutamylcysteine ligase catalytic subunit (GCLC), at aspartate residue 499. Intriguingly, these truncated GCLC fragments compete with full-length GCLC in forming a heterocomplex with the modifier subunit (GCLM) but exhibit markedly reduced enzymatic activity. Crucially, using an aspartate-to-glutamate mutation knock-in (D499E-KI) mouse model that blocks GCLC truncation, we observed a notable delay in ARC formation compared to WT mice: Nearly 50% of D499E-KI mice remained cataract-free versus ~20% of the WT mice at their age of 20 months. Our findings concerning age-related GCLC truncation might be the key to understanding the profound reduction in lens GSH with age. By halting GCLC truncation, we can rejuvenate lens GSH levels and considerably postpone cataract onset.

Inhibition of Crystallin Ascorbylation by Nucleophilic Compounds in the hSVCT2 Mouse Model of Lenticular Aging

Senile cataracts are associated with oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. This process is partially explained by advanced glycation end products (AGEs) from ascorbic acid (ASA), as the authors unequivocally demonstrated in an hSVCT2 transgenic mouse. The authors present the first pharmacologic intervention study against ascorbylation in these mice. Five groups of mice from 2 to 9 months of age (10 mice/group) were fed a diet containing 0.1% (wt/wt) aminoguanidine, pyridoxamine, penicillamine, and nucleophilic compounds NC-I and NC-II. AGEs were determined in crystallin digests using high-performance liquid chromatography, liquid chromatography-mass spectrometry, or gas chromatography-mass spectrometry. Lens protein extract was incubated in vitro with ASA or dehydroascorbic acid. The ASA level increased approximately 10-fold in all groups and was unaffected by treatment. AGEs were increased several-fold in transgenic compared with control lenses. Body weight, food intake, lenticular glutathione, and glycated lysine level were unaltered. In vitro, all compounds inhibited AGE formation. In vivo, NC-I and NC-II significantly decreased protein fluorescence at lambda(ex)335/(em)385 (P = 0.045, P = 0.017, respectively) and lambda(ex)370/(em)440 (P = 0.029, P = 0.007, respectively). Other inhibitors had no effect. After 7 months, only NC-I and NC-II induced a 50% reduction in pentosidine (P = NS for NC-I; P = 0.035 for NC-II). NC-I also decreased carboxymethyllysine (P = 0.032) and carboxyethyllysine (P = NS). Fluorescent cross-link K2P was decreased by NC-I, NC-II, aminoguanidine, and pyridoxamine (P = NS). Pharmacologically blocking protein ascorbylation with absorbable guanidino compounds is feasible and may represent a new strategy for the delay of age-related nuclear sclerosis of the lens.

The Effect of N-Acetyl Serotonin on Ultraviolet-Radiation-Induced Cataracts in Rats

Aims: To investigate the effects of ultraviolet radiation (UVR) on cataract development in rat lenses and whether or not N-acetyl serotonin has an effect on changes in these lenses. Material and Methods: The study was performed using 5 groups of Sprague-Dawley albino rats, with each group consisting of 15 rats. The 5th group being the control group did not receive any applications, whilst the other 4 groups received a daily dose of 0.2 J/cm²/day UVR (305 nm wavelength) for 60 days. A dose of 4 mg/kg/0.1 ml N-acetyl serotonin was injected intraperitoneally to group 1 and group 2 every day and on alternate days, respectively. Group 3 received an intraperitoneal injection of 0.1 ml phosphate buffer solution every day, whilst group 4 received no injection. On the 60th day, an intracardiac withdrawal of blood was performed, after the rats had been anesthetized with ether. Following the withdrawal of blood, the rats were killed using a high dose of ether and their eyes were enucleated. The lens fresh weights, plasma malondialdehyde (P-MDA), erythrocyte glutathione peroxidase (E-GSHPx), erythrocyte glutathione reductase, blood reduced glutathione (B-RGSH), erythrocyte catalase (E-CAT), lenticular malondialdehyde, lenticular superoxide dismutase (L-SOD), lenticular glutathione peroxidase (L-GSHPx) and lenticular glutathione (L-GSH) levels were all assessed. Results: The lens fresh weights were determined to be lower in group 1 and in the control group in comparison with the other groups (p < 0.01). Whilst the P-MDA level was found to be lower (p < 0.001), the E-GSHPx level was higher (p < 0.01) in the control group than in the other groups. The E-GSHPx level was higher in groups 1 and 2 than in groups 3 and 4 (p < 0.01). The B-RGSH level was higher in the control group than in the other groups (p < 0.001). The E-CAT level was higher in both the control group and group 1 than in groups 2, 3 and 4 (p < 0.01), whilst it was higher in group 2 when compared to groups 3 and 4 (p < 0.01). The L-SOD levels were found to be higher in the control group and group 1 than in groups 2, 3 and 4 (p < 0.001). Whilst the L-GSHPx levels were determined to be higher only in the control group (p < 0.001), the L-GSH levels were higher in the control group and group 1 than in the other groups (p < 0.001). Conclusions: In recent years, the depletion of the atmospheric ozone layer has resulted in the penetration of more UVR to the earth, which causes various effects on different tissues of organisms. N-acetyl serotonin, a melatonin precursor, may well be effective in the prevention of the negative effects induced by the UVR upon the lens tissue, in which case the capability of melatonin to capture free radicals as well as its antioxidative properties should be taken into consideration.

Effect of melatonin against oxidative stress in ultraviolet-B exposed rat lens

To investigate the defensive effect of melatonin against oxidative stresses in ultraviolet-B (UVB) radiation induced cataract development. Young rats received 8 kJ/m(2) UVB for 15 min. For the intervention of cataract development intraperitoneal injection of melatonin (4 mg/kg daily for 1 week) following UVB exposure was performed. Lenticular glutathione peroxidase (GSHPx), catalase (CAT), superoxide dismutase (SOD) activities and glutathione (GSH), malondialdehyde (MDA) levels were determined in UVB-melatonin, UVB, and control groups. One week after exposure in the UVB group, lens opacities were observed and CAT, SOD, and GSHPx activities, and GSH level were lower than control and MDA level was higher than control (p < 0.05). In the UVB-melatonin group CAT and SOD activities were lower than control (p < 0.05), and the MDA level was lower than the UVB group (p < 0.05). These results suggest that melatonin may protect against the UVB-induced cataract development by directly quenching lipid peroxides and indirectly by enhancing the production of the endogenous antioxidant GSH.

Effect of melatonin against oxidative stress in ultraviolet-B exposed rat lens

Purpose. To investigate the defensive effect of melatonin against oxidative stresses in ultraviolet-B (UVB) radiation induced cataract development. Methods. Young rats received 8 kJ/m 2 UVB for 15 min. For the intervention of cataract development intraperitoneal injection of melatonin (4 mg/kg daily for 1 week) following UVB exposure was performed. Lenticular glutathione peroxidase (GSHPx), catalase (CAT), superoxide dismutase (SOD) activities and glutathione (GSH), malondialdehyde (MDA) levels were determined in UVB-melatonin, UVB, and control groups. Results. One week after exposure in the UVB group, lens opacities were observed and CAT, SOD, and GSHPx activities, and GSH level were lower than control and MDA level was higher than control (p < 0.05). In the UVB-melatonin group CAT and SOD activities were lower than control (p < 0.05), and the MDA level was lower than the UVB group (p < 0.05). Conclusions. These results suggest that melatonin may protect against the UVB-induced cataract development by directly quenching lipid peroxides and indirectly by enhancing the production of the endogenous antioxidant GSH.

Liver and lens glutathione and cysteine regulation in galactose-fed guinea pigs

PURPOSE. To study the mechanism of lenticular glutathione (GSH) depletion in galactose-fed guinea pigs, with particular reference to correlations between liver and lens GSH, precursor (cysteine) status and GSH synthetic capacities. METHODS. Guinea pigs in the ad libitum-fed state were fed powdered guinea pig chow containing 50% galactose for 3 and 14–16 days. Plasma GSH and GSH levels in lens, liver and freshly isolated hepatocytes were determined. Maximal rates of GSH synthesis in liver and lens as well as steady state levels of precursor cysteine were also determined. In separate experiments, linear rate of 35 S-cysteine uptake was studied in isolated hepatocytes from control and galactose-fed animals. Lens and liver GSH decreased significantly with galactose feeding. Hepatic GSH showed a dramatic decrease (~83%) as early as day 3 whereas ~43% decrease was observed in lens. The maximal GSH synthetic rates (GSH-SR) in the whole lens and liver on days 3 and 14–16 were not different from those of controls. Steady-state levels of cysteine also decreased in both tissues with galactose feeding, and the magnitude of decrease was higher in the liver as compared to the lens. The rate of cysteine uptake in hepatocytes isolated from galactose-fed guinea pigs was significantly lower for the cysteine concentrations studied (10 µM to 1 µM) as compared to control uptake. The decreased steady-state liver GSH and cysteine levels in galactose-fed guinea pigs caused a significant decrease in plasma (and aqueous) GSH concentrations. CONCLUSIONS. We concluded that the decrease in lens GSH due to galactose occurs without alterations in the capacity of GSH synthesis, in either lens or liver. It is suggested that decreased hepatic GSH, resulting in reduced plasma GSH levels due to decreased GSH efflux into plasma, may contribute to impairment in plasma to lens GSH transport with galactose. Thus, the functional role of recently identified lens GSH transporters, particularly that of Na + -dependent GSH transporter, in galactose-induced cataract formation will be worthy of investigation.

Prevention of Naphthalene-Induced Cataract and Hepatic Glutathione Loss by the L-Cysteine Prodrugs, MTCA and PTCA

Rapid-onset cataracts were induced in SPF C57 bl/6 mice by intraperitoneal administration of naphthalene following cytochrome P-450 isozyme induction with phenobarbital. Several l-cysteine prodrugs with masked sulfhydryl groups in the form of thiazolidine-4-carboxylic acids, as well as N-acetyl- l-cysteine, N,S- bis-acetyl- l-cysteine and glutathione ethyl ester, were evaluated for their ability to maintain hepatic and lenticular glutathione at near-normal levels and to prevent naphthalene-induced cataract formation. Each prodrug was administered at three specified times to a cumulative total of 1.5 mole equivalents of the single dose of naphthalene. Three l-cysteine prodrugs delayed but did not prevent cataract formation in 40–60% of the mice over a 72-hr period, while eight of the 13 compounds produced cataract yields similar to the naphthalene control animals, i.e. 83% in 72hr. However, two l-cysteine prodrugs, 2( R,S)-methylthiazolidine-4( R)-carboxylic acid (MTCA) and 2( R,S)- n-propylthiazolidine-4( R)-carboxylic acid (PTCA), prevented cataract formation in 20 of 21 and 12 of 12 mice, respectively, and maintained hepatic reduced glutathione levels at 82% and 51% of untreated controls. In contrast, glutathione was depressed to 3% of the normal value in those animals treated with naphthalene alone. Lenticular glutathione values were depressed, albeit minimally, in all naphthalene-treated mice regardless of administration of either MTCA or PTCA. The mice protected with either MTCA or PTCA showed no visible effects of naphthalene toxicity or lens opacities at any time. It can be concluded that these l-cysteine prodrugs were effective in preventing naphthalene-induced cataract and maintaining near-normal hepatic glutathione levels.

Augmentation of human and rat lenticular glutathione in vitro by prodrugs of gamma-L-glutamyl-L-cysteine.

A marked age-related decrease in glutathione (GSH) levels as well as depression of gamma-glutamylcysteine synthetase activity are factors that are believed to render the aged lens more susceptible to oxidative stress and, therefore, to cataractogenesis. Providing gamma-L-glutamyl-L-cysteine, the dipeptide precursor of GSH, would effectively bypass the compromised first step in its biosynthesis and should protect the lens from GSH depletion. Accordingly, some bioreversible sulfhydryl-, amino-, and C-terminal carboxyl-protected prodrug forms of this dipeptide were prepared. Sulfhydryl protection was in the form of an acetyl thioester, while the carboxyl group was protected as the ethyl ester. These prodrugs were evaluated for their GSH-enhancing activity in cultured human and rat lenses in vitro using an assay that measured the incorporation of [14C]glycine into lens GSH. Ethyl S-acetyl-gamma-L-glutamyl-L-cysteinate (2) raised GSH levels in human lenses by 25% and in rat lenses by >150%. These data suggest that 2 may have potential as an anticataract agent since ethyl gamma-L-glutamyl-L-cysteinate (1a), the des-S-acetyl analog of 2, had been shown (by others) to protect against experimental rodent cataracts. GSH augmentation by 1a was 2% in human lenses and 25% in rat lenses, considerably less than that shown by 2.

13C-NMR spectroscopic studies of 2-mercaptoethanol-stimulated glutathione synthesis in the intact ocular lens

13C-NMR spectroscopy was employed non-invasively for the real-time measurement of the rates of glutathione synthesis in intact rabbit lenses supported in organ culture containing l-[3- 13C]cysteine. Supplementation of the organ culture medium with 2-mercapto-ethanol resulted in a dose- dependent enhancement of lenticular glutathione synthesis rates (dose for 50% maximal effect = 125 μM). At the most effective concentration (400 μM) 2-mercaptoethanol increased the rate of glutathione synthesis 163% relative to the rate observed under control conditions. The mechanism of action for this effect was investigated in intact lenses using antagonists of specific amino acid uptake systems. These experiments demonstrated that the enhanced rates of glutathione synthesis observed in the presence of 2-mercaptoethanol were due to the affinity of the mixed-disulfide formed between cysteine and 2-mercaptoethanol for L system amino acid uptake, thereby providing a mechanism for increasing intracellular cysteine levels by circumventing normal cellular cysteine uptake pathways in the lens. Because of the role of cysteine as the rate limiting substrate in lenticular glutathione biosynthesis, these results suggest a potential strategy to prevent tissue opacification associated with depleted glutathione levels.

Why does the human visual system age in the way it does?

The tentative observation that the decrease of lenticular glutathione in man and in cattle may be under genetic control is extended to other biological functions which show a systematic reduction with age. Ocular and visual parameters are shown to decline consistently with the view that the human eye has evolved in keeping with other biological functions sustaining a life-span of approximately 120 years. Analysis of the data suggests that presbyopia represents an outlier in the distribution of ocular attributes, and should not be used as a biomarker for ageing.

Desferal-Mn(III) in the therapy of diquat-induced cataract in rabbit

In rabbit lenses subjected to oxidative stress, induced by 1 m m diquat in vitro, there were 7- to 10-fold increases ( p < 0.001) in malondialdehyde, conjugated dienes, and carbonyl dienes, indicating extensive peroxidation of cellular membrane lipids, and approximately a 60% decrease in reduced glutathione. In the presence of 0.1–5 m m Desferal-Mn(III) these changes were diminished by 50–70%. In an experimental group of 12 rabbits having diquat-induced cataract, Desferal-Mn(III) ( 5% w v ) applied topically as a 50-μl eye drop four times per day and a single intraperitoneal dose of 64 mg/kg body wt daily for 5 weeks (including pretreatment for 1 week) retarded the progression of lens opacities, whereas, in a control group of 6 rabbits treated with the vehicle (0.15 m NaCl) cataract progressed to an advanced grade. Treatment with Desferal-Mn(III) also significantly diminished production of O ∸ 2 and OH· in the lens, aqueous humor, and vitreous humor, and of H 2O 2 in the aqueous humor and vitreous humor. It also suppressed lipid peroxidation and oxidation of protein-SH of the lens and restored lenticular glutathione and ascorbate to normal levels.

Glutathione and related indices in rat lenses, liver and red cells during riboflavin deficiency and its correction

Biochemical changes in lenses and at other sites in adult rats were investigated during the induction and correction of riboflavin deficiency. Riboflavin deficient (D), 1-day-repleted (R1), 2-days-repleted (R2), 16-days-repleted (R3), food-restricted, weight-matched controls (CFR) and ad libitum-fed controls (CAL) were compared. Activation coefficients of erythrocyte and lens glutathione reductase, which became abnormal in the deficient (D) animals, were corrected to varying extents in the repleted (R) groups. Hepatic flavin concentrations were lowered in the groups with raised glutathione. Inter-group differences in hepatic glutathione concentrations were not simply related to tissue flavin depletion or its reversal, but were complicated by changes in liver: body-weight ratios. Inter-group differences in lenticular glutathione levels were very small. In both liver and lens, sorbitol concentrations were lowest in group R3 and highest in groups D, R1 and R2. Lens ascorbate levels and the lens enzymes, aldose reductase, sorbitol dehydrogenase, glutathione peroxidase and superoxide dismutase, were not significantly affected by diet. Thiobarbituric acid-reactive substances were increased in riboflavin-deficient rat lenses but were lowered in riboflavin-deficient plasma samples. The results suggest overall that while riboflavin deficiency may affect certain biochemical indices, such as sorbitol and thiobarbituric-reactive substances, in the lens and other tissues, these changes are not the result of lowered glutathione levels. They also clearly demonstrate the importance of inanition as a confounding factor in the interpretation of changes resulting from riboflavin deficiency in experimental animals.

Thermal inactivation study of glutathione peroxidase and glutathione reductase activities in lenses of primates and non-primates

Heat lability studies of glutathione peroxidase and glutathione reductase activities were conducted on rabbit, sheep, rat, human, galago, cat and rhesus monkey lens supernatants. These species represent five mammalian orders. Incubation periods were 10.0 minutes in duration, with temperatures ranging from 25-100 degrees C (depending on which enzyme was being investigated). Results obtained for glutathione peroxidase activity demonstrated nearly identical heat lability profiles for human and rhesus monkey lenses. Both species were extremely labile to heat, losing activity at 30 degrees C and becoming totally inactive at temperatures of 50 degrees C (rhesus monkey) and 55 degrees C (human). Their profiles were very dissimilar to those of the other five species investigated, providing evidence for the existence of an evolutionary break. Glutathione reductase activity was extremely stable under conditions of highly elevated temperature for all seven species investigated. The human lens enzyme, the most stable of the species, maintained nearly 100% of its original activity up to 65 degrees C. Lenticular glutathione reductase activity did not reach zero levels in any of the seven species until a temperature of at least 80 degrees C was attained.

Influence Dietary Riboflavin Deficiency on Lenticular Glutathione Redox Cycle, Lipid Peroxidation, and Free Radical Scavengers in the Rat

Effect of dietary riboflavin deficiency on rat lens glutathione, glutathione redox cycle, and oxidative-antioxidative processes was investigated to understand the possible biochemical mechanism(s) responsible for aggregation of lens proteins. Induction of riboflavin deficiency resulted in a marked reduction in the activity of glutathione reductase (GSH-R). Levels of glutathione (both reduced and oxidised) and sulfhydryl groups (total and non-protein), however, were unchanged. Activity of glucose 6-phosphate dehydrogenase, which generates the reducing equivalents, NADPH, necessary for maintenance of normal GSH-R activity, was found to be unaltered. On the other hand, glutathione dependent glutathione peroxidase, a potent peroxide scavenging enzyme, showed a significant rise in its activity with a concomitant increase in lipid peroxidation. Among the other antioxidative systems studied, superoxide dismutase and catalase remained unaltered, while levels of ascorbate declined.These results indicate impairments in the glutathione redox cycle and antioxidative defence mechanisms, in addition to enhanced lipid peroxidation in the lens of riboflavin deficient rats.

Pharmacological action of hachimijiogan (Ba-wei-wan) on the metabolism of aged subjects.

In order to elucidate the active mechanism of traditional Chinese medicine, we investigated the effects of Hachimijiogan which has been alleged to restore the function of old degenerated organs. After administering Hachimijiogan to aged rats and mice, we examined the lipid and glutathione metabolism. The cholesterol and triglyceride levels in the mice liver were significantly decreased in the Hachimijiogan treated group. The cholesterol turnover was examined using labeled cholesterol in aged mice, and Hachimijiogan may increase the turnover of cholesterol metabolism. The lenticular glutathione levels and enzyme activities concerned with glutathione metabolism were investigated in aged rats given Hachimijiogan, suggesting that Hachimijiogan may offer protective and therapeutic effects against senile cataract. From our study, Hachimijiogan is considered to improve the lipid and glutathione metabolism as a result of its complicated in vivo compound effects.

FAD-induced in vitro activation of glutathione reductase in the lens of B2 deficient rats.

We studied the FAD-induced in vitro stimulation of lenticular glutathione reductase in riboflavin-deficient rats. The stimulatory effect of FAD on lenticular glutathione reductase in rats fed a B2-deficient diet for 4 weeks was remarkably higher than in paired control rats fed a B2-supplemented basal diet and control rats had ad libitum access to a B2-supplemented basal diet. The in vitro FAD stimulation effect on rat lenticular glutathione reductase represents a sensitive indicator of the B2 deficient status.

Net proteolysis in glucose-deprived rat lenses incubated in amino acid-free medium

A study of proteolysis was undertaken in rat lenses cultured in balanced salt solution, without amino acids. When glucose was present, slow loss of amino acids to the medium occurred, but sodium and potassium levels remained normal for at least 48 hr. Lenticular glutathione slowly disappeared, with matching increases of glycine and cysteine in the lens plus medium. Lenticular ATP gradually decreased, with matching increase of free phosphate. Ascorbate decreased steadily during incubation, and lactate loss by diffusion exceeded replacement via glycolysis. Lens taurine decreased steadily, but the total in lens plus medium remained constant. In the absence of glucose, the initial loss of ATP and glutathione were faster and some exchange of sodium and potassium occurred. Starting abruptly after 24 hr, when ATP was depleted, rapid net proteolysis was observed. The quantities of proteogenic amino acids in lens plus medium increased linearly to several times the initial values by 48 hr. Increased lens levels of proteogenic amino acids were accompanied by marked influx of sodium and water. Calcium also increased in the lens. Loss of lens taurine was accelerated and depletion of lactate was faster. The results obtained suggest that disappearance of ATP in glucose-deprived lenses is followed by onset of net proteolysis.

Cataract and gamma-glutamyl cycle in myotonic dystrophy.

Gamma-Glutamyl transpeptidase (gamma-GT) may be responsible for the rapid catabolism and low levels of lenticular glutathione often associated with cataract formation. Elevated levels of serum gamma-GT in patients with myotonic dystrophy suggest that since the defect could be present in all tissues it might be responsible for the cataracts frequently observed in this genetic disease.

Transport of l-glutamic acid and l-glutamine and their incorporation into lenticular glutathione

The dicarboxylic amino acids penetrate the cells of the lens (calf, rat) with difficulty. l-Aspartic acid was confined to the extracellular space of the bovine lens. In contrast, l-glutamine was transported as a typical neutral amino acid in both species. This amino acid was found to equilibrate readily with lenticular glutamate in the rat and the calf, and the latter amino acid was then incorporated into glutathione (γ-glutamylcysteinylglycine). A turnover rate of 2% per hr was calculated for the tripeptide in the calf lens. Since twice as much glutamine as glutamic acid was found in bovine aqueous humor, the amide appears to be the natural source of glutamine, glutamic acid, and the glutamyl residue of glutathione in the lens. l-Glutamic acid entered the cells of the rodent lens primarily by a special class of transporters (Glu −). Influx of the dicarboxylic amino acids was 20% or less than that of l-glutamine. Incorporation into glutathione was noted of both l-glutamate and l-glutamine in the rat, with glutamine giving about four times the specific activity of glutamate in this compound. A turnover rate of 6% per hr was obtained. Conversion of both glutamine and glutamate to proline was observed in these lenses.