An HPLC radiotracer method for assessing the ability of L-cysteine prodrugs to maintain glutathione levels in the cultured rat lens.

PURPOSE: To apply a high performance liquid chromatographic radiotracer method to test a variety of L-cysteine prodrugs and one dipeptide prodrug for their ability to synthesize glutathione in cultured rat lenses. METHODS: Rat lenses were incubated for 48 h in a medium containing [14C(U)]-glycine and prodrugs. Following homogenization and derivatization, lens extracts were analyzed to determine the extent of biosynthetic incorporation of this labeled amino acid into [14C]-glutathione using high performance liquid chromatography with radioisotope and ultraviolet absorption detection. All of the thiazolidine prodrugs contained masked sulfhydryl groups to stabilize them against air oxidation. L-buthionine-(S,R)-sulfoximine-an inhibitor of the first step in glutathione biosynthesis-was present in media containing the dipeptide prodrug. RESULTS: In all cases, a large [14C]-labeled peak eluted just prior to [14C]-glutathione. This peak had some characteristics of the mixed disulfide of glutathione and L-cysteine, viz., L-cysteine/glutathione disulfide, but requires further investigation in order to be positively identified. Of the eleven L-cysteine prodrugs investigated, the most effective was 2(R,S)-methylthiazolidine-4(R)-carboxylic acid, which increased the rate of [14C]-glutathione biosynthesis 35% over that of the controls. A number of other L-cysteine prodrugs were somewhat effective, increasing glutathione synthesis 5-30% over the controls, while several L-cysteine prodrugs were totally ineffective. The only dipeptide prodrug investigated, viz., gamma-L-glutamyl-L-cysteine ethyl ester, increased the biosynthesis of [14C]-glutathione 18% over control. Biosynthetic rates based on ultraviolet absorption of the derivatized glutathione demonstrated a similar pattern, the compounds most effective in synthesizing [14C]-glutathione generally yielding the highest ultraviolet glutathione concentrations and the ineffective compounds showing the lowest concentrations. CONCLUSIONS: 2(R,S)-methylthiazolidine-4(R)-carboxylic acid, 2(R,S)-n-propylthiazolidine-4(R)-carboxylic acid and N-acetyl-L-cysteine were the only compounds that were statistically significant in yielding higher levels of both ultraviolet and radioactive glutathione as compared to their respective controls. Thus, these prodrugs have very promising anti-cataract potential.

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 beta-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.

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 72 hr. 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.

Prevention of acetaminophen- and naphthalene-induced cataract and glutathione loss by CySSME.

PURPOSES: To assess the efficacy of 2-mercaptoethanol/L-cysteine mixed disulfide (CySSME) as an L-cysteine prodrug suitable for glutathione biosynthesis in rat lenses in vitro, as an agent for the prevention of acetaminophen- and naphthalene-induced murine cataract in genetically-susceptible mice, and as an agent for maintenance of near-normal glutathione levels in lenses and livers of mice subjected to acetaminophen and naphthalene at cataractogenic doses. METHODS: Synthetic CySSME was added as a prodrug to rat lens culture medium devoid of L-cystine and L-methionine but containing [14C(U)]-glycine. After a 48-hour period of incubation, extracts of rat lenses were prepared for separation of [14C]-glutathione by high-performance liquid chromatography (HPLC) with a radioisotope detector to determine the extent of its biosynthesis. Cytochrome P-450 isozymes were induced in C57 bl/6 mice by either beta-naphthoflavone or phenobarbital. Cataracts were induced by administration of either acetaminophen or naphthalene to the pretreated mice. CySSME was coadministered with either acetaminophen or naphthalene to other groups of mice. Both oxidized and reduced glutathione were determined in extracts of livers and lenses using the HPLC method above. RESULTS: CySSME served as an effective L-cysteine precursor for glutathione biosynthesis in cultured rat lenses. This L-Cysteine prodrug was also highly effective in preventing acetaminophen- and naphthalene-induced cataract in mice and in maintaining near-normal glutathione levels in lenses and livers of such treated animals. CONCLUSIONS: This investigation demonstrates that maintenance of adequate physiological levels of glutathione in the presence of specific known cataractogenic agents by pharmacologic intervention with CySSME, an L-cysteine prodrug, is sufficient to prevent cataract formation.

Protection against acetaminophen-induced hepatotoxicity by L-CySSME and its N-acetyl and ethyl ester derivatives.

We have recently observed that S-(2-hydroxyethylmercapto)-L-cysteine (L-CySSME), the mixed disulfide of L-cysteine and 2-mercaptoethanol, prevented cataracts induced in mice by acetaminophen (ACP) by functioning as a prodrug of L-cysteine and protecting the liver. This prompted the evaluation of the more lipophilic N-acetyl (Ac-CySSME) and ethyl ester (Et-CySSME) derivatives of L-CySSME as proprodrug forms, as well as the "D" enantiomer, as hepatoprotective agents. Serum ALT levels were measured at 24 hours after a toxic but nonlethal dose of ACP that insured 48 hour survival of the animals. Since the increases in ALT produced were highly variable (even after log transformation) and complicated the statistical analyses, we calculated confidence intervals for the mean ALT levels for each treatment group. This enabled comparisons to be made of the efficacy of L-CySSME as well as Ac-CySSME and Et-CySSME with other representative prodrugs of L-cysteine, namely, 2(RS)-methylthiazolidine-4(R)-carboxylic acid (MTCA), L-2-oxothiazolidine-4-carboxylic acid (OTCA), and N-acetyl-L-cysteine (NAC), in protecting the liver. It was shown that L-CySSME and MTCA administered intraperitoneally at 2.5 mmol/kg were superior to the other cysteine prodrugs at equimolar doses in protecting mice from hepatotoxicity elicited by a 400 mg/kg (2.65 mmol/kg) dose of ACP given i.p. 30 minutes prior to the prodrugs. The "D" form of CySSME was totally without protective effect. Oral doses of the prodrugs even at 2x the i.p. dose were less effective, although MTCA was the most protective.

Influence on lenticular glutathione research.

Otto Hockwin influenced the early research career in ophthalmic biochemistry of William Rathbun in several ways. These included multiple exposures via symposia to the European research community, providing editorial experience and encouragement to expand fundamental glutathione enzyme research to the problems of aging, species variation and anti-cataract drug development.

The effects of age on glutathione peroxidase and glutathione reductase activities in lenses of Old World simians and prosimians.

The effects of age on the activities of the two enzymes of the glutathione redox cycle, glutathione peroxidase and glutathione reductase, were studied in lenses of primates. Three species were Old World simians (orangutan, olive baboon and pigtail monkey) and two were prosimians (galago and mouse lemur). Glutathione peroxidase activity of the olive baboon lens increased steadily with age while that of the pigtail monkey increased during the first 6-8 years and then plateaued. Enzyme activity in the orangutan increased steadily from 9 months to 28 years. This enzyme activity decreased steadily in the galago but increased only slightly in the mouse lemur lens. The lenticular glutathione reductase activity profiles showed decreases with age for all three simian species. Enzyme activity in the galago decreased gradually from birth to the age of 16.5 years. The enzyme activity values of the mouse lemur lens did not yield a comprehensible pattern. Lens weight increased with age in all five primate species, particularly in the infant and juvenile years and leveled off in adulthood. The current investigation demonstrated that the responses of these enzyme activities to aging were very different in Old World simians as compared to prosimians. These studies are consistent with earlier enzyme activity and thermolability data and are indicative of probable critical differences in the primary structures of the enzymes between the two primate groups.

Activity loss of glutathione synthesis enzymes associated with human subcapsular cataract.

PURPOSE: To assess the activities of the two enzymes required for glutathione synthesis, gamma-glutamylcysteine synthetase and glutathione synthetase, in various forms of human cataracts. METHODS: The Cooperative Cataract Research Group cataract classification method and standard enzyme assay procedures were used. RESULTS: An inverse relationship was shown between residual activity of each of the glutathione synthesis enzymes and degree of subcapsular cataract. A weaker inverse relationship existed between glutathione synthetase activity and supranuclear and nuclear cataracts. No other parameters yielded comparable correlations with the activity of either enzyme. CONCLUSIONS: Activity loss of the glutathione synthesis enzymes is associated with human subcapsular cataract formation.

The effects of age on glutathione synthesis enzymes in lenses of Old World simians and prosimians.

The activities of gamma-glutamylcysteine synthetase and glutathione synthetase, the two enzymes required for glutathione synthesis, were determined as a function of age in lenses of three species of Old World higher primates: orangutan, pigtail monkey and olive baboon. These were compared to enzyme activities in lenses of two prosimians: mouse lemur and galago. gamma-Glutamylcysteine synthetase activity decreased as a function of age in all three Old World simians. The rate of decrease was greatest in the juvenile lenses. In contrast, the enzyme activity increased continuously with age in the galago lens. In the mouse lemur the enzyme activity increased per lens, but was constant when expressed as specific activity or as units per gram of lens. The loss of enzyme activity with age was limited to Old World higher primates apparently representing genetic change. Glutathione synthetase activity decreased logarithmically with age in the lenses of all five species.

Age-related cysteine uptake as rate-limiting in glutathione synthesis and glutathione half-life in the cultured human lens.

The study included human lenses of ages ranging from newborn to 92 years. Protein-free reduced glutathione decreased 14-fold, whereas protein-free oxidized glutathione increased 2.6-fold with increasing age. L-Cyst(e)ine uptake g-1 lens of very old cultured lenses decreased 70% from that exhibited in newborn lenses, demonstrating a marked decline of L-cyst(e)ine uptake as a function of age. In these same lenses the synthesis of reduced glutathione (mumol g-1 lens) decreased 73% with age. It was concluded that the glutathione decrease observed in the aging human lens was associated with decreased uptake of L-cyst(e)ine, decreased glutathione synthesis and possibly an increase in protein-free oxidized glutathione. The high correlation of L-cyst(e)ine uptake and glutathione synthesis supports the hypothesis that L-cyst(e)ine uptake is a rate-limiting factor of glutathione synthesis in the intact human lens. By the use of buthionine sulfoximine, the half-life of glutathione was estimated to be 90 hr in the cultured human lens.

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.

Naphthalene-induced cataract in the rat. II. Contrasting effects of two aldose reductase inhibitors on glutathione and glutathione redox enzymes.

This investigation compared the effects of two types of aldose reductase inhibitors on several biochemical parameters in naphthalene-induced cataract of the rat over a time span of 102 days of treatment. Feeding of naphthalene daily to brown Norway rats resulted in gradual, progressive development of zonular opacities. As compared to control animals, the values of soluble protein, soluble glutathione (total of oxidized plus reduced) and activities of glutathione peroxidase and glutathione reductase were decreased in rats fed either naphthalene or naphthalene + FK366, a carboxylic-acid-type aldose reductase inhibitor. In marked contrast, treatment with A11576, a hydantoin-type aldose reductase inhibitor, maintained the values of most parameters (with one exception) at levels that were similar to those of the controls, and all lenses remained clear. A decline of glutathione was noted in all naphthalene-fed rats, irrespective of whether these animals had been treated with an aldose reductase inhibitor. The great decrease of glutathione with A11576 suggests that this inhibitor acts at some step in naphthalene metabolism following formation of naphthalene epoxide.

Cysteine and glutathione metabolism in organ-cultured rat corneas.

The capability of organ-cultured rat corneas to metabolize cysteine and synthesize glutathione was assessed by use of radioactive L-cyst(e)ine. Metabolites from protein-free tissue extracts were separated and quantified by HPLC, using radioisotope and ultraviolet detectors. Most of the radioactivity detected in the rat cornea was found in three areas of the HPLC elution profile: 1) reduced glutathione, 2) cystine and 3) in a rapidly eluting area (the 'five-minute' area) consisting of several unidentified peaks. The proportion of radioactivity found within the five-minute area increased with time of incubation and became the dominant radioactive peak area by 48 hours of incubation. In contrast to rat lens extracts, the synthesis of glutathione in rat cornea formed a minor portion of the L-cysteine metabolic products. The unlabeled reduced glutathione concentration was relatively stable over a 48-hour incubation period. Synthesis of glutathione was prevented by 0.3 mM buthionine sulfoximine, resulting in more than 90% of the radioactivity being contained in the five-minute peak area. Inhibition of glutathione synthesis allowed the estimation of glutathione's half-life to approximate 10.5 hours in the organ-cultured rat cornea.

Amino acid sequence from degu islet amyloid-derived insulin shows unique sequence characteristics.

The main protein of enriched and purified amyloid from Octodon degus pancreatic islets was identified as insulin. The material was reduced and alkylated and the A- and the B-chain were separated by reversed phase chromatography and subjected to Edman degradation and amino acid analysis. It was shown that the A-chain contains two additional C-terminal amino acid residues (i.e. a total of 23 residues) and that the B-chain has a deletion in the C-terminal part (i.e. a total of 29 residues). The obtained sequence follows: A-chain: GIVDQCCNNICTFNQLQNYCNVP B-chain: YSSQHLCGSNLVEALYMTCGRSGFYRPHD.

Conditions for maximizing and inhibiting synthesis of glutathione in cultured rat lenses: an application of HPLC with radioisotope detection.

This investigation was concerned with factors which would maximize and inhibit L-cyst(e)ine uptake and glutathione synthesis of rat lenses cultured for 24 hours in a medium containing [35S]-L-cystine. The lenticular protein-free extract was separated and quantified by use of HPLC equipment which included an in-line radioisotope detector coupled with extensive post-run computer analysis. Six thiols were assessed for their ability to increase the uptake of L-cyst(e)ine and its utilization for glutathione synthesis. The most successful was 2-mercaptoethanol, which increased the L-cyst(e)ine uptake 3.6-fold and glutathione synthesis 2.9-fold. This work demonstrated that the rate of glutathione synthesis was directly proportional to the rate of uptake of L-cyst(e)ine. An inhibitor of glutathione synthesis, buthionine sulfoximine, in a concentration range of 0.11-3 mM, decreased uptake of the [35S]-label by one-third. The 3.0 mM concentration inhibited glutathione synthesis 98.7% and decreased glutathione 51% in 24 hours. The data indicate that half the glutathione disappeared in 23 hours in these cultured rat lenses. Because determination of this value did not depend upon the variable rate of cysteine transport, the value may approximate the in vivo value.

Glutathione synthesis and glutathione redox pathways in naphthalene cataract of the rat.

This investigation examined many parameters during the course of early development of naphthalene-induced cataract in a time span of 0 to 79 days of treatment. Feeding naphthalene daily to Black-Hooded rats resulted in gradual progressive development of cataract. The first faint opacities were detectable after 7 days. Free soluble total glutathione (oxidized and reduced) of these lenses was shown to gradually decrease to a maximum loss of about 20%, a value reached by day 30 of treatment. No activity loss of either enzyme required for glutathione synthesis (gamma-glutamylcysteine synthetase or glutathione synthetase) was observed in homogenates of naphthalene versus control lenses. There was also neither impairment of [35S]-L-cystine uptake nor of [35S]-glutathione synthetic capacity in lenses cultured from rats after 12, 24 or 36 days of naphthalene feeding when compared to control lenses. Hence, glutathione loss cannot be explained by a damaged glutathione synthesis system. Progressive activity loss of glutathione peroxidase and glutathione reductase was observed. The loss of glutathione peroxidase activity was especially remarkable. Thus, the defense system against oxidative damage is impaired and may be a significant factor in naphthalene-induced cataract of the rat.

Inhibition of Na,K-ATPase by sodium selenite and reversal by glutathione.

Sodium selenite has been shown to inhibit Na,K-ATPase. Glutathione, at sufficient excess, is able to prevent or reverse the inhibition. Dithiothreitol can also reverse much of the inhibition, but KCN cannot. Selenomethionine does not inhibit Na,K-ATPase. The interactions of sodium selenite with Na,K-ATPase and glutathione may aid in understanding the early events in selenium cataractogenesis.

Activity of glutathione peroxidase and glutathione reductase in the human lens related to age.

Lenses from 42 eye bank eyes were assayed for glutathione peroxidase and glutathione reductase activities. The activity of glutathione peroxidase, when considered as a function of age, was lowest in the neonate lens, increasing with age to reach maximal values in young adult lenses, and thereafter progressively decreasing with ages greater than 40 years. Glutathione reductase activity was little affected by age when expressed as activity per lens, per gram lens or per mg soluble protein, indicating that activity of this enzyme did not increase with lens size as would a representative lenticular protein. However, the activity of this enzyme per gram lens was among the highest of any species yet examined.

Glutathione metabolism in lenses of Emory and cataract-resistant mice: activity of five enzymes.

The activities of five enzymes of glutathione metabolism were determined in lenses from cataract-resistant and cataract-prone (Emory) mouse variants at three different ages (5 weeks, 10 weeks and 6 months). The enzymes included those required for glutathione synthesis, gamma-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione-S-transferase, glutathione peroxidase and glutathione reductase. The differences in the activities of the five enzymes in the two mouse variants were not remarkable at any of the three ages. Activity of each enzyme was noted to be in excess of the preceding one in this integrated metabolic pathway, with the exception of glutathione reductase. gamma-Glutamylcysteine synthetase appears to be the pacesetting enzyme of this metabolic scheme in the mouse lens. The activity of each enzyme was compared with that earlier reported for human, rabbit and dog lenses.