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.

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.

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. Method.

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.

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.

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.

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.

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.

Glutathione metabolism in primate lenses: A phylogenetic study of glutathione synthesis and glutathione redox cycle enzyme activities.

Lens wet weights, soluble protein, and activities of γ-glutiamylcysteine synthetase, glutathione synthetase, glutathione peroxidase, and glutathione reductase were determined in primate lenses. The primary sources of lenses were middle-aged adult animals. The Primates, from 23 genera, were categorized into six superfamilies: hominoids (five species), Old World monkeys (seven species), New World monkeys (five species), tarsiers (two species), lemurs (six species), and lorisids (three species).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Glutathione peroxidase, glutathione reductase and glutathione-S-transferase activities in the rhesus monkey lens as a function of age.

The activities of glutathione peroxidase, glutathione reductase and glutathione-S-transferase were determined in lenses from rhesus monkeys (Macaca mulatta) as a function of age. The ages ranged from 137 day old embryos to a 34 year old. Glutathione peroxidase activity (units/g lens) occurred at a very low level in lenses of fetuses and neonates, but increased dramatically with age, peaking in the adult of about 12 to 20 years of age and declining thereafter.

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 glutathione synthesis enzymes in the rhesus monkey lens related to age: a model for the human lens.

The low activity level of lenticular gamma-glutamylcysteine synthetase appears to be an evolutionary phenomenon restricted to higher primates. Rapid reduction with age of the activity of both enzymes (gamma-glutamylcysteine synthetase and glutathione synthetase) required for glutathione synthesis in the human lens was demonstrated in an earlier study. The activities of gamma-glutamylcysteine synthetase and glutathione synthetase, the two enzymes responsible for glutathione synthesis, were determined in 39 lenses from the rhesus monkey (Macaca mulatta) as a function of age.

Species survey of glutathione peroxidase and glutathione reductase: search for an animal model of the human lens.

Lenses from representative species of eight mammalian orders were assayed for glutathione peroxidase and glutathione reductase activities. Wide variation of glutathione peroxidase activity was noted, the lowest activity being that of the prosimian galago while the highest activities were from three Old World monkey species of the genera Macaca and Papio. The hominoids, including the human, all exhibited lower activities of this enzyme.