Development of a HPLC-MS/MS method for assessment of thiol redox status in human tear fluids.

Oxidative stress is reported to be part of the pathology of many ocular diseases. For the diagnosis of ocular diseases, tear fluid has unique advantages. Although numerous analytical methods exist for the measurement of different types of biomolecules in tear fluid, few have been reported for comprehensive understanding of oxidative stress-related thiol redox signaling. In this study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to determine a panel of twelve metabolites that systematically covered several thiol metabolic pathways. With optimization of MS/MS parameters and HPLC mobile phases, this method was sensitive (LOQ as low as 0.01 ng/ml), accurate (80-125% spike recovery) and precise (<10% RSD). This LC-MS/MS method combined with a simple tear fluid collection with Schirmer test strip followed by ultrafiltration allowed the high-throughput analysis for efficient determination of metabolites associated with thiol redox signaling in human tear fluids. The method was then applied to a small cohort of tear fluids obtained from healthy individuals. The method presented here provides a new technique to facilitate future work aiming to determine the complex thiol redox signaling in tear fluids for accurate assessment and diagnosis of ocular diseases.

Continuous measurement of reactive oxygen species inside and outside of a residential house during summer.

Reactive oxygen species (ROS) are an important contributor to adverse health effects associated with ambient air pollution. Despite infiltration of ROS from outdoors, and possible indoor sources (eg, combustion), there are limited data available on indoor ROS. In this study, part of the second phase of Air Composition and Reactivity from Outdoor aNd Indoor Mixing campaign (ACRONIM-2), we constructed and deployed an online, continuous, system to measure extracellular gas- and particle-phase ROS during summer in an unoccupied residence in St. Louis, MO, USA. Over a period of one week, we observed that the non-denuded outdoor ROS (representing particle-phase ROS and some gas-phase ROS) concentration ranged from 1 to 4 nmol/m3 (as H2 O2 ). Outdoor concentrations were highest in the afternoon, coincident with peak photochemistry periods. The indoor concentrations of particle-phase ROS were nearly equal to outdoor concentrations, regardless of window-opening status or air exchange rates. The indoor/outdoor ratio of non-denuded ROS (I/OROS ) was significantly less than 1 with windows open and even lower with windows closed. Combined, these observations suggest that gas-phase ROS are efficiently removed by interior building surfaces and that there may be an indoor source of particle-phase ROS.

Simultaneous determination of tiopronin and its primary metabolite in plasma and ocular tissues by HPLC.

Tiopronin, formally 2-mercaptopropionylglycine (MPG), is currently prescribed to treat cystinuria and rheumatoid arthritis, and its antioxidant properties have led to its investigation as a treatment for cataracts, a condition in which oxidative stress is strongly implicated. To study its accumulation in the eye, a reliable, isocratic HPLC method was developed for the determination of MPG and its primary metabolite 2-mercaptopropionic acid (MPA) in plasma and relevant ocular tissues. This method utilizes pre-column derivatization and fluorescence detection. The 3.5 min separation enables high-throughput analysis, and validation experiments demonstrated that this method is suitable for evaluating ocular accumulation of MPG and MPA at concentrations as low as 66 and 33 nm, respectively. Excellent linearity was achieved over the working concentration range with R2 > 0.997. Extraction recovery was reproducible within each matrix and exceeded 97%. Accuracy was within 13.3% relative error, and intra- and inter-day precisions were within 6% CV and 7% CV, respectively. Sample stability was demonstrated under various storage conditions, and the use of an internal standard conferred exceptional ruggedness. This method has been successfully applied for the determination of MPG and MPA in plasma, cornea, lens and retina following intraperitoneal administration of the drug in Wistar rats.

Effect of Nanodiamond Surface Chemistry on Adsorption and Release of Tiopronin.

Tiopronin is an FDA-approved thiol drug currently used to treat cystinuria and rheumatoid arthritis. However, due to its antioxidant properties, it may be beneficial in a variety of other conditions. One primary obstacle to its wider application is its limited bioavailability, which necessitates administration of high systemic doses to achieve localized therapeutic effects. Incorporation of a drug delivery vehicle can solve this dilemma by providing a means of controlled, targeted release. Functionalized nanodiamond is a promising theranostic platform that has demonstrated great potential for biomedical applications, including drug delivery. Design of nanodiamond theranostic platforms requires comprehensive understanding of drug-platform interactions, and the necessary physical chemical investigations have only been realized for a limited number of compounds. Towards the long-term goal of developing a nanodiamond-tiopronin treatment paradigm, this study aims to shed light on the effects of nanodiamond surface chemistry on adsorption and release of tiopronin. Specifically, adsorption isotherms were measured and fit to Langmuir and Freundlich models for carboxylated, hydroxylated, and aminated nanodiamonds, and release was monitored in solutions at pH 4.0, 5.8, 7.3, and 8.1. Our results indicate that aminated nanodiamonds exhibit the highest loading capacity while hydroxylated nanodiamonds are the most effective for sustained release. Therefore, a high degree of flexibility may be afforded by the use of nanodiamonds with different surface chemistries optimized for specific applications.

Prevention and reversal of selenite-induced cataracts by N-acetylcysteine amide in Wistar rats.

BACKGROUND: The present study sought to evaluate the efficacy of N-acetylcysteine amide (NACA) eye drops in reversing the cataract formation induced by sodium selenite in male Wistar rat pups. METHODS: Forty male Wistar rat pups were randomly divided into a control group, an N-acetylcysteine amide-only group, a sodium selenite-induced cataract group, and a NACA-treated sodium selenite-induced cataract group. Sodium selenite was injected intraperitoneally on postpartum day 10, whereas N-acetylcysteine amide was injected intraperitoneally on postpartum days 9, 11, and 13 in the respective groups. Cataracts were evaluated at the end of week 2 (postpartum day 14) when the rat pups opened their eyes. N-acetylcysteine amide eye drops were administered beginning on week 3 until the end of week 4 (postpartum days 15 to 30), and the rats were sacrificed at the end of week 4. Lenses were isolated and examined for oxidative stress parameters such as glutathione, lipid peroxidation, and calcium levels along with the glutathione reductase and thioltransferase enzyme activities. Casein zymography and Western blot of m-calpain were performed using the water soluble fraction of lens proteins. RESULTS: Morphological examination of the lenses in the NACA-treated group indicated that NACA was able to reverse the cataract grade. In addition, glutathione level, thioltransferase activity, m-calpain activity, and m-calpain level (as assessed by Western blot) were all significantly higher in the NACA-treated group than in the sodium selenite-induced cataract group. Furthermore, sodium selenite- injected rat pups had significantly higher levels of malondialdehyde, glutathione reductase enzyme activity, and calcium levels, which were reduced to control levels upon treatment with NACA. CONCLUSIONS: The data suggest that NACA has the potential to significantly improve vision and decrease the burden of cataract-related loss of function. Prevention and reversal of cataract formation could have a global impact. Development of pharmacological agents like NACA may eventually prevent cataract formation in high-risk populations and may prevent progression of early-stage cataracts. This brings a paradigm shift from expensive surgical treatment of cataracts to relatively inexpensive prevention of vision loss.

Antioxidant potential of Sutherlandia frutescens and its protective effects against oxidative stress in various cell cultures.

BACKGROUND: Sutherlandia frutescens (L.) R.Br. (SF) is a South African plant that is widely used to treat stress, infections, cancer, and chronic diseases, many of which involve oxidative stress. The aim of the study was to quantitatively assess the antioxidant potential of SF extracts in cell-free system as well as in cell lines. METHODS: Dried SF vegetative parts were extracted using six different solvents, and the extracts were assessed for total phenolic and flavonoid contents, total reducing power, iron chelating capacity, and free radical scavenging power, including, scavenging of hydroxyl radicals, superoxide anions, nitric oxide, and hydrogen peroxide. We further investigated the freeze-dried hot water extract of SF (SFE) to assess its effect against oxidative stress induced by tert-butyl hydroperoxide (t-BHP), an organic peroxide. Three different cell lines: Chinese hamster ovary (CHO), human hepatoma (HepaRG), and human pulmonary alveolar carcinoma (A549) cells, were employed to determine cell viability, intracellular reactive oxygen species (ROS) levels, and reduced to oxidized glutathione levels (GSH/GSSG). RESULTS: The results indicated that: (1) SF extracts have significant antioxidant potential that is dependent upon the nature of the extraction solvent and (2) SFE protects against tBHP-induced oxidative stress in cells by scavenging ROS and preserving intracellular GSH/GSSG. CONCLUSION: Oxidative stress is implicated in a number of disorders, and due to the public's concerns about synthetic antioxidants, various natural antioxidants are being explored for their therapeutic potential. Our findings support claims for S. frutescens being a promising adjunctive therapeutic for oxidative stress-related health problems.

Pharmacological inhibition of mitochondrial carbonic anhydrases protects mouse cerebral pericytes from high glucose-induced oxidative stress and apoptosis.

Diabetes-associated complications in the microvasculature of the brain are caused by oxidative stress, generated by overproduction of reactive oxygen species from hyperglycemia-induced accelerated oxidative metabolism of glucose. Pericytes, essential for the viability of the microvasculature, are especially susceptible to oxidative stress. Mitochondrial carbonic anhydrases, regulators of the oxidative metabolism of glucose, determine the rate of reactive oxygen species production and inhibition of mitochondrial carbonic anhydrases rescues glucose-induced pericyte loss in the diabetic mouse brain. We hypothesized that high glucose induces intracellular oxidative stress and pericyte apoptosis and that inhibition of mitochondrial carbonic anhydrases protects pericytes from oxidative stress-induced apoptosis. To validate our hypothesis, conditionally immortalized cerebral pericyte (IPC) cultures were established from Immortomice to investigate the effect of high glucose on oxidative stress and pericyte apoptosis. The IPCs expressed pericyte markers and induced high transendothelial electrical resistance and low permeability in brain endothelial cell monolayers comparable with pericytes in primary cultures. The IPCs also secreted cytokines constitutively and in response to lipopolysaccharide similar to pericytes. High glucose caused oxidative stress and apoptosis of these cells, with both oxidative stress and apoptosis significantly reduced after mitochondrial carbonic anhydrase inhibition. These results provide the first evidence that pharmacological inhibition of mitochondrial carbonic anhydrases attenuates pericyte apoptosis caused by high glucose-induced oxidative stress. Carbonic anhydrase inhibitors have a long history of safe clinical use and can be immediately evaluated for this new indication in translational research. Thus, mitochondrial carbonic anhydrases may provide a new therapeutic target for oxidative stress-related illnesses of the brain.

Effects of a growth hormone-releasing hormone antagonist on telomerase activity, oxidative stress, longevity, and aging in mice.

Both deficiency and excess of growth hormone (GH) are associated with increased mortality and morbidity. GH replacement in otherwise healthy subjects leads to complications, whereas individuals with isolated GH deficiency such as Laron dwarfs show increased life span. Here, we determined the effects of treatment with the GH-releasing hormone (GHRH) receptor antagonist MZ-5-156 on aging in SAMP8 mice, a strain that develops with aging cognitive deficits and has a shortened life expectancy. Starting at age 10 mo, mice received daily s.c. injections of 10 µg/mouse of MZ-5-156. Mice treated for 4 mo with MZ-5-156 showed increased telomerase activity, improvement in some measures of oxidative stress in brain, and improved pole balance, but no change in muscle strength. MZ-5-156 improved cognition after 2 mo and 4 mo, but not after 7 mo of treatment (ages 12, 14 mo, and 17 mo, respectively). Mean life expectancy increased by 8 wk with no increase in maximal life span, and tumor incidence decreased from 10 to 1.7%. These results show that treatment with a GHRH antagonist has positive effects on some aspects of aging, including an increase in telomerase activity.

Release of N-acetylcysteine and N-acetylcysteine amide from contact lenses.

OBJECTIVE: To investigate the use of contact lenses to release N-acetylcysteine (NAC) and N-acetylcysteine amide (NACA) that have frequently used for the treatment of some eye diseases. METHODS: Three commercial contact lenses were used: Soflens Multi-Focal, 1-Day ACUVUE TruEye, and Frequency 55. All contact lenses were individually kept for 3 days in 10 mL of 3 mM NAC or NACA solutions in phosphate-buffered saline (PBS). After the loading period, the lenses were removed from the solution and put into 5 mL of PBS for 3 days (static mode). During this period, samples were taken at specified times and analyzed by high-pressure liquid chromatography. RESULTS: From the release profiles of NAC and NACA, it was found that both NAC and NACA could be released from the lenses within 72 hours. Frequency 55 released 95.9%±2.7% of loaded NAC and 60.0%±2.1% of loaded NACA in 24 hours, whereas 1-Day ACUVUE TruEye released 80.9%±1.2% of loaded NAC and 54.0%±1.9% of loaded NACA and Soflens Multi-Focal released 72.8%±2.8% of loaded NAC and 51.9%±2.3% of loaded NACA during that same period. CONCLUSIONS: The lenses could achieve the appropriate delivery of drugs during their intended time of wear. The amount of released NACA was less than that of NAC because of the more hydrophobic structure of NACA. According to the power law, the values of the exponential constant n were found to be below 0.5, indicating that the behavior observed was "less Fickian".

N-acetylcysteine amide (NACA) prevents retinal degeneration by up-regulating reduced glutathione production and reversing lipid peroxidation.

Oxidative stress plays a critical role in accelerating retinal pigment epithelial dysfunction and death in degenerative retinal diseases, including age-related macular degeneration. Given the key role of oxidative stress-induced retinal pigment epithelial cell death and secondary photoreceptor loss in the pathogenesis of age-related macular degeneration, we hypothesized that a novel thiol antioxidant, N-acetylcysteine amide (NACA), might ameliorate cellular damage and subsequent loss of vision. Treatment of human retinal pigment epithelial cells with NACA protected against oxidative stress-induced cellular injury and death. NACA acted mechanistically by scavenging existing reactive oxygen species while halting production of reactive oxygen species by reversing lipid peroxidation. Furthermore, NACA functioned by increasing the levels of reduced glutathione and the phase II detoxification enzyme glutathione peroxidase. Treatment of mice exposed to phototoxic doses of light with NACA maintained retinal pigment epithelial cell integrity and prevented outer nuclear layer cell death as examined by histopathologic methods and rescued photoreceptor function as measured by electroretinography. These observations indicate that NACA protects against oxidative stress-induced retinal pigment epithelial and photoreceptor cell death in vitro and in vivo. The data suggest that NACA may be a novel treatment in rescuing retinal function and preventing vision loss secondary to retinal degenerative diseases, including age-related macular degeneration.

Thiol antioxidants protect human lens epithelial (HLE B-3) cells against tert-butyl hydroperoxide-induced oxidative damage and cytotoxicity.

Oxidative damage to lens epithelial cells plays an important role in the development of age-related cataract, and the health of the lens has important implications for overall ocular health. As a result, there is a need for effective therapeutic agents that prevent oxidative damage to the lens. Thiol antioxidants such as tiopronin or N-(2-mercaptopropionyl)glycine (MPG), N-acetylcysteine amide (NACA), N-acetylcysteine (NAC), and exogenous glutathione (GSH) may be promising candidates for this purpose, but their ability to protect lens epithelial cells is not well understood. The effectiveness of these compounds was compared by exposing human lens epithelial cells (HLE B-3) to the chemical oxidant tert-butyl hydroperoxide (tBHP) and treating the cells with each of the antioxidant compounds. MTT cell viability, apoptosis, reactive oxygen species (ROS), and levels of intracellular GSH, the most important antioxidant in the lens, were measured after treatment. All four compounds provided some degree of protection against tBHP-induced oxidative stress and cytotoxicity. Cells treated with NACA exhibited the highest viability after exposure to tBHP, as well as decreased ROS and increased intracellular GSH. Exogenous GSH also preserved viability and increased intracellular GSH levels. MPG scavenged significant amounts of ROS, and NAC increased intracellular GSH levels. Our results suggest that both scavenging ROS and increasing GSH may be necessary for effective protection of lens epithelial cells. Further, the compounds tested may be useful for the development of therapeutic strategies that aim to prevent oxidative damage to the lens.

Glutathione is essential for early embryogenesis--analysis of a glutathione synthetase knockout mouse.

Glutathione (GSH) is present in all mammalian tissues and plays a crucial role in many cellular processes. The second and final step in the synthesis involves the formation of GSH from gamma-glutamylcysteine (γ-GC) and glycine and is catalyzed by glutathione synthetase (GS). GS deficiency is a rare autosomal recessive disorder, and is present in patients with a range of phenotypes, from mild hemolytic anemia and metabolic acidosis to severe neurologic disorders or even death in infancy. The substrate for GS, γ-GC, has been suggested as playing a protective role, by substituting for GSH as an antioxidant in GS deficient patients. To examine the role of GS and GSH metabolites in development, we generated mice deficient in GSH by targeted disruption of the GS gene (Gss). Homozygous mice died before embryonic day (E) 7.5, but heterozygous mice survived with no distinct phenotype. GS protein levels and enzyme activity, as well as GSH metabolites, were investigated in multiple tissues. Protein levels and enzyme activity of GS in heterozygous mice were diminished by 50%, while GSH levels remained intact. γ-GC could not be detected in any investigated tissue. These data demonstrate that GSH is essential for mammalian development, and GSH synthesis via GS is an indispensable pathway for survival.

Extensive Thiol Profiling for Assessment of Intracellular Redox Status in Cultured Cells by HPLC-MS/MS.

Oxidative stress may contribute to the pathology of many diseases, and endogenous thiols, especially glutathione (GSH) and its metabolites, play essential roles in the maintenance of normal redox status. Understanding how these metabolites change in response to oxidative insult can provide key insights into potential methods of prevention and treatment. Most existing methodologies focus only on the GSH/GSH disulfide (GSSG) redox couple, but GSH regulation is highly complex and depends on several pathways with multiple redox-active sulfur-containing species. In order to more fully characterize thiol redox status in response to oxidative insult, a high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method was developed to simultaneously determine seven sulfur-containing metabolites, generating a panel that systematically examines several pathways involved in thiol metabolism and oxidative stress responses. The sensitivity (LOQ as low as 0.01 ng/mL), accuracy (88-126% spike recovery), and precision (≤12% RSD) were comparable or superior to those of existing methods. Additionally, the method was used to compare the baseline thiol profiles and oxidative stress responses of cell lines derived from different tissues. The results revealed a previously unreported response to oxidative stress in lens epithelial (B3) cells, which may be exploited as a new therapeutic target for oxidative-stress-related ocular diseases. Further application of this method may uncover new pathways involved in oxidative-stress-related diseases and endogenous defense mechanisms.

Genotype, Age, Genetic Background, and Sex Influence Epha2-Related Cataract Development in Mice.

Purpose: Age-related cataract is the leading cause of blindness worldwide. Variants in the EPHA2 gene increase the disease risk, and its knockout in mice causes cataract. We investigated whether age, sex, and genetic background, risk factors for age-related cataract, and Epha2 genotype influence Epha2-related cataract development in mice. Methods: Cataract development was monitored in Epha2+/+, Epha2+/-, and Epha2-/- mice (Epha2Gt(KST085)Byg) on C57BL/6J and FVB:C57BL/6J (50:50) backgrounds. Cellular architecture of lenses, endoplasmic reticulum (ER) stress, and redox state were determined using histological, molecular, and analytical techniques. Results: Epha2-/- and Epha2+/- mice on C57BL/6J background developed severe cortical cataracts by 18 and 38 weeks of age, respectively, compared to development of similar cataract significantly later in Epha2-/- mice and no cataract in Epha2+/- mice in this strain on FVB background, which was previously reported. On FVB:C57BL/6J background, Epha2-/- mice developed severe cortical cataract by 38 weeks and Epha2+/- mice exhibited mild cortical cataract up to 64 weeks of age. Progression of cataract in Epha2-/- and Epha2+/- female mice on C57BL/6J and mixed background, respectively, was slower than in matched male mice. N-cadherin and ß-catenin immunolabeling showed disorganized lens fiber cells and disruption of lens architecture in Epha2-/- and Epha2+/- lenses, coinciding with development of severe cataracts. EPHA2 immunolabeling showed intracellular accumulation of the mutant EPHA2-ß-galactosidase fusion protein that induced a cytoprotective ER stress response and in Epha2+/- lenses was also accompanied by glutathione redox imbalance. Conclusions: Both, Epha2-/- and Epha2+/- mice develop age-related cortical cataract; age as a function of Epha2 genotype, sex, and genetic background influence Epha2-related cataractogenesis in mice.

Protective effects of tiopronin on oxidatively challenged human lung carcinoma cells (A549).

Tiopronin (MPG) is a thiol antioxidant drug that has been explored as a treatment for various oxidative stress-related disorders. However, many of its antioxidant capabilities remain untested in well-validated cell models. To more thoroughly understand the action of this promising pharmaceutical compound against acute oxidative challenge, A549 human lung carcinoma cells were exposed to tert-butyl hydroperoxide (tBHP) and treated with MPG. Analyses of cell viability, intracellular glutathione (GSH) levels, and the prevalence of reactive oxygen species (ROS) and mitochondrial superoxide were used to examine the effects of MPG on tBHP-challenged cells. MPG treatment suppressed intracellular ROS and mitochondrial superoxide and prevented tBHP-induced GSH depletion and apoptosis. These results indicate that MPG is effective at preserving redox homeostasis against acute oxidative insult in A549 cells if present at sufficient concentrations during exposure to oxidants such as tBHP. The effects of treatment gleaned from this study can inform experimental design for future in vivo work on the therapeutic potential of MPG.

Postharvest application of thiol compounds affects surface browning and antioxidant activity of fresh-cut potatoes.

The aim of this study was to compare the effects of sodium metabisulphite and the thiol compounds, glutathione (GSH), L-cysteine (CYS), and N-acetylcysteine (NAC), on the enzymatic browning, antioxidant activities, total phenolic, and ascorbic acid content of potatoes after 1, 24, and 48 hr. Three different concentrations (0.5%, 1.0%, and 2.0%) of each thiol compound were tested. While sulphite solution inhibited polyphenol oxidase as expected, NAC and CYS also decreased its activity. CYS-treated samples exhibited the highest residual thiol content, while the amount of residual thiol in GSH-treated samples was the lowest. The 2.0% NAC and 2.0% CYS solutions were the most effective at increasing antioxidant activity and ascorbic acid content; however, the results of total phenolic content assays were complicated. In summary, solutions containing 2.0% NAC, 1.0% CYS, and 2.0% CYS prevented enzymatic browning and increased the residual thiol content, ascorbic acid, and antioxidant activities of fresh-cut potatoes significantly, but GSH did not significantly inhibit browning. PRACTICAL APPLICATIONS: Fresh-cut potatoes are susceptible to enzymatic browning, which significantly reduces their commercial value. In literature, there have been several methods to protect the enzymatic browning of fruits and vegetables. Among these methods, thiols are good inhibitors of enzymatic browning. So, GSH, CYS, and NAC were used in this study. The outcomes of current work may help to inhibit polyphenol oxidase activity and increase the ascorbic acid content, residual thiol content, and antioxidant activity of fresh-cut potatoes. Both CYS and NAC may be useful alternatives to sulphite anti-browning agents, which may have adverse health effects.

Medicinal Thiols: Current Status and New Perspectives.

The thiol (-SH) functional group is found in a number of drug compounds and confers a unique combination of useful properties. Thiol-containing drugs can reduce radicals and other toxic electrophiles, restore cellular thiol pools, and form stable complexes with heavy metals such as lead, arsenic, and copper. Thus, thiols can treat a variety of conditions by serving as radical scavengers, GSH prodrugs, or metal chelators. Many of the compounds discussed here have been in use for decades, yet continued exploration of their properties has yielded new understanding in recent years, which can be used to optimize their clinical application and provide insights into the development of new treatments. The purpose of this narrative review is to highlight the biochemistry of currently used thiol drugs within the context of developments reported in the last five years. More specifically, this review focuses on thiol drugs that represent the standard of care for their associated conditions, including N-acetylcysteine, 2,3-meso-dimercaptosuccinic acid, British anti-Lewisite, D-penicillamine, amifostine, and others. Reports of novel dosing regimens, delivery strategies, and clinical applications for these compounds were examined with an eye toward emerging approaches to address a wide range of medical conditions in the future.

N-acetylcysteineamide (NACA) prevents inflammation and oxidative stress in animals exposed to diesel engine exhaust.

Diesel exhaust particles (DEPs), a by-product of diesel engine exhaust (DEE), are one of the major components of air borne particulate matter (PM) in the urban environment. DEPs are composed of soot, polycyclic aromatic hydrocarbons (PAHs), redox active semi-quinones, and transition metals, which are known to produce pro-oxidative and pro-inflammatory effects, thereby leading to oxidative stress-induced damage in the lungs. The objective of this study was to determine if N-acetylcysteineamide (NACA), a novel thiol antioxidant, confers protection to animals exposed to DEPs from oxidative stress-induced damage to the lung. To study this, male C57BL/6 mice, pretreated with either NACA (250mg/kg body weight) or saline, were exposed to DEPs (15mg/m(3)) or filtered air (1.5-3h/day) for nine consecutive days. The animals were sacrificed 24h after the last exposure. NACA-treated animals exposed to DEP had significant decreases in the number of macrophages and the amount of mucus plug formation in the lungs, as compared to the DEP-only exposed animals. In addition, DEP-exposed animals, pretreated with NACA, also experienced significantly lower oxidative stress than the untreated group, as indicated by the glutathione (GSH), and malondialdehyde (MDA) levels and catalase (CAT) activity. Further, DEP-induced toxicity in the lungs was reversed in NACA-treated animals, as indicated by the lactate dehydrogenase levels. Taken together, these data suggest that the thiol-antioxidant, NACA, can protect the lungs from DEP-induced inflammation and oxidative stress related damage.

N-acetylcysteine amide decreases oxidative stress but not cell death induced by doxorubicin in H9c2 cardiomyocytes.

BACKGROUND: While doxorubicin (DOX) is widely used in cancer chemotherapy, long-term severe cardiotoxicity limits its use. This is the first report of the chemoprotective efficacy of a relatively new thiol antioxidant, N-acetylcysteine amide (NACA), on DOX-induced cell death in cardiomyocytes. We hypothesized that NACA would protect H9c2 cardiomyocytes from DOX-induced toxicity by reducing oxidative stress. Accordingly, we determined the ability of NACA to mitigate the cytotoxicity of DOX in H9c2 cells and correlated these effects with the production of indicators of oxidative stress. RESULTS: DOX at 5 microM induced cardiotoxicity while 1) increasing the generation of reactive oxygen species (ROS), 2) decreasing levels and activities of antioxidants and antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase) and 3) increasing lipid peroxidation. NACA at 750 microM substantially reduced the levels of ROS and lipid peroxidation, as well as increased both GSH level and GSH/GSSG ratio. However, treating H9c2 cells with NACA did little to protect H9c2 cells from DOX-induced cell death. CONCLUSION: Although NACA effectively reduced oxidative stress in DOX-treated H9c2 cells, it had minimal effects on DOX-induced cell death. NACA prevented oxidative stress by elevation of GSH and CYS, reduction of ROS and lipid peroxidation, and restoration of antioxidant enzyme activities. Further studies to identify oxidative stress-independent pathways that lead to DOX-induced cell death in H9c2 are warranted.

Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol.

A reverse-phase HPLC method incorporating dithiothreitol (DTT) reduction for quantitative determination of oxidized glutathione (GSSG) in biological samples is described here. This method is based on our previous enzymatic reduction technique that uses N-1-(pyrenyl) maleimide (NPM) as a derivatizing agent. In our earlier method, glutathione disulfide (GSSG) was measured by first reducing it to GSH with glutathione reductase (GR) in the presence of NADPH. However, this is a very costly and time-consuming technique. The method described here employs a common and inexpensive thiol-disulfide exchanging agent, DTT, for reduction of GSSG to GSH, followed by derivatization with NPM. The calibration curves are linear over a concentration range of 25-1250 nm (r(2) > 0.995). The coefficients of variations for intra-run precision and inter-run precision range from 0.49 to 5.10% with an accuracy range of 1.78-6.15%. The percentage of relative recovery ranges from 97.3 to 103.2%. This new method provides a simple, efficient, and cost-effective way of determining glutathione disulfide levels with a 2.5 nm limit of detection per 5 microL injection volume.