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

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

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

Separation and Quantification of N-acetylcysteine-amide (NACA) by HPLC with Fluorescence Detection

N-acetyl-l-cysteine (NAC) is a well-known antioxidant that is capable of facilitating glutathione (GSH) biosynthesis and replenishing intracellular GSH under oxidatively challenging circumstances. N-acetyl-cysteine-amide (NACA), the amide form of NAC, is a newly designed and synthesized thiol-containing compound which is believed to be more lipophilic and permeable through cell membranes than NAC. The metabolic and antioxidant effects of these compounds in vitro and in vivo are under investigation. However, an analytical method that can separate and quantify both compounds simultaneously is not yet available, to the best of our knowledge. Because of their structural similarities, the two compounds are difficult to separate using earlier HPLC methods which were designed for NAC quantification. Therefore, the goal of this work was to develop an HPLC method with fluorescence detection for simultaneous quantification of NAC and NACA in biological blood and tissue samples. A gradient HPLC program with fluorescence detection (λex = 330 nm, λem = 376 nm) using N-(1-pyrenyl)maleimide (NPM) as the derivatizing agent was developed. The calibration curves were linear over a concentration range of 25-5000 nm (r2 \u3e 0.997). The coefficients of variation for within-run precision and between-run precision ranged from 0.67 to 5.23% and for accuracy ranged from 0.98 to 10.54%; the percentage relative recovery ranged from 94.5 to 102.8%. This new method provides satisfactory separation of NAC and NACA, along with other biological thiols, in 20 min with a 5 nm limit of detection (LOD) per 5 µL injection volume

Antioxidant Potential of Sutherlandia Frutescens and its Protective Effects against Oxidative Stress in Various Cell Cultures

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. 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). 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. Oxidative stress is implicated in a number of disorders, and due to the public\u27s 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

The Protective Effect of N-Acetylcysteine Amide Against Paraquat-Induced Neurotoxicity

N-acetylcysteine amide (NACA) is a new antioxidant molecule with powerful radical scavenging properties. The aim of this study was to investigate neuroprotective effects of NACA against paraquat (PQ) toxicity in the midbrains of rats by using motor coordination tests and biochemical and histological analysis. Thirty adult Wistar albino rats were divided into three groups: Group 1: control (n = 10), Group 2: PQ (10 mg/kg) (n = 10), and Group 3: PQ (10 mg/kg) + NACA (100 mg/kg) (n = 10). NACA was administrated intraperitoneally 30 min before PQ injection. Performance was measured for a period of 28 days. The rotarod and accelerod tests were performed prior to and after the experimental period. After the experimental period, rats were sacrificed and midbrain tissues were removed. According to biochemical data, malondialdehyde levels exhibited a significant increase (P \u3c 0.05) when the PQ group was compared to the control group, whereas the NACA-treated group showed a significant decline (P \u3c 0.05). The total glutathione levels (P \u3c 0.01) and the glutathione peroxidase and butyrylcholinesterase activities (P \u3c 0.05) in the NACA treatment group were significantly raised compared with the PQ group. The main finding in the rotarod and accelerod tests was that the PQ+NACA group had improved motor coordination functions, whereas the PQ group had lost motor coordination (P \u3c 0.05). Our histological data were also outstanding and were consistent with biochemical and motor coordination results in terms of the protective role of NACA against PQ-induced neurotoxicity

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 μM 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 μM 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

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

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 H2O2). 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

Acute and subacute pulmonary toxicity and mortality in mice after intratracheal instillation of ZnO nanoparticles in three laboratories

Inhalation is the main pathway of ZnO exposure in the occupational environment but only few studies have addressed toxic effects after pulmonary exposure to ZnO nanoparticles (NP). Here we present results from three studies of pulmonary exposure and toxicity of ZnO NP in mice. The studies were prematurely terminated because interim results unexpectedly showed severe pulmonary toxicity. High bolus doses of ZnO NP (25 up to 100μg; ≥1.4mg/kg) were clearly associated with a dose dependent mortality in the mice. Lower doses (≥6μg; ≥0.3mg/kg) elicited acute toxicity in terms of reduced weight gain, desquamation of epithelial cells with concomitantly increased barrier permeability of the alveolar/blood as well as DNA damage. Oxidative stress was shown via a strong increase in lipid peroxidation and reduced glutathione in the pulmonary tissue. Two months post-exposure revealed no obvious toxicity for 12.5 and 25μg on a range of parameters. However, mice that survived a high dose (50μg; 2.7mg/kg) had an increased pulmonary collagen accumulation (fibrosis) at a similar level as a high bolus dose of crystalline silica. The recovery from these toxicological effects appeared dose-dependent. The results indicate that alveolar deposition of ZnO NP may cause significant adverse health effects