Thiocyl (sodium thiosalicylate) belongs to a salicylate group of drugs, thus it has analgesic, an... more Thiocyl (sodium thiosalicylate) belongs to a salicylate group of drugs, thus it has analgesic, antipyretic and anti-inflammatory effects. It possesses metal chelating function because it also belongs to a thiol-containing group of compounds which are well-known chelators. The studies of our research group showed that thiocyl is a promising chelator of lead poisoning due to its antioxidant and metal-chelating abilities. To the best of our knowledge, no methods were currently available for measuring thiocyl in biological samples. Therefore, we developed a reversed-phase HPLC method using fluorescence detection (λ ex = 365 nm, λ em = 445 nm) with a one-step derivatizing reaction between thiocyl and a derivatizing agent-ThioGlo TM 3 (9-acetoxy-2-(4-(2, 5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)pyenyl)-3-oxo-3H-naphtho[2,1-b]pyran). Most biological thiols (such as N-acetylcysteine (NAC), cysteine (CYS), glutathione (GSH) and homocysteine (HCYS)) do not interfere with the detection of thiocyl by using this technique. The linear range of its calibration curve was determined to be 25-2500 nM, and the detection limit of thiocyl was found to be 3 nM with 20 L injection volume. The coefficients of variation (CV) for within-run precision and between-run precision ranged from 0.93 to 7.21%. This assay proved to be a rapid, sensitive and simple method for determining thiocyl in biological samples.
Oxidative stress plays an important role in neurodegenerative disorders such as Parkinson's Disea... more Oxidative stress plays an important role in neurodegenerative disorders such as Parkinson's Disease and Alzheimer's Disease. Methamphetamine (METH) is an amphetamine analog that causes degeneration of the dopaminergic system in mammals and subsequent oxidative stress. In our present study, we have used immortalized human brain microvascular endothelial (HBMVEC) cells to test whether N-Acetylcysteineamide (NACA), a novel antioxidant, prevents METH-induced oxidative stress in vitro. Our studies showed that NACA protects against METH-induced oxidative stress in HBMVEC cells. NACA significantly protected the integrity of our blood brain barrier (BBB) model, as shown by permeability and trans-endothelial electrical resistance (TEER) studies. NACA also significantly increased the levels of intracellular glutathione (GSH) and glutathione peroxidase (GPx). Malondialdehyde (MDA) levels increased dramatically after METH exposure, but this increase was almost completely prevented when the cells were treated with NACA. Generation of reactive oxygen species (ROS) also increased after METH exposure, but was reduced to control levels with NACA treatment, as measured by dichlorofluorescin (DCF). These results suggest that NACA protects the BBB integrity in vitro, which could prevent oxidative stress-induced damage; therefore, the effectiveness of this antioxidant should be evaluated for the treatment of neurodegenerative diseases in the future.
Annals of the New York Academy of Sciences, Jun 1, 1988
Vasoactive intestinal peptide (VIP) mediated inhibition of platelet aggregation has been demonstr... more Vasoactive intestinal peptide (VIP) mediated inhibition of platelet aggregation has been demonstrated in rabbit platelets.' While the mechanism by which VIP inhibits aggregation is thought to be via a specific receptor linked to adenylate cyclase, platelet receptors for VIP have not yet been demonstrated. The current studies were designed to test whether VIP regulates aggregation in human platelets and to determine whether specific, high-affinity VIP receptors are expressed on platelet membranes. Platelet-rich plasma was isolated from peripheral blood of normal human subjects. Platelets were disrupted in lysis buffer (10 mM Tris, 5 mM EDTA, pH 7.4) with a polytron. Plasma membranes were isolated by differential centrifugation' and stored in Buffer A (20 mM Hepes, 150 m M NaCl, 2 mM MgCl,, 5 m M EDTA, 1 mM-& mercaptoethanol, 5 0 pg/ml phenylmethyl sulfonyl fluoride, pH 7.4).
Oxidative stress is seen in various metabolic disorders for unknown reasons. Oxidative stress is ... more Oxidative stress is seen in various metabolic disorders for unknown reasons. Oxidative stress is defined as an imbalance between pro-oxidant and antioxidant status in favor of the former. This study investigated whether oxidative stress exists in phenylketonuria (PKU) using the BTBR-Pah enu2 animal model for PKU. Animals (14 -24 weeks old) were sacrificed and brain and red blood cells (RBCs) were obtained aseptically. The lipid peroxidation by-product, evaluated as malondialdehyde (MDA), was significantly higher in the brains and RBCs of PKU animals (n ϭ 6) than in controls (n ϭ 6). Glutathione/glutathione disulfide, a good indicator for tissue thiol status, was significantly decreased both in the brains and RBCs. Some antioxidant enzymes were also analyzed in RBCs, including glucose-6-phosphate dehydrogenase (G6PD), which provides the RBC's main reducing power, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and catalase detoxifies H 2 O 2 by catalyzing its reduction to O 2 and H 2 O. Both catalase and G6PD were significantly increased in the RBCs of PKU animals.
Archives of Environmental Contamination and Toxicology, Dec 1, 1999
The toxicities and oxidative stress-inducing actions of (Ϫ)-nicotine and smokeless tobacco extrac... more The toxicities and oxidative stress-inducing actions of (Ϫ)-nicotine and smokeless tobacco extract (STE), containing equivalent amounts of nicotine, were studied. Toxicities were determined by colony formation assays using Chinese hamster ovary (CHO) cells. Results indicated that nicotine is less toxic than smokeless tobacco extract that contained the same amount of nicotine. The generation of reactive oxygen species, following treatment with smokeless tobacco extract and nicotine, was assessed by measurement of changes in glutathione (GSH) and malondialdehyde (MDA) levels. CHO cells (5 ϫ 10 5 cells/5 ml media) were incubated with 4, 0.8, and 0.08 mg of nicotine and STE containing the same amounts of nicotine. All preparations of smokeless tobacco extract significantly decreased GSH levels and increased MDA generation. However, 0.08 mg of nicotine treatment did not result in a significant change in GSH level, and only 4 mg of nicotine were sufficient to increase MDA generation. Addition of free radical scavenging enzymes, superoxide dismutase (SOD) and catalase (CAT), and an intracellular GSH precursor, N-acetyl-L-cysteine (NAC), replenished the GSH levels in nicotine-treated cells. GSH levels in cells exposed to smokeless tobacco extract containing 4 and 0.8 mg nicotine remained significantly lower than the control with the addition of SOD and CAT. However, co-addition of NAC with smokeless tobacco extract preparations returned the GSH levels to the control level. Lactate dehydrogenase (LDH) activities were measured in the media to establish the membrane damage following exposure to smokeless tobacco extract and nicotine. Treatment of cells with 4 mg nicotine caused a significant increase in LDH activity, which was returned to control level in the presence of the antioxidant enzymes and NAC. Smokeless tobacco extract did not change the LDH activity.
A new method for the determination of reduced and oxidized glutathione by using capillary zone el... more A new method for the determination of reduced and oxidized glutathione by using capillary zone electrophoresis was developed and compared with the N-(1-pyreny1)maleimide high-performance liquid chromotography method. The tissue samples were obtained from C57BL/6 mice and homogenized in deionized water. The detection wavelength was set at 214 nm. A 50-cm effective length X 75 pm i.d. uncoated fused silica capillary with a 10 mM phosphate buffer at pH 7.5 was used for the experiment. Samples were introduced by pressure injection for 5 s. The voltage for separation was at 20 kV. Between runs, the capillary was regularly washed with 0.1 N NaOH and deionized water, followed by reconditioning with running buffer. The present method was shown to be reproducible and is convenient in that a derivatization step is not required. The method has been used to measure glutathione levels in biological samples and the results show good agreement with those obtained by high-performance liquid chromotography.
The thiol (-SH) functional group is found in a number of drug compounds and confers a unique comb... more 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.
Oxidative stress, which is the loss of balance between antioxidant defense and oxidant production... more Oxidative stress, which is the loss of balance between antioxidant defense and oxidant production in the cells, is implicated in the molecular mechanism of heavy metal-induced neurotoxicity. Given the key role of lead (Pb) and cadmium (Cd) in inducing oxidative stress, we investigated their role in disrupting the integrity and function of immortalized human brain microvascular endothelial cells (hCMEC/D3). To study this, hCMEC/D3 cells were exposed to control media or to media containing different concentrations of Pb or Cd. Those exposed to Pb or Cd showed significantly higher oxidative stress than the untreated group, as indicated by cell viability, reactive oxygen species (ROS), glutathione (GSH) levels, and catalase enzyme activity. Pb also induced oxidative stress-related disruption of the hCMEC/D3 cell monolayer, as measured by trans-endothelial electrical resistance (TEER), the dextran permeability assay, and the level of tight junction protein, zona occluden protein (ZO-2). However, no significant disruption in the integrity of the endothelial monolayer was seen with cadmium at the concentrations used. Taken together, these results show that Pb and Cd induce cell death and dysfunction in hCMEC/D3 cells and, in the case of Pb, barrier disruption. This suggests blood brain barrier (BBB) dysfunction as a contributing mechanism in Pb and Cd neurotoxicities.
The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disor... more The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disorders considerably; however, prevalence of HIV-1-associated neurocognitive disorders (HAND) has been on the rise in the post-HAART era. In view of these developments, we investigated whether a HAART drug combination of 3'-Azido-2', 3'-deoxythymidine (AZT) and Indinavir (IDV) can alter the functionality of the blood-brain barrier (BBB) endothelial cells, thereby exacerbating this condition. Viability of hCMEC/D3 cells (in vitro model of BBB) that were exposed to these drugs was significantly reduced after 72 hr treatment, in a dose-dependent manner. Reactive oxygen species (ROS) were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione (GSH) and malondialdehyde (MDA), were found to be altered in the treated groups. Loss of mitochondrial membrane potential (Ψ m ), as assessed with fluorescent microscopy and decreased levels of ATP, indicated that cytoxicity was mediated through mitochondrial dysfunction. Furthermore, AZT + IDV treatment caused apoptosis in endothelial cells, as assessed by the expression of cytochrome c and procaspase-3 proteins. Pretreatment with thiol antioxidant N-acetylcysteine amide (NACA) reversed some of the pro-oxidant effects of AZT+IDV. Results from our in vitro studies indicate that the AZT + IDV combination may affect the BBB in HIV infected individuals treated with HAART drugs.
This study examined whether lead-induced alterations in selected parameters that are indicative o... more This study examined whether lead-induced alterations in selected parameters that are indicative of oxidative stress accompany the toxic effects of lead in red blood cells (RBCs) in vivo. It also explored the possibility that treatment with N-acetylcysteine (NAC) or succimer (meso-2,3-dimercaptosuccinic acid) was capable of reversing parameters indicative of lead-induced oxidative stress. Fisher 344 rats were given 2000 ppm lead acetate in their drinking water for 5 weeks. The lead was then removed and the animals were given NAC (800 mg/kg/day) or succimer (90 mg/kg/day) in their drinking water for 1 week, after which the RBCs were harvested. Animals not given lead and those given lead, but not NAC or succimer, served as negative and positive controls, respectively. At the end of the experiment, blood-lead levels were 35 94 vg/dl in lead-treated animals, which were reduced to 2.5 9 1 vg/dl by treatment with succimer and to 25 93 vg/dl by treatment with NAC. Lead-exposed animals demonstrated signs of anemia as evidenced by anisocytosis, poikilocytosis, and alterations in hemoglobin, hematocrit, and mean corpuscular volume. Lipid peroxidation, as evidenced by increased malondialdehyde (MDA) content, as well as decreases in reduced glutathione (GSH) and increases in catalase and glucose 6-phosphate dehydrogenase (G6PD) activity were noted in RBCs from lead-treated rats, suggesting that the lead induced oxidative stress. In addition, a significant reduction in blood l-aminolevulinic acid dehydratase (ALAD) activity suggested that accumulation and autooxidation of l-aminolevulinic acid might contribute to lead-induced oxidative stress. Treatment with either NAC or succimer reversed lead-induced alterations in MDA and GSH content, but only succimer appeared to partially restore ALAD activity. These results provide in vivo evidence supporting the hypothesis that lead induces oxidative stress in RBCs, which is reversible by treatment with a thiol antioxidant (NAC), as well as a chelating agent (succimer).
Oxidative stress may contribute to the pathology of many diseases, and endogenous thiols, especia... more 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 oxidativestress-related ocular diseases. Further application of this method may uncover new pathways involved in oxidative-stress-related diseases and endogenous defense mechanisms.
Archives of Environmental Contamination and Toxicology, Apr 1, 2003
Lead poisoning has been extensively studied over the years. Many adverse physiological and behavi... more Lead poisoning has been extensively studied over the years. Many adverse physiological and behavioral impacts on the human body have been reported due to the entry of this heavy metal. It especially causes the hematological effects to people of all ages. However, its molecular mechanisms of action remain largely unknown. Hence, the aim of the present study was to evaluate the cytotoxicity and oxidative stress induced by lead nitrate in a human leukemia cell line using the MTT [3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], and lipid hydroperoxide assays, respectively. HL-60 cells were treated with different doses of lead nitrate for 24 h prior to cytotoxicity oxidative stress assessment. The results obtained from the MTT assay indicated that lead nitrate significantly decreases the viability of HL-60 cells in a dose-dependent manner. Similar result was obtained with the trypan blue exclusion test. Data generated from lipid hydroperoxide assay resulted in a significant increase (p < 0.05) in the production of hydroperoxides (degradation products of lipid peroxidation) with increasing doses of lead nitrate. Upon 24 h of exposure, the hydroperoxide concentrations in the sample [μM] (mean ±SE, n = 3) compared to untreated control were 6.7 ± 2, 7.1 ± 1, 14.7 ± 2, 15.7 ± 1, 16.2 ± 1, and 15.2 ± 1 in 0, 10, 20, 30, 40, and 50. μg/mL of lead nitrate, respectively. In summary, findings from this study demonstrated that lead nitrate is cytotoxic to HL-60 cells. This cytotoxicity is found to be associated with oxidative stress. Although there are many studies showing evidence that lead is a multi-targeted toxicant, causing effects in the gastrointestinal tract, cardiovascular system, central and peripheral nervous systems, immune system, and reproductive system, little is known about the effect of lead on the hematopoietic system. Hence, the present study was designed to use human leukemia (HL-60) cells as models to determine whether exposure to lead is associated with the
Background: Age-related macular degeneration (AMD) is a leading cause of blindness in the United ... more Background: Age-related macular degeneration (AMD) is a leading cause of blindness in the United States among adults age 60 and older. While oxidative stress is implicated in the pathogenesis of AMD, dietary antioxidants have been shown to delay AMD progression in clinical studies. We hypothesized that N-acetylcysteine amide (NACA), a thiol antioxidant, would protect retinal pigment epithelium and impede progression of retinal degeneration. Methods: tert-Butyl hydroperoxide (TBHP) was used to induce oxidative stress in cell cultures. The goal was to evaluate the efficacy of NACA in an in vitro model of AMD in primary human retinal pigment epithelial cells (HRPEpiC). Results: Our data indicates that TBHP generated reactive oxygen species (ROS), which reduced cell viability, depleted glutathione (GSH) levels, and compromised glutathione reductase (GR) activity. Pretreatment with NACA significantly reduced ROS generation, restored GSH levels and GR activity, and recovered transepithelial electrical resistance. Pretreatment with NACA did not decrease the number of dying cells as determined by flow cytometry analysis. However, survival was significantly improved when cells were co-exposed to NACA and TBHP after a shortened pretreatment period. Conclusion: Our data suggest that pretreatment with NACA reduces sublethal but not lethal effects of TBHP in HRPEpiC. NACA significantly improves cell survival when administered prior to and during oxidative damage similar to that observed in the development of dry AMD. These results indicate that continuation of a thiol antioxidant regimen for treatment of AMD is beneficial throughout the course of the disease, and NACA is a potent antioxidant that should be further evaluated for this purpose.
Chinese Hamster Ovary (CHO) cells were propagated in vitro and exposed to varying doses of ionizi... more Chinese Hamster Ovary (CHO) cells were propagated in vitro and exposed to varying doses of ionizing radiation. The surviving fraction of cells was determined, being found to be a function of the radiation dose. The cell survival curves obtained as a function of radiation dose were modified by the inclusion of varying doses of glutamine in the medium with glutamine demonstrating a radioprotective effect. The radioprotectant effect of glutamine for CHO cells was more pronounced at higher radiation doses. Glutamine has been categorized as a non-essential amino acid in that it can be synthesized in some tissues; however, a number of cell lines require glutamine to survive and grow in vitro and supplementation of glutamine has been found to ameliorate the stress of surgery or irradiation to the gastrointestinal tract. It may be appropriate to consider glutamine as a conditionally essential amino acid.
Thiol antioxidants protect human lens epithelial (HLE B-3) cells against tert-butyl hydroperoxide-induced oxidative damage and cytotoxicity
Biochemistry and Biophysics Reports, 2022
Oxidative damage to lens epithelial cells plays an important role in the development of age-relat... more 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.
Data were analyzed using multiple linear regression, and all models controlled for age, gender, a... more Data were analyzed using multiple linear regression, and all models controlled for age, gender, and BMI. Effect sizes were evaluated by examining squared semi-partial correlations (sr 2 ). Results: Contrary to our hypothesis, inflammatory cytokines did not mediate the relationship between TST and TL. Instead, TST and AHI both directly predicted TL independent of covariates (TST: b = 892.11, p < .01; AHI: b = 117.07, p < .01). However TST had a stronger effect (sr 2 = .55) on TL than age (sr 2 = .42), gender (sr 2 = .06), BMI (sr 2 = .19), and AHI (sr 2 = .23). PTSD, combat status, perceived stress, and sleep quality did not predict TL. Greater levels of fatigue predicted higher hsCRP (b = 0.02, p < .01, sr 2 = .41), but neither fatigue nor hsCRP was associated with TL. This study was the first to examine inflammation as a mediator between sleep and TL in veterans. Results suggest that objectively measured TST is the most robust predictor of telomere length, independent of several health related and psychosocial variables. The AHI-TL relationship may be complex. Results should be replicated in a larger sample, but this pilot study suggests the need for further research examining sleep duration as a unique risk factor for premature aging in veteran populations. Support (If Any): Metropolitan Detroit Research and Education Foundation.
Development of a HPLC-MS/MS method for assessment of thiol redox status in human tear fluids
Analytical Biochemistry, 2021
Oxidative stress is reported to be part of the pathology of many ocular diseases. For the diagnos... more 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.
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