Thiol-containing Antioxidants Research Articles

Toxic metals and oxidative stress part I: mechanisms involved in metal-induced oxidative damage.

Toxic metals (lead, cadmium, mercury and arsenic) are widely found in our environment. Humans are exposed to these metals from numerous sources, including contaminated air, water, soil and food. Recent studies indicate that transition metals act as catalysts in the oxidative reactions of biological macromolecules therefore the toxicities associated with these metals might be due to oxidative tissue damage. Redox-active metals, such as iron, copper and chromium, undergo redox cycling whereas redox-inactive metals, such as lead, cadmium, mercury and others deplete cells' major antioxidants, particularly thiol-containing antioxidants and enzymes. Either redox-active or redox-inactive metals may cause an increase in production of reactive oxygen species (ROS) such as hydroxyl radical (HO.), superoxide radical (O2.-) or hydrogen peroxide (H2O2). Enhanced generation of ROS can overwhelm cells' intrinsic antioxidant defenses, and result in a condition known as "oxidative stress". Cells under oxidative stress display various dysfunctions due to lesions caused by ROS to lipids, proteins and DNA. Consequently, it is suggested that metal-induced oxidative stress in cells can be partially responsible for the toxic effects of heavy metals. Several studies are underway to determine the effect of antioxidant supplementation following heavy metal exposure. Data suggest that antioxidants may play an important role in abating some hazards of heavy metals. In order to prove the importance of using antioxidants in heavy metal poisoning, pertinent biochemical mechanisms for metal-induced oxidative stress should be reviewed.

Induction of vascular endothelial growth factor by nitric oxide in cultured human articular chondrocytes

We investigated the role of nitric oxide (NO) in the control of vascular endothelial growth factor A (VEGF) gene expression in cultured human articular chondrocytes. Cell treatment with the NO-generating compound nitrosoglutathione (GSNO) caused a significant accumulation of 4.4 kb VEGF mRNA, a major VEGF mRNA isoform expressing in chondrocytes. This is the first demonstration that NO can induce VEGF mRNA expression in chondrocytes. VEGF mRNA level was not affected in cells exposed to dibutyryl cGMP, a non-hydrolyzable analog of cGMP, suggesting that the cGMP system is not involved in NO-dependent transcriptional activation of VEGF gene. The GSNO-stimulated induction of VEGF mRNA was slightly attenuated by MAP protein kinase inhibitors PD98058 and SB203580, but was completely blocked in cells incubated with GSNO in the presence of catalase and superoxide dismutase, enzymes scavenging reactive oxygen species (ROS), or in the presence of thiol-containing antioxidants, N-acetyl cysteine and reduced glutathione. These results suggest that in articular chondrocytes the GSNO-induced VEGF gene transcriptional activation is dependent on endogenous ROS production and oxidative thiol modifications.

Inhibition of oxidative DNA repair in cadmium-adapted alveolar epithelial cells and the potential involvement of metallothionein

This study evaluated the effects of cadmium (Cd) adaptation in cultured alveolar epithelial cells on oxidant-induced DNA damage and its subsequent repair. Using the comet assay, we determined that lower levels of DNA damage occurred in Cd-adapted cells compared with non-adapted cells following treatment of cells with hydrogen peroxide (H 2O 2). This may be a consequence of increased thiol-containing antioxidants that were observed in adapted cells, including metallothionein and glutathione. Cd-adapted cells were, however, less efficient at repairing total oxidative DNA damage compared with non-adapted cells. Subsequently, we investigated the effect of Cd adaptation on the repair of particular oxidized DNA lesions by employing lesion-specific enzymes in the comet assay, namely formamidopyrimidine DNA glycosylase (Fpg), an enzyme that predominantly repairs 8-oxoguanine (8-oxoG), and endonuclease III, that is capable of repairing oxidized pyrimidines. The data demonstrated that adaptation to Cd results in significantly impaired repair of both Fpg- and endonuclease III-sensitive lesions. In addition, in situ detection of 8-oxoG using a recombinant monoclonal antibody showed that Cd-adaptation reduces the repair of this oxidative lesion after exposure of cells to H 2O 2. Activities of 8-oxoG-DNA glycosylase and endonuclease III were determined in whole cell extracts using 32P-labeled synthetic oligonucleotides containing 8-oxoG and dihydrouracil sites, respectively. Cd adaptation was associated with an inhibition of 8-oxoG-DNA glycosylase and endonuclease III enzyme activity compared with non-adapted cells. In summary, this study has shown that Cd adaptation: (1) reduces oxidant-induced DNA damage; (2) increases the levels of key intracellular antioxidants; (3) inhibits the repair of oxidative DNA damage.

The molecular mechanism of dopamine-induced apoptosis: identification and characterization of genes that mediate dopamine toxicity.

Parkinson's disease (PD) is a progressive neurological disorder caused by rather selective degeneration of the dopaminergic (DA) neurons in the substantia nigra. Though subject to intensive research, the etiology of this nigral neuronal loss is still enigmatic and treatment is basically symptomatic. The current major hypothesis suggests that nigral neuronal death in PD is due to excessive oxidative stress generated by auto- and enzymatic oxidation of the endogenous neurotransmitter dopamine (DA), the formation of neuromelanin and presence of high concentrations of iron. We have found that DA toxicity is mediated through its oxidative metabolites. Whereas thiol-containing antioxidants provided marked protection against DA toxicity, ascorbic acid accelerated DA-induced death. Using the differential display approach, we sought to isolate and characterize genes whose expression is altered in response to DA toxicity. We found an upregulation of the collapsin response mediator protein (CRM) and TCP-1delta in sympathetic neurons, which undergo dopamine-induced apoptosis. The isolation of these genes led us to examine the expression and activity of CRM and TCP-1delta related genes. Indeed, we found a significant induction of mRNAs of the secreted collapsin-1 and the mitochondrial stress protein HSP60. Antibodies directed against collapsin-1 provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. In a parallel study, using antisense technology, we found that inhibition of TCP-1delta expression significantly reduced DA-induced neuronal death. These findings suggest a functional role for collapsin-1 and TCP-1delta as positive mediators of DA-induced neuronal apoptosis.

Inhibition of nitric oxide synthesis in primary cultured mouse hepatocytes by α-lipoic acid

Recent work shows that septic or endotoxic shock is associated with lipopolysaccharide and cytokine mixture-induced nitric oxide (NO) synthesis in liver. Here we found that dl-α-lipoic acid inhibited but other thiol-containing antioxidants such as glutathione and N-acetylcysteine enhanced lipopolysaccharide and cytokine mixture (referred as LPS/CM)-induced NO synthesis in hepatocytes. The inhibitory action of α-lipoic acid on hepatocyte NO synthesis was as potent as that of N G-monomethyl- l-arginine without obvious cytotoxicity. Deletion by diethylmaleate or inhibition by buthionine sulfoximine of intracellular glutathione caused a significant decrease in hepatocyte NO synthesis, implying that increased intracellular reduced glutathione levels could not be the reason for α-lipoic acid inhibited NO synthesis. α-Lipoic acid inhibition of NO synthesis seems to be from α-lipoic acid improved carbohydrate metabolism in hepatocytes. Since α-lipoic acid is an essential compound existing naturally in physiological systems, it may serve as both a research and therapeutic agent for sepsis.

Rheological changes in human red blood cells under oxidative stress: effects of thiol-containing antioxidants

Thiol-compounds, dithiothreitol (DTT) and N-acetylcysteine (NAC), were examined for their antioxidant effects on Fe 2+/ascorbate-treated and diamide-treated red blood cells (RBCs). Both of the compounds protected RBCs against diamide-induced spectrin crosslinking; they also prevented loss of deformability and aggregation impairment in diamide-treated cells. Neither DTT nor NAC were effective in suppressing lipid peroxidation induced by Fe 2+/ascorbate treatment. DTT showed a limited protective effect on iron-mediated band 3 fragmentation and RBC deformability, but it was capable of preventing the decrease of rouleaux formation rate to some extent. NAC exhibited a dose-dependent band 3 protective activity, partially prevented Fe 2+/ascorbate-induced deformability impairment and the decrease of rouleux formation rate. These findings suggest that thiol compounds are effective in modulating diamide-induced oxidative alterations of RBCs membrane constituents and rheological properties. NAC is more efficacious than DTT in protecting RBCs against iron-mediated oxidative injury, which can be attributed to its potent reductant and radical scavenging abilities.

4-hydroxynonenal inhibits glutathione peroxidase: protection by glutathione

4-Hydroxy-2,3- trans-nonenal, a lipid peroxidation product, inhibits glutathione peroxidase in a concentration-dependent manner. The concentration providing 50% inhibition is 0.12 mM. This inhibition can be almost completely (89%) prevented by 1 mM glutathione added to the incubation mixture 30 min before 4-hydroxy-2,3- trans-nonenal or 2,3- trans-nonenal, but not by other thiol-containing antioxidants such as 0.5 mM dithiothreitol or β-mercaptoethanol. Again the addition of 1 mM glutathione, and not of 0.5 mM dithiothreitol or β-mercaptoethanol, to the enzyme 30 min after incubation with 4-hydroxy-2,3- trans-nonenal restores activity to the same extent as does the preincubation with GSH. In view of the known reactivity of 4-hydroxy-2,3- trans-nonenal with lysine residues and the reversibility of the inhibition, the involvement of a lysine residue in GSH binding to glutathione peroxidase is proposed. The potential relevance of the inhibition of glutathione peroxidase by 4-hydroxy-nonenal to oxidative tissue damage is discussed with particular emphasis on neurological disorders.

The Redox State as a Correlate of Senescence and Wasting and as a Target for Therapeutic Intervention

The loss of body cell mass (bcm) in senescence and wasting is poorly understood. We now show that the plasma cystine/acid soluble thiol ratio, ie, an indicator of the redox state, is increased in old age and cancer patients and correlated with a decrease in bcm and plasma albumin. A cause/effect relationship was suggested by two independent studies with N-acetyl-cysteine (NAC). NAC caused an increase in the bcm of healthy persons with high plasma cystine/thiol ratios, and treatment of cancer patients with NAC plus interleukin-2 caused an increase in bcm, plasma albumin, and functional capacity. Albumin levels below 680 micromol/L were associated with an increase in body water. Our studies suggest that the shift in the redox state may contribute to the loss of bcm and may provide a quantitative guideline for therapeutic intervention. Treatment of cancer patients with thiol-containing antioxidants may improve the quality of life.

In Vitro Antioxidant Treatment Recovers Proliferative Responses of Anergic CD4+ Lymphocytes From Human Immunodeficiency Virus–Infected Individuals

Oxidative stress has been proposed to be involved in the immunologic defeat observed in effector calls of the immune system as well as in lymphocyte cell death and viral replication in human immunodeficiency virus (HIV)-infected patients. Because thiol-containing antioxidants such as N-acetyl-L-cysteine have been shown to have beneficial effects on CD4+ lymphocyte survival and to inhibit programmed cell death and HIV-1 replication, they may play a role in therapeutic strategies of this disease. In this work we have studied the cellular thiol levels and the affect of in vitro antioxidant treatment of purified CD4+ lymphocytes from HIV-infected patients, and correlated these parameters to proliferative responses and programmed cell death. We show that CD4+ lymphocytes from HIV-infected patients display impaired proliferative responses and a significant decrease in cellular thiol levels, indicating a disturbed redox status. Interestingly, antioxidant treatment succeeded to restore defective proliferative responses to CD3-mediated activation in 8 of 11 patients (high antioxidant responders). In contrast to high responders, patients failing to respond to antioxidant treatment (low antioxidant responders), were characterized by an abnormal ratio of apoptotic cells, which was not affected by N-acetyl-L-cysteine and/or 2-beta-mercaptoethanol preincubation. These results demonstrate for the first time that antioxidant treatment is able to revert the impaired proliferative activity of CD4 cells from HIV-infected patients and could help designing therapeutic strategies with antioxidant drugs. However, this action is not observed in cells undergoing programmed cell death.

Antioxidant inhibition of thymocyte apoptosis by dihydrolipoic acid

Recent findings suggest that intracellular oxidants are involved in the induction of apoptosis, and that this type of cell death can be inhibited by various thiol-containing antioxidants such as N-acetyl cysteine. To study the effects of a physiologically important thiol reductant, rat thymocytes were preincubated with either lipoic acid, dihydrolipoic acid, or lipoamide and then exposed to methylprednisolone or etoposide, two stimuli known to induce apoptosis in these cells. Dihydrolipoic acid and lipoamide both exerted an inhibitory effect on apoptosis induced by the two stimuli, while lipoic acid was inactive. Inhibition of apoptosis was evident as (a) reduced formation of condensed, pyknotic nuclei; (b) a prevention of cell shrinkage; and (c) decreased chromatin degradation. Furthermore, the depletion of reduced glutathione that occurs as thymocytes undergo apoptosis was also prevented in the presence of DHLA. Investigation of the pattern of chromatin fragmentation revealed that DNA in the antioxidant-loaded thymocytes remained above 50 kb pairs in size, indicating that inhibition by DHLA was operative at an early step in the apoptotic pathway. These results suggest that intracellular oxidation is an obligate, early component of thymocyte apoptosis.

Effects of N-acetyl- l-cysteine on T-cell apoptosis are not mediated by increased cellular glutathione

Thiol-containing antioxidants such as N-acetyl- l-cysteine (NAC) are known to inhibit apoptosis, although it is unclear whether this effect is direct or mediated through modulation of intracellular glutathione (GSH). In the present study, NAC treatment of the murine T-cell hybridoma DO-11.10 was found to inhibit apoptosis triggered by anti-CD3 antibody but enhance the process when induced by 6-α-methylprednisolone. HPLC measurements showed that these effects were not correlated with the levels of GSH or glutathione disulfide (GSSG) in the cells. Similar effects on DNA fragmentation were obtained when the experiments were repeated in the presence either of a specific inhibitor of GSH biosynthesis (buthionine sulfoximine) or the isomer N-acetyl- d-cysteine which cannot be enzymatically converted into GSH. We conclude that NAC can have divergent effects on apoptosis independent of changes in either the amount or redox state of intracellular GSH.

Diene Rubber Modification Using Thiol-Type Derivatives

Abstract Thiol-containing antioxidants such as 4-(mercaptoacetamido)-diphenylamine (MADA) undergo ready addition of-SH groups to the double bonds in diene rubbers in the presence of initiators. The reaction is a radical chain process leading to rubbers with improved thermooxidative resistance. The modification reaction has been carried out on high cis-polyisoprene and polybutadiene, and conditions under which the physicochemical properties (e.g., inherent viscosity, gel content, and microstructure) are less affected have been found. An oxygen absorption test and DSC were used as rapid methods for evaluation of thermooxidative stability of modified rubbers. The superiority of rubbers with MADA chemically attached (ranging from 1 to 4 phr) was shown by a circulating air oven test and by an extraction process which simulates the aggressive environments experienced by many rubbers under practical conditions. The results obtained show that a small degree of modification (1 phr MADA) is large enough to provide good thermooxidative stability without alteration of the molecular characteristics of the parent rubbers. At higher degrees, cistrans isomerization can occur.

Mechanisms of antioxidant action: Mechanochemical addition of thiol antioxidants to ABS

It is shown that thiol-containing antioxidants and stabilisers can be chemically reacted with ABS in high yields and in substantial concentrations under conditions of high temperature and high shear during processing. The resulting antioxidant concentrates are shown to be very effective thermal and photo-oxidative stabilisers for ABS when used as masterbatch additives for unstabilised ABS. Synergistic combinations of a bound antioxidant (BHBM (I(a))) and a bound uv stabiliser (EBHPT (II)) confer a high level of uv stability on ABS when incorporated into ABS. All the antioxidants show a high resistance to removal from the polymer by solvent extraction. The effect of the mechano-chemical process on the efficiency of adduct formation and on the effectiveness of the stabilisers so formed is discussed.