Combined administration of N-acetylcysteine and monoisoamyl DMSA on tissue oxidative stress during arsenic chelation therapy

The present study deals with the therapeutic potential of combined administration of N-acetylcysteine (NAC) along with monoisoamyl DMSA (MiADMSA) against chronic arsenic poisoning in guinea pigs. Animal were exposed to 50 ppm arsenic in drinking water for 8 mo and subsequently treated for 5 consecutive days with 100 mg/kg NAC (orally) and MiADMSA (intraperitoneally), individually or in combination (50 mg/kg each). Arsenic exposure produced a significant depletion of blood δ-aminolevulinic acid dehydrate (ALAD) activity, increased the blood zinc protoporphyrin (ZPP) level, and reduced blood and liver glutathione (GSH) levels in guinea pigs. Hepatic oxidized glutathione (GSSG) and thiobarbituric acid reactive substance (TBARS) levels showed a marked increase, whereas hepatic alkaline phosphatase (ALP) activity decreased and acid phosphatase (ACP) activity increased on arsenic exposure. Significant depletion of liver transaminase activities on arsenic exposure suggests organ injury. Administration of MiADMSA, alone and in combination with NAC after arsenic exposure, was able to significantly enhance hepatic GSH and to reduce GSSG and TBARS levels compared to the arsenic control. Biochemical variables indicative of liver injury generally remained insensitive to any of these treatments. The recoveries in parameters indicative of oxidative stress were more marked in guinea pigs treated with combined administration of NAC and MiADMSA than monotherapy. Interestingly, there was a more pronounced depletion of arsenic from blood and tissues after combined treatment with NAC plus MiADMSA than MiADMSA. Blood and tissues copper, zinc, iron, and calcium concentrations showed a significant increase after arsenic exposure, which showed improvement, particularly after combined administration of MiADMSA and NAC. Based on these data, a proposal can be made that greater effectiveness in chelation treatment against chronic arsenic poisoning (i.e., turnover in the oxidative stress and removed of arsenic from the system) could be achieved by combined administration of an antioxidant (preferably having a thiol moiety) with MiADMSA.     

Skin resistance to oxidative stress induced by resveratrol: From Nrf2 activation to GSH biosynthesis

Skin is particularly exposed to oxidative stress, either from environmental insults such as sunlight or pollution or as a consequence of specific impairments in antioxidant status resulting from pathologies or aging. Traditionally, antioxidant products are exogenously provided to neutralize pro-oxidant species. However, another approach based on stimulation of endogenous antioxidant defense pathways is more original. Resveratrol (RSV) was reported to display such a behavior in various tissues, but data about the mechanisms of action in skin are scarce. We show here that, in primary culture of normal human keratinocytes (NHKs) or in full-thickness reconstructed human skin, RSV activated the Nrf2 pathway at nontoxic doses, from 20 µM up to 100 µM. Among the Nrf2 downstream genes, glutamylcysteinyl ligase and glutathione peroxidase-2 were induced at the mRNA and protein levels. In parallel, a significant increase in glutathione content, assessed by LC/MS analysis, was observed in both models. Nrf2 gene silencing experiments performed in NHKs confirmed that Nrf2 was involved in RSV-induced modulation of cellular antioxidant status, in part by increasing cellular glutathione content. Finally, improvement of endogenous defenses induced in RSV-pretreated reconstructed skin ensured protection against the toxic oxidative effects of cumene hydroperoxide (CHP). In fact after RSV pretreatment, in response to CHP stress, glutathione content did not decrease as in unprotected samples. Cellular alterations at the dermal–epidermal junction were clearly prevented. Together, these complementary experiments demonstrated the beneficial effects of RSV on skin, beyond its direct antioxidant properties, by upregulation of a cutaneous endogenous antioxidant pathway.     

Vitamin C prevents hyperbaric oxygen-induced growth retardation and lipid peroxidation and attenuates the oxidation-induced up-regulation of glutathione in guinea pigs

Hyperbaric oxygen therapy is used to treat various clinical conditions, but it also causes oxidative damage. The objectives of this study are to determine if increased vitamin C intake can prevent hyperbaric oxygen-induced damage and to determine interactions among vitamin C, glutathione and vitamin E in response to oxidative stress. The growth rates of unexposed guinea pigs fed 1.25 mg vitamin C/day were indistinguishable from that of guinea pigs fed 50 mg vitamin C/day. In contrast, hyperbaric oxygen exposure resulted in growth retardation in guinea pigs fed 1.25 mg vitamin C/day, but it had little effect on the growth rates of guinea pigs fed 50 mg vitamin C/day. Increased vitamin C intake also prevented hyperbaric oxygen-induced lipid peroxidation in the liver. In guinea pigs not exposed to hyperbaric oxygen, levels of vitamin C in tissues were closely related to vitamin C intake, but tissue levels of glutathione and vitamin E were not related to vitamin C intake. However, interactions between vitamin C and glutathione were observed upon chronic hyperbaric oxygen exposure. Chronic hyperbaric oxygen exposure resulted in >2-fold increases in the levels of glutathione in liver and lung of guinea pigs fed 1.25 mg vitamin C/day. In comparison, the oxidation-induced increases in glutathione were significantly attenuated in guinea pigs fed 50 mg vitamin C/day. These data show that increased intake of vitamin C can prevent or alleviate the hyperbaric oxygen-induced damage. The interactions between vitamin C and glutathione upon hyperbaric oxygen exposure indicate that there is a homeostatic regulation of antioxidant capacity in guinea pig tissues.     

The preventive effect of Se-methylselenocysteine on γ-radiation-induced oxidative stress in rat lungs

We investigated the preventive effect of Se-methylselenocysteine (MSC) administration on γ-radiation (whole body irradiation, single 10-Gy dose)-induced oxidative damage in rat lungs. Rats were pretreated with MSC (0.75 mg/rat/day) for 1 week before γ-irradiation. The MSC pretreatment prevented the irradiation-induced increase in lipid peroxidation and the concomitant decrease in cellular glutathione content. The prevention of irradiation-induced oxidative damage in MSC-pretreated rat lungs appeared to be associated with increased antioxidant capacity, particularly in the glutathione system. The 1-week MSC treatment resulted in an increase in glutathione peroxidase, glutathione reductase, and glucose 6-phosphate dehydrogenase activities, which are involved in glutathione redox cycling. An increase in catalase activity was also observed in the rat lungs. Additionally, a significantly increased level of nuclear factor erythroid 2-related factor 2 (Nrf2) was exhibited in the MSC-treated rat lungs. Heme oxygenase 1, glutathione S-transferase pi, and peroxiredoxin 1, which are known target proteins of Nrf2, were also increased in MSC-treated lungs. These results implicate Nrf2 signaling in the MSC-induced activation of the antioxidant system.     

Thiol supplementation in aged animals alters antioxidant enzyme activity after heat stress.

Declines in oxidative and thermal stress tolerance are well documented in aging systems. It is thought that these alterations are due in part to reductions in antioxidant defenses. Although intracellular thiols are major redox buffers, their role in maintaining redox homeostasis is not completely understood, particularly during aging, where the reliance on antioxidant enzymes and proteins may be altered. To determine whether thiol supplementation improved the antioxidant enzyme profile of aged animals after heat stress, young and old Fischer 344 rats were treated with N-acetylcysteine (NAC; 4 mmol/kg ip) 2 h before heat stress. Liver tissue was collected before and 0, 30, and 60 min after heat stress. Aging was associated with a significant decline in tissue cysteine and glutathione (GSH) levels. There was also an age-related decrease in copper-zinc superoxide dismutase activity. Heat stress did not alter liver GSH, glutathione disulfide, or antioxidant enzyme activity. With NAC treatment, old animals took up more cysteine than young animals as reflected in an increase in liver GSH and a corresponding decrease in glutamate cysteine ligase activity. Catalase activity increased after NAC treatment in both age groups. Copper-zinc superoxide dismutase activity did not change with heat stress or drug treatment, whereas manganese superoxide dismutase activity was increased in old animals only. These data indicate that GSH synthesis is substrate limited in old animals. Furthermore, aged animals were characterized by large fluctuations in antioxidant enzyme balance after NAC treatment, suggesting a lack of fine control over these enzymes that may leave aged animals susceptible to subsequent stress.     

Effect of exogenous and endogenous antioxidants on 3-nitropionic acid-induced in vivo oxidative stress and striatal lesions: insights into Huntington's disease

3-Nitropropionic acid (3-NP) is an irreversible inhibitor of complex II in the mitochondria. 3-NP toxicity has gained acceptance as an animal model of Huntington's disease (HD). In the present study, we confirmed that rats injected with 3-NP (20 mg/kg, i.p., daily for 4 days) exhibit increased oxidative stress in both striatum and cortical synaptosomes as well as lesions in the striatum. Synaptosomal membrane proteins from rats injected with 3-NP exhibited a decrease in W/S ratio, the relevant electron paramagnetic resonance (EPR) parameter used to determine levels of protein oxidation, and western blot analysis for protein carbonyls revealed direct evidence of increased synaptosomal protein oxidation. Treatment of rats with the brain-accessible free radical spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO; 30 mg/kg, i.p., daily 2 h before 3-NP injection) or with N-acetylcysteine (NAC; 100 mg/kg, i.p., daily 2 h before 3-NP injection), a known glutathione precursor, before 3-NP treatments protects against oxidative damage induced by 3-NP as measured by EPR and western blot analysis for protein carbonyls. Furthermore, both DEMPMPO and NAC treatments before 3-NP administration significantly reduce striatal lesion volumes. These data suggest oxidative damage is a prerequisite for striatal lesion formation and that antioxidant treatment may be a useful therapeutic strategy against 3-NP neurotoxicity and perhaps against HD as well.     

LL202 ameliorates colitis against oxidative stress of macrophage by activation of the Nrf2/HO-1 pathway

LL202, a newly synthesized flavonoid derivative, has been confirmed to inhibit the mitogen-activated protein kinase pathway and activation protein-1 activation in monocytes; however, the anti-inflammatory mechanism has not been clearly studied. Uncontrolled overproduction of reactive oxygen species (ROS) has involved in oxidative damage of inflammatory bowel disease. In this study, we investigated that LL202 reduced lipopolysaccharide (LPS)-induced ROS production and malondialdehyde levels and increased superoxide dismutase, glutathione, and total antioxidant capacity in RAW264.7 cells. Mechanically, LL202 could upregulate heme oxygenase-1 (HO-1) via promoting nuclear translocation of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) to regulate LPS-induced oxidative stress in macrophages. In vivo, we validated the role of LL202 in dextran sulfate sodium- and TNBS-induced colitis models, respectively. The results showed that LL202 decreased the proinflammatory cytokine expression and regulated colonic oxidative stress by activating the Nrf2/HO-1 pathway. In conclusion, our study showed that LL202 exerts an anti-inflammatory effect by enhancing the antioxidant capacity of the Nrf2/HO-1 pathway to macrophages.     

Salvianolic acid B inhibits myofibroblast transdifferentiation in experimental pulmonary fibrosis via the up-regulation of Nrf2

Salvianolic acid B (SalB) is one of the most bioactive components extracted from Salvia miltiorrhiza, and its antioxidant capacity corresponds with its protective effects against cell injury from oxidative stress. The aim of the present study was to evaluate the effect of SalB on experimental pulmonary fibrosis and its ability to ameliorate the oxidative/antioxidative imbalance during fibrosis pathogenesis. The anti-fibrotic activity of SalB was first confirmed in Transforming growth factor β1(TGF-β1)-stimulated MRC-5 cells. The protection of SalB against oxidative stress during fibrogenesis in vitro was verified by detecting ROS production, the levels of glutathione (GSH) and malondialdehyde (MDA). The Western blot and PCR results indicated that SalB could up-regulate nuclear factor erythroid-derived 2-like 2 (Nrf2) at both the protein and mRNA levels and induce Nrf2 nuclear translocation in vitro, which may be the mechanism underlying the anti-fibrotic capacity of SalB. Furthermore, the anti-fibrotic and antioxidant capacities of SalB in vivo were confirmed in rats with BLM-induced pulmonary fibrosis. The immunohistochemistry results showed that Nrf2 was absent in fibroblastic foci (FF) areas, while the SalB treatment could increase the expression of Nrf2 in lung tissues, especially in FF areas.     

Dual role of glutathione in selenite-induced oxidative stress and apoptosis in human hepatoma cells

It is well known that glutathione, the major intracellular antioxidant, is closely involved in the metabolism and bioactivity of selenium. In the present study, glutathione was demonstrated to play a dual role on selenite (Se)-induced oxidative stress and apoptosis in human hepatoma HepG(2) cells. The experiment was carried out in two different modes to modulate intracellular reduced glutathione (GSH) content. In Mode A (pretreatment), cells were pretreated with N-acetylcysteine (NAC), buthionine sulfoximine (BSO), or GSH prior to Se exposure. In Mode B (simultaneous treatment), cells were treated with Se and NAC, BSO, or GSH simultaneously. It was found that Se-induced oxidative stress and apoptosis are closely related to the intracellular level of GSH. Both the increase and depletion of GSH content significantly enhanced Se-induced oxidative stress and apoptosis in HepG(2) cells. Results from this study clearly demonstrated that GSH has a dual role in the effects of Se on cancer cells: (i) GSH acts as a pro-oxidant, facilitating Se-induced oxidative stress, and (ii) GSH acts as an antioxidant, protecting against Se-induced oxidative stress and apoptosis. Understanding such a unique association between GSH and Se may help to explain the controversy in the literature over the complex relationship between selenium and glutathione, and ultimately the capability of selenium to prevent cancer.     

Oxidative stress is not required for the induction of apoptosis upon glutamine starvation of Sp2/0-Ag14 hybridoma cells

L-glutamine (Gln) withdrawal rapidly triggers apoptosis in the murine hybridoma cell line Sp2/0-Ag14 (Sp2/0). In this report, we examined the possibility that Gln deprivation of Sp2/0 cells triggers an oxidative stress which would contribute to the activation of apoptotic pathways. Gln withdrawal triggered an oxidative stress in Sp2/0 cells, as indicated by an increased accumulation of reactive oxygen species (ROS) and an increase in the intracellular content in protein carbonyl groups. Gln starvation also caused a decrease in the intracellular levels of glutathione (GSH). However, a decrease in GSH was not sufficient to induce Sp2/0 cell death since reducing GSH levels with DL-buthionine-[S,R]-sulfoximine did not affect cell viability. The antioxidant N-acetyl-L-cysteine (NAC), while effective in inhibiting ROS accumulation and oxidative stress, did not prevent the loss in cell viability or the processing and activation of caspase-3 triggered by Gln starvation. On the other hand, NAC did reduce the formation of apoptotic bodies in dying cells. Altogether these results indicate that in Sp2/0 cells, Gln deprivation leads to the induction of an oxidative stress which, while involved in the formation of apoptotic bodies, is not essential to the activation of the cell death program.     

Effects of N-acetyl-l-cysteine on bleomycin induced oxidative stress in malignant testicular germ cell tumors

Testicular cancer is a very common cancer in males aged 15–44 years. Bleomycin is used in chemotherapy regimens in the treatment of patients having testicular germ-cell tumor. Bleomycin generates oxygen radicals, induces oxidative cleavage of DNA strand and induces apoptosis in cancer cells. There is no study in the literature investigating effects of N-Acetyl-l-Cysteine (NAC) on bleomycin-induced oxidative stress in testicular germ cell tumors. For this reason, we studied effects of NAC on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testis cancer cells incubated with bleomycin and compared the results with H₂O₂ which directly produces oxidative stress. We determined protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide levels and total antioxidant capacity in both testicular cancer cells. Bleomycin and H₂O₂ significantly increased 8-isoprostane, TBARS, protein carbonyl and lipid hydroperoxide levels in NTera-2 and NCCIT cells. Bleomycin and H₂O₂ significantly decreased antioxidant capacity and GSH levels in both cell lines. Co-incubation with NAC significantly decreased lipid hydroperoxide, 8-isoprostane, protein carbonyl content and TBARS levels increased by bleomycin and H₂O₂. NAC enhanced GSH levels and antioxidant capacity in the NTera-2 and NCCIT cells. It can be concluded that NAC diminishes oxidative stress in human testicular cancer cells induced by bleomycin and H₂O₂.     

Bardoxolone-methyl inhibits migration and metabolism in MCF7 cells

Bardoxolone-methyl (BAR) is reported to have anti-inflammatory, anti-proliferative and anti-fibrotic effects. BAR activates Nrf2 and may ameliorate oxidative stress through induction of antioxidant genes. However, off-target effects, probably concentration and NFkB-dependent, have limited the clinical use of BAR. Nrf2 regulates expression of antioxidant and mitochondrial genes and has been proposed as a target for both obesity and breast cancer. Therefore, we explored whether BAR can alter migration and proliferation in the MCF7 cell line and whether metabolic function is affected by BAR. Incubation with BAR caused a time-dependent migratory inhibition and an associated decrease in mitochondrial respiration. Both migratory and mitochondrial inhibition by BAR were further enhanced in the presence of fatty acids. In addition to the activation of Nrf2, BAR altered the expression of target mRNA GCLC and UCP1. After 24?h, BAR inhibited both glycolytic capacity, reserve (p?p?N-acetyl cysteine. The fatty acid, palmitate, increased mitochondrial ROS, impaired migration and oxidative phosphorylation but palmitate toxicity towards MCF7 could not be inhibited by N-acetyl cysteine suggesting that they exert effects through different pathways. BAR-activated AKT, induced DNA damage and inhibited cell proliferation. When the proteasome was inhibited, there was loss of BAR-mediated changes in p65 phosphorylation and SOD2 expression suggesting non-canonical NFkB signaling effects. These data suggest that BAR-induced ROS are important in inhibiting MCF7 migration and metabolism by negatively affecting glycolytic capacity and mitochondrial function.     

Involvement of Nrf2 and Keap1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent peptides from soft-shelled turtle

The antioxidant response element (ARE) is a cis-acting enhancer sequence located in the region containing genes related to antioxidant and detoxification. Under oxidative stress, the induction of nuclear factor-E2-related factor 2 (Nrf2)/ARE is considered as a fundamental process involved in defending reactive oxygen species (ROS) and providing protection against toxic xenobiotics. In this study, we obtained seven antioxidant peptides from soft-shelled turtle and concluded that Glu-Asp-Tyr-Gly-Ala (EDYGA) is the most potent ARE-luciferase inducer. To gain fundamental insights into the role of EDYGA in oxidative stress, we evaluated the effects of EDYGA on the Nrf2/Keap1 system in HepG₂ cells. The results revealed that EDYGA modulated the Nrf2/ARE pathway by enhancing Nrf2 level through the stabilization of Nrf2, which was accomplished by a decrease in the level of Keap1. These actions eventually led to an increase in nuclear Nrf2 accumulation and ARE-binding activity. Moreover, silencing Nrf2 markedly reduced ARE-driven activity induced by EDYGA. Docking results proved that glutamate residues of peptide EDYGA directly bind to Arg 415 of Kelch domain receptor pocke. The results were helpful in understanding the antioxidant activity of peptides from soft-shelled turtle which have potential to be used in foods and drugs as functional ingredients.     

Location of intra- and extracellular M. tuberculosis populations in lungs of mice and guinea pigs during disease progression and after drug treatment

The lengthy treatment regimen for tuberculosis is necessary to eradicate a small sub-population of M. tuberculosis that persists in certain host locations under drug pressure. Limited information is available on persisting bacilli and their location within the lung during disease progression and after drug treatment. Here we provide a comprehensive histopathological and microscopic evaluation to elucidate the location of bacterial populations in animal models for TB drug development.To detect bacilli in tissues, a new combination staining method was optimized using auramine O and rhodamine B for staining acid-fast bacilli, hematoxylin QS for staining tissue and DAPI for staining nuclei. Bacillary location was studied in three animal models used in-house for TB drug evaluations: C57BL/6 mice, immunocompromised GKO mice and guinea pigs. In both mouse models, the bacilli were found primarily intracellularly in inflammatory lesions at most stages of disease, except for late stage GKO mice, which showed significant necrosis and extracellular bacilli after 25 days of infection. This is also the time when hypoxia was initially visualized in GKO mice by 2-piminidazole. In guinea pigs, the majority of bacteria in lungs are extracellular organisms in necrotic lesions and only few, if any, were ever visualized in inflammatory lesions. Following drug treatment in mice a homogenous bacillary reduction across lung granulomas was observed, whereas in guinea pigs the remaining extracellular bacilli persisted in lesions with residual necrosis. In summary, differences in pathogenesis between animal models infected with M. tuberculosis result in various granulomatous lesion types, which affect the location, environment and state of bacilli. The majority of M. tuberculosis bacilli in an advanced disease state were found to be extracellular in necrotic lesions with an acellular rim of residual necrosis. Drug development should be designed to target this bacillary population and should evaluate drug regimens in the appropriate animal models.     

In vivo and in vitro evidence for the involvement of Nrf2-antioxidant response element signaling pathway in the inflammation and oxidative stress induced by particulate matter (PM10): the effective role of gallic acid

Environmental pollution is one of the risk factors for respiratory diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2) is the major mechanisms contributing to cellular defense against oxidative damage. Gallic acid (GA) is regarded as potent anti-inflammatory and antioxidant agents. The aim was to evaluate the role of Nrf2 pathway in particulate matter (PM₁₀) exposure on lung and epithelial cells with an emphasis on the role of GA. In in vivo part, the rats were divided as control, GA (30?mg/kg), particulate matter (PM) (0.5, 2.5, and 5?mg/kg), and PM?+?GA. In in vitro study, the cells were divided as control, PM₁₀ (100, 250, and 500?µg/ml), GA (50 µmol/L) and PM₁₀+GA. Inflammation, oxidative stress and Nrf2-pathway factors were assessed. PM₁₀ groups showed a considerable increase in the epithelial permeability and inflammatory parameters. We also found a significant decrease in the expression of Nrf2 and its up-stream regulators genes. Accordingly, the biosynthesis of glutathione (GSH) and other antioxidant activities significantly decreased. Gallic acid was identified to restore the antioxidant status to the normal levels. Our findings approved that Nrf2 is involved in PM₁₀-induced oxidative damages and showed that Nrf2 activation by natural agents could ameliorate respiratory injuries induced by PM₁₀.