Selenium protects against Pb-induced renal oxidative injury in weaning rats and human renal tubular epithelial cells through activating NRF2.

BACKGROUND: Lead (Pb) poisoning posing a crucial health risk, especially among children, causing devastating damage not only to brain development, but also to kidney function. Thus, an urgent need persists to identify highly effective, safe, and low-toxicity drugs for the treatment of Pb poisoning. The present study focused on exploring the protective effects of Se on Pb-induced nephrotoxicity in weaning rats and human renal tubular epithelial cells, and investigated the possible mechanisms. METHODS: Forty weaning rats were randomly divided into four groups in vivo: control, Pb-exposed, Pb+Se and Se. Serum creatinine (Cr), urea nitrogen (BUN) and hematoxylin and eosin (H&E) staining were performed to evaluate renal function. The activities of antioxidant enzymes in the kidney tissue were determined. In vitro experiments were performed using human renal tubular epithelial cells (HK-2 cells). The cytotoxicity of Pb and Se was detected by 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Inverted fluorescence microscope was used to investigate cell morphological changes and the fluorescence intensity of reactive oxygen species (ROS). The oxidative stress parameters were measured by a multi-detection reader. Nuclear factor-erythroid-2-related factor (NRF2) signaling pathways were measured by Western blot and reverse transcription polymerase chain reaction (RT-PCR) in HK-2 cells. RESULTS: We found that Se alleviated Pb-induced kidney injury by relieving oxidative stress and reducing the inflammatory index. Se significantly increased the activity of the antioxidant enzymes glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), whereas it decreased the excessive release of malondialdehyde (MDA) in the kidneys of weaning rats and HK-2 cells. Additionally, Se enhanced the antioxidant defense systems via activating the NRF2 transcription factor, thereby promoting the to downstream expression of heme oxygenase 1. Furthermore, genes encoding glutamate-cysteine ligase synthetase catalytic (GCLC), glutamate-cysteine ligase synthetase modifier (GCLM) and NADPH quinone oxidoreductase 1 (NQO1), downstream targets of NRF2, formed a positive feedback loop with NRF2 during oxidative stress responses. The MTT assay results revealed a significant decrease in cell viability with Se treatment, and the cytoprotective role of Se was blocked upon knockdown of NRF2 by small interfering RNA (siRNA). MDA activity results also showed that NRF2 knockdown inhibited the NRF2-dependent transcriptional activity of Se. CONCLUSIONS: Our findings demonstrate that Se ameliorated Pb-induced nephrotoxicity by reducing oxidative stress both in vivo and in vitro. The molecular mechanism underlying Se's action in Pb-induced kidney injury is related to the activation of the NRF2 transcription factor and the activity of antioxidant enzymes, ultimately suppressing ROS accumulation.

Identification of Nrf2 Activators from the Roots of Valeriana officinalis.

Various age-related chronic diseases have been linked to oxidative stress. The cellular antioxidant response pathway is regulated by the transcription factor nuclear erythroid factor 2. Therefore, plant-derived nuclear erythroid factor 2 activators might be useful therapeutics to stimulate the body's defense mechanisms. Our study focused on the discovery of potent nuclear erythroid factor 2 activators from medicinal plants. Initially, a variety of medicinal plant extracts were screened for nuclear erythroid factor 2 activity using a nuclear erythroid factor 2 luciferase reporter cell line. Among these, Valerian (Valeriana officinalis) root was identified as a potent candidate. Sequential extraction and bioassay-guided fractionation led to the isolation of four nuclear erythroid factor 2-active compounds, which were structurally identified by NMR and LC/HRMS as the known compounds isovaltrate, valtrate, jatamanvaltrate-P, and valerenic acid. These four compounds were then tested in relevant biological assays. Firstly, their effects on the expression of glutathione S-transferase, glutamate-cysteine ligase catalytic subunit, glutathione peroxidase, and heme oxygenase 1 were determined in HepG2 cells. Glutathione S-transferase P1 and glutamate-cysteine ligase catalytic subunit were upregulated by isovaltrate, valtrate, and jatamanvaltrate-P, while heme oxygenase 1 was upregulated by isovaltrate, jatamanvaltrate-P, and valerenic acid. The four compounds also increased the levels of glutathione and its metabolite, CysGly. As glutathione aids in the detoxification of hydrogen peroxide, cytoprotective effects of these four nuclear erythroid factor 2 activators against hydrogen peroxide toxicity were investigated, and indeed, the compounds significantly improved cell survival. This study provides evidence that four valepotriates from the roots of V. officinalis are activators of nuclear erythroid factor 2-mediated antioxidant and detoxification pathways. Our data might expand the medical use of this plant beyond its current application as a sleep aid.

The C-glucosyl flavone isoorientin pretreatment attenuates the methylglyoxal-induced mitochondrial dysfunction in the human neuroblastoma SH-SY5Y cells: role for the AMPK-PI3K/Akt/Nrf2/γ-GCL/GSH axis.

The reactive dicarbonyl methylglyoxal (MG) behaves as a pro-oxidant agent, causing redox dysfunction and cell death by different mechanisms in mammalian cells. MG is also a mitochondrial toxicant, impairing the oxidative phosphorylation (OXPHOS) system and leading to bioenergetics and redox collapses. MG induces glycation and exerts an important role in neurodegenerative and cardiovascular diseases. Isoorientin (ISO), a C-glucosyl flavone found in Aspalathus linearis, Fagopyrum esculentum, and Passiflora edulis, among others, is an antioxidant and anti-inflammatory molecule. ISO is a potent inducer of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), the master modulator of the redox environment in mammals. We investigated here whether ISO would prevent the mitochondria-related redox and bioenergetics impairments induced by MG in the human neuroblastoma SH-SY5Y cells. The cells were administrated with ISO at 20 µM for 18 h prior to the exposure to MG at 500 µM for further 24 h. It was observed that ISO efficiently prevented the mitochondrial impairments caused by MG. ISO upregulated the activity of the enzyme γ-glutamate-cysteine ligase (γ-GCL), consequently stimulating the synthesis of glutathione (GSH). The inhibition of γ-GCL, adenosine monophosphate-activated protein kinase (AMPK), and phosphoinositide 3-kinase/Akt (PI3K/Akt) suppressed the beneficial effects induced by ISO on the MG-challenged cells. Moreover, silencing of Nrf2 blocked the ISO-dependent γ-GCL and GSH upregulation and the effects on the mitochondria of the MG-challenged cells. Then, ISO caused mitochondrial protection by an AMPK-PI3K/Akt/Nrf2/γ-GCL/GSH-dependent manner in MG-administrated SH-SY5Y cells.

Role of Zerumbone, a Phytochemical Sesquiterpenoid from Zingiber zerumbet Smith, in Maintaining Macrophage Polarization and Redox Homeostasis.

Macrophages and microglia are highly versatile cells that can be polarized into M1 and M2 phenotypes in response to diverse environmental stimuli, thus exhibiting different biological functions. In the central nervous system, activated resident macrophages and microglial cells trigger the production of proinflammatory mediators that contribute to neurodegenerative diseases and psychiatric disorders. Therefore, modulating the activation of macrophages and microglia by optimizing the inflammatory environment is beneficial for disease management. Several naturally occurring compounds have been reported to have anti-inflammatory and neuroprotective properties. Zerumbone is a phytochemical sesquiterpenoid and also a cyclic ketone isolated from Zingiber zerumbet Smith. In this study, we found that zerumbone effectively reduced the expression of lipocalin-2 in macrophages and microglial cell lines. Lipocalin-2, also known as neutrophil gelatinase-associated lipocalin (NGAL), has been characterized as an adipokine/cytokine implicated in inflammation. Moreover, supplement with zerumbone inhibited reactive oxygen species production. Phagocytic activity was decreased following the zerumbone supplement. In addition, the zerumbone supplement remarkably reduced the production of M1-polarization-associated chemokines CXC10 and CCL-2, as well as M1-polarization-associated cytokines interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α. Furthermore, the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 and the production of NO were attenuated in macrophages and microglial cells supplemented with zerumbone. Notably, we discovered that zerumbone effectively promoted the production of the endogenous antioxidants heme oxygenase-1, glutamate-cysteine ligase modifier subunit, glutamate-cysteine ligase catalytic subunit, and NAD(P)H quinone oxidoreductase-1 and remarkably enhanced IL-10, a marker of M2 macrophage polarization. Endogenous antioxidant production and M2 macrophage polarization were increased through activation of the AMPK/Akt and Akt/GSK3 signaling pathways. In summary, this study demonstrated the protective role of zerumbone in maintaining M1 and M2 polarization homeostasis by decreasing inflammatory responses and enhancing the production of endogenous antioxidants in both macrophages and microglia cells. This study suggests that zerumbone can be used as a potential therapeutic drug for the supplement of neuroinflammatory diseases.

Emodin Alleviates Sodium Taurocholate-Induced Pancreatic Ductal Cell Damage by Inhibiting the S100A9/VNN1 Signaling Pathway.

OBJECTIVES: Because the pathogenesis of the disease is unclear, the treatment of patients with acute pancreatitis, especially severe acute pancreatitis, is still a major challenge for clinicians. Emodin is an anthraquinone compound extracted from rhubarb that can alleviate the damage to pancreatic ductal epithelial cells induced by adenosine triphosphate, but whether it has a similar protective effect on sodium taurocholate (STC)-stimulated pancreatic ductal cells and the underlying mechanism has not yet been reported. METHODS: A model of STC-induced HPDE6-C7 human pancreatic ductal epithelial cell injury was established, and then apoptosis and the levels of reactive oxygen species (ROS), glutathione, gamma-glutamylcysteine synthetase, and inflammatory cytokines were assessed in the presence or absence of emodin pretreatment. S100 calcium binding protein A9 (S100A9) and Vanin1 (VNN1) protein expression was also measured. RESULTS: Emodin significantly increased HPDE6-C7 cell viability, inhibited apoptosis and ROS release, and elevated glutathione levels and gamma-glutamylcysteine synthetase activity. Furthermore, emodin downregulated S100A9 and VNN1 protein expression and inhibited the production of inflammatory factors, such as interleukin (IL)-1ß, IL-6, IL-8, and IL-18. CONCLUSIONS: Emodin attenuates STC-induced pancreatic ductal cell injury possibly by inhibiting S100A9/VNN1-mediated ROS release. This finding provides evidence for the future development of emodin as a therapeutic agent.

Antioxidant activity and protective effect of wheat germ peptides in an in vitro celiac disease model via Keap1/Nrf2 signaling pathway.

Celiac disease (CD) is an allergic intestinal disease caused primarily by gliadin and is widespread in the population. Alpha gliadin peptide causes cellular damage by substantially increasing cellular reactive oxygen species (ROS) levels. In this study, we examined the protective effect of 25 wheat germ peptides (WGPs) on the ɑ-gliadin peptide (P31-43)-treated Caco-2 cells. The experimental results showed that three peptides, WGP2, WGP7, and WGP11, significantly promoted cell viability and greatly alleviated the damage of Caco-2 cells by P31-43. According the assay of ROS, The three WGPS significantly reduced ROS to normal levels, which were elevated by P31-43 peptide. The results in terms of antioxidant-related enzymes showed that WGPs significantly increased catalase (CAT), Glutathione Reductases (GR), Glutathione peroxidase (GPx), and Glutathione (GSH)/ oxidized Glutathione (GSSG) levels, thus significantly enhancing the antioxidant level of cells. By studying the key protein expression levels of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2) pathway, the results show that WGPs could activate Nrf2 and glutamate-cysteine ligase catalytic subunit (GCLC) up-regulation. For glutamate-cysteine ligase modifier (GCLM), WGP2 and WGP7 lead to its down-regulation, while WGP11 leads to its significant up-regulation.The present study found that peptides from wheat germ can effectively mitigate the cellular damage induced by the ɑ-gliadin peptide, which provides a new perspective for the prevention and treatment of CD.

N-cystaminylbiguanide MC001 prevents neuron cell death and alleviates motor deficits in the MPTP-model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN), which is highly associated with oxidative stress. Antioxidants are therefore considered as potential therapies in PD treatment. In this study, we examined the neuroprotective effect of a cysteamine-based biguanide N-cystaminylbiguanide (MC001) in the MPTP mouse model of PD. The results showed that MC001 prevented neuron cell death and alleviated motor deficits in the MPTP mouse model of PD. Both in vitro and in vivo data indicated that MC001 may exert its neuroprotective effect by alleviating ROS production, suppressing neuroinflammation, and upregulating BDNF expression. Further mechanistic studies revealed that MC001 promoted GSH synthesis by inducing the expression of Glutamate-cysteine ligase catalytic subunit (Gclc) and enhancing the activity of Glutamate-cysteine ligase (Gcl). Our results suggest that MC001 warrants further investigation as a potential candidate for the treatment of PD.

[Mechanism of Tibetan medicine Ershiwuwei Songshi Pills against liver injury induced by acetaminophen in mice based on Keap1/Nrf2 and TLR4/NF-κB p65 signaling pathways].

The present study investigated the mechanism of the Tibetan medicine Ershiwuwei Songshi Pills(ESP) against the liver injury induced by acetaminophen(APAP) in mice based on the kelch-like ECH-associated protein 1(Keap1)/nuclear transcription factor E2 related factor 2(Nrf2) and Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) p65 signaling pathways. Kunming mice were randomly divided into a blank control group, a model group, an N-acetyl-L-cysteine(NAC) group, and high-(400 mg·kg~(-1)), medium-(200 mg·kg~(-1)), and low-dose(100 mg·kg~(-1)) ESP groups. After 14 days of continuous administration, except for those in the control group, the mice were intraperitoneally injected with 200 mg·kg~(-1) APAP. After 12 h, the serum and liver tissues of mice were collected. Hematoxylin-eosin(HE) staining was performed on pathological sections of the liver, and the levels of aspartate aminotransferase(AST) and alanine aminotransferase(ALT) in the serum and the levels of glutathione(GSH), malondialdehyde(MDA), superoxide dismutase(SOD), catalase(CAT), myeloperoxidase(MPO), and total antioxidant capacity(T-AOC) in liver tissue homogenate were detected to observe and analyze the protective effect of ESP on APAP-induced liver injury in mice. The serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1ß), and interleukin-6(IL-6) were determined by enzyme-linked immunosorbent assay(ELISA). The protein expression of Nrf2, Keap1, TLR4, and NF-κB p65 in the liver was determined by Western blot. Quantitative real-time was used to determine the mRNA expression of glutamate-cysteine ligase catalytic subunit(GCLC), glutamate-cysteine ligase regulatory subunit(GCLM), heme oxygenase-1(HO-1), and NAD(P)H dehydrogenase quinone 1(NQO-1) in the liver to explore the mechanism of ESP in improving APAP-induced liver damage in mice. As revealed by results, compared with the model group, the ESP groups showed improved liver pathological damage, decreased ALT and AST levels in the serum and MDA and MPO content in the liver, increased GSH, SOD, CAT, and T-AOC in the liver, reduced TNF-α and IL-6 levels in the serum, down-regulated expression of Keap1 in the liver cytoplasm and NF-κB p65 in the liver nucleus, up-regulated expression of Nrf2 in the liver nucleus, insignificant change in TLR4 expression, and elevated relative mRNA expression levels of antioxidant genes GCLC, GCLM, HO-1, and NQO-1. ESP can reduce the oxidative damage and inflammation caused by APAP, and the mechanism may be related to the Keap1/Nrf2 signaling pathway and the signal transduction factors on the TLR4/NF-κB p65 pathway.

Astragaloside-IV alleviates high glucose-induced ferroptosis in retinal pigment epithelial cells by disrupting the expression of miR-138-5p/Sirt1/Nrf2.

Astragaloside-IV (AS-IV) (C41H68O14) is a high-purity natural product extracted from Astragalus, which has demonstrated biological activities. However, the effect of AS-IV on retinal pigment epithelial (RPE) cells in diabetic retinopathy (DR) remains unclear. In this study, high glucose (HG) was shown to promote ARPE-19 RPE cell death, increase the contents of reactive oxygen species (ROS) and oxidized glutathione (GSSG), and enhance lipid peroxidation density of mitochondrial membrane. In contrast, AS-IV decreased glutathione (GSH) content, mitochondria size and ridge. Addition of iron death inhibitor Ferrostatin-1 (Fer-1) to RPE cells decreased cell dead rate, thus indicating that HG-induced mitochondrial damage occurred due to ferroptosis. AS-IV alleviated HG-induced RPE cell damage. Furthermore, HG decreased levels of silent information regulator 1 (Sirt1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the nucleus of RPE cells; AS-IV could alleviate these effects and increased expression of glutathione peroxidase 4 (GPX4), glutamate cysteine ligase (GCLM) and glutamate cysteine ligase catalytic subunit (GCLC), which are Nrf2 downstream genes. Mechanistically, AS-IV was shown to alleviate the effects of HG by increasing mir-138-5p expression in RPE cells and promoting expression of Sirt1 and Nrf2 in the nucleus. Transfection of mir-138-5p agonist inhibited the regulatory effects of AS-IV on Sirt1 and Nrf2, accompanied by decreased GPX4, GCLM and GCLC levels, and restoration of ferroptosis-related changes. Collectively, HG increased ferroptosis rate in RPE cells. In addition, AS-IV inhibited miR-138-5p expression, subsequently increasing Sirt1/Nrf2 activity and cellular antioxidant capacity to alleviate ferroptosis, resulting decreased cell death, which potentially inhibits the DR pathological process.

Mechanism of Tibetan medicine Ershiwuwei Songshi Pills against liver injury induced by acetaminophen in mice based on Keap1/Nrf2 and TLR4/NF-κB p65 signaling pathways / 中国中药杂志

The present study investigated the mechanism of the Tibetan medicine Ershiwuwei Songshi Pills(ESP) against the liver injury induced by acetaminophen(APAP) in mice based on the kelch-like ECH-associated protein 1(Keap1)/nuclear transcription factor E2 related factor 2(Nrf2) and Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) p65 signaling pathways. Kunming mice were randomly divided into a blank control group, a model group, an N-acetyl-L-cysteine(NAC) group, and high-(400 mg·kg~(-1)), medium-(200 mg·kg~(-1)), and low-dose(100 mg·kg~(-1)) ESP groups. After 14 days of continuous administration, except for those in the control group, the mice were intraperitoneally injected with 200 mg·kg~(-1) APAP. After 12 h, the serum and liver tissues of mice were collected. Hematoxylin-eosin(HE) staining was performed on pathological sections of the liver, and the levels of aspartate aminotransferase(AST) and alanine aminotransferase(ALT) in the serum and the levels of glutathione(GSH), malondialdehyde(MDA), superoxide dismutase(SOD), catalase(CAT), myeloperoxidase(MPO), and total antioxidant capacity(T-AOC) in liver tissue homogenate were detected to observe and analyze the protective effect of ESP on APAP-induced liver injury in mice. The serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1β), and interleukin-6(IL-6) were determined by enzyme-linked immunosorbent assay(ELISA). The protein expression of Nrf2, Keap1, TLR4, and NF-κB p65 in the liver was determined by Western blot. Quantitative real-time was used to determine the mRNA expression of glutamate-cysteine ligase catalytic subunit(GCLC), glutamate-cysteine ligase regulatory subunit(GCLM), heme oxygenase-1(HO-1), and NAD(P)H dehydrogenase quinone 1(NQO-1) in the liver to explore the mechanism of ESP in improving APAP-induced liver damage in mice. As revealed by results, compared with the model group, the ESP groups showed improved liver pathological damage, decreased ALT and AST levels in the serum and MDA and MPO content in the liver, increased GSH, SOD, CAT, and T-AOC in the liver, reduced TNF-α and IL-6 levels in the serum, down-regulated expression of Keap1 in the liver cytoplasm and NF-κB p65 in the liver nucleus, up-regulated expression of Nrf2 in the liver nucleus, insignificant change in TLR4 expression, and elevated relative mRNA expression levels of antioxidant genes GCLC, GCLM, HO-1, and NQO-1. ESP can reduce the oxidative damage and inflammation caused by APAP, and the mechanism may be related to the Keap1/Nrf2 signaling pathway and the signal transduction factors on the TLR4/NF-κB p65 pathway.

Stimulation of de novo glutathione synthesis by nitrofurantoin for enhanced resilience of hepatocytes.

Toxicity is not only a function of damage mechanisms, but is also determined by cellular resilience factors. Glutathione has been reported as essential element to counteract negative influences. The present work hence pursued the question how intracellular glutathione can be elevated transiently to render cells more resistant toward harmful conditions. The antibiotic nitrofurantoin (NFT) was identified to stimulate de novo synthesis of glutathione in the human hepatoma cell line, HepG2, and in primary human hepatocytes. In intact cells, activation of NFT yielded a radical anion, which subsequently initiated nuclear-factor-erythroid 2-related-factor-2 (Nrf2)-dependent induction of glutamate cysteine ligase (GCL). Application of siRNA-based intervention approaches confirmed the involvement of the Nrf2-GCL axis in the observed elevation of intracellular glutathione levels. Quantitative activation of Nrf2 by NFT, and the subsequent rise in glutathione, were similar as observed with the potent experimental Nrf2 activator diethyl maleate. The elevation of glutathione levels, observed even 48 h after withdrawal of NFT, rendered cells resistant to different stressors such as the mitochondrial inhibitor rotenone, the redox cycler paraquat, the proteasome inhibitors MG-132 or bortezomib, or high concentrations of NFT. Repurpose of the antibiotic NFT as activator of Nrf2 could thus be a promising strategy for a transient and targeted activation of the endogenous antioxidant machinery. Graphical abstract.

Vitamin D and L-cysteine levels correlate positively with GSH and negatively with insulin resistance levels in the blood of type 2 diabetic patients.

BACKGROUND/OBJECTIVES: Vitamin D, L-cysteine (LC) and glutathione (GSH) levels are lower in the blood of diabetic patients. This study examined the hypothesis that the levels of vitamin D and LC correlate with those of GSH in the blood of type 2 diabetic patients (T2D), and that vitamin D and LC upregulate glutamate-cysteine ligase (GCLC), which catalyzes GSH biosynthesis, in cultured monocytes. SUBJECTS/METHODS: Fasting blood was obtained after written informed consent from T2D (n=79) and healthy controls (n=22). U937 monocytes were pretreated with 1,25 (OH)2 vitamin D (0-25 nM) or LC (0-500 µM) for 24 h and then exposed to control or high glucose (25 mM) for 4 h. RESULTS: Plasma levels of vitamin D, LC, GSH and GCLC protein were significantly lower in T2D versus those in age-matched healthy controls. Multiple linear regression analyses and adjustment for body weight showed a significant positive correlation between plasma levels of vitamin D (r=0.26, P=0.05) and LC (r=0.81, P=0.001) and that of GSH, and between LC and vitamin D (r=0.27, P=0.045) levels. Plasma levels of GSH (r=-0.34, P=0.01) and LC (r=-0.33, r=0.01) showed a negative correlation with triglyceride levels. Vitamin D correlated inversely with HbA1C (-0.30, P=0.01) and homeostatic model assessment insulin resistance (r=-0.31, P=0.03), which showed a significant positive correlation with triglycerides (r=0.44, P=0.001) in T2D. Cell culture studies demonstrate that supplementation with vitamin D and LC significantly increased GCLC expression and GSH formation in control and high-glucose-treated monocytes. CONCLUSIONS: This study suggests a positive relationship between the concentrations of the micronutrients vitamin D and LC and that of GSH. Some of the beneficial effects of vitamin D and LC supplementation may be mediated by an increase in the levels of GSH and a decrease in triglyceride levels in T2D patients.

Ability of transgenic poplars with elevated glutathione content to tolerate zinc(2+) stress.

Phytoremediation potentials of four poplar lines, Populus nigra (N-SL clone), Populus canescens, and two transgenic P. canescens clones were investigated using in vitro leaf discs cultures. The transgenic poplars overexpressed a bacterial gene encoding gamma-glutamylcysteine synthetase in the cytosol (11ggs) or in the chlopoplasts (6LgI), and therefore, they contained an elevated level of glutathione. Leaf discs of poplar clones were exposed to different concentrations of ZnSO(4) for 21 days. Zinc(2+) was phytotoxic only at high concentrations (10(-2) to 10(-1) M) at all P. canescens lines, but P. nigra was more sensitive. Transgenic poplars showed elevated heavy metal uptake as compared to the nontransformed clones. Treatments with zinc(2+) strongly induced the activity of glutathione S-transferase enzyme in untransformed poplar lines but to a lesser extent in the transgenic clones. These results suggest that transgenic poplars are more suitable for phytoremediation of soils contaminated with zinc(2+) than wild-type plants.

Glutathione depletion is associated with augmenting a proinflammatory signal: evidence for an antioxidant/pro-oxidant mechanism regulating cytokines in the alveolar epithelium.

Chemioxyexcitation [deltapO2/reactive oxygen species (ROS)] constitutes a potential signaling mechanism for regulating an inflammatory signal associated with oxidative stress. Exposure of fetal alveolar type II epithelial cells to an ascending deltaPO2 regimen with or without the hydroxyl radical (OH) or the superoxide radical anion (O2*-) induces a dose-dependent release of pro-inflammatory cytokines. Similarly, the Escherichia coli-derived lipopolysaccharide (LPS) upregulates cytokine biosynthesis in a dose- and time-dependent manner. Irreversible inhibition by L-buthionine-(S,R)-sulfoximine (BSO) of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of glutathione (GSH), induces intracellular accumulation of ROS and augments chemioxyexcitation and LPS-mediated release of interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha). Analysis of the molecular mechanism implicated reveals an inhibitory kappaB (IkappaB-alpha)/nuclear factor kappaB (NF-kappaB)-independent pathway mediating the redox-dependent regulation of inflammatory cytokines. Although BSO stabilizes cytosolic IkappaB-alpha and downregulates its phosphorylation, thereby blockading NF-kappaB activation, it augments cytokine biosynthesis in a dose-dependent manner. These results indicate that glutathione depletion is associated with augmentation of an oxidative stress-mediated pro-inflammatory state in an ROS-dependent mechanism and that the IkappaB-alpha/NF-kappaB pathway is otherwise not necessarily indispensable for redox-mediated regulation of cytokines.

Basic fibroblast growth factor stimulation of glial cells protects dopamine neurons from 6-hydroxydopamine toxicity: involvement of the glutathione system.

Neurotrophic factors have been shown to support the survival and promote the recovery of injured neurons both in vivo and in vitro. Here, we investigated whether glial cell line-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) could modify the damage to dopamine (DA) neurons in mesencephalic cultures caused by the neurotoxin 6-hydroxydopamine (6-OHDA). The data show that bFGF, but not GDNF, effectively protected DA neurons from 6-OHDA toxicity. Because bFGF is a glial mitogen, whereas GDNF is not, we tested whether glial cells participated in bFGF neuroprotection. Inhibition of glial cell proliferation completely prevented the protective effect of bFGF. Because oxidative events have been associated with 6-OHDA-induced damage, we examined the levels of glutathione (GSH) in control and bFGF-treated cultures. Cultures treated with bFGF had higher levels of GSH, which increased even further in response to 6-OHDA exposure. Control cultures failed to up-regulate GSH levels after 6-OHDA, suggesting a relationship between increased GSH levels and protection from 6-OHDA. Inhibition of glial cell proliferation prevented the rise in GSH in bFGF-treated cultures and abolished the increase after 6-OHDA treatment. Protection from 6-OHDA by bFGF was also diminished when GSH levels were decreased by the GSH synthesis inhibitor L-buthionine sulfoximine. Our study shows that stimulation of glial cells by bFGF allows the up-regulation of antioxidant defenses and supports cell survival during oxidative stress.

Alpha-lipoic acid prevents buthionine sulfoximine-induced cataract formation in newborn rats.

We investigated the effect of alpha-lipoic acid, a powerful antioxidant, on cataract formation in L-buthionine(S,R)-sulfoximine (BSO)-treated newborn rats and found that a dose of 25 mg/kg b.w. protected 60% of animals from cataract formation. L-buthionine(S,R)-sulfoximine is an inhibitor of glutathione synthesis, whose administration to newborn animals leads to the development of cataracts; this is a potential model for studying the role of therapeutic antioxidants in protecting animals from cataract formation. Major biochemical changes in the lens associated with the protective effect of alpha-lipoic acid were increases in glutathione, ascorbate, and vitamin E levels, loss of which are effects of BSO administration. Treatment with alpha-lipoic acid also restored the activities of glutathione peroxidase, catalase, and ascorbate free radical reductase in lenses of L-buthionine(S,R)-sulfoximine-treated animals but did not affect glutathione reductase or superoxide dismutase activity. We conclude that alpha-lipoic acid may take over some of the functions of glutathione (e.g., maintaining the higher level of ascorbate, indirect participation in vitamin E recycling); the increase of glutathione level in lens tissue mediated by lipoate could be also due to a direct protection of protein thiols. Thus, alpha-lipoic acid could be of potential therapeutic use in preventing cataracts and their complications.