Integrated approach to the mechanisms of thyroid toxins: electron transfer,reactive oxygen species,oxidative stress,cell signaling,receptors, and antioxidants

Although considerable numbers of reviews are available on toxicity of major body organs based on electron transfer (ET), reactive oxygen species (ROS), and oxidative stress (OS), the integrated concept has been less applied to glands. This review represents an interdisciplinary approach to thyroid toxicity, involving ET, ROS, OS, cell signaling, receptors, toxicants, and beneficial effects of antioxidants (AOs). The introductory portion includes general function of the thyroid as well as the mechanism of thyroxine synthesis entailing participation of oxidative events, including the role of iodine. Various ROS, both endogenous and exogenous, are importantly involved in the diverse toxic manifestations. Discussion is centered on hydrogen peroxide and lipid peroxides. There is also treatment of receptor-ligand activity. Cell signaling participates in the various biochemical events taking place in the thyroid, both beneficial and adverse. In addition, the mechanism of cell signaling is discussed based on radicals, ET, relays, conduits, and electrochemistry. In addition to endogenous toxins, various exogenous ones are addressed, falling in diverse classes. Data indicate involvement of ET-ROS-OS in the toxic manifestations. Large numbers of reports reveal the beneficial effects of AOs in countering the toxicity, which is in accord with the mechanistic framework.     

Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor

Chloroquine, a widely used anti-malarial and anti-rheumatoid agent, has been reported to induce apoptotic and non-apoptotic cell death. Accumulating evidence now suggests that chloroquine can sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis by inhibiting autophagy. However, chloroquine is reported to induce GM1 ganglioside accumulation in cultured cells at low μM concentrations and prevent damage to the blood brain barrier in mice. It remains unknown whether chloroquine has neuroprotective properties at concentrations below its reported ability to inhibit lysosomal enzymes and autophagy. In the present study, we demonstrated that chloroquine protected mouse hippocampal HT22 cells from glutamate-induced oxidative stress by attenuating production of excess reactive oxygen species. The concentration of chloroquine required to rescue HT22 cells from oxidative stress was much lower than that sufficient enough to induce cell death and inhibit autophagy. Chloroquine increased GM1 level in HT22 cells at low μM concentrations but glutamate-induced cell death occurred before GM1 accumulation, suggesting that GM1 induction is not related to the protective effect of chloroquine against glutamate-induced cell death. Interestingly, BD1047 and NE-100, sigma-1 receptor antagonists, abrogated the protective effect of chloroquine against glutamate-induced cell death and reactive oxygen species production. In addition, cutamesine (SA4503), a sigma-1 receptor agonist, prevented both glutamate-induced cell death and reactive oxygen species production. These findings indicate that chloroquine at concentrations below its ability to inhibit autophagy and induce cell death is able to rescue HT22 cells from glutamate-induced cell death by reducing excessive production of reactive oxygen species through sigma-1 receptors. These results suggest potential use of chloroquine, an established anti-malarial agent, as a neuroprotectant against oxidative stress, which occurs in a variety of neurodegenerative diseases.     

Interrelation of active oxygen species,membrane damage and altered calcium functions

Incubation of freshly isolated rat liver mitochondria in the presence of oxygen free radical generating hypoxanthine —xanthine oxidase system led to swelling of mitochondria as measured by the change in optical density, which was reversed by the addition of superoxide dismutase. O₂ ^– in the presence of CaCl₂ enhanced the peroxidative decomposition of mitochondrial membrane lipids along with swelling of the organelle. Free radical generation led to enhancement of monoamine oxidase activity while glutathione peroxidase and cytochrome c oxidase were inhibited. Tertbutyl hydroperoxide (t-BHP) caused mitochondrial swelling through oxidative stress. Incorporation of ruthenium red, which is a Ca²⁺ transport blocker, during assay abolished peroxidative membrane damage and swelling. Dithiothreitol (DTT) accorded protection against t-BHP induced mitochondrial swelling. The above in vitro data suggest a possible interrelationship of active oxygen species, membrane damage and calcium dynamics.     

The three Rs along the TRAIL: Resistance,re-sensitization and reactive oxygen species (ROS)

Abstract

Ligation of the Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors has been associated with cancer specific apoptotic execution in a number of model systems. This has generated tremendous interest in the use of TRAIL as a potential therapeutic modality. However, recent evidence indicates that resistance to TRAIL might present with a therapeutic challenge. In this short report, we review the basic biology of TRAIL signalling in cancer cells, highlight the mechanisms underlying resistance to TRAIL and the ability of small molecule compounds to re-sensitize cells to TRAIL-mediated apoptosis. In particular, we provide evidence that intracellular reactive oxygen species could be critical in regulating the response of cancer cells to TRAIL.     

p38 MAPK: A dual role in hepatocyte proliferation through reactive oxygen species

Abstract

p38 MAPKs are important mediators of signal transduction that respond to a wide range of extracellular stressors such as UV radiation, osmotic shock, hypoxia, pro-inflammatory cytokines, and oxidative stress. The most abundant family member is p38α, which helps to couple cell proliferation and growth in response to certain damaging stimuli. In fact, increased proliferation and impaired differentiation are hallmarks of p38α-deficient cells. It has been reported that reactive oxygen species (ROS) play a critical role in cytokine-induced p38α activation. Under physiological conditions, p38α can function as a mediator of ROS signaling and either activate or suppress cell cycle progression depending on the activation stimulus. The interplay between cell proliferation, p38 MAPK activation, and ROS production plays an important role in hepatocytes. In fact, low levels of ROS seem to be needed to activate several signaling pathways in response to hepatectomy and to orchestrate liver regeneration. p38 MAPK works as a sensor of oxidative stress and cells that have developed mechanisms to uncouple p38 MAPK activation from oxidative stress are more likely to become tumorigenic. So far, p38α influences the redox balance, determining cell survival, terminal differentiation, proliferation, and senescence. Further studies would be necessary in order to clarify the precise role of p38 MAPK signaling as a redox therapeutical target.     

Scavenging of reactive oxygen species by a novel glucurinated flavonoid antioxidant isolated and purified from spinach

NAO is a natural water soluble antioxidant that was isolated and purified from spinach leaves. Using HPLC, NMR, and CMR spectroscopy, the main components were identified as flavonoids and p-coumaric acid derivatives. The NAO was found to be a very effective antioxidant in several in vivo and in vitro biological systems. In the present study, the antioxidant activity of the novel antioxidant glucurinated flavonoid (GF) isolated and characterized from NAO, is compared to well-known antioxidants. In addition, the direct free radical scavenging properties of the purified component GF were studied using the electron spin resonance (ESR) technique. GF and NAO were found to be superior to EGCG and NAC and to the Vitamin E homologue Trolox in inhibiting reactive oxygen species (ROS) formation in the autooxidation system of linoleic acid and in fibroblasts exposed to metal oxidation. GF and NAO were found to inhibit the ESR signal intensity of DMPO-O(2) radical formation during the riboflavin photodynamic reaction. 10 mM GF caused approximately 90% inhibition in the intensity of the ESR signal, while NAO at a concentration of 60 microg/ml caused an inhibition of about 50%. Using the Fenton reaction, GF and NAO were found to inhibit DMPO-OH radical formation. A concentration of 2 mM GF caused a 70% inhibition in the intensity of the DMPO-OH radical ESR signal, while propyl gallate at the same concentration caused only 50% inhibition. Furthermore, both GF and NAO also inhibited the (1)O(2) dependent TEMPO radical generated in the photoradiation TPPS4 system. About 80% inhibition was obtained by 4 mM GF. The results obtained indicate that the natural antioxidants derived from spinach may directly affect the scavenging of ROS and, as a consequence, may be considered as effective sources for combating oxidative damage.     

Protection against reactive oxygen species during mouse preimplantation embryo development: role of EDTA, oxygen tension, catalase, superoxide dismutase and pyruvate

Oxidative damage due to the production of reactive oxygen species (ROS) is one of a number of culture-induced stresses which may compromise preimplantation embryo development in vitro. Ethylenediaminetetraacetic acid (EDTA), reduced oxygen tension, superoxide dismutase (SOD) and catalase (CAT) offer protection against oxidative stress, but few attempts have been made to determine which of these agents, or which combination, is the most effective. In particular, no systematic investigation of their actions and interactions has been made using a multifactorial experimental design. Murine zygotes were cultured in the presence or absence of 10 miccroM EDTA, SOD (100-7,000 U/ml) and CAT (50-100 U/ml) at atmospheric (20%) and reduced (5%) oxygen tensions. Blastocyst formation and hatching rates (at various time points), and cell numbers were recorded, whilst parallel groups of embryos had their consumption of pyruvate, a hydrogen peroxide scavenger, measured. All parameters interacted significantly and affected blastocyst formation, hatching rate and cell numbers but the effect of EDTA was the most pronounced. There were beneficial effects of 5% O2, CAT and SOD, while 20% O2 had a deleterious effect on development. EDTA improved blastocyst formation and hatching rates but paradoxically led to a reduction in cell number. 5% O2 was the next most significant parameter to enhance embryo development and also increased cell numbers. No differences in pyruvate uptake were apparent between the various treatment groups. The results suggest that embryo culture in EDTA-free medium under 5% O2 provides the most practical and physiological conditions for in vitro murine embryo culture.     

Induction of oxidative cell damage by photo-treatment with zinc meta N-methylpyridylporphyrin

We have previously reported that isomeric Zn(II) N-methylpyridylporphyrins (ZnTM-2(3,4)-PyP^4 + ) can act as photosensitizers with efficacy comparable to that of hematoporphyrin derivative (HpD) in preventing cell proliferation and causing cell death in vitro. To better understand the biochemical basis of this activity, the effects of photo-activated ZnTM-3-PyP^4 +  on GSH/GSSG ratio, lipid peroxidation, membrane permeability, oxidative DNA damage, and the activities of SOD, catalase, glutathione reductase, and glutathione peroxidase were evaluated. Light exposure of ZnTM-3-PyP^4 + -treated colon adenocarcinoma cells caused a wide spectrum of oxidative damage including depletion of GSH, inactivation of glutathione reductase and glutathione peroxidase, oxidative DNA damage and peroxidation of membrane lipids. Cell staining with Hoechst-33342 showed morphological changes consistent with both necrotic and apoptotic death sequences, depending upon the presence of oxygen.     

Effects of unaccustomed downhill running on muscle damage,oxidative stress,and leukocyte apoptosis

[Purpose]

The purpose of this study was to investigate the effect of unaccustomed downhill running on muscle damage, oxidative stress, and leukocyte apoptosis.

[Methods]

Thirteen moderately trained male subjects performed three 40 min treadmill runs at ~70% VO_2max on separate days: a level run (L) followed by two downhill runs (DH1 and DH2). Blood samples were taken at rest (PRE) and immediately (POST), 2 h, 24 h, and 48 h after each run. Data were analyzed using 2-way repeated measures ANOVA with post hoc Tukey tests.

[Results]

Creatine kinase (CK) activity and oxidative stress level were significantly elevated at 24 h and 48 h following DH1 (P < 0.05). The level of oxidative stress at the POST measurement following DH1 and DH2 was greater than PRE. The rate of leukocyte apoptosis was significantly increased at the POST measurement following all three runs, and remained elevated for up to 48 h following DH1 (P < 0.01).

[Conclusion]

CK activity and oxidative stress were elevated following an acute bout of moderate intensity downhill running, resulting in a greater apoptotic response at 24 h and 48 h post-exercise in comparison with level grade running or a second downhill run. These elevations were blunted following DH2. Although the link between exercise-induced muscle damage and leukocyte apoptosis is currently unknown, the differential response to DH1 vs. L and DH2 indicates that it may be mediated by the elevation of oxidative stress.     

Chikungunya-induced cell death is limited by ER and oxidative stress-induced autophagy

It has been recognized that macroautophagy constitutes an important survival mechanism that allows both the maintenance of cellular homeostasis and the regulation of programmed cell death pathways (e.g., apoptosis). Although several pathogens have been described to induce autophagy, the prosurvival function of this process in infectious models remains poorly characterized. Our recent studies on chikungunya virus (CHIKV), the causative agent of major epidemics in India, Southeast Asia and southern Europe, reveal a novel mechanism by which autophagy limits the cytopathic effects of CHIKV by impinging upon virus-induced cell death pathways.     

Modulation of the GABA(A)-gated chloride channel by reactive oxygen species

The accumulation of reactive oxygen species during cellular injury leads to oxidative stress. This can have profound effects on ionic homeostasis and neuronal transmission. Gamma-aminobutyric acid (GABA) neurotransmission is sensitive to reactive oxygen species, but most studies have indicated that this is due to alterations in GABA release. Here, we determined whether reactive oxygen species can alter GABA(A) receptor-gated Cl- channels in the adult hippocampus. First, we measured the effects of hydrogen peroxide on intracellular Cl- using UV laser scanning confocal microscopy and the Cl(-)-sensitive probe, 6-methoxy-N-ethylquinolium iodide (MEQ). Superfusion of adult rat hippocampal slices with hydrogen peroxide for 10 min decreased MEQ fluorescence (elevation in [Cl-]i) significantly in area CA1 pyramidal cell soma. Alterations in [Cl-]i were prevented by the vitamin E analog Trolox, an antioxidant that scavenges free radicals. After exposure of slices to hydrogen peroxide, the ability of the GABA agonist muscimol to increase [Cl-]i was attenuated. To determine if GABA(A) receptors were sensitive to oxidative insults, the effect of hydrogen peroxide on the binding of [35S]t-butylbicyclophosphorothionate (TBPS) to GABA-gated Cl- channels was measured using receptor autoradiography and homogenate binding assays. Hydrogen peroxide inhibited [35S]TBPS binding in a regionally selective manner, with the greatest inhibition in cerebral cortex, hippocampus and striatum, areas vulnerable to oxidative stress. Similarly, xanthine and xanthine oxidase, which generate superoxide radicals, reduced [35S]TBPS binding in these regions. The effect of hydrogen peroxide on [35S]TBPS binding was non-competitive and was prevented by Trolox and the iron chelator, deferoxamine. We conclude that reactive oxygen species may compromise GABA(A)-mediated neuronal inhibition via interaction with pre and postsynaptic sites. A reduction in GABA(A)-gated Cl- channel function during periods of oxidative stress may contribute to the development of neuronal damage.     

Fabrication of nanofiber coated with l-arginine via electrospinning technique: a novel nanomatrix to counter oxidative stress under crosstalk of co-cultured fibroblasts and satellite cells

The objective of this study was to synthesize and characterize novel polyurethane (PU)-nanofiber coated with l-arginine by electrospinning technique. This study determined whether l-arginine conjugated with PU-nanofiber could stimulate cell proliferation and prevent H₂O₂-induced cell death in satellite cells co-cultured with fibroblasts isolated from Hanwoo (Korean native cattle). Our results showed that l-arginine conjugated with PU nanofiber could reduce cytotoxicity of co-cultured satellite cells. Protein expression levels of bcl-2 were significantly upregulated whereas those of caspase-3 and caspase-7 were significantly downregulated in co-culture of satellite cells compared to those of monoculture cells after treatment with PU-nanofiber coated with l-arginine and which confirmed by Confocal microscope. These results suggest that co-culture of satellite cells with fibroblasts might be able to counter oxidative stress through translocation/penetration of antioxidant, collagen, and molecules secreted to satellite cells. Therefore, this nanofiber might be useful as a wound dressing in animals to counter oxidative stresses.     

cAMP/protein kinase A signalling pathway protects against neuronal apoptosis and is associated with modulation of Kv2.1 in cerebellar granule cells

Previously, we have reported that apoptosis of cerebellar granular neurons induced by incubation in 5 mm K(+) and serum-free medium (LK-S) was associated with an increase in the delayed rectifier K(+) current (I(K)). Here, we show that I(K) associated with apoptotic neurons is mainly encoded by a Kv2.1 subunit. Silencing Kv2.1 expression by small interfering RNA reduces I(K) and increases neuron viability. Forskolin is able to decrease the I(K) amplitude recording from neurons of both the LK-S and control group, and prevents apoptosis of granule cells that are induced by LK-S. Dibutyryl cAMP mimicks the effect of forskolin on the modulation of I(K) and, accordingly, the inhibitor of protein kinase A, H-89, aborts the neuron-protective effect induced by forskolin. Whereas the expression of Kv2.1 was silenced by Kv2.1 small interfering RNA, the inhibition of forskolin on the current amplitude was significantly reduced. Quantitative RT-PCR and whole-cell recording revealed that the expression of Kv2.1 was elevated in the apoptotic neurons, and forskolin significantly depressed the expression of Kv2.1. We conclude that the protection against apoptosis via the protein kinase A pathway is associated with a double modulation on I(K) channel properties and its expression of alpha-subunit that is mainly encoded by the Kv2.1 gene.     

Chemiluminescence assay for reactive oxygen species scavenging activities and inhibition on oxidative damage of DNA in Deinococcus radiodurans.

Free radical scavenging effects of the cellular protein extracts from two strains of Deinococcus radiodurans and Escherichia coli against O2-, H2O2 and *OH were investigated by chemiluminescence (CL) methods. The cellular protein extracts of D. radiodurans R1 and KD8301 showed higher scavenging effects on O2- than that of E. coli. D. radiodurans R1 and KD8301 also strongly scavenged H2O2 with an EC50 (50% effective concentration) of 0.12 and 0.2 mg/mL, respectively, compared to that of E. coli (EC50 = 3.56 mg/mL). The two strains of D. radiodurans were effective in scavenging *OH generated by the Fenton reaction, with EC50 of 0.059 and 0.1 mg/mL, respectively, compared to that of E. coli (EC50 > 1 mg/mL). Results from the chemiluminescence assay of *OH-induced DNA damage and the plasmid pUC18 DNA double-strand break (DSB) model in vitro showed that D. radiodurans had remarkably inhibitory effect on the *OH-induced oxidative damage of DNA. The scavenging effects of D. radiodurans on reactive oxygen species (ROS) played an important role in the response to oxidation stress and preventing against DNA oxidative damage, and may be attributed to intracellular scavenging proteins, including superoxide dismutase (SOD) and catalase.     

H2O2 at physiological concentrations modulates Leydig cell function inducing oxidative stress and apoptosis

H₂O₂ is one of the active reactive oxygen species secreted by macrophages that are seen closely aligned with Leydig cells in the testicular interstitium. The present study was initiated to investigate the role of H₂O₂ on Leydig cell function in vitro at physiological concentrations. Significant decrease in both testosterone production (p < 0.05) and 3 β-hydroxysteroid dehydrogenase activity (p < 0.05) in adult Leydig cells were observed even with H₂O₂ at low concentrations (30 – 50 μM). H₂O₂ exposure increased oxidative stress in Leydig cells with the rise in lipid peroxidation and fall in the activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT) & glutathione-s-transferase (GST). There was also a marginal increase (∼8%) in cell apoptosis accompanied by rise in FasL expression and caspase-3 activation. The above findings indicate that H₂O₂ as a bio-molecule modulates Leydig cell function at or below physiological concentrations through a variety of actions like decrease in steroidogenic enzyme activity and increase in oxidative stress and apoptosis.