Intracellular Redox Environment Research Articles

Low Intensity Exercise Attenuates Acute Lipid Loading‐Induced Alterations in Mitochondrial Function in Rat Skeletal Muscle

A single high‐fat meal acutely increases skeletal muscle mitochondrial H2O2 emitting potential (mEH2O2), shifts the intracellular redox environment to a more oxidized state, and increases circulating markers of oxidative stress. Bioenergetically, this implies an acute lipid load may elevate the reducing pressure/membrane potential (Δ Ψm) within mitochondria and, conversely, that even a mild increase in energy expenditure may be sufficient to prevent these effects. To test this hypothesis, male Sprague‐Dawley rats received an oral lipid gavage (20% intralipid, 45 Kcal/kg lean body mass) or water followed either by 2h of rest or 1h of rest plus 1h of low intensity treadmill exercise (15 m/min, 0% grade). Permeabilized fiber bundles were prepared from red gastrocnemius muscle for testing mitochondrial function. In rats receiving lipid, Δ Ψm and mEH2O2 were higher (P<0.05) and calcium retention capacity (mCa2+ RC, an index of resistance to mitochondrial permeability transition) was lower under state IV and/or “clamped” ADP‐stimulated state III conditions. All three effects were prevented when lipid gavage was followed by low‐intensity exercise. Respiratory capacity was unaffected by any of the interventions. These findings provide evidence that mitochondrial Δ Ψm, mEH2O2, and mCa2+ RC are acutely affected by nutritional overload in skeletal muscle, but can be prevented by low intensity exercise. NIH DK073488

Palmitate Improves Redox Balance and Enhances Contractility While Offsetting Adverse Effects of Hyperglycemia in the Diabetic Cardiomyocyte

High blood glucose and fatty acid levels constitute main hallmarks of diabetes. In heart from diabetic animals, wide variations in plasmatic glucose combined with sympathetic hyperactivation represent a challenge to mitochondria that have to supply energy and maintain redox balance. Our previous data in db/db mice unveiled a significant redox imbalance in heart cells under combined hyperglycemia (HG) and beta adrenergic stimulation (via isoproterenol, ISO), leading to cardiomyocyte dysfunction. Herein, we address the role of palmitate (Palm) upon the adverse effects of HG+ISO on diabetic cardiomyocyte contractility and intracellular redox status. The ISO (10nM) response of WT vs db/db myocytes subjected to 5mM and 30mM glucose, in the absence and presence of 0.4mM (WT) and 0.8mM (db/db) Palm, was examined comparatively with respect to: i) fractional sarcomere shortening (FS) and Ca2+ transients, and ii) the intracellular status of NADH, reduced glutathione (GSH) and reactive oxygen species (ROS). Both WT and db/db myocytes exhibited a significant increase in ISO response with Palm under 5mM glucose: FS increased by 320% and 323% in WT and db/db, p<0.01 vs 193% and 178%, respectively, no Palm. Under HG, FS was similarly enhanced in WT with Palm (372% vs 181% with no Palm, p<0.01). Diabetic myocytes showed lower contractility in HG+ISO (94%, p<0.01 vs WT) with Palm enhancing the ISO response to 380%. Under every condition, Palm strongly decreased ROS (15- to 60-fold) and significantly increased GSH (20% to 40%) in both WT and db/db. NADH was significantly increased in WT (40% to 50%) but decreased with db/db (30% to 35%). These findings reveal that Palm enhances contractility of db/db myocytes via reduction of the intracellular redox environment, thus overcoming the adverse effects of HG under ISO stimulation.

Highly sensitive electrochemical detection of potential cytotoxicity of CdSe/ZnS quantum dots using neural cell chip

Cell chip was recently developed as a simple and highly sensitive tool for the toxicity assessment of various kinds of chemicals or nano-materials. Here, we report newly discovered potential cytotoxic effects of CdSe/ZnS quantum dots (QDs) on intracellular redox environment of neural cancer cells at very low concentrations which can be only detected by cell chip technology. Green (2.1 nm in diameter) and red (6.3 nm in diameter) QDs capped with cysteamine (CA) or thioglycolic acid (TA) were found to be toxic at 100 μg/mL when assessed by trypan blue and differential pulse voltammetry (DPV). However, in case of concentration-dependent cytotoxicity, toxic effects of TA-capped QDs on human neural cells were only measured by DPV method when conventional MTT assay did not show toxicity of TA-capped QDs at low concentrations (1–10 μg/mL). Red-TA QDs and Green-TA QDs were found to decrease electrochemical signals from cells at 10 μg/mL and 5 μg/mL, respectively, while cell viability decreased at 100 μg/mL and 50 μg/mL when assessed by MTT assay, respectively. The relative decreases of cell viability determined by MTT assay were 15% and 11.9% when cells were treated with 5–50 μg/mL of Red-TA QDs and 5–30 μg/mL of Green-TA QDs, respectively. However, DPV signals decreased 37.5% and 39.2% at the same concentration range, respectively. This means that redox environment of cells is more sensitive than other components and can be easily affected by CdSe/ZnS QDs even at low concentrations. Thus, our proposed neural cell chip can be applied to detect potential cytotoxicity of various kinds of molecular imaging agents simply and accurately.

Hydrogen peroxide signaling is required for glucocorticoid-induced apoptosis in lymphoma cells

Glucocorticoid-induced apoptosis is exploited clinically for the treatment of hematologic malignancies. Determining the required molecular events for glucocorticoid-induced apoptosis will identify resistance mechanisms and suggest strategies for overcoming resistance. In this study, we found that glucocorticoid treatment of WEHI7.2 murine thymic lymphoma cells increased the steady-state [H 2O 2] and oxidized the intracellular redox environment before cytochrome c release. Removal of glucocorticoids after the H 2O 2 increase resulted in a 30% clonogenicity; treatment with PEG–CAT increased clonogenicity to 65%. Human leukemia cell lines also showed increased H 2O 2 in response to glucocorticoids and attenuated apoptosis after PEG–CAT treatment. WEHI7.2 cells that overexpress catalase (CAT2, CAT38) or were selected for resistance to H 2O 2 (200R) removed enough of the H 2O 2 generated by glucocorticoids to prevent oxidation of the intracellular redox environment. CAT2, CAT38, and 200R cells showed a 90–100% clonogenicity. The resistant cells maintained pERK survival signaling in response to glucocorticoids, whereas the sensitive cells did not. Treating the resistant cells with a MEK inhibitor sensitized them to glucocorticoids. These data indicate that: (1) an increase in H 2O 2 is necessary for glucocorticoid-induced apoptosis in lymphoid cells, (2) increased H 2O 2 removal causes glucocorticoid resistance, and (3) MEK inhibition can sensitize oxidative stress-resistant cells to glucocorticoids.

Calcium and Reactive Oxygen Species in Acute Pancreatitis: Friend or Foe?

Acute pancreatitis (AP) is a debilitating and, at times, lethal inflammatory disease, the causes and progression of which are incompletely understood. Disruption of Ca(2+) homeostasis in response to precipitants of AP leads to loss of mitochondrial integrity and cellular necrosis. While oxidative stress has been implicated as a major player in the pathogenesis of this disease, its precise roles remain to be defined. Recent developments are challenging the perception of reactive oxygen species (ROS) as nonspecific cytotoxic agents, suggesting that ROS promote apoptosis that may play a vital protective role in cellular stress since necrosis is avoided. Fresh clinical findings have indicated that antioxidant treatment does not ameliorate AP and may actually worsen the outcome. This review explores the complex links between cellular Ca(2+) signaling and the intracellular redox environment, with particular relevance to AP. Recent publications have underlined the importance of both Ca(2+) and ROS within the pathogenesis of AP, particularly in the determination of cell fate. Future research should elucidate the subtle interplay between Ca(2+) and redox mechanisms that operate to modulate mitochondrial function, with a view to devising strategies for the preservation of organellar function.

Plastidic Phosphoglycerate Kinase from Phaeodactylum tricornutum: On the Critical Role of Cysteine Residues for the Enzyme Function

Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in photosynthetic organisms, catalyzing a key step in the Calvin cycle. We performed the molecular cloning of the gene encoding chloroplastidic PGKase-1 in the diatom Phaeodactylum tricornutum. The recombinant enzyme was expressed in Escherichia coli, purified and characterized. Afterward, it showed similar kinetic properties than the enzyme studied from other organisms, although the diatom enzyme displayed distinctive responses to sulfhydryl reagents. The activity of the enzyme was found to be dependent on the redox status in the environment, determined by different compounds, including some of physiological function. Treatment with oxidant agents, such as diamide, hydrogen peroxide, glutathione and sodium nitroprusside resulted in enzyme inhibition. Recovery of activity was possible by subsequent incubation with reducing reagents such as dithiothreitol and thioredoxins (from E. coli and P. tricornutum). We determined two midpoint potentials of different regulatory redox centers, both values indicating that PGKase-1 might be sensitive to changes in the intracellular redox environment. The role of all the six Cys residues found in the diatom enzyme was analyzed by molecular modeling and site-directed mutagenesis. Results suggest key regulatory properties for P. tricornutum PGKase-1, which could be relevant for the functioning of photosynthetic carbon metabolism in diatoms.

Plant Glutathione Biosynthesis: Diversity in Biochemical Regulation and Reaction Products

In plants, exposure to temperature extremes, heavy metal-contaminated soils, drought, air pollutants, and pathogens results in the generation of reactive oxygen species that alter the intracellular redox environment, which in turn influences signaling pathways and cell fate. As part of their response to these stresses, plants produce glutathione. Glutathione acts as an anti-oxidant by quenching reactive oxygen species, and is involved in the ascorbate–glutathione cycle that eliminates damaging peroxides. Plants also use glutathione for the detoxification of xenobiotics, herbicides, air pollutants (sulfur dioxide and ozone), and toxic heavy metals. Two enzymes catalyze glutathione synthesis: glutamate–cysteine ligase, and glutathione synthetase. Glutathione is a ubiquitous protective compound in plants, but the structural and functional details of the proteins that synthesize it, as well as the potential biochemical mechanisms of their regulation, have only begun to be explored. As discussed here, the core reactions of glutathione synthesis are conserved across various organisms, but plants have diversified both the regulatory mechanisms that control its synthesis and the range of products derived from this pathway. Understanding the molecular basis of glutathione biosynthesis and its regulation will expand our knowledge of this component in the plant stress response network.

Imaging in real-time with FRET the redox response of tumorigenic cells to glutathione perturbations in a microscale flow

Despite the potential benefits of selective redox-modulating strategies for cancer therapy, an efficacious methodology for testing therapies remains elusive because of the difficulty in measuring intracellular redox potentials over time. In this report, we have incorporated a new FRET-based biosensor to follow in real time redox-sensitive processes in cells transformed to be tumorigenic and cultured in a microfluidic channel. A microfluidic network was used to control micro-scale flow near the cells and at the same time deliver drugs exogenously. Subsequently, the response of a redox homeostasis circuit was tested, namely reduced glutathione (GSH)/oxidized glutathione(GSSG), to diamide, a thiol oxidant, and two drugs used for cancer therapies: BSO (L-buthionine-[SR]-sulfoximine) and BCNU (carmustine). The main outcome from these experiments is a comparison of the temporal depletion and recovery of GSH in single living cells in real-time. These data demonstrate that mammalian cells are capable of restoring a reduced intracellular redox environment in minutes after an acute oxidative insult is removed. This recovery is significantly delayed by (i) the inhibition of GSH biosynthesis by BSO; (ii) the inactivation of glutathione reductase by BCNU; and (iii) in tumorigenic cells relative to an isogenic non-tumorigenic control cell line.

Improvement of Physiological Characteristic of Selenium-Enriched Candida utilis with Amino Acids Addition

The effects of amino acids addition on cell growth, glutathione biosynthesis, glutathione distribution, and the intracellular oxidation-reduction environment of Candida utilis SZU 07-01 during selenium enrichment were investigated in this study. Most amino acids under appropriate concentrations have positive effects on cell growth of the yeast strain, except for phenylalanine and proline, compared with the control without amino acid addition. The bioconversion of selenite to organic selenium induced the reduction of glutathione synthesis and intracellular distribution of glutathione. However, amino acids including cysteine, glutamine, glutamic acid, isoleucine, leucine, and tyrosine could effectively promote the selenium-enriched yeast to elevate glutathione production, especially increasing the intracellular glutathione content. Moreover, addition of these six different amino acids apparently decreased malondialdehyde concentration and recovered the normal intracellular redox environment of the selenium-enriched C. utilis SZU 07-01. The improvement of physiological characteristic of the selenium-enriched yeast by increasing intracellular glutathione content and lowering malondialdehyde content will undoubtedly help to widen application of selenium-enriched yeast as food or feed additives.

Regulatory Effects of Nitric Oxide on Src Kinase, FAK, p130Cas, and Receptor Protein Tyrosine Phosphatase Alpha (PTP-α): A Role for the Cellular Redox Environment

The role of NO in regulating the focal adhesion proteins, Src, FAK, p130 Cas, and PTP-alpha, was investigated. Fibroblasts expressing PTP-alpha (PTP-alpha(WT) cells), fibroblasts "knockout" for PTP-alpha (PTP-alpha(-/-) cells), and "rescued" "knockout" fibroblasts (PTP-alpha A5/3 cells) were stimulated with either S-nitroso-N-acetylpenicillamine (SNAP) or fetal bovine serum (FBS). FBS increased inducible NO synthase in both cell lines. Activation of Src mediated either by SNAP or by FBS occurred independent of dephosphorylation of Tyr527 in PTP-alpha(-/-) cells. Both stimuli promoted dephosphorylation of Tyr527 and activation of Src kinase in PTP-alpha(WT) cells. NO-mediated activation of Src kinase affected the activities of FAK and p130Cas and was dependent on the expression of PTP-alpha. Analogous to tyrosine phosphorylation, SNAP and FBS stimulated differential generation of NO and S-nitrosylation of Src kinase in both cell lines. Incubation with SNAP resulted in higher levels of NO and S-nitrosylation of immunoprecipitated Src in PTP-alpha(-/-) cells (oxidizing redox environment) as compared with the levels of NO and S-nitrosylated Src in PTP-alpha(WT) cells (reducing redox environment). SNAP differentially stimulated cell proliferation of both cell lines is dependent on the intracellular redox environment, Src activity, and PTP-alpha expression. This dependence also is observed with FBS-stimulated cell migration.

Peroxisomal Plant 3-Ketoacyl-CoA Thiolase Structure and Activity Are Regulated by a Sensitive Redox Switch

The breakdown of fatty acids, performed by the beta-oxidation cycle, is crucial for plant germination and sustainability. beta-Oxidation involves four enzymatic reactions. The final step, in which a two-carbon unit is cleaved from the fatty acid, is performed by a 3-ketoacyl-CoA thiolase (KAT). The shortened fatty acid may then pass through the cycle again (until reaching acetoacetyl-CoA) or be directed to a different cellular function. Crystal structures of KAT from Arabidopsis thaliana and Helianthus annuus have been solved to 1.5 and 1.8 A resolution, respectively. Their dimeric structures are very similar and exhibit a typical thiolase-like fold; dimer formation and active site conformation appear in an open, active, reduced state. Using an interdisciplinary approach, we confirmed the potential of plant KATs to be regulated by the redox environment in the peroxisome within a physiological range. In addition, co-immunoprecipitation studies suggest an interaction between KAT and the multifunctional protein that is responsible for the preceding two steps in beta-oxidation, which would allow a route for substrate channeling. We suggest a model for this complex based on the bacterial system.

Arginase 2 and nitric oxide synthase: Pathways associated with the pathogenesis of thyroid tumors

We have previously shown that ARG2 expression was increased in most malignant thyroid tumors, but absent in benign lesions and normal tissues. Small interfering RNA knockdown was used to investigate the role of ARG2 in a thyroid carcinoma cell line. ARG2 knockdown decreased eNOS expression as well as the expression of eNOS-related genes ( p21, Akt1, HIF-1, VEGF, and CAV1). ARG2 silencing changed tumor properties of thyroid cancer cells promoting apoptosis and reduced expression of cell proliferation markers. These results, coupled with enhanced nitric oxide production and elevated reactive oxygen species (ROS) levels, account for the altered intracellular redox environment. Genes related to either production ( DUOX1 and NOX4) or catabolism (SODs) of ROS and reactive nitrogen species were negatively modulated by ARG2 knockdown. Additionally, a positive correlation of ARG2 with eNOS and related genes was investigated in thyroid tumors, further substantiating our in vitro findings. Our results suggest that ARG2 and eNOS may work in a coordinated manner and the underlying mechanism might be of major significance for thyroid tumorigenesis and/or tumor progression pathways. Fine modulation of ARG2, eNOS, and related genes may represent a potential source for targeted therapy of several cancer types.

The iron chelator, desferrioxamine, reduces inflammation and atherosclerotic lesion development in experimental mice

Vascular inflammation and monocyte recruitment are initiating events in atherosclerosis that have been suggested to be caused, in part, by iron-mediated oxidative stress and shifts in the intracellular redox environment of vascular cells. Therefore, the objective of this study was to investigate whether the intracellular iron chelator, desferrioxamine (DFO), reduces inflammation and atherosclerosis in experimental mice. Treatment of C57BL/6J mice with DFO (daily intraperitoneal injection of 100 mg/kg body weight for two weeks) strongly inhibited lipopolysaccharide-induced increases of soluble cellular adhesion molecules and monocyte chemoattractant protein-1 (MCP-1) in the serum and activation of the redox-sensitive transcription factors, nuclear factor-kappaB and activator protein-1, in the aorta. Furthermore, treatment of apolipoprotein E-deficient (apoE-/-) mice with DFO (100 mg/kg, intraperitoneal, daily for 10 weeks) attenuated aortic atherosclerotic lesion development by 26% (P < 0.05). DFO treatment of apoE-/- mice also lowered serum levels of MCP-1 and gene expression of proinflammatory and macrophage markers in the aorta and heart, in parallel with increased protein expression of the transferrin receptor in the heart and liver. In contrast, DFO treatment had no effect on serum cholesterol and triglyceride levels. These data show that DFO inhibits inflammation and atherosclerosis in experimental mice, providing the proof-of-concept for an important role of iron in atherogenesis. Whether eliminating excess iron is a useful adjunct for the prevention or treatment of atherosclerosis in humans remains to be investigated.

Effect of glutathione redox state on Leydig cell susceptibility to acute oxidative stress

The free radical, or oxidative stress, theory posits that imbalance in cells between prooxidants and antioxidants results in an altered redox state and, over time, an accumulation of oxidative damage. We hypothesized herein that cells with an increasingly prooxidant intracellular environment also might be particularly susceptible to acute oxidative stress. To test this hypothesis, MA-10 cells were used as a model because of their well-defined, measurable function, namely progesterone production. We first experimentally altered the redox environment of the cells by their incubation with buthionine sulfoximine (BSO) or diethyl maleate (DEM) so as to deplete glutathione (GSH), and then exposed the GSH-depleted cells acutely to the prooxidant tert-butyl hydroperoxide (t-BuOOH). Neither BSO nor DEM by themselves affected progesterone production. However, when the GSH-depleted cells subsequently were exposed acutely to t-BuOOH, intracellular reactive oxygen species concentration was significantly increased, and this was accompanied by significant reductions in progesterone production. In striking contrast, treatment of control cells with t-BuOOH had no effect. Depletion of GSH and subsequent treatment of the cells with t-BuOOH-induced the phosphorylation of each of ERK1/2, JNK and p38, members of the MAPK family. Inhibition of p38 phosphorylation largely prevented the t-BuOOH-induced down-regulation of progesterone production in GSH-depleted cells. These results suggest that, as hypothesized, alteration of the intracellular GSH redox environment results in the increased sensitivity of MA-10 cells to oxidative stress, and that this is mediated by activation of one or more redox-sensitive MAPK members.

A Non-invasive Tool for Examining the Intracellular Redox Environment of Mycobacterium tuberculosis

A Non-invasive Tool for Examining the Intracellular Redox Environment of Mycobacterium tuberculosis

Hypoxia-induced redox alterations and their correlation with 99mTc-MIBI and 99mTc-HL-91 uptake in colon cancer cells

Colorectal cancer is one of the most common malignancies in the Western world and is an example of a solid tumour in which hypoxia is a common feature and develops because of the inability of the vascular system to supply adequate amounts of oxygen to growing tumours. Hypoxia effects on tumour cell biology can be detected and characterized using different methods. The use of imaging with gamma-emitting radionuclides to detect hypoxic tissue was first suggested by Chapman in 1979 [N Engl J Med 301 (1979) 1429-1432]. (99m)Tc-4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime, also known as (99m)Tc-HL-91, has been among the most studied hypoxia markers. The objective of this study was to correlate the uptake of (99m)Tc-HL-91 and (99m)Tc-MIBI in colon cancer cells under normoxic and hypoxic conditions and to compare this information with some parameters such as oxidative stress and mitochondrial dysfunction of the cells analyzed by flow cytometry. Our results show that the in vitro (99m)Tc-HL-91 uptake is higher in hypoxic conditions, which is confirmed by the decreased uptake of (99m)Tc-MIBI. Flow cytometry results demonstrate that hypoxic conditions used are not enough to induce cellular death, but are responsible for the alterations in the intracellular redox environment, namely, increase of ROS production, proteic pimonidazol-derived adduct formation and alteration in the mitochondrial membrane permeability. Therefore, these results confirm that (99m)Tc-HL-91 is a radiopharmaceutical with favourable characteristics for detecting hypoxia.

Redox-cleavable star cationic PDMAEMA by arm-first approach of ATRP as a nonviral vector for gene delivery

In this work, we synthesized a star cationic polymer(s-PDMAEMA) consisting of cleavable poly[ N, N-bis(acryloyl) cystamine](PBAC) crosslinked core and poly( N, N-dimethyl-ethylamine methacrylate) (PDMAEMA) arms by atomic transfer radical polymerization using one-pot “arm first” method. The s-PDMAEMA that was degradable in a mimic intracellular redox environment was more efficient in condensing DNA. It was shown that s-PDMAEMA achieved higher gene transfection levels relative to their linear precursors and s-PDMAEMA200 with longer and more arms exhibited superior transfection efficiencies and lower cytotoxicity compared to PEI25K. The buffer capacities were examined by acid–base titration; the pH-dependent morphological evolution and enzyme stability of PDMAEMA/DNA complexes were investigated by atomic force microscopy (AFM) and time-resolved fluorescence spectroscopy, respectively. The results indicated that the star polymers exhibited a stronger buffering ability than their linear precursors due to the increased inner osmotic pressure. By decreasing the pH from 7.4 to 5.0, the linear PDMEMA/DNA complexes became more compact; in contrast, s-PDMAEMA200/DNA complex adopted a loose morphology due to the steric barrier of inter-arms and outward extension of positively charged arms. Analysis of the fluorescence life times of free and intercalated ethidium bromide unveiled more effective protection of DNA afforded by s-PDMAEMA. The effect of medium pH on the star PDMAEMA system was smaller owing to the ability of densely tertiary amino groups along multiple arms to absorb more protons, which was favorable for endosomolytic escape of complexes.

Redox-Dependent Translocation of p53 to Mitochondria or Nucleus in Human Melanocytes after UVA- and UVB-Induced Apoptosis

The p53 protein is an important transcription factor and tumor suppressor that is induced in response to many forms of cellular stress. UVA irradiation of human melanocytes caused generation of reactive oxygen species, which altered the intracellular redox balance and was accompanied by translocation of p53 to mitochondria. In contrast, UVB did not affect the redox status and p53 was translocated to the nucleus. Although different intracellular location of p53, UVA/B induced apoptosis through the intrinsic pathway detected as translocation of Bax to mitochondria, release of cytochrome c, and activation of caspases. These events were all prevented by inhibition of p53 with pifithrin-alpha. Furthermore, inhibition of p53 prevented lysosomal membrane permeabilization, detected as translocation of cathepsins to the cytosol, after UVB exposure, whereas UVA-induced lysosomal release was unaffected by inhibition of p53. In control cells, p53 coimmunoprecipitated with the antiapoptotic proteins Bcl-2 and Bcl-x(L) and upon UVA exposure the interaction was replaced by binding to the proapoptotic proteins Bax, Noxa, and Puma. Our findings suggest that UVA-induced apoptosis is caused by extensive oxidative damage leading to p53-regulated mitochondrial release, whereas UVB induces DNA damage and apoptosis signaling upstream of lysosomal membrane permeabilization.

Phytochemical Induction of Cell Cycle Arrest by Glutathione Oxidation and Reversal by N-Acetylcysteine in Human Colon Carcinoma Cells

Cancer prevention by dietary phytochemicals has been shown to involve decreased cell proliferation and cell cycle arrest. However, there is limited understanding of the mechanisms involved. Previously, we have shown that a common effect of phytochemicals investigated is to oxidize the intracellular glutathione (GSH) pool. Therefore, the objective of this study was to evaluate whether changes in the glutathione redox potential in response to dietary phytochemicals was related to their induction of cell cycle arrest. Human colon carcinoma (HT29) cells were treated with benzyl isothiocyanate (BIT) (BIT), diallyl disulfide (DADS), dimethyl fumarate (DMF), lycopene (LYC) (LYC), sodium butyrate (NaB) or buthione sulfoxamine (BSO, a GSH synthesis inhibitor) at concentrations shown to cause oxidation of the GSH: glutathione disulfide pool. A decrease in cell proliferation, as measured by [ 3 H]-thymidine incorporation, was observed that could be reversed by pretreatment with the GSH precursor and antioxidant N-acetylcysteine (NAC). Cell cycle analysis on cells isolated 16 h after treatment indicated an increase in the percentage (ranging from 75–30% for benzyl isothiocyanate and lycopene, respectively) of cells at G2/M arrest compared to control treatments (dimethylsulfoxide) in response to phytochemical concentrations that oxidized the GSH pool. Pretreatment for 6 h with N-acetylcysteine (NAC) resulted in a partial reversal of the G2/M arrest. As expected, the GSH oxidation from these phytochemical treatments was reversible by NAC. That both cell proliferation and G2/M arrest were also reversed by NAC leads to the conclusion that these phytochemical effects are also mediated, in part, by intracellular oxidation. Thus, one potential mechanism for cancer prevention by dietary phytochemicals is inhibition of the growth of cancer cells through modulation of their intracellular redox environment.

Reciprocal amplification of ROS and Ca2+ signals in stressed mdx dystrophic skeletal muscle fibers

Muscular dystrophies are among the most severe inherited muscle diseases. The genetic defect is a mutation in the gene for dystrophin, a cytoskeletal protein which protects muscle cells from mechanical damage. Mechanical stress, applied as osmotic shock, elicits an abnormal surge of Ca(2+) spark-like events in skeletal muscle fibers from dystrophin deficient (mdx) mice. Previous studies suggested a link between changes in the intracellular redox environment and appearance of Ca(2+) sparks in normal mammalian skeletal muscle. Here, we tested whether the exaggerated Ca(2+) responses in mdx fibers are related to oxidative stress. Localized intracellular and mitochondrial Ca(2+) transients, as well as ROS production, were assessed with confocal microscopy. The rate of basal cellular but not mitochondrial ROS generation was significantly higher in mdx cells. This difference was abolished by pre-incubation of mdx fibers with an inhibitor of NAD(P)H oxidase. In addition, immunoblotting showed a significantly stronger expression of NAD(P)H oxidase in mdx muscle, suggesting a major contribution of this enzyme to oxidative stress in mdx fibers. Osmotic shock produced an abnormal and persistent Ca(2+) spark activity, which was suppressed by ROS-reducing agents and by inhibitors of NAD(P)H oxidase. These Ca(2+) signals resulted in mitochondrial Ca(2+) accumulation in mdx fibers and an additional boost in cellular and mitochondrial ROS production. Taken together, our results indicate that the excessive ROS production and the simultaneous activation of abnormal Ca(2+) signals amplify each other, finally culminating in a vicious cycle of damaging events, which may contribute to the abnormal stress sensitivity in dystrophic skeletal muscle.