Role of some growth regulators on cytogenetic activity of barley under salt stress

The effects of gibberellic acid (GA₃), kinetin (KIN), benzyladenine and ethylene (E) on mitotic activity and chromosomal aberrations in root tips of barley seeds (Hordeum vulgare L. cv. “Bülbül 89”) germinated under salt stress were investigated. It was determined that all of these plant growth regulators (PGRs) decreased mitotic index in root tips of barley seeds germinated at 20 °C and in distilled water. Furthermore, some of the PGRs studied increased significantly the frequency of chromosomal aberrations. The frequency of chromosomal aberrations in seeds treated with E and KIN was considerably higher than in the seeds germinated under nonstress conditions. The inhibitory effect of salt stress on mitotic index increased with increasing salt concentration (0.30, 0.35, 0.40 and 0.45 molal, m). GA₃ and KIN pretreatments showed a successful performance in ameliorating the negative effects of increasing salinity on mitotic activity. The number of chromosomal aberrations also increased with increasing NaCl concentration. However, most of the PGR pretreatments studied alleviated the detrimental effects of increasing salinity on chromosomal aberrations. KIN pretreatment at 0.30 and 0.35 m salinity could not rescued the cytogenetic activity of salt stress on this parameter.     

Protective roles of exogenous polyamines on chromosomal aberrations in <Emphasis Type="Italic">Hordeum vulgare</Emphasis> exposed to salinity

The effects of exogenous polyamines (PAs): spermine (Spm), spermidine (Spd), cadaverine (Cad) and putrescine (Put) on mitotic activity and chromosomal aberrations in root meristem cells of Hordeum vulgare L. (barley) seeds exposed to salinity were analyzed. The PAs significantly inhibited cell division in distilled water. Furthermore, most of these PAs (except for Spd) caused a significant increase in the frequency of chromosomal aberrations as compared to control group. Seeds treated with Put caused the highest percentage of mitotic abnormalities in total. The negative effect of salinity on mitotic index and the frequency of chromosomal aberrations increased with increasing salt concentration. PAs studied could not be successful in ameliorating of the negative effect of salinity on mitotic activity. Particularly, exposure to Cad and 0.40 M NaCl caused a complete block of cell division in total. However, most of the PA studied showed a perfectly performance in alleviating the detrimental effects of increasing salinity on chromosomal aberrations.     

Pre-treatment with H<Subscript>2</Subscript>O<Subscript>2</Subscript> induces salt tolerance in Barley seedlings

Barley seedlings were pre-treated with 1 and 5 μM H₂O₂ for 2 d and then supplied with water or 150 mM NaCl for 4 and 7 d. Exogenous H₂O₂ alone had no effect on the proline, malondialdehyde (MDA) and H₂O₂ contents, decreased catalase (CAT) activity and had no effect on peroxidase (POX) activity. Three new superoxide dismutase (SOD) isoenzymes appeared in the leaves as a result of 1 μM H₂O₂ treatment. NaCl enhanced CAT and POX activity. SOD activity and isoenzyme patterns were changed due to H₂O₂ pre-treatment, NaCl stress and leaf ageing. In pre-treated seedlings the rate of ¹⁴CO₂ fixation was higher and MDA, H₂O₂ and proline contents were lower in comparison to the seedlings subjected directly to NaCl stress. Cl⁻ content in the leaves 4 and 7 d after NaCl supply increased considerably, but less in pre-treated plants. It was suggested that H₂O₂ metabolism is involved as a signal in the processes of barley salt tolerance.     

Cytogenetic response of 24-epibrassinolide on the root meristem cells of barley seeds under salinity

Cytogenetic response of 24-epibrassinolide (EBR) and different NaCl concentrations (0.30, 0.35 and 0.40 M, molar) on root meristem cells of barley were analysed. Plants grown on media containing 0.30, 0.35 and 0.40 M NaCl showed a significant decrease of mitotic index and higher number of chromosomal abnormalities as compared to those of control conditions. Also, the mitotic index approximately 50% decreased in EBR- treated samples and chromosomal abnormalities almost tipled those of control. EBR pretreatment in higher concentrations of salt (0.40 M NaCl) caused total inhibition of mitotic activity in root-tip cells. However, comparison of all concentrations of salt and control revealed to have a successful performance in ameliorating of the detrimental effects of salinity on chromosomal abnormalities.     

The control of mitochondrial succinate-dependent H<Subscript>2</Subscript>O<Subscript>2</Subscript> production

In brain mitochondria succinate activates H₂O₂ release, concentration dependently (starting at 15 μM), and in the presence of NAD dependent substrates (glutamate, pyruvate, β-hydroxybutyrate). We report that TCA cycle metabolites (citrate, isocitrate, α-ketoglutarate, fumarate, malate) individually and quickly inhibit H₂O₂ release. When they are present together at physiological concentration (0.2, 0.01, 0.15, 0.12, 0.2 mM respectively) they decrease H₂O₂ production by over 60% at 0.1–0.2 mM succinate. The degree of inhibition depends on the concentration of each metabolite. Acetoacetate is a strong inhibitor of H₂O₂ release, starting at 10 μM and acting quickly. It potentiates the inhibition induced by TCA cycle metabolites. The action of acetoacetate is partially removed by β-hydroxybutyrate. Removal is minimal at 0.1 mM acetoacetate, and is higher at 0.5 mM acetoacetate. We conclude that several inhibitors of H₂O₂ release act jointly and concentration dependently to rapidly set the required level of H₂O₂ generation at each succinate concentration.     

Cinnamtannin B-1, a natural antioxidant that reduces the effects of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on CCK-8-evoked responses in mouse pancreatic acinar cells

This work was designed in order to gain an insight on the mechanisms by which antioxidants prevent pancreatic disorders. We have examined the properties of cinnamtannin B-1, which belongs to the class of polyphenols, against the effect of hydrogen peroxide (H₂O₂) in mouse pancreatic acinar cells. We have studied Ca²⁺ mobilization, oxidative state, amylase secretion, and cell viability of cells treated with cinnamtannin B-1 in the presence of various concentrations of H₂O₂. We found that H₂O₂ (0.1–100 μM) increased CM-H₂DCFDA-derived fluorescence, reflecting an increase in oxidation. Cinnamtannin B-1 (10 μM) reduced H₂O₂-induced oxidation of CM-H₂DCFDA. CCK-8 induced oxidation of CM-H₂DCFDA in a similar way to low micromolar concentrations of H₂O₂, and cinnamtannin B-1 reduced the oxidant effect of CCK-8. In addition, H₂O₂ induced a slow and progressive increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_c). Cinnamtannin B-1 reduced the effect of H₂O₂ on [Ca²⁺]_c, but only at the lower concentrations of the oxidant. H₂O₂ inhibited amylase secretion in response to cholecystokinin, and cinnamtannin B-1 reduced the inhibitory action of H₂O₂ on enzyme secretion. Finally, H₂O₂ reduced cell viability, and the antioxidant protected acinar cells against H₂O₂. In conclusion, the beneficial effects of cinnamtannin B-1 appear to be mediated by reducing the intracellular Ca²⁺ overload and intracellular accumulation of digestive enzymes evoked by ROS, which is a common pathological precursor that mediates pancreatitis. Our results support the beneficial effect of natural antioxidants in the therapy against oxidative stress-derived deleterious effects on cellular physiology.     

Effects of salt-tolerant rootstock grafting on ultrastructure,photosynthetic capacity,and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-scavenging system in chloroplasts of cucumber seedlings under NaCl stress

Plant growth, photosynthetic parameters, chloroplast ultrastructure, and the ascorbate-glutathione cycle system in chloroplasts of self-grafted and rootstock-grafted cucumber leaves were investigated. Grafted plants were grown hydroponically and were exposed to 0, 50, and 100 mM NaCl concentrations for 10 days. Under NaCl stress, the hydrogen peroxide (H₂O₂) content in cucumber chloroplasts increased, the chloroplast ultrastructure was damaged, and the gas stomatal conductance, intercellular CO₂ concentration, as well as shoot dry weight, plant height, stem diameter, leaf area, and leaf relative water content were inhibited, whereas these changes were less severe in rootstock-grafted plants. The activities of ascorbate peroxidase (APX; EC 1.11.1.11), glutathione reductase (GR; EC 1.6.4.2), and dehydroascorbate reductase (DHAR EC 1.8.5.1) were higher in the chloroplasts of rootstock-grafted plants compared with those of self-grafted plants under 50 and 100 mM NaCl. Similar trends were shown in leaf net CO₂ assimilation rate and transpiration rate, as well as reduced glutathione content under 100 mM NaCl. Results suggest that rootstock grafting enhances the H₂O₂-scavenging capacity of the ascorbate–glutathione cycle in cucumber chloroplasts under NaCl stress, thereby protecting the chloroplast structure and improving the photosynthetic performance of cucumber leaves. As a result, cucumber growth is promoted.     

Protective Effects of Asiatic Acid on Rotenone- or H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Injury in SH-SY5Y Cells

Parkinson’s disease (PD) is a progressive neurodegenerative disorder with a prevalence of 1–2% in people over the age of 50. Mitochondrial dysfunction occurred in PD patients showing a 15–30% loss of activity in complex I. Asiatic acid (AA), a triterpenoid, is an antioxidant and used for depression treatment, but the effect of AA against PD-like damage has never been reported. In the present study, we investigated the protective effects of AA against H₂O₂ or rotenone-induced cellular injury and mitochondrial dysfunction in SH-SY5Y cells. Mitochondrial membrane potential (MMP) and the expression of voltage-dependent anion channel (VDAC) were detected with or without AA pretreatment following cellular injury to address the possible mechanisms of AA neuroprotection. The results showed that pre-treatment of AA (0.01–100 nM) protected cells against the toxicity induced by rotenone or H₂O₂. In addition, MMP dissipation occurred following the exposure of rotenone, which could be prevented by AA treatment. More interestingly, pre-administration of AA inhibited the elevation of VDAC mRNA and protein levels induced by rotenone(100 nM) or H₂O₂ (300 μM).These data indicate that AA could protect neuronal cells against mitochondrial dysfunctional injury and suggest that AA might be developed as an agent for PD prevention or therapy. Special issue article in honor of Dr. Akitane Mori.     

Ca<Superscript>2+</Superscript> and CaM are Involved in NO- and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Adventitious Root Development in Marigold

Our previous results have demonstrated that both nitric oxide (NO) and hydrogen peroxide (H₂O₂) are involved in the promotion of adventitious root development in marigold (Tagetes erecta L.). However, not much is known about the intricate molecular network of adventitious root development triggered by NO and H₂O₂. In this study, the involvement of calcium (Ca²⁺) and calmodulin (CaM) in NO- and H₂O₂-induced adventitious rooting in marigold was investigated. Exogenous Ca²⁺ was capable of promoting adventitious rooting, with a maximal biological response at 50 μM CaCl₂. Ca²⁺ chelators and CaM antagonists prevented NO- and H₂O₂-induced adventitious rooting, indicating that both endogenous Ca²⁺ and CaM may play crucial roles in the adventitious rooting induced by NO and H₂O₂. NO and H₂O₂ treatments increased the endogenous content of Ca²⁺ and CaM, suggesting that NO and H₂O₂ enhanced adventitious rooting by stimulating the endogenous Ca²⁺ and CaM levels. Moreover, treatment with Ca²⁺ enhanced the endogenous levels of NO and H₂O₂. Additionally, Ca²⁺ might be involved as an upstream signaling molecule for CaM during NO- and H₂O₂-induced rooting. Altogether, the results suggest that both Ca²⁺ and CaM are two downstream signaling molecules in adventitious rooting induced by NO and H₂O₂.     

MEK/ERK inhibitor U0126 enhanced salt stress-induced programmed cell death in <Emphasis Type="Italic">Thellungiella halophila</Emphasis> suspension-cultured cells

Programmed cell death (PCD) is an active cellular suicide that occurs both in animals and plants throughout development and in response to abiotic or biotic stress. In contrast to plant hypersensitive response-like cell death, little is known about the molecular machinery that regulates the halophyte plant PCD under high salinity stress. Since mitogen-activated protein kinases (MAPKs) are involved in plant response/tolerance to salt stress, and plant MAPK genes belong to the extracellular signal-regulated kinase (ERK) subfamily, we have investigated the role of ERK-like enzymes in high salinity stress-induced cell death in Thellungiella halophila. The data showed that ERK-like enzymes were early (10 min) and transiently activated under 300 mM NaCl stress. Pretreatment with 10 μM U0126, a special MEK/ERK inhibitor, resulted in a small but statistically significant increase of the percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive nuclei in contrast to salt alone. The effects of U0126 on H₂O₂ production and cytochrome c (cyt c) release were also investigated. We found that the pretreatment with U0126 accelerated H₂O₂ production as well as cyt c release, and eventually enhanced cell death. The results suggest that ERK-like enzymes in Thellungiella halophila may act as a positive regulator of salt tolerance, as illustrated by pretreatment with U0126 which enhanced cell death under high salinity stress.     

Insulin increases H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced pancreatic beta cell death

Insulin resistance results, in part, from impaired insulin signaling in insulin target tissues. Consequently, increased levels of insulin are necessary to control plasma glucose levels. The effects of elevated insulin levels on pancreatic beta (β) cell function, however, are unclear. In this study, we investigated the possibility that insulin may influence survival of pancreatic β cells. Studies were conducted on RINm, RINm5F and Min-6 pancreatic β-cells. Cell death was induced by treatment with H₂O₂, and was estimated by measurements of LDH levels, viability assay (Cell-Titer Blue), propidium iodide staining and FACS analysis, and mitochondrial membrane potential (JC-1). In addition, levels of cleaved caspase-3 and caspase activity were determined. Treatment with H₂O₂ increased cell death; this effect was increased by simultaneous treatment of cells with insulin. Insulin treatment alone caused a slight increase in cell death. Inhibition of caspase-3 reduced the effect of insulin to increase H₂O₂-induced cell death. Insulin increased ROS production by pancreatic β cells and increased the effect of H₂O₂. These effects were increased by inhibition of IR signaling, indicative of an effect independent of the IR cascade. We conclude that elevated levels of insulin may act to exacerbate cell death induced by H₂O₂ and, perhaps, other inducers of apoptosis.     

Exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> improves the chilling tolerance of manilagrass and mascarenegrass by activating the antioxidative system

The effect of foliar pretreatment by hydrogen peroxide (H₂O₂) at low concentrations of 0, 5, 10, and 15 mM on the chilling tolerance of two Zoysia cultivars, manilagrass (Zoysia matrella) and mascarenegrass (Zoysia tenuifolia), was studied. The optimal concentration for H₂O₂ pretreatment was 10 mM, as demonstrated by the lowest malondialdehyde (MDA) content and electrolyte leakage (EL) levels and higher protein content under chilling stress (7°C/2°C, day/night). Prior to initiation of chilling, exogenous 10 mM H₂O₂ significantly increased catalase (CAT), ascorbate peroxidase (APX), glutathione-dependent peroxidases (GPX), and glutathione-S-transferase (GST) activities in manilagrass, and guaiacol peroxidase (POD), APX, and glutathione reductase (GR) activities in mascarenegrass, suggesting that H₂O₂ may act as a signaling molecule, inducing protective metabolic responses against further oxidative damage due to chilling. Under further stress, optimal pretreatments alleviated the increase of H₂O₂ level and the decrease of turfgrass quality, and improved CAT, POD, APX, GR, and GPX activities, with especially significant enhancement of APX and GPX activities from the initiation to end of chilling. These antioxidative enzymes were likely the important factors for acquisition of tolerance to chilling stress in the two Zoysia cultivars. Our results showed that pretreatment with H₂O₂ at appropriate concentration may improve the tolerance of warm-season Zoysia grasses to chilling stress, and that manilagrass had better tolerance to chilling, as evaluated by lower MDA and EL, and better turfgrass quality, regardless of the pretreatment applied.     

Effect of NaCl and CaCl<Subscript>2</Subscript> on the antioxidant mechanism of leaves and stems of the rootstock CAB-6P (<Emphasis Type="Italic">Prunus cerasus</Emphasis> L.) under in vitro conditions

The effect of salinity on the non-enzymic and enzymic antioxidant activity, shoot proliferation and nutrient accumulation was studied in in vitro cultures of the rootstock CAB-6P (Prunus cerasus L.). Three concentrations (0, 30 and 60 mM) of NaCl or CaCl₂ were added to a modified MS medium. Between the two salt treatments used, only the explants treated with CaCl₂ presented significant decrease in growth parameters. The concentrations of Na⁺ and Cl⁻ in the explants treated with NaCl were increased, as NaCl in the culture medium increased. Furthermore, in the explants treated with CaCl₂ the concentrations of Ca²⁺ and Cl⁻ were increased while that of K⁺ decreased, as CaCl₂ concentration increased. The activity of peroxidase in leaves as well as the number of its anionic isoforms was increased under 30 mM CaCl₂ as well as 60 mM NaCl or CaCl₂. On the contrary, increasing salinity, from 0 to 60 mM CaCl₂, resulted in a reduction of the catalase activity in leaves followed by disappearance of the only one catalase isoform that was detected in leaves (60 mM CaCl₂). In the stems of the explants treated with NaCl the peroxidase activity was reduced. In the stems and leaves of the explants grown in saline substrate the non-enzymic antioxidant activity was significantly increased. The results suggest that the stems and leaves of CAB-6P explants presented variable antioxidant responses that were depended on the salt form used. The contribution of enzymic and non-enzymic protection mechanisms to the adaptation of CAB-6P explants under salinity stress is discussed.     

Hydrogen peroxide (H<Subscript>2</Subscript>O<Subscript>2</Subscript>) supply significantly improves xanthan gum production mediated by <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> in vitro

To improve xanthan gum productivity, a strategy of adding hydrogen peroxide (H₂O₂) was studied. The method could intensify oxygen supply through degradation of H₂O₂ to oxygen (O₂). In shake flask testing, the xanthan gum yield reached 2.8% (improved by 39.4%) when adding 12.5 mM H₂O₂ after 24 h of fermentation. In fermentor testing, it was obvious that the oxygen conditions varied with the H₂O₂ addition time. Eventually, gum yield of 4.2% (w/w) was achieved (increased by 27.3%). Compared with the method of intense mixing and increasing the air flow rate, adding H₂O₂ to improve the dissolved oxygen concentration was more effective and much better. Moreover, addition of H₂O₂ improved the quality of xanthan gum; the pyruvate content of xanthan was 4.4% (w/w), higher than that of the control (3.2%).     

Characterization of <Emphasis Type="Italic">Brassica juncea</Emphasis> antioxidant potential under salinity stress

Present study characterizes the anti-oxidative defense potential of four Brassica juncea varieties, Pusa Jaikisan, Varuna, RLM-198, and CS-52, differing in their ability to withstand salinity stress. 7-day-old seedlings raised in MS medium supplemented with 0, 50, 100, and 150 mM NaCl were used to monitor changes in the growth profile, level of stress marker molecules, and activities of important antioxidant enzymes. Increasing NaCl concentration resulted in a significant (P ≤ 0.05) reduction of shoot fresh and dry mass and vigor index in all the varieties tested. Maximum reduction in growth was recorded for RLM-198 while CS-52 maintained better growth characteristics. Varuna and RLM-198 exhibited a limited increase in superoxide dismutase, ascorbate peroxidase, and total peroxidase activity under increasing salinity. These varieties also recorded maximum salt stress-induced damage in terms of increased lipid peroxidation, H₂O₂ content, and electrolyte leakage. On the other hand, CS-52 recorded maximum proline accumulation with minimum levels of H₂O₂, electrolyte leakage, and malondialdehyde contents. With increasing salinity stress, CS-52 recorded maximal increase in the activity of antioxidant enzymes. However, catalase activity did not correlate with alterations in H₂O₂ levels under stress. Interestingly, a lower superoxide dismutase:ascorbate peroxidase ratio in CS-52 correlated with stress tolerance trait, while a comparatively higher superoxide dismutase:ascorbate peroxidase ratio in RLM-198 marked the susceptible nature of the variety. Our results propose that superoxide dismutase:ascorbate peroxidase ratio is the critical factor, determining the degree of stress tolerance in Brassica juncea.     

Variations of vinblastine accumulation and redox state affected by exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don

Effects of exogenous H₂O₂ application on vinblastine (VBL) and its precursors, vindoline (VIN), catharanthine (CAT) and α-3′,4′-anhydrovinblastine (AVBL), were measured in Catharanthus roseus seedlings in order to explore possible correlation of VBL formation with oxidative stress. VBL accumulation has previously been shown to be regulated by an in vitro H₂O₂-dependent peroxidase (POD)-like synthase. Experimental exposure of plants to different concentrations of H₂O₂ showed that endogenous H₂O₂ and alkaloid concentrations in leaves were positively elevated. The time-course variations of alkaloid concentrations and redox state, reflected by the concentrations of H₂O₂, ascorbic acid (AA), oxidative product of glutathione (GSSG) and POD activity, were significantly altered due to H₂O₂ application. The further correlation analysis between alkaloids and redox status indicated that VBL production was tightly correlated with redox status. These results provide a new link between VBL metabolisms and redox state in C. roseus.     

Differential Effects of Methionine and Cysteine Oxidation on [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in Cultured Hippocampal Neurons

Methionine and cysteine residues in proteins are the major targets of reactive oxygen species (ROS). The present work was designed to characterize the impact of methionine and cysteine oxidation upon [Ca²⁺]_i in hippocampal neurons. We investigated the effects of H₂O₂ and chloramine T(Ch-T) agents known to oxidize both cysteine and methionine residues, and 5, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB)—a cysteine-specific oxidant, on the intracellular calcium in hippocampal neurons. The results showed that these three oxidants, 1 mM H₂O₂, 1 mM Ch-T, and 500 μM DTNB, induced an sustained elevation of [Ca²⁺]_i by 76.1 ± 3.9%, 86.5 ± 5.0%, and 24.4 ± 3.2% over the basal level, respectively. The elevation induced by H₂O₂ and Ch-T was significantly higher than DTNB. Pretreatment with reductant DTT at 1 mM for 10 min completely prevented the action of DTNB on [Ca²⁺]_i, but only partially reduced the effects of H₂O₂ and Ch-T on [Ca²⁺]_i, the reductions were 44.6 ± 4.2% and 29.6 ± 6.1% over baseline, respectively. The elevation of [Ca²⁺]_i induced by H₂O₂ and Ch-T after pretreatment with DTT were statistically higher than that induced by single administration of DTNB. Further investigation showed that the elevation of [Ca²⁺]_i mainly resulted from internal calcium stores. From our data, we propose that methionine oxidation plays an important role in the regulation of intracellular calcium and this regulation may mainly be due to internal calcium stores.     

Vanadate-induced cell death is dissociated from H<Subscript>2</Subscript>O<Subscript>2</Subscript> generation

Vanadium is an environmentally toxic metal with peculiar and sometimes contradictory cellular effects. It is insulin-mimetic, it can either stimulate cell growth or induce cell death, and it has both mutagenic and antineoplastic properties. However, the mechanisms involved in those effects are poorly understood. Several studies suggest that H₂O₂ is involved in vanadate-induced cell death, but it is not known whether cellular sensitivity to vanadate is indeed related to H₂O₂ generation. In the present study, the sensitivity of four cell lines from different origins (K562, K562-Lucena 1, MDCK, and Ma104) to vanadate and H₂O₂ was evaluated and the production of H₂O₂ by vanadate was analyzed by flow cytometry. We show that cell lines very resistant to H₂O₂ (K562, K562-Lucena 1, and Ma104 cells) are much more sensitive to vanadate than MDCK, a cell line relatively susceptible to H₂O₂, suggesting that vanadate-induced cytotoxicity is not directly related to H₂O₂ responsiveness. In accordance, vanadate concentrations that reduced cellular viability to approximately 60–70% of the control (10 μmol/L) did not induce H₂O₂ formation. A second hypothesis, that peroxovanadium (PV) compounds, produced once vanadate enters into the cells, are responsible for the cytotoxicity, was only partially confirmed because MDCK cells were resistant to both vanadate and PV compounds (10 μmol/L each). Therefore, our results suggest that vanadate toxicity occurs by two distinct pathways, one dependent on and one independent of H₂O₂ production.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript> production within tumor microenvironment inversely correlated with infiltration of CD56<Superscript>dim</Superscript> NK cells in gastric and esophageal cancer: possible mechanisms of NK cell dysfunction

Human NK cells can be divided into two subsets, CD56^dimCD16(+)NK and CD56^brightCD16(−)NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed the relationship between CD56^dim/CD56^bright NK cells and H₂O₂ in tumor-infiltrating NK cells in patients with gastric (n = 50) and esophageal (n = 35) cancer. The ratio of CD56^dim NK cells infiltrating tumors gradually decreased according to disease progression. H₂O₂ was abundantly produced within tumor microenvironments, and there was an inverse correlation between CD56^dim NK cell infiltration and H₂O₂ production. CD56^dim NK cells are more sensitive to apoptosis induced by physiological levels of H₂O₂ than CD56^bright NK cells. Furthermore, the exposure of NK cells to H₂O₂ resulted in the impairment of ADCC activity. In conclusion, H₂O₂ produced within tumor microenvironments inversely correlated with the infiltration of CD56^dim NK cells, possibly due to their preferentially induced cell death. These observations may explain one of the mechanisms behind NK cell dysfunction frequently observed in tumor microenvironments.