The effects of MAPK inhibitors on antimycin A-treated Calu-6 lung cancer cells in relation to cell growth, reactive oxygen species, and glutathione

Antimycin A (AMA) inhibits succinate oxidase, NADH oxidase, and mitochondrial electron transport chain between cytochrome b and c. We recently demonstrated that AMA inhibited the growth of Calu-6 lung cancer cells through apoptosis. Here, we investigated the effects of AMA and/or MAPK inhibitors on Calu-6 lung cancer cells in relation to cell growth, cell death, reactive oxygen species (ROS), and GSH levels. Treatment with AMA inhibited the growth of Calu-6 cells at 72 h. AMA-induced apoptosis was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). While ROS were decreased in AMA-treated Calu-6 cells, O ₂ ^?? among ROS was increased. AMA also induced GSH depletion in Calu-6 cells. Treatment with MEK inhibitor intensified cell death, MMP (ΔΨ_m) loss, and GSH depletion in AMA-treated Calu-6 cells. JNK inhibitor also increased cell death, MMP (ΔΨ_m) loss, and ROS levels in these cells. Treatment with p38 inhibitor magnified cell growth inhibition by AMA and increased cell death, MMP (ΔΨ_m) loss, ROS level, and GSH depletion in AMA-treated cells. Conclusively, all the MAPK inhibitors slightly intensified cell death in AMA-treated Calu-6 cells. The changes of ROS and GSH by AMA and/or MAPK inhibitors were in part involved in cell growth and death in Calu-6 cells.     

Homocysteine modulates the proteolytic potential of human arterial smooth muscle cells through a reactive oxygen species dependant mechanism

Suberoyl bishydroxamic acid (SBHA) as a histone deacetylase (HDAC) inhibitor has various cellular effects such as cell growth and apoptosis. In the present study, we evaluated the effects of SBHA on the growth and death of A549 lung cancer cells. SBHA inhibited the growth of A549 cells with an IC₅₀ of approximately 50 μM at 72 h in a dose-dependent manner. DNA flow cytometric analysis indicated that SBHA induced a G2/M phase arrest of the cell cycle. This agent also induced apoptosis, as evidenced by sub-G1 cells and annexin V-FITC staining cells. SBHA-induced apoptosis was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m), Bcl-2 decrease, Bax increase, and the activation of caspase-3. All of the tested caspase inhibitors significantly rescued some cells from SBHA-induced A549 cell death. However, none of the caspase inhibitors prevented the loss of MMP (ΔΨ_m) induced by SBHA. Intracellular reactive oxygen species (ROS) levels including O ₂ ^?? were increased in 50 μM SBHA-treated A549 cells. None of the caspase inhibitors attenuated ROS levels in these cells. SBHA also elevated the number of glutathione (GSH)-depleted cells in A549 cells, which was reduced by treatment with caspase inhibitors. In conclusion, this is the first report that SBHA inhibited the growth of A549 lung cancer cells via caspase-dependent apoptosis, which was related to GSH depletion rather than changes in ROS level.     

Gallic acid-induced lung cancer cell death is accompanied by ROS increase and glutathione depletion

Gallic acid (GA) is generally distributed in a variety of plants and foods, and its various biological effects have been reported. Here, we investigated the effects of GA and/or caspase inhibitors on Calu-6 and A549 lung cancer cells in relation to cell death and reactive oxygen species (ROS). The growths of Calu-6 and A549 cells were diminished with an IC(50) of approximately 30 and 150 μM GA at 24 h, respectively. GA also inhibited the growth of primary human pulmonary fibroblast (HPF) cells with an IC(50) of about 300 μM. GA induced apoptosis and/or necrosis in lung cancer cells, which was accompanied by the loss of mitochondrial membrane potential (MMP, ΔΨ(m)). The percents of MMP (ΔΨ(m)) loss and death cells by GA were lower in A549 cells than in Calu-6 cells. Caspase inhibitors did not significantly rescued lung cancer cells from GA-induced cell death. GA increased ROS levels including O(2) (?-) and induced GSH depletion in both lung cancer cells. Z-VAD (pan-caspase inhibitor) did not decrease ROS levels and GSH depleted cell number in GA-treated lung cancer cells. In conclusion, GA inhibited the growth of lung cancer and normal cells. GA-induced lung cancer cell death was accompanied by ROS increase and GSH depletion.     

Biochemical analysis of reactive oxygen species production and antioxidative responses in unripe avocado (Persea americana Mill var Hass) fruits in response to wounding

We analyzed the production of reactive oxygen species (ROS) and of detoxifying enzymes and enzymes of the ascorbate (ASC) acid cycle in avocado fruit (Pesea Americana Mill cv Hass) in response to wounding. The levels of superoxide anion (O₂ ^?), hydroxyl radicals (OH^.) and hydrogen peroxide (H₂O₂) increased at 15 min and 2 and 15 h post-wounding. Peroxidase (POD) activity had increased to high levels 24 h after wounding; in contrast, catalase and superoxide dismutase (SOD) levels hat decreased significantly at 24 h post-treatment. Basic POD was the major POD form induced, and the levels of at least three apoplastic POD isozymes –increased following wounding. Using specific inhibitors, we characterized one MnSOD and two CuZnSOD isozymes. CuZnSOD activities decreased notably 12 h after treatment. The activities of dehydroascorbate reductase and glutathione reductase increased dramatically following the wounding treatment, possibly as a means to compensate for the redox changes due to ROS production.     

Suberoyl bishydroxamic acid-induced apoptosis in HeLa cells via ROS-independent, GSH-dependent manner

Suberoyl bishydroxamic acid (SBHA) is a HDAC inhibitor that can regulate many biological functions including apoptosis and proliferation in various cancer cells. Here, we evaluated the effect of SBHA on the growth of HeLa cervical cancer cells in relation to apoptosis, reactive oxygen species (ROS) and glutathione (GSH) levels. Dose-dependent inhibition of cell growth was observed in HeLa cells with an IC₅₀ of approximately 15 μM at 72 h. SBHA also induced apoptosis in HeLa cells, as evidenced by sub-G1 cells, annexin V-FITC staining cells, activations of caspase 3 and 8, and the loss of mitochondrial membrane potential (ΔΨ_m). In addition, all of the tested caspase inhibitors rescued some cells from SBHA-induced HeLa cell death. SBHA increased ROS levels including O ₂ ^?? and induced GSH depletion in HeLa cells. Generally, caspase inhibitors did not affect ROS levels in SBHA-treated HeLa cells, but they significantly prevented GSH depletion in these cells. Furthermore, while the well-known antioxidants, N-acetyl cysteine and vitamin C, did not affect cell death, ROS level or GSH depletion in SBHA-treated HeLa cells, l-buthionine sulfoximine, a GSH synthesis inhibitor, enhanced cell death and GSH depletion in these cells. In conclusion, SBHA inhibits the growth of HeLa cervical cancer cells via caspase-dependent apoptosis, and the inhibition is independent of ROS level changes, but dependent on GSH level changes.     

Glutathione improves survival of cryopreserved embryogenic calli of <Emphasis Type="Italic">Agapanthus praecox</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis>

The effect of supplementation of reduced glutathione (GSH) to cryoprotectant solution on the generation of reactive oxygen species (ROS) (e.g., H₂O₂, OH^·, and O ₂ ^·? ) and antioxidants (e.g., SOD, POD, CAT, AsA, and GSH), as well as membrane lipid peroxidation (i.e., MDA content) mitigation in cryopreserving of embryogenic calli (EC) of Agapanthus praecox subsp. orientalis was investigated. The vitrification-based cryopreservation method was used in this study. The addition of GSH at a final concentration of 0.08 mM to the cryoprotectant solution has significantly improved cryotolerance of A. praecox EC. The EC post-thaw survival rate increased by 68.34 % using the cryoprotectant solution containing 0.08 mM GSH as compared to the control (GSH-free). EC treated with GSH displayed the reduction in  OH^· generation activity and the contents of H₂O₂ and MDA, as well as enhancement in the inhibition of O ₂ ^·? generation and the antioxidant activity. Treatment with exogenous GSH also increased endogenous AsA and GSH contents after dehydration step. Expression of stress-responsive genes, e.g., peroxidase (POD), peroxiredoxin, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione peroxidase (GPX), was also increased during cryopreservation processes. The expression of DAD1 (Defender against apoptotic cell death) was elevated, while cell death-related protease SBT was suppressed. These results demonstrated that the addition of GSH to cryoprotectant solution affects the ROS level and could effectively improve survival of A. praecox EC through enhancing antioxidant enzyme activities and decreasing cell death.     

Exogenous salicylic acid regulates reactive oxygen species metabolism and ascorbate–glutathione cycle in <Emphasis Type="Italic">Nitraria tangutorum</Emphasis> Bobr. under salinity stress

The effect of 0.5–1.5 mM salicylic acid (SA) on modulating reactive oxygen species metabolism and ascorbate–glutathione cycle in NaCl-stressed Nitraria tangutorum seedlings was investigated. The individual plant fresh weight (PFW) and plant dry weight (PDW) significantly increased under 100 mM NaCl while remained unchanged or decreased under 200–400 mM NaCl compared to the control. Superoxide anion (O ₂ ^·? ), hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS), reduced ascorbate (AsA), dehydroascorbate (DHA), reduced glutathione (GSH) and oxidized glutathione (GSSG) increased whereas the ratios of AsA/DHA and GSH/GSSG decreased under varied NaCl treatments. Ascorbate peroxidase (APX) and glutathione reductase (GR) activities were enhanced while dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) activities remained unvaried under 100–400 mM NaCl stresses. In addition, exogenous SA further increased PFW, PDW and root/shoot ratio. SA effectively diminished O ₂ ^·? accumulation. H₂O₂ and TBARS decreased under 0.5 and 1.0 mM SA treatments compared to those without SA. 0.5 mM of SA increased while 1.0 and 1.5 mM SA decreased APX activities. DHAR activities were elevated by 0.5 and 1.0 mM SA but not by 1.5 mM SA. MDHAR and GR activities kept constant or significantly increased at varying SA concentrations. Under SA treatments, AsA and GSH contents further increased, DHA and GSSG levels remained unaltered, while the decreases in AsA/DHA and GSH/GSSG ratios were inhibited. The above results demonstrated that the enhanced tolerance of N. tangutorum seedlings conferred by SA could be attributed mainly to the elevated GR and DHAR activities as well as the increased AsA/DHA and GSH/GSSG ratios.     

Cyclophilin A inhibits A549 cell oxidative stress and apoptosis by modulating the PI3K/Akt/mTOR signaling pathway

The excessive and inappropriate production of reactive oxygen species (ROS) can cause oxidative stress and is implicated in the pathogenesis of lung cancer. Cyclophilin A (CypA), a member of the immunophilin family, is secreted in response to ROS. To determine the role of CypA in oxidative stress injury, we investigated the role that CypA plays in human lung carcinoma (A549) cells. Here, we showed the protective effect of human recombinant CypA (hCypA) on hydrogen peroxide (H₂O₂)-induced oxidative damage in A549 cells, which play crucial roles in lung cancer. Our results demonstrated that hCypA substantially promoted cell viability, superoxide dismutase (SOD), glutathione (GSH), and GSH peroxidase (GSH-Px) activities, and attenuated ROS and malondialdehyde (MDA) production in H₂O₂-induced A549 cells. Compared with H₂O₂-induced A549 cells, Caspase-3 activity in hCypA-treated cells was significantly reduced. Using Western blotting, we showed that hCypA facilitated Bcl-2 expression and inhibited Bax, Caspase-3, Caspase-7, and PARP-1 expression. Furthermore, hCypA activates the PI3K/Akt/mTOR pathway in A549 cells in response to H₂O₂ stimulation. Additionally, peptidyl-prolyl isomerase activity was required for PI3K/Akt activation by CypA. The present study showed that CypA protected A549 cells from H₂O₂-induced oxidative injury and apoptosis by activating the PI3K/Akt/mTOR pathway. Thus, CypA might be a potential target for lung cancer therapy.     

Exogenous sodium nitroprusside and glutathione alleviate copper toxicity by reducing copper uptake and oxidative damage in rice (Oryza sativa L.) seedlings

Nitric oxide (NO) and glutathione (GSH) regulate a variety of physiological processes and stress responses; however, their involvement in mitigating Cu toxicity in plants has not been extensively studied. This study investigated the interactive effect of exogenous sodium nitroprusside (SNP) and GSH on Cu homeostasis and Cu-induced oxidative damage in rice seedlings. Hydroponically grown 12-day-old seedlings were subjected to 100 μM CuSO₄ alone and in combination with 200 μM SNP (an NO donor) and 200 μM GSH. Cu exposure for 48 h resulted in toxicity symptoms such as stunted growth, chlorosis, and rolling in leaves. Cu toxicity was also manifested by a sharp increase in lipoxygenase (LOX) activity, lipid peroxidation (MDA), hydrogen peroxide (H₂O₂), proline (Pro) content, and rapid reductions in biomass, chlorophyll (Chl), and relative water content (RWC). Cu-caused oxidative stress was evident by overaccumulation of reactive oxygen species (ROS; superoxide (O₂ ^?–) and H₂O₂). Ascorbate (AsA) content decreased while GSH and phytochelatin (PC) content increased significantly in Cu-stressed seedlings. Exogenous SNP, GSH, or SNP?+?GSH decreased toxicity symptoms and diminished a Cu-induced increase in LOX activity, O₂ ^?–, H₂O₂, MDA, and Pro content. They also counteracted a Cu-induced increase in superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and glyoxalase I and glyoxalase II activities, which paralleled changes in ROS and MDA levels. These seedlings also showed a significant increase in catalase (CAT), glutathione peroxidase (GPX), dehydroascorbate reductase (DHAR), glutathione S-transferase (GST) activities, and AsA and PC content compared with the seedlings stressed with Cu alone. Cu analysis revealed that SNP and GSH restricted the accumulation of Cu in the roots and leaves of Cu-stressed seedlings. Our results suggest that Cu exposure provoked an oxidative burden while reduced Cu uptake and modulating the antioxidant defense and glyoxalase systems by adding SNP and GSH play an important role in alleviating Cu toxicity. Furthermore, the protective action of GSH and SNP?+?GSH was more efficient than SNP alone.     

Effects of Selenium and Topiramate on Cytosolic Ca2+ Influx and Oxidative Stress in Neuronal PC12 Cells

It has been widely suggested that selenium (Se) deficiency play an important role in the pathophysiology of epilepsy. It has been reported that Se provides protection against the neuronal damage in patients and animals with epilepsy by restoring the antioxidant defense mechanism. The neuroprotective effects of topiramate (TPM) have been reported in several studies but the putative mechanism of action remains elusive. We investigated effects of Se and TPM in neuronal PC12 cell by evaluating Ca²⁺ mobilization, lipid peroxidation and antioxidant levels. PC12 cells were divided into eight groups namely control, TPM, Se, H₂O₂, TPM + H₂O₂, Se + H₂O₂, Se + TPM and Se + TPM + H₂O₂. The toxic doses and times of H₂O₂, TPM and Se were determined by cell viability assay which is used to evaluate cell viability. Cells were incubated with 0.01 mM TPM for 5 h and 500 nM Se for 10 h. Then, the cells were exposed to 0.1 mM H₂O₂ for 10 h before analysis. The cells in all groups except control, TPM and Se were exposed to H₂O₂ for 15 min before analysis. Cytosolic Ca²⁺ release and lipid peroxidation levels were higher in H₂O₂ group than in control, Se and TPM combination groups although their levels were decreased by incubation of Se and TPM combination. However, there is no difference on Ca²⁺ release in TPM group. Glutathione peroxidase activity, reduced glutathione and vitamin C levels in the cells were lower in H₂O₂ group than in control, Se and TPM groups although their values were higher in the cells incubated with Se and TPM groups than in H₂O₂ groups. In conclusion, these results indicate that Se induced protective effects on oxidative stress in PC12 cells by modulating cytosolic Ca²⁺ influx and antioxidant levels. TPM modulated also lipid peroxidation and glutathione and vitamin C concentrations in the cell system.     

An Integrated Mitochondrial ROS Production and Scavenging Model: Implications for Heart Failure

It has been observed experimentally that cells from failing hearts exhibit elevated levels of reactive oxygen species (ROS) upon increases in energetic workload. One proposed mechanism for this behavior is mitochondrial Ca²⁺ mismanagement that leads to depletion of ROS scavengers. Here, we present a computational model to test this hypothesis. Previously published models of ROS production and scavenging were combined and reparameterized to describe ROS regulation in the cellular environment. Extramitochondrial Ca²⁺ pulses were applied to simulate frequency-dependent changes in cytosolic Ca²⁺. Model results show that decreased mitochondrial Ca²⁺uptake due to mitochondrial Ca²⁺ uniporter inhibition (simulating Ru360) or elevated cytosolic Na⁺, as in heart failure, leads to a decreased supply of NADH and NADPH upon increasing cellular workload. Oxidation of NADPH leads to oxidation of glutathione (GSH) and increased mitochondrial ROS levels, validating the Ca²⁺ mismanagement hypothesis. The model goes on to predict that the ratio of steady-state [H₂O₂]_m during 3Hz pacing to [H₂O₂]_m at rest is highly sensitive to the size of the GSH pool. The largest relative increase in [H₂O₂]_m in response to pacing is shown to occur when the total GSH and GSSG is close to 1 mM, whereas pool sizes below 0.9 mM result in high resting H₂O₂ levels, a quantitative prediction only possible with a computational model.     

An Integrated Mitochondrial ROS Production and Scavenging Model: Implications for Heart Failure

It has been observed experimentally that cells from failing hearts exhibit elevated levels of reactive oxygen species (ROS) upon increases in energetic workload. One proposed mechanism for this behavior is mitochondrial Ca²⁺ mismanagement that leads to depletion of ROS scavengers. Here, we present a computational model to test this hypothesis. Previously published models of ROS production and scavenging were combined and reparameterized to describe ROS regulation in the cellular environment. Extramitochondrial Ca²⁺ pulses were applied to simulate frequency-dependent changes in cytosolic Ca²⁺. Model results show that decreased mitochondrial Ca²⁺uptake due to mitochondrial Ca²⁺ uniporter inhibition (simulating Ru360) or elevated cytosolic Na⁺, as in heart failure, leads to a decreased supply of NADH and NADPH upon increasing cellular workload. Oxidation of NADPH leads to oxidation of glutathione (GSH) and increased mitochondrial ROS levels, validating the Ca²⁺ mismanagement hypothesis. The model goes on to predict that the ratio of steady-state [H₂O₂]_m during 3Hz pacing to [H₂O₂]_m at rest is highly sensitive to the size of the GSH pool. The largest relative increase in [H₂O₂]_m in response to pacing is shown to occur when the total GSH and GSSG is close to 1 mM, whereas pool sizes below 0.9 mM result in high resting H₂O₂ levels, a quantitative prediction only possible with a computational model.     

Pyrogallol inhibits the growth of human pulmonary adenocarcinoma A549 cells by arresting cell cycle and triggering apoptosis

Pyrogallol (PG) is a polyphenol compound and has been known to be an O generator. We evaluated the effects of PG on the growth of human pulmonary A549 cells in relation to the cell cycle and apoptosis. Treatment with 50 or 100 μM PG significantly inhibited the cell growth of A549 for 72 h. DNA flow cytometric analysis indicated that PG slightly induced a G1 phase arrest of the cell cycle at 24 or 48 h, but did not induce the specific cell cycle arrest at 72 h. Intracellular GSH depletion was observed in PG‐treated cells. PG induced apoptosis in A549 cells, as evidenced by sub‐G1 cells, annexin V staining cells, and the loss of mitochondrial membrane potential (Δ Ψ_m). The intracellular ROS (reactive oxygen species) level including O increased in PG‐treated A549 cells at 24 and 48 h, and persisted at 72 h. The changes in GSH as well as ROS levels by PG affected the cell viability in A549 cells. In conclusion, PG inhibited the growth of human pulmonary A549 cells by inducing cell cycle arrest as well as triggering apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:36–42, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20263     

The changes of reactive oxygen species and glutathione by MG132, a proteasome inhibitor affect As4.1 juxtaglomerular cell growth and death

The proteasome inhibitor MG132 has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). Here, we evaluated the effects of MG132 on the growth and death of As4.1 juxtaglomerular cells in relation to ROS and glutathione (GSH) levels. MG132 inhibited the growth of As4.1 cells with an IC₅₀ of approximately 0.3–0.4 μM at 48 h and induced cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m), Bcl-2 decrease, activation of caspase-3 and -8, and PARP cleavage. MG132 increased intracellular ROS levels including O₂^? and GSH depleted cell numbers. N-acetyl cysteine (NAC, a well-known antioxidant) significantly decreased ROS level and GSH depleted cell numbers in MG132-treated As4.1 cells, along with the prevention of cell growth inhibition, cell death and MMP (ΔΨ_m) loss. NAC also decreased the caspase-3 activity of MG132. l-Buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) or diethyldithiocarbamate (DDC; an inhibitor of Cu/Zn-SOD) did not affect cell growth, death, ROS and GSH levels in MG132-treated As4.1 cells. Conclusively, MG132 reduced the growth of As4.1 cells via apoptosis. The changes of ROS and GSH by MG132 were involved in As4.1 cell growth and death.     

The role of insulin against hydrogen peroxide-induced oxidative damages in differentiated SH-SY5Y cells

Abstract

Exogenous hydrogen peroxide (H₂O₂) can easily penetrate into biological membranes and enhance the formation of other reactive oxygen species (ROS). In the present study, we have investigated the neuroprotective effects of insulin on H₂O₂-induced toxicity of retinoic acid (RA)-differentiated SH-SY5Y cells. To measure the changes in the cell viability of SH-SY5Y cells at different concentrations of H₂O₂ for 24?h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based assay was used and a 100?µM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 24?h of 100?µM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), ROS, and calcium ion (Ca²⁺) in neuronal cells, but insulin can effectively diminish the H₂O₂-induced oxidative damages to these cells. Moreover, cells treated with insulin increased H₂O₂-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of insulin treatment with SH-SY5Y cells increased the Bcl-2 levels and decreased the Akt levels. The treatment of insulin had played a protective effect on H₂O₂-induced oxidative stress related to the Akt/Bcl-2 pathways.     

Enhancement of antioxidant enzymes activity and expression of CAT and PAL genes in hazel (Corylus avellana L.) cells in response to low-intensity ultrasound

Induction of antioxidant systems of hazel cells by low-energy ultrasound, the potential role of hydrogen peroxide (H₂O₂) as a signaling molecule in regulation of activity of stress-related enzymes, and expression of catalase (CAT) and phenylalanine ammonialyase (PAL) genes were investigated. Suspension-cultured Corylus avellana L. cells were agitated by an ultrasonic device at 29 kHz with the power of 4 mW/cm², for 8–40 min. The activities of CAT, superoxide dismutase (SOD), and ascorbate peroxidase (APX) of treated cells increased by 4, 1.7 and 7 times of the control ones, respectively. Induction of increase in the expression of CAT gene started 24 h after the treatment with ultrasound. Significant increase also was observed in the expression of PAL gene, 6 h after exposure to ultrasound, which resulted in turn to increase of total contents of soluble phenolics, 24 h of the treatment. Exposure to ultrasound up to 20 min had no adverse effects on cell viability although it slightly increased the accumulation of H₂O₂. However, it is likely that this level of increased H₂O₂ was not deteriorative for hazel cells, but rather triggered antioxidant system and provided hazel cells a sustainable growth after ultrasound treatment.     

Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.     

Functional roles of superoxide and hydrogen peroxide generated by mitochondrial DNA mutation in regulating tumorigenicity of HepG2 cells

Mitochondria are a major source of reactive oxygen species (ROS). Recent studies have estimated that mitochondrial DNA mutations inducing the overproduction of ROS are associated with human cancer. However, a substantial challenge in elucidating their diverse roles in regulating tumorigenesis is the lack of methods for probing ROS in living systems with molecular specificity. In this study, we reported the application of two fluorescent probes, 2‐chloro‐1,3‐dibenzothiazolinecyclohexene and naphthofluorescein disulfonate, which showed high selectivity for superoxide (O₂^•−) and hydrogen peroxide (H₂O₂). They were capable of detecting and visualizing O₂^•− and H₂O₂ overproduction caused by a mutation in the gene encoding nicotinamide adenine dinucleotide dehydrogenase subunit 6 (ND6) in HepG2 cells. The levels of O₂^•− and H₂O₂ in mitochondria isolated from HepG2 cells were found to be 0·63 ± 0·07 and 1·13 ± 0·05 μM, respectively. Using assays of tumorigenesis in mouse models, we found that treatment of the mice with different ROS scavengers suppressed tumour growth. These findings suggested that ROS generated by ND6 gene mutation do play an important role in regulating tumorigenesis and H₂O₂ may be a key modulator. Copyright © 2011 John Wiley & Sons, Ltd.