Altered Reactive Oxygen Species Production Research Articles

SNTA1 altered reactive oxygen species production is associated with J wave syndromes

BackgroundJ wave syndromes (JWS) are life-threatening arrhythmias characterized by a prominent J wave in electrocardiograms. MethodsWhole exome sequencing was utilized to uncover genetic variants associated with JWS. Pathogenicity score and mode of inheritance through bioinformatics tools were undertaking to identify the possible disease-causing mutation. ROS production was stained with red fluorescence by Dihydroethidium. ResultsSNTA1 gene mutation (Ser495Leu), an extremely rare and putatively pathogenic heterozygous mutation site was first reported associated with a JWS family. Ser495Leu heterozygous mutation of SNTA1 was noted in the patient and his mother. C/T hybridization of the patient and his mother were confirmed and his father was recognized as C/C homozygous by Sanger sequencing. Both support vector machine and neural network analyses suggested that the mutation site (c.1484C>T) could reduce SNTA1 stability. Their confidence score was −0.8718 and −0.9934, respectively, which means that primarily reduce SNTA1 protein stability. SNTA1 Overexpression could decrease ROS production, while knockdown could reverse in H9C2 cells in vitro. ConclusionsThese findings imply that SNTA1 mutation may be associated with JWS. SNTA1, as a heterozygous mutation site in JWS, is important as a potential target for screening in prospective clinical practice.

Reactive oxygen species metabolism and photosynthetic performance in leaves of Hordeum vulgare plants co-infested with Heterodera filipjevi and Aceria tosichella

Key messageDefence responses of cyst nematode and/or wheat curl mite infested barley engage the altered reactive oxygen species production, antioxidant machinery, carbon dioxide assimilation and photosynthesis efficiency.The primary aim of this study was to determine how barley responds to two pests infesting separately or at once; thus barley was inoculated with Heterodera filipjevi (Madzhidov) Stelter (cereal cyst nematode; CCN) and Aceria tosichella Keifer (wheat curl mite; WCM). To verify hypothesis about the involvement of redox metabolism and photosynthesis in barley defence responses, biochemical, photosynthesis efficiency and chlorophyll a fluorescence measurements as well as transmission electron microscopy were implemented. Inoculation with WCM (apart from or with CCN) brought about a significant suppression in the efficiency of electron transport outside photosystem II reaction centres. This limitation was an effect of diminished pool of rapidly reducing plastoquinone and decreased total electron carriers. Infestation with WCM (apart from or with CCN) also significantly restricted the electron transport on the photosystem I acceptor side, therefore produced reactive oxygen species oxidized lipids in cells of WCM and double infested plants and proteins in cells of WCM-infested plants. The level of hydrogen peroxide was significantly decreased in double infested plants because of glutathione–ascorbate cycle involvement. The inhibition of nitrosoglutathione reductase promoted the accumulation of S-nitrosoglutathione increasing antioxidant capacity in cells of double infested plants. Moreover, enhanced arginase activity in WCM-infested plants could stimulate synthesis of polyamines participating in plant antioxidant response. Infestation with WCM (apart from or with CCN) significantly reduced the efficiency of carbon dioxide assimilation by barley leaves, whereas infection only with CCN expanded photosynthesis efficiency. These were accompanied with the ultrastructural changes in chloroplasts during CCN and WCM infestation.

Virodhamine, an endocannabinoid, induces megakaryocyte differentiation by regulating MAPK activity and function of mitochondria.

Endocannabinoids are well-known regulators of neurotransmission by activating the cannabinoid (CB) receptors. Endocannabinoids are being used extensively for the treatment of various neurological disorders such as Alzheimer's and Parkinson's diseases. Although endocannabinoids are well studied in cell survival, proliferation, and differentiation in various neurological disorders and several cancers, the functional role in the regulation of blood cell development is less examined. In the present study, virodhamine, which is an agonist of CB receptor-2, was used to examine its effect on megakaryocytic development from a megakaryoblastic cell. We observed that virodhamine increases cell adherence, cell size, and cytoplasmic protrusions. Interestingly, we have also observed large nucleus and increased expression of megakaryocytic marker (CD61), which are the typical hallmarks of megakaryocytic differentiation. Furthermore, the increased expression of CB2 receptor was noticed in virodhamine-induced megakaryocytic cells. The effect of virodhamine on megakaryocytic differentiation could be mediated through CB2 receptor. Therefore, we have studied virodhamine induced molecular regulation of megakaryocytic differentiation; mitogen-activated protein kinase (MAPK) activity, mitochondrial function, and reactive oxygen species (ROS) production were majorly affected. The altered mitochondrial functions and ROS production is the crucial event associated with megakaryocytic differentiation and maturation. In the present study, we report that virodhamine induces megakaryocytic differentiation by triggering MAPK signaling and ROS production either through MAPK effects on ROS-generating enzymes or by the target vanilloid receptor 1-mediated regulation of mitochondrial function.

Sappanchalcone, a flavonoid isolated from Caesalpinia sappan L., induces caspase-dependent and AIF-dependent apoptosis in human colon cancer cells

The present study examined the apoptotic effects and the underlying mechanism of sappanchalcone, a major bioactive compound isolated from Caesalpinia sappan L. on human colon cancer cells. To achieve this, we used two different colon cancer cell lines, namely HCT116 (as wild-type p53 cells) and SW480 (as p53-mutant cells) cells. Our results illustrated that sappanchalcone treatment decreased the proliferation and further promoted apoptosis in HCT116 cells compared with the findings in SW480 cells. Sappanchalcone triggered phosphorylation of p53, which is involved in the activation of caspases and increased expression of Bax in HCT116 cells. Conversely, sappanchalcone-treated SW480 cells displayed no change in p53 phosphorylation or caspase activation. In addition, sappanchalcone further increased reactive oxygen species (ROS) levels and apoptosis-inducing factor (AIF) release in both HCT116 and SW480 cells. These data suggest that sappanchalcone induces apoptosis through caspase-dependent and caspases-independent mechanisms that were characterized by decreased Bcl-2 expression, mitochondrial targeting, and altered ROS production and AIF translocation to the nuclei.

Metformin promotes susceptibility to experimental Leishmania braziliensis infection.

BACKGROUND Metformin (MET) is a hypoglycemic drug used for the treatment of diabetes, despite interference in host immunity against microorganisms. Cutaneous infection caused by pathogens such as Leishmania braziliensis (Lb), the agent responsible for cutaneous leishmaniasis (CL) in Brazil, represents an interesting model in which to evaluate the effects associated with MET.OBJECTIVE To evaluate the modulatory effect of MET in Lb infection.MATERIAL AND METHODS Experimental study of Lb infection and MET treatment in BALB/c mice and Raw 264.7 macrophages.FINDINGS MET treatment interfered with lesion kinetics, increased parasite load and reduced macrophage proliferation. Low concentrations of MET in Lb culture allow for the maintenance of stationary parasite growth phase. Lb-infected cells treated with MET exhibited increased parasite load. While both MET and Lb infection alone promoted the production of intracellular reactive oxygen species (ROS), reduced levels of ROS were seen in MET-treated Lb-infected macrophages.MAIN CONCLUSION Experimental treatment with MET interfered with the kinetics of cutaneous ulceration, increased Lb parasite load, altered ROS production and modulated cellular proliferation. Our experimental results indicate that MET interfere with the evolution of CL.

Effect of Arsenic on Intracellular Calcium & Redox Homeostasis

A range of environmental agents causes tissue injury that has been attributed to reactive oxygen species (ROS). Altered ROS production is commonly originated from the mitochondria and is associated with dysregulation of calcium (Ca2+) homeostasis and mitochondrial apoptosis. However, the causative pathways remain largely unknown because the fundamental roles of mitochondria in cell survival, cell signaling and dynamics are commonly mediated through local communication between mitochondria and other organelles, which have been difficult to directly monitor. Our aim is to develop fluorescent protein-based tools allowing sensitive and specific measurements of ROS and [Ca2+] at the mitochondria-endoplasmic reticulum (ER) interface. Applying these novel tools, we have studied the effects of environmental stress caused by arsenicon local mitochondria-ER communication and its impact for cell signaling, and cell physiology in primary mouse hepatocytes. Either acute addition (30 µM) or prolonged, overnight exposure (3 µM) to sodium arsenite has induced an increase in ROS. Upon prolonged arsenic exposure, these alterations were accompanied by a significant decrease in the cytoplasmic calcium signal to an IP3-linked agonist: a decrease was observed in the fraction of responsive cells, in the amplitude of the response as well as in the sustained phase. Regarding the acute effect of arsenic, we observed a progressive increase of the basal cytoplasmic [Ca2+] with a decrease in the amplitude of the IP3-linked calcium signal. Neither acute nor prolonged arsenic exposure impaired mitochondrial calcium uptake. Nevertheless, we documented increased mitochondrial calcium content in the prolonged arsenic exposure group. In conclusion, the results suggest that an initial component of the arsenic-induced hepatocyte injury is increased ROS, which is associated with impaired local mitochondria-ER calcium communication.

Respiration and substrate transport rates as well as reactive oxygen species production distinguish mitochondria from brain and liver.

BackgroundAberrant mitochondrial function, including excessive reactive oxygen species (ROS) production, has been implicated in the pathogenesis of human diseases. The use of mitochondrial inhibitors to ascertain the sites in the electron transport chain (ETC) resulting in altered ROS production can be an important tool. However, the response of mouse mitochondria to ETC inhibitors has not been thoroughly assessed. Here we set out to characterize the differences in phenotypic response to ETC inhibitors between the more energetically demanding brain mitochondria and less energetically demanding liver mitochondria in commonly utilized C57BL/6J mice.ResultsWe show that in contrast to brain mitochondria, inhibiting distally within complex I or within complex III does not increase liver mitochondrial ROS production supported by complex I substrates, and liver mitochondrial ROS production supported by complex II substrates occurred primarily independent of membrane potential. Complex I, II, and III enzymatic activities and membrane potential were equivalent between liver and brain and responded to ETC. inhibitors similarly. Brain mitochondria exhibited an approximately two-fold increase in complex I and II supported respiration compared with liver mitochondria while exhibiting similar responses to inhibitors. Elevated NADH transport and heightened complex II–III coupled activity accounted for increased complex I and II supported respiration, respectively in brain mitochondria.ConclusionsWe conclude that important mechanistic differences exist between mouse liver and brain mitochondria and that mouse mitochondria exhibit phenotypic differences compared with mitochondria from other species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0051-8) contains supplementary material, which is available to authorized users.

Cilostazol does not improve peripheral arterial disease-linked oxidative stress.

Cilostazol is a drug widely used to treat peripheral arterial disease (PAD) in patients with advanced atherosclerosis. It is an inhibitor of platelet aggregation and causes arterial vasodilatation. It has been speculated that cilostazol might act to improve oxidative stress in these patients. We analyzed 25 patients with demonstrated lower limb peripheral arterial disease to determine whether lipid peroxidation (LPO) or total antioxidant capacity (TAC) were modified after 6 months of cilostazol treatment (postintervention phase) with respect to the basal phase. Analysis of plasma samples determined that LPO levels decreased significantly over the postintervention phase (26±15 vs. 11±7 pM, P=0.0003). However, when TAC levels were analyzed, no significant differences were observed (0.80±0.21 vs. 0.85±0.17 mM, P=0.42). Under basal conditions, LPO showed a positive correlation to treatment as judged by the ankle-brachial index (r=0.800, P=0.002) as well as uric acid (r=0.508, P=0.03) and CRP (r=0.481, P=0.05) levels. In contrast, TAC negatively correlated with triglycerides (r=-0.879, P<0.0001) and microalbuminuria (r=-0.868, P<0.0001). In the postintervention phase, TAC negatively correlated with HbA1c (r=-0.570, P=0.02) and triglycerides (r=-0.864, P<0.0001). Our data indicate that cilostazol treatment does not improve oxidative stress in PAD patients after 6 months of treatment. However, lipid peroxidation and total antioxidant capacity were affected by cilostrazol treatment, which could be related to altered reactive oxygen species production. Further research may be needed to determine the critical dose of cilostazol to clarify the protective role of this drug in PAD.

Abstract 4750: Development of potent NADPH oxidase inhibitors with significant activity against colon cancer

Abstract The NADPH oxidase (NOX) gene family (NOX1-5 and Duox1/2) plays an important role in host defense and the development of inflammation-induced carcinogenesis. We recently found that two iodonium-class NOX inhibitors, diphenyleneiodonium (DPI) and di-2-thienyliodonium (DTI), block NOX-dependent reactive oxygen species (ROS) formation, NOX1 mRNA expression, and growth of human colon cancer cells in vitro and in vivo (Free Rad. Biol. Med. 57: 162, 2013). To improve the efficacy and bioavailability of DPI and DTI, we synthesized 48 new analogs of these compounds (21 DPI analogs, 12 DTI analogs, and 15 DPI and/or DTI hybrids; structures to be presented at the meeting) and prioritized the agents for further study based on inhibition of: HT-29 growth (MTT and clonogenic assay), ROS generation (by chemiluminescence and flow cytometry), and NOX1 mRNA expression (by qRT-PCR). The compounds were also characterized for lack of altered mitochondrial ROS production, and inhibitory effects on ROS generated by cell-based assays specific for NOX1, NOX2, NOX4, NOX5, and Duox2. DPI and five DPI analogs, NSCs 740104, 734428, 742837, 751140, 737392, strongly inhibited HT-29 cell growth in a dose dependant manner (IC50's after 48 hr drug exposure of 300, 50, 74, 87, 148, and 202 nM, respectively). Treatment for 24 hr with 10 nM DPI, 740104, 734428, or 751140, or with 100 nM 737392 significantly decreased ROS production in HT-29 cells. In addition, 60% or greater inhibition of NOX1 mRNA expression was observed following treatment with 250 nM 740104 and 734428 for 4 hr; 751140 and DPI for 24 hr; and 737392 for 48 hr. Analog 742837 demonstrated no significant inhibition of either ROS production or NOX1 expression in HT-29 cells; it was not analyzed further. Growth inhibitory properties of 734958, a DTI analog, were similar to the parent molecule. To evaluate compound specificity, a luminescence assay for O2•¯ was used with: HEK293 cells expressing all components of the NOX1 complex; fully differentiated HL-60 cells expressing active NOX2; and UACC257 melanoma cells that exclusively express active NOX5. H2O2 was measured by Amplex Red assay to determine NOX4 or Duox2 activity in fully reconstituted HEK293 cells. DPI, 734428, 740104, 737392, or 751140 exposure for 30 min inhibited NOX1, 2, 4, and 5 with low nM IC50's; analogs 740104, 734428, and 751140 were more potent inhibitors of NOX1, 2, and 4, respectively, than DPI; each was broadly active against these NOX isoforms. The DTI analog 734958 was more potent than its parent molecule. NSC737392 and 734428 were &amp;gt;10-fold more active than the other compounds examined against Duox2 (200-400 nM IC50's). In summary, we developed novel iodonium analogs with anticancer activity that possess enhanced potency against human NOX species, including the first agents with specificity against Duox2. These tool compounds will be evaluated in vivo for their therapeutic and anti-inflammatory activities in NOX-containing tumor xenograft models. Citation Format: Jiamo Lu, Prabhakar Risbood, Charles T. Kane, Md Tafazzal Hossain, Yongzhong Wu, Smitha Antony, Jennifer L. Meitzler, Agnes Juhasz, Han Liu, Guojian Jiang, Krishnendu Roy, James H. Doroshow. Development of potent NADPH oxidase inhibitors with significant activity against colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4750. doi:10.1158/1538-7445.AM2014-4750

Loss of UCP2 Attenuates Mitochondrial Dysfunction without Altering ROS Production and Uncoupling Activity

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.

Relationships between human vitality and mitochondrial respiratory parameters, reactive oxygen species production and dNTP levels in peripheral blood mononuclear cells

Low vitality (a component of fatigue) in middle-aged and older adults is an important complaint often identified as a symptom of a disease state or side effect of a treatment. No studies to date have investigated the potential link between dysfunctional mitochondrial ATP production and low vitality. Therefore, we measured a number of cellular parameters related to mitochondrial activity in peripheral blood mononuclear cells (PBMCs) isolated from middle-aged men, and tested for association with vitality. These parameters estimate mitochondrial respiration, reactive oxygen species (ROS) production, and deoxyribonucleotide (dNTP) balance in PBMCs. The population was drawn from the Metropolit cohort of men born in 1953. Vitality level was estimated from the Medical Outcomes Study Short Form 36 (SF-36) vitality scale. We found that vitality score had no association with any of the mitochondrial respiration parameters. However, vitality score was inversely associated with cellular ROS production and cellular deoxythymidine triphosphate (dTTP) levels and positively associated with deoxycytidine triphosphate (dCTP) levels. We conclude that self-reported persistent low vitality is not associated with specific aspects of mitochondrial oxidative phosphorylation capacity in PBMCs, but may have other underlying cellular dysfunctions that contribute to dNTP imbalance and altered ROS production.

Loss of Stem Cell Quiescence and Impaired Stem Cell Function of CD34+/CD38low Cells One Year Following Autologous Stem Cell Transplantation

Abstract 1893Autologous stem cell transplantation (ASCT) allows the application of high-dose chemotherapy and is included in the standard treatment regimens for multiple myeloma and relapsing lymphoma. The application of ASCT has considerably improved treatment outcome but in 30–50% of the patients the underlying malignant disorder relapses. In that case treatment options are limited, in part due to a diminished capacity of the transplanted bone marrow to tolerate chemotherapy. The transplanted bone marrow seems to be more vulnerable to chemotoxic stress. This is supported by our recent observations that progenitor cells from post-ASCT bone marrow demonstrate altered phenotypic and functional properties (Woolthuis et al. BMT 2011:110-115). In the present study, a more detailed examination of the hematopoietic stem cell (HSC) compartment post-ASCT was performed. The examined bone marrow cells were obtained from patients with normal peripheral blood cell counts one year after ASCT. First it was studied whether stem cell quiescence had been changed due to the ASCT procedure. Therefore the percentage of cells in the G0 phase was measured by staining the cells with Hoechst and Pyronin Y followed by flowcytometric analysis. Both the stem cell (CD34+/CD38low) and progenitor (CD34+/CD38+) fractions were analyzed. Post-ASCT bone marrow cells (n=6) were compared with normal bone marrow cells (n=9) and mobilized peripheral blood stem cells (PBSC) (n=7). Interestingly, post-ASCT bone marrow contained a significantly lower percentage of quiescent cells in the CD34+/CD38low fraction compared to normal bone marrow (mean percentage 23.6% (95%CI: 6.5–40.8) vs. 48.6% (95%CI: 31.4–65.9), p=0.045). In contrast no differences were observed in the CD34+/CD38+ fraction. To examine whether the loss of stem cell quiescence is associated with loss of stem cell function, stem cell frequency was analyzed in vitro by the long-term colony-initiating cell assay. These analyses revealed a strongly decreased stem cell frequency in post-ASCT CD34+ cells compared to normal CD34+ bone marrow cells (mean frequency 0.0016 (95%CI: 0.0003–0.0028) vs. 0.0206 (95%CI: 0.0162–0.0250), p=0.002). Importantly, CD34+ PBSC cells demonstrated a stem cell frequency comparable with the frequency observed in normal bone marrow, suggesting that the decrease in stem cell frequency observed in post-ASCT bone marrow can not simply be explained by the previous chemotherapy and the mobilization procedure. To obtain more insight into the mechanisms explaining our observations, levels of reactive oxygen species (ROS) were measured by flowcytometry. Comparable ROS levels were observed in post-ASCT and normal bone marrow CD34+/CD38low cells while the CD34+/CD38+ post-ASCT cells demonstrated significantly higher ROS levels compared to normal bone marrow (p=0.01). In addition significant higher ROS levels were observed in CD34+/CD38low PBSC compared to normal bone marrow (p=0.043). To define pathways associated with altered quiescence and/or ROS production, gene expression analysis was performed comparing CD34+ cells from post-ASCT bone marrow (n=6), normal bone marrow (n=31) and PBSC (n=5). Preliminary analysis reveals an upregulation in post-ASCT CD34+ cells of genes involved in cell cycle compared to normal bone marrow. In contrast, cell cycle genes appeared to be downregulated in CD34+ PBSC cells. In conclusion, our data indicate that the diminished regenerative capacity of bone marrow post-ASCT is likely related to a loss of stem cell quiescence and enhanced ROS production by progenitor cells which will make the bone marrow cells more vulnerable for the effects of cytotoxic stress. Disclosures:No relevant conflicts of interest to declare.

Evaluation of correct endogenous reactive oxygen species content for human sperm capacitation and involvement of the NADPH oxidase system

Generation of controlled amounts of reactive oxygen species (ROS) and phosphorylation of protein tyrosine residues (Tyr) are two closely related changes involved in sperm capacitation. This study investigated the effect of altered endogenous ROS production on Tyr-phosphorylation (Tyr-P), acrosome reaction (AR) and cell viability during sperm capacitation. The possible origin of the altered ROS production was also evaluated by apocynin (APO) or oligomycin (Oligo) addition. A total of 63 samples of purified sperm were analysed for ROS production by enhanced chemiluminescence, Tyr-P pattern by immunocytochemistry, and AR and viability by fluorochrome fluorescein isothiocyanate (FITC)-labelled peanut (Arachis hypogaea) agglutinin and propidium iodide positivity, respectively. Samples were divided into four categories depending on the ability of sperm to produce ROS, expressed as Relative Luminescence Units (RLU), in capacitating conditions: low ROS production (LRP), range about 0.0-0.05 RLU; normal (NRP), 0.05-0.1 RLU; high (HRP), 0.1-0.4 RLU; very high (VHRP) 0.4-2.0 RLU. In NRP sperm heads, capacitation induced Tyr-P in 87.9 ± 4.3%, and the AR occurred in 62.5 ± 5.4% of cells; in LRP, HRP and VHRP Tyr-P labelling rarely spread over the head, acrosome-reacted cells only accounted for a small number of sperm, and the non-viable cells (NVC) were increased. The addition of APO, but not Oligo, drastically decreased ROS production in analysed samples. This study proposes the optimal threshold for endogenous ROS production for correct sperm viability and functioning, and indicates the direct involvement of APO-sensitive NADPH oxidase in ROS production.

Ethylene signaling regulates accumulation of the FLS2 receptor and is required for the oxidative burst contributing to plant immunity.

Reactive oxygen species (ROS) are potent signal molecules rapidly generated in response to stress. Detection of pathogen-associated molecular patterns induces a transient apoplastic ROS through the function of the NADPH respiratory burst oxidase homologs D (RbohD). However, little is known about the regulation of pathogen-associated molecular pattern-elicited ROS or its role in plant immunity. We investigated ROS production triggered by bacterial flagellin (flg22) in Arabidopsis (Arabidopsis thaliana). The oxidative burst was diminished in ethylene-insensitive mutants. Flagellin Sensitive2 (FLS2) accumulation was reduced in etr1 and ein2, indicating a requirement of ethylene signaling for FLS2 expression. Multiplication of virulent bacteria was enhanced in Arabidopsis lines displaying altered ROS production at early but not late stages of infection, suggesting an impairment of preinvasive immunity. Stomatal closure, a mechanism used to reduce bacterial entry into plant tissues, was abolished in etr1, ein2, and rbohD mutants. These results point to the importance of flg22-triggered ROS at an early stage of the plant immune response.

γ‐H2AX Formation is not Coupled to Elevations in Intracellular Ca2+, and Hyperactivation of PARP does not Facilitate AIF Translocation During ROS‐induced Cell Death.

2,3,5‐tris(Glutathion‐S‐yl)hydroquinone (TGHQ) is a redox‐active metabolite of hydroquinone, and causes non‐apoptotic cell death in renal proximal tubule epithelial cells (HK‐2) via the generation of reactive oxygen species (ROS). TGHQ induced ROS generation and DNA strand breaks leads to the overactivation of poly(ADP‐ribose) polymerase‐1 (PARP‐1), the rapid depletion of nicotinamide adenine dinucleotide (NAD+), and the appearance of γ‐H2AX foci. Inhibition of PARP‐1 completely blocked TGHQ‐mediated poly(ADP‐ribosyl)ation (PAR), NAD+ consumption, and the consequent necrotic cell death. Moreover, although Ca2+ chelation inhibited PAR and significantly abrogated cell death, NAD+ depletion was only delayed and not prevented. Despite their ability to inhibit cell death, neither PARP inhibition nor Ca2+ chelation altered ROS production or the initial DNA damage (γ‐H2AX foci). Interestingly, PARP‐1 hyperactivation was not accompanied by the translocation of apoptosis‐inducing factor (AIF) from mitochondria to the nucleus, a process usually associated with PARP‐dependent cell death. In conclusion, PARP activation and Ca2+ elevation are critical responses to ROS‐mediated cell death, but at least in HK‐2 cells γ‐H2AX formation is not coupled to elevations in intracellular Ca2+, and PARP activation does not facilitate AIF translocation. (DK59491, P30 ES 06694).

Possible role of avian uncoupling protein in down-regulating mitochondrial superoxide production in skeletal muscle of fasted chickens

Little is known about the precise physiological roles of uncoupling protein 1 (UCP1) homologs (UCP2, UCP3, avian UCP) whose levels are up-regulated during fasting. UCPs in skeletal muscle are thought to play a role in the regulation of lipids as fuel substrates, and/or in controlling the production of reactive oxygen species (ROS). The aim of this investigation, using skeletal muscle from fasted chickens, was to examine alterations in the expression of genes encoding for avian UCP and key enzymes relevant to lipid flux across the mitochondrial β-oxidation pathway. We also clarified whether an increase in avUCP content could be associated with altered ROS production by mitochondria. Transcription levels of avUCP and CPT-I genes were increased 7.7- and 9.5-fold after a 24 h fast and slightly diminished but remained about 5.0- and 7.7-fold higher than baseline levels, respectively, after 48 h of fasting. In contrast, members of the β-oxidation pathway, LCAD and 3HADH, were gradually up-regulated from 12 to 48 h of fasting. This suggests that processes involved in the transfer and oxidation of fatty acids are up-regulated differently during the initial stage of fasting. Analysis of ROS production by lucigenin-derived chemiluminescence showed that the FFA-sensitive portion of carboxyatractyloside-upregulated ROS production was greater in skeletal muscle mitochondria from 24 h-fasted chickens compared with control, which leads us to postulate that ROS production is potentially down-regulated by UCP. The possible involvement of a backlog of fatty acid for oxidation, observed in chickens after a 24 h fast, in a transmembrane gradient of free non-oxidized fatty acids is also discussed.

OxLDL enhances L-type Ca2+ currents via lysophosphatidylcholine-induced mitochondrial reactive oxygen species (ROS) production

To examine the mechanisms underlying oxidised LDL- (oxLDL)-induced alterations in Ca(2+) currents, an effect which underlies altered vascular contractility and cardiac myocyte function. Ca(2+) currents (I(Ca)) were recorded by whole-cell patch-clamp in HEK293 cells expressing L-type Ca(2+) channel alpha(1C) subunits or isolated rat ventricular myocytes. oxLDL (but not native LDL) significantly enhanced recombinant I(Ca), an effect mimicked by 1 microM lysophosphatidylcholine (LPC). LPC failed to enhance I(Ca) either in mitochondrial electron transport chain-depleted rho(0) cells, or in the presence of rotenone (1 microM), or MPP(+) (10 microM). The LPC response was similarly ablated by ascorbate (200 microM) or TROLOX (500 microM) and by the mitochondria-targeted antioxidant, MitoQ (250 nM). In myocytes, enhancement of I(Ca) due to LPC was similarly abrogated with rotenone and MitoQ. These data suggest that LPC enhanced recombinant Ca(2+) currents due to increased mitochondrial ROS production. In support with this, LPC enhanced fluorescence in HEK293 cells and cardiac myocytes loaded with a ROS-sensitive mitochondrial dye, reduced mitotracker red. LPC up-regulates L-type Ca(2+) currents due to altered mitochondrial ROS production, an effect which mediates the response of the native I(Ca) in cardiac myocytes to oxLDL.

The chlorinated AHR ligand 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) promotes reactive oxygen species (ROS) production during embryonic development in the killifish ( Fundulus heteroclitus)

Exposure to dioxin-like chemicals that activate the aryl hydrocarbon receptor (AHR) can result in increased cellular and tissue production of reactive oxygen species (ROS). Little is known of these effects during early fish development. We used the fish model, Fundulus heteroclitus, to determine if the AHR ligand and pro-oxidant 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) can increase ROS production during killifish development, and to test a novel method for measuring ROS non-invasively in a living organism. The superoxide-sensitive fluorescent dye, dihydroethidium (DHE), was used to detect in ovo ROS production microscopically in developing killifish exposed to PCB126 or vehicle. Both in ovo CYP1A activity (ethoxyresorufin- o-deethylase, EROD) and in ovo ROS were induced by PCB126. In ovo CYP1A activity was inducible by PCB126 concentrations as low as 0.003 nM, with maximal induction occurring at 0.3 nM PCB126. These PCB126 concentrations also significantly increased in ovo ROS production in embryonic liver, ROS being detectable as early as 5 days post-fertilization. These data demonstrate that the pro-oxidant and CYP1A inducer, PCB126, increases both CYP1A activity and ROS production in developing killifish embryos. The superoxide detection assay (SoDA) described in this paper provides a semi-quantitative, easily measured, early indicator of altered ROS production that can be used in conjunction with simultaneous in ovo measurements of CYP1A activity and embryo development to explore functional relationships among biochemical, physiological and developmental responses to AHR ligands.

Decreased reactive oxygen species concentration in the elongation zone contributes to the reduction in maize leaf growth under salinity

Reactive oxygen species (ROS) in the apoplast of cells in the growing zone of grass leaves are required for elongation growth. This work evaluates whether salinity-induced reductions in leaf elongation are related to altered ROS production. Studies were performed in actively growing segments (SEZ) obtained from leaf three of 14-d-old maize (Zea mays L.) seedlings gradually salinized to 150 mM NaCl. Salinity reduced elongation rates and the length of the leaf growth zone. When SEZ obtained from the elongation zone of salinized plants (SEZs) were incubated in 100 mM NaCl, the concentration where growth inhibition was approximately 50%, O2*- production, measured as NBT formazan staining, was lower in these than in similar segments obtained from control plants. The NaCl effect was salt-specific, and not osmotic, as incubation in 200 mM sorbitol did not reduce formazan staining intensity. SEZs elongation rates were higher in 200 mM sorbitol than in 100 mM NaCl, but the difference could be cancelled by scavenging or inhibiting O2*- production with 10 mM MgCl2 or 200 microM diphenylene iodonium, respectively. The actual ROS believed to stimulate growth is *OH, a product of O2*- metabolism in the apoplast. SEZ(s) elongation in 100 mM NaCl was stimulated by a *OH-generating medium. Fusicoccin, an ATPase stimulant, and acetate buffer pH 4, could also enhance elongation in these segments, although both failed to increase ROS activity. These results show that decreased ROS production contributes to the salinity-associated reduction in grass leaf elongation, acting through a mechanism not associated with pH changes.