Characterization of oxidation of glutathione by cytochrome c

Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) cytochrome c can be reduced by gluthatione as well as ascorbic acid. We investigated the effect of pH, Ca2+, Mg2+ and anionic phospholipids on the reduction of cytochrome c by glutathione.The reduction of cytochrome c by thiols was measured using photometry. Mitochondrial oxygen consumption was detected by use of oxygen electrode. Glutathione does not reduce cytochrome c at pH = 7.0 in the absence of Ca2+ and Mg2+. The reduction of cytochrome c by glutathione is inhibited by anionic lipids, especially cardiolipin. The typical conditions of apoptosis—elevated pH, Ca2+ level and Mg2+—increases the reduction of cytochrome c. Glutathione (5 mM) causes increased mitochondrial O2 consumption at pH = 8.0, in the presence of ADP either 1 mM Mg2+ or 1 mM Ca2+. Our results suggest that membrane bound cyt c does not oxidize glutathione. Free (not membrane bound) cytochrome c can oxidize glutathione. In mitochondria, O2 is depleted only in the presence of ADP, so the O2 depletion observed in the presence of glutathione can be related to the respiratory chain. Decreased glutathione levels play a role in apoptosis. Therefore, membrane unbound cyt c can contribute to apoptosis by oxidation of glutathione.

A Study of C2C12 Myoblast Bioenergetics in Response to the CCG-1423 Rho A Inhibitor

CCG-1423 is a Rho A pathway inhibitor which has been reported to inhibit Rho/SRF-mediated transcriptional regulation. SRF and SRF cofactors, which include ternary complex factors (TCFs) and myocardin-related transcription factor (MRTF), regulate various cellular functions. The Rho/SRF signaling pathway also regulates the sirtuin 2 (SIRT2) gene that contains a classic serum response element (SRE) sequence. Current research on CCG-1423 focuses on gene expression levels of SRF in response to CCG-1423 and how SRF levels affect the cells; the studies are focused on cell morphology, migration, viability/reproduction, and overall function. The pathways of this inhibitor have yet to be fully elucidated, but several have been suggested with good evidence. Our goal is to study the effect of CCG-1423 on mitochondrial function and gene expression of cells. In this work C2C12 myoblast cells have been used as an in-vitro model to study cellular bioenergetics and variations in gene expressions induced by CCG-1423. The effect of CCG-1423 on mitochondrial function was determined by measuring the mitochondrial oxygen consumption rate and glycolysis rate after treating C2C12 cells with varying concentrations of CCG-1423 overnight. In C2C12 myoblast cells, CCG-1423 treatment significantly reduced mitochondrial oxygen consumption rate (OCR) in a dose-dependent manner. However, treatment of C2C12 cells with CCG-1423 overnight increased the extracellular acidification rate (ECAR) in a dose-dependent manner. By indicating that CCG-1423 represses mitochondrial respiration via the Rho-SRF signaling pathway, the results of this study may enable a better understanding of the bioenergetics of the cell in the aging body.

Hypothermia Suppresses Uncoupling of Oxidative- Phosphorylation after Neonatal Cerebral Hypoxia-Ischemia

Hypothermia is the best available therapy for neonatal hypoxia ischemia (HI) brain injury, but its primary mechanisms remain uncertain. We hypothesize that HI induces, whereas hypothermia represses, uncoupling of oxidative phosphorylation (OXPHOS), an increase of the cerebral metabolic rate of oxygen (CMRO2) despite reduction of the mitochondrial energy output. We used a multiparametric photoacoustic microscopy (PAM) system to compare the effects of HI and post HI hypothermic treatment on CMRO2 in awake 10 day old (P10) mice. Here we show that hypoxia (10% O2) elevated CMRO2, but the addition of unilateral carotid artery ligation suppressed CMRO2 and sparked a rapid overshoot of post HI CMRO2 in the ipsilateral cerebral cortex for at least 2 hours. The post HI surge of CMRO2 was linked to an increase of mitochondrial oxygen consumption and superoxide outburst, despite reduction of the mitochondrial membrane potential. Notably, post HI hypothermia blocked the surge of superoxide and CMRO2, primarily by limiting oxygen extraction fraction (OEF), leading to better preservation of adenosine triphosphate (ATP), creatine (Cr) and N acetylaspartate (NAA) after HI. Mice that did not receive hypothermia exhibited ~80% reduction of CMRO2 at 24 h post HI, coupled to a large cortical infarction. These results suggest that mitigation of post HI uncoupling of OXPHOS is an early and/or pivotal effect of hypothermia. Further, optical measurement of CMRO2 may be a sensitive and noninvasive method to monitor brain damage in hypoxic ischemic encephalopathy (HIE).

Intracellular oxygen metabolism during bovine oocyte and preimplantation embryo development

We report a novel method to profile intrcellular oxygen concentration (icO2) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO2 associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO2 and decreasing oxygen consumption to increase icO2. Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO2 measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO2 for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO2 of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO2 was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9–4 × 106 copies and did not correlate with icO2. These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.

Aerobic exercise training prevents skeletal myopathy by myomiRs regulation in heart failure

Introduction Heart failure (HF) is the endpoint of systemic arterial hypertension. Exercise intolerance is a common symptom, partly due, to changes in the skeletal muscle mass (SM) and fiber type profile. Otherwise, aerobic exercise training (ET) has been used as an important non-pharmacological therapy in HF. MyomiRs are a muscle-specific class of miRNAs, which regulate genes that inhibiting the expression of proteins in pathological and physiological conditions controlling phenotypic changes in the SM, however little is known about these changes in ET-induced HF Purpose To elucidate the molecular mechanisms of ET involved in the metabolic alterations of SM in HF rats of hypertensive etiology. Methods The study was approved by the animal ethics committee (USP-No. 2020/01). 20 male rats, spontaneously hypertensive (SHR), and 10 Wistar Kyoto rats (WKY), SHR controls, nine-months-old, were divided into three groups: sedentary WKY (WKY-S), sedentary SHR (SHR-S), and trained (SHR-T). The ET consisted of swimming sessions with 60 minutes, 1x/day, 5x/week, for 10 weeks, with 5% of body overload. After ET protocol, blood pressure (BP), cardiac morphology and function (Echocardiography), exercise tolerance test, maximal oxygen uptake (VO2 peak), mitochondrial oxygen consumption (Oroboros), immunohistochemistry of the SM, expression of miRNAs (RT-qPCR) were evaluated. Statistical analyzes were performed by one-way ANOVA followed by the Tukey test. The results were expressed as mean ± standard error. Results ET reduced blood pressure levels and cardiac dysfunction in SHR-T compared to SHR-S. The SHR-S group covered smaller distance in the exercise tolerance test (255±22 meters) compared to the WKY-S (419±19 meters, p<0.0001), however ET reestablished the exercise tolerance (SHR-T: 365±20 meters; SHR-S: p<0.001 and WKY-S: p>0.05). The HF induced changes in type I and II fibers composition (I: 73±0.6% and II: 24±0.9%), VO2 peak (50±1.5 mL kg–1 min–1), mitochondrial oxygen consumption (State 3: 3.0±0.2 nmol O2 min–1 mg protein–1) and myomiRs expression (miRNA-208b: 65±4%, -499: 73±5%, -1: 153±10%) in the soleus muscle of SHR-S compared to WKY-S (I: 94±0.6%, II: 6±0.6%, p<0.001; VO2 peak: 59±2.3 mL kg–1 min–1, p<0.01; State 3: 4.0±0.2 nmol O2 min–1 mg protein–1, p<0.05; miRNAs: p<0.01). ET minimized changes in metabolic profile by counteract the muscle fiber type switching, and the oxygen consumption impairment, and myomiRs expression dysregulation (I: 90±0.5%, II: 9±0.6%, SHR-S p<0.01; VO2 peak: 79±2.4 mL kg–1 min–1, SHR-S: p<0.0001; State 3: 5.45±0.32 nmol O2 min–1 mg protein–1, SHR-S: p<0.0001; miRNA-208b: 91±5%, -499: 106±8%, -1: 100±9%; SHR-S: p<0.01). Conclusions ET reestablished structural and metabolic changes in SM, resulting from the progression of HF, through the regulation of myomiRs, improving exercise tolerance. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): The Coordination for the Improvement of Higher Education Personnel (CAPES): Academic Excellence Program (Proex).

Effect of Radiation Emitted by Wireless Devices on Male Reproductive Hormones: A Systematic Review

Exposure to radiofrequency electromagnetic radiation (RF-EMR) from various wireless devices has increased dramatically with the advancement of technology. One of the most vulnerable organs to the RF-EMR is the testes. This is due to the fact that testicular tissues are more susceptible to oxidative stress due to a high rate of cell division and mitochondrial oxygen consumption. As a result of extensive cell proliferation, replication errors occur, resulting in DNA fragmentation in the sperm. While high oxygen consumption increases the level of oxidative phosphorylation by-products (free radicals) in the mitochondria. Furthermore, due to its inability to effectively dissipate excess heat, testes are also susceptible to thermal effects from RF-EMR exposure. As a result, people are concerned about its impact on male reproductive function. The aim of this article was to conduct a review of literature on the effects of RF-EMR emitted by wireless devices on male reproductive hormones in experimental animals and humans. According to the findings of the studies, RF-EMR emitted by mobile phones and Wi-Fi devices can cause testosterone reduction. However, the effect on gonadotrophic hormones (follicle-stimulating hormone and luteinizing hormone) is inconclusive. These findings were influenced by several factors, which can influence energy absorption and the biological effect of RF-EMR. The effect of RF-EMR in the majority of animal and human studies appeared to be related to the duration of mobile phone use. Thus, limiting the use of wireless devices is recommended.

Cysteine induces mitochondrial reductive stress in glioblastoma through hydrogen peroxide production

SummaryGlucose and amino acid metabolism are critical for glioblastoma (GBM) growth, but little is known about the specific metabolic alterations in GBM that are targetable with FDA-approved compounds. To investigate tumor metabolism signatures unique to GBM, we interrogated The Cancer Genome Atlas for alterations in glucose and amino acid signatures in GBM relative to other human cancers and found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers. Treatment of patient-derived GBM cells with the FDA-approved cysteine compound N-acetylcysteine (NAC) reduce GBM cell growth and mitochondrial oxygen consumption, which was worsened by glucose starvation. Mechanistic experiments revealed that cysteine compounds induce rapid mitochondrial H2O2 production and reductive stress in GBM cells, an effect blocked by oxidized glutathione, thioredoxin, and redox enzyme overexpression. These findings indicate that GBM is uniquely susceptible to NAC-driven reductive stress and could synergize with glucose-lowering treatments for GBM.

Sodium Thiosulfate Improves Intestinal and Hepatic Microcirculation Without Affecting Mitochondrial Function in Experimental Sepsis

IntroductionIn the immunology of sepsis microcirculatory and mitochondrial dysfunction in the gastrointestinal system are important contributors to mortality. Hydrogen sulfide (H2S) optimizes gastrointestinal oxygen supply and mitochondrial respiration predominantly via K(ATP)-channels. Therefore, we tested the hypothesis that sodium thiosulfate (STS), an inducer of endogenous H2S, improves intestinal and hepatic microcirculation and mitochondrial function via K(ATP)-channels in sepsis.MethodsIn 40 male Wistar rats colon ascendens stent peritonitis (CASP) surgery was performed to establish sepsis. Animals were randomized into 4 groups (1: STS 1 g • kg-1 i.p., 2: glibenclamide (GL) 5 mg • kg-1 i.p., 3: STS + GL, 4: vehicle (VE) i.p.). Treatment was given directly after CASP-surgery and 24 hours later. Microcirculatory oxygenation (µHBO2) and flow (µflow) of the colon and the liver were continuously recorded over 90 min using tissue reflectance spectrophotometry. Mitochondrial oxygen consumption in tissue homogenates was determined with respirometry. Statistic: two-way ANOVA + Dunnett´s and Tukey post - hoc test (microcirculation) and Kruskal-Wallis test + Dunn’s multiple comparison test (mitochondria). p < 0.05 was considered significant.ResultsSTS increased µHbO2 (colon: 90 min: + 10.4 ± 18.3%; liver: 90 min: + 5.8 ± 9.1%; p < 0.05 vs. baseline). Furthermore, STS ameliorated µflow (colon: 60 min: + 51.9 ± 71.1 aU; liver: 90 min: + 22.5 ± 20.0 aU; p < 0.05 vs. baseline). In both organs, µHbO2 and µflow were significantly higher after STS compared to VE. The combination of STS and GL increased colonic µHbO2 and µflow (µHbO2 90 min: + 8.7 ± 11.5%; µflow: 90 min: + 41.8 ± 63.3 aU; p < 0.05 vs. baseline), with significantly higher values compared to VE. Liver µHbO2 and µflow did not change after STS and GL. GL alone did not change colonic or hepatic µHbO2 or µflow. Mitochondrial oxygen consumption and macrohemodynamic remained unaltered.ConclusionThe beneficial effect of STS on intestinal and hepatic microcirculatory oxygenation in sepsis seems to be mediated by an increased microcirculatory perfusion and not by mitochondrial respiratory or macrohemodynamic changes. Furthermore, the effect of STS on hepatic but not on intestinal microcirculation seems to be K(ATP)-channel-dependent.

The Role of Dietary Saturated Fat in Associations between Exposure to Ambient Fine Particulate Matter and Mitochondrial Respiratory Functions in Circulating Platelets

Objectives Exposure to ambient fine particulate matter (PM2.5) is associated with platelet activation and increased mitochondrial respiration. The impact of dietary saturated fat on the circulating platelets is not understood. This project aimed to determine whether dietary saturated fatty acids moderate mitochondrial respiratory function in circulating platelets after short-term exposure to PM2.5. Methods Platelets were isolated from 22 healthy male volunteers (mean age ± SD, 37 ± 8.2) in a panel study and measured for mitochondrial oxygen consumption rates using an extracellular flux analyzer. Intakes of saturated fat were determined from 24 hr dietary recalls the day before the assay. Daily ambient PM2.5 concentrations during the study period were obtained from ambient air quality monitoring stations. Data were fitted with a moderation model, where the level of ambient PM2.5 was the independent variable, saturated fat intake was the moderator, and mitochondrial respiratory functions in circulating platelets were the dependent variables. Results After controlling for age, dietary consumption of saturated fat moderated the mitochondrial oxygen consumption rates of non-mitochondrial respiration, basal respiration, maximum respiration, ATP production, and spare respiratory capacity after exposure to ambient PM2.5 with 2 days lag. Specifically, the negative associations between the above mentioned mitochondrial respiratory measurements and PM2.5 levels reached statistical significance (95% Confident Intervals did not include 0) in subjects with a high intake of total saturated fat. Further, results for individual saturated fatty acid showed similar patterns, specifically that negative association between mitochondrial oxygen consumption rates of non-mitochondrial respiration, basal respiration and ATP production and levels of exposed PM2.5 was moderated by intakes of short-chain (C4:0), medium-chain (C6:0, C8:0, C10:0, C12:0), long-chain (C14:0, C16:0) saturated fatty acids. Conclusions Taken together, these preliminary findings suggest that consumption of saturated fat moderates platelet mitochondrial respiration after exposure to PM2.5. THIS ABSTRACT OF A PROPOSED PRESENTATION DOES NOT NECESSARILY REFLECT EPA POLICY. Funding Sources This project was supported by the U.S. EPA Intramural Research Program.