Electron Transport Chain Enzymes Research Articles

Mycobacterium tuberculosis protein MoxR1 enhances virulence by inhibiting host cell death pathways and disrupting cellular bioenergetics

ABSTRACT Mycobacterium tuberculosis (M. tb) utilizes the multifunctionality of its protein factors to deceive the host. The unabated global incidence and prevalence of tuberculosis (TB) and the emergence of multidrug-resistant strains warrant the discovery of novel drug targets that can be exploited to manage TB. This study reports the role of M. tb AAA+ family protein MoxR1 in regulating host-pathogen interaction and immune system functions. We report that MoxR1 binds to TLR4 in macrophage cells and further reveal how this signal the release of proinflammatory cytokines. We show that MoxR1 activates the PI3K-AKT-MTOR signalling cascade by inhibiting the autophagy-regulating kinase ULK1 by potentiating its phosphorylation at serine 757, leading to its suppression. Using autophagy-activating and repressing agents such as rapamycin and bafilomycin A1 suggested that MoxR1 inhibits autophagy flux by inhibiting autophagy initiation. MoxR1 also inhibits apoptosis by suppressing the expression of MAPK JNK1/2 and cFOS, which play critical roles in apoptosis induction. Intriguingly, MoxR1 also induced robust disruption of cellular bioenergetics by metabolic reprogramming to rewire the citric acid cycle intermediates, as evidenced by the lower levels of citric acid and electron transport chain enzymes (ETC) to dampen host defence. These results point to a multifunctional role of M. tb MoxR1 in dampening host defences by inhibiting autophagy, apoptosis, and inducing metabolic reprogramming. These mechanistic insights can be utilized to devise strategies to combat TB and better understand survival tactics by intracellular pathogens.

VDAC1 Knockout Affects Mitochondrial Oxygen Consumption Triggering a Rearrangement of ETC by Impacting on Complex I Activity.

Voltage-Dependent Anion-selective Channel isoform 1 (VDAC1) is the most abundant isoform of the outer mitochondrial membrane (OMM) porins and the principal gate for ions and metabolites to and from the organelle. VDAC1 is also involved in a number of additional functions, such as the regulation of apoptosis. Although the protein is not directly involved in mitochondrial respiration, its deletion in yeast triggers a complete rewiring of the whole cell metabolism, with the inactivation of the main mitochondrial functions. In this work, we analyzed in detail the impact of VDAC1 knockout on mitochondrial respiration in the near-haploid human cell line HAP1. Results indicate that, despite the presence of other VDAC isoforms in the cell, the inactivation of VDAC1 correlates with a dramatic impairment in oxygen consumption and a re-organization of the relative contributions of the electron transport chain (ETC) enzymes. Precisely, in VDAC1 knockout HAP1 cells, the complex I-linked respiration (N-pathway) is increased by drawing resources from respiratory reserves. Overall, the data reported here strengthen the key role of VDAC1 as a general regulator of mitochondrial metabolism.

Constitutive Expression of Hif2α Confers Acute O2 Sensitivity to Carotid Body Glomus Cells.

Acute oxygen (O2) sensing and adaptation to hypoxia are essential for physiological homeostasis. The prototypical acute O2 sensing organ is the carotid body, which contains chemosensory glomus cells expressing O2-sensitive K+ channels. Inhibition of these channels during hypoxia leads to cell depolarization, transmitter release, and activation of afferent sensory fibers terminating in the brain stem respiratory and autonomic centers. Focusing on recent data, here we discuss the special sensitivity of glomus cell mitochondria to changes in O2 tension due to Hif2α-dependent expression of several atypical mitochondrial electron transport chain subunits and enzymes. These are responsible for an accelerated oxidative metabolism and the strict dependence of mitochondrial complex IV activity on O2 availability. We report that ablation of Epas1 (the gene coding Hif2α) causes a selective downregulation of the atypical mitochondrial genes and a strong inhibition of glomus cell acute responsiveness to hypoxia. Our observations indicate that Hif2α expression is required for the characteristic metabolic profile of glomus cells and provide a mechanistic explanation for the acute O2 regulation of breathing.

Inhibition of hepatic energy metabolizing enzymes in murine model exposed to diisononyl phthalate

Background and objectives: Diisononyl phthalate (DINP) is a class of phthalates and phthalates are known to be metabolism disrupting chemicals (MDCs). Numerous MDCs, to which humans are exposed, have an effect on every aspect of energy transduction. They affect the liver by impairing insulin secretion in pancreatic cells and altering the liver’s insulin-dependent glucose metabolism. Methods: For this study, eighteen male albino rats weighing 200±20g were randomly assigned to three groups (of six rats each) and followed for a 14-days period. The groups were: group A or control which was given Tween-80 orally, group B or DINP1 group which was given 20 mg/kg b.wt. DINP, and Group C or DINP2 group which received 200 mg/kg b.wt. DINP. The rats were then sacrificed, their livers were removed, and the glycolytic and oxidative phosphorylation enzyme activities were evaluated. Results: Activities of the glycolytic, tricarboxylic acid cycle and electron transport chain enzymes under investigation were significantly down-regulated with severity observed in decreased activities of hepatic oxidative phosphorylation enzymes when compared with control (P<0.05). Hepatic tissue sections of 20 and 200mg/kg DiNP group revealed distorted cytoarchitecture of hepatocytes ranging from histocellular disarrangement to vaocular changes suggestive of loss of liver integrity or fibrosis. Conclusions: Finally, DINP exposure impairs hepatic energy transduction enzymes as evident in down-regulation of the various enzymes of energy metabolism under investigation and this may invariably be a good tool for the diagnosis of hepatic energy impairment as seen in some disease conditions.

Effect of geranylated dihydrochalcone from Artocarpus altilis leaves extract on Plasmodium falciparum ultrastructural changes and mitochondrial malate: Quinone oxidoreductase

Nearly half of the world's population is at risk of being infected by Plasmodium falciparum, the pathogen of malaria. Increasing resistance to common antimalarial drugs has encouraged investigations to find compounds with different scaffolds. Extracts of Artocarpus altilis leaves have previously been reported to exhibit in vitro antimalarial activity against P. falciparum and in vivo activity against P. berghei. Despite these initial promising results, the active compound from A. altilis is yet to be identified. Here, we have identified 2-geranyl-2′, 4′, 3, 4-tetrahydroxy-dihydrochalcone (1) from A. altilis leaves as the active constituent of its antimalarial activity. Since natural chalcones have been reported to inhibit food vacuole and mitochondrial electron transport chain (ETC), the morphological changes in food vacuole and biochemical inhibition of ETC enzymes of (1) were investigated. In the presence of (1), intraerythrocytic asexual development was impaired, and according to the TEM analysis, this clearly affected the ultrastructure of food vacuoles. Amongst the ETC enzymes, (1) inhibited the mitochondrial malate: quinone oxidoreductase (PfMQO), and no inhibition could be observed on dihydroorotate dehydrogenase (DHODH) as well as bc1 complex activities. Our study suggests that (1) has a dual mechanism of action affecting the food vacuole and inhibition of PfMQO-related pathways in mitochondria.

Differential effects of 808-nm light on electron transport chain enzymes in isolated mitochondria: Implications for photobiomodulation initiation

Photobiomodulation is a term for using low-power red to near-infrared light to stimulate a variety of positive biological effects. Though the scientific and clinical acceptance of PBM as a therapeutic intervention has increased dramatically in recent years, the molecular underpinnings of the effect remain poorly understood. The putative chromophore for PBM effects is cytochrome c oxidase. It is postulated that light absorption at cytochrome c oxidase initiates a signaling cascade involving ATP and generation of reactive oxygen species (ROS), which subsequently results in improved cellular robustness. However, this hypothesis is largely based on inference and indirect evidence, and the precise molecular mechanisms that govern how photon absorption leads to these downstream effects remain poorly understood. We conducted low-power PBM-type light exposures of isolated mitochondria to 808 nm NIR light, at a number of irradiances. NIR exposure was found to enhance the activity of complex IV, depress the activity of complex III, and had no effect on the activity of complex II. Further, examining the dose-response of complex IV we found NIR enhancement did not exhibit irradiance reciprocity, indicating the effect on complex IV may not have direct photochemical basis. In summary, this research presents a novel method to interrogate the earliest stages of PBM in the mitochondria, and a unique window into the corresponding molecular mechanism(s) of induction.

Indole-3-carbinol ameliorated the neurodevelopmental deficits in neonatal anoxic injury in rats.

Neonatal anoxia is linked to long-lasting neurodevelopmental deficits. Due to the lack of pharmacological intervention to treat neonatal anoxia, there is interest in finding new molecules for its treatment. Indole-3-carbinol (I3C) has shown neuroprotective effects in some disease conditions. However, the neuroprotective role of I3C in neonatal anoxia has not been explored. Consequently, we have investigated the effect of I3C on neonatal anoxia-induced brain injury and neurodevelopmental deficits. Rat pups after 30 h of birth were subjected to two episodes of anoxia (10min in each) at a time interval of 24 h by flowing 100% nitrogen. I3C was administered within 30 min of the second episode of anoxia on a postnatal day (PND) 3 and continued for PND 9. Neurodevelopmental deficits, cortical mitochondrial membrane potential (MMP), opening of mitochondrial permeability transition pore (MPTP), electron transport chain (ETC) enzyme activities, oxidative stress, hypoxia-inducible factor-1α (HIF-1α) levels, histopathological changes, and apoptosis were measured. I3C treatment dose-dependently ameliorated the neurodevelopmental deficits and somatic growth in anoxic pups. I3C improved mitochondrial function by enhancing the MMP, mitochondrial ETC enzymes, and antioxidants. It blocked the MPTP opening and release of cytochrome C in anoxic pups. Further, I3C reduced the elevated cortical HIF-1α in neonatal anoxic pups. Furthermore, I3C ameliorated histopathological abnormalities and mitochondrial-mediated apoptotic indicators Cyt C, caspase-9, and caspase-3. Our study concludes that I3C improved neuronal development in anoxic pups by enhancing mitochondrial function, reducing HIF-1α, and mitigating apoptosis.

Regional heterogeneity in mitochondrial function underlies region specific vulnerability in human brain ageing: Implications for neurodegeneration.

Selective neuronal vulnerability (SNV) of specific neuroanatomical regions such as frontal cortex (FC) and hippocampus (HC) is characteristic of age-associated neurodegenerative diseases (NDDs), although its pathogenetic basis remains unresolved. We hypothesized that physiological differences in mitochondrial function in neuroanatomical regions could contribute to SNV. To investigate this, we evaluated mitochondrial function in human brains (age range:1-90y) in FC, striatum (ST), HC, cerebellum (CB) and medulla oblongata (MD), using enzyme assays and quantitative proteomics. Striking differences were noted in resistant regions- MD and CB compared to the vulnerable regions- FC, HC and ST. At younger age (25±5y), higher activity of electron transport chain enzymes and upregulation of metabolic and antioxidant proteins were noted in MD compared to FC and HC, that was sustained with increasing age (≥65y). In contrast, the expression of synaptic proteins was higher in FC, HC and ST (vs. MD). In line with this, quantitative phospho-proteomics revealed activation of upstream regulators (ERS, PPARα) of mitochondrial metabolism and inhibition of synaptic pathways in MD. Microtubule Associated Protein Tau (MAPT) showed overexpression in FC, HC and ST both in young and older age (vs. MD). MAPT hyperphosphorylation and the activation of its kinases were noted in FC and HC with age. Our study demonstrates that regional heterogeneity in mitochondrial and other cellular functions contribute to SNV and protect regions such as MD, while rendering FC and HC vulnerable to NDDs. The findings also support the "last in, first out" hypothesis of ageing, wherein regions such as FC, that are the most recent to develop phylogenetically and ontogenetically, are the first to be affected in ageing and NDDs.

Baseline oxygen consumption decreases with cortical depth.

The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase-the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. It has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we tested the above hypothesis using direct measurements of the partial pressure of O2 (pO2) in cortical tissue by means of 2-photon phosphorescence lifetime microscopy (2PLM). We revisited our previously developed method for extraction of the cerebral metabolic rate of O2 (CMRO2) based on 2-photon pO2 measurements around diving arterioles and applied this method to estimate baseline CMRO2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO2 from layer I to layer IV. This decrease of CMRO2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O2 reserve during surges of neuronal activity or certain metabolically active brain states rather than reflecting baseline energy needs. Our study provides to our knowledge the first quantification of microscopically resolved CMRO2 across cortical layers as a step towards better understanding of brain energy metabolism.

Aminooxyacetic acid hemihydrochloride inhibits osteoclast differentiation and bone resorption by attenuating oxidative phosphorylation.

Osteoclasts undergo active metabolic reprogramming to acquire the energy needed during differentiation and bone resorption. Compared with immature osteoclasts, mature osteoclasts comprise higher levels of electron transport chain enzymes and more metabolically active mitochondria. Of all energy metabolism pathways, oxidative phosphorylation is considered to be the most efficient in supplying energy to osteoclasts. We found that the malate-aspartate shuttle inhibitor aminooxyacetic acid hemihydrochloride inhibits osteoclastogenesis and bone resorption by inhibiting exchange of reducing equivalents between the cytosol and the mitochondrial matrix and attenuating mitochondrial oxidative phosphorylation in vitro. The weakening of the oxidative phosphorylation pathway resulted in reduced mitochondrial function and inadequate energy supply along with reduced reactive oxygen species production. Furthermore, treatment with aminooxyacetic acid hemihydrochloride helped recover bone loss in ovariectomized mice. Our findings highlight the potential of interfering with the osteoclast intrinsic energy metabolism pathway as a treatment for osteoclast-mediated osteolytic diseases.

Journal-related Activities and Other Special Activities at the 2022 American Society of Anesthesiologists Meeting

Journal-related Activities and Other Special Activities at the 2022 American Society of Anesthesiologists Meeting

Favipiravir induces oxidative stress and genotoxicity in cardiac and skin cells

Favipiravir (T-705), used against influenza viruses, is approved for emergency use in many countries for the treatment of COVID-19. The frequent adverse effects of favipiravir are related with the gastrointestinal system, however, studies suggest a positive association of favipiravir on QTc prolongation, which can cause cardiotoxicity. Also, there are reports of skin reactions such as angioedema due to favipiravir. Despite the several adverse effects, studies examining the drug’s effects at the molecular level are insufficient, e.g., the genotoxic and oxidative stress-inducing effects of favipiravir, which are among the primary mechanisms of drug-induced toxicity. The cytotoxicity of favipiravir was analyzed with the measurement of the ATP content in H9c2 cardiomyoblasts and CCD-1079Sk skin fibroblasts. The ATP level decreased starting from 200 µM. The inhibitory effect on the mitochondrial electron transport chain enzymes complex I and complex V was also evaluated where favipiravir showed significant enzyme inhibitory effects in the highest concentration studied. A molecular docking study evaluating the interaction between favipiravir-RTP and mitochondrial DNA polymerase (POLG1) was done. The relationship of favipiravir with oxidative stress was examined by measuring glutathione (GSH) and protein carbonyl levels which were observed higher after drug treatment compared to the control group. The genotoxicity study was done using the Comet assay and increase in DNA tail has been detected. Furthermore, 8-OHdG levels were measured higher in favipiravir treated cells indicating oxidative DNA damage. Favipiravir induced oxidative stress leading to DNA damage in cardiomyoblast cells and fibroblastic skin cells. Oxidative stress and DNA damage might eventually lead to organ-specific damage such as cardiotoxicity and dermal toxicity. Considering the increased use of favipiravir in recent years, and that oxidative stress and genotoxicity are two important indicators of drug-induced toxicity, the obtained results are worth attention.

The aquatic macrophyte Salvinia molesta mitigates herbicides (glyphosate and aminomethylphosphonic acid) effects to aquatic invertebrates.

We evaluated the individual and combined effects of different environmentally representative concentrations of glyphosate (0, 25, 50, 75, and 100µgl-1) and aminomethylphosphonic acid (AMPA; 0, 12.5, 25, 37.5, and 50µgl-1) on the physiology of Aedes aegypti larvae, as well as the capacity of the aquatic macrophyte Salvinia molesta to attenuate those compounds' toxicological effects. Larvae of Ae. aegypti (between the third and fourth larval stages) were exposed for 48h to glyphosate and/or AMPA in the presence or absence of S. molesta. Glyphosate and AMPA induced sublethal responses in Ae. aegypti larvae during acute exposures. Plants removed up to 49% of the glyphosate and 25% of AMPA from the water, resulting in the exposure of larvae to lower concentration of those compounds in relation to media without plants. As a result, lesser effects of glyphosate and/or AMPA were observed on larval acetylcholinesterase, P450 reductase, superoxide dismutase, mitochondrial electron transport chain enzymes, respiration rates, and lipid peroxidation. In addition to evidence of deleterious effects by media contamination with glyphosate and AMPA on aquatic invertebrates, our results attest to the ability of S. molesta plants to mitigate the toxicological impacts of those contaminants.

Abstract P3054: Alpha-1a Adrenergic Receptor Activation Increases Electron Transport Chain Activity And Preserves Cardiac Contractility Following Myocardial Infarction

Heart failure features decreased electron transport chain (ETC) activity in cardiomyocyte mitochondria. Previous studies have demonstrated that alpha-1A adrenergic receptor (α1A-AR) activation increases cardiac contractility in the failing heart. However, α1A-AR activation as a method of ETC regulation following cardiac injury has not been studied. Here, we assessed the hypothesis that α1A-AR activation increases ETC activity following myocardial infarction (MI) induced by left coronary artery ligation. We measured cardiac contractility using echocardiography at baseline and 7 days after MI in male CL57Bl/6J mice receiving the selective α1A-AR agonist A61603 (10 ng/kg/d) (n = 8) or vehicle (0.9% saline) (n = 9) via subcutaneous osmotic minipumps (Panel A). We then profiled citrate synthase (CS), a key enzyme in the citric acid cycle and common marker for mitochondrial mass, and ETC Complex I-IV activities in ventricular lysates. ETC enzyme activities were normalized to CS to define ETC capacity on a per mitochondrion basis. On Day 0, there were no differences in fractional shortening between groups (Panel B, 46 ± 3% vs. 49 ± 3%, p = 0.27) (mean ± SEM). On Day 7, fractional shortening was preserved in A6-treated mice compared to vehicle controls (Panel B, 24 ± 3% vs. 14 ± 1%, p = 0.01). A6 decreased CS activity compared to vehicle controls (Panel C, 11,024 ± 1,035 vs. 15,966 ± 1,068 nmol/mg/min, p < 0.01). When normalized to CS activity, A6 increased Complex I (16 ± 2% vs. 7 ± 1%, p < 0.01) (1,584 ± 116 vs. 1,106 ± 154 nmol/min/mg), Complex III (82 ± 8% vs. 56 ± 5%, p = 0.01) (8,435 ± 461 vs. 8,542 ± 327 nmol/min/mg), and Complex IV (121 ± 12% vs. 92 ± 9.%, p = 0.04) (12,523 ± 673 vs. 14,174 ± 1,156) activities (Panel D), suggesting enhanced ETC activity on a per mitochondrion basis. In conclusion, we show that a low dose of a selective α1A-AR agonist enhances cardiac contractility following MI. Increased ETC enzyme activity, particularly Complex I, may contribute to this beneficial effect. Ours is the first study in which selective α1A-AR activation has been linked to enhanced cardiac contractility and mitochondrial metabolism following MI. These findings may outline a novel mechanism through which α1A-AR activation preserves cardiac function in the injured and failing heart.

Abstract P3106: Trametinib Decreases Electron Transport Chain Activity In The Uninjured Mouse Heart

Trametinib (Trm) is a highly selective inhibitor of the tyrosine kinases MEK1/2 used to treat patients with melanoma and non-small cell lung cancer featuring BRAF mutations. Although Trm is generally well tolerated, it is associated with adverse cardiovascular effects including heart failure. Furthermore, the mechanisms underlying this cardiotoxicity are unclear. Here, we assessed the hypothesis that Trm decreases oxidative phosphorylation in the mouse heart. Female FVB mice were treated with Trm (3 mg/kg/d) or vehicle (DMSO) via oral gavage (n = 10, each group). We assessed cardiac contractility on Day 0 and Day 7 using echocardiography. We then performed electron transport chain (ETC) enzyme assays in isolated cardiac mitochondria and RNA sequencing of cardiac tissue to assess ETC Complex I-IV enzyme activity and ETC transcript abundance, respectively. Additionally, we measured citrate synthase (CS) activity as a marker for mitochondrial abundance. Mice treated with Trm had decreased fractional shortening on Day 7 compared to Day 0 (Panel A, 47.7 ± 1.6% vs. 55.5 ± 0.8%, p < 0.001) (mean ± SEM), confirming the cardiotoxicity of our treatment model. Additionally, mice treated with Trm had increased lung weight to heart weight (LW/HW) ratios compared to vehicle controls (Panel B, 1.69 ± 0.11 vs. 1.39 ± 0.03, p = 0.01), indicating heart failure. Mice treated with Trm had decreased CS (7,235 ± 586 vs. 8,562 ± 192 nmol/mg/min, p = 0.024), Complex II (1,659 ± 207 vs. 2,239 ± 239, p = 0.04), and Complex IV (3,979 ± 460 vs. 5,576 ± 615, p = 0.03) activities with a trend towards decreased Complex III (2,703 ± 452 vs. 3,554 ± 434, p = 0.09) activity (Panel C). Mice treated with Trm had decreased transcript abundance of Complex I (41 of 43), Complex II (6 of 6), Complex III (8 of 8), and Complex IV (18 of 23) transcripts (Panel D). Gene Ontology cellular compartment analysis of transcriptomic data revealed that Trm treatment primarily alters abundance of transcripts related to the mitochondrion (Panel E, p adj = 3.2 x 10 -113 ). KEGG Pathway analysis revealed that oxidative phosphorylation was the most significantly altered process following Trm treatment (Panel F, p adj = 2.9 x 10 -27 ). Collectively, these findings suggest that Trm causes widespread decreases in ETC activity that may hinder cardiomyocyte oxidative phosphorylation and contractility.

Physiological responses and phytoremediation capacity of floating and submerged aquatic macrophytes exposed to ciprofloxacin.

Ciprofloxacin (Cipro) water contamination is a global concern, having reached disturbing concentrations and threatening the aquatic ecosystems. We investigated the physiological responses and Cipro-phytoremediation capacity of one floating (Salvinia molesta D.S. Mitchell) and one submerged (Egeria densa Planch.) species of aquatic macrophytes. The plants were exposed to increased concentrations of Cipro (0, 1, 10, and 100µg.Cipro.L-1) in artificially contaminated water for 96 and 168 h. Although the antibiotic affected the activities of mitochondrial electron transport chain enzymes, the resulting increases in H2O2 concentrations were not associated with oxidative damage or growth reductions, mainly due to the activation of antioxidant systems for both species. In addition to being tolerant to Cipro, after only 96h, plants were able to reclaim more than 58% of that from the media. The phytoremediation capacity did not differ between the species, however, while S. molesta bioaccumulate, E. densa appears to metabolize Cipro in their tissues. Both macrophytes are indicated for Cipro-phytoremediation projects.

Dynamics of pentavalent inorganic arsenic effects on some glycolytic and mitochondrial energy metabolizing enzymes in male Wistar rats

Exposure to environmental toxicants such as arsenic presents serious health challenges to humans. The effects of arsenic have been extensively evaluated, but there is a dearth of scientific findings on its effects on some key enzymes of glycolysis, Krebs cycle, and electron transport chain. This study, therefore, is aimed at investigating the time-course effects of arsenic exposure on these key enzymes in male Wistar rats. Rats (n = 45) were exposed to arsenic (100, 150, and 200 ppm) for 4, 8, and 12 weeks respectively in their drinking water. Control rats (n = 5) received drinking water for 12 weeks, after which blood and liver were removed from the animals and analyzed for some energy metabolizing enzymes spectrophotometrically. Data were analyzed using one-way analysis of variance followed by Tukey's post hoc test and p < 0.05 was considered significant. Our findings showed that arsenic exposure reduced the activities of plasma hexokinase at weeks 4, 8, and 12. Meanwhile, erythrocyte and liver homogenate hexokinase were significantly (p < 0.05) enhanced only at week 4. Plasma and liver aldolase activities were significantly (p < 0.05) induced at week 4, while aldolase activities of other compartments were significantly (p < 0.05) reduced at weeks 4, 8, and 12. In addition, arsenic significantly (p < 0.05) increased the activity of lactate dehydrogenase in various tissue compartments, effects of arsenic exposure were characterized by a significant (p < 0.05) decrease in the activities of malate dehydrogenase, complexes of the electron transport chain when compared with the control animals in all the tissues. These findings indicated that arsenic exposure induced various compartment-specific modulation of key enzymes of glycolysis, Krebs cycle and electron transport chain. This might be one of the mechanisms through which arsenic mediates its toxicity and its associated disease endpoints.

No Effects of Photobiomodulation on Prefrontal Cortex and Hippocampal Cytochrome C Oxidase Activity and Expression of c-Fos Protein of Young Male and Female Rats.

The role of light in our biological processes and systems is extensively known. In addition, the use of light devices has been introduced in the field of healthcare as an opportunity to administer power light at specific wavelengths to improve our body functions and counteract light deficiency. One of these techniques is photobiomodulation (PBM), which uses red to infrared light in a non-invasive way to stimulate, heal, regenerate, and protect tissue. The main proposed mechanism of action is the stimulation of the cytochrome c oxidase (CCO), the terminal enzyme in the mitochondrial electron transport chain. PBM has achieved positive effects on brain activity and behavioral function of several adult animal models of health and disease, the potential use of this technique in developing stages is not surprising. This research aims to examine the effects of PBM on the prefrontal cortex and hippocampus of 23 day-old healthy male (n = 31) and female (n = 30) Wistar rats. Three groups of each sex were used: a PBM group which received 5 days of PBM, a device group submitted to the same conditions but without light radiation, and a control basal group. CCO histochemistry and c-Fos immunostaining were used to analyze brain metabolic activity and immediate early genes activation, respectively. Results displayed no metabolic differences between the three groups in both sexes. The same results were found in the analysis of c-Fos positive cells, reporting no differences between groups. This research, in contrast to the PBM consequences reported in healthy adult subjects, showed a lack of PBM effects in the brain markers we examined in young healthy rat brains. At this stage, brain function, specifically brain mitochondrial function, is not disturbed so it could be that the action of PBM in the mitochondria may not be detectable using the analysis of CCO activity and c-Fos protein expression. Further studies are needed to examine in depth the effects of PBM in brain development, cognitive functions and postnatal disorders, along with the exploration of the optimal light parameters.

Assessment by Multi‐Distance Hyperspectral NIRS of Changes in the Oxidation State of Cytochrome C Oxidase (oxCCO) to Carotid Artery Compressions

IntroductionThe occlusion of the common carotid artery (CCA) during carotid endarterectomy (CEA) is associated with a risk of cerebral ischemia due to inadequate collateral blood flow1. By monitoring tissue oxygen saturation (StO2), near‐infrared spectroscopy (NIRS) is frequently employed during CEA2 to determine sufficient oxygen delivery. However, StO2 is only a proxy of oxygen metabolism and an unreliable marker of ischemic injury. In contrast, cytochrome c oxidase (oxCCO) is an enzyme in the mitochondrial electron transport chain and therefore a specific marker of aerobic metabolism3. Thus, the aim of this study was to assess the ability of a multi‐distance hyperspectral (hs) NIRS to monitor changes in StO2 and oxCCO during CCA compressions in healthy participants. We hypothesized that changes in oxCCO concentration would be greater in the brain (i.e., long source‐detector distance, SDD) than in the extracerebral tissue (i.e., short SDD), and this difference would be larger than that of StO2.MethodsWe modified an in‐house built hsNIRS4 to include a second channel for acquiring data at two SDD (1 and 3 cm) to detect potential changes in the extracerebral tissue. Data from 10 young, healthy participants was acquired continuously during one 30‐second unilateral digital CCA compression, performed ipsilateral to the location of the optic probes. In addition, cerebral blood flow index was determined using diffuse correlation spectroscopy and mean arterial blood pressure by finger photoplethysmography.ResultsDuring CCA compression, the change in oxCCO at 3 cm vs. 1 cm (0.4 ± 0.3 µM vs. 0.06 ± 0.1 µM, p = 0.027) was approximately 50% greater than the change in StO2 at 3 cm vs. 1cm (‐4.0 ± 2.2% vs. 1.2 ± 0.7% p = 0.007). The corresponding decrease in cerebral blood flow was 57 ± 14% (p &lt; 0.001 vs. baseline). Mean arterial blood pressure increased 4 ± 1 mmHg (p &lt; 0.001 vs. baseline).DiscussionA larger difference between the two distances was observed for oxCCO than StO2, likely due to the greater concentration of mitochondria in the brain compared to extracerebral tissues. Collecting multi‐distance hsNIRS demonstrated the enhanced sensitivity of oxCCO to the brain. Lastly, this study demonstrates the utility of momentary unilateral CCA compressions as a fast, easy, and strong perturbation for transiently altering cerebral inflow and metabolism.References(1) Aceto, Eur. J. Anestesiol.(2020); (2) Yu, Cochrane Database Syst. Rev.(2014); (3) Bale, J. Biomed. Opt. (2020); (4) Rajaram, Brain Sci (2020).

Investigating the role of DNMT1 gene expression on myocardial ischemia reperfusion injury in rat and associated changes in mitochondria

Altered DNA methylation and mitochondrial dysfunction are the two key features of myocardial ischemia reperfusion injury (I/R), but their association with I/R remains unknown. In the present study, the relationship between DNA methyl transferase1 (DNMT1), the key methylation gene, and the mitochondrial quality control genes in rat heart during I/R was explored. We used the Langendorff rat heart model with 30 min of ischemia followed by 60 min of reperfusion and subsequent inhibition of DNMT1 with 5-azacytidine to evaluate the role of DNA methylation in I/R. Reperfusion significantly increased the expression of the DNMT1 gene, enzyme activity, and global DNA methylation levels, along with decreased mitochondrial copy, electron transport chain (ETC) activities, and ATP level. This was in agreement with the significant downregulation of 11 mitochondrial genes PGC-1α, TFAM, POLG, MFN1 and MFN2, FIS1, PARKIN, OPTN, ND1, ND4L, Cyt B and COX1 in I/R induced rat hearts. The expression pattern of the mitochondrial genes PGC-1α, TFAM, ND1 and Cyt B showed a significant negative correlation with DNMT1 expression. Rate pressure product, index of cardiac performance negatively correlated with DNMT1 expression (r = -0.8231, p = 0.0456). However, DNMT1 inhibited rat hearts via 5-azacytidine significantly improved the heart from I/R injury and reversed the I/R associated changes in the gene expression of TFAM, POLG, PGC-1α, ND1, COX1 and Cyt B, and improved the overall mtDNA copies, with a subsequent improvement in the ETC enzyme activity and ATP levels. To conclude, I/R augmented the DNMT1 activity with a subsequent increase in cardiac injury via downregulating the mitochondrial functional genes.