Toxicity and Sublethal Effects of Piper hispidinervum Essential Oil on Behavioral and Physiological Responses of Sitophilus zeamais Populations.

This study aimed to evaluate the toxicity of Piper hispidinervum essential oil (PHEO) against 11 Brazilian populations of Sitophilus zeamais (Coleoptera: Curculionidae). The effects of sublethal doses of PHEO on the behavior (walking and flying), respiration, and population growth (ri) of the insect populations were investigated. PHEO toxicity was determined through concentration-mortality bioassays, with mortality curves established using increasing PHEO concentrations ranging from 140.00 to 1000.00 µL kg-1. Behavior was evaluated based on walking distance, walking time, walking speed, walking time proportion, flight height, and flight takeoff success. Respiration was measured via the respiratory rate, while population growth (ri) was assessed through the instantaneous growth rate. All 11 populations of S. zeamais were susceptible to PHEO, showing no signs of resistance. The populations exhibited varying behavioral and physiological responses to sublethal exposure to PHEO, indicating different mitigation strategies. The results confirm that PHEO possesses insecticidal potential for controlling S. zeamais populations. However, the observed behavioral and physiological responses should be considered when establishing control measures in pest management programs for stored products.

Diatomaceous earth as insecticide: physiological and morphological evidence of its underlying mechanism.

BACKGROUND: Wheat grain containers or silos can be perfect habitats for insects, which generate large economic losses to grain production. Natural alternatives to synthetic insecticides have grown in popularity because of health, economic and ecological issues. Diatomaceous earth is a natural compound that has an insecticide effect by enhancing an insect's dehydration with no toxicity on mammals including humans. The aim of this study is to confirm the effect of diatomaceous earth as an insecticide for the wheat grain pest, the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae) and demonstrate its underlying mechanisms as an insecticide by open-flow respirometry and scanning electron microscopy. RESULTS: Survival bioassays of T. castaneum revealed a dose-dependent insecticide effect of diatomaceous earth. Gravimetric measurements showed that 2 days exposure to diatomaceous earth produces a significant increase of mass loss. Open-flow respirometry measurements showed an increase of total water emission rate on insects due to an increase of both, respiratory and cuticular water loss. Our study revealed that diatomaceous earth produces an increase of insect's cuticle permeability, which is responsible for elevated cuticular water loss. Scanning electron microscopy images provided visual evidence of the lipid absorbent properties of diatomaceous earth particles, and showed a tendency for higher, although not significant, damaged area of the cuticle's surface from diatomaceous earth treated insects compared to control ones. CONCLUSION: With state-of-the art techniques like open-flow respirometry and scanning electron microscopy, we demonstrated the underlying mechanism of diatomaceous earth as an insecticide and provided new cues for understanding the properties of the cuticle and its ecological importance. © 2024 Society of Chemical Industry.

Mitochondrial function and reactive oxygen species production during human sperm capacitation: Unraveling key players.

Sperm capacitation is a critical process for male fertility. It involves a series of biochemical and physiological changes that occur in the female reproductive tract, rendering the sperm competent for successful fertilization. The precise mechanisms and, specifically, the role of mitochondria, in sperm capacitation remain incompletely understood. Previously, we revealed that in mouse sperm mitochondrial activity (e.g., oxygen consumption, membrane potential, ATP/ADP exchange, and mitochondrial Ca2+ ) increases during capacitation. Herein, we studied mitochondrial function by high-resolution respirometry (HRR) and reactive oxygen species production in capacitated (CAP) and non-capacitated (NC) human spermatozoa. We found that in capacitated sperm from normozoospermic donors, the respiratory control ratio increased by 36%, accompanied by a double oxygen consumption rate (OCR) in the presence of antimycin A. Extracellular hydrogen peroxide (H2 O2 ) detection was three times higher in CAP than in NC sperm cells. To confirm that H2 O2 production depends on mitochondrial superoxide ( O 2 · - $$ {\mathrm{O}}_2^{\cdotp -} $$ ) formation, we evaluated mitochondrial aconitase (ACO2) amount, activity, and role in the metabolic flux from the sperm tricarboxylic acid cycle. We estimated that CAP cells produce, on average by individual, (59 ± 22)% more O 2 · - $$ {\mathrm{O}}_2^{\cdotp -} $$ in the steady-state compared to NC cells. Finally, we analyzed two targets of oxidative stress: lipid peroxidation by western blot against 4-hydroxynonenal and succinate dehydrogenase (SDH) activity by HRR. We did not observe modifications in lipoperoxidation nor the activity of SDH, suggesting that during capacitation, the increase in mitochondrial H2 O2 production does not damage sperm and it is necessary for the normal CAP process.

Ecotoxicological assessment of natural soil amended with sewage sludge: the impacts on soil edaphic organisms and microbial community.

Sewage sludge usage as agricultural soil amendment is a well-known practice employed worldwide. However, certain components may pose risks to the soil ecosystem. For a better verification of the potential adverse effects on the soil biota, biological assays have become an indispensable tool for an accurate understanding of the residue's behavior on soil, as well as its potential toxicity. Accordingly, to properly assess the effects of natural tropical soil (Oxisoil) amended with sewage sludge, we conducted toxicological tests with edaphic organisms (Enchytraeus crypticus and Folsomia candida) and microbial biomass (through respirometric assessment). Results indicate that E. crypticus and F. candida present similar reproduction sensitivity behavior to sewage sludge. For the microbiological analysis, the results suggest that microbial activity was stimulated by sludge application. For further evaluation of respiration of the microbial community and CO2 stabilization values behavior, Ford-Walford modeling was applied and presented limit values for sludge application in soil for 1.5 g kg-1 and 15.0 g kg-1 of, approximately, 55 mg and 88 mg, respectively. CO2 releases were faster and reached stability within 18 weeks for the soil with higher sludge content (15.0 g kg-1 of dry soil). In contrast, CO2 releases were slower for the soil with lower sludge content (1.5 g kg-1 of dry soil), and until the experiment's final period (21 weeks) respiration behavior did not reach stability. This study indicates that the stabilized sewage sludge, at the considered recommended application rate, presents a low toxicity risk for the studied bioindicators, being suitable for agricultural use.

Sepsis-induced mitochondrial dysfunction: A narrative review.

Sepsis represents a deranged and exaggerated systemic inflammatory response to infection and is associated with vascular and metabolic abnormalities that trigger systemic organic dysfunction. Mitochondrial function has been shown to be severely impaired during the early phase of critical illness, with a reduction in biogenesis, increased generation of reactive oxygen species and a decrease in adenosine triphosphate synthesis of up to 50%. Mitochondrial dysfunction can be assessed using mitochondrial DNA concentration and respirometry assays, particularly in peripheral mononuclear cells. Isolation of monocytes and lymphocytes seems to be the most promising strategy for measuring mitochondrial activity in clinical settings because of the ease of collection, sample processing, and clinical relevance of the association between metabolic alterations and deficient immune responses in mononuclear cells. Studies have reported alterations in these variables in patients with sepsis compared with healthy controls and non-septic patients. However, few studies have explored the association between mitochondrial dysfunction in immune mononuclear cells and unfavorable clinical outcomes. An improvement in mitochondrial parameters in sepsis could theoretically serve as a biomarker of clinical recovery and response to oxygen and vasopressor therapies as well as reveal unexplored pathophysiological mechanistic targets. These features highlight the need for further studies on mitochondrial metabolism in immune cells as a feasible tool to evaluate patients in intensive care settings. The evaluation of mitochondrial metabolism is a promising tool for the evaluation and management of critically ill patients, especially those with sepsis. In this article, we explore the pathophysiological aspects, main methods of measurement, and the main studies in this field.

Effect of brief exposures of anesthesia on thermotolerance and metabolic rate of the spotted-wing fly, Drosophila suzukii: Differences between sexes?

The spotted-wing fly, Drosophila suzukii, is a world-wide pest insect for which there is increasing interest in its physiological traits including metabolism and thermotolerance. Most studies focus only on survival to different time exposures to extreme temperatures, mainly in female flies. In addition, it has not been tested yet how anesthesia affects these measurements. We analyzed the effects of anesthesia by brief exposures to cold, anoxia by CO2 or N2 on three standard thermotolerance assays, as well as the aerobic metabolic rate in both sexes. For heat tolerance we measured CTmax by thermolimit respirometry, and CTmin and chill-coma recovery time for cold tolerance. Aerobic metabolism was calculated by CO2 production of individual flies in real time by open flow respirometry. Results showed that females have a significantly higher V̇CO2 for inactive (at 25 °C) and maximum metabolic rate than males. This difference is mainly explained by body mass and disappears after mass correction. Males had a more sensitive MR to temperature than females showed by a significantly higher Q10 (2.19 vs. 1.98, for males and females, respectively). We observed a significantly lower CTmin (X2 = 4.27, P = 0.03) in females (3.68 ± 0.38 °C) than males (4.56 ± 0.39 °C), although we did not find significant effects of anesthesia. In contrast, anesthesia significantly modifies CTmax for both sexes (F3,62 = 7.86, P < 0.001) with a decrease of the CTmax in cold-anesthetized flies. Finally, we found a significantly higher CTmax in females (37.87 ± 0.07 °C) than males (37.36 ± 0.09 °C). We conclude that cold anesthesia seems to have detrimental effects on heat tolerance, and females have broader thermotolerance range than males, which could help them to establish in invaded temperate regions with more variable environmental temperatures.

Caffeine improves mitochondrial function in PINK1B9-null mutant Drosophila melanogaster.

Mitochondrial dysfunction plays a central role in Parkinson's disease (PD) and can be triggered by xenobiotics and mutations in mitochondrial quality control genes, such as the PINK1 gene. Caffeine has been proposed as a secondary treatment to relieve PD symptoms mainly by its antagonistic effects on adenosine receptors (ARs). Nonetheless, the potential protective effects of caffeine on mitochondrial dysfunction could be a strategy in PD treatment but need further investigation. In this study, we used high-resolution respirometry (HRR) to test caffeine's effects on mitochondrial dysfunction in PINK1B9-null mutants of Drosophila melanogaster. PINK1 loss-of-function induced mitochondrial dysfunction in PINK1B9-null flies observed by a decrease in O2 flux related to oxidative phosphorylation (OXPHOS) and electron transfer system (ETS), respiratory control ratio (RCR) and ATP synthesis compared to control flies. Caffeine treatment improved OXPHOS and ETS in PINKB9-null mutant flies, increasing the mitochondrial O2 flux compared to untreated PINKB9-null mutant flies. Moreover, caffeine treatment increased O2 flux coupled to ATP synthesis and mitochondrial respiratory control ratio (RCR) in PINK 1B9-null mutant flies. The effects of caffeine on respiratory parameters were abolished by rotenone co-treatment, suggesting that caffeine exerts its beneficial effects mainly by stimulating the mitochondrial complex I (CI). In conclusion, we demonstrate that caffeine may improve mitochondrial function by increasing mitochondrial OXPHOS and ETS respiration in the PD model using PINK1 loss-of-function mutant flies.

Methylmalonic Acid Impairs Cell Respiration and Glutamate Uptake in C6 Rat Glioma Cells: Implications for Methylmalonic Acidemia.

Methylmalonic acidemia is an organic acidemia caused by deficient activity of L-methylmalonyl-CoA mutase or its cofactor cyanocobalamin and it is biochemically characterized by an accumulation of methylmalonic acid (MMA) in tissue and body fluids of patients. The main clinical manifestations of this disease are neurological and observable symptoms during metabolic decompensation are encephalopathy, cerebral atrophy, coma, and seizures, which commonly appear in newborns. This study aimed to investigate the toxic effects of MMA in a glial cell line presenting astrocytic features. Astroglial C6 cells were exposed to MMA (0.1-10 mM) for 24 or 48 h and cell metabolic viability, glucose consumption, and oxygen consumption rate, as well as glutamate uptake and ATP content were analyzed. The possible preventive effects of bezafibrate were also evaluated. MMA significantly reduced cell metabolic viability after 48-h period and increased glucose consumption during the same period of incubation. Regarding the energy homeostasis, MMA significantly reduced respiratory parameters of cells after 48-h exposure, indicating that cell metabolism is compromised at resting and reserve capacity state, which might influence the cell capacity to meet energetic demands. Glutamate uptake and ATP content were also compromised after exposure to MMA, which can be influenced energy metabolism impairment, affecting the functionality of the astroglial cells. Our findings suggest that these effects could be involved in the pathophysiology of neurological dysfunction of this disease. Methylmalonic acid compromises mitochondrial functioning leading to reduced ATP production and reduces glutamate uptake by C6 astroglial cells.

Heart rate response and bimodal gas exchange in three developmental stages of the bullfrog Lithobates catesbeianus (Anura: Ranidae)

ABSTRACT Measuring cardiorespiratory variables can be challenging in developing animals, especially when they use bimodal gas exchange to maintain metabolic activity. In tadpoles, gas exchange may occur through the integument and gills when breathing in the water and through the lungs when breathing air, with varying contributions of each respiratory structure during development. The interaction between aquatic and air breathing results in a complex physiological response that may affect the cardiac cycle. Measuring the heart rate (fH) together with aquatic and aerial gas exchange in anurans during their development can be challenging, since it may involve handling small animals and/or a certain degree of invasiveness (i.e., surgery to implant electrodes). Here, we evaluated concomitantly aquatic and aerial gas exchange, lung ventilation, and fH in three stages of development of the bullfrog Lithobates catesbeianus (Shaw, 1802). We built a novel, noninvasive, closed respirometry system capable of measuring fH, aerial and aquatic gas exchange simultaneously in animals of different sizes. Our integrative analysis revealed a decrease in the heart rate and an increase in oxygen consumption during the developmental stages of the bullfrog, but there was no adjustment of heart rate after or during air breathing. Moreover, tadpoles in metamorphosis showed higher oxygen consumption in air than in water, while aquatic breathing was responsible for releasing CO2. Our results are consistent with those found in the literature, yet our study represents the first non-invasive investigation to evaluate bimodal gas exchange and heart rate simultaneously. Moreover, our setup holds potential for further advancements that would allow for controlled water and air composition. This tool could greatly facilitate the investigation of how cardiorespiratory physiology responds to varying environmental conditions.

Mitochondrial metabolism determines the functional status of human sperm and correlates with semen parameters.

The diagnosis of male infertility is based essentially on the patient's medical history and a standard semen analysis. However, the latter rarely provides information on the causes of a possible infertility, emphasizing the need to extend the analysis of the sperm function. Mitochondrial function has been associated with sperm function and dysfunction, the latter primarily through the production of excessive amounts of reactive oxygen species (ROS). We hypothesized that analysis of sperm mitochondrial metabolism together with sperm ROS production could be an additional tool to improve routine semen analysis, after appropriate validations. To test our hypothesis, we performed several experiments using a non-routine method (high-resolution respirometry, HRR) to access mitochondrial function. First, we investigated whether mitochondrial function is related to human sperm motility and morphology. When mitochondrial metabolism was challenged, sperm motility decreased significantly. Additionally, morphological abnormalities in the sperm mid-piece and mitochondria were associated with global sperm defects evaluated by routine methods. Subsequently, sperm mitochondrial function was assessed by HRR. Respiratory control ratio (RCR) was determined and evaluated in the context of classical sperm analysis. In parallel, sperm hydrogen peroxide (H2O2) production and seminal plasma (SP) antioxidant capacity were measured. The percentage of sperm with progressive motility correlated positively with RCR, SP antioxidant capacity, and negatively with the concentration of extracellular H2O2 production ([H2O2]). The percentage of normal sperm morphology correlated positively with RCR and negatively with [H2O2]. Sperm morphology did not correlate with seminal plasma antioxidant capacity. Furthermore, Receiver Operating Characteristic curves were used for the first time to test the diagnostic ability of RCR, [H2O2], and SP antioxidant capacity as binary classifiers. An RCR cut off value of 3.2 was established with a sensitivity of 73% and a specificity of 61%, using reference values considered normal or abnormal in routine semen analysis. The cut off value for [H2O2] was 0.2 µM/106 sperm (sensitivity = 65%, specificity = 60%). There were no reference values for SP antioxidant capacity that distinguished between abnormal and normal sperm samples. We conclude that sperm mitochondrial function indices in combination with [H2O2] may be useful tools to complement the routine semen analysis.

Energy expenditure of red-footed tortoises (Chelonoidis carbonaria) fed kibble diets with high levels of fibre, starch, or fat.

Knowledge of energy requirements is necessary to optimise the nutritional management of animals. For tortoises, very little is known about their nutrient and energy requirements. Data on substrate oxidation and the implications of starch or fat intake on the energy metabolism are lacking. The present study compared the daily energy expenditures (DEE) of red-footed tortoises (Chelonoidis carbonaria) that were fed three extruded diets: a control diet high in fibre and two test diets, one with high starch content and another with high fat content. A total of 18 tortoises (5.5 ± 1.18 kg) were used in a completely randomised design, with 6 animals per diet. After 14 months of experimental diet intake and 48 h of preliminary fasting, the animals were kept for 12 h in 70-l respiratory chambers. An open "push mode" system was used to determine the carbon dioxide production and oxygen consumption levels for the subsequent calculations of DEE. The data were analysed with ANOVA, and the means were compared by using Tukey's test (p < 0.05). The body weights, chamber temperatures and food intakes of the tortoises were similar among the treatments (p > 0.05). There were no significant differences in oxygen consumption (21.7 ± 3.16 ml · kg-1 · h-1), carbon dioxide production (18.1 ± 1.96 ml · kg-1 · h-1), or DEE (9.7 ± 1.04 kJ · kg-1 d-1) between diets or sex (p > 0.05). The respiratory quotients (0.84 ± 0.11) were also similar among the diets (p > 0.05). The DEE of red footed tortoises did not differ after a long-term starch or fat intake.

Microbial activity and carbon rates the soil in response to the application of potassium sources

The continuous use of KCl may not be sustainable in the long term in agricultural systems. High doses used in crops accumulate in the soil and plants, hindering the metabolic processes of soil organisms. This study assessed the soil microbial activity in response to the application of K sources in banana crop and effects on microbial C. The experimental design was completely randomized with four K sources: potassium nitrate (KNO3), potassium chloride (KCl), potassium sulfate (K2SO4), and monopotassium phosphate (KH2PO4) at 200 mg kg-1of K2O, besides the control (without K) and combinations KCl:K2SO4. KCl application increased microbial activity 7 days after incubation, with gradual reduction over time. The isolated application of K2SO4and the combination KCl: K2SO4at the ratio 60: 40% increased total CO2released by the microbiota. K2SO4source had the highest microbial biomass C (MBC), as well as the 60: 40 combinations. Isolated application of K sources, especially with high chloride concentration, reduces the soil microbial activity and MBC.(AU)

Comparison between Aliivibrio fischeri and activated sludge microorganisms in the evaluation of the toxic pollutants of leachates from Brazilian landfills.

Ecotoxicological assessment of landfill leachate has become a priority to determine its impacts on the ecosystem. Toxicity assays with microorganisms stand out due to their quick response, low cost and ease of testing. In this context, the present study evaluated the acute toxic effects of leachates from two landfills of different ages and modes of operation to bacterium Aliivibrio fischeri and activated sludge microorganisms and the ammonia nitrogen and humic substances (HS) sensitivity to these organisms. Reductions greater than 30% in leachate toxicity were observed after ammonia removal for A. fischeri and activated sludge microorganisms. After 97% removal of HS, the greater reductions in toxicity (44.28 to 79.82%) were verified for microbial species studied, indicating that the organic compounds (measured as chemical oxygen demand, total organic carbon and humic substances) were the primary pollutants responsible for the toxicity of the leachates. Concerning the organisms studied, A. fischeri showed greater sensitivity to the leachates' pollutants compared to the activated sludge microorganisms. Nevertheless, a strong correlation was observed between A. fischeri and activated sludge microorganisms' toxicity responses, suggesting that respirometry assay can be used to determine leachate toxicity.

Methylmalonic Acid Compromises Respiration and Reduces the Expression of Differentiation Markers of SH-SY5Y Human Neuroblastoma Cells.

Methylmalonic acidemia is a rare metabolic disorder caused by the deficient activity of l-methylmalonyl-CoA mutase or its cofactor 5-deoxyadenosylcobalamin and is characterized by accumulation of methylmalonic acid (MMA) and alternative metabolites. The brain is one of the most affected tissues and neurologic symptoms, characterized by seizures, mental retardation, psychomotor abnormalities, and coma, commonly appear in newborns. The molecular mechanisms of neuropathogenesis in methylmalonic acidemia are still poorly understood, specifically regarding the impairments in neuronal development, maturation, and differentiation. In this study, we investigated the effects of MMA in both undifferentiated and differentiated phenotypes of SH-SY5Y human neuroblastoma cells. We observed an increase in glucose consumption and reduction in respiratory parameters of both undifferentiated and differentiated cells after exposition to MMA, suggesting that differentiated cells are slightly more prone to perturbations in respiratory parameters by MMA than undifferentiated cells. Next, we performed qPCR of mature neuronal-specific gene markers and measured mitochondrial functioning to evaluate the role of MMA during differentiation. Our results showed that MMA impairs the respiratory parameters only at the late stage of differentiation and downregulates the transcriptional gene profile of mature neuronal markers neuron-specific enolase (ENO2) and synaptophysin (SYP). Altogether, our findings point out important changes observed during neuronal maturation and energetic stress vulnerability that can play a role in the neurological clinical symptoms at the newborn period and reveal important molecular mechanisms that could help the screening of targets to new approaches in the therapies of this disease.

The Potent Trypanocidal Effect of LQB303, a Novel Redox-Active Phenyl-Tert-Butyl-Nitrone Derivate That Causes Mitochondrial Collapse in Trypanosoma cruzi.

Chagas disease, which is caused by Trypanosoma cruzi, establishes lifelong infections in humans and other mammals that lead to severe cardiac and gastrointestinal complications despite the competent immune response of the hosts. Furthermore, it is a neglected disease that affects 8 million people worldwide. The scenario is even more frustrating since the main chemotherapy is based on benznidazole, a drug that presents severe side effects and low efficacy in the chronic phase of the disease. Thus, the search for new therapeutic alternatives is urgent. In the present study, we investigated the activity of a novel phenyl-tert-butyl-nitrone (PBN) derivate, LQB303, against T. cruzi. LQB303 presented trypanocidal effect against intracellular [IC50/48 h = 2.6 µM] and extracellular amastigotes [IC50/24 h = 3.3 µM] in vitro, leading to parasite lysis; however, it does not present any toxicity to host cells. Despite emerging evidence that mitochondrial metabolism is essential for amastigotes to grow inside mammalian cells, the mechanism of redox-active molecules that target T. cruzi mitochondrion is still poorly explored. Therefore, we investigated if LQB303 trypanocidal activity was related to the impairment of the mitochondrial function of amastigotes. The investigation showed there was a significant decrease compared to the baseline oxygen consumption rate (OCR) of LQB303-treated extracellular amastigotes of T. cruzi, as well as reduction of "proton leak" (the depletion of proton motive force by the inhibition of F1Fo ATP synthase) and "ETS" (maximal oxygen consumption after uncoupling) oxygen consumption rates. Interestingly, the residual respiration ("ROX") enhanced about three times in LQB303-treated amastigotes. The spare respiratory capacity ratio (SRC: cell ability to meet new energy demands) and the ATP-linked OCR were also impaired by LQB303 treatment, correlating the trypanocidal activity of LQB303 with the impairment of mitochondrial redox metabolism of amastigotes. Flow cytometric analysis demonstrated a significant reduction of the ΔΨm of treated amastigotes. LQB303 had no significant influence on the OCR of treated mammalian cells, evidencing its specificity against T. cruzi mitochondrial metabolism. Our results suggest a promising trypanocidal activity of LQB303, associated with parasite bioenergetic inefficiency, with no influence on the host energy metabolism, a fact that may point to an attractive alternative therapy for Chagas disease.

Acetyl-CoA-driven respiration in frozen muscle contributes to the diagnosis of mitochondrial disease.

BACKGROUND: Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. PATIENTS AND METHODS: We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O2 consumption rates were normalized using citrate synthase activity. RESULTS: Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O2 consumption rates and enzyme activity assays of individual ETC complexes. CONCLUSIONS: We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans.

Biological removal of hexavalent chromium: evaluation of the metabolic activity of native and Cr(VI)-acclimated activated sludge using a respirometric method.

Hexavalent chromium becomes in one of the tops internationally concern environmental issues due to its wide usage in several industrial activities. There are two stable oxidation states of chromium in the environment which differ significantly on its toxicity; Cr(III) has lower solubility, mobility and lesser biological toxicity in comparison with Cr(VI). While Cr(VI) is a well-known carcinogen, Cr(III) is an essential dietary element. For this reason, most technologies focus attention on the reduction of Cr(VI) to Cr(III). On this context, the ability of microorganisms to reduce Cr(VI) to Cr(III) has gained attention. The objectives of the present work were to analyze the effect of Cr(VI) on the activated sludge community in a continuous reactor, and to evaluate the differences on the metabolic activity of native (NAS) and Cr(VI)-acclimated activated sludge (CrAAS) using a respirometric method. Results showed that the activated sludge community had the capability to acclimate to the presence of Cr(VI). On the other hand, the increase of the initial Cr(VI) concentration from 0 to 100 mgCr/L leads to a decrease in the specific exogenous respiration rate (qEx ) values, but this reduction was more noticeably in the case of NAS in comparison with CrAAS. The respirometric curves were well described by the proposed mathematical model. It was concluded that the CrAAS tolerated a Cr(VI) concentration about one order of magnitude higher than NAS, which was positively reflected in the respiration rate first-order decay constant (kd ), the specific maximum exogenous respiration rate (qExm ), and the observed oxidation coefficient (YO/S ) values.

A Protocol to Study Mitochondrial Function in Human Neural Progenitors and iPSC-Derived Astrocytes.

Mitochondrial dysfunction is a central component in the pathophysiology of multiple neuropsychiatric and degenerative disorders. Evaluating mitochondrial function in human-derived neural cells can help characterize dysregulation in oxidative metabolism associated with the onset of brain disorders, and may also help define targeted therapies. Astrocytes play a number of different key roles in the brain, being implicated in neurogenesis, synaptogenesis, blood-brain-barrier permeability, and homeostasis, and, consequently, the malfunctioning of astrocytes is related to many neuropathologies. Here we describe protocols for generating induced pluripotent stem cell (iPSC)-derived astrocytes and evaluating multiple aspects of mitochondrial function. We use a high-resolution respirometry assay that measures real-time variations in mitochondrial oxygen flow, allowing the evaluation of cellular respiration in the context of an intact intracellular microenvironment, something not possible with permeabilized cells or isolated mitochondria, where the cellular microenvironment is disrupted. Given that an impairment in the mitochondrial regulation of intracellular calcium homeostasis is involved in many pathologic stresses, we also describe a protocol to evaluate mitochondrial calcium dynamics in human neural cells, by fluorimetry. Lastly, we outline a mitochondrial function assay that allows for the measurement of the enzymatic activity of mitochondrial hexokinase (mt-HK), an enzyme that is functionally coupled to oxidative phosphorylation and is involved in redox homeostasis, particularly in the brain. In all, these protocols allow a detailed characterization of mitochondrial function in human neural cells. High-resolution respirometry, calcium dynamics, and mt-HK activity assays provide data regarding the functional status of mitochondria, which may reflect mitochondrial stress or dysfunction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of iPSC-derived human astrocytes Basic Protocol 2: Measuring real-time oxygen flux in human iPSC-derived astrocytes using a high-resolution OROBOROS Oxygraph 2k (O2k) Basic Protocol 3: Measuring mitochondrial calcium dynamics fluorometrically in permeabilized human neural cells Basic Protocol 4: Measuring OXPHOS-dependent activity of mitochondrial hexokinase in permeabilized human neural cells using a spectrophotometer.

Relevance of Mitochondrial Dysfunction in the Reserpine-Induced Experimental Fibromyalgia Model.

Fibromyalgia (FM) is one of the most common musculoskeletal pain conditions. Although the aetiology of FM is still unknown, mitochondrial dysfunction and the overproduction of reactive oxygen intermediates (ROI) are common characteristics in its pathogenesis. The reserpine experimental model can induce FM-related symptoms in rodents by depleting biogenic amines. However, it is unclear whether reserpine causes other pathophysiologic characteristics of FM. So far, no one has investigated the relevance of mitochondrial dysfunction in the reserpine-induced experimental FM model using protection- and insult-based mitochondrial modulators. Reserpine (1 mg/kg) was subcutaneously injected once daily for three consecutive days in male Swiss mice. We carried out analyses of reserpine-induced FM-related symptoms, and their modulation by using mitochondrial insult on ATP synthesis (oligomycin; 1 mg/kg, intraperitoneally) or mitochondrial protection (coenzyme Q10; 150 mg/kg/5 days, orally). We also evaluated the effect of reserpine on mitochondrial function using high-resolution respirometry and oxidative status. Reserpine caused nociception, loss in muscle strength, and anxiety- and depressive-like behaviours in mice that were consistent with clinical symptoms of FM, without inducing body weight and temperature alterations or motor impairment. Reserpine-induced FM-related symptoms were increased by oligomycin and reduced by coenzyme Q10 treatment. Reserpine caused mitochondrial dysfunction by negatively modulating the electron transport system and mitochondrial respiration (ATP synthesis) mainly in oxidative muscles and the spinal cord. These results support the role of mitochondria in mediating oxidative stress and FM symptoms in this model. In this way, reserpine-inducing mitochondrial dysfunction and increased production of ROI contribute to the development and maintenance of nociceptive, fatigue, and depressive-like behaviours.

Atorvastatin Improves Mitochondrial Function and Prevents Oxidative Stress in Hippocampus Following Amyloid-ß1-40 Intracerebroventricular Administration in Mice.

Amyloid-ß (Aß) peptides play a significant role in the pathogenesis of Alzheimer's disease (AD). Neurotoxic effects promoted by Aß peptides involve glutamate transmission impairment, decrease of neurotrophic factors, mitochondrial dysfunction, oxidative stress, synaptotoxicity, and neuronal degeneration. Here, we assessed the early events evoked by Aß1-40 on the hippocampus. Additionally, we sought to unravel the molecular mechanisms of atorvastatin preventive effect on Aß-induced hippocampal damage. Mice were treated orally (p.o.) with atorvastatin 10 mg/kg/day during 7 consecutive days before the intracerebroventricular (i.c.v.) infusion of Aß1-40 (400 pmol/site). Twenty-four hours after Aß1-40 infusion, a reduced content of mature BDNF/proBDNF ratio was observed in Aß-treated mice. However, there is no alteration in synaptophysin, PSD-95, and doublecortin immunocontent in the hippocampus. Aß1-40 promoted an increase in reactive oxygen species (ROS) and nitric oxide (NO) generation in hippocampal slices, and atorvastatin prevented this oxidative burst. Mitochondrial OXPHOS was measured by high-resolution respirometry. At this time point, Aß1-40 did not alter the O2 consumption rates (OCR) related to phosphorylating state associated with complexes I and II, and the maximal OCR. However, atorvastatin increased OCR of phosphorylating state associated with complex I and complexes I and II, maximal OCR of complexes I and II, and OCR associated with mitochondrial spare capacity. Atorvastatin treatment improved mitochondrial function in the rodent hippocampus, even after Aß infusion, pointing to a promising effect of improving brain mitochondria bioenergetics. Therefore, atorvastatin could act as an adjuvant in battling the symptoms of AD to preventing or delaying the disease progression.