The modulatory role of fermented Rooibos on oxidative stress and its impact on submaximal exercise performance in adult males

Energy uptake and utilization in eukaryotic cells is a dynamic process regulated by a series of interacting metabolic networks. Interrogation of this complex network relies on rapid, sensitive approaches that do not require extensive sample handling and are easily adaptable to 96- and 384-well plates. Previously, using the Ultra-Glo luciferase reaction we developed a panel of bioluminescent assays that can be used to monitor numerous aspects of cellular metabolism and mitochondrial function including ATP production, glucose and amino acid metabolism, and the TCA cycle. Here, we extend the use of the luciferase reaction and report on the development of novel bioluminescence probes for studying two important metabolic cellular responses: fatty acid β-oxidation (FAO) and production of reactive oxygen and nitrogen species. For studying FAO, we developed a cell permeable probe with caged-luciferin attached to a fatty acid chain. The probe enters the cells and following FAO cycling, free luciferin is released and detected using Luciferin Detection Reagent. We validated the approach using known FAO activators and inhibitors and used it to monitor the changes in FAO during T-cell activation. For measuring reactive oxygen and nitrogen species, highly selective bioluminescence probes were developed. Upon reaction of the probes with their corresponding ROS target, they form the stable D-luciferin reporter molecule, causing luciferin to accumulate. Upon treatment with luciferase in the detection step, the generated light allows for quantification of the superoxide or nitric oxide formed. Both probes are suitable for in vitro and cell-based detection of ROS in an “add and read” format, providing for a simple workflow amenable to high throughput experimentation. Citation Format: Kim Haupt, Matt Larsen, Hui Wang, Natasha Karassina, Mike Valley, Wenhui Zhou, Jolanta Vidugiriene. Novel bioluminescence approaches for measuring fatty acid β-oxidation and production of reactive oxygen and nitrogen species. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4778.

Oxidative stress is the outcome of an imbalance between the production and neutralization of reactive oxygen and nitrogen species (RONS) such that the antioxidant capacity of cell is overwhelmed. The present review briefly summarized the underlying role of overwhelming levels of RONS in the pathophysiology of diabetes mellitus (DM). The primary causative factor of oxidative stress in DM is hyperglycemia, which operates via several mechanisms. However, the individual contribution of other intermediary factors to hyperoxidative stress remains undefined, in terms of the dose response relationship between hyperglycemia and overall oxidative stress in DM. Intuitively, the inhibition and/or scavenging of intracellular free radical formation provide a therapeutic strategy to prevent oxidative stress and ensuing pathologic conditions. Therefore, the integration of antioxidants formulations into conventional therapeutic interventions, either by ingestion of natural antioxidants or through dietary supplementation, should be encouraged for a holistic approach to the management and prevention of DM and the complications associated with the pathology. Introduction Oxidative stress is the outcome of an imbalance between the production and neutralization of reactive oxygen and nitrogen species (RONS) such that the antioxidant capacity of cell is overwhelmed [1-4]. Ordinarily, the peculiar molecular configuration of oxygen (O2) confers a very slow reactivity between O2 and biomolecules. Two main factors make O2 kinetically insert; the spin restriction imposed by its triplet state, and the negative standard potential for one electron reduction of O2 to superoxide radical (O2 •−). However, O2 possesses the attributes of free radicals in that it has two unpaired electrons with parallel spin in different π-anti-bonding orbitals that is responsible for its paramagnetic properties and relative stability [4,5]. Spin restriction can be overcome by single electron exchange that converts it to strong oxidizing agent [6,7]. Therefore, the activation of O2 by specific enzymes is achieved by the presence, at the active site, of either flavins or reduced transition metals such as iron (Fe2+) and copper (Cu2+), which donates single electron to O2 [6]. In the case of metalloproteins, a varying degree of electron transfer from the metallic moiety to O2 is possible. On this basis, metalloproteins can behave either as O2 carriers (hemoglobin, hemocyanin, hemerythrin, myoglobin), where reversible interaction with O2 occurs, or as O2 reductants. Studies showed that autoxidation of oxy-hemoglobin elicit the generation of free radicals [8]. Free radical production and oxidative stress Electron transfer to O2 is catalyzed by oxidases for production of chemical energy or oxidation of substrates. These enzymes, located in different subcellular compartments (mitochondria, endoplasmic reticulum, peroxisomes) are potential sources of partially reduced Cu2+ derivatives in biological milieu. Cytosolic enzymes {xanthine oxidase, NADPH oxidases, lipoxygenase, cyclooxygenase (COX), cytochrome P450 enzymes and aldehyde oxidase}, uncoupled endothelial nitric oxide synthase (eNOS), and other hemoproteins also produce O2 •− during catalysis [2,9,10]. The mitochondrial electron transport chain reduces O2 to O2 •− at ubiquinone and NADH dehydrogenase sites whereas; microsomal cytochrome P450 and its reductases produce O2 •− during xenobiotic biotransformation [11-14]. The “leaky” inner mitochondrial membrane electron transport chain reacts with O2 directly to generate O2 •−, which dismutates to form hydrogen peroxide (H2O2), which can further react to form the hydroxyl radical ( •−OH) [2,5,10]. Additionally, the mitochondrial outer membrane enzyme monoamine oxidase catalyzes the oxidative deamination of biogenic amines and is a quantitatively large source of H2O2 that contributes to increase in the steady state concentrations of reactive species within both the mitochondrial matrix and cytosol [15]. Specifically, O2 •− is the primary radical formed by the reduction of O2 leading to secondary radicals or reactive oxygen species (ROS) such as H2O2 and •−OH in the mitochondria [2,5]. Although the cause-effect relationship remains tentative, there appears to be a strong association between mitochondrial dysfunction and chronic metabolic diseases such as Type II diabetes mellitus (T2DM) and obesity [10]. The origin, enzymatic pathways of ROS and their oxidized products, as well as their enzymatic inactivation pathways in T2DM have previously been summarized [16]. RONS have been implicated in the pathophysiology of various disease states, including diabetes mellitus (DM) and long-term development of associated complications [10,12,14,16,17]. Oxidative tissue damage is mediated byactivating a number of cellular stresssensitive pathways, which include nuclear factor-ĸB (NFĸB), p38 Correspondence to: Paul C. Chikezie, Department of Biochemistry, Imo State University, PMB 2000,Owerri, Imo State, Nigeria, Tel: +2348038935327; E-mail: p_chikezie@yahoo.com

Anti-oxidant strategies

Cardiac Metallothionein Synthesis in Streptozotocin-Induced Diabetic Mice, and Its Protection against Diabetes-Induced Cardiac Injury

A more complete understanding of reactive chemistry generated by atmospheric pressure plasma jets (APPJs) is critical to many emerging medical, agricultural, and water treatment applications. Adding molecular gases to the noble working gas which flows through the jet is a common method to tailor the resulting production of reactive oxygen and nitrogen species (RONS). In this paper, results are discussed from a computational investigation of the consequences of H2O and O2 admixtures on the reactive chemistry of He APPJs flowing into humid air. This investigation, performed with a 2-dimensional plasma hydrodynamics model, addresses the RONS that are initially produced and the evolution of that chemistry on longer time scales. Without an admixture, the impurities in 99.999% pure helium are a major source of RONS. The addition of H2O decreases the production of reactive nitrogen species (RNS) and increases the production of reactive oxygen species (ROS). The addition of O2 significantly decreases the production of RNS, as well as hydrogen-containing ROS, but increases the production of ROS without hydrogen. This selectivity comes from the lower ionization energy of O2 compared to N2 and H2O, which then allows for charge exchange reactions. These charge exchange reactions change the RONS which are produced in the afterglow by dissociative recombination. The consequences of impurities were also examined. Humid air impurities as low as 10 ppm in the helium can account for 79%-98% of the production of most RONS in the absence of an intentional admixture. The degree to which the impurities affect the RONS production depends on the electrode configuration and can be reduced by molecular admixtures.

Radical innate regulation of autoimmune diabetes

Brain cancer is known as one of the deadliest cancers globally. One of the causative factors is the imbalance between oxidative and antioxidant activities in the body, which is referred to as oxidative stress (OS). As part of regular metabolism, oxygen is reduced by electrons, resulting in the creation of numerous reactive oxygen species (ROS). Inflammation is intricately associated with the generation of OS, leading to the increased production and accumulation of reactive oxygen and nitrogen species (RONS). Glioma stands out as one of the most common malignant tumors affecting the central nervous system (CNS), characterized by changes in the redox balance. Brain cancer cells exhibit inherent resistance to most conventional treatments, primarily due to the distinctive tumor microenvironment. Oxidative stress (OS) plays a crucial role in the development of various brain-related malignancies, such as glioblastoma multiforme (GBM) and medulloblastoma, where OS significantly disrupts the normal homeostasis of the brain. In this review, we provide in-depth descriptions of prospective targets and therapeutics, along with an assessment of OS and its impact on brain cancer metabolism. We also discuss targeted therapies.

The physiological demands of soccer challenge the entire spectrum of the response capacity of the biological systems and fitness requirements of the players. In this review we examined variations and evolutionary trends in body composition, neuromuscular and endurance-related parameters, as well as in game-related physical parameters of professional players. Further, we explore aspects relevant for training monitoring and we reference how different training stimulus and situational variables (e.g., competition exposure) affect the physiological and performance parameters of players. Generally, improvements of small magnitude in non- (non-CMJ) and countermovement-based jumps (CMJBased) and in the sprint acceleration (ACCPhase) and maximal velocity phase (MVPhase) are observed from start of preparation phase (PPS) to beginning of competition phase (BCP). A greater magnitude of increases is observed in physiological and endurance performance measures within this period; moderate magnitude in sub-maximal intensity exercise (velocity at fixed blood lactate concentrations; V2–4mmol/l) and large magnitude in VO2max, maximal aerobic speed (MAS) and intense intermittent exercise performance (IE). In the middle of competition phase (MCP), small (CMJBased and ACCPhase), moderate (non-CMJ; MVPhase; VO2max; sub-maximal exercise) and large (MAS and IE) improvements were observed compared to PPS. In the end of competition period (ECP), CMJBased and MVPhase improve to a small extent with non-CMJ, and ACCPhase, VO2max, MAS, sub-maximal intensity exercise and IE revealing moderate increments compared to PPS. Although less investigated, there are generally observed alterations of trivial magnitude in neuromuscular and endurance-related parameters between in-season assessments; only substantial alterations are examined for IE and sub-maximal exercise performance (decrease and increase of small magnitude, respectively) from BCP to MCP and in VO2max and IE (decrements of small magnitude) from MCP to ECP. Match performance may vary during the season. Although, the variability between studies is clear for TD, VHSR and sprint, all the studies observed substantial increments in HSR between MCP and ECP. Finally, studies examining evolutionary trends by means of exercise and competition performance measures suggests of a heightened importance of neuromuscular factors. In conclusion, during the preseason players “recover” body composition profile and neuromuscular and endurance competitive capacity. Within in-season, and more robustly towards ECP, alterations in neuromuscular performance seem to be force-velocity dependent, and in some cases, physiological determinants and endurance performance may be compromised when considering other in-season moments. Importantly, there is a substantial variability in team responses that can be observed during in-season. Consequently, this informs on the need to both provide a regular training stimulus and adequate monitorization throughout the season.

Antioxidative nanomaterials and biomedical applications

Persistent inflammation and the generation of reactive oxygen and nitrogen species play pivotal roles in tissue injury during disease pathogenesis and as a reaction to toxicant exposures. The associated oxidative and nitrative stress promote diverse pathologic reactions including neurodegenerative disorders, atherosclerosis, chronic inflammation, cancer, and premature labor and stillbirth. These effects occur via sustained inflammation, cellular proliferation and cytotoxicity and via induction of a proangiogenic environment. For example, exposure to the ubiquitous air pollutant ozone leads to generation of reactive oxygen and nitrogen species in lung macrophages that play a key role in subsequent tissue damage. Similarly, studies indicate that genes involved in regulating oxidative stress are altered by anesthetic treatment resulting in brain injury, most notable during development. In addition to a role in tissue injury in the brain, inflammation, and oxidative stress are implicated in Parkinson's disease, a neurodegenerative disease characterized by the loss of dopamine neurons. Recent data suggest a mechanistic link between oxidative stress and elevated levels of 3,4-dihydroxyphenylacetaldehyde, a neurotoxin endogenous to dopamine neurons. These findings have significant implications for development of therapeutics and identification of novel biomarkers for Parkinson's disease pathogenesis. Oxidative and nitrative stress is also thought to play a role in creating the proinflammatory microenvironment associated with the aggressive phenotype of inflammatory breast cancer. An understanding of fundamental concepts of oxidative and nitrative stress can underpin a rational plan of treatment for diseases and toxicities associated with excessive production of reactive oxygen and nitrogen species.

To assess the influence of co-culture with mineral trioxide aggregate (MTA) on phagocytosis and the production of reactive oxygen intermediates (ROI) and nitrogen (NO) species and the arginase activity by M1 and M2 peritoneal macrophages. Cellular viability, adherence and phagocytosis of Saccharomyces boulardii were assayed in the presence of MTA. Macrophages were stimulated with zymosan for ROI assays and with Fusobacterium nucleatum and Peptostreptococcus anaerobius and IFN-gamma for NO production and arginase activity, when in contact with capillaries containing MTA. Data were analysed by T, anova, Kruskall-Wallis and Mann-Whitney tests. M2 macrophages displayed greater cellular viability in polypropylene tubes, greater ability to ingest yeast and smaller production of ROI and higher arginase activity when compared with M1 macrophages. Both macrophages, M1 and M2, presented similar cell adherence and NO production. The addition of bacterial preparations to macrophages interfered with NO and arginase productions. MTA did not interfere with any of the parameters measured. Phagocytosis and the ability of the two macrophage subtypes to eliminate microbes were not affected by MTA.

Myoglobin causes oxidative stress, increase of NO production and dysfunction of kidney's mitochondria

Oxidative and nitrosative stress play an important role in the complications of diabetes mellitus. Free radicals are produced when there is an electron leak in the mito-chondria and a change in the mitochondrial membrane potential. The present study was undertaken to investigate the role of Semecarpus anacardium in protecting the mito-chondria by modulating the production of reactive oxygen species and reactive nitrogen species in diabetic rats. Diabetes was induced using streptozotocin at a dose of 50 mg/kg body weight and, starting 3 days after the induction, Semecarpus anacardium nut milk extract was administered for 21 days. The same duration of study was used for control, diabetes-induced and drug control groups, together with a group treated with metformin. After the experimental period, the animals were sacrificed and the levels of superoxide, hydrogen peroxide, nitrate and nitrite were estimated. Changes in mitochondrial membrane potential, intracellular reactive oxygen species and intracellular calcium were also determined. Confocal laser microscopic images were taken for mitochondria isolated from the liver and kidneys. The results of the study revealed that Semecarpus anacardium was able to decrease the production of reactive oxygen and nitrogen species, and reverse the changes in mitochondrial membrane potential and the influx of calcium into the mitochondria. The mitochondrial protective effect may be mediated by scavenging of free radicals and complexing of metal ions by virtue of the antioxidative effect of Semecarpus anacardium.

Our understanding of the mechanisms involved in the development of alcohol-induced liver disease has increased substantially in recent years. Specifically, reactive oxygen and nitrogen species have been identified as key components in initiating and possibly sustaining the pathogenic pathways responsible for the progression from alcohol-induced fatty liver to alcoholic hepatitis and cirrhosis. Ethanol has been demonstrated to increase the production of reactive oxygen and nitrogen species and decrease several antioxidant mechanisms in liver. However, the relative contribution of the proposed sites of ethanol-induced reactive species production within the liver is still not clear. It has been proposed that chronic ethanol-elicited alterations in mitochondria structure and function might result in increased production of reactive species at the level of the mitochondrion in liver from ethanol consumers. This in turn might result in oxidative modification and inactivation of mitochondrial macromolecules, thereby contributing further to mitochondrial dysfunction and a loss in hepatic energy conservation. Moreover, ethanol-related increases in reactive species may shift the balance between pro- and anti-apoptotic factors such that there is activation of the mitochondrial permeability transition, which would lead to increased cell death in the liver after chronic alcohol consumption. This article will examine the critical role of these reactive species in ethanol-induced liver injury with specific emphasis on how chronic ethanol-associated alterations to mitochondria influence the production of reactive oxygen and nitrogen species and how their production may disrupt hepatic energy conservation in the chronic alcohol abuser.

Hypoxia and certain chronic diseases can increase the work of breathing, potentially impacting the muscle hyperemic response during exercise and overall exercise performance. Respiratory muscle training (RMT) has been observed to decrease perceived effort under hypoxic conditions; however, it remains unclear if RMT affects exercise performance in acute hypoxia. We investigated whether 4 weeks of RMT could enhance submaximal and maximal exercise performance in hypoxia (barometric pressure = 425 mmHg, ~4876 m). Seven adult males underwent a 250 kJ submaximal (target RPE 15–17) cycling trial in hypoxia, followed by an incremental test to maximal exertion. After 4 weeks of RMT under normoxic conditions, these tests were repeated in hypoxia. After RMT, RPE showed no significant difference during the submaximal test (Pre‐RMT: 16 ± 0.45, Post‐RMT: 16 ± 0.45, p > 0.05) despite an increase in HR (Pre‐RMT:154 ± 16, Post‐RMT: 161 ± 12, p < 0.05). There were no significant differences in submaximal exercise performance. However, RMT did raise maximal oxygen uptake (V̇O2max) peak power (Pre‐RMT: 243 ± 35, Post‐RMT: 252 ± 38 W, p = 0.04, Cohen's dz. = 0.97). Future investigations should explore the potential effects of RMT on perceived exertion and exercise tolerance at altitude. The results of this pilot study suggest that RMT may improve peak power at V̇O2max, however given the limited sample size of this investigation, further research is required to determine if RMT impacts performance in other domains of exercise intensity. Furthermore, whether RMT impacts performance at altitude in females remains unknown.