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Mitochondrial free radical generation, oxidative stress, and aging1
Abstract
Mitochondria have been described as “the powerhouses of the cell” because they link the energy-releasing activities of electron transport and proton pumping with the energy conserving process of oxidative phosphorylation, to harness the value of foods in the form of ATP. Such energetic processes are not without dangers, however, and the electron transport chain has proved to be somewhat “leaky.” Such side reactions of the mitochondrial electron transport chain with molecular oxygen directly generate the superoxide anion radical (O2•−), which dismutates to form hydrogen peroxide (H2O2), which can further react to form the hydroxyl radical (HO). In addition to these toxic electron transport chain reactions of the inner mitochondrial membrane, 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 an increase in the steady state concentrations of reactive species within both the mitochondrial matrix and cytosol. In this article we review the mitochondrial rates of production and steady state levels of these reactive oxygen species. Reactive oxygen species generated by mitochondria, or from other sites within or outside the cell, cause damage to mitochondrial components and initiate degradative processes. Such toxic reactions contribute significantly to the aging process and form the central dogma of “The Free Radical Theory of Aging.” In this article we review current understandings of mitochondrial DNA, RNA, and protein modifications by oxidative stress and the enzymatic removal of oxidatively damaged products by nucleases and proteases. The possible contributions of mitochondrial oxidative polynucleotide and protein turnover to apoptosis and aging are explored.
... These highly reactive species can disrupt the normal function and structure of essential cellular molecules like DNA, RNA, lipids, and proteins [84,85]. This disruption can initiate various complications, including metabolic dysfunction and consequently, which lead to cell death [86,87]. ...
... This can cause damage to cells and tissues, known as oxidative damage. Oxidation is important in the chicken production industry because it directly impacts meat quality, leading to spoilage and degradation [86,107]. Several factors can increase a broiler's susceptibility to oxidative stress, including high ambient temperature, exposure to toxins, and various pathological conditions [76,108]. ...
... Most oxidants are produced during cellular metabolism in the mitochondria of living cells. Cellular metabolism is not the only source of oxidants; some external sources, including feed comprised of oxidized lipids and fats, are responsible for producing reactive oxygen species [86]. According to [94], leakage of electrons from the mitochondrial respiratory chain during oxidative phosphorylation is the primary ROS source. ...
Broiler breeders’ problems arise from various factors, such as management, stress, nutrition, immunosuppression, heat and cold stress, feed restriction, stocking density, pollutants, and exposure to disease agents. Stress can have a significant impact on both performance and overall health, making individuals more vulnerable to disease. Research has shown that chickens are no exception to this, as their performance, welfare, and health can all be negatively affected by stress. This can result in a variety of issues, such as changes in behavior, decreased meat quality, damage to tissues and intestines, and even a higher risk of mortality. Managing stress is crucial for the success of breeding programs in broiler chickens. Stressors can be tackled by supplementing chicken diets with vitamins and antioxidants. Poultry birds cannot produce enough vitamins during stressful periods, and therefore, it is recommended to supplement their diets with a combination of vitamins or antioxidants. This approach is more effective than using individual vitamins to alleviate stress in chickens. This chapter discusses stress in broilers and specific causes of stress in broiler breeders. It also covers management practices and strategies to prevent and alleviate the negative effects of stress.
... Infection, hemorrhage, and retinal detachment are among the hazards associated with cataract surgery; nevertheless, the chances of all those consequences are less than 1%. The majority of patients are discharged from the hospital on the same day [8]. ...
... A decrease in ATP synthesis may be the cause of a decrease in RPE phagocytic activity, which leads to RPE hyperpigmentation, an AMD risk factor since one of the primary functions of RPE is the phagocytosis of photoreceptor outer segments following photoactivation [23]. Furthermore, by activating NF-κB, UV light causes the overexpression of pro-inflammatory chemicals, which could hasten the production of drusen [8]. ...
... The aging process is linked to an increase in macromolecule oxidative alterations and a corresponding decline in antioxidant defenses. Age-related increases in ROS formation may be explained by an increase in electron leakage from the electron transport chain [8]. Furthermore, it has been noted that aging is associated with a decreased capacity for antioxidants as well as a compromised adaptive induction of antioxidants [7]. ...
One of the main causes of blindness is the multifactorial condition known as cataract. It is believed that oxidative stress plays a significant role in starting the cataractogenesis process. Today, it is a well-established fact that oxidative stress plays a role in both diabetes-induced cataract (diabetic) and age-related cataract (senile). The most likely cause of oxidative damage to the lens is a compromised antioxidant defense system brought on by age and diabetes-related increases in reactive oxygen species (ROS) production. The main factor contributing to cataract formation is systemic oxidative stress, which is produced externally to the lens. An imbalance between pro- and antioxidant-oxidants leads to oxidative stress. It is essential to eliminate hazardous free radicals because they are a byproduct of normal metabolism. Globally, cataracts are the primary cause of blindness. Oxidative stress is the direct cause of the lens’s opacity. Although age is the main cause of cataracts, diabetes is also a common cause, as higher superoxide levels in the mitochondria arise from hyperglycemia. This review will look into ultraviolet (UV) light, diabetes, and diet (fat, alcohol, and vitamins) as risk factors for cataracts.
... Antioxidants are generally the chemicals for assessment of the oxidative stress status that can be divided into reactive oxygen species, ROS diluents or antioxidant chemicals and transcription factors regulating ROS production. It is difficult to evaluate oxidative stress in different diseases by similar markers or biomarkers because the markers used in a particular disease are limited and must always be carefully filtered (14). They contain a variety of antioxidants as counter-attacking agents on both sides of their membranes. ...
... Journal of New Findings in Health and Educational Sciences (IJHES), 1(1):[7][8][9][10][11][12][13][14][15][16][17] 2023 ...
The production of reactive oxygen species (ROS) can alter macromolecules in living organisms and can result in a wide range of injuries. Recently, oxidative stress has been known as a key mechanism in insulin resistance. Today, oxidative stress (OS) status assessment is performed using circulating markers such as malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPX). Polycystic ovary syndrome (PCOS) with a prevalence of 4-12 % is the most common endocrine-metabolic disorder in the reproductive age of women. PCOS is now recognized as an important metabolic disorder. Insulin resistance (IR) independent of obesity in PCO women has been identified as a predisposing factor for type2 diabetes and cardiovascular disease (CVD). Oxidative stress index is strongly associated with PCOS. The role of oxidative stress is very important but not considered but it plays an important role in the development of IR. In this mini review, we presented a viewpoint about the key role of brain̕s IR/OS in the brain-ovarian axis in the women with PCOS. These review articles helps us to better understanding of the PCO etiology.
... Interleukin-1β IL- 6 Interleukin-6 ITCs Isothiocyanates SFE Sulforaphene SFN Sulforaphane Nrf2 ...
... Sirtuin 1 PGC-1α Peroxisome proliferator-activated receptor γ coactivator 1α ERK Extracellular regulated protein kinases JNK c-Jun N-terminal kinase T-AOC Total antioxidant capacity HYP Hydroxyproline HA Hyaluronic acid 4-HNE 4-Hydroxynonenal LPS Lipopolysaccharide MMP-3 Matrix metallopeptidase-3 MMP-1 Matrix metallopeptidase-1 TGF-β Transforming growth factor-β IL- 10 Interlenkin-10 mitochondrial dysfunction, telomere wear, and protein balance disruption) and extrinsic factors (e.g., ultraviolet B, ultraviolet A, ionizing radiation, chemicals, and toxins) [3,4]. According to the mitochondrial free radical theory of aging, progressive mitochondrial dysfunction during aging promotes reactive oxygen species (ROS) generation, leading to deteriorating mitochondrial function and overall cellular damage [5,6]. Injecting d-galactose (d-gal) in mice induces an imbalance in the ROS and antioxidant system, resulting in oxidative stress, neuron damage, mitochondrial dysfunction, and compromised antioxidant defense system function [7]. ...
d-Galactose (d-gal) accumulation triggers the generation of oxygen free radicals, resulting in skin aging. Sulforaphene (SFE), an isothiocyanate compound derived from radish seeds, possesses diverse biological activities, including protective effects against inflammation and oxidative damage. This investigation delves into the antioxidant impact of SFE on age-related skin injury. In vivo experiments demonstrate that SFE treatment significantly improves the macro- and micro-morphology of dorsal skin. It effectively diminishes the elevation of oxidative stress biomarkers in mice skin tissue treated with d-gal, concurrently enhancing the activity of antioxidant enzymes. Additionally, SFE mitigates collagen mRNA degradation, lowers pro-inflammatory cytokine levels, and downregulates MAPK-related protein expression in the skin. Moreover, SFE supplementation reduces lipid metabolite levels and elevates amino acid metabolites, such as l-cysteine and l-histidine. These findings suggest that SFE holds promise as a natural remedy to mitigate aging induced by oxidative stress.
... This review details how MT made from the intestines interacts in the gut-liver axis to prevent liver diseases. We have explained that MT produced in intestinal mitochondria targets hepatic mitochondria by regulating oxidative stress mainly generated by oneelectron leakage from the electron transport chain [6] as well as from the activities of cytochrome P450-2E1 (CYP2E1), inducible nitric oxide synthetase (iNOS), and NADPHdependent oxidases (NOXs). Consequently, MT attenuates mitochondrial dysfunction, and mitigates metabolic alterations and inflammatory processes that are involved in the progression of liver diseases. ...
... MT is primarily localized in the mitochondria of many cells [115][116][117][118], maintaining energy homeostasis [116,119,120] and reducing oxidative stress. Reactive oxygen species (ROS) are mainly produced from the one-electron leakage from the electron transport chain [6]. ROS may also be produced by the ethanol-inducible CYP2E1 enzyme [62,121], which is typically localized in microsomes but may translocate to mitochondria [122][123][124], causing mitochondrial dysfunction and fat accumulation [62,125]. ...
Melatonin (MT) has often been used to support good sleep quality, especially during the COVID-19 pandemic, as many have suffered from stress-related disrupted sleep patterns. It is less known that MT is an antioxidant, anti-inflammatory compound, and modulator of gut barrier dysfunction, which plays a significant role in many disease states. Furthermore, MT is produced at 400–500 times greater concentrations in intestinal enterochromaffin cells, supporting the role of MT in maintaining the functions of the intestines and gut–organ axes. Given this information, the focus of this article is to review the functions of MT and the molecular mechanisms by which it prevents alcohol-associated liver disease (ALD) and metabolic dysfunction-associated steatotic liver disease (MASLD), including its metabolism and interactions with mitochondria to exert its antioxidant and anti-inflammatory activities in the gut–liver axis. We detail various mechanisms by which MT acts as an antioxidant, anti-inflammatory compound, and modulator of intestinal barrier function to prevent the progression of ALD and MASLD via the gut–liver axis, with a focus on how these conditions are modeled in animal studies. Using the mechanisms of MT prevention and animal studies described, we suggest behavioral modifications and several exogenous sources of MT, including food and supplements. Further clinical research should be performed to develop the field of MT in preventing the progression of liver diseases via the gut–liver axis, so we mention a few considerations regarding MT supplementation in the context of clinical trials in order to advance this field of research.
... About 0.4-4% of oxygen is converted to superoxide via normal cell respiration, mainly in the mitochondria 33 . These ROS should normally be further reduced to water by cytochrome oxidase of complex IV to overcome their harmful effects 34,35 . Another source of hydrogen peroxide is a direct two electron reduction of oxygen by monoamine oxidase in the outer membrane of the mitochondria 34 . ...
... These ROS should normally be further reduced to water by cytochrome oxidase of complex IV to overcome their harmful effects 34,35 . Another source of hydrogen peroxide is a direct two electron reduction of oxygen by monoamine oxidase in the outer membrane of the mitochondria 34 . The hydroxyl radical is one of the main ROS produced in vivo that has attracted a lot of interest due to its high reactivity. ...
Acute kidney injury is characterised by abrupt failure of kidney function, sometimes leading to chronic kidney disease, and is associated with significant morbidity and mortality. However, there is no clear effective therapeutic solution and treatment is mainly based on either alleviation or removal of the possible cause and/or renal replacement therapy. Oxidative stress has been indicated as one of the main pathophysiological pathways in the development of acute kidney injury. Various treatments including antioxidants, inflammatory mediators and genetic modifiers have been proposed to for the treatment of this condition. Epidemiological studies show lower incidence of kidney failure with higher consumption of antioxidants. However, the data is inconclusive due to their physicochemical properties, bioavailability or toxicity. Novel drug delivery systems such as liposomes and nanoparticles have been proposed to overcome the pharmacodynamic and pharmacokinetic barriers. This review provides a brief introduction to acute kidney injury and the different factors involved in its pathology, focusing on oxidative stress. It also covers details of antioxidant use as preventive and/or treatment option. It will summarise their limitations as free drugs and the possible improvement in their bioavailability by two main novel drug delivery systems: liposomes and polymeric nanoparticles. Other therapies such as inflammatory mediators and genetic modifiers are also discussed briefly.
... 1,3,7-TMUA, a major metabolite of coffee, 13 is a potent antioxidant that is effective at scavenging hydroxyl radicals and inhibiting lipid peroxidation, 14 which are associated with immunosenescence. 15 Ferulic acid 4-sulfate, a ferulic acid (FA), is also a major coffee metabolite with antioxidant and anti-inflammatory effects. 16,17 FA supplementation can effectively delay immunosenescence. ...
Coffee, a widely consumed beverage, has shown benefits for human health but lacks sufficient basic and clinical evidence to fully understand its impacts and mechanisms. Here, we conducted a cross‐sectional observational study of coffee consumption and a 1‐month clinical trial in humans. We found that coffee consumption significantly reshaped the immune system and metabolism, including reduced levels of inflammatory factors and a reduced frequency of senescent T cells. The frequency of senescent T cells and the levels of the senescence‐associated secretory phenotype were lower in both long‐term coffee consumers and new coffee consumers than in coffee nondrinking subjects, suggesting that coffee has anti‐immunosenescence effects. Moreover, coffee consumption downregulated the activities of the The Janus kinase/signal transduction and activator of transcription (JAK/STAT) and mitogen‐activated protein kinases (MAPK) signaling pathways and reduced systemic proinflammatory cytokine levels. Mechanistically, coffee‐associated metabolites, such as 1‐methylxanthine, 3‐methylxanthine, paraxanthine, and ceramide, reduced the frequency of senescent CD4⁺CD57+ T cells in vitro. Finally, in vivo, coffee intake alleviated inflammation and immunosenescence in imiquimod‐induced psoriasis‐like mice. Our results provide novel evidence of the anti‐inflammatory and anti‐immunosenescence effects of coffee, suggesting that coffee consumption could be considered a healthy habit.
... Some studies have reported that the ingestion of H 2 O 2 could result in a dysfunction of mitochondria. 56 It has proved that vanillic acid protected against mitochondrial dysfunction by increasing complex I, complex II, and complex-III activities in quinolinic acid-induced rat model of Huntington's disease. 57 MFN2 has distinct function to promote mitochondrial fusion. ...
Oxidative stress is proposed as a regulatory element in various neurological disorders, which is involved in the progress of several neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Antioxidant drugs are widely used to alleviate neurodegenerative disorders. Astragalus membranaceus (Huangqi, AM) is a commonly used medicinal herb with a wide range of pharmacological effects. Here, the protective effect and mechanism of AM extract (AME) and its bioactive compounds against neurodegenerative disorders via alleviating oxidative stress were detected using adult Drosophila melanogaster. The drug safety was measured by development analysis; oxidative stress resistance ability was detected by survival rate under H2O2 environment; ROS level was detected by DHE staining and gstD1‐GFP fluoresence assay; antioxidative abilitiy was represent by measuring antioxidant enzyme activity, antioxidative‐related gene expression, and ATP and MFN2 levels. The neuroprotective effect was evaluated by lifespan and locomotion analysis in Aβ42 transgenic and Pink1B9 mutants. AME dramatically increased the survival rates, improved the CAT activity, restored the decreased mRNA expressions of Sod1, Cat, and CncC under H2O2 stimulation, and ameliorated the neurobehavioral defects of the AD and PD. Thirteen small molecules in AM had antioxidant function, in which vanillic acid and daidzein had the most potent antioxidant effect. Vanillic acid and daidzein could increase the activities of SOD and CAT, GSH level, and the expressions of antioxidant genes. Vanillic acid could improve the levels of ATP and MFN2, and mRNA expressions of ND42 and SDHC to rescue mitochondrial dysfunction. Furthermore, vanillic acid ameliorated neurobehavioral defects of PD. Daidzein ameliorated neurobehavioral defect of Aβ‐induced AD mode. Taken together, AM plays a protective role in oxidative damage, thereby as a potential natural drug to treat neurodegenerative disorders.
... The reduced oxidative damage may be correlated with the preserved mitochondrial respiratory enzyme activity. Studies have shown that mitochondrial ROS production exhibits an inverse relationship with oxidative phosphorylation rates [56,57]. Our results revealed a notably higher activity of Complex I within the precooling group than control group, suggesting that precooling likely mitigates the impairment of respiratory enzymes induced by IRI. ...
Transplanted organs experience several episodes of ischemia and Ischemia-reperfusion. The graft injury resulting from ischemia-reperfusion (IRI) remains a significant obstacle to the successful survival of transplanted grafts. Temperature plays an important role in cellular metabolic rates since biochemical reactions are highly temperature dependent. Therefore, ischemia-triggered degradative reactions could be mitigated by lowering temperature. Whether a local hypothermia on liver before blockage of blood flow protects liver grafts against IRI has not been investigated. In this study, we applied local hypothermia to mouse donor livers for a specific duration before stopping blood flow to liver lobes, a procedure called "liver precooling." Mouse donor liver temperature in control groups was controlled at 37ºC. Subsequently, the liver donors were preserved in cold University of Wisconsin solution for various durations followed by orthotopic liver transplantation. Liver graft injury, function and inflammation were assessed at 1- and 2-days post-transplantation. Liver precooling exhibited a significant improvement in graft function, revealing more than a 47% decrease in plasma aspartate transaminase (AST) and alanine aminotransferase (ALT) levels, coupled with a remarkable reduction of approximately 50% in liver graft histological damage compared to the control group. The protective effects of liver precooling were associated with the preservation of mitochondrial function, substantial reduction in hepatocyte cell death, and a significantly attenuated inflammatory response. A retrospective analysis of patient data revealed a close correlation between hypothermia and enhanced liver graft function. Taken together, reduction of the cellular metabolism and enzymatic activity to a minimum level before ischemia protects against IRI during transplantation.
... We and others have previously reported that mitochondrial dysfunction with oxidative stress and intermediary metabolite concentrations contributes to accelerated senescence, including post-mitotic senescence in skeletal muscle (Cadenas & Davies, 2000;Kumar et al., 2021;Mycielska et al., 2022;Welch et al., 2021;Zheng et al., 2015). Interestingly, our data suggest that Hif1a KO results in higher mitochondrial efficiency and an increase in αKG in myotubes, which protects against senescence (Asadi Shahmirzadi et al., 2020). ...
Hypoxia‐inducible factor (HIF)‐1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex‐specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post‐natal muscle‐specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography–mass spectrometry; and post‐mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1β protein expression and muscle fibre diameter were sex‐dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post‐mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. image
Key points
Hypoxia‐inducible factor ‐1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption.
In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function.
To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays.
HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post‐mitotic senescence markers, some of which were differentially impacted by sex.
The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post‐mitotic senescence during normoxia.
... When tissues, cells, and biological macromolecules are exposed to these excessive oxidants over an extended period, a series of biochemical reactions are triggered, causing oxidative damage and consequently, impairing normal cellular functions. Prolonged oxidative stress is regarded as a pivotal factor in instigating various diseases such as cardiovascular diseases (10), cancer (11), neurodegenerative diseases (12), diabetes (13), and aging (14). To prevent oxidative damage, an antioxidative system exists within the organism, consisting of antioxidative enzymes (such as superoxide dismutase and catalase) and nonenzymatic antioxidants (such as vitamin C, vitamin E, and glutathione). ...
Background
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease globally, with the potential to progress to non-alcoholic steatohepatitis (NASH), cirrhosis, and even hepatocellular carcinoma. Given the absence of effective treatments to halt its progression, novel molecular approaches to the NAFLD diagnosis and treatment are of paramount importance.
Methods
Firstly, we downloaded oxidative stress-related genes from the GeneCards database and retrieved NAFLD-related datasets from the GEO database. Using the Limma R package and WGCNA, we identified differentially expressed genes closely associated with NAFLD. In our study, we identified 31 intersection genes by analyzing the intersection among oxidative stress-related genes, NAFLD-related genes, and genes closely associated with NAFLD as identified through Weighted Gene Co-expression Network Analysis (WGCNA). In a study of 31 intersection genes between NAFLD and Oxidative Stress (OS), we identified three hub genes using three machine learning algorithms: Least Absolute Shrinkage and Selection Operator (LASSO) regression, Support Vector Machine - Recursive Feature Elimination (SVM-RFE), and RandomForest. Subsequently, a nomogram was utilized to predict the incidence of NAFLD. The CIBERSORT algorithm was employed for immune infiltration analysis, single sample Gene Set Enrichment Analysis (ssGSEA) for functional enrichment analysis, and Protein-Protein Interaction (PPI) networks to explore the relationships between the three hub genes and other intersecting genes of NAFLD and OS. The distribution of these three hub genes across six cell clusters was determined using single-cell RNA sequencing. Finally, utilizing relevant data from the Attie Lab Diabetes Database, and liver tissues from NASH mouse model, Western Blot (WB) and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) assays were conducted, this further validated the significant roles of CDKN1B and TFAM in NAFLD.
Results
In the course of this research, we identified 31 genes with a strong association with oxidative stress in NAFLD. Subsequent machine learning analysis and external validation pinpointed two genes: CDKN1B and TFAM, as demonstrating the closest correlation to oxidative stress in NAFLD.
Conclusion
This investigation found two hub genes that hold potential as novel targets for the diagnosis and treatment of NAFLD, thereby offering innovative perspectives for its clinical management.
... Superoxide and byproducts of lipid peroxidation are radicals that are strong stimulators of mitochondrial uncoupling proteins, autophagic engulfment, and signaling molecules that have important functions in differentiation, adhesion, migration, and cell survival. The superoxide level was reported to be higher in mitochondria than in the cytoplasm (Cadenas and Davies, 2000). The superoxide anion is short-lived and membrane-impermeable, bearing a strong capacity to damage lipids, proteins, and mitochondrial DNA. ...
Humans and wildlife, including domesticated animals, are exposed to a myriad of environmental contaminants that are derived from various human activities, including agricultural, household, cosmetic, pharmaceutical, and industrial products. Excessive exposure to pesticides, heavy metals, and phthalates consequently causes the overproduction of reactive oxygen species. The equilibrium between reactive oxygen species and the antioxidant system is preserved to maintain cellular redox homeostasis. Mitochondria play a key role in cellular function and cell survival. Mitochondria are vulnerable to damage that can be provoked by environmental exposures. Once the mitochondrial metabolism is damaged, it interferes with energy metabolism and eventually causes the overproduction of free radicals. Furthermore, it also perceives inflammation signals to generate an inflammatory response, which is involved in pathophysiological mechanisms. A depleted antioxidant system provokes oxidative stress that triggers inflammation and regulates epigenetic function and apoptotic events. Apart from that, these chemicals influence steroidogenesis, deteriorate sperm quality, and damage male reproductive organs. It is strongly believed that redox signaling molecules are the key regulators that mediate reproductive toxicity. This review article aims to spotlight the redox toxicology of environmental chemicals on male reproduction function and its fertility prognosis. Furthermore, we shed light on the influence of redox signaling and metabolism in modulating the response of environmental toxins to reproductive function. Additionally, we emphasize the supporting evidence from diverse cellular and animal studies.
... Oxidative stress has been thought to be one of the main contributors in the progression of AMD. An imbalance in the generation and clearance of oxygen-derived free radicals as a result of decreased activity of endogenous antioxidant enzymes or increased accumulation of reactive oxygen species (ROS) can result in oxidative stress [4,5]. Studies have revealed that oxidative stress expedites the progression of neurodegenerative disorders that cause cognitive impairment, among other symptoms [6]. ...
Purpose:
Among aging adults, age-related macular degeneration (AMD), is a prevalent cause of blindness. Nevertheless, its progression may be halted by antioxidation in retinal pigment epithelium (RPE). The primary effective constituent of Tripterygium wilfordii Hook. F., triptolide (TP), has demonstrated anti-inflammatory, antiproliferative, and antioxidant properties. The mechanics of the protective effect of triptolide against the oxidative damage in retinal pigment epithelial (RPE) were assessed in this study.
Methods:
ARPE-19 cells were pretreated with TP, and then exposed to sodium iodate (SI). First, cell viability was assessed using CCK-8. Subsequently, we measured indicators for cell oxidation including reactive oxygen species (ROS), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA). Then, we used network pharmacological analysis and molecular docking to explore the signaling pathway of TP. Last, we used western blot, ELISA, and immunofluorescence assays to clarify the potential mechanistic pathways.
Results:
The network pharmacology data suggested that TP may inhibit AMD by regulating the PI3K/Akt signaling pathway. Experimental results showed that the potential mechanism is that it regulates the PI3K/Akt pathway and promotes Nrf2 phosphorylation and activation, thereby raising the level of antioxidant factors (HO-1, NQO1) and reducing the generation of ROS, which inhibit oxidative damage.
Conclusion:
Our findings suggested that the effect of TP on SI-exposed RPE cells principally relies on the regulation of oxidative stress through the PI3K/Akt/Nrf2 signaling pathway.
... The electrons transported by NADH and FADH2 flow through the electron transport chain (ETC), which then creates an electrochemical gradient on the inner mitochondrial membrane (IMM) and ultimately synthesizes adenosine triphosphate (ATP) [36]. In general, 0.2 % to 2 % of the electrons are not correctly transferred in the ETC but spill off the chain and interact with oxygen to form ROS [37,38]. Under normal physiological conditions, the ROS generated along with ETC are cleared by enzymatic and nonenzymatic antioxidants to keep them at low levels and steady-state without causing cytotoxicity [39]. ...
Background: Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease
worldwide, whereas there is no approved drug therapy due to its complexity. Studies are emerging to discuss the role of selective autophagy in the pathogenesis of NAFLD, because the specificity among the features of selective autophagy makes it a crucial process in mitigating hepatocyte damage caused by
aberrant accumulation of dysfunctional organelles, for which no other pathway can compensate.
Aim of review: This review aims to summarize the types, functions, and dynamics of selective autophagy
that are of particular importance in the initiation and progression of NAFLD. And on this basis, the review
outlines the therapeutic strategies against NAFLD, in particular the medications and potential natural
products that can modulate selective autophagy in the pathogenesis of this disease.
Key scientific concepts of review: The critical roles of lipophagy and mitophagy in the pathogenesis of
NAFLD are well established, while reticulophagy and pexophagy are still being identified in this disease
due to the insufficient understanding of their molecular details. As gradual blockage of autophagic flux
reveals the complexity of NAFLD, studies unraveling the underlying mechanisms have made it possible
to successfully treat NAFLD with multiple pharmacological compounds that target associated pathways.
... In direct comparisons at equivalent concentrations, both glutathione and myo-inositol consistently yielded superior results compared to the other three antioxidants. Glutathione functions to suppress ROS and shield cells from oxidative stress-induced damage [64]. Its efficacy has been demonstrated across various fish sperm, including in depik fish Rasbora tawarensis [39], seurukan fish Osteochilus vittatus [43], and African catfish C. gariepinus [42]. ...
... Even when synthesized during normal respiration, they might cause cumulative damage, eventually leading to cell and tissue dysfunction and disease. Their production rises above natural antioxidant levels with progressive disease and aging [124]. Hypoxic tissues generate excessive ROS during sepsis, increasing GSH consumption [125]. ...
Acute kidney injury (AKI) is caused by a sudden loss of renal function, resulting in the build-up of waste products and a significant increase in mortality and morbidity. It is commonly diagnosed in critically ill patients, with its occurrence estimated at up to 50% in patients hospitalized in the intensive critical unit. Despite ongoing efforts, the death rate associated with AKI has remained high over the past half-century. Thus, it is critical to investigate novel therapy options for preventing the epidemic. Many studies have found that inflammation and Toll-like receptor-4 (TLR-4) activation have a significant role in the pathogenesis of AKI. Noteworthy, challenges in the search for efficient pharmacological therapy for AKI have arisen due to the multifaceted origin and complexity of the clinical history of people with the disease. This article focuses on kidney injury's epidemiology, risk factors, and pathophysiological processes. Specifically, it focuses on the role of TLRs especially type 4 in disease development.
... On day 14 of the organotypic hippocampal slice culture, the substance treatment was conducted at 37 • C and 5% CO 2 for the duration of 24 h (Figure 4). For induction of oxidative stress, slices were incubated with 100 µM H 2 O 2 (positive control condition; Sigma-Aldrich, St. Louis, MO, USA) [87]. The selection of this specific concentration was based on previous ex vivo studies on mitochondrial dynamics [47][48][49]88], while the time of incubation was adapted to our long-term treatment approach. ...
In multiple sclerosis (MS), mitochondrial alterations appear to contribute to disease progression. The sphingosine-1-phosphate receptor modulator siponimod is approved for treating secondary progressive MS. Its preceding compound fingolimod was shown to prevent oxidative stress-induced alterations in mitochondrial morphology. Here, we assessed the effects of siponimod, compared to fingolimod, on neuronal mitochondria in oxidatively stressed hippocampal slices. We have also advanced the model of chronic organotypic hippocampal slices for live imaging, enabling semi-automated monitoring of mitochondrial alterations. The slices were prepared from B6.Cg-Tg(Thy1-CFP/COX8A)S2Lich/J mice that display fluorescent neuronal mitochondria. They were treated with hydrogen peroxide (oxidative stress paradigm) ± 1 nM siponimod or fingolimod for 24 h. Afterwards, mitochondrial dynamics were investigated. Under oxidative stress, the fraction of motile mitochondria decreased and mitochondria were shorter, smaller, and covered smaller distances. Siponimod partly prevented oxidatively induced alterations in mitochondrial morphology; for fingolimod, a similar trend was observed. Siponimod reduced the decrease in mitochondrial track displacement, while both compounds significantly increased track speed and preserved motility. The novel established imaging and analysis tools are suitable for assessing the dynamics of neuronal mitochondria ex vivo. Using these approaches, we showed that siponimod at 1 nM partially prevented oxidatively induced mitochondrial alterations in chronic brain slices.
... το ανιόν του υπεροξειδίου του οξυγόνου (Ο -), που προκύπτει από διαφεύγοντα ηλεκτρόνια της οξειδωτικής φωσφορυλίωσης τα οποία ενώνονται με μοριακό οξυγόνο, ή το υπεροξείδιο υδρογόνου (Η Ο ), 12 που προκύπτει είτε από τη δράση της NADPH-οξειδάσης στα φαγοσώματα των έξω τμημάτων των φωτοϋποδοχέων, είτε από την οξείδωση πεφθέντων λιπιδίων των έξω τμημάτων των φωτοϋποδοχέων και τη δημιουργία καρβοξυαιθυλπυρρόλης (carboxyethylpyrrole, CEP). 17 Οξειδωτική δράση και δημιουργία δραστικών μορφών οξυγόνου προάγουν και εξωγενείς παράγοντες, όπως η ηλιακή ακτινοβολία κυρίως με το κυανό μήκος κύματος και τη δράση της επί των φωτοϋποδοχέων, καθώς και το κάπνισμα. Δραστικές μορφές οξυγόνου δημιουργούνται επίσης από την υπορρύθμιση διαφόρων αντιοξειδωτικών ενζύμων του τύπου της καταλάσης. ...
Η ηλικιακή εκφύλιση της ωχράς κηλίδας (ΗΕΩ) είναι μια προοδευτική εκφυλιστική νόσος του οφθαλμού. Προσβάλλει πρωτοπαθώς το μελάγχρουν επιθήλιο και δευτερευόντως τους φωτοϋποδοχείς. Περιλαμβάνει δύο κλινικές μορφές, την ξηρά και τη νεοαγγειακή μορφή, οι οποίες στο τελικό στάδιο μπορούν να επιφέρουν μερική ή ολική (νομική) τύφλωση. Η αιτιοπαθογένεση είναι πολύπλοκη, πολυπαραγοντική και ατελώς γνωστή, και εμπλέκει τη γενετική ευαισθησία καθώς και την επίδραση διαφόρων κλινικών, περιβαλλοντικών και μη περιβαλλοντικών παραγόντων. Στην πορεία εξέλιξης της ΗΕΩ στον εξώτερο αμφιβληστροειδή λαμβάνουν χώρα τέσσερα ανατομο-παθολογικά γεγονότα: η λιποφουσκινογένεση, η γένεση των drusen, η χρόνια φλεγμονή, και η νεοαγγείωση μόνο στη νεοαγγειακή μορφή. Η υψηλότερη συχνότητα της ΗΕΩ παρουσιάζεται στις ηλικίες ≥80 ετών, με επιπολασμό από 8,8–12,33%, ενώ υπάρχει τάση αύξησής της στις ηλικίες από 55 ετών και άνω. Η θεραπεία της νόσου αφορά κυρίως στη νεοαγγειακή μορφή. Αναμένεται σημαντικό κοινωνικό και οικονομικό κόστος στα υγειονομικά συστήματα λόγω της αύξησης του προσδόκιμου επιβίωσης στις επόμενες δεκαετίες.
... Along with ATP production, mitochondria continuously generate ROS from the electron transport chain (ETC) that are physiologically important under normal conditions [21]. The redox homeostasis of mitochondria is maintained by an antioxidant system that includes superoxide dismutase 1 (SOD1) in the intermembrane space and SOD2 in the matrix. ...
Pulmonary hypertension (PH) resulting from chronic hypoxia (CH) occurs in patients with chronic obstructive pulmonary diseases, sleep apnea, and restrictive lung diseases, as well as in residents at high altitude. Previous studies from our group and others demonstrate a detrimental role of reactive oxygen species (ROS) in the pathogenesis of CH-induced PH, although the subcellular sources of ROS are not fully understood. We hypothesized that mitochondria-derived ROS (mtROS) contribute to enhanced vasoconstrictor reactivity and PH following CH. To test the hypothesis, we exposed rats to 4 weeks of hypobaric hypoxia (PB ≈ 380 mmHg), with control rats housed in ambient air (PB ≈ 630 mmHg). Chronic oral administration of the mitochondria-targeted antioxidant MitoQ attenuated CH-induced decreases in pulmonary artery (PA) acceleration time, increases in right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary arterial remodeling. In addition, endothelium-intact PAs from CH rats exhibited a significantly greater basal tone compared to those from control animals, as was eliminated via MitoQ. CH also augmented the basal tone in endothelium-disrupted PAs, a response associated with increased mtROS production in primary PA smooth muscle cells (PASMCs) from CH rats. However, we further uncovered an effect of NO synthase inhibition with Nω–nitro-L-arginine (L-NNA) to unmask a potent endothelial vasoconstrictor influence that accentuates mtROS-dependent vasoconstriction following CH. This basal tone augmentation in the presence of L-NNA disappeared following combined endothelin A and B receptor blockade with BQ123 and BQ788. The effects of using CH to augment vasoconstriction and PASMC mtROS production in exogenous endothelin 1 (ET-1) were similarly prevented by MitoQ. We conclude that mtROS participate in the development of CH-induced PH. Furthermore, mtROS signaling in PASMCs is centrally involved in enhanced pulmonary arterial constriction following CH, a response potentiated by endogenous ET-1.
The provided texts emphasize the critical importance of glutathione as a super antioxidant, highlighting its essential role in maintaining cellular health, supporting the immune system, and protecting against oxidative stress. These texts collectively contribute to the literature by:
Detoxification and Immune Support: Detailing how glutathione aids in detoxifying harmful substances and bolstering the immune system, thereby preventing various diseases.
Oxidative Stress and Disease Prevention: Explaining how glutathione neutralizes free radicals and reactive oxygen species (ROS), reducing oxidative stress and preventing cellular damage, which is linked to numerous chronic conditions including infertility, eczema, ADHD, and accelerated aging.
Supplementation Methods: Comparing different glutathione supplementation methods—oral, liposomal, intravenous, and transdermal—highlighting the superior benefits of transdermal delivery due to its sustained release and high bioavailability.
Genetic Variations and Health Implications: Discussing the impact of genetic variations in glutathione-related genes (e.g., GPX1, GSTM1) on the body's antioxidant capacity and the increased need for glutathione in such cases.
Broad Health Impact: Illustrating the wide-ranging health impacts of glutathione deficiency, from exacerbating conditions like sickle cell disease and osteoporosis to playing a role in the pathophysiology of neurodevelopmental disorders like ADHD.
This comprehensive approach not only underscores the multifaceted benefits of glutathione but also advocates for greater awareness and optimal supplementation practices to leverage its full health potential.
Human manganese superoxide dismutase (MnSOD) plays a crucial role in controlling levels of reactive oxygen species (ROS) by converting superoxide (O 2 ●− ) to molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ) with proton-coupled electron transfers (PCETs). The reactivity of human MnSOD is determined by the state of a key catalytic residue, Tyr34, that becomes post-translationally inactivated by nitration in various diseases associated with mitochondrial dysfunction. We previously reported that Tyr34 has an unusual pK a due to its proximity to the Mn metal and undergoes cyclic deprotonation and protonation events to promote the electron transfers of MnSOD. To shed light on the role of Tyr34 MnSOD catalysis, we performed neutron diffraction, X-ray spectroscopy, and quantum chemistry calculations of Tyr34Phe MnSOD in various enzymatic states. The data identifies the contributions of Tyr34 in MnSOD activity that support mitochondrial function and presents a thorough characterization of how a single tyrosine modulates PCET catalysis.
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystem disabling disease with unclear etiology and pathophysiology, whose typical symptoms include prolonged debilitating recovery from fatigue or postexertional malaise (PEM). Disrupted production of adenosine triphosphate (ATP), the intracellular energy that fuels cellular activity, is a cause for fatigue. Here, we present a long-term case of ME/CFS: a 75-year-old Caucasian female patient, whose symptoms of ME/CFS were clearly triggered by an acute infection of the Epstein–Barr virus 24 years ago (mononucleosis). Before then, the patient was a healthy professional woman. A recent DNA sequence analysis identified missense variants of mitochondrial respiratory chain enzymes, including ATP6 (ChrMT: 8981A > G; Q152R) and Cox1 (ChrMT: 6268C > T; A122V). Protein subunits ATP6 and Cox1 are encoded by mitochondrial DNA outside of the nucleus: the Cox1 gene encodes subunit 1 of complex IV (CIV: cytochrome c oxidase) and the ATP6 gene encodes subunit A of complex V (CV: ATP synthase). CIV and CV are the last two of five essential enzymes that perform the mitochondrial electron transport respiratory chain reaction to generate ATP. Further analysis of the blood sample using transmission electron microscopy demonstrated abnormal, circulating, extracellular mitochondria. These results indicate that the patient had dysfunctional mitochondria, which may contribute directly to her major symptoms, including PEM and neurological and cognitive changes. Furthermore, the identified variants of ATP6 (ChrMT: 8981A > G; Q152R) and Cox1 (ChrMT: 6268C > T; A122V), functioning at a later stage of mitochondrial ATP production, may play a role in the abnormality of the patient’s mitochondria and the development of her ME/CFS symptoms.
Recent advancements in neuroendocrinology challenge the long‐held belief that hormonal effects are confined to perivascular tissues and do not extend to the central nervous system (CNS). This paradigm shift, propelled by groundbreaking research, reveals that synthetic hormones, notably in anti‐inflammatory medications, significantly influence steroid psychosis, behavioural, and cognitive impairments, as well as neuropeptide functions. A seminal development in this field occurred in 1968 with McEven's proposal that rodent brains are responsive to glucocorticoids, fundamentally altering the understanding of how anxiety impacts CNS functionality and leading to the identification of glucocorticosteroids and mineralocorticoids as distinct corticotropic receptors. This paper focuses on the intricate roles of the neuroendocrine, immunological, and CNS in fostering stress resilience, underscored by recent animal model studies. These studies highlight active, compensatory, and passive strategies for resilience, supporting the concept that anxiety and depression are systemic disorders involving dysregulation across both peripheral and central systems. Resilience is conceptualized as a multifaceted process that enhances psychological adaptability to stress through adaptive mechanisms within the immunological system, brain, hypothalamo–pituitary–adrenal axis, and ANS Axis. Furthermore, the paper explores oxidative stress, particularly its origin from the production of reactive oxygen species (ROS) in mitochondria. The mitochondria's role extends beyond ATP production, encompassing lipid, heme, purine, and steroidogenesis synthesis. ROS‐induced damage to biomolecules can lead to significant mitochondrial dysfunction and cell apoptosis, emphasizing the critical nature of mitochondrial health in overall cellular function and stress resilience. This comprehensive synthesis of neuroendocrinological and cellular biological research offers new insights into the systemic complexity of stress‐related disorders and the imperative for multidisciplinary approaches in their study and treatment.
Background
Increasing evidence suggests an association between mitochondrial function and ischemic stroke (IS). However, whether this association might be causal or explained by reverse causal association/residual confounding is unclear. Therefore, we designed this study to evaluate the causal association of mitochondrial function with IS risk.
Methods
Mitochondrial proteins were considered the exposure factor, and IS was considered the outcome variable. Exposures and outcomes were obtained from the IEU Open GWAS database. First, we obtained 66 mitochondrial protein genome-wide association studies data sets from European populations, as well as IS data. We then performed two-sample Mendelian randomization (MR) analysis to determine associations between mitochondrial proteins and IS. We additionally performed bidirectional MR analysis to examine the directions of the causal associations.
Results
IVW indicated that three mitochondrial proteins were associated with IS: ribosome-recycling factor (mtRRF) was negatively associated with IS [OR = 0.93, 95%CI (0.88–0.98), P = 0.005]; malonyl-CoA decarboxylase (MLYCD) was negatively associated with IS [OR = 0.89, 95%CI (0.82–0.97), P = 0.005]; and mitochondrial Lon protease homolog (LONP1) was positively associated with IS [OR = 1.06, 95%CI (1.02–1.10), P = 0.004]. Sensitivity analysis indicated no evidence of reverse causality, pleiotropy, or heterogeneity, thus suggesting that MR was an effective method for causal inference in this study.
Conclusion
Our MR analysis indicated that three mitochondrial proteins are causally associated with IS, and may aid in early detection and prevention of IS at the microscopic molecular level. Our findings provide new insights into IS microscopic mechanisms and clinical research.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are highly reactive molecules produced naturally by the body and by external factors. When these species are generated in excessive amounts, they can lead to oxidative stress, which in turn can cause cellular and tissue damage. This damage is known to contribute to the aging process and is associated with age-related conditions, including cardiovascular and neurodegenerative diseases. In recent years, there has been an increased interest in the development of compounds with antioxidant potential to assist in the treatment of disorders related to oxidative stress. In this way, compounds containing sulfur (S) and/or selenium (Se) have been considered promising due to the relevant role of these elements in the biosynthesis of antioxidant enzymes and essential proteins with physiological functions. In this context, studies involving heterocyclic nuclei have significantly increased, notably highlighting the indolizine nucleus, given that compounds containing this nucleus have been demonstrating considerable pharmacological properties. Thus, the objective of this research was to evaluate the in vitro antioxidant activity of eight S- and Se-derivatives containing indolizine nucleus and different substituents. The in vitro assays 1,1-diphenyl-2-picryl-hydrazil (DPPH) scavenger activity, ferric ion (Fe ³⁺ ) reducing antioxidant power (FRAP), thiobarbituric acid reactive species (TBARS), and protein carbonylation (PC) were used to access the antioxidant profile of the compounds. Our findings demonstrated that all the compounds showed FRAP activity and reduced the levels of TBARS and PC in mouse brains homogenates. Some compounds were also capable of acting as DPPH scavengers. In conclusion, the present study demonstrated that eight novel organochalcogen compounds exhibit antioxidant activity.
Stress oxy hóa là tình trạng mất cân bằng giữa gốc tự do hay gốc có oxy hoạt động và chất chống oxy hóa. Cơ thể cần một số lượng gốc tự do, gốc có oxy hoạt động cho hoạt động bình thường. Cân bằng giữa sinh và loại bỏ gốc có oxy hoạt động được duy trì bởi chất chống oxy hóa. Chất chống oxy hóa có tác dụng làm chậm hay ức chế hiện tượng oxy hóa. Nguyên nhân chính của stress oxy hóa là nguyên nhân ngoại sinh, như chế độ dinh dưỡng, rượu, stress, môi trường ô nhiễm, khói thuốc, tia xạ, thuốc điều trị, vận động thể lực quá mức hay không đủ. Stress oxy mạn tính tác động tới phân tử sinh học, như peroxide lipid, oxid hóa protein, bất hoạt enzyme, biến đổi DNA, rối loan điều hòa oxy hóa khử, làm tổn thương tế bào, mô, dẫn đến viêm nhiễm, nhiều bệnh lý và lão hóa. Stress oxy hóa liên quan tới bệnh sinh nhiều bệnh, như ,bệnh thoái hóa thần kinh, ung thư, bệnh tim mạch, bệnh xương-cơ- khớp, rối loan chuyển hóa, viêm nhiễm mạn tính, bệnh da, bệnh mắt, ở người lớn và trẻ em. Biểu hiện lâm sàng stress oxy hóa đang xảy ra không đặc hiệu, như mỏi mệt, giảm trí nhớ, đau đầu, đau mỏi cơ, khớp, mắt nhìn mờ, hình thành nếp nhăn và bạc tóc. Để phát hiện stress oxy hóa phải dựa vào một số xét nghiệm, như đo lường tiêu thụ oxy, tìm các dấu ấn oxy hóa, phát hiện gốc tự do, và thử nghiệm chất chống oxy hóa. Tránh oxy hóa không cần thiết và tăng cường chất chống oxy hóa đã trở thành chiến lược cho điều trị chống lại tổn thương oxy hóa và dự phòng stress oxy hóa.
Aging is considered part of the natural process of life, however in recent years medical literature has started to show that specific facets of aging are beginning to be understood and those factors may even be considered preventable with various measures.
Aging is also considered the number one cause of poor quality of life, disease, disability, and death, so the importance of understanding the aging process and how to control certain aspects of it cannot be underestimated when age related suffering is factored in. The causes of aging are now becoming well understood, and in recent years many therapies have already become available to the public to attenuate specific corridors of aging. The heterogeneity of the aging process and the biological drivers involved is examined here in parallel with various compounds and therapies to combat biological decline.
The benefits for governments in keeping their populations healthy and vibrant are vast, and at the same time offer a great incentive to invest into newly emerging technologies that may prevent the onset of preventable disease.
Whilst this paper only discusses nine pathways to the aging process, many more exist.
Reactive oxygen species (ROS) are associated with various pathological conditions, including atherosclerosis and cancer. Photoaging, mainly caused by UVB-induced ROS, accelerates skin aging and collagen degradation. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates antioxidant enzymes and has demonstrated protective effects against chronic diseases. Jeju lava seawater (JLS), which is rich in minerals, is attracting attention for its health benefits. The current study investigates the antioxidant properties of JLS in human dermal fibroblasts (HDFs). experiments were conducted by culturing HDFs in JLS with different water hardness levels and irradiating UVB. The results show that JLS does not affect HDF viability, especially at high water hardness. JLS treatment enhances collagen production and upregulates Nrf2 and antioxidant enzymes such as NQO1 and HO-1. This mechanism involves the translocation of Nrf2 to the cell nucleus. JLS shows promise as an antioxidant, potentially mitigating the effects of oxidative stress and promoting collagen synthesis.
Ageing is an inevitable process that increases the probability of chronic disease that involves an evolving decline in organism function over time. The length of human life is closely related to ageing, which can cause a deterioration of physiological functions and lead to the development of various chronic diseases. Many theories have been put forward to illustrate the ageing mechanisms, with the free oxidative stress theory being the most well-known. Aging is caused by the excessive accumulation of cells or tissues related to oxidative damage caused by ROS. As everyone knows that oxidative stress is closely related to aging, antioxidants may be potentially valuable in the treatment of senescence disease. Hydrogen (H2) is a colorless, tasteless small molecule that plays a major role in eliminating harmful free radicals and reducing oxidative damage in vivo, such as anti-oxidation, anti-inflammation, and anti-apoptosis. Additionally, H2 can be utilized to prevent and cure several aging-related diseases, including cancer, Alzheimer’s disease, and gastrointestinal diseases. Understanding the aging process is crucial for comprehending how to slow the aging process and the development of aging-related diseases. This chapter summarized the preventive and treatment applications of molecular H2 in anti-ageing and underlying mechanisms in aging-related diseases.
Human manganese superoxide dismutase (MnSOD) is a crucial oxidoreductase that maintains the vitality of mitochondria by converting O 2 •− to O 2 and H 2 O 2 with proton-coupled electron transfers (PCETs). Since changes in mitochondrial H 2 O 2 concentrations are capable of stimulating apoptotic signaling pathways, human MnSOD has evolutionarily gained the ability to be highly inhibited by its own product, H 2 O 2 . A separate set of PCETs is thought to regulate product inhibition, though mechanisms of PCETs are typically unknown due to difficulties in detecting the protonation states of specific residues that coincide with the electronic state of the redox center. To shed light on the underlying mechanism, we combined neutron diffraction and X-ray absorption spectroscopy of the product-bound, trivalent, and divalent states to reveal the all-atom structures and electronic configuration of the metal. The data identifies the product-inhibited complex for the first time and a PCET mechanism of inhibition is constructed.
The present study investigated the potential role of Bacillus subtilis as probiotic in striped catfish ( Pangasius hypophthalmus ). Fish (initial weight = 150.00±2.63g n = 180) were stocked in circular tanks. Four isonitrogenous (30%) and isolipidic (3.29%) diets were formulated having supplementation of B . subtilis at four different levels (P0; 0, P1: 1×10 ⁶ , P2: 1×10 ⁸ and P3: 1×10 ¹⁰ CFU/g). Each treatment had three replicates, while each replicate had fifteen fish. The trial started on second week of July and continued for eight weeks. Growth, feed conversion ratio, crude protein content, the concentration of amylase and protease, the profile of both dispensable and non-dispensable amino acids in all four dietary groups increased with a gradual increase of B . subtilis in the diet. At the end of growth experiment, fish in all four groups were exposed to Staphylococcus aureus (5×10 ⁵ CFU/ml). After S . aureus challenge, fish fed with B . subtilis responded better to damage caused by reactive oxygen species and lipid peroxidation and better survival rate. The catalase and superoxide dismutase level also increased in response to bacterial challenge in B . subtilis fed groups. On the other hand, the concentration of malondialdehyde gradually decreased in these groups (+ve P0 >P1>P2>P3). It is concluded that supplementation of B . subtilis as a probiotic improved the growth, protein content, antioxidant response and immunocompetency against S . aureus in striped catfish. The optimum dosage of B . subtilis , at a concentration of 1×10 ¹⁰ CFU/g, resulted in the most favorable outcomes in striped catfish. This single bacterial strain can be used as an effective probiotic in large scale production of aquafeed for striped catfish. Future studies can investigate this probiotic’s impact in the intensive culture of the same species.
Human manganese superoxide dismutase (MnSOD) is a crucial oxidoreductase that maintains the vitality of mitochondria by converting O 2 ·– to O 2 and H 2 O 2 with proton-coupled electron transfers (PCETs). Since changes in mitochondrial H 2 O 2 concentrations are capable of stimulating apoptotic signaling pathways, human MnSOD has evolutionarily gained the ability to be highly inhibited by its own product, H 2 O 2 . A separate set of PCETs is thought to regulate product inhibition, though mechanisms of PCETs are typically unknown due to difficulties in detecting the protonation states of specific residues that coincide with the electronic state of the redox center. To shed light on the underlying mechanism, we combined neutron diffraction and X-ray absorption spectroscopy of the product-bound, trivalent, and divalent states to reveal the all-atom structures and electronic configuration of the metal. The data identifies the product-inhibited complex for the first time and a PCET mechanism of inhibition is constructed.
Excessive reactive oxygen species production can detrimentally impact skin cell physiology, resulting in cell growth arrest, melanogenesis, and aging. Recent clinical studies have found that lactic acid bacteria have a special effect directly or indirectly on skin organs, but the exact mechanism has not been elucidated. In this study, we investigated the mechanisms underlying the antioxidant protective effect and the inhibitory effect on melanin synthesis of Lactobacillus kunkeei culture supernatant (CSK), isolated from Apis mellifera Linnaeus (the Western honeybee). CSK exhibited notable efficacy in promoting cell migration and wound healing under oxidative stress, surpassing the performance of other strains. CSK pretreatment significantly upregulated the expression of Nrf2/HO-1 (nuclear factor erythroid 2-related factor 2/heme oxygenase-1), a key player in cellular defenses against oxidative stress, relative to the control H2O2-treated cells. The DCF-DA (dichloro-dihydro-fluorescein diacetate) assay results confirmed that CSK’s ability to enhance Nrf2 and HO-1 expression aligns with its robust ability to remove H2O2-induced reactive oxygen species. Furthermore, CSK upregulated MAPK (mitogen-activated protein kinase) phosphorylation, an upstream signal for HO-1 expression, and MAPK inhibitors compromised the wound-healing effect of CSK. Additionally, CSK exhibited inhibitory effects on melanin synthesis, downregulating melanogenesis-related genes in B16F10 cells. Thus, the present study demonstrated that CSK exhibited antioxidant effects by activating the Nrf2/HO-1 pathway through MAPK phosphorylation, thereby restoring cell migration and demonstrating inhibitory effects on melanin production. These findings emphasize the antioxidant and antimelanogenic potential of CSK, suggesting its potential use as a therapeutic agent, promoting wound healing, and as an active ingredient in skin-lightening cosmetics.
Mitochondria serve as essential organelles that play a key role in regulating stem cell fate. Mitochondrial dysfunction and stem cell exhaustion are two of the nine distinct hallmarks of aging. Emerging research suggests that epigenetic modification of mitochondria-encoded genes and the regulation of epigenetics by mitochondrial metabolites have an impact on stem cell aging or differentiation. Here, we review how key mitochondrial metabolites and behaviors regulate stem cell fate through an epigenetic approach. Gaining insight into how mitochondria regulate stem cell fate will help us manufacture and preserve clinical-grade stem cells under strict quality control standards, contributing to the development of aging-associated organ dysfunction and disease.
Salivary glands are indirectly damaged during radiotherapy for head and neck cancer, resulting in acute and chronic hyposalivation. Current treatments for radiation-induced hyposalivation do not permanently restore function to the gland; therefore, more mechanistic understanding of the damage response is needed to identify therapeutic targets for lasting restoration. Energy metabolism reprogramming has been observed in cancer and wound healing models to provide necessary fuel for cell proliferation; however, there is limited understanding of alterations in energy metabolism reprogramming in tissues that fail to heal. We measured extracellular acidification and oxygen consumption rates, assessed mitochondrial DNA copy number, and tested fuel dependency of irradiated primary salivary acinar cells. Radiation treatment leads to increases in glycolytic flux, oxidative phosphorylation, and ATP production rate at acute and intermediate time points. In contrast, at chronic radiation time points there is a significant decrease in glycolytic flux, oxidative phosphorylation, and ATP production rate. Irradiated salivary glands exhibit significant decreases in spare respiratory capacity and increases in mitochondrial DNA copy number at days 5 and 30 post-treatment, suggesting a mitochondrial dysfunction phenotype. These results elucidate kinetic changes in energy metabolism reprogramming of irradiated salivary glands that may underscore the chronic loss of function phenotype.
Purpose:
Diabetic retinopathy (DR) is one of the most common reasons for blindness. uncoupling protein 2 (UCP2), an uncoupling protein located in mitochondria, has been reported to be related to metabolic and vascular diseases. This research aimed to illustrate the function and mechanism of UCP2 in the pathogenesis of DR.
Methods:
Human epiretinal membranes were collected to investigate the expression of UCP2 by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence. Primary human retinal microvascular endothelial cells (HRECs) were cultured in high glucose (HG) to establish an in vitro cell model for DR. Flow cytometry analysis was used to measure intracellular reactive oxygen species (ROS). Senescence levels were evaluated by the senescence-associated beta-galactosidase (SA-β-gal) assay, the expression of senescence marker P21, and cell-cycle analysis. Adenovirus-mediated UCP2 overexpression or knockdown and specific inhibitors were administered to investigate the underlying regulatory mechanism.
Results:
Proliferative fibrovascular membranes from patients with DR illustrated the downregulation of UCP2 and sirtuin 3 (SIRT3) by qRT-PCR and immunofluorescence. Persistent hyperglycemia-induced UCP2 downregulation in the progress of DR and adenovirus-mediated UCP2 overexpression protected endothelial cells from hyperglycemia-induced oxidative stress and senescence. Under hyperglycemic conditions, UCP2 overexpression attenuated NAD+ downregulation; hence, it promoted the expression and activity of SIRT3, an NAD+-dependent deacetylase regulating mitochondrial function. 3-TYP, a selective SIRT3 inhibitor, abolished the UCP2-mediated protective effect against oxidative stress and senescence.
Conclusions:
UCP2 overexpression relieved oxidative stress and senescence based on a novel mechanism whereby UCP2 can regulate the NAD+-SIRT3 axis. Targeting oxidative stress and senescence amelioration, UCP2-SIRT3 signaling may serve as a method for the prevention and treatment of DR and other diabetic vascular diseases.
Reactive oxygen species (ROS) are closely related to oxidative stress, aging, and the onset of human diseases. To mitigate ROS-induced damages, extensive research has focused on examining the antioxidative attributes of various synthetic/natural substances. Coordination compounds serving as synthetic antioxidants have emerged as a promising approach to attenuate ROS toxicity. Herein, we investigated the antioxidant potential of a series of Fe(III) (1), Mn(III)Mn(II) (2) and Cu(II) (3) coordination compounds synthesized with the ligand N-(2-hydroxybenzyl)-N-(2-pyridylmethyl)[(3-chloro)(2-hydroxy)]-propylamine in Saccharomyces cerevisiae exposed to oxidative stress. We also assessed the antioxidant potential of these complexes in the alternative model of study, Galleria mellonella. DPPH analysis indicated that these complexes presented moderate antioxidant activity. However, treating Saccharomyces cerevisiae with 1, 2 and 3 increased the tolerance against oxidative stress and extended yeast lifespan. The treatment of yeast cells with these complexes decreased lipid peroxidation and catalase activity in stressed cells, whilst no change in SOD activity was observed. Moreover, these complexes induced the Hsp104 expression. In G. mellonella, complex administration extended larval survival under H2O2 stress and did not affect the insect's life cycle. Our results suggest that the antioxidant potential exhibited by these complexes could be further explored to mitigate various oxidative stress-related disorders.
An earlier commentary (Wardman P, Radiat Res. 2020; 194:607-617) discussed possible chemical reaction pathways that might be involved in the differential responses of tissues to high- vs. low-dose-rate irradiation, focusing on reactions between radicals, and radiolytic depletion of a chemical influencing radiosensitivity. This brief postscript updates discussion to consider recent modeling and experimental studies, and presents more detail to support the earlier suggestion that rapid depletion of nitric oxide will certainly occur after a radiation pulse of a few grays, underlining the need to include the consequences of such a change when considering FLASH effects.
Salivary glands are indirectly damaged during radiotherapy for head and neck cancer, resulting in acute and chronic hyposalivation. Current treatments for radiation-induced hyposalivation do not permanently restore function to the gland; therefore, more mechanistic understanding of the damage response is needed to identify therapeutic targets for lasting restoration. Energy metabolism reprogramming has been observed in cancer and wound healing models to provide necessary fuel for cell proliferation; however, there is limited understanding of alterations in energy metabolism reprogramming in tissues that fail to heal. We measured extracellular acidification and oxygen consumption rates, assessed mitochondrial DNA copy number, and tested fuel dependency of irradiated primary salivary acinar cells. Radiation treatment leads to increases in glycolytic flux, oxidative phosphorylation, and ATP production rate at acute and intermediate time points. In contrast, at chronic radiation time points there is a significant decrease in glycolytic flux, oxidative phosphorylation, and ATP production rate. Irradiated salivary glands exhibit significant decreases in spare respiratory capacity and increases in mitochondrial DNA copy number at days 5 and 30 post-treatment, suggesting a mitochondrial dysfunction phenotype. These results elucidate kinetic changes in energy metabolism reprogramming of irradiated salivary glands that may underscore the chronic loss of function phenotype.
We report a two- to three-fold increase in free radical (R•) concentrations of muscle and liver following exercise to exhaustion. Exhaustive exercise also resulted in decreased mitochondrial respiratory control, loss of sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) integrity, and increased levels of lipid peroxidation products. Free radical concentrations, lipid peroxidation, and SR, ER, and mitochondrial damage were similar in exercise exhausted control animals and non-exercised vitamin E deficient animals, suggesting the possibility of a common R• dependent damage process. In agreement with previous work showing that exercise endurance capacity is largely determined by the functional mitochondrial content of muscle (1–4), vitamin E deficient animals endurance was 40% lower than that of controls. The results suggest that R• induced damage may provide a stimulus to the mitochondrial biogenesis which results from endurance training.
The reversible inhibitory effects of nitric oxide (·NO) on mitochondrial cytochrome oxidase and O2uptake are dependent on intramitochondrial ·NO utilization. This study was aimed at establishing the mitochondrial pathways for ·NO utilization that regulate O⨪2 generation via reductive and oxidative reactions involving ubiquinol oxidation and peroxynitrite (ONOO–) formation. For this purpose, experimental models consisting of intact mitochondria, ubiquinone-depleted/reconstituted submitochondrial particles, and ONOO–-supplemented mitochondrial membranes were used.
The results obtained from these experimental approaches strongly suggest the occurrence of independent pathways for ·NO utilization in mitochondria, which effectively compete with the binding of ·NO to cytochrome oxidase, thereby releasing this inhibition and restoring O2 uptake. The pathways for ·NO utilization are discussed in terms of the steady-state levels of ·NO and O⨪2 and estimated as a function of O2 tension. These calculations indicate that mitochondrial ·NO decays primarily by pathways involving ONOO– formation and ubiquinol oxidation and, secondarily, by reversible binding to cytochrome oxidase.
The rapid inactivation of aconitase by O2-, previously seen to occur in vitro, was explored in vivo. A fraction of the aconitase in growing, aerobic, Escherichia coli is inactive at any instant but can be activated by imposition of anaerobic conditions. This reactivation occurred in the absence of protein synthesis and was inhibited by the ferrous chelator alpha,alpha'-dipyridyl. This fraction of inactive, but activatable, aconitase was increased by augmenting O2- production with paraquat, decreased by elevation of superoxide dismutase, and increased by inhibiting reactivation with alpha,alpha'-dipyridyl. The balance between inactive and active aconitase thus represented a pseudoequilibrium between inactivation by O2- and reactivation by restoration of Fe(II), and it provided, for the first time, a measure of the steady-state concentration of O2- within E. coli. On this basis, [O2-] was estimated to be approximately 20-40 pM in aerobic log phase E. coli containing wild type levels of superoxide dismutase and approximately 300 pM in a mutant strain lacking superoxide dismutase.
The presence of heme-containing catalase in rat heart mitochondria (20 +/- 5 units/mg) was demonstrated by biochemical and immunocytochemical analysis. Intact rat heart mitochondria efficiently consumed exogenously added H2O2. The rate of H2O2 consumption was not influenced by succinate, glutamate/malate, or N-ethylmaleimide but was significantly inhibited by cyanide. Hydrogen peroxide decomposition by mitochondria yielded molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism. Mitochondrial fractionation studies and quantitative electron microscopic immunocytochemistry revealed that most catalase was matrix-associated. Electrophoretic analysis and Western blotting of the mitochondrial matrix fraction indicated the presence of a protein with similar electrophoretic mobility to bovine and rat liver catalase and immunoreactive to anti-catalase antibody. Myocardial tissue has a lower catalase-specific activity and a greater mitochondrial H2O2 production/g of tissue than most organs. Thus catalase, representing 0.025% of heart mitochondrial protein, is important for detoxifying mitochondrial derived H2O2 and represents a key antioxidant defense mechanism for myocardial tissue.
Superoxide production has been measured in subcellular fractions of SOD-deficient Escherichia coli provided with physiological reductants. Although cytosolic enzyme(s) do generate O2-., the larger portion is produced by autoxidation of components of the respiratory electron-transport chain. At 37 degrees C and with pO2, NADH, and NAD+ levels matching those in vivo, respiring membrane vesicles generate 3 O2-./10,000 electrons transferred. This corresponds to intracellular O2-. production, in glucose-fed cells, of 5 microM/s. The high SOD content of normal cells restricts O2-. accumulation to 2.10(-10) M, with a moderate gradient from the membrane to the center of the cell. SOD-deficient mutants achieve a much higher steady-state content of O2-.. Rates of superoxide-mediated inactivation of certain enzymes are sufficiently rapid that even 10(-10) M O2-. imposes a significant oxidative stress.
In the accompanying paper (Davies, K. J. A., and Doroshow, J. A. (1986) J. Biol. Chem. 261, 3060-3067), we have demonstrated that anthracycline antibiotics are reduced to the semiquinone form at Complex I of the mitochondrial electron transport chain. In the experiments presented in this study we examined the effects of doxorubicin (Adriamycin), daunorubicin, and related quinonoid anticancer agents on superoxide, hydrogen peroxide, and hydroxyl radical production by preparations of beef heart submitochondrial particles. Superoxide anion formation was stimulated from (mean +/- S.E.) 1.6 +/- 0.2 to 69.6 +/- 2.7 or 32.1 +/- 1.5 nmol X min-1 X mg-1 by the addition of 90 microM doxorubicin or daunorubicin, respectively. However, the anthracycline 5-iminodaunorubicin, in which an imine group has been substituted in the C ring quinone moiety, did not increase superoxide production over control levels. In the presence of rotenone, initial rates of oxygen consumption and superoxide formation were identical under comparable experimental conditions. Furthermore, H2O2 production increased from undetectable control levels to 2.2 +/- 0.3 nmol X min-1 X mg-1 after treatment of submitochondrial particles with doxorubicin (200 microM). The hydroxyl radical, or a related chemical oxidant, was also detected after the addition of an anthracycline to this system by both ESR spectroscopy using the spin trap 5,5-dimethylpyrroline-N-oxide and by gas chromatographic quantitation of CH4 produced from dimethyl sulfoxide. Hydroxyl radical production, which was iron-dependent in this system, occurred in a nonlinear fashion with an initial lag phase due to a requirement for H2O2 accumulation. We also found that two quinonoid anti-cancer agents which produce less cardiotoxicity than the anthracyclines, mitomycin C, and mitoxantrone, stimulated significantly less or no hydroxyl radical production by submitochondrial particles. These experiments suggest that injury to cardiac mitochondria which is produced by anthracycline antibiotics may result from the generation of the hydroxyl radical during anthracycline metabolism by NADH dehydrogenase.
In the present study we have used beef heart submitochondrial preparations (BH-SMP) to demonstrate that a component of mitochondrial Complex I, probably the NADH dehydrogenase flavin, is the mitochondrial site of anthracycline reduction. During forward electron transport, the anthracyclines doxorubicin (Adriamycin) and daunorubicin acted as one-electron acceptors for BH-SMP (i.e. were reduced to semiquinone radical species) only when NADH was used as substrate; succinate and ascorbate were without effect. Inhibitor experiments (rotenone, amytal, piericidin A) indicated that the anthracycline reduction site lies on the substrate side of ubiquinone. Doxorubicin and daunorubicin semiquinone radicals were readily detected by ESR spectroscopy. Doxorubicin and daunorubicin semiquinone radicals (g congruent to 2.004, signal width congruent to 4.5 G) reacted avidly with molecular oxygen, presumably to produce O2-, to complete the redox cycle. The identification of Complex I as the site of anthracycline reduction was confirmed by studies of ATP-energized reverse electron transport using succinate or ascorbate as substrates, in the presence of antimycin A or KCN respiratory blocks. Doxorubicin and daunorubicin inhibited the reduction of NAD+ to NADH during reverse electron transport. Furthermore, during reverse electron transport in the absence of added NAD+, doxorubicin and daunorubicin addition caused oxygen consumption due to reduction of molecular oxygen (to O2-) by the anthracycline semiquinone radicals. With succinate as electron source both thenoyltrifluoroacetone (an inhibitor of Complex II) and rotenone blocked oxygen consumption, but with ascorbate as electron source only rotenone was an effective inhibitor. NADH oxidation by doxorubicin during BH-SMP forward electron transport had a KM of 99 microM and a Vmax of 30 nmol X min-1 X mg-1 (at pH 7.4 and 23 degrees C); values for daunorubicin were 71 microM and 37 nmol X min-1 X mg-1. Oxygen consumption at pH 7.2 and 37 degrees C exhibited KM values of 65 microM for doxorubicin and 47 microM for daunorubicin, and Vmax values of 116 nmol X min-1 X mg-1 for doxorubicin and 114 nmol X min-1 X mg-1 for daunorubicin. In marked contrast with these results, 5-iminodaunodrubicin (a new anthracycline with diminished cardiotoxic potential) exhibited little or no tendency to undergo reduction, or to redox cycle with BH-SMP. Redox cycling of anthracyclines by mitochondrial NADH dehydrogenase is shown, in the accompanying paper (Doroshow, J. H., and Davies, K. J. A. (1986) J. Biol. Chem. 261, 3068-3074), to generate O2-, H2O2, and OH which may underlie the cardiotoxicity of these antitumor agents.
We report that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide- and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite. S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50-60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.
We have studied the effects of hyperoxia and of cell loading with artificial lipofuscin or ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversible senescence-like growth arrest after about 4 wk and shortened postmitotic life span from 1-1/2 years down to 3 months. During the first 8 wk of hyperoxia-induced 'aging', overall protein degradation (breakdown of [(35)S]methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lysosomal cathepsin-specific activity (using the fluorogenic substrate z-FR-MCA) and cytoplasmic proteasome-specific activity (measured with suc-LLVY-MCA) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to lipofuscin/ceroid loading under normoxic conditions. Lipofuscin/ceroid-loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity decreased by 25% over this period, which is important since proteasome is normally responsible for degrading oxidized cell proteins. In contrast, an apparent increase in lysosomal cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether lipofuscin/ceroid could in fact directly inhibit proteasome activity, thus causing oxidized proteins to accumulate, we incubated purified proteasome with lipofuscin/ceroid preparations in vitro. We found that proteasome is directly inhibited by lipofuscin/ceroid. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin/ceroid may actually cause further increases in damage accumulation during aging by inhibiting the proteasome.
Monoamine oxidases A/B (EC 1.4.3.4, MAO), flavoenzymes located on the outer mitochondrial membrane, catalyze the oxidative deamination of biogenic amines, such as dopamine, serotonin, and norepinephrine. In this study, we examined whether the H2O2formed during the two-electron oxidation of tyramine [4-(2-aminoethyl)phenol] (a substrate for monoamine oxidases A/B) may contribute to the intramitochondrial steady-state concentration of H2O2([H2O2]ss) and, thus, be involved in the oxidative impairment of mitochondrial matrix components. Supplementation of intact, coupled rat brain mitochondria with benzylamine, β-phenylethylamine, or tyramine showed initial rates of H2O2production ranging from 0.4- to 1.6 nmol H2O2/min/mg protein. ESR analysis of the oxidative deamination of tyramine by intact rat brain mitochondria revealed the formation of hydroxyl (HO) and carbon-centered radical adducts—the latter probably originating by the HO-mediated oxidation of mannitol. The signals were substantially enhanced upon addition of FeSO4and were abolished by catalase. The intramitochondrial [H2O2]sscalculated in terms of glutathione peroxidase activity during the metabolism of tyramine was 48-fold higher (7.71 ± 0.25 × 10−7M) than that obtained during the oxidation of succinate via complex II in the presence of antimycin A (1.64 ± 0.2 × 10−8M). Oxidative damage to the brain mtDNA was assessed by single strand breakage. The ratio of nicked DNA for the preparations treated with tyramine and those without the amine was 1.5 ± 0.29 (n= 4), 2.12 ± 0.28 (n= 8,P≤ 0.05), and 3.12 ± 0.69 (n= 3,P≤ 0.05) at 15, 30, and 60 min, respectively. Preincubation of mitochondria with tranylcypromine (trans-2-phenylcyclopropylamine), an inhibitor to MAO A/B, abolished mtDNA oxidative damage. Catalase inhibited mtDNA strand breakage by approximately 60%. Incubation of intact, coupled rat brain mitochondria with chlorodinitrobenzene (CDNB) depleted mitochondrial GSH by 72%. Tyramine-dependent damage of mtDNA was decreased by 68% in CDNB-treated mitochondria (with 28% remaining GSH). The [H2O2]sswas slightly increased in CDNB-treated mitochondria: 1.38- and 1.28-fold increase during the oxidation of succinate in the presence of antimycin A and during the oxidation of tyramine, respectively. These results suggest that the H2O2generated during the MAO-catalyzed oxidation of biogenic amines and possibly certain neurotransmitters at the outer mitochondrial membrane contributes to the intramitochondrial [H2O2]ssand may cause oxidative damage to mtDNA. This is effected by the intramitochondrial concentration of GSH and might have potential implications for aging and neurodegenerative processes.
Reactive oxygen species (ROS: superoxide radical, O2−; hydrogen peroxide, H2O2; hydroxyl radical, OH), which arise from the univalent reduction of dioxygen are formed in mitochondria. We summarize here results which indicate that ROS, and also the radical nitrogen monoxide (‘nitric oxide’, NO), act as physiological modulators of some mitochondrial functions, but may also damage mitochondria. Hydrogen peroxide, which originates in mitochondria predominantly from the dismutation of superoxide, causes oxidation of mitochondrial pyridine nucleotides and thereby stimulates a specific Ca2+ release from intact mitochondria. This release is prevented by cyclosporin A (CSA). Hydrogen peroxide thus contributes to the maintenance of cellular Ca2+ homeostasis. A stimulation of mitochondrial ROS production followed by an enhanced Ca2+ release and re-uptake (Ca2+ ‘cycling’) by mitochondria causes apoptosis and necrosis, and contributes to hypoxia/reperfusion injury. These kinds of cell injury can be attenuated at the mitochondrial level by CSA. When ROS are produced in excessive amounts in mitochondria nucleic acids, proteins, and lipids are extensively modified by oxidation. Physiological (sub-micromolar) concentrations of NO potently and reversibly deenergize mitochondria at oxygen tensions that prevail in cells by transiently binding to cytochrome oxidase. This is paralleled by mitochondrial Ca2+ release and uptake. Higher NO concentrations or prolonged exposure of cells to NO causes their death. It is concluded that ROS and NO are important physiological reactants in mitochondria and become toxic only when present in excessive amounts.
Several mitochondrial functions decline with age. The contributing factors include, the intrinsic rate of proton leakage across the inner mitochondrial membrane (a correlate of oxidant formation), decreased membrane fluidity, and decreased levels and function of cardiolipin, which supports the function of many of the proteins of the inner mitochondrial membrane. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. Evidence supports the suggestion that age-associated accumulation of mitochondrial deficits due to oxidative damage is likely to be a major contributor to cellular, tissue, and organismal aging.
The cell employs several lines of defense against the toxic products of oxygen reduction. The first is systemic protection against high oxygen tensions at the cellular level. The second is the intracellular localization of the enzymes appropriate to the decomposition of the toxic intermediates at or near the site where they are generated, together with steep gradients of the reactive species themselves. A third line of defense is provided by radical scavengers such as α-tocopherol and β-carotene, which also have the advantage of being appropriately distributed in the membranes where lipid peroxidation might occur. A fourth level of protection is provided by glutathione peroxidase, which reacts directly with lipid peroxides. Finally, recent understanding of the beneficial action of H(2)O(2) in phagocytosis and in ethanol oxidation suggests caution in condemning any metabolite as useless until its functions in toto are thoroughly understood.
1. A polarographic assay of superoxide (O2--) dismutase (EC 1.15.1.1) activity is described, in which the ability of the enzyme to inhibit O2---dependent sulphite oxidation, initiated by xanthine oxidase activity, is measured. The assay was used in a study of the intracellular distribution of superoxide dismutase in rat liver. Both cyanide-sensitive cupro-zinc dismutase (92% of the total activity) and cyanide-insensitive mangano-dismutase (8%) were measured. 2. Rat liver homogenates contained both particulate (16%y and soluble (84%) dismutase activity. The particulate activity contained both types of dismutase, whereas nearly all the soluble dismutase was a cupro-zinc enzymes. The distribution pattern of mangano-dismutase was similar to that of cytochrome oxidase and glutamate dehydrogenase, indicating that the enzyme was probably present exclusively in the mitochondria. 3. Superoxide dismutase activity in the heavy-mitochondrial (M) fraction was latent and was activated severalfold and largely solubilized by sonication. Treatment of the M fraction with digitonin or a hypo-osmotic suspending medium indicated that most of the cupro-zinc dismutase was located in the mitochondrial intermembrane space, whereas the mangano-enzyme was located in the inner-membrane and matrix space. 4. A small amount of dismutase activity appeared to be present in the nuclei and microsomal fraction, but little or no activity in the lysosomes or peroxisomes. 5. The results are discussed in relation to the intracellular location of known O2---generating enzymes, the possible role of superoxide dismutase activity in intracellular H2O2 formation, and to current views on the physiological function of the enzyme.
The subcellular distributions of glutathione peroxidase and 75Se in rat liver were determined. Approximately 75% of the enzyme and 58% of the 73Se were contained in the cytosolic fraction. Rat liver cytosol glutathione peroxidase was purified 1029-fold from homogenate to yield a sample with a specific activity of 278 μmol of NADPH oxidized/ min/mg of protein. The purified enzyme was subjected to disc-gel and sodium dodecyl sulfate-disc-gel electrophoresis, which confirmed the purity of the enzyme as well as the existence of multiple electrophoretic forms. Glutathione peroxidase existed as a large aggregate after homogenization, and means of dissociating the aggregate were investigated. The enzyme was isolated as a neutrally charged protein and became negatively charged upon storage, a phenomenon that was independent of the aggregation.
Oxidant stress, due to the formation of hydrogen peroxide and oxygen-derived free radicals, can cause cell damage due to chain reactions of membrane lipid peroxidation. Because the substantia nigra is rich in dopamine, which can undergo both enzymatic oxidation via monoamine oxidase and nonenzymatic autoxidation, hydrogen peroxide and oxyradicals (superoxide anion radical and hydroxyl radical) are generated in this midbrain nucleus. Although proof that oxidant stress actually causes the loss of monoaminergic neurons in patients with Parkinson's disease is lacking, there is a considerable body of evidence from studies in both animals and humans that support the concept. (1) Neurotoxins that selectively destroy the dopaminergic neurons in the nigra, such as 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), appear to act via oxidant stress. (2) The substantia nigra of patients with Parkinson's disease reveals evidence of oxidant stress by the findings of increased lipid peroxidation and decreased reduced glutathione. (3) Total iron is increased and ferritin is reduced in the substantia nigra pars compacta in patients with Parkinson's disease. This combination suggests that this transition metal is in a low molecular weight form, capable of catalyzing nonenzymatic oxidative reactions, especially the conversion of hydrogen peroxide to hydroxyl radical, which is the most reactive of the oxygen radicals. (4) Neuromelanin, a product of dopamine autoxidation, can serve as a reservoir for iron, promoting the generation of oxyradicals. (5) Antioxidant defense mechanisms appear to be reduced in the parkinsonian substantia nigra with the findings of decreased activities of glutathione peroxidase and catalase.(ABSTRACT TRUNCATED AT 250 WORDS)
The biologically occurring thiols, glutathione, cysteamine and cysteine, significantly enhance the single-strand breaks in plasmid pBR322 DNA induced by singlet molecular oxygen (1O2) generated by the thermodissociation of the endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate. The enhancing effect was also observed with chemically related sulfhydryl compounds but not by disulfides. In contrast, dihydrolipoate and its disulfide lipoate protected the plasmid DNA. Metal chelators as well as superoxide dismutase or catalase had no effect, whereas mannitol or sodium azide, decreased the thiol-1O2-induced strand breaks. It is concluded that the observed effects are mediated by reactive oxidation products arising from the 1O2-oxidation of thiols.
Aerobic organisms are constantly exposed to oxygen radicals and related oxidants. The antioxidant compounds and enzymes they have evolved remove most of the potentially damaging radicals/oxidants; however, damage to cellular proteins, lipids, nucleic acids and carbohydrates can be observed even under normal physiological conditions. Re-reduction of cellular components (direct repair) may be important for some biomolecules. In most cases studied to date, however, enzymatic degradation (by proteases, lipases, nucleases) appears to release damaged elements for excretion and conserve undamaged components for reutilization (indirect repair). In addition, the removal of damaged components appears to prevent or diminish the potential cytotoxicity of oxidized macromolecules. Several studies have reported an accumulation of oxidatively damaged cellular components with age (e.g., cataract formation, lipofuscin). Such reports are evidence that oxidant damage is one of several factors which contribute to the aging process, and provide at least partial support for the free-radical theory of aging. Studies of age-related changes in the activities, or levels of antioxidant enzymes and antioxidant compounds, however, have not provided complete understanding of the putative role of free radicals/oxidants in the aging process. In this review, we present the hypothesis that decreased activities or constitutive levels of oxidant repair enzymes may contribute to a progressive accumulation of oxidant damage with aging. Furthermore, the ability to mount an effective response to oxidative stress (induction of oxidant stress genes and proteins) may decline with age, thus predisposing older cells and organisms to oxidant damage.
Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (.OH) alone, superoxide anion radical (O2-) alone, or mixtures of .OH and O2-, by gamma radiolysis in the presence of 100% N2O (.OH exposure), 100% O2 + formate (O2- exposure), or 100% O2 alone (.OH + O2- exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by .OH (10% inactivation at 15-40 nmol of .OH/mg of SMP protein, 50-90% inactivation at 600 nmol of .OH/mg of SMP protein) and by .OH + O2- (10% inactivation at 20-80 nmol of .OH + O2-/mg of SMP protein, 45-75% inactivation at 600 nmol of .OH + O2-/mg of SMP protein). Importantly, O2- was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O2-/mg of SMP protein, 40% inactivation at 600 nmol of O2-/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O2-/mg of SMP protein, 30% inactivation at 600 nmol of O2-/mg of SMP protein), and a poor inactivator of succinate oxidase (less than 10% inactivation at 600 nmol of O2-/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at .OH, O2-, OH + O2-, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of .OH/mg of SMP protein. The .OH-dependent inactivations reported above were largely inhibitable by the .OH scavenger mannitol. In contrast, the O2(-)-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with .OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all.(ABSTRACT TRUNCATED AT 400 WORDS)
Progress in identifying the important endogenous processes damaging DNA and developing methods to assay this damage in individuals is presented. This approach may aid studies on modulation of cancer and aging. The endogenous background level of oxidant-induced DNA damage in vivo has been assayed by measuring 8-hydroxydeoxyguanosine (oh8dG), thymine glycol and thymidine glycol in urine and oh8dG in DNA. oh8dG is one of about 20 adducts found on oxidizing DNA, e.g., by radiation. The level of oxidative DNA damage as measured by oh8dG in normal rat liver is shown to be extensive, especially in mtDNA (1/130,000 bases in nuclear DNA and 1/8,000 bases in mitochondrial DNA). We also discuss three hitherto unrecognized antioxidants in man.
The quinonoid anthracycline, doxorubicin (Adriamycin) is a potent anti-neoplastic agent whose clinical use is limited by severe cardiotoxicity. Mitochondrial damage is a major component of this cardiotoxicity, and rival oxidative and non-oxidative mechanisms for inactivation of the electron transport chain have been proposed. Using bovine heart submitochondrial preparations (SMP) we have now found that both oxidative and non-oxidative mechanisms occur in vitro, depending solely on the concentration of doxorubicin employed. Redox cycling of doxorubicin by Complex I of the respiratory chain (which generates doxorubicin semiquinone radicals, O2-, H2O2, and .OH) caused a 70% decrease in the Vmax. for NADH dehydrogenase during 15 min incubation of SMP, and an 80% decrease in NADH oxidase activity after 2 h incubation. This inactivation required only 25-50 microM-doxorubicin and represents true oxidative damage, since both NADH (for doxorubicin redox cycling) and oxygen were obligatory participants. The damage appears localized between the NADH dehydrogenase flavin (site of doxorubicin reduction) and iron-sulphur centre N-1. Succinate dehydrogenase, succinate oxidase, and cytochrome c oxidase activities were strongly inhibited by higher doxorubicin concentrations, but this phenomenon did not involve doxorubicin redox cycling (no NADH or oxygen requirement). Doxorubicin concentrations of 0.5 mM were required for 50% decreases in these activities, except for cytochrome c oxidase which was only 30% inhibited following incubation with even 1.0 mM-doxorubicin. Our results indicate that low concentrations of doxorubicin (50 microM or less) can catalyse a site-specific oxidative damage to the NADH oxidation pathway. In contrast, ten-fold higher doxorubicin concentrations (or more) are required for non-oxidative inactivation of the electron transport chain; probably via binding to cardiolipin and/or generalized membrane chaotropic effects. The development of agents to block doxorubicin toxicity in vivo will clearly require detailed clinical studies of doxorubicin uptake in the heart.
When exposed to oxidative stress, by oxygen radicals or H2O2, E. coli exhibited decreased growth, decreased protein synthesis, and dose-dependent increases in protein degradation. The quinone menadione induced proteolysis when cells were incubated in air, but was not effective when cells were incubated without oxygen. Anaerobically grown cells also exhibited significantly lower proteolytic capacity than did cells that were grown aerobically. Xanthine plus xanthine oxidase (which generate O2- and H2O2) caused a stimulation of proteolysis which was inhibitable by catalase, but not by superoxide dismutase: Indicating that H2O2 was responsible for the increased protein degradation. Indeed, H2O2 alone was effective in inducing increased intracellular proteolysis. Two-dimensional polyacrylamide gel electrophoresis of [3H]leucine labeled E. coli revealed greater than 50% decreases in the concentrations of 10-15 cell proteins following H2O2 or menadione exposure, while several other proteins were less severely affected. To test for the presence of soluble proteases, we prepared cell-free extracts of E. coli and incubated them with radio-labeled protein substrates. E. coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase. When these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E. coli extracts. Studies with albumin revealed correlations greater than 0.95 between the degree of oxidative denaturation and proteolytic susceptibility. Pretreatment of E. coli with menadione or H2O2 did not increase the proteolytic capacity of cell extracts; indicating that neither protease activation, nor protease induction were required.(ABSTRACT TRUNCATED AT 250 WORDS)
E. coli contains a soluble proteolytic pathway which can recognize and degrade oxidatively denatured proteins and protein fragments, and which may act as a "secondary antioxidant defense." We now provide evidence that this proteolytic pathway is distinct from the previously described ATP-dependent, and protease "La"-dependent, pathway which may degrade other abnormal proteins. Cells (K12) which were depleted of ATP, by arsenate treatment or anaerobic incubation (after growth on succinate), exhibited proteolytic responses to oxidative stress which were indistinguishable from those observed in cells with normal ATP levels. Furthermore, the proteolytic responses to oxidative damage by menadione or H2O2 were almost identical in the isogenic strains RM312 (a K12 derivative) and RM1385 (a lon deletion mutant of RM312). Since the lon (or capR) gene codes for the ATP-dependent protease "La," these results indicate that neither ATP nor protease "La" are required for the degradation of oxidatively denatured proteins. We next prepared cell-free extracts of K12, RM312, and RM1385 and tested the activity of their soluble proteases against proteins (albumin, hemoglobin, superoxide dismutase, catalase) which had been oxidatively denatured (in vitro) by exposure to .OH, .OH + O2- (+O2), H2O2, or ascorbate plus iron. The breakdown of oxidatively denatured proteins was several-fold higher than that of untreated proteins in extracts from all three strains, and ATP did not stimulate degradation. Incubation of extracts at 45 degrees C, which inactivates protease "La," actually stimulated the degradation of oxidatively denatured proteins. Although Ca2+ had little effect on proteolysis, serine reagents, transition metal chelators, and hemin effectively inhibited the degradation of oxidatively denatured proteins in both intact cells and cell-free extracts. Degradation of oxidatively denatured proteins in cell-free extracts was maximal at pH 7.8, and was unaffected by dialysis of the extracts against membranes with molecular weight cutoffs as high as 50,000. Our results indicate the presence of a neutral, ATP- and calcium- independent proteolytic pathway in the E. coli cytosol, which contains serine- and metallo- proteases (with molecular weights greater than 50,000), and which preferentially degrades oxidatively denatured proteins.
When incubated with mitochondria in an air atmosphere, menadione and doxorubicin (which redox cycle with the respiratory chain to produce oxygen radicals), as well as xanthine oxidase plus xanthine (which generate superoxide and H2O2), stimulated the degradation of newly-synthesized [( 3H]leucine-labelled) mitochondrial polypeptides. No stimulation was observed in an N2 atmosphere, ATP was not required, and xanthine oxidase was not effective without xanthine. Various forms of oxidative stress induced varying degrees of protein cross-linking, protein fragmentation and proteolysis, as judged by gel electrophoresis and amino acid analysis. To learn more about the proteolytic enzymes involved in degradation, we undertook studies with purified protein substrates which had been exposed to oxidative stress (OH or H2O2) in vitro. Despite mitochondrial contamination with acid proteases of lysosomal (and other) origin, pH profiles revealed distinct proteolytic activities at both pH 4 and pH 8. The pH 8 activity preferentially degraded the oxidatively-denatured forms of haemoglobin, albumin and superoxide dismutase; was unaffected by digitonin; and exhibited a several-fold increase in activity upon mitochondrial disruption (highest activity being found in the matrix). In contrast, the pH 4 activity was dramatically decreased by digitonin treatment (to reduce lysosomal contamination); was unaffected by mitochondrial disruption; and showed no preference for oxidatively-denatured proteins. The pH 8 activity was not stimulated by ATP, but was inhibited by EDTA, haemin and phenylmethylsulphonyl fluoride. In contrast, the contaminating pH 4 activity was only inhibited by pepstatin and leupeptin. Thus, our experiments reveal a distinct mitochondrial (matrix) proteolytic pathway which can preferentially degrade oxidatively-denatured proteins.
Oxidative damage to DNA can be caused by excited oxygen species, which are produced by radiation or are by-products of aerobic metabolism. The oxidized base, 8-hydroxydeoxyguanosine (oh8dG), 1 of approximately 20 known radiation damage products, has been assayed in the DNA of rat liver. oh8dG is present at a level of 1 per 130,000 bases in nuclear DNA and 1 per 8000 bases in mtDNA. Mitochondria treated with various prooxidants have an increased level of oh8dG. The high level of oh8dG in mtDNA may be caused by the immense oxygen metabolism, relatively inefficient DNA repair, and the absence of histones in mitochondria. It may be responsible for the observed high mutation rate of mtDNA.
1. The enzyme-substrate complex of yeast cytochrome c peroxidase is used as a sensitive, specific and accurate spectrophotometric H(2)O(2) indicator. 2. The cytochrome c peroxidase assay is suitable for use with subcellular fractions from tissue homogenates as well as with pure enzyme systems to measure H(2)O(2) generation. 3. Mitochondrial substrates entering the respiratory chain on the substrate side of the antimycin A-sensitive site support the mitochondrial generation of H(2)O(2). Succinate, the most effective substrate, yields H(2)O(2) at a rate of 0.5nmol/min per mg of protein in state 4. H(2)O(2) generation is decreased in the state 4-->state 3 transition. 4. In the combined mitochondrial-peroxisomal fraction of rat liver the changes in the mitochondrial generation of H(2)O(2) modulated by substrate, ADP and antimycin A are followed by parallel changes in the saturation of the intraperoxisomal catalase intermediate. 5. Peroxisomes supplemented with uric acid generate extraperoxisomal H(2)O(2) at a rate (8.6-16.4nmol/min per mg of protein) that corresponds to 42-61% of the rate of uric acid oxidation. Addition of azide increases these H(2)O(2) rates by a factor of 1.4-1.7. 6. The concentration of cytosolic uric acid is shown to vary during the isolation of the cellular fractions. 7. Microsomal fractions produce H(2)O(2) (up to 1.7nmol/min per mg of protein) at a ratio of 0.71-0.86mol of H(2)O(2)/mol of NADP(+) during the oxidation of NADPH. H(2)O(2) is also generated (6-25%) during the microsomal oxidation of NADH (0.06-0.025mol of H(2)O(2)/mol of NAD(+)). 8. Estimation of the rates of production of H(2)O(2) under physiological conditions can be made on the basis of the rates with the isolated fractions. The tentative value of 90nmol of H(2)O(2)/min per g of liver at 22 degrees C serves as a crude approximation to evaluate the biochemical impact of H(2)O(2) on cellular metabolism.
1. Pigeon heart mitochondria produce H(2)O(2) at a maximal rate of about 20nmol/min per mg of protein. 2. Succinate-glutamate and malate-glutamate are substrates which are able to support maximal H(2)O(2) production rates. With malate-glutamate, H(2)O(2) formation is sensitive to rotenone. Endogenous substrate, octanoate, stearoyl-CoA and palmitoyl-carnitine are by far less efficient substrates. 3. Antimycin A exerts a very pronounced effect in enhancing H(2)O(2) production in pigeon heart mitochondria; 0.26nmol of antimycin A/mg of protein and the addition of an uncoupler are required for maximal H(2)O(2) formation. 4. In the presence of endogenous substrate and of antimycin A, ATP decreases and uncoupler restores the rates of H(2)O(2) formation. 5. Reincorporation of ubiquinone-10 and ubiquinone-3 to ubiquinone-depleted pigeon heart mitochondria gives a system in which H(2)O(2) production is linearly related to the incorporated ubiquinone. 6. The generation of H(2)O(2) by pigeon heart mitochondria in the presence of succinate-glutamate and in metabolic state 4 has an optimum pH value of 7.5. In states 1 and 3u, and in the presence of antimycin A and uncoupler, the optimum pH value is shifted towards more alkaline values. 7. With increase of the partial pressure of O(2) to the hyperbaric region the formation of H(2)O(2) is markedly increased in pigeon heart mitochondria and in rat liver mitochondria. With rat liver mitochondria and succinate as substrate in state 4, an increase in the pO(2) up to 1.97MPa (19.5atm) increases H(2)O(2) formation 10-15-fold. Similar pO(2) profiles were observed when rat liver mitochondria were supplemented either with antimycin A or with antimycin A and uncoupler. No saturation of the system with O(2) was observed up to 1.97MPa (19.5atm). By increasing the pO(2) to 1.97MPa (19.5atm), H(2)O(2) formation in pigeon heart mitochondria with succinate as substrate increased fourfold in metabolic state 4, with antimycin A added the increase was threefold and with antimycin A and uncoupler it was 2.5-fold. In the last two saturation of the system with oxygen was observed, with an apparent K(m) of about 71kPa (0.7-0.8atm) and a V(max.) of 12 and 20nmol of H(2)O(2)/min per mg of protein. 8. It is postulated that in addition to the well-known flavin reaction, formation of H(2)O(2) may be due to interaction with an energy-dependent component of the respiratory chain at the cytochrome b level.
In intact liver slices and liver homogenates ∼ 85% of the 25°C, g=2.004 EPR signal is now identified as stabilized mitochondrial ubisemiquinone radicals. Redox poised liver slices and liver homogenates demonstrate ubisemiquinone stability constants of 1.6×10−3 and 2.2×10−3 respectively. Such high ubisemiquinone concentrations in liver demonstrate the physiologic feasibility of “Q cycle” mechanisms for mitochondrial electron transport.
This study examined the effects of various anthracycline antibiotics and mitoxantrone, bisantrene, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), and neocarzinostatin on oxygen radical formation by cardiac sarcoplasmic reticulum and submitochondrial particles. Doxorubicin, daunorubicin, rubidazone, and aclacinomycin A stimulated superoxide production by both heart fractions in a dose-dependent fashion that appeared to follow saturation kinetics. The anthracycline drugs also significantly increased hydrogen peroxide production by heart sarcosomes and submitochondrial particles. On the other hand, mitoxantrone, bisantrene, m-AMSA, and neocarzinostatin did not significantly enhance cardiac reactive oxygen metabolism. Thus, it is unlikely that the mechanism of the cardiac toxicity produced by mitoxantrone and m-AMSA in patients previously treated with anthracycline drugs can be directly related to oxidation-reduction cycling catalyzed by cardiac flavin dehydrogenases.
The intracellular generation of reactive forms of reduced oxygen, namely, hydrogen peroxide, superoxide and hydroxyl radical, can damage dopamine neurons. Oxy-radicals, and hydrogen peroxide generated by monoamine oxidase, can contribute to increased rates of senescence of dopamine neurons in Parkinson's disease. The evidence that oxy-radicals and monoamine oxidase are potentially cytotoxic is reviewed, and a pathobiology of dopamine neuron senescence in Parkinson's disease is proposed.
Modifications at two points in the sequence of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) analysis have contributed to a more accurate and simplified determination of 8-OH-dG in DNA. The first was an improvement in the detection limit for 8-OH-dG in high-performance liquid chromatography analysis and the second was a pronase digestion and ethanol precipitation method (pronase/ethanol method) for DNA isolation which could minimize artificial formation of 8-OH-dG. Since the changes in background current from electrochemical detection are regularly periodical, it was possible to reduce this background change by connecting a pressure damper, degassing the eluent before use and finally subtracting its theoretical function. After this background correction, the detection limit for 8-OH-dG was improved one order of magnitude, from 20 fmol (5.68 pg) to 1.76 fmol (0.5 pg). Therefore, 0.005 8-OH-dG/10(5) dG can be detected from 50 micrograms DNA. This improvement will allow the analysis of small samples, tissues from needle biopsies, < 5 ml whole blood, etc., and will contribute to the accuracy of 8-OH-dG measurements. The pronase/ethanol method resulted in lower levels of 8-OH-dG than the phenol method in analyses of both rat liver and calf thymus DNA, even after 6 h incubation at 45 degrees C. The level obtained by the pronase/ethanol method with butylated hydroxytoluene was approximately equal to or lower than the 8-OH-dG levels reported in normal rat liver. The pronase/ethanol method for DNA isolation can replace the phenol or other methods in 8-OH-dG analysis. This method also omits the use of highly toxic organic solvents.
Production of hydroxyl radicals (HO.) by substrate-supplemented beef heart submitochondrial particles was studied by electron paramagnetic resonance in conjunction with the spin trap 5,5'-dimethyl-1-pirroline-N-oxide (DMPO). Supplementation of submitochondrial particles with NADH or succinate in the presence of antimycin resulted in the formation hydroxyl-, alpha-hydroxyethyl-, and methyl radical adducts. The latter two adducts were derived from HO. attack of ethanol or dimethyl sulfoxide (DMSO), respectively, the solvents used for the inhibitors of the respiratory chain. These ESR signals were slightly increased by superoxide dismutase and abolished by catalase. Further support for the production of HO. during mitochondrial electron transfer was furnished by kinetic competition experiments with DMSO as the HO. scavenger. This approach yielded a kappa SCAVENGER/kappa DMPO value of 1.7, in agreement with a competitive spin trapping of free HO. using DMSO as a scavenger. The scission of H2O2 to HO. requires consideration of a Fenton chemistry, i.e., the participation of metals or redox active metal pools in mitochondria to drive this reaction. The effect of several metal chelators on the formation of both HO. and H2O2 was examined. Bathophenantroline, bathocuproine, and desferrioxamine decreased the DMPO-HO. signal and increased accumulation of H2O2. Conversely, EDTA or diethylenetriaminepentaacetic acid substantially increased the DMPO-HO. signal intensity and decreased H2O2 accumulation. These different results were rationalized in terms of the reduction potential of the redox couples involved, i.e., that of the ligated metal and those encompassed in the one-electron reduction of superoxide radical and of hydrogen peroxide. The formation of 8-hydroxydesoxyguanosine in mitochondrial DNA was examined under experimental conditions in which H2O2 production by isolated mitochondria was enhanced. The formation of 8-hydroxydesoxyguanosine increased with increasing rates of H2O2 formation. The biological significance of H2O2 and HO. formation during mitochondrial electron transfer is discussed in terms of oxidative damage of mitochondrial DNA and the implications for mitochondrial functions and aging.
Nitric oxide (.NO) released by S-nitrosoglutathione (GSNO) inhibited enzymatic activities of rat heart mitochondrial membranes. Cytochrome oxidase activity was inhibited to one-half at an effective .NO concentration of 0.1 microM, while succinate- and NADH-cytochrome-c reductase activities were half-maximally inhibited at 0.3 microM .NO. Submitochondrial particles treated with .NO (either from GSNO or from a pure solution) showed increased O(-)(2) and H202 production when supplemented with succinate alone, at rates that were comparable to those of control particles with added succinate and antimycin. Rat heart mitochondria treated with .NO also showed increased H2O2 production. Cytochrome spectra and decreased enzymatic activities in the presence of .NO are consistent with a multiple inhibition of mitochondrial electron transfer at cytochrome oxidase and at the ubiquinone-cytochrome b region of the respiratory chain, the latter leading to the increased O2- production. Electrochemical detection showed that the buildup of a .NO concentration from GSNO was interrupted by submitochondrial particles supplemented with succinate and antimycin and was restored by addition of superoxide dismutase. The inhibitory effect of .NO on cytochrome oxidase was also prevented under the same conditions. Apparently, mitochondrial O2- reacts with .NO to form peroxynitrate and, by removing .NO, reactivates the previously inhibited cytochrome oxidase. It is suggested that, at physiological concentrations of .NO, inhibition of electron transfer, .NO-induced O2- production, and ONOO- formation participate in the regulatory control of mitochondrial oxygen uptake.
The paradox of aerobic life, or the 'Oxygen Paradox', is that higher eukaryotic aerobic organisms cannot exist without oxygen, yet oxygen is inherently dangerous to their existence. This 'dark side' of oxygen relates directly to the fact that each oxygen atom has one unpaired electron in its outer valence shell, and molecular oxygen has two unpaired electrons. Thus atomic oxygen is a free radical and molecular oxygen is a (free) bi-radical. Concerted tetravalent reduction of oxygen by the mitochondrial electron-transport chain, to produce water, is considered to be a relatively safe process; however, the univalent reduction of oxygen generates reactive intermediates. The reductive environment of the cellular milieu provides ample opportunities for oxygen to undergo unscheduled univalent reduction. Thus the superoxide anion radical, hydrogen peroxide and the extremely reactive hydroxyl radical are common products of life in an aerobic environment, and these agents appear to be responsible for oxygen toxicity. To survive in such an unfriendly oxygen environment, living organisms generate--or garner from their surroundings--a variety of water- and lipid-soluble antioxidant compounds. Additionally, a series of antioxidant enzymes, whose role is to intercept and inactivate reactive oxygen intermediates, is synthesized by all known aerobic organisms. Although extremely important, the antioxidant enzymes and compounds are not completely effective in preventing oxidative damage. To deal with the damage that does still occur, a series of damage removal/repair enzymes, for proteins, lipids and DNA, is synthesized. Finally, since oxidative stress levels may vary from time to time, organisms are able to adapt to such fluctuating stresses by inducing the synthesis of antioxidant enzymes and damage removal/repair enzymes. In a perfect world the story would end here; unfortunately, biology is seldom so precise. The reality appears to be that, despite the valiant antioxidant and repair mechanisms described above, oxidative damage remains an inescapable outcome of aerobic existence. In recent years oxidative stress has been implicated in a wide variety of degenerative processes, diseases and syndromes, including the following: mutagenesis, cell transformation and cancer; atherosclerosis, arteriosclerosis, heart attacks, strokes and ischaemia/reperfusion injury; chronic inflammatory diseases, such as rheumatoid arthritis, lupus erythematosus and psoriatic arthritis; acute inflammatory problems, such as wound healing; photo-oxidative stresses to the eye, such as cataract; central-nervous-system disorders, such as certain forms of familial amyotrophic lateral sclerosis, certain glutathione peroxidase-linked adolescent seizures, Parkinson's disease and Alzheimer's dementia; and a wide variety of age-related disorders, perhaps even including factors underlying the aging process itself. Some of these oxidation-linked diseases or disorders can be exacerbated, perhaps even initiated, by numerous environmental pro-oxidants and/or pro-oxidant drugs and foods. Alternatively, compounds found in certain foods may be able to significantly bolster biological resistance against oxidants. Currently, great interest centres on the possible protective value of a wide variety of plant-derived antioxidant compounds, particularly those from fruits and vegetables.
We have identified an RNA species that appears to be induced by oxidative stress in hamster HA-1 fibroblasts using the differential display technique, but instead is found to be degraded when evaluated by Northern blot hybridization. Cloning and subsequent sequencing identified the partially degraded RNA as 16S ribosomal RNA (rRNA), a major component of mitochondrial ribosomes. Degradation, and associated decreases in the levels of the mature- and precursor-species of 16S rRNA, appear to be dependent upon calcium, but not cytoplasmic protein synthesis nor nuclear transcription. Other decreased mitochondrial RNAs were also identified, including 12S rRNA, NADH dehydrogenase subunit 6, ATPase subunit 6, and cytochrome oxidase subunits I and III. A significant part of many, if not all, of these RNA decreases was due to degradation. As compared with 16S rRNA, significantly less degradation was observed for cytoplasmic 28S/18S rRNAs, even at very high peroxide concentration. Analysis of 21 cytoplasmic mRNAs revealed little or no decrease in mature band signal in response to peroxide, and several cytoplasmic mRNAs were actually up-regulated. Thus, a preferential down-regulation of mitochondrial RNAs occurs in HA-1 fibroblasts in response to hydrogen peroxide. Subcellular fractionation analysis, using 16S rRNA degradation as a gauge, indicates that this down-regulation is specific to mitochondria. The down-regulation of mitochondrial RNAs may represent a general mechanism by which cells protect themselves against oxidative stress.
The use of mitochondrial RNA as an indicator of apoptosis was investigated. Exposure of HA-1 fibroblastic cells to 10 micromol H(2)O(2) per 10(7) cells induced nuclear fragmentation, cell shrinkage, and internucleosomal DNA fragmentation, all characteristics of apoptosis. RNA extracted from control and apoptotic cultures, and analyzed by Northern blot hybridization, revealed a significant increase in the degradation of mitochondrial 16S ribosomal RNA (rRNA) that was associated with apoptosis. Conversely, minimal, if any, degradation of glyceraldehyde-3-phosphate dehydrogenase or actin mRNAs was observed. Similar results were obtained for HA-1 cells treated with the protein kinase inhibitor staurosporine, and for HT-2 T-lymphocytes induced to undergo apoptosis by interleukin-2 withdrawal. In addition, 16S rRNA degradation was an early event that was discernable well before chromatin condensation in hydrogen peroxide-treated HA-1 cells. These observations suggest that degradation of mitochondrial 16S ribosomal RNA is a new marker of mammalian cell apoptosis.
Protein oxidation in vivo is a natural consequence of aerobic life. Oxygen radicals and other activated oxygen species generated as by-products of cellular metabolism or from environmental sources cause modifications to the amino acids of proteins that generally result in loss of protein function/enzymatic activity. Oxidatively modified proteins can undergo direct chemical fragmentation or can form large aggregates due to covalent cross-linking reactions and increased surface hydrophobicity. Mammalian cells exhibit only limited direct repair mechanisms and most oxidized proteins undergo selective proteolysis. The proteasome appears to be largely responsible for the degradation of soluble intracellular proteins. In most cells, oxidized proteins are cleaved in an ATP-and ubiquitin-independent pathway by the 20 S "core" proteasome. The proteasome complex recognizes hydrophobic amino acid residues, aromatic residues, and bulky aliphatic residues that are exposed during the oxidative rearrangement of secondary and tertiary protein structure: increased surface hydrophobicity is a feature common to all oxidized proteins so far tested. The recognition of such (normally shielded) hydrophobic residues is the suggested mechanism by which proteasome catalyzes the selective removal of oxidatively modified cell proteins. By minimizing protein aggregation and cross-linking and by removing potentially toxic protein fragments, proteasome plays a key role in the overall antioxidant defenses that minimize the ravages of aging and disease.
The objective of this study was to elucidate the role of mitochondrial GSH in the reactions leading to mitochondrial DNA oxidative damage in terms of 8-hydroxy-desoxyguanosine (8-HOdG) accumulation. With this purpose, tightly coupled mitochondria depleted of matrix GSH were used and the effects of H2O2 (generated during the oxidation of substrates) on 8-HOdG levels were investigated. Mitochondrial integrity, assessed by O2 uptake, respiratory control and P/O ratios, was conserved upon depletion of GSH up to 95%. The rates of H2O2 production linked to the oxidation of endogenous substrates by control and GSH-depleted mitochondria were similar. Succinate (in the absence or presence of antimycin A) enhanced the rate H2O2 production to a similar extent in both control and GSH-depleted mitochondria. These rates of H2O2 production accounted for 1.5-2.5% of the rate of O2 uptake. The levels of 8-HOdG in GSH-depleted mitochondria were 35-50% lower than those in control mitochondria, when measured at different H2O2 production rates. Conversely, in experiments carried out with calf thymus DNA with different Cu/Fe content, GSH increased 1.4-2.4-fold the accumulation of 8-HOdG. These values were further enhanced (44-50%) by superoxide dismutase and decreased by catalase. The lower levels of 8-HOdG in GSH-depleted mitochondria and the higher levels in GSH-supplemented calf thymus DNA suggest a role for the non-protein thiol in the reactions leading to mtDNA oxidative damage. These findings are interpreted in terms of the redox transitions involving O2, GSH, and metal catalysts bound to DNA. A mechanism is proposed by which GSH plays a critical role in the reduction of DNA-Cu complexes and decays by free radical pathways kinetically regulated by superoxide dismutase.
Oxidative stress has many effects on biological cells, including the modulation of gene expression. Reactive oxygen species are known to up-regulate and down-regulate RNA expression in prokaryotic and eukaryotic cells. We have previously reported that a preferential and calcium-dependent down-regulation of mitochondrial RNAs occurs when HA-1 hamster fibroblasts are exposed to hydrogen peroxide. Here we extend these studies to determine whether this down-regulation is specific to mitochondria RNA or involves general polynucleotide degradation. Degradation and associated decreases in the levels of 16S mitochondrial rRNA following exposure of cells to 400 microM hydrogen peroxide were found to be dependent on calcium at 2 and 5 h. Degradation of mitochondrial genomic DNA was also observed following peroxide exposure, and occurred at similar time points as for mitochondrial RNA degradation. As with mitochondrial RNA degradation, this mitochondrial genomic DNA degradation was dependent on calcium. These results indicate that there is a general, calcium-dependent degradation of mitochondrial polynucleotides following exposure of HA-1 fibroblasts to oxidative stress, and suggest that a dramatic shut-down in mitochondrial biosynthesis is an early-stage response to oxidative stress.
This chapter discusses the regulation of mitochondrial respiration by adenosine diphosphate, oxygen, and nitric oxide (NO). Oxygen is required in adequate steady-state concentrations to sustain mitochondrial respiration and ATP production. The reaction of reduced cytochrome oxidase, the oxygen acceptor, and terminal enzyme of the mitochondrial respiratory chain, with O2 is very and fast the rate of electron transfer to cytochrome oxidase by the respiratory chain is the key factor to define the operational O2 concentration for half-maximal rate of O2 uptake. The intracellular oxygen concentration in mammalian organs and tissues, in the 5–25/μM O2 range, is close and partially overlaps with the critical concentration, in the 2–6/μM O2 range, that limits the rate of mitochondrial respiration. Nitric oxide, the product of the NO synthase of vascular endothelium, with estimated steady state concentrations in mammalian tissues in the 0.05–1/μM NO range has been recognized as a high affinity inhibitor of cytochrome oxidase activity and mitochondrial respiration in a competitive way with O2.
The 20S proteasome is localized in the cytosol and nuclei of mammalian cells. Previous work has shown that the cytosolic 20S proteasome is largely responsible for the selective recognition and degradation of oxidatively damaged cytosolic proteins. Since nuclear proteins are also susceptible to oxidative damage (e.g., from metabolic free radical production, ionizing radiation, xenobiotics, chemotherapy) we investigated the degradation of oxidatively damaged histones, in the presence and in the absence of DNA, by the 20S proteasome. We find that both soluble histones and DNA-bound histones are susceptible to selective proteolytic degradation by the 20S proteasome following mild oxidative damage. In contrast, more severe oxidative damage actually decreases the proteolytic susceptibility of histones. Soluble H1 showed the highest basal and maximal absolute proteolytic rates. Histone fraction H4 exhibited the greatest relative increase in proteolytic susceptibility following oxidation, almost 14-fold, and this occurred at a peroxide exposure of 5 mM. At the other end of the spectrum, histone H2A exhibited a maximal proteolytic response to H2O2 of only 6-fold, and this required an H2O2 exposure of 15 mM. An oxidation of reconstituted linear DNA plasmid-histone complex makes up to 95% of the histones bound to DNA susceptible to degradation, whereas undamaged protein-DNA complexes are not substrates for the proteasome. Severe oxidation by high concentrations of H2O2 appears to decreases the proteolytic susceptibility of histones due to the formation of cross-linked histone-DNA aggregates which appear to inhibit the proteasome. We conclude that the degradation of nuclear proteins is highly selective and requires prior damage of the substrate protein, such as that caused by oxidation.
The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the cytoplasm. Nuclear proteins are also subject to oxidation, and the nucleus of mammalian cells contains proteasome. In human beings, tumor cells frequently are subjected to oxidation as a consequence of antitumor chemotherapy, and K562 human myelogenous leukemia cells have a higher nuclear proteasome activity than do nonmalignant cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs and the mechanisms of inducible tumor resistance to oxidation are of obvious importance. After hydrogen peroxide treatment of K562 cells, degradation of the model proteasome peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increases significantly within minutes. Both increased proteolytic susceptibility of the histone substrates (caused by modification by oxidation) and activation of the proteasome enzyme complex occur independently during oxidative stress. This rapid up-regulation of 20S proteasome activity is accompanied by, and depends on, poly-ADP ribosylation of the proteasome, as shown by inhibitor experiments, 14C-ADP ribose incorporation assays, immunoblotting, in vitro reconstitution experiments, and immunoprecipitation of (activated) proteasome with anti-poly-ADP ribose polymerase antibodies. The poly-ADP ribosylation-mediated activated nuclear 20S proteasome is able to remove oxidatively damaged histones more efficiently and therefore is proposed as an oxidant-stimulatable defense or repair system of the nucleus in K562 leukemia cells.
Proliferating mammalian cells exhibit a broad spectrum of responses to oxidative stress, depending on the stress level encountered. Very low levels of hydrogen peroxide, e.g., 3 to 15 microM, or 0.1 to 0.5 micromol/10(7) cells, cause a significant mitogenic response, 25% to 45 % growth stimulation. Greater concentrations of H2O2, 120 to 150 microM, or 2 to 5 micromol/10(7) cells, cause a temporary growth arrest that appears to protect cells from excess energy use and DNA damage. After 4-6 h of temporary growth arrest, many cells will exhibit up to a 40-fold transient adaptive response in which genes for oxidant protection and damage repair are preferentially expressed. After 18 h of H2O2 adaptation (including the 4-6 h of temporary growth arrest) cells exhibit maximal protection against oxidative stress. The H2O2 originally added is metabolized within 30-40 min, and if no more is added the cells will gradually de-adapt, so that by 36 h after the initial H2O2 stimulus they have returned to their original level of H2O2 sensitivity. At H2O2 concentrations of 250 to 400 microM, or 9 to 14 micromol/10(7) cells, mammalian fibroblasts are not able to adapt but instead enter a permanently growth-arrested state in which they appear to perform most normal cell functions but never divide again. This state of permanent growth arrest has often been confused with cell death in toxicity studies relying solely on cell proliferation assays as measures of viability. If the oxidative stress level is further increased to 0.5 to 1.0 mM H2O2, or 15 to 30 micromol/10(7) cells, apoptosis results. This oxidative stress-induced apoptosis involves nuclear condensation, loss of mitochondrial transmembrane potential, degradation/down-regulation of mitochondrial mRNAs and rRNAs, and degradation/laddering of both nuclear and mitochondrial DNA. At very high H2O2 concentrations of 5.0 to 10.0 mM, or 150 to 300 micromol/10(7) cells and above, cell membranes disintegrate, proteins and nucleic acids denature, and necrosis swiftly follows. Cultured cells grown in 20% oxygen are essentially preadapted or preselected to survive under conditions of oxidative stress. If cells are instead grown in 3% oxygen, much closer to physiological cellular levels, they are more sensitive to an oxidative challenge but exhibit far less accumulated oxidant damage. This broad spectrum of cellular responses to oxidant stress, depending on the amount of oxidant applied and the concentration of oxygen in the cell culture system, provides for a new paradigm of cellular oxidative stress responses.
Oxidative stress is known to modulate RNA expression in both prokaryotic and eukaryotic cells. We have previously determined that a preferential and calcium-dependent downregulation of mitochondrial RNA occurs in HA-1 hamster fibroblasts in response to hydrogen peroxide, and that this is accompanied by the degradation of mitochondrial genomic DNA. Here we extended these studies to determine whether downregulation is specific to transcripts derived from mitochondrial-encoded genes; to determine whether genomic DNA degradation occurs in the nucleus; and to compare overall polynucleotide stress response with cellular growth arrest and apoptosis. We observed that nuclear genome-encoded mRNAs whose protein products are targeted for the electron transport chain of mitochondria were not degraded. Furthermore, early stage degradation of genomic DNA, assessed within the first 5 h of peroxide exposure, was specific to mitochondria, as nuclear genomic DNA was not degraded under the same treatment conditions. These differential degradations occurred under conditions where extensive growth-arrest and moderate apoptosis were observed, and were accompanied by significant induction of the growth arrest mRNAs gadd45, gadd153, and adapt15/gadd7. Combined, these results indicate that there is a general degradation of mitochondrial- but not nuclear-polynucleotides during early stage response of HA-1 fibroblasts to oxidative stress.