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A New Paradigm: Manganese Superoxide Dismutase Influences the Production of H2O2 in Cells and Thereby Their Biological State
Abstract
The principal source of hydrogen peroxide in mitochondria is thought to be from the dismutation of superoxide via the enzyme manganese superoxide dismutase (MnSOD). However, the nature of the effect of SOD on the cellular production of H(2)O(2) is not widely appreciated. The current paradigm is that the presence of SOD results in a lower level of H(2)O(2) because it would prevent the non-enzymatic reactions of superoxide that form H(2)O(2). The goal of this work was to: a) demonstrate that SOD can increase the flux of H(2)O(2), and b) use kinetic modelling to determine what kinetic and thermodynamic conditions result in SOD increasing the flux of H(2)O(2). We examined two biological sources of superoxide production (xanthine oxidase and coenzyme Q semiquinone, CoQ(*-) that have different thermodynamic and kinetic properties. We found that SOD could change the rate of formation of H(2)O(2) in cases where equilibrium-specific reactions form superoxide with an equilibrium constant (K) less than 1. An example is the formation of superoxide in the electron transport chain (ETC) of the mitochondria by the reaction of ubisemiquinone radical with dioxygen. We measured the rate of release of H(2)O(2) into culture medium from cells with differing levels of MnSOD. We found that the higher the level of SOD, the greater the rate of accumulation of H(2)O(2). Results with kinetic modelling were consistent with this observation; the steady-state level of H(2)O(2) increases if K<1, for example CoQ(*-)+O(2)-->CoQ+O(2)(*-). However, when K>1, e.g. xanthine oxidase forming O(2)(*-), SOD does not affect the steady state-level of H(2)O(2). Thus, the current paradigm that SOD will lower the flux of H(2)O(2) does not hold for the ETC. These observations indicate that MnSOD contributes to the flux of H(2)O(2) in cells and thereby is involved in establishing the cellular redox environment and thus the biological state of the cell.
... Moreover, sBE enhanced oxidation of Amplex Red, an indicator of steady-state levels of H 2 O 2 at 3.0 mT but not 0.5 mT compared to untreated cells ( Figure 5A and S5A). These findings are consistent with the ratio of production being altered by disproportionation of O 2 •− to H 2 O 2 (Buettner et al., 2006). We then assessed the effects of sBE on markers of O 2 ...
... •− disproportionation (Buettner et al., 2006;Güler et al., 2006;Lin et al., 2018;Usselman et al., 2016). Due to the diversity of interactions and the high degree of reactivity, the biological effects of a given set of ROS signals are challenging to decode. ...
... Furthermore, preclinical data show that MnSOD enzyme overexpression has antitumor effects that are abrogated by overexpression of H 2 O 2 -metabolizing enzymes including catalase and glutathione peroxidases (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Last, recent work has provided a mathematical model describing a possible mechanism by which MnSOD overexpression could selectively enhance H 2 O 2 flux in cancer cell mitochondria when acting as a tumor suppressor (17). ...
... Second, tumor cells exhibit fundamental differences in mitochondrial metabolism leading to higher steady-state concentrations of O 2 •− . It has also been suggested that higher steady-state concentrations of O 2 •− in tumor cells relative to normal cells occur as a result of alterations in the stoichiometry of the assembly of electron transport chains (42), allowing AVA in cancer cells to increase the flux of electrons to form H 2 O 2 via Le Chatelier's principle (17). In addition, transient increased reactive oxygen species (ROS) generation via collapse of mitochondrial membrane potential can trigger ROSinduced ROS release (RIRR), releasing ROS into the cytosol, which induces RIRR in neighboring mitochondria, leading to a cascade of enhanced ROS production and the potential for further mitochondrial or cellular injury (43). ...
Avasopasem manganese (AVA or GC4419), a selective superoxide dismutase mimetic, is in a phase 3 clinical trial ( NCT03689712 ) as a mitigator of radiation-induced mucositis in head and neck cancer based on its superoxide scavenging activity. We tested whether AVA synergized with radiation via the generation of hydrogen peroxide, the product of superoxide dismutation, to target tumor cells in preclinical xenograft models of non–small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, and pancreatic ductal adenocarcinoma. Treatment synergy with AVA and high dose per fraction radiation occurred when mice were given AVA once before tumor irradiation and further increased when AVA was given before and for 4 days after radiation, supporting a role for oxidative metabolism. This synergy was abrogated by conditional overexpression of catalase in the tumors. In addition, in vitro NSCLC and mammary adenocarcinoma models showed that AVA increased intracellular hydrogen peroxide concentrations and buthionine sulfoximine– and auranofin-induced inhibition of glutathione- and thioredoxin-dependent hydrogen peroxide metabolism selectively enhanced AVA-induced killing of cancer cells compared to normal cells. Gene expression in irradiated tumors treated with AVA suggested that increased inflammatory, TNFα, and apoptosis signaling also contributed to treatment synergy. These results support the hypothesis that AVA, although reducing radiotherapy damage to normal tissues, acts synergistically only with high dose per fraction radiation regimens analogous to stereotactic ablative body radiotherapy against tumors by a hydrogen peroxide–dependent mechanism. This tumoricidal synergy is now being tested in a phase I-II clinical trial in humans ( NCT03340974 ).
... The biochemistry leading to injurious ROS production by mitochondria, and oxidative distress, is best imagined with an analogy, proposed and diagramed in [77]: if the ETC is a bathtub where electrons are held and complex I represents the inflowing faucet of individual electrons from NADH, then the coordinated transfer by complex IV of four electrons onto oxygen to make two water molecules represents the outflowing electron drain. Electrons enter individually and collect at the drain to make water from oxygen without much risk of damage. ...
... Further, the combination of low flux to complex IV and high oxygen tension places chondrocyte ETCs at a high risk for aberrant one-electron reductions of oxygen in any instance where NADH:NAD is high. Martin has demonstrated that ROS production from complex I is the crucial mechanism of acute injury after trauma to articular cartilage [45,46,70,74,75,[77][78][79][83][84][85][86]; however, a detailed description of the relationship between NADH:NAD and ROS production by complex I has not been made. Mechanotransductive calcium signals sensitive to modeling conditions [47][48][49][50][51][52][53][54][55][56]87] also appear likely to be linked to mitochondrial polarization or depolarization, known determinates of ROS production, but precise mechanisms are undescribed. ...
Purpose of Review
This review will cover foundational studies and recent findings that established key concepts for understanding the importance of redox biology to chondrocyte mitochondrial function and osteoarthritis pathophysiology after injury.
Recent Findings
Articular chondrocyte mitochondria can be protected with a wide variety of antioxidants that will be discussed within a framework suggested by classic studies. These agents not only underscore the importance of thiol metabolism and associated redox function for chondrocyte mitochondria but also suggest complex interactions with signal transduction pathways and other molecular features of osteoarthritis that require more thorough investigation. Emerging evidence also indicates that reductive stress could occur alongside oxidative stress.
Summary
Recent studies have shed new light on historic paradoxes in chondrocyte redox and mitochondrial physiology, leading to the development of promising disease-modifying therapies for posttraumatic osteoarthritis.
... Moreover, sBE enhanced oxidation of Amplex Red, an indicator of steady-state levels of H 2 O 2 at 3.0 mT but not 0.5 mT compared to untreated cells ( Figure 5A and S5A). These findings are consistent with the ratio of production being altered by disproportionation of O 2 •− to H 2 O 2 (Buettner et al., 2006). We then assessed the effects of sBE on markers of O 2 ...
... •− disproportionation (Buettner et al., 2006;Güler et al., 2006;Lin et al., 2018;Usselman et al., 2016). Due to the diversity of interactions and the high degree of reactivity, the biological effects of a given set of ROS signals are challenging to decode. ...
... Moreover, sBE enhanced oxidation of Amplex Red, an indicator of steady-state levels of H 2 O 2 at 3.0 mT but not 0.5 mT compared to untreated cells ( Figure 5A and S5A). These findings are consistent with the ratio of production being altered by disproportionation of O 2 •− to H 2 O 2 (Buettner et al., 2006). We then assessed the effects of sBE on markers of O 2 ...
... •− disproportionation (Buettner et al., 2006;Güler et al., 2006;Lin et al., 2018;Usselman et al., 2016). Due to the diversity of interactions and the high degree of reactivity, the biological effects of a given set of ROS signals are challenging to decode. ...
... Moreover, sBE enhanced oxidation of Amplex Red, an indicator of steady-state levels of H 2 O 2 at 3.0 mT, but not 0.5 mT, compared to untreated cells ( Figures 5A and S5A). These findings are consistent with the ratio of production being altered by disproportionation of O 2 ,À to H 2 O 2 (Buettner et al., 2006). We then assessed the effects of sBE on markers of O 2 ,À in vivo using HFD mice. ...
... ROS vary in terms of their reactivity and molecular targets; thus, modestly altering ROS stoichiometries may result in profoundly different biological effects (Brandes et al., 2018;Jones and Sies, 2015). In line with this concept and prior work on EMFs, we demonstrated that sBE alters ROS homeostasis in a manner consistent with the altered metabolism of O 2 ,À (Buettner et al., 2006;G€ uler et al., 2006;Lin et al., 2018;Usselman et al., 2016). Due to the diversity of interactions and the high degree of reactivity, the biological effects of a given set of ROS signals are challenging to decode. ...
Aberrant redox signaling underlies the pathophysiology of many chronic metabolic diseases, including type 2 diabetes (T2D). Methodologies aimed at rebalancing systemic redox homeostasis have had limited success. A noninvasive, sustained approach would enable the long-term control of redox signaling for the treatment of T2D. We report that static magnetic and electric fields (sBE) noninvasively modulate the systemic GSH-to-GSSG redox couple to promote a healthier systemic redox environment that is reducing. Strikingly, when applied to mouse models of T2D, sBE rapidly ameliorates insulin resistance and glucose intolerance in as few as 3 days with no observed adverse effects. Scavenging paramagnetic byproducts of oxygen metabolism with SOD2 in hepatic mitochondria fully abolishes these insulin sensitizing effects, demonstrating that mitochondrial superoxide mediates induction of these therapeutic changes. Our findings introduce a remarkable redox-modulating phenomenon that exploits endogenous electromagneto-receptive mechanisms for the noninvasive treatment of T2D, and potentially other redox-related diseases.
... h and 6 h after termination of the tNMR treatment ( Fig. 2A and B), while mitochondrial H 2 O 2 peaked at time points 0 h and 4 h (Fig. 2, C). It is known that the enzymes of the , but also the flux and thus the amount of H 2 O 2 produced [45,46]. Our data (Fig. 2) appear to support the hypothesis of [21], in which a major role in magnetic signalling is assigned to O 2 .itself. ...
Cellular magnetic field effects are assumed to base on coherent singlet-triplet interconversion of radical pairs that are sensitive to applied radiofrequency (RF) and weak magnetic fields (WEMFs), known as radical pair mechanism (RPM). As a leading model, the RPM explains how quantum effects can influence biochemical and cellular signalling. Consequently, radical pairs generate reactive oxygen species (ROS) that link the RPM to redox processes, such as the response to hypoxia and the circadian clock. Therapeutic nuclear magnetic resonance (tNMR) occupies a unique position in the RPM paradigm because of the used frequencies, which are far below the range of 0.1–100 MHz postulated for the RPM to occur. Nonetheless, tNMR was shown to induce RPM like effects, such as increased extracellular H2O2 levels and altered cellular bioenergetics. In this study we compared the impact of tNMR and intermittent hypoxia on the circadian clock, as well as the role of superoxide in tNMR induced ROS partitioning. We show that both, tNMR and intermittent hypoxia, exert on/off effects on cellular clocks that are dependent on the time of application (day versus night). In addition, our data provide further evidence that superoxide plays a central role in magnetic signal transduction. tNMR used in combination with scavengers, such as Vitamin C, led to strong ROS product redistributions. This discovery might represent the first indication of radical triads in biological systems.
... So it is possible that the produced ROS could not oxidize the lipid content because of the lower concentration as a lower TBARS level was observed in the present study in the rainy season. The SOD enzyme converts superoxide radicals into H 2 O 2 (Buettner et al., 2006). The elevation of SOD activity in the crab tissue during the winter-summer season might explain the involvement of this enzyme in the neutralization of superoxide radicals to increase the cellular H 2 O 2 level. ...
The oxidative stress (OS) condition and antioxidant level as a function of pH, few major elements, temperature, turbidity, organic carbon, sediment, and water salinity are vital to understanding the redox homeostasis of inhabiting animals. These parameters are also used to monitor environmental health. A spatiotemporal redox antioxidant system, followed by discriminant function analysis about the aforementioned abiotic factors, was investigated in the muscle, gill, and hepatopancreas of the mud crab, Scylla serrata, sampled from the Indian coastal belt along the Bay of Bengal (Tamil Nadu and Odisha) and the Arabian Sea (Gujarat) as a measure of environmental health assessment. Results revealed that the redox homeostasis of mud crabs significantly varied with seasonal fluctuations of abiotic factors and sediment chemistry. The level of superoxide dismutase and the non-protein-SH group were negatively correlated, whereas other antioxidant molecules with lipid peroxidation levels were positively correlated with abiotic factors. Only the activities of glutathione peroxidase and glutathione reductase were strongly correlated with all the abiotic factors. The hepatopancreas was found to be the most susceptible organ to OS. The lipid peroxidation level was 20–25 times higher in hepatopancreatic tissue than that in other tissues. The antioxidant level was elevated to 200% during the summer compared to the rainy season. Thus, the results of redox homeostasis in S. serrata may be useful for monitoring the ecotoxic effects of estuarine and marine environments and managing the inhabiting species.
... As an essential trace element for human body, manganese (Mn) plays an important role in promoting the normal growth and development of bones, maintaining normal glucose, lipid metabolism and the function of central nervous system. Manganese is also an important component and activator of some critical enzymes in the body that regulate oxidative stress (1), antioxidant status (2), mitochondrial function (3) and neurotransmitter synthesis (4). Moreover, manganese ions (Mn 2+ ) has also shown promises as a functional intracellular MR imaging contrast agent through its ability to evaluate cellular integrity, activity, and neural connectivity (5,6). ...
Manganese (Mn), a nutrient inorganic trace element, is necessary for a variety of physiological processes of animal body due to their important roles in oxidative regulation effects and other aspects of activities. Moreover, manganese ion (Mn ²⁺ ) has widely reported to be crucial for the regulations of different immunological responses, thus showing promising application as potential adjuvants and immunotherapeutics. Taking the advantages of Mn-based biological and immunological activities, Manganese dioxide nanoparticles (MnO 2 NPs) are a new type of inorganic nanomaterials with numerous advantages, including simple preparation, low cost, environmental friendliness, low toxicity, biodegradable metabolism and high bioavailability. MnO 2 NPs, as a kind of drug carrier, have also shown the ability to catalyze hydrogen peroxide (H 2 O 2 ) to produce oxygen (O 2 ) under acidic conditions, which can enhance the efficacy of radiotherapy, chemotherapy and other therapeutics for tumor treatment by remodeling the tumor microenvironment. More importantly, MnO 2 NPs also play important roles in immune regulations both in innate and adaptive immunity. In this review, we summarize the biological activities of Manganese, followed by the introduction for the biological and medical functions and mechanisms of MnO 2 NPs. What’s more, we emphatically discussed the immunological regulation effects and mechanisms of MnO 2 NPs, as well as their potentials to serve as adjuvants and immunomodulators, which might benefit the development of novel vaccines and immunotherapies for more effective disease control.
... Manganese (Mn) is an essential trace element in various cellular functions in the body, serving as a cofactor of several enzymes such as glutamine synthetase (Wedler and Denman, 1984) and Mn superoxide dismutase (MnSOD) (Buettner et al., 2006). The daily reference intake (2.3 mg/day for men and 1.8 mg/ day for women) of Mn is required for physiological functions and acquired through a regular diet . ...
Chronic exposure to elevated levels of manganese via occupational or environmental settings causes a neurological disorder known as manganism, resembling the symptoms of Parkinson's disease, such as motor deficits and cognitive impairment. Numerous studies have been conducted to characterize manganese's neurotoxicity mechanisms in search of effective therapeutics, including natural and synthetic compounds to treat manganese toxicity. Several potential molecular targets of manganese toxicity at the epigenetic and transcriptional levels have been identified recently, which may contribute to develop more precise and effective gene therapies. This review updates findings on manganese-induced neurotoxicity mechanisms on intracellular insults such as oxidative stress, inflammation, excitotoxicity, and mitophagy, as well as transcriptional dysregulations involving Yin Yang 1, RE1-silencing transcription factor, transcription factor EB, and nuclear factor erythroid 2-related factor 2 that could be targets of manganese neurotoxicity therapies. This review also features intracellular proteins such as PTEN-inducible kinase 1, parkin, sirtuins, leucine-rich repeat kinase 2, and α-synuclein, which are associated with manganese-induced dysregulation of autophagy/mitophagy. In addition, newer therapeutic approaches to treat manganese's neurotoxicity including natural and synthetic compounds modulating excitotoxicity, autophagy, and mitophagy, were reviewed. Taken together, in-depth mechanistic knowledge accompanied by advances in gene and drug delivery strategies will make significant progress in the development of reliable therapeutic interventions against manganese-induced neurotoxicity. CITATION Pajarillo E, Nyarko-Danquah I, Digman A, Multani HK, Kim S, Gaspard P, Aschner M and Lee E (2022), Mechanisms of manganese-induced neurotoxicity and the pursuit of neurotherapeutic strategies.
... Indeed, in AD and PD, MAO activity is enhanced [379,380], and MAO inhibitors have been proposed as therapeutic agents for AD [381] and are currently a therapeutic option for PD [382]. MAOs can also keep ROS generation under control, preventing neurotransmitter oxidation; in fact, while the MAO-catalyzed reaction produces a single hydrogen peroxide molecule, the oxidation of amine neurotransmitters would generate peroxide at high rates [383]. The MAO reaction is coupled with aldehyde dehydrogenase (ADH), which converts the aldehydes to the corresponding acids, detoxifying them. ...
Brain metabolism is comprised in Alzheimer’s disease (AD) and Parkinson’s disease (PD). Since the brain primarily relies on metabolism of glucose, ketone bodies, and amino acids, aspects of these metabolic processes in these disorders—and particularly how these altered metabolic processes are related to oxidative and/or nitrosative stress and the resulting damaged targets—are reviewed in this paper. Greater understanding of the decreased functions in brain metabolism in AD and PD is posited to lead to potentially important therapeutic strategies to address both of these disorders, which cause relatively long-lasting decreased quality of life in patients.
... The microenvironment of tumors (TME) is characterized by a high content of reactive oxygen species (ROS), which is a collective term referring to unstable, reactive, partially reduced oxygen derivatives, such as hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 − ), singlet oxygen ( 1 O 2 ), and hydroxyl radical (·OH) [1,2]. The elevated ROS in the TME is mainly due to the dismutation of superoxide dismutase in mitochondria [3,4]. Additionally, the production of ROS is also attributed to the rapid proliferation of tumor cells [5]. ...
Cancer is one of the diseases that threatens human health and is a leading cause of mortality worldwide. High levels of reactive oxygen species (ROS) have been observed in cancer tissues compared with normal tissues in vivo, and it is not yet known how this influences chemotherapeutic drug action. Cationic porphyrin 5,10,15,20-tetra-(N-methyl-4-pyridyl) porphyrin (TMPyP4) is a photosensitizer used in photodynamic therapy (PDT) and a telomerase inhibitor used in the treatment of telomerase-positive cancer. Here, we investigated the anticancer activity of TMPyP4 in A549 and PANC cells cultured in H2O2. The results showed that compared to TMPyP4 alone, the combination of TMPyP4 and H2O2 exhibited sensitization effects on cell viability and colony formation inhibition and apoptosis in A549 and PANC cells, but had no effect in human normal MIHA cells. Mechanistically, the combination of TMPyP4 and H2O2 activates high ROS and mitochondrial membrane potential in A549 and PANC cells, resulting in intense DNA damage and DNA damage responses. Consequently, compared to TMPyP4 alone, TMPyP4 and H2O2 combined treatment upregulates the expression of BAX, cleaved caspase 3, and p-JNK and downregulates the expression of Bcl-2 in A549 and PANC cells. Taken together, these data suggested that H2O2 enhanced the anticancer activity of TMPyP4-mediated ROS-dependent DNA damage and related apoptotic protein regulation, revealing that the high ROS tumor microenvironment plays an important role in chemotherapeutic drug action.
... Induced by the disproportionation of superoxide dismutase in mitochondria, H 2 O 2 in the TME is overproduced compared with that in normal tissues but endogenous H 2 O 2 in the TME is still at the microscale of 100 µM-1 mM [15,16]. For those POD-mimicking nanozymes utilized in organisms, in cases of low POD-like catalytic activity, endogenous H 2 O 2 in the TME is insufficient. ...
Nanozymes are emerging as a promising strategy for the treatment of tumors. Herein, to cope with the tumor microenvironment (TME), weak acidity (pH 5.6 to 6.8) and trace amounts of overexpressed hydrogen peroxide (H2O2) (100 µM–1 mM), we report nitrogen-doped graphene nanomaterials (N-GNMs), which act as highly efficient catalytic peroxidase (POD)-mimicking nanozymes in the TME for tumor-specific treatment. N-GNMs exhibit POD catalytic properties triggered by a weakly acidic TME and convert H2O2 into highly toxic hydroxyl radicals (•OH) thus causing the death of tumor cells while in the neutral pH surroundings of normal tissues, such catalysis is restrained and leaves normal cells undamaged thereby achieving a tumor-specific treatment. N-GNMs also display a high catalytic activity and can respond to the trace endogenous H2O2 in the TME resulting in a high efficiency of tumor therapy. Our in vitro chemical and cell experiments illustrated the POD-like activity of N-GNMs and in vivo tumor model experiments confirmed the significant inhibitory effect of N-GNMs on tumor growth.
... In the present study, Mn caused significant elevation of tissue levels of MDA associated with a marked decrease in the activities of antioxidant enzymes, namely, SOD and GSH, which coincided with the results of Nadeem et al. (2018). SOD and GSH are crucial components of the antioxidant defense mechanism, playing a principal role in balancing the intracellular redox status by acting as an antioxidant (Buettner et al. 2006). The significant decrease in SOD and GSH levels could be attributed to the Mn accumulation in mitochondria that inhibits the oxidative phosphorylation and encourages free radical generation. ...
Despite being important in the body’s mechanisms, excessive accumulation of manganese (Mn) can induce severe toxicity in vital organs of the body. Thymoquinone (TQ) is extracted from Nigella sativa seeds which recently gained popularity as dietary supplements and plant-based antioxidants. Vildagliptin (VLD) is a dipeptidyl peptidase IV (DPPIV) inhibitor, approved as anti-hyperglycemic agents with cardioprotective and renoprotective effects. The present study aimed to investigate the nephrotoxicity of Mn and the potential protective effects of thymoquinone and vildagliptin. Sixty-four adult male albino rats were equally divided into 8 groups: group I (control, received no medication), group II (vehicle, received normal saline), group III (TQ, 50 mg/kg/day), group IV (VLD, 10 mg/kg/day), group V (MnCl2, 50 mg/kg/day), group VI (Mn+TQ), group VII (Mn+VLD), and group VIII (Mn+TQ+VLD). Groups VI, VII, and VIII, received the same previously mentioned doses. All drugs were orally gavaged for 12 weeks. Manganese administration resulted in an elevation in the levels of serum and tissues Mn, blood glucose, serum urea, creatinine, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and reduction in insulin, kidney superoxide dismutase (SOD), glutathione (GSH), and interleukin-10. Histopathological structural renal damage was detected associated with strong positive immunoexpression of caspase-3. On the other hand, individual or combined TQ and VLD administration with Mn significantly decreased the serum and tissue levels of Mn, declined the blood glucose, inflammatory markers, oxidative stress markers, ameliorated the histopathological effects, and down-regulated the immunoexpression of caspase-3. In conclusion, TQ and VLD co-administration elicited protective effects against Mn-induced nephrotoxicity.
... The microenvironment of tumors (TME) is characterized by a high content of reactive oxygen species (ROS), which is a collective term referring to unstable, reactive, partially reduced oxygen derivatives, such as hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 − ), singlet oxygen ( 1 O 2 ) and hydroxyl radical (·OH) [1,2]. The elevated ROS in the TME is mainly due to the dismutation of superoxide dismutase in mitochondria [3,4]. Additionally, the production of ROS is also attributed to the rapid proliferation of tumor cells [5]. ...
Cancer is one of the diseases that threatens human health and is a leading cause of mortality worldwide. High levels of reactive oxygen species (ROS) have been observed in cancer tissues compared with normal tissues in vivo , and it is not yet known how this influences chemotherapeutic drug action. Cationic porphyrin 5,10,15,20-tetra-(N-methyl-4-pyridyl) porphyrin (TMPyP4) is a photosensitizer used in photodynamic therapy (PDT) and a telomerase inhibitor used in the treatment of telomerase-positive cancer. Here, we investigated the anticancer activity of TMPyP4 in A549 and PANC cells cultured in H 2 O 2 . The results showed that compared to TMPyP4 alone, the combination of TMPyP4 and H 2 O 2 exhibited sensitization effects on cell viability and colony formation inhibition and apoptosis in A549 and PANC cells but had no effect in human normal MIHA cells. Mechanistically, the combination of TMPyP4 and H 2 O 2 activates high ROS and mitochondrial membrane potential in A549 and PANC cells, resulting in intense DNA damage and DNA damage responses. Consequently, compared to TMPyP4 alone, TMPyP4 and H 2 O 2 combined treatment upregulates the expression of BAX, cleaved caspase 3, and p-JNK, and downregulates the expression of Bcl-2 in A549 and PANC cells. Taken together, these data suggested that H 2 O 2 enhanced the anticancer activity of TMPyP4-mediated ROS-dependent DNA damage and related apoptotic protein regulation, revealing that the high ROS tumor microenvironment plays an important role in chemotherapeutic drug action.
... The antioxidant system in the body can also produce ROS. For example, the scavenging activity of SOD involves the conversion of the superoxide anion radical (O 2 − ) into H 2 O 2 [22]. However, H 2 O 2 is detoxified by CAT and GSH peroxidase (GPx), which are the two most important enzymes that regulate intracellular H 2 O 2 levels in biological systems [23]. ...
Radon inhalation activates antioxidative functions in mouse organs, thereby contributing to inhibition of oxidative stress-induced damage. However, the specific redox state of each organ after radon inhalation has not been reported. Therefore, in this study, we evaluated the redox state of various organs in mice following radon inhalation at concentrations of 2 or 20 kBq/m3 for 1, 3 or 10 days. Scatter plots were used to evaluate the relationship between antioxidative function and oxidative stress by principal component analysis (PCA) of data from control mice subjected to sham inhalation. The results of principal component (PC) 1 showed that the liver and kidney had high antioxidant capacity; the results of PC2 showed that the brain, pancreas and stomach had low antioxidant capacities and low lipid peroxide (LPO) content, whereas the lungs, heart, small intestine and large intestine had high LPO content but low antioxidant capacities. Furthermore, using the PCA of each obtained cluster, we observed altered correlation coefficients related to glutathione, hydrogen peroxide and LPO for all groups following radon inhalation. Correlation coefficients related to superoxide dismutase in organs with a low antioxidant capacity were also changed. These findings suggested that radon inhalation could alter the redox state in organs; however, its characteristics were dependent on the total antioxidant capacity of the organs as well as the radon concentration and inhalation time. The insights obtained from this study could be useful for developing therapeutic strategies targeting individual organs.
... It is produced from superoxide via dismutation (i.e. reactions catalyzed by superoxide dismutase), reactions with electron donors and chain reactions [15]. Hydrogen peroxide is involved in many physiological processes such as hypoxic signal transduction, cell differentiation, proliferation and mediating immune response [14,16]. ...
Commelina benghalensis is a troublesome but exotic weed native to the African and Asian subregions used traditionally for the treatment and management of various disorders. The aim of this study was to investigate the potential antioxidant activity of the methanolic leaf extract of Commelina benghalensis using various in vitro models. This was done by investigating the ability of the extract to scavenge hydrogen peroxide and hydroxyl radical. Other activities assessed were the reducing ability, ability to inhibit erythrocyte damage and reduce ferrous-ascorbate induced lipid peroxidation on bovine liver and egg yolk homogenates. The results revealed that the plant extract possessed significant hydrogen peroxide and hydroxyl radical scavenging abilities. The extract also possessed significant ability to reduce ferric ions and molybdate VI. The methanolic extract also significantly inhibited hydrogen peroxide-induced erythrocyte hemolysis and lipid peroxidation. Lipid peroxidation in bovine liver and egg yolk homogenates induced by the ferrous-ascorbate system was also reduced by the extract. In many instances, the effect of the extract was concentration-dependent. (p < 0.05). This antioxidant activity of the extract is ascribed to the phytochemicals which probably acted in synergy thus the Commelina benghalensis leaves could be exploited both nutraceutically and pharmacologically.
... A fragmentary understanding of ART-induced oxidative stress calls for a chemically defined two-step bifurcation model for ART-induced mitochondrial oxidative stress (see Figure 6). The first bifurcation stipulates that the nature of the oxidative stress observed depends, in part, on the ability of superoxide to evade MnSOD to directly react with its matrix interactome [208,213]. Specifically, matrix NO • to produce peroxynitrite (and subsequently free radicals, reactions 10-11), protein bound (e.g., aconitase) or "free" transition metal ions [214][215][216]. The alkaline pH of the mitochondrial matrix means that MnSOD controls the direct reactivity of superoxide, because uncatalyzed dismutation (reaction 12) proceeds at a negligible rate without HO 2 • (reaction 13), because two anions electrostatically repel one another [55,56,[217][218][219]. ...
The consensus that assisted reproduction technologies (ART), like in vitro fertilization, to induce oxidative stress (i.e., the known) belies how oocyte/zygote mitochondria—a major presumptive oxidative stressor—produce reactive oxygen species (ROS) with ART being unknown. Unravelling how oocyte/zygote mitochondria produce ROS is important for disambiguating the molecular basis of ART-induced oxidative stress and, therefore, to rationally target it (e.g., using site-specific mitochondria-targeted antioxidants). I review the known mechanisms of ROS production in somatic mitochondria to critique how oocyte/zygote mitochondria may produce ROS (i.e., the unknown). Several plausible site-and mode-defined mitochondrial ROS production mechanisms in ART are proposed. For example, complex I catalyzed reverse electron transfer-mediated ROS production is conceivable when oocytes are initially extracted due to at least a 10% increase in molecular dioxygen exposure (i.e., the intriguing). To address the term oxidative stress being used without recourse to the underlying chemistry, I use the species-specific spectrum of biologically feasible reactions to define plausible oxidative stress mechanisms in ART. Intriguingly, mitochondrial ROS-derived redox signals could regulate embryonic development (i.e., their production could be beneficial). Their potential beneficial role raises the clinical challenge of attenuating oxidative damage while simultaneously preserving redox signaling. This discourse sets the stage to unravel how mitochondria produce ROS in ART, and their biological roles from oxidative damage to redox signaling.
... Superoxide dismutase is a critical antioxidant in the mammalians. It has a vital role in preserving the basic cellular physiology (Fridovich, 1997;Waddington et al., 2000;Buettner et al., 2006). ...
... As is known, SOD is the enzyme catalyzing the conversion of superoxide to hydrogen peroxide and molecular oxygen. SOD is present in almost all living organisms and catalyzes the dismutation reaction of superoxide 23 . ...
... When superoxides are broken down, hydrogen peroxide is formed. (35) The hydrogen peroxide formed continues to break down with catalase. (36) During these events, glutathione s-transferases act as catalysts. ...
Objective:
We aimed to show that endometrial and tubes epithelium suffered free radical damage during HSG in line with previously made many studies on different systems.
Material and methods:
A total of forty rats were evaluated in five different groups. Only ionized radiation was applied to the two groups. In another two groups, iohexol was applied together with ionizing radiation. One group was determined as the control group. Groups were evaluated after seven and forty -two days. Inflammation and cellular changes were evaluated histopathologically. Cellular activities of antioxidant enzymes were measured immunohistochemically.
Results:
Ionizing radiation and iohexol had serious negative effects on endometrium and tubes in both periods, especially in the late period. Although there is a lot of research done before, there is no definitive method yet to protect against the harmful effects of iodinated contrast agents and ionizing radiation.
Conclusion:
New methods need to be explored to protect cells and tissues from reactive oxygen radical damage caused by HSG.
... Arginases II [130] Mostly in kidneys Mitochondria Amino acid metabolism Pyruvate Carboxylase [131] Liver, kidney and adipose tissue Mitochondria Gluconeogenesis and lipogenesis Mn-superoxide dismutase [132] ubiquitous Mitochondria Antioxidant activity Glutamine Synthetase [133] Brain, kidneys, and liver Both Amino acid metabolism protein serine/threonine phosphatase-1 (PP1) [134] ubiquitous cytosol cell survival and differentiation ...
Background
Manganese (Mn) is a metal ubiquitously present in nature and essential for many living organisms. As a trace element, it is required in small amounts for the proper functioning of several important enzymes, and reports of Mn deficiency are indeed rare.
Methods
This mini-review will cover aspects of Mn toxicokinetics and its impact on brain neurotransmission, as well as its Janus-faced effects on humans and other animal’s health.
Results
The estimated safe upper limit of intracellular Mn for physiological function is in anarrow range of 20 to 53 μM.Therefore, intake of higher levels of Mn and the outcomes have been well documented.
Conclusion
The metal affects mostly the brain by accumulating in specific brain areas, altering cognitive functions and locomotion, thus severely impacting the health of the exposed organisms.
... A recent study has shown that cisplatin reduces SOD activity in blood serum [32]. All types of SODs are enzymes that catalyzing the dismutation of superoxide and the conversion of superoxide to hydrogen peroxide and molecular oxygen [33]. The data obtained from the literature indicates that SOD is an endogenous antioxidant enzyme inhibiting ROS production and protecting tissues from ROS damage. ...
Rutin (Vitamin P1) is an antioxidant and anti-inflammatory flavonoid. This study examined the effect of rutin on the potential of cisplatin to cause oxidative and proinflammatory damage in blood serum, ureter, bladder and urethra in rats. Animals were divided into four groups: (i) healthy (HG), (ii) 50 mg/kg of rutin + 5 mg/kg of cisplatin (C + R50), (iii) 100 mg/kg of rutin + 5 mg/kg of cisplatin (C + R100) and (iv) only cisplatin (CG). Rutin and distilled water were applied once a day for eight days. Cisplatin was injected intraperitoneally at a dose of 5 mg/kg for eight days, once every two days. Finally, animals were sacrificed by high -dose anesthesia after blood samples were taken. Ureter, bladder and urethra tissues were removed and malondialdehyde (MDA), myeloperoxidase (MPO), total glutathione (tGSH), superoxide dismutase (SOD) interleukin beta (IL-1β) and tumor necrosis factor alpha (TNF-α) levels were measured. The results showed that the levels of MDA, MPO, IL-1β and TNF-α in the blood serum of the CG group increased significantly (p < 0.0001) and the tGSH and SOD levels decreased compared to the HG. Rutin administration reversed the effect as seen in C + R50 and C + R100 groups. However, there was no statistically significant difference in the levels of MDA, MPO, tGSH, SOD, IL-1β and TNF-α in ureter, bladder and urethra tissue (p > 0.05).This suggests that cisplatin produces oxidative stress in the blood serum but not in the ureter, bladder and urethra tissues. It was observed that rutin prevented the cisplatin-related oxidative and proinflammatory damage in the blood serum.
... The H 2 O 2 content and SOD activity were promoted by the 0.125 mM NaHS treatment compared with the HT treatment. Therefore, the SOD activity might have contributed to the accumulation of H 2 O 2 [39]. Studies have reported that H 2 S contributes to higher activities of reactive oxygen species (ROS)-scavenging enzymes under abiotic stress [40][41][42]. ...
Hydrogen sulfide (H2S) is endogenously produced in plant cells and plays an essential role in root development. Given its potential for future agricultural applications, the optimal concentration of sodium hydrosulfide (NaHS, an H2S donor) and the potential mechanisms for root development in the strawberry ‘Seolhyang’ were investigated in this study. The results showed that NaHS with a concentration of 1.250 mM had a positive effect on root development in strawberry. Further experiments showed that exogenous NaHS elevated the H2S content in the root. The dry root weight was increased by the 1.250 mM NaHS treatment, but was reduced by the hypotaurine (an H2S scavenger) treatment. Similar changes were found between H2S and soluble sugar contents, indicating that H2S enhanced the accumulation of soluble sugar. Therefore, it is suggested that the accumulation of soluble sugar induced by H2S is either directly or indirectly involved in root development in strawberry during plug production. Moreover, superoxide dismutase was shown to have contributed to the elevated H2O2 contents. These results contribute to our understanding of the role that H2S plays and some of the relevant mechanisms in which H2S regulates root development.
... As observed from the CL kinetics of the system, it can be possibly concluded that the catalytic reduction of artesunate is similar to that of hydrogen peroxide. The effects on CL intensity after addition of varying concentrations of sodium azide-scavengers of singlet oxygen ( 1 O 2 ) [32], superoxide dismutase-scavengers of superoxide radical anion (O 2 •− ) [33], mannitol scavengers of ROS [17,34,35] and thiourea-scavengers of hydroxyl radical (HO • ) [36] are studied to reveal the CL mechanism. As shown in Fig. S1 A S2) consistent with the typical spectrum of luminol [22,37]. ...
... The latter displays specific catalytic competence toward peroxidation of PUFA-CLs and several other anionic phospholipids, such as phosphatidylserine (PS) and phosphatidylglycerol (PG). 28 The supply of the oxidizing equivalents, H 2 O 2 , is provided by the disrupted electron transport leading to the re-direction of the electron flow to O 2 to yield O 2 •-that dismutates to H 2 O 2. 74 Initially, the rate constant of the reaction of H 2 O 2 with cyt c/CL heme is very low (k ~ 46 M −1 s −1 ). However, after several catalytic cycles, accumulated CL hydroperoxides react with the cyt c/CL complex much more effectively (10 3 -10 5 M −1 s −1 ). ...
In addition to the known prominent role of polyunsaturated (phospho)lipids as structural blocks of biomembranes, there is an emerging understanding of another important function of these molecules as a highly diversified signaling language utilized for intra- and extracellular communications. Technological developments in high-resolution mass spectrometry facilitated the development of a new branch of metabolomics, redox lipidomics. Analysis of lipid peroxidation reactions has already identified specific enzymatic mechanisms responsible for the biosynthesis of several unique signals in response to inflammation and regulated cell death programs. Obtaining comprehensive information about millions of signals encoded by oxidized phospholipids, represented by thousands of interactive reactions and pleiotropic (patho)physiological effects, is a daunting task. However, there is still reasonable hope that significant discoveries, of at least some of the important contributors to the overall overwhelmingly complex network of interactions triggered by inflammation, will lead to the discovery of new small molecule regulators and therapeutic modalities. For example, suppression of the production of AA-derived pro-inflammatory mediators, HXA3 and LTB4, by an iPLA2 γ inhibitor, R-BEL, mitigated injury associated with the activation of pro-inflammatory processes in animals exposed to whole-body irradiation. Further, technological developments promise to make redox lipidomics a powerful approach in the arsenal of diagnostic and therapeutic instruments for personalized medicine of inflammatory diseases and conditions.
... MnSOD is present in the mitochondrial matrix and together with CuZn-SOD, which is located in the cytosol and mitochondrial intermembrane space, catalyzes the transformation of superoxide anion in hydrogen peroxide and molecular oxygen [48]. The mitochondrial isoenzyme maintains the levels of this superoxide at nanomolar concentration in physiological conditions, but also controls the cellular accumulation of hydrogen peroxide [49]. In our experiment, the variation of Nrf2 expression is indirectly correlated with MnSOD activity (Figure 6). ...
Silica nanoparticles (SiO2 NPs) represent environmentally born nanomaterials that are used in multiple biomedical applications. Our aim was to study the amorphous SiO2 NP-induced inflammatory response in MRC-5 human lung fibroblasts up to 72 hours of exposure. The intracellular distribution of SiO2 NPs was measured by transmission electron microscopy (TEM). The lactate dehydrogenase (LDH) test was used for cellular viability evaluation. We have also investigated the lysosomes formation, protein expression of interleukins (IL-1β, IL-2, IL-6, IL-8, and IL-18), COX-2, Nrf2, TNF-α, and nitric oxide (NO) production. Our results showed that the level of lysosomes increased in time after exposure to the SiO2 NPs. The expressions of interleukins and COX-2 were upregulated, whereas the expressions and activities of MMP-2 and MMP-9 decreased in a time-dependent manner. Our findings demonstrated that the exposure of MRC-5 cells to 62.5 µg/mL of SiO2 NPs induced an inflammatory response.
1) 郑州大学医学院基础医学院生物化学与分子生物学系，郑州 450001； 2) 郑州大学体育学院，郑州 450001； 3) 郑州大学第一附属医院妇科，郑州 450001； 4) 郑州大学医学院临床医学系，郑州 450001； 5) 郑州大学化学学院，郑州 450001； 6) 河南应用技术职业学院，郑州 450042) 摘要 锰超氧化物歧化酶 (MnSOD) 催化两分子超氧自由基歧化为分子氧和过氧化氢。超氧自由基被 Mn 3+ SOD 氧化成分子 氧的反应以扩散的方式进行。超氧自由基被 Mn 2+ SOD 还原为过氧化氢的反应以快循环和慢循环两条途径平行进行。在慢循 环途径中，Mn 2+ SOD 与超氧自由基形成产物抑制复合物，然后该复合物被质子化而缓慢释放出过氧化氢。在快循环途径中， 超氧自由基直接被 Mn 2+ SOD 转化为产物过氧化氢，快速循环有利于酶的复活与周转。本文提出温度是调节锰超氧化物歧化 酶进入慢速或者快速循环催化途径的关键因素。随着在生理温度范围内的温度升高，慢速循环成为整个催化反应的主流， 因而生理范围内的温度升高反而抑制该酶的活性。锰超氧化物歧化酶的双相酶促动力学特性可以用该酶保守活性中心的温 度依赖性配位模型进行合理化解释。当温度降低时，1 个水分子 (或者 OH-) 接近 Mn、甚至与 Mn 形成配位键，从而干扰超 氧自由基与 Mn 形成配位键而避免形成产物抑制。因此在低温下该酶促反应主要在快循环通路中进行。最后阐述了几种化学 修饰模式对该酶的调节，说明锰超氧化物歧化酶受到多种形式的快速调节 (变构调节与化学修饰) 。这些快速调节直接改变 酶的活化状态，进而调节细胞中超氧自由基和过氧化氢的平衡与流量，为揭示锰超氧化物歧化酶和超氧自由基的生理作用 提供新理论。 关键词 锰超氧化物歧化酶，变构调节，共价修饰，活性氧，生物氧化，温度，酶催化机制 中图分类号 Q5，Q6
This chapter entitled “Cell Cycle, DNA Damage Repair Systems, and Impact of Redox Regulation in Cancer” introduces the cell cycle components in normal cells and further discusses regulators involvement in cell cycle checkpoints, cyclin-CDK interaction, and activation. Cell cycle inhibitors, transcription regulatory network, redox oscillation, and metabolic activity in cell cycle were discussed. All these events are further examined in the context of cancer cells, highlighting the differences from normal cell situations.
Further, the DNA damage repair system with repair pathways involving various enzymatic systems has been described. Oxidative stress inducing genomic instability and cancer development has been introduced. Apurinic/apyrimidinic 1 (APE1) and oxidative DNA damage repair have been discussed, focusing on their dual action as repair and redox reactions. The impact of redox reaction in DNA repair, DNA damage response (DDR), and cancer therapy strategies has been discussed at length. PARP inhibitors of DDR, other inhibitors such as ATM, ATR, and DNA-PKCs (regulation of DDR signaling), and further involvement of synthetic lethality concept for cancer therapy have been discussed.
Current study aimed to research the effect of Hippophae rhamnoides (HRE) on potantial oral oxidative and
inflammatory damage of 5-FU in rats. The rats were assigned to three groups; healthy (HG), 5-FU 100mg/kg (FUG) and
HRE 50mg/kg +5-FU 100mg/kg (HRFU). The 5-FU was injected in the FUG group intraperitoneally. The HRFU was
injected 5-FU at 100mg/kg IP one hour after the 50mg/kg HRE was given orally. Olive oil was used as a solvent for the
HG. HRE was given to the rats three times a day for ten days. 5-FU was given one dose on the 1st, 3rd and 5th days. On
the 10th day, the tissues removed from the animals were euthanized with high-dose anaesthesia and were
macroscopically examined. The levels of the oxidant, antioxidant and proinflammatory cytokines were investigated.It
was seen that HRE alleviated the symptoms of severe mucositis by antagonizing the effects of 5-FU on oxidant,
antioxidant and proinflammatory cytokines such as malondialdehyde, total glutathione, superoxide dismutase, catalase,
nuclear factor kappa-B and interleukin-6 in inner cheek and tongue tissue. These results recommend that HRE may be
benefical in the cure of 5-FU-associated oral mucositis.
Background and Objective: Ischemia-reperfusion (I/R) injury is seen in many conditions, including ovarian torsion/detorsion. Ovarian ischemia is an emergency that occurs as a result of torsion of the ovaries. This study aimed to investigate the extent to which brain, heart, lung, kidney and liver tissues were affected by ovarian ischemia-reperfusion damage in albino Wistar-type female rats and to evaluate them by comparison. Materials and Methods: Animals were divided into two groups: The group to be applied sham operation (SG) and the group to be applied the ischemia-reperfusion procedure to the ovaries (OIR). As 2 hrs of ischemia and 6 hrs of reperfusion were conducted in the right ovaries of OIR group rats. After reperfusion, the ovarian, brain, heart, lung, kidney and liver tissues of the animals sacrificed were removed. The severity of tissue damage was assessed by the degree of significance of the increase in oxidative and proinflammatory parameters and the decrease in antioxidants. Results: The organs obtained from the OIR group in which MDA and IL-6 levels were highest, while tGSH, SOD and CAT levels were the lowest, were ovary>kidney>lung>heart, respectively. The levels of these parameters in the brain and liver tissues were found to be almost the same as in the SG group. Current findings showed that ovarian ischemia-reperfusion damage did not affect brain and liver tissues, but the heart was mildly affected, the lung was moderately and kidney tissue was severely affected. Conclusion: Current experimental results showed that it may be useful to consider the possibility of damage to organs such as the heart, lungs and kidneys before and after the ovarian ischemia-reperfusion procedure and take the necessary precautions.
Objective:
This study aims to investigate possible preventive effect of ATP on optic nerve damage caused by amiodarone in rats.
Material and method:
Thirty albino male Wistar rats weighing between 265 and 278 grams were used in the study. Before the experiment, the rats were housed at 22 °C in a 12-hour light/dark cycle under appropriate condition. The rats were equally divided into five groups of six animals each: healthy group, 50 mg/kg amiodarone (AMD-50), 100 mg/kg amiodarone (AMD-100), 25 mg/kg ATP + 50 mg/kg amiodarone (ATAD-50), and 25 mg/kg ATP + 100 mg/kg amiodarone (ATAD-100). At the end of 14th day, the animals were sacrificed using cardiac puncture under deep thiopental anesthesia, and optic nerve tissues were harvested to measure superoxide dismutase (SOD), total glutathione (tGSH), malondialdehyde (MDA), and catalase (CAT) levels.
Results:
The MDA levels were found to be significantly higher in the AMD-50 and AMD-100 groups compared to the healthy group (p˂0.001). There was also a significant difference between the AMD-50 and ATAD-50 groups, and between the AMD-100 and ATAD-100 groups regarding MDA levels (p˂0.001). tGSH, SOD, and CAT levels were significantly lower in the AMD-50 and AMD-100 groups compared to the healthy group (p˂0.001). ATP was found to partially inhibit amiodarone-induced optic neuropathy.
Conclusion:
The biochemical and histopathological results of this study demonstrated that amiodarone at high doses caused more severe optic neuropathy inducing oxidative damage, but ATP could relatively antagonize these negative effects on the optic nerve. Therefore, we believe that ATP may be beneficial in preventing amiodarone-induced optic neuropathy.
The aim of this study is to investigate the effect of metyrosine on ischemia-reperfusion (I/R) induced ovarian injury in rats in terms of biochemistry and histopathology. Rats were divided into: ovarian I/R (OIR), ovarian I/R+50 mg/kg metyrosine (OIRM) and sham (SG) operations. OIRM group received 50 mg/kg metyrosine one hour before the application of the anesthetic agent, OIR and SG group rats received equal amount of distilled water to be used as a solvent orally through cannula. Following the application of the anesthetic agent, ovaries of OIRM and OIR group rats were subjected to ischemia and reperfusion, each of which took two hours. This biochemical experiment findings revealed high levels of malondialdehyde (MDA) and cyclo-oxygenase-2 (COX-2) and low levels of total glutathione (tGSH), superoxide dismutase (SOD) and cyclo-oxygenase-1 (COX-1) in the ovarian tissue of OIR group, with significant histopathological injury. In metyrosine group, MDA and COX-2 levels were lower than the OIR group whereas tGSH, SOD and COX-1 levels were higher, with slighter histopathological injury. Our experimental findings indicate that metyrosine inhibits oxidative and pro-inflammatory damage associated with ovarian I/R in rats. These findings suggest that metyrosine could be useful in the treatment of ovarian injury associated with I/R.
Essential metals including iron (Fe) and manganese (Mn) with known physiological functions in human body play an important role in cell homeostasis. Excessive exposure to these essential as well as non-essential metals including mercury (Hg) and Aluminum (Al) may contribute to pathological conditions, including PD. Each metal could be toxic through specific pathways. Epidemiological evidences from occupational and ecological studies besides various in vivo and in vitro studies have revealed the possible pathogenic role and neurotoxicity of different metals. Pesticides are substances that aim to mitigate the harm done by pests to plants and crops, and are extensively used to boost agricultural production. This review provides an outline of our current knowledge on the possible association between metals and PD. We have discussed the potential association between these two, furthermore the chemical properties, biological and toxicological aspects as well as possible mechanisms of Fe, Mn, Cu, Zn, Al, Ca, Pb, Hg and Zn in PD pathogenesis. In addition, we review recent evidence on deregulated microRNAs upon pesticide exposure and possible role of deregulated miRNA and pesticides to PD pathogenesis.
Liver and muscle health are intimately connected. Nutritional strategies that support liver detoxification are beneficial to muscle recovery. Computational–in silico–molecular systems’ biology analysis of supplementation of calcium and potassium glucarate salts and their metabolite D-glucaric acid (GA) reveals their positive effect on mitigation of liver detoxification via four specific molecular pathways: (1) ROS production, (2) deconjugation, (3) apoptosis of hepatocytes, and (4) β-glucuronidase synthesis. GA improves liver detoxification by downregulating hepatocyte apoptosis, reducing glucuronide deconjugates levels, reducing ROS production, and inhibiting β-Glucuronidase enzyme that reduces re-absorption of toxins in hepatocytes. Results from this in silico study provide an integrative molecular mechanistic systems explanation for the mitigation of liver toxicity by GA.
In the last few years, numerous types of cancers are increasing that represents the second leading cause of deaths after cardiovascular diseases. Though many chemotherapeutic drugs are available, but they show toxicity and possess multiple side effects. Moreover, in case of brain cancer, the blood–brain barrier (BBB) is the major hurdle that resists the entry of larger and hydrophobic molecules to reach the tumor region in the brain. This will hamper the treatment to brain tumor and affect the patient’s recovery. Additionally, tumor cells also sometimes can become chemo-resistant and many chemotherapeutic drugs have adverse effect on normal healthy cells results in chemotoxicity. The level of reactive oxygen species (ROS) is also a major determining factor in tumorigenesis and hence targeting ROS is important in cancer therapy. Thus, recent combinational nanotherapy targeting ROS and the biological processes involved in ROS production represent a crucial strategy for cancer treatment. In this chapter, we present the conflicting roles of ROS in oncogenesis and chemotherapy using ROS targeting molecules as nanotherapeutics. Additionally, the biological processes involved in ROS generation within the tumor cells, their effect on tumor cells and ROS detoxification mechanisms have also been discussed. Moreover, recent advances in the application of nanoparticles and nanoencapsulated agents alone or in combination with the anticancer drugs for improving the patient’s health have been highlighted.
The quantification of synergistic effects of multi-combination compounds is critical in developing “cocktails” that are efficacious. In this research, a method for in silico modeling and the quantification of synergistic effects of multi-combination compounds is applied for assessing a combination of phytonutrients for joint pain. Joint pain is the most prominent and disabling symptom of arthritis. Arthritic pain leads to a reduced quality of life. This research explores the efficacy of a synergistic combination of two plant-based flavonoids—apigenin and hesperidin—on joint pain. The study employs computational systems biology: (1) to identify biomolecular mechanisms of joint pain; (2) to identify the specific effects of apigenin and hesperidin, individually and in combination, on the mechanisms of joint pain; and (3) to predict the quantitative effects of apigenin and hesperidin, individually and in combination, on joint pain and whether these combination effects are synergistic or additive. Four molecular pathways that are affected by apigenin and hesperidin include the following: (1) arachidonic acid metabolism, (2) PGE2 signaling, (3) COX-2 synthesis, and (4) oxidative stress. The combination of apigenin and hesperidin significantly lowered PGE2 production, CGRP production, TRVP-1 synthesis, COX-2 production, and reactive oxygen species (ROS) production. Our results indicate that the apigenin and hesperidin combination synergistically affected four of the five modalities to attenuate joint pain.
A self-preservation Pt(IV) nanoplatform, amorphous ferric oxide-coating selenium core-shell nanoparticles (iAIO@NSe-Pt), was developed for H2O2 depletion-mediated tumor anti-angiogenesis, apoptosis, and ferroptosis. Upon entry into the blood, the ferric oxide shell effectively blocked the contact Pt(IV) prodrug with reduced molecules, then avoided the inactivation of the Pt(IV) prodrug and increased its accumulation in the tumor. After entering cancer cells, iAIO@NSe-Pt caused a series of cascade reactions: (1) AIO on the surface of iAIO@NSe-Pt quickly dissolved, released an abundance of Fe(II) because of the weakly acidic tumor microenvironment, and then catalyzed cellular H2O2 into highly toxic ˙OH, resulting in cellular H2O2 deficiency and cell ferroptosis. (2) The platinum(IV) prodrugs were exposed and quickly reduced to highly toxic Pt(II) by depleting GSH. This process inactivated GPX4, promoted ROS accumulation, and further accelerated ferroptosis. In addition, the generated Pt(II) quickly inhibited DNA replication, achieving effective apoptotic cell death. Meanwhile, Pt(II) inactivated SOD1, which blocked the synthesis of cellular H2O2 and accelerated ROS (superoxide anion radical) accumulation. (3) The deficiency of cellular H2O2 significantly inhibited the expression of vascular endothelial growth factor-A (VEGF-A), blocking tumor angiogenesis and then improving the anticancer effect. (4) After such a cascade reaction, the exposed NSe successively disrupted mitochondrial respiration and inhibited cancer angiogenesis, further inducing cancer cell death. Collectively, our functional and mechanical investigation suggested that iAIO@NSe-Pt exhibits excellent tumor targeting, biocompatibility and anti-tumor efficiency in vitro and in vivo, and provides a novel example of a self-preservation Pt(IV) nanoplatform for H2O2 depletion-mediated tumor anti-angiogenesis, apoptosis, and ferroptosis, showing great promise for future clinical use.
A conservative characteristic of manganese superoxide dismutase is the rapid formation of product inhibition at high temperatures. At lower temperatures, the enzyme is less inhibited and undergoes more catalytic fast cycles before being product-inhibited. The temperature-dependent kinetics could be rationalized by the temperature-dependent coordination in the conserved center of manganese superoxide dismutase. As temperature decreases, a water molecule (WAT2) approaches or even coordinates Mn as the sixth ligand to interfere with O2•--Mn coordination and reduce product inhibition, so the dismutation should mainly proceed in the fast outer-sphere pathway at low temperatures. Cold-activation is an adaptive response to low temperature rather than a passive adaptation to excess superoxide levels since the cold-activated dismutase activity significantly exceeds the amount of superoxide in the cell or mitochondria. Physiologically speaking, cold activation of manganese superoxide dismutase mediates cold stress signaling and transduces temperature (physical signal) degree into H2O2 fluxes (chemical signal), which in turn may act as a second messenger to induce a series of physiological responses such as cold shock.
MnSOD catalyzes superoxide dismutation via temperature-dependent 'fast cycle' and 'slow cycle' pathways. Higher temperature promotes the formation of the product inhibition complexes, and the slow cycle pathway becomes mainstream in the overall dismutation. Decreasing the temperature reversely induces the dismutation into the fast cycle pathway. Cold-induced activation is a specific response to temperature and is a conservative feature of all MnSODs from microorganisms to humans. Cold-activation is an adaptive response to low temperature rather than a passive adaptation to excess superoxide. The cold-activated activity of MnSOD significantly exceeds the steady-state level of superoxide in the cell. Cold-activation is a regulatory mechanism for the prompt release of hydrogen peroxide fluxes which may act as a second messenger involving the regulation of adaptive thermogenesis. MnSOD may be a catalytic thermoreceptor playing a central role in adaptive thermogenesis.
Microtubules consisting of α/β-tubulin dimers play critical roles in cells. More than seven genes encode α-tubulin in vertebrates. However, the property of microtubules composed of different α-tubulin isotypes is largely unknown. Here, we purified recombinant tubulin heterodimers of mouse α-tubulin isotypes including α1A and α1C with β-tubulin isotype β2A. In vitro microtubule reconstitution assay detected that α1C/β2A microtubules grew faster and underwent catastrophe less frequently than α1A/β2A microtubules. Generation of chimeric tail-swapped and point-mutation tubulins revealed that the carboxyl-terminal (C-terminal) tails of α-tubulin isotypes largely accounted for the differences in polymerization dynamics of α1A/β2A and α1C/β2A microtubules. Kinetics analysis showed that in comparison to α1A/β2A microtubules, α1C/β2A microtubules displayed higher on-rate, lower off-rate, and similar GTP hydrolysis rate at the plus-end, suggesting a contribution of higher plus-end affinity to faster growth and less frequent catastrophe of α1C/β2A microtubules. Furthermore, EB1 had a higher binding ability to α1C/β2A microtubules than to α1A/β2A ones, which could also be attributed to the difference in the C-terminal tails of these two α-tubulin isotypes. Thus, α-tubulin isotypes diversify microtubule properties, which, to a great extent, could be accounted by their C-terminal tails.
Oxidative stress is a component of many diseases, including atherosclerosis, chronic obstructive pulmonary disease, Alzheimer disease and cancer. Although numerous small molecules evaluated as antioxidants have exhibited therapeutic potential in preclinical studies, clinical trial results have been disappointing. A greater understanding of the mechanisms through which antioxidants act and where and when they are effective may provide a rational approach that leads to greater pharmacological success. Here, we review the relationships between oxidative stress, redox signalling and disease, the mechanisms through which oxidative stress can contribute to pathology, how antioxidant defences work, what limits their effectiveness and how antioxidant defences can be increased through physiological signalling, dietary components and potential pharmaceutical intervention. Although oxidative stress is associated with a broad range of diseases, therapeutic antioxidant approaches have so far been disappointing. Here, Forman and Zhang review the roles of oxidative stress and redox signalling in disease, assess antioxidant therapeutic strategies and highlight key limitations that have challenged their clinical application.
Manganese in the diet is nutritionally essential for normal physiologic functioning. However, excessive exposure to manganese has been associated with developmental, neurodegenerative and other disorders.
The book comprehensively covers the toxicology of manganese. Leading investigators provide perspectives from toxicology, neuroscience, nutrition, molecular biology and risk assessment disciplines and chapters cover the toxicokinetics, toxicodynamic interactions and health effects of manganese, as well as its potential role in neurodegenerative diseases. A large section devoted to health effects presents the latest research that associates manganese exposure to potential human diseases.
Any scientists, health professional or regulator involved with metal exposure and toxicology should find this volume essential reading. Students and researchers in neurotoxicology will also find this book a useful reference.
Mitochondria are highly dynamic organelles that are found in most eukaryotic organisms. It is broadly accepted that mitochondria originally evolved from prokaryotic bacteria, e.g., proteobacteria. The mitochondrion has its independent genome that encodes 37 genes, including 13 genes for oxidative phosphorylation. Accumulative evidence demonstrates that mitochondria are not only the powerhouse of the cells by supplying adenosine triphosphate, but also exert roles as signaling organelles in the cell fate and function. Numerous factors can affect mitochondria structurally and functionally. Carotenoids are a large group of fat-soluble pigments commonly found in our diets. Recently, much attention has been paid in carotenoids as dietary bioactives in mitochondrial structure and function in human health and disease, though the mechanistic research is limited. Here, we update the recent progress in mitochondrial functioning as signaling organelles in human health and disease, summarize the potential roles of carotenoids in regulation of mitochondrial redox homeostasis, biogenesis, and mitophagy, and discuss the possible approaches for future research in carotenoid regulation of mitochondrial function.
Fluorescent quantum dots (QDs) modified with polyethylene glycol (PEG) and albumin bovine serum (BSA) have profound application in the detection and treatment of hepatocellular carcinoma (HCC) cells. In the present study, the effects and mechanism of PEG and BSA modification on the cytotoxicity of QDs have been explored. It was found that the diameter of the as-prepared QDs, PEG@QDs, BSA@QDs is 3–5 nm, 4–5 nm, and 4–6 nm, respectively. With increase of the treatment time from 0 to 24 h, the HCC cell viability treated with QDs, PEG@QDs, and BSA@QDs obviously decreases, showing a certain time-dependent manner. When the concentration of several nanomaterials is increased from 10 to 90 nM, the cell viability decreases accordingly, exhibiting a certain concentration-dependent manner. Under the same concentration change conditions, the reactive oxygen species contents of cells treated by QDs, PEG@QDs, and BSA@QDs also rise from 7.9 × 103, 6.7 × 103, and 4.7 × 103 to 13.2 × 103, 14.3 × 103, and 12.3 × 103, respectively. In these processes, superoxide dismutase does not play a major role. This study provides strong foundation and useful guidance for QD applications in the diagnosis and treatment of HCC.
Purpose:
Oral mucositis (OM) remains a common, debilitating toxicity of radiation therapy (RT) for head and neck cancer. The goal of this phase IIb, multi-institutional, randomized, double-blind trial was to compare the efficacy and safety of GC4419, a superoxide dismutase mimetic, with placebo to reduce the duration, incidence, and severity of severe OM (SOM).
Patients and methods:
A total of 223 patients (from 44 institutions) with locally advanced oral cavity or oropharynx cancer planned to be treated with definitive or postoperative intensity-modulated RT (IMRT; 60 to 72 Gy [≥ 50 Gy to two or more oral sites]) plus cisplatin (weekly or every 3 weeks) were randomly assigned to receive 30 mg (n = 73) or 90 mg (n = 76) of GC4419 or to receive placebo (n = 74) by 60-minute intravenous administration before each IMRT fraction. WHO grade of OM was assessed biweekly during IMRT and then weekly for up to 8 weeks after IMRT. The primary endpoint was duration of SOM tested for each active dose level versus placebo (intent-to-treat population, two-sided α of .05). The National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03, was used for adverse event grading.
Results:
Baseline patient and tumor characteristics as well as treatment delivery were balanced. With 90 mg GC4419 versus placebo, SOM duration was significantly reduced (P = .024; median, 1.5 v 19 days). SOM incidence (43% v 65%; P = .009) and severity (grade 4 incidence, 16% v 30%; P = .045) also were improved. Intermediate improvements were seen with the 30-mg dose. Safety was comparable across arms, with no significant GC4419-specific toxicity nor increase of known toxicities of IMRT plus cisplatin. The 2-year follow-up for tumor outcomes is ongoing.
Conclusion:
GC4419 at a dose of 90 mg produced a significant, clinically meaningful reduction of SOM duration, incidence, and severity with acceptable safety. A phase III trial (ROMAN; ClinicalTrials.gov identifier: NCT03689712) has begun.
It is well known that tumors have an acidic pH microenvironment and contain a high content of hydrogen peroxide (H2O2). These features of the tumor microenvironment may provide physiochemical conditions that are suitable for selective tumor therapy and recognition. Here, for the first time, we demonstrate that a type of graphene oxide nanoparticle (N-GO) can exhibit peroxidase-like activities (i.e., can increase the levels of reactive oxygen species (ROS)) under acidic conditions and catalyze the conversion of H2O2 to ROS-hydroxyl radicals (HO·) in the acidic microenvironment in Hela tumors. The concentrated and highly toxic HO· can then trigger necrosis of tumor cells. In the microenvironment of normal tissues, which has a neutral pH and low levels of H2O2, N-GOs exhibit catalase-like activity (scavenge ROS) that splits H2O2 into O2 and water (H2O), leaving normal cells unharmed. In the recognition of tumors, an inherent redox characteristic of dopamine is that it oxidizes to form dopamine-quinine under neutral (pH 7.4) conditions, quenching the fluorescence of N-GOs; however, this characteristic has no effect on the fluorescence of N-GOs in an acidic (pH 6.0) medium. This pH-controlled response provides an active targeting strategy for the diagnostic recognition of tumor cells. Our current work demonstrates that nanocatalytic N-GOs in an acidic and high-H2O2 tumor microenvironment can provide novel benefits that can reduce drug resistance, minimize side effects on normal tissues, improve antitumor efficacy, and offer good biocompatibility for tumor selective therapeutics and specific recognition.
One of the urgent tasks at the present time is to obtain and widely use functional foods that have antioxidant and anticarcinogenic effects, which serve not only to meet human needs in proteins, fats, carbohydrates, micro - and macronutrients, but also contribute to the immune system, improve the heart and other human organs. For food plants, rich in antioxidant protection components, include perennial onions, from the variety of wild species which are in demand by modern medicine are only very few. The Republic of Komi is classified as the most uncomfortable territories for human habitation. In the flora of the Komi Republic there are three types of perennial bows – Allium angulosum L., A. schoenoprasum L. and A. strictum Schrad. The collection of the Botanical garden of the Institute of biology, Komi scientific center includes more than 150 species. Comparison of the chemical composition of four types of onions from the Botanical gardens of the Komi scientific center and Moscow state University (MSU) showed that the total content of flavonoids in onions, Komi scientific center of the BS is much higher, and the content of selenium is lower than in the same types of BS, Moscow state University. In samples of onions sticking out in the Republic of Tuva, the Republic of Buryatia, in the Altai Mountains, the Se content is also much higher than in regional species. The soils of the Komi Republic are depleted Se, however, as shown by our research, wild and cultivated species of the genus Allium, are batteries of this trace element. According to our calculations, the use of 100 g of fresh herbs of chives may meet up to 60% of the daily needs of the human body in ascorbic acid, up to 94% manganese, 20% copper, 12% zinc.
The reaction of 6-electron reduced chicken liver xanthine dehydrogenase (XDH) with molecular oxygen was studied using both stopped flow and steady-state turnover techniques at pH 7.8, 4°C. Oxidation of fully reduced XDH proceeded via four phases, three of which were detected with the stopped flow spectrophotometer. The fastest phase was second order in oxygen (1900 M⁻¹ s⁻¹), resulted in the appearance of flavin semiquinone and yielded no superoxide. The next phase was also second order in oxygen (260 M⁻¹ s⁻¹), involved the loss of flavin semiquinone and yielded, on average, 1 mol of superoxide/mol of XDH oxidized. The last 2 electron equivalents were located in the iron-sulfur centers. They were released one equivalent at a time in the form of superoxide.
Steady-state kinetics were found to be critically dependent on temperature and oxygen concentration. When these factors were carefully controlled, both the xanthine-oxygen and NADH-oxygen reductase reactions gave linear Lineweaver-Burk plots. The xanthine-oxygen data yielded a turnover number of 43 min⁻¹, which was 42% of that for xanthine-NAD turnover. During turnover, with xanthine and O2, 40–44% of the electron equivalents introduced by xanthine appeared as superoxide. Reduced pyridine nucleotides, NAD and 3-aminopyridine adenine dinucleotide, dramatically reduced the formation of superoxide at levels which did not seriously inhibit oxygen reactivity.
The reaction of reduced xanthine oxidase with oxygen occurs in two distinct kinetic phases with the rate of the fast phase being about 10 times greater than that of the slow phase. Comparison of transient state and steady state kinetics demonstrates that only the fast phase of oxidation is catalytically significant. The time course of oxidation is quantitatively independent of the following factors: (a) the activity to flavin ratio; (b) presence of competitive inhibitors; (c) pretreatment of enzyme with either cyanide or allopurinol; and (d) addition of superoxide dismutase. Removal of flavin eliminates both oxidase activity and the reactivity of the reduced enzyme with molecular oxygen (Komai, H., Massey, V., and Palmer, G. (1969) J. Biol. Chem. 244, 1692–1700).
Both optical and EPR kinetic data support the interpretation that 5 electrons are consumed in the fast phase and 1 in the slow phase. The spectral properties of the slowly reacting species suggest that the residual electron is present principally as reduced iron-sulfur center II at pH 8.5. The simplest interpretation of the kinetic data requires that rapid reaction of the enzyme with oxygen occurs via fully reduced flavin and that the slow phase observed for 1-electron reduced enzyme is a consequence of both the poor reactivity and small equilibrium concentration of flavin semiquinone. The absence of any effect of superoxide dismutase on both the optical and EPR data and the small amount of superoxide generated indicates that hydrogen peroxide is the major product formed during oxidation of fully reduced xanthine oxidase.
The rate of autoxidation of epinephrine and the sensitivity of this autoxidation to inhibition by superoxide dismutase were both augmented, as the pH was raised from 7.8 → 10.2. O2⁻, generated by the xanthine oxidase reaction, caused the oxidation of epinephrine to adrenochrome and the yield of adrenochrome produced per O2⁻ introduced, increased with increasing pH in the range 7.8 → 10.2 and also increased with increasing concentration of epinephrine. These results, in conjunction with complexities in the kinetics of adrenochrome accumulation, lead to the proposal that the autoxidation of epinephrine proceeds by at least two distinct pathways, only one of which is a free radical chain reaction involving O2⁻ and hence inhibitable by superoxide dismutase. This chain reaction accounted for a progressively greater fraction of the total oxidation as the pH was raised. The ability of superoxide dismutase to inhibit the autoxidation of epinephrine at pH 10.2 has been used as the basis of a convenient and sensitive assay for this enzyme.
Oxidants are toxic, but at low doses they can stimulate rather than inhibit the growth of mammalian cells and play a role in the etiology of cancer and fibrosis. The effect of oxidants on cells is modulated by multiple interacting antioxidant defense systems. We have studied the individual roles and the interaction of Cu,Zn-superoxide dismutase (SOD) and catalase (CAT) in transfectants with human cDNAs of mouse epidermal cells JB6 clone 41. Since only moderate increases in these enzymes are physiologically meaningful, we chose the following five clones for in-depth characterization: CAT 4 and CAT 12 with 2.6-fold and 4.2-fold increased catalase activities, respectively, SOD 15 and SOD 3 with 2.3-fold and 3.6-fold increased Cu,Zn-SOD activities, respectively, and SOCAT 3 with a 3-fold higher catalase activity and 1.7-fold higher Cu,Zn-SOD activity than the parent JB6 clone 41. While the increases in enzyme activities were moderate, the human cDNAs were highly expressed in the transfectants. As demonstrated for the clone SOD 15, this discordance between message concentrations and enzyme activities may be due to the low stability of the human Cu,Zn-SOD mRNA in the mouse recipient cells. According to immunoblots the content of Mn-SOD was unaltered in the transfectants. While the activities of glutathione peroxidase were comparable in all strains, the concentrations of reduced glutathione (GSH) were significantly lower in SOD 3 and SOD 15. This decrease in GSH may reflect a chronic prooxidant state in these Cu,Zn-SOD overproducers.(ABSTRACT TRUNCATED AT 250 WORDS)
It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43 degrees C, 1 h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H2O2 formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.
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.
At pH 7.0, in air, 20% of the total electron flux through xanthine oxidase can be accounted for in terms of the univalent reduction of oxygen. The fraction of the total flux of electrons which traversed the univalent pathway to oxygen was increased by raising the pH and by raising the oxygen tension. It was further shown that at any given pH and oxygen tension, the amount of univalently reduced oxygen, which was detectable in terms of the reduction of cytochrome c, rose as the turnover rate of the enzyme was decreased by decreasing the concentration of xanthine. This effect of xanthine was more pronounced at pH 7.0 than at pH 10.0. Another reflection of this same phenomenon was a difference in Km for xanthine measured in terms of urate production as compared to Km for xanthine measured in terms of cytochrome c reduction. Here too the differences were diminished as the pH and the oxygen tension were raised. The quantitative aspects of these phenomena are presented as well as an explanation which is consistent with all of the observations and which was, in fact, predictive of several of them.
Production of superoxide radical during oxidation of dihydroorotate in rat liver mitochondria was not affected by antimycin A, thenoyltrifluoroacetone, or added ubiquinone but was inhibited by orotate, a product inhibitor of dihydroorotate dehydrogenase. It appears likely that superoxide is generated at the primary dehydrogenase. Dihydroorotate dehydrogenase differs from succinate dehydrogenase both in its utilization of ubiquinone and in the mechanism of cytochrome b reduction. Thenoyltrifluoroacetone completely inhibits fumarate synthesis and reduction of cytochrome b by succinate. Formation of orotate is only partially inhibited by thenolytrifluoroacetone and the inhibitor does not prevent reduction of cytochrome b by dihydroorotate. It is proposed that several pathways exist for linkage of the primary dihydrorotate dehydrogenase with the electron transport chain. One route involves electron transfer from ubiquinone to cytochrome c and is inhibited by thenoyltrifluoroacetone. A second route bypasses ubiquinone and is inhibited by antimycin A. A third pathway utilizes both ubiquinone and cytochrome b and is partiayly inhibited by either thenoyltrifluoroacetone or antimycin A.
Diminished amounts of manganese-containing superoxide dismutase have been found in all the tumors examined to date. Lowered amounts of the copper-zinc-containing superoxide dismutase have been found in many, but not all, tumors. At the same time, tumors have been shown to produce superoxide radicals. It is shown how diminished enzyme activities along with radical production may lead to many of the observed properties of cancer cells. The apparent exploitation of the differences between normal and cancer cell superoxide dismutase activity in the treatment of cancer is discussed.
Electron transfer within milk xanthine oxidase has been examined by the technique of pulse radiolysis. Radiolytically generated N-methylnicotinamide radical or 5-deazalumiflavin radical has been used to rapidly and selectively introduce reducing equivalents into the enzyme so that subsequent equilibration among the four redox-active centers of the enzyme (a molybdenum center, two iron-sulfur centers, and FAD) could be monitored spectrophotometrically. Experiments have been performed at pH 6 and 8.5, and a comprehensive scheme describing electron equilibration within the enzyme at both pH values has been developed. All rate constants ascribed to equilibration between specific pairs of centers in the enzyme are found to be rapid relative to enzyme turnover under the same conditions. Electron equilibration between the molybdenum center and one of the iron-sulfur centers of the enzyme (tentatively assigned Fe/S I) is particularly rapid, with a pH-independent first-order rate constant of approximately 8.5 x 10(3) s-1. The results unambiguously demonstrate the role of the iron-sulfur centers of xanthine oxidase in mediating electron transfer between the molybdenum and flavin centers of the enzyme.
Although increased superoxide dismutase (SOD) activity is often associated with enhanced resistance of cells and organisms to oxidant challenges, few direct tests of the antioxidant importance of this enzyme have been carried out. To assess the importance of SOD in defending against gamma-radiation, we employed Escherichia coli with deficient, normal, and super-normal enzyme activities. Surprisingly, the radiation sensitivity of E. coli actually increases as bacterial SOD activity increases. Elevated intracellular SOD activity sensitizes E. coli to radiation-induced mortality, whereas SOD-deficient bacteria show normal or decreased radiosensitivity. Toxic effects of activated oxygen species are involved in this phenomenon; bacterial SOD activity has no effect on radiation sensitivity under anaerobic conditions or on the lethality of other, non-oxygen-dependent, toxins such as ultraviolet radiation.
Immunolocalization studies of hamster kidney development were performed using polyclonal antibodies to antioxidant enzymes, including antibodies to copper, zinc and manganese superoxide dismutases, catalase, glutathione peroxidase and glutathione S-transferases and their subunits. Antibodies to extracellular matrix proteins were also studied to determine the temporal sequence between expression of immunoreactive protein for basement membrane proteins, which serve as markers of embryonic induction of nephron development, and antioxidant enzyme expression in kidney development. Immunoreactive proteins for antioxidant enzymes were not detectable in the developing kidney until after extracellular matrix proteins had been deposited. However, immunoreactive proteins for the antioxidant enzymes copper, zinc and manganese superoxide dismutases, catalase, and α class glutathione S-transferase Ya subunit were detected in renal tubules before birth. μ class glutathione S-transferase subunits Yb1 and Yb2 stained transitional epithelium at high levels before birth. Our results indicate: (1) each type of kidney cell has a unique antioxidant enzyme profile, (2) antioxidant enzymes are expressed in different types of cell at different times during development, but antioxidant enzyme immunoreactive protein was not present until after immunoreactive proteins for extracellular matrix molecules were detected, and (3) certain antioxidant enzymes are present before birth, indicating that high oxygen tension present at birth is not crucial for induction of immunoreactive protein.
This paper describes mathematical and software developments for a suite of programs for solving ordinary differential equations in MATLAB.
Kinetic data for the superoxide radical (HOâarrow - right - leftOâ»â +H/sup +/, pK = 4.8) in aqueous solution have been critically assessed. Rate constants for reactions of Oâ»â and HOâ with more than 300 organic and inorganic ions, molecules and other transient species have been tabulated.
The absorption characteristics and acid dissociation constants of the semiquinone radicals of several methyl derivatives of benzoquinone and naphthoquinone have been determined by pulse radiolysis. Absolute rate constants for electron transfer from the superoxide-ion O2- to mono- and di-methyl benzoquinones have been determined. In the case of duroquinone, evidence has been obtained for the occurrence of the equilibrium reaction: O2- + duroquinone ⇌ O2 + durosemiquinone with Kc = 2.3 ± 0.2 × 10-2.
Redox state is a term used widely in the research field of free radicals and oxidative stress. Unfortunately, it is used as a general term referring to relative changes that are not well defined or quantitated. In this review we provide a definition for the redox environment of biological fluids, cell organelles, cells, or tissue. We illustrate how the reduction potential of various redox couples can be estimated with the Nernst equation and show how pH and the concentrations of the species comprising different redox couples influence the reduction potential. We discuss how the redox state of the glutathione disulfide-glutathione couple (GSSG/2GSH) can serve as an important indicator of redox environment. There are many redox couples in a cell that work together to maintain the redox environment; the GSSG/2GSH couple is the most abundant redox couple in a cell. Changes of the half-cell reduction potential (E(hc)) of the GSSG/2GSH couple appear to correlate with the biological status of the cell: proliferation E(hc) approximately -240 mV; differentiation E(hc) approximately -200 mV; or apoptosis E(hc) approximately -170 mV. These estimates can be used to more fully understand the redox biochemistry that results from oxidative stress. These are the first steps toward a new quantitative biology, which hopefully will provide a rationale and understanding of the cellular mechanisms associated with cell growth and development, signaling, and reductive or oxidative stress.
Reduction of an electron acceptor (oxidant), A, or oxidation of an electron donor (reductant), A2−, is often achieved stepwise via one-electron processes involving the couples A/A⋅− or A⋅−/A2− (or corresponding prototropic conjugates such as A/AH⋅ or AH⋅/AH2). The intermediate A⋅−(AH⋅) is a free radical. The reduction potentials of such one-electron couples are of value in predicting the direction or feasibility, and in some instances the rate constants, of many free-radical reactions. Electrochemical methods have limited applicability in measuring these properties of frequently unstable species, but fast, kinetic spectrophotometry (especially pulse radiolysis) has widespread application in this area. Tables of ca. 1200 values of reduction potentials of ca. 700 one-electron couples in aqueous solution are presented. The majority of organic oxidants listed are quinones, nitroaryl and bipyridinium compounds. Reductants include phenols, aromatic amines, indoles and pyrimidines, thiols and phenothiazines. Inorganic couples largely involve compounds of oxygen, sulfur, nitrogen and the halogens. Proteins, enzymes and metals and their complexes are excluded.
The catalysis of superoxide dismutation (2O2- + 2H+ --> H2O2 + O2) by manganese superoxide dismutase (MnSOD) from Thermus Thermophilus was examined by stopped-flow spectrophotometry. As found earlier by McAdam et al. [McAdam, M.E.; Fox, R.A.; Lavelle, F.; Fielden, E.M. Biochem J. 1977, 165, 81-87], decay curves of O2- in the presence of MnSOD from Bacillus Stearothermophilus are characterized by three distinct phases: rapid disappearance of O2- (the ''burst'' phase), a period of approximately zero-order disappearance of O2- (the ''steady-state'' phase), and a very rapid depletion of O2- toward the end of the reaction. The enzyme from T. Thermophilus shows a similar kinetic pattern, and our data provide a chemical explanation for this behavior: The molar consumption of O2- in the burst phase is ([O2-]B/[Mn]T) approximately 80. The magnitude of the burst is decreased approximately 2.5-fold in D2O, whereas the zero-order phase is the same in both solvent. This indicates that proton transfer is probably the rate-limiting step when the enzyme is saturated with O2- and that the reaction by which inactive enzyme returns to active enzyme is not limited by proton transfer. At low temperatures (2-6-degrees-C) in D2O, the overall reaction was sufficiently slow to allow observation of spectral changes associated with the metal chromophore during the steady state, and we were able to obtain an absorption spectrum of the enzyme during this period. This was assigned to the inactive form of the enzyme and is characterized by a band near 650 nm (epsilon approximately 230 [Mn]-1 cm-1) and a band near 410 nm (epsilon approximately 700 [Mn]-1 cm-1). We speculate that inactivation of the enzyme occurs by oxidative addition of O2- to Mn(II), within a Michaelis complex, forming a cyclic peroxo complex of Mn(III) with the reverse of this reaction yielding active enzyme.
Living organisms obtain energy from the oxidation of various biomolecules, including carbohydrates, lipids and the carbon
skeletons of amino acids. Under aerobic conditions, the reducing coenzymes produced during these reactions are re-oxidized
in the electron transport chain, transferring electrons to molecular oxygen (E°= +800mV) through a series of electron carriers
in the respiratory chain. This electrochemical energy is converted into a proton gradient which, in turn, operates a rotor-type
enzymatic complex (ATP synthase or Complex V), inducing conformational changes which cause ADP and inorganic phosphate to
bind to the active site and ATP to be released (Noji and Yoshida 2001).
1.1.In unbuffered alkaline solutions, autoxidizing adrenalin generates superoxide anions: both the scavenging by adrenalin itself, leading to adrenochrome, and the formation of the nitrite from hydroxylamine are inhibited by superoxide dismutase. No hydroxyl radical could be detected.2.2.The yield of hydrogen peroxide increases with pH in a way similar to that of adrenochrome and nitrite. The dissociated form of adrenalin (pK = 8.5) is proposed as the source of superoxide anions.3.3.Superoxide dismutase delays rather than inhibits the reaction. In addition to the diminished formation of adrenochrome due to the scavenging of superoxide anions and re-reduction of the semiquinone by hydrogen peroxide, respectively, adrenochrome is further removed by hydrogen peroxide, with final products absorbing at 310 nm.4.4.The diminished inhibitory effect of superoxide dismutase above pH 10 is due to superoxide-independent reactions. This effect is masked by the alkaline conversion of adrenochrome to indole compounds.5.5.It is concluded that monitoring the absorption of adrenochrome in alkaline solutions does not procedure reliable evidence for superoxide anions.
Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O2− at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H2O2 at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H2O2 was essentially produced by disproportionation of O2−, which constitutes the precursor of H2O2. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O2− and 0.63 mol of H2O2/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O2 to O2−, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H2O2 generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H2O2 generation. The efficiency of added quinones as peroxide generators decreased in the order Q1 > Q0 > Q2 > Q6 = Q10, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymatically by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O2− and H2O2 during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q0H2), benzoquinol, duroquinol and menadiol generated O2− with k3 values of 0.1 to 1.4 m− · s−1 and H2O2 with k4 values of 0.009 to 4.3 m−1 · s−1.
A computational model for the mitochondrial respiratory chain that appropriately balances mass, charge, and free energy transduction is introduced and analyzed based on a previously published set of data measured on isolated cardiac mitochondria. The basic components included in the model are the reactions at complexes I, III, and IV of the electron transport system, ATP synthesis at F(1)F(0) ATPase, substrate transporters including adenine nucleotide translocase and the phosphate-hydrogen co-transporter, and cation fluxes across the inner membrane including fluxes through the K/H antiporter and passive H and K permeation. Estimation of 16 adjustable parameter values is based on fitting model simulations to nine independent data curves. The identified model is further validated by comparison to additional datasets measured from mitochondria isolated from rat heart and liver and observed at low oxygen concentration. To obtain reasonable fits to the available data, it is necessary to incorporate inorganic-phosphate-dependent activation of the dehydrogenase activity and the electron transport system. Specifically, it is shown that a model incorporating phosphate-dependent activation of complex III is able to reasonably reproduce the observed data. The resulting validated and verified model provides a foundation for building larger and more complex systems models and investigating complex physiological and pathophysiological interactions in cardiac energetics.
1. In unbuffered alkaline solutions, autoxidizing adrenalin generates superoxide anions: both the scavenging by adrenalin itself, leading to adrenochrome, and the formation of nitrite from hydroxylamine are inhibited by superoxide dismutase. No hydroxyl radical could be detected. 2. The yield of hydrogen peroxide increases with pH in a way similar to that of adrenochrome and nitrite. The dissociated form of adrenalin (pK = 8.5) is proposed as the source of superoxide anions. 3. Superoxide dismutase delays rather than inhibits the reaction. In addition to the diminished formation of adrenochrome due to the scavenging of superoxide anions and re-reduction of the semiquinone by hydrogen peroxide, respectively, adrenochrome is further removed by hydrogen peroxide, with final products absorbing at 310 nm. 4. The diminished inhibitory effect of superoxide dismutase above pH 10 is due to superoxide-independent reactions. This effect is masked by the alkaline conversion of adrenochrome to indole compounds. 5. It is concluded that monitoring the absorption of adrenochrome in alkaline solutions does not produce reliable evidence for superoxide anions.
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.
Dihydroorotate dehydrogenase in rat brain mitochondria is capable of producing superoxide. The presence of a superoxide dismutase activity in brain mitochondria, similar to that found in mitochondria from chicken liver, suggests that production of superoxide may occur in vivo. Formation of superoxide is not dependent upon reduction of cytochrome b, rather, superoxide production is competitive with cytochrome b reduction. Phenazine methosulfate apparently competes with both oxygen (superoxide production) and cytochrome b as an electron carrier but does not enhance reduction of dichlorophenolindophenol or cytochrome c.
Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.
The development and application of sensitive methods for the determination of hydrogen peroxide led, a few years ago in the laboratories of the Johnson Research Foundation, to the recognition, of intact mitochondria as an effective source of H2O2 (Fig. 1; refs. 1–4). Previous observations by Jensen (5) and by Hinkle et al. (6) had indicated that the mitochondrial respiratory chain was capable of producing H2O2. However, these results were taken with caution, in the sense that they might reflect an artificial activity induced by the ultrasonic or alkaline treatment used in the preparation of the submitochondrial particles. In 1971, Chance and Oshino (1) demonstrated variations in the level of the catalase intermediate of the peroxisomal-mitochondrial fraction of rat liver following the addition of mitochondrial substrates and uncouplers. In the same year, Loschen et al. (2) showed H2O2 formation in pigeon heart mitochondria and its relationship to the mitochondrial metabolic state by using the peroxidase-scopoletin method. It was realized that this assay could be easily interfered by endogenous hydrogen donor of the horseradish peroxidase and by exogenous hydrogen donors in the mitochondrial preparations, and consequently, an alternative method was developed.
Univalent oxidation-reduction reactions coupled with the oxygen-superoxide system were investigated in the reactions shown in eq 3 and 8, where Q and Q.- stand for p-benzoquinone and p-benzosemiquinone, respectively. From kinetic experiments the following rate constants were obtained at pH 7.0:k3 = 4.5 x 10(4) M-1 sec-1 and k8 = 3 x 10(-2) M-1 sec-1. With known values of k-3 and k-8, and of E0' for the systems Q-Q.- (0.10 V) and Cyt c3+ - Cyt c2+ (0.255 V), the calculated values of E0(O2-O2.-) were found to lie in the range between -0.27 and -0.33 V.
The present knowledge of glutathione (GSH) peroxidase is briefly reviewed: GSH peroxidase has a molecular weight of about 85,000, consists of four apparently-identical subunits and contains four g atom of selenium/mol. The enzyme-bound selenium can undergo a substrate-induced redox change and is obviously essential for activity. In accordance with the assumption that a selenol group is reversibly oxidized during catalysis, ping-pong kinetics are observed. Limiting maximum velocities and Michaelis constants, indicating the formation of an enzyme-substrate complex, are not detectable. The enzyme is highly specific for GSH but reacts with many hydroperoxides. It can be deduced from the kinetic analysis of GSH peroxidase that in physiological conditions removal of hydroperoxide is largely independent of fluctuations in the cellular concentration of GSH. However, the system will abruptly collapse if the rate of hydroperoxide formation exceeds that of regeneration of GSH. By these considerations, the pathophysiological manifestation of disorders in GSH metabolism and pentose-phosphate shunt may be explained. With regard to its low specificity for hydroperoxides, GSH peroxidase could be involved in various metabolic events such as H2O2 removal in compartments low in catalase, hydroperoxide-mediated mutagenesis, protection of unsaturated lipids in biomembranes, prostaglandin biosynthesis, and regulation of prostacyclin formation.
A thenoyl trifluoroacetone-sensitive and antimycin-insensitive ubisemiquinone radical (Qs) is readily detected in purified succinate-cytochrome c reductase. When this reductase is resolved into succinate-Q and ubiquinol-cytochrome c reductases, Qs was not detected in either reductase. The difficulty in detecting such a radical in purified succinate-Q reductase has puzzled investigators for years. A deficiency of Q in the isolated complex is the reason for the failure to detect Qs. Upon addition of exogenous Q, a thenoyl trifluoroacetone-sensitive Q-radical is readily detectable in isolated succinate-Q reductase under a controlled redox potential. Maximum radical concentration is observed when 5 mol of exogenous Q, per mole of flavin, is added. The radical gives an EPR signal with a g-value of 2.005 and a line-width of 12 G. The Em of Qs is 84 mV at pH 7.4, with half-potentials of E1 = 40 mV and E2 = 128 mV. The Qs-radical does not show power saturation, even at 200 mW.
Plasma and blood xanthine and hypoxanthine levels were assayed using a sensitive and specific method involving gas chromatography-mass spectrometry, associated with an optimized sample preparation procedure. Physiological variation was studied in 224 subjects with no purine metabolism disorders. An age dependency for both compounds was found, comparable with that known for uric acid. The mean plasma levels for the 224 subjects were 0.65 +/- 0.24 microM for xanthine and 1.65 +/- 0.78 microM for hypoxanthine. Corresponding mean blood levels were 0.59 +/- 0.21 microM for xanthine and 1.72 +/- 0.74 microM for hypoxanthine. Plasma and blood levels were significantly different, by ca. 10%. Rapid in vitro release of hypoxanthine from erythrocytes and continuation of intraerythrocytal metabolism lead to overestimation exceeding 10% within half an hour after sample blood collection. Hence samples must be deproteinized promptly. Blood can therefore be conveniently used for oxypurine assay instead of plasma when prompt spinning of samples is difficult to manage, as is usually encountered in clinical practice.
Suspensions of human neuroblastoma cells consume oxygen at a constant rate when the oxygen pressure is greater than approximately 11 Torr. The rate of oxygen consumption, however, becomes dependent on the oxygen pressure below this level. The falling respiratory rate at the lower pressures gives rise to an oxygen pressure for half-maximal respiration (P50) of approximately 0.8 Torr, which is consistent with the 0.5 Torr value for suspensions of isolated mitochondria in the presence of ATP (J. Biol. Chem. 263: 2712-2718, 1988). When the cellular metabolic energy state is lowered by addition of an uncoupler of mitochondrial oxidative phosphorylation, the respiratory rate increases up to fivefold, but the P50 decreases to approximately 0.6 Torr. In the cells treated with uncoupler, the P50 decreases further when the mitochondrial respiratory rate is inhibited with amobarbital (amytal), an inhibitor of the respiratory chain. The additional decrease in P50 is proportional to the decrease in respiratory rate. Thus, for cells treated with uncoupler, the P50 appears to be limited by oxygen diffusion from the external medium to the mitochondria. When the respiratory rate of the uncoupled cells is inhibited to the level of coupled cells, the P50 for the former is less than 0.15 Torr. This indicates that for coupled cells the difference in oxygen pressure from the external medium to the mitochondria is less than 0.15 Torr at half-maximal respiratory rate and does not significantly affect the P50 for oxygen that occurs at 0.8 Torr.(ABSTRACT TRUNCATED AT 250 WORDS)
O2- generation in mitochondrial electron transport systems, especially the NADPH-coenzyme Q10 oxidoreductase system, was examined using a model system, NADPH-coenzyme Q1-NADPH-dependent cytochrome P-450 reductase. One electron reduction of coenzyme Q1 produces coenzyme Q1-. and O2- during enzyme-catalyzed reduction and O2+ coenzyme Q1-. are in equilibrium with O2- + coenzyme Q1 in the presence of enough O2. The coenzyme Q1-. produced can be completely eliminated by superoxide dismutase, identical to bound coenzyme Q10 radical produced in a succinate/fumarate couple-KCN-submitochondrial system in the presence of O2. Superoxide dismutase promotes electron transfer from reduced enzyme to coenzyme Q1 by the rapid dismutation of O2- generated, thereby preventing the reduction of coenzyme Q1 by O2-. The enzymatic reduction of coenzyme Q1 to coenzyme Q1H2 via coenzyme Q1-. is smoothly achieved under anaerobic conditions. The rate of coenzyme Q1H2 autoxidation is extremely slow, i.e., second-order constant for [O2][coenzyme Q1H2] = 1.5 M-1.s-1 at 258 microM O2, pH 7.5 and 25 degrees C.
The reaction of 6-electron reduced chicken liver xanthine dehydrogenase (XDH) with molecular oxygen was studied using both stopped flow and steady-state turnover techniques at pH 7.8, 4 degrees C. Oxidation of fully reduced XDH proceeded via four phases, three of which were detected with the stopped flow spectrophotometer. The fastest phase was second order in oxygen (1900 M-1 s-1), resulted in the appearance of flavin semiquinone and yielded no superoxide. The next phase was also second order in oxygen (260 M-1 s-1), involved the loss of flavin semiquinone and yielded, on average, 1 mol of superoxide/mol of XDH oxidized. The last 2 electron equivalents were located in the iron-sulfur centers. They were released one equivalent at a time in the form of superoxide. Steady-state kinetics were found to be critically dependent on temperature and oxygen concentration. When these factors were carefully controlled, both the xanthine-oxygen and NADH-oxygen reductase reactions gave linear Lineweaver-Burk plots. The xanthine-oxygen data yielded a turnover number of 43 min-1, which was 42% of that for xanthine-NAD turnover. During turnover, with xanthine and O2, 40-44% of the electron equivalents introduced by xanthine appeared as superoxide. Reduced pyridine nucleotides, NAD and 3-aminopyridine adenine dinucleotide, dramatically reduced the formation of superoxide at levels which did not seriously inhibit oxygen reactivity.
Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.