Mitochondrial Radical Generation Research Articles

Quantum Sensing of Free Radical Generation in Mitochondria of Human Keratinocytes during UVB Exposure.

Ultraviolet (UV) radiation is known to cause skin issues, such as dryness, aging, and even cancer. Among UV rays, UVB stands out for its ability to trigger problems within cells, including mitochondrial dysfunction, oxidative stress, and DNA damage. Free radicals are implicated in these cellular responses, but they are challenging to measure due to their short lifetime and limited diffusion range. In our study, we used a quantum sensing technique (T1 relaxometry) involving fluorescent nanodiamonds (FNDs) that change their optical properties in response to magnetic noise. This allowed us to monitor the free radical presence in real time. To measure radicals near mitochondria, we coated FNDs with antibodies, targeting mitochondrial protein voltage-dependent anion channel 2 (anti-VDAC2). Our findings revealed a dynamic rise in radical levels on the mitochondrial membrane as cells were exposed to UVB (3 J/cm2), with a significant increase observed after 17 min.

Quantum Sensing of Free Radical Generation in Mitochondria of Single Heart Muscle Cells during Hypoxia and Reoxygenation.

Cells are damaged during hypoxia (blood supply deprivation) and reoxygenation (oxygen return). This damage occurs in conditions such as cardiovascular diseases, cancer, and organ transplantation, potentially harming the tissue and organs. The role of free radicals in cellular metabolic reprogramming under hypoxia is under debate, but their measurement is challenging due to their short lifespan and limited diffusion range. In this study, we employed a quantum sensing technique to measure the real-time production of free radicals at the subcellular level. We utilize fluorescent nanodiamonds (FNDs) that exhibit changes in their optical properties based on the surrounding magnetic noise. This way, we were able to detect the presence of free radicals. To specifically monitor radical generation near mitochondria, we coated the FNDs with an antibody targeting voltage-dependent anion channel 2 (anti-VDAC2), which is located in the outer membrane of mitochondria. We observed a significant increase in the radical load on the mitochondrial membrane when cells were exposed to hypoxia. Subsequently, during reoxygenation, the levels of radicals gradually decreased back to the normoxia state. Overall, by applying a quantum sensing technique, the connections among hypoxia, free radicals, and the cellular redox status has been revealed.

Fluorescent probe disclosing hydroxyl radical generation in mitochondria and nucleoli of cells during ferroptosis

Ferroptosis, a recently discovered form of cell death driven by iron-dependent lipid peroxidation (LP), significantly contributes to the inhibition of cancer proliferation and chemo-resistance. The hydroxyl radical (•OH), generated by a Fe2+-catalyzed Fenton reaction in cells, can result in LP and is intrinsically involved in ferroptosis. However, the number of •OH generation sites in the cells remains unclear. Herein, we report a pyronine-based fluorescent probe for •OH with phenothiazine as the reactive group. The probe responded very rapidly to •OH (< 1 s), and exhibited excellent selectivity. The probe can simultaneously target the mitochondria and nucleoli of cells, and thus shed light on •OH generation at both sites during ferroptosis. By comparison, drugs that could not induce cell ferroptosis, such as β-lapachone and rapamycin, only stimulated •OH generation in the mitochondria of cells. •OH production at both sites may be a new biochemical characteristic of ferroptosis, which provides a distinct insight into the mechanisms of drug action.

Cranberry (Vaccinium macrocarpon) peel polyphenol-rich extract attenuates rat liver mitochondria impairments in alcoholic steatohepatitis in vivo and after oxidative treatment in vitro

Abstract Alcoholic steatohepatitis is an important medical problem but its effective therapies are still not available. Plant polyphenols are widely used for prevention of toxic liver damages. The aim of the study was to evaluate the hepatoprotective mechanism(s) of a cranberry peel polyphenol-rich extract, focusing on the effects of the polyphenols on the mitochondrial function. The main components of cranberry peel phenolic compounds were anthocyanins, flavan-3-ols, procyanidins, flavonols, phenolic acids, as was detected using UHPLC-ESI-QTOF-MS. The treatment of rats receiving ethanol (4 g/kg bw, 8 weeks) with cranberry polyphenols (daily, 4 mg/kg bw) partially prevented alcoholic liver damage, ameliorating steatosis and inflammatory signs in the liver, decreasing serum and liver triglyceride contents, ALT and AST activities, as well as diminishing TNFα and TGFβ levels in serum. The polyphenols inhibited Ca2+ - induced mitochondrial permeability transition, free radical generation in mitochondria during intoxication. The polyphenols (25 µg/ml) prevented mitochondrial oxidative impairments in vitro. In conclusion, the cranberry peel polyphenols with antioxidant properties exerted a hepatoprotective and anti-inflammatory effects in the model of alcoholic steatohepatitis via prevention of liver mitochondria dysfunction.

Low-level lasers affect uncoupling protein gene expression in skin and skeletal muscle tissues

Wavelength, frequency, power, fluence, and emission mode determine the photophysical, photochemical, and photobiological responses of biological tissues to low-level lasers. Free radicals are involved in these responses acting as second messengers in intracellular signaling processes. Irradiated cells present defenses against these chemical species to avoid unwanted effects, such as uncoupling proteins (UCPs), which are part of protective mechanisms and minimize the effects of free radical generation in mitochondria. In this work UCP2 and UCP3 mRNA gene relative expression in the skin and skeletal muscle tissues of Wistar rats exposed to low-level red and infrared lasers was evaluated. Samples of the skin and skeletal muscle tissue of Wistar rats exposed to low-level red and infrared lasers were withdrawn for total RNA extraction, cDNA synthesis, and the evaluation of gene expression by quantitative polymerase chain reaction. UCP2 and UCP3 mRNA expression was differently altered in skin and skeletal muscle tissues exposed to lasers in a wavelength-dependent effect, with the UCP3 mRNA expression dose-dependent. Alteration on UCP gene expression could be part of the biostimulation effect and is necessary to make cells exposed to red and infrared low-level lasers more resistant or capable of adapting in damaged tissues or diseases.

Biochemical and Molecular Alterations Following Arsenic-Induced Oxidative Stress and Mitochondrial Dysfunction in Rat Brain.

Oxidative stress is associated with the generation of reactive oxygen species (ROS), which is supposed to be one of the mechanisms of arsenic-induced neurodegeneration. Mitochondria, being the major source of ROS generation may present an important target of arsenic-mediated neurotoxicity. Hence, we planned the study to elucidate the possible biochemical and molecular alterations induced by arsenic exposure in rat brain mitochondria. Chronic sodium arsenite treatment (25ppm for 12weeks) resulted in decreased activity of mitochondrial complexes I, II, and IV followed by increased ROS generation. There was decrease in mitochondrial superoxide dismutase (MnSOD) activity in arsenic-treated rat brain further showing increased superoxide radical generation in mitochondria. The decrease in MnSOD activity might be responsible for the increased protein and lipid oxidation as observed in our study. Protein and messenger RNA (mRNA) levels of MnSOD and mitochondrial uncoupling protein 2 (UCP-2) were downregulated suggesting decreased removal of ROS in rat brain. Fourier transform infrared (FTIR) spectroscopy analysis revealed significant decrease in amide A, amide I, amide II, and Olefinic = CH stretching band area suggesting molecular alteration in proteins and lipids after arsenic treatment. The results of present study indicate that arsenic-induced disturbed mitochondrial metabolism, decreased removal of ROS, decrease in protein synthesis, and altered membrane lipid polarity and fluidity may be responsible for the mitochondrial oxidative damage in rat brain that may further be implicated as contributing factor in arsenic-induced neurodegeneration.

Mitochondrial free radical generation and lifespan control in the fungal aging model Podospora anserina

In the filamentous fungus Podospora anserina a central role of mitochondria in the control of aging has been repeatedly demonstrated. Interestingly, impairments in cytochrome c oxidase (COX) activity induce an enhancement in the expression of the quinol-oxygen alternative oxidoreductase (AOX) correlating with an extension of lifespan. This effect is thought to be determined by a reduction of the free radical generation in mitochondria. In the current investigation we have analyzed the electron transport chain composition of P. anserina and the superoxide generation rate in wild type s and in mutant grisea, a long-lived mutant with complex IV deficiency. Here we report that, similarly to other fungi, mitochondrial respiration in P. anserina is a combination of standard and alternative routes. A switch in the COX/AOX respiration balance affects the mitochondrial free radical generation. Lower mitochondrial rates of superoxide generation were found in the long-lived mutant, supporting the central role of mitochondrial free radical generation in the lifespan control of P. anserina. The question of how the activity of the alternative respiratory pathway influences the rate of free radical generation in P. anserina mitochondria is discussed.

Cephaloridine Induces Translocation of Protein Kinase C δ Into Mitochondria and Enhances Mitochondrial Generation of Free Radicals in the Kidney Cortex of Rats Causing Renal Dysfunction

We have previously reported that the enhancement of free radical generation in mitochondria isolated from the kidney cortex of rats exposed to cephaloridine (CER) is probably mediated by the activation of protein kinase C (PKC). We examined which isoenzymes of PKC might be involved in the development of nephrotoxicity induced by CER in rats. The CER-induced renal dysfunction observed 24 h after its injection was prevented by a potent antioxidant DPPD and well-known PKC inhibitors like H-7 and rottlerin. At 1.5 and 3.5 h after the CER injection, the free radical generation was increased markedly and this was associated with translocation of PKCδ into the mitochondria of renal cortex tissue. Pretreatment of rats with H-7, a PKC inhibitor, significantly inhibited the CER-derived increase in mitochondrial generation of free radicals, suggesting that H-7 probably gets into the mitochondria and inhibits the activity of translocated PKC within the mitochondria. It was also shown that pretreatment of rats with rottlerin, a specific inhibitor of PKCδ, suppressed the early translocation of PKCδ into mitochondria and inhibited the CER-derived development of renal dysfunction. These results suggest that the CER-derived early translocation of PKCδ into mitochondria probably leads to the enhanced production of free radicals through the mitochondrial respiratory chain during the development of the nephrotoxicity caused by CER. Understanding the role of PKCδ in mitochondria may provide an important clue to the molecular mechanisms of mitochondrial production of reactive oxygen species and the free radical-induced renal failure in rats treated with CER.

Impaired mitochondrial respiratory chain and bioenergetics during chagasic cardiomyopathy development.

In this study, we evaluated the activities of respiratory chain complexes and oxidative phosphorylation (OXPHOS) capacity of the heart to gain insights into the pathological significance of mitochondrial dysfunction in chagasic cardiomyopathy (CCM). In a murine model of Trypanosoma cruzi infection, biochemical and histochemical analysis of the cardiac mitochondria revealed deficiency of the respiratory chain complexes (CI-CV) in infected mice; the inhibition of CI activity was more pronounced in the acute infection phase, CIII was constitutively repressed throughout the infection and disease phase, and the CV defects appeared in chronic phase only. A substantial decline in cardiac mtDNA content (54-60%) and mitochondria-encoded transcripts (50-65%) with disease development indicated that the alterations in mtDNA contribute to the quantitative deficiencies in respiratory chain activity in chagasic hearts. The observations of a selective inhibition of redox-sensitive CI and CIII complexes that are also the site of free radical generation in mitochondria, and the decline in cardiac mtDNA content in infected mice, all support the free radical hypothesis of mitochondria dysfunction in CCM. Consequently, OXPHOS-mediated ATP synthesis capacity of the cardiac mitochondria in infected mice was substantially reduced (37-50%), suggesting an energy homeostasis in the affected tissue.

Hydroxyl radical generation in mitochondria requires a soluble iron chelate: [formula omitted], Melodie Weller and Julia Knowles. Department of Chemistry, Central Washington University, Ellensburg, WA 98926, USA

Hydroxyl radical generation in mitochondria requires a soluble iron chelate: [formula omitted], Melodie Weller and Julia Knowles. Department of Chemistry, Central Washington University, Ellensburg, WA 98926, USA

The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice.

Adriamycin (ADR) is a potent anticancer drug known to cause severe cardiac toxicity. Although ADR generates free radicals, the role of free radicals in the development of cardiac toxicity and the intracellular target for ADR-induced cardiac toxicity are still not well understood. We produced three transgenic mice lines expressing increased levels of human manganese superoxide dismutase (MnSOD), a mitochondrial enzyme, as an animal model to investigate the role of ADR-mediated free radical generation in mitochondria. The human MnSOD was expressed, functionally active, and properly transported into mitochondria in the heart of transgenic mice. The levels of copper-zinc SOD, catalase, and glutathione peroxidase did not change in the transgenic mice. Electron microscopy revealed dose-dependent ultrastructural alterations with marked mitochondrial damage in nontransgenic mice treated with ADR, but not in the transgenic littermates. Biochemical analysis indicated that the levels of serum creatine kinase and lactate dehydrogenase in ADR-treated mice were significantly greater in nontransgenic than their transgenic littermates expressing a high level of human MnSOD after ADR treatment. These results support a major role for free radical generation in ADR toxicity as well as suggesting mitochondria as the critical site of cardiac injury.