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Vascular calcification is a prevalent hallmark of cardiovascular risk in elderly and diabetic individuals. Senescent vascular smooth muscle cells (VSMCs) participate in calcification; however, the associated underlying mechanisms remain unknown. Aberrant activation of the cytosolic DNA sensing adaptor stimulator of interferon gene 1 (STING1) caused by cytosolic DNA, particularly that leaked from damaged mitochondria, is a catalyst for aging-related diseases. Although oleoylethanolamide (OEA) is an endogenous bioactive lipid mediator with lipid overload-associated vasoprotective effects, its benefit in diabetic vascular calcification remains uncharacterized. This study focused on the role of STING1 in mitochondrial dysfunction-mediated calcification and premature VMSC senescence in diabetes and the effects of OEA on these pathological processes. In diabetic in vivo rat/mouse aorta calcification models and an in vitro VSMC calcification model induced by Nε-carboxymethyl-lysine (CML), senescence levels, STING1 signaling activation, and mitochondrial damage markers were significantly augmented; however, these alterations were markedly alleviated by OEA, partially in a nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent manner, and similar anti-calcification and senescence effects were observed in STING1-knockout mice and STING1-knockdown VSMCs. Mechanistically, mitochondrial DNA (mtDNA) damage was aggravated by CML in a reactive oxygen species-dependent manner, followed by mtDNA leakage into the cytosol, contributing to VSMC senescence-associated calcification via STING1 pathway activation. OEA treatment significantly attenuated the aforementioned cytotoxic effects of CML by enhancing cellular antioxidant capacity through the maintenance of Nrf2 translocation to the nucleus. Collectively, targeting STING1, a newly defined VSMC senescence regulator, contributes to anti-vascular calcification effects. [Display omitted] • OEA ameliorates senescence-associated diabetic vascular calcification. • OEA attenuates STING1-induced VSMC senescence-associated calcification by alleviating CML-triggered mtDNA damage. • OEA maintains mtDNA homeostasis by improving the Nrf2-dependent antioxidative destruction of CML-treated VSMCs. • STING1 knockout mitigates VSMC senescence-associated diabetic mouse vascular calcification. [ABSTRACT FROM AUTHOR]

Pesticide treatment leads to a significant increase in wheat yield and enhances pest resistance. However, pesticides, particularly the insecticide cypermethrin, which is used for wheat Triticum aestivum L. field and grain storage treatment, are toxic xenobiotics. To reduce their toxicity, it is possible to pre-treat seeds with plant growth-promoting rhizobacteria (PGPR), which can enhance plant resistance to adverse environmental factors. In this study, we used a previously unused as PGPR representative of the genus Azospirillum, A. thiophilum BV-ST, isolated in Russia, which makes it interesting for agricultural use in the same climatic zone. We optimized the detection method of A. thiophilum using PCR. Then, we demonstrated that this bacterium successfully inoculates to wheat roots. We showed that cypermethrin treatment of one- and two-week-old wheat seedlings caused an increase in mtDNA damage, and in one-week-old seedlings, it induced a decrease in chlorophyll levels. However, three- and four-week-old seedlings were more resistant to damage, possibly due to their approximately 5-fold higher expression of NDOR-1 and GST genes, which play an important role in xenobiotic detoxification. Pre-treatment of wheat seeds with A. thiophilum contributed to the upregulation of NDOR-1 and GST gene expression in one- and two-week-old seedlings, resulting in the protection of mtDNA from cypermethrin-induced damage and prevented the decrease in chlorophyll levels. [ABSTRACT FROM AUTHOR]

Interstitial lung diseases (ILDs) are lethal lung diseases characterized by pulmonary inflammation and progressive lung interstitial scarring. We previously developed a mouse model of ILD using vanadium pentoxide (V2O5) and identified several gene candidates on chromosome 4 associated with pulmonary fibrosis. While these data indicated a significant genetic contribution to ILD susceptibility, they did not include any potential associations and interactions with the mitochondrial genome that might influence disease risk. To conduct this pilot work, we selected the two divergent strains we previously categorized as V2O5-resistant C57BL6J (B6) and -responsive DBA/2J (D2) and compared their mitochondrial genome characteristics, including DNA variants, heteroplasmy, lesions, and copy numbers at 14- and 112-days post-exposure. While we did not find changes in the mitochondrial genome at 14 days post-exposure, at 112 days, we found that the responsive D2 strain exhibited significantly fewer mtDNA copies and more lesions than control animals. Alongside these findings, mtDNA heteroplasmy frequency decreased. These data suggest that mice previously shown to exhibit increased susceptibility to pulmonary fibrosis and inflammation sustain damage to the mitochondrial genome that is evident at 112 days post-V2O5 exposure. [ABSTRACT FROM AUTHOR]

Introduction: Mitochondrial dysfunction is postulated to be a central event in fetal alcohol spectrum disorders (FASD). People with the most severe form of FASD, fetal alcohol syndrome (FAS) are estimated to live only 34 years (95% confidence interval, 31 to 37 years), and adults who were born with any form of FASD often develop early aging. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage, hallmarks of aging, are postulated central events in FASD. Ethanol (EtOH) can cause mtDNA damage, consequent increased oxidative stress, and changes in the mtDNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1). Studies of molecular mechanisms are limited by the absence of suitable human models and non-invasive tools. Methods: We compared human and rat EtOH-exposed fetal brain tissues and neuronal cultures, and fetal brain-derived exosomes (FB-Es) from maternal blood. Rat FASD was induced by administering a 6.7% alcohol liquid diet to pregnant dams. Human fetal (11-21 weeks) brain tissue was collected and characterized by maternal self-reported EtOH use. mtDNA was amplified by qPCR. OGG1 and Insulin-like growth factor 1 (IGF-1) mRNAs were assayed by qRT-PCR. Exosomal OGG1 was measured by ddPCR. Results: Maternal EtOH exposure increased mtDNA damage in fetal brain tissue and FB-Es. The damaged mtDNA in FB-Es correlated highly with small eye diameter, an anatomical hallmark of FASD. OGG1-mediated mtDNA repair was inhibited in EtOH-exposed fetal brain tissues. IGF-1 rescued neurons from EtOH-mediated mtDNA damage and OGG1 inhibition. Conclusion: The correlation between mtDNA damage and small eye size suggests that the amount of damaged mtDNA in FB-E may serve as a marker to predict which at risk fetuses will be born with FASD. Moreover, IGF-1 might reduce EtOH-caused mtDNA damage and neuronal apoptosis. [ABSTRACT FROM AUTHOR]

IntroductionMitochondrial dysfunction is postulated to be a central event in fetal alcohol spectrum disorders (FASD). People with the most severe form of FASD, fetal alcohol syndrome (FAS) are estimated to live only 34 years (95% confidence interval, 31 to 37 years), and adults who were born with any form of FASD often develop early aging. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage, hallmarks of aging, are postulated central events in FASD. Ethanol (EtOH) can cause mtDNA damage, consequent increased oxidative stress, and changes in the mtDNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1). Studies of molecular mechanisms are limited by the absence of suitable human models and non-invasive tools.MethodsWe compared human and rat EtOH-exposed fetal brain tissues and neuronal cultures, and fetal brain-derived exosomes (FB-Es) from maternal blood. Rat FASD was induced by administering a 6.7% alcohol liquid diet to pregnant dams. Human fetal (11–21 weeks) brain tissue was collected and characterized by maternal self-reported EtOH use. mtDNA was amplified by qPCR. OGG1 and Insulin-like growth factor 1 (IGF-1) mRNAs were assayed by qRT-PCR. Exosomal OGG1 was measured by ddPCR.ResultsMaternal EtOH exposure increased mtDNA damage in fetal brain tissue and FB-Es. The damaged mtDNA in FB-Es correlated highly with small eye diameter, an anatomical hallmark of FASD. OGG1-mediated mtDNA repair was inhibited in EtOH-exposed fetal brain tissues. IGF-1 rescued neurons from EtOH-mediated mtDNA damage and OGG1 inhibition.ConclusionThe correlation between mtDNA damage and small eye size suggests that the amount of damaged mtDNA in FB-E may serve as a marker to predict which at risk fetuses will be born with FASD. Moreover, IGF-1 might reduce EtOH-caused mtDNA damage and neuronal apoptosis.

T-2 toxin is produced by different Fusarium species and belongs to the group of type A trichothecene mycotoxins. T-2 toxin contaminates various grains, such as wheat, barley, maize, or rice, thus posing a risk to human and animal health. The toxin has toxicological effects on human and animal digestive, immune, nervous and reproductive systems. In addition, the most significant toxic effect can be observed on the skin. This in vitro study focused on T-2 toxicity on human skin fibroblast Hs68 cell line mitochondria. In the first step of this study, T-2 toxin's effect on the cell mitochondrial membrane potential (MMP) was determined. The cells were exposed to T-2 toxin, which resulted in dose- and time-dependent changes and a decrease in MMP. The obtained results revealed that the changes of intracellular reactive oxygen species (ROS) in the Hs68 cells were not affected by T-2 toxin. A further mitochondrial genome analysis showed that T-2 toxin in a dose- and time-dependent manner decreased the number of mitochondrial DNA (mtDNA) copies in cells. In addition, T-2 toxin genotoxicity causing mtDNA damage was evaluated. It was found that incubation of Hs68 cells in the presence of T-2 toxin, in a dose- and time-dependent manner, increased the level of mtDNA damage in both tested mtDNA regions: NADH dehydrogenase subunit 1 (ND1) and NADH dehydrogenase subunit 5 (ND5). In conclusion, the results of the in vitro study revealed that T-2 toxin shows adverse effects on Hs68 cell mitochondria. T-2 toxin induces mitochondrial dysfunction and mtDNA damage, which may cause the disruption of adenosine triphosphate (ATP) synthesis and, in consequence, cell death. [ABSTRACT FROM AUTHOR]

Background Mitochondrial abnormalities exist in gastrocnemius muscle of people with peripheral artery disease (PAD). Whether abnormalities in mitochondrial biogenesis and autophagy are associated with greater ischemia or walking impairment in PAD is unknown. Methods and Results Protein markers of mitochondrial biogenesis and autophagy and the abundance of mitochondrial electron transport chain complexes were quantified in gastrocnemius muscle biopsies from people with and without PAD. Their 6‐minute walk distance and 4‐m gait speed were measured. Sixty‐seven participants (mean age 65.0 years [±6.8], 16 [23.9%] women, 48 [71.6%] Black) were enrolled, including 15 with moderate to severe PAD (ankle brachial index [ABI]

Abstract Background Hepatocellular carcinoma (HCC) is the leading cause of primary liver cancers. Surveillance of individuals at specific risk of developing HCC, early diagnostic markers, and new therapeutic approaches are essential to obtain a reduction in disease-related mortality. Apurinic/apyrimidinic endonuclease 1 (APE1) expression levels and its cytoplasmic localization have been reported to correlate with a lower degree of differentiation and shorter survival rate. The aim of this study is to fully investigate, for the first time, the role of the mitochondrial form of APE1 in HCC. Methods As a study model, we analyzed samples from a cohort of patients diagnosed with HCC who underwent surgical resection. Mitochondrial APE1 content, expression levels of the mitochondrial import protein Mia40, and mtDNA damage of tumor tissue and distal non-tumor liver of each patient were analyzed. In parallel, we generated a stable HeLa clone for inducible silencing of endogenous APE1 and re-expression of the recombinant shRNA resistant mitochondrially targeted APE1 form (MTS-APE1). We evaluated mtDNA damage, cell growth, and mitochondrial respiration. Results APE1’s cytoplasmic positivity in Grades 1 and 2 HCC patients showed a significantly higher expression of mitochondrial APE1, which accounted for lower levels of mtDNA damage observed in the tumor tissue with respect to the distal area. In the contrast, the cytoplasmic positivity in Grade 3 was not associated with APE1’s mitochondrial accumulation even when accounting for the higher number of mtDNA lesions measured. Loss of APE1 expression negatively affected mitochondrial respiration, cell viability, and proliferation as well as levels of mtDNA damage. Remarkably, the phenotype was efficiently rescued in MTS-APE1 clone, where APE1 is present only within the mitochondrial matrix. Conclusions Our study confirms the prominent role of the mitochondrial form of APE1 in the early stages of HCC development and the relevance of the non-nuclear fraction of APE1 in the disease progression. We have also confirmed overexpression of Mia40 and the role of the MIA pathway in the APE1 import process. Based on our data, inhibition of the APE1 transport by blocking the MIA pathway could represent a new therapeutic approach for reducing mitochondrial metabolism by preventing the efficient repair of mtDNA.

Mitochondria are primarily involved in energy production through the process of oxidative phosphorylation (OXPHOS). Increasing evidence has shown that mitochondrial function impacts a plethora of different cellular activities, including metabolism, epigenetics, and innate immunity. Like the nucleus, mitochondria own their genetic material, but this organellar genome is circular, present in multiple copies, and maternally inherited. The mitochondrial DNA (mtDNA) encodes 37 genes that are solely involved in OXPHOS. Maintenance of mtDNA, through replication and repair, requires the import of nuclear DNA-encoded proteins. Thus, mitochondria completely rely on the nucleus to prevent mitochondrial genetic alterations. As most cells contain hundreds to thousands of mitochondria, it follows that the shear number of organelles allows for the buffering of dysfunction--at least to some extent--before tissue homeostasis becomes impaired. Only red blood cells lack mitochondria entirely. Impaired mitochondrial function is a hallmark of aging and is involved in a number of different disorders, including neurodegenerative diseases, diabetes, cancer, and autoimmunity. Although alterations in mitochondrial processes unrelated to OXPHOS, such as fusion and fission, contribute to aging and disease, maintenance of mtDNA integrity is critical for proper organellar function. Here, we focus on how mtDNA damage contributes to cellular dysfunction and health outcomes. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA (mtDNA) is a circular double-stranded genome that exists independently of the nucleus. In recent years, research on mtDNA has significantly increased, leading to a gradual increase in understanding of its physiological and pathological characteristics. Reactive oxygen species (ROS) and other factors can damage mtDNA. This damaged mtDNA can escape from the mitochondria to the cytoplasm or extracellular space, subsequently activating immune signaling pathways, such as NLR family pyrin domain protein 3 (NLRP3), and triggering inflammatory responses. Numerous studies have demonstrated the involvement of mtDNA damage and leakage in the pathological mechanisms underlying various diseases including infectious diseases, metabolic inflammation, and immune disorders. Consequently, comprehensive investigation of mtDNA can elucidate the pathological mechanisms underlying numerous diseases. The prevention of mtDNA damage and leakage has emerged as a novel approach to disease treatment, and mtDNA has emerged as a promising target for drug development. This article provides a comprehensive review of the mechanisms underlying mtDNA-induced inflammation, its association with various diseases, and the methods used for its detection. [ABSTRACT FROM AUTHOR]

Abstract Background Decreased heart rate variability (HRV) leads to cardiovascular diseases and increased mortality in clinical studies. However, the underlying mechanisms are still inconclusive. Systemic inflammation-induced neuroinflammation is known to impair the autonomic center of cardiovascular regulation. The dynamic stability of blood pressure and heart rate (HR) is regulated by modulation of the reciprocal responses of sympathetic and parasympathetic tone by the baroreflex, which is controlled by the nucleus of the solitary tract (NTS). Methods Systemic inflammation was induced by E. coli lipopolysaccharide (LPS, 1.2 mg/kg/day, 7 days) peritoneal infusion via an osmotic minipump in normotensive Sprague-Dawley rats. Systolic blood pressure (SBP) and HR were measured by femoral artery cannulation and recorded on a polygraph under anesthesia. The low-frequency (LF; 0.25–0.8 Hz) and high-frequency (HF; 0.8–2.4 Hz) components of SBP were adopted as the indices for sympathetic vasomotor tone and parasympathetic vasomotor tone, while the baroreflex effectiveness index (BEI) was adopted from the analysis of SBP and pulse interval (PI). The plasma levels of proinflammatory cytokines and mitochondrial DNA (mtDNA) oxidative damage were analyzed by ELISA. Protein expression was evaluated by Western blot. The distribution of oxidative mtDNA was probed by immunofluorescence. Pharmacological agents were delivered via infusion into the cisterna magna with an osmotic minipump. Results The suppression of baroreflex sensitivity was concurrent with increased SBP and decreased HR. Neuroinflammatory factors, including TNF-α, CD11b, and Iba-1, were detected in the NTS of the LPS group. Moreover, indices of mtDNA damage, including 8-OHdG and γ-H2AX, were significantly increased in neuronal mitochondria. Pentoxifylline or minocycline intracisternal (IC) infusion effectively prevented mtDNA damage, suggesting that cytokine and microglial activation contributed to mtDNA damage. Synchronically, baroreflex sensitivity was effectively protected, and the elevated blood pressure was significantly relieved. In addition, the mtDNA repair mechanism was significantly enhanced by pentoxifylline or minocycline. Conclusion These results suggest that neuronal mtDNA damage in the NTS induced by neuroinflammation could be the core factor in deteriorating baroreflex desensitization and subsequent cardiovascular dysfunction. Therefore, the enhancement of base excision repair (BER) signaling in mitochondria could be a potential therapeutic strategy for cardiovascular reflex dysregulation.

Abstract Atherosclerosis with associated cardiovascular diseases remains one of the main causes of disability and death worldwide, requiring development of new solutions for prevention and treatment. Macrophages are the key effectors of a series of events involved in atherogenesis, such as inflammation, plaque formation, and changes in lipid metabolism. Some of these events were shown to be associated with mitochondrial dysfunction and excessive mitochondrial DNA (mtDNA) damage. Moreover, macrophages represent a promising target for novel therapeutic approaches that are based on the expression of various receptors and nanoparticle uptake. Lipid‐based gene delivery to mitochondria is considered to be an interesting strategy for mtDNA damage correction. To date, several nanocarriers and their modifications have been developed that demonstrate high transfection efficiency and low cytotoxicity. This review discusses the possibilities of lipid‐based gene delivery to macrophage mitochondria for atherosclerosis therapy.

Atherosclerosis with associated cardiovascular diseases remains one of the main causes of disability and death worldwide, requiring development of new solutions for prevention and treatment. Macrophages are the key effectors of a series of events involved in atherogenesis, such as inflammation, plaque formation, and changes in lipid metabolism. Some of these events were shown to be associated with mitochondrial dysfunction and excessive mitochondrial DNA (mtDNA) damage. Moreover, macrophages represent a promising target for novel therapeutic approaches that are based on the expression of various receptors and nanoparticle uptake. Lipid-based gene delivery to mitochondria is considered to be an interesting strategy for mtDNA damage correction. To date, several nanocarriers and their modifications have been developed that demonstrate high transfection efficiency and low cytotoxicity. This review discusses the possibilities of lipid-based gene delivery to macrophage mitochondria for atherosclerosis therapy. [ABSTRACT FROM AUTHOR]

Atherosclerosis is a multifactorial disease of the cardiovascular system associated with aging, inflammation, and oxidative stress. An important role in the development of atherosclerosis play elevated plasma lipoproteins. A number of external factors (smoking, diabetes, infections) can also contribute to the development of the disease. For a long time, atherosclerosis remains asymptomatic, therefore, the search for early markers of the disease is critical for the timely management and better outcomes for patients. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage appear to connect different aspects of atherosclerosis pathogenesis. To date, multiple lines of research have demonstrated the strong association of mitochondrial dysfunction with the development of various human diseases. Therapies aimed at restoring the mitochondrial function are being actively developed, and are expected to broaden the therapeutic possibilities for several chronic human diseases. The development of such therapies depends on our understanding of the functional roles of different mtDNA variants associated with one or another disorder, and the molecular mechanisms linking mitochondrial dysfunction with a given pathological feature. These questions are, however, challenging and require future intensive research. This review summarizes the recent studies and describes the central processes of the development of atherosclerosis, and shows their relationship with mitochondrial dysfunction. One of the promising therapeutic approaches for future atherosclerosis treatments is the use of mitochondria-targeted antioxidants. Future studies should focus on characterizing the mechanisms of mitochondrial involvement in cardiovascular pathologies to better direct the search for novel therapies.

Key points: Breast cancer 1 early onset gene codes for the DNA repair enzyme, breast cancer type 1 susceptibility protein (BRCA1). The gene is prone to mutations that cause a loss of protein function.BRCA1/Brca1 has recently been found to regulate several cellular pathways beyond DNA repair and is expressed in skeletal muscle.Skeletal muscle specific knockout of Brca1 in mice caused a loss of muscle quality, identifiable by reductions in muscle force production and mitochondrial respiratory capacity.Loss of muscle quality was associated with a shift in muscle phenotype and an accumulation of mitochondrial DNA mutations.These results demonstrate that BRCA1 is necessary for skeletal muscle function and that increased mitochondrial DNA mutations may represent a potential underlying mechanism. Recent evidence suggests that the breast cancer 1 early onset gene (BRCA1) influences numerous peripheral tissues, including skeletal muscle. The present study aimed to determine whether induced‐loss of the breast cancer type 1 susceptibility protein (Brca1) alters skeletal muscle function. We induced genetic ablation of exon 11 in the Brca1 gene specifically in the skeletal muscle of adult mice to generate skeletal muscle‐specific Brca1 homozygote knockout (Brca1KOsmi) mice. Brca1KOsmi exhibited kyphosis and decreased maximal isometric force in limb muscles compared to age‐matched wild‐type mice. Brca1KOsmi skeletal muscle shifted toward an oxidative muscle fibre type and, in parallel, increased myofibre size and reduced capillary numbers. Unexpectedly, myofibre bundle mitochondrial respiration was reduced, whereas contraction‐induced lactate production was elevated in Brca1KOsmi muscle. Brca1KOsmi mice accumulated mitochondrial DNA mutations and exhibited an altered mitochondrial morphology characterized by distorted and enlarged mitochondria, and these were more susceptible to swelling. In summary, skeletal muscle‐specific loss of Brca1 leads to a myopathy and mitochondriopathy characterized by reductions in skeletal muscle quality and a consequent kyphosis. Given the substantial impact of BRCA1 mutations on cancer development risk in humans, a parallel loss of BRCA1 function in patient skeletal muscle cells would potentially result in implications for human health. Key points: Breast cancer 1 early onset gene codes for the DNA repair enzyme, breast cancer type 1 susceptibility protein (BRCA1). The gene is prone to mutations that cause a loss of protein function.BRCA1/Brca1 has recently been found to regulate several cellular pathways beyond DNA repair and is expressed in skeletal muscle.Skeletal muscle specific knockout of Brca1 in mice caused a loss of muscle quality, identifiable by reductions in muscle force production and mitochondrial respiratory capacity.Loss of muscle quality was associated with a shift in muscle phenotype and an accumulation of mitochondrial DNA mutations.These results demonstrate that BRCA1 is necessary for skeletal muscle function and that increased mitochondrial DNA mutations may represent a potential underlying mechanism. [ABSTRACT FROM AUTHOR]

Reduced cellular response to insulin in skeletal muscle is one of the major components of the development of type 2 diabetes (T2D). Mitochondrial dysfunction involves in the accumulation of toxic reactive oxygen species (ROS) that leads to insulin resistance. The aim of this study was to verify the involvement of mitochondrial DNA damage at ROS generation in skeletal muscle during development of T2D. Wistar rats were fed a diet containing 60% fat over 8 weeks and at day 14 a single injection of STZ (25 mg/kg) was administered (T2D-induced). Control rats received standard food and an injection of citrate buffer. Blood and soleus muscle were collected. Abdominal fat was quantified as well as glucose, triglyceride, LDL, HDL, and total cholesterol in plasma and mtDNA copy number, cytochrome b (cytb) mRNA, 8-hydroxyguanosine, and 8-isoprostane (a marker of ROS) in soleus muscle. T2D-induced animal presented similar characteristics to humans that develop T2D such as changes in blood glucose, abdominal fat, LDL, HDL and cholesterol total. In soleus muscle 8-isoprostane, mtDNA copy number and 8-hydroxyguanosine were increased, while cytb mRNA was decreased in T2D. Our results suggest that in the development of T2D, when risks factors of T2D are present, intracellular oxidative stress increases in skeletal muscle and is associated with a decrease in cytb transcription. To overcome this process mtDNA increased but due to the proximity of ROS generation, mtDNA remains damaged by oxidation leading to an increase in ROS in a vicious cycle accounting to the development of insulin resistance and further T2D. [ABSTRACT FROM AUTHOR]

Although chemotherapeutics can be highly effective at targeting malignancies, their ability to trigger cardiovascular morbidity is clinically significant. Chemotherapy can adversely affect cardiovascular physiology, resulting in the development of cardiomyopathy, heart failure and microvascular defects. Specifically, anthracyclines are known to cause an excessive buildup of free radical species and mitochondrial DNA damage (mtDNA) that can lead to oxidative stress-induced cardiovascular apoptosis. Therefore, oncologists and cardiologists maintain a network of communication when dealing with patients during treatment in order to treat and prevent chemotherapy-induced cardiovascular damage; however, there is a need to discover more accurate biomarkers and therapeutics to combat and predict the onset of cardiovascular side effects. Telomerase, originally discovered to promote cellular proliferation, has recently emerged as a potential mechanism to counteract mitochondrial defects and restore healthy mitochondrial vascular phenotypes. This review details mechanisms currently used to assess cardiovascular damage, such as C-reactive protein (CRP) and troponin levels, while also unearthing recently researched biomarkers, including circulating mtDNA, telomere length and telomerase activity. Further, we explore a potential role of telomerase in the mitigation of mitochondrial reactive oxygen species and maintenance of mtDNA integrity. Telomerase activity presents a promising indicator for the early detection and treatment of chemotherapy-derived cardiac damage. [ABSTRACT FROM AUTHOR]

Anti-synthetase syndrome is an autoimmune disease characterized by autoantibodies toward amino acyl-tRNA synthetases (ARS), anti-Jo 1 being the most commonly detected. Muscle damage develops in up to 90% of ARS-positive patients, characterized by a necrotizing myositis restricted to the perifascicular region. This topographic distribution of muscle damage may lead to a misdiagnosis of dermatomyositis (DM) at muscle biopsy. We compared morphological, immunohistochemical, and histoenzymatic features of muscle from ARS-positive patients (n = 11) with those of DM (n = 7) providing clues for their differential diagnosis. In addition, we evaluated markers of mitochondrial damage to provide a further distinction between these two entities. Necrosis occurred in the majority of ARS patients, mainly located in the perifascicular region. It was often limited to small foci of fibers, always associated with myocyte regeneration. This last often overwhelmed necrosis, representing occasionally the main finding. In DM, necrosis/regeneration was scarce while the peculiar feature was a diffuse atrophy of perifascicular fibers. These last showed decreased cytochrome c oxidase (COX) stain and mitochondrial DNA depletion, consistent with mitochondrial dysfunction. In contrast to DM, ARS displayed scattered COX-deficient fibers, not restricted to the perifascicular region. This feature occurred in up to 91% of patients, being prominent only in two. [ABSTRACT FROM AUTHOR]

Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins. Our results not only help in the understanding of how mitochondria cope with replicative stress but can also explain some controversial features of the lagging-strand replication. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction is a relevant mechanism in cardiac aging. Here, we investigated the effects of late-life enalapril administration at a non-antihypertensive dose on mitochondrial genomic stability, oxidative damage, and mitochondrial quality control (MQC) signaling in the hearts of aged rats. The protein expression of selected mediators (i.e., mitochondrial antioxidant enzymes, energy metabolism, mitochondrial biogenesis, dynamics, and autophagy) was measured in old rats randomly assigned to receive enalapril (n = 8) or placebo (n = 8) from 24 to 27 months of age. We also assessed mitochondrial DNA (mtDNA) content, citrate synthase activity, oxidative lesions to protein and mtDNA (i.e., carbonyls and the abundance of mtDNA4834 deletion), and the mitochondrial transcription factor A (TFAM) binding to specific mtDNA regions. Enalapril attenuated cardiac hypertrophy and oxidative stress-derived damage (mtDNA oxidation, mtDNA4834 deletion, and protein carbonylation), while increasing mitochondrial antioxidant defenses. The binding of mitochondrial transcription factor A to mtDNA regions involved in replication and deletion generation was enhanced following enalapril administration. Increased mitochondrial mass as well as mitochondriogenesis and autophagy signaling were found in enalapril-treated rats. Late-life enalapril administration mitigates age-dependent cardiac hypertrophy and oxidative damage, while increasing mitochondrial mass and modulating MQC signaling. Further analyses are needed to conclusively establish whether enalapril may offer cardioprotection during aging.

Oxidative stress and mitochondrial dysfunction are acute consequences of status epilepticus (SE). However, the role of mitochondrial oxidative stress and genomic instability during epileptogenesis remains unknown. Using the kainate animal model of temporal lobe epilepsy, we investigated oxidative mitochondrial DNA (mtDNA) damage and changes in the mitochondrial base excision repair pathway (mtBER) in the rat hippocampus for a period of 3 months after SE. Acute seizure activity caused a time-dependent increase in mitochondrial, but not nuclear 8-hydroxy-2-deoxyguanosine (8-OHdG/2dG) levels and a greater frequency of mtDNA lesions. This was accompanied by increased mitochondrial H2O2 production and a transient decrease in mtDNA repair capacity. The mtBER proteins 8-oxoguanine glycosylase (Ogg1) and DNA polymerase gamma (Pol γ) demonstrated elevated expression at mRNA and protein levels shortly after SE and this was followed by a gradual improvement in mtDNA repair capacity. Recurrent seizures associated with the chronic phase of epilepsy coincided with the accumulation of mtDNA damage, increased mitochondrial H2O2 levels, decreased expression of Ogg1 and Pol γ and impaired mtDNA repair capacity. Together, increased oxidative mtDNA damage, mitochondrial H2O2 production and alterations in the mtBER pathway provide evidence for mitochondrial oxidative stress in epilepsy and suggest that mitochondrial injury may contribute to epileptogenesis.

Aims: The present study was designed to investigate a possible interaction between vascular smooth muscle cell (SMC)-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) and mitochondrial DNA (mtDNA) damage. Results: Treatment of cultured SMCs with the proinflammatory stimulus lipopolysaccharide (LPS) stimulated PCSK9 release and induced mtDNA damage. PCSK9 inhibition by its siRNA reduced, and its enhancement increased, mtDNA damage. Induction of mitochondria-derived reactive oxygen species (mtROS) (by rotenone, thenoyltrifluoroacetone, or antimycin A) enhanced mtDNA damage as well as PCSK9 release, suggesting a role of mtROS in PCSK9-mtDNA damage interplay. Induction of mtDNA damage (with the autophagy inhibitor, 3-methyladenine, or DNase II inhibition) enhanced PCSK9 expression, and inhibition of mtDNA damage (with the autophagy inducer, rapamycin) reduced PCSK9 expression, indicating bidirectional interplay between PCSK9 and mtDNA damage. Other studies showed that p38 MAPK is involved in PCSK9-induced mtDNA damage, and mammalian target of rapamycin activation plays a role in mtDNA damage-induced PCSK9 release. Functional impact of PCSK9-mtDNA damage cross-talk was evident in the form of SMC apoptosis, which was enhanced in cells treated with recombinant human PCSK9, but inhibited in cells treated with PCSK9 siRNA. Last, LPS administration in wild-type mice resulted in simultaneous PCSK9 release and mtDNA damage, but mtDNA damage was minimal in PCSK9-null mice given LPS. Innovation: Vascular SMC-derived PCSK9 induces mtDNA damage, and damaged mtDNA fragments stimulate PCSK9 release mediated, at least in part, by mtROS. Conclusions: These observations suggest positive feedback interplay between SMC-derived PCSK9 and mtDNA damage in the proinflammatory milieu involving mtROS. This interaction results in cellular injury, characterized by apoptosis-a hallmark of atherosclerosis. Antioxid. Redox Signal. 25, 997-1008. [ABSTRACT FROM AUTHOR]

Mitochondrial genome maintenance plays a central role in preserving brain health. We previously demonstrated accumulation of mitochondrial DNA damage and severe neurodegeneration in transgenic mice inducibly expressing a mutated mitochondrial DNA repair enzyme (mutUNG1) selectively in forebrain neurons. Here, we examine whether severe neurodegeneration in mutUNG1-expressing mice could be rescued by feeding the mice a ketogenic diet, which is known to have beneficial effects in several neurological disorders. The diet increased the levels of superoxide dismutase 2, and mitochondrial mass, enzymes, and regulators such as SIRT1 and FIS1, and appeared to downregulate N -methyl-D-aspartic acid (NMDA) receptor subunits NR2A/B and upregulate γ-aminobutyric acid A (GABA A ) receptor subunits α 1 . However, unexpectedly, the ketogenic diet aggravated neurodegeneration and mitochondrial deterioration. Electron microscopy showed structurally impaired mitochondria accumulating in neuronal perikarya. We propose that aggravation is caused by increased mitochondrial biogenesis of generally dysfunctional mitochondria. This study thereby questions the dogma that a ketogenic diet is unambiguously beneficial in mitochondrial disorders. [ABSTRACT FROM AUTHOR]

[Display omitted] Vascular calcification, a prevalent pathological alteration in metabolic syndromes, is tightly related with cardiometabolic risk events. Ferroptosis, a newly iron-dependent programmed cell death, induced by palmitic acid (PA), the major saturated free fatty acid in hyperlipidemia, is a vital mechanism of vascular calcification. Recent studies reported that ferroptosis is a distinctive type of cell death dependent on autophagy, with the lipotoxicity of PA on cell viability being closely linked with autophagy. Oleoylethanolamide (OEA), an endogenous bioactive mediator of lipid homeostasis, exerts vascular protection against intimal calcification, atherosclerosis; however, its beneficial effect on vascular smooth muscle cell (VSMC)-associated medial calcification has not been investigated. Our aim was to characterize the effect of OEA on vascular calcification and ferroptosis of VSMCs under hyperlipidaemia/PA exposure. In vivo, vascular calcification model was induced in rats by high-fat diet and vitamin D3 plus nicotine; in vitro , VSMCs ferroptosis was induced by PA or plus β-glycerophosphate mimicking vascular calcification. The calcium deposition in hyperlipidaemia-mediated rat thoracic aortas, the PA-induced ferroptosis and subsequent calcium deposition in VSMCs, were suppressed by OEA treatment. Additionally, CGAS-STING1-induced ferritinophagy, the main molecular mechanism of PA-triggered ferroptosis of VSMCs, was activated by mitochondrial DNA damage; however, early administration of OEA alleviated these phenomena. Intriguingly, overexpression of peroxisome proliferator activated receptor alpha (PPARα) contributed to a decrease in PA-induced ferroptosis, whereas PPARɑ knockdown inhibited the OEA-mediated anti-ferroptotic effects. Collectively, our study demonstrated that OEA serves as a prospective candidate for the prevention and treatment of vascular calcification in metabolic abnormality syndromes. [ABSTRACT FROM AUTHOR]

To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)-Cas9-mediated knockout of TFAM or other genes essential for mtDNA replication. Support protocols detail approaches for several processes: (1) genotyping ρ 0 cells of human, mouse, and rat origin by polymerase chain reaction (PCR); (2) quantification of mtDNA by quantitative PCR (qPCR); (3) preparation of calibrator plasmids for mtDNA quantification; and (4) quantification of mtDNA by direct droplet digital PCR (dddPCR). © 2023 Wiley Periodicals LLC. Basic Protocol: Inducing mtDNA loss with mUNG1 Alternate Protocol 1: Generation of ρ 0 cells by mtDNA depletion with EtBr and ddC Alternate Protocol 2: Generation of ρ 0 cells by knocking out genes critical for mtDNA replication Support Protocol 1: Genotyping ρ 0 cells by DirectPCR Support Protocol 2: Determination of mtDNA copy number by qPCR Support Protocol 3: Preparation of calibrator plasmid for qPCR Support Protocol 4: Determination of mtCN by direct droplet digital PCR (dddPCR)., (© 2023 Wiley Periodicals LLC.)

Mitochondrial dysfunction has been associated with late onset neurodegenerative disorders, among which is Machado-Joseph disease (MJD/SCA3). In a previous study, using a transgenic mouse model of MJD, we reported a decrease in mitochondrial DNA (mtDNA) copy number and an accumulation of the 3876-bp deletion with age and with phenotype development. We extended this study by analyzing the pattern of mtDNA depletion and the accumulation of the 3876-bp deletion in 12 older transgenic (TG) and 4 wild-type (wt) animals, and by investigating the accumulation of somatic mutations in the D-loop region in 76 mice (42 TG and 34 wt). mtDNA damage was studied in TG and wt mice at different ages and tissues (blood, pontine nuclei, and hippocampus). Results for older mice demonstrate an accumulation of the mtDNA 3867-bp deletion with age, which was more pronounced in TG animals. Furthermore, the tendency for mtDNA copy number decrease with age, in all analyzed tissues of TG and wt animals, was also confirmed. No point mutations were detected in the D-loop, neither in TG nor wt animals, in any of the tissues analyzed. Due to the absence of mtDNA somatic mutations, we can suggest that mtDNA point mutation accumulation cannot be used to monitor the development and progression of the phenotype in this mouse model and likely in any MJD mice model. The present results further confirm not only the association between mtDNA alterations (copy number and deletions) and age, but also between such alterations and the expression of the mutant ataxin-3 in TG mice. [ABSTRACT FROM AUTHOR]

Highlights: [•] DNA damage distributes in a heterogeneous pattern under normal physiological conditions. [•] Mitochondrial oxidative stress induces damage in a gene-specific manner, but promoter and coding regions are equally susceptible. [•] The rate of repair of induced DNA damage is independent of site and recovery time depends on the initial insult. [•] Repair of mtDNA is slower and more sensitive to ATP depletion than nDNA repair. [ABSTRACT FROM AUTHOR]

Abstract: Degenerative conditions are associated with free radical-induced oxidative damages in the mitochondrial paraphernalia. Antimycin A (AMA) treatment of cells mimics such conditions in vitro by augmentation in ROS levels, thus causing injury to mitochondrial DNA (mtDNA), proteins and lipids, along with depolarization of mitochondrial membrane, activation of pro-apoptotic factors, resulting in apoptosis. This study investigates the potential of aqueous and methanolic extracts of Lawsonia inermis in prevention of such oxidative damage to the cell homeostasis. The extracts significantly mitigated membrane damages induced by peroxide, along with substantial decline in AMA-induced degeneration of mitochondrial proteins and lipids in hepatic carcinoma (Hep3B) cells. mtDNA analyzed for oxidative damage by assaying for 8-OHdG revealed considerable protective effect of both extracts against AMA-induced mtDNA damage. SQ-PCR of selected mtDNA genes confirmed that both extracts alleviated amplitudes of mtDNA injury. FACS analysis with JC-1 dye established that both extracts maintained homeostasis of mitochondrial membrane potential in AMA-treated cells. Extract treatments caused decline in AMA-induced discharge of cytochrome c and AIF into the cytoplasm along with consequent subjugation of apoptosis. All activities of the extracts reported in the present study significantly (P <0.05) correlated to their total phenolic contents, thereby proving that polyphenolic constituents of the extracts alleviate cells from oxidative stress-induced injury. [Copyright &y& Elsevier]

Abstract: Mitochondrial DNA (mtDNA) damage and the generation of reactive oxygen species have been associated with and implicated in the development and progression of Alzheimer's disease. To study how mtDNA damage affects reactive oxygen species and amyloid beta (Aβ) pathology in vivo, we generated an Alzheimer's disease mouse model expressing an inducible mitochondrial-targeted endonuclease (Mito-PstI) in the central nervous system. Mito-PstI cleaves mtDNA causing mostly an mtDNA depletion, which leads to a partial oxidative phosphorylation defect when expressed during a short period in adulthood. We found that a mild mitochondrial dysfunction in adult neurons did not exacerbate Aβ accumulation and decreased plaque pathology. Mito-PstI expression altered the cleavage pathway of amyloid precursor protein without increasing oxidative stress in the brain. These data suggest that mtDNA damage is not a primary cause of Aβ accumulation. [Copyright &y& Elsevier]

Highlights: [•] Two enzymatic systems for regulated and targeted mtDNA damage are characterized. [•] mtDNA damage is inflicted by mitochondrial targeting of either exoIII or mUNG1 mutant. [•] mtDNA damage is immediately followed by degradation. [•] mtDNA damage by mUNG1 and exoIII has low mutagenicity. [•] Observed physiological changes are secondary to reduced expression of mitochondrial polypeptides. [Copyright &y& Elsevier]

Abstract: Tacrine (THA) was the first drug licensed for the treatment of Alzheimer's disease. Unfortunately, reversible hepatotoxicity is evident in about 30% of patients and limits its clinical use. The intracellular mechanisms have not yet been elucidated. Phellinus linteus (PL) is a mushroom that has long been used as a folk medicine. In our previous study, we found that PL could decrease the reactive oxygen species (ROS) formation in HepG2 cells. Presently, protective effects of PL on tacrine-induced ROS formation and mitochondria dysfunction were evaluated. The results showed that PL significantly reduced tacrine-induced ROS production, disruption of ΔΨm, 8-OHdG formation in mitochondrial DNA, and cytotoxicity in HepG2 cells. These data suggest that PL could attenuate the cytotoxicity and mitochondria dysfunction induced by tacrine in HepG2 cells. The protection is probably mediated by an antioxidant protective mechanism. Consumption of PL may be a plausible way to prevent tacrine-induced hepatotoxicity. [Copyright &y& Elsevier]

Abstract: HBx is strongly associated with hepatocellular carcinoma development through transcription factor activation and reactive oxygen species (ROSs) production. However, the exact role of HBx during hepatocellular carcinogenesis is not fully understood. Recently, it was reported that C-terminal truncated HBx is associated with tumor metastasis. In the present study, we confirmed that the C-terminal region of HBx is required for ROS production and 8-oxoguanine (8-oxoG) formation, which is considered as a reliable biomarker of oxidative stress. These results suggest ROS production induced by the C-terminal region of HBx leads to mitochondrial DNA damage, which may play a role in HCC development. [Copyright &y& Elsevier]

The current study examined the effects of ochratoxin A (OTA) and methanol on mitochondrial DNA (mtDNA). The results showed that methanol application caused approximately 29 % more damage than the control group, and that there were no significant differences among the other groups in terms of mtDNA damage. The application of 0.04 ng/mL OTA and 0.04 ng/mL OTA with methanol increased mtDNA copy number compared with the control and 0.005 ng/mL OTA with methanol groups. The copy number in the 0.04 ng/mL OTA group was approximately 16 % greater than in the control. [ABSTRACT FROM AUTHOR]

Abstract: In the pathogenesis of diabetic retinopathy, an increase in retinal oxidative stress precedes mitochondrial dysfunction and capillary cell apoptosis. This study is designed to understand the mechanism responsible for the protection of mitochondria damage in the early stages of diabetic retinopathy. After 15 days–12 months of streptozotocin-induced diabetes in rats, retina was analyzed for mitochondria DNA (mtDNA) damage by extended length PCR. DNA repair enzyme and replication machinery were quantified in the mitochondria, and the binding of mitochondrial transcriptional factor A (TFAM) with mtDNA was analyzed by ChIP. Key parameters were confirmed in the retinal endothelial cells incubated in 20mM glucose for 6–96h. Although reactive oxygen species (ROS) were increased within 15 days of diabetes, mtDNA damage was observed at 6 months of diabetes. After 15 days of diabetes DNA repair/replication enzymes were significantly increased in the mitochondria, but at 2 months, their mitochondrial accumulation started to come down, and mtDNA copy number and binding of TFAM with mtDNA became significantly elevated. However, at 6 months of diabetes, the repair/replication machinery became subnormal and mtDNA copy number significantly decreased. A similar temporal relationship was observed in endothelial cells exposed to high glucose. Thus, in the early stages of diabetes, increased mtDNA biogenesis and repair compensates for the ROS-induced damage, but, with sustained insult, this mechanism is overwhelmed, and mtDNA and electron transport chain (ETC) are damaged. The compromised ETC propagates a vicious cycle of ROS and the dysfunctional mitochondria fuels loss of capillary cells by initiating their apoptosis. [Copyright &y& Elsevier]

Intrinsic oxidative stress through enhanced production of reactive oxygen species (ROS) in prostate and other cancers may contribute to cancer progression due to its stimulating effect on cancer growth. In this study, we investigate differential responses to exogenous oxidative stimuli between aggressive prostate cancer and normal cell lines and explore potential mechanisms through interactions between cytotoxicity, cellular ROS production and oxidative DNA damage. The circular, multi-copy mitochondrial DNA (mtDNA) is used as a sensitive surrogate to oxidative DNA damage. We demonstrate that exogenous H2O2 induces preferential cytotoxicity in aggressive prostate cancer than normal cells; a cascade production of cellular ROS, composed mainly of superoxide (O2ꔷ−), is shown to be a critical determinant of H2O2-induced selective toxicity in cancer cells. In contrast, mtDNA damage and copy number depletion, as measured by a novel two-phase strategy of the supercoiling-sensitive qPCR method, are very sensitive to exogenous H2O2 exposure in both cancer and normal cell lines. Moreover, we demonstrate for the first time that the sensitive mtDNA damage response to exogenous H2O2 is independent of secondary cellular ROS production triggered by several ROS modulators regardless of cell phenotypes. These new findings suggest different mechanisms underpinning cytotoxicity and DNA damage induced by oxidative stress and a susceptible phenotype to oxidative injury associated with aggressive prostate cancer cells in vitro. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondria provide most of the adenosine triphosphates (ATP) necessary for the maturation of oocytes. Various environmental toxicants would lead damage to mitochondrial DNA (mtDNA) and hence interfere with oocytes development. In the current study, the effect of arsenic trioxide (As2O3) on the common 3867bp deletion and the copy number of mtDNA in mitochondria of mouse oocytes in vivo or in vitro, as well as the molecular pathway leading to the damage were investigated. PCR strategy was used to detect the damage of mtDNA. Reactive oxygen species (ROS) and ATP content in oocytes were checked to determine the influence of As2O3 on oxidative stress and activity of mitochondria. The results showed that As2O3 could obviously decrease the copy number of mtDNA and cause severe 3867bp deletion in mitochondria together with elevated ROS level, while ATP content was decreased. Co-treatment with N-Acetyl-Cysteine (NAC) efficiently eliminated ROS induced by As2O3, lessened the mtDNA damage and enhanced ATP content in mouse oocytes both in vivo and in vitro. Taken together, the present study revealed that As2O3 could cause severe mtDNA damage and decrease ATP content by inducing excessive ROS, and this damage would then probably restrain the further development of mouse oocytes. [Copyright &y& Elsevier]