• DNA polymerase
• DNA repair
• long-range PCR
• mitochondrial DNA

• DNA, Mitochondrial/metabolism
• DNA, Mitochondrial/genetics
• Humans
• DNA Damage
• Polymerase Chain Reaction/methods
• Animals
• DNA Repair

• DNA base damage
• FLARE-coupled rt-qPCR
• Gene regulatory sequences

• Animals
• DNA Repair
• Base Sequence
• DNA Damage
• DNA/genetics
• DNA/metabolism
• Mutagenesis
• Mutagens
• Mammals/metabolism

• base excision repair
• chemoptogenetics
• mitochondria
• mitochondrial DNA
• mitochondrial dysfunction
• reactive oxygen species

• R33 ES025606 NIEHS NIH HHS
• R35 ES031638 NIEHS NIH HHS
• R35ES031638 NIH HHS

• Co-culture setup
• Human adipose-derived mesenchymal stromal cells
• Microenvironment
• Neuroprotection
• Neuroretina
• RPE cells
• Trophic factor

• extracellular vesicles
• microglia
• mitochondrial DNA
• oxidative stress
• retinal pigment epithelial

• DNA, Mitochondrial/genetics
• DNA, Mitochondrial/metabolism
• DNA-Binding Proteins/metabolism
• Epithelial Cells/metabolism
• Humans
• Inflammation/metabolism
• Microglia/metabolism
• Oxidative Stress/genetics
• RNA-Binding Proteins/metabolism
• Retinal Pigment Epithelium/metabolism
• Retinal Pigments/metabolism

• T32 ES007254 NIEHS NIH HHS

• Rhesus monkeys
• astrocytes activation
• diabetic retinopathy
• neurodegeneration
• reactive gliosis

Ethoxyquin (EQ), a quinolone-based antioxidant, has demonstrated neuroprotective properties against several neurotoxic drugs in a phenotypic screening and is shown to protect axons in animal models of chemotherapy-induced peripheral neuropathy. We assessed the effects of EQ on peripheral nerve function in the db/db mouse model of type II diabetes. After a 7 week treatment period, 12-week-old db/db-vehicle, db/+ -vehicle and db/db-EQ treated animals were evaluated by nerve conduction, paw withdrawal against a hotplate, and fiber density in hindlimb footpads. We found that the EQ group had shorter paw withdrawal latency compared to vehicle db/db group. The EQ group scored higher in nerve conduction studies, compared to vehicle-treated db/db group. Morphology studies yielded similar results. To investigate the potential role of mitochondrial DNA (mtDNA) deletions in the observed effects of EQ, we measured total mtDNA deletion burden in the distal sciatic nerve. We observed an increase in total mtDNA deletion burden in vehicle-treated db/db mice compared to db/+ mice that was partially prevented in db/db-EQ treated animals. These results suggest that EQ treatment may exert a neuroprotective effect in diabetic neuropathy. The prevention of diabetes-induced mtDNA deletions may be a potential mechanism of the neuroprotective effects of EQ in diabetic neuropathy.

• 4-hydroxynonenal (4HNE), infertility
• lipid peroxidation
• male germ cells
• oxidative stress
• reactive oxygen species
• sperm capacitation
• sperm motility
• spermatozoa

• Antioxidant system
• Iron
• Membrane enzymes

As the primary cellular location for respiration and energy production, mitochondria serve in a critical capacity to the cell. Yet, by virtue of this very function of respiration, mitochondria are subject to constant oxidative stress that can damage one of the unique features of this organelle, its distinct genome. Damage to mitochondrial DNA (mtDNA) and loss of mitochondrial genome integrity is increasingly understood to play a role in the development of both severe early-onset maladies and chronic age-related diseases. In this article, we review the processes by which mtDNA integrity is maintained, with an emphasis on the repair of oxidative DNA lesions, and the cellular consequences of diminished mitochondrial genome stability.

• aging
• base excision repair
• metabolic syndrome
• mitochondrial DNA
• neurodegenerative diseases

• Parkinson's disease
• QPCR-based assay
• mitochondrial DNA damage
• neurodegeneration
• nuclear DNA damage

Apurinic/apyrimidinic (AP) sites are frequently generated in the genome by spontaneous depurination/depyrimidination or after removal of oxidized/modified bases by DNA glycosylases during the base excision repair (BER) pathway. Unrepaired AP sites are mutagenic and block DNA replication and transcription. The primary enzyme to repair AP sites in mammalian cells is AP endonuclease (APE1), which plays a key role in this repair pathway. Although overexpression of APE1 in diverse cancer types and its association with chemotherapeutic resistance are well documented, alteration of posttranslational modification of APE1 and modulation of its functions during tumorigenesis are largely unknown. Here, we show that both classical histone deacetylase HDAC1 and NAD⁺-dependent deacetylase SIRT1 regulate acetylation level of APE1 and acetylation of APE1 enhances its AP-endonuclease activity both in vitro and in cells. Modulation of APE1 acetylation level in cells alters AP site repair capacity of the cell extracts in vitro. Primary tumor tissues of diverse cancer types have higher level of acetylated APE1 (AcAPE1) compared to adjacent non-tumor tissue and exhibit enhanced AP site repair capacity. Importantly, in the absence of APE1 acetylation, cells accumulate AP sites in the genome and show increased sensitivity to DNA damaging agents. Together, our study demonstrates that elevation of acetylation level of APE1 in tumor could be a novel mechanism by which cells handle the elevated levels of DNA damages in response to genotoxic stress and maintain sustained proliferation.

• BER
• DNA damage repair
• acetylation
• apurinic/apyrimidinic endonuclease 1 (APE1)

• R01 AG052978 NIA NIH HHS
• R33 ES025606 NIEHS NIH HHS

This report identifies mitochondrial DNA (mtDNA) as a target and active mediator that links low-level oxidative stress to inflammatory response in pulmonary epithelial cells. Extrusion of mtDNA into the bronchoalveolar lavage fluid occurs as an early event in mice subjected to cigarette smoke injury, concomitantly with the depletion of mtDNA in the lung tissue. In cultured lung epithelial cells, prolonged, low-level oxidative stress damages the mtDNA, without any detectable damage to the nuclear DNA. In turn, cellular depletion of the mtDNA occurs, together with a transient remodeling of cellular bioenergetics and morphology - all without any detectable impairment in overall cell viability. Damaged mtDNA first enters the cytoplasm, where it binds to Z-DNA binding protein 1 (ZBP1) and triggers inflammation via the TANK-binding kinase 1 /interferon regulatory factor 3 signaling pathway. Fragments of the mtDNA are subsequently released into the extracellular space via exosomes. MtDNA-containing exosomes are capable of inducing an inflammatory response in naïve (non-oxidatively stressed) epithelial cells. In vivo, administration of isolated mtDNA into the in lungs of naïve mice induces the production of pro-inflammatory mediators, without histopathologic evidence of tissue injury. We propose that mtDNA-specific damage, and subsequent activation of the ZBP1 pathway, is a mechanism that links prolonged, low-level oxidative stress to autocrine and paracrine inflammation during the early stages of inflammatory lung disease.

• Alzheimer’s disease
• Huntington’s disease
• Melatonin
• Mitochondria
• Neurodegeneration
• Parkinson’s disease

• Animals
• Antioxidants/pharmacology
• Humans
• Melatonin/pharmacology
• Mitochondria/drug effects
• Mitochondria/metabolism
• Mitochondrial Diseases/metabolism
• Mitochondrial Diseases/prevention & control
• Neurodegenerative Diseases/metabolism
• Neurodegenerative Diseases/prevention & control

• Antioxidants
• Cornea
• Fuchs endothelial dystrophy
• Kearns Sayre Syndrome
• Keratoconus
• Mitochondria
• Oxidative stress

Ascorbate (AscH⁻) functions as a versatile reducing agent. At pharmacological doses (P-AscH⁻; [plasma AscH⁻] ≥≈20 mM), achievable through intravenous delivery, oxidation of P-AscH⁻ can produce a high flux of H₂O₂ in tumors. Catalase is the major enzyme for detoxifying high concentrations of H₂O₂. We hypothesize that sensitivity of tumor cells to P-AscH⁻ compared to normal cells is due to their lower capacity to metabolize H₂O₂. Rate constants for removal of H₂O₂ (k_cell) and catalase activities were determined for 15 tumor and 10 normal cell lines of various tissue types. A differential in the capacity of cells to remove H₂O₂ was revealed, with the average k_cell for normal cells being twice that of tumor cells. The ED₅₀ (50% clonogenic survival) of P-AscH⁻ correlated directly with k_cell and catalase activity. Catalase activity could present a promising indicator of which tumors may respond to P-AscH⁻.

•

Ascorbate oxidizes in cell culture medium to generate a flux of H₂O₂.

Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10⁻²⁴), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10⁻³). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10⁻³; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10⁻³; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10⁻³). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10⁻⁴) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10⁻⁵). KC corneas also had increased mtDNA damage (P = 3.63×10⁻¹⁰), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC.

Many environmental and physiological stresses are chronic. Thus, cells are constantly exposed to diverse types of genotoxic insults that challenge genome stability, including those that induce oxidative DNA damage. However, most in vitro studies that model cellular response to oxidative stressors employ short exposures and/or acute stress models. In this study, we tested the hypothesis that chronic and repeated exposure to a micromolar concentration of hydrogen peroxide (H₂O₂) could activate DNA damage responses, resulting in cellular adaptations. For this purpose, we developed an in vitro model in which we incubated mouse myoblast cells with a steady concentration of ~50 μM H₂O₂ for one hour daily for seven days, followed by a final challenge of a 10 or 20X higher dose of H₂O₂ (0.5 or 1 mM). We report that intermittent long-term exposure to this oxidative stimulus nearly eliminated cell toxicity and significantly decreased genotoxicity (in particular, a >5-fold decreased in double-strand breaks) resulting from subsequent acute exposure to oxidative stress. This protection was associated with cell cycle arrest in G2/M and induction of expression of nine DNA repair genes. Together, this evidence supports an adaptive response to chronic, low-level oxidative stress that results in genomic protection and up-regulated maintenance of cellular homeostasis.

•

Repetitive stimulus with subtoxic H₂O₂ resulted in cellular adaptive response.

• Adaptation
• DNA damage response
• G2/M arrest
• Genotoxicity
• ROS
• Up-regulation of DNA repair genes

• Antioxidant
• Mitochondria
• Mouse
• Neurodegeneration
• Neuroprotection
• Oxidative stress

• Amyotrophic Lateral Sclerosis/genetics
• Amyotrophic Lateral Sclerosis/metabolism
• Amyotrophic Lateral Sclerosis/pathology
• Animals
• Antioxidants/metabolism
• Cell Survival/genetics
• Central Nervous System/metabolism
• Central Nervous System/pathology
• Cytoplasm/metabolism
• Dynamins/genetics
• Gene Expression Regulation/genetics
• Humans
• Mice
• Mitochondria/genetics
• Mitochondria/metabolism
• Mitochondria/ultrastructure
• Mitochondrial Membranes/metabolism
• Mitochondrial Membranes/pathology
• Mitochondrial Proteins/genetics
• Mitochondrial Proteins/metabolism
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism

• 311394 European Research Council
• MC_U137761449 Medical Research Council
• MC_UU_12021/2 Medical Research Council

• Animals
• Bacteria/metabolism
• Biochemistry/history
• DNA/metabolism
• DNA Repair
• Eukaryota/metabolism
• History, 20th Century
• History, 21st Century
• Humans
• Nobel Prize
• United States

• R01 CA174713 NCI NIH HHS
• 1R21/33ES025606 NIEHS NIH HHS
• 1R01CA174713 NCI NIH HHS
• 1R01ES019566 NIEHS NIH HHS
• R01 ES019566 NIEHS NIH HHS
• R33 ES025606 NIEHS NIH HHS

• Age
• Dentin
• Free radicals
• HV2
• mtDNA mutations

Neurodegeneration has been correlated with mitochondrial DNA (mtDNA) damage and exposure to environmental toxins, but causation is unclear. We investigated the ability of several known environmental genotoxins and neurotoxins to cause mtDNA damage, mtDNA depletion, and neurodegeneration in Caenorhabditis elegans. We found that paraquat, cadmium chloride and aflatoxin B₁ caused more mitochondrial than nuclear DNA damage, and paraquat and aflatoxin B₁ also caused dopaminergic neurodegeneration. 6-hydroxydopamine (6-OHDA) caused similar levels of mitochondrial and nuclear DNA damage. To further test whether the neurodegeneration could be attributed to the observed mtDNA damage, C. elegans were exposed to repeated low-dose ultraviolet C radiation (UVC) that resulted in persistent mtDNA damage; this exposure also resulted in dopaminergic neurodegeneration. Damage to GABAergic neurons and pharyngeal muscle cells was not detected. We also found that fasting at the first larval stage was protective in dopaminergic neurons against 6-OHDA-induced neurodegeneration. Finally, we found that dopaminergic neurons in C. elegans are capable of regeneration after laser surgery. Our findings are consistent with a causal role for mitochondrial DNA damage in neurodegeneration, but also support non mtDNA-mediated mechanisms.

• Adrenergic Agents/toxicity
• Animals
• Caenorhabditis elegans/drug effects
• Caenorhabditis elegans/genetics
• Caenorhabditis elegans/growth & development
• DNA Damage/drug effects
• DNA, Mitochondrial/genetics
• Dopamine/toxicity
• Dopamine Agents/toxicity
• Dopaminergic Neurons/drug effects
• Dopaminergic Neurons/pathology
• Mitochondria/drug effects
• Mitochondria/genetics
• Mitochondria/pathology
• Mutagens/toxicity
• Nerve Degeneration/drug therapy
• Nerve Degeneration/pathology
• Oxidopamine/toxicity
• Paraquat/toxicity

• T32 GM007223 NIGMS NIH HHS
• P40 OD010440 NIH HHS
• R01-ES017540-01A2 NIEHS NIH HHS
• R21 NS065468 NINDS NIH HHS
• R01 ES017540 NIEHS NIH HHS

• Mitochondrial DNA replication
• Mitochondrial disease
• Mutagenesis
• POLG
• mtDNA

• DNA Damage/genetics
• DNA Replication/genetics
• DNA, Mitochondrial/genetics
• Genome, Mitochondrial
• Humans
• Mitochondrial Diseases/genetics
• Mitochondrial Diseases/pathology
• Mutagenesis
• Mutation
• Oxidative Phosphorylation

• Z01 ES065078 Intramural NIH HHS
• Z01 ES065080 Intramural NIH HHS
• ZIA ES065078-20 Intramural NIH HHS
• ZIA ES065080-19 Intramural NIH HHS

• Bioenergetics
• Cytoprotection
• DNA repair
• Mitochondria
• Oxidative stress

• Cardiomyopathies/etiology
• Cardiomyopathies/metabolism
• Humans
• Iron/metabolism
• Iron Overload/complications
• Iron Overload/metabolism
• Oxidative Stress/physiology
• Signal Transduction/physiology

• mtdna damage
• mtdna degradation
• mtdna repair
• exoiii
• ung1 y147a
• reactive oxygen species

• Cell Respiration
• Cell Survival
• Cyclooxygenase 2/genetics
• Cyclooxygenase 2/metabolism
• DNA Breaks, Single-Stranded
• DNA Fragmentation
• DNA, Mitochondrial/drug effects
• DNA, Mitochondrial/genetics
• DNA, Mitochondrial/metabolism
• Doxycycline/toxicity
• Exodeoxyribonucleases/metabolism
• HeLa Cells
• Humans
• Membrane Potential, Mitochondrial
• Mitophagy
• Mutation
• Reactive Oxygen Species/metabolism
• Uracil-DNA Glycosidase/genetics
• Uracil-DNA Glycosidase/metabolism

• R21 RR023961 NCRR NIH HHS
• RR031286 NCRR NIH HHS
• P01 HL66299 NHLBI NIH HHS
• P01 HL066299 NHLBI NIH HHS
• S10RR027535 NCRR NIH HHS
• R01 RR031286 NCRR NIH HHS
• R01 OD010944 NIH HHS
• ES03456 NIEHS NIH HHS
• R01 ES003456 NIEHS NIH HHS
• S10 RR027535 NCRR NIH HHS
• OD010944 NIH HHS

Insulin receptor substrate (IRS)-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS) are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels.

In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO) provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3), aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress-induced autophagy and cell death.

Our results suggest that overexpression of IRS-1 promotes cells growth, inhibits basal autophagy, reduces oxidative stress-induced autophagy, and diminishes oxidative stress-mediated autophagy-dependent cell death. ROS-mediated autophagy may occur via inhibition of IRS-1/phosphatidylinositol 3-kinase/mTOR signaling. Our data afford a plausible explanation for IRS-1 involvement in tumor initiation and progression.

• mitochondrial dna
• iron overload
• cardiomyopathy

• Animals
• Cardiomyopathies/etiology
• DNA, Mitochondrial/drug effects
• DNA, Mitochondrial/metabolism
• Electron Transport Complex I/metabolism
• Electron Transport Complex IV/metabolism
• Iron Overload/complications
• Male
• Mice
• Mitochondria, Heart/drug effects
• Mitochondria, Heart/metabolism
• Myocardium/metabolism

• R01 HL099014 NHLBI NIH HHS
• R01 DK073586 NIDDK NIH HHS
• R01 AA018016 NIAAA NIH HHS
• R01 DK073586-01 NIDDK NIH HHS
• R01AA018016 NIAAA NIH HHS

• mitochondrial dna
• dna damage
• dna repair
• genotoxicity
• biomarker

• Animals
• Cell Nucleus/metabolism
• DNA Damage
• DNA, Mitochondrial/metabolism
• Ecotoxicology/methods
• Environmental Monitoring
• Genetic Markers
• Humans
• Mutagenicity Tests
• Polymerase Chain Reaction
• Reproducibility of Results
• Risk Assessment
• Species Specificity

• P42 ES010356 NIEHS NIH HHS
• P42 ES010356-08 NIEHS NIH HHS
• P42 ES10356 NIEHS NIH HHS

• Adolescent
• Adult
• Cells, Cultured
• Child
• Cohort Studies
• DNA Damage
• Female
• Friedreich Ataxia/genetics
• Friedreich Ataxia/pathology
• Gene Expression
• Humans
• Iron-Binding Proteins/genetics
• Male
• Middle Aged
• RNA/blood
• RNA/genetics
• Young Adult
• Frataxin

• Intramural NIH HHS