Simplified qPCR method for detecting excessive mtDNA damage induced by exogenous factors

Gureev, Artem P.; Shaforostova, Ekaterina A.; Starkov, Anatoly A.; Popov, Vasily N.

doi:10.1016/j.tox.2017.03.010

Cited by 42 publications

(42 citation statements)

References 46 publications

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“…The increase of the reduced NADH can explain why 1 μM ETC inhibitor causes more mtDNA lesions than 100 μM MB. Also, inhibition of ETC was shown to cause more mtDNA damage than exogenous addition of 500 μM H 2 O 2 in our past experiments (Gureev et al, 2017).…”

Section: Discussionmentioning

confidence: 56%

“…mtDNA isolation was performed using the Plasmid Miniprep Kit (Evrogen, Russia), as described in our earlier papers (Gureev et al, 2017). Number of lesions was measured for six mtDNA fragments.…”

Section: Initiation and The Quantitative Evaluation Of The Mtdna Lesimentioning

confidence: 99%

“…The 1st fragment corresponded to 12S and 16S rRNAs, the 2nd fragment corresponded to 16S rRNA and ND1, the 3rd fragment corresponded to ND1 and ND2, the 4th fragment corresponded to ND5, the 5th fragment corresponded to ND6 and CytB, the 6th fragment corresponded to D-loop. Primers for amplification of six mtDNA fragments used in this study were done according to the same protocol (Gureev et al, 2017). The extent of excessive mtDNA damage which was induced by rotenone, antimycin A and MB was estimated using the ΔΔCq method.…”

Section: Initiation and The Quantitative Evaluation Of The Mtdna Lesimentioning

confidence: 99%

“…Both the control and the experimental groups were sacrificed 24 hours later. The mtDNA was isolated from both the midbrain and the cerebral cortex according to the methods described earlier (Gureev et al, 2017).…”

Section: Assessment Of Mb Toxicity In Vivomentioning

confidence: 99%

“…mtDNA is localized in the mitochondrial matrix and it is in the close proximity to ETC and MB, both of which produce H 2 O 2 . Our modification of the qPCR method used for detecting the mtDNA lesions can also show the regions of the mtDNA which are most sensitive to damage (Gureev et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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Methylene blue elicits non-genotoxic H2O2 production and protects brain mitochondria from rotenone toxicity

Gureev¹,

Shaforostova²,

Laver³

et al. 2019

J Appl Biomed

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Methylene blue (MB) is a promising compound with a broad range of neuroprotective activity. One of therapeutic effects is the activation of mitochondrial biogenesis via Nrf2/ARE signaling cascade. Probably, mild oxidative stress caused by MB-depended H 2 O 2 production is a trigger for activation of this signaling cascade. So mechanistically, MB can be regarded as prooxidant. We investigated the dose-dependent H 2 O 2 production in intact brain mitochondria and showed the increase in the H 2 O 2 production after adding as little as 50 nM MB. We have not found genotoxic effect of therapeutic concentration of MB to mitochondrial genome. 100 μM MB selectively damaged fragments of mitochondrial DNA, which correlated with the number of purine-T-G-purine (RTGR)-sequences in studied fragments. Furthermore, 20 μM MB combined with the red light caused the formation of singlet oxygen, which strongly damaged mitochondrial DNA in all studied fragments. We did not observe mitochondrial DNA lesions in brain after single intraperitoneal injection of MB in the concentration of 50 mg/kg. Furthermore, we showed the neuroprotective properties of MB pretreatments after rotenone injection. Therefore, we suggest that MB-induced mild oxidative stress does not have genotoxic effect on mitochondrial DNA.

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Section: Discussionmentioning

confidence: 56%

Section: Initiation and The Quantitative Evaluation Of The Mtdna Lesimentioning

confidence: 99%

Section: Initiation and The Quantitative Evaluation Of The Mtdna Lesimentioning

confidence: 99%

Section: Assessment Of Mb Toxicity In Vivomentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Methylene blue elicits non-genotoxic H2O2 production and protects brain mitochondria from rotenone toxicity

Gureev¹,

Shaforostova²,

Laver³

et al. 2019

J Appl Biomed

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NEK5 interacts with LonP1 and its kinase activity is essential for the regulation of mitochondrial functions and mtDNA maintenance

et al. 2021

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Little is known about Nima‐related kinase (NEKs), a widely conserved family of kinases that have key roles in cell‐cycle progression. Nevertheless, it is now clear that multiple NEK family members act in networks, not only to regulate specific events of mitosis, but also to regulate metabolic events independently of the cell cycle. NEK5 was shown to act in centrosome disjunction, caspase‐3 regulation, myogenesis, and mitochondrial respiration. Here, we demonstrate that NEK5 interacts with LonP1, an AAA+ mitochondrial protease implicated in protein quality control and mtDNA remodeling, within the mitochondria and it might be involved in the LonP1‐TFAM signaling module. Moreover, we demonstrate that NEK5 kinase activity is required for maintaining mitochondrial mass and functionality and mtDNA integrity after oxidative damage. Taken together, these results show a new role of NEK5 in the regulation of mitochondrial homeostasis and mtDNA maintenance, possibly due to its interaction with key mitochondrial proteins, such as LonP1.

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Role of Mitochondrial DNA in Inflammatory Airway Diseases

Snyder

Kleeberger

2021

Comprehensive Physiology

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The mitochondrial genome is a small, circular, and highly conserved piece of DNA which encodes only 13 protein subunits yet is vital for electron transport in the mitochondrion and, therefore, vital for the existence of multicellular life on Earth. Despite this importance, mitochondrial DNA (mtDNA) is located in one of the least-protected areas of the cell, exposing it to high concentrations of intracellular reactive oxygen species (ROS) and threat from exogenous substances and pathogens. Until recently, the quality control mechanisms which ensured the stability of the nuclear genome were thought to be minimal or nonexistent in the mitochondria, and the thousands of redundant copies of mtDNA in each cell were believed to be the primary mechanism of protecting these genes. However, a vast network of mechanisms has been discovered that repair mtDNA lesions, replace and recycle mitochondrial chromosomes, and conduct alternate RNA processing for previously undescribed mitochondrial proteins. New mtDNA/RNA-dependent signaling pathways reveal a mostly undiscovered biochemical landscape in which the mitochondria interface with their host cells/organisms. As the myriad ways in which the function of the mitochondrial genome can affect human health have become increasingly apparent, the use of mitogenomic biomarkers (such as copy number and heteroplasmy) as toxicological endpoints has become more widely accepted.In this article, we examine several pathologies of human airway epithelium, including particle exposures, inflammatory diseases, and hyperoxia, and discuss the role of mitochondrial genotoxicity in the pathogenesis and/or exacerbation of these conditions.

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Simplified qPCR method for detecting excessive mtDNA damage induced by exogenous factors

Cited by 42 publications

References 46 publications

Methylene blue elicits non-genotoxic H<sub>2</sub>O<sub>2</sub> production and protects brain mitochondria from rotenone toxicity

Methylene blue elicits non-genotoxic H<sub>2</sub>O<sub>2</sub> production and protects brain mitochondria from rotenone toxicity

NEK5 interacts with LonP1 and its kinase activity is essential for the regulation of mitochondrial functions and mtDNA maintenance

Role of Mitochondrial DNA in Inflammatory Airway Diseases

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