PCR‐Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage

Gonzalez‐Hunt, Claudia P.; Rooney, John P.; Ryde, Ian T.; Anbalagan, Charumathi; Joglekar, Rashmi; Meyer, Joel N.

doi:10.1002/0471140856.tx2011s67

Cited by 82 publications

(90 citation statements)

References 36 publications

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“…A LA‐PCR assay (Gonzalez‐Hunt et al, ) was used to measure mtDNA damage. This assay detects DNA damage such as bulky adducts and single‐strand breaks that can detectably inhibit the progression of the DNA polymerase used in the PCR amplification reaction (Ponti et al, ).…”

Section: Methodsmentioning

confidence: 99%

Predictors of mitochondrial DNA copy number and damage in a mercury‐exposed rural Peruvian population near artisanal and small‐scale gold mining: An exploratory study

Berky

Ryde

Feingold

et al. 2018

Environ and Mol Mutagen

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Mitochondrial DNA (mtDNA) copy number (CN) and damage in circulating white blood cells have been proposed as effect biomarkers for pollutant exposures. Studies have shown that mercury accumulates in mitochondria and affects mitochondrial function and integrity; however, these data are derived largely from experiments in model systems, rather than human population studies that evaluate the potential utility of mitochondrial exposure biomarkers. We measured mtDNA CN and damage in white blood cells (WBCs) from 83 residents of nine communities in the Madre de Dios region of the Peruvian Amazon that vary in proximity to artisanal and small‐scale gold mining. Prior research from this region reported high levels of mercury in fish and a significant association between food consumption and human total hair mercury level of residents. We observed that mtDNA CN and damage were both associated with consumption of fruit and vegetables, higher diversity of fruit consumed, residential location, and health characteristics, suggesting common environmental drivers. Surprisingly, we observed negative associations of mtDNA damage with both obesity and age. We did not observe any association between total hair mercury or, in contrast to previous results, age, with either mtDNA damage or CN. The results of this exploratory study highlight the importance of combining epidemiological and laboratory research in studying the effects of stressors on mitochondria, suggesting that future work should incorporate nutritional and social characteristics, and caution should be taken when applying conclusions from epidemiological studies conducted in the developed world to other regions, as results may not be easily translated. Environ. Mol. Mutagen. 60: 197–210, 2019. © 2018 Wiley Periodicals, Inc.

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Section: Methodsmentioning

confidence: 99%

Predictors of mitochondrial DNA copy number and damage in a mercury‐exposed rural Peruvian population near artisanal and small‐scale gold mining: An exploratory study

Berky

Ryde

Feingold

et al. 2018

Environ and Mol Mutagen

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“…Mitochondrial (mtDNA) and nuclear (nucDNA) genome copy number were determined as previously described (Gonzalez – Hunt et al 2016; Rooney et al 2015). Briefly, 6 nematodes were added to 90μl proteinase K-containing lysis buffer using a platinum worm pick and frozen at −80°C.…”

Section: Methodsmentioning

confidence: 99%

Deficiencies in mitochondrial dynamics sensitize Caenorhabditis elegans to arsenite and other mitochondrial toxicants by reducing mitochondrial adaptability

et al. 2017

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Mitochondrial fission, fusion, and mitophagy are interlinked processes that regulate mitochondrial shape, number, and size, as well as metabolic activity and stress response. The fundamental importance of these processes is evident in the fact that mutations in fission (DRP1), fusion (MFN2, OPA1), and mitophagy (PINK1, PARK2) genes can cause human disease (collectively >1/10,000). Interestingly, however, the age of onset and severity of clinical manifestations varies greatly between patients with these diseases (even those harboring identical mutations), suggesting a role for environmental factors in the development and progression of certain mitochondrial diseases. Using the model organism Caenorhabditis elegans, we screened ten mitochondrial toxicants (2, 4-dinitrophenol, acetaldehyde, acrolein, aflatoxin B1, arsenite, cadmium, cisplatin, doxycycline, paraquat, rotenone) for increased or decreased toxicity in fusion (fzo-1, eat-3)-, fission (drp-1)-, and mitophagy (pdr-1, pink-1)-deficient nematodes using a larval growth assay. In general, fusion-deficient nematodes were the most sensitive to toxicants, including aflatoxin B1, arsenite, cisplatin, paraquat, and rotenone. Because arsenite was particularly potent in fission- and fusion-deficient nematodes, and hundreds of millions of people are chronically exposed to arsenic, we investigated the effects of these genetic deficiencies on arsenic toxicity in more depth. We found that deficiencies in fission and fusion sensitized nematodes to arsenite-induced lethality throughout aging. Furthermore, low-dose arsenite, which acted in a “mitohormetic” fashion by increasing mitochondrial function (in particular, basal and maximal oxygen consumption) in wild-type nematodes by a wide range of measures, exacerbated mitochondrial dysfunction in fusion-deficient nematodes. Analysis of multiple mechanistic changes suggested that disruption of pyruvate metabolism and Krebs cycle activity underlie the observed arsenite-induced mitochondrial deficits, and these disruptions are exacerbated in the absence of mitochondrial fusion. This research demonstrates the importance of mitochondrial dynamics in limiting arsenite toxicity by permitting mitochondrial adaptability. It also suggests that individuals suffering from deficiencies in mitodynamic processes may be more susceptible to the mitochondrial toxicity of arsenic and other toxicants.

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“…Total nuclear DNA was isolated from the nuclear fraction using QIAamp DNA Mini Kit (Qiagen), according to the manufacturer’s protocol. The purified mitochondrial DNA was quantified by quantitative PCR with SYBR Green master mix (Quanta Biosystems) as described previously (Gonzalez-Hunt et al, 2016; Rooney et al, 2015). Mitochondrial DNA content was represented by primers towards two mtDNA- encoded genes, mitochondrial cyclooxygenase II (CoxII), and NADH dehydrogenase subunit 1 (ND1, Realtimesprimer.com) normalized to a nuclear intron of β-globin.…”

Section: Methodsmentioning

confidence: 99%

“…The primer sequences were as follows: Cox2, 5’-GCCGACTAAATCAAGCAACA-3’ (forward) and 5’-CAATGGGCATAAAGCTATGG-3’ (reverse); and β-globin, 5 ‘-GAAGCGATTCTAGGGAGCAG-3’ (forward) and 5’-GGAGCAGC GATTCTGAGTAGA-3’ (reverse). The relative mtDNA to nuclear DNA copy number ratio was determined using the comparative DDCT method (Ballista-Hemandez et al, 2017; Gonzalez-Hunt et al, 2016; Lien et al, 2017), in which NDl/β-globin and Cox2/β-globin ratios were calculated.…”

Section: Methodsmentioning

confidence: 99%

Activation of cyclin D1 affects mitochondrial mass following traumatic brain injury

Saha

Gupta

Sen

et al. 2018

Neurobiology of Disease

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Cell cycle activation has been associated with varying types of neurological disorders including brain injury. Cyclin D1 is a critical modulator of cell cycle activation and upregulation of Cyclin D1 in neurons contributes to the pathology associated with traumatic brain injury (TBI). Mitochondrial mass is a critical factor to maintain the mitochondrial function, and it can be regulated by different signaling cascades and transcription factors including NRF1. However, the underlying mechanism of how TBI leads to impairment of mitochondrial mass following TBI remains obscure. Our results indicate that augmentation of CyclinD1 attenuates mitochondrial mass formation following TBI. To elucidate the molecular mechanism, we found that Cyclin D1 interacts with a transcription factor NRF1 in the nucleus and prevents NRF1’s interaction with p300 in the pericontusional cortex following TBI. As a result, the acetylation level of NRF1 was decreased, and its transcriptional activity was attenuated. This event leads to a loss of mitochondrial mass in the pericontusional cortex following TBI. Intranasal delivery of Cyclin D1 RNAi immediately after TBI rescues transcriptional activation of NRF1 and recovers mitochondrial mass after TBI.

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PCR‐Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage

Cited by 82 publications

References 36 publications

Predictors of mitochondrial DNA copy number and damage in a mercury‐exposed rural Peruvian population near artisanal and small‐scale gold mining: An exploratory study

Predictors of mitochondrial DNA copy number and damage in a mercury‐exposed rural Peruvian population near artisanal and small‐scale gold mining: An exploratory study

Deficiencies in mitochondrial dynamics sensitize Caenorhabditis elegans to arsenite and other mitochondrial toxicants by reducing mitochondrial adaptability

Activation of cyclin D1 affects mitochondrial mass following traumatic brain injury

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