Aspects of design and statistical analysis in the Comet assay

Wiklund, Stig Johan; Agurell, Eva

doi:10.1093/mutage/18.2.167

Cited by 86 publications

(36 citation statements)

References 5 publications

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“…Comet, q-p53, and p-H2AX data from irradiation experiments were analyzed by fitting a mixed, nested linear model with R statistical software (53). The following R code was used: "fit ¼ glmer(score $ genotype * treatment þ (treatmentjmouse), family ¼ Poisson)" (54). Comet, q-P53, and p-H2AX data from the etoposide and 100 lg/mL MMS experiments did not have nesting and were analyzed by the nonparametric Mann-Whitney test using GraphPad Prism software.…”

Section: Discussionmentioning

confidence: 99%

DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy

Fayzullina

Martin

2016

J Neuropathol Exp Neurol

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We studied DNA damage response (DDR) and DNA repair capacities of skeletal muscle cells from a mouse model of infantile spinal muscular atrophy (SMA) caused by loss-of-function mutation of survival of motor neuron (Smn). Primary myocyte cultures derived from skeletal muscle satellite cells of neonatal control and mutant SMN mice had similar myotube length, myonuclei, satellite cell marker Pax7 and differentiated myotube marker myosin, and acetylcholine receptor clustering. DNA damage was induced in differentiated skeletal myotubes by c-irradiation, etoposide, and methyl methanesulfonate (MMS). Unexposed control and SMA myotubes had stable genome integrity. After c-irradiation and etoposide, myotubes repaired most DNA damage equally. Control and mutant myotubes exposed to MMS exhibited equivalent DNA damage without repair. Control and SMA myotube nuclei contained DDR proteins phosphop53 and phospho-H2AX foci that, with DNA damage, dispersed and then re-formed similarly after recovery. We conclude that mouse primary satellite cell-derived myotubes effectively respond to and repair DNA strand-breaks, while DNA alkylation repair is underrepresented. Morphological differentiation, genome stability, genome sensor, and DNA strand-break repair potential are preserved in mouse SMA myocytes; thus, reduced SMN does not interfere with myocyte differentiation, genome integrity, and DNA repair, and faulty DNA repair is unlikely pathogenic in SMA.

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Section: Discussionmentioning

confidence: 99%

DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy

Fayzullina

Martin

2016

J Neuropathol Exp Neurol

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“…The procurement of a suitable surrogate tissue is therefore still a major challenge (67). Apart from the target tissue, the definition of a suitable outcome variable for DNA strand breaks with Comet assay is another uncertainty because the segmentation of DNA fragments in head and tail regions can vary (68,69). Further, it is common practice to prepare two slides per subject and to analyze only f50 single cells per slide.…”

Section: Discussionmentioning

confidence: 99%

Dose-Response Modeling of Occupational Exposure to Polycyclic Aromatic Hydrocarbons with Biomarkers of Exposure and Effect

Pesch

Kappler

Straif³

et al. 2007

Cancer Epidemiology, Biomarkers &Amp; Prevention

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In regulatory toxicology, the dose-response relationship between occupational exposure and biomarkers is of importance in setting threshold values. We analyzed the relationships between occupational exposure to polycyclic aromatic hydrocarbons (PAH) and various biomarkers of internal exposure and DNA damage with data from 284 highly exposed male workers. Personal exposure to phenanthrene and other PAHs was measured during shift and correlated with the sum of 1À, 2+9À, 3À, and 4-hydroxyphenanthrenes in post-shift urine. PAHs and hydroxyphenanthrenes were associated with DNA damage assessed in WBC as 8-oxo-7,8-dihydro-2 ¶-deoxyguanosine/10 6 dGuo and strand breaks by Comet assay as Olive tail moment. Hydroxyphenanthrenes correlated with phenanthrene (Spearman r s = 0.70; P < 0.0001). No correlations could be found between strand breaks and exposure (r s = 0.01, P < 0.0001 for PAHs; r s = À0.03, P = 0.68 for hydroxyphenanthrenes). Correlations with 8-oxo-7,8-dihydro-2 ¶-deoxyguanosine/10 6 dGuo were weakly negative (r s = À0.22, P = 0.004 for PAHs) or flat (r s = À0.08, P = 0.31 for hydroxyphenanthrenes). Linear splines were applied to assess the relationships between the log-transformed variables. All regression models were adjusted for smoking and type of industry. For hydroxyphenanthrenes, 51.7% of the variance could be explained by phenanthrene and other predictors. Up to 0.77 Mg/m 3 phenanthrene, no association could be found with hydroxyphenanthrenes. Above that point, hydroxyphenanthrenes increased by a factor of 1.47 under a doubling of phenanthrene exposure (slope, 0.56; 95% confidence interval, 0.47-0.64). Hydroxyphenanthrenes may be recommended as biomarker of occupational PAH exposure, whereas biomarkers of DNA damage in blood did not show a dose-response relation to PAH exposure. (Cancer Epidemiol Biomarkers Prev 2007;16(9):1863 -73)

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“…The unit to be used for the analysis of data is the animal (Hartmann et al, 2003). Different statistical approaches for data analysis have been proposed (Lovell et al, 1999;Hartmann et al, 2003;Wiklund and Agurell, 2003;Rothfuss et al, 2010).…”

Section: Evaluation and Interpretation Of Resultsmentioning

confidence: 99%

Minimum Criteria for the acceptance ofin vivoalkaline Comet Assay Reports

2012

EFSA Journal

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The in vivo alkaline Comet assay detects primary DNA damage in various organs and tissues of exposed animals and can be used to assess the genotoxicity of a great variety of chemical compounds, which include food additives, flavourings, food contact materials, foodborne by-products, pesticides, contaminants, etc. Among the various versions of the assay, the alkaline method (pH of DNA unwinding and electrophoresis buffer > 13) identifies the broadest spectrum of DNA damage and is, therefore, recommended for regulatory purposes. It can detect double-and single-strand breaks, alkalilabile lesions that are expressed as single-strand breaks and single-strand breaks arising as DNA repair intermediates. No OECD Test Guideline yet exists for the Comet assay but internationally agreed protocols are available for performing this test. Since establishing of an OECD Test Guideline for this assay will require some further time, there is a need for an agreed approach on the minimum requirements on conduction and reporting of the in vivo Comet assay, which should be fulfilled for the purposes of EFSA during this transition period.

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Aspects of design and statistical analysis in the Comet assay

Cited by 86 publications

References 5 publications

DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy

DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy

Dose-Response Modeling of Occupational Exposure to Polycyclic Aromatic Hydrocarbons with Biomarkers of Exposure and Effect

Minimum Criteria for the acceptance ofin vivoalkaline Comet Assay Reports

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