Technical recommendations to perform the alkaline standard and enzyme-modified comet assay in human biomonitoring studies

Azqueta, Amaya; Muruzabal, Damián; Boutet-Robinet, Élisa; Milić, Mirta; Dušinská, Maria; Brunborg, Gunnar; Möller, Peter; Collins, Andrew

doi:10.1016/j.mrgentox.2019.04.007

Cited by 62 publications

(41 citation statements)

References 66 publications

(109 reference statements)

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“…Some technical aspects related specifically to biomonitoring are also covered, such as sampling and storing of specimens, as well as statistical and ethical issues that should be addressed before and after the biomonitoring study. However, technical issues related specifically to variability in assay conditions and efforts to produce standard procedures have been reviewed elsewhere [4]. Inter-laboratory variation in DNA damage levels has been described since the early 2000s through ring-trials involving multiple laboratories [5][6][7].…”

Section: Introductionmentioning

confidence: 99%

Application of the comet assay in human biomonitoring: An hCOMET perspective

Azqueta

Ladeira

Giovannelli

et al. 2020

Mutation Research/Reviews in Mutation Research

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114

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The comet assay is a well-accepted biomonitoring tool to examine the effect of dietary, lifestyle, environmental and occupational exposure on levels of DNA damage in human cells. With such a wide range of determinants for DNA damage levels, it becomes challenging to deal with confounding and certain factors are interrelated (e.g. poor nutritional intake may correlate with smoking status). This review describes the effect of intrinsic (i.e. sex, age, tobacco smoking, occupational exposure and obesity) and extrinsic (season, environmental exposures, diet, physical activity and alcohol consumption) factors on the level of DNA damage measured by the standard or enzyme-modified comet assay. Although each factor influences at least one comet assay endpoint, the collective evidence does not indicate single factors have a large impact. Thus, controlling for confounding may be necessary in a biomonitoring study, but none of the factors is strong enough to be regarded a priori as a confounder. Controlling for confounding in the comet assay requires a case-by-case approach. Inter-laboratory variation in levels of DNA damage and to some extent also reproducibility in biomonitoring studies are issues that have haunted the users of the comet assay for years. Procedures to collect specimens, and their storage, are not standardized. Likewise, statistical issues related to both sample-size calculation (before sampling of specimens) and statistical analysis of the results vary between studies. This review gives guidance to statistical analysis of the typically complex exposure, co-variate, and effect relationships in human biomonitoring studies.

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Section: Introductionmentioning

confidence: 99%

Application of the comet assay in human biomonitoring: An hCOMET perspective

Azqueta

Ladeira

Giovannelli

et al. 2020

Mutation Research/Reviews in Mutation Research

Self Cite

114

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“…В руководстве OECD со ссылкой на результаты исследований японского центра по валидации альтернативных методов (JaCVAM) рекомендуется проводить электрофорез в течение 20 мин при напряженности 0,7 В/см [18]. По мнению ряда авторитетных специалистов оптимально проведение электрофореза при 1,15 В/см в течение 20 мин или при 0,7 В/см в течение 30 мин [3,4,15,19]. Согласно данным литературы большинство исследователей использует напряженность 1 В/см, ориентируясь на рекомендации, выпущенные в 2000 г. R.R.…”

Section: Discussionunclassified

“…Оценка уровней первичных повреждений ДНК в качестве биомаркера в медико-биологических и генотоксикологических исследованиях in vitro и in vivo получила широкое распространение с внедрением в практику метода гель-электрофореза отдельных клеток (SCGE, метод ДНК-комет) [ Важными характеристиками метода, в особенности при использовании в качестве экспертного теста в генотоксикологии, является его точность и воспроизводимость. Меж-и внутрилабораторная вариабельность данных, получаемых методом ДНК-комет, остается нерешенной проблемой, порождающей получение неопределенных или противоречивых данных [3,4].…”

Section: Introductionunclassified

“…Результаты исследований продемонстрировали высокую вариабельность данных между лабораториями, причиной которой, по заключению специалистов, явилось использование различных протоколов проведения метода. Было установлено, что критическими методическими факторами, определяющими результаты метода ДНК-комет, являются концентрация агарозного геля, длительность щелочной денатурации/электрофореза и напряженность электрического поля [3,4,7]. Температура раствора в ходе электрофореза также оказывает значительное влияние на оцениваемые показатели [8,9].…”

Section: Introductionunclassified

“…При этом напряженность электрического поля, как и для гель-электрофореза нуклеиновых кислот, определяется от-ношением подаваемого напряжения к расстоянию между электродами [15,16]. Неоднократно высказывалось мнение, что такой принцип расчета напряженности при щелочном электрофорезе ДНКкомет может быть ошибочным [4,15,17]. Вместе с тем, только в единственной работе на примере единичной электрофоретической камеры была проведена оценка соответствия между расчетной и фактической напряженностью электрического поля [3].…”

Section: Introductionunclassified

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Technical aspects of the electrophoresis stage in the comet assay

Жанатаев

Anisina

Pligina

et al. 2020

Ecological genetics

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Inter- and intralaboratory variability of results is still a serious issue in the comet assay. There are several technical conditions of procedure, which may critically affect the results and electrophoresis terms were identified as main. A comparative assessment of the expected and actual field strength in five electrophoretic tanks and the contribution of the revealed differences to the variability in DNA damage carried out. Only for one tank, the measured field strength coincided with the expected 1 V/cm, while for four it ranged from 0.6 to 2.0 V/cm. The values of DNA damage assessed in the same samples of mouse kidney cells differed between tanks up to 4.7-fold for induced and up to 10-fold for spontaneous DNA damage. High local variations in the field strength and solution temperature across the platform as well as in %DNA in the tail of identical cell samples within electrophoresis runs also revealed. These variations were reduced by recirculation of electrophoresis solution. The results show that discrepancy between the estimated and the actual field strength can be reason of inter-laboratory variation of the comet assay results. Recirculation of the solution during electrophoresis will be useful to control of intra-laboratory and intra-assay variations.

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