Optimization of the alkaline comet assay for easy repair capacity quantification of oxidative DNA damage in PBMC from human volunteers using aphidicolin block

Grace A Odongo; Ivan Skatchkov; Corinna Herz; Evelyn Lamy

doi:10.1016/j.dnarep.2019.03.005

Optimization of the alkaline comet assay for easy repair capacity quantification of oxidative DNA damage in PBMC from human volunteers using aphidicolin block

DNA Repair (Amst). 2019 May:77:58-64. doi: 10.1016/j.dnarep.2019.03.005. Epub 2019 Mar 14.

Authors

Grace A Odongo¹, Ivan Skatchkov², Corinna Herz², Evelyn Lamy³

Affiliations

¹ Molecular Preventive Medicine, University Medical Center and Faculty of Medicine, University of Freiburg, Breisacher Straße 115b, 79106 Freiburg im Breisgau, Germany; Institute of Food Chemistry, Hamburg School of Food Science, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
² Molecular Preventive Medicine, University Medical Center and Faculty of Medicine, University of Freiburg, Breisacher Straße 115b, 79106 Freiburg im Breisgau, Germany.
³ Molecular Preventive Medicine, University Medical Center and Faculty of Medicine, University of Freiburg, Breisacher Straße 115b, 79106 Freiburg im Breisgau, Germany. Electronic address: evelyn.lamy@uniklinik-freiburg.de.

PMID: 30889507
DOI: 10.1016/j.dnarep.2019.03.005

Abstract

Assessment of DNA repair capacity (DRC) upon ex vivo challenge of peripheral blood mononuclear cells (PBMC) with oxidative damage inducing agents, as evaluated by the comet assay, is widely used as biomarker to assess the antioxidant status in human studies. Here, the alkaline comet assay was now optimized for easy and time saving detection of repair capacity upon oxidative stress-induced DNA damage using the DNA polymerase inhibitor aphidicolin (APC) to block repair of hydrogen peroxide (H₂O₂) induced DNA damage. Addition of a DMSO-containing DNA damage stop solution was found suitable to replace washing steps for H₂O₂ removal before APC block. Cell treatment with APC at 6 μM did not impact baseline DNA damage but could reliably block DNA repair after H₂O₂ challenge in both fresh and cryopreserved samples thus omitting the use of a starting time point control. Under the conditions used, frozen cells, with or without an additional 4 h rest, showed the same repair capacity as their fresh counterpart. The intra assay coefficient of variation (CV) was 3.3%. To provide proof of principle, the modified assay was applied to cryopreserved PBMC from 19 participants of a short-term Brassica diet intervention study investigating potential health promoting effects of the food intervention. Then, a 33% increase in DRC (p ≤ 0.01) could be shown in samples after intervention (mean ± SD: 5.82 ± 1) as compared to baseline (mean ± SD: 4.38 ± 1.21). Individual samples from baseline and intervention showed an inter-individual CV of 27.65% (baseline) and 17.26% (intervention). Taken together this modified comet assay protocol allows the facilitated detection of DNA repair in fresh or cryopreserved human PBMC samples with a good sensitivity and reliability and could be useful in human studies addressing the antioxidant status and repair capacity of PBMC.

Keywords: Aphidicolin block; Brassica; Comet assay; DNA repair; Fresh and cryopreserved PBMC; Oxidative damage.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adult
Aphidicolin / pharmacology*
Comet Assay / methods*
Cryopreservation
DNA Damage*
DNA Repair / drug effects*
DNA-Directed DNA Polymerase / metabolism
Dose-Response Relationship, Drug
Female
Healthy Volunteers
Humans
Hydrogen Peroxide / metabolism
Leukocytes, Mononuclear / drug effects*
Leukocytes, Mononuclear / metabolism*
Male
Nucleic Acid Synthesis Inhibitors / pharmacology
Oxidative Stress / drug effects
Oxidative Stress / genetics*
Time Factors
Young Adult

Substances

Nucleic Acid Synthesis Inhibitors
Aphidicolin
Hydrogen Peroxide
DNA-Directed DNA Polymerase