Vol.:(0123456789) 1 3 Archives of Toxicology https://doi.org/10.1007/s00204-020-02881-5 PROTOCOLS Expanded usage of the Challenge‑Comet assay as a DNA repair biomarker in human populations: protocols for fresh and cryopreserved blood samples, and for diferent challenge agents Vanessa Valdiglesias 1,2  · María Sánchez‑Flores 2,3  · Natalia Fernández‑Bertólez 2,3  · William Au 4,5  · Eduardo Pásaro 2,3  · Blanca Lafon 2,3 Received: 2 July 2020 / Accepted: 12 August 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract Defciencies in DNA damage response and repair (DDRR) can cause serious pathological outcomes; therefore, having an ability to determine individual DDRR would enhance specifcities in health risk assessment and in determining individual’s response to cancer therapies. However, most methods for evaluating DDRR are not fully appropriate for population studies. The Challenge-Comet assay has gained acceptance for this purpose. The assay has traditionally used X-rays as challenge agent and isolated peripheral blood mononuclear cells (PBMC) as cell specimen. To enhance the usefulness of the assay, the objectives of this investigation were to use diferently processed blood samples, to employ other challenge agents with diferent mechanisms of induction of DNA damage/repair, and to generate protocols for detecting diferent DDRR capaci- ties. Fresh and frozen blood samples were challenged with bleomycin, methyl methanesulfonate (MMS) and ultraviolet light. Signifcant induction of damage after all treatments, and progressive and time-dependent DDRR were observed. No signifcant diferences were obtained in the DDRR capacities of fresh or frozen whole blood samples as compared to PBMC, except that fresh blood samples showed higher MMS-induced DDRR capacity than PBMC. Results from this study show that the Challenge-Comet assay can be used as routine biomarker of DDRR capacity in human biomonitoring studies, and that whole blood is also a useful biomatrix for this assay. The collected data allow us to recommend diferent protocols for the Challenge-Comet assay which are useful for evaluating DDRR capacities in several key DNA repair pathways. Conse- quently, the usefulness of the Challenge-Comet assay can be greatly expanded. Keywords Biomonitoring · Cellular repair assay · Challenge-Comet assay · DNA repair · Genotoxicity · Health hazard assessment Introduction Excessive exposure to environmental or occupational muta- genic agents can cause DNA damage and serious health consequences, but such outcomes are crucially depend- ent upon an exposed individual’s DNA repair capacity. Therefore, having an ability to determine individual’s DNA repair capacity would be very useful in providing more pre- cise health risk assessment and disease prevention among exposed populations. Several methods and strategies have been attempted to evaluate individual’s functional DNA repair capacity, including both genetic and phenotypic approaches (reviewed in Valdiglesias et al. 2011 and Figueroa-González and Pérez- Plasencia 2017), but most of these assays are neither precise enough nor easily adopted for population studies when many * Vanessa Valdiglesias vvaldiglesias@udc.es 1 Centro de Investigaciones Científcas Avanzadas (CICA), Departamento de Biología, Facultad de Ciencias, Universidade da Coruña, Grupo DICOMOSA, Campus A Zapateira s/n, 15071 A Coruña, Spain 2 AE CICA-INIBIC. Oza, Instituto de Investigación Biomédica de A Coruña (INIBIC), 15071 A Coruña, Spain 3 Centro de Investigaciones Científcas Avanzadas (CICA), Departamento de Psicología, Facultad de Ciencias de La Educación, Universidade da Coruña, Grupo DICOMOSA, Campus Elviña s/n, 15071 A Coruña, Spain 4 University of Medicine, Pharmacy, Science and Technology, Gheorghe Marinescu nr. 38, 540139 Targu Mures, Romania 5 University of Texas Medical Branch, Galveston, TX, USA