Expanding the comet assay toolbox for human biomonitoring using frozen whole blood.

The comet assay is a widely used tool for evaluating DNA damage in various biological samples, with frozen whole blood (WB) as an attractive biomatrix, due to its ease of collection, handling, and storage. Although frozen WB has been successfully applied in the comet assay to study DNA damage, its application in the comet assay to study potential protective mechanisms, such as DNA repair or antioxidant capacity, has not been fully established yet. In this study, we set up a protocol using frozen WB for assessing base excision repair (BER) activity and further optimized the protocol to assess the cellular antioxidant defense capacity against oxidation-induced DNA damage. White blood cells (WBCs) were isolated to extract proteins, and a protein concentration of 2 mg/mL enabled reliable detection of BER activity, even in samples stored for up to 27 months. This optimized assay was subsequently applied in a pilot study involving 28 participants with either a family tumor history (FTH, n = 10) or not (NFTH, n = 18). The FTH group exhibited significantly less BER activity compared to the NFTH group (p = 0.005). In addition, the hydrogen peroxide (H₂O₂) challenge comet assay was further optimized to assess the cellular antioxidant defense capacity against oxidation-induced DNA damage in frozen WB and subsequently applied to a cohort study (n = 140). Frequent intake of seeds (p = 0.025) and vegetables (p = 0.003) was observed to protect against H2O2-induced DNA damage. Together, these innovations expand the applications of the comet assay in frozen blood samples, providing a more comprehensive biomonitoring toolbox. By enabling reliable measurements of DNA damage, antioxidant capacity, and DNA repair activity from a single frozen blood sample, these assays are ideally suited for large-scale human biomonitoring and clinical studies.