In a previous study of the effects of intermittent extremely low frequency (ELF) magnetic fields (MF) on umbilical cord blood lymphocytes (UCBL), we evaluated MF amplitudes between 6 µT and 24 µT and found an effect only for those below 13 µT. This suggested the existence of an amplitude window. In this brief communication, we further tested this hypothesis. UCBLs from healthy newborns were isolated and exposed for 72 h to an intermittent ELF-MF (triangular, 7.8 Hz, 250 s ON/250 s OFF) with 6 different amplitudes between 3 µT and 12 µT, utilizing an oblong coil. Percentage of viable, early apoptotic (EA), and late apoptotic/necrotic (LAN) cells were determined by flow cytometry. Moreover, reactive oxygen species (ROS) were determined at 1 h and 3 h of the exposure. Like in our previous work, neither EA, nor LAN, nor ROS were statistically significantly affected by the intermittent ELF-MF. However, the percentage of viable cells was decreased by exposure to the fields with intensities of 6.5 µT and 12 µT (p < 0.05; and p = 0.057 for 8.5 µT). ELF-MF decreased the percentage of viable cells for fields down to 6.5 µT, but not for 5 µT, 4 µT, or 3 µT. Combined with our previous findings, the results reported here indicate an amplitude window effect between 6 µT and 13 µT. The obtained data are in line with a notion of amplitude and frequency windows, which request scanning of both amplitude and frequency while studying the ELF-MF effects.

Dephosphorylation inhibitor calyculin A (cal A) has been reported to inhibit the disappearance of radiation-induced γH2AX DNA repair foci in human lymphocytes. However, other studies reported no change in the kinetics of γH2AX focus induction and loss in irradiated cells. While apoptosis might interplay with the kinetics of focus formation, it was not followed in irradiated cells along with DNA repair foci. Thus, to validate plausible explanations for significant variability in outputs of these studies, we evaluated the effect of cal A (1 and 10 nM) on γH2AX/53BP1 DNA repair foci and apoptosis in irradiated (1, 5, 10, and 100 cGy) human umbilical cord blood lymphocytes (UCBL) using automated fluorescence microscopy and annexin V-FITC/propidium iodide assay/γH2AX pan-staining, respectively. No effect of cal A on γH2AX and colocalized γH2AX/53BP1 foci induced by low doses (≤10 cGy) of γ-rays was observed. Moreover, 10 nM cal A treatment decreased the number of all types of DNA repair foci induced by 100 cGy irradiation. 10 nM cal A treatment induced apoptosis already at 2 h of treatment, independently from the delivered dose. Apoptosis was also detected in UCBL treated with lower cal A concentration, 1 nM, at longer cell incubation, 20 and 44 h. Our data suggest that apoptosis triggered by cal A in UCBL may underlie the failure of cal A to maintain radiation-induced γH2AX foci. All DSB molecular markers used in this study responded linearly to low-dose irradiation. Therefore, their combination may represent a strong biodosimetry tool for estimation of radiation response to low doses. Assessment of colocalized γH2AX/53BP1 improved the threshold of low dose detection.

DNA double-strand breaks (DSB) represent one of the most severe forms of genomic damage. Thus, cells have evolved a complex network of DSB repair pathways, including homologous recombination, classical and alternative end joining, and single-strand annealing, which are tightly regulated by genetic and epigenetic factors. The selection and efficiency of these pathways influence genome integrity, oncogenesis, and therapeutic response. This comprehensive review synthesizes recent findings on the genetic regulation of DSB repair, with emphasis on pathway-specific regulators, chromatin context, and post-translational modifications. Moreover, this review integrates primary research from mammalian systems, including CRISPR-based studies, proteomics, and imaging, with a focus on publications from 2020 to 2025. We discuss the role of key players, such as MRE11–RAD50–NBS1 (MRN), ataxia telangiectasia mutated (ATM), mediator tumor suppressor p53-binding protein 1 (53BP1), breast cancer type 1 susceptibility protein (BRCA1), anti-silencing function 1 (ASF1), ring finger protein (RNF)8/168, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and RAD51 recombinase (RAD51), in orchestrating the associated pathway choice. Epigenetic modifications, RNA-mediated mechanisms, and chromatin remodeling dynamically influence the efficiency and fidelity of repair. Particular attention is provided to emerging regulators, including thyroid hormone receptor interactor 13 (TRIP13), ubiquitin-like with plant homeodomain (PHD) and RING finger domains 1 (UHRF1), Shieldin, and polymerase theta. This review highlights novel insights into transcription-associated DSB repair, the interplay of replication stress with repair pathway engagement, and context-dependent synthetic lethality. We also examine implications for cancer biology, including therapy resistance and biomarker development. Ultimately, understanding the genetic regulation of DSB repair pathways can provide critical insights into genome stability maintenance and reveal new therapeutic opportunities in cancer. Future work should focus on pathway crosstalk, phase-specific regulation, and integrating repair modulation into personalized medicine.

Purpose : Ionizing radiation induced foci (IRIF) known also as DNA repair foci represent the most sensitive endpoint for assessing DNA double strand breaks (DSB). IRIF are usually visualized and enumerated with the aid of fluorescence microscopy using antibodies to γH2AX and 53BP1. Although several approaches and software packages were developed for the quantification of IRIF, not one of them was commonly accepted and inter-laboratory variability in the outputs was reported. In this study, the sensitization of Metafer software to counting also small appearing IRIF was validated. Materials and Methods : Human lymphocytes were γ-irradiated at a dose of 2 Gy. The cells were fixed at 0.5, 1, 2, and 18 hours post-irradiation, permeabilized and IRIF were immunostained using appropriate antibodies. Cell images were acquired with the automatic Metafer system. Radiation-induced γH2AX and 53BP1 foci were enumerated using either manual counting (JCountPro program) or the Metafer software (after its classifier optimization has been done) and compared. The statistical analysis was performed using One-way ANOVA. Results : The enumeration of 53BP1, γH2AX foci manually by JCountPro did not statistically significantly differ from the automatic one performed with the optimized Metafer classifier. A detailed step-by-step protocol of this successful optimization is described in this study. Conclusions : We concluded that the Metafer software after the optimization was efficient in objectively enumerating IRIF, having a potential for usage in clinics and molecular epidemiology.