Analysis of DNA double-strand breaks induced by DNA damaging agents in trophoblast organoids.

DNA double-strand break (DSB) is one of the most genotoxic lesions, and unrepaired DSBs can lead to chromosomal aberrations and eventually cause carcinogenesis, ageing and dysfunctions of organs. Therefore, studies of DNA repairs and DNA damage responses (DDRs) corresponding to DSBs has been gathered intense interest. Meanwhile, understanding of the clinical symptoms how dysfunction and accelerated ageing of various organs caused by the effects of DNA damages has been less accomplished. To address this, we have developed a new method that we could investigate DSB formation in trophoblast organoids. Organoids are self-organized three-dimensional tissues that mimic the complex structures and functions of an organ but are not widely used in nucleic acids research field. Using trophoblast organoids, we have examined the DSB formation induced by the treatments with various DNA damaging agents by pulsed-field gel electrophoresis. Organoids and 2D cultured cells were treated with bleomycin, mitomycin C, camptothecin, etoposide, gemcitabine, and 5-fluorouracil, and the patterns of DSB formation in the organoids and conventional two-dimensional (2D) cultured cells were compared. The pattern of DSB formation induced by DNA damaging agents in organoids was different from that appeared in 2D cultures. To further enhance versatility, we have also established a mouse organoid system and have examined its validity. We succeeded to develop a new method that can detect damage-induced DSB formation in organoids. Application of this method would be used not only for DDRs in a tissue model but also for the risk assessment of genotoxicity.