Replication stress induced exposure to methotrexate in root meristem cells of Vicia faba

Replication stress (RS) and oxidative stress (OS) are two main types of endogenous stress (ES) which, by inducing various forms of DNA damage lead to genome destabilization and disruption of cell division control mechanisms. Methotrexate (MTX) is a compound that inhibits dihydrofolate reductase (DHFR), thereby blocking DNA replication and exhibiting antiproliferative effects. The aim of the study was to investigate how 72-h exposure to 0.75 mM MTX on meristematic cells of Vicia faba roots affects the morphology of cell nuclei and mitotic chromosomes, population change of cells in interphase, DNA replication dynamics, cell viability, and hydrogen peroxide (H₂O₂) production. Furthermore, in order to assess epigenetic changes induced by MTX, associated with DNA damage and the replication process, histone H3 acetylation at lysine 56 (H3K56Ac) and histone H4 acetylation at lysine 5 (H4K5Ac) were examined. It was demonstrated that root meristematic cells treated with MTX exhibited abnormal chromosome structure, sustained DNA biosynthesis, and elevated intracellular H₂O₂ levels. Immunocytochemical studies revealed an increased number of fluorescent H3K56Ac foci. It was observed that in the case of H4K5Ac, MTX significantly reduced the frequency of cell populations characterized by euchromatin immunofluorescence and limited the occurrence of heterochromatin-type nuclei. Furthermore, in cells treated with MTX, a significant increase in the number of nuclei with marked nucleoli was observed in addition to the gap 1 (G1) phase. In summary, continuous 3-day exposure to low concentrations of MTX induced a cellular response to reactive oxygen species and DNA replication stress conditions.