Visualization of acetamide-induced large micronucleus formation via nuclear atrophy in rat primary hepatocyte

Micronuclei are small nuclei that form when chromosomes or fragments fail to segregate into daughter nuclei during cell division. We previously suggested that acetamide induces the formation of large micronuclei through nuclear atrophy in binuclear hepatocytes. In this study, to confirm large micronuclei formation from atrophic nuclei within identical hepatocytes, we established an in vitro model. Large micronuclei formed from rat primary hepatocytes treated with 30 mM acetamide showing molecular features identical to those observed in vivo, including loss of nuclear lamina, accumulation of phosphorylated histone H2AX, and heterochromatinization. Time-lapse imaging demonstrated that large micronuclei were specifically formed due to unilateral nuclear atrophy in binuclear hepatocytes. Although nuclear atrophy was also observed in mononuclear hepatocytes, most hepatocytes with atrophic nuclei underwent subsequent cell death, whereas only a portion of binuclear hepatocytes generated large micronuclei and survived. These findings indicate that nuclear atrophy occurs in both mononuclear and binuclear hepatocytes and generally leads to cell death, whereas a subset of binuclear hepatocytes survives through large micronucleus formation. Atrophic nuclei exhibited a partial loss of nuclear lamina accompanied by DNA damage accumulation and chromatin condensation, whereas heterochromatinization was observed only in some of them, suggesting the involvement of nuclear rupture in large micronucleus formation. Overall, these data demonstrate a novel mechanism of large micronuclei formation through unilateral nuclear atrophy in binuclear hepatocytes independent of cell division. We propose the term "atrophic micronucleus" for this large micronucleus, which may mediate chromosomal rearrangements that contribute to acetamide hepatocarcinogenesis.