Cyclophosphamide-induced apoptosis induces phocomelia in the mouse.

ABSTRACT

Phocomelia (absence of upper fore and/or hind limbs) was induced in mouse fetuses using cyclophosphamide. On day 11 of gestation, pregnant mice were injected intraperitoneally with 10 ml/kg of saline containing cyclophosphamide (CP) at a dosage of 20 mg/kg body weight. On day 18, the fetuses were removed by Caesarean section from dams given CP on day 11 and were examined for external anomalies. Of 22 fetuses from CP-treated dams, 13 were dead or absorbed, but the surviving 9 fetuses were found to have phocomelia with various other external anomalies. In order to examine the direct cytotoxic effect of CP on fetal limb buds, fetuses were removed at 8, 16, 24, and 48 h after CP administration on day 11, revealing the presence of frequent pyknotic nuclei and apoptotic bodies in hematoxylin and eosin (H & E) preparations. Cell-nuclei and apoptotic bodies were frequently observed by nick end-labeling in limb buds. Transmission electron microscopy demonstrated the typical changes of apoptosis. DNA extracted from the fetal limb buds submitted to CP was analysed by agarose gel electrophoresis, showing the ladder pattern characteristic of internucleosomal cleavage. These findings suggest that cyclophosphamide causes apoptosis in mouse fetal limb buds and that this process induces the external anomalies of phocomelia.