Cyclophosphamide‐induced teratogenesis in ICR mice: The role of apoptosis

Abstract

It is known that programmed cell death (apoptosis) is an important physiological determinant of embryonic development. In parallel, it may be one of the major events involved in induced teratogenesis. The present study was designated to evaluate to what extent is apoptosis involved in the formation of some final abnormalities induced by cyclophosphamide (CP) in ICR mice. The level of apoptosis in limbs, tail, liver, and whole embryo was assessed 24 h after administration of various doses of CP (day 12 of pregnancy) by flow cytometric analysis and by DNA fragmentation assay. In parallel, the rate of limb and tail malformations, resorptions, and growth retardation induced by various doses of CP was evaluated in animals sacrificed on day 19 of pregnancy using routine teratological methods. A striking correlation between the rate of CP-induced apoptosis in limb and tail cells and the severity of limb and tail anomalies was found after administration of CP ranging from 10 to 40 mg/kg. Thus, the percent of apoptotic cells collected from limbs and tails increased from 18 to 78%. In parallel, the severity of limb and tail anomalies increased from digit anomalies to amely and from crooked to short or absent tail. CP-induced embryolethality and fetal growth retardation also correlated with the level of apoptosis in cells collected from whole embryos but to a lesser extent. These results claim that CP-induced apoptosis is one of the inevitable events in the pathway leading to the formation of CP-induced abnormalities and also suggest that the extent of the involvement of apoptosis in the formation of different types of final abnormalities, may be different.

p53 regulates cyclophosphamide teratogenesis by controlling caspases 3, 8, 9 activation and NF-κB DNA binding

“…A body of studies suggests that the pathogenesis of CP-induced developmental structural anomalies includes intermediate steps such as excessive apoptosis and suppression of cell proliferation (Chernoff et al 1989, Francis et al 1990, Little & Mirkes 1992, Torchinsky et al 1995a. In this work, CP-induced apoptosis was evaluated by the TUNEL assay and by measuring the levels of active caspases 3, 8, and 9.…”

“…The choice of the brain was based on our previous studies (Torchinsky et al 1995a) suggesting that the intensity of CP-induced apoptosis in this organ directly correlates with the incidence (or severity) of nearly all CP-induced structural anomalies tested in the study. As to the limbs, they were chosen because in this study the limbs of p53 knockout and wild-type embryos were found to differ dramatically in the sensitivity to CP-induced teratogenic insult.…”

Cellular events and the pattern of p53 protein expression following cyclophosphamide-initiated cell death in various organs of developing embryo

“…at 8:00 AM on day 12 of pregnancy at a dose of 40 mg/kg (in 0.5 ml saline per 20 g body weight). The results of previous studies [3,4,13] indicated that this dose of CP will affect all tested embryonic organs in all embryos.…”

“…We have shown that in litters of mice treated with a dose of 40 mg/kg of cyclophosphamide (CP) and tested on day 19 of pregnancy, all fetuses had craniofacial (agnathia, exencephaly, cleft palate, open eyes), limb reduction anomalies (amelia or phocomelia), and severe growth retardation [3]. In parallel, it has been observed [4] that this dose of CP initiates apoptosis in the limbs, head, and liver of murine embryos 24 h after CP administration and that limb reduction and craniofacial anomalies are clearly seen by 48 h after CP treatment. The liver, however, did not differ macroscopically from that of control embryos either at this time point or 24 h later.…”

“…Implantation sites, resorptions, live, and dead embryos were recorded and live embryos were examined for external anomalies, as described previously [4]. To characterize dysmorphic events in the liver, indices such as malpositioned liver, misshapen liver, mottled or pale liver, and abnormal liver lobation were used [14].…”