Effects of 4‐hydroperoxycyclophosphamide (4‐OOH‐CP) and 4‐hydroperoxydechlorocyclophosphamide (4‐OOH‐deCICP) on the cell cycle of post implantation rat embryos

Abstract

In this study, we used preactivated forms of cyclophosphamide (CP) and dechlorocyclophosphamide (deClCP) to examine the effects of phosphoramide mustard (PM) and acrolein, respectively, on the cell cycle of postimplantation rat embryos. The percentage distribution of cells in the G1/G0, S, and G2/M phases of the cell cycle was determined by flow-cytometric analysis. At embryotoxic concentrations, 4-OOH-CP (PM) induced major cell cycle perturbations whereas 4-OOH-deClCP (acrolein) caused no major perturbation of the cell cycle. These data support the hypothesis that the mechanism of the embryotoxic action of PM involves alkylation of DNA, whereas the mechanism of action of acrolein does not. The primary effect of PM on the cell cycle was an initial delay in the S phase followed by a G2/M arrest. At low embryotoxic concentrations of 4-OOH-CP, there was apparent reversal of the G2/M arrest; at higher embryotoxic concentrations there was little recovery from the G2/M arrest. The high level of cell death found at higher drug concentrations suggests that prolonged G2/M arrest leads to cell death. Using radiolabeled CP and cell sorting, it was determined that PM predominantly alkylated DNA in the S phase of the cell cycle. Overall, the data from this study support the hypothesis that DNA cross-links, induced by the alkylation of DNA by PM, induce cell cycle perturbations. Furthermore, these cell cycle alterations may be one of the early steps in the mechanism leading to the embryotoxicity of PM.

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.…”

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

“…Cell cycle alterations, including cell death, have been reported to be an early effect in the pathway leading to CP-induced structural anomalies [19,20,21]. CP induces these alterations in a dose-and tissue-dependent fashion and, if of sufficient magnitude, is followed by cell death and gross malformations.…”

PCR‐RFLP to detect codon 248 mutation in exon 7 of p53 tumor suppressor gene

“…In view of the low activity of DNA repair enzymes (reviewed in Vinson and Hales, 2002), and the unusually rapid cell cycles in embryos (Mac Auley et al, 1993), it is interesting to speculate that the embryo may also be readily prompted to undergo apoptosis in lieu of initiating DNA repair. Indeed, a number of studies show exposure to activated CPA metabolites induced apoptosis in embryos (Chernoff et al, 1989; Little and Mirkes, 1992; Chen et al, 1994; Moallem and Hales, 1995; Torchinsky et al, 1995) and much effort has been made to understand the pathways involved, although all these will not be discussed presently because this would require an entire review dedicated to this theme alone (Francis et al, 1990; Hosako et al, 2007; Huang and Hales, 2002; Little and Mirkes, 2002; Mirkes and Little, 2000; Mirkes et al, 2000; Mirkes, 2002; Moallem and Hales, 1996, 1998; Torchinsky et al, 1995, 1999).…”