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Cell cycle alterations and cell death in cyclophosphamide teratogenesis
N Chernoff1, J M Rogers, A J Alles
1Developmental Toxicology Division, Environmental Protection Agency, Research Triangle Park, North Carolina.
Teratogenesis, Carcinogenesis, and Mutagenesis
|January 1, 1989
Summary
Maternal exposure to cyclophosphamide (CP) causes limb malformations in mice by inducing cell death and cell cycle disruption in developing embryos. These effects correlate with the severity of fetal toxicity and malformations observed.
Area of Science:
- Developmental toxicology
- Teratology
- Molecular toxicology
Background:
- Cyclophosphamide (CP) exposure during pregnancy is known to cause limb malformations in mouse offspring.
- Previous research indicates that programmed cell death occurs in embryonic limb buds following maternal CP treatment.
- The precise mechanisms linking CP exposure to developmental toxicity, including cell death and cell cycle perturbation, require further investigation.
Purpose of the Study:
- To investigate the relationship between cyclophosphamide-induced cell death, cell cycle perturbation, and embryo/fetal toxicity in developing mouse limbs.
- To quantify the dose-dependent effects of CP on cell viability and cell cycle progression in embryonic limb buds.
- To correlate early cellular events with the incidence of malformations and fetal mortality at term.
Main Methods:
- Pregnant Swiss-Webster mice were administered varying doses of cyclophosphamide (CP) on day 10 of gestation.
- Embryos were collected at 4, 8, and 28 hours post-exposure for analysis.
- Cell death was assessed using Nile blue sulphate vital staining; cell cycle progression was analyzed by flow cytometry (FCM) of propidium iodide-stained nuclei from isolated limb buds.
Main Results:
- Nile blue sulphate staining revealed a dose-related increase in cell death in limb buds by 8 hours post-exposure, primarily in areas of DNA synthesis.
- Flow cytometry demonstrated a dose-dependent retardation of S-phase progression starting at 4 hours, persisting through 8 hours post-exposure.
- At 28 hours, cell cycle histograms normalized in low-dose embryos but remained perturbed in intermediate and high-dose groups. Limb malformations and fetal mortality were observed at term, correlating with the observed cellular effects.
Conclusions:
- Cyclophosphamide exposure induces significant cell death and cell cycle perturbation in developing mouse limb buds.
- The magnitude and timing of these cellular events correlate directly with the observed fetal toxicity, including limb malformations and mortality.
- These findings suggest a mechanistic link between CP-induced cell cycle disruption, subsequent cell death, and the teratogenic outcomes observed.