Contribution of mesenchymal cell death and mitotic alteration to asymmetric limb malformations induced by MNNG

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces fetal asymmetric limb malformations with exposure of pregnant mice to 50 mg/kg on day 11 of gestation. Hindlimbs were more frequently malformed than forelimbs, and a fourfold greater incidence of postaxial ectrodactyly was found in left forelimbs than in right forelimbs, and a two fold excess in left hindlimbs compared to right hindlimbs. The level of cell death and mitotic index were measured in forelimbs and hindlimbs from treated and control embryos at 1, 4, 18, 24, 48, and 72 hr after exposure to ascertain if these parameters could be correlated with the differential teratogenic susceptibility of the limbs. An increase in necrotic index was first detected in treated limbs at 4 hr, increased at 18 hr, peaked at 24 hr, and began declining at 48 hr to reach the control baseline at 72 hr. At 24 hr, the correlation between the level of cell death and susceptibility of malformation was the strongest, with the left hindlimb having a necrotic index of 58%, the right hindlimb 47%, the left forelimb 30% and the right forelimb 12%. In both forelimbs and hindlimbs, MNNG treatment initially depressed mitotic activity followed by an elevation at 48 hr relative to controls. The magnitude of the depression, extent of the elevation, and overall pattern of mitotic activity could not be uniformly related to limb defects. These results indicate that the amount of cell death in limb buds at 24 hr after MNNG exposure may predict target organ susceptibility. Depressions in mitotic activity and alterations in the pattern of mitosis were also observed which were not as clearly correlated with the incidence of malformations as was the amount of cell death.