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5-AZA-2'-DEOXYCYTIDINE INDUCED CYTOTOXICITY AND LONG BONE REDUCTION DEFECTS IN THE MURINE LIMB
Rosen, M B. AND N Chernoff. 5-AZA-2'-DEOXYCYTIDINE INDUCED CYTOTOXICITY AND LONG BONE REDUCTION DEFECTS IN THE MURINE LIMB. TERATOLOGY. Alan R Liss, Inc, New York, NY, 65(4):180-190, (2002).
The antineoplastic drug 5-aza-2'-deoxycytidine (dAZA) is a DNA hypomethylating agent that can be used to induce hind limb phocomelia in the offspring of CD-1 Swiss Webster mice. Previously, our laboratory investigated the possibility that dAZA induced alterations in gene expression may uniquely affect hind limb pattern formation. The present studies test an alternative hypotheses, namely that dAZA may act as a "universal" teratogen by inducing general cytotoxicity among rapidly proliferating cells such as those of the limb bud mesenchyme. Initially, gestation day (GD) 10 CD-1 Swiss Webster mice were dosed with 1 mg/kg dAZA (i.p.) at either 9 a.m., 2 p.m., or 7 p.m. and fetuses were collected for hind limb skeletal necropsy following staining with alcian blue. Hind limb defects were not limited to a specific set of skeletal elements or limb segment but consisted of a range of temporally related anomalies such that the more proximal skeletal elements were affected by early dosing and the more distal elements by later dosing. This is consistent with a general insult to the limb bud mesenchyme since limb outgrowth proceeds in a proximal to distal direction. Additional mice were then dosed at midnight (0000 hrs) on GD 10 and fetuses were collected for fore limb skeletal examination. In contrast to published data, modest defects of the radius were observed. This argues against the hypothesis that a limb specific mechanism, such as a unique change in gene expression, underlies the long bone reduction anomalies observed in the hind limb. By 12 hrs after dosing, histological examination of limb bud sections revealed acute mesenchymal cell death in both fore and hind limbs. Using bromodeoxyuridine immunohistochemistry, reduced cell proliferation was also found by 24 hrs after dosing. The timing and location of these changes strongly suggests a role for cytotoxicity in the etiology of dAZA induced long bone reductions. Since these effects appeared to be less pronounced in the fore limb they are consistent with the earlier finding of increased teratogenicity in the murine hind limb. The expression of scleraxis, a marker gene for early chondrogenesis, was reduced 12 hrs after exposure to dAZA as was the emergence of alcian blue stained long bone anlagen on GD 11. These results indicate that dAZA affects long bone development between GD 10 and 11. This likely represents a crucial stage of limb outgrowth during which the mesenchymal condensations that shape the prospective long bones begin to form. These findings support the hypothesis that reduced mesenchymal proliferation during early limb outgrowth can induce the proximal limb truncations characteristic of phocomelia.