ABSTRACT
Further delineation of mechanisms by which 6-aminonicotinamide (6-AN) induces micro-melia in the chick embryo was investigated by studies on rates of incorporation of thymidine, proline, glucosamine and sulfate as precursors to DNA, collagen and mucopolysaccharide, respectively. Twentyfour hours after in ovo administration of the vitamin antagonist, 6-AN, to day-4 chick embryos, hind limbs from experimental and control groups were excised and incubated for 1 h in medium containing 3 × 10 − 6 M radioactive precursor. Molar incorporation of precursors into the TCA-precipitable fraction showed, in isolated limb buds, (a) that 6-AN enhanced incorporation of thymidine, (b) that 6-AN inhibited utilization of sulfate, and (c) that 6-AN did not significantly alter utilization of glucosamine and proline.
Rates of incorporation of thymidine, glucosamine and proline indicate that 6-AN is not cytotoxic to the isolated limb bud. Enhanced incorporation of thymidine suggests expression of compensatory change 24 h after initial effects of 6-AN on DNA synthesis. Rate of in-corporation of proline suggests that, under the influence of 6-AN, tropocollagen was syn-thesized in normal quantities by limb cells. Similarly, rate of incorporation of glucosamine suggests that under the influence of 6-AN normal amounts of hexosamine sugars were being attached to the nascent core-protein of chondroitin. Inhibition of sulfation and failure to complete the chondroitin sulfate molecule seem to account for 6-AN-induced micromelia. This suggests that sulfation depends upon specific NAD-dependent dehydrogenase reactions. As far as can be established by rates of incorporation of labeled precursors, 5-day limb buds, at 24 h after exposure to teratogenic levels of 6-AN, synthesize matrix proteins and hexo-samine polysaccharides at normal rates.