The Teratogenic Mechanism of 6-Aminonicotinamide on Limb Formation of Chick Embryos: Abnormalities in the Biosynthesis of Glycosaminoglycans and Proteoglycans in Micromelia1

Abstract

After a dose of 10 micrograms of 6-aminonicotinamide (6-AN) was administered to day-4- chick embryo in ovo, micromelia was obviously observed in the hind limbs of 7-day chick embryos. We examined the teratogenic mechanism of 6-AN by using the normal or micromelial hind limbs (buds) from day 5 to day 7, with special attention to the biosynthesis of glycosaminoglycan (GAG) and proteoglycan as an index of limb chondrogenesis. The present study provides evidence for abnormalities in the levels of GAG or proteoglycan biosynthesis in the micromelial hind limbs (buds). 1) Both [35S]sulfate and [3H]glucosamine incorporation into GAG per 10 limbs or mg DNA of the micromelia were inhibited, suggesting a decrease of GAG synthesis. 2) The micromelial limbs synthesized low-sulfated chondroitin sulfate (chondroitin) as judged by the 35S/3H ratio, the proportion of unsulfated disaccharide (delta Di-0S), and the result of cellulose acetate electrophoresis, although there were no significant differences in the approximate molecular size of 35S-chondroitin sulfates synthesized between the normal and micromelial limbs. 3) PAPS-synthesizing activity in the micromelial limbs was markedly inhibited, and this may result in the production of low-sulfated proteoglycan. 4) The transition from mesenchymal- to cartilage-specific proteoglycan synthesis did not appear in the micromelial limbs as judged by the sedimentation profiles. 5) 6-AN caused marked reductions in the oxygen consumption and ATP level of the micromelial limbs, thereby causing the defect in PAPS formation. We suggest that these 6-AN-induced sequential molecular defects (the reduction of respiratory activity, ATP and PAPS level, and concomitant interference with GAG and proteoglycan biosynthesis) in the limbs (buds) during the critical period of limb morphogenesis must be major factors resulting in the cartilage growth retardation or disorder, i.e., micromelia.

Abnormal synthesis of cartilage-characteristic proteoglycan in azaserine-induced micromelial limbs

“…Also, an abnormal form of PG-H is produced in azaserine-induced micromelial limbs during limb formation in chick embryos. Considering our previous results obtained with micromelial limbs induced by 6-aminonicotinamide [19,20], the retardation of chondrogenesis and the abnormalities of PG-H synthesis at a critical stage of limb morphogenesis must be key factors resulting in both growth retardation and disordered formation of cartilage.…”

“…Fractions (3 ml) were collected and assayed for radioactivity. PG-H was obtained from 19 (a) or 26 (b) limbs. For further experimental details, see the text.…”

“…Labelled proteoglycans were extracted from normal or micromelial hind limbs by the method described previously [19,20]. Briefly, the labelled tissues were extracted with 5 vol.…”

“…For the assay of PG-H core protein, the hind limbs (30 limbs) of day-7 normal and azaserine-treated embryos were extracted with I ml of solution A and the extracts were centrifuged on a glycerol-density-gradient under dissociative conditions as above. The faster sedimenting proteoglycan fractions (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) were precipitated with 3 vol. of ice-cold 9500 (v/v) ethanol containing 1.30%…”

Hyaluronate synthetase inhibition by normal and transformed human fibroblasts during growth reduction

“…50, Toyo Roshi, Tokyo, Japan) with 40 txl of a mixture of propanoic acid and water (1:1, vol/vol) (or 1% SDS), which was used to wash the sample tube. Descending paper chromatography was carried out overnight with the first solvent (1-butanol/ethanol/water [52:32:16, vol/vol/vol]) to remove salts in the sample (18) and then again overnight with the second solvent (1-butanol/propanoic acid/1 N NI-I4OH (1:2:2, vol/vol/vol]). After air drying the paper, the spot at the origin was cut off for liquid scintillation counting.…”