The teratogenic mechanism of 6-aminonicotinamide on limb formation of chick embryos: abnormalities in the biosynthesis of glycosaminoglycans and proteoglycans in micromelia

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.     

Time of onset and selective response of chondrogenic core of 5-day chick limb after treatment with 6-aminonicotinamide.

Exposure of the chick embryo to the nicotinamide analog, 6-aminonicotinamide (6-AN), causes specific changes in chondrogenic cells that result in limb deformity. Autoradiography has further delineated these changes and relates them to altered utilization of molecular precursors of cartilage matrix and DNA. With ³⁵SO₄ to monitor synthesis of glycosaminoglycan, it was shown that, at 6 hr and persisting until 24 hr after treatment, 6-AN inhibited utilization of sulfate by cells in the chondrogenic core while having no detectable effect on cells in the chondrogenic periphery. Similarly, 6-AN suppressed incorporation of [³H]thymidine into core cells while having no effect to a slight enhancement effect on chondrogenic and nonchondrogenic cells surrounding the core. These observations support the view that, in response to 6-AN-inhibited NAD(P)-dependent reactions, limb chondrogenic cells (CORE) cease to produce matrix glycosaminoglycan, cease to synthesize DNA, and ultimately succumb. Conversely, presumably as a result of more efficient energy production because they lie closer to a vascular supply of oxygen, cells in the chondrogenic periphery withstand the teratogenic insult and continue proliferating to become the source where subsequent partial repair of the limb occurs.     

Ascorbate inhibition of 6-aminonicotinamide teratogenesis in chicken embryos.

Chicken eggs of 4 or 6 days of incubation were injected with 10 mug 6-aminonicotinamide (6-AN) or 6-AN plus various doses of sodium ascorbate, calcium ascorbate, or ascorbic acid; 11-day embryos were examined grossly and histologically. 6-AN-treated embryos had various degrees of micromelia and were reduced in overall size. All three ascorbates inhibited 6-AN teratogenesis but not completely. The extent of inhibition was dose related. Increased amounts of intercellular matrix and decreased necrosis of chondrocytes in the limb cartilage of protected embryos correlated with the gross findings.     

Enhanced chondrocytic differentiation in chick limb bud cell cultures by inhibitors of poly(ADP-ribose) synthetase

Inhibitors of poly(ADP-ribose) synthetase, namely nicotinamide, benzamide, m-methoxybenzamide and 3-aminobenzamide, augmented chondrocytic differentiation chick embryo limb bud mesenchymal cells, in culture. These inhibitors stimulated early appearance and massive formation of cartilage nodules in micromass cultures stage 23-24 chick embryos. They also induced nodule formation in micromass and cartilage colonies at micromass plating densities from stage 18-19 embryo Benzamide, however, did not prevent differentiated chondrocytes from undergoing a pleiotypic change in cell type. These results are compatible with the putative regulatory function of poly(ADP-ribose) on cell differentiation.     

The site and sequence of action of 6-aminonicotinamide in causing bone malformations of embryonic chick limb and its relationship to normal development

The results reported here include the following findings: (1) 6-aminonicotinamide (6AN) affects cartilage-producing cells, resulting in subsequent bone malformations. (2) 6AN causes chondrogenic cells to degenerate, a process morphologically similar to that previously described for the action of 6AN on chick limb mesodermal cells in vitro. (3) 6AN acts on the process of phenotypic expression or the newly determined chondrocyte as opposed to acting on mature chondrocytes. Degenerative effects in the limb are not observed until stage 25 corresponding to the first appearance of stainable cartilaginous matrix material. Also, 6AN acts on older embryos to produce zones of degeneration of cartilage involving a process, again, morphologically similar to that described for the action of 6AN on chick limb mesodermal cells in vitro, and again this process does not seem to affect mature chondrocytes. (4) 6AN is effective for a relatively short period, about 36 hr after its introduction into the egg, as shown by the inability of nicotinamide to completely counteract 6AN-caused effects. These observations and others are used to discuss the mode of action of nicotinamide-sensitive teratogens as well as the role of nicotinamide in influencing the expression of limb mesodermal cells into myogenic and chondrogenic phenotypes.     

Replacement of proteoglycans in embryonic chick cartilage in organ culture after treatment with testicular hyaluronidase

Explants of cartilage from tibiae of 11-12 days chick embryos were grown in organ culture. To one group hyaluronidase was added to the medium during the first 2 days of culture; the treated tissue was then cultured in medium without enzyme for a further 4 days. Control explants grown in hyaluronidase-free medium for 6 days grew rapidly in size and the total hexosamine content more than doubled during this time. After exposure to hyaluronidase, much of the hexosamine was lost from treated cartilage and appeared in the culture medium, but it was mostly replaced in the tissue during the subsequent recovery period. Analysis of cartilage and medium showed that net synthesis of hexosamine increased greatly in treated cartilage. The proteoglycans were extracted by two procedures from control and treated cartilage after 2, 4 and 6 days in culture. The hydrodynamic sizes of the purified proteoglycans were compared by gel chromatography and the composition of the gel-chromatographic fractions was determined. The proteoglycans from controls did not change during culture, but after exposure to hyaluronidase the proteoglycans from treated cartilage were of much smaller size and lower chondroitin sulphate content. During recovery, even though new proteoglycans were formed, they were nevertheless of smaller size and lower chondroitin sulphate content than control proteoglycans. They gradually became more like control proteoglycans during recovery from treatment, but even after 4 days they were not yet the same. After 2 days of treatment with the enzyme, the chondroitin sulphate in the cartilage was of shorter chain length than in controls but during recovery after 4 and 6 days in culture, the chain lengths in control and treated cartilage were similar. It is concluded that the proteoglycans formed in embryo cartilage in response to their depletion by enzyme treatment contained fewer chondroitin sulphate chains attached to the protein moiety of proteoglycans. This may have resulted from a failure under stress to glycosylate the protein moiety to the usual extent; alternatively the synthesis of normal proteoglycans of low chondroitin sulphate content may have increased, thus changing the proteoglycan population.     

Normal development of the skeleton in chick limb buds devoid of dorsal ectoderm

It has been suggested that the ectoderm on the dorsal and ventral faces of the limb bud plays a part in controlling the pattern of cartilage differentiation. To test this, the dorsal wing bud ectoderm in the chick embryo was destroyed by irradiation with ultraviolet light at stage 17-19, at the very beginning of limb bud development, but the apical ectodermal ridge was spared. The irradiated ectoderm disappeared within 24 hr (by stage 23-24) and did not regenerate thereafter; thus the dorsal surface of the limb bud was kept denuded throughout most of the period of skeletal pattern formation. By 6 or 7 days after the irradiation (stage 35), when the rudiments of all the adult skeletal elements are normally present in recognizable form, the irradiated wings could be placed into two categories, those that were approximately normal in shape and those that had curled dorsally. All of these limbs were reduced in size, to varying degrees, when compared to their controls and lacked dorsal soft tissues. The limbs that were normal in shape, however, even though sometimes denuded over practically the whole extent of their dorsal surface, almost always had a complete and normally proportioned cartilage pattern, suggesting that ectoderm (other than the apical ectodermal ridge) does not exert any direct control over the development of the limb cartilage pattern. However, many of those limbs that had curled as a result of the irradiation did have major pattern deformities, suggesting that the topology of cartilage differentiation does depend on the shape of the limb bud.     

0.2 T magnetic field inhibits angiogenesis in chick embryo chorioallantoic membrane

Inhibition of angiogenesis is a major target in the fight against cancer and other diseases. Although the effects of static magnetic fields on cancer development and cell growth have been investigated, effects on angiogenesis have received no attention so far. In this study we report the effects on angiogenesis of exposure to 0.2 T static magnetic field. Angiogenesis was analyzed using the chick embryo chorioallantoic membrane assay. Exposure to 0.2 T static magnetic field was achieved by placing the eggs for 3 hr in the isocentre of the magnet of a sectorial magnetic resonance tomograph used in clinical practice. In sham exposed specimens treated with phosphate buffered saline (negative control), no significant vascular reaction was detectable; 3 hr exposure to 0.2 T static magnetic field did not affect the basal pattern of vascularization or chick embryo viability. Prostaglandin E1 and fetal calf serum elicited a strong angiogenic response in sham exposed eggs. This angiogenic response was significantly inhibited by 3 hr exposure to 0.2 T static magnetic field. These findings point to possible use of static magnetic field in inhibiting angiogenesis; this effect could be exploited for treatment of cancer and other diseases where excessive angiogenesis is involved.     

Chronic ethanol exposure in the embryonic chick heart: effect on myocardial function and structure

To investigate the effect of chronic ethanol exposure on the embryonic chick heart, chick embryos were exposed daily to one of seven graded doses of ethanol or to saline only (shams) from 0 to 96 hr of incubation. One hour before and after exposure at 72 hr, and 1 hr before and after exposure at 96 hr, embryos were analyzed for changes in heart function, embryo tissue ethanol content, occurrence of anomalies, and embryo weights. At both 71 and 73 hr of incubation (during cardiogenesis), when compared to shams, heart rate (HR) in embryos receiving ethanol doses greater than 0.0375 ml increased significantly (P less than .05) with commensurate increases in injected ethanol. Additionally, at 73 hr, depressed cardiac contractility, measured as shortening fraction, was noted at doses greater than or equal to .0375 when compared to shams. While slight increases in shortening fraction (SF) across dose were noted at 95 and 97 hr, only random doses were statistically significant from shams, with no specific trend in either HR or SF at this postcardiogenesis stage. Within each time group, gas chromatography analysis of embryo tissue ethanol content demonstrated a linear relationship between dose injected and tissue ethanol content retrieved. With increasing dose and stage, viability decreased. Weights of ethanol-injected embryos were not significantly different from shams within each time group. Our studies of the response of the embryonic chick heart to ethanol indicate both dose and stage susceptibility, with greater susceptibility to ethanol injury during active cardiogenesis.     

Absence of O6-alkylguanine-DNA alkyltransferase induction in chick embryo liver and brain following X-irradiation or treatment with bleomycin.

1. The presence of O6-alkylguanine-DNA alkyltransferase (AT) in liver and brain of chick embryos, chicks and hens was demonstrated. An induction of AT activity has only been found in the liver of chicks and hens 48 hr after X-irradiation (5, 10 or 12 Gy). 2. The administration of methylmethanesulphonate to the chick embryo resulted 3-24 hr later in strong inhibition of AT activity accompanied by DNA alkylation. Under the same conditions, X-irradiation, dimethylnitrosamine and bleomycin exhibited no effect. 3. The results are compared with those obtained in mouse, rat and human foetal tissues.     

Development of the mandibular skeleton in the embryonic chick as evaluated using the DNA-inhibiting agent 5-fluoro-2'-deoxyuridine

Mandibular development was examined in embryonic chicks following administration of 5-fluoro-2'-deoxyuridine (FUDR, 0.001-1.0 microgram/egg), an inhibitor of both DNA synthesis and of cell division. FUDR was injected in ovo at one of three developmental stages corresponding to 1) the migration of mandible-destined, midbrain-level neural crest cells (Hamburger and Hamilton [H.H.] stage 10); 2) midway through the epithelial-mesenchymal interaction required to initiate mandibular osteogenesis (H.H. stage 22), which is also after the epithelial-neural crest cell interaction required for the initiation of chondrogenesis in Meckel's cartilage; and 3) when prechondroblasts of Meckel's cartilage are beginning to differentiate (H.H. stage 25). Micromelia was induced following the administration of FUDR at either H.H. stages 22 or 25 but not when FUDR was given at H.H. stage 10. Although the micromelic mandibles were shorter than normal, Meckel's cartilage and the mandibular membrane bones both differentiated and grew along the full proximodistal length of the shortened mandibles. In contrast to the situation previously described by Ferguson for alligator embryos exposed to FUDR, the migration of neural crest cells in the embryonic chick was not inhibited by FUDR. In contrast to the situation previously described for rat embryos exposed to FUDR, differentiation of Meckel's cartilage was not inhibited in embryonic chicks exposed to FUDR. Differentiation of the membrane bones was also normal following either in ovo administration of FUDR or when mandibular processes were maintained in FUDR in vitro. Therefore, FUDR does not produce micromelia in the embryonic chick by interfering with the epithelial-mesenchymal/neural crest cell interactions, which are prerequisites or differentiation of cartilage or bone, nor by inhibiting the differentiation of chondrogenic or osteogenic mesenchymal cells after completion of these tissue interactions. Neither did the growth-inhibiting action of FUDR result from an inhibition of growth of Meckel's cartilage during the several days following initial chondrogenic differentiation. Rather, subsequent growth of the entire mandibular process was delayed. This mechanism of action differs from that in the alligator embryo, in which FUDR inhibits mandibular growth by removing mandible-destined, migrating neural crest cells, and in the rat, in which FUDR inhibits the differentiation of Meckel's cartilage but catch-up growth restores growth of the mandible to normal.     

Absence of O6-alkylguanine-DNA alkyltransferase induction in chick embryo liver and brain following X-irradiation or treatment with bleomycin

1. The presence of O⁶-alkylguanine-DNA alkyltransferase (AT) in liver and brain of chick embryos, chicks and hens was demonstrated. An induction of AT activity has only been found in the liver of chicks and hens 48 hr after X-irradiation (5, 10 or 12 Gy).2. The administration of methylmethanesulphonate to the chick embryo resulted 3–24 hr later in strong inhibition of AT activity accompanied by DNA alkylation. Under the same conditions, X-irradiation, dimethylnitrosamine and bleomycin exhibited no effect.3. The results are compared with those obtained in mouse, rat and human foetal tissues.     

Affinity of intramitochondrial granules for ruthenium red accompanying induced cell death in chick embryos.

To test the hypothesis that ruthenium red binding of intramitochondrial granules might reflect an altered or pathological state of membranes associated with degeneration, embryos were treated with 6-AN to induce cell death in cartilaginous skeletons of chick embryos. Cervical cartilage from normal, 6-AN-treated and nicotinamide-alleviated 6-AN embryos was examined ultrastructurally for presence of IM RR-positive granules. Mitochondria of normal cervical chondroblasts which undergo normal phenotypic expression acquire RR-positive granules, although few mature cells are observed in young embryos. Necrotic chondroblasts, chondroblasts in various stages of degeneration, and proliferating chondrogenic cells of 6-AN-treated embryos all demonstrated induced RR-positive IM granules. Foci of degenerating chondroblasts, with mitochondria demonstrating RR granules, were observed infrequently in teratogen-alleviated tissue. The cytological features induced by 6-AN confirm its lethal effect and the degenerative effect on membranes presumably "unmasks" mitochondrial Ca-affinity sites which then become RR-positive. Cytochemical observations correspond with the biochemical and structural changes induced by 6-AN and confirm the hypothesis that RR-positive sites are the result of pathological changes.     

Studies on the cytosolic DNA of chick embryo fibroblasts and its uptake by recipient cultured cells

Cytosol and extruded DNA complexes from cultured chick embryo fibroblast cells have been separated by agarose gel chromatography at intervals after pulse labelling with [3H]thymidine. The proportion of the various cytosol components changed markedly with time: there was a lag period of 3 hr before the major labelled (5 X 10(5) dalton) DNA complex appeared in the cytosol, and a further lag period of 5 hr before it was extruded from the cell. Cultured chick embryo fibroblast, and rat spleen, cells rapidly and very efficiently import their own or each others cytosolic DNA complexes into their respective cytosol fractions: the material recovered from the cytosol of recipient cells is characteristic of the presented material. Homologous cytosolic DNA complex presented to chick embryo fibroblast cells also becomes associated with the nucleus. The rat at which this occurs is comparable with the rate of incorporation of [3H]thymidine into nuclear DNA.     

In vitro stimulation of cartilage in embryonic chick neural crest cells by products of rentinal pigmented epithelium.

The retinal pigmented epithelium of the chick embryo influences head neural crest mesenchymal cells to form the scleral cartilage of the eye. The possible role of extracellular matrix in this interaction was studied. Extracellular matrix was deposited on Millipore filters in vitro by pigmented epithelial cells which were then killed by distilled water lysis. When grown on the Millipore filters which had carried pigmented epithelium, clonal neural crest and periocular mesenchyme “target” cells formed cartilage in 61 of 155 experiments. Cartilage was not formed when the cells were grown on naked filters nor did gels of purified Type I and Type II collagen promote chondrogenesis. It is concluded that extracellular matrix deposited by the pigmented epithelium in vitro is a potent stimulus for the induction of chondrogenesis in competent mesenchyme, and that living pigmented epithelial cells need not be present for such induction.     

The enhancement of in vitro survival and chondrogenesis of limb bud cells by cartilage conditioned medium

Dissociated stage 21–28 chick embryo limb bud cells showed an increasing ability to produce cartilage colonies in vitro with in vivo maturation. In addition dissociated stage 21–28 chick embryo limb bud cells exposed to cartilage conditioned medium continuously or only for 48 hr prior to subculture showed an enhanced (as much as 15-fold) ability to form differentiated cartilage colonies. By this criterion, cells were more responsive to conditioned medium prior to stage 25. Conditioned medium from fibroblast cultures caused an inhibition of cartilage colony formation, suggesting that the effect is cell-type specific. Besides increasing cartilage colony formation by enhanced cell survival, the incorporation of S³⁵O₄ into isolated glycosaminoglycans is also stimulated when limb bud cells are exposed to cartilage conditioned medium. The results support a model for cell differentiation which involves the enhancement of a particular differentiated capacity by a diffusible cell-type-specific macromolecule.     

Ultrastructural reaction of the multipotential glia in the cerebellum of the rat after treatment with 6-aminonicotinamide]

6-Aminonicotinamide (6-AN), an antimetabolite of nicotinamide, damages the astrocytes and oligodendrocytes through a blockade of the pentose phosphate pathway. Both types of glia cells become hydropic. A third type of glia cell, described by VAUGHN and PETERS, the multipotential glia, is affected to a lesser extent. These cells phagocytize and form pseudopodia after treatment with 6-AN. Thus the multipotential glia cells are 'marked' by the action of 6-AN, since they are obviously less dependent on the pentose phosphate pathway in the carbohydrate metabolism.     

METABOLITES AND ENZYMES OF THE PENTOSE PHOSPHATE PATHWAY IN ISOLATED NERVE ENDINGS1

Abstract— The activities of each enzyme associated with the pentose phosphate pathway as well as the non-enzymatic intermediates in this pathway were measured in synaptosomes isolated from rat cerebral cortex. The specific activities of transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) were significantly lower in synaptosomes than cerebral cortex; however, the specific activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribosephosphate isomerase (EC 5.3.1.6) and ribulosephosphate epimerase (EC 5.1.3.1.) were comparable in homogenates of synaptosomal fractions and cerebral cortex. Concentrations of most intermediates of the pentose pathway were also similar in extracts of synaptosomes and brain homogenates. Six hours after treatment of rats with the nicotinamide analog, 6-aminonicotinamide (6-AN), 6-phosphogluconate levels in synaptosomes were increased 5-fold; however, glucose-6-phosphate levels remained unchanged. During a 30 min in uitro incubation 6-phosphogluconate levels increased approx 2-fold in synaptosomes obtained from 6-AN treated rats but did not change in synaptosomes from untreated rats. During the same period glucose-6-phosphate levels decreased in synaptosomes from both control and 6-AN treated rats. The conversion of both [1-¹⁴C]glucose and [6-¹⁴C]glucose to ¹⁴CO₂ was depressed in synaptosomes from 6-AN treated rats; however, the ratio of the two isotopes converted to ¹⁴CO₂ was essentially the same. It is concluded that the pentose phosphate pathway is active in nerve endings both in vivo and in vitro.