ABSTRACT
The analogue and antagonist of nicotinamide, 6-aminonicotinamide (6-AN), impairs cartilage formation and results in shortening of the limbs when administered to chick embryos. Studies have shown that 6-AN forms an abnormal NAD analogue which inhibits the activity of NAD-dependent enzymes associated with production of ATP. To determine if an effect on ATP synthesis might be associated with the mechanism of teratogenesis in the chick embryo, ATP levels of cartilage from day-8 chick embryos treated in vitro were assayed in relation to biosynthesis of protein, DNA and chondroitin sulfate. Incorporation of 35SO4= was inhibited by 6 h of treatment with 10μg/ml of 6-AN, whereas incorporation of [3H]thymidine and [3H]amino acid was not inhibited until 12 h. Incorporation of [3H]- glucosamine was increased at all treatment times. A decrease in the level of ATP preceded any detectable inhibition of precursor incorporation. These results are consistent with the hypothesis that 6-AN inhibits chondroitin sulfate synthesis through a reduction in the level of ATP in chondrocytes.
INTRODUCTION
One of the most noticeable effects produced by the nicotinamide analogue 6-aminonicotinamide (6-AN) in the chick embryo is micromelia (Landauer, 1957). The deformity results from inhibition of matrix production by chondro-cytes of the developing endochondral skeleton (Seegmiller, Overman & Runner, 1972; Overman, Seegmiller & Runner, 1972; Seegmiller & Runner, 1974; Seegmiller, 1977; Seegmiller, Horwitz & Dorfman, 1980). Although the sequence of molecular events leading to micromelia is not fully understood, it has been reported that 6-AN competes with nicotinamide in forming NAD cofactors. By isolating the dinucleotide analogues from 6-AN treated mice, Dietrich, Friedland & Kaplan (1958) demonstrated that 6-AN competes with nicotinamide in the formation of NAD(P). The 6-AN analogues of NAD are incapable of functioning in NAD(P)-dependent enzymic reactions, including the dehydrogenase reactions of glucose catabolism which eventually lead to the synthesis of ATP (Coper & Neubert, 1964; Dietrich et al. 1958; Lange & Proft, 1970). In addition to isolating structurally altered cofactors, Dietrich et al. (1958) demonstrated that 6-ANAD decreases the levels of ATP and ADP while increasing the level of AMP. Ritter, Scott & Wilson (1975) provided further evidence that 6-AN inhibits ATP synthesis by demonstrating an immediate decrease in the level of ATP in whole rat embryos treated in utero with 6-AN.
We have proposed that chondrocytes of the developing limb, by virtue of being relatively anaerobic and less efficient at synthesizing ATP than neighboring non-chondrogenic cells, are sensitive to 6-AN (Seegmiller et al. 1972; Seegmiller, 1977). When treated, chondrocytes presumably do not produce sufficient ATP to continue synthesis of cartilage matrix and maintenance of cell viability. The present study was undertaken to determine, in isolated limb cartilage, the effect of 6-AN on ATP production in relation to its effect on incorporation of molecular precursors of DNA, protein and chondroitin sulfate.
MATERIALS AND METHODS
Whole tibias were removed from day-8 chick embryos of the Babcock strain (Rigtrup Hatchery, Spanish Fork, Utah) and cultured in groups of five in 2 ml Waymouth’s medium (Grand Island Biological). At time zero 6-AN (Sigma) was added to the treatment group at a concentration of 10 μg/ml. Experimental and control groups were incubated for 2-24 h in 5 % CO2 in air at 38 °C.
Tibias assayed for ATP were quick-frozen, weighed and homogenized in 0·5 M perchloric acid. The mixture was neutralized with 1·0 M-KOH, centrifuged, and an aliquot of the supernatant was combined with luciferin-luciferase (Du Pont) for determination of ATP content based on wet weight.
The effect of 6-AN on sulfate incorporation was monitored by terminally labeling treated and control tibias for 2 h in the presence of 10 μCi/ml of Na235SO4 (700 mCi/mM). Other radioisotopes, viz. D[63H]glucosamine hydrochloride (20·7 Ci/mM), [3H]methyl thymidine (6·7 Ci/mM) and a mixture of [3H]amino acids, were separately added to the media (5μCi/ml) to monitor general biosynthetic activity. All labeled compounds were obtained from New England Nuclear. Following the labeling period, the tibias were rinsed in saline, fixed in acetone, desiccated, weighed, solubilized with Protosol (New England Nuclear) and counted in a liquid scintillation system. Greater than 90 percent of the sulfate and glucosamine counts were cetylpyridinium chloride precipitable and chondroitinase ABC (Miles) digestible, according to the method of Yamagata, Saito, Habuchi & Sazuki (1968). Similarly, greater than 90 % of thymidine and amino acid counts remaining in the tissue after processing were TCA precipitable and DNAse or protease digestible. Total incorporation was based on dry weight. Differences between experimental and control groups were tested for significance by the t-test.
RESULTS
Level of ATP in response to 6-AN treatment (Table 1)
The level of ATP in control cartilage ranged from 3·8–5·4x 10−3μg/μg wet weight. Treatment for 2h with 10/6g/ml of 6-AN decreased the level of ATP to approximately 50 % of control. Treatment for longer periods further decreased the level of ATP. The inhibitory effect of 6-AN was statistically significant for all treatment periods.
Macromolecular synthesis following treatment with 6-AN (Table 2)
The level of 35SO4= incorporated by control cartilage after 2 h of culture was 177 c.p.m./μg dry weight. The activity in controls decreased to 74 c.p.m./μg dry weight after 12 h of culture and remained at approximately this level for the next 6 h.
Treatment for 2 h with 10 μg/ml of 6-AN did not significantly affect sulfate utilization. At 6 h, incorporation of sulfate was significantly decreased to 60 % of control. Treatment for longer periods further decreased sulfate incorporation such that by 18 h treated groups were 30 % of control activity.
Incorporation of [3H]glucosamine into untreated cartilage ranged from 62-86 c.p.m./μg dry weight. The level of [3H]glucosamine incorporated by 6-AN treated cartilage was significantly increased at treatment times 6-24 h. The average increase was 17 %.
Incorporation of [3H]amino acids into untreated cartilage ranged from 114-157 c.p.m./μg dry weight. Treatment with 6-AN for 6h did not significantly affect the utilization of amino acids. Treatment for 12 and 24 h significantly inhibited the utilization of amino acids, but not below 80 % of control.
Control levels for [3H]thymidine ranged from 234-378 c.p.m./μg dry weight. Treatment with 6-AN for 6 h did not inhibit utilization of thymidine. Treatment for 12 and 24 h significantly inhibited incorporation of thymidine to 73 and 60 % of control, respectively.
DISCUSSION
The present study demonstrates that within 2 h 6-AN decreases ATP levels in isolated cartilage, the target tissue for 6-AN teratogenesis (Table 1). The prompt inhibitory effect on ATP preceded any significant effect on chondroitin sulfate, protein, or DNA synthesis, as indicated by normal levels of incorporation of sulfate at 2 h and of amino acids and thymidine at 6 h after treatment (Table 2). Normal synthetic rates for protein and DNA at 6 h suggest that the immediate decrease in cellular levels of ATP were not due to generalized cell death.
We have reported that utilization of sulfate by 6-AN-treated cartilage is inhibited (Seegmiller et al. 1972; Overman et al. 1972; Seegmiller & Runner, 1974; Seegmiller, 1977). The decreased rate of sulfate incorporation by treated cartilage is taken as a measure of the rate of synthesis of chondroitin sulfate (Seegmiller et al. 1980). The effect on synthesis of chondroitin sulfate, a major constituent of the extracellular matrix of cartilage, apparently is due to decreased synthesis of the glycosaminoglycan side chains of the proteoglycan. The enhanced utilization of [3H]glucosamine that coincided with the inhibitory effect on sulfate incorporation at 6 h may be due to pool size effects of UDP-N-acetylhexosamine (Seegmiller et al. 1980).
Since the inhibitory effect on ATP preceded any detectable effect on chondroitin sulfate synthesis, it is suggested that ATP levels were decreased below that required to sustain tissue-specific synthesis of chondroitin sulfate. As a possible mechanism, 6-AN may decrease the level of nucleotide sugar available for synthesis of chondroitin sulfate chains by lowering the level of ATP and other nucleotide triphosphates available for production of nucleotide sugars. In other words, since the synthesis of nucleotide sugars from glucose requires both ATP and UTP, nucleotide sugar production may be promptly inhibited by a decrease in nucleotide triphosphate pools.
An ATP mediated mechanism for 6-AN teratogenesis may explain the differential effect of 6-AN on cartilage, as compared with other tissues. Cartilage exists as an avascular tissue under conditions of reduced oxygen tension (Pawlek, 1969) and thereby resorts to the less efficient anaerobic synthesis of ATP (Whitehead & Weidmann, 1959). A decrease in tissue level of ATP following treatment with 6-AN, therefore may preferentially affect cartilage.
In summary, this study demonstrates that inhibition of ATP synthesis by 6-AN is both prompt and marked in isolated chick cartilage, the target tissue associated with 6-AN induced micromelia. The decrease in ATP levels in treated cartilage is temporally associated with a decrease in chondroitin sulfate biosynthesis (Fig. 1). Although a cause-and-effect relationship between levels of ATP and inhibition of sulfate incorporation has yet to be determined, the results of the present study are consistent with such a relationship and demonstrate a direct and prompt effect of 6-AN on cartilage levels of ATP.
Relative to control limb cartilage, tissue levels of ATP (Δ——Δ) in cartilage exposed to 6-aminonicotinamide (10μg/ml of culture medium) were decieased by approximately 50 % at 2 h and 90 % at 18 h of treatment. Utilization of 35SO4 = (○——○) by cultured tibias was not inhibited by 6-AN at 2 h but was inhibited 40 % at 6 h and 70 % at 18 h of treatment.
Relative to control limb cartilage, tissue levels of ATP (Δ——Δ) in cartilage exposed to 6-aminonicotinamide (10μg/ml of culture medium) were decieased by approximately 50 % at 2 h and 90 % at 18 h of treatment. Utilization of 35SO4 = (○——○) by cultured tibias was not inhibited by 6-AN at 2 h but was inhibited 40 % at 6 h and 70 % at 18 h of treatment.
Acknowledgement
This work was supported by grants from the March of Dimes Birth Defects Foundation and the Pharmaceutical Manufactures Association Foundation. Appreciation is extended to Dr William Bradshaw for helpful suggestions offered during the course of this work.