/ . Embryol. exp. Morph. Vol. 59, pp. 217-222, 1980
Printed in Great Britain © Company of Biologists Limited 1980
217
Impaired energy metabolism as an initial step in
the mechanism for 6-aminonicotinamide-induced
limb malformation
By YAL C. SHEFFIELD AND ROBERT E. SEEGMILLER 1
From the Department of Zoology, Brigham Young University, Provo
SUMMARY
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 10 /*g/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 chondrocytes 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
1
Author's address: 593 WIDB, Department of Zoology, Brigham Young University,
Provo, Utah 84602, U.S.A.
218
V. C. SHEFFIELD AND R. E. SEEGMILLER
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/*g/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 - K O H , 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 /tCi/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 (5 /tCi/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 r-test.
Inhibition of cartilage ATP by 6-AN
Table 1. Effect o/6-ANt
219
on ATP content
ATP (/*g//*g wet weight x 10~3)
Piiltnr
V^UHUL
time (hr)
2
6
12
18
•
t
6-AN treated
Control
52
5-4±0-8
2-8 ± 0 1 *
14
4-5 ±0-6
0-6±01*
28
1-1+0-1*
40 ±0-2
12
3-8±10
0-5±01*
Significantly different (P < 0-05) from control value.
Concentration ini the medium was 1(
Table 2. Effect of 6-AN-f on precursor
Culture
(hr)
DF
6
8
5
6
incorporation
CPM/ftg dry weight Avg. ± S.E.
f.
firnp
11111C
6-AN, %
control
Control
6-AN treated
6-AN, %
control
DF
95
60
42
30
30
30
32
24
121
118
113
16
13
34
95
82
84
11
14
26
[*5S]Sulfate
2
6
12
18
177 ±6
112 + 3
74±4
98±3
169 + 8
67 ±3*
31 ±2*
29 ±2*
[3H]Glucosamine
6
12
24
62±2
76+3
86±2
75 ±4*
90 ±5*
97 ±4*
[3H]Amino acids
6
12
24
157 + 4
158±3
114±3
149 ±5
130 + 2*
96 ±2*
[3H]Thymidine
6
12
24
107
23
378 ±10
404 ±18
73
8
325±16
236±18*
60
8
141 ±13*
234 ±21
* Significantly different (P < 0-05) from control value.
t Concentration in the medium was 10/tg/ml.
RESULTS
Level of ATP in response to 6-AN treatment (Table 1)
The level of ATP in control cartilage ranged from 3-8-5-4 x 10~3/*g//tg wet
weight. Treatment for 2 h with 10 /^g/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.//*g dry weight. The activity in controls decreased to 74 c.p.m.//ig
dry weight after 12 h of culture and remained at approximately this level for
the next 6 h.
220
V. C. SHEFFIELD AND R. E. SEEGMILLER
Treatment for 2 h with 10 ^g/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.//*g 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.//Ag dry weight. Treatment with 6-AN for 6 h 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.//tg 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 UDPN-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
221
Inhibition of cartilage ATP by 6-AN
100
80
60
8
40
20
6
8
10
12
14
16
18
Treatment time (hr)
Fig. .1. Relative to control limb cartilage, tissue levels of ATP (A
A) in cartilage
exposed to 6-aminonicotinamide (10/tg/ml of culture medium) were decieased by
approximately 50 % at 2 h and 90 % at 18 h of treatment. Utilization of 35SO4 =
(O——O) 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.
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.
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.
EMB 59
222
V. C. SHEFFIELD AND R. E. SEEGMILLER
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{Received 7 November 1979, revised 17 March 1980)