/ . Embryol. exp. Morph. Vol. 31, 2, pp. 305-312,1974
Printed in Great Britain
305
Normal incorporation rates
for precursors of collagen and mucopolysaccharide
during expression of micromelia induced by
6 -aminonicotinamide
By ROBERT E. SEEGMILLER 1 AND MEREDITH N. RUNNER
From the Department of Molecular, Cellular
and Developmental Biology
University of Colorado, Boulder, Colorado
SUMMARY
Further delineation of mechanisms by which 6-aminonicotinamide (6-AN) induces micromelia 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. Twenty-four 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 x 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 incorporation of proline suggests that, under the influence of 6-AN, tropocollagen was synthesized 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 hexosamine polysaccharides at normal rates.
INTRODUCTION
Reduced capability for limb chondrogenesis induced by 6-aminonicotinamide
(6-AN) in the embryonic chick has been biochemically (Overman, Seegmiller &
Runner, 1971) and ultrastructurally (Seegmiller, Overman & Runner, 1971)
related to the causal chain of teratogenic events leading to micromelia. Changes
1
Author's address: Department of Zoology, Brigham Young University, Provo, Utah
84601, U.S.A.
306
R. E. SEEGMILLER AND M. N. RUNNER
in chondrocytes and limits of protection with nicotinamide have in part been
confirmed by Caplan (1972). It is generally believed that the effects of 6-AN
are the consequence of the vitamin antagonist becoming incorporated into an
analogue of NAD. As such the false NAD has impaired capability to serve as a
coenzyme in at least some of the dehydrogenase reactions (Landauer, 1957;
Dietrich, Friedland & Kaplan, 1958; Verrusio, Pollard & Fraser, 1968;
Kohler, Barrach &Neubert, 1970; Landauer & Sopher, 1970). Herken, Lange &
Kolbe (1969) have pointed out that dehydrogenase reactions early in catabolism
of glucose are selectively inhibited by 6-AN. This has led Seegmiller, Overman &
Runner (1972) to suggest that lowered levels of high energy phosphate, resulting from defective dehydrogenase(s), could interfere with production of chondrogenic matrix in the chick limb during development of micromelia.
Overman, Seegmiller & Runner (1972) have reported that, in limbs and
cartilage rudiments of embryos treated with 6-AN, incorporation of 35S-sulfate
is inhibited while incorporation of amino acids is not. Significant experiments
would be to determine whether a teratogenic dose of 6-AN would differentially
(a) inhibit incorporation of precursors of chondroitin sulfate assembled prior
to sulfation, (b) inhibit incorporation of precursors of collagen, a primary
constituent of cartilage produced by chondrocytes, or (c) act as a cytotoxin.
In pursuit of these objectives chick embryos were treated with a teratogenic
dose of 6-AN at day 4 of incubation. Limbs were excised at day 5 and assayed
for capability to incorporate labeled precursors. This experimental design provided net rates of incorporation of sulfate, glucosamine, proline and thymidine
in isolated limb buds at a time when Seegmiller et al. (1972) showed that cells
were expressing the initial fine structural changes leading to micromelia.
Results from 5-day limb buds excised after 24 h exposure to 6-AN (a)
confirmed our previous reports of impaired capability for incorporation of
35
S-sulfate, i.e. sulfation was defective, (b) showed enhanced capability to
incorporate thymidine, i.e. 6-AN at this time was not cytotoxic and (c) showed
incorporation of proline and glucosamine that was similar to controls, i.e.
synthesis by chondrocytes was normal at a time when matrix was visibly deficient
(Seegmiller et al. 1972).
MATERIALS AND METHODS
Chick embryos (Hy-Line Poultry Farms) were incubated at 37 °C for 4 days
(to approximately stage 23, Hamburger & Hamilton, 1951). Ten micrograms of
6-AN (Sigma) were injected into the extra-embryonic coelom. Twenty-four
hours later (120 h of incubation) hind limbs were excised from both treated and
control embryos and were incubated for 1 h at 37 °C in 1 ml chick Ringer solution containing phosphate-bicarbonate buffer, 0-9 % glucose and 3 x 10~6 M
isotopically labeled precursor. The precursors used were 3H-methyl-thymidine
(17 Ci/mM), 3H-proline (500mCi/mM), 14C-glucosamine (40mCi/mM) and
35
S-Na2SO4 (65 Ci/mM, before adjusting for decay). Isotopes were purchased
Mechanism
of 6-AN teratogenesis
307
from New England Nuclear and Schwarz Bioresearch. The cpm per mole of
precursor in the medium enabled conversion of counts in limb extracts to moles
of precursor incorporated per hour per ju,g protein nitrogen. Following the
terminal exposure to label in vitro for 1 h, uptake of precursor by limbs was
stopped by 4 rinses with non-radioactive medium at 4 °C. The limbs were then
transferred in pairs to test tubes containing 0-5 ml glucose-saline at 0 °C and
were homogenized by sonic vibration. Centrifugation of homogenates at 4 °C,
after precipitation in 10 % trichloracetic acid (TCA) at 4 °C for 1-2 h, separated
acid-soluble and -insoluble fractions. Aliquots of the fractions were counted
using Nuclear Chicago Solubilizer and PPO and POPOP in toluene.
An aliquot of the TCA-soluble extract was counted in order to assess the
intracellular, unincorporated pool. Radioactivity of labeled precursor in the
TCA-insoluble fraction, determined after hydrolysis in lN-NaOH, provided a
measure for rate of incorporation. Protein nitrogen was determined by the
Folin phenol method (Lowry, Rosebrough, Farr & Randall, 1951) at a wavelength of 750 nm using a Zeiss spectrophotometer and crystalline bovine serum
albumen as a reference.
Incorporation of thymidine into bulk DNA was estimated, in the hot PCA
extract of the TCA-insoluble fraction, by the diphenylamine reaction (Burton,
1956). Digestion of homogenized limbs with DNase (Worthington Biochemical),
buffered at pH 5, prevented 90 to 95 % of counts from appearing in the PCA
extract. After liquid scintillation counting, specific activity was calculated as
moles of thymidine incorporated per hour per [ig DNA. Significance of the
differences in amounts of incorporated precursor was tested using the t test and
the non-parametric Mann-Whitney U test (Siegel, 1956). Table 1 shows the
mean values after combining the 37 to 46 determinations from three treatmentlabeling-extraction experiments.
RESULTS
Incorporation of precursors into mucopolysaccharide and collagen. Rear limbs
from embryos treated in ovo at day 4, excised 24 h later and labeled in vitro for
one hour incorporated proline and glucosamine into TCA-precipitable materials
at respective average rates of 299 x 10~12 and 29 x 10~12 moles/h//tg protein
nitrogen, i.e. 88 and 97 % of the control levels (Table 1). Sulfate was incorporated
into the TCA-insoluble fractions of treated limbs at 25 x 10~12 moles/h//*g
protein nitrogen which is 29 % of control value. This specific and drastic
reduction in rate of incorporation of 35S into the TCA-insoluble fraction was
accompanied by a less severe reduction of 50 % of 35S into the TCA-soluble
fraction. The effect of 6-AN on the amount of 35S in the TCA-soluble fraction
is particularly meaningful because the soluble fractions derived from other
labeled precursors, proline, glucosamine and thymidine, showed no significant
difference as a consequence of exposure to 6-AN.
Quantitation of precursor on the basis of micrograms of protein nitrogen was
308
R. E. SEEGMILLER AND M. N. RUNNER
Table 1. Effect of 6-AN on incorporation into the TCA precipitate
by day-5 limbs
Moles x 10~12 of precuisor/h//*g of protein
nitrogen
Precursor
Number of
determinations
Control,
avg. ± S.E.
37
339 ± 24
299 ±24
41
46
30±4
86±4
29 + 4
25±4
Prohne
Glucosamine
Sulfate
6-AN treated, 6-AN treated,
avg. ± S.E.
% control
Probability
0-23 N.S.
97
29
0-69 N.S.
0003*
N.S. The differences in incorporation of proline and glucosamine into TCA-insoluble
fraction are not significant (Mann-Whitney U and t tests, two-tailed).
* The difference in incorporation of sulfate into TCA-insoluble fraction is highly significant (Mann-Whitney U and t tests, two-tailed).
Table 2. Protein and DNA content of control and 6-AN treated limb buds
Assay
Control,
avg. ± S.E.
(/tg/limb)
6-AN treated,
avg. ± S.E.
(/tg/limb)
Treated,
control
Probability
(/ test)
57
24-2 ±0-7
23-8 ±0-7
98
0-5
24
9-8 ±0-7
10-2 ±0-7
104
0-5
Number of
determinations
Protein nitrogen
(Lowry Folin
phenol)
DNA (Burton
diphenylamine)
/o
an effort to obviate variability resulting from differences in size of excised limbs.
The average amount of protein per excised limb bud was in fact lower in treated
limbs (Table 2), although the difference in protein content was not statistically
significant.
Incorporation of thymidine into DNA. Treatment with 6-AN at 96 h had no
detectable effect on bulk DNA content of 120-h rear limbs (Table 2). The rate
of incorporation of thymidine in the TCA-insoluble fraction, by rear limbs
treated with 6-AN, was 60% above the average control level of 124 x 10~12
moles/h//tg protein nitrogen (Table 3). The finding of an increased rate of incorporation following exposure to 6-AN was further substantiated by determining
the specific activity of labeled thymidine in the DNA extracted by hot PCA
from the TCA-insoluble fraction (Table 4).
The data-in Table 4 show that after treatment with 6-AN the rate of incorporation of thymidine into extracted DNA increased to 58 % above the control
value of 473 x 10~12 moles/h//4g DNA. The observation that treatment with
6-AN at 96 h resulted in an increased rate of incorporation of thymidine at 120 h
Mechanism of 6-AN teratogenesis
309
Table 3. Effect of 6-AN on incorporation of thymidine into the TCA-insoluble
fraction by day-5 limbs {moles x 10~x2 of thymidine \h\'jug of protein nitrogen)
Control
6-AN treated
Number of
determinations
Avg. ± S.E.
Treated,
% control
Probability
(7 test)
24
24
124 ±23
198±23
—
160
—
0004*
* The difference in incorporation of thymidine into TCA-insoluble fraction is highly
significant (Mann-Whitney U and / tests, two-tailed).
Table 4. Effect of 6-AN on incorporation of thymidine into DNA extracted from
the TCA-insoluble fraction {moles x 10~12 of thymidine I h/jag of DNA)
Control
6-AN-treated
Number of
determinations
Avg. ± S.E.
Treated,
% control
Probability
(t test)
12
12
473 ±49
747 ±49
—
158
—
0001*
* The difference in incorporation of thymidine into PCA-extractable, DNase-digestible
DNA is highly significant according to both Mann-Whitney U and t tests (one-tailed).
was further validated by the finding that more than 90 % of radioactivity was
removed from limb buds after digestion with deoxyribonuclease. Thus, measuring both specific activity of label in the DNA extract and moles of thymidine
per jug protein nitrogen, the rate of incorporation of thymidine by limbs
treated with 6-AN was found to be about 60 % greater than the rate of incorporation by control limb buds.
DISCUSSION
6-aminonicotinamide has been shown to modify the structural relationships
of central cells of the chick limb bud (Seegmiller et al. 1971). These central cells
are destined to become chondrogenic cells and to produce chondroitin sulfate
as a major extracellular product. The observations reported on fractions extracted from isolated limb buds characterize the 'average' cell of the 5-day
limb bud. The mechanism by which 6-AN causes a deficiency of cartilage
matrix and produces micromelia has been more precisely established.
The normal to above-normal rates of incorporation of proline, glucosamine
and thymidine at day 5 (Tables 1 and 3) indicate integrity of the isolated 5-day
limb bud and preclude the possibility that, at the time studied, early teratogenic
effects of 6-AN result from cytotoxicity of the average cell within the limb bud.
The observed normal rate of incorporation of glucosamine by limbs subjected
to 6-AN suggests but does not prove a normal rate of synthesis of mucopoly-
310
R. E. SEEGMILLER AND M. N. RUNNER
saccharide. Either labeled hexosamine is attached at a normal rate as synthesis
of the core-protein proceeds, or mucopolysaccharide synthesis is inhibited but
labeled, exogenous hexosamine is more readily available in the 6-AN-treated
limbs. Availability of hexosamine as compensatory for inhibition of net synthesis of mucopolysaccharide can be explained if permeability to transport of
hexosamine increased and/or endogenous sources of hexosamine diminished.
Although altered availability must realistically be considered, the probability of
precise compensation by altering pool and/or transport to the equivalent of
control rates of incorporation seems improbable. The first alternative seems
more reasonable, viz. that, under the influence of 6-AN, mucopolysaccharide
is synthesized at an approximately normal rate. This apparent unrestricted
assembly of mucopolysaccharide in the presence of 6-AN reinforces a previous
report (Overman et ah 1972) that amino acid incorporation, and presumably
chondroitin core-protein synthesis, continues under the influence of 6-AN.
The incorporation rate of 35S-Na2SO4 in limbs treated with 6-AN was seen
(Table 1) to be 29 % of the rate of incorporation into control limbs, i.e. a reduction of 71 %. Two concomitant observations assist interpretation of possible
actions of 6-AN. First, the rates of incorporation of the precursors proline and
glucosamine were unaltered by 6-AN, and second, the quantity of 35S in the
TCA-soluble fraction from treated limbs was lowered, albeit less drastically
than the incorporation into the macromolecules precipitated by TCA. The
normal rates of incorporation of proline and glucosamine demonstrate specificity of effect of 6-AN upon 35S incorporation. The lowered level of counts of
35
S in the TCA-soluble fraction could indicate low rate of transport into cells
or high rate of incorporation of intracellular 35S, or both. That the rate of
incorporation was lowered, not raised, shows that the low value for 35S in the
soluble fraction is a consequence of lowered rate of transport. Since accumulation of 35S in the TCA-soluble fraction was depressed less than the 35S in
macromolecules, the observations suggest that both the source of intracellular
supply (transport) and the rate of incorporation of available 35S were inhibited
by 6-AN. Having uncoupled this apparent dual effect of 6-AN causing reduced
availability inside the cell and causing lowered rate of incorporation of available
35
S-Na2SO4, several lines of investigation are opened for subsequent study.
Overman et ah 1972 reported inhibition of incorporation of sulfate by limbs
taken from chick embryos that were both treated and labeled in ovo. This finding
has now been confirmed with the isolated 5-day limb. Dorfman and co-workers
(Perlman, Telser & Dorfman, 1964; Horowitz & Dorfman, 1968) have reported
that, after hexosamines have been added one at a time during assembly of the
core-protein, sulfation of chondroitin occurs at the Golgi site. The finding
reported here that, concomitant with inhibition of sulfate, limb buds exposed to
6-AN incorporate normal amounts of glucosamine, is particularly significant.
Subnormal incorporation of sulfate concomitant with a normal rate of incorporation of glucosamine indicates that, under the influence of 6-AN, chondroitin
Mechanism of 6-AN teratogenesis
311
synthesis proceeds past the addition of hexose sugars to form mucopolysaccharide but is stopped before the usual quantity of sulfate is added. The
consequence is subnormal accumulation of sulfated mucopolysaccharide in the
matrix.
The relatively normal rate of labeling with proline indicates that, 24 h after
administering 6-AN, limb cells are synthesizing collagen. This protein, like the
core-protein for chondroitin, is a major component of intercellular matrix and
is also produced by chondrocytes. Incorporation of proline at a control rate
reinforces the interpretation that under the influence of 6-AN matrix proteins
continue to be synthesized but are not exported.
Previous fine-structural studies (Seegmiller et al. 1972) gave visual evidence
for failure of export of precursors of extracellular matrix by the Golgi apparatus
and for accumulation of electron-dense material in the unusually enlarged
cisternae of the rough endoplasmic reticulum. The observation that protein
synthesis appears to continue under the influence of 6-AN supports an earlier
suggestion that the electron-dense material in the rough endoplasmic reticulum
may indicate a typical intracellular accumulation of proteinaceous precursors
of mucopolysaccharides and collagen.
The meaning of a 60 % increase in rate of incorporation of thymidine awaits
additional studies. The data are consistent with one or all of the following
possibilities: (a) a tendency for limb cells to synchronously synthesize DNA
following release from a transitory block by 6-AN, (b) an increase in availability
(uptake and incorporation) of exogenous thymidine and (c) a tendency for limb
cells to de-emphasize special synthesis-for-export and to reinstitute synthesisfor-proliferation. The augmented rate of incorporation of thymidine seen here
correlates with the transitory, heterodisperse nuclear chromatin reported
earlier (Seegmiller et al. 1972) in nuclei of 6-AN-treated chondrocytes at the
same age, day 5. The possibility that the observed rates of incorporation of
thymidine at day 5, 24 h after administering 6-AN, may be a transitory compensation is being investigated.
The mechanism by which 6-AN induces micromelia in the 4- to 5-day chick
embryo, as revealed at 24 h after treatment, includes (1) integrity of synthetic
capabilities of isolated limb buds, (2) normal rates of incorporation of precursors to synthesis of collagen and mucopolysaccharide, (3) a deficient amount of
extracellular chondrogenic matrix, and (4) intracellular accumulation of unfinished products. The hypothesis that 6-AN interferes with specific NADdependent dehydrogenase reactions, which in turn accounts for subnormal
sulfation of mucopolysaccharide and for failure of export of proteinaceous
products, can now be explored at more precisely defined loci, viz. intracellular
accumulation of Na2SO4 and its assembly into mucopolysaccharide.
EM B 31
312
R. E. SEEGMILLER AND M. N. R U N N E R
This work was supported by grants from The National Institutes of Health, National
Institute of Child Health and Human Development (HD 02282) and from the National
Science Foundation (GB 14662), and by a fellowship from the Pharmaceutical Manufacturers
Association Foundation. Special thanks are extended to Mrs Karen Howard and Miss Susan
McAfterty for technical assistance.
REFERENCES
K. (1956). A study of the conditions and mechanism of the diphenylamine reaction
for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-322.
CAPLAN, A. I. (1972). The site and sequence of action of 6-amino-nicotinamide in causing
bone malformation of embryonic chick limb and its relationship to normal development.
Devi Biol 28, 71-83.
DIETRICH, L. S., FRIEDLAND, I. M. & KAPLAN, L. A. (1958). Pyridine nucleotide metabolism:
mechanism of action of the niacin antagonist, 6-aminonicotinamide. /. biol. Chem. 233,
964-968.
HAMBURGER, V. & HAMILTON, H. (1951). A series of normal stages in the development of the
chick embryo. /. Morphol. 88, 49-92.
HERKEN, H., LANGE, K. & KOLBE, H. (1969). Brain disorders induced by pharmacological
blockade of the pentose phosphate pathway. Biochem. biophys. Res. Comm. 36, 93-100.
HOROWITZ, A. L. & DORFMAN, A. (1968). Subcellular sites for synthesis of chondromucoprotein of cartilage. /. Cell Biol. 38, 358-368.
KOHLER, E., BARRACH, H. & NEUBERT, D. (1970). Inhibition of NADP dependent oxidoreductase by the 6-aminonicotinamide analogue of NADP. FEBS Lett. 6, 225-228.
LANDAUER, W. (1957). Niacin antagonists and chick development. /. exp. Zool. 136, 509-530.
LANDAUER, W. & SOPHER, D. (1970). Succinate, glycerophosphate and ascorbate as sources
of cellular energy and as antiteratogens. /. Embryol. exp. Morph. 24, 187-202.
LOWRY, O. H., ROSEBROUGH, J. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with folin phenol reagent. /. biol. Chem. 193, 265-275.
OVERMAN, D. O., SEEGMILLER, R. E. & RUNNER, M. N. (1971). Biochemical processes in
micromelia of the chick induced by 6-aminonicotinamide. Teratology 4, 237-238.
OVERMAN, D. O., SEEGMILLER, R. E. & RUNNER, M. N. (1972). Coenzyme competition and
precursor specificity during teratogenesis induced by 6-aminonicotinamide. Devi Biol. 28,
573-582.
PERLMAN, R. L., TELSER, A. & DORFMAN, A. (1964). The biosynthesis of chondroitin sulfate
by a cell free preparation. /. biol. Chem. 239, 3623-3629.
SEEGMILLER, R. E., OVERMAN, D. O. & RUNNER, M. N. (1971). Cartilage differentiation of
micromelia induced in the chick by 6-amino-nicotinamide: light and electron microscopy.
Teratology 4, 241.
SEEGMILLER, R. E., OVERMAN, D. O. & RUNNER, M. N. (1972). Histological and fine structural changes during chondrogenesis in micromelia induced by 6-aminonicotinamide.
Devi Biol. 28, 555-574.
SIEGEL, S. (1956). Nonparametric statistics for the Behavioral Sciences. New York: McGrawHill Book Co.
VERRUSIO, A. C, POLLARD, D. R. & FRASER, F. C. (1968). A cytoplasmically transmitted
diet-dependent differences in response to the teratogenic effects of 6-aminonicotinamide.
Science, N.Y. 160, 206-207.
BURTON,
{Received 10 May 1973)