[CANCER RESEARCH 34, 1381-1384, June 1974]
ß-Aminoisobutyric Acid, a New Probe for the Metabolism of DNA
and RNA in Normal and Tumorous Tissue
Henrik Rist Nielsen,1-2 Knud-Erik Sj^lin,1 Kaare Nyholm,1
B. S. Baliga, Rosemary Wong, and Ernest Borek
Departments of Clinical Chemistry, Pathology, and Surgery, Sundby Hospital, Copenhagen, Denmark [H. R. N., K-E. S., K. N.}, and Department of
Microbiology, University of Colorado Medical Center, Denver, Colorado 80220 [fi. S. B., R. W., E. B.]
SUMMARY
i8-Aminoisobutyric acid, a catabolite of thymine metabo
lism, originates from the thymine of both DNA and transfer
RNA. The /3-aminoisobutyric acid originating from the
thymine of DNA and transfer RNA can be distinguished by
specific labeling of the former with 14Cformate and of the
latter with methionine-methyl-3H. Therefore, it can serve as
a probe for the metabolism of each macromolecule. In an
animal with a rapidly growing tumor (Novikoff hepatoma),
the excretion of 0-aminoisobutyric acid diminishes, proba
bly because of the salvage of thymine and its reutilization
for DNA synthesis. Such a salvage path is suggested by the
increased presence of tritium-labeled thymine in the DNA
of the tumor.
AIB8 is a catabolic product that is normally excreted in
low levels in human urine (1), and this excretion is elevated
in many patients with urothelial tumors (9). Until recently,
the origin of this metabolic product was thought to be the
thymine of DNA. However, since thymine is present in low
levels in tRNA as well, it seemed of interest to ascertain
whether AIB may also stem from this source. In a
'Supported
by Grant
CA-14047-02
from the NIH,
Institute, and by the Danish Cancer Society.
"Supported by Grant j.nr. 512-2115 and -2125.
3The abbreviation used is: AIB, i8-aminoisobutyric
Received January
14, 1974; accepted
National
Cancer
acid.
March 4, 1974.
cooperative study in our 2 laboratories, it was determined
that the urinary AIB indeed has a dual origin in the normal
rat (8). Such a demonstration could be accomplished by
taking advantage of the known difference in the pathway of
synthesis of the thymine of DNA and of tRNA. The former
is synthesized from the 1-carbon pool, with formyltetrahydrofolic acid as the intermediate donor (4). This thymine
can, therefore, be labeled by formate-14C. On the other
hand, it was shown by Mandel and Borek (5) that the
thymine of tRNA is synthesized by the direct addition of an
intact methyl group to uracil which had been previously
incorporated into the macromolecule. The methyl donor in
this reaction is 5-adenosylmethionine (3), and the methyl
group of the thymine of tRNA can, therefore, be readily
labeled with tritium. If the tritiated methyl group should
enter the 1-carbon pool, most of its tritium would be ex
changed and diluted.
With the availability of such specific markers for DNA
and tRNA, a number of hitherto moot questions in the
metabolism of these macromolecules can be approached. In
this communication we report that AIB is derived, in part,
from the thymine of tRNA in normal rats and rats bearing
slow-growing Morris hepatomas. However, in rats with the
rapidly growing Novikoff hepatoma, excretion of AIB is
lower than in the animal prior to the tumor implant. This
finding confirms an observation made in vitro by Ferdinandus et al. (2) in their studies of enzyme activities in tumor
tissues. They had reported a marked decline in the degrada-
Table 1
Total excretion of AIB and its dual origin in normal and Novikoff hepatoma-bearing
rats
The mixture of iostopes was injected i.p. on Day 0 as described in "Materials and Methods." On Day 5, all animals
received
homogenized Novikoff hepatoma implants. Collection of the urine was resumed on Day 8; Rat D died on Day 10. AIB is expressed
as ¿¿moles
per 24 hr. The 14C and 3H counts are expressed as counts per 20 min in the 24-hr pool of AIB.
Rat A
Day
2 AIB
RatC
Rat B
2AIB
•¿H
Rat D
2 AIB
2 AIB
14C
12348910110.801.251.151.451.450.300.700.801505185135956351230152105055409
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.4030
.5515
.8060
.3035
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JUNE
1974
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1381
H. R. Nielsen et al.
tion of thymine via the dihydrothymine and AI B pathway in
rapidly growing tumors. Weber has suggested a salvage
pathway for thymine in rapidly growing tumors. Our
findings of the diminished excretion of AI B in rats bearing
Novikoff hepatoma confirm this salvage pathway in the
whole animals. However, the diminshed excretion of AI B
with both labels implies a salvage pathway of thymine from
both DNA and RNA with the possible reincorporation
of
the thymine into DNA. Such a pathway is implied by data
obtained from analysis of 5-methylcytosine and thymine in
isolated DNA of the tumor and compared with the same
components
isolated from the DNA of hepatectomized
normal livers.
MATERIALS
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For the experiments
with the Novikoff hepatoma, 4
female Holtzman rats weighing 150 to 200 g were used.
They received injections of 0.2 ml of a solution containing
100 ¿tCieach of L-methionine-methyl-3H
(6.4 Ci/mmole)
and formic acid-14C:sodium salt (59 mCi/mmole), and each
animal was placed in an individual metabolic cage. Urine
was collected at 24-hr intervals for 4 consecutive days. All
the animals received i.p. injections of 0.2 ml 0.9% NaCl
homogenate
of a 5- to 6-day-old Novikoff hepatoma,
consisting of l g tissue : 3 ml 0.9% NaCl solution. Three days
later or 7 days after the 1st radioactive injection, when the
tumor was growing rapidly, the animals were given injec
tions of identical amounts of the original radioactive
mixture. Urine was collected for 4 additional days. One of
the rats died 1 day prior to termination of the experiment.
The urine samples collected from normal animals during the
1st part of the experiment served as controls for the urine
collected later from the same animals with hepatoma.
For the experiments with Morris hepatomas, 7-monthold Buffalo rats that had been inoculated with Morris
hepatoma 8999 when they were 3 months old were made
available to us by Dr. George Weber. All tumors matured
approximately
2.5 months after the transplantation.
Four
animals were inoculated with 0.2 ml of a solution containing
67 /¿Cieach of L-methionine-methyl-3H
(9 Ci/mmole) and
formic acid-14C:sodium
salt (59 mCi/mmole).
Twentyfour-hr urine samples were collected for 4 successive days,
as in the case of rats bearing Novikoff hepatoma.
Urine samples collected from 4 normal rats of the same
age, bearing no tumor, served as controls for this experi
ment.
The urine samples in all cases were filtered and lyophilized, and AI B was isolated by thin-layer chromatography
on silica gel of the dinitrophenyl derivative with chloro
form : py ridine : glacial acetic acid ( 100:100:2) as the solvent
system. Spots containing dinitrophenyl-AIB
were scraped
off and counted in a Packard liquid scintillation counter in a
dioxane:naphthalene
medium and quantitated
spectrophotometrically
at 366 nm in a Beckman DU photometer.
All determinations
were done in duplicate samples (for
details, see Ref. 7).
For studies on the utilization of the thymine of RNA for
DNA synthesis, female Holtzman rats weighing 150 to 200
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CANCER
^
RESEARCH
VOL. 34
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AfB, a New Probe for Metabolism of DNA and RNA
Table 3
The origin of the methyl group in 5-methylcylosines and in some thymine in DNA
3\\ counts in
DNAanalyzed(Mg)No.
ofsamplesanalyzed
DayGuano-sineCyto-sineAdenineand5-methyl-cytosine
ThymineThymine/5-methyl-cytosmeAmountof
Novikoff hepatoma DNA
50
55
30
30
18
36
86
214
923
1178
558
836
498
561
302
472
0.54
0.47
0.54
0.56
580
584
583
565
Hepateclomized rat liver DNA
85
69
75
55
54
42
462
289
419
119
93
97
0.25
0.32
0.23
565
573
573
1For 2 days, rats were given injections of 400 ^Ci i -methionine-3H.
' For 3 days, rats were given injections of 400 pCi i-methionine-3H.
g were used. Novikoff hepatoma was grown in the omentum
of rats and the hepatoma was ready for collection 5 to 6
days following the injection of the tumor. Partial hepatectomy on normal rats was performed by Dr. Martinez of the
Pathology Department of the University of Colorado
Medical Center. i.-Methionine-3H, specific activity, 2.6
Ci/mmole, was obtained from Schwarz/Mann, Orangeburg, N. Y. 5-Methylcytosine and 6-methylaminopurine
were purchased from Calbiochem, San Diego, Calif.
Hepatectomized rats and animals that 3 days earlier were
given injections of Novikoff hepatoma, each received i.p.
injections of 400 ¿tCiof i.-methionine-3H. Three days later,
the rats were sacrificed and the livers and hepatoma were
removed and either used immediately or frozen at -15°.
Isolation and Purification of DNA. The fresh or frozen
tissue was homogenized in a Potter-Elvehjem homogenizer
with 5 volumes of buffer containing 0.15 M NaCl:0.015 M
sodium citrate:0.0015 M EDTA: 1% sodium lauryl sulfate.
Sodium chloride was then added to the homogenate to a
Final concentration of 1 M. Deproteinization was carried out
at room temperature by 2 extractions with equal amounts of
chloroform :isoamyl alcohol (24: 1). The DNA was subse
quently isolated according to the procedures of Marmur (6).
After the extractions with chloroform : isoamyl alcohol and
the addition of 2 volumes of 95% ethanol, the DNA was
spooled from the interface of the aqueous layer. It was
dissolved in 0.15 M NaCl and 0.015 M sodium citrate and
exposed to RNase (30 /¿g/ml)and Pronase (200 //g/ml)
successively for 1-hr periods at 37°.The solution was again
extracted with chloroform: isoamyl alcohol and the DNA
was spooled again from the aqueous layer after the addition
of 95% ethanol. Further purification of the DNA was
accomplished through 2 isopropyl alcohol precipitation
steps.
For the analysis of DNA, 1 to 4 mg of the various DNA
samples were dissolved in water, the absorbance was
determined in a Beckman spectrophotometer, and the
solutions were subjected to treatment with 0.5 N NaOH for
18 hr. The DNA was then collected by trichloroacetic acid
precipitation and washed with 95% ethanol and ether.
About 650 to 700 /¿gof DNA were hydrolyzed in
formic acid for 1 hr at 170°.The hydrolysate was dried over
KOH in a desiccator. The residue was dissolved in 30 n\ of
water and spotted on Whatman No. 1 paper strips (56 x 8
cm), along with authentic standards of 5-methylcytosine
and 6-methylaminopurine. The strips were then chromatographed in 1-butanol: water concentrated ammonium hy
droxide (258:39:1) for 34 hr. The UV-absorbing spots were
located, cut out, and eluted with water. Bray's solution was
then added to the eluates, and the 3H counts associated with
the various bases were determined in a Nuclear-Chicago
liquid scintillation counter. The recovery of DNA after
NaOH treatment and formic acid hydrolysis was about
90%.
RESULTS
In Table 1, the patterns of excretion of AI B by rats before
and after implantation of Novikoff hepatoma are presented.
The constancy of daily total AIB excretion for each rat is
notable. A decrease in the daily excretion is particularly
notable 6 to 7 days after the tumor implant. Of course, by
then the tumors are 10 to 15 g in size.
The pattern of excretion reveals an unexpected phenome
non. One day after the injection of formate-14C and
methionine-3H, there is a massive excretion of labeled newly
synthesized AIB. During the 2nd day there is a large
diminution in the excreted label but, in subsequent days, the
excretion diminished at a much slower rate. The only
plausible explanation of this phenomenon is that there may
be a hitherto unsuspected synthesis of thymine by direct
methylation of some uracil derivative and the resulting
thymine is then degraded to AIB and excreted. An enzyme
that methylates thiouracil is known to be present in the
kidney.
The continuing excretion of labeled AIB by the rats with
Morris hepatomas is markedly different (Table 2). The level
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1383
H. R. Nielsen et al.
of excretion is essentially constant and is similar in extent to
that in the normal animal.
If a salvage pathway of thymine in a rapidly growing
tumor is functioning in vivo, then this should be reflected by
the incorporation of thymine from tRNA which had been
labeled at the macromolecular level by tritium. In Table 3,
the results of experiments designed to test such a hypothesis
are presented. 5-Methylcytosine of the DNA is, as expected,
extensively labeled via the methionine-methyl-3H. It is well
established that this methyl group originates from Sadenosylmethionine which methylates some cytosine resi
dues in the preformed polymer. The label from S-adenosylmethionine in the thymine from the DNA of the tumor is
also very high. This was unexpected, since thymine of DNA
is synthesized, as stated earlier, from formate.
Extensive labeling although not as high as in the tumor,
was also observed in the thymine of the DNA in regenerat
ing liver.
Two different mechanisms may account for the high level
of tritium in thymine. Some of the 5-methylcytosines may
be deaminated at the macromolecular level, or the thymine
of tRNA that becomes available from the turnover of the
macromolecule finds its way into DNA.
With the available data, no resolution can be made as to
which of these 2 pathways contributes to the label in
thymine. The resolution of this problem will await more
sophisticated approaches.
ACKNOWLEDGMENTS
We are grateful to Dr. George Weber for fruitful discussions and for
providing the animals described in the text. We thank the Institute of
1384
Experimental Endocrinology, University of Copenhagen, for providing the
Packard liquid scintillation counter. The authors are also grateful to B.
Vingaard for her careful technical assistance.
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2. Ferdinandus, J. A., Morris, H. P.. and Weber, G. Behavior of
Opposing Pathways of Thymidine Utilization in Differentiating,
Regenerating, and Neoplastic Liver. Cancer Res., 31: 550 556, 1971.
3. Fleissner, E., and Borek, E. Studies on the Enzymatic Methylation of
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7. Nielsen. H. R. Variability in Urinary /3-Aminoisobutyric Acid and
Creatinine in a Human Control Group. Danish Med. Bull., 19:
144-147, 1972.
8. Nielsen, H. R., Borek, E., Sjolin, K. E., and Nyholm, K. Dual Origin
of ^-aminoisobutyric Acid, a Thymine Catabolite. Acta Pathol.
Microbiol. Scand., SO:687 688, 1972.
9. Nielsen. H. R., Nyholm, K., and Sj01in, K. E. /3-Aminoisobutyric Acid
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D'études
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CANCER RESEARCH VOL. 34
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1974 American Association for Cancer Research.
β-Aminoisobutyric Acid, a New Probe for the Metabolism of DNA
and RNA in Normal and Tumorous Tissue
Henrik Rist Nielsen, Knud-Erik Sjølin, Kaare Nyholm, et al.
Cancer Res 1974;34:1381-1384.
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