[CANCER RESEARCH 30, 1677—1680,June 1970]
Transfer RNA Patterns in Livers of Rats Fed Diets Containing
3'-. Methyl-4-dimethylaminoazobenzene'
Michele Goldman and A. Clark Griffin2
Department ofBiochemistry, The Universityof TexasM. D. Anderson Hospitaland Tumor Institute at Houston, Houston, Texas 77025
SUMMARY
isolated
Albino rats were fed diets containing the hepatocarcinogen
3'-methyl-4-dimethylaminoazobenzene
for approximately
8
weeks. At this time, tRNA was prepared from the livers of
these animals and control animals. The tRNA's were charged
with labeled amino acids and cochromatographed on reversed
phase columns. Some major differences were observed in the
lysyl, leucyl, phenylalanyl,
and tyrosyl patterns of the
dye-treated animals. The comparative patterns of the arginyl-,
seryl-, methionyl-, valyl-, histidyl-, and threonyl-tRNA's of
normal and precancerous livers were almost identical.
The interaction of carcinogens with proteins, nucleic acids,
and other components of cells has been observed by many
investigators. However, the significance of these interactions in
terms of the transformation of the normal cells to cancerous
cells has not been ascertained. Farber et al. (4) reported that
ethionine combines with liver RNA and to an even greater
extent with the tRNA's. It was shown further that this
interaction is mostly through the ethylation of 1 or more of
the bases and ribose. A loss of I of the leucine tRNA's was
observed in the livers of rats fed diets containing ethionine (I).
Many of the nitrosamines and nitrosamides are highly active
carcinogens and have been shown to react with nucleic acids
and other cellular components. These compounds result in the
alkylation of DNA and RNA with 7-methyl- or 7-ethyl guanine
as 1 of the major products formed. The significance of
alkylation of nucleic acids in the origin of cancer has been
reviewed by Magee et al. (8).
N-Hydroxylation
appears to be an essential step in the
metabolic
activation
of several of the important
carcinogens:
a cetylaminofluorene , acetylaminostilbene , aminoazo
car
cinogens, etc. (9, 10). The N-hydroxy metabolites are more
carcinogenic than the parent compounds and will react in vitro
with protein, RNA, and DNA. Acetylaminofluorene
or its
metabolic derivatives will react in vitro or in vivo with nucleic
acids, and acetylaminofluorene-tRNA
complexes have been
1Supported
by
benzoylated
diethylaminoethyl
cellulose
this agent. In view of the high resolution
of tRNA's
be attained with reversed phase chromatography
that may
(6, 16), the
present study was initiated to ascertain the effects of the
highly active hepatocarcinogen,
3'-Me-DAB,3
upon specific
tRNA's of the liver.
MATERIALS AND METHODS
Materials
INTRODUCTION
@
with
chromatography ( 15). Tada et al. ( 14) have reported the
formation of specific complexes between the carcinogen,
4-nitroquinoline-l-oxide,
and DNA or RNA in cells exposed to
American
Cancer
Society
Grant
P-SOlD
Robert A. Welch Foundation Grant G-035.
2American Cancer Society Professorof Biochemistry.
ReceivedOctober 2, 1969; accepted January 30, 1970.
and
the
14 C-Labeled
Amino
Acids.
Uniformly
labeled
‘
â€C̃-labeled
L
amino acids were purchased from the following sources: New
England
Nuclear
(Boston,
Mass.), methionine,
165
mCi/mmole; International Chemical and Nuclear Corp. (Irvine,
Calif.), isoleucine, 230 mCi/mmole, leucine, 240 mCi/mmole,
serine, 135 mCi/mmole, threonine, 132 mCi/mmole, valine,
190 mCi/mmole; Schwarz BioResearch Inc. (Orangeburg,
N. Y.), arginine, 236 mCi/mmole, glutamic acid, 155 mCi/
mmole,
lysine,
175 mCi/mmole,
phenylalanine,
468 mCi/
mmole, and tyrosine, 450 mCi/mmole.
3 H-Labeled
Amino
Acids.
Uniformly
labeled
H-labeled
L
amino acids were purchased from the following sources: New
England
Nuclear,
arginine,
2.5 Ci/mmole,
glutamic
acid,
1.0
Ci/mmole, histidine, 4.1 Ci/mmole, and serine, 3.73 Ci/mmole;
Schwarz BioResearch, Inc., phenylalanine, 1.65 Ci/mmole,
threonine, 225 mCi/mmole, and valine, 340 mCi/mmole;
Nuclear-Chicago
Corp.
(Des
Plaines,
Ill.),
tyrosine,
250
mCi/mmole.
Preparation of in Vitro Methylated tRNA's. Diazomethane
was used to methylate normal rat liver tRNA in vitro. The
methylated tRNA's were charged with ‘
4C- or 3H-labeled
amino acids in the usual manner as above (6) and cochroma
tographed
with similarly charged tRNA's from rat liver or
from the livers of the dye-fed animals. Several preliminary
studies were performed to determine the conditions for
methylation
of the tRNA fractions. Two ml of 0.1 M
dia.zomethane (in diethyl ether) were added to 5 mg liver
tRNA and 0.25 ml of a buffer of 0.05 M NaCl, 0.01 M
magnesium acetate, and 0.005 M EDTA, pH 7.0. This mixture
was shaken for 10 mm at 0°and allowed
to stand for 1 to 2
mm. The ether layer was removed and the methylated
was charged immediately.
JUNE 1970
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1970 American Association for Cancer Research.
tRNA
1677
Michele Goldman and A. Clark Griffin
The reversed phase column chromatography of Weiss and
Kelmers (16) was utilized in this study. The column prepara
tion, procedures, and assays were performed as described
previously (6). Combinations
of ‘
4C- and 3H-labeled
aminoacyl-tRNA's were applied to these columns, so that
direct comparisons could be made of the tRNA patterns of
liver and precancerous liver or the methylated tRNA on the
@
@
@
@
same column. These comparisonswere repeated for several
amino acids. The 4C- and 3H-labeledamino acids were
reversed in the
patterns could
chromatographic
aration (6) was
study.
above combinations to verify that similar
be obtained. The label did not change the
profiles. A normal rat liver synthetase prep
used for all of the aminoacylations in this
profiles
specific
@
@
@
I
——
.55.-S
rat liver and the
and
the
precancerous
1$,
I@
1@w *18
LEUCYI lRP@@
ITS_il.
IRNA
I
4
I
A@
___ 02
02
0I
01
057
050
tRNA's
were
prepared
from
L_
3@-*SD.8
I.@
80
)
57
70
,,@
-u*
,**
.55555 5 3@*-c188
*8*
80
110
‘@
I.*,
‘*1
g
TYROSYL II@M
I
I
@00
02
02
0I
050
--
050
01
025
50
70
90
TI.1(
NLN@ER
025
80
30
50
70
90
T1.$(
MM@R
80
Chart 1. Comparison of aminoacyl-tRNA patterns from livers of
normal rats and livers of rats fed diets containing 3'-Me-DAB.
a re
action mixture containing 5 to 7 mg normal liver tRNA, or
from tRNA from the livers of the dye@fedrats, 2.5 ml diethyl
aminoethyl cellulose-stripped synthetase protein, 0.017 mmole
ATP. 25 pCi ‘4C-or 160 @iCi3H-labeled amino acid, and
buffer (6). The mixture was incubated for 30 mm at 37°,
followed by phenol extraction and ethanol precipitation as
described above. The resulting aminoacyl-tRNA was dissolved
in 1.0 ml H2O, pH 4.5.
70
57
1@( *@
025
a
-U.
*1*
555—55 8-@s-*8
M@
-L*@ *18
@NA
*260
u._
18M
————S
3@—Ns-18I
1*8 *18
I
a
g
@w&
*1A
I
02
0I
050
050 400
08
03
)
50
70
90
T@( @MNER
--1*8*18
.5.55 5- 5-*-c*8
‘
025
50
I@
70
io
no
-——
8*1
Li.,
*18
@-Ms-l18@ 18*
@0LEUc@
,ais*
@A
a
I
fibrotic,
but without
evidence
of visual tumors.
There
was
some variation among animals in the extent of carcinogen
induced liver damage. However, the livers from 6 to 10 rats
were pooled for each of the several preparations of tRNA used
in this study. Approximately 60 mg of tRNA were obtained
from 100 g of liver (fresh weight). The amino acid acceptor
activity
of the tRNA's
prepared
3The abbreviation used is: 3'-Me-DAB,3'-methyl-4-dimethylamino
1678
@260
02
02
0I
050
400 08
@400
025
30
50
10
,@
90
18@R
PlO
30
50
10
90
110
@.*8 *@R
Chart 2. Comparison of aminoacyl-tRNA patterns from livers of
normal rats and livers of rats fed diets containing 3'-Me-DAB.
from the normal rat liver and
from the livers of the dye-fed animals were approximately the
same. Comparisons of the reversed phase chromatography
azobenzene.
so
,.*8
a@
As indicated in “Methods,―
the rats were maintained on the
diet containing the azo carcinogen for a period of 8 weeks.
The livers at this time were grossly enlarged, cirrhotic, and
8110
1
02
RESULTS
@
liver
@IA
--
3H-aminoacyl
of the
3:.*_@
57
and
of normal
025
Female albino rats ( I 50 to 175 g) of the Sprague-Dawley
strain were fed a semisynthetic diet containing 0.06% 3'-Me
DAB3 (5). The animals were fed ad libitum for 8 weeks. At
this time the livers were enlarged , cirrhotic , and fibrotic , but
without evidence of visual tumors. Livers of the dye-fed
animals and also livers from normal rats were excised and the
tRNA and aminoacyl-tRNA
synthetase
fractions were
prepared as described below.
tRNA fractions were prepared from the liver by the
procedure of Brunngraber (2), and an average yield of 60
ing! 100 g of liver, wet weight , was obtained. The aminoacyl
synthetases were prepared with the pH 5-insoluble material
from normal rat liver, as reported previously (6).
1 4C-
aminoacyl-tRNA's
--Lw
@
@
tRNA's
liver were cochromatographed.
Methods
@
of the aminoacyl
“precancerous―
livers are presented in Charts 1 to 3 . Each
graph represents the composite of several runs wherein the
The tRNA patterns of the precancerous livers exhibiting the
greatest deviation from normal liver are shown in Chart 1. It
may be observed that portions of the lysyl-, leucyl-,
phenylalanyl-, and tyrosyl-tRNA peaks from precancerous
livers emerged earlier from the reversed phase columns than
did corresponding aminoacyl-tRNA of normal liver. Some
minor changes in the chromatography patterns of glutamyl
CANCER RESEARCH VOL.30
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1970 American Association for Cancer Research.
3'-Me-DAB on Liver tRNA
—— 1*8 ‘818*
‘55555' 3@@Ms-@ 1*8
g
VALYL8RPM
@
-Uwi 188*
5555-55 3@_8*-o*8
*8*
1*1
@**
g
HISTRY6.
8RPM
•200
a
@400 02
02
000
02
0I
@400
68
054 400
025
P025
so
so
i@
so
i@o
)
50
TheE
@O
@0
TubM@
0I
I@7
Chart 3. Comparison of aminoacyl-tRNA patterns from livers of
normal rats and livers ofrats fed dietscontaining 3'-Me-DAB.
TUBENUMBER
and isoleucyl-tRNA's were observed (Chart 2). The com
parative patterns of the arginyl-, seryl-, methionyl-, valyl-,
histidyl-,
and threonyl-tRNA's
of normal
chart 4. Effect of in vitro methytation upon the chromatographic
behavior of rat liver phenylalanyl-and tyrosyl-tRNA's.
and precancerous
livers were almost identical (Charts 2 and 3).
Methylation of the tRNA's in the livers of rats exposed to
the 3'-Me-DAB was considered as one possible explanation for
the altered tRNA patterns. A preliminary study was carried
out to ascertain if in vitro methylation of liver tRNA would
or other metabolites of the 3'-Me-DAB. Direct methylation of
liver tRNA with diazomethane (7) prior to charging with
phenylalanine or tyrosine and followed by reversed phase
chromatography resulted in profiles quite similar to those
alter the chromatographic
behavior. Following treatment with
obtained from the precancerous livers. Capra and Peterkofsky
diazomethane under conditions where the amino acid acceptor
(3) obtained a new species of leucine@tRNA from Escherichia
activity of the tRNA was not markedly reduced, the
coli grown on a methionine-deficient
medium. When this
phenylalanyl- and tyrosyl-tRNA profiles were altered. One of methyl-deficient
leucyl tRNA was methylated
and
rechromatographed
on reversed phase columns,
the
the phenylalanyl peaks emerged approximately
10 tubes
earlier than the corresponding normal peak and the entire
methylated species emerged earlier from the column. However,
tyrosyl-tRNA also eluted much earlier than the control
the specific alteration or change in the liver tRNA's of the
counterpart (Chart 4).
dye-fed animals is still unknown and wifi require further
investigation.
What role alterations in tRNA's resulting from chemicals or
DISCUSSION
other agents may play in the mechanism of cancer induction is
only speculative at present. It would appear quite feasible to
It would appear that the prolonged administration of diets
study the effects of carcinogenic agents upon the tRNA
containing the hepatocarcinogen,
3'-Me-DAB, altered several
of the liver aminoacyl-tRNAprofilesresultingfrom reversed synthetase reaction, the interaction with ribosomes, the
coding, the peptide chain elongation, and, finally, the
phase chromatography.
Two possible explanations
for these
regulatory functions of tRNA proposed by Stent and Brenner
changes in the tRNA patterns may be offered. The livers of the
(13), Roth et al. (1 2), and other investigators. Finally, it
dye-fed animals exhibited gross morphological changes. There
should be possible to correlate
some of these carcinogen
was evidence of enlargement, extensive nodular formation,
induced changes with altered structural configurations.
cirrhosis, etc. Price et a!. (1 1) observed that after 7 to 8 weeks
of dye ingestion there was a considerable distortion of normal
liver architecture. They reported a nodular replacement of
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proliferated bile duct cells by cells that closely resembled
normal
liver cells. However,
types were present
large numbers
of other
cellular
in these livers. It is possible that the
tRNA's of these other cellular types differ from normal liver
tRNA's.
Another possible explanation
is that the altered tRNA's have
undergone methylation,
or have reacted with N-hydroxylated
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JUNE 1970
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1970 American Association for Cancer Research.
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CANCER
RESEARCH
VOL.30
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1970 American Association for Cancer Research.
Transfer RNA Patterns in Livers of Rats Fed Diets Containing 3′
-Methyl-4-dimethylaminoazobenzene
Michele Goldman and A. Clark Griffin
Cancer Res 1970;30:1677-1680.
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