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Occurrence of 7-methylguanine in nucleic acids of rat liver

Authors:

Abstract

1. Microsomal and soluble RNA of rat liver have been studied by column and paper chromatography after administration of [Me-(14)C]methionine; evidence was obtained for the occurrence of 7-methylguanine, the methyl group being derived from methionine. 2. No evidence was obtained for the occurrence of 7-methylguanine in DNA.
Biochem.
J. (1968)
107,
179
Printed
in
Great
Britain
Occurrence
of
7-Methylguanine
in
Nucleic
Acids
of
Rat
Liver
By
VALDA
M.
CRADDOCK,
S.
VILLA-TREVINO*
AND
P.
N.
MAGEE
Toxicology
Re8earch
Unit,
Medical
Re8earch
Council
Laboratorie8,
Car8halton,
Surrey
(Received
6
October
1967)
1.
Microsomal
and
soluble
RNA
of
rat
liver
have
been
studied
by
column
and
paper
chromatography
after
administration
of
[Me-14C]methionine;
evidence
was
obtained
for
the
occurrence
of
7-methylguanine,
the
methyl
group
being
derived
from
methionine.
2.
No
evidence
was
obtained
for
the
occurrence
of
7-methyl-
guanine
in
DNA.
Though
nine
or
more
methylated
bases
are
known
to
be
minor
components
of
RNA
isolated
from
various
sources,
there
is
less
information
about
the
RNA
of
rat
liver.
Special
interest
in
the
methylated
bases
in
this
tissue
arose
during
a
study
of
the
biochemical
reactions
of
those
carcinogens
that
have been
shown
to
alkylate
nucleic
acids.
Certain
of
these
compounds,
including
dimethylnitrosamine
and
nitrosomethylurea,
methylate
rat
liver
nucleic
acids
to
form
predominantly
7-methylguanine,
and
it
is
conceivable
that
this
reaction
is
involved
in
their
carcinogenic
actions
(Magee
&
Barnes,
1967).
To
help
assess
this
possibility,
it
was
decided
to
test
whether
7-methylguanine
occurs
in
normal
rat
liver
RNA
or
DNA.
This
methylated
base
is
of
special
interest
for
the
additional
reason
that
it
appears
to
have
a
turnover
rate
higher
than
that
of
other
methylated
purines
in
the
intact
animal
(Mandel,
Srinivasan
&
Borek,
1966).
The
extensive
work
of
Smith
and
Dunn
had
shown
that
a
number
of
methylated
bases
are
present
in
rat
liver
RNA
(Dunn,
1959;
Smith
&
Dunn,
1959a),
including
thymine
(Price,
Hinds
&
Brown,
1963),
but
7-methylguanine
had
been
shown
to
occur
only
in
pig
liver
soluble
RNA
(Dunn,
1963)
and
in
calf
liver
soluble
RNA
(D.
B.
Dunn,
personal
communication),
although
there
was
suggestive
evidence
for
its
occurrence
in
rat
liver
soluble
RNA
(Sluyser
&
Bosch,
1962).
The
occurrence
of
this
base
in
DNA
does
not
appear
to
have
been
reported.
However,
methylation
of
guanine
in
DNA
in
the
7-position
is
known
to
labilize
the
glycosidic
bond,
and
the
methylated
base
is
fairly
rapidly
split
out
of
the
nucleic
acid
(Lawley
&
Wallick,
1957).
It
therefore
seemed
possible
that 7-methylguanine
might
have
a
temporary
existence
in
DNA
and
have
escaped
detection.
Also,
if
it
is
present
at
a
*
Present
address:
Departamento
Biologia
Celular,
Centro
de
Investigacion
y
de
Estudios
Avanzados
del
Instituto
Politecnico
Nacional,
Apartado
Postal
14-740,
Mexico
14,
D.F.,
Mexico.
very
low
concentration,
it
would
be
advantageous
to
use
isotopic
methods
for
its
investigation.
The
effect
of
methylation
on
the
stability
of
RNA
is
in
dispute
(Kriek
&
Emmelot,
1963;
Lawley
&
Brookes,
1963).
There
is
much
evidence
that
the
methylated
bases
occurring
in
nucleic
acids
in
vivo
are
formed
by
enzymic
methylation
of
the
preformed
polymer,
rather
than
by
incorporation
of
the
methylated
base
itself,
and
that
the
reactions
are
catalysed
by
specific
methylases
that
use
S-adenosylmethionine
as
the
methyl
donor
(Mandel
&
Borek,
1963;
Borek
&
Srinivasan,
1966).
An
approach
to
the
problem
was
therefore
to
administer
[Me-14C]methionine
to
rats,
and
after
a
suitable
time
to
kill
the
animals,
isolate
the
nucleic
acids
and
analyse
the
radioactive
bases
present.
A
preliminary
account
of
some
of
this
work
has
been
published
(Villa-Trevino
&
Magee,
1966).
There
is
evidence
(Bergquist
&
Matthews,
1962)
that
there
is
an
increased
proportion
of
methylated
bases
in
nucleic
acids
of
tumour
tissues.
The
experiments
described
below
are
the
first
stage
in
a
more
detailed
study
of
the
effect
of
carcinogens
and
other
toxic
agents
on
the
methylated
bases
of
nucleic
acids.
EXPERIMENTAL
Animal8
and
materials
Wistaralbino
rats
ofthe
Porton
strain
were
maintained
on
M.R.C.
diet
41B
(Bruce
&
Parkes,
1956).
Female
rats,
200g.
body
wt.,
were
used.
[Me-l4C]Methionine
was
purchased
from
The
Radiochemical
Centre,
Amersham,
Bucks.
Treatment
of
animale
and
Beparation
of
nucleic
acide
Expt.
1.
Two
rats,
starved
overnight,
were
placed
in
metabolism
units
for
the
separate
collection
of
urine
and
faeces.
Each
animal
was
given
a
series
of
five
intraperitoneal
injections
of
20,uc
(0.1mg.)
of
[14C]methionine,
each
in
1
ml.
of
0
9
m-NaCl,
at
hourly
intervals.
One
hour
after
the
final
injection
the
rats
were
killed
By
a
blow
on
the
head
and
179
V.
M.
CRADDOCK,
S.
VILLA-TREVINO
AND
P.
N.
MAGEE
decapitation,
the
livers
removed
and
RNA
and
DNA
isolated
by
a
modification
of
the
method
of
Kirby
(1962)
as
suggested
by
Professor
K.
S.
Kirby
(personal
communica-
tion).
The
tissue
was
homogenized
in
a
solution
of
0.5%
naphthalene-1,5-disulphonic
acid
(sodium
salt),
containing
0-1ml.
of
2-5M-K2HPO4
(pH7
3)/100ml.
of
solution,
adjusted
to
pH6-5
with
dilute
H3PO4.
The
RNA
was
extracted
with
phenol
-
m-
cresol
-
g-
hydroxyquinoline-
water
(500:
70:
0
5:
55,
by
wt.)
mixture.
DNA
was
freed
of
RNA
by
treatment
with
ribonuclease,
and
polysaccharide
was
removed
from
both
nucleic
acids
by
the
methoxy-
ethanol-phosphate
procedure
(Kirby,
1956).
DNA
and
RNA
were
precipitated
by
addition
of
an
equal
volume
of
1%
cetyltrimethylammonium
bromide,
and
after
the
precipitates
had
been
washed
the
nucleic
acids
were
converted
into
the
sodium
salts
by
treatment
with
2%
(w/v)
sodium
acetate
in
70%
(v/v)
ethanol,
and
washed
with
ethanol,
ethanol-ether
(1:1,
v/v)
and
ether.
Expt.
2.
Two
rats,
starved
overnight,
were
given
a
series
of
four
intraperitoneal
injections
of
[14C]methionine
at
hourly
intervals,
the
first
of
40
juc
(0-2mg.)
in
0-8ml.
of
0-
9
m-NaCl,
the
remaining
three
injections
of
20
uc
(0.1
mg.)
in
0-4ml.
of
0
9M-NaCl.
One
hour
after
the
final
injection
the
rats
were
killed
by
a
blow,
the
livers
removed,
and
microsomal
and
pH5
RNA
(referred
to
below
as
'soluble
RNA')
isolated
by
a
modification
of
the
method
of
Hoagland,
Stephenson,
Scott,
Hecht
&
Zamecnik
(1958).
The
micro-
somal
and
soluble
fractions
were
resuspended
in
15ml.
of
solution
A
(0-25M-sucrose-4mM-MgCl2-25mM-KCl-50mM-
tris-HCl
buffer,
pH
7-6)
and
15
ml.
of
water,
0-3ml.
of
10%
sodium
dodecyl
sulphate
solution
and
an
equal
volume
of
90%
phenol
were
added,
and
the
mixture
was
shaken
for
lhr.
at
0-5'.
After
centrifugation
at
17000g
for
25min.
the
supernatant
was
aspirated
and
the
phenol
layer
was
washed
with
0-25
vol.
of
the
solution
A-water-sodium
dodecyl
sulphate
solution
and
recentrifuged.
To
the
pooled
aqueous
extracts
were
added
01
vol.
of
2
M-potassium
acetate
and
2-5vol.
of
ethanol,
and
the
mixtures
were
left
overnight
at
-25°.
The
RNA
precipitates
that
formed
were
dried
in
ethanol-ether
and
ether.
Analy8i8
of
nucleic
acid8
Expt.
1.
Samples
of
liver
RNA
(32
mg.)
and
DNA
(28.5mg.)
were
hydrolysed
with
HC104
by
the
method
of
Wyatt
(1952).
The
hydrolysates
were
diluted
to
3ml. with
water
and
centrifuged,
carriers
were
added
as
appropriate,
and
the
supernatant
solutions
were
chromatographed
on
columns
(1
cm.
x
1Ocm.)
of
Dowex
50
at
a
flow
rate
of
12-5ml./hr.,
with
N-HCI
for
the
first
300ml.
and
then
a
linear
gradient
approaching
4N-HCI.
The
extinction
at
260mix
was
recorded.
Expt.
2.
Microsomal
RNA
(a).
To
act
as
an
internal
marker
for
any
7[14C]-methylguanine
in
the
RNA,
a
sample
of
RNA
containing
7[3H]-methylguanine
was
added
to
the
preparation.
The
tritiated
material
had
been
prepared
previously
from
rats
given
[3H]dimethylnitrosamine,
and
was
known
to
contain
more
than
85%
of
the
radioactivity
as
7-methylguanine
(Lee,
Lijinsky
&
Magee,
1964).
The
microsomal
RNA
(14
mg.)
from
the
rat
treated
with
[14C]methionine
was
mixed
with
the
tritium-labelled
methylated
RNA
(0.7mg.),
and
the
mixture
was
hydrolysed
with
4ml.
of
N-HCI
for
60min.
at
1000
and
analysed
on
a
column
(20
cm.
x
2
cm.)
of
Dowex
50
at
a
flow
rate
of
25m1.Jhr.
with
an
exponential
gradient
of
0-25-3N-HCI.
Microsomal
RNA
(b).
A
sample
of
microsomal
RNA
(14mg.)
was
mixed
with
total
liver
RNA
(0-65mg.)
prepared
from
a
rat
treated
with
[3H]iimethylnitrosamine
as
described
above.
The
mixture
was
hydrolysed
with
0-3N-KOH
(lml.)
for
18hr.
at
370,
adjusted
to
pHl
with
HC104
and
centrifuged,
the
precipitate
was
washed,
and
the
first
supernatant
and
washings
were
pooled,
adjusted
to
pH7
with
N-KOH
and
analysed
on
a
column
(20
cm.
x
2
cm.)
of
Dowex
1
(formate
form).
For
the
elution
procedure,
250ml.
of
water
was
placed
in
the
mixing
chamber
of
the
gradient
device.
In
the
reservoir
were
water
(tubes
1-8),
N-formic
acid
(tubes
9-28)
and
then
4N-formic
acid.
Soluble
RNA.
The
preparation
(4-5mg.)
was
treated
as
for
the
acid
hydrolysis
of
microsomal
RNA
(a)
described
above.
Determination
of
radioactivity
Samples
of
fractions
from
column
chromatography
were
evaporated
to
dryness
in
a
stream
of
air,
and
radioactivity
was
determined
as
described
previously
(Craddock
&
Magee,
1966),
9
ml.
of
the
10
ml.
fractions
being
used
in
Expt.
1,
and
5ml.
of
the
10ml.
fractions
in
Expt.
2.
Paper
chromatography
The
remainders
of
the
fractions
from
column
chromato-
graphy
were
pooled,
corresponding
to
peaks
of
radioactivity,
and
were
concentrated
by
the
use
of
a
rotary
evaporator.
Paper
chromatography
was
carried
out
in
methanol-
conc.
HCl-water
(7:2:1,
by
vol.),
butan-l-ol-aq.
NH3
(sp.gr.
0-88)-water
(85:2:13,
by
vol.)
and
propan-2-ol-water
(7:3,
v/v)
with
NH3
in
the
gaseous
phase
(Markham
&
Smith,
1952).
The
chromatograms
were
cut
into
narrow
strips
crosswise,
the
pieces
eluted
with
N-HCI
and
the
eluates
evaporated
to
dryness,
and
radioactivity
was
determined
as
described
above.
Treatment
of
urine
The
urine
collected
during
Expt.
1
was
fractionated
as
described
by
Craddock
&
Magee
(1967)
and
the
purine
fraction
analysed
by
column
chromatography
on
a
column
(1cm.
x
10cm.)
of
Dowex
50
with
a
linear
gradient
of
1-4N-HCI.
RESULTS
The
result
of
column
chromatography
of
liver
RNA,
Expt.
1,
is
shown
in
Fig.
1.
The
compounds
responsible
for
the
extinction
profile
are
uracil,
xanthine,
hypoxanthine,
guanine,
7-methylguanine
and
adenine.
The
relative
positions
of
the
radio-
active
peaks,
and
the
results
of
paper
chromato-
graphy,
are
consistent
with
peak
1
being
thymine,
peak
2
5-methylcytosine,
peak
3
N2-methylguanine
and
N2-dimethylguanine,
peak
4
1-methylguanine,
peak
5
7-methylguanine
and
peak
6
1-methyl-
adenine.
Some
of
the
radioactivity
in
peaks
3
and
6
is,
as
expected,
in
guanine
and
adenine
as
a
result
of
purine
biosynthesis.
The
result
of
column
chromato-
graphy
of
DNA
is
shown
in
Fig.
2.
The
radioactive
peak
1
is
presumably
thymine
and
peak
2
is
5-methylcytosine.
The
result
of
column
chromatography
of
the
180
1968
7-METHYLGUANINE
IN
NUCLEIC
ACIDS
1-0
0-8
oo
0-6
cq
0-4
0-2
0
200 400
600
800
Vol.
of
effluent
(ml.)
1000
1200
1400
Fig.
1.
Ion-exchange
chromatography
of
an
HCl04
hydrolysate
of
total
RNA
of
rat
liver,
after
administration
of
a
total
of
lOOgtc
of
[14C]methionine/rat.
Details
are
given
in
the
text.
Carrier
xanthine
(X),
hypoxanthine
(H)
and
7-methylguanine
(M)
were
added.
Other
peaks
shown
by
E260
readings
are
uracil
(U),
cytosine
(C),
guanine
(G)
and
adenine
(A).
o,
E260;
A,
radioactivity.
*
200
02
a
160
0
0
0
0
c.)
c3
40
0
0
200
400
600
800
lo0w
1200
1400
Vol.
of
effluent
(ml.)
Fig.
2.
Ion-exchange
chromatography
of
an
HC104
hydrolysate
of
rat
liver
DNA,
after
administration
of
a
total
of
lOOuc
of
[14C]methionine/rat.
Details
are
given
in
the
text.
Carrier
7-methylguanine
(M)
was
added.
Other
peaks
shown
by
E260
readings
are
thymine
(T),
cytosine
(C),
guanine
(G)
and
adenine
(A).
o,
E260;
A,
radioactivity.
acid
hydrolysates
of
microsomal
RNA
(Fig.
3)
and
soluble
RNA
(Fig.
4)
shows
a
peak
of
14C
radio-
activity
in
the
position
of
7-methylguanine,
the
profile
of
which
corresponds
to
the
profile
of
7[3H]-methylguanine
originating
in
the
RNA
added
to
act
as
a
marker.
Paper
chromatography
of
samples
from
the
pooled
samples
of
this
peak
from
the
microsomal
RNA
shows
that
all
the
14C
and
3H
radioactivity
moves
with
the
same
RF
as
authentic
7-methylguanine.
Colunm
chromatography
of
the
alkaline
hydro-
lysate
of
microsomal
RNA
with
added
RNA
from
a
rat
treated
with
[3H]dimethylnitrosamine
is
shown
in
Fig.
5.
The
3H
radioactivity
profile
shows
only
one
major
peak,
which
is
presumably
2,4-diamino-6-
hydroxy
-
5
-
methylformamidopyrimidine,
formed
from
7-methylguanine
in
RNA
by
treatment
with
alkali
(Haines,
Rees
&
Todd,
1962).
The
14C
radio-
activity
profile
also
shows
a
peak
in
this
position.
The
increased
number
of
radioactive
peaks
com-
pared
with
the
column
chromatography
of
bases
resulting
from
acid
hydrolysis
is
possibly
due
to
the
presence
of
2'[14C]-O-methyl-ribonucleotides
(Smith
&
Dunn,
1959b).
The
urine
analysis
(Fig.
6)
showed
a
peak
of
radioactivity
in
the
position
of
7-methyl-
guanine.
The
initial
high
peak
of
radioactivity
is
presumably
due
to
14C
incorporated
in
the
mixture
of
uric
acid,
xanthine,
hypoxanthine
and
1-methylhypoxanthine.
DISCUSSION
The
experiments
shown
in
Figs.
1-5
give
evidence
that
rat
liver
RNA,
including
microsomal
and
soluble
RNA,
contains
7-methylguanine,
although
Vol.
107
1-:
z
1000
m
4-i
0
800
O
0
;C,
600
.wk
c
400
Ca
0
Cas
200
S
,S
181
uI
to
c4
V.
M.
CRADDOCK,
S.
VILLA-TREVINO
AND
P.
N.
MAGEE
E
0
._
._.
*S
0
V'
0
'5!
1968
2400
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S
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.3
Ca
.9-4
1200
;a
0.
600
Ce
0
0a
co
Vol.
of
effluent
(ml.)
Fig.
3.
Ion-exchange
chromatography
of
an
HCI
hydrolysate
of
a
mixture
of
liver
microsomal
RNA
from
a
rat
treated
with
[14C]methionine
and
total
liver
RNA
from
animals
treated
with
[H3]dimethylnitrosamine,
the
latter
having
been
added
to
act
as
a
marker
for
7-methylguanine.
For
details
see
the
text.
Peaks
shown
by
E260
readings
are
UMP,
CMP,
guanine
(G)
and
adenine
(A).
0,
E260,
o,
140
radioactivity;
A,
3H
radioactivity.
G
A
4000
.a
3000
ea
$
._'.
m
2000
!:,
.,.
1000
ro
Ca
v
t
-
I
8000
.9
0
00
.C3
6000
b
t
0
._4
2000
;8
45
cQ
O0
600
800
1000,
Vol.
of
effluent
(ml.)
Fig.
4.
Ion-exchange
chromatography
of
an
HCI
hydrolysate
of
a
mixture
of
liver
soluble
RNA
from
a
rat
treated
with
[14C]methionine
and
total
liver
RNA
from
animals
treated
with
[3H]dimethylnitrosamine,
the
latter
having
been
added
to
act
as
a
marker
for
7-methylguanine.
For
details
see
the
text.
Peaks
shown
by
E260
readings
are
UMP,
CMP,
guanine
(G)
and
adenine
(A).
it
is
not
excluded
that
the
much
smaller
amount
of
the
base
found
in
microsomal
RNA
occurs
in
soluble
RNA
bound
to
it.
However,
the
difference
in
the
relative
heights
of
the
radioactive
peaks
in
the
two
profiles
argues
against
this.
Messenger
RNA
probably
occurs
in
the
microsomal
fraction,
but
there
is
evidence
that
messenger
RNA
does
not
contain
methylated
bases
(Moore,
1966).
The
results
show
also
that
methionine
is
a
methyl
donor
during
the
biosynthesis
of
7-methylguanine
and
of
certain
other
methylated
bases
in
rat
liver
in
vivo.
It
has
been
shown
by
Rodek,
Feldman
&
Littauer
(1967)
that
an
RNA
methylase
preparation
from
rat
liver
methylates
E8cherichia
coli
soluble
RNA
to
form,
among
other
methylated
bases,
7-methyl-
guanine.
However,
this
does
not
necessarily
imply
182
0
Vol.
107
0
to
ec
7-METHYLGUANINE
IN
NUCLEIC
ACIDS
183
12400
1-:
.._
-4
co
-4
*E
1800
;:
.o
1200
:'
;a
P-Z
-0
0>
Vol.
of
effluent
(ml.)
Fig.
5.
Ion-exchange
chromatography
of
alkaline
hydrolysate
of
a
mixture
of
liver
microsomal
RNA
from
a
rat
treated
with
[14C]methionine
and
total
liver
RNA
from
animals
treated
with
[3H]dimethylnitrosamine,
the
latter
having
been
added
to
act
as
a
marker
for
7-methy]guanine.
For
details
see
the
text.
Peaks
shown
by
E260
readings
are
CMP,
AMP,
GMP
and
UMP.
*,
E260;
0,
14C
radioactivity;
A,
3H
radioactivity.
5000
._-
-4
4000
9
0
3oo
I.-
3000
._
eC
1000
Ca
0.I
0.8
a
w
c4
0.6
0-4
Asnhe
pos;
0-2
A
aK
0
200
400
600
800
1000
Vol.
of
effluent
(ml.)
1200
1400
Fig.
6.
Ion-exchange
chromatography
of
purine
fraction
of
urine
collected
for
5
hr.
during
administration
of
[L4C]methionine.
Carrier
guanine
(G)
was
added.
Peaks
shown
by
E260
readings
are
the
initial
mixture
of
purine
metabolites,
chiefly
uric
acid
(Ur),
and
guanine
(G).
o,
E260;
A,
radioactivity.
that
7-methylguanine
is
present
in
rat
liver
RNA,
as
methylation
is
believed
to
depend
on
the
presence
of
the
appropriate
base
sequence
in
the
RNA
substrate,
which
may
have
been
present
in
E.
coli
RNA
but
not
in
rat
liver
RNA.
Normal
urine
is
known
to
contain
7-methyl-
guanine
(Kruger
&
Salomon,
1
898a,b;
Weissmann,
Bromberg
&
Gutman,
1957),
and
there
is
evidence,
based
on
the
excretion
of
radioactive
7-methyl-
guanine
by
rats
whose
nucleic
acids
had
been
pre-
labelled
in
the
neonatal
period
with
[14C]formate,
that
the
methylated
base
originates
in
the
nucleic
acids
(Craddock
&
Magee,
1967).
Mandel
et
at.
(1966)
showed
that
methionine
is
the
methyl
donor
of
the
7-methylguanine
excreted
in
urine.
Thus
it
seems
reasonable
to
assume
that
catabolism
of
RNA,
or
release
of
the
methylated
base
by
depurina-
tion,
accounts
for
some
7-methylguanine
in
urine.
The
other
methylated
bases
found
in
RNA
occur
to
a
much
smaller
extent,
if
at
all,
in
the
5
hr.
urine
sample
analysed,
and
therefore
either
are
further
metabolized
or
have
a
slower
turnover
rate
than
184
V.
M.
CRADDOCK,
S.
VILLA-TREVINO
AND
P.
N.
MAGEE
1968
7-methylguanine.
The
short
time
period
of
the
experiment,
designed
primarily
to
detect
methyla-
tion
of
RNA,
probably
explains
why
no
labelled
1-methyladenine
was
detected
in
urine,
although
it
had
been
found
by
Mandel
et
al.
(1966).
These
authors
found
that
most
of
the
radioactivity
of
the
urinary
purines
occurred
in
7-methylguanine
after
administration
of
[14C]methionine
to
mice;
as
this
base
also
had
a
comparatively
higher
specific
radioactivity,
it
was
suggested
that
it
has
a
higher
rate
of
turnover
than
other
methylated
bases.
The
possible
functions
of
the
methylated
bases
remain
obscure,
although
a
role
in
protein
synthesis
and
in
cell
differentiation
has
often
been
postulated
(Borek
&
Srinivasan,
1966).
The
experiments
give
no
evidence
for
the
occurrence
of
7-methylguanine
in
DNA,
although
conceivably the
methyl
group
could
be
derived
from
a
methyl
donor
other
than
methionine.
Alkylation
on
the
7-position
of
guanine
labilizes
the
glycosidic
bond,
and
the
methylated
base
splits
out
of
the
DNA
spontaneously
(Lawley
&
Wallick,
1957).
However,
the
rate
of
this
non-enzymic
excision
under
physiological
conditions in
vitro
(Lawley
&
Brookes,
1963)
and
when
7-methyl-
guanine
is
formed
by
the
action
of
a
low
dose
of
dimethylnitrosamine
in
vivo
(V.
M.
Craddock,
unpublished
work)
is
such
that
loss
of
7-methyl-
guanine
would
be
unlikely
to
occur
to
a
considerable
extent
during
the
5hr.
period
of
the
experiments
described
here.
In
addition,
colunm
chromato-
graphy
of
a
large
preparation
of
rat
liver
DNA
(386mg.)
gave
no
evidence
for
the
presence
of
7-methylguanine
from
extinction
determinations
(P.
F.
Swann,
personal
communication;
Shank
&
Magee,
1967).
Thus
rat
liver
DNA
differs
from
mammalian
sperm
DNA,
where
1-methylguanine
has
been
reported
in
human
sperm
DNA
and
N2-dimethylguanine
in
bull
sperm
DNA
(Unger
&
Venner,
1966).
When
7-methylguanine
is
formed
in
rat
liver
DNA
after
administration
of
certain
carcinogens,
it
can
therefore
be
regarded
as
being
an
abnormal
base,
whose
presence
might
be
expected
to
exert
an
effect
on
the
biological
function-
ing
of
DNA.
The
authors
thank
Mr
J.
A.
E.
Jarvis,
Mr
R.
Hunt
and
Miss
A.
Taylor
for
skilled
technical
assistance.
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Chapter
This chapter discusses the low-molecular-weight constituents of nucleic acids nucleosides, nucleotides, and their analogues. Nucleic acids are composed of phosphoric acid, a sugar component, (deoxyribose or ribose) and purine and pyrimidine bases (adenine, guanine and cytosine, thymine, or uracil). These basic components can be isolated from the total hydrolyzates of polymers, while partial hydrolysis of the polynucleotide chain leads to fragments in the form of nucleosides, nucleotides, and oligonucleotides. In addition to the four fundamental nucleosides for each type of nucleic acid, six so-called minor components are found in DNA (mostly from bacteriophages) and about 35 in RNA (mainly tRNA). Most column-type fractionation techniques are nowadays carried out with the use of automatic fraction collectors and instruments recording the substance analyzed. In an attempt to shorten as much as possible the time needed for separation, to enhance the sensitivity and resolution of the column (nanomoles of substances separated), etc., special chromatographic instruments and systems have been developed even for the separation of nucleic acid components (in analogy with amino acid analyzers).
Article
Two of the major conceptual advances in chemical carcinogenesis made during the past several years have been the discovery of (1) the enzymatic activation of procarcinogens to reactive ultimate carcinogens and (2) the chemical interactions of the active metabolites of many carcinogens with a variety of tissue nucleophils including RNA, DNA, and protein. This chapter is concerned with the interactions of chemical carcinogens and their activated metabolites, proximate and ultimate carcinogens, with nucleic acids, both DNA and RNA.
Article
Combined oral administration of dimethylamine and sodium nitrite to mice yielded a marked dose dependent inhibition of liver nuclear RNA synthesis. Inhibition of nuclear RNA synthesis was still significant when sodium nitrite was administered 1 h subsequent to dimethylamine; no inhibition of nuclear RNA synthesis was observed when sodium nitrite was given 30 min prior to the dimethylamine. Furthermore, radioactivity was observed in 7‐methyl guanine in liver RNA from mice treated with dimethylamine‐C and sodium nitrite but not with di‐methylamine‐C alone. This study is of import because of the in vivobiosynthesis of carcinogenic nitrosamines in man following suggestion of nitrites and secondary amines present in food.
Article
The long-term feeding of ethionine, the ethyl analog of methionine, leads to hepatic carcinoma (Farber, 1963). The intraperitoneal injection of [ethyl-1-14C-] L-ethionine into rats also leads to the rapid ethylation of both hepatic protein and nucleic acid fractions (Natori, 1963; Stekol , 1960; Stekol, 1965; Farber , 1967). Stekol (1965) reported that DNA is ethylated and 7-ethylguanine was the only labeled nucleic acid component that could be isolated following acid hydrolysis. However, others (Farber , 1967; Ortwerth and Novelli, 1968) demonstrated that RNA, particularly tRNA, is the most significantly labeled nucleic acid fraction and that a more complex pattern of RNA ethylation was observed (Farber , 1967) than indicated by Stekol (1965).In the present paper, the major portion of the labeled ethylpurines obtained from rat liver tRNA following the injection of ethyl-labeled ethionine is identified as -ethyl-, 7-ethyl- and ,-diethylguanine, which together account for 23%, 10%, and 2% respectively of the total radioactivity found in tRNA. This accounts for two-thirds of the purine ethylation observed.
DNA methylase activity was studied in the intact animal, first to determine whether any methylated base is formed in addition to 5-methylcytosine, and second to compare the extent of methylation of cytosine in DNA isolated from normal and from precancerous liver.Experiments in which radioactive methionine, formate and adenine were given separately or simultaneously to normal rats, and to animals at the time when DNA synthesis was occurring at the maximum rate after partial hepatectomy, gave no evidence for the formation of any minor methylated base other than 5-methylcytosine in liver or testis DNA.Experiments using animals fed a diet containing the carcinogen dimethylnitrosamine showed that the increase in methylation of DNA, measured by incorporation of 3H from [3H]methionine into 5-methylcytosine, was proportional to the increase in DNA synthesis, measured simultaneously by incorporation of [14C]adenine into the adenine fraction of DNA. This suggests that methylation of DNA as mediated by DNA methylase occurs normally during carcinogenesis induced by dimethylnitrosamine.Experiments in which [14C]methionine was injected into rats previously fed a diet containing ethionine showed the presence of two abnormal labelled components in liver DNA. Administration of [14C]ethionine to rats after partial hepatectomy gave no evidence for the formation of 5-ethylcytosine, the alkylated base which would have been formed if DNA methylase used S-adenosylethionine in place of S-adenosylmethionine as an alkyl donor. Thus while abnormal alkylation of DNA may occur during carcinogenesis induced by ethionine, it is probably not brought about by utilisation of S-adenosylethionine in place of S-adenosylmethionine by DNA methylase.
Previous studies of the mechanism of induction of liver cancer by dimethylnitrosamine have been carried out by investigating the reactions of the carcinogen with cellular macromolecules after giving normal animals a single injection of the labelled compound. The fact that dimethylnitrosamine induces liver cancer when fed in the diet, but very rarely when administered as a single dose to a normal adult animal, made the results of these experiments difficult to evaluate. However, when it was found that one single dose given after partial hepatectomy, at the time of DNA replication, does induce liver cancer, it was then possible to study reactions of dimethylnitrosamine with liver cell components under conditions known to induce cancer.Di[14C]methylnitrosamine was given to normal rats and also 24 h after partial hepatectomy, and liver DNA was isolated 5–7 h and 24 h after treatment. had been formed. This base had not been detected previously in DNA of animals treated with dimethylnitrosamine owing to its acid lability. No was detected after treatment with methyl methanesulphonate, a methylating agent found not to be a liver carcinogen even when given after partial hepatectomy.The amounts of 7-methylguanine, , 3-methyladenine, 1-methyladenine and 3-methylguanine were similar in DNA isolated from intact and from regenerating liver. The results suggest that, for carcinogenesis to ensue, must be present in DNA at the time of DNA replication. The methylated base is then likely to cause miscoding during transcription, so that the transient abnormality of methylation would be converted into a permanent inherited change in the genetic material.The methylated bases had largely disappeared from DNA by 2 days after treatment. The fact that 3-methyladenine disappears faster than 7-methylguanine or suggests that specific enzymic excision of this base is taking place.
Article
Single urine specimens from 72 normal adult males, including Jews from Israel, Arabs from Gaza, and Africans from Kenya, were analyzed for purines (excluding uric acid). These groups showed similar results for adenine, 7-methylguanine, hypoxanthine, and xanthine, and differences observed for the minor methylated purines were probably artefactual. The results were similar to those found in the United States and Britain, and do not suggest that such analyses may be useful for detecting exposure to carcinogenic alkylating agents, except at large doses. Urine from 30 subjects with cirrhosis of the liver or cancer of various organs (excluding leukemia) were also examined. These showed significant changes in various ratios indicating a depressed excretion of hypoxanthine and xanthine (and also creatinine), relative to that of adenine and 7-methylguanine. Some of these subjects also showed an increased excretion of 8-hydroxy-7-methylguanine and (less frequently) 1-methylhypoxanthine and 1-methyladenine. Similar effects have previously been observed in gout and in leukemia.
Article
The effects of ACTH on testicular enzymes was studied using human testicular tumors from patients with a genetic deficiency of 21-hydroxylase and a resulting unsuppressed secretion of ACTH from the pituitary. The human tumor cells from patients off glucocorticoid therapy were capable of forming glucocorticoids in vitro, whereas tissue from patients whose ACTH production was suppressed with adequate glucocorticoid therapy (17-ketosteroid excretion maintained at normal values) showed no glucocorticoid formation in vitro. Patients off glucocorticoid therapy had very high 11β-hydroxylase activity in their testicular tumors whereas patients suppressed with glucocorticoids had tumor cells with little or no 11β-hydroxylase activity. Non-tumorous testicular cells from patients on or off therapy formed primarily 17-hydroxyprogesterone and testosterone in vitro with no glucocorticoid formation.The administration of 20 IU ACTH twice daily to rats over a 10-day period altered the kind of RNA formed within certain testicular cells. This RNA enhanced glucocorticoid formation when added to mouse adrenals in vitro. Similar results were obtained using testicular RNA from ACTH-treated adrenalectomized rats, showing that endogenous glucocorticoids could not be mediating the effect. Thus ACTH responsive cells exist in testicular tissues from humans and rats, suggesting that under appropriate circumstances the tropic hormone of the adrenal may influence the biosynthetic processes of certain cells in the testis.
Article
The possibility that carcinogens may affect methylase-mediated methylation of replicating DNA was investigated. A system eminently suitable for this purpose is liver regenerating after partial hepatectomy, as one injection of dimethylnitrosamine (DMN) given during the ensuing period of increased DNA synthesis induces hepatocellular carcinoma. Methylation of DNA by DNA methylase normally occurs only in proportion to DNA synthesis. Therefore simultaneous measurements were made of synthesis (incorporation of [14C]adenine into DNA adenine, or of d[5-3H]cytidine into DNA cytosine), and of methylation (incorporation of [methyl-3H]methionine into 5-methylcytosine of DNA) in liver regenerating after partial hepatectomy. After treatment with DMN, the ratio of methylation: synthesis remained within the normal range. Methyl methanesulphonate (MMS), a compound which damages DNA in regenerating liver in a similar but not identical way to DMN and which does not induce tumors in liver even when given after partial hepatectomy, caused an increase in methylation in relation to synthesis. These experiments therefore do not support the view that altered DNA methylase activity is involved in carcinogenesis.
The methylation of transfer RNA (tRNA) and of ribosomal RNA (rRNA) was studied in normal animals and in animals fed diets containing the carcinogens dimethylnitrosamine, aflatoxin or ethionine. The rats were injected with [14C]methionine, and the nucleic acids were isolated, hydrolysed with HClO4 and analysed by column chromatography on Dowex-50.The level of labelling of each methylated base in tRNA was increased by the carcinogenic treatment. The increase probably does not reflect merely an increased rate of synthesis of tRNA, as each methylated base increases to a different extent in precancerous liver, in contrast to the situation in intestine, where the labelling of the different methylated bases is higher than in liver, but each is increased to a similar extent. Also, in precancerous liver, the increase in protein synthesis, judged by the incorporation of [14C]methionine into protein, is in most cases much less than the increased methylation of tRNA, while in intestine the incorporation of [14C]-methionine into protein and the methylation of tRNA exceed the values in liver in equal proportions. There also appears to be a change in composition of the major bases of tRNA during carcinogenesis produced by dimethylnitrosamine and by aflatoxin. These results add evidence to the view that there is a change in the relative abundance of different species of tRNA synthesised during carcinogenesis, the change being towards more highly methylated species.In the case of rRNA there is an increase in labelling of each methylated base, but the increase may correlate with an increased synthesis of rRNA. There is evidence for a change in the kinetics of the reactions leading to the formation of rRNA precursor.
Article
1. Rats were given the hepatotoxin and carcinogen cycasin by stomach tube. In one experiment, rats whose RNA had previously been labelled with [(14)C]-formate were given the acetate ester of the aglycone form of cycasin, methylazoxymethanol, by intraperitoneal injection. 2. Incorporation of (14)C from l-[U-(14)C]leucine into the proteins of some organs was measured in cycasin-treated rats. Cycasin inhibited leucine incorporation into liver proteins but not into kidney, spleen or ileum proteins. This inhibition was not evident until about 5hr. after cycasin administration, but once established it persisted for the next 20hr. 3. Methylation of nucleic acids was detected in some organs of rats treated with cycasin or methylazoxymethanol. The purine bases of RNA and DNA were isolated by acid hydrolysis followed by ion-exchange column chromatography. The resulting chromatograms showed an additional purine base that was identified as 7-methylguanine. It was shown that, in animals treated with the toxin, liver RNA was methylated to a greater extent than was either kidney or small-intestine RNA. Also, as a result of cycasin administration, liver DNA guanine was methylated to a greater extent than was RNA guanine. 4. These results are discussed in relation to comparable experiments with dimethylnitrosamine. It is suggested that cycasin and dimethylnitrosamine are metabolized to the same biochemically active compound, perhaps diazomethane, but that various tissues differ in their capacity to metabolize the two carcinogens.
Article
Dimethylnitrosamine is metabolized to form an alkylating intermediate, which may have significance for its carcinogenic action. However, certain other compounds that are known to be highly mutagenic, including nitrous acid and hydroxylamine, might also be formed. Owing to the general reactivity of these compounds, it would be difficult to detect their formation in the intact animal. Instead, the nucleic acids of carcinogen-treated animals were examined for products of reaction with nitrous acid and hydroxylamine, i.e. for deamination of adenine and guanine, and formation of N(6)-hydroxycytosine, respectively. A double-labelling technique was used to detect very small amounts of the abnormal bases. The purine moieties of DNA in adult rat liver were labelled either with (14)C or with (3)H, by treating the neonatal animals with [(14)C]formate or with [(3)H]formate, and then allowing a period for normal growth. During this time, in liver, the labels were largely lost by metabolic turnover from cell components other than DNA. The pyrimidine moieties in DNA were labelled by treating the neonatal animals with [(14)C]orotate. The purine constituents of RNA of adult rat liver were labelled by repeated administration of [(14)C]- or [(3)H]-formate to the adult rats. The [(14)C]nucleic acid-labelled rat could then be treated with the carcinogen, and the [(3)H]nucleic acid-labelled animal could be used as a control. By this means the experimental and control tissues could be homogenized together in a single preparation, and the nucleic acids from the two tissues could be isolated, hydrolysed and analysed in a single sample. It was therefore possible to have an internal control for artifacts due to changes taking place in the nucleic acid bases during the experimental procedures. With this technique, the formation in vivo of 7-methylguanine in rat-liver DNA and RNA after administration of dimethylnitrosamine was confirmed, and no evidence was found for the formation of xanthine, hypoxanthine, N(6)-hydroxycytosine, or any other abnormal base.
Article
1. Evidence is presented for the excretion of 7-methylguanine in normal rat urine at a rate of approx. 65mug./day. Experiments with animals in which the nucleic acids had been prelabelled by treatment of the neonatal rats with [(14)C]-formate gave evidence that the methylated base originated in the nucleic acids of the rat. 2. Injection of [(14)C]dimethylnitrosamine leads to an increased excretion of 7-methylguanine, and the base becomes labelled in the methyl group. The disappearance of labelled 7-methylguanine formed in nucleic acids of rats treated with the carcinogen therefore does not take place by an N-demethylation reaction, but by liberation of the intact methylated base.