[CANCER RESEARCH 36, 2073-2079, June 1976]
Nonrandom Nature of in Vivo Methylation by
Dimethylnitrosamine and the Subsequent
Removal of Methylated Products from
Rat Liver Chromatin DNA1
R. Ramanathan,2
S. Rajalakshmi,
D. S. R. Sarma,3 and E. Farber'
Fels Research Institute(R. R., S. R., 0. S. R. S., E. F.Jand Departments of Pathology(D. S. A. S., E. F.Jand Biochemistry(S. R., E. F.J, Temple University
MedicalSchool, Philadelphia,Pennsylvania19140
SUMMARY
This investigation was designed to study whether methyl
ation of liver chromatin DNA by dimethylnitrosamine (DMN)
and the subsequent in vivo removal of DNA-bound methyl
ated products are random. Liver chromatin DNA was frac
tionated into nuclease-digestible and nondigestible mate
nab 4 hr following the administration of [3H]DMN (0.5 mgI
250 @Ci/100g body weight). Digestion of such methylated
liver chromatin with pancreatic DNase I or micrococcab
nuclease and analysis of nuclease-digested acid-soluble
products revealed a discrepancy between the radioactivity
released (72%) and the nucleotides released (50%) as mea
sured by the absorbance at 260 nm. This discrepancy disap
peared, and the rate and extent of release of both the
radioactivity and the absorbance at 260 nm were identical
when the total purified DNA isolated from methylated chro
matin was used as the substrate instead of chromatin DNA
in the nuclease reaction. These results, together with the
fact that guanine contents of the DNA ofthe two fractions of
the chromatin isolated by nuclease digestion were identical,
suggest that methylation of the nuclease-accessible region
of hepatic chromatin DNA is relatively greater than that of
the inaccessible region.
The study of the removal of methylated products in the
accessible region of the chromatin DNA further reveals that,
of the methylated products present at 4 hr, 62% is lost by 3
days, 87% is lost by 1 week and 94% is lost by 2 weeks.
However, loss from the nuclease-inaccessible region of
chromatin DNA is only 27% by 3 days, 49% by 1 week, and
86% by 2 weeks, thereby suggesting that the removal of
methylated products from this region of chromatin DNA is
relatively slower compared with that from the nuclease
accessible region of chmomatin-DNA. The results of this
I This
investigation
was
supported
in part
by
USPHS
Research
Grants
CA
14689, CA 12218, and CA 12227 from the National Cancer Institute and
American Cancer Society Grant BC-7P.
This work was presented at the annual meeting of American Association
for Cancer Research held at San Diego, Calif., May 8 to 11, 1975 (29).
2 Present
address:
Frederick
Cancer
Research
Center,
Post
Box
“B,―
Frederick, Md. 21701.
3 To
whom
4 Present
requests
address:
for
reprints
Department
should
of
be
Pathology,
addressed.
University
College Street, Toronto, Ontario, Canada M5G 1L5.
Received December 11, 1975; accepted March 10, 1976.
of
Toronto,
study thus indicated (a) an increased methylation and faster
rate of removal of DMN-induced methylated products in
nuclease-accessible regions of chromatin DNA and (b) de
creased methylation and slower rate of removal from the
nuclease-inaccessible regions of chromatin DNA. It is con
cluded that the distribution and removal of DMN-induced
methybated products in liver chromatin DNA is nonrandom
as measured by this technique.
INTRODUCTION
Chemical carcinogens either by themselves or after being
activated interact with cellular DNA, RNA, and protein (8,
13, 17, 27,32). Extensive work has been carried out on the
characterization of the nature of the interaction between
DNA and carcinogens and the rates of in vivo removal of
DNA-bound carcinogens or their metabolites (6, 12, 14, 15,
32, 36, 37). However, very little or nothing is known regard
ing the intragenomic distribution of carcinogens and the
rates of their removal from different regions of chromo
somab DNA. This aspect of carcinogen-DNA interaction is of
importance in view of the fact that certain regions of mam
malian DNA are covered with acidic and basic proteins (3,
16) and are thus inaccessible to some enzymes such as RNA
pobymerase (34, 35), staphybococcal nuclease (3), and DNA
bigase (40).
In this investigation we have studied the intragenomic
distribution of alkylated products and the rate of their sub
sequent removal from liver chromatin DNA following the
administration of the hepatocarcinogen DMN.5
Several methods are available for the fractionation of
chromatin DNA (3, 24, 30). In the present study we have
fractionated the liver chromatin DNA by making use of the
interesting observation reported by Clark and Felsenfeld (3)
that about 50% of the chromatin DNA is digestible by micro
coccal nuclease.
In this communication we report that, in liver chromatin
DNA, the in vivo alkylation and the subsequent removal of
alkylated products following the administration of DMN are
nonrandom. The alkylation is more and the subsequent
100
SThe abbreviations used are: DMN, dimethylnitrosamine;
PCA, perchloric
acid.
JUNE 1976
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2073
A. Ramanathanet a!.
removal of DNA-bound alkylated products is faster from the
nuclease-accessible regions of liver chromatin DNA in com
parison to that from nuclease-inaccessible regions.
MATERIALS AND METHODS
Animals. Male Wistar albino rats weighing about 110 to
130 g were obtained from Carworth Farms. The animals had
free access to water and food and were housed under
controlled temperature and lighting conditions.
Chemicals. [3H-methy!]Thymidine(specific activity, 20 Ci!
mmole), [3H]DMN (39 and 390 mCi/mmobe) (New England
Nuclear, Boston, Mass.), Protease type VI (purified) (Sigma
Chemical Co., St. Louis, Mo.), DMN (K and K Laboratories,
Plainview, N. J.), micrococcal nuclease and pancreatic
DNase I (electrophoretically pure) (Worthington Biochemi
cals Freehold, N. J.), and cellulose-coated thin-layer chro
matography plates (Brinkman Instruments, Westbury, N. Y.)
were used in these studies.
Labeling of Liver DNA. Liver DNA was labeled by inject
ing [3H-methy!]thymidine during the period of DNA synthe
sisfollowing
partial
hepatectomy(5,10).
Carcinogen Treatment. DMN was injected i.p. in an
aqueous solution at 2.5, 5, or 10 mglkg body weight. In
some experiments [3HJDMNwas suitably diluted with nonra
dioactive DMN and was injected at 0.5 mg/250 @Ci/100g
body weight. In matsgiven injections of [3H]DMN, the liver
DNA was not prelabeled with radioactively labeled thymi
dine.
Preparation of Liver ChromatIn. Hepatic chromatin was
prepared essentially by the method described by Mc
Conaughy and McCarthy (24) with a few minor modifica
tions. The liver was suspended in a buffer (3 g/18 ml)
containing 0.075 M NaCI, 0.024 M EDTA (pH 8), and 0.1%
Triton X-100 and homogenized using a Potter-Elvehjem
glass homogenizer fitted with a Teflon pestle. After an inter
val of 15 to 20 mm on ice, the homogenate was centrifuged
in an International Model PA-6 refrigerated centrifuge at
3000 rpm for 10 mm. The nuclei were washed 3 times with
0.075 M NaCI:0.024 M EDTA (pH 8.0) containing 0.1% Triton
x-100 and 3 times with 0.05 M Tris-HCI (pH 7.5) without
Triton X-100, using 20 ml of the buffer each time. The nuclei
were finally washed once with 0.01 M glycine (pH 6.0) and
resuspended in 35 ml of the same gbycine buffer. The sus
pension was maintained at 4°in 0.01 M glycine (pH 6.0) for
16 hr to solubilize the chromatin. The chromatin was then
sheared using a VirTis Model 45 homogenizer, at a setting
of 40, for 90 sec in 3 bursts of 30 sec each at 4°.The
preparation was then centrifuged for 30 mm at 10,000 x g in
a Sorvall Model RC2B centrifuge using an 55-34 rotor to
pellet any insoluble material. This pellet contained less than
5% of DNA. In some experiments liver chromatin was pre
pared by the method described by Marushige and Bonner
(23). Identical patterns of results were obtained whether the
chromatin was prepared by the method described by Maru
shige and Bonner (23) or that of McConaughy and McCarthy
(24).
Preparation of Liver DNA. Liver DNA was prepared by
homogenizing the liver in a buffer containing Triton X-100
as used in the preparation of chromatin. The resulting nu
2074
clear pellet was used for the extraction of DNA by the
method of Marmum (22).
Digestion of Liver Chromatin DNA with DNase. The incu
bation conditions were adopted from those described by
Clark and Febsenfeld (3) and Mirsky (28). The sheared chro
matin DNA was incubated with micrococcab nuclease (4 @g/
ml) or pancreatic DNase I (4 pg/mI) in an incubaticxn mixture
that contained chromatin DNA (150 to 200 @g/ml),and 5 mM
sodium phosphate buffer, pH 6.7. In addition, 2.5 x 10@ M
CaCI2 was included in experiments where micrococcab
nuclease was used and 1 mM MgCI2 was used when pan
creatic DNase I was used. Aliquots were withdrawn at differ
ent time intervals, and the reaction was stopped by adding
cold 1.0 N PCA to give a final concentration of 0.25 N. Cold
acid-soluble absorbance at 260 nm (A2,@
nm)and radioactiv
ity were measured (28). The zero time and the endogenous
reactions were carried out simultaneously. The results were
expressed as the percentage of total DNA of the chromatin.
This value was obtained by digesting the chromatin (150 to
200 @tg
of chromatin DNA per ml) initially with autodigested
(5) Pronase (250 @g/ml)in the presence of EDTA for 30 mm
at 30°.The reaction was arrested with 0.25 N cold PCA and
centrifuged. The pellet was hydrolyzed with 0.50 N PCA at
70°for45 mm. After centrifugation at 3000 rpm for 15 mm in
an International Model PA-6 refrigerated centrifuge, the
clear supernatant was used for the measurement of absorb
ance at 260 nm and for radioactivity determination. Since
the content of RNA after alkaline hydrolysis was only 2 to
3%, the data have not been corrected for RNA contamina
tion.
Radioactivity Measurements. The radioactivity in the
acid-soluble fractions was determined with a Triton :tobuene
scintillation mixture at an efficiency of 30% using an Inter
technique liquid scintillation spectrometer.
Determination of Protein, DNA, and RNA. Protein, DNA,
and RNA were determined by the methods described by
Lowry et a!. (21), Burton (1), and Schneider (33), mespec
tively.
Determination of Guanine Content. The guanine content
was determined in total chromatin DNA, in acid-soluble
supemnatant after 90 mm of enzymic hydrolysis, and in un
hydrolyzed chromatin by acid hydrolysis (0.1 N HCI, 1 hr at
100°).The bases and the nucleotides were separated by
thin-layer chromatography on cellulose plates (MN 300 G)
using a solvent system consisting of isopropyl alco
hol:HCI:water [65:17.2:17.8, v/v/v (11)].
Determination of 7-Methylguanine in Enzyme Digests of
Chromatin DNA. The nuclease-released acid-soluble oligo
nucleotides were acid hydrolyzed at 70°for 30 mm, a treat
ment that converts the oligonucleotides to pyrimidine nu
cleotides and purine bases (20). In some experiments, the
amount of 7-methylguanine in the enzyme digests was de
termined without the acid hydrolysis. The hydrolysate was
transferred to a Dowex 50- X8 column equilibrated with 0.5
N PCA and eluted
with
batches
of 0.5,
1 .0, and 2.0 N PCA.
Standard 7-methylguanine was eluted in the 3rd fraction
(2.0 N PCA fraction). The earlier fractions contained the
pyrimidine nucleotides. The amount of methyl label ebuted
in the 1st 2 fractions comprised of at beast20% of the total
DNA methyl label, and the rest was eluted in the fraction
CANCER RESEARCH VOL'.36
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Nonrandom Methy!ation of Liver Chromatin DNA
with standard 7-methylguanine. In a few experiments 7methylguanine was eluted with a linear gradient of 0.5 to 4 N
HCI from the Dowex 50-X8 columns (25) which were equibi
brated with 0.5 N HCI.
Table 1
Content of guanine inDNARelative
the DNaseI digests of chromatin
concentration
(@tmole)in of guanine
chromatin
DNA90
RESULTS
TreatmentTotal
DNAControl0.126
In this communication, the terms “nuclease
accessible―
or “digestible'
‘
and “nuclease
inaccessible' ‘
or ‘
‘nondigesti
ble―are used operationally to represent those regions that
are rendered acid soluble and acid insoluble, respectively,
by nuclease digestion of chromatin DNA. Both pancreatic
DNase I and micrococcal nuclease were used in our initial
studies. Since essentially the same patterns of results were
obtained with either of the enzymes, the data with pan
creatic DNase I are presented. Chart 1 shows some of the
properties of the liver chromatin DNA methylated in vivo by
DMN for 4 hr. The results show that methylation of chroma
tin DNA has not affected its susceptibility towards nuclease
digestion. Further, the kinetics of nuclease digestion was
identical whether the digestion was followed by measuring
the absorbance at 260 nm of the released acid-soluble nu
cleotides or by measuring radioactivity when chromatin
DNA was prelabeled with [3H-methy!]thymidine. The
DNA:pmotein ratio of the methylated chromatin was the
same as that of the nonmethylated chromatin . In Table 1 are
shown the guanines content of nuclease-accessible and
-inaccessible regions of chromatin DNA. The results reveal
that both these regions have the same guanine content.
Having established that (a) the methylation of chromatin
DNA by DMN does not alter its susceptibility towards nu
clease digestion, (b) under the experimental conditions
about 50% of the methylated chromatin DNA is nuclease
susceptible and the other 50% is nuclease resistant, and (c)
(50.8)DMM0.093
a Numbers
in total
in
b Rats
chromatin
digestion.
chromatin
were
@
50
@
E 40
-0
@
CC
a@'J 30
0.5
@
0
20
@
IC
0
10
20
30
40
50
6@'
90
20
TIME OF INCUBATION(MIN)
Chart 1. Kinetics of digestion of [3H]thymidine-labeled liver chromatin
DNA of control rats and of rats given injections of DMN (5 mg/kg; 4 hr) by
pancreatic DNase I. The incubation medium contained 3 to 4 A2RO
units of
DNA in 5 mM phosphate buffer (pH 6.7), 1 mM MgCI2, and 4 @.og
of the enzyme
per ml. Reaction was monitored by measuring the absorbance at 260 nm (A,
control; & DMN) and the radioactivity (•,control; 0. DMN) of the ice-cold
0.25 N PCA-soluble material at different times during the course of the
incubation. At zero time, only 2 to 3% and at 90-mm incubation period 7 to
10% of the digestion due the endogenous reaction was obtained. All the
results presented are corrected for endogenous values.
JUNE
90-mm lef
tover
(100)―
0.062
(49.2)
0.046
0.047
(100)
(49.5)
(50.5)
percentages
of
total
0.064
guanine
present
DNA.
given
DMN
(5
mg/kg)
and
were
killed
4 hr
later.
Liver
was isolated and an aliquot was subjected to nuclease
The guanine (+ 7-methylguanine)
content of the total
DNA after 90 mm of enzyme digestion was determined
after mild acid hydrolysis and chromatography on thin-layer chro
matographyon cellulose layers (11). Results are the averageof 5
animals.
the guanine content of the susceptible and the resistant
regions is identical, we proceeded to study the distribution
of methylated products in these 2 regions of chromatin
DNA. This is achieved by labeling the chromatin DNA with
radioactive methyl groups from [3H]DMN for 4 hr in vivo , the
time at which maximal methylation occurs, and then sub
jecting the chromatin DNA to nuclease digestion. The radio
activity present in the acid-soluble fraction of the nuclease
digest would be a measure of methybated products, and the
absorbance at 260 nm in the same fraction represents the
total nucleotides released during nuclease digestion of the
accessible regions of the chromatin DNA. Since we already
know (Chart 1) that the nucleotides released during the
enzyme incubation are about 50%, any deviation from this
figure for the methylated products released would suggest a
nonrandom methylation of chmomatin DNA. The results pre
sented in Chart 2 indicate that the release of radioactivity,
i.e. , methylated
60
parentheses,
chromatin
DNA
ofenzyme
mm
hydrolysate
products,
is always higher than the absorb
ance at 260 nm during the entire period of digestion. 5ev
enty-two % of the methylated products is released in con
trast to the 50% release of nucleotides (A260nm)
during an
incubation of 120 mm. In 4experiments micrococcal nude
ase also released 50% of nucleotides (A2,@
nm)and 71% of the
radioactivity. Increased release of radioactivity was ob
tamed even when a bower concentration of nucbease was
used (Chart 3). When total DNA methylated in vivo and pun
fied from liver chromatin was used instead of chromatin as
the substrate in the nuclease digestion reaction, the rates of
release of nucleotides and the methylated products were
identical (Chart 4). This rules out the possibility that pan
creatic DNase I preferentially attacks at or near the methyl
ated site. Such an increased release of methylated products
can also occur if the pancreatic DNase I used in the expeni
ment is contaminated with an activity capable of excising
preferentially methylated bases. In order to assay whether
such an activity is present in the DNase I preparation, the
enzyme digest was analyzed for the free 7-methylguanine
(the major alkylated base formed in DNA by DMN adminis
tration) using Sephadex G-10 columns according to the
procedure of Lawley and Shah (18) and also using Dowex
1976
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1976 American Association for Cancer Research.
2075
A. Ramanathanet a!.
The enhanced release of radioactive methylated products
in the nuclease digest could not in part be due to the release
of the radioactivity trapped within the chromatin complex
because of 2 reasons: (a) the radioactivity found in the
nuclease-resistant DNA plus the radioactivity released in the
supernatant (nuclease-accessibbe DNA) after digestion
equalled the total acid-precipitable radioactivity in the onigi
nabchromatin DNA following extensive deproteinization by
Pronase (see Table 2); (b) the percentage of release of
radioactivity was calculated after subtracting the blank
value derived by incubating the chromatin under identical
conditions to that of the reaction but in the absence of the
added pancreatic DNase I. Cold acid-soluble blank value
represents both the radioactivity due to endogenous en
zyme and that trapped within the chromatin complex. Since
4.
C@.
; .@
at
TIME OFINCUBATION
(MIN)
@
@
Chart 2. Nuclease digestion of liver chromatin DNA from rats given injec
tions of rH]DMN. Four hr after @H)DMN
injection (0.5 mg/250 @.tCi/l00g
body weight), rats were killed, and liver chromatin was purified and sub
jected to pancreatic DNase I digestion as detailed in the legend to Chart 1 and
“Materials
and Methods.―The 0.25 N PCA (ice-cold)-soluble AN,,,...@
(•)was
measured to determine the nucleotides released, and the acid-soluble radio
activity ( 0) was monitored to estimate the carcinogen radioactivity bound to
the released nucleotides. The results were expressed as the percentage of
total DNA nucleotides and of DNA-bound carcinogen radioactivity, respec
tively. Suitable blank reactions were run and taken into account for the
computation of values.
@
z .@
;
00
80
q 60
z
40
20
4.
z.
0
0
20
30
40
50
60
TIME OFINCUBATION
IMIN)
Chart 4. Digestion of purified in vivo alkylated liver DNA by pancreatic
DNase I. Four hr after [‘H]DMN
(0.5 mg/250 @zCi/100
g body weight) injection,
liver DNA was purified from the nuclear pellet by the method of Marmur (22).
The purified DNA was subjected to DNase I digestion under the conditions
described in the legend to Chart 1. The cold acid-soluble A2@nm(•)
and the
radioactivity (x) were measured. The results were expressed as the percent
ages of total DNA nucbeotides and DNA-bound carcinogen radioactivity,
respectively.
00
z
Table 2
Specific
fractionsLiver
radioactivity of methyl groups in chromatin DNA
periodsafter
chromatin was isolated from rats at different time
andsubjected
[3H]DMN(0.5 mg/250 .oCi/100g body weight) injection
TIME OF INCUBATION(MIN)
Chart 3. Nuclease digestion of liver chromatin DNA from rats given injec
tions of [‘H]DMN.
The details are the same as described in the legend to
Chart 2 except that the enzyme concentration was only 0.8 @tg/mlinstead of 4
@&g/ml.
Enzyme reaction was monitored by measuring the absorbance at 260
nm (•)
and the radioactivity (x), in the ice-cold 0.25 N PCA-soluble material.
ofDNase-digestible
to nucbease digestion. From the specific radioactivity
chromatin,the
and -nondigestible
DNA segments of
amount of methyl groups incorporated was calculated. Nu
totalchromatin
dlease“nondigestible―
DNAsegmentsof chromatin DNAand
DNAwereextensivelydeproteinizedand the PCAprecip
itates were hydrolyzedin the 0.5 N PCAat 70°
for45 mm. Radioac
tivity
50-X8 columns.
From 7 to 10% of total
radioactivity
of
enzymatic digest appeared as 7-methylguanine; this may be
due to the depunination during the acid precipitation of
chromatin at the termination of reaction, because this
amount of radioactivity, i.e., 7 to 10% was released as 7methylguanine even when DNase I was omitted from the
reaction mixture. However, in 3 experiments, analysis of
acid hydrolysate of the enzymatic digest revealed that 72%
of the total 7-methylguanine present in DMN-treated rat liver
chromatin DNA was hydrolyzed by pancreatic DNase I in 120
mm.
2076
and absorbance
at 260 nm were determined
in the acid
hydrob
ysates.The results are
experiments.Methyl
the averageof 4 separate
ofDNA)Time
groups incorporated (pmobes/mg
DNase-resistantDMN
after [3H]- Total chromatin
fraction4
injection
DNA
DNase-digesti-
ble fraction
2883
hr
462
673
(27)1
days
244 (47)―
255 (62)
210
(49)2
wk
106 (77)
87 (87)
147
wk
a Numbers
34 (93)
in
parentheses,
40 (94)
percentages
of
loss
40 (86)
of
DNA-bound
methyl groups as compared to the 4-hr value.
CANCER RESEARCHVOL. 36
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Nonrandom Methy!ation of Liver Chromatin DNA
the increased release of methylated bases in the accessible
region of the chromatin is explainable neither on the basis
of a preferential attack at or near an alkylated base in the
DNA nor to a contaminating enzyme capable of selectively
removing methylated bases, it may be concluded that the
nuclease-accessible regions of chromatin DNA are selec
tively methylated to a greater extent than that of the mac
cessible regions.
Hav'ng established an increased level of alkylation in the
accessible region of chromatin DNA, we examined the pat
@emns
of in vivo removal of methylated products in these 2
fractions of chromatin. To study this, the amount of methyl
ated products present in the 2 regions was assyed using
DNase I in the same manner as described above at different
periods of time such as 4 hr, 3 days, 1 week, and 2 weeks
following [:IH]DMN administration. The results are summa
nized in Table 2 and Chart 5. At 4 hr 673 pmoles of methyl
groups per mg of DNA were incorporated in the accessible
regions and 288 pmoles were incorporated in the inaccessi
ble regions of chromatin DNA after DMN administration.
Similar, higher methylation of accessible regions compared
to that of inaccessible regions was observed whether we
injected a lower (0.25 mg/100 g) or a higher (1.0 mg/100 g)
dose of DMN. Four hr after DMN (0.25 mg/100 g) adminis
tration 341 pmobes of methyl groups per mg of DNA were
incorporated in the accessible regions and 162 pmoles were
incorporated in the inaccessible regions of liver chromatin
DNA. When a higher dose of DMN (1.0 mg/100 g; 4 hr) was
injected , 1179 pmoles of methyl groups per mg of DNA were
incorporated in the accessible regions and 641 pmobeswere
incorporated in the inaccessible regions of liver chromatin
DNA. The removal of the methylated products from the
chromatin DNA was studied using 0.5 mg of DMN per 100 g
body weight. The data shown in Table 2 and Chart 5 reveal
that of the methylated products present at 4 hr in the acces
sible regions 62% was lost by 3 days, 87% was lost by 1
week, and 94% was lost by 2 weeks. However, from the
inaccessible regions of chromatin DNA boss of methylated
products was only 27% by 3 days, 49% by 1 week, and 86%
by 2 weeks. Thus, not only is the methylation of the macdes
sible regions of chromatin DNA relatively bower than that of
the accessible regions but also the rate of removal is slower.
Further, the kinetics of the loss of methylated products from
these 2 regions are different.
DISCUSSION
The results described in this communication indicate
clearly that digestion of liver chromatin DNA either by pan
creatic DNase I or by staphylococcal nuclease results in the
release of 50% of DNA as acid-soluble oligomems as mea
sured by the absorbance at 260 nm. Neverthbess, when the
chromatin DNA was methylated in vivo by [3H]DMN and
subjected to nudlease digestion, the radioactivity associ
ated with the released acid-soluble material was found to be
72% and not 50% as given by the absorbance measure
ments of the same acid-soluble material. This increased
release of radioactive methylated nucleotides could not be
ascribed to either a preferential attack by DNase I at or near
the alkylated products or to an increased guanine content in
I-.
>
IC)
‘4
0
L@
0
Id
0.
On
z
Id
0
Id
0.
TIME AFTER[3H]DMN
INJECTION
Chart 5. in vivo loss of methyl groups from methylated liver chromatin
DNA. Rats were given injections of [3H]DMN (0.5 mg/250 @Ci/10O
g body
weight) and were sacrificed at 4 hr, 3 days, 1 week, and 2 weeks after the
carcinogen injection. Liver chromatin was purified and subjected to nuclease
digestion
for 90 mm. The specific
radioactivity
of the DNase-digestible
( 0),
and- nondigestible (A) DNA segments of chromatin and of the total chroma
tin DNA after extensive deproteinization (•)were determined. The specific
activity (dpm/mg DNA) at 4 hr has been taken as 100°!.,and the radioactivity
at other time intervals was computed as percentage of specific radioactivity
at 4 hr.
the nudlease-accessible regions. Thus the results are
strongly in favor of a preferential methylation of the DNA in
the nuclease-accessible regions of chromatin DNA. Further,
the rate of removal of alkylated products in vivo is faster in
this region than in the nuclease-inaccessible regions.
It has been
known
that certain
carcinogens
remain
bound
to DNA for long periods of time following their interaction
with cellular DNA. The persistence of the derivatives of 2acetybammnofluomene(6, 14, 36), dimethylaminoazobenzene
(37), and 7-12 dimethybbenz(a)anthracene (15) in the cebbu
lamDNA has been reported. It would be of great interest now
to study whether, in these instances, the carcinogen is
associated with inaccessible or accessible regions of chro
matin DNA.
Our knowledge regarding the activation of carcinogen
and the nature and extent of interaction of the active forms
of carcinogens with DNA and ANA is abundant. However,
the intragenomic distribution of the carcinogen or its deny
atives is rather rare. Using cytochemical techniques Cas
persson et a!. (2) have reported that the binding of quina
crine mustards was limited to certain defined regions of
chromosomes. Similarly, a nonmandom intrachromosomal
distribution of chromatid aberrations induced by X-rays and
alkylating agents has been reported in Vicia faba (31). How
ever, Zeiger et a!. (39) have reported that the extent of
binding of 9,10-dimethyb-1 ,2-benz(a)anthracene to mouse
skin satellite and main band DNA is identical.
Few studies were reported on the intragenomic distribu
tion of repair of DNA damage. Measurement of repair may
not represent the intragenomic distribution of the carcino
JUNE 1976
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2077
R. Ramanathanet a!.
gen. Meltz and Painter (26) using HeLa cells and Leiberman
and Poinier (19) using human dipboid fibrobbasts found simi
barrates of repair as measured by unscheduled DNA synthe
sis in the repetitious and unique sequences of DNA when
either UV or chemical carcinogens are used as DNA damag
ing agents. On the contrary, Harris et a!. (9) reported a
nonrandom distribution of repair, namely an enhanced rate
of repair in euchnomatin as compared to heterochromatin.
In this study human diploid fibroblasts treated with UV or
chemical carcinogens N -acetoxy-2 - acetylaminofluorene
and 7-bromo-methylbenz(a)anthracene were used , and the
repair was measured by unscheduled DNA synthesis using
an autoradiographic
technique. Similarly, Fahmy and
Fahmy (7) have reported mainly r-gene mutations in Dro
sophila on exposure to a variety of carcinogens. The dis
crepancies in the results reported in the above studies may
be due to (a) the difference in the chemical and physical
nature of the agents used in different studies, (b) the sensi
tivity and the nature of the techniques used to fractionate
the genetic material, or (C) a combination of both.
The utilization of the susceptibility of chromatin contain
ing DNA prelabebed in vivo with a carcinogen to nuclease
and analyzing the released acid-soluble products for radio
activity and absorbance at 260 nm as is done in the present
study seem to offer promise in the elucidation of the mecha
nisms involved in the intragenomic distribution of the car
cinogen and its subsequent removal. As pointed out under
“Introduction,―
certain regions of mammalian DNA are coy
ered with proteins, and hence the 2 types of DNA segments
may exhibit differential reactivity towards chemical carcino
gens as well as repair enzymes. The availability of the 2
types of the regions of the mammalian DNA in vivo towards
carcinogen interaction may to a large extent depend on the
chemical nature of the carcinogen.
It is not yet known whether the slow rate of removal of
methylated products from the nuclease-inaccessible me
gions of chromatin DNA is due to the chemical nature of the
methylated product present such as O-6-methylguanmne, N7-methylguanine, N-3-methyladenine, and phosphotniester
or that the inaccessible regions of the chromatin DNA are
relatively less accessible to repair enzymes in vivo . In a brief
report Wilkins and Hart (38) have suggested that nuclear
proteins may mask some areas of DNA damage from repair
enzymes.
The biological significance of either the enhanced rate of
removal of the methylated products from the nudlease-ac
cessible regions of the liver chromatin DNA or the slower
rate of removal from the nuclease-inaccessible regions of
the chromatin DNA is not clear at present.
The existence of a relationship between the extent of
alkylation of nuclear DNA and carcinogenic potential of the
monofunctional alkylating agent could not be established. It
is likely that a study of the intragenomic distribution of
alkybated products and the rate of their subsequent removal
from different regions of chromatin DNA may throw some
light on the relationship between the carcinogen-DNA inter
action and carcinogenic potential of the agent.
Since the submission of this manuscript, Cooper et a!. (4)
have also reported a nonrandom distribution of DMN-in
duced methyl groups in rat liver chromatin DNA. However,
2078
the pattern of distribution is different from that reported in
this manuscript, possibly because they have used a slightly
different procedure to fractionate the liver chromatin DNA.
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2079
Nonrandom Nature of in Vivo Methylation by
Dimethylnitrosamine and the Subsequent Removal of
Methylated Products from Rat Liver Chromatin DNA
R. Ramanathan, S. Rajalakshmi, D. S. R. Sarma, et al.
Cancer Res 1976;36:2073-2079.
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