[CANCER RESEARCH 38, 2135-2143.
0008-5472/78/0038-0000$02.00
July 1978]
De Novo and Repair Replication of DMA in Liver of Carcinogen-treated
Animals
V. M. Craddock and A. R. Henderson
Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carsha/ton, Surrey, SM5 4EF, England
ABSTRACT
The effect of certain carcinogens on DNA replication in
the intact animal was studied for the determination of
whether repair replication was induced and whether there
was a change in the rate of de novo replication. Carcino
gens were selected to include those known to react with
DNA and those for which there was apparently no pre
vious evidence for reaction with DNA in vivo. The method
used depends on the increase in nuclear size that occurs
in replicating cells. Replicating and nonreplicating diploid
and tetraploid nuclei were fractionated in a sucrose gra
dient in a zonal rotor. Incorporation of [mef/iy/-3H]thymidine
into replicating nuclei measured de novo replication of
DNA, and hydroxyurea-resistant incorporation into nonreplicating nuclei measured repair replication.
Diethylnitrosamine, ethyl methanesulfonate, aflatoxin,
and retrorsine were shown to induce DNA repair replica
tion in vivo but not to alter de novo synthesis two hr after
injection. Carbon tetrachloride and ethionine also induced
repair replication but only after a delay period. This
suggests that the repair was that of damage caused by an
indirect mechanism, such as by deoxyribonuclease activ
ity resulting from lysosomal damage (carbon tetrachlo
ride) or from nonenzymic reaction of DNA with a metabo
lite of the carcinogen that is slow to accumulate in liver
(S-adenosylethionine after ethionine). Thioacetamide did
not cause detectable repair replication, a result that
correlates with the lack of evidence for a reaction between
thioacetamide and DNA.
INTRODUCTION
Over the last 10 years, it has become apparent that the
potential of a chemical to induce cancer depends not only
on the nature of the genetic damage it causes but also on
the rate of cell replication in the target tissue at the time of
treatment and on the rate at which the cell repairs DNA
damage. Much evidence has accumulated to suggest that
replicating cells are especially sensitive to carcinogens (10,
52). There is also good evidence that the ability of the cell
to repair damage to DNA is an important consideration in
carcinogenesis (51). For certain chemicals to induce can
cer, it is possible that DNA replication must occur to convert
a transitory abnormality in DNA caused by reaction with the
carcinogen into an inheritable change such as an alteration
in base sequence. Obviously, for this to take place, the DNA
must replicate before the damage caused by reaction with
the carcinogen has been repaired by any of the repair
mechanisms in the cell. Therefore, information concerning
Received June 27, 1977; accepted March 9, 1978.
JULY
de novo and repair replication of DNA after treatment with
carcinogens is of much interest.
In the case of liver, a single treatment with any carcinogen
very rarely causes cancer. This may be because liver nor
mally has a low mitotic index (24) and a high capacity for
repair of DNA damage (22, 29, 34). Although certain aspects
of repair have previously been studied in the intact animal
(7, 8, 16, 35), there is little information concerning resynthesis of the excised stretch of DNA, i.e., unscheduled
synthesis.
For measurement of repair replication in vivo, some use
has been made of the technique involving prelabeling of
DNA with bromodeoxyuridine (21). However, there is evi
dence that bromodeoxyuridine itself causes repair replica
tion (4), and it is also possible that the presence of the base
analog in DNA might affect repair of damage caused by
other agents. An alternative method for studying unsched
uled synthesis in vivo is based on the fact that an increase
in nuclear size occurs before de novo replication of DNA
takes place (25) while apparently there is no evidence that
repair replication is associated with nuclear swelling.
Therefore, nonreplicating nuclei can be separated from
replicating nuclei in a sucrose gradient in a zonal centri
fuge, and HU1-resistant incorporation of [3H]dThd into non
replicating nuclei indicates the presence of repair replica
tion (12). As in other systems, HU was shown to reduce de
novo synthesis to a very low level without inhibiting repair
replication (12). Repair synthesis occurred after treatment
of rats with dimethylnitrosamine or methyl methanesulfo
nate, compounds known to react with DNA in vivo but not
after treatment with cycloheximide, a compound that re
duces DNA synthesis indirectly by inhibition of protein
synthesis but that is not known to cause direct damage to
DNA (12).
This technique has now been used to study de novo and
repair replication of DNA in vivo after treatment of animals
with other carcinogens. The compounds ranged from those
that were known to react with DNA and that appeared to be
likely to induce repair synthesis to those for which there
was apparently no evidence for reaction with DNA. Inas
much as 1 injection of DENA results in ethylation of guanine
residues in liver DNA (47), it was of interest to determine
whether DENA induced unscheduled synthesis in the intact
animal. EMS was studied because this compound also
ethylates DNA in vivo (47), but it has not been shown to
induce liver cancer by a single treatment (11). Aflatoxin also
reacts with DNA in vivo (49), but the evidence that it induces
repair replication in the intact animal is limited (27). Metab
olites of certain pyrrolizidine alkaloids react with DNA in
1The abbreviations used are: HU, hydroxyurea; [3H]dThd, [methyl3H]thymidine; DENA, diethylnitrosamine; EMS, ethyl methanesulfonate.
1978
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
2135
V. M. Craddock and A. R. Henderson
vitro (53), but there was apparently no evidence for reaction
of pyrrolizidines with DMA in liver of the intact animal or for
the repair of DNA damage in vivo. The occurrence of
unscheduled synthesis of DNA after treatment with retrorsine, a pyrrolizidine shown to induce liver cancer (42), was
investigated.
A few liver carcinogens are anomalous in that they have
not been shown to react with DNA in vivo or in vitro.
However, these compounds could damage DNA by forming
unstable adducts that rapidly decompose, thereby eluding
detection. Alternatively, they could damage DNA indirectly,
as by activation of nuclear or lysosomal nuclease. In the
case of CCI4, there is evidence that the compound reacts
with a nucleic acid fraction in rat liver (40) but the reaction
occurs only with rRNA and not with DNA (41). With ethionine, the level of reaction with DNA/'n vivo was exceedingly
small (48). In the case of thioacetamide, there was appar
ently no evidence for any reaction with DNA.
The occurrence and timing of de novo and repair repli
cation after administration of these carcinogens has been
studied.
MATERIALS
AND METHODS
Animals. Female (200-g) 9-week-old Wistar rats of the
Portónstrain were used.
Chemicals. Carcinogens were obtained from the follow
ing sources: DENA, Eastman Kodak, Liverpool, England;
EMS, Koch-Light Laboratory Ltd., Colnbrook, Bucking
hamshire, England; retrorsine, a gift from Dr. R. Mattocks
of the Toxicology Unit; aflatoxin B,, Makor Inc., Jerusalem,
Israel; thioacetamide, British Drug Houses, Poole, Dorset,
England; CCU, Fisons Scientific Apparatus Ltd., Loughborough, Leicestershire, England. L-ethionine, Calbiochem,
Bishops Stortford, Herfordshire, England. [methyl-3H]Thymidine (2.0 Ci/mmol) was purchased from The Radiochemical Centre, Amersham, Buckinghamshire, England.
Measurement of de Novo and Repair Replication. Four
rats were used in each experiment. Animals were given a
single i.p. injection of carcinogen dissolved in 0.9% NaCI
solution (DENA, EMS, retrorsine, thioacetamide, and Lethionine) or in dimethyl sulfoxide (100 /¿I)(aflatoxin B,).
CCI, was mixed with an equal volume of liquid paraffin and
administered by stomach tube. Either 2, or 17 hr later in
experiments in which repair replication was being studied,
the animals were given an i.p. injection of HU (500 mg/kg)
followed 10 min later by an i.p. injection of [3H]dThd (100
/iCi/animal). In experiments measuring de novo replication,
the injection of HU was omitted, and [3H]dThd was injected
2 hr after the carcinogen. Animals were killed 1 hr after
injection of [3H]dThd, the livers were removed, and 4 g of
each of the 4 livers were pooled for isolation and fractionation of nuclei by zonal centrifugation as previously de
scribed (12), except that centrifugation on the zonal rotor
was for a period of 25 min.
From the 10-ml fractions of effluent collected from the
zonal rotor, duplicate 3-ml samples were precipitated with
perchloric acid, washed, dried, and solubilized from the
Millipore filter with Nuclear Chicago solubilizer as previ
ously described (12). For determination of radioactivity, 1
of each duplicate sample was treated with 5 ml PPO, 0.6%
2136
in toluene, and the duplicate sample was treated with 5 ml
Dimilume (Packard Instrument Company, Inc., Reading,
England). Use of Dimilume avoided the need to keep the
samples cold and dark for 24 hr to abolish chemiluminescence before counting. Efficiency of counting in each case
was 35 to 37%. At least 1000 counts were measured in each
of the samples over the radioactive regions of the effluent.
The duplicate samples gave essentially identical results.
Each zonal profile shown represents the result of 1 experi
ment. Evidence has been given previously (12) that labeling
observed after treatment with carcinogen, HU, and
[3H]dThd was not found if the samples were incubated with
DNase.
The DNA content of peak fractions of effluent was deter
mined with the modification of Giles and Myer, (19) of the
method of Burton (5). Concentrations of nuclei were mea
sured on an improved Neubauer hemacytometer under
phase contrast. Counting of nuclei was begun soon (within
approximately 20 min) after collecting the diploid and tetraploid samples. At least 1000 nuclei were counted initially in
each case. Microphotographs of the diploid and tetraploid
nuclei were then taken under phase contrast to establish
that aggregation or damage had not occurred. Fresh nu
clear suspensions were then prepared on a hemacytometer,
and the counting was repeated (approximately 2 hr later).
The concentration of nuclei had usually decreased by 10 to
15%, presumably as a result of lysis. The initial count was
used in calculation of dpm/108 nuclei.
RESULTS
AND DISCUSSION
One injection of a carcinogen rarely induces liver cell
cancer unless it is preceded by an independent stimulus for
mitosis (10). The explanation may be that the DNA damage
caused by the carcinogen is repaired before DNA replica
tion takes place, so that there is little replication of dam
aged DNA. Experiments were carried out for the determi
nation of whether 1 injection of the carcinogen caused a
rapid onset of repair replication and whether the rate of de
novo replication was affected.
De novo replication was studied by measuring incorpo
ration of [3H]dThd into replicating diploid and tetraploid
nuclei that were separated from nonreplicating nuclei on
the basis of their faster sedimentation in a sucrose gradient.
Chart ÃŒA
shows the de novo replication as occurring during
the hr following injection of [3H]dThd. The light-scattering
profile locates the bulk of the nuclei, which are nonreplicat
ing diploids and nonreplicating tetraploids. In Chart 1, the
results obtained with normal animals are shown for com
parison with carcinogen-treated animals. Fig. 1 shows that
the nuclei in the tetraploid peak are larger than those in the
diploid peak. If approximately 0.01% of nuclei in adult rat
liver is in mitosis (24) and if S phase is approximately 10
times the duration of mitosis (6),,then only 0.1% of the
nuclei would be in S phase. This is not a sufficient number
to be apparent on the light-scattering profile. However,
nuclei in S phase are larger and faster sedimenting, so that
diploids in S phase are separated from noncycling diploids,
and tetraploids in S phase have sedimented further than
have non-S-phase tetraploids. The 3H profile shows the
location of replicating diploids and replicating tetraploids.
CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
Repair and de Novo Synthesis of DNA in Carcinogenesis
gests that there may be more repair replication in tetraploid
nuclei per unit DNA (Table 1).
J400
With reference to the repair observed after treatment with
different doses of DENA, it is seen that the intermediate
dose induced the largest amount of repair replication (Table
1). In dimethylnitrosamine, a dose dependence had been
found, especially in the case of tetraploid nuclei (12), and
later work showed that the curve flattened out at higher
dose levels (V. M. Craddock and A. R. Henderson, unpub
lished results). These results correlate with other evidence
for the nonlinearity of dose response for unscheduled
synthesis after treatment with alkylating agents (50). The
fall
off at higher dose levels may result from the alkylation
40
30
20
of protein including, presumably, enzymes involved in DNA
Fraction numtwr
répairand from the saturation of some or all of the enzymes
involved in the process (28).
EMS. De novo DNA synthesis was not significantly af
60 3
fected in diploid or tetraploid nuclei 2 hr after injection of
EMS (500 mg/kg) (Chart 2C; cf. Chart 1A). However, exper
iments with HU and EMS showed that repair replication was
taking place at this time (Chart 20). Diploids and tetraploids
were approximately equally affected; twice as much
[3H]dThd was incorporated into the tetraploid nuclei with
twice the DNA content (Table 1). Rapid onset of repair of
DNA damage, together with the fact that probably less of
this mispairing base, O6-ethylguanine, is formed in relation
00
30
20
40
to 7-ethylguanine after treatment with EMS (44) than after
Fraction number
DENA, could explain why EMS is not a liver carcinogen.
Chart 1. Zonal profile of liver nuclei prepared from normal rat, 200 g body
weight, fractionated as described in text. Animals were treated with (A) The results obtained with EMS show that the induction of
[3H]dThd and (8) HU followed 10 min later by [3H]dThd and were killed 1 hr
repair synthesis should be taken as evidence that the
after injection of [3H]dThd.
, light scattering at 254 nm in arbitrary units.
compounds concerned cause repairable genetic damage
U, membrane fraction; D, nonreplicating diploid nuclei; T, nonreplicating
tetraploid nuclei. Stepwise profile, cpm acid-insoluble 3H in 3 ml of 10-ml
but not that they are necessarily capable of inducing can
fractions, showing diploids in S phase (OS) and tetraploids in S phase (TS).
cer.
Arrow, direction of sedimentation (republished from Ref. 12 by permission
Aflatoxin. Aflatoxin (0.5 or 2.0 mg/kg) did not inhibit de
of Elsevier Publishing Corp., Amsterdam, The Netherlands).
novo DNA synthesis (Chart 3A). Botti dose levels caused
repair replication (Chart 30); the extent of unscheduled
The membrane fraction probably represents contamination
synthesis was higher after the larger dose (Table 1). As
of the original nuclear preparation by plasma membranes.
mentioned previously, the extent of repair replication per
The radioactivity between the membrane fraction and the nucleus in tetraploids would be double that in diploids if
diploid nuclei has not been characterized (12), but no DMA DNA damage and repair were uniformly distributed
was detectable by the diphenylamine reaction in fractions
throughout the DNA. While the higher dose of aflatoxin
from this region of the gradient. Treatment with HU (500 affects both cell types equally, selectivity of action on
mg/kg) reduced the incorporation of [3H]dThd to a very low tetraploid nuclei was suggested by the low-dose experi
ment, in which the tetraploid level is more than twice the
level (Chart 18).
DENA. Chart 2A shows the de novo replication that diploid level (Table 1). This correlates with the fact that
occurs when [3H]dThd was given 2 hr after injection of aflatoxin apparently has a selective toxic action on the
DENA (250 mg/kg). The extent of incorporation of [3H]dThd tetraploid population (33). The female Porton-derived Wisinto replicating nuclei (diploids and tetraploids in S phase) tar rats used in these experiments are less sensitive to
was within the normal range (cf. Chart ^A). When de novo aflatoxin than are the male Fischer rats used in the experi
replication was inhibited by injection of HU 10 min before ments quoted (33) so that dose levels are not comparable.
the injection of [3H]dThd, the isotope was incorporated
Retrorsine. Treatment with retrorsine at approximately
specifically into nonreplicating diploids and tetraploids
the dose lethal to 50% of the female rats (160 mg/kg) (30),
(Chart 20) Inasmuch as HU-resistant non-S phase incorpo
did not significantly inhibit DNA synthesis in diploid or
ration of [3H]dThd is an operational definition of repair tetraploid nuclei (Chart 3C). This is in keeping with the fact
replication, the results show that repair is taking place 2 hr that the inhibitory effect of pyrrolizidines on cell replication
after injection of DENA. If damage and repair were uni
is apparently not due to gross inhibition of DNA synthesis.
formly distributed throughout the DNA in diploid and tetra
Thus, injection of lasiocarpine (26) or of retrorsine (2)
ploid nuclei, tetraploids would be expected to incorporate
resulted in the formation of cells with large nuclei in which
twice as much [3H]dThd as would diploids as a result of DNA synthesis but not cell division had taken place. There
their containing twice the amount of DNA (Table 1). Calcu
is evidence that treatment of mouse (13) or sheep (14) cells
lation of [3H]dThd dpm incorporated per 10" nuclei sug
in culture specifically inhibits synthesis of satellite DNA.
JULY 1978
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
2137
V. M. Craddock
and A. P. Henderson
30
30
20
fraction numb«
20
fraction
runber
600
-400
ZOO
so
40
30
20
IO
Fraction number
Chart 2. Zonal profiles of liver nuclei prepared from animals treated with DENA (250 mg/kg) or EMS (500 mg/kg). A, DENA, and 2 hr later [3HJdThd; B,
DENA, 2 hr later HU, and 10 min later [3H]dThd; C, EMS, and 2 hr later [3H]dThd. D, EMS, 2 hr later HU, and 10 min later [3H]dThd. Further description is
given in the legend to Chart 1.
Table 1
Inasmuch as satellite forms only a small proportion of the
Repair replication of DNA in diploid and tetraploid nuclei
total DNA, this effect would not be seen in these experi
Animals were treated with carcinogen, and after 2 (DENA, EMS,
ments.
aflatoxin, and retrorsine) or 17 hr (CCL, and ethionine), they
Repair replication was taking place 2 hr after injection of
received an injection of HU followed by one of [3H]dThd. Repair
retrorsine
(Chart 3D). The reproducibility of the data can be
replication was studied as described in the text. Nuclear fractions
assessed from the duplicate experiment with retorsine
from the peaks of the nonreplicating diploids and tetraploids were
used for calculation of the extent of repair replication, expressed
shown in Table 1. Pyrrolizidines have been shown to induce
as dpm [3H]dThd incorporated per 10s nuclei. The light-scattering
repair replication in cells in culture (15) and in bacteria after
profile was used only for the determination of the positions of the
incubation with alkaloid in the presence of rat liver microdifferent classes of nuclei. Nuclear counts were made with a
somes
(23). The experiments described show that repair of
hemacytometer as described in the text.
damage
occurs in vivo. This is of special interest because
nucleiCompound
dpm/108
(pg/nucleus)Diploid8.56.0
certain alkaloids have a prolonged antimitotic action on
liver in the intact animal. Thus, the regenerative response
administeredHU
(mg/kg)100
ploid7283458
ploid18.212.4
to partial hepatectomy is still inhibited when the operation
is carried out 4 weeks after injection of pyrrolizidine (17).
aloneDiethylnitrosaIt is possible that the alkaloids produce at least 2 types
of DNA damage, 1 of which is rapidly repaired and accounts
mineEthyl
200
1667
4974
18.9
9.4
250300 470528 11941217 6.09.8 11.115.2 for the repair synthesis that is seen in these experiments
and in work with cells in culture and the other of which
methaneis repaired slowly, if at all. As the persistent lesion is anti
sulfonateAflatoxinRetrorsineDose
5000.5 910785 22772856 7.05.6 10.413.7 mitotic, it is possible that the lesion that is rapidly repaired
is carcinogenic.
Carbon Tetrachloride. No evidence was found for repair
2.0160 15931032 34932384 6.1Tetra 11.9
replication 2 hr after treatment with CCI, (Chart 4/4). The
very low level of [3H]dThd incorporation remaining after
160Diploid2281282
995Tetra 1939DNA
treatment with HU and CCI4 is in the location of replicating
diploid nuclei. However, repair replication was taking place
Carbon tetra4000
793
1322
16.0
7.8
17 hr after treatment (Chart 46). Because the incorporation
chloride
of [3H]dThd into tetraploids was approximately twice that
Ethionine
773
1206
8.2
12.1
500
into diploids (Table 1), the extent of incorporation corre2138
CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
Repair and de Novo Synthesis of DNA in Carcinogenesis
40
30
2O
Fraction number
-i80
40
30
20
10
30
20
Fraction number
Chart 3. Zonal profiles of liver nuclei prepared from animals treated with (A) aflatoxin (260 mg/kg) and 2 hr later [3H]dThd; (B) aflatoxin (0.5 mg/kg), 2 hr
later HU, and 10 min later [3H]dThd, (C) retrorsine (160 mg/kg), and 2 hr later [3H]dThd; and (D) retrorsine (160 mg/kg), 2 hr later HU, and 10 min later
[3H]dThd. Further description of chart given under Chart 1.
Fraction
number
lates with the DMA content of the nuclei, and damage and
repair are equally distributed between both sets of chro
mosomes in the tetraploid nuclei.
The dose of CCU used (2.5 ml/kg) causes necrosis. Al
though this is apparent only at a later time than the period
studied here (36), the biochemical events leading to necro
sis may already be in progress. However, there is no appar
ent reason why HU-resistant incorporation of [3H]dThd
into nonreplicating nuclei should be associated with the
approach of cell death, and it is probable that incorporation
of [3H]dThd is due to repair replication. The explanation of
the DMA repair synthesis may be that CCU causes lysosomal
damage, with the result that nucleases pass from the lysosomes into the nuclei and cause DNA damage which is
repairable. Although it has been shown that repair replica
tion is produced in isolated nuclei by treatment in vitro with
DNase I (46), apparently there was no evidence for the
occurrence of unscheduled synthesis resulting from DNA
damage caused by nuclease action in the intact cell. The
concept that lysosomal damage can result in nuclease
action on DNA in the nucleus and that this may be relevant
in carcinogenesis was suggested by Allison and Patón(3).
These results give more evidence for this theory. The
damage could cause mutation if repaired by an error-prone
system. Repair occurs at the time of an increased de novo
DNA replication (Chart 4C). If both processes occur in the
same cell, error-prone postreplication repair may be in
volved. Such a mechanism for mutagenicity correlates with
the fact that CCL,is not mutagenic in the Ames test (31).
Ethionine. Ethionine produced a result similar to that
given by CCL,, i.e., repair was taking place at 17 hr but not
at 2 hr after treatment (Chart 4, D and E). The residual
[3H]dThd incorporated at 2 hr in the presence of HU is in
the location of replicating diploid and tetraploid nuclei.
Damage and repair appear to be equally distributed
throughout the DNA of diploid and tetraploid nuclei be
cause there is approximately twice the incorporation of
[3H]dThd in the tetraploids (Table 1). The fact that repair
occurs at 17 hr but not at 2 hr suggests that the damage
being repaired is produced indirectly rather than by a direct
reaction of DNA with ethionine or with a rapidly formed
metabolite. S-Adenosylethionine accumulates in liver after
injection of ethionine and is still at a high level 24 hr after
1000 mg/kg were given (18). It is possible that when a
sufficiently high concentration is reached this compound
may react nonenzymatically with DNA. In fact, a very small
amount of 7-ethylguanine has been detected in rat liver
DNA 18 hr after treatment with ethionine (48). The occur
rence of repair replication is additional evidence that DNA
is in fact damaged by ethionine.
De novo replication of DNA was not stimulated 17 hr after
treatment with ethionine (Chart 4F). This is in keeping with
the fact that the microscopically visible damage to cell
organelles is not irreversible and does not lead to cell death
(43) and that consequently there is no restorative hyperplasia.
Thioacetamide. The rate of sedimentation of nuclei iso
lated from animals treated with thioacetamide is within the
normal range (Chart 4 G to /). Although there is much
evidence that thioacetamide causes nuclear enlargement,
JULY 1978
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
2139
V. M. Craddock and A. R. Henderson
Froctton
numb«'
-300
so
30
Fraction
number
Fraction number
Chart 4. Zonal profiles of liver nuclei prepared from animals treated with CCI, (2.5 ml/kg), ethionine (500 mg/kg), or thioacetamide (150 mg/kg). A, CCI«,
2 hr later HU, and 10 min later [3H]dThd; B, CCI.,, 17 hr later HU, and 10 min later [3H]dThd; C, CCI,, 17 hr later [3H]dThd; D, ethionine, 2 hr later HU, and 10
min later [3H]dThd; E, ethionine, 17 hr later HU, and 10 min later [3H]dThd; F, ethionine, 17 hr later [3H]dThd; G, thioacetamide, 2 hr later HU, and 10 min
later [3H]dThd; H, thioacetamide, 17 hr later HU, and 10 min later [3H]dThd; /, thioacetamide, 17 hr later [3H]dThd. Further description is given in the legend
to Chart 1.
the dose used in these experiments (150 mg/kg) results in
maximum nuclear swelling after 4 hr (32). At 2 hr, enlarge
ment had not yet begun and by 6 hr the size had returned to
normal (32). Therefore, at the times studied in these exper
iments, 2 and 17 hr after injection, the nuclei would be
expected to be of normal size. Chart 41shows that there is
no increase in de novo DNA replication at 17 hr although
this may have increased at a later time (39).
No evidence was obtained for significant HU-resistant
incorporation of [3H]dThd into nonreplicating nuclei at 2 or
17 hr after thioacetamide (Chart 4, G and H). This suggests
either that thioacetamide does not damage rat liver DNA or
that any damage produced is not repaired. This result,
2140
together with the apparent lack of evidence for a reaction
between thioacetamide and DNA and also with the nonmutagenicity of thioacetamide in the Ames test (31), suggests
that in this case cancer may be brought about by a nonmutagenic mechanism. Zonal fractionation of nuclei isolated
from rats after chronic treatment with thioacetamide re
vealed a sequence of complex changes in the nucleus (20),
but the molecular mechanisms responsible are not under
stood. Changes in transport of RNA from nucleus to cyto
plasm may well be relevant (45). Another possibility is that
the growths induced by thioacetamide in Porton-derived
Wistar rats are not in fact malignant liver cell tumors.
Animals fed a diet containing thioacetamide developed very
CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
Repair and de Novo Synthesis
of DNA in Carcinogenesis
H
60
50
40
30
20
90
Fraction number
40
30
20
Fraction number
0.2
40
3O
Fraction
number
Chart 4-G-l.
large abnormal livers, but there was no evidence for métas
tases or any other sign of cancer, and the animals lived a
normal life span (V. M. Craddock, unpublished results). Cell
damage caused by the thioacetamide could have led to
reduced liver function, and this in turn could have led to
restorative hyperplasia, which in time would produce a
large abnormal liver.
The experiments described show that DENA, aflatoxin,
and retrorsine induce repair replication in liver DNA in the
intact animal. This unscheduled synthesis is occurring 2 hr
after treatment of the animal with the carcinogen and is pre
sumably repair of damage caused by reaction of DNA with
rapidly formed metabolites of the chemicals. This rapid on
set of repair replication may mean that repair of carcinogeninduced damage occurs before much de novo replication
has taken place so that extensive replication of damaged
DNA does not occur. Inasmuch as there is much evidence
to suggest that replication of damaged DNA is necessary
for "fixation" of the transformed state (38), this may explain
why a single treatment with a carcinogen rarely causes liver
cancer. One treatment does induce liver cancer if there is
also a simultaneous stimulus for replication, as by partial
hepatectomy (9, 10). In support of this concept is the fact
that replication of damaged DNA can take place in the
regenerating liver (1, 37, 54).
CCI, and ethionine induce repair replication after a delay
period. This suggests that DNA damage is caused by an
indirect process, such as DNase activity resulting from
lysosomal damage (CCI4),or from nonenzymic reaction with
a metabolite of the carcinogen that is slow to reach a high
concentration in liver (S-adenosylethionine in the case of
ethionine). Error-prone repair of such damage could be
relevant in carcinogenesis.
Thioacetamide was found not to induce repair replica
tion. Inasmuch as there is apparently no evidence for
reaction of thioacetamide with DNA, the initiation stage of
carcinogenesis may not in this case be a result of repairable
DNA damage.
ACKNOWLEDGMENTS
We wish to thank C. M. Ansley and D. Wilkinson for nuclear counting.
REFERENCES
1. Abanobi, S. E., Mulivor, R. A., Rajalakshmi, S., and Sarma, D. S. R.
Characterisation of Dimethylnitrosamine (DMN)-induced Methylated
Products in Rat Liver DNA Replicated In Vivo. Proc. Am. Assoc. Cancer
Res., 17: 103, 1976.
2. Afzelius, B. A., and Schoental, R. Ultrastructure of the Enlarged Hepatocytes Induced in Rats with a Single Oral Dose of Retrorsine, a
Pyrrolizidine (Senecio) Alkaloid. J. Ultrastruct. Res., 20: 328-345, 1967.
3. Allison, A. C., and Patón,G. R. Chromosome Damage in Human Diploid
Cells Following Activation of Lysosomal Enzymes. Nature, 207: 11701173,1965.
4. Arfellini, G., Prodi, G.. and Grilli, S. Removal of 5-Bromodeoxyuridine
Incorporation in DNA of Regenerating Rat Liver. Nature, 265: 337-379,
1977.
5. Burton, K. A Study of the Conditions and Mechanism of the Diphenylamine Reaction for the Colorimetrie Estimation of DNA. Biochem J.. 62:
315-323, 1956.
6. Cameron, I. L. Cell Proliferations and Renewal in the Mammalian Body.
In: I. L. Cameron and J. D. Thrasher (eds.). Cellular and Molecular
Renewal in the Mammalian Body, Chap. 3, New York: Academic Press,
Inc., 1971.
7. Cox, R., Damjanov, I., Abanobi, S. E., and Sarma, D. S. R. A Method for
JULY 1978
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
2141
V. M. Craddock
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
and A. R. Henderson
Measuring DNA Damage and Repair in the Liver in Vivo. Cancer Res.,
33: 2114-2121, 1973.
Craddock, V. M. The Pattern of Methylated Purines Formed in DMA of
Intact and Regenerating Liver of Rats Treated with the Carcinogen
Dimethylnitrosamine. Biochim. Biophys. Acta, 3J2: 202-210, 1973.
Craddock, V. M. Induction of Liver Tumors in Rats by a Single Treatment
with Nitroso Compounds Given after Partial Hepatectomy. Nature, 245.
386-388, 1973.
Craddock, V. M. Cell Proliferation and Experimental Liver Cancer. In: H.
M. Cameron, C. A. Linsell, and G. P. Warwick (eds.), Liver Cell Cancer,
pp. 153-201. Amsterdam: Elsevier Publishing Corp., 1976.
Craddock, V. M., and Frei, J. V. Induction of Tumors in Intact and
Partially Hepatectomised Rats with Ethyl Methanesulphonate. Brit. J.
Cancer, 34: 207-209, 1976.
Craddock, V.M., Henderson, A. R., and Ansley, C. M. Repair Replication
of DNA in the Intact Animal following Treatment with Dimethylnitrosa
mine and with Methyl Methanesulphonate, Studied by Fractionation of
Nuclei in a Zonal Centrifuge. Biochim. Biophys. Acta, 447: 53-64, 1976.
Curtain, C. C. Selective Inhibition of the Synthesis of Mouse Satellite
DNA by Dehydroheliotridine. Chem.-Biol. Interactions, 4: 421-425,1971.
Curtain, C. C. Decreased Synthesis of Hepatic Satellite DNA in Pyrrolizidine Alkaloidosis of the Sheep. Chem.-Biol. Interactions, 10: 133-139,
1975.
Curtain, C. C., and Edgar, J. A. Binding of Dehydroheliotridine to DNA
and the Effect of It and Other Compounds on Repair Synthesis in Main
and Satellite Band DNA. Chem.-Biol. Interactions, 73: 243-256, 1976.
Damjanov, I., Cox, R., Sarma, D. S. R., and Farber, E. Patterns of
Damage and Repair of Liver DNA Induced by Carcinogenic Methylating
Agents in Vivo. Cancer Res., 33. 2122-2128, 1973.
Downing, D. T., and Peterson, J. E. Quantitative Assessment of the
Persistent Antimitotic Effect of Certain Hepatotoxic Pyrrolizidine Alka
loids on Rat Liver. Australian J. Exptl. Biol. Med. Sci, 46: 493-502, 1968.
Farber, E., Shull, K. H., Villa-Trevino, S., Lombardi, B., and Thomas, M.
Biochemical Pathology of Acute Hepatic Adenosine-triphosphate Defi
ciency. Nature, 203: 34-40, 1964.
Giles, K. W., and Myers, A. An Improved Diphenylamine Method for
Estimation of DNA. Nature, 206: 93,1965.
Gonzalez-Mujica, F., and Mathias, A. P. Studies of Nuclei Seperated by
Zonal Centrifugation from Liver of Rats Treated with Thioacetamide.
Biochem. J., 732: 163-183, 1973.
Goodman, J. I. Hepatic DNA Repair Synthesis in Rats Fed 3'-Methyl-4dimethylaminoazobenzene. Biochem. Biophys. Res. Commun., 56: 5259, 1974.
Goth, R., and Rajewsky, M. F. Persistence of O'-Ethylguanine in Rat
Brain DNA: Correlation with Nervous System-specific Carcinogenesis by
Ethylnitrosourea. Proc. Nati. Acad. Sei. U. S., 71: 639-643,1974.
Green, M. H. L., and Muriel, W. J. Use of Repair-deficient Strains of E.
coli and Liver Microsomes to Detect and Characterise DNA Damage
Caused by Pyrrolizidine Alkaloids Heliotrine and Monocrotaline. Muta
tion Res.,28: 331-336, 1975.
Grisham, J. W. A Morphologic Study of DNA Synthesis and Cell Prolif
eration in Regenerating Rat Liver; Autoradiography with Thymidine-H3.
Cancer Res., 22: 842-849, 1962.
Haines, M. E., Johnston. I. R., and Mathias, A. P. The Role of Rat Liver
Nuclear DNA Polymerase and its Distribution in Various Classes of Liver
Nuclei. Federation European Biochem. Soc. Letters, 10: 113-116, 1970.
Jago, M. V. Development of Hepatic Megalocytosis after Chronic Pyrrol
izidine Alkaloid Poisoning. Am. J. Pathol., 56: 405-422,1969.
Keefe, D. A., and Edwards, G. S. Aflatoxin B, Does Not Break Hepatic
DNA but Inhibits the Repair of Alkylated DNA. Mutation Res., 37: 330,
1975.
Kleihues, P., and Margison, G. P. Exhaustion and Recovery of Repair
Excision of O6-Methylguanine from Rat Liver DNA. Nature, 259: 153-155,
1976.
Margison, G. P., and Kleihues, P. Chemical 'Carcinogenesis in the
Nervous System. Preferential Accumulation of O'-Methyl-Guanine in Rat
Brain DNA during Repetitive Administration of N-Methyl-fV-Nitrosourea.
Biochem. J.. Õ48:521-525, 1975.
Mattocks, A. R. Acute Hepatotoxicity and Pyrrolic Metabolites in Rats
dosed with Pyrrolizidine Alkaloids. Chem.-Biol. Interactions, 5: 227-242.
1972.
McCann, J.. Choi, E., Yamasaki, E., and Ames, B. N. Detection of
Carcinogens as Mutagens in the Salmonella/Microsome Test: Assay of
2142
300 Chemicals. Proc. Nati. Acad. Sei. U. S., 72. 5135-5139, 1975.
32. Moraru, I., Dragomir, C. T., Tomescu, E., Cotutiu, C., and Ungureanu,
D. Similitudes and Differences in the Reactivity of the Liver Cell Chromatin to Thioacetamide and Hydrocortisone. Electron Microscopic and
Cytochemical Quantitative Data Concerning the Chromatin Condensa
tion-Dispersion Cycle. Pathol. Europaea, 9: 317-323,1974.
33. Neal. G. E., Godoy. H. M., Judah, D. J.. and Butler, W. H. Some Effects
of Acute and Chronic Dosing with Aflatoxin B, on Rat Liver Nuclei.
Cancer Res., 36: 1771-1778, 1976.
34. Nicoli, J. W., Swann, P. F., and Pegg, A. E. Effect of Dimethylnitrosa
mine on Persistence of Methylated Guanines in Rat Liver and Kidney
DNA. Nature, 254: 261-262, 1975.
35. O'Connor, P. J., Capps, M. J., and Craig, A. W. Comparative Studies of
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
the Hepatocarcinogen Dimethylnitrosamine in Vivo: Reaction Sites in
Rat Liver DNA and the Significance of Their Relative Stabilities. Brit. J.
Cancer, 27: 153-166,1973.
Pound, A. W., Horn, L., and Lawson, T. A. Decreased Toxicity of
Dimethylnitrosamine in Rats after Treatment with Carbon Tetrachloride.
Pathology, 5: 233-242, 1973.
Rajalakshmi, S., and Sarma, D. S. R. Replication of Hepatic DNA in Rats
treated with Dimethylnitrosamine. Chem.-Biol. Interactions, 77: 245252, 1975.
Rajewsky, M. F. Proliferative Parameters of Mammalian Cell Systems
and Their Role in Tumor Growth and Carcinogenesis. Z. Krebsforsch..
78: 12-30,1972.
Reddy, J., Chiga, M., and Svoboda, D. Initiation of the Division Cycle of
Rat Hepatocytes following a Single Injection of Thioacetamide. Lab.
Invest.,20: 405-411, 1969.
Reynolds, E. S. Liver Parenchymal Cell Injury IV Pattern of Incorporation
of Carbon and Chlorine from Carbon Tetrachloride into Chemical Con
stituents of Liver in Vivo. J. Pharmacol. Exptl. Therap., 755: 117-126,
1967.
Rocchi, P., Prodi, G., Grilli, S., and Ferreri, A. M. In Vivo and in Vitro
Binding of Carbon Tetrachloride with Nucleic Acids and Proteins in Rat
and Mouse Liver. Intern. J. Cancer, 77: 419-425, 1973.
Schoental, R., and Bensted, J. P. M. Effects of Whole Body Irradiation
and of Partial Hepatectomy on the Liver Lesions Induced in Rats by a
Single Dose of Retrorsine, a Pyrrolizidine (Senecio) Alkaloid. Brit. J.
Cancer, 77:242-251, 1963.
Shinozuka, H., Lombardi, B., and Farber, E. Dynamics of Injury and
Repair in Hepatic Cells. II. Association of Membranes with Lipid during
Recovery from Ethionine-induced Fatty Liver. Am. J. Pathol., 63: 161172, 1971.
Singer, B. The Chemical Effects of Nucleic Acid Alkylation and Their
Relation to Mutagenesis and Carcinogenesis. Prog. Nucleic Acid Res.,
75: 219-284,1975.
Smuckler. E. A., and Koplitz, M. Thioacetamide-induced Alterations in
Nuclear RNA Transport. Cancer Res., 34: 827-838, 1974.
Stenstrom, M. L., Edelstein, M., and Grisham, J. W. Effect of araCTP on
DNA Replication and Repair in Isolated Hepatocyte Nuclei. Exptl. Cell
Res., 89.:439-442, 1974.
Swann, P. F., and Magee, P. N. The Alkylation of N-7 of Guanine of
Nucleic Acids of the Rat by Diethylnitrosamine, W-Ethyl-AV-Nitrosourea
and Ethyl Methanesulphonate. Biochem. J., 725: 841-847, 1971.
Swann, P. F., Pegg, A. E., Hawks, A., Farber, E., and Magee, P. N.
Evidence for Ethylation of Rat Liver DNA following Ethionine Administra
tion to Rats. Biochem. J., 723: 175-181, 1971.
Swenson, D. H., Lin, J. K., Miller, E. C., and Miller, J. A. Aflatoxin B.-2.3oxide as a Probable Intermediate in the Covalent Binding of Aflatoxins
B, and B, to Rat Liver DNA and Ribosomal RNA/'n Vivo. Cancer Res.,37:
172-181, 1977.
Thielmann, H. W., Vosberg, H., and Reygers, U. Carcinogen-induced
DNA Repair in Nucleotide-permeable Escherichia coli Cells. European J.
Biochem., 56: 433-447, 1975.
Trosko, J. E., and Chu, E. H. Y. The Role of DNA Repair and Somatic
Mutation in Carcinogenesis. Advan. Cancer Res., 2Õ.391-425, 1975.
Warwick, G. P. Effects of the Cell Cycle on Carcinogenesis. Fed. Proc.
30: 1760-1765, 1971.
White, I. N. H., and Mattocks, A. R. Reaction of Dihydropyrrolizines with
Deoxyribonucleic Acids in Vitro. Biochem. J., 728: 291-297, 1972.
Zahner, A. J., Rajalakshmi, S., and Sarma, D. S. R. Isolation of in Vivo
Replicated Rat Liver DNA Containing N-OH-2-AAF Metabolite(s). Proc.
Am. Assoc. Cancer Res., 78: 99, 1977.
CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
Repair and de Nova Synthesis of DNA in Carcina genesis
@
0
:@
c@
p
@
C
(
-.
--@
C
(@@)
@
C;
-
(:
L
a
-
©@
\‘
‘I.
Fig. I . Suspensions of nuclei from diploid (a) and tetraplold (b) peaks isolated by zonal centrifugatlon. Phase contrast, x 370.
JULY 1978
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.
2143
De Novo and Repair Replication of DNA in Liver of
Carcinogen-treated Animals
V. M. Craddock and A. R. Henderson
Cancer Res 1978;38:2135-2143.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/38/7/2135
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1978 American Association for Cancer Research.