Download Inability of Methapyrilene to Induce Sister

[CANCER RESEARCH 42, 4614-4618,
November 1982]
Inability of Methapyrilene to Induce Sister Chromatid Exchanges in
Vitro and in Vivo'
P. Thomas lype,2 Ratna Ray-Chaudhuri, William Lijinsky, and Susan P. Kelley
Chemical Carcinogenesis
Program, NCI-Frederick
Cancer Research Facility, Frederick,
ABSTRACT
The induction of sister chromatid exchanges (SCE) by the
hepatocarcinogen
methapyrilene hydrochloride was investi
gated using appropriate in vitro and in vivo mammalian cell
systems. Methapyrilene, even at the maximum tolerated dose,
did not induce SCE in Chinese hamster ovary cells (CHO) or
when CHO cells or hamster lung fibroblasts, V-79, were cocultivated with early cultures of rat liver epithelial cells, which
are known to metabolize different classes of chemical carcin
ogens to active forms. Moreover, a hybrid clone of cells (formed
by fusion of CHO cells with rat liver epithelial cells), which is
highly sensitive to SCE formation by a number of xenobiotics,
failed to produce SCE after treatment with methapyrilene.
Experiments in vivo with bone marrow cells and in vitro with
CHO cells cocultivated with primary hepatocytes from rats also
confirmed the inability of methapyrilene to induce SCE in the
indicator cells. Since aflatoxin Bi induced SCE in the in vitro
and in vivo models, it may be concluded that methapyrilene
does not induce SCE at a concentration which is not cytotoxic
to the indicator cells in the different systems described. Autoradiographic studies in cultured rat liver cells with tritiated
methapyrilene showed that the label was localized in the cyto
plasm but not in the interphase nuclei or in the metaphase
chromosomes, indicating a lack of interaction of methapyrilene
with the nuclear macromolecules of the putative target cells for
methapyrilene.
The antihistaminic methapyrilene is reported to be a potent
hepatocarcinogen (16) which does not significantly react with
rat liver DNA, but soluble proteins isolated from liver contain
considerable bound material (15). It is also known that SCE3 in
different indicator cells types are induced by carcinogenic
chemicals which interact with DNA (3, 21). Despite its nonmutagenic property in the Salmonella mutagenicity assay of Ames
(2) and its inability to induce unscheduled DNA synthesis in
freshly isolated rat liver cells (23), methapyrilene or its metab
olites might induce SCE indirectly (e.g., through reaction with
nuclear proteins or through the production of free radicals).
Moreover, Althaus et al. (1) recently published data which
suggest that unscheduled DNA synthesis is indeed induced by
methapyrilene in primary cultures of freshly isolated rat liver
cells. They also found that this drug may be a direct-acting
carcinogen. We investigated the SCE-inducing properties of
by Contract
NO1-CO-75380
with the National
Cancer Institute, NIH. Bethesda, Md. 20205.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: SCE, sister chromatid exchanges; DMBA, 7,12dimethylbenz[a]anthracene;
CHO. Chinese hamster ovary; BrdUrd, 5-bromo-2'deoxyuridine.
Received March 8, 1982; accepted August 6, 1982.
4614
methapyrilene, using appropriate in vitro and in vivo mammalian
cell systems. SCE were not induced in any of the systems and
radioactivity from [3H]methapyrilene was localized primarily in
the cytoplasm of the early cultures of liver epithelial cells.
MATERIALS
AND METHODS
Test Chemicals. Methapyrilene hydrochloride (Lot 48C-0022) was
purchased from Sigma Chemical Co. (St. Louis, Mo.). [3H]Methapyrilene (specific activity, 1.3 Ci/mmol) was obtained from a sample
prepared as described elsewhere (15). [3H]DMBA (specific activity, 37
Ci/mmol), purchased from the Radiochemical Center, Amersham/
Searle Corp. (Arlington Heights, III.), was chromatographically
purified
(5) before use. Mitomycin C and aflatoxin B, were obtained from
Calbiochem (San Diego, Calif.) and Sigma, respectively.
Cell Lines. Chinese hamster ovary cell line CHO was purchased
from the American Type Culture Collection, Rockville, Md. (CCI
61CHO-K1). Chinese hamster lung fibroblasts, V-79, were obtained
from Dr. B. Myhr, Litton Bionetics, Kensington, Md. Rat liver epithelial
cell lines LNRL and FNRL were prepared in our laboratory from Lewis
and Fischer strains of rats, respectively, by previously described meth
ods (8) and were used between the 10th and 15th passages. The
hybrid clone 3.1.9 derived from fusion of LNRL and CHO cells (10)
was also used in these studies.
Culture Methods. All the cell lines were cultured routinely in Ham's
F-10 medium (6) supplemented
with 10% fetal bovine serum (K. C.
Biologicals, Inc., Lenexa, Kans.). The cells were grown on plastic Petri
dishes (Falcon Plastics, Inc., Oxnard, Calif.) and incubated at 37° in
humidity cabinets with a gas phase of 5% CO2 in air.
Viability of Cells with Methapyrilene. Single-cell suspensions of the
cell line were plated at a cell density of 20 cells/sq cm (500 cells in 5
ml of medium in 60-mm dishes). After 1 day, the medium was removed
INTRODUCTION
' This work was supported
Maryland 21701
and an equal volume of either the control medium or media with a
different concentration of methapyrilene was added to the cultures.
The cells were refed on the 6th day with appropriate media, and on the
11th day cells were fixed in methanol and stained with Giemsa. The
colonies were counted with an automatic colony counter (a minimum of
3 dishes/dose).
SCE Induction in Vitro. Single-cell suspensions trypsinized from
monolayers were plated at a density of 1 x 105 cells/dish in 5 ml of
culture medium. In some experiments, the CHO and LNRL or freshly
isolated rat liver cells, or V-79 and FNRL cells, were mixed at a ratio of
1:1 and plated as above. After 48 hr, the culture medium was removed,
and an equal volume of medium containing BrdUrd (10 fig/ml; Sigma)
and varying concentrations of the test chemicals was added to the
culture. Control cultures received medium containing BrdUrd. The
cultures were kept in complete darkness to minimize the induction of
exchanges caused by photolysis of BrdUrd-containing
DNA (7) and
were handled under a yellow safelight. After an additional 24 hr of
incubation, during which the cells went through 2 rounds of DNA
replication, Colcemid (0.02/tg/ml;
Grand Island Biological Co., Grand
Island, N. Y.) were added to arrest the cells in mitosis, and 2 hr later
the cells were harvested using 0.05% trypsin. The cells were sus
pended in a hypotonie solution (0.075 M KCI) for 20 to 30 min and
then fixed in methanohacetic acid (3:1) for a minimum of 30 min. After
3 changes of fixative, the cells were spread on microscope slides and
air dried. Some of the slides were stained for 15 min in 5% Giemsa
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1982 American Association for Cancer Research.
VOL. 42
Lack of SCE Induction by Methapyrilene
R66 diluted in a pH 6.8 buffer, both obtained from G. T. Gurr, Hopkin
and Williams, Chadwell Heath, Essex, United Kingdom. Other slides
were stained by a modified fluorochrome-plus-Giemsa
technique (22,
25).
SCE Induction in Vivo. Male Fischer rats (6 weeks old; average
weight, 125 g) were given i.p. injections of BrdUrd (30 jtg/g body
weight) every 30 min for a 10-hr period. Fifteen min after the last
injection, the test chemicals were administered by gavage. Colchicine
was injected i.p. (2 ng/g body weight) 11 hr after treatment with the
carcinogen, and the rats were killed 1 hr later. The marrow cells were
isolated by aspirating the hypotonie solution (0.075 M KCI) through the
femur bones with a 21-gauge needle. The cells were processed as
described above.
Metaphase plates of the various cells containing differentially stained
chromosomes were examined with an oil immersion objective using a
Zeiss Universal microscope. The SCE present in 50 metaphase plates
selected at random were counted for each concentration of the test
compounds in all the cell systems. Every experiment had its own
control SCE, and the data were analyzed statistically.
Autoradiographic Studies. The rat liver cell line FNRL was plated at
a concentration 2x10"
cells/ml in 5 ml of culture medium either in
60-mm Retri dishes or on glass coverslips.
The culture medium was
changed on the second day after plating, and an equal volume of
medium containing either [3H]methapyrilene
(4.3 /¿Ci,1 fig/ml) or
[3H]DMBA (3.7 /uCi, 0.025 ng/ml) was added, and the cells were
incubated for 24 hr. The coverslips were rinsed three times with
Dulbecco's phosphate-buffered
saline (Grand Island Biological Co.)
containing an excess of the appropriate unlabeled carcinogens, methapyrilene or DMBA. The cells were then fixed with methanol:acetic acid
(3:1 ); and the coverslips were washed repeatedly with water to remove
the noncovalently bound methapyrilene and/or its metabolites or, in
the case of DMBA samples, additionally washed with benzene to
remove unbound DMBA and air dried. The coverslips were attached to
glass slides with the cells facing upwards, using Permount (Fisher
Scientific Co., Fair Lawn, N. J.). The cells grown on the dishes were
treated with Colcemid for the last 3 hr of incubation, and chromosomes
were prepared as described above. The coverslips and the chromo
some preparations were dipped in NTB 2 nuclear track emulsion
(Kodak, Arlington, Va.), dried and exposed for 4 to 8 weeks, and then
developed with Kodak D-19 developer. Controls (cells not incubated
with the labeled compounds) for the intact cells and the chromosome
preparations were also used for autoradiography.
The slides were
lightly counterstained with Giemsa and analyzed with a Zeiss Universal
microscope.
RESULTS
The effect of methapyrilene on cell viability was determined
in the rat liver cell line FNRL as well as in the indicator cell
lines CHO and a hybrid clone derived from fusion of rat liver
cells with CHO cells. Treatment of the cells for 10 days with
methapyrilene did not produce toxicity up to a dose of 10 jug/
ml (Table 1). At a dose of 50 fig/mi, the relative plating
lines. Although the inhibition of plating efficiency in CHO cells
was also significant, it was lower than that seen in FNRL and
the hybrid cell line. All the cell lines were very sensitive to a
100-/ug/ml dose of methapyrilene (Table 1).
Methapyrilene did not show any induction of SCE in CHO
cells cultured alone at any of the concentrations used (Table
2). An upper limit of 80 /¿g/ml was set for methapyrilene,
because this concentration inhibited about 50% of the cells
from entering into the second mitosis in the presence of
BrdUrd. No induction of SCE was seen in those cells which did
undergo 2 rounds of DNA synthesis and which showed clear
NOVEMBER
Table 1
Ellect oÃ-methapyrilene
lineFNRLHybrid
Cell
on the viability of mammalian cell lines
of colonies/
plat
ing efficiency100819713"2"10010
(jig/ml)011050100011050100011050100No.
dish149
8a121
±
8145±
1220
±
33±
268±
1.9°CHODose
clone 3.
374±
873±
101
±
1082
±
1088
±
1586
±
1357
±
146
±
± 3Relative
•¿
Mean ±S.E.
b The inhibition in these samples was highly significant (p < 0.001).
0 See Ref. 10 for nomenclature of the hybrid clone of rat liver and hamster
ovary cells.
d The inhibition (p < 0.01) seen in CHO at this dose of methapyrilene
is much
smaller than that seen in FNRL and the hybrid clone.
differentiation of sister chromatids. Since the direct-acting
agent mitomycin C (21) produced a 7-fold increase in SCE
over the control, it seemed likely that methapyrilene may need
metabolic activation before SCE can be induced. Since the
commonly used indicator cell types (CHO; V-79) have little or
no capacity to metabolically activate chemicals to derivatives
that interact with cellular macromolecules, we have used ap
propriate activating cell systems. In experiments in which the
indicator cells are cocultivated with the rat liver cells from the
early passages of the cell lines known to metabolize a variety
of chemical carcinogens (11, 25), or where the hybrid cells
(10) which are very sensitive to SCE induction by directly or
indirectly acting mutagenic carcinogens are used as the indi
cator cells, methapyrilene showed no effect on SCE production
(Table 2). Under the same experimental conditions, the appro
priate positive agent, aflatoxin B,, did induce SCE. Early cul
tures of the nonmalignant liver cells could not be used as
indicator cells for assessing SCE, since they do not undergo 2
rounds of DNA synthesis in the presence of BrdUrd (25), which
is a prerequisite for visualizing sister chromatid differentiation.
We have therefore used primary cultures of rat liver cells for
cocultivation with CHO cells, but SCE were not induced in the
indicator cells by methapyrilene (Table 2). Moreover, metha
pyrilene was ineffective even in bone marrow cells in rats
gavaged with the chemical, whereas aflatoxin Bi showed a
significant induction of SCE in this system (Table 2). No in
creased frequencies of chromosome breaks over control val
ues were seen in any of the cell systems used. However, with
the higher dose used (80 /ig/g body weight), there was a
reduction in the percentage of cells showing differentiation of
sister chromatids.
Since a high level of binding of [3H]methapyrilene to proteins
isolated from liver was reported (15), we have used autoradiographic techniques to localize the covalently bound radioac
tivity in one of the rat liver cell lines (FNRL) used for the
cocultivation experiments. The results are shown in Figs. 1 to
3. In the intact rat liver cell line FNRL, grown on coverslips and
treated with [3H]methapyrilene, the label was found mainly in
1982
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1982 American Association for Cancer Research.
4615
P. T. type et al.
Table 2
Effect of methapyrilene
compoundCHO
Indicator cells
cultured alone
Test compound
on SCE induction in different indicator cells and experimental
systems
of
of
compound
treated vs.
S.E.0.580.530.500.580.550.584.080.570.510.580.520.540.550.571.440.470.460.
±
(pig/ml)0520408000.00305208004003040030408003040*8003SCE"Mean
control0.910.871.000.956.950.901.01
Positive
MethapyrileneMitomycin
CCHO
MethapyrileneV79
+ LNRL mixed culture
cultureCHO
+ FNRL mixed
B,Hybrid
+ LNRL mixed culture
clone 3.1 .9o
Aflatoxin
MethapyrileneAflatoxin
B,CHO
Methapyrilenemixed
+ primary hepatocytes
cultureAflatoxin
BBone
marrow cells in vivo
-0
-0
-0
MethapyrileneAflatoxin
B,Dose
-3
-0.800.770.830.750.720.804.730.890.600.730.680.950.9
a Results are expressed as SCE/cell for the freshly isolated bone marrow cells in which there was no variation in the diploid number of chromosomes within the
samples examined. In the case of cultured cells and especially in the hybrid clone, there was considerable variation in the number of chromosomes within the
samples; hence, the results are expressed as SCE/chromosome for these samples.
b Probability associated with tests of control versus treated values using Student's f test.
c NS, not significant; p > 0.25 in these samples.
d See Ref. 10 for nomenclature of the hybrid clone of rat liver and hamster ovary cells.
8 jig/g body weight.
the cytoplasm (Fig. 1). The nucleus contained hardly any grains
over that of the background. Similarly, in the chromosome
preparations, the metaphase chromosomes (Fig. 2A) as well
as the interphase nuclei (Fig. 2ß)did not show grains over the
control. In contrast, in [3H]DMBA-treated cells, both the meta
phase chromosomes (Fig. 3A) and the interphase nuclei (Fig.
36) contained the grains.
DISCUSSION
It is becoming increasingly clear that SCE in different indi
cator cell types (3, 21 ) are induced by carcinogenic chemicals
which interact with nuclear DNA. However, certain chemicals
which do not interact directly with DNA (e.g., tumor-promoting
agents) are also known to enhance SCE production (13, 20,
24, 26). Therefore, even though methapyrilene gave negative
results in mutagenicity tests (2) and gave conflicting results as
to the ability to induce unscheduled DNA synthesis in freshly
isolated liver cells (1, 23), we investigated the effects of various
doses of this hepatocarcinogen
on the viability of and SCE
production in the various cell culture systems.
4616
Although methapyrilene appears to be more cytotoxic to rat
liver cells and to a rat liver-CHO hybrid cell clone than to CHO
cells (Table 1), this result does not prove that metabolism of
methapyrilene is carried out by any of the cells or that metab
olism is required for the induction of cytotoxicity. The latter
could well be the result of direct action of this compound with
any of the cellular organelles or macromolecules which may or
may not include the nucleus and/or nuclear DNA. Cytotoxicity
was observed by others (23) when 500- to 1000-nmol/ml
(150- to 300-jug/ml) doses of the drug were added to freshly
isolated rat liver cells. The colony assay reported here gives a
precise end point for cell viability, and a concentration of 50
jug/ml is shown to produce a significant inhibition of cell viabil
ity.
Methapyrilene did not induce or enhance SCE over the
control value in any of the in vitro systems whether the indicator
cells were cultured alone or cocultivated with early cultures of
rat liver epithelial cells (Table 2). These results suggest that (a)
methapyrilene may not be a direct DNA-damaging agent, (b)
the cultured liver cells and the liver cell/CHO hybrid cell clones
may be incapable of producing reactive metabolites from meth-
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1982 American Association for Cancer Research.
VOL.
42
Lack of SCE Induction by Methapyrilene
apyrilene (or the metabolites themselves may not induce SCE),
or (c) none of the in vitro systems may be adequate for testing
methapyrilene, which may need whole body metabolism such
as bioactivation by gut flora (19).
Since a large number of DMA-damaging agents have been
shown to induce SCE in different in vitro cell systems (inter alia
Refs. 10 and 21), it seems likely that methapyrilene must be
metabolically activated before SCE can be induced. We used
early cultures of the diploid rat liver epithelial cells as the
metabolizing cells, since they were shown to have the ability to
metabolize polycyclic hydrocarbons (11) and a number of other
classes of xenobiotics (25). Moreover, these liver cells with
proliferative capacity also retain many liver-specific functions
such as the ability to synthesize bile acids (9) and tyrosine
aminotransferase (17) for several weeks after isolation from
the animal. Despite their having these liver-specific properties,
the liver cell cultures have not been proven to be capable of
metabolizing methapyrilene, although they are likely to have
this capacity. Therefore, we have also cocultivated CHO cells
with primary hepatocytes (12, 14) freshly isolated from rat liver
which have been shown to metabolize methapyrilene (18).
Even in this cocultivation system, SCE were not induced in
CHO cells by methapyrilene (Table 2), which strongly suggests
that the metabolites themselves may not induce SCE.
The autoradiography
results have shown that unlike
[3H]DMBA, which was reported by others to bind to the nucleus
(4), the label from [3H]methapyrilene was not localized in the
metaphase chromosomes. If there is a level of binding in the
nucleus below the threshold of detection, the condensed chromatin of the metaphase chromosomes would be expected to
have concentrated it considerably. However, no grains were
seen in the chromosomes either. These results clearly show
that methapyrilene does not bind to the nuclear macromolecules without metabolic activation and that, if metabolism does
take place, the metabolites themselves do not react with the
nuclear macromolecules. We have then standardized the con
ditions for producing sister chromatid differentiation in bone
marrow cells of Fischer rats and examined the effects of
methapyrilene and aflatoxin Bi in this in vivo system. It is clearly
demonstrated that methapyrilene does not induce SCE (even
at 80 /xg/g body weight, a dose which interferes with cell
proliferation) in this system, while aflatoxin B, does show a
significant induction (Table 2).
An important point which merits discussion is the relationship
between the dose of the drug and the expression of the
different parameters being studied in the various in vitro
screening systems. The tests used for the determination of
mutagenic capacity, transformation frequency, and SCE induc
tion all have a "built-in" control that removes the nonviable
target cells from being evaluated, and only the cells which
actually divide after treatment with the maximum tolerated dose
of the drug will be available for the assessment of the appro
priate biological effects. Methapyrilene was shown to be
"negative" in all of the above systems, but cell death was
observed as the dose was increased (Ref. 24; Table 1). In the
protocols used for determining the induction of unscheduled
DNA synthesis, there are no such built-in controls, and some
independent methods must be used to determine the maximum
tolerated dose of the drug. This might account for the discrep
ancy in the results obtained when methapyrilene was tested
with these different types of in vitro assay systems.
NOVEMBER
1982
The lack of SCE induction or chromosome breaks by meth
apyrilene seen in this study may be attributed to the inability of
this drug to react with the nuclear macromolecules; therefore,
this hepatocarcinogen does not appear to manifest one of the
characteristics of genotoxic carcinogens such as aflatoxin B,.
The mechanism by which methapyrilene produces hepatocellular carcinomas is not clear at present and merits further
research.
ACKNOWLEDGMENTS
The authors wish to thank Albert Herring and Michael Currens for their help in
some of the experiments reported.
REFERENCES
1. Althaus, F. R., Lawrence, S. D., Sattler, G. L., and Pilot, H. C. DNA damage
induced by the antihistaminic drug methapyrilene hydrochloride.
Mutât.
Res., 103: 213-218, 1982.
2. Andrews, A. W., Fornwald, J. A., and Lijinsky, W. Nitrosation and mutagenicity of some amine drugs. Toxicol. Appi. Pharmacol., 52: 237-244, 1980.
3. Bradley, M. O., Hsu, I. C., and Harris, C. C. Relationships between sister
chromatid exchange and mutagenicity, toxicity and DNA damage. Nature
(Lond.), 282. 318-320, 1979.
4. Diamond, L., Defendi, V., and Brookes, P. The interaction of 7.12-dimethylbenz(a)anthracene with cells sensitive and resistant to the toxicity induced
by this carcinogen. Cancer Res., 27. 890-897, 1967.
5. Dipple, A., Tomaszewski, J. E., Moschel, R. C., Bigger, C. A. H., Nebzydoski,
J. A., and Egan. M. Comparison of metabolism-mediated binding to DNA of
7-hydroxymethyl-12-methylbenz(a)anthracene
and 7,12-dimethylbenz(a)
anthracene. Cancer Res., 39. 1154-1158,
1979.
6. Ham, R. G. An improved nutrient solution for diploid Chinese hamster and
human cell lines. Exp. Cell Res.. 29: 515-520, 1965.
7. Ikushima, T., and Wolff, S. Sister chromatid exchanges induced by light
flashes to 5-bromodeoxyuridine and 5-iododeoxyuridine substituted Chinese
hamster chromosomes. Exp. Cell Res., 87: 15-19, 1974.
8. lype, P. T. Cultures from adult rat liver cells. I. Establishment of monolayer
cell cultures from normal liver. J. Cell Physiol., 78: 281-288, 1971.
9. lype, P. T., Issaq, H. J., and Shaikh, B. Synthesis of primary bile acids by rat
liver epithelial cell lines. J. Steroid Biochem., 16: 333-337, 1982.
10. lype, P. T., Raychaudhuri. R., and Kelley, S. Hybrid clones of rat liver and
hamster ovary cells as targets for carcinogen screening. Carcinogenesis, 2.
49-54, 1981.
11. lype, P. T., Tomaszewski, J. E., and Dipple, A. Biochemical basis for
cytotoxicity of 7,12-dimethylbenz[a]anthracene
in rat liver epithelial cells.
Cancer Res., 39: 4925-4929.
1979.
12. Jones, C. A., and Huberman, E. A sensitive hepatocyte-mediated
assay for
the metabolism of nitrosamines to mutagens for mammalian cells. Cancer
Res., 40: 406-411, 1980.
13. Kinsella, A. R., and Radman. M. Tumor promoter induces sister chromatid
exchanges: relevance to mechanisms of carcinogenesis. Proc. Nati. Acad.
Sei. U. S. A., 75: 6149-6153,
1978.
14. Kligerman, A. D., Strom. S. C.. and Michalopoulos, G. Sister chromatid
exchange studies in human fibroblast-rat hepatocyte co-cultures: a new in
vitro system to study SCEs. Environ. Mutagen., 2: 157-165, 1980.
15. Lijinsky, W., and Muschik, G. M. Distribution of the liver carcinogen metha
pyrilene in Fischer rats and its interaction with macromolecules. J. Cancer
Res. Clin. Oncol., 103: 69-73, 1982.
16. Lijinsky, W., Reuber, M. D., and Blackwell, B. N. Liver tumors induced in
rats by oral administration of the antihistaminic methapyrilene hydrochloride.
Science (Wash. D. C.), 209: 817-819, 1980.
17. Malan-Shibley, L., and lype, P. T. The influence of culture conditions on cell
morphology and tyrosine aminotransferase levels in rat liver epithelial cell
lines. Exp. Cell Res., 131: 363-371, 1981.
18. Marin-Rosé, J., and Castagnoli, N. Methapyrilene: metabolism and macromolecular binding. Fed. Proc., 41: 1570, 1982.
19. Mirsalis, J. C., and Butterworth, B. E. Detection of unscheduled DNA
synthesis in hepatocytes isolated from rats treated with genotoxic agents:
an in vivo-in vitro assay for potential carcinogens and mutagens. Carcino
genesis, Õ:621-625, 1980.
20. Nagasawa, H., and Little, J. B. Factors influencing the induction of sister
chromatid exchanges in mammalian cells by 12-O-tetradecanoylphorbol-13acetate. Carcinogenesis, 2: 601 -607, 1981.
21. Perry, P., and Evans, H. J. Cytological detection of mutagen-carcinogen
exposure by sister chromatid exchange. Nature (Lond.), 258. 121-125,
1975.
22. Perry, P., and Wolff, S. New Giemsa method for the differential staining of
sister chromatids. Nature (Lond.), 257: 156-158, 1974.
4617
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1982 American Association for Cancer Research.
P. T. lype et al.
23. Probst, G. S., and Neal, S. B. The induction of unscheduled DNA synthesis
by antihistamines in primary hepatocyte culture. Cancer Lett., 10: 67-73,
1980.
24. Raychadhuri, R., Currens, M., and lype, P. T. Enhancement of sister chromatid exchanges by tumor promoting chemicals. Br. J. Cancer, 45:
769-777, 1982.
25. Raychaudhuri, R., Kelley, S., and lype. P. T. Induction of sister chromatid
exchanges by carcinogens mediated through cultured rat liver epithelial
cells. Carcinogenesis, 1: 779-786, 1980.
26. Wolff, S., and Rodin, B. Saccharin-induced
sister chromatid exchanges in
Chinese hamster and human cells. Science (Wash. D. C.), 200. 543-545,
1978.
Fig. 1. Autoradiograph of rat liver cells in
cubated with [3H]methapyrilene (4.3/xCi; 1 fig/
ml) for 24 hr and exposed for 8 weeks. The
grains are localized in the cytoplasm of these
intact cells. Giemsa, x 1000.
1
Fig. 2. Autoradiograph
of chromosome
preparations from rat liver cells treated with
[3H]methapyrilene and exposed for 8 weeks.
Metaphase
chromosomes
(A:
Giemsa,
x 1250) and interphase nuclei and surround
ing cytoplasm (B: Giemsa, x 1000) do not
show any grains over the background.
»
Fig. 3. Autoradiographs
of chromosome
preparations from rat liver cells treated with
[3H]DMBA (3.7 /iCi; 0.025 /xg/ml) for 24 hr
/
«>.,
2A % ^
.«..
2B
and exposed for 4 weeks. A number of grains
are located on the metaphase chromosomes
(A: Giemsa, x 1250). There is a preferential
accumulation of grains on the interphase nu
clei (B: Giemsa, x 1000). The surrounding
cytoplasm also has more grains over the back
ground.
.'
kr* •¿*•%
7ft.
4618
'.-'
C v •¿*•
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1982 American Association for Cancer Research.
VOL.
42
Inability of Methapyrilene to Induce Sister Chromatid Exchanges
in Vitro and in Vivo
P. Thomas Iype, Ratna Ray-Chaudhuri, William Lijinsky, et al.
Cancer Res 1982;42:4614-4618.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/42/11/4614
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. © 1982 American Association for Cancer Research.