Download Comparison of the Mutagenic and Clastogenic

[CANCER RESEARCH 44, 4420-4431,
October 1984]
Comparison of the Mutagenic and Clastogenic Activity of Amsacrine and
Other DMA-intercalating Drugs in Cultured V79 Chinese Hamster Cells1
William R. Wilson,2 Noelene M. Harris, and Lynnette R. Ferguson
Section of Oncology, Department of Pathology [W. R. W.] and Cancer Research Laboratory [N. M. H., L R. F.], University of Auckland School of Medicine, Private Bag,
Auckland, New Zealand
ABSTRACT
The acridine derivative amsacrine (m-AMSA) is used clinically
for the treatment of acute leukemias. The mutagenic activity of
this drug has been evaluated at the 6-thioguanine (6-TG) and
ouabain resistance loci in cultured Chinese hamster fibroblasts
(V79-171b cell line). m-AMSA was found to have weak but
significant mutagenic activity at the 6-TG but not at the ouabain
resistance locus, after either 1- or 45-hr exposures at concentra
tions causing up to 90% cell kill. Two other intercalating agents
with antitumor activity, Adriamycin and actinomycin D, provided
essentially identical results. All three drugs were potent inducers
of micronuclei in V79-171b cells, indicating high clastogenic
activity. For these intercalating agents, the yield of 6-TG-resistant
mutants was approximately 100-fold lower than that for ethyl
methanesulfonate after exposures causing equivalent toxicity or
equivalent chromosome breakage. The acridine half-mustard
ICR-191 resembled ethyl methanesulfonate rather than the other
intercalating agents in providing a high yield of 6-TG-resistant
mutants relative to its clastogenic activity. The tumor-inactive
intercalator 9-aminoacridine demonstrated only low clastogenic
activity with a lack of significant mutagenic activity at toxic
concentrations. These results suggest that, for m-AMSA, Adri
amycin, and actinomycin D, both cell killing and mutagenesis
could be direct consequences of chromosome breakage, while
9-aminoacridine may kill cells by a different mechanism. In view
of its mutagenic and clastogenic activity at clinically achievable
exposures and the similarity of its genotoxic properties to Adri
amycin, m-AMSA should be considered a potential carcinogen.
INTRODUCTION
Many antitumor drugs in current use are mutagenic in mam
malian cells (37). Such activity presents 2 potential problems for
their clinical use: (a) mutagenic drugs may act as carcinogens in
inducing new neoplasms after successful induction of remission
(23, 24); (b) mutagenic agents may induce drug resistance, thus
limiting the activity of other drugs used in combination protocols
(38). m-AMSA3 is a 9-anilinoacridine derivative developed as an
antileukemia agent in the laboratory of the late B. F. Cain (8). Its
clinical activity in the treatment of acute leukemia (28) and
lymphomas (46) has been established recently, and it is now
1This work was supported by grants from the Cancer Society of New Zealand,
Inc., by its Auckland Division, and by the Medical Research Council of New Zealand.
2To whom requests for reprints should be addressed.
' The abbreviations used are: m-AMSA, amsacrine; 9-AA, 9-aminoacridine; ACTD, actinomycin D; ADRIA, Adriamycin; EMS, ethyl methanesulfonate; PCS, fetal
calf serum; HGPRT; hypoxanthine-guanine phosphoribosyltransferase;
10»,drug
concentration causing 50% inhibition of growth (decrease in cell density relative to
controls); MF, mutation frequency; MN, micronucteus; NCS, neonatal calf serum;
QUA, ouabain; 6-TG, 6-thioguanine; ICR-191,2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine
dihydrochloride; o-MEM, a-minimal essential me
dium; Da?,drug concentration to reduce survival to 37% of controls.
Received January 9. 1984; accepted June 7, 1984.
4420
finding increasing acceptance as a less cardiotoxic substitute for
daunorubicin in remission induction therapy for acute myelocytic
leukemia (2). The present study has been undertaken with 2
major objectives: to assess the mutagenic potential of m-AMSA;
and to seek insights into the mechanism of cytotoxicity of this
and other intercalating agents.
m-AMSA binds reversibly to nucleic acids, with pronounced
specificity for native DMA (47), by intercalation (45). Comparison
of the biological activities of a series of m-AMSA analogues with
differing DMA-binding affinities suggests this interaction to be a
necessary, although not sufficient, condition for antitumor activity
(3). m-AMSA causes breakage of both DMA (20, 49) and chro
mosomes (14), but, as for other intercalators, the relationship of
these events to cell killing is uncertain. The mutagenic activity of
m-AMSA has not been investigated previously in mammalian
cells, but it is known to induce frameshift mutations in Salmonella
typhimurium (16).
In this study, we have quantitated the mutagenic activity of mAMSA, using the induction of 6-TG and ODA resistance in
cultured Chinese hamster fibroblasts (V79 cells). The clastogenic
activity of m-AMSA has been assessed in the same experiments
by scoring MN in interphase cells. Other intercalators have been
shown previously to be efficient inducers of MN in cultured
mammalian cells (36). MN usually arise from broken chromo
somes, the acentric fragments failing to segregate normally at
mitosis and remaining in the cytoplasm rather than becoming
incorporated into the daughter nuclei (22). It should be noted
that MN can also be generated without chromosome breakage
as a result of direct interference with segregation of (intact)
chromosomes during mitosis, although their production is in most
cases a consequence of clastogenic activity (48).
We have compared m-AMSA to EMS as a reference mutagen
and clastogen and to ADRIA and ACT-D as representative
intercalating drugs used clinically as antineoplastic agents. In
addition, 2 acridine derivatives lacking antitumor activity but
having known mutagenic properties have been investigated. ICR191 is a potent bacterial (12) and mammalian cell mutagen (1,
19) which appears to act as an alkylating agent (12), while 9-AA
binds to DNA reversibly by intercalation and causes frameshift
mutations in bacteria (17, 32).
MATERIALS AND METHODS
Drugs. EMS, ACT-D, 9-AA hydrochloride, and QUA were purchased
from Sigma Chemical Co., St. Louis, MO; ADRIA was from Farmitalia,
Milan, Italy; 6-TG was from Aldrich Chemical Co., Milwaukee, Wl; and
ICR-191 was from Polysciences, Ltd. The isethionate salt of m-AMSA
was kindly provided by Dr. B. C. Baguley, Auckland Division Cancer
Society of New Zealand. Sterile stock solutions of ICR-191 (1 mw in
0.01 N HCI), ACT-D (1 mw in dimethyl sulfoxide), 6-TG (1 mg/ml in 0.5%
NazCOa), and ADRIA (1 rtiM in 50% ethanol-water, v/v) were stored at
-20°. The purity of stock solutions was checked periodically by thin-
CANCER
RESEARCH
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VOL. 44
Mutagenicity and Clastogenicity of m-AMSA
layer chromatography on silica gel or cellulose. Fresh solutions of EMS,
m-AMSA, 9-AA, and QUA were prepared immediately before use.
Cells. The Chinese hamster fibroblast cell line V79-171 b was originally
obtained from Dr. W. R. Inch, London, Ontario, Canada. It is maintained
in this laboratory in a-MEM without nucleosides or antibiotics, containing
10% (v/v) heat-inactivated PCS (Gioco, New Zealand) by trypsinization
and subculture to 104 cells/T-25 flask twice weekly.
Preliminary experiments indicated a high level of spontaneous 6-TGresistant cells in our stocks of V79-171b (frequency, approximately 3 x
10~4with 6-TG at 5 ng/m\). This frequency was not decreased by dialysis
of serum or by increasing the 6-TG concentration from 5 to 10 /¿g/ml.In
the present study, cultures were therefore maintained by subculturing to
103 cells/flask once weekly to eliminate spontaneous 6-TG-resistant
mutants by limiting dilution (43). The V79-171b cell line maintained in this
manner is here designated V79-K. The frequency of background 6-TGresistant mutants in V79-K was 8.2 x 10"* ±6.3 (S.D.) x 10~* over the
course of this study. The frequency of OUA-resistant variants in V79-K
(4.8 x 10"* ±4.3 x 10"6) was not significantly different from that for
V79-171b.
V79-171b and V79-K cultures were reinitiated from frozen
stocks after not more than 12 weeks. The 2 cell lines showed no
divergence of properties with respect to growth rate (doubling time, 8.5
hr), cell morphology (by electron microscopy), colony morphology, cloning
efficiency, spontaneous MN frequency, or sensitivity to the growthinhibitory and clastogenic effects of the drugs studied. Both lines were
free of Mycoplasma as judged by cytochemical staining (9). All experi
ments described here were performed with V79-K unless otherwise
indicated.
A polyclonal OUA-resistant V79 cell line, V79-Oua-59, was selected
by mutagenizing V79-K with EMS (4 muÃ-for 60 min), subculturing in
nonselective medium for 4 days, and selecting 59 separate clones after
plating in 3 mw OUA. The pooled mutants (103 cells each) were grown
in bulk culture for 7 doublings in the presence of 3 mM OUA and then
maintained in the absence of OUA without loss of mutant phenotype. A
polyclonal 6-TG-resistant subline, V79-6TG-74, was developed in a
similar manner by pooling 74 separate clones arising from nonmutagenized V79-171b plated in 6-TG (5 >ig/ml).
Drug Exposure and Determination of Growth Inhibition and Cell
Killing. Exponential-phase cultures initiated 24 hr previously at 1.5 x 10s
cells/ml (for 60 min drug exposure) or 2.5 x 104 cells/ml (for 45 hr drug
exposure) in 10 ml growth medium [a-MEM containing 10% PCS,
penicillin (100 units/ml), and streptomycin (100 fig/ml)] per 100-mm Retri
dish were treated by addition of drug in 5 ml of prewarmed growth
medium containing 40 mw 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic
acid, pH 7.3. A typical experiment included 2 solvent controls and 5 drug
concentrations, chosen on the basis of a preliminary toxicity experiment
using log-phase cells in 24-well culture dishes. Retri dishes were imme
diately placed on a submerged grill in a 37°water bath, covered with a
Lucite dome, and flushed with 5% CO2 (60 min exposure) or returned to
the CO2 incubator (45 hr exposure). Drug treatment was terminated by
washing 3 times with phosphate-buffered saline (NaCI, 8 g/liter; KCI, 0.2
g/liter; KH2PO4, 0.2 g/liter; Na2HPO4, 1.15 g/liter; CaCI2, 0.1 g/liter,
MgCI?, 0.1 g/liter), and a single-cell suspension was obtained by trypsin
ization for 10 min at room temperature in 0.07% Difco Bacto-trypsin in
citrate:saline (trisodium citrate, 4.4 g/liter; KCI, 10 g/liter; pH 7.3). Cell
densities were determined with an electronic particle counter (Coulter
Electronics). The IDso following 45-hr drug exposure was determined.
Cell survival was assessed by plating up to 10" cells in 5 ml plating
medium of [a-MEM containing 15% NCS, penicillin (100 units/ml), and
streptomycin (100 Mg/ml)] in 60-mm Retri dishes and assessing colony
formation 8 days later by staining with 0.5% méthylèneblue in 50%
ethanol. The surviving fraction was determined relative to the plating
efficiency of the controls, which was in the range of 60 to 90%.
Expression and Selection of Mutants. After drug treatment, cells
were subcultured in growth medium to give approximately 106 clonogenic
cells (estimated on the basis of previous survival curve determinations),
but not more than 4 x 106 cells in total, per 100-mm Retri dish. Cells
were subsequently
OCTOBER
maintained in exponential-phase
growth by subcul-
turing to 10s cells/100-mm
dish every 2 days to allow expression
of
mutant phenotype. After an expression period of (typically) 6 days
following 60 min drug treatment or 4 days after 45 hr treatment, mutants
were selected by plating 6 x 10s cells (unless otherwise indicated) in
100-mm Retri dishes (4 replicates/culture) containing 15 ml of plating
medium with OUA (3 mM) or 6-TG (5 ^g/ml). At the same time, 100-mm
dishes containing 15 ml plating medium were seeded with 120 cells (in
triplicate for each culture) to determine the plating efficiency at the time
of mutant selection. Dishes were incubated for 11 days (selection dishes)
or 8 days (nonselective dishes) to allow colony formation. The MF was
calculated as the ratio of the plating efficiency in selective media to that
under nonselective conditions. The errors shown for MF are root mean
square averaged standard errors based on colony counts in replicate
selective and nonselective plates.
The statistical significance of drug-induced mutagenesis was tested
by 2 independent methods. Significant mutagenic activity was required
simultaneously In both tests for a result to be classed as positive. In
Method A, trends in MF with drug concentration were tested by linear
regression analysis. Significance of individual values of the correlation
coefficient, r, were ascertained using tables derived from Fisher's nor
malizing arc-tanh transformation
with n - 2 d.f. Using this test, agents
were classed as mutagenic if the gradient of the regression line was
significantly greater than zero at p = 0.05. In method B, MF values at
the highest drug concentration were tested to determine whether these
were above the 95th percentile of the controls. The values of all controls
over the course of these experiments (47 determinations for 6-TG
resistance and 42 determinations for OUA resistance) were not normally
distributed. However, for both markers, the upper 80% of the distribution
was fitted well by the lognormal distribution, with the 95th percentile at
a MF of 20.5 x 10"6 and 13 x 10"* (99th percentiles at 35 and 23.5 x
10"6) for 6-TG and OUA, respectively.
Determination of MN. Drug-induced chromosome breakage was as
sessed by counting MN in Giemsa-stained preparations prepared either
2 days after 60 min drug treatment or immediately
treatment. Trypsinized single-cell suspensions were
Camoy's fixative (methanohacetic acid, 3:1, v/v) after
tonic KCI (0.075 M) for 6 min at 37°,and were dropped
after 45 hr drug
fixed in ¡ce-cokJ
swelling in hypo20 cm onto clean
glass slides. Cytoplasmic structures were scored as MN if they showed
the same Giemsa staining reaction as the nucleus, were clearly resolved
from the nucleus (to distinguish from nuclear blebs), and had diameters
in the range 2.5 to 10 /im. The range of nuclear diameters in these slides
was 12.5 to 27.5 Mm for control cells. Either 100 cells with MN or 2000
cells in total were scored for each data point.
RESULTS
Reconstruction of Mutant Selection
Most quantitative studies on mutagenesis at the HGPRT locus
in V79 cells have used a selective medium containing 5 or 10%
PCS with 6-TG at approximately 5 //g/ml. Such selections are
performed using not more than 2 x 10s cells/100-mm Retri dish
to ensure efficient recovery of mutants (7). We evaluated the
efficiency of mutant recovery at a range of seeding densities
using selective medium containing 15% NCS and 6-TG (5 ¿¿g/ml)
(Chart 1). To avoid generalization on the basis of a single mutant
phenotype, a 6-TG-resistant cell line of polyclonal origin, V796TG-74, was used. The efficiency of selection of 6-TG-resistant
cells and the size of the resulting colonies, were not diminished
by the presence of up to 106 wild-type cells (V79-K) per 100-mm
dish. An analogous experiment with a polyclonal OUA-resistant
subline, V79-Oua-59, again demonstrated efficient selection of
these mutants against a background of up to 106 wild-type cells
in medium containing 15% NCS and 3 mw OUA (Chart 1). In
subsequent experiments, seeding densities of 6 x 10s cells/100-
1984
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4421
W.R. Wilsonet al.
Ô5>•
1000
ICR-191
6TG".
,fT
ozLU
EMS, Qua"
üü_u_
'
too-
LUOZ^lUU80604020n•
CELLS PLATED x 1CT5
Chart 1. Reconstruction of mutant selection. Variable numbers of V79-K cells
were seeded with 100 V79-6TG-74 (O) or 100 V79-Oua-59 (•)
cells in 100-mm
diameter Retri dishes containing 15 ml of selective medium [plating medium
containing 6-TG (5 ^g/ml) or 3 rnw QUA, respectively]. Plating efficiencies were
determined by scoring colonies after 11 days. Bars (SE)were smaller than the size
of the plotted points when not shown. Plating efficiencies in nonselective plating
medium were 64 ±2% for V79-6TG-74 and 87 ±4% for V79-Oua-59. The
background frequency of mutants in V79-K in this experiment (1.3 x 10"* for 6-TG
resistance, 2.0 x 10"* for QUA resistance) has not been subtracted.
Control,
6TG*
10
12
10-
EXPRESSION TME (d)
mm selection dish were used, with this inoculum being reduced
when necessary to ensure less than 100 mutant colonies per
plate.
We will present results first for the alkylating agents used as
reference mutagens and then for the intercalating agents mAMSA, ADRIA, ACT-D, and 9-AA.
Alkylating Agents
Phenotypic Expression Times. Followingexposureto EMS
for 1 hr, the expression of the 6-TG resistance mutant phenotype
was incomplete after 2 days of growth under nonselective con
ditions but appeared to be fully established by Day 4. For
example, mutation frequencies were 2.2 (r = 0.67), 36.5 (r =
0.90), and 46.6 (r = 0.95) mutants/106 clonogenic cells/mM on
Days 2, 4, and 6, respectively, after 60 min exposure (gradients
of linear regression curves relating MF to drug concentration for
the pooled data of 2 experiments with 6 dose levels each). In
contrast, expression of the OUA-resistant phenotype occurred
more rapidly and was complete by Day 2, with MF of 3.4 (r =
0.96), 4.1 (r = 0.81), and 3.2 (r = 0.82) mutants/106 clonogenic
cells/mw on Days 2, 4, and 6 in the same experiments. Data
were fitted acceptably by a linear relationship between MF and
concentration in these and other experiments (see correlation
coefficients in Tables 1 and 2) but, in order to accommodate a
wide range of values, are plotted on a logarithmic scale in Charts
2 to 9.
Expression of QUA resistance was again complete by 2 days
after termination of a 45-hr exposure to EMS (Chart 2). 6-TG
resistance was also fully expressed by 2 days after 45 hr
treatment with EMS (data not shown) or ICR-191 (Chart 2).
Linear regression analysis of the data of Chart 2 indicated the
lack of a significant trend in mutant frequency with time after
mutagenesis with EMS or ICR-191, suggesting that there was
no marked differential in growth of either 6-TG- or OUA-resistant
mutants relative to wild-type V79-K. In subsequent experiments,
an expression time of 6 days after 60 min drug treatment and 4
days after the end of 45 hr exposure was generally used. In
4422
Chart 2. Dependenceof MF on expression time following 45 hr exposure to
EMS at 0.5 mu (•)
or ICR-191 at 1.5 ,.M(•).
Controls (O, D) were parallelcultures
receiving no drug treatment. O. •,selection in 6-TG: D, •selection in QUA.
Expression time is recorded from the end of the drug treatment period, d, days;
67G", 6-TG-resistant; Oua", OUA-resistant.
most experiments, at least one other expression time was tested.
No significant difference between 4-, 6-, or 8-day expression
was observed for any drug.
Cytotoxic, Clastogenic, and Mutagenic Potency. Exposure
of V79-K cells to EMS for 60 min resulted in a high yield of
mutants resistant to 6-TG or OUA at concentrations causing little
cell killing (Chart 3). Over this same range of drug concentrations,
chromosome breakage was readily detected as an increase in
the frequency of MN, which rose to 10 times the control fre
quency when scored 2 days after treatment with 30 mw EMS
(Chart 3).
Mutagenesis was detected with greater sensitivity after 45 hr
exposure, under which conditions the molar potency of EMS as
a mutagen was approximately 10-fold higher than when using 1
hr exposure (Chart 3; Tables 1 and 2). However, the relationship
between clastogenesis and mutagenesis was similar using the 2
exposure times. Thus, in both cases, EMS induced approxi
mately 20 mutants at the HGPRT locus for every thousand cells
in which MN were induced, while approximately 1.5 mutants
were induced at the Na+/K+-ATPase locus for this extent of
chromosome breakage (Tables 1 and 2). These estimates were
derived from the ratios of the gradients of the linear regression
lines giving best fit to the mutation frequency and MN frequency
as a function of drug concentration. Drug concentrations giving
MN frequencies higher than 9% were excluded from analysis
since there is clear evidence for nonlinearity at high doses as a
result of inhibition of progression of damaged cells through
mitosis.4
The effects of treatment with ICR-191 are shown in Chart 4
and Tables 1 and 2. ICR-191 was highly mutagenic at the HGPRT
locus after either acute or chronic exposure. As with EMS, the
4W. R. Wilson, S. M. Tapp, and J. C. Probert, manuscript in preparation.
CANCER RESEARCH
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VOL. 44
Mutagenicity and Clastogenicity of m-AMSA
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CANCER RESEARCH
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VOL. 44
Mutagenicity and Clastogenicity of m-AMSA
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Chart 3. Dose-response curves for cell killing, clastogenesis, and mutagenesisfollowing exposure to EMS
for 60 min (left) or 45 hr (right). The surviving fraction
(O) was determined by plating cells at the end of drug
treatment, and the percentage of cells with MN was
assessed at the same time (right) or 2 days later (left).
MF is expressed with respect to surviving (clonoqenic)
cells at the time of selection (see 'Materials and Meth
ods"). Left, 6-TG-resistant mutants, expression time 4
(3) or 6 days (•);
OUA-resistant mutants, expression
time 4 (U) or 6 days (•).
Right, 6-TG-resistant mutants,
expression time 8 days (•);OUA-resistant mutants,
expression time 4 days (•).
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Chart 4. Dose-response curves for cell killing, cte
togenesis, and mutagenesisfollowing exposure to 1C
191 for 60 min (left) or 45 hr (right). Symoo/s are
defined for Chart 3, left. Bars have been omitted frc
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CONCENTRATION
molar potency of ICR-191 as a mutagen was increased approx
imately 10-fold using 45 hr exposure. Activity at the ODA resist
ance locus was much lower and did not achieve consistent
statistical significance. Although relatively little cell killing was
observed in these experiments, significant induction of MN was
observed, enabling comparison of mutagenic and clastogenic
activity. The average number of 6-TG-resistant mutants induced
per thousand cells with MN was 28 for a 60-min exposure and
25 for 45 hr exposure. Thus, the relationship between clasto
genic activity and mutagenesis at the HGPRT locus is similar for
the 2 alkylating agents.
Amsacrine
m-AMSA is at least 100 times more potent than is the acridine
half-mustard ICR-191 as a cytotoxic agent in V79-K cultures. In
OCTOBER 1984
O
OS
io
15
(pM)
the experiment illustrated in Chart 5, a 1-hr exposure to m-AMSA
caused exponential cell killing with a D37 of 0.19 ^M and pro
nounced chromosome breakage as evidenced by a marked
increase in MN after an expression time of 2 days. This experi
ment provided clear evidence of mutagenesis to 6-TG resistance
when selected using either 2x105or6x105
cells/100-mm
dish, while no mutation to QUA resistance was observed. Similar
results were obtained at approximately 20-fold lower drug con
centrations using 45 hr exposure (Chart 5).
In the above experiment, mutagenesis expression times of 6
days after 60 min drug exposure and 4 days after 45 hr drug
exposure were used. In 2 other experiments, the dose-response
relationship for mutagenesis after 1 hr exposure to m-AMSA
was not significantly different when expression times of both 4
and 6 days were compared, these data being pooled to calculate
the mutagenic activities shown in Table 1. A more systematic
4425
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W. R. Wilson et al.
too
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OC 10
Charts. Dose-response curves for Å“il killing, clastogenesis,
and mutagenesis following exposure to m-AMSA. The surviving
fraction (O) was determined by plating cells at the end of drug
treatment, and the percentage of cells with MN was assessed at
the same time (45 hr drug exposure (right)] <* 2 daVs later I60 min
drug exposure (left)]. Selections were performed 6 days after 60
min exposure and 4 days after 45 hr exposure in QUA (•)
or 6-TG
(»,2x10» cells/dish; •,6 x 10s cells/dish).
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CONCENTRATION
examination of expression time dependence after 45 hr exposure
to m-AMSA at 20 nM revealed significant mutagenesis on each
occasion when assayed every 2 days from 2 to 12 days after
treatment, with no significant trend in mutant yield over this time.
Although a much more dose-potent drug than either of the 2
alkylating agents tested, m-AMSA was only weakly mutagenic
relative to its cytotoxic or clastogenic activity. Thus, in the
experiment of Chart 5, the frequency of drug-induced mutations
at the HGPRT locus was only 2.5% of that with EMS when both
drugs were compared at the D37(Table 1). The yield of mutants
at equivalent clastogenic activity was also markedly lower for mAMSA with 0.56 mutation/thousand cells with MN, compared to
a value of 20 for this ratio with EMS (Table 1).
In repeat experiments, the mutagenic potency of m-AMSA
was somewhat variable, ranging from 65 to 146 6-TG-resistant
mutants/106 clonogenic cells/juM for 1-hr exposure and from 615
to 2790 mutants/106 clonogenic cells/Mmol for 45-hr exposure
(Tables 1 and 2). In only 4 of the 7 experiments summarized in
Tables 1 and 2 was mutagenesis at the HGPRT locus statistically
significant when assessed by determining whether the gradient
of the linear regression line was significantly different from zero
(Method A). However, in 2 of the remaining 3 experiments,
mutagenesis was statistically significant at the highest drug
concentration when tested using Method B. These 2 experi
ments each included less than 6 dose levels, so that a very high
r value would be required to establish significance by Method A.
(
relationship between cell killing, chromosome breakage, and
mutagenesis was observed following 45 hr drug treatment with
approximately 0.7 6-TG-resistant mutant/103 induced MN in each
case (Tables 1 and 2), but the dose potency of the drug was
higher using the longer exposure time (Chart 6).
Exposure to ACT-D for 45 hr provided a quite different result.
Little clastogenic activity could be detected and no significant
mutagenesis was observed at either locus (Chart 7; Tables 1
and 2), even when concentrations extended to 3 times the IDso.
However, over this range of concentrations, ACT-D caused little
or no cell killing (Chart 7). Thus, in contrast to m-AMSA and
ADRIA, ACT-D at very low concentrations inhibited cell growth
during prolonged drug exposure by a reversible cytostatic proc
ess without significant killing. Higher drug concentrations could
not be used in attempting to obtain cell killing during 45 hr
exposure, since the marked growth inhibiton precluded recovery
of sufficient cells for subsequent study.
9-Aminoacridine
The cytotoxic, clastogenic, and mutagenic activities of ADRIA
and ACT-D resulting from 1-hr exposures were very similar to
those of m-AMSA, both in absolute (molar) potency and in the
The survival curve for treatment of V79-K with 9-AA for 1 hr
demonstrated a marked difference from the other 3 intercalating
drugs, with a larger threshold and a D37approximately 200-fold
higher (Chart 8). 9-AA was peculiar among the agents in this
study in demonstrating mutagenic activity at neither the HGPRT
or QUA resistance locus, even at cytotoxic drug concentrations
(Tables 1 and 2).
The clastogenic activity of 9-AA was also low, not only in
terms of absolute potency but also in relation to cell killing. Thus,
after treatment with 9-AA for 1 hr, the frequency of cells with
drug-induced MN was markedly less than after treatment by
EMS, m-AMSA, ADRIA, or ACT-D at equivalent cytotoxicity
relationship between these end points (Tables 1 and 2). As
illustrated in Charts 6 (ADRIA) and 7 (ACT-D), pronounced
induction of MN was evident over the range of drug concentra
tions causing cell kill after 60 min exposure, and this was
accompanied by significant mutagenesis at the HGPRT locus,
but not at the ODA resistance locus. For ADRIA, a similar
(Chart 9). Since MN can be detected only after damaged cells
pass though mitosis, it is possible that the relative lack of
induction of M N by 9-AA could be a consequence of a pro
nounced inhibition of cell division after treatment, delaying ap
pearance of MN. We found that such inhibition of cycle progres
sion was more pronounced with 9-AA than with the other 3
ADRIA and ACT-D
4426
CANCER RESEARCH
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VOL. 44
Mutagenicìtyand Clastogenicity of m-AMSA
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RIA. Left, 60 min exposure, 6-day expresston; right, 45
hr exposure, 4-day expression. Symbols as for Chart
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Chart 7. Dose-response curves for cell killing, clastogenesis, and mutagenesis following exposure to
ACT-D. ¿.eft,60 min exposure, selection in QUA (•,6day expression) or 6-TG (9, 4-day expression; •,6day expression); right, 45 hr exposure, selection in OUA
P, 4-day expression) or 6-TG (<».
2-day expression; •,
4-day expresston). Other symbols as for Chart 5.676",
6-TG-resistant; Oua", OUA-resistant.
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intercalators when cells were treated to equivalent cytotoxicity.
However, when MN were scored daily for 4 days after treatment
with 9-AA or m-AMSA, frequencies decreased markedly with
either drug after 2 days, and the summed frequencies at times
of equivalent cell division were much lower for 9-AA. These
experiments, which will be reported in detail elsewhere,4 estab
lish that the apparent low clastogenic activity of 9-AA is not an
artifact resulting from retarded progression through mitosis.
After continuous exposure for 45 hr, 9-AA resembled ACT-D
in causing little cell killing at concentrations in the vicinity of the
IDso (Chart 8). Weak clastogenic activity was observed in this
experiment, and some indication of an increased frequency of 6TG-resistant mutants was obtained. The trend towards in
creased MF was significant by the criterion of Method A, but not
by that of Method B (Table 2). Repetition of this experiment did
not provide a significant trend in MF with dose using either
statistical method. We therefore consider 9-AA to be nonmutagenic at both the 6-TG and OUA resistance loci.
OCTOBER 1984
0-001
O-OO2
0-003
(pM)
DISCUSSION
Validation of Methodology. The procedure adopted in this
study for quantitating mutagenesis at the HGPRT and OUA
resistance loci in V79 cells is a minor modification of the method
in use in many other laboratories (for a review, see Ref. 7). In
our system, 6-TG- or OUA-resistant mutants can be selected
with high efficiency against a background of 6 x 105 wild-type
cells in a 100-mm Retri dish (Chart 1). Although other workers
have found that cell densities of 2 x IC^/IOO-mm dish should
not be exceeded for selection of 6-TG-resistant mutants (33,
44), we have confirmed that the higher density is acceptable for
the quantitation of m-AMSA-induced mutants (Chart 5), as well
as in selection reconstruction experiments using spontaneous
mutants in the V79-171b cell line (Chart 1). Efficient selection at
high cell density is presumably a feature of the V79-K cell line,
rather than the use of NCS in place of FCS since we have
subsequently obtained similar results using 3% FCS in the
4427
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W. R. Wilson et al.
¿ 1OO -
O)
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Chart 8. Dose-response curves for cell killing, clastogenesis, and mutagenesis following exposure to 9AA. Symbols as for Chart 5. The expression ame before
mutant selection was 6 days after 60 min drug treat
ment (left) and 4 days after 45 hr drug treatment (right).
6TGP, 6-TG-resistant; CW, OUA-resistant.
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CONCENTRATION
a
4
(yM)
to EMS, while expression of the 6-TG-resistant phenotype oc
curs more slowly. Following more protracted exposure to EMS
or ICR-191 (for 45 hr), the expression of 6-TG resistance appears
to be more rapid, being complete by 2 days after drug removal
(Chart 2), presumably because of the additional time for expres
sion available during the exposure period.
Mutagenesis by Intercalating Agents. No clear consensus
has emerged in the literature regarding the mutagenic activity of
DMA-intercalating agents. 9-AA has been reported to be nonmutagenic at the HGPRT locus in human fibroblasts (15) but
significantly mutagenic at the thymidine kinase locus in L5178Y
lymphoblasts (1). ACT-D was found to be mutagenic at the
HGPRT locus in Chinese hamster ovary cells (21) but not in V79
cells (31), in contrast to ADRIA which was strongly mutagenic in
the same system despite control MFs of approximately 10~4.
10
06
04
02
01
10
06
04
SURVIVING FRACTION
Chart 9. Relationship between MN frequency and cell killing for 60 min (left) and
45 hr (right) drug exposure. Each point represents one drug-treated culture.
. 2 S.D. above the mean for the controls. Separate symbols represent different
experiments for exposure to EMS (x, +), m-AMSA (O, D, A, V), ADRIA (C, 9)
ACT-D (E), or 9-AA (•,•A). Curves were fitted by inspection.
selection media. Variations in cross-feeding, and hence in effi
ciency of selection of 6-TG-resistant mutants, have been noted
in different V79 cell sublines (34).
The results obtained with the reference mutagens EMS and
ICR-191 are in good agreement with those reported by others
with respect to mutagenic specificities, absolute mutagenic po
tencies, and kinetics of expression of the mutant phenotype.
Thus, EMS was strongly mutagenic at the HGPRT locus with
lower activity at the ODA resistance locus (Chart 3), while ICR191 was active only at the former locus (Chart 4; Tables 1 and
2). The absolute value of mutagenic potency following 60 min
exposure to EMS (0.041 mutant/106 clonogenic cells/MM) is
similar to previous estimates for V79 cells (18, 47). The muta
genic activity of ICR-191 in V79 cells has not been reported
previously. However, its activity at the HGPRT locus (19,25,27)
and lack of activity at the QUA resistance locus (19, 29, 30) has
been demonstrated in a variety of other mammalian cells.
Like others (7), we find expression of the OUA-resistant mutant
phenotype to be complete within 2 days after a 60-min exposure
4428
Other workers have described ADRIA as a weak mutagen at the
HGPRT locus in V79 (6,39), or Chinese hamster ovary (37) cells.
We find ADRIA and ACT-D to be consistently mutagenic at the
HGPRT locus, although the activity of ACT-D is not seen when
the duration of drug exposure is extended from 1 to 45 hr.
Neither agent causes mutation to QUA resistance. These drugs
induced only low levels of mutants relative to their cytotoxic
activities, with mutagenic activities at the D37in the order of 100fold lower than for EMS (Tables 1 and 2).
For m-AMSA, the relationship between toxicity and mutagene
sis at the HGPRT locus, although somewhat variable, was similar
to that for ADRIA and ACT-D (Tables 1 and 2). In 4 of 7
experiments with m-AMSA, the statistical significance of the
apparent mutagenesis could be established by determining that
the gradient of the linear regression line fitted to the data was
significantly different from zero and was positive (Method A). In
2 of the 3 experiments where this condition was not met, the
yield of mutants at the highest dose was sufficient for the
compound to be classed as a mutagen by the criterion of Method
B, which assesses MF in relation to the distribution of control
values over all experiments. The statistical evaluation of mam
malian cell mutagenesis by weak mutagens is an acknowledged
problem (7,13) which awaits a satisfactory solution. The use of
the 2 independent methods used here offers a partial solution to
some of the limitations in available tests. Regression analysis
offers the advantage of utilizing data throughout the drug conCANCER RESEARCH
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VOL. 44
Mutagenìcityand Clastogenicity of m-AMSA
centration range and of providing objective interpolation, but it
suffers several defects in the present context, particularly when
the number of data points is low. In the latter case, a true
mutagen may fail to demonstrate a significant correlation despite
a high r value. Further, regression analysis fails to take into
account the considerable interexperiment variability of controls.
The marked positive skewness in the latter distribution suggests
that high MF values can occur relatively frequently with nonmutagens and could give rise to spurious correlations by regression
analysis. Determination of the upper 5 and 1% confidence limits
for this skewed control distribution thus provides valuable addi
tional information with which to check the conclusions from
regression analysis.
Mutagenesis by m-AMSA was not evident at the QUA resist
ance locus. In fact, although not statistically significant, the
frequency of OUA-resistant mutants tended to decline with dose
of this or the other ¡ntercalators. This trend, if real, cannot
properly be considered as an antimutagenic effect since most of
the mutants in question were presumably already present in the
cell stocks prior to drug treatment. Elimination of these preexist
ing mutants by limiting dilution could have caused this apparent
antimutagenic effect, since the total number of clonogenic cells
used to initiate expression plates was reduced at high drug
concentrations.
One- versus 45-hr Drug Exposure. To our knowledge, no
systematic comparison of mammalian cell mutagenesis under
conditions of acute high-dose versus chronic low-dose drug
treatment has been reported, although one study has shown
that the mutagenic activity of certain platinum compounds is
seen only after prolonged exposure (40). We have shown that
exposing V79 cells to intercalating or alkylating agents for 45 hr
provides a test system approximately 10-fold more sensitive
than 1-hr exposure with respect to dose potency (Tables 1 and
2) but that the relationship between mutagenesis and cell killing
is similar under both conditions. However, continuous low dose
exposure is not satisfactory for the evaluation of drugs which
inhibit cell growth under such conditions by a reversible mecha
nism rather by causing cell killing. For 4 of the 6 agents tested
in the present study (EMS, ICR-191, ACT-D, and 9-AA), the
concentration required for 90% cell kill was well in excess of the
ID50, making it impractical to recover sufficient cells treated at
cytotoxic concentrations. Thus, despite its lowered sensitivity,
pulse exposure to test agents for periods which are short in
relation to the doubling time appears to be preferable.
The observation that ACT-D is mutagenic when V79-K cells
are exposed for 1 hr but not for 45 hr is presumably a reflection
of the lack of cytotoxicity of the drug at concentrations providing
sufficient cells for analysis under the latter conditions. This
suggests that mutagenesis by ACT-D is a consequence of the
same lesion responsible for cell killing and that the lesion respon
sible for growth inhibition is different. ACT-D differs from the
other ¡ntercalatorstested in its interference with nucleolar struc
ture and RNA synthesis at concentrations which are low relative
to its cytotoxic potency.5 Inhibition of RNA synthesis could
therefore be responsible for this reversible cytostatic effect, while
cell killing at higher drug concentrations may result from chro
mosome breakage (see below).
Clastogenesis: The Cause of Cell Killing and Mutagenesis
by the Antitumor Intercalating Agents? Quantitation of MN in
5C. G. Jensen, W. R. Wilson, and A. R. Bleumink, manuscript in preparation.
OCTOBER
1984
the same experiments in which mutagenesis was assessed has
enabled a direct comparison of Clastogenesis and mutagenic
effect at the HGPRT locus for all agents. These comparisons
suggest a simple hypothesis relating Clastogenesis, cell killing,
and mutagenesis for the antitumor intercalators, namely, that
chromosome breakage is directly responsible both for loss of
reproductive potential (through generation of postmitotic genetic
defects) and for functional inactivation of the HGPRT gene
(through deletion and/or translocation).
The following observations can be cited as evidence in support
of this view.
Induction of MN by the 3 antitumor intercalators, m-AMSA,
ADRIA, and ACT-D shows a close correlation with cell killing
(Chart 9), consistent with both end points being a consequence
of the same lesion. The alkylating agents EMS and ICR-191
demonstrate a similar relationship, suggesting that they may also
cause cell killing by this mechanism.
The magnitude of MN induction is consistent with chromosome
breakage being a major contributor to the loss of reproductive
potential caused by these drugs. Time lapse studies of individual
irradiated mammalian cells under phase contrast have convinc
ingly demonstrated that formation of a MN represents a lethal
event, at least for ionizing radiation (26). Although in the present
study only approximately 6% of cells contained MN 2 days after
a 1-hr treatment which caused 50% cell kill (Chart 9), there are
several reasons why the frequency of cells with MN is expected
to be substantially less than the proportion of cells killed. Thus,
each mitosis generating a daughter cell with a MN will also
produce a genetically deficient sister cell of normal appearance.
In addition, the culture at 2 days after treatment may contain
some killed cells which have failed to pass through mitosis
precluding display of acentric fragments as MN and may suffer
from dilution of MN-bearing cells as a result of overgrowth by
viable cells. Further, the stringent criteria used for scoring MN in
this study (see "Materials and Methods") excluded many pre
sumptive MN, less stringent criteria providing estimates up to
30% higher than those reported.
In each instance where an intercalator caused efficient induc
tion of MN (m-AMSA and ADRIA after 1 or 45 hr exposure, ACTD after 1 hr exposure), a weak mutagenic effect of similar
magnitude was observed at the HGPRT locus (Tables 1 and 2).
In contrast, 9-AA was a relatively inefficient inducer of MN even
at cytotoxic concentrations and failed to elicit significant muta
genesis.
Ionizing radiation resembles the antitumor intercalators in in
ducing MN at high frequency (5, 10) and in its weak mutagenic
activity at the HGPRT locus and lack of activity at the Na+-K+ATPase locus (19, 41). The latter authors, and Cox and Massen
(11), have suggested these mutations to be due to gross genetic
damage rather than point mutations. The reported (42) MF for
6-TG resistance after 7-irradiation of V79 cells (approximately
35 mutants/106 clonogenic cells at the D37)is in the same range
as our estimates for m-AMSA, ADRIA, and ACT-D at equivalent
toxicity (29 ±14 mutants/106 clonogenic cells). Thus, the mu
tagenic action of the antitumor intercalating agents could be a
direct consequence of chromosome breakage, resulting in dele
tion of the HGPRT locus or its functional inactivation through
translocation. The lack of mutagenic activity at the OUA locus is
consistent with the view that these compounds do not cause
point mutations since mutagenesis at this site requires a localized
lesion leading to inactivation of the OUA-binding site of the Na+4429
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1984 American Association for Cancer Research.
W. R. Wilson et al.
K*-ATPase without loss of catalytic activity.
It is of interest that the classical bacterial frameshift mutagen,
9-AA, has less mutagenic activity than the antitumor intercalators
in V79 cells and is also conspicuously less active as a clastogen
at equivalent toxicity. The mechanism of cell killing by 9-AA
would therefore appear to be fundamentally different from the
other intercalators and is probably not a consequence of chro
mosome breakage. The clear difference in genotoxicity between
9-AA and the antitumor intercalators in the V79 cell line offers
an excellent opportunity for evaluating the mechanism(s) of
action of the intercalating agents, and in particular for testing the
role of DNA breakage and inhibition of nucleic acid biosynthesis
in cell killing and mutagenesis.
Implications for the Therapeutic Use of m-AMSA. In view of
the potent clastogenic activity of m-AMSA and its weak but
significant mutagenic activity in mammalian cells, this new clinical
antileukemia agent must be considered a potential carcinogen.
In this context, we note that ADRIA causes mammary tumors in
rats after a single i.v. dose (4, 31, 35). When used clinically in
the treatment of adult leukemia, m-AMSA is usually administered
i.v. as a daily dose of approximately 90 mg/sq m for 5 days.
Since a single tracer dose of [14C]-m-AMSA at 6 mg/sq m
resulted in peak plasma levels in humans in the vicinity of 0.1
iiM, with a terminal half-life of approximately 7 hr (22), we
estimate that clinical exposures [concentration (C) x time (f)] will
be in the order of 10 //M-hr. In contrast, C x f values for mAMSA in the present study did not exceed 1 /iw-hr. Thus,
exposures resulting in significant mutagenesis in mammalian cell
cultures are well within the range attainable in humans at thera
peutic doses.
In addition to its potential as a carcinogen, the mutagenic
action of m-AMSA is of concern in relation to possible induction
of drug resistance when used in combination with other cytotoxic
drugs. Since 6-TG has been used in combination with m-AMSA
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ACKNOWLEDGMENTS
The authors wish to thank S. M. Tapp for expert technical assistance, S. Hill for
typing the manuscript, L. Logan for preparing the figures, P. Mullins for assistance
with statistical analysis, and Drs. B. C. Baguley, W. A. Denny, and J. C. Proben
for their support and interest.
22.
26.
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Comparison of the Mutagenic and Clastogenic Activity of
Amsacrine and Other DNA-intercalating Drugs in Cultured V79
Chinese Hamster Cells
William R. Wilson, Noelene M. Harris and Lynnette R. Ferguson
Cancer Res 1984;44:4420-4431.
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