Mutation Research, 266 (1992) 17 ! - 180 © 1992 Elsevier Science Publishers B.V. All rights reserved 002%5107/92/$05.00
171
MUT 05065
Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells H. Kojima a, H. Konishi
a
and Y. Kuroda b
a Biochemical Research Institute, Nippon Menard Cosmetic Co. Ltd., Ogaki, Gila 503 and b Laboratory of Phenogenetics, National Institute of Genetics, Mishima, Shizuoka 4 i l (Japan) (Received 2 April 1990) (Revision received 18 October 1991) (Accepted 18 October 1991)
Keywords: Methyl methanesulfonate; Ethyl methanesulfonate; Combined mutagenicity; Mammalian cells
Summary The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D2o and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of Dzo and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.
Many chemicals present in our environment have been examined for their mutagenic activity with various test systems. Most of these tests have dealt with the mutagenic effect of single chemi-
Correspondence: Prof. Dr. Yukiaki Kuroda, Research Institute of Biosciences, Azabu University, !-17-71, Fuchinobe, Sagamihara, Kanagawa 229 (Japan). Tel.: 0427-54-7111 (Ext. 430); Fax: 0427-54-7661. Abbreviations: MMS, methyl methanesulfonate; EMS, ethyl methanesulfonate; 6TG, 6-thioguanine; OUA, ouabain.
cals on organisms. In our environment, however, many chemicals are present together. It is necessary to examine the combined effects of different chemicals. The combination of more than two chemicals may produce various modification of the mutagenicity of each chemical involved. One of the modifications of the mutagenicity of chemicals involves the enhanced or synergistic effects of more than two chemicals. The other modification is the inhibition of one chemical by another one. The inhibition of mutagenicity of one chemical by
another one can be divided into desmutagenesis, which directly inactivates the mutagens, and bioantimutagenesis, which suppresses the cellular mutagenesis induced by other mutagens (Kada et al., 1985). Antimutagenesis and anticarcinogenesis have been studied (Shankel et al., 1986; Kuroda et al., 1990). There have, however, been few studies concerning additional or enhanced effects of more than two mutagens. Vericat et al. (1984) reported the expression of the SOS response following a simultaneous treatment with N-methyl-N'-nitroN-nitrosoguanidine (MNNG) and mitomycin C in Escherichia coll. Kawazoe et al. (1986) reported on the mutation frequency of Salmonella typhimurium TA100 after a simultaneous treatment of alkylating agents. These reports concerned the combined effects of more than two chemicals on induced mutations in bacterial and yeast cells. Information on the combined effects of more than two mutagens on mammalian cells is extremely limited. Recently Ager and Haynes (1987, 1988) presented a general mathematical technique for the description and classification of the lethal interactive effects that occur when cells are treated with two or more toxic agents. They applied this mathematical technique to the interaction between heat and ultraviolet light (UV) and between EMS and UV for cell killing, mutation and gene conversion in the yeast Saccharomyces cere. risiae (Agar and Haynes, 1990a,b). In the present study, the effects of simultaneous treatments of MMS and EMS on the induction of 6TG-resistant mutants and chromosome aberrations were examined in cultured Chinese hamster V79 cells. Some of the results obtained have been reported previously (Kojima et al., 1986). Materials and methods
Cell culture The cell line used was the strain of Chinese hamster lung V79 cells (Ford and Yerganian, 1958). The cells were maintained in Eagle's minimum essential medium (Nissui Seiyaku Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (Gibed, New York) in 60-ram petri
dishes (Lux, Newbury Park, CA, No. 5216, 2-mm grid) under a controlled atmosphere of 5% CO 2 and 95% air at 37.5°C. The cells were mycoplasma-free, gave a colony-forming ability of more than 80% and a population doubling time of 15.3 h under the above culture conditions. The cells were grown in large mass, distributed in many ampoules, and frozen at -80°C. Before use, cells were thawed at 37.5°C, suspended in fresh medium, and cultured in GHAT medium (1.6 x 10 -~ M glycine, 10 -4 M hypoxanthine, 4 × 10 - 7 M amethopterin, and 1.6 x 10 -s M thymidine) at 37.5°C for 24 h, to remove the pre-existing 6TG-resistant cells in the cell population. During further subcultivation in normal medium, the appearance of spontaneous 6TG-resistant mutants was occasionally noted. For this reason, stock cultures were discarded at the tenth subculture and replaced from frozen stocks. Cells at an exponential growth phase in monolayer were dissociated by treatment with 0.25% trypsin (Difco, Detroit, MI, 1:250) solution, and centrifuged at 1500 rpm for 5 rain. The cells were then resuspended in the culture medium and used for experiments.
Mutagens tested Ethyl me~hanes,alfonate (EMS) (Aldrich) and methyl methaa,:~ulfonate (MMS) (Aldrich) were tested for their combined effects on the induction of 6TG-resistant mutations. They were diluted in Hanks' (Nissui Seiyaku Co., Tokyo) solution, sterilized through a filter with 0.45/~m pore size, and used for treatments of cells at various concentrations. Cytotoxicity assay The cytotoxic effect of chemicals was examined by determining the colony-forming ability of ceils, as described previously (Kuroda, 1984; Kuroda et al., 1985). Triplicate inocula of 2 x 102 cells were incubated in 60-mm petri dishes in 5 ml of normal medium for 20 h, washed twice with Hanks' salt solution, and treated with MMS at a concentration of D2o and EMS at various concentrations simultaneously or with EMS at a concentration of D.,0 and MMS at various concentrations in Hanks' salt solution at 37.5°C (the values of D2, of EMS and MMS are shown in Table 1).
173 TABLE 1 THE D2o VALUES OF MMS AND EMS ON SURVIVAL OF CHINESE HAMSTER V79 CELLS Time of treatment
D2o value (p.g/ml) MMS
EMS
3 6 9
30 25 15
400 200 100
(h)
After incubation for 3, 6 or 9 h, cells were washed twice with Hanks' salt solution, and incubated in normal medium for 7 days. The colonies formed were fixed in absolute methanol and stained with May-Griinwald-Giemsa (Merck, Darmstadt) and Giemsa (Merck). The number of colonies containing more than 50 cells was scored under a binocular microscope.
Mutagenicity assay 6TC-resistant mutation assay. 2 × l0 S cells were incubated in 100-mm petri dishes in 10 ml of normal medium for 20 h, washed twice in Hanks' salt solution, and treated simultaneously with MMS and EMS in Hanks' salt solution at 37.5°C. After incubation t'or 3, 6 or 9 h, cells were again washed and incubated in normal medium for an expression time of 6 days. The cells were dissociated by treatment with 0.25% trypsin solution, centrifuged and inoculated into fresh dishes. 2 × l0 s cells were replated in each of five 100-ram petri dishes and incubated in culture medium containing 5 p,g/ml 6TG (Wako Pure Chem. Ind., Osaka) for 10 days. Colonies formed were fixed and stained. The number of colonies containing more than 50 cells was scored by the same procedure as described in the cytotoxicity assay. In parallel experiments, triplicate inocula of 2 x 102 dissociated cells were incubated in normal medium for 7 days. The colonies formed were fixed, stained and scored. The number of mutant colonies was corrected by the colony-forming ability of replated cells cultured in normal medium. The induced mutation frequency was calculated from the number of
mutant colonies of treated cells reduced by the number of mutant colonies in untreated control cultures. Induced mutation frequency was expressed as the number of induced mutant colonies per 10 5 survivors.
Chromosome aberration assay. 2 × 105 cells were incubated in normal medium for 20 h, washed twice in Hanks" salt solution, and treated simultaneously with MMS and EMS in Hanks' salt solution at 37.5°C. After incubation for 3 h, cells were again washed and incubated in normal medium for 18-21 h. Colcemid (Wako) was added to the cultures at a concentration of 5/~g/ml and incubated for 2 h before cells were harvested. The cells were dissociated by treatment with 0.25% trypsin solution, centrifuged and fixed with methanol-acetic acid (3: 1) after hypotonic treatment with 0.075 M KCI solution at 37.5°C for 15 rain. Chromosome preparations were made according to the routine air-drying procedure. The next day, they were stained with Giemsa. Scoring of the chromosome aberrations was done according to the method of Cohen and Hirschhorn (1971). One hundred metaphases were scored for chromosome aberrations in cells treated at each concentration. Mathematical analysis. The interactive effects between MMS and EMS for cytotoxicity and 6TG-resistant mutation induction were analyzed with the mathematical procedure described by A g e r and Haynes (1990a,b). Consider a homogeneous population of cells that is treated uniformly with doses x and y of agents X and Y. We denote the average, or expected number of lethal and mutational hits per cell by the functions Hk(X) and Hm(X). respectively. On the basis of single-event Poisson probabilities, the probability of survival S(x) is exp[-Hk(X)]. Thus, the expected number of lethal hits per cell can be written as: - I n S(x) =Hk(X)
(1)
If a cell sustains a mutational hit, but no lethal hit, it will survive as a mutant. The expected
174
The interaction functions h(x, y) and m(x, y) describe the interactive effects between agents X and Y for cell killing and mutation, respectively. The interaction may be synergistic, zero, or antagonistic depending on whether each interaction function is greater than, equal to, or less than
induced mutant yield is the joint probability of these two events and is: Ym(x)
=
[1
-- e t/mix)] - e -ukt~) --
Hm(x )
e -/'/kt~)
(2)
We
zero.
assume stochastic independence of mutation and killing, that is, the probability of survival of the induced mutation in the assay procedures is the same as that for cells in the population as a whole. By definition the induced mutation frequency (mutants per survival) is given by:
M ( x ) = Ym(X)/S(x) = 1 -- enm(X)- Hm(x ) (3) Extending this approach to describe the interactive effects that may occur when cells are treated with two mutagenic agents, we can write:
Hk( X, y ) = - I n S( x, y)
=Hk(X ) +Hk(Y ) +h(x, y)
(4)
Hm(x, y) tiM(x, y) = M ( x ) + M ( y ) +re(x, y)
l'°°r'~"-~ 3h ~o m
O.lO
(5)
Results
Lethal interaction between MMS and EMS The combined effects on cell survival of simultaneous treatments of EMS and MMS were examined. Fig. 1 indicates the surviving fraction of V79 cells treated simultaneously with MMS at a concentration of D2, and EMS at various concentrations for 3, 6 or 9 h. In the presence of MMS, the surviving fraction of V79 cells treated with EMS was markedly decreased. Fig. 2 shows the surviving fraction of V79 cells with simultaneous treatments with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In the presence of EMS, the surviving fraction of V79 cells treated with MMS was also decreased. The number of lethal hits arising entirely from interactive effects, h(x, can be calculated ac-
y),
6h
1.00~
9h
1.00
b
0.1G
!
e..
0'010
400
800
i , i 1200 1000
0.01
4~0
I
800
I
1200
I
IG00
0.Ol
0
I
400
I
800
!
1200
I
IO00
C o n c e n t r a t i o n of E M S ( p g / m l ) Fig. 1. Combined effects of MMS and EMS on survival of Chinese hamster V79 cells. V79 cells were treated simultaneously with MMS at a D2o concentration and EMS at various concentrations for 3, 6 or 9 h. Open circles show treatment with EMS alone and closed circles show simultaneous treatment with MMS and EMS.
175 cording to Eqn. (4). Under the transformation w - - x y , the surface represented by h(x, y) maps, within experimental error, to a single curve in the (h, w) plane (Fig. 3). Least-squares analysis of the combined data shown in Figs. 1 and 2 indicates that the specific interaction function for the M M S - E M S interaction in V79 cells may be described by functions of the forms: for 3-h treatment,
D20 and EMS at various concentrations for 3, 6 or 9 h, mutation frequencies are shown in Fig. 4. In cells treated simultaneously with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h, the induced mutation frequencies are shown in Fig. 5. The mutation frequency in V79 cells treated with EMS at a concentration of D2o was 5-10 × 10 -5. In the presence of EMS, MMS-induced mutation frequencies were higher than in cells treated with EMS alone. The number of mutational hits arising entirely from interactive effects can be calculated according to Eqn. (5). Under the transformation, w = cy, the surface represented by re(x, y) maps to a single curve in the (m, w) plane (Fig. 6). Leastsquares analysis of the data reveals that the mutational interaction between MMS and EMS in V79 cells is described by the forms:
h(x, y) = - 0 . 3 3 + 0.05 xy
(6) for 6-h treatment,
h(x, y )
for 9-h treatment,
h(x, y) ~ - 0 . 1 7 + 0.74 xy
--
0.10 + 0.38 xy
(7)
(8) In the low-dose region, no synergistic interaction between MMS and EMS was observed. At higher doses increasing synergism was found in cells treated for 3, 6 and 9 h. For cells treated for 9 h, a more rapid increase in synergism was found.
for 3-h treatment,
re(x, y) = - 7 . 2 1 + 0.72 xy
(9) for 6-h treatment,
re(x, y) = - 0 . 7 6 + 0.71 xy
Mutational interaction between MMS and EMS
(10)
In the 6TG-resistant mutation assay, V79 cells treated with MMS at a concentration of D20 showed low mutation frequencies. In a simultaneous treatment with MMS at a concentration of
for 9-h treatment,
(ii) 1.00[
1.00
1.00
re(x, y) = - 6 . 9 + 2.86 xy
6h
9h
I
I] e
.2 0.10
0.1(]
0.10
..~ .~.
/ 0.01
0
I 20
I 40
' 60
' 80
~ 100
0.01 [ 0
I 20
I 40
I 60
! 80
0.01
I 20
I 40
I 60
! 80
Concentration of MMS (pg/ml) Fig. 2. Combined effects of EMS and MMS on survival of Chinese hamster V79 cells. V79 cells were treated simultaneouslywith EMS at a D20 concentration and MMS at various concentrations for 3, 6 or 9 h. Open squares show treatment with MMS alone and closed squares show simultaneous treatment with EMS and MMS.
176
ah
J
EMS
x
MMS (pg/ml) a x 10~
6 II
.e
2
J
00
,'--
;
1
,'o
EMS x MM.q (pg/ml) I x 10a 4
¢
oh
m
In the low-dose region, no syngergistic interaction between MMS and EMS was found. At higher doses, mutational synergism was found in cells treated for 3, 6 and 9 h. Stronger dose-dependent increases in synergistic interaction were observed in cells treated for 9 h. Chromosome aberrations are the biologically different endpoints from 6TG-resistant mutations. Table 2 shows the results of a chromosome aberration assay in cells treated with EMS alone, and treated simultaneously with MMS at a concentration of D2o and EMS at various concentrations for 3 h. The number of aberrations produced by treatment with MMS alone at a concentration of D2o was low. In the presence of MMS, however, chromosome aberrations in cells treated with EMS increased significantly. All aberrant types of chromosome aberrations increased by simultaneous treatments with MMS and EMS. On the other hand, the results were different in simultaneous treatments with EMS at a concentration of D2o and MMS at various concentrations. Table 3 shows the results of a chromosome aberration assay in cells treated with EMS alone, and treated simultaneously with EMS and MMS for 3 h. The number of chromosome aberrations in ceils treated with EMS alone at a concentration of D, 0 was low. In the presence of EMS, chromosome aberrations in cells treated with MMS increased slightly. No significant difference was observed in all aberrant types of chromosome aberrations in the absence and presence of EMS at a concentration of D20.
Discussion
I !
I II
I 3
! 4
EMS x MMS Olg/ml)I x lO:l Fig. 3, Interactive lethal-hi1 9lot for the lethal interaction between MMS and EMS in Chinese hamster V79 cells treated with MMS and EMS simultaneously for ~, 6 or 9 h, Closed circles show the value calculated from the surviving fraction of cells treated with M M S at a D:o concentration and EMS at
various concentrations, Closed squares show the value calculated from the surviving fraction of cells treated with EMS at a Dz0 concentration and MMS at various concentrations. The solid lines represent the least-squares fit with parameters given in Eqns, 6, 7 and 8.
MMS and EMS are known as alkTlating agents which cause alkylation of cellular substances. These two alkylating agents differ only in having o methyl or an ethyl group, respectively, in their chsmical structures. MMS may react by a SN 2type mechanism (substitution, nucleophilic bi-
molecular), and EMS may react by mixed types of SN I- (substitution, nucleophilic unimolecular) and SN2-typo mechanisms, As for the difference in biological activity between MMS and EMS, MMS has a strong cytotoxicity and induces chromosome aberrations and SCE (sister-chromatid exchanges) at high frequency compared with EMS.
177 p~
50
50
50
3h
7 •~
6h
9h
40
40
40
30
30
30
20
20
20
|
/
.= )O
o
'~-'
0
200
10
400
600
01
800
I 200
I 40Q
I 6OO
I 800
200
-~
'
I
400
GO0
I
800
Concentration of EMS (#g/ml) Fig. 4. Combined effects of MMS and EMS on 6TG-resistant mutations in Chinese hamster V79 cells. V79 cells were treated simultaneously with MMS at a D2o concentration and EMS at various concem~ations for 3, 6 or 9 h. Open circles show treatment with EMS alone and closed ciTcles show simultaneous treatment with MMS and EMS.
On the other hand, EMS shows a high mutagenicity which may be correlated with the formation of O6-alkylation (Sega, 1984). In the present study, the effects of simultaneous treatments with MMS and EMS on surviving fraction, 6TG-resistant mutations and chromo-
some aberrations were examined in Chinese hamster V79 cells. At a low concentration (D2o). MMS produced much cellular and macromolecular damage which resulted in high cytotoxicity and the induction of many chromosome aberrations. On the other
30
3o
ah
~" o
30
9h
6h
20
20
;--,J 0
20
40
60
80
100
o
i
,
60
80
O
U
I
I
I
I
20
40
60
80
Concentration of MMS (#g/ml) Fig. 5. Combined effects of EMS and MMS on 6TG-resistant mutations in Chinese hamster V79,cclls. V79 cells were treated simultaneously with EMS at a D20 concentration and MMS at various concentrations for 3, 6 or 9 h. Open squares show treatment with MMS alone and closed squares show simultaneous treatment with EMS and MMS.
178 TABLE 2 COMBINED EFFECTS OF MMS AND EMS ON CHROMOSOME ABERRATIONS IN CHINESE HAMSTER V79 CELLS Concentration of EMS MMS (~.g/ml) (/zg/ml) 0 400 600 800
0 0 0 0
Number of cells examined
Cells" with aberrations (%)
Number of aberrations per cell b cdg cdb icdg icdb
0.0 2.0 3.0 9.0 17.0 21.0
4 5 9 16 20 22
0 0 1 7 10 22
0 2 2 3
0 0 0 0
5
1
2
1
1
3
4 7 12 27 38 49
0.0 1.0 12.0 14.0 20.0 30.0
2 7 9 10 19 33
0 I 11 10 15 30
0 1 0 2 0 3
0 0 1 1 2 1
0 0 0 2 3 13
2 9 21 25 39 80
1000
0
1200
0
100 100 100 100 100 100
0 0 200 400 600 800
0 30 30 30 30 30
I00 100 100 100 100 100
cdx 0 0 0 1
Totals
cdg, chromatid gap; cdb, chromatid break; icdg, isochromatid gap; icdb, isochromatid break; cdx, chromatid exchange. u Cells with gaps only were excluded. b Acentric fragments not associated with dicentrics and rings were scored as isochromatid breaks.
SN~-type mechanism in cells treated for 3 h and do by an SN2-type mechamism in a similar way tO the reaction observed with MMS in cells treated for more than 6 h. These results indicate that some alkylating
hand, at a low concentration (D20), EMS produced much genetic or DNA damage which resulted in high 6TG-resistant mutation induction in cells treated for 3 h and cytotoxicity in cells treated for 6 and 9 h. EMS may react by a strong
TABLE 3 COMBINED EFFECTS OF EMS AND MMS ON CHROMOSOME ABERRATIONS IN CHINESE HAMSTER V79 CELLS Concentration of MMS (pg/ml)
Number
EMS (/~g/ml)
Ceils" with
Number of aberrations per cell b
of cells examined
aberrations (%)
cdg
cdb
icdg
icdb
cdx
Totals
0 20 40 60 80 100
0 0 0 0 0 0
I00 IO0 I00 100 100 100
0.0 1.0 10.0 20.0 49.0 77.0
I 6 17 17 32 37
0 1 6 15 64 104
0 I 4 2 2 9
0 0 0 3 12 6
0 0 0 2 20 40
I 8 27 39 130 196
0 0 20 40 60 80
0 400 400 400 400 400
I00 I00 100 100 100 100
0.0 0.0 11.0 13.0 21.0 52.0
I 6 8 17 28 37
0 0 8 8 23 79
0 0 2 1 3 3
0 0 2 2 2 7
0 0 0 2 8 15
I 6 20 30 64 141
cdg, chromatid gap; cdb, chromatid break; icdg, isochromatid gap; icdb, isochromatid break~ cdx, chromatid exchange. " Cells with gaps only were excluded. t, Acentric fragments not associated with dicentrics and rings were scored as isochromatid breaks.
179 21
A
18 15 12 m
9
3 =: .2
!
5
i.f
, 10
•
• 15
, 20
, 35
, 30
. 35
3.0
a.5
E M S x M M S (itg/ml) s x 1O s 12
B
+'9
6h
a. w
/ o ,,,i
EMS x MMS
21
(Fg/ml)
~
x
In a
C
, ! 18 18
I
O 0 S 0 -0 EMS
x MMS
Otg/ml) a x lO s
Fig. 6. re(w) plot for the MMS=EMS interaction for 6TG-resistant mutations in Chinese hamster V79 cells treated with MMS and EMS simultaneously for 3, 6 or 9 h. Closed circles show the value calculated from the induced mutatiol~ frequency of cells treated with MMS at a D20 concentration and EMS at various concentrations. Closed squares show the value calculated from the induced mutation frequency of cells treated with EMS at a D20 concentration and MMS at various concentrations. The solid lines represent the computer fit with parameters given in Eqns. 9, 10 and 11.
agents act more effectively when a small amount of the other alkylating agents is present simultaneously. It is suggested that damages produced in the cellular and macromolecular substances by some al~lating agent may easily be increased in the presence of a small amount of another. There are few reports on the combined effects of more than two mutagens on mutation induction, as described in the introduction. Among them several reports deal with the effects of simultaneous treatments with two mutagens on the induction of mutations and DNA damages. Vericat et al. (1984) showed that the mutagens causing different types of damage in DNA appeared to have no interfering effects on the mutagenicity of each mutagen in E. coll. But Kawazoe et al. (1986) showed that the mutagenic interaction as 'equivalent' or 'independent' appeared to be different among al~lating agents in S. typhimurium. The methylation and ethylation phases may be recognized as partially, but not entirely equivalent damages of DNA leading to methylation. Ager and Haynes (1990a) found a strong synergistic interaction for cell killing between heat and UV in S. cereuisiae. They also found that heat-UV synergism in gene conversion and mutation arises via the inhibition of two different repair pathways. They z!so found a strong synergistic interaction between EMS and UV for cell killing, but mutation and gene conversion frequencies induced by EMS and UV did not deviate significantly from that expected on the basis of simple additivity. Our results on two chemicals producing methylation and ethylation in Chinese hamster V79 cells showed synergistic interactions between MMS and EMS for cell killing and 6TG-resistant mutations. Synergism between the two chemicals was observed more strongly in cells treated for a longer period, 9 h. It is suggested that the mutations produced by simultaneous treatments of mutagens may be not produced by a simple reaction. In dealing with alkylating agents such as MMS and EMS, an adaptation response and repair mechanism may inevitably occur. The combined effects of successive treatments of two alk~iating agents at different times with incubation in nor-
180
mai medium between the two treatments have to be examined in a future study.
Acknowledgements The authors acknowledge helpful advice from Dr. K. Tachi-Shinkawa, H. Tezuka, A. Yokoiyama and Dr. T. Kada in the National Institute of Genetics. The authors also wish to thank Y. Takada and E. Asakura for their technical assistance during this work. References Ager, D.D., and R.H. Haynes (1987) Mathematical description of the interactions between cellular inactivating agents, Mutation Res., II0, 129-141. Ager, D.D., and R.H. Haynes (1988) Quantitative aspects of the interactive killing effects between X rays and other mutagens in microorganisms, Mutation Res., 115, 124-140. Ager, D.D., and R.H. Haynes (1990a) Analysis of interactions between mutagens, I. Heat and ultraviolet light in Saccha. romyces ceretqsiae, Mutation Res., 232, 313-326. Ager, D,D., and R.H. Haynes (1990b) Analysis of interactions between mutagens, ll. Ethyl methanesulfonate and ultravi. diet light in Saccharomyces ceret'isia¢, Mutation Res., 232, 327-336. Cohen, M.M,, and K, Hirschhorn O971) Cytogenetic studies in animals, in: A, Ho;laender (Ed.), Chemical Mutagens, Principles and Methods for Their Detection, Vol, 2, Plenum, New York, pp, 515-534. Ford, D.K., and O, Yerganian (1958) Observations on the chromosomes of Chinese hamster cells in tissue culture, J. Natl, Cancer lnst,, 21,393-425, Kada, T., K, Kaneko, b. Matsuzaki, T. Matsuzaki and Y, Hara
(1985) Detection and chemical induction of natural bio-antimutagens. A case of the green tea factor, Mutation Res., 150, 127-132. Kawazoe, Y., A. Hakura and K. Kohda (1986) Studies on chemical carcinogens and mutagens. XXXIII. Mutation frequencies induced by combinations of methylating and/or ethylating mutagens, Chem. Pharm. Bull., 34, 250255. Kojima, H., H. Konishi and Y. Kuroda (1986) Combined effects of chemicals on mammalian cells in culture. I. Effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the mutation induction, Mutation Res., 164, 272 (Abstract). Kuroda, Y. (1984) Dose-rate effects of chemicals on mutation induction in mammalian cells in culture, in: Y. Tazima, S. Kondo and Y. Kuroda.(Eds.), Problems of Threshold in Chemical Mutagenesis, Environmental Mutagen Society of Japan, Mishima, pp. 99-108. Kuroda, Y. (1990) Antimutagenic ac:ivity of vitamins in cultured mammalian cells, in: Y. Kuroda, D.M. Shankel and M.D. Waters (Eds.), Antimutagenesis and ~mticareinogenesis Mechanisms !I, Plenum, Hew York, pp. 233-256. Kuroda, Y., A. Yokoiyama and T. Kada (1985) Assays for the induction of mutations to 6-thioguanine resistance in Chinese hamster V79 cells in culture, in: J. Ashby, FJ. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, World Health Organization, Eise. vier, Amsterdam, pp. 537-542, Sega, G.A. (1984) A review of the genetic effects of ethyl methanesulfonate, Mutation Res,, 134, 113-142. Shankel, D.M., P.E, Hartman, T, Kada and A, Hollaender (Eds.) (1986) Antimutagenesis and Anticarcinogenesis Mechanisms, Plenum, New York, pp. 1-605. Vericat, J.A,, J, Barhe and R. Ouerrero (1984) Expression of th~ SOS response following simultaneous treatment with methylnitrosoguanidine and mitomycin C in £scherichia coil, Mutation Res,, 132, 15-20.
171
MUT 05065
Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells H. Kojima a, H. Konishi
a
and Y. Kuroda b
a Biochemical Research Institute, Nippon Menard Cosmetic Co. Ltd., Ogaki, Gila 503 and b Laboratory of Phenogenetics, National Institute of Genetics, Mishima, Shizuoka 4 i l (Japan) (Received 2 April 1990) (Revision received 18 October 1991) (Accepted 18 October 1991)
Keywords: Methyl methanesulfonate; Ethyl methanesulfonate; Combined mutagenicity; Mammalian cells
Summary The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D2o and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of Dzo and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.
Many chemicals present in our environment have been examined for their mutagenic activity with various test systems. Most of these tests have dealt with the mutagenic effect of single chemi-
Correspondence: Prof. Dr. Yukiaki Kuroda, Research Institute of Biosciences, Azabu University, !-17-71, Fuchinobe, Sagamihara, Kanagawa 229 (Japan). Tel.: 0427-54-7111 (Ext. 430); Fax: 0427-54-7661. Abbreviations: MMS, methyl methanesulfonate; EMS, ethyl methanesulfonate; 6TG, 6-thioguanine; OUA, ouabain.
cals on organisms. In our environment, however, many chemicals are present together. It is necessary to examine the combined effects of different chemicals. The combination of more than two chemicals may produce various modification of the mutagenicity of each chemical involved. One of the modifications of the mutagenicity of chemicals involves the enhanced or synergistic effects of more than two chemicals. The other modification is the inhibition of one chemical by another one. The inhibition of mutagenicity of one chemical by
another one can be divided into desmutagenesis, which directly inactivates the mutagens, and bioantimutagenesis, which suppresses the cellular mutagenesis induced by other mutagens (Kada et al., 1985). Antimutagenesis and anticarcinogenesis have been studied (Shankel et al., 1986; Kuroda et al., 1990). There have, however, been few studies concerning additional or enhanced effects of more than two mutagens. Vericat et al. (1984) reported the expression of the SOS response following a simultaneous treatment with N-methyl-N'-nitroN-nitrosoguanidine (MNNG) and mitomycin C in Escherichia coll. Kawazoe et al. (1986) reported on the mutation frequency of Salmonella typhimurium TA100 after a simultaneous treatment of alkylating agents. These reports concerned the combined effects of more than two chemicals on induced mutations in bacterial and yeast cells. Information on the combined effects of more than two mutagens on mammalian cells is extremely limited. Recently Ager and Haynes (1987, 1988) presented a general mathematical technique for the description and classification of the lethal interactive effects that occur when cells are treated with two or more toxic agents. They applied this mathematical technique to the interaction between heat and ultraviolet light (UV) and between EMS and UV for cell killing, mutation and gene conversion in the yeast Saccharomyces cere. risiae (Agar and Haynes, 1990a,b). In the present study, the effects of simultaneous treatments of MMS and EMS on the induction of 6TG-resistant mutants and chromosome aberrations were examined in cultured Chinese hamster V79 cells. Some of the results obtained have been reported previously (Kojima et al., 1986). Materials and methods
Cell culture The cell line used was the strain of Chinese hamster lung V79 cells (Ford and Yerganian, 1958). The cells were maintained in Eagle's minimum essential medium (Nissui Seiyaku Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (Gibed, New York) in 60-ram petri
dishes (Lux, Newbury Park, CA, No. 5216, 2-mm grid) under a controlled atmosphere of 5% CO 2 and 95% air at 37.5°C. The cells were mycoplasma-free, gave a colony-forming ability of more than 80% and a population doubling time of 15.3 h under the above culture conditions. The cells were grown in large mass, distributed in many ampoules, and frozen at -80°C. Before use, cells were thawed at 37.5°C, suspended in fresh medium, and cultured in GHAT medium (1.6 x 10 -~ M glycine, 10 -4 M hypoxanthine, 4 × 10 - 7 M amethopterin, and 1.6 x 10 -s M thymidine) at 37.5°C for 24 h, to remove the pre-existing 6TG-resistant cells in the cell population. During further subcultivation in normal medium, the appearance of spontaneous 6TG-resistant mutants was occasionally noted. For this reason, stock cultures were discarded at the tenth subculture and replaced from frozen stocks. Cells at an exponential growth phase in monolayer were dissociated by treatment with 0.25% trypsin (Difco, Detroit, MI, 1:250) solution, and centrifuged at 1500 rpm for 5 rain. The cells were then resuspended in the culture medium and used for experiments.
Mutagens tested Ethyl me~hanes,alfonate (EMS) (Aldrich) and methyl methaa,:~ulfonate (MMS) (Aldrich) were tested for their combined effects on the induction of 6TG-resistant mutations. They were diluted in Hanks' (Nissui Seiyaku Co., Tokyo) solution, sterilized through a filter with 0.45/~m pore size, and used for treatments of cells at various concentrations. Cytotoxicity assay The cytotoxic effect of chemicals was examined by determining the colony-forming ability of ceils, as described previously (Kuroda, 1984; Kuroda et al., 1985). Triplicate inocula of 2 x 102 cells were incubated in 60-mm petri dishes in 5 ml of normal medium for 20 h, washed twice with Hanks' salt solution, and treated with MMS at a concentration of D2o and EMS at various concentrations simultaneously or with EMS at a concentration of D.,0 and MMS at various concentrations in Hanks' salt solution at 37.5°C (the values of D2, of EMS and MMS are shown in Table 1).
173 TABLE 1 THE D2o VALUES OF MMS AND EMS ON SURVIVAL OF CHINESE HAMSTER V79 CELLS Time of treatment
D2o value (p.g/ml) MMS
EMS
3 6 9
30 25 15
400 200 100
(h)
After incubation for 3, 6 or 9 h, cells were washed twice with Hanks' salt solution, and incubated in normal medium for 7 days. The colonies formed were fixed in absolute methanol and stained with May-Griinwald-Giemsa (Merck, Darmstadt) and Giemsa (Merck). The number of colonies containing more than 50 cells was scored under a binocular microscope.
Mutagenicity assay 6TC-resistant mutation assay. 2 × l0 S cells were incubated in 100-mm petri dishes in 10 ml of normal medium for 20 h, washed twice in Hanks' salt solution, and treated simultaneously with MMS and EMS in Hanks' salt solution at 37.5°C. After incubation t'or 3, 6 or 9 h, cells were again washed and incubated in normal medium for an expression time of 6 days. The cells were dissociated by treatment with 0.25% trypsin solution, centrifuged and inoculated into fresh dishes. 2 × l0 s cells were replated in each of five 100-ram petri dishes and incubated in culture medium containing 5 p,g/ml 6TG (Wako Pure Chem. Ind., Osaka) for 10 days. Colonies formed were fixed and stained. The number of colonies containing more than 50 cells was scored by the same procedure as described in the cytotoxicity assay. In parallel experiments, triplicate inocula of 2 x 102 dissociated cells were incubated in normal medium for 7 days. The colonies formed were fixed, stained and scored. The number of mutant colonies was corrected by the colony-forming ability of replated cells cultured in normal medium. The induced mutation frequency was calculated from the number of
mutant colonies of treated cells reduced by the number of mutant colonies in untreated control cultures. Induced mutation frequency was expressed as the number of induced mutant colonies per 10 5 survivors.
Chromosome aberration assay. 2 × 105 cells were incubated in normal medium for 20 h, washed twice in Hanks" salt solution, and treated simultaneously with MMS and EMS in Hanks' salt solution at 37.5°C. After incubation for 3 h, cells were again washed and incubated in normal medium for 18-21 h. Colcemid (Wako) was added to the cultures at a concentration of 5/~g/ml and incubated for 2 h before cells were harvested. The cells were dissociated by treatment with 0.25% trypsin solution, centrifuged and fixed with methanol-acetic acid (3: 1) after hypotonic treatment with 0.075 M KCI solution at 37.5°C for 15 rain. Chromosome preparations were made according to the routine air-drying procedure. The next day, they were stained with Giemsa. Scoring of the chromosome aberrations was done according to the method of Cohen and Hirschhorn (1971). One hundred metaphases were scored for chromosome aberrations in cells treated at each concentration. Mathematical analysis. The interactive effects between MMS and EMS for cytotoxicity and 6TG-resistant mutation induction were analyzed with the mathematical procedure described by A g e r and Haynes (1990a,b). Consider a homogeneous population of cells that is treated uniformly with doses x and y of agents X and Y. We denote the average, or expected number of lethal and mutational hits per cell by the functions Hk(X) and Hm(X). respectively. On the basis of single-event Poisson probabilities, the probability of survival S(x) is exp[-Hk(X)]. Thus, the expected number of lethal hits per cell can be written as: - I n S(x) =Hk(X)
(1)
If a cell sustains a mutational hit, but no lethal hit, it will survive as a mutant. The expected
174
The interaction functions h(x, y) and m(x, y) describe the interactive effects between agents X and Y for cell killing and mutation, respectively. The interaction may be synergistic, zero, or antagonistic depending on whether each interaction function is greater than, equal to, or less than
induced mutant yield is the joint probability of these two events and is: Ym(x)
=
[1
-- e t/mix)] - e -ukt~) --
Hm(x )
e -/'/kt~)
(2)
We
zero.
assume stochastic independence of mutation and killing, that is, the probability of survival of the induced mutation in the assay procedures is the same as that for cells in the population as a whole. By definition the induced mutation frequency (mutants per survival) is given by:
M ( x ) = Ym(X)/S(x) = 1 -- enm(X)- Hm(x ) (3) Extending this approach to describe the interactive effects that may occur when cells are treated with two mutagenic agents, we can write:
Hk( X, y ) = - I n S( x, y)
=Hk(X ) +Hk(Y ) +h(x, y)
(4)
Hm(x, y) tiM(x, y) = M ( x ) + M ( y ) +re(x, y)
l'°°r'~"-~ 3h ~o m
O.lO
(5)
Results
Lethal interaction between MMS and EMS The combined effects on cell survival of simultaneous treatments of EMS and MMS were examined. Fig. 1 indicates the surviving fraction of V79 cells treated simultaneously with MMS at a concentration of D2, and EMS at various concentrations for 3, 6 or 9 h. In the presence of MMS, the surviving fraction of V79 cells treated with EMS was markedly decreased. Fig. 2 shows the surviving fraction of V79 cells with simultaneous treatments with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In the presence of EMS, the surviving fraction of V79 cells treated with MMS was also decreased. The number of lethal hits arising entirely from interactive effects, h(x, can be calculated ac-
y),
6h
1.00~
9h
1.00
b
0.1G
!
e..
0'010
400
800
i , i 1200 1000
0.01
4~0
I
800
I
1200
I
IG00
0.Ol
0
I
400
I
800
!
1200
I
IO00
C o n c e n t r a t i o n of E M S ( p g / m l ) Fig. 1. Combined effects of MMS and EMS on survival of Chinese hamster V79 cells. V79 cells were treated simultaneously with MMS at a D2o concentration and EMS at various concentrations for 3, 6 or 9 h. Open circles show treatment with EMS alone and closed circles show simultaneous treatment with MMS and EMS.
175 cording to Eqn. (4). Under the transformation w - - x y , the surface represented by h(x, y) maps, within experimental error, to a single curve in the (h, w) plane (Fig. 3). Least-squares analysis of the combined data shown in Figs. 1 and 2 indicates that the specific interaction function for the M M S - E M S interaction in V79 cells may be described by functions of the forms: for 3-h treatment,
D20 and EMS at various concentrations for 3, 6 or 9 h, mutation frequencies are shown in Fig. 4. In cells treated simultaneously with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h, the induced mutation frequencies are shown in Fig. 5. The mutation frequency in V79 cells treated with EMS at a concentration of D2o was 5-10 × 10 -5. In the presence of EMS, MMS-induced mutation frequencies were higher than in cells treated with EMS alone. The number of mutational hits arising entirely from interactive effects can be calculated according to Eqn. (5). Under the transformation, w = cy, the surface represented by re(x, y) maps to a single curve in the (m, w) plane (Fig. 6). Leastsquares analysis of the data reveals that the mutational interaction between MMS and EMS in V79 cells is described by the forms:
h(x, y) = - 0 . 3 3 + 0.05 xy
(6) for 6-h treatment,
h(x, y )
for 9-h treatment,
h(x, y) ~ - 0 . 1 7 + 0.74 xy
--
0.10 + 0.38 xy
(7)
(8) In the low-dose region, no synergistic interaction between MMS and EMS was observed. At higher doses increasing synergism was found in cells treated for 3, 6 and 9 h. For cells treated for 9 h, a more rapid increase in synergism was found.
for 3-h treatment,
re(x, y) = - 7 . 2 1 + 0.72 xy
(9) for 6-h treatment,
re(x, y) = - 0 . 7 6 + 0.71 xy
Mutational interaction between MMS and EMS
(10)
In the 6TG-resistant mutation assay, V79 cells treated with MMS at a concentration of D20 showed low mutation frequencies. In a simultaneous treatment with MMS at a concentration of
for 9-h treatment,
(ii) 1.00[
1.00
1.00
re(x, y) = - 6 . 9 + 2.86 xy
6h
9h
I
I] e
.2 0.10
0.1(]
0.10
..~ .~.
/ 0.01
0
I 20
I 40
' 60
' 80
~ 100
0.01 [ 0
I 20
I 40
I 60
! 80
0.01
I 20
I 40
I 60
! 80
Concentration of MMS (pg/ml) Fig. 2. Combined effects of EMS and MMS on survival of Chinese hamster V79 cells. V79 cells were treated simultaneouslywith EMS at a D20 concentration and MMS at various concentrations for 3, 6 or 9 h. Open squares show treatment with MMS alone and closed squares show simultaneous treatment with EMS and MMS.
176
ah
J
EMS
x
MMS (pg/ml) a x 10~
6 II
.e
2
J
00
,'--
;
1
,'o
EMS x MM.q (pg/ml) I x 10a 4
¢
oh
m
In the low-dose region, no syngergistic interaction between MMS and EMS was found. At higher doses, mutational synergism was found in cells treated for 3, 6 and 9 h. Stronger dose-dependent increases in synergistic interaction were observed in cells treated for 9 h. Chromosome aberrations are the biologically different endpoints from 6TG-resistant mutations. Table 2 shows the results of a chromosome aberration assay in cells treated with EMS alone, and treated simultaneously with MMS at a concentration of D2o and EMS at various concentrations for 3 h. The number of aberrations produced by treatment with MMS alone at a concentration of D2o was low. In the presence of MMS, however, chromosome aberrations in cells treated with EMS increased significantly. All aberrant types of chromosome aberrations increased by simultaneous treatments with MMS and EMS. On the other hand, the results were different in simultaneous treatments with EMS at a concentration of D2o and MMS at various concentrations. Table 3 shows the results of a chromosome aberration assay in cells treated with EMS alone, and treated simultaneously with EMS and MMS for 3 h. The number of chromosome aberrations in ceils treated with EMS alone at a concentration of D, 0 was low. In the presence of EMS, chromosome aberrations in cells treated with MMS increased slightly. No significant difference was observed in all aberrant types of chromosome aberrations in the absence and presence of EMS at a concentration of D20.
Discussion
I !
I II
I 3
! 4
EMS x MMS Olg/ml)I x lO:l Fig. 3, Interactive lethal-hi1 9lot for the lethal interaction between MMS and EMS in Chinese hamster V79 cells treated with MMS and EMS simultaneously for ~, 6 or 9 h, Closed circles show the value calculated from the surviving fraction of cells treated with M M S at a D:o concentration and EMS at
various concentrations, Closed squares show the value calculated from the surviving fraction of cells treated with EMS at a Dz0 concentration and MMS at various concentrations. The solid lines represent the least-squares fit with parameters given in Eqns, 6, 7 and 8.
MMS and EMS are known as alkTlating agents which cause alkylation of cellular substances. These two alkylating agents differ only in having o methyl or an ethyl group, respectively, in their chsmical structures. MMS may react by a SN 2type mechanism (substitution, nucleophilic bi-
molecular), and EMS may react by mixed types of SN I- (substitution, nucleophilic unimolecular) and SN2-typo mechanisms, As for the difference in biological activity between MMS and EMS, MMS has a strong cytotoxicity and induces chromosome aberrations and SCE (sister-chromatid exchanges) at high frequency compared with EMS.
177 p~
50
50
50
3h
7 •~
6h
9h
40
40
40
30
30
30
20
20
20
|
/
.= )O
o
'~-'
0
200
10
400
600
01
800
I 200
I 40Q
I 6OO
I 800
200
-~
'
I
400
GO0
I
800
Concentration of EMS (#g/ml) Fig. 4. Combined effects of MMS and EMS on 6TG-resistant mutations in Chinese hamster V79 cells. V79 cells were treated simultaneously with MMS at a D2o concentration and EMS at various concem~ations for 3, 6 or 9 h. Open circles show treatment with EMS alone and closed ciTcles show simultaneous treatment with MMS and EMS.
On the other hand, EMS shows a high mutagenicity which may be correlated with the formation of O6-alkylation (Sega, 1984). In the present study, the effects of simultaneous treatments with MMS and EMS on surviving fraction, 6TG-resistant mutations and chromo-
some aberrations were examined in Chinese hamster V79 cells. At a low concentration (D2o). MMS produced much cellular and macromolecular damage which resulted in high cytotoxicity and the induction of many chromosome aberrations. On the other
30
3o
ah
~" o
30
9h
6h
20
20
;--,J 0
20
40
60
80
100
o
i
,
60
80
O
U
I
I
I
I
20
40
60
80
Concentration of MMS (#g/ml) Fig. 5. Combined effects of EMS and MMS on 6TG-resistant mutations in Chinese hamster V79,cclls. V79 cells were treated simultaneously with EMS at a D20 concentration and MMS at various concentrations for 3, 6 or 9 h. Open squares show treatment with MMS alone and closed squares show simultaneous treatment with EMS and MMS.
178 TABLE 2 COMBINED EFFECTS OF MMS AND EMS ON CHROMOSOME ABERRATIONS IN CHINESE HAMSTER V79 CELLS Concentration of EMS MMS (~.g/ml) (/zg/ml) 0 400 600 800
0 0 0 0
Number of cells examined
Cells" with aberrations (%)
Number of aberrations per cell b cdg cdb icdg icdb
0.0 2.0 3.0 9.0 17.0 21.0
4 5 9 16 20 22
0 0 1 7 10 22
0 2 2 3
0 0 0 0
5
1
2
1
1
3
4 7 12 27 38 49
0.0 1.0 12.0 14.0 20.0 30.0
2 7 9 10 19 33
0 I 11 10 15 30
0 1 0 2 0 3
0 0 1 1 2 1
0 0 0 2 3 13
2 9 21 25 39 80
1000
0
1200
0
100 100 100 100 100 100
0 0 200 400 600 800
0 30 30 30 30 30
I00 100 100 100 100 100
cdx 0 0 0 1
Totals
cdg, chromatid gap; cdb, chromatid break; icdg, isochromatid gap; icdb, isochromatid break; cdx, chromatid exchange. u Cells with gaps only were excluded. b Acentric fragments not associated with dicentrics and rings were scored as isochromatid breaks.
SN~-type mechanism in cells treated for 3 h and do by an SN2-type mechamism in a similar way tO the reaction observed with MMS in cells treated for more than 6 h. These results indicate that some alkylating
hand, at a low concentration (D20), EMS produced much genetic or DNA damage which resulted in high 6TG-resistant mutation induction in cells treated for 3 h and cytotoxicity in cells treated for 6 and 9 h. EMS may react by a strong
TABLE 3 COMBINED EFFECTS OF EMS AND MMS ON CHROMOSOME ABERRATIONS IN CHINESE HAMSTER V79 CELLS Concentration of MMS (pg/ml)
Number
EMS (/~g/ml)
Ceils" with
Number of aberrations per cell b
of cells examined
aberrations (%)
cdg
cdb
icdg
icdb
cdx
Totals
0 20 40 60 80 100
0 0 0 0 0 0
I00 IO0 I00 100 100 100
0.0 1.0 10.0 20.0 49.0 77.0
I 6 17 17 32 37
0 1 6 15 64 104
0 I 4 2 2 9
0 0 0 3 12 6
0 0 0 2 20 40
I 8 27 39 130 196
0 0 20 40 60 80
0 400 400 400 400 400
I00 I00 100 100 100 100
0.0 0.0 11.0 13.0 21.0 52.0
I 6 8 17 28 37
0 0 8 8 23 79
0 0 2 1 3 3
0 0 2 2 2 7
0 0 0 2 8 15
I 6 20 30 64 141
cdg, chromatid gap; cdb, chromatid break; icdg, isochromatid gap; icdb, isochromatid break~ cdx, chromatid exchange. " Cells with gaps only were excluded. t, Acentric fragments not associated with dicentrics and rings were scored as isochromatid breaks.
179 21
A
18 15 12 m
9
3 =: .2
!
5
i.f
, 10
•
• 15
, 20
, 35
, 30
. 35
3.0
a.5
E M S x M M S (itg/ml) s x 1O s 12
B
+'9
6h
a. w
/ o ,,,i
EMS x MMS
21
(Fg/ml)
~
x
In a
C
, ! 18 18
I
O 0 S 0 -0 EMS
x MMS
Otg/ml) a x lO s
Fig. 6. re(w) plot for the MMS=EMS interaction for 6TG-resistant mutations in Chinese hamster V79 cells treated with MMS and EMS simultaneously for 3, 6 or 9 h. Closed circles show the value calculated from the induced mutatiol~ frequency of cells treated with MMS at a D20 concentration and EMS at various concentrations. Closed squares show the value calculated from the induced mutation frequency of cells treated with EMS at a D20 concentration and MMS at various concentrations. The solid lines represent the computer fit with parameters given in Eqns. 9, 10 and 11.
agents act more effectively when a small amount of the other alkylating agents is present simultaneously. It is suggested that damages produced in the cellular and macromolecular substances by some al~lating agent may easily be increased in the presence of a small amount of another. There are few reports on the combined effects of more than two mutagens on mutation induction, as described in the introduction. Among them several reports deal with the effects of simultaneous treatments with two mutagens on the induction of mutations and DNA damages. Vericat et al. (1984) showed that the mutagens causing different types of damage in DNA appeared to have no interfering effects on the mutagenicity of each mutagen in E. coll. But Kawazoe et al. (1986) showed that the mutagenic interaction as 'equivalent' or 'independent' appeared to be different among al~lating agents in S. typhimurium. The methylation and ethylation phases may be recognized as partially, but not entirely equivalent damages of DNA leading to methylation. Ager and Haynes (1990a) found a strong synergistic interaction for cell killing between heat and UV in S. cereuisiae. They also found that heat-UV synergism in gene conversion and mutation arises via the inhibition of two different repair pathways. They z!so found a strong synergistic interaction between EMS and UV for cell killing, but mutation and gene conversion frequencies induced by EMS and UV did not deviate significantly from that expected on the basis of simple additivity. Our results on two chemicals producing methylation and ethylation in Chinese hamster V79 cells showed synergistic interactions between MMS and EMS for cell killing and 6TG-resistant mutations. Synergism between the two chemicals was observed more strongly in cells treated for a longer period, 9 h. It is suggested that the mutations produced by simultaneous treatments of mutagens may be not produced by a simple reaction. In dealing with alkylating agents such as MMS and EMS, an adaptation response and repair mechanism may inevitably occur. The combined effects of successive treatments of two alk~iating agents at different times with incubation in nor-
180
mai medium between the two treatments have to be examined in a future study.
Acknowledgements The authors acknowledge helpful advice from Dr. K. Tachi-Shinkawa, H. Tezuka, A. Yokoiyama and Dr. T. Kada in the National Institute of Genetics. The authors also wish to thank Y. Takada and E. Asakura for their technical assistance during this work. References Ager, D.D., and R.H. Haynes (1987) Mathematical description of the interactions between cellular inactivating agents, Mutation Res., II0, 129-141. Ager, D.D., and R.H. Haynes (1988) Quantitative aspects of the interactive killing effects between X rays and other mutagens in microorganisms, Mutation Res., 115, 124-140. Ager, D.D., and R.H. Haynes (1990a) Analysis of interactions between mutagens, I. Heat and ultraviolet light in Saccha. romyces ceretqsiae, Mutation Res., 232, 313-326. Ager, D,D., and R.H. Haynes (1990b) Analysis of interactions between mutagens, ll. Ethyl methanesulfonate and ultravi. diet light in Saccharomyces ceret'isia¢, Mutation Res., 232, 327-336. Cohen, M.M,, and K, Hirschhorn O971) Cytogenetic studies in animals, in: A, Ho;laender (Ed.), Chemical Mutagens, Principles and Methods for Their Detection, Vol, 2, Plenum, New York, pp, 515-534. Ford, D.K., and O, Yerganian (1958) Observations on the chromosomes of Chinese hamster cells in tissue culture, J. Natl, Cancer lnst,, 21,393-425, Kada, T., K, Kaneko, b. Matsuzaki, T. Matsuzaki and Y, Hara
(1985) Detection and chemical induction of natural bio-antimutagens. A case of the green tea factor, Mutation Res., 150, 127-132. Kawazoe, Y., A. Hakura and K. Kohda (1986) Studies on chemical carcinogens and mutagens. XXXIII. Mutation frequencies induced by combinations of methylating and/or ethylating mutagens, Chem. Pharm. Bull., 34, 250255. Kojima, H., H. Konishi and Y. Kuroda (1986) Combined effects of chemicals on mammalian cells in culture. I. Effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the mutation induction, Mutation Res., 164, 272 (Abstract). Kuroda, Y. (1984) Dose-rate effects of chemicals on mutation induction in mammalian cells in culture, in: Y. Tazima, S. Kondo and Y. Kuroda.(Eds.), Problems of Threshold in Chemical Mutagenesis, Environmental Mutagen Society of Japan, Mishima, pp. 99-108. Kuroda, Y. (1990) Antimutagenic ac:ivity of vitamins in cultured mammalian cells, in: Y. Kuroda, D.M. Shankel and M.D. Waters (Eds.), Antimutagenesis and ~mticareinogenesis Mechanisms !I, Plenum, Hew York, pp. 233-256. Kuroda, Y., A. Yokoiyama and T. Kada (1985) Assays for the induction of mutations to 6-thioguanine resistance in Chinese hamster V79 cells in culture, in: J. Ashby, FJ. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, World Health Organization, Eise. vier, Amsterdam, pp. 537-542, Sega, G.A. (1984) A review of the genetic effects of ethyl methanesulfonate, Mutation Res,, 134, 113-142. Shankel, D.M., P.E, Hartman, T, Kada and A, Hollaender (Eds.) (1986) Antimutagenesis and Anticarcinogenesis Mechanisms, Plenum, New York, pp. 1-605. Vericat, J.A,, J, Barhe and R. Ouerrero (1984) Expression of th~ SOS response following simultaneous treatment with methylnitrosoguanidine and mitomycin C in £scherichia coil, Mutation Res,, 132, 15-20.
