Mutation Research, 282 (1992) 31-37
31
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 0652
Application of the standard mutagenesis assay results in underestimation of ethyl methanesulphonate-induced mutations to ouabain-resistance in Chinese hamster cells * Farideh Mirzayans a, James
M. Parry b
and Razmik Mirzayans
c
'~ Division of Infectious Diseases, Department of Medicine, University of Alberta, Edmonton, Alb. T6G 2R7 (Canada), I, Department of Genetics, University College of Swansea, Swansea SA2 8PP (Great Britain) and ~ Molecular Genetics and Carcinogenesis Laboratory, Department of Medicine, Cross Cancer Institute, Edmonton, Alb. T6G lZ2 (Canada)
(Received 30 September 1991) (Revision received 20 December 1991) (Accepted 23 December 1991) Keywords: Ethyl methanesulphonate; 8-Azaguanine; 6-Thioguanine; Ouabain; Chinese hamster V79 cells
Summary Chinese hamster V79 cells were exposed to ethyl methanesulphonate (EMS) and the incidence of mutant cells resistant to 8-azaguanine (8AZG), 6-thioguanine (6TG) or ouabain ( O U A ) was determined both by the respreading and the in situ techniques. In the former assay, the mutagen-treated cultures were grown for several days to permit the expression of mutations after which the cells were trypsinized, replated (10 5 cells/100-mm dish), and grown in medium supplemented with a selective agent. In the in situ assay, cultures were left undisturbed between EMS treatment and incubation in the presence of the selective agents. The yield of 8AZG-resistant mutants observed at optimal expression times after EMS treatment was comparable for both techniques; the induced mutation frequency (corrected for spontaneous mutation frequency) was estimated to be 82 x 10 -6 mutations per viable cell per unit dose (mM) of EMS. The frequency of 6TG-resistant mutants equalled 45 and 4 x 1 0 - 6 / m M EMS as determined by the respreading and the in situ assays, respectively. In sharp contrast to that observed with 6TG, the frequency of OUA-resistant mutants scored by the in situ assay (30 x 1 0 - 6 / r a M EMS) proved to be an order of magnitude greater than that determined by the respreading assay (3 x 1 0 - 6 / m M EMS). Our data therefore indicate that, when O U A is used for mutant selection, the application of the respreading technique, which has been widely adopted as the standard mammalian mutational assay over the past decade, may result in a marked underestimation of the actual mutation frequency ( ~ 10-fold in V79 cells).
* Dedicated to the memoryof Dr. George Mirzayans. Correspondence: Dr. Razmik Mirzayans, Molecular Genetics and Carcinogenesis Laboratory, Department of Medicine, Cross Cancer Institute, Edmonton, Alb. T6G 1Z2 (Canada).
Since the development of mammalian cell mutagenesis techniques over two decades ago (Chu and Malling, 1968), numerous laboratories have directed considerable effort to optimising the experimental conditions for measuring the induc-
32 tion of forward mutations at specific loci in mammalian cells treated with physical and chemical genotoxic agents. Although a variety of mutant phenotypes may be selected for (Cole and Arlett, 1984), resistance to 8-azaguanine (8AZG), 6thioguanine (6TG) or ouabain (OUA) are the most extensively studied and well defined mutational systems utilized with cultured human and Chinese hamster (V79 and CHO) cells (Cole and Arlett, 1984; Nestmann et al., 1991). The guanine analogues, 8AZG and 6TG, select variants harbouring mutations at the X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. The function of the H G P R T enzyme is to convert the purines, hypoxanthine and guanine, to nucleotides via a purine nucleotide salvage pathway. This enzyme also converts 6TG and 8AZG into their respective nucleotides which cause cellular inactivation after incorporation in place of normal nucleotides into DNA (Cole and Arlett, 1984). The steroid compound O U A is a potent inhibitor of the membrane-bound Na+/K+-dependent ATPase (Glynn, 1964; Dunham and Hoffman, 1970), an enzyme which is necessary for the maintenance of ionic balance in the cell, and is thus vital for cell survival (Thacker et al., 1978). In OUA-resistant mutants a portion of the N a + / K + ATPase molecule is believed to be altered such that the cells retain the ability to control ionic balance but lose sensitivity to the drug (Thacker et al., 1978). Selection of mutants in a population of somatic cell cultures has been performed by either the in situ or the respreading protocols. The former protocol, introduced by Chu and Malling in 1968, was almost universally adopted as the method of choice in the 1970's. In this approach, mutagen-treated cultures are plated at known densities and incubated in non-selective medium for various 'expression' times, usually 24-72 h, so that the pre-existing enzyme, and its mRNA, are diluted to a negligible level and the mutant phenotype becomes fully expressed. Selective agents are then added to the culture medium and the number of drug-resistant colonies determined. A major shortcoming of the in situ technique concerns the overcrowding that may occur at late expression times (Davies and Parry, 1974; Arlett et al., 1975). If overcrowding occurs, the
H G P R T - and H G P R T + cells remain in close proximity and as a result of metabolic cooperation between them, the H G P R T - cells may acquire the H G P R T enzyme and thus cease to grow in the presence of 8AZG or 6TG. In addition, a high density of wild-type cells present in the dishes at late expression times may physically prevent the formation of mutant colonies (Buchwald, 1977). To circumvent the overcrowding problem associated with the in situ technique, O'Neill and associates (1977a,b) introduced the respreading (also called replating) procedure for mutant selection. In this approach, the mutagen-treated cultures are dispersed and replated at appropriate cell densities prior to the addition of selective drugs. The respreading technique is currently accepted as the standard mammalian mutagenesis assay for genetic toxicology studies (Cole and Arlett, 1984; Nestmann et al., 1991). It has been proposed that the newly arising mutants may grow at a slower rate than wild-type cells (Buchwald, 1977; Chang et al., 1978) and that some mutants may be hypersensitive to the action of trypsin (Turnbull, 1975; Barrett et al., 1978). If so, then the yield of mutations measured by the standard respreading protocol is likely to be underestimated because a proportion of mutant cells may be lost or selected against during the several rounds of trypsinization and subculturing following genotoxin treatment. In the work described here we tested this possibility by comparing the frequencies of 8AZG-, 6TG- and OUA-resistant mutations induced by ethyl methanesulphonate (EMS) in Chinese hamster V79 cells, as determined by the in situ and the respreading techniques. Materials and methods
Cells and their cultiuation The particular Chinese hamster V79 cell line used in this study was provided by Dr. John Thacker (MRC Radiobiology Unit, Harweli, Didcot, U.K.). The cells were routinely cultured at 37°C in a humidified atmosphere (5% CO 2 in air) in Dulbecco's modification of Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 1 mM L-glutamine, 100 I U / m l penicillin G and
33 100 p~g/ml streptomycin sulfate (complete culture medium). All cell culture supplies were purchased from Flow Laboratories (Ayrshire, U.K.).
Selecti~'e agents The chemicals used for mutant selection were obtained from Sigma (Dorset, U.K.) and stored as a solid at - 2 0 ° C . A 100-times concentrated stock solution of each chemical was prepared in either 0.5% sodium carbonate solution ( 8 A Z G and 6TG) or in complete medium (OUA). The stock solutions were prepared fresh prior to each experiment and stored at 4°C for the duration of the experiment. 8AZG, 6TG and O U A were used at final concentrations of 0.2 mM, 15 p~M and 1 mM, respectively (Arlett et al., 1975; Cole and Arlett, 1984). Reduction of spontaneous mutation frequencies The frequency of spontaneously arising drugresistant mutants observed in the V79 cell line used in this study equalled < 2/105 clonable cells when 6TG or O U A were used for selection, whereas in the case of 8 A Z G the background mutation frequency was as high as 50/105 clonable cells. The maintenance of a relatively low level of pre-existing 8AZG-resistant variants ( < 10/105 clonable cells) was accomplished by serial subculturing from small inocula (Thompson and Baker, 1973). The resultant cultures displaying satisfactory frequencies of spontaneous mutations were used for post-EMS mutagenesis experiments or frozen down for future use. We avoided the use of dialysed sera to reduce the number of background 8AZG-resistant colonies (Nestmann et al., 1991) because it also led to a decrease in the plating efficiency of the cells (also see Thacker et al., 1976). EMS treatment protocol Exponentially growing cultures were incubated for 1 h at 37°C with complete medium containing the desired concentrations of EMS (Sigma). The cells were then rinsed twice with pre-warmed (37°C) phosphate-buffered saline, trypsinized, and assayed for cytotoxicity and mutagenesis.
densities (100-1000 cells per dish; 5 - 1 0 dishes per density) and incubated at 37°C with complete medium. 7 days later the cultures were stained with a 1% methylene blue solution (dissolved in 50% methanol) and colonies containing at least 50 ceils were counted. The ratio (as a percentage) of the number of colonies obtained in the treated compared to control dishes was taken as the extent of survival of colony-forming ability following EMS treatment.
Mutation measurement by the in situ assay Immediately after mutagen treatment, cells were plated into a number of 100-mm dishes at a density of 105 cells/dish and incubated in growth medium. At specific times thereafter the selective agents were added to the medium. Incubations were continued for a further 10-14 days after which the cells were stained with methylene blue solution and the colonies counted. The mutation frequencies were calculated from the mean number of mutant colonies recovered per dish corrected for the cloning efficiency of the cells determined in non-selective medium. Mutation measurement by the respreading assay U p o n completion of EMS treatment, cultures were incubated in complete medium for 2 days and then trypsinized. A portion of the cells was seeded in 100-mm dishes at 105/dish (10 plates for each mutagen treatment schedule and for each selective agent) and after incubation in medium supplemented with a particular selective agent for 10-14 days, the cells were stained with methylene blue solution and the number of drug-resistant colonies counted. The remainder of the cells were grown in the absence of selective drugs until the subsequent expression time, at which the cells were again trypsinized and incubated in the presence of selective agents as above. The cloning efficiency of the cells at each expression time was also determined in non-selective medium; the cloning efficiency values were used to correct the observed mutation frequency. Results and discussion
Cytotoxicity assay EMS-treated and sham-treated cells were plated into 60-mm diameter petri dishes at cloning
V79 cells were exposed to EMS (5-25 mM) and assayed concomitantly for induction of cell
34
tO0
I
I
I
I
I
TA B LE 1
9O 8O
M U T A G E N I C E F F E C T O F EMS IN V79 CELLS AS DET E R M I N E D BY T H E IN SITU ASSAY
70 6O ¢0
50
.>
EMS dose
Original plating
Number of mutant c o l o n i e s / 1 0 s cells plated
(raM)
efficiency
8AZG r
6TG r
OUA r
0 10
0.92 0.89
13 " 90
2 8
0 29
¢' Number of mutant colonies observed at the expression time of 45 h.
10
I
I
I
I
10
I
20
EMS concentration
30
(mM)
Fig. 1. Cytotoxicity of EMS in V79 cells. The mean ( + SE) of 4 independent experiments are presented.
killing and mutagenesis. Averaged results of multiple survival experiments are shown in Fig. 1. The survival curve contained an initial resistant shoulder at low doses ( < 10 mM) followed by an exponential region such that the colony-forming ability of cells treated with 25 mM EMS was decreased to 15%.
(n 12o o
.> >
I
I
20
411
I
I
60
80
100
>., 0 C G)
8O
o" 6o v.. P 0
~.
40
E '0 4) 0
2O
"0
_c
o
Expression
100
time (hrs)
Fig. 2. Induction of mutations to 8 A Z G r (I), 6TG r ( * ) and O U A r ( D ) by EMS (10 mM) as determined by the in situ assay. Each datum point represents the mean ( + SE) of the values obtained in 10 dishes.
Fig. 2 and Table 1 present the outcome of an in situ mutagenesis experiment performed with cultures exposed to a non-toxic concentration (10 mM) of EMS. Irrespective of the selective agent used, the absolute number of mutant colonies observed in the treated cultures was significantly greater than that arising in sham-treated controls (Table 1). The frequency of induced mutations (corrected for spontaneous levels) was greatest when 8AZG was used for mutant selection and smallest when 6TG was used for selection. The mutation frequencies observed at optimal expression times equalled 97/105 clonable cells for 8AZG, 34/105 for O U A and 6/105 for 6TG (Fig. 2). As mentioned in the Introduction, the decline in mutation frequencies observed at late expression times (Fig. 2; also see Davies and Parry, 1974) is known to reflect overcrowding of wild-type ceils prior to addition of selective agents to culture medium (see Discussion in Buchwald, 1977). When the mutation frequencies were determined by the respreading technique, the incidence of 6TG-resistant mutants proved to be markedly greater compared to OUA-resistant mutants and up to half of the frequency of 8AZG-resistant mutants (Fig. 3). The number of 8AZG- and OUA-resistant colonies reached the maximum values at early expression times (day 2), whereas full expression of 6TG-resistant cells required ~ 5 days of incubation after mutagen treatment. It should be noted that the maximum frequency of OUA-resistant mutants detected by the respreading technique upon exposure to 25 mM EMS (Fig. 3) was only one third of the
35 3oo
I
0
._> .> 250
% 200 ¢J
O"
150
e'. . 0m
100
E "0
O = "O C
50
0 0
2
4
6
8
10
Expression time (days) Fig. 3. Induction of mutations to 8AZG 4 (e), 6TG r ( A ) and O U A ([]) by EMS (25 mM) as determined by the respreading assay. Each datum point represents the mean ( + SE) of the values obtained in 10 dishes.
frequency measured by the in situ technique in cultures exposed to 10 mM EMS (Fig. 2). The results of independent mutation experiments are averaged in Fig. 4. For each dose of EMS employed, complete mutation profiles simi-
LU
E .
i E
8AZG r
6TG r
OUA r
Fig. 4. Comparison of the in situ and respreading assays for the measurement of mutation frequencies induced by EMS. Muta:ion frequencies observed at optimal expression times in the in situ method and those obtained on the plateau of the corresponding profiles of the respreading method were used for calculation of the data. The results are expressed as mutation frequencies ( × 1 0 6 survivors) induced per unit dose (mM) of EMS.
lar to those illustrated in Figs. 2 and 3 were generated and the values observed at optimum expression times in the in situ assays and values on the "plateau" of the corresponding profiles of the respreading assays were used for calculation of the data. The respreading experiments were usually conducted following damage with relatively high doses of EMS (20-25 mM) because the number of OUA-resistant colonies observed in cultures treated with < 10 mM EMS was at most 2 times greater than that seen in shamtreated cultures (data not shown). For accuracy of comparison of results obtained in different experiments, the data in Fig. 4 are expressed as mutation frequencies induced per unit dose (mM) of EMS. The two methods proved to yield comparable frequencies of 8AZG-resistant mutants in EMS treated cells (Fig. 4). On the other hand, the respreading assay afforded ~ 10 times greater yield of 6TG-resistant mutants than the in situ assay. These latter results were not unexpected, however, since the optimal expression of 6TG-resistant mutations following damage with genotoxic agents is known to require a higher degree of dilution of preexisting H G P R T enzyme as compared to when 8AZG is used for selection (Abbondandolo, 1977; Morrow, 1977; McMillan and Fox, 1979). In sharp contrast to that found when 6TG was used for mutant selection, the frequency of EMS-induced mutants resistant to O U A was ~ 10 times greater when determined by the in situ than the respreading assay. Consistent with this observation, following exposure of V79 cells to equicytotoxic concentrations of EMS, the frequencies of OUA-resistant mutations obtained by Arlett et al. (1975), who employed the in situ assay, were at least an order of magnitude greater than the frequencies observed by Thacker et al. (1978), who used the respreading assay. Likewise, Chang et al. (1978) reported that the frequency of ultraviolet (UV) light-induced mutations to OUA-resistance in V79 cells was maximal (induced mutation frequency, ~ 35/105 survivors obtained upon exposure to 20 J / m 2) at expression time of 48 h, which required no subculturing following irradiation, but the mutation frequency decreased to background levels when the irradi-
36
ated cells were subcultured 8 times between UV treatment and growth in the presence of OUA. The phenotypic stability of the three kinds of drug-resistant variants isolated by the respreading and the in situ methods were routinely examined. This was accomplished by growing cultures derived from individual clones in the absence of selective agents for at least 10 population doublings, followed by incubation in the presence of corresponding selective agents. All of the clones tested (at least 10 clones per selective agent per assay) proved to retain the drug-resistant phenotypes (data not shown). According to a recent survey (Nestmann et al., 1991), although 6TG (a marker for the HGPRT gene) coupled with the respreading technique is universally employed as the standard mammalian mutagenesis assay in genetic toxicology studies, several laboratories also adopt the OUA/respreading system to evaluate mutation induction at the N a + / K + ATPase locus (also see Buchwald, 1977; Baker et al., 1979; Nairn et al., 1989; Eldridge and Gould, 1991). The results of the present study indicate that the in situ assay is much preferable to the respreading assay for optimal yield of genotoxin-induced OUA-resistant mutants in Chinese hamster cells.
References Abbondandolo, A. (1977) Prospects for evaluating genetic damage in mammalian cells in culture, Mutation Res., 42, 279-298. Arlett, C.F., D. Turnbull, S.A. Harcourt, A.R. Lehmann and C.M. Colella (1975) A comparison of the 8-azaguanine and ouabain-resistance systems for the selection of induced mutant Chinese hamster cells, Mutation Res., 33, 261-278. Baker, R.M., W.C. Van Voorhis and L.A. Spencer (1979) HeLa cell variants that differ in sensitivity to monofunctional alkylating agents, with independence of cytotoxic and mutagenic responses, Proc. Natl. Acad. Sci. (U.S.A.), 76, 5249-5253. Barrett, J.C., N.E. Bias and P.O.P. Ts'o (1978) A mammalian cellular system for the concomitant study of neoplastic transformation and somatic mutation, Mutation Res., 50, 121-136. Buchwald, M. (1977) Mutagenesis at the ouabain-resistance locus in human diploid fibroblasts, Mutation Res., 44, 401-412. Chang, C.-C., J.E. Trosko and T. Akera (1978) Characteriza-
tion of ultraviolet light-induced ouabain-resistant mutations in Chinese hamster cells, Mutation Res., 51, 85-98. Chu, E.H.Y., and H.V. Mailing (1968) Mammalian cell genetics, II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro, Proc. Natl. Acad. Sci. (U.S.A.), 61, 1306-1312. Cole, J., and C.F. Arlett (1984) The detection of gene mutations in cultured mammalian cells, in: S. Venin amd J.M. Parry (Eds.), Mutagenicity Testing: A Practical Approach, IRL Press, Oxford, pp. 233-273. Davies, P.J., and J.M. Parry (1974) The induction of ouabainresistant mutations by N-methyl-N'-nitro-N-nitrosoguanidine in Chinese hamster cells, Genet. Res., 24, 311-314. Dunham, P.B., and J.F. Hoffman (1970) Partial purification of the ouabain-binding component and of Na,K-ATPase from human red cell membranes, Proc. Natl. Acad. Sci. (U.S.A.), 66, 936-943. Eldridge, S.R., and M.N. Gould (1991) Specific locus mutagenesis of human mammary epithelial cells by ultraviolet radiation, Int. J. Radiat. Biol., 59, 807-814. Glynn, I.M. (1964) The action of cardiac glycosides on ion movements, Pharmacol. Rev., 16, 381-407. McMillan, S., and M. Fox (1979) Failure of caffeine to influence induced mutation frequencies and the independence of cell killing and mutation induction in V79 Chinese hamster cells, Mutation Res., 60, 91-107. Morrow, L. (1977) Gene inactivation as a mechanism for the generation of variability in somatic cells cultivated in vitro, Mutation Res., 44, 391-400. Nairn, R.S., D.L. Mitchell, G.M. Adair, L.H. Thompson, M.J. Siciliano and R.M. Humphrey (1989) UV mutagenesis, cytotoxicity and split-dose recovery in human-CHO cell hybrid having intermediate (6-4) photoproduct repair, Mutation Res., 217, 193-201. Nestmann, E.R., R.L Brillinger, J.P.W. Gilman, C.J. Rudd and S.H.H. Swierenga (1991) Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: II. Mutation in Chinese hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells, Mutation Res., 246, 255-284. O'Neill, J.P., P.A. Brimer, R. Machanoff, G.P. Hirsch and A.W. Hsie (1977a) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system, Mutation Res., 45, 91-101. O'Neill, J.P., D.B. Couch, R. Machanoff, J.R. San Sebastian, P.A. Brimer and A.W. Hsie (1977b) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/GPRT system): Utilization with a variety of mutagenic agents, Mutation Res., 45, 103-109. Thacker, J., M.A. Stephens and A. Stretch (1976) Factors affecting the efficiency of purine analogues as selective agents for mutants of mammalian cells induced by ionising radiation, Mutation Res., 35, 465-478. Thacker, J., M.A. Stephens and A. Stretch (1978) Mutation to ouabain-resistance in Chinese hamster cells: induction by
37 ezhyl methanesulphonate and lack of induction by ionising radiation, Mutation Res., 51,255-270. Thompson, L.H., and R.M. Baker (1973) Isolation of mutants of cultured mammalian cells, in: D.M. Prescott (Ed.), Methods in Cell Biology, Vol. 6, Academic Press, New York, pp. 209-281.
Turnbull, D. (1975) Factors affecting the response of cultured Chinese hamster cells to mutagenic alkylating agents, D. Phil. Thesis, University of Sussex, U.K.
Communicated by J. Little
31
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
MUTLET 0652
Application of the standard mutagenesis assay results in underestimation of ethyl methanesulphonate-induced mutations to ouabain-resistance in Chinese hamster cells * Farideh Mirzayans a, James
M. Parry b
and Razmik Mirzayans
c
'~ Division of Infectious Diseases, Department of Medicine, University of Alberta, Edmonton, Alb. T6G 2R7 (Canada), I, Department of Genetics, University College of Swansea, Swansea SA2 8PP (Great Britain) and ~ Molecular Genetics and Carcinogenesis Laboratory, Department of Medicine, Cross Cancer Institute, Edmonton, Alb. T6G lZ2 (Canada)
(Received 30 September 1991) (Revision received 20 December 1991) (Accepted 23 December 1991) Keywords: Ethyl methanesulphonate; 8-Azaguanine; 6-Thioguanine; Ouabain; Chinese hamster V79 cells
Summary Chinese hamster V79 cells were exposed to ethyl methanesulphonate (EMS) and the incidence of mutant cells resistant to 8-azaguanine (8AZG), 6-thioguanine (6TG) or ouabain ( O U A ) was determined both by the respreading and the in situ techniques. In the former assay, the mutagen-treated cultures were grown for several days to permit the expression of mutations after which the cells were trypsinized, replated (10 5 cells/100-mm dish), and grown in medium supplemented with a selective agent. In the in situ assay, cultures were left undisturbed between EMS treatment and incubation in the presence of the selective agents. The yield of 8AZG-resistant mutants observed at optimal expression times after EMS treatment was comparable for both techniques; the induced mutation frequency (corrected for spontaneous mutation frequency) was estimated to be 82 x 10 -6 mutations per viable cell per unit dose (mM) of EMS. The frequency of 6TG-resistant mutants equalled 45 and 4 x 1 0 - 6 / m M EMS as determined by the respreading and the in situ assays, respectively. In sharp contrast to that observed with 6TG, the frequency of OUA-resistant mutants scored by the in situ assay (30 x 1 0 - 6 / r a M EMS) proved to be an order of magnitude greater than that determined by the respreading assay (3 x 1 0 - 6 / m M EMS). Our data therefore indicate that, when O U A is used for mutant selection, the application of the respreading technique, which has been widely adopted as the standard mammalian mutational assay over the past decade, may result in a marked underestimation of the actual mutation frequency ( ~ 10-fold in V79 cells).
* Dedicated to the memoryof Dr. George Mirzayans. Correspondence: Dr. Razmik Mirzayans, Molecular Genetics and Carcinogenesis Laboratory, Department of Medicine, Cross Cancer Institute, Edmonton, Alb. T6G 1Z2 (Canada).
Since the development of mammalian cell mutagenesis techniques over two decades ago (Chu and Malling, 1968), numerous laboratories have directed considerable effort to optimising the experimental conditions for measuring the induc-
32 tion of forward mutations at specific loci in mammalian cells treated with physical and chemical genotoxic agents. Although a variety of mutant phenotypes may be selected for (Cole and Arlett, 1984), resistance to 8-azaguanine (8AZG), 6thioguanine (6TG) or ouabain (OUA) are the most extensively studied and well defined mutational systems utilized with cultured human and Chinese hamster (V79 and CHO) cells (Cole and Arlett, 1984; Nestmann et al., 1991). The guanine analogues, 8AZG and 6TG, select variants harbouring mutations at the X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. The function of the H G P R T enzyme is to convert the purines, hypoxanthine and guanine, to nucleotides via a purine nucleotide salvage pathway. This enzyme also converts 6TG and 8AZG into their respective nucleotides which cause cellular inactivation after incorporation in place of normal nucleotides into DNA (Cole and Arlett, 1984). The steroid compound O U A is a potent inhibitor of the membrane-bound Na+/K+-dependent ATPase (Glynn, 1964; Dunham and Hoffman, 1970), an enzyme which is necessary for the maintenance of ionic balance in the cell, and is thus vital for cell survival (Thacker et al., 1978). In OUA-resistant mutants a portion of the N a + / K + ATPase molecule is believed to be altered such that the cells retain the ability to control ionic balance but lose sensitivity to the drug (Thacker et al., 1978). Selection of mutants in a population of somatic cell cultures has been performed by either the in situ or the respreading protocols. The former protocol, introduced by Chu and Malling in 1968, was almost universally adopted as the method of choice in the 1970's. In this approach, mutagen-treated cultures are plated at known densities and incubated in non-selective medium for various 'expression' times, usually 24-72 h, so that the pre-existing enzyme, and its mRNA, are diluted to a negligible level and the mutant phenotype becomes fully expressed. Selective agents are then added to the culture medium and the number of drug-resistant colonies determined. A major shortcoming of the in situ technique concerns the overcrowding that may occur at late expression times (Davies and Parry, 1974; Arlett et al., 1975). If overcrowding occurs, the
H G P R T - and H G P R T + cells remain in close proximity and as a result of metabolic cooperation between them, the H G P R T - cells may acquire the H G P R T enzyme and thus cease to grow in the presence of 8AZG or 6TG. In addition, a high density of wild-type cells present in the dishes at late expression times may physically prevent the formation of mutant colonies (Buchwald, 1977). To circumvent the overcrowding problem associated with the in situ technique, O'Neill and associates (1977a,b) introduced the respreading (also called replating) procedure for mutant selection. In this approach, the mutagen-treated cultures are dispersed and replated at appropriate cell densities prior to the addition of selective drugs. The respreading technique is currently accepted as the standard mammalian mutagenesis assay for genetic toxicology studies (Cole and Arlett, 1984; Nestmann et al., 1991). It has been proposed that the newly arising mutants may grow at a slower rate than wild-type cells (Buchwald, 1977; Chang et al., 1978) and that some mutants may be hypersensitive to the action of trypsin (Turnbull, 1975; Barrett et al., 1978). If so, then the yield of mutations measured by the standard respreading protocol is likely to be underestimated because a proportion of mutant cells may be lost or selected against during the several rounds of trypsinization and subculturing following genotoxin treatment. In the work described here we tested this possibility by comparing the frequencies of 8AZG-, 6TG- and OUA-resistant mutations induced by ethyl methanesulphonate (EMS) in Chinese hamster V79 cells, as determined by the in situ and the respreading techniques. Materials and methods
Cells and their cultiuation The particular Chinese hamster V79 cell line used in this study was provided by Dr. John Thacker (MRC Radiobiology Unit, Harweli, Didcot, U.K.). The cells were routinely cultured at 37°C in a humidified atmosphere (5% CO 2 in air) in Dulbecco's modification of Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 1 mM L-glutamine, 100 I U / m l penicillin G and
33 100 p~g/ml streptomycin sulfate (complete culture medium). All cell culture supplies were purchased from Flow Laboratories (Ayrshire, U.K.).
Selecti~'e agents The chemicals used for mutant selection were obtained from Sigma (Dorset, U.K.) and stored as a solid at - 2 0 ° C . A 100-times concentrated stock solution of each chemical was prepared in either 0.5% sodium carbonate solution ( 8 A Z G and 6TG) or in complete medium (OUA). The stock solutions were prepared fresh prior to each experiment and stored at 4°C for the duration of the experiment. 8AZG, 6TG and O U A were used at final concentrations of 0.2 mM, 15 p~M and 1 mM, respectively (Arlett et al., 1975; Cole and Arlett, 1984). Reduction of spontaneous mutation frequencies The frequency of spontaneously arising drugresistant mutants observed in the V79 cell line used in this study equalled < 2/105 clonable cells when 6TG or O U A were used for selection, whereas in the case of 8 A Z G the background mutation frequency was as high as 50/105 clonable cells. The maintenance of a relatively low level of pre-existing 8AZG-resistant variants ( < 10/105 clonable cells) was accomplished by serial subculturing from small inocula (Thompson and Baker, 1973). The resultant cultures displaying satisfactory frequencies of spontaneous mutations were used for post-EMS mutagenesis experiments or frozen down for future use. We avoided the use of dialysed sera to reduce the number of background 8AZG-resistant colonies (Nestmann et al., 1991) because it also led to a decrease in the plating efficiency of the cells (also see Thacker et al., 1976). EMS treatment protocol Exponentially growing cultures were incubated for 1 h at 37°C with complete medium containing the desired concentrations of EMS (Sigma). The cells were then rinsed twice with pre-warmed (37°C) phosphate-buffered saline, trypsinized, and assayed for cytotoxicity and mutagenesis.
densities (100-1000 cells per dish; 5 - 1 0 dishes per density) and incubated at 37°C with complete medium. 7 days later the cultures were stained with a 1% methylene blue solution (dissolved in 50% methanol) and colonies containing at least 50 ceils were counted. The ratio (as a percentage) of the number of colonies obtained in the treated compared to control dishes was taken as the extent of survival of colony-forming ability following EMS treatment.
Mutation measurement by the in situ assay Immediately after mutagen treatment, cells were plated into a number of 100-mm dishes at a density of 105 cells/dish and incubated in growth medium. At specific times thereafter the selective agents were added to the medium. Incubations were continued for a further 10-14 days after which the cells were stained with methylene blue solution and the colonies counted. The mutation frequencies were calculated from the mean number of mutant colonies recovered per dish corrected for the cloning efficiency of the cells determined in non-selective medium. Mutation measurement by the respreading assay U p o n completion of EMS treatment, cultures were incubated in complete medium for 2 days and then trypsinized. A portion of the cells was seeded in 100-mm dishes at 105/dish (10 plates for each mutagen treatment schedule and for each selective agent) and after incubation in medium supplemented with a particular selective agent for 10-14 days, the cells were stained with methylene blue solution and the number of drug-resistant colonies counted. The remainder of the cells were grown in the absence of selective drugs until the subsequent expression time, at which the cells were again trypsinized and incubated in the presence of selective agents as above. The cloning efficiency of the cells at each expression time was also determined in non-selective medium; the cloning efficiency values were used to correct the observed mutation frequency. Results and discussion
Cytotoxicity assay EMS-treated and sham-treated cells were plated into 60-mm diameter petri dishes at cloning
V79 cells were exposed to EMS (5-25 mM) and assayed concomitantly for induction of cell
34
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I
I
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TA B LE 1
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M U T A G E N I C E F F E C T O F EMS IN V79 CELLS AS DET E R M I N E D BY T H E IN SITU ASSAY
70 6O ¢0
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EMS dose
Original plating
Number of mutant c o l o n i e s / 1 0 s cells plated
(raM)
efficiency
8AZG r
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0 10
0.92 0.89
13 " 90
2 8
0 29
¢' Number of mutant colonies observed at the expression time of 45 h.
10
I
I
I
I
10
I
20
EMS concentration
30
(mM)
Fig. 1. Cytotoxicity of EMS in V79 cells. The mean ( + SE) of 4 independent experiments are presented.
killing and mutagenesis. Averaged results of multiple survival experiments are shown in Fig. 1. The survival curve contained an initial resistant shoulder at low doses ( < 10 mM) followed by an exponential region such that the colony-forming ability of cells treated with 25 mM EMS was decreased to 15%.
(n 12o o
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411
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60
80
100
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8O
o" 6o v.. P 0
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100
time (hrs)
Fig. 2. Induction of mutations to 8 A Z G r (I), 6TG r ( * ) and O U A r ( D ) by EMS (10 mM) as determined by the in situ assay. Each datum point represents the mean ( + SE) of the values obtained in 10 dishes.
Fig. 2 and Table 1 present the outcome of an in situ mutagenesis experiment performed with cultures exposed to a non-toxic concentration (10 mM) of EMS. Irrespective of the selective agent used, the absolute number of mutant colonies observed in the treated cultures was significantly greater than that arising in sham-treated controls (Table 1). The frequency of induced mutations (corrected for spontaneous levels) was greatest when 8AZG was used for mutant selection and smallest when 6TG was used for selection. The mutation frequencies observed at optimal expression times equalled 97/105 clonable cells for 8AZG, 34/105 for O U A and 6/105 for 6TG (Fig. 2). As mentioned in the Introduction, the decline in mutation frequencies observed at late expression times (Fig. 2; also see Davies and Parry, 1974) is known to reflect overcrowding of wild-type ceils prior to addition of selective agents to culture medium (see Discussion in Buchwald, 1977). When the mutation frequencies were determined by the respreading technique, the incidence of 6TG-resistant mutants proved to be markedly greater compared to OUA-resistant mutants and up to half of the frequency of 8AZG-resistant mutants (Fig. 3). The number of 8AZG- and OUA-resistant colonies reached the maximum values at early expression times (day 2), whereas full expression of 6TG-resistant cells required ~ 5 days of incubation after mutagen treatment. It should be noted that the maximum frequency of OUA-resistant mutants detected by the respreading technique upon exposure to 25 mM EMS (Fig. 3) was only one third of the
35 3oo
I
0
._> .> 250
% 200 ¢J
O"
150
e'. . 0m
100
E "0
O = "O C
50
0 0
2
4
6
8
10
Expression time (days) Fig. 3. Induction of mutations to 8AZG 4 (e), 6TG r ( A ) and O U A ([]) by EMS (25 mM) as determined by the respreading assay. Each datum point represents the mean ( + SE) of the values obtained in 10 dishes.
frequency measured by the in situ technique in cultures exposed to 10 mM EMS (Fig. 2). The results of independent mutation experiments are averaged in Fig. 4. For each dose of EMS employed, complete mutation profiles simi-
LU
E .
i E
8AZG r
6TG r
OUA r
Fig. 4. Comparison of the in situ and respreading assays for the measurement of mutation frequencies induced by EMS. Muta:ion frequencies observed at optimal expression times in the in situ method and those obtained on the plateau of the corresponding profiles of the respreading method were used for calculation of the data. The results are expressed as mutation frequencies ( × 1 0 6 survivors) induced per unit dose (mM) of EMS.
lar to those illustrated in Figs. 2 and 3 were generated and the values observed at optimum expression times in the in situ assays and values on the "plateau" of the corresponding profiles of the respreading assays were used for calculation of the data. The respreading experiments were usually conducted following damage with relatively high doses of EMS (20-25 mM) because the number of OUA-resistant colonies observed in cultures treated with < 10 mM EMS was at most 2 times greater than that seen in shamtreated cultures (data not shown). For accuracy of comparison of results obtained in different experiments, the data in Fig. 4 are expressed as mutation frequencies induced per unit dose (mM) of EMS. The two methods proved to yield comparable frequencies of 8AZG-resistant mutants in EMS treated cells (Fig. 4). On the other hand, the respreading assay afforded ~ 10 times greater yield of 6TG-resistant mutants than the in situ assay. These latter results were not unexpected, however, since the optimal expression of 6TG-resistant mutations following damage with genotoxic agents is known to require a higher degree of dilution of preexisting H G P R T enzyme as compared to when 8AZG is used for selection (Abbondandolo, 1977; Morrow, 1977; McMillan and Fox, 1979). In sharp contrast to that found when 6TG was used for mutant selection, the frequency of EMS-induced mutants resistant to O U A was ~ 10 times greater when determined by the in situ than the respreading assay. Consistent with this observation, following exposure of V79 cells to equicytotoxic concentrations of EMS, the frequencies of OUA-resistant mutations obtained by Arlett et al. (1975), who employed the in situ assay, were at least an order of magnitude greater than the frequencies observed by Thacker et al. (1978), who used the respreading assay. Likewise, Chang et al. (1978) reported that the frequency of ultraviolet (UV) light-induced mutations to OUA-resistance in V79 cells was maximal (induced mutation frequency, ~ 35/105 survivors obtained upon exposure to 20 J / m 2) at expression time of 48 h, which required no subculturing following irradiation, but the mutation frequency decreased to background levels when the irradi-
36
ated cells were subcultured 8 times between UV treatment and growth in the presence of OUA. The phenotypic stability of the three kinds of drug-resistant variants isolated by the respreading and the in situ methods were routinely examined. This was accomplished by growing cultures derived from individual clones in the absence of selective agents for at least 10 population doublings, followed by incubation in the presence of corresponding selective agents. All of the clones tested (at least 10 clones per selective agent per assay) proved to retain the drug-resistant phenotypes (data not shown). According to a recent survey (Nestmann et al., 1991), although 6TG (a marker for the HGPRT gene) coupled with the respreading technique is universally employed as the standard mammalian mutagenesis assay in genetic toxicology studies, several laboratories also adopt the OUA/respreading system to evaluate mutation induction at the N a + / K + ATPase locus (also see Buchwald, 1977; Baker et al., 1979; Nairn et al., 1989; Eldridge and Gould, 1991). The results of the present study indicate that the in situ assay is much preferable to the respreading assay for optimal yield of genotoxin-induced OUA-resistant mutants in Chinese hamster cells.
References Abbondandolo, A. (1977) Prospects for evaluating genetic damage in mammalian cells in culture, Mutation Res., 42, 279-298. Arlett, C.F., D. Turnbull, S.A. Harcourt, A.R. Lehmann and C.M. Colella (1975) A comparison of the 8-azaguanine and ouabain-resistance systems for the selection of induced mutant Chinese hamster cells, Mutation Res., 33, 261-278. Baker, R.M., W.C. Van Voorhis and L.A. Spencer (1979) HeLa cell variants that differ in sensitivity to monofunctional alkylating agents, with independence of cytotoxic and mutagenic responses, Proc. Natl. Acad. Sci. (U.S.A.), 76, 5249-5253. Barrett, J.C., N.E. Bias and P.O.P. Ts'o (1978) A mammalian cellular system for the concomitant study of neoplastic transformation and somatic mutation, Mutation Res., 50, 121-136. Buchwald, M. (1977) Mutagenesis at the ouabain-resistance locus in human diploid fibroblasts, Mutation Res., 44, 401-412. Chang, C.-C., J.E. Trosko and T. Akera (1978) Characteriza-
tion of ultraviolet light-induced ouabain-resistant mutations in Chinese hamster cells, Mutation Res., 51, 85-98. Chu, E.H.Y., and H.V. Mailing (1968) Mammalian cell genetics, II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro, Proc. Natl. Acad. Sci. (U.S.A.), 61, 1306-1312. Cole, J., and C.F. Arlett (1984) The detection of gene mutations in cultured mammalian cells, in: S. Venin amd J.M. Parry (Eds.), Mutagenicity Testing: A Practical Approach, IRL Press, Oxford, pp. 233-273. Davies, P.J., and J.M. Parry (1974) The induction of ouabainresistant mutations by N-methyl-N'-nitro-N-nitrosoguanidine in Chinese hamster cells, Genet. Res., 24, 311-314. Dunham, P.B., and J.F. Hoffman (1970) Partial purification of the ouabain-binding component and of Na,K-ATPase from human red cell membranes, Proc. Natl. Acad. Sci. (U.S.A.), 66, 936-943. Eldridge, S.R., and M.N. Gould (1991) Specific locus mutagenesis of human mammary epithelial cells by ultraviolet radiation, Int. J. Radiat. Biol., 59, 807-814. Glynn, I.M. (1964) The action of cardiac glycosides on ion movements, Pharmacol. Rev., 16, 381-407. McMillan, S., and M. Fox (1979) Failure of caffeine to influence induced mutation frequencies and the independence of cell killing and mutation induction in V79 Chinese hamster cells, Mutation Res., 60, 91-107. Morrow, L. (1977) Gene inactivation as a mechanism for the generation of variability in somatic cells cultivated in vitro, Mutation Res., 44, 391-400. Nairn, R.S., D.L. Mitchell, G.M. Adair, L.H. Thompson, M.J. Siciliano and R.M. Humphrey (1989) UV mutagenesis, cytotoxicity and split-dose recovery in human-CHO cell hybrid having intermediate (6-4) photoproduct repair, Mutation Res., 217, 193-201. Nestmann, E.R., R.L Brillinger, J.P.W. Gilman, C.J. Rudd and S.H.H. Swierenga (1991) Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: II. Mutation in Chinese hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells, Mutation Res., 246, 255-284. O'Neill, J.P., P.A. Brimer, R. Machanoff, G.P. Hirsch and A.W. Hsie (1977a) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system, Mutation Res., 45, 91-101. O'Neill, J.P., D.B. Couch, R. Machanoff, J.R. San Sebastian, P.A. Brimer and A.W. Hsie (1977b) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/GPRT system): Utilization with a variety of mutagenic agents, Mutation Res., 45, 103-109. Thacker, J., M.A. Stephens and A. Stretch (1976) Factors affecting the efficiency of purine analogues as selective agents for mutants of mammalian cells induced by ionising radiation, Mutation Res., 35, 465-478. Thacker, J., M.A. Stephens and A. Stretch (1978) Mutation to ouabain-resistance in Chinese hamster cells: induction by
37 ezhyl methanesulphonate and lack of induction by ionising radiation, Mutation Res., 51,255-270. Thompson, L.H., and R.M. Baker (1973) Isolation of mutants of cultured mammalian cells, in: D.M. Prescott (Ed.), Methods in Cell Biology, Vol. 6, Academic Press, New York, pp. 209-281.
Turnbull, D. (1975) Factors affecting the response of cultured Chinese hamster cells to mutagenic alkylating agents, D. Phil. Thesis, University of Sussex, U.K.
Communicated by J. Little
