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Influence of experimental conditions and DNA repair ability on EMS-induced mutagenesis and DNA binding in Escherichia coli K12
Mutation Research, 92(1982) 15-27 Elsevier Biomedical Press
15
Influence of experimental conditions and D N A repair ability on EMS-induced mutagenesis and D N A binding in Escherichia coli K12 Comparison With mammalian cell mutagenesis G.R. Mohn l A.A. van Zeeland 1 and B.W. Glickman 2 t Department of Radiation Genetics and Chemical Mutagenesis, The.State University of Leiden, Leiden (The Netherlands); and : Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC (U.S.A.)
(Received 15 September 1981) (Accepted 17 September 1981)
Summary In continuation of previous experiments aimed at quantitatively comparing the magnitude of ethyl methanesulfonate (EMS)-induced DNA binding and mutagenesis in bacterial and mammalian cells, the present studies were undertaken to determine the influence of various experimental conditions on EMS-induced changes in E. coli K12/343/113. The sensitivity to EMS mutagenesis in this strain was assayed for various genetic endpoints, namely gal + , MTW, NAL r, VALr and arg + . EMS mutagenesis and DNA binding in the repair-proficient wild-type was, furthermore, compared to that in derivatives deficient in excision repair ( A u v r B l O 1 ) and postreplication repair ( r e c A l 3 ) . Attempts were, finally, undertaken to determine the shape of the dose-response curve for the most sensitive genetic systems found, namely NAU and VALr mutations, at relatively low EMS-exposure conditions of 0-7 mM for 120 rain at 37°C. From the present experiments and from previous results obtained under identical EMS treatment conditions in E. coli 343/113 and in the mammalian cell lines V79 and L5178Y, the following conclusions can" be drawn: (i) At EMS exposure concentrations ranging from 2.5 to 50 mM and treatment periods of 120 min at 37°C in buffer pH 7.2, the levels of DNA alkylation at termination of the treatment are similar in E. coli and mammalian cells and increase from 1.7 x 10 -4 to 1.3 × 10 -3 ethylations per nucleotide. (ii) The presence of nutrients such as 1-arginine, o-glucose and fetal calf serum, during EMS treatment of stationary E. coli cells does not influence the level of DNA alkylation but greatly increases EMS-induced mutation Abbreviations: 3H-EMS, 1[3H]ethyl methanesulfonate; LEC, lowest effective concentration.
0027-5107/82/0000-0000/$02.75 © Elsevier Biomedical Press
16 yields, probably because of the accumulation of these nutrients into the cells, thereby leading to increased capability of genetic fixation and/or phenotypic expression of the premutational DNA changes. (iii) The mammalian cells are more sensitive to the killing action of EMS than E. coli repair-proficient cells (survival at 10 mM of 12 and 100%, respectively), which indicates that the genetic and/or physiologic target for inactivation of colony-forming ability is larger in the mammalian cells than in bacteria, or alternatively that these cells have lower ability to repair EMS-induced DNA lesions. (iv) The 2 new mutation systems tested in the present series of experiments, namely N A U and VALr mutations appear to be quite comparable to those tested in mammalian cells, such as thioguanine resistance ( H G P R T - ) and BUdR resistance ( T K - / - ) , in the sense that at low doses, i.e. EMS-exposure concentrations of up to 7 mM for 120 min at 37°C in buffer, pH 7.2, the induction kinetics in all systems are compatible with a linear relationship between dose and mutation frequency, with no indication of a threshold. In the case of N A U in E. coli and H G P R T - in V79 mammalian cells the Lowest Effective Concentrations of EMS are 1-2 mM for treatment periods of 120 min at 37°C. It remains to be determined if the same EMS-induced DNA adducts are responsible in both cell types for the genetic effects observed; further comparative experiments are also required to assess the general suitability of bacteria in quantifying mutagenic and tumor-initiating potency of chemicals.
Sobels (1976, 1977, 1980) has identified and described several phases in detecting, quantifying, and extrapolating to man the mutagenic and tumor-initiating potency of environmental chemicals, by making use of different genetic endpoints and test organisms ranging from microbes to mammals. Extensive comparable studies have shown that, qualitatively, there is a high degree of parallelism between the mutagenicity of chemicals in microorganisms and their mutagenic and carcinogenic effects in animals (McCann et al., 1975; Hollstein et al., 1979), depending on the reactivitY class of chemicals considered. Because reports about newly detected mutagens in the environment accumulate, based on results obtained with microorganisms, and because a substantial lag period remains for obtaining quantitative mutagenicity a n d / o r carcinogenicity results in experimental animals, methodologies have to be developed for a more quantitative and representative use of these microbial systems. The question then to be answered is which genetic endpoints and which genetic backgrounds are best suited for an accurate and quantitative prediction of genetic efficiency. Of special interest in this context is the determination of the extent to which the response to chemical mutagens is influenced by intrinsic cellular factors, such as chromosome structure and DNA content per cell, and by metabolic and physiologic differences between test organisms. This can be done by performing quantitative comparisons of the magnitude of genetic effects induced in various organisms by known mutagens/carcinogens. Such a comparison necessitates a precise determination of the actual dose at the target cell or the target molecule, e.g. DNA. The reason for carrying out dose measurements is that one cannot, a priori,
17 assume that identical exposure to a chemical will lead to identical dose at the DNA level. Instead, one expects that penetration and metabolism in general will influence the extent of the reaction with and the binding to DNA. Previous experiments were performed to quantitatively compare in microorganisms and in mammalian cell lines the magnitude of genetic effects induced by the alkylating agent ethyl methanesulfonate (EMS) on the basis of DNA dose, measured as ethylation per nucleotide (Aaron et al., 1978, 1980; Mohn et al., 1980; van Zeeland et al., 1982). They showed that standardization of test methodology and DNA dose measurements are possible with prokaryotic and eukaryotic cells. They also showed that, on the basis of equal DNA dose, the variation of EMS-induced mutagenic effects within vastly differing species (E. coli, Saccharomyces cerevisiae, Neurospora crassa, Chinese hamster cells, and mouse lymphoma cells) was not greater than the variation between different genetic endpoints (forward mutations) within one species or one cdl line. Some quantitative differences between the microbes (of both prokaryotic and eukaryotic cell type) and the mammalian cell lines remained, however, in the following points: (i) the wild-type microorganisms are more resistant than the mammalian cells to EMS-induced inactivation; (ii) the mutation systems studied in the mammalian cell lines (thioguanine resistance, BUdR resistance, ouabain resistance) show linear EMS-induced kinetics, whereas the microbial systems usually have biphasic or exponential kinetics with exponents of up to 2.6; and (iii) at low exposure concentrations the frequencies of mutations induced in the microbial systems are lower than in the mammalian cell lines. These differences are not due to differences in dose to the DNA, since dosimetry measurements have shown that at identical EMS exposures (from 2.5 to 50 mM for 120 min at 37°C in buffer, pH 7.2) the level of ethylation per nucleotide is similar in microbial and in mammalian cells (Aaron et al., 1978, 1980; van Zeeland et al., 1982). The experiments reported in this paper were designed to gain further insight into the role of factors responsible for the above-mentioned differences by varying, in a bacterial indicator, the conditions of EMS treatment, the genetic endpoints, and the DNA repair ability of the tester strain. It has been shown previously by using EMS and other alkylating agents that the growing state of the cells has a profound influence on mutagen sensitivity (Howell-Saxton et al., 1973; Hince and Neale, 1977) as has the DNA repair ability of these cells (Kondo et al., 1970; Lawley, 1974; Todd et al., 1981). The influence of these parameters and the mutagenic response of additional genetic endpoints were determined in a bacterial strain previously used in the comparative studies mentioned above, namely E. coli K12 343/113. After conditions for optimal expression of EMS-induced premutations were assessed, attempts were made to determine the linearity or non-linearity of the dose-response at low EMS exposures in these bacterial cells. As in the previous experiments the dose to the DNA was measured with 3H-labeled EMS and expressed as ethylation per nucleotide.
18 Material and methods
Chemicals 1[ 3 H]Ethyl methanesulfonate (3H-EMS) was obtained from New England Nuclear at a specific activity of 4.8 Ci/mmole dissolved in diethyl ether. Unlabeled EMS was obtained from Eastman Chemical. 10 mCi of 3H-EMS in 10 ml diethyl ether were mixed with 2-4 ml phosphate-buffered saline (PBS), pH 7.2, and the ether was evaporated under N 2. The amount of 3H-EMS left was determined by diluting 20q~l samples and counting in a liquid scintillation counter. Unlabeled EMS was added to a final concentration of 100 mM, resulting in a specific activity of 10-30 m C i / mmole. Samples of this stock solution were added immediately to bacterial cell suspensions for detection of the ethylation level in DNA. For the detection o f mutagenic effects, stock solutions of unlabeled EMS were prepared in PBS. immediately before the beginning of the experiment. Growth and suspension media Phosphate-buffered saline (PBS), pH 7.2, nutrient broth (NB), complete agar (COA), and the agar media for detection of MTRr, gal +, and arg + mutants were prepared as described previously; the composition of agar media for scoring valineresistant (VAU) and nalidixic acid-resistant (NAU) mutants was the same as MTW except that, instead of 5-methyltryptophan, VAU contained 10 #g 1-valine per ml medium and NAU contained 15 /~g nalidixic acid per ml medium (Mohn et al., 1981). In some mutation-induction experiments the bacteria were treated with EMS while suspended in various media, namely, PBS, PBS supplemented with 1% D-glucose, PBS supplemented with 30 /~g 1-arginine per ml, and the synthetic medium F10 supplemented with 15% calf serum, in which usually mammalian cells are grown before harvest and EMS treatment (van Zeeland, 1978). Bacterial strains For detection of various mutation endpoints, strain E. coli 343//113 was used (Mohn and Ellenberger, 1977; Mohn et al., 1981). A deletion including the uvrB gene was introduced by Pl-mediated cotransduction with chl and bio (Kato and Shinoura, 1977) and the recA13 allele was introduced after transduction and selection for sorbitol utilization (Howard-Flanders, 1967). Mutagenicity tests Standard EMS treatment was performed as described previously (Aaron et al., 1978, 1980). Briefly, suspensions of stationary-phase cells of E. coli 343/113 or its repair-deficient derivatives were exposed to various concentrations of EMS in a final volume of 2.5 ml PBS (pH 7.2) for 120 min at 37°C in the dark and under rotary shaking. After treatment the cells were washed to remove excess mutagen and, upon appropriate dilution, were plated on various agar media for the determination of cell survival and mutation frequencies. Since some mutation types, e.g. NAU, VAU, and MTR r, require post-treatment incubation in non-selective growth medium for opti-
19 mum expression of induced premutations, experiments involving these mutations included dilution of EMS-treated and washed cells into nutrient broth (final titer 1-2 × 108 cells per ml) and overnight incubation in the dark at 37°C, thereby leading to a mean number of 3-4 cell divisions. In some experiments the cells were suspended in media supplemented with growth factors during EMS treatment, as indicated later in the text. In those experiments involving treatment of growing cells, log-phase cells were obtained by diluting, 120 min before EMS treatment, stationary-phase cells into nutrient broth and incubating at 37°C; slaortly before treatment the cells were washed and resuspended either in buffer, or in supplemented minimal medium, and further handled as described before. DNA-binding experiments Treatment of E. coli 343/113 with 3H-labeled EMS was performed in a manner
identical to and in parallel with the mutation-induction experiments. After the completion of the treatment, the cells were washed and incubated with lysozyme, then lysed with sarcosyl and incubated with proteinase K. The DNA was isolated by CsCI density-gradient centrifugation and the amount of radio-labeled mutagen bound to DNA was measured as described previously (Aaron et al., 1978, 1980).
Results and discussion
A number of experimental conditions during EMS treatment was varied to determine their influence on induced mutant yields. First, since mammalian cells were grown in the previous comparative experiments in F10 medium+ 15% calf serum (CS), EMS-induced mutation frequencies were compared in the present studies while E. coli 343/113 cells were suspended in PBS and in F10 + 15% CS, respectively. The results presented in Fig. 1 show that E. coil cells suspended in F10 + 15% CS exhibit higher EMS-induced arg + mutation frequencies than the cells suspended in PBS. Analogous results were obtained with gal + mutations (data not shown). This may indicate that accumulation of nutrients into the - - otherwise stationary - - bacterial cells increases the ability to phenotypically express part of the EMS-induced premutations; alternatively, treatment in nutrient medium might lead to an increased binding of EMS to the cellular DNA, thereby leading to an increased dose at apparently identical exposure. That the latter is not the case is demonstrated by data summarized in Table 1: In an experiment in which both mutation induction and DNA binding after EMS treatment of stationary E. coli suspended in different media were measured, it could be shown (i) that the mutation yield-enhancing effect of F10 + 15% CS for gal + and arg + mutations is probably due to the presence of D-glucose and 1-arglnine, since these compounds alone can produce effects analogous to that of F10 + 15% CS in the respective mutation systems, and (ii) that this enhancement is not paralleled by an increase in the level of EMS-induced D N A alkylations. The influence of growth rate of the cells on mutability after EMS treatment is further demonstrated in Fig. 2, in which the
20
i IL~
400
!fl00
350
750
~d !o-
SERUM
5
500
300 /
25fl
250 i i'
/
000
200
/ /
/
751~
150
i
/
/
/
/
/
/
/
7
1o9
II
/ //
500
250
J
0
5
10
15
20 conc.
25
30
EMS (mM)
5
lfl ~
i
i
~
L
i
15
2fl
25
30
35
co~c. EMS (mM)
Fig. 1. Induction of arg + mutations by EMS in stationary cells of strain E. coil 343/113 suspended in phosphate-buffered saline (PBS) or in FI0 medium supplemented with 15% calf serum (SERUM). The experimental points represent mean values of arg + mutant-colony number per plate (3 plates in parallel) of 1 typical experiment. At the EMS-exposure concentrations used there was no decrease in cell survival, nor did the titer of the cells suspended in supplemented medium increase during the 120-min treatment period (I-2X 109/ml). Fig. 2. Induction of gal + mutations by EMS in E. coli cells of different growth phase. The data are those of I typical of several performed experiments. EMS treatment was as described in Material and Methods, i.e. 120 min at 37°C, of stationary cells (stat.); growing cells suspended in PBS immediately before the beginning of EMS treatment (log I), and growing cells suspended in fully supplemented growth medium before the beginning of EMS treatment (log II). After termination of treatment, there was no change in viable cell titer in the stationary cells, the log l-type cells had grown from 1.2 x 109 per ml to 1.7 x 109 per ml, and the log II-type cells from 1.1X 10 9 per ml to 2.6X 10 9 per ml. After treatment, the cells were washed and resuspended in such a way that inoculum per plate for the detection of gal + mutants was in all cases about I-2X 10 6 viable cells.
i n d u c t i o n of g a l + is c o m p a r e d i n stationary a n d i n growing cells; a n a l o g o u s results were o b t a i n e d with a r g + m u t a t i o n s (data n o t shown). Again, the largely e n h a n c e d m u t a t i o n i n d u c t i o n i n growing cells indicates that the a c c u m u l a t i o n of n u t r i e n t s i n b a c t e r i a increases their ability to fix a n d express E M S - i n d u c e d p r e m u t a t i o n s , i n c o n c o r d a n c e with previous results o b t a i n e d using other E. coil strains (Howell-Saxton et al., 1973; H i n c e a n d Neale, 1977). This also m a y explain why m a m m a l i a n cells which in the previous experiments were always allowed for o p t i m u m p h e n o t y p i c expression i n F10 + 15% CS, showed i n some cases higher E M S - i n d u c e d m u t a t i o n frequencies t h a n the E . coli cells. I n s u b s e q u e n t experiments, therefore, precautions
21
were taken to allow optimum phenotypic expression of premutations in both cell types, by post-treatment incubation of the cells in non-selective growth media. To assay whether further genetic endpoints in E. coil would be more sensitive to EMS mutagenesis than those tested so far, namely g a l + , MTW, and a r g + , comparative experiments were performed in which, additionally, NAU and VAU mutations were scored. The results of one typical experiment are shown in Fig. 3. They demonstrate and confirm the large variability of response of different systems to EMS mutagenesis, a phenomenon which is also found in mammalian cell lines, and show that even under optimum mutation expression conditions the g a l + (forward) mutation system is still less sensitive to EMS mutagenesis than the H G P R T - and TK - / - systems in mammalian cells (see Aaron et al., 1978, 1980; and Mohn et al., 1980). It is also clear from these results that there is no apparent correlation between the spontaneous and the EMS-induced mutation frequencies, as can be seen by comparing the g a l ÷ mutation induction data with that of the remaining systems. Furthermore, it is clear that, for EMS mutagenesis, NAL r is the most sensitive system so far described in E. coli (Howell-Saxton et al., 1973; Kondo
400
/ 250
/
/ /?MTF
/ J/ 0
2 4 6
8
10 12 14 IG 18 20 cone. EMS(mM)
Fig. 3. Induction by EMS of mutations in various genes of E. coli 3 4 3 / I 13. The experimental points represent mean values of 3 plates in parallel. EMS treatment was performed while the stationary cells were suspended in PBS for 120 min at 37°C. N o decrease in viable cell titer occurred. After treatment the cells were washed and resuspended in nutrient broth at a titer of about 1 - 2 x l0 s per ml and incubated overnight at 37°C for expression of m u t a n t phenotypes. Thereafter, the cells were again washed and upon appropriate dilution aliquots were spread over the different mutation media. Inocula of cells were about l - 2 X 106 per plate in the gal + and M T R r mutation systems, while in the N A L r and VAL r systems inoculum was about I - 2 X 10 s viable cells per plate.
TABLE 1 I N F L U E N C E OF N U T R I E N T S A D D E D K12/343/113 EMS treatment (raM)
D N A binding ( E / N ) PBS
0 10 20
0 2.12X 10-4 n.d.
DURING
T R E A T M E N T ON E M S - I N D U C E D
MUTAGENESIS
AND
DNA BINDING
gal + m u t a t i o n s X 10 6 plated cells
arg + mutations)< 10
F10+CS
PBS
FI0+CS
PBS+GLU
PBS
F10+CS
PBS+ARG
0 2.4X 10 4 n.d.
19.3 23.7 39.0
20.7 49.4 326
17.0 56.0 304
25.0 34.3 53.7
29.3 98.0 293
22.0 116 316
IN E. coil
6 plated cells
Concomitant treatment with non-radioactive and 3H-labeled EMS was performed as described in Material and Methods. Stationary cells (titer 10 l° per ml) were suspended in various media and incubated for 120 min at 37°C with either l0 or 20 m M of EMS; after treatment the cells were washed and the a m o u n t of gal + and arg + mutations and the level of D N A alkylation were determined separately. Abbreviations: E / N , ethylations per nucleotide; PBS, phosphate-buffered saline, pH 7.2; F 1 0 + C S , minimal essential medium supplemented with 15% calf serum; P B S + G L U , PBS supplemented with 1% o-glucose; P B S + A R G , PBS supplemented with 1-arginine ( 3 0 / t g per ml).
TABLE 2 I N F L U E N C E OF E X C I S I O N - R E P A I R C A P A C I T Y OF TESTER STRAINS ON E M S - I N D U C E D M U T A G E N E S I S A N D D N A K12/343/113 EMS treatment (mM)
D N A binding ( E / N ) Wild-type
0 20
0 2.12X 10 - 4
B I N D I N G IN E. coli
gal + m u t a t i o n s x 10 6
arg + m u t a t i o n s x 1 0 - 6
Cell survival (%)
uvrB
Wild-type
uvrB
Wild-type
uvrB
Wild-type
uvrB
0 2.07x 10 - 4
34.3 42.0
176 712
22.0 37.7
55.3 219
100 104.0
100 42.7
The treatment with cold and with radioactive EMS was performed as described in Material and Methods, using stationary cells (titer 101° per ml) of both strains, suspended in PBS during 120 rain at 37°C and the EMS concentrations indicated. After treatment the cells were washed and the a m o u n t of D N A binding, the cell survival, and the gal + and arg + mutation frequencies were determined.
23 et al., 1970; Todd et al., 1981; Turtocky and Ehrenberg, 1969), with a Lowest Effective Concentration (LEC) of about 1-2 mM of EMS, which is quite similar to the LEC reported for the induction of H G P R T - and TK - / - mutations in mammalian cells (Aaron et al., 1978, 1980; van Zeeland, 1978). A further point of interest is the comparison of the kinetics of mutation induction in E. coli and mammalian cells by EMS in the low dose range, i.e. a t exposure concentrations lower than 10 mM. While linear relationships between dose and genetic effect are usually observed in mammalian cells, previous results in the gal + system of E. coli 343/113 could not be used unambiguously, mainly because of large variability in mutant colony numbers per plate and relatively high spontaneous mutant frequencies (Aaron et al., 1980). This point was again addressed to in the present studies by using VALr and NAL r mutations, since variability and spontaneous frequencies are low compared to gal + mutations. Results of an experiment in which stationary E. coli 343/113 cells suspended in PBS were treated with up to 7 mM of EMS for 120 min at 37°C, and then assayed for induced NAL r and VALr mutants, are shown in Fig. 4a and b, respectively. 10 mutation plates were spread concurrently for each mutation system per experimental point. The data obtained, when examined by regression analysis show that in both systems, there is a proportionality between induced effects and EMS-exposure concentration. In fact linear regression gives a better fit for dose-effect kinetics of N A U and VALr mutations (correlation coefficients of 0.93 and 0.87) than does exponential regression (coefficients of 0.89 and 0.83, respectively); in neither system there is an indication of threshold for mutagenicity. These results again are similar to those obtained in mammalian cells. At EMS exposures higher than 10 mM, the bacterial systems tested so far exhibit mutation induction kinetics in which the increments in mutant frequencies are up to 2.5 times higher than the corresponding increments in EMS-exposure concentration, as can be seen from the results of Fig. 3 and from other data (Turtocky and Ehrenberg, 1969; Aaron et al., 1978, 1980; Todd et al., 1981). This effect might be due to a progressive saturation of an error-free DNA repair system or, alternatively, to the induction of an error-prone repair pathway. To partly test this hypothesis, EMS-induced mutation frequencies were compared: (i) in strain E. coli 343/113 which has wild-type DNA repair ability, (ii) in an excision-repair-deficient derivative (AuvrBlO1), and (iii) in a derivative deficient in post-replication repair carrying the f e c A l 3 allele. The results obtained in the arg + mutation system are shown in Fig. 5. They confirm those of Kondo et al. (1970) and Todd et al. (1981) and demonstrate that EMS-induced mutagenesis is increased in the AuvrB derivative and decreased, but not abolished, in the recA strain. The 2 repair-deficient derivatives also showed decreased resistance to EMS-induced cell kilting; while in the experiments depicted in Fig. 5, survival of wild-type E. coli 343/113 was 100% at 40 mM EMS, it was only 36% in the recA derivative (at 40 mM EMS) and 65% in the uvrB derivative (at 10 mM EMS). The overall shape of the mutation induction curve in the recA strain is consistent with proportionality between dose and effect throughout the EMS concentration range tested, but this cannot be precisely ascertained with the present experimental values.. The uvrB data strongly indicate a non-linearity of dose and
24
NALr MUTANTS PER PLATE
12@
o B
100 80 °
60 40 20 0
0
1
2
3 corqc.
4
5
EMIS
(mM)
6
7
VAILr MUTANTS PER PLATE
5~
40 30
20 10
0
o
0
g
I
I
I
I
I
I
I
1
2
3
4
5
5
7
EMS
(raM)
comc.
~"
Fig. 4. Induction of NAL r and VAL r mutations in E. coli 343/113 at relatively low EMS-exposure concentrations. The experimental points represent the absolute mutant-colony numbers per plate at the given EMS concentrations; 10 plates were spread in parallel in this experiment. EMS treatment of the stationary cells was for 120 min at 37°C in PBS, pH 7.2. After treatment the cells were washed and allowed for phenotypic expression as described previously.
effect, as did previous results with another K12 strain (Todd et al., 1981). Taken together, the present results show that uvrB- and recA-mediated repair pathways are partly involved in the avoidance or in the production of EMS-induced mutations, respectively; they also suggest that other factors may be responsible for the biphasic or exponential dose-effect kinetics observed in the wild-type and in the uvrB derivative. To determine whether the differences in EMS sensitivity shown in Fig. 5 between wild-type and uvrB strain were due to differences in DNA binding, a control experiment was performed, the results of which are shown in Table 2. Stationary
25
250
;wB
200
/
150
/
100 cA I
i
~
*
0
5
10
i
15
i
i
i
i
J
2fl
25
30
35
40
cor, c.
EMS (mM)
Fig. 5. Induction of arg + mutations by EMS in E. coil 3 4 3 / I 13 derivatives varying in D N A repair ability. The experimental points represent mean values of mutant colony numbers of 3 plates in parallel. EMS treatment was performed while stationary cells (titer about 1.-2 × l0 I° per ml) were suspended in PBS for 120 min at 37°C. Under these conditions, survival of wild-type strain was about 100% at 40 mM, that of r e c A was 36.3% at 40 m M , and that of the tmrB strain was 65.0% at i0 m M EMS. After treatment the cells were washed and resuspended in PBS in such a way that inoculum per plate for the detection of a r g + mutants was about 1-2 x l06 viable cells.
cells of the repair-proficient strain and the uvrB derivative were treated in parallel with either non-radioactive or 3H-labeled EMS in standard procedures (20 mM exposure concentration in PBS, pH 7.2, for 120 min at 37°C) and the amount of radioactivity bound to DNA, the induced mutation frequencies in the gal + and arg + systems, and cell survival determined. The uvrB derivative clearly shows decreased cell survival and increased mutability, but there is no significant difference in the amount of EMS bound to D N A when compared with the wild-type situation. This suggests that the increased mutation induction by EMS in the uvrB strain is due to a decreased ability to repair premutagenic D N A alterations subsequently to the EMS treatment, rather than an increased susceptibility of its D N A to be ethylated by EMS during the 120-min treatment period.
Conclusions These and previous experiments show that at identical EMS-exposure concentrations and under identical treatment conditions, E. coli and mammalian cells in
26 culture have similar levels of D N A ethylation in a range extending from 2.5 to 50 m M of EMS, i.e. dose values ranging from 1.7 X 10-4 to 1.5 X 10 -3 ethylations per nucleotide. The present E. coil data also show that nutrients present in the treatment medium have no influence on total D N A dose; similarly, the excision-repair-deficient background during EMS treatment does not have an influence on levels of D N A alkylation. A strong increase in EMS-induced mutation yields in the various genetic systems studied in these series of experiments ( g a l + , a r g ÷ , M T R r, N A L r, and V A U ) is observed when growth factors or nutrients, such as arginine, glucose, and calf serum, are added during EMS treatment. Under conditions in which optimum expression of premutations is allowed, certain mutation systems in E. coli 343/113, such as N A L r, VAL r, and MTW, and M T R r have similar mutation induction kinetics and the Lowest Effective Concentrations of EMS ( 1 - 2 mM) are similar to those found in m a m m a l i a n cells, with no indication of threshold values for mutagenicity. These results indicate that, at least for monofunctional alkylating agents such as EMS, mutation-induction data obtained in bacteria can be used with a certain degree of confidence for extrapolation to mammalian cell systems. It remains to be determined, however, whether the analogous responses to EMS mutagenesis and D N A binding in the present series of experiments are fortuitous or due to common premutagenic D N A changes and to similar probabilities of these D N A adducts to lead to mutations in both prokaryotic and eukaryotic cell types. Further investigations on the basis of D N A dose and involving different classes of mutagenic/carcinogenic chemicals will be required to assess the general suitability of bacterial systems in quantifying and ranking the genetic potential of chemicals.
Acknowledgements The authors would like to thank Prof. F.H. Sobels very warmly for his constant support and encouragement during this work. Financial support from the Koningin Wilhelmina Fonds, grants Nos. SG 81.89 and SG 81.92 is acknowledged. The competent technical assistance of Susan Bouter and Peter de Knijff is greatly appreciated.
References Aaron, C.S., A.A. van Zeeland, G.R. Mohn and A.T. Natarajan (1978) Molecular dosimetry of the chemical mutagen ethyl methanesulfonate iaa Escherichia coli and in V79 Chinese hamster cells, Mutation Res., 50, 419-426. Aaron, C.S., A.A. van Zeeland, G.R. Mohn, A.T. Natarajan, A.G.A.C. Knaap, A.D. Tates and B.W. Glickman (1980) Molecular dosimetry of the chemical mutagen ethyl methanesulfonate, Quantitative comparison of mutation induction in Escherichia coli, V79 Chinese hamster cells and L5178Y mouse lymphoma cells, and some cytologicalresults in vitro and in vivo, Mutation Res., 69, 201-216. Hince, T.A., and S. Neale (1977) Physiologicalmodifications of alkylating agent-induced mutagenesis, I. Effect of growth rate and repair capacity of nitromethylurea-induced mutation of Escherichia col& Mutation Res., 46, 1-I0.
27 Hollstein, M., J. McCann, F.A. Angelosanto and W.W. Nichols (1979) Short-term tests for carcinogens and mutagens, Mutation Res., 65, 133-226. Howard-Flanders, P. (1967) DNA repair, Annu. Rev. Biochem., 37, 175-200. Howell-Saxton, E., S. Zamenhof and P.J. Zamenhof (1973) Response of Escherichia coli to ethyl methanesulfonate; Influence of growth phase and repair ability on survival and mutagenesis, Mutation Res., 20, 327-337. Kato, T., and Y. Shinoura (1977) Isolation and characterization of mutants of Escherichia coli deficient in the induction of mutations by ultraviolet light, Mol. Gen. Genet., 156, 121-131. Kondo, S., H. Ichikawa, K. Iwo and T. Kato (1970) Base-change mutagenesis and prophage induction in strains of Escherichia coli with different DNA repair capacities, Genetics, 66, 187-217. Lawley, P.D. (1974) Some chemical aspects of dose-response relationships in alkylation mutagenesis, Mutation Res., 23,283-295. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 979-983. Mohn, G.R., and J. Ellenberger (1977) The use of Escherichia coil K-12-343/I 13 as a multi-purpose indicator strain in various mutagenicity testing procedures, in: B.J. Kilbey et al. (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 95-107. Mohn, G.R., A.A. van Zeeland, A.G.A.C. Knaap, B.W. Glickman, A.T. Natarajan, M. Brendel, F.J. de Serres and C.S. Aaron (1980) Quantitative molecular dosimetry of ethyl methanesulfonate (EMS) in several genetic test systems, in: K.H. Norpoth et al. (Eds.), Short-Term Test Systems for Detecting Carcinogens, Springer, Berlin, pp. 160-169. Mohn, G.R., P. Kerklaan, P. de Knijff and S. Bouter (1981) Influence of phenotypic expression lag and division delay on apparent frequencies of induced mutations in Escherichia coli KI2, Mutation Res., 91,419-425. Sobels, F.H. (1976) Some thoughts on the evaluation of environmental mutagens, Mutation Res., 38, 361-366. Sobels, F.H. (1977) Some problems associated with the testing of environmental mutagens and a perspective for studies in 'Comparative Mutagenesis', Mutation Res., 46, 245-260. Sobels, F.H. (1980) Evaluating the mutagenic potential of chemicals, The minimal battery and extrapolation problems, Arch. Toxicol., 46, 21-30. Todd, P.A., J. Brouwer and B.W. Glickman (1981) Influence of DNA-repair deficiencies on MMS- and EMS-induced mutagenesis in Escherichia coli KI2, Mutation Res., 82, 239-250. Turtocky, I., and L. Ehrenberg (1969) Reaction rates and biological action of alkylating agents, Preliminary report on bactericidal and mutagenic action in E. coil, Mutation Res., 8, 229-238. van Zeeland, A.A. (1978) Post-treatment with caffeine and the induction of gene mutations by ultraviolet irradiation and ethyl methanesulfonate in V79 Chinese hamster cells in culture, Mutation Res., 50, 145-151. van Zeeland, A.A., G.R. Mohn, C.S. Aaron, B.W. Glickman, M. Brendel, F.J. de Serres, C.Y. Hung and H.E. Brockman (1982) Molecular dosimetry of the chemical mutagen ethyl methanesulphonate, Quantitative comparison of the mutagenic potency in Neurospora crassa and Saccharomyces cerevisiae, Mutation Res., submitted.
15
Influence of experimental conditions and D N A repair ability on EMS-induced mutagenesis and D N A binding in Escherichia coli K12 Comparison With mammalian cell mutagenesis G.R. Mohn l A.A. van Zeeland 1 and B.W. Glickman 2 t Department of Radiation Genetics and Chemical Mutagenesis, The.State University of Leiden, Leiden (The Netherlands); and : Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC (U.S.A.)
(Received 15 September 1981) (Accepted 17 September 1981)
Summary In continuation of previous experiments aimed at quantitatively comparing the magnitude of ethyl methanesulfonate (EMS)-induced DNA binding and mutagenesis in bacterial and mammalian cells, the present studies were undertaken to determine the influence of various experimental conditions on EMS-induced changes in E. coli K12/343/113. The sensitivity to EMS mutagenesis in this strain was assayed for various genetic endpoints, namely gal + , MTW, NAL r, VALr and arg + . EMS mutagenesis and DNA binding in the repair-proficient wild-type was, furthermore, compared to that in derivatives deficient in excision repair ( A u v r B l O 1 ) and postreplication repair ( r e c A l 3 ) . Attempts were, finally, undertaken to determine the shape of the dose-response curve for the most sensitive genetic systems found, namely NAU and VALr mutations, at relatively low EMS-exposure conditions of 0-7 mM for 120 rain at 37°C. From the present experiments and from previous results obtained under identical EMS treatment conditions in E. coli 343/113 and in the mammalian cell lines V79 and L5178Y, the following conclusions can" be drawn: (i) At EMS exposure concentrations ranging from 2.5 to 50 mM and treatment periods of 120 min at 37°C in buffer pH 7.2, the levels of DNA alkylation at termination of the treatment are similar in E. coli and mammalian cells and increase from 1.7 x 10 -4 to 1.3 × 10 -3 ethylations per nucleotide. (ii) The presence of nutrients such as 1-arginine, o-glucose and fetal calf serum, during EMS treatment of stationary E. coli cells does not influence the level of DNA alkylation but greatly increases EMS-induced mutation Abbreviations: 3H-EMS, 1[3H]ethyl methanesulfonate; LEC, lowest effective concentration.
0027-5107/82/0000-0000/$02.75 © Elsevier Biomedical Press
16 yields, probably because of the accumulation of these nutrients into the cells, thereby leading to increased capability of genetic fixation and/or phenotypic expression of the premutational DNA changes. (iii) The mammalian cells are more sensitive to the killing action of EMS than E. coli repair-proficient cells (survival at 10 mM of 12 and 100%, respectively), which indicates that the genetic and/or physiologic target for inactivation of colony-forming ability is larger in the mammalian cells than in bacteria, or alternatively that these cells have lower ability to repair EMS-induced DNA lesions. (iv) The 2 new mutation systems tested in the present series of experiments, namely N A U and VALr mutations appear to be quite comparable to those tested in mammalian cells, such as thioguanine resistance ( H G P R T - ) and BUdR resistance ( T K - / - ) , in the sense that at low doses, i.e. EMS-exposure concentrations of up to 7 mM for 120 min at 37°C in buffer, pH 7.2, the induction kinetics in all systems are compatible with a linear relationship between dose and mutation frequency, with no indication of a threshold. In the case of N A U in E. coli and H G P R T - in V79 mammalian cells the Lowest Effective Concentrations of EMS are 1-2 mM for treatment periods of 120 min at 37°C. It remains to be determined if the same EMS-induced DNA adducts are responsible in both cell types for the genetic effects observed; further comparative experiments are also required to assess the general suitability of bacteria in quantifying mutagenic and tumor-initiating potency of chemicals.
Sobels (1976, 1977, 1980) has identified and described several phases in detecting, quantifying, and extrapolating to man the mutagenic and tumor-initiating potency of environmental chemicals, by making use of different genetic endpoints and test organisms ranging from microbes to mammals. Extensive comparable studies have shown that, qualitatively, there is a high degree of parallelism between the mutagenicity of chemicals in microorganisms and their mutagenic and carcinogenic effects in animals (McCann et al., 1975; Hollstein et al., 1979), depending on the reactivitY class of chemicals considered. Because reports about newly detected mutagens in the environment accumulate, based on results obtained with microorganisms, and because a substantial lag period remains for obtaining quantitative mutagenicity a n d / o r carcinogenicity results in experimental animals, methodologies have to be developed for a more quantitative and representative use of these microbial systems. The question then to be answered is which genetic endpoints and which genetic backgrounds are best suited for an accurate and quantitative prediction of genetic efficiency. Of special interest in this context is the determination of the extent to which the response to chemical mutagens is influenced by intrinsic cellular factors, such as chromosome structure and DNA content per cell, and by metabolic and physiologic differences between test organisms. This can be done by performing quantitative comparisons of the magnitude of genetic effects induced in various organisms by known mutagens/carcinogens. Such a comparison necessitates a precise determination of the actual dose at the target cell or the target molecule, e.g. DNA. The reason for carrying out dose measurements is that one cannot, a priori,
17 assume that identical exposure to a chemical will lead to identical dose at the DNA level. Instead, one expects that penetration and metabolism in general will influence the extent of the reaction with and the binding to DNA. Previous experiments were performed to quantitatively compare in microorganisms and in mammalian cell lines the magnitude of genetic effects induced by the alkylating agent ethyl methanesulfonate (EMS) on the basis of DNA dose, measured as ethylation per nucleotide (Aaron et al., 1978, 1980; Mohn et al., 1980; van Zeeland et al., 1982). They showed that standardization of test methodology and DNA dose measurements are possible with prokaryotic and eukaryotic cells. They also showed that, on the basis of equal DNA dose, the variation of EMS-induced mutagenic effects within vastly differing species (E. coli, Saccharomyces cerevisiae, Neurospora crassa, Chinese hamster cells, and mouse lymphoma cells) was not greater than the variation between different genetic endpoints (forward mutations) within one species or one cdl line. Some quantitative differences between the microbes (of both prokaryotic and eukaryotic cell type) and the mammalian cell lines remained, however, in the following points: (i) the wild-type microorganisms are more resistant than the mammalian cells to EMS-induced inactivation; (ii) the mutation systems studied in the mammalian cell lines (thioguanine resistance, BUdR resistance, ouabain resistance) show linear EMS-induced kinetics, whereas the microbial systems usually have biphasic or exponential kinetics with exponents of up to 2.6; and (iii) at low exposure concentrations the frequencies of mutations induced in the microbial systems are lower than in the mammalian cell lines. These differences are not due to differences in dose to the DNA, since dosimetry measurements have shown that at identical EMS exposures (from 2.5 to 50 mM for 120 min at 37°C in buffer, pH 7.2) the level of ethylation per nucleotide is similar in microbial and in mammalian cells (Aaron et al., 1978, 1980; van Zeeland et al., 1982). The experiments reported in this paper were designed to gain further insight into the role of factors responsible for the above-mentioned differences by varying, in a bacterial indicator, the conditions of EMS treatment, the genetic endpoints, and the DNA repair ability of the tester strain. It has been shown previously by using EMS and other alkylating agents that the growing state of the cells has a profound influence on mutagen sensitivity (Howell-Saxton et al., 1973; Hince and Neale, 1977) as has the DNA repair ability of these cells (Kondo et al., 1970; Lawley, 1974; Todd et al., 1981). The influence of these parameters and the mutagenic response of additional genetic endpoints were determined in a bacterial strain previously used in the comparative studies mentioned above, namely E. coli K12 343/113. After conditions for optimal expression of EMS-induced premutations were assessed, attempts were made to determine the linearity or non-linearity of the dose-response at low EMS exposures in these bacterial cells. As in the previous experiments the dose to the DNA was measured with 3H-labeled EMS and expressed as ethylation per nucleotide.
18 Material and methods
Chemicals 1[ 3 H]Ethyl methanesulfonate (3H-EMS) was obtained from New England Nuclear at a specific activity of 4.8 Ci/mmole dissolved in diethyl ether. Unlabeled EMS was obtained from Eastman Chemical. 10 mCi of 3H-EMS in 10 ml diethyl ether were mixed with 2-4 ml phosphate-buffered saline (PBS), pH 7.2, and the ether was evaporated under N 2. The amount of 3H-EMS left was determined by diluting 20q~l samples and counting in a liquid scintillation counter. Unlabeled EMS was added to a final concentration of 100 mM, resulting in a specific activity of 10-30 m C i / mmole. Samples of this stock solution were added immediately to bacterial cell suspensions for detection of the ethylation level in DNA. For the detection o f mutagenic effects, stock solutions of unlabeled EMS were prepared in PBS. immediately before the beginning of the experiment. Growth and suspension media Phosphate-buffered saline (PBS), pH 7.2, nutrient broth (NB), complete agar (COA), and the agar media for detection of MTRr, gal +, and arg + mutants were prepared as described previously; the composition of agar media for scoring valineresistant (VAU) and nalidixic acid-resistant (NAU) mutants was the same as MTW except that, instead of 5-methyltryptophan, VAU contained 10 #g 1-valine per ml medium and NAU contained 15 /~g nalidixic acid per ml medium (Mohn et al., 1981). In some mutation-induction experiments the bacteria were treated with EMS while suspended in various media, namely, PBS, PBS supplemented with 1% D-glucose, PBS supplemented with 30 /~g 1-arginine per ml, and the synthetic medium F10 supplemented with 15% calf serum, in which usually mammalian cells are grown before harvest and EMS treatment (van Zeeland, 1978). Bacterial strains For detection of various mutation endpoints, strain E. coli 343//113 was used (Mohn and Ellenberger, 1977; Mohn et al., 1981). A deletion including the uvrB gene was introduced by Pl-mediated cotransduction with chl and bio (Kato and Shinoura, 1977) and the recA13 allele was introduced after transduction and selection for sorbitol utilization (Howard-Flanders, 1967). Mutagenicity tests Standard EMS treatment was performed as described previously (Aaron et al., 1978, 1980). Briefly, suspensions of stationary-phase cells of E. coli 343/113 or its repair-deficient derivatives were exposed to various concentrations of EMS in a final volume of 2.5 ml PBS (pH 7.2) for 120 min at 37°C in the dark and under rotary shaking. After treatment the cells were washed to remove excess mutagen and, upon appropriate dilution, were plated on various agar media for the determination of cell survival and mutation frequencies. Since some mutation types, e.g. NAU, VAU, and MTR r, require post-treatment incubation in non-selective growth medium for opti-
19 mum expression of induced premutations, experiments involving these mutations included dilution of EMS-treated and washed cells into nutrient broth (final titer 1-2 × 108 cells per ml) and overnight incubation in the dark at 37°C, thereby leading to a mean number of 3-4 cell divisions. In some experiments the cells were suspended in media supplemented with growth factors during EMS treatment, as indicated later in the text. In those experiments involving treatment of growing cells, log-phase cells were obtained by diluting, 120 min before EMS treatment, stationary-phase cells into nutrient broth and incubating at 37°C; slaortly before treatment the cells were washed and resuspended either in buffer, or in supplemented minimal medium, and further handled as described before. DNA-binding experiments Treatment of E. coli 343/113 with 3H-labeled EMS was performed in a manner
identical to and in parallel with the mutation-induction experiments. After the completion of the treatment, the cells were washed and incubated with lysozyme, then lysed with sarcosyl and incubated with proteinase K. The DNA was isolated by CsCI density-gradient centrifugation and the amount of radio-labeled mutagen bound to DNA was measured as described previously (Aaron et al., 1978, 1980).
Results and discussion
A number of experimental conditions during EMS treatment was varied to determine their influence on induced mutant yields. First, since mammalian cells were grown in the previous comparative experiments in F10 medium+ 15% calf serum (CS), EMS-induced mutation frequencies were compared in the present studies while E. coli 343/113 cells were suspended in PBS and in F10 + 15% CS, respectively. The results presented in Fig. 1 show that E. coil cells suspended in F10 + 15% CS exhibit higher EMS-induced arg + mutation frequencies than the cells suspended in PBS. Analogous results were obtained with gal + mutations (data not shown). This may indicate that accumulation of nutrients into the - - otherwise stationary - - bacterial cells increases the ability to phenotypically express part of the EMS-induced premutations; alternatively, treatment in nutrient medium might lead to an increased binding of EMS to the cellular DNA, thereby leading to an increased dose at apparently identical exposure. That the latter is not the case is demonstrated by data summarized in Table 1: In an experiment in which both mutation induction and DNA binding after EMS treatment of stationary E. coli suspended in different media were measured, it could be shown (i) that the mutation yield-enhancing effect of F10 + 15% CS for gal + and arg + mutations is probably due to the presence of D-glucose and 1-arglnine, since these compounds alone can produce effects analogous to that of F10 + 15% CS in the respective mutation systems, and (ii) that this enhancement is not paralleled by an increase in the level of EMS-induced D N A alkylations. The influence of growth rate of the cells on mutability after EMS treatment is further demonstrated in Fig. 2, in which the
20
i IL~
400
!fl00
350
750
~d !o-
SERUM
5
500
300 /
25fl
250 i i'
/
000
200
/ /
/
751~
150
i
/
/
/
/
/
/
/
7
1o9
II
/ //
500
250
J
0
5
10
15
20 conc.
25
30
EMS (mM)
5
lfl ~
i
i
~
L
i
15
2fl
25
30
35
co~c. EMS (mM)
Fig. 1. Induction of arg + mutations by EMS in stationary cells of strain E. coil 343/113 suspended in phosphate-buffered saline (PBS) or in FI0 medium supplemented with 15% calf serum (SERUM). The experimental points represent mean values of arg + mutant-colony number per plate (3 plates in parallel) of 1 typical experiment. At the EMS-exposure concentrations used there was no decrease in cell survival, nor did the titer of the cells suspended in supplemented medium increase during the 120-min treatment period (I-2X 109/ml). Fig. 2. Induction of gal + mutations by EMS in E. coli cells of different growth phase. The data are those of I typical of several performed experiments. EMS treatment was as described in Material and Methods, i.e. 120 min at 37°C, of stationary cells (stat.); growing cells suspended in PBS immediately before the beginning of EMS treatment (log I), and growing cells suspended in fully supplemented growth medium before the beginning of EMS treatment (log II). After termination of treatment, there was no change in viable cell titer in the stationary cells, the log l-type cells had grown from 1.2 x 109 per ml to 1.7 x 109 per ml, and the log II-type cells from 1.1X 10 9 per ml to 2.6X 10 9 per ml. After treatment, the cells were washed and resuspended in such a way that inoculum per plate for the detection of gal + mutants was in all cases about I-2X 10 6 viable cells.
i n d u c t i o n of g a l + is c o m p a r e d i n stationary a n d i n growing cells; a n a l o g o u s results were o b t a i n e d with a r g + m u t a t i o n s (data n o t shown). Again, the largely e n h a n c e d m u t a t i o n i n d u c t i o n i n growing cells indicates that the a c c u m u l a t i o n of n u t r i e n t s i n b a c t e r i a increases their ability to fix a n d express E M S - i n d u c e d p r e m u t a t i o n s , i n c o n c o r d a n c e with previous results o b t a i n e d using other E. coil strains (Howell-Saxton et al., 1973; H i n c e a n d Neale, 1977). This also m a y explain why m a m m a l i a n cells which in the previous experiments were always allowed for o p t i m u m p h e n o t y p i c expression i n F10 + 15% CS, showed i n some cases higher E M S - i n d u c e d m u t a t i o n frequencies t h a n the E . coli cells. I n s u b s e q u e n t experiments, therefore, precautions
21
were taken to allow optimum phenotypic expression of premutations in both cell types, by post-treatment incubation of the cells in non-selective growth media. To assay whether further genetic endpoints in E. coil would be more sensitive to EMS mutagenesis than those tested so far, namely g a l + , MTW, and a r g + , comparative experiments were performed in which, additionally, NAU and VAU mutations were scored. The results of one typical experiment are shown in Fig. 3. They demonstrate and confirm the large variability of response of different systems to EMS mutagenesis, a phenomenon which is also found in mammalian cell lines, and show that even under optimum mutation expression conditions the g a l + (forward) mutation system is still less sensitive to EMS mutagenesis than the H G P R T - and TK - / - systems in mammalian cells (see Aaron et al., 1978, 1980; and Mohn et al., 1980). It is also clear from these results that there is no apparent correlation between the spontaneous and the EMS-induced mutation frequencies, as can be seen by comparing the g a l ÷ mutation induction data with that of the remaining systems. Furthermore, it is clear that, for EMS mutagenesis, NAL r is the most sensitive system so far described in E. coli (Howell-Saxton et al., 1973; Kondo
400
/ 250
/
/ /?MTF
/ J/ 0
2 4 6
8
10 12 14 IG 18 20 cone. EMS(mM)
Fig. 3. Induction by EMS of mutations in various genes of E. coli 3 4 3 / I 13. The experimental points represent mean values of 3 plates in parallel. EMS treatment was performed while the stationary cells were suspended in PBS for 120 min at 37°C. N o decrease in viable cell titer occurred. After treatment the cells were washed and resuspended in nutrient broth at a titer of about 1 - 2 x l0 s per ml and incubated overnight at 37°C for expression of m u t a n t phenotypes. Thereafter, the cells were again washed and upon appropriate dilution aliquots were spread over the different mutation media. Inocula of cells were about l - 2 X 106 per plate in the gal + and M T R r mutation systems, while in the N A L r and VAL r systems inoculum was about I - 2 X 10 s viable cells per plate.
TABLE 1 I N F L U E N C E OF N U T R I E N T S A D D E D K12/343/113 EMS treatment (raM)
D N A binding ( E / N ) PBS
0 10 20
0 2.12X 10-4 n.d.
DURING
T R E A T M E N T ON E M S - I N D U C E D
MUTAGENESIS
AND
DNA BINDING
gal + m u t a t i o n s X 10 6 plated cells
arg + mutations)< 10
F10+CS
PBS
FI0+CS
PBS+GLU
PBS
F10+CS
PBS+ARG
0 2.4X 10 4 n.d.
19.3 23.7 39.0
20.7 49.4 326
17.0 56.0 304
25.0 34.3 53.7
29.3 98.0 293
22.0 116 316
IN E. coil
6 plated cells
Concomitant treatment with non-radioactive and 3H-labeled EMS was performed as described in Material and Methods. Stationary cells (titer 10 l° per ml) were suspended in various media and incubated for 120 min at 37°C with either l0 or 20 m M of EMS; after treatment the cells were washed and the a m o u n t of gal + and arg + mutations and the level of D N A alkylation were determined separately. Abbreviations: E / N , ethylations per nucleotide; PBS, phosphate-buffered saline, pH 7.2; F 1 0 + C S , minimal essential medium supplemented with 15% calf serum; P B S + G L U , PBS supplemented with 1% o-glucose; P B S + A R G , PBS supplemented with 1-arginine ( 3 0 / t g per ml).
TABLE 2 I N F L U E N C E OF E X C I S I O N - R E P A I R C A P A C I T Y OF TESTER STRAINS ON E M S - I N D U C E D M U T A G E N E S I S A N D D N A K12/343/113 EMS treatment (mM)
D N A binding ( E / N ) Wild-type
0 20
0 2.12X 10 - 4
B I N D I N G IN E. coli
gal + m u t a t i o n s x 10 6
arg + m u t a t i o n s x 1 0 - 6
Cell survival (%)
uvrB
Wild-type
uvrB
Wild-type
uvrB
Wild-type
uvrB
0 2.07x 10 - 4
34.3 42.0
176 712
22.0 37.7
55.3 219
100 104.0
100 42.7
The treatment with cold and with radioactive EMS was performed as described in Material and Methods, using stationary cells (titer 101° per ml) of both strains, suspended in PBS during 120 rain at 37°C and the EMS concentrations indicated. After treatment the cells were washed and the a m o u n t of D N A binding, the cell survival, and the gal + and arg + mutation frequencies were determined.
23 et al., 1970; Todd et al., 1981; Turtocky and Ehrenberg, 1969), with a Lowest Effective Concentration (LEC) of about 1-2 mM of EMS, which is quite similar to the LEC reported for the induction of H G P R T - and TK - / - mutations in mammalian cells (Aaron et al., 1978, 1980; van Zeeland, 1978). A further point of interest is the comparison of the kinetics of mutation induction in E. coli and mammalian cells by EMS in the low dose range, i.e. a t exposure concentrations lower than 10 mM. While linear relationships between dose and genetic effect are usually observed in mammalian cells, previous results in the gal + system of E. coli 343/113 could not be used unambiguously, mainly because of large variability in mutant colony numbers per plate and relatively high spontaneous mutant frequencies (Aaron et al., 1980). This point was again addressed to in the present studies by using VALr and NAL r mutations, since variability and spontaneous frequencies are low compared to gal + mutations. Results of an experiment in which stationary E. coli 343/113 cells suspended in PBS were treated with up to 7 mM of EMS for 120 min at 37°C, and then assayed for induced NAL r and VALr mutants, are shown in Fig. 4a and b, respectively. 10 mutation plates were spread concurrently for each mutation system per experimental point. The data obtained, when examined by regression analysis show that in both systems, there is a proportionality between induced effects and EMS-exposure concentration. In fact linear regression gives a better fit for dose-effect kinetics of N A U and VALr mutations (correlation coefficients of 0.93 and 0.87) than does exponential regression (coefficients of 0.89 and 0.83, respectively); in neither system there is an indication of threshold for mutagenicity. These results again are similar to those obtained in mammalian cells. At EMS exposures higher than 10 mM, the bacterial systems tested so far exhibit mutation induction kinetics in which the increments in mutant frequencies are up to 2.5 times higher than the corresponding increments in EMS-exposure concentration, as can be seen from the results of Fig. 3 and from other data (Turtocky and Ehrenberg, 1969; Aaron et al., 1978, 1980; Todd et al., 1981). This effect might be due to a progressive saturation of an error-free DNA repair system or, alternatively, to the induction of an error-prone repair pathway. To partly test this hypothesis, EMS-induced mutation frequencies were compared: (i) in strain E. coli 343/113 which has wild-type DNA repair ability, (ii) in an excision-repair-deficient derivative (AuvrBlO1), and (iii) in a derivative deficient in post-replication repair carrying the f e c A l 3 allele. The results obtained in the arg + mutation system are shown in Fig. 5. They confirm those of Kondo et al. (1970) and Todd et al. (1981) and demonstrate that EMS-induced mutagenesis is increased in the AuvrB derivative and decreased, but not abolished, in the recA strain. The 2 repair-deficient derivatives also showed decreased resistance to EMS-induced cell kilting; while in the experiments depicted in Fig. 5, survival of wild-type E. coli 343/113 was 100% at 40 mM EMS, it was only 36% in the recA derivative (at 40 mM EMS) and 65% in the uvrB derivative (at 10 mM EMS). The overall shape of the mutation induction curve in the recA strain is consistent with proportionality between dose and effect throughout the EMS concentration range tested, but this cannot be precisely ascertained with the present experimental values.. The uvrB data strongly indicate a non-linearity of dose and
24
NALr MUTANTS PER PLATE
12@
o B
100 80 °
60 40 20 0
0
1
2
3 corqc.
4
5
EMIS
(mM)
6
7
VAILr MUTANTS PER PLATE
5~
40 30
20 10
0
o
0
g
I
I
I
I
I
I
I
1
2
3
4
5
5
7
EMS
(raM)
comc.
~"
Fig. 4. Induction of NAL r and VAL r mutations in E. coli 343/113 at relatively low EMS-exposure concentrations. The experimental points represent the absolute mutant-colony numbers per plate at the given EMS concentrations; 10 plates were spread in parallel in this experiment. EMS treatment of the stationary cells was for 120 min at 37°C in PBS, pH 7.2. After treatment the cells were washed and allowed for phenotypic expression as described previously.
effect, as did previous results with another K12 strain (Todd et al., 1981). Taken together, the present results show that uvrB- and recA-mediated repair pathways are partly involved in the avoidance or in the production of EMS-induced mutations, respectively; they also suggest that other factors may be responsible for the biphasic or exponential dose-effect kinetics observed in the wild-type and in the uvrB derivative. To determine whether the differences in EMS sensitivity shown in Fig. 5 between wild-type and uvrB strain were due to differences in DNA binding, a control experiment was performed, the results of which are shown in Table 2. Stationary
25
250
;wB
200
/
150
/
100 cA I
i
~
*
0
5
10
i
15
i
i
i
i
J
2fl
25
30
35
40
cor, c.
EMS (mM)
Fig. 5. Induction of arg + mutations by EMS in E. coil 3 4 3 / I 13 derivatives varying in D N A repair ability. The experimental points represent mean values of mutant colony numbers of 3 plates in parallel. EMS treatment was performed while stationary cells (titer about 1.-2 × l0 I° per ml) were suspended in PBS for 120 min at 37°C. Under these conditions, survival of wild-type strain was about 100% at 40 mM, that of r e c A was 36.3% at 40 m M , and that of the tmrB strain was 65.0% at i0 m M EMS. After treatment the cells were washed and resuspended in PBS in such a way that inoculum per plate for the detection of a r g + mutants was about 1-2 x l06 viable cells.
cells of the repair-proficient strain and the uvrB derivative were treated in parallel with either non-radioactive or 3H-labeled EMS in standard procedures (20 mM exposure concentration in PBS, pH 7.2, for 120 min at 37°C) and the amount of radioactivity bound to DNA, the induced mutation frequencies in the gal + and arg + systems, and cell survival determined. The uvrB derivative clearly shows decreased cell survival and increased mutability, but there is no significant difference in the amount of EMS bound to D N A when compared with the wild-type situation. This suggests that the increased mutation induction by EMS in the uvrB strain is due to a decreased ability to repair premutagenic D N A alterations subsequently to the EMS treatment, rather than an increased susceptibility of its D N A to be ethylated by EMS during the 120-min treatment period.
Conclusions These and previous experiments show that at identical EMS-exposure concentrations and under identical treatment conditions, E. coli and mammalian cells in
26 culture have similar levels of D N A ethylation in a range extending from 2.5 to 50 m M of EMS, i.e. dose values ranging from 1.7 X 10-4 to 1.5 X 10 -3 ethylations per nucleotide. The present E. coil data also show that nutrients present in the treatment medium have no influence on total D N A dose; similarly, the excision-repair-deficient background during EMS treatment does not have an influence on levels of D N A alkylation. A strong increase in EMS-induced mutation yields in the various genetic systems studied in these series of experiments ( g a l + , a r g ÷ , M T R r, N A L r, and V A U ) is observed when growth factors or nutrients, such as arginine, glucose, and calf serum, are added during EMS treatment. Under conditions in which optimum expression of premutations is allowed, certain mutation systems in E. coli 343/113, such as N A L r, VAL r, and MTW, and M T R r have similar mutation induction kinetics and the Lowest Effective Concentrations of EMS ( 1 - 2 mM) are similar to those found in m a m m a l i a n cells, with no indication of threshold values for mutagenicity. These results indicate that, at least for monofunctional alkylating agents such as EMS, mutation-induction data obtained in bacteria can be used with a certain degree of confidence for extrapolation to mammalian cell systems. It remains to be determined, however, whether the analogous responses to EMS mutagenesis and D N A binding in the present series of experiments are fortuitous or due to common premutagenic D N A changes and to similar probabilities of these D N A adducts to lead to mutations in both prokaryotic and eukaryotic cell types. Further investigations on the basis of D N A dose and involving different classes of mutagenic/carcinogenic chemicals will be required to assess the general suitability of bacterial systems in quantifying and ranking the genetic potential of chemicals.
Acknowledgements The authors would like to thank Prof. F.H. Sobels very warmly for his constant support and encouragement during this work. Financial support from the Koningin Wilhelmina Fonds, grants Nos. SG 81.89 and SG 81.92 is acknowledged. The competent technical assistance of Susan Bouter and Peter de Knijff is greatly appreciated.
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