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Mutagenicity testing experiments with the Cobas Bact
Mutation Research, 172 (1986) 1 9
1
Elsevier MTR08625
Mutagenicity testing experiments with the Cobas Bact Elmar Gocke and Martin Schiapbach Biological Pharmaeeutical Research Department, F. Hoffmann-La Roche and Co., Ltd.. CH-4002 Basel (Switzerland)
(Received 3 December 1985) (Revision received 4 April 1986) (Accepted 17 April 1986)
Summary The feasibility of mutagenicity assays with the Cobas Bact Automatic analyser was explored using selected model mutagens. The reduction of the latency period (the period until the growth of the mutant cells becomes optically measurable) was found to be a valid measure for the mutagenic activity of strong mutagens. For weaker mutagens an evaluation analogous to the fluctuation test seemed the more appropriate approach. The influence of various variables, such as concentration of histidine, size of inoculum, medium composition and $9 concentration, is described. Adaptation of the Cobas Bact system to the differential growth inhibition test is also mentioned.
During the last decade bacterial mutagenicity tests have been more and more widely used as screening tests for the detection of chemicals which might pose a threat to the genetic material of man. The Salmonella/microsome assay (Ames test) (Maron and Ames, 1983; Hollstein et al., 1979; de Serres and Ashby, 1981) has been especially successful as a short-term alternative to the animal test for potential carcinogenicity. Although the correlation between Ames test results and animal carcinogenicity experiments depends largely on the class of substances being tested (e.g. Bartsch et al., 1980; McCann and Ames, 1976), the test has nevertheless filled a vacuum stemming from the need to assess the genotoxicity of an enormous number of newly synthesized chemicals as well as the identification of potentially cancer causing substances in our everyday diet. The Salmonella test is usually performed as an agar plate incorporation assay but an incubation of the cells in liquid medium during mutagen exposure is often advantageous (Matsushima et
al., 1980; de Serres and Shelby, 1979). Complete performance in liquid (as fluctuation test) has been advocated for specific conditions (Hubbard et al., 1981: Green et al., 1977; Hollstein et al., 1979). More recently the automation of the measurement of cell growth in suspension (Arni, 1985; Falck et al. 1985; Arni et al., 1985) has opened possibilities to reduce further the manual workload of the genetic toxicology laboratory. The Cobas Bact Analyser, originally developed for standard microbiological procedures such as susceptibility testing or pathogen identification seems well suited for routine microbial mutagenicity testing. However, as the sensitivity of the Ames test against only small variations of protocol is extensively documented (e.g. Bridges et al., 1981; Seiler, 1983), the equivalence of the results of the Cobas Bact system with the standard plate tests needs to be verified. In this report we describe experiments with selected model mutagens which are performed to explore the advantages and limitations of the test
0165-1218/86/$03.50 ~v;1986 Elsevier Science Publishers B.V. (Biomedical Division)
system in comparison to the plate assay. Adaptation of the Cobas Bact to other mutagenicity tests is also mentioned. Material and methods
Bacteria The bacterial strains were originally obtained from B.N. Ames and H.B. Maruyama. Subcultures are stored in liquid nitrogen. Overnight cultures are inoculated from master plates or from stock cultures kept at - 8 0 ° C . Strain identity is checked regularly. Media For overnight culture the cells were grown in nutrient broth (Difco). In the Cobas Bact rotors Vogel-Bonner medium, supplemented with 0.12 /xg/ml biotin and histidine, was used. The plateau value of the initial growth is dependent on the amount of histidine. At a concentration of 0.1 ~ g / m l the optical density increases by about 0.02-0.03 and at 0.8 ~ g / m l by about 0.2-0.3. This is about the maximum which can be used because at higher concentrations the mutant growth will become hidden by the growth of the non-mutant cells. The concentration of histidine is indicated in the figure legends. Glucose concentration was 0.8%. Addition of amino acids other than histidine (Arimoto et al., 1981) increased the growth velocity of the bacteria and can be used to shorten the test period. In the experiments shown here, except the one shown in Fig. 1, the medium was not supplemented with amino acids. Metabolic activation. $9 from phenobarbitone//~-naphthoflavone induced male F~llinsdorf albino rats was prepared by standard procedures (Maron and Ames, 1983). The amounts of $9 added to the cultures are detailed in the figure legends. Mutagens. Mitomycin C and Na-azide were obtained from Serva, 2-aminoanthracene from Aldrich. Dinitropyrene (a mixture of the isomers) was a gift of K. Eckhardt. IQ (2-amino-3-methylimidazo[4,5-f]quinoline) was a gift of D. Wild. Description of the Cobas Bact The Cobas Bact Automatic Analyser is a tabletop machine developed by Diagnostica Roche for
standard microbiological tests such as susceptibility testing and pathogen identification. The unit culture container is a plastic rotor with 16 peripheral measuring compartments which are connected to a central well via a circuitous path. The cell suspension is pipetted into the center of the rotor and the test substances are pipetted directly into the measuring compartments. After sealing, the rotor is transferred to the Cobas Bact and the cell suspension is injected into the measuring compartments by an initial 3000 rpm centrifugation. Determination of the optical density is made every 20 min or at predetermined longer intervals during a low speed spin (500 rpm), which presses the fluid between two clear plastic windows. This ensures that surface effects do not disturb the measurement. Between the O.D. determinations the rotor is deposited in an incubator on a continuously rotating paternoster elevator. Incubation temperature, wavelength of the O.D. measurement, centrifugation speeds, shaking periods, etc., can be selected by the operator. The machine holds up to 50 rotors which can be run after different programs. We used the internal computer to collect and print the data. Additionally an external computer can be connected. In this report we have not attempted to calculate the number of induced mutants from the reduction of the latency period. We feel that the variability of the time when mutants are able to start growing as well as possible differences of the doubling times make such a calculation hazardous and generate deceptions. Likewise we have not replaced the O.D. values in the figures with calculated cell numbers. The turbidity of suspensions is not only a function of the cell density but also of the size and form of the cells which might vary depending on the growth status and toxicity of the test compound. Since quantification of the mutagenic effects does not depend on such calculated numbers, the usefulness of the machine is not at stake.
Experimental procedure The test substances, dissolved in appropriate solvents, were pipetted into the measuring compartments and the Vogel-Bonner medium, biotin, histidine, $9 mix and bacteria were pipetted into the central well. The rotor was completely sealed and transferred into the machine. The initial
centrifugation distributed the cell suspension into the compartments containing the mutagen. Usually it was sufficient to measure the optical density about every 3 h. After the end of the run, growth data for each culture were printed. Growth curves can also be plotted. As detailed in the figure legends, each experiment was carried out with some modification of this procedure. The latency period is defined as the incubation period until the culture reaches an O.D. value of 0.3. Results and discussion
Occurrence of a mutation such as reversion from histidine auxotrophy to prototrophy can be detected by the formation of a visible colony on selective semi-solid medium or by the development of visible turbidity in selective liquid medium. While on the agar plate two or more independent mutations can be scored as separate colonies, the question of whether more than one mutation has occurred in the liquid assay cannot be answered a priori. In the fluctuation test this problem of quantitation is solved by limiting the growth of the inoculum to values where spontaneous reversions occur only occasionally, i.e. in only a few tubes. Thus an increase of the mutant frequency is determined by an increase of the number of turbid tubes. Measurement of the optical density needs to be performed only once after a predetermined optimal incubation period, usually a few days. A somewhat different approach is made possible by the use of machines able to monitor periodically the optical density of the bacterial cultures. In this way the kinetic growth curve of the culture c a r be determined. An increase of the number of mutants per culture vessel should cause a reduction of the period until the prototrophic growth becomes measurable. Obviously this method (in the following called the latency period reduction approach) works if the number of mutants per tube is larger than 1. Compared to the fluctuation test the cell numbers per tube can be higher and fewer tubes are needed. Furthermore the growth curves should give further information concerning toxicity or presence of growth enhancing ingredients.
To verify these predictions we performed reconstruction experiments by adding varying numbers of established his + revertants to the inoculum of his- cells. As can be seen in Fig. 1, the culture which was not spiked did not show prototrophic growth while the latency period of the spiked cultures decreased logarithmically with increasing numbers of mutant cells added: an increase by the factor 2 translated itself into a reduction of the latency period by about 45 min. This therefore was the doubling time of the his + cells in the Arimoto medium. We then wanted to test whether newly induced mutants grow similarly to the established mutants. Fig. 2 shows an experiment comparing the growth of reconstructed cultures to the growth of cultures exposed to the mutagen dinitropyrene. At appropriate times aliquots of the cultures were taken out of the Cobas Bact and plated on VB medium to determine the number of his + cells or on NB medium to determine the total number of cells. It is obvious that the optical densities of the cultures (upper panel) increased parallel to the increase of the total number of cells (lower panel; broken lines) and the secondary (prototrophic) growth became optically measurable at those times when the densities of the his + cells (lower panel; solid lines) approached the densities of the nonmutated cells. From the growth curves one can determine that the plateau value of the his cells (at the applied histidine concentration of 0.8 ~ g / m l ) was about 1.5 × l0 s cells per ml or 5 × 107 cells/cuvette. When compared to the growth of the established mutant cells, the dinitropyrene induced his + cells grew quite similarly (curves a, b, c versus curves 1, 2; solid lines), except for a slower growth at around 2 8 h of incubation. This delay might have been due to a division delay of the not yet fully expressed mutant cells or more likely to an artefact resulting from formation of mutants on the agar plates after plating. The higher dinitropyrene concentration (curve 2) induced about 10-fold more mutants than the lower concentration (curve 1). This fits well to the observation that the time differences of the latency periods between the mutagen-treated cultures are approximately the same as between the reconstituted cultures because the numbers of the inoculated
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Fig. 1. Reconstitution experiment. To the cultures of TA1535 ( - 3 x 1 0 ~ cells/cuvette) the indicated numbers of cells of an established revertant of TA1535 were added. The his + cell numbers were calculated from a titer determination by plating. Vogel-Bonner minimal medium supplemented with 0.12 /~g/ml biotin and 1 m g / m l amino acids (total) other than histidine (Arimoto et al., 1981 ) was used. This medium allows a considerably quicker growth of the cells as compared to non-supplemented VB medium, which is demonstrated by the shorter latency periods of the cultures when compared to Fig. 2.
his + cells were chosen to differ by a factor of 10. Taking the reconstitution experiments together, one can conclude that the theoretical predictions are met very well. The sensitivity of the latency period reduction approach is demonstrated in Fig. 3. The potent mutagen 2-amino-3-methylimidazo[4,5]quinoline (IQ) was tested in TA1538 both in the presence and absence of $9. As little as 15 pg per culture (50 p g / m l ) gave a well measurable effect in the presence of $9. The latency period was the shortest around 5-50 n g / m l . At 500 n g / m l toxic effects prolong the latency period and growth was
almost completely abolished at 5/~g/ml. The toxic effect was also seen by the reduced initial growth. In the absence of metabolic activation IQ showed mutagenic effects at the comparatively high concentration of 5 /~g/ml. This is in line with a relatively weak direct mutagenicity of IQ in the plate incorporation assay as recently reported by Wild et al. (1985). The dependency of the mutagenic effect of 2-aminoanthracene (2-AA) on the concentration of $9 is shown in Fig. 4. Two doses of the promutagen were used. Without metabolic activation no mutagenic effect was seen. In the presence
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Fig. 2. Titer determination of the cells growing in the cuvettes. Cultures of TA98 ( -107 cells/ml) were either spiked with 15, 150 or 1500 established revertant cells of TA98 per ml (analogously to the experiment shown in Fig. 1) or exposed to 0, 0.3 or 6 ng/ml dinitropyrene. Several identical rotors were prepared. The upper panel shows the optical densities of one set of cultures. At the times indicated in the lower panel, sets of rotors were taken out of the machine. The number of cells (broken lines) and the number of revertant cells (solid lines) were determined by plating appropriate dilutions on nutrient broth agar or Vogel-Bonner agar, respectively. The growth medium in the rotors was Vogel-Bonner minimal supplemented with 0.12 #g/ml biotin and 0.8 ~g/ml histidine.
o f 0.25% $9 in the $9 mix only the higher 2 - A A dose caused prototrophic growth. At higher $9 concentrations the latency period b e c a m e progressively shorter for both 2 - A A doses with the curves for the lower dose generally lagging behind. The plateau value of the initial growth of the nonmutated cells increased with increasing $9 concentrations presumably due to the presence of growth e n h a n c i n g factors (e.g. histidine) in the liver extract. Higher a m o u n t s than 25% $9 in $9 mix (final concentration of $9: 1.25%) therefore
Fig. 3. Mutagenicity of IQ (2-amino-3-imidazo[4,5-/]quinoline). Cultures of TA1538 ( - 107 cells/ml) were exposed to the indicated concentrations of IQ both in the presence (solid lines) and absence (broken lines) of $9. For each concentration two independent cultures were prepared. Vogel-Bonner minimal medium plus 0.12 /~g biotin and 0.8/~g/ml histidine and 50 ~tl/ml of 5% $9 mix (or buffer) was used.
could not be used because the prototrophic growth b e c o m e s h i d d e n in the auxotrophic growth. T h e data of this experiment are replotted in Fig. 5 (upper panel). Here the inverse of the latency period is plotted against the $9 concentration. The activating effect of $9 and the difference between the cultures treated with the different 2 - A A concentration are obvious. As a c o m p l e m e n tary experiment the inactivating effect of $9 on the mutagenicity of dinitropyrene is demonstrated in the lower panel of Fig, 5. A n a m o u n t of 5% $9 in $9 mix was sufficient to negate the mutagenic action of 1 n g / m l of dinitropyrene, while $9 mix c o n t a i n i n g 25% $9 was necessary for a concentration of 10 n g / m l of the mutagen. It should be noted that the reduction of the latency period seen w h e n $9 was increased from 0 to 1% or 2.5% was not due to activation of the c o m p o u n d , but was
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properties of $9, as heat-inactivated $9 showed very similar effects (data not shown). Taking the experiments shown so far, the feasibility of the latency period reduction approach to assess strong mutagens could be demonstrated. The fluctuation test approach is better suited for mutagens not eliciting strong responses and is the only possibility to test such compounds with strains showing low spontaneous reversion frequencies. This becomes apparent from the following experiments. Table 1 shows the incidence of prototrophic growth as a function of the histidine concentration for several of the Ames tester strains. The highest histidine concentration (0.8 /~g/ml) was chosen to be close to the maximally possible concentration at which the density of the auxotrophic cells will not interfere with the growth of the his + cells. At this concentration the strain TA102, with the highest spontaneous mutation frequency, showed prototrophic growth in 12 out of 16 wells. This means that on average there was already more than 1 mutant per well and an increase of the number of mutants should in principle be detectable by a reduction of the latency period as discussed above. On the other side, only 3 of 80 wells showed prototrophic growth for the strain TA1538. Thus only rather strong mutagens will be able to increase the mutant frequency to more than 1 mutant per well, so that a reduction of the latency period could be expected. Table 1 also
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more probably caused by reduction of the toxic effect (either enzymatically or by unspecific protein binding) a n d / o r by the growth-stimulating TABLE l
EFFECT OF THE H1STID1NE C O N C E N T R A T I O N ON THE M U T A N T NUMBERS Cultures of the indicated 5 tester strains were grown in VB medium at different concentrations of histidine. 16 (or more) cultures per sample condition were examined. The ratio of the histidine concentration to the size of the inoculum was kept constant, to allow always the same number of cell divisions during the incubation (thus at 0.8/~g/ml histidine - 10 v cells/ml were added, at 0.4/~g/ml 5 x 106 cells/ml etc.). From the fraction of cuvettes not showing prototrophic growth the number of mutants per cuvette ( = 0.3 ml) were calculated with the aid of the Poisson distribution. With the number of his cells (the plateau value of the initial growth as determined by plating, see Fig. 2) the average mutant frequency was estimated. Strain
Number of wells with prototrophic growth Number of wells tested
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Concentration of histidine (~ g / m l )
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Fig. 5. Dependence of the latency period on the $9 concentration. The upper panel is a replotting of the data shown in Fig. 4, the lower panel shows an experiment with dinitropyrene performed analogously to the experiment with 2-aminoanthracene. The latency period is defined in this case as the incubation period (in hours) until the cultures reach an O.D. of 0.4. The inverse value of the latency period is plotted against the concentration of $9 in $9 mix. Each point represents one culture. The inverse latency period value of a culture not showing prototrophic growth (i.e. not reaching an O.D. of 0.4) was set to be zero. This plotting mode was chosen to associate increasing mutagenic activity with increasing abscissa values.
shows that r e d u c i n g the histidine c o n c e n t r a t i o n l o w e r e d - - as expected - - the fraction of t u r b i d wells, W i t h the a i d of the Poisson d i s t r i b u t i o n one can estimate the average m u t a n t f r e q u e n c y for the different strains. O b v i o u s l y the r a n k i n g of the different strains with respect to their s p o n t a n e o u s r e v e r t a n t frequencies is the s a m e as for the agar p l a t e assay (TA102 > T A 9 7 > T A 1 0 0 >> T A 9 8 > TA1538). T h e influence of histidine c o n c e n t r a t i o n on the p e r f o r m a n c e of the test is also d e m o n s t r a t e d in Fig. 6. The fraction of t u r b i d cultures of T A 1 0 0 i n d u c e d b y different doses of s o d i u m azide is given for each of 4 histidine c o n c e n t r a t i o n s . F u r thermore, the step curves shown for each set of d a t a give the d i s t r i b u t i o n of the l a t e n c y p e r i o d s for the cultures with p r o t o t r o p h i c growth. Several conclusions can be d r a w n : A t the low histidine c o n c e n t r a t i o n the m u t a g e n i c action is easily d e m o n s t r a t e d b y the increasing n u m b e r s of t u r b i d wells. T h u s e v a l u a t i o n of the results can be d o n e by the fluctuation test a p p r o a c h . Since the average n u m b e r of m u t a n t s p e r well d i d n o t increase m u c h
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Fig. 6. Effect of the histidine concentration on the mutagenic effect of Na-azide. Cultures of TA100 were grown at different histidine concentrations as described in Fig. 5. Sodium azide was added at the indicated concentrations. The number of cuvenes (per 16) showing prototrophic growth are shown for each sample condition along with step curves indicating the latency periods of the positive cultures (i.e. at 0.2 p,g/ml histidine the 3 control cultures showing prototrophic growth had latency periods of 34, 35 and 42 h).
a b o v e a value of 1 (acc. to the Poisson d i s t r i b u tion), there was no obvious c h a n g e of the average l a t e n c y period. Therefore, the latency p e r i o d red u c t i o n a p p r o a c h is n o t applicable. However, at the highest histidine c o n c e n t r a t i o n the n u m b e r of positive wells j u m p e d to 16 (out of 16) a l r e a d y at the lowest N a - a z i d e dose (20 n g / m l ) . O n average there was a r e d u c t i o n of the l a t e n c y p e r i o d b y 5 h when increasing the N a - a z i d e c o n c e n t r a t i o n from 20 n g / m l to 200 n g / m l . 5 h c o r r e s p o n d to a b o u t 3 - 4 d o u b l i n g times (as e s t i m a t e d from Fig. 2). T h u s an increase of the m u t a n t frequency by a b o u t a factor of 10 (23 to 24) was indicated. A t still higher azide c o n c e n t r a t i o n s the latency p e r i o d was further r e d u c e d ( d a t a not shown). Fig. 6 shows also that the v a r i a b i l i t y of the l a t e n c y p e r i o d s d e c r e a s e d with increasing n u m b e r s of m u t a n t s per well (i.e. for the highest N a - a z i d e concentrations). This was to be e x p e c t e d since the differences in time of e m e r g e n c e a n d d o u b l i n g times of the m u t a n t cell lines were m o r e likely to average out if m a n y m u t a n t s p e r well were i n d u c e d t h a n if only o n e or a few m u t a n t s p e r well were induced. C o n t r a r y to e x p e c t a t i o n the average latency p e r i o d of the c o n t r o l cultures (at 0.8 / ~ g / m l histidine) was shorter than that of the cultures treated with 20 n g / m l N a - a z i d e . P r e s u m a b l y this was caused b y a later e m e r g e n c e of the a z i d e - i n d u c e d m u t a n t s as c o m p a r e d to the s p o n t a n e o u s mutants. This
effect might be specific to sodium azide as it was not observed with MMC (in TA102, data not shown). From these considerations is becomes obvious that the successful application of the latency period reduction approach is sometimes difficult, since recognition or quantification of the mutagenic effect can be problematic. With more experience, however, this approach should be a very quick and inexpensive method to ascertain mutagenic activity of environmental chemicals. At present the use of the fluctuation test approach seems better suited to fulfil the needs of routine testing. The Cobas Bact has enough capacity to perform a full test with the strains containing the p K M 101 plasmid (the strains showing relatively high mutant frequencies) which recently have been recommended by Ames for standard use.
Furthermore, the Cobas Bact can be adapted to other test systems with microbial cells. A very straightforward application is the differential toxicity test with repair proficient and deficient bacteria. As an example, Fig. 7 summarizes an experiment showing that a 10 times larger concentration of MMC was needed to produce the same reduction of growth in a pol + strain as MMC 1
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compared to a pol strain. The quantitative relationship of this differential growth inhibition can be easily visualized by plotting the relative O.D. after a certain period of growth in a "survival type" plot (Fig. 7, right panel). Currently, we are investigating the adaptation of the Cobas Bact to other mutagenicity test systems. The computer program allows that rotors can be loaded completely independently using different programs. Thus different tests can be run in parallel. Conclusion
The data presented indicate that automation of microbial mutagenicity test systems by repeated optical density measurement of liquid cultures is a promising way to reduce the manual workload of the test lab. With more experience some uncertainties of interpretation of test results should become clarified. Our results with an array of different model mutagens have shown a very good analogy so far between the standard plate test and the Cobas Bact assay. Of course validation of the system requires the evaluation of many more substances. While the properties of the agar plate tests have been thoroughly explored in decades of microbiological works, the experience with liquid tests is much more limited. Obviously, the approach to the problem of mutagenicity evaluation is somewhat different when using methodologies such as described in this report. Therefore, the diverse aspects of the bacterial response are highlighted differently than in the plate assay. In this context we would like to contend that the Cobas Bact might become a very useful tool not only for routine but also for basic toxicological investigations. References
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10 100 I000 dose(ng/m;)
Fig. 7. Differential growth inhibition by MMC. Cultures of E. coil w3110 or p3478 (both inoculated at - 104 cells/ml) were exposed to the indicated concentrations of mitomycin C. The left panels show the growth curves, the right panel the dose dependency of the relative optical densities, defined as the O.D. of the treated culture divided by the O.D. of the control culture. Nutrient broth medium was used.
Arimoto, S., K. Negishi and H. Hayatsu (1981) A modification of the Ames test procedure: accelerated growth of His + revertants, Mutation Res., 91,407-411. Arni, P. (1985) Automatisierte Durchf't~hrung einer Variante des Ames Tests und anderer mikrobieller Mutagenit~tstests mit Cobas Bact, Swiss Chem., 7, 55-57. Arni, P., P. Dollenmeier and D. Mialler (1985) Automated modification of the Ames test with Cobas Bact, Mutation Res., 144, 137 140. Bartsch, H., C. Malaveille, A.M. Camus, G. Martel Planche, G.
Brun, A. Hautefeuille, N. Sabadie, A. Barbin, T. Kuroki, C. Drevon, C. Piccoli and R. Montesato (1980) Validation and comparative studies on 180 chemicals with S. typhimurium strains and V79 Chinese hamster cells in the presence of various metabolizing systems, Mutation Res., 76, 1-50. Bridges, B.A., E. Zeiger and D.B. McGregor (1981) Summary report on the performance of bacterial mutation assays in evaluation of short-term test for carcinogens, in: F. de Serres and J. Ashby (Eds.), Elsevier, New York, pp. 49-67. de Serres, F., and J. Ashby (1981) Evaluation of short-term tests for carcinogens: Report of the international collaborative program, Elsevier, New York, 808 pp. de Serres, F., and M. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay, Mutation Res., 64, 159-165. Falck, K., P. Partanen, M. Sorsa, O. Suovaniemi and H. Vainio (1985) Mutascreen, an automated bacterial mutagenicity assay, Mutation Res., 150, 119-125. Green, M.H.L., B.A. Bridges, A.M. Rogers, G. Hospool, W.J. Muriel, J.W. Bridges and J.R. Fry (1977) Mutagen screening by a simplified bacterial fluctuation test, Mutation Res., 48, 287-294. Hollstein, M., J. McCann, F. Angelosanto and W. Nichols (1979) Short-term tests for carcinogens and mutagens, Mutation Res., 65, 133-226.
Hubbard, S.A., M.H.C. Green, B.A. Bridges, A.J. Wain and J.W. Bridges (1981) Fluctuation test with $9 and hepatocyte activation, in: F. de Serres and J. Ashby (Eds.), Evaluation of short-term tests for carcinogens, Elsevier, New York, pp. 361-370, Maron, D.M., and B. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Matsushima, T., T. Sugimura, N. Nagao, T. Yahagi, A. Shirai and M. Sawamura (1980) Factors modulating mutagenicity in microbial tests, in: K.H. Norpoth and R.C. Garner (Eds.), Short-Term Test Systems for Detecting Carcinogens, Springer, Berlin, pp. 273-285, McCann, J., and B. Ames (1976) Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals: Discussion, Proc. Natl. Acad, Sci. (U.S.A.), 73, 950-954. Seiler, J.P. (1983) Criteria for standardisation of Salmonella mutagenicity test, Results of a collaborative study, Teratogen. Carcinog. Mutagen., 3, 183ff. Wild, D., E. Gocke, D. Harnasch, G. Kaiser and M.-T. King (1985) Differential mutagenic activity of IQ in Salmonella typhimurium strains in vitro and in vivo, in Drosophila and in mice, Mutation Res., 156, 93-102.
1
Elsevier MTR08625
Mutagenicity testing experiments with the Cobas Bact Elmar Gocke and Martin Schiapbach Biological Pharmaeeutical Research Department, F. Hoffmann-La Roche and Co., Ltd.. CH-4002 Basel (Switzerland)
(Received 3 December 1985) (Revision received 4 April 1986) (Accepted 17 April 1986)
Summary The feasibility of mutagenicity assays with the Cobas Bact Automatic analyser was explored using selected model mutagens. The reduction of the latency period (the period until the growth of the mutant cells becomes optically measurable) was found to be a valid measure for the mutagenic activity of strong mutagens. For weaker mutagens an evaluation analogous to the fluctuation test seemed the more appropriate approach. The influence of various variables, such as concentration of histidine, size of inoculum, medium composition and $9 concentration, is described. Adaptation of the Cobas Bact system to the differential growth inhibition test is also mentioned.
During the last decade bacterial mutagenicity tests have been more and more widely used as screening tests for the detection of chemicals which might pose a threat to the genetic material of man. The Salmonella/microsome assay (Ames test) (Maron and Ames, 1983; Hollstein et al., 1979; de Serres and Ashby, 1981) has been especially successful as a short-term alternative to the animal test for potential carcinogenicity. Although the correlation between Ames test results and animal carcinogenicity experiments depends largely on the class of substances being tested (e.g. Bartsch et al., 1980; McCann and Ames, 1976), the test has nevertheless filled a vacuum stemming from the need to assess the genotoxicity of an enormous number of newly synthesized chemicals as well as the identification of potentially cancer causing substances in our everyday diet. The Salmonella test is usually performed as an agar plate incorporation assay but an incubation of the cells in liquid medium during mutagen exposure is often advantageous (Matsushima et
al., 1980; de Serres and Shelby, 1979). Complete performance in liquid (as fluctuation test) has been advocated for specific conditions (Hubbard et al., 1981: Green et al., 1977; Hollstein et al., 1979). More recently the automation of the measurement of cell growth in suspension (Arni, 1985; Falck et al. 1985; Arni et al., 1985) has opened possibilities to reduce further the manual workload of the genetic toxicology laboratory. The Cobas Bact Analyser, originally developed for standard microbiological procedures such as susceptibility testing or pathogen identification seems well suited for routine microbial mutagenicity testing. However, as the sensitivity of the Ames test against only small variations of protocol is extensively documented (e.g. Bridges et al., 1981; Seiler, 1983), the equivalence of the results of the Cobas Bact system with the standard plate tests needs to be verified. In this report we describe experiments with selected model mutagens which are performed to explore the advantages and limitations of the test
0165-1218/86/$03.50 ~v;1986 Elsevier Science Publishers B.V. (Biomedical Division)
system in comparison to the plate assay. Adaptation of the Cobas Bact to other mutagenicity tests is also mentioned. Material and methods
Bacteria The bacterial strains were originally obtained from B.N. Ames and H.B. Maruyama. Subcultures are stored in liquid nitrogen. Overnight cultures are inoculated from master plates or from stock cultures kept at - 8 0 ° C . Strain identity is checked regularly. Media For overnight culture the cells were grown in nutrient broth (Difco). In the Cobas Bact rotors Vogel-Bonner medium, supplemented with 0.12 /xg/ml biotin and histidine, was used. The plateau value of the initial growth is dependent on the amount of histidine. At a concentration of 0.1 ~ g / m l the optical density increases by about 0.02-0.03 and at 0.8 ~ g / m l by about 0.2-0.3. This is about the maximum which can be used because at higher concentrations the mutant growth will become hidden by the growth of the non-mutant cells. The concentration of histidine is indicated in the figure legends. Glucose concentration was 0.8%. Addition of amino acids other than histidine (Arimoto et al., 1981) increased the growth velocity of the bacteria and can be used to shorten the test period. In the experiments shown here, except the one shown in Fig. 1, the medium was not supplemented with amino acids. Metabolic activation. $9 from phenobarbitone//~-naphthoflavone induced male F~llinsdorf albino rats was prepared by standard procedures (Maron and Ames, 1983). The amounts of $9 added to the cultures are detailed in the figure legends. Mutagens. Mitomycin C and Na-azide were obtained from Serva, 2-aminoanthracene from Aldrich. Dinitropyrene (a mixture of the isomers) was a gift of K. Eckhardt. IQ (2-amino-3-methylimidazo[4,5-f]quinoline) was a gift of D. Wild. Description of the Cobas Bact The Cobas Bact Automatic Analyser is a tabletop machine developed by Diagnostica Roche for
standard microbiological tests such as susceptibility testing and pathogen identification. The unit culture container is a plastic rotor with 16 peripheral measuring compartments which are connected to a central well via a circuitous path. The cell suspension is pipetted into the center of the rotor and the test substances are pipetted directly into the measuring compartments. After sealing, the rotor is transferred to the Cobas Bact and the cell suspension is injected into the measuring compartments by an initial 3000 rpm centrifugation. Determination of the optical density is made every 20 min or at predetermined longer intervals during a low speed spin (500 rpm), which presses the fluid between two clear plastic windows. This ensures that surface effects do not disturb the measurement. Between the O.D. determinations the rotor is deposited in an incubator on a continuously rotating paternoster elevator. Incubation temperature, wavelength of the O.D. measurement, centrifugation speeds, shaking periods, etc., can be selected by the operator. The machine holds up to 50 rotors which can be run after different programs. We used the internal computer to collect and print the data. Additionally an external computer can be connected. In this report we have not attempted to calculate the number of induced mutants from the reduction of the latency period. We feel that the variability of the time when mutants are able to start growing as well as possible differences of the doubling times make such a calculation hazardous and generate deceptions. Likewise we have not replaced the O.D. values in the figures with calculated cell numbers. The turbidity of suspensions is not only a function of the cell density but also of the size and form of the cells which might vary depending on the growth status and toxicity of the test compound. Since quantification of the mutagenic effects does not depend on such calculated numbers, the usefulness of the machine is not at stake.
Experimental procedure The test substances, dissolved in appropriate solvents, were pipetted into the measuring compartments and the Vogel-Bonner medium, biotin, histidine, $9 mix and bacteria were pipetted into the central well. The rotor was completely sealed and transferred into the machine. The initial
centrifugation distributed the cell suspension into the compartments containing the mutagen. Usually it was sufficient to measure the optical density about every 3 h. After the end of the run, growth data for each culture were printed. Growth curves can also be plotted. As detailed in the figure legends, each experiment was carried out with some modification of this procedure. The latency period is defined as the incubation period until the culture reaches an O.D. value of 0.3. Results and discussion
Occurrence of a mutation such as reversion from histidine auxotrophy to prototrophy can be detected by the formation of a visible colony on selective semi-solid medium or by the development of visible turbidity in selective liquid medium. While on the agar plate two or more independent mutations can be scored as separate colonies, the question of whether more than one mutation has occurred in the liquid assay cannot be answered a priori. In the fluctuation test this problem of quantitation is solved by limiting the growth of the inoculum to values where spontaneous reversions occur only occasionally, i.e. in only a few tubes. Thus an increase of the mutant frequency is determined by an increase of the number of turbid tubes. Measurement of the optical density needs to be performed only once after a predetermined optimal incubation period, usually a few days. A somewhat different approach is made possible by the use of machines able to monitor periodically the optical density of the bacterial cultures. In this way the kinetic growth curve of the culture c a r be determined. An increase of the number of mutants per culture vessel should cause a reduction of the period until the prototrophic growth becomes measurable. Obviously this method (in the following called the latency period reduction approach) works if the number of mutants per tube is larger than 1. Compared to the fluctuation test the cell numbers per tube can be higher and fewer tubes are needed. Furthermore the growth curves should give further information concerning toxicity or presence of growth enhancing ingredients.
To verify these predictions we performed reconstruction experiments by adding varying numbers of established his + revertants to the inoculum of his- cells. As can be seen in Fig. 1, the culture which was not spiked did not show prototrophic growth while the latency period of the spiked cultures decreased logarithmically with increasing numbers of mutant cells added: an increase by the factor 2 translated itself into a reduction of the latency period by about 45 min. This therefore was the doubling time of the his + cells in the Arimoto medium. We then wanted to test whether newly induced mutants grow similarly to the established mutants. Fig. 2 shows an experiment comparing the growth of reconstructed cultures to the growth of cultures exposed to the mutagen dinitropyrene. At appropriate times aliquots of the cultures were taken out of the Cobas Bact and plated on VB medium to determine the number of his + cells or on NB medium to determine the total number of cells. It is obvious that the optical densities of the cultures (upper panel) increased parallel to the increase of the total number of cells (lower panel; broken lines) and the secondary (prototrophic) growth became optically measurable at those times when the densities of the his + cells (lower panel; solid lines) approached the densities of the nonmutated cells. From the growth curves one can determine that the plateau value of the his cells (at the applied histidine concentration of 0.8 ~ g / m l ) was about 1.5 × l0 s cells per ml or 5 × 107 cells/cuvette. When compared to the growth of the established mutant cells, the dinitropyrene induced his + cells grew quite similarly (curves a, b, c versus curves 1, 2; solid lines), except for a slower growth at around 2 8 h of incubation. This delay might have been due to a division delay of the not yet fully expressed mutant cells or more likely to an artefact resulting from formation of mutants on the agar plates after plating. The higher dinitropyrene concentration (curve 2) induced about 10-fold more mutants than the lower concentration (curve 1). This fits well to the observation that the time differences of the latency periods between the mutagen-treated cultures are approximately the same as between the reconstituted cultures because the numbers of the inoculated
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his + cells were chosen to differ by a factor of 10. Taking the reconstitution experiments together, one can conclude that the theoretical predictions are met very well. The sensitivity of the latency period reduction approach is demonstrated in Fig. 3. The potent mutagen 2-amino-3-methylimidazo[4,5]quinoline (IQ) was tested in TA1538 both in the presence and absence of $9. As little as 15 pg per culture (50 p g / m l ) gave a well measurable effect in the presence of $9. The latency period was the shortest around 5-50 n g / m l . At 500 n g / m l toxic effects prolong the latency period and growth was
almost completely abolished at 5/~g/ml. The toxic effect was also seen by the reduced initial growth. In the absence of metabolic activation IQ showed mutagenic effects at the comparatively high concentration of 5 /~g/ml. This is in line with a relatively weak direct mutagenicity of IQ in the plate incorporation assay as recently reported by Wild et al. (1985). The dependency of the mutagenic effect of 2-aminoanthracene (2-AA) on the concentration of $9 is shown in Fig. 4. Two doses of the promutagen were used. Without metabolic activation no mutagenic effect was seen. In the presence
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o f 0.25% $9 in the $9 mix only the higher 2 - A A dose caused prototrophic growth. At higher $9 concentrations the latency period b e c a m e progressively shorter for both 2 - A A doses with the curves for the lower dose generally lagging behind. The plateau value of the initial growth of the nonmutated cells increased with increasing $9 concentrations presumably due to the presence of growth e n h a n c i n g factors (e.g. histidine) in the liver extract. Higher a m o u n t s than 25% $9 in $9 mix (final concentration of $9: 1.25%) therefore
Fig. 3. Mutagenicity of IQ (2-amino-3-imidazo[4,5-/]quinoline). Cultures of TA1538 ( - 107 cells/ml) were exposed to the indicated concentrations of IQ both in the presence (solid lines) and absence (broken lines) of $9. For each concentration two independent cultures were prepared. Vogel-Bonner minimal medium plus 0.12 /~g biotin and 0.8/~g/ml histidine and 50 ~tl/ml of 5% $9 mix (or buffer) was used.
could not be used because the prototrophic growth b e c o m e s h i d d e n in the auxotrophic growth. T h e data of this experiment are replotted in Fig. 5 (upper panel). Here the inverse of the latency period is plotted against the $9 concentration. The activating effect of $9 and the difference between the cultures treated with the different 2 - A A concentration are obvious. As a c o m p l e m e n tary experiment the inactivating effect of $9 on the mutagenicity of dinitropyrene is demonstrated in the lower panel of Fig, 5. A n a m o u n t of 5% $9 in $9 mix was sufficient to negate the mutagenic action of 1 n g / m l of dinitropyrene, while $9 mix c o n t a i n i n g 25% $9 was necessary for a concentration of 10 n g / m l of the mutagen. It should be noted that the reduction of the latency period seen w h e n $9 was increased from 0 to 1% or 2.5% was not due to activation of the c o m p o u n d , but was
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properties of $9, as heat-inactivated $9 showed very similar effects (data not shown). Taking the experiments shown so far, the feasibility of the latency period reduction approach to assess strong mutagens could be demonstrated. The fluctuation test approach is better suited for mutagens not eliciting strong responses and is the only possibility to test such compounds with strains showing low spontaneous reversion frequencies. This becomes apparent from the following experiments. Table 1 shows the incidence of prototrophic growth as a function of the histidine concentration for several of the Ames tester strains. The highest histidine concentration (0.8 /~g/ml) was chosen to be close to the maximally possible concentration at which the density of the auxotrophic cells will not interfere with the growth of the his + cells. At this concentration the strain TA102, with the highest spontaneous mutation frequency, showed prototrophic growth in 12 out of 16 wells. This means that on average there was already more than 1 mutant per well and an increase of the number of mutants should in principle be detectable by a reduction of the latency period as discussed above. On the other side, only 3 of 80 wells showed prototrophic growth for the strain TA1538. Thus only rather strong mutagens will be able to increase the mutant frequency to more than 1 mutant per well, so that a reduction of the latency period could be expected. Table 1 also
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more probably caused by reduction of the toxic effect (either enzymatically or by unspecific protein binding) a n d / o r by the growth-stimulating TABLE l
EFFECT OF THE H1STID1NE C O N C E N T R A T I O N ON THE M U T A N T NUMBERS Cultures of the indicated 5 tester strains were grown in VB medium at different concentrations of histidine. 16 (or more) cultures per sample condition were examined. The ratio of the histidine concentration to the size of the inoculum was kept constant, to allow always the same number of cell divisions during the incubation (thus at 0.8/~g/ml histidine - 10 v cells/ml were added, at 0.4/~g/ml 5 x 106 cells/ml etc.). From the fraction of cuvettes not showing prototrophic growth the number of mutants per cuvette ( = 0.3 ml) were calculated with the aid of the Poisson distribution. With the number of his cells (the plateau value of the initial growth as determined by plating, see Fig. 2) the average mutant frequency was estimated. Strain
Number of wells with prototrophic growth Number of wells tested
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shows that r e d u c i n g the histidine c o n c e n t r a t i o n l o w e r e d - - as expected - - the fraction of t u r b i d wells, W i t h the a i d of the Poisson d i s t r i b u t i o n one can estimate the average m u t a n t f r e q u e n c y for the different strains. O b v i o u s l y the r a n k i n g of the different strains with respect to their s p o n t a n e o u s r e v e r t a n t frequencies is the s a m e as for the agar p l a t e assay (TA102 > T A 9 7 > T A 1 0 0 >> T A 9 8 > TA1538). T h e influence of histidine c o n c e n t r a t i o n on the p e r f o r m a n c e of the test is also d e m o n s t r a t e d in Fig. 6. The fraction of t u r b i d cultures of T A 1 0 0 i n d u c e d b y different doses of s o d i u m azide is given for each of 4 histidine c o n c e n t r a t i o n s . F u r thermore, the step curves shown for each set of d a t a give the d i s t r i b u t i o n of the l a t e n c y p e r i o d s for the cultures with p r o t o t r o p h i c growth. Several conclusions can be d r a w n : A t the low histidine c o n c e n t r a t i o n the m u t a g e n i c action is easily d e m o n s t r a t e d b y the increasing n u m b e r s of t u r b i d wells. T h u s e v a l u a t i o n of the results can be d o n e by the fluctuation test a p p r o a c h . Since the average n u m b e r of m u t a n t s p e r well d i d n o t increase m u c h
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30 40 0 3 (hrs)
Fig. 6. Effect of the histidine concentration on the mutagenic effect of Na-azide. Cultures of TA100 were grown at different histidine concentrations as described in Fig. 5. Sodium azide was added at the indicated concentrations. The number of cuvenes (per 16) showing prototrophic growth are shown for each sample condition along with step curves indicating the latency periods of the positive cultures (i.e. at 0.2 p,g/ml histidine the 3 control cultures showing prototrophic growth had latency periods of 34, 35 and 42 h).
a b o v e a value of 1 (acc. to the Poisson d i s t r i b u tion), there was no obvious c h a n g e of the average l a t e n c y period. Therefore, the latency p e r i o d red u c t i o n a p p r o a c h is n o t applicable. However, at the highest histidine c o n c e n t r a t i o n the n u m b e r of positive wells j u m p e d to 16 (out of 16) a l r e a d y at the lowest N a - a z i d e dose (20 n g / m l ) . O n average there was a r e d u c t i o n of the l a t e n c y p e r i o d b y 5 h when increasing the N a - a z i d e c o n c e n t r a t i o n from 20 n g / m l to 200 n g / m l . 5 h c o r r e s p o n d to a b o u t 3 - 4 d o u b l i n g times (as e s t i m a t e d from Fig. 2). T h u s an increase of the m u t a n t frequency by a b o u t a factor of 10 (23 to 24) was indicated. A t still higher azide c o n c e n t r a t i o n s the latency p e r i o d was further r e d u c e d ( d a t a not shown). Fig. 6 shows also that the v a r i a b i l i t y of the l a t e n c y p e r i o d s d e c r e a s e d with increasing n u m b e r s of m u t a n t s per well (i.e. for the highest N a - a z i d e concentrations). This was to be e x p e c t e d since the differences in time of e m e r g e n c e a n d d o u b l i n g times of the m u t a n t cell lines were m o r e likely to average out if m a n y m u t a n t s p e r well were i n d u c e d t h a n if only o n e or a few m u t a n t s p e r well were induced. C o n t r a r y to e x p e c t a t i o n the average latency p e r i o d of the c o n t r o l cultures (at 0.8 / ~ g / m l histidine) was shorter than that of the cultures treated with 20 n g / m l N a - a z i d e . P r e s u m a b l y this was caused b y a later e m e r g e n c e of the a z i d e - i n d u c e d m u t a n t s as c o m p a r e d to the s p o n t a n e o u s mutants. This
effect might be specific to sodium azide as it was not observed with MMC (in TA102, data not shown). From these considerations is becomes obvious that the successful application of the latency period reduction approach is sometimes difficult, since recognition or quantification of the mutagenic effect can be problematic. With more experience, however, this approach should be a very quick and inexpensive method to ascertain mutagenic activity of environmental chemicals. At present the use of the fluctuation test approach seems better suited to fulfil the needs of routine testing. The Cobas Bact has enough capacity to perform a full test with the strains containing the p K M 101 plasmid (the strains showing relatively high mutant frequencies) which recently have been recommended by Ames for standard use.
Furthermore, the Cobas Bact can be adapted to other test systems with microbial cells. A very straightforward application is the differential toxicity test with repair proficient and deficient bacteria. As an example, Fig. 7 summarizes an experiment showing that a 10 times larger concentration of MMC was needed to produce the same reduction of growth in a pol + strain as MMC 1
p 3478 05
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compared to a pol strain. The quantitative relationship of this differential growth inhibition can be easily visualized by plotting the relative O.D. after a certain period of growth in a "survival type" plot (Fig. 7, right panel). Currently, we are investigating the adaptation of the Cobas Bact to other mutagenicity test systems. The computer program allows that rotors can be loaded completely independently using different programs. Thus different tests can be run in parallel. Conclusion
The data presented indicate that automation of microbial mutagenicity test systems by repeated optical density measurement of liquid cultures is a promising way to reduce the manual workload of the test lab. With more experience some uncertainties of interpretation of test results should become clarified. Our results with an array of different model mutagens have shown a very good analogy so far between the standard plate test and the Cobas Bact assay. Of course validation of the system requires the evaluation of many more substances. While the properties of the agar plate tests have been thoroughly explored in decades of microbiological works, the experience with liquid tests is much more limited. Obviously, the approach to the problem of mutagenicity evaluation is somewhat different when using methodologies such as described in this report. Therefore, the diverse aspects of the bacterial response are highlighted differently than in the plate assay. In this context we would like to contend that the Cobas Bact might become a very useful tool not only for routine but also for basic toxicological investigations. References
10
20 30 t ime(hrs)
10 100 I000 dose(ng/m;)
Fig. 7. Differential growth inhibition by MMC. Cultures of E. coil w3110 or p3478 (both inoculated at - 104 cells/ml) were exposed to the indicated concentrations of mitomycin C. The left panels show the growth curves, the right panel the dose dependency of the relative optical densities, defined as the O.D. of the treated culture divided by the O.D. of the control culture. Nutrient broth medium was used.
Arimoto, S., K. Negishi and H. Hayatsu (1981) A modification of the Ames test procedure: accelerated growth of His + revertants, Mutation Res., 91,407-411. Arni, P. (1985) Automatisierte Durchf't~hrung einer Variante des Ames Tests und anderer mikrobieller Mutagenit~tstests mit Cobas Bact, Swiss Chem., 7, 55-57. Arni, P., P. Dollenmeier and D. Mialler (1985) Automated modification of the Ames test with Cobas Bact, Mutation Res., 144, 137 140. Bartsch, H., C. Malaveille, A.M. Camus, G. Martel Planche, G.
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