Mutagenesis, Vol. 14, No. 4, 411-415,
July 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Development of a new bioluminescent mutagenicity assay based on the Ames test
Centro de Ciencias Medioambientales, CSIC, c/Serrano 115 dpdo, 28006 Madrid and 1 R&D Laboratory, Gomensoro/Biotech, Madrid, Spain
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Abstract |
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A newly developed rapid mutagenicity assay based on the adenosine triphosphate (ATP)-bioluminescence technique and the Ames test is described. Salmonella typhimurium strains TA98 and TA100 were exposed in an appropriate liquid medium to the direct mutagens 4-nitroquinoline-N-oxide and methyl methanesulphonate, respectively, and to the indirect mutagen 2-aminoanthracene. Both auxotrophic and prototrophic growth were monitored throughout the incubation period as variations in the intracellular ATP levels by means of the luciferin–luciferase assay. After 9–12 h of incubation a dose–response increase in the levels of ATP was readily detected. In order to demonstrate that this increase was due to the growth of revertant bacteria, aliquots from each culture were plated on minimal agar plates. A very good correlation between the changes in ATP levels and the appearance of revertant colonies on the plates was found. Given the rapidity of this method as compared with conventional mutagenicity assays, it has potential for industrial and environmental applications. Other potential applications are also discussed.
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Introduction |
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The relevance of genetic testing relies on its ability to detect genotoxic carcinogens employing endpoints other than neoplasia. Short-term genetic bioassays have been routinely applied to screen both pure compounds and complex environmental mixtures for genotoxic activities. These assays should be rapid, sensitive, inexpensive and simple to use. The most popular and validated mutagenicity bacterial reversion screening test is the Salmonella/microsoma test (Ames test) (Maron and Ames, 1983
). Histidine-requiring strains of Salmonella typhimurium are exposed to the test sample and grown under selective conditions (minimal media with a trace amount of histidine) in the presence and absence of a metabolic activation system. Only those auxotrophic bacteria which revert to histidine independence are able to form colonies. The use of a set of strains with known point mutations in the histidine operon provides information about the mechanisms of mutagenicity of a given sample. However, this assay presents some disadvantages: (i) it is time-consuming (a 2 day incubation period is needed as the end-point is the growth of revertants on minimal medium); (ii) it is labour-intensive (preparation of chemically defined media and pouring onto plates); (iii) the test sample or soluble components of the metabolising system may diffuse into the bottom-agar thus varying the final concentration over time; and (iv) it does not provide direct toxicity measurements (toxicity is determined by subjective criteria such as background clearing or a reduction in the number of revertants on treated plates compared with the solvent control). Therefore, alternative short-term mutagenicity bacterial tests able to measure other endpoints are needed.
The use of bioluminescence-based techniques is rapidly growing due to their high sensitivity. Amongst them the luciferin–luciferase assay for determining ATP concentration is one of the most widely used (Lundin et al., 1986). This assay employs the firefly luciferase enzyme which uses ATP and the heterocyclic cofactor luciferin to produce light. Detection of bioluminescence with a photomultiplier gives a sensitive measurement of ATP concentration. Under optimum conditions one photon of light is produced for each molecule of ATP. Cellular ATP can be released by direct lysis of the cells with a suitable detergent thus reacting with the luciferin–luciferase and leading to light emission.
The intracellular ATP content depends on the cellular growth phase, being then a measurement of the metabolically active biomass (McEntee et al., 1989). On the other hand, it is rapidly degraded when cells die and it is transiently depressed by many forms of cell stress. Previous work has shown the ATP-luminescent assay to be a reproducible and reliable technique with applications in areas such as quality control of food, beverages, cosmetics, environmental control, clinical studies, hygiene monitoring and in biological and medical research (Lundin, 1989).
In the present study, we examine the feasibility of the luciferin–luciferase-based ATP technique to develop a kinetic method to monitor the growth in a minimal liquid medium of TA98 and TA100, two of the S.typhimurum strains used in the Ames test, after exposure to standard mutagens in the presence and absence of a metabolic activation system. Such a method would be able to detect the appearance of revertant bacteria in a shorter period of time than that required for the Ames test since the endpoint is a biochemical parameter rather than counting the visible prototrophic colonies. This method will increase the sensitivity because less prototrophic revertants will be needed to produce a scorable positive. Since it is liquid-based it has all the advantages described for the fluctuation test (Hubbard et al., 1984). Additionally, it has the potential of providing information on the toxicity of a given sample based on the decrease of intracellular ATP levels.
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Materials and methods |
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Chemicals
The positive control chemicals, methyl methanesulphonate (MMS; CAS no. 66-27-3), 4-nitroquinoline-N-oxide (4NQO; CAS no. 56-57-5) and 2-aminoanthracene (2AA; CAS no. 613-13-8) were purchased from Sigma (St Louis, MO). The positive controls were dissolved in DMSO. NADP and D-glucose-6-phosphate were obtained from Boehringer Mannheim (Mannheim, Germany). Phenobarbitone sodium salt (PB) and 5,6-benzoflavone (ß-naphthoflavone) were from Sigma. All other chemicals were of reagent grade.
Bacterial strains
Salmonella typhimurium strains TA98 and TA100 were kindly supplied by Professor B.N.Ames, University of California, Berkeley, CA, USA. Their genotypes have already been described (Maron and Ames, 1983). Their genetic markers and other characteristics, such as response to positive controls and the number of spontaneous revertants, were routinely verified as described by Maron and Ames (1983).
Metabolic activation system
The post-mitochondrial supernatant from a liver homogenate (S9) from Wistar male rats weighing ~200 g was used as the metabolic activation system. For induction a combined injection of PB and ß-naphthoflavone was used (Matsushima et al., 1976). The rats were injected i.p. with (i) PB (30 mg/kg), (ii) PB (60 mg/kg), (iii) PB (60 mg/kg) and ß-naphthoflavone (80 mg/kg), and (iv) PB (60 mg/kg), 4, 3, 2 and 1 day(s), respectively, before they were killed. The S9 liver fraction was prepared according to the method described by Maron and Ames (1983). The protein concentration was 47 mg/ml as determined by the Lowry et al. (1951) procedure. Prior to the mutagenicity tests, the S9 mix was prepared by the addition of NADP and glucose-6-phosphate to S9.
Assay protocol
Bacterial incubation.
Overnight cultures of the tester strains were prepared by inoculation from the stock cultures into Difco Bacto nutrient broth and incubated for 12 h at 37°C with shaking. Cells were then diluted 1:10 in nutrient broth and incubated for an additional 2 h in order to maintain the cells in log phase. Cells were then centrifuged at 3000 r.p.m. for 4 min in a bench top centrifuge and resuspended to an optical density of 0.2 (600 nm) in minimal growth medium that consisted of (per 100 ml) 72.2 ml Davis–Mingoli salts (5.5x), 15.8 ml D-glucose 40%, 0.45 ml D-biotin 0.1%, 0.25 ml L-histidine 0.1% and 11.3 ml deionized water. The assay was initiated by adding 2 ml of the above bacterial culture to 23 ml of Davis–Mingoli salts (0.79x). The standard mutagens were dissolved in dimethylsulfoxide (DMSO). The final DMSO concentration in the assay never exceeded 1% v/v. Two aliquots of 100 µl were taken at different time intervals to measure intracellular ATP concentrations as described below and to perform the standard plate incorporation test according to Maron and Ames (1983). When needed, an appropriate dilution of the cultures was made in order to obtain a suitable number of colonies to be counted by eye. Results are expressed as revertants/plate after being corrected by the dilution factor.
For each experiment, untreated (spontaneous mutation), solvent (DMSO) and positive (response to a standard mutagen) controls were included to ensure the validity of the test. The positive controls used were (with corresponding strains in parentheses) MMS (TA100) and 4NQO (TA98). When assays were performed in the presence of S9 mix, 2AA was used.
ATP assay.
The firefly (Photinus pyralis) luciferase was used for ATP determination. The luminescent reaction produced by the luciferase uses ATP and the organic cofactor luciferine as co-substrates:
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The luminescent reaction was performed using an ATP Kit and a photon-counting luminometer `Optocomp' both supplied by Gomensoro/Biotech (Madrid, Spain). Briefly, the protocol consisted of the sequential automatic addition, by the luminometer, of two reactives directly to the samples. First a micro-organism lysing and ATP releaser agent was added for a 20 s incubation period. Next a mixture of luciferin–luciferase was automatically added and after a 10 s incubation period, light emission was recorded for 1 s. Results are presented as percent of increase in relative light units (RLU) over the respective values at time 0 h for comparison purposes.
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Results |
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Preliminary experiments included the determination of the optimal initial cell density and histidine concentration in the medium that provided adequate sensitivity. We performed a series of experiments with different cell densities and histidine concentrations using TA98 and TA100 in the presence and absence of 4NQO and MMS, respectively, and measured growth turbidimetrically at 600 nm at regular intervals throughout the incubation period. An initial bacteria population ranging from 106 to 107 cells/ml and an initial histidine concentration of 2 µg/ml proved to be the most satisfactory. The results for TA98 with different concentrations of 4NQO are shown in Figure 1
. The growth curve obtained could be divided into three different sections: in the first 5 h there was auxotrophic growth due to the trace amount of histidine present in the medium; the next 19 h of incubation showed a plateau where no growth was observed as histidine was depleted; thereafter, only revertants were able to grow. A dose–response increase in bacterial growth was observed with increasing concentrations of 4NQO. Similar results were obtained for TA100 in the presence of MMS (data not shown).
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The turbidity increase caused by the growth of revertant bacteria was observed only after their population had reached the detection level. Therefore, the higher sensitivity of the ATP-based measurement should allow for earlier detection of revertants. To test this hypothesis we exposed TA98 and TA100 to different concentrations of the above mentioned direct mutagens and to the indirect mutagen 2AA, in the presence of a metabolic activation system. RLU were recorded at different time intervals throughout the incubation period.
Figure 2A shows the results of the ATP assay with TA98 in the presence of 4NQO. The shape of the curve was found to be very similar to that obtained when turbidity was recorded. As expected, the detection time of the revertants and the effect of the mutagen were significantly different for the two methods. Instead of the 24 h needed for the turbidity test, in the ATP-based assay a statistically significant difference (t-test; P 
0.05) between the three treatments and their respective controls was found starting from the 12th hour of incubation with a clear dose–response effect of the mutagen. In order to demonstrate that the observed increase in RLU was due to the growth of revertants, aliquots from each culture were transferred onto minimal agar plates at different time intervals to test their his+ genotype. These aliquots were shown to be real revertant bacteria capable of growing in histidine-free medium (Figure 2B).
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Figure 3A
shows the results of the ATP assay with TA100 in the presence of different concentrations of MMS. The data followed a similar pattern to that obtained with TA98, with increasing ATP levels after 12–14 h of incubation, this increase being statistically significant as of the 14th hour of incubation for the two highest concentrations of MMS used (t-test; P 0.05). As before, there was a good correlation between the observed increase in RLU and the number of colonies on minimal agar plates (Figure 3B).
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The RLU values for TA98 and TA100 in the presence of 2AA and S9 mix are shown in Figures 4A and 5A
. The shape of the growth curves was very similar but slightly different to those obtained in the absence of the metabolic activation system. The increase in revertant growth started earlier, between the 9th and 10th hour of incubation, reaching its maximum level at the 12th hour. The difference between the treatments and their respective controls was found to be statistically significant as of the 9th hour of incubation for both strains (t-test; P 0.05). This earlier detection of revertants was also observed on minimal agar plates (Figures 4B and 5B).
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Discussion |
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There is a widespread need for new fast and sensitive short-term genetic bioassays to evaluate the mutagenic properties of both pure compounds and complex environmental mixtures. Alternative methods to the Ames test that use electrical impedance, luminescent bacteria or colorimetric techniques have been proposed (Forsythe, 1990
; van der Lelie et al., 1997; Mzibri et al., 1996; Côté et al., 1995). Most of these assays are not mutagenicity assays but SOS induction assays where different SOS-responsive gene fusions are constructed and the activity of the reporter genes (generally lacZ and lux) monitored. The main advantage of these assays over the Ames test is their rapidity. On the other hand, these assays do not detect mutagenic samples that are not SOS inducing, they do not provide information on the mechanism of DNA damage and most of them do not provide information on the toxicity of the sample. Based on the theoretical principles of the Ames test, we aimed to develop a new mutagenicity method to monitor the growth of revertant bacteria by means of the ATP-bioluminescence technique. The rationale of using the same bacteria and metabolic system as in the Ames test is that it would allow the data collected with the ATP-based assay to be compared with the current database of chemical mutagens already tested with the Ames test.
The determination of ATP by means of the firefly luciferase (P.pyralis) reaction is a highly sensitive (detection range of fentomoles) and rapid method. It is also a well accepted technique for a broad range of applications in the monitoring of bacterial cultures: antibiotic susceptibility test, biocides and bacteriostatic effects, monitoring of fermentations, biofilms, microbia consortia, etc. (Stanley, 1989).
The results obtained in the present study show that, with the use of the ATP-bioluminescence technique, it is possible to obtain kinetic data in a fast and sensitive manner on both the auxotrophic and prototrophic growth of bacteria throughout the incubation period after their exposure to a chemical. The advantage of this technique is that it provides not only qualitative information on the mutagenic activity of a given sample but also a complete picture of its mutagenic effect over time. As with the Ames test, the use of a set of different S.typhimurium strains provides additional information on the mechanism of action underlying the mutagenic process.
To prove that the changes observed in the intracellular ATP levels in response to the different mutagens used were due to the induction of revertant bacteria, we plated aliquots of the same cultures on minimal media plates at different times. The results obtained showed that when visible revertant colonies appeared on the plates there was a parallel increase in the levels of ATP, suggesting that this increase was due to the proliferation of revertant bacteria. A good correlation between the levels of ATP and the number of revertant colonies at different times of exposure to the mutagens was obtained even with different bacterial strains.
Mention should be made of the different pattern of data obtained with the direct and indirect mutagens assayed. In the case of the indirect mutagen 2AA, both the appearance of revertant colonies and the increase in ATP levels were observed between the 9th and 12th hour of incubation while in the case of the direct mutagens assayed (MMS and 4NQO) the induction of revertant bacteria was observed after 12–14 h of incubation. The same profile was seen in their respective controls. This effect could be due to the presence of growth promoting substances in the S9 fraction able to increase the number of auxotrophic bacteria in the first hours of culture which, in turn, induced an earlier appearance of revertants.
Comparing the ATP and revertant results it can be seen that, following the exponential phase of increase in ATP levels, a maximum is reached (between the 12th and 15th hour depending on the presence or not of S9 mix) while if revertants are studied there is a greater increase in the number of revertant bacteria over time. This result seems to indicate that there is a dramatic increase in the intracellular ATP concentration at the expense of the ATP and ADP pools during the exponential growth phase. At the end of the growth phase, the intracellular ATP levels start to decrease and this effect is offset by the increase in the number of cells, resulting in a constant ATP concentration. Finally, from the 13th or 17th hour of incubation (in the presence or absence of S9 mix, respectively) a decrease in ATP levels is observed, presumably due to a slowing of cell growth leading to a decrease in the average levels of intracellular ATP, as previously described (Lee and Colston, 1986; Holm-Hansen and Karl, 1987).
An additional application of the above described method could be the simultaneous monitoring of toxicity and mutagenicity. A toxic compound that interferes with cellular metabolism or integrity would lead to a decrease in the intracellular ATP concentration as previously described (Bitton and Koopman, 1992). It is important to note the different time course of both effects: a toxic effect would be detected within the first hours of exposure while incubation times of at least 12 h are needed for evaluating a mutagenic effect. Therefore, it would be feasible to monitor both parameters in the same sample and in the same assay.
In summary, this work describes the development of a mutagenicity method based on the Ames test and the ATP-bioluminescence technique that has the advantages of being less time-consuming and laborious than conventional mutagenicity tests and has the potential for providing toxicity data on a given sample. The results obtained parallel those of the induction of revertant bacteria measured by means of classic microbiology techniques.
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Acknowledgments |
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We gratefully acknowledge Mr S.Carlin for language revision and Dr P.Gasco from the Spanish Institute of Toxicology for providing us with the facilities for S9 purification. We also thank Mrs Antonia Martinez for help with the preparation of the manuscript.
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Notes |
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2 To whom correspondence should be addressed. Tel: +34 91 5625020; Fax: +34 91 5640800; Email: aguadano{at}ccma.csic.es
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Received on December 8, 1998; accepted on April 13, 1999.
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