microsome multiple indicator mutagenicity test

Mutation Research, 121 (1983) 33-37

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Elsevier MRLett 0407

Salmonella/microsome multiple indicator mutagenicity test A n t o n y J. V i t h a y a t h i l , C h e r y l M c C l u r e a n d J o h n W . M y e r s Chemistry Department, Portland State University, Portland, OR 97207 (U.S.A.)

(Accepted 22 March 1983)

The Ames Salmonella/microsome mutagenesis test is now widely used for the rapid and inexpensive screening of chemical carcinogens (Ames et al., 1975). This test detects mutation caused by chemicals by detecting reversion to histidine prototrophy in a set of His S. t y p h i m u r i u m strains. In the standard protocol of the Ames test, the scoring is strictly limited to specific backmutations in the histidine operon. It is necessary to carry out independent tests against more than one tester strain to detect different classes of frameshift and base-pair substitution types of mutagens. In this paper we report a different protocol, which allows scoring of mutations in more than one locus. With this multiple indicator test, it is possible to use a single tester strain such as S. t y p h i m u r i u m TA98 for the detection of different classes of mutagens. This new test also has the advantage that it can be carried out even in the presence of histidine and the results can be expressed, if necessary, in a more quantitative f o r m as number of mutant cells produced per survivors. The multiple indicator test equals and in some cases exceeds the sensitivity of the standard plate test. In the multiple indicator test, the assays are carried out in the following three steps: (i) mutation step; (ii) removal of test c o m p o u n d and growing surviving cells to steady state population; and (iii) scoring mutations in selective media. In the first step, a 0.5-ml suspension of tester strain TA98 in minimal media supplemented with histidine and biotin is incubated in a test tube with the test c o m p o u n d with or without the presence o f the microsome ($9) preparation. The mutation process is then stopped by addition of ice-cold nutrient broth. It is then centrifuged and the supernatants which contain excess test compounds are removed. The bacterial cells (which contain the tester strain along with any mutant cells produced by the test compound) are then resuspended in fresh nutrient broth and allowed to grow overnight to a steady-state population o f approximately 10 9 cells/ml. In the final step, using standard plating techniques, mutant ceils are scored on selective media plates. Histidine revertants are scored on minimal media plates which lack histidine. For-

0165-7992/83/$ 03.00 © 1983 Elsevier Science Publishers B.V.

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TABLE 1 D E T E C T I O N OF D I R E C T - A C T I N G M U T A G E N S BY S. typhimurium TA98 Compound

N-Methylnitrosonitroguanidine (MNNG) Methyl methanesulfonate (MMS) 1,3-Dichloropropene Captan Ethylethanenitrosocarbamate fl-Propiolactone Sodium nitrite 4-Nitrobiphenyl 4-Nitroquinoline-N-oxide 2-Nitrofluorene

BASE-PAIR

SUBSTITUTION

AND

FRAMESHIFT

Amount

N u m b e r o f m u t a n t s / p l a t e o r / ( 1 0 s survivors)

tested (~g)

Histidine revertant

Rifampicin resistant

Control Test (no compound)

Control Test (no compound)

25 (19)

19 (22)

22 (17)

832 (981)

18 10 10

22 56 85

29 24 24

234 385 589

10 9 (33) 20 (25)

26 5 (13) 24 (33)

24 16 (58) 12 (15)

650 290 (753) 102 (142)

6 6 14 (17)

21 85 283 (430)

2 2 5 (6)

76 703 214 (325)

2.5 200 100 5

10 10 5000 1.0 0.5 5.0

The following stock solutions were used in test: (A) Tester strain, TA98 grown overnight at 37°C in Difco nutrient broth (0.8% broth, 0.5% NaCI) with 100 p.g/ml ampicillin. (B) Minimal media, 2 x concentrated V o g e l - B o n n e r minimal m e d i u m E, with 4% glucose, 2 m g / m l histidine and 25 # g / m l biotin. (C) Test c o m p o u n d s , in DMSO or sterile water such that the desired dose could be delivered in 10 #1. (D) Bacterial test suspension, mixture of A, B and sterile water (4:10:6) incubated at 37°C for 30 min prior to test.

Procedure Test c o m p o u n d s are delivered to sterile 18 m m x 150 m m test tubes. To each tube 500 #1 of stock D is added. The tubes are placed on rotator and incubated at 37°C for 2 h. 10 ml of ice-cold nutrient b r o t h / D M S O (9.1:0.9) is then added to each tube and centrifuged at 5°C for 10 min at 1000 g. The supernatant is decanted off a n d the cells resuspended in 10 ml nutrient broth (with 100 # g / m l ampicillin) and allowed to grow overnight at 37°C to steady state population. 0.1-ml aliquots were then assayed for mutants. Histidine revertants were scored on minimal media plates without histidine (Ames et al., 1975) using standard plating procedures with the aid o f 2 ml top agar (0.7% Difco) with 125 # g / m l biotin. The same plating procedure (the plates used were not more than 2 weeks old) was used to score rifampicinresistant mutants, except the top agar was supplemented with 125 ttg/ml biotin, 1 m g / m l histidine and 1.25 m g / m l rifampicin. To score total bacteria, a 0.1 ml of 250000-fold diluted sample in ice-cold nutrient broth was assayed on nutrient agar (1.5% Difco) plates. These values were used to determine the exact number of m u t a n t cells produced/108 total survivors and are reported here in parentheses next to the actual experimental number of m u t a n t colonies counted per plate. These two numbers vary because bacterial cell density m a y vary somewhat from sample to sample and is not always exactly 108 cells/0.1 ml.

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ward mutants of the various drug-resistant types are scored on minimal media plates supplemented with the appropriate drug as well as histidine and biotin. A m o n g the various forward mutations we have studied, one in particular has shown excellent promise for screening mutagens. This is the R N A polymerase mutation resulting in rifampicin resistance which yielded very well-defined colonies (Wehrli et al., 1968). We have utilized this forward mutation and the histidine backmutation as indicators in the mutagenicity tests reported here. S. typhimurium TA98 was used as the tester strain. In the Ames test, TA98, like its predecessor TA1538, detects a large number of frameshift mutagens, but is not sensitive to mutagens of the base pair substitution type (McCann et al., 1975a). The latter require either TA100 or TA1535 for their detection. However, using the multiple indicator protocol described here, it is possible to detect - as well as distinguish - both these classes of compounds. The results of multiple indicator tests of several direct-acting (which do not require microsomal activation) mutagens are shown on Table 1. A m o n g these, all except 2-nitrofluorene, respond primarily towards TA100 or TA1535 in the Ames test.

TABLE 2 DETECTION OF ACTIVATABLE MUTAGENS BY TA98 IN THE SALMONELLA/M1CROSOME MULTIPLE INDICATOR TEST Compound

Amount tested Og)

Number of mutants/plate or /(108 survivors) Histidine revertant

Rifampicin resistant

Control

Control

Test

(no

(no

compound)

compound)

2-Acetamidofluorene 6-Aminochrysene 2-Aminoanthracene Aflatoxin B~

I0 0.5 1.0 0.1

17 13 25 23

(29) (25) (20) (25)

Benzo[a]pyrene 3-Methylcholanthrene 3,3-Dichlorobenzidine 'Difco' beef extract

25 10 10 20 mg

17 (29) 17 (16) 47 15 (12)

351 193 366 195

Test

(471) (377) (572) (282)

17 (29) 18 (34) 15 (12) 10 (11)

236 768 965 200

(451) (1499) (1507) (289)

61 (90) 17 (19) 682 116 (85)

17 (29) 15 (16) 25 (20)

108 (159) 43 (47) 52 (38)

The following stocks were used: (E) Rat-liver homogenate, Aroclor-induced rat-liver homogenate ($9) prepared according to procedures used in standard Ames test (Ames et al., 1975) and tested for sterility on standard nutrient broth plates. (F) Cofactors, 10 mg/ml glucose 6-phosphate, 10 mg/ml NADP, and 6.7 mg/ml MgC1.6H20 made up in ice-cold solution of B (footnote Table 1) sterilized by Millipore filtration. (G) Microsomal mix, ice cold mixture E, F and sterile water (0.2:1.8:1). (H) Bacterial suspension, mixture of A, B (footnote Table 1) and sterile water (8:10:2) and incubated at 37°C for 30 min prior to use. (I) Bacteria/microsomal mix, mixture of G and H (1:1). Procedure: The test procedure is exactly the same as described under Table 1, except D is replaced by H.

36 In the same test, to a lesser degree, 1,3-dichloropropene, captan, 4-nitrobiphenyl, and 4-NQO are also positive towards TA98 (McCann et al., 1975b). As expected, we also get similar results in our tests in the histidine backmutation scoring. However, all these compounds show forward mutations to a significant degree in TA98 which can be easily detected by rifampicin-resistance scoring. 2-Nitrofluorene, which is sensitive to TA98 in Ames test, but not to TA100 or TA1535, is positive in both the histidine-reversion and rifampicin-resistance parts of the assay. Like the Ames test, the multiple indicator test can easily be adapted for detection o f mutagens which require metabolic activation. The results of multiple indicator tests in the presence of Aroclor-induced rat-liver microsomes ($9) are shown in Table 2. All these mutagens are easily detected in the presence o f microsomal activation by forward a n d / o r backmutation. Finally, when using Ames test to assay mutagens in samples which contain excess histidine, such as commercial beef extract, it is necessary to use extraction procedures to remove histidine, prior to the mutagenicity testing. However, as shown in Table 2, a simple Millipore filter sterilized aqueous solution o f commercial beef extract can be successfully tested for mutagenic activity in the multiple indicator test without any prior extraction procedures to remove histidine. The multiple indicator (MI) test retains almost all the advantages of the Ames test. At the same time, it overcomes some of its drawbacks. Although the MI test requires a few extra steps and takes one additional day to complete, the overall simplicity of the two tests is similar. The MI test is carried out in 0.5-ml bacterial suspension as opposed to approximately 20 ml agar in the Ames test. Because of this we have found that the overall sensitivity (minimum amount of mutagen required for detection) of our test is at least equal to or - in some cases, better than the Ames test. (Since the MI test can be carried out in the presence o f histidine, it is potentially useful for testing body fluids like blood and urine.) The MI test requires the use of only one tester strain; the results obtained are more quantitative (mutants/same number o f survivors); and mutations are detected in more than one locus. In addition to rifampicin resistance, this protocol can be easily adapted to score simultaneously other forward mutations including 8-azaguanine resistance (Skopek et al., 1978). All these features, we believe, will make the Salmonella/ microsome multiple indicator test particularly suitable for screening mutagenicity of chemicals and environmental carcinogens.

Acknowledgements We are grateful to the Chiles Foundation, Portland, Oregon for providing financial support for this work. We also thank Dr. Bruce Ames for supplying the tester strains, Dr. L. Akileswaran, J.I. Henry, B. Agum, M. Strasik, L. Strasik and P. Pankratz for their assistance.

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References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with Salmonella/mammalian microsome mutagenicity test, Mutation Res., 31, 347-364. McCann, J., N.E. Spingarn, J. Kobori and B.N. Ames (1975a) Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids, Proc. Natl. Acad. Sci. (U.S.A.), 72, 979-983. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975b) Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Skopek, T.R., H.L. Liber, J.J. Krolewski and W.G. Thilly (1978) Quantitative forward mutation assay in Salmonella typhimurium using 8-azaguanine resistance as a genetic marker, Proc. Natl. Acad. Sci. (U.S.A.), 75,410-414. Wehrli, W., F. Knusel, K. Schmid and M. Staehelin (1968) Interaction of rifamycin with bacterial RNA polymerase, Proc. Natl. Acad. Sci. (U.S.A.), 61, 667-673.