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An assay for mutagenic activity of 4CMB, 4HMB and BC, using the “microtitre” fluctuation test
Mutation Research, 100 (1982) 81-85 Elsevier Biomedical Press
81
AN ASSAY FOR MUTAGENIC ACTIVITY OF 4CMB, 4HMB AND BC, U S I N G T H E " M I C R O T I T R E " FLUCTUATION TEST
M.S.M. POUR, C. MERRILL and JAMES M. PARRY Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP (Great Britain)
(Received 9 April 1981) (Accepted 1 June 1981)
SUMMARY 4CMB, 4 H M B and BC were assayed for mutagenic activity using the 'microtitre' bacterial fluctuation test without metabolic activation. 4CMB was positive in strains of Salmonella typhimurium detecting both base-substitution and frameshift mutation. BC was weakly positive only in the strain which detects basesubstitution mutation. 4 H M B was negative in both strains. 4CMB and 4 H M B were equally toxic to the strains, whilst BC was comparatively less toxic.
4CMB, 4 H M B and BC were tested using the 'microtitre' bacterial fluctuation test (Gatehouse, 1978). The strains used were Salmonella typhimurium strains TA98 and TA100, which are sensitive to frameshift and base-substitution mutagens respectively (Ames et al., 1975). In order to establish a suitable dose range for each c o m p o u n d , a toxicity test was carried out. This was performed by determining the concentration at which, each c o m p o u n d inhibited the growth of strain TA98 in microtitre plates containing supplemented medium which allowed cell growth. MATERIALS AND METHODS Strains TA98 and T A I 0 0 were obtained f r o m Dr. David Tweats. TA98 contains the his D3052 mutation, and is reverted from histidine auxotrophy to histidine independence by frameshift mutation. TA100 contains the his G46 mutation,-and is revertible by base-substitution mutation. Both strains contain the rfa deep-rough mutation, the PKM101 R factor, and a deletion through uvr B, which also includes the biotin gene. They were maintained and checked according to the method of Ames et al. (1975). Chemicals. 4CMB, 4 H M B and BC were all kept at 4°C and were dissolved in 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
82
dimethyl sulphoxide (DMSO) at 4, 5 and 10 mg/ml respectively just before use. These stock solutions were dissolved in the test medium to the required concentration of chemical. Fluctuation test. This was performed according to the method of Gatehouse (1978), with the following modifications. 1. An overnight culture of the bacterial strain was grown up in Davis-Mingioli salts buffer supplemented with histidine (20/~g/ml), biotin (10/~g/ml) and glucose (0.40/o w/v) to 109 cells/ml. The test medium was inoculated directly from this at a concentration of 5 × 105 cells/ml. 2. Bromocresol purple (5 txg/ml) was used as an indicator, and was added at the beginning of the incubation period. The colour change scored was from purple to yellow. Doubtful wells were scored as negative. 3. The wells were scored at 4 days and significance was determined by the x 2 test using the formula described by Green et al. (1976). X 2 --
2 n ( t - c - 1/2)2 (t + c)(2n - t - c)
where n = n u m b e r of wells/plate, t = n u m b e r of positive wells in treated plates, c = number of positive wells in control plates. For significance at 5°7o level x 2> 3.84; for significance at 1°7o level x 2 > 6.63. Toxicity test. This was carried out on TA98 only. A fluctuation test procedure was performed using medium supplemented with histidine (20/zg/ml), and biotin (10/zg/ml) which allowed growth of the cells so that all of the wells in the control plates turned yellow. After 4 days survival at each dose was scored as (number of yellow wells)/96. Toxicity was also assayed on solid plates but no essential differences were observed. Controls. In both fluctuation tests and toxicity tests 4 controls were used per dose range, 2 blank controls and 2 controls containing the amount of DMSO present in the highest dose of compound. The experiments have been performed at least twice, and representative results are shown. RESULTS
Table 1 shows the number of yellow wells present after incubation with increasing doses of the 3 compounds for 4 days. 4CMB and 4HMB inhibited growth completely at a concentration of 150/zg/ml, and BC at 375/~g/ml. Therefore, dose ranges were chosen of 0-125/zg/ml for 4CMB and 4HMB and 0-250 #g/ml for BC. Table 2 shows the results obtained with 4CMB in the fluctuation test using TA98 and TA100. A dose-related increase in the number of positive wells occurred with both TA98 and TAI00. With both strains, increases above control values occurred which were significant at the 1070 level. Table 3 shows the results obtained with 4HMB. No significant increase in positive
83 TABLE 1 DETERMINATION OF SURVIVAL OF TA98 IN MICROTITRE PLATES WITH 4CMB, 4HMB AND BC 4CMB (#g/ml) 0 (dH20) 0 (DMSO)
Number of yellow wells
4HMB (#g/ml)
96, 96 96, 96
Number of yellow wells
0 (dH20) 0 (DMSO)
BC (#g/ml)
96, 96 96, 96
0 (dH20) 0 (DMSO)
Number of yellow wells 96, 96 96, 96
25 50
96 96
25 50
96 96
25 50
96 96
75 100
96 96
75 100
96 96
75 100
96 96
125 150
56 0
125 150
68 0
150 250
96 96
375 500
0 0
TABLE 2 RESULTS OF THE FLUCTUATION TEST WITH 4CMB USING TA98 AND TAI00 TAI00
Number of +ve wells
X 2 significance
4CMB (#g/ml)
TA98
Number of +ve wells
X2 significance
4CMB (/zg/ml)
0 (dH20) 0 (DMSO)
16, 1~}12 12,
1 5
2i 11
10 20
22 31
35 50
55 19
75 lO0 125
1%
0 (dH20) 0 (DMSO)
2,~} 1, 3
1 5
8 8
10 20
21 26
+ +
35 50
38 53
+ +
2 0
75 lO0
46 30
+ +
0
125
0
+ +
5%
1%
5%
+ , significant.
wells above the control values was observed with either strain. Table 4 shows the results obtained with BC. With TA100, 2 o f the doses gave weakly positive responses, the increases over the control being significant at the 5% level. There was no significant increase with TA98.
84 TABLE 3 RESULTS OF T H E F L U C T U A T I O N TEST W I T H 4 H M B USING TA98 A N D TA100 TA100
Number of +ve wells
X 2 significance
4HMB (#g/ml)
TA98
Number of +ve wells
x 2 significance
4HMB (#g/ml)
0 (dH20) 0 (DMSO)
13, 13/14 12, 18/
0 (dH20) 0 (DMSO)
3, ~}2 2,
l 5
14 12
l 5
5. 4
10 20
12 15
10 20
6 2
35 50
l0 12
35 50
4 3
75 100 125
16 0 0
75 100 125
0 0 0
1%
5%
1%
5%
TABLE 4 RESULTS OF T H E F L U C T U A T I O N TEST W I T H BC USING TA98 A N D TA100 TAI00
Number of +ve wells
X 2 significance
BC (tzg/ml) 0 (dH20) 0 (DMSO)
Number of +ve wells
X2 significance
BC (~g/ml) 22, 27 ~ 22 17, 23 !
1 5 l0
11 27 25
20 35 50
23 37 29
75 100 125
33 30 28
150 175 200
38 19 29
225 250
0 0
+ , significant.
TA98
1%
5%
+
+
0 (dH20) 0 (DMSO)
6, 8 4 2, 2 /
1 5 10
5 0 5
20 35 50
3 3 6
75 100 125
7 4 5
150 175 200
2 0 0
225 250
0 0
1%
5%
85 CONCLUSION The method of toxicity testing used here, although useful in determining the maximum dose level for the fluctuation test, is not an efficient method of making comparisons of the toxic effects of the 3 compounds. It indicates, however, that 4CMB and 4HMB were equally toxic to strains TA98, and that BC was comparatively less toxic. 4CMB showed clear positive dose responses with both TA98 and T A 1 0 0 . 4 H M B , although apparently equally toxic was negative in both strains. The positive result obtained with BC in TA100 was for 2 non-adjacent doses, and only at the 5°7o significance level, and can therefore be regarded as doubtful. In repeat experiments (data not shown) BC did not show any positive results with either strain, and the results for 4CMB and 4HMB were similar to those shown. The results show 4CMB to be a potent base-substitution and frameshift mutagen. ACKNOWLEDGEMENT Part of this work was supported by a grant from the EEC Environmental Programme.
REFERENCES Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res., 31,347-364. Gatehouse, D. (1978) Detection of mutagenic derivations of cyclophosphamideand a variety of other mutagens in a 'microtitre' fluctuation test, without microsomal activation, Mutation Res., 53, 289-308. Green, M.H.L., W.J. Muriel and B.A. Bridges (1976) Use of a simplified fluctuation test to detect low levels of mutagens, Mutation Res., 38, 33-41.
81
AN ASSAY FOR MUTAGENIC ACTIVITY OF 4CMB, 4HMB AND BC, U S I N G T H E " M I C R O T I T R E " FLUCTUATION TEST
M.S.M. POUR, C. MERRILL and JAMES M. PARRY Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP (Great Britain)
(Received 9 April 1981) (Accepted 1 June 1981)
SUMMARY 4CMB, 4 H M B and BC were assayed for mutagenic activity using the 'microtitre' bacterial fluctuation test without metabolic activation. 4CMB was positive in strains of Salmonella typhimurium detecting both base-substitution and frameshift mutation. BC was weakly positive only in the strain which detects basesubstitution mutation. 4 H M B was negative in both strains. 4CMB and 4 H M B were equally toxic to the strains, whilst BC was comparatively less toxic.
4CMB, 4 H M B and BC were tested using the 'microtitre' bacterial fluctuation test (Gatehouse, 1978). The strains used were Salmonella typhimurium strains TA98 and TA100, which are sensitive to frameshift and base-substitution mutagens respectively (Ames et al., 1975). In order to establish a suitable dose range for each c o m p o u n d , a toxicity test was carried out. This was performed by determining the concentration at which, each c o m p o u n d inhibited the growth of strain TA98 in microtitre plates containing supplemented medium which allowed cell growth. MATERIALS AND METHODS Strains TA98 and T A I 0 0 were obtained f r o m Dr. David Tweats. TA98 contains the his D3052 mutation, and is reverted from histidine auxotrophy to histidine independence by frameshift mutation. TA100 contains the his G46 mutation,-and is revertible by base-substitution mutation. Both strains contain the rfa deep-rough mutation, the PKM101 R factor, and a deletion through uvr B, which also includes the biotin gene. They were maintained and checked according to the method of Ames et al. (1975). Chemicals. 4CMB, 4 H M B and BC were all kept at 4°C and were dissolved in 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
82
dimethyl sulphoxide (DMSO) at 4, 5 and 10 mg/ml respectively just before use. These stock solutions were dissolved in the test medium to the required concentration of chemical. Fluctuation test. This was performed according to the method of Gatehouse (1978), with the following modifications. 1. An overnight culture of the bacterial strain was grown up in Davis-Mingioli salts buffer supplemented with histidine (20/~g/ml), biotin (10/~g/ml) and glucose (0.40/o w/v) to 109 cells/ml. The test medium was inoculated directly from this at a concentration of 5 × 105 cells/ml. 2. Bromocresol purple (5 txg/ml) was used as an indicator, and was added at the beginning of the incubation period. The colour change scored was from purple to yellow. Doubtful wells were scored as negative. 3. The wells were scored at 4 days and significance was determined by the x 2 test using the formula described by Green et al. (1976). X 2 --
2 n ( t - c - 1/2)2 (t + c)(2n - t - c)
where n = n u m b e r of wells/plate, t = n u m b e r of positive wells in treated plates, c = number of positive wells in control plates. For significance at 5°7o level x 2> 3.84; for significance at 1°7o level x 2 > 6.63. Toxicity test. This was carried out on TA98 only. A fluctuation test procedure was performed using medium supplemented with histidine (20/zg/ml), and biotin (10/zg/ml) which allowed growth of the cells so that all of the wells in the control plates turned yellow. After 4 days survival at each dose was scored as (number of yellow wells)/96. Toxicity was also assayed on solid plates but no essential differences were observed. Controls. In both fluctuation tests and toxicity tests 4 controls were used per dose range, 2 blank controls and 2 controls containing the amount of DMSO present in the highest dose of compound. The experiments have been performed at least twice, and representative results are shown. RESULTS
Table 1 shows the number of yellow wells present after incubation with increasing doses of the 3 compounds for 4 days. 4CMB and 4HMB inhibited growth completely at a concentration of 150/zg/ml, and BC at 375/~g/ml. Therefore, dose ranges were chosen of 0-125/zg/ml for 4CMB and 4HMB and 0-250 #g/ml for BC. Table 2 shows the results obtained with 4CMB in the fluctuation test using TA98 and TA100. A dose-related increase in the number of positive wells occurred with both TA98 and TAI00. With both strains, increases above control values occurred which were significant at the 1070 level. Table 3 shows the results obtained with 4HMB. No significant increase in positive
83 TABLE 1 DETERMINATION OF SURVIVAL OF TA98 IN MICROTITRE PLATES WITH 4CMB, 4HMB AND BC 4CMB (#g/ml) 0 (dH20) 0 (DMSO)
Number of yellow wells
4HMB (#g/ml)
96, 96 96, 96
Number of yellow wells
0 (dH20) 0 (DMSO)
BC (#g/ml)
96, 96 96, 96
0 (dH20) 0 (DMSO)
Number of yellow wells 96, 96 96, 96
25 50
96 96
25 50
96 96
25 50
96 96
75 100
96 96
75 100
96 96
75 100
96 96
125 150
56 0
125 150
68 0
150 250
96 96
375 500
0 0
TABLE 2 RESULTS OF THE FLUCTUATION TEST WITH 4CMB USING TA98 AND TAI00 TAI00
Number of +ve wells
X 2 significance
4CMB (#g/ml)
TA98
Number of +ve wells
X2 significance
4CMB (/zg/ml)
0 (dH20) 0 (DMSO)
16, 1~}12 12,
1 5
2i 11
10 20
22 31
35 50
55 19
75 lO0 125
1%
0 (dH20) 0 (DMSO)
2,~} 1, 3
1 5
8 8
10 20
21 26
+ +
35 50
38 53
+ +
2 0
75 lO0
46 30
+ +
0
125
0
+ +
5%
1%
5%
+ , significant.
wells above the control values was observed with either strain. Table 4 shows the results obtained with BC. With TA100, 2 o f the doses gave weakly positive responses, the increases over the control being significant at the 5% level. There was no significant increase with TA98.
84 TABLE 3 RESULTS OF T H E F L U C T U A T I O N TEST W I T H 4 H M B USING TA98 A N D TA100 TA100
Number of +ve wells
X 2 significance
4HMB (#g/ml)
TA98
Number of +ve wells
x 2 significance
4HMB (#g/ml)
0 (dH20) 0 (DMSO)
13, 13/14 12, 18/
0 (dH20) 0 (DMSO)
3, ~}2 2,
l 5
14 12
l 5
5. 4
10 20
12 15
10 20
6 2
35 50
l0 12
35 50
4 3
75 100 125
16 0 0
75 100 125
0 0 0
1%
5%
1%
5%
TABLE 4 RESULTS OF T H E F L U C T U A T I O N TEST W I T H BC USING TA98 A N D TA100 TAI00
Number of +ve wells
X 2 significance
BC (tzg/ml) 0 (dH20) 0 (DMSO)
Number of +ve wells
X2 significance
BC (~g/ml) 22, 27 ~ 22 17, 23 !
1 5 l0
11 27 25
20 35 50
23 37 29
75 100 125
33 30 28
150 175 200
38 19 29
225 250
0 0
+ , significant.
TA98
1%
5%
+
+
0 (dH20) 0 (DMSO)
6, 8 4 2, 2 /
1 5 10
5 0 5
20 35 50
3 3 6
75 100 125
7 4 5
150 175 200
2 0 0
225 250
0 0
1%
5%
85 CONCLUSION The method of toxicity testing used here, although useful in determining the maximum dose level for the fluctuation test, is not an efficient method of making comparisons of the toxic effects of the 3 compounds. It indicates, however, that 4CMB and 4HMB were equally toxic to strains TA98, and that BC was comparatively less toxic. 4CMB showed clear positive dose responses with both TA98 and T A 1 0 0 . 4 H M B , although apparently equally toxic was negative in both strains. The positive result obtained with BC in TA100 was for 2 non-adjacent doses, and only at the 5°7o significance level, and can therefore be regarded as doubtful. In repeat experiments (data not shown) BC did not show any positive results with either strain, and the results for 4CMB and 4HMB were similar to those shown. The results show 4CMB to be a potent base-substitution and frameshift mutagen. ACKNOWLEDGEMENT Part of this work was supported by a grant from the EEC Environmental Programme.
REFERENCES Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res., 31,347-364. Gatehouse, D. (1978) Detection of mutagenic derivations of cyclophosphamideand a variety of other mutagens in a 'microtitre' fluctuation test, without microsomal activation, Mutation Res., 53, 289-308. Green, M.H.L., W.J. Muriel and B.A. Bridges (1976) Use of a simplified fluctuation test to detect low levels of mutagens, Mutation Res., 38, 33-41.