APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1978, p. 794-797 0099-2240/78/0036-0794$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 36, No. 6
Printed in U.S.A.
Improvement in the Sensitivity of DNA Polymerase IDeficient Escherichia coli for Detecting Mutagens and Carcinogens SANDRA VENTURINI* AND C. MONTI-BRAGADIN Institute ofMicrobiology, University of Trieste, Trieste, Italy Received for publication 18 September 1978
The sensitivity of a polA strain to the antibacterial activity of mutagens and carcinogens may be increased by inserting one or both of the following characteristics, a lexA mutation or the R391 bacterial plasmid. The effects of the lexA mutation and the plasmid appear to be additive. The differential sensitivity of a polA lexA (R391) strain could be adapted as a preliminary screening test for mutagens and potential carcinogens. The differential sensitivity of repair-deficient Sigma, except cis-platinum dichlorodiamine, which Escherichia coli strains has been proposed as was a gift of G. Mestroni, University of Trieste. Antibacterial plate test. Substances under test an alternative to mutagenesis tests for inferring the interaction between different compounds were diluted in water or dimethyl sulfoxide. Samples of 50 p1 of appropriate dilutions were used to fill wells, and bacterial DNA (1, 7, 13' 1,5, 16). mmn in diameter, cut into poured plates of 20 ml of Strains lacking polymerase I activity (15, 16), 8minimal agar plus tryptophan (20 Ag/ml) with excision repair (1), or recombination repair (7, a lawn ofmedium bacteria. Plates were incubated for 16 h at strains In have been used. 13) principle, carrying 37°C, and the zone of growth inhibition was measured. multiple defects could be very suitable for this Antibacterial test in liquid medium. A 0.1-ml kind of test. Some combinations of defects, how- sample of an overnight culture of tester strain in ever, are not viable, like recA polA (19), whereas Davis-Mingioli liquid minimal medium with tryptophan (20 jg/ml) was added to 10 ml of the same others are not additive, like uvrA poLA (10). In the present paper it is shown that the medium with appropriate concentrations of the chemsensitivity of a poL4 strain may be increased by icals under test. Percentage of growth relative to conwithout added chemicals was estimated by optiinserting one or both of the following character- trols density reading after 16 h of incubation at 37°C. istics, a lexA mutation or the R391 bacterial calMetabolic activation. Bacteria were inoculated at plasmid (4). a concentration of approximately 5 x 104 bacteria per lexA strains lack the function of one sector of ml in liquid minimal medium plus tryptophan (20 postreplication repair (12), and the R391 plasmid ,ug/ml) with the addition of S9-mix (1:10, vol/vol) and was chosen because it is the only one, among an appropriate concentration of chemicals. Survivors more than 70 plasmids tested, that has a mutator were counted as colony-forming units per milliliter effect and increases the UV sensitivity of strains after 3 h of incubation at 37°C. S9-mix was prepared as described by Ames et al. (2) and contained a rat carrying it (unpublished data). liver fraction plus the following cofactors: nicotinamide adenine dinucleotide phosphate, 0.1 mM; glucose-6-phosphate, 5 mM; KCl, 33 mM; and MgCl2, 8 mM, in 0.1 M phosphate buffer (pH 7.4). The mix was added in a proportion of 1:10 to the incubation mixture.
MATERIALS AND METHODS Bacterial strains. The derivation and characters of the bacterial strains used are listed in Table 1. The R391 plasmid received from N. Datta (Hammersmith Hospital, London) determines kanamycin resistance and was transferred to the different strains by conjugation with selection of transconjugants on DavisMingioli (5) minimal medium agar with added tryptophan (10 ,ug/ml) and kanamycin (20 ,ig/ml). The polA mutation was introduced into metE strains by P1 transduction from the polymerase I-deficient strain p3478. Sonic extracts of the so-derived strains, TM930 (WP2poLA) and TM970 (WP2 lexA polA), were shown to lack any DNA polymerase activity when tested by the technique of De Lucia and Cairns (6). Chemicals. All chemicals used were obtained from
RESULTS AND DISCUSSION Figure 1 shows the diameter of the halo of inhibition produced with the above strains by various amounts of known mutagens and carcinogens (8) (methyl methane sulfonate, ethyl methane sulfonate, mitomycin C, metronidazole). As expected, according to previous reports (15, 16), strain TM930 showed larger inhibition zones than the wild-type strain, due to the higher 794
SENSITIVITY OF polA E. COLI
VOL. 36, 1978
795
40
E E
1
0 c 0
._
1.25
.
2.5
5
5
15
45
Q
~0 40
E a~
6
30
000
Drug amount: jig FIG. 1. Antibacterial activity of various drugs on E. coli strains WP2 (0), TM930 polA (O), TM970 polA lexA (A), TM1079poUA (R391) (A), and TM1080 polA lexA (R391) (0). (A) Methyl methane sulfonate; (B) ethyl methane sulfonate; (C) mitomycin C; (D) metronidazole. Amounts of methyl methane sulfonate are in micromoles; amounts of ethyl methane sulfonate are in milligrams.
APPL. ENVIRON. MICROBIOL.
VENTURINI AND MONTI-BRAGADIN
796
sensitivity of strains lacking polymerase I activity to DNA modifying agents. This effect was much more pronounced if the strain also carried, in addition to the poA defect, a lexA mutation, the R391 plasmid, or both. The effects of the lexA mutation and the plasmid thus appear to be additive. It is also evident that larger halos are produced on TM970, TM1079, and TM1080 than on TM930. In preliminary tests it was shown that our test using TM1080 was more sensitive than similar tests using uvrA recA and uvrA lexA strains. Although addition of the R391 plasmid to E. coli p3478 increases the sensitivity of this strain to chemicals interacting with DNA, it was found that E. coli p3478 (R391) is less sensitive than TM1080. Moreover, drug amounts too low to produce any halo on WP2 or TM930 gave quite discernible zones of inhibition on the other TABLE 1. Characteristics of strains used Genetic markers mid) (plas-
Source and reference
WP2 CM561 p3478 TM900
trp trp lexA thy polA trp metE
PT16
trp lexA metE
TM930
trp poUA
TM970
trp lexA polA
Venitt (3) Venitt (3) Cairns (6) EMSa mutagenesis from WP2 EMS mutagenesis from CM561 Plp3478 x TM900 (select met+) Plp3478 x PT16 (select met+) TM930 x J53 (R391) TM970 x J53 (R391)
Strain
TM1079 trp polA (R391) TM1080 trp lexA polA (R391) a EMS, Ethyl methane sulfonate.
strains. Similar results (not shown) were obtained with other mutagens and carcinogens, such as 4-nitroquinoline-1-oxide, hycanthone, ethidium bromide, 1-(5-nitrofurfurylidene)amino-hydantoin (8), K2Cr2O7 (17), cis-platinum dichlorodiamine (11), and daunomycin (8, 14). It is worth noting at this point that the antibacterial activity of a drug not mediated by DNA interaction generated equal inhibition zones in all strains including WP2 wild type. TABLE 2. Killing activity of 2-amino-fluorene with metabolic activation on various E. coli strains CFU/mla with E. coli strain: TM1080
WP2
TM930 poL4 polA
lexA plA (R391)
Control (no addition) Control (S9-mix) 2-AFb (0.2 to 4 lug/ml) 2-AF (0.2 tig/ml) +
2.5 x 105
3 x 105
3 x 105
ND
ND
4 x 105
2-AF (0.5 yg/ml) +
ND
ND
6.2 x 104
S9-mix 2-AF (1 ug/ml) + S9-mix
ND
Addition to the culture
4 x 105 3.6 x 105 4.5 x 105 2.7 x 105 2.8 x 105 2.9 x 105
S9-mix
2-AF (2 ,ug/ml) +
4 x 105
4 x 104
4.2 x 105 3.1 x 105
2 x 103
3.9 x 105 5.4 x 104
1 x
S9-mix 2-AF (4 ,ug/ml) +
102
S9-mix aAfter 3 h of incubation at 37°C. CFU, Colony-
forming units. ND, Not done. b 2-AF, 2-Amino-fluorene. -I
100
in
bl
-C
0 h.
501 *1
0 u
0
I 0
I
20
llI
II
II
60
100
50
I
100
150
200
Drug concentration : jig/ml FIG. 2. Antibacterial activity of methyl methane sulfonate (A) and metronidazole (B) in liquid medium on E. coli strains WP2 (0), TM930poA (Ol), and TM1080poLA lexA (R391) (0).
VOL. 36, 1978
This has been found to hold true for the following antibiotics and disinfectants: streptomycin, chloramphenicol, nalidixic acid, ethanol, and benzalkonium chloride. DNA-modifying agents show their preferential antibacterial activity on TM930 and, in a much more marked manner, in TM1080 even in liquid culture, as shown in Fig. 2. In this way, the effect of drugs requiring metabolic microsomal activation may be tested, as shown in Table 2 for 2-amino-fluorene. Tests based on differential sensitivity of strains lacking repair functions have two major advantages over tests for mutagenicity, e.g., the Salmonella/microsome assay (1, 2). First, being more simple to perform, at least when carried out as a spot test, they may be used as prescreening tests for identifying substances to be submitted for more sophisticated test systems. Second, they may offer the main evidence for the interaction of a substance with bacterial DNA for strongly bactericidal agents. From the above results it is clear that, using a strain such as TM1080 with multiple defects and carrying the mutator plasmid R391, a quick and easy test could be adopted as a preliminary screening test for mutagens and potential carcinogens. Validation of this test on a wider range of substances is in progress. ACKNOWLEDGMENTS This study was supported by C. N. R., progetto finalizzato "Promozione della qualita dell'ambiente." S. V. is the recipient of a fellowship from Anna Villa Rusconi Foundation, Varese, Italy. LITERATURE CITED 1. Ames, B. N., F. D. Lee, and W. E. Durston. 1973. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. U.S.A. 70:782-786. 2. Ames, B. N., J. McCann, and E. Yamasaki. 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31:347-364.
SENSITIVITY OF polA E. COLI
797
3. Bridges, B. A., R. P. Mottershead, M. A. Rothwell, and M. H. L. Green. 1972. Repair deficient bacterial strains suitable for mutagenic screening: test with the fungicide captan. Chem. Biol. Interact. 5:77-84. 4. Coetzee, J. N., N. Datta, and R. W. Hedges. 1972. R factors from Proteus rettgeri. J. Gen. Microbiol. 72: 543-552. 5. Davis, B., and E. S. Mingioli. 1950. Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60:17-28. 6. De Lucia, P., and J. Cairns. 1969. Isolation of an Escherichia coli strain with a mutation affecting DNA polymerase. Nature (London) 224:1164-1166. 7. Kada, T., M. Moriya, and Y. Shirasu. 1974. Screening of pesticides for DNA interactions by "Rec-assay" and mutagenesis testing, and frameshift mutagens detected. Mutat. Res. 26:243-248. 8. McCann, J., E. Choi, E. Yamasaki, and B. N. Ames. 1975. Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A. 72:5135-5139. 9. Monk, M., and J. Kinrose. 1972. Conditional lethality of recA and recB derivatives of a strain of Escherichia coli K-12 with a temperature-sensitive deoxyribonucleic acid polymerase I. J. Bacteriol. 109:971-978. 10. Monk, M., M. Peacey, and J. D. Gross. 1971. Repair of damage induced by ultraviolet light in DNA polymerase-defective Escherichia coli cells. J. Mol. Biol. 58: 623-630. 11. Monti-Bragadin, C., M. Tamaro, and E. Banfi. 1975. Mutagenic activity of platinum and rutherium complex. Chem. Biol. Interact. 11:469-472. 12. Moody, E. E. M., K. B. Low, and D. W. Mount. 1973. Properties of strains of Escherichia coli K12 carrying mutant lex and rec alleles. Mol. Gen. Genet. 121: 197-205. 13. Nishioka, H. 1975. Mutagenic activities of metal compounds in bacteria. Mutat. Res. 31:185-189. 14. Pani, B., C. Monti-Bragadin, and L. Samer. 1975. Effect of excision repair system on antibacterial and mutagenic activity of daunomycin and other intercalating agents in Salmonella typhimurium. Experientia 31: 787-788. 15. Rosenkranz, H. S., B. Gutter, and W. T. Speck. 1976. Mutagenicity and DNA-modifying activity: a comparison of two microbial assays. Mutat. Res. 41:61-70. 16. Slater, E. E., M. D. Anderson, and H. S. Rosenkranz. 1971. Rapid detection of mutagens and carcinogens. Cancer Res. 31:970-973. 17. Venitt, S., and L. S. Levy. 1974. Mutagenicity of chromates in bacteria and its relevance to chromate carcinogenesis. Nature (London) 250:493-495.
Vol. 36, No. 6
Printed in U.S.A.
Improvement in the Sensitivity of DNA Polymerase IDeficient Escherichia coli for Detecting Mutagens and Carcinogens SANDRA VENTURINI* AND C. MONTI-BRAGADIN Institute ofMicrobiology, University of Trieste, Trieste, Italy Received for publication 18 September 1978
The sensitivity of a polA strain to the antibacterial activity of mutagens and carcinogens may be increased by inserting one or both of the following characteristics, a lexA mutation or the R391 bacterial plasmid. The effects of the lexA mutation and the plasmid appear to be additive. The differential sensitivity of a polA lexA (R391) strain could be adapted as a preliminary screening test for mutagens and potential carcinogens. The differential sensitivity of repair-deficient Sigma, except cis-platinum dichlorodiamine, which Escherichia coli strains has been proposed as was a gift of G. Mestroni, University of Trieste. Antibacterial plate test. Substances under test an alternative to mutagenesis tests for inferring the interaction between different compounds were diluted in water or dimethyl sulfoxide. Samples of 50 p1 of appropriate dilutions were used to fill wells, and bacterial DNA (1, 7, 13' 1,5, 16). mmn in diameter, cut into poured plates of 20 ml of Strains lacking polymerase I activity (15, 16), 8minimal agar plus tryptophan (20 Ag/ml) with excision repair (1), or recombination repair (7, a lawn ofmedium bacteria. Plates were incubated for 16 h at strains In have been used. 13) principle, carrying 37°C, and the zone of growth inhibition was measured. multiple defects could be very suitable for this Antibacterial test in liquid medium. A 0.1-ml kind of test. Some combinations of defects, how- sample of an overnight culture of tester strain in ever, are not viable, like recA polA (19), whereas Davis-Mingioli liquid minimal medium with tryptophan (20 jg/ml) was added to 10 ml of the same others are not additive, like uvrA poLA (10). In the present paper it is shown that the medium with appropriate concentrations of the chemsensitivity of a poL4 strain may be increased by icals under test. Percentage of growth relative to conwithout added chemicals was estimated by optiinserting one or both of the following character- trols density reading after 16 h of incubation at 37°C. istics, a lexA mutation or the R391 bacterial calMetabolic activation. Bacteria were inoculated at plasmid (4). a concentration of approximately 5 x 104 bacteria per lexA strains lack the function of one sector of ml in liquid minimal medium plus tryptophan (20 postreplication repair (12), and the R391 plasmid ,ug/ml) with the addition of S9-mix (1:10, vol/vol) and was chosen because it is the only one, among an appropriate concentration of chemicals. Survivors more than 70 plasmids tested, that has a mutator were counted as colony-forming units per milliliter effect and increases the UV sensitivity of strains after 3 h of incubation at 37°C. S9-mix was prepared as described by Ames et al. (2) and contained a rat carrying it (unpublished data). liver fraction plus the following cofactors: nicotinamide adenine dinucleotide phosphate, 0.1 mM; glucose-6-phosphate, 5 mM; KCl, 33 mM; and MgCl2, 8 mM, in 0.1 M phosphate buffer (pH 7.4). The mix was added in a proportion of 1:10 to the incubation mixture.
MATERIALS AND METHODS Bacterial strains. The derivation and characters of the bacterial strains used are listed in Table 1. The R391 plasmid received from N. Datta (Hammersmith Hospital, London) determines kanamycin resistance and was transferred to the different strains by conjugation with selection of transconjugants on DavisMingioli (5) minimal medium agar with added tryptophan (10 ,ug/ml) and kanamycin (20 ,ig/ml). The polA mutation was introduced into metE strains by P1 transduction from the polymerase I-deficient strain p3478. Sonic extracts of the so-derived strains, TM930 (WP2poLA) and TM970 (WP2 lexA polA), were shown to lack any DNA polymerase activity when tested by the technique of De Lucia and Cairns (6). Chemicals. All chemicals used were obtained from
RESULTS AND DISCUSSION Figure 1 shows the diameter of the halo of inhibition produced with the above strains by various amounts of known mutagens and carcinogens (8) (methyl methane sulfonate, ethyl methane sulfonate, mitomycin C, metronidazole). As expected, according to previous reports (15, 16), strain TM930 showed larger inhibition zones than the wild-type strain, due to the higher 794
SENSITIVITY OF polA E. COLI
VOL. 36, 1978
795
40
E E
1
0 c 0
._
1.25
.
2.5
5
5
15
45
Q
~0 40
E a~
6
30
000
Drug amount: jig FIG. 1. Antibacterial activity of various drugs on E. coli strains WP2 (0), TM930 polA (O), TM970 polA lexA (A), TM1079poUA (R391) (A), and TM1080 polA lexA (R391) (0). (A) Methyl methane sulfonate; (B) ethyl methane sulfonate; (C) mitomycin C; (D) metronidazole. Amounts of methyl methane sulfonate are in micromoles; amounts of ethyl methane sulfonate are in milligrams.
APPL. ENVIRON. MICROBIOL.
VENTURINI AND MONTI-BRAGADIN
796
sensitivity of strains lacking polymerase I activity to DNA modifying agents. This effect was much more pronounced if the strain also carried, in addition to the poA defect, a lexA mutation, the R391 plasmid, or both. The effects of the lexA mutation and the plasmid thus appear to be additive. It is also evident that larger halos are produced on TM970, TM1079, and TM1080 than on TM930. In preliminary tests it was shown that our test using TM1080 was more sensitive than similar tests using uvrA recA and uvrA lexA strains. Although addition of the R391 plasmid to E. coli p3478 increases the sensitivity of this strain to chemicals interacting with DNA, it was found that E. coli p3478 (R391) is less sensitive than TM1080. Moreover, drug amounts too low to produce any halo on WP2 or TM930 gave quite discernible zones of inhibition on the other TABLE 1. Characteristics of strains used Genetic markers mid) (plas-
Source and reference
WP2 CM561 p3478 TM900
trp trp lexA thy polA trp metE
PT16
trp lexA metE
TM930
trp poUA
TM970
trp lexA polA
Venitt (3) Venitt (3) Cairns (6) EMSa mutagenesis from WP2 EMS mutagenesis from CM561 Plp3478 x TM900 (select met+) Plp3478 x PT16 (select met+) TM930 x J53 (R391) TM970 x J53 (R391)
Strain
TM1079 trp polA (R391) TM1080 trp lexA polA (R391) a EMS, Ethyl methane sulfonate.
strains. Similar results (not shown) were obtained with other mutagens and carcinogens, such as 4-nitroquinoline-1-oxide, hycanthone, ethidium bromide, 1-(5-nitrofurfurylidene)amino-hydantoin (8), K2Cr2O7 (17), cis-platinum dichlorodiamine (11), and daunomycin (8, 14). It is worth noting at this point that the antibacterial activity of a drug not mediated by DNA interaction generated equal inhibition zones in all strains including WP2 wild type. TABLE 2. Killing activity of 2-amino-fluorene with metabolic activation on various E. coli strains CFU/mla with E. coli strain: TM1080
WP2
TM930 poL4 polA
lexA plA (R391)
Control (no addition) Control (S9-mix) 2-AFb (0.2 to 4 lug/ml) 2-AF (0.2 tig/ml) +
2.5 x 105
3 x 105
3 x 105
ND
ND
4 x 105
2-AF (0.5 yg/ml) +
ND
ND
6.2 x 104
S9-mix 2-AF (1 ug/ml) + S9-mix
ND
Addition to the culture
4 x 105 3.6 x 105 4.5 x 105 2.7 x 105 2.8 x 105 2.9 x 105
S9-mix
2-AF (2 ,ug/ml) +
4 x 105
4 x 104
4.2 x 105 3.1 x 105
2 x 103
3.9 x 105 5.4 x 104
1 x
S9-mix 2-AF (4 ,ug/ml) +
102
S9-mix aAfter 3 h of incubation at 37°C. CFU, Colony-
forming units. ND, Not done. b 2-AF, 2-Amino-fluorene. -I
100
in
bl
-C
0 h.
501 *1
0 u
0
I 0
I
20
llI
II
II
60
100
50
I
100
150
200
Drug concentration : jig/ml FIG. 2. Antibacterial activity of methyl methane sulfonate (A) and metronidazole (B) in liquid medium on E. coli strains WP2 (0), TM930poA (Ol), and TM1080poLA lexA (R391) (0).
VOL. 36, 1978
This has been found to hold true for the following antibiotics and disinfectants: streptomycin, chloramphenicol, nalidixic acid, ethanol, and benzalkonium chloride. DNA-modifying agents show their preferential antibacterial activity on TM930 and, in a much more marked manner, in TM1080 even in liquid culture, as shown in Fig. 2. In this way, the effect of drugs requiring metabolic microsomal activation may be tested, as shown in Table 2 for 2-amino-fluorene. Tests based on differential sensitivity of strains lacking repair functions have two major advantages over tests for mutagenicity, e.g., the Salmonella/microsome assay (1, 2). First, being more simple to perform, at least when carried out as a spot test, they may be used as prescreening tests for identifying substances to be submitted for more sophisticated test systems. Second, they may offer the main evidence for the interaction of a substance with bacterial DNA for strongly bactericidal agents. From the above results it is clear that, using a strain such as TM1080 with multiple defects and carrying the mutator plasmid R391, a quick and easy test could be adopted as a preliminary screening test for mutagens and potential carcinogens. Validation of this test on a wider range of substances is in progress. ACKNOWLEDGMENTS This study was supported by C. N. R., progetto finalizzato "Promozione della qualita dell'ambiente." S. V. is the recipient of a fellowship from Anna Villa Rusconi Foundation, Varese, Italy. LITERATURE CITED 1. Ames, B. N., F. D. Lee, and W. E. Durston. 1973. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. U.S.A. 70:782-786. 2. Ames, B. N., J. McCann, and E. Yamasaki. 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31:347-364.
SENSITIVITY OF polA E. COLI
797
3. Bridges, B. A., R. P. Mottershead, M. A. Rothwell, and M. H. L. Green. 1972. Repair deficient bacterial strains suitable for mutagenic screening: test with the fungicide captan. Chem. Biol. Interact. 5:77-84. 4. Coetzee, J. N., N. Datta, and R. W. Hedges. 1972. R factors from Proteus rettgeri. J. Gen. Microbiol. 72: 543-552. 5. Davis, B., and E. S. Mingioli. 1950. Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60:17-28. 6. De Lucia, P., and J. Cairns. 1969. Isolation of an Escherichia coli strain with a mutation affecting DNA polymerase. Nature (London) 224:1164-1166. 7. Kada, T., M. Moriya, and Y. Shirasu. 1974. Screening of pesticides for DNA interactions by "Rec-assay" and mutagenesis testing, and frameshift mutagens detected. Mutat. Res. 26:243-248. 8. McCann, J., E. Choi, E. Yamasaki, and B. N. Ames. 1975. Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A. 72:5135-5139. 9. Monk, M., and J. Kinrose. 1972. Conditional lethality of recA and recB derivatives of a strain of Escherichia coli K-12 with a temperature-sensitive deoxyribonucleic acid polymerase I. J. Bacteriol. 109:971-978. 10. Monk, M., M. Peacey, and J. D. Gross. 1971. Repair of damage induced by ultraviolet light in DNA polymerase-defective Escherichia coli cells. J. Mol. Biol. 58: 623-630. 11. Monti-Bragadin, C., M. Tamaro, and E. Banfi. 1975. Mutagenic activity of platinum and rutherium complex. Chem. Biol. Interact. 11:469-472. 12. Moody, E. E. M., K. B. Low, and D. W. Mount. 1973. Properties of strains of Escherichia coli K12 carrying mutant lex and rec alleles. Mol. Gen. Genet. 121: 197-205. 13. Nishioka, H. 1975. Mutagenic activities of metal compounds in bacteria. Mutat. Res. 31:185-189. 14. Pani, B., C. Monti-Bragadin, and L. Samer. 1975. Effect of excision repair system on antibacterial and mutagenic activity of daunomycin and other intercalating agents in Salmonella typhimurium. Experientia 31: 787-788. 15. Rosenkranz, H. S., B. Gutter, and W. T. Speck. 1976. Mutagenicity and DNA-modifying activity: a comparison of two microbial assays. Mutat. Res. 41:61-70. 16. Slater, E. E., M. D. Anderson, and H. S. Rosenkranz. 1971. Rapid detection of mutagens and carcinogens. Cancer Res. 31:970-973. 17. Venitt, S., and L. S. Levy. 1974. Mutagenicity of chromates in bacteria and its relevance to chromate carcinogenesis. Nature (London) 250:493-495.
