Chem.-Al&d. httefact&ms.1I (1975) 63-65 0 Elwvicr ScimtitlcPublbhin~Company, Armtcdm -RintcdinThcNcthufad
Uw
of mpalr-dofkbnt
mubgamia
Ed;
m&a
and Uvw nboaomm
63
to cbmcbdm
bydimothylnhtwa~
M. H. L. GREENANDW.J. MURIEL MRC Cdl Mwtorton Udt, lJntw&y o/Sussex, Fakr,
(Reded
lb&hmn BNi 9QG (Great B&ah)
October 14th. 1974)
(Revisionwived January20th. 1975) (Accepted February20th. 1975)
DMN is a metabolically activated carcinogenl, and it is mutagenic to Solmonello in the presence of a liver microsomal extra&s. It is thought that DMN is metabolized to monomethylnitrosamine which is unstable and alkylates DNA via the “carbonium ion” (CHs;) (ref. 4). If so, DMN should be identical to MNNG or MNU in its effect as a bacterial mutagem. Such a prediction can be tested using repairdeficient strains of Escherichio cd. MNNG and MNU cause mutation by misreplication as well as misrepair (i.e., they mutate exrA strains of E.coli nearly as efficiently as they mutate exrA+ strainW). Moreover the mutagenic lesions are not subject to excision by urr endonuclease (i.e., mutation is equal in a uvr and a uvr+ straitW). Other alkylating agents give different patterns of response; for instance mutations arise almost entirely by misrepair of non-excisable damage in the case of methylmethanesulphonate’, by misrepair of excisable damage in the case of captans, and by misreplication of excisable damage in the case of the volatile breakdown product of captan*. Finally the difunctional alkylating agent mitomycin C is mutagenic only in typhimurium
an excision-proficient strain@. We have, therefore, examined DMN mutagenesis using suitable E.coli strains. We have confirmed the observation of Ames ef al.‘0 that DMN is not mutagenic when incubated with liver microsomes in soft agar, although it is mutagenic using the
Mailing* system of incubation with microsomes in liquid and bubbling air through the mixture. This aeration did not seem helpful and we adapted the method described in Table 1. Escherichlo calf strain WP2 and its derivatives WP2 uvrA and CM611 WA cxrA were tested (Table I). There was considerable variation probably due to differences in the efficiency of liver activation in individual samples. Nevertheless, all three strains were mutated significantly by DMN in all experiments (probability less than 1% using Student’s r test). The mean induced mutation frequencies were not significantly different (probability greater than 10% using Student’s 1 test). Abbreviations: DMN, dimethylnitrosamine;MNNG, N-methyl-N’aitro-Ntr~~nidine: MNU, N-nitroso-N-methyl urea;NADP, nicotinamideadeninedinuckotidephosphate.
64 TABLE I INDUCTION BY ACTIVATED DMN OF REVERSIONTO fr’+ IN CIENT DERIVATIVESWPP UUrdiAND
E.coli WP* uvr+ exr+ AND ITS RBPA~R-DEFI-
CM611 UvrAeXrA
I&at livers were prepared, homogenized, centrifuged and tho supcrnatant frozen as described by Ames et ul.10. The reaction mixturecontainedper 10 ml: Liver fraction 3 ml; NasHPOd 12 Ha0 (11.8 me/ml pH 7.4) 3 ml; MgCls 2 Ha0 (16.3 mg/ml) 1 ml; KCl(24.6 me/ml) 1 ml; Glucose 6phosphate (14.1 mg/ml) 1 ml; NADP (32 mg/ml) 1 ml. Logarithmic 37” bacterial cultures in M9 i- 1% casamino acids + 0.4% glucose + 10 [cg/ml tryptophan were filtered, concentrated x 10, and 0.1 ml added to 1 ml of reaction mixture in a universal bottle. All subsequent operations were performed in a well ventilated fume cupboard. DMN (lOa) was added, the bottle closed and incubated 30 min at 37” with shaking. In experiments 1 and 2 quantities were doubled and air was blown vigorously through the sample. Aliquots of 0.1 ml were plated on minimal Davis Mingioli agar supplemented with casamino acids 0.25 %; glucose 0.4% and tryptophan 0.75 Icg/ml. Dilutions of 10-a were plated on the same medium to determine viability’. Plates were incubated at 37” in sealed containers for 48 h. No induced mutants wepe observed in any experiment where the cells and reaction mixture were poured as a sott agat layer, or where the microsome fraction was omitted. The repair-deficient mutants and Trp+ reversion system in E.coli strain WP2 have been described previously7. Struin
Experiment number
Aeration
Average no. Trp+ revertants/p/ate Control
WP2 uvr+
exrf
I
+
2
-t
3 4 5
WP2 uvrA
:
exr+
3 4 5
CM611 uvrA exrA
:
Calculated induced mutation fieq.llO’ cells
Treated
35 74
88 589
20.1 64.4
28 30 110
II8 571 497
11.6 78.0 94.2
26
106
32.2
42
92
7.9
31 50 27
226 414 705
23.8 45.4 61.5
1
+
18
43
19.3
2
+
28
294
56.5
17 I9 I8
177 33 221
17.4 5.0 38.9
3 4 5 ---
average 53.6
average 34.0
average 27.4
-_.
It seems reasonable to conclude that the three strains were mutated to about the same extent. No mutagenicity was found in the presence of soft agar to the absence of the liver fraction (data not shown). The results therefore confirm the prediction that activated DMN induces mutations in a similar way to MNNG or MNU. Repair-deficient strains of E.coli offer a convenient method of characterizing such mutagens. We would like to thank Professor B. A. Bridges for his advice and assistance.
65 1 P. N. Magee and 1. hi. Barnes, Carcinogenic nitroso compounds, A&n. CuncerRes,, 10 (1967) 163-246. 2 H. V. Mailing, Dimethylnitrosamine; formation of mutagenic compounds by interaction with mouse liver microsomes, MururlonRes., 13 (1971) 425-429. 3 T. Nakajtma and S. Iwahara, Mutagenicity of dimethylnitrosaminein the metabolic process by rat liver microsomes, Mrtlution Res., 18 (1973) 121-127. 4 P. D. Lawley, The action of alkylating mutagens and carcinomns on nucleic acids: N-methyl-Nnitroso compounds as methylating agents, in W. Nakahara, Topics in Chetnicui Curcinogenesis, S. Takayama and S. Gdashima (Eds.), University of Tokyo Press, 1972, pp. 237-253. 5 T. A. Hince and S. Neale, E&t of IV-nitroso-IV-methylu on viability and mutagenic response of repair-deficientstrains of Escherichiu coli, Mururion Res., 22 (1974) 235-242. 6 E. M. Witkin, Mutation-proof and mutation-prone modes of survivalin derivativesof Ewkerichiu coli B differingin sensitivity to ultraviolet light, Brookhaven Symp. Biol., 20 (1967) 17-55. 7 B. A. Bridges,R. P. Mottershead, M. H. L. Green and W. J. H. Gray, Mutagenicity of dichlorvos and methyl methanesulphonate for Escher&u coli WP2 and some derivatives deficient in DNA repair, Mutation Res., 19 (1973) 295-303. 8 B. A. Bridges, R. P. Mottershead, M. A. Rothwell and M. H. L. Green, Repair deficientbacterial strains suitable for mutagenicity screening: tests with the fungicide captan, Chem.-Biol. fnrerucr., 5 (1972) 77-84. 9 I. Murayamaand N. Otsuji, Mutation by mitomycins in the ultraviolet light sersitive mutant of Escherichiu co/i, Mururion Res., I8 (1973) 117-119.
10 B. N. Ames, W. E. Durston, E. Yamasuki and F. D. Lee, Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection, Proc. NurL Acad. Sci. (US.),
70 (1973) 2281-2285.
Uw
of mpalr-dofkbnt
mubgamia
Ed;
m&a
and Uvw nboaomm
63
to cbmcbdm
bydimothylnhtwa~
M. H. L. GREENANDW.J. MURIEL MRC Cdl Mwtorton Udt, lJntw&y o/Sussex, Fakr,
(Reded
lb&hmn BNi 9QG (Great B&ah)
October 14th. 1974)
(Revisionwived January20th. 1975) (Accepted February20th. 1975)
DMN is a metabolically activated carcinogenl, and it is mutagenic to Solmonello in the presence of a liver microsomal extra&s. It is thought that DMN is metabolized to monomethylnitrosamine which is unstable and alkylates DNA via the “carbonium ion” (CHs;) (ref. 4). If so, DMN should be identical to MNNG or MNU in its effect as a bacterial mutagem. Such a prediction can be tested using repairdeficient strains of Escherichio cd. MNNG and MNU cause mutation by misreplication as well as misrepair (i.e., they mutate exrA strains of E.coli nearly as efficiently as they mutate exrA+ strainW). Moreover the mutagenic lesions are not subject to excision by urr endonuclease (i.e., mutation is equal in a uvr and a uvr+ straitW). Other alkylating agents give different patterns of response; for instance mutations arise almost entirely by misrepair of non-excisable damage in the case of methylmethanesulphonate’, by misrepair of excisable damage in the case of captans, and by misreplication of excisable damage in the case of the volatile breakdown product of captan*. Finally the difunctional alkylating agent mitomycin C is mutagenic only in typhimurium
an excision-proficient strain@. We have, therefore, examined DMN mutagenesis using suitable E.coli strains. We have confirmed the observation of Ames ef al.‘0 that DMN is not mutagenic when incubated with liver microsomes in soft agar, although it is mutagenic using the
Mailing* system of incubation with microsomes in liquid and bubbling air through the mixture. This aeration did not seem helpful and we adapted the method described in Table 1. Escherichlo calf strain WP2 and its derivatives WP2 uvrA and CM611 WA cxrA were tested (Table I). There was considerable variation probably due to differences in the efficiency of liver activation in individual samples. Nevertheless, all three strains were mutated significantly by DMN in all experiments (probability less than 1% using Student’s r test). The mean induced mutation frequencies were not significantly different (probability greater than 10% using Student’s 1 test). Abbreviations: DMN, dimethylnitrosamine;MNNG, N-methyl-N’aitro-Ntr~~nidine: MNU, N-nitroso-N-methyl urea;NADP, nicotinamideadeninedinuckotidephosphate.
64 TABLE I INDUCTION BY ACTIVATED DMN OF REVERSIONTO fr’+ IN CIENT DERIVATIVESWPP UUrdiAND
E.coli WP* uvr+ exr+ AND ITS RBPA~R-DEFI-
CM611 UvrAeXrA
I&at livers were prepared, homogenized, centrifuged and tho supcrnatant frozen as described by Ames et ul.10. The reaction mixturecontainedper 10 ml: Liver fraction 3 ml; NasHPOd 12 Ha0 (11.8 me/ml pH 7.4) 3 ml; MgCls 2 Ha0 (16.3 mg/ml) 1 ml; KCl(24.6 me/ml) 1 ml; Glucose 6phosphate (14.1 mg/ml) 1 ml; NADP (32 mg/ml) 1 ml. Logarithmic 37” bacterial cultures in M9 i- 1% casamino acids + 0.4% glucose + 10 [cg/ml tryptophan were filtered, concentrated x 10, and 0.1 ml added to 1 ml of reaction mixture in a universal bottle. All subsequent operations were performed in a well ventilated fume cupboard. DMN (lOa) was added, the bottle closed and incubated 30 min at 37” with shaking. In experiments 1 and 2 quantities were doubled and air was blown vigorously through the sample. Aliquots of 0.1 ml were plated on minimal Davis Mingioli agar supplemented with casamino acids 0.25 %; glucose 0.4% and tryptophan 0.75 Icg/ml. Dilutions of 10-a were plated on the same medium to determine viability’. Plates were incubated at 37” in sealed containers for 48 h. No induced mutants wepe observed in any experiment where the cells and reaction mixture were poured as a sott agat layer, or where the microsome fraction was omitted. The repair-deficient mutants and Trp+ reversion system in E.coli strain WP2 have been described previously7. Struin
Experiment number
Aeration
Average no. Trp+ revertants/p/ate Control
WP2 uvr+
exrf
I
+
2
-t
3 4 5
WP2 uvrA
:
exr+
3 4 5
CM611 uvrA exrA
:
Calculated induced mutation fieq.llO’ cells
Treated
35 74
88 589
20.1 64.4
28 30 110
II8 571 497
11.6 78.0 94.2
26
106
32.2
42
92
7.9
31 50 27
226 414 705
23.8 45.4 61.5
1
+
18
43
19.3
2
+
28
294
56.5
17 I9 I8
177 33 221
17.4 5.0 38.9
3 4 5 ---
average 53.6
average 34.0
average 27.4
-_.
It seems reasonable to conclude that the three strains were mutated to about the same extent. No mutagenicity was found in the presence of soft agar to the absence of the liver fraction (data not shown). The results therefore confirm the prediction that activated DMN induces mutations in a similar way to MNNG or MNU. Repair-deficient strains of E.coli offer a convenient method of characterizing such mutagens. We would like to thank Professor B. A. Bridges for his advice and assistance.
65 1 P. N. Magee and 1. hi. Barnes, Carcinogenic nitroso compounds, A&n. CuncerRes,, 10 (1967) 163-246. 2 H. V. Mailing, Dimethylnitrosamine; formation of mutagenic compounds by interaction with mouse liver microsomes, MururlonRes., 13 (1971) 425-429. 3 T. Nakajtma and S. Iwahara, Mutagenicity of dimethylnitrosaminein the metabolic process by rat liver microsomes, Mrtlution Res., 18 (1973) 121-127. 4 P. D. Lawley, The action of alkylating mutagens and carcinomns on nucleic acids: N-methyl-Nnitroso compounds as methylating agents, in W. Nakahara, Topics in Chetnicui Curcinogenesis, S. Takayama and S. Gdashima (Eds.), University of Tokyo Press, 1972, pp. 237-253. 5 T. A. Hince and S. Neale, E&t of IV-nitroso-IV-methylu on viability and mutagenic response of repair-deficientstrains of Escherichiu coli, Mururion Res., 22 (1974) 235-242. 6 E. M. Witkin, Mutation-proof and mutation-prone modes of survivalin derivativesof Ewkerichiu coli B differingin sensitivity to ultraviolet light, Brookhaven Symp. Biol., 20 (1967) 17-55. 7 B. A. Bridges,R. P. Mottershead, M. H. L. Green and W. J. H. Gray, Mutagenicity of dichlorvos and methyl methanesulphonate for Escher&u coli WP2 and some derivatives deficient in DNA repair, Mutation Res., 19 (1973) 295-303. 8 B. A. Bridges, R. P. Mottershead, M. A. Rothwell and M. H. L. Green, Repair deficientbacterial strains suitable for mutagenicity screening: tests with the fungicide captan, Chem.-Biol. fnrerucr., 5 (1972) 77-84. 9 I. Murayamaand N. Otsuji, Mutation by mitomycins in the ultraviolet light sersitive mutant of Escherichiu co/i, Mururion Res., I8 (1973) 117-119.
10 B. N. Ames, W. E. Durston, E. Yamasuki and F. D. Lee, Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection, Proc. NurL Acad. Sci. (US.),
70 (1973) 2281-2285.
