following Activation with Rat Liver Microsomes ...

Mutagenicity of Cyclic Nitrosamines in Escherichia coli following Activation with Rat Liver Microsomes Rosalie K. Elespuru and William Lijinsky Cancer Res 1976;36:4099-4101. Published online November 1, 1976.

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ICANCER RESEARCH 36,4099-4101 . November 1976]

Mutagenicity of Cyclic Nitrosamines in Escherichia coil following Activation with Rat Liver Microsomes Rosalie K. Elespuru1 and William Lijinsky2 Institutefor Toxicologyand Chemotherapy(Director: Prof. Dr. Med.D. Schmähl), GermanCancerResearchCenter(DeutschesKrebsforschungszentrum), Heidelberg, Germany

SUMMARY

tagenicity assay because of its suitability for studies of the genetics of mutagenesis.

Ten cyclic nitnosamines were tested for mutagenicity in Escherichia

coli

after

incubation

in vitro

with 9000 x g

microsomal supennatants prepared from nat liver, and the results were compared with carcinogenicity data from the same species. None of the compounds was mutagenic in the absence of microsomes. Seven carcinogenic corn pounds, nitnosopyrnolidine ‘ nitnosopipenidine, nitrosohexa methyleneimine, nitrosoheptamethyleneimine, nitrosomor pholine, dinitnosopipenazine ‘and dinitnosohomopipena zine, were mutagenic after microsomal activation. One compound, nitrosoheptamethyleneimine, was toxic to the bacteria. Two noncarcinogens, 1-nitrosopiperazine and 1methyl-4-nitnosopiperazine, and 1 strong carcinogen, 2,6dimethyldinitrosopiperazine, were not mutagenic with or without microsomal incubation. The liven micnosome prepa ration activated equally well those compounds that are liver carcinogens in Spnague-Dawley rats, and compounds for which the liver is not a target organ. INTRODUCTION

MATERIALS AND METHODS The 10 cyclic nitrosamines used were prepared and pun fied according to well-established methods described previ ously in connection with their chronic toxicity tests (4, 8, 10, 13). The 2 mononitrosopipenazines

—80°.One ml of supernatant

That many carcinogens can be activated to mutagens by liver microsomal enzyme preparations is now well estab lished(1,2,6,7,15,16).Thisfindinghas stimulated a great deal of work and speculation concerning the relationship between carcinogenesis and mutagenesis. Much effort has gone into the development of mutagenicity assays for car cinogens, which have great promise for monitoring carcin ogens in the environment or in tissues and organs of expeni mental animals. The usefulness of mutagenicity assays would be greatly enhanced if it could be established that a quantitative come lation exists between mutagenicity and cancinogenicity of a group of similar compounds. Such data are difficult to assemble because of the diversity of systems used for muta genicity and carcinogenicity testing. We present here the results of mutagenicity testing of a group of 10 cyclic nitnos amines for which quantitative carcinogenicity data in the rat have been obtained. Escherichia coli was used for the mu

‘Present

address;

Basic

Research

Program,

Frederick

Cancer

Program,

Frederick

Research

Center, Frederick, Md. 21701. 2 Present

address:

Chemical

Carcinogenesis

Research Center, Frederick, Md. 21701. Received January 26, 1976; accepted August 11, 1976.

Cancer

were used as the hydro

chlorides, since they are strong bases and might upset the buffering of the incubation system. Crude microsome fractions were made according to stan dand procedures (1) from the livers of Sprague-Dawley rats fed 1% phenobanbitab in their drinking water for 3 days prior to sacrifice by cervical dislocation. The livers were washed with sterile 0.15 M KCI by injection into the hepatic portal vein and were excised under aseptic conditions. The livers were homogenized in 3 volumes of cold KCI solution, with a Potter-Elvehjem homogenizer, followed by centnifugation in the cold at 9000 x g for 10 mm. Supennatants of homoge nates were mixed with an equal volume of 0.01 M phosphate buffer, pH 7.4, and were frozen in 5- on 10-mI portions at is derived

from

166 mg of

liven. The bacteria, E. coli WU 3610 (tyr , leu), obtained from Dr. D. L. George (originally from Dr. E. M. Witkin), contain nonsense mutations at both sites (17). Stationary overnight cultures were diluted 20-fold into Difco nutrient broth and grown for 1 hr, to early bogphase (1 to 2 x 10@bacteria/mb). Twenty ml of bacterial suspension were pebleted, and the pellet was resuspended in 10 to 20 ml of a solution of cofactons (1 ml contains 7.5 mg NADP, 10 mg glucose 6phosphate, 10 mg nicotinamide, and 6.5 mg MgCb2in 0.01 M sodium-potassium phosphate buffer, pH 7.4). The suspen sion containing bacteria and cofactons was warmed to 37°, and glucose-6-phosphate dehydnogenase, 1 MI/mI, was added. The solution was kept warm and was dispensed rapidly into tubes containing the nitroso compounds to be tested, in volumes appropriate to make 2 to 4 ml of 50 mM solution. When the nitroso compound was dissolved, 0.5-mb ali quots were dispensed into 4 tubes, and volumes of freshly thawed microsomal suspension were added, ranging from 0.1 to 1.0 ml (see Table 1). Phosphate buffer was added to each of the solutions containing less than 1.5 ml so as to make the volumes equal. The volume in each tube was 1.5 ml and the concentration of each test compound was 16.7

NOVEMBER 1976

4099

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R. K. E!espuru and W. Lijinsky Table 1 microsomesNitrosamines Mutagenicity of cyclic nitrosamines in E. coli with and without rat liver plusbacteria.

were incubated

for 1 hr with aliquots

of a 9000 x g rat liver supernatant

The days.No. suspensionswere plated on selective agar and incubated 2 of mutants°/dishfollowing incu bation with different

amounts

of mi

crosomal supernatant

Carcino genicity in

ComenceNM pound

0mb

0.2ml

3,11NPy 4 13 3NP― 4 40 12esophagusNHex 5 25 8NHepr 13MNP

7 3

33 28

0.5mb

1.Oml

Refer

rats

Site

59 92 82

130 240 250

++++ ++ +++

Liver Liver Nasalcavity,

150 46

270 79

++++ ++++

Liver,esophagus Lung,esophagus

4MeNP

4

7

6

8

0

3DNP

3 5

8 8

9 23

9 43

0 +++

cavityDNHP 10esophagusMe2DNP 4 18

47

82

++++

Nasalcavity,

4

9

6

++++

Nasalcavity,

31 3

65 8

++++

Liver

9

10esophagusDMN 5 18None

5 4

Esophagus, 10nasal liver,

3,

3,

3,

a Numbers are the average of 3 or more experiments, except NHep, which is the average of 2.

Mutants are tyr —* tyr@. b NP,

nitrosopiperidine;

dinitrosopiperazine;

NHex,

nitrosohexamethyleneimine;

MNP,

1-nitrosopiperazine;

DNP,

14-

DNPH, 1 ,4-dinitrosohomopiperazine.

CCompound was toxic at 0.0167 M even in the absence of microsomes. Concentration was

reduced 10-fold. concentration used. Few or no survivors were seen among the bacteria plated. At a 10-fold lower concentration, how bacteria) were platedon minimum medium E (19)sup even, survival was 70%. None of the compounds showed any plemented with 2.5% nutrient broth + 0.4% glucose + 20 @tg mutagenic activity without activation by microsomal en L-leucine pen ml. Plates were incubated for 2 days at 37°and zymes. With nat liver microsomes, 7 of the compounds were mutagenic and 3 had no detectable mutagenic activity. then counted. Under these conditions, bacteria divide 5ev enabtimes, allowing expression of mutations and the ap The almost linear increase in the number of mutants peanance of a background lawn of bacteria (5). The beucine (Table 1) with increasing volume of microsomal suspen present supplements 1 deficiency, and the mutants that sions is evidence of a dose response to the mutagen formed. These kinetics indicate that the amount of activat appear as large colonies are phenotypic nevertants from tyr to tyn@,most likely due to a suppressor (5, 17). In some ing enzyme is the limiting factor in the reaction. In animals, cases, the samples were diluted and plated for measure as well, the effective dose of carcinogen might be inde pendent of the amount administered, above the level that ment of survival. saturates the activating enzymes. The relatively low effec tiveness of barge single doses of nitnosamines in inducing RESULTS AND DISCUSSION tumors might be explained on this basis. In kinetic studies of mutation induction, Gletton et a!. (6) Most of the cyclic nitnosamines were more active in this system than DMN, although the latter is readily converted showed that the enzymatic activation of DMN3 was propor into an alkybatingmoiety (14),while most of the cyclic tional to the concentration of compound and to the concen tration of microsomal protein. Both dose-response curves compounds examined gave rise to little on no detectable saturated at the same level of induced mutations. In our alkylation of nucleic acids in rat liven (9). It seems possible, experiments, we have kept the nitrosamine concentration then, that the mutagenic product made by the micnosomes is not an alkybating moiety, but some other type of structure constant in order to compare the microsome-dependent which is common to both cyclic and aliphatic nitnosamines. activation of the compounds. The results of the mutagenicity tests of the 10 cyclic From our data it appears that liven microsomes convert nitrosamines are given in Table 1 as number of mutants per the various nitroso compounds into mutagens to different plate. Survival as determined by dilution and plating was extents, on that the products vary in their mutagenic effec 70% on more, except with NHep. NHep was toxic or growth tiveness. The pipenazines as a group were consistently the inhibitory, with or without microsomes, at the 16.7 mM least effective among the cyclic nitnosamines tested. NM, which was included in every experiment as a standard, was intermediate in activity, and NPy, nitrosopipenidine, and ni heptamethyleneimine; NM , nitrosomorpholine; NPy, nitrosopyrrolidine; trosohexamethybeneimine were similar and the most active. Me@DNP,2,6-dimethyl-1 ,4-dinitnosopipenazine; MeNP, 1-nitnoso-4-methylpi There seems to be no quantitative relationship between perazine. mM. The tubes

were

incubated

with shaking

at 37°for 1 hr.

Aliquotsof 0.2 ml of each solution(containing2 x 10@

3 The

abbreviations

used

are:

DMN,

dimethylnitrosamine:

NHep,

nitroso

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CANCER

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RESEARCH

VOL. 36

Mutagenesis by Cyclic Nitrosamines mutagenicity in these tests and carcinogenicity in rats, whether to liven on to other organs. DMN and NM are about equally strong liven carcinogens (affecting different cell types), but show quite different mutagenicities. NPy is a much weaken liven carcinogen (by an order of magnitude on more) than DMN, but is a more effective mutagen. NHep and 1,4-dinitrosohomopipenazine are not liver carcinogens in rats but are effectively activated to a mutagenic form by rat liven microsomes. Among the nitnosopiperazines there is a positive conrela tion between mutagenicity and carcinogenicity, with 1 ex ception, that being the negative response of the strongly carcinogenic Me2DNP in the mutagenicity test. One possi ble explanation is that Me2DNP is not metabolized to a mutagenic form by liver microsomes, but would be muta genic if incubated with micnosomes from an organ in which it induces tumors. However, Bartsch et al. (2) found that microsomes from a target organ other than the liven did not activate diethylnitrosamine to a bacterial mutagen. The 2 compounds negative for cancinogenicity in this series, 1nitrosopipenazine and MeNP, are also negative for mutagen icity. In the host-mediated assay, by contrast, one of these compounds, MeNP, gave positive results (20). In our experiments a reasonable quantitative correlation between the mutagenicity and cancinogenicity of a group of cyclic nitnosamines is not demonstrated. Even among corn pounds carcinogenic for rat liver, a conrelatable mutagenic response to these compounds after activation by micro somes from this organ is not seen. Equably perplexing is the absence of liver cancinogenicity of compounds that are activated by liven microsomes to the same extent as liver carcinogens. An explanation of these results would seem to be important in understanding the relationship between cancinogenesis and mutagenesis. The lack of a quantitative relationship raises the possibil ity that a carcinogenic compound might not be detected in a mutagenicity test, as was the case with Me2DNP in our system. It is possible that some other mutagenicity system might be better at predicting the carcinogenicity of a group of similar compounds. However, the complexities of biologi cabsystems make it quite probable that a quantitative come lation between mutagenicity and carcinogenicity is not achievable. Further exploration into these differences is needed as the basis for development of better assay sys tems for potentially carcinogenic compounds.

NOVEMBER

REFERENCES 1. Ames, B. A., Durston, W. E.. Yamasaki, E., and Lee, F. D. Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for Activation and Bacteria for Detection. Proc. NatI. Acad. Sci. U. S. , 70: 2281-2285. 1973.

2. Bartsch,H.. Malaveille,C., and Montesano,R. In Vitro Metabolismand Microsome-mediated Mutagenicity of Dialkylnitrosamines in Rat, Ham ster, and Mouse Tissues. Cancer Res. , 35: 644—651 , 1975. 3. Dnuckrey,H., Preussmann, R., Ivankovic, S. . and Schmáhl,0. Organo trope cancinogene Winkungen bei 65 venschiedenen N-Nitroso-Verbin dungen an BD-Ratten. Z. Krebsforsch., 69: 103-201 . 1967. 4. Garcia, H., Keefen,L. , Lijinsky, W. , and Wenyon, C. L. Carcinogenicity of Nitrosothiomorpholine and 1-Nitrosopiperazine in Rats. Z. Krebsforsch., 74: 179-184, 1970.

5. George,D. L.. and Witkin, E. M. UltravioletLight-inducedResponsesof an mid Mutant of E. coli B/n Having a Slow Rate of Dimer Excision. Mutation Res., 28: 347-354, 1975.

6. Gletten, H., Weekes,U., and Brusick, D. In Vitro MetabolicActivity of Chemical Mutagens. I. Development of an in Vitro Mutagenicity Assay using Liver Microsomal Enzymes for Activation of Dimethylnitrosamine to a Mutagen. Mutation Res., 28: 113-122, 1975. 7. Gomez, R. F.. Johnston, M., and Sinskey, A. J. Activation of Nitrosomor pholine and Nitrosopyrrolidine to Bacterial Mutagens. Mutation Res., 24: 5-7, 1974.

8. Goodall, C. M., Lijinsky, W., and Tomatis, L. Tumonigenicityof N-Nitro sohexamethyleneimine. Cancer Res.. 28: 1217-1222, 1968. 9. Lijinsky. W., Keefer, L., Loo, J., and Ross, A. E. Studies of Alkylation of Nucleic Acids in Rats by Cyclic Nitrosamines. Cancer Res., 33: 16341641, 1973. 10. Lijinsky. W., and Taylor. H. W. Carcinogenicity of Methylated Dinitroso piperazines in Rats. Cancer Res., 35: 1270-1273, 1975. 11. Lijinsky, W., and Taylor, H. W. Increased Carcinogenicity of 2,6-Di methylnitrosomorpholine Compared with Nitrosomorpholine in Rats. Cancer Res., 35: 2123-2125, 1975. 12. Lijinsky, W., and Taylor. H. W. Carcinogenicity of Methylated Nitrosopi penidines. Intern. J. Cancer, 16: 318-322, 1975. 13. Lijinsky, W., Tomatis, L. , and Wenyon, C. E. M. Lung Tumors in Rats Treated with N-Nitrosoheptamethyleneimine and N-Nitrosooctamethyl eneimine. Proc. Soc. Exptl. Biol. Med. , 130: 945-949, 1969. 14. Magee, P. N., and Farber, E. Toxic Liven Injury and Carcinogenesis. Methylation of Rat-Liver Nucleic Acids by Dimethylnitrosamine in Vivo. Biochem.J.,83: 114-124, 1962. 15. MaIling, H. V. Mutagenic Activation of Dimethylnitrosarnine and Diethyl nitrosamine in the Host-Mediated Assay and the Microsomal System. Mutation Res., 26: 465-472, 1974. 16. McCann, J., Spingarn, N. E.. Koboni, J., and Ames, B. N. Detection of Carcinogens as Mutagens: Bacterial Tester Strains with A Factor Plas mids. Proc. NatI. Aced. Sci. U. S., 72: 979-983, 1975. 17. Osborn, M. , and Person, S. Characterization of Revertants of E. coli WU 3610 and WP 2 using Amber Mutants and an Ochne Mutant of Bacterio phage T,. Mutation Res., 4: 504-505, 1967. 18. Taylor, H. W., Lijinsky, W., Nettesheim, P., and Snyder, C. M. Alteration of Tumor Response in Rat Liver by Carbon Tetrachlonide. Cancer Res., 34: 3391-3395, 1974. 19. Vogel, H. J., and Bonner, D. M. Acetylornithinase of Escherichia coli: Partial Purification and Some Properties. J. Biol. Chem., 218: 97-106, 1956. 20. Zeiger, E., Legator, M. S., and Lijinsky, W. Mutagenicity of N-Nitrosopi perazines for Salmonella typhimurium in the Host-mediated Assay. Can cer Res., 32: 1598-1599, 1972.

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