This article was downloaded by: [New York University] On: 15 May 2015, At: 08:52 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Mutagenicity and mutagenic specificity of metronidazole and niridazole in neurospora crassa Tong‐man Ong a
a b
& Barbara Slade
a
National Institute of Environmental Health Sciences , Research Triangle Park, North Carolina
b
National Institute for Occupational Safety and Health , ALOSH , Morgantown, West Virginia, 26505 Published online: 20 Oct 2009.
To cite this article: Tong‐man Ong & Barbara Slade (1978) Mutagenicity and mutagenic specificity of metronidazole and niridazole in neurospora crassa , Journal of Toxicology and Environmental Health: Current Issues, 4:5-6, 815-824, DOI: 10.1080/15287397809529702 To link to this article: http://dx.doi.org/10.1080/15287397809529702
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
MUTAGENICITY AND MUTAGENIC SPECIFICITY OF METRONIDAZOLE AND NIRIDAZOLE IN NEUROSPORA CRASSA Tong-man Ong, Barbara Slade
Downloaded by [New York University] at 08:52 15 May 2015
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
Mutagenicity and mutagenic specificity of niridazole and metronidazole, two chemo~ therapeutic agents used in the treatment of human parasitic diseases, were studied with the ad-3 test system of Neurospora crassa. The results show that neither compound is mutagenic in resting conidia. In growing vegetative cells, however, both compounds are mutagenic in N. crassa. Genetic analysis of the mutants indicated that niridazole induces predominantly base-pair substitution mutations. None of the niridazole-induced mutants resulted from multilocus deletions. The spectra of genetic alterations induced by metronidazole are similar to those induced by the monofunctional aikyiating agents ethy/eneimine (El), ethylmethanesulfonate (EMS), and ICR-177. It is therefore suggested that the mechanism of mutation induction by metronidazole in Neurospora is similar to that of monofunctional aikyiating agents.
INTRODUCTION Niridazole and metronidazole (Table 1) are chemotherapeutic drugs used in the treatment of parasitic disease in humans. Niridazole has been used for the treatment of schistosomiasis, and metronidazole (Flagyl) is widely used for the treatment of Trichomonas vaginalis. It has been estimated that more than 200,000 humans have been treated with niridazole (World Health Organization, 1972) and that more than 2 million prescriptions a year for Flagyl have been written by physicians (Mintz, 1974). Recent studies in bacteria have shown both compounds to be mutagenic. Niridazole is mutagenic in TA 1538, TA 98, and TA 100 of Salmonella typhimurium and WP2 uvrA ~ of Escherichia coll (Legator et al., 1975; McCann et al., 1975; McCalla et al., 1975). Metronidazole is active in the induction of reverse mutations in G46 and the testers derived from G46 of S. typhimurium (Legator et al., 1975; Rosenkranz and Speck, 1975; Voogd et al., 1974). This compound is also mutagenic in several other bacterial species (Voogd et al., 1974). Metronidazole has also been found to be carcinogenic in mice (Rustia and Shubik, 1972). Requests for reprints should be sent to Tong-man Ong, National Institute for Occupational Safety and Health, ALOSH, Morgantown, West Virginia 26505. 815 Journal of Toxicology and Environmental Health, 4:815-824,1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/040815-10$2.25
816
T. ONG AND B. SLADE
TABLE 1. Chemical Names and Structures of Niridazole and Metronidazole Common name
Niridazole
Metronidazole
Chemical name
Chemical structure
1-(5-Nitro-2-thiazolyl)-2-imidazolidinone
1-(2-Hydroxyethyl)-2-methyl-5nitroimidazole
Downloaded by [New York University] at 08:52 15 May 2015
CHJ-CHJOH
Studies of the mutagenicity and mutagenic specificity have been conducted with the adenine-3 (ad-3) system of Neurospora crassa, a eukaryotic microorganism. With this system, mutants resulting from either point mutations or recessive chromosomal deletions can be recovered at the ad-3A or ad-3B locus and the forward mutation frequencies can be accurately determined (de Serres and Mailing, 1971). Mutants recovered from forward mutation experiments can be characterized by a series of simple genetic tests to determine the spectrum of genetic alterations (de Serres, 1964a). The data presented here show that neither niridazole nor metronidazole is mutagenic in the resting conidia but both are mutagenic in the growing vegetative cells of N. crassa. Genetic analyses of mutants induced by both agents indicate that the spectra of genetic alterations induced by niridazole and metronidazole are similar to those induced by base analogs and monofunctional alkylating agents, respectively.
MATERIALS AND METHODS Strains A genetically marked two-component heterokaryon of N. crassa consisting of strain 74-OR60-29A (component I) and strain 74-OR31-16A (component II) (de Serres and Webber, 1963) was used in these studies. The ad-3 mutation was induced in component II of this heterokaryon. Chemicals Niridazole was kindly donated by Dr. Bueding, Johns Hopkins University, Baltimore, Md. Metronidazole was obtained from a local pharmacy. Both Flagyl tablets and an extract of metronidazole from tablets were used.
MUTAGENICITY OF METRONIDAZOLE AND NIRIDAZOLE IN NEUROSPORA
817
Mutation Induction in Resting Conidia Conidia from vegetative cultures of the heterokaryon were harvested in 0.067 M phosphate buffer (pH 7). The conidial suspension was filtered through a 250-mesh platinum wire gauze filter to remove any mycelia. The conidia were washed twice with phosphate buffer by centrifugation and were then treated ( 6 X 1 0 6 conidia/ml) with different concentrations of niridazole or metronidazole for 2 or 24 h at 30°C. A t the end of the treatment, the conidia were washed with Fries' basal medium, pH 8 (Horowitz and Beadle, 1943). The ad-3 mutation frequency was determined after samples of treated and untreated conidia were incubated in the medium at 30°C in dark for 7 d as described by de Serres and Mailing (1971).
Downloaded by [New York University] at 08:52 15 May 2015
Mutation Induction in Vegetative Cultures Conidial suspensions from vegetative cultures of the heterokaryon were prepared as described in the previous section. Conidia ( 6 X 1 0 6 ) were inoculated into 10 ml of Fries' basal medium supplemented with 1% sucrose, in 2 0 X 1 5 0 mm test tube. Specific amounts of niridazole or metronidazole were added to the experimental tubes. Niridazole was dissolved in dimethyl sulfoxide (DMSO), and metronidazole was dissolved in Fries' basal medium. The concentration of DMSO in each tube did not exceed 5%. Cultures were kept at 35°C in the dark for I d and then at 25°C in the light for 7-12 d. Conidia from each of these new vegetative cultures were then harvested and analyzed for the presence of ad-3 mutations. Complementation Test The complementation pattern and the genotype (ad-3A; ad-3B; or ad-3A, ad-3B) of each mutant induced by niridazole or metronidazole was determined by heterokaryon tests for complementation. Nine standard testers were used (Ong et al., 1975). On the basis of the complementation patterns, ad-3B mutants were classified as noncomplementing, nonpolarized complementing, and polarized complementing. Dikaryon and Trikaryon Tests Dikaryon tests were carried out by plating conidia from each mutant in Fries' minimal medium supplemented with adenine and pantothenate. The plates were incubated at 35°C for 2 d and were then examined for the presence or absence of colonial temperature-sensitive [cot) colonies. Mutants that gave no viable cot colonies were subjected to the trikaryon test. In the trikaryon test, trikaryons were formed by growing each mutant with each of the three testers. Tester 12-17-215 carries a deletion covering the ad-3A locus, tester 12-5-182 carries a deletion covering the ad-3B
818
T. ONG AND B. SLADE
locus, and tester 12-1-8 carries a deletion covering the ad-3A, ad-3B and nie loci (de Serres, 1968). The presence or absence of cot colonies from each trikaryon was determined by plating conidia into Fries' minimal medium supplemented with adenine and pantothenate. Dikaryon and trikaryon tests determine whether the induced mutant resulted from a point mutation at the ad-3 locus, a point mutation at this locus with a recessive lethal mutation elsewhere in the genome, or a deletion covering ad-3A and/or ad-3B and the adjacent genes. The methods of these tests and the interpretation of the results have been described in detail by de Serres (1964b, 1968).
Downloaded by [New York University] at 08:52 15 May 2015
RESULTS Forward Mutation Experiments in Resting Conidia No significant increase in the mutation frequencies or decrease in survival was found after conidia were treated with 0.2 mg/ml niridazole for 4 h or with 8.8 mg/ml metronidazole for 24 h. Mutation Induction in Vegetative Cultures The results summarized in Table 2 indicate that niridazole is mutagenic in the vegetative cultures of N. crassa. The ad-3 mutation frequency increased to 25.4 mutants per 106 survivors in the first experiment and 22.4 mutants per 106 survivors in the second experiment. Only 81-86% of conidia from niridazole-treated vegetative cultures were viable. Table 3 shows that both the commercial tablets of Flagyl and the pure extract of metronidazole are mutagenic. The ad-3 mutation frequency increased to 41.2 and 50.5 mutants per 106 survivors after vegetative cells were treated with 8.8 mg/ml metronidazole extract. When vegetative cells were treated with a Flagyl tablet in 10 ml medium, the ad-3 mutation frequency increased to 47.0 per 106 survivors. The survival of the cells TABLE 2. Forward Mutation Experiments with Niridazole in Vegetative Cultures of Neurospora crassa
Experiment
1° 2b
(%)
No. of colonies assayed (X 10 6 )
No. of purple colonies
No. of purple colonies per 106 survivors
100 86 100 81
3.8 3.3 3.6 11.5
0 83 0 251b
0 25.4 0 22.4
Concentration of niridazole (mg/ml)
Survivals
0 0.2 0 0.2
"Data published previously (Ong and de Serres, 1975). Mutants from this experiment were subjected to genetic analysis.
MUTAGENICITY OF METRONIDAZOLE AND NIRIDAZOLE IN NEUROSPORA
819
TABLE 3. Mutation Induction by Metronidazole in Vegetative Cultures in Neurospora crassa
Experiment 1
Concentration of metronidazole (mg/ml) 0 2.2 4.4 8.8
25.0°
Downloaded by [New York University] at 08:52 15 May 2015
2
0 2.2 4.4 8.8
(%)
No. of colonies assayed (X 10 s )
100 99 74 55 48 100 53 46 87
1.2 4.7 2.7 2.0 2.3 3.0 1.6 1.8 3.5
Survival
No. of purple colonies 0 12 64 81 108 0 1 35
178 6
No. of purple colonies per 10* survivors 0
2.5 24.1 41.2 47.0 0 0.6
19.3 50.3
One Flagyl tablet was added in 10 ml medium. These mutants were genetically analyzed.
was only 48% of the control value. Both survival and mutation frequencies appear to be dose-dependent with one exception. The reason that a high survival was found in experiment 2 after cells were treated with 8.8 mg/ml metronidazole is not known. Complementation Test It was found that 199 of 257 niridazole-induced and 142 of 178 metronidazole-induced mutants were made homokaryotic for the adenine requirement, and they were tested for phenotype and complementation patterns. Among the 199 niridazole-induced mutants, 40 (20%) were ad-3A and 159 (80%) were ad-3B. Of the 142 metronidazole-induced mutants, 61 (43%) were ad-3A and the remaining 81 (57%) were ad-3B. None of the niridazole- or metronidazole-induced mutants tested were ad-3A, ad-3B double mutants (Table 4). The frequencies of ad-3B complementation patterns induced by both compounds are given in Table 5. Niridazole induces a high frequency of complementing ad-3B mutants (70%). Almost all of the complementing ad-3B mutants (99%) induced by niridazole have a nonpolarized complementation pattern. TABLE 4. Frequencies of Genotypes among Nindazole- and Metronidazole-induced ad-3 Mutants
Mutants induced by
No. of mutants tested
No. of od-3A mutants
No. of ad-3B mutants
Niridazole Metronidazole
199 142
40 (20%) 61 (43%)
159 (80%) 81 (57%)
820
T. ONG AND B. SLADE
TABLE 5. Frequencies of Complementation and of Nonpolarized Complementation Patterns among Niridazole- and Metronidazole-lnduced ad-3B Mutants Mutants induced by
Complementing ad-3B's among ad-3B's
Nonpolarized od-3B's among complementing ad-3B's
Niridazole Metronidazole
110/159(70%) 37/81 (46%)
110/111(99%) .25/37 (68%)
Downloaded by [New York University] at 08:52 15 May 2015
Approximately half of the metronidazole-induced mutants show allelic complementation; among the complementing ad-3B mutants, 68% have nonpolarized complementation patterns. Dikaryon and Trikaryon Tests All 199 niridazole-induced and 142 metronidazole-induced mutants were tested with the dikaryon test. Twenty-three metronidazole-induced mutants failed to give cot colonies in the dikaryon test; these were further tested in the trikaryon test. Three mutants gave cot colonies from only one trikaryon; these were classified as multilocus deletion mutants. The remaining 20 mutants gave cot colonies from all three trikaryons and were classified as mutants carrying a point mutation at the ad-3A or ad-3B locus and a recessive mutation elsewhere in the genome. Mutants giving cot colonies in the dikaryon test were ad-3 point mutation mutants. Results of the dikaryon and trikaryon tests are summarized in Table 6. DISCUSSION The mutagenicity of niridazole and metronidazole at the ad-3 region of N. crassa was studied by assaying for the induction of recessive lethal mutations in resting conidia and vegetative cells. Under the conditions studied, neither compound increased the ad-3 mutation frequency in conidia; both compounds, however, were toxic and mutagenic in the vegetative cells. Approximately a 50-fold and more than a 100-fold increase in the ad-3 mutation frequency over the spontaneous frequency (0.4 mutant per 106 survivors) (Brockman and de Serres, 1963). were found when the vegetative cells were treated with 0.2 mg/ml of niridazole TABLE 6. Results of Dikaryon and Trikaryon Tests
Mutants induced by
No. of mutants tested
No. of mutations
No. of multilocus deletions
Niridazole Metronidazole
199 142
199 (100%) 139(98%)
0 3 (2%)
Downloaded by [New York University] at 08:52 15 May 2015
MUTAGENICITY OF METRONIDAZOLE AND NIRIDAZOLE IN NEUROSPORA
821
and 8.8 mg/ml metronidazole, respectively. When growing cells were treated with a Flagyl tablet in 10 ml Fries' minimal medium, the ad-3 mutation frequency increased more than 100-fold over the spontaneous frequency. It appears, therefore, that both niridazole and metronidazole are very effective mutagens in Neurospora. The recommended human dosage of Flagyl for the treatment of trichomoniasis is one 250-mg tablet orally 3 times daily for 10 d. Simultaneous treatment with vaginal inserts of 500 mg/d for 10 d is also recommended. It seems that Flagyl-treated individuals are exposed to high dose levels of this compound. Studies of Legator et al. (1975) showed that substances mutagenic in bacteria can be recovered from the urine of patients treated with niridazole or metronidazole. These and other data therefore suggest the need for more extensive work related to the possible mutagenic, carcinogenic, and teratogenic hazards of these agents in humans. Why niridazole and metronidazole are not mutagenic in the resting conidia but are mutagenic in the metabolically active growing cells is not known. One possible explanation is that both compounds require metabolic activation, which can occur in the growing cells. In yeast, niridazole is found to be recombinogenic only when cells are treated in a rich medium in which vegetative growth is occurring (Shahin, 1975). In bacteria, niridazole acts as a direct mutagen.. It is likely, however, that niridazole is not active per se but is converted to an active form by bacterial cells (McCalla et al., 1975). Studies have shown that niridazole can be metabolically reduced, possibly by NADPH-cytochrome c reducíase (Feller et al., 1971) or by xanthine oxidase (Monta et al., 1971), to a hydroxylamine derivative. It is not known whether hydroxyaminothiamidazole is responsible for the mutagenic activity of niridazole. Enzymatic reduction of metronidazole to the hydroxylamine or amino derivative is probably an important step for the mutagenicity of this agent. It has been shown that metronidazole is mutagenic in TA 100 of S. typhimurium but not mutagenic in a derivative of this strain that is deficient in nitroreductase (Rosenkranz and Speck, 1975). Metronidazole is mutagenic in this nitroreductase-deficient strain of Salmonella if the treatment includes a 9000 X ^ supernatant fraction of a mammalian liver homogenate (Rosenkranz and Speck, 1975). Genetic analyses of niridazole- and metronidazole-induced ad-3 mutants show that niridazole induces a low frequency of ad-3A (20%) mutants whereas metronidazole induces a high frequency of ad-3A mutants. In general, approximately one-third of the mutants induced in the ad-3 system of Neurospora are ad-3A. The ratio of ad-3A to ad-3B, however, varies from mutagen to mutagen. In this test system, it is known that the frequencies of allelic complementation and nonpolarized complementation patterns in ad-3B mutants depend on mutagenic origin. If the mutants are induced by 2-aminopurine (AP), which results
822
T. ONG AND B. SLADE
TABLE 7. Comparison of ad-3B Mutants Induced by Various Mutagens Frequency (%)
Mutagenic origin Spontaneous" ICR-17O0 Metronidazole ICR-1770 EMS"
AP° Niridazole
Complementing ad-3B's among ad-3B'%
Nonpolarized ad-3B's among complementing ad-3B's
38.1 23.8 46 51.1 53.2 77.1 70
25.0 35.5 68 70.8 78.0 92.6 99
Downloaded by [New York University] at 08:52 15 May 2015
°Data from de Serres (1964b), de Serres et al. (1971), Brockman and Goven (1965), Ong (1970), and H. V. Mailing (personal communication).
predominantly in base-pair substitutions, high frequencies of allelic complementation and nonpolarized complementation patterns are observed (Brockman and de Serres, 1963). On the other hand, if the mutants are induced by ICR-170, an agent that causes predominantly frameshifts in N. crassa (Mailing, 1967; Brusick, 1969), low frequencies of allelic complementation and nonpolarized complementation patterns are found (Brockman and Goben, 1965). Studies of the reversions of ad-3 mutants with various mutagens have further confirmed the correlation between complementation characteristics and the predominant type of genetic alteration (Mailing and de Serres, 1968a, 1968b). Frequencies of allelic complementation and nonpolarized complementation patterns are very similar in niridazole-induced and AP-induced mutants (Table 7). None of the niridazole-induced mutants tested are multilocus deletion mutants. These data suggest that niridazole induces predominantly base-pair substitution mutations in Neurospora. In 5. typhimurium, niridazole reverts both base-pair substitution and frameshift mutants (McCann et al., 1975). In E. coli, this compound probably induces base-pair substitution mutations (McCalla et al., 1975). The frequencies of allelic complementation observed at comparable forward mutation frequencies are similar to those induced by the monofunctional alkylating agents ethyleneimine (El), ethylmethanesulfonate (EMS), and ICR-177 (Table 7). Metronidazole induces a low frequency of multilocus deletions. These results suggest that the mechanisms of mutation induction by metronidazole in N. crassa are similar to those of monofunctional alkylating agents.
REFERENCES Brockman, H. E. and de Serres, F. J. 1963. Induction of ad-3 mutants of Neurospora crassa by 2-aminopurine. Genetics 48:597-604.
Downloaded by [New York University] at 08:52 15 May 2015
MUTAGENICITY OF METRONIDAZOLE AND NIRIDAZOLE IN NEUROSPORA
823
Brockman, H. E. and Goben, W. 1965. Mutagenicity of a monofunctional alkylating agent derivative of acridine in Neurosporo. Science 147:750-751. Brusick, D. J. 1969. Revision of acridine mustard-induced ad-3 mutants of Neurospora crassa. Mutat. Res. 8:247-254. de Serres, F. J. 1964a. Mutagenesis and chromosome structure. J. Cell. Comp. Physio/. 64(Suppl. 1):33-42. de Serres, F. J. 1964b. Genetic analysis of the structure of the ad-3 region of Neurosporo crassa by means of irreparable recessive lethal mutations. Genetics 50:21-30. de Serres, F. J. 1968. Genetic analysis of the extent and type of functional ¡nactivation in irreparable recessive lethal mutations in the ad-3 region of Neurospora crassa. Genetics 58:69-77. de Serres, F. J. and Brockman, H. E. 1968. Homology tests on presumed multilocus deletions in the ad-3 region of Neurospora crassa induced by the acridine mustard ICR-170. Genetics 58:79-83. de Serres, F. J. and Mailing, H. V. 1971. Measurement of recessive lethal damage over entire genomes and at two specific loci in the ad-3 region of a two-component heterokaryon of Neurospora crassa. In Chemical Mutagens: Principles and Methods for Their Detection, ed. A. Hollaender, vol. 2, pp. 311-342. New York: Plenum, de Serres, F. J. and Webber, B. B. 1963. Recessive lethal mutations resulting from deletion of closely linked loci in balanced heterokaryons of Neurospora crassa. Neurospora Newslett. 3:3-5. de Serres, F. J., Brockman, H. E., Barnett, W. E., and Kølmark, H. G. 1971. Mutagen specificity in Neurospora crassa. Mutat. Res. 12:129-142. Feller, D. R., Monta, M., and Gillette, J. R. 1971. Enzymatic reduction of niridazole by rat liver microsomes. Biochem. Pharmacol. 20:203-215. Horowitz, N. H. and Beadle, G. W. 1943. A microbiological method for the determination of choline by use of a mutant of Neurospora. J. Biol. Chem. 150:325-333. Legator, M. S., Connor, T. H., and Stockel, M. 1975. Detection of mutagenic activity of metronidazole and niridazole in body fluids of humans and mice with Salmonella typhimurium. Science 188:1118-1119. Mailing, H. V. 1967. The mutagenicity of acridine mustard (ICR-170) and the structurally related compounds in Neurospora. Mutat. Res. 4:265-274. Mailing, H. V. and de Serres, F. J. 1968a. Correlation between base-pair transition and complementation pattern in nitrous acid-induced ad-3B mutants of Neurospora crassa. Mutat. Res. 5:359-371. Mailing, H. V. and de Serres, F. J. 1968b. Identification of genetic alterations induced by ethyl methanesulfonate in Neurospora crassa. Mutat. Res. 6:181-193. McCalla, D. R., Voutsinos, D., and Olive, P. L. 1975. Mutagen screening with bacteria: Niridazole and nitrofurans. Mutat. Res. 31:31-37. McCann, J., Spingarn, N. E., Kobori, J., and Ames, B. N. 1975. Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids. Proc. Natl. Acad. Science U.S.A.
72:979-983. Mintz, M. 1974. Controversial drug okayed for tests. Washington Post, June 30. Morita, M., Feller, D. R., and Gillette, J. R. 1971. Reduction of niridazole by rat liver xanthine oxidase. Biochem. Pharmacol. 20:217-226. Ong, T. 1970. Characterization of ICR-177-induced ad-3 mutants of Neurospora crassa. Mutat. Res. 9:183-191. Ong, T. and de Serres, F. J. 1975. Mutagenic evaluation of antischistosomal drugs and their derivatives in Neurospora crassa. J. Toxicol. Environ. Health 1:271-279. Ong, T., Matter, B. E., and de Serres, F. J. 1975. Genetic characterization of adenine-3 mutants induced by 4-nitroquinoline 1-oxide and 4-hydroxyamino-quinoline 1-oxide in Neurospora crassa. Cancer Res. 35:291-295. Rosenkranz, H. S. and Speck, W. T. 1975. Mutagenicity of metronidazole: Activation by mammalian liver microsomes. Biochem. Biophys. Res. Commun. 66:520-525.
824
T. ONG AND B. SLADE
Rustía, M. and Shubik, P. 1972. Induction of lung tumors and malignant lymphomas in mice by metronidazole. J. Natl. Cancer Inst. 48:721-729. Shahin, M. M. 1975. Genetic activity of niridazole in yeast. Mutat. Res. 30:191-198. Voogd, C. E., Van der Stel, J. J., and Jacobs, J. J. J. A. A. 1974. The mutagenic action of nitroimidazoles. 1. Metronidazole, niridazole, dimetridazole, and ronidazole. Mutat. Res. 26:483-490.
Downloaded by [New York University] at 08:52 15 May 2015
Received November 2, 1977 Accepted December 1, 1977
Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Mutagenicity and mutagenic specificity of metronidazole and niridazole in neurospora crassa Tong‐man Ong a
a b
& Barbara Slade
a
National Institute of Environmental Health Sciences , Research Triangle Park, North Carolina
b
National Institute for Occupational Safety and Health , ALOSH , Morgantown, West Virginia, 26505 Published online: 20 Oct 2009.
To cite this article: Tong‐man Ong & Barbara Slade (1978) Mutagenicity and mutagenic specificity of metronidazole and niridazole in neurospora crassa , Journal of Toxicology and Environmental Health: Current Issues, 4:5-6, 815-824, DOI: 10.1080/15287397809529702 To link to this article: http://dx.doi.org/10.1080/15287397809529702
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
MUTAGENICITY AND MUTAGENIC SPECIFICITY OF METRONIDAZOLE AND NIRIDAZOLE IN NEUROSPORA CRASSA Tong-man Ong, Barbara Slade
Downloaded by [New York University] at 08:52 15 May 2015
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
Mutagenicity and mutagenic specificity of niridazole and metronidazole, two chemo~ therapeutic agents used in the treatment of human parasitic diseases, were studied with the ad-3 test system of Neurospora crassa. The results show that neither compound is mutagenic in resting conidia. In growing vegetative cells, however, both compounds are mutagenic in N. crassa. Genetic analysis of the mutants indicated that niridazole induces predominantly base-pair substitution mutations. None of the niridazole-induced mutants resulted from multilocus deletions. The spectra of genetic alterations induced by metronidazole are similar to those induced by the monofunctional aikyiating agents ethy/eneimine (El), ethylmethanesulfonate (EMS), and ICR-177. It is therefore suggested that the mechanism of mutation induction by metronidazole in Neurospora is similar to that of monofunctional aikyiating agents.
INTRODUCTION Niridazole and metronidazole (Table 1) are chemotherapeutic drugs used in the treatment of parasitic disease in humans. Niridazole has been used for the treatment of schistosomiasis, and metronidazole (Flagyl) is widely used for the treatment of Trichomonas vaginalis. It has been estimated that more than 200,000 humans have been treated with niridazole (World Health Organization, 1972) and that more than 2 million prescriptions a year for Flagyl have been written by physicians (Mintz, 1974). Recent studies in bacteria have shown both compounds to be mutagenic. Niridazole is mutagenic in TA 1538, TA 98, and TA 100 of Salmonella typhimurium and WP2 uvrA ~ of Escherichia coll (Legator et al., 1975; McCann et al., 1975; McCalla et al., 1975). Metronidazole is active in the induction of reverse mutations in G46 and the testers derived from G46 of S. typhimurium (Legator et al., 1975; Rosenkranz and Speck, 1975; Voogd et al., 1974). This compound is also mutagenic in several other bacterial species (Voogd et al., 1974). Metronidazole has also been found to be carcinogenic in mice (Rustia and Shubik, 1972). Requests for reprints should be sent to Tong-man Ong, National Institute for Occupational Safety and Health, ALOSH, Morgantown, West Virginia 26505. 815 Journal of Toxicology and Environmental Health, 4:815-824,1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/040815-10$2.25
816
T. ONG AND B. SLADE
TABLE 1. Chemical Names and Structures of Niridazole and Metronidazole Common name
Niridazole
Metronidazole
Chemical name
Chemical structure
1-(5-Nitro-2-thiazolyl)-2-imidazolidinone
1-(2-Hydroxyethyl)-2-methyl-5nitroimidazole
Downloaded by [New York University] at 08:52 15 May 2015
CHJ-CHJOH
Studies of the mutagenicity and mutagenic specificity have been conducted with the adenine-3 (ad-3) system of Neurospora crassa, a eukaryotic microorganism. With this system, mutants resulting from either point mutations or recessive chromosomal deletions can be recovered at the ad-3A or ad-3B locus and the forward mutation frequencies can be accurately determined (de Serres and Mailing, 1971). Mutants recovered from forward mutation experiments can be characterized by a series of simple genetic tests to determine the spectrum of genetic alterations (de Serres, 1964a). The data presented here show that neither niridazole nor metronidazole is mutagenic in the resting conidia but both are mutagenic in the growing vegetative cells of N. crassa. Genetic analyses of mutants induced by both agents indicate that the spectra of genetic alterations induced by niridazole and metronidazole are similar to those induced by base analogs and monofunctional alkylating agents, respectively.
MATERIALS AND METHODS Strains A genetically marked two-component heterokaryon of N. crassa consisting of strain 74-OR60-29A (component I) and strain 74-OR31-16A (component II) (de Serres and Webber, 1963) was used in these studies. The ad-3 mutation was induced in component II of this heterokaryon. Chemicals Niridazole was kindly donated by Dr. Bueding, Johns Hopkins University, Baltimore, Md. Metronidazole was obtained from a local pharmacy. Both Flagyl tablets and an extract of metronidazole from tablets were used.
MUTAGENICITY OF METRONIDAZOLE AND NIRIDAZOLE IN NEUROSPORA
817
Mutation Induction in Resting Conidia Conidia from vegetative cultures of the heterokaryon were harvested in 0.067 M phosphate buffer (pH 7). The conidial suspension was filtered through a 250-mesh platinum wire gauze filter to remove any mycelia. The conidia were washed twice with phosphate buffer by centrifugation and were then treated ( 6 X 1 0 6 conidia/ml) with different concentrations of niridazole or metronidazole for 2 or 24 h at 30°C. A t the end of the treatment, the conidia were washed with Fries' basal medium, pH 8 (Horowitz and Beadle, 1943). The ad-3 mutation frequency was determined after samples of treated and untreated conidia were incubated in the medium at 30°C in dark for 7 d as described by de Serres and Mailing (1971).
Downloaded by [New York University] at 08:52 15 May 2015
Mutation Induction in Vegetative Cultures Conidial suspensions from vegetative cultures of the heterokaryon were prepared as described in the previous section. Conidia ( 6 X 1 0 6 ) were inoculated into 10 ml of Fries' basal medium supplemented with 1% sucrose, in 2 0 X 1 5 0 mm test tube. Specific amounts of niridazole or metronidazole were added to the experimental tubes. Niridazole was dissolved in dimethyl sulfoxide (DMSO), and metronidazole was dissolved in Fries' basal medium. The concentration of DMSO in each tube did not exceed 5%. Cultures were kept at 35°C in the dark for I d and then at 25°C in the light for 7-12 d. Conidia from each of these new vegetative cultures were then harvested and analyzed for the presence of ad-3 mutations. Complementation Test The complementation pattern and the genotype (ad-3A; ad-3B; or ad-3A, ad-3B) of each mutant induced by niridazole or metronidazole was determined by heterokaryon tests for complementation. Nine standard testers were used (Ong et al., 1975). On the basis of the complementation patterns, ad-3B mutants were classified as noncomplementing, nonpolarized complementing, and polarized complementing. Dikaryon and Trikaryon Tests Dikaryon tests were carried out by plating conidia from each mutant in Fries' minimal medium supplemented with adenine and pantothenate. The plates were incubated at 35°C for 2 d and were then examined for the presence or absence of colonial temperature-sensitive [cot) colonies. Mutants that gave no viable cot colonies were subjected to the trikaryon test. In the trikaryon test, trikaryons were formed by growing each mutant with each of the three testers. Tester 12-17-215 carries a deletion covering the ad-3A locus, tester 12-5-182 carries a deletion covering the ad-3B
818
T. ONG AND B. SLADE
locus, and tester 12-1-8 carries a deletion covering the ad-3A, ad-3B and nie loci (de Serres, 1968). The presence or absence of cot colonies from each trikaryon was determined by plating conidia into Fries' minimal medium supplemented with adenine and pantothenate. Dikaryon and trikaryon tests determine whether the induced mutant resulted from a point mutation at the ad-3 locus, a point mutation at this locus with a recessive lethal mutation elsewhere in the genome, or a deletion covering ad-3A and/or ad-3B and the adjacent genes. The methods of these tests and the interpretation of the results have been described in detail by de Serres (1964b, 1968).
Downloaded by [New York University] at 08:52 15 May 2015
RESULTS Forward Mutation Experiments in Resting Conidia No significant increase in the mutation frequencies or decrease in survival was found after conidia were treated with 0.2 mg/ml niridazole for 4 h or with 8.8 mg/ml metronidazole for 24 h. Mutation Induction in Vegetative Cultures The results summarized in Table 2 indicate that niridazole is mutagenic in the vegetative cultures of N. crassa. The ad-3 mutation frequency increased to 25.4 mutants per 106 survivors in the first experiment and 22.4 mutants per 106 survivors in the second experiment. Only 81-86% of conidia from niridazole-treated vegetative cultures were viable. Table 3 shows that both the commercial tablets of Flagyl and the pure extract of metronidazole are mutagenic. The ad-3 mutation frequency increased to 41.2 and 50.5 mutants per 106 survivors after vegetative cells were treated with 8.8 mg/ml metronidazole extract. When vegetative cells were treated with a Flagyl tablet in 10 ml medium, the ad-3 mutation frequency increased to 47.0 per 106 survivors. The survival of the cells TABLE 2. Forward Mutation Experiments with Niridazole in Vegetative Cultures of Neurospora crassa
Experiment
1° 2b
(%)
No. of colonies assayed (X 10 6 )
No. of purple colonies
No. of purple colonies per 106 survivors
100 86 100 81
3.8 3.3 3.6 11.5
0 83 0 251b
0 25.4 0 22.4
Concentration of niridazole (mg/ml)
Survivals
0 0.2 0 0.2
"Data published previously (Ong and de Serres, 1975). Mutants from this experiment were subjected to genetic analysis.
MUTAGENICITY OF METRONIDAZOLE AND NIRIDAZOLE IN NEUROSPORA
819
TABLE 3. Mutation Induction by Metronidazole in Vegetative Cultures in Neurospora crassa
Experiment 1
Concentration of metronidazole (mg/ml) 0 2.2 4.4 8.8
25.0°
Downloaded by [New York University] at 08:52 15 May 2015
2
0 2.2 4.4 8.8
(%)
No. of colonies assayed (X 10 s )
100 99 74 55 48 100 53 46 87
1.2 4.7 2.7 2.0 2.3 3.0 1.6 1.8 3.5
Survival
No. of purple colonies 0 12 64 81 108 0 1 35
178 6
No. of purple colonies per 10* survivors 0
2.5 24.1 41.2 47.0 0 0.6
19.3 50.3
One Flagyl tablet was added in 10 ml medium. These mutants were genetically analyzed.
was only 48% of the control value. Both survival and mutation frequencies appear to be dose-dependent with one exception. The reason that a high survival was found in experiment 2 after cells were treated with 8.8 mg/ml metronidazole is not known. Complementation Test It was found that 199 of 257 niridazole-induced and 142 of 178 metronidazole-induced mutants were made homokaryotic for the adenine requirement, and they were tested for phenotype and complementation patterns. Among the 199 niridazole-induced mutants, 40 (20%) were ad-3A and 159 (80%) were ad-3B. Of the 142 metronidazole-induced mutants, 61 (43%) were ad-3A and the remaining 81 (57%) were ad-3B. None of the niridazole- or metronidazole-induced mutants tested were ad-3A, ad-3B double mutants (Table 4). The frequencies of ad-3B complementation patterns induced by both compounds are given in Table 5. Niridazole induces a high frequency of complementing ad-3B mutants (70%). Almost all of the complementing ad-3B mutants (99%) induced by niridazole have a nonpolarized complementation pattern. TABLE 4. Frequencies of Genotypes among Nindazole- and Metronidazole-induced ad-3 Mutants
Mutants induced by
No. of mutants tested
No. of od-3A mutants
No. of ad-3B mutants
Niridazole Metronidazole
199 142
40 (20%) 61 (43%)
159 (80%) 81 (57%)
820
T. ONG AND B. SLADE
TABLE 5. Frequencies of Complementation and of Nonpolarized Complementation Patterns among Niridazole- and Metronidazole-lnduced ad-3B Mutants Mutants induced by
Complementing ad-3B's among ad-3B's
Nonpolarized od-3B's among complementing ad-3B's
Niridazole Metronidazole
110/159(70%) 37/81 (46%)
110/111(99%) .25/37 (68%)
Downloaded by [New York University] at 08:52 15 May 2015
Approximately half of the metronidazole-induced mutants show allelic complementation; among the complementing ad-3B mutants, 68% have nonpolarized complementation patterns. Dikaryon and Trikaryon Tests All 199 niridazole-induced and 142 metronidazole-induced mutants were tested with the dikaryon test. Twenty-three metronidazole-induced mutants failed to give cot colonies in the dikaryon test; these were further tested in the trikaryon test. Three mutants gave cot colonies from only one trikaryon; these were classified as multilocus deletion mutants. The remaining 20 mutants gave cot colonies from all three trikaryons and were classified as mutants carrying a point mutation at the ad-3A or ad-3B locus and a recessive mutation elsewhere in the genome. Mutants giving cot colonies in the dikaryon test were ad-3 point mutation mutants. Results of the dikaryon and trikaryon tests are summarized in Table 6. DISCUSSION The mutagenicity of niridazole and metronidazole at the ad-3 region of N. crassa was studied by assaying for the induction of recessive lethal mutations in resting conidia and vegetative cells. Under the conditions studied, neither compound increased the ad-3 mutation frequency in conidia; both compounds, however, were toxic and mutagenic in the vegetative cells. Approximately a 50-fold and more than a 100-fold increase in the ad-3 mutation frequency over the spontaneous frequency (0.4 mutant per 106 survivors) (Brockman and de Serres, 1963). were found when the vegetative cells were treated with 0.2 mg/ml of niridazole TABLE 6. Results of Dikaryon and Trikaryon Tests
Mutants induced by
No. of mutants tested
No. of mutations
No. of multilocus deletions
Niridazole Metronidazole
199 142
199 (100%) 139(98%)
0 3 (2%)
Downloaded by [New York University] at 08:52 15 May 2015
MUTAGENICITY OF METRONIDAZOLE AND NIRIDAZOLE IN NEUROSPORA
821
and 8.8 mg/ml metronidazole, respectively. When growing cells were treated with a Flagyl tablet in 10 ml Fries' minimal medium, the ad-3 mutation frequency increased more than 100-fold over the spontaneous frequency. It appears, therefore, that both niridazole and metronidazole are very effective mutagens in Neurospora. The recommended human dosage of Flagyl for the treatment of trichomoniasis is one 250-mg tablet orally 3 times daily for 10 d. Simultaneous treatment with vaginal inserts of 500 mg/d for 10 d is also recommended. It seems that Flagyl-treated individuals are exposed to high dose levels of this compound. Studies of Legator et al. (1975) showed that substances mutagenic in bacteria can be recovered from the urine of patients treated with niridazole or metronidazole. These and other data therefore suggest the need for more extensive work related to the possible mutagenic, carcinogenic, and teratogenic hazards of these agents in humans. Why niridazole and metronidazole are not mutagenic in the resting conidia but are mutagenic in the metabolically active growing cells is not known. One possible explanation is that both compounds require metabolic activation, which can occur in the growing cells. In yeast, niridazole is found to be recombinogenic only when cells are treated in a rich medium in which vegetative growth is occurring (Shahin, 1975). In bacteria, niridazole acts as a direct mutagen.. It is likely, however, that niridazole is not active per se but is converted to an active form by bacterial cells (McCalla et al., 1975). Studies have shown that niridazole can be metabolically reduced, possibly by NADPH-cytochrome c reducíase (Feller et al., 1971) or by xanthine oxidase (Monta et al., 1971), to a hydroxylamine derivative. It is not known whether hydroxyaminothiamidazole is responsible for the mutagenic activity of niridazole. Enzymatic reduction of metronidazole to the hydroxylamine or amino derivative is probably an important step for the mutagenicity of this agent. It has been shown that metronidazole is mutagenic in TA 100 of S. typhimurium but not mutagenic in a derivative of this strain that is deficient in nitroreductase (Rosenkranz and Speck, 1975). Metronidazole is mutagenic in this nitroreductase-deficient strain of Salmonella if the treatment includes a 9000 X ^ supernatant fraction of a mammalian liver homogenate (Rosenkranz and Speck, 1975). Genetic analyses of niridazole- and metronidazole-induced ad-3 mutants show that niridazole induces a low frequency of ad-3A (20%) mutants whereas metronidazole induces a high frequency of ad-3A mutants. In general, approximately one-third of the mutants induced in the ad-3 system of Neurospora are ad-3A. The ratio of ad-3A to ad-3B, however, varies from mutagen to mutagen. In this test system, it is known that the frequencies of allelic complementation and nonpolarized complementation patterns in ad-3B mutants depend on mutagenic origin. If the mutants are induced by 2-aminopurine (AP), which results
822
T. ONG AND B. SLADE
TABLE 7. Comparison of ad-3B Mutants Induced by Various Mutagens Frequency (%)
Mutagenic origin Spontaneous" ICR-17O0 Metronidazole ICR-1770 EMS"
AP° Niridazole
Complementing ad-3B's among ad-3B'%
Nonpolarized ad-3B's among complementing ad-3B's
38.1 23.8 46 51.1 53.2 77.1 70
25.0 35.5 68 70.8 78.0 92.6 99
Downloaded by [New York University] at 08:52 15 May 2015
°Data from de Serres (1964b), de Serres et al. (1971), Brockman and Goven (1965), Ong (1970), and H. V. Mailing (personal communication).
predominantly in base-pair substitutions, high frequencies of allelic complementation and nonpolarized complementation patterns are observed (Brockman and de Serres, 1963). On the other hand, if the mutants are induced by ICR-170, an agent that causes predominantly frameshifts in N. crassa (Mailing, 1967; Brusick, 1969), low frequencies of allelic complementation and nonpolarized complementation patterns are found (Brockman and Goben, 1965). Studies of the reversions of ad-3 mutants with various mutagens have further confirmed the correlation between complementation characteristics and the predominant type of genetic alteration (Mailing and de Serres, 1968a, 1968b). Frequencies of allelic complementation and nonpolarized complementation patterns are very similar in niridazole-induced and AP-induced mutants (Table 7). None of the niridazole-induced mutants tested are multilocus deletion mutants. These data suggest that niridazole induces predominantly base-pair substitution mutations in Neurospora. In 5. typhimurium, niridazole reverts both base-pair substitution and frameshift mutants (McCann et al., 1975). In E. coli, this compound probably induces base-pair substitution mutations (McCalla et al., 1975). The frequencies of allelic complementation observed at comparable forward mutation frequencies are similar to those induced by the monofunctional alkylating agents ethyleneimine (El), ethylmethanesulfonate (EMS), and ICR-177 (Table 7). Metronidazole induces a low frequency of multilocus deletions. These results suggest that the mechanisms of mutation induction by metronidazole in N. crassa are similar to those of monofunctional alkylating agents.
REFERENCES Brockman, H. E. and de Serres, F. J. 1963. Induction of ad-3 mutants of Neurospora crassa by 2-aminopurine. Genetics 48:597-604.
Downloaded by [New York University] at 08:52 15 May 2015
MUTAGENICITY OF METRONIDAZOLE AND NIRIDAZOLE IN NEUROSPORA
823
Brockman, H. E. and Goben, W. 1965. Mutagenicity of a monofunctional alkylating agent derivative of acridine in Neurosporo. Science 147:750-751. Brusick, D. J. 1969. Revision of acridine mustard-induced ad-3 mutants of Neurospora crassa. Mutat. Res. 8:247-254. de Serres, F. J. 1964a. Mutagenesis and chromosome structure. J. Cell. Comp. Physio/. 64(Suppl. 1):33-42. de Serres, F. J. 1964b. Genetic analysis of the structure of the ad-3 region of Neurosporo crassa by means of irreparable recessive lethal mutations. Genetics 50:21-30. de Serres, F. J. 1968. Genetic analysis of the extent and type of functional ¡nactivation in irreparable recessive lethal mutations in the ad-3 region of Neurospora crassa. Genetics 58:69-77. de Serres, F. J. and Brockman, H. E. 1968. Homology tests on presumed multilocus deletions in the ad-3 region of Neurospora crassa induced by the acridine mustard ICR-170. Genetics 58:79-83. de Serres, F. J. and Mailing, H. V. 1971. Measurement of recessive lethal damage over entire genomes and at two specific loci in the ad-3 region of a two-component heterokaryon of Neurospora crassa. In Chemical Mutagens: Principles and Methods for Their Detection, ed. A. Hollaender, vol. 2, pp. 311-342. New York: Plenum, de Serres, F. J. and Webber, B. B. 1963. Recessive lethal mutations resulting from deletion of closely linked loci in balanced heterokaryons of Neurospora crassa. Neurospora Newslett. 3:3-5. de Serres, F. J., Brockman, H. E., Barnett, W. E., and Kølmark, H. G. 1971. Mutagen specificity in Neurospora crassa. Mutat. Res. 12:129-142. Feller, D. R., Monta, M., and Gillette, J. R. 1971. Enzymatic reduction of niridazole by rat liver microsomes. Biochem. Pharmacol. 20:203-215. Horowitz, N. H. and Beadle, G. W. 1943. A microbiological method for the determination of choline by use of a mutant of Neurospora. J. Biol. Chem. 150:325-333. Legator, M. S., Connor, T. H., and Stockel, M. 1975. Detection of mutagenic activity of metronidazole and niridazole in body fluids of humans and mice with Salmonella typhimurium. Science 188:1118-1119. Mailing, H. V. 1967. The mutagenicity of acridine mustard (ICR-170) and the structurally related compounds in Neurospora. Mutat. Res. 4:265-274. Mailing, H. V. and de Serres, F. J. 1968a. Correlation between base-pair transition and complementation pattern in nitrous acid-induced ad-3B mutants of Neurospora crassa. Mutat. Res. 5:359-371. Mailing, H. V. and de Serres, F. J. 1968b. Identification of genetic alterations induced by ethyl methanesulfonate in Neurospora crassa. Mutat. Res. 6:181-193. McCalla, D. R., Voutsinos, D., and Olive, P. L. 1975. Mutagen screening with bacteria: Niridazole and nitrofurans. Mutat. Res. 31:31-37. McCann, J., Spingarn, N. E., Kobori, J., and Ames, B. N. 1975. Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids. Proc. Natl. Acad. Science U.S.A.
72:979-983. Mintz, M. 1974. Controversial drug okayed for tests. Washington Post, June 30. Morita, M., Feller, D. R., and Gillette, J. R. 1971. Reduction of niridazole by rat liver xanthine oxidase. Biochem. Pharmacol. 20:217-226. Ong, T. 1970. Characterization of ICR-177-induced ad-3 mutants of Neurospora crassa. Mutat. Res. 9:183-191. Ong, T. and de Serres, F. J. 1975. Mutagenic evaluation of antischistosomal drugs and their derivatives in Neurospora crassa. J. Toxicol. Environ. Health 1:271-279. Ong, T., Matter, B. E., and de Serres, F. J. 1975. Genetic characterization of adenine-3 mutants induced by 4-nitroquinoline 1-oxide and 4-hydroxyamino-quinoline 1-oxide in Neurospora crassa. Cancer Res. 35:291-295. Rosenkranz, H. S. and Speck, W. T. 1975. Mutagenicity of metronidazole: Activation by mammalian liver microsomes. Biochem. Biophys. Res. Commun. 66:520-525.
824
T. ONG AND B. SLADE
Rustía, M. and Shubik, P. 1972. Induction of lung tumors and malignant lymphomas in mice by metronidazole. J. Natl. Cancer Inst. 48:721-729. Shahin, M. M. 1975. Genetic activity of niridazole in yeast. Mutat. Res. 30:191-198. Voogd, C. E., Van der Stel, J. J., and Jacobs, J. J. J. A. A. 1974. The mutagenic action of nitroimidazoles. 1. Metronidazole, niridazole, dimetridazole, and ronidazole. Mutat. Res. 26:483-490.
Downloaded by [New York University] at 08:52 15 May 2015
Received November 2, 1977 Accepted December 1, 1977
