Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Mutagenicity testing of antischistosomal thioxanthenones and indazoles on yeast R. C. vonBorstel & Sándor Igali To cite this article: R. C. vonBorstel & Sándor Igali (1975) Mutagenicity testing of antischistosomal thioxanthenones and indazoles on yeast, Journal of Toxicology and Environmental Health, 1:2, 281-291, DOI: 10.1080/15287397509529327 To link to this article: http://dx.doi.org/10.1080/15287397509529327
Published online: 20 Oct 2009.
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Date: 17 June 2016, At: 06:50
MUTAGENICITY TESTING OF ANTISCHISTOSOMAL THIOXANTHENONES AND INDAZOLES ON YEAST R. C. von Borstel, Sándor Igali
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Department of Genetics, University of Alberta, Edmonton, Alberta, Canada
Two antischistosomal thioxanthenones, lucanthone and hycanthone, and four antischistosomal indazoles, IA-3, IA-4, IA-5, and IA-6, have been tested for mutagenicity on stationary phase cells of the yeast Saccharomyces cerevisiae. It was shown that, although there are some gaps in the data, hycanthone and IA-6 are mutagenic at pH 7.0, hycanthone is mutagenic at 5.9, and none of the other compounds is mutagenic at either pH. (Because mutagenicity of these compounds at pH 7.0 appears to be related to the presence of a methoxy group at position 5 of the poiycydic ring, it is possible that IA-4 will be mutagenic on yeast when it is tested at pH 7.0.) An excision-repair-deficient strain of yeast is no more sensitive than other strains. It was found from timeconcentration studies on lethality that an inverse relation held: cells exposed to a mutagenic compound are more sensitive when time of exposure was varied and concentration of the compound was held constant, and cells exposed to a nonmutagenic compound are more sensitive when concentration is varied and time of exposure held constant. When the compounds were tested on growing cells of yeast in rich media, none of the compounds is mutagenic, although some are lethal. The kinetic behavior in reversion of yeast exposed to these compounds shows marked departures from similar reversion studies where yeast is exposed to radiation, implicating different physiological mechanisms for the alteration of responses of yeast cells exposed to the different mutagens.
INTRODUCTION There are several reasons why the yeast Saccharomyces cerevisiae is a particularly good organism for testing compounds such as antischistosomal agents that might constitute genetic hazards (Brusick and Mayer, 1973). In the first place, Saccharomyces is an organism in which a great deal of genetic research has already been carried out, and both classical and molecular analyses of mutations are possible (Mortimer and Hawthorne, 1971). This study was supported by Contract NIH-74-C-798 of the National Institute of Environmental Health Science, Research Triangle Park, North Carolina, and a grant from the National Research Council of Canada. We appreciate receiving gifts of the compounds from Dr. Ernest Bueding and Dr. F. J. de Serres. Dr. Siew-Keen Quah, Dr. P. J. Hastings, and Dr. M. Shahin were very helpful through our many discussions. We are grateful to Dr. Siew-Keen Quah and Theresa Brychcy for the strains and to Soo-JeetTeh for technical assistance. Sándor Igali's present address is Frédéric Joliot-Curie National Research Institute for Radiobiology and Radiohygiene, Budapest, Hungary. Requests for reprints should be sent to R. C. von Borstel, Department of Genetics, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. 281 Journal of Toxicology and Environmental Health, 1:281-291,1975 Copyright © 1975 by Hemisphere Publishing Corporation
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Second, it is one of the very few organisms in which molecular lesions induced by mutagenic compounds are identifiable (Sherman and Stewart, 1973); thus it is possible to resolve particular or peculiar mutagen specificities. Third, yeast is a eukaryotic organism with both haploid and diploid phases of vegetative growth and a meiotic cycle that takes place in individual diploid cells without growth and differentiation of a germinal tissue; thus quantitation of events occurring in both mitotic and meiotic cells is straightforward. Finally, the techniques for handling yeast are simple and research is inexpensive; moreover, the results are reliable and reproducible from laboratory to laboratory and from person to person. Classically, mutation frequencies induced by mutagens are determined on static (stationary phase) cells of yeast rather than on growing ones. It was found two decades ago that yeast in different stages of the mitotic cell cycle can exhibit as much as 20-fold sensitivity differences to ionizing radiation (Beam et al., 1954). In order to treat cells under easily reproducible conditions in one phase of the cell cycle, it became a common practice to harvest cells for mutagenization at the end of log phase when growth had ceased. The first comparisons that are normally carried out for mutagenic agents are varying time of exposure to a mutagen and holding constant the concentration, and the reverse, varying the concentration and holding constant the time of exposure. These experiments can most easily be carried out on stationary phase cell cultures. Our first experiments with the thioxanthenones and the indazoles were all done on stationary phase cell cultures (Meadows, 1973; Meadows et al., 1972, 1973; von Borstel and Quah, 1973; Igali and von Borstel, 1974). It was disconcerting to find that only the thioxanthenones, lucanthone and hycanthone, were soluble at pH 7.0 without the use of solubilizing agents. So the first experiments were done at pH 7.0 and 5.9 for the hycanthone, pH 7.0 for lucanthone, and pH 5.9 for the indazoles. The mutagenic testing of indazoles at pH 7.0 in the presence of solubilizing agents such as dimethylsulfoxide or ethyl alcohol was done later. A summary of the work is presented in Table 1. MUTAGENICITY OF THIOXANTHENONES AND INDAZOLES At pH 5.9 only hycanthone (Meadows, 1973; Meadows et al., 1973) induces all types of mutations (base substitutions and frameshift mutations); IA-3, IA-4, IA-5, and IA-6 do not induce any types of mutation. Nevertheless, at pH 5.9 all of the compounds except IA-6 kill both haploid and diploid cells, and cell inactivation, as percent of the control, follows an exponential decline after an initial shoulder at the very lowest dose levels. IA-6 is relatively innocuous at pH 5.9 on stationary phase cell cultures even after 96 hr of treatment. It is of interest to note that hycanthone is more active mutagenically at the higher pH [Meadows, 1973; Meadows et al., 1973; see also Shahin and de Serres (1974) for recombinogenic experiments
TABLE 1. Structure and Mutagenicity of the Thioxanthenones and the Indazoles Tested on Saccharomyces Stationary phase cells pH 5.9 O
pH 7.0
NH-CH2CH2-N(C2HS)2 Nonmut.
Nonmut.
Mut.
Mut.
Nonrnut.
Nonmut.
Nonmut.
Nonmut.
Mut.
Nonmut.
Lucanthone
O
NH-CH2CH2-N(C2HS)2
Hycanthone
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pH 7.0
Growing cells
CH2OH N
N-CH 2 CH 2 -N(CjH 5 ) 2
IA-3
N
N-CH 2 CH 2 - N (C2 H5 )2 Nonmut.
IA-4
CH2OH N
N-CH 2 CH 2 - N (C2 Hs )2 Nonmut.
IA-5
N
N-CH 2 CH2 - N (C2 Hs )2 Nonmut.
IA-6
Nonmut. = Nonmutagenic. Mut. = Mutagenic. 283
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on hycanthone in yeast where a similar effect of pH was found]. The lethal effect of hycanthone is also relatively greater at the higher pH (Meadows et al., 1973; Shahin and de Serres, 1974). At pH 7.0 lucanthone does not induce mutations, but IA-6 becomes powerfully mutagenic. IA-3 is not mutagenic at this pH; IA-4 and IA-5 have not yet been tested. The lack of mutagenicity of lucanthone and IA-3 on yeast at pH 7.0 is significant. It confirms that our test strain of yeast is like Salmonella typhimurium in its inability to respond to the methyl-substituted derivatives of thioxanthenones and indazoles, for example, lucanthone and IA-3 (Hartman et al., 1973; Mayer, 1972). The hydroxylating enzymes that could convert these compounds into mutagens (Hartman and Hulbert, 1976) must be lacking in the strain of Saccharomyces we are using as a test system.
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EFFECT OF VARYING TIME OF EXPOSURE AND CONCENTRATION OF COMPOUNDS One of the more startling discoveries came in the time-concentration series of experiments (Meadows, 1973; Igali and von Borstel, 1974; S. Igali and R. C. von Borstel, unpublished data). Here it was found that, for hycanthone, when the time of exposure was varied and the concentration held constant, the cells were approximately 3 times as sensitive as when time of exposure was held constant and concentration of the hycanthone was varied. This effect was noted both for cell killing and mutation (Meadows, 1973). This is most simply explained by assuming that hycanthone is activated into a more mutagenic state over a period of time. P. J. Hastings (personal communication) suggested that the results can also be explained by assuming that hycanthone can induce a series of enzymes that activate hycanthone to a more mutagenic state. With this notion in mind, an attempt was made to mutagenize with hycanthone at different temperatures. It was found that survival is not depressed nor are mutations induced by hycanthone when cells are incubated at 4°C with hycanthone, for times varying from 8 to 96 hr (S. Igali and R. C. von Borstel, unpublished data). Two alternatives come to mind: either hycanthone is not absorbed at 4°C or enzymes that might activate hycanthone are prevented from being activated at this temperature. The reverse of the hycanthone time-concentration effect was found for lucanthone at pH 7.0, and IA-3 and IA-5 at pH 5.9 (Igali and von Borstel, 1974; S. Igali and R. C. von Borstel, unpublished data). That is, when time of exposure is varied and concentration of the indazole is held constant, the killing is approximately 8 times less than when concentration is varied and time of exposure is held constant. In this respect the indazoles appear to act like radiation in that protracted doses are not as damaging as acute doses. As with radiation the effect of lucanthone and the indazoles can be explained most simply by assuming that, with time, repair systems come into play that repair the lesions caused by the indazoles, or that detoxifying enzyme systems begin to act.
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Again, as has been suggested for ultraviolet radiation (Defais et al., 1971), lucanthone and the indazoles may be acting by inducing the repair systems. One experiment was done to investigate the actions of indazoles on a repair-deficient strain of yeast. The mutant used was rad2-2. It is in the excision-repair pathway for mutational lesions induced by ultraviolet radiation, but it is not the first step (Game and Cox, 1972). This strain shows no increase in sensitivity to either hycanthone or the indazoles whereas it shows a marked increase in sensitivity to ultraviolet radiation (S. Igali and R. C. von Borstel, unpublished data). So at least one of the radiation-repair pathways is not involved in repair of thioxanthenone- or indazole-induced lesions. This response is quite unlike that of Salmonella where defectiveness in excision-repair increases sensitivity to the mutagenicity of these compounds (Hartman and Hulbert, 1976). EFFECTS OF THIOXANTHENONES AND INDAZOLES ON GROWING CELLS Except for the stationary phase cells of yeast, the conidia of Neurospora, the sperm of Drosophila and Habrobracon, and the young oocytes of a mouse, virtually all other test systems for measuring induced mutation frequencies utilize growing and dividing cells. Certainly, the comprehensive studies of Hartman and Ames and their collaborators on 5. typhimurium with antischistosomal compounds and carcinogenic compounds (Hartman et al., 1971, 1973; Ames et al., 1972a,b, 1973), and the careful studies of Clive (1973) with mammalian cells have been done on growing and dividing systems. We have just begun studies with antischistosomal compounds on growing cell cultures, but our first experiments clearly indicate that we may expect some surprises. Hycanthone is much less active mutagenically on growing cell cultures than it is on stationary phase cells (S. Igali and R. C. von Borstel, unpublished data; M. Shahin, unpublished data). In the one experiment carried out to date, no mutations were induced at all with a concentration of 250 jug/ml of hycanthone in complete medium buffered to pH 7.0. Growing cells are not killed as easily as stationary phase cells, again implicating repair, or detoxification, of uptake systems. An indication of nonmutagenicity of growing cells came from spot test studies of hycanthone and other compounds on yeast (Brusick and Zeiger, 1972; R. C. von Borstel, unpublished data), but at the time we merely attributed the finding to the insensitivity of spot tests as a method for measuring mutagenicity. The reverse is certain to be the case as well. For example, it is well known that the anticancer agent ICR-170 is not particularly active on stationary phase cells of yeast, but it is very active on growing yeast cells (Brusick, 1970, 1972), and 5-amino acridine is inactive or antimutagenic on stationary phase cells but quite mutagenic on meiotic cells of yeast (Magni et al., 1964). Shahin (1975) has now shown that the antischistosomal agent
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niridazole is relatively inactive recombinogenetically on stationary phase cells of yeast, but it is quite active on growing cells. On the other hand, one reversion test system, hisl-7, responds to niridazole in stationary phase cells, but hisJ-7 appears not to revert under growing conditions (S. Igali and R. C. von Borstel, unpublished data). So it is mandatory that these studies be continued and growing cell cultures of yeast also be used as a test system. KINETIC ANALYSES OF MUTATION INDUCTION BY HYCANTHONE
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Hycanthone induces both base substitutions and frameshift mutations in stationary phase cells of yeast, yet it is specific for frameshift mutations in cultures of growing cells of 5. typhlmurium. The reason for this difference is not understood, but it may have to do with either unique reactions of different species or the differences in responses of growing and stationary
1
10 HOURS
OF EXPOSURE
FIGURE 1. Dose-response curves of frameshift mutations induced by hycanthone methanesulfonate in a stationary Saccharomyces cerevisiae test strain in phosphate buffer at pH 7.0 (open circles) and at pH 5.9 (solid circles). From Meadows et al. (1973).
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100 Hycanthone Added (>ig/ml) FIGURE 2. Dose-response curves of frameshift mutations induced by hycanthone furoate in a growing Salmonella typhimurium test strain in liquid medium at pH 7.0 where the bacteria were exposed to the mutagen for 2 and 4 hr. From Hartman et al. (1973).
phase cells of yeast. An indication that it may be the latter is suggested by the findings of Brusick (1970, 1972) that ICR-170 seems to induce predominantly frameshift mutations in growing yeast, while G. E. Magni (unpublished data) and Sherman and Stewart (1973) found that ICR-170 induces a high frequency of base substitutions in stationary phase cell cultures. If extended times or higher concentrations of hycanthone are used in cultures of growing cells of yeast, it may be that frameshift mutations will predominate. It is of interest to note in both the work of Meadows et al. (1973) and Hartman et al. (1973) that induction of frameshift events by hycanthone follows one-hit kinetics. Yet in both yeast and Salmonella at high doses, the proportion of mutants per survivor declines and the similarity is striking (Figs. 1 and 2). Classically, this decline of mutation frequencies at high doses has been interpreted as being due to two populations, one sensitive and one resistant: as the sensitive population is depleted, the mutant cells of this population are also killed, and then induction of mutants in the resistant
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population can be seen in a secondary rise in the frequency of mutants per survivor (von Borstel, 1966). Yet for the declines at higher doses shown in both Figs. 1 and 2, alternative explanations are more likely to be the correct ones. The decline at high doses in Salmonella shown in Fig. 2 is most likely to be essentially a technical expectation of the experiment (P. E. Hartman, personal communication). The experiment on Salmonella was really one of mutants per milliliter of growing cells rather than mutants per survivor, and at high doses the cell lethality (or inhibition of cell division) begins to outrun the new mutants; that is, the mutants themselves are being killed (or the total cell population is smaller). If both killing and mutation induction are single but separate events, the number of mutants per milliliter where the mutants reach a peak would be at an exposure of mutagen where the population of bacteria surviving is e~x (von Borstel, 1966). Since the Salmonella are growing continuously in these experiments, numerous parameters are interacting, such as rates of killing, rates of cell division, and phenotypic lag. The explanation for the data in yeast shown in Fig. 1 is a different story, for here the data are truly mutants per surviving cell, and the mutant frequency decline at high doses is a real phenomenon. Two different explanations for the yeast data are worth considering in detail because they take into account phenomena not before observed with radiation mutagenesis. Also, and perhaps more importantly, these considerations indicate that a good deal more phenomenology will need to be explored before kinetic analysis of data derived from chemical mutagen experiments will have the significance that is inherent in kinetic analysis of data derived from radiation experiments. Explanation 1 A puzzling feature of results obtained with hycanthone on yeast is that the mutant decline may not begin at the same dose for different mutants (Fig. 3), a result that, by classical kinetic theory, could only be explained by assuming that sets and subsets of cell populations are present as a mixture. This result appears to be unlike results so far obtained with ionizing radiation. Holliday (1971) has provided evidence that recombination-repair is not taking place in cells that die, implying that a proportion of the cells live because they have a recombination-repair ability. The same phenomenon may explain the decline of the dose-mutation curve, namely, that some of the cells have a mutation-repair ability and consequently live to higher doses of the toxic effect than those cells that lack this ability. The observation that decline of the dose-mutation curve occurs at different doses for different types of mutation induction may imply that there are several subpopulations of cells each having a different mutational repair pathway operative or induced in them.
MUTAGENICITY TESTING OF ANTISCHISTOSOMAL THIOXANTHENONES
Oj 02
0.6 1
2
289
4 6 10 20 40 60
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EXPOSURE TO HYCANTHONE (mgh) FIGURE 3. Dose-response curves for cells of Saccharomyces cerevisioe for the reversion from auxotrophy to prototrophy for hisl-7 (open circles), Iys1-1 (open triangles), and hom3-10 (open squares). The dashed portions of the curves correspond to the spontaneous revertant frequency. The cells were exposed to hycanthone at pH 7.0 at a concentration of hycanthone of 0.250 mg/ml.This concentration is multiplied times the hours of exposure to provide the milligram-hour scale of the abscissa.
Explanation 2 Alternatively, one can consider an interesting effect that was observed with both the thioxanthenones and the indazoles. Prototrophic revertants of both spontaneous and induced origin seem to be more susceptible to killing than are cells of the original population of auxotrophic cells. Particularly, the revertants of /ysJ-7 show this sensitivity (Fig. 3). Prototroph sensitivity might cause the decline in mutants per survivor at high dose levels through an enhancement of metabolism of prototrophic cells whereby hycanthone is enactivated into a more lethal state or transported to an intracellular site where it could be more effective in cellular killing. Clearly, more experiments must be done to determine the sensitivities of different genetic strains of yeast under different physiological conditions before the kinetics of mutagenesis can be understood profoundly. CONCLUSIONS The thioxanthenones and indazoles are mutagenic agents with interesting specificities. Some of the specificities are characteristics of species or strains, such as the mutagenic ineffectiveness of methyl-substituted derivatives on both Salmonella and Saccharomyces but not in Neurospora or mammalian cells. On the other hand, Salmonella cells deficient in excision-repair are
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more sensitive to these agents, but yeast cells deficient in excision-repair exhibit no such sensitization. Some specificities are physiological, determining the action when certain clusters of enzymes are acting; these very likely cause the phase sensitivities during the cell cycle seen for hycanthone and IA-6.
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REFERENCES Ames, B. N., Gurney, E. G., Miller, J. A. and Bartsch, H. 1972a. Carcinogens as frameshift mutagens: Metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens. Proc Nat. Acad. Sci. U.S. 69:3128-3132. Ames, B. N., Sims, P. and Grover, P. L. 1972b. Epoxides of carcinogenic polycyclic hydrocarbons are frameshift mutagens. Science 176:47-49. Ames, B. N., Lee, F. D. and Durston, W. E. 1973. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Nat. Acad. Sci. U.S. 70:782-788. Beam, C. A., Mortimer, R. K., Wolfe, R. G. and Tobias, C. A. 1954. The relation of radioresistance to budding in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 49:110-122. Brusick, D. J. 1970. The mutagenic activity of ICR-170 in Saccharomyces cerevisiae. Mutation Res. 10:11-19. Brusick, D. J. 1972. Induction of cyclohexamide-resistant mutants in Saccharomyces cerevisiae with N-methyl-N'-nitro-N-nitrosoguanidine and ICR-170. J. Bacteriol. 109:1134-1138. Brusick, D. J. and Mayer, V. M. 1973. New developments in mutagenicity screening techniques with yeast. Environ. Health Perspect. no. 6:83-96. Brusick, D. J. and Zeiger, E. 1972. A comparison of chemically induced reversion patterns of Salmonella typhimurium and Saccharomyces cerevisiae, using in vitro plate tests. Mutation Res. 14:271-275. Clive, D. 1973. Recent developments with the L5178Y TK heterozygote mutagen assay system. Environ. Health Perspect. no. 6:119-125. Defais, M., Fauquet, M., Radman, M. and Errera, M. 1971. Ultraviolet reactivation and ultraviolet mutagenesis of λ in different genetic systems. Virology 43:495-503. Game, J. C. and Cox, B. S. 1972. Epistatic interactions between four rad loci in yeast. Mutation Res. 16:352-362. Hartman, P. E. and Hulbert, P. B. 1976. Genetic activity spectra of some antischistosomal agents, with particular emphasis on thioxanthenones and indazoles.y. Toxicol. Environ. Health. In press. Hartman, P. E., Levine, K., Hartman, Z. and Berger, H. 1971. Hycanthone: A frameshift mutagen. Science 172:1058-1060. Hartman, P. E., Berger, H. and Hartman, Z. 1973. Comparison of hycanthone ("etrenol"), some hycanthone analogs, myxin, and 4-nitroquinoline-1-oxide as frameshift mutagens. J. Pharmacol. Exp. Ther. 186:390-398. Holliday, R. 1971. Biochemical measure of the time of frequency of radiation-induced allelic recombination in Ustilago. Nature New Biol. 232:233-236. Igali, S. and von Borstel, R. C. 1974. Induction of mutations and lethality in Saccharomyces cerevisiae after exposure to several indazole analogs of hycanthone. Mutation Res. 26:452. Magni, G. E., von Borstel, R. C. and Sora, S. 1964. Mutagenic action during meiosis and anti-mutagenic action during mitosis by 5 amino-acridine in yeast. Mutation Res. 1:227-230. Mayer, V. W. 1972. Mutagenic effects induced in Saccharomyces cerevisiae by breakdown products of 1-naphthylamine and 2-naphthylamine formed in an enzyme-free hydroxylation system. Mutation Res. 15:147-153. Meadows, M. G. 1973. Mutagenic action of two indazole analogs on Saccharomyces cerevisiae. M.S. thesis, University of Alberta. Meadows, M. G., Quah, S.-K. and von Borstel, R. C. 1972. Mutation induction in Saccharomyces by hycanthone and a related compound. Genetics 71 (3/2):s39.
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Meadows, M. G., Quah, S.-K. and von Borstel, R. C. 1973. Mutagenic action of hycanthone and IA-4 on yeast. J. Pharmacol. Exp. Ther. 187:444-450. Mortimer, R. K. and Hawthorne, D. C. 1971. Yeast genetics. In The yeasts, ed. A. H. Rose and J. S. Harrison, vol. 1, pp. 386-459. London: Academic Press. Shahin, M. 1975. Genetic activity of niridazole in yeast. Mutation Res. In press. Shahin, M. M. and de Serres, F. J. 1974. The effect of pH on hycanthone methanesulfonate induced inactivation and mitotic recombination in Saccharomyces. Mutation Res. 26:377-384. Sherman, F. and Stewart, J. W. 1973. Mutations at the end of the iso-1-cytochrome c gene of yeast. In The
biochemistry
of gene
expression
in higher
organisms,
e d . J . K . Pollak and J . W . L e e , p p .
56-86. Sydney: Australian and New Zealand Book Co. von Borstel, R. C. 1966. Effects of radiation on cells. In The biological basis of radiation therapy, ed. E. E. Schwartz, pp. 60-125. Philadelphia: Lippincott. von Borstel, R. C. and Quah, S.-K. 1973. Induction of mutations in Saccharomyces with hycanthone. Mutation Res. 21:52.
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Received March 28, 1975 Accepted June 20, 1975
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Mutagenicity testing of antischistosomal thioxanthenones and indazoles on yeast R. C. vonBorstel & Sándor Igali To cite this article: R. C. vonBorstel & Sándor Igali (1975) Mutagenicity testing of antischistosomal thioxanthenones and indazoles on yeast, Journal of Toxicology and Environmental Health, 1:2, 281-291, DOI: 10.1080/15287397509529327 To link to this article: http://dx.doi.org/10.1080/15287397509529327
Published online: 20 Oct 2009.
Submit your article to this journal
View related articles
Citing articles: 4 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh19 Download by: [University of Florida]
Date: 17 June 2016, At: 06:50
MUTAGENICITY TESTING OF ANTISCHISTOSOMAL THIOXANTHENONES AND INDAZOLES ON YEAST R. C. von Borstel, Sándor Igali
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Department of Genetics, University of Alberta, Edmonton, Alberta, Canada
Two antischistosomal thioxanthenones, lucanthone and hycanthone, and four antischistosomal indazoles, IA-3, IA-4, IA-5, and IA-6, have been tested for mutagenicity on stationary phase cells of the yeast Saccharomyces cerevisiae. It was shown that, although there are some gaps in the data, hycanthone and IA-6 are mutagenic at pH 7.0, hycanthone is mutagenic at 5.9, and none of the other compounds is mutagenic at either pH. (Because mutagenicity of these compounds at pH 7.0 appears to be related to the presence of a methoxy group at position 5 of the poiycydic ring, it is possible that IA-4 will be mutagenic on yeast when it is tested at pH 7.0.) An excision-repair-deficient strain of yeast is no more sensitive than other strains. It was found from timeconcentration studies on lethality that an inverse relation held: cells exposed to a mutagenic compound are more sensitive when time of exposure was varied and concentration of the compound was held constant, and cells exposed to a nonmutagenic compound are more sensitive when concentration is varied and time of exposure held constant. When the compounds were tested on growing cells of yeast in rich media, none of the compounds is mutagenic, although some are lethal. The kinetic behavior in reversion of yeast exposed to these compounds shows marked departures from similar reversion studies where yeast is exposed to radiation, implicating different physiological mechanisms for the alteration of responses of yeast cells exposed to the different mutagens.
INTRODUCTION There are several reasons why the yeast Saccharomyces cerevisiae is a particularly good organism for testing compounds such as antischistosomal agents that might constitute genetic hazards (Brusick and Mayer, 1973). In the first place, Saccharomyces is an organism in which a great deal of genetic research has already been carried out, and both classical and molecular analyses of mutations are possible (Mortimer and Hawthorne, 1971). This study was supported by Contract NIH-74-C-798 of the National Institute of Environmental Health Science, Research Triangle Park, North Carolina, and a grant from the National Research Council of Canada. We appreciate receiving gifts of the compounds from Dr. Ernest Bueding and Dr. F. J. de Serres. Dr. Siew-Keen Quah, Dr. P. J. Hastings, and Dr. M. Shahin were very helpful through our many discussions. We are grateful to Dr. Siew-Keen Quah and Theresa Brychcy for the strains and to Soo-JeetTeh for technical assistance. Sándor Igali's present address is Frédéric Joliot-Curie National Research Institute for Radiobiology and Radiohygiene, Budapest, Hungary. Requests for reprints should be sent to R. C. von Borstel, Department of Genetics, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. 281 Journal of Toxicology and Environmental Health, 1:281-291,1975 Copyright © 1975 by Hemisphere Publishing Corporation
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Second, it is one of the very few organisms in which molecular lesions induced by mutagenic compounds are identifiable (Sherman and Stewart, 1973); thus it is possible to resolve particular or peculiar mutagen specificities. Third, yeast is a eukaryotic organism with both haploid and diploid phases of vegetative growth and a meiotic cycle that takes place in individual diploid cells without growth and differentiation of a germinal tissue; thus quantitation of events occurring in both mitotic and meiotic cells is straightforward. Finally, the techniques for handling yeast are simple and research is inexpensive; moreover, the results are reliable and reproducible from laboratory to laboratory and from person to person. Classically, mutation frequencies induced by mutagens are determined on static (stationary phase) cells of yeast rather than on growing ones. It was found two decades ago that yeast in different stages of the mitotic cell cycle can exhibit as much as 20-fold sensitivity differences to ionizing radiation (Beam et al., 1954). In order to treat cells under easily reproducible conditions in one phase of the cell cycle, it became a common practice to harvest cells for mutagenization at the end of log phase when growth had ceased. The first comparisons that are normally carried out for mutagenic agents are varying time of exposure to a mutagen and holding constant the concentration, and the reverse, varying the concentration and holding constant the time of exposure. These experiments can most easily be carried out on stationary phase cell cultures. Our first experiments with the thioxanthenones and the indazoles were all done on stationary phase cell cultures (Meadows, 1973; Meadows et al., 1972, 1973; von Borstel and Quah, 1973; Igali and von Borstel, 1974). It was disconcerting to find that only the thioxanthenones, lucanthone and hycanthone, were soluble at pH 7.0 without the use of solubilizing agents. So the first experiments were done at pH 7.0 and 5.9 for the hycanthone, pH 7.0 for lucanthone, and pH 5.9 for the indazoles. The mutagenic testing of indazoles at pH 7.0 in the presence of solubilizing agents such as dimethylsulfoxide or ethyl alcohol was done later. A summary of the work is presented in Table 1. MUTAGENICITY OF THIOXANTHENONES AND INDAZOLES At pH 5.9 only hycanthone (Meadows, 1973; Meadows et al., 1973) induces all types of mutations (base substitutions and frameshift mutations); IA-3, IA-4, IA-5, and IA-6 do not induce any types of mutation. Nevertheless, at pH 5.9 all of the compounds except IA-6 kill both haploid and diploid cells, and cell inactivation, as percent of the control, follows an exponential decline after an initial shoulder at the very lowest dose levels. IA-6 is relatively innocuous at pH 5.9 on stationary phase cell cultures even after 96 hr of treatment. It is of interest to note that hycanthone is more active mutagenically at the higher pH [Meadows, 1973; Meadows et al., 1973; see also Shahin and de Serres (1974) for recombinogenic experiments
TABLE 1. Structure and Mutagenicity of the Thioxanthenones and the Indazoles Tested on Saccharomyces Stationary phase cells pH 5.9 O
pH 7.0
NH-CH2CH2-N(C2HS)2 Nonmut.
Nonmut.
Mut.
Mut.
Nonrnut.
Nonmut.
Nonmut.
Nonmut.
Mut.
Nonmut.
Lucanthone
O
NH-CH2CH2-N(C2HS)2
Hycanthone
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pH 7.0
Growing cells
CH2OH N
N-CH 2 CH 2 -N(CjH 5 ) 2
IA-3
N
N-CH 2 CH 2 - N (C2 H5 )2 Nonmut.
IA-4
CH2OH N
N-CH 2 CH 2 - N (C2 Hs )2 Nonmut.
IA-5
N
N-CH 2 CH2 - N (C2 Hs )2 Nonmut.
IA-6
Nonmut. = Nonmutagenic. Mut. = Mutagenic. 283
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on hycanthone in yeast where a similar effect of pH was found]. The lethal effect of hycanthone is also relatively greater at the higher pH (Meadows et al., 1973; Shahin and de Serres, 1974). At pH 7.0 lucanthone does not induce mutations, but IA-6 becomes powerfully mutagenic. IA-3 is not mutagenic at this pH; IA-4 and IA-5 have not yet been tested. The lack of mutagenicity of lucanthone and IA-3 on yeast at pH 7.0 is significant. It confirms that our test strain of yeast is like Salmonella typhimurium in its inability to respond to the methyl-substituted derivatives of thioxanthenones and indazoles, for example, lucanthone and IA-3 (Hartman et al., 1973; Mayer, 1972). The hydroxylating enzymes that could convert these compounds into mutagens (Hartman and Hulbert, 1976) must be lacking in the strain of Saccharomyces we are using as a test system.
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EFFECT OF VARYING TIME OF EXPOSURE AND CONCENTRATION OF COMPOUNDS One of the more startling discoveries came in the time-concentration series of experiments (Meadows, 1973; Igali and von Borstel, 1974; S. Igali and R. C. von Borstel, unpublished data). Here it was found that, for hycanthone, when the time of exposure was varied and the concentration held constant, the cells were approximately 3 times as sensitive as when time of exposure was held constant and concentration of the hycanthone was varied. This effect was noted both for cell killing and mutation (Meadows, 1973). This is most simply explained by assuming that hycanthone is activated into a more mutagenic state over a period of time. P. J. Hastings (personal communication) suggested that the results can also be explained by assuming that hycanthone can induce a series of enzymes that activate hycanthone to a more mutagenic state. With this notion in mind, an attempt was made to mutagenize with hycanthone at different temperatures. It was found that survival is not depressed nor are mutations induced by hycanthone when cells are incubated at 4°C with hycanthone, for times varying from 8 to 96 hr (S. Igali and R. C. von Borstel, unpublished data). Two alternatives come to mind: either hycanthone is not absorbed at 4°C or enzymes that might activate hycanthone are prevented from being activated at this temperature. The reverse of the hycanthone time-concentration effect was found for lucanthone at pH 7.0, and IA-3 and IA-5 at pH 5.9 (Igali and von Borstel, 1974; S. Igali and R. C. von Borstel, unpublished data). That is, when time of exposure is varied and concentration of the indazole is held constant, the killing is approximately 8 times less than when concentration is varied and time of exposure is held constant. In this respect the indazoles appear to act like radiation in that protracted doses are not as damaging as acute doses. As with radiation the effect of lucanthone and the indazoles can be explained most simply by assuming that, with time, repair systems come into play that repair the lesions caused by the indazoles, or that detoxifying enzyme systems begin to act.
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Again, as has been suggested for ultraviolet radiation (Defais et al., 1971), lucanthone and the indazoles may be acting by inducing the repair systems. One experiment was done to investigate the actions of indazoles on a repair-deficient strain of yeast. The mutant used was rad2-2. It is in the excision-repair pathway for mutational lesions induced by ultraviolet radiation, but it is not the first step (Game and Cox, 1972). This strain shows no increase in sensitivity to either hycanthone or the indazoles whereas it shows a marked increase in sensitivity to ultraviolet radiation (S. Igali and R. C. von Borstel, unpublished data). So at least one of the radiation-repair pathways is not involved in repair of thioxanthenone- or indazole-induced lesions. This response is quite unlike that of Salmonella where defectiveness in excision-repair increases sensitivity to the mutagenicity of these compounds (Hartman and Hulbert, 1976). EFFECTS OF THIOXANTHENONES AND INDAZOLES ON GROWING CELLS Except for the stationary phase cells of yeast, the conidia of Neurospora, the sperm of Drosophila and Habrobracon, and the young oocytes of a mouse, virtually all other test systems for measuring induced mutation frequencies utilize growing and dividing cells. Certainly, the comprehensive studies of Hartman and Ames and their collaborators on 5. typhimurium with antischistosomal compounds and carcinogenic compounds (Hartman et al., 1971, 1973; Ames et al., 1972a,b, 1973), and the careful studies of Clive (1973) with mammalian cells have been done on growing and dividing systems. We have just begun studies with antischistosomal compounds on growing cell cultures, but our first experiments clearly indicate that we may expect some surprises. Hycanthone is much less active mutagenically on growing cell cultures than it is on stationary phase cells (S. Igali and R. C. von Borstel, unpublished data; M. Shahin, unpublished data). In the one experiment carried out to date, no mutations were induced at all with a concentration of 250 jug/ml of hycanthone in complete medium buffered to pH 7.0. Growing cells are not killed as easily as stationary phase cells, again implicating repair, or detoxification, of uptake systems. An indication of nonmutagenicity of growing cells came from spot test studies of hycanthone and other compounds on yeast (Brusick and Zeiger, 1972; R. C. von Borstel, unpublished data), but at the time we merely attributed the finding to the insensitivity of spot tests as a method for measuring mutagenicity. The reverse is certain to be the case as well. For example, it is well known that the anticancer agent ICR-170 is not particularly active on stationary phase cells of yeast, but it is very active on growing yeast cells (Brusick, 1970, 1972), and 5-amino acridine is inactive or antimutagenic on stationary phase cells but quite mutagenic on meiotic cells of yeast (Magni et al., 1964). Shahin (1975) has now shown that the antischistosomal agent
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niridazole is relatively inactive recombinogenetically on stationary phase cells of yeast, but it is quite active on growing cells. On the other hand, one reversion test system, hisl-7, responds to niridazole in stationary phase cells, but hisJ-7 appears not to revert under growing conditions (S. Igali and R. C. von Borstel, unpublished data). So it is mandatory that these studies be continued and growing cell cultures of yeast also be used as a test system. KINETIC ANALYSES OF MUTATION INDUCTION BY HYCANTHONE
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Hycanthone induces both base substitutions and frameshift mutations in stationary phase cells of yeast, yet it is specific for frameshift mutations in cultures of growing cells of 5. typhlmurium. The reason for this difference is not understood, but it may have to do with either unique reactions of different species or the differences in responses of growing and stationary
1
10 HOURS
OF EXPOSURE
FIGURE 1. Dose-response curves of frameshift mutations induced by hycanthone methanesulfonate in a stationary Saccharomyces cerevisiae test strain in phosphate buffer at pH 7.0 (open circles) and at pH 5.9 (solid circles). From Meadows et al. (1973).
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287
100 Hycanthone Added (>ig/ml) FIGURE 2. Dose-response curves of frameshift mutations induced by hycanthone furoate in a growing Salmonella typhimurium test strain in liquid medium at pH 7.0 where the bacteria were exposed to the mutagen for 2 and 4 hr. From Hartman et al. (1973).
phase cells of yeast. An indication that it may be the latter is suggested by the findings of Brusick (1970, 1972) that ICR-170 seems to induce predominantly frameshift mutations in growing yeast, while G. E. Magni (unpublished data) and Sherman and Stewart (1973) found that ICR-170 induces a high frequency of base substitutions in stationary phase cell cultures. If extended times or higher concentrations of hycanthone are used in cultures of growing cells of yeast, it may be that frameshift mutations will predominate. It is of interest to note in both the work of Meadows et al. (1973) and Hartman et al. (1973) that induction of frameshift events by hycanthone follows one-hit kinetics. Yet in both yeast and Salmonella at high doses, the proportion of mutants per survivor declines and the similarity is striking (Figs. 1 and 2). Classically, this decline of mutation frequencies at high doses has been interpreted as being due to two populations, one sensitive and one resistant: as the sensitive population is depleted, the mutant cells of this population are also killed, and then induction of mutants in the resistant
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population can be seen in a secondary rise in the frequency of mutants per survivor (von Borstel, 1966). Yet for the declines at higher doses shown in both Figs. 1 and 2, alternative explanations are more likely to be the correct ones. The decline at high doses in Salmonella shown in Fig. 2 is most likely to be essentially a technical expectation of the experiment (P. E. Hartman, personal communication). The experiment on Salmonella was really one of mutants per milliliter of growing cells rather than mutants per survivor, and at high doses the cell lethality (or inhibition of cell division) begins to outrun the new mutants; that is, the mutants themselves are being killed (or the total cell population is smaller). If both killing and mutation induction are single but separate events, the number of mutants per milliliter where the mutants reach a peak would be at an exposure of mutagen where the population of bacteria surviving is e~x (von Borstel, 1966). Since the Salmonella are growing continuously in these experiments, numerous parameters are interacting, such as rates of killing, rates of cell division, and phenotypic lag. The explanation for the data in yeast shown in Fig. 1 is a different story, for here the data are truly mutants per surviving cell, and the mutant frequency decline at high doses is a real phenomenon. Two different explanations for the yeast data are worth considering in detail because they take into account phenomena not before observed with radiation mutagenesis. Also, and perhaps more importantly, these considerations indicate that a good deal more phenomenology will need to be explored before kinetic analysis of data derived from chemical mutagen experiments will have the significance that is inherent in kinetic analysis of data derived from radiation experiments. Explanation 1 A puzzling feature of results obtained with hycanthone on yeast is that the mutant decline may not begin at the same dose for different mutants (Fig. 3), a result that, by classical kinetic theory, could only be explained by assuming that sets and subsets of cell populations are present as a mixture. This result appears to be unlike results so far obtained with ionizing radiation. Holliday (1971) has provided evidence that recombination-repair is not taking place in cells that die, implying that a proportion of the cells live because they have a recombination-repair ability. The same phenomenon may explain the decline of the dose-mutation curve, namely, that some of the cells have a mutation-repair ability and consequently live to higher doses of the toxic effect than those cells that lack this ability. The observation that decline of the dose-mutation curve occurs at different doses for different types of mutation induction may imply that there are several subpopulations of cells each having a different mutational repair pathway operative or induced in them.
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Oj 02
0.6 1
2
289
4 6 10 20 40 60
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EXPOSURE TO HYCANTHONE (mgh) FIGURE 3. Dose-response curves for cells of Saccharomyces cerevisioe for the reversion from auxotrophy to prototrophy for hisl-7 (open circles), Iys1-1 (open triangles), and hom3-10 (open squares). The dashed portions of the curves correspond to the spontaneous revertant frequency. The cells were exposed to hycanthone at pH 7.0 at a concentration of hycanthone of 0.250 mg/ml.This concentration is multiplied times the hours of exposure to provide the milligram-hour scale of the abscissa.
Explanation 2 Alternatively, one can consider an interesting effect that was observed with both the thioxanthenones and the indazoles. Prototrophic revertants of both spontaneous and induced origin seem to be more susceptible to killing than are cells of the original population of auxotrophic cells. Particularly, the revertants of /ysJ-7 show this sensitivity (Fig. 3). Prototroph sensitivity might cause the decline in mutants per survivor at high dose levels through an enhancement of metabolism of prototrophic cells whereby hycanthone is enactivated into a more lethal state or transported to an intracellular site where it could be more effective in cellular killing. Clearly, more experiments must be done to determine the sensitivities of different genetic strains of yeast under different physiological conditions before the kinetics of mutagenesis can be understood profoundly. CONCLUSIONS The thioxanthenones and indazoles are mutagenic agents with interesting specificities. Some of the specificities are characteristics of species or strains, such as the mutagenic ineffectiveness of methyl-substituted derivatives on both Salmonella and Saccharomyces but not in Neurospora or mammalian cells. On the other hand, Salmonella cells deficient in excision-repair are
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R. C. VONBORSTELANDS. IGALI
more sensitive to these agents, but yeast cells deficient in excision-repair exhibit no such sensitization. Some specificities are physiological, determining the action when certain clusters of enzymes are acting; these very likely cause the phase sensitivities during the cell cycle seen for hycanthone and IA-6.
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REFERENCES Ames, B. N., Gurney, E. G., Miller, J. A. and Bartsch, H. 1972a. Carcinogens as frameshift mutagens: Metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens. Proc Nat. Acad. Sci. U.S. 69:3128-3132. Ames, B. N., Sims, P. and Grover, P. L. 1972b. Epoxides of carcinogenic polycyclic hydrocarbons are frameshift mutagens. Science 176:47-49. Ames, B. N., Lee, F. D. and Durston, W. E. 1973. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Nat. Acad. Sci. U.S. 70:782-788. Beam, C. A., Mortimer, R. K., Wolfe, R. G. and Tobias, C. A. 1954. The relation of radioresistance to budding in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 49:110-122. Brusick, D. J. 1970. The mutagenic activity of ICR-170 in Saccharomyces cerevisiae. Mutation Res. 10:11-19. Brusick, D. J. 1972. Induction of cyclohexamide-resistant mutants in Saccharomyces cerevisiae with N-methyl-N'-nitro-N-nitrosoguanidine and ICR-170. J. Bacteriol. 109:1134-1138. Brusick, D. J. and Mayer, V. M. 1973. New developments in mutagenicity screening techniques with yeast. Environ. Health Perspect. no. 6:83-96. Brusick, D. J. and Zeiger, E. 1972. A comparison of chemically induced reversion patterns of Salmonella typhimurium and Saccharomyces cerevisiae, using in vitro plate tests. Mutation Res. 14:271-275. Clive, D. 1973. Recent developments with the L5178Y TK heterozygote mutagen assay system. Environ. Health Perspect. no. 6:119-125. Defais, M., Fauquet, M., Radman, M. and Errera, M. 1971. Ultraviolet reactivation and ultraviolet mutagenesis of λ in different genetic systems. Virology 43:495-503. Game, J. C. and Cox, B. S. 1972. Epistatic interactions between four rad loci in yeast. Mutation Res. 16:352-362. Hartman, P. E. and Hulbert, P. B. 1976. Genetic activity spectra of some antischistosomal agents, with particular emphasis on thioxanthenones and indazoles.y. Toxicol. Environ. Health. In press. Hartman, P. E., Levine, K., Hartman, Z. and Berger, H. 1971. Hycanthone: A frameshift mutagen. Science 172:1058-1060. Hartman, P. E., Berger, H. and Hartman, Z. 1973. Comparison of hycanthone ("etrenol"), some hycanthone analogs, myxin, and 4-nitroquinoline-1-oxide as frameshift mutagens. J. Pharmacol. Exp. Ther. 186:390-398. Holliday, R. 1971. Biochemical measure of the time of frequency of radiation-induced allelic recombination in Ustilago. Nature New Biol. 232:233-236. Igali, S. and von Borstel, R. C. 1974. Induction of mutations and lethality in Saccharomyces cerevisiae after exposure to several indazole analogs of hycanthone. Mutation Res. 26:452. Magni, G. E., von Borstel, R. C. and Sora, S. 1964. Mutagenic action during meiosis and anti-mutagenic action during mitosis by 5 amino-acridine in yeast. Mutation Res. 1:227-230. Mayer, V. W. 1972. Mutagenic effects induced in Saccharomyces cerevisiae by breakdown products of 1-naphthylamine and 2-naphthylamine formed in an enzyme-free hydroxylation system. Mutation Res. 15:147-153. Meadows, M. G. 1973. Mutagenic action of two indazole analogs on Saccharomyces cerevisiae. M.S. thesis, University of Alberta. Meadows, M. G., Quah, S.-K. and von Borstel, R. C. 1972. Mutation induction in Saccharomyces by hycanthone and a related compound. Genetics 71 (3/2):s39.
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Meadows, M. G., Quah, S.-K. and von Borstel, R. C. 1973. Mutagenic action of hycanthone and IA-4 on yeast. J. Pharmacol. Exp. Ther. 187:444-450. Mortimer, R. K. and Hawthorne, D. C. 1971. Yeast genetics. In The yeasts, ed. A. H. Rose and J. S. Harrison, vol. 1, pp. 386-459. London: Academic Press. Shahin, M. 1975. Genetic activity of niridazole in yeast. Mutation Res. In press. Shahin, M. M. and de Serres, F. J. 1974. The effect of pH on hycanthone methanesulfonate induced inactivation and mitotic recombination in Saccharomyces. Mutation Res. 26:377-384. Sherman, F. and Stewart, J. W. 1973. Mutations at the end of the iso-1-cytochrome c gene of yeast. In The
biochemistry
of gene
expression
in higher
organisms,
e d . J . K . Pollak and J . W . L e e , p p .
56-86. Sydney: Australian and New Zealand Book Co. von Borstel, R. C. 1966. Effects of radiation on cells. In The biological basis of radiation therapy, ed. E. E. Schwartz, pp. 60-125. Philadelphia: Lippincott. von Borstel, R. C. and Quah, S.-K. 1973. Induction of mutations in Saccharomyces with hycanthone. Mutation Res. 21:52.
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Received March 28, 1975 Accepted June 20, 1975
