POINT-MUTATION RESEARCH: RELEVANCE FOR HUMANS Bernd A. Herbold, G. Röhrborn Institut für Humangenetik und Anthropologie der Medizinischen Einrichtungen der Universität Düsseldorf, Düsseldorf, West Germany W. Buselmaier Institut für Anthropologie und Humangenetik der Universität Heidelberg, Heidelberg, West Germany
The host-mediated assay and Salmonella/microsome test are reviewed and critically evaluated. Their methodological problems and relevance for humans are considered.
INTRODUCTION In recent years it has been demonstrated that ionizing rays and various environmental agents—pharmaceutical products, pesticides, cosmetic ingredients, and certain food additives—have mutagenic activities in mammals and humans that are potentiated by additive and/or synergistic effects of those substances. These findings emphasize the necessity of relevant mutagenicity tests because mutations frequently produce pathologic effects in humans. These alterations may be various polymorphisms but they may also be grave deformities. Therefore mutagenicity assays are necessary despite the great expense they entail. Methods of studying different types of mutations have been found which differ in their relevance for humans. Some substances from the groups named above have already been tested in such assays. We believe that tests in mammals have the greatest relevance for humans. One of these is the dominant-lethal test, an indirect method for detecting chromosome aberrations that cause dominant lethal mutations. In this test male or female mice are treated with test compounds and mated with untreated animals. Lethal effects are seen as dead implantations in F j . Concerning the mating time and sex of the treated animals, the sensitive stages in spermatogenesis or oogenesis can be determined, which is of great importance for benefit-risk equations. There are also direct tests that make it possible to demonstrate genetic Requests for reprints should be sent to Dr. Bernd A. Herbold, Institut für Humangenetik und Anthropologie der Medizinischen Einrichtungen der Universität Düsseldorf, Universitätsstrasse 1, 4000 Düsseldorf, West Germany. 1183 Journal of Toxicology and Environmental Health, 2:1183-1191,1977 Copyright © 1977 by Hemisphere Publishing Corporation
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damage in cytogenetic assays by light microscopy. This can be done in somatic and germ cells of mammals. However, only visible damage (gross alterations of the DNA) can be diagnosed with these assays. Problems arise in finding proof of genetic damage in submicroscopic regions; that is, proof of point mutations. The best-known direct method is the specific locus tests, but in most cases it is not possible to use this test because of its cost and the large number of animals needed to ascertain the small number of point-mutation increases with weak mutagens. Nevertheless, it is important to prove point mutations because these alterations may be more compatible with life than are chromosome aberrations. Proof of point mutations is very difficult in mammals but it is very easy in bacteria in vitro. These tests, however, are of little relevance for humans. To increase their relevance, various types of assays have been developed in recent years which still use microorganisms as indicators but also include part of the mammalian metabolism. The fundamental principles involved in the systems of Legator (Gabridge and Legator, 1969) and Ames (Ames et al., 1973a) have been shown to be most suitable. In Legator's test (host-mediated assay) microorganisms are injected into mammals (mostly mice), followed by sc, iv, or oral administration of the test compound. The metabolites of the agent then reach the microorganisms in the body fluid. Mutagenic substances may cause point mutations in indicators. After a suitable incubation time, the microorganisms are removed and checked for genetic alterations by common bacteriological methods. The principal ideas in the system of Ames are a logical extension of this idea. It is well known that 80% or more of metabolism is accomplished by liver microsomes, so it seems obvious to use microorganisms for in vitro metabolic activation rather than whole animals. This is possible because microsomes retain enzymatic activity in vitro (Brock and Hohorst, 1963). Therefore isolated liver microsomes, the test substance, and the indicators are mixed in vitro and plated on culture medium, assuming that the reaction is identical to in vivo metabolism. The simple Ames technique was believed to be a perfect pointmutation assay long before it was tested in everyday use. In this article we want to show some positive and negative aspects of this system in routine screening.
MATERIALS AND METHODS Compounds and Solvents We tested the following substances by liver microsomal assay (Salmonella/microsome test) and compared the results with those obtained by host-mediated assay. Dimethylnitrosamine (DMN), diethylnitrosamine
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1185
(DEN), cyclophosphamide, amethopterin, hydrazine, isoniazid, and methylnitronitrosoguanidine (MNNG) were dissolved in distilled water (pH 7). Agents not sufficiently soluble in polar medium were dissolved in dimethylsulfoxide (DMSO); these were 6-mercaptopurine, azathioprine, trypaflavine, and captan. Bacteria As indicator organisms mutants of Salmonella typhimurium LT-2 were used; those selected by Ames (Ames et al., 1973b) are called 5. typhimurium TA 1535-TA 1538, and those selected by Legator (Gabridge and Legator, 1969) S. typhimurium G 46. Procedures The livers used were extracted from 8-12 wk old male NMRI mice, pretreated with sodium phenobarbital (0.1% in drinking water for 8 days). On the experimental day the animals were killed by cervical dislocation and their livers were removed under sterile conditions. The livers were washed with an equivalent amount (g = ml) of 0.15 M KG solution and homogenized in three volumes of 0.15 M KG. To purify the microsomes and remove the larger cell fragments, the homogenate was centrifuged at 3,000 rpm for 10 min. The supernatant was saved and mixed with substrate solution in the proportion 3:7. The substrate solution contained MgCI2, KG, glucose 6-phosphate, and NADP, and the dilution gave the following concentrations in the mixture: 8 m/W MgCI2, 33 mM KG, 5 m/W glucose 6-phosphate, and 4 vnM NADP. It was buffered at pH 7.4 with 100 m/W sodium phosphate. To test 0.5 ml of this mixture, 0.1 ml test solution and 0.1 ml fully grown bacterial culture were added to 2 ml soft agar (45°C), immediately plated on minimal medium, and examined after 48 hr of incubation at 37°C. RESULTS The compounds used were selected from four groups of agents grouped according to their mode of action. These were alkylating agents (DMN, DEN, cyclophosphamide, captan, and IVINNG), antimetabolites (azathioprine, 6-mercaptopurine, and amethopterin), acridines (trypaflavin), and agents that form radicals in the cell (isoniazid and hydrazine). All chemicais of the first group caused mutations. DMN, DEN, and cyclophosphamide reverted S. typhimurium TA 1535 and G 46. Captan and IVINNG, however, gave rise to a significant increase of mutation rate for all strains except TA 1536. In TA 1536 no compound produced any mutagenic effect. Among the antimetabolites azathioprine and 6-mercaptopurine showed
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genetic activity in S. typhimurium TA 1535 and G 46; amethopterin was the only substance to remain ineffective in this test. The acridine derivative trypaflavine caused frame shift mutations, that is, reverted 5. typhimurium TA 1537 and TA 1538. Both radical-forming agents had mutagenic effects on TA 1535 and G 46, proving INH (isoniazid) to be the weakest mutagen. DISCUSSION Standard mutagens selected from four essential groups were detected with high sensitivity in the Salmonella /microsome test. The bacterial spectrum used seems to be suitable for routine testing. The greater sensitivity of liver microsomal assay compared to host-mediated assay is demonstrated in Table 1. A comparison with cytogenetic results (Table 2) demonstrates the necessity for using point-mutation tests. However, point-mutation systems do not seem to be sufficient to detect all mutagens, as has been postulated elsewhere. Amethopterin was found to be mutagenic in cytogenetic tests (Table 2) but was ineffective in liver microsomal and host-mediated assays. Therefore this system for the detection of mutagens should not be overestimated. However, it seems pointless to expand the spectrum of point-mutation tests for routine mutagenicity testing, especially by using more point-mutation assays in vitro. This is further discussed below. Advantages of the Salmonella /Microsome Test Compared to Other Assays Because this assay is very easy to perform, we can test many compounds at low cost in a short time. Therefore it seems to be the ideal TABLE 1. Comparison of Results Obtained with Two Point-Mutation Substance
Salmonel/a/microsome test"
Host-mediated Assay
DMN
+
+b
DEN MMNG Captan Cyclophosphamide Azathioprine 6-Mercaptopurine Amethopterin Trypaflavine INH Hydrazine
+ + + + + + — + + +
-b>c +b'c +b'c +b —c —c —b>c —c +b>c +b>c
"Herbold and Buselmaier, 1976. ^Proppingetal., 1973. c Pool, 1973.
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TABLE 2. Comparison of Results Obtained with the Salmonella/Mlcrosome Mammalian Cytogenetic Tests Substance DMN DEN MNNG Captan Cyclophosphamide Azathioprine 6-Mercaptopurine Amethopterin Trypaflavin INH Hydrazine
Sa/mone//a/microsome + + + + + + + — + + +
test 0
Test and
Mammalian in vivo systems +b - c +d +e>f +^ +^ +' +' +* -' —m
"Herbold and Buselmaier, 1976. ^Brooks and Cregger, 1973. c Proppinget al., 1972. ^Generoso, 1969. e Collins, 1972a, 1972b. ^Mandzhgaladze and Vashakidze, 1972. ^Hess et al., 1973; Sotomeyor and Cumming, 1975. ''Clark, 1975; Krogh-Jensen, 1970. 'Frohberg and Schulze Schencking, 1975; Generoso et al., 1975; Holden et al., 1973a, 1973b. -'Haas, 1970; Locher and Frantz, 1965; Melnyke et al., 1971; Rohrborn and Hansmann, 1971. ^Baldermann etal., 1967; Brucklacher, 1968. ; Adler and Miiller, 1975; Muller et al., 1975; Rohrborn et al., 1972. '"Epstein and Schafner, 1968.
test system for screening the large number of environmental agents. This is further supported by the small number of mammals needed as liver donors. By using indicators that differ genetically, a wide spectrum of types of point mutations may be detected. Mechanisms of reaction and correlation between causes and effects of mutagenic agents can be examined. Compared to host-mediated assay, liver microsomal assay is more sensitive for the substances shown in Table 1. It was able to demonstrate the mutagenic effects of compounds that were not found to be mutagenic in other systems but were suspected of effecting genetic alterrations. Thus we succeeded in identifying diethylnitrosamine, azathioprine, 6-mercaptopurine, and trypaflavine as point mutagens by liver microsomal assay, whereas they remained ineffective in host-mediated assay. In contrast to their lack of effect in mammalian systems in vivo, isoniazid, hydrazine, and diethylnitrosamine gave rise to mutagenic effects in the Salmonella! microsome test (Table 2). However, we must point out that there are great problems in
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B. A. HERBOLDETAL.
comparing the doses used in these systems, and it may be impossible to solve these problems. Disadvantages of the Sa/mone//a/Microsome Test Compared to Other Systems An essential parameter in extrapolating data to humans is the tolerable dose. In contrast to toxicological evaluations, we cannot use the threshold dose. A very low dosage may produce irreparable genetic defects that are potentiated by cell divisions, whereas killing of some cells or other toxic effects at low concentrations are easily compensated by organisms (Vogel et al., 1974). Extrapolation of the tolerable dose can be done with higher probability when mammals are used to demonstrate mutagenic effects (in cytogenetic assays or the dominant-lethal test, for example) than when indicator systems are used. But this effect is of great relevance in benefit-risk estimations. From this point of view it is our opinion that these assays will only be used in pretesting because it is well known in toxicology that, as Paracelsus said, "All things are poisons and nothing is without poison, only the dose makes that thing not poison." It is our opinion that this common interpretation has to be restricted for chromosome interactions. For the induction of mutations special chemical constitutions seem to be necessary. Nevertheless the critical or tolerable dose is of great importance for benefit-risk estimations in monitoring genetic hazards. But the advantages mentioned above should not be left out of consideration. We should remember that many important medicines would not have been marketed without consideration of these dose effects. Based on considerations about tolerable dose, safety factors should be introduced. They may be small for essential drugs,, such as cytostatin, but may be greater according to the exposed population and lesser effect of the disease. The same considerations apply to other environmental agents. Further essential problems arise from the fact that in liver microsomal assay only soluble substances can be investigated. Even if substances are sufficiently soluble in nonpolar medium, there may be complications because they precipitate in the polar experimental medium. Turbidity may occur after the mixture is plated, making it impossible to examine the plates. Volatile agents evaporate from experimental mixtures before they can be plated. An example is acrolein, a metabolite of cyclophosphamide. This compound is mutagenic per se, but proving this in the Salmonellal microsome test is very difficult because of its high volatility (B. A. Herbold, unpublished data). The effect of mode of application cannot be demonstrated in liver microsomal assay. In host-mediated assay MNNG is not mutagenic when administered orally. It may not reach bacteria in the peritoneum because it is not absorbed by the intestines (Propping and Buselmaier, 1971). In
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contrast, cycasin appears mutagenic only after sc injection (Legator, 1970). Differences in mutagenesis observed with different modes of application must be included in benefit-risk considerations. A further disadvantage in this system is the background due to the addition of substrates for which bacteria are auxotrophic. The quantity is calculated to bring out small bacterial colonies invisible to the naked eye. This produces a milky turbidity on plates, which Ames used to demonstrate the inactivating effects of compounds, but which may falsify the data. The background forms large colonies from a low total amount of bacteria. If the bacteria/substrate ratio is altered, the indicators will receive more substrate per bacterium, form colonies, and demonstrate a pseudomutagenic effect. The background would not be necessary if a titer determination could be performed to determine exact toxic effects. Lowering the amino acid concentrations also presents some problems. To demonstrate mutagenic effects, indicators need a few cell divisions to mutate. Therefore one is forced to add minimal quantities of needed substrates. The level to which these concentrations can be diminished has not yet been determined, but if it is impossible to lower this background growth it will be very difficult or even impossible to examine the cultures. Compounds may be transformed to mutagens by mammalian metabolism and vice versa. But this inactivation can not be demonstrated in the Salmonella/m'icrosome test because of the experimental setup. This setup also prevents use of chronic exposure. CONCLUSIONS Because it is not practical to test point mutagens directly in mammals in vivo, it is still necessary to use the indicator systems described above. The results obtained have to be interpreted taking into account that the data were gathered from microorganisms even if mammalian metabolism was involved. The Sa/monel/a/microsome test has some advantages compared to other available methods. It cannot be used exclusively, but it is an improvement on the other present methods. For all relevant mutagenicity tests, point-mutation assays must be complemented by in vivo cytogenetic methods in mammals. REFERENCES Adler, I. D. and Müller, D. 1975. Chromosome analysis in spermatocytes of mice and Chinese hamsters. Mutat. Res. 29:256. Ames, B. N., Durston, W. E., Yamasaki, E. and Lee, F. D. 1973a. Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. U.S.A. 70:2281-2285. Ames, B. N., Lee, F. D. and Durston, W. E. 1973b. An improved bacterial system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. U.S.A. 70:782-786. Baldermann, K. H., Röhrborn, G. and Schroeder, T. M. 1967. Mutagenitätsuntersuchungen mit
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Trypaflavin und Hexamethylentetramin am Säuger in vivo und in vitro. Humangenetik 4:112-126. Brock, N. and Hohorst, H. J. 1963. Über die Aktivierung von Cyclophosphamid in vivo und in vitro. Arzneim. Forsch. 13:1021-1031. Brookes, A. L. and Cregger, V. 1973. Production of chromosome type aberrations in the liver cells of the Chinese hamster by dimethylnitrosamine (DMN). Mutat. Res. 21:214. Brucklacher, M. 1968. Untersuchungen zur Induktion prä- und postimplantativer Eiverluste durch Behandlung virginer weiblicher C3H Mäuse mit Trypaflavin. Ph.D. thesis, RuprechtKarl-University, Heidelberg, Germany. Clark, M. 1975. The mutagenicity of azathioprine in mice, Drosophila melanogaster and Neurospora crassa. Mutat. Res. 28:87-99. Collins, T. F. X. 1972a. Dominant lethal assay. I. Captan. Food Cosmet. Toxicol. 10:253-262. Collins, T. F. X. 1972b. Effect of captan and triethylenemelamine (TEM) on reproductive fitness of DBA/2J mice. Toxicol. Appl. Pharmacol. 23:277-287. Epstein, S. S. and Schafner, H. 1968. Chemical agents in the human environment. Nature (Lond.) 219:385-387. Frohberg, H. and Schulze Schencking, M. 1975. In vivo cytogenetic investigations in bone marrow cells of rats, Chinese hamsters and mice treated with 6-mercaptopurine. Arch. Toxicol. 33:209-224. Gabridge, M. G. and Legator, M. S. 1969. A host-mediated microbial assay for the detection of mutagenic compounds. Proc. Soc. Exp. Biol. Med. 130:831-834. Generoso, W. M. 1969. Chemical induction of dominant lethals in female mice. Genetics 61:461-470. Generoso, W. M., Preston, R. J. and Brewen, J. G. 1975. 6-Mercaptopurine, an inducer of cytogenetic and dominant-lethal effects in premeiotic and early meiotic germ cells of male mice. Mutat. Res. 28:437-447. Haas, E. 1970. Die Wirkung von Methotrexat auf die Spermatogenese der Maus. Ph.D. thesis, Ruprecht-Karl-University, Heidelberg, Germany. Herbold, B. and Buselmaier, W. 1976. Induction of point mutations by different chemical mechanisms in the liver microsomal assay. Mutat. Res. 40:73-84. Hess, R., Langauer, M., Rathenberg, R., Strasser, F. and Müller, D. 1973. Evaluation of the micronucleus test in comparison with chromosome studies on germ cells and on somatic cells of Chinese hamsters treated with cyclophosphamide. Mutat. Res. 21:36. Holden, H. E., Ray, V. A., Wahrenburg, M. G. and Zelenski, J. D. 1973a. Mutagenicity studies with 6-mercaptopurine. I. Cytogenetic activity in vivo. Mutat. Res. 20:257-263. Holden, H. E., Ray, V. A., Wahrenburg, M. G. and Zelenski, J. D. 1973b. In vitro cytogenetic studies with 6-mercaptopurine. Mutat. Res. 21:222. Krogh-Jensen, M. 1970. Effect of azathioprine on the chromosome complement of human bone marrow cells. Int. J. Cancer 5:147-151. Legator, M. S. 1970. The host-mediated assay, a practical procedure for evaluating potential mutagenic agents. In Chemical mutagenesis in mammals and man, eds. F. Vogel and G. Röhrborn. Berlin: Springer. Locher, H. and Fräntz, J. 1965. Chromosomenveränderungen nach Methotrexatbehandlung. Med. Welt 34. Mandzhgaladze, R. N. and Vashakidze, V. I. 1972. Action of some chemical compounds on rat progeny and sex ratio. Soobshch. Akad. Nauk. Gruz. SSR 65:485-488. Melnyk, J., Duffy, D. M. and Sparkes, R. S. 1971. Human mitotic and meiotic chromosome damage following in vivo exposure to methotrexate. Clin. Genet. 2 : 2 8 - 3 1 . Müller, D., Grafe, A., Miltenburger, H., Röhrborn, G. and Schulze Schencking, M. 1975. Chromosome analysis of bone marrow and nucleus anomaly test in mammals after treatment with INH. Mutat. Res. 29:256. Pool, B. L. 1973. Untersuchungen zur Eignung eines repairfähigen und eines repairdefizienten Escherichia coli Stammes im Host-mediated Assay. Thesis, Ruprecht-Karl-University, Heidelberg, Germany.
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Propping, P. and Buselmaier, W. 1971. The influence of metabolism on mutagenic activity in the host-mediated assay. Arch. Toxicol. 28:129-134. Propping, P., Röhrborn, G. and Buselmaier, W. 1972. Comparative investigations on the chemical induction of point mutations and dominant lethal mutations in mice. Mol. Gen. Genet. 117:197-209. Propping, P., Buselmaier, W. and Röhrborn, G. 1973. Kritische Betrachtung über die intra-animale Kultur von Mikroorganismen, eine Methode zum Nachweis chemisch induzierter Mutationen. Arzneim. Forsch. 23:746-749. Röhrborn, G. and Hansmann, I. 1971. induced chromosome aberrations in unfertilized oocytes of mice. Humangenetik 13:184-198. Röhrborn, G., Propping, P. and Buselmaier, W. 1972. Mutagenic activity of isoniazid and hydrazine in mammalian test systems. Mutat. Res. 16:189-194. Sotomayor, R. E. and Cumming, R. B. 1975. Induction of translocations by cyclophosphamide in different germ cell stages of male mice. Cytological characterization and transmission. Mutat. Res. 27:375-388. Vogel, F., Röhrborn, G. and Hansmann, I. 1974. Die Testung von Fremdstoffen auf Mutagenität. Arzneim. Forsch. 24:1665-1677. Received January 21, 1977 Accepted February 75, 7977
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The host-mediated assay and Salmonella/microsome test are reviewed and critically evaluated. Their methodological problems and relevance for humans are considered.
INTRODUCTION In recent years it has been demonstrated that ionizing rays and various environmental agents—pharmaceutical products, pesticides, cosmetic ingredients, and certain food additives—have mutagenic activities in mammals and humans that are potentiated by additive and/or synergistic effects of those substances. These findings emphasize the necessity of relevant mutagenicity tests because mutations frequently produce pathologic effects in humans. These alterations may be various polymorphisms but they may also be grave deformities. Therefore mutagenicity assays are necessary despite the great expense they entail. Methods of studying different types of mutations have been found which differ in their relevance for humans. Some substances from the groups named above have already been tested in such assays. We believe that tests in mammals have the greatest relevance for humans. One of these is the dominant-lethal test, an indirect method for detecting chromosome aberrations that cause dominant lethal mutations. In this test male or female mice are treated with test compounds and mated with untreated animals. Lethal effects are seen as dead implantations in F j . Concerning the mating time and sex of the treated animals, the sensitive stages in spermatogenesis or oogenesis can be determined, which is of great importance for benefit-risk equations. There are also direct tests that make it possible to demonstrate genetic Requests for reprints should be sent to Dr. Bernd A. Herbold, Institut für Humangenetik und Anthropologie der Medizinischen Einrichtungen der Universität Düsseldorf, Universitätsstrasse 1, 4000 Düsseldorf, West Germany. 1183 Journal of Toxicology and Environmental Health, 2:1183-1191,1977 Copyright © 1977 by Hemisphere Publishing Corporation
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B. A. HERBOLD ETAL.
damage in cytogenetic assays by light microscopy. This can be done in somatic and germ cells of mammals. However, only visible damage (gross alterations of the DNA) can be diagnosed with these assays. Problems arise in finding proof of genetic damage in submicroscopic regions; that is, proof of point mutations. The best-known direct method is the specific locus tests, but in most cases it is not possible to use this test because of its cost and the large number of animals needed to ascertain the small number of point-mutation increases with weak mutagens. Nevertheless, it is important to prove point mutations because these alterations may be more compatible with life than are chromosome aberrations. Proof of point mutations is very difficult in mammals but it is very easy in bacteria in vitro. These tests, however, are of little relevance for humans. To increase their relevance, various types of assays have been developed in recent years which still use microorganisms as indicators but also include part of the mammalian metabolism. The fundamental principles involved in the systems of Legator (Gabridge and Legator, 1969) and Ames (Ames et al., 1973a) have been shown to be most suitable. In Legator's test (host-mediated assay) microorganisms are injected into mammals (mostly mice), followed by sc, iv, or oral administration of the test compound. The metabolites of the agent then reach the microorganisms in the body fluid. Mutagenic substances may cause point mutations in indicators. After a suitable incubation time, the microorganisms are removed and checked for genetic alterations by common bacteriological methods. The principal ideas in the system of Ames are a logical extension of this idea. It is well known that 80% or more of metabolism is accomplished by liver microsomes, so it seems obvious to use microorganisms for in vitro metabolic activation rather than whole animals. This is possible because microsomes retain enzymatic activity in vitro (Brock and Hohorst, 1963). Therefore isolated liver microsomes, the test substance, and the indicators are mixed in vitro and plated on culture medium, assuming that the reaction is identical to in vivo metabolism. The simple Ames technique was believed to be a perfect pointmutation assay long before it was tested in everyday use. In this article we want to show some positive and negative aspects of this system in routine screening.
MATERIALS AND METHODS Compounds and Solvents We tested the following substances by liver microsomal assay (Salmonella/microsome test) and compared the results with those obtained by host-mediated assay. Dimethylnitrosamine (DMN), diethylnitrosamine
POINT-MUTATION RESEARCH
1185
(DEN), cyclophosphamide, amethopterin, hydrazine, isoniazid, and methylnitronitrosoguanidine (MNNG) were dissolved in distilled water (pH 7). Agents not sufficiently soluble in polar medium were dissolved in dimethylsulfoxide (DMSO); these were 6-mercaptopurine, azathioprine, trypaflavine, and captan. Bacteria As indicator organisms mutants of Salmonella typhimurium LT-2 were used; those selected by Ames (Ames et al., 1973b) are called 5. typhimurium TA 1535-TA 1538, and those selected by Legator (Gabridge and Legator, 1969) S. typhimurium G 46. Procedures The livers used were extracted from 8-12 wk old male NMRI mice, pretreated with sodium phenobarbital (0.1% in drinking water for 8 days). On the experimental day the animals were killed by cervical dislocation and their livers were removed under sterile conditions. The livers were washed with an equivalent amount (g = ml) of 0.15 M KG solution and homogenized in three volumes of 0.15 M KG. To purify the microsomes and remove the larger cell fragments, the homogenate was centrifuged at 3,000 rpm for 10 min. The supernatant was saved and mixed with substrate solution in the proportion 3:7. The substrate solution contained MgCI2, KG, glucose 6-phosphate, and NADP, and the dilution gave the following concentrations in the mixture: 8 m/W MgCI2, 33 mM KG, 5 m/W glucose 6-phosphate, and 4 vnM NADP. It was buffered at pH 7.4 with 100 m/W sodium phosphate. To test 0.5 ml of this mixture, 0.1 ml test solution and 0.1 ml fully grown bacterial culture were added to 2 ml soft agar (45°C), immediately plated on minimal medium, and examined after 48 hr of incubation at 37°C. RESULTS The compounds used were selected from four groups of agents grouped according to their mode of action. These were alkylating agents (DMN, DEN, cyclophosphamide, captan, and IVINNG), antimetabolites (azathioprine, 6-mercaptopurine, and amethopterin), acridines (trypaflavin), and agents that form radicals in the cell (isoniazid and hydrazine). All chemicais of the first group caused mutations. DMN, DEN, and cyclophosphamide reverted S. typhimurium TA 1535 and G 46. Captan and IVINNG, however, gave rise to a significant increase of mutation rate for all strains except TA 1536. In TA 1536 no compound produced any mutagenic effect. Among the antimetabolites azathioprine and 6-mercaptopurine showed
1186
B. A. HERBOLD ETAL.
genetic activity in S. typhimurium TA 1535 and G 46; amethopterin was the only substance to remain ineffective in this test. The acridine derivative trypaflavine caused frame shift mutations, that is, reverted 5. typhimurium TA 1537 and TA 1538. Both radical-forming agents had mutagenic effects on TA 1535 and G 46, proving INH (isoniazid) to be the weakest mutagen. DISCUSSION Standard mutagens selected from four essential groups were detected with high sensitivity in the Salmonella /microsome test. The bacterial spectrum used seems to be suitable for routine testing. The greater sensitivity of liver microsomal assay compared to host-mediated assay is demonstrated in Table 1. A comparison with cytogenetic results (Table 2) demonstrates the necessity for using point-mutation tests. However, point-mutation systems do not seem to be sufficient to detect all mutagens, as has been postulated elsewhere. Amethopterin was found to be mutagenic in cytogenetic tests (Table 2) but was ineffective in liver microsomal and host-mediated assays. Therefore this system for the detection of mutagens should not be overestimated. However, it seems pointless to expand the spectrum of point-mutation tests for routine mutagenicity testing, especially by using more point-mutation assays in vitro. This is further discussed below. Advantages of the Salmonella /Microsome Test Compared to Other Assays Because this assay is very easy to perform, we can test many compounds at low cost in a short time. Therefore it seems to be the ideal TABLE 1. Comparison of Results Obtained with Two Point-Mutation Substance
Salmonel/a/microsome test"
Host-mediated Assay
DMN
+
+b
DEN MMNG Captan Cyclophosphamide Azathioprine 6-Mercaptopurine Amethopterin Trypaflavine INH Hydrazine
+ + + + + + — + + +
-b>c +b'c +b'c +b —c —c —b>c —c +b>c +b>c
"Herbold and Buselmaier, 1976. ^Proppingetal., 1973. c Pool, 1973.
POINT-MUTATION RESEARCH
1187
TABLE 2. Comparison of Results Obtained with the Salmonella/Mlcrosome Mammalian Cytogenetic Tests Substance DMN DEN MNNG Captan Cyclophosphamide Azathioprine 6-Mercaptopurine Amethopterin Trypaflavin INH Hydrazine
Sa/mone//a/microsome + + + + + + + — + + +
test 0
Test and
Mammalian in vivo systems +b - c +d +e>f +^ +^ +' +' +* -' —m
"Herbold and Buselmaier, 1976. ^Brooks and Cregger, 1973. c Proppinget al., 1972. ^Generoso, 1969. e Collins, 1972a, 1972b. ^Mandzhgaladze and Vashakidze, 1972. ^Hess et al., 1973; Sotomeyor and Cumming, 1975. ''Clark, 1975; Krogh-Jensen, 1970. 'Frohberg and Schulze Schencking, 1975; Generoso et al., 1975; Holden et al., 1973a, 1973b. -'Haas, 1970; Locher and Frantz, 1965; Melnyke et al., 1971; Rohrborn and Hansmann, 1971. ^Baldermann etal., 1967; Brucklacher, 1968. ; Adler and Miiller, 1975; Muller et al., 1975; Rohrborn et al., 1972. '"Epstein and Schafner, 1968.
test system for screening the large number of environmental agents. This is further supported by the small number of mammals needed as liver donors. By using indicators that differ genetically, a wide spectrum of types of point mutations may be detected. Mechanisms of reaction and correlation between causes and effects of mutagenic agents can be examined. Compared to host-mediated assay, liver microsomal assay is more sensitive for the substances shown in Table 1. It was able to demonstrate the mutagenic effects of compounds that were not found to be mutagenic in other systems but were suspected of effecting genetic alterrations. Thus we succeeded in identifying diethylnitrosamine, azathioprine, 6-mercaptopurine, and trypaflavine as point mutagens by liver microsomal assay, whereas they remained ineffective in host-mediated assay. In contrast to their lack of effect in mammalian systems in vivo, isoniazid, hydrazine, and diethylnitrosamine gave rise to mutagenic effects in the Salmonella! microsome test (Table 2). However, we must point out that there are great problems in
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comparing the doses used in these systems, and it may be impossible to solve these problems. Disadvantages of the Sa/mone//a/Microsome Test Compared to Other Systems An essential parameter in extrapolating data to humans is the tolerable dose. In contrast to toxicological evaluations, we cannot use the threshold dose. A very low dosage may produce irreparable genetic defects that are potentiated by cell divisions, whereas killing of some cells or other toxic effects at low concentrations are easily compensated by organisms (Vogel et al., 1974). Extrapolation of the tolerable dose can be done with higher probability when mammals are used to demonstrate mutagenic effects (in cytogenetic assays or the dominant-lethal test, for example) than when indicator systems are used. But this effect is of great relevance in benefit-risk estimations. From this point of view it is our opinion that these assays will only be used in pretesting because it is well known in toxicology that, as Paracelsus said, "All things are poisons and nothing is without poison, only the dose makes that thing not poison." It is our opinion that this common interpretation has to be restricted for chromosome interactions. For the induction of mutations special chemical constitutions seem to be necessary. Nevertheless the critical or tolerable dose is of great importance for benefit-risk estimations in monitoring genetic hazards. But the advantages mentioned above should not be left out of consideration. We should remember that many important medicines would not have been marketed without consideration of these dose effects. Based on considerations about tolerable dose, safety factors should be introduced. They may be small for essential drugs,, such as cytostatin, but may be greater according to the exposed population and lesser effect of the disease. The same considerations apply to other environmental agents. Further essential problems arise from the fact that in liver microsomal assay only soluble substances can be investigated. Even if substances are sufficiently soluble in nonpolar medium, there may be complications because they precipitate in the polar experimental medium. Turbidity may occur after the mixture is plated, making it impossible to examine the plates. Volatile agents evaporate from experimental mixtures before they can be plated. An example is acrolein, a metabolite of cyclophosphamide. This compound is mutagenic per se, but proving this in the Salmonellal microsome test is very difficult because of its high volatility (B. A. Herbold, unpublished data). The effect of mode of application cannot be demonstrated in liver microsomal assay. In host-mediated assay MNNG is not mutagenic when administered orally. It may not reach bacteria in the peritoneum because it is not absorbed by the intestines (Propping and Buselmaier, 1971). In
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contrast, cycasin appears mutagenic only after sc injection (Legator, 1970). Differences in mutagenesis observed with different modes of application must be included in benefit-risk considerations. A further disadvantage in this system is the background due to the addition of substrates for which bacteria are auxotrophic. The quantity is calculated to bring out small bacterial colonies invisible to the naked eye. This produces a milky turbidity on plates, which Ames used to demonstrate the inactivating effects of compounds, but which may falsify the data. The background forms large colonies from a low total amount of bacteria. If the bacteria/substrate ratio is altered, the indicators will receive more substrate per bacterium, form colonies, and demonstrate a pseudomutagenic effect. The background would not be necessary if a titer determination could be performed to determine exact toxic effects. Lowering the amino acid concentrations also presents some problems. To demonstrate mutagenic effects, indicators need a few cell divisions to mutate. Therefore one is forced to add minimal quantities of needed substrates. The level to which these concentrations can be diminished has not yet been determined, but if it is impossible to lower this background growth it will be very difficult or even impossible to examine the cultures. Compounds may be transformed to mutagens by mammalian metabolism and vice versa. But this inactivation can not be demonstrated in the Salmonella/m'icrosome test because of the experimental setup. This setup also prevents use of chronic exposure. CONCLUSIONS Because it is not practical to test point mutagens directly in mammals in vivo, it is still necessary to use the indicator systems described above. The results obtained have to be interpreted taking into account that the data were gathered from microorganisms even if mammalian metabolism was involved. The Sa/monel/a/microsome test has some advantages compared to other available methods. It cannot be used exclusively, but it is an improvement on the other present methods. For all relevant mutagenicity tests, point-mutation assays must be complemented by in vivo cytogenetic methods in mammals. REFERENCES Adler, I. D. and Müller, D. 1975. Chromosome analysis in spermatocytes of mice and Chinese hamsters. Mutat. Res. 29:256. Ames, B. N., Durston, W. E., Yamasaki, E. and Lee, F. D. 1973a. Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. U.S.A. 70:2281-2285. Ames, B. N., Lee, F. D. and Durston, W. E. 1973b. An improved bacterial system for the detection and classification of mutagens and carcinogens. Proc. Natl. Acad. Sci. U.S.A. 70:782-786. Baldermann, K. H., Röhrborn, G. and Schroeder, T. M. 1967. Mutagenitätsuntersuchungen mit
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Trypaflavin und Hexamethylentetramin am Säuger in vivo und in vitro. Humangenetik 4:112-126. Brock, N. and Hohorst, H. J. 1963. Über die Aktivierung von Cyclophosphamid in vivo und in vitro. Arzneim. Forsch. 13:1021-1031. Brookes, A. L. and Cregger, V. 1973. Production of chromosome type aberrations in the liver cells of the Chinese hamster by dimethylnitrosamine (DMN). Mutat. Res. 21:214. Brucklacher, M. 1968. Untersuchungen zur Induktion prä- und postimplantativer Eiverluste durch Behandlung virginer weiblicher C3H Mäuse mit Trypaflavin. Ph.D. thesis, RuprechtKarl-University, Heidelberg, Germany. Clark, M. 1975. The mutagenicity of azathioprine in mice, Drosophila melanogaster and Neurospora crassa. Mutat. Res. 28:87-99. Collins, T. F. X. 1972a. Dominant lethal assay. I. Captan. Food Cosmet. Toxicol. 10:253-262. Collins, T. F. X. 1972b. Effect of captan and triethylenemelamine (TEM) on reproductive fitness of DBA/2J mice. Toxicol. Appl. Pharmacol. 23:277-287. Epstein, S. S. and Schafner, H. 1968. Chemical agents in the human environment. Nature (Lond.) 219:385-387. Frohberg, H. and Schulze Schencking, M. 1975. In vivo cytogenetic investigations in bone marrow cells of rats, Chinese hamsters and mice treated with 6-mercaptopurine. Arch. Toxicol. 33:209-224. Gabridge, M. G. and Legator, M. S. 1969. A host-mediated microbial assay for the detection of mutagenic compounds. Proc. Soc. Exp. Biol. Med. 130:831-834. Generoso, W. M. 1969. Chemical induction of dominant lethals in female mice. Genetics 61:461-470. Generoso, W. M., Preston, R. J. and Brewen, J. G. 1975. 6-Mercaptopurine, an inducer of cytogenetic and dominant-lethal effects in premeiotic and early meiotic germ cells of male mice. Mutat. Res. 28:437-447. Haas, E. 1970. Die Wirkung von Methotrexat auf die Spermatogenese der Maus. Ph.D. thesis, Ruprecht-Karl-University, Heidelberg, Germany. Herbold, B. and Buselmaier, W. 1976. Induction of point mutations by different chemical mechanisms in the liver microsomal assay. Mutat. Res. 40:73-84. Hess, R., Langauer, M., Rathenberg, R., Strasser, F. and Müller, D. 1973. Evaluation of the micronucleus test in comparison with chromosome studies on germ cells and on somatic cells of Chinese hamsters treated with cyclophosphamide. Mutat. Res. 21:36. Holden, H. E., Ray, V. A., Wahrenburg, M. G. and Zelenski, J. D. 1973a. Mutagenicity studies with 6-mercaptopurine. I. Cytogenetic activity in vivo. Mutat. Res. 20:257-263. Holden, H. E., Ray, V. A., Wahrenburg, M. G. and Zelenski, J. D. 1973b. In vitro cytogenetic studies with 6-mercaptopurine. Mutat. Res. 21:222. Krogh-Jensen, M. 1970. Effect of azathioprine on the chromosome complement of human bone marrow cells. Int. J. Cancer 5:147-151. Legator, M. S. 1970. The host-mediated assay, a practical procedure for evaluating potential mutagenic agents. In Chemical mutagenesis in mammals and man, eds. F. Vogel and G. Röhrborn. Berlin: Springer. Locher, H. and Fräntz, J. 1965. Chromosomenveränderungen nach Methotrexatbehandlung. Med. Welt 34. Mandzhgaladze, R. N. and Vashakidze, V. I. 1972. Action of some chemical compounds on rat progeny and sex ratio. Soobshch. Akad. Nauk. Gruz. SSR 65:485-488. Melnyk, J., Duffy, D. M. and Sparkes, R. S. 1971. Human mitotic and meiotic chromosome damage following in vivo exposure to methotrexate. Clin. Genet. 2 : 2 8 - 3 1 . Müller, D., Grafe, A., Miltenburger, H., Röhrborn, G. and Schulze Schencking, M. 1975. Chromosome analysis of bone marrow and nucleus anomaly test in mammals after treatment with INH. Mutat. Res. 29:256. Pool, B. L. 1973. Untersuchungen zur Eignung eines repairfähigen und eines repairdefizienten Escherichia coli Stammes im Host-mediated Assay. Thesis, Ruprecht-Karl-University, Heidelberg, Germany.
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Propping, P. and Buselmaier, W. 1971. The influence of metabolism on mutagenic activity in the host-mediated assay. Arch. Toxicol. 28:129-134. Propping, P., Röhrborn, G. and Buselmaier, W. 1972. Comparative investigations on the chemical induction of point mutations and dominant lethal mutations in mice. Mol. Gen. Genet. 117:197-209. Propping, P., Buselmaier, W. and Röhrborn, G. 1973. Kritische Betrachtung über die intra-animale Kultur von Mikroorganismen, eine Methode zum Nachweis chemisch induzierter Mutationen. Arzneim. Forsch. 23:746-749. Röhrborn, G. and Hansmann, I. 1971. induced chromosome aberrations in unfertilized oocytes of mice. Humangenetik 13:184-198. Röhrborn, G., Propping, P. and Buselmaier, W. 1972. Mutagenic activity of isoniazid and hydrazine in mammalian test systems. Mutat. Res. 16:189-194. Sotomayor, R. E. and Cumming, R. B. 1975. Induction of translocations by cyclophosphamide in different germ cell stages of male mice. Cytological characterization and transmission. Mutat. Res. 27:375-388. Vogel, F., Röhrborn, G. and Hansmann, I. 1974. Die Testung von Fremdstoffen auf Mutagenität. Arzneim. Forsch. 24:1665-1677. Received January 21, 1977 Accepted February 75, 7977
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