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Mutation Research, 41 (1976) 163--170
© Elsevier/North-Holland Biomedical Press
PANEL DISCUSSION ON SUBMAMMALIAN SYSTEMS F.K. ZIMMERMANN Technische Hochschule Darmstadl, Schnittpahnstrasse 61, Darmstadt (BRD)
(Received April 23rd, 1976) Participants: R.C. yon Borstel, D.J. Brusick, H. Kurata, G.R. Mohn, A.T. Natarajan, N.K. Notani, J. SchSneich, J. Velemfnsk:~, F.H. W/irgler and F.K. Zimmermann (co-ordinator) Submammalian systems, whose development started some t w o decades ago, have turned out to be useful and powerful for a qualitative evaluation of genetic hazards exerted by environmental chemicals. They have been used to investigate the induction of generative genetic damage which contributes to the genetic load of natural populations, and also induction of genetic damage in somatic cells which is thought to be a cause of carcinogenesis [3,17,38] and premature senility [8]. The inclusion of submammalian systems in screening programs for environmental mutagens provides the following advantages. (1) All known genetic alterations can be scored and the genetic nature of the effects readily established, thus meeting the transmissibility criterion. (2) Metabolic activation of promutagens by mammalian, and in certain cases human, metabolism can be combined in several ways with submammalian indicator test systems. In addition, metabolic activation of chemicals by agricultural crop plants can also be detected. In Drosophila, metabolic activation is believed to be similar to that in mammals. (3) All tests can be performed at low cost and within a short time. The only major limitation of submammalian systems in environmental screening programs is that they allow only for qualitative assessments of genetic hazards but not necessarily for a quantitative evaluation in the sense of possible threshold or tolerance doses in man. The following end points have to be scored by a complete screening program for environmental mutagens: (1) point mutations; (2) structural chromosomal aberrations; (3) numerical chromosomal aberrations; (4) genetic alterations in cytoplasmic genetic determinants and in the case of somatic genetic damage, relevant in respect to carcinogenieity; (5) mitotic recombination. Evaluation of presently available test systems for the different genetic end points Systems for the detection of point mutations have reached a high degree of sophistication. Through the use of various microbial strains, e.g. S a l m o n e l l a
164 typhimurium, mutagens can be classified as base-pair substitution {including base specificity) and frame-shift mutagens [2,3]. These systems are based on the mutational restoration of p r o t o t r o p h y in auxotrophic strains. Permeability problems have partly been overcome by introduction into the test strains of mutations affecting the bacterial cell envelope. In addition, the strains have been made very sensitive to mutagenic action by introducing mutationally determined defects in repair activities. A similar possibility of detailed analysis of mutational events is provided by the unicellular eukaryote Saccharomyces cerevisiae {baker's yeast) which Sherman and collaborators used to develop a reverse mutation system to a degree of sophistication that monitors not only plain mutation induction but also transitions, transversion and base specificity [22]. The sensitivity of this system can still be enhanced by introducing certain defects in repair systems; and certain cell wall or cell membrane defects may be introduced to circumvent permeation barriers. However, this system and its further potential have not been exploited for environmental mutagenesis work. Reverse mutation systems, in spite of their great sensitivity, have limitations because they are based on the restoration of a functional allele or genotype from a non-functional condition. Such restorations usually require very specific mutational events which cannot be induced by all mutagens, a phenomenon called mutagen specificity. This limitation can partly be compensated by using a whole battery of the different mutant alleles sensitive to as many known mutagens as possible. Nevertheless, it became increasingly clear that strain and mutagen specificity can best be overcome by using forward mutation tests in which mutations in many sites within one or several genes can be detected. The best established test system is provided by the classical test for recessive lethal mutations in the X-chromosome of Drosophila [1,31]. Using Escherichia coli as an indicator organism, Mohn et al. [19] have developed a multi-purpose strain in which several types of mutation can be detected simultaneously including deletions extending to two adjacent genes, and furthermore allowing the testing for induction of prophage lambda (Mohn and Ellenberger, to be published). Selective forward mutation systems are also available in the yeast Saccharomyces cerevisiae. One such system has been elaborated by Brusick [7]. This is based on the inactivation of a permease gene which results in a resistance to canavanine. Such mutants were induced by ICR-170 which is a poor inducer of simple base-pair substitution mutation. Consequently, canavanine resistance provides a test for a wide range of mutational alterations. Alper and Ames [4] have devised a test system in Salmonella which specifically monitors induction of deletions several genes long. Another system for the detection of large size deletions -- in addition to point mutations -- in all parts of the genome of Neurospora crassa has been developed by de Serres and Malling [26] using a heterokaryon. Test systems in yeast designed to detect mitotic crossing over also respond to deletions in specific parts of the genome, but they do not allow one to identify the observed effects as unambiguously due to deletion [40,41]. Drosophila provides systems for the detection of various types of structural chromosomal aberrations, not only deletions as in the microbial systems, but also inversions, duplications and translocations [ 31 ]. Meiotic numerical aberrations can also be scored in Drosophila, and systems
165 have been described by Wiirgler in the panel. Systems for chromosomal loss in mitotic cells of Aspergillus nidulans [ 6] and in yeast [21 ] are available. The systems for the detection of chromosomal structural and numerical aberrations described so far are based on genetic rather than microscopic methods of detection. Of course, there is a bulk of experience with cytogenetic studies discussed by Natarajan in the panel. However, these tests do not automatically involve the demonstration of genetic transmissibility; they have to be classified as indirect correlation tests. There is urgency for the inclusion of a test for numerical chromosomal aberrations in environmental mutagenicity screening programs. All other genetic end points are based on DNA as the final genetic target. Changes in chromosome numbers can also be caused by chemicals that interfere with the components of the spindle fiber apparatus, and such chemicals may not interact at all with DNA or the enzymic machinery involved in DNA metabolism. Ommission of this genetic end point may lead to a systematic failure in detecting a rather unique class of mutagens. It is well established that a eukaryotic cell has genetic components not only in the nuclear genetic compartment but also in the cytoplasm, notably so in mitochondria and chloroplasts [24]. If it should turn out that there are mutagens specific for prokaryotes, then they should also be tested in eukaryotes for their ability to induce mitochondrial mutations because of certain essential similarities between bacterial and mitochondrial genetic systems. No systems for the detection of mutations in mitochondrial or other cytoplasmic genetic determinants have been extensively used in environmental mutagenicity screening. This gap has also to be closed. Environmental mutagenesis is not only revelant for the germ line but also for somatic cells in respect to short term carcinogenicity testing. Somatic genetic damage can arise not only through the genetic end points dealt with up to here, but also through recombination processes occurring in mitotic cells. A diploid organism can be heterozygous for recessive lethal or detrimental alleles which can become homozygous and expressed through mitotic recombination and also mitotic non-disjunction. This can result in developmental disorders and deleterious alterations in rapidly dividing tissues. There are two types of mitotic recombination: reciprocal mitotic crossing over and non-reciprocal mitotic gene conversion. Yeast has been a widely used organism for the detection of mitotic crossing over and gene conversion, and strains for their detection are now available and in wide use [ 39--41]. Aside from screening for this particular end point, such strains provide a basis for convenient and rapid detection of genetically active chemicals in general. Further submammalian systems are available and already in use in environmental mutagenicity screening programs. The increased sensitivity of m u t a n t strains with defects in certain repair functions as compared with the respective wild-type strains can be taken as a first indication of genetic activity of a chemical. Sch5neich reported that the screening program in the German Democratic Republic includes such a system. Slater et al. [29] were first to test the validity of such an approach on a large number of chemicals. Direct effects on chemically pure DNA can be studied by using the trans-
166 forming principle [15], and this has been discussed by Notani. The mouse dominant-lethal test [5] is a popular and relatively economical mammalian test used in environmental mutagenicity screening programs. The genetic basis for the observed effects is still incompletely understood. The wasp Habrobracon is an organism well suited for investigating the genetic mechanisms involved in dominat lethality [30]. It should be used as a model organism to elucidate the genetic basis of this phenomenon. An important aspect in mutagenesis is phase specificity in respect to the cell cycle {e.g. S-period) and life cycle {meiosis and gametogenesis). Microorganisms can be synchronized so that various stages of the cell cycle can be studied in their response to mutagenic action. In respect to meiosis and gametogenesis, Drosophila is a well suited organism as discussed by Wfirgler. Dramatic differences in sensitivity may be observed. In yeast, certain acridine dyes are only mutagenic during meiosis [23]. These important points can be investigated in submammalian systems. The predictive value of data obtained in submammalian systems for man can be increased by including in the tier I part of a screening program the full spectrum of genetic end points and also a variety of organisms (e.g. bacteria, fungi, Drosophila). Metabolic activation and submammalian systems It is well established that many mutagens and carcinogens are not active per se; they are pro-mutagens or pro-carcinogens converted to the ultimate active form in cellular metabolism. Clearly, mammals metabolize most chemicals and Drosophila is similar to mammals in its activating capacities. In contrast with this, microorganisms usually lack the enzymic systems required for metabolism of foreign compounds. This requires a combination of a microbial indicator system with a system of mammalian activation. This has been achieved in two ways: the classical host-mediated assay in vivo of Gabridge and Legator [12] and microsomal preparations in vitro [16], an approach successfully exploited by Ames et al. [3]. In vitro, activating systems m'e not only of relevance for the detection of indirect mutagens using microbial indicator organisms. They also allow the determination of the site of activation within an entire organism. Weekes and Brusick [37] prepared microsomal fractions from various organs from different strains of mice, and they found organ and even organ-specific sex differences and differences between different strains. Moreover, Weeks [36] showed that the ability of kidney microsomal enzymes to activate carcinogenic dimethylnitrosamines correlates well with the induction of kidney tumors by this agent. The fascinating possibility is that such preparations can be made from human biopsy and autopsy material. This allows one to determine whether a certain chemical is activated to a mutagenic form in humans as detected by microbial indicator organisms. Such a combination falls only slightly short of the ideal solution of directly demonstrating mutagenic activity in man. What can be demonstrated is the formation of a mutagenic derivative by human metabolism. The host-mediated assay in its original form [12] was based on the injection
167 of microbial indicator organisms into the peritoneal cavity of animals treated with mutagens. Metabolic activation of pro-mutagens occurs only in certain tissues and organs, and the ultimate mutagens formed there have to reach the indicator cells in the peritoneal cavity. For very short-lived ultimate mutagens, this way may be too long and no activity would be detected. Mohn and Ellenberger [18] injected bacterial indicators into the blood stream of mice and found a wide distribution of bacteria in many locations especially in the liver. This puts the microbial indicator into close proximity to potential sites of activation. Mohn reported, to the panel, that phage T4 remains active for at least a week and allows for long-term exposure to chemicals in the host. This approach opens an avenue for screening directly the intact organism for activation of a given chemical and also the site of activation and distribution of the ultimate mutagen. Another feature of the use of the host-mediated assay is highlighted by reports by Fahrig [10,11] who injected yeast as a microbial indicator into testis of rats treated with mutagens. He demonstrated that certain mutagens did not reach the gonads. Even though this does not provide a direct demonstration of a lack of mutation induction or the presence of mutation induction in germ line cells or gametes, this approach allows one to test for the absence or presence of ultimate mutagens in this crucial location. Another approach to harness the mammalian and even human metabolic activation potential is provided by the urinary assay as exemplified by Siebert and Simon [27] who, with yeast as a microbial indicator organism, demonstrated the formation of ultimate mutagen in cyclophosphamide-treated patients. This approach can be more sensitive than all other systems because m a n y metabolites are concentrated up to a 1000-fold in the urine, and consequently are much easier to detect than at the site of activation. The limitation is that only long-lived ultimate mutagens can be detected. On the other hand, Stoltz and Miller [33] showed that isoniazid was not active in the Ames test with microsomal activation, but was active in the urine test with rats. In conclusion, microbial indicator organisms can be used to screen the entire body of the mammalian species chosen for metabolic activation for the presence of ultimate mutagens after administration of a chemical to be tested. But it is not only the presence but also the absence of ultimate mutagens that can be established with microbial indicators. Entirely neglected in all screening programs for environmental mutagens is the possibility of metabolic activation in plants. Plants are grown on soils well fertilized for higher yields, shielded against weeds by herbicides and protected against many kinds of parasites by pesticides. It is possible that some of these chemicals are converted to ultimate mutagens by plants but not by mammalian organisms. Certain plants convert, under special conditions, nitrate into nitrite, and this can occur to such an extent that nitrite levels in spinach are high enough to cause severe methemoglobinemia in babies fed with spinach [25,28]. Under the acid conditions in the stomach, nitrous acid is formed which is a classical mutagen in microorganisms and a co-mutagen in mammals as shown by the host-mediated assay [9]. Atrazine used as a herbicide in corn farming is converted in corn plants into a mutagen for yeast [13] and also for corn itself as reported by de Serres in the panel. SchSneich also reported on the mutagenic activation of a pesticide in plants. Mutagenicity in plants of herbicides,
168 fungicides and pesticides has been reviewed by Gottschalk [14]. A direct test for mutagenic activity of a chemical is well possible in plants (Arabidopsis [20], soybean [35], Tradescantia [32,34]). Various aspects of the use of plants were discussed by Natarajan and Veleminsk~ in the panel discussion. A special tier I loop may be indicated for fertilizers, herbicides and pesticides as pointed out by Bridges during the discussion. The predictive value of submammalian systems The predictive value of submammalian systems is very high because mammalian and even human metabolic activation capacities can be harnessed for combined assays. The problem left is whether an ultimate mutagen reacting with DNA is mutagenic in all organisms. Veleminsk:~ reported in the panel that the classical point mutagen 1-methyl-3-nitro-l-nitrosoguanidine does not induce point mutations in barley. A few more examples were mentioned by Mohn for bacterial species. Not many mutagens have been investigated on such a broad spectrum of organisms, but the possibility has to be considered that there are more mutagens like that. To prevent the erroneous classification of a chemical as negative, one has to include in the submammalian part of a screening program several organisms: bacteria, fungi and Drosophila. If this is taken into account, it should be possible to demonstrate reliably enough the mutagenic activity of a chemical or its conversion to a mutagen by mammalian or plant metabolism. The value of submammalian systems is clearly predictive, not absolute. What can be demonstrated unequivocally is the basic capactiy of a chemical to induce genetic alterations directly or after activation. This reduces the limitations of submammalian systems to the problem of the universality of genetic activity of a given mutagen. This is less critical with mutagens reacting directly with DNA. It might be very crucial however, with chemicals interfering with the enzymic machinery of DNA replication, repair and recombination on one hand and with the spindle fiber apparatus on the other. In such cases, the mutagenic activity might reside in a rather specific interference with the function of enzymes and other proteins. The susceptibility of a given enzyme to such an interference could vary from species to species, e.g. be different in men and mice, and beyond that between individuals of the same species. R e c o m m e n d a t i o n for further efforts It is clear that Drosophila and Salmonella and to a certain extent yeast provide well-established systems for mutagenicity screening, and support for screening of large numbers of chemicals is well warranted. On the other hand, further developments are still necessary, and they badly need support. There is only very limited experience with testing for induction of cytoplasmic mutation. Numerical chromosomal aberrations is another neglected end point. Finally, the metabolic activation capacity of crop plants in respect to fertilizers, herbicides and pesticides deserves considerable attention and support for development.
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28 S i m o n , C., N i t r a t e p o i s o n i n g f r o m s p i n a c h , T h e L a n c e t , 1, ( 1 9 6 6 ) 872. 29 Slater, E.E., M.D. A n d e r s o n a n d H.S. R o s e n k r a n z , R a p i d d e t e c t i o n of m u t a g e n s a n d c a r c i n o g e n s , C a n c e r Res., 31 ( 1 9 7 1 ) 9 7 0 - - 9 7 3 . 30 S m i t h , R.H. a n d R.C. y o n Borstel, I n d u c i n g m u t a t i o n s w i t h c h e m i c a l s in H a b r o b r a c o n , in A. Hollaend e r (ed.), C h e m i c a l Mutagens. Principles a n d M e t h o d s for t h e i r D e t e c t i o n , Vol. 2, P l e n u m Press, N e w Y o r k , 1 9 7 1 , pp. 445---460. 31 Sobels, F . H . , T h e a d v a n t a g e s of D r o s o p h i l a for m u t a t i o n studies, M u t a t i o n Res., 26 ( 1 9 7 4 ) 2 7 7 - - 2 8 4 . 3 2 S p a r r o w , A . H . , L.A. S c h a i r e r a n d R. Vfllalobos-Pietrini, C o m p a r i s o n of s o m a t i c m u t a t i o n rates ind u c e d in T r a d e s c a n t i a by c h e m i c a l a n d p h y s i c a l m u t a g e n s , M u t a t i o n Res., 26 ( 1 9 7 4 ) 2 6 5 - - 2 7 6 . 33 S t o l t z , D.R. a n d C.T. Miller, M u t a g e n i e a c t i v i t y in u r i n e c o n c e n t r a t e s f r o m rats orally d o s e d with a c h e m i c a l w h i c h is inactive in t h e A m e s test with m i c r o s o m a l a c t i v a t i o n , M u t a t i o n Res., 31 ( 1 9 7 5 ) 325--326. 34 U n d e r b r i n k , A . G . , L.A. Schairer a n d A . H . S p a r r o w , T r a d e s c a n t i a s t a m e n hairs: a r a d i o b i o l o g i c a l test s y s t e m a p p l i c a b l e to c h e m i c a l m u t a g e n e s i s , in A. H o l l a e n d e r (ed.), C h e m i c a l M u t a g e n s . Principles a n d M e t h o d s for t h e i r Detection~ Vol. 3, P l e n u m Press, N e w Y o r k , 1 9 7 3 , pp. 1 7 1 - - 2 0 7 . 3 5 Vig, B.K., S o y b e a n ( G l y c i n e m a x ) : a n e w test s y s t e m for s t u d y of genetic p a r a m e t e r s as a f f e c t e d by e n v i r o n m e n t a l m u t a g e n s , M u t a t i o n Res., 31 ( 1 9 7 5 ) 4 9 - - 5 6 . 36 Weekes, U.Y., M e t a b o l i s m of d i m e t h y l n i t r o s a r n i n e to m u t a g e n i c i n t e r m e d i a t e s by k i d n e y m i c r o s o m a l e n z y m e s a n d c o r r e l a t i o n with r e p o r t e d h o s t susceptibility to k i d n e y t u m o r s , J. Natl. C a n c e r lnst., 55 (1975) 1199--1201. 37 Weekes, U. a n d D. Brusick, In v i t r o m e t a b o l i c a c t i v a t i o n of c h e m i c a l m u t a g e n s . II. T h e r e l a t i o n s h i p s a m o n g m u t a g e n f o r m a t i o n , m e t a b o l i s m a n d c a r c i n o g e n i c i t y for d i m e t h y l n i t r o s a m i n e in the liver, kidn e y s a n d lungs of Balb/cJ, C 5 7 1 3 6 / 6 J a n d R F / J m i c e , M u t a t i o n Res., 31 ( 1 9 7 5 ) 1 7 5 - - 1 8 3 . 38 Z i m m e r m a n n , F.K., G e n e t i c a s p e c t s of c a r c i n o g e n e s i s , Biochern. P h a r m o c o l . , 20 ( 1 9 7 1 a ) 9 8 5 - - 9 9 5 . 39 Z i m m c r m a n n , F.K., I n d u c t i o n of m i t o t i c gcne c o n v e r s i o n by m u t a g e n s , M u t a t i o n Res., 11 ( 1 9 7 1 b ) 327--337. 40 Z i m m e r m a n n , F.K., A strain for visual screening for the t w o r e c i p r o c a l p r o d u c t s of m i t o t i c crossing o v e r , M u t a t i o n Res., 21 ( 1 9 7 3 ) 2 6 3 - - 2 6 9 . 41 Z i m m e r m a n n , F . K . , R. K e r n a n d H. R a s e n b e r g e r , A strain for s i m u l t a n e o u s d e t e c t i o n of i n d u c e d m i t o t i c crossing o v e r , m i t o t i c gene c o n v e r s i o n a n d reverse m u t a t i o n , M u t a t i o n Res., 28 ( 1 9 7 5 ) 3 8 1 - 388.
Mutation Research, 41 (1976) 163--170
© Elsevier/North-Holland Biomedical Press
PANEL DISCUSSION ON SUBMAMMALIAN SYSTEMS F.K. ZIMMERMANN Technische Hochschule Darmstadl, Schnittpahnstrasse 61, Darmstadt (BRD)
(Received April 23rd, 1976) Participants: R.C. yon Borstel, D.J. Brusick, H. Kurata, G.R. Mohn, A.T. Natarajan, N.K. Notani, J. SchSneich, J. Velemfnsk:~, F.H. W/irgler and F.K. Zimmermann (co-ordinator) Submammalian systems, whose development started some t w o decades ago, have turned out to be useful and powerful for a qualitative evaluation of genetic hazards exerted by environmental chemicals. They have been used to investigate the induction of generative genetic damage which contributes to the genetic load of natural populations, and also induction of genetic damage in somatic cells which is thought to be a cause of carcinogenesis [3,17,38] and premature senility [8]. The inclusion of submammalian systems in screening programs for environmental mutagens provides the following advantages. (1) All known genetic alterations can be scored and the genetic nature of the effects readily established, thus meeting the transmissibility criterion. (2) Metabolic activation of promutagens by mammalian, and in certain cases human, metabolism can be combined in several ways with submammalian indicator test systems. In addition, metabolic activation of chemicals by agricultural crop plants can also be detected. In Drosophila, metabolic activation is believed to be similar to that in mammals. (3) All tests can be performed at low cost and within a short time. The only major limitation of submammalian systems in environmental screening programs is that they allow only for qualitative assessments of genetic hazards but not necessarily for a quantitative evaluation in the sense of possible threshold or tolerance doses in man. The following end points have to be scored by a complete screening program for environmental mutagens: (1) point mutations; (2) structural chromosomal aberrations; (3) numerical chromosomal aberrations; (4) genetic alterations in cytoplasmic genetic determinants and in the case of somatic genetic damage, relevant in respect to carcinogenieity; (5) mitotic recombination. Evaluation of presently available test systems for the different genetic end points Systems for the detection of point mutations have reached a high degree of sophistication. Through the use of various microbial strains, e.g. S a l m o n e l l a
164 typhimurium, mutagens can be classified as base-pair substitution {including base specificity) and frame-shift mutagens [2,3]. These systems are based on the mutational restoration of p r o t o t r o p h y in auxotrophic strains. Permeability problems have partly been overcome by introduction into the test strains of mutations affecting the bacterial cell envelope. In addition, the strains have been made very sensitive to mutagenic action by introducing mutationally determined defects in repair activities. A similar possibility of detailed analysis of mutational events is provided by the unicellular eukaryote Saccharomyces cerevisiae {baker's yeast) which Sherman and collaborators used to develop a reverse mutation system to a degree of sophistication that monitors not only plain mutation induction but also transitions, transversion and base specificity [22]. The sensitivity of this system can still be enhanced by introducing certain defects in repair systems; and certain cell wall or cell membrane defects may be introduced to circumvent permeation barriers. However, this system and its further potential have not been exploited for environmental mutagenesis work. Reverse mutation systems, in spite of their great sensitivity, have limitations because they are based on the restoration of a functional allele or genotype from a non-functional condition. Such restorations usually require very specific mutational events which cannot be induced by all mutagens, a phenomenon called mutagen specificity. This limitation can partly be compensated by using a whole battery of the different mutant alleles sensitive to as many known mutagens as possible. Nevertheless, it became increasingly clear that strain and mutagen specificity can best be overcome by using forward mutation tests in which mutations in many sites within one or several genes can be detected. The best established test system is provided by the classical test for recessive lethal mutations in the X-chromosome of Drosophila [1,31]. Using Escherichia coli as an indicator organism, Mohn et al. [19] have developed a multi-purpose strain in which several types of mutation can be detected simultaneously including deletions extending to two adjacent genes, and furthermore allowing the testing for induction of prophage lambda (Mohn and Ellenberger, to be published). Selective forward mutation systems are also available in the yeast Saccharomyces cerevisiae. One such system has been elaborated by Brusick [7]. This is based on the inactivation of a permease gene which results in a resistance to canavanine. Such mutants were induced by ICR-170 which is a poor inducer of simple base-pair substitution mutation. Consequently, canavanine resistance provides a test for a wide range of mutational alterations. Alper and Ames [4] have devised a test system in Salmonella which specifically monitors induction of deletions several genes long. Another system for the detection of large size deletions -- in addition to point mutations -- in all parts of the genome of Neurospora crassa has been developed by de Serres and Malling [26] using a heterokaryon. Test systems in yeast designed to detect mitotic crossing over also respond to deletions in specific parts of the genome, but they do not allow one to identify the observed effects as unambiguously due to deletion [40,41]. Drosophila provides systems for the detection of various types of structural chromosomal aberrations, not only deletions as in the microbial systems, but also inversions, duplications and translocations [ 31 ]. Meiotic numerical aberrations can also be scored in Drosophila, and systems
165 have been described by Wiirgler in the panel. Systems for chromosomal loss in mitotic cells of Aspergillus nidulans [ 6] and in yeast [21 ] are available. The systems for the detection of chromosomal structural and numerical aberrations described so far are based on genetic rather than microscopic methods of detection. Of course, there is a bulk of experience with cytogenetic studies discussed by Natarajan in the panel. However, these tests do not automatically involve the demonstration of genetic transmissibility; they have to be classified as indirect correlation tests. There is urgency for the inclusion of a test for numerical chromosomal aberrations in environmental mutagenicity screening programs. All other genetic end points are based on DNA as the final genetic target. Changes in chromosome numbers can also be caused by chemicals that interfere with the components of the spindle fiber apparatus, and such chemicals may not interact at all with DNA or the enzymic machinery involved in DNA metabolism. Ommission of this genetic end point may lead to a systematic failure in detecting a rather unique class of mutagens. It is well established that a eukaryotic cell has genetic components not only in the nuclear genetic compartment but also in the cytoplasm, notably so in mitochondria and chloroplasts [24]. If it should turn out that there are mutagens specific for prokaryotes, then they should also be tested in eukaryotes for their ability to induce mitochondrial mutations because of certain essential similarities between bacterial and mitochondrial genetic systems. No systems for the detection of mutations in mitochondrial or other cytoplasmic genetic determinants have been extensively used in environmental mutagenicity screening. This gap has also to be closed. Environmental mutagenesis is not only revelant for the germ line but also for somatic cells in respect to short term carcinogenicity testing. Somatic genetic damage can arise not only through the genetic end points dealt with up to here, but also through recombination processes occurring in mitotic cells. A diploid organism can be heterozygous for recessive lethal or detrimental alleles which can become homozygous and expressed through mitotic recombination and also mitotic non-disjunction. This can result in developmental disorders and deleterious alterations in rapidly dividing tissues. There are two types of mitotic recombination: reciprocal mitotic crossing over and non-reciprocal mitotic gene conversion. Yeast has been a widely used organism for the detection of mitotic crossing over and gene conversion, and strains for their detection are now available and in wide use [ 39--41]. Aside from screening for this particular end point, such strains provide a basis for convenient and rapid detection of genetically active chemicals in general. Further submammalian systems are available and already in use in environmental mutagenicity screening programs. The increased sensitivity of m u t a n t strains with defects in certain repair functions as compared with the respective wild-type strains can be taken as a first indication of genetic activity of a chemical. Sch5neich reported that the screening program in the German Democratic Republic includes such a system. Slater et al. [29] were first to test the validity of such an approach on a large number of chemicals. Direct effects on chemically pure DNA can be studied by using the trans-
166 forming principle [15], and this has been discussed by Notani. The mouse dominant-lethal test [5] is a popular and relatively economical mammalian test used in environmental mutagenicity screening programs. The genetic basis for the observed effects is still incompletely understood. The wasp Habrobracon is an organism well suited for investigating the genetic mechanisms involved in dominat lethality [30]. It should be used as a model organism to elucidate the genetic basis of this phenomenon. An important aspect in mutagenesis is phase specificity in respect to the cell cycle {e.g. S-period) and life cycle {meiosis and gametogenesis). Microorganisms can be synchronized so that various stages of the cell cycle can be studied in their response to mutagenic action. In respect to meiosis and gametogenesis, Drosophila is a well suited organism as discussed by Wfirgler. Dramatic differences in sensitivity may be observed. In yeast, certain acridine dyes are only mutagenic during meiosis [23]. These important points can be investigated in submammalian systems. The predictive value of data obtained in submammalian systems for man can be increased by including in the tier I part of a screening program the full spectrum of genetic end points and also a variety of organisms (e.g. bacteria, fungi, Drosophila). Metabolic activation and submammalian systems It is well established that many mutagens and carcinogens are not active per se; they are pro-mutagens or pro-carcinogens converted to the ultimate active form in cellular metabolism. Clearly, mammals metabolize most chemicals and Drosophila is similar to mammals in its activating capacities. In contrast with this, microorganisms usually lack the enzymic systems required for metabolism of foreign compounds. This requires a combination of a microbial indicator system with a system of mammalian activation. This has been achieved in two ways: the classical host-mediated assay in vivo of Gabridge and Legator [12] and microsomal preparations in vitro [16], an approach successfully exploited by Ames et al. [3]. In vitro, activating systems m'e not only of relevance for the detection of indirect mutagens using microbial indicator organisms. They also allow the determination of the site of activation within an entire organism. Weekes and Brusick [37] prepared microsomal fractions from various organs from different strains of mice, and they found organ and even organ-specific sex differences and differences between different strains. Moreover, Weeks [36] showed that the ability of kidney microsomal enzymes to activate carcinogenic dimethylnitrosamines correlates well with the induction of kidney tumors by this agent. The fascinating possibility is that such preparations can be made from human biopsy and autopsy material. This allows one to determine whether a certain chemical is activated to a mutagenic form in humans as detected by microbial indicator organisms. Such a combination falls only slightly short of the ideal solution of directly demonstrating mutagenic activity in man. What can be demonstrated is the formation of a mutagenic derivative by human metabolism. The host-mediated assay in its original form [12] was based on the injection
167 of microbial indicator organisms into the peritoneal cavity of animals treated with mutagens. Metabolic activation of pro-mutagens occurs only in certain tissues and organs, and the ultimate mutagens formed there have to reach the indicator cells in the peritoneal cavity. For very short-lived ultimate mutagens, this way may be too long and no activity would be detected. Mohn and Ellenberger [18] injected bacterial indicators into the blood stream of mice and found a wide distribution of bacteria in many locations especially in the liver. This puts the microbial indicator into close proximity to potential sites of activation. Mohn reported, to the panel, that phage T4 remains active for at least a week and allows for long-term exposure to chemicals in the host. This approach opens an avenue for screening directly the intact organism for activation of a given chemical and also the site of activation and distribution of the ultimate mutagen. Another feature of the use of the host-mediated assay is highlighted by reports by Fahrig [10,11] who injected yeast as a microbial indicator into testis of rats treated with mutagens. He demonstrated that certain mutagens did not reach the gonads. Even though this does not provide a direct demonstration of a lack of mutation induction or the presence of mutation induction in germ line cells or gametes, this approach allows one to test for the absence or presence of ultimate mutagens in this crucial location. Another approach to harness the mammalian and even human metabolic activation potential is provided by the urinary assay as exemplified by Siebert and Simon [27] who, with yeast as a microbial indicator organism, demonstrated the formation of ultimate mutagen in cyclophosphamide-treated patients. This approach can be more sensitive than all other systems because m a n y metabolites are concentrated up to a 1000-fold in the urine, and consequently are much easier to detect than at the site of activation. The limitation is that only long-lived ultimate mutagens can be detected. On the other hand, Stoltz and Miller [33] showed that isoniazid was not active in the Ames test with microsomal activation, but was active in the urine test with rats. In conclusion, microbial indicator organisms can be used to screen the entire body of the mammalian species chosen for metabolic activation for the presence of ultimate mutagens after administration of a chemical to be tested. But it is not only the presence but also the absence of ultimate mutagens that can be established with microbial indicators. Entirely neglected in all screening programs for environmental mutagens is the possibility of metabolic activation in plants. Plants are grown on soils well fertilized for higher yields, shielded against weeds by herbicides and protected against many kinds of parasites by pesticides. It is possible that some of these chemicals are converted to ultimate mutagens by plants but not by mammalian organisms. Certain plants convert, under special conditions, nitrate into nitrite, and this can occur to such an extent that nitrite levels in spinach are high enough to cause severe methemoglobinemia in babies fed with spinach [25,28]. Under the acid conditions in the stomach, nitrous acid is formed which is a classical mutagen in microorganisms and a co-mutagen in mammals as shown by the host-mediated assay [9]. Atrazine used as a herbicide in corn farming is converted in corn plants into a mutagen for yeast [13] and also for corn itself as reported by de Serres in the panel. SchSneich also reported on the mutagenic activation of a pesticide in plants. Mutagenicity in plants of herbicides,
168 fungicides and pesticides has been reviewed by Gottschalk [14]. A direct test for mutagenic activity of a chemical is well possible in plants (Arabidopsis [20], soybean [35], Tradescantia [32,34]). Various aspects of the use of plants were discussed by Natarajan and Veleminsk~ in the panel discussion. A special tier I loop may be indicated for fertilizers, herbicides and pesticides as pointed out by Bridges during the discussion. The predictive value of submammalian systems The predictive value of submammalian systems is very high because mammalian and even human metabolic activation capacities can be harnessed for combined assays. The problem left is whether an ultimate mutagen reacting with DNA is mutagenic in all organisms. Veleminsk:~ reported in the panel that the classical point mutagen 1-methyl-3-nitro-l-nitrosoguanidine does not induce point mutations in barley. A few more examples were mentioned by Mohn for bacterial species. Not many mutagens have been investigated on such a broad spectrum of organisms, but the possibility has to be considered that there are more mutagens like that. To prevent the erroneous classification of a chemical as negative, one has to include in the submammalian part of a screening program several organisms: bacteria, fungi and Drosophila. If this is taken into account, it should be possible to demonstrate reliably enough the mutagenic activity of a chemical or its conversion to a mutagen by mammalian or plant metabolism. The value of submammalian systems is clearly predictive, not absolute. What can be demonstrated unequivocally is the basic capactiy of a chemical to induce genetic alterations directly or after activation. This reduces the limitations of submammalian systems to the problem of the universality of genetic activity of a given mutagen. This is less critical with mutagens reacting directly with DNA. It might be very crucial however, with chemicals interfering with the enzymic machinery of DNA replication, repair and recombination on one hand and with the spindle fiber apparatus on the other. In such cases, the mutagenic activity might reside in a rather specific interference with the function of enzymes and other proteins. The susceptibility of a given enzyme to such an interference could vary from species to species, e.g. be different in men and mice, and beyond that between individuals of the same species. R e c o m m e n d a t i o n for further efforts It is clear that Drosophila and Salmonella and to a certain extent yeast provide well-established systems for mutagenicity screening, and support for screening of large numbers of chemicals is well warranted. On the other hand, further developments are still necessary, and they badly need support. There is only very limited experience with testing for induction of cytoplasmic mutation. Numerical chromosomal aberrations is another neglected end point. Finally, the metabolic activation capacity of crop plants in respect to fertilizers, herbicides and pesticides deserves considerable attention and support for development.
169
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