DISCUSSION PAPER: THE USE OF DROSOPHILA FOR MUTAGENESIS SCREENING Ruby Allen Valencia Mirtagenesis Laboratory W A R F Institute, Inc. Madison. Wisconsin 53701
In the time allotted to me. I will simply make a few comments based on my current experience with a Drosophila mutagenesis testing laboratory. During the past year, I have been involved in installing and operating such a laboratory. We are currently supported by the Environmental Protection Agency and are testing pesticides. We plan, however, to test other types of compounds for other agencies and for industry. Although a certain amount of investigation is done, mainly in methodology, the laboratory is not primarily a research laboratory. I point this fact out because I think it is important. There are very many environmental chemical agents that need to be tested for mutagenicity, and I think that to do the job, we must think of many large efficient factorylike testing facilities, not only for Drosophila testing but also for other types of tests. (Of course, the different test types will have to be specified, limited, and coordinated somehow, and this is one of the big problems we are facing.) During the past few months, I have satisfied myself that mutagenesis screening with Drosophila can indeed be performed on a large scale in an efficient and effective manner. I have heard it said and seen it written that Drosophila genetic schemes are too sophisticated and complex for use by any but the trained Drosophila geneticist. It is true that serious and costly errors have been made (resulting in false negatives or false positives for mutagenicity of compounds) when tests have been performed by insufficiently trained and inexperienced persons. I point out, however, that only one trained and experienced person is needed in any particular testing laboratory. This person should plan, initiate, and oversee the tests and interpret the results. The technical procedures that are performed in the laboratory, however. are not difficult and can be taught to any good technician in a short time. We are exposing our flies in the laboratory. but flies could be exposed elsewhere, such as in some occupational environment, and brought into the laboratory for the genetic tests. They could be exposed nearby or at some considerable distance from the laboratory, because Drosophila can be transported or sent by mail quite successfully. This has been done many times, and, in fact, we are presently carrying flies from Madison to Brookhaven National Laboratory, Long Island, New York, irradiating them, and returning them to Madison for genetic studies. We are currently geared to screen for three types of genetic end-points: dominant lethals, chromosome breakage, and rearrangement; nondisjunction and loss; and sex-linked recessive lethals. We d o not, however, feel bound to perform these same tests indefinitely. We allow some time for experimentation and are always looking for tests that might be faster or easier or more diagnostic. I agree with Dr. Zimmering that some type of selective system might
34
Valencia: Drosophila & Mutagenesis Screening
35
be very useful. In fact, we are presently using such a scheme. Devised by a graduate student, Loring Craymer, at the University of Wisconsin, it is a scheme that selects for chromosome damage. Males bearing a translocation between the X and Y chromosomes (one of several obtained from John Merriam) are mated to females with normal X chromosomes. All regular progeny of this cross are aneuploid and fail to reach maturity. (Many do reach larval and pupal stages, however, and thus “work” the medium, providing for healthy culture conditions.) Certain kinds of chromosome loss and nondisjunction, however, result in euploid zygotes, which do reach maturity. Thus, one simply counts emerging flies to score for damage. I have baptized this scheme the “lifesaver” scheme. Preliminary runs indicate that this technique may indeed be useful for screening, and we are presently testing it further. I would like to insert a word of caution with respect to procedure when dealing with chemicals that. if mutagenic. are likely to be weak mutagens, that must be selected from among many nonmutagens, and that might be “spotty” in effect. I believe that these circumstances require some modification of standard Drosophila test procedures. The classic procedure in testing for sex-linked recessive lethals, for example, is to treat males, mass-mate them to appropriate females, and then mate the resultant F, daughters individually to apropriate males (usually brothers) to test for recessive lethals. We feel that this procedure is not sufficiently discriminating for our purpose, and therefore, despite the need for speedy testing, we are using the following more elaborate method: Treated and control males are mated individually, and the F , female matings from each male are kept grouped. Each male is transferred sequentially through four broods in order to sample the entire range of germ cell developmental stages. We aim for a total of 7000 treated chromosome tests and 4000 untreated. These numbers, according to a formula devised by Dr. James Crow, are sufficient to detect a mutation frequency equal to four times the spontaneous frequency. The total desired number is iisually obtained in two or three repeat experiments. This procedure allows for a qualitative analysis of results with the following benefits: Chemicals that affect some germ cell stages and not others would not be missed ( i.e.. erroneously called nonmutagenic) . Groupings of lethals can be identified (see explanation below). A smaller number of tests is often diagnostic or suggestive of effect and allows efficient planning of further tests. Mutations that occur (either spontaneously or by induction) in gonad cells may be duplicated and reduplicated during spermatogenesis to yield several sperm cells that bear the mutation. This is a “run” or “cluster.” When dealing with potent mutagens, such as high doses of x rays, clusters are no great source of error. With low-level mutagens, however, clusters can lead to erroneous conclusions. For example, one of the pesticides tested by us yielded 27 lethals in 2803 tests, which is a frequency of 0.963%, decidedly high when compared to the parallel control frequency of 0.144% or the overall control frequency of 0.145%. In a standard mass-mating procedure, this pesticide would have
36
Annals New York Academy of Sciences
been called mutagenic. Our procedure, however, showed that 21 of the 27 mutations were from a single male, and they came from all stages of gametogenesis. Thus, they probably represent one large cluster that arose from a spontaneous mutation, and the pesticide is probably not mutagenic. On the other hand, the chance occurrence of clustering in a control group can raise the control frequency sufficiently to mask a slight positive effect in the treated group. If, however, such clusters can be identified, the compound will at least be read as suspect and the test repeated. Mutations could also be grouped if different males were by chance exposed to different amounts of the chemical (quite a likely occurrence when exposure is by ingestion) or if the mutagen reached certain gonads and not others or reached only certain spots in the gonads. Data obtained by the “family” method described above can be tested statistically to determine whether apparent groupings are abnormal. Obviously, if enough chromosomes were tested, the “noise” caused by clustering and uneven exposure would become insignificant. I simply suggest that perhaps it is easier and faster to use a more analytic procedure and test fewer chromosomes. As I mentioned at the beginning, we are presently running not only recessive lethals but also dominant Iethals and are testing for chromosome breakage, loss, rearrangement, and nondisjunction. The dominant lethal test is very rapid in the Drosophila system and should be just as meaningful as dominant lethals in mice. For those not familiar with the test, it consists of treating flies, mating them, then allowing the female to lay eggs for 24 hr on the surface of a special medium in special containers. (I will not describe the exact technical procedure.) Eggs are counted, incubated, and at 48 hr observed for hatching. In our experiments, we transfer treated individual males sequentially to new virgin females daily for 7 days. Nineteen males and females (the capacity of each egg-laying chamber) produce aproximately 2000 tests, and the results are available in 10 days. We hope that this test may serve as a fast preliminary screen for each new compound. The second test scheme is a standard one that scores for loss of X or Y chromosome or loss of markers from a specially constructed Y chromosome. It is also relatively rapid, because it is an F, test. With sequential brooding, results are obtained in about 3% weeks. The plan that is being followed, then, is one that aims at a relatively rapid detection of mutagenicity. There is a fast “probe” (dominant lethals) , then a still quite rapid but more analytic screen (chromosome breakage, and so o n ) , followed by the recessive lethal test, which requires (with sequential brooding and confirmation of lethals) about 6 weeks. The recessive lethal test is probably the best, because sex-linked recessive lethals involve many loci (200 or more) and many types of original lesion (“point” mutations or chromosome rearrangements). Except for nondisjunction, which is revealed by the second test, the first two tests do not screen for any genetic damage that would not be shown by the recessive lethal test. Their main advantage is time. In conclusion, I believe that mutagenesis screening of environmental chemicals through the use of Drosophila can be performed in a rapid, economical, and practical manner. We should, however, look for new and/or improved procedures, keeping in mind the special circumstances of mutagen screening.
In the time allotted to me. I will simply make a few comments based on my current experience with a Drosophila mutagenesis testing laboratory. During the past year, I have been involved in installing and operating such a laboratory. We are currently supported by the Environmental Protection Agency and are testing pesticides. We plan, however, to test other types of compounds for other agencies and for industry. Although a certain amount of investigation is done, mainly in methodology, the laboratory is not primarily a research laboratory. I point this fact out because I think it is important. There are very many environmental chemical agents that need to be tested for mutagenicity, and I think that to do the job, we must think of many large efficient factorylike testing facilities, not only for Drosophila testing but also for other types of tests. (Of course, the different test types will have to be specified, limited, and coordinated somehow, and this is one of the big problems we are facing.) During the past few months, I have satisfied myself that mutagenesis screening with Drosophila can indeed be performed on a large scale in an efficient and effective manner. I have heard it said and seen it written that Drosophila genetic schemes are too sophisticated and complex for use by any but the trained Drosophila geneticist. It is true that serious and costly errors have been made (resulting in false negatives or false positives for mutagenicity of compounds) when tests have been performed by insufficiently trained and inexperienced persons. I point out, however, that only one trained and experienced person is needed in any particular testing laboratory. This person should plan, initiate, and oversee the tests and interpret the results. The technical procedures that are performed in the laboratory, however. are not difficult and can be taught to any good technician in a short time. We are exposing our flies in the laboratory. but flies could be exposed elsewhere, such as in some occupational environment, and brought into the laboratory for the genetic tests. They could be exposed nearby or at some considerable distance from the laboratory, because Drosophila can be transported or sent by mail quite successfully. This has been done many times, and, in fact, we are presently carrying flies from Madison to Brookhaven National Laboratory, Long Island, New York, irradiating them, and returning them to Madison for genetic studies. We are currently geared to screen for three types of genetic end-points: dominant lethals, chromosome breakage, and rearrangement; nondisjunction and loss; and sex-linked recessive lethals. We d o not, however, feel bound to perform these same tests indefinitely. We allow some time for experimentation and are always looking for tests that might be faster or easier or more diagnostic. I agree with Dr. Zimmering that some type of selective system might
34
Valencia: Drosophila & Mutagenesis Screening
35
be very useful. In fact, we are presently using such a scheme. Devised by a graduate student, Loring Craymer, at the University of Wisconsin, it is a scheme that selects for chromosome damage. Males bearing a translocation between the X and Y chromosomes (one of several obtained from John Merriam) are mated to females with normal X chromosomes. All regular progeny of this cross are aneuploid and fail to reach maturity. (Many do reach larval and pupal stages, however, and thus “work” the medium, providing for healthy culture conditions.) Certain kinds of chromosome loss and nondisjunction, however, result in euploid zygotes, which do reach maturity. Thus, one simply counts emerging flies to score for damage. I have baptized this scheme the “lifesaver” scheme. Preliminary runs indicate that this technique may indeed be useful for screening, and we are presently testing it further. I would like to insert a word of caution with respect to procedure when dealing with chemicals that. if mutagenic. are likely to be weak mutagens, that must be selected from among many nonmutagens, and that might be “spotty” in effect. I believe that these circumstances require some modification of standard Drosophila test procedures. The classic procedure in testing for sex-linked recessive lethals, for example, is to treat males, mass-mate them to appropriate females, and then mate the resultant F, daughters individually to apropriate males (usually brothers) to test for recessive lethals. We feel that this procedure is not sufficiently discriminating for our purpose, and therefore, despite the need for speedy testing, we are using the following more elaborate method: Treated and control males are mated individually, and the F , female matings from each male are kept grouped. Each male is transferred sequentially through four broods in order to sample the entire range of germ cell developmental stages. We aim for a total of 7000 treated chromosome tests and 4000 untreated. These numbers, according to a formula devised by Dr. James Crow, are sufficient to detect a mutation frequency equal to four times the spontaneous frequency. The total desired number is iisually obtained in two or three repeat experiments. This procedure allows for a qualitative analysis of results with the following benefits: Chemicals that affect some germ cell stages and not others would not be missed ( i.e.. erroneously called nonmutagenic) . Groupings of lethals can be identified (see explanation below). A smaller number of tests is often diagnostic or suggestive of effect and allows efficient planning of further tests. Mutations that occur (either spontaneously or by induction) in gonad cells may be duplicated and reduplicated during spermatogenesis to yield several sperm cells that bear the mutation. This is a “run” or “cluster.” When dealing with potent mutagens, such as high doses of x rays, clusters are no great source of error. With low-level mutagens, however, clusters can lead to erroneous conclusions. For example, one of the pesticides tested by us yielded 27 lethals in 2803 tests, which is a frequency of 0.963%, decidedly high when compared to the parallel control frequency of 0.144% or the overall control frequency of 0.145%. In a standard mass-mating procedure, this pesticide would have
36
Annals New York Academy of Sciences
been called mutagenic. Our procedure, however, showed that 21 of the 27 mutations were from a single male, and they came from all stages of gametogenesis. Thus, they probably represent one large cluster that arose from a spontaneous mutation, and the pesticide is probably not mutagenic. On the other hand, the chance occurrence of clustering in a control group can raise the control frequency sufficiently to mask a slight positive effect in the treated group. If, however, such clusters can be identified, the compound will at least be read as suspect and the test repeated. Mutations could also be grouped if different males were by chance exposed to different amounts of the chemical (quite a likely occurrence when exposure is by ingestion) or if the mutagen reached certain gonads and not others or reached only certain spots in the gonads. Data obtained by the “family” method described above can be tested statistically to determine whether apparent groupings are abnormal. Obviously, if enough chromosomes were tested, the “noise” caused by clustering and uneven exposure would become insignificant. I simply suggest that perhaps it is easier and faster to use a more analytic procedure and test fewer chromosomes. As I mentioned at the beginning, we are presently running not only recessive lethals but also dominant Iethals and are testing for chromosome breakage, loss, rearrangement, and nondisjunction. The dominant lethal test is very rapid in the Drosophila system and should be just as meaningful as dominant lethals in mice. For those not familiar with the test, it consists of treating flies, mating them, then allowing the female to lay eggs for 24 hr on the surface of a special medium in special containers. (I will not describe the exact technical procedure.) Eggs are counted, incubated, and at 48 hr observed for hatching. In our experiments, we transfer treated individual males sequentially to new virgin females daily for 7 days. Nineteen males and females (the capacity of each egg-laying chamber) produce aproximately 2000 tests, and the results are available in 10 days. We hope that this test may serve as a fast preliminary screen for each new compound. The second test scheme is a standard one that scores for loss of X or Y chromosome or loss of markers from a specially constructed Y chromosome. It is also relatively rapid, because it is an F, test. With sequential brooding, results are obtained in about 3% weeks. The plan that is being followed, then, is one that aims at a relatively rapid detection of mutagenicity. There is a fast “probe” (dominant lethals) , then a still quite rapid but more analytic screen (chromosome breakage, and so o n ) , followed by the recessive lethal test, which requires (with sequential brooding and confirmation of lethals) about 6 weeks. The recessive lethal test is probably the best, because sex-linked recessive lethals involve many loci (200 or more) and many types of original lesion (“point” mutations or chromosome rearrangements). Except for nondisjunction, which is revealed by the second test, the first two tests do not screen for any genetic damage that would not be shown by the recessive lethal test. Their main advantage is time. In conclusion, I believe that mutagenesis screening of environmental chemicals through the use of Drosophila can be performed in a rapid, economical, and practical manner. We should, however, look for new and/or improved procedures, keeping in mind the special circumstances of mutagen screening.
