Document rBVjZMYvkQ13eKmjMZnMQLLVa

Environmental and Molecular Mutagenesis 2 5 2 1 1-215 (1995) Induction of Delayed Mutations by Benzene and Ethylene Dibromide in Drosophila Purushottam Kale and Ranjini Kale Department of Biology, Alabama A. & M . University, Normal, Alabama Two carcinogens, ethylene dibromide a n d ben- zene, were used to induce delayed (germinal m e saic)sex-linked recessive lethal mutations in spermotozoo a n d spermatids of adult Drosophila males. Significant numbers of delayed mutations (in F3) were scored in a b s e n c e of conventional (in Fz) muta- tions. A lorge proportion of nonlethal F Z cultures corried delayed mutations, so much so thot, in some cultures, oll FZ females w e r e carriers of mutotions. The mechanism through which single strand dam- oge to treated X chromosomes can result in such deloyed lethals is discussed. These observations indicate thot the delayed mutation test should be used for testing the mutagenicity of environmental compounds, especially corcinogens, which tested nego- tive in the conventionol sex-linked recessive lethol mutotion test. The d a t a will support the relotionship between mutagenesis a n d carcinogenesis and, olso will further enhance the sensitivity of the Drosophila mutation assay. o 1995~ i l e y - ~ i sIsn,c. Key words: Drosophila, mosaic, delayed, mutations, carcinogens I treatment, 10 ml of saturated vapor was injected in the treatment jar. After 30 min the jar was ventilated in a hood and the males were Genetic tests using Drosophila have long been recog- b e d as indicators of the potential of chemicals to 'ntabie gene mutations and chromosome in mima1 g e m cells. Among the several tests available in transferred to fresh food for 1 day before first matings. The exposure was approximately 125 ppni ;is calculated for our previous EDB experiments [Kale and Baum, 19791. No dose-frequency response was required and therefore precise dose was not a factor in these treatments. The procedure was similar for benzene treatment except instead of Drosophila, the sex-linked recessive lethal test (SLRL) is saturated vapor, 0.I ml liquid benzene was dropped on a blotting paper hemost sensitive. If a chemical is negative in this test, the in the jar. Benzene is volatile and forms vapor immediately. After I hr of its being a carcinogen is lowe~ ~ ~ the ja~r was ve~ntilated.hThe ma~les wereltransfer~red to fr~esh food~and the , potent carcinogens have indeed been shown survivors were stored for 1 day. We had used a similar exposure previously [Kale and Baum, 19831 and found that this amount of benzene lo be negative in this test and this reflects negatively on generates 27,000 ppm by volume. he utility of Drosophila in carcinogen prediction. Thus, The treated males were individually mated to six Eosc virgins for 2 der screening hundreds of chemicals, Foureman et al. 11941 conclude that heS L ~ is specific but not as unsitive*The test has been removed from the revised ffice of Toxic Sllbstances test Xheme days to obtain brood 1. Three such broods were cultured in a period of 6 days. The F, progeny was pair-mated separately for each male in order to identify clusters. The F2 cultures were scored as usual for complete lethals. These data are given in Table 1. In EDB experiments, Fz non-lethal cultures were chosen randomly and from each of these, ntley et al., 19941. However, the Drosophila literature three to ten pair-matings were set up for scoring the delayed mutations uments examples where chemicals negative in the d in a conventional manner may still e mutagenswhen tested for their action in F, generation (Table 11). In the case of benzene (Table 111). a different experimental method was used. In order to test if any genetic (heritable) instability can be induced in some of the treated males, the mutation induction was followed in each serially numbered individual male for the three broods. Unlike EDB experiments where between three and ten pair matings were made from one random F2vial. in the case of benzene rids that tested negative may induce delayed muta- s. similarly, very low doses ofmutagens or carcino- ns found negative in the conventional SLRL may delaye(fmutations which appear in the F3 genera- only one Fz vial was chosen from each treated male. From this F2 vial, three to twenty F, vials were cultured. As can be seen in Table 111 for spermatozoa, male No. 1 produced 65 F2pair-matings. None of these was a lethal. One of these was used to generate twenty pair-matings for F,. For spermatids this male produced 21 F2 vials and one of these was tested this hypothesis using ethylene dibromide used to generate twenty F, vials. The purpose was to see if the same nd benzene and report that this indeed is true. male produces delayed lethals in each successive brood. Indeed, this appears to be the case in males Nos. 2 and 7. The number of F?cultures ERIALS AND METHODS ith a small aperture in the lid. For ethylene dibromide (EDB) Received September 19. 1994; revised and accepted December 17. 1994. Address reprint requests to Dr. P.G. Kale, Alabarna A. & M. University. Department of Biology, P.O. Box 610, Normal, AL 35762. 212 Kale and Kale TABLE I. Induced Sex-Linked Recessive Lethal Mutations in Three Successive 2-Day Broods From D. melanoaaster I Males Treated With EDB and Benzene Germ cell stage Males brooded NO. of- Males sterile Chs. tested in F2 Mutations in F2 -re-r-.c.e-n.t mutations in F2 NO. of- Chs. tested in F, Mutations in F, rercem mutaliua in F, EDB Spermatozoa Late spermatids 24 21 2. 0 816 1,251 00 2,738 4 0.32 603 '29 I .w 9 1.49 Early spermatids 20 3 637 21 3.29 1.659 101 6.09 Benzene Spermatozoa 40 14 1,967 3 0.15 483 4 0.83 Late spermatids 39 14 1.046 I 0.09 455 5 109 , Early spermatids 38 20 420 0 0.00 162 2 1.23 Control Spermatozoa Late spermatids 24 24 0 0 59I 580 I 0.16 00 482 528 0 01 00 Early spermatids 23 0 707 1 0.14 616 I 0.I6 TABLE II. Delayed Mutations (recovered in F3) Induced by Ethylene Dibromide in Drosophila Spermatozoa and Spermatids* (Mutations; N = total number of chromosomes tested) Brood I FLvial Spermatozoa No.of F3 vials cultured N No. of F3 vials L 1 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 to 313 7 6 4 IO IO IO 7 IO 10 IO IO 10 7 IO 8 2.609 7 6 4 1 1 1 I 1 1 1 1 1 1 I I 0 Total Percent delayed mutations 2,738 29 1.6 Late spermatids Brood 2 Fz vial No. of F3vials cultured N No. of F3 vials lethal L 1 2 3 4 5 6 to76 6 7 10 9 8 563 5 1 1 1 1 0 76 603 9 1.49 Early Spermatids Brood 3 F2vial No. of F3 vials cultured N No. of F3 vials lethal L 1 IO I O 2 99 3 10 8 4 77 5 IO 6 6 IO 6 755 8 IO 5 954 10 10 4 I 1 10 4 12 IO 3 13 IO 3 14 10 3 15 10 16 10 17 IO 18 10 2 2 2 2 Brood 3 F2vial 19 20 21 22* 23 24 -25 26 27 28 29 30 31 32 33 34 35 to 178 No. of F3 vials cultured N 1N0.d F, vi& IethJ 7 L' 1 130 7 10 10 10 IO IO IO I1O0 10 10 IO IO IO 1,303 1I 1 ;I I 1: I' I' 1, I 0 1,659 101 6.09 *L. lethal mutations; N, total number of chromosomes tested. used (only one for each treated male) is small. However, the proportion of these cultures carrying a delayed lethal is quite high. In Table IV, we have summarized the data from both the chemicals and also provide the frequencies of F2cultures which carry delayed lethals. We perfom one control experiment every month. One ofthem coincided with the EDB and one with the benzene experiment. These data were used as concurrent controls. The frequency in these controls were not significantly different from each other or from our historical control (0.067%).The control data are given in Table I. RESULTS AND DlSCUSSlON In our previous work on chemical mutagenesis [Kale and Baum, 1979, 19831, we used both EDB and benzene. In the present investigation our intent was to use very Ilow in Order that significant !lumberOsf con. ventional SLRL mutations are not induced. This was 214 Kale a,id Kale achieved in the first two broods of EDB and for benzene substance like hydroxylamine strongly supports the s treated flies (Table I). gestion that, in tests for potential mutagens in Drosoph We have used our earlier experiments, which were es- a negative or doubtful result in F2 should be tested pecially performed to study the differential germ cell sen- delayed lethals in F3. After all, if lethals are cons sitivity [Kale and Baum, 19791, to relate the broods with a genetic risk, it makes no difference whether they germ cell stages. We equate brood 1 to treated spermato- first as completes. zoa, brood 2 to late spermatids, and brood 3 to early The mechanism underlying the induGtion of dela spermatids. Table I gives the F3 mutation frequencies in mutations has been well illustrated by Jenkins [I9 the three cell types for both the compounds which appear Lethal hits in both complementary DNA strands to be quite high. However, these numbers should be kept quired to induce a conventional SLRL mutation. How in perspective for EDB since up to ten pair-matings were ever, if only one strand is hit and mutates, upon replicalia made from each F2 culture. Therefore, the underlying and division, two DNA molecules will be generated,00 detailed data are given in Table 11. A total of 313 ran- mutant and one non-mutant. Thus in an F, daughter ofc domly chosen F, cultures from treated spermatozoa pro- treated male there are two kinds of cells: 1) cells wib duced 29 F3 (delayed) lethals in 2,738 vials. Out of the the mutant chromosome which are mutant, and 2) Ilr 313analyzed, 298 were non-lethal and fifteen were lethal. cells with the non-mutant chromosome which are n o d Of these 15, twelve (Nos. 4-15) produced one lethal in In the next (F,) generation, this female will produce wiy ten F, cultures, whereas in the remaining three, all F3 type males and the culture will be scored as non-lethd cultures were lethal (see Table 11). In the next two broods, even when several females in the culture carry a mutada especially in early spermatids, the frequency of such high If tested for an additional (F,) generation, cultures numbers (from one F2 culture) is very high, so much so these females will be scored as mutations. It is like1 that 18 Fz cultures (Nos. 1-18) produced two or more weak mutagens and low doses of strong mutagen delayed lethals. In several of these cases all F, females the case of benzene and EDB, can induce single stnd in the vial carried a delayed lethal which was scored as damage which can be scored only in the F3 generation. a full lethal in F,. Table IV shows the proportion of At higher doses, the probability of hitting both strant$ Fz cultures carrying one or more delayed lethals. These is more than at lower doses. This sliould result in low numbers are quite high (4.79%, 6.58%. and 20%) and frequency of delayed lethals at higher doses. Epler [1!Mj demonstrate that a large number of conventional F2 CUI- testing ethyl methanesulphonate tested the dose-respow tures may appear normal (non-lethal) but can indeed carry of complete and mosaic mutations induced in the X chm delayed lethals. mosomes of Drosophila. Indeed, the frequency of corn The results clearly indicate that 1) low doses of EDB plete mutations increased linearly with the dose but he which do not induce conventional SLRL mutations do frequency of mosaics declined sharpb at higher doses. induce a highly significant number of delayed mutations Although the Drosophila SLRL assay is being used in F3 and 2) potent carcinogens such as benzene, which successfully to predict carcinogenicity of chemicals,some has been declared negative in the conventional SLRL potent carcinogens and other important environmental test, do induce a highly significant number of delayed chemicals have been shown to be negative in this assay. mutations. This has resulted in the recent removal of the assay from There is evidence in the literature [Lee et al., 1967a, the battery of short-term tests [Bentley et al., 19941. How. 1970; Shukla and Auerbach, 19791 demonstrating that ever, this apparent insensitivity may be due to an inappm delayed lethals can be detected in F, in the absence of priate end-point chosen to test some chemicals. We have significant numbers of conventional lethals in F2. Thus provided evidence that the conventional SLRL assay (car- Lee et al. [1967b] observed that incorporation of P3*in ried up to the F2 generation) fails to identify the mutage. Drosophila spermatozoa resulted in significant increase nicity of some potent carcinogens and weak mutagens in the frequency of complete mutations. Similarly, Shukla unless the test is extended for one more generation. At and Auerbach [19791documented the delayed mutagenic present, our evidence includes only the two chernica!s action of hydroxylamine which produces mainly one- being reported here. It will be worthwhile to test addi- strand mutations. The sex-linked recessive lethal muta- tional so-called ``negative compounds" to re-establish tions in this experiment were scored in the usual way. In the sensitivity of Drosophila in genotoxicity testing and order to test for delayed lethals, as in the case of our carcinogen prediction. experiments, one female from each of a large number of apparently lethal-free F2 vials was mated to a Mueller-5 REFERENCES male and the progeny of this cross (F3) was scored for lethals. The frequency of lethals in F3 was clearly more than that in the control. According to the authors, the Auerbach C (1946): Chemically induced mosaicism in D mduttogufrr Proc R Soc Edin B 62:211-222. Auerbach C (1976): "Mutation Research: Problems. Results and Prr. preponderance of delayed lethals after treatment with a spectives." 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