Potent Mutagenicity of Urethan (Ethyl Carbamate) Gas in Drosophila Melanogaster^

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[CANCER RESEARCH 39. 4224-4227, October 1979) 0008-5472/79/0039-OOOOS02.00 Potent Mutagenicity of Urethan (Ethyl Carbamate) Gas in Drosophila melanogaster^

Taisei Nomura2

Department of Medical Genetics. University of Wisconsin. Madison. Wisconsin 53 706

ABSTRACT an enormous number of patents. However, potent carcinogenic and teratogenic activities had been reported in experimental Exposure of spermatozoa to urethan (ethyl carbamate) gas animals before the usage of urethan in humans (see Ref. 21). induced a high frequency of X-linked recessive lethal mutations On the other hand, mutagenicity of urethan has not been detected by the Muller-5 technique in Drosophila melanogas- detected in Salmonella, even with the use of enzymatic acti ter. and a clear dose response was observed. Mutagenic vation by liver homogenates or S-9 fraction, whereas recently activity of urethan gas on mature spermatozoa following 1.5 most carcinogens have turned out to be mutagens when tested (2.4% mutations per chromosome) and 5.5 hr (5.5%) exposure by these methods (16). In Drosophila melanogaster, Vogt (31) is approximately equal to that induced by 1000 (2.3%) and reported that injection of urethan solution into adult males 2000 (5.2%) rads of y-radiation. Urethan gas also induced a induced significant yields of X-linked recessive lethal mutations significant yield of mutations (1.2%) when the spermatogonia by the CIB technique. The frequency was about 5.5 times that stage was treated for 5.5 hr. However, exposure of spermato in untreated controls. However, an extensive study by Fahmy zoa to urethan gas did not induce a significant yield of trans- and Fahmy (7) showed only very weak mutagenicity by the locations, even when the treated sperm was stored in females Muller-5 technique. In the present study, adult males of Dro for more than 10 days, whereas y-rays which produced a sophila were exposed to urethan gas, because urethan pos comparable frequency of X-linked recessive lethal mutations sesses a unique characteristic of high volatility. Two genetic induced translocations with high frequency. Thus, urethan can effects, gene mutation and translocation, were tested. In order barely induce translocation but can strongly induce point mu to assess the type of DMA damage produced by urethan, we tation in Drosophila germ cells. This may be the major cause of also studied the effects of caffeine on urethan-initiated muta the lack of dominant lethals. Mature spermatozoa were ex genesis, since it is known that in Eschehchia coli mutagenesis posed to mutagens and eggs were exposed to caffeine at initiated by UV light and the UV-mimetic compound 4-nitroqui- immature stages when eggs are permeable. X-linked recessive noline 1-oxide is reduced by the presumed error-prone post- lethal mutations induced by 3.5 hr exposure to urethan gas replication DNA repair inhibitor, caffeine (13). were not reduced by caffeine feeding to females at a low dose (0.5 mg/ml) but significantly reduced at the 5% level by caf MATERIALS AND METHODS feine feeding at a high dose (2.0 mg/ml). When mature sper matozoa were exposed to urethan gas for a longer period (5.5 X-Linked Recessive Lethal Mutations. One g of powdered hr), urethan-induced mutations were slightly reduced by a high urethan (Wako Pure Chemical Industry, Ltd., Osaka, Japan) dose (2.0 mg/ml) of caffeine, but the difference was not wrapped in cotton was covered with cheesecloth and pressed significant. The patterns of caffeine effects on urethan-initiated to the bottom of a 22-mm vial. Fifty adult Oregon R males mutagenesis in Drosophila are similar to those on UV- or 4- (collected 5 days after emergence) were placed in the vial, and nitroquinoline 1-oxide-initiated mutagenesis in Eschehchia coli. the vial was tightly stoppered with a wad of cotton. Although the flies are quickly anesthetized, they continue to take up urethan gas. Males were exposed to urethan for 1.5, 3.5, and INTRODUCTION 5.5 hr in the vial at 22Â°in a chemical hood; 24 hr after urethan Urethan (ethyl carbamate) has long been used as a sedative, treatment, 25 males were mass-mated for 24 hr with 25 virgin a hypnotic, an anesthetic, and a cosolvent for more than 200 females (48 to 54 hr after emergence) of a non-Bar FM7 strain, Ins (1) SCB15D-E; 20A-B dl-49, y3'" sc8 vf v01, in a 0.25-pint drugs commonly used in humans (until 1976) as well as for pesticides and cosmetics (6, 22, 33). Urethan is also contained bottle containing cornmeal-molasses medium. Males and fe in fermented beverages (27) and has proved to be useful as a males were discarded after 24 hr. X-linked recessive lethal finishing agent for cotton fabric (1), as an ingredient in hair mutations were detected by the Muller-5 technique. As positive wave compounds (10, 26) and colored-flame-producing can controls, adult Oregon R males (5 days after emergence) were exposed to 1000, 2000, and 3000 rads of y-rays from a 137Cs dles (8), for making porous tablets (9) and molds (11), for printing photographs (18, 32), for the prevention of formalde source at a dose rate of 435 rads/min, and mutations were hyde odor from aminoplast-coated wood substrates (19), for detected by the same procedure. In order to detect mutations the stabilization of soil (28), and for many other purposes under at the spermatogonia stage, urethan-treated Oregon R males were again mated similarly with FM7 females 14 days after urethan gas exposure (5.5 hr). For description of stocks and ' The work was performed during the tenure of the International Agency for mutants, see the paper of Lindsley and Grell (15). Research on Cancer (WHO) Fellowship at the Department of Medical Genetics. University of Wisconsin. This is Paper 2275 from the Genetics Laboratory Translocations. Adult Oregon R males (5 days after emer â€¢'Presentaddress: Department of Fundamental Radiology and Institute for gence) were exposed to urethan gas for 3.5 and 5.5 hr; 24 hr Cancer Research. Osaka University Medical School. Nakanoshima, Kita-ku, after treatment, they were mated with virgin females of the Osaka 530. Japan constitution, y se5' In 49 sc8; dp bw; st p", in 0.25-pint bottles Received September 22. 1978; accepted July 10. 1979

4224 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. Potent Mutagenicity of Urethan Gas in Drosophila containing cornmeal-molasses medium. Treated males were Average Hours discarded 24 hr after mating. The inseminated females were Egg Stage after Emergence then transferred to fresh 0.25-pint bottles and discarded after I 0 48 hr. For storage study, inseminated females were fed on 7 Kleenex tissue paper saturated with 3% concentration of mo lasses to discourage egg-laying during the 10-day storage period and transferred to fresh 0.25-pint bottles containing cornmeal-molasses medium. The medium was changed at 3- day intervals, and females were transferred to cornmeal-molas ses medium for 12 hr at each change of molasses medium. With this procedure, very few eggs were deposited during the storage period. Ft males were placed individually in vials with 2 or 3 virgin females of y scs/ In 49 sc8; dp bw; st p" to detect Egg Membrane translocations (30). As positive controls, males were exposed to 1000 and 2000 rads of y-rays (137Cs), and translocations X linked Recessive Lethals

were detected by the same method. Chart 1. Schematic diagram of experimental procedure and egg development. Caffeine Treatment to Females in Relation to Egg Devel In virgin. 24-hr-old females, all stages of eggs are found, but Stages 1 to 12 are opment. As shown in Chart 1, mature spermatozoa were known to make up 97% of the total (12). During the period (24 to 48 hr after treated with urethan or EMS,3 and eggs were treated with emergence) of caffeine treatment, eggs develop to Stage 14 (12). Caffeine can reach the egg freely during Stages 1 to 9, slightly during stages 9 to 12. and various doses of caffeine following Mendelson's method (1 7), remain in the egg after the completion of the chorion (Stage 12) because it is difficult for chemicals to pass through the chorion. When caffeine-treated females but at immature stages when the eggs are permeable (12). As were mated with mutagen-treated males 48 hr after emergence, females start to for EMS treatment, 50 adult Oregon R males (48 to 72 hr after deposit eggs. Consequently, about 97% of eggs are considered to contain emergence) were fed on Kleenex tissue paper saturated with caffeine at the time of fertilization. Mature spermatozoa with DNA damage produced by the mutagen enter caffeine-containing eggs and X-linked recessive 3.5 ml of 2.5 mw EMS (Sigma Chemical Co., St. Louis, Mo.) in lethal mutations derived only from sperm DNA were detected by the Muller-5 1% sucrose solution for 24 hr. Twenty-five FM7 virgin females technique. (24 to 30 hr after emergence) were fed on Kleenex tissue paper saturated with 0, 0.5, 2.0, 5.0, and 10.0 mg concentra tion of caffeine per ml in 1% sucrose solution for 24 hr and mated with 25 mutagen-treated males for 24 hr in a 0.25-pint IO.O P bottle containing cornmeal-molasses medium. All procedures Urethan Gas /'52530I were performed at 25Â°except for treatment of adult males with O---0 mutagens. X-linked recessive lethal mutations were again de 7.5 ZT Rays tected by the Muller-5 technique. By treating the sperm and egg separately before fertilization, we can exclude interaction â€¢Z 5.0 between caffeine and mutagen before the mutagen reaches (36/1.0121 the chromosomes and also any influence of caffeine on the metabolism of urethan. FM7 females were highly resistant to SÃ¤ II7Ã•707I V p-'IIO'44H caffeine, and killing was not observed even with 10.0 mg CÂ£ 2.5 concentration per ml. The ratio of the number of eggs deposited 1 I'8501 .â€¢'Â¡( Ray Exposure ( R i in caffeine-treated groups to those in the caffeine-less control 1.000 ?.000 3.000 group was 72.5, 71.9, 65.7, and 61.4% at 0.5, 2.0, 5.0, and 0.0 0 1.5 3.5 5.5 10.0 mg concentration of caffeine per ml, respectively. Urethan Gas ExposureiHoursi Chart 2. X-linked recessive lethal mutations in the progeny of males exposed RESULTS to urethan gas and -y-radiation at the mature spermatozoa stage. Numbers in X-Linked Recessive Lethal Mutations and Translocations. parentheses, X-linked recessive lethal mutations per chromosomes tested. De Urethan gas induced a high frequency of X-linked recessive tails of the experimental procedure are given in the text. lethal mutations when the mature spermatozoa stage was (p < 0.01 versus untreated controls). As for translocations, treated, and a clear dose response was observed as shown in exposure of spermatozoa to urethan gas did not induce signif Chart 2. Mutagenic activity of urethan gas on mature sperma icant yields of translocations even if the treated sperm was tozoa following 1.5 and 5.5 hr exposure is approximately equal stored in females for more than 10 days, whereas y-rays which to that induced by 1000 and 2000 rads of y-radiation, respec produced a comparable frequency of X-linked recessive lethal tively. Feeding of 20 and 50% urethan in 1% sucrose solution mutations induced translocations with high frequency (Table also induced mutations at a frequency of 1 of 143 and 67 of 1). In order to examine the volatility of urethan, Whatman No. 1549. The difference between the latter and its control is highly 1 filter papers (1x2 cm) were moistened with 0.4 /iCi of significant (p

OCTOBER1979 4225

Table 1 Transloca lions involving the Y. II, and III chromosomes in the progeny of males exposed to urethan gas or y-rays at the mature spermatozoa stage

translocationsTreatment No of

quency/1020.170.200.01.136.190.0PaNS"NSNSNSÂ«0001 malesUrethanto gas3.5 hr5.5 hr5.5 hr(stored)0rrays(137Cs)1000

R2000 RUntreatedTested590494464442452367r<2;3>1102190r

" The x2 test with Yates' correction was applied in comparisons with untreated controls. b NS. not significant. c The treated sperm was stored in females for 10 to 12 days Details are given in the text.

Table 2 CÃ¡lleme effects on urethan gas and EMS-initiated X-linked recessive lethals (frequency per IO1) in D melanogaster The x2 test was used to compare with untreated controls. Yates correction was done when expected values in the cells were less than 5

lethal0.0 of X-linked recessive /chromosome2.0 caffeinefemales5.0 doses to Treatment to malesUrethan mg/ml36/1012 mg/ml49/1241 mg/ml31/1498 mg/ml77/442 mg/ml46/334 gas" (3.56)" (3.95)Â°80/450 (2.07)" 3.5hr 39/866(4.50)c76/426 5.5hrEMS 28/513(5.46)98/620 (17.8)c2/523 (17.8)c4/796 (17.4)c2/312 (13.8)c2/359 (2.5IHM)UntreatedNo. (15.8)1/850 (0.38)cmutations (0.50)cin (0.64)c10.0 (0.56)c (0.12)0.5 " Urethan gas was given to 5-day-old Oregon R males. 6 Numbers in parentheses, percentage. c NS. not significant. p < 0.05. than-initiated X-linked recessive lethal mutations detected by increase (2). Failure of the induction of translocations by ure the Muller-5 technique were significantly reduced by 2.0 mg of than may be the major cause of the lack of dominant lethals in caffeine per ml when mature spermatozoa were exposed to Drosophila and mice (4, 25), and urethan-induced DMA lesions urethan gas for 3.5 hr and slightly, but not significantly, re responsible for tumor induction may not necessarily be a large duced when exposed for 5.5 hr (Table 2). However, EMS- chromosomal change, but as small as those for point mutation. initiated mutations were not reduced. As for the caffeine effects on urethan-initiated mutagenesis, we cannot conclude that the caffeine effect is established, since only one of 3 comparisons was significant at the 5% level. The DISCUSSION inhibitory effect of caffeine on urethan-initiated mutagenesis in D. melanogaster seems to be less than that on urethan-initiated There is no doubt to the potent mutagenicity of urethan in D. carcinogenesis and teratogenesis in mice (23, 24). In E. coli, melanogaster, whereas its mutagenicity has not been detected caffeine has little suppressive effect on mutagenesis initiated in Salmonella even with the use of enzymatic activation by liver by a high level of mutagen (13, 14). Furthermore, the patterns homogenates or S-9 fraction (16). The complicated metabolism of caffeine effects on urethan-initiated mutagenesis in Dro of the in vivo system may be transforming urethan into an sophila (slight increase by a low dose of caffeine and inhibition active form, since none of the toxic effects of urethan have by a high dose) appear to be similar to those on UV- or 4- been detected in the in vitro system (16, 29). Dominant lethals nitroquinoline 1-oxide-initiated mutagenesis in the wild type of (data not shown) and translocations were not significantly E. coli (13). Since the inhibitory effect of caffeine is apparent increased by urethan, nor are they in mice (4, 25), while both in the mutant-strain of E. coli, which lacks the ability of excision of them are easily induced by X-rays or y-rays in Drosophila repair of UV-induced DMA damage (13,14), the problem could (2) and mice (3, 25). Because some mutagens which did not be elucidated by the use of similar mutants of Drosophila (5). induce translocations in Drosophila by the usual method have When the sperm and egg were treated separately before ferti turned out to induce translocations by storing the treated sperm lization, no interaction between mutagens and caffeine nor an in females (2, 20, 30), a storage study was carried out. How influence of caffeine on the metabolism of the mutagens ap ever, increased yields of translocations were not observed peared, while in bacterial and mouse experiments, both chem even if the treated sperm were stored for more than 10 days. icals are present in the same cells or individuals. Except for a However, the spontaneous rate of translocations in Drosophila possible carry-over of small amounts of urethan in the sperm, is so low that even 2 translocations may represent a slight it could be shown that the effect of caffeine must be on the

4226 CANCER RESEARCH VOL. 39

urethan-induced damage to DNA. 1977. 14. Kondo, S., and Nomura, T. Antimutagenic action of caffeine and protease This work is a warning of the possible hazards (mutation as inhibitor "antipain" in relation to tumorigenesis. Proc. Jpn. Cancer Assoc., well as cancer and malformation) of using products that contain 37. 12. 1978. urethan and to the workers who deal with urethan, because 15. Lindsley, D. L.. and Grell, E. H. Genetic variation of Drosophila melanogaster. urethan gas is able to induce mutations at both spermatozoa Carnegie Inst. Wash. Pubi. No. 627, 1972 16. McCann, J. E., Yamasaki, C. E.. and Ames, B. N. Detection of carcinogens and spermatogonia stages. as mutagens in the Sa/mone//a/microsome test: assay of 300 chemicals. Proc. Nati. Acad. Sei. U. S. A., 72. 5135-5139, 1975. 17. Mendelson, D. The effect of caffeine on repair systems in oocytes of Drosophila melanogaster. I. MutÃ¢t.Res., 22. 145-156, 1974. ACKNOWLEDGMENTS 18. Miller, H. A., and Henn, R. Photographic elements, compositions and proc esses using eutectic compositions. Defense Publication. United States Pat I would like to thank Dr. J. F. Crow, Dr. S. Abrahamson, Dr. J. K. Lim, Dr. L. ent Office No. 896.024, 1971. Craymer. and Dr. G. A. Dahl for their advice and help and C. Preston for her 19. Miwa, T., and Tomokawa, H. Prevention of formaldehyde odor from amon- invaluable assistance. oplast-coated wood substrates. Jpn. Kokai. 74: 42, 736, 1974. 20. MuÃ±oz,E. R., and Mazar Barnett, B. ll-lll translocations induced by diethyl sulfate in mature sperm of Drosophila melanogaster MutÃ¢t Res 45 355- 357, 1977. REFERENCES 21. Nomura, T. An analysis of the changing urethan response of the developing mouse embryo in relation to mortality, malformation and neoplasm. Cancer 1. Arceneaux, R. L., Frick, J. G., Jr., Reid, J. D.. and Gautreaux, G. A. A Res.. 34: 2217-2231. 1974. carbamate finish for wrinkled-resistant and "wash-and-ware" cottons. Am. 22. Nomura, T. 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Franmeni, Jr. melanogaster increases mutagen sensitivity and decreases excision repair. (eds.), Tumors of Early Life in Man and Animals, pp. 873-891. Perugia. Genetics, 34: 527-544, 1976. Italy: Grafica di Salvi & Co., 1978. 6. Crout, J. R. Drug product containing urethan. Opportunity for hearing on 26. Ono, K., Torii, K., Ozawa, T., Iwabe. K., and Horikawa. H. Hair wave proposal to withdraw approval of new drug application for profenil injection. compositions. German Offenbarungsschiift. 2,421,248, 1974. United States Federal Registry Document No. 76-5599. 1976. 27. Ough. C. S. Ethyl carbamate in fermented beverages and foods. I. Naturally 7. Fahmy. O. G., and Fahmy, M. J. Gene elimination in carcinogenesis: rein- occurring ethyl carbamate. Agrie. Food Chem., 24: 323-328, 1976. terpretation of the somatic mutation theory. Cancer Res . 30. 195-205, 28. Sano. K. Stabilization of soil with chemicals. Secchaku/Adhesion and Ad- 1970. hesives, 75: 751-766, 1971. 8. Fujiwara, S. 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Taisei Nomura

Cancer Res 1979;39:4224-4227.

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