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 22°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 •'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 25°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.