Clastogenic activity of urethane in mice

Mutation Research, 281 (1992) 99-103

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© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00

MUTLET 00616

Clastogenic activity of urethane in mice Roumen Balansky, Penka Blagoeva and Zwetana Mircheva Laboratory of Chemical Mutagenesis and Carcinogenesis, National Centre of Oncology, Sofia-1756 (Bulgaria) ,2eceived 27 September 1991) ~i ~.~epted 27 September 1991)

Keywords: Urethane; Clastogenicity; Micronuclei; Bone marrow; Peripheral blood; Transplacental genotoxicity; (Mice)

Summary Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 x DBA2) mice with urethane (0.5 or 1.0 g / k g ) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6-8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female B a l b / c mice with urethane (3 g / l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% ( p < 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% ( p < 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.

Recently, some contradictory data concerning the clastogenic effects of urethane studied in vivo with the micronucleus assay in mouse bone marrow have been published (Holmstrom, 1990; Ashby et al., 1990). Previously, urethane had been shown to be a strong clastogen in mouse bone marrow when applied intraperitoneally (i.p.)

Correspondence: Dr. Roumen Balansky, c / o Prof. S. De Flora, Institute of Hygiene and Preventive Medicine, University of Genoa, via Pastore 1, 1-16132, Genoa (Italy).

(Wild, 1978; Bruce and Heddle, 1979; Heddle et al., 1983). However, not long ago Holmstrom (1990) reported that an increased frequency of micronucleated polychromatic erythrocytes (MNPCE) occurs in mouse bone marrow after 1-3 i.p. injections with ethyl carbamate but not after its oral administration. In their immediate response prompted by the surprising diversity of urethane effects in dependence on the route of administration, Ashby et al. (1990) established an increased number of MNPCE in mouse bone marrow when urethane was administered either i.p. or orally. It

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should be mentioned that earlier Trzos et al. (1978) had failed to detect any urethane-induced clastogenic effect in rat bone marrow. These controversial data as well as the lack of mutagenicity in S. typhirnurium (McCann et al., 1975) or in mammalian cells treated with urethane in vitro (Borenfreund et al., 1964; Bateman, 1976; Allen et al., 1982a) raised interest in further investigation of urethane-induced genotoxicity, especially in vivo and including transplacentally, as this chemical is a well known carcinogen. In addition, the presence of urethane in some drugs, pesticides, beverages as well as in tobacco smoke increases the possibility of industrial or environmental exposure of humans to urethane (IARC, 1974; Allen et al., 1982b).

02

7

14

21

37

52

Time ( days )

Materials

and methods

Animals In total 80 male and female (some of them pregnant on day 17 of gestation) BDFl (C57Bl x DBA2) as well as 20 female Balb/c mice aged 6-8 weeks, weighing 20-24 g were used in the present investigations. In some series of experiments 6-month-old BDFl mice weighing 30-32 g were also used to study their age-dependent sensitivity to the urethane clastogenicity. The animals had free access to standard rodent chow and water.

Treatment of animals Some of the BDFl mice were injected i.p. with a single dose of urethane (0.5 or 1.0 g/kg) and 24 h later the animals were killed by cervical dislocation to analyze the frequency of MNPCE in their bone marrow. In addition, 10 female Balb/c mice were treated orally with urethane added to the drinking water at a concentration of 3 g/l for 3 consecutive weeks. At different time points (see Fig. 1) during carcinogen exposure and the subsequent month, peripheral blood samples from the tail vein were taken to study the micronucleated normochromatic erythrocyte (MNNCE) frequency in their blood circulation.

Fig. 1. Frequency of micronucleated cytes (MNNCE) (10,000 NCE scored blood of female Balb/c mice, either with urethane (0.3% in the drinking

normochromatic erythroper mouse) in peripheral untreated or treated p.o. water for 21 days).

Furthermore, the transplacental clastogenic effect of urethane was investigated in the liver and peripheral blood of mouse fetuses. For this purpose 5 pregnant BDFl mice (the animals were mated overnight and the morning after mating was considered day 0 of pregnancy) were injected i.p. with urethane (1.0 g/kg) on day 17 of gestation and the animals were killed 24 h later. Micronucleus assay in mouse bone marrow After the mice were killed bone marrow smears were prepared, air-dried and stained with MayGriinwald Giemsa according to Schmid (1975). From each mouse (10 mice per group) 1000 PCE (10,000 PCE per group in total) were scored for micronuclei (Heddle et al., 1983). Micronucleus assay in mouse peripheral erythrocytes After being air-dried, the blood smears were stained with May-Griinwald Giemsa stain (MacGregor, 19801. From each mouse (10 mice per group) 10,000 peripheral NCE (in total 100,000 NCE per group) were scored for micronuclei.

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Processing of fetal fiver and neonatal peripheral blood Single-cell suspensions of fetal livers were prepared in fetal calf serum by mild pipetting (Cole et al., 1981). The smears were air-dried and stained with May-Griinwald Giemsa (Cole et al., 1981). At least 2000 PCE per fetus (in total 72,000 PCE per group) were examined for micronuclei. Fetal blood smears directly prepared from the decapitated fetuses were air-dried and stained with May-Griinwald Giemsa stain. At least 2000 PCE per fetus (in total 72,000 PCE per group) were screened for micronuclei. Only PCE with a homogeneously blue staining cytoplasm and no signs of RNA reticulum were scored in fetal liver and in fetal blood (Cole et al., 1981).

Statistical analysis The data obtained were analyzed according to Student's t-test. Results and discussion

The data summarized in Table 1 confirm the strong clastogenic activity of urethane in mouse bone marrow after its i.p. administration reported earlier (Wild, 1978; Bruce and Heddle, 1979; Balansky et al., 1987; Holmstrom, 1990; Ashby et al., 1990). The chromosome-damaging effect of ethyl carbamate when applied i.p. in BDF1 mice was

dose-, sex-, and age-dependent. Thus, a single dose of urethane (1.0 g/kg) induced twice as many MNPCE per 1000 PCE as a single dose of 0.5 g/kg, in mice of both sexes. At the same time, the 6-8-week-old animals were roughly twice as sensitive to the urethane-induced clastogenicity as the 6-month-old mice. In addition, the clastogenic response observed in male mice was about 2-fold greater that that observed in female mice, regardless of the doses used or of the age of treated animals (Table 1). These data confirm the general conclusion drawn by Holmstrom (1990) that the clastogenic activity of urethane in mouse bone marrow is a sex-dependent event. However, in the BDF1 mice used in the present experiments the male mice were more susceptible than the females, while Holmstrom (1990) found the female CD-1 mice to have a greater clastogenic response to urethane than the male animals. Previously, no significant sex differences in the formation of MNPCE in bone marrow of BDF1 mice treated with mitomycin C (MMC) or cyclophosphamide (CP) had been established (Blagoeva et al., 1991). According to the test report of US EPA Gene-Tox Program concerning the practical use of the micronucleus assay, 10 out of 30 evaluated clastogens studied by different investigators elicited sex-dependent activity, 7 of them being more effective in male mice (Mavournin et al., 1990). The spontaneous level of M N P C E in mouse bone marrow was not influenced by the sex and the age of animals and no signs of urethane-in-

TABLE 1 F R E Q U E N C Y OF MNPCE PER 1000 PCE IN BONE M A R R O W OF BDF1 MICE 24 h A F T E R T R E A T M E N T WITH U R E T H A N E : I N F L U E N C E OF A G E AND SEX Group

Male

PCE/NCE

Female

PCE/NCE

Untreated controls Urethane, 0.5 g / k g , i.p. Urethane, 1.0 g / k g , i.p.

2.5 _+0.48 a 30.8 _+2.18 * 53.6 _+3.21 *

1.10 _+0.03 b 0.98 _+0.08 0.93 _+0.07

1.5 ± 0.27 18.8 +_1.99 * 36.6 _+2.25 *

1.07 + 0.07 1.02 ± 0.03 0.90 + 0.11

Untreated Untreated Urethane, Urethane,

2.3 _+0.78 2.8 _+0.65 63.3 ± 1.23 * 35.2 ± 4.76 *

1.15 _+0.09 0.95 + 0.05 1.05 + 0.04 0.85 + 0.05

2.5 _+0.32 2.4 + 0.46 37.4 + 2.45 * 21.2 + 1.53 *

0.97 ± 0.04 1.11 ± 0.07 1.02 ± 0.05 0.89 ± 0.09

controls, 1.5 months controls, 6 months 1.0 g/kg, i.p., 1.5 months 1.0 g / k g , i.p., 6 months

a M e a n ± S E , data from 10 mice, 1000 PCE scored per mouse. h Mean ± SE. * p < 0.001.

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duced toxicity were found in mouse bone marrow according to the PCE/NCE ratio (Table 1). The 3-week oral treatment of Balb/c mice with urethane added to the drinking water significantly increased (up to 3-4-fold) the level of MNNCE circulating in the mouse peripheral blood (Fig. 1). A slight elevation of the MNNCE number could be detected as early as 48-72 h after the start of carcinogen exposure. The number of MNNCE steadily increased up to weeks 2-3 of exposure when the MNNCE level plateaued. A month after urethane exposure was stopped, the level of MNNCE dropped to the control values (Fig. 11, which is consistent with data showing the life span of MNNCE to be about 1 month (Schlegel and MacGregor, 1982; Mavournin et al., 1990). These data confirm the results published by Ashby et al. (1990) as well as the data recently reported by Westmoreland et al. (1991) that urethane is an effective clastogen in mouse bone marrow also when given orally as the MNNCE detected in the peripheral blood were induced in bone marrow (Mavournin et al., 1990). Obviously, the micronucleus test in mouse peripheral blood could be used successfully when studying the clastogenic activity of urethane in mice treated for long periods of time with this carcinogen. Transplacental clastogenic effect of urethane could also easily be detected by means of the micronucleus test in the liver and peripheral blood of mouse fetuses 24 h after the i.p. treatment of pregnant mice on day 17 of gestation with ethyl carbamate (1.0 g/kg) (Table 2). These data confirm and further extend our preliminary observa-

TABLE

tions showing the transplacental activity of urethane in mouse fetal liver (Balansky et al., 1989). Neeper-Bradley and Conner (1989) also demonstrated the transplacental genotoxicity of urethane by measuring the level of sister chromatid exchanges (SCE) in mouse fetal liver cells. The clastogenic effect of ethyl carbamate established in the present investigations (24 h after the i.p. administration of urethane at a dose of 1.0 g/kg on day 17 of gestation) was less pronounced in fetal erythroblasts than in those induced at the same time in maternal bone marrow, probably due to the possible protective role of the placenta, differences in fetal and maternal metabolism or differences in fetal erythropoiesis compared to those processes in adult mice. A similar trend was observed by Neeper-Bradley and Conner (1989) with regard to urethane’s ability to induce SCE in maternal bone marrow and in mouse fetal liver. However, it could not be excluded that at other time intervals (15-18 h after urethane administration, for example) the number of MNPCE induced in fetal liver could be higher (Cole et al., 1981). In addition, it seemed that pregnancy had no significant effect on the mother’s sensitivity to urethane-induced clastogenicity in bone marrow (Table 2). The data obtained allow the conclusion that urethane applied i.p. or orally induces in bone marrow and in peripheral blood of mice a clear dose-, sex-, and age-dependent clastogenic effect. Furthermore, the chromosome-damaging activity of urethane measured as micronucleus formation could also be detected in the liver and peripheral blood of mouse fetuses. The micronucleus assay

2

FREQUENCY OF MNPCE PER 1000 PCE IN THE LIVER AND PERIPHERAL BLOOD THE MATERNAL BONE MARROW 24 h AFTER TREATMENT WITH URETHANE GKllIp

Untreated Urethane,

controls 1.0 g/kg,

i.p.

Number of mice

Maternal bone marrow ”

Number fetuses

5 5

2.2 f 0.37 33.6+3.26 +1427O/o ***

41 36

‘I Mean f SE, 1000 PCE scored per mouse. ” Mean f SE, 2000 PCE scored per fetus. * p < 0.05; * * p < 0.01; *** p < 0.001.

of

OF MOUSE

FETUSES

Fetal liver ”

Fetal peripheral blood h

3.5 f 0.25 21.5 * 0.87 +514% **

4.6&0.30 11.7k0.63 + 154% *

AND

IN

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applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic genotoxic activity of urethane, its transplacental effects as well as the influence of different modifying factors on these processes.

Acknowledgement The authors acknowledge the support of the Italian Association for Cancer Research (IACRItalian Fellowship granted to R.B. and spent at Laboratory of Mutagenesis and Primary Prevention of Cancer, Institute of Hygiene and Preventive Medicine, University of Genoa, 1-16132, Genoa, Italy). References Allen, J.W., R. Langenbach, S. Nesnow, K. Sasseville, S. Leavitt, J. Campbell, K. Brock and Y. Sharief (1982a) Comparative genotoxicity studies of ethyl carbamate and related chemicals: further support for vinyl carbamate as a proximate carcinogenic metabolite, Carcinogenesis, 3, 1437-1441. Allen, J.W., Y. Sharief and R. Langenbach (1982b) An overview of ethyl carbamate (urethane) and its genotoxic activity, in: R. Tice, D. Costa and K. Shaich (Eds.), Genotoxic Effects of Airborne Agents, Plenum, New York, pp. 443-460. Ashby, J., H. Tinwell and R.D. Callander (1990) Activity of urethane and N,N-dimethylurethane in the mouse bonemarrow micronucleus assay: equivalence of oral and intraperitonel routes of exposure, Mutation Res., 245, 22723O. Balansky, R., and P. Blagoeva (1989) Tobacco-smoke-induced clastogenicity in mouse fetuses and in newborn mice, Mutation Res., 223, 1-6. Balansky, R., P. Blagoeva and Z. Mircheva (1987) Investigation of the mutagenic activity of tobacco smoke, Mutation Res., 188, 13-19. Blagoeva, P., R. Balansky and Z. Mircheva (1991) Potentiation by caffeine of the frequencies of micronuclei induced by mitomycin C and cyclophosphamide in young mice, Mutation Res., 246, 123-127. Borenfreund, E., M. Kriun and A. Bendich (1964) Chromosomal aberrations induced by hyponitrite and hydroxylamine derivatives, J. Natl. Cancer Inst., 32, 667-679. Bruce, W.R., and J.A. Heddle (1979) The mutagenic activity of 61 agents as determined by the micronucleus,

Salmonella, and sperm abnormality assay, Can. J. Genet. Cytol., 21,319-334. Cole, R.J., N. Taylor, J. Cole and C. Arlett (1981) Short-term test for transplacentally active carcinogens. I. Micronucleus formation in fetal and maternal mouse erythroblasts, Mutation Res., 80, 141-157. Heddle, J., M. Hite, B. Kirkhart, K. Mavournin, J. MacGregor, G. Newell and M. Salamone (1983) The induction of micronuclei as a measure of genotoxicity, Mutation Res., 123, 61-118. Holmstrom, M. (1990) Induction of micronuclei in bone marrow of mice exposed to 1, 2 or 3 daily doses of urethane, Mutation Res., 234, 147-154. IARC (1974) Some antithyroid and related substances, nitrofurans and industrial chemicals, in: IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man, Urethane, Vol. 7, International Agency for Research on Cancer, Lyon, pp. 111-140. MacGregor, J.T., C.M. Wehr and D.H. Gould (1980) Clastogen-induced micronuclei in peripheral blood erytbrocytes: the basis of an improved micronucleus test, Environ. Mutagen., 2, 509-514. Mavournin, K., D.H. Blakey, M.C. Cimino, M.F. Salamone and J.A. Heddle (1990) The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 239, 29-80. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/ microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Neeper-Bradley, T.L., and M.K. Conner (1989) lntralitter variation in murine fetal sister chromatid exchange responses to the transplacental carcinogen ethyl carbamate, Environ. Mol. Mutagen., 14, 90-97. Schlegel, R., and J.T. MacGregor (1982) The persistence of micronuclei in peripheral blood erythrocytes: detection of chronic chromosome breakage in mice, Mutation Res., 104, 367-369. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Trzos, R.J., G.L. Petzold, M.N. Brunden and J.A. Swenberg (1978) The evaluation of sixteen carcinogens in the rat using the micronucleus test, Mutation Res., 58, 79-86. Westmoreland, C., M. Plumstead and D. Gatehouse (1991) Activity of urethane in rat and mouse micronucleus test after oral administration~ Mutation Res., 262, 247-251. Wild, D. (1978) Cytogenetic effects in the mouse of 17 chemical mutagens and carcinogens evaluated by the micronucleus test, Mutation Res., 56, 319-327. Communicated by K. Sankaranarayanan