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Amitrole-induced cell transformation and gene mutations in Syrian hamster embryo cells in culture
Mutation Research, 140 (1984) 205-207
205
Elsevier MRLett 0583
Amitrole-induced cell transformation and gene mutations in Syrian
hamster embryo cells in culture Takeki Tsutsui, Heiji Maizumi and J. Carl Barrett* *Environmental Carcinogenesis Group, Laboratory of Pulmonary Function and Toxicology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.) (JCB) and Nippon Dental University Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo (Japan) (TT and HM)
(Accepted 10 March 1984)
Summary Amitrole, a widely used herbicide, is an animal carcinogen and an inducer of cell transformation. However, it is inactive as a mutagen in bacterial test systems. Thus, it has been suggested that amitrole is a non-mutagenic carcinogen. Over the dose range that induces morphological transformation of Syrian hamster embryo cells in culture, amitrole induced gene mutations at the Na ÷ / K + ATPase and hypoxanthine phosphoribosyl transferase loci measured concomitantly in the same cells. These results indicate that amitrole may act via a mutational mechanism.
Amitrole (3-amino-l,2,4-triazole), a widely used herbicide, induces thyroid and liver tumors in both mice and rats following oral or subcutaneous administration [11]. Pienta et al. [14] and Inoue et al. [12] have reported that amitrole induces morphological transformation of Syrian hamster embryo cells in culture. Styles [15] has also reported transformation of BHK cells by amitrole although this finding was not confirmed in another laboratory [10]. Negative results for the mutagenicity of amitrole in bacterial systems have been found in several laboratories [9,12,13]. These findings have been interpreted to indicate a nonmutagenic mechanism for the carcinogenicity and cell transforming abilities of amitrole [1]. * To whom reprint request should be addressed. Abbreviations: amitrole, 3-amino-l,2,4-triazole; B(a)P, ben-
zo[a]pyrene; 6TG, 6-thioguanine; HPRT, hypoxanthine phosphoribosyl transferase. o165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
For a number of years we have been interested in the relationship between cell transformation and mutagenicity. Using the Syrian hamster embryo cell transformation system which can be used to concomitantly measure transformation and gene mutation in the same cells, we have examined chemicals which are reported exceptions to the correlation between mutagenicity and carcinogenicity [2-8,16,17]. Some of the chemicals have shown mutagenic activity in the Syrian hamster cells under conditions that induce cell transformation [2,6], whereas other chemicals can induce cell transformation in the absence of measurable induction of gene mutations [5,8,16,17]. Understanding the mechanism of action of the latter group of compounds may provide new insight into the mechanisms of chemical carcinogenesis [3,5]. Therefore, we have compared the ability of amitrole to induce cell transformation and specific-locus mutations in Syrian hamster embryo cells.
206
Materials and methods
Cells and growth medium. Cell cultures were established from 13-day embryos of Syrian hamsters, strain LVG: LAK (Lakeview Hamster Colony, Charles River, Newfield, N J). Primary cultures of pooled littermates were cryopreserved in liquid nitrogen, secondary cultures were initiated from frozen stocks, and all experiments were performed with tertiary cultures [4,7]. Culture medium employed was IBR modified Dulbecco's Eagles's reinforced medium (Gibco, Grand Island, NY) supplemented with 3.7 g/1 sodium bicarbonate, 10% v/v Hy-Clone fetal bovine serum (Sterile Systems Inc. Logan, UT) and 100 units/ml penicillin and 100 /zg/ml streptomycin (Gibco). Cells were grown in a humidified atmosphere of 10% COz in air at 37°C. Cultures were passaged by gently trypsinization with 0.1 °70 trypsin solution (1:250, Gibco) in Dulbecco's phosphate-buffered saline for 5 min at 37°C. Cultures were routinely tested and found to be free of mycoplasma. Chemicals. 3-Amino-l,2,4-triazole (amitrole) and benzo[a]pyrene B(a)P were acquired from Aldrich Chemical Company (Milwaukee, WI). Amitrole was dissolved in complete medium at 1 mg/ml and filter sterilized. B(a)P was dissolved in dimethyl sulfoxide at 1 mg/ml. 6-Thioguanine (6TG) and ouabain were purchased from Sigma Chemical Co. (St. Louis, MO). Morphological transformation and somatic mutation assays. Cells (2.5 × l0 s) were inoculated into 75-cm 2 flasks, incubated overnight and treated
with 0.3-10 ~g/ml amitrole or 1 t~g/ml B(a)P for 48 h. After washing 3 times with complete medium, the cells were harvested by trypsinization and were assayed for cytotoxicity and morphological transformation by plating 3 x 103 cells (for amitrole) or 5 x 103 cells (for B(a)P) in each of twenty 100-mm dishes (Falcon) with complete medium. The remaining cells were plated in 75-cm 2 flasks at the density of 4 × 105 cells per flask. Following growth for an additional 4 days in the absence of the test substance, the cells (1 × l0 s) were then replated for the assay of gene mutation at H P R T and Na +/K + ATPase loci in each of ten 100-mm dishes with medium containing 3.3 ~g/ml 6TG or 1.1 mM ouabain. In addition, 3 x 103 cells were seeded into each of five 100-mm dishes to examine the cell survival 4 days after treatment. 7 days later, all cells were fixed with absolute methanol, stained with 3% Giemsa solution, and the number of morphologically transformed colonies and the number of mutant colonies on dishes were scored. The frequency of morphological transformation and mutation at two loci was calculated as described previously [4,7]. There was no significant difference in cell survival after 4 days expression time between control and experimental groups. Results and discussion
Exposure of Syrian hamster embryo cells to 0.3-10 tzg/ml amitrole for 48 h resulted in a dosedependent increase in morphological transformation (Table 1) in confirmation of the results of
TABLE 1 AM1TROL-INDUCED Treatment
CELL TRANSFORMATION
Dose
Survival
(%)
AND GENE MUTATIONS
Number of morphologically Transformation transformed colonies/ (%) total colonies
DMSO Amitrol Amitrol
0.1 °70 0.3 # g / m l 1 #g/ml
100 111 111
1/3028 4/3378 4/3360
0.03 0.12 0.12
Amitrol Amitrol B(a)P
3 #g/ml 10 # g / m l 1 /~g/ml
98 117 56
5/2980 7/3552 27/3332
0.18 0.20 0.81
M u t a t i o n frequency 6TG r
Oua r
< 10 -6
<10 6
5.0X 10 6 10× 10 6
5.6 x 10 6 3 4 x 10 -6
15xlO 6
45x10 6 l l O x 10 -6 5 0 × 10 6
1 l O x 10 -6
2 0 0 × 10 6
207
Inoue et al. [12] and Pienta et al. [14]. When gene mutations at the Na ÷ / K ÷ ATPase or H P R T loci were measured concomitantly in the same cells by induction of cells resistant to ouabain or 6TG respectively, dose-dependent increases in mutation were observed over the same concentration range used for cell transformation (Table 1). These results clearly indicate that amitrole is mutagenic at two genetic loci in mammalian cells and this contrasts with the negative results observed with bacterial mutation assays [9,12,13]. While a variety of mechanisms may account for differences in bacterial versus mammalian cell mutagenesis [3,5], the most likely explanation is the necessity for metabolism of amitrole for its activity. Syrian hamster embryo cells are able to metabolize a variety o f chemical carcinogens to active mutagens and transforming intermediates [5]. Further studies with these cells may provide insight into the nature of the ultimate carcinogenic form o f this widely used herbicide. Although our results indicate that amitrole is a mutagenic carcinogen, other chemicals are able to induce cell transformation and are nonmutagenic at specific genetic loci [5,8,16,17]. Many o f these c o m p o u n d s however, induce c h r o m o s o m e mutations [6,17]. The ability of amitrole to induce numerical or structural c h r o m o s o m e changes in Syrian hamster embryo cells is u n k n o w n . While our observations on the mutagenic activity o f amitrole support a mutational mechanism, it is not possible at present to conclude whether amitroleinduced gene or c h r o m o s o m a l mutations are involved in the carcinogenicity o f this herbicide.
References 1 Ashby, J. (1981) Overview of study and test chemicals, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of ShortTerm Test for Carcinogens; Report of the International Collaborative Program, Elsevier/North Holland, New York, pp. 112-171. 2 Barrett, J.C. (1981) Gene mutation and cell transformation of mammalian cells induced by two modified purines, 2-aminopurine and 6-N-hydroxyl-aminopurine, Proc. Natl. Acad. Sci. (U.S.A.), 78, 5685-5689. 3 Barrett, J.C. (1981) Cell transformation, mutation, and cancer, Gann, Monograph on Cancer Research, 27, 195-200.
4 Barrett, J.C., N.E. Bias and P.O.P. Ts'o (1978) A mammalian cellular system for the concomitant study of neoplastic transformation and somatic mutation, Mutation Res., 50, 121-136. 5 Barrett, J.C., T.W. Hesterberg and D.G. Thomassen (1981) The use of cell transformation systems for carcinogenicity testing and mechanistic studies of carcinogenesis, Pharmacol. Rev., in press. 6 Barrett, J.C., D.G. Thomassen and T.W. Hesterberg (1983) Role of gene and chromosomal mutations in cell transformation, N.Y. Acad. Sci., 407, 291-300. 7 Barrett, J.C., and P.O.P. Ts'o (1978) The relationship between somatic mutation and neoplastic transformation, Proc. Natl. Acad. Sci. (U.S.A.), 75, 3297-3301. 8 Barrett, J.C., A. Wong and J.A. McLachlan (1981) Diethylstilbestrol induces neoplastic transformation of cells in culture without measurable somatic mutation at two loci, Science, 212, 1402-1404. 9 Bridges, B.A. (1981) Summary report on the Performances of Bacterial Mutation Assays, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens: Report of the International Collaborative Program, Elsevier/North Holland, New York, pp. 626-637. 10 Daniel, M.R., and J.M. Daniel (1981) Cell transformation test with baby hamster kidney cell, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens: Report of the International Collaborative Program, Elsevier/North Holland, New York, pp. 626-637. 11 IARC (1974) Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, Some anti-thyroid and related substances, nitrofurans and industrial chemicals, 7, 31-43. 12 lnoue, K., Y. Katoh and S. Takayama (1981) In vitro transformation of hamster embryo cells by 3-(Nsalicyloyl)amino1,2,4-triazole, Toxicol. Lett., 7, 211-215. 13 McCann, J.E., 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, 5135-5140. 14 Pienta, R.J., J.A. Poiley and W.B. Lebherz (1977) Morphological transformation of early passage golden Syrian hamster embryo cells from cryopreserved cultures as a reliable in vitro bioassay for identifying diverse carcinogens, Int. J. Cancer, 199, 642-649. 15 Styles, J.A. (1981) Activity of 42 coded compounds in the BHK-21 cell transformation tests, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens: Report of the International Collaborative Program, Elsevier/North-Holland, New York, pp. 626-637. 16 Tsutsui, T., H. Maizumi and J.C. Barrett (1984) Colcemidinduced neoplastic transformation and aneuploidy in Syrian hamster embryo cells, Carcinogenesis, in press. 17 Tsutsui, T., H. Maizumi, J.A. McLachlan and J.C. Barrett (1983) Aneuploidy induction and cell transformation by diethylstilbestrol: a possible chromosome mechanism in carcinogenesis, Cancer Res., 43, 3814-3821.
205
Elsevier MRLett 0583
Amitrole-induced cell transformation and gene mutations in Syrian
hamster embryo cells in culture Takeki Tsutsui, Heiji Maizumi and J. Carl Barrett* *Environmental Carcinogenesis Group, Laboratory of Pulmonary Function and Toxicology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.) (JCB) and Nippon Dental University Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo (Japan) (TT and HM)
(Accepted 10 March 1984)
Summary Amitrole, a widely used herbicide, is an animal carcinogen and an inducer of cell transformation. However, it is inactive as a mutagen in bacterial test systems. Thus, it has been suggested that amitrole is a non-mutagenic carcinogen. Over the dose range that induces morphological transformation of Syrian hamster embryo cells in culture, amitrole induced gene mutations at the Na ÷ / K + ATPase and hypoxanthine phosphoribosyl transferase loci measured concomitantly in the same cells. These results indicate that amitrole may act via a mutational mechanism.
Amitrole (3-amino-l,2,4-triazole), a widely used herbicide, induces thyroid and liver tumors in both mice and rats following oral or subcutaneous administration [11]. Pienta et al. [14] and Inoue et al. [12] have reported that amitrole induces morphological transformation of Syrian hamster embryo cells in culture. Styles [15] has also reported transformation of BHK cells by amitrole although this finding was not confirmed in another laboratory [10]. Negative results for the mutagenicity of amitrole in bacterial systems have been found in several laboratories [9,12,13]. These findings have been interpreted to indicate a nonmutagenic mechanism for the carcinogenicity and cell transforming abilities of amitrole [1]. * To whom reprint request should be addressed. Abbreviations: amitrole, 3-amino-l,2,4-triazole; B(a)P, ben-
zo[a]pyrene; 6TG, 6-thioguanine; HPRT, hypoxanthine phosphoribosyl transferase. o165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
For a number of years we have been interested in the relationship between cell transformation and mutagenicity. Using the Syrian hamster embryo cell transformation system which can be used to concomitantly measure transformation and gene mutation in the same cells, we have examined chemicals which are reported exceptions to the correlation between mutagenicity and carcinogenicity [2-8,16,17]. Some of the chemicals have shown mutagenic activity in the Syrian hamster cells under conditions that induce cell transformation [2,6], whereas other chemicals can induce cell transformation in the absence of measurable induction of gene mutations [5,8,16,17]. Understanding the mechanism of action of the latter group of compounds may provide new insight into the mechanisms of chemical carcinogenesis [3,5]. Therefore, we have compared the ability of amitrole to induce cell transformation and specific-locus mutations in Syrian hamster embryo cells.
206
Materials and methods
Cells and growth medium. Cell cultures were established from 13-day embryos of Syrian hamsters, strain LVG: LAK (Lakeview Hamster Colony, Charles River, Newfield, N J). Primary cultures of pooled littermates were cryopreserved in liquid nitrogen, secondary cultures were initiated from frozen stocks, and all experiments were performed with tertiary cultures [4,7]. Culture medium employed was IBR modified Dulbecco's Eagles's reinforced medium (Gibco, Grand Island, NY) supplemented with 3.7 g/1 sodium bicarbonate, 10% v/v Hy-Clone fetal bovine serum (Sterile Systems Inc. Logan, UT) and 100 units/ml penicillin and 100 /zg/ml streptomycin (Gibco). Cells were grown in a humidified atmosphere of 10% COz in air at 37°C. Cultures were passaged by gently trypsinization with 0.1 °70 trypsin solution (1:250, Gibco) in Dulbecco's phosphate-buffered saline for 5 min at 37°C. Cultures were routinely tested and found to be free of mycoplasma. Chemicals. 3-Amino-l,2,4-triazole (amitrole) and benzo[a]pyrene B(a)P were acquired from Aldrich Chemical Company (Milwaukee, WI). Amitrole was dissolved in complete medium at 1 mg/ml and filter sterilized. B(a)P was dissolved in dimethyl sulfoxide at 1 mg/ml. 6-Thioguanine (6TG) and ouabain were purchased from Sigma Chemical Co. (St. Louis, MO). Morphological transformation and somatic mutation assays. Cells (2.5 × l0 s) were inoculated into 75-cm 2 flasks, incubated overnight and treated
with 0.3-10 ~g/ml amitrole or 1 t~g/ml B(a)P for 48 h. After washing 3 times with complete medium, the cells were harvested by trypsinization and were assayed for cytotoxicity and morphological transformation by plating 3 x 103 cells (for amitrole) or 5 x 103 cells (for B(a)P) in each of twenty 100-mm dishes (Falcon) with complete medium. The remaining cells were plated in 75-cm 2 flasks at the density of 4 × 105 cells per flask. Following growth for an additional 4 days in the absence of the test substance, the cells (1 × l0 s) were then replated for the assay of gene mutation at H P R T and Na +/K + ATPase loci in each of ten 100-mm dishes with medium containing 3.3 ~g/ml 6TG or 1.1 mM ouabain. In addition, 3 x 103 cells were seeded into each of five 100-mm dishes to examine the cell survival 4 days after treatment. 7 days later, all cells were fixed with absolute methanol, stained with 3% Giemsa solution, and the number of morphologically transformed colonies and the number of mutant colonies on dishes were scored. The frequency of morphological transformation and mutation at two loci was calculated as described previously [4,7]. There was no significant difference in cell survival after 4 days expression time between control and experimental groups. Results and discussion
Exposure of Syrian hamster embryo cells to 0.3-10 tzg/ml amitrole for 48 h resulted in a dosedependent increase in morphological transformation (Table 1) in confirmation of the results of
TABLE 1 AM1TROL-INDUCED Treatment
CELL TRANSFORMATION
Dose
Survival
(%)
AND GENE MUTATIONS
Number of morphologically Transformation transformed colonies/ (%) total colonies
DMSO Amitrol Amitrol
0.1 °70 0.3 # g / m l 1 #g/ml
100 111 111
1/3028 4/3378 4/3360
0.03 0.12 0.12
Amitrol Amitrol B(a)P
3 #g/ml 10 # g / m l 1 /~g/ml
98 117 56
5/2980 7/3552 27/3332
0.18 0.20 0.81
M u t a t i o n frequency 6TG r
Oua r
< 10 -6
<10 6
5.0X 10 6 10× 10 6
5.6 x 10 6 3 4 x 10 -6
15xlO 6
45x10 6 l l O x 10 -6 5 0 × 10 6
1 l O x 10 -6
2 0 0 × 10 6
207
Inoue et al. [12] and Pienta et al. [14]. When gene mutations at the Na ÷ / K ÷ ATPase or H P R T loci were measured concomitantly in the same cells by induction of cells resistant to ouabain or 6TG respectively, dose-dependent increases in mutation were observed over the same concentration range used for cell transformation (Table 1). These results clearly indicate that amitrole is mutagenic at two genetic loci in mammalian cells and this contrasts with the negative results observed with bacterial mutation assays [9,12,13]. While a variety of mechanisms may account for differences in bacterial versus mammalian cell mutagenesis [3,5], the most likely explanation is the necessity for metabolism of amitrole for its activity. Syrian hamster embryo cells are able to metabolize a variety o f chemical carcinogens to active mutagens and transforming intermediates [5]. Further studies with these cells may provide insight into the nature of the ultimate carcinogenic form o f this widely used herbicide. Although our results indicate that amitrole is a mutagenic carcinogen, other chemicals are able to induce cell transformation and are nonmutagenic at specific genetic loci [5,8,16,17]. Many o f these c o m p o u n d s however, induce c h r o m o s o m e mutations [6,17]. The ability of amitrole to induce numerical or structural c h r o m o s o m e changes in Syrian hamster embryo cells is u n k n o w n . While our observations on the mutagenic activity o f amitrole support a mutational mechanism, it is not possible at present to conclude whether amitroleinduced gene or c h r o m o s o m a l mutations are involved in the carcinogenicity o f this herbicide.
References 1 Ashby, J. (1981) Overview of study and test chemicals, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of ShortTerm Test for Carcinogens; Report of the International Collaborative Program, Elsevier/North Holland, New York, pp. 112-171. 2 Barrett, J.C. (1981) Gene mutation and cell transformation of mammalian cells induced by two modified purines, 2-aminopurine and 6-N-hydroxyl-aminopurine, Proc. Natl. Acad. Sci. (U.S.A.), 78, 5685-5689. 3 Barrett, J.C. (1981) Cell transformation, mutation, and cancer, Gann, Monograph on Cancer Research, 27, 195-200.
4 Barrett, J.C., N.E. Bias and P.O.P. Ts'o (1978) A mammalian cellular system for the concomitant study of neoplastic transformation and somatic mutation, Mutation Res., 50, 121-136. 5 Barrett, J.C., T.W. Hesterberg and D.G. Thomassen (1981) The use of cell transformation systems for carcinogenicity testing and mechanistic studies of carcinogenesis, Pharmacol. Rev., in press. 6 Barrett, J.C., D.G. Thomassen and T.W. Hesterberg (1983) Role of gene and chromosomal mutations in cell transformation, N.Y. Acad. Sci., 407, 291-300. 7 Barrett, J.C., and P.O.P. Ts'o (1978) The relationship between somatic mutation and neoplastic transformation, Proc. Natl. Acad. Sci. (U.S.A.), 75, 3297-3301. 8 Barrett, J.C., A. Wong and J.A. McLachlan (1981) Diethylstilbestrol induces neoplastic transformation of cells in culture without measurable somatic mutation at two loci, Science, 212, 1402-1404. 9 Bridges, B.A. (1981) Summary report on the Performances of Bacterial Mutation Assays, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens: Report of the International Collaborative Program, Elsevier/North Holland, New York, pp. 626-637. 10 Daniel, M.R., and J.M. Daniel (1981) Cell transformation test with baby hamster kidney cell, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens: Report of the International Collaborative Program, Elsevier/North Holland, New York, pp. 626-637. 11 IARC (1974) Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, Some anti-thyroid and related substances, nitrofurans and industrial chemicals, 7, 31-43. 12 lnoue, K., Y. Katoh and S. Takayama (1981) In vitro transformation of hamster embryo cells by 3-(Nsalicyloyl)amino1,2,4-triazole, Toxicol. Lett., 7, 211-215. 13 McCann, J.E., 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, 5135-5140. 14 Pienta, R.J., J.A. Poiley and W.B. Lebherz (1977) Morphological transformation of early passage golden Syrian hamster embryo cells from cryopreserved cultures as a reliable in vitro bioassay for identifying diverse carcinogens, Int. J. Cancer, 199, 642-649. 15 Styles, J.A. (1981) Activity of 42 coded compounds in the BHK-21 cell transformation tests, in: F.J. de Serres and J. Ashby (Eds.), Evaluation of Short-Term Tests for Carcinogens: Report of the International Collaborative Program, Elsevier/North-Holland, New York, pp. 626-637. 16 Tsutsui, T., H. Maizumi and J.C. Barrett (1984) Colcemidinduced neoplastic transformation and aneuploidy in Syrian hamster embryo cells, Carcinogenesis, in press. 17 Tsutsui, T., H. Maizumi, J.A. McLachlan and J.C. Barrett (1983) Aneuploidy induction and cell transformation by diethylstilbestrol: a possible chromosome mechanism in carcinogenesis, Cancer Res., 43, 3814-3821.