337
Mutation Research, 48 (1977) 337--354
© Elsevier/North-Holland Biomedical Press
CHROMOSOME TESTS WITH 134 COMPOUNDS ON CHINESE HAMSTER CELLS IN VITRO -- A SCREENING FOR CHEMICAL CARCINOGENS
MOTOI ISHIDATE, Jr. and SHIGEYOSHI ODASHIMA Department of Chemical Pathology, National Institute of Hygienic Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158 (Japan)
(Received July 7th, 1976) (Revision received December 12th, 1976) (Accepted December 14th, 1976)
Summary Chromosomal aberration tests in vitro were carried out on Chinese hamster cells grown in culture with various chemicals, including carcinogenic N-nitroso compounds and their related derivatives, food additives, medical drugs, pesticides and other chemicals c o m m o n l y used in laboratories or industries. Sixty-three of the 134 chemicals gave negative results in our test system even with doses at which the cell growth was markedly inhibited. Nearly all compounds known to be mutagenic in bacteria were also positive in our tests. Both urethane and diethylstilbestrol were positive, even though they are known to be carcinogenic but not mutagenic in bacteria. Compounds such as N-alkyl-N'nitroguanidines, barbital, sodium benzoate, saccharin sodium, sodium nitrite, sodium nitrate and 4-aminoquinoline-l-oxide were positive in our chromosome tests, but they have n o t been conclusively tested for their carcinogenicity.
Introduction The detection of chemicals that may pose a genetic hazard in our environment is now both feasible and necessary [10,14]. Several assay systems with rapid and reliable methods for this purpose have been introduced: mutation assays in microorganisms [1,5,15,16], cytogenetic assays in both plant [6] and mammalian cells in vivo as well as in vitro [3,8,9,12]. These assay systems can also be applied as a screening probe for the detection of possible carcinogenic substances in our environment. This is based on the premise that neoplastic changes may be due to somatic m u t a t i o n in the cells that have been exposed to certain mutagens. There is also evidence that most carcinogens are mutagenic if the compounds undergo metabolic activation by microsomal enzymes in the bacterial assay system [1]. Carcinogens may be regarded as potential mutagens;
338 however not all mutagens are carcinogenic. At present such assay systems cannot replace animal tests for carcinogenicity in vivo. It is important, however, to devise a rapid screening method to detect possible environmental carcinogens which can later be affirmed with tests in vivo. This report presents our screening data on 134 chemicals, concerning the induction of chromosomal aberrations in Chinese hamster cells grown in culture, and data obtained from mutation assays in bacteria, which were carried our at the Dept. of Microbiol. of our institute using the same compounds. Information on their carcinogenic activities was obtained from data from the Natl. Inst. of Health, U.S.A. [13]. Materials and methods
Chemicals tested Compounds listed in Table II were supplied in part from the project teams supported by the Ministry of Health and Welfare, Japan (indicated as W in the table) [11]. Others were supplied from various departments of our institute (S, C, M, F or Ph), Tokyo Biochemical Research Institute, T o k y o (T), or Shizuoka Pharmaceutical College, Shizuoka, Japan (U). A few samples were obtained commercially (P). The purity of most of these chemicals was checked at the Dept. of Drugs of our institute. All samples were kept in the dark in a refrigerator until used. Solvents used for the dilution of the compounds were physiological saline (S), ethanol (E), aqueous solution of albumin bovine (A) or DMSO (D) as indicated in the table. Cell cultures A clonal sub-line of a Chinese hamster fibroblast cell line (CHL) was used. This cell line was originally established from the lung of a young adult by Dr. T. Utakoji, Cancer Institute, Tokyo. The karyotype consisted of 25 chromosomes. The cells had been maintained by 5-day passages and grown in a monolayer in petri dishes with Eagle's MEM (GIBCO F-11) supplemented with 10% calf serum. Their doubling time was estimated as 18.2 h at their exponential growth at 37°C in a 5% CO2 atmosphere. Estimation of the 50% growth inhibition dose of each sample Growth inhibition tests were carried out on each sample before the chromosome tests were started. The 50% inhibition dose was estimated as follows. Several different doses of each agent were separately added to the 3-day-old cultures (about 6 × 103 cells/3-cm dish}. The doses were prepared by a factor of 2 from the maximal dose, estimated from the data on LDs0, which appeared in references for each agent. The cells in a monolayer were washed, fixed with 10% formalin solution, and then stained with 0.1% crystal violet solution for 3 min. After washing and drying each dish was placed under a photodensitometer, Model CDM-X, which was designed in our laboratory and is being manufactured by Olympus Co., Japan, to measure the color absorption values from which relative cell densities on the dishes were easily calculated. The color absorption values obtained by this apparatus reflected well the actual number of cells that survived at the b o t t o m of each petri dish.
339
Chromosome test Three different doses, including the 50% inhibition dose of each agent, which was estimated by the growth inhibition test described above, were prepared and separately added to 3-day-old cultures (about 10 s cells/6-cm dish). Chromosome preparations were made, as a rule at 24 and 48 h, or when necessary at 6 h, after the treatment. Cells were treated with colcemid (0.2 pg/ml) for 2 h, and after trypsinization, they were incubated in 0.075 M KC1 hypotonic solution for 15 rain at 37°C. The cells were fixed with ice-cold fixative (methanol : glacial acetic acid, 3 : 1 v/v) which was changed 3 times. A few drops of the suspension were then placed on clean dry slides which were held horizontally under an electric heater. The slides were stained with 1% Giemsa's buffered solution (pH 6.8) for 20 min. The number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases at the magnification of 700. Types of aberration were classified into 5 groups: chromatid gaps (g), chromatid breaks (b), chromatid or chromosomal translocation (t), ring formation (r) and fragmentation or pulverization (f). Breaks less than the width of a sister chromatid were designated as gaps in our criteria. The incidence of polyploid cells was also calculated. Untreated cells and cells treated only with solvent served as controls. Evaluation of effects as the final judgement CHL cells c o m m o n l y have less than 3.0% cells with chromosomal aberrations. Therefore, the final judgement given to all experimental groups was as follows. Negative (--) if less than 4.9% of the aberration was detected even when doses of t h e agent were elevated to sub-lethal amounts, where almost no mitosis was observed; suspicious (+) if between 5.0 and 9.9%, and positive if between 10.0 and 19.9% (+), 20.0 and 49.9% (++) or more than 50.0% (+++). When no reasonable dose response was obtained, additional experiments with different doses were carried out to confirm its reproducibility. Results
Incidences of polyploid cells and chromosomal aberrations in control cells The incidence of polyploid cells and chromosomal aberrations observed in untreated cells and cells treated only with the solvents used for the dilution of chemicals are shown in Table I. No incidence was more than 2.0%, indicating t h a t there are no significant increases in numbers of polyploid cells or of chromosomal aberrations after treatment by solvent alone. Screening data of chromosome tests on 134 compounds The manes of compounds tested, their origins, solvents used for dilutioPL, doses and time for maximal effects, type of aberration and the final judgement according to our criteria, are listed in Table II. For convenience, the compounds are arranged alphabetically and divided into 3 groups; negative (N), suspicious (S) and positive (P), from the results obtained in chromosome tests. The last column shows whether the c o m p o u n d was mutagenic in bacteria of carcinogenic in animals.
340 TABLE I INCIDENCE OF POLYPLOID CELLS AND CELLS WITH CHROMOSOMAL CELLS OF DIFFERENT CONTROL GROUPS
Group
Untreated Saline Ethanol DMSO Albumin bovine
Final dose
--1.0% 0.5% 0.2%
ABERRATIONS
IN CHL
P o l y p l o i d c e l l s (%)
C h r o m o s o m a l aberration (%) a
24 h
4a h
24 h
48 h
0.8 1.0 0.4 0.8 NT
0.8 0.8 0.6 0.0 0.8
0.9 0.8 1.4 1.0 NT
1.1 0.6 1.2 1.0 1.8
+- 0 . 8 -+ 0 . 7 +- 0 . 5 ,+ 1 . 0
± 0.6 ± 0.7 -+ 0 . 5 -+ 0 . 0 ,+ 0 . 7
± 0.8 -+ 0 . 4 -+ 0 . S +- 0 . 9
-+ 1 . 0 -+ 0 . 8 ,+ 0 . 7 + 0.6 -+ 1 . 2
a M e a n _+ S . D . b N T , n o t tested.
Compounds negative in our chromosome tests (N group) Among 134 compounds tested, 63 were negative. Because of different cytotoxicities of each compound, the doses to which cells were exposed were variable: e.g. 0.004 mg/ml (0.1 X 10 -4 M) for ethynylestradiol (N-31) to the maximum 15.0 mg/ml for dextran (N-20). Even at such high doses that the cells were about to be killed, no significant increases of chromosomal aberration were detected, although cytolysis as well as karyorrhexis were frequently observed. This group included 9 compounds known to be mutagens or carcinogens. Amoung these false negative compounds, however, dibutylnitrosamine (N-22), dimethylnitrosamine (N-27), 2-methyl-4-dimethylaminoazobenzene (N-40), quinoline (N-57) and 4-o-tolylazo-o-toluidine (N-61) are mutagenic to bacteria only when they are activated by metabolic enzymes. Carcinogenic ethynylestradiol (N-31) and Tween 60 (N-62) were both negative in our test and in mutation assays with bacteria. Nitrofurazone (N-43) and phenanthrene (N-45) were negative, b u t they were both carcinogenic and mutagenic. Compounds suspicious in our chromosome tests (S group) Seventeen compounds were ranked in this group. According to our criteria they were n o t judged to be negative, because they produced more than 5.0% of chromosomal aberration, and translocation type of aberrations were more frequently observed as compared with those observed in the cells of controls and the N-group. Among these, 3 compounds such as 7-benzene hexachloride (S-2), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) (S-9) and 3-hydroxy-anthranilic acid (S-10) are included, which have been shown to be carcinogenic. The AF-2, known as a potential mutagen, was effective at a dose as low as 0.01 mg/ml (0.4 X 10 -4 M) which induced mainly chromatid gaps only 6 h after treatment. Amaranth (S-l), butylphthalyl-butylglycollate (S-5), dibutylphthalate (S-8) and phenacetin (S-14) were slightly positive, but they were not thoroughly investigated for their carcinogenicity, although they were negative in mutation assays with bacteria. Compounds positive in our chromosome tests (P group) Fifty-four out of 134 compounds were positive in our tests. Almost all Nnitroso compounds that have been proved to be both carcinogenic and muta-
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Mutation Research, 48 (1977) 337--354
© Elsevier/North-Holland Biomedical Press
CHROMOSOME TESTS WITH 134 COMPOUNDS ON CHINESE HAMSTER CELLS IN VITRO -- A SCREENING FOR CHEMICAL CARCINOGENS
MOTOI ISHIDATE, Jr. and SHIGEYOSHI ODASHIMA Department of Chemical Pathology, National Institute of Hygienic Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158 (Japan)
(Received July 7th, 1976) (Revision received December 12th, 1976) (Accepted December 14th, 1976)
Summary Chromosomal aberration tests in vitro were carried out on Chinese hamster cells grown in culture with various chemicals, including carcinogenic N-nitroso compounds and their related derivatives, food additives, medical drugs, pesticides and other chemicals c o m m o n l y used in laboratories or industries. Sixty-three of the 134 chemicals gave negative results in our test system even with doses at which the cell growth was markedly inhibited. Nearly all compounds known to be mutagenic in bacteria were also positive in our tests. Both urethane and diethylstilbestrol were positive, even though they are known to be carcinogenic but not mutagenic in bacteria. Compounds such as N-alkyl-N'nitroguanidines, barbital, sodium benzoate, saccharin sodium, sodium nitrite, sodium nitrate and 4-aminoquinoline-l-oxide were positive in our chromosome tests, but they have n o t been conclusively tested for their carcinogenicity.
Introduction The detection of chemicals that may pose a genetic hazard in our environment is now both feasible and necessary [10,14]. Several assay systems with rapid and reliable methods for this purpose have been introduced: mutation assays in microorganisms [1,5,15,16], cytogenetic assays in both plant [6] and mammalian cells in vivo as well as in vitro [3,8,9,12]. These assay systems can also be applied as a screening probe for the detection of possible carcinogenic substances in our environment. This is based on the premise that neoplastic changes may be due to somatic m u t a t i o n in the cells that have been exposed to certain mutagens. There is also evidence that most carcinogens are mutagenic if the compounds undergo metabolic activation by microsomal enzymes in the bacterial assay system [1]. Carcinogens may be regarded as potential mutagens;
338 however not all mutagens are carcinogenic. At present such assay systems cannot replace animal tests for carcinogenicity in vivo. It is important, however, to devise a rapid screening method to detect possible environmental carcinogens which can later be affirmed with tests in vivo. This report presents our screening data on 134 chemicals, concerning the induction of chromosomal aberrations in Chinese hamster cells grown in culture, and data obtained from mutation assays in bacteria, which were carried our at the Dept. of Microbiol. of our institute using the same compounds. Information on their carcinogenic activities was obtained from data from the Natl. Inst. of Health, U.S.A. [13]. Materials and methods
Chemicals tested Compounds listed in Table II were supplied in part from the project teams supported by the Ministry of Health and Welfare, Japan (indicated as W in the table) [11]. Others were supplied from various departments of our institute (S, C, M, F or Ph), Tokyo Biochemical Research Institute, T o k y o (T), or Shizuoka Pharmaceutical College, Shizuoka, Japan (U). A few samples were obtained commercially (P). The purity of most of these chemicals was checked at the Dept. of Drugs of our institute. All samples were kept in the dark in a refrigerator until used. Solvents used for the dilution of the compounds were physiological saline (S), ethanol (E), aqueous solution of albumin bovine (A) or DMSO (D) as indicated in the table. Cell cultures A clonal sub-line of a Chinese hamster fibroblast cell line (CHL) was used. This cell line was originally established from the lung of a young adult by Dr. T. Utakoji, Cancer Institute, Tokyo. The karyotype consisted of 25 chromosomes. The cells had been maintained by 5-day passages and grown in a monolayer in petri dishes with Eagle's MEM (GIBCO F-11) supplemented with 10% calf serum. Their doubling time was estimated as 18.2 h at their exponential growth at 37°C in a 5% CO2 atmosphere. Estimation of the 50% growth inhibition dose of each sample Growth inhibition tests were carried out on each sample before the chromosome tests were started. The 50% inhibition dose was estimated as follows. Several different doses of each agent were separately added to the 3-day-old cultures (about 6 × 103 cells/3-cm dish}. The doses were prepared by a factor of 2 from the maximal dose, estimated from the data on LDs0, which appeared in references for each agent. The cells in a monolayer were washed, fixed with 10% formalin solution, and then stained with 0.1% crystal violet solution for 3 min. After washing and drying each dish was placed under a photodensitometer, Model CDM-X, which was designed in our laboratory and is being manufactured by Olympus Co., Japan, to measure the color absorption values from which relative cell densities on the dishes were easily calculated. The color absorption values obtained by this apparatus reflected well the actual number of cells that survived at the b o t t o m of each petri dish.
339
Chromosome test Three different doses, including the 50% inhibition dose of each agent, which was estimated by the growth inhibition test described above, were prepared and separately added to 3-day-old cultures (about 10 s cells/6-cm dish). Chromosome preparations were made, as a rule at 24 and 48 h, or when necessary at 6 h, after the treatment. Cells were treated with colcemid (0.2 pg/ml) for 2 h, and after trypsinization, they were incubated in 0.075 M KC1 hypotonic solution for 15 rain at 37°C. The cells were fixed with ice-cold fixative (methanol : glacial acetic acid, 3 : 1 v/v) which was changed 3 times. A few drops of the suspension were then placed on clean dry slides which were held horizontally under an electric heater. The slides were stained with 1% Giemsa's buffered solution (pH 6.8) for 20 min. The number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases at the magnification of 700. Types of aberration were classified into 5 groups: chromatid gaps (g), chromatid breaks (b), chromatid or chromosomal translocation (t), ring formation (r) and fragmentation or pulverization (f). Breaks less than the width of a sister chromatid were designated as gaps in our criteria. The incidence of polyploid cells was also calculated. Untreated cells and cells treated only with solvent served as controls. Evaluation of effects as the final judgement CHL cells c o m m o n l y have less than 3.0% cells with chromosomal aberrations. Therefore, the final judgement given to all experimental groups was as follows. Negative (--) if less than 4.9% of the aberration was detected even when doses of t h e agent were elevated to sub-lethal amounts, where almost no mitosis was observed; suspicious (+) if between 5.0 and 9.9%, and positive if between 10.0 and 19.9% (+), 20.0 and 49.9% (++) or more than 50.0% (+++). When no reasonable dose response was obtained, additional experiments with different doses were carried out to confirm its reproducibility. Results
Incidences of polyploid cells and chromosomal aberrations in control cells The incidence of polyploid cells and chromosomal aberrations observed in untreated cells and cells treated only with the solvents used for the dilution of chemicals are shown in Table I. No incidence was more than 2.0%, indicating t h a t there are no significant increases in numbers of polyploid cells or of chromosomal aberrations after treatment by solvent alone. Screening data of chromosome tests on 134 compounds The manes of compounds tested, their origins, solvents used for dilutioPL, doses and time for maximal effects, type of aberration and the final judgement according to our criteria, are listed in Table II. For convenience, the compounds are arranged alphabetically and divided into 3 groups; negative (N), suspicious (S) and positive (P), from the results obtained in chromosome tests. The last column shows whether the c o m p o u n d was mutagenic in bacteria of carcinogenic in animals.
340 TABLE I INCIDENCE OF POLYPLOID CELLS AND CELLS WITH CHROMOSOMAL CELLS OF DIFFERENT CONTROL GROUPS
Group
Untreated Saline Ethanol DMSO Albumin bovine
Final dose
--1.0% 0.5% 0.2%
ABERRATIONS
IN CHL
P o l y p l o i d c e l l s (%)
C h r o m o s o m a l aberration (%) a
24 h
4a h
24 h
48 h
0.8 1.0 0.4 0.8 NT
0.8 0.8 0.6 0.0 0.8
0.9 0.8 1.4 1.0 NT
1.1 0.6 1.2 1.0 1.8
+- 0 . 8 -+ 0 . 7 +- 0 . 5 ,+ 1 . 0
± 0.6 ± 0.7 -+ 0 . 5 -+ 0 . 0 ,+ 0 . 7
± 0.8 -+ 0 . 4 -+ 0 . S +- 0 . 9
-+ 1 . 0 -+ 0 . 8 ,+ 0 . 7 + 0.6 -+ 1 . 2
a M e a n _+ S . D . b N T , n o t tested.
Compounds negative in our chromosome tests (N group) Among 134 compounds tested, 63 were negative. Because of different cytotoxicities of each compound, the doses to which cells were exposed were variable: e.g. 0.004 mg/ml (0.1 X 10 -4 M) for ethynylestradiol (N-31) to the maximum 15.0 mg/ml for dextran (N-20). Even at such high doses that the cells were about to be killed, no significant increases of chromosomal aberration were detected, although cytolysis as well as karyorrhexis were frequently observed. This group included 9 compounds known to be mutagens or carcinogens. Amoung these false negative compounds, however, dibutylnitrosamine (N-22), dimethylnitrosamine (N-27), 2-methyl-4-dimethylaminoazobenzene (N-40), quinoline (N-57) and 4-o-tolylazo-o-toluidine (N-61) are mutagenic to bacteria only when they are activated by metabolic enzymes. Carcinogenic ethynylestradiol (N-31) and Tween 60 (N-62) were both negative in our test and in mutation assays with bacteria. Nitrofurazone (N-43) and phenanthrene (N-45) were negative, b u t they were both carcinogenic and mutagenic. Compounds suspicious in our chromosome tests (S group) Seventeen compounds were ranked in this group. According to our criteria they were n o t judged to be negative, because they produced more than 5.0% of chromosomal aberration, and translocation type of aberrations were more frequently observed as compared with those observed in the cells of controls and the N-group. Among these, 3 compounds such as 7-benzene hexachloride (S-2), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) (S-9) and 3-hydroxy-anthranilic acid (S-10) are included, which have been shown to be carcinogenic. The AF-2, known as a potential mutagen, was effective at a dose as low as 0.01 mg/ml (0.4 X 10 -4 M) which induced mainly chromatid gaps only 6 h after treatment. Amaranth (S-l), butylphthalyl-butylglycollate (S-5), dibutylphthalate (S-8) and phenacetin (S-14) were slightly positive, but they were not thoroughly investigated for their carcinogenicity, although they were negative in mutation assays with bacteria. Compounds positive in our chromosome tests (P group) Fifty-four out of 134 compounds were positive in our tests. Almost all Nnitroso compounds that have been proved to be both carcinogenic and muta-
341 o
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