- Email: [email protected]
A method to detect tumors and presumed somatic mutations in mice
131
Cancer Letters, 18 (1983) 131-135 Elsevier Scientific Publishers Ireland Ltd.
A METHOD TO DETECT MUTATIONS IN MICE
TUMORS AND PRESUMED
SOMATIC
TAISEI NOMURA*, KEIKO SHIBATA and SHINSUKE HATA Institute for Cancer Research and Department of Fundamental Radiology, Osaka University Medical School, l-l-50, Fukushima, Fukushima-ku, Osaka 553 (Japan) (Received 15 September 1982) (Revised version received 9 November 1982) (Accepted 24 November 1982)
SUMMARY
A method was tested to detect both tumors and somatic mutations in mice. When HT-A/J F1 embryos were treated with ethylnitrosourea on day 11 of gestation, a single injection was enough to induce tumors and presumed somatic mutations of coat color in significantly high incidence. Urethane, treated in a similar way, induced somatic mutations and malformations in the offspring. Tumor incidence was also increased but was not significantly different from controls. Treatment on day 13 was too late to induce somatic mutations and malformations, while high incidence of tumors were induced.
INTRODUCTION
Although several investigators have reported that the initial process of carcinogenesis is closely related to mutagenesis [2-4,7,11], such studies are based on data from micro-organisms which do not develop cancer. To analyse the mechanism of carcinogenesis more accurately, it is important to detect somatic mutations in mammals and further to establish a method to detect both cancer and somatic mutations in the same body of animals. In this paper, we tested such a method using mice with mutant coat-color genes. MATERIALS
AND METHODS
HT and A/J mice were used. HT was kindly provided by Drs. M.F. Lyon and A.G. Searle (Radiobiology Unit,, Medical Research Council, Harwell, U.K.) and A/J by Dr. E.S. Russell (Jackson Laboratory, Bar Harbor, ME, *To whom all reprint requests should be addressed. 0304-3835/83/000+0000/$03.00 0 1983 Elsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland
132
U.S.A.) in 1978. HT is homozygous for the following recessives: a (nonagouti); bp (brachypodism);pa (pallid); fz (fuzzy); In (1eaden);pe (pearl). A/J is a tumor-susceptible strain and homozygous for the following recessive alleles: a, b (brown); c (albino). HT and A/J mice were maintained with mouse diet CRF-1 (Charles River Japan, Kanagawa, Japan) in a complete barrier system at 21--23&C. An estrous HT female was mated with A/J males in the evening, and the next morning, a vaginal plug was checked to determine day 1 of gestation [ 91. Mice were exposed to light from 4 : 00 h to 18: 00 h, which resulted in ovulation at about 2 : 00 h [ 11, indicating that fertilization occurs at about 2 : 00 h [l] of day 1. Pregnant HT received a single subcutaneous injection of 1.0 mg of urethane (ethyl carbamate, Wako Pure Chemical Ind., Osaka, Japan) per g of body weight, or received a single intraperitoneal injection of 30 pg of N-ethyl-N-nitrosourea (ENU) (Sigma Chemical Co., St. Louis, MO) per g of body weight at 14 : 00 h of day 11. Consequently, F1 embryos at 10.5 days after fertilization were treated with urethane or ENU. This stage of the embryo was selected for treatment, because day 11 is an optimum stage for detecting presumed somatic mutations [ 5,8,10] and is the earliest stage for detecting tumors in the lung and liver in mice [ 6,9]. Furthermore, day 11 is a critical stage for the induction of malformation [6]. At later stages, it becomes difficult to detect somatic mutations [ 51 and malformations [ 61, while tumors are induced in higher incidence [ 6,9]. To confirm it, pregnant mice were treated with 1.0 mg/g of urethane on day 13. F1 embryos are heterozygous for 5 recessive coat color loci, b/+, c/+, In/+, pa/+ and pe/+ , and homozygous for a. When forward mutation or deletion is induced in the wild allele, presumed somatic mutation is detected as a colored spot on the black coat, which derived from a mutated pigment cell [ 5,8,10], F, offspring were examined for spots and malformations every week and killed 1 year after birth to examine neoplasms. Specimens were submitted to microscopical examinations. Malformations of skeleton and internal organs were also examined at the time of killing. Skeletal anomalies were examined by soft X-ray apparatus (SOFRON, Tokyo, Japan). RESULTS
AND DISCUSSION
Results are summarized in Table 1. When urethane was given on day 11, significant numbers of colored spots (P < 0.02) and malformations (P < 0.05) were induced in the offspring. Although an increased incidence of tumors was observed, there were no statistical differences from the control value. Treatment with urethane on day 13 resulted in high incidence of tumors (P < O.OOl), but did not induce significant numbers of colored spots and malformations in the offspring. Certainly, day 11 seems an optimum stage for detecting presumed somatic mutations, as is the case with different mutagens [ 5,8,10], and the earliest stage for detecting tumors [ 6,9]. Day 13 is later for detecting somatic mutations [ 51 and malformations [ 6,9]. If
1
43 (4.8)
94 (5.9)
17 (16)
37 (9)
16 (16)
13
11
14/138’ P < l/98 NS 2142 NS 2187 (2.3)
(4.8)
(10.1) 0.05 (1.0)
2PD
2 T
8 T, 5 PD. 1 DW 1 T
17/138 P c l/98 NS 11/42e P < 2187
(96)
spots
(26.2) 0.001 (2.3)
(12.3) 0.02 (1.0)
Incidence
Incidence
(%)
Colored
(0.0)
(2.2)
0,:: (0.0) NS 1187 (1.1)
31138 0,::
Incidence
Tumors
32/138 P + 36/87d P < 16/40d P < 12187
(23.2) 0.085 (41.4) 0.001 (40.0) 0.002 (13.8)
(96)
MUTATIONS
(%a) Incidence
SOMATIC
WMVS
AND PRESUMED
Malformations Details
MALFORMATIONS,
10 LT. 3 He 2 G,l IT.1 SpT 12 LT. 1 He
27 LT. 4 He. 1 ST, 1 OC 30 LT. 8 He
Details
PER SURVIVORS
Incidence of tumors malformations, and presumed somatic mutations was given by the numbers of affected offspring per survivors. Statistical analysis was performed by xz -test against untreated controls. x2-test was applied with Yates’ correction when expected value in a cell was less than 5. The abbreviations used are. WMVS, white midventral spots caused by kiIIing or specific division delay of melanoblasts [ 5,101: T, tail anomaly; PD. polydactyly; DW, dwarf: LT. lung tumor (adenoma and adenocarcinoma); He. hepatoma; ST, squamous ceII carcinoma of the stomach: OC, ovarian cystoma; G, granulosa cell tumor: IT. adenocarcinoma of the ileum; SpT, splenic tumor. Some mice developed 2 kinds of tumors. aNos. of pregnant mice which delivered live offspring. The remainder resulted in abortion or cannibalism bAverage nos. of live births per pregnant mouse which delivered live offspring. ‘Nos. of offspring which survived more than 4 weeks. Differenre from live births shows neonates which died a few days after birth. dSeverd mice died before killing at 1 year of age. eOne developed 2 colored spots.
111 (6.9)
153 (5.7)
40 (27)a
11
Live births No. (Av.)b
Urethane (1.0 mglg) Urethane (1.0 mglg) ENU (30 wglg) None
No. of pregnant mice
Treated day
Carcinogen (dose)
INCIDENCE OF HT-A/J F, OFFSPRING BEARING TUMORS. AFTER PRENATAL EXPOSURE TO URETHANE OR ENU
TABLE
134 embryos are treated continuously on day 11 and after, both tumors and somatic mutations will be more effectively induced. When a potent carcinogen ENU was given on day 11, a large number of pregnancies resulted in abortion and cannibalism. However, a single injection was enough to induce both tumors and somatic mutations in significantly high incidence (P < 0.002 and P < 0.001, respectively). The incidence of malformations was not significantly increased by ENU from the control value, although urethane induced significant numbers of polydactylies and tail anomalies (P< 0.05). This is due to the fact that the dose of ENU (30 pg/g) was less than the threshold dose for teratogenicity, because high incidence of malformations are observed with doses more than 50 pg/g (Nomura et al., unpublished data), while that-of urethane was 0.8 mg/g
[61. Using this method, we can exclude the contradiction of using bacteria for studying carcinogenesis in animals and avoid the problems of using different strains of mice for detecting mutation and cancer. This is critical for assessing accurately the relationship between mutagenesis and carcinogenesis. Furthermore, the method is useful for studying mutagenesis, teratogenesis and carcinogenesis, because we can detect all of them at the same time by 1 experiment. Further improvement is elucidated for detecting mutations and tumors which derive not only from the same body but also from the same cell. ACKNOWLEDGEMENTS
We thank Drs. M.F. Lyon and A.G. Searle for providing the HT strain, Dr. J.F. Crow for his support to carry out this project, Drs. Y. Sakamoto and S. Kondo for their encouragement, and E. Tada and M. Fujita for their assistance. The work was supported by the Isabelle DeCazes de Neue Prize, by grants from the Japanese Ministry of Education, Culture, and Science and from the Osaka Cancer Society, and by the Mitsuhisa Memorial Cancer Research Fund. REFERENCES Hoppe, P.C. (1975) Fertilizing ability of mouse sperm from different epididymal regions and after washing and centrifugation. J. Exp. Zool., 192, 219-222. Kakunaga, T. (1975) Caffeine inhibits cell transformation by 4nitroquinoline loxide, Nature (London), 258,248-250. Kondo, S. (1977) A test for mutation theory of cancer: carcinogenesis by misrepair of 4-nitroquinoline l-oxide DNA damage. Br. J. Cancer, 35, 595-601. McCann, J.E., Yamasaki, C.E. and Ames, B.N. (1975) Detection of carcinogens as mutagens in the Salmonelia/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A., 72,5135-5139. NeuhCuser-Klaus, A. (1981) An approach towards the standardization of the mammalian spot test. Arch Toxicol., 48, 229-243.
135 6 Nomura, T. (1974) An analysis of the changing urethan response of the developing mouse embryo in relation to mortality, malformation and neoplasm. Cancer Res., 34,2217-2231. 7 Nomura, T. (1978) Mutagenesis, teratogenesis and carcinogenesis; evidence obtained by caffeine post-treatment after carcinogens. In: Tumors of Early Life in Man and Animals, pp. 821-841. Editor: L. Severi. Grafica de Salvi & Co., Perugia, Italy. 8 Nomura, T. (1982) Linear dose-response relationship of urethan-induced somatic mutations in PT-HT F, mice. 3rd ICEM, 96, September 20-23,1981, Tokyo, Japan. 9 Nomura, T. and Okamoto, E. (1972) Transplacental carcinogenesis by urethan in mice; teratogenesis and carcinogenesis in relation to organogenesis. Gann, 63, 7 31742. 10 Russell, L.B. and Major, M.H. (1957) Radiation-induced presumed somatic mutations in the house mouse. Genetics, 42,161-175. 11 Zajdela, F. and Latarjet, R. (1973) Effect inhibiteur de la cafeine sur l’induction de cancers cutanes par les rayons ultraviolets chez la souris. C.R. Hebd. Seances Acad. Sci., Ser. D. 277,1073-1076.
Cancer Letters, 18 (1983) 131-135 Elsevier Scientific Publishers Ireland Ltd.
A METHOD TO DETECT MUTATIONS IN MICE
TUMORS AND PRESUMED
SOMATIC
TAISEI NOMURA*, KEIKO SHIBATA and SHINSUKE HATA Institute for Cancer Research and Department of Fundamental Radiology, Osaka University Medical School, l-l-50, Fukushima, Fukushima-ku, Osaka 553 (Japan) (Received 15 September 1982) (Revised version received 9 November 1982) (Accepted 24 November 1982)
SUMMARY
A method was tested to detect both tumors and somatic mutations in mice. When HT-A/J F1 embryos were treated with ethylnitrosourea on day 11 of gestation, a single injection was enough to induce tumors and presumed somatic mutations of coat color in significantly high incidence. Urethane, treated in a similar way, induced somatic mutations and malformations in the offspring. Tumor incidence was also increased but was not significantly different from controls. Treatment on day 13 was too late to induce somatic mutations and malformations, while high incidence of tumors were induced.
INTRODUCTION
Although several investigators have reported that the initial process of carcinogenesis is closely related to mutagenesis [2-4,7,11], such studies are based on data from micro-organisms which do not develop cancer. To analyse the mechanism of carcinogenesis more accurately, it is important to detect somatic mutations in mammals and further to establish a method to detect both cancer and somatic mutations in the same body of animals. In this paper, we tested such a method using mice with mutant coat-color genes. MATERIALS
AND METHODS
HT and A/J mice were used. HT was kindly provided by Drs. M.F. Lyon and A.G. Searle (Radiobiology Unit,, Medical Research Council, Harwell, U.K.) and A/J by Dr. E.S. Russell (Jackson Laboratory, Bar Harbor, ME, *To whom all reprint requests should be addressed. 0304-3835/83/000+0000/$03.00 0 1983 Elsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland
132
U.S.A.) in 1978. HT is homozygous for the following recessives: a (nonagouti); bp (brachypodism);pa (pallid); fz (fuzzy); In (1eaden);pe (pearl). A/J is a tumor-susceptible strain and homozygous for the following recessive alleles: a, b (brown); c (albino). HT and A/J mice were maintained with mouse diet CRF-1 (Charles River Japan, Kanagawa, Japan) in a complete barrier system at 21--23&C. An estrous HT female was mated with A/J males in the evening, and the next morning, a vaginal plug was checked to determine day 1 of gestation [ 91. Mice were exposed to light from 4 : 00 h to 18: 00 h, which resulted in ovulation at about 2 : 00 h [ 11, indicating that fertilization occurs at about 2 : 00 h [l] of day 1. Pregnant HT received a single subcutaneous injection of 1.0 mg of urethane (ethyl carbamate, Wako Pure Chemical Ind., Osaka, Japan) per g of body weight, or received a single intraperitoneal injection of 30 pg of N-ethyl-N-nitrosourea (ENU) (Sigma Chemical Co., St. Louis, MO) per g of body weight at 14 : 00 h of day 11. Consequently, F1 embryos at 10.5 days after fertilization were treated with urethane or ENU. This stage of the embryo was selected for treatment, because day 11 is an optimum stage for detecting presumed somatic mutations [ 5,8,10] and is the earliest stage for detecting tumors in the lung and liver in mice [ 6,9]. Furthermore, day 11 is a critical stage for the induction of malformation [6]. At later stages, it becomes difficult to detect somatic mutations [ 51 and malformations [ 61, while tumors are induced in higher incidence [ 6,9]. To confirm it, pregnant mice were treated with 1.0 mg/g of urethane on day 13. F1 embryos are heterozygous for 5 recessive coat color loci, b/+, c/+, In/+, pa/+ and pe/+ , and homozygous for a. When forward mutation or deletion is induced in the wild allele, presumed somatic mutation is detected as a colored spot on the black coat, which derived from a mutated pigment cell [ 5,8,10], F, offspring were examined for spots and malformations every week and killed 1 year after birth to examine neoplasms. Specimens were submitted to microscopical examinations. Malformations of skeleton and internal organs were also examined at the time of killing. Skeletal anomalies were examined by soft X-ray apparatus (SOFRON, Tokyo, Japan). RESULTS
AND DISCUSSION
Results are summarized in Table 1. When urethane was given on day 11, significant numbers of colored spots (P < 0.02) and malformations (P < 0.05) were induced in the offspring. Although an increased incidence of tumors was observed, there were no statistical differences from the control value. Treatment with urethane on day 13 resulted in high incidence of tumors (P < O.OOl), but did not induce significant numbers of colored spots and malformations in the offspring. Certainly, day 11 seems an optimum stage for detecting presumed somatic mutations, as is the case with different mutagens [ 5,8,10], and the earliest stage for detecting tumors [ 6,9]. Day 13 is later for detecting somatic mutations [ 51 and malformations [ 6,9]. If
1
43 (4.8)
94 (5.9)
17 (16)
37 (9)
16 (16)
13
11
14/138’ P < l/98 NS 2142 NS 2187 (2.3)
(4.8)
(10.1) 0.05 (1.0)
2PD
2 T
8 T, 5 PD. 1 DW 1 T
17/138 P c l/98 NS 11/42e P < 2187
(96)
spots
(26.2) 0.001 (2.3)
(12.3) 0.02 (1.0)
Incidence
Incidence
(%)
Colored
(0.0)
(2.2)
0,:: (0.0) NS 1187 (1.1)
31138 0,::
Incidence
Tumors
32/138 P + 36/87d P < 16/40d P < 12187
(23.2) 0.085 (41.4) 0.001 (40.0) 0.002 (13.8)
(96)
MUTATIONS
(%a) Incidence
SOMATIC
WMVS
AND PRESUMED
Malformations Details
MALFORMATIONS,
10 LT. 3 He 2 G,l IT.1 SpT 12 LT. 1 He
27 LT. 4 He. 1 ST, 1 OC 30 LT. 8 He
Details
PER SURVIVORS
Incidence of tumors malformations, and presumed somatic mutations was given by the numbers of affected offspring per survivors. Statistical analysis was performed by xz -test against untreated controls. x2-test was applied with Yates’ correction when expected value in a cell was less than 5. The abbreviations used are. WMVS, white midventral spots caused by kiIIing or specific division delay of melanoblasts [ 5,101: T, tail anomaly; PD. polydactyly; DW, dwarf: LT. lung tumor (adenoma and adenocarcinoma); He. hepatoma; ST, squamous ceII carcinoma of the stomach: OC, ovarian cystoma; G, granulosa cell tumor: IT. adenocarcinoma of the ileum; SpT, splenic tumor. Some mice developed 2 kinds of tumors. aNos. of pregnant mice which delivered live offspring. The remainder resulted in abortion or cannibalism bAverage nos. of live births per pregnant mouse which delivered live offspring. ‘Nos. of offspring which survived more than 4 weeks. Differenre from live births shows neonates which died a few days after birth. dSeverd mice died before killing at 1 year of age. eOne developed 2 colored spots.
111 (6.9)
153 (5.7)
40 (27)a
11
Live births No. (Av.)b
Urethane (1.0 mglg) Urethane (1.0 mglg) ENU (30 wglg) None
No. of pregnant mice
Treated day
Carcinogen (dose)
INCIDENCE OF HT-A/J F, OFFSPRING BEARING TUMORS. AFTER PRENATAL EXPOSURE TO URETHANE OR ENU
TABLE
134 embryos are treated continuously on day 11 and after, both tumors and somatic mutations will be more effectively induced. When a potent carcinogen ENU was given on day 11, a large number of pregnancies resulted in abortion and cannibalism. However, a single injection was enough to induce both tumors and somatic mutations in significantly high incidence (P < 0.002 and P < 0.001, respectively). The incidence of malformations was not significantly increased by ENU from the control value, although urethane induced significant numbers of polydactylies and tail anomalies (P< 0.05). This is due to the fact that the dose of ENU (30 pg/g) was less than the threshold dose for teratogenicity, because high incidence of malformations are observed with doses more than 50 pg/g (Nomura et al., unpublished data), while that-of urethane was 0.8 mg/g
[61. Using this method, we can exclude the contradiction of using bacteria for studying carcinogenesis in animals and avoid the problems of using different strains of mice for detecting mutation and cancer. This is critical for assessing accurately the relationship between mutagenesis and carcinogenesis. Furthermore, the method is useful for studying mutagenesis, teratogenesis and carcinogenesis, because we can detect all of them at the same time by 1 experiment. Further improvement is elucidated for detecting mutations and tumors which derive not only from the same body but also from the same cell. ACKNOWLEDGEMENTS
We thank Drs. M.F. Lyon and A.G. Searle for providing the HT strain, Dr. J.F. Crow for his support to carry out this project, Drs. Y. Sakamoto and S. Kondo for their encouragement, and E. Tada and M. Fujita for their assistance. The work was supported by the Isabelle DeCazes de Neue Prize, by grants from the Japanese Ministry of Education, Culture, and Science and from the Osaka Cancer Society, and by the Mitsuhisa Memorial Cancer Research Fund. REFERENCES Hoppe, P.C. (1975) Fertilizing ability of mouse sperm from different epididymal regions and after washing and centrifugation. J. Exp. Zool., 192, 219-222. Kakunaga, T. (1975) Caffeine inhibits cell transformation by 4nitroquinoline loxide, Nature (London), 258,248-250. Kondo, S. (1977) A test for mutation theory of cancer: carcinogenesis by misrepair of 4-nitroquinoline l-oxide DNA damage. Br. J. Cancer, 35, 595-601. McCann, J.E., Yamasaki, C.E. and Ames, B.N. (1975) Detection of carcinogens as mutagens in the Salmonelia/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A., 72,5135-5139. NeuhCuser-Klaus, A. (1981) An approach towards the standardization of the mammalian spot test. Arch Toxicol., 48, 229-243.
135 6 Nomura, T. (1974) An analysis of the changing urethan response of the developing mouse embryo in relation to mortality, malformation and neoplasm. Cancer Res., 34,2217-2231. 7 Nomura, T. (1978) Mutagenesis, teratogenesis and carcinogenesis; evidence obtained by caffeine post-treatment after carcinogens. In: Tumors of Early Life in Man and Animals, pp. 821-841. Editor: L. Severi. Grafica de Salvi & Co., Perugia, Italy. 8 Nomura, T. (1982) Linear dose-response relationship of urethan-induced somatic mutations in PT-HT F, mice. 3rd ICEM, 96, September 20-23,1981, Tokyo, Japan. 9 Nomura, T. and Okamoto, E. (1972) Transplacental carcinogenesis by urethan in mice; teratogenesis and carcinogenesis in relation to organogenesis. Gann, 63, 7 31742. 10 Russell, L.B. and Major, M.H. (1957) Radiation-induced presumed somatic mutations in the house mouse. Genetics, 42,161-175. 11 Zajdela, F. and Latarjet, R. (1973) Effect inhibiteur de la cafeine sur l’induction de cancers cutanes par les rayons ultraviolets chez la souris. C.R. Hebd. Seances Acad. Sci., Ser. D. 277,1073-1076.