Carcinogenesis by Urethan Via Mother's Milk and Its Enhancement of Transplacental Carcinogenesis in Mice1

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[CANCER RESEARCH 33, 1677-1683, July 1973] Carcinogenesis by Urethan Via Mother's Milk and Its Enhancement of Transplacental Carcinogenesis in Mice1

Taisei Nomura2

First Department of Surgery, Osaka university Medicai School, Dojima-hamadori 3-1~2. Fukushima-ku, Osaka. 553. Japan

SUMMARY (29, 35). The author induced tumors in suckling mice whose mothers were treated with urethan during lactation (23), Pulmonary tumors were induced in mice that were and urethan was detected in mother's milk and also in the suckled by mothers that received urethan during lactation. blood, lung, and liver of sucklings (25). In this experiment, More induced tumors appeared in sucklings when urethan further investigation was conducted to induce tumors in was injected into lactating mothers soon after delivery. higher incidence. When pregnant mice were treated with urethan during Transplacental Carcinogenesis was studied by several gestation and lactation their offspring had a significantly investigators (1, 4, 10, 11, 14, 15, 21 25, 27, 31 37); how increased incidence of pulmonary tumors, and the number ever, the author also administered urethan to pregnant mice of tumors per mouse was compatible with the sum of on various days of gestation by single injection, utilizing its tumors in mice who received either prenatal or postnatal fast action and complete placental permeability. Tumors ap treatment. However, when prenatal urethan treatment was peared in the offspring only after the appearance of fetal coupled with postnatal exposure via mother's milk, off organ buds. Before the appearance of fetal organ buds, spring of mothers treated on Day 9 or 15 of gestation bore tumors did not appear, although there was a high incidence more tumors than the sum. These two days correspond to of malformation (23 25). Urethan, however, distributes the most sensitive stages to urethan for lung malformation itself equally in all organs, tissues, and body fluid of fetuses and Carcinogenesis. Induction of ovarian cystadenomas irrespective of the stage of pregnancy (25). Therefore, addi might also suggest that subtumorigenic effects of urethan on tional treatments with urethan via mother's milk were in embryonic cells of the developing female gonad on Day 11 stituted after transplacental exposure to urethan on various and 13 were made manifest by the postnatal exposure. days of gestation in order to induce more and different However, prenatal and postnatal exposure to urethan may tumors and malformations which were not induced by suppress the induction of lymphosarcomas (node type). transplacental exposure or postnatal exposure only. Mothers developed endometrial hyperplasias and uterine hemangiomas when treated during pregnancy. Additional MATERIALS AND METHODS treatment after delivery increased the incidence of uterine hemangiomas. Animals. ICR/Jcl female mice (Japan Central Labora tory for Experimental Animals, Tokyo, Japan), 10 to 12 INTRODUCTION weeks old and weighing 30 to 35 g were used. They were Recently, several chemical agents have been detected in placed in a cage with a breeder male, and a vaginal plug was mother's milk and their effects on newborns have been checked daily to determine Day 1 of gestation. Details were noted (2, 3, 6 8, 12, 13, 16 18, 25, 26, 28, 30). There have reported previously (23). been 2 reports on induction of tumors via mother's milk by Urethan. Crystalline ethyl carbamate, m.p. 48 51Â°,was DeBenedictis el al. (9) with urethan and by Mohr and used (Wako Pure Chemical Industries Ltd., Tokyo, Japan). Althoff (20) with diethylnitrosamine and dibutylnitrosa- A 10% solution of urethan in distilled water was prepared mine. However, these carcinogens were not detected in just before use. sucklings. Although 3-methylcholanthrene could be de Treatment during Pregnancy. Pregnant mice were given tected in mother's milk when administered during lactation urethan s.c., 1.0 mg/g body weight, on Days 7, 13, 15, or 17 (7, 8, 28), tumors were not induced significantly in sucklings of gestation. On Day 9 or 11, pregnant mice received urethan, 0.5 mg/g body weight because a high incidence of 1This is Paper 4 of the series entitled "Transplacental Carcinogene embryonal death was observed when 1.0 mg/g was adminis sis by Urethan in Mice." This investigation was supported by a grant-in-aid tered in these fetal stages (23). Control animals were treated for Cancer Research from the Japanese Ministry of Education. This report with distilled water only during pregnancy. was presented at the 29th and 31st Annual Meeting of the Japanese Cancer Treatment during Lactation. Lactating mothers were Association (1970 and 1972). 'Present address: Institute for Cancer Research, Osaka University given urethan s.c., 1.0 mg/g body weight as follows: on the Medical School. Dojima-hamadori 3-1~2, Fukushima-ku, Osaka, 553, 2nd day postpartum; on the 2nd, 4th, 6th, and 8th days Japan. postpartum; on the 2nd, 7th, 12th, and 17th days post Received November 20, 1972; accepted March 14, 1973. partum; or on the 14th, 16th, 18th, and 20th days post-

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1973 American Association for Cancer Research. Taisei Nomura partum. Offspring were suckled by these urethan-treated high incidence via mother's milk. The incidence was signifi mothers. The control was treated on the 2nd day post- cantly higher than the control (Table 2, t test A). The earlier partum with distilled water. the treatment while nursing, the greater the number of Treatment during Pregnancy and Lactation. Pregnant tumors induced in sucklings (t test B in Table 2). Ovarian mice were treated with urethan on the same time and dose cysts did not appear in sucklings via mother's milk. schedule as described above. The same mothers received 4 Fetuses transplacentally exposed to urethan were exposed additional exposures to urethan on the 2nd, 7th, 12th, and again postnatally during their suckling stage via mother's 17th days postpartum, and suckled their offspring while milk. Incidence of induced tumors is shown in Table 3. nursing. Pulmonary Tumors. Pulmonary tumors appeared in sig Mothers and their offspring were maintained on mouse nificantly higher incidence in offspring that had urethan via diet CA-1 (CLEA Japan, Tokyo, Japan) and water. Off mother's milk and also that had been exposed transplacen spring were separated from mothers 4 weeks after birth, and tally during their fetal stages than in those that were only females were maintained as virgins. They were sacrificed 32 transplacentally exposed (p < 0.01, Table 3). Tumors per weeks after birth, and gross pathological lesions were mouse were compatible with the sum of both prenatal examined. Specimens were fixed in 20% neutral formalde treatment and postnatal treatment. The offspring of moth hyde solution, and submitted to microscopic examinations. ers treated on Day 9 and postpartum or on Day 15 and In the case of the lungs, the number of pulmonary tumors postpartum, however, bore more tumors than the sum was counted after fixation. (Tables 1 to 3). Hepatomas. Postnatal exposure to urethan via mother's milk did not increase the incidence of hepatomas except in RESULTS the group treated on Day 9 and postpartum (Tables 1 and 3). Urethan injected into pregnant mice only after Day 11 of Ovarian Cysts. A few ovarian cysts developed in the gestation induced pulmonary tumors in significantly higher offspring of mothers treated on Day 13 of gestation. incidence in offspring (p < 0.01, Table 1). Hepatomas also However, there was no significant difference with the developed in the offspring of mothers treated from Days 11 control (p -=Â¡0.08,Table 1). When pregnant mice were to 15 of gestation. When urethan was injected into lactating treated on Day 11 or 13 of gestation and then additional treatments were given to their offspring via mother's milk, mothers, pulmonary tumors appeared in their sucklings in

Table I Tumors in the offspring of mothers receiving ureihan during pregnancy

in cyst Treated tumorsInci maleInci infemaleInci (day of ofoff gesta test(P)>0.7= tion)Day?Dayspring2253103395636212Pulmonarydence2/225/5315/10312/3936/5625/3611/212%9.19.414.630.864.369.45.2Tumors/mouse0.091dence0/140/262/543/202/280/180/101%0.00.03.715.07.10.00.0Ovariandence0/80/270/492/190/280/181/111%0.00.00.010.50.00.00.9 Â±0.019"0.1 9Day 32Â±0.0570.1 0.2<0.01< 11Day 94Â±0.0510.520 13Day Â±0.2001.460 0.001

Table 2 Tumors in the offspring of mothers receiving urethan during lactation

Pulmonary tumor

of test(p)A< Treateddayspostpartum22,4,6,82,7,off spring3410312741Incidence14/3410/1015/26"15/271/41%41.210057.755.62.4Tumors/mouse0.618

Â±0.158"13.600 0.001

'Mean Â±S.E. ' Five offspring were excluded for pneumonia.

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Table 3 Tumors in the offspring of mothers receiving ureihan during pregnancy and lactation

cyst TreatmentDay tumor%59.190.576.978.178.687.0Tumors/mouse1.364inmaleInci infemaleInci

of of gesta off tionDay spring222126352X23Incidence13/2219/2120/2625/32"22/2820/23Pulmonarydence0/82/81/153/151/170/13%0.025.06.720.05.90.0Ovariandence0/140/133/119/201/110/10%0.00.027.345.09.10.0 7Day Â±0.339Â°2.476 9Day Â±0.5061.385 0gland (Fig. and the latter to the most rapidly growing stage of fetal 3/4); another developed a malignant mesenchymoma in the bronchial elements (23). These facts suggest that embryonic small pelvic cavity (Fig. 4/Ã•);both conditions are very rare. cells just differentiating toward fetal lung buds are sensitive The former consisted mainly of adenocarcinoma which was to subteratogenic doses of urethan and the retained effects obviously malignant and metastasized to the liver. The are manifested by the additional exposure to urethan during squamous element was observed in every part (Fig. 3fi). The suckling periods. Rapidly growing fetal lung on Day 15 malignant mesenchymoma consisted of immature tissues, might receive the same effects. Ovarian cysts were not including 2 sarcomatous elements of different histogenesis, induced significantly by the prenatal exposure or postnatal reticulum cell sarcoma, and chondrosarcoma. A part of exposure only, and I have not been able to find any report of reticulum cell sarcoma (Fig. 4B) was established in tissue this tumor in experimental animals in the literature. A culture and has been serially transplanted. significantly high incidence of ovarian cysts, when animals

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Table 4 Tumors in mothers receiving urelhan during pregnancy and lactation

heman- hyperplasiaNo. giomaNo. cystNo. tumorNo.45 of TreatmentDuring mothers48 %14 %4 %3

pregnancy 29.15 8.3 6.2 During pregnancy 2225Endometrial22.72 522.71 313.60 224%93.710016.0 and lactation Control"No. 8.0Uterine 4.0Ovarian 0.0Pulmonary " Control mothers received distilled water only during pregnancy. were treated on Day 11 or 13 and postpartum, also suggests the Progeny. Proc. Soc. Exptl. Biol. Med., 135: 84 86, 1970. that subtumorigenic effects of urethan on embryonic cells of 5. Cividalli, G.. Mirvish, S. S., and Berenblum. I. The Catabolism of the genital ridge just differentiating toward the female Urethan in Young Mice of Varying Age and Strain, and in X- gonad are manifested by the postnatal exposure. The same irradiated Mice, in Relation to Urethan Carcinogenesis. Cancer Res.. was observed in induction of cystic kidneys on Day 11 of 25: 853 858, 1965. 6. Curley, A., and Kimbrough, R. Chlorinated Hydrocarbon Insecticides gestation. However, manifestation was not observed so in Plasma and Milk of Pregnant and Lactating Women. Arch. clearly in induction of hepatomas. It may be assumed that Environ. Health, 18: 156-164, 1969. transplacental exposure to urethan from Days 11 to 15 is a 7. Dao, T. L. Studies on Mechanism of Carcinogenesis in the Mammary major cause of hepatocarcinogenesis in mice. These fetal Gland. Progr. Exptl. Tumor Res.. //. 235 261, 1969. days correspond to the rapidly growing stage of fetal liver 8. Dao, T. L., Bock, F. G., and Crouch, S. Level of 3-methylcholanthrene (23). This finding in hepatomas is opposite to that of in Mammary Glands of Rats after Intragastric Instillation of Carcino Vesselinovitch (37), possibly because of the smaller dose of gen. Proc. Soc. Exptl. Biol. Med., 102: 635 638, 1959. urethan exposed postnatally in this experiment. Contrary to 9. DeBenedictis, G., Maiorano, G., Chieco-Bianchi, L., and Fiore- the results with the above-mentioned tumors, induction of Donati. L. Lung Carcinogenesis by Urethane in Newborns, Sucklings, lymphosarcomas (node type) was suppressed in the off and Adult Swiss Mice. Brit. J. Cancer, 16: 686 689, 1962. 10. Druckrey, H., Ivankovic, S., and Preussmann, R. Teratogenic and spring by the prenatal and postnatal exposure to urethan. Carcinogenic Effects in the Offspring after Single Injection of Exposure to urethan in these stages might suppress the Ethylnitrosourea to Pregnant Rats. Nature, 210: 1378 1379, 1966. development of lymphocytes. If dysgenesis of lymphatic 11. Druckrey, H., Ivankovic, S., Preussmann, Râ€žLandschutz,C., Stekar, apparatus might result in the defective change of im- J.. Brunnar, U.. and Schagen, B. Transplacentar Induction of Neuro- munosurveillance, it might be a cause of enhancement of genie Malignomas by 1,2-Diethyl-hydrazine, Azo-, and Azoxy-ethane carcinogenesis in other organs and tissues. in Rats. Experientia, 24: 561-562, 1968. As for the mothers, it is a specific finding that tumors are 12. Havelka, J., Hejzlar, M., Popov, V., Viktorinova, D., and Prochazka, observed commonly in reproductive organs. These organs J. Excretion of Chloramphenicol in Human Milk. Chemotherapy, 13: 204-211, 1968. may be susceptible to urethan during pregnancy and 13. Jukes, T. H. DDT in Mother's Milk. Nature, 226: 194, 1970. lactation. 14. Klein, M. The Transplacental Effect of Urethan on Lung Tumorigene- sis in Mice. J. Nati. Cancer Inst., 12: 1003 1010, 1952. 15. Lar iÂ».C. D. Pulmonary-tumor Induction by Transplacental Expo ACKNOWLEDGMENTS sure to Urethan. J. Nati. Cancer Inst., 8: 63-69, 1947. 16. Laumas, K. R., Malkoni. P. K., Bhatnagar, S., and Laumas, V. We would like to thank Dr. H. Manabe and Dr. E. Okamoto of this Radioactivity in the Breast Milk of Lactating Women after Oral department. Dr. T. Miyaji, Department of Pathology of this University, Administration of 3H-norethynodrel. Am. J. Obstet. Gynecol., 98: and Dr. T. Tanaka, Aichi Cancer Center Research Institute, Nagoya. 411 413, 1967. Japan, for their advice and assistance, and K. Murazumi for her technical 17. Masri, M. S., Garcia, V. C., and Page, J. R. The Aflatoxin M Content assistance. of Milk from Cows Fed Known Amounts of Aflatoxin. Vet. Record, 83: 146 147, 1969. REFERENCES 18. Mayer, A. M.. Polijakoff-Mayber. A.. Robinson, P., and Slowatizky, I. A Simple Bioassay for Detection of Aflatoxin in Milk. Toxicon, 7: 1. Alexandrov, V. A. Blastomogenic Effects of Dimethylnitrosamine on 13-14, 1069. Pregnant Rats and their Offspring. Nature, 218: 280-281, 1968. 19. Mirvish, S. S., Cividalli, G., and Berenblum, I. Slow Elimination of 2. Buchanan, R. A., Eaton, C. J., Koeff, S. T., and Kinkel, A. W. The Urethan in Relation to Its High Carcinogenicity in Newborn Mice. Breast Milk Excretion of Mefenamic Acid. Current Therapy Res., 10: Proc. Soc. Exptl. Biol. Med., Â¡16:265 268, 1964. 592-596, 1968. 20. Mohr, U., and Althoff, J. Carcinogenic Activity of Aliphatic Nitrosa- 3. Buchanan, R. A., Eaton, C. J., Koeff, S. T., and Kinkel, A. W. The mines Via the Mother's Milk in the Offspring of Syrian Golden Breast Milk Excretion of Flufenamic Acid. Current Therapy Res., //: Hamsters. Proc. Soc. Exptl. Biol. Med., 136: 1007 1009, 1971. 533-538, 1969. 21. Mohr. U., Althoff. J., and Authaler, A. Diaplacental Effect of the 4. Bulay, O. M., and Wattenberg, L. W. Carcinogenic Effects of Carcinogen Diethylnitrosamine in the Golden Hamster. Cancer Res., Subcutaneous Administration of Benzo[a]pyrene during Pregnancy on 26: 2349-2352, 1966.

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22. Napalkov, N. P., and Alexandrov, V. A. On the Effects of Blastomo- 30. Simpson, W. J., and Tuba, J. An Investigation of Fluoride Concentra genic Substances on the Organism durng Embryogenesis. Z. Krebs- tion in the Milk of Nursing Mothers. J. Oral Med.,23: 104 106, 1968. forsch., 71: 32 50, 1968. 31. Smith. W. E., and Rous. P. The Neoplastic Potentialities of Mouse 23. Nomura, T., and Okamoto, E. Transplacental Carcinogenesis by Embryo Tissues. IV. Lung Adenomas in Baby Mice as Result of Urethan in Mice; Teratogenesis and Carcinogenesis in Relation to Prenatal Exposure to Urethan. J. Exptl. Med., 88: 529-555, 1948. OrganogÃ©nesis.Gann, 63: 731 742, 1972. 32. Spatz, M., and Laqueur. L. Transplacental Induction of Tumors in 24. Nomura, T., Okamolo, E., and Manabe, H. Ovarian Teratoma Found Sprague-Dawley Rats with Crude Cycad Material. J. Nati. Cancer in an Offspring of Mothers (ICR-JCL Mice) Treated with Urethane. Inst., 38: 233-245, 1967. Med. J. Osaka Univ., 23: 121 131, 1972. 33. Tanaka, T. Transplacental Induction of Tumours and Malformations 25. Nomura, T., Takebe, H., and Okamoto, E. Long Retention of in Rats Treated with Some Chemical Carcinogens. UniÃ³ Intern. Urethan Transferred into Newborns Transplacentally: A Possible Contra Cancrum Monograph, in press. Cause of High Carcinogenesis. Gann, 64: 29 40. 1973. 34. Tomatis, L.. and Goodal. C. M. The Occurrence of Tumors in F,, F2, 26. Ottoboni, A., and Ferguson, J. I. Excretion of DDT Compounds in F3 Descendants of Pregnant Mice Injected with 7,12-Dimelhylbenz- Rat Milk. Toxicol. Appi. Pharmacol., 15: 56 61, 1969. [a-]anthracene. Intern. J. Cancer, 4: 219-225, 1969. 27. Rice, J. M. Transplacental Carcinogenesis in Mice by 1-Ethyl-l- 35. Tomatis, L., Turusov, V., Guibbert, D., Duperray, B., Malaveille, C., nitrosourea. Ann. N. Y. Acad. Sci., 163: 813 827, 1969. and Pacheco, H. Transplacental Carcinogenic Effect of 3-Methyl- 28. Shay, H., Friedmann. B.. Gruenstein, M., and Weinhouse, S. Mam cholanthrene in Mice and Its Quantitation in Fetal Tissues. J. Nati. mary Excretion of 20-Methylcholanthrene. Cancer Res.. 10: 797-800, Cancer Inst., 47: 645-651, 1971. 1950. 36. Vesselinovitch, S. D., Mihailovich, N.. and Pietra, G. The Prenatal 29. Shay, H., Weinhouse, S.. Gruenstein, M., Marx, H. E., and Fried- Exposure of Mice to Urethan and the Consequent Development of mann, B. Development of Malignant Lymphoma in Some of the Tumors in Various Tissues. Cancer Res., 27: 2333-2337, 1967. Young Rats Suckled by Mothers Receiving Methylcholanthrene by 37. Vesselinovitch, S. D., Mihailovich, N., Rao, K. V. N., and Itze, L. Stomach Tube Only during the Lactating Period: Preliminary Report. Perinatal Carcinogenesis of Urethan. Cancer Res., 31: 2143 2147, Cancer, 3: 891 895, 1950. 1971.

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Fig. 1. A, ovarian cyst. Many blood vessels were observed on the cyst wall. The cyst was multilocular and contained clear, serous fluid, fi. low-power microscopic view of ovarian cyst. Ciliated cuboidal or columnar epithelial cells were protruding into the cavity with papillary proliferation. H & E, x 200. C, high-power microscopic view of ciliated epithelium of ovarian cyst. H & E, x 2000. Fig. 2. Cystic kidney. Ureter and pelvic cavity were not dilated.

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Fig. 3. /l, adenoacanthoma of Bartholin's gland. A firm, nodular tumor which originated from Bartholin's gland was found in urethan-treated mother, compressing the vagina to the opposite site, a, multiple mÃ©tastasestothe liver: 6, urinary bladder; c, uterus. B, squamous element of adeno- canthoma. H & E, x 300. Fig. 4. A, malignant mesenchyrnoma in the small pelvic cavity. B, a part of reticulum cell sarcoma of A. H & E x 300.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1973 American Association for Cancer Research. Carcinogenesis by Urethan Via Mother's Milk and Its Enhancement of Transplacental Carcinogenesis in Mice

Taisei Nomura

Cancer Res 1973;33:1677-1683.

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