Int. J . Cancer: 18, 530-535 (1976)

LUNG TUMOUR INCIDENCE IN MlCE TREATED WITH HYDRAZINE SULPHATE S. V. BHlDE l, R. A. D’SOUZA*, M. M. SAWAIa and Kamal J. RANADIVE Biology Division, Cancer Research Institute, Tata Memorial Centre, Parel, Bombay 400 012; and Pathology Department, Tata Memorial Hospital, Tata Memorial Centre, Parel, Bombay 400 012, India.

Swiss strain mice

SUMMARY

Lung tumour incidence in mice fed with hydrazine sirlphate (1.1 mgldaylmouse) was studied in male and female mice of Swiss, Strong ‘‘ A ” and F, cross of ICRC (female) x C3H(Jax) (male), as weN as in C17 males. Swiss strain mice showed 100% lung adenocarcinomas. None of the treated mice of different strains had liver tumours. Hydrazine sulphate also induced adenocarcinornas of lung in Strong ’‘ A ” and F, cross of ICRC females x C3H (Jax) males but it produced lymphomas of lung in C17 strain. Female mice of Swiss strain and F1 hybrids showed greater susceptibility to hydrazine sulphate than the males. It was interesting to observe that protein and vitamin B deficiency in the diet shortened the triniour induction period in Swiss strain mice. Hydrazine sulphate (HS) an immediate metabolite

of isoniazid (El Masri et al., 1958) is reported to induce lung tumours in rats (Severi and Biancifiori, 1968), BALB/c/Cb/Se mice (Biancifiori, 1970) and Swiss mice (Toth, 1969). In view of the extensive use of isoniazid as an antitubercular drug, it seemed interesting to study the tumorigenicity of HS under different experimental conditions in Swiss strain mice as well as in othcr strains of mice which either (1) have spontaneous lung tumours or (2) have an organ other than lung, susceptible to chemical carcinogens and (3) which have an inherent viral factor. The present paper reports salient observations on HS tumorigenicity in the Strong “ A ” strain, C17 strain, F, cross of ICRC (female)/C3H(Jax) (male) as well as in Swiss strain mice subjected to partial hepatectoiny and diets deficient in the Vitamin B complex or in protein.

Male and female mice of the inbred Swiss strain were used for these studies. Intact Swiss mice. Eight-week-old Swiss strain male mice were fed with hydrazine sulphate and killed at the ages of 4, 9, 11, 13 and 15 months. Eight-week-old female virgin and breeder mice were fed with hydrazine sulphate and killed at the ages of 4, 9, 11 and 13 months. Partially hepatectomized Swiss male mice. Eightweek-old male mice were partially hepatectomized by the method of Higgins and Anderson (1930). Seventy-two hours after the operation, the mice were put on hydrazine sulphate treatment and killed at the ages of 4,9, 11, 13 and 15 months. Partially hepatectomized mice fed with distilled water served as controls. Swiss mice on vitamin B complex deficient diet. Seven-week-old male mice were put on a diet containing 1 %yeast instead of 4% as in the standard diet (Ranadive, 1957). One week after, the mice on vitamin B complex deficient diet were put on hydrazine sulphate treatment. Male mice of the same age and kept on vitainin B complex deficient diet and fed with distilled water served as controls. Both control and treated mice were killed at the ages of 4, 9 and 11 months. Swiss mice on protein-deficient diet. Seven-week-old mice were fed with a diet containing 10.88 % protein instead of 15.82 ”/,as in standard diet. After one week the mice on the deficient diet were put on hydrazine sulphate treatment. Male mice kept on the proteindeficient diet and fed with distilled water were used as controls. Both control and treated mice were killed at the ages of 4, 9 and 1 1 months. Strong

MATERIAL AND METHODS

1 Mice of the different strains used

in the present

work were obtained from the Animal Colony of the Cancer Research Institute, Bombay. Experiniental mice were fed with HS (1.1 mglniouselday) 6 times a week by stomach tube until they were killed. Mice of the corresponding age, Sex and strain fed with distilled water were used as controls.



A



strain

Stock mice of this strain were originally brought from the Department of Anatomy, Yale University, USA in 1940 and subsequently maintained in the Animal Colony of the Cancer Research Institute,

Received: April 5 , 1976 and in revised form July IS, 1916.

LUNG TUMOURS IN HYDRAZINE-TREATED MICE

53 1

by brotherlsister mating. Eight-week-old normal male mice of this strain were put on hydrazine sulphate treatment and killed at the ages of 4, 9 and 11 months. CI 7 strain

This strain was originally started in our laboratory by crossing two inbred resistant strains, namely C57(BL) and XVII (Paris). The inbred line was developed by strict brother/sister ma!ing of the breeding nucleus of these hybrid mice (Ranadive et al., 1969). Eigh:-week-old male mice of this strain were fed with hydrazine sulphate and some were killed at 4 , 9 and 11 months. A total of 24 mice were killed before I 1 months of age. The remaining 20 were grouped into 13- to 14-month and 16- to 18-month-old groups and killed when they became weak. Fl cross between inbredlCRC(fema1e) and C3H (Jax) male mice

Eight-week-old male mice of C3H(Jax) strain (obtained from Roscoe B. Jackson Memorial Laboratories, Bar Harbor (Maine), USA in 1955) were mated with 8-week-old ICRC virgins [a strain developed at the Cancer Research Institute and which is susceptible to breast cancer and leukemia (Ranadive and Kanekar, 1963)l. The progeny was weaned at the age of 3 weeks. At the age of 8 weeks both male and female mice were fed with hydrazine sulphate and killed when they were weak.

FIGURE 1

Tumour shows papillae with delicate stroma and low columnar lining epithelial cells. H. & E, x 140.

RESULTS

Control groups and 4-month-old treated mice of different strains showed no abnormality in the lung and liver tissues at the gross or microscopic level. Intact Swiss mice treated with hydrazine sulphate

Treated male mice up to the age of 9 months did not show any gross abnormality (Table I).

TABLE I INCIDENCE OF LUNG TUMOURS I N INTACT A N D PARTIALLY HEPATECTOMIZED MICE FED WITH HYDRAZINE SULPHATE ( 1 . 1 MG/DAY/MOUSE) SIX TIMES WEEKLY Age (months)

Intact Partially hepatectomized

4

9

11

0/7

0/15

2/12 10/10 10/10

0/6

0/13

2/10 10/10

13

15

919

In the 11-month-old group, two out of 12 mice showed lung tumours. All the treated mice aged 13 and 15 months showed lung tumours. The lung tissue of 9-month-old mice showed normal histology. Gross observation of the lung tumours showed that these occupied almost the entire lung. The bronchi did not reveal any significant changes. The tumours displayed a papillary structure (Fig. 1). They were thus designated as papillary adenocarcinoma and could be easily transplanted. One of the tumours is in the 11th generation. Microscopic studies of the liver of 13-month-old treated mice showed that the changes occurred mainly in the nuclei. They were larger and hyperchromatic and the number of binucleate cells was increased. Occasional mitotic figures were discerned, and the cytoplasm showed early fatty changes in a few cases. Kupffer cells appeared prominent. I n another group of Swiss mice, hydrazine sulphate feeding was discontinued after 7 months’ treatment and these mice were kept under observation until death. All treated mice developed 100 % lung adenocarcinomas (10/10) at the age of 16 months.

532

BHIDE ET AL. TABLE 11

LUNG TUMOUR INCIDENCE I N HYDRAZINE SULPHATE (1.1 MG/DAY/MOUSE)-TREATED SWISS MICE KEPT ON PROTEIN AND VITAMIN B DEFICIENT DIET AND NORMAL DIET Age (months)

Normal diet Protein-deficient diet Vitamin B deficient diet

4

9

I1

014 0/4

017

218

419

416

014

216

616

hepatectomy promotes hepatocarcinogenesis induced by urethane (Lane et al., 1970) and dimethylnitrosamine (Craddock, 1971). None of the partially hepatectomized mice treated with hydrazine sulphate developed liver tumours. U p to the age of 9 months no abnormality was observed in treated mice (Table I). In the 11-month-old group two out of 10 mice showed lung adenocarcincnias. I n the 13- and 15-month-old groups all the treated mice showed lung adenocarcinomas. The histology of

Partially hepatectornized Swiss male mice Riancifiori and his co-workers have reported a hepatoma incidence of 62% in male and 71 % in female CBA/Cb/Se mice (Biancifiori et a/., 1964). However, we did not observe any liver tumours. Subsequently it seemed worthwhile to see if hydrazine sulphate induced liver tumours in partially hepatectomized mice because it is known that partial

FIGURE 3 Tumour consists of cords of cubiodal cells with hyperchromatic nuclei. H. & E., )i560.

the lung adenocarcinomas and the liver of the treated 13- and 15-month-old mice showed the same structure as that seen in the previous group.

Vitamin B cornplex and protein-deficienf Swiss tnak mice

FIGURE 2 Section of the whole lung shows three non-encapsulated umoiirs. H. & E., x 11. LUJ

At the age of 9 months all mice of both groups showed multiple nodules i n the lung. In four out of nine protein-deficient mice and two out of six vitamin B complex deficient mice single or mu1W e non-encapsulated malignallt nodules were observed (Table 11) at the periphery of the lung

533

LUNG TUMOURS IN HYDRAZINE-TREATED MICE

(Fig. 2). These tumours consisted of compactly arranged cords of uniform cuboidal cells supported by a minimal stroma (Fig. 3). The cytoplasm of the cells was acidophilic. The nuclei were rounded, hyperchromatic and exhibited occasional mitosis. At the age of 11 months all the vitamin R complex deficient and four out of six protein-deficient mice treated with hydrazine sulphate showed focal adenocarcinonias with papillary structure.

TABLE IV TUMOUR INCIDENCE IN STRONG < ( A nA N D C17 STRAIN MALE MICE TREATED WITH HYDRAZINE SULPHATE (1.1 MG/DAY/MOUSE) ~

Age (months) Strain

4

9

11

Strong “ A ’’

0/8

7/8

12/12

C17

O/S

0/8

4/8

13-14

~

6/10

16-18 -

8/10

Swiss female mice

At the age of 9 months all the treated mice showed lung nodules. However, histological exaniination showed that 3 out of 12 breeders and 4 out of I2 virgin mice showed evidence of adenocarcinonia of the lung (Table 111). At the age of 11 months 6 out of 12 breeders and 8 out of 12 virgin mice showed evidence of adenocarcinonia of the lung. At the agc of 13 months all the treated virgin and breeder TABLE I l l LUNG TUMOUR INCIDENCE IN SWISS VIRGIN AND BREEDER MICE TREATED WITH HYDRAZINE SULPHATE ( 1 . 1 MG/DAY/MOUSE) Age (months)

Virgin Breeder

4

9

II

0/8 O/S

4/12 3/12

8/12 6/12

13

8/8 8/8

mice showed well-developed papillary lung tumours. Microscopic studies of the liver of I 1-month-old treated mice showed [he same structure as that seen in intact Swiss mice treated with hydrazine sulphate. Subsequen+ly, experiments here carried out to study strain specificity in hydrazine sulphate tuniorigenesis. Strong ‘‘ A



arid C17 strain mice

Seven out of eight 9-month-old S u x ~ i ~“g A ” strain mice showed multiple malignant, lung tumours (Table JV). In the 11-month-old group all the mice (l2/12) showed multiple adenocarcinonias of the lung. Though we have observed that 10% normal Slrong “ A ” strain mice develop lung tuniours at the age of 18-23 months, no tumours were observed in normal mice at the age of 1 1 months. Four out of eight I I-month-old C17 strain mice had lymphomas. In older mice (13-14 mon!hs) six out of 10 had lymphomas, while in 16- to 18month-old mice, eight out of 10 had large lung lymphomas. The histology of the lymphomas was ill defined. Tumour cells infiltrated the interalveolar septae, as well as the long neurovascular bundles.

Fl cross of ICRC x C3H( Jax) treated with hydrazine sirlphate The killed mice could be divided into two agegroups, 11-12 and 18-19 months old. In 11- to 12-month-old control females neither breast nor lung tumours were observed, but in treated females of the same age a11 10 mice showed mammary adenocarcinonias and only 4 mice in this age group had large lung tumours (Table V). I n 18- to 19-monthold treated females only 4 out of 14 showed breast tumours but large lung tuniours were observed in 13/14 mice. It is very difficult to explain the relatively low incidence of breast tumour in 18to 19-month-old treated female mice, although a high incidence of breast tumours was observed at the corresponding age in the control group (four out of six). No lung tummrs were observed in con’rol mice of this age. In treated male mice no tuniours were observed in the 11- to 12-month-old group but in the 18- to 19-month-old group two out of eight treated mice showed lung adenocarciiiomas. They occupied almost the entire lung and displayed a papillary structure. Control male mice of both age groups showed no lung tumour. DISCUSSION

The foregoing data clearly indicate that hydrazine sulphateinduced lung tumours in 100%of the treated mice. Unlike other workers (Biancifiori et al., 1964)

TABLE V TUMOUR INCIDENCE IN I-, H Y B R I D S OF ICRC FEMALE )iC3H (JAX) MALE MICE TREATED WITH HYDRAZINE SULPHATE ( 1 . 1 MGIDAYIMOUSE) 11-12 months Sex

Untreated

Male Female

18-19 months

Tuniour Treated

Untreated

Treated

Lung

0/6

Oj8

0/6

2/8

Breast

016

10110

4/6

4/14

Lung

0/6

4/10

0/6

13/14

534

BHlDE ET AL.

we did not obtain any hepatomas in treated mice. Even partial hepatectomy did not induce malignant changes in liver tissue of treated mice. This clearly showed that in Swiss mice hydrazine sulphate does not act even as a weak hepatocarcinogen. It is further evident that it acts as a carcinogen in all strains tested by us and lung tissue is the target tissue. In the C17 strain it induced lymphomas but in other strains it produced adenocarcinomas. Further, it is interesting to note that hydrazine sulphate shortened the period of tumour induction in Strong “ A ” strain mice which develop spontaneous lung tumours. We obtained multiple lung tumours in Strong “ A ” strain mice much earlier, i.e. at the age of 9 months, than in Swiss strain mice. The present data on female Swiss mice and the F, cross of ICRC (female) x C3H(Jax) (male) show that female mice are more susceptible to hydrazine sulphate treatment, supporting earlier reports in literature. Biancifiori (1 971 ) has reported that female mice of the C3H b/Cb/Se strain are more susceptible than the male mice to hydrazine sulphate treatment. However, our observations on Strong “ A ” strain male and female mice do not show any variations in their susceptibility to hydrazine sulphate. This suggests that no such sex difference may be evident in a strain that shows inherent susceptibility to lung carcinogenesis. Another noteworthy observation is that both vitamin B complex and protein deficiency shortened

the tumour induction period by 2 months. An accelerating effect of vitamin B complex deficiency (Kensler et al., 1974) on chemical carcinogenesis has been reported but the studies dealing with the effect of protein deficiency on chemical carcinogenesis are contradictory. McLean and Magee (1970) as well as Rogers et al. (1974) have shown an increased carcinogenicity of nitrosamines in animals on a protein-deficient diet, whereas Tannenbaum and Silverstone (1953) have shown an accelerating effect of protein deficiency on chemical carcinogenesis. Available literature on the carcinogenicity of isoniazid which is widely used as an antituberculosis drug is conflicting. Same reports show increased incidence of lung cancer in patients treated with isoniazid (Hammond et a[., 1967), whereas others have failed to show any significant increase in the incidence of cancer among patients treated with isoniazid (Jung, 1971 ; Pompe, 1956). However, all these epideniiological reports are not as yet based on 15-year studies hence it is premature to rule out the carcinogenicity of isoniazid in man. Regarding the widespread use of isoniazid as a prophylactic and therapeutic drug in the Indian population where malnutrition is prevalent to a great extent, the observed effect of a deficiency of vitamin B complex and protein on hydrazine sulphate carcinogenesis is noteworthy and warrants further work.

L’INCIDENCE DES TUMEURS DES POUMONS CHEZ LES SOURIS TRAITEES A L’HYDRAZTNE SULFATE L’incidence des tumeurs des poumons induites par le traitement tr l’hydrazine sulJate a ktk ktudike chez les souris mdles et femelles de la souche Swiss, Strong A et les mdles C17. On a observk que l‘administration d’hydrazine sulfate (1.1 mgljour) six fois par semaine produisait cent pour cent d’adknocarcinomes chez les mciles et femelles Swiss, mais aucun hkpatome. L’hydrazine sulfate a aussi induit des adknocarcinomes des poumom chez les souris Strong A et les hybrides des ICRC 0 Y C3H ( J a x ) 6, et des lyrnphomes despoumons chez les souris C17. Les femelles de la souche Swiss et les hybrides fernelles sont plus sensibles que les rnciles de la me‘me souche. I1 est intCressant d’observer qu’un rkgime pauvre en vitamine B ou en protkines raccourcit lu pkriode d’induction des tunieurs chez les souris Swiss. REFERENCES

BIANCIFIORI, C., Lung and liver tumours induced by small amounts o f hydrazine sulfate in BALB/c/Ch/Se mice. Lnv. Anat. Par. Perugia, 30 (2), 89-99 (1970). BIANCIFIORI, C., Effects of ovarian hormones on pulmonary carcinogenesis by hydrazine sulfate in C3H b/Cb/Se mice. Lav. Anat. Pat. Perrcgia, 31 (I), 5-17 (1971). BIANCIFIORI, C., BUCCIARELLI, E., CLAYSON,D. B., and SANTILLI,F. E., Induction of hepatomas in CBA/Cb/Se mice by hydrazine sulfate and lack of croton oil on tumour induction in BALB/c/Cb/Se mice. Brit. J. Cancer, 18, 543-549 (1964). CRADDOCK, V. M., Liver carcinomas induced in rats by single administration of dimethylnitrosamine after partial hepatectomy. J . nat. Cancer Inst., 47, 899-905 (1971).

EL MASRI, A . M., SMITH, J. N., and WILLIAMS, R. T., Studies in detoxication 75. Further observations on the metabolism of hydrazides of aromatic acids. Biochem. J., 68, 587-592 (1958). HAMMOND,E. C., SELIKOFF, I. J., and ROBITZEK, E. H . , Isoniazid therapy in relation t o later occurrence of cancer in adults and in infants. Brit. med. J . , ii, 792-796 (1967). HIGGINS,G . M., and ANDERSON, R. M., Experimental pathology of the liver of the white rat following partial surgical removal. Arch. Path., 12, 186-190 (1930). JUNG, H. D., Zur fraglichen Induzierung von Bronchial und Hautkarzinomen durch INH (Isoniazid) am Buspiel der Tuberculosis luposa. 2. Erkr. Atmungsorgane, 135, 3 1-37 (1971).

LUNG TUMOUKS IN HYDRAZINE-TREATED MICE

KENSLER, C. J., SUGIURA, K., YOUNG,N. F., HALTER,C. R., and RHOADS,C. P., Partial protection of rats by riboflavin with casein against liver cancer caused by dimethylaminoazobenzene. Science, 93, 308-310 (1974). LANE, M., LIEBELT, A., CALVERT, J., and LIEBELT, R., Effect of partial hepatectomy on tumour incidence of BALB/c mice treated with urethane. Cancer Res., 30, 1812-1816 (1970).

MCLEAN,A. E. M., and MAGEE,P. W., Increased renal carcinogenesis by dimethylnitrosamine in protein deficient rats. Brit.J. exp. Path., 51, 587-590 (1970). POMPE,K., Einfluss von Isonicotinhydrazid auf die Lupuskarzinomentstehung. Derm. Wschr., 133, 105-109 (1956). RANADIVE, K . I., Diet formula evolved at the Indian Cancer Research Centre, L.A.C. Collected papers, 5 , 39-41 (1957). RANADIVE,K . J., GOTHOSKAR, S. V., and FERNANDES, G., A new inbred strain of mouse C17/1CRC developed for testing weak carcinogens. fnd. J . med. Res., 57, 521-526 (1969).

535

RANADIVE, K. J., and KANEKAR, S. S., Biological studies on the new albino mouse inbred at the Indian Cancer Research Centre. fnd. J. med. Res., 51, 1005-1014 (1963). ROGERS,A. E., SANCHER, O., FEINSOD, F. M., and NEWBORNE,P. M., Dietary enhancement of nitrosamine carcinogenesis. Cancer Res., 34, 96-99 (1974). SEVERI,L., and BIANCIFIORI, C., Hepatic carcinogenesis in CBA/Cb/Se mice and Cb/Se rats by isonicotinic acid hydrazide and hydrazine sulfate. J . nut. Cancer fnst., 41, 331-349 (1968).

TANNENBAUM, A., and SILVERSTONE, H., The effects of varying the level of minerals in the diet. Cancer Res., 460-463 (1953).

TOTH,B., Lung tumour induction and inhibition of breast adenocarcinomas by hydrazine sulfate in mice. J . n a f . Cancer fnst., 42, 469-475 (1969).

Lung tumour incidence in mice treated with hydrazine sulphate.

Int. J . Cancer: 18, 530-535 (1976) LUNG TUMOUR INCIDENCE IN MlCE TREATED WITH HYDRAZINE SULPHATE S. V. BHlDE l, R. A. D’SOUZA*, M. M. SAWAIa and Kam...
603KB Sizes 0 Downloads 0 Views