Cancer Letters, 1 (1975) 69--74 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands

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CARCINOGENIC EFFECTS OF NIRIDAZOLE*

HUSEYIN K. URMAN, ORHAN BULAY, DAVID B. CLAYSON and PHILIPPE SHUBIK

Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105 (U.S.A.)

(Received 21 July 1975)

SUMMARY

Swiss mice received chronic treatment with the schistosomacide, niridazole, at 3 dose levels. Tumors were found in several organs, including stomach, lung, mammary gland, ovary and bladder. Niridazole is, without doubt, car- cinogenic to mice.

INTRODUCTION

Niridazole, 1.(5-nitro-2-thiazolyl)-2-imidazolidinone, also known as Ambilhar, was introduced in 1964 for the treatment of schistosomiasis and amebiasis [6]. The compound subsequently presented mutagenic activity [4,7]. Biocidal activity was attributed to the 5-nitrothiazole ring [1].

The carcinogenicity of nitrofuran [10] and nitroimidazole [11] deriva- tives has been demonstrated. The 5-nitrothiazole ring of niridazole is chem- ically analogous to these ring systems. This led us to follow the advice of a WHO consultant group [ 12] on the comparative evaluation of new schisto- somicidal drugs, which recommended that the carcinogenic activity of niri- dazole be assessed. This report describes the carcinogenicity of niridazole in mice.

MATERIALS AND METHODS

Randomly bred, 10-week-old male and female Swiss mice from the Eppley colony were housed in plastic cages on San-i-cel bedding in groups of 5 by sex. Food and water were given ad libitum. The animals were inspected regu- larly, palpated for internal and external growths and weighed weekly. All animals were autopsied and bladders and stomachs were distended with ice- cold buffered formalin; 24 hr later, after longitudinal bisection, bladders were

*Supported by Public Health Service Contract NO1 CP33278 from the National Cancer Institute, NIH.

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examined under a stereo-microscope to detect the presence of small lesions. Tissues were taken from all organs, including the lungs, liver, kidneys, testes, ovaries and bone marrow. Histologic sections were stained routinely with hematoxylin and eosin. The bladder, lungs and stomach were examined in serial section.

Niridazole (Ciba-Geigy, Summit, N.J.) was incorporated into Wayne Lab- Blox (Allied Mills, Chicago, Ill.) by a roller-type Versa-Mill (Fisher Scientific Co., Fair Lawn, N.J.) and stored at 3--5°C. The powdered food was checked frequently to determine the stability of the niridazole.

Following subacute toxicity tests, which showed 0.4 and 0.2% niridazole in powdered food caused body weight loss and severe atrophy of the testes, the maximum tolerated dose was established as 0.1%.

Groups of 30 male and 30 female mice were initially given 0.1%, 0.05% and 0.025% niridazole in the diet. An untreated group of 25 males and 25 fe- males was established. Because significant growth retardation was noted in mice receiving 0.1% niridazole, t reatment was stopped in all 3 groups at the 30th week. Animals were then fed the basal diet for a 6-week recovery period. Treatment was resumed by feeding one-half the amount of the initial dose of niridazole in each group for the remainder of the animals' lives.

RESULTS

Male mice receiving the highest level of niridazole had a higher mortal i ty than did other groups (Fig. 1). Niridazole-treated mice developed tumors with a greater incidence and shorter latency than did untreated mice (Table 1). Female mice were generally more susceptible than male mice to the carci- nogen. In female mice of groups 2 and 3, there were 97 and 97% tumor bear- ing animals, whereas in male mice there were 67 and 80%, respectively. Com- parisons in group 1 are vitiated by the greater mortali ty of the male mice.

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Fig. 1. Survival rates of mice treated ~vith different doses of niridazole (see text). Control (straight and broken lines); 0.10% niridazole (open and closed circles); 0.050% niridazole (open and closed triangles); and, 0.025% niridazole in the diet (open and closed squares).

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TABLE 2

INCIDENCE OF URINARY BLADDER NEOPLASMS

Group Number of Type of Latency in instances lesions weeks (range) and sex

4 f Transitional-cell carcinoma 55 (40--65) 1

3 f Hyperplasia 64 (54--71)

1 f Transitional-cell carcinoma 61 2

1 m Submucosal atypical cellular masses 72

3 1 m Submucosal atypical cellular masses 52

Control None -

Except in group 1 male mice, the mean number of tumors/animal was pro- portional to the dose given. The mean average numbers of tumors for female mice in groups 1--4 were 3.0, 2.7, 2.4 and 1.4 respectively. In males, the corresponding values were 1.5, 2.0, 2.0 and 1.0.

Benign and malignant tumors were induced in a variety of organs, princi- pally in the forestomach, lung, breast, ovary and urinary bladder and occa- sionally in the subcutaneous tissue, Harderian gland and peripheral nervous system (Schwannomas) (Tables 1 and 2). Malignant lymphomas were observed infrequently in male and more commonly in female mice, but the difference in incidence between treated and control male or female mice was not suffi- cient to demonstrate an effect of the chemical on their induction. In female mice, the reduction in latency from 71 to 55 weeks indicates the chemical may have had an effect.

In the highest dose group, 4 female mice developed urinary bladder carci- nomas and 3, hyperplasia of the bladder epithelium. One intermediate dose female mouse developed bladder carcinoma (Table 2). Some bladders were almost occluded by the carcinomatous mass. Most of these transitional cell carcinomas contained squamous cell components. One carcinoma metasta- sized to the lung. One in the intermediate and one in the lowest dose treated groups revealed atypical cellular masses under the mucosa as described by Bonser and JuU [2].

Testicular atrophy was a major finding in the subchronic feeding studies with 0.4 and 0.2% niridazole. It was less marked in the chronic studies at lower dosage levels and occurred in 19, 14 and 10% of male mice in groups 1, 2 and 3.

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DISCUSSION

This e x p e r i m e n t leaves no d o u b t t ha t n i r idazole is a p o t e n t carc inogen in Swiss mice. This conc lus ion is s u p p o r t e d by the increased incidence and de- creased l a tency of benign and ma l ignan t t u m o r s and b y the va r ie ty o f a f f ec t ed tissues.

Mutagen ic i ty s tudies [3] suggest t h a t n i r idazole does n o t require m e t a b o l i c ac t iva t ion b y liver mic rosomes . T h e induc t ion o f f o r e s t o m a c h pap i l lomas and ca rc inomas in the p resen t e x p e r i m e n t conf i rms the idea t h a t n i r idazole m a y in te rac t d i rec t ly wi th tissues. The m e t a b o l i s m o f the subs tance which is main- ly c o n c e n t r a t e d in the liver [ 4 ] , an organ n o t a f f ec t ed by the carc inogenic ac- t ion o f nir idazole , is poss ib ly largely de tox i f i ca t ion , Never theless , if n i r idazole is me tabo l i ca l l y ac t iva ted b y r educ t ion to the co r re spond ing N - h y d r o x y deri- vative, l ike the a rom a t i c amines [ 8 ] , the r educ t ion o f the drug b y a NADPH- c y t o c h r o m e c reductase , poss ib ly xan t h i ne oxidase [5 , 9 ] , m a y be significant.

T h e carc inogenic ac t iv i ty o f ni r idazole in Swiss mice means t ha t its use as a sch i s tosomac ide in m a n m u s t be reassessed [12 ] .

REFERENCES

1 Bond, H.W. (1969): Chemistry of schistosomicides. Ann. N.Y. Acad. Sci., 160, 519--529.

2 Bonser, G.M. and Jull, J.W. (1956): The histological changes in the mouse bladder following surgical implantations of paraffin wax pellets containing various chemicals. J. Path. Bact., 72,489--495.

3 Connor, T., Stoeckel, M. and Legator, M.S. (1974): Niridazole, a direct acting frame- shift mutagen, not affected by microsomal enzyme preparation which can be detected in the host-mediated assay. Mutat. Res., 26, 456--457.

4 Faigle, J.W. and Keberle, H. (1969): Metabolism of niridazole in various species, in- cluding man. Ann. N.Y. Acad. Sci., 160,544--557.

5 Feller, D.R., Morita, M. and Gillette, J.R. (1971): Enzymatic reduction of niridazole by rat liver microsomes. Biochem. Pharmacology, 20, 203--215.

6 Lambert, C.R., Wilhelm, M., Striebel, H., Kradolfer, F. and Schmidt, P. (1964): Eine neue gegen Bilharziose und Amoebiase Wirksame Verbindung. Experientia, 20, 452--453.

7 McCaUa, D.R., Voutsinos, D. and Olive, P.L. (1975): Mutagen screening with bacteria: niridazole and nitrofurans. Mutat. Res.; 31, 31--37.

8 Miller, J.A. (1970): Carcinogenesis by chemicals: An overview -- G.H.A. Clowes Memo- rial Lecture. Cancer Res., 30, 559--576.

9 Morita, M., Feller, D.R. and Gillette, J.R. (1971): Reduction of niridazole by rat liver xanthine oxidase. Biochem. Pharmacol., 20, 217--226.

10 Price, J.M., Morris, J.E. and Lalich, J.J. (1966): Evaluation of the carcinogenic activity of 5-nitrofuran derivatives in the rat. Fed. Proc., 25,419.

11 Rustia, M. and Shubik, P. (1972): Induction of lung tumors and malignant lymphomas in mice by metronidazole. J. Nail. Cancer Inst., 48, 721--729.

12 WHO reports on schistosomicidal drugs (1972): I. Report of a WHO consultant group on hycanthone and II. Report of a WHO consultant group on the comparative evalua- tion of new schistosomicidal drugs for use in treatment campaigns. WHO documents WHO]SCHISTO/72.19 and 72.20. In: Boletin de la Oficina Saniforia Panamericana, Vol. 2, pp. 82--100.