Fd Cormer. Toxirol. Vol. 19. pp. 353 lo 355. 1981 Printed in Great Britain. All rights reserved

0015-6264/81/03035M3SO2.00/0 Copyright 0 1981 Pergsmon Press Ltd

SHORT PAPERS

NON-CARCINOGENIC RESPONSE TO COUMARIN IN SYRIAN GOLDEN HAMSTERS

I. UENO and I. HIRONO Department of Carcinogenesis and Cancer Susceptibility, Institute of Medical Science,

University of Tokyo, Minato-ku , Tokyo 108, Japan

(Received 13 June 1980)

Abstract-Groups of 11-12 male and lo-13 female Syrian golden hamsters received diets containing 00 (controls), 0.1 or 0.5% coumarin for up to 2 yr. In these three groups, survivors of the initial totals of 24, 24 and 21 hamsters, respectively, numbered 17. 9, and 18 at 18 months, 12, 1 and 12 at 22 months and 10, 1 and 5 at 24 months. There was no evidence that coumarin had induced fatty change or cysts in the liver, bile-duct proliferation, cholangiofibrosis or cholangiocarcinoma in animals dying during the study or in those killed terminally. Tumours observed at extra-hepatic sites were randomly distributed between the control and the test groups. On the basis of this study, coumarin does not appear to be hepatotoxic or hepatocarcinogenic in the Syrian golden hamster.

Introduction

Coumarin occurs naturally in various plant products and is widely used as a flavouring ingre- dient, although its usage as a food additive is restric- ted. Although its hepatotoxicity has been demon- strated unequivocally in the rat (Hagen, Hansen, Fitz- hugh, Jenner, Jones, Taylor, Long, Nelson & Brouwer, 1967; Hazleton, Tusing, Zeitlin, Thiessen & Murer, 1956), conflicting evidence has been obtained on the carcinogenicity of coumarin in that species. BIr & Griepentrog (1967) and Griepentrog (1973) reported the occurrence of bile-duct carcinoma in rats fed coumarin at 5000 or 6000 ppm in the diet for 2 yr. In contrast, Hagan et al. (1967) detected neither benign nor malignant tumours in the liver or any other organs of rats fed dietary levels up to 5000 ppm for 2 yr, although they did observe slight fatty change, bile-duct proliferation and minimal focal necrosis at 2500 and 5COOppm and cholangiofibrosis at the 5000-ppm level. In a mouse-skin bioassay, coumarin showed no tumour-initiating activity (Roe & Sala- man, 1955).

Metabolic studies on coumarin in the rat (Booth, Masri, Robbins, Emerson, Jones & DeEds, 1959; Kaighen & Williams, 1961; Van Sumere & Teuchy, 1971), the hamster (Gangolli, Shilling, Grass0 & Gaunt, 1974), the baboon (Gangolli et al. 1974) and man (Shilling, Crampton & Longland, 1969) indicated considerable species differences in its metabolic fate (Cohen, 1979). This suggests that different species may have different levels of susceptibility to the chronic hepatotoxicity of coumarin.

The Syrian golden hamster has been reported to develop bile-duct proliferation and liver cysts spon- taneously, together with other lesions and malignant turnours (Della Porta, Shubik & Scortessi, 1959; Fortner, 1957). A continuum of lesions from bile-duct hyperplasia to cholangioadenoma and cholangiocar- cinema was induced in the hamster by a chemical

carcinogen (tacetamidofluorene) in the study reported by Della Porta et al. (1959). The subject of chemical carcinogenesis in hamsters has been compre- hensively reviewed by Homburger (1979a,b). In view of the susceptibility of the hamster to known liver carcinogens, a study on coumarin was undertaken in this species.

Experimental

Crystalline coumarin (99% pure), from Tokyo Kasei Kogyo Co. Ltd, Tokyo, was administered at 0.1 and 0.5% in the diet to Syrian golden hamsters (from Hoshino Co., Tokyo). The composition of the basal diet (CE-2 powder; Nihon CLEA Co. Ltd, Tokyo) has been described by Hirono, Mori, Yamada, Hirata, Haga, Tatematsu & Kanie (1977).

When 8 wk old, the hamsters were divided into three groups and housed five or six per cage, separ- ated by sex and treatment. The basal (control) diet was fed to 12 males and 12 females, @loA coumarin to 11 males and 13 females and 0.5% to 11 males and 10 females, for up to 2 yr. Food and water was available ad lib. Food intake was measured weekly for the first 6 months of treatment and body weight was recorded monthly. All animals that died during the experimdnt or that were killed when moribund or at termination were autopsied. After macroscopic examination, tissues were fixed in 10% buffered formalin, sectioned and stained with haematoxylin and eosin for histo: pathological study.

The chi-square test was used for statistical analysis of the incidence of tumours, cystic liver lesions and fatty degeneration.

Results and Discwioa

Hamsters in the 0.1 and 0.5% groups showed a transient reduction in food intake, this being about

353

354 I. UENO and I. HIRONO

Table 1. Surricul und in&ewe of ucrrious tumours und hepatic lesions in hamsters fed coumarin at 0-0~5~~ in the diet for 2 pr

Values for groups of hamsters fed coumarin at dietary levels (“/A) of:

0 (Control) 0.1 0.5

M F M F M F

Initial size group 12 12 11 13 11 10 No. surviving at month 15 11 8 10 5 11 8

18 10 7 1 2 11 1 22 9 3 2 0 10 2 24 8 2 1 0 4 1

No. of hamsters with: Forestomach papilloma 1 1 2 Adrenal cortical adenoma I 2 2 4 2 Adrenal phaeochromocytoma 1 Adrenal haemangioma 1 Pancreatic islet-cell carcinoma 2 Uterine leiomyoma 1 1 Thymic lymphoma (histiocytic) 1 Haemangioma 1 Leukaemia 1 Mesenteric lymph-node sarcoma 1 Sarcoma in inguinal region 1 Hepatic fatty degeneration 8 4 5 7 8 3 Cystic liver lesion 3 3 2 6

Total tumour-bearing animals (“,)t 8 42 18 23 45* 4i

tExpressed as a percentage of the initial group size. Some animals developed more than one type of tumour.

The value marked with an asterisk differs significantly (*P c 0.05 by the chi-square test) from that for the male control group.

20% lower than in controls at 1 month but returning to normality by month 5. Growth retardation was not apparent in either coumarin-treated group. Although survival in the 05% group was similar to that in con- trols, animals fed 0.1% coumarin showed poor sur- vival (Table 1).

The incidence of hepatic lesions (fatty degeneration or cysts) and of tumours at various sites in the control and test groups is also shown in Table 1. Although the number of tumour-bearing animals in the males fed 0.5% coumarin was greater than in control males (45 r. 8%, expressed as a percentage of initial group size), it was of the same order as that in control females (42%). There was no consistent difference in the incidence of any given tumour between test and control hamsters.

Of particular note was the absence of cholangio- adenoma or cholangiocarcinoma in test animals and the fact that the minimal bile-duct proliferation ob- served in some animals on @I or 0.5% coumarin was also seen in the controls. These negative findings con- trast strikingly with observations in coumarin-fed rats. notably the bile-duct carcinomas reported by Bar & Griepentrog (1967) and Griepentrog (1973) and the focal bile-duct proliferation of atypical appear- ance with fibrosis (cholangiofibrosis) observed by Hagan et al. (1967).

Liver cysts develop spontaneously in Syrian golden hamsters (Fortner. 1957: Della Porta et al. 1959). In the present study cysts, all with the same histological structure, occurred with similar frequency in test and control animals (25, 18 and 557; in males fed 0.0, 0.1 and 0.57; coumarin and 25, 0 and SO”, in the corre-

sponding female groups). Liver cysts appeared earlier in females on 0.5% coumarin than in other groups of either sex.

Fatty degeneration has been induced in rats fed coumarin at and above 2500 ppm for 90 days (Hazle- ton et al. 1956) or 2 yr (Hagan et al. 1967). In the hamster, the magnitude and frequency of this lesion were similar in the control and test groups, the inci- dence being 67, 45 and 73% in males on 0.0, 0.1 and 0.5% coumarin and 33, 54 and 30% in females on the corresponding levels.

Only limited conclusions can be drawn from this study because of the relatively small number of ani- mals used. Nevertheless, it does suggest that the Syr- ian golden hamster is refractory to the hepatotoxic action of coumarin, and in particular fails to react like the rat in the development of cholangiofibrosis or supposed cholangiocarcinoma.

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