399

Mutation Research, 259 (1991) 399-410 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100071V

MUTGEN000~

Carcinogenicities of heterocyclic amines in cooked food Hiroko Ohgaki *, Shozo Takayama and Takashi Sugimura National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo (Japan) (Received 15 September 1990) (Accepted 17 October 1990)

Keywords: Heterocyclic amines; Carcinogenicity

Contents Summary ................................................................................... Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carcinogenic effects of long-term oral administration of heterocyclic amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MelQx .................................................................................. IQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MelQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trp-P-1 and Trp-P-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GIu-P-1 and Glu-P-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M e A a C and A a C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PhlP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of combinations of heterocyclic amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Long-term subcutaneous carcinogenicity experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Initiating activities of heterocyclic amines in 2-stage carcinogenesis models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

399 400 d00 401 401 402 404 405 405 406 406 406 407 407

Summary Mutagenic heterocyclic amines in cooked foods were carcinogenic to mice, rats and/or monkeys, when they were given orally continuously. The most common target organ was the liver, but in CDF~ mice lung tumors, forestomach tumors, lymphomas/leukemias, and blood vessel tumors in the brown adipose tissues were also induced. In F344 rats, in addition to liver tumors, tumors in the Zymbal gland, skin, clitoral gland, small and large intestines, oral cavity, and mammary gland were also induced. Monkeys given IQ developed metastatic hepatocellular carcinomas.

Correspondence: Dr. T. Sugimura, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo (Japan). * Present Address: Abteilung Neuropathologie, Institut f'tir Pathologic, Universit~itsspital, CH-8091 Zurich (Switzerland). Abbreviations: MelQx, 2-amino-3,8-dimethylimidazo[4,5-f]-

quinoxaline; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MelQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline; Trp-P-1, 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole; Trp-P-2, 3amino-l-methyl-5H-pyrido[4,3-b]indole; Glu-P-1, 2-amino-6methyldipyrido[1,2-a : 3',2'-d]imidazole; GIu-P-2, 2-aminodipyrido[1,2-a : 3',2'-d]imidazole; M e A a C , 2-amino-3-methyl9H-pyrido[2,3-b]indole; A a C , 2-amino-9H-pyrido[2,3-b]indole; PhlP, 2-amino-1 - methyl-6-phenylimidazo[4,5-b ]pyridine.

400

Introduction

[4,5-b]pyridine (PhlP) from fried ground beef (Felton et al., 1986). These heterocyclic amines were also present in cigarette smoke condensates (Sugimura et al., 1988, 1989). These compounds were found to be metabolically activated by the P448 type of cytochrome P450 (Kato and Yamazoe, 1987). DNA adducts were detected in the liver of rats after administration of various heterocyclic amines using 32p-postlabeling methods (Yamashita et al., 1988). In this article, we summarize all the available data of the carcinogenicities of heterocyclic amines in rats, mice and monkeys, and also discuss the risk of these chemicals to humans.

Epidemiologically, it has been clearly demonstrated that life style, especially foods eaten daily, greatly modulates the frequency of human cancers. This finding suggests that environmental carcinogens, especially those in the ordinary diet, play an important role in the development of human cancer. In 1977, we found that the charred parts of broiled fish and meat were mutagenic in the Ames Salmonella assay (Sugimura et al., 1977a; Nagao et al., 1977). After that, a series of highly mutagenic compounds, the heterocyclic amines, have been isolated and identified from pyrolysates of amino acids, protein, and protein-rich foods. They were 2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MelQx) from broiled beef (Kasai et al., 1981), 2-amino-3-dimethylimidazo[4,5-f ]quinoline (IQ) and 2-amino-3,4-methylimidazo[4,5-f]quinoline (MelQ) from broiled fish (Kasai et al., 1980a, b), 3-amino- 1,4-dimethyl-5H-pyrido[4,3-b ]indole (Trp-P-1) and 3-amino-l-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) from tryptophan pyrolysates (Sugimura et al., 1977b), 2-amino-6-methyldipyrido [1,2-a : 3',2'-d]imidazole (Glu-P-1) and 2-aminodipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-2) from glutamic acid pyrolysate (Yamamoto et al., 1978), 2-amino-3-methyl-9H-pyrido[2,3-b ]indole (MeA~C), and 2-amino-9H-pyrido[2,3-b]indole (AaC) from soybean globulin pyrolysate (Yoshida et al., 1978), and 2-amino-l-methyl-6-phenylimidazo-

Carcinogenic effects of long-term oral administration of heterocyclic amines Synthetic MelQx, IQ, MelQ, Trp-P-1 acetate, Trp-P-2 acetate, Glu-P-1 • HC1 and Glu-P-2 • HC1, MeAaC acetate, A a C acetate and PhlP- HC1 were added to a pellet diet (CE-2; CLEA Japan, Tokyo) at appropriate concentrations of 0.01-0.08%, judged from the results of preliminary experiments on the effects of their administration at various concentrations for 4 weeks. The purities of the heterocyclic amines were determined by elementary analysis, infrared and mass spectrometry and HPLC. The presence of more than 83% of the added heterocyclic amines in the pellet diet was confirmed by HPLC after their extraction with methanol.

TABLE 1 I N C I D E N C E S OF T U M O R S IN C D F 1 M I C E A N D F344 RATS G I V E N Me l Q x Sex

C D F 1 mice

F344 rats

MeIQx (0.06%) None

MelQx (0.04%) None

Effective number

N u m b e r of animals with tumors (%) Liver

Lung

L y m p h o m a and leukemia

M F M F

37 35 36 39

16 (43) * 32 (91) * 6 (17) 0 (0)

16 (43) 16 (43) * 10(28) 4(10)

11 10 2 11

M F M F

20 19 19 20

20 (100) * 10 (53) * 0 0

0 0 0 0

0 0 0 0

(29) * (28) (5) (28)

Zymbal gland 0 0 0 0 15 (75) * 10 (53) * 0 0

Clitoral gland 0 0 12 (63) 0

Skin 0 0 0

1 (3) 7 (35) * 1 (5) 0 0

* Statistically significantly higher than the respective controls ( p < 0.05) (based on Ohgaki et al., 1987; Kato et al., 1988).

401 Most long-term experiments were performed on C D F 1 ( ( B A L B / c A n N × DBA/2N)F1) mice or F344 rats. C D F 1 mice and F344 rats of both sexes were obtained from Charles River Japan (Atsugi, Kanagawa). Mice were 6-7 weeks old and rats were 8 weeks old at the start of the experiments. All animals were given pellet diets containing a heterocyclic amine continuously during the experiment. Control animals were given basal diets. All animals that died or became moribund during the experiment were carefully autopsied. All major organs were fixed in 15% neutralized formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined histologically. The significance of the differences in the incidences of tumors in experimental and control groups was examined by the X2 test.

MelQx (a) Carcinogenicity in mice. In CDF 1 mice given 0.06% MelQx in the diet, liver tumors developed with a high incidence. These tumors were multiple yellowish-white nodules and most were identified histologically as hepatocellular carcinomas and hepatocellular adenomas. Females were more susceptible than males to the hepatocarcinogenic effect of MelQx. The incidences of lung tumors, and lymphomas and leukemias were also significantly higher in females and males, respectively, in test groups than in controls (Table 1). Although the incidence of lymphomas and leukemias was not significantly different in experimental and control groups of females, the time of their appearance in the female experimental group was much earlier than that in the female control group. Lung tumors were solid, white nodules which were identified histologically as adenocarcinomas and adenomas. In mice with lymphoma or leukemia, splenomegaly and marked enlargement of lymph nodes at various sites were frequently observed. Histological examination showed that the tissues of the spleen and lymph nodes were often replaced by diffusely proliferating lymphoblastic cells. Infiltration of neoplastic lymphoid cells into the liver and various other organs and marked invasion of neoplastic

lymphoid cells into the blood stream were also seen in many animals (Ohgaki et al,, 1987). The effect of the dose of MelQx on the incidence of cancer was also examined. The incidences of liver tumors in mice given diets containing 0.06%, 0.02%, 0.006% and 0.002% MelQx for 83 weeks were 82%, 3%, 0% and 0%, respectively. Although no tumors developed in the livers of mice in the 0.006% group, the level of D N A adducts was about 1 / 1 0 of that in the livers of mice in the 0.06% group (Ohgaki et al., 1990).

(b) Carcinogenicity in rats. On administration of MelQx at 0.04% in the diet to F344 rats ~for 61 weeks, tumors developed in the liver, Zymbal gland, clitoral gland and skin (Table 1). The incidence of liver tumors was significantly higher in males than in females. Moreover, 95% of the liver tumors in males were hepatocellular carcinomas and 30% of the males with hepatocellular carcinomas were found to have metastases to the lung, whereas all the liver tumors in females were neoplastic nodules. Thus, in contrast to the results with mice, results with rats showed that males were more susceptible than females to the hepatocarcinogenic effect of MelQx. Most tumors in the Zymbal gland, clitoral gland and skin were identified as squamous cell carcinomas (Kato et al., 1988).

I0 (a) Carcinogenicity in mice. High incidences of liver, forestomach and lung tumors were observed in C D F 1 mice of both sexes given 0.03% IQ in the diet for 96 weeks (Table 2). The liver tumors were hepatocellular carcinomas or hepatocellular adenomas. Most hepatocellular carcinomas had a trabecular pattern. The incidence of liver tumors was higher in females than in males. The forestomach tumors were papillomas or squamous cell carcinomas. The lung tumors were solid, white nodules, identified histologically as adenomas or adenocarcinomas (Ohgaki et al., 1984a). (b) Carcinogenicity in rats. IQ was given to male and female F344 rats for 55 and 72 weeks, respectively. IQ (0.03% in the diet) induced high

402 TABLE 2 INCIDENCES OF TUMORS IN CDF1 MICE, F344 RATS AND SPRAGUE-DAWLEY RATS GIVEN IQ Sex Effective Number of animals with tumors (%) number Liver Lung ForeIntestine stomach Small Large CDF1 mice IQ (0.03%) None

M 39 F 36 M 33 F 38

16 (41)* 27 (75) * 3 (9) 3 (8)

F344rats

IQ (0.03%) None

M F M F

27 (68)* 18 (45)* 1 (2) 0

SpragneDawley rats

Gastric a F intubation Vehicle F

40 40 50 50

27 (69)* 15 (42) * 7(21) 7(18)

16 (41)* 11 (31) * 1 (3) 0 (0)

3 (8) 0 1 (3) 0

0 0 0 0

Clitoral gland 0 0 0 0

Skin 0 0 0 1 (3)

0 0 0 0

0 0 0 0

32

6(19) * 0

0

0

0

0

0

27

0

0

0

0

0

0

0

Zymbal Mammary gland gland 0 0 0 0

12 (30)* 25 (63)* 17 (43)* 36 (90)* 1 (3) 9(23)* 20 (50)* 3 (8) 27 (68)* 0 0 0 0 0 0 0 0 0 0

0 1 (3) 0 0 0 0 0 0

11 (34) * 14 (44) * 0 (0)

2 (74)

a IQ was given by gavage (0.4 mmole/kg b.w.) weekly for 31 weeks starting with 6-week-old animals. * Statistically significantly higher than the respective controls ( p < 0.05) (based on Ohgaki et al., 1984a; Takayama et al., 1984b; Tanaka et al., 1985).

incidences of t u m o r s in the liver, s m a l l a n d large intestines, Z y m b a l gland, clitoral g l a n d a n d skin in F 3 4 4 rats ( T a b l e 2). M o s t liver t u m o r s were h e p a t o c e l l u l a r c a r c i n o m a s . T h e t u m o r s in the small a n d large intestines s h o w e d m u l t i p l e p o l y p o i d growth, a n d m o s t of t h e m were a d e n o c a r c i n o m a s , r e s e m b l i n g h u m a n colon c a n c e r in histological features. Small i n t e s t i n a l t u m o r s were m a i n l y l o c a t e d in the t e r m i n a l ileum, a n d c o l o n t u m o r s were m a i n l y l o c a t e d in the m u c o s a 3 - 8 c m a b o v e the a n o r e c t a l j u n c t i o n . M o s t t u m o r s in the Z y m b a l g l a n d , clitoral g l a n d a n d skin were s q u a m o u s cell carcinomas, and metastases of squamous cell c a r c i n o m a s o f the Z y m b a l g l a n d s to the lung were f o u n d in 4 m a l e a n d 2 female rats ( T a k a y a m a et al., 1984b). I Q (0.4 m m o l e / k g b o d y weight) was given to f e m a l e S p r a g u e - D a w l e y rats b y gavage ( T a n a k a et al., 1985). Rats, 6 weeks o l d at the start o f the e x p e r i m e n t , were given I Q 3 times a week in e x p e r i m e n t a l weeks 1 - 4 , twice a week in weeks 5 - 8 , a n d once a week in weeks 9 - 3 1 . A l l rats were killed in week 52. H i g h incidences of m a m m a r y tumors, liver t u m o r s a n d ear d u c t t u m o r s were o b s e r v e d in I Q - t r e a t e d a n i m a l s ( T a b l e 2).

(c) Carcinogenicity in monkeys. T w o g r o u p s of 20 C y n o m o l g u s m o n k e y s a r e n o w b e i n g given I Q b y gastric i n t u b a t i o n 5 times a w e e k at doses o f 10 a n d 20 m g / k g b o d y weight, respectively, c o n t i n u ously. T o date, 1 female given 20 m g o f I Q / k g for 27 m o n t h s ( t o t a l dose 19.4 g), 1 m a l e given 20 m g o f I Q / k g for 32 m o n t h s (total d o s e 26.5 g) a n d 1 m a l e given 10 m g / k g o f I Q for 37 m o n t h s (total d o s e 22.2 g) h a v e d e v e l o p e d h e p a t o c e l l u l a r c a r c i n o m a s with m e t a s t a s e s to the r e g i o n a l l y m p h n o d e s a n d lungs. T h e time p e r i o d for t u m o r i n d u c tion was similar to that for i n d u c t i o n o f t u m o r s in C y n o m o l g u s m o n k e y s receiving o r a l doses o f Nn i t r o s o d i e t h y l a m i n e . Since the r e m a i n i n g m o n k e y s in this e x p e r i m e n t have revealed liver a b n o r m a l i ties, it is p r e d i c t e d that a high p e r c e n t a g e of m o n k e y s given I Q will d e v e l o p m a l i g n a n t t u m o r s ( A d a m s o n et al., 1990). MelQ (a) Carcinogenicity in mice. C D F 1 mice were given M e l Q at c o n c e n t r a t i o n s of 0.01% a n d 0.04% in the diet. T h e average b o d y weights o f the 0.04% a n d 0.01% g r o u p s were 7 0 - 8 0 % a n d 90%, respec-

403 IOC Male

Female

"-~ 2!

4o

6o

80

40

6o

8o

Weeks

Fig. 1. Cumulative incidences of liver and forestomach tumors induced by MeIQ in CDF1 mice. • •, 0.01% MelQ; • m, 0.04% MelQ.

tively, of those of c o n t r o l s t h r o u g h o u t the experiment. Since the f o o d i n t a k e of mice in the 0.04% g r o u p was less t h a n that in the 0.01% group, the a c t u a l M e l Q i n t a k e in the 0.04% g r o u p was a b o u t 3 times that in the 0.01% group. T h e target organs o f M e l Q in C D F 1 mice were the f o r e s t o m a c h a n d liver ( T a b l e 3). D o s e - r e s p o n s e relationships were o b s e r v e d in the d e v e l o p m e n t o f f o r e s t o m a c h t u m o r s in b o t h sexes a n d in that of liver t u m o r s in females. Liver a n d f o r e s t o m a c h t u m o r s a p p e a r e d earlier in the 0.04% g r o u p t h a n in the 0.01% g r o u p (Fig. 1). F o r e s t o m a c h t u m o r s were multiple, white p o l y p o i d t u m o r s t h a t d e v e l o p e d at various sites in the f o r e s t o m a c h . I n m a n y cases, a l m o s t the whole f o r e s t o m a c h was r e p l a c e d b y t u m o r o u s masses.

Histologically, m o s t f o r e s t o m a c h t u m o r s were dia g n o s e d as p a p i l l o m a s a n d s q u a m o u s cell c a r c i n o m a s . I n a d v a n c e d cases, these t u m o r s ext e n d e d b e y o n d the l i m i t i n g r i d g e a n d i n v a d e d the g l a n d u l a r s t o m a c h . It is n o t e w o r t h y that a b o u t 40% of the s q u a m o u s cell c a r c i n o m a s t h a t develo p e d in the 0.04% g r o u p s m e t a s t a s i z e d to the liver. L y m p h a t i c s p r e a d of n e o p l a s t i c cells in the gland u l a r s t o m a c h was also f r e q u e n t l y o b s e r v e d ( O h g a k i et al., 1986). W e are n o w e s t a b l i s h i n g m e t a s t a t i c cell lines f r o m these m e t a s t a t i c fores t o m a c h s q u a m o u s cell c a r c i n o m a s .

(b) Carcinogenicity in rats. M e I Q (0.03% in diet) i n d u c e d t u m o r s in the Z y m b a l g l a n d , oral cavity, colon, skin a n d m a m m a r y g l a n d s in F 3 4 4 rats b y week 40 ( T a b l e 3). T h e first t u m o r s o f the Z y m b a l gland, oral c a v i t y a n d skin were recognized in 3 s e p a r a t e rats in week 20. M o s t t u m o r s o f the o r a l c a v i t y initially d e v e l o p e d o n the lip, a n d then g r a d u a l l y e x p a n d e d to the entire m u c o s a of the oral c a v i t y a n d o u t e r surface of the cheek. Histologically, these t u m o r s were i d e n t i f i e d as s q u a m o u s cell c a r c i n o m a s o r s e b a c e o u s s q u a m o u s cell c a r c i n o m a s . C o l o n t u m o r s were a d e n o c a r c i n o m a s o r a d e n o m a s . Skin t u m o r s were localized o n the back, f r e q u e n t l y f o r m i n g ulcers a n d were m o s t l y multiple. T h e y were m a i n l y i d e n t i f i e d as s q u a m o u s cell c a r c i n o m a s . M a m m a r y g l a n d t u m o r s were i d e n t i f i e d as a d e n o c a r c i n o m a s ( K a t o et al., 1989).

TABLE 3 INCIDENCES OF TUMORS IN CDF1 MICE AND F344 RATS GIVEN MelQ MelQ

Sex

Effective number

Number of animals with tumors (%)

Liver

Forestomach

CDF1 mice

MelQ (0.04%) MeIQ (0.01%) None

M F M F M F

38 38 38 26 29 40

7 (18) 27 (71) * 11 (29) 4 (11) * 4 (14) 0

35 (92) * 34 (89) * 7 (18) * 9 (53) * 0 0

F344 rats

MelQ (0.03%) None

M F M F

20 20 20 20

1 (5) 0 0 0

1 (5) 0 0 0

Zymbal gland 0 0 0 0 0 0 19 (95) * 17 (85) * 0 0

Oral cavity

Colon

0 0 0 0 0 0

0 0 0 0 0 0

7 (35) * 7 (35) * 0 0

7 (35) * 5 (25) * 0 0

Skin 0 0 0 0 0 0 10 (50) * 1 (5) 0 0

Mammary gland 0 0 0 1 (4) 0 1 (3) 0 5 (25) * 0 0

* Statistically significantly higher than the respective controls (p < 0.05) (based on Ohgaki et al., 1986; Kato et al., 1989).

404 TABLE 4 I N C I D E N C E S OF T U M O R S IN C D F 1 MICE, A N D F344 RATS A N D ACI RATS G I V E N Trp-P-1 A N D Trp-P-2 Dose (% in the diet)

Sex

Effective number

N u m b e r of animals with liver tumors (%)

Trp-P-1

0.02%

Trp-P-2

0.02%

M F M F M F

24 26 25 24 25 24

5 16 4 22 1 0

0.015% 0.02%

M F M F

40 40 50 50

30 (75) * 37 (93) * 1 (2) 0

0.01

M F M F

10 9 30 30

0 7 (78) * 0 0

C D F 1 mice

None

F344 rats

Trp-P-1 None

ACI rats

Trp-P-2 None

(21) (62) * (16) (92) * (4) (0)

* Statistically significantly higher than the respective control ( p < 0.05) (based on Matsukura et al., 1981; T a k a y a m a et al., 1985b; Hosak a et al., 1981).

Trp-P-1 and Trp,P-2 (a) Carcinogenicity in mice. Trp-P-1 and TrpP-2 induced hepatocellular carcinomas and hepatocellular adenomas in C D F 1 mice (Table 4)

(Matsukura et al., 1981). Females were more susceptible than males to the hepatocarcinogenic effects of both Trp-P-1 and Trp-P-2, and the incidence of liver tumors in female mice given Trp-P-2 was higher than that in females given Trp-P-1.

TABLE 5 I N C I D E N C E S OF T U M O R S IN C D F 1 M I C E A N D F344 RATS G I V E N Glu-P-1 A N D Glu-P-2 Sex

C D F 1 mice

Glu-P-1

0.05%

GIu-P-2

0.05%

No ne

F344 rats

GIu-P-1

0.05%

GIu-P-2

0.05%

None

Effective number

N u m b e r of animals with tumors (%) Liver

Blood

Intestine

vessels

Small

M F M F M F

34 38 37 36 39 40

4 37 10 36 0 0

(12) (97) * (27) * (100) *

30 31 27 20 0 0

M F M F M F

42 42 42 42 50 50

35 (83) * 24 (57) * 11 (26) * 2 (5) 2 (4) 0

0 0 0 0 0 0

(88) (82) (73) (56)

* * * *

Large

0 0 0 0 0 0 26 10 14 8 0 0

0 0 0 0 0 0 (62) (24) (35) (19)

* * * *

19 7 6 8 0 0

Zymbal

Clitoral

gland

gland

0 0 0 0 0 0 (45) (17) (14) (19)

* * * *

18 18 1 7 0 0

0 0 0 (43) * (43) * (2) (17) *

5 (12) * 11 (26) * 0

* Statistically significantly higher than the respective controls ( p < 0.05) (based on Ohgaki et al., 1984b; T a k a y a m a et al., 1984a).

405

(b) Carcinogenicity in rats.

Trp-P-1 also induced liver tumors in F344 rats (Table 4) ( T a k a y a m a et al., 1985b). A n experiment on the effect of Trp-P-2 was also carried out for 2 years, but results were negative (unpublished data). Thus, in F344 rats, the carcinogenic effects of Trp-P-1 and Trp-P-2 were different. A long-term experim e n t on the carcinogenicity of Trp-P-2 at higher concentrations to F344 rats is in progress. H o s a k a et al. (1981) reported that on treatment with 0.01% Trp-P-2 for 9 5 - 1 2 4 weeks, 6 neoplastic nodules and a hemangioendothelial sarcoma of the liver developed in 9 female A C I rats, but no liver tumors developed in 10 male A C I rats (Table

4).

TABLE 6 INCIDENCE OF TUMORS IN CDF1 MICE GIVEN MeAaC AND AaC Sex Effective Number of animals number with tumors (%) Liver CDF1 mice MeAaC (0.08%) AaC (0.08%) None

M F M F M F

37 33 38 34 39 40

Blood vessels

21 (57) * 35 (95) * 28 (85) * 28 (85) * 15 (39) * 20 (53) * 33 (97) * 6 (18) * 0 0 0 0

* Statistically significantly higher than the respective controls ( p < 0.05) (based on Ohgaki et al., 1984b).

Glu-P-1 and Glu-P-2 (a) Carcinogenicity in mice.

A t concentrations of 0.05% in the diet, Glu-P-1 and Glu-P-2 induced high incidences of liver tumors and blood vessel tumors in C D F a mice (Table 5). A b o u t 70-90% of the blood vessel tumors in each group were located in the interscapular b r o w n adipose tissue, but some were observed in the abdominal cavity, pleural cavity and axilla. MacroscopicaUy, the tumors were soft, hemorrhagic masses of 5 - 3 0 m m in diameter. Histologically, most tumors were identified as hemangioendothelial sarcomas and a few as hemangioendotheliomas. Hemangioendothelial sarcomas in the blood vessels were c o m p o s e d of typical endothelial cells which formed b o t h vascular channels and solid areas. Fig. 2 shows the times of appearance of liver and blood vessel IOC Male

Female

tumors though tumors than in

induced b y Glu-P-1 and Glu-P-2. Althe incidences of tumors were similar, appeared sooner in mice given Glu-P-1 those given Glu-P-2 (Ohgaki et al., 1984b).

(b) Carcinogenicity in rats. Glu-P-1 and GluP-2 induced t u m o r s in the liver, small and large intestines, ' Z y m b a l gland and clitoral gland of F344 rats (Table 5). The incidences of tumors at all sites except the clitoral gland were higher in rats given Glu-P-1 than in those given Glu-P-2. Most small intestinal tumors were located in the terminal i l e u m and most colon t u m o r s were located 2 and 1 2 c m above the anorectal j u n c t i o n and were frequently multiple. A s t r o c y t o m a s were found in the brain of 3 females given GIu-P-1 and 2 males given Glu-P-2. The increase in their incidence in these groups was not significant, but as spontaneous brain tumors are very rare in F344 rats, the appearance of these tumors m a y be related to treatment with these heterocyclic amines ( T a k a y a m a et al., 1984a). MeAetC and AaC

8 2~

Weeks

Fig. 2. Cumulative incidences of liver and blood vessel tumors induced by Glu-P-1 (O O) and Glu-P-2 (m II) in CDF] mice.

(a) Carcinogenicity in mice., M e A a C and A a C induced high incidences of liver tumors and blood vessel tumors when given to CDF~ mice , at a concentration of 0.08% in the diet (Table 6). The liver tumors induced were hepatocellular c a r c i n o mas and hepatocellular adenomas. Most hepatocellular carcinomas had a trabecular structure. In

406

females given AaC, 30% of hepatocellular carcinomas that developed metastasized to the lung. Most blood vessel tumors induced by MeAaC were located in the interscapular brown adipose tissue, like those induced by Glu-P-1 and Glu-P-2, but about half the blood vessel tumors induced by AaC were located in the abdominal cavity. These blood vessel tumors were hemangioendothelial sarcomas or hemangioendotheliomas (Ohgaki et al., 1984b). Fig. 3 shows the time of appearance of liver or blood vessel tumors induced by AaC and MeAaC. Tumors developed earlier in mice given MeAaC than those given AaC.

(b) Carcinogenicity in rats. Long-term experiments were made on the carcinogenicities to F344 rats of MeAaC and AaC at concentrations of 0.08% in the diet, but results were negative (Takayama et al., 1985a). Rats given MeAaC showed severe emaciation after month 8. At autopsy, salivary glands and pancreas were found to be severely atrophic and lesions were observed in about 70% of the rats fed MeAaC until the end of the experiment. Atrophy of the salivary glands was particularly apparent in the submaxillary glands. Histologically, marked atrophy of the cells in mucous and serous alveoli was most prominent. In the pancreas, marked atrophy of acinar cells, islets of Langerhans and pancreatic ducts was found. In rats given AaC, no atrophy of the salivary glands or pancreas were observed. The incidence of tumors in rats given MeAaC or AaC was not different from that in control rats. The

ioc

Femole

Merle

-

/

2!

~" 4o

60

80

ioo

~' ,;o

6O

Weeks

Fig. 3. Cumulative incidences of liver and blood vessel tumors induced by M e A a C ( e e ) and A a C (11 II) in C D F 1 mice.

TABLE 7 I N C I D E N C E O F T U M O R S IN CDF] M I C E GIVEN PhlP

C D F 1 mice

PhlP (0.04%) None

Sex

Effective number

N u m b e r of animals with l y m p h o m a (%)

M F M F

35 38 36 40

11 26 2 6

(31) * (68) * (6) (15)

• Statistically significantly higher than the respective controls ( p < 0.05) (based on Esumi et al., 1989).

levels of DNA adducts measured by the 32p-postlabeling method were high in salivary glands and pancreas of rats given MeAaC but they were low in those organs of rats given AaC, suggesting that atrophy in salivary glands and pancreas of rats given MeAaC was related to the DNA damage induced by this chemical (Yamashita et al., 1986).

PhlP Carcinogenicity in mice. CDF 1 mice were given PhlP at a concentration of 0.04% in the diet for 83 weeks. 31% of the males and 68% of the females fed PhlP developed lymphomas, which were of significantly higher frequencies than those in the respective controls. The most frequent sites of lesions were the mesenteric and mediastinal lymph nodes and spleen (Esumi et al., 1989) (Table 7). Effects of combinations of heterocyclic amines Takayama et al. (1987) administered Trp-P-1, Trp-P-2, Glu-P-2, AaC and IQ simultaneously to F344 rats. These compounds were added to the diet at 1/5 of the doses used in long-term carcinogenicity experiments on each compound alone (i.e., Trp-P-1, 0.003%; Trp-P-2, 0.004%; GIu-P-2, 0.01%; AaC, 0.016%; IQ, 0.006%). This combined treatment resulted in high incidences of tumors in the liver, colon, Zymbal gland, skin and clitoral gland, suggesting that the carcinogenic effects of these 5 heterocyclic amines were additive. Long-term subcutaneous carcinogenicity ments

experi-

Ishikawa et al. (1979) injected Trp-P-1 and Trp-P-2 s.c. into Syrian golden hamsters and F344

407

rats (1.5 mg/animal, once a week for 20 weeks), and terminated the experiment after 10 months. Subcutaneous sarcomas were induced at the site of injection of Trp-P-1 in 3 of 8 Syrian golden hamsters. Tumors were also induced in 5 female rats among 20 rats (10 males and 10 females) treated with Trp-P-1. No tumors were observed in animals treated with Trp-P-2. Newborn ICR mice were treated s.c. with either GIu-P-1 or Glu-P-2 (25 mg and 12.5 m g / k g b.w.) within 24 h after birth and were observed for 1 year. The incidences of lung tumors in the mice given Glu-P-1 or Glu-P-2 and of liver tumors in male mice given Glu-P-2 were significantly higher than those in controls (Fujii et al., 1988).

Initiating activities of heterocydic amines in 2-stage carcinogenesis models The initiating activities of heterocyclic amines were studied (Sato et al., 1987; Takahashi et al., 1986). The compounds were applied topically to the dorsal skin of female CD-1 mice twice weekly for 5 weeks in total doses of 20 mg of Trp-P-1, Trp-P-2, Glu-P-1 or Glu-P-2, 40 mg of MeAaC or

AaC, 5 mg of Lys-P-1, 7.5 mg of IQ, or 100 mg Phe-P-1, and then 12-O-tetradecanoylphorbol-13acetate (TPA) as a promoter was applied in the same way for 47 weeks. In groups treated with heterocyclic amines and TPA, tumors developed in 5-30% of the mice, whereas no tumors developed in groups treated with heterocyclic amines or TPA only. Trp-P-1, Trp-P-2, M e A a C and Phe-P-1 showed significant initiating activity in this experiment. IQ did not show initiating activity in female Sencar mice given TPA for 20 weeks after topical application of IQ (Barnes et al., 1985). Administration of an IQ diet at concentrations of 0.1, 0.05 or 0.025% for 2 weeks combined with partial hepatectomy and followed by phenobarbital or 3'-methyl-4-dimethylaminoazobenzene resulted in dose-dependent development of liver and thyroid tumors in F344 rats (Tsuda et al., 1988).

Discussion All 10 heterocyclic amines tested were shown to be carcinogenic to mice, rats a n d / o r monkeys.

TABLE 8 SUMMARY OF CARCINOGENICITIES OF HETEROCYCLIC AMINES IN CDF1 MICE Heterocyclic amine

Sex

Concentration in diet

Experimental period (weeks)

Target organs

References

84 84 96 96 91 91 89 89 89 89 57 68 84 83 73 84 98 98 82 82

Liver, hematopoietic system Liver, lung, hematopoietic system Liver, forestomach, lung Liver, forestomach, lung Forestomach Liver, forestomach

Ohgaki et al., 1987

Liver

Matsukura et al., 1981

Liver Blood vessels Liver, blood vessels Liver, blood vessels Liver, blood vessels Liver, blood vessels Liver, blood vessels Liver, blood vessels Liver, blood vessels Hematopoietic system Hematopoietie system

Matsukura et al., 1981 Ohgaki et al., 1984b

(~) MelQx IQ MelQ Trp-P-1 Trp-P-2 Glu-P-1 Giu-P-2 MeAaC AaC PhlP

M F M F M F M F M F M F M F M F M F M F

0.06 0.06 0.03 0.03 0.04 and 0.01 0.04 and 0.01 0.02 0.02 0.02 0.02 0.05 0.05 0.05 0.05 0.08 0.08 0.08 0.08 0.04 0.04

Ohgaki et al., 1984a Ohg~ki et al., 1986

Ohgaki et al., 1984b Ohgaki et al., 1984b Ohgaki et al., 1984b Esumi et al., 1989

408 This fact indicates the validity of a short-term m u t a t i o n assay with Salmonella typhimurium TA98 and TA100 as a screening test for carcinogens. As described in the text and summarized in Tables 8 and 9, heterocyclic amines were carcinogenic to various organs: the liver, forestomach, lung, hematopoietic system a n d blood vessels of mice and the liver, small and large intestines, Z y m b a l gland, clitoral gland, skin, oral cavity and m a m m a r y gland of rats. These results suggest that various kinds of cancers might also be induced by heterocyclic amines in humans. The liver was a c o m m o n target organ, but the other target organs of the respective heterocyclic amines differed in mice and rats. In m o n k e y s given IQ, hepatocellular carcinomas developed ( A d a m s o n et al., 1990), suggesting that I Q also induces liver cancer in humans. It is also possible that administration Of lower doses of IQ, which allow m o n k e y s a longer life span, p r o d u c e cancers in different organs in addition to the liver. In carcinogenicity experiments, sex differences in susceptibility were also observed. Female C D F 1 mice were more susceptible than males to the hepatocarcinogenic effects of all heterocyclic

amines tested. O n the other hand, male F344 rats were more susceptible than females to hepatocarcinogenesis induced b y IQ, MeIQx, Glu-P-1 and Glu-P-2. Therefore, the susceptibilities of men and w o m e n to heterocyclic amines m a y also be different. These species, sex and organ differences in susceptibility are considered to be at least partly related to differences in the a m o u n t and inducibility of c y t o c h r o m e P450, which is responsible for the activation of heterocyclic amines ( D e g a w a et al., 1987). The carcinogenic p o t e n c y of a chemical can be expressed as its TDs0 value, the daily dose that induces tumors in 50% of the animals tested when given throughout their lives ( G o l d et al., 1984). The TDs0 values of heterocyclic amines were 2 . 7 14.7 m g / k g / d a y in C D F 1 mice and 0.1-5.7 m g / k g / d a y in F344 rats. These TDs0 values are within a m o d e r a t e range c o m p a r e d with those of other carcinogens and are similar to those of N, N-dimethylnitrosamine and dibenz[ a, h ]anthracene. It is difficult to estimate the risk of heterocyclic amines for h u m a n s directly f r o m carcinogenicity experiments, in which doses several t h o u s a n d times

TABLE 9 SUMMARY OF CARCINOGENICITIES OF HETEROCYCLIC AMINES 1N RATS Heterocyclic amine

Sex

Concentration in diet (%)

MelQx

M F M

0.06 0.06 0.03

61 61 55

F

0.03

72

M

0.04

40

F

0.04

40

M F M F M

0.15 0.02 0.02 0.02 0.05

52 52 124 124 64

F

0.05

67

M F

0.05 0.05

104 104

IQ

MelQ

Trp-P-1 Trp-P-2 Glu-P-1

GIu-P-2

Experimental period (weeks)

Target organs

References

Liver, Zymbal gland, skin Liver, Zymbal gland, clitoral gland Liver, small and large intestine, Zymbal gland, skin Liver, large intestine, Zymbal gland, clitoral gland Large intestine, Zymbal gland, skin, oral cavity Large intestine, Zymbal gland, oral cavity, mammary gland Liver Liver

Kato et al.,

1988

Takayama et al., 1984b

Kato et al., 1989

Takayama et al., 1985b Hosaka et al., 1981

Liver Liver, small and large intestine, Zymbal gland Liver, small and large intestine, Zymbal gland, clitoral gland Liver, small and large intestine Small and large intestine, Zymbal gland, clitoral gland

Takayama et al., 1984b

Takayama et al., 1984b

409 higher were used t h a n the actual a m o u n t s in the h u m a n diet: the e s t i m a t e d d a i l y a m o u n t of heterocyclic a m i n e s p e r m a n , a s s u m i n g t h a t a p e r s o n c o n s u m e s 200 g o f fried b e e f a n d smokes 20 cigarettes, is a p p r o x i m a t e l y 3.5 /~g. I n our prel i m i n a r y study, the incidences of liver t u m o r s in m i c e given 0.06%, 0.02%, 0.006% a n d 0.002% of M e l Q x were 82%, 3%, 0% a n d 0%, respectively ( O h g a k i et al., 1990). Thus, from the results o f this e x p e r i m e n t , it s e e m e d that there was a t h r e s h o l d in carcinogenicity, i.e., a d m i n i s t r a t i o n o f low doses of c a r c i n o g e n s does n o t d e v e l o p cancers. However, the level of D N A a d d u c t s in various organs of mice given 0.006% M e l Q x was a p p r o x i m a t e l y 10% of those in the 0.06% group. T h e d i s c r e p a n c y in the presence of a d d u c t s a n d the a b s e n c e of c a r c i n o g e n i c i t y in the 0.006% g r o u p might b e exp l a i n e d b y the l i m i t e d n u m b e r s of mice used. T h e p r o b a b i l i t y o f o c c u r r e n c e o f m u t a t i o n s in oncogenes in a n i m a l s fed low doses of M e l Q x m i g h t h a v e b e e n t o o low to d e t e c t carcinogenicity in small n u m b e r s of animals. A similar p h e n o m e n o n was r e p o r t e d b y Y a m a s h i t a et al. (1990), that the level o f D N A a d d u c t s in the liver o f rats given M e l Q x in the diet at 0.00004%, 0.0004%, 0.004% a n d 0.04% for 1 week, were 0.04, 0.28, 3.34, 39.0 a d d u c t s / 1 0 7 nucleotides, showing the linear relat i o n s h i p b e t w e e n the dose a n d the level of adducts, suggesting t h a t heterocyclic a m i n e s c a n p r o d u c e D N A a d d u c t s at m u c h lower e x p o s u r e levels t h a n those usually used in c a r c i n o g e n i c i t y experiments, p r o b a b l y w i t h o u t a threshold. I n h u m a n cases, it is also p o s s i b l e that small n u m b e r s of D N A a d d u c t s are p r o d u c e d b y heterocyclic a m i n e s in c o o k e d f o o d s e a t e n daily.

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