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Induction of sister-chromatid exchanges by mutagens from amino acid and protein pyrolysates
65
Mutation Research, 77 (1980) 65--69 © Elsevier/North-Holland Biomedical Press
INDUCTION OF SISTER-CHROMATID EXCHANGES BY MUTAGENS FROM AMINO ACID AND PROTEIN P Y R O L Y S A T E S *
HIROKO TOHDA 1, ATSUSHI OIKAWA 1, TAKASHI KAWACHI 2 and TAKASHI SUGIMURA 2 1 Department o f Pharmacology, Research Institute for Tuberculosis and Cancer, Tohoku University, 4-1 Seiryo-machi, Sendai 980, and 2 National Cancer Center Research Institute, 5-1 Tsukiji, Chuo-ku, Tokyo 104 (Japan)
(Received 20 May 1979) (Revision received 28 July 1979) (Accepted 6 August 1979}
Summary Sister-chromatid exchanges (SCEs) in a permanent cell line of human lymphoblastoid cells were induced by 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-l-methyl-5H-pyrido [ 4,3-b ] indole (Trp-P-2), 2-amino6-methyldipyrido[ 1,2-a :3',2'-d] imidazole (Glu-P-1) and 2-amino-9H-pyrido[2, 3-b]indole (2-amino~-carboline). The first two compounds were found in tryptophan pyrolysates, the third in a glutamic acid pyrolysate and the last in a globulin pyrolysate. All these c o m p o u n d s required the metabolic activation system ($9 mix) for induction of SCE. Trp-P-2 had the highest SCE-inducing activity of these chemicals (approximately equivalent to that of aflatoxin B1), followed b y Trp-P-1, Glu-P-1 and then 2-amino-~-carboline.
Potent mutagenic activities have been found in pyrolysates of amino acids and proteins with S a l m o n e l l a t y p h i m u r i u m b y Sugimura et al. [7,8,10,14] and Matsumoto et al. [6]. The active substances were isolated from tryptophan pyrolysates and identified as 3-amino-l,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) by Sugimura and co-workers [5,13]. Similarly, the active substance in a glutamic acid pyrolysate was identified as 2-amino-6-methyl-dipyrido[1,2-a:3',2'~]imidazole (Glu-P-1) [21]. These 3 c o m p o u n d s are all newly identified chemicals. 2-Amino-9H-pyrido[2,3-b]indole (2-amino<~-carboline) was found in a pyrolysate of soybean globulin [22]. Trp-P-1 and Trp-P-2 could transform cryo-pre~erved * P a r t l y s u p p o r t e d b y grants f r o m the Ministry o f E d u c a t i o n a n d C u l t u r e , t h e M i n i s t r y o f H e a l t h a n d Welfare, a n d t h e A s s o c i a t i o n f o r t h e A d v a n c e m e n t o f C a n c e r R e s e a r c h .
66 Syrian hamster embryo cells in vitro [16] and these transformed cells could be transferred successively in the cheek pouch of Syrian hamsters [15]. Furthermore, Trp-P-1 and Trp-P-2 produced malignant fibrosarcomas at the site of injection in Syrian hamsters [4]. The mutagenic substances from pyrolysates of amino acids and proteins seem to be carcinogenic. Therefore, in the present study, sister-chromatid exchanges (SCEs) induced by these chemicals were examined with a permanent cell line of human lymphoblastoid cells, which we established previously [18,19]. All 4 chemicals induced SCEs. Materials and methods
Chemicals Synthetic Trp-P-1, Trp-P-2, and 2-amino~-carboline were obtained from Nard Institute Ltd., Osaka. Synthetic Glu-P-1 was obtained from Katsura Chemical Co., Tokyo. The syntheses of Trp-P-1 and Trp-P-2 were reported by Akimoto et al. [1]. The preparations were confirmed to be over 99% pure by thin-layer chromatography, paper chromatography, high-pressure liquid chromatography and mass spectrometry. Cell cultures A permanent human lymphoblastoid cell line, NL3 [18], was used. Culture medium RPMI 1640 supplemented with 20% foetal bovine serum (Flow Laboratory, Rockville, MD), 2 mM L-glutamine and kanamycin (50 pg/ml) was used throughout. Metabolic activation system $9 was prepared from the liver of rats treated with polychlorinated biphenyl (Kanechlor 500, 54% chlorinated) and supplied by courtesy of Dr. T. Matsushima of the Institute of Medical Science, University of Tokyo. Modified $9 mix was used, consisting of $9 (2.5% as liver homogenate), 8 mM MgC12, 33 mM KC1, 5 mM glucose 6-phosphate, 4 mM NADPH, 4 mM NADH and 0.1 M sodium phosphate buffer (pH 7.4) [9]. Induction o f SCEs Exponentially growing cells were inoculated at a density of 3--4 X 10 s cells per ml into culture medium containing 10 pM bromodeoxyuridine and cultured for 86 h to permit two cell cycles of growth. At the end of the first cell doubling the test compounds dissolved in an appropriate volume of dimethylsulphoxide (DMSO) and one tenth volume of $9 mix were added. The final concentration of DMSO was 0.5% or less, and the a m o u n t did not interfere with SCE induction. After 2-h incubation, the medium was removed and the cells were washed once with the medium. Incubation was then continued in fresh medium containing bromodeoxyuridine. At the end of culture, 1.3 pM colcemid was added, and 4 h later the cells were collected, treated with 0.05 M KC1 for 10 min, and fixed in methanol : acetic acid (3 : 1, v/v). For SCE analysis, fixed cells mounted on slides were processed by the "fluorescent plus Giemsa" technique with Hoechst 33258 as the fluorescent dye [11].
67 Results and discussion The dose responses of SCE induction by Trp-P-1, Trp-P-2, Glu-P-1 and 2-amino~-carboline in the presence and absence of $9 mix are shown in Table 1. SCE induction was greatly enhanced in all cases by metabolic activation catalysed by $9 mix. The slight SCE induction seen on treatment with Trp-P-1 or Trp-P-2 in the absence of $9 mix was probably due to weak metabolic activation by the cells themselves. The approximate concentrations of chemicals that induced 5 SCEs per cell more than the spontaneous number were estimated from the results, and are expressed in pg/ml and --log M in Table 2. The latter value can be regarded as approximately related to the SCE-inducing activity of the chemicals on a logarithmic scale. The values for 4-nitroquinoline-l-oxide, aflatoxin BI, N-nitroso-N-methylurea, benzo[a]pyrene and methylurea are also given in Table 2 for comparison. Many carcinogenic and mutagenic chemicals induce SCEs in mammalian cells [20], although the molecular mechanisms of spontaneous and induced SCE are n o t fully understood. The present results show that chemicals found in pyrolysates of amino acids and protein induced SCEs in eukaryotic cells. These SCEs could be produced by interaction of metabolically activated forms of these chemicals with DNA in h u m a n cell nuclei. It might be thought that humans may have become resistant to these pyrolytic mutagenic substances, because they have been exposed to these substances for a very long time by eating broiled foods and so they may have acquired the ability to induce less activation and more inactivation of these mutagens, and also may have developed an efficient mechanism for repair of DNA after its damage by these mutagens. However, it has been reported that Trp-P-1, Trp-
TABLE 1 INDUCTION OF SCEs BY Trp-P-I, Trp-P-2, GIu-P-1 AND 2-AMINO-<~-CARBOLINE Concentration (M)
SCEs/ceU
a
Trp-P-1
Trp-P-2
GIu-P-1
2-Amino-ce-c~Lrb oline
In the a b s e n c e o f $ 9 m i x 0 1 × 10 -7 1 X 10 -6
9.2 + 0.7 ---
8.4 + 0.6 8 . 0 -+ 0 . 5 8 . 7 _+ 0 . 7
9 . 6 -+ 1 . 0 ---
7 . 6 +_ 0 . 6 ---
1 × i 0 -$ 5 )< 1 0 - 5 1 X 10 -4
12.0 + 0.9 1 2 . 7 _+ 1 . 2 toxic b
10.3 + 1.0 -1 0 . 9 _+ 0 . 6
9.5 + 0.9 1 0 . 2 _+ 0 . 8 1 0 . 2 _+ 0 . 8
8.0 +_0.6 7 . 5 _+ 0 . 7 7 . 2 _+ 0 . 6
7 . 4 _+ 0 . 4 1 2 . 7 _+ 1 . 1
10.3 --
-+ 0 . 9
8 . 1 _+ 0 . 7 --
1 7 . 0 -+ 1 . 1 2 2 . 1 _+ 1 . 2 -toxic b
11.7 16.9 19.3 toxic
_+ 0 . 9 -+ 1 . 3 +_ 1.3 b
In the p r e s e n c e o f $ 9 m i x 0 1 X 10 -7 1 1 5 1
X X X ×
10 -6 I0 -5 I 0 -S 1 0 .-4
9 . 8 +_ 1 . 0 -12.6 20.1 20.8 toxic
_+ 1 . 1 -+ 1 . 3 _+ 1.3 b
9.7 10.5 15.7 17.3
a S C E s o f m o r e t h a n 3 0 c e l l s w e r e s c o r e d , a n d e x p r e s s e d as m e a n s _+ s t a n d a r d e r r o r s . b Chemicals were so toxic that SCEs could not be scored. s
_+ 1 . 0 +_ 0 . 8 + 1.6 + 1.7
68 TABLE 2 SCE-INDUCING A C T I V I T Y OF M U T A G E N S Carcinomutagens
$9 m i x
4 - N i t r o q u i n o l i n e 1-oxide c A f l a t o x i n B 1 c,d T r p -P-2 Trp-P-1 Nitrosomethylurea c Benzo[a]pyrene c Glu-P-1 2-Amino-(~-carboline M e t h y l u r e a ( c o n t r o l ) c,e
-+ + + _ + + + _
a b c d e
[ p g / m l ] 5s a 0.005 0.013 0.016 0.40 0.41 0.5 1.0 4.0 >>100
PM5s b 7.6 7.4 7.1 5.7 5.4 5.7 5.3 4.6 <<2.9
[ # g / m l ] 5s = c o n c e n t r a t i o n in p g / m l giving 5 i n d u c e d SCEs/cell. PM5s = - - t o g [ m o l a r c o n c e n t r a t i o n giving 5 i n d u c e d SCEs/ceU]. Ref. 17. I n t h e a b s e n c e of $9 m i x , [ ~ g / m l ] 5 s and PM5s are 6.2 and 4.7, resp. 1.33 m M m e t h y l u r e a ( 1 0 0 # g / m l ) d o e s n o t i n d u c e SCE.
P-2, Glu-P-1 and amino~-carboline were activated by human $9 preparation [12]. Moreover, the present results demonstrate that the human genetic apparatus is susceptible to these mutagens. Previous papers have reported the mutagenicity of these compounds in microbes [13,21,22], their carcinogenicity in vivo and in vitro [4,15], their DNAbinding ability [2,3], their ability to induce sperm abnormalities [12] and to form adducts with guanine [12]. The present paper provides further evidence of possible genetic/carcinogenic risk by these environmental mutagens to humans. References 1 A k i m o t o , H., A. K a w a i , H. N o m u r a , M. N a g a o , T. K a w a c h i a n d T. S u g i m u r a , S y n t h e s i s of P o t e n t m u t a g e n s in t r y p t o p h a n p y r o l y s a t e s , C h e m . L e t t . , ( 1 9 7 7 ) 1 0 6 1 - - 1 0 6 4 . 2 H a s h i m o t o , Y., K. S h u d o a n d T. O k a m o t o , Structt~ral i d e n t i f i c a t i o n o f a m o d i f i e d base in D N A c o v a l c n t l y b o u n d w i t h m u t a g e n i c 3-amino-l-methyl-5It-pyrido[4,3-b]indole, C h e m . Pharra. Bull., 27 (1979) 1058--1060. 3 H a s h i m o t o , Y., K. T a k e d a , K. S h u d o , T. O k a m o t o , T. S u g i m u r a a n d T. Kosuge, R a t liver m i c r o s o m e m e d i a t e d b i n d i n g t o D N A o f 3 - a r n i n o - l - m e t h y l - 5 H - p y r i d o [ 4 , 3 - b ] i n d o l e , a p o t e n t m u t a g e n isolated f r o m t r y p t o p h a n p y r o l y s a t e , Chem.-Biol. I n t e r a c t . , 23 ( 1 9 7 8 ) 1 3 7 - - 1 4 0 . 4 I s h i k a w a , T., S. T a k a y a m a , T. K a w a c h i , M. K i n e b u c h i , N. M a t s u k u r a , E. U c h i d a a n d T. S u g i m u r a , In vivo e x p e r i m e n t s o n t r y p t o p h a n p y r o l y s i s p r o d u c t s , in: I. H i r o n o , E.C. Miller, J . A . Miller, T. S u g i m u r a a n d S. T a k a y a m a (Eds.), N a t u r a l l y O c c u r r i n g C a r e i n o g e n s - - M u t a g e n s a n d M o d u l a t o r s o f Carcinogenesis, J a p a n Scientific Assoc. Press, T o k y o a n d Univ. Park Press, B a l t i m o r e , in press. 5 K o s u g e , T., K. Tsuji, K. W a k a b a y a s h i , T. O k a m o t o , K. S h u d o , Y. Iitaka, A. Itai, T. S u g i m u r a , T. K a w a c h i , M. N a g a o , T. Y a h a g i a n d T. Seino, I s o l a t i o n a n d s t r u c t u r e s t u d i e s o f m u t a g e n i c p r i n c i p l e s in a m i n o acid p y r o l y s a t e , C h e m . P h a r m . Bull., 26 ( 1 9 7 8 ) 6 1 1 - - 6 1 9 . 6 M a t s u m o t o , T., D. Y o s h i d a , S. Mizusaki a n d H. O k a m o t o , M u t a g e n i c a c t i v i t y o f a m i n o acid p y r o l y . z a t e s in Salmonella t y p h i m u r i u m T A 9 8 , M u t a t i o n Res., 4 8 ( 1 9 7 7 ) 2 7 9 - - 2 8 6 . 7 N a g a o , M., M. H o n d a , Y. Seino, T. Yahagi, T. K a w a c h i a n d T. S u g i m u r a , M u t a g e n i c i t i e s o f p r o t e i n pyrolysates, Cancer Lett., 2 (1977) 335--340. 8 N a g a o , M., M. H o n d a , Y. Seino, T. Y a h a g i a n d T. S u g i m u r a , M u t a g e n i c i t i e s of s m o k e c o n d e n s a t e s a n d the c h a r r e d s u r f a c e o f f i s h a n d m e a t , C a n c e r L e t t . , 2 ( 1 9 7 7 ) 2 2 1 - - 2 2 6 . 9 N a g a o , M., E. S u z u k i , K. Y a s u o , T. Yahagi, Y. Seino, T. S u g i m u r a a n d M. O k a d a , M u t a g e n i c i t y of N - b u t y l - N - ( 4 - h y d r o x y b u t y l ) n i t r o s a m i n e , a b l a d d e r c a r c i n o g e n , a n d r e l a t e d c o m p o u n d s , C a n c e r Res., 37 ( 1 9 7 7 ) 3 9 9 - - 4 0 7 . 10 N a g a o , M., T. Yahagi, T. K a w a c h i , Y. Seino, M. H o n d a , N. M a t s u k u r a , T. S u g i m u r a , K. W a k a b a y a s h i ,
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K. Tsuji a n d T. K o s u g e , M u t a g e n s in f o o d s , a n d e s p e c i a l l y p y r o l y s i s p r o d u c t s o f p r o t e i n , in: D. S c o t t , B.A. B r i d g e s a n d F . H . S o b e l s ( E d s . ) , P r o g r e s s in G e n e t i c T o x i c o l o g y , E l s e v i e r / N o r t h - H o l l a n d , A m sterdam, 1977, pp. 259--264. P e r r y , P., a n d S. W o l f f , N e w G i e m s a m e t h o d f o r t h e d i f f e r e n t i a l s t a i n i n g o f sister c h r o m a t i d s , N a t u r e (London), 251 (1974) 156--158. S u g l m u r a , T., N a t u r a l l y o c c u r r i n g g e n o t o x i e c a r c i n o g e n s , in: I. H i r o n o , E.C. Miller, J . A . Miller, T. S u g i m u r a a n d S. T a k a y a m a ( E d s . ) , N a t u r a U y O c c u r r i n g C a x c i n o g e n s - - M u t a g e n s a n d M o d u l a t o r s o f C a r c i n o g e n e s i s , J a p a n S c i e n t i f i c A s s o c . Press, T o k y o a n d Univ. P a r k Press, B a l t i m o r e , in p r e s s . S u g i m u r a , T., T. K a w a c h i , M. N a g a o , T. Y a h a g i , Y. S e i n o , T. O k a m o t o , K. S h u d o , T. K o s u g e , K. Tsuji, K. W a k a b a y a s h i , Y. I i t a k a a n d A. Itai, M u t a g e n i c p r i n c i p l e ( s ) in t r y p t o p h a n a n d p h e n y l a l a n i n e p y r o lysis p r o d u c t s , P r o c . J p n . A c a d . , 5 3 ( 1 9 7 7 ) 5 8 - - 6 1 . S u g i m u r a , T., M. N a g a o , T. K a w a c h i , M. H o n d a , T. Y a h a g i , Y. S e i n o , S. S a t o , N. M a t s u k u r a , T. Mats u s h i m a , A. S h i r a l , M. S a w a m u r a a n d H . M a t s u m o t o , M u t a g e n - - c a r e i n o g e n s in f o o d , w i t h s p e c i a l r e f e r e n c e t o h i g h l y m u t a g e n i c p y r o l y t i c p r o d u c t s in b r o i l e d f o o d s , in: H . H . H i a t t , J . D . W a t s o n a n d J.A. Winsten (Eds.), Origins of Human Cancer, Cold Spring Harbor Lab., Cold Spring Harbor, 1977, pp. 1561--1577. T a k a y a m a , S., T. H i x a k a w a a n d T. S u g i m u r a , M a l i g n a n t t r a n s f o r m a t i o n in v i t r o b y t r y p t o p h a n p y r o lysis p r o d u c t s , P r o c . J p n . A c a d . , 5 4 ( B ) ( 1 9 7 8 ) 4 1 8 - - 4 2 2 . T a k a y a m a , S., Y. K a t o h , M. T a n a k a , M. N a g a o , K. W a k a b a y a s h i a n d T. S u g l m u r a , In v i t r o t r a n s f o r m a t i o n o f h a m s t e r e m b r y o cells w i t h t r y p t o p h a n p y r o l y s i s p r o d u c t s , P r o c . J p n . A c a d . , 5 3 ( B ) ( 1 9 7 7 ) 126--129. T o h d a , H., a n d A. O i k a w a , 4 N Q O - a n d M N N G - i n d u c e d u n s c h e d u l e d D N A s y n t h e s e s a n d sister c h r o m a t i d e x c h a n g e s in X P l y m p h o b l a s t o i d cell lines, P r o c e e d i n g s o f t h e J a p a n e s e C a n c e r A s s o c i a t i o n , 3 6 t h A n n u a l M e e t i n g , 1 9 7 7 , p. 1 1 3 . T o h d a , H., A . O i k a w a , T. K a t s u k i , Y. H i n u m a a n d M. Seiji, A c o n v e n i e n t m e t h o d o f e s t a b l i s h i n g perm a n e n t l i n e s o f x e r o d e r m a p i g m e n t o s u m cells, C a n c e r Res., 3 8 ( 1 9 7 8 ) 2 5 3 - - 2 5 6 . T o h d a , H., A. O i k a w a , T. K u d o a n d T. T a c h i b a n a , A g r e a t l y s i m p l i f i e d m e t h o d o f e s t a b l i s h i n g B - l y m p h o b l a s t o i d cell lines, C a n c e r R e s . , 3 8 ( 1 9 7 8 ) 3 5 6 0 - - 3 5 6 2 . Wolff, S., Sister c h r o m a t i d e x c h a n g e , A n n u . R e v . G e n e t . , 11 ( 1 9 7 7 ) 1 8 3 - - 2 0 1 . Y a m a x n o t o , T., K. Tsuji, T. K o s u g e , T. O k a m o t o , K . S h u d o , K. T a k e d a , Y. I i t a k a , K . Y a m a g u c h i , Y. S e i n o , T. Y a h a g i , M. N a g a o a n d T. S u g l m u r a , I s o l a t i o n a n d s t r u c t u r e d e t e r m i n a t i o n o f m u t a g e n l c s u b s t a n c e s in L - g l u t a m i c a c i d p y r o l y s a t e , P r o c . J p n . A c a d . , 5 4 ( B ) ( 1 9 7 8 ) 2 4 8 - - 2 5 0 . Y o s h i d a , D., T. M a t s u m o t o , R. Y o s h i m u r a a n d T. M a t s u z a k l , M u t a g e n i e i t y o f a m i n o - ~ - c a r b o l i n e s in pyrolysis products of soybean globulin, Biochem. Biophys. Res. Commun., 83 (1978) 915--920.
Mutation Research, 77 (1980) 65--69 © Elsevier/North-Holland Biomedical Press
INDUCTION OF SISTER-CHROMATID EXCHANGES BY MUTAGENS FROM AMINO ACID AND PROTEIN P Y R O L Y S A T E S *
HIROKO TOHDA 1, ATSUSHI OIKAWA 1, TAKASHI KAWACHI 2 and TAKASHI SUGIMURA 2 1 Department o f Pharmacology, Research Institute for Tuberculosis and Cancer, Tohoku University, 4-1 Seiryo-machi, Sendai 980, and 2 National Cancer Center Research Institute, 5-1 Tsukiji, Chuo-ku, Tokyo 104 (Japan)
(Received 20 May 1979) (Revision received 28 July 1979) (Accepted 6 August 1979}
Summary Sister-chromatid exchanges (SCEs) in a permanent cell line of human lymphoblastoid cells were induced by 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-l-methyl-5H-pyrido [ 4,3-b ] indole (Trp-P-2), 2-amino6-methyldipyrido[ 1,2-a :3',2'-d] imidazole (Glu-P-1) and 2-amino-9H-pyrido[2, 3-b]indole (2-amino~-carboline). The first two compounds were found in tryptophan pyrolysates, the third in a glutamic acid pyrolysate and the last in a globulin pyrolysate. All these c o m p o u n d s required the metabolic activation system ($9 mix) for induction of SCE. Trp-P-2 had the highest SCE-inducing activity of these chemicals (approximately equivalent to that of aflatoxin B1), followed b y Trp-P-1, Glu-P-1 and then 2-amino-~-carboline.
Potent mutagenic activities have been found in pyrolysates of amino acids and proteins with S a l m o n e l l a t y p h i m u r i u m b y Sugimura et al. [7,8,10,14] and Matsumoto et al. [6]. The active substances were isolated from tryptophan pyrolysates and identified as 3-amino-l,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) by Sugimura and co-workers [5,13]. Similarly, the active substance in a glutamic acid pyrolysate was identified as 2-amino-6-methyl-dipyrido[1,2-a:3',2'~]imidazole (Glu-P-1) [21]. These 3 c o m p o u n d s are all newly identified chemicals. 2-Amino-9H-pyrido[2,3-b]indole (2-amino<~-carboline) was found in a pyrolysate of soybean globulin [22]. Trp-P-1 and Trp-P-2 could transform cryo-pre~erved * P a r t l y s u p p o r t e d b y grants f r o m the Ministry o f E d u c a t i o n a n d C u l t u r e , t h e M i n i s t r y o f H e a l t h a n d Welfare, a n d t h e A s s o c i a t i o n f o r t h e A d v a n c e m e n t o f C a n c e r R e s e a r c h .
66 Syrian hamster embryo cells in vitro [16] and these transformed cells could be transferred successively in the cheek pouch of Syrian hamsters [15]. Furthermore, Trp-P-1 and Trp-P-2 produced malignant fibrosarcomas at the site of injection in Syrian hamsters [4]. The mutagenic substances from pyrolysates of amino acids and proteins seem to be carcinogenic. Therefore, in the present study, sister-chromatid exchanges (SCEs) induced by these chemicals were examined with a permanent cell line of human lymphoblastoid cells, which we established previously [18,19]. All 4 chemicals induced SCEs. Materials and methods
Chemicals Synthetic Trp-P-1, Trp-P-2, and 2-amino~-carboline were obtained from Nard Institute Ltd., Osaka. Synthetic Glu-P-1 was obtained from Katsura Chemical Co., Tokyo. The syntheses of Trp-P-1 and Trp-P-2 were reported by Akimoto et al. [1]. The preparations were confirmed to be over 99% pure by thin-layer chromatography, paper chromatography, high-pressure liquid chromatography and mass spectrometry. Cell cultures A permanent human lymphoblastoid cell line, NL3 [18], was used. Culture medium RPMI 1640 supplemented with 20% foetal bovine serum (Flow Laboratory, Rockville, MD), 2 mM L-glutamine and kanamycin (50 pg/ml) was used throughout. Metabolic activation system $9 was prepared from the liver of rats treated with polychlorinated biphenyl (Kanechlor 500, 54% chlorinated) and supplied by courtesy of Dr. T. Matsushima of the Institute of Medical Science, University of Tokyo. Modified $9 mix was used, consisting of $9 (2.5% as liver homogenate), 8 mM MgC12, 33 mM KC1, 5 mM glucose 6-phosphate, 4 mM NADPH, 4 mM NADH and 0.1 M sodium phosphate buffer (pH 7.4) [9]. Induction o f SCEs Exponentially growing cells were inoculated at a density of 3--4 X 10 s cells per ml into culture medium containing 10 pM bromodeoxyuridine and cultured for 86 h to permit two cell cycles of growth. At the end of the first cell doubling the test compounds dissolved in an appropriate volume of dimethylsulphoxide (DMSO) and one tenth volume of $9 mix were added. The final concentration of DMSO was 0.5% or less, and the a m o u n t did not interfere with SCE induction. After 2-h incubation, the medium was removed and the cells were washed once with the medium. Incubation was then continued in fresh medium containing bromodeoxyuridine. At the end of culture, 1.3 pM colcemid was added, and 4 h later the cells were collected, treated with 0.05 M KC1 for 10 min, and fixed in methanol : acetic acid (3 : 1, v/v). For SCE analysis, fixed cells mounted on slides were processed by the "fluorescent plus Giemsa" technique with Hoechst 33258 as the fluorescent dye [11].
67 Results and discussion The dose responses of SCE induction by Trp-P-1, Trp-P-2, Glu-P-1 and 2-amino~-carboline in the presence and absence of $9 mix are shown in Table 1. SCE induction was greatly enhanced in all cases by metabolic activation catalysed by $9 mix. The slight SCE induction seen on treatment with Trp-P-1 or Trp-P-2 in the absence of $9 mix was probably due to weak metabolic activation by the cells themselves. The approximate concentrations of chemicals that induced 5 SCEs per cell more than the spontaneous number were estimated from the results, and are expressed in pg/ml and --log M in Table 2. The latter value can be regarded as approximately related to the SCE-inducing activity of the chemicals on a logarithmic scale. The values for 4-nitroquinoline-l-oxide, aflatoxin BI, N-nitroso-N-methylurea, benzo[a]pyrene and methylurea are also given in Table 2 for comparison. Many carcinogenic and mutagenic chemicals induce SCEs in mammalian cells [20], although the molecular mechanisms of spontaneous and induced SCE are n o t fully understood. The present results show that chemicals found in pyrolysates of amino acids and protein induced SCEs in eukaryotic cells. These SCEs could be produced by interaction of metabolically activated forms of these chemicals with DNA in h u m a n cell nuclei. It might be thought that humans may have become resistant to these pyrolytic mutagenic substances, because they have been exposed to these substances for a very long time by eating broiled foods and so they may have acquired the ability to induce less activation and more inactivation of these mutagens, and also may have developed an efficient mechanism for repair of DNA after its damage by these mutagens. However, it has been reported that Trp-P-1, Trp-
TABLE 1 INDUCTION OF SCEs BY Trp-P-I, Trp-P-2, GIu-P-1 AND 2-AMINO-<~-CARBOLINE Concentration (M)
SCEs/ceU
a
Trp-P-1
Trp-P-2
GIu-P-1
2-Amino-ce-c~Lrb oline
In the a b s e n c e o f $ 9 m i x 0 1 × 10 -7 1 X 10 -6
9.2 + 0.7 ---
8.4 + 0.6 8 . 0 -+ 0 . 5 8 . 7 _+ 0 . 7
9 . 6 -+ 1 . 0 ---
7 . 6 +_ 0 . 6 ---
1 × i 0 -$ 5 )< 1 0 - 5 1 X 10 -4
12.0 + 0.9 1 2 . 7 _+ 1 . 2 toxic b
10.3 + 1.0 -1 0 . 9 _+ 0 . 6
9.5 + 0.9 1 0 . 2 _+ 0 . 8 1 0 . 2 _+ 0 . 8
8.0 +_0.6 7 . 5 _+ 0 . 7 7 . 2 _+ 0 . 6
7 . 4 _+ 0 . 4 1 2 . 7 _+ 1 . 1
10.3 --
-+ 0 . 9
8 . 1 _+ 0 . 7 --
1 7 . 0 -+ 1 . 1 2 2 . 1 _+ 1 . 2 -toxic b
11.7 16.9 19.3 toxic
_+ 0 . 9 -+ 1 . 3 +_ 1.3 b
In the p r e s e n c e o f $ 9 m i x 0 1 X 10 -7 1 1 5 1
X X X ×
10 -6 I0 -5 I 0 -S 1 0 .-4
9 . 8 +_ 1 . 0 -12.6 20.1 20.8 toxic
_+ 1 . 1 -+ 1 . 3 _+ 1.3 b
9.7 10.5 15.7 17.3
a S C E s o f m o r e t h a n 3 0 c e l l s w e r e s c o r e d , a n d e x p r e s s e d as m e a n s _+ s t a n d a r d e r r o r s . b Chemicals were so toxic that SCEs could not be scored. s
_+ 1 . 0 +_ 0 . 8 + 1.6 + 1.7
68 TABLE 2 SCE-INDUCING A C T I V I T Y OF M U T A G E N S Carcinomutagens
$9 m i x
4 - N i t r o q u i n o l i n e 1-oxide c A f l a t o x i n B 1 c,d T r p -P-2 Trp-P-1 Nitrosomethylurea c Benzo[a]pyrene c Glu-P-1 2-Amino-(~-carboline M e t h y l u r e a ( c o n t r o l ) c,e
-+ + + _ + + + _
a b c d e
[ p g / m l ] 5s a 0.005 0.013 0.016 0.40 0.41 0.5 1.0 4.0 >>100
PM5s b 7.6 7.4 7.1 5.7 5.4 5.7 5.3 4.6 <<2.9
[ # g / m l ] 5s = c o n c e n t r a t i o n in p g / m l giving 5 i n d u c e d SCEs/cell. PM5s = - - t o g [ m o l a r c o n c e n t r a t i o n giving 5 i n d u c e d SCEs/ceU]. Ref. 17. I n t h e a b s e n c e of $9 m i x , [ ~ g / m l ] 5 s and PM5s are 6.2 and 4.7, resp. 1.33 m M m e t h y l u r e a ( 1 0 0 # g / m l ) d o e s n o t i n d u c e SCE.
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