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Relation between chemical constituents of tobacco and mutagenic activity of cigarette smoke condensate
319
Mutation Research, 48 (1977) 319--326 © Elsevier/North-Holland Biomedical Press
RELATION BETWEEN CHEMICAL CONSTITUENTS OF TOBACCO AND MUTAGENIC ACTIVITY OF CIGARETTE SMOKE CONDENSATE
S. MIZUSAKI, H. O K A M O T O , A. A K I Y A M A and Y. F U K U H A R A Central Research Institute, The Japan Tobacco and Salt Public Corporation 6-2, Umegaoka, Midori-ku, Yokohama, Kanagawa 227 (Japan)
(Received December 22nd, 1976) (Accepted February 2nd, 1977)
Summary Mutagenic activities of cigarette smoke condensate were assayed in the presence of S-9 Mix using Salmonella typhimurium TA 98. The results were examined in relation to chemical uata of tobacco leaves. Among the nitrogenous constituents examined, the contents of total nitrogen and protein nitrogen and the soluble nitrogenous fraction were positively and significantly related to an increase in mutagenic activity of the smoke condensate, whereas nicotine and nitrate were not important in contributing to mutagenic potency of such condensates. The age of tobacco leaves influenced the mutagenic potency of the condensate, which was lowest in leaves from the lower stalk position and increased with ascending leaf position on the stalk. Smoke condensate from tobacco with higher sugar content resulted in lower mutagenic activity. The present results, together with the previous study on the mutagenicity of the amino acid pyrolyzates, suggest that potent mutagens in cigarette smoke condensate are nitrogen-containing compounds, which may be formed from proteins and amino acids during the burning of a cigarette.
Introduction
Since a close relation between carcinogenicity and mutagenicity was demonstrated [10,16], the microbial mutagenicity test has become a practically useful means for screening of environmental carcinogens. Cigarette smoke condensate has long been known to be carcinogenic for experimental animals, and polycyclic aromatic hydrocarbons are regarded as the main tumor initiators in such condensates [19,20]. However, recent reports by Sugimura et al. [16], Mizusaki et al. [11] and Matsumoto et al. [9] suggested the presence of other potent mutagens besides benzo(a)pyrene in cigarette smoke condensates. Kier et al. [6] and Hutton et al. [4] have recently reported that cigarette smoke
320 condensate has mutagenic activity towards Salmonella typhimurium TA 1538 after metabolic activation and that most of the activity of the w h o l e smoke condensate was found in basic and weakly acidic fractions with no detectable activity in the neutral fraction containing polycyclic aromatic hydrocarbons. In the previous paper [ 11 ], we described the wide variation in the mutagenic potency of the smoke condensates from various brands of commercial cigarettes. This suggests that the mutagenic activity of such condensates is related to the difference in smoke composition which is dependent on the types of tobacco used in cigarettes. This study is to examine the effect of chemical constituents of tobaccos on the mutagenic potency of the resulting smoke condensates. Data in this report show that the mutagenic p o t e n c y of the smoke condensate is closely related to the content of the nitrogenous constituents of tobacco. Materials and methods
Tobacco sample Leaf samples, including 12 bright tobaccos, 2 Burley tobaccos and 6 Japanese domestic tobaccos, were obtained from conventionally flue-cured and air-cured materials. To examine the effect of the stalk position on mutagenic activity o f smoke condensate, 3 different varieties of Japanese domestic tobacco were used. These tobaccos were grown at the Utsunomiya Tobacco Experiment Station during 1975 and dried by the conventional air-curing schedule. These samples included 3 different leaves from the base to the top of the stalk. Experimental cigarettes (70 mm in length) were made from each sample of leaf tobacco and selected to a draw resistance of 65 +- 5 mm H20 and had weights within +20 mg of the average weight after moisture equilibration at 60% relative humidity and 20 ° C.
Preparation of smoke condensate Each cigarette was smoked on an automatic smoking machine under standard conditions: a single puff of 2 sec duration once a min; a p u f f volume of 35 ml; a butt length of 30 mm. The particulate matter from 3 cigarettes was collected on a glass-fiber filter (Toyo Roshi GA 200). Dimethylsulfoxide was added to the filter pad to a concentration of 1% total particulate matter. After being shaken for 20 min, the solution of smoke condensate was filtered through a fritted glass filter at reduced pressure and the filtrate was used for the detection of mutagenicity. Mutagenesis assay was performed within 2 h after preparation of the condensate.
Mutagenesis assay The histidine-requiring strain of Salmonella typhimurium TA 98 was kindly supplied b y Dr. B.N. Ames. Mutagenic activity of smoke condensate was assayed according to the method of Ames et al. [1,2]. In this procedure, the tester strain, properly diluted samples of smoke condensate and S-9 Mix were incorporated into an agar overlayer. After incubation at 37°C for 2 days, histidine revertant colonies were counted. Results were expressed as the average of triplicate plates. Liver microsomal fraction (S-9) was prepared from rats that were injected with PCB as described by Ames et al. [1,2].
321
Analy tical methods The total and protein nitrogen were determined by the Kjeldahl method [8]. The protein fraction was obtained as the insoluble matter after treatment with 5% trichloroacetic acid. Soluble nitrogen was calculated as total nitrogen minus protein nitrogen. Nicotine was steam~listilled from an alkaline suspension of the tobacco sample and determined spectrophotometrically according to the procedure of Willits et al. [18]. Nitrate was determined by the nitrate-ion electrode method according to the procedure of Ogata et al. [13]. Soluble sugar was determined with the Technicon auto-analyzer according to a modified procedure of Yamazaki et al. [21]. Results and discussion Data on the mutagenic activity are presented as the number of revertant colonies induced by 0 . 5 mg of the smoke condensate, since a linear dose response was observed up to this concentration of each condensate. The relationship between total nitrogen content of tobacco leaves and the mutagenic activities of the smoke condensates is shown in Fig. 1. The nitrogen content of the leaves ranged from 1.41 to 3.92% on a dry weight basis. Burley tobacco and Japanese domestic tobacco had higher nitrogen contents than bright tobacco. The smoke condensate from Burley and the domestic tobacco had much higher mutagenic activity than that from bright tobacco as reported previously [11]. An increase in mutagenic activity was found in the smoke condensate from the tobacco having higher nitrogen content, and a high correlation was observed between the mutagenic potency of the smoke condensate
r=o.94
r=o.88
_~1000
~ooo 0
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.~ 500
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100
Total nitrogen(%)
I 1
Protein nitrogen(~o1
F i g . 1. R e l a t i o n s h i p b e t w e e n n i t r o g e n c o n t e n t o f t o b a c c o leaves a n d m u t a g e n i c a c t i v i t y o f s m o k e c o n d e n sate. F i g . 2. R e l a t i o n s h i p b e t w e e n condensate.
p r o t e i n n i t r o g e n c o n t e n t o f t o b a c c o leaves a n d m u t a g e n i c a c t i v i t y o f s m o k e
322 and the nitrogen content of tobacco leaves. This suggests that nitrogenous constituents of tobacco leaves may contribute to the formation of the mutagens by smoking. These results also suggest that the mutagenic potency of the smoke condensate may be estimated by the chemical analysis of the total nitrogen content of cigarettes. Nitrogenous constituents of tobacco consist mainly of proteins, amino acids and tobacco alkaloids such as nicotine [17]. To examine the possible effect of such components on the mutagenic activity of the resulting smoke condensate, the results of chemical analysis of proteins, soluble nitrogenous fraction and nicotine were plotted against the mutagenic activities of the smoke condensates. As shown in Figs. 2, 3 and 4, the mutagenic activities of such condensates were positively and significantly related to the content of these constituents in the leaves. We have previously demonstrated that pyrolysis of amino acids or proteins formed mutagens, which required metabolic activation for induction of the his* mutant in S. typhimurium TA 98 [9]. Nagao et al. [12] and Kawachi et al. [5] have recently reported that the smoke condensate obtained by the roasting of raw fish or meat exhibited mutagenic activity towards S. typhimurium TA 98 and TA 100 after metabolic activation. They also confirmed that the mutagenic activity was derived from pyrolysis of amino acids. These observations indicate that proteins and amino acids in tobacco are important in contributing to an increase in the mutagenic activity of the cigarette smoke condensate. Nicotine, an important component of tobacco, has not been shown to be mutagenic on bacteria [10]. The analytical data showed that nicotine content of leaves followed a similar trend in the levels of the other nitrogenous frac-
0
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~100C
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o•
•
=E
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500
•
•8
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i I 1 2 Soluble nitrogen(%)
10[
I
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I
4
I
6
Nicotine(~,)
Fig. 3. R e l a t i o n s h i p b e t w e e n s o l u b l e n i t r o g e n c o n t e n t o f t o b a c c o leaves a n d m u t a g e n i c a c t i v i t y o f s m o k e condensate. Fig. 4. R e l a t i o n s h i p b e t w e e n n i c o t i n e c o n t e n t o f t o b a c c o leaves a n d m u t a g e n i c activity o f s m o k e c o n d e n sate.
323
tions such as total nitrogen, protein nitrogen and soluble nitrogenous fraction, and that they were positively correlated to one another. In this experiment, therefore, it appears unlikely that nicotine is strictly related to an increase in the mutagenic activity of such condensates. This possibility may be derived from the fact that nicotine readily distilled from tobacco behind the burning zone without chemical modification because of its high volatility [7]. Fig. 5 shows a relation between the nitrate content of tobacco and the mutagenic activity o f the smoke condensate. We have previously reported that the mutagenic activity of smoke condensate tended to increase with increasing amount of nitrate in cigarettes [11]. In this experiment, however, such high correlation was not observed between them. The reason for this difference is n o t obvious but may be due to the limited number of cigarettes used in the previous experiment. Kier et al. [6] have reported that the smoke condensate from the nitrate-treated cigarette was mutagenic towards S. typhimurium TA 1 5 3 8 and TA 1 5 3 5 even in the absence of S-9 Mix for metabolic activation and suggested that nitrate may produce new types of mutagenic agents not found in the condensate from non-treated cigarettes. Therefore, more detailed study may be required to define the effect of nitrate in tobacco on the mutagenic potency of smoke condensate. The effect of the leaf position on the mutagenic activity of the smoke condensate is shown in Table I. The mutagenic potency of the condensate was lowest in leaves from the lower stalk position and increased with ascending leaf position on the stalk. Levels of the nitrogenous constituents, except for nitrate, followed a trend similar to the mutagenic activity with the tobacco from different stalk positions. The wide differences in the content of the nitrogenous
r = 0.27 100C
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,
~100{3
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o.
i@
o
E =
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*
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5.
-~ 50[ -° E =
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R e l a t i o n s h i p b e t w e e n nitrate c o n t e n t o f t o b a c c o leaves and m u t a g e n i c a c t i v i t y o f s m o k e c o n d e n -
sate.
Fig. 6, Relationship between soluble sugar content of tobacco leaves and mutagenic activity of smoke condensate.
324 TABLE I CONTENT OF NITROGENEOUS CONSTITUENTS TIVITY OF SMOKE CONDENSATE
IN T O B A C C O L E A V E S A N D M U T A G E N I C A C -
T o b a c c o leaves f r o m d i f f e r e n t stalk p o s i t i o n s w e r e o b t a i n e d f r o m J a p a n e s e d o m e s t i c t o b a c c o . T h e m u t a genic a c t i v i t y o f s m o k e c o n d e n s a t e w a s a s s a y e d w i t h Sa/molella typhimurium T A 9 8 in the p r e s e n c e o f S-9 Mix. T h e n u m b e r o f s p o n t a n e o u s revertants ( 3 0 per p l a t e ) w a s s u b t r a c e d f r o m the n u m b e r o f r e v e t a n t s ind u c e d b y 0 . 5 m g o f the c o n d e n s a t e . Tobacco sample
A
Leaf position
N i t r o g e n o u s c o n s t i t u e n t s (%)
Mutagenic activity o f smoke condensate (revertants/ plate)
Total nitrogen
Protein nitrogen
Nicotine
Nitrate
Upper Middle Lower
1069 693 511
3.76 3.51 2.44
1.20 1.11 0.92
6.63 5.01 1.19
0.05 0.30 1.65
Upper Middle Lower
1355 1281 721
3.92 3.89 2.93
1.35 1.21 0.99
5.21 4.32 1.52
0.14 0.14 3.20
Upper Middle Lower
893 619 370
3.50 2.48 2.34
1.13 0.91 0.85
2.98 2.01 1.58
0.63 0.45 0.83
constituents reflect the age of tobacco leaves, which in turn affect the mutagenic activity of the smoke condensate. Fig. 6 shows the effect of soluble sugar content of tobacco on the mutagenic activity of the resulting smoke condensate. The mutagenic activity tended to decrease with the smoke condensate from the tobacco having increasing amounts of sugar. Bright tobacco is characterized by the high sugar content in comparison with air-cured tobacco. This may be merely because tobacco types with high sugar contents are usually low in nitrogenous constituents, resulting in lower mutagenic activity of smoke condensate. In the carcinogenesis experiment, the bulk of the tumor-initiating activity of cigarette smoke condensate detected on mouse skin is presumed to be due to polycyclic aromatic hydrocarbons [19,20], although the carcinogenic hydrocarbons are not alone responsible for the total carcinogenic activity [3,14,15]. Our study indicates that the mutagenic potency depends on the nitrogenous components in tobacco. Kier et al. [6] and Hutton et al. [4] examined the mutagenic activity of various fractions of smoke condensate; they found most of the activity of the whole smoke condensate in basic and weakly acidic fractions and no detectable activity in the neutral fraction containing polycyclic aromatic hydrocarbons. The previous study on the mutagenicity of the amino acid pyrolyzates, together with the present results, suggests that potent mutagens in cigarette smoke condensate are nitrogen-containing compounds, which may be formed from proteins and amino acids in tobacco by smoking. It is now o f importance to identify such mutagens and reduce the mutagenic potency of the smoke condensate.
325
Acknowledgement The authors thank Drs. K. Tomaru and D. Yoshida, of this Institute, for helpful discussions and comments on the manuscript. We also thank the staff of the Utsunomiya Tobacco Experiment Station for the supply of tobacco leaves. References 1 Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee, Carcinogens are mutagens: a simple test system combining liver h o m o g e n a t e s for activation and bacteria for detection, Proc. Natl. Acad. Sci. (U.S.A.), 70 (1973) 2281--2285. 2 Ames, B.N., J. McCann and E. Yamasaki, Method for detecting carcinogens and mut a ge ns with the S a l m o n e l l a / m a m m a l i a n mutagenieity test, Mutation Res., 31 (1975) 347--364. 3 Bock, F.G., A.P. Swain and R.L. Stedman, Bioassay of major fractions of cigarette s moke condensate by an accelerated technic, Cancer Res., 29 (1969) 584--587. 4 Hu tton, J J . and C. Hackney, Metabolism of cigarette smoke condensates by h u m a n and rat homogenates to form mutagen detectable by Salmonella t y p h i m u r i u m TA 1538, Cancer Res., 35 (1975) 2461--2468. 5 Kawachi, T., M. Nagao, N. Matsukura, Y. Yahagi, Y. Seino and T. Sugimura, Presented at the 5th Annual Meeting of the Japanese Environmental Mutagen Society, October, Tokyo, 1976. 6 Kier, L.D., E. Yamasaki and B.N. Ames, Detection of mutagenic activity in cigarette smoke condensate, Proc. Natl. Acad. Sci. (U,S.A.), 71 (1974) 4159--4163. 7 Kuhn, H., Tobacco alkaloids and their pyrolysis p r o d u c t in the smoke, in U.S. yon Euler (ed.) Tobacco alkaloids and related c o m p o u n d s , Vol. 4, Pergamon Press, 1964, pp. 37--51. 8 Lepper, H.A. (ed.), Official Methods of Analysis of the A.O.A.C., 7th ed., pp. 12--13. 9 Matsumoto, T., D. Yoshida, S. Mizusaki and H. O k a m o t o , Mutagenic activity of amino acid pyrolyzate in Salmonella t y p h i m u r i u m TA 98, Mutation Res., 48 (1976) 279--286. 10 McCann, J., E. Choi, E. Yamasaki and B.N. Ames, De t e c t i on of carcinogens as mut a ge ns in the Salm o n e l l a / m i c r o s o m e test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 5135-5139. 11 Mizusaki, S., T. Takashima and K. Tomaru, Factors affecting mutagenic activity of cigarette smoke condensate in Salmonella t y p h i m u r i u m TA 1538, Mutation Res., 48 (1977) 29--36. 12 Nagao, M., M. Honda, N. Matsukura, K. Yahagi, Y. Seino, T. Kawachi and T. Sugimura, Presented at the 35th Annual Meeting of the Japanese Cancer Association, October, 1976. 13 Ogata, N. and A. Fukazawa, D e t e r m i n a t i o n of nitrate by ion selective electrode, Sci. Paper, Cent. Res. Inst., Japan Tobacco and Salt Pub. Corp., 116 (1974) 71--74. 14 Orris, L., B.L. Vann Durren, A.I. Kosak, N. Nelson and F.L. Schmitt, The carcinogenicity for mouse skin and the aromatic h y d r o c a r b o n c o n t e n t of cigarette-smoke condensate, J. Natl. Cancer. Inst., 21 (1958) 557--561. 15 Roe, E.E., M.H. Salaman and J. Cohen, I n c o m p l e t e carcinogens in cigarette smoke condensate: tumorp r o m o t i o n by a phenolic fraction, Brit. J. Cancer, 13 (1959) 623--633. 16 Sugimura, T., S. Sato, M. Nagao, T. Yahagi, T. Matsushima, Y. Seino, M. Takeuchi and T. Kawachi, Overlapping of carcinogens and mutagens, in P.N. Magee et al. (cds.) F u n d a m e n t a l s in Cancer Prevention, Univ. of T o kyo Press, Tokyo]Univ. Park Press. Baltimore, 1976. 17 Stedman , R.L., The chemical c o m p o s i t i o n of tobacco and tobacco smoke, Chem Rev., 68 (1968) 153--207. 18 Willits, C.O., L.M. Swain, J. A. Connely and B.A. Brice, S p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n of nicotine, Anal. Chem., 22 (1950) 430--433. 19 Wynder, E.L. and D. Hoffman, Tobacco and tobacco smoke, Studies in e x p e r i m e n t a l carcinogenesis, Academic Press, 1967. 20 Wynder, E.L. and D. Hoffman, E x p e r i m e n t a l carcinogenesis, Science, 162 (1968) 862--871. 21 Yamasaki, M., H. Kimura and T. Namiki, A u t o m a t e d d e t e r m i n a t i o n of t ot a l and reducing sugars in tobacco (I), D e t e r m i n a t i o n of total sugar in deproteinized solution of tobacco by auto analyzer, Sci. Paper, Cent. Res. Inst., Japan Tobacco and Salt Pub. Corp., 111 (1969) 77--83.
Mutation Research, 48 (1977) 319--326 © Elsevier/North-Holland Biomedical Press
RELATION BETWEEN CHEMICAL CONSTITUENTS OF TOBACCO AND MUTAGENIC ACTIVITY OF CIGARETTE SMOKE CONDENSATE
S. MIZUSAKI, H. O K A M O T O , A. A K I Y A M A and Y. F U K U H A R A Central Research Institute, The Japan Tobacco and Salt Public Corporation 6-2, Umegaoka, Midori-ku, Yokohama, Kanagawa 227 (Japan)
(Received December 22nd, 1976) (Accepted February 2nd, 1977)
Summary Mutagenic activities of cigarette smoke condensate were assayed in the presence of S-9 Mix using Salmonella typhimurium TA 98. The results were examined in relation to chemical uata of tobacco leaves. Among the nitrogenous constituents examined, the contents of total nitrogen and protein nitrogen and the soluble nitrogenous fraction were positively and significantly related to an increase in mutagenic activity of the smoke condensate, whereas nicotine and nitrate were not important in contributing to mutagenic potency of such condensates. The age of tobacco leaves influenced the mutagenic potency of the condensate, which was lowest in leaves from the lower stalk position and increased with ascending leaf position on the stalk. Smoke condensate from tobacco with higher sugar content resulted in lower mutagenic activity. The present results, together with the previous study on the mutagenicity of the amino acid pyrolyzates, suggest that potent mutagens in cigarette smoke condensate are nitrogen-containing compounds, which may be formed from proteins and amino acids during the burning of a cigarette.
Introduction
Since a close relation between carcinogenicity and mutagenicity was demonstrated [10,16], the microbial mutagenicity test has become a practically useful means for screening of environmental carcinogens. Cigarette smoke condensate has long been known to be carcinogenic for experimental animals, and polycyclic aromatic hydrocarbons are regarded as the main tumor initiators in such condensates [19,20]. However, recent reports by Sugimura et al. [16], Mizusaki et al. [11] and Matsumoto et al. [9] suggested the presence of other potent mutagens besides benzo(a)pyrene in cigarette smoke condensates. Kier et al. [6] and Hutton et al. [4] have recently reported that cigarette smoke
320 condensate has mutagenic activity towards Salmonella typhimurium TA 1538 after metabolic activation and that most of the activity of the w h o l e smoke condensate was found in basic and weakly acidic fractions with no detectable activity in the neutral fraction containing polycyclic aromatic hydrocarbons. In the previous paper [ 11 ], we described the wide variation in the mutagenic potency of the smoke condensates from various brands of commercial cigarettes. This suggests that the mutagenic activity of such condensates is related to the difference in smoke composition which is dependent on the types of tobacco used in cigarettes. This study is to examine the effect of chemical constituents of tobaccos on the mutagenic potency of the resulting smoke condensates. Data in this report show that the mutagenic p o t e n c y of the smoke condensate is closely related to the content of the nitrogenous constituents of tobacco. Materials and methods
Tobacco sample Leaf samples, including 12 bright tobaccos, 2 Burley tobaccos and 6 Japanese domestic tobaccos, were obtained from conventionally flue-cured and air-cured materials. To examine the effect of the stalk position on mutagenic activity o f smoke condensate, 3 different varieties of Japanese domestic tobacco were used. These tobaccos were grown at the Utsunomiya Tobacco Experiment Station during 1975 and dried by the conventional air-curing schedule. These samples included 3 different leaves from the base to the top of the stalk. Experimental cigarettes (70 mm in length) were made from each sample of leaf tobacco and selected to a draw resistance of 65 +- 5 mm H20 and had weights within +20 mg of the average weight after moisture equilibration at 60% relative humidity and 20 ° C.
Preparation of smoke condensate Each cigarette was smoked on an automatic smoking machine under standard conditions: a single puff of 2 sec duration once a min; a p u f f volume of 35 ml; a butt length of 30 mm. The particulate matter from 3 cigarettes was collected on a glass-fiber filter (Toyo Roshi GA 200). Dimethylsulfoxide was added to the filter pad to a concentration of 1% total particulate matter. After being shaken for 20 min, the solution of smoke condensate was filtered through a fritted glass filter at reduced pressure and the filtrate was used for the detection of mutagenicity. Mutagenesis assay was performed within 2 h after preparation of the condensate.
Mutagenesis assay The histidine-requiring strain of Salmonella typhimurium TA 98 was kindly supplied b y Dr. B.N. Ames. Mutagenic activity of smoke condensate was assayed according to the method of Ames et al. [1,2]. In this procedure, the tester strain, properly diluted samples of smoke condensate and S-9 Mix were incorporated into an agar overlayer. After incubation at 37°C for 2 days, histidine revertant colonies were counted. Results were expressed as the average of triplicate plates. Liver microsomal fraction (S-9) was prepared from rats that were injected with PCB as described by Ames et al. [1,2].
321
Analy tical methods The total and protein nitrogen were determined by the Kjeldahl method [8]. The protein fraction was obtained as the insoluble matter after treatment with 5% trichloroacetic acid. Soluble nitrogen was calculated as total nitrogen minus protein nitrogen. Nicotine was steam~listilled from an alkaline suspension of the tobacco sample and determined spectrophotometrically according to the procedure of Willits et al. [18]. Nitrate was determined by the nitrate-ion electrode method according to the procedure of Ogata et al. [13]. Soluble sugar was determined with the Technicon auto-analyzer according to a modified procedure of Yamazaki et al. [21]. Results and discussion Data on the mutagenic activity are presented as the number of revertant colonies induced by 0 . 5 mg of the smoke condensate, since a linear dose response was observed up to this concentration of each condensate. The relationship between total nitrogen content of tobacco leaves and the mutagenic activities of the smoke condensates is shown in Fig. 1. The nitrogen content of the leaves ranged from 1.41 to 3.92% on a dry weight basis. Burley tobacco and Japanese domestic tobacco had higher nitrogen contents than bright tobacco. The smoke condensate from Burley and the domestic tobacco had much higher mutagenic activity than that from bright tobacco as reported previously [11]. An increase in mutagenic activity was found in the smoke condensate from the tobacco having higher nitrogen content, and a high correlation was observed between the mutagenic potency of the smoke condensate
r=o.94
r=o.88
_~1000
~ooo 0
$
0
t:
ee .,~ 50C
e• ee
.~ 500
"% ee
100.
100
Total nitrogen(%)
I 1
Protein nitrogen(~o1
F i g . 1. R e l a t i o n s h i p b e t w e e n n i t r o g e n c o n t e n t o f t o b a c c o leaves a n d m u t a g e n i c a c t i v i t y o f s m o k e c o n d e n sate. F i g . 2. R e l a t i o n s h i p b e t w e e n condensate.
p r o t e i n n i t r o g e n c o n t e n t o f t o b a c c o leaves a n d m u t a g e n i c a c t i v i t y o f s m o k e
322 and the nitrogen content of tobacco leaves. This suggests that nitrogenous constituents of tobacco leaves may contribute to the formation of the mutagens by smoking. These results also suggest that the mutagenic potency of the smoke condensate may be estimated by the chemical analysis of the total nitrogen content of cigarettes. Nitrogenous constituents of tobacco consist mainly of proteins, amino acids and tobacco alkaloids such as nicotine [17]. To examine the possible effect of such components on the mutagenic activity of the resulting smoke condensate, the results of chemical analysis of proteins, soluble nitrogenous fraction and nicotine were plotted against the mutagenic activities of the smoke condensates. As shown in Figs. 2, 3 and 4, the mutagenic activities of such condensates were positively and significantly related to the content of these constituents in the leaves. We have previously demonstrated that pyrolysis of amino acids or proteins formed mutagens, which required metabolic activation for induction of the his* mutant in S. typhimurium TA 98 [9]. Nagao et al. [12] and Kawachi et al. [5] have recently reported that the smoke condensate obtained by the roasting of raw fish or meat exhibited mutagenic activity towards S. typhimurium TA 98 and TA 100 after metabolic activation. They also confirmed that the mutagenic activity was derived from pyrolysis of amino acids. These observations indicate that proteins and amino acids in tobacco are important in contributing to an increase in the mutagenic activity of the cigarette smoke condensate. Nicotine, an important component of tobacco, has not been shown to be mutagenic on bacteria [10]. The analytical data showed that nicotine content of leaves followed a similar trend in the levels of the other nitrogenous frac-
0
r=o.89
1"=0.56
~100C
-Ciooo
==
o•
•
=E
•e
soG
3
Z
500
•
•8
eo
I00
i I 1 2 Soluble nitrogen(%)
10[
I
2
I
4
I
6
Nicotine(~,)
Fig. 3. R e l a t i o n s h i p b e t w e e n s o l u b l e n i t r o g e n c o n t e n t o f t o b a c c o leaves a n d m u t a g e n i c a c t i v i t y o f s m o k e condensate. Fig. 4. R e l a t i o n s h i p b e t w e e n n i c o t i n e c o n t e n t o f t o b a c c o leaves a n d m u t a g e n i c activity o f s m o k e c o n d e n sate.
323
tions such as total nitrogen, protein nitrogen and soluble nitrogenous fraction, and that they were positively correlated to one another. In this experiment, therefore, it appears unlikely that nicotine is strictly related to an increase in the mutagenic activity of such condensates. This possibility may be derived from the fact that nicotine readily distilled from tobacco behind the burning zone without chemical modification because of its high volatility [7]. Fig. 5 shows a relation between the nitrate content of tobacco and the mutagenic activity o f the smoke condensate. We have previously reported that the mutagenic activity of smoke condensate tended to increase with increasing amount of nitrate in cigarettes [11]. In this experiment, however, such high correlation was not observed between them. The reason for this difference is n o t obvious but may be due to the limited number of cigarettes used in the previous experiment. Kier et al. [6] have reported that the smoke condensate from the nitrate-treated cigarette was mutagenic towards S. typhimurium TA 1 5 3 8 and TA 1 5 3 5 even in the absence of S-9 Mix for metabolic activation and suggested that nitrate may produce new types of mutagenic agents not found in the condensate from non-treated cigarettes. Therefore, more detailed study may be required to define the effect of nitrate in tobacco on the mutagenic potency of smoke condensate. The effect of the leaf position on the mutagenic activity of the smoke condensate is shown in Table I. The mutagenic potency of the condensate was lowest in leaves from the lower stalk position and increased with ascending leaf position on the stalk. Levels of the nitrogenous constituents, except for nitrate, followed a trend similar to the mutagenic activity with the tobacco from different stalk positions. The wide differences in the content of the nitrogenous
r = 0.27 100C
r=-0.74
,
~100{3
o
o.
i@
o
E =
o
500
*
*
Nitrate(%) Fig.
5.
-~ 50[ -° E =
•
Soluble sugar(%)
R e l a t i o n s h i p b e t w e e n nitrate c o n t e n t o f t o b a c c o leaves and m u t a g e n i c a c t i v i t y o f s m o k e c o n d e n -
sate.
Fig. 6, Relationship between soluble sugar content of tobacco leaves and mutagenic activity of smoke condensate.
324 TABLE I CONTENT OF NITROGENEOUS CONSTITUENTS TIVITY OF SMOKE CONDENSATE
IN T O B A C C O L E A V E S A N D M U T A G E N I C A C -
T o b a c c o leaves f r o m d i f f e r e n t stalk p o s i t i o n s w e r e o b t a i n e d f r o m J a p a n e s e d o m e s t i c t o b a c c o . T h e m u t a genic a c t i v i t y o f s m o k e c o n d e n s a t e w a s a s s a y e d w i t h Sa/molella typhimurium T A 9 8 in the p r e s e n c e o f S-9 Mix. T h e n u m b e r o f s p o n t a n e o u s revertants ( 3 0 per p l a t e ) w a s s u b t r a c e d f r o m the n u m b e r o f r e v e t a n t s ind u c e d b y 0 . 5 m g o f the c o n d e n s a t e . Tobacco sample
A
Leaf position
N i t r o g e n o u s c o n s t i t u e n t s (%)
Mutagenic activity o f smoke condensate (revertants/ plate)
Total nitrogen
Protein nitrogen
Nicotine
Nitrate
Upper Middle Lower
1069 693 511
3.76 3.51 2.44
1.20 1.11 0.92
6.63 5.01 1.19
0.05 0.30 1.65
Upper Middle Lower
1355 1281 721
3.92 3.89 2.93
1.35 1.21 0.99
5.21 4.32 1.52
0.14 0.14 3.20
Upper Middle Lower
893 619 370
3.50 2.48 2.34
1.13 0.91 0.85
2.98 2.01 1.58
0.63 0.45 0.83
constituents reflect the age of tobacco leaves, which in turn affect the mutagenic activity of the smoke condensate. Fig. 6 shows the effect of soluble sugar content of tobacco on the mutagenic activity of the resulting smoke condensate. The mutagenic activity tended to decrease with the smoke condensate from the tobacco having increasing amounts of sugar. Bright tobacco is characterized by the high sugar content in comparison with air-cured tobacco. This may be merely because tobacco types with high sugar contents are usually low in nitrogenous constituents, resulting in lower mutagenic activity of smoke condensate. In the carcinogenesis experiment, the bulk of the tumor-initiating activity of cigarette smoke condensate detected on mouse skin is presumed to be due to polycyclic aromatic hydrocarbons [19,20], although the carcinogenic hydrocarbons are not alone responsible for the total carcinogenic activity [3,14,15]. Our study indicates that the mutagenic potency depends on the nitrogenous components in tobacco. Kier et al. [6] and Hutton et al. [4] examined the mutagenic activity of various fractions of smoke condensate; they found most of the activity of the whole smoke condensate in basic and weakly acidic fractions and no detectable activity in the neutral fraction containing polycyclic aromatic hydrocarbons. The previous study on the mutagenicity of the amino acid pyrolyzates, together with the present results, suggests that potent mutagens in cigarette smoke condensate are nitrogen-containing compounds, which may be formed from proteins and amino acids in tobacco by smoking. It is now o f importance to identify such mutagens and reduce the mutagenic potency of the smoke condensate.
325
Acknowledgement The authors thank Drs. K. Tomaru and D. Yoshida, of this Institute, for helpful discussions and comments on the manuscript. We also thank the staff of the Utsunomiya Tobacco Experiment Station for the supply of tobacco leaves. References 1 Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee, Carcinogens are mutagens: a simple test system combining liver h o m o g e n a t e s for activation and bacteria for detection, Proc. Natl. Acad. Sci. (U.S.A.), 70 (1973) 2281--2285. 2 Ames, B.N., J. McCann and E. Yamasaki, Method for detecting carcinogens and mut a ge ns with the S a l m o n e l l a / m a m m a l i a n mutagenieity test, Mutation Res., 31 (1975) 347--364. 3 Bock, F.G., A.P. Swain and R.L. Stedman, Bioassay of major fractions of cigarette s moke condensate by an accelerated technic, Cancer Res., 29 (1969) 584--587. 4 Hu tton, J J . and C. Hackney, Metabolism of cigarette smoke condensates by h u m a n and rat homogenates to form mutagen detectable by Salmonella t y p h i m u r i u m TA 1538, Cancer Res., 35 (1975) 2461--2468. 5 Kawachi, T., M. Nagao, N. Matsukura, Y. Yahagi, Y. Seino and T. Sugimura, Presented at the 5th Annual Meeting of the Japanese Environmental Mutagen Society, October, Tokyo, 1976. 6 Kier, L.D., E. Yamasaki and B.N. Ames, Detection of mutagenic activity in cigarette smoke condensate, Proc. Natl. Acad. Sci. (U,S.A.), 71 (1974) 4159--4163. 7 Kuhn, H., Tobacco alkaloids and their pyrolysis p r o d u c t in the smoke, in U.S. yon Euler (ed.) Tobacco alkaloids and related c o m p o u n d s , Vol. 4, Pergamon Press, 1964, pp. 37--51. 8 Lepper, H.A. (ed.), Official Methods of Analysis of the A.O.A.C., 7th ed., pp. 12--13. 9 Matsumoto, T., D. Yoshida, S. Mizusaki and H. O k a m o t o , Mutagenic activity of amino acid pyrolyzate in Salmonella t y p h i m u r i u m TA 98, Mutation Res., 48 (1976) 279--286. 10 McCann, J., E. Choi, E. Yamasaki and B.N. Ames, De t e c t i on of carcinogens as mut a ge ns in the Salm o n e l l a / m i c r o s o m e test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 5135-5139. 11 Mizusaki, S., T. Takashima and K. Tomaru, Factors affecting mutagenic activity of cigarette smoke condensate in Salmonella t y p h i m u r i u m TA 1538, Mutation Res., 48 (1977) 29--36. 12 Nagao, M., M. Honda, N. Matsukura, K. Yahagi, Y. Seino, T. Kawachi and T. Sugimura, Presented at the 35th Annual Meeting of the Japanese Cancer Association, October, 1976. 13 Ogata, N. and A. Fukazawa, D e t e r m i n a t i o n of nitrate by ion selective electrode, Sci. Paper, Cent. Res. Inst., Japan Tobacco and Salt Pub. Corp., 116 (1974) 71--74. 14 Orris, L., B.L. Vann Durren, A.I. Kosak, N. Nelson and F.L. Schmitt, The carcinogenicity for mouse skin and the aromatic h y d r o c a r b o n c o n t e n t of cigarette-smoke condensate, J. Natl. Cancer. Inst., 21 (1958) 557--561. 15 Roe, E.E., M.H. Salaman and J. Cohen, I n c o m p l e t e carcinogens in cigarette smoke condensate: tumorp r o m o t i o n by a phenolic fraction, Brit. J. Cancer, 13 (1959) 623--633. 16 Sugimura, T., S. Sato, M. Nagao, T. Yahagi, T. Matsushima, Y. Seino, M. Takeuchi and T. Kawachi, Overlapping of carcinogens and mutagens, in P.N. Magee et al. (cds.) F u n d a m e n t a l s in Cancer Prevention, Univ. of T o kyo Press, Tokyo]Univ. Park Press. Baltimore, 1976. 17 Stedman , R.L., The chemical c o m p o s i t i o n of tobacco and tobacco smoke, Chem Rev., 68 (1968) 153--207. 18 Willits, C.O., L.M. Swain, J. A. Connely and B.A. Brice, S p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n of nicotine, Anal. Chem., 22 (1950) 430--433. 19 Wynder, E.L. and D. Hoffman, Tobacco and tobacco smoke, Studies in e x p e r i m e n t a l carcinogenesis, Academic Press, 1967. 20 Wynder, E.L. and D. Hoffman, E x p e r i m e n t a l carcinogenesis, Science, 162 (1968) 862--871. 21 Yamasaki, M., H. Kimura and T. Namiki, A u t o m a t e d d e t e r m i n a t i o n of t ot a l and reducing sugars in tobacco (I), D e t e r m i n a t i o n of total sugar in deproteinized solution of tobacco by auto analyzer, Sci. Paper, Cent. Res. Inst., Japan Tobacco and Salt Pub. Corp., 111 (1969) 77--83.