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Carcinogenic tryptophan pyrolysis products in cigarette smoke condensate and cigarette smoke-polluted indoor air
Environmental Pollution 64 (1990) 121-132
Carcinogenic Tryptophan Pyrolysis Products in Cigarette Smoke Condensate and Cigarette Smoke-Polluted Indoor Air Shigeo M a n a b e & O s a m u W a d a Department of Hygiene and PreventiveMedicine,Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,Tokyo 113, Japan (Received 1l August 1989; accepted 24 November 1989) ABSTRACT The carc&ogenic tryptophan pyrolys& products, 3-am&o-l,4-dimethyl5H-pyrido[4,3-b]indole (Trp-P-l) and 3-amino-l-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), have been measured in condensate of cigarette mainstream smoke by high-performance liquid chromatography. These carcinogens have been detected in indoor air as well as in the air of the outdoor environment. Levels of these carcinogens in indoor air were much higher than those in outdoor air. The souree of these carcinogens in indoor air was determined to be cigarette smoke by the application of smoking machine studies. Concentrations of these carcinogens in indoor air increased markedly with an increase in cigarettes smoked. The results in this investigation suggest that cigarette smoking is a source of carcinogenic tryptophan pyrolysis products in the indoor environment. Our data also suggest that smokers are persistently exposed to the carcinogenic heterocyclic amines together with potent carcinogens such as polynuclear aromatic hydrocarbons and N-nitroso compounds.
NOTATION A~C Glu-P-1 GIu-P-2 IQ MeA~C Trp-P-1 Trp-P-2
2-Amino-9H-pyrido[2,3-b]indole 2-Amino-6-methyldipyrido [ 1,2-a: 3',2'-d] imidazole 2-Aminodipyrido[ 1,2-a: 3',2'-d]imidazole 2-Amino-3-methylimidazo [4,5-f]quinoline 2-Amino-3-methyl-9H-pyridol-2,3-b]indole 3-Amino- 1,4-dimethyl- 5H-pyrido [4,3-b]indole 3-Amino- 1-methyl- 5H-pyrido [4,3-b]indole 121 Environ. Pollut. 0269-7491/90/$03-50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
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Shigeo Manabe, Osamu Wada
INTRODUCTION Carcinogenic tryptophan pyrolysis products 3-amino-l,4-dimethyl-5Hpyrido[4,3-b]indole (Trp-P-1) and 3-amino-l-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) were first isolated from tryptophan pyrolysates as compounds with potent mutagenic effects on Salmonella t y p h i m u r i u m tester strains (Sugimura et al., 1977). Later, a new series ofheterocyclic amines were isolated as potent mutagens from pyrolysates of amino acids and proteins (Sugimura & Sato, 1983; Sugimura, 1986). All the mutagenic heterocyclic amines so far tested have been shown to be carcinogenic in experimental animals (Sugimura, 1986). The mutagenic and carcinogenic heterocyclic amines such as A~C, MeA~C, Glu-P-1, Glu-P-2, IQ, Trp-P-1 and Trp-P-2 (see Notation) have been reported to be present in several cooked foods (Sugimura & Sato, 1983). Recent investigations indicated that humans are actually exposed to these carcinogenic heterocyclic amines (Baker et al., 1982; Dolara et al., 1984; Felton & Healy, 1984; Hayatsu et al., 1985; Manabe et al., 1987a, b; Manabe & Wada, 1988; Kanai et al., 1988; Murray et al., 1989). Our recent investigation revealed that Trp-P-1 and Trp-P-2 are present in airborne particles and rain water, indicating that these carcinogens are ubiquitous environmental components (Manabe et al., 1989b). In the course of the previous study, we found that the levels of Trp-P-1 and Trp-P-2 in indoor air were higher than those in outdoor air. This suggested to us that cigarette smoke may be the origin or one of the origins of these carcinogens in indoor air, although the presence of these compounds in cigarette smoke had not been confirmed.
METHODS AND MATERIALS
Preparation of cigarette smoke condensate Commercial cigarettes (three Japanese brands, one American and one English brand) were used in this study. The filter-tipped cigarettes were smoked by the Filtrona Model 302 smoking machine under standard conditions (puff frequency: 1 puff/60 s; puff volume: 35 ml/2 s; butt length: 30 mm). The cigarette smoke condensate was collected on glass fiber filters (Toyo Roshi type GA 200, Toyo Roshi Co., Tokyo), and extracted twice with 100 ml of methanol/28% ammonia water in an ultrasonic bath. The extract was evaporated to dryness under a nitrogen stream. The cigarette smoke condensate thus obtained was dissolved in 20mM HaPO 4, pH 2"0/ acetonitrile (90/10, by vol.) and filtered with a disposable filter unit (0.45/~m, Gelman Science Japan Ltd, Tokyo). The filtrate served for HPLC analyses.
TrkTtophan pyrolysates in cigarette smoke
123
Preparation of particulate matter in indoor air before and after cigarette smoking In order to study indoor air pollution by carcinogenic heterocyclic amines through cigarette smoking, we used a representative room in our laboratory (Fig. 1) for smoking experiments. The test space was approximately 57.2 m 3, and the laboratory r o o m was equipped with an air-conditioner (ventilation rate: 5"2 m 3 air/min). A smoking machine and an air sampler were placed in the r o o m as shown in Fig. 1. Particulate matter in indoor and outdoor air was collected by a high-volume air sampler Kimoto Model-120 (Kimoto Electronics Co., Ltd., Osaka, Japan) on quartz microfiber filters (20"32cm × 25.4cm, W h a t m a n QM-A). Before and after smoking of Japanese filter cigarette C (Table i) with an automatic smoking machine with standard parameters as described above, the particulate matter in the air was collected with the air sampler by continuous suction for 60 min at a flow rate of 1.5 m3/min. One-fourth of the 20.32 cm × 25-4 cm filter was used for the determination o f carcinogenic tryptophan pyrolysis products. These carcinogens in the filter bearing the collected particulate matter were extracted once with methanol/28% a m m o n i a water (50/1, by vol.). Onefourth o f the filter was soaked in 200 ml o f methanol/28% a m m o n i a water
Fig. 1. A sketch of a room used for determining the pollution of indoor air by cigarette smoking. A: an automatic smoking machine, B: a high-volume air sampler.
Shigeo Manabe, Osamu Wada
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TABLE 1 Carcinogenic T r y p t o p h a n Pyrolysis Products in Condensate of Cigarette Mainstream Smoke
Tobacco product a
Japanese filter cigarette A (2) Japanese filter cigarette B (2) Japanese filter cigarette C (4) US filter cigarette (3) UK filter cigarette (2) Mean _ SD
Mainstream condensate (mg/cig)
Carcinogenic tryptophan pyrolysates (ng/cig) Trp-P-1 b
Trp-P-2
7.8 6.6 17.5 9.6 10.1
0'19 c 0'29 0'31 0"20 0"24
0-65 0.61 0-66 0-51 0-53
10'3 _+ 4-3
0"25 _+ 0'05
0'59 _+ 0-07*
a In parentheses, n u m b e r of cigarettes tested. h Abbreviations: Trp-P- 1, 3-amino- 1,4-dimethyl-5H-pyrido[4,3-b]indole; Trp-P-2, 3-amino- l-met hyl-5H-pyrido[4,3-b]indole. c Values were not corrected for recoveries. * p < 0"001 as compared with Trp-P-1 levels.
for 30 min at room temperature in the ultrasonic bath. The methanol/28% ammonia water was condensed under a nitrogen stream. The condensed extract ( ~ 10 ml) was filtered with a disposable filter unit (0.45 #m, Gelman Science Japan, Ltd). The filtrate was further dried under a nitrogen stream and served for HPLC analyses.
HPLC analyses Reversed-phase HPLC analyses were performed by means of a Hitachi 655A chromatograph (Hitachi, Tokyo) equipped with an F-1000 fluorometric detector (Hitachi). Assay conditions for HPLC analyses were according to our previous method (Manabe et al., 1989b). Partial purification was carried out by using an Asahipack ES-502C column (9"0/~m particle size, 7.6 x 100 mm, Asahi Chemical Industries, Ltd, Kawasaki, Japan) under the following conditions: mobile phase, 20mM H3PO 4, pH 2-0/acetonitrile (90/10, by vol.); flow rate, 1.0ml/min at 40°C. Fluorescence was monitored at 399nm when excited at 266nm. The fractions corresponding to Trp-P-1 and Trp-P-2 were collected separately. Then, the volumes of these fractions were reduced to ~ l m l with a Centrifugal Evaporator EC-57 (Sakuma Seisakusho Co., Ltd, Tokyo). The condensed fractions (,--1 ml) were further purified by means of a Nucleosile 5C8 column (10ktm particle size, 4 x 150nm; Union Co., Ltd, Takasaki, Japan). The solvent system for the fraction corresponding to Trp-
Tryptophan pyrolysates in cigarette smoke
125
P-2 on the first-step purification was 20mM H3PO4, pH2-0/acetonitrile (90/10, by vol.) while the eluate for the Trp-P-1 fraction was 20 mM H3PO4, pH 2.0/acetonitrile (85/15, by vol.). A flow rate of 1.0ml/min at 50°C was used. Fluorescence was monitored at 399 nm when excited at 266 nm. The fraction corresponding to Trp-P-1 or Trp-P-2 was collected, and condensed to ,-~2 ml with a Centrifugal Evaporator EC-57. Final HPLC analysis was carried out on a Kaseisorb LC ODS-300-5 column (5/~m particle size and 300,~ pore size, 7"5 × 250mm; Tokyo Chemical Industries, Ltd, Tokyo), as described previously (Manabe & Wada, 1988; Manabe et al., 1989b). The mobile phase was a gradient (0-30%) of acetonitrile in 10mM H3PO 4 over a course of 35 min. The flow rate was 3.0ml/min at 50°C. Fluorescence was monitored as described above. The fraction corresponding to each authentic compound was collected separately. The contents of the fractions were extracted with chloroform after the acetonitrile in the fractions was evaporated and the pH was adjusted to ~ 10-0 with 28% ammonia/water (Manabe & Wada, 1988).
Spectrophotometric analyses Spectrometric measurements were performed on a Shimadzu UV 260 spectrophotometer (Shimadzu, Kyoto) and a Hitachi F-3000 fluorescence spectrophotometer (Hitachi, Tokyo) after dissolving the samples in methanol. The mass spectral analyses were conducted in the electron impact (El) mode using the direct insertion probe on a double-focusing JEOL JMSDx 300 mass spectrometer. The mass spectra were recorded with JMA-3500 mass data analysis system, employing 70eV ionization voltage, 300/~A ionization current and 3 kV accelerating voltage with a course temperature of 200°C.
RESULTS
Amounts of Trp-P-1 and Trp-P-2 in cigarette smoke condensate Typical charts of HPLC for cigarette smoke condensate are shown in Fig. 2 when one-fourth of the extract from the mainstream smoke of a Japanese filter cigarette was analyzed. On the final-step analyses, sharp peaks corresponding to Trp-P-1 and Trp-P-2 were clearly identifiable (Fig. 2(C)). As reported previously (Manabe & Wada, 1988), peak heights of Trp-P-1 and Trp-P-2 on the final chromatogram obtained by using an ODS-300-5 column showed linear relationships with the amounts of authentic carcinogens. The amounts of Trp-P-1 and Trp-P-2 in cigarette smoke
126
Shigeo Manabe, Osamu Wada
(B)
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.
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Fig. 2. HPLC chromatographic profiles of Trp-P-I and Trp-P-2 in cigarette smoke condensate. Fluorescencewas monitored at 399 nm when excited at 266 nm. (A) Preparative HPLC chromatogram on an ES-502C column.The fractions corresponding to authentic TrpP-1 and Trp-P-2 were collected separately.(B) Chromatograms of the second step purification on a Nucleosile 5C8 column. Upper: Chromatogram of the fraction corresponding to Trp-P-2 on the first step purification(A). Lower:Chromatogram of the fraction corresponding to TrpP-1 on the first step purification (A). (C) Final chromatographic profiles on an ODS-300-5 column. condensate were calculated on the basis of these linear relationships. As shown in Table 1, the mean level o f Trp-P-2 in cigarette smoke condensate was approximately twice that of Trp-P-1.
Recoveries The glass fiber filters absorbed with methanol solution o f Trp-P-1 and TrpP-2 (each 100pg) were treated by the same procedure as that for the analytical sample. The percentages o f Trp-P-1 and Trp-P-2 recovered by this method were 71.0% and 66-4%, respectively. The chemical forms of these
127
Tryptophan pyrolysates in cigarette smoke
compounds in cigarette smoke condensate have not been determined. If parts of these compounds in cigarette smoke condensate are bound to other materials, those compounds may not be extracted by the method we used (Manabe et al., 1989a). Therefore, the data indicated were not corrected for the recoveries (Table 1).
Identification of Trp-P-1 and Trp-P-2 Two hundred cigarettes were smoked by an tutomatic smoking machine. The smoke condensate was treated by the same procedure as that for the analytical sample. For confirmation of the compounds purified with HPLC, ultraviolet (UV) absorbance and fluorescence and mass spectra were determined. The absorbance and fluorescence spectra of the fraction corresponding to Trp-P-1 and Trp-P-2 on the final purification step were identical with the UV absorbance and fluorescence spectra of authentic compounds (Fig. 3). Mass spectra of the fractions corresponding to Trp-P-1 and Trp-P-2 revealed molecular ion peaks at m/z 211 and m/z 197, respectively, which were identical with those observed for authentic Trp-P- 1 and Trp-P-2 (not shown).
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Fig. 3. Fluorescence emission spectra excited at 266 nm (upper) and UV absorption spectra (lower) of Trp-P-1 and Trp-P-2 fractions. (A) The fraction corresponding to Trp-P-I in cigarette smoke condensate ( ) and authentic Trp-P-1 (0"2/~M) (..... ). (B) The fraction corresponding to Trp-P-2 in cigarette smoke condensate ( ) and authentic Trp-P-2 (0"6/~M) (..... ).
Shigeo Manabe, Osamu Wada
128
Levels of Trp-P-1 and Trp-P-2 in indoor air before and after cigarette smoking In order to determine the effects of cigarette smoking on Trp-P-1 and Trp-P-2 levels in indoor air, levels of these carcinogens in the air of the test space (Fig. 1) were determined before and after cigarette smoking. Trp-P-1 and Trp-P-2 were detected in the indoor air before smoking, and concentrations of Trp-P-1 and Trp-P-2 were 0-416pg/m 3 air and 0.610pg/m 3 air, respectively (Fig. 4), whereas concentrations of Trp-P-1 and Trp-P-2 in the outdoor air determined on the same day were 0"06 pg/m 3 air and 0.05 pg/m 3 air, respectively. The results indicate that levels of these carcinogens in the indoor air of the test room were much higher than those in the air of the outdoor environment. Trp-P-1 and Trp-P-2 levels in the indoor air were remarkably increased with the number of cigarettes smoked (Fig. 4). After this experiment, the door of the room used in this study was left open and the indoor air was ventilated by an airconditioner for 1 h at a flow rate of 5.2 m 3 air/min. Then, the particulate matter in the air of the indoor environment was collected by a high-volume air sampler for 12h: at a flow rate of 1"5 m3/min. Concentrations of Trp-P-1 and Trp-P-2 in the indoor air after L.
"~ 100
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/// :/ 0
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Cigarette Smoked
Fig. 4. Increases of concentrations of Trp-P-1 ( 0 - - 0 ) and Trp-P-2 ( 0 - - 0 ) in indoor air with the number of cigarettes smoked with an automatic smoking machine.
129
Tryptophan pyrolysates in cigarette smoke
ventilation were reduced to 0.78pg/m 3 and 1.67pg/m 3, respectively. However, levels of these carcinogens in the indoor air were apparently higher than those before cigarette smoking. These results indicate that the indoor air of the test space can be polluted by carcinogenic tryptophan pyrolysis products of cigarette smoking.
DISCUSSION Epidemiological studies have shown that cigarette smoking is associated with the development of human cancers (Doll, 1977; Wynder & Gori, 1977; Doll & Peto, 1981; Brinton et al., 1984). The role of tobacco smoke as a causative agent in various types of cancer has been frequently reaffirmed by many researchers (IARC Monograph, 1986). Known carcinogens in tobacco smoke include polynuclear aromatic hydrocarbons (PAH), N-nitroso compounds, polycyclic aza-arenes and miscellaneous nitrogen compounds (IARC Monograph, 1986). Recent investigations have revealed that carcinogenic heterocyclic amines such as IQ, A0~Cand MeA~C are present in cigarette smoke condensate (Yoshida & Matsumoto, 1980; Matsumoto et al., 1981; Yamashita et al., 1986). However, the presence of carcinogenic tryptophan pyrolysis products in cigarette smoke has not been confirmed. In this investigation, we have clearly demonstrated that Trp-P-1 and Trp-P-2 are present in cigarette smoke condensate. The mean levels of Trp-P-1 and Trp-P-2 in the condensate of mainstream smoke were 0.25ng/cig and 0.59 ng/cig, respectively (Table 1). The mean level of Trp-P-2 in cigarette smoke condensate was approximately twice that of Trp-P-1. So far as is known, the presence of three carcinogenic heterocyclic amines (A~C, MeA~C and IQ) has been confirmed in previous reports (Table 2). As TABLE 2
Summary of CarcinogenicHeterocyclicAmine Levels in Cigarette Mainstream Smoke Carcinogenic heterocyclic amines ~
A~C MeA~C IQ Trp-P-1 Trp-P-2
Range (ng/cig)
Mean (ng/cig)
25-258 9-37 -0"19-0"31 0'51-0-66
101'1 18'2 0.26 0"25 0'59
ReJerences
Yoshida& Matsumoto (1980) Yoshida& Matsumoto (19801 Yamashitaet al. (1986) This study This study
a Abbreviations: A~C, 2-amino-9H-pyrido[2,3-b]indole; MeA~C, 2-amino-3-methyl-9Hpyrido[2,3-b]indole; IQ, 2-amino-3-methylimidazo[4,5-f-Jquinoline;Trp-P-l, 3-amino-l,4dimethyl-5H-pyrido[4,3-b]indole;Trp-P-2, 3-amino-l-methyl-5H-pyrido[4,3,-b]indole.
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Shigeo Manabe, Osamu Wada
compared with the reported values of these carcinogenic heterocyclic amines in cigarette smoke condensate (Table 2), it seems that Trp-P-1 levels in cigarette smoke condensate are almost the same as the IQ level. It has been shown that air in indoor environment with extreme tobacco smoke pollution can contain several times higher concentrations of polynuclear aromatic hydrocarbons and N-nitrosamines than polluted urban air (IARC Monograph, 1983; Brunnemann & Hoffman, 1978). A similar phenomenon was observed in this study. Levels of carcinogenic tryptophan pyrolysis products in the indoor air of the test space were apparently higher than those in the outdoor air observed on the same day that the indoor air was sampled. This turned our attention to the origin(s) of these carcinogens in the indoor air. In order to determine that cigarette smoke is one of the origins of the carcinogenic tryptophan pyrolysis products in indoor air, levels of these carcinogens in the air of the indoor environment were measured before and after cigarette smoking by an automatic smoking machine. As shown in Fig. 4, levels of these carcinogens were increased with an increase in cigarettes smoked. Furthermore, even after ventilation of the air in the test room (Fig. 1), levels of these carcinogens in the indoor air of the test space were apparently higher than those before cigarette smoking. The results indicate that the indoor air of the test space can be polluted by carcinogenic tryptophan pyrolysis products in cigarette smoke. The carcinogenic heterocyclic amines were originally isolated from pyrolysates of proteins and amino acids (Sugimura & Sato, 1983; Sugimura, 1986). These carcinogens have been thought to be formed during cooking of foods. Therefore, these compounds are called 'dietary carcinogens'. However, the presence of carcinogenic heterocyclic amines including Trp-P-1 and Trp-P-2 in cigarette smoke condensate might suggest that these carcinogens are produced by combustion of various materials such as foods, garbage, wood and grass. The hypothesis seems to be supported by the fact that carcinogenic tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, are present in airborne particles and rain water (Manabe et al., 1989b). While it is, of course, important to investigate the mechanism of formation of these carcinogens, it is also necessary to determine the distributions of these heterocyclic amines in the environment.
ACKNOWLEDGEMENT This research was supported in part by grants A-63440029 and B-63480174 from the Ministry of Education, Science and Culture of Japan.
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REFERENCES Baker, R., Arlauskas, A. & Bonin, A. (1982). Detection of mutagenic activity in human urine following fried pork or bacon meals. Cancer Lett., 16, 81 9. Brinton, L. A., Blot, W. J., Becher, J. A., Winn, D. M., Browder, J. P., Farmer, J. C. Jr & Fraumeni, J. F. Jr (1984). A case-control study of cancers of the nasal cavity and paranasal sinuses. Am. J. Epidemiol., 119, 896-906. Brunnemann, K. D. & Hoffmann, D. (1978). Chemical studies on tobacco smoke. LIX. Analysis of volatile nitrosamines in tobacco smoke and polluted indoor environments. In Environmental Aspects of N-Nitroso Compounds (IARC Scientific Publications No. 19), ed. Walker, E. A., Castegnaro, M., Griciute, L. & Lyle, E. R., Lyon, International Agency for Research on Cancer, pp. 343-56. Dolara, P., Caderni, G., Salvadorl, M., Tringale, L. & Lodovici, M. (1984). Urinary mutagens in humans after fried pork and bacon meals. Cancer Lett., 22, 275-80. Doll, R. (1977). Strategy for detection of cancer hazards to man. Nature, 265, 589-96. Doll, R. & Peto, R. (1981). The causes of cancer: Quantitative estimates of avoidable risks of cancer in the United States today. J. Natl. Cancer Inst., 66, 1191 308. Felton, J. S. & Healy, S. K. (1984). Mutagenic activation of cooked ground beef by human liver microsomes. Mutation Res., 140, 61-5. Hayatsu, H., Hayatsu, T., Wataya, Y. & Mower, H. F. (1985). Fecal mutagenicity arising from ingestion of fried ground beef in the human. Mutation Res., 143, 207-11. IARC (1983). IARC Monograph on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 32, Polynuclear Aromatic Compounds, Part 1, Chemical, Environmental and Experimental Data, Lyon, pp. 37-8, 122 34, 277-88, 315-20. IARC (1986). IARC Monographs on the Evlauation of the Carcinogenic Risk of Chemicals to Humans, Vol. 38, Tobacco Smoking, Epidemiological Studies of Cancer in Humans, Lyon, pp. 199-314, 389-95. Kanai, Y., Manabe, S. & Wada, O. (1988). In vitro and in vivo N-acetylation of carcinogenic glutamic acid pyrolysis products in humans. Carcinogenesis, 9, 2179-84. Manabe, S. & Wada, O. (1988). Analysis of human plasma as an exposure level monitor for carcinogenic tryptophan pyrolysis products. Mutation Res., 209, 33 8. Manabe, S., Kanai, Y. & Wada, O. (1989a). Exposure level monitor of 3-amino- 1,4dimethyl-5H-pyrido[4,3-b]indole, a dietary carcinogen, in rabbits. Environ. Mutagenesis, 14, 3441. Manabe, S., Uchino, E. & Wada, O. (1989b). Carcinogenic tryptophan pyrolysis products in airborne particles and rainwater. Mutation Res., 226, 215-21. Manabe, S., Yanagisawa, H., Guo, S., Abe, S., Ishikawa, S. & Wada, O. (1987a). Detection of Trp-P-I and Trp-P-2, carcinogenic tryptophan pyrolysis products, in dialysis fluid of patients with uremia. Mutation Res., 179, 33~40. Manabe, S., Yanagisawa, H., Ishikawa, S., Kitagawa, Y., Kanai, Y. & Wada, O. (1987b). Accumulation of GIu-P-1 and GIu-P-2, carcinogenic glutamic acid pyrolysis products, in plasma of patients with uremia. Cancer Res., 47, 615~5. Matsumoto, T., Yoshida, D. & Tomita, H. (1981). Determination of mutagens, amino-~-carbolines in grilled foods and cigarette smoke condensate. Cancer Lett., 12, 105 10.
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Murray, S., Gooderham, N. J., Boobis, A. R. & Davis, D. S. (1989). Detection and measurement of MeIQx in human urine after ingestion of a cooked meat meal. Carcinogenesis, 10, 763-5. Sugimura, T. (1986). Studies on environmental chemical carcinogenesis in Japan. Science, 233, 312-18. Sugimura, T. & Sato, S. (1983). Mutagens-carcinogens in foods. Cancer Res., 43 (Suppl.), 2415s-21s. Sugimura, T., Kawachi, T., Nagao, M., Yahagi, T., Seino, Y., Okamoto, T., Shudo, K., Kosuge, T., Tsuji, K., Wakabayashi, K., Iitaka, Y. & Itai, A. (1977). Mutagenic principle(s) in tryptophan and phenylalanine pyrolysis products. Proc. Jpn. Acad., 53, 58-61. Wynder, E. L. & Gori, G. B. (1977). Contribution of the environment to cancer: an epidemiologic exercise. J. Natl. Cancer Inst., 58, 825-32. Yamashita, M., Wakabayashi, K., Nagao, M., Sato, S. Yamaizumi, Z., Takahashi, M., Kinae, N., Tomita, I. & Sugimura, T. (1986). Detection of 2-amino-3methyl-amidazo[4,5-f]quinoline in cigarette smoke condensate. Jpn. J. Cancer Res. (Gann), 77, 419-22. Yoshida, D. & Matsumoto, T. (1980). Amino-~t-carbolines as mutagenic agents in cigarette smoke condensate. Cancer Lett., 10, 141-9.
Carcinogenic Tryptophan Pyrolysis Products in Cigarette Smoke Condensate and Cigarette Smoke-Polluted Indoor Air Shigeo M a n a b e & O s a m u W a d a Department of Hygiene and PreventiveMedicine,Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,Tokyo 113, Japan (Received 1l August 1989; accepted 24 November 1989) ABSTRACT The carc&ogenic tryptophan pyrolys& products, 3-am&o-l,4-dimethyl5H-pyrido[4,3-b]indole (Trp-P-l) and 3-amino-l-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), have been measured in condensate of cigarette mainstream smoke by high-performance liquid chromatography. These carcinogens have been detected in indoor air as well as in the air of the outdoor environment. Levels of these carcinogens in indoor air were much higher than those in outdoor air. The souree of these carcinogens in indoor air was determined to be cigarette smoke by the application of smoking machine studies. Concentrations of these carcinogens in indoor air increased markedly with an increase in cigarettes smoked. The results in this investigation suggest that cigarette smoking is a source of carcinogenic tryptophan pyrolysis products in the indoor environment. Our data also suggest that smokers are persistently exposed to the carcinogenic heterocyclic amines together with potent carcinogens such as polynuclear aromatic hydrocarbons and N-nitroso compounds.
NOTATION A~C Glu-P-1 GIu-P-2 IQ MeA~C Trp-P-1 Trp-P-2
2-Amino-9H-pyrido[2,3-b]indole 2-Amino-6-methyldipyrido [ 1,2-a: 3',2'-d] imidazole 2-Aminodipyrido[ 1,2-a: 3',2'-d]imidazole 2-Amino-3-methylimidazo [4,5-f]quinoline 2-Amino-3-methyl-9H-pyridol-2,3-b]indole 3-Amino- 1,4-dimethyl- 5H-pyrido [4,3-b]indole 3-Amino- 1-methyl- 5H-pyrido [4,3-b]indole 121 Environ. Pollut. 0269-7491/90/$03-50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
122
Shigeo Manabe, Osamu Wada
INTRODUCTION Carcinogenic tryptophan pyrolysis products 3-amino-l,4-dimethyl-5Hpyrido[4,3-b]indole (Trp-P-1) and 3-amino-l-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) were first isolated from tryptophan pyrolysates as compounds with potent mutagenic effects on Salmonella t y p h i m u r i u m tester strains (Sugimura et al., 1977). Later, a new series ofheterocyclic amines were isolated as potent mutagens from pyrolysates of amino acids and proteins (Sugimura & Sato, 1983; Sugimura, 1986). All the mutagenic heterocyclic amines so far tested have been shown to be carcinogenic in experimental animals (Sugimura, 1986). The mutagenic and carcinogenic heterocyclic amines such as A~C, MeA~C, Glu-P-1, Glu-P-2, IQ, Trp-P-1 and Trp-P-2 (see Notation) have been reported to be present in several cooked foods (Sugimura & Sato, 1983). Recent investigations indicated that humans are actually exposed to these carcinogenic heterocyclic amines (Baker et al., 1982; Dolara et al., 1984; Felton & Healy, 1984; Hayatsu et al., 1985; Manabe et al., 1987a, b; Manabe & Wada, 1988; Kanai et al., 1988; Murray et al., 1989). Our recent investigation revealed that Trp-P-1 and Trp-P-2 are present in airborne particles and rain water, indicating that these carcinogens are ubiquitous environmental components (Manabe et al., 1989b). In the course of the previous study, we found that the levels of Trp-P-1 and Trp-P-2 in indoor air were higher than those in outdoor air. This suggested to us that cigarette smoke may be the origin or one of the origins of these carcinogens in indoor air, although the presence of these compounds in cigarette smoke had not been confirmed.
METHODS AND MATERIALS
Preparation of cigarette smoke condensate Commercial cigarettes (three Japanese brands, one American and one English brand) were used in this study. The filter-tipped cigarettes were smoked by the Filtrona Model 302 smoking machine under standard conditions (puff frequency: 1 puff/60 s; puff volume: 35 ml/2 s; butt length: 30 mm). The cigarette smoke condensate was collected on glass fiber filters (Toyo Roshi type GA 200, Toyo Roshi Co., Tokyo), and extracted twice with 100 ml of methanol/28% ammonia water in an ultrasonic bath. The extract was evaporated to dryness under a nitrogen stream. The cigarette smoke condensate thus obtained was dissolved in 20mM HaPO 4, pH 2"0/ acetonitrile (90/10, by vol.) and filtered with a disposable filter unit (0.45/~m, Gelman Science Japan Ltd, Tokyo). The filtrate served for HPLC analyses.
TrkTtophan pyrolysates in cigarette smoke
123
Preparation of particulate matter in indoor air before and after cigarette smoking In order to study indoor air pollution by carcinogenic heterocyclic amines through cigarette smoking, we used a representative room in our laboratory (Fig. 1) for smoking experiments. The test space was approximately 57.2 m 3, and the laboratory r o o m was equipped with an air-conditioner (ventilation rate: 5"2 m 3 air/min). A smoking machine and an air sampler were placed in the r o o m as shown in Fig. 1. Particulate matter in indoor and outdoor air was collected by a high-volume air sampler Kimoto Model-120 (Kimoto Electronics Co., Ltd., Osaka, Japan) on quartz microfiber filters (20"32cm × 25.4cm, W h a t m a n QM-A). Before and after smoking of Japanese filter cigarette C (Table i) with an automatic smoking machine with standard parameters as described above, the particulate matter in the air was collected with the air sampler by continuous suction for 60 min at a flow rate of 1.5 m3/min. One-fourth of the 20.32 cm × 25-4 cm filter was used for the determination o f carcinogenic tryptophan pyrolysis products. These carcinogens in the filter bearing the collected particulate matter were extracted once with methanol/28% a m m o n i a water (50/1, by vol.). Onefourth o f the filter was soaked in 200 ml o f methanol/28% a m m o n i a water
Fig. 1. A sketch of a room used for determining the pollution of indoor air by cigarette smoking. A: an automatic smoking machine, B: a high-volume air sampler.
Shigeo Manabe, Osamu Wada
124
TABLE 1 Carcinogenic T r y p t o p h a n Pyrolysis Products in Condensate of Cigarette Mainstream Smoke
Tobacco product a
Japanese filter cigarette A (2) Japanese filter cigarette B (2) Japanese filter cigarette C (4) US filter cigarette (3) UK filter cigarette (2) Mean _ SD
Mainstream condensate (mg/cig)
Carcinogenic tryptophan pyrolysates (ng/cig) Trp-P-1 b
Trp-P-2
7.8 6.6 17.5 9.6 10.1
0'19 c 0'29 0'31 0"20 0"24
0-65 0.61 0-66 0-51 0-53
10'3 _+ 4-3
0"25 _+ 0'05
0'59 _+ 0-07*
a In parentheses, n u m b e r of cigarettes tested. h Abbreviations: Trp-P- 1, 3-amino- 1,4-dimethyl-5H-pyrido[4,3-b]indole; Trp-P-2, 3-amino- l-met hyl-5H-pyrido[4,3-b]indole. c Values were not corrected for recoveries. * p < 0"001 as compared with Trp-P-1 levels.
for 30 min at room temperature in the ultrasonic bath. The methanol/28% ammonia water was condensed under a nitrogen stream. The condensed extract ( ~ 10 ml) was filtered with a disposable filter unit (0.45 #m, Gelman Science Japan, Ltd). The filtrate was further dried under a nitrogen stream and served for HPLC analyses.
HPLC analyses Reversed-phase HPLC analyses were performed by means of a Hitachi 655A chromatograph (Hitachi, Tokyo) equipped with an F-1000 fluorometric detector (Hitachi). Assay conditions for HPLC analyses were according to our previous method (Manabe et al., 1989b). Partial purification was carried out by using an Asahipack ES-502C column (9"0/~m particle size, 7.6 x 100 mm, Asahi Chemical Industries, Ltd, Kawasaki, Japan) under the following conditions: mobile phase, 20mM H3PO 4, pH 2-0/acetonitrile (90/10, by vol.); flow rate, 1.0ml/min at 40°C. Fluorescence was monitored at 399nm when excited at 266nm. The fractions corresponding to Trp-P-1 and Trp-P-2 were collected separately. Then, the volumes of these fractions were reduced to ~ l m l with a Centrifugal Evaporator EC-57 (Sakuma Seisakusho Co., Ltd, Tokyo). The condensed fractions (,--1 ml) were further purified by means of a Nucleosile 5C8 column (10ktm particle size, 4 x 150nm; Union Co., Ltd, Takasaki, Japan). The solvent system for the fraction corresponding to Trp-
Tryptophan pyrolysates in cigarette smoke
125
P-2 on the first-step purification was 20mM H3PO4, pH2-0/acetonitrile (90/10, by vol.) while the eluate for the Trp-P-1 fraction was 20 mM H3PO4, pH 2.0/acetonitrile (85/15, by vol.). A flow rate of 1.0ml/min at 50°C was used. Fluorescence was monitored at 399 nm when excited at 266 nm. The fraction corresponding to Trp-P-1 or Trp-P-2 was collected, and condensed to ,-~2 ml with a Centrifugal Evaporator EC-57. Final HPLC analysis was carried out on a Kaseisorb LC ODS-300-5 column (5/~m particle size and 300,~ pore size, 7"5 × 250mm; Tokyo Chemical Industries, Ltd, Tokyo), as described previously (Manabe & Wada, 1988; Manabe et al., 1989b). The mobile phase was a gradient (0-30%) of acetonitrile in 10mM H3PO 4 over a course of 35 min. The flow rate was 3.0ml/min at 50°C. Fluorescence was monitored as described above. The fraction corresponding to each authentic compound was collected separately. The contents of the fractions were extracted with chloroform after the acetonitrile in the fractions was evaporated and the pH was adjusted to ~ 10-0 with 28% ammonia/water (Manabe & Wada, 1988).
Spectrophotometric analyses Spectrometric measurements were performed on a Shimadzu UV 260 spectrophotometer (Shimadzu, Kyoto) and a Hitachi F-3000 fluorescence spectrophotometer (Hitachi, Tokyo) after dissolving the samples in methanol. The mass spectral analyses were conducted in the electron impact (El) mode using the direct insertion probe on a double-focusing JEOL JMSDx 300 mass spectrometer. The mass spectra were recorded with JMA-3500 mass data analysis system, employing 70eV ionization voltage, 300/~A ionization current and 3 kV accelerating voltage with a course temperature of 200°C.
RESULTS
Amounts of Trp-P-1 and Trp-P-2 in cigarette smoke condensate Typical charts of HPLC for cigarette smoke condensate are shown in Fig. 2 when one-fourth of the extract from the mainstream smoke of a Japanese filter cigarette was analyzed. On the final-step analyses, sharp peaks corresponding to Trp-P-1 and Trp-P-2 were clearly identifiable (Fig. 2(C)). As reported previously (Manabe & Wada, 1988), peak heights of Trp-P-1 and Trp-P-2 on the final chromatogram obtained by using an ODS-300-5 column showed linear relationships with the amounts of authentic carcinogens. The amounts of Trp-P-1 and Trp-P-2 in cigarette smoke
126
Shigeo Manabe, Osamu Wada
(B)
(A)
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.
rl
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Fig. 2. HPLC chromatographic profiles of Trp-P-I and Trp-P-2 in cigarette smoke condensate. Fluorescencewas monitored at 399 nm when excited at 266 nm. (A) Preparative HPLC chromatogram on an ES-502C column.The fractions corresponding to authentic TrpP-1 and Trp-P-2 were collected separately.(B) Chromatograms of the second step purification on a Nucleosile 5C8 column. Upper: Chromatogram of the fraction corresponding to Trp-P-2 on the first step purification(A). Lower:Chromatogram of the fraction corresponding to TrpP-1 on the first step purification (A). (C) Final chromatographic profiles on an ODS-300-5 column. condensate were calculated on the basis of these linear relationships. As shown in Table 1, the mean level o f Trp-P-2 in cigarette smoke condensate was approximately twice that of Trp-P-1.
Recoveries The glass fiber filters absorbed with methanol solution o f Trp-P-1 and TrpP-2 (each 100pg) were treated by the same procedure as that for the analytical sample. The percentages o f Trp-P-1 and Trp-P-2 recovered by this method were 71.0% and 66-4%, respectively. The chemical forms of these
127
Tryptophan pyrolysates in cigarette smoke
compounds in cigarette smoke condensate have not been determined. If parts of these compounds in cigarette smoke condensate are bound to other materials, those compounds may not be extracted by the method we used (Manabe et al., 1989a). Therefore, the data indicated were not corrected for the recoveries (Table 1).
Identification of Trp-P-1 and Trp-P-2 Two hundred cigarettes were smoked by an tutomatic smoking machine. The smoke condensate was treated by the same procedure as that for the analytical sample. For confirmation of the compounds purified with HPLC, ultraviolet (UV) absorbance and fluorescence and mass spectra were determined. The absorbance and fluorescence spectra of the fraction corresponding to Trp-P-1 and Trp-P-2 on the final purification step were identical with the UV absorbance and fluorescence spectra of authentic compounds (Fig. 3). Mass spectra of the fractions corresponding to Trp-P-1 and Trp-P-2 revealed molecular ion peaks at m/z 211 and m/z 197, respectively, which were identical with those observed for authentic Trp-P- 1 and Trp-P-2 (not shown).
t ~o
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2'00
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Fig. 3. Fluorescence emission spectra excited at 266 nm (upper) and UV absorption spectra (lower) of Trp-P-1 and Trp-P-2 fractions. (A) The fraction corresponding to Trp-P-I in cigarette smoke condensate ( ) and authentic Trp-P-1 (0"2/~M) (..... ). (B) The fraction corresponding to Trp-P-2 in cigarette smoke condensate ( ) and authentic Trp-P-2 (0"6/~M) (..... ).
Shigeo Manabe, Osamu Wada
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Levels of Trp-P-1 and Trp-P-2 in indoor air before and after cigarette smoking In order to determine the effects of cigarette smoking on Trp-P-1 and Trp-P-2 levels in indoor air, levels of these carcinogens in the air of the test space (Fig. 1) were determined before and after cigarette smoking. Trp-P-1 and Trp-P-2 were detected in the indoor air before smoking, and concentrations of Trp-P-1 and Trp-P-2 were 0-416pg/m 3 air and 0.610pg/m 3 air, respectively (Fig. 4), whereas concentrations of Trp-P-1 and Trp-P-2 in the outdoor air determined on the same day were 0"06 pg/m 3 air and 0.05 pg/m 3 air, respectively. The results indicate that levels of these carcinogens in the indoor air of the test room were much higher than those in the air of the outdoor environment. Trp-P-1 and Trp-P-2 levels in the indoor air were remarkably increased with the number of cigarettes smoked (Fig. 4). After this experiment, the door of the room used in this study was left open and the indoor air was ventilated by an airconditioner for 1 h at a flow rate of 5.2 m 3 air/min. Then, the particulate matter in the air of the indoor environment was collected by a high-volume air sampler for 12h: at a flow rate of 1"5 m3/min. Concentrations of Trp-P-1 and Trp-P-2 in the indoor air after L.
"~ 100
i/I
.o 0..
.~_ 10 el c ¢Q.
o
c
/// :/ 0
C
u 0.1
Number of
Cigarette Smoked
Fig. 4. Increases of concentrations of Trp-P-1 ( 0 - - 0 ) and Trp-P-2 ( 0 - - 0 ) in indoor air with the number of cigarettes smoked with an automatic smoking machine.
129
Tryptophan pyrolysates in cigarette smoke
ventilation were reduced to 0.78pg/m 3 and 1.67pg/m 3, respectively. However, levels of these carcinogens in the indoor air were apparently higher than those before cigarette smoking. These results indicate that the indoor air of the test space can be polluted by carcinogenic tryptophan pyrolysis products of cigarette smoking.
DISCUSSION Epidemiological studies have shown that cigarette smoking is associated with the development of human cancers (Doll, 1977; Wynder & Gori, 1977; Doll & Peto, 1981; Brinton et al., 1984). The role of tobacco smoke as a causative agent in various types of cancer has been frequently reaffirmed by many researchers (IARC Monograph, 1986). Known carcinogens in tobacco smoke include polynuclear aromatic hydrocarbons (PAH), N-nitroso compounds, polycyclic aza-arenes and miscellaneous nitrogen compounds (IARC Monograph, 1986). Recent investigations have revealed that carcinogenic heterocyclic amines such as IQ, A0~Cand MeA~C are present in cigarette smoke condensate (Yoshida & Matsumoto, 1980; Matsumoto et al., 1981; Yamashita et al., 1986). However, the presence of carcinogenic tryptophan pyrolysis products in cigarette smoke has not been confirmed. In this investigation, we have clearly demonstrated that Trp-P-1 and Trp-P-2 are present in cigarette smoke condensate. The mean levels of Trp-P-1 and Trp-P-2 in the condensate of mainstream smoke were 0.25ng/cig and 0.59 ng/cig, respectively (Table 1). The mean level of Trp-P-2 in cigarette smoke condensate was approximately twice that of Trp-P-1. So far as is known, the presence of three carcinogenic heterocyclic amines (A~C, MeA~C and IQ) has been confirmed in previous reports (Table 2). As TABLE 2
Summary of CarcinogenicHeterocyclicAmine Levels in Cigarette Mainstream Smoke Carcinogenic heterocyclic amines ~
A~C MeA~C IQ Trp-P-1 Trp-P-2
Range (ng/cig)
Mean (ng/cig)
25-258 9-37 -0"19-0"31 0'51-0-66
101'1 18'2 0.26 0"25 0'59
ReJerences
Yoshida& Matsumoto (1980) Yoshida& Matsumoto (19801 Yamashitaet al. (1986) This study This study
a Abbreviations: A~C, 2-amino-9H-pyrido[2,3-b]indole; MeA~C, 2-amino-3-methyl-9Hpyrido[2,3-b]indole; IQ, 2-amino-3-methylimidazo[4,5-f-Jquinoline;Trp-P-l, 3-amino-l,4dimethyl-5H-pyrido[4,3-b]indole;Trp-P-2, 3-amino-l-methyl-5H-pyrido[4,3,-b]indole.
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Shigeo Manabe, Osamu Wada
compared with the reported values of these carcinogenic heterocyclic amines in cigarette smoke condensate (Table 2), it seems that Trp-P-1 levels in cigarette smoke condensate are almost the same as the IQ level. It has been shown that air in indoor environment with extreme tobacco smoke pollution can contain several times higher concentrations of polynuclear aromatic hydrocarbons and N-nitrosamines than polluted urban air (IARC Monograph, 1983; Brunnemann & Hoffman, 1978). A similar phenomenon was observed in this study. Levels of carcinogenic tryptophan pyrolysis products in the indoor air of the test space were apparently higher than those in the outdoor air observed on the same day that the indoor air was sampled. This turned our attention to the origin(s) of these carcinogens in the indoor air. In order to determine that cigarette smoke is one of the origins of the carcinogenic tryptophan pyrolysis products in indoor air, levels of these carcinogens in the air of the indoor environment were measured before and after cigarette smoking by an automatic smoking machine. As shown in Fig. 4, levels of these carcinogens were increased with an increase in cigarettes smoked. Furthermore, even after ventilation of the air in the test room (Fig. 1), levels of these carcinogens in the indoor air of the test space were apparently higher than those before cigarette smoking. The results indicate that the indoor air of the test space can be polluted by carcinogenic tryptophan pyrolysis products in cigarette smoke. The carcinogenic heterocyclic amines were originally isolated from pyrolysates of proteins and amino acids (Sugimura & Sato, 1983; Sugimura, 1986). These carcinogens have been thought to be formed during cooking of foods. Therefore, these compounds are called 'dietary carcinogens'. However, the presence of carcinogenic heterocyclic amines including Trp-P-1 and Trp-P-2 in cigarette smoke condensate might suggest that these carcinogens are produced by combustion of various materials such as foods, garbage, wood and grass. The hypothesis seems to be supported by the fact that carcinogenic tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, are present in airborne particles and rain water (Manabe et al., 1989b). While it is, of course, important to investigate the mechanism of formation of these carcinogens, it is also necessary to determine the distributions of these heterocyclic amines in the environment.
ACKNOWLEDGEMENT This research was supported in part by grants A-63440029 and B-63480174 from the Ministry of Education, Science and Culture of Japan.
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