Clinical Investigator
Clin Investig (1992) 70:290-294
Original ArtJcJe
© Springer-Verlag 1992
Tobacco-specific nitrosamines- metabolism and biological monitoring of exposure to tobacco products E. Richter, G. Sch/iffler, A. Malone, and J. Schulze Walther Straub-Institut f/Jr Pharmakologie und Toxikologie, Ludwig-Maximilians-Universitfit Mfinchen
Summary. Tobacco-specific nitrosamines are derived from nicotine and related tobacco alkaloids and can be detected in tobacco products as well as in mainstream and sidestream smoke. Two of them, N-nitrosonornicotine and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone, are strong carcinogens in laboratory animals. Because of its organospecificity for the lung, the latter is considered to be a causative factor in tobacco-related human lung cancer. U p o n metabolic activation both nitrosamines give rise to a common reactive intermediate binding to macromolecules such as D N A and haemoglobin and hydrolysing to 4-hydroxy-l-(3pyridyl)-l-butanone. Because of easy access to large quantities of haemoglobin from blood sampies, it is most suitable for biomonitoring human exposure to tobacco-specific nitrosamines. A highly sensitive analytical method for determination of femtogram amounts of 4-hydroxy-l-(3-pyridyl)-lbutanone provides an approach to assess individual exposure to active and passive smoking.
Key words: Tobacco - Tobacco-specific nitrosamines - Metabolism - Biomonitoring
The correlation between smoking and lung cancer is well known from epidemiological studies [13]. However, the ultimate lung carcinogen has still not been identified, since tobacco and tobacco smoke NNK = 4-(methylnitrosamino)-l-(3-pyridyl)-lbutanone; TSNA=tobacco-specific nitrosamines; ETS=environmental tobacco smoke; NDMA = N-nitrosodimethylamine; NNN = N-nitrosonornicotine; NAB = N-nitrosoanabasine; NAT = N-nitrosoanatabine; HPB = 4-hydroxy-l-(3-pyridyl)-1butanone; NNAL = 4-(methylnitrosamino)-l-(3-pyridyl)-1-butan-l-ol; NICI-MS=negative ion chemical ionization mass spectrometry; 4-ABP= 4-aminobiphenyl Abbreviations:
contain a variety of cancer causing agents, including nitrosamines, polycyclic aromatic hydrocarbons, aromatic amines, 21°po, formaldehyde and others. Of these substances, the nicotine derived nitrosamino ketone ( N N K ) is organospecific for the lung in animal experiments and therefore has been suspected as being the substance responsible for the high incidence of lung cancer in cigarette smokers [7].
Occurrence of TSNA N N K , 4-(methylnitrosamino)- 1-(3-pyridyl)- l-butanone, belongs to the group of so-called tobaccospecific nitrosamines (TSNA), which are nitrosation products of nicotine and other tobacco alkaloids regularly found both in tobacco products and in tobacco smoke in high concentrations (Fig. 1, Table 1). Sidestream smoke often contains higher concentrations of T S N A than mainstream smoke, raising the question of lung cancer induction by environmental tobacco smoke (ETS) [7]. In all tobacco products other nitrosamines also occur, e.g. N-nitrosodimethylamine (NDMA). However, tobacco products are not the only sources of these nitrosamines.
Carcinogenic properties of TSNA Animal experiments to clarify the carcinogenic properties of T S N A have clearly pointed to N N K as a strong lung carcinogen (Fig. 2). After subcutaneous administration to Fischer 344 rats, N N K elicited a dose-dependent incidence of lung cancers. In addition, tumours in the liver and nose were observed. However, pancreatic tumours were only seen after oral administration of N N K with the drinking water [14]. N N K was found to be more potent than N D M A , an extensively assayed
291
NICOTINE
NORNICOTINE
ANATABINE
ANABASINE
1
l N,.-O 4-(METHYI-NITROSAMINO)-1(3-PYRIDYL)-I-BUTANONE
N'-NITROSONORNICOTINE N'-NITROSOANABASINE
(NNN)
(NAB)
(NNK)
Table 1. Tobacco-specific nitrosamines in mainstream (MS) and sidestream smoke (SS) of four US commercial brands of cigarettes [1], in MS and cigarette tobacco (CT) of 20 German commercial brands of cigarettes [19], and in smokeless tobacco (ST) of either 20 products of different countries [18] or eight US commercial brands of snuff [4]. The values are presented as nanograms per cigarette for the cigarette products and as micrograms per gram of tobacco for the smokeless tobacco products Sample
NNN
NNK
NAT/NAB
Reference
MS SS MS CT
66-1007 185-857 19 855 400-5340
17425 386-1444 21-470 100 960
102-744 125-783 18 520 220-2330
1 1 19 19
ST ST
0.3-79 4.2 129
0.1-24 0.6-15.2
0.4-99 3.7 184
18 4
N'-NITROSOANATABINE
(NAT)
Fig. 1. Structures of tobacco alkaloids and the corresponding nitrosamines
nitrosamine [8]. Lung tumours were also observed in Syrian golden hamsters and A/J mice. In Sencar mice topical application of N N K resulted in the induction of lung tumours, as well as skin tumours. After oral swabbing of N N K together with N-nitrosonornicotine (NNN), rats developed oral cavity tumours [7]. NNN, the second toxicologically important TSNA, is as potent a carcinogen as NNK, producing mainly nasal tumours. On the other hand Nnitrosoanatabine (NAT) and N-nitrosoanabasine (NAB) have little or no carcinogenic activity: NAT was inactive in rats in lifetime doses up to 9 mmol/ kg, and NAB was inactive in Syrian hamsters and a weak oesophageal carcinogen in rats [7].
Metabolismof NNKandNNN lOO]
8O
j'° t 80 501 40
30t 20
10 0
B 0.09
0.17
0.33
Total dose of ~
0.68
0.98
(mmol/kg b.w.)
Fig. 2. Organospecificity of N N K in F344 rats. N N K was either injected subcutaneously three times a week for 20 weeks to give total doses of 0.09 [3], 0.33 [9] and 0.98 mmol/kg [8], or given lifelong orally with the drinking water [14]. ~ nose; I lung; ~ liver; ~ pancreas
The metabolism of N N K and N N N has been extensively investigated in vivo and in vitro [7, 10]. Metabolic activation for binding to DNA and other cellular macromolecules occurs by hydroxylation in the methyl and methylene carbon adjacent to the N-nitroso nitrogen (c~-hydroxylation) (Fig. 3). 2'-Hydroxylation of N N N and hydroxylation of the methyl carbon of N N K lead to a common hypothetical pyridyloxobutylating intermediate. Upon hydrolysis of DNA or haemoglobin adducts 4-hydroxy-l-(3-pyridyl)-l-butanone (HPB) is released. Because of its specific structure this adduct has been proposed as a suitable dosimeter for exposure to TSNA [5]. Another common metabolic pathway of N N N and N N K is the oxidation of the pyridine nitrogen. w i t h NNN, hydroxylation of the 3'- and 4'-carbons, as well as denitrosation, have also been ob-
292
OH N=O .,,,*"~J N~. CH~ N" NNAL
O
~
I
'~'~'J
NNK
/
o
N=O
N=O
\
-7
/ O
N=O
-1
L,:~',,.m
1O
\ DNA N ' ~ O I , CH30H
NNN
N=O
]
/
1
[ DNAor globlrl
7-melhylguinlne OI.melhylguanlne
I
H20
adducts
Ol-methylthymldlnll
J hydrolysis
H20
~ O H
0 ~~HPB
OH =
Fig. 3. Metabolic activation of N N K and NNN. Substances in brackets are chemically unstable in aqueous solution, reacting with electrophiles like water, proteins or nucleic acids
served. An important initial metabolic pathway of N N K is the reduction of the carbonyl group to 4-(methylnitrosamino)-l-(3-pyridyl)-l-butan-l-ol (NNAL), itself a potent carcinogen with organospecificity for the lung, liver and pancreas [14]. Recently, the glucuronide of N N A L was characterized as a N N K metabolite in the urine of rats and mice [12]. This metabolite is also excreted into the bile of rats [Binder and Richter, unpublished observation]. N N K undergoes a substantial first-pass metabolism during absorption in isolated perfused small intestinal segments of rats [11] and mice [15]. NOxidation was a major metabolic pathway in rats but was nearly absent in mice. The first-pass metabolism of N N K could be induced by starvation and/or acetone pretreatment. To our knowledge this is the first example of the induction of xenobiotic metabolism in small intestine by ketone bodies, and is in contrast to the reduced activity of arylhydrocarbon hydroxylase after starvation [20]. Biomonitoring of TSNA exposure Monitoring the exposure to tobacco smoke, especially to passive smoking, has proved to be a complicated procedure. To monitor exposure of humans to tobacco smoke the test has to fulfil the following conditions:
-
sensitivity to reliably detect the exposure to even small amounts of tobacco smoke; - s e l e c t i v i t y to distinguish between tobaccosmoke-related effects and similar effects with other causes; - availability of samples for analytical determinations.
Haemoglobin adducts have been proposed as internal dosimeters for carcinogens because of the ease of isolation of large quantities of haemoglobin from blood, the long lifetime of erythrocytes allowing the estimation of chronic exposure, and evidence from animal experiments showing a correlation with D N A adducts [6, 16]. To detect exposure to tobacco-smoke products selectively, the measurement of HPB, the common adduct from the decay of N N K and N N N , seems to be most promising because of its unique chemical structure (Fig. 3) [5]. It might be preferred over measuring the more easily detectable adducts formed by aromatic amines, aromatic hydrocarbons and ethylene oxide which also have a variety of other sources. The haemoglobin adduct HPB resulting from in vivo N N K and N N N degradation is therefore a selective parameter for environmental exposure to tobacco products. We changed the protocol described by Carmella et al. [5] to avoid extensive extraction of the aqueous haemoglobin hydrolysate. This streamlined the procedure and simplified
293
Collection of erythrocytes from blood samples ( ~ 20 ml); haemolysis by addition of distilled water (store at - 2 0 ° C if desired). HB-quantification. Addition of deuterated internal standard; protein cleavage by sonication in 0.1 N NaOH for 1 h with ultrasound. Protein precipitation with 10% w/v ammonium sulphate; centrifugation Clear supernatant over C18 Bond Elut (1 ml bed volume); wash with 150 mM NaxPO4, pH 9.0 Elution of HPB with < 1 ml acetonitrile; evaporate acetonitrile; resuspend sample in 600 gl CH2C12 and 600 gl 1 M triethylamine in hexane Add 2 gl pentafluorobenzoyl chloride for derivatization; heat at 60° C for 2 h Prepurification of HPB on HPLC. Addition of the retention time markers pentano- and hexanophenone (12.5 cm, C18; eluent 50% acetonitrile/water, 0.7 ml/min); retention time of HPB ~ 20 min Evaporate solvent completely; resuspend in 20 gl hexane; analyse 2 gl on GC-MS
Fig. 4. Analytical procedure for the analysis of haemoglobin
[2, 16]. Although a large interindividual variability is observed in smokers and non-smokers, no overlap of adduct levels between the two groups is found. In a smoking cessation study the concentration of the 4-ABP adduct decreased from 120_+ 7 pg/g to 82_+ 6 pg/g within 3 weeks and to 34_+ 7 pg/g within 2-3 months after stopping smoking. Various factors have been shown to affect 4-ABP adduct levels including the number of cigarettes smoked per day, type of cigarette tobacco (blond versus black), metabolic phenotype of the smoker, and possibly the extent of ETS exposure. Preliminary studies in our laboratory have shown that 4-ABP haemoglobin adducts can be analysed in parallel with HPB. The only modification to the scheme shown in Fig. 4 is the use of a second reagent, pentafluoropropionic anhydride, for derivatization of 4-ABP. Both derivatives are most sensitively detected by NICI-MS [5, 17]. Just having received the necessary equipment for this technique we are planning to measure the adduct levels of HPB and 4-ABP in healthy male volunteers with different, carefully controlled exposures to active and passive smoking as well as to other tobacco products. Acknowledgement. This study was supported by a grant from the Forschungsrat Rauchen und Gesundheit.
adducts
References
the handling, thus allowing the processing of a larger number of samples. The principle of the procedure used in our laboratory is outlined in Fig. 4. Another major difference from the method of Carmella et al. [5] is the detection by electron impact mass spectrometry (HP-MSD; Hewlett-Packard, Frankfurt) instead of negative ion chemical ionization mass spectrometry (NICI-MS). The latter detection method is not only two orders of magnitude more sensitive but is also more specific. With our present instrumentation we were able to detect HPB haemoglobin adducts in blood samples from heavy smokers and users of nasal snuff. However, our current threshold for the detection of tobaccospecific haemoglobin adducts is not sufficiently sensitive to detect HPB reliably in very low concentrations that might be expected in non-smokers exposed to ETS [5]. Although the haemoglobin adduct of the bladder carcinogen 4-aminobiphenyl (4-ABP) should be less characteristic for exposure to tobacco smoke because of other possible sources, recent results have shown that 4-ABP exposure is indeed predominantly the result of cigarette smoking
1. Adams JD, O'Mara-Adams KJ, Hoffmann D (1987) Toxic and carcinogenic agents in undiluted mainstream smoke and sidestream smoke of different types of cigarettes. Carcinogenesis 8:729 731 2. Bartsch H, Caporaso N, Coda M, Kadlubar F, Malaveille C, Skipper P, Talaska G, Tannenbaum SR, Vineis P (1990) Carcinogen hemoglobin adducts, urinary mutagenicity, and metabolic phenotype in active and passive cigarette smokers. J Natl Cancer Inst 82:1826-1831 3. Belinsky SA, Foley JF, White CM, Anderson MW, Maronpot RR (1990) Dose-response relationship between 0 6methylguanine formation in Clara cells and induction of pulmonary neoplasia in the rat by 4-(methylnitrosamino)-l(3-pyridyl)-l-butanone. Cancer Res 50:3772-3780 4. Brunnemann KD, Genoble L, Hoffmann D (1987) Identification and analysis of a new tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-4-(3-pyridyl)-l-butanol.Carcinogenesis 8 :465-469 5. Carmella SG, Kagan SS, Foiles PG, Palladino G, Quart AM, Quart E, Hecht SS (1990) Mass spectrometric analysis of tobacco-specific nitrosamine hemoglobin adducts in snuff-dippers, smokers, and nonsmokers. Cancer Res 50:5438 5445 6. Ehrenberg L, Osterman-Golkar S (1980) Alkylation of macromolecules for detecting mutagenie agents. Teratogenesis Carcinog Mutagen 1 : 105 127 7. Hecht SS, Hoffmann D (1988) Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis 9 : 875-884
294 8. Hecht SS, Trushin N, Castonguay A, Rivenson A (1986) Comparative tumorigenicity and DNA methylation in F344 rats by 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanoneand N-nitrosodimethylamine. Cancer Res 46: 498-502 9. Hecht SS, Lin D, Castonguay A, Rivenson A (1987) Effects of e-deuterium substitution on the tumorigenicity of 4(methylnitrosamino)-l-(3-pyridyl)-l-butanonein F344 rats. Carcinogenesis 8:291-294 10. Hoffmann D, Hecht SS (1985) Nicotine derived N-nitrosamines and tobacco related cancer: current status and future directions. Cancer Res 45:935-944 11. Malone A (1991) First-Pass-Metabolismus von 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanon, NNK, am Dfinndarm der Ratte. Dissertation, Tier/irztliche Fakult/it der Ludwig-Maximilians-Universit/it,Mfinchen 12. Morse MA, Eklind KI, Toussaint M, Amin SG, Chung F-L (1990) Characterization of a glucuronide metabolite of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone(NNK) and its dose-dependent excretion in the urine of mice and rats. Carcinogenesis 11:1819-1823 13. Ockene JK, Kuller LH, Svendsen KH, Meilahn E (1990) The relationship of smoking cessation to coronary heart disease and lung cancer in the multiple risk factor intervention trial (MRFIT). Am J Publ Health 80:954-958 14. Rivenson A, Hoffmann D, Prokopczyk B, Amin S, Hecht SS (1988) Induction of lung and exocrine pancreas tumors in F344 rats by tobacco-specific and Areca-derived N-nitrosamines. Cancer Res 48:6912-6917 15. Schulze J, Hunder G, Richter E (1990) Intestinal first pass metabolism of NNK in the mouse. In : Adlkofer F, Thurau
K (eds) Effects of nicotine on biological systems. Birkh/iuser Basel, pp 123-127 16. Skipper PL, Tannenbaum SR (1990) Protein adducts in the molecular dosimetry of chemical carcinogens. Carcinogenesis 11:507-518 17. Stillwell WG, Bryant MS, Wishnok JS (1987) GC/MS analysis of biologically important aromatic amines. Application to human dosimetry. Biomed Environ Mass Spectrom 14:221-227 18. Tricker AR, Haubner R, Spiegelhalder B, Preussmann R (1988) The occurrence of tobacco-specific nitrosamines in oral tobacco products and their potential formation under simulated gastric conditions. Food Chem Toxicol 26:861865 19. Tricker AR, Ditrich C, Preussmann R (1991) N-Nitroso compounds in cigarette tobacco and their occurrence in mainstream tobacco smoke. Carcinogenesis 12:257-261 20. Wattenberg LW (1972) Dietary modification of intestinal and pulmonary arylhydrocarbon hydroxylase activity. Toxicol Appl Pharmacol 23 : 741-747 Received: July 15, 1991 Accepted: September 3, 1991 Priv.-Doz. Dr. E. Richter Walther Straub-Institut fiir Pharmakologie und Toxikologie Ludwig-Maximilians-Universitfit Miinchen Nussbaumstrasse 26 W-8000 Miinchen 2, FRG
Clin Investig (1992) 70:290-294
Original ArtJcJe
© Springer-Verlag 1992
Tobacco-specific nitrosamines- metabolism and biological monitoring of exposure to tobacco products E. Richter, G. Sch/iffler, A. Malone, and J. Schulze Walther Straub-Institut f/Jr Pharmakologie und Toxikologie, Ludwig-Maximilians-Universitfit Mfinchen
Summary. Tobacco-specific nitrosamines are derived from nicotine and related tobacco alkaloids and can be detected in tobacco products as well as in mainstream and sidestream smoke. Two of them, N-nitrosonornicotine and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone, are strong carcinogens in laboratory animals. Because of its organospecificity for the lung, the latter is considered to be a causative factor in tobacco-related human lung cancer. U p o n metabolic activation both nitrosamines give rise to a common reactive intermediate binding to macromolecules such as D N A and haemoglobin and hydrolysing to 4-hydroxy-l-(3pyridyl)-l-butanone. Because of easy access to large quantities of haemoglobin from blood sampies, it is most suitable for biomonitoring human exposure to tobacco-specific nitrosamines. A highly sensitive analytical method for determination of femtogram amounts of 4-hydroxy-l-(3-pyridyl)-lbutanone provides an approach to assess individual exposure to active and passive smoking.
Key words: Tobacco - Tobacco-specific nitrosamines - Metabolism - Biomonitoring
The correlation between smoking and lung cancer is well known from epidemiological studies [13]. However, the ultimate lung carcinogen has still not been identified, since tobacco and tobacco smoke NNK = 4-(methylnitrosamino)-l-(3-pyridyl)-lbutanone; TSNA=tobacco-specific nitrosamines; ETS=environmental tobacco smoke; NDMA = N-nitrosodimethylamine; NNN = N-nitrosonornicotine; NAB = N-nitrosoanabasine; NAT = N-nitrosoanatabine; HPB = 4-hydroxy-l-(3-pyridyl)-1butanone; NNAL = 4-(methylnitrosamino)-l-(3-pyridyl)-1-butan-l-ol; NICI-MS=negative ion chemical ionization mass spectrometry; 4-ABP= 4-aminobiphenyl Abbreviations:
contain a variety of cancer causing agents, including nitrosamines, polycyclic aromatic hydrocarbons, aromatic amines, 21°po, formaldehyde and others. Of these substances, the nicotine derived nitrosamino ketone ( N N K ) is organospecific for the lung in animal experiments and therefore has been suspected as being the substance responsible for the high incidence of lung cancer in cigarette smokers [7].
Occurrence of TSNA N N K , 4-(methylnitrosamino)- 1-(3-pyridyl)- l-butanone, belongs to the group of so-called tobaccospecific nitrosamines (TSNA), which are nitrosation products of nicotine and other tobacco alkaloids regularly found both in tobacco products and in tobacco smoke in high concentrations (Fig. 1, Table 1). Sidestream smoke often contains higher concentrations of T S N A than mainstream smoke, raising the question of lung cancer induction by environmental tobacco smoke (ETS) [7]. In all tobacco products other nitrosamines also occur, e.g. N-nitrosodimethylamine (NDMA). However, tobacco products are not the only sources of these nitrosamines.
Carcinogenic properties of TSNA Animal experiments to clarify the carcinogenic properties of T S N A have clearly pointed to N N K as a strong lung carcinogen (Fig. 2). After subcutaneous administration to Fischer 344 rats, N N K elicited a dose-dependent incidence of lung cancers. In addition, tumours in the liver and nose were observed. However, pancreatic tumours were only seen after oral administration of N N K with the drinking water [14]. N N K was found to be more potent than N D M A , an extensively assayed
291
NICOTINE
NORNICOTINE
ANATABINE
ANABASINE
1
l N,.-O 4-(METHYI-NITROSAMINO)-1(3-PYRIDYL)-I-BUTANONE
N'-NITROSONORNICOTINE N'-NITROSOANABASINE
(NNN)
(NAB)
(NNK)
Table 1. Tobacco-specific nitrosamines in mainstream (MS) and sidestream smoke (SS) of four US commercial brands of cigarettes [1], in MS and cigarette tobacco (CT) of 20 German commercial brands of cigarettes [19], and in smokeless tobacco (ST) of either 20 products of different countries [18] or eight US commercial brands of snuff [4]. The values are presented as nanograms per cigarette for the cigarette products and as micrograms per gram of tobacco for the smokeless tobacco products Sample
NNN
NNK
NAT/NAB
Reference
MS SS MS CT
66-1007 185-857 19 855 400-5340
17425 386-1444 21-470 100 960
102-744 125-783 18 520 220-2330
1 1 19 19
ST ST
0.3-79 4.2 129
0.1-24 0.6-15.2
0.4-99 3.7 184
18 4
N'-NITROSOANATABINE
(NAT)
Fig. 1. Structures of tobacco alkaloids and the corresponding nitrosamines
nitrosamine [8]. Lung tumours were also observed in Syrian golden hamsters and A/J mice. In Sencar mice topical application of N N K resulted in the induction of lung tumours, as well as skin tumours. After oral swabbing of N N K together with N-nitrosonornicotine (NNN), rats developed oral cavity tumours [7]. NNN, the second toxicologically important TSNA, is as potent a carcinogen as NNK, producing mainly nasal tumours. On the other hand Nnitrosoanatabine (NAT) and N-nitrosoanabasine (NAB) have little or no carcinogenic activity: NAT was inactive in rats in lifetime doses up to 9 mmol/ kg, and NAB was inactive in Syrian hamsters and a weak oesophageal carcinogen in rats [7].
Metabolismof NNKandNNN lOO]
8O
j'° t 80 501 40
30t 20
10 0
B 0.09
0.17
0.33
Total dose of ~
0.68
0.98
(mmol/kg b.w.)
Fig. 2. Organospecificity of N N K in F344 rats. N N K was either injected subcutaneously three times a week for 20 weeks to give total doses of 0.09 [3], 0.33 [9] and 0.98 mmol/kg [8], or given lifelong orally with the drinking water [14]. ~ nose; I lung; ~ liver; ~ pancreas
The metabolism of N N K and N N N has been extensively investigated in vivo and in vitro [7, 10]. Metabolic activation for binding to DNA and other cellular macromolecules occurs by hydroxylation in the methyl and methylene carbon adjacent to the N-nitroso nitrogen (c~-hydroxylation) (Fig. 3). 2'-Hydroxylation of N N N and hydroxylation of the methyl carbon of N N K lead to a common hypothetical pyridyloxobutylating intermediate. Upon hydrolysis of DNA or haemoglobin adducts 4-hydroxy-l-(3-pyridyl)-l-butanone (HPB) is released. Because of its specific structure this adduct has been proposed as a suitable dosimeter for exposure to TSNA [5]. Another common metabolic pathway of N N N and N N K is the oxidation of the pyridine nitrogen. w i t h NNN, hydroxylation of the 3'- and 4'-carbons, as well as denitrosation, have also been ob-
292
OH N=O .,,,*"~J N~. CH~ N" NNAL
O
~
I
'~'~'J
NNK
/
o
N=O
N=O
\
-7
/ O
N=O
-1
L,:~',,.m
1O
\ DNA N ' ~ O I , CH30H
NNN
N=O
]
/
1
[ DNAor globlrl
7-melhylguinlne OI.melhylguanlne
I
H20
adducts
Ol-methylthymldlnll
J hydrolysis
H20
~ O H
0 ~~HPB
OH =
Fig. 3. Metabolic activation of N N K and NNN. Substances in brackets are chemically unstable in aqueous solution, reacting with electrophiles like water, proteins or nucleic acids
served. An important initial metabolic pathway of N N K is the reduction of the carbonyl group to 4-(methylnitrosamino)-l-(3-pyridyl)-l-butan-l-ol (NNAL), itself a potent carcinogen with organospecificity for the lung, liver and pancreas [14]. Recently, the glucuronide of N N A L was characterized as a N N K metabolite in the urine of rats and mice [12]. This metabolite is also excreted into the bile of rats [Binder and Richter, unpublished observation]. N N K undergoes a substantial first-pass metabolism during absorption in isolated perfused small intestinal segments of rats [11] and mice [15]. NOxidation was a major metabolic pathway in rats but was nearly absent in mice. The first-pass metabolism of N N K could be induced by starvation and/or acetone pretreatment. To our knowledge this is the first example of the induction of xenobiotic metabolism in small intestine by ketone bodies, and is in contrast to the reduced activity of arylhydrocarbon hydroxylase after starvation [20]. Biomonitoring of TSNA exposure Monitoring the exposure to tobacco smoke, especially to passive smoking, has proved to be a complicated procedure. To monitor exposure of humans to tobacco smoke the test has to fulfil the following conditions:
-
sensitivity to reliably detect the exposure to even small amounts of tobacco smoke; - s e l e c t i v i t y to distinguish between tobaccosmoke-related effects and similar effects with other causes; - availability of samples for analytical determinations.
Haemoglobin adducts have been proposed as internal dosimeters for carcinogens because of the ease of isolation of large quantities of haemoglobin from blood, the long lifetime of erythrocytes allowing the estimation of chronic exposure, and evidence from animal experiments showing a correlation with D N A adducts [6, 16]. To detect exposure to tobacco-smoke products selectively, the measurement of HPB, the common adduct from the decay of N N K and N N N , seems to be most promising because of its unique chemical structure (Fig. 3) [5]. It might be preferred over measuring the more easily detectable adducts formed by aromatic amines, aromatic hydrocarbons and ethylene oxide which also have a variety of other sources. The haemoglobin adduct HPB resulting from in vivo N N K and N N N degradation is therefore a selective parameter for environmental exposure to tobacco products. We changed the protocol described by Carmella et al. [5] to avoid extensive extraction of the aqueous haemoglobin hydrolysate. This streamlined the procedure and simplified
293
Collection of erythrocytes from blood samples ( ~ 20 ml); haemolysis by addition of distilled water (store at - 2 0 ° C if desired). HB-quantification. Addition of deuterated internal standard; protein cleavage by sonication in 0.1 N NaOH for 1 h with ultrasound. Protein precipitation with 10% w/v ammonium sulphate; centrifugation Clear supernatant over C18 Bond Elut (1 ml bed volume); wash with 150 mM NaxPO4, pH 9.0 Elution of HPB with < 1 ml acetonitrile; evaporate acetonitrile; resuspend sample in 600 gl CH2C12 and 600 gl 1 M triethylamine in hexane Add 2 gl pentafluorobenzoyl chloride for derivatization; heat at 60° C for 2 h Prepurification of HPB on HPLC. Addition of the retention time markers pentano- and hexanophenone (12.5 cm, C18; eluent 50% acetonitrile/water, 0.7 ml/min); retention time of HPB ~ 20 min Evaporate solvent completely; resuspend in 20 gl hexane; analyse 2 gl on GC-MS
Fig. 4. Analytical procedure for the analysis of haemoglobin
[2, 16]. Although a large interindividual variability is observed in smokers and non-smokers, no overlap of adduct levels between the two groups is found. In a smoking cessation study the concentration of the 4-ABP adduct decreased from 120_+ 7 pg/g to 82_+ 6 pg/g within 3 weeks and to 34_+ 7 pg/g within 2-3 months after stopping smoking. Various factors have been shown to affect 4-ABP adduct levels including the number of cigarettes smoked per day, type of cigarette tobacco (blond versus black), metabolic phenotype of the smoker, and possibly the extent of ETS exposure. Preliminary studies in our laboratory have shown that 4-ABP haemoglobin adducts can be analysed in parallel with HPB. The only modification to the scheme shown in Fig. 4 is the use of a second reagent, pentafluoropropionic anhydride, for derivatization of 4-ABP. Both derivatives are most sensitively detected by NICI-MS [5, 17]. Just having received the necessary equipment for this technique we are planning to measure the adduct levels of HPB and 4-ABP in healthy male volunteers with different, carefully controlled exposures to active and passive smoking as well as to other tobacco products. Acknowledgement. This study was supported by a grant from the Forschungsrat Rauchen und Gesundheit.
adducts
References
the handling, thus allowing the processing of a larger number of samples. The principle of the procedure used in our laboratory is outlined in Fig. 4. Another major difference from the method of Carmella et al. [5] is the detection by electron impact mass spectrometry (HP-MSD; Hewlett-Packard, Frankfurt) instead of negative ion chemical ionization mass spectrometry (NICI-MS). The latter detection method is not only two orders of magnitude more sensitive but is also more specific. With our present instrumentation we were able to detect HPB haemoglobin adducts in blood samples from heavy smokers and users of nasal snuff. However, our current threshold for the detection of tobaccospecific haemoglobin adducts is not sufficiently sensitive to detect HPB reliably in very low concentrations that might be expected in non-smokers exposed to ETS [5]. Although the haemoglobin adduct of the bladder carcinogen 4-aminobiphenyl (4-ABP) should be less characteristic for exposure to tobacco smoke because of other possible sources, recent results have shown that 4-ABP exposure is indeed predominantly the result of cigarette smoking
1. Adams JD, O'Mara-Adams KJ, Hoffmann D (1987) Toxic and carcinogenic agents in undiluted mainstream smoke and sidestream smoke of different types of cigarettes. Carcinogenesis 8:729 731 2. Bartsch H, Caporaso N, Coda M, Kadlubar F, Malaveille C, Skipper P, Talaska G, Tannenbaum SR, Vineis P (1990) Carcinogen hemoglobin adducts, urinary mutagenicity, and metabolic phenotype in active and passive cigarette smokers. J Natl Cancer Inst 82:1826-1831 3. Belinsky SA, Foley JF, White CM, Anderson MW, Maronpot RR (1990) Dose-response relationship between 0 6methylguanine formation in Clara cells and induction of pulmonary neoplasia in the rat by 4-(methylnitrosamino)-l(3-pyridyl)-l-butanone. Cancer Res 50:3772-3780 4. Brunnemann KD, Genoble L, Hoffmann D (1987) Identification and analysis of a new tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-4-(3-pyridyl)-l-butanol.Carcinogenesis 8 :465-469 5. Carmella SG, Kagan SS, Foiles PG, Palladino G, Quart AM, Quart E, Hecht SS (1990) Mass spectrometric analysis of tobacco-specific nitrosamine hemoglobin adducts in snuff-dippers, smokers, and nonsmokers. Cancer Res 50:5438 5445 6. Ehrenberg L, Osterman-Golkar S (1980) Alkylation of macromolecules for detecting mutagenie agents. Teratogenesis Carcinog Mutagen 1 : 105 127 7. Hecht SS, Hoffmann D (1988) Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis 9 : 875-884
294 8. Hecht SS, Trushin N, Castonguay A, Rivenson A (1986) Comparative tumorigenicity and DNA methylation in F344 rats by 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanoneand N-nitrosodimethylamine. Cancer Res 46: 498-502 9. Hecht SS, Lin D, Castonguay A, Rivenson A (1987) Effects of e-deuterium substitution on the tumorigenicity of 4(methylnitrosamino)-l-(3-pyridyl)-l-butanonein F344 rats. Carcinogenesis 8:291-294 10. Hoffmann D, Hecht SS (1985) Nicotine derived N-nitrosamines and tobacco related cancer: current status and future directions. Cancer Res 45:935-944 11. Malone A (1991) First-Pass-Metabolismus von 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanon, NNK, am Dfinndarm der Ratte. Dissertation, Tier/irztliche Fakult/it der Ludwig-Maximilians-Universit/it,Mfinchen 12. Morse MA, Eklind KI, Toussaint M, Amin SG, Chung F-L (1990) Characterization of a glucuronide metabolite of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone(NNK) and its dose-dependent excretion in the urine of mice and rats. Carcinogenesis 11:1819-1823 13. Ockene JK, Kuller LH, Svendsen KH, Meilahn E (1990) The relationship of smoking cessation to coronary heart disease and lung cancer in the multiple risk factor intervention trial (MRFIT). Am J Publ Health 80:954-958 14. Rivenson A, Hoffmann D, Prokopczyk B, Amin S, Hecht SS (1988) Induction of lung and exocrine pancreas tumors in F344 rats by tobacco-specific and Areca-derived N-nitrosamines. Cancer Res 48:6912-6917 15. Schulze J, Hunder G, Richter E (1990) Intestinal first pass metabolism of NNK in the mouse. In : Adlkofer F, Thurau
K (eds) Effects of nicotine on biological systems. Birkh/iuser Basel, pp 123-127 16. Skipper PL, Tannenbaum SR (1990) Protein adducts in the molecular dosimetry of chemical carcinogens. Carcinogenesis 11:507-518 17. Stillwell WG, Bryant MS, Wishnok JS (1987) GC/MS analysis of biologically important aromatic amines. Application to human dosimetry. Biomed Environ Mass Spectrom 14:221-227 18. Tricker AR, Haubner R, Spiegelhalder B, Preussmann R (1988) The occurrence of tobacco-specific nitrosamines in oral tobacco products and their potential formation under simulated gastric conditions. Food Chem Toxicol 26:861865 19. Tricker AR, Ditrich C, Preussmann R (1991) N-Nitroso compounds in cigarette tobacco and their occurrence in mainstream tobacco smoke. Carcinogenesis 12:257-261 20. Wattenberg LW (1972) Dietary modification of intestinal and pulmonary arylhydrocarbon hydroxylase activity. Toxicol Appl Pharmacol 23 : 741-747 Received: July 15, 1991 Accepted: September 3, 1991 Priv.-Doz. Dr. E. Richter Walther Straub-Institut fiir Pharmakologie und Toxikologie Ludwig-Maximilians-Universitfit Miinchen Nussbaumstrasse 26 W-8000 Miinchen 2, FRG
