Tobacco Specific N-Nitrosamines: Occurrence, Carcinogenicity, and

8 Tobacco Specific N-Nitrosamines: Occurrence, Carcinogenicity, and Metabolism

Downloaded by MONASH UNIV on February 29, 2016 | http://pubs.acs.org Publication Date: June 20, 1979 | doi: 10.1021/bk-1979-0101.ch008

S T E P H E N S. H E C H T , C H I - H O N G B. C H E N , G . D A V I D and D I E T R I C H H O F F M A N N

McCOY,

Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY 10595

It i s now widely accepted that cigarette smoking causes lung cancer (1,2). It i s less widely known that smoking i s also correlated with an increased incidence of cancer of the oral cavity, esophagus, pancreas and bladder (2,3,4,5,6). Tobacco chewing can also cause oral cavity and esophageal cancer (3,4,7). In fact, oral cavity cancer i s a major cancer among men i n India, where the habit of chewing the betel quid containing tobacco i s widespread (8). Cigarette smoke i s known to contain tumor i n i t i a t o r s such as the polynuclear aromatic hydrocarbons, and tumor promoters and cocarcinogens, such as catechol (9). These agents can explain many of the observed effects of cigarette smoke condensates i n experimental animals and almost certainly are involved in some of the human cancers associated with smoking. However, nitrosamines may also be causative factors i n the tobacco related cancers, especially in those organs which are remote from direct contact with tobacco or tobacco smoke. Thus it i s known that nitrosamines can cause esophageal, pancreas and bladder cancer i n experimental animals, as well as affecting the lung and oral cavity (10,11,12). Since tobacco and tobacco smoke have specific carcinogenic effects i n man, it i s tempting to speculate that there may be unique carcinogenic agents in tobacco and tobacco smoke. The tobacco specific nitrosamines are such a group. These nitrosamines are derived from the tobacco alkaloids (see Figure 1). The most prevalent alkaloid i s nicotine, which occurs i n general i n concentrations of 1-2% in commercial tobacco products. Both nicotine and nornicotine could give rise to the prototype of tobacco specific nitrosamines, N'-nitrosonornicotine (NNN). Nicotine could also be nitrosated to form 4-(N-methyl-N-nitrosamino)-1(3-pyridyl)-1-butanone (NNK) or 4-(N-methyl-N-nitrosamino)-4(3-pyridyl)butanal (NNA). In addition, N-nitrosopyrrolidine (NPy) could also be derived from nicotine and nornicotine. Nitrosation of anabasine would give nitrosoanabasine (NAB). The structures of these nitrosamines, which w i l l be considered i n this review, are shown i n Figure 2. Of course, inspection of Figure 1 reveals other interesting p o s s i b i l i t i e s for nitrosation of the tobacco 0-8412-0503-5/79/47-101-125$07.00/0 © 1979 A m e r i c a n C h e m i c a l Society

In N-Nitrosamines; Anselme, Jean-Pierre; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


N-NITROSAMINES

ANABASINE

Figure 1.

Figure 2.

ANATABINE

BIPYRIDYL

Common tobacco alkaloids in tobacco and/or tobacco smoke

Some nitrosamines which can be derived from the tobacco alkaloids


8.

HECHT

ET AL.

Tobacco Specific N-Nitrosamines

127

a l k a l o i d s ; some o f these w i l l be the s u b j e c t o f future s t u d i e s . Occurrence And Formation Of Tobacco S p e c i f i c Nitrosamines The prototype o f the tobacco s p e c i f i c nitrosamines, NNN, has been detected i n both tobacco smoke and unburned tobacco. Various a n a l y t i c a l methods have been used i n c l u d i n g gas chromatography (GLC) (_13,1£, 15^,16) combined GLC-mass spectrometry (Γ7) , t h i n l a y e r chromatography ( 1 8 ) h i g h pressure l i q u i d chromatography (HPLC) (19^20), and combined HPLOthermal energy a n a l y s i s (21). NNN l e v e l s i n c i g a r e t t e smoke t y p i c a l l y range from 140-240 n g / c i g i n a t y p i c a l American 85mm n o n - f i l t e r c i g a r e t t e . Surprisingly high l e v e l s o f NNN were found i n unburned tobacco (0.3-9.0 ppm i n c i g a r e t t e tobacco, 3.0-45.3 ppm i n c i g a r tobacco, 3.5-90.6 ppm i n chewing tobacco, and 12.1-29.1 ppm i n s n u f f ) . These l e v e l s are among the h i g h e s t f o r an environmental nitrosamine i n terms o f occurrence and human exposure (22). Thus, r a t h e r d e t a i l e d s t u d i e s were c a r r i e d out t o determine the o r i g i n s o f NNN i n tobacco and tobacco smoke.


f

To study the formation o f NNN i n tobacco, p l a n t s were an alyzed a t v a r i o u s étages o f growth and c u r i n g (23). NNN was not detected p r i o r t o harvest o r i n f r e s h l y harvested Burley tobacco but only during and a f t e r a i r c u r i n g (0.5-1.1 ppm). Since e i t h e r n i c o t i n e o r n o r n i c o t i n e c o u l d have been a^grecursor t o NNN i n tobacco, tobacco leaves were f e d n i c o t i n e - 2 ' - C o r n o r n i c o t i n e - 2 - l c and cured (24). The cured leaves were then analyzed for ΝΝΝ-2·- 0. The y i e l d o f NNN from n i c o t i n e was 0.009% and from n o r n i c o t i n e , 0.007%. These r e s u l t s showed t h a t both n i c o t i n e and n o r n i c o t i n e could be p r e c u r s o r s t o NNN i n tobacco. How ever, the g r e a t e r abundance o f n i c o t i n e i n tobacco l e a f (20-100 times the c o n c e n t r a t i o n o f n o r n i c o t i n e ) favored n i c o t i n e as the major p r e c u r s o r o f NNN i n tobacco. The t r a n s f e r o f NNN from c i g a r e t t e tobacco t o mainstream smoke was studied (20). F o r t h i s purpose, NNN-2'- C was added to c i g a r e t t e s and the smoke was analyzed. The t r a n s f e r r a t e was found t o be 11.3%. Since, i n t h i s experiment, the tobacco column smoked contained 974 ng NNN, 110 ng were t r a n s f e r e d t o the main stream smoke. A n a l y s i s o f the mainstream smoke r e v e a l e d 238 ng NNN; thus the remaining 128 ng were formed during smoking. There f o r e , about 50% o f the NNN i n mainstream smoke o r i g i n a t e d by t r a n s f e r from tobacco while the remainder was formed during smok ing. E i t h e r n i c o t i n e o r n o r n i c o t i n e c o u l d be a p r e c u r s o r t o NNN formed d u r i n g smoking. To examine t h i s q u e s t i o n , n i c o t i n e o r n o r n i c o t i n e was added t o c i g a r e t t e s and the smoke was analyzed f o r NNN (13). In each case, NNN c o n c e n t r a t i o n i n smoke i n c r e a s e d i n d i c a t i n g t h a t both a l k a l o i d s are p r e c u r s o r s t o NNN formed dur i n g smoking. However, n i c o t i n e i s considered the more important precursor due t o i t s higher c o n c e n t r a t i o n i n tobacco. The r e s u l t s of these s t u d i e s on the formation o f NNN during c u r i n g , i t s ,

4

14

14



128

N-NITROSAMINES

t r a n s f e r to smoke, and i t s formation during smoking are summarized i n F i g u r e 3. In tobacco samples examined so f a r , the l e v e l s of NAB were s i g n i f i c a n t l y l e s s than those of NNN. In f a c t , NAB has not y e t been detected with c e r t a i n t y i n unburned tobacco (15). These f i n d i n g s are i n l i n e with the major r o l e o f n i c o t i n e r a t h e r than n o r n i c o t i n e as a p r e c u r s o r to NNN s i n c e k i n e t i c s t u d i e s showed t h a t n o r n i c o t i n e and anabasine were n i t r o s a t e d a t s i m i l a r r a t e s (25). These r a t e s are r e l a t i v e l y high, which suggests t h a t the formation o f NNN and NAB could be favored in vivo. When chewing tobacco was incubated with human s a l i v a f o r 3 hours a t 37° and the mixture analyzed f o r NNN, the concentrations o f NNN increased by 44% over t h a t i n the chewing tobacco, presumably as a r e s u l t o f f u r t h e r n i t r o s a t i o n (15). Thus, in vivo formation o f NNN and NAB could c o n s t i t u t e an a d d i t i o n a l exposure of smokers or chewers to these tobacco s p e c i f i c nitrosamines. Since n i c o t i n e i s the major p r e c u r s o r to NNN i n tobacco and tobacco smoke, the r e a c t i o n o f n i c o t i n e with sodium n i t r i t e was s t u d i e d to provide i n f o r m a t i o n on formation o f other tobacco s p e c i f i c nitrosamines, e s p e c i a l l y NNK and NNA, which could a r i s e by o x i d a t i v e cleavage o f the l ' - 2 ' bonds or l ' - 5 ' bond o f n i c o t i n e followed by n i t r o s a t i o n (26). The r e a c t i o n was i n v e s t i g a t e d under a v a r i e t y o f c o n d i t i o n s as summarized i n Table I . A l l three nitrosamines were formed when the r e a c t i o n was done under r e l a t i v e l y m i l d c o n d i t i o n s (17 h r s , 20°). The y i e l d s are t y p i c a l o f the formation of nitrosamines from t e r t i a r y amines (27). At 90°, with a f i v e f o l d excess o f n i t r i t e , only NNN and NNK were detected. Under these c o n d i t i o n s , both NNK and NNA gave secondary products. NNK was n i t r o s a t e d α t o the carbonyl to y i e l d 4-(N-methyl-Nnitrosamino) -2-oximino-l- (3-pyridyl) -1-butanone while NNA under went c y c l i z a t i o n followed by o x i d a t i o n , d e c a r b o x y l a t i o n and de h y d r a t i o n to give l - m e t h y l - 5 - ( 3 - p y r i d y l ) p y r a z o l e , as shown i n F i g u r e 4. Extensive fragmentation and o x i d a t i o n o f the p y r r o l i dine r i n g was a l s o observed under these c o n d i t i o n s . The products of the r e a c t i o n o f n i c o t i n e and n i t r i t e a t 90° are summarized i n Table I I . The formation o f NNN, NNK, and NNA from n i c o t i n e probably i n volved the intermediacy o f c y c l i c iminium s a l t s , as shown i n F i g ure 5 (_28) . These s a l t s can undergo h y d r o l y s i s to the f r e e amines which are n i t r o s a t e d , or a t near n e u t r a l pH, can be d i r e c t l y n i t r o s a t e d to give nitrosamines. The formation o f nitrosamines from iminium s a l t s under n e u t r a l c o n d i t i o n s has been demonstrated i n a t l e a s t two s t u d i e s and i s o f i n t e r e s t because iminium s a l t s are known to be intermediates i n the mammalian metabolism o f n i c o t i n e (26,29,30,31). The p o s s i b i l i t y t h a t tobacco b a c t e r i a could n i t r o s a t e n i c o t i n e v i a t h i s pathway i s c u r r e n t l y under i n vestigation. The formation o f NNK and NNA from n i c o t i n e i n these model s t u d i e s encouraged us to search f o r these nitrosamines i n tobacco and tobacco smoke. In s t u d i e s undertaken so f a r , NNK but not NNA


8.

HECHT E T A L .

Tobacco Specific

FRESH HARVESTED TOBACCO

Ν-Nitrosamines

129

NICOTINE

NORNICOTINE

\0.009% NITRITE

CURED TOBACCO

\

0.007%/ /

NICOTINE

NORNICOTINE

NITROGEN OXIDES


NITRITE

NITROGEN OXIDES

MAINSTREAM CIGARETTE SMOKE International Agency for Research on Cancer

Figure 3.

Origins of NNN in tobacco and tobacco smoke (22)

Table I Formation o f NNN,NNK, and NNA from N i c o t i n e and NaNO„

[NaNQ2] [Nicotine] 1.4 1.4 1.4 1.4 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

Conditions T(°C) pH c

2.0 3.4 4.5 7.0 3.4-4.2 3.4-4.2 3.4-4.2 5.4-5.9 5.4-5.9 5.4-5.9 7.0-7.3 7.0-7.3 7.0-7.3

a

20 20 20 20 90 90 90 90 90 90 90 90 90

t (hrs) 17 17 17 17 0. 3. 6. 0.3 3.0 6.0 0.3 3.0 6.0

Yields (%) NNK NNN 0.1 0.5 0.5 0.2 8.0 8.8 8.0 9.0 13. 11. 1, 4. 5.5

ND 0.1 0.5 0.1 0.7 2.3 1.5 2.7 4.3 2.6 0.1 0.2 0.2

a

NNA 0.2 2.8 2.3 0.1 ND ND ND ND ND ND ND ND ND

. Determined by GC and based on s t a r t i n g n i c o t i n e .

^ B u f f e r systems: pH2, KC1-HC1; pH 3.4-7, c i t r a t e - p h o s p h a t e . ND=not detected.


130

N-NITROSAMINES

Table I I a »b Products Formed i n the Reaction o f N i c o t i n e and NaNO^


Product

Y i e l d (%)

NNN NNK 4- (N-methyl-N-nitrosamino)2-oximino-l- C 3 - p y r i d y 1 ) 1-butanone l-methyl-5-(3-pyridyl)pyrazole and trans 3-Pyr-CH=CHCN 3- Pyr-CONHCH 3-Pyr-COOH cotinine 3-Pyr-CH=CH-COOH 3-Pyr-COCH 3-Pyr-CN 3-Pyr-C0 CH 3-Pyr-CHO myosmine^ 3-Pyr-CH CN

c

Method o f

Tobacco Specific N-Nitrosamines: Occurrence, Carcinogenicity, and

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