Formation, Occurrence, and Carcinogenicity of N-Nitrosamines in

18 Formation, Occurrence, and Carcinogenicity of N-Nitrosamines in Tobacco Products DIETRICH HOFFMANN, JOHN D. ADAMS, KLAUS D. BRUNNEMANN,

and STEPHEN S. HECHT Naylor Dana Institute for Disease Prevention, American Health Foundation, Downloaded by UNIV OF ARIZONA on March 11, 2017 | http://pubs.acs.org Publication Date: December 9, 1981 | doi: 10.1021/bk-1981-0174.ch018

Valhalla, NY 10595 Tobacco and tobacco smoke contain three types of N-nitrosamines. These are the volatile nitrosamines (VNA), nitrosamines deriving from residues of agricultural chemicals on tobacco, and the tobacco specific nitrosamines (TSNA). A l l three types of nitrosamines are formed during tobacco processing and during smoking. The VNA can be selectively reduced in cigarette smoke by cellulose acetate filter tips. Of the nitrosamines deriving from agricultural chemicals, we have so far only detected the animal carcinogen nitrosodiethanolamine in concentrations of 0.1-6.8 ppm in tobacco and 10-40 ng per cigarette in the smoke. TSNA are formed during tobacco processing and smoking from nicotine, nornicotine and anatabine. Their concentrations range from 1-100 ppm in tobacco and from 1-10 µg/cig. in cigarette smoke. Selective removal of TSNA from mainstream smoke by cellulose acetate filters was demonstrated and can be enhanced by perforation of the filter tip. In mice, rats and hamsters, N'-nitrosonornicotine is a moderately active carcinogen while 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone is a strong carcinogen. N-nitrosoanatabine is being bioassayed at this time. N-Nitrosamines are formed during processing and smoking of tobacco products. Proteins, agricultural chemicals and alkaloids in tobacco products serve as major precursors for v o l a t i l e , non v o l a t i l e , and tobacco-specific nitrosamines (Figure 1). In this review we w i l l summarize the progress achieved i n respect to to bacco nitrosamines since the last ACS symposium i n Boston i n June of 1978 O ) . Additional papers w i l l review the metabolism of c y c l i c N-nitrosamines, including that of N -nitrosonornicotine (2) and the correlation between tobacco and alcohol consumption and cancer of the upper alimentary tract (3). 1

©

1981

c

S| CH

0

CARBARYL(SEVIN )

3

DIMETHYLDODECYLAMINE -ACETATE (PENAR) OH , I l

cΟ

N

, + "

H

MALEIC HYDRAZIDE Figure 3.

„CH -CH -0H HN CH2-CH -0H c

2

c

2

N

2

DIETHANOLAMINE ( MH-30)

Agricultural chemicals for tobacco cultivation.

Scanlan and Tannenbaum; N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

253

Downloaded by UNIV OF ARIZONA on March 11, 2017 | http://pubs.acs.org Publication Date: December 9, 1981 | doi: 10.1021/bk-1981-0174.ch018

254

N-NITROSO COMPOUNDS

3%0V-225

W-HP

OVEN: 2 1 0 C E

INJ.PT: 2 5 0 C E

0

5

Κ) 0

5

10

15

20

MINUTES Figure 4.

NDELA

analysis via direct

GC-TEA.



18.

HOFFMANN ET AL.

N-Nitrosamines

in Tobacco

Products

255

225 on Chromosorb W HP 80-100, operated at 130°C (Figure 5). NDELA i n tobacco smoke was determined by directing the mainstream smoke of 100 cigarettes or l i t t l e cigars through 2 gas wash bottles i n sequence which were f i l l e d with ethyl acetate and ascorbic acid. Cigars were smoked under appropriate standard conditions developed for these products (20). Enrichment steps and GC-TEA procedures were similar to those described for the tobacco extracts. Table I I I l i s t s some of our results for NDELA in commercial as well as i n experimental tobacco products. In tobacco, we found the highest NDELA values for fresh and aged snuff with 6.8 and 3.2 ppm, respectively. The fermentation process appears to increase NDELA, as was also observed for VNA concentrations. There was clear evidence that tobaccos which had not been treated with MH-30, and cigarette smoke obtained from these tobaccos, were free of NDELA, whereas a l l MH-30 treated tobaccos and cigarette smoke derived from them showed measurable quantities of this nitrosamine (19). In order to ascertain that the NDELA formation does not oc cur as a result of trapping of the smoke or during the analysis, we added diethanolamine to tobacco prior to extraction with ethyl acetate i n the presence of ascorbic acid. The control value for NDELA was 121 ppb and the experiment with 5.5 mg diethanolamine addition yielded 113 ppb NDELA. For control of the smoke analy sis we added 5.5 mg of DELA in the solvent trap and smoked cigar ettes known to be free of DELA. Analysis of the trapped material showed no significant quantities of NDELA, so that a r t i f a c t u a l formation of this nitrosamine during smoke collection and analy sis can be ruled out. Thus, we conclude that the diethanolamine in MH-30 i s the major precursor for NDELA i n processed U.S. tobaccos and tobacco smoke. In fact, NDELA concentrations of 600-1,900 ppb were already present i n five agricultural spray formulations of MH-30 which we analyzed (19). Currently, we are studying the transfer rate of NDELA from tobacco into smoke as well as the potential of DELA and NDELA to serve as precursors for other nitrosamines i n smoke. Carcinogenicity of NDELA. Our special interest i n NDELA as a constituent of tobacco products and as an environmental agent relates to the observation that this nitrosamine induces carcino ma of the l i v e r as well as of the kidney in rats (21,22) and car cinoma of the nasal cavity and papillomas of the trachea in ham sters (23). Recently, L i j i n s k y et a l reported that NDELA admini stration i n drinking water for 34 weeks i n concentrations of 3,900 ppm induces hepat ο carcinomas i n a l l rats in the test. Thus, the authors consider NDELA to be a potent carcinogen i n rats (22). In an ongoing bioassay i n Syrian golden hamsters, we observe that NDELA induces carcinomas of the nasal cavity inde pendent of route of administration, i . e . subcutaneous injection, skin painting or oral swabbing i n minimum doses of 58 mg/kg (24).



256

N-NITROSO COMPOUNDS

3%0V-225W-HP| 0VEN:I30 C N-NO IN J P T : I 6 0 C (CH ) Si-0-(CH2) "" #

(CH ) Si-0-(CH ) , 3

3

3

3

2

2

#

2

TOBACCO EXTRACT

STANOARD

TOBACCO SMOKE (100 CIGARETTES)

(40g)

(CH } S i - 0 - ( C H ) 3

(CH ) Si-0-(CH2) 4 3

3

(CH ) Si-0-(CH ) ' 3

lo

3

15 θ"

Figure 5.

N DELA

(CH )

2

2

3

3

3

2

2

N-NO

2

10 15 MINUTES analysis via silylation

2

Si-0-(CH )2

10 15

GC-TEA.


N-NO

18.

HOFFMANN ET AL.

N-Nitrosamines

in Tobacco

257

Products

Table I I I .


NITROSODIETHANOLAMINE (NDELA) INTOBACCOAND TOBACCO SMOKE*

Tobacco Product

a. Experimental C i g a r e t t e s Kentucky 1R1, NF, 85 mm Kentucky 2R1, NF, 85 mm Handsuckered 1970 Burley, NF, 85 mm MH-30 t r e a t e d 1970 Burley, NF, 85 mm USDA M-6, B r i g h t , p e s t i c i d e treated, NF, 85 mm USDA L-8, B r i g h t , p e s t i c i d e f r e e * * * , NF, 85 mm b. Commercial Products C i g a r e t t e A, NF, 85 mm C i g a r e t t e B, F, 85 mm L i t t l e Cigar, F, 85 mm Large Cigar (7.7 g ) , NF, 125 mm Snuff (Fine C u t ) , aged Snuff (Fine C u t ) , fresh Chewing Tobacco A Chewing Tobacco Β

Tobacco** (ppb)

Mainstream (ng/cig.)

86 84 n.d. 227

30 51 n.d. 290

19

20

n.d.

n.d.

115 194 419 108 3,180 6,840 285 224

36 24 68 10

-

* Data corrected f o r recovery using isotope d i l u t i o n method. ** Tobacco data are reported per dry weight. *** Grown i n P r i n c e Edward I s l a n d , Canada. n.d. = not detected (below d e t e c t i o n l i m i t ) , NF - n o n - f i l t e r , F - filter.


258

iV-NJTROSO COMPOUNDS

This then confirms the fact that NDELA penetrates the skin to act as an organ specific carcinogen as was also evident from the studies by L i j i n s k y et4 a l on rats (25), from the reports by Edwards et al4 on cosmetics i n man (18) and from studies with Syrian golden hamsters in our laboratory (24).


Tobacco-Specific N-Nitrosamines Precursors and Formation. Tobaccos used for commercial pro ducts i n the U.S.A. contain between 0.5 and 2.7% alkaloids. Nic otine constitutes 85-95% of the t o t a l alkaloids (14,26,27). Important minor alkaloids are nornicotine, anatabine, anabasine, cotinine and N'-formylnornicotine (Figure 6). Several of these alkaloids are secondary and tertiary amines and, as such, amenable to N-nitrosation. The N-nitrosated alkaloids identified to date i n tobacco and tobacco smoke include Ν -nitrosonornico tine (NNN), 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) and N -nitrosoanatabine (NAT; Figure 7). In model experiments, nitrosation of nicotine also yielded 4-(methylnitrosamino)-4-(3pyridyDbutanal (NNA; 28). In a study for precursor determination, we stem-fed i n d i v i d ual Bur ley leaves with nicotine-2 '-4C or nomicotine-2'-4C (29). Subsequently, the leaves were a i r cured, dried and anal yzed for NNN and NNN-4C. Recovery of the β-activity in the form of NNN- C amounted to 0.009% and 0.007%, respectively of the stem-fed label. This demonstrates that both alkaloids give r i s e to NNN. More importantly, i t points to the fact that the ter tiary amine, nicotine, which constitutes 0.5-2.6% of commercial tobaccos (26,27), i s the major precursor for the carcinogenic to bacco-specific NNN, while the secondary amine, nornicotine i s of lesser importance because i t amounts to only 0.005-0.06% i n to bacco (Figure 8). We thus assume, that the biochemical processes during tobac co curing may be different from the in v i t r o N-nitrosation of secondary and t e r t i a r y amines (30). 1

1

14

Nonvolatile Nitrosamines In Tobacco. A method which we developed several years ago for the analysis of tobacco-specific nitrosamines (TSNA; 31) involves extraction of tobacco with buf fered ascorbic acid~TpH 4.5) followed by p a r t i t i o n with ethyl acetate, chromatographic clean-up on s i l i c a gel, and analysis by HPLC-TEA (Figure 9). Results obtained with this method for a large spectrum of tobacco products (Table IV), strongly support the concept that the levels of nitrate and alkaloids, and especi a l l y the methods for curing and fermentation, determine the yields of TSNA i n tobacco products. Recent and as yet prelimin ary data from snuff analyses indicate that aerobic bacteria play a role i n the formation of TSNA during a i r curing and fermenta tion.


HOFFMANN ET AL.

N-Nitrosamines

in Tobacco

(οβ' Downloaded by UNIV OF ARIZONA on March 11, 2017 | http://pubs.acs.org Publication Date: December 9, 1981 | doi: 10.1021/bk-1981-0174.ch018

NICOTINE

(§fi

( Νc / ?

N'-FORMYLNORNICOTINE

Figure 6.

Figure 7.

COTININE

ANABASINE

259

Products

1

(o) ? NORNICOTINE

(ô/? ANATABINE

Common tobacco alkaloids in tobacco and/or smoke.

Some nitrosamines which can be derived from the tobacco alkaloids.



260

N-NITROSO COMPOUNDS

Figure 8.

Conversion of labeled nicotine and nornicotine during curing.

NAT

N

N

N

LU CO Ζ

Ο

û. CO LU

Formation, Occurrence, and Carcinogenicity of N-Nitrosamines in

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