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0 .., . . . ~'t. ~ . '. frotn Banbu~ R .A Sc~c CigdrQte , ~ 980 Cbld 3'piing 1Ie[fipCi ~?= 5;..~-..,~e'~!`.e~.-v..;.. ,.. .*..-'r~aY'. `TN~e ~Role of Voiatille and Nonvolatile N-Ni~trosamilnes 1. _ k Tobacco Carcinogeniesis DIETRICH!HOFFMANN. CHI-HONG B. CHEN, and STEPHEN S. HECHT D~~ivision,of~EnviironrnentallCarcinoge~~nesis~~. Naylor Dana Institute for Disease Prevention Arnerican Health Found'ation Valhalla, New Y©~rk~110585' +2 ~ , (ls43~3P4i~i~3~3'f`ti'': ~ri 1 Epidemiologicall studies have correlated smoking of cigarettes with cancer of' - 1, F riSs~siii'23

The Role of Volatile and Nonvolatile N-Nitrosarrnines in Tobacco Carcinogienesis. , `St!ti?S~Skl3"?i°ftjt~ift ytn ~;2ii^? DIETRICH HOFFMANiN, CHI-HONG B. CHEN, and STEPHEN S. HECHT Division of'EnviironmentallCarcinogenesis Naylor Dana Institute for Disease Prevention American Health Foundation Valhalla, New York 10595 Epidemiological studies~ have correlated smoking of cigarettes with cancer of the oral cavity; pharynx, esophagus, pancreas, renal pelivis, andi urinary bladder (Wynder et al. 1!957; Wynder and Bross 1961; Wynder et al. 1973; Royal College of Physicians 1977; Wynder and Goldsmith 1977; Public Health Service 1979). The increased risk of cancer at these sites in cigarette smokers may be due to several factors. One of these is the likelihood that inhalation of' tobacco smoke leads tio, enzyme: inductions that, in turn, aetivate certain en- vironmental agents to their ultimate carcinogenic forms (Jasko 1979). Other risk factors may be due to the presenl organ-specific carcinogens in tobacco smoke or to: smoke compounds that constitute precursors of' organ-specific carcinogens that can be formed in vivo (Hoffmann et al. 1'978). Druckrey and Preussmann (1962) had already suggested' the possibility that tobacco smoke contains organ-specific carcinogens such as N=nitrosamines. However, inten- sive and systematic studies of N-nitrosamines, in tobaccocarc':inogQnesisdid not begin until 11973. . V()LATILE N NITPiOSAN9INES. The volatile amines in tobacco and in, its smoke (Hecht et al. 1977) may undergo partial nitrosation to N-nitrosamines. To assess the nature and quan- tities of such compounds in tobacco smoke, we developedl a rapid analytical method, taking precautions against artifactual nitrosamine formation during, trapping and aging, of the smoke ('Brunnemanni et al. 1'977). The volatile N-nitrosamines (VNAs) were enrichedlby solvent distribution and chromatog- raphy, separated by gas liquid chromatography (G':LC),and detected and quantitated with~~ ai thermal energy analyzer (TEA)'. This detector is highly sensitive and specific for N-nitrosamines(':Fineetal:. 1975). The influence of nitrate fertil'izersin, the soil onVNIA formation inn cigarette smoke was demonstrated by comparing smoke from tobaccos growni under varyi~ng conditions. Figure I represents GLC-TEA data for VNA con-113

114/ D; Hoffmann, C.B. Chen„and S.S. Hecht ` 60 CATTERTON ~' HIGH NITNATE. 0 W N Z N Ui Q W H lOE t y;lI1tP4~Stft~t>21Pitsf y<n lai~?~,~ yi> t;, A 80iCATTERTON LOw NRRATE I I I I I 0 5 10 15 20 MdNUTES Figure 1 Gas ctiromatograms of'mainstneam smoke M-nittosamines as affected by nitrate levels in cigarette -sxy5 ~rr tobacco centrates obtained in this comparison. All four of'the V NAs identified here are carcinogenic in the experimental animal (Magee et al. 1976). Table 1 presents analytical data for the major VNAs in the mainstream smoke of tobacco. Since tobacco itself contains only negligible amounts of VNAs, it is clear that the VNA yield in the smoke depends primarily on the nitrate content of tobacco, the combustibility of the producty and the proteini content of tobacco. Celllalose acetate filter tips can selectively reduce VNAs by at least, 70% (Table 1)„ Thus, optimal means for redttct2on' of carcinogenic. VNAs in mainstream smoke are selection of low-nitrate tobaccos and use of filter tips with potential for selective reduction. Most present+-day commercial filter cigarettes are effectively reducing VNA. Like ammonia (Schmeltz and HIoffirtann 1!977), VNAs are formed in greater proportions during, smoldering of a cigarette or cigar between puffs than dwring,puff drawing, so that VNA levels in sidestream smoke are signifiicantlyy higher~ tha'nin, mainstream smoke. Thi'sis reflected in data that we established for indoor' environments polluted withi cigarette smoke. Measurable quantities

Nitrosamines in Tobacco Products /116 Table 11 Volatile N-Nitrosamines in Cigarette Smoke Cigarette Mainstream smoke Burley, NFa' Briglit, NF Commercial, NF Commerieal, FAb Kentucky 1R1, NF' Catterton, high NO;,, NIF Catterton, low NIO~„ NF' Frenchs NF French, FA French, FP ' Commerciall,,FA Commercial 1, minus FA Commercial 11, FCd Commercial Il, minus FA Sidestream smoke Commercial, NF Commercial, NIF~ Commercial, FA . Commercial, NF aNonfilter. ''Cellulose acetate filter:, °Paper fil ter. °Charcoal~cellulose acetate filter. eNnitrosodi methylamine. 'N-nitrosoethylmethy,lamine: KN-n itrosAd iethylami ne: °N-nitrosopyrroi idine: Volatile nitrosamines (nglt:igt) NDMhA e' NEIU1i4 ` NDEAg NPYRh 75.9 9.1 2.5: 51.7. 13.2 <0! 1 1.8 6:2 13.0 1.8 1.5 11.0 5.7 0.4 1.3 5.1 9.0 1'_5 2.0 6.6 97.0 8.0 4.8 42.0 20.0' 1.2 2.3' 4.1 29ff 2:7 0.6 25!0 4.3 0!481 0.1 10!5 13.5 2.1 0.4 111.0 6.8 0.5 0~8 8.5 27.0 2.2 1.2 33.0 14.0 0.6 7.6 7.6' 1!9:0 1.2' 8.3' 14.0 680.0 9.4 53:0' 300.0 820.0 30.0 8.2 205.0 730.0 10.0 73.0 390.0 1040.0 10.0 63.0 210.0 of dimethylnitrosamine (D11rIh1)were detected in air samples collected under conditions that precluded artifacts (Table 2; Brunnemann and Hoffmann 1978), In highly polluted indoor environments, a nonsmoker may be exposed to~ an equivalent of the mainstream smoke VNAs of 9- 19nonfilter cigarettes,, or 17-35 filter cigarettesduring~ 1 hour of normal respiration. At thisttime, however; there are no epiderniologie studies suggesting a measurable incre'ased risk for tobacco-related diseases among nonsmokers andl for people working in srnoke-polluted environments (Public Health Service 1979).

116 [1). Hoffmannl C.B: Chen, and S.S: Hecht I Kl3t1?3rftSS#itfly;;lf, f<r, ;•ii:f ? ; > 1.; Table 2 Dirmethylnitrosarrline in Polluted Indoor Air Environment Concentration a (rng/l) Expmsurelhr b (ng) 'Ilcaia I (bar car): 0.13 62-110 Ttain1 2(bar car) 0.11i 53-6'2' Bar 0.24 115-200 Sports hall 0.09 43-76 Betting,parlor 0.05 24-42 Betdng; parlor 0:05 ~ 24 -42 Discotheque 0:09 43 -76 Large room of bank 0:01 5-9 Suburban residenceC ' <0;005 - Urban residencec <0.003 - Blank <0.00'1 - e'Isolated amounts. b Respiratory rate 8-14! l/min. e Nonsmoker's residence.. NONVOLATILE N-NITRQSAMINES In general, cominercial tobacco products contain between 1 and 2% of specif'ic', alkalbids. The prevalent compound among, these is nicotine, constituting at least 90% of the alkaloids in mostl tobacco varieties. Nornicot'ine, anatabine„ anabasine, and cotinine' are the' other common tobacco alkaloids (Figure 2; Schmeltz and! Hoffmann 1977). There also are 5' 10 minor alkaloids, as well N N©RNOCOTINE ANATABINE ANABASINE OXYNdCOTTNE COTINI'NE N 3;2' 91PYNIDYL 03, N N Figure 2. Chemical structures of major tobacco alkaloids

Illitrosamines iniTobacco Products /1'17 . , f ;~tlfi?3tStfi~itf.ttsi ~r, . ?;21 as their oxidation products (Enzell et al. 1977; Schmeltz and Hoffrnann 1977): During, curing and fermentation, tobacco-specific N-nirtrosarni'nes aree formed from these alkaloids (Hecht et, al. 1978b). Im vitro nicotine is nitrosated to N"-nitrosonornicotine (NNN)s 4(~N-methyl-N-nitrosarnino)+l-(3'-pyridyl)-1- butanone (NNIC); and 4-(N-methyl-N-nitrosa'mino)-4-(3'-pyridyl)}butanal ('NNA) (Hecht et al. 1978a). As shown in Figure 3, NNN and NNK have been identified initobacco and initobacco smoke, as has N-nitrosoanatabine (NAtB). For the analysis of these tobacco-specific N-nitrosamines, we developed a specific method~ (Hoffmann et al. 1979) that uses high-pressure liquid chrorna} tography (HPI1,C) with a TEA detector in the final step, resulting, in a clear separation of'the tobacco-specific N-nitrosamines (see Fig. 4). Table 3 sum- marizes analytical findings that demonstrate the relatively high concentration of these N-nitrosamines in tobacco products, ranging from 0i2-90 ppm in to- bacco, 0! 1-4.6 Ftg, in the mainstream smoke, and 0.1-6' F,ug, in the sidestrearn smoke of a cigarette (Hmflfmann et al. 1'979; J.Ji. Piade ehal., in prep.). Studies withi [14C]NNN have shown that 40-50% of the NNN in the smoke is produced by transfer from the tobacco and the remainder is synthe- sized during smoking (Hbffmann et' al. 1977; J.J!. Piade et al., in prep.). Thus, a significant reduction in the formation of these tobacco-specific nitrosamines diaring, tobacco processing shou'ld also be reflected in the rediictioni of these compounds in the smoke. Reducing these cyclic nitrosatnines in tfJbacco is especialQyianportant because they may not beamenabletb, selective filtration at a pH below 6.5. Nornicotine. Nicotine Anatabine NNN NNtf . NAtB Figure 3 Formation ot major tobacco specific N nitrosamines from nicotine. nornicotine, and anatabine during tobaceo process.ing and smoking

~S?3l3tS#f~?i?f 3'n . #it~ii ,r > r t~.,' 1181 D. Hoffmann, C.B. Chen, and S.S. Hacht NYttB NNN N N` CH3 NO NNK T -i I 0 10 24 30, M I NUif E5 Figure 4 High-performance liquid chromatogram ofl nomolatile nitrosamines from tobacco CARCINOGENICITY OF THE'TOBACCO-SPECIFIC'N 11rIITROSAMINES The detection of alkaloid-derived nitrosamines in tobacco products raised thee question of their earcinogeniaity. Results from several bioassays are presented in Table 4. In mice, NNN and NNK induced lung adenomas andl adenocar- cinoma and, in some instances, tumors of the salivary glands (Boyland et all. 1964; Hbffmann et! al. 1976; Hecht et al. 197~9')t When given subcutaneously in rats, NNN inducedl priinarily carcinoma of the nasaU cavirty,, whereas NNK elicited tumprsoflthe nasal cavity„as,well as of the lung and liNer(Hdcht et al. 1980). When administered in, the drinking water, NNN induced primariPycancer of theesophagutsand also some nasal tu~mors, (Hoffmann et al. 1975; Singer and Taylor19716). These results indicated that tobacco-specific nitros- amines may be contact carcinogens as well as organ-specific carcinogens. This observation may offeroneexplanatyorn fbrtheincreased risk for cancer of the oral cavi'tyand esophagus, not, only to cigarette, cigar, and pipe smokers, but also to tbbaceochewers (PublicHealft Service197'9). In Syrian golden hamsters, NNN indiDCestracheal tumors(H'ilifrich et al. 1977). N11dIKis currently under study, and so far it has been shown to,indwce ttacheall tumors. Currently we are painting theora'l eavitiesof aleohpl-treated

z Table 3 Tobacco-Specific N-Nitrosamines in Tobacco Products - - Tobacco (ppm) Mainstream (µg/cigt) Sidestream (µg/cigt) Tobacco Producte NAtB NNN NNK NAtB NNN NNK NAtB NNN NNK Burley cigarette, NF 3.2 7.0 n.dt. b 4.6 3.7 0.32 1.5 6.1 0.66 Bright cigarette, NF 0.44 0.22 0.37 0.41 0.62 0.42 0.39 1.7 0.50 Commercial cigarette, NF 1.6 1.7 0.74 0.33 0.24 0.11 0.27 1.7 0.41 Commercial cigarette, FA 1.3 1.4 0.70 0.37 0.31 0.15 0.15 0.15 0.19 Kentucky IRI, NF 0.62 0.63 0.13 0.53 0.39 0.16 0.19 0.21 0.24 Commercial French cigarette NF, 70 mm 1.8 2.9 0.526 0.18 0.48 0.44 - - -- Commercial French cigarette FA, 70 mm 1.5 2.7 0.37 0.18 0.49 0.36 - French cigarette, NF 2.0 11.9 1.1 0.68 3.2 0.43 - - - French cigarette, FA 2.0 11.9 1.1 0.19 .0 0.19 - - - French cigarette, FP 2.0 11.9 1.1 0.16 0.73 0.12 - - - Little cigar, FA 13.0 45.0 35.0 1.7 5.5 4.2 0.57 0.88 0.81 Cigar (Colombia tobacco) (5.7 g) 3.3 10.7 1.1 1.9 3.2. 1.9 n.d. `' 16.6 15.7 Fine-cut chewing tobacco 44.0 39.0 2.4 aAil cigarettes ana the little cigar were 85 mm long, _ng, except otherwise stated. ''Not detected. 'Not deterrnined (interference by unknowns). tE6fi09Tzaz ~. .x. ~

J o TabIe 4 Carcinogenic Activity of Tobacco-Specific N-Nitrosamines Compounds Species Application Principal organs affected NNN mouse i.p.'' lung (adenoma, adenocar- cinoma) salivary glands (?) NNN NNN NNN NNK rat hamster mouse rat s.c. b p.o.C (water), nasal cavity (carcinoma) liver esophagus (papilloma, carcinoma) pharynx (papilloma) nasal cavity (carcinoma) trachea (papilloma) nasal cavity (carcinoma) %ntraperitoneal. °Subcutaneous. °Per os. SCsVo9zzoz lung (adenoma, adenocar- cinoma) nasal cavity (carcinoma) liver lung (adenoma, carcinoma) ,i;5~ ~. References Boyland et al. (1964) Hoffmann et al. (1976) Hecht et al. (1978b) Hecht et al. (197tfb) Hecht et al. (1980) Hoffmann et al. (1975) Singer and Taylor(1976) HilPrich et al. (1977) Hecht et al. (1978b) Hecht et al. (1980)

Nitrosamines in Tobacco ProductsJ72'1 Syrian hamsters and hamsters on a normal dia wsthi olive oil, solutions of NNN and NNK. Sasedl on macroscopic examination, these applications induce tumors of the:mouth. METABOLIC ACTIUATION OF'TOBACCO-SPECIFIC N-NITROSAMINE& ILike most~ N~nitrosamines, the tobacco-specific nitrosamines are procarcino- gens, which requim in vivo metabollc activation to their ultimate carcinogenic forms. Figure 5 summarizes our present knowledge about the metabolic activa- tion of NNN and NNK (Cheniet a1_ 1978'y 1979). In both cases, the initial step appears to be a-hydroxylatiom The:resulting,a-nitrosaminoalcohols are unsta- blb and decompose with formation of diazohydroxides and, subsequently, carboniumi ions. The latter are possibNy the ultimate carcinogenic forms of these t;obacco-speci'fic carcinogens.. In the case of NNN, the a-hydroxylation occurs in both a-positions leading to two different carbonium ions. These carbonium ions react primarily with intracellular water, forming, a keto alcohol or a hydboxyaldehyde. The latter are oxidieed in vivo to the corresponding~ ketio~acidk. NNK is a,hydroxylated au the methyl group and at the a-methylbne group, respectively. These compounds decompose with formationi of a metliylcar- bonium ion and a 4-(/3-pyridyi)-4-ketobutylcarbonium ion. The butylcarbonium ion rmay, be correlated in rats with tumor formation in the nasal cavity, whereas ~M•0 0 M~ 0 / M ~N3 Figure 5 !vletabolism oE NNN and'NNK LM I ON