Volatile nitrosamines and tobacco-specific nitrosamines in the smoke of Thai cigarettes: a risk factor for lung cancer and a suspected risk factor for liver cancer in Thailand

Eugene J. Mitacek1,6, Klaus D. Brunnemann2, Dietrich Hoffmann2, Thira Limsila3, Maitree Suttajit4, Nimit Martin4 and Lee S. Caplan1,5

1 Department of Preventive Medicine, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8036 and New York Institute of Technology, Old Westbury, NY 11568,
2 American Health Foundation, Valhalla, NY 10595, USA,
3 School of Medicine, Chiang Mai University, Chiang Mai, Thailand and
4 Department of Oncology and Surgery, Siriraj Hospital, Faculty of Medicine, Mahidol University, Bangkok, Thailand


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In Thailand, smoking of commercial cigarettes and of handmade cigarettes has drastically increased in recent decades. Cancer of the lung and of the upper aerodigestive tract have also increased in Thailand as they have in many other countries. It is our working hypothesis that the increase of primary cancer of the liver, especially of cholangiocarcinoma in the north-eastern provinces of Thailand is associated with the use of tobacco in men infested with the liver fluke Opisthorchis viverrini (OV). Bioassays have shown that volatile nitrosamines and tobacco-specific nitrosamines induce cholangiocarcinoma in laboratory animals and that the hepatocarcinogenic action of nitrosodimethylamine in hamsters is significantly increased by infestation with the liver fluke OV. The endogenous formation of nitrosamines is significantly increased by OV infestation. This report presents analytical data on the concentration of volatile nitrosamines and tobacco-specific nitrosamines in mainstream smoke of nine leading brands of commercially produced Thai cigarettes which represent ~85% of the market share in Thailand. Observed ranges (ng/cigarette) were 8.5–31.9 for nitrosodimethylamine, 8.8–49.6 for nitrosopyrrolidine and 4.2–18.9 for nitrosodi-n-butylamine. These values are exceptionally high compared with the smoke of light and blended cigarettes from North America and Western Europe. Among the tobacco-specific nitrosamines, the range was 28–730 for nitrosonornicotine and 16–370 for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. There was a correla-tion between volatile and tobacco-specific nitrosamines, and tar and nicotine deliveries in the mainstream smoke. The analytical data are in line with the rate for lung cancer and support our working hypothesis that nitrosamines, and especially the tobacco-specific nitrosamines, are associated with the increased risk for primary liver cancer among those Thai people who smoke cigarettes and also carry OV infestation.

Abbreviations: CCA, cholangiocarcinoma; HCC, hepatocellular carcinoma; NAB, nitrosoanabasine; NAT, nitrosoanatabine; NDBA, nitrosodi-n-butylamine; NDMA, nitrosodimethylamine; NNN, nitrosonornicotine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NPYR, nitrosopyrrolidine; OV, Opisthorchis viverrini; PAH, polynuclear aromatic hydrocarbons; TSNA, tobacco-specific nitrosamines; VNA, volatile nitrosamines.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In Thailand the annual consumption per adult (>=15 years of age) of manufactured cigarettes has increased from 810 in 1970–1972 to 1050 in 1990–1992 and of unmanufactured cigarettes to ~1000 in 1994 (1). The age-adjusted lung cancer mortality rate (100 000/year) in Thailand has increased from ~11.0 to 25.0 in males and from 6.3 to 12.1 in females (2). For comparison the cigarette consumption per adult in the USA in 1970–1972 was 3700 and gradually decreased to 2670 in 1990–1992, while the age-adjusted lung cancer mortality rate in males increased from 46.8 in 1970–1971 to 57.1 in 1988–1991 and in women from 11.0 to 24.7, respectively (1,3,4).

Each year 250 000 new cases of primary liver cancer occur worldwide. Sub-Saharan Africa, Melanesia, and East and South-East Asia report especially high incidence rates of this cancer (2). Among men in Thailand, the estimated mortality rate from liver cancer is 10.6/100 000/year (18.5% of all cancer deaths) and is the leading cause of cancer deaths; among Thai women liver cancer is the second leading cause of cancer deaths at 4.3/100 000/year (11.0% of all cancer deaths). The estimated average age-standardized incidence rates per 100 000 per year for primary liver cancer in 1988–1991 for all of Thailand were 40.5 among men and 16.5 among women, while the incidence rates in the Khon Kaen region in north-eastern Thailand (Figure 1Go) were 94.8 and 39.4, respectively. These incidence rates are among the highest in the world (5,6). On the basis of the analysis of a limited number of tumor specimens, an average of 48.5% of the liver tumors in Thailand were cholangiosarcoma (CCA) and 46.5% were hepatocarcinoma (HCC), whereas in the Khon Kaen region 89% were CCA and 7.6% HCC (5).



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Fig. 1. A map of Thailand showing the major regions for a comparison of geographical differences on the age-standardized incidence rate of the three most common cancers in males and in females. The population of Thailand is 62 million. Data compiled from Vatanasapt et al. (5).

 
Chemical and epidemiological studies in Thailand support the concept that CCA is causally associated with chronic infestation by the liver fluke Opisthorchis viverrini (OV). In humans, the parasite most likely induces free radical production, leading to the formation of mutagens, DNA lesions and cellular proliferation of the intrahepatic bile duct epithelium (79).

Cigarette smoking has been associated with an increased risk for liver cancer in several epidemiological surveys (1012). On reviewing the epidemiological evidence, Doll (13) concluded that cigarette smoking is causally associated with liver cancer.

Nitrosodimethylamine (NDMA) is a liver carcinogen in mice, rats, Syrian golden hamsters, Chinese and European hamsters, as well as in rabbits and mink (14). NDMA has been identified in cigarette mainstream smoke at levels of 6–100 ng/cigarette, while up to 1000 ng/cigarette was generated in the sidestream smoke (15). Syrian golden hamsters with OV infestation that were given sodium nitrite and the NDMA precursor aminopyrine, developed a high incidence rate of CCA; none of the hamsters treated with only one of the three agents, or with a combination of two of them, developed a significant number of CCA. This bioassay supports the concept that the OV infestation potentiates the endogenous formation of nitrosamines, in this case that of NDMA (16). Nitrosamines present in food are also a risk factor for liver cancer (17). Both men and women in Thailand who were positive for OV antibody and who received 500 mg proline supplements to their diet, excreted in the urine significantly more nitrosoproline (12.3 ± 18.7 µg/12 h) than those who were negative for OV antibody (3.5 ± 3.2 µg/12 h) (18). These findings indicate that people infested with OV have an increased potential for endogenous formation of nitrosamines. A recent study by Satarug et al. (19) indicates that during the infection with the OV NO is formed and that the latter may serve as nitrosating species for amines giving rise to carcinogenic nitrosamines.

The major carcinogenic nitrosamines in cigarette smoke are the tobacco-specific nitrosamines (TSNA). These include the nicotine-derived 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and nitrosonornicotine (NNN), which is formed by nitrosation of nicotine or nornicotine. NNK is a powerful carcinogen that induces in rats primary lung and liver tumors (20) and is a stronger carcinogen than NDMA (21); NNN is also a strong carcinogen as it induces benign and malignant tumors in the lung and in the upper aerodigestive tract of mice, rats, hamsters and mink.

Cigarette smoke also contains per cigarette 100–600 µg nitrogen oxides, 4–10 µg dimethylamine, 100–3000 µg nicotine and 5–150 µg nornicotine (22). The nitrosation potential of cigarette smoke has been demonstrated in smokers by the formation of nitrosoproline from dietary proline (23). These findings underscore the findings by Srianujata et al. (18) that the Thai smoker infested with OV has an increased risk for the endogenous formation of carcinogenic nitrosamines.

It is our working hypothesis that the OV-infested cigarette smokers in Thailand have an especially high risk for CCA. To challenge this hypothesis, we report here a quantitative assessment of volatile nitrosamines (VNA) and TSNA in the smoke of the leading commercial cigarettes in Thailand.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cigarettes
Two hundred cigarettes each of nine brands of Thai cigarettes were purchased on the open market in Bangkok in 1993 and 1994 and in Chiang Mai (Figure 1Go) during 1995. Prior to smoking, the cigarettes were selected by weight (±20 mg of average weight), and were kept in a chamber at a relative humidity of 60 ± 2% and at 22 ± 2°C for at least 24 h.

Apparatus
VNA and TSNA were separated and quantified with the aid of a Hewlett-Packard Model 5890 gas chromatograph (GC) interfaced with a Model 3390A integrator (Hewlett-Packard, Paramus, NJ) and a model 610 Thermal Energy Analyser (TEA; Thermedics, Waltham, MA) using a modification described earlier (24). Nicotine was analysed on a Hewlett-Packard Model 5890 GC equipped with a flame ionization detector (FID) and interfaced with a Hewlett-Packard Model 3390A integrator. Mainstream cigarette smoke was generated with a Borgwaldt RM 20/CS 20-port smoking machine with rotating head (H.Borgwaldt, Hamburg, Germany) with modifications described in the literature (25).

Reagents
Reference VNA were synthesized as described as were the TSNA (2628). The purity (>99%) of the synthesized compounds was verified by GC-TEA and by GC with a flame ionization detector. Nitroso-n-pentylpicolylamine, serving as an internal standard for the GC analysis of TSNA, was a gift from Dr B.Spiegelhalder of the German Cancer Center. Three per cent XE-60 on GCQ (100–120 mesh) used for the gas chromatographic analysis of VNA and TSNA was purchased from Alltech (Deerfield, IL).

Analyses of VNA and TSNA in mainstream smoke
The VNA and TSNA determinations in cigarette smoke were performed according to a method initially described by Adams et al. (29) which was subsequently modified (30). The cigarettes were smoked under standard conditions of the Federal Trade Commission (31) with a 35 ml puff volume, a 2 s puff duration, a frequency of one puff per minute, and butt lengths of 23 mm or filter plus overwrap plus 3 mm (32).

Forty cigarettes of each brand were smoked and the mainstream smoke was collected on a Cambridge filter (9.0 cm diameter), after passing through two consecutive wash bottles, each containing 100 ml of citrate buffer (pH 4.5), as well as 20 mM ascorbic acid. NPePicA was added as an internal standard to the buffer solution. The Cambridge filter was changed after every 10 cigarettes. The total particulate matter (TPM) trapped on the Cambridge filters was extracted with dichloromethane and the pooled dichloromethane extracts were chromatographed on 90 g basic alumina (Woelm) act. II–III. The VNA and TSNA fractions were concentrated and analysed by GC-TEA using a 3.6 mx2 mm i.d. glass column packed with 3% XE-60 on GCQ. The column temperature was programmed from 150 to 210°C at 6°C/min with an initial 10 min hold, and was finally held at 210°C for 25 min. Each brand was analysed at least twice.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Table IGo presents the comparison and ranking of tar and nicotine levels of Thai cigarettes in relation to total VNA and total TSNA. The best selling commercial cigarette brands, Khrong Thip 90, Falling Rain 90 and Gold City 90 delivered average values per cigarette for the highest smoke yields of tar (24.7 mg), nicotine (2.31 mg), total VNA (76.7 ng) and total TSNA (1,055 ng). By comparison with smoke analyses of the leading US cigarette in 1995 (which have yielded 16 mg tar, 1.1 mg nicotine, 12.5 ng VNA and 600 ng TSNA per cigarette), the smoke yields for these Thai cigarettes are regarded as very high. The current findings complement earlier studies (33,34) which have indicated very high levels of nicotine and tar in the smoke of major brands of commercially produced Thai cigarettes.


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Table I. Comparison and ranking of tar, nicotine, total VNA and total TSNA in the mainstream smoke of Thai cigarettes
 
The concentrations of individual VNA and TSNA in the mainstream smoke of all Thai cigarette brands are summarized in Table IIGo. The highest concentrations of VNA and TSNA were recorded for the filter-tipped brand Gold City 90. They averaged 82 ng total VNA and 1580 ng total TSNA per cigarette; in fact, the latter value exceeded the highest TSNA concentration ever reported for a cigarette, which was 1285 ng total TSNA in the smoke of a French, black tobacco cigarette without filter tip (25). On the other hand, the total TSNA concentrations of the Thai filter cigarettes Royal Standard 90, Royal Standard 90 Deluxe and Samit 90, were relatively low, i.e. 198, 182 and 134 ng, respectively.


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Table II. Volatile nitrosamines and tobacco-specific nitrosamines in mainstream smoke of Thai cigarettes (ng/cigarette)
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The high yields of tar and nicotine in the smoke of Thai cigarettes, except for Royal Standard 90 Deluxe, can be explained, in part, by the use of local tobaccos that are grown in the northern provinces and by the fact that Thai cigarette blends incorporate only a small proportion of USA-grown tobacco (35). The level of tar in cigarette smoke is widely accepted as a measure of its carcinogenic potential (36). In the USA and in several other countries, the tar and nicotine levels of commercial cigarettes have to be reported (31). So far, Thailand has no legislation that regulates cigarette package labelling in respect to smoke yields (33).

There was no apparent difference in smoke yields between filter and non-filter cigarettes. In fact, Gold City 90 with filter delivered the highest levels of NNN (730 ng/cigarette) and NNK (369 ng/cigarette), and very high NDMA (29.8 ng/cigarette) and nitrosopyrrolidine (NPYR) (37.0 ng/cigarette) levels. A similar observation was made for the other four brands with high tar yields. There is an observed quantitative correlation between the VNA, TSNA and the tar delivery, and it was demonstrated that the filter does not significantly impact on the levels of tar, TSNA and VNA in mainstream smoke of Thai cigarettes. Russian cigarettes with slow burning rates that resulted in high yields of tar also generated high TSNA levels in mainstream smoke, even though the nicotine content of such cigarettes is relatively low (37). The high nitrate content of dark tobaccos is a major contributor for the high yields of NDMA, NPYR and NNK in the smoke of these cigarettes; NNK is especially high in the smoke of dark tobaccos which are also rich in nicotine (38,39). Hand-rolled cigarettes require the use of highly tensile cigarette paper which has a low porosity. Therefore, the yields of tar, nicotine and carbon monoxide in the smoke of hand-rolled cigarettes are significantly higher than in the smoke of machine-made cigarettes containing the same tobacco (40).

In many countries the carcinogenic potential of cigarette smoke is determined by the tar content, but because its mechanism of cancer induction is different for nitrosamines from that of other carcinogens, we have suggested that the abundant carcinogenic TSNA in the mainstream smoke of commercial cigarettes ought to be declared as an additional risk factor for cancer (41). A similar proposal has been made for the VNA in tobacco smoke (42).

Estimated tobacco sales in Thailand reached ~42.6 billion pieces in 1990, which does not include hand-rolled cigarettes, cigars and imported cigarettes. Increases have been observed in terms of per capita sales (34). However, ~50% of all cigarettes and cigars consumed in Thailand are hand-rolled. Earlier studies have shown that the tobacco in these hand-rolled products ranked especially high in smoke yields of tar and nicotine (33,34). A recent case-control study reported a close relationship between infection with the liver fluke OV, smoking and CCA (43). In that study, the majority (73.7%) reported smoking hand-rolled, home-made cigarettes containing local tobacco. Research needs to be undertaken to determine VNA and TSNA levels in these tobacco products, as well as in betel nut mixtures that contain tobacco. The habit of chewing betel nut with tobacco has emerged as a risk factor for CCA and possibly also for HCC (44).

Commercially manufactured cigarettes utilize blends of Thai and small amounts of USA-grown tobacco. The best selling US cigarette brands in Thailand amount to ~10% of the market. We have reported that the US cigarettes currently sold in Thailand have similar or lower yields of tar and less nicotine than manufactured Thai cigarettes (34). However, the levels of TSNA and VNA are comparable with those of Thai products classified as high-yield cigarettes. Both NNN and NNK are powerful carcinogens in laboratory animals that induce benign and malignant tumors; NNN induces lung cancer in mice and upper aerodigestive tract cancer in rats and hamsters, while NNK induces cancer of the lung, upper aerodigestive tract, pancreas and liver in mice, rats and hamsters (22). The carcinogenic potential of the VNA, NDMA and NPYR, has been determined in a multitude of bioassays (42).

Cancer mortality data from Thailand indicate a trend towards increasing mortality rates from lung and liver cancer (5). Examination of the histological types of lung cancer in Thailand pointed to the predominance of adenocarcinoma (45). Several studies have shown that liver carcinoma can be induced with NNK and NDMA in mice and rats (46) and this organ-specific effect occurs regardless of site or form of application (47).

In several countries, the changes in the make-up of filter cigarettes have brought about increased smoke yields of VNA and TSNA, whereas the yields of carcinogenic polynuclear aromatic hydrocarbons (PAH) have decreased (22). Future investigations should lead to practical measures towards reducing the exposure to both carcinogenic PAH and nitrosamines. However, abstention from tobacco or cessation of tobacco use are the only measures of certainty with regard to risk reduction. Nevertheless, reduction of exposure to tobacco carcinogens can be accomplished by product modification. VNA and TSNA can be reduced by selecting lighter tobacco blends with low levels of preformed TSNA, and by selecting tobacco laminae and ribs that are low in nitrate, and thus have reduced nitrosation potential for amines in tobacco (48). Yet, using tobacco blends with lower nitrate levels for manufacturing cigarettes may lead to higher levels of PAH in the mainstream smoke (22). More research is needed to develop methods for inhibiting the formation of VNA and TSNA.

The data presented in this report suggest tobacco smoke to be also a risk factor for cancer of the liver. This concept reemphasizes the need for cigarettes which generate smoke low in tar and carcinogens, including VNA and TSNA.

An epidemiological case–control study is under way in North Thailand, initiated by our preliminary results, to correlate the incidence rates of HCC and CCA with the use of tobacco-containing betel quid and with cigarette smoking. In addition, biomarkers are needed as to the uptake, and the extent of endogenous formation of VNA and TSNA in cigarette smokers and non-smokers. In Thailand, such studies should include persons with and without OV infestation. Such data will enable us to evaluate the contribution of cigarette smoking and betel quid tobacco chewing to the high risk of CCA, especially in the north-eastern part of the country. Research is also needed to establish analytical data for smoke carcinogens and toxins produced by the hand-rolled cigarettes.


    Notes
 
5 Present address: Division of Cancer Prevention and Control, Center for Disease Control and Prevention, Atlanta, GA 30341, USA Back

6 To whom correspondence should be addressed at NYIT Back


    Acknowledgments
 
We thank Dr Songsak Srianujata (Institute of Nutrition at Mahidol University), Dr Petcharin Srivatanakul (National Cancer Institute), Dr Panata Migasena (Institute of Tropical Medicine at Mahidol University), Dr Mathuros Ruchirawat and Dr Jutamaad Satayavivad (Chulabhorn Research Institute), Dr Vanchai Vatanasapt (Khon Kaen University), Dr Vinai Suvatte, Dr Natee Rukspollmuang, Dr Pradit Chareonthaitawee (Siriraj Hospital Mahidol University) and Dr Pongsiri Prathnadi (Chiang Mai University) for help given to E.J.M. while he was a visiting research professor at the Faculty of Medicine, Mahidol University, Bangkok, and at the Faculty of Medicine, Chiang Mai University, Chiang Mai, during the summer semesters in 1988–1996. The laboratory assays on smoke yields of the tobacco products were carried out at the American Health Foundation and were supported by grants CA-25980 and CA-70972 from the US National Cancer Institute.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 7, 1998; revised August 20, 1998; accepted September 21, 1998.