A tobacco-specific lung carcinogen: From basic research to tobacco control.

LE02-01

It is imperative that we continue to find new approaches to the prevention of tobacco-related cancer. This perhaps can best be accomplished through basic research. In this lecture, I will attempt to demonstrate how the discovery of one tobacco carcinogen led to preventive strategies, some of which have arguably affected tobacco-related cancer death. While great progress has been made in tobacco control, and smoking prevalence in the U.S. continues to decline, the overall tobacco facts are still ugly. There are 1.2 billion smokers in the world and hundreds of millions of smokeless tobacco users (1). Tobacco smoking is a cause of at least fourteen types of cancer and smokeless tobacco use is a cause of two (2,3). Overall, tobacco use accounts for at least 30% of cancer mortality in developed countries (4). Thanks to the work of many talented researchers, we have a solid framework for understanding the mechanisms by which tobacco products cause cancer (5,6). Tobacco use generally begins in the teen age years and, due to the addictive characteristics of nicotine, often persists throughout life. Nicotine is not a carcinogen, but each puff of a cigarette, or dip of smokeless tobacco, contains a mixture of carcinogens. Many of these require an enzymatically catalyzed metabolic activation process to be converted into forms that can react with DNA, forming covalently bound products called DNA adducts. DNA adducts are absolutely critical in the carcinogenic process. If they persist unrepaired, they can cause permanent mutations during DNA replication, when adducts are read incorrectly by DNA polymerases leading to insertion of the wrong complementary base. DNA repair and apoptosis oppose this mutagenic mechanism by removing adducts or cells that have DNA damage. If the mutations occur in critical regions of oncogenes such as K-ras or tumor suppressor genes such as p53, the result can be loss of normal cellular growth control mechanisms, genomic instability, and ultimately, cancer. Tobacco and tobacco smoke are rich in carcinogens that can form DNA adducts. One group of carcinogens in tobacco products, and the focus of this lecture, is the tobacco-specific nitrosamines (7). Fifty years ago, Magee and Barnes, in a landmark study, demonstrated the strong hepatocarcinogenicity of dimethylnitrosamine in rats (8). This led to intensive investigation of the carcinogenic properties of nitrosamines in general. The impetus for this work was the realization that nitrosamines could be readily formed from amines and nitrite, and that this process could occur in vivo. Many nitrosamines are powerful carcinogens, demonstrated in at least 30 different animal species (9). They are the standard model compounds for induction of various types of cancer in laboratory animals. Against this background, and in view of literature precedent, it seemed reasonable to ask whether nicotine and related tobacco alkaloids could also be nitrosated to form carcinogens (10,11). Thus, Dietrich Hoffmann and I, together with others at the American Health Foundation, investigated the reactions of nicotine with nitrite, and the carcinogenicity and occurrence in tobacco of the resulting nitrosamines. The result of this work was the discovery of 7 tobacco-specific nitrosamines in tobacco products (7). Two of these- N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, for nicotine derived nitrosamino ketone) - are considered to be the most important of the group due to their levels in tobacco products and carcinogenic activities. This lecture will focus on NNK. The lung is the main target for the carcinogenicity of NNK based on experiments carried out in rats, mice, hamsters, and ferrets (12,13). Other affected tissues are the nasal mucosa, liver, and pancreas in rats, the nasal mucosa and trachea of hamsters, and the nasal mucosa of mink (12). Adenocarcinoma is the main type of lung cancer induced by NNK. Lung tumors are induced by NNK independent of the route of administration, in experiments carried out in rats and multiple strains of mice (12). Clearly, NNK is a systemic lung carcinogen. Its cancer causing activity is particularly strong in the rat, in which lung tumors have been induced by total doses of only a few mg/kg body weight given by subcutaneous injection or in the drinking water. When these observations are combined with its common presence in tobacco products, both in unburned tobacco and tobacco smoke, and related mechanistic and biomarker data, the inescapable conclusion is that it must play a significant role in tobacco-induced cancer. The International Agency for Research on Cancer evaluates NNK and NNN as "carcinogenic to humans", Group 1 (3). The finding that NNK was a tobacco-specific lung carcinogen spurred work on this compound. Many studies were carried out on the analysis of tobacco and tobacco smoke for this carcinogen, on the development of new animal models for lung tumor induction, on NNK metabolism, DNA adduct formation and repair, and on its impact on cellular signaling pathways and oncogene/suppressor gene activation or inactivation (12,14-17). These basic research studies continue to the present and have had practical consequences with respect to tobacco control. For example, the tobacco and smoke analysis studies were in part responsible for the labeling of smokeless tobacco products as potential causes of oral cancer and for process modifications leading to reduced nitrosamine levels in some tobacco products. The carcinogenicity studies led to the development of new animal models for lung adenoma/adenocarcinoma which in turn have been used for the preclinical evaluation of chemopreventive agents against lung cancer. The metabolic studies led to the development of specific biomarkers for tobacco carcinogen uptake, that have been applied in studies of tobacco product evaluation and secondhand smoke exposure, research that will be discussed here. Pathways of NNK metabolism have been extensively documented (12). A common pathway in most systems examined is conversion to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides (NNAL-Glucs). NNAL has carcinogenic activity similar to that of NNK while NNAL-Glucs are detoxification products (18). We reasoned that analysis of NNAL and NNAL-Glucs in human urine could provide a specific biomarker of tobacco carcinogen uptake. My colleague Steven Carmella took the lead in developing a highly sensitive and specific method for the analysis of NNAL and NNAL-Glucs in human urine (19-21). The technical aspects of this methodology have evolved considerably, and it is now possible to quantify NNAL and NNAL-Glucs routinely in fairly large studies. The sum of NNAL and NNAL-Glucs, termed "total NNAL", has emerged as a very useful biomarker for assessing tobacco carcinogen uptake in humans (22,23). This is due in part to its tobacco-specificity. Tobacco products are the only known source of NNK, and therefore NNAL. Total NNAL in urine can come only from tobacco product exposure, not from the diet or other sources. Therefore, there is little or no background, a common problem with other tobacco carcinogen biomarkers for which there are also dietary or environmental exposures. In collaboration with Professor Dorothy Hatsukami and the University of Minnesota Transdisciplinary Tobacco Use Research Center, we have used total NNAL to evaluate carcinogen uptake in a variety of clinical studies. The first of these asked a simple question (24). Would a reduction in smoking, as measured by fewer cigarettes per day, lead to a corresponding reduction in carcinogen uptake? Subjects gradually reduced their smoking by 25% in the first 2 weeks, 50% in the next two weeks, 75% in the next two weeks, and then were asked to maintain this reduced level through week 26. Urine samples were collected at these intervals and analyzed for total NNAL. The results demonstrated that there were significant reductions in total NNAL levels at these various time points compared to baseline, but that the reductions were modest and transient compared to reduction in cigarettes per day. When smokers smoke fewer cigarettes per day, they compensate by smoking these cigarettes differently (25). Our conclusion was that reduction in cigarettes smoked per day would have only a modest effect on carcinogen uptake and probably is not a viable strategy for substantially reducing risk. In a second study, we investigated levels of urinary total NNAL in smokers of regular, light, and ultra-light cigarettes (26). These are classifications of cigarettes, used for many years, based on their "tar" levels, as measured on a smoking machine. When we carried out this study in 175 smokers, there had been no previous studies to determine whether carcinogen uptake was actually different in smokers of these cigarettes. There was no difference in levels of total NNAL, 1-hydroxypyrene, or cotinine in the urine of these smokers, indicating that there should not be any protection against lung cancer in smokers of light cigarettes, a finding that has been confirmed in epidemiologic studies. Our next study involved a new product - the Omni cigarette (27). Many new tobacco products are appearing on the market now with claims of reduced harm. These so called "potential reduced exposure products" or PREPS require evaluation. The Omni cigarette was advertised as "significantly reducing carcinogens that are among the major causes of lung cancer", according to machine measurements. In our study, smokers used their usual brand for 2 weeks, then either stopped smoking and switched to the nicotine patch for 4 weeks, or switched to Omni for 4 weeks. The results demonstrated that there was a modest but significant 25% reduction in urinary total NNAL in smokers who switched to Omni, while cessation with the nicotine patch resulted in a 65% reduction. These results indicated that Omni provided only modest protection against carcinogen uptake. Omni is no longer on the market. We have also carried out studies evaluating smokeless tobacco products as alternatives to cigarette smoking (27,28). Our general conclusion from these studies is that some smokeless tobacco products may lead to lower tobacco-specific carcinogen uptake compared to cigarette smoking but that cessation with nicotine replacement therapy is a better option. Most smokeless tobacco products currently on the market are still contaminated with tobacco-specific nitrosamines, in levels much higher than those of carcinogens found in other products designed for oral consumption. The strongest impact of the total NNAL biomarker has probably been in studies of secondhand tobacco smoke exposure. While there is complete agreement now that secondhand smoke exposure (involuntary smoking) is a cause of lung cancer in non-smokers, this was not always the case, and the epidemiologic data have never been as compelling as those connecting smoking and lung cancer (29). Total NNAL is an ideal biomarker to investigate this phenomenon because of the sensitivity of the analytical method and the tobacco-specificity and lung carcinogenicity of NNK. We have carried out a series of studies from 1994 to the present, examining the uptake of NNK, as measured by total NNAL in urine, in non-smokers exposed to secondhand tobacco smoke (30,31). These studies have clearly demonstrated NNK uptake throughout life in non-smokers exposed to secondhand tobacco smoke. We have quantified total NNAL in amniotic fluid of mothers who smoke, in the urine of newborns of mothers who smoke, in infants exposed to parental cigarette smoke, in elementary school children, in women living with men who smoke, in hospital workers exposed to patients who smoke, in people who frequented smoking sections of gambling casinos, and in restaurants and bars where smoking was permitted. The results of these studies consistently show uptake of NNK greater than in non-exposed non-smokers, with levels of total NNAL about 1-5% as great as in smokers. These data provide very strong supporting evidence for the epidemiologic studies showing elevated risk of lung cancer in non-smokers exposed to cigarette smoke. These studies also have impact, because they inevitably attract media attention. This work has contributed to the legislative momentum for smoke free restaurants and bars, which, along with taxation and anti-tobacco advertising, is a mainstay of current tobacco control strategies. The regulation of indoor smoking can reduce cues for smoking, reduce the amount smoked, and ultimately can change social norms. In summary, some basic research questions involving nicotine chemistry and mechanisms of carcinogenesis of the tobacco-specific lung carcinogen NNK led to more applied studies which have had an impact on tobacco control. It is hoped that continued fundamental research on mechanisms of tobacco induced cancer will similarly lead to new insights for cancer prevention, and to continued reduction of cancer incidence. References 1. International Agency for Research on Cancer (2004) Tobacco Smoke and Involuntary Smoking. In IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 83 pp 53-119, IARC, Lyon, FR. 2. 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