The global burden of cancer continues to increase largely because of the aging and growth of the world population alongside an increasing adoption of cancer-causing behaviors, particularly smoking, in economically developing countries. Based on the GLOBOCAN 2008 estimates, about 12.7 million cancer cases and 7.6 million cancer deaths are estimated to have occurred in 2008; of these, 56% of the cases and 64% of the deaths occurred in the economically developing world. Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females, accounting for 23% of the total cancer cases and 14% of the cancer deaths. Lung cancer is the leading cancer site in males, comprising 17% of the total new cancer cases and 23% of the total cancer deaths. Breast cancer is now also the leading cause of cancer death among females in economically developing countries, a shift from the previous decade during which the most common cause of cancer death was cervical cancer. Further, the mortality burden for lung cancer among females in developing countries is as high as the burden for cervical cancer, with each accounting for 11% of the total female cancer deaths. Although overall cancer incidence rates in the developing world are half those seen in the developed world in both sexes, the overall cancer mortality rates are generally similar. Cancer survival tends to be poorer in developing countries, most likely because of a combination of a late stage at diagnosis and limited access to timely and standard treatment. A substantial proportion of the worldwide burden of cancer could be prevented through the application of existing cancer control knowledge and by implementing programs for tobacco control, vaccination (for liver and cervical cancers), and early detection and treatment, as well as public health campaigns promoting physical activity and a healthier dietary intake. Clinicians, public health professionals, and policy makers can play an active role in accelerating the application of such interventions globally.

Each year, the American Cancer Society estimates the numbers of new cancer cases and deaths expected in the United States in the current year and compiles the most recent data on cancer incidence, mortality, and survival based on incidence data from the National Cancer Institute, the Centers for Disease Control and Prevention, and the North American Association of Central Cancer Registries and mortality data from the National Center for Health Statistics. A total of 1,660,290 new cancer cases and 580,350 cancer deaths are projected to occur in the United States in 2013. During the most recent 5 years for which there are data (2005-2009), delay-adjusted cancer incidence rates declined slightly in men (by 0.6% per year) and were stable in women, while cancer death rates decreased by 1.8% per year in men and by 1.5% per year in women. Overall, cancer death rates have declined 20% from their peak in 1991 (215.1 per 100,000 population) to 2009 (173.1 per 100,000 population). Death rates continue to decline for all 4 major cancer sites (lung, colorectum, breast, and prostate). Over the past 10 years of data (2000-2009), the largest annual declines in death rates were for chronic myeloid leukemia (8.4%), cancers of the stomach (3.1%) and colorectum (3.0%), and non-Hodgkin lymphoma (3.0%). The reduction in overall cancer death rates since 1990 in men and 1991 in women translates to the avoidance of approximately 1.18 million deaths from cancer, with 152,900 of these deaths averted in 2009 alone. Further progress can be accelerated by applying existing cancer control knowledge across all segments of the population, with an emphasis on those groups in the lowest socioeconomic bracket and other underserved populations.

Over the past decade, comprehensive sequencing efforts have revealed the genomic landscapes of common forms of human cancer. For most cancer types, this landscape consists of a small number of "mountains" (genes altered in a high percentage of tumors) and a much larger number of "hills" (genes altered infrequently). To date, these studies have revealed ~140 genes that, when altered by intragenic mutations, can promote or "drive" tumorigenesis. A typical tumor contains two to eight of these "driver gene" mutations; the remaining mutations are passengers that confer no selective growth advantage. Driver genes can be classified into 12 signaling pathways that regulate three core cellular processes: cell fate, cell survival, and genome maintenance. A better understanding of these pathways is one of the most pressing needs in basic cancer research. Even now, however, our knowledge of cancer genomes is sufficient to guide the development of more effective approaches for reducing cancer morbidity and mortality.

BACKGROUND

The aggressive and heterogeneous nature of lung cancer has thwarted efforts to reduce mortality from this cancer through the use of screening. The advent of low-dose helical computed tomography (CT) altered the landscape of lung-cancer screening, with studies indicating that low-dose CT detects many tumors at early stages. The National Lung Screening Trial (NLST) was conducted to determine whether screening with low-dose CT could reduce mortality from lung cancer.

METHODS

From August 2002 through April 2004, we enrolled 53,454 persons at high risk for lung cancer at 33 U.S. medical centers. Participants were randomly assigned to undergo three annual screenings with either low-dose CT (26,722 participants) or single-view posteroanterior chest radiography (26,732). Data were collected on cases of lung cancer and deaths from lung cancer that occurred through December 31, 2009.

RESULTS

The rate of adherence to screening was more than 90%. The rate of positive screening tests was 24.2% with low-dose CT and 6.9% with radiography over all three rounds. A total of 96.4% of the positive screening results in the low-dose CT group and 94.5% in the radiography group were false positive results. The incidence of lung cancer was 645 cases per 100,000 person-years (1060 cancers) in the low-dose CT group, as compared with 572 cases per 100,000 person-years (941 cancers) in the radiography group (rate ratio, 1.13; 95% confidence interval [CI], 1.03 to 1.23). There were 247 deaths from lung cancer per 100,000 person-years in the low-dose CT group and 309 deaths per 100,000 person-years in the radiography group, representing a relative reduction in mortality from lung cancer with low-dose CT screening of 20.0% (95% CI, 6.8 to 26.7; P=0.004). The rate of death from any cause was reduced in the low-dose CT group, as compared with the radiography group, by 6.7% (95% CI, 1.2 to 13.6; P=0.02).

CONCLUSIONS

Screening with the use of low-dose CT reduces mortality from lung cancer. (Funded by the National Cancer Institute; National Lung Screening Trial ClinicalTrials.gov number, NCT00047385.).

Cancers arise owing to mutations in a subset of genes that confer growth advantage. The availability of the human genome sequence led us to propose that systematic resequencing of cancer genomes for mutations would lead to the discovery of many additional cancer genes. Here we report more than 1,000 somatic mutations found in 274 megabases (Mb) of DNA corresponding to the coding exons of 518 protein kinase genes in 210 diverse human cancers. There was substantial variation in the number and pattern of mutations in individual cancers reflecting different exposures, DNA repair defects and cellular origins. Most somatic mutations are likely to be 'passengers' that do not contribute to oncogenesis. However, there was evidence for 'driver' mutations contributing to the development of the cancers studied in approximately 120 genes. Systematic sequencing of cancer genomes therefore reveals the evolutionary diversity of cancers and implicates a larger repertoire of cancer genes than previously anticipated.

Cancer is driven by mutation. Worldwide, tobacco smoking is the principal lifestyle exposure that causes cancer, exerting carcinogenicity through >60 chemicals that bind and mutate DNA. Using massively parallel sequencing technology, we sequenced a small-cell lung cancer cell line, NCI-H209, to explore the mutational burden associated with tobacco smoking. A total of 22,910 somatic substitutions were identified, including 134 in coding exons. Multiple mutation signatures testify to the cocktail of carcinogens in tobacco smoke and their proclivities for particular bases and surrounding sequence context. Effects of transcription-coupled repair and a second, more general, expression-linked repair pathway were evident. We identified a tandem duplication that duplicates exons 3-8 of CHD7 in frame, and another two lines carrying PVT1-CHD7 fusion genes, indicating that CHD7 may be recurrently rearranged in this disease. These findings illustrate the potential for next-generation sequencing to provide unprecedented insights into mutational processes, cellular repair pathways and gene networks associated with cancer.

The complexity of tobacco smoke leads to some confusion about the mechanisms by which it causes lung cancer. Among the multiple components of tobacco smoke, 20 carcinogens convincingly cause lung tumors in laboratory animals or humans and are, therefore, likely to be involved in lung cancer induction. Of these, polycyclic aromatic hydrocarbons and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone are likely to play major roles. This review focuses on carcinogens in tobacco smoke as a means of simplifying and clarifying the relevant information that provides a mechanistic framework linking nicotine addiction with lung cancer through exposure to such compounds. Included is a discussion of the mechanisms by which tobacco smoke carcinogens interact with DNA and cause genetic changes--mechanisms that are reasonably well understood--and the less well defined relationship between exposure to specific tobacco smoke carcinogens and mutations in oncogenes and tumor suppressor genes. Molecular epidemiologic studies of gene-carcinogen interactions and lung cancer--an approach that has not yet reached its full potential--are also discussed, as are inhalation studies of tobacco smoke in laboratory animals and the potential role of free radicals and oxidative damage in tobacco-associated carcinogenesis. By focusing in this review on several important carcinogens in tobacco smoke, the complexities in understanding tobacco-induced cancer can be reduced, and new approaches for lung cancer prevention can be envisioned.

We report the results of whole-genome and transcriptome sequencing of tumor and adjacent normal tissue samples from 17 patients with non-small cell lung carcinoma (NSCLC). We identified 3,726 point mutations and more than 90 indels in the coding sequence, with an average mutation frequency more than 10-fold higher in smokers than in never-smokers. Novel alterations in genes involved in chromatin modification and DNA repair pathways were identified, along with DACH1, CFTR, RELN, ABCB5, and HGF. Deep digital sequencing revealed diverse clonality patterns in both never-smokers and smokers. All validated EFGR and KRAS mutations were present in the founder clones, suggesting possible roles in cancer initiation. Analysis revealed 14 fusions, including ROS1 and ALK, as well as novel metabolic enzymes. Cell-cycle and JAK-STAT pathways are significantly altered in lung cancer, along with perturbations in 54 genes that are potentially targetable with currently available drugs.

The devastating link between tobacco products and human cancers results from a powerful alliance of two factors - nicotine and carcinogens. Without either one of these, tobacco would be just another commodity, instead of being the single greatest cause of death due to preventable cancer. Nicotine is addictive and toxic, but it is not carcinogenic. This addiction, however, causes people to use tobacco products continually, and these products contain many carcinogens. What are the mechanisms by which this deadly combination leads to 30% of cancer-related deaths in developed countries, and how can carcinogen biomarkers help to reveal these mechanisms?

It is estimated that cigarette smoking kills over 1 000 000 people each year by causing lung cancer as well as many other neoplasmas. p53 mutations are frequent in tobacco-related cancers and the mutation load is often higher in cancers from smokers than from nonsmokers. In lung cancers, the p53 mutational patterns are different between smokers and nonsmokers with an excess of G to T transversions in smoking-associated cancers. The prevalence of G to T transversions is 30% in smokers' lung cancer but only 12% in lung cancers of nonsmokers. A similar trend exists, albeit less marked, in laryngeal cancers and in head and neck cancers. This type of mutation is infrequent in most other tumors aside from hepatocellular carcinoma. At several p53 mutational hotspots common to all cancers, such as codons 248 and 273, a large fraction of the mutations are G to T events in lung cancers but are almost exclusively G to A transitions in non-tobacco-related cancers. Two important classes of tobacco smoke carcinogens are the polycyclic aromatic hydrocarbons (PAH) and the nicotine-derived nitrosamines. Recent studies have indicated that there is a strong coincidence of G to T transversion hotspots in lung cancers and sites of preferential formation of PAH adducts along the p53 gene. Endogenously methylated CpG dinucleotides are the preferred sites for G to T transversions, accounting for more than 50% of such mutations in lung tumors. The same dinucleotide, when present within CpG-methylated mutational reporter genes, is the target of G to T transversion hotspots in cells exposed to the model PAH compound benzo[a]pyrene-7,8-diol-9,10-epoxide. As summarized here, a number of other tobacco smoke carcinogens also can cause G to T transversion mutations. The available data suggest that p53 mutations in lung cancers can be attributed to direct DNA damage from cigarette smoke carcinogens rather than to selection of pre-existing endogenous mutations.

Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small-cell lung carcinomas in smokers being the predominant form of the disease. Although previous studies have identified important common somatic mutations in lung cancers, they have primarily focused on a limited set of genes and have thus provided a constrained view of the mutational spectrum. Recent cancer sequencing efforts have used next-generation sequencing technologies to provide a genome-wide view of mutations in leukaemia, breast cancer and cancer cell lines. Here we present the complete sequences of a primary lung tumour (60x coverage) and adjacent normal tissue (46x). Comparing the two genomes, we identify a wide variety of somatic variations, including >50,000 high-confidence single nucleotide variants. We validated 530 somatic single nucleotide variants in this tumour, including one in the KRAS proto-oncogene and 391 others in coding regions, as well as 43 large-scale structural variations. These constitute a large set of new somatic mutations and yield an estimated 17.7 per megabase genome-wide somatic mutation rate. Notably, we observe a distinct pattern of selection against mutations within expressed genes compared to non-expressed genes and in promoter regions up to 5 kilobases upstream of all protein-coding genes. Furthermore, we observe a higher rate of amino acid-changing mutations in kinase genes. We present a comprehensive view of somatic alterations in a single lung tumour, and provide the first evidence, to our knowledge, of distinct selective pressures present within the tumour environment.

Tobacco use remains the single largest preventable cause of death and disease in the United States. According to the 2010 U.S. Surgeon General's report, approximately 443,000 U.S. adults die from smoking-related illnesses each year. In addition, smoking has been estimated to cost the United States $96 billion in direct medical expenses and $97 billion in lost productivity annually. To assess progress toward the Healthy People 2020 (HP2020) objective to reduce cigarette smoking by adults (objective TU-1.1),* CDC's Office on Smoking and Health used data from the 2011 National Health Interview Survey (NHIS) to estimate current national cigarette smoking prevalence. The findings indicate that 19.0% of adults smoked cigarettes in 2011 and no statistically significant change in current adult smoking prevalence occurred from 2010 (19.3%) to 2011 (19.0%). Among daily smokers, the proportion who smoked ≥30 cigarettes per day (CPD) declined significantly, from 12.6% in 2005 to 9.1% in 2011, whereas the proportion of those who smoked 1-9 CPD increased significantly, from 16.4% to 22.0%. To help reduce the national prevalence of cigarette smoking among adults to the HP2020 target of 12%, population-based prevention strategies (e.g., increasing prices of tobacco products, antitobacco media campaigns featuring graphic personal stories on the adverse health impact of smoking, smoke-free laws for workplaces and public places, and barrier-free access to help quitting) will need to be implemented more extensively. Such evidence-based tobacco control interventions can help adults quit and prevent the initiation of tobacco use.

Methods were developed for the efficient routine degradation and fractionation of ethylated and methylated DNA. Alkylated DNA was hydrolyzed by a neutral thermal method to yield 3- and 7- alkylpurines and O2-alkylcytosines. The partially apurinic DNA was separated from the bases by precipitation in 0.1 N HCl. Portions of the DNA precipitate were further hydrolyzed either by 0.1 N HCl to yield purine bases, or by enzymes to yield nucleosides and phosphotriesters. The chemical and enzymic digests were fractionated by a combination of high pressure liquid chromatography systems to yield quantitative estimates of the following products from methylated or ethylated DNA: 1-, 3-, and 7-alkyladenines, O2-alkylcytosines, 3-, O6-, and 7- alkylguanines and O2-, 3-, and O4-alkylthymines. N6-Alkyladenines, 1-alkylguanines and N2-alkylguanines were not detected and the 3- alkylcytosines were detected but not quantified. Phosphotriesters were estimated from the amounts of recovered alkyl phosphotriesters of thymidylyl (3'-5') thymidine. Using these methods, it was possible to account for 98, 81, 98, and 92% of the DNA bound alkyl groups obtained from DNA reacted with [14C]methyl methanesulfonate, [3H]ethyl methanesulfonate, N-[3H]-methyl-N-nitrosourea, and N-[14C]ethyl-N-nitrosourea, respectively. The methods described provide reproducible and quantitative methods of analysis for all the known methylated or ethylated products in a single DNA sample.

Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.

Polycyclic aromatic hydrocarbons (PAH) are believed to be among the principal causative agents for lung cancer in smokers, but no epidemiologic studies have evaluated the relationship of PAH uptake and metabolism to lung cancer. In this study, we quantified prediagnostic urinary levels of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), a validated biomarker of PAH uptake and metabolism, as well as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL), and cotinine and its glucuronides (total cotinine), validated biomarkers of uptake of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and nicotine, respectively, in relation to lung cancer risk among current smokers in a nested case-control study within a cohort of 18,244 Chinese men in Shanghai, China. Urinary levels of PheT, total NNAL, and total cotinine were significantly higher in cases than controls (N = 476 matched pairs). ORs (95% confidence intervals) for lung cancer in the second, third, fourth, and fifth quintiles of PheT were 1.70 (1.00-2.88), 1.07 (0.62-1.84), 1.48 (0.86-2.53), and 2.34 (1.33-4.11), respectively, relative to the lowest quartile (P(trend) = 0.023) after adjustment for self-reported smoking intensity and duration and urinary total NNAL and total cotinine. This study also confirmed that urinary total NNAL and total cotinine are independently related to lung cancer risk.

N'-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are tobacco-specific nitrosamines. NNN and NNK can induce cancers of the esophagus and lung, respectively, in laboratory animals, but data on human esophageal cancer are lacking. The association between levels of NNN and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), an NNK metabolite, in urine samples collected before diagnosis and risk of esophageal cancer was examined in 77 patients with esophageal cancer and 223 individually matched controls, all current smokers, from a cohort of 18244 Chinese men in Shanghai, China, followed from 1986 to 2008. Urinary total NNN (free NNN plus NNN-N-glucuronide) was significantly higher, whereas the percentage of its detoxification product NNN-N-glucuronide was significantly lower in cases than controls. Odds ratios (95% confidence intervals) of esophageal cancer for the second and third tertiles of total NNN were 3.99 (1.25-12.7) and 17.0 (3.99-72.8), respectively, compared with the first tertile after adjustment for urinary total NNAL and total cotinine and smoking intensity and duration (P(trend) < 0.001). The corresponding figures for the percentage of NNN-N-glucuronides were 0.37 (0.17-0.80) and 0.27 (0.11-0.62) (P(trend) = 0.001). Urinary total NNN and the percentage of NNN-N-glucuronides almost completely accounted for the observed association for urinary total NNAL (free NNAL plus its glucuronides), urinary total cotinine and smoking intensity with esophageal cancer risk. These findings along with results of previous studies in laboratory animals support a significant and unique role of NNN in esophageal carcinogenesis in humans.

BACKGROUND

Environmental tobacco smoke has been classified by the Environmental Protection Agency as a carcinogen causally associated with lung cancer in adults, but there have been no reports of lung carcinogens or their metabolites in the body fluids or tissues of nonsmokers exposed to environmental tobacco smoke.

METHODS

Five male nonsmokers were exposed to sidestream cigarette smoke generated by machine smoking of reference cigarettes for 180 minutes on each of two days, six months apart. Sidestream smoke is the smoke that originates from the smoldering end of a cigarette between puffs. Twenty-four-hour urine samples were collected before and after exposure. The urine samples were analyzed for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide, which are metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a powerful lung carcinogen in rodents. NNAL is also a lung carcinogen in rodents.

RESULTS

The urinary excretion of the metabolites increased after exposure to sidestream smoke in all the men. The mean (+/- SD) amount of NNAL and NNAL glucuronide was significantly higher after exposure than at base line (33.9 +/- 20.0 vs. 8.4 +/- 11.2 ng per 24 hours [127 +/- 74 vs. 31 +/- 41 pmol per day], P < 0.001) and was correlated with urinary cotinine excretion (r = 0.89, P < 0.001). The nicotine concentrations in the air to which the men were exposed were comparable to those in a heavily smoke-polluted bar.

CONCLUSIONS

Nonsmokers exposed to sidestream cigarette smoke take up and metabolize a lung carcinogen, which provides experimental support for the proposal that environmental tobacco smoke can cause lung cancer.

NNN (1) is an esophageal carcinogen in rats. 2'-Hydroxylation of NNN is believed to be the major bioactivation pathway for NNN tumorigenicity. (S)-NNN is preferentially metabolized by 2'-hydroxylation in cultured rat esophagus, whereas there is no preference for 2'-hydroxylation versus 5'-hydroxylation in the metabolism of (R)-NNN. 2'-Hydroxylation of NNN generates the reactive intermediate 4-oxo-4-(3-pyridyl)butanediazohydroxide (8), resulting in the formation of pyridyloxobutyl (POB)-DNA adducts. On the basis of these observations, we hypothesized that (S)-NNN treatment would produce higher levels of POB-DNA adducts than that by (R)-NNN in the rat esophagus. We tested this hypothesis by treating male F344 rats with 10 ppm of (R)-NNN or (S)-NNN in drinking water. After 1, 2, 5, 10, 16, or 20 weeks of treatment, POB-DNA adducts in esophageal, liver, and lung DNA were quantified by HPLC-ESI-MS/MS. In the rat esophagus, (S)-NNN treatment generated levels of POB-DNA adducts 3-5 times higher than (R)-NNN treatment, which supports our hypothesis. 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 14) was the major adduct detected, followed by O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 11) and O2-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (POB-Cyt, 15). O6-[4-(3-Pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O6-POB-dGuo, 10) was not detected. The total POB-DNA adduct levels in the esophagus were 3-11 times higher than those in the liver for (R)-NNN and 2-6 times higher than those for (S)-NNN. In contrast to the esophagus and liver, (R)-NNN treatment produced more POB-DNA adducts than (S)-NNN treatment in the rat lung, which suggested an important role for cytochrome P450 2A3 in NNN metabolism in the rat lung. In both the liver and lung, O2-POB-dThd was the predominant adduct and accumulated during the experiment. The results of this study demonstrate that individual POB-DNA adducts form and persist in the esophagi, livers, and lungs of rats chronically treated with NNN enantiomers and demonstrate that (S)-NNN produces higher levels of POB-DNA adducts in the esophagus than (R)-NNN, suggesting that (S)-NNN is more tumorigenic than (R)-NNN to the rat esophagus.

The formation of tobacco-specific nitrosamines from the major tobacco alkaloid nicotine was examined. Detached leaf tobacco was fed either [2'-14C]nicotine or [2'-14C]nornicotine and air cured. The cured leaf was then analyzed for [2'-14C]N'-nitrosonornicotine ([2'-14C]NNN). The yield of [2'-14C]NNN was 0.007% from nornicotine and 0.009% from nicotine. Because the ratio of nicotine to nornicotine in conventional nicotine-type tobacco is 20-100:1, nicotine is considered to be the major precursor for the carcinogen NNN in tobacco. The formation of other nitrosamines from nicotine in vitro was then studied. Reaction of nicotine with NaNO2 gave rise to NNN, as well as to two other nitrosamines, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(N-methyl-N-nitrosamino)-4-(3-pyridyl)butanal (NNA). Analysis of market products revealed the presence of NNK (0.6-24 microgram/g) in chewing tobacco and snuff. The tumorigenic activity of NNN, NNK, and NNA in strain A mice was studied. NNK induced more lung adenomas per mouse than did NNN, whereas NNA was less active than NNN. In addition, two cases of undifferentiated carcinoma of the salivary glands occurred in the NNN experimental groups.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are potent pulmonary carcinogens in rats. NNK and NNAL require metabolic activation to express their carcinogenicity. Cytochrome P450-catalyzed alpha-hydroxylation at the methyl position of NNK or NNAL generates reactive intermediates, which alkylate DNA to form pyridyloxobutyl (POB)-DNA adducts or pyridylhydroxybutyl (PHB)-DNA adducts. NNK is metabolized to NNAL in a reversible and stereoselective manner, and the tissue-specific retention of (S)-NNAL is believed to be important to the carcinogenicity of NNK. In the present study, we investigated the formation of POB- and PHB-DNA adducts in extrahepatic tissues of F344 rats treated chronically with NNK and (R)- and (S)-NNAL (10 ppm in the drinking water, 1-20 weeks). POB- and PHB-DNA adducts were quantified in nasal olfactory mucosa, nasal respiratory mucosa, oral mucosa, and pancreas of treated rats. Adduct formation in the nasal respiratory mucosa exceeded that in the other tissues. O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd) or O(2)-[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine (O(2)-PHB-dThd) was the major adduct, followed by 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua) or 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]guanine (7-PHB-Gua). There was a remarkable similarity in adduct formation between the NNK and the (S)-NNAL groups, both of which were distinctively different from that in the (R)-NNAL group. For example, in the nasal olfactory mucosa, POB-DNA adduct levels in the NNK and (S)-NNAL groups were not significantly different from each other, while (R)-NNAL treatment generated 6-33 times lower amounts of POB-DNA adducts than did NNK treatment. In contrast, (R)-NNAL treatment produced significantly higher levels of PHB-DNA adducts than did NNK or (S)-NNAL treatment. Similar trends were observed in the nasal respiratory mucosa, oral mucosa, and pancreas. These results suggest extensive retention of (S)-NNAL in various tissues of NNK-treated rats and support a mechanism in which the preferential metabolism of NNK to (S)-NNAL, followed by sequestration of (S)-NNAL in the target tissues and reoxidation to NNK, is important to NNK tumorigenesis.

Metabolites of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a potent pulmonary carcinogen, have been quantified in the urine of 11 smokers. They were not detected in nonsmokers' urine. The metabolites, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide, were detected in quantities of 0.23-1.0 and 0.57-6.5 micrograms/24 h, respectively. The results of this study provide the first evidence for metabolites of tobacco-specific nitrosamines in human urine.

N'-Nitrosonornicotine (NNN) is one of the most important strong carcinogens in tobacco products and is believed to play a significant role in the induction of esophageal cancer in smokers and oral cavity cancer in snuff dippers. NNN is metabolically activated through cytochrome P450-catalyzed alpha-hydroxylation. 2'-Hydroxylation produces a reactive intermediate 4-(3-pyridyl)-4-oxobutanediazohydroxide (7), which alkylates DNA to form pyridyloxobutyl (POB)-DNA adducts. DNA pyridyloxobutylation from NNN treatment, as measured by released 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 8), has been observed in vitro and in vivo. In the present study, we have used liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to analyze specific POB-DNA adducts in the nasal olfactory, nasal respiratory, and oral mucosa of F344 rats treated chronically with (R)-NNN or (S)-NNN in the drinking water (10 ppm, 1-20 weeks). Adduct levels in the nasal respiratory mucosa exceeded those in the nasal olfactory and oral mucosa. (R)-NNN treatment generated 2-4 times more adducts in the nasal olfactory and respiratory mucosa than did (S)-NNN at most time points. O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd, 11) predominated in the nasal olfactory and respiratory mucosa, followed by 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 14). Levels of O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O(2)-POB-Cyt, 13) and O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O(6)-POB-dGuo, 12) were significantly lower. In the oral mucosa, the opposite stereoselectivity was observed, with (S)-NNN treatment producing 3-5 times more POB-DNA adducts than did (R)-NNN. O(2)-POB-dThd and 7-POB-dGuo were the two major adducts, and their levels were similar. Overall, POB-DNA adduct formation in the nasal olfactory and nasal respiratory mucosa was similar to that previously observed in the lung, whereas in the oral mucosa, the trend resembled that in the esophagus. These results indicate that different mechanisms are involved in NNN metabolism and tumorigenesis in rat nasal and oral tissues. NNN enters the nasal mucosa through the circulation, and tissue-specific metabolism is important, while in the oral mucosa, direct exposure and local activation both play significant roles. Our results also support the potential importance of NNN as an oral carcinogen in people who use smokeless tobacco products.