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part 1. PART 1

concordance between cancer in humans and in experimental CHAPTER 5 animals

chapter 5. and its constituents Stephen S. Hecht and David M. DeMarini

This chapter addresses the con- Coherence: carcinogenicity the induction of lung tumours, both cordance in studies of - of tobacco smoke in humans benign and malignant, by icity and mechanisms between versus experimental animals smoke. experimental animals and humans Although these studies have Volume 100E and previous IARC established animal models for the for tobacco smoke and its constit- Monographs and certain reviews on study of tobacco smoke carcino- uents. Volume 100E of the IARC tobacco smoke have summarized genesis and, in aggregate, support Monographs updated the literature the literature on animal studies of the the epidemiological findings that on tobacco smoke and the evalua- carcinogenicity of tobacco smoke is a cause of cancers of the tions of its carcinogenicity (IARC, (IARC, 1986, 2004, 2012b; Hecht, lung and larynx in humans, specif- 2012b). It concluded that there was 2005). These studies demonstrated ic problems associated with animal sufficient evidence that tobacco that cigarette smoke can induce tu- studies of tobacco smoke expo- smoking causes multiple types of mours of the lung and nasal cavity in sure have been recognized. Many cancer in humans, including (to var- mice and rats and tumours of the lar- of these issues result from the fact ying extents) cancers of the lung, ynx in hamsters. Some recent stud- that most laboratory animals are ob- oral cavity, pharynx, oesophagus, ies not included in the evaluations ligate nose breathers and, thus, do in Volume 100E have consistently stomach, colorectum, liver, pancre- not inhale tobacco smoke voluntari- established the carcinogenicity of ly and habitually in the same way in as, nasal cavity and paranasal si- cigarette smoke to the lung in the which humans smoke tobacco prod- nuses, larynx, uterine cervix, ovary A/J mouse, where it produces ade- ucts (Wynder and Hoffman, 1967). (mucinous), urinary bladder, kidney, noma and adenocarcinoma, as well Constant whole-body exposure of ureter, and bone marrow (myeloid as causing emphysema (Stinn et al., rodents to cigarette smoke, often at leukaemia). 2010, 2013). The A/J mouse, which relatively high concentrations, pro- is highly susceptible to lung tumour duces avoidance reactions, stress, development, appears to present weight loss, and other indicators of a relatively reproducible system for toxicity that are widely different from

Part 1 • Chapter 5. Tobacco smoke and its constituents 47 the human responses to voluntary imals (IARC, 2010). Similarly, NNK is in urine, collected years before di- inhalation driven by the desire for re- a powerful lung carcinogen, inducing agnosis, and oesophageal cancer, curring small doses of . adenocarcinoma of the lung in rats, but not , in smokers, after There are some mechanistic dif- mice, and hamsters independent of correction for duration and intensity ferences as well. For example, muta- the route of administration and fre- of smoking (Yuan et al., 2011). This tions in the KRAS gene are frequent- quently at very low doses (Hecht, indicates considerable concordance ly observed in lung adenocarcinoma 1998). Thus, PAHs and NNK are between target tissues of NNN and in humans, as are K-ras mutations in widely considered to be causes of NNK in rats and observations of can- lung adenocarcinoma in mice; how- lung cancer in smokers. PAHs – cer incidence in smokers (Stepanov ever, the mutation frequency is not and their diol epoxide metabolites et al., 2014). increased and the mutation spec- in particular – produce mutations in Carcinogenicity studies in lab- trum is not altered in mice exposed TP53 and KRAS that are similar to oratory animals and studies in hu- to cigarette smoke (Hutt et al., 2005; those observed in lung tumours from mans exposed occupationally have DHHS, 2010; Stinn et al., 2013). smokers (DHHS, 2010). In recent established aromatic amines such Taken together, there is only moder- nested case–control studies within as 4-aminobiphenyl and 2-naphthyl­ ate concordance between the carci- prospective cohorts, biomarkers of amine as human bladder carcino- nogenic and mechanistic effects of PAH and NNK exposure were asso- gens (IARC, 1987, 2012a). These tobacco smoke evident in laboratory ciated with risk of lung cancer, after and other aromatic amines are com- animals and epidemiological obser- correction for duration and intensity ponents of mainstream cigarette vations in humans (Witschi, 2007). of smoking (Hecht et al., 2013). smoke (Xie et al., 2013). There is 1,3- is another com- also considerable mechanistic evi- Concordance: carcinogenicity pound likely to be involved in the dence from studies of haemoglobin of tobacco smoke in humans etiology of lung cancer in smokers. It adducts consistent with the propos- versus carcinogenicity of is found in relatively high concentra- al that 4-aminobiphenyl is respon- tobacco smoke constituents in experimental animals tions in tobacco smoke and is a pow- sible for bladder cancer in smokers erful lung carcinogen in mice, but not (Castelao et al., 2001; IARC, 2012a). There is considerable concordance in rats (IARC, 2008). is a leukaemogen in hu- between the known carcinogenic Other tobacco smoke compounds mans, and it occurs in considerable properties of many tobacco smoke with the lung as a target tissue/organ quantities in cigarette smoke (IARC, constituents and the multiple target in some animal studies include iso- 1987, 2012a). The uptake of benzene tissues of tobacco smoke as demon- prene, ethylene oxide, ethyl carba- by smokers has been demonstrated strated in epidemiological studies mate, benzene, and various metals conclusively by biomarker studies (IARC, 2012b). (Hecht, 2011). (Hecht et al., 2010). Thus, it is like- It should be noted that carcino- The oral cavity, pharynx, and ly that benzene is responsible for genicity assays of pure compounds oesophagus of rats are established leukaemia in smokers, although it generally do not suffer from the target tissues of the tobacco-specif- does not cause leukaemia in rodents above-mentioned operational dif- ic nitrosamine N′-nitrosonornicotine (IARC, 2012a). ficulties with respect to tobacco (NNN), and in particular its (S) enan- Multiple tobacco smoke carcino- smoke (Witschi, 2007). tiomer (Hecht, 1998; Balbo et al., gens have produced tumours of the With respect to lung cancer, 2013). NNN is found in the smoke of upper respiratory tract. Tumours of multiple polycyclic aromatic hy- all tobacco products (IARC, 2007). the larynx, nose, and trachea as well drocarbons (PAHs) and the tobac- N-nitrosodiethylamine is another as of the pharynx and oesophagus co-specific nitrosamine 4-(methylni- tobacco smoke constituent that in- were induced in inhalation studies trosamino)-1-(3-pyridyl)-1-butanone duces oesophageal tumours in rats, with benzo[a], an archetyp- (NNK) are found in the smoke of although its levels in smoke are con- al PAH, in hamsters (IARC, 2010). all tested to date (IARC, siderably lower than those of NNN. Tumours of the nose have also been 2004). There is abundant evidence One nested case–control study observed in rats treated with tobac- attesting to the ability of PAHs to found a strong relationship between co-specific nitrosamines (Hecht, induce lung cancer in laboratory an- levels of NNN and its glucuronides 1998; Balbo et al., 2013). Inhalation

48 studies with and ac- lead to the eventual development of and cause DNA replication errors etaldehyde produced nasal tumours lung cancer (IARC, 2004; DHHS, that may lead to mutations. If these (IARC, 1985, 2006, 2012a). 2010, 2014). This scheme is for lung mutations occur in important regions Tobacco smoke contains com- cancer because it is for this disease of critical growth control genes, such PART 1

pounds that are carcinogenic to that the most data are available. as the oncogene KRAS or the tu- CHAPTER 5 the colorectum in rats, most nota- The central pathway of Fig. 5.1 mour suppressor gene TP53, cellular bly certain heterocyclic aromatic in particular shows great coherence growth processes become severely amines (IARC, 2004, 2012b; Hecht, with established genotoxic mecha- dysregulated, resulting in uncon- 2012). With respect to induction nisms by which many , trolled cell proliferation and cancer. of liver cancer by tobacco smoke, including most of the more than 70 There are convincing data from there is coherence with furan and established carcinogens in cigarette studies of smokers and lung cancer N-nitrosodimethylamine, which are smoke, drive the process of cancer patients that illustrate coherence of liver carcinogens in rats (Peto et al., induction. Thus, exhaustive mecha- these observations in humans with 1991; NTP, 19 93), whereas NNK nistic studies carried out both in vitro the results observed in the plethora and its major metabolite 4-(methyl- and in laboratory animals since the of mechanistic studies noted above. nitrosamino)-1-(3-pyridyl)-1-butanol middle of the 20th century and con- Carcinogen uptake by smokers has (NNAL) induce pancreatic cancer in tinuing today provide solid evidence been unequivocally demonstrated rats (Hecht, 1998). that most carcinogens, either direct- by biomarker studies that compare Collectively, there is considerable ly or after metabolism catalysed by levels of carcinogens and their me- concordance between established multiple cytochrome P450 enzymes, tabolites in the urine of smokers sites of tobacco smoke carcinogen- react with nucleophilic sites in DNA and non-smokers (Hecht et al., icity in humans and target tissues to form covalent binding products 2010). These studies leave no doubt in experimental animals of individ- called DNA adducts. that exposure to multiple carcino- ual carcinogens present in tobacco There are cellular repair systems gens, including tobacco-specific ni- smoke. that have evolved to repair these trosamines, PAHs, aromatic amines, DNA adducts and restore the nor- and various volatile compounds in- Concordance: overall mechanism of cancer mal structure of DNA. These repair cluding benzene and 1,3-butadiene, induction in humans versus systems are crucial because cer- is significantly higher for smokers laboratory studies tain rare DNA repair-deficiency syn- than for non-smokers. dromes, such as xeroderma pigmen- These and related studies also Fig. 5.1 presents a widely accepted tosum, lead to a high susceptibility to show that virtually all of these car- mechanistic framework describing cancer development. Thus, DNA ad- cinogens are metabolized by cy- the events that occur in smokers and ducts, if left unrepaired, can persist tochrome P450 enzymes, resulting

Fig. 5.1. Mechanistic framework describing events involved in lung carcinogenesis in smokers. Adapted with permission from DHHS (2010).

Part 1 • Chapter 5. Tobacco smoke and its constituents 49 in the formation of highly reactive in 163 of the 188 tumours, including genes important in cancer. Overall, metabolites that react with DNA to 915 point mutations, 12 dinucleotide these results are coherent with the produce adducts. The induction of mutations, 29 insertions, and 57 de- induction of multiple mutations in the cytochrome P450 1A1 enzyme in letions. The analysis identified 26 critical growth control genes by met- the lungs of smokers via activation of genes mutated at significantly ele- abolically activated carcinogens in the aryl hydrocarbon receptor, result- vated frequencies, including TP53, cigarette smoke, although it should ing in the conversion of benzo[a]py­ KRAS, CDKN2A, and STK11, con- be recognized that other processes rene and related compounds to their sistent with other studies and with downstream of carcinogen exposure DNA-reactive forms, is a frequently the known involvement of these probably also contribute to the muta- observed and consistent finding in genes in growth control. Mutations tion load. the literature on the effects of ciga- were found most frequently in TP53 In aggregate, these studies pres- rette smoking (DHHS, 2010). and KRAS (Ding et al., 2008). ent a coherent mechanism based on Many studies have demonstrated Another study examined a small multiple studies, including chemical the presence of multiple DNA ad- cell lung cancer cell line. More than analyses, measurements of muta- ducts in the lungs of smokers, gen- 22 000 somatic substitutions were tion induction, and tests in labora- erally at higher levels than those in identified, among which were 134 in tory animals as well as biochemical non-smokers. Although there is still coding exons. G → T transversions and molecular biological evaluations room for further elaboration of the were the most common (34%), fol- of human tissues, blood, and urine. specific DNA adducts involved in lowed by G → A transitions (21%) The data are consistent and convinc- this process, there can be little doubt and A → G transitions (19%), sim- ing with respect to the central track about the higher levels of DNA dam- ilar to earlier data in many studies of Fig. 5.1. age in the lung tissue of smokers (Pleasance et al., 2010). compared with non-smokers (IARC, Another investigation focused It is clear that other processes 2004; Phillips and Venitt, 2012). on a non-small cell lung cancer are involved. Certain compounds in Consistent with these data are the from a patient aged 51 years who tobacco smoke, or their metabolites, common findings of mutagenicity in had smoked 25 cigarettes per day may interact directly with cellular re- urine of smokers and sister chroma- for 15 years before excision of the ceptors. This can lead to activation tid exchange in peripheral lympho- tumour, which was histologically of protein kinases, growth recep- cytes of smokers (IARC, 2004). characterized as an adenocarcino- tors, and other molecules that can Multiple recent studies with cur- ma. Single nucleotide variants were contribute to carcinogenesis (Chen rently available DNA sequencing common, mostly at GC base pairs, et al., 2011). It is well established that methods have demonstrated that frequently G → T transversions. tobacco smoke contains inflammato- DNA adducts in the lungs of smokers Approximately 17.7 mutations per ry substances, resulting in enhanced result in mutations. megabase were observed, for a total pneumocyte proliferation, activation of Greenman et al. (2007) stud- of more than 50 000 single nucleo- nuclear factor kappa-light-chain-en- ied mutations in the coding exons tide variants. At least eight genes hancer of activated B cells (NF-κB), of multiple protein kinase genes in in the EGFR-RAS-RAF-MEK-ERK and tumour promotion (Takahashi lung cancer and other cancers. Lung pathway were either mutated or am- et al., 2010). There are also co-car- cancers were among those with the plified (Lee et al., 2010). cinogens that undoubtedly con- most somatic mutations (4.21 per These results are fully consis- tribute to the overall mechanism megabase); the authors attributed tent with those reported earlier of tobacco smoke carcinogenesis. this to frequent exposure to exoge- (DHHS, 2010) and with data in the nous mutagens (Greenman et al., Catalogue of Somatic Mutations in Furthermore, cigarette smoke induc- 2007). Cancer (COSMIC) database (http:// es oxidative damage, altered gene In another study, 188 primary lung www.sanger.ac.uk/genetics/CGP/ promoter methylation, dysregulation adenocarcinomas were sequenced. cosmic/) and the IARC TP53 data- of gene expression by microRNAs, Analysis of 247 megabases of tu- base (http://www-p53.iarc.fr/), which and chronic cell injury and cytotoxic- mour DNA sequence identified 1013 store and display somatic mutations ity with regenerative proliferation as non-synonymous somatic mutations in TP53 and KRAS as well as other an amplifying factor, all of which can

50 contribute to the overall carcinogen- Disclaimer the contents reflect the views of the ic effect (IARC, 2012b; Milara and agency, nor does mention of trade We thank J.A. Ross and C.E. Wood for Cortijo, 2012; Momi et al., 2014). names or commercial products con- their helpful comments on this manu- In summary, cigarette smoking stitute endorsement or recommen- script. This article was reviewed by the PART 1 represents a potent combination of dation for use. CHAPTER 5 National Health and Environmental biological effects associated with Effects Research Laboratory, United carcinogenesis, coherent with land- States Environmental Protection mark publications dating back more Agency and approved for publica- than 60 years (Hecht and Szabo, tion. Approval does not signify that 2014).

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