Other Data Relevant to an Evaluation of Carcinogenicity and Its Mechanisms
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pp1005-1080-Section 4.qxd 30/04/2004 11:03 Page 1005 4. Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms 4.1 Absorption, distribution, metabolism and excretion 4.1.1 Humans The Working Group attempted to provide extensive coverage of the published lite- rature since 1985, in some cases referring to recent reviews. (a) Introduction Most carcinogens are enzymatically transformed to a series of metabolites as the exposed organism attempts to convert them to forms that are more readily excreted. The initial steps are usually carried out by cytochrome P450 (P450) enzymes that oxygenate the substrate (Guengerich, 1997). Other enzymes such as lipoxygenases, cyclooxyge- nases, myeloperoxidase and monoamine oxidases may also be involved, but less com- monly. If the oxygenated intermediates formed in these initial reactions are electrophilic, they may react with DNA or other macromolecules to form covalent binding products known as adducts. This process is called metabolic activation. Alternatively, these meta- bolites may undergo further transformations catalysed by glutathione S-transferases, uri- dine-5′-diphosphate (UDP)-glucuronosyltransferases, epoxide hydrolase (EH), N-acetyl- transferases (NATs) (Kadlubar & Beland, 1985), sulfotransferases and other enzymes (Armstrong, 1997; Burchell et al., 1997; Duffel, 1997). Such reactions frequently, but not always, result in detoxification. Figure 4.1 presents an overview of the metabolism of the six tobacco smoke carci- nogens for which the formation of DNA adducts has been demonstrated in human tissues, namely, benzo[a]pyrene (IARC, 1983a, 1987), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1- butanone (NNK) (IARC, 1985a; Hecht et al., 1994), N-nitrosodimethylamine (NDMA) (IARC, 1978a; Shuker & Bartsch, 1994), N′-nitrosonornicotine (NNN) (IARC, 1985b; Hecht et al., 1994), ethylene oxide (IARC, 1994a), and 4-aminobiphenyl (4-ABP) (IARC, 1972; Kadlubar, 1994). The major metabolic activation pathway of benzo[a]pyrene is con- version to a 7,8-diol-9,10-epoxide, which is highly carcinogenic and reacts with DNA to form adducts with the exocyclic N2 of guanine (Cooper et al., 1983). In competition with this process are detoxification pathways leading to phenols, diols and their conju- –1005– pp1005-1080-Section 4.qxd 30/04/2004 11:03 Page 1006 1006 IARC MONOGRAPHS VOLUME 83 Figure 4.1. Metabolism of six tobacco smoke carcinogens which produce DNA adducts that have been identified in the lungs of smokers 12 1 11 2 10 NO 3 BaP P450s 9 O epoxides N NNAL NNAL- 8 4 Gluc EH 7 6 5 CH3 P450s NIH N NNAL-N- UGTs sulfates NNK glucuronides diols shift P450s oxide GSTs NNK-N-oxide α P450s -hydroxyNNALs P450s ADP sulfates phenols quinones adducts GSTs diol glutathione UGTs α-hydroxyNNKs epoxides conjugates tetraols phenol glucuronides epoxides diazonium ions + aldehydes CH3 CH3 N −hydroxy NDMA DNA Adducts P450s OH N O diazonium ions P450s NDMA NHAC NAc + CH2 O + aldehydes NO NATs 2 CH3NH2 NATs CH O P450s diazonium ions 2 NH2 NHOH NHOAc NATs 4-ABP α-hydroxyNNNs phenols P450s norcotinine NHOX −hydroxyNNNs N NNN-N-oxide mercapturic CH2 CH2 acids NO GSTs O NNN ethylene oxide ethylene CO2 glycol Clockwise from top left: benzo[a]pyrene (BaP), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosodimethylamine (NDMA), N′-nitrosonornicotine (NNN), ethylene oxide and 4-aminobiphenyl (4-ABP) P450s, cytochrome P450s; EH, epoxide hydrolase; UGTs, uridine-5′-diphosphate-glucuronosyl transferases; GSTs, glutathione S-transferases; NNAL, 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol; NATs, N-acetyl- transferases; gluc, glucuronide; ‘NIH shift’, phenomenon of hydroxylation-induced intramolecular migration; ADP, adenosine diphosphate; Ac, acetyl In the 4-ABP scheme, X represents conjugates such as glucuronide or sulfate. Adapted from Cooper et al. (1983); Preussmann & Stewart (1984); Kadlubar & Beland (1985); IARC (1994a); Hecht (1998, 1999) gates as well as other metabolites. The major metabolic activation pathways of NNK and its main metabolite, 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL), involve hydroxylation of the carbons adjacent to the N-nitroso group (α-hydroxylation) which leads, via diazonium ions, to the formation of two types of DNA adduct: methyl adducts such as 7-methylguanine and O6-methylguanine (IARC, 1985a), and pyridyloxobutyl adducts (Hecht, 1998). Glucuronidation of NNAL and pyridine-N-oxidation of NNK and NNAL are detoxification pathways. The metabolic activation of NDMA occurs by α- hydroxylation leading, via methyl diazonium ions, to the formation of 7-methylguanine pp1005-1080-Section 4.qxd 30/04/2004 11:03 Page 1007 TOBACCO SMOKE 1007 and O6-methylguanine. Denitrosation, producing nitrite and methylamine, is considered to be a detoxification pathway (Preussmann & Stewart, 1984). Aldehydes are also formed in the metabolism of NNK and NDMA. Their role in carcinogenesis is unclear. α-Hydroxy- lation of NNN can lead to the formation of pyridyloxobutyl adducts whereas detoxification occurs by β-hydroxylation, pyridine-N-oxidation and denitrosation/oxidation to produce norcotinine (Hecht, 1998). Ethylene oxide reacts directly with DNA to form 7-(2-hydroxyethyl)guanine and other adducts. There are competing detoxification path- ways involving glutathione conjugation (IARC, 1994b). 4-ABP is metabolically activated by N-hydroxylation. Conjugation of the resulting hydroxylamine with acetate or other groups such as sulfate ultimately produces nitrenium ions that react with DNA to produce adducts mainly at C-8 of guanine. Acetylation of 4-ABP can be a detoxification pathway if it is not followed by N-hydroxylation. Ring hydroxylation and conjugation of the phenols result in detoxification (Kadlubar & Beland, 1985). The balance between metabolic activation and detoxification varies between indivi- duals exposed to these genotoxic components of tobacco smoke and is likely to affect cancer risk because DNA adducts are absolutely central to the carcinogenic process induced by these agents (Hecht, 1999; Tang et al., 2001). DNA adducts, if unrepaired, can cause miscoding during replication resulting in permanent mutation. Cells have DNA repair systems that can remove adducts and restore the DNA to its normal structure (Memisoglu & Samson, 2000; Pegg, 2000; Hanawalt, 2001; Norbury & Hickson, 2001). There are interindividual differences in their capacity for DNA repair that can affect cancer risk (Wei et al., 2000). Moreover, DNA repair systems are not com- pletely efficient or error-free, and some adducts escape repair and persist in DNA. These persistent DNA adducts can cause miscoding. For example, when the O6-position of guanine in DNA is methylated following metabolic activation of NNK, the resulting DNA adduct, O6-methylguanine, is misread by DNA polymerases as adenine, and thymine is inserted during replication (Loechler et al., 1984). The consequence is the permanent con- version of a G:C base pair to an A:T base pair. This mutation and others can activate onco- genes such as KRAS or inactivate tumour-suppressor genes such as TP53. There are consi- derable data to indicate that mutations in KRAS and TP53 result directly from the reaction of these genes with metabolically activated carcinogens (Hecht, 1999). Many genetic ab- normalities occur during the process of lung cancer induction. These include loss of heterozygosity, microsatellite alterations, mutations in RAS oncogenes, MYC amplifi- cation, BCL-2 expression, mutations in the TP53, RB, CDKN2A and FHIT tumour-sup- pressor genes, expression of telomerase activity and others (Figure 4.2) (Wistuba et al., 1997; Sekido et al., 1998). Although the temporal sequence of mutations is somewhat unclear, we do know that carcinogens can, through the process just described, cause irre- versible damage to critical genes involved in the control of cellular growth. Smokers are subjected to a chronic barrage of metabolically activated carcinogens that cause these multiple changes (Figure 4.2). This constant assault on genes is entirely consistent with genetic derangements that lead to six proposed hallmarks of cancer: — self-sufficiency in growth signals; pp1005-1080-Section 4.qxd 30/04/2004 11:03 Page 1008 1008 IARC MONOGRAPHS VOLUME 83 — insensitivity to anti-growth signals; — evasion of apoptosis; — tissue invasion and metastasis; — sustained angiogenesis; and — limitless replicative potential (Hanahan & Weinberg, 2000). Figure 4.2. Scheme linking cigarette smoke carcinogens with genetic changes in lung and lung cancer development Adapted from Hecht (2002a) This scheme may also apply to other cancers. A key aspect is the chronic exposure of DNA to metabolically activated carcinogens resulting in the formation of DNA adducts and consequent genetic changes. This chronic barrage of DNA damage, taking place daily over a period of many years, is fully consistent with multiple genetic changes in lung cancer (it does not always take 30 years to get lung cancer; it may take only five years). Some cigarette smoke carcinogens may operate through other mechanisms. The time periods and sequence of genetic changes are uncertain. For further details see Sekido et al. (1998); Hecht (1999, 2002a). pp1005-1080-Section 4.qxd 30/04/2004 11:03 Page 1009 TOBACCO SMOKE 1009 Urinary carcinogen metabolites, carcinogen-protein adducts, and carcinogen-DNA adducts have been used as biomarkers to assess the uptake, metabolic activation and detoxification of tobacco carcinogens in humans. These are discussed in the following sections. (b) Effects