Mechanisms Of Tumour Induction

The mechanisms by which tobacco causes cancer can best be illustrated by considering the relationship between cigarette smoking and lung cancer, because it is here that the most information is available. The overall framework for discussing this information is illustrated in Figure 4 (Hecht, 1999). Carcinogens form the link between nicotine addiction and cancer. Nicotine addiction is the reason why people continue to smoke. While nicotine itself is not considered to be carcinogenic, each cigarette contains a mixture of carcinogens, including a small dose of PAHs and NNK among other lung carcinogens, tumour promoters and co-carcinogens (Hecht, 1999). Carcinogens such as NNK and PAHs require metabolic activation, that is, they must be enzymatically transformed by the host into reactive intermediates, in order to exert their carcinogenic effects. There are competing detoxification pathways which result in harmless excretion of the carcinogen. The balance between metabolic activation and detoxification differs among individuals and will affect cancer risk.

We know a great deal about mechanisms of carcinogen metabolic activation and detoxification (Hecht, 1999). The metabolic activation process leads to the formation of

Excretion Normal DNA Apoptosis

Figure 4 Scheme linking nicotine addiction and lung cancer via tobacco smoke carcinogens and their induction of multiple mutations in critical genes. PAHs = polycyclic aromatic hydrocarbons; NNK = 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone.

Excretion Normal DNA Apoptosis

Figure 4 Scheme linking nicotine addiction and lung cancer via tobacco smoke carcinogens and their induction of multiple mutations in critical genes. PAHs = polycyclic aromatic hydrocarbons; NNK = 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone.

DNA adducts, which are carcinogen metabolites bound covalently to DNA, usually at guanine or adenine. There have been major advances in our understanding of DNA adduct structure and its consequences in the past two decades and we now have a large amount of mechanistic information (Hecht, 1999). If DNA adducts escape cellular repair mechanisms and persist, they may lead to miscoding, resulting in a permanent mutation. This occurs when DNA polymerase enzymes read an adducted DNA base incorrectly, resulting in the insertion of the wrong base, or other errors. As a result of clever strategies that combine DNA adduct chemistry with the tools of molecular biology, we know a great deal about the ways in which carcinogen DNA adducts cause mutations. Cells with damaged DNA may be removed by apoptosis, or programmed cell death (Sekido et al., 1998). If a permanent mutation occurs in a critical region of an oncogene or tumour-suppressor gene, it can lead to activation of the oncogene or deactivation of the tumour-suppressor gene. Oncogenes and tumour-suppressor genes play critical roles in the normal regulation of cellular growth. Changes in multiple tumour-suppressor genes or oncogenes lead to aberrant cells with loss of normal growth control and ultimately to lung cancer. Although the sequence of events has not been well defined, there can be little doubt that these molecular changes are important (Sekido et al., 1998). There is now a large amount of data on mutations in the human K-ras oncogene and p53 tumour-suppressor gene in lung tumours from smokers (Hecht, 1999).

Blocking any of the horizontal steps in Figure 4 may lead to decreased lung cancer, even in people who continue to smoke. In the following discussion, some of these steps will be considered in more detail.

Upon inhalation, cigarette smoke carcinogens are enyzmatically 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 (CYP) enzymes which oxygenate the substrate (Guengerich and Shimada, 1998). These enzymes typically are responsible for metabolism of drugs, other foreign compounds and some endogenous substrates. Other enzymes such as lipoxygenases, cyclooxygenases, myeloperoxidase and monoamine oxidases may also be involved, but less frequently. The oxygenated intermediates formed in these initial reactions may undergo further transformations by glutathione-^-transferases, uridine-5'-diphosphate glucuronosyltrans-ferases, sulfatases and other enzymes which are typically involved in detoxification (Hecht, 1999). Some of the metabolites produced by the CYPs react with DNA or other macromolecules to form covalent binding products known as adducts. This is referred to as metabolic activation (see Figure 4). Metabolic pathways of BaP and NNK, representative pulmonary carcinogens in cigarette smoke, have been extensively defined through studies in rodent and human tissues. The major metabolic activation pathway of BaP is conversion to a reactive diol epoxide metabolite called BPDE; one of the four isomers produced is highly carcinogenic and reacts with DNA to form adducts with the ^2-atom of deoxyguanosine (Hecht, 1999). The major metabolic activation pathways of NNK and its main metabolite, 4-(methylnitrosamino)-1-(3-pyr-idyl)-1-butanol (NNAL), occur by hydroxylation of the carbons adjacent to the ^-nitroso group (a-hydroxylation), which leads to the formation of two types of DNA adducts: methyl adducts such as 7-methylguanine or O6-methyl-guanine, and pyridyloxobutyl adducts (Hecht, 1998a, 1999).

Considerable information is available on pulmonary carcinogen metabolism in vitro, in both animal and human tissues, but fewer studies have been carried out on uptake, metabolism and adduct formation of cigarette smoke lung carcinogens in smokers (Hecht, 1999). Various measures of cigarette smoke uptake in humans have been used, including exhaled carbon monoxide, carboxyhaemoglobin, thiocyanate and urinary mutagenicity. However, the most specific and widely used biochemical marker is the nicotine metabolite cotinine (IARC, 1986; Hecht, 1999). While continine and other nicotine metabolites are excellent indicators of tobacco smoke constituent uptake by smokers, the NNK metabolites NNAL and its O-glucuronide (NNAL-Gluc) are excellent biomarkers of tobacco smoke lung carcinogen uptake (Hecht, 1999). NNAL is a potent pulmonary carcinogen like NNK, whereas NNAL-Gluc is a detoxified metabolite of NNK (Hecht, 1998a, 1999). Since NNK is a tobacco-specific carcinogen, its metabolites NNAL and NNAL-Gluc are found only in the urine of individuals exposed to tobacco products. Urinary NNAL and NNAL-Gluc have been quantified in several studies of smokers and in nonsmokers exposed to environmental tobacco smoke. The latter results demonstrate that uptake of NNAL-Gluc is 1-3% of that in smokers, consistent with the weaker epidemiological evidence for a role of environmental tobacco smoke, compared with mainstream cigarette smoke, as a cause of lung cancer (Hecht, 1999).

BaP has been detected in human lung; no differences between smokers and nonsmokers were noted (Hecht, 1999). 1-Hydroxypyrene and its glucuronide, urinary metabolites of the noncarcinogen pyrene, have been widely used as indicators of PAH uptake. 1-Hydroxy-pyrene levels in smokers are generally higher than in nonsmokers (Hecht, 1999). Overall, there is considerable evidence that pulmonary carcinogens in cigarette smoke are taken up and metabolized by smokers as well as by nonsmokers exposed to environmental tobacco smoke.

Less than 20% of smokers will get lung cancer (IARC, 1986). Susceptibility will depend in part on the balance between carcinogen metabolic activation and detoxification in smokers. This is an important area requiring further study. Most investigations have focused on the metabolic activation pathways by quantifying DNA or protein adducts. There are considerable data demonstrating activation of BaP to DNA adducts in the lungs of smokers. Earlier investigations demonstrated that cigarette smoke induces aryl hydrocarbon hydroxylase (AHH) activity and proposed a relationship between AHH activity and lung cancer (IARC, 1986; Hecht, 1999). AHH metabolizes BaP to 3-hydroxyBaP, and is equivalent to CYP 1A1. Cigarette smoking induces expression of this enzyme. Cancer patients who stopped smoking within 30 days of surgery had elevated levels of AHH activity compared with nonsmoking cancer patients (Hecht, 1999). Lung tissue from recent smokers with elevated AHH activity also metabo-lically activated BaP to a greater extent than lung tissue from nonsmokers or ex-smokers. DNA adduct levels correlated with AHH activity in the same samples. Collectively, these results support the existence of a cigarette smoke-inducible pathway leading to BaP--DNA adducts in smokers' lungs, as illustrated in Figure 4.

A large number of studies have used immunoassays and 32P postlabelling, which are sensitive but relatively nonspecific techniques, to estimate levels of 'PAH-DNA adducts' or 'hydrophobic DNA adducts' in white blood cells and other human tissues including lung (Hecht, 1999). Many of these have shown elevated adduct levels in smokers. However, none of the studies using immuno-assays and 32P postlabelling has identified the structures of the compounds leading to DNA adduct formation. Probably some are PAHs, but individual PAH--DNA adducts have not been characterized in these studies.

Several studies have detected 7-methylguanine in human lung (Hecht, 1999). Levels were higher in smokers than in nonsmokers in two studies, suggesting that NNK may be one source of these adducts. While 7-methylgua-nine is not generally considered as an adduct that would lead to miscoding in DNA and the introduction of a permanent mutation, other methyl adducts which do have miscoding properties such as 06-methylguanine are formed at the same time, but at lower levels. Pyridyloxo-butylated DNA also has been detected in lung tissue from smokers in one study, reflecting metabolic activation of NNK or NNN. The detection of methyl and pyridyloxo-butyl adducts in DNA from smokers' lungs is consistent with the ability of human lung tissue metabolically to activate NNK, but the quantitative aspects of the relationship of metabolism to DNA adduct levels are unclear (Hecht, 1998a, 1999).

DNA repair processes are important in determining whether DNA adducts persist. Because smoking is a chronic habit, one would expect a steady-state DNA adduct level to be achieved by the opposing effects of damage and repair. There are three mechanisms of DNA repair: direct repair, base excision repair and nucleotide excision repair. With respect to smoking and lung cancer, direct repair of 06-methylguanine by 06-methylguanine-DNA alkyltransferase and nucleotide excision repair of PAH--DNA adducts would appear to be the most relevant processes (Hecht, 1999).

As indicated in Figure 4, the direct interaction of metabolically activated carcinogens with critical genes such as the p53 tumour-suppressor gene and the K-ras oncogene is central to the hypothesis that specific carcinogens form the link between nicotine addiction and lung cancer (Hecht, 1999). The p53 gene plays a central role in the delicate balance of cellular proliferation and death. It is mutated in about half of all cancer types, including over 50% of lung cancers, leading to loss of its activity for cellular regulation. Point mutations at guanine (G) are common. In a sample of 550 p53 mutations in lung tumours, 33% were G ^ T transversions, and 26% were G ^ A transitions. (A purine ^ pyrimidine or pyrimidine ^ purine mutation is referred to as a transversion, and a purine ^ purine or pyrimidine ^ pyrimidine mutation is called a transition.) A positive relationship between lifetime cigarette consumption and the frequency ofp53 mutations and of G ^ T transversions on the nontranscribed DNA strand has also been noted. These observations are generally consistent with the fact that most activated carcinogens react predominantly at G, and that repair of the resulting adducts would be slower on the nontranscribed strand, and thus support the hypothesis outlined in Figure 4.

Mutations in codon 12 of the K-ras oncogene are found in 24-50% of human primary adenocarcinomas but are rarely seen in other lung tumor types (Hecht, 1999). When K-ras is mutated, a complex series of cellular growth signals are initiated. Mutations in K-ras are more common in smokers and ex-smokers than in nonsmokers, which suggests that they may be induced by direct reaction with the gene of an activated tobacco smoke carcinogen. The most commonly observed mutation is GGT ^ TGT, which typically accounts for about 60% of the codon 12 mutations, followed by GGT ^ GAT (20%), and GGT ^ GTT (15%).

The pl6INK4a tumour-suppressor gene is inactivated in more than 70% of human non-small cell lung cancers, via homozygous deletion or in association with aberrant hypermethylation of the promoter region (Hecht, 1999). In the rat, 94% of adenocarcinomas induced by NNK were hypermethylated at the pl6 gene promoter. This change was frequently detected in hyperplastic lesions and adenomas which are precursors to the adenocarcinomas induced by NNK. Similar results were found in human squamous cell carcinomas of the lung. The pl6 gene was coordinately methylated in 75% of carcinoma in situ lesions adjacent to squamous cell carcinomas which had this change. Methylation ofpl6 was associated with loss of expression in tumours and precursor lesions indicating functional inactivation of both alleles. Aberrant methyl-ation of pl6 has been suggested as an early marker for lung cancer. The expression of cell cycle proteins is related to the pl6 and retinoblastoma tumour-suppressor genes; NNK-induced mouse lung tumours appear to resemble human non-small-cell lung cancer in the expression of cell cycle proteins. The oestrogen receptor gene is also inactivated through promoter methylation. There was concordance between the incidence of promoter methylation in this gene in lung tumours from smokers and from NNK-treated rodents.

Loss of heterozygosity and exon deletions within the fragile histidine triad (FHIT) gene are associated with smoking habits in lung cancer patients and have been proposed as a target for tobacco smoke carcinogens (Hecht, 1999). However, point mutations within the coding region of the FHIT gene were not found in primary lung tumours.

Collectively, the evidence favouring the sequence of steps illustrated in Figure 4 as an overall mechanism of tobacco-induced cancer is extremely strong, although there are important aspects of each step that require further study. These include carcinogen metabolism and DNA binding in human lung, the effects of cigarette smoke on DNA repair and adduct persistence, the relationship between specific carcinogens and mutations in critical genes and the sequence of gene changes leading to lung cancer.

Using a weight-of-the-evidence approach, specific PAHs and the tobacco-specific nitrosamine NNK can be identified as probable causes of lung cancer in smokers, but the contribution of other agents cannot be excluded (Table 9). The chronic exposure of smokers to the DNA-damaging intermediates formed from these carcinogens is consistent with our present understanding of cancer induction as a process which requires multiple genetic changes. Thus, it is completely plausible that the continual barrage of DNA damage produced by tobacco smoke carcinogens causes the multiple genetic changes that are associated with lung cancer. While each dose of carcinogen from a cigarette is extremely small, the cumulative damage produced in years of smoking will be substantial.

Aspects of the scheme illustrated in Figure 4 are well understood for PAHs and NNK. A great deal is known about the metabolic activation and detoxification of these compounds. There is a good general understanding of the mechanisms by which they interact with DNA to form adducts and considerable information is available about the repair, persistence and miscoding properties of these adducts. There are many aspects of these processes that require further study, however. In particular, little is known about the levels, persistence, and repair of specific carcinogen DNA adducts in the lungs of smokers or the effects of chronic smoking on these factors. The location of carcinogen adducts at specific sites in human DNA has not been studied, mainly owing to limitations on sensitivity. Nevertheless, one can reasonably conclude that metaboli-cally activated tobacco smoke carcinogens directly cause mutations observed in tumour-suppressor genes and onco-genes, although details remain elusive since numerous DNA-damaging agents in tobacco smoke cause similar mutations. (See also chapter The Formation of DNA Adducts.)

The Smoker's Sanctuary

The Smoker's Sanctuary

Save Your Lungs And Never Have To Spend A Single Cent Of Ciggies Ever Again. According to a recent report from the U.S. government. Centers for Disease Control and Prevention, more than twenty percent of male and female adults in the U.S. smoke cigarettes, while more than eighty percent of them light up a cigarette daily.

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