Advanced Drug Delivery Reviews 54 (2002) 1245–1256 www.elsevier.com / locate / drugdeliv
CYP2A6 genetic variation and potential consequences Chun Xu, Shari Goodz, Edward M. Sellers, Rachel F. Tyndale* Centre for Addiction and Mental Health and Department of Pharmacology, University of Toronto, Toronto M5 S 1 A8, Canada
Abstract Human cytochrome P450 2A6 (CYP2A6) has been shown to have large interindividual and interethnic variability in levels of expression and activity. This is thought to be largely due to genetic polymorphisms. In recent years, 13 genetic variants (CYP2 A6*1 – *11 and the gene duplication, *1 3 2 ) of CYP2 A6 have been identified and a number of these have been shown to result in altered CYP2A6 enzyme activity. For example, there are alleles which result in variants that are in inactive (e.g. due to a gene deletion), have decreased activity (e.g. altered enzyme structure or transcriptional activity) or have increased activity (e.g. due to gene duplications). The resulting interindividual variation in metabolic activity may affect the metabolism of CYP2A6 substrates including nicotine, cotinine (the major metabolite of nicotine), several tobaccospecific procarcinogens, coumarin and many toxins. The frequencies of the CYP2 A6 alleles vary considerably among different ethnic populations, which may partially explain the interethnic variability found in CYP2A6-related metabolic activity (e.g. nicotine metabolism), behaviors (i.e. smoking) and disease (i.e. lung cancer). Investigations of the genetic variation of CYP2 A6 and its resulting effects on metabolism and health consequences are still fairly early; this review summarizes what is presently known about CYP2A6, its genetic variants and their clinical consequences. 2002 Elsevier Science B.V. All rights reserved. Keywords: Pharmacogenetics; Nicotine; Drug metabolism; Smoking; Cancer; Coumarin
Contents 1. Introduction ............................................................................................................................................................................ 2. The cytochrome P450 superfamily............................................................................................................................................ 3. CYP2A6 substrates, inhibitors and inducers .............................................................................................................................. 3.1. Substrates ........................................................................................................................................................................ 3.2. Inhibitors and inducers ..................................................................................................................................................... 4. Structural organization of the CYP2A6 gene.............................................................................................................................. 5. CYP2A6 and pharmacogenetics ............................................................................................................................................... 5.1. Interindividual variability in CYP2A6 activity.................................................................................................................... 5.2. Known CYP2A6 allelic variants........................................................................................................................................ 5.3. CYP2A6 allele frequencies ............................................................................................................................................... 6. CYP2A6 genetic variation and potential consequences ............................................................................................................... 6.1. CYP2A6 and the metabolism of pharmaceuticals / toxins ..................................................................................................... 6.2. CYP2A6 and nicotine metabolism ..................................................................................................................................... *Corresponding author. Tel.: 1 1-416-978-6374; fax: 1 1-416-978-6395. E-mail address:
[email protected] (R.F. Tyndale). 0169-409X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 02 )00065-0
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6.3. CYP2A6 and smoking ...................................................................................................................................................... 6.4. CYP2A6 and cancer ......................................................................................................................................................... 6.5. Nicotine treatments .......................................................................................................................................................... 7. Concluding remarks and future directions ................................................................................................................................. References ..................................................................................................................................................................................
1. Introduction Cytochromes P450 (CYPs) are a superfamily of enzymes involved in the metabolism of endogenous and exogenous compounds. A great deal of interindividual and interethnic variability in the activity and levels of these enzymes exist which may lead to differences in the effects and toxicities of many drugs (clinical and recreational) and environmental compounds (reviewed in Ref. [1]). The causes of this variability include both genetic and environmental factors. It has been estimated that | 20–40% of the interindividual variability in drug metabolism and drug response can be attributed to the existence of polymorphisms in CYP [1]. Many allelic variants of CYP2 A6, which is a member of the CYP2 A subfamily, have been identified over the past 5 years. CYP2A6 participates in the metabolism of several compounds including drugs, toxins and procarcinogens whose blood levels, actions and duration of effects may be altered by polymorphisms in this gene. This review will summarize the current knowledge on CYP2 A6, its genetic variants and their potential for clinical consequences.
2. The cytochrome P450 superfamily CYPs are heme-containing enzymes which catalyse the metabolism of a wide variety of compounds including environmental pollutants and dietary chemicals. CYP genes are designated by a family number, a subfamily letter then a number and an asterisk followed by a number(s) for each allelic variant (i.e. CYP2 A6*2 ) [2]. A CYP allele database has been constructed and is available online [3]. CYPs are categorised into their families and subfamilies based on their sequence similarities. Those with amino acid sequences that are greater than 40% identical belong to the same family while sequences that are greater than 55% identical also belong to the
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same subfamily. In humans, 18 CYP families and 43 subfamilies exist [4]. The CYP1, CYP2 and CYP3 families contain the enzymes that are mainly involved in the metabolism of exogenous compounds such as drugs. To date, 57 known human CYP genes and 29 known CYP pseudogenes (do not produce functional proteins) have been sequenced [4].
3. CYP2A6 substrates, inhibitors and inducers
3.1. Substrates The CYP2A6 enzyme, which is primarily expressed in the liver, was first recognized for its involvement in the metabolism of coumarin [5], a naturally occurring plant compound. CYP2A6 is selective for coumarin 7-hydroxylation making coumarin a selective in vitro and in vivo probe for measuring CYP2A6 activity [6]. In recent years, the role of CYP2A6 in nicotine metabolism has become of interest. On average, 80% of nicotine is inactivated to cotinine in vivo by C-oxidation [7]. CYP2A6 is the major enzyme catalysing this reaction ( | 90%) although in some individuals there are also minor contributions from other CYPs including CYP2B6 [8]. CYP2A6 also catalyses the further metabolism of cotinine to its primary metabolite 39-hydroxycotinine [9] (see Fig. 1) as well as its conversion to 59-hydroxycotinine and possibly norcotinine [10]. CYP2A6’s substrate specificity is somewhat narrow and apart from nicotine, cotinine and SM-12502, a platelet activating factor antagonist [11], it is not known to be important in the metabolism of many other clinically used drugs. CYP2A6 is however involved in the activation of many toxic and / or procarcinogenic compounds (refer to Table 1 for selected examples). Large species differences exist in the metabolism of human CYP2A6 substrates by animal CYP2A members, restricting most
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Fig. 1. Human nicotine metabolism. The major enzyme involved in the metabolism of nicotine to cotinine is CYP2A6 (as determined in vitro and in vivo). The further metabolism of cotinine to 3-hydroxycotinine is also thought to be mainly catalysed by CYP2A6 (demonstrated in vitro) [7–9].
Table 1 Selected examples of human CYP2A6 substrates, inhibitors and inducers [5–7,10,12,14,18,25,45,90–99]. Some substrates may be used as inhibitors Substrates
Specific inhibitors
Inducers
Coumarin, nicotine, cotinine, 7-hydroxycoumarin, nitrosodimethylamine, aflatoxin B1, SM-12502, halothane, methyl tert-butyl ether, butadiene, nitrosamines such as 4-(methylnitrosamino)-1(3-pyridyl)-1-butanol and 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone, methoxylfurance, acetaminophen, hexamethylphosphoramide, and valproic acid
Tranylcypromine, psoralen, naphthoflavone, pilocarpine, methoxsalen, menthofuran
Phenobarbital, rifampin, antiepileptics
studies in this field to human or non-human primate tissues [12,13].
3.2. Inhibitors and inducers Numerous compounds have been investigated for their inhibitory effects on the CYP2A6 enzyme. Most studies were done in vitro with human liver microsomes or expressed CYP2A6 and tested for the inhibition of coumarin 7-hydroxylation [14]. Methoxsalen, first tested in vitro, has also proven to be a useful in vivo inhibitor of CYP2A6 activity and can therefore be used to phenocopy slower CYP2 A6 genotypes [14–16]. In addition, CYP2A6 is an inducible enzyme [17]. This is important to consider since ingestion of inducers or potential repressors can lead to altered enzyme activity in individuals with fully functional alleles (refer to Table 1 for selected examples).
4. Structural organization of the CYP2A6 gene The human CYP2 A6 gene was cloned and sequenced in 1990 [18,19]. Its gene locus spans a region of 6 kbp, contains nine exons and has been physically mapped to the long arm of chromosome 19 between 19q12 and 19q13.2 [20,21]. It is located within a 350-kbp gene cluster containing the CYP2 A7 and CYP2 A13 genes which show high sequence homology to the CYP2 A6 gene [12,20–22]. CYP2A13 is mostly expressed in the nasal mucosa, lung and trachea. It has been shown to be only one-tenth as active as CYP2A6 in catalysing coumarin 7-hydroxylation but has high metabolic activity towards 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen [23]. CYP2A7 is thought to be non-functional [18]. Previously, two CYP2 A7 pseudogenes, CYP2 A7 PC and CYP2 A7 PT, were described in this
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region. They were found to have identical sequences that were most similar to CYP2A7 exons 1–5 [12,20,21]. Recently, it was discovered that this finding was probably a sequence assembly error and that only one of these pseudogenes is present [22]. A second pseudogene, representing the other half of CYP2 A7 (exons 6–9), has been described in this region [4,24]. The split pseudogene has been named CYP2 A18 in which the N-terminus part is designated CYP2 A18 PN (59 half including exon 1 to 5) and the C-terminus part is designated CYP2 A18 PC (39 half including exons 6 though 9) [22]. Members of the CYP2 B, CYP2 F, CYP2 G and CYP2 S subfamilies have also been mapped to this chromosome 19 gene cluster; it is thought that this grouping of CYP genes is likely the result of a series of tandem duplications and at least one inverted duplication [12,20–22,24].
5. CYP2A6 and pharmacogenetics In the past several years, the role of CYP2A6 and the consequences of its genetic variants have become the focus of numerous studies. To date, 13 allelic variants of the CYP2 A6 gene have been discovered (*1 – *11 and the gene duplication). Several of these variants result in altered enzyme activity that may consequently affect the metabolism of CYP2A6 substrates including clinically and non-clinically used drugs, toxins and procarcinogens; understanding the genetic variation and clinical consequences is therefore important.
5.1. Interindividual variability in CYP2 A6 activity CYP2A6 activity shows great interindividual variation. This has been demonstrated both in vitro and in vivo using coumarin, nicotine and cotinine [8,9,18,25,26]. Polymorphisms in the CYP2 A6 gene are thought to be largely responsible for the observed variation in CYP2A6 activity; however, other factors such as environmental inducers and disease may also contribute (reviewed in Ref. [12]). As mentioned, coumarin is a selective CYP2A6 substrate and has been used regularly to assess CYP2A6 activity in vivo. We have defined the following groups: poor metabolizers (PMs) are individuals with two copies of inactive gene variants
and no enzymatic function (i.e. homozygous for two inactive alleles CYP2 A6*4 /*4 or CYP2 A6*2 /*2 ). Extensive metabolizers (EMs) have one or two copies of active gene alleles (i.e. wildtype homozygous CYP2 A6*1 /*1 ) and fast metabolizers (FMs) have two copies of the active gene (i.e. gene duplication CYP2 A6*1 /*1 3 2 ) [27]. Phenotyping studies have shown that CYP2A6 PMs excrete very little ( , 0.1% of EMs) 7-hydroxycoumarin when coumarin is used as the probe drug [28] and also produce little cotinine (0–15% of EMs) when nicotine is used as the probe substrate [29–31]. In contrast, CYP2A6 FMs (carrying more than two copies of active CYP2 A6 alleles) metabolize nicotine more rapidly [27,32].
5.2. Known CYP2 A6 allelic variants The wide variation described in CYP2A6 activity and levels can be largely attributed to polymorphisms in the CYP2 A6 gene. A change in the gene sequence (i.e. point mutation, deletion, gene conversion) may lead to the overproduction, underproduction, malfunction or absence of the protein. This may result in positive effects (i.e. protection against disease due to lack of procarcinogen activation) or negative effects (i.e. adverse reaction to a drug that is not metabolized). To date, 13 genetic variants of the CYP2 A6 gene have been identified (Fig. 2) using different methods such as restriction fragment length polymorphisms with Southern blotting (i.e. CYP2 A6*4 ) [11] and polymerase chain reaction– single stranded conformation polymorphisms (i.e. CYP2 A6*7, *8, *10 ) [33]. Once identified, the variants were either characterized in vitro (e.g. by site-directed mutagenesis) and / or in vivo (e.g. by phenotyping using substrates) [33–35]. Initially, the wildtype allele (CYP2 A6*1 ), which has full CYP2A6 activity, and two defective alleles (CYP2 A6*2 and CYP2 A6*3 ) were discovered [18,21]. CYP2 A6*2 encodes a protein with a Leu160His substitution that is believed to be unable to incorporate heme [18]. This variant has been shown to be inactive both in vitro [36] and in vivo (tested using coumarin and nicotine) [37,38]. CYP2 A6*3 is thought to be created by a gene conversion with CYP2 A7 in exons 3, 6 and 8 and is also thought to be inactive [21]. It has been demon-
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Fig. 2. Schematic diagram of the CYP2 A6 gene structure and variants. (A) CYP2 A6 point mutation variants. Exons are represented by hatched boxes. E, exon; I, intron; E1:180 indicates the length of exon 1 is 180 bp; 59UTR:9 indicates the length of 59 untranslated region is 9 bp; 39UTR:255 means the length of 39 untranslated region is 255 bp. The length of exons and introns were obtained from GenBank accession number NG 000008. CYP2 A6*10 consists of the combination of variants CYP2 A6*7 and CYP2 A6*8. The CYP2 A6*3 allele is not ] listed in this table since it is either extremely rare or is an artifact. (B) Additional CYP2 A6 variants resulting from large domain changes.
strated that the original genotyping methods for CYP2 A6*2 and CYP2 A6*3 provide erroneous results. These assays were shown to create false positives by misidentifying CYP2 A7 and CYP2 A6*1 B as CYP2 A6*2 and CYP2 A6*3 [28,39]. The CYP2 A6*1 B variant is created by a 58-bp gene conversion with CYP2 A7 in its 39 untranslated region and maintains normal enzyme activity [39]. Its high frequency and the location of genotyping PCR primers within this 58-bp region contributed to the overestimation of CYP2 A6*3 alleles [39] while using the original methods [21]. It is clear now that CYP2 A6*3 either does not exist, or is very rare [39–41]. The CYP2 A6 gene deletion variants, known collectively as the CYP2 A6*4 alleles, lack activity towards coumarin and nicotine (in vitro and in vivo) [29,30,39]. These variants are thought to have resulted from unequal crossover events between CYP2 A7 and CYP2 A6 [41,42]. Multiple crossover positions have been identified in the highly homologous regions of CYP2 A6 and CYP2 A7 (intron 8—39 untranslated region) creating different deletion var-
iants (A–D) and likely duplication variants as well [27,30,31,39]. This has been seen previously with another CYP, CYP2 D6 (review in Ref. [42]). One duplication variant has been identified whose crossover region is consistent with the opposite crossover position in the deletion variant CYP2 A6*4 D; individuals with this variant are expected to be CYP2A6 FMs [27,32]. CYP2 A6*5 was identified in a coumarin PM; it has a Gly479Val substitution in exon 9 (within substrate recognition site 6) and is thought to encode an unstable protein [39]. Very recently, additional variants of the CYP2 A6 gene have been discovered. CYP2 A6*6 contains a single nucleotide polymorphism in exon 3 resulting in an Arg128Gln substitution and has decreased activity when expressed in vitro [35]. CYP2 A6*7 was shown to have decreased activity towards nicotine and full activity for coumarin and CYP2 A6*8 appears to be fully functional [33,34]. Both these variants contain amino acid substitutions (Ile471Thr and Arg485Leu, respectively) resulting from nucleotide changes in exon 9 of the CYP2 A6 gene [33,34]. When both
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variants are found on the same allele, named CYP2 A6*10 (containing both Ile471Thr and Arg485Leu), preliminary evidence indicates that it has dramatically reduced enzyme function for nicotine and coumarin in vivo and may in fact be inactive for some or all substrates [33]. CYP2 A6*9 contains a mutation in the 59 flanking region of CYP2 A6 resulting in 48T → G substitution in the TATA box (TAGA) and decreased enzyme transcription (tested in vitro) [43]. The latest discovery, CYP2 A6*11 (Ser224Pro), is unpublished, but listed on the CYP web site as possessing decreased enzyme activity both in vivo and in vitro [3]. Although 13 CYP2 A6 genetic variants have been discovered it is likely that further variants remain to be identified as phenotyped PMs exist who do not have the known CYP2 A6 variants indicating the presence of unidentified variant alleles.
5.3. CYP2 A6 allele frequencies
Table 2 CYP2 A6 allelic frequencies
*1A *1B *2 *4 *5 *6 *7 *8 *9 *10 *1 3 2
66.5 30.0 1.1–3.0 0.5–4.9 0.0–0.2 1.0 0.0 5.2 0.0 0.7
Japanese (%)
6. CYP2A6 genetic variation and potential consequences In the past several decades, techniques have evolved which allow us to study the role of genetic factors in human diseases and disorders. As mentioned, CYP2A6 is involved in the metabolism of nicotine, some procarcinogens and several toxins. Variation in this gene leads to altered enzyme activity and consequently to changes in the metabolism of these compounds. This section summarizes how the CYP2 A6 variants may affect smoking, cancer, as well as the treatment of cigarette smoking and other disorders.
6.1. CYP2 A6 and the metabolism of pharmaceuticals /toxins
Recent studies have demonstrated that the frequencies of the known CYP2 A6 alleles vary substantially between different ethnic groups. Table 2 displays the current frequencies for the published CYP2 A6 variants. Differences in the frequency of these alleles may partially explain the interethnic variability in the metabolism of CYP2A6 substrates as well as the observed interethnic differences in
CYP2 A6 Caucasian Chinese allele (%) (%)
CYP2A6-related behaviors (i.e. smoking) and disease (i.e. lung cancer).
African American (%)
43.2 40.0–42.0 40.6 38.0–41.0 0.0–0.7 0.0 0.3 6.6–15.1 20.0–31.0 1.0 0.0 0.4 2.2 6.3 3.5 1.6 15.7 0.4 1.6 0.4 0.0
The frequencies of these variants are based on new techniques [27,28,31,33,35,39–41,43,51,79,100–102]. The CYP2 A6*3 allele is not listed in this table since it is either extremely rare or is an artifact. No data is available for CYP2 A6*11.
CYP2A6 plays a role in the metabolism of pharmaceutical agents and the activation of toxic compounds (refer to Table 1) [44]. This indicates that CYP2 A6 variants may influence the dosing, action and side effects of a prescribed drug as well as the adverse effects associated with exposure to certain potentially toxic compounds. For example, CYP2 A6 slow metabolizers (with defective CYP2 A6 alleles) may be partially protected from hepatotoxicity caused by certain drugs such as valproic acid [45] and halothane [46] that are metabolically activated by CYP2A6. On the other hand, individuals with extra CYP2 A6 copies may be at greater risk.
6.2. CYP2 A6 and nicotine metabolism It is known that the frequency of the CYP2 A6 alleles vary between different ethnic groups which may result in interethnic differences in the average ability to metabolize CYP2A6 substrates [33,47] (see also review in Ref. [48]). Studies have demonstrated that ethnic differences in nicotine and cotinine metabolism (both CYP2A6 substrates) do exist. It was found that African-Americans have a significantly reduced clearance of cotinine, fractional conversion of nicotine to cotinine and metabolic clear-
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ance of nicotine to cotinine compared with Caucasians [49,50]. The metabolic ratio of cotinine to nicotine has also been shown to be elevated in Korean versus Japanese populations [51]. In addition, it was recently found that Chinese-Americans metabolize nicotine 25% slower than Caucasians and Latinos [47]. Interethnic as well as interindividual variation (discussed in Section 5.1) in nicotine and / or cotinine metabolism is of particular interest since this may have several important clinical implications. The altered rates of metabolism may affect smoking behaviour, risk of tobacco-related cancers and treatment of nicotine addiction as well as other disorders with nicotine replacement therapies. The following sections will address these issues.
6.3. CYP2 A6 and smoking It has been reported that approximately one third of the global population 15 years or older smokes and that the number of smoking-associated deaths is estimated at 3,000,000 people per year worldwide [52]. It has been demonstrated that smoking behaviour is influenced by both genetic and environmental factors [53–55]. Many studies have shown that genetics contributes to | 50% of smoking initiation and . 70% of maintenance and amount smoked [55–57]. Nicotine is known to be the primary compound in tobacco that establishes and maintains tobacco dependence [58]. In addition, tobacco-dependent individuals are known to adjust their smoking behaviour to maintain constant blood and brain nicotine levels [59,60]. This is controlled both by the intake of nicotine (i.e. smoking) and its removal (i.e. rate of metabolism). The CYP2 A6 variants can alter the rate of nicotine metabolism and consequently the amount of nicotine maintained in the body. Therefore, it can be hypothesized that individuals who are slow metabolizers (SM, one or more inactive CYP2 A6 alleles) differ in their risk for various aspects of smoking behaviour such as rates and severity of tobacco dependence, amount smoked regularly, and success at quitting. We hypothesized that SMs would be less likely to become tobaccodependent since nicotine levels would remain in these individuals for longer periods of time therefore prolonging the initial adverse effects of nicotine (this could have had the opposite effect). Once tobacco-
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dependent, SMs were hypothesized to smoke less since they will experience nicotine withdrawal at greater intervals of time (reviewed in Ref. [61]). Pianezza et al. reported reduced risk of tobacco dependence and reduced total consumption of cigarettes in individuals carrying one or two inactive alleles [62]. Due to the occurrence of multiple gene and pseudogene duplications, high sequence homology and gene conversion events in the CYP2 A6 locus, the original genotyping methods [21] used for these studies were found to be erroneous. Using new and improved techniques, it has been demonstrated that individuals with inactive CYP2 A6 alleles compensate for decreased nicotine metabolism by decreasing the number of cigarettes smoked per day [27] and individuals with extra copies of the CYP2 A6 gene compensate for increased nicotine metabolism by inhaling their cigarettes with greater intensity [27]. Furthermore, it has been demonstrated that individuals with inactive CYP2 A6 alleles start smoking at a later age (3 years later), smoke for a shorter duration (9 years less) and are 1.75 times more likely to quit in comparison with wildtype individuals [63]. These studies present evidence that CYP2A6 is involved in the regulation of smoking behaviour; however, not all studies using validated genotyping assays have found an association [64]. The discrepancies may be due to several factors including the genotyping techniques used, statistical power, the presence of unknown variants, use of mixed ethnic groups as well as variable definitions of tobacco dependence / smokers.
6.4. CYP2 A6 and cancer It is well-known that cigarette smoking is associated with lung cancer as well as other forms of cancers [65–67]. It has been demonstrated that the major risk for lung cancer is cigarette smoking and that this risk is increased with a higher number of cigarettes / day and with more years of smoking [66,68,69]. There are hundreds of chemicals present in tobacco smoke of which the polycyclic aromatic hydrocarbons and the tobacco-specific nitrosamine, NNK, are likely to be the major compounds causing lung cancer [70,71]. Cytotoxicity and mutagenicity studies have revealed that CYP2A6, which is present in the liver and in small amounts in the lung [23],
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can efficiently activate NNK to carcinogenic intermediates [72]. CYP2A6 can also activate other nitrosamines present in tobacco smoke including N9nitrosonornicotine (NNN), and N-nitrosodiethylamine (NDEA) [73,74]. In addition, it has been demonstrated that methoxsalen treatment causes the re-routing of NNK from its mutagenic hydroxylation pathway to a non-mutagenic glucuronidation pathway (by 30%) [75]. This indicates that CYP2A6 PMs may have a reduced activation of procarcinogens. CYP2A6 PMs may be at lower risk of lung cancer development for several reasons including reduced efficiency at bioactivating certain tobacco smoke procarcinogens, decreased risk of becoming tobacco-dependent and less smoking if tobacco-dependent (note that smoke exposure is exponentially related to cancer risk [76,77]). In genetic studies of CYP2 A6, a decreased rate of lung cancer among individuals carrying inactive CYP2 A6 variants in comparison with homozygote wildtype individuals has been found in Japanese subjects [78]. In contrast, one study in France found no relationship between CYP2 A6 and lung cancer [79] although it may not have had sufficient statistical power. Another study performed in Chinese has reported an elevated lung and esophageal cancer risk in subjects who were CYP2A6 PMs [80]. The latter results are postulated to be due to several factors including reduced firstpass clearance of ingested nitrosamines in the liver resulting in higher levels of these compounds reaching the lung [80], disease status of the patients, sampling bias, statistical power, the genotyping technique and lack of controls for environmental factors such as smoking. CYP2A6 may also influence susceptibility to esophageal (see above) and liver cancer. It has been demonstrated that these NNN nitrosamines can induce tumors in the rat esophagus [81] (see also review in Ref. [82]), however it has not yet been shown to be a causative agent for esophageal cancer in tobacco users. N-Nitrosobenzylmethylamine is also known to be a potent esophageal carcinogen in rodents [83]. As mentioned, CYP2A6 can activate these nitrosamines and therefore individuals with decreased activity may have some protection against cancers caused by these procarcinogens. The liver has high CYP2A6 expression and may be a target organ since it is involved in the metabolism of many
procarcinogens. Clearly a greater understanding of the genetic variation of CYP2 A6 and its role in both smoking and the activation of procarcinogens is needed before conclusions can be reached about a role for CYP2A6 in the etiology of cancer.
6.5. Nicotine treatments Nicotine addiction via cigarette smoking is a serious medical condition that needs to be treated like any other chronic disease or dependency. Many smokers try to quit smoking each year but few are successful [84]. Today, the most widely used smoking cessation aids are the nicotine replacement therapies (NRTs); these treatments show significant efficacy (6–18% abstinence at 1 year versus placebo) but there is a clear need for new and improved therapies (see review in Ref. [85]). At present, no oral pill formulation of nicotine is available due to nicotine’s high first-pass metabolism by CYP2A6 and gastrointestinal distress caused by elevated nicotine doses. However, this route of treatment may prove to be an additionally useful NRT since it may produce sufficient nicotine levels using a route / formulation that traditionally has higher rates of compliance. Due to CYP2A6’s role in nicotine metabolism and studies indicating that inactive CYP2 A6 variants may decrease smoking, it can be expected that CYP2A6 inhibition may work to help reduce smoking. In this situation, a much lower nicotine dose can be given orally (avoiding gastrointestinal distress) since first-pass metabolism will be blocked; this should act like other NRTs in smoking cessation as well as exposure reduction. One study demonstrated that methoxsalen and tranylcypromine (CYP2A6 inhibitors) in combination with oral nicotine were both capable of increasing nicotine plasma levels and decreasing self-reported cigarette cravings [15]. In addition, this study also showed that methoxsalen plus nicotine treatment causes a significant reduction of carbon monoxide levels (measure of smoke exposure and exhalation) and a significant decrease in cigarettes smoked per day compared with nicotine plus placebo inhibitor treatment [15]. This suggests that CYP2A6 inhibitors combined with NRTs may provide a more powerful quitting tool than NRTs alone. It is important to note that CYP2A6 inhibition is possible since CYP2A6 has
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been shown to have a highly constrained catalytic acceptance site and is not known to be important in the metabolism of many clinically used drugs. Furthermore, since NRTs are being used today without CYP2A6 inhibition, it may be important to consider the effect of the variants on the success of this treatment. It is likely that rapid CYP2A6 metabolizers of nicotine have lower plasma nicotine levels compared to slow metabolizers which may result in altered efficacy of NRT treatment. Smoking has been observed to alter aspects of a number of neurological disorders. As a result NRTs are being tested as new treatments for several degenerative neurologic and cognitive disorders such as Tourettes, Down syndrome, Alzheimer’s and Parkinson’s disease [86–88] (see review in Ref. [89]). As nicotine plasma and brain levels will alter the success of treatment [86], it is likely that the CYP2 A6 genotype will also affect the success rates.
[3] [4] [5]
[6]
[7]
[8]
[9]
7. Concluding remarks and future directions [10]
Large advancements in our understanding of CYP2A6 have occurred in the past several years, however it is obvious that major scientific gaps still exist. Further research concentrating on the CYP2 A6 gene, its variants and their clinical consequences will improve our understanding of the role that CYP2A6 genetic variation plays in interindividual differences in metabolism and clarify some of the incomplete and / or conflicting data. We believe that this will also provide us with new options for prevention and treatment of CYP2A6-related behaviors and diseases. In addition, these studies may also provide insight into the reasons for ethnic differences in smoking behaviors and incidences of tobacco-related diseases.
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