Cytochrome P450 2A6 (CYP2A6) is one of the most important enzymes in human nicotine metabolism. Genetic polymorphisms in the CYP2A6 gene causes important interindividual variability in CYP2A6 activity and this variation can explain some of the interindividual variability in nicotine metabolism previously reported. Here I summarise the current knowledge about the CYP2A6 polymorphism, and also discuss the potential importance of this polymorphism for differences in smoking behaviour.
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INTRODUCTION FOR NICOTINE ADDICTION WORKSHOP BY MARCUS MUNAFO AND MIKE MURPHY: The ICRF General Practice Research Group in Oxford recently hosted a workshop on the theme of "Neuroscience, molecular genetics and nicotine addiction". It was chaired by Professor Neal Benowitz from the University of California at San Francisco, a specialist in the field of nicotine pharmacology spending an invited week in the University to work with the Group, and included about 30 other experts from groups around the UK and Sweden, including the ICRF Molecular Pharmacology Unit in Dundee and the ICRF Health Behaviour unit in London. Their backgrounds included neuroscience, molecular genetics, genetic epidemiology, pharmacology, psychology and clinical medicine, but they were united in a belief about the importance of tobacco control.
It is now recognised that addiction to nicotine lies at the heart of the tobacco control problem. Without the marketing of tobacco its use would dwindle. But while it is so widely promoted smoking research focuses heavily on factors affecting why people continue smoking after trying it and how to get them to stop with psychological support and the use of effective drugs. Although tobacco use is rightly viewed as a social and psychological problem we also need to see it as a pharmacological one with a potential genetic contribution to the addictive process. The need to integrate knowledge about the brain chemistry underlying addiction, the molecular genetics of these cellular processes, and how the drugs which are effective work, was the workshop's subject.
Each of the meeting's 4 sessions kicked off with a brief introduction by speakers who had precirculated draft papers. The sessions considered in turn: (1)how nicotine is metabolised and cleared from the body (2)the brain receptors in which nicotine "docks" to exert its effects (3)the brain pathways involving the neurotransmitter,dopamine,which are thought to contribute to the rewarding properties of nicotine which reinforce a smoker's desire to smoke (4)other non-dopamine pathways that are activated by nicotine and may underly such effects as nicotine withdrawal symptoms.
Understanding how the drugs which are effective (Nicotine Replacement Therapy and some antidepressants) interact with the expression of individual genotypes in the brain and elsewhere to predict success in quitting smoking will help to shed light on which bits of the brain are important in nicotine addiction at both the anatomical and cellular level. The workshop participants emerged pleased to have caught up with advances in fields which were not their speciality and with a strong sense that more enduring closer contact and cooperation will be possible in the future.
1. There is a pronounced interindividual and interethnic variability in the levels and activity of many drug metabolising enzymes. This might cause interindividual differences in the sensitivity to the effects and toxicity of many clinically used drugs and environmental compounds such as nicotine and precarcinogens. In many cases the basis for this variability is genetic polymorphisms in the genes encoding these enzymes (Ingelman-Sundberg et al, 1999). During the last couple of years, a lot of attention has been focused on cytochrome P450 2A6 (CYP2A6), which is one of the major enzymes involved in human nicotine metabolism (see below). This enzyme also contributes to the metabolism of some pharmaceuticals including SM-12502, methoxyflurane,halothane, valproic acid and disulfiram, and can activate a number of precarcinogens e.g., aflatoxin B1, 1,3-butadiene, 2,6-dichlorobenzo- nitrile and the nitrosoamines NNK [4-(methylnitrosoamino)-1-(3-pyridyl) -1-butanone], NNAL [4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol], NDEA [N-nitrosodiethylamine] and NNN [N'-nitrosonor-nicotine](Pelkonen et al, 2000).
2. The CYP2A6 gene is located on the long arm of chromosome 19 (between 19q12 and 19q13.2). It is located within a 350 kb gene cluster together with the CYP2A7 and CYP2A13 genes, two CYP2A7 pseudogenes as well as genes in the CYP2B and CYP2F subfamilies (Hoffman et al, 1995). CYP2A7 has been shown not to incorporate heme and is therefore not active (Yamano et al, 1990; Ding et al, 1995), whereas the possible activity of CYP2A13 still remains unknown.
3. Early studies using human liver microsomes as well as in vivo phenotyping studies with the probe drug coumarin revealed a pronounced interindividual variability in CYP2A6 levels and activity. Most of this variability can be attributed to genetic polymorphisms in the CYP2A6 gene, and two variant alleles (CYP2A6*2 and CYP2A6*3) were described several years ago (Yamano et al, 1990; Fernandez-Salguero et al, 1995). Using the genotyping method described to detect these alleles, we noticed a poor correlation between the CYP2A6 genotype and coumarin phenotype. We therefore designed a more specific method for the detection of these alleles, which gave a much better correlation with the phenotype (Oscarson et al, 1998). Using this method we detected a CYP2A6*2 allele frequency of 1-3 % in European populations, much lower than previously reported. We also failed to detect any true CYP2A6*3 alleles (Oscarson et al, 1999), a finding which has been supported by several other groups (Chen et al, 1999, Kitagawa et al, 1999). A gene conversion with the closely related CYP2A7 gene in the 3' flanking region of the CYP2A6 gene that abolishes the binding site for one of the primers used in the earlier method is the likely explanation for most of these misclassifications (Oscarson et al, 1999).
4. Studies using human liver microsomes suggested that the CYP2A6 PM phenotype was more common in Asian populations compared to Caucasians (Shimada et al, 1996), and studies by Nunoya and co-workers suggested that a partial or whole deletion of the CYP2A6 gene could contribute to the PM phenotype in Japanese individuals (Nunoya et al, 1998; 1999). We studied the molecular basis for this, and in a Chinese population we characterised a common deletion of the whole CYP2A6 gene, that was designated CYP2A6*4 (Oscarson et al, 1999). This allele was most likely generated through an unequal cross-over event in the 3' flanking region of the CYP2A6 and CYP2A7 genes. A PCR-based genotyping method was developed to detect this allele which revealed an allele frequency of 15 % in the Chinese population but only 1 % in Europeans, suggesting an important interethnic variability in CYP2A6 activity (Oscarson et al, 1999).
5. In humans, nicotine is primarily C-oxidised by cytochrome P450 to a nicotine (delta-1'(5')-iminium ion, which is further metabolised to cotinine by a cytosolic aldehyde oxidase, but nicotine is also N-oxidised by flavine monooxygenases to nicotine N'-oxide. Cotinine is subsequently converted to a number of hydroxylated products including trans-3'- hydroxycotinine, 5'-hydroxycotinine and norcotinine. In addition, nicotine, cotinine and trans-3'-hydroxycotinine are glucuronidated (Benowitz and Jacob, 1997). In vitro studies using human liver microsomes and recombinant P450s have shown that CYP2A6 is the most important P450 responsible for both the C-oxidation of nicotine (Nakajima et al, 1996; Messina et al, 1997; Cashman et al, 1992) as well as the subsequent oxidation of cotinine (Murphy et al 1999, Nakajima et al, 1996). In vivo studies have confirmed the important contribution of CYP2A6 to nicotine metabolism, as individuals homozygous for a CYP2A6 gene deletion displayed only15% of urinary cotinine levels compared to individuals carrying at least one active CYP2A6 gene, when smoking the same number of cigarettes (Kitagawa et al, 1999).
6. Because of the important contribution of CYP2A6 to nicotine metabolism, it has been suggested that CYP2A6 activity would be an important factor in determining an individual's smoking behaviour. Pianezza and co-workers (Pianezza et al, 1998) presented data showing that a lower number of individuals carrying the CYP2A6*2 and CYP2A6*3 alleles were found in a tobacco-dependent group as compared to a never tobacco-dependent group, and that smokers carrying a defective CYP2A6 allele smoked fewer cigarettes. This is an interesting finding that implies that the CYP2A6 activity would influence the risk for nicotine dependence. These findings have however been questioned. First of all, the original non- specific genotyping method (Fernandez-Salguero et al, 1995) was used and a total allele frequency of 6-10 % was found for the CYP2A6*2 and CYP2A6*3 alleles. As discussed above, most of the "alleles" represents PCR artefacts. Secondly, several groups have failed to reproduce these findings using both the original as well as more specific genotyping methods (Pianezza et al, 1998, London et al, 1999). It would be interesting to carry out this kind of study in Asian populations, where the frequency of defective alleles is considerably higher and the impact on interindividual variability in nicotine metabolism should be much greater. Furthermore, it would be beneficial to phenotype the individuals with coumarin as an indicator of CYP2A6 activity, because there are probably still a number of CYP2A6 alleles which have not been identified, and this would elucidate the predictive value of CYP2A6 genotyping with the alleles known today.
7. The CYP2A6 genotype has also been suggested to modulate an individual's risk of getting lung cancer. Theoretically, absence of CYP2A6 could potentially have a dual protective effect against lung cancer. First, if the hypothesis raised by Pianezza and co-workers is correct, individuals lacking CYP2A6 activity would not become smokers, or at least smoke less. Secondly, as a number of precarcinogens found in tobacco smoke such as NNK, NDEA and NNN are metabolically activated by this enzyme, absence of CYP2A6 would be protective. In line with this, Japanese subjects with a CYP2A6 gene deletion were shown to be at reduced risk of lung cancer (Miyamoto et al, 1999). These findings could however not be reproduced in a French population (Loriot et al, 2001), indicating that the relationship between the CYP2A6 polymorphism and lung cancer is more complex.
8. Our increased knowledge concerning the different alleles in the highly polymorphic locus CYP2A will facilitate conclusions regarding the genetic influence of CYP2A6 on interindividual variation in tobacco use and lung cancer incidence. Several well-designed studied are needed to fully clarify this potentially important aspect of the CYP2A6 polymorphism.
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