CYP2A6: genetics, structure, regulation, and function

Drug Metab Drug Interact 2012;27(2):73–88 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/dmdi-2012-0001

Review

CYP2A6: genetics, structure, regulation, and function

Hannu Raunio * and Minna Rahnasto-Rilla*

Faculty of Health Sciences , School of Pharmacy, University of Eastern Finland, Kuopio , Finland

Abstract

The human CYP2A gene subfamily consists of three mem-bers, CYP2A6 , CYP2A7 , and CYP2A13 . The CYP2A6 gene is highly polymorphic with approximately 40 annotated allelic variants. Individuals homozygous for some of these alleles have a total lack of CYP2A6 activity. The CYP2A6 protein is most abundant in liver and is expressed, although at much lower levels, in some other tissues, especially nasal muco-sa. CYP2A6 differs from other human liver CYP forms in that it participates in the metabolism of very few currently used drugs. The two most relevant substrates for CYP2A6 are coumarin and nicotine. Coumarin is the marker sub-stance for determining CYP2A6 activity both in vitro and in vivo. Approximately 80 % of a nicotine dose is eliminated by CYP2A6, and there is a clear link between CYP2A6 geno-types, smoking behavior, and lung cancer risk.

Keywords: cigarette smoking; CYP2A6; genetic polymor-phisms; nicotine.

Introduction

Of all the major human liver xenobiotic-metabolizing CYP enzymes, CYP2A6 plays a minor role in terms of relative abundance and number of clinically used drugs metabolized by this enzyme. Nevertheless, two features of CYP2A6 have maintained considerable interest in this CYP form: (i) it is the major catalyst of metabolic elimination of nicotine, and (ii) CYP2A6 and another member in the human CYP2A sub-family, CYP2A13, effi ciently activate procarcinogenic nitro-samines, especially those present in tobacco smoke. CYP2A6 genetic polymorphism and other factors affecting enzyme activity have been shown to affect smoking behavior and sus-ceptibility to lung cancer.

The purpose of this review is to highlight the major fea-tures of human CYP2A6, from genes in the CYP2A subfamily

to the practical implications of interindividual and intereth-nic differences in CYP2A6 expression. Several animal spe-cies also possess genes in the CYP2A subfamily. The closest structural and functional ortholog to CYP2A6 is the mouse CYP2A5 form. There is a considerable database available for CYP2A5, and some of this information can be extrapolated to clarify CYP2A6 function. There are comprehensive reviews available on the CYP2A enzymes in animals (1 – 5) .

CYP2A genes

CYP2 gene cluster

The human CYP2 gene cluster on human chromosome 19 was elucidated around 10 years ago (6, 7) . The cluster is made up of six different subfamilies ( CYP2A , CYP2B , CYP2F , CYP2G , CYP2S , CYP2T ), which are more closely related among themselves than to the other CYP2 subfami-lies on other chromosomes. These subfamilies have evolved through numerous gene duplications and other rearrange-ments. The CYP2A cluster contains three complete genes ( CYP2A6 , CYP2A7 , CYP2A13 ) and several variant alleles and pseudogenes. The CYP2A6 gene consists of nine exons, which are defi ned by consensus splicing sequences (GT and AG) on the boundaries between intron and exon regions. The transcribed genes within the human CYP2A subfamily share very high genomic sequence similarities. CYP2A6 is 97 % identical to CYP2A7 in its exonic sequence, and 85 % identical to CYP2A13 , whereas CYP2A7 and CYP2A13 are 90 % identical. CYP2A6 and CYP2A13 proteins share 93 % similarity.

CYP2A6 protein is mainly expressed in the liver, where it represents approximately 10 % of the total liver CYP con-tent, although with marked interindividual variability. Several sensitive detection methods such as reverse transcriptase-polymerase chain reaction (RT-PCR) can be used to detect CYP2A6 mRNA in extrahepatic tissues, most notably nasal mucosa and other areas of the respiratory tract. CYP2A7 is a true gene that produces mRNA in human liver, but no active protein seems to be generated (8 – 12) . CYP2A13 is predomi-nantly expressed in the nasal mucosa and respiratory tract (13, 14) . A recent study revealed that CYP2A6 and CYP2A13 activity is conserved in polarized primary tracheobronchial epithelial cells cultured in the presence of retinoic acid (15) . In the nasal mucosa and other respiratory tissues, CYP2A13 and presumably also CYP2A6 may contribute to short- and long-term toxicity by compounds that are metabolized by these enzymes (16, 17) .

*Corresponding authors: Hannu Raunio and Minna Rahnasto-Rilla, Faculty of Health Sciences, School of Pharmacy, University of Eastern Finland, Box 1627, 70211 Kuopio, FinlandE-mail: [email protected] Received January 18, 2012; accepted February 24, 2012;previously published online May 5, 2012

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74 Raunio and Rahnasto-Rilla: CYP2A6

Table 1 CYP2A6 alleles affecting CYP2A6 function.

Allele Change in cDNA Activity of protein

CYP2A6*1A Wild type Normal CYP2A6*1X2 Gene duplication Increased CYP2A6*2 479T > A None CYP2A6*4 Gene deletion (8 types) None CYP2A6*5 1436G > T, gene conversion in the 3 ′ region None CYP2A6*6 383G > A Reduced CYP2A6*7 1412T > C, gene conversion in the 3 ′ region Reduced CYP2A6*9A SNP in the TATA box Reduced CYP2A6*10 1412T > C, 1454G > T, gene conversion in the 3 ′ region Reduced CYP2A6*11 670T > C Reduced CYP2A6*12A exons 1 – 2 of CYP2A7 origin, exons 3 – 9 of CYP2A6 origin Reduced CYP2A6*17 459G > A, 1093G > A, 1224C > T Reduced CYP2A6*18A 1175A > T Reduced CYP2A6*18B 51G > A, 1175A > T, 1191T > C, 1209T > C Reduced CYP2A6*19 1175A > T, 1412T > C, gene conversion in the 3 ′ region Reduced CYP2A6*20 Frameshift SNP None CYP2A6*21 51G > A; 1427A > G Reduced CYP2A6*23 607C > T Reduced CYP2A6*24A 312A > G; 328G > C; 1312A > T Reduced CYP2A6*26 22C > T, 352T > C, 383G > T, 390C > T, 391T > G, 1440T > C Reduced CYP2A6*27 22C > T, 352T > C, 608_609GC > A, 1440T > C Reduced CYP2A6*35A 1312A > T Reduced CYP2A6*35B 22C > T, 1312A > T Reduced

Source: www.cypalleles.ki.se.

Very little is known about the ontogeny of CYP2A pro-teins in humans, but it appears that only a limited degree of CYP2A6 and CYP2A13 expression occurs in liver before birth, after which CYP2A6 abundance gradually increases. The level of CYP2A6 activity, as measured by coumarin 7-hydroxylation capacity, reaches adult levels between 6 and 13 years of age (18, 19) .

The human CYP2A6 and mouse Cyp2a5 genes are con-sidered orthologous because they have similar functions and regulation; CYP2A6 has 86 % amino acid sequence similarity to CYP2A5 (3) . CYP enzymes orthologous to CYP2A6 have been detected at relatively high concentrations in the olfac-tory tissues of several animal species, indicating that these enzymes could participate in the metabolism of odorants and the processing of olfactory signals (5, 17) .

CYP2A6 genetic polymorphism

CYP2A6 is a highly polymorphic gene. There are currently (February 2012) 38 numbered CYP2A6 allelic variants and many additional single nucleotide polymorphisms (SNPs) in the Home Page of the Human Cytochrome P450 Allele Nomenclature Committee ( www.cypalleles.ki.se ). Many of the genetic variants have been shown to alter the expression, stability, and function of the CYP2A6 enzyme (Table 1 ).

Many CYP2A6 alleles originate from unequal crossover events and/or gene conversions between the CYP2A6 and CYP2A7 genes. For example, CYP2A6*2 is a SNP at a critical site in the enzyme and CYP2A6*4 is a gene deletion variant. Both alleles result in a complete lack of CYP2A6 catalytic ac-tivity (20) . Individuals with different CYP2A6 variants can be

grouped according to CYP2A6 enzyme activity as normal, in-termediate (approx. 75 % of normal), or slow ( < 50 % of normal) metabolizers. Substantial ethnic variations exist in the popula-tion frequencies of CYP2A6 alleles. CYP2A6*4 , CYP2A6*7 , CYP2A6*9 , and CYP2A6*10 alleles are more prevalent in the Japanese population than Caucasians. The CYP2A6*4 allele is present in approximately 20 % of Japanese. Some rare alleles, such as CYP2A6*18B in Caucasians, are found only in specifi c populations (21) .

Regulation of CYP2A6 expression

Host factors

Several CYP2A genes in different species have been shown to be regulated by various physiological factors. One notable example is the marked sex dependent differences in hepatic CYP2A enzyme levels in many species (22) . Constitutive CYP2A6 activity is higher in women than in men, as deter-mined by phenotyping with coumarin (23) , rates of nicotine and cotinine metabolism (24) , metabolism of caffeine (25) , and amounts of hepatic CYP2A6 protein and mRNA (26) . The accelerated nicotine metabolism appears to be a result of estrogen, as the use of oral estrogen containing contraceptives increases enzymatic activity (24, 25) , and nicotine and coti-nine clearances are increased by as much as 140 % in preg-nancy compared with the postpartum situation (27) . However, some studies have failed to observe this gender difference, and a recent genome-wide analysis did not detect baseline female-biased hepatic expression of CYP2A6 (28) .



Raunio and Rahnasto-Rilla: CYP2A6 75

Higashi and coworkers (29) showed convincingly that CYP2A6 is directly induced by estrogen in an estrogen receptor α (ER α )-dependent manner. Interestingly, CYP2A6 mRNA was found to be expressed at much higher levels in ER α -positive breast tumors compared with ER α -negative tu-mors (30) . Nevertheless, menstrual cycle (follicular phase vs. luteal phase) has no effect on nicotine and cotinine pharma-cokinetics in healthy non-smoking women (31) .

Expression of CYP2A6 has been shown to be elevated in specifi c areas of cirrhotic liver, particularly in those areas im-mediately next to fi brotic and infl amed sites (32 – 34) . Local elevation of CYP2A6 protein occurs also during hepatitis caused by hepatitis B or C virus infection (33) and infestation of the parasite Opisthorchiasis viverrini (35) . An increase in the rate at which coumarin is 7-hydroxylated was observed in patients infected with O. viverrini and having biliary fi brosis (36) . Overexpression of CYP2A6 protein also occurs in he-patocellular carcinoma, where it is associated with chronic infl ammation and cirrhosis (37) . This local upregulation is in stark contrast with other xenobiotic-metabolizing CYP forms; their activities are repressed during infection and infl amma-tion (38) . Patients with varying degrees of non-alcoholic or alcoholic liver disease exhibit reduced CYP2A6 activity measured by systemic coumarin 7-hydroxylation rate (23) . Hepatitis A markedly reduces coumarin 7-hydroxylation in adults and children (39) .

Exogenous factors

CYP2A6 has been shown to be upregulated by classical CYP inducers, such as the antiepileptic drugs phenytoin, Phenobarbital, and carbamazepine in vitro (40 – 47) and in vivo (9, 26, 48, 49) . The potent CYP3A4 inducer rifampicin elevates CYP2A6 mRNA and catalytic activity to variable de-grees in primary human hepatocytes (43, 45, 47) . Induction by phenobarbital may be mediated through the constitutive active receptor (CAR) and the pregnane X receptor (PXR) (50) . In one study (51) , administration of the antimalarial drug artemisinin to healthy volunteers increased renal ex-cretion of coumarin metabolites, indicative of induction of CYP2A6 activity.

Hepatocyte nuclear factor 4 α (HNF4 α , NR2A1) is required for the development of the liver as well as for con-trolling the expression of many genes specifi cally expressed in the liver and it is associated with a number of critical metabolic pathways. Among the genes regulated by HNF4 α are the xenobiotic-metabolizing CYPs, especially CYP3A4. HNF4 α acts as a direct transactivator of several CYP genes, suggesting that this factor is a global regulator which supports CYP transcription in the liver. HNF4 α expression displays signifi cant variability in human liver which may account for a proportion of the interindividual variability in the expres-sion of xenobiotic-metabolizing genes and the clearance rate of a wide variety of drugs (52, 53) . Introduction of HNF4 α antisense DNA into primary human hepatocytes markedly downregulated the expression of CYP3A4, CYP3A5, and CYP2A6, evidence for a decisive role of HNF4 α in main-taining constitutive levels of CYP2A6 in liver (54) . HNF4 α

is also crucial for the regulation of constitutive expression of mouse liver Cyp2a5 (55) .

The CAR (NR1I2) and PXR (NR1I3) are ligand-activated transcription factors that regulate numerous CYP and conju-gating enzymes (56) . In a study examining 20 liver samples (57) , correlations were identifi ed among mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, P450 oxidoreductase, UDP-glucuronosyltransferase 1A1, and some transporters, suggesting that these xenobiotic metabolism genes are co-regulated at the transcriptional level, presum-ably by PXR. CYP2A6 was shown to be induced via PXR and PGC-1 α (peroxisome proliferator-activated receptor- γ co-activator 1 α ) through the DR4-like elements at the dis-tal response region. These elements are located at – 6698, – 5476, and – 4618 in the CYP2A6 gene, to which PXR and CAR could bind after dimerization with the retinoid X recep-tor (58) .

Dexamethasone increases CYP2A6 expression in human hepatocytes in primary culture (59) . This effect is attenu-ated by the glucocorticoid receptor (GR) antagonist, mife-pristone. Truncations of the CYP2A6 promoter pinpointed dexamethasone responsiveness to the region containing an HNF4 α response element. Mutation of this element sup-pressed transactivation of CYP2A6, whereas exposure to dexamethasone increased binding of HNF4 α to the response element. Histone H4 acetylation of the CYP2A6 proximal promoter chromatin was increased by dexamethasone; this may allow for increased binding of HNF4 α to the response element. These fi ndings indicated that increased expression of CYP2A6 by dexamethasone is mediated by the GR via a non-conventional transcriptional mechanism involving the inter-action of HNF4 α with an HNF4 α response element rather than a glucocorticoid response element (59) .

Cigarette smoking can affect the rate of metabolism of nicotine. The clearance of nicotine is signifi cantly slower in cigarette smokers compared with non-smokers, and pharma-cokinetic studies with coumarin also support the reducing effect of smoking on CYP2A6-mediated metabolism (31) . These fi ndings suggest that there are as yet unidentifi ed sub-stances in tobacco smoke that can inhibit the metabolism of nicotine. One candidate inhibitor is β -nicotyrine, a minor tobacco alkaloid (60) , but further studies will be needed to clarify its role. Cotinine clearly does not inhibit CYP2A6, and a recent randomized crossover study indicates that high-dose nicotine does not affect nicotine pharmacokinetics (61) . Another possibility is that the reduced CYP2A6 activity is due to downregulation of CYP2A6 expression and not to in-hibition as suggested by the ability of nicotine to decrease CYP2A6 activity by suppressing CYP2A6 mRNA and pro-tein in monkey liver (62) .

Mouse models

Regulation of the mouse CYP2A5 has been extensively inves-tigated. Liver CYP2A5 is upregulated in various conditions in which other xenobiotic-metabolizing enzymes are suppressed. The types of stimuli capable of evoking this effect include toxic chemicals such as carbon tetrachloride, pyrazole, heavy




metals, and porphyrinogenic compounds, pathophysiological conditions such as infections caused by viruses, bacteria and parasites, and liver tumors. This upregulation has been pro-posed to represent a cytoprotective mechanism against oxida-tive stress [reviewed in (1, 3, 5, 23, 63) ].

The mechanisms of induction include transcriptional and post-transcriptional events. Several stress activated transcrip-tion factors, including the Nrf2, AhR, PGC-1 α , and hnRNP A1 control induction of the Cyp2a5 gene during stress situa-tions. Nuclear factor-E2 p45-related factor 2 (Nrf2; NFE2L2) is a transcription factor activated by changes in the cellular redox state. Nrf2 is a major regulator of the Cyp2a5 gene and activation of the Nrf2 pathway may explain CYP2A5 upregulation by many different types of hepatotoxic stimuli [reviewed in (64) ]. Recent studies have indicated that activa-tion of Nrf2 also induces CYP2A6 expression in human liver (65) . In addition, CYP2A5 and CYP2A6 are inducible biliru-bin oxidases possibly controlling intracellular bilirubin levels in transient oxidative stress situations (64) .

The multifunctional protein heterogeneous nuclear ribo-nucleoprotein A1 (hnRNP A1), which is involved in various stages of mRNA turnover, plays an important role in post-transcriptional and putatively also in transcriptional regula-tion of CYP2A5 (64) . The 3 ′ -UTR of CYP2A6 mRNA has been shown to interact with a cytosolic protein which is most probably hnRNP A1. The transcript was stabilized by this interaction in conditions where CYP2A6 gene transcription was impaired (66, 67) . Wang and coworkers (68) identifi ed polymorphic motifs in the 3 ′ -untranslated region of CYP2A6 infl uencing CYP2A6 mRNA stabilization and enzyme expression. However, these motifs are not involved in binding hnRNP A1.

It has long been known that basal and induced rates of hepatic metabolism of CYP2A5-mediated coumarin 7-hy-droxylation are markedly higher in DBA/2J mice than they are in the AKR/J, C57BL/6J, and C3H/HeJ strains (69) . In the rat, very little coumarin 7-hydroxylation occurs in liver, and what activity is present, is not mediated by CYP2A enzymes (70) . A Cyp2a5 knockout mouse model was re-cently described (71) . Mice homozygous for the disrupted allele were indistinguishable from their wild-type litter-mates, or from wild-type B6 mice, in terms of growth rate

and reproductive ability. The phenotypes also indicated that CYP2A5 is not critical for embryonic development and normal growth. While Cyp2a5 knockout mice did not exhibit changes in systemic clearance of testosterone or pro-gesterone, nicotine clearance was signifi cantly reduced in Cyp2a5 -null mice confi rming the role of hepatic CYP2A5 in cotinine formation.

Substrates and inhibitors of CYP2A6

Substrates

The CYP2A6 enzyme participates in the metabolism of sev-eral compounds including drugs and toxic chemicals. Usually CYP2A6 substrates are relatively polar, low to medium molecular weight compounds. For the majority of clinically used drugs, CYP2A6 represents a minor metabolic pathway, or is a high K m , low V max enzyme for the reaction, contri-buting to a minor degree to total metabolic clearance at rele-vant substrate concentrations. There are actually very few drug substrates whose clearance depends exclusively or even mainly on CYP2A6, with coumarin and nicotine being the best examples. These two compounds also have more general toxicological signifi cance, and will be dealt with in more de-tail in separate sections below. Table 2 lists the most relevant CYP2A6 drug substrates.

The relatively minor role of CYP2A6 in the metabolism of drugs becomes apparent when one compares it with the CYP2D6 and especially CYP3A4 forms, which are respon-sible for the metabolism of hundreds of clinically used drugs (91, 92) . Apart from coumarin, nicotine, and perhaps tegafur, the other drugs listed in Table 2 are only partially metabo-lized by CYP2A6, and in most cases, the in vivo kinetics of metabolism has not been elucidated (5) . More detailed lists of CYP2A6 substrates can be found in comprehensive reviews of CYP ligands (21, 93) . Coumarin is the preferred probe agent for determining CYP2A6 activity in vitro and in vivo, as 7-hydroxylation of coumarin is mediated almost exclusive-ly by CYP2A6. Owing to its very low abundance in human liver, CYP2A13 does not play a major role in the systemic clearance of any drug.

Table 2 Selected CYP2A6 drug substrates.

Substrate Therapeutic class or indication References

Coumarin Constituent in many herbal medicinal products (8, 72) Nicotine Smoking reduction products (73 – 75) Methoxyfl urane Anesthetic (76) Halothane Anesthetic (77) Valproic acid Antiepileptic drug (78, 79) Losigamone Antiepileptic drug (experimental) (80) Pilocarpine Cholinergic drug (81) Letrozole Aromatase inhibitor (82, 83) Efavirenz Antiretroviral drug (84, 85) Tegafur Anticancer drug (86 – 89) S-1 (tegafur, 5-chloro-2,4-dihydroxypyridine, potassium oxonate) Anticancer drug (90)




Tegafur, a prodrug of 5-fl uorouracil (5-FU), has been used for over 30 years in cancer chemotherapy. The novel drug S-1 consists of tegafur and two biomodulators: 5-chloro-2,4-dihydroxypyridine and potassium oxonate. Both tegafur and S-1 are widely used in Japan and China (90) . Tegafur has been shown to be converted to 5-FU by CYP2A6 and thymidine phosphorylase in vitro (86 – 88) . Consistent with this, individuals with the CYP2A6*4 deletion allele have reduced 5-FU formation from tegafur in the Japanese (94) and Chinese populations (89) . With regard to S-1, initial clinical trials in the USA and Europe observed diarrhea as the dose-limiting toxic symptom. However, initial Japanese studies reported myelosuppression as the dose-limiting toxi city. The differing dose tolerance in these two popula-tions may be attributable to polymorphisms in the CYP2A6 gene (90) .

Current non-clinical development of drugs involves detailed studies on the metabolic pathways of candidate drugs. These studies aim at identifying specifi c CYP forms catalyz-ing metabolism and potential for drug interactions. CYP2A6 is usually not included in the selection of the CYP forms included in these types of metabolism screens; these lists typically include CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 (95, 96) .

In comparison with drugs, a greater number of compounds of more general toxicological signifi cance are either activated or inactivated by CYP2A6. Examples of such compounds are listed in Table 3 .

Of particular interest are several carcinogenic N -nitrosamines that are metabolized by CYP2A6 and CYP2A13. Especially the tobacco-specifi c 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolically activated to intermediates that form adducts with DNA. The activation pathway is α -hydroxylation, which is more effi ciently cata-lyzed by CYP2A13 than CYP2A6 (13, 114) . This metabolic activation may initiate a cascade of events leading to cancer

formation in tissues exposed to tobacco smoke, especially lung and larynx (115) .

Mentholated cigarettes has been claimed to inhibit the metabolism of nicotine, but the consequences of this addic-tion with regard to addiction or health effects is unclear (116) . This issue is worth exploring, as menthol is legally added to cigarettes, and mentholated cigarettes have a market share of approximately 30 % in the USA (117) .

CYP2A6 is also involved in the metabolism of several endogenous compounds, such as retinoic acid, testosterone, estradiol, and progesterone, but the contribution of CYP2A6 is minor in their overall metabolic pathways (21) . Nothing is known about the possible consequences of the altered metabolism of endogenous compounds due to CYP2A6 ge-netic polymorphism. Recent studies have suggested that bili-rubin might be an endogenous substrate to mouse CYP2A5 (118) .

Inhibitors

Several compounds have been shown to inhibit CYP2A6 activity (Table 4 ). Whereas the inhibitory activity of most of these compounds has been assessed only in vitro, some agents, including some drugs, have been elucidated also in vivo.

The most thoroughly investigated inhibitor of CYP2A6 is methoxsalen, which acts as a mechanism-based (suicide) inhibitor with in vitro K i values of 0.04 – 1.9 μ M. Methoxsalen is not very selective as it also inhibits other CYP forms, es-pecially CYP1A2 and CYP2B6 with low K i values (92) . Presumably because of the effi cient mechanism of action, moderate doses (30 – 50 mg) of methoxsalen are able to sig-nifi cantly reduce CYP2A6 activity in vivo (119, 144, 145) . An elegant chimeric mouse model with a humanized liver has been developed (146) . This model was used to test benzalde-hyde and lactone derivatives as specifi c inhibitors of human CYP2A6 and mouse CYP2A5, respectively (147) . Efforts

Table 3 Examples of toxic and other compounds metabolized by CYP2A6.

Compound Assay/end-point References

Coumarin 7-Hydroxylation, 3,4-epoxidation (8, 72, 97) Nicotine C-oxidation (73 – 75) Cotinine 3 ′ -Hydroxylation (98) NDEA, NDMA Mutagenicity (99) NNK α -Hydroxylation, mutagenicity (100) AFB 1 Epoxide formation, mutagenicity (99) 1,3-Butadiene Monoxide formation (101) Quinoline 1-Oxidation (102) DCBN Protein adduct formation (103) MTBE O -demethylation (104, 105) Chloroform Trichloromethanol formation (106) 1,7-Dimethylxanthine 1,7-Dimethylurate formation (107 – 109) Indole Indoxyl formation (110) Fenchol Oxidation (111) Menthol Hydroxylation (112) Camphor Hydroxylation (113)

AFB 1 , afl atoxin B 1 ; DCBN, 2,6-dichlorobenzonitrile; MTBE, methyl tert-butyl ether; NDEA, N -nitrosodiethylamine; NDMA, N -nitrosodimethylamine; NNK, 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone.




to rationally develop selective CYP2A6 inhibitors will be discussed below.

CYP2A6 and risk assessment of coumarin

Coumarin is a component in some herbal medicinal products used for treating diverse disorders (148, 149) . Along with sa-frole and estragole, coumarin belongs to the group of ingre-dients in spices and herbs that are listed by the Council of Europe as “ active principles ” (150) . People are also exposed to coumarin through diet and from its use in perfumes pres-ent in personal care products. Exposure varies from approxi-mately 1 mg/day from consumption of coumarin-containing food to as high as 7 g/day for short periods during clinical coumarin treatment (97) .

High doses of coumarin administered by the oral route evoke liver toxicity in rodents, especially in rats and some strains of mice. In vitro and in vivo tests have indicated that coumarin is not genotoxic and the induction of tumors at high doses in rodents is attributed to cytotoxicity (97) . Extensive studies have shown that coumarin is rapidly metabolized to the non-toxic 7-hydroxycoumarin by CYP2A6 humans and CYP2A5 in mouse strains expressing high levels of this enzyme in liver. In many other species including the rat,

coumarin is converted to the proximate hepatotoxic metabolite coumarin 3,4-epoxide, this type of reaction being catalyzed by other CYP enzymes (23, 97, 151) . Thus, the species-specifi c liver toxicity is related to the pathways of coumarin metabolism (Figure 1 ).

Administration of high doses of coumarin to patients has revealed subgroups that are more susceptible to the hepato-toxic effect. Although there are many possible explanations, including CYP2A6 genetic polymorphism and effects of liver diseases of CYP2A6 activity, no conclusive links between these putative mechanisms and coumarin toxicity have been established (150) . A physiologically based biokinetic model predicted that 24-h exposure in liver (AUC 0 – 24 h ) of the aver-age CYP2A6 wild-type and CYP2A6*2 homozygote human would be 1000- and 10-fold lower, respectively, than the AUC 0 – 24 h in rat liver (152) . This result is in good agreement with the observed differences in coumarin hepatotoxicity between rat and man.

CYP2A6, nicotine metabolism, and cigarette

smoking

It is nicotine that establishes and then maintains tobacco de-pendence. Nicotine-dependent smokers adjust their cigarette

Table 4 Selected inhibitors of CYP2A6.

Inhibitor Description/source References

Methoxsalen a Psoriasis drug (119 – 122) Pilocarpine Cholinergic alkaloid (121, 123) Tranylcypromine Monoamine oxidase inhibitor (120, 122, 124) Menthofuran a Natural plant constituent (125, 126) Organosulfur compounds Natural plant constituents (127) Tryptamine Endogenous neurotransmitter (122, 128) Isoniazid a Antituberculosis drug (129) Menthol Natural plant constituent (130, 131) 4-Methoxybenzaldehyde Synthetic (132) 2-Bromonaphthalene Synthetic (133) Grapefruit juice Natural plant constituents (134) Genistein Natural isofl avone (135) PPM Synthetic nicotine derivative (136, 137) MIP Synthetic nicotine derivative (136, 137) Decursinol angelate Natural furanocoumarin (138) Selegiline a Monoamine oxidase inhibitor (139) N1-(4-fl uorophenyl) cyclopropane-1-carboxamide Synthetic (140) 1 H-substituted imidazoles Synthetic (141) Chalepensin Natural furanocoumarin (142) 1-Benzothiophene-3-carbaldehyde Synthetic (143)

a Mechanism-based inhibition. PPM, 3-(pyridin-3-yl)- 1 H-(pyrazol-5-yl)methenamine; MIP, 3-(2-methyl- 1 H-imidazol-1-yl)pyridine.

Other CYPsO

O O O O O O OH

Coumarin 3,4-epoxide Coumarin 7-Hydroxycoumarin

CYP2A

Figure 1 A simplifi ed scheme of the initial steps in activation (3,4-epoxide) and detoxifi cation (7-hydroxycoumarin) pathways of coumarin.




consumption to maintain steady levels of nicotine in the brain. Factors infl uencing nicotine plasma levels, by either intake or removal, affect smoking behavior (153) . Approximately 80 % of nicotine is eliminated by CYP2A6-mediated metabolism to cotinine in the liver. In this two-step reaction, nicotine is fi rst oxidized to nicotine iminium ion and subsequently to coti-nine by cytosolic aldehyde oxidase (Figure 2 ). The CYP2A6-mediated formation of nicotine iminium ion is the rate-limiting step in this reaction. Cotinine is further metabolized to trans-3-hydroxycotinine, mediated solely by CYP2A6 (31) . In mice, the majority of nicotine is metabolized by CYP2A5 (154) .

Slow metabolism CYP2A6 alleles are associated with a reduction in the rates of nicotine inactivation. Individuals who are homozygous for CYP2A6 deletion alleles ( CYP2A6*4 / CYP2A6*4 ) excrete reduced amounts of cotinine and hydroxycotinine. The plasma ratio of hydroxycotinine to cotinine has been used as a proxy for CYP2A6 activity, and is correlated with oral nicotine clearance (155) . A recent study has suggested that among European-Americans, seven polymorphisms in the CYP2A6 gene explain the majority of variability in the metabolism of nicotine to cotinine after oral administration (156) . Several studies have shown that slow nicotine inactivators smoke fewer cigarettes per day than in-dividuals with normal metabolic rates. Slower metaboli zers also smoke for shorter durations and are more likely to quit smoking. Rapid metabolism of nicotine is also associated with more severe withdrawal symptoms. These fi ndings sug-gest that nicotine levels remain elevated longer in smokers with slow-metabolizing CYP2A6 gene alleles in compari-son with the situation in individuals with fast-metabolizing alleles, resulting in a decreased need to smoke to avoid with-drawal (157) .

In addition, some studies have indicated that reduced CYP2A6 activity may also alter the rate of acquisition of nicotine dependence and the rate of escalation of dependence. During smoking initiation, slower nicotine metabolism may be associated with greater vulnerability to addiction, per-haps because the prolonged presence of higher brain nicotine concentrations produces more reinforcement. For the same reason, slower metabolism could also enhance the reinforc-ing effects of smoking cigarettes with low nicotine content in adults and adolescents. Slow metabolism could therefore lower the nicotine reinforcement threshold in smoking initia-tion and maintenance (158, 159) .

Several recent studies have evaluated the impact of genetic polymorphisms on smoking behaviors. A genome-wide asso-ciation study (GWAS) in large mainly Caucasian populations (160) found variants in three genomic regions to be associated

with numbers of cigarettes smoked per day, including SNPs at chromosomal loci 15q25, 8p11, and 19q13. These regions contain genes encoding acetylcholine nicotinic receptor sub-units (several CHRN genes in 15q25 and 8p11) as well as CYP2A6 and CYP2B6 (19q13), all of which have been previ-ously linked with smoking and nicotine dependence. The most signifi cant association in the 19q13 region was found with a SNP (rs4105144[C]) that is in linkage disequilibrium with the CYP2A6*2 reduced function allele. A candidate gene study targeting CHRN and CYP2A6 (161) suggested that the slow metabolizer CYP2A6 genotype increases adolescent likeli-hood of being a regular smoker but increases later life quit-ting likelihood and reduces average consumption. The effects of CYP2A6 and CHRNA5 were found to be independent, with each trait doubling the odds of being a regular smoker. Two other candidate gene studies in Spanish Caucasians (162) and Han Chinese (163) found an association between CYP2A6 non-functional alleles and smoking-related phenotypes. Two GWASs in patients with chronic obstructive pulmonary dis-ease (164, 165) confi rmed the associations of the CYP2A6 and some CHRNA loci with smoking intensity. Taken collectively, these studies strengthen the hypothesis that CYP2A6 function can affect smoking behavior.

CYP2A6 genetic variation may also affect the response to nicotine replacement therapy. The reduced CYP2A6 activity has been found in many studies to be associated with higher plasma nicotine levels and substantially greater quitting suc-cess when nicotine was administered as a patch (157) . By contrast, slow metabolizers have equal quit rates relative to normal metabolizers in the group that used nicotine nasal spray. One probable explanation is that nicotine spray, simi-lar to cigarette smoking, allows for adjustment in differences in the nicotine need and rates of metabolism. In one study (166) comparing placebo with bupropion, slow CYP2A6 metabolizers achieved superior quit rates during treatment with placebo compared with fast metabolizers. In addi-tion, when bupropion was compared with placebo, only fast CYP2A6 metabolizers had any additional benefi t. These fi nd-ings suggest that CYP2A6 slow metabolizers have superior quit rates even in the absence of active drug, and this effect is enhanced by the nicotine patch. By contrast, CYP2A6 fast metabolizers respond poorly in the absence of pharmacother-apy and respond relatively well to bupropion.

As nicotine elimination is a key factor in determining the number of cigarettes smoked, a strategy that may be useful for smoking cessation is to reduce the elimination of nico-tine. The decreasing effect on smoking by genetically slow CYP2A6 metabolism can be mimicked by phenocopying,

CYP2A6 Aldehydeoxidase

Continine

N N NO

HH H

N NNNicotine Nicotine-Δ1’(5’)-iminium ion

H3C H2C H3C

+

Figure 2 The fi rst steps in the major pathway of nicotine metabolism.




i.e., suppressing CYP2A6 activity with chemical inhibi-tors. Tyndale and coworkers (167, 168) have suggested that CYP2A6 inhibition could be used as a component of treatment for tobacco dependence, simultaneously reducing exposure to the harmful components of tobacco smoke. Pharmacokinetic studies with the CYP2A6 inhibitors methoxsalen and tranyl-cypromine demonstrated that the inhibitors increase nicotine plasma concentration (144) . In a small proof-of-concept study (169) , oral nicotine and methoxsalen signifi cantly reduced the number of cigarettes smoked, number of puffs, and inhala-tion intensity compared with placebo. These studies were the fi rst evidence that slowing nicotine elimination could be one way to reduce smoking behavior. In addition, blocking fi rst-pass metabolism also makes it possible to administer nicotine orally, which is not an option with present products during nicotine replacement therapy. Administration of methoxsalen to smokers was shown to reduce activation of NNK (145) . Thus, in addition to augmenting smoking cessation, inhibition of nicotine metabolism may provide reduction in exposure to tobacco-specifi c procarcinogens.

The prospect that CYP2A6 inhibitors could be used as drugs in nicotine replacement therapy has prompted ef-forts to devise new inhibitor molecules. Examples of novel inhibitor structures include derivatives of nicotine (136, 137) , naphthalene (133) , and benzothiophene (143) . However, all these compounds are at very early stages of development. To test new inhibitors in vivo, mouse is the preferred model, as CYP2A5 catalyzes nicotine to cotinine (71, 170, 171) .

CYP2A6 and cancer

After the discovery of fi rst variant alleles of the CYP2A6 gene, it was hypothesized that slow CYP2A6 catalyzed metabolism could be protective against tobacco smoke-induced lung cancer. There is a plausible link between CYP2A6 genetic polymorphism and lung cancer risk because the enzyme metabolizes compounds relevant in the carcinogene-sis process, i.e., nicotine and many procarcinogens, especially tobacco-specifi c nitrosamines. Several studies have indicated that slow CYP2A6 metabolizers are indeed at a lower risk for lung cancer. A summary of studies in Asian populations reveals that the defective CYP2A6 alleles appear to protect against lung and head and neck cancers. A meta-analysis yielded an odds ratio (OR) of 0.25 [95 % confi dence interval (CI) 0.16 – 0.39] for CYP2A6*4 compared with CYP2A6*1 homozygotes for tobacco-related cancers [reviewed in (172) ]. A recent study in Caucasians (173) indicated that cigarette consumption and nicotine dependence were greatest in the combined CYP2A6 normal metabolizers and the at-risk group for nicotine receptor polymorphism. The combined risk group also exhibited the greatest lung cancer risk (OR = 2.03; 95 % CI 1.21 – 3.40). Variation in CYP2A6 and nicotine receptors was independently and additively associated with increased cigarette consumption, nicotine dependence, and lung cancer risk. In a GWAS on smoking behaviors in Caucasians (160) , nominal associations were also observed with lung cancer and CYP2A6 slow metabolism genotype, but the effect was rather

weak (OR = 1.09; 95 % CI 1.00 – 1.18). As discussed above, the underlying reason for the association between CYP2A6 slow metabolism and reduced risk for lung cancer could be a lower rate of activation of tobacco procarcinogens and/or lower lev-els of tobacco consumption. However, more work is needed to pinpoint the causal factor(s) for the association.

However, some studies have failed to observe any asso-ciations between CYP2A6 genotype and smoking or cancer risk. This may be due to the variable number of alleles being assessed in different studies and especially the failure to include a suffi cient spectrum of variant alleles in the early studies (168) . Notably, Africa-American smokers are more likely to be CYP2A6 slow metabolizers (155) but have a signifi cantly higher cancer risk compared with Caucasians (174) . Several studies have also been published on CYP2A6 polymorphism and cancers at other sites, such as esopha-gus, stomach, liver, and nasopharynx, usually suggesting no associations (175) .

Experimental animal evidence also supports the involve-ment of CYP2A enzymes in lung cancer initiation. In mouse models, the potent CYP2A5 inhibitor, methoxsalen, very effi -ciently blocks mutagenesis and lung tumor formation caused by NNK (126) . Targeted deletion of the protein kinase Akt2 gene created linkage to a reduced activity Cyp2a5 allele (Ala117Val) that decreased activation of NNK in vitro. Mice with this Cyp2a5 allele had decreased NNK-induced DNA adduct formation in vivo and decreased NNK-induced lung tumor formation (176) .

Structure and modeling of CYP2A6 protein

The CYP structures consist of approximately 500 amino acids organized in two main domains, the α -domain which usually contains 13 α -helices (A – L and B ′ ) and the β -domain with fi ve β -sheets ( β 1 – β 5). The heme binding pocket is located in the α -domain (177) . Elucidation of two CYP2A6 crys-tal structures provided detailed information about CYP2A6 protein (137, 178) . CYP2A6 exhibits the typical mammalian CYP protein fold with two short helices, F ′ and G ′ , in the F – L loop region. There is also a channel from the surface of the protein to the active site between helices F, G and B ′ ; this channel is closed by Glu221 on the F ′ helix. A π - π stacking interaction between Trp109 on the B ′ -helix and Phe238 on the G-helix also stabilizes the compact, “ closed ” conforma-tion of the enzyme. The volume of CYP2A6 active is 260 Å 3 , which is approximately four times smaller than those of CYP2C8, CYP2C9, and CYP3A4. The presence of large aro-matic residues, especially phenylalanines, reduces the volume of the substrate binding site. Residue Asn297 forms a hydro-gen bond in the crystal structures with the ligands coumarin, methoxsalen, and amine derivatives. Residues Phe118 and Thr305 also participate in ligand orientation. Figure 3 illus-trates the tertiary structure and key features of the CYP2A6 active site.

Mutagenesis studies have highlighted the importance of the hydrophobic residues at the CYP2A6 active site. For example, the mutation of Val117 to Ala signifi cantly




decreases enzyme activity (179) . The change of residue size and polarity in the active site may modify the interaction between protein and substrate and affect substrate binding and orientation. Mutation of Asn297 alters the K m and K s val-ues of many substrates such as coumarin. Residue Ala481 is located in the active site of CYP2A6 and the Ala481Thr substitution displayed decreased coumarin binding affi nity (180) . The substrate specifi city of CYP2A6 seems to be ex-panded by Ile300Val and Asn297Gln mutations. Interestingly, the Ile300Val change opens an additional substrate pocket (110) .

Crystal structures of CYP2A6 Asn297Gln, Leu240Cys/Asn297Gln, and Asn297Gln/Ile300Val mutants have also been resolved (181) . Inclusion of four CYP2A13 amino acids resulted in a CYP2A6 mutant protein with binding af-fi nities for substrates much more similar to those observed for CYP2A13 than to those for CYP2A6 without altering coumarin binding (182) . The crystal structures of CYP2A6, CYP2A13, CYP2E1, and the CYP2A6 mutant enzyme were

recently determined with pilocarpine in the active site (183) . Although pilocarpine coordinated to the heme iron in all four structures, the binding orientation was different for CYP2A6, CYP2A13, and CYP2E1. Comparisons revealed how indi-vidual amino acids lining the active sites of these three dis-tinct human enzymes interact differently with the inhibitor pilocarpine.

Mouse CYP2A enzymes have not been crystallized. However, mutation experiments have revealed several key amino acid residues for catalytic specifi city of CYP2A4 and CYP2A5 enzymes indicating that they have undergone selec-tion pressure during evolution. Residues 117, 209, and 365 are critical for substrate specifi city. A single amino acid change, Phe209Leu, is suffi cient to convert the specifi city of CYP2A5 from coumarin to CYP2A4-like steroid hydroxylation (184) . Three simultaneous amino acid substitutions in the CYP2A4 protein to a CYP2A5-like protein (Ala117Val, Leu209Phe, Leu365Met) converts the testosterone hydroxylation of CYP2A4 to coumarin 7-hydroxylation activity (185) .

B’ HelixF Helix

Phe107Phe111

Oxygen pocketPhe209

I Helix

Thr305

Hema

CoumarinVal117

Asn297

Figure 3 CYP2A6 tertiary structure (above) and active site (bottom). The α -helices and β -sheets are marked in red and light blue, respectively. Coumarin (dark blue) is oriented towards heme (gray). Some key amino acid residues in the I, F and B ′ helices are shown in the active site. An oxygen pocket is formed by a local helical distortion in the I helix. Hydrogen bonding between coumarin and Asn297 and three phenylalanine residues are shown.




Several laboratories around the world have employed molecular modeling ( in silico ) approaches to characterize the interactions between CYP2A6 ligands and the active site of the protein. Before the availability of the fi rst crystal structure (178) , homology models with docking were used to defi ne the CYP2A6 active site (186 – 188) . Subsequently, several laboratories have applied different modeling ap-proaches, including ligand-based and target-based methods, to rationally design CYP2A6 inhibitors. We constructed sev-eral different 3-dimensional quantitative structure-activity relationship (3D-QSAR) models to identify the most impor-tant features of CYP2A6 of inhibitors and to identify novel CYP2A6 inhibitors by screening chemical databases. In par-ticular, comparative molecular fi eld analysis (CoMFA) has proved to be valuable in designing novel CYP2A6 inhibitors and predicting the inhibitory activities of test compounds. Several novel alkyl amine and benzaldehyde derivatives were identifi ed during this work (132, 133, 140) .

We recently applied molecular docking and CoMFA combined with virtual screening to design novel CYP2A6 inhibitors (143) . The colors of the CoMFA contour map de-scribed the desired features of CYP2A6 inhibitors (Figure 4 ). Electrostatic fi elds near Asn297 were associated with the presence of hydrogen bonds between the inhibitor and pro-tein. The sterically favored and disfavored regions provided indications about the optimal volume and shape of a potent inhibitor. The steric fi elds (green) suggested that inhibitory potency should increase if the inhibitor is occupied by a hy-drophobic group in these regions. This knowledge will help in synthesizing inhibitors with an optimal fi t in the enzyme active site.

Phe480

Phe107

Leu370

Phe118

Ans297

Figure 4 CoMFA fi elds of the model based on CYP2A6 crystal structure with coumarin as the template. CoMFA electrostatic fi elds: blue, negative charge disfavored area; red, negative-charge favored area. CoMFA steric fi eld: green, bulk favored area; yellow, bulk disfavored area.

Conclusions

In comparison with the other human liver xenobiotic-metabolizing CYP forms, CYP2A6 plays a minor role in the metabolism and total clearance of currently available drugs. Nevertheless, coumarin, nicotine, and tegafur are examples of drugs that are metabolized mainly by CYP2A6. Coumarin is not a widely used drug in most countries, although it may be present in some herbal remedies. Derivatives of coumarin are metabolized by other CYP forms, e.g., warfarin by CYP2C9. Tegafur is an anticancer prodrug activated by CYP2A6 to 5-FU, and its pharmacokinetics appears to be dependent on CYP2A6 activity. However, the clinical consequences of this variability in systemic 5-FU exposure have not been evaluated.

Although CYP2A6 activity is regulated by several end o-genous and exogenous factors, a major part of interindividual variability appears to be explained by genetic polymorphisms in the CYP2A6 gene. Two key pieces of evidence indicate that CYP2A6 in man and CYP2A5 in mouse do not participate in any fundamental physiological functions: (i) CYP2A6 null genotype and phenotype is very prevalent, up to 20 % in some populations; these individuals seem to exhibit no alterations in any other body function except for slower metabolism of CYP2A6 substrates, and (ii) the CYP2A5 knockout mouse has a perfectly normal phenotype. It may well be that both enzymes contribute to coping with endogenous and ex o-genous stress. This situation is analogous to the CYP2D6 in that this enzyme function is lacking in approximately 7 % of Caucasians with no adverse consequences except when these individuals are exposed to drugs that are cleared through CYP2D6.

Much effort has been expended on attempts to elucidate relationships between CYP2A6 function and smoking hab-its. Reduced CYP2A6 activity in smokers appears to be associated with fewer cigarettes being smoked, shorter smok-ing duration, and increased likelihood to quit. Phenocopying genetically slow CYP2A6-mediated nicotine inactivation is possible by administering chemical inhibitors. Some inhibi-tors have been tested in small-scale studies, and the results suggest that cigarette consumption can be reduced in this way. Molecular modeling methods are being used to ratio-nally design novel CYP2A6 inhibitors.

CYP2A6 and especially CYP2A13 also play a role in activating tobacco-specifi c nitrosamines; thus, there is a fascinating link between the metabolism of nicotine, the addictive component in tobacco, and the metabolism of nitrosamines, important etiological factors in lung cancer. In populations where CYP2A6 non-functional alleles are common (e.g., Japanese), the protecting effect of CYP2A6 slow metabolism on lung cancer risk is evident. By con-trast, this effect is less clear in Caucasians and other popu-lations in which there is a low prevalence of CYP2A6 slow metabolizers. It is still unclear whether the risk for lung cancer is mediated through the effect on smoking behav-ior or whether it also involves increased vulnerability to the carcinogenic effects of compounds present in cigarette smoke.




Acknowledgments

We thank Risto Juvonen and Ewen MacDonald for help on preparing the manuscript.

Confl ict of interest statement

Authors ’ confl ict of interest disclosure: The authors stated that there are no confl icts of interest regarding the publication of this article. Research support played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Research funding: The work by the authors is supported by the Academy of Finland (grants 118434 and 137589). Employment or leadership: None declared. Honorarium: None declared.

References

1. Fernandez-Salguero P, Gonzalez FJ. The CYP2A gene subfam-ily: species differences, regulation, catalytic activities and role in chemical carcinogenesis. Pharmacogenetics 1995;5:S123 – 8.

2. Chang TK, Waxman DJ. CYP2A subfamily. In: Ioannides C, editor. Cytochrome P450 – metabolic and toxicological aspects. Boca Raton, FL: CRC Press, 1996:99 – 134.

3. Honkakoski P, Negishi M. The structure, function, and regu-lation of cytochrome P450 2A enzymes. Drug Metab Rev 1997;29:977 – 96.

4. Su T, Ding X. Regulation of the cytochrome P450 2A genes. Toxicol Appl Pharmacol 2004;199:285 – 94.

5. Raunio H, Hakkola J, Pelkonen O. The CYP2A subfamily. In: Ioannides C, editor. Cytochrome P450 – role in metabolism and toxicity of drugs and other xenobiotics. Cambridge: RSC, Advancing the Chemical Sciences Publishing, 2008:151 – 71.

6. Hoffman SM, Fernandez-Salguero P, Gonzalez FJ, Mohrenweiser HW. Organization and evolution of the cytochrome P450 CYP2A-2B-2F subfamily gene cluster on human chromosome 19. J Mol Evol 1995;41:894 – 900.

7. Hoffman SM, Nelson DR, Keeney DS. Organization, structure and evolution of the CYP2 gene cluster on human chromosome 19. Pharmacogenetics 2001;11:687 – 98.

8. Miles JS, McLaren AW, Forrester LM, Glancey MJ, Lang MA, Wolf CR. Identifi cation of the human liver cytochrome P-450 responsible for coumarin 7-hydroxylase activity. Biochem J 1990;267:365 – 71.

9. Yamano S, Tatsuno J, Gonzalez FJ. The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochemistry 1990;29:1322 – 9.

10. Yun CH, Shimada T, Guengerich FP. Purifi cation and character-ization of human liver microsomal cytochrome P-450 2A6. Mol Pharmacol 1991;40:679 – 85.

11. Koskela S, Hakkola J, Hukkanen J, Pelkonen O, Sorri M, Saranen A, et al. Expression of CYP2A genes in human liver and extrahe-patic tissues. Biochem Pharmacol 1999;57:1407 – 13.

12. Redlich G, Zanger UM, Riedmaier S, Bache N, Giessing AB, Eisenacher M, et al. Distinction between human cytochrome P450 (CYP) isoforms and identifi cation of new phosphorylation sites by mass spectrometry. J Proteome Res 2008;7:4678 – 88.

13. Su T, Bao Z, Zhang QY, Smith TJ, Hong JY, Ding X. Human cytochrome P450 CYP2A13: predominant expression in the respiratory tract and its high effi ciency metabolic activation

of a tobacco-specifi c carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res 2000;60:5074 – 9.

14. Chen Y, Liu YQ, Su T, Ren X, Shi L, Liu D, et al. Immunoblot analysis and immunohistochemical characterization of CYP2A expression in human olfactory mucosa. Biochem Pharmacol 2003;66:1245 – 51.

15. Newland N, Baxter A, Hewitt K, Minet E. CYP1A1/1B1 and CYP2A6/2A13 activity is conserved in cultures of differentiated primary human tracheobronchial epithelial cells. Toxicol In vitro 2011;25:922 – 9.

16. Hukkanen J, Pelkonen O, Hakkola J, Raunio H. Expression and regulation of xenobiotic-metabolizing cytochrome P450 (CYP) enzymes in human lung. Crit Rev Toxicol 2002;32:391 – 411.

17. Ding X, Kaminsky LS. Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts. Annu Rev Pharmacol Toxicol 2003;43:149 – 73.

18. Hakkola J, Tanaka E, Pelkonen O. Developmental expression of cytochrome P450 enzymes in human liver. Pharmacol Toxicol 1998;82:209 – 17.

19. Hines RN, McCarver DG. The ontogeny of human drug-metab-olizing enzymes: phase I oxidative enzymes. J Pharmacol Exp Ther 2002;300:355 – 60.

20. Oscarson M. Genetic polymorphisms in the cytochrome P450 2A6 (CYP2A6) gene: implications for interindividual differ-ences in nicotine metabolism. Drug Metab Dispos 2001;29:91 – 5.

21. Di YM, Chow VD, Yang LP, Zhou SF. Structure, function, regu-lation and polymorphism of human cytochrome P450 2A6. Curr Drug Metab 2009;10:754 – 80.

22. Waxman DJ, Holloway MG. Sex differences in the expres-sion of hepatic drug metabolizing enzymes. Mol Pharmacol 2009;76:215 – 28.

23. Pelkonen O, Rautio A, Raunio H, Pasanen M. CYP2A6: a human coumarin 7-hydroxylase. Toxicology 2000;144:139 – 47.

24. Benowitz NL, Lessov-Schlaggar CN, Swan GE, Jacob P III. Female sex and oral contraceptive use accelerate nicotine me-tabolism. Clin Pharmacol Ther 2006;79:480 – 8.

25. Sinues B, Fanlo A, Mayayo E, Carcas C, Vicente J, Arenaz I, et al. CYP2A6 activity in a healthy Spanish population: effect of age, sex, smoking, and oral contraceptives. Hum Exp Toxicol 2008;27:367 – 72.

26. Al Koudsi N, Hoffmann EB, Assadzadeh A, Tyndale RF. Hepatic CYP2A6 levels and nicotine metabolism: impact of genetic, physiological, environmental, and epigenetic factors. Eur J Clin Pharmacol 2010;66:239 – 51.

27. Dempsey D, Jacob P III, Benowitz NL. Accelerated metabolism of nicotine and cotinine in pregnant smokers. J Pharmacol Exp Ther 2002;301:594 – 8.

28. Zhang Y, Klein K, Sugathan A, Nassery N, Dombkowski A, Zanger UM, et al. Transcriptional profi ling of human liver identi-fi es sex-biased genes associated with polygenic dyslipidemia and coronary artery disease. PLoS One 2011;6:e23506.

29. Higashi E, Fukami T, Itoh M, Kyo S, Inoue M, Yokoi T, et al. Human CYP2A6 is induced by estrogen via estrogen receptor. Drug Metab Dispos 2007;35:1935 – 41.

30. Bi è che I, Girault I, Urbain E, Tozlu S, Lidereau R. Relationship between intratumoral expression of genes coding for xenobiotic-metabolizing enzymes and benefi t from adjuvant tamoxifen in estrogen receptor alpha-positive postmenopausal breast carci-noma. Breast Cancer Res 2004;6:R252 – 63.

31. Hukkanen J, Jacob P III, Benowitz NL. Metabolism and disposi-tion kinetics of nicotine. Pharmacol Rev 2005;57:79 – 115.




32. Palmer CN, Coates PJ, Davies SE, Shephard EA, Phillips IR. Localization of cytochrome P-450 gene expression in normal and diseased human liver by in situ hybridization of wax-embedded archival material. Hepatology 1992;16:682 – 7.

33. Kirby GM, Batist G, Alpert L, Lamoureux E, Cameron RG, Alaoui-Jamali MA. Overexpression of cytochrome P-450 iso-forms involved in afl atoxin B1 bioactivation in human liver with cirrhosis and hepatitis. Toxicol Pathol 1996;24:458 – 67.

34. Niemel ä O, Parkkila S, Juvonen RO, Viitala K, Gelboin HV, Pasanen M. Cytochromes P450 2A6, 2E1, and 3A and production of protein-aldehyde adducts in the liver of patients with alcoholic and non-alcoholic liver diseases. J Hepatol 2000;33:893 – 901.

35. Yongvanit P, Phanomsri E, Namwat N, Kampan J, Tassaneeyakul W, Loilome W, et al. Hepatic cytochrome P450 2A6 and 2E1 sta-tus in peri-tumor tissues of patients with Opisthorchis viverrini-associated cholangiocarcinoma. Parasitol Int 2012;61:162 – 6.

36. Satarug S, Lang MA, Yongvanit P, Sithithaworn P, Mairiang E, Mairiang P, et al. Induction of cytochrome P450 2A6 expression in humans by the carcinogenic parasite infection, Opisthorchiasis viverrini. Cancer Epidemiol Biomarkers Prev 1996;5:795 – 800.

37. Raunio H, Juvonen R, Pasanen M, Pelkonen O, P ä ä kk ö P, Soini Y. Cytochrome P4502A6 (CYP2A6) expression in human hepa-tocellular carcinoma. Hepatology 1998;27:427 – 32.

38. Renton KW. Regulation of drug metabolism and disposition dur-ing infl ammation and infection. Expert Opin Drug Metab Toxicol 2005;1:629 – 40.

39. Pasanen M, Rannala Z, Tooming A, Sotaniemi EA, Pelkonen O, Rautio A. Hepatitis A impairs the function of human hepatic CYP2A6 in vivo. Toxicology 1997;123:177 – 84.

40. Maurice M, Emiliani S, Dalet-Beluche I, Derancourt J, Lange R. Isolation and characterization of a cytochrome P450 of the IIA subfamily from human liver microsomes. Eur J Biochem 1991;200:511 – 7.

41. Dalet-Beluche I, Boulenc X, Fabre G, Maurel P, Bonfi ls C. Purifi cation of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papio papio) liver microsomes. Cross reactivity with human forms. Eur J Biochem 1992;204:641 – 8.

42. Mattes WB, Li AP. Quantitative reverse transcriptase/PCR assay for the measurement of induction in cultured hepatocytes. Chem Biol Interact 1997;107:47 – 61.

43. Donato MT, Viitala P, Rodriguez-Antona C, Lindfors A, Castell JV, Raunio H, et al. CYP2A5/CYP2A6 expression in mouse and human hepatocytes treated with various in vivo inducers. Drug Metab Dispos 2000;28:1321 – 6.

44. Meunier V, Bourri é M, Julian B, Marti E, Guillou F, Berger Y, et al. Expression and induction of CYP1A1/1A2, CYP2A6 and CYP3A4 in primary cultures of human hepatocytes: a 10-year follow-up. Xenobiotica 2000;30:589 – 607.

45. Rae JM, Johnson MD, Lippman ME, Flockhart DA. Rifampin is a selective, pleiotropic inducer of drug metabolism genes in human hepatocytes: studies with cDNA and oligonucleotide expression arrays. J Pharmacol Exp Ther 2001;299:849 – 57.

46. Edwards RJ, Price RJ, Watts PS, Renwick AB, Tredger JM, Boobis AR, et al. Induction of cytochrome P450 enzymes in cultured precision-cut human liver slices. Drug Metab Dispos 2003;31:282 – 8.

47. Madan A, Graham RA, Carroll KM, Mudra DR, Burton LA, Krueger LA, et al. Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human he-patocytes. Drug Metab Dispos 2003;31:421 – 31.

48. Sotaniemi EA, Rautio A, B ä ckstrom M, Arvela P, Pelkonen O. CYP3A4 and CYP2A6 activities marked by the metabolism of

lignocaine and coumarin in patients with liver and kidney dis-eases and epileptic patients. Br J Clin Pharmacol 1995;39:71 – 6.

49. Williams JM, Gandhi KK, Benowitz NL. Carbamazepine but not valproate induces CYP2A6 activity in smokers with mental ill-ness. Cancer Epidemiol Biomarkers Prev 2010;19:2582 – 9.

50. Maglich JM, Parks DJ, Moore LB, Collins JL, Goodwin B, Billin AN, et al. Identifi cation of a novel human constitutive androstane receptor (CAR) agonist and its use in the identifi cation of CAR target genes. J Biol Chem 2003;278:17277 – 83.

51. Asimus S, Hai TN, Van Huong N, Ashton M. Artemisinin and CYP2A6 activity in healthy subjects. Eur J Clin Pharmacol 2008;64:283 – 92.

52. Gonzalez FJ. Regulation of hepatocyte nuclear factor 4 alpha-mediated transcription. Drug Metab Pharmacokinet 2008;23:2 – 7.

53. Jover R, Moya M, G ó mez-Lech ó n MJ. Transcriptional regula-tion of cytochrome p450 genes by the nuclear receptor hepato-cyte nuclear factor 4-alpha. Curr Drug Metab 2009;10:508 – 19.

54. Jover R, Bort R, G ó mez-Lech ó n MJ, Castell JV. Cytochrome P450 regulation by hepatocyte nuclear factor 4 in human hepa-tocytes: a study using adenovirus-mediated antisense targeting. Hepatology 2001;33:668 – 75.

55. Ulvila J, Arpiainen S, Pelkonen O, Aida K, Sueyoshi T, Negishi M, et al. Regulation of Cyp2a5 transcription in mouse primary hepatocytes: roles of hepatocyte nuclear factor 4 and nuclear fac-tor I. Biochem J 2004;381:887 – 94.

56. Timsit YE, Negishi M. CAR and PXR: the xenobiotic-sensing receptors. Steroids 2007;72:231 – 46.

57. Wortham M, Czerwinski M, He L, Parkinson A, Wan YJ. Expression of constitutive androstane receptor, hepatic nuclear factor 4 alpha, and P450 oxidoreductase genes determines inter-individual variability in basal expression and activity of a broad scope of xenobiotic metabolism genes in the human liver. Drug Metab Dispos 2007;35:1700 – 10.

58. Itoh M, Nakajima M, Higashi E, Yoshida R, Nagata K, Yamazoe Y, et al. Induction of human CYP2A6 is mediated by the preg-nane X receptor with peroxisome proliferator-activated receptor- γ coactivator 1 α . J Pharmacol Exp Ther 2006;319:693 – 702.

59. Onica T, Nichols K, Larin M, Ng L, Maslen A, Dvorak Z, et al. Dexamethasone-mediated up-regulation of human CYP2A6 involves the glucocorticoid receptor and increased binding of he-patic nuclear factor 4 α to the proximal promoter. Mol Pharmacol 2008;73:451 – 60.

60. Denton TT, Zhang X, Cashman JR. Nicotine-related alkaloids and metabolites as inhibitors of human cytochrome P-450 2A6. Biochem Pharmacol 2004;67:751 – 6.

61. Hukkanen J, Jacob P, Peng M, Dempsey D, Benowitz NL. Effects of nicotine on cytochrome P450 2A6 and 2E1 activities. Br J Clin Pharmacol 2010;69:152 – 9.

62. Schoedel KA, Sellers EM, Palmour R, Tyndale RF. Down-regulation of hepatic nicotine metabolism and a CYP2A6-like enzyme in African green monkeys after long-term nicotine ad-ministration. Mol Pharmacol 2003;63:96 – 104.

63. Kirby GM, Nichols KD, Antenos M. CYP2A5 induction and he-patocellular stress: an adaptive response to perturbations of heme homeostasis. Curr Drug Metab 2011;12:186 – 97.

64. Abu-Bakar A, Hakkola J, Juvonen R, Rahnasto-Rilla M, Raunio H, Lang MA. Function and regulation of the Cyp2a5 / CYP2A6 genes in response to toxic insults. Curr Drug Metab 2012;Mar 19, in press.

65. Yokota S, Higashi E, Fukami T, Yokoi T, Nakajima M. Human CYP2A6 is regulated by nuclear factor-erythroid 2 related factor 2. Biochem Pharmacol 2011;81:289 – 94.




66. Gilmore J, Rotondo F, Pelletier AM, LaMarre J, Alaoui-Jamali M, Kirby GM. Identifi cation of a 43-kDa protein in human liver cytosol that binds to the 3 ′ -untranslated region of CYP2A6 mRNA. Biochem Pharmacol 2001;62:669 – 78.

67. Christian K, Lang M, Maurel P, Raffalli-Mathieu F. Interaction of heterogeneous nuclear ribonucleoprotein A1 with cyto-chrome P450 2A6 mRNA: implications for post-transcription-al regulation of the CYP2A6 gene. Mol Pharmacol 2004;65:1405 – 14.

68. Wang J, Pitarque M, Ingelman-Sundberg M. 3 ′ -UTR poly-morphism in the human CYP2A6 gene affects mRNA stabil-ity and enzyme expression. Biochem Biophys Res Commun 2006;340:491 – 7.

69. Wood AW, Conney AH. Genetic variation in coumarin hy-droxylase activity in the mouse (Mus musculus). Science 1974;185:612 – 4.

70. Honkakoski P, M ä enp ä ä J, Leikola J, Pasanen M, Juvonen R, Lang MA, et al. Cytochrome P4502A-mediated coumarin 7-hydroxylation and testosterone hydroxylation in mouse and rat lung. Pharmacol Toxicol 1993;72:107 – 12.

71. Zhou X, Zhuo X, Xie F, Kluetzman K, Shu YZ, Humphreys WG, et al. Role of CYP2A5 in the clearance of nicotine and cotinine: insights from studies on a Cyp2a5-null mouse model. J Pharmacol Exp Ther 2010;332:578 – 87.

72. Yamano S, Tatsuno J, Gonzalez FJ. The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochemistry 1990;29:1322 – 9.

73. Cashman JR, Park SB, Yang ZC, Wrighton SA, Jacob P III, Benowitz NL. Metabolism of nicotine by human liver mi-crosomes: stereoselective formation of trans-nicotine N ′ -oxide. Chem Res Toxicol 1992;5:639 – 46.

74. Nakajima M, Yamamoto T, Nunoya K, Yokoi T, Nagashima K, Inoue K, et al. Role of human cytochrome P4502A6 in C-oxidation of nicotine. Drug Metab Dispos 1996;24:1212 – 7.

75. Messina ES, Tyndale RF, Sellers EM. A major role for CYP2A6 in nicotine C-oxidation by human liver microsomes. Pharmacol Exp Ther 1997;282:1608 – 14.

76. Kharasch ED, Hankins DC, Thummel KE. Human kidney meth-oxyfl urane and sevofl urane metabolism. Intrarenal fl uoride production as a possible mechanism of methoxyfl urane nephro-toxicity. Anesthesiology 1995;82:689 – 99.

77. Kharasch ED, Hankins DC, Fenstamaker K, Cox K. Human halot-hane metabolism, lipid peroxidation, and cytochromes P(450)2A6 and P(450)3A4. Eur J Clin Pharmacol 2000;55:853 – 9.

78. Sadeque AJ, Fisher MB, Korzekwa KR, Gonzalez FJ, Rettie AE. Human CYP2C9 and CYP2A6 mediate formation of the hepatotoxin 4-ene-valproic acid. J Pharmacol Exp Ther 1997;283:698 – 703.

79. Tan L, Yu JT, Sun YP, Ou JR, Song JH, Yu Y. The infl uence of cytochrome oxidase CYP2A6, CYP2B6, and CYP2C9 polymor-phisms on the plasma concentrations of valproic acid in epileptic patients. Clin Neurol Neurosurg 2010;112:320 – 3.

80. Luszczki JJ. Third-generation antiepileptic drugs: mechanisms of action, pharmacokinetics and interactions. Pharmacol Rep 2009;61:197 – 216.

81. Endo T, Ban M, Hirata K, Yamamoto A, Hara Y, Momose Y. Involvement of CYP2A6 in the formation of a novel metabo-lite, 3-hydroxypilocarpine, from pilocarpine in human liver mi-crosomes. Drug Metab Dispos 2007;35:476 – 83.

82. Murai K, Yamazaki H, Nakagawa K, Kawai R, Kamataki T. Deactivation of anti-cancer drug letrozole to a carbinol me-tabolite by polymorphic cytochrome P450 2A6 in human liver microsomes. Xenobiotica 2009;39:795 – 802.

83. Desta Z, Kreutz Y, Nguyen AT, Li L, Skaar T, Kamdem LK, et al. Plasma letrozole concentrations in postmenopausal women with breast cancer are associated with CYP2A6 genetic vari-ants, body mass index, and age. Clin Pharmacol Ther 2011;90:693 – 700.

84. di Iulio J, Fayet A, Arab-Alameddine M, Rotger M, Lubomirov R, Cavassini M, et al.; Swiss HIV Cohort Study. In vivo analysis of efavirenz metabolism in individuals with impaired CYP2A6 function. Pharmacogenet Genomics 2009;19:300 – 9.

85. Kwara A, Lartey M, Sagoe KW, Rzek NL, Court MH. CYP2B6 (c.516G → T) and CYP2A6 (*9B and/or *17) polymorphisms are independent predictors of efavirenz plasma concentra-tions in HIV-infected patients. Br J Clin Pharmacol 2009;67:427 – 36.

86. Ikeda K, Yoshisue K, Matsushima E, Nagayama S, Kobayashi K, Tyson CA, et al. Bioactivation of tegafur to 5-fl uorouracil is catalyzed by cytochrome P-450 2A6 in human liver microsomes in vitro. Clin Cancer Res 2000;6:4409 – 15.

87. Komatsu T, Yamazaki H, Shimada N, Nakajima M, Yokoi T. Roles of cytochromes P450 1A2, 2A6, and 2C8 in 5-fl uorouracil formation from tegafur, an anticancer prodrug, in human liver microsomes. Drug Metab Dispos 2000;28:1457 – 63.

88. Kajita J, Fuse E, Kuwabara T, Kobayashi H. The contribution of cytochrome P450 to the metabolism of tegafur in human liver. Drug Metab Pharmacokinet 2003;18:303 – 9.

89. Wang H, Bian T, Liu D, Jin T, Chen Y, Lin A, et al. Association analysis of CYP2A6 genotypes and haplotypes with 5-fl uo-rouracil formation from tegafur in human liver microsomes. Pharmacogenomics 2011;12:481 – 92.

90. Blum M, Suzuki A, Ajani JA. A comprehensive review of S-1 in the treatment of advanced gastric adenocarcinoma. Future Oncol 2011;7:715 – 26.

91. Wienkers LC, Heath TG. Predicting in vivo drug interac-tions from in vitro drug discovery data. Nat Rev Drug Discov 2005;4:825 – 33.

92. Pelkonen O, Turpeinen M, Hakkola J, Honkakoski P, Hukkanen J, Raunio H. Inhibition and induction of human cytochrome P450 enzymes: current status. Arch Toxicol 2008;82:667 – 715.

93. Rendic S. Summary of information on human CYP en-zymes: human P450 metabolism data. Drug Metab Rev 2002;34:83 – 448.

94. Fujita K, Yamamoto W, Endo S, Endo H, Nagashima F, Ichikawa W, et al. CYP2A6 and the plasma level of 5-chloro-2, 4-dihydroxypyridine are determinants of the pharmacokinetic vari-ability of tegafur and 5-fl uorouracil, respectively, in Japanese pa-tients with cancer given S-1. Cancer Sci 2008;99:1049 – 54.

95. European Medicines Agency. Guideline on the investigation of drug interactions (Draft). EMA/CHMP/EWP/125211/2010.

96. Zhang L, Reynolds KS, Zhao P, Huang SM. Drug interactions evaluation: an integrated part of risk assessment of therapeutics. Toxicol Appl Pharmacol 2010;243:134 – 45.

97. Felter SP, Vassallo JD, Carlton BD, Daston GP. A safety assess-ment of coumarin taking into account species-specifi city of toxi-cokinetics. Food Chem Toxicol 2006;44:462 – 75.

98. Nakajima M, Yamamoto T, Nunoya K, Yokoi T, Nagashima K, Inoue K, et al. Characterization of CYP2A6 involved in 3 ′ -hydroxylation of cotinine in human liver microsomes. J Pharmacol Exp Ther 1996;277:1010 – 5.

99. Crespi CL, Penman BW, Leakey JA, Arlotto MP, Stark A, Parkinson A, et al. Human cytochrome P450IIA3: cDNA se-quence, role of the enzyme in the metabolic activation of pro-mutagens, comparison to nitrosamine activation by human cytochrome P450IIE1. Carcinogenesis 1990;11:1293 – 300.




100. Yamazaki H, Inui Y, Yun CH, Guengerich FP, Shimada T. Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes. Carcinogenesis 1992;13:1789 – 94.

101. Duescher RJ, Elfarra AA. Human liver microsomes are effi cient catalysts of 1,3-butadiene oxidation: evidence for major roles by cytochromes P450 2A6 and 2E1. Arch Biochem Biophys 1994;311:342 – 9.

102. Reigh G, McMahon H, Ishizaki M, Ohara T, Shimane K, Esumi Y, et al. Cytochrome P450 species involved in the metabolism of quinoline. Carcinogenesis 1996;17:1989 – 96.

103. Ding X, Spink DC, Bhama JK, Sheng JJ, Vaz AD, Coon MJ. Metabolic activation of 2,6-dichlorobenzonitrile, an olfactory-specifi c toxicant, by rat, rabbit, and human cytochromes P450. Mol Pharmacol 1996;49:1113 – 21.

104. Hong JY, Wang YY, Bondoc FY, Lee M, Yang CS, Hu WY, et al. Metabolism of methyl tert-butyl ether and other gasoline ethers by human liver microsomes and heterologously expressed human cytochromes P450: identifi cation of CYP2A6 as a major catalyst. Toxicol Appl Pharmacol 1999;160:43 – 8.

105. Shamsipur M, Miran Beigi AA, Teymouri M, Poursaberi T, Mostafavi SM, Soleimani P, et al. Biotransformation of methyl tert-butyl ether by human cytochrome P450 2A6. Biodegradation 2011;Mar 19:Epub ahead of print.

106. Gemma S, Vittozzi L, Testai E. Metabolism of chloroform in the human liver and identifi cation of the competent P450s. Drug Metab Dispos 2003;31:266 – 74.

107. Sweeney C, Coles BF, Nowell S, Lang NP, Kadlubar FF. Novel markers of susceptibility to carcinogens in diet: associations with colorectal cancer. Toxicology 2002;27:183 – 7.

108. Nowell S, Sweeney C, Hammons G, Kadlubar FF, Lang NP. CYP2A6 activity determined by caffeine phenotyping: associa-tion with colorectal cancer risk. Cancer Epidemiol Biomarkers Prev 2002;11:377 – 83.

109. Kimura M, Yamazaki H, Fujieda M, Kiyotani K, Honda G, Saruwatari J, et al. CYP2A6 is a principal enzyme involved in hydroxylation of 1,7-dimethylxanthine, a main caffeine metab-olite, in humans. Drug Metab Dispos 2005;33:1361 – 6.

110. Wu ZL, Podust LM, Guengerich FP. Expansion of substrate specifi city of cytochrome P450 2A6 by random and site-direct-ed mutagenesis. J Biol Chem 2005;280:41090 – 100.

111. Miyazawa M, Gyoubu K. Roles of human CYP2A6 and rat CYP2B1 in the oxidation of ( + )-fenchol by liver microsomes. Xenobiotica 2007;37:943 – 53.

112. Miyazawa M, Marumoto S, Takahashi T, Nakahashi H, Haigou R, Nakanishi K. Metabolism of ( + )- and ( – )-menthols by CYP2A6 in human liver microsomes. J Oleo Sci 2011;60:127 – 32.

113. Nakahashi H, Miyazawa M. Biotransformation of ( – )-cam-phor by Salmonella typhimurium OY1002/2A6 expressing human CYP2A6 and NADPH-P450 reductase. J Oleo Sci 2011;60:545 – 8.

114. Jalas JR, Hecht SS, Murphy SE. Cytochrome P450 enzymes as catalysts of metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco specifi c carcinogen. Chem Res Toxicol 2005;18:95 – 110.

115. Hecht SS. Progress and challenges in selected areas of tobacco carcinogenesis. Chem Res Toxicol 2008;21:160 – 71.

116. Benowitz NL. Clinical pharmacology of nicotine: implications for understanding, preventing, and treating tobacco addiction. Clin Pharmacol Ther 2008;83:531 – 41.

117. Benowitz NL, Samet JM. The threat of menthol cigarettes to US public health. N Engl J Med 2011;364:2179 – 81.

118. Abu-Bakar A, Arthur DM, Aganovic S, Ng JC, Lang MA. Inducible bilirubin oxidase: a novel function for the mouse cy-tochrome P450 2A5. Toxicol Appl Pharmacol 2011;257:14 – 22.

119. M ä enp ä ä J, Juvonen R, Raunio H, Rautio A, Pelkonen O. Metabolic interactions of methoxsalen and coumarin in humans and mice. Biochem Pharmacol 1994;48:1363 – 9.

120. Draper AJ, Madan A, Parkinson A. Inhibition of coumarin 7-hydroxylase activity in human liver microsomes. Arch Biochem Biophys 1997;341:47 – 61.

121. Koenigs LL, Trager WF. Mechanism-based inactivation of P450 2A6 by furanocoumarins. Biochemistry 1998;37:10047 – 61.

122. Zhang W, Kilicarslan T, Tyndale RF, Sellers EM. Evaluation of methoxsalen, tranylcypromine, and tryptamine as specifi c and selective CYP2A6 inhibitors in vitro. Drug Metab Dispos 2001;29:897 – 902.

123. Kimonen T, Juvonen RO, Alhava E, Pasanen M. The inhibition of CYP enzymes in mouse and human liver by pilocarpine. Br J Pharmacol 1995;114:832 – 6.

124. Taavitsainen P, Juvonen R, Pelkonen O. In vitro inhibition of cytochrome P450 enzymes in human liver microsomes by a po-tent CYP2A6 inhibitor, trans-2-phenylcyclopropylamine (tra-nylcypromine), and its nonamine analog, cyclopropylbenzene. Drug Metab Dispos 2001;29:217 – 22.

125. Khojasteh-Bakht SC, Koenigs LL, Peter RM, Trager WF, Nelson SD. (R)-( + )-Menthofuran is a potent, mechanism-based inactivator of human liver cytochrome P450 2A6. Drug Metab Dispos 1998;26:701 – 4.

126. Miyazaki M, Yamazaki H, Takeuchi H, Saoo K, Yokohira M, Masumura K, et al. Mechanisms of chemopreventive ef-fects of 8-methoxypsoralen against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mouse lung adenomas. Carcinogenesis 2005;26:1947 – 55.

127. Fujita K, Kamataki T. Screening of organosulfur com-pounds as inhibitors of human CYP2A6. Drug Metab Dispos 2001;29:983 – 9.

128. Higashi E, Nakajima M, Katoh M, Tokudome S, Yokoi T. Inhibitory effects of neurotransmitters and steroids on human CYP2A6. Drug Metab Dispos 2007;35:508 – 14.

129. Wen X, Wang JS, Neuvonen PJ, Backman JT. Isoniazid is a mechanism-based inhibitor of cytochrome P450 1A2, 2A6, 2C19 and 3A4 isoforms in human liver microsomes. Eur J Clin Pharmacol 2002;57:799 – 804.

130. MacDougall JM, Fandrick K, Zhang X, Serafi n SV, Cashman JR. Inhibition of human liver microsomal (S)-nicotine oxi-dation by ( – )-menthol and analogues. Chem Res Toxicol 2003;16:988 – 93.

131. Benowitz NL, Herrera B, Jacob P III. Mentholated cigarette smoking inhibits nicotine metabolism. J Pharmacol Exp Ther 2004;310:1208 – 15.

132. Rahnasto M, Raunio H, Poso A, Juvonen RO. More potent in-hibition of human CYP2A6 than mouse CYP2A5 enzyme ac-tivities by derivatives of phenylethylamine and benzaldehyde. Xenobiotica 2003;33:529 – 39.

133. Rahnasto M, Raunio H, Poso A, Wittekindt C, Juvonen RO. Quantitative structure-activity relationship analysis of inhibi-tors of the nicotine metabolizing CYP2A6 enzyme. J Med Chem 2005;48:440 – 9.

134. Hukkanen J, Jacob P III, Benowitz NL. Effect of grapefruit juice on cytochrome P450 2A6 and nicotine renal clearance. Clin Pharmacol Ther 2006;80:522 – 30.

135. Nakajima M, Itoh M, Yamanaka H, Fukami T, Tokudome S, Yamamoto Y, et al. Isofl avones inhibit nicotine C-oxidation cat-alyzed by human CYP2A6. J Clin Pharmacol 2006;46:337 – 44.




136. Denton TT, Zhang X, Cashman JR. 5-Substituted, 6-substituted, and unsubstituted 3-heteroaromatic pyridine analogues of nico-tine as selective inhibitors of cytochrome P-450 2A6. J Med Chem 2005;48:224 – 39.

137. Yano JK, Denton TT, Cerny MA, Zhang X, Johnson EF, Cashman JR. Synthetic inhibitors of cytochrome P-450 2A6: inhibitory activity, difference spectra, mechanism of inhibition, and pro-tein cocrystallization. J Med Chem 2006;49:6987 – 7001.

138. Yoo HH, Lee MW, Kim YC, Yun CH, Kim DH. Mechanism-based inactivation of cytochrome P450 2A6 by decursinol angelate isolated from Angelica gigas. Drug Metab Dispos 2007;35:1759 – 65.

139. Siu EC, Tyndale RF. Selegiline is a mechanism-based inactiva-tor of CYP2A6 inhibiting nicotine metabolism in humans and mice. J Pharmacol Exp Ther 2008;324:992 – 9.

140. Rahnasto M, Wittekindt C, Juvonen RO, Turpeinen M, Petsalo A, Pelkonen O, et al. Identifi cation of inhibitors of the nico-tine metabolising CYP2A6 enzyme – an in silico approach. Pharmacogenomics J 2008;8:328 – 38.

141. Chougnet A, Woggon WD, Locher E, Schilling B. Synthesis and in vitro activity of heterocyclic inhibitors of CYP2A6 and CYP2A13, two cytochrome P450 enzymes present in the respi-ratory tract. Chembiochem 2009;10:1562 – 7.

142. Ueng YF, Chen CC, Chung YT, Liu TY, Chang YP, Lo WS, et al. Mechanism-based inhibition of cytochrome P450 (CYP) 2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo. Br J Pharmacol 2011;163:1250 – 62.

143. Rahnasto MK, Raunio HA, Wittekindt C, Salminen KA, Lepp ä nen J, Juvonen RO, et al. Identifi cation of novel CYP2A6 inhibitors by virtual screening. Bioorg Med Chem 2011;19:7186 – 93.

144. Sellers EM, Tyndale RF. Mimicking gene defects to treat drug dependence. Ann NY Acad Sci 2000;909:233 – 46.

145. Sellers EM, Ramamoorthy Y, Zeman MV, Djordjevic MV, Tyndale RF. The effect of methoxsalen on nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) me-tabolism in vivo. Nicotine Tob Res 2003;5:891 – 9.

146. Katoh M, Matsui T, Nakajima M, Tateno C, Kataoka M, Soeno Y, et al. Expression of human cytochromes P450 in chime-ric mice with humanized liver. Drug Metab Dispos 2004;32:1402 – 10.

147. Aoki K, Kashiwagura Y, Horie T, Sato H, Tateno C, Ozawa N, et al. Characterization of humanized liver from chimeric mice using coumarin as a human CYP2A6 and mouse CYP2A5 probe. Drug Metab Pharmacokinet 2006;21:277 – 85.

148. Egan D, O ’ Kennedy R, Moran E, Cox D, Prosser E, Thornes RD. The pharmacology, metabolism, analysis, and applications of coumarin and coumarin-related compounds. Drug Metab Rev 1990;22:503 – 29.

149. Riveiro ME, De Kimpe N, Moglioni A, V á zquez R, Monczor F, Shayo C, et al. Coumarins: old compounds with novel promising therapeutic perspectives. Curr Med Chem 2010;17:1325 – 38.

150. Abraham K, W ö hrlin F, Lindtner O, Heinemeyer G, Lampen A. Toxicology and risk assessment of coumarin: focus on human data. Mol Nutr Food Res 2010;54:228 – 39.

151. Raunio H, Rautio A, Gullst é n H, Pelkonen O. Polymorphisms of CYP2A6 and its practical consequences. Br J Clin Pharmacol 2001;52:357 – 63.

152. Rietjens IM, Punt A, Schilter B, Scholz G, Delatour T, van Bladeren PJ. In silico methods for physiologically based bioki-netic models describing bioactivation and detoxifi cation of cou-marin and estragole: implications for risk assessment. Mol Nutr Food Res 2010;54:195 – 207.

153. Benowitz NL. Nicotine addiction. N Engl J Med 2010;362:2295 – 303.

154. Murphy SE, Raulinaitis V, Brown KM. Nicotine 5 ′ -oxidation and methyl oxidation by P450 2A enzymes. Drug Metab Dispos 2005;33:1166 – 73.

155. Mwenifumbo JC, Tyndale RF. Genetic variability in CYP2A6 and the pharmacokinetics of nicotine. Pharmacogenomics 2007;8:1385 – 402.

156. Bloom J, Hinrichs AL, Wang JC, von Weymarn LB, Kharasch ED, Bierut LJ, et al. The contribution of common CYP2A6 alleles to variation in nicotine metabolism among European-Americans. Pharmacogenet Genomics 2011;21:403 – 16.

157. Mroziewicz M, Tyndale RF. Pharmacogenetics: a tool for iden-tifying genetic factors in drug dependence and response to treat-ment. Addict Sci Clin Pract 2010;5:17 – 29.

158. Audrain-McGovern J, Al Koudsi N, Rodriguez D, Wileyto EP, Shields PG, Tyndale RF. The role of CYP2A6 in the emergence of nicotine dependence in adolescents. Pediatrics 2007;119:e264 – 74.

159. O ’ Loughlin J, Paradis G, Kim W, DiFranza J, Meshefedjian G, McMillan-Davey E, et al. Genetically decreased CYP2A6 and the risk of tobacco dependence: a prospective study of novice smokers. Tob Control 2004;13:422 – 8.

160. Thorgeirsson TE, Gudbjartsson DF, Surakka I, Vink JM, Amin N, Geller F, et al. Sequence variants at CHRNB3-CHRNA6 and CYP2A6 affect smoking behavior. Nat Genet 2010;42:448 – 53.

161. Rodriguez S, Cook DG, Gaunt TR, Nightingale CM, Whincup PH, Day IN. Combined analysis of CHRNA5, CHRNA3 and CYP2A6 in relation to adolescent smoking behaviour. J Psychopharmacol 2011;25:915 – 23.

162. Verde Z, Santiago C, Rodr í guez Gonz á lez-Moro JM, de Lucas Ramos P, L ó pez Mart í n S, Bandr é s F, et al. ‘ Smoking genes ’ : a genetic association study. PLoS One 2011;6:e26668.

163. Liu T, David SP, Tyndale RF, Wang H, Zhou Q, Ding P, et al. Associations of CYP2A6 genotype with smoking behaviors in southern China. Addiction 2011;106:985 – 94.

164. Siedlinski M, Cho MH, Bakke P, Gulsvik A, Lomas DA, Anderson W, et al; the COPDGene Investigators and ECLIPSE Investigators. Genome-wide association study of smok-ing behaviours in patients with COPD. Thorax 2011;66:894 – 902.

165. Cho MH, Castaldi PJ, Wan ES, Siedlinski M, Hersh CP, Demeo DL, et al., on behalf of the ICGN, ECLIPSE, and COPDGene Investigators. A genome-wide association study of COPD iden-tifi es a susceptibility locus on chromosome 19q13. Hum Mol Genet 2012;21:947 – 57.

166. Patterson F, Schnoll RA, Wileyto EP, Pinto A, Epstein LH, Shields PG, et al. Toward personalized therapy for smoking ces-sation: a randomized placebo-controlled trial of bupropion. Clin Pharmacol Ther 2008;84:320 – 5.

167. Tyndale RF, Sellers EM. Variable CYP2A6-mediated nicotine metabolism alters smoking behavior and risk. Drug Metab Dispos 2001;29:548 – 52.

168. Khokhar JY, Ferguson CS, Zhu AZ, Tyndale RF. Phar-macogenetics of drug dependence: role of gene variations in susceptibility and treatment. Annu Rev Pharmacol Toxicol 2010;50:39 – 61.

169. Sellers EM, Kaplan HL, Tyndale RF. Inhibition of cytochrome P450 2A6 increases nicotine ’ s oral bioavailability and decreases smoking. Clin Pharmacol Ther 2000;68:35 – 43.

170. Siu EC, Tyndale RF. Non-nicotinic therapies for smoking cessa-tion. Annu Rev Pharmacol Toxicol 2007;47:541 – 64.




171. Raunio H, Pokela N, Puhakainen K, Rahnasto M, Mauriala T, Auriola S, et al. Nicotine metabolism and urinary elimination in mouse: in vitro and in vivo. Xenobiotica 2008;38:34 – 47.

172. Rodriguez-Antona C, Gomez A, Karlgren M, Sim SC, Ingelman-Sundberg M. Molecular genetics and epigenetics of the cyto-chrome P450 gene family and its relevance for cancer risk and treatment. Hum Genet 2010;127:1 – 17.

173. Wassenaar CA, Dong Q, Wei Q, Amos CI, Spitz MR, Tyndale RF. Relationship between CYP2A6 and CHRNA5-CHRNA3-CHRNB4 variation and smoking behaviors and lung cancer risk. J Natl Cancer Inst 2011;103:1342 – 6.

174. Haiman CA, Stram DO, Wilkens LR, Pike MC, Kolonel LN, Henderson BE, et al. Ethnic and racial differences in the smoking-related risk of lung cancer. N Engl J Med 2006;354:333 – 42.

175. Rossini A, de Almeida Sim ã o T, Albano RM, Pinto LF. CYP2A6 polymorphisms and risk for tobacco-related cancers. Pharmacogenomics 2008;9:1737 – 52.

176. Hollander MC, Zhou X, Maier CR, Patterson AD, Ding X, Dennis PA. A Cyp2a polymorphism predicts susceptibility to NNK-induced lung tumorigenesis in mice. Carcinogenesis 2011;32:1279 – 84.

177. Otyepka M, Skopal í k J, Anzenbacherov á E, Anzenbacher P. What common structural features and variations of mam-malian P450s are known to date ? Biochim Biophys Acta 2007;1770:376 – 89.

178. Yano JK, Hsu MH, Griffi n KJ, Stout CD, Johnson EF. Structures of human microsomal cytochrome P450 2A6 com-plexed with coumarin and methoxsalen. Nat Struct Mol Biol 2005;12:822 – 3.

179. He XY, Shen J, Hu WY, Ding X, Lu AY, Hong JY. Identifi cation of Val117 and Arg372 as critical amino acid residues for the activity difference between human CYP2A6 and CYP2A13

in coumarin 7-hydroxylation. Arch Biochem Biophys 2004;427:143 – 53.

180. Kim D, Wu ZL, Guengerich FP. Analysis of coumarin 7-hy-droxylation activity of cytochrome P450 2A6 using random mutagenesis. J Biol Chem 2005;280:40319 – 27.

181. Sansen S, Hsu MH, Stout CD, Johnson EF. Structural insight into the altered substrate specifi city of human cytochrome P450 2A6 mutants. Arch Biochem Biophys 2007;464:197 – 206.

182. DeVore NM, Smith BD, Wang JL, Lushington GH, Scott EE. Key residues controlling binding of diverse ligands to human cytochrome P450 2A enzymes. Drug Metab Dispos 2009;37:1319 – 27.

183. Devore NM, Meneely KM, Bart AG, Stephens ES, Battaile KP, Scott EE. Structural comparison of cytochromes P450 2A6, 2A13, and 2E1 with pilocarpine. FEBS J 2011;doi: 10.1111/j.1742-4658.2011.08412.x.

184. Lindberg RL, Negishi M. Alteration of mouse cytochrome P450coh substrate specifi city by mutation of a single amino-acid residue. Nature 1989;339:632 – 4.

185. Negishi M, Uno T, Darden TA, Sueyoshi T, Pedersen LG. Structural fl exibility and functional versatility of mammalian P450 enzymes. FASEB J 1996;10:683 – 9.

186. Poso A, Gynther J, Juvonen R. A comparative molecular fi eld analysis of cytochrome P450 2A5 and 2A6 inhibitors. J Comput Aided Mol Des 2001;15:195 – 202.

187. Asikainen A, Tarhanen J, Poso A, Pasanen M, Alhava E, Juvonen RO. Predictive value of comparative molecular fi eld analysis modelling of naphthalene inhibition of human CYP2A6 and mouse CYP2A5 enzymes. Toxicol In vitro 2003;17:449 – 55.

188. Lewis DF, Ito Y, Goldfarb PS. Structural modelling of the human drug-metabolizing cytochromes P450. Curr Med Chem 2006;13:2645 – 52.



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CYP2A6: genetics, structure, regulation, and function

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