The Pharmacogenomics Journal (2010) 10, 478–488 & 2010 Macmillan Publishers Limited. All rights reserved 1470-269X/10 www.nature.com/tpj ORIGINAL ARTICLE Functional characterization of eight human cytochrome P450 1A2 gene variants by recombinant protein expression B Brito Palma1,2, M Silva e Sousa1, Inter-individual variability in cytochrome P450 (CYP)-mediated xenobiotic 2 2 metabolism is extensive. CYP1A2 is involved in the metabolism of drugs CR Vosmeer , J Lastdrager , and in the bioactivation of carcinogens. The objective of this study was 1 2 JRueff,NPEVermeulen to functionally characterize eight polymorphic forms of human CYP1A2, and M Kranendonk1 namely T83M, S212C, S298R, G299S, I314V, I386F, C406Y and R456H. cDNAs of these variants were constructed and coexpressed in Escherichia coli 1Department of Genetics, Faculty of Medical with human NADPH cytochrome P450 oxidoreductase (CYPOR). All variants Sciences, Universidade Nova de Lisbon, Lisbon, showed similar levels of apoprotein and holoprotein expression, except for Portugal and 2Division of Molecular Toxicology, Department of Pharmacochemistry, LACDR, Vrije I386F and R456H, which showed only apoprotein, and both were functionally Universiteit Amsterdam, Amsterdam, The inactive. The activity of CYP1A2 variants was investigated using 8 substrates, Netherlands measuring 16 different activity parameters. The resulting heterogeneous activity data set was analyzed together with CYP1A2 wild-type (WT) form, applying Correspondence: Dr M Kranendonk, Department of Genetics, multivariate analysis. This analysis indicated that variant G299S is substantially Faculty of Medical Sciences, Universidade Nova altered in catalytic properties in comparison with WT, whereas variant T83M is de Lisbon, Rua da Junqueira 96, 1349-008 slightly but significantly different from the WT. Among CYP1A2 variants, out of Lisbon, Portugal. the heterogeneous set of eight substrates, carcinogen 4-(methylnitrosamino)-1- E-mail: [email protected] (3-pyridyl)-1-butanone (NNK) was the most discriminative compound. In addition, R456 could be identified as an important residue for proper heme binding and stabilization. The Pharmacogenomics Journal (2010) 10, 478–488; doi:10.1038/tpj.2010.2; published online 2 February 2010 Keywords: cytochrome P450; CYP1A2; pharmacogenomics; pharmacokinetics; polymorphisms Introduction Inter-individual variability in xenobiotic and drug metabolism is extensive. Plasma levels of a drug can vary more than 1000-fold between individuals upon the same drug dosage.1 These variations can be caused by induction or inhibition and genetic polymorphisms of metabolizing enzymes, or from physiological, pathophysiological or environmental factors.2 In fact, genetic factors are considered to be responsible for 20–40% of the differences between individuals in the response and susceptibility to drugs and xenobiotics.3 Variability among individuals in drug metabolism can result in reduced drug efficacies or undesirable toxic side effects, which are phenomena that may compromise drug therapy and development. Cytochrome P450s (CYPs) are responsible for the metabolism of a wide variety Received 28 July 2009; revised 16 October 4 2009; accepted 21 December 2009; of clinically, physiologically and toxicologically important compounds. CYP published online 2 February 2010 families 1, 2 and 3 are responsible for 70–80% of all phase I metabolism of Characterization of 8 human CYP 1A2 gene variants BB Palma et al 479 clinically used drugs5 and are involved in the biotransfor- NADPH cytochrome P450 oxidoreductase (CYPOR) interac- mation of a large number of xenobiotics. Biotransformation tion zone. As such, these polymorphic forms concern T83M can lead to bioactivation of pre-carcinogens and drugs, (CYP1A2*9), S212C (CYP1A2*12), S298R (no allele designa- which can result in carcinogenic or other toxic effects. The tion), G299S (CYP1A2*13), I314V (no allele designation), majority of CYP-mediated xenobiotic metabolism is carried I386F (CYP1A2*4), C406Y (CYP1A2*5) and R456H out by polymorphic enzymes.6 Human CYP polymorphisms (CYP1A2*8). For this purpose, the cDNA of the WT allele, include mutations both in non-coding regions (promoter, present in a bacterial expression vector,25 was adapted intron) and protein-coding sequences. Mutations in non- through genetic engineering to generate the eight different coding regions may lead to altered expression levels, CYP1A2 forms. CYP1A2 alleles were coexpressed with whereas those in protein-coding sequences may change human CYPOR in a bacterial cell model,26 and the activity the structure and catalytic properties of the enzyme.7 Many of the eight allelic variants and WT was investigated. The drugs that can cause adverse reactions are metabolized in enzyme activity of these variants was analyzed together with processes catalyzed by polymorphic CYP enzymes. There- WT and scrutinized through multivariate analysis. Results of fore, the understanding of the nature of CYP polymorph- this analysis were interpreted using the CYP1A2 crystal isms is fundamental for the comprehension of inter- structure.24 individual differences in chemical exposure, adverse drug 8 effects and toxicity in general. Materials and methods The human CYP1A family consists of CYP1A1 and CYP1A2. Their genes are located on chromosome 15 and Reagents 9 are oriented head-to-head, 23.3 kb apart. The spacer L-arginine, ampicillin, kanamycin sulfate, chloramphenicol, sequence may contain distinct regulatory regions of each cytochrome c, isopropyl b-D-thiogalactoside (dioxane-free), gene or, alternatively, the regulatory regions of both genes.10 thiamine, 2-aminoanthracene (2AA), glucose-6-phosphate, CYP1A1 is mainly expressed in extrahepatic tissues, whereas NADPH, ethoxyresorufin (ER), methoxyresorufin (MR), CYP1A2 is almost exclusively expressed in the liver, resorufin (R), phenylmethanesulfonyl fluoride and a-naphtho- representing B15% of its total CYP content.11 CYP1A flavone (a-NF) were obtained from Sigma Chemical (St enzymes catalyze the bioactivation of pre-carcinogens, such Louis, MO, USA). 2-Amino-3-methylimidazo(4,5-f)quinoline as polycyclic aromatic hydrocarbons (CYP1A1), as well as (IQ) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone aromatic and heterocyclic amines (CYP1A2).12 CYP1A2 is (NNK) were obtained from Toronto Research Chemicals also responsible for the metabolism of drugs, such as (North York, ON, Canada). Both 3-cyano-7-ethoxycoumarin phenacetin,13 clozapine,14,15 naproxen,16 propranolol17 (CEC) and 3-cyano-7-hydroxycoumarin (CHC) were ob- and tizanidine.18 tained from BD Biosciences (Bedford, MA, USA). Phenacetin The CYP1A2 gene consists of seven exons, including non- was obtained from Brocades-ACF (Maarssen, The Nether- coding exon 1.19 Inter-individual differences in CYP1A2 lands) and clozapine from Duchefa Farma bv. (Haarlem, The activity are well known; up to 60-fold variations have been Netherlands). Bacto agar, bacto peptone, bacto tryptone and reported.11,20 In addition, B15- and 40-fold variations in bacto yeast extract were obtained from Difco (Detroit, MI, mRNA and protein expression levels have been observed in USA). All other chemicals were of the highest quality. the human liver.19,21 To date, 36 CYP1A2 haplotypes have been published on the Human Cytochrome P450 Allele Site-directed mutagenesis Nomenclature Committee home page (http://www.cypalleles. Site-directed mutagenesis was performed using ‘Quick- ki.se/cyp1a2.htm). Of these, 21 are in the non-coding region change XL’ (Cedar Creek, TX, USA) and applied according and 15 are in the coding region, all causing amino-acid to the manufacture’s instructions. All variants, except I386F, changes of the CYP1A2 protein. Apart from studies that were constructed using plasmid pCWh1A226 as DNA characterized gene polymorphisms in the non-coding template, encoding human CYP1A2*1 (isoform 2) (NCBI region, few reports described non-synonymous protein NM_000761) with the N terminus modified as described by polymorphisms. Mostly, these concern the identification Fisher et al.25 In the case of the variant I386F, the template of genetic polymorphisms and their frequencies, but not was pUC18_h1A2. This plasmid was constructed by cloning their effects on protein level and activities. Murayama the cDNA of CYP1A2 from pCWh1A2 as an NdeI-XbaI et al.,22 Saito et al.23 and Zhou et al.7 characterized most fragment in pUC18. Forward and reverse mutagenesis of the non-synonymous polymorphisms with a limited primers for the creation of the desired mutation for each number of substrates. The primary objective of this study is variant are presented in Supplementary Table 1. to investigate polymorphic forms, representing the majority The introduced mutation for each variant was confirmed of alleles with the non-synonymous polymorphic CYP1A2 by sequencing the full extent of the CYP1A2 cDNA. The protein, and to characterize their activity with a diverse pCWh1A2 plasmid has a considerable size (6.9 kb), and it is group of substrates. known that, for a site-directed mutagenesis procedure, large On the basis of a crystal structure published by Sansen plasmids have a reduced but significant risk for incorpora- et al.24 of CYP1A2 wild-type (WT) (CYP1A2*1), we selected tion of undesired mutations. Therefore, mutated cDNAs mutants that seemed to be localized in the vicinity of were re-cloned into the pCW vector to ensure that plasmid the active center, heme, entrance/exit channel or the sequences remained unaltered. For this, the restriction