Single nucleotide polymorphism of CYP3A4 intron 2 and its influence on CYP3A4 mRNA expression and liver enzymatic activity in human liver

Abstract

In adult liver, CYP3A4 plays an important role in the metabolism of a wide range of endogenous and exogenous compounds. To investigate whether there is a single nucleotide polymorphism (SNP) of CYP3A4 intron 2 in the liver and its effects on the mRNA expression and enzymatic activity of CYP3A4, genomic DNA was extracted from 96 liver tissue samples obtained from patients who had undergone liver surgery. An SNP of CYP3A4 intron 2 was identified by polymerase chain reaction (PCR)-single-strand confirmation polymorphism and DNA sequencing. The mRNA expression of CYP3A4 was determined by the fluorescence quantitative PCR technique. The enzymatic activity of CYP3A4 was measured using erythromycin and testosterone as probe substrates. Twelve patients were found to have the SNP/T4127G CYP3A4 within intron 2. The mRNA levels of CYP3A4 in wild-type and SNP/T4127G samples were 2.62±1.09 and 2.79±1.63, respectively (P>0.05). Erythromycin N-demethylase activity in wild-type and SNP/T4127G samples were 121.2±32.8 and 124.7±61.6 nmol·mg⁻¹·min⁻¹, respectively (P>0.05). The activity of testosterone 6β-hydroxylase was significantly different between wild-type (648±173 pmol·mg⁻¹·min⁻¹) and SNP/T4127G samples (540±196 pmol·mg⁻¹·min⁻¹; P<0.05). In conclusion, the SNP/T4127G of CYP3A4 intron 2 exists in the liver. This SNP does not affect the mRNA expression of CYP3A4 but significantly decreases the hepatic microsomal testosterone 6β-hydroxylase activity of CYP3A4. Furthermore, this study indicates that the appropriate selection of probe substrates is very important in studying the relationship between the genotype and phenotype of CYP3A4.

Keywords

CYP3A4 / single nucleotide polymorphism / mRNA expression / enzymatic activity / human liver

References

[1] NelsonDR, KamatakiT, WaxmanDJ, et al. . The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol, 1993, 12(1): 1-51 PMID: 7678494

[2] ThummelKE, WilkinsonGR. In vitro and in vivo drug interactions involving human CYP3A. Annu Rev Pharmacol Toxicol, 1998, 38(1): 389-430 PMID: 9597161

[3] GuengerichFP. Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu Rev Pharmacol Toxicol, 1999, 39(1): 1-17 PMID: 10331074

[4] YamaoriS, YamazakiH, IwanoS, et al. . Ethnic differences between Japanese and Caucasians in the expression levels of mRNAs for CYP3A4, CYP3A5 and CYP3A7: lack of co-regulation of the expression of CYP3A in Japanese livers. Xenobiotica, 2005, 35(1): 69-83 PMID: 15788369

[5] DaiD, TangJ, RoseR, et al. . Identification of variants of CYP3A4 and characterization of their abilities to metabolize testosterone and chlorpyrifos. J Pharmacol Exp Ther, 2001, 299(3): 825-831 PMID: 11714865

[6] Rodriguez-AntonaC, DonatoMT, ParejaE, et al. . Cytochrome P-450 mRNA expression in human liver and its relationship with enzyme activity. Arch Biochem Biophys, 2001, 393(2): 308-315 PMID: 11556818

[7] RebbeckTR, JaffeJM, WalkerAH, et al. . Modification of clinical presentation of prostate tumors by a novel genetic variant in CYP3A4. J Natl Cancer Inst, 1998, 90(16): 1225-1229 PMID: 9719084

[8] WestlindA, LofbergL, TindbergN, et al. . Interindividual differences in hepatic expression of CYP3A4: relationship to genetic polymorphism in the 5'-upstream regulatory region. Biochem Biophys Res Commun, 1999, 259(1): 201-205 PMID: 10334940

[9] HuYF, HeJ, ChenGL, et al. . CYP3A5*3 and CYP3A4*18 single nucleotide polymorphisms in a Chinese population. Clin Chim Acta, 2005, 353(1–2): 187-192 PMID: 15698606

[10] SachseC, BrockmollerJ, BauerS, et al. . Functional significance of a CA polymorphism in intron 1 of the cytochrome P450 CYP1A2 gene tested with caffeine. Br J Clin Pharmacol, 1999, 47(4): 445-449 PMCID: 2014233 PMID: 10233211

[11] BorroniB, ArchettiS, AgostiC, et al. . Intronic CYP46 polymorphism along with ApoE genotype in sporadic Alzheimer disease: from risk factors to disease modulators. Neurobiol Aging, 2004, 25(6): 747-751 PMID: 15165699

[12] IbeanuGC, BlaisdellJ, FergusonRJ, et al. . A novel transversion in the intron 5 donor splice junction of CYP2C19 and a sequence polymorphism in exon 3 contribute to the poor metabolizer phenotype for the anticonvulsant drug S-mephenytoin. J Pharmacol Exp Ther, 1999, 290(2): 635-640 PMID: 10411572

[13] BourrieM, MeunierV, BergerY, et al. . Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. J Pharmacol Exp Ther, 1996, 277(1): 321-332 PMID: 8613937

[14] EaglingVA, TjiaJF, BackDJ. Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes. Br J Clin Pharmacol, 1998, 45(2): 107-114 PMCID: 1873357 PMID: 9491822

[15] HickmanD, WangJP, WangY, et al. . Evaluation of the selectivity of in vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. Drug Metab Dispos, 1998, 26(3): 207-215 PMID: 9492382

[16] DresserGK, SpenceJD, BaileyDG. Pharmacokinetic-pharmacodynamic consequences and clinical relevance of cytochrome P450 3A4 inhibition. Clin Pharmacokinet, 2000, 38(1): 41-57 PMID: 10668858

[17] FotiRS, RockDA, WienkersLC, et al. . Selection of alternative CYP3A4 probe substrates for clinical drug interaction studies using in vitro data and in vivo simulation. Drug Metab Dispos, 2010, 38(6): 981-987 PMID: 20203109

[18] PengRX, WangYS, LeiSB. Characterization of monooxygenase system in Chinese fetal adrenal gland. Asia Pac J Pharmacol, 1994, 9: 195-200

[19] LowryOH, RosebroughNJ, FarrAL, et al. . Protein measurement with the Folin phenol reagent. J Biol Chem, 1951, 193(1): 265-275 PMID: 14907713

[20] YooJS, SmithTJ, NingSM, et al. . Modulation of the levels of cytochromes P450 in rat liver and lung by dietary lipid. Biochem Pharmacol, 1992, 43(12): 2535-2542 PMID: 1632812

[21] IyerKR, SinzMW. Characterization of Phase I and Phase II hepatic drug metabolism activities in a panel of human liver preparations. Chem Biol Interact, 1999, 118(2): 151-169 PMID: 10359459

[22] LambaJK, LinYS, SchuetzEG, et al. . Genetic contribution to variable human CYP3A-mediated metabolism. Adv Drug Deliv Rev, 2002, 54(10): 1271-1294 PMID: 12406645

[23] KuehlP, ZhangJ, LinY, et al. . Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat Genet, 2001, 27(4): 383-391 PMID: 11279519

[24] Fukushima-UesakaH, SaitoY, WatanabeH, et al. . Haplotypes of CYP3A4 and their close linkage with CYP3A5 haplotypes in a Japanese population. Hum Mutat, 2004, 23(1): 100 PMID: 14695543

[25] XieHG, WoodAJ, KimRB, et al. . Genetic variability in CYP3A5 and its possible consequences. Pharmacogenomics, 2004, 5(3): 243-272 PMID: 15102541

[26] JohnsonAD, WangD, SadeeW. Polymorphisms affecting gene regulation and mRNA processing: broad implications for pharmacogenetics. Pharmacol Ther, 2005, 106(1): 19-38 PMID: 15781120

[27] GibsonGG, PlantNJ, SwalesKE, et al. . Receptor-dependent transcriptional activation of cytochrome P4503A genes: induction mechanisms, species differences and interindividual variation in man. Xenobiotica, 2002, 32(3): 165-206 PMID: 11958559

[28] ModrekB, LeeC. A genomic view of alternative splicing. Nat Genet, 2002, 30(1): 13-19 PMID: 11753382

[29] Rodriguez-AntonaC, SayiJG, GustafssonLL, et al. . Phenotype-genotype variability in the human CYP3A locus as assessed by the probe drug quinine and analyses of variant CYP3A4 alleles. Biochem Biophys Res Commun, 2005, 338(1): 299-305 PMID: 16171783

[30] FloydMD, GervasiniG, MasicaAL, et al. . Genotype-phenotype associations for common CYP3A4 and CYP3A5 variants in the basal and induced metabolism of midazolam in European- and African-American men and women. Pharmacogenetics, 2003, 13(10): 595-606 PMID: 14515058

[31] WrightonSA, StevensJC. The human hepatic cytochromes P450 involved in drug metabolism. Crit Rev Toxicol, 1992, 22(1): 1-21 PMID: 1616599

[32] YuanR, MadaniS, WeiXX, et al. . Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos, 2002, 30(12): 1311-1319 PMID: 12433797

[33] GaletinA, ClarkeSE, HoustonJB. Multisite kinetic analysis of interactions between prototypical CYP3A4 subgroup substrates: midazolam, testosterone, and nifedipine. Drug Metab Dispos, 2003, 31(9): 1108-1116 PMID: 12920166

[34] GaletinA, ItoK, HallifaxD, et al. . CYP3A4 substrate selection and substitution in the prediction of potential drug-drug interactions. J Pharmacol Exp Ther, 2005, 314(1): 180-190 PMID: 15784650

[35] SchragML, WienkersLC. Covalent alteration of the CYP3A4 active site: evidence for multiple substrate binding domains. Arch Biochem Biophys, 2001, 391(1): 49-55 PMID: 11414684

[36] HoseaNA, MillerGP, GuengerichFP. Elucidation of distinct ligand binding sites for cytochrome P450 3A4. Biochemistry, 2000, 39(20): 5929-5939 PMID: 10821664