Skip to main content

Advertisement

SpringerLink
Log in

Cytochrome P450 mRNA expression in peripheral blood lymphocytes as a predictor of enzyme induction

  • Pharmacokinetics and Disposition
  • Published:
European Journal of Clinical Pharmacology Aims and scope Submit manuscript

Abstract

Objective

Previous reports have supported the concept that messenger ribonucleic acid (mRNA) concentrations for cytochrome P450 (CYP) enzymes in peripheral blood mononuclear cells may be predictive of systemic enzyme activity. We investigated whether changes in mRNA expression for CYP1A2,CYP2C19, CYP2D6 and CYP3A4 in peripheral blood lymphocytes (PBLs) may serve as surrogate markers for changes in CYP enzyme activity following the administration of rifampin.

Methods

On day 1 and day 9 of the study, 12 healthy volunteers were administered caffeine 100 mg, debrisoquine 10 mg and omeprazole 40 mg orally, along with midazolam 0.025 mg/kg intravenously. Blood samples and urine were collected for 8 h after drug administration. The subjects took rifampin 300 mg (n=6) or 600 mg (n=6) daily on days 2–8. Total RNA was isolated from PBLs on day 1 and day 9, and mRNA expression for the CYP enzymes and hGAPDH were determined by means of quantitative, real-time, reverse-transcriptase polymerase chain reaction. CYP1A2 activity was estimated by calculating the plasma paraxanthine to caffeine AUC ratio (caffeine metabolic ratio; CMR), CYP2C19 activity by the 2-h omeprazole hydroxylation index (HI), CYP2D6 activity by the urinary debrisoquine recovery ratio (DBRR) and CYP3A4 activity by midazolam clearance.

Results

Median midazolam clearance (0.362 to 0.740 l/kg/h), omeprazole HI (0.752 to 0.214), CMR (0.365 to 0.450) and DBRR (0.406 to 0.479) all changed significantly following rifampin, consistent with the expected enzyme induction. CYP1A2,CYP2D6 and CYP3A4 mRNA content were measurable in all samples. CYP2C19 mRNA was inconsistently detectable. There were no significant correlations between changes in enzyme activity and mRNA expression by Spearman’s rank order correlation.

Conclusion

The results do not support the use of mRNA expression assays for CYP1A2, CYP2C19, CYP2D6 and CYP3A4 enzymes in PBLs as surrogates for quantifying changes in systemic enzyme activity in the setting of enzyme induction.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Asghar A, Gorski JC, Haehner-Daniels B, Hall SD (2002) Induction of multidrug resistance-1 and cytochrome P450 mRNAs in human mononuclear cells by rifampin. Drug Metab Dispos 30:20–26

    Google Scholar 

  2. Balian JD, Sukhova N, Harris JW, Hewett J, Pickle L, Goldstein JA, Woosley RL, Flockhart DA (1995) The hydroxylation of omeprazole correlates with S-mephenytoin metabolism: a population study. Clin Pharmacol Ther 57:662–669

    Google Scholar 

  3. Bauer LA, Gibaldi M (1983) Computation of model-independent pharmacokinetic parameters during multiple dosing. J Pharm Sci 72:978–979

    Google Scholar 

  4. Branch RA, Adedoyin A, Frye RF, Wilson JW, Romkes M (2000) In vivo modulation of CYP enzymes by quinidine and rifampin. Clin Pharmacol Ther 68:401–411

    Google Scholar 

  5. Caraco Y, Sheller J, Wood AJ (1997) Pharmacogenetic determinants of codeine induction by rifampin: the impact on codeine’s respiratory, psychomotor and miotic effects. J Pharmacol Exp Ther 281:330–336

    Google Scholar 

  6. Carcillo JA, Parise RA, Adedoyin A, Frye R, Branch RA, Romkes M (1996) CYP2D6 mRNA expression in circulating peripheral blood mononuclear cells correlates with in vivo debrisoquine hydroxylase activity in extensive metabolizers. Res Commun Mol Pathol Pharmacol 91:149–159

    Google Scholar 

  7. Carcillo JA, Adedoyin A, Burckart GJ, Frye RF, Venkataramanan R, Knoll C, Thummel K, Roskos L, Wilson JW, Sereika S, Romkes M, Bebia Z, Branch RA (2003) Coordinated intrahepatic and extrahepatic regulation of cytochrome p4502D6 in healthy subjects and in patients after liver transplantation. Clin Pharmacol Ther 73:456–467

    Google Scholar 

  8. Chalasani N, Gorski JC, Asghar MS, Asghar A, Foresman B, Hall SD, Crabb DW (2003) Hepatic cytochrome P450 2E1 activity in nondiabetic patients with nonalcoholic steatohepatitis. Hepatology 37:544–550

    Google Scholar 

  9. Cosma GN, Toniolo P, Currie D, Pasternack BS, Garte SJ (1992) Expression of the CYP1A1 gene in peripheral lymphocytes as a marker of exposure to creosote in railroad workers. Cancer Epidemiol Biomarkers Prev 1:137–142

    Google Scholar 

  10. Cotreau MM, von Moltke LL, Beinfeld MC, Greenblatt DJ (2000) Methodologies to study the induction of rat hepatic and intestinal cytochrome P450 3A at the mRNA, protein, and catalytic activity level. J Pharmacol Toxicol Methods 43:41–54

    Google Scholar 

  11. Eichelbaum M, Mineshita S, Ohnhaus EE, Zekorn C (1986) The influence of enzyme induction on polymorphic sparteine oxidation. Br J Clin Pharmacol 22:49–53

    Google Scholar 

  12. Finnstrom N, Thorn M, Loof L, Rane A (2001) Independent patterns of cytochrome P450 gene expression in liver and blood in patients with suspected liver disease. Eur J Clin Pharmacol 57:403–409

    Google Scholar 

  13. Finnstrom N, Ask B, Dahl ML, Gadd M, Rane A (2002) Intra-individual variation and sex differences in gene expression of cytochromes P450 in circulating leukocytes. Pharmacogenomics J 2:111–116

    Google Scholar 

  14. Frye R, Stiff D, Branch RA (1998) A sensitive method for the simultaneous determination of caffeine and its dimethylxanthine metabolites in human plasma: application to CYP1A2 phenotyping. J Liq Chrom Rel Tech 21:1161–1171

    Google Scholar 

  15. Fromm MF, Kauffmann HM, Fritz P, Burk O, Kroemer HK, Warzok RW, Eichelbaum M, Seigmund W, Schrenk D (2000) The effect of rifampin treatment on intestinal expression of human MRP transporters. Am J Pathol 157:1575–1580

    Google Scholar 

  16. Frye RF, Branch RA (1996) Improved high-performance liquid chromatographic determination of debrisoquine and 4-hydroxydebrisoquine in human urine following direct injection. J Chromatogr B Biomed Appl 677:178–182

    Google Scholar 

  17. Frye RF, Matzke GR, Adedoyin A, Porter JA, Branch RA (1997) Validation of the five-drug “Pittsburgh cocktail” approach for assessment of selective regulation of drug-metabolizing enzymes. Clin Pharmacol Ther 62:365–376

    Google Scholar 

  18. Fuhr U, Rost KL (1994) Simple and reliable CYP1A2 phenotyping by the paraxanthine/caffeine ratio in plasma and in saliva. Pharmacogenetics 4:109–116

    Google Scholar 

  19. Gashaw I, Kirchheiner J, Goldammer M, Bauer S, Seidemann J, Zoller K, Mrozikiewicz PM, Roots I, Brockmoller J (2003) Cytochrome P450 3A4 messenger ribonucleic acid induction by rifampin in human peripheral blood mononuclear cells: correlation with alprazolam pharmacokinetics. Clin Pharmacol Ther 74:448–457

    Google Scholar 

  20. Gerbal-Chaloin S, Pascussi JM, Pichard-Garcia L, Daujat M, Waechter F, Fabre JM, Carrere N, Maurel P (2001) Induction of CYP2C genes in human hepatocytes in primary culture. Drug Metab Dispos 29:242–251

    Google Scholar 

  21. Giessmann T, Modess C, Hecker U, Zschiesche M, Dazert P, Kunert-Keil C, Warzok R, Engel G, Weitschies W, Cascorbi I, Kroemer HK, Siegmund W (2004) CYP2D6 genotype and induction of intestinal drug transporters by rifampin predict presystemic clearance of carvedilol in healthy subjects. Clin Pharmacol Ther 75:213–222

    Google Scholar 

  22. Gorski JC, Hall SD, Jones DR, VandenBranden M, Wrighton SA (1994) Regioselective biotransformation of midazolam by members of the human cytochrome P450 3A (CYP3A) subfamily. Biochem Pharmacol 47:1643–1653

    Google Scholar 

  23. Hukkanen J, Hakkola J, Anttila S, Piipari R, Karjalainen A, Pelkonen O, Raunio H (1997) Detection of mRNA encoding xenobiotic-metabolizing cytochrome P450s in human bronchoalveolar macrophages and peripheral blood lymphocytes. Mol Carcinog 20:224–230

    Google Scholar 

  24. Kaisary A, Smith P, Jaczq E, McAllister CB, Wilkinson GR, Ray WA, Branch RA (1987) Genetic predisposition to bladder cancer: ability to hydroxylate debrisoquine and mephenytoin as risk factors. Cancer Res 47:5488–5493

    Google Scholar 

  25. Kalow W, Tang BK (1993) The use of caffeine for enzyme assays: a critical appraisal. Clin Pharmacol Ther 53:503–514

    Google Scholar 

  26. Kashuba AD, Bertino JS Jr, Rocci ML Jr, Kulawy RW, Beck DJ, Nafziger AN (1998) Quantification of 3-month intraindividual variability and the influence of sex and menstrual cycle phase on CYP3A activity as measured by phenotyping with intravenous midazolam. Clin Pharmacol Ther 64:269–277

    Google Scholar 

  27. Kauffmann HM, Keppler D, Gant TW, Schrenk D (1998) Induction of hepatic mrp2 (cmrp/cmoat) gene expression in nonhuman primates treated with rifampicin or tamoxifen. Arch Toxicol 72:763–768

    Google Scholar 

  28. Kim MJ, Bertino JS Jr, Gaedigk A, Zhang Y, Sellers EM, Nafziger AN (2002) Effect of sex and menstrual cycle phase on cytochrome P450 2C19 activity with omeprazole used as a biomarker. Clin Pharmacol Ther 72:192–199

    Google Scholar 

  29. Kim RB, Wandel C, Leake B, Cvetkovic M, Fromm MF, Dempsey PJ, Roden MM, Belas F, Chaudhary AK, Roden DM, Wood AJ, Wilkinson GR (1999) Interrelationship between substrates and inhibitors of human CYP3A and P-glycoprotein. Pharm Res 16:408–414

    Google Scholar 

  30. Kronbach T, Mathys D, Umeno M, Gonzalez FJ, Meyer UA (1989) Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4. Mol Pharmacol 36:89–96

    Google Scholar 

  31. Krovat BC, Tracy JH, Omiecinski CJ (2000) Fingerprinting of cytochrome P450 and microsomal epoxide hydrolase gene expression in human blood cells. Toxicol Sci 55:352–360

    Google Scholar 

  32. Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA (1998) The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest 102:1016–1023

    Google Scholar 

  33. Lin YS, Dowling ALS, Quigley SD, Farin FM, Zhang J, Lamba J, Schuetz EG, Thummel KE (2002) Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism. Mol Pharmacol 62:162–172

    Google Scholar 

  34. Madan A, Graham RA, Carroll KM, Mudra DR, Burton LA, Krueger LA, Downey AD, Czerwinski M, Forster J, Ribadeneira MD, Gan LS, LeCluyse EL, Zech K, Robertson P Jr, Koch P, Antonian L, Wagner G, Yu L, Parkinson A (2003) Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes. Drug Metab Dispos 31:421–431

    Google Scholar 

  35. Marinac JS, Balian JD, Foxworth JW, Willsie SK, Daus JC, Owen R, Flockhart DA (1996) Determination of CYP2C19 phenotype in black Americans with omeprazole: correlation with genotype. Clin Pharmacol Ther 60:138–144

    Google Scholar 

  36. McConnachie LA, Phillips B, Bajpai M, Shen DD, Ho RJ (2003) Only truncated, not complete cytochrome p450 2D6 RNA transcript and not detectable enzyme activity are expressed in human lymphocytes. Drug Metab Dispos 31:1103–1107

    Google Scholar 

  37. Miners JO, Birkett DJ (1996) The use of caffeine as a metabolic probe for human drug metabolizing enzymes. Gen Pharmacol 27:245–249

    Google Scholar 

  38. Nakamoto T, Hase I, Imaoka S, Hiroi T, Oda Y, Asada A, Funae Y (2000) Quantitative RT-PCR for CYP3A4 mRNA in human peripheral lymphocytes: induction of CYP3A4 in lymphocytes and in liver by rifampicin. Pharmacogenetics 10:571–575

    Google Scholar 

  39. Nguyen LT, Ramanathan M, Weinstock-Guttman B, Dole K, Miller C, Planter M, Patrick K, Brownscheidle C, Jacobs LD (2000) Detection of cytochrome P450 and other drug-metabolizing enzyme mRNAs in peripheral blood mononuclear cells using DNA arrays. Drug Metab Dispos 28:987–993

    Google Scholar 

  40. Palmer JL, Scott RJ, Gibson A, Dickins M, Pleasance S (2001) An interaction between the cytochrome P450 probe substrates chlorzoxazone (CYP2E1) and midazolam (CYP3A). Br J Clin Pharmacol 52:555–561

    Google Scholar 

  41. Pauli-Magnus C, Rekersbrink S, Klotz U, Fromm MF (2001) Interaction of omeprazole, lansoprazole and pantoprazole with P-glycoprotein. Naunyn Schmiedebergs Arch Pharmacol 364:551–557

    Google Scholar 

  42. Raucy JL, Schultz ED, Wester MR, Arora S, Johnston DE, Omdahl JL, Carpenter SP (1997) Human lymphocyte cytochrome P450 2E1, a putative marker for alcohol-mediated changes in hepatic chlorzoxazone activity. Drug Metab Dispos 25:1429–1435

    Google Scholar 

  43. Raucy JL, Ingelman-Sundberg M, Carpenter S, Rannug A, Rane A, Franklin M, Romkes M (1999) Drug metabolizing enzymes in lymphocytes. J Biochem Mol Toxicol 13:223–226

    Google Scholar 

  44. Raunio H, Hakkola J, Hukkanen J, Pelkonen O, Edwards R, Boobis A, Anttila S (1998) Expression of xenobiotic-metabolizing cytochrome P450s in human pulmonary tissues. Arch Toxicol Suppl 20:465–469

    Google Scholar 

  45. Rendic S, Di Carlo FJ (1997) Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab Rev 29:413–580

    Google Scholar 

  46. Rogers JF, Rocci ML Jr, Haughey DB, Bertino JS Jr (2003) An evaluation of the suitability of intravenous midazolam as an in vivo marker for hepatic cytochrome P4503A activity. Clin Pharmacol Ther 73:153–158

    Google Scholar 

  47. Sempoux C, Starkel P, Stevens M, Van Den Berge V, Horsmans Y (1999) Cytochrome P450 3A proteins are expressed in B lymphocytes but not in T lymphocytes. Pharmacogenetics 9:263–265

    Google Scholar 

  48. Shitara Y, Sugiyama D, Kusuhara H, Kato Y, Abe T, Meier PJ, Itoh T, Sugiyama Y (2002) Comparative inhibitory effects of different compounds on rat Oatp1 (slc21a1)- and Oatp2 (slc21a5)-mediated transport. Pharm Res 19:147–153

    Google Scholar 

  49. Slaughter RL, Edwards DJ (1995) Recent advances: the cytochrome P450 enzymes. Ann Pharmacother 29:619–624

    Google Scholar 

  50. Smirlis D, Muangmoonchai R, Edwards M, Phillips IR, Shephard EA (2001) Orphan receptor promiscuity in the induction of cytochromes p450 by xenobiotics. J Biol Chem 276:12822–12826

    Google Scholar 

  51. Starkel P, Sempoux C, Van Den Berge V, Stevens M, De Saeger C, Desager JP, Horsmans Y (1999) CYP 3A proteins are expressed in human neutrophils and lymphocytes but are not induced by rifampicin. Life Sci 64:643–653

    Google Scholar 

  52. Streetman DS, Bertino JS Jr, Nafziger AN (2000) Phenotyping of drug-metabolizing enzymes in adults: a review of in-vivo cytochrome P450 phenotyping probes. Pharmacogenetics 10:187–216

    Google Scholar 

  53. Streetman DS, Bleakley JF, Kim JS, Nafziger AN, Leeder JS, Gaedigk A, Gotschall R, Kearns GL, Bertino JS Jr (2000) Combined phenotypic assessment of CYP1A2, CYP2C19, CYP2D6, CYP3A, N-acetyltransferase-2, and xanthine oxidase with the “Cooperstown cocktail”. Clin Pharmacol Ther 68:375–383

    Google Scholar 

  54. Sumida A, Kinoshita E, Fukuda T, Matsuda H, Yamamoto I, Inaba T, Azuma J (1999) Relationship between mRNA levels quantified by reverse transcription-competitive PCR and metabolic activity of CYP3A4 and CYP2E1 in human liver. Biochem Biophys Res Commun 262:499–503

    Google Scholar 

  55. Tanaka E, Ishikawa A, Yamamoto Y, Osada A, Tsuji K, Fukao K, Misawa S, Iwasaki Y (1992) A simple useful method for the determination of hepatic function in patients with liver cirrhosis using caffeine and its three major dimethylmetabolites. Int J Clin Pharmacol 30:336–341

    Google Scholar 

  56. Thummel KE, Shen DD, Podoll TD, Kunze KL, Trager WF, Bacchi CE, Marsh CL, McVicar JP, Barr DM, Perkins JD, Carithers RL (1994) Use of midazolam as a human cytochrome P450 3A probe: II. Characterization of inter- and intra-individual hepatic CYP3A variability after liver transplantation. J Pharmacol Exp Ther 271:557–566

    Google Scholar 

  57. Thummel KE, Shen DD, Podoll TD, Kunze KL, Trager WF, Hartwell PS, Raisys VA, Marsh CL, McVicar JP, Barr DM et al (1994) Use of midazolam as a human cytochrome P450 3A probe: I. In vitro–in vivo correlations in liver transplant patients. J Pharmacol Exp Ther 271:549–556

    Google Scholar 

  58. Vanden Heuvel JP, Clark GC, Thompson CL, McCoy Z, Miller CR, Lucier GW, Bell DA (1993) CYP1A1 mRNA levels as a human exposure biomarker: use of quantitative polymerase chain reaction to measure CYP1A1 expression in human peripheral blood lymphocytes. Carcinogenesis 14:2003–2006

    Google Scholar 

  59. Vavricka SR, Van Montfoort J, Ha HR, Meier PJ, Fattinger K (2002) Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver. Hepatology 36:164–172

    Google Scholar 

  60. Wang Z, Hall SD, Maya JF, Li L, Asghar A, Gorski JC (2003) Diabetes mellitus increases the in vivo activity of cytochrome P450 2E1 in humans. Br J Clin Pharmacol 55:77–85

    Google Scholar 

  61. Watkins PB (1994) Noninvasive tests of CYP3A enzymes. Pharmacogenetics 4:171–184

    Google Scholar 

  62. Westlind-Johnsson A, Malmebo S, Johansson A, Otter C, Andersson TB, Johansson I, Edwards RJ, Boobis AR, Ingelman-Sundberg M (2003) Comparative analysis of CYP3A expression in human liver suggests only a minor role for CYP3A5 in drug metabolism. Drug Metab Dispos 31:755–761

    Google Scholar 

  63. Xie W, Barwick JL, Simon CM, Pierce AM, Safe S, Blumberg B, Guzelian PS, Evans RM (2000) Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR. Genes Dev 14:3014–3023

    Google Scholar 

  64. Zhuge J, Yu YN (2004) Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue. World J Gastroenterol 10:3356–3360

    Google Scholar 

Download references

Acknowledgements

We are grateful to Jessica A. Haas and Diane Letina B.S. for their technical assistance with the conduct of the real-time quantitative RT-PCR assays. This research was completed in compliance with the current laws of the U.S., inclusive of ethics (IRB) approval. None of the authors has any conflict of interest to disclose with respect to the manuscript. This study was supported by a Kapoor Foundation Grant, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo. The mass spectrometer was obtained by Shared Instrumentation Grant #S10RR14572 from the National Center for Research Resources, National Institutes of Health. At the time of this study, Dr. Haas and Ms. Cloen were partially supported by the VISN-2 Research Development Fund, Veteran’s Administration.

Author information

Authors and Affiliations

  1. School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, 311 Hochstetter Hall, Buffalo, NY, 14260, USA

    Curtis E. Haas, Daniel Brazeau, Denise Cloen, Brent M. Booker, Valerie Frerichs & Colleen Zaranek

  2. Veteran’s Administration Western New York Healthcare System, Buffalo, NY, USA

    Curtis E. Haas, Denise Cloen & Thomas Kufel

  3. College of Pharmacy, University of Florida, Gainesville, FL, USA

    Reginald F. Frye

  4. School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY, USA

    Thomas Kufel

Authors
  1. Curtis E. Haas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Brazeau
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Denise Cloen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Brent M. Booker
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Valerie Frerichs
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Colleen Zaranek
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Reginald F. Frye
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Thomas Kufel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Curtis E. Haas.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Haas, C.E., Brazeau, D., Cloen, D. et al. Cytochrome P450 mRNA expression in peripheral blood lymphocytes as a predictor of enzyme induction. Eur J Clin Pharmacol 61, 583–593 (2005). https://doi.org/10.1007/s00228-005-0971-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00228-005-0971-0

Keywords

Search

Navigation