Measurement of Human Cytochrome P4501A2 (CYP1A2) Activity In Vitro

Thomas M. Polasek1, David J. Elliot1, John O. Miners1

1 Flinders Medical Centre and Flinders University School of Medicine, Bedford Park
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.19
DOI:  10.1002/0471140856.tx0419s27
Online Posting Date:  March, 2006
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Abstract

Cytochrome P4501A2 (CYP1A2) is responsible for the metabolism of a diverse range of clinically used drugs and dietary and environmental chemicals (including many procarcinogens). CYP1A2 expression is influenced by numerous factors, and hence wide interindividual variability is a characteristic feature of this enzyme in humans. Phenacetin represents a convenient probe for the assessment of human CYP1A2 activity in vitro (hepatic microsomes and recombinant enzyme). It is a relatively high‐turnover substrate that forms only one major primary metabolite, the O‐deethylated derivative acetaminophen. Acetaminophen formation in incubations of phenacetin with a CYP1A2 source is readily measured by HPLC with UV detection. The assay has a low requirement for human liver microsomes or recombinant enzyme, and is both selective and sensitive without the requirement for a solvent extraction step. Overall assay reproducibility is excellent, with coefficients of variation <4%.

Keywords: cytochrome P4501A2; CYP1A2; phenacetin O‐deethylation; high performance liquid chromatography

     
 
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Table of Contents

  • Basic Protocol 1: Quantification of Acetaminophen Formed in Incubations of Phenacetin with Microsomal CYP1A2
  • Alternate Protocol 1: Quantification of Acetaminophen Formed in Incubations of Phenacetin with Recombinant CYP1A2
  • Support Protocol 1: Quantitative Analysis of Acetaminophen by HPLC
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantification of Acetaminophen Formed in Incubations of Phenacetin with Microsomal CYP1A2

  Materials
  • 200 mM phenacetin [(N‐4‐ethoxyphenyl)acetamide]: prepare by dissolving 35.8 mg phenacetin (Sigma‐Aldrich) in 1.00 ml methanol on the day of use
  • Human liver microsomes (see Polasek et al., )
  • 1 M potassium phosphate buffer, pH 7.4 (see recipe)
  • 1 M MgCl 2
  • NADPH‐generating system (see recipe)
  • Acetonitrile (HPLC grade), ice cold
  • 12 × 75–mm borosilicate glass culture tubes
  • 37°C shaking water bath
  • Centrifuge
  • Additional reagents and equipment for determination of protein concentration ( appendix 3G or appendix 3I)

Alternate Protocol 1: Quantification of Acetaminophen Formed in Incubations of Phenacetin with Recombinant CYP1A2

  • Recombinant CYP1A2 (BD Biosciences)

Support Protocol 1: Quantitative Analysis of Acetaminophen by HPLC

  Materials
  • HPLC mobile phase A (see recipe)
  • HPLC mobile phase B: prepared by filtering HPLC grade acetonitrile through a 0.45‐µm membrane filter and stored in a tightly closed container up to 1 month at room temperature
  • 1 M phosphate buffer, pH 7.4
  • 100 µM acetaminophen (Sigma‐Aldrich; see recipe)
  • HPLC column, Ultrasphere ODS 4.6 × 250–mm (Beckman Instruments)
  • 12 × 75–mm glass culture tubes
  • Gradient HPLC system with UV detector and data system
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Figures

Videos

Literature Cited

Literature Cited
   Butler, M.A., Iwasaki, M., Guengerich, F.P., and Kadlubar, F.F. 1989. Human cytochrome P450PA (P4501A2), the phenacetin O‐deethylase, is primarily responsible for the hepatic 3‐demethylation of caffeine and N‐oxidation of carcinogenic arylamines. Proc. Natl. Acad. Sci. U.S.A. 86:7696‐7700.
   Chauret, N., Gauthier, A., and Nicoll‐Griffith, D.A. 1998. Effect of common organic solvents and in vitro cytochrome P450‐mediated metabolic activities in human liver microsomes. Drug Metab. Dispos. 26:1‐4.
   Eaton, D.L., Gallagher, E.P., Bammler, T.K., and Kunze, K.L. 1995. Role of cytochrome P4501A2 in chemical carcinogenesis: Implications for human variability in expression and enzyme activity. Pharmacogenetics 5:259‐274.
   Miners, J.O. and McKinnon, R.A. 2000. CYP1A. In Metabolic Drug Interactions (R.H. Levy, K.E. Thummel, W.F. Trager, P.D. Hansten, and M.E. Eichelbaum, eds.) pp. 61‐74. Lippincott Williams & Wilkins, Philadelphia.
   Omura, T. and Sato, R. 1964. The carbon monoxide binding pigment of liver microsomes. J. Biol. Chem. 293:2379‐2385.
   Polasek, T.M., Elliot, D.J., Lewis, B.C., and Miners, J.O. 2004. Mechanism‐based inactivation of human cytochrome P4502C8 by drugs in vitro. J. Pharmacol. Exp. Ther. 311:996‐1007.
   Rasmussen, B.B., Brix, T.H., Kyvik, K.O., and Brosen, K. 2002. The interindividual differences in the 3‐demethylation of caffeine alias CYP1A2 is determined by both genetic and environmental factors. Pharmacogenetics 12:473‐478.
   Sasche, C., Bhambra, U., Smith, G., Lightfoot, T.J., Barrett, J.H., Scollay, J., Garner, R.C., Boobis, A.R., Wolf, C.R., Gooderham, N.J., and the Colorectal Cancer Study Group. 2003. Polymorphisms in the cytochrome P4501A2 gene (CYP1A2) in colorectal cancer patients and controls: Allele frequencies, linkage disequilibrium and influence on caffeine metabolism. Br. J. Clin. Pharmacol. 55:68‐76.
   Schweikl, H., Taylor, J.A., Kitareewan, S., Linko, P., Nagorney, D., and Goldstein, J.A. 1993. Expression of CYP1A1 and CYP1A2 genes in human liver. Pharmacogenetics 3:239‐249.
   Tassaneeyakul, W., Mohamed, Z., Birkett, D.J., McManus, M.E., Veronese, M.E., Tukey, R.H., Quattrochi, L.C., Gonzalez, F.J., and Miners, J.O. 1992. Caffeine as a probe for human cytochromes P450: Validation using cDNA‐expression, immunoinhibition and microsomal kinetic and inhibitor techniques. Pharmacogenetics 2:173‐183.
   Tassaneeyakul, W., Birkett, D.J., Veronese, M.E., McManus, M.E., Tukey, R.H., Quattrochi, L.C., Gelboin, H.V., and Miners, J.O. 1993. Specificity of substrate and inhibitor probes for human cytochromes P450 1A1 and 1A2. J. Pharmacol. Exp. Ther. 265:401‐407.
   Tassaneeyakul, W., Birkett, D.J., McManus, M.E., Tassaneeyakul, W., Veronese, M.E., Andersson, T., Tukey, R.H., and Miners, J.O. 1994. Caffeine metabolism by human hepatic cytochromes P450: Contributions of 1A2, 2E1 and 3A isoforms. Biochem. Pharmacol. 47:1767‐1776.
   Walsky, R.L. and Obach, R.S. 2004. Validated assays for human cytochrome P450 activities. Drug Metab. Dispos. 32:647‐660.
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