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Cytochrome P450 (P450) isoenzyme specific dealkylation of alkoxyresorufins in rat brain microsomes

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Abstract

Characterization of xenobiotic metabolizing cytochrome P450s (P450s) was carried out in rat brain microsomes using the specific substrates, 7-pentoxy- and 7-ethoxyresorufin (PR and ER), metabolized in the liver by P450 2B1/2B2 and 1A1/1A2 respectively and 7-benzyloxyresorufin (BR), a substrate for both the isoenzymes. Brain microsomes catalysed the O-dealkylation of PR, BR and ER in the presence of NADPH. The ability to dealkylate alkoxyresorufins varied in different regions of the brain. Microsomes from the olfactory lobes exhibited maximum pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD) and ethoxyresorufin-O-dealkylase (EROD) activities. The dealkylation was found to be inducer selective. While pretreatment with phenobarbital (PB; 80 mg/kg; i.p. × 5 days) resulted in significant induction in PROD (3-4 fold) and BROD (4-5 fold) activities, 3-methylcholanthrene (MC; 30 mg/kg; i.p. × 5 days) had no effect on the activity of PROD and only a slight effect on that of BROD (1.4 fold). MC pretreatment significantly induced the activity of EROD (3 fold) while PB had no effect on it. Kinetic studies have shown that this increase in the activities following pretreatment with P450 inducers was associated with a significant increase in the velocity of the reaction (Vmax) of O-dealkylation. In vitro studies using organic inhibitors and antibodies have further provided evidence that the O-dealkylation of alkoxyresorufins is isoenzyme specific. While in vitro addition of a-naphthoflavone (ANF), an inhibitor of P450 1A1/1A2 catalysed reactions and antibody for hepatic P450 1A1/1A2 isoenzymes produced a concentration-dependent inhibition of EROD activity, metyrapone, an inhibitor of P450 2B1/2B2 and antibody for hepatic P450 2B1/2B2 significantly inhibited the activity of PROD and BROD in vitro. The data suggest that, as in the case of liver, dealkylation of alkoxyresorufins can be used as a biochemical tool to characterise the xenobiotic metabolising P450s and substrate selectivity of P450 isoenzymes in rat brain microsomes.

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References

  1. Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR, Gotoh O, Coon MJ, Estabrook RW, Gunsalus IC, Nebert DW: P450 superfamily: Update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6: 1–42, 1996

    PubMed  Google Scholar 

  2. Lewis DFW: Cytochromes P450: Structure, function and mechanism. Taylor and Francis Ltd., Great Britain, 1996, pp 169–208

    Google Scholar 

  3. Nebert DW: Proposed role of drug metabolising enzyme: Regulation of steady state levels of the ligands that effect growth, homeostasis, differentiation and neuroendocrine functions. Mol Endocrinol 5: 1203–1214, 1991

    PubMed  Google Scholar 

  4. Alland P, Marcusson JO, Ross SB: [3H] GBR 12935 binding to cytochrome P450 in the human brain. J Neurochem 62: 342–348, 1994

    PubMed  Google Scholar 

  5. Stromestedt M, Warner M, Banner CD, MacDonald PC, Gustafsson JA: Role of brain cytochrome P450 in regulation of the level of anesthetic steroids in the brain. Mol Pharmacol 44: 1077–1083, 1993

    PubMed  Google Scholar 

  6. Stapleton G, Steel M, Richardson M, Mason JO, Rose KA, Morris RGM, Lathe R: A novel cytochrome P450 expressed primarily in brain. J Biol Chem 270: 29739–29745, 1995

    PubMed  Google Scholar 

  7. Warner M, Gustafsson JA: Effect of ethanol on cytochrome P450 in the rat brain. Proc Natl Acad Sci USA 91: 1019–1023, 1994

    PubMed  Google Scholar 

  8. Kalow W, Tyndale R: Debrisoquine/Sparteine monooxygenase and other P450s in the brain. In: W. Kalow (ed). Pharmacogenetics of Drug Metabolism. The International Encyclopaedia of Pharmacology and Therapeutics. Pergamon Press Inc., New York, 1992, pp 649–656

    Google Scholar 

  9. Warner M, Kohler C, Hansson T, Gustafsson JA: Regional distribution of cytochrome P450 in the rat brain: Spectral quantitation and contribution of P450 b, e and P450 c,d. J Neurochem 50: 1057–1065, 1988

    PubMed  Google Scholar 

  10. Farin FM, Omiecinski CJ: Regiospecific expression of cytochrome P-450s and microsomal epoxide hydrolase in human brain tissue. J Toxicol Environ Health 40: 317–335, 1993

    PubMed  Google Scholar 

  11. Schilter B, Omiecinski CJ: Regional distribution and expression modulation of cytochrome P450 and epoxide hydrolase mRNAs in the rat brain. Mol Pharmacol 44: 990–996, 1993

    PubMed  Google Scholar 

  12. Kawashima H, Strobel HW: cDNA cloning of a novel rat brain cytochrome P450 belonging to the CYP2D subfamily. Biochem Biophys Res Commun 209: 535–540, 1995

    PubMed  Google Scholar 

  13. Kawashima H, Strobel HW: cDNA cloning of the new forms of rat brain cytochrome P450 belonging to the CYP4F subfamily. Biochem Biophys Res Commun 217: 1137–1144, 1995

    PubMed  Google Scholar 

  14. Wang H, Kawashima H, Strobel HW: cDNA cloning of a novel CYP3A from rat brain. Biochem Biophys Res Commun 221: 157–162, 1996

    PubMed  Google Scholar 

  15. Das M, Seth PK, Mukhtar H: Characterization of microsomal aryl hydrocarbon hydroxylase of rat brain. J Pharmacol Exp Ther 216: 156–161, 1981

    PubMed  Google Scholar 

  16. Das M, Seth PK, Dixit R, Mukhtar H: NADH-dependent inducible arylhydrocarbon hydroxylase activity in rat brain mitochondria. Arch Biochem Biophys 217: 205–215, 1982

    PubMed  Google Scholar 

  17. Dhawan A, Parmar D, Das M, Seth PK: Characterization of cerebral 7-ethoxycoumarin-O-deethylase: Evidence for multiplicity of cytochrome P450 in brain. Biochem Med Metab Biol 41: 184–192, 1989

    PubMed  Google Scholar 

  18. Parmar D, Dhawan A, Dayal M, Seth PK: Immunochemical and biochemical evidences for expression of phenobarbital (PB) and 3-methylcholanthrene (MC) inducible isoenzymes of cytochrome P450 in rat brain. Int J Toxicol 17: 619–630, 1998

    Google Scholar 

  19. Dhawan A, Parmar D, Das M, Seth PK: 7-Ethoxyresorufin-O-deethylase: A cytochrome P448 dependent enzyme in mammalian brain. Eur J Pharmacol (Brief Communication) 183: 1509–1510, 1990

    Google Scholar 

  20. Parmar D, Dhawan A, Seth PK: Evidence for O-dealkylation of 7-pentoxyresorufin by cytochrome P450 2B1/2B2 isoenzymes in brain. Mol Cell Biochem 189: 201–205, 1998

    PubMed  Google Scholar 

  21. Burke MD, Thomson S, Elcombe CR, Halpert J, Haaparanta T, Mayer RT: Ethoxy-, pentoxy-and benzyloxyphenoxazones and homologues: A series of substrates to distinguish between different induced cytochrome P450. Biochem Pharmacol 34: 3337–3345, 1985

    PubMed  Google Scholar 

  22. Ravindranath V, Anandatheerthavarada HK: High concentration of cytochrome P-450 linked aminopyrine N-demethylase in mouse brain, and associated sex related difference. Biochem J 261: 769–773, 1989

    PubMed  Google Scholar 

  23. Anandatheerthavarada HK, Shankar SK, Ravindranath V: Rat brain cytochrome P-450: Catalytic, immunochemical properties and inducibility of multiple forms. Brain Res 536: 339–343, 1990

    PubMed  Google Scholar 

  24. Glowinski J, Iverson JL: Regional studies of catecholamines in the rat brain – the deposition of [3H]-norepinephrine, [3H]-dopamine and [3H]-DOPA in various regions of the brain. J Neurochem 13: 655–670, 1966

    PubMed  Google Scholar 

  25. Rutten AAJJL, Falke HE, Catsburg JF, Wortelboer HM, Blaauboer BJ, Doorn L, Van Leeuwen EXR, Theelen R, Rietjens IMCM: Interlaboratory comparison of microsomal ethoxyresorufin and pentoxyresorufin-O-dealkylation determinations: standardization of assay conditions. Arch Toxicol 66: 237–244, 1992

    PubMed  Google Scholar 

  26. Pohl RJ, Fouts JR: A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions. Anal Biochem 107: 150–155, 1980

    PubMed  Google Scholar 

  27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951

    PubMed  Google Scholar 

  28. Walther B, Ghersi-Egea JF, Minn A, Siest G: Subcellular distribution of cytochrome P-450 in the brain. Brain Res 375: 338–344, 1986

    PubMed  Google Scholar 

  29. Perrin R, Minn A, Ghersi-Egea JF, Grassiot MC, Siest G: Distribution of cytochrome P450 activities towards alkoxyresorufin derivatives in rat brain regions, subcellular fractions and isolated cerebral microvessels. Biochem Pharmacol 40: 2145–2151, 1990

    PubMed  Google Scholar 

  30. Waxman DJ, Azaroff L: Phenobarbital induction of cytochrome P-450 gene expression. Biochem J 281: 577–592, 1992

    PubMed  Google Scholar 

  31. Nakajima T, Elovaara E, Park SS, Gelboin HV, Hietnen E, Vaino H: Monoclonal antibody-directed characterization of benzene, ethoxyresorufin and pentoxyresorufin metabolism in rat liver microsomes. Biochem Pharmacol 40: 1255–1261, 1990

    PubMed  Google Scholar 

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  1. Industrial Toxicology Research Centre, P.O. Box-80, M.G. Marg, Lucknow -, 226 001, India

    Alok Dhawan, Devendra Parmar, Monika Dayal & Prahlad K. Seth

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  1. Alok Dhawan
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  2. Devendra Parmar
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  3. Monika Dayal
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  4. Prahlad K. Seth
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Dhawan, A., Parmar, D., Dayal, M. et al. Cytochrome P450 (P450) isoenzyme specific dealkylation of alkoxyresorufins in rat brain microsomes. Mol Cell Biochem 200, 169–176 (1999). https://doi.org/10.1023/A:1007026800114

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