Elsevier

Brain Research

Volume 855, Issue 2, 14 February 2000, Pages 235-243
Brain Research

Research report
Cytochrome P-450 activities in human and rat brain microsomes

https://doi.org/10.1016/S0006-8993(99)02354-9Get rights and content

Abstract

The role of cytochrome P450 in the metabolism of dextromethorphan, amitriptyline, midazolam, S-mephenytoin, citalopram, fluoxetine and sertraline was investigated in rat and human brain microsomes. Depending on the parameters, the limit of quantification using gas chromatography–mass spectrometry methods was between 1.6 and 20 pmol per incubation, which generally contained 1500 μg protein. Amitriptyline was shown to be demethylated to nortriptyline by both rat and human microsomes. Inhibition studies using ketoconazole, furafylline, sulfaphenazole, omeprazole and quinidine suggested that CYP3A4 is the isoform responsible for this reaction whereas CYP1A2, CYP2C9, CYP2C19 and CYP2D6 do not seem to be involved. This result was confirmed by using a monoclonal antibody against CYP3A4. Dextromethorphan was metabolized to dextrorphan in rat brain microsomes and was inhibited by quinidine and by a polyclonal antibody against CYP2D6. Only the addition of exogenous reductase allowed the measurement of this activity in human brain microsomes. Metabolites of the other substrates could not be detected, possibly due to an insufficiently sensitive method. It is concluded that cytochrome P450 activity in the brain is very low, but that psychotropic drugs could undergo a local cerebral metabolism which could have pharmacological and/or toxicological consequences.

Introduction

Cytochrome P-450 (P450) is the most important xenobiotic metabolizing enzyme system. It is also involved in the metabolism of endogenous compounds such as steroids and fatty acids. P450 activity requires the presence of cytochrome P450 reductase which transfers reducing equivalents from NADPH to P450. Even if the liver is the organ that plays the essential role in drug metabolism, the extrahepatic presence and activity of P450 is now a major area of interest. Brain has become one of the most studied organs because of the possible pharmacological and toxicological implications of cerebral drug metabolism. Several isoforms of P450 (1A1, 1A2, 2B1, 2B2, 2D, 2E1, 3A) have been described in the brain, mostly in rodents 8, 34, 45 but also in humans 9, 19, 20, 32, 37. These investigations have been performed using immunohistochemistry, polymerase chain reaction (PCR) and metabolism studies. The first two approaches aim to demonstrate the presence of the enzymes or its mRNA while metabolic studies show the activity of the enzymatic system. However, discrepancies exist in the results. The differences in the principle and in the sensitivity of the methods and the antibodies used are thought to be partly responsible for the differences [22]. The levels and activities of P450 found in cerebral tissues are very low and make metabolic studies difficult. However, metabolism of several substrates has been shown using brain microsomes. Recently, biotransformation of imipramine to desipramine and hydroxy-imipramine [41], and of dextromethorphan to dextrorphan has been described in rat brain microsomes. The latter reaction is thought to be mediated by CYP2D1 [26]. Its human equivalent form, CYP2D6, is known to be polymorphic and is involved in the metabolism of many psychotropic drugs 7, 11, 33, 42. According to some highly discussed hypotheses 13, 38, subjects who have a genetic deficiency of CYP2D6 (« poor metabolizers », PM) are more likely to develop neurodegeneratives diseases such as Parkinson's disease or Alzheimer's disease 31, 36, 39.

The aim of this study was to describe the metabolic activity of P450 isoforms in rat and human brain microsomes, and especially of CYP2D6, against specific substrates including some psychotropic drugs. The determination of the presence of P450 or its mRNA by immunohistochemistry or by PCR was not investigated in this study.

Section snippets

Chemicals

Dextromethorphan, dextrorphan and midazolam were gifts from Roche (Basel, Switzerland), 1′-hydroxy-midazolam and ketoconazole were gifts from Prof. P. Dayer (Geneva, Switzerland), amitriptyline, nortriptyline, citalopram and demethyl–citalopram from Lundbeck (Copenhagen, Denmark), maprotiline and methyl–maprotiline from Novartis (Basel, Switzerland), sertraline and norsertraline from Pfizer (Zurich, Switzerland), fluoxetine and norfluoxetine from Elli Lilly (Indianapolis, USA), clovoxamine from

Dextromethorphan-O-demethylase activity

A production of dextrorphan was detected in incubations with rat brain microsomes with dextromethorphan concentrations ranging from 100 to 600 μM (Fig. 1) (No production was measurable with human brain microsomes using HB1-5 and HB12-13).The activity remained linear over the tested time points (30–120 min) (Fig. 2). The production increased proportionally with the amount of protein in the incubation tubes (Fig. 3). A smaller production of dextrorphan was also observed in rat brain mitochondria

Discussion

This study shows some, but in comparison to the liver very low, activity of P450 in rat and even considerably lower activity in human brain microsomes.

The metabolic pathways investigated in this study are the main ones for the molecules tested. In human liver, dextromethorphan is specifically N-demethylated by the CYP2D6, S-mephenytoin and midazolam are hydroxylated by the CYP2C19 and 3A4, respectively while amitriptyline, citalopram, sertraline and fluoxetine are not specific substrates of a

Acknowledgements

This study was supported by a grant from the foundation from the Basle Chemical Industry for PhD candidates and from the Swiss National Foundation for Scientific Research (32-42076.94, 32-53717.98). We are grateful to Mrs M-F Hamou, Mrs M-J Voirol, Mrs E. Kovari, Mr. M. Giacomini and Mr. A. Lobrinus, MD, for their help in collecting samples. For the preparation of the manuscript, we appreciated the help of Mrs. K. Powell Golay, C. Bertschi, M. Gobin, and T. Bocquet.

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