Research reportCytochrome P-450 activities in human and rat brain microsomes
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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