Research reportBrain microsomal metabolism of phencyclidine in male and female rats
Introduction
Phencyclidine (PCP) is a psychotomimetic agent that exerts its pharmacological effects at several sites within the central nervous system (CNS). Among these sites are the PCP receptor which is a binding site in the ion channel associated with the N-methyl-d-aspartate (NMDA) receptor complex 25, 44, 53, and the dopamine transporter which is inhibited by PCP 9, 43. Although the CNS plays a key role in mediating PCP effects, its role in metabolism of PCP is not known.
Metabolism is the major mechanism for termination of the pharmacological effects of PCP 16, 26, however mammalian metabolism of PCP has mostly been studied in liver and lung tissue (for review see [17]). These studies show PCP metabolism leads to the formation of a monohydroxylated metabolite of PCP (1-(1-phenylcyclohexyl)-4-hydroxypiperidine, PCHP) that has about one-third the pharmacological potency of PCP [10]. In addition, PCP metabolism in these tissues leads to in vitro and in vivo irreversible binding of a PCP metabolite 13, 23, 24, 52. Direct toxicity and/or pharmacological effects of this metabolite are not known. Nevertheless, it has been postulated that if highly reactive PCP metabolites were formed in the human brain, it could play a role in the long-lasting PCP induced psychotic behavior which is sometimes observed 14, 23.
Although several enzyme systems are responsible for PCP metabolism, the cytochrome P450 (CYP) system is of major importance [17]. Law 23, 24reports that pretreatment of rats with PCP results in a significant decrease in benzo(a)pyrene hydroxylase activity. This substrate is associated with CYP1A1 activity (previously known as P-450c) [37]. Phenobarbital induction of liver metabolism suggests the involvement of the phenobarbital-inducible CYP isoforms in PCP metabolism 8, 20, 35and irreversible binding in the liver 13, 14, 20. Studies from Owens et al. [33]and Hiratsuka et al. [12]suggest a possible role for CYP2D isoforms in the formation of PCP metabolites. These investigators 12, 39also find that CYP2C11 (a male-specific CYP isoform [19]) is a major enzyme responsible for PCP metabolism and irreversible binding in normal male rat livers.
Although immunochemical and reverse transcriptase-polymerase chain reaction techniques have demonstrated the presence of multiple CYP forms in the brain (e.g. 21, 38, 45, 47), relatively few studies have examined the role of these enzymes in the formation of metabolites of xenobiotics. Furthermore, PCP and/or PCP metabolites persist in rat brain for up to 3 weeks following a single dose [27]and brain concentrations of PCP are significantly higher than serum concentrations at all times after intravenous PCP administration in male Sprague–Dawley (SD) rats [42]. Perrin et al. [35]suggest that PCP can interact with the active site of CYP enzymes in the brain since PCP causes a type I spectral modification when incubated with rat brain mitochondrial or microsomal fractions. Another study [51]shows that a PCP-derived tetrahydropyridinium metabolite is formed in the presence of rat brain mitochondria.
Although these studies suggest PCP can be metabolized by the brain, the results are unclear. Therefore, the purpose of these studies was to examine the metabolism of PCP in rat brain microsomes. Since previous studies find sexually dimorphic PCP-induced behavior [28], neurotoxicity [30]and liver metabolism 28, 32, 39, we examined in vitro PCP metabolism in male and female rat brain homogenates.
Section snippets
Materials
Phencyclidine hydrochloride (PCP), cis-1-(1-phenyl-4-hydroxycyclohexyl)piperidine (c-PPC), trans-(1-phenyl-4-hydroxycyclohexyl)piperidine (t-PPC), 1-(1-phenylcyclohexyl)-4-hydroxypiperidine (PCHP), 5-[N-(1′-phenylcyclohexyl)amino]pentanoic acid (PCHAP), and 1-(1-[phenyl-3-3H(n)]cyclohexyl)piperidine ([3H]PCP, 15.69 Ci/mmol labeled at a metabolically stable site) were obtained from the National Institute on Drug Abuse (Rockville, MD). All other chemicals were obtained from Fisher Scientific
Characteristics of rat brain microsomal protein
For each pool of five rat brains, approximately 40 mg of microsomal protein was recovered. We attempted to measure the total CYP content by the methods described by Omura and Sato [31]and the more sensitive method for brain microsomes described by Ghersi-Egea et al. [11]; but, we were unable to determine the cytochrome P450 content of these preparations due to the low levels of CYP and a high spectral background (results not shown). Nevertheless, a study by Perrin et al. [36]reports equal
Discussion
These studies examined the in vitro metabolism of PCP by male and female rat brain microsomes. We detected the formation of several major metabolites of PCP in brain that are also formed in rat liver (Table 1). These PCP metabolites were all monohydroxylated metabolites (c-PPC, t-PPC and PCHP) (Fig. 1). The formation of these metabolites was time, protein and NADP+ dependent (Fig. 2, Fig. 3, Fig. 4). The highly polar amino pentanoic acid metabolite (PCHAP) was not detected. This metabolite is
Acknowledgements
This work was supported by a grant from the National Institute on Drug Abuse (DA04136), a Research Scientist Development Award to S.M.O. (K02 DA00110), and a predoctoral fellowship to E.M.L. (T32 DA07260). We wish to thank Dr. H.V. Gelboin for his generous gift of the anti-CYP2C11 monoclonal antibody and Dr. L.M. Arnold for the control monoclonal antibody. Shannon L. Sorrels and Melinda Gunnell also provided helpful technical assistance and advice. A preliminary report of these studies was
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Current address: Department of Environmental Health, School of Public Health, University of Washington, 4225 Roosevelt Way NE, #100, Seattle, WA 98105-6099, USA.