cDNA Cloning of a Novel Rat Brain Cytochrome P450 Belonging to the CYP2D Subfamily

doi:10.1006/bbrc.1995.1534

Biochemical and Biophysical Research Communications

Volume 209, Issue 2, 17 April 1995, Pages 535-540

https://doi.org/10.1006/bbrc.1995.1534 Get rights and content

Abstract

Two cDNA clones designated 2d-29 and 2d-35 encoding a novel P450 belonging to the P450 2D subfamily were isolated from control and imipramine-treated rat train Libraries,respectively. cDNA 2d-29 and 2d-35 contained 1786 and 2754 bp nucleotides each. Although their 5′- and 3′-untranslated sequences were different from each other, their open reading frames were identical, encoding a protein which had 5 substitutions of amino acids in the C-terminal region compared with P450 2D4. Northern blot analysis showed that the expression levels of 2d-29 and 2d-35 in the brain were lower than that of P450 2D4 in the liver. However, it is noteworthy that the expression of 2d-35 was not detected in the fiver but in the brain. These results suggest that a novel P450 is expressed in the brain under different regulation.

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In rats, six homologs of CYP2D6 have been isolated: CYP2D1, CYP2D2, CYP2D3, CYP2D4, CYP2D5, and CYP2D18 (Nelson et al., 1996). The mRNA similarities range from 76–83% among these isoforms, and the amino acid sequences display between 72% and 99.5% similarity (Gonzalez et al., 1987; Kawashima and Strobel, 1995). There are 22 amino acid differences between CYP2D5 and CYP2D1, and only 2 residues are different among the active site residues.
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Cytochromes P450 (P450), a family of heme-containing proteins, is involved in the oxidative metabolism of both foreign and endogenous compounds. Although liver is quantitatively the major organ involved in the metabolism of most xenobiotics, there is increasing evidence that these enzymes are present in extrahepatic tissues, such as lung, kidney, brain, etc and they may contribute to the in situ metabolism of xenobiotics in these organs. The possible relationship between genetic polymorphism seen in P4502D6 and incidence of neurodegenerative diseases, such as Parkinson's disease, has prompted the characterization of P4502D enzymes in rat brain. In the present study, we demonstrate that P4502D1 (the rat homologue of human P4502D6) is constitutively expressed in rat brain and the mRNA and protein are localized predominantly in neuronal cell population in the olfactory bulb, cortex, cerebellum, and hippocampus. An alternate spliced transcript of CYP2D1 having exon 3 deletion was detected in rat brain but not in liver. Deletion of exon 3 causes frame shift and generates a stop codon at 391 bp relative to the start codon ATG leading to premature termination of translation. Thus, Northern blotting and in situ hybridization represent contributions from functional transcripts and alternate spliced variants that do not translate into functional protein. Further, the splice variant having partial inclusion of intron 6 detected in human brain was not detected in rat brain indicating that alternate spliced gene products of P450 enzymes are generated in species-specific and tissue-specific manner.
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The CYP2D subfamily has evolved differently in humans and rats. Isoenzymes of the human CYP2D subfamily are encoded by one active CYP2D6 gene and two pseudogenes, while six genes, CYP2D1–5 and CYP2D18, have been identified in rats [24,27,38,46]. It is still unclear which of these six known rat CYP2D subfamily members is/are homologous to human CYP2D6 [40,53,65].
Direct evidence for accumulation of 1,2,3,4-tetrahydroisoquinoline (TIQ), an endo- and exogenous substance suspected of producing Parkinsonism in humans, has not yet been shown. This study aimed to examine TIQ disposition in the whole rat brain and in the striatum and substantia nigra (SN). TIQ was administered to male Wistar and Dark Agouti rats (20, 40 and 100 mg/kg i.p.) alone or jointly with specific CYP2D inhibitor quinine (20, 40, 80 mg/kg i.p.), acutely or chronically. TIQ concentration in brain of both strains was several-fold higher than in plasma. The level of its metabolite, 4-OH-TIQ, was very low in the brain and plasma of TIQ-treated Wistar while in those receiving additionally quinine or in Dark Agouti rats, 4-OH-TIQ was absent or negligible. Inhibition of CYP2D catalyzing TIQ 4-hydroxylation in the liver had no influence on TIQ accumulation in the brain. Exogenous TIQ was actively transported from periphery into the brain by the organic cation transporter system, mainly OCT3, and quickly eliminated from it by P-glycoprotein. TIQ accumulation after chronic injection to Wistar rats was short-lasting and limited to SN. High concentration of TIQ in SN induces while in the liver inhibits the nigral and hepatic activity CYP2D, respectively.
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2003, Food and Chemical Toxicology
Oral administration of lindane (2.5, 5, 10 and 15 mg/kg, body weight) for 5 days was found to produce a dose-dependent increase in the activity of P450 dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) in rat brain and liver. A significant increase in the hepatic and brain P450 monooxygenases was also observed when the duration of exposure of low dose (2.5 mg/kg) of lindane was increased from 5 days to 15 or 21 days. As observed with different doses, the magnitude of induction in the activity of P450 monooxygenases was several fold higher in liver microsomes when compared with the brain. Western blotting studies have indicated that the increase in the P450 enzymes could be due to the increase in the expression of P450 1A1/1A2, 2B1/2B2 and 2E1 isoenzymes. In vitro studies using organic inhibitors specific for individual P450 isoenzymes and antibody inhibition experiments have further demonstrated that the increase in the activity of PROD, EROD and NDMA-d are due to the increase in the levels of P450 2B1/2B2, 1A1/1A2 and 2E1 isoenzymes, respectively. Induction studies have further shown that while pretreatment of 3-methylcholanthrene (MC), an inducer of P4501A1/1A2, did not produce any significant effect in the incidence of lindane induced convulsions, pretreatment with phenobarbital (PB), an inducer of P450 2B1/2B2 or ethanol, an inducer of P450 2E1 catalysed reactions, significantly increased the incidence of lindane induced convulsions. Similarly, when the P450-mediated metabolism of lindane was blocked by cobalt chloride incidence of convulsions was increased in animals treated with lindane indicating that lindane per se or its metabolites formed by PB or ethanol inducible P450 isoenzymes are involved in its neurobehavioral toxicity.
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Citation Excerpt :
In humans, only one CYP2D isoform, CYP2D6, has been found [1]. On the other hand, five CYP2D isoforms, CYP2D1 through CYP2D5, have been found in the rat liver [29–31]. The present results showed that BLH activity was extensively metabolized by CYP2D2, whereas CYP2D1 barely catalyzed the activity (Fig. 2C).
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Regular ArticlecDNA Cloning of a Novel Rat Brain Cytochrome P450 Belonging to the CYP2D Subfamily

Abstract

Regular Article
cDNA Cloning of a Novel Rat Brain Cytochrome P450 Belonging to the CYP2D Subfamily