Expression of Multiple Forms of Cytochrome P450 and Associated Mono-oxygenase Activities in Rat Brain Regions

doi:10.1016/S0006-2952(98)00036-7

Biochemical Pharmacology

Volume 56, Issue 3, 1 August 1998, Pages 371-375

https://doi.org/10.1016/S0006-2952(98)00036-7 Get rights and content

Abstract

Cytochrome P450 (P450) content and P450-mediated mono-oxygenase activities were measured in microsomes prepared from various regions of rat brain. The regional P450 content in brain varied between 0.1 and 0.15 nmol/mg of protein, with the brainstem and cerebellum showing the highest levels. NADPH cytochrome c reductase activity was highest in the cortex followed by cerebellum and brainstem as compared with the whole brain. Mono-oxygenase activities also varied among the various brain regions. Southern blot analysis of the cDNA synthesized from the poly(A)RNA isolated from rat brain regions and hybridized with cDNA to rat liver P4502B or P4502E1 revealed the presence of a transcript in untreated rat brain that had a molecular mass similar to that of the corresponding transcript from rat liver. Immunoblot analyses using antisera to purified rat liver P4502E1, P450(2B1/2B2), and a phenobarbital-inducible form of rat brain P450 revealed the presence of corresponding immunoreactive protein bands in all the brain regions examined. The present study demonstrated the diversity in the distribution of P450 and associated mono-oxygenase activities in brain and thus may reflect the differential capability of various regions of the brain to detoxify or bioactivate diverse xenobiotics.

Section snippets

Materials and Methods

Male Wistar rats (3–4 months old) were obtained from the NIMHANS Animal Research Facility. Animals had access to pelleted diet (Lipton India Ltd.) and water ad lib. Animals were anesthetized with ether and perfused transcardially with 30 mL of 0.1 M Tris–HCl (pH 7.4) prior to decapitation. The brain was removed immediately and six regions, namely the cortex, cerebellum, brainstem, striatum, hippocampus, and thalamus, were dissected as described [16].

Microsomes were prepared from whole brain and

Results

P450 content and various P450-associated mono-oxygenase activities in rat brain regions are given in Table 1. P450 levels were significantly higher in brainstem and cerebellum as compared with the whole brain. NADPH cytochrome c reductase activity in the brain was approximately 30% of the hepatic level (data not shown), and its activity was significantly higher in the cerebral cortex, cerebellum, and brainstem than in the whole brain.

P450-mediated mono-oxygenase activities also showed regional

Discussion

The present study revealed some distinct regionality in the distribution of P450 and associated mono-oxygenase activities in rat brain. Although the P450 levels were significantly higher in cerebellum and brainstem, it is interesting that the brainstem region contains relatively low levels of glutathione compared with other brain regions 29, 30. A relatively high P450 content combined with low glutathione levels might render this region particularly vulnerable to damage through P450-mediated

Acknowledgements

The authors wish to thank Dr. B. J. Song for the gift of antisera and cDNA to rat liver P4502E1 and Dr. F. Gonzalez for the cDNA to rat liver P4502B1. P.S.T. and S.C.U. thank CSIR, India for the award of a Senior Research Associateship and a Junior Research Fellowship, respectively. This work was funded in part by a research grant from the Third World Academy of Sciences, Italy.

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Moreover, cytoarchitectonic organization and cell functions are extremely variable in brain and, when compared cell to cell, the levels of CYPs in specific neurons can be as important or more so than levels in hepatocytes [7]. Differential expression according to the cerebral region has been described, with the highest CYP content in the brain stem and cerebellum and the lowest values in the striatum and hippocampus, showing some degree of similarity with rat brain [8,9]. Every CYP isoform is also distributed among different regions with a specific expression pattern.
The metabolism of xenobiotics in human brain constitutes a field of recent intensive research in relation to the potential implications in the pharmacological effect of drugs acting on the central nervous system. Cytochrome P450 enzymes (CYPs) play a crucial role in these metabolic pathways and the existence of functional CYP monooxygenases in brain is now well established. These enzymes are preferentially localized in the neuronal cells within the microsomal fraction and the inner membrane of mitochondria. Although low, the metabolism in situ could influence individual response to xenobiotics or produce reactive, toxic metabolites causing irreversible damage in the neuronal cells. The abundant presence of CYPs in selective cell populations within different regions of the brain has also suggested a role for these enzymes in brain physiology thus not restricted to xenobiotic-induced neurotoxicity. For instance, CYPs participate in the regulation of neurotransmitters and steroids and brain maintenance of cholesterol homeostasis. Recent advances support an additional role for these enzymes in the pathogenesis of psychiatric and neurodegenerative disorders such as depression, schizophrenia, and Alzheimer's and Parkinson's diseases. The characterization of brain CYP isoforms and their localization, the identification of their substrates and metabolic end-products will allow better understanding of the role of these enzymes in brain physiology, development and diseases.
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Expression and monooxygenase activity of various cytochrome P450 (CYP) enzymes along with constitutive androstane (CAR) and the pregnane X (PXR) receptors were investigated in the brain of control and phenobarbital-treated rabbits (80 mg/kg for 4 days). RT-PCR analysis, using specific primers, demonstrated that in control rabbits mRNAs of CYP 2A10, 2B4/5 and 3A6 were expressed, though to a different extent, in the liver, as well as in brain cortex, midbrain, cerebellum, striatum, hippocampus and hypothalamus, whilst CYP2A11 and 4B1 were not expressed in the hypothalamus. CAR was expressed in liver and all the brain regions examined, whereas the PXR was expressed only in liver and cortex. Real time RT-PCR analysis demonstrated that in vivo treatment with phenobarbital, in contrast with what happened in liver, did not induce the expression of CYP 2B4/5 mRNA in cortex, midbrain and cerebellum. NADPH cytochrome c reductase and some other enzymatic activities markers of CYP 2A, 2B, 3A and 4B activities were studied in liver microsomes as well as in microsomes and mitochondria of brain cortex, midbrain and cerebellum of control and phenobarbital-treated rabbits. In contrast to what was observed in liver, phenobarbital treatment did not induce the aforementioned monooxygenase activities in brain. However, we cannot exclude that a longer phenobarbital treatment may lead to a significant induction of CYP activities in brain. These findings indicated that brain CYPs, despite the presence of CAR, were resistant to phenobarbital induction, indicating a possible different regulation of these enzymes between brain and liver.
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Toxicological consequences of differential regulation of cytochrome P450 isoforms in rat brain regions by phenobarbital
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Cytochrome P4502B is an isoform of cytochrome P450 (P450) that is induced by the anticonvulsant drug phenobarbital. Here, we demonstrate the constitutive expression and predominant localization of CYP2B in neurons of rat brain. Administration of phenobarbital to rats resulted in selective induction of P450 levels in cortex and midbrain, while other regions were unaffected. Immunohistochemical localization of P4502B in brains of phenobarbital treated rats revealed localization of P4502B in neuronal cells, most predominantly the reticular neurons in midbrain. The anticancer agent 9-methoxy-N²-methylellipticinium acetate (MMEA) has been shown to exhibit preferential neuronal toxicity in vitro. Pretreatment of rats with phenobarbital potentiated the toxicity of intrathecally administered MMEA in vivo, as seen by the degeneration of reticular neurons. Thus, induction of P450 in selective regions of brain by phenobarbital would profoundly influence xenobiotic metabolism in these regions, especially in clinical situations where phenobarbital is coadministered with other psychoactive drugs/xenobiotics.
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We found that CYP2J9 expression is particularly abundant in the cerebellum and highly localized to Purkinje cells, neurons that integrate information from within and outside the cerebellum, and communicate with motor nuclei. Tirumalai and co-workers (22) have shown that brain P450 content varies depending on the region, with the brainstem and cerebellum showing the highest levels. P450RAI-2 appears to be highly localized to the cerebellum, although its cellular localization within the cerebellum is unknown (65).
A cDNA encoding a new cytochrome P450 was isolated from a mouse brain library. Sequence analysis reveals that this 1,958-base pair cDNA encodes a 57–58-kDa 502-amino acid polypeptide that is 70–91% identical to CYP2J subfamily P450s and is designated CYP2J9. Recombinant CYP2J9 was co-expressed with NADPH-cytochrome P450 oxidoreductase (CYPOR) in Sf9cells using a baculovirus system. Microsomes of CYP2J9/CYPOR-transfected cells metabolize arachidonic acid to 19-hydroxyeicosatetraenoic acid (HETE) thus CYP2J9 is enzymologically distinct from other P450s. Northern analysis reveals that CYP2J9 transcripts are present at high levels in mouse brain. Mouse brain microsomes biosynthesize 19-HETE. RNA polymerase chain reaction analysis demonstrates that CYP2J9 mRNAs are widely distributed in brain and most abundant in the cerebellum. Immunoblotting using an antibody raised against human CYP2J2 that cross-reacts with CYP2J9 detects a 56-kDa protein band that is expressed in cerebellum and other brain segments and is regulated during postnatal development. In situ hybridization of mouse brain sections with a CYP2J9-specific riboprobe and immunohistochemical staining with the anti-human CYP2J2 IgG reveals abundant CYP2J9 mRNA and protein in cerebellar Purkinje cells. Importantly, 19-HETE inhibits the activity of recombinant P/Q-type Ca²⁺ channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release. Based on these data, we conclude that CYP2J9 is a developmentally regulated P450 that is abundant in brain, localized to cerebellar Purkinje cells, and active in the biosynthesis of 19-HETE, an eicosanoid that inhibits activity of P/Q-type Ca²⁺ channels. We postulate that CYP2J9 arachidonic acid products play important functional roles in the brain.AF336850

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NeuroscienceExpression of Multiple Forms of Cytochrome P450 and Associated Mono-oxygenase Activities in Rat Brain Regions

Abstract

Section snippets

Materials and Methods

Results

Discussion

Acknowledgements

Cancer Lett

Brain Res

Brain Res

Anal Biochem

Anal Biochem

J Biol Chem

Methods Enzymol

J Mol Biol

Anal Biochem

Methods Enzymol

Neurosci Lett

Neurosci Lett

Arch Biochem Biophys

Pharmacol Res

Multiplicity of mammalian microsomal cytochrome P-450

Pharmacol Res

Hepatic cytochrome P-450 linked drug metabolizing system

Neuroscience
Expression of Multiple Forms of Cytochrome P450 and Associated Mono-oxygenase Activities in Rat Brain Regions