Brain Cytochrome P450 Research Articles

Cytochrome P450 in normal human brain and brain tumours.

Cytochrome P450 in normal human brain and brain tumours.

43-2 - Cytochrome P-450 in animal and human brain

43-2 - Cytochrome P-450 in animal and human brain

Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7alpha-hydroxy dehydroepiandrosterone and 7alpha-hydroxy pregnenolone.

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (Km, 13.6 microM) and pregnenolone (Km, 4.0 microM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17beta-estradiol and 5alpha-androstane-3beta,17beta-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7alpha-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7alpha-hydroxy DHEA but not with 7beta-hydroxy DHEA; when [7alpha-3H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7alpha position. Brain extracts also efficiently liberated tritium from [7alpha-3H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7alpha-hydroxy DHEA. We conclude that Cyp7b is a 7alpha-hydroxylase participating in the synthesis, in brain, of neurosteroids 7alpha-hydroxy DHEA, and 7alpha-hydroxy pregnenolone.

Transient sex differences of aromatase (CYP19) mRNA expression in the developing rat brain.

Sex differences in the activity of aromatase cytochrome P450 (CYP19) in the rat brain have been reported during pre- and postnatal development. It is unclear, however, whether these differences are reflected by corresponding differences in specific mRNA levels. To address this question, we have examined aromatase mRNA levels in specific regions of male and female rat brains by means of in situ hybridization (ISH). At prenatal stages of development, i.e. at gestational day 18 (GD18) and GD20, aromatase mRNA was detected in several preoptic, hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal sex differences in any of these regions. In contrast, clear-cut sex differences were determined at postnatal day (PN) 2; male animals expressed significantly more aromatase mRNA in the bed nucleus of stria terminalis (BST) and the sexually dimorphic nucleus of the preoptic area (SDN). Smaller but still significant differences (females > males) were obtained in the medial preoptic area (MPO). At PN6, sex differences of aromatase mRNA signals (males > females) were still present in the BST, but were no longer detectable in the SDN and the MPO. At PN15 and in adult animals, aromatase mRNA levels were similar in BST and medical amygdaloid nucleus of male and female rats. Since aromatase mRNA expression decreases during postnatal development, no ISH signals could be detected anymore in MPO, SDN and ventromedial hypothalamic nucleus. Our results are consistent with the concept that differential regulation of aromatase mRNA expression might be important for the establishment of different neuronal circuitry in male and female animals.

Genomic Cloning and Protein Expression of a Novel Rat Brain Cytochrome P-450 CYP2D18* Catalyzing Imipramine N-Demethylation

We have previously reported the isolation of two cDNA clones, designated 2d-29 and 2d-35, which have identical open reading frames and code for a novel brain cytochrome P-450 (P-450) belonging to the CYP2D subfamily, and noted that the mRNA of clone 2d-35 seems to be expressed in the brain but not in the liver (1). Although the deduced amino acid sequence of these clones differs from that of the liver CYP2D4 by only 5 amino acids distributed in the C-terminal region, this new P-450 cDNA clone contained a unique 5'-extension, and we posit in this report by analysis of a genomic clone that this 5'-untranslated sequence is derived from a gene distinct from that of CYP2D4. Thus, this novel P-450 was named P-450 2D18 according to the recommended nomenclature (2). The expressibility of this cDNA was confirmed by in vitro translation using a reticulocyte system, and protein expression was performed using COS-M6 cells. Immunoblot analysis showed a cross-reacting band of the predicted size range with anti-P-450 2D6 antiserum, which was not seen in control cells. Furthermore, the CYP2D18-expressed COS cell lysate showed N-demethylation activity toward imipramine, whereas another brain P-450 CYP4F6-expressed COS cell lysate showed 10-hydroxylation activity. This is the first report that associates an individual P-450 isozyme in brain with a particular metabolic alteration of the antidepressant imipramine.

Brain cytochrome P450 in the rat.

Brain cytochrome P450 in the rat.

Expression of multiple forms of brain cytochrome P450

Multiple forms of cytochrome P450 (P450) in brain tissue have been demonstrated to be expressible in brain tissue using polymerase chain reaction (PCR) techniques, Northern blotting, hydroxylation activity assessment and cloning approaches. The antidepressant drug imipramine is metabolized by brain microsomes to multiple products by pathways inhibitable by quinidine, 7,8-benzoflavone, and ketoconazole, well-known inhibitors of P450-catalyzed reactions. Moreover, PCR studies revealed that a number of P450s are expressible in brain tissue and in glioma C6 cells. Quantitative PCR studies further demonstrated the response of many of these forms to induction in agreement with hydroxylation activity results.

The effect of protoporphyrinogen oxidase inhibitors on microsomal and mitochondrial cytochromes.

Neurological dysfunction is a characteristic feature of acute porphyrias, unexplained until now. One of the possible explanations is a deficiency of heme in the central nervous system, caused by a block in porphyrin biosynthesis. To test this possibility, the content of brain mitochondrial cytochrome a3 was determined after intracerebroventricular administration of the protoporphyrinogen oxidase-inhibiting herbicide fomesafen. It was established in in vitro experiments that fomesafen is a potent inhibitor of brain mitochondrial protoporphyrinogen oxidase (PROTOOX). Addition of 10(-6) M fomesafen to incubation mixture decreased PROTOOX from 1.02 nmol/mg/h (controls) to 0.42 nmol/mg/h. 10(-5)M of fomesafen decreased this activity to 0.27 nmol/mg/h. In in vivo experiments, 5 microleters of fomesafen solution containing 0.2 M fomesafen/l was administered to male rats by intracerebroventricular injection. The content of brain mitochondrial cytochrome a3 was determined 72 hours later. A slight decrease of the a3 content was observed (control rats 0.25 nmol/mg protein, treated rats 0.22 nmol/mg). Brain cytochrome P450 activities were below detection limits in both control and treated groups. In a separate experiment, male ICR mice were fed 1000 ppm of the protoporphyrinogen oxidaseinhibiting herbicide oxadiazon in the diet for 10 days. Liver microsomal cytochrome P450 content was decreased and liver porphyrins increased. An increase of porphyrin content was also observed in the testes of oxadiazon-fed mice, but testicular cytochrome a3 content was unchanged. The results indicate that, contrary to liver microsomal cytochromes P450, the mitochondrial cytochromes are not susceptible to changes in heme biosynthesis.

CDNA Cloning of Three New Forms of Rat Brain Cytochrome P450 Belonging to the CYP4F Subfamily

Three cDNA clones, designated 4f-8, 4f-34 and 4f-41, coding for three new forms of cytochrome P450 belonging to the CYP4F subfamily were isolated from an untreated rat brain cDNA library. cDNA 4f-8, 4f-34 and 4f-41 coded for proteins of 522, 526 and 537 amino acids, respectively, and their amino acid sequence similarity to CYP4F1 ranged from 71 to 80%. These new P450s were thus named CYP4F4, 4F5 and 4F6, respectively. Northern blot analysis revealed that the expression levels of these forms of P450 in the brain were somewhat low and that similar forms of subfamily 4F P450 were expressed in liver and kidney at a relatively high level compared with brain. No CYP4A expression was detected by Northern blot analysis in untreated rat brain mRNA. All three clones were in vitro-translatable using a reticulocyte lysate system. These results show that multiple forms of subfamily 4F P450 exist in the brain and that the subfamily 4F P450 may be one of the major forms of P450 in the brain.

Cytochrome P450 and associated monooxygenase activities in the rat and human spinal cord: induction, immunological characterization and immunocytochemical localization.

We have discovered cytochrome P450 and associated monooxygenase activities in microsomes prepared from spinal cord tissues from rats and a human. Cytochrome P450 levels and nicotinamide adenine dinucleotide phosphate cytochrome c reductase activities in microsomes from rat spinal cord were similar to those observed from the whole brain. However, certain monooxygenase activities were significantly lower in the rat spinal cord microsomes as compared to the corresponding activities observed in the whole brain. Cytochrome P450-mediated monooxygenase activities were also detectable in microsomes prepared from human spinal cord. Immunoblot analyses of rat and human spinal cord microsomes using antisera to various forms of hepatic cytochrome P450 namely (2B1 + 2B2), 1A1, 1A2 and 2E1 revealed the presence of immunologically similar forms. The spinal cord microsomes also cross-reacted with the antiserum to the phenobarbital-inducible form of rat brain cytochrome P450. Immunocytochemical stain was predominant in the gray horns of the rat spinal cord. At the cervical level, lamina 1 and 2 representing the substantia gelatinosa were intensely stained. In the ventral horns, lamina 7, 8 and 9 containing the large motor neurons were strongly labelled, while small neurons revealed variable staining. In the white matter, the glial cells were stained but the axons remained non-reactive.(ABSTRACT TRUNCATED AT 250 WORDS)

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

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.

Effect of ethanol on cytochrome P450 in the rat brain.

After a single dose of ethanol (0.8 ml/kg) administered intraperitoneally, the P450 content of the rat brain increased from 62 +/- 19 to 230 +/- 97 pmol/g (wet weight) of tissue (mean +/- SD, n = 5). Most of this increase could be accounted for by a 10- to 20-fold increase in the olfactory lobes and hypothalamic preoptic area. The P450s were identified by Western blot analysis and by microsequencing of the N-terminal ends after resolution of the proteins on SDS gels. They were identified as P450 2C7, 2C11, 2E1, 4A3, 4A8, and a member of the P450 2D family. In P450 extracted from the brains of control rats, P450 2C and 4A were also detectable but at a much lower concentration. P450 1A1, 2A1, 2B1, or 3A was not detected in the brains of either control or ethanol-treated rats. Oral administration of the same dose of ethanol resulted in a similar increase in the whole brain but smaller effects in the olfactory lobes. This effect of ethanol on the P450 in the brain has implications for the mechanism of toxicity and the development of tolerance to ethanol and for the effects of other drugs and environmental pollutants that act on the central nervous system.

3H] GBR‐12935 Binding to Cytochrome P450 in the Human Brain

The presence of multiple [3H]GBR-12935 binding sites in the human brain has been revealed in several recent studies. One site represents the dopamine uptake site. In rat brain it was demonstrated that [3H]GBR-12935 also binds to nondopaminergic "piperazine acceptor sites." One of these sites has been identified as cytochrome P450IID1 in canine brain. [3H]GBR-12935 binding to the piperazine acceptor sites in the human brain was investigated in the present study. A pharmacological definition of the piperazine acceptor sites is presented: the [3H]GBR-12935 binding fraction that could be discriminated by 10 microM GBR-12909 in the presence of 0.3 microM mazindol. This binding fraction was saturable, with binding affinity in the range of 3-8 nM. It was also demonstrated that the piperazine acceptor or cytochrome P450-sensitive drugs cis-flupentixol and proadifen (SKF 525 A) compete for the same binding sites, suggesting the cytochrome P450 nature of the binding. The findings presented support the proposal that at least part of this fraction represents cytochrome P450IID6, the human form of P450IID1. The distribution of [3H]GBR-12935 binding to the suggested P450IID6-site in 12 brain regions was examined, without significant differences in binding densities between the regions. The significance of the present findings on the cytochrome P450 system in brain is discussed.

The Chronic Administration of Nicotine Induces Cytochrome P450 in Rat Brain

The objective of these studies was to determine whether chronic administration of nicotine altered the cytochrome P450 (P450) monooxygenase system in rat brain. Male Sprague-Dawley rats received injections of nicotine bitartrate (1.76 mg/kg, s.c., twice daily for 10 days), and total cytochrome P450 content, the activity of NADPH-cytochrome c reductase, and the activities and relative abundance of P4502B1 and P4502B2 (P4502B1/2) were determined in microsomal fractions from rat brain. The content of P450 increased significantly (p < 0.02) in all brain regions examined from nicotine-injected rats; the largest increase (208% of control) was in frontal cortex and the smallest increase (122% of control) in cerebellum. The activity of NADPH-cytochrome c reductase was unaltered by nicotine administration. Benzyloxyresorufin O-dealkylase (BROD) and pentoxyresorufin O-dealkylase (PROD) activities, mediated by P4502B1/2, increased significantly (p < 0.02) following nicotine administration; the largest increase (213-227% of control) was in frontal cortex. Western blots of microsomal proteins indicated that the increase in enzymatic activity was associated with an increase in content of P4502B1/2 immunoreactive proteins. In contrast to brain, total P450 content, activities of NADPH-cytochrome c reductase, BROD, and PROD, and levels of P4502B1/2 immunoreactive proteins in liver were unaffected by chronic nicotine administration. Results indicate that chronic nicotine administration regulates the expression of P4502B1/2 in brain and that at the dose schedule used this effect occurs without a demonstrable effect on the hepatic P450 monooxygenase system.

Purification of Multiple Forms of Cytochrome P450 from a Human Brain and Reconstitution of Catalytic Activities

The present study demonstrates the presence of multiple forms of cytochrome P450 (P450) in human brain obtained at autopsy, the purification of various isoforms to apparent homogeneity, and the monooxygenase activities in reconstituted systems. Sequential chromatography on octylamino-Sepharose 4B, DEAE-Sephacel, and DEAE-cellulose yielded four isoforms of P450 (A, B, C, and D) with specific contents of 11.0, 9.4, 12.5, and 8.3 nmol of P450/mg protein, respectively. While the forms A, B, and C were apparently homogeneous as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the P450D was not homogeneous. The apparent molecular masses of the four forms of P450 were 60,200 Da (P450A), 60,900 Da (P450B), 60,200 Da (P450C), and 61,000 Da (P450D), respectively. NADPH cytochrome P450 reductase (reductase) was also partially purified from the brain microsomes. Immunoblot analysis of the four forms of human purified P450, using antisera to purified rat liver P450 (IIBL + 11B2), rat liver P450 (1A1 + 1A2), phenobarbital-inducible rat brain P450, human liver P450 IIE 1, P450 1A2, P450 IIC, and P450 IIIA4, indicated differential immunological cross-reactivity. The monooxygenase activities of the purified human brain P450s were demonstrated with various substrates (aminopyrine, morphine, aniline, 7-ethoxycoumaria, and nifedipine) as examined in reconstituted systems consisting of purified human brain P450, purified rat brain NADPH cytochrome P450 reductase, deoxycholate, phospholipid, and NADPH.

Induction of brain cytochrome P-450IIE1 by chronic ethanol treatmen

Cytochrome P-450 mediated metabolism is potentially involved in the expression of the pharmacological and/or toxicological effects of a wide variety of drugs and environmental chemicals upon tissues which contain this metabolic system. In the present investigation, the presence of cytochrome P-450IIE1 and associated mono-oxygenase activities in brain and the effect of chronic ethanol treatment on brain cytochrome P-450 (P-450) were studied. Aniline hydroxylase, N-nitroso-dimethylamine N-demethylase and p-nitrophenol hydroxylase activities (known to be mediated by P-450IIE1) were detectable in brain microsomes from untreated rats and were about 5%, 125% and 8.3%, respectively, of the corresponding hepatic levels. Chronic ethanol treatment resulted in induction of the above enzyme activities in brain microsomes by 243%, 496% and 155%, respectively. Intake of ethanol for a prolonged period also resulted in the induction of total P-450 in the brain (150% of the control). Addition of the antisera raised against rat liver cytochrome P-450IIE1 markedly inhibited brain microsomal p-nitrophenol hydroxylase activity. Immunoblot analysis of rat brain microsomes using the above antisera also revealed the induction of brain cytochrome P-450IIE1 following chronic ethanol administration. Immunocytochemical localization of cytochrome P-450IIE1 using the above antisera, revealed the preferential localization of the enzyme in the neuronal cell bodies in the cortex, hippocampus, basal ganglia, hypothalamic nuclei and reticular nuclei in the brainstem of rats treated chronically with ethanol. Based upon these studies, it is conceivable that chronic alcohol ingestion could enhance the sensitivity of certain regions of the brain to environmental chemicals that are metabolized to more toxic derivatives by the P-450 system.

Brain cytochrome P450 and testosterone metabolism by rat brain subcellular fractions: Presence of cytochrome P450 3A immunoreactive protein in rat brain mitochondria

The hydroxylation of testosterone by rat brain subcellular fractions has been studied using an HPLC method with an enhanced resolution for the separation of testosterone and its monohydroxy derivatives. Although the analysis time is longer than that reported for earlier methods, a baseline separation was obtained between all hydroxytestosterones, excepting 6α-hydroxytestosterone and 15β-hydroxytestosterone, which were separated using a second chromatography system. This separation was important as rat brain microsomes metabolized testosterone to 15α-, 6β-, 15β-, 16β-, 2β-, 1β-hydroxytestosterone and androstenedione. Testosterone metabolism was found to be linear with time and protein concentration. The rat brain mitochondrial fraction metabolized testosterone to androstenedione. Small amounts of immunoreactive bands comigrating with purified cytochromes P450j, P450b, and P450p were detected by Western blot analysis in rat brain microsomes, while only an immunoreactive protein related to cytochrome P450p was found in the mitochondrial fractions. Immunoinhibition studies showed that BEA33, a monoclonal antibody to cytochrome P450b and simultaneously recognizing cytochromes P450e and P450a, was able to inhibit the metabolism of testosterone to the 1β-, 15α-, 2β-, and 6α-hydroxylated metabolites, whereas polyclonal anticytochrome P450p did not inhibit the formation of the 6β-hydroxytestosterone by rat brain microsomes. The metabolism of testosterone by rat brain microsomal or mitochondrial fractions was refractory to induction by 3-methylcholanthrene or pregnenolone-16α-carbonitrile. Thus, in the brain multiple isozymes of cytochrome P450 are constitutively expressed in different subcellular fractions, which suggests that brain cytochrome P450 may play an important role in the metabolism of endogenous compounds. The significance and role of cytochrome P450p-related protein in the rat brain mitochondrial fraction are yet to be determined.

Reconstitution of the brain mixed function oxidase system: purification of NADPH-cytochrome P450 reductase and partial purification of cytochrome P450 from whole rat brain.

NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot techniques. Kinetic studies using cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P450c (P450IA1) as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. Our results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.

Immunocytochemical staining of the RLM6 form of cytochrome P-450 in oligodendrocytes and myelin of rat brain.

We used immunocytochemical staining to localize the RLM6 form of cytochrome P-450 in rat brain. Immunofluorescence staining in vibratome sections was positive in cells that resembled oligodendrocytes, which are the cells that synthesize and maintain myelin. Double immunofluorescence staining with anti-RLM6, plus mouse monoclonal antibodies (MAb) against 2',3'-cyclic nucleotide-3'-phosphohydrolase or galactocerebrosides, showed localization of each of these oligodendrocyte "markers" in the same cells as RLM6. In vibratome sections from brains of adult rats there was faint RLM6 immunostaining in some of the myelinated fibers as well as in oligodendrocytes. In paraffin sections from adult rat brains, myelinated tracts were RLM6 positive, as were oligodendrocytes and myelinated fibers in the gray matter. Oligodendrocytes were also shown to contain glucose-6-phosphate dehydrogenase. We suggest that RLM6, which is constitutive to liver, is also constitutive to brain and, via the acetone monooxygenase reaction, which also utilizes NADPH, may contribute to the conversion of ketone bodies to substrates that could provide energy for the synthesis and maintenance of myelin.

Administration of testosterone alleviates the constitutive sex difference in rat brain cytochrome P-450

Sex-related difference was observed in the levels of total cytochrome P-450 (P-450) and the mono-oxygenase activity mediated by P-450 (b,e), namely, aminopyrine N-demethylase and morphine N-demethylase activity in rat brain microsomes. Male rat brain had higher activity of the above enzymes as compared to the female rat brain. On the other hand, P-450 (c,d) mediated 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase activity showed no sex-related difference in rat brain. Administration of testosterone elevated the levels of total P-450, aminopyrine N-demethylase and morphine N-demethylase in female rat brain to levels comparable with that of the male rat brain. No significant change was observed in the levels of 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase and NADPH cytochrome c reductase. All of the above enzyme levels were unaffected in the male rat brain following the treatment with testosterone. These results indicate that testosterone may regulate the forms of cerebral P-450 that are associated with the sex-related difference observed in rat brain.