Forms Of Cytochrome P-450 Research Articles

Drug-mediated inactivation of cytochrome P450.

1. Multiple forms of cytochrome P450 (CYP) catalyse the oxidation of chemicals of endogenous and exogenous origin, including drugs, carcinogens, steroids and eicosanoids. However, this unusual low substrate specificity also makes CYP susceptible to inhibition by a wide range of drugs, leading to pharmacokinetic interactions of potential clinical significance. 2. Some drugs are converted by CYP to reactive metabolites that bind covalently to sites within the active centre of the same CYP. Such mechanism-based inhibition leads to CYP inactivation or complexation. These processes give rise to long-term effects on drug pharmacokinetics, as the inactivated or complexed CYP must be replaced by newly synthesized CYP protein. 3. Drugs that inactivate CYP generally possess recognizable functional groups that are oxidized to reactive products. Thus, drugs with side chains containing unsaturated carbon-carbon bonds and furan ring systems are associated with CYP inactivation. Nitrogen-containing systems may also inactivate CYP. 4. Metabolites formed from drugs containing alkylamino and methylenedioxy functionalities can trap CYP as inert complexes without eliciting inactivation. However, the functional effects of inactivation and complexation on drug pharmacokinetics are indistinguishable. Drugs that elicit CYP complexation include the first generation macrolide antibiotics, but newer analogues appear much safer. Some antidepressants, antiepileptics and tuberculostatic agents have been associated with CYP complexation.

Metabolic activation of toxins: tissue-specific expression and metabolism in target organs.

Cytochrome P450 (CYP) enzymes catalyze the generation of reactive species capable of binding with cellular macromolecules, leading to acute and delayed toxicity. Since individual CYP forms differ markedly in their substrate preferences and regulation, the expression profiles of CYP in various cell types are important determinants in tissue-specific toxicity. The highest concentrations of most forms of CYP are found in liver, but they are also present in many extrahepatic organs. Liver is also a target organ in which CYP-mediated activation and toxic outcome have been most convincingly linked. Prime examples are paracetamol-induced hepatotoxicity and aflatoxin B1-associated hepatic cancer. In contrast to liver, most extrahepatic tissues are composed of multiple call types, which make experimental approaches difficult. Also the low abundance of individual forms is a challenge in the study of extrahepatic CYP-related toxicity. Recent years have witnessed the emergence of molecular biological techniques, e.g., reverse transcriptase-polymerase chain reactions, which facilitate the study of low abundant CYP forms in human tissues. Nevertheless, in the end we need definite information on the expression of activity, and for this purpose enzyme-specific substrates, reactions, and inhibitors and other methods to detect proteins and associated activities are needed. In humans, it is important to measure activities of specific enzymes in vivo. For this purpose, two approaches are currently available. Metabolism and/or elimination of enzyme-specific drugs can be employed. In cases in which genetic background determines the presence or absence of a specific enzyme, phenotyping and genotyping tests can be devised, e.g., for CYP2D6 (debrisoquine hydroxylation) polymorphism.

Purification and characterization of two new cytochrome P-450 related to CYP2C subfamily from rabbit small intestine microsomes

Two forms of cytochrome P-450, designated P-450id and P-450ie, were purified to specific contents of 14.3 and 15.0 nmol of P-450/mg of protein, respectively, from small intestine mucosa microsomes of rabbits. P-450id and P-450ie showed apparent molecular weights of 50 and 49 kDa, respectively, on SDS-PAGE. Both P-450s catalyzed N-demethylation of nitrosodimethylamine. The NH 2-terminal amino acid sequence (first 19 residues) of P-450id exhibited 74–90% identity with those of six members of the rabbit P-450 2C subfamily, except for P-450 2C3. Similarly, the NH 2-terminal sequence (first 22 residues) of P-450ie showed 73–86% identity with those of the same members of the rabbit P-450 2C subfamily. The peptide mapping patterns of the two P-450s were quite different from each other. In addition, P-450id did not cross-react with the guinea-pig antibodies against P-450ie. The results indicate that rabbit small intestine mucosa contain two new distinct forms of P-450s, both of which may be classified into the 2C subfamily.

Putative oxidative metabolites of 1-methyl-6-hydroxy-1,2,3,4-tetrahydro-β-carboline of potential relevance to the addictive and neurodegenerative consequences of ethanol abuse

Ethanol is metabolized in the brain by catalase/H 2O 2 to yield acetaldehyde and by an ethanol-inducible form of cytochrome P450 (P450 IIE1) in a reaction that yields oxygen radicals. Within the cytoplasm of serotonergic axon terminals these metabolic pathways together provide conditions for the endogenous synthesis of 1-methyl-6-hydroxy-1,2,3,4-tetrahydro-β-carboline ( 1), by reaction of acetaldehyde with unbound 5-hydroxytryptamine (5-HT), and for the oxygen radical-mediated oxidation of this alkaloid. The major initial product of the hydroxyl radical (HO·)-mediated oxidation of 1 in the presence of free glutathione (GSH), a constituents of nerve terminals and axons, is 8- S-glutathionyl-1-methyl-1,2,3,4,-tetrahydro-β-carboline-5,6-dione ( 6). When administered into the brains of mice, 6 is a potent toxin (LD 50 = 2.9μg) and evokes episodes of hyperactivity and tremor. Compound 6 binds at the GABA B receptor and evokes elevated release and turnover of several neurotransmitters. Furthermore, the GABA B receptor antagonist phaclofen attenuates the behavioral response caused by intracerebral administration of 6. These observations suggest that 6 might be an inverse agonist at the GABA B receptor site. Accordingly, it is speculated that ethanol drinking might potentiate formation of 6 that contributes to elevated release of several neurotransmitters including dopamine (DA) and endogenous opioids in regions of the brain innervated by serotonergic axon terminals. Subsequent interactions of DA and opioids with their receptors might be related to the initial development of dependence on ethanol. Redox cycling of 6 (and of several putative secondary metabolites) in the presence of intraneuronal antioxidants and molecular oxygen to produce elevated fluxes of cytotoxic reduced oxygen species might contribute to the degeneration of serotonergic pathways. Low levels of 5-HT in certain brain regions of the rat predisposes these animals to drink or augments drinking. Accordingly, 6, formed as a result of ethanol metabolism in the cytoplasm of certain serotonergic axon terminals, might contribute to the initial development of dependence on ethanol, by mediating DA and opioid release, and long-term preference and addiction to the fluid as a result of the progressive degeneration of these neurons.

Purification and characterization of three constitutive cytochrome P-450 isoforms from bovine olfactory epithelium.

Three constitutive forms of cytochrome P-450 (P-450s) were isolated from olfactory microsomes of cattle. The purified P-450s, designated P-450bov1, P-450bov2 and P-450bov3, were electrophoretically nearly homogeneous by SDS/PAGE and their apparent relative molecular masses were estimated to be 50000, 53000 and 51000 respectively. As indicated by several criteria including the N-terminal sequence and absorption spectra, the three olfactory forms of P-450 were distinct from each other and from all the other P-450s currently known in cattle. P-450bov1 and P-450bov2 were purified in the low-spin state, whereas P-450bov3 was in the high-spin state. Studies to evaluate, by Western blot analysis, the reactivity of these purified P-450s with antibodies raised against rat hepatic P-450 2E1, 2B, 1A and 3A and rabbit olfactory P-450NMa and P-450NMb showed that P-450bov3 strongly cross-reacted with anti-P-450NMb IgG, and P-450bov1 moderately with anti-P-450NMa IgG. As determined by immunoblots, P-450bov1 and P-450bov3 represented a great portion of the total olfactory P-450. In a reconstituted system with NADPH:cytochrome P-450 reductase and phospholipids, P-450bov1 was more active in the metabolism of xenobiotic compounds (i.e. O-de-ethylation of ethoxycoumarin and N-demethylation of hexamethylphosphoramide) than towards endogenous substrates (testosterone and progesterone). Conversely, P-450bov3 metabolized the xenobiotics at lower rates but exhibited total oxidation rates of the above sex hormones higher than those of P-450bov1. From the comparison of the catalytic, immunochemical and structural properties, it was inferred that P-450bov1 and P-450bov3 are the bovine orthologues of P-450NMa (2A) and P-450NMb (2G1) respectively, the only two olfactory P-450s previously purified from rabbit. P-450bov2, which showed low activity toward some exogenous and endogenous compounds, represents a novel purified olfactory hemoprotein possibly belonging to the 3A subfamily. These results are consistent with a specific presence of catalytically and structurally similar P-450s, at least for the major ones, in the olfactory mucosa of mammals.

Effect of pyridine on the expression of cytochrome P450 isozymes in primary rat hepatocyte culture.

In vivo administration of pyridine has been shown to increase the activity and content of several forms of cytochrome P450 by transcriptional and posttranscriptional mechanisms. The effect of pyridine on CYP1A and CYP2E1 isozymes was studied in a rat hepatocyte culture model. Hepatocytes were isolated from non-induced rats and seeded onto matrigel-coated dishes and incubated in William's medium E containing 10% fetal calf serum, hormones, and essential metals. Cultures were treated with 0, 10 or 25 mM pyridine for 1-3 days and microsomes were isolated to determine catalytic activity and for immunoblot analysis, and total RNA was isolated for mRNA determinations. CYP2E1 content, CYP2E 1 mRNA, and CYP2E1 catalyzed oxidation of p-nitrophenol declined during culture to values of 3, 30 and 19% that of initial, non-cultured controls by day 3 of culture. Pyridine prevented this decline of CYP2E1 protein and activity such that 60-80% original activity remained after 3 days of culture in the presence of 25 mM pyridine. However, pyridine did not prevent the fall in CYP2E1 mRNA levels, nor did pyridine increase the content or activity of CYP2E1 above initial values of microsomes from freshly isolated hepatocytes. Pyridine increased the content of CYP1A2 and the oxidation of ethoxyresorufin 2-4 fold compared to cultures incubated without pyridine over the 3 day culture period. CYP1A1 levels, which rapidly declined, were induced and maintained in the presence of pyridine. Pyridine increased CYP1A content and activity 2-3 fold over initial values of freshly isolated hepatocytes. These increases were associated with corresponding increases in CYP1A mRNA levels. CYP1A2, but not CYP1A1, mRNA levels increased in the cultures incubated in the absence of pyridine. These results indicate that pyridine has different effects on CYP1A and CYP2E1 in this hepatocyte culture model. Pyridine appears to modulate CYP2E1 levels by posttranscriptional mechanisms as CYP2E1 activity and content were maintained in the presence of pyridine under conditions in which CYP2E1 mRNA levels declined. These mechanisms may involve increased translational efficiency of existing CYP2E1 mRNA or stabilization of CYP2E1 protein against degradation. Pyridine increased CYP1A1 and CYP1A2 content, activity and mRNA levels, either inducing CYP1A transcription or stabilizing CYP1A mRNA. Hepatocyte cultures may be a useful model to study the interaction of pyridine with P450 isozymes and their associated drug-mediated toxicity.

PHARMACOKINETIC DRUG INTERACTION WITH GRAPEFRUIT JUICE

Simultaneous intake of grapefruit juice, but not other fruit beverages, is now known to alter pharmacokinetics and efficacies of various drugs.. Although drugs described above do not relate structurally with each other, a form of cytochrome P450 (CYP) 3A4, is shown to be responsible for the metabolisms of these drugs. The exact mechanism of this interaction remains yet unclear, but the suppression of drug metabolisms has been suggested as a major mechanism. We have isolated four components from grapefruit juice that inhibit human CYP3Amediated drug oxidation. The structures of these components were identified as furocoumarin derivatives by absorption spectra, APCI-LC/MS/MS and NMR after their purification by a reversed-phase HPLC. These furocoumarins are strong candidates for causative agents of grapefruit juice-mediated drug interaction, because of their inhibition equal or stronger than did a typical CYP3A4 inhibitor, ketoconazole, on microsomal testosterone 6β-hydroxylation in human livers.

OMEPRAZOLE HYDROXYLATION BY CYP2C19 AND CYP3A4: PREDICTION TOWARDS HUMAN LIVER ACTIVITIES USING THE DATA OF RECOMBINANT P450 ENZYMES

Omeprazole 5-hydroxylation and sulfoxidation activities were determined in liver microsomes of different humans whose levels of individual forms of cytochrome P450 (P450 or CYP) varied. Omeprazole 5-hydroxylation by liver microsomes of a human sample which contained relatively high levels of CYP3A4 and low levels of CYP2C19 were inhibited very significantly by ketoconazole and anti-CYP3A4 antibodies, while a human sample having high in CYP2C19 and low in CYP3A4 was found to be sensitive towards fluvoxamine and anti-CYP2C9 antibodies. Both recombinant human CYP2C19 and CYP3A4 enzymes had activities for omeprazole 5-hydroxylation, with low Km and high Vmax values for the former enzyme and high Km and low Vmax values for the CYP3A4. Omeprazole 5-hydroxylation activities of different human samples were found to be related to predicted values calculated from the kinetic parameters of recombinant enzymes and the levels of liver microsomal P450 enzymes. When recombinant human CYP2C19 and CYP3A4 were mixed at levels found in different human samples, relatively similar profiles of omeprazole oxidation by the recombinant and microsomal enzyme systems were determined. These results suggest that both CYP2C19 and CYP3A4 are involved in the 5-oxidation of omeprazole (at a substrate concentration of 10 μM) in human liver microsomes and that contributions of these P450 enzymes depend on the compositions of CYP2C19 and CYP3A4 in liver. Different contributions of CYP2C19 and CYP3A4 In the oxidation of testosterone and progesterone by human liver microsomes are also reported.

Alternatively spliced transcripts of the aromatase cytochrome P450 (CYP19) gene in adipose tissue of women.

Estrogen biosynthesis in adipose tissue has assumed great significance in terms of a number of estrogen-related diseases. The biosynthesis of estrogens from C19 steroids is catalyzed by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). The human CYP19 gene comprises nine coding exons, II-X, and its transcripts are expressed in the ovary, placenta, testes, adipose tissue, and brain. Tissue-specific expression of the CYP19 gene is determined at least in part by the use of tissue-specific promoters, which give rise to transcripts with unique 5'-noncoding termini. Thus, the distal promoter I.1 is responsible for expression uniquely in placenta. On the other hand, the proximal promoter II, which regulates expression via a cAMP-dependent signaling pathway, is responsible for expression in the gonads. Transcripts in breast adipose tissue contain 5'-termini corresponding to expression derived from promoters I.4, II, and I.3, with I.4-specific termini predominating. The latter are derived from promoter I.4, which contains a glucocorticoid response element and an interferon-gamma activation site element and is responsible for expression in the presence of glucocorticoids and members of the class I cytokine family. The object of the present study was to determine the distribution of these various transcripts in adipose tissue from abdomen, buttocks, and thighs of women, as this would provide important clues to the factors regulating aromatase expression in these sites. To achieve this, we employed competitive reverse transcription-PCR to amplify unique 5'-ends of each of the transcripts of the CYP19 gene that are expressed in adipose tissue as well as for the coding region to evaluate total CYP19 gene (P450arom) transcript levels. We observed that exon I.4-specific transcripts were predominantly present in adipose tissue samples obtained from women regardless of the tissue site or the age of the individual. In these tissues, promoter II- and exon I.3-specific transcripts were present in lower copy numbers. We also demonstrated that in these sites total or exon-specific P450arom transcripts levels increased in direct proportion to advancing age and that transcript levels were the highest in buttocks, followed by thighs, and lowest in the abdomen. These results suggest that in normal human adipose tissue, aromatase expression is mainly under local control by a number of cytokines via paracrine and autocrine mechanisms in the presence of systemic glucocorticoids.

Effect of guaiazulene on some cytochrome P450 activities. Implication in the metabolic activation and hepatotoxicity of paracetamol.

The in vitro and in vivo effect of guaiazulene, a natural azulene derivative, on rat hepatic cytochrome P450 (CYP) is investigated. Furthermore, paracetamol hepatotoxicity is induced in rats and the activity of specific cytochrome P450 forms, involved in the metabolic activation of paracetamol to the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI) is examined, after the administration of guaiazulene, using diagnostic cytochrome P450 substrates. It is found that guaiazulene inhibited considerably CYP1A2 and CYP2B1 and had a weak effect on CYP1A1 in rat hepatic microsomal fractions. Guaiazulene administered to rats did not produce any macroscopic toxic effect and caused no change of liver weight, microsomal protein and total cytochrome P450 content. Guaiazulene inhibited CYP1A2 activity in rats with or without paracetamol intoxication. Considering that CYP1A2 participates in the formation of NAPQI, as well as in the metabolic activation of several toxic and carcinogenic compounds, these results, in combination with the antioxidant activity of guaiazulene that we have found in previous investigations, indicate potential useful applications of guaiazulene.

Use of cDNA-Expressed Human Cytochrome P450 Enzymes to Study Potential Drug-Drug Interactions

Complementary DNA (cDNA)-expressed human cytochrome P450 enzymes provide a reproducible, consistent source of single enzymes for many types of studies. The use of single enzymes systems, relative to multienzyme systems, has distinct advantages and disadvantages depending on the specific application. cDNA-expressed materials have advantages in the analysis of cytochrome P450 form-selective metabolism of a drug or drug candidate. This analysis can be accomplished by direct incubation of the drug with microsomes prepared from cells expressing a single cytochrome P450 form coupled with analysis of either metabolite formation or loss of parent compound. This approach allows the unambiguous assignment of specific biotransformations to specific enzymes. However, extending these data to the balance of enzymes present in human liver microsomes can be problematic. New approaches for relating rates of metabolism for cDNA-expressed enzymes to human liver microsomes metabolism are being developed (Crespi, 1995). In addition, cDNA-expressed enzymes can be used to study the cytochrome P450 form-selective inhibition by drugs or drug candidates. This analysis is accomplished through the study of the inhibition of the metabolism of a model substrate by the drug or drug candidate. Through these analyses, apparent Ki values can be obtained and compared to Ki values for known, clinically significant inhibitors of the same enzyme. For this application, cDNA-expressed, single enzyme systems have distinct advantages because of greater flexibility in the choice of model substrates and the lack of competing pathways of metabolism. Specific data for the use of cDNA-expressed CYP2C9, CYP2D6, and CYP3A4 are presented.

Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients.

Estrogen biosynthesis in adipose tissue has assumed great significance in terms of a number of estrogen-related diseases. Recent evidence suggests that estrogen synthesized locally in the breast is of singular significance in the development of breast cancer in elderly women. The biosynthesis of estrogen from C19 steroids is catalyzed by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). The human CYP19 gene comprises nine coding exons, II-X, and its transcripts are expressed in the ovary, placenta, testes, adipose tissue, and brain. Tissue-specific expression of the CYP19 gene is determined, at least in part, by the use of tissue-specific promoters, which give rise to transcripts with unique 5'-noncoding termini. Transcripts in adipose tissue contain 5'-termini derived from specific untranslated exons, corresponding to expression derived from the proximal promoter II and its splice variant I.3, as well as a distal promoter, I.4. The object of the present study was to determine the distribution of these various exon-specific transcripts in breast adipose tissues from cancer-free women undergoing reduction mammoplasty and from patients with breast cancer, because this would provide important clues as to the nature of the factors regulating aromatase expression in these sites. To achieve this, we employed competitive RT-PCR, utilizing an internal standard for each exon-specific transcript of the CYP19 gene, as well as for the coding region, to evaluate total CYP19 gene transcripts. In cancer patients (n = 18), total CYP19 gene transcript levels were significantly higher in adipose tissue proximal to a tumor in comparison with adipose tissue distal to a tumor, in agreement with previous findings. Moreover, total transcript levels were higher in breast adipose tissue of cancer patients in comparison with those of cancer-free individuals (n = 9), even when the adipose tissue from the cancer patient was taken from a quadrant with no detectable tumor. We observed that exon I.4-specific transcripts were predominant in breast adipose obtained from cancer-free women. In this tissue, promoter-II-specific and exon I.3-specific transcripts were present in low copy number. On the other hand, in breast cancer patients, CYP19 gene transcripts from breast adipose tissue had primarily promoter-II-specific and exon I.3-specific sequence, whereas comparatively few transcripts had exon I.4-specific sequence at the 5'-terminus. We conclude that CYP19 gene transcription in breast adipose tissue of cancer-free individuals uses preferably promoter I.4, implicating a role of glucocorticoids and members of the IL-6 cytokine family in the regulation of this expression. On the other hand, the increased expression in breast adipose tissue bearing a carcinoma results from expression from promoters II and I.3, which are regulated by unknown factors acting via increased cAMP formation, which are presumably secreted by the tumor or associated cells.

Effect of cytochrome P450 inducers on expression of oncogenes and a tumor suppressor gene in human colon cancer cells

To examine the possibility that chemical inducers of cytochrome P450 may have effects on the expression of oncogenes and a tumor suppressor gene, human colon LS174T cells were treated with different cytochrome P450 inducers such as benzanthracene (Ba), pyrazole (Pyr) and phenobarbital-hydrocortisone (Pb-Hc). Three forms of cytochrome P450 (CYP1A1, CYP1A2 and CYP2E1) were detected in the colon cell line and all of them were induced following chemical treatments. Altered expression of c-fos, c-myc, c-jun, was observed following the treatments. c-src, K-ras and p53 were not significantly affected by any of the treatments at the time-point selected. Interestingly, c-fos expression was the most dramatically altered by different treatments, showing repression with all of the treatments. These data show that some oncogenes are responsive to inducers of cytochrome P450, the enzyme system by which many carcinogens are activated.

Enzyme-Specific Transport of Rat Liver Cytochrome P450 to the Golgi Apparatus

It has been reported that cytochrome P450 is expressed in the plasma membrane of hepatocytes isolated from human and rat. Cytochrome P450s expressed on the cell surface are potential targets for the immune response of drug-induced and autoimmune hepatitis. However, the mechanisms behind transport of cytochrome P450 to the plasma membrane are obscure. The present investigation aimed at identifying cytochrome P450 expressed in the Golgi apparatus. Golgi membrane fractions from rat liver were prepared and characterized: one enriched withcis-Golgi, one highly enriched withtrans-Golgi, and one intermediate Golgi fraction representingmedial-Golgi. In these three fractions, significant amounts of cytochrome P450 and NADPH cytochrome P450 reductase were present, which could not be accounted for by contamination with endoplasmic reticulum. A marked difference between the relative content of different cytochrome P450 enzymes was found. CYP4A1 was found at the highest concentration, CYP2E1 at an intermediary level, and CYP1A2 at low levels, whereas no Golgi-specific CYP3A1 was detectable. It was also shown that the CYP2E1 present in the Golgi fractions was catalytically active. It is suggested that various forms of hepatic cytochrome P450 are transported to the plasma membrane through the Golgi apparatus in an enzyme-specific manner.

Immunochemical relationships of cytochrome P4503A-like proteins in teleost fish

Multiple P450 proteins have been purified from several teleost species, including rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chrysops) and Atlantic cod (Gadus morhua). Identity, relationships and/or functions have been established in these fish species for the cytochrome P4501 As. Information about the structure, function, regulation and relationships of other piscine cytochrome P450 (CYP) proteins is sparse. In the present study we have focused on constitutively expressed CYP forms, P450con and LMC5 isolated from rainbow trout, P450A from scup, and P450b from Atlantic cod, and we consider evidence for the relationship of these proteins to mammalian members of the CYP3A subfamily. Reciprocal western blot analysis shows that P450con and LMC5, isolated from rainbow trout in two different laboratories, are closely related and ostensibly identical proteins. These trout proteins show specific reciprocal cross-reactivity with scup P450A, and polyclonal antibodies (PAb) to the trout and scup proteins both recognize cod P450b, indicating that rainbow trout P450con/LMC5, scup P450A and cod P450b are immunochemically-related proteins. In analyses of liver microsomes of trout, scup and cod, PAb to trout P450con/LMC5 and scup P450A recognize only bands that are identical in migration to the CYP proteins purified from these species, and which were used as immunogens. These CYP proteins purified from fish are each immunochemically-related to mammalian CYP3A proteins, showing recognition by PAb to human CYP3A4 and to rat CYP3A1. PAb to the mammalian CYP3As also recognize the same bands in liver microsomes from these fish species as seen by PAb to the fish proteins. These results strongly suggest that these fish proteins are members of theCYP3 gene family and probably theCYP3A subfamily. Although sequence analysis is required before their designation in the CYP3A subfamily can be confirmed and specified, we refer to these as CYP3A-like. Immunoblot analyses of hepatic microsomes from other fish species with PAb to scup P450A and trout P450con show that multiple CYP3A-like proteins are expressed in liver of several species, including killifish (Fundulus heteroclitus) and winter flounder (Pleuronectes americanus). Important questions still remain to be addressed concerning CYP3A structure, multiplicity, physiological function, regulation and metabolism of endogenous as well as exogenous substrates in fish.

Something completely different?

Three cDNAs 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 CYP4F 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.

Peroxidase Activity of Liver Microsomal Vitamin D 25-Hydroxylase and Cytochrome P450 1A2 Catalyzes 25-Hydroxylation of Vitamin D3and Oxidation of Dopamine to Aminochrome

Purified liver microsomal vitamin D 25-hydroxylase, a cytochrome P450, catalyzes 25-hydroxylation of vitamin D3in the absence of NADPH and NADPH–cytochrome P450 reductase by usingt-butyl hydroperoxide as electron donors. The rate of 25-hydroxylation was approximately the same when NADPH/NADPH–cytochrome P450 reductase ort-butyl hydroperoxide was used as electron donor. TheKmvalue for vitamin D3in the presence oft-butyl hydroperoxide was found to be 30 μM. The rates of 25-hydroxylation of 1α-hydroxyvitamin D3and 5β-cholestane-3α, 7α-diol catalyzed by 25-hydroxylase were significantly higher when the reaction proceeded in the presence of NADPH/NADPH–cytochrome P450 reductase than in the presence oft-butyl hydroperoxide. Other liver microsomal cytochrome P450 forms such as taurochenodeoxycholic acid 6α-hydroxylase and cytochromes P450 1A2 and 2B4 did not catalyze 25-hydroxylation of vitamin D3in the presence of NADPH/NADPH–cytochrome P450 reductase ort-butyl hydroperoxide. The peroxidase activity of the 25-hydroxylase also catalyzed oxidation of dopamine to aminochrome. A linear correlation between increase in aminochrome formation and increase in the amount of 25-hydroxylase was observed in the oxidation of dopamine. TheKmvalues for dopamine andt-butyl hydroperoxide were 8.6 μMand 1 mMwhen 25-hydroxylase catalyzes the formation of aminochrome in the presence oft-butyl hydroperoxide. Oxidation of dopamine to aminochrome catalyzed by the peroxidase activity of cytochrome P-450 1A2 was observed in the presence oft-butyl hydroperoxide. A linear correlation between formation of aminochrome and the amount of cytochrome P450 1A2 was found.

Distinct Effects of Phenobarbital and Its N-Methylated Derivative on Liver Cytochrome P450 Induction

The relationship between barbiturate structures and their effects on induction of rat cytochrome P450 forms was studied in primary cultured hepatocytes. Treatment of hepatocytes cultured on matrigel with 1 mMbarbital,N-methylbarbital, cyclobarbital, hexobarbital, phenobarbital (PB), or mephobarbital (N-methyl-PB) resulted in increased amounts of CYP2B1/2 and CYP2C6 forms. Microsomal CYP3A content was also enhanced by treatment with these barbiturates, except for barbital. Although no relationship was observed between the levels of CYP2B1/2 and CYP3A, ratios of CYP3A/CYP2B1 plus CYP2B2 contents were invariably higher with hepatocytes treated with N-methylated barbiturates than with the nonmethylated analogs. Consistent results were also observedin vivoin rats treated with PB andN-methyl-PB. These results indicate the difference in the structure requirement for induction of CYP2B and CYP3A. In addition,N-methyl-PB was found to suppress PB-mediated induction of CYP2B1. Hepatic levels of CYP2B1 mRNA and protein were increased by treatment with PB orN-methyl-PB alone, but decreased by cotreatment with 1 mMPB andN-methyl-PB. The suppression has been shown to occur at the transcriptional level of the CYP2B1 gene by using a chloramphenicol acetyltransferase reporter–CYP2B1 fused gene system.

Regional distribution of cytochrome P450 2D1 in the rat central nervous system.

Cytochrome P450s are enzymes involved in the oxidative metabolism of numerous endogenous and exogenous molecules. The enzyme cytochrome debrisoquine/sparteine-type monoxygenase is a specific form of cytochrome P450 and is found in the liver and the brain (in the rat the enzyme is known as CYP2D1). CYP2D1 has no established role in the brain; however, it has been shown to share substrate and inhibitor specificities with the dopamine transporter and the enzyme monoamine oxygenase B. Using CYP2D-specific deoxyoligonucleotide probes and a polyclonal antibody to CYP2D1, we have mapped the distribution of CYP2D mRNA and CYP2D1-like immunoreactivity in the rat central nervous system. CYP2D1 immunoreactivity and the CYP2D1 mRNA signal were heterogenously distributed between brain areas. There were moderate to high levels of immunoreactivity and mRNA signal in the olfactory bulb, olfactory tubercle, cerebral cortex, hippocampus, dentate gyrus, piriform cortex, caudate putamen, supraoptic nucleus, medial habenula, hypothalamus, thalamus, medial mammilliary nucleus and superior colliculus. In the brainstem, strong CYP2D1 immunoreactivity and CYP2D mRNA signal were observed in the substantia nigra compacta, red nucleus, interpeduncular nucleus, pontine grey, locus coeruleus, cerebellum, and the ventral horn of the spinal cord. This study indicates that CYP2D1 is widely and constitutively expressed in neuronal and some glial populations in the rat brain. The localization of CYP2D1 in several regions known to harbor catecholamines and serotonin may suggest a role for CYP2D1 in the metabolism of monoamines.

Reduction in Hepatic Microsomal P-450 and Related Catalytic Activity in Farm-Raised Red Drum

Hepatic microsomal factors were investigated in red drum Sciaenops ocellatus, an important aquaculture and sportfish species. Constitutive and induced levels of microsomal cytochrome P-450 and P-420, catalytic activities of 7-ethoxyresorufin O-deethylase (EROD) and NADPH–cytochrome-c reductase, and the extent of membrane lipid peroxidation were compared in juvenile farm-raised and wild fish. We detected smaller amounts of active cytochrome P.450 and more cytochrome P-420, the denatured form of cytochrome P-450, in farm-raised than in wild red drum. Lipid peroxide formation was significantly higher and EROD catalytic activity was decreased in farm-raised red drum, whereas wild fish exhibited significantly higher levels of cytochrome P-450 and EROD activity. No significant alterations in NADPH–cytochrome-c reductase activity were observed in farm-raised fish. The livers from farm-raised red drum had higher levels of cellular lipid, which correlated with higher levels of lipid peroxides and were apparently associated with the loss of active cytochrome P-450. These data indicate that farm-raised fish fed a commercial, potentially lipogenic, diet may be less capable of metabolizing endobiotics, drugs, and hydrocarbon pollutants, and suggest that substantially increased hepatic lipid deposition may have potentially dangerous toxicological implications for these animals.