P450 Subfamily Research Articles

Stereoselective metabolism of bisoprolol enantiomers in dogs and humans

To clarify the mechanism of the species difference in the metabolism of bisoprolol enantiomers, in vitro metabolic studies were performed using dog liver microsomes and human cytochrome P450 (CYP) isoforms. The O-deisopropylation of bisoprolol enantiomers showed biphasic kinetics in dog liver microsomes. The intrinsic clearance (Vmax/Km) for O-deisopropylation of R(+)-bisoprolol was higher than S(−)-isomer in both high-affinity and low-affinity components. The R S ratio of the intrinsic clearance in high- and low-affinity components was 1.34 and 1.65, respectively. The inhibition studies in dog liver microsomes using CYP isoform-selective inhibitors indicated that the O-deisopropylation of both bisoprolol enantiomers was mediated via the CYP2D and CYP3A subfamily, and suggested that high-affinity oxidation was dependent on CYP2D. The kinds of CYP subfamilies in dogs, which contribute to the metabolism of bisoprolol enantiomers, were the same as those in humans. The intrinsic clearance for O-deisopropylation of R(+)-bisoprolol by human recombinant CYP2D6 was also different from that of S(−)-enantiomers ( R S : 1.50). However, unlike the dog microsomes, the intrinsic clearance by the human recombinant CYP3A4 did not show a stereoselective difference. Therefore, the species difference in the R S ratio of metabolic clearance for the oxidation of bisoprolol enantiomers (dog > human) is mainly due to the species difference in the stereoselectivity of one of the cytochrome P450 subfamilies (CYP3A).

Characterization of members of the novel cytochrome P450 subfamilies CYP2N and CYP2P from the fish Fundulus heteroclitus

To understand the current situation of job burnout among coal miners in Xinjian. The effects of occupational stress, GCCR and SLC6A4 gene polymorphisms, and gene-environment interactions on job burnout in coal miners were analyzed. This study provides a scientific basis for formulating health strategies to combat job burnout in the future.The job burnout scale and job content questionnaire (JCQ) were used to investigate the general situation of job burnout among coal miners and its influencing factors. The imLDRTM multiple SNP typing kit was used to type 300 samples (150 samples from the burnout case group and 150 from the control group). The relationship between the occurrence of job burnout, and the GCCR and SLC6A4 genes was analyzed.There were significant differences in the rate of burnout among miners of different sexes, ages, working years, shifts, working types, and marital status (P < 0.05). The difference in occupational stress between the different job burnout groups was statistically significant (P < 0.05). The GG genotype at rs41423247 increased the risk of burnout (OR=3.224, 95% CI:1.425-7.294). Similarly, compared to the TT genotype at rs11080122, the CC genotype increased the susceptibility of job burnout (OR =2.614, 95% CI:1.047-6.527). The results of gene-environment interaction regression analyses showed that the interaction between rs41423247, rs17209237, and occupational stress increased the risk of job burnout (OR = 5.049, 95% CI = 2.371-10.750).In addition to demographic characteristics, occupational stress was also a risk factor for job burnout. The interaction between rs41423247 and rs17209237 of the GCCR gene and occupational stress increased the risk of job burnout.

Covalent binding of carbamazepine reactive metabolites to P450 isoforms present in the skin

Carbamazepine is an anticonvulsant associated with a high risk for severe cutaneous reactions. Upon metabolism by cytochrome P450, carbamazepine may produce reactive metabolites. We evaluated in vitro the covalent binding of carbamazepine reactive metabolites on human P450s and then the presence of these P450s in human epidermis. Carbamazepine reactive metabolites covalent binding to human liver microsomes involved P450 subfamilies 1A, 2C and 3A. Specific covalent binding to yeasts expressing different P450s showed that carbamazepine reactive metabolites bound specifically to P450 1A2 and 3A4. We confirmed the constitutive presence of P450 3A in human epidermis and after induction with coaltar of P450 1A. Consequently, the production in epidermis of carbamazepine reactive metabolites is theoretically possible with formation of P450 adduct metabolites.

CYP2M1: Cloning, Sequencing, and Expression of a New Cytochrome P450 from Rainbow Trout Liver with Fatty Acid (ω-6)-Hydroxylation Activity

A cDNA clone was isolated from a female rainbow trout liver λgτ11 library using polyclonal antibodies raised against rainbow trout cytochrome P450 LMC1. This 2149-nucleotide clone contained an open reading frame encoding a protein of 499 amino acids with a calculatedMrof 56,850 Da. On the basis of cytochrome P450 (P450) amino acid sequence comparisons, this rainbow trout P450 was assigned by the P450 Nomenclature Committee to a new P450 subfamily designated as CYP2M1. Northern blot results suggest that the expression of CYP2M1 at the transcriptional level was generally sex, tissue, and age specific. By use of a full-length CYP2M1 cDNA probe, it was observed that this cDNA hybridized strongly to a single 2.2-kb transcript in juvenile female rainbow trout trunk kidney and in liver from juvenile and sexually mature trout from both sexes. Negligible amounts of mRNA hybridizable to CYP2M1 cDNA were found in the juvenile and sexually mature male trunk kidney. cDNA-directed expression in COS-7 cells and of recombinant baculovirus in insect cells produced a protein that was reactive with rabbit anti-trout P450 LMC1 polyclonal antibody and exhibited the unique (ω-6)-hydroxylation toward lauric acid previously observed with rainbow trout P450 LMC1.

Mapping of porcine genes belonging to two different cytochrome P450 subfamilies.

Three cDNA clones from a porcine small intestine cDNA library, tentatively identified as transcripts of two different cytochrome P450 (CYP) gene subfamilies, were sequenced and mapped. One of the clones was found to comprise an open reading frame of 503 amino acids and showed up to an 80% similarity to genes from the CYP3A subfamily. Translation of the other two cDNA clones revealed high similarity to genes from the CYP2C subfamily. One of these clones was truncated, lacking a part of the 5' end of the coding region, whereas the other encodes a putative pseudogene. The CYP3A gene was mapped cytogenetically by means of a pig/rodent somatic hybrid cell panel to pig chromosome 3, in the region p16-p17 or p11. The CYP2C pseudogene was mapped genetically to pig chromosome 14, using the PiGMaP shared reference pedigrees. The localisations provide information for the human/pig comparative map.

Identification of a functional glucocorticoid response element in the CYP3A1/IGC2 gene.

The rat CYP3A subfamily of cytochrome P450 consists of steroid- and drug-metabolizing enzymes inducible by pregnenolone 16alpha-carbonitrile and by supra-physiological doses of dexamethasone. The induction of CYP3A by dexamethasone has been proposed to be mediated by a mechanism distinct from the glucocorticoid receptor mediated response. However, a synergistic induction of CYP3A has been observed with physiological doses of glucocorticoids and other CYP3A inducers. We have identified the presence of a glucocorticoid-responsive element in the CYP3A1/IGC2 gene that mediates the induction with physiological doses of glucocorticoids. A 219-bp dexamethasone responsive fragment of the CYP3A1/IGC2 gene localized at -2100/-1882 bp upstream of the transcription initiation site was identified in transfection experiments with HepG2 cells. Maximum induction was achieved with 50-100 nM dexamethasone. DNase I footprinting analysis revealed two glucocorticoid receptor-protected sequences in the 5' flank of the CYP3A1/IGC2 gene. Point mutations in footprint I (-1982/-1960-bp) completely abolished binding and transcription activation whereas a mutation in footprint II (-2001/-1986-bp) only decreased the binding and had no effect on transcription activation. These results led to the conclusion that the glucocorticoid response element present in footprint I mediated the dexamethasone response in transfection experiments with HepG2 cells. Pregnenolone 16alpha-carbonitrile failed to induce any transcriptional effect mediated by this response element in the HepG2 cells.

Cytochrome P450 nomenclature.

AbstractWith genome projects spewing forth DNA sequence at tens of Megabases per year, the problem of genetic nomenclature becomes daunting. In the field of cytochrome P450 (P450), there are more than 750 sequences and they are accumulating rapidly. At the current rate, there will be more than 1000 P450 sequences by late 1998 or early 1999. For these data to be accessible and useful they must be sorted and categorized in some meaningful way. To that end, a cytochrome P450 nomenclature system was devised (1). This system relied on evolutionary relationships as depicted in phylogenetic trees or dendrograms derived from the P450 protein sequences. The whole collection of sequences represents the P450 superfamily, with families and subfamilies being arbitrarily defined as distinct clusters on the tree. Orrgmally, 40% identity was used as a cutoff for family membership and 55% was used for subfamily membership. Since the inception of the system, both numbers have crept downward. The actual decision to mclude a sequence in an existing group largely depends on how it clusters on a tree and not so much on the absolute percent identity, which is more or less a rule of thumb. The most recent published compilation of the nomenclature is given in Nelson et al. (1996) (2). More up-to-date information is available at .KeywordsP450 GenePercent IdentityBacterial SequenceAllene OxideP450 SequenceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Catalytic assays for human cytochrome P450.

Cytochrome P450 (P450) enzymes catalyze the blotransformatlon of a broad range of structurally diverse foreign chemicals and endogenous substances. Many P450 enzymes have been isolated and identified in liver and other tlssues, including kidney, lung, intestines, and brain. Although these enzymes have a high degree of similarity in their ammo-acid sequences, many of them are subject to differential regulation and have distinct catalytic functions With the development of P450 form-specific (or P450 subfamlly-specific) inhlbltory antibodies, the discovery of enzyme-selective chemical inhibitors, and the general availability of catalytically active, mdivldual P450 cDNA-expressed proteins, several substrates, including drugs and endogenous substances, have been identified as useful catalytic monitors for specific P450 enzymes or subfamilies (see Table 1). These catalytic monitors can serve as useful expenmental tools in a variety of studies. These include studies designed: 1 To identify individual P45Os that catalyze the actlvatlon or detoxification of a particular compound, 2 To quantify the expression and catalytic activity of an mdlvrdual P450 in tissue samples and In cultured cells, 3. To analyze the enzyme kinetics of a particular P450; and 4 To eluctdate the mechanism of interaction between xenoblotlcs and mdlvldual P450 enzymes

Induction of liver monooxygenases by β-myrcene

β-Myrcene (MYR) is an acyclic monoterpene found in the essential oils of a variety of useful plants such as lemongrass ( Cymbopogon citratus), hop, verbena, bay and others. MYR and essential oils containing this olefinic monoterpene are widely used as flavoring food additives, as fragrances in cosmetics and as scents in household products. The present study was undertaken to investigate the induction of liver monooxygenases by MYR. Female Wistar rats were treated by gavage with MYR (1000 mg/kg body weight) or corn oil (vehicle) for 1 or 3 consecutive days. Activities of ethoxycoumarin- O-deethylase (ECOD) and alkoxy-resorufin O-dealkylases (methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD) and benzyloxy-resorufin- O-dealkylation (BROD)) were determined fluorimetrically in the hepatic microsomal fraction. Exposure to MYR, either for 1 or 3 days, produced marked (13–34-fold) increases in the activities of PROD and BROD and only minor changes in ECOD, EROD and MROD. Since PROD and BROD are metabolized mainly by CYP2B isoenzymes, these results suggest that MYR induces this phenobarbital-inducible P450 subfamily. The induction of CYP2B isoenzymes was confirmed by SDS-PAGE and immunoblotting. Levels of apoproteins CYP2B1/2B2 were increased 8.2-fold after treatment with MYR (1000 mg/kg body wt, 3 days). Results from this study therefore indicate that MYR is an inducer of isoenzymes belonging to CYP2B subfamily.

Sensitivity of CYP1A1 mRNA Inducibility by Dioxin Is the Same in Cyp1a2(/) Wild-type and Cyp1a2(−/−) Null Mutant Mice

In mammals, the induction of experimental porphyria by halogenated aromatic hydrocarbons (HAHs) seems to be influenced by the levels of hepatic CYP1A2. The pharmacokinetics and relative rates of uptake and storage of HAHs in the liver are correlated with hepatic CYP1A2 concentrations. It is possible that these rates of HAH uptake and storage might affect the expression of other HAH-inducible genes. The differential inducibility of liver CYP1A1 mRNA by dioxin was therefore compared in Cyp1a2(+/+) wild-type mice, Cyp1a2(+/−) heterozygotes, and Cyp1a2(−/−) homozygous null mutants. Using doses of dioxin over eight orders of magnitude (from 10 −12 to 10 −4 g/kg), we could detect no differences in the sensitivity of CYP1A1 mRNA inducibility. These data indicate that the complete absence of the microsomal CYP1A2 enzyme has no measurable effect on hepatic expression of the Cyp1a1, gene, the only other known member of the mammalian CYP1A cytochrome P450 subfamily.

Barbiturate-induced expression of neuronal nitric oxide synthase in the rat cerebellum

Neuronal nitric oxide synthase (nNOS) is known to share some structural and functional similarities with the cytochrome P450 mixed function oxidase system. Unlike P450, it does not require a second enzyme, reductase, to transfer electrons. This characteristic is similar to P450 BM-3 of Bacillus megaterium. P450 BM-3 and certain mammalian subfamilies of P450, such as P4502B, are known to be induced by phenobarbital (PB), and these P450s share a consensus sequence called the Barbie box. Because of the similarities nNOS shares with P450 BM-3 and other mammalian P450s, we have examined whether nNOS also responds to PB treatment. We have used semi-quantitative PCR, Western blot analysis, a functional assay, and immunohistochemistry in order to answer this question. These data show a threefold increase in nNOS mRNA expression, more modest nNOS protein and activity induction, and no discernible changes in localization of nNOS within the cerebellum following PB treatment.

Changes in three hepatic cytochrome P450 subfamilies during a reproductive cycle in turbot (Scophthalmus maximus L.)

Changes in three hepatic cytochrome P450 subfamilies during a reproductive cycle in turbot (Scophthalmus maximus L.)

Changes in three hepatic cytochrome P450 subfamilies during a reproductive cycle in turbot (Scophthalmus maximus L.)

Changes in three hepatic cytochrome P450 subfamilies during a reproductive cycle in turbot (Scophthalmus maximus L.)

Changes in three hepatic cytochrome P450 subfamilies during a reproductive cycle in turbot (Scophthalmus maximus L.)

Sexual differentiation of the hepatic cytochrome P450 system was characterized in 2-year-old farmed turbot (Scophthalmus maximus L.) during their first spawning period (January–September). The fish were kept in tanks supplied with continuously flowing seawater (34.5%, ppt) at a constant temperature of 16°C and natural photoperiod (60°N). Sampling of liver samples (n = 4–6) was performed once every month for 9 months. Pronounced sex differences were recorded in the activities of 7-ethoxyresorufin O-deethylase (EROD), 7-ethoxycoumarin O-deethylase (ECOD), and NADPH-cytochrome P450 reductase during spawning (May–July). EROD activity in female fish decreased gradually towards the onset of ovulation in May–July to rise again in the postspawning period. The decrease correlated with increasing gonadosomatic index and estradiol17β (E2) levels in plasma. Immunochemical detection of CYP1A (58 kDa), CYP2K-like (47 and 52 kDa), and CYP3A-like (58 and 60 kDa) proteins in Western blotting, and ELISA showed higher protein levels in male compared to female fish from April/May-June, and significant differences were observed in June (CYP2K-like also in April and May). Analysis of monthly variations within sexes during the reproductive cycle shows significant monthly changes in all parameters in both female and male fish. Both CYP2K- and CYP3A-like protein levels were significantly elevated in male fish during spawning in June. To study the induction response during spawning, β-naphthoflavone (BNF) 75 mg/kg body weight) was administered intraperitoneally to both sexes in June. BNF caused a significant increase in EROD and ECOD activities and CYP1A protein levels but had no effect on NADPH-cytochrome P450 reductase activity or CYP2K-like/CYP3A-like proteins. This study documents, for the first time in any fish species or lower vertebrate, the sexual differentiation in the liver of three different CYP subfamilies during sexual maturation and spawning. J. Exp. Zool. 277:313–325, 1997. © 1997 Wiley-Liss, Inc.

Pseudomonas aeruginosa lipopolysaccharides and pathogenesis.

Pseudomonas aeruginosa lipopolysaccharide (LPS) plays a key role in pathogenesis. In acute infections, a smooth LPS protects the organism from complement-mediated killing and, during chronic lung infections, an altered rough LPS helps the organism evade host defense mechanisms.

Bell pepper fruit fatty acid hydroperoxide lyase is a cytochrome P450 (CYP74B)

Fatty acid hydroperoxide lyases cleave a CC bond adjacent to a hydroperoxide group in lipoxygenase derived lipid hydroperoxides to form short-chain aldehydes and oxo-acids. Previously, we showed that fatty acid hydroperoxide lyase from bell pepper fruits is a heme protein whose spectrophotometric properties greatly resemble a cytochrome P450. In order to ascertain the relationship of it to the P450 gene family, we have cloned cDNA encoding fatty acid hydroperoxide lyase from immature bell pepper fruits. The cDNA encodes 480 amino acids, and shares homology with P450s mostly at the C terminus. The heme binding cysteine is recognizable at position 441. The most closely related P450 is allene oxide synthase (CYP74A), with which it has 40% identity. It qualifies the lyase as a member of a new P450 subfamily, CYP74B. From this finding, the enzyme is thought to be a novel member of P450 specialized for the metabolism of lipid peroxides.

Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases.

The fah1 mutant of Arabidopsis is defective in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. Earlier results indicated that the FAH1 locus encodes ferulate-5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase (P450) of the general phenylpropanoid pathway. We have cloned the gene encoding this P450 by T-DNA tagging and have confirmed the identity of the cloned gene by complementation of the mutant phenotype. F5H shows 34% amino acid sequence identity with the avocado ripening-induced P450 CYP71A1 and 32% identity with the flavonoid-3',5'-hydroxylases of Petunia hybrida. In contrast, it shares much less homology with cinnamate-4-hydroxylase, a P450 that catalyzes the hydroxylation of cinnamic acid three steps earlier in the general phenylpropanoid pathway. Since the highest degree of identity between F5H and previously sequenced P450s is only 34%, F5H identifies a new P450 subfamily that has been designated CYP84.

Highly specific cytochrome P450-like enzymes for all-trans-retinoic acid in T47D human breast cancer cells.

When human breast cancer T47D cells were treated with all-trans-retinoic acid (RA), the RA 4- and 18-hydroxylase activities were induced in microsomes in a time-dependent manner, indicating that these cells readily metabolized RA into more polar compounds, such as all-trans-4-hydroxy-RA and all-trans-18-hydroxy-RA. In contrast, T47D cells treated for 12 h with xenobiotics, such as phenobarbital, beta-naphthoflavone, 3-methylcholanthrene, and dimethylsulfoxide, showed lower levels of catalytic activities for 4- and 18-hydroxylases. The induction of 4- and 18-hydroxylase activities appears to be regulated at the level of transcriptional control (basal level). Competitive assays demonstrated that inhibitors and substrates for 1A, 2A, 3A, 2B, and 2C cytochrome P450 (P450 subfamilies), all-trans-retinol, and all-trans-retinal showed no inhibition of RA metabolism, but other retinoic acid derivatives competed highly with RA. The RA-inducible 4- and 18-hydroxylases showed high specificity for RA and high levels of catalytic activities, with Km and maximum velocity values for 4-hydroxylase equal to 99 nmol/L and 0.26 pmol/min.mg protein, respectively, and those for 18-hydroxylase equal to 65 nmol/L and 0.18 pmol/min.mg protein. Cell-free metabolism of RA required microsomes from RA-treated cells and NADPH, and was inhibited by liarozole, an inhibitor of P450. These data suggest that RA-inducible 4- and 18-hydroxylases may be novel P450 isozymes.

CDNA and Protein Sequence of a Major Form of P450, CYP2L, in the Hepatopancreas of the Spiny Lobster,Panulirus argus

A P450 fraction was previously isolated from spiny lobster hepatopancreas microsomes and shown in reconstitution experiments to be efficient in catalyzing the monooxygenation of benzphetamine, aminopyrine, benzo(a)pyrene, progesterone, and testosterone. In this study, N-terminal sequence information up to residue 39 was obtained from this P450 and used to design degenerate primers for screening a cDNA library constructed from hepatopancreas mRNA. Clones were obtained that contained part of the coding region of a P450 protein. Further exact primers were designed that permitted the isolation of clones containing coding information for other parts of the P450 sequence, as well as a clone that coded for the complete P450 protein sequence. The open reading frame of the complete coding region corresponded to a protein of 492 amino acids. The deduced amino acid sequence of this P450 was about 36% similar to individual mammalian P450s in the 2 family and did not show strong matches with other proteins in the data base. Based on sequence and the previously determined function, this spiny lobster P450 was assigned by the P450 nomenclature committee to a new P450 subfamily, CYP2L. This is the first description of a P450 primary sequence from a marine crustacean species and the first assignment of an invertebrate P450 into the 2 family.

Modulation of constitutive and inducible hepatic cytochrome(s) P-450 by interferon β in mice

Aims/Methods: Interferon β is used as a therapeutic agent, but its effects on the hepatic cytochrome P-450-dependent drug metabolizing system have not yet been characterized. We investigated the effect of interferon β on cytochrome P-450 in mice. Results: Interferon β (2×10 5 units/mouse) significantly reduced total hepatic cytochrome P-450 (20%) and the activity of NADPH cytochrome C reductase (12%) 24 h after administration; lower doses had no such effect. Various monooxygenase activities were slightly reduced, the one most affected being 7-ethoxycoumarin O-deethylase (29%). In phenobarbital-treated mice, interferon β reduced the induction of total cytochrome P-450 (22%), the activities of pentoxyresorufin O-dealkylase (38%), benzyloxyresorufin O-dealkylase (30%), erythromycin N-demethylase (30%), 7-ethoxycoumarin O-deethylase (16%) and cytochrome P-450 2B1 (33%) and 3A (45%) proteins. In β-naphthoflavone-treated mice, interferon β lowered the induction of total cytochrome P-450 (18%), the activities of ethoxyresorufin O-deethylase (31%) and of 7-ethoxycoumarin O-deethylase (25%) and of cytochrome P-450 1A1 protein (31%). Conclusions: Thus it appears that induced cytochrome(s) P-450 were susceptible to interferon β, this effect not being influenced by the type of inducer. Since various members of the same cytochrome P-450 subfamilies catalyze oxidation of drugs in humans, our findings have potential significance as regards the fate of drugs or exogenous compounds given to patients receiving interferon β.