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PubMed1995

Identification of a common cytochrome P450 epitope near the conserved heme-binding petide with antibodies raised against recombinant cytochrome P450 family 2 proteins.

The cytochrome P450 (P450) proteins constitute a superfamily of enzymes involved in various oxidations and related activities. Polyclonal antibodies raised against bacterial recombinant human P450s varied in specificity, depending upon the individual rabbits used. Several of the antisera raised against P450s 2C10 and 2E1 recognized a number of P450 family 1, 2, and 3 proteins, and two of the less selective antibodies were used to identify cross-reactive epitopes. P450 2C10 peptides reacting with anti-P450 2E1 and P450 2E1 peptides reacting with anti-P450 2C10 were isolated after electrophoresis/immunoblotting and analyzed by Edman degradation. Several of these were in a region near the highly conserved Cys that is a putative axial ligand to the heme. Peptides corresponding to the most conserved regions in this area were synthesized. Anti-P450 2C10 sera did not recognize 14-mer peptides corresponding to the heme-binding region (2C10 410-423 or 2E1 409-422) or the 14-mer peptides immediately C-terminal to these (2C10 425-438 or 2E1 424-437), but anti-P450 2E1 sera showed weak reaction with the latter two synthetic peptides. A longer peptide (29-mer) of P450 2E1 containing parts of both regions (412-440) reacted with both anti-P450 2C10 and anti-P450 2E1 antisera. Antibodies raised against a conjugate of the 29-mer peptide (with hemocyanin) recognized this antigen, the more C-terminal 14-mer peptides (2C10 425-438 and 2E1 424-437), P450s 2C10 and 2E1, and P450s 1A1, 11A1, and 17A. The 29-mer peptide showed considerable alpha-helix structure as judged by CD spectroscopy, in contrast to any of the 14-mers.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences.

The substrate recognition regions in cytochrome P450 family 2 (CYP2) proteins were inferred by group-to-group alignment of CYP2 sequences and those of bacterial P450s, including Pseudomonas putida P450 101A (P450cam), whose substrate-binding residues have been definitely identified by x-ray crystallography of a substrate-bound form (Poulos T. L., Finzel, B. C., and Howard, A. J. (1987) J. Mol. Biol. 195, 687-700). The six putative substrate recognition sites, SRSs, thus identified are dispersively located along the primary structure and constitute about 16% of the total residues. All the reported point mutations and chimeric fragments that significantly affect the substrate specificities of the parental CYP2 enzymes fell within or overlapped some of the six SRSs. Analysis of nucleotide substitution patterns in closely related members in four subfamilies, CYP2A, 2B, 2C, and 2D, consistently indicated that the SRSs have accumulated more nonsynonymous (amino acid-changing) substitutions than the rest of the sequence. This observation supports the idea that diversification of duplicate genes of drug-metabolizing P450s occurs primarily in substrate recognition regions to cope with an increasing number of foreign compounds.

Amino Acid Sequence↗
PubMed2001

Different expression of hepatic and renal cytochrome P450s between the streptozotocin-induced diabetic mouse and rat.

1. Since limited information is available about alterations of cytochrome P450 levels in diabetic animals other than rat, expression of P450s in the liver and kidney of the streptozotocin (STZ)-induced diabetic mouse was investigated. 2. The mRNA levels of CYP2B10, 3A11, 4A10 and 4A14 in the liver were increased in the STZ-induced diabetic mouse of both sexes. The CYP2B9 mRNA level was increased in the liver of the male diabetic mouse. These alterations were observed even at 2 weeks after administration. Insulin treatment restored these changes. The findings were consistent with changes reported in rat. 3. The levels of hepatic CYP1A2 and 2E1 and renal 2E1 and 4A did not change in the diabetic mouse at any time-point examined. No changes were seen in CYP2A- or 2C-related proteins in the diabetic mouse. These findings were in contrast to those in rat. 4. The results indicate that mouse P450s respond to insulin-dependent diabetes mellitus differently from those of the rat, and suggest that the expression of P450s in diabetes is not generally the same across animal species.

Animals↗
PubMed2005

Renal and hepatic accumulation of cadmium and lead in the expression of CYP4F2 and CYP2E1.

The present study examined accumulation of the metal toxins cadmium (Cd) and lead (Pb) in relation to the abundance of cytochrome P450 4F2 (CYP4F2), CYP2E1 and concentrations of zinc and copper in liver and kidney samples using immunoblotting coupled with metal analysis. The post mortem liver and kidney cortex samples were from 23 males and 8 females aged 3-89 years. All were Caucasians who had not been exposed to metals in the workplace. Average kidney cortex Cd load of 17.4 microg/g w.w. was 17 times greater than average liver Cd load (1.1 microg/g w.w.). In contrast, average kidney cortex Pb load of 0.09 microg/g w.w. was two times lower than liver Pb load of 0.19 microg/g w.w. Average Zn and Cu concentrations in the kidney cortex samples were 67% and 33% lower than those in the liver. Liver and kidney Cd loads, but not liver or kidney Pb loads, correlated positively with donors' age. After controlling for liver Cd load, an inverse correlation was seen between Zn and age (partial r=-0.39, P=0.02), suggesting reduction in liver Zn levels in old age. Liver CYP2E1 protein abundance correlated with age-adjusted Cd load (partial r=0.37, P=0.02) whereas kidney CYP4F2 protein abundance showed a positive correlation with age-adjusted Cd loads (partial r=0.40, P=0.02). These findings suggest that Cd may be an inducer of renal CYP4F2 and hepatic CYP2E1 and that increased renal CYP4F2 expression may implicate in Cd-linked renal tubular dysfunction and high blood pressure, involving CYP4F2-dependent arachidonic acid metabolism.

Adolescent↗
PubMed2004

Estrogen signaling in livers of male mice with hepatocellular carcinoma induced by exposure to arsenic in utero.

BACKGROUND: Exposure of pregnant mice to inorganic arsenic induces a spectrum of tumors, including hepatocellular carcinoma (HCC), in their adult offspring similar to that induced by exposing adult mice to estrogenic compounds. To investigate whether arsenic exposure in utero causes altered estrogen signaling, we examined expression of estrogen receptor-alpha (ER-alpha), cyclin D1 (an estrogen-responsive hepatic oncogene), and several cytochrome P450 genes (with sexually dimorphic liver expression patterns) in livers from adult male mice with in utero arsenic-induced HCC. METHODS: Quantitative real-time reverse transcription-polymerase chain reaction was used to evaluate gene expression in livers of adult male mice that had (i.e., exposed mice; n = 8) or had not (i.e., control mice; n = 5) been exposed to arsenic in utero. DNA methylation status of portions of the ER-alpha and cyclin D1 gene promoters in liver tissue was measured using methylation-specific polymerase chain reaction. Statistical tests were two-sided. RESULTS: ER-alpha mRNA levels were 3.1-fold (95% confidence interval [CI] = 2.0-fold to 4.3-fold) higher in livers of exposed mice than in those of control mice, and cyclin D1 levels were 3.0-fold (95% CI = 1.7-fold to 4.3-fold) higher. Exposed mice showed a feminized expression pattern of several cytochrome P450 genes, expressing the female-dominant CYP2A4 (P =.017 versus control) and CYP2B9 (P<.001) genes at 8.7 and 10.5 times, respectively, the level in control mice and expressing the male-dominant CYP7B1 at approximately one-fourth the level in control mice(P =.0012). Exposed mice exhibited reduced (by approximately 90%) methylation of the ER-alpha gene promoter in liver DNA as compared with control mice; the cyclin D1 gene promoter was not methylated in either exposed or control mice. CONCLUSION: Altered estrogen signaling may play a role in induction of HCC by arsenic exposure in utero. Specifically, overexpression of ER-alpha, potentially through promoter region hypomethylation, in livers of such mice may be linked to the hepatocarcinogenicity of arsenic.

Animals↗
PubMed2004

Differential expression of cytochrome P450 isoforms in the lungs of septic animals.

OBJECTIVE: Sepsis is characterized by an early, hyperdynamic phase and a late, hypodynamic phase. Although studies have shown that cytochrome P450 (CYP) plays an important role in the regulation of vascular reactivity, alterations of vascular CYP isoforms in sepsis remain unknown. Since CYP2C11 and CYP2J4 convert arachidonic acid to vasodilative epoxyeicosatrienoic acids, and CYP4A3 metabolizes arachidonic acid to both epoxyeicosatrienoic acids and vasoconstrictive 19,20-hydroxyeicosatetraenoic acid, the aim of this study was to examine the expression of these isoforms in sepsis and their association with hemodynamic changes. DESIGN: Prospective, controlled, and randomized animal study. SETTING: An institute research laboratory. SUBJECTS: Male adult Sprague-Dawley rats were subjected either to polymicrobial sepsis by cecal ligation and puncture or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation). INTERVENTIONS: At 5 hrs (early sepsis) or 20 hrs (late sepsis) after cecal ligation and puncture, blood vessel-rich tissues (i.e., lungs) were harvested. The expression of CYP isoforms at both messenger RNA and protein levels was determined by reverse transcription polymerase chain reaction and Western blot analysis (CYP2C11), respectively. Hemodynamic variables were measured by radioactive microspheres. MAIN RESULTS: The results indicate that the gene expression of CYP2C11 and CYP2J4 was significantly down-regulated at 20 hrs after cecal ligation and puncture, whereas the expression of CYP4A3 was markedly up-regulated at 5 hrs. The protein concentrations of CYP2C11 also decreased significantly at 20 hrs after cecal ligation and puncture. Although total peripheral resistance markedly increased, mean arterial pressure did not change significantly at 20 hrs after the onset of sepsis. In contrast, cardiac output and pulmonary perfusion markedly decreased in late sepsis. CONCLUSIONS: Since the up-regulated CYP4A3 is associated with the early, hyperdynamic phase of sepsis and the down-regulated CYP2C11 and CYP2J4 are associated with the late, hypodynamic phase, vascular CYP isoforms that metabolize arachidonic acid may be involved in regulating the cardiovascular response during the progression of sepsis.

Animals↗
PubMed2004

The unique regulation of brain cytochrome P450 2 (CYP2) family enzymes by drugs and genetics.

Cytochrome P450 (CYP) enzymes in the brain may have a role in the activation or inactivation of centrally acting drugs, in the metabolism of endogenous compounds, and in the generation of damaging toxic metabolites and/or oxygen stress. CYPs are distributed unevenly among brain regions, and are found in neurons, glial cells and at the blood-brain interface. They have been observed in mitochondrial membranes, in neuronal processes and in the plasma membrane, as well as in endoplastic reticulum. Brain CYPs are inducible by many common hepatic inducers, however many compounds affect liver and brain CYP expression differently, and some CYPs which are constitutively expressed in liver are inducible in brain. CYP induction is isozyme-, brain region-, cell type- and inducer-specific. While it is unlikely that brain CYPs contribute to overall clearance of xenobiotics, their punctate, region- and cell-specific expression suggests that CNS CYPs may create micro-environments in the brain with differing drug and metabolite levels (not detected or predicted by plasma drug monitoring). Coupled with the sensitivity of CNS CYPs to induction, this may in part account for inter-individual variation in response to centrally acting drugs and neurotoxins, and may have implications for individual variation in receptor adaptation and cross-tolerance to different drugs. In addition, genetic variation in brain CYPs, depending on the type of polymorphism (structural versus regulatory), will alter enzyme activity. These aspects of brain CYP expression regulation and genetic influences are illustrated in this review using mRNA, protein, and enzyme activity data for CYP2D1/6, CYP2E1 and CYP2B1/6 in rat and human brain. The role of CYP-mediated metabolism in the brain, a highly heterogeneous and complex organ, is a new and relatively unexplored field of scientific enquiry. It holds promise for furthering our undestanding of inter-individual variability in response to centrally acting drugs as well as risk for neurological diseases and pathogies.

Animals↗
PubMed2004

Identification of metastasis-related genes in a mouse model using a library of randomized ribozymes.

Libraries of randomized ribozymes have considerable potential as tools for the identification of functional genes critically involved in a biological phenotype of interest in vitro. We have used a ribozyme library in an in vivo mouse model to identify genes related to metastasis. We injected weakly metastatic melanoma cells that had been treated with the library intravenously into mice. We then isolated ribozymes that accelerated metastasis from pulmonary tumors that had developed from metastasizing cells. As candidates for metastasis-related genes that were targets of the isolated ribozymes, we identified five unknown and three known genes: stromal interaction molecule 1 (STIM1), polymerase gamma2 accessory subunit (Polg2), and cytochrome P450, family 2, subfamily d, polypeptide 22 (Cyp2d22). Repression of four of these by small interfering RNAs indeed resulted in the accelerated mobility of cells in in vitro scratch-wound assay. The further characterization of these candidate genes would provide clues to the complex mechanism(s) of metastasis.

Animals↗
PubMed2006

Assessment of stress-related gene expression in the heavy metal-exposed nematode Caenorhabditis elegans: a potential biomarker for metal-induced toxicity monitoring and environmental risk assessment.

The toxicity of cadmium, lead, chromium, and arsenite on Caenorhabditis elegans was investigated to identify sensitive biomarkers for environmental monitoring and risk assessment. Effects of these metals on stress-related gene expression, growth, reproduction, and mortality of C. elegans were investigated under laboratory conditions. The possibility of using C. elegans as a biosensor for environmental toxicity monitoring was also tested using a green fluorescent protein transgenic nematode. The 24-h median lethal concentrations of cadmium, lead, chromium, and arsenite in C. elegans were 846, 34, 115, and 92 mg/L, respectively. Cadmium exposure led to an increase in the expression of most of the genes tested. The degree of increase was more than threefold compared to control in heat shock protein 16.2, heat shock protein 70, metallothionein 2, cytochrome P450 family protein 35A2, glutathione-S-transferase 4, superoxide dismutase 1, catalase 2, C. elegans p53-like protein 1, and apoptosis enhancer 1 genes. The lead-, chromium-, and arsenite-exposed nematode, on the other hand, showed little change in gene expression. Alterations in growth and reproduction were observed in cadmium- and chromium-exposed worms. To consider a transgenic nematode as a biosensor for toxicity monitoring, the responses of stress-related gene promoters need to be tested with a variety of metals. The overall results suggest that cadmium exhibits a high level of tolerance compared to the other metals tested. Use of the responses of stress-related gene expression therefore has considerable potential as a sensitive biomarker for the diagnosis of cadmium contamination, and C. elegans seems to be a good biological model for this approach.

Animals↗
PubMed2005

Functional role of residues in the helix B' region of cytochrome P450 2B1.

Comparison of several recently determined X-ray crystal structures of mammalian cytochrome P450 family 2 enzymes suggests considerable movement of helix B' when ligands bind. To investigate the functional role of helix B' in P450 2B1, residues 100-109 were substituted with alanine and phenylalanine. Kinetic properties were examined with the typical 2B substrates 7-benzyloxyresorufin, 7-ethoxy-4-trifluoromethylcoumarin, benzphetamine, and testosterone. Several mutants showed 2- to 3-fold changes in k(cat) values and significant differences in catalytic efficiencies among the substrates examined, consistent with structural information suggesting that the helix B' region can adopt multiple conformations with different contact residues depending on the substrate. Homology modeling of P450 2B1 was performed based on an inhibitor-bound P450 2B4 structure, and the docking analyses were consistent with experimental results. The findings suggest that residues in the helix B' region affect regio- and stereoselective oxidation in P450 family 2 enzymes as well as substrate entry.

Amino Acid Substitution↗
PubMed2006

Across-tissue expression and evolution of genes controlled by the Aire transcription factor.

Aire (autoimmune regulatory protein) enhances expression of certain genes in thymic medullary epithelial cells (MECs). Using publicly available data, we examined expression patterns, across 82 distinct tissue types, of genes previously identified as Aire-activated, Aire-repressed, and Aire-independent. Consistent with the hypothesis that the effect of Aire in MECs is to increase expression of tissue-specific genes, Aire-activated genes had a low overall level of expression but a large range between the lowest and the highest levels of expression in different tissues. By contrast, Aire-repressed genes tended to have a high overall level of expression and less marked differences between the highest and the lowest levels of expression. Nonetheless, the expression scores of Aire-repressed genes showed broader ranges of values than those of Aire-independent genes. Phylogenetic analyses of members of two gene families that included two Aire-activated genes illustrated two contrasting patterns of the relationship of Aire-activated genes within the same family. The two Aire-activated members of the major urinary protein family arose through a recent gene duplication (after the rat-mouse divergence), whereas the most recent common ancestor of the two Aire-activated members of cytochrome p450 family 2 duplicated prior to the radiation of the eutherian orders. In the latter family, the Aire-activated Cyp2a4 gene and the Aire-independent Cyp2a5 gene arose through a recent duplication, after the rat-mouse divergence. Thus the set of Aire-activated genes is subject to change over evolutionary time and includes genes of recent origin.

Animals↗
PubMed1998

Nutritional status modulates rat liver cytochrome P450 arachidonic acid metabolism.

Alterations in nutritional status affect hepatic cytochrome P450 levels. Since cytochromes P450 participate in the metabolism of arachidonic acid, we hypothesized that changes in liver P450 arachidonic acid metabolism occur during fasting and refeeding. Male Fisher 344 rats were either fed, fasted 48 hr (F48), fasted 48 hr and then refed 6 hr (F48/R6), or fasted 48 hr and then refed 24 hr (F48/R24). F48 rats had reduced body weight, increased plasma beta-hydroxybutyrate, and reduced plasma insulin compared with the other groups. Although there was no significant change in total liver P450 content, there was a significant 20%, 48%, and 24% reduction in total hepatic microsomal arachidonic acid metabolism in F48, F48/R6, and F48/R24 rats, respectively, compared with fed rats. Epoxygenase activity decreased by 28%, 51%, and 26% in F48, F48/R6, and F48/R24 rats, respectively. In contrast, omega-1 hydroxylase activity increased by 126% in F48 rats compared with fed rats. Immunoblotting revealed that levels of CYP2C11 protein were markedly reduced, whereas levels of CYP2E1 protein were markedly increased in the F48 and F48/R6 groups. In contrast, levels of CYP1A1, CYP1A2, CYP2B1, CYP2J3, CYP4A1, and CYP4A3 were unchanged with fasting/refeeding. Northern blots revealed that levels of CYP2C11 mRNAs were decreased, whereas CYP2E1 mRNAs were increased in F48 and F48/R6 rats. Recombinant CYP2C11 metabolized arachidonic acid primarily to epoxides with preference for the 14(S),15(R)-, 11(R), 12(S)-, and 8(S),9(R)- epoxyeicosatrienoic acid enantiomers. We conclude that (1) nutritional status affects hepatic microsomal arachidonic acid metabolism, (2) reduced epoxygenase activity in F48 and F48/R6 rats is accompanied by decreased levels of CYP2C11, (3) increased omega-1 hydroxylase activity is accompanied by augmented levels of CYP2E1, and (4) the effects of fasting on CYP2C11 and CYP2E1 expression occur at the pretranslational level.

8,11,14-Eicosatrienoic Acid↗
PubMed1995

Isoflurane and cytochrome b5 stimulation of 2-chloro-1,1-difluoroethene metabolism by reconstituted rat CYP2B1 and CYP2C6.

Isoflurane stimulates the metabolism of 2-chloro-1,1-difluoroethene (CDE) in liver microsomes from phenobarbital-treated rats or rabbits. The P450 isozymes involved and the mechanism by which such stimulation occurs have not been clarified. The present study examined the effects of isoflurane and cytochrome b5 on CDE metabolism in reconstituted systems containing purified rat CYP2B1 or CYP2C6. Under similar incubation conditions, CYP2B1 defluorinated CDE at approximately five times the rate of CYP2C6. Isoflurane was a potent stimulator of CDE metabolism, increasing it nearly 5-fold when catalyzed by CYP2B1, but only 2-fold when catalyzed by CYP2C6. Isoflurane had no stimulatory effect on benzphetamine metabolism by CYP2B1 or CYP2C6. Cytochrome b5 was not required for isoflurane-facilitated CDE metabolism; however, the addition of cytochrome b5 to CYP2B1 increased CDE metabolism 71 and 44%, in the absence and presence of isoflurane, respectively. In reconstituted CYP2B1, isoflurane generated a type I difference spectrum of approximately twice the magnitude of CDE and stimulated NADPH consumption more so than CDE. The same quantity of NADPH was consumed when CDE was present with isoflurane as compared with isoflurane alone. These data support the hypothesis that isoflurane stimulates CDE metabolism by a mechanism involving increased P450 reduction via direct isoflurane interaction with P450.

Animals↗
PubMed2000

Metabolic activation of o-phenylphenol to a major cytotoxic metabolite, phenylhydroquinone: role of human CYP1A2 and rat CYP2C11/CYP2E1.

1. The in vitro metabolic activation of o-phenylphenol has been evaluated as yielding a toxic metabolite, 2,5-dihydroxybiphenyl (phenylhydroquinone), by p-hydroxylation in liver microsomes of rat and human. The involvement of rat CYP2C11, CYP2E1 and human CYP1A2 in the p-hydroxylation of o-phenylphenol is suggested. 2. 2,3- and phenylhydroquinone, which induced DNA single-strand scission in the presence of 1 microM CuCl2, were the most cytotoxic chemicals examined to cultured mammalian cell lines among o-phenylphenol, m-phenylphenol, p-phenylphenol, 2,2'-, 4,4'-, 2,3- and phenylhydroquinone. 3. Rat and human liver microsomes catalysed the formation of phenylhydroquinone, but not 2,3-dihydroxybiphenyl, using o-phenylphenol as a substrate. A higher rate of metabolic activation of o-phenylphenol was observed with livers of the male than the female rats by 5.6- and 2.6-fold respectively. 4. Inhibitory antibodies against the male-specific CYP2C11 inhibited hepatic o-phenylphenol p-hydroxylation in the male F344 and Sprague-Dawley rat by > 70%. Liver microsomes from the isoniazid-treated rats produced 1.8- and 3-fold induction of o-phenylphenol p-hydroxylation and chlorzoxazone 6-hydroxylation (a CYP2E1-dependent activity) respectively. 5. Human CYP1A2, expressed by baculovirus-mediated cDNA expression systems, exhibited a remarkably higher capacity for o-phenylphenol p-hydroxylation at concentrations of 5 (> 5-fold), 50 (> 2-fold) and 500 microM (> 2-fold) than CYP2A, CYP2B, CYP2Cs, CYP2D6, CYP2E1 and CYP3A4 on the basis of pmol P450. 6. Among various CYP inhibitors tested here, 7,8-benzoflavone and furafylline, typical human CYP1A2 inhibitors, inhibited the microsomal p-hydroxylation of o-phenylphenol in human livers most potently by 70 and 50% respectively. 7. The results thus indicate the involvement of rat CYP2C11/CYP2E1 and human CYP1A2 in the hepatic p-hydroxylation of o-phenylphenol.

Animals↗
PubMed1993

Induction of CYP2B1 and 3A1, and associated monoxygenase activities by tamoxifen and certain analogues in the livers of female rats and mice.

Previous studies suggest long-term feeding of tamoxifen (Z-1-[4-(2-dimethylamino-ethoxy)phenyl]1,2-diphenyl-1-butane) to rats gives rise to liver tumours, while mice are resistant. The effects of tamoxifen on cytochrome P450 isoenzymes and associated monoxygenase activities in the livers of female Fischer rats and C57Bl/6 and DBA/2 mice have been compared. Total microsomal cytochrome P450 was not induced in the livers of rats given tamoxifen (45 mg/kg daily for 4 days) and was in fact significantly reduced after 3 days treatment. In contrast, there was a 30-60-fold increase in the metabolism of benzyloxy- and pentoxyresorufins to resorufin. Ethoxyresorufin O-deethylase was induced only 2.5-fold. The regio- and stereo-specific hydroxylation of testosterone following tamoxifen pretreatment of rats showed a general time- and dose-dependent induction. 6 beta- and 16 alpha-hydroxylation of testosterone together with oxidation to androstenedione were increased 2-3-fold while 2 beta-hydroxylation was induced only marginally, suggesting that tamoxifen produces a mixed pattern of induction with a significant phenobarbitone-like component. No induction of the 2 beta- or 6 beta-hydroxylation pathway occurred in either mouse strain. In rats, immunoblotting experiments with polyclonal antibodies raised against CYP2B1 or 3A1 showed that tamoxifen pretreatment resulted in 2-3-fold increases in both CYP2B1, 2B2 and 3A1 proteins, relative to controls. Immunohistochemistry of rat liver sections showed a centrilobular localization of these induced proteins. Similar patterns of induction as measured by immunoblotting experiments and testosterone hydroxylation were seen following the administration of structurally related analogues, toremifene and droloxifene (3-hydroxytamoxifen), thought to be non-carcinogenic in the rat. No induction of these monooxygenase activities was seen in C57Bl/6 mice and only small increases in benzyloxy and pentoxyresorufin metabolism were in DBA/2 mice. It is suggested that the induction of cytochrome P450-dependent activities by tamoxifen may result in accelerated liver metabolism of this drug with important implications for the disposition of tamoxifen in vivo and also for its metabolic conversion to genotoxic metabolite(s). The difference in inducibility of cytochrome P450-dependent monooxygenase activities between rats and mice offers a plausible and testable hypothesis that the difference in tamoxifen metabolism between the two species may contribute to their carcinogenic response to tamoxifen.

Animals↗
PubMed2003

Determination of the rate of reduction of oxyferrous cytochrome P450 2B4 by 5-deazariboflavin adenine dinucleotide T491V cytochrome P450 reductase.

The use of 5-deazaFAD T491V cytochrome P450 reductase has made it possible to directly measure the rate of electron transfer to microsomal oxyferrous cytochrome (cyt) P450 2B4. In this reductase the FMN moiety can be reduced to the hydroquinone, FMNH(2), while the 5-deazaFAD moiety remains oxidized [Zhang, H., et al. (2003) Biochemistry 42, 6804-6813]. The rate of electron transfer from 5-deazaFAD cyt P450 reductase to oxyferrous cyt P450 was determined by rapidly mixing the ferrous cyt P450-2-electron-reduced 5-deazaFAD T491V reductase complex with oxygen in the presence of substrate. The 5-deazaFAD T491V reductase which can only donate a single electron reduces the oxyferrous cyt P450 and oxidizes to the air-stable semiquinone, with rate constants of 8.4 and 0.37 s(-1) at 15 degrees C. Surprisingly, oxyferrous cyt P450 turns over more slowly with a rate constant of 0.09 s(-1), which is the rate of catalysis under steady-state conditions at 15 degrees C (k(cat) = 0.08 s(-1)). In contrast, the rate constant for electron transfer from ferrous cyt b(5) to oxyferrous cyt P450 is 10 s(-1) with oxyferrous cyt P450 and cyt b(5) simultaneously undergoing spectral changes. Quantitative analyses by LC-MS/MS revealed that the product, norbenzphetamine, was formed with a coupling efficiency of 52% with cyt b(5) and 32% with 5-deazaFAD T491V reductase. Collectively, these results suggest that during catalysis a relatively stable reduced oxyferrous intermediate of cyt P450 is formed in the presence of cyt P450 reductase but not cyt b(5) and that the rate-limiting step in catalysis follows introduction of the second electron.

Aryl Hydrocarbon Hydroxylases↗
PubMed2006

Downregulation of hepatic cytochrome P-450 isoforms and PPAR-gamma: their role in hepatic injury and proinflammatory responses in a double-hit model of hemorrhage and sepsis.

BACKGROUND: The "double-hit" model of hemorrhage and sepsis mimics the critically ill patient admitted to the surgical intensive care unit. Although the protein expression of a cytochrome (CYP) P-450 isoform CYP1A2 is reduced in the late stage of sepsis, the effect of hemorrhage on CYP isoforms and the anti-inflammatory nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has not been investigated. We hypothesized that hemorrhage down-regulates CYP isoforms and PPAR-gamma in the liver, which plays an important role in producing tissue injury and proinflammatory responses after the subsequent sepsis (i.e., double-hit). MATERIALS AND METHODS: Male Sprague Dawley rats were divided into four groups. Animals in the double-hit group underwent hemorrhage (40 +/- 2 mmHg for 90 min) followed by fluid resuscitation. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) 20 h after hemorrhage, and the animals were sacrificed 4 h after CLP. Rats in the hemorrhage-alone group were sacrificed 20 h after the insult. Rats in the CLP-alone group were sacrificed 4 h after the onset of sepsis. Animals in the sham-operated group underwent neither hemorrhage nor CLP. The gene expression of P-450 isoforms (i.e., CYP1A2 and 2C11) and PPAR-gamma in the liver was determined using RT-PCR. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate, and proinflammatory cytokines (i.e., IL-6, TNF-alpha) were also assessed. RESULTS: In the hemorrhage-alone group, hepatic mRNA expression of CYP1A2, CYP2C11, and PPAR-gamma was significantly down-regulated 20 h after the initial stress compared with sham-operated rats. Double-hit did not appear to further decrease CYP and PPAR-gamma gene expression. In contrast, serum levels of AST, ALT, lactate, IL-6, and TNF-alpha did not change significantly in either hemorrhaged or septic animals. Those organ injury indicators and cytokines, however, were significantly elevated after the double-hit of hemorrhage and sepsis. CONCLUSIONS: Hepatic CYP1A2, CYP2C11, and PPAR-gamma were down-regulated after the initial stress (hemorrhage). These down-regulated CYPs and PPAR-gamma seem to work as important factors contributing to the progression of organ injury and proinflammatory responses after the second stress (CLP).

Alanine Transaminase↗
PubMed2000

Effect of 4-tert-octylphenol on cytochrome P450 enzymes in rat liver.

The effects were studied of 4-tert-octylphenol (OP) on hepatic cytochrome P450 enzymes in rats. Rats were treated intraperitoneally with OP twice, at doses of 5, 10, and 20 mg/kg. Among the cytochrome P450-dependent monooxygenase activities, testosterone 2alpha-hydroxylase activity, which is associated with CYP2C11, was significantly decreased by OP at all doses. The level relative to control activity was 67-22%. CYP3A2-dependent monooxygenase, testosterone 6beta-hydroxylase activity was also decreased by 51% by OP at 20 mg/kg. Furthermore, immunoblotting showed that OP (10 or 20 mg/kg) significantly decreased CYP2C11/6 and CYP3A2/1 protein levels. However, the reduction ratio of CYP2C11/6 and CYP3A2/1 protein levels by OP treatment was lower than that of testosterone 2alpha-hydroxylase and testosterone 6beta-hydroxylase activities. The Cl(int) (Vmax/Km) value for testosterone 2alpha-hydroxylase was significantly decreased by OP at all doses, whereas the Cl(int) value for testosterone 6beta-hydroxylase was only decreased by OP at 20 mg/kg. In addition, 7-ethoxycoumarin O-deethylase activity was significantly decreased by 32% by the highest dose of OP. By contrast, CYP1A1-, CYP1A2-, CYP2A1-, CYP2B1/2-, CYP2D1-, CYP2E1- and CYP4A1/2/3-dependent monooxygenase activities were not affected by OP at any dose. These results suggest that OP changes the male-specific cytochrome P450 isoforms in rat liver, and that these changes closely relate to the toxicity of OP.

Animals↗