Cytochrome P450IIB1 Research Articles

Propofol-induced cytochrome P450 inhibition: An in vitro and in vivo study in rats

Propofol, a widely used anesthetic drug, is known to inhibit cytochrome P450 activities in vitro. The goal of this study was to compare cytochrome P450 activities in vitro and in vivo in presence of propofol. In vitro (liver microsomes and freshly isolated hepatocytes), cytochrome P450 IA2 and IIB1 activities were measured as the production of resorufin from ethoxy- and pentoxyresorufin, respectively, in presence of various concentrations of propofol. In vivo, cytochrome P450 activities were assessed as the production of 13CO 2 from 13C-aminopyrine injected intravenously, during acute administration or after chronic treatment. In vitro results confirmed the dose-dependent inhibitory effect of propofol on cytochrome P450 activities, both on liver microsomes and isolated hepatocytes. In vivo, the acute administration of propofol induced a significant decrease of 13C-aminopyrine metabolism. Chronic treatment with propofol induced a significant inhibition of 13C-aminopyrine metabolism only after 2 weeks. No enzyme induction was observed. In conclusion, our results demonstrate that propofol inhibits cytochrome P450 also in vivo. Drug interactions may thus occur during propofol administration.

Posttranscriptional regulation of mRNA levels in rat liver associated with deoxycholic acid feeding.

We investigated the effects of bile acid feeding on the mRNA levels and transcriptional activity of genes involved in various facets of hepatic cell function. Rats were maintained for 10 days on standard diet supplemented with combinations of 1 and 0.4% deoxycholic acid and ursodeoxycholic acid. Significant reductions in mRNA levels for liver fatty acid binding protein, albumin, the asialoglycoprotein receptor, connexins 32 and 26, and cytochromes P-450IIB1 and P-450IIE1 were associated with 1% deoxycholic acid feeding. Conversely, the 1% deoxycholic acid-fed animals exhibited increased mRNA levels for cholesterol 7 alpha-hydroxylase, 3-hydroxy-3-methylglutaryl-CoA reductase, multidrug resistance, procollagens, extracellular matrix, protooncogenes, tumor suppressors, and cyclins. The 0.4% deoxycholic acid-fed animals exhibited increased mRNA levels for c-jun, H-ras, p53, cyclins D1 and D3, fibronectin, and procollagens alpha 1(I) and alpha 1(III). Transcriptional rate changes could not account for the observed changes in steady-state mRNA levels. Ursodeoxycholic acid feeding had no significant effect on gene expression and almost completely inhibited the changes associated with 1% deoxycholic acid when coadministered. The results indicate that dietary ingestion of deoxycholic acid profoundly affects hepatic gene expression in the rat, and regulation occurs primarily at the posttranscriptional level.

CB pattern in the harbour porpoise: Bioaccumlation, metabolism and evidence for cytochrome p450 IIB activity

Metabolism of chlorobiphenyls (CBs) was studied in harbour porpoise by comparing patterns of CB-X/CB-153 ratios in blood, brain, liver and blubber with the patters in herring, the main food source. The CBs were classified in five groups, based on the presence/absence of vicinal H-atoms (vic. Hs) in meta,para (m,p) and/or ortho, meta (o,m) positions and the number of ortho-Cl-atoms (ortho-Cls). Plots of CB-X/CB-153 ratios in porpoise tissue vs the ratios in herring appeared to be linear for each CB group in all tissues. Slopes of these plots (metabolic slopes) were used as quantitative indicators of metabolic activity. In this way, activity of PB-type isozymes of the P450 monooxygenase system was apparent: in contrast to existing literature data, harbour porpoise appears to be able to metabolize congeners with m,p vic. Hs, even in the presence of more than 2 ortho-Cls. The presence of 3-MC-type (MC-type) isozymes was also detected. The metabolic slopes were also used as basis for risk assessment. Due to their metabolism the most toxic non-ortho CBs were not present in the tissues at detectable levels. We suggest a risk assessment approach which takes this into account. It is considered to be an alternative and more reliable basis for risk assessment than the use of toxic equivalent factors. The results support the model of equilibrium distribution of CBs in harbour porpoise and the role of blood as central transport medium. The model has been developed for persistent compounds; it appears to hold for metabolizable CB congeners as well.

The effect of varying halogen substituent patterns on the cytochrome P450 catalysed dehalogenation of 4-halogenated anilines to 4-aminophenol metabolites

The cytochrome P450 catalysed biotransformation of 4-halogenated anilines was studied in vitro with special emphasis on the dehalogenation to 4-aminophenol metabolites. The results demonstrated that a fluorine substituent at the C4 position was more easily eliminated from the aromatic ring than a chloro-, bromo- or iodo-substituent. HPLC analysis of in vitro biotransformation patterns revealed that the dehalogenation of the C4-position was accompanied by formation of non-halogenated 4-aminophenol, without formation of NIH-shifted metabolites. Changes in the apparent V maxfor the microsomal oxidative dehalogenation appeared to correlate with the electronegativity of the halogen substituent at C4, the fluorine substituent being the one most easily eliminated. A similar decrease in the rate of dehalogenation from a fluoro- to a chloro- to a bromo- to an iodo-substituent was observed in a system with purified reconstituted cytochrome P450 IIB1, in a tertiair butyl hydroperoxide supported microsomal cytochrome P450 system as well as in a system with microperoxidase 8. This microperoxidase 8 is a haem-based mini-enzyme without a substrate binding site, capable of catalysing cytochrome P450-like reaction chemistry. Together, these results excluded the possibility that the difference in the rate of dehalogenation with a varying C4-halogen substituent arose from a change in the contribution of cytochrome P450 enzymes involved in oxidative dehalogenation with a change in the halogen substituent. Rather, they strongly suggested that the difference was indeed due to an intrinsic electronic parameter of the various C4 halogenated anilines dependent on the type of halogen substituent. Additional in vitro experiments with polyfluorinated anilines demonstrated that elimination of the C4-fluorine substituent became more difficult upon the introduction of additional electron withdrawing fluorine substituents in the aniline-ring. 19F-NMR analysis of the metabolite patterns showed that the observed decrease in 4-aminophenol formation was accompanied by a metabolic switch to 2-aminophenols and N-hydroxyanilines, while products resulting from NIH-type mechanisms were not observed. For a C4chloro-, bromo-, or iodo-substituted 2-fluoroaniline the V max for the oxidative dehalogenation was reduced by the additional electron withdrawing fluorine substituent at the C2 position in a similar way. In conclusion, the results of the present study strongly indicate that the possibilities for cytochrome P450 mediated dehalogenation of 4-halogenated anilines to 4-aminophenol metabolites are dependent on: (i) the characteristics of the halogen that has to be eliminated, the most electronegative and smallest halogen being the one most easily eliminated, and (ii) the electron-withdrawing capacities of other substituents in the aromatic ring, electron-withdrawing substituents decreasing the relative rate of the reaction. Together these data lead to the conclusion that the halogen is eliminated as a halogen anion.

Cytochrome P450 in rat astrocytes in vivo and in vitro: intracellular localization and induction by phenytoin.

Cytochrome P450IIB1,2 (nomenclature according to Nelson et al., DNA Cell Biol 12:1-51, 1993 and Volk et al., Neuroscience 42:215-235, 1991) immunoreactivity (P450-IR) is associated with astrocytes both in vivo and in vitro. Although they are unevenly distributed throughout the brain with a preference for phylogenetically elder parts, no significant differences between astrocytes prepared from different brain regions were observed in astrocyte cultures. The percentage of strongly immunoreactive astrocytes decreased from 40% after 7 days in culture to 15% after 21 days. Essentially all astrocytes have a low but significant P450-IR within this interval. Preembedding immunoelectron microscopy revealed peroxidase reaction products on the endoplasmic reticulum and on the outer membranes of mitochondrial and nuclear envelopes. Phenytoin (1 microM) added to the medium for 7 days significantly (1.22-fold) increased the amount of total P450 in astrocyte homogenates as measured by spectrophotometry. Considerably more immunoreactive cells (1.5-fold) were found in treated cultures than in controls.

Transcriptional regulation of rat alpha 1-acid glycoprotein gene by phenobarbital.

Phenobarbital induces gene transcription of both cytochrome P450IIB (the barbiturate-inducible cytochrome P450 in mammals) and alpha 1-acid glycoprotein, one of the major acute-phase proteins in rats and humans. Analysis of the 5'-regulatory sequences of cytochrome P450IIB and alpha 1-acid glycoprotein genes in rats revealed the presence of a consensus sequence of 10 base pairs, termed the phenobarbital-responsive element or Barbie box, located in a region extending from positions -136 to -127 from the transcription start site of the alpha 1-acid glycoprotein gene. A 17-base pair oligonucleotide probe specific for alpha 1-acid glycoprotein and including the consensus sequence showed, in mobility shift assays, slight binding to liver nuclear protein from untreated animals. This binding was strongly and specifically increased with protein extracts from phenobarbital-treated rats. Transfection of rat primary hepatocytes with the pAGPcat construct induced basal expression of chloramphenicol acetyltransferase activity, which was increased by phenobarbital and dexamethasone treatment of cells. Induction of chloramphenicol acetyltransferase activity by phenobarbital was abolished when hepatocytes were transfected by constructs with a mutation or deletion of the Barbie box sequence. These results strongly suggest that the Barbie box sequence is involved in alpha 1-acid glycoprotein gene regulation by phenobarbital.

The effect of colupulone (a hops β-acid) on hepatic cytochrome P-450 enzymatic activity in the rat

Colupulone, a component of hops, was examined for its ability to alter rat hepatic cytochrome P-450 enzymatic activity, expression of hepatic cytochrome P-450 mRNA, and in vitro promutagen activation. Colupulone was fed to male Sprague-Dawley rats for 5 days at 0.36% in the modified AIN 76 diet. Three cytochrome P-450 enzymatic activities were measured, and the corresponding steady-state mRNA levels were examined by Northern blot hybridization. Colupulone increased cytochrome P450IIB and P450IIIA steady-state mRNA levels. In vitro promutagen activation was measured in the Ames assay using liver homogenates from each treatment group. Colupulone treatment did not alter the ex vivo cytochrome P-450-mediated activation of aflatoxin B 1 or benzo[ a]pyrene to their mutagenic forms. The effect of long-term colupulone administration on in vivo cytochrome P-450 enzymatic activity remains to be determined.

Effects of induction and age-dependent enzyme expression on lung bioavailability, metabolism, and DNA binding of urban air particulate-absorbed benzo[a]pyrene, 2-nitrofluorene, and 3-amino-1,4-dimethyl-5H-pyridol-(4,3)-indole.

The effect of interactions between urban air particulates (UAP) and carcinogens on bioavailability, metabolism, and DNA binding was studied in the isolated perfused and ventilated rat lung. The availability of benzo[a]pyrene (B[a]P) varied from 29 to 60% after intratracheal doses of carcinogen particulates dissolving extremely slow and fast, respectively. Several cytochrome P450 enzyme (P450) inducers acting as 2,3,7,8-tetrachlorodibenzo[p]dioxin-receptor ligands have been identified in UAP extracts. beta-Naphthoflavone (BNF) was used to study how P450 induction alters the lung metabolism of carcinogens. Pretreatment increased the lung clearance for B[a]P 8-fold and for 2-nitrofluorene (2NF) by a factor of four from 0.55 +/- 0.06 ml/min to 2.37 +/- 0.62 ml/min. Studies with the intact lung and with isolated lung cells show that carcinogen metabolism and pharmacokinetics depend both on the route of exposure and dosage and on the distribution of specific enzymes. A cytochrome P450IIB1 enzyme was detected in lung epithelial cells where it catalyzes 9-hydroxylation of 2NF. This rat lung 2NF-9-hydroxylation capacity increases in parallel with the age dependent up-regulation of lung P450IIB1 expression. Both human and rat lung tissue have the capacity to form 9-hydroxy-2-nitrofluorene (9-OH-2NF) that is mutagenic. A BNF-inducible P450IA1 was detected in endothelial and alveolar type II cells. Consequently, aromatic hydroxylation dominated when 2NF was dosed directly into the lung circulation. Pretreatment of rats with BNF before intratracheal B[a]P dosage induced lung cytochrome P450IA1. The 7,8-dihydroxy-9,10-oxybenzo[a]pyrene-deoxyguanosine adduct and the total lung DNA adduct levels increased significantly from a peak level of 75 +/- 8 to 151 +/- 19 fmole/mg DNA in lungs from control and BNF pretreated rats, respectively. 3-Amino-1,4-dimethyl-5H-pyridol-(4,3)-indole (TRP-P1) is a potent mutagen and carcinogen identified in UAP extracts. Dietary BNF pretreatment of rats altered the [14C]TRP-P1 distribution as analyzed by whole-body autoradiography. An enhanced retention was observed in the small intestine, forestomach, esophagus, and lung. UAP catalyzes oxygen radical formation and deoxyguanosine-8-hydroxylation, which were inhibited when the UAP samples were extracted with organic solvents or when they were incubated in the presence of desferroxamine. We therefore postulate that a polycyclic aromatic hydrocarbon autooxidation pathway may be responsible for generation of hydrogen peroxide, which may be further converted to hydroxyl radicals through an iron-dependent reaction.

Hydrazine Genotoxicity in the Neonatal Rat

The neonatal rat, because of its relatively rapid rate of liver DNA replication without chemical or surgical induction, was used to assess the genotoxicity of the carcinogen hydrazine. Hydrazine is a more potent acute toxicant in the neonate than in the adult rat. Administration of hydrazine sc (1.5-50 mg/kg body wt) to newborn rats during the period of rapid liver DNA synthesis, 72-96 hr after birth, resulted in the formation of 7-methylguanine and O 6-methylguanine in hepatic DNA; O 6-methylguanine was seen only in animals given near-lethal doses of the carcinogen. Methylguanines were detectable in liver DNA only when the dose of hydrazine was necrogenic, but lethal doses of hydrazine to neonates produced more methylguanines in liver DNA than in adult rats given equal doses. Southern analyses were performed on liver DNA from neonates treated with 25 or 50 mg hydrazine/kg, doses which were necrogenic to the liver. The results indicated that one or more MspI restriction sites (5′-C↓CGG-3′) were lost or blocked in liver DNA from hydrazine-treated animals and that these sites were located at or near the genes for γ-glutamyl transpeptidase and cytochrome P450 IIB1. Restriction sites near albumin, H- ras, and cytochrome P450 IIE1 genes cut by MspI, HpaII, or HhaI did not appear to be affected by hydrazine treatment. The results suggest that hydrazine-induced damage is not random in the DNA molecule. The neonate shows less DNA adduct formation at low doses of hydrazine, but higher levels at high doses.

σRecognition sites in brain and peripheral tissues Characterization and effects of cytochrome P450 inhibitors

Binding to σ sites in subcellular fractions of brain and in crude homogenates from peripheral tissues of the guinea pig was characterized with the [ 3H]ligands (+)pentazocine and di(2-tolyl)guanidine (DTG). The inhibitory effects of representative σ compounds and cytochrome P450 inhibitors were evaluated in guinea pig tissues, and the effects of cytochrome P450 induction σ binding in the rat were investigated. For both ligands, the majority of sites were localized to the microsomal fractions. The K D values for [ 3H](+)pentazocine- or [ 3H]DTG-labeled by sites in guinea pig liver and testes were 2-fold lower than those in brain and heart. The number of sites labeled by [ 3H](+)pentazocine varied, with an order of liver>testes>brain>heart. In contrast, the B MAX values for [ 3H]DTG-defined σ sites were greatest in testes, followed by liver, brain and heart. The rank order of potency for representative σ and P450 compounds was similar in brain, liver and testes for both [ 3H]ligands, and the potency of selective compounds to displace σ binding in guinea pig liver failed to correlate with their abilities to inhibit cytochrome P450IID1 activity in human liver. Following induction of cytochrome P450IIB1 with phenobarbital or cytochrome P450IA1 with β-naphthoflavone, neither the affinity nor the number of σ sites was altered in rat brain or liver. These results suggest that σ sites in the periphery are similar to those in the brain, and that the σ binding site is not identical with cytochrome P450IIB1, P450IA1 or P450IID1.

Biomonitoring of pollution: the hepatic cytochrome P-450 enzyme system in the feral mouse Apodemus flavicollis as indicator

The aim of the present study was to find a suitable wild-rodent to be used as terrestrial bioindicator. The wild mice were trapped at three sites around Vienna and at a fourth location in the countryside. Apodemus flavicollis, the yellow-necked mouse, was the most frequently captured mouse at all four sites. For each sample hepatic microsomal protein content, cytochromes b5 and P450, and enzyme catalytic activities-ECOD, EROD, BROD and PROD were measured. Except for one male urban group no significant differences between rural and urban groups in ECOD activity were observed. Increased cytochrome b5 and P450 contents per mg of microsomal protein were found in one of the male and all the three female urban groups. However the alkoxyresorufin O-dealkylase activities were significantly increased in both sexes of the three city groups. These results suggest that induction of cytochrome P450IA and P450IIB families has occurred in urban populations, indicating a possible pollution with halogenated dioxins and furans, PCBs, PAHs etc. The data show that induction of hepatic mixed function oxidase (HMFO) activity in the feral mouse Apodemus flavicollis can be successfully used as a terrestrial bioindicator for pollution monitoring.

A specific interaction between NADPH-cytochrome reductase and phosphatidylserine and phosphatidylinositol.

In the present study the interaction of NADPH-cytochrome reductase with phospholipids was investigated using 31P-NMR, thin-layer chromatography combined with chemical analysis, fluorescence spectroscopy and kinetic studies with purified rat liver cytochrome P450 IIB1. 31P-NMR analysis demonstrates that the composition of the phospholipids that remain associated to NADPH-cytochrome reductase upon its purification is significantly different from the phospholipid composition of the microsomal membrane. Thin-layer chromatography followed by chemical analysis of the phospholipid composition demonstrates that the isolated NADPH-cytochrome reductase was enriched in L-alpha-1,2-diacyl-sn-glycero-3-phosphoserine (acyl2GroPSer) and L-alpha-1,2-diacyl-sn-glycero-3-phosphoinositol (acyl2GroPIns) compared to the microsomal membrane. The observed preference of NADPH-cytochrome reductase for acyl2GroPSer and acyl2GroPIns appeared not to be a result of the procedure for solubilisation and/or purification of the protein. The specific interaction of NADPH-cytochrome reductase with acyl2GroPSer and acyl2GroPIns was further investigated by comparison of the effect of acyl2GroPSer and acyl2GroPIns with that of acyl2GroPCho and acyl2GroPEtn on the 2-[3-(diphenylhexatrienyl)propanoyl]-1-hexadecanoyl-sn-glycero-3- phosphocholine-(DphPamGroPCho)-dependent quenching of the tryptophan fluorescence of purified NADPH-cytochrome reductase. The results demonstrate that the addition of acyl2GroPSer or acyl2GroPIns affects the DphPamGroPCho-dependent quenching of the tryptophan fluorescence in a manner significantly different from the addition of acyl2GroPCho or acyl2GroPEtn. The relatively larger DphPamGroPCho-induced quenching of the tryptophan fluorescence of NADPH-cytochrome reductase in the presence of acyl2GroPSer and acyl2GroPIns must result from a change in the conformation of NADPH-cytochrome reductase induced by the latter two lipids. Finally, the possible consequences of this special interaction of acyl2GroPSer and acyl2GroPIns with NADPH-cytochrome reductase on the kinetic characteristics of the cytochrome P450 system were studied using cytochrome-P450-IIB1-dependent O-dealkylation of pentoxyresorufin as the model reaction. These studies demonstrate that a 1:1 mixture of acyl2GroPCho and acyl2GroPSer results in a significantly higher apparent maximum rate (V) of O-dealkylation than a 1:1 mixture of acyl2GroPCho and acyl2ProPEtn or acyl2GroPCho alone. This increase in the apparent V can be ascribed to an acyl2GroPSer-dependent improvement of the interaction of NADPH-cytochrome reductase with cytochrome P450. This improvement of the interaction of the proteins cannot, however, be exclusively ascribed to the negative charge of acyl2GroPSer, since the other negatively charged phospholipid investigated, namely acyl2GroPIns, resulted in a significant decrease in the apparent V.(ABSTRACT TRUNCATED AT 400 WORDS)

Biotransformation of methylxanthines in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms. Allocation of metabolic pathways to isoforms and inhibitory effects of quinolones

V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2, P450IIB1, human P450IA2, and rat liver epithelial cells expressing murine P450IA2 were used to allocate metabolic pathways of methylxanthines to specific isoforms and to test the suitability of such cell lines for investigations on drug interactions occurring at the cytochrome expressed. The cell lines were exposed to caffeine and/or theophylline and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2, resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-demethylation and 1-demethylation of caffeine to a similar extent. The results support the hypothesis that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity. Addition of the quinolone antibiotic agents pipemidic acid or pefloxacin, both known to inhibit caffeine metabolism in vivo and in human liver microsomes, reduced formation rates of all metabolites of caffeine in cells expressing rat and human P450IA2. Theophylline was mainly metabolized via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or human P450IA2, or rat P450IA1.

A Microassay for Measuring Cytochrome P450IA1 and Cytochrome P450IIB1 Activities in Intact Human and Rat Hepatocytes Cultured on 96-Well Plates

This study deals with the development of a sensitive and simple microassay for measuring cytochrome P4501A1 and P45011B1 activities in intact human and rat hepatocytes. The method is based on the metabolism by cells cultured in microwells of appropriate substrates at noncytotoxic concentrations (8 μM 7-ethoxy-resorufin and 15 μM 7-pentoxyresorufin). After incubation of the probes with the cells, the dealkylated resorufin formed and released into culture medium was quantified. To ensure the hydrolysis of the resorufin conjugates eventually formed, culture supernatants were incubated in the microwells with β-glucuronidase and arylsulfatase. The fluorescence was then read using a microplate fluorescence reader. A high correlation between the monooxygenase activity measured by this procedure and that measured by conventional procedures in the microsomal fraction of the same cells was found. The major advantages of this method are: (1) the small number of cells required; (2) a drastic reduction in assay time; (3) that the assay is performed in intact cells; and (4) the possibility of performing repeated assays with the same cell monolayer over a period of several days since no injury to cells is detectable during the assay. This method proved to be very convenient for studying cytochrome P450 induction by xenobiotics in primary cultures of human hepatocytes.

Kinetics of cytochromes P-450 IA1 and IIB1 in reconstituted systems with dilauroyl- and distearoyl-glycerophosphocholine.

In the present study the effect of changing the fatty acyl moiety of phosphatidylcholine from dilauroyl to distearoyl on the kinetic parameters of O-dealkylation of alkoxyresorufins and ethoxycoumarin dependent on reconstituted cytochromes P-450 IA1 and IIB1 has been investigated. The results demonstrate that (a) the maximum rate of O-dealkylation (V) for both P-450 enzymes was about two times higher in the L-alpha-dilauroyl-sn-glycero-3-phosphocholine (Lau2GroPCho) system and (b) changes in the fatty acyl moiety of phosphatidylcholine (acyl2GroPCho) from dilauroyl to distearoyl affected the apparent Km for the substrate (Kms) of P-450 IA1 and IIB1 in a different way. In addition, (c) the kinetic parameters appeared to be dependent on the acyl2GroPCho/P-450 ratio and a change in this ratio affected the kinetic parameters of P-450 IA1 and IIB1 in a different manner. From these last two observations it was concluded that the mechanism by which phospholipids influence P-450-IIB1-dependent O-dealkylation of ethoxycoumarin is different from that by which they influence P-450-IIB1-dependent O-dealkylation of this substrate. Furthermore, the results of the present study demonstrate that the increase in the rate of O-dealkylation of ethoxycoumarin, reported in the literature for reconstituted systems in the presence of Lau2GroPCho, results from an effect of Lau2GroPCho on both the Kms and the V. In a number of additional experiments possible mechanisms underlying the observed differential effect of Lau2GroPCho and Ste2GroPCho on the Kms and V of P-450 IA1 and IIB1 were investigated. This was done by studying the effect of the two acyl2GroPCho species on the kinetic parameters of some of the different steps of the P-450 cycle, namely substrate binding, oxygen binding and the rate of electron transfer. The results demonstrate an influence of Lau2GroPCho and Ste2GroPCho on (a) substrate binding to cytochrome P-450, (b) the affinity of cytochromes P-450 for NADPH-cytochrome reductase and thus on (c) the electron flow through the reconstituted system. Based on the results from these experiments it was concluded that the increased V of P-450 IA1 and IIB1 in the presence of Lau2GroPCho compared to the systems with Ste2GroPCho was at least in part due to an increased affinity of both P-450 enzymes for NADPH-cytochrome reductase in the presence of Lau2GroPCho compared to Ste2GroPCho.(ABSTRACT TRUNCATED AT 400 WORDS)

L-gulono-gamma-lactone oxidase is not induced in rats by xenobiotics stimulating L-ascorbic acid biosynthesis.

L-Ascorbic acid (AsA) is synthesized from D-glucose in rats; the terminal step of this synthetic pathway is catalyzed by L-gulono-gamma-lactone oxidase (GLO). In this study, we examined the effects of phenobarbital (PB) and 3-methylcholanthrene (MC), both of which are known to stimulate AsA biosynthesis in rats, on the hepatic levels of GLO activity, GLO mRNA, and AsA. Firstly, the existence of GLO mRNA was examined in the liver, kidney, lung, small intestine, spleen, testis, and prostate from a male rat; and GLO mRNA was found to be present only in the liver, in which GLO activity was also detected. The intraperitoneal injection with PB (100 mg/day/kg body weight, once a day for 2 days) or MC (20 mg/day/kg body weight, once) significantly elevated the hepatic level of AsA and the urinary excretion of AsA in rats (5-week-old males). The hepatic levels of cytochrome P-450IIB1 mRNA and cytochrome P-450IIB2 mRNA and those of cytochrome P-450IA1 mRNA and cytochrome P-450IA2 mRNA were also elevated in the rats treated with PB and MC, respectively, indicating a normal response of these animals to these compounds. However, the level of GLO mRNA and the activity of GLO in the liver tended to be slightly decreased by the administration of PB or MC, though the differences were not significant. Thus it is clear that the treatment with PB or MC stimulates the biosynthesis of AsA by increasing the activity of some enzyme(s) participating in the synthesis prior to GLO.

Selective induction of murine liver cytochrome p450 IIB1 by Halogenated hydrocarbons

Thirteen halogenated hydrocarbons, 1,1,1,2‐tetrachloroethane (1,1,1,2‐TCE), 1,1,2,2‐tetrachloroethane (1,1,2,2‐TCE), pentachloroethane (PCE), 1,2‐dibromoethane (DBE), 1,1,2,2‐tetrabromoethane (1,1,2,2‐TBE), iodoethane (IE), 1,1‐dichloroethane (1,1‐DCE), 1,1,1‐trichloroethane (1,1,1‐TCE), 1,1,2‐trichloro‐ethane (1,1,2‐TCE), dichloromethane (DCM), 1,1‐dichloroethylene (1,1‐DCEE) and trans 1,2‐dichloro‐ethylene (t1,2‐DCE) were evaluated for their ability to modulate specific cytochrome P450 (cyt P450) isoforms following in vivo administration in male mice. Each chemical was administered (i.p.) in equivalent doses (50, 25, 12.5 and 6.25% of the respective LD50). Total cyt P450 and selected microsomal monoxygenase activities towards different P450 isoenzymes (classes IA1, UBI, IIE1 and IIIA P450) were examined in hepatic microsomes. With the exception of 1,1‐DCE, all considered hydrocarbons were inducers of Cyp2B1 genes, as exemplified by the enhancement of pentoxyresorufin O‐dealkylation. A 1.5‐(DBE, DCM) to ...

Differential cumene hydroperoxide sensitivity of cytochrome P-450 enzymes IA1 and IIB1 determined by their way of membrane incorporation

The cytochrome P-450-dependent O-dealkylation of all alkoxyresorufins was used to study the effect of cumene hydroperoxide on cytochrome P-450 IIB1 and IA1 in microsomal and reconstituted systems. In liver microsomal systems from respectively phenobarbital and 3-methylcholanthrene pretreated male Wistar rats, cytochrome P-450 IIB1-dependent pentoxyresorufin- O-dealkylation appeared to be more sensitive to cumene hydroperoxide treatment than cytochrome P-450 IA1-dependent ethoxyresorufin- O-dealkylation. This phenomenon was also observed when the cumene hydroperoxide sensitivity of P-450 IIB1 and IA1 was studied in an isosafrole pretreated rat liver microsomal system. The decrease in alkoxy- O-dealkylating activities appeared to proceed by destruction of the cytochrome P-450 component of the enzyme system. Purification and reconstitution of the enzyme system components in a system in which the isolated proteins were not incorporated into a membrane resulted in the disappearance of the difference in sensitivity between the two P-450 enzymes. However, in a reconstituted system with membrane incorporated proteins, again cytochrome P-450 IIB1 expressed a higher sensitivity towards cumene hydroperoxide than cytochrome P-450 IA1. From this it was concluded tha the differential cumene hydroperoxide sensitivity of cytochrome P-450 IIB1 and IA1 is not caused by an intrinsic difference in their sensitivity but by a differential effect of membrane incorporated on their cumene hydropeoxide sensitivity.

Structure-activity relationship of kynurenate analogues for interaction with the glycine site on the n-methyl-o-aspartate (NMDA) receptor

Propofol, a widely used anesthetic drug, is known to inhibit cytochrome P450 activities in vitro. The goal of this study was to compare cytochrome P450 activities in vitro and in vivo in presence of propofol. In vitro (liver microsomes and freshly isolated hepatocytes), cytochrome P450 IA2 and IIB1 activities were measured as the production of resorufin from ethoxy- and pentoxyresorufin, respectively, in presence of various concentrations of propofol. In vivo, cytochrome P450 activities were assessed as the production of 13CO2 from 13C-aminopyrine injected intravenously, during acute administration or after chronic treatment. In vitro results confirmed the dose-dependent inhibitory effect of propofol on cytochrome P450 activities, both on liver microsomes and isolated hepatocytes. In vivo, the acute administration of propofol induced a significant decrease of 13C-aminopyrine metabolism. Chronic treatment with propofol induced a significant inhibition of 13C-aminopyrine metabolism only after 2 weeks. No enzyme induction was observed. In conclusion, our results demonstrate that propofol inhibits cytochrome P450 also in vivo. Drug interactions may thus occur during propofol administration.

Selective induction of murine cytochromes P450 IIB1 and IIEI by fungicides

Selective induction of murine cytochromes P450 IIB1 and IIEI by fungicides