Rat Hepatic Microsomal Cytochrome Research Articles

The relationship between the binding of 2- n-alkylbenzimidazoles to rat hepatic microsomal cytochrome P-450 and the inhibition of monooxygenation

The binding of a homologous series of 2-n-alkylbenzimidazoles to rat hepatic microsomal cytochrome P-450 has been examined. Type I, Type RI and mixed Type I/RI spectra were observed with control, phenobarbitone or 20-methylcholanthrene-induced microsomal preparations. In general short chain (C1-C4) substituted compounds elicited Type RI spectra, whereas C5-C9 substituted benzimidazoles gave rise to Type RI/I or Type I spectra. The type of binding spectrum observed was dependent upon the substrate concentration, the source of microsomes and the length of the substituent alkyl chain. As the lipophilic character of the substituent was increased a corresponding increase in Type I nature was noted. However, an optimal chain length of C7-C8 carbon atoms was observed for Type I binding; compounds with longer side chains showed a decreased affinity for the Type I site. The apparent spectral binding constants (Ks values) for the Type I site (but not the Type RI site) were closely associated with the Ki and I50 values for the inhibition of cytochrome P-450-dependent monooxygenation. From their inhibition properties it seems that even the short chain (C1-C4) substituted benzimidazoles also bind toi the Type I site and thus compete for the substrate binding site of cytochrome P-450.

Purification, characterization and regulation of five rat hepatic microsomal cytochrome P-450 isozymes.

1. Five hepatic microsomal cytochrome P-450 isozymes (cytochromes P-450a, P-450b, P-450c, P-450d, P-450e) have been purified to apparent homogeneity from immature male rats treated with various xenobiotics. 2. The unique electrophoretic properties, substrate specificities and spectral characteristics of these haemoproteins have been compared and contrasted. 3. Structural studies of these cytochrome P-450 isozymes have included NH2-terminal amino acid sequence analyses, as well as electrophoretic profiles of limited proteolytic digests and cyanogen bromide fragments of the haemoproteins. 4. Specific antibodies have been prepared against four of the isozymes and used to evaluate immunochemical relationships among these cytochrome P-450s by Ouchterlony double-diffusion analyses. 5. The levels of some of these cytochrome P-450 isozymes have been quantified immunologically in hepatic microsomal preparations from untreated rats and following induction by phenobarbital, 3-methylcholanthrene or Aroclor 1254. 6. Antibodies directed against cytochromes P-450a and P-450b have been used to establish the presence of more than one 7 alpha- and 16 alpha-testosterone hydroxylase in rat hepatic microsomes. The relative contributions of cytochromes P-450c and P-450d to the overall microsomal metabolism of benzo[a]pyrene have been evaluated using antibodies to these haemoproteins.

Metyrapone - reduced cytochrome P-450 complex: A specific method for the determination of the phenobarbital inducible form of rat hepatic microsomal cytochrome P-450

Abstract Using homogeneous cytochrome P-450, we have shown that the well-known metyrapone-dithionite reduced cytochrome P-450 complex is specific for the cytochrome P-450b induced by phenobarbital. A linear relationship was observed between the absorbance of metyrapone-reduced cytochrome P-450 complex and the one of CO-reduced cytochrome P-450 complex, the usual method for the determination of cytochrome P-450. A method has been proposed for the specific determination of the cytochrome P-450b.

Interaction of isosafrole in vivo with rat hepatic microsomal cytochrome P-450 following treatment with phenobarbitone or 20-methylcholanthrene

Interaction of isosafrole in vivo with rat hepatic microsomal cytochrome P-450 following treatment with phenobarbitone or 20-methylcholanthrene

Potentiation of fluroxene (2,2,2-trifluoroethyl vinyl ether) toxicity with polychlorinated biphenyls

The effects of induction of rat hepatic microsomal cytochrome P-450 by commercial mixtures of polychlorinated biphenyls (PCBs) (100 mg/kg/day for 3 days) in potentiating the toxicity of the anesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) have been determined. The PCB mixtures used were Aroclors 1221, 1016, 1254, and 1260. All Aroclors significantly induced cytochrome P-450 relative to controls (1.4–2.1), and the more highly chlorinated PCB mixtures produced the greatest induction. The cytochrome P-450 composition of the Aroclor-induced microsomes was probed using R warfarin metabolism. Aroclor 1254-induced microsomes comprised mainly cytochrome P-448 of the 3-methylcholanthrene-inducible type, and Aroclor 1260-induced microsomes comprised mainly cytochrome P-450 of the phenobarbital-inducible type, although each contained lesser quantities of the other forms. Only Aroclor 1254 and 1260 induction produced death in rats (12/20 and 11/13 respectively) following ip administration of fluroxene (2.5 g/kg body wt). Fluroxene potentiated the destruction of cytochrome P-450 in vivo and this destruction was enhanced by Aroclor induction. Cytochrome P-448 suffered the greatest destruction in Aroclor 1221- and 1254-induced microsomes, and cytochrome P-450 was most extensively destroyed in Aroclor 1260-induced microsomes. The Aroclor 1254-induced cytochrome P-450 concentration did not return to the corresponding control value 24 hr after clearance of the fluroxene dose which implies that fluroxene metabolism interferes with the biosynthesis of cytochrome P-450. It is concluded that some Aroclors, through induction of cytochrome P-450 of the phenobarbital-inducible type, increase the rates of metabolism of fluroxene, which results in the production of toxic metabolites leading to liver necrosis, destruction of cytochrome P-450, and death.

Inhibition of rat hepatic microsomal cytochrome P-450 system by cobaltous chloride and reversal of inhibition by iron in vivo.

The effects of preexposure of rats to cobaltous chloride (CO) mixed in iron-sufficient (I.S) and -deficient (I.D) diets on hepatic microsomal electron transfer system was investigated. Male Sprague-Dawley rats were fed for 4 weeks on I.D diets mixed with 0, 100 and 200 ppm CO. At the end of 4 weeks three rats from each group were transferred to I.S diets mixed with the same amount of CO. Liver microsomal NADPH - Cytochrome C reductase, NADPH - dehydrogenase, cytochrome P-450 and aniline binding were determined in both batches of rats. The rats receiving CO in the I.D diets showed a 35, 60, 75 and 40% decrease in NADPH - Cyt. C reductase, dehydrogenase, Cyt. P-450 and aniline binding respectively. The rats transferred from I.D diet to I.S diet showed a complete recovery of the inhibition of the microsomal electron transfer system. The rats receiving 200 ppm mixed with I.S diets did not show any changes in any biochemical parameter measured.

Rat hepatic microsomal cytochrome(s) P-450 induced by polybrominated biphenyls.

Rat hepatic microsomal cytochrome(s) P-450 induced by polybrominated biphenyls.

Interaction of pargyline with rat hepatic microsomes.

When administered by intraperitoneal injection daily for 3, 7 or 14 days, pargyline (75 mg/kg) significantly reduced rat hepatic microsomal ethylmorphine N-demethylase activity and cytochrome P-450 content. Injection of a lower dose of pargyline (15 mg/kg) failed to alter significantly either ethylmorphine N-demethylase activity or cytochrome P-450 content. Studies performed in vitro reveal that pargyline is metabolized to at least three compounds by rat hepatic microsomes. Thin-layer chromatography and other analyses suggest that one metabolite is an N-demethylated form, norpargyline.

Ligand binding to human placental cytochrome P-450: Interaction of steroids and heme-binding ligands

Abstract Type II ligands such as n -octylamine, 1-(2-isopropylphenyl)-imidazole, cyanophenylimidazole, aniline, ethylisocyanide, metyrapone, nicotinamide and aminoglutethimide bind to microsomal cytochrome P-450 from human placenta to give rise to optical difference spectra which are similar in form to those observed with rat hepatic microsomal cytochrome P-450 but which are more intense relative to the CO-spectrum. Addition of the type I ligands androstenedione or 19-norandrostenedione cause changes in both magnitude and λ max of the difference spectra produced by type II ligands. These changes appear to be due to the displacement of the type II ligand and are not shown with rat liver cytochrome P-450. Differences between the two steroids are related to their binding constants. Recent studies with π-acceptor ligands such as tin and germanium dihalides are extended to placental cytochrome P-450 with which CO-like spectra may be formed. Type II ligands, which bind to the heme iron, are inhibitors of placental aromatase activity while inhibitors that require metabolic activation, such as the methylenedioxyphenyl compounds, are not.

The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 [formula omitted

In the presence of hepatic microsomes, vinyl chloride produces a ‘type I’ difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome b5 or NADPH-cytochrome c reductase in vitro.

Albumin and cytochrome P 450 binding characteristics of juvenile hormone and its analogs

Abstract Binding data were gathered for the cecropia juvenile hormone (methyl(E, E cis)-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate) and two of its analogs {isopropyl(2E, 4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate; (E)-4-[(6,7-epoxy-3,7-dimethyl-2-nonenyl)-oxyl]-1,2-(methylenedioxy)benzene} with bovine serum albumin and rat hepatic microsomal cytochrome P450. The proteins were found to bind the juvenile hormone and juvenile hormone analogs with affinity constants ranging from 105 to 106 M−1. Thermodynamic calculations suggest that the binding of all three compounds is electrostatic in nature and that the size of the ether and ester substituents can greatly influence the binding to proteins. The juvenile hormone and its analogs all formed spectrally apparent Type I complexes with oxidized cytochrome P450; one of the juvenile hormone analogs formed a spectrally observable product adduct with reduced cytochrome P450. The product complex may contribute many of the hormonal effects observed for this compound.

Temperature dependence of cytochrome P-450 reduction. A model for NADPH-cytochrome P-450 reductase:cytochrome P-450 interaction.

The NADPH-dependent reduction of rat hepatic microsomal cytochrome P-450 has been studied as a function of temperature. In the temperature range 4-37 degrees the reduction reaction was found to be biphasic and composed of two concurrent first order processes. This phenomenon was observed with microsomes from untreated and phenobarbital-induced animals in the presence or absence of exogenous Type I substrates. The amount of cytochrome P-450 reduced in the fast phase comprised approximately 70% of the total cytochrome P-450 at temperatures above 20 degrees. The temperature dependence of the fast phase was unusual for a membrane-bound enzyme system in that it lacked a discontinuity in the Arrhenius plot at a presumed phase transition temperature for the microsomal membrane. The slow phase of reduction behaved in a normal fashion for a membrane-bound enzyme system with a break in the Arrhenius plot at about 20 degrees. The data presented here combined with previous observations which include (a) the ratio of cytochrome P-450 to NADPH cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) is 20:1, (b) the catalytic portion of the reductase molecule probably protrudes above the surface of the membrane, and (c) the cytochrome P-450 molecules are presumably embedded in the membrane support the hypothesis that the hepatic microsomal drug-metabolizing system exists as clusters with most of the cytochrome P-450 molecules arranged about a central reductase molecule. This central flavoprotein reductase is able to randomly reduce those cytochrome P-450 molecules within the cluster without translational motion through the microsomal membrane. The slow phase of reduction represents the reduction of those molecules not directly associated with the clusters.

Interaction of volatile anesthetics with rat hepatic microsomal cytochrome P-450.

The addition of halothane, methoxyflurane, or enflurane to hepatic microsomes caused the appearance of a trough at 420 mμ and simultaneous appearance of an absorption band at 385 mμ (type I), indicative of an interaction between cytochrome P-450 and the anesthetics. Diethyl ether had no effect Halot