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E Hodgson

Publications and source records attributed to E Hodgson.

At least 73 records · Page 4Linked to original sources
PubMed1986

Interactions of diethylphenylphosphine with purified, reconstituted mouse liver cytochrome P-450 monooxygenase systems.

Purified mouse liver cytochrome P-450 reconstituted with purified NADPH-cytochrome P-450 reductase and phosphatidylcholine metabolized diethylphenylphosphine to diethylphenylphosphine oxide. NADPH was required for the reaction and the amount of oxide formed was time and cytochrome P-450 dependent. Purified phenobarbital-induced cytochrome P-450 produced more oxide per nmole enzyme than any of the purified uninduced cytochrome P-450s. the phosphine oxide was also formed in lesser amounts in incubation mixtures containing only NADPH-cytochrome P-450 reductase and NADPH. Diethylphenylphosphine bound to oxidized purified phenobarbital-induced cytochrome P-450 and uninduced cytochrome P-450 with Ks values of 16 microM and 11-18 microM respectively. Diethylphenylphosphine was also a competitive inhibitor of p-nitroanisole O-demethylation catalyzed by a reconstituted phenobarbital-induced cytochrome P-450-dependent monooxygenase system, with a Ki value of 5 microM. The phosphine oxide produced no observable optical difference spectrum with oxidized phenobarbital-induced cytochrome P-450 and caused no inhibition of p-nitroanisole O-demethylation.

Animals↗
PubMed1986

Cytochrome P-450 induction by 3-methylcholanthrene and its antagonism by 2,2-dimethyl-5-t-butyl-1,3-benzodioxole.

Previous studies in this laboratory have shown 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBBD) to antagonize 3-methylcholanthrene induction of cytochrome P-450 in Dub:ICR mice yet have no effect on phenobarbital induction. In the present experiments, C57BL/6 mice, an Ah responsive strain, produced a similar response under the same experimental conditions. The hypothesis that DBBD, although not a cytochrome P-450 inducer, competes with 3-methylcholanthrene for binding to the Ah receptor was tested. Using sucrose density gradients, the Ah receptor was measured in hepatic cytosol from Dub:ICR and C57BL/6 male mice. DBBD was unable to displace either 2,3,7,8-tetra-chlorodibenzo-p-dioxin or 3-methylcholanthrene from the Ah receptor, in vitro. However, in in vivo experiments, DBBD treatment of Dub:ICR mice caused Ah receptor depression at 6 and 24 hr with complete recovery in between, while 3-methylcholanthrene treatment caused a 2-fold Ah receptor reduction at 2 hr followed by complete recovery after 12 hr. When 3-methylcholanthrene and DBBD were coadministered, the depression of the Ah receptor was additive. DBBD-pretreated mice had a 2.25-fold reduction in Ah receptor level, effectively blocking the ability of 3-methylcholanthrene to increase the cytochrome P-450 content and either benzo[a]pyrene hydroxylase or ethoxyresorufin O-deethylase activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that 3-methylcholanthrene induction of cytochrome P-450 was inhibited by DBBD pretreatment. Hence, although DBBD does not displace 3-methylcholanthrene from the Ah receptor in vitro, it does antagonize 3-methylcholanthrene induction of cytochrome P-450 and also reduces the amount of available receptor in vivo. This interaction may be due either to antagonism or to downregulation of the Ah receptor.

Animals↗
PubMed1986

Formation of hydrogen peroxide and N-hydroxylated amines catalyzed by pulmonary flavin-containing monooxygenases in the presence of primary alkylamines.

In atypical reaction, incubation of purified rabbit pulmonary flavin-containing monooxygenase with certain primary alkylamines results in the oxidation of NADPH and the formation of hydrogen peroxide. In addition, significant amounts of N-hydroxylated primary amine are also generated, as determined by colorimetric assay and GC/MS analysis of n-octylamine metabolites. Similar reactions appear to be catalyzed by the mouse pulmonary enzyme. In contrast, incubation of primary alkylamines with hepatic flavin-containing monooxygenases from rabbit, mouse, or pig does not result in NADPH oxidation or metabolism. Another effect of primary alkylamines is marked activation of the mouse pulmonary and pig hepatic flavin-containing monooxygenases with some substrates. The structural requirements for primary alkylamines to elicit NADPH oxidation by the rabbit pulmonary enzyme or to activate the mouse pulmonary and pig hepatic enzymes are identical. This indicates that different flavin-containing monooxygenases probably have a conserved alkylamine-binding site of defined specificity. In the case of the rabbit pulmonary enzyme, this binding may occur very close to or at the catalytic site resulting in some N-hydroxylation of the alkylamine.

Amines↗
PubMed1986

Induction of cytochrome P-450 in congenic C57BL/6J mice by isosafrole: lack of correlation with the Ah locus.

Isosafrole induction of cytochrome P-450 was compared in congenic strains of C57BL/6J mice, one of which expresses normal levels of the Ah receptor [B6(Ahb)], and another that does not contain a measurable receptor concentration [B6(Ahd)]. Using sucrose gradient analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding, an Ah receptor concentration of 69.1 +/- 3.8 fmol/mg protein was measured in the hepatic cytosol from B6(Ahb) mice, while no receptor could be detected in the cytosol from B6(Ahd) mice. Isosafrole treatment (75 mg/kg X 3 days) increased the total hepatic microsomal cytochrome P-450 content to the same extent in the two congenic strains. The level of microsomal monooxygenase induction in the isosafrole-treated B6(Ahd) mice was greater than that of B6(Ahb) mice for ethylmorphine N-demethylase and isosafrole metabolite-complex formation, the latter a measure of cytochrome P2-450. In the case of 7-ethoxycoumarin O-deethylase only the isosafrole-treated B6(Ahd) mice had elevated microsomal activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) also revealed a similar induction pattern for the two congenic strains, following isosafrole treatment. Thus, the isosafrole treated B6(Ahd) mice produced an equivalent or slightly larger induction of cytochrome P-450 than the B6(Ahb) mice, suggesting that there is no direct role for the Ah receptor in the regulation of these cytochrome P-450 monooxygenase activities by isosafrole.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1985

Metabolism of phosphorus-containing compounds by pig liver microsomal FAD-containing monooxygenase.

Oxidative desulfuration of phosphonate insecticides such as fonofos (S-phenyl ethyl ethylphosphonodithioate) and its analogs is catalyzed by pig liver microsomal FAD-containing monooxygenase, although desulfuration of phosphorodithioates, such as parathion, is not. Substitution of an alkyl group for the remaining alkoxy group, as in S-phenyl diethylphosphinodithioate, did not increase its oxidation rate. Diethylphenylphosphine sulfide, containing 3 phosphorus-carbon bonds, was actually a poorer substrate than fonofos. Replacement of the S-phenyl group of fonofos with an O-phenyl group increased the Km value above the solubility limit. Trivalent phosphorus-containing compounds were also excellent substrates for this enzyme. Diethylphenylphosphine had a Km value lower than 2.5 microM. Diethyl phenylphosphonite also appeared to be an excellent substrate but its rapid nonenzymatic hydrolysis and/or oxidation precluded accurate Km determinations. Stoichiometry studies with diethylphenylphosphine and its sulfide showed that O2 and NADPH consumption were approximately equal to the substrate consumed. The major metabolite of both diethylphenylphosphine and its sulfide was the phosphine oxide. These results show that microsomal FAD-containing monooxygenase of pig liver has activity as a phosphorus-oxidase, in addition to its well characterized nitrogen- and sulfur-oxidase roles.

Animals↗
PubMed1985

Identification of distinct hepatic and pulmonary forms of microsomal flavin-containing monooxygenase in the mouse and rabbit.

The flavin-containing monooxygenase has been purified from mouse and rabbit lung microsomes and shown to be distinct from the flavin-containing monooxygenase found in the liver of the same species. The mouse and rabbit lung monooxygenases have a unique ability to N-oxidize the primary aliphatic amine, n-octylamine, commonly included in microsomal incubations to inhibit cytochrome P-450. In the mouse lung, this compound not only serves as a substrate but is also a positive effector of metabolism. The mouse and rabbit lung enzymes have unusual pH optimum, near 9.8, compared to the liver enzymes which have peaks near pH 8.8. Using antibodies raised in goats, Ouchterlony immunodiffusion analysis indicates that the liver and lung proteins are immunochemically dissimilar.

Animals↗
PubMed1985

Catalytic activity and substrate specificity of the flavin-containing monooxygenase in microsomal systems: characterization of the hepatic, pulmonary and renal enzymes of the mouse, rabbit, and rat.

Inhibitory antibodies against NADPH-cytochrome P-450 reductase, detergent solubilization to dissociate functional interaction between the reductase and cytochrome P-450, and selective trypsin degradation have been used to characterize flavin-containing monooxygenase activity in microsomes from different tissues and species. A comparison of assay methods is reported. The native microsome-bound flavin-containing monooxygenase of mouse, rabbit, and rat liver, lung, and kidney can metabolize compounds containing thiol, sulfide, thioamide, secondary and tertiary amine, hydrazine, and phosphine substituents. Therefore, this enzyme from these common experimental animals has catalytic capabilities similar to those of the well-characterized porcine liver enzyme. True allosteric activation by n-octylamine does not appear to be a property of either the mouse, rabbit, or rat liver enzymes, but is a property of the pig liver and mouse lung enzymes. The microsomal pulmonary flavin-containing monooxygenase of the rabbit has some unique substrate preferences which differ from the mouse lung enzyme. Both the rabbit and mouse pulmonary enzymes have recently been shown to be distinct enzyme forms. However, the rat pulmonary flavin-containing monooxygenase appears to be catalytically identical to the rat liver enzyme, and does not have any of the unusual catalytic properties of either the rabbit or mouse lung enzymes. Enzyme activity of mouse, rabbit, and rat kidney microsomes is qualitatively similar to the hepatic activities. Substrates which saturate the microsome-bound flavin-containing monooxygenase at 1.0 mM, including thiourea, thioacetamide, methimazole, cysteamine, and thiobenzamide, are metabolized at common maximal velocities. This suggests that the kinetic mechanism of the native enzyme is similar to that established for the isolated porcine liver enzyme in that the rate-limiting step of catalysis occurs after substrate binding, and that all substrates capable of saturating the microsomal enzyme should be metabolized at a common maximal velocity.

Animals↗
PubMed1985

Induction of specific cytochrome P-450 isozymes by methylenedioxyphenyl compounds and antagonism by 3-methylcholanthrene.

Two methylenedioxyphenyl compounds, isosafrole (5-propenyl-1,3-benzodioxole) and an analog, 5-t-butyl-1,3-benzodioxole (BD), differ markedly as inducers of cytochrome P-450 isozymes in rat liver microsomes. Isosafrole is a mixed-type inducer, inducing P-450b, P-450c, and P-450d. In contrast, BD is a phenobarbital-type inducer, increasing P-450b, but producing little or no increase in P-450c or P-450d. Similarly, isosafrole increases the amount of translatable mRNA for P-450b, c and d, while BD induces only the mRNA for P-450b. Dimethylation of the methylene bridge carbon of BD to give 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBD) blocks the formation of NADPH-reduced Type III metabolite-P-450 complexes in vitro, and diminishes but does not abolish the ability of the compound to induce P-450b. Western blots of microsomes from isosafrole and BD-treated rat livers confirm that in contrast to isosafrole, BD does not induce P-450d or P-450c. However, the antibody to P-450d recognizes two new polypeptides (approximately 50K Mr) from sodium dodecyl sulfate-polyacrylamide gels of liver microsomes from BD-treated rats. These polypeptides are not observed in control, isosafrole, 3-methylcholanthrene (3-MC), or DBD-treated rats. They are intensified by coadministration of 3-MC with BD and may represent either modified isozyme-metabolite adducts or degradation products of P-450d. However, the polypeptides could not be generated in vitro by addition of BD to 3-MC-induced microsomes with NADPH under conditions which produced spectral metabolite complexes, or in a reconstituted system with P-450d. The two methylenedioxyphenyl compounds do not form stable metabolite complexes with the same P-450 isozymes. BD formed distinct spectral metabolite complexes in vitro with both P-450b and P-450c but not with P-450d in a reconstituted system. In contrast, isosafrole forms metabolite complexes with all three isozymes. Coadministration of 3-MC with BD blocked induction of P-450b by 80% and produced a similar repression of its translatable mRNA. This finding indicates that 3-MC type inducers not only induce certain cytochrome P-450 isozymes, but also repress synthesis of other isozymes.

Animals↗
PubMed1985

Partial purification and characterization of cytochrome P-450 from human placenta.

Two isozymes of cytochrome P-450 were partially purified to specific contents of 7.0 and 0.5 nmol/mg of protein, respectively, from placenta of non-smoking women by chromatography on octyl Sepharose, hydroxylapatite, DEAE-cellulose and CM-cellulose. NADPH-cytochrome P-450 reductase was purified from phenobarbital-induced mouse liver and from human placenta and was combined with cytochrome P-450 and dilauroylphosphatidylcholine to reconstitute the cytochrome P-450 monooxygenase system. Substrates investigated were benzo[a]pyrene, 7-ethoxycoumarin and delta 4-androstene-3,17-dione.

Androstenedione↗
PubMed1985

The binding of isocyanides to cytochrome P-450 from mouse hepatic microsomes.

The spectral interactions of a number of isocyanides with cytochrome P-450 were investigated. An interaction between the hydrophobic nature of the side chain and the spectral interaction was apparent. The concentration of the isocyanide affected the stability of the complex. Phenyl isocyanide dichloride was determined to be an acceptable replacement for ethyl isocyanide for the characterization of cytochrome P-450.

Animals↗
PubMed1985

Spectral interactions of piperonyl butoxide and isocyanides with purified hepatic cytochrome P-450 from uninduced mice.

The binding of isocyanides and the metabolites of piperonyl butoxide (PBO) to reduced cytochrome P-450 in intact microsomes gives rise to the type III optical difference spectrum which is characterized by two pH dependent peaks in the Soret region. Each of the purified cytochrome P-450 isozymes (A1, B1, B2, B3) metabolized PBO and produced a spectrum in the Soret region. Only the A1 fraction produced the pH dependent type III spectrum. The B1 fraction produced a spectrum with only one peak at 430 nm while the spectrum produced by both the B2 and B3 fractions contained only the 455 nm peak. Each of the isozymes produce pH dependent type III spectra with both ethyl isocyanide and phenyl isocyanide dichloride.

Animals↗
PubMed1984

2,2-Dimethyl-5-t-butyl-1,3-benzodioxole: an unusual inducer of microsomal enzymes.

Our previous studies have shown that 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBBD), a methylenedioxyphenyl (MDP) analog in which the methylene hydrogens have been replaced by methyl groups, does not form an inhibitory complex with cytochrome P-450 nor induce this cytochrome. However, in the present experiments, DBBD-treated male Dub:ICR mice showed an increase in NADPH-dependent cytochrome c (P-450) reductase and epoxide hydrolase activity. This separation of cytochrome P-450 induction from the induction of epoxide hydrolase and NADPH-dependent cytochrome c (P-450) reductase appears to be unique among inducers of xenobiotic metabolizing enzymes. In similar experiments, mice were treated with phenobarbital + DBBD or 3-methylcholanthrene + DBBD and the following parameters were measured: cytochrome P-450 content; NADPH-dependent reduction of cytochrome c; ethylmorphine and benzphetamine N-demethylase; 7-ethoxycoumarin O-deethylase; benzo[a]pyrene hydroxylase; and ethoxyresorufin O-deethylase. The microsomal proteins were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Phenobarbital + DBBD treatment gave results which did not differ significantly from those obtained with phenobarbital alone. In contrast, cytochrome P-450 content and benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase activities were less in mice treated with 3-methylcholanthrene + DBBD than in animals treated with 3-methylcholanthrene alone. SDS-PAGE confirmed that induction of cytochrome P-450 by 3-methylcholanthrene was reduced by DBBD, suggesting that the latter compound may be an antagonist to the Ah cytosolic receptor.

Animals↗
PubMed1984

Characterization of the purified microsomal FAD-containing monooxygenase from mouse and pig liver.

The FAD-containing monooxygenase (FMO) has been purified from both mouse and pig liver microsomes by similar purification procedures. Characterization of the enzyme from these two sources has revealed significant differences in catalytic and immunological properties. The pH optimum of mouse FMO is slightly higher than that of pig FMO (9.2 vs. 8.7) and, while pig FMO is activated 2-fold by n-octylamine, mouse FMO is activated less than 20%. Compounds, including primary, secondary and tertiary amines, sulfides, sulfoxides, thiols, thioureas and mercaptoimidazoles were tested as substrates for both the mouse and pig liver FMO. Km- and Vmax-values were determined for substrates representative of each of these groups. In general, the mouse FMO had higher Km-values for all of the amines and disulfides tested. Mouse FMO had Km-values similar to those of pig FMO for sulfides, mercaptoimidazoles, thioureas, thiobenzamide and cysteamine. Vmax-values for mouse FMO with most substrates was approximately equal, indicating that as with pig FMO, breakdown of the hydroxyflavin is the rate limiting step in the reaction mechanism. Either NADPH or NADH will serve as an electron donor for FMO, however, NADPH is the preferred donor. Pig and mouse FMOs have similar affinity for NADPH (Km = 0.97 and 1.1 microM, respectively) and for NADH (Km = 48 and 73 microM, respectively). An antibody, prepared by immunizing rabbits with purified pig liver FMO, reacts with purified pig liver FMO but not with mouse liver FMO, indicating structural differences between these two enzymes. This antibody inhibited pig FMO activity up to 60%.

Animals↗
PubMed1984

Biochemical mechanisms of resistance to insecticides.

The principal biochemical mechanisms of resistance to insecticides involve either modified, less sensitive cholinesterase, esterase action, glutathione S-transferase action or cytochrome P-450-dependent monooxygenation. Both quantitative and qualitative differences in cytochrome P-450 isozymes are under genetic control and both are related to resistance. Recent characterization studies involving ligand binding and multiplicity of isozymes in Musca domestica (the housefly) are discussed in relation to resistance. The recent demonstration that multiple isozymes of glutathione S-transferase exist in susceptible and resistant insects is of interest, and some re-examination of their role in the mechanism of resistance is required. Esterases are a heterogeneous group of enzymes whose role in resistance has often been suggested but seldom rigorously defined. Purification studies in the green rice leafhopper, Nephotettix cincticeps, have involved an enzyme with carboxylesterase, phosphotriesterase and pyrethroid esterase activities. A similar enzyme, but without pyrethroid esterase activity, is also found in the housefly. In resistance such enzymes may serve either to catalyse hydrolysis or as binding proteins. It has been suggested, from time to time, that regulator genes, enzyme induction and gene magnification all play a part in controlling biochemical mechanisms of resistance, although clearly defined evidence has not always been brought forward. These hypotheses are re-examined.

Acetylcholinesterase↗
PubMed1984

Purification of the flavin-containing monooxygenase from mouse and pig liver microsomes.

The microsomal flavin-containing monooxygenase has been purified from mouse and pig liver utilizing Cibacron-Blue Sepharose, Procion-Red agarose, and 2'5'-ADP Sepharose. The enzymes had a final specific activity of 1200 and 954 nmol/min/mg protein from mouse and pig liver respectively. The enzyme from both mouse and pig liver displayed typical flavoprotein spectra and appeared homogeneous by denaturing polyacrylamide gel electrophoresis.

Animals↗
PubMed1984

The metabolism of insecticides: the role of monooxygenase enzymes.

In summary, it can be said that advances have been made in understanding the range and significance of monooxygenase reactions involving insecticides in both target and non-target species and that some specific aspects emerge that should command the attention of insecticide toxicologists and others in the immediate future. The role of co-oxidation of insecticides during prostaglandin synthesis has just emerged as an issue but, if we can extrapolate from other xenobiotics (126), we can predict with some confidence that it will assume major proportions. It is also apparent that both activation and detoxication reactions are catalyzed by the FAD-containing monooxygenase and that many of these reactions were formerly attributed to the cytochrome P-450-dependent monooxygenase system. Since any particular substrate may be oxidized by either or both of these two routes, it is essential that studies be conducted to define their relative contributions to xenobiotic oxidation in microsomal preparations and ultimately in vivo. The area of interactions and the mechanisms behind them is also of importance, from the public health point of view in the case of multiple exposures, and from the practical viewpoint in the case of insecticide synergists. While the above aspects are not the only ones of future importance, they should contribute to the ultimate goal of insecticide toxicology: practical, safe use of chemicals for the control of insect pests.

Amines↗
PubMed1984

The measurement of FAD-containing mono-oxygenase activity in microsomes containing cytochrome P-450.

Antibodies to NADPH-cytochrome P-450 reductase have been used to essentially abolish the contribution of cytochrome P-450 to xenobiotic metabolism by mammalian microsomes. This permits the determination of the activity of the FAD-containing mono-oxygenase and the stoichiometry between substrate, O2 and NADPH, in the microsomal membrane, and in the absence of cytochrome P-450-dependent activity. FAD-containing mono-oxygenase oxidation rates were determined for sulphur- and nitrogen-containing substrates, including: thiols; sulphides; thioamides; primary, secondary and tertiary amines; hydrazines. Although the enzyme in mouse, rabbit, rat and pig microsomes displays similar substrate specificity, some catalytic characteristics are different between species and tissues.

Animals↗