Induction of the mixed-function oxidase system in the liver of the barn owl Tyto alba by PCBs

Injection of 30 mg/kg body wt of polychlorinated biphenyl (Aroclor 1254) into liver parenchymal tissue of nestling and adult barn owls Tyto alba resulted in increases in the level of cytochrome P-450. Concomitantly, there were increases in catalytic activity of the microsomal enzyme system as measured by aldrin epoxidation and aminopyrine N-demethylation. However, the ratio $\text{455}\text{430}\text{nm}$ in the ethylisocyanide-difference spectrum remained unchanged. Of particular interest is the sudden drop in the level and catalytic activity of cytochrome P-450 in nestling owls at age 40 days. Treatment with Aroclor 1254 produced small hemorrhages in the liver of nestling owls and the liver appeared much enlarged (hepatomegaly), indicating a toxic effect and resulting in little induction of microsomal enzymes. In adult owls the inductive effect was much greater. Aroclor 1254 produced a spectral shift in the cytochrome P-450-difference spectrum from 450 to 448 nm and in the ethylisocyanide-difference spectrum from 455 to 453 nm and from 430 to 427 nm.     

Hepatic microsomal oxidative metabolism of pesticides and other xenobiotics in pregnant CD1 mice

Pregnancy-related changes in oxidative metabolism of several xenobiotics including pesticides were examined in the hepatic microsomes of CD₁ mice. The effect of pregnancy on hepatic microsomal cytochrome P-450-catalyzed substrate oxidation was found to be dependent upon the type of reaction examined. Not all substrates undergoing the same reaction showed identical changes during pregnancy. Those enzyme activities which exhibited a decline in specific activity during pregnancy generally exhibited no change in total hepatic capacity. Enzymes posting no change in specific activity throughout gestation generally showed large increases in total hepatic activity. Phorate S-oxidation was catalyzed by both microsomal flavin-containing monooxygenase (MFMO) and cytochrome P-450. Moreover, there was no pregnancy-related change in either MFMO or total enzymatic (MFMO plus cytochrome P-450) phorate S-oxidation.     

Prochloraz,a potent inducer of the microsomal cytochrome P-450 system

Prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-imidazole-1-carboxamide), a recently developed agricultural fungicide, is a potent inducer of microsomal enzymes. Rats fed 7 days with a prochloraz-contaminated diet (2500 ppm) showed an increase in hepatic cytochrome P-450, cytochrome b₅, and microsomal protein level; aniline hydroxylase, 7-ethoxycoumarin dealkylase, 7-ethoxyresorufin dealkylase, NADPH-cytochrome c reductase, and epoxide hydrolase were significantly induced. At a lower dose (100 ppm), only an increase in cytochrome P-450 and 7-ethoxyresorufin dealkylase was noticed. As shown with aniline hydroxylase and 7-ethoxycoumarin dealkylase, prochloraz is also a potent inhibitor of drug-metabolizing enzymes. The interaction of prochloraz with hepatic microsomal fraction from rat liver was also studied. Prochloraz binds to oxidized cytochrome P-450 to produce a type II spectral change; the compound also binds to reduced cytochrome P-450. The binding of some ligands (7-ethoxycoumarin, n-octylamine, aniline, and imidazole) to oxidized cytochrome P-450 was determined after induction by prochloraz. Japanese quails (Coturnix coturnix) fed 7 days with a prochloraz-contaminated diet (2000 ppm) showed a dramatic increase in liver weight (2.5-fold) and both hepatic and duodenal cytochrome P-450 (9- and 12-fold, respectively).     

Influences of sub-acute exposure to paraquat on cytochrome P450 3A2 expression in rat liver and lungs

This study was designed to investigate the possible effects of paraquat sub-acute poisoning on cytochrome P450 3A2 expression in the liver and lungs. Twenty adult male rats (150-200 g) were exposed either against saline normal as control group or various doses of paraquat (3.5, 7 and 10 mg/kg, s.c.) as test groups for 7 consecutive days. Paraquat-exposed animals showed loss of body weight and elevation in serum levels of alkaline phosphates and alanine aminotransferase in a dose-dependent fashion. Moreover, animals in the test groups demonstrated a significant (P < 0.05) increase of malondialdehyde content in both the liver and lung tissues. Ultimately, by using RT-PCR method it became clear that 7 days exposure to paraquat resulted in a total suppression of cytochrome P450 3A2 at the mRNA level in the lungs. By contrast, a considerable up regulation of the same gene occurred in the liver. This data suggest that paraquat not only affect the lungs as a main target tissue but also up regulates the predominant cytochrome P450 gene in the liver which may induce detoxification processes.     

Cytochrome P-450 in insects. 6. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase,and monooxygenase activities in susceptible and resistant strains of Musca domestica

Development and phenobarbital (PB) induction of microsomal cytochrome P-450, cytochrome P-450 reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of insecticide-susceptible (NAIDM) and -resistant (Rutgers) house flies. Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Untreated insects of both strains had comparable reductase levels, whereas cytochrome P-450 and associated monooxygenase activities were 1.5-fold or more higher in Rutgers. Maximum induction, as well as toxicity, occurred at a lower PB concentration in NAIDM than Rutgers. The drug caused consistently higher increases in enzymes and activities within 12 hr of starting treatment in both strains. When PB was withdrawn from treated flies (both strains) 48 hr after treatment began, specific activities (product min^?1 mg protein^?1) in all enzymes returned to control values in 24 hr while metabolic capacity (product min^?1 insect^?1) achieved control values within 48 hr. The changes in turnover numbers (pmol product min^?1 pmol P-450^?1), in conjunction with the differences in the monooxygenation of the four substrates, suggest that PB treatment induced both a quantitative and qualitative change in NAIDM monooxygenation but only a quantitative change in Rutgers monooxygenation.     

The role of tyrosinase in the inactivation of house fly microsomal mixed-function oxidases

The low mixed-function oxidase activity of house fly microsomes has been associated with low cytochrome P-450 content and NADPH-cytochrome c reductase activity. The microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity could be decreased by the addition of catechol and increased by the addition of cyanide to the homogenates. Similar results were obtained with rat liver microsomes treated with tyrosinase and catechol. During the inactivation of rat liver microsomal enzymes by tyrosinase and catechol, crosslinking of microsomal proteins occurred. These results suggest that the instability of house fly microsomal mixed-function oxidase may be due in part to the action of contaminating tyrosinase on endogenous substrates.     

Cytochrome P-450 in insects: 4. Reconstitution of cytochrome P-450-dependent monooxygenase activity in the house fly

Two cytochrome P-450-containing fractions were isolated from detergent-solubilized house fly microsomes by hydrophobic chromatography on a tryptamine-Sepharose gel. These fractions (designated P-450-1 and P-450-2) were distinctive in their spectral characteristics and in their profiles following electrophoresis in the presence of sodium dodecyl sulfate. Both fractions exhibited NADPH-dependent epoxidase activity when reconstituted with purified house fly cytochrome P-450 reductase and phospholipid. The aldrin epoxidase activity of fraction P-450-1 was twice that of P-450-2 even though heptachlor epoxidase activity of the fractions was equivalent. O-Demethylase activity with 7-methoxy-4-methylcoumarin was detectable only in the P-450-2 fraction.     

Polysubstrate monooxygenases (cytochrome P-450) in larvae of susceptible and resistant strains of house flies

The polysubstrate monooxygenases (PSMO or cytochrome P-450) of house fly larvae were studied at the mature larval or “clear gut” stage. Fat body and gut tissues were most efficient in the conversion of aldrin to dieldrin. Microsomal fractions of larval homogenates had the highest PSMO activities, with lower PSMO activities also found associated with mitochondrial fractions. Microsomes from Rutgers (resistant) larvae had higher levels of NADPH:cytochrome c reductase (2×), cytochrome P-450 (2×), aldrin (4×), and heptachlor (9×) epoxidases than microsomes from CSMA (susceptible) larvae. Cytochrome P-450 of Rutgers larvae had an absorption maximum at 449 nm, 2 nm lower than the cytochrome P-450 of CSMA larvae. n-Octylamine spectra showed that the level of high-spin cytochrome P-450 was higher in Rutgers larvae. NADPH:cytochrome c reductase, cytochrome P-450, and aldrin epoxidase were induced by phenobarbital, and Rutgers larvae were shown to be more sensitive to this inducer than CSMA larvae. Induction of larval PSMO by phenobarbital did not affect the expression or the inducibility of PSMO in adults.     

Cytochrome P-450 in insects. 7. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase,and monooxygenase activities in the black blow fly (Phormia regina,Meigen)

Development and phenobarbital (PB) induction of microsomal cytochrome P-450, NADPH-cytochrome c (P-450) reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of female black blow flies (Phormia regina, Meigen). Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Induction occurred in all enzymes, even at 0.005% PB, and was maximum at 0.15%. Dramatic induction of the O-demethylation of 7-methoxy-4-methylcoumarin was observed in flies dosed with the maximum concentration of the drug. This monooxygenase activity increased to nearly 1400 times the level in control flies, whereas the other O-demethylation (methoxyresorufin) and the two epoxidation reactions exhibited considerably less change. Induction of the structural enzymes of this enzyme system were 10-fold for cytochrome P-450 and 5-fold for NADPH-cytochrome c (P-450) reductase. These data suggest that PB induces several P-450's in the blow fly, particularly one bearing a high degree of specificity for 7-methoxy-4-methycoumarin.     

Cytochrome P-450 content and aldrin epoxidation to dieldrin in isolated rat hepatocytes

A rat hepatocyte suspension effectively epoxidized aldrin to dieldrin with a V_max of 7.19 mol/mol P-450/min and a K_m of 9.27 μM. Viability and metabolic activity were stable for 6 hr after isolation when cells were maintained at room temperature (20°C) with the gentle introduction of $\text{O}{}_2\text{CO}{}_2$ onto the surface of the suspension. The cytochrome P-450 content of the suspension was 303 pmol/10⁶ cells. Primary maintenance culture of the cells also epoxidized aldrin. During culture for 3 days, metabolic activity decreased slowly day by day. Metabolic activity of microsomal fraction from rat liver was also examined. Microsomes epoxidized aldrin with a V_max of 5.11 mol/mol P-450/min and a K_m of 1.64 μM. Significant loss of some subspecies of cytochrome P-450 during fractionation of liver homogenate was indicated.     

Albumin and cytochrome P450 binding characteristics of juvenile hormone and its analogs

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 10⁶M^?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.     

Cytochrome P-450 in insects: 1. Differences in the forms present in insecticide resistant and susceptible house flies

Soluble cytochrome P-450 prepared from the microsomal fraction of abdomen homogenates of an insecticide resistant strain (Rutgers) and a susceptible strain (NAIDM) of the house fly, Musca domestica L., was characterized by spectral and electrophoretic methods. Six chromatographically distinct fractions were obtained after chromatography on DEAE-cellulose and hydroxylapatite. Examination of the six fractions by difference spectrophotometry indicated that the wave lengths for maximum absorption of the cytochrome P-450-carbon monoxide complexes were at 450, 451, and 452 nm for the NAIDM fractions and at 449, 450, and 451 nm for the Rutgers fractions. The type II binding spectra of the cytochrome P-450 in each fraction were measured with n-octylamine. Several of these resembled spectra which, in studies of hepatic cytochrome P-450, have been shown to be due to the presence of the high spin form of this hemoprotein. Four of the fractions from the resistant strain were of this type compared to one from the susceptible strain. Electrophoresis experiments indicated that there were at least three hemoproteins in the 40,000–60,000 molecular weight range in the fractions from the resistant strain while four could be detected in those from the susceptible strain. The specific aldrin epoxidase activity of the most active Rutgers fractions was considerably higher than that of similar fractions from the NAIDM microsomes in reconstitution experiments.     

Components of the electron transport system in the microsomal mixed-function oxidase system in wild birds

Notable differences were found among six species of wild-caught birds in the levels of cytochrome P-450, cytochrome b₅, NADPH-cytochrome c reductase, and NADH-cytochrome c reductase. Ethyl isocyanide difference spectra showed significant variations among the species in peak height and in the ratios of the $\text{430}\text{455}\text{-}\text{nm}$ peaks. Substantial aldrin epoxidase activity was found in all species, and the amounts of dieldrin produced compared favorably with pigeon and rat liver microsomes. Higher content of cytochrome P-450 was not always accompanied by a similar rise in specific catalytic activity. Thus, no correlation could be established between these two parameters. Aldrin epoxidase activity with NADH as the sole electron donor was 25–49% as effective as with the NADPH-generating system. Addition of both NADH and NADPH-generating systems to the incubation mixture produced a synergistic effect with liver microsomes of two species but not with two other species. DDE and polychlorinated biphenyls residues were found in the heart tissue of all species examined, and this might indicate a possible inductive effect on the microsomal mixed-function oxidase system by environmental contaminants.     

Cytochrome P-450 in insects: 2. Multiple forms in the flesh fly (Sarcophaga bullata,Parker), and the blow fly (Phormia regina (Meigen))

Microsomes prepared from the abdomens of the flesh fly (Sarcophaga bullata, Parker) and the blow fly (Phormia regina (Meigen)) contain approximately one-fifth and one-eighth as much cytochrome P-450, respectively, as those prepared from house fly (Musca domestica, L.) abdomens. These values correlate well with the microsomal aldrin epoxidase activity of the three species and with their respective susceptibilities to the insecticide, propoxur. When the microsomes of the flesh fly and the blow fly are solubilized by treatment with deoxycholate and resolved by ion-exchange chromatography on DEAE-cellulose and hydroxylapatite, four chromatographically distinct fractions containing cytochrome P-450 are obtained. Spectrophotometric assays of the cytochrome P-450 in these fractions indicate purifications of two-to sixfold for the flesh fly hemoprotein and two-to eightfold for that of the blow fly. SDS-Polyacrylamide gel electrophoresis of the four column fractions from the flesh fly microsomes indicates that six hemoproteins in the 40,000–60,000 molecular weight range are present. In similar experiments with blow fly fractions containing approximately the same amount of cytochrome P-450 no high molecular weight hemoproteins could be detected. This result is interpreted, with other evidence, as an indication of the greater instability of the blow fly hemoprotein. The results indicate that multiple forms of cytochrome P-450 are present in both species but there is insufficient data on which to estimate the number of such forms.     

Studies on a fluorescent insect growth regulator metabolite complex with house-fly microsomal cytochrome P-450

The fluorescent insect growth regulator 5[[[5-(dimethylamino)-1-naphthalenyl]amino]-1,3-benzodioxole (DNSAB) forms a metabolite complex with house-fly microsomal cytochrome P-450. Formation of the metabolite complex is dependent on the presence of NADPH and O₂; NADH supports the reaction at a reduced rate. The presence of antibodies to house-fly cytochrome c (P-450) reductase in reaction mixtures inhibits the complex formation, indicating that the reductase is necessary for transfer of electrons from NADPH to cytochrome P-450 to complete the reaction. In the oxidized form, the metabolite complex has a single absorbance maximum at 431 nm, whereas the reduced form has two absorbance maxima at 426 (major) and 455 nm (minor). The pH of the media affects the extinction of the 426- and 455-nm Soret bands; increased pH decreases the extinction of the 426-nm band and increases the extinction of 455-nm band. Formation of the DNSAB metabolite-cytochrome P-450 complex decreases the amount of CO-reactive cytochrome P-450 by 24%. The metabolite complex is not dissociable by treatment with ferricyanide or by using centrifugation techniques. Dissociation is accomplished by addition of DNSAB to the oxidized metabolite complex. Kinetic analysis of the complex formation gives apparent K_m and V_max values at 2.55 ± 1.0 μM and 1.1 ± 0.4 × 10^?2 ΔA min^?1 nmol^?1 cytochrome P-450, respectively. Addition of juvenile hormone [(E,E)-cis-methyl-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate; JH] to the reaction medium competitively inhibits the formation of the metabolite complex giving an inhibition constant of 16 μM. DNSAB synergized the lethal effects of JH against Aedes aegypti larvae threefold; however, JH did not synergize DNSAB. These data suggest that DNSAB may acquire its hormonal qualities by complexing a species of cytochrome P-450 that metabolizes JH, thereby prolonging the in vivo lifetime of this hormone.     

In vitro effects of chlordecone (Kepone) on hepatic microsomal cytochrome P-450

The interaction of chlordecone (decachlorooctahydro-1,3,4-metheno-2H-cyclobuta[cd]-pentalene-2-one, Kepone) with hepatic microsomal cytochrome P-450 was studied. Chlordecone binds to cytochrome P-450 to produce a Type I spectral change the magnitude of which is dependent upon the chemical pretreatment of the animal. In kinetic studies of chlordecone was found to be a competitive inhibitor of aminopyrine N-demethylase and p-nitroanisole O-demethylase and a noncompetitive inhibitor of aniline p-hydroxylase.     

Conversion of α-pinene to α-pinene oxide by rat liver and the bark beetle Dendroctonus terebrans microsomal fractions

The metabolism of α-pinene, a major monoterpene in Pinus spp. in the United States, has been examined utilizing microsomal fractions from larval and adult Dendroctonus terebrans and rat liver. Under hydroxylating conditions, both insect and rat liver microsomes convert α-pinene into α-pinene oxide and several other undentified products. α-Pinene oxide was identified by mass spectrometry. α-Pinene is an inducer of cytochrome P-450 in rat liver microsomes and its effect on the pattern of α-pinene metabolism is very similar to β-naphthoflavone. No increase in cytochrome P-450 was observed when insects were treated with α-pinene; however, the quantity of α-pinene metabolic products was increased by α-pinene pretreatment. The role of cytochrome P-450 linked reactions in the production of insect pheromones via the α-pinene epoxide intermediate is discussed.     

Alterations in microsomal cytochrome P-450-catalyzed reactions as a function of chlordecone (Kepone) induction

The effects of chlordecone treatment on the hepatic microsomal monooxygenase system of male rats were investigated. Chlordecone increased the microsomal content of cytochrome P-450, NADPH-cytochrome P-450 (c) reductase and, to a lesser extent, cytochrome b₅ in a time- and dose-dependent manner. The content of NADH-cytochrome b₅ (c) reductase was reduced. The turnover of seven substrates was studied in detail and, with the exception of aniline, was found to be increased between 1.3- and 2.2-fold. The apparent K_m's for these substrates were increased 2.1- to 16.7-fold. In addition, zoxazolamine paralysis time was reduced as a result of chlordecone treatment. These kinetic changes are explained on the basis of alterations in the cytochrome P-450 pool together with residual chlordecone acting as an inhibitor of substrate turnover. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein pattern of microsomes isolated from chlordecone-treated rats more closely resembled that of microsomes isolated from untreated rats than that of microsomes isolated following phenobarbital or 3-methylcholanthrene treatment.     

Isolation of insect microsomal oxidases by rapid centrifugation

Only about 60% of the total relative gravitational force conventionally used to sediment microsomes is needed to prepare highly active microsomes from the midgut tissues of an insect larva. A rapid preliminary centrifugation for 2 min at 39,000g_max effectively removed contaminating microorganisms, tissue debris, nuclei, and mitochondria. The supernatant was recentrifuged for 20 min to 210,000g to sediment the microsomes. There were no losses of microsomal oxidase activities or degradation of cytochrome P-450 to the inactive form (P-420) resulting from the application of the higher gravitational force. Incorporation of 1 mM EDTA in the buffer and washing the microsomes resulted in an improved yield of the cytochrome compared to that in microsomes prepared in sucrose. Yields of microsomal protein, cytochrome P-450, and NADPH-cytochrome c reductase in the rapidly isolated microsomes were as good as those in conventionally prepared microsomes. The apparent kinetic characteristics of several microsomal oxidation activities and optical difference spectra of Types 1 and 2 ligands were identical in the rapidly and conventionally prepared microsomes. The morphological appearance of the microsomes was examined by electron microscopy. Microsomal pellets prepared by either method were indistinguishable. The rapid procedure saves significant time in microsome preparation and yields microsomal oxidase activities as good or slightly better than those prepared by usual centrifuged procedures.     

Metabolism and toxicity of metribuzin in mouse liver

Metribuzin was hepatotoxic in mice when administered intraperitoneally (ip) at sublethal doses of 150 to 250 mg/kg. Four dose-dependent abnormalities were evident. Histopathological examination revealed a fulminant centrilobular hepatic necrosis. The serum glutamic-pyruvic transaminase (GPT) activity was elevated. The liver glutathione (GSH) content was almost completely depleted. There was extensive covalent binding of radiocarbon from [carbonyl-¹⁴C]metribuzin to liver proteins and also high blood levels of metribuzin fragments. Each of these four effects of metribuzin on the liver or blood was alleviated or blocked in mice pretreated with piperonyl butoxide (PB), which inhibits the cytochrome P-450-dependent monooxygenase. PB also reduced the lethality of metribuzin by three-fold. In contrast, pretreatment with diethyl maleate to suppress the liver GSH content increased the lethality of metribuzin by twofold. The hepatotoxicity and acute lethality of metribuzin were probably due to reactive intermediates which are normally detoxified by GSH conjugation. The principal urinary metabolites of metribuzin in mice and rats are mercapturic acids, which arise via metribuzin sulfoxide or deaminometribuzin sulfoxide reacting with GSH. Sulfoxidation therefore appears to activate metribuzin to an electrophilic metabolite which, in the absence of GSH, binds to tissue proteins producing hepatotoxicity.