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.     

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.     

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.     

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.     

Microsomal oxidases in the flesh fly (Sarcophaga bullata Parker) and the black blow fly [Phormia regina (Meigen)]

The microsomal oxidase system in the flesh fly (Sarcophaga bullata Parker) and the black blow fly [Phormia regina (Meigen)] was examined using aldrin as substrate. In both species the oxidase requires NADPH and is inhibited by CO and by piperonyl butoxide. The enzyme activity changes significantly during larval development, reaching a maximum shortly before puparium formation then declining during the pupal stage and increasing again in adults. Dietary sodium phenobarbital at 0. 1– 0.5% increases the oxidase activity up to 60- and 56-fold in the larvae of blow flies and flesh flies, respectively.     

Methylenedioxyphenyl complexes with microsomal cytochrome P-450: In vivo complex formation in rat liver and in midgut tissues of the Southern armyworm (Spodoptera eridania)

The capacity of several methylenedioxyphenyl insecticide synergists to generate metabolite complexes with cytochrome P-450 was studied in midgut tissues of the Southern armyworm (Spodoptera eridania). Examination of the NADH-reduced versus oxidized spectra from methylene-dioxyphenyl-induced midgut indicated that isosafrole, dihydrosafrole, and 4-ethoxy-1,2-methylenedioxybenzene generated metabolite complexes with a principal absorbance maximum at 427 nm and smaller absorbance maxima near 460 and 556 nm. Further studies with 2-n-heptylbenzimidazole showed that the complex between insect cytochrome P-450 and dihydrosafrole was unusually resistant to displacement. Initial rates of complex displacement in insect microsomes were found to be approximately an order of magnitude slower than those of the corresponding complexes in rat hepatic microsomes. Nevertheless, with the exception of the dihydrosafrole complex in insect microsomes, the “time to half-maximal displacement” parameter was found to be very similar for each complex. These findings indicate that the formation of dissociable complexes between cytochrome P-450 and the methylenedioxyphenyl metabolite occurs in both insect midgut and rat hepatic microsomes after in vivo exposure. From the present study it would appear that dihydrosafrole may constitute a useful probe to distinguish binding sites within insect and mammalian cytochrome P-450.     

Juvenile hormone analogs: In vitro metabolism in relation to biological activity in blow flies and flesh flies

Six juvenile hormone analogs of the alkyl 3,7,11-trimethyl-2,4-dodecadienoate type were compared as substrates for esterases and oxidases prepared from homogenates of the flesh fly (Sarcophaga bullata) and blow fly (Phormia regina). The esterase system was able to hydrolyze all of the analogs except the isopropyl ester, known commercially as methoprene or ZR-515. This result was consistent with the biological activity of the analogs, methoprene being more effective in preventing pupal-adult ecdysis. The esterases were present in all life stages in both species with the adult (abdomens) containing the highest titers. According to their reaction with paraoxon, the enzymes are classified as C-type esterases. Microsomal oxidases prepared from adult abdomens metabolized all of the juvenile hormone analogs.     

Blood meal and cytochrome P-450 monooxygenases in the northern house mosquito,Culex pipiens

Conditions for the measurement of aldrin epoxidation by microsomes prepared from abdominal tissues (fat body + integument) of adult female Culex pipiens were characterized. The enzyme activity had a pH optimum of 7.2 and an apparent K_m of 3.4 μM. Aldrin epoxidation and NADPH-cytochrome c reductase had similar patterns of inhibition by a rabbit antiserum to house fly NADPH-cytochrome P-450 reductase, thus implicating cytochrome P-450 monooxygenase(s) in the epoxidation of aldrin. Low (71 pmol/mg protein) levels of cytochrome P-450 were detected in abdominal tissue microsomes. In non-blood-fed insects, aldrin epoxidation and NADPH-cytochrome c reductase activities did not change between Day 1 and Day 12 after adult emergence, except for a small peak on Day 2. In insects fed a blood meal on Day 6 after emergence both activities increased (two- to threefold) to a plateau maintained between 2 and 4 days after the blood meal. Aldrin epoxidation and NADPH-cytochrome c reductase activities decreased to normal values between 4 and 6 days after the blood meal.     

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.     

Monooxygenases from soybean root nodules: Aldrin epoxidase and cinnamic acid 4-hydroxylase

The metabolism of aldrin and trans-cinnamic acid within the root nodules of soybean (Glycine max. cv. Forrest) was investigated. In vitro studies with microsomal preparations revealed the presence of two distinct monooxygenase enzymes requiring NADPH and molecular oxygen. This was shown by different distributions in membrane fractions from sucrose density gradients. In addition, cinnamic acid hydroxylase was sensitive to carbon monoxide (CO), similar to cytochrome P-450-dependent monooxygenases, whereas aldrin epoxidation was not. Cyanide ion, strongly inhibited the epoxidase, without affecting hydroxylase activity. The superoxide radical (O₂⁻) scavengers, superoxide dismutase and norepinephrine, inhibited aldrin epoxidation but allowed greater cinnamic acid 4-hydroxylase activity. These results indicate aldrin epoxidase, in contrast to cinnamic acid hydroxylase, does not involve cytochrome P-450, and may instead utilize a peroxidase-like hemoprotein.     

Spectral and inhibitory interactions of methylenedioxyphenyl compounds with southern armyworm (Spodoptera eridania) midgut microsomes

Characteristics of the Type III optical difference spectra of 13 methylenedioxyphenyl compounds in NADPH-fortified armyworm midgut microsomes varied with the nature of the substituents in the aromatic ring. Compounds with electron-donating substituents yielded spectra with large $\text{427}\text{458}\text{nm}$ peak ratios, whereas those with electron-withdrawing groups exhibited low $\text{427}\text{458}\text{nm}$ peak ratios. Small amounts of carbon monoxide were generated during incubation of the 4,5-dihalo derivatives with midgut microsomes, and cis- and trans-methylenedioxycyclohexanes exhibited spectra with a major Soret peak at about 430 nm and a very weak absorbance maximum at about 480 nm. Formation of the Type III spectral complex occurred very rapidly and was associated with a marked decrease (up to 72%) in cytochrome P-450 levels as measured by carbon monoxide binding. Although a 24% reduction of cytochrome P-450 was observed in the absence of any measureable 458-nm spectral complex a linear relationship existed between further decreases in the cytochrome and the increase in Type III complex formation (458 nm). Inhibitory potencies of the compounds towards aldrin epoxidase and benzopyrene hydroxylase activities were not clearly correlated with either spectral complex formation or decrease in cytochrome P-450 and it is apparent that different factors are involved in the inhibition of different monooxygenase reactions.     

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.     

Ecdysone metabolism by soluble enzymes from three species of diptera and its inhibition by the insect growth regulator TH-6040

Homogenates of larvae, pupae, and adults of house flies (Musca domestica L.), flesh flies (Sarcophaga bullata Parker), and blow flies (Phormia regina (Meigen)) have been examined for enzymes which convert α- and β-ecdysone to apolar products. Most of the activity was found in the soluble fraction from house flies and flesh flies but none of the blow fly fractions was active. Two enzymes seem to be involved in the ecdysone metabolism, one requiring NADPH and the other functioning without this cofactor. The product of the latter enzyme is thought to be the 3-dehydro-ecdysone. This product is further converted to the 3α-hydroxy isomer of ecdysone by the NADPH-requiring enzyme. On feeding the insect growth regulator TH-6040 (1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)-urea) to larvae at dietary levels ranging from 0.3 to 10 ppm, the activity of the enzyme producing the 3-dehydro product is reduced by 20 to 82%. It is suggested that the growth regulator exerts its effect on pupal-adult ecdysis through its inhibition of ecdysone metabolism.     

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.     

Biochemical genetics of oxidative resistance to diazinon in the house fly

The genetics and biochemistry of oxidative resistance to diazinon were investigated in a diazinon-resistant strain of the house fly, Musca domestica L. The resistant strain was crossed with a multimarker susceptible strain and substrains containing portions of the resistant strain genome were prepared. Resistance, microsomal oxidase, and cytochrome P-450 spectral characteristics were then compared in the different strains. The major gene for resistance to diazinon is semidominant and is located on chromosome II, 13 crossing over units from the recessive mutant stubby wing. Additional resistance genes occur on chromosome II and on other chromosomes as well. Resistance to diazinon was introduced into a susceptible mutant-marked strain via genetic crossing over. Increases in parathion oxidase, total and P-450-specific N- and O-demethylase activity, and resistant strain type I binding spectrum were introduced along with resistance, indicating genes controlling these parameters and resistance are either identical or closely linked. No increase in activity of cytochrome P-450 itself was introduced into the mutant strain. Additional genes controlling the amount of cytochrome P-450 and several spectral changes characteristic of the resistant strains are apparently controlled by genes located at different loci on chromosome II. Resistance factors on other chromosomes are also present, but were not characterized.     

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.     

Metabolism of juvenile hormone I by microsomal oxidase,esterase, and epoxide hydrase of Musca domestica and some comparisons with Phormia regina and Sarcophaga bullata

House fly (Musca domestica L.) microsomes prepared from larvae, pupae, or adults contain three enzyme system which can metabolize juvenile hormone I: an esterase, an oxidase, and epoxide hydrase. The presence of the oxidase is indicated by the increased metabolism when microsomes are supplemented with NADPH and by the occurrence of additional metabolites tentatively identified as products arising from oxidation of the 6, 7 double bond. Additional evidence of the activity of the oxidase system is the increased metabolism of juvenile hormone I by the NADPH-dependent system from phenobarbital-induced insects, by inhibition of the oxidation by piperonyl butoxide and carbon monoxide, and by the greater metabolism of the hormone by microsomes from insecticide-resistant (high oxidase) strains. In vivo studies of house fly adults treated with ³H-labeled juvenile hormone I reveal a pattern of metabolism similar to that seen during NADPH-supplemented in vitro metabolism. The three enzymes have somewhat different patterns of activity during the larval stage of the house fly, juvenile hormone esterase and epoxide hydrase beginning at a high level of activity in the young larvae while the juvenile hormone oxidase is low at this stage. In the late larval stage all three enzymes show increased activity followed by declines during the pupal stage and further increases in the adult stage. Comparison of in vitro enzyme levels of the house fly, flesh fly (Sarcophaga bullata Parker), and blow fly [Phormia regina (Meigen)] showed that, although the enzymes were present in the latter two species, their activity on a per insect basis was considerably less than that of the house fly.     

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.     

Enzyme activities and cytochrome P-450 levels of some Japanese quail tissues with respect to their metabolism of several pesticides

In the Japanese quail, cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in postmitochondrial centrifugal fractions, in microsomes, and supernatant fractions of liver, lungs, kidneys, and testes. Liver microsomes contained the highest A-esterase activity and P-450 levels. B-esterase was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in quail lung and kidney, and lowest in the liver (per mg protein). Very little difference in glutathione S-aryl transferase activity was noted among the tissues assayed. In vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed that the production of 1-naphthyl-N-hydroxymethylcarbamate and 1-naphthol, the major metabolites, was greatest in the postmitochondrial fraction of the liver. The major carbaryl metabolite in all other quail tissue fractions was 1-naphthol. Phosphamidon metabolism in postmitochondrial preparations of quail liver was higher than in the supernatant and microsomes. Chlorotoluron metabolism occurred only in the postmitochondrial fractions of quail liver. The major products were the oxidative metabolites, N-(3-chloro-4-methylphenyl)-N′-methylurea and N-(3-chloro-4-hydroxymethylphenyl)-N′-methylurea.