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 When PB was withdrawn from treated flies (both strains) 48 hr after treatment began, specific activities (product min^?1 mg protein^?1) in all enzymes returned to control values in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.     

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.     

Temperature and diet modulate cytochrome P-450 activities in southern armyworm,Spodoptera eridania (Cramer), caterpillars

Larvae of the southern armyworm, Spodoptera eridania (Cramer), grew well in the 15–30°C temperature range. Pupae survived poorly at 15°C but moths emerged from 85% of the pupae at 30°C. The time for development was prolonged at 15°C and larvae grew significantly bigger than at 30°C. Cytochrome P-450 content, cytochrome P-450 reductase, p-chloro N-methylaniline N-demethylation, methoxyresorufin 0-demethylation, and aldrin epoxidation activities were higher at 15°C than at 30°C. All cytochrome P-450 activities were more inducible by dietary pentamethylbenzene at 30°C than at 15°C. High cytochrome P-450-catalyzed activities were associated with increases in microsomal protein rather than with changes in membrane lipid or phospholipid content. Phosphatidylcholine was the major midgut membrane phospholipid. There was only a tendency towards increased unsaturation of the phospholipid fatty acyl moieties and lowered membrane phase transition temperature in cold-adapted larvae. Acute oral carbaryl toxicity was generally inversely correlated with cytochrome P-450 catalyzed activities. Carbaryl toxicity was decreased about 10-fold by pentamethylbenzene induction and about 3-fold by the lower acclimatization temperature.     

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.     

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.     

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).     

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.     

An evaluation of the role of oxidative enzymes in Colorado potato beetle resistance to carbamate insecticides

Carbofuran and carbaryl LD₅₀ values were determined with and without piperonyl butoxide pretreatment for a resistant (New Jersey) and two susceptible (Utah and Netherland) populations of Colorado potato beetle larvae. Similar bioassays were conducted with carbofuran for resistant (Rutgers) and susceptible (NAIDM) adult house flies. The degree of resistance development by New Jersey Colorado potato beetles (RR = 848) was greater than that of the laboratory-selected colony of Rutgers house flies (RR = 583). Comparisons of synergist difference calculations including “percentage synergism” (%S), “log percentage synergism” (L%S), and “relative percentage synergism (R%S) for the resistant (R) and the susceptible (S) populations indicated the possibility that monooxygenases and other resistance mechanisms may be involved in Colorado potato beetle resistance to these carbamates. Monooxygenase involvement in resistance of Rutgers house flies was demonstrated in vitro by a 4-fold enhancement of p-nitroanisole O-demethylation over that of NAIDM house flies. O-demethylation of p-nitroanisole could not be demonstrated for potato beetle larvae. Colorado potato beetle resistance was associated with increases in microsomal levels of NADPH-cytochrome c reductase (ca. 2-fold) and NADPH oxidation (1.2-fold). The inability to measure O-demethylation in Colorado potato beetles may have been due to the solubilization of NADPH-cytochrome c reductase during microsomal preparation. Significant differences between resistant and susceptible Colorado potato beetle larvae were not observed in the penetration of [¹⁴C]carbaryl. Excretion of the radiocarbon may have been significantly greater in the resistant New Jersey population, but some of the insecticide may have also rubbed off the cuticle. This increased capacity for excretion, combined with increased levels of monooxygenase enzymes, could account for the high resistance level of this population.     

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: 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.     

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.     

Absence of enhanced detoxication of permethrin in pyrethroid-resistant house flies

The mechanisms of resistance to pyrethroids were studied in a permethrin-selected (147-R) strain of the house fly, Musca domestica L. Approximately 12-fold synergism was obtained with a mixture of (1R)-trans-permethrin:piperonyl butoxide (1:5) so that the resistance decreased from 97-fold to 22-fold. Tests with the esterase inhibitor S,S,S-tributyl phosphorotrithioate produced very little synergism in either the resistant (R) strain (1.6-fold) or the susceptible (S) strain (1.9-fold). An investigation of the microsomal components revealed that compared to the S strain, the R strain demonstrated twice as much cytochrome P-450 and cytochrome b₅ and double the rate of NADPH-cytochrome c reductase activity. In addition, the rate of p-nitroanisole O-demethylation was found to be six times greater in the R strain. An in vivo accumulation study showed that the R strain displayed a decreased rate of penetration of trans-[¹⁴C]permethrin. When treated at equitoxic doses the R strain was found to tolerate 50-fold more internal permethrin than the S strain. An in vitro metabolism study indicated that there was no difference between strains in the overall rate of metabolism of trans-[¹⁴C]permethrin. The evidence obtained supports the conclusion that several resistance factors are involved but that decreased sensitivity of the nervous system to the action of pyrethroids is the principal mechanism of resistance in the 147-R strain.     

Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi

The fungicides miconazole, fenarimol, and etaconazole block ergosterol biosynthesis in fungi by inhibiting sterol 14α-demethylation, which is mediated by a cytochrome P-450 enzyme. The sensitivity of cytochrome P-450-dependent hydroxylation or demethylation of several substrates to these fungicides and similar compounds was compared to that of fungal growth and sterol 14α-demethylation. Demethylation of p-chloro-N-methylaniline (PCMA) by sporidia of Ustilago maydis and 11α-hydroxylation of progesterone by Aspergillus nidulans were relatively insensitive to these compounds and to metyrapone. The ability of a sterol 14α-demethylation-deficient mutant to demethylate PCMA indicates that this substrate is not demethylated by the sterol 14α-demethylation system of U. maydis. The 14α-hydroxylation of progesterone by cells of Curvularia lunata was quite sensitive to the three fungicides, and also to metyrapone and isopropylphenylimidazole. This system was less sensitive to the three fungicides than sterol 14α-demethylation, but was appreciably more sensitive than PCMA demethylation. A study of progesterone 14α-hydroxylation in cell-free preparations of C. lunata showed the reaction to be inhibited by CO, and to be competitively inhibited by low concentrations of miconazole. These data suggest that the primary action of sterol biosynthesis-inhibiting (SBI) fungicides is competitive inhibition of sterol/steroid-type cytochrome P-450 enzymes rather than interference with the function of sterol carrier proteins or enzyme-modulating phospholipids.     

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.     

Inducibility of metabolic insecticide defenses in boll weevils and tobacco budworm caterpillars

Of five cotton allelochemicals, (+)-α-pinene, β-caryophyllene, gossypol, umbelliferone, and scopoletin, all except gossypol induced cytochrome P-450 content, N-demethylation, and epoxidation activities more in tobacco budworm larval midguts than in adult or larval boll weevils. All except gossypol also induced glutathione transferase activity in tobacco budworm larval midguts and adult boll weevils. Microsomal esterase activity was unaffected or suppressed by all five allelochemicals. Soluble esterase activities were unaffected, or induced only in boll weevil larvae. The contrast in insecticide resistance development between the tobacco budworm and the boll weevil may be, in part, related to superior inducibility of cytochrome P-450 and associated activities in the former species in addition to overall higher uninduced activities (26).     

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.     

Biochemical characteristics of insecticide resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith)

A strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from corn in Citra, Florida, showed high resistance to carbaryl (562-fold) and methyl parathion (354-fold). Biochemical studies revealed that various detoxification enzyme activities were higher in the field strain than in the susceptible strain. In larval midguts, activities of microsomal oxidases (epoxidases, hydroxylase, sulfoxidase, N-demethylase, and O-demethylase) and hydrolases (general esterase, carboxylesterase, β-glucosidase) were 1.2- to 1.9-fold higher in the field strain than in the susceptible strain. In larval fat bodies, various activities of microsomal oxidases (epoxidases, hydroxylase, N-demethylase, O-demethylases, and S-demethylase), glutathione S-transferases (CDNB, DCNB, and p-nitrophenyl acetate conjugation), hydrolases (general esterase, carboxylesterase, β-glucosidase, and carboxylamidase) and reductases (juglone reductase and cytochrome c reductase) were 1.3- to 7.7-fold higher in the field strain than in the susceptible strain. Cytochrome P450 level was 2.5-fold higher in the field strain than in the susceptible strain. In adult abdomens, their detoxification enzyme activities were generally lower than those in larval midguts or fat bodies; this is especially true when microsomal oxidases are considered. However, activities of microsomal oxidases (S-demethylase), hydrolases (general esterase and permethrin esterase) and reductases (juglone reductase and cytochrome c reductase) were 1.5- to 3.0-fold higher in the field strain than in the susceptible strain. Levels of cytochrome P450 and cytochrome b₅ were 2.1 and 1.9-fold higher, respectively, in the field strain than in the susceptible strain. In addition, acetylcholinesterase from the field strain was 2- to 85-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, propoxur, carbofuran, bendiocarb, thiodicarb) and organophosphates (methyl paraoxon, paraoxon, dichlorvos), insensitivity being highest toward carbaryl. Kinetics studies showed that the apparent K_m value for acetylcholinesterase from the field strain was 56% of that from the susceptible strain. The results indicated that the insecticide resistance observed in the field strain was due to multiple resistance mechanisms, including increased detoxification of these insecticides by microsomal oxidases, glutathione S-transferases, hydrolases and reductases, and target site insensitivity such as insensitive acetylcholinesterase. Resistance appeared to be correlated better with detoxification enzyme activities in larval fat bodies than in larval midguts, suggesting that the larval fat body is an ideal tissue source for comparing detoxification capability between insecticide-susceptible and -resistant insects.     

抗阿维菌素小菜蛾的细胞色素P450酶系研究

通过对不同发育时期敏感和抗阿维菌素小菜蛾品系细胞色素P450含量的测定,以及使用不同模式底物对P450单加氧酶活性的比较研究发现:除成虫期外,不同发育时期抗性品系小菜蛾中P450和细胞色素b5的含量都高于敏感品系;抗性品系还原型辅酶Ⅱ(NADPH)-细胞色素P450还原酶活性是敏感品系的1.97倍;同时发现抗性品系中甲氧试卤灵-O-脱甲基酶(MROD)、乙氧试卤灵-O-脱乙基酶(EROD)、乙氧基香豆素-O-脱乙基酶(ECOD)以及对硝基苯甲醚-O-脱甲基酶(PNOD)的活性均明显高于敏感品系,分别为敏感品系的9.41、4.15、1.67和2.94倍。研究结果表明,细胞色素P450含量和单加氧酶活性的增高是小菜蛾对阿维菌素产生抗性的一个重要机制。     

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.