Role of ascorbate in biological hydroxylations: Origin of life considerations and the nature of the oxenoid species in oxygenase reactions

The proposition is made that the enzymes that catalyze ascorbate-dependent hydroxylation reactions in metabolism make use of a primordial dioxygen fixation system, in which a ferrous ascorbate complex is the central and permanent catalytic unit. It is suggested that dioxygen on bonding to the complex becomes a nucleophile that will react with an electrophilic and easily oxidizable compound, such as an α-ketoacid. This gives rise to a presumably fairly stable intermediate ferryl ascorbate complex that acts as an electrophilic oxenoid both in ascorbate-dependent oxygenase reactions and in chemical models thereof, such as Udenfriend's system (ascorbate and Fe^2?, with or without EDTA in water). A similar ferryl species with easily oxidizable ligands is suggested to be the oxenoid in other oxygenase reactions such as the tetrahydrobiopterin and the cytochrome P-450-dependent monooxygenases.     

Hydroxylation of steroids in a dienzyme system consisting of cytochrome P-450 and immobilized adrenodoxin

Experimental systems for the hydroxylation of steroids (11-deoxycorticosterone and cholesterol) with reduced electron transfer chain, in which flavoprotein was omitted, were investigated. Incubation of chemically reduced immobilized adrenodoxin either with cytochrome P-45011 beta or cytochrome P-450scc in the presence of substrate of hydroxylation and oxygen yields the specific reaction products, corticosterone or pregnenolone. The catalytic activity of the experimental dienzyme systems proves the possibility of the steroid hydroxylation mechanism based exclusively on dissociation and reassociation of the electron transporting protein complexes.     

Metabolic conditions determining the composition and catalytic activity of cytochrome P-450 monooxygenases in Candida tropicalis.

In the microsomal fraction of Candida tropicalis cells, two distinct monooxygenases were detected, depending on the growth conditions. The distinction of the two monooxygenases was evident from: (i) the absorption maxima in the reduced CO difference spectra of the terminal oxidases (cytochromes P-450 and P-448); (ii) the contents of the monooxygenase components (cytochromes P-450/P-448, NADPH-cytochrome c (P-450) reductase, and cytochrome b5) and (iii) the catalytic activity of the complete system (aliphatic hydroxylation and N-demethylation activity). The occurrence of the respective monooxygenases could be related to the carbon source (n-alkanes or glucose). Oxygen limitation led to a significant increase of cytochrome P-450/P-448 content, independent of the carbon source utilized by the cells. An improved method for the isolation of microsomes enabled us to demonstrate the presence of cytochrome P-448 in glucose-grown cells.     

Substrate specificity of soluble methane monooxygenase. Mechanistic implications

Following the example set by studies of the mechanistic aspects of the substrate specificity of various cytochrome P-450 enzymes, we have undertaken a parallel investigation of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme with a broad substrate specificity. Using substrates previously tested with cytochrome P-450 enzymes and using purified enzyme preparations, this work indicates that soluble methane monooxygenase has a similar oxidative reaction mechanism to cytochrome P-450 enzymes. The evidence suggests that soluble methane monooxygenase oxidizes substrates via a nonconcerted reaction mechanism (hydrogen abstraction preceding hydroxylation) with radical or carbocation intermediates. Aromatic hydroxylation proceeds by epoxidation followed by an NIH shift.     

Stabilization of microbial cytochrome P-450 activity by creation of station-phase conditions in a continuously operated immobilized-cell reactor.

Bacillus megaterium (ATCC 13368) exhibits cytochrome P-450 monooxygenase activity (referred to herein as Cyt P-450 meg) catalyzing 15 beta-steroid hydroxylation. This activity belongs to the widespread ferredoxin reductase-ferredoxin-Cyt P-450 type of monooxygenases, providing a representative model system for this type of activity. The level of Cyt P-450 meg activity reaches its maximum in the cells during the stationary phase of the growth curve and is not affected by Cyt P-450 inducers. Here we present the development of an approach for stabilizing the Cyt P-450 meg system so that it performs continuous steroid hydroxylation and will be a model system for Cyt P-450-based detoxification. It is based on cell immobilization and simulation of stationary-phase conditions in a continuously operated fluidized-bed bioreactor. The combination of an appropriate immobilization technique, operational conditions, and medium composition provided a stabilized cell environment resulting in "freezing" of a physiological steady-state analog under stationary phase conditions, allowing stable performance of continuous hydroxylation for several weeks. It is suggested that this approach may be extended for use with other environmentally induced enzymatic activities.     

Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.     

Fatty acid hydroxylation in rat kidney cortex microsomes

Rat kidney microsomes have been found to catalyze the hydroxylation of medium-chained fatty acids to the omega- and (omego-1)-hydroxy derivatives. This reaction, which requires NADPH and molecular oxygen, is a function of monooxygenase system present in the kidney microsomes, containing NADPH-cytochrome c reductase and cytochrome P-450K. NADH is about half as effective as an electron donor as NADPH and there is an additive effect in the presence of both nucleotides. Cytochrome P-450K absorbs light maximally at 452-3 nm, when it is reduced and bound to carbon monoxide. The extinction coefficient of this complex is 91 mM(-1) cm(-1). Electrons from NADPH are transferred to cytochrome P-450K via the NADPH-cytochrome c reductase. The reduction rate of cytochrome P-450K is stimulated by added fatty acids and the reduction kinetics reveal the presence of endogenous substrates bound to cytochrome P-450K. Both cytochrome P-450K concentration and fatty acid hydroxylation activity in kidney microsomes are increased by starvation. On the other hand, phenobarbital treatment of the rats has no effect on either the hemoprotein or the overall hydroxylation reaction and 3,4-benzpyrene administration induces a new species of cytochrome P-450K not involved in fatty acid hydroxylation. Cytochrome P-450K shows, in contrast to liver P-450, high substrate specificity. The only substances forming enzyme-substrate complexes with cytochrome P-450K are the medium-chained fatty acids and certain derivatives of these acids. The chemical requirements for substrate binding include a carbon chain of medium length and at the end of the chain a carbonyl group and a free electron pair on a neighbouring atom. The distance between the binding site for the carbonyl group and the active oxygen is suggested to be in the order of 16 A. This distance fixes the ratio of omega- and (omega-1)-hydroxylated products formed from a certain fatty acid by the single species of cytochrome P-450K involved. The membrane microenvironment seems also to be of importance for the substrate specificity of cytochrome P-450K, since removal of the cytochrome from the membrane lowers its binding specificity to some extent. A comparison between the liver and kidney cytochrome P-450 systems suggests that the kidney cytochrome P-450K system is specialized for fatty acid hydroxylation.     

Hydroxylation products of hydrophobic xenobiotics--stabilizers of cytochrome P-450 in the hepatocytes

The effects of the hydroxylation product 3,4-benzo(a)pyrene and the free radical scavenger 1,2,3-trioxybenzene on cytochrome P-450 degradation in isolated rat hepatocytes induced by the Fe2+-ADP + NADPH system activating lipid peroxidation (LPO) were investigated. During incubation of hepatocytes, cytochrome P-450 is destroyed due to accumulation of LPO products. Addition of the free radical scavenger 1,2,3-trioxybenzene and the monoxygenase substrate 3,4-benzo(a)pyrene to the incubation medium induces inhibition of LPO and simultaneous stabilization of cytochrome P-450. Deceleration of malonic dialdehyde production by the free radical scavenger of the monoxygenase substrate suggests that both the compounds stabilize cytochrome P-450. It is assumed that in liver hepatocytes, exogenous free radical scavengers of the phenolic type and the products of their decarboxylation protect cytochrome P-450 against the LPO-induced destruction via oxidative metabolism of hydrophobic substrates.     

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.     

Induction and catalytic activities of cytochromes P-450b/e and P-450c in the liver microsomes of neonatal rats

The activities of cytochrome P-450-dependent monooxygenases has been investigated in the liver microsomes of newborn rats (3-16 days after birth) induced with PB or 3-MC. It has been shown that the induction by PB and 3-MC results in the increase of both the total amount of cytochrome P-450 as determined by the CO-reduced spectrum and the amount of induced forms P-450b/e and P-450c respectively. In the course of induction of the specific forms of cytochrome P-450 BP-hydroxylase and 7-ER-O-deethylase activities increased at 3-MC-induction, while BPh-N-demethylase and BP-hydroxylase increased at PB-induction. Analysis of inhibition of monooxygenase reactions with antibodies has showed that only P-450c was involved in metabolism of BP and 7-ER. Participation of P-450b/e in BPh N-demethylation was notably lower in the neonates in comparison to the adult rats. In the one-week-old rats induced with 3-MC a considerable rate of BP hydroxylation and 7-ER O-deethylation (2-4.5 nmol of product min-1 mg-1) has been observed despite a small amount of P-450 (0.02-0.1 nmol/mg of protein). This fact shows the higher catalytic activity of this cytochrome P-450 in the neonates compared to similar characteristics of P-450c in the 3-MC-induced microsomes. Metabolism of BP in the PB-microsomes of the neonatal rats was inhibited neither by anti-P-450b/e nor anti-P-450c in contrast to the adults, where this reaction was inhibited by antibodies against P-450b/e.     

Model systems for the coordination chemistry of cytochrome P-450.

Studies on model complexes have supported the presence of a mercaptide as the fifth ligand of cytochrome P-450 monooxygenases. When alcohol or thiol ligands are added to the sixth coordination position of a five-coordinated 4-nitrobenzene thiolate complex of FeIII protoporphyrin IX dimethyl ester chloride low spin complexes with optical and EPR-spectra very similar to cytochrome P-450 are obtained. From a comparison with all ligands of cytochrome P-450 and the model complexes it is concluded that a hard ligands must occupy the sixth coordination position of cytochrome P-450. An imidazole group is less likely, also in view of the ligand field parameters. The significance of the fifth and sixth ligand of cytochrome P-450 is discussed with respect to the monooxygenase mechanism.     

Hydroxylation of prostaglandins by inducible isozymes of rabbit liver microsomal cytochrome P-450. Participation of cytochrome b5

The hydroxylation of prostaglandin (PG) E1, PGE2, and PGA1 was investigated in a reconstituted rabbit liver microsomal enzyme system containing phenobarbital-inducible isozyme 2 or 5,6-benzoflavone-inducible isoenzyme 4 of P-450, NADPH-cytochrome P-450 reductase, phosphatidylcholine, and NADPH. Significant metabolism of prostaglandins by isozyme 2 occurred only in the presence of cytochrome b5. Under these conditions, PGE1 hydroxylation was linear with time (up to 45 min) and protein concentration, and maximal rates were obtained with a 1:1:2 molar ratio of reductase: cytochrome b5:P-450LM2. Moreover, P-450LM2 catalyzed the conversion of PGE1, PGE2, and PGA1 to the respective 19- and 20-hydroxy metabolites in a ratio of about 5:1, and displayed comparable activities toward the three prostaglandins based on the total products formed in 60 min. Apocytochrome b5 or ferriheme could not substitute for intact cytochrome b5, while reconstitution of apocytochrome b5 with ferriheme led to activities similar to those obtained with the native cytochrome. Isozyme 4 of P-450 differed markedly from isozyme 2 in that it catalyzed prostaglandin hydroxylation at substantial rates in the absence of cytochrome b5, was regiospecific for position 19 of all three prostaglandins, and had an order of activity of PGA1 greater than PGE1 greater than PGE2. P-450LM4 preparations from untreated and induced animals had similar activities with PGE1 and PGE2, respectively. Addition of cytochrome b5 resulted in a 20 to 30% increase in the rate of PGE1 hydroxylation and an appreciably greater enhancement in the extent of all the P-450LM4-catalyzed reactions, the stimulation being greatest with PGE2 (3-fold) and least with PGA1 (1.6-fold). Cytochrome b5 was thus required for maximal metabolism of all three prostaglandins, but did not alter the regiospecificity or the order of activity of P-450 isozyme 4 with the individual substrates. In the presence of cytochrome b5, the prostaglandin hydroxylase activities of isozyme 4 were two to six times higher than those of isozyme 2.     

Regioselectivity of hydroxylation of prostaglandins by liver microsomes supported by NADPH versus H2O2 in methylcholanthrene-treated and control rats: formation of novel prostaglandin metabolites

The effects of methylcholanthrene (MC) treatment of male rats on the regioselectivity of hydroxylation of prostaglandins E1 and E2 (PGE1 and PGE2) by liver microsomes, supplemented with NADPH or H2O2, was examined. In the presence of NADPH, control microsomes catalyzed the hydroxylation at omega-1 (C19) and at omega-(C20) sites with minimal formation of novel monohydroxy metabolites of PGE1 and PGE2, referred to as compounds X1 and X2, respectively. Similarly, H2O2 supported the 19-hydroxylation and the formation of compounds X1 and X2, but yielded only minimal amounts of 20-hydroxy products. With NADPH, MC-treated microsomal incubations demonstrated only minor quantitative change in the 19- and 20-hydroxylation as compared with controls, but showed a 7- to 11-fold increase in formation of compound X1 and a 10-fold increase in formation of X2. By contrast with H2O2, MC-treatment increased by about 3-fold the 19- and 20-hydroxylation of PGE1 and by 35- to 46-fold the formation of X1; similarly, there was an approximate 2-fold increase in 19- and 20-hydroxylation of PGE2 and a 10-fold increase in formation of X2. These findings suggest that several monooxygenases are involved in catalyzing the hydroxylation at the various sites of the PGE molecule. Inhibitors of monooxygenases (SKF 525A, alpha-naphthoflavone, and imidazole derivatives) provided further evidence that the hydroxylation at the three sites of PGEs is catalyzed by different P-450 monooxygenases. It is striking that the inhibitors had a much lesser effect on the 20-hydroxylation of PGE1 as compared with other sites of hydroxylation. Structural identification of compounds X1 and X2 was elucidated as follows. Resistance of the PGB derivative of X1 to periodate oxidation and mass fragmentation analysis of the t-butyldimethylsilyl ether methyl ester, placed the hydroxylation at C17 or C18. Finally, mass fragmentation of trimethylsilyl ether methyl ester PGB derivatives of X1 and X2 provided conclusive evidence that X1 and X2 are 18-hydroxy-PGE1 and 18-hydroxy-PGE2, respectively. The above findings indicate that the high regioselectivity of hydroxylation of PGE1 and PGE2, resulting in the formation of 18-hydroxy-PGE1 and 18-hydroxy-PGE2, respectively, is catalyzed by P-450 isozyme(s) which are induced by MC, possibly by P-450c.     

Effects of acetylenic and olefinic pyrenes upon cytochrome P-450 dependent benzo[a]pyrene hydroxylase activity in liver microsomes

1-Ethynylpyrene, trans-, & cis-1-(2-bromovinyl)pyrene, methyl 1-pyrenyl acetylene, and phenyl 1-pyrenyl acetylene are substrates for cytochrome P-450 dependent monooxygenases and also inhibitors of cytochrome P-450 dependent benzo[a]pyrene hydroxylase activities in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene, trans-1-(2-bromovinyl)pyrene, and methyl 1-pyrenyl acetylene cause a mechanism based inhibition (suicide inhibition) of the benzo[a]pyrene hydroxylase activities in microsomes from 5,6-benzoflavone or phenobarbital pretreated rats, while cis-1-(2-bromovinyl)pyrene only causes suicide inhibition of the hydroxylse activities in the 5,6-benzoflavone induced microsomes and phenyl 1-pyrenyl acetylene does not cause a detectable suicide inhibition of these activities in either type of microsome. Incubation with NADPH and 1-ethynylpyrene, trans-, or cis-1-(2-bromovinyl)pyrene causes a loss of the P-450 content in the microsomes from 5,6-benzoflavone or phenobarbital pretreated rats, but incubations with methyl 1-pyrenyl acetylene or phenyl 1-pyrenyl acetylene did not cause a loss of the P-450 content of either microsomal preparation.     

Induction of cytochrome P-450-dependent sulfonylurea metabolism in Streptomyces griseolus

Inducible cometabolism of several sulfonylurea herbicides by Streptomyces griseolus has been shown to occur by hydroxylation, O-dealkylation, or deesterification reactions. Only after growth of the bacterium in the presence of sulfonylurea did cell-free extracts exhibit NAD(P)H-dependent sulfonylurea metabolism. These extracts were shown to contain elevated levels of soluble cytochrome P-450 and exhibit sulfonylurea induced difference spectra consistent with binding of substrate to cytochrome(s) P-450. These results establish the presence of an inducible cytochrome P-450-dependent sulfonylurea metabolizing system in S. griseolus.     

Interaction between cytochrome P-448 and NADP-cytochrome P-450 reductase in reconstituted microsomal membranes

The regularities of changes in the functional activity of the microsomal monooxygenase system reconstituted by self-assembly from intact rat liver microsomes solubilized with 4% sodium cholate were studied at variable levels of NADPH-cytochrome P-450 reductase and the 3-methylcholanthrene-induced form of cytochrome P-450. Using antibodies against cytochrome P-448, the role of cytochrome P-448 in the overall reaction of benzopyrene hydroxylation induced in the microsomal membrane by a set of molecular forms of cytochrome P-450 was investigated. The effect of NADPH-cytochrome P-450 reductase and cytochrome P-448 incorporation into reconstituted microsomal membranes on benzpyrene metabolism suggests that in intact microsomal membranes benzopyrene metabolism induced by different forms of cytochrome P-450, with the exception of P-448, is limited by reductase is not the limiting component; however, cytochrome P-448 reveals its maximum activity at the cytochrome to reductase optimal molar ratio of 5:1; above this level, the catalytic activity of cytochrome P-448 is lowered.     

Interaction of coumarin-hydroxylating cytochrome P-450coh from liver microsomes of mice induced by pyrazole with cytochrome b5

Cytochrome P-450coh from pyrazole-treated mice was shown to form a tight and specific complex with cytochrome b5 from mouse liver microsomes. The complex formation was found to result in type I spectral changes indicating a spin shift from the low to the high spin form. When added to a reconstituted system containing cytochrome P-450coh, NADPH-cytochrome P-450 reductase and phospholipid, cytochrome b5 stimulates hydroxylation of coumarin and O-deethylation of 7-ethoxycoumarin. The maximal stimulating effect is reached at a 1:1 stoichiometry. Mouse liver cytochrome b5 stimulates hydroxylation and deethylation by 100% and 60%, respectively. The stimulating effect of cytochrome b5 was found to result from the increase of the maximal rate of oxidation, being practically without effect on Km. Cytochrome b5 purified from rat and rabbit liver microsomes interacts with cytochrome P-450coh but fails to stimulate the oxidation reaction. At large excess, cytochrome b5 inhibits the oxidations catalyzed by cytochrome P-450coh. Immobilized cytochrome b5 either from mouse or rat and rabbit microsomes proved to be an efficient affinity matrix for cytochrome P-450coh purification.     

Hepatic microsomal metabolism of androst-4-ene-3,17-dione: Relative importance of ring hydroxylation and aromatization in control and induced rat liver

The purpose of these studies was to determine whether oestrogen production is a quantitatively important pathway in the hepatic microsomal metabolism of androst-4-ene-3,17-dione. The effects of the enzyme inducing agents phenobarbitone and β-naphthoflavone on microsomal cytochrome P-450-mediated androst-4-ene-3,17-dione hydroxylation and aromatization was investigated in the rat in vitro. In microsomal fractions from untreated rats the ratio of hydroxylated products to aromatized (oestrogenic) metabolites was 33:1. Phenobarbitone pretreatment of rats increased total hydroxylation by about 20% but did not change the ratio of hydroxylated to aromatized products (27:1). In contrast, β-naphthoflavone induction decreased total hydroxylation to about 35% of control but did not affect total aromatization. Thus the ratio of hydroxylation to aromatization was significantly lower than in control microsomes (17:1).The principal aromatized products were oestriol and 2-hydroxyoestradiol-17β, with oestradiol-17β and its 4-hydroxy metabolite as minor products; no oestrone was observed. In further studies of the microsomal metabolism of oestrone, the major product was oestradiol-17β whereas hydroxylated metabolites were only minor products. Oestradiol-17β, in contrast, was hydroxylated to a considerable extent. These findings suggest that oestrone is a better substrate for the microsomal 17β-oxidoreductase than it is for cytochrome P-450. It therefore appears likely that any oestrone formed from the aromatization of androst-4-ene-3,17-dione would be readily converted to oestradiol-17β which, in turn, is subject to cytochrome P-450-mediated hydroxylation. Although the liver is a site of C₁₉-steroid aromatization, it appears unlikely that this organ could contribute significantly to serum oestrogen levels since microsomal hydroxylases are readily able to convert aromatized products to biologically inactive metabolites.     

1-chloro-2,2,2-trifluoroethyl radical: Formation from halothane by human cytochrome P-450 in reconstituted vesicles and binding to phospholipids

We report the first complete structural characterization of a metabolically-produced halocarbon radical bound to a phospholipid. Human cytochrome P-450 and NADPH cytochrome P-450 reductase were reconstituted into vesicles composed of dioleoylphosphatidylcholine and egg phosphatidylethanolamine. The vesicles were incubated under argon with NADPH and [¹⁴C]-halothane (1-[¹⁴C]-2-bromo-2-chloro-1,1,1-trifluoroethane), the dioleoylphosphatidylcholine fraction was isolated and subjected to transesterification. Separation of the resulting fatty acid methyl esters resulted in one radioactive fraction which gas chromatography-mass spectrometry revealed to be a mixture of 9- and 10-(1-chloro-2,2,2-trifluoroethyl)-stearate methyl ester.     

Enzyme systems of insecticide detoxication in the Colorado beetle

The activity of three enzymatic systems of xenobiotic metabolism (cytochrome P-450-dependent monooxygenases, non-specific esterases and glutathione S-transferases) was studied at different stages of development of the Colorado potato beetle (Leptinotarsa decemlineata). Significant sex and ontogenetic differences in the content of cytochrome P-450, position of maxima of CO-difference spectra of the cytochrome P-450 reduced form and substrate specificity of cytochrome P-450 were revealed. The activity of non-specific esterases was shown to increase depending on the age of larvae. The insecticide 1-naphtholenol methylcarbamate which is metabolized by the system of cytochrome P-450-dependent monooxygenases is more toxic in the larvae than at the imago stage, which is correlated with the activity of this system at different stages of ontogenesis. The microsomal monooxygenase inhibitor, piperonylbutoxide, increases the insecticide toxicity in the Colorado beetle imago more than 2-fold. The experimental results testify to different contribution of the detoxication systems to the sensitivity of the Colorado beetle to insecticides at various metamorphosis stages.