Krystyna Hetnarski

Substituted imidazoles as inhibitors of microsomal oxidation and insecticide synergists

Abstract The inhibitory activity of forty-seven 1-, 2-, and 4(5)-substituted imidazoles toward microsomal epoxidation, hydroxylation, and N -demethylation in enzyme preparations from rat liver and armyworm gut has been evaluated. Many of the 1- and 4(5)-substituted compounds are among the most potent inhibitors of microsomal expoxidase and hydroxylase yet reported but are uniformly less active toward N -demethylation. The armyworm gut enzymes are generally more susceptible to inhibition than those from rat liver. Inhibition follows either competitive or mixed type kinetics depending on both the inhibitor and substrate involved. The imidazoles bind powerfully to cytochrome P-450 and yield type II difference spectra in oxidized and NADPH-reduced microsomes. Since spectral dissociation constants ( K s ) appear to closely parallel I 50 values, the inhibitory activity of the imidazoles is considered to result mainly from their capacity to bind to both cytochrome P-450 and closely related substrate binding sites. Several of the 1-arylimidazoles are potent in vivo synergists of carbaryl against NAIDM insecticide susceptible houseflies.

Structure-activity relationships in the effects of 1-alkylimidazoles on microsomal oxidation in vitro and in vivo

The inhibitory activity of a series of 13 1-alkylimidazoles toward microsomal epoxidation of aldrin in enzyme preparations from rat liver and armyworm (Prodenia eridania) gut is optimal in compounds with a chain length of 8–10 carbon atoms. The capacity of the imidazoles to bind to cytochrome P-450 (type II) appears to be closely related to their inhibitory activity. The activity of the compounds in vivo in synergizing the toxicity of carbaryl to houseflies and potentiating pentobarbital sleeping time in mice closely parallels the data in vitro. Regression analyses clearly establish that both activity patterns in vitro and in vivo can be satisfactorily described by linear equations in terms of the hydrophobic bonding constant (π and π2) indicating a close correlation between biological activity and lipophilic character.

Selective inhibitory interactions of alkoxymethylenedioxybenzenes towards mono-oxygenase activity in rat-hepatic microsomes

A series of eight 4-n-alkoxymethylenedioxybenzene (AMDB) derivatives were evaluated for their inhibitory effects on several mono-oxygenase reactions and their capacity to form metabolite complexes with cytochrome P-450 in vitro in hepatic microsomes from phenobarbital (PB)-and Beta-naphthoflavone (Beta NF)-induced rats. Ethoxyresorufin O-deethylase in Beta NF-induced microsomes and aminopyrine N-demethylase in PB-induced microsomes were most susceptible to inhibition by the test compounds. In contrast, aldrin epoxidation and arylhydrocarbon hydroxylase in PB-and Beta NF-induced microsomes, respectively, were not inhibited by derivatives of AMDB. All AMDB derivatives elicited spectral complexes with cytochrome P-450, the characteristics of which were influenced by the microsomes employed and by the length of the AMDB alkoxy side-chain. Derivatives containing short-chain alkoxy substituents (C1 to C3) formed unstable metabolite complexes and generated substantial quantities of carbon monoxide (CO), those with intermediate length alkoxy groups (C4 to C6) generated little CO and rapidly formed intense spectral complexes (large delta A max), and those with the largest alkoxy groups (C7 and C8) formed no CO and elicited complexes of high stability. Quantitative structure-activity analyses showed that the biological data could be described by parabolic equations in II, the hydrophobic constant of the alkoxy substituent, and suggested the importance to AMDB interactions of a lipophilic-binding region at the active centre of the cytochrome P-450. The alkoxy chain length for optimal mono-oxygenase inhibition and complex formation with cytochrome P-450 appeared to be about five or six carbon atoms. The data suggest that the capacity of AMDB compounds to form stable inhibitory complexes with cytochrome P-450 may not always be associated with their ability to inhibit mono-oxygenase activity.

Aryl hydrocarbon hydroxylase in larvae of the southern armyworm (Spodoptera eridania)

Abstract Aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity in midgut microsomes of southern armyworm ( Spodoptera eridania ) larvae was induced 11-fold and 5.6-fold respectively following three days of feeding on diets containing pentamethylbenzene or naphthalene (both 0.2% w/v). β-Naphthoflavone and Aroclor 1254 were less effective inducers of AHH activity, phenobarbital was only slightly active and 3-methylcholanthrene caused a decrease in enzyme activity. AHH activity in microsomes from untreated and induced larvae was susceptible to inhibition by α-naphthoflavone, 1-phenylimidazole and piperonyl butoxide. Equilibrium dialysis studies with 1-(4′- 3 H-phenyl)imidazole showed that control and induced armyworm midgut microsomes contained a class of cytochrome(s) P-450 with a uniformly high affinity for phenylimidazole. It is concluded that AHH activity in the armyworm is catalyzed by a class of cytochrome(s) P-450 with characteristics intermediate between mammalian cytochrome(s) P-450 and P-448.

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

Abstract 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 427 458 nm peak ratios, whereas those with electron-withdrawing groups exhibited low 427 458 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.

Cytochrome P-450 and monooxygenase activity in hepatic microsomes from N-phenylimidazole-treated rats

Repeated administration of N-phenylimidazole (PI) to rats (3 daily doses of 200 mumol/kg/day) enhanced hepatic microsomal cytochrome P-450 levels (approx. 130%) and aminopyrine N-demethylase (APDM) and aniline p-hydroxylase (APH) activities (approx. 140%); aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD) activities were not enhanced over control values under similar conditions. Spectral studies with PI-induced microsomes indicated that although type II PI-binding characteristics were similar to those observed in controls, the 427 nm/455 nm absorbance ratio of the type III dihydrosafrole metabolite-cytochrome P-450 complex was lower than that in control microsomes. The results suggest that the inducing characteristics of PI bear some resemblance to those of phenobarbital (PB).

The functional role of nadph-cytochrome c reductase in southern armyworm (Spodoptera eridania) midgut microsomes

A rabbit antibody was prepared against a purified protease (bromelain)-solubilized fragment of NADPH-cytochrome c reductase from microsomes of southern armyworm (Spodoptera eridania) larval midguts. The antibody cross-reacted with the reductase from both control and pentamethylbenzene-induced midgut microsomes and inhibited the NADPH-dependent, but not the NADH-dependent, reduction of artificial electron acceptors. It also inhibited microsomal epoxidation, N-demethylation and O-demethylation, thus establishing NADPH-cytochrome c reductase as an obligatory component of the electron transport pathway leading to cytochrome P-450 in midgut microsomes. SDS-polyacrylamide gel electrophoresis of the immunoprecipitate obtained by treatment of deoxycholate-solubilized microsomes with the antibody indicated that the native reductase has a mol. wt. of 80,500. The antibody to the insect enzyme exhibited no cross-reactivity towards rat liver NADPH-cytochrome c reductase and an antibody prepared to the latter was similarly inactive towards the insect enzyme.

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

Abstract 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.