Kinetics of uptake, clearance, transfer, and metabolism of hexaflumuron by eastern subterranean termites (Isoptera: Rhinotermitidae).

The rates of uptake, clearance, insect-to-insect transfer, and metabolism of [14C] hexaflumuron [N-(((3,5-dichloro-4-(1,1,2,2-tetrafluroethoxy)phenyl)- amino)carbonyl)-2,6-diflurobenzamide] were measured in eastern subterranean termite workers, Reticulitermes flavipes (Kollar), fed cellulose diets containing either 0.1 or 0.5% (wt:wt) hexaflumuron. The rate of uptake, level of maximum uptake, and amount of insect-to-insect transfer were concentration dependent. The clearance rate constant for hexaflumuron was independent of concentration, with a mean value of 3.2 x 10(-3)/h. This corresponds to a mean half-life for hexaflumuron inside termites of 9 d. No evidence of metabolism of hexaflumuron to additional products was detected when extracting and examining the radioactivity contained in the fecal and regurgitated material within the termite holding apparatus 40 d after exposure to the chemical. Hexaflumuron was efficiently transferred from treated to untreated termites, through trophallaxis, resulting in spread of the toxicant throughout the insect population. The combination of uptake and efficient transfer of hexaflumuron between treated and untreated termites ensures broad distribution of the material even to insects not directly exposed to the toxicant. The distribution of hexaflumuron by termite workers, along with their minimal ability to metabolize the compound to other metabolites, and their slow ability to clear the material from the termite population results in death of the entire group of termites contained within the holding apparatus.

Human red blood cell acetylcholinesterase inhibition as the appropriate and conservative surrogate endpoint for establishing chlorpyrifos reference dose.

Chlorpyrifos (O,O-diethyl O-(3,5, 6-trichloro-2-pyridinyl)- phosphorothioate) is an organophosphorus (OP) insecticide used for controlling insect pests. Currently, the reference dose (RfD) used by the Environmental Protection Agency (EPA) to establish acceptable human exposure tolerances for chlorpyrifos is based upon inhibition of blood butyrylcholinesterase (BuChE), which is not the target enzyme of chlorpyrifos, and does not play any role in cholinergic transmission. Data are presented showing that inhibition of acetylcholinesterase (AChE) associated with red blood cells (RBC), an enzyme similar to or identical with that in the nervous system, is a more appropriate endpoint on which to base the RfD. Basing an acceptable level of human exposure (e.g., RfD) on inhibition of RBC AChE provides a significant margin of safety, since it is 12- to 14-fold more sensitive as an indicator of chlorpyrifos exposure than the AChE in the most sensitive relevant neurological tissues (brain or retina). Inhibition of RBC AChE activity is consistently exhibited at lower dosages of chlorpyrifos than those required to result in clinical symptoms of OP toxicity, or alterations in cognitive functional responses. There is no unique sensitivity of the fetus or neonates to chlorpyrifos when administered by an appropriate oral dose. Thus, inhibition of RBC AChE activity is an appropriate surrogate measurement of chlorpyrifos exposure and provides a conservative endpoint for establishing appropriate margins of safety for both adults and infants.

Inhibition of rat liver microsomal cytochrome P-450 steroid hydroxylase reactions by imidazole antimycotic agents.

The imidazole antimycotic agents ketoconazole, miconazole and clotrimazole were tested for their abilities to inhibit the reactions involved in the oxidative metabolism of androst-4-ene-3,17-dione by rat liver microsomal cytochromes P-450. All three compounds were found to function as potent inhibitors of steroid hydroxylase reactions, producing 50% inhibition of 6 beta-, 16 beta-, and 16 alpha-hydroxylase activities at concentrations between 10(-7) and 10(-5) M. The antimycotic agents, when added to liver microsomes, bound to cytochrome P-450 with high affinity to produce a "type II" spectral complex. These agents showed differential inhibition of the various steroid hydroxylases and were found not to affect the activities of the liver microsomal steroid 5 alpha-reductase or the androst-4-ene-3,17-dione 17-oxidoreductase. The results presented demonstrate an interaction of these imidazole antimycotic agents with the various cytochromes P-450 of liver microsomes, resulting in selective inhibition of monooxygenase activity.

Multiple sites of steroid hydroxylation by the liver microsomal cytochrome P-450 system: primary and secondary metabolism of androstenedione.

To investigate the potential interaction of the various pathways of androgen hydroxylation, we have conducted studies to identify the profile of products formed during the time course of metabolism of androst-4-ene-3,17-dione (AD). Incubates containing AD, NADPH, and liver microsomes (from rats pretreated with phenobarbital) were sampled at times between 0 and 20 min and the metabolites resolved by reverse-phase (C18) high-performance liquid chromatography. By this method, the pattern of formation and of utilization of eight major primary and secondary metabolites of AD was determined. We report here the formation of two previously unidentified major metabolites of AD: 6 beta,16 alpha-dihydroxyandrost-4-ene-3,17-dione and 6 beta,16 beta-dihydroxyandrost-4-ene-3,17-dione. We propose that liver microsomal cytochromes P-450 can sequentially hydroxylate a single molecule of AD at multiple sites. These hydroxylase activities are presumably a result of multiple cytochrome P-450 isozymes acting on AD resulting in a transient time course for the appearance of some monohydroxylated metabolites. In addition, a unidirectional conversion of the metabolite 16 alpha-hydroxyandrost-4-ene-3,17-dione to 16 beta-hydroxyandrost-4-ene-3,17-dione is described. Evidence is provided to support the role of cytochrome P-450 in catalyzing this reaction.

Discriminatory inhibition of adrenocortical 17 alpha-hydroxylase activity by inhibitors of cholesterol side chain cleavage cytochrome P-450.

Two inhibitors of the cholesterol side chain cleavage reaction were tested for their ability to inhibit bovine adrenocortical 17 alpha-hydroxylase and 21-hydroxylase activities. One inhibitor, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), was found to be a potent inhibitor of 17 alpha-hydroxylation of either progesterone or pregnenolone but was inactive on 21-hydroxylase activity. 22-ABC was found to be a competitive inhibitor of 17 alpha-hydroxylase (cytochrome P-45017 alpha) activity, having an apparent inhibitor constant of 29 nM when using pregnenolone as the substrate. Spectral binding studies showed that 22-ABC produces a type II difference spectrum when added to a bovine adrenocortical microsomal preparation, due presumably to a coordination of its amine nitrogen atom to the heme-iron of cytochrome P-45017 alpha. The second cholesterol side chain cleavage inhibitor tested, (20R)-20-phenyl-5-pregnene-3 beta,20-diol (20-PPD), was found not to inhibit either the 21- or 17 alpha-hydroxylase activities. It is proposed that the phenyl group projecting from C-20 of 20-PPD prevents this steroid from binding to cytochrome P-45017 alpha. The discriminatory interaction of these two steroids with adrenocortical cytochromes P-450 provides some insight with respect to possible structural features of the active-site regions of these enzymes.

Imidazole antimycotics: inhibitors of steroid aromatase.

Miconazole and clotrimazole, members of a class of imidazole agents which have broad spectrum antimycotic activity, were shown to be potent inhibitors of steroid aromatase activity of human placental microsomes. The I50 values for the inhibition of aromatase activity by miconazole, clotrimazole, ketoconazole, and aminoglutethimide were 0.6, 1.8, 60 and 44 microM respectively. The most effective compound, miconazole, exhibited competitive kinetics with respect to androstenedione, the aromatase substrate. The apparent inhibitory constant (Ki) was 55 nM, under assay conditions where the apparent Km for androstenedione was 220 nM. The inhibition of aromatase activity by miconazole was shown to be reversible by dilution. Miconazole was a relatively poor inhibitor of the cholesterol side chain cleavage activity of a placental mitochondria-enriched fraction, while both clotrimazole and ketoconazole markedly inhibited this mitochondrial monooxygenase activity. Spectrophotometric studies revealed that miconazole bound to the cytochrome P-450 component of the placental microsomal aromatase complex and had negligible effect on NADPH-cytochrome c (P-450) reductase activity. These results strongly support direct interaction of miconazole with microsomal cytochrome P-450 in human placental microsomes with high affinity resulting in the inhibition of aromatase activity.

Ketoconazole: a potent inhibitor of cytochrome P-450-dependent drug metabolism in rat liver.

Ketoconazole, an orally active imidazole antimycotic agent, is shown to be a potent inhibitor of drug N-demethylase activities of liver microsomes from rats pretreated with phenobarbital or pregnenolone-16 alpha-carbonitrile, and an inhibitor of 7-ethoxyresorufin O-deethylase activity of liver microsomes from rats pretreated with 5,6-benzoflavone. Spectrophotometric studies reveal that the imidazole compound binds to the cytochrome P-450 component of the monooxygenase complex, and has little effect on NADPH-cytochrome c (P-450) reductase activity. These results are strongly suggestive that cytochrome P-450 is the site of action of this potent inhibitor of drug metabolism in liver microsomes.

Amino-steroids as inhibitors and probes of the active site of cytochrome P-450scc. Effects on the enzyme from different sources.

A series of analogues of cholesterol, each having a primary amine attached to a shortened side chain, were tested for their effects on cytochrome P-450scc from several different sources. Reconstituted enzyme systems using disrupted mitochondria from bovine adrenal and placenta, adult human adrenal and placenta, neonatal human adrenal, and rat adrenal and testis were used to assay for inhibitory effects on the side chain cleavage of cholesterol to pregnenolone. Two of the derivatives tested, 22-amino-23,24-bisnor-5-cholen-3 beta-ol and 23-amino-24-nor-5-cholen-3 beta-ol, were found to be potent inhibitors of this reaction; the derivatives in which the amine was attached closer to or further from the steroid ring, (20 R and S)-20-amino-5-pregnen-3 beta-ol and 24-amino-5-cholen-3 beta-ol, were much weaker inhibitors. In addition, spectral studies with rat adrenal mitochondria and a soluble preparation of human placental cytochrome P-450scc showed that binding of the 22-amine derivative to the enzyme produces difference spectra characteristic of nitrogen bonding to the heme; this indicates that the heme is positioned close to C-22 in the steroid-enzyme complex. These findings on the relative effectiveness of the amino-steroid inhibitors and the type of complex formed are similar to results obtained with purified bovine adrenocortical cytochrome P-450scc. This establishes that the proximity of the substrate binding site and the heme-iron catalytic site is a feature common to the enzyme from several sources and is therefore likely to be a necessary property of the active site structure.

Active site-directed inhibitors of cytochrome P-450scc. Structural and mechanistic implications of a side chain-substituted series of amino-steroids.

A series of analogues of cholesterol, each having a shortened side chain and a primary amine group, were prepared and tested for their effects on bovine adrenocortical cholesterol side chain cleavage cytochrome P-450 (P-450scc). A previous study had shown that one derivative, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, is a potent competitive inhibitor of the enzyme and forms a complex in which the steroid ring binds to the cholesterol site and the side chain amine forms a bond with the heme iron (Sheets, J. J., and Vickery, L. E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5773-5777). In the studies reported here, the 23-amine derivative, 23-amino-24-nor-5-cholen-3 beta-ol, was found to be an equally potent inhibitor and to be competitive with respect to cholesterol (Ki = 38 nM). Binding of the 23-amine to P-450scc also caused formation of a low spin complex with an absorption maximum at 422 nm, indicative of a nitrogen-donor ligand. Other derivatives in which the side chain amine was linked closer to the steroid, 17 beta-amino-5-androsten-3 beta-ol and (20 R + S)-20-amino-5-pregnen-3 beta-ol, were found to be only very weak inhibitors (I50 greater than 100 microM) and did not produce the 422 nm spectral form when bound. Derivatives in which the amine was attached a greater distance from the steroid ring, 24-amino-5-cholen-3 beta-ol and 25-amino-26,27-bisnor-5-cholesten-3 beta-ol, caused a progressive decrease in inhibitory potency and a failure to produce the 422 nm form on binding. The dependence of the type of interaction of these amino-steroids with P-450scc upon the amine position establishes that the steroid binding site and the heme catalytic site of the enzyme are fixed within a specific distance of one another. The heme appears to be located sufficiently close to the position that the side chain of cholesterol would occupy to allow for direct attack of an iron-bound oxidant to occur during hydroxylation and side chain cleavage.

Inhibition of steroidogenesis in cultured Leydig tumor cells by 22-amino-23,24-bisnor-5-cholen-3 beta-O1 and (20R) 20-phenyl-5-pregnene-3 beta,20-diol.

Using the cholesterol side-chain cleavage enzyme purified from bovine adrenals, we have previously shown that 22-amino-23,24-bisnor-5-cholen-3-beta-ol (22-ABC) and (20R) 20-phenyl-5-pregnene-3 beta, 20 diol (20-PPD) are potent competitive inhibitors of this enzyme. The studies presented herein were designed to characterize the effects of these new inhibitors on steroid production by intact cells. Using cultured Leydig tumor cells, we compared the effects of 22-ABC, 20-PPD, and aminoglutethimide (a well-known inhibitor of the cholesterol side-chain cleavage enzyme) on hormone-stimulated steroidogenesis. Our results show that these compounds inhibit steroid production in a dose-dependent manner and that 20-PPD and 22-ABC are about 100 and 4 times more active, respectively, than aminoglutethimide. In these cells, the inhibitory effects of the new compounds seem to be localized exclusively at the conversion of cholesterol to pregnenolone.

C-22-substituted steroid derivatives as substrate analogues and inhibitors of cytochrome P-450scc.

Spectral and kinetic studies are reported for the effects of C-22-substituted steroids on purified bovine adrenocortical cytochrome P-450scc. The results are consistent with the recent proposal that the potency of 22-amino-23,24-bisnor-5-cholen-3 beta-ol as an inhibitor of the enzyme arises from a dual interaction, the binding of the steroid ring to the cholesterol site and bonding of the amine to the heme iron (Sheets, J.J., and Vickery, L.E., (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5773-5777). An analogue of the inhibitor with the 5,6 double bond reduced, 22-amino-23,24-bisnor-5 alpha-cholan-3 beta-ol, was synthesized by a similar procedure. A complex of this form with P-450scc produced a 422 nm Soret absorption maximum as found for the parent compound, indicating nitrogen coordination to the heme iron. A decrease in the spectral dissociation constant and inhibitory potency was also observed and is consistent with binding of the steroid ring to the cholesterol site on the enzyme. The 22-hydroxy analogue, 23,24-bisnor-5-cholene-3 beta,22-diol, was also prepared. This derivative produced a complex with P-450scc having a Soret peak at 417 nm as in the substrate-free form of the enzyme; the diol was also a competitive inhibitor, but exhibited decreased potency relative to the amine form. These results provide additional support for the role of amine coordination in producing the 422 nm species and in contributing to tight binding.

Proximity of the substrate binding site and the heme-iron catalytic site in cytochrome P-450scc.

As an approach to "mapping" the active site of the cytochrome P-450 that catalyzes cholesterol side-chain cleavage, designated cytochrome P-450scc, we have synthesized steroid derivatives with the potential to interact with both the substrate binding site and the heme-iron catalytic site of the enzyme. The effects of these substrate analogs were studied with cytochrome P-450scc purified from bovine adrenal cortex. One derivative, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, was found to be a potent inhibitor of pregnenolone formation in a reconstituted enzyme system, and a kinetic analysis of the inhibition showed that binding of the derivative is competitive with respect to cholesterol. The spectral properties of a stable complex formed between the steroidal amine and the purified cytochrome suggest that the 22-amine group coordinates directly to the heme-iron. A model for the structure of this inhibitor-enzyme complex is proposed in which the 5-androstene ring system of the steroid occupies the substrate binding site, and the amine group of the side chain occupies an axial coordination position of the Fe(III) center. This places limits on the distance between these two domains in the enzyme and offers support for proposed mechanisms of cytochrome P-450-catalyzed oxygen-insertion reactions in which an iron-bound oxidant directly attacks the substrate.

Effects of aminoglutethiumide and its metabolite, N-acetylaminoglutethimide, on bovine adrenocortical and human placental cytochromes P-450scc.

The ability of aminoglutethimide to inhibit cholesterol conversion to pregnenolone was lost upon acetylation of the arylamine nitrogen. This appears to be due to failure of N-acetyl-d-aminoglutethimide to bind to cytochrome P-450scc, since it does not produce the altered low spin form of the enzyme formed upon binding of d-aminoglutethimide. These findings provide further evidence for a role of the free arenamine function in aminoglutethimide and related inhibitors.