1H nuclear magnetic resonance and magnetic circular dichroism studies of ferric low-spin cytochrome P-450scc

400 MHz ¹H NMR of ferric low-spin cytochrome P-450_scc purified from bovine adrenal cortex was measured for the first time. As compared with ¹H NMR spectra of low-spin P-450_cam and metMb- mercaptan complexes, paramagnetic shifts of low-spin P-450_scc complexes were more divergent, suggesting that there is a subtle difference in the heme environment between P-450_scc and P-450_cam [1]. The paramagnetic shifts of low-spin complexes of P-450_scc caused by adding nitrogenous inhibitors, aminoglutethimide and metyrapone, were different from those caused by adding an intermediate, 20α-hydroxycholesterol, and a detergent, Tween 20 [2]. The paramagnetic shifts of the metMb-mercaptan complexes were convergent compared with those of ferric low-spin P-450_scc and P-450_cam, suggesting that the electronic character and/or the conformation of the internal thiolate ligand in P-450_scc and P-450_cam are different from those of the external thiolate ligand in metMb-thiolate complexes [3]. The paramagetic shifts of the metMb-mercaptan complexes were dependent on the electron donating factor of the alkyl group of the bound mercaptans [4].Magnetic CD(MCD) spectra of ferric low-spin P-450_scc, rabbit liver P-450 complexes and metMb- mercaptan complexes were also observed at various temperatures. The temperature dependences of the Soret MCD bands for the low-spin P-450 and metMb- mercaptan complexes were decidedly less pronounced than those for the low-spin metMb-CN? or imidazole complexes, suggesting that thiolate ligands markedly influence the Soret MCD band of the ferric low-spin complexes [1]. The suggestion described in [2] implied by the ¹H NMR study was reconfirmed from the temperature dependence study of the Soret MCD [2]. The temperature dependences of the Soret MCD bands for low-spin P-450 complexes having a non-nitrogenous ligand were more pronounced than for those having a nitrogenous ligand.     

Synergism of diacylhydrazine insecticides with metyrapone and diethylmaleate

Abstract:  The two metabolic synergists, metyrapone (2-methyl 1,2-di-3-pyridyl-1-propanone) and diethylmaleate could increase the toxicity of the three most recent diacylhydrazines, representing a novel group of insect growth regulators (IGRs). These components are known as respective inhibitors of oxidative and glutathione S-transferase enzymes. Larvicidal toxicity was tested in the laboratory by oral treatment against last-instar larvae of the Colorado potato beetle Leptinotarsa decemlineata (Say) and of the beet armyworm Spodoptera exigua (Hübner). The obtained results should enable to improve the activity of tebufenozide, methoxyfenozide and halofenozide, and it may be used to suggest potential mechanisms for resistance against this novel group of IGRs, that is important to counteract cross-resistance with other insecticide groups.     

Inhibitory effect of an in vivo activated soft analogue of A-phenyl-B-triazolium metyrapone during pupariation in Sarcophaga bullata

Experiments with the ovoviviparous fleshfly Sarcophaga bullata (Parker) (Dipt., Sarcophagidae), showed that the compound 4-(dodecanoyloxymethyl)-1-(2-methyl-1-oxo-1-phenyl-2-propyl)-(1,2,4-triazolium) chloride (NKI-42002) was an in vivo inhibitor of the cytochrome P-450-dependent monooxygenase system (MFO) during pupariation. The hard analogue of A-phenyl-B-triazolium metyrapone and the soft analogue of A-phenyl-B-imidazolium metyrapone showed no specific activity. Our results suggest that the soft quaternary group may help the transport of A-phenyl-B-triazolium metyrapone to the MFO system.A dose of 0.8 nmole/spec. of NKI-42002 had both a specific (delay in pupariation:1.64 ratio) and toxic (perc. of mortality:56) effect. The effects of the 0.4 nmole/spec. NKI-42002 can be eliminated by the simultaneous injection of 0.4 pmole/spec. of 20-OH ecdysone. These reversal studies support the hypothesis that NKI-42002 interferes with the biosynthesis of 20-OH ecdysone.

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Résumé Au cours d'études sur la mouche ovovivipare Sarcophaga bullata, on a constaté que le composé 4-(dodécanoyloxyméthyl)-1-(2-méthyl-1-oxo-1-phenyl-2-propyl)-(1,2,4-triazolium) chlorure (NKI-42002) était un inhibiteur in vivo du système cyt. P-450 dépendant mono-oxygénase (MFO) lors de la formation des pupes. Les analogues, solide du A-phényl-B-triazolium métyrapone et fluide du A-phényl-B-imidazolium métyrapone, n'ont présenté aucune activité spéciale. Ces résultats suggèrent que le groupe quaternaire fluide peut faciliter le transfert du A-phényl-B-triazolium métyrapone au système MFO.La dose de 0,8 nmole/ind. de NKI-42002 a présenté une action tant spécifique (retard à la pupaison; ratio 1,64), que toxique (mortalité 56%). On peut éliminer l'influence d'une dose de 0,4 nmole/ind. de NKI-42002 par injection simultanée d'une dose de 0,4 pmole/ind. de 20-OH ecdysone. Ces résultats confortent l'hypothèse suivant laquelle le NKI-42002 interfère avec la biosynthèse de la 20-OH ecdysone.

     

Evidence of Metyrapone Reduction by two Mycobacterium strains shown by 1H NMR

In situ 1H NMR monitoring of metyrapone incubations with resting-cells of two strains of Mycobacterium, Mycobacterium aurum MO1 and Mycobacterium sp. RP1, showed the biotransformation of this compound, and more precisely the carbonyl-reduction of metyrapone into the corresponding alcohol, metyrapol. This reduction produced both enantiomers. The use of inhibitors allowed us to show the multiple enzymatic activities involved in this biotransformation including carbonyl reductase (EC 1.1.1.1.84) from the short-chain dehydrogenase superfamily and aldehyde reductase (EC 1.1.1.2) from the aldo-keto reductase superfamily.     

Thiomorpholine and morpholine oxidation by a cytochrome P450 in Mycobacterium aurum MO1. Evidence of the intermediates by in situ 1H NMR

Spectrophotometric assays of Mycobacterium aurum MO1 cells extracts gave evidence of a soluble cytochrome P450, involved in the degradative pathway of morpholine, a waste product from the chemical industry. In order to get further information, the kinetics of the biodegradation of the sulfur analogue thiomorpholine was monitored by using in situ nuclear magnetic resonance (NMR). This technique allowed the identification of two intermediates: the sulfoxide of thiomorpholine resulting from S-oxidation and thiodiglycolic acid owing to ring cleavage. The S-oxidation (S SO) represents one of the well-known reactions catalyzed by cytochromes P450. The inhibitory effect of metyrapone, a cytochrome P450 inhibitor, on the thiomorpholine and morpholine degradative abilities of M. aurum MO1 confirmed the involvement of a cytochrome P450. These results and the decrease of the rate of formation of the first intermediate during the morpholine degradation, 2-(2-aminoethoxy) acetate, proved the key role of the cytochrome P450 in the early events of the biodegradation, i.e, in the C–-N bond cleavage.     

Species differences in the biochemical properties of liver microsomal arylamine and arylamide N-hydroxylases

Cytochrome P-448 dependent microsomal N-hydroxylases are key enzymes in the metabolic activation of both arylamides and arylamines.Using 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF) as substrates, the present report compares the biochemical properties of rat, hamster and mouse liver N-hydroxylases. There are marked species differences both in terms of the affinity for the two substrates and in terms of maximum velocity of the enzymes. The rat and hamster liver arylamide N-hydroxylases are induced by pretreatment with 2-AAF which also significantly increases their affinity for the substrate. In mouse liver neither arylamide nor arylamine N-hydroxylases are modified or induced. With 2-AF as substrate, arylamide treatment never enhances N-hydroxylation but it reduces the K_m-value of the rat and hamster liver enzymes.Among the effectors tested in vitro, 3-methylcholanthrene (3-MC), 7,8-benzoflavone (BF), benzo[α]pyrene (B[α]P) and miconazole (MN) inhibit hepatic arylamide N-hydroxylase in the submicromolar range. Harman (H) and paraoxon (PX) act in a dose-dependent manner in the micromolar range and metyrapone (MP) is not an inhibitor even at 50-μM concentration.Among the position isomers, 1- and 3-AAF are inhibitors of the N-hydroxylating enzymes whereas 4-AAF is not.These data are discussed in relationship to the toxic effects (mutagenicity and hepatocarcinogenicity) of arylamides and arylamines with respect to the role and the complexity of their microsomal metabolism.     

Effect of restraint stress on rat brain serotonin

Restraint-induced stress in rats was found to enhance steady state concentrations of whole brain and hypothalamic serotonin, at 1,2 and 4 h after immobilization. The increase was maximal at 1 h and tended to decline thereafter. The rate of accumulation of rat brain serotonin, in pargyline pretreated animals, was significantly enhanced after restraint stress. Bilateral adrenalectomy and metyrapone, an endogenous corticoid synthesis inhibitor, failed to affect restraint stress (1h)-induced increase in rat brain serotonin levels. Thus restraint stress-induced autoanalgesia and potentiation of the pharmacological actions of several centrally acting drugs, in rats, are serotonin-mediated responses. The results also indicate that restraint stress-induced effects on rat brain serotonin are not dependent on endogenous corticoid activity.     

Effects of cholinesterase inhibitors and metyrapone on wandering and pupation in the tobacco hornworm, Manduca sexta

Eserine, BW 284c51 and neostigmine inhibit larval cholinesterase activity in vitro. Injection of eserine, neostigmine or metyrapone into feeding fifth instar larve had no effect on the normal onset of wandering. Eserine injection during the prepupal stage interfered with the development of the pupa.