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Abstract Background The role of Aspergillus fumigatus mycotoxins in the colonization of the respiratory tract by conidia has not been studied extensively, even though patients at risk from invasive aspergillosis frequently exhibit respiratory epithelium damage. In a previous study, we found that filtrates of A. fumigatus cultures can specifically alter the electrophysiological properties of human nasal epithelial cells (HNEC) compared to those of non pathogenic moulds. Results We fractionated the organic phase of filtrate from 3-day old A. fumigatus cultures using high-performance liquid chromatography. The different fractions were tested for their ability to modify the electrophysiological properties of HNEC in an in vitro primary culture model. The fraction collected between 20 and 30 min mimicked the effects of the whole filtrate, i.e. decrease of transepithelial resistance and increase of potential differences, and contained secondary metabolites such as helvolic acid, fumagillin, and verruculogen. Only verruculogen (10-8 M) had effects similar to the whole filtrate. We verified that verruculogen was produced by a collection of 67 human, animal, plant and environmental A. fumigatus isolates. Using MS-MS analysis, we found that verruculogen was associated with both mycelium and conidia extracts. Conclusion Verruculogen is a secondary metabolite that modifies the electrophysiological properties of HNEC. The role of these modifications in the colonization and invasion of the respiratory epithelium by A. fumigatus on first contact with the epithelium remains to be determined.

In the present study, we synthesized 14C-labelled 5-nitro-l.2.4-triazol-3-one (NTO) and investigated its hepatic metabolism by dexamethasone-induced murine hepatic microsomes. Under the nitrogen atmosphere. 5-amino-l.2.4-triazol-3-one was the only detected metabolite of NTO. The microsomal nitroreductase activity was dependent on NADPH, totally inhibited by carbon monoxide and partially inhibited by oxygen. In aerobic conditions, beside a low amount of amine, the major metabolite formed is the 5-hydroxy-triazolone, urazole. This compound resulted from the oxidative denitrification of NTO, which produced equivalent amount of nitrite. This reaction, like the nitroreductase activity, was dependent on NADPH and totally inhibited by carbon monoxide. Both nitroreduction and oxidative denitrification were inhibited by imidazole-related inhibitors: miconazole and methimazole, and to a less extent by N-octylamine. The microsomal denitrification was induced by the treatment of mrs with dexamethasone and phenobarbital. The microsomal reductase activity is present in untreated rat microsomes, and recovered with various inducers. The results of this study indicate the role played by cytochrome P-450 in the metabolism of NTO. supported by its transformation with reconstituted cytochrome P-450 systems. [ABSTRACT FROM AUTHOR]

Cytochrome P-450cam oxidises 1,2,3,4-tetrahydro-β-carboline, an indolic alkaloid. We report here measurements of the product distribution of this oxidation. To rationalise the experimental results, ab initio quantum-chemistry calculations of the product stabilities and molecular-dynamics calculations of the substrate-binding mode in the active site were performed. The calculations suggest that the product distribution is influenced by both the relative intrinsic gas-phase stabilities of the monohydroxy products and by conformational rearrangement of the active site on substrate binding. [ABSTRACT FROM AUTHOR]

The reaction of p-chlorophenylhydrazine with prostaglandin H synthase (PGHS) Fe(III) under aerobic conditions leads to a partial destruction of the heme and to a new complex absorbing at 436 nm. This complex is also obtained by reaction of p-chlorophenyldrazne (pClPhN = NH) with PGHS Fe(III) under anaerobic conditions and by oxidation of the PGHS Fe(II)(pClPhN - NH) diazene complex by Fe(CN)6K3. The similarity between those reactions and those of arylhydrazines and aryldiazenes with other hemoproteins such as cytochrome P450 and hemoglobin and myoglobin, as well as the similarities between the spectroscopic and chemical properties of this complex and those of the c-aryl complexes of other hemoproteins such as hemoglobin and myoglobin, strongly suggested a PGHS Fe(III)-pClPh structure for this complex. It was completely established after the extraction of its heme, by butan-2-one at 0°C under neutral or acidic conditions, which led to the σ-aryl PGHS-Fe(III)-pClPh complex and to N-phenylprotopoiphyrin IX, respectively. A mechanism is proposed for the formation of the PGHS Fe(III) pClPh complex; it includes the reduction of PGHS Fe(III) into PGHS Fe{II) with formation of the diazene pClPhN - NH. This diazene can bind to PGHS Fe(II) or be oxidized with formation of pClPh free radicals. These radicals can react with PGHS Fe(II) to form the PGHS Fe(III)-pClPh complex or with the protein, or may initiate free radical oxidations which could lead to destruction of the heme or of the protein. Other alkylhydrazines or arylhydrazines also react with PGHS Fe(III) under aerobic conditions with the formation of PGHS Fe(III)-R or aryl (Ar) complexes and heme destruction. Alkylhydrazines such as methylhydrazine, which lead to very reactive alkyl radicals, lead to very low amounts of PGHS Fe(III)-R complex and high amounts of heme destruction, whereas arylhydrazines bearing electron-withdrawing substituents such as 3.4-dichlorophenylhydrazine, which lead to stabilized aryl radicals, lead to a high amounts of PGHS Fe(III)-Ar complex and low amounts of heme destruction. Some information concerning the structure of the active site of PGHS has been drawn from those studies as follows: (a) the formation of σ-aryl PGHS Fe(III)-Ar and PGHS Fe(II)(ArN = NH) complexes bearing bulky ligands on the iron is in agreement with a large hydrophobic active site with an easily accessible iron atom and, (b) a comparison of the UV-visible characteristics of PGHS Fe(III)-R or Ar complexes with those of other hemoprotein-Fe(III)-R or hemoprotein-Fe(UI)-Ar complexes is in agreement with a histidine as the axial proximal ligand of the iron of PGHS. Finally, the formation of iron(III)-R or iron (III)-Ar complexes and the free radical oxidative degradation of the heme could both explain why monosubstituted hydrazines are powerful inhibitors of PGHS and very bad reducing cosubstrates of the peroxidase function of PGHS. [ABSTRACT FROM AUTHOR]

The alkyldiazenes RN = NH (R = CH3 or C2H5) react with reduced microsomal cytochrome P450 leading to complexes exhibiting a Sorer peak at 446 nm. Upon oxidation of the [cytochrome P450-Fe(II)(CH3N = NH)] complex with limited amounts of dioxygen, a new complex characterized by a Sorer peak at 486 nm is formed. The hitter complex was also formed upon slow reaction of methyldiazene with microsomal cytochrome P450-Fe(III) or in situ oxidation of methylhydrazine by limited amounts of O2 or ferricyanide. This complex is rapidly destroyed by O2 or ferricyanide in excess and more slowly by excess dithionite in the presence of CO. Reactions of ethyldiazene or benzyldiazene with cytochrome P450-Fe(III) afforded similar° complexes characterized by Sorer peaks around 480 nm. These results, when compared to those recently described on reactions of monosubstituted hydrazines RNHNH2 and diazenes RN=NH with hemoglobin and iron-porphyrins, are consistent with a [cytochrome P450-Fe(II)(P,N = NH)] structure for the 446-nm-absorbing complexes and a σ-alkyl cytochrome P450-Fe(III-R structure for the complexes characterized by a Soret peak around 480 nm. They also suggest a σ-cytochrome P450-Fe(III)-Ph structure for the complex derived from phenylhydrazine oxidation, recently described in the literature. Finally, they provide the first evidence that cytochrome P450-Fe(III)-R complexes are formed upon microsomal oxidation of alkyl or phenylhydrazines. [ABSTRACT FROM AUTHOR]

Cytochrome P-450cam oxidises 1,2,3,4-tetrahydro-β-carboline, an indolic alkaloid. We report here measurements of the product distribution of this oxidation. To rationalise the experimental results, ab initio quantum-chemistry calculations of the product stabilities and molecular-dynamics calculations of the substrate-binding mode in the active site were performed. The calculations suggest that the product distribution is influenced by both the relative intrinsic gas-phase stabilities of the monohydroxy products and by conformational rearrangement of the active site on substrate binding. [ABSTRACT FROM AUTHOR]