Iván Bélai

Antibiotic resistance and enhanced insecticide catabolism as consequences of steroid induction in the gram-negative bacterium Comamonas testosteroni.

The effects of steroid induction on antibiotic resistance against the fungal steroid fusidic acid (ramycin; 16-(acetyloxy)-3 alpha,11 alpha-dihydroxy-29-dammara-17(20), 24-dien-21-oic-acid) as well as on carbonyl reduction and degradation of the novel anti-insect agent NKI 42255 (2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanone) were studied in the Gram-negative soil bacterium Comamonas testosteroni strain ATCC 11996. Cells grown with testosterone as inducing agent showed a 5-6-fold elevation of antibiotic resistance against the fungal steroid fusidic acid. Furthermore, testosterone induction caused a faster uptake and different metabolism of the anti-insect agent NKI 42255 compared to control cultures, revealing carbonyl reduction of the substrate keto group as an initial degradation step in induced cells. It is concluded that the formerly described steroid inducible hydroxysteroid dehydrogenases/carbonyl reductases present in Comamonas testosteroni contribute to these altered phenotypes, thus establishing steroid-inducible catabolic pathways as important defense processes against natural and synthetic toxicants in certain bacteria, which are present in the intestinal microflora of mammalian species as well as in soil samples.

Biotransformation and detoxification of insecticidal metyrapone analogues by carbonyl reduction in the human liver

1. The carbonyl reduction of insecticidal metyrapone analogues to their hydroxyl metabolites by human liver microsomes and cytosol was examined. Metabolite quantification was performed by means of hplc determination and inhibition experiments, using specific carbonyl reductase inhibitors, were conducted. 2. The cytotoxicity of the ketones and their hydroxy metabolites was assessed with the MTT test, using Chang liver cells. 3. It was found that the alcohol derivatives are the major metabolite, both in microsomes and cytosol. The microsomal reductive metabolism, considered to be mediated by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) (EC 1.1.1.146), was more extensive than the cytosolic carbonyl reduction. In each case, this metabolism was inhibited significantly by equimolar concentrations of the microsomal 11 beta-HSD inhibitor glycyrrhetinic acid and the cytosolic carbonyl reductase inhibitor quercitrin, respectively. 4. The parent ketones were more cytotoxic than their alcohol metabolites. 5. These results demonstrate that the metyrapone analogues are extensively metabolized by human liver microsomes, presumably by 11 beta-HSD, to the less cytotoxic and readily excretable alcohols. 6. Since the metyrapone analogues can inhibit ecdysone 20-monooxygenase (EC 1.14.99.22), our results indicate potential application of these compounds as insecticides, which would be safer for humans, due to their reductive detoxification, mainly by the hepatic microsomal 11 beta-HSD, to the less toxic hydroxy metabolites.

11β-Hydroxysteroid dehydrogenase type 1: tissue-specific expression and reductive metabolism of some anti-insect agent azole analogues of metyrapone

The azole analogues of metyrapone are novel candidates for selective anti-insect agents that inhibit the synthesis of 20-hydroxyecdysone (20E), the moulting hormone of insects. Metyrapone, which is a model substrate for studying the reductive properties of oxidoreductases, is itself effectively reduced to the corresponding alcohol by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD 1). For this reason, the ability of 11beta-HSD 1 to metabolize the metyrapone analogues as well was studied. In addition, the expression (by Western blots) and activity (reduction/oxidation of dehydrocorticosterone/corticosterone) of 11beta-HSD 1 in different male and female mouse tissues were investigated. Xenobiotic carbonyl reductase activities in these tissues were assessed with metyrapone as a model substrate. The kinetic parameters of 11beta-HSD 1 with metyrapone analogues as substrates were calculated after high-pressure liquid chromatography (HPLC) determination of the product alcohols. Our results indicate that the novel insecticides are extensively metabolized by mouse 11beta-HSD 1. Moreover, the resulting alcohols are not only less toxic than the parent ketones but also have the potential, owing to the newly formed hydroxyl group, to be eliminated from the body by consecutive phase II reactions. Thus, the new metyrapone analogues may be potential anti-insect agents, safer for humans due to their reductive detoxification, mainly by the hepatic 11beta-HSD 1, and selectively affecting insect development by inhibiting ecdysone 20-monooxygenase (E-20-M).

Comparison of Components from Red and White Wines for Antimicrobial Activity by Biodetection after OPLC Separation

Abstract Using OPLC separation and subsequent biodetection with Pseudomonas savastanoi pv. phaseolicola antibacterial compounds have been detected in both red and white wine extracts. However, the microbicidal power of red wine extracts was, in all cases, higher than that of white wines. The red wines contain different types of antimicrobial compounds but trans-resveratrol (TR) and its reduced form (bibenzyl-3,5,4′-triol, BB) in some cases are determining components. When the formaldehyde (HCHO) capturing molecule was used in culture medium (BioArena system) the antimicrobial activity of all antibiotic-like compounds was decreased characteristically, that is, HCHO plays a role in the antibacterial activity of these known and unknown compounds.

Theoretical molecular descriptors relevant to the uptake of persistent organic pollutants from soil by zucchini. A QSAR study.

The uptake of persistent organic pollutants (POPs) from soil by plants allows the development of phytoremediation protocols to rehabilitate contaminated areas. The use of diverse theoretical descriptors has been reported in the literature for developing quantitative structure-activity relationship (QSAR) models for predicting the bioconcentration factors (BCFs) of POPs in different plants. In this paper an evaluation is given on the molecular properties of POPs in terms of theoretical molecular descriptors that are relevant to the uptake and accumulation of these persistent pollutants from soil by two zucchini varieties. Statistically significant and predictive linear regression models have been developed for the BCF values of 20 polychlorinated dibenzo-p-dioxins/dibenzofurans and 14 polyhalogenated biphenyls in two zucchini varieties based on retrospective data. The relevant parameters have been selected from a set of 1660 DRAGON, 150 VolSurf, and 11 quantum chemical descriptors. The two most significant regression models, containing VolSurf, DRAGON GETAWAY, and quantum chemical descriptors, displayed the following statistical parameters: (eq 3) n = 27, R(2) = 0.940, q(2) = 0.922, SE = 0.155, F = 392.1; (eq 4) n = 27, R(2) = 0.921, q(2) = 0.898, SE = 0.161, F = 140.4. Predictive capabilities of the equations have been validated by using external validation sets. The QSAR models proposed might contribute to the development of viable soil remediation strategies.

Interpretation of Scoring Functions Using 3D Molecular Fields. Mapping the Diacyl-Hydrazine-Binding Pocket of an Insect Ecdysone Receptor

A method is presented for the interpretation of receptor docking score values (rough measures of binding affinities) of ligands in terms of 3D molecular field interaction contributions. The FlexX and FlexX-Pharm methods were used to dock the structures of designed sets of ligands into the ligand-binding pocket of a selected receptor. In the next step the relationship was investigated between the FlexX and CScore scores and 3D molecular fields obtained for the docked conformations of the ligands, using the CoMFA (Comparative Molecular Field Analysis) and CoMSIA (Comparative Molecular Similarity Indices Analysis) methods. The approach yielded highly significant CoMFA and CoMSIA models demonstrating that a high portion of the variance in the docking score values of the ligands can be explained by steric, electrostatic, hydrophobic, and hydrogen bond donor and acceptor molecular field interaction contributions. The approach was exemplified by using the crystal structure of the ligand-binding domain of the ecdysone receptor (EcR) of the moth Heliotis virescens as well as virtual molecule libraries of analogues of known diacyl-hydrazine (DAH) type ecdysteroid agonists. By docking appropriately designed virtual compound libraries into the DAH binding pocket of EcR followed by CoMFA and CoMSIA of the docked conformations, hitherto unexplored regions of the receptor cavity could be mapped. By mapping the significant molecular field interaction contributions onto the model of the receptor-ligand complex, important receptor-ligand interactions could be highlighted that may help the design of novel highly scored receptor ligands. An advantage of the method is that no experimental biological activity data are required to exhaustively map the receptor-binding site.

Projuvenoids: Synthesis and biological evaluation of sulfenylated, sulfinylated, and sulfonylated carbamates

Applying the proinsecticide principle developed earlier for neurotoxic carbamate insecticides, a series of new N-sulfenylated, N-sulfinylated, and N-sulfonylated derivatives of fenoxycarb were synthesized and evaluated for juvenile hormone mimicking activity. Laboratory evaluations of the compounds using Pieris brassicae and Sitophilus oryzae, as well as field experiments using Bemisia tabaci, showed that several symmetrical biscarbamates with either a sulfenyl or sulfinyl bridge possessed higher activity than the parent carbamate. From the unsymmetrical compounds containing biologically inert derivatizing moieties, one of the sulfenylated biscarbamates also showed improved activity against P. brassicae. The changes in the biological activity of the sulfur-containing derivatives compared to that of the parent compound are attributed to the modified physicochemical characteristics, i.e., increased lipophilicity facilitating penetration, transport, as well as protection of the compound from metabolism.

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.

An enzyme-linked immunosorbent assay (ELISA) for the detection of acetochlor

Competitive enzyme-linked immunosorbent assays (ELISAs) were developed in hapten-homologous and hapten-heterologous formats for the detection of the chloroacetanilide herbicide acetochlor. ELISA systems were devised using antibodies generated against acetochlor conjugated to carrier proteins through a thioether moiety replacing the chlorine atom in the parent structure, while haptens modified both on the chloroacetyl moiety and on the ethoxymethyl group of acetochlor have been used for coating antigens. The optimized ELISA systems allowed the detection of acetochlor 0.2-65 µg/L, and cross-reactivity studies revealed high specificity of the immunoassay: only four (propisochlor, butachlor, alachlor and metolachlor) among 18 structurally related acetanilide herbicides, fungicides and intermediates showed significant (> 1%) cross-reactivity, with even the highest value (propisochlor) being below 10%. Assay performance was not affected detrimentally by methanol up to 10% (v/v) and ethanol up to 5% (v/v). Assay performance was tested by measuring acetochlor concentration in water samples and compared favorably ( r 2 = 0.976) with those detected by gas chromatographic method coupled with mass spectrometry (GC-MS) using solid-phase microextraction (SPME) for sample preparation.

Carbonyl reduction of an anti-insect agent imidazole analogue of metyrapone in soil bacteria, invertebrate and vertebrate species.

Carbonyl reduction to the respective alcohol metabolites of the anti-insect agent imidazole analogue of metyrapone, NKI 42255 (2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanone) and its parent compound metyrapone was characterized in subcellular fractions previously described bacterial and mammalian hydroxysteroid dehydrogenases/carbonyl from soil bacteria, as well as insect, invertebrate and teleost species. The enzymes involved in this metabolic step were characterized with respect to their cosubstrate specificities, inhibitor susceptibilities, and immunological crossreactivities with antibodies directed against reductases (HSD/CR). All fractions investigated rapidly reduced metyrapone, with highest specific activities found in insect, invertebrate and vertebrate fractions. Except for the insect fractions, all species examined reduced the NKI compound. Cosubstrate dependence and inhibitor specificities suggest that the enzymes described belong to the protein superfamilies of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR). Immunological crossreactions to the previously established subgroup of HSD/CRs were found in trout liver microsomes and insect homogenates, but not in all bacterial extracts or earthworm microsomes. These findings suggest that the high CR activities found in these fractions belong to different subgroups of SDR or AKR.