Total mineralization of 2-ethylhexyl nitrate by bacterial cocultures

2-Ethylhexyl nitrate (2-EHN) is a widely–used chemical which is commonly added to diesel oil to boost its cetane index. The 2-EHN molecule is recalcitrant to biodegradation but still utilized as sole carbon source by Mycobacterium austroafricanum IFP 2173. The incomplete degradation of 2-EHN by this strain results in the accumulation of an intermediary metabolite i.e. 4-ethyldihydrofuran-2(3H)-one (4-EDF). The study aimed at isolating 4-EDF degraders in order to achieve total mineralization of 2-EHN in cocultures with M. austroafricanum IFP 2173. Bacterial isolates were obtained from diesel-contaminated soil by enrichment in serial cultures supplemented with 4-EDF, the degradation of which was monitored by CO₂ measurements. Two strains were isolated and identified as Bacillus cereus and Burkholderia sp., respectively. Complete mineralization of 2-EHN was achieved by associating M. austroafricanum IFP 2173 with either bacterial isolate in cocultures. In the context of environmental acceptability, efficient degradation of a potentially persistent pollutant by a bacterial consortium is demonstrated.     

Role of undecan-2-one on ethanol-induced apoptosis in HepG2 cells

Based on the reduced expression of ethanol-oxidizing enzymes in human hepatocellular carcinoma (HepG2) cells, we analyzed the role of nonoxidative metabolites in ethanol-induced apoptosis in HepG2 cells. For this purpose, an analysis of volatile metabolites of ethanol using ion-mobility spectrometry and gas chromatography–mass spectrometry was performed. HepG2 cells exposed to 1 mmol/L ethanol exhibited significant synthesis of undecan-2-one compared to untreated cells. Undecan-2-one is a fatty acid ethyl ester metabolite synthesized through a nonoxidative pathway. Undecan-2-one had a dose-dependent cytotoxic effect on HepG2 cells as shown by release of lactate dehydrogenase (LDH). The most notable finding of this study was the potentiation of ethanol-induced apoptosis demonstrated by an increased apoptotic rate induced by undecan-2-one in ethanol-treated HepG2 cells. The data presented in this study contribute to the better understanding of the molecular mechanisms of ethanol exposure at low concentration in HepG2 cells, a human hepatocellular carcinoma-derived cell line.     

Isomer of antiaggregative pheromone identified from male Douglas-fir beetle: 3-Methylcyclohex-3-en-1-one

A compound previously detected in volatile material released by Dendroctonus pseudotsugae was identified by coupled gas chromatography/mass spectrometry as 3-methylcyclohex-3-en-1-one (3,3-MCH). This is an isomer of 3-methylcyclohex-2-en-1-one (3,2-MCH, formerly called MCH), a multifunctional pheromone of this species. Not previously reported in natural products, it was collected from live male beetles in larger quantities than 3,2-MCH and frontalin, which were also released.     

A global proteome study of Mycobacterium gilvum PYR-GCK grown on pyrene and glucose reveals the activation of glyoxylate, shikimate and gluconeogenetic pathways through the central carbon metabolism highway

Various hydrocarbons have been released into the environment as a result of industrialization. An effective way of removing these materials without further environmental contamination is microbial bioremediation. Mycobacterium gilvum PYR-GCK, a bacteria isolated from a PAH polluted estuary, was studied using comparative shotgun proteomics to gain insight on its molecular activity while using pyrene and glucose as sole carbon and energy sources. Based on annotated genomic information, a confirmation analysis was first performed to confirm its pyrene degradation activity, using gas chromatography–mass spectrometry technology. One dimensional gel electrophoresis and liquid chromatography–mass spectrometry technologies employed in the proteomics analysis revealed the expression of pyrene degrading gene products along with upregulated expression of proteins functioning in the glyoxylate and shikimate pathways, in the pyrene-induced cells. The study also revealed the pathway of pyrene degraded intermediates, via partial gluconeogenesis, into the pentose phosphate pathway to produce precursors for nucleotides and amino acids biosynthesis.     

Biodegradability of 2-ethylhexyl nitrate (2-EHN), a cetane improver of diesel oil

The 2-ethyhexyl nitrate (2-EHN) is currently added to diesel oil to improve ignition and boost cetane number. The biodegradability of this widely used chemical needed to be assessed in order to evaluate the environmental impact in case of accidental release. In aerobic liquid cultures, biodegradation of 2-EHN was assessed in biphasic liquid cultures using an inert non-aqueous phase liquid such as 2,2,4,4,6,8,8-heptamethylnonane (HMN) as solvent for the hydrophobic substrate. 2-EHN was found to be biodegradable by microbial communities from refinery wastewater treatment plants, but was recalcitrant to those of urban wastewater treatment facilities. Out of eighteen hydrocarbon-polluted or non-polluted soil samples, six microbial populations were also able to degrade 2-EHN. However, strain isolation from these microbial populations was rather difficult suggesting close cooperation between members of the microbial communities. Specific axenic bacterial strains selected for their ability to catabolize recalcitrant-hydrocarbons were also tested for their capacity to degrade 2-EHN. In liquid cultures with HMN phase as non-aqueous phase liquid, some Mycobacterium austroafricanum strains were found to degrade and mineralize 2–EHN significantly.     

A new sulphate metabolite as a long-term marker of metandienone misuse

Metandienone is one of the most frequently detected anabolic androgenic steroids in sports drug testing. Metandienone misuse is commonly detected by monitoring different metabolites excreted free or conjugated with glucuronic acid using gas chromatography mass spectrometry (GC–MS) and liquid chromatography tandem mass spectrometry (LC–MS/MS) after hydrolysis with β-glucuronidase and liquid–liquid extraction. It is known that several metabolites are the result of the formation of sulphate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulphation is an important phase II metabolic pathway of metandienone that has not been comprehensively studied. The aim of this work was to evaluate the sulphate fraction of metandienone metabolism by LC–MS/MS. Seven sulphate metabolites were detected after the analysis of excretion study samples by applying different neutral loss scan, precursor ion scan and SRM methods. One of the metabolites (M1) was identified and characterised by GC–MS/MS and LC–MS/MS as 18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one sulphate. M1 could be detected up to 26 days after the administration of a single dose of metandienone (5 mg), thus improving the period in which the misuse can be reported with respect to the last long-term metandienone metabolite described (18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one excreted in the glucuronide fraction).     

Decolourization of 4-Chloro-2-Nitrophenol by a Soil Bacterium,Bacillus subtilis RKJ 700

A 4-Chloro-2-nitrophenol (4C2NP) decolourizing strain RKJ 700 was isolated from soil collected from a pesticide contaminated site of India and identified as Bacillus subtilis on the basis of the 16S rRNA gene sequence analysis. Bacillus subtilis RKJ 700 decolourized 4C2NP up to concentration of 1.5 mM in the presence of additional carbon source. The degradation pathway of 4C2NP was studied and 4-chloro-2-aminophenol, 4-chloro-2-acetaminophenol and 5-chloro-2-methylbenzoxazole (5C2MBZ) were identified as metabolites by high performance liquid chromatography and gas chromatography-mass spectrometry. Resting cell studies showed that Bacillus subtilis RKJ 700 depleted 4C2NP completely with stoichiometric formation of 5C2MBZ. This is the first report of (i) the degradation of 4C2NP at high concentration (1.5 mM) and, (ii) the formation of 5C2MBZ by a soil bacterium.     

Determination of hydralazine metabolites: 4-hydrazinophthalazin-1-one and n-acetylhydrazinophthalazin-1-one by gas chromatography and s-triazolo[3,4-a]phthalazine and phthalazinone by high-performance liquid chromatography

Methods are described for the determination of 4-N-acetylhydrazinophthalazin-1-one, 4-hydrazinophthalazin-1-one, phthalazinone and s-triazolo[3,4-a]phthalazine in human urine.4-Hydrazinophthalazin-1-one and 4-N-acetylhydrazinophthalazin-1-one (following acid hydrolysis) are reacted with acetylacetone to give a distinctive pyrazole derivative which can be determined by gas chromatography using a nitrogen-specific detector.Phthalazinone and s-triazolo[3,4-a]phthalazine are measured underivatised by high-performance liquid chromatography.     

Formation of 5-[17beta-2H]androstene-3beta,17alpha-diol from 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one by the microsomal fraction of boar testis

After incubation of 3beta-hydroxy-5-[17,21,21,21-2H]-pregnen-20-one with the microsomal fraction of boar testis, the metabolites were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following metabolites were identified: 3beta,17alpha-dihydroxy-5-[21,21,21-3H]pregnen-20-one, 3beta-hydroxy-5-androsten-17-one, 5-androstene-3beta,17beta-diol, and 5-[17beta-2H]androstene-3beta,17alpha-diol. The presence of a 2H atom at the 17beta position of 5-androstene-3beta,17alpha-diol was confirmed by oxidizing the steroid with 3beta-hydroxy-steroid dehydrogenase of Pseudomonas testosteroni to obtain 17alpha-hydroxy-4-[2H]androsten-3-one and then by oxidizing the latter steroid with chromic acid to obtain nonlabeled 4-androstene-3,17-dione. Among these metabolites, the first three can be interpreted to be synthesized by a well documented pathway, including 17alpha-hydroxylation followed by side chain cleavage as follows: 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one leads to 3beta,17alpha-dihydroxy-2-[21,21,212H]-pregnen-20-one leads to 3beta-hydroxy-5-androsten-17-one leads to 5-androstene-3beta,17beta-diol. On the other hand, 5-androstene-3beta,17alpha-diol, which contained a 2H atom at the 17beta position, is not likely to be synthesized via above mentioned pathway in which nonlabeled 3beta-hydroxy-5-androsten-17-one is formed as the first C19-steroid. It seems that an alternate side chain cleavage mechanism leading from pregnenolone to 17alpha-hydroxy-C19-steroid exists in boar testis.     

Derivatization of bioactive carbazoles by the biphenyl-degrading bacterium <Emphasis Type="Italic">Ralstonia</Emphasis> sp. strain SBUG 290

Different 9H-carbazole derivatives have been investigated within the last decades due to their broad range of pharmacological applications. While the metabolism of 9H-carbazole has previously been reported, nothing was known about the bacterial transformation of 2,3,4,9-tetrahydro-1H-carbazole and 9-methyl-9H-carbazole. Thus, for the first time, the bacterial biotransformation of 2,3,4,9-tetrahydro-1H-carbazole and 9-methyl-9H-carbazole was analyzed using biphenyl-grown cells of Ralstonia sp. strain SBUG 290 expressing biphenyl 2,3-dioxygenase. This strain accumulated 3-hydroxy-1,2,3,5,6,7,8,9-octahydrocarbazol-4-one and 6′-iminobicyclohexylidene-2′,4′-dien-2-one as major products during the incubation with 2,3,4,9-tetrahydro-1H-carbazole. Carbazol-9-yl-methanol was verified as the primary oxidation product of 9-methyl-9H-carbazole. In addition, 9H-carbazol-1-ol, 9H-carbazol-3-ol, and 3-hydroxy-1,2,3,9-tetrahydrocarbazol-4-one where detected in lower concentrations during the transformation of carbazol-9-yl-methanol and 9-methyl-9H-carbazole. Products were identified by high-performance liquid chromatography, gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry, as well as ¹H and ¹³C nuclear magnetic resonance analyses.     

Oxysterols from human bile induce apoptosis of canine gallbladder epithelial cells in monolayer culture

Oxysterols have been detected in various mammalian organs and blood. Biliary epithelium is exposed to high concentrations of cholesterol, and we have identified three keto-oxysterols (cholest-4-en-3-one, cholesta-4,6-dien-3-one, cholesta-3,5-dien-7-one) in human bile and gallstones. Because the effects of oxysterols on biliary physiology are not well defined, we investigated their biological effects on dog gallbladder epithelial cells. Enriched medium (culture medium containing taurocholate and lecithin and cholesterol +/- various oxysterols) was applied to confluent monolayers of dog gallbladder epithelial cells in culture. Cytotoxicity and apoptosis were studied by morphological analysis and flow cytometry. Oxysterols in the mitochondrial fraction were identified by gas chromatography/mass spectrometry, whereas release of cytochrome c from mitochondria was assayed by spectrophotometry and Western blot analysis. Compared with cells treated with culture medium or with enriched medium containing cholesterol, oxysterol-treated cells showed significantly increased apoptosis (P < 0.05). Exogenously applied oxysterols were recovered from the mitochondrial fraction. Cytochrome c release from mitochondria was increased significantly by cholest-4-en-3-one, cholesta-4,6-dien-3-one, and 5beta-cholestan-3-one (all P < 0.05). Thus oxysterols recovered from human bile and gallstones induce apoptosis of biliary epithelium via a mitochondrial-dependent pathway and may play a role in the pathogenesis of chronic inflammation and carcinogenesis in the gallbladder.     

Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one

A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4-cholesten-3-one as a substrate and extra- or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA-COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4-cholesten-6-ol-3-one based on comparisons of its high-performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton-nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4-cholesten-6-ol-3-one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6-hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3β-hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6-hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4-cholesten-3-one and 4-chotesten-6-ol-3-one by cholesterol oxidase, and the observed differences in the rate of formation of the 6-hydroxy-ketosteroid by the enzymes of different bacterial sources are also discussed.     

Macrocyclic trichothecene toxins produced by a strain of Stachybotrys atra from Hungary.

A strain of Stachybotrys atra isolated from a field case of stachybotryotoxicosis in Hungary was cultured in Hungary. All of the compounds toxic to brine shrimp were separated from the culture extract by solvent partition, column chromatography, and preparative thin-layer chromatography. Two of the toxic compounds were identified as verrucarin J and satratoxin H by comparison with pure standards resolved by high-pressure liquid chromatography and characterized by mass spectrometry. Two other toxic components were identified as roriden E and satratoxin G on the basis of their mass spectra. The fifth toxic compound was identified as a macrocyclic trichothecene based on the following findings: a positive 4-(p-nitrobenzyl)pyridine color reaction, hydrolysis resulting in verrucarol verified by combined gas chromatography-mass spectrometry, and a characteristic trichothecene proton-nuclear magnetic resonance spectrum. This macrocyclic trichothecene has a molecular ion (528) identical to satratoxin H, and its mass spectrum is similar; however, its Rf value on Silica Gel G differs.     

Degradation of the multiple branched alkane 2,6,10,14-tetramethyl-pentadecane (pristane) in Rhodococcus ruber and Mycobacterium neoaurum

Pristane, a highly branched hydrocarbon that also contains iso-branched termini, was used as a substrate for several alkane-metabolizing bacteria. Rhodococcus ruber and Mycobacterium neoaurum were able to utilize pristane for growth effectively. The intermediates produced by these bacteria during incubation with pristane were analyzed by gas chromatography (GC) and gas chromatography/mass spectra (GC/MS). The products revealed as products of 4-methyl pentanoic acid; methyl butanedioic acid; 2-methyl pentadioic acid; methyl propanedioic acid; 4-methyl heptanedioic acid; and 2,6,10,14-tetramethyl-pentadecan-3-one were detected in M. neoaurum cultures. In R. ruber, methyl butanedioic acid; 2-methyl pentadioic acid; 4,8-dimethylnonanoic acid, 4-methyl heptanedioic acid; 2,6,10-trimethylundecanoic acid; 3,7-dimethyl decanedioic acid; and 2,6,10,14-tetramethyl-pentadecan-3-one were detected. The occurrence of these intermediates showed that pristane could be catabolized not only via mono- but also by a di-terminal oxidation pathway. Furthermore, the presence of 2,6,10,14-tetramethyl-pentadecan-3-one; 3,7-dimethyldecandioate; and 2-methylbutandioate established a third pathway initiated by sub-terminal oxidation at the third carbon atom of pristane. Novel intermediates detected suggest simultaneous sub-terminal and di-terminal oxidation pathways.     

Quantification of steroid conjugates using fast atom bombardment mass spectrometry

Fast atom bombardment/mass spectrometry or liquid secondary ion mass spectrometry provides the capability for direct analysis of steroid conjugates (sulfates, glucuronides) without prior hydrolysis or derivatization. During the analysis of biologic extracts, limitations on the sensitivity of detection arise from the presence of co-extracted material which may suppress or obscure the analyte signal. A procedure is described for the quantitative determination of dehydroepiandrosterone sulfate in serum which achieved selective isolation of the analyte using immunoadsorption extraction and highly specific detection using tandem mass spectrometry. A stable isotope-labeled analog [( 2H2]dehydroepiandrosterone sulfate) was used as internal standard. Fast atom bombardment of dehydroepiandrosterone sulfate yielded abundant [M-H]- ions that fragmented following collisional activation to give HSO4-; m/z 97. During fast atom bombardment/tandem mass spectrometry of serum extracts, a scan of precursor ions fragmenting to give m/z 97 detected dehydroepiandrosterone sulfate and the [2H2]-labeled analog with a selectivity markedly superior to that observed using conventional mass spectrometry detection. Satisfactory agreement was observed between quantitative data obtained in this way and data obtained by gas chromatography/mass spectrometry of the heptafluorobutyrates of dehydroepiandrosterone sulfate and [2H2]dehydroepiandrosterone sulfate obtained by direct derivatization.     

Screening of eltanolone metabolites in dog urine by anion-exchange/reversed-phase liquid chromatography and mass spectrometry

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated ¹⁴C-labelled eltanolone (5β-Pregnan-3α-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of ¹⁴C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC–ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography–mass spectrometry with an electron impact ion source (GC–MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).     

Detection of designer steroid methylstenbolone in “nutritional supplement” using gas chromatography and tandem mass spectrometry: Elucidation of its urinary metabolites

The use of “nutritional supplements” containing unapproved substances has become a regular practice in amateur and professional athletes. This represents a dangerous habit for their health once no data about toxicological or pharmacological effects of these supplements are available. Most of them are freely commercialized online and any person can buy them without medical surveillance. Usually, the steroids intentionally added to the “nutritional supplements” are testosterone analogues with some structural modifications.In this study, the analyzed product was bought online and a new anabolic steroid known as methylstenbolone (2,17α-dimethyl-17β-hydroxy-5α-androst-1-en-3-one) was detected, as described on label. Generally, anabolic steroids are extensively metabolized, thus in-depth knowledge of their metabolism is mandatory for doping control purposes. For this reason, a human excretion study was carried out with four volunteers after a single oral dose to determine the urinary metabolites of the steroid. Urine samples were submitted to enzymatic hydrolysis of glucuconjugated metabolites followed by liquid–liquid extraction and analysis of the trimethylsilyl derivatives by gas chromatography coupled to tandem mass spectrometry. Mass spectrometric data allowed the proposal of two plausible metabolites: 2,17α-dimethyl-16ξ,17β-dihydroxy-5α-androst-1-en-3-one (S1), 2,17α-dimethyl-3α,16ξ,17β-trihydroxy-5α-androst-1-ene (S2). Their electron impact mass spectra are compatible with 16-hydroxylated steroids O-TMS derivatives presenting diagnostic ions such as m/z 231 and m/z 218. These metabolites were detectable after one week post administration while unchanged methylstenbolone was only detectable in a brief period of 45 h.     

Biochemical studies of tick embryogenesis. Free sugars in adult haemolymph and during embryogenesis of Dermacentor andersoni

Free sugars in eggs, embryos and adult haemolymph of the tick Dermacentor andersoni have been estimated by gas liquid chromatography and their identity confirmed by mass spectrometry. Glucose is the major, but not exclusive sugar in the haemolymph, whereas mannose is the principal free sugar in the eggs. The developmental profiles of mannose, glucose and myo-inositol during embryogenesis suggest their possible interconversion, and/or release from a bound form.     

Identification of metabolites of methylprednisolone in equine urine

Methylprednisolone and three metabolites, 17,21-dihydroxy-6 alpha-methyl-1,4-pregnadiene-3,11,20-trione, 6 alpha-methyl-17,20 beta,21-trihydroxy-1,4-pregnadiene-3,11-dione, and 6 alpha-methyl-11 beta,17,20 beta,21-tetrahydroxy-1,4-pregnadien-3-one were detected in equine urine after intraarticular administration of methylprednisolone acetate. All four compounds were excreted both in the unconjugated form and as glucuronic acid conjugates. They were identified by comparing data obtained from analyses by high performance liquid chromatography, thin-layer chromatography, ultraviolet spectroscopy and gas chromatography/mass spectrometry to those of the synthesized standards. The presence of trace amounts of a fourth metabolite, 6 alpha-methyl-11 beta,17,20 alpha,21-tetrahydroxy-1,4-pregnadien-3-one, was indicated by high performance liquid chromatography but confirmation has not been attained by the other methods.