Stereoselective degradation of tebuconazole in rat liver microsomes

The aim of this study was to assess the stereoselectivity of two tebuconazole [(RS)-1-p-chlorophenyl-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol] enantiomers in in vitro system (rat liver microsomes). The analytes were extracted with acetic ether and concentrations were determined by high performance liquid chromatography (HPLC) with a cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase. The degradation of rac-tebuconazole (15 μM) followed first-order kinetics, and the degradation of the S-tebuconazole (t(1/2) = 22.31 min) was faster than that of the R-tebuconazole (t(1/2) = 48.76 min), but no significant difference between the enantiomers was found in the respective incubation (7.5 μM for each). Kinetic assays showed that the K(m) was different between the two enantiomers (K(mR) = 14.83 ± 2.19, K(mS) = 12.23 ± 2.72). The interaction results revealed that there was competitive inhibition between S- and R-form, and there was a significant difference between the IC(50) of R- to S-tebuconazole and S- to R-tebuconazole (IC(50R/S)/IC(50S/R) = 4.98).     

Mutagenicity of metronidazole: Activation by mammalian liver microsomes

The mutagenicity of metronidazole, a widely used chemotherapeutic agent, for $\text{Salmonella typhimurium}$ was confirmed. Moreover using a mutant of of $\text{S. typhimurium}$ unable to activate metronidazole to a genetically active metabolite, it is shown that this activation can be carried out by a microsomal preparation devived from rat liver. Heretofore it had been postulated that this metabolic event was catalyzed solely by enzymes present in protozoa and anaerobic bacteria. The present findings which indicate that mammalian enzymes can activate metronidazole to a genetically active intermediate may have a direct relevance to the carcinogenicity of this agent.     

Activation of branched and other long-chain fatty acids by rat liver microsomes

Acyl coenzyme A synthetase (EC 6.2.1.3) of rat liver microsomes activates iso- and anteiso-branched long-chain fatty acids containing 12 to 20 carbon atoms. Fatty acid chain length appears to be the major determinant of the maximum rate of acyl CoA biosynthesis of branched, or saturated, or cis monounsaturated long-chain fatty acids. Based on activation studies conducted at 22-45 degrees C, it is concluded that the rate of activation is a function of long-chain fatty acid solubility. The shape of the in vitro activation curve with respect to fatty acid concentration appears to be determined by fatty acid melting point as well as by the presence and position of double bonds. Differently shaped activation curves were observed for cis or trans Delta(6) to Delta(12) central positional isomers of octadecenoic acid and for Delta(3), Delta(4), Delta(13) to Delta(15) terminal isomers of octadecenoic acid. The relationships between fatty acid structure, melting point, solubility, and shape of the activation curve observed during in vitro measurement of acyl CoA formation are discussed.     

Tracheobronchial washings from seven vertebrate species as growth media for the four species of Bordetella

Abstract The four species of Bordetella differed in their ability to grow at 37°C in membrane-filtered tracheobronchial washings (TBW) from seven vertebrate species, including their natural hosts. From washed inocula of approximately 2×10³ colony-forming units per ml (cfu ml⁻¹), Bordetella bronchiseptica and B. avium grew much better than the other two bordetellae and yielded stationary-phase cultures containing 10⁸−10⁹ cfu ml⁻¹ in most of the TBW samples. These counts were only moderately lower than those attained in CL medium which contains about a 450-times higher concentration of amino acids. B. bronchiseptica and B. avium also grew to a limited extent in phosphate-buffered saline without nutrient supplements. B. parapertussis grew in TBW from man, sheep, rabbit, mouse and chicken, but not in TBW from a dog and a horse or in PBS. B. pertussis grew well in CL medium, but not in PBS or in any of 13 samples of TBW from the seven vertebrate species, which included three samples of lung lavage fluid from human patients. Analysis of the TBW samples for known Bordetella nutrients revealed concentrations of amino acids and nicotinic acid averaging 0.35 mM and 0.56 μg ml⁻ respectively.     

Evaluation of alpha-cyanoesters as fluorescent substrates for examining interindividual variation in general and pyrethroid-selective esterases in human liver microsomes

Carboxylesterases hydrolyze many pharmaceuticals and agrochemicals and have broad substrate selectivity, requiring a suite of substrates to measure hydrolytic profiles. To develop new esterase substrates, a series of alpha-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays. The use of these substrates as surrogates for Type II pyrethroid hydrolysis was tested. The results suggest that these novel analogs are appropriate for the development of high-throughput assays for pyrethroid hydrolase activity. A set of human liver microsomes was then used to determine the ability of these substrates to report esterase activity across a small population. Results were compared against standard esterase substrates. A number of the esterase substrates showed correlations, demonstrating the broad substrate selectivity of these enzymes. However, for several of the substrates, no correlations in hydrolysis rates were observed, suggesting that multiple carboxylesterase isozymes are responsible for the array of substrate hydrolytic activity. These new substrates were then compared against alpha-naphthyl acetate and 4-methylumbelliferyl acetate for their ability to detect hydrolytic activity in both one- and two-dimensional native electrophoresis gels. Cyano-2-naphthylmethyl butanoate was found to visualize more activity than either commercial substrate. These applications demonstrate the utility of these new substrates as both general and pyrethroid-selective reporters of esterase activity.     

Reduction of Nomega-hydroxy-L-arginine to L-arginine by pig liver microsomes, mitochondria, and human liver microsomes

Nomega-Hydroxy-L-arginine, the intermediate in nitric oxide formation from L-arginine catalyzed by NO synthase, can be released into the extracellular space. It has been suggested that it can circulate and exert paracrine effects. Since it cannot only be used as substrate by NO synthases, but can also be oxidized by cytochrome P450 and other hemoproteins in a superoxide-dependent manner, it has been proposed that it can serve as NO donor. In the present study, the in vitro reduction of Nomega-hydroxy-L-arginine was examined. Pig and human liver microsomes as well as pig liver mitochondria were capable of reducing Nomega-hydroxy-L-arginine to L-arginine in an oxygen-insensitive enzymatic reaction. These results demonstrate that this metabolic pathway has to be considered when suggesting Nomega-hydroxy-L-arginine as NO-precursor. The reconstituted liver microsomal system of a pig liver CYP2D enzyme, the benzamidoxime reductase, was unable to replace microsomes to produce L-arginine from Nomega-hydroxy-L-arginine.     

Interference of UDP-glucuronyltransferase and beta-glucuronidase activity in rat liver microsomes at pH 7.5 with p-nitrophenol and p-nitrophenylglucuronide as substrates.

Microsomal fraction contains the whole of hepatic UDP-glucuronyltransferase as well as part of beta-glucuronidase. The activities of the two enzymes were assayed under identical conditions using untreated male rat liver microsomes at pH 7.5. In a 30-min incubation with p-nitrophenol and UPD-glucuronic acid, a net glucuronide formation of 0.010 mumol.min-1.g.liver-1 was measured. In the presence of saccharolactone at concentrations selectively blocking beta-glucuronidase, the glucuronidation rate was 0.015 mumol.min-1.g.liver-1. Using the kinetic parameters of beta-glucuronidase (Km = 0.06 mmol/l p-nitrophenylglucuronide, Vm = 0.075 mumol pNP formed.h-1.g.liver-1) determined in the absence of UDP-glucuronic acid, to correct for the beta-glucuronidase's interference on the glucuronidation process, a glucuronide formation of 0.011 mumol.min-1.g.liver-1 was calculated.     

Identification and comparative oridonin metabolism in different species liver microsomes by using UPLC-Triple-TOF-MS/MS and PCA

Oridonin (ORI) is an active natural ent-kaurene diterpenoid ingredient with notable anti-cancer and anti-inflammation activities. Currently, a strategy was developed to identify metabolites and to assess the metabolic profiles of ORI in vitro using ultra-high-performance liquid chromatography-Triple/time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). Meanwhile, the metabolism differences of ORI in the liver microsomes of four different species were investigated using a principal component analysis (PCA) based on the metabolite absolute peak area values as the variables. Based on the proposed methods, 27 metabolites were structurally characterized. The results indicate that ORI is universally metabolized in vitro, and the metabolic pathway mainly includes dehydration, hydroxylation, di-hydroxylation, hydrogenation, decarboxylation, and ketone formation. Overall, there are obvious inter-species differences in types and amounts of ORI metabolites in the four species. These results will provide basic data for future pharmacological and toxicological studies of ORI and for other ent-kauranes diterpenoids. Meanwhile, studying the ORI metabolic differences helps to select the proper animal model for further pharmacology and toxicological assessment.     

In vivo rates of protein synthesis in brain,muscle, and liver of five vertebrate species

To compare cerebral protein metabolism rates in vivo, protein synthesis rates of three organs of five vertebrate species were measured after a single i.p. injection of a flooding dose of [1-¹⁴C]valine. In muscle, brain, and liver, the respective average protein synthesis rates, expressed as percent of total protein-bound valine replaced per hour, that is, percent synthesis per hour, in goldfish at 22°C body temperature, were 0.07, 0.23, and 0.57%; in the bullfrog at 20°C, 0.06, 0.18, and 0.55%; in the white Leghorn chicken at 39°C, 0.24, 0.70, and 2.17%; and in the mouse at 38°C, 0.22, 0.65, and 2.0%. In the Tokay lizard at different body temperatures, the synthesis rates were 0.04, 0.13, and 0.43% at 26°C; 0.05, 0.20, and 0.63% at 32°C; and 0.07, 0.27, and 0.81% at 38°C. The results demonstrate differences in protein synthesis rates in organs of the various species examined. The differences among the species seem to be due, to a major extent, to differences in body temperature; rates in lizard are below those in other species at temperatures tried. Protein synthesis rates in brain in all species are almost three times lower than those in liver and almost three times higher than those in muscle.     

Novel extremely acidic lipases produced from Bacillus species using oil substrates

The extremely acidophilic microorganisms Bacillus pumilus and Bacillus subtilis were isolated from soil collected from the commercial edible oil and fish oil extraction industry. Optimization of conditions for acidic lipase production from B. pumilus and B. subtilis using palm oil and fish oil, respectively, was carried out using response surface methodology. The extremely acidic lipases, thermo-tolerant acidic lipase (TAL) and acidic lipase (AL), were produced by B. pumilus and B. subtilis, respectively. The optimum conditions for B. pumilus obtaining the maximum activity (1,100 U/mL) of TAL were fermentation time, 96 h; pH, 1; temperature, 50 °C; concentration of palm oil, 50 g/L. After purification, a 7.1-fold purity of lipase with specific activity of 5,173 U/mg protein was obtained. The molecular weight of the TAL was 55 kDa. The AL from B. subtilis activity was 214 U/mL at a fermentation time of 72 h; pH, 1; temperature, 35 °C; concentration of fish oil, 30 g/L; maltose concentration, 10 g/L. After purification, an 11.4-fold purity of lipase with specific activity of 2,189 U/mg protein was obtained. The molecular weight of the extremely acidic lipase was 22 kDa. The functional groups of lipases were determined by Fourier transform-infrared (FT-IR) spectroscopy.     

Purification of ethanolaminephosphotransferase from bovine liver microsomes

CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) has been purified to electrophoretic homogeneity and in a catalytically active form from bovine liver microsomes. The purification method is based on the high hydrophobicity of the protein whose charged sites appear to be masked from the interaction with the chromatographic stationary phases when membranes are solubilized with an excess of non-ionic detergent.The isolated protein has a molecular mass of about 38 kDa, as estimated by SDS-PAGE mobility, and exhibits both ethanolaminephosphotransferase and cholinephosphotransferase activities. Evidence is given that both activities are Mn²⁺-dependent and that the same catalytic site is involved in cholinephosphotransferase and ethanolaminephosphotransferase reactions. Mg²⁺-dependent CDP-choline:diacylglycerol cholinephosphotransferase (EC 2.7.8.2) is completely inactivated during the solubilization and purification steps.     

Cytochrome P450 enzyme-mediated degradation of Echinacea alkylamides in human liver microsomes

Echinacea preparations are widely used herbal remedies for the prevention and treatment of colds. In this study we have investigated the metabolism by human liver microsomes of the alkylamide components from an Echinacea preparation as well as that of pure synthetic alkylamides. No significant degradation of alkylamides was evident in cytosolic fractions. Time- and NADPH-dependent degradation of alkylamides was observed in microsomal fractions suggesting they are metabolised by cytochrome P450 (P450) enzymes in human liver. There was a difference in the susceptibility of 2-ene and 2,4-diene pure synthetic alkylamides to microsomal degradation with (2E)-N-isobutylundeca-2-ene-8,10-diynamide (1) metabolised to only a tenth the extent of (2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (3) under identical incubation conditions. Markedly less degradation of 3 was evident in the mixture of alkylamides present in an ethanolic Echinacea extract, suggesting that metabolism by liver P450s was dependent both on their chemistry and the combination present in the incubation. Co-incubation of 1 with 3 at equimolar concentrations resulted in a significant decrease in the metabolism of 3 by liver microsomes. This inhibition by 1, which has a terminal alkyne moiety, was found to be time- and concentration-dependent, and due to a mechanism-based inactivation of the P450s. Alkylamide metabolites were detected and found to be the predicted epoxidation, hydroxylation and dealkylation products. These findings suggest that Echinacea may effect the P450-mediated metabolism of other concurrently ingested pharmaceuticals.     

Comparative metabolism of sildenafil in liver microsomes of different species by using LC/MS-based multivariate analysis

Sildenafil metabolism in liver microsomes obtained from different species was studied in vitro and compared using liquid chromatography-mass spectrometry (LC/MS) and multivariate statistical analysis. Sildenafil (1, 5, and 25 μM) was incubated with rat, mouse, dog, monkey, and human liver microsomes along with NADPH, and the reaction mixtures were analyzed by LC/MS to obtain species-specific metabolic profiles of sildenafil. A total of 12 metabolites were detected and their peak area ratio values were used as variables for multivariate analyses to evaluate the interspecies differences in sildenafil metabolism. Principal components analysis of the metabolic profiles showed that the mouse samples were generally clustered closer to the human samples on the principal component score plot. Similarity index (SI) indicated that sildenafil metabolism in mice, compared to the other animals, was highly analogous (SI=0.764 at 25 μM) to that in humans. These results suggest that LC/MS-based multivariate analytical approaches are useful for the evaluation of interspecies differences in the metabolism of xenobiotics.