In vitro metabolism of testosterone in the horse liver and involvement of equine CYPs 3A89, 3A94 and 3A95

Testosterone (TES) 6-β-hydroxylation is a significant metabolic step in the biotransformation of TES in human liver microsomes and reflects cytochrome P450 (CYP) 3A4/5 specific metabolic activity. Several CYP3A enzymes have been annotated in the horse genome, but functional characterization is missing. This descriptive study investigates TES metabolism in the horse liver in vitro and the qualitative contribution of three CYP3A isoforms of the horse. Metabolism of TES was investigated by using equine hepatocyte primary cultures and liver microsomes. Chemical inhibitors were used to determine the CYPs involved in TES biotransformation in equine microsomes. Single CYPs 3A89, 3A94, and 3A95, recombinantly expressed in V79 hamster lung fibroblasts, were incubated with TES and the fluorescent metabolite 7-benzyloxy-4-trifluoromethylcoumarin (BFC). The effect of ketoconazole and troleandomycin was evaluated on single CYPs. Testosterone metabolites were analyzed by HPLC and confirmed by GC/MS. In hepatocyte primary cultures, the most abundant metabolite was androstenedione (AS), whereas in liver microsomes, 6-β-hydroxytestosterone showed the largest peak. Formation of 6-β-hydroxytestosterone and 11-β-hydroxytestosterone in liver microsomes was inhibited by ketoconazole, troleandomycin, and quercetin. Equine recombinant CYP3A95 catalyzed 11-β-hydroxylation of testosterone (TES). Metabolism of BFC was significantly inhibited by ketoconazole in CYP3A95, whereas troleandomycin affected the activities of CYP3A94 and CYP3A95. Both inhibitors had no significant effect on CYP3A89. Metabolic reactions and effects of inhibitors differed between the equine CYP3A isoforms investigated. This has to be considered in future in vitro studies.

Inhibition of in vitro metabolism of testosterone in human, dog and horse liver microsomes to investigate species differences.

Testosterone hydroxylation was investigated in human, canine and equine liver microsomes and in human and canine single CYPs. The contribution of the CYP families 1, 2 and 3 was studied using chemical inhibitors. Testosterone metabolites were analyzed by HPLC. The metabolites androstenedione, 6β- and 11β-hydroxytestosterone were found in microsomes of all species, but the pattern of metabolites varied within species. Androstenedione was more prominent in the animal species, and an increase over time was seen in equines. Testosterone hydroxylation was predominantly catalyzed by the CYP3A subfamily in all three species. While CYP2C9 did not metabolise testosterone, the canine ortholog CYP2C21 produced androstenedione. Quercetin significantly inhibited 6β- and 11β-hydroxytestosterone in all species investigated, suggesting that CYP2C8 is involved in testosterone metabolism, whereas sulfaphenazole significantly inhibited the formation of 6β- and 11β-hydroxytestosterone in human microsomes, at 60min in equine microsomes, but not in canine microsomes. A contribution of CYP2B6 in testosterone metabolism was only found in human and equine microsomes. Inhibition of 17β-hydroxysteroid dehydrogenase 2 indicated its involvement in androstenedione formation in humans, increased androstenedione formation was found in equines and no involvement in canines. These findings provide improved understanding of differences in testosterone biotransformation in animal species.

Metabolomics reveals the metabolic map of procainamide in humans and mice

Procainamide, a type I antiarrhythmic agent, is used to treat a variety of atrial and ventricular dysrhythmias. It was reported that long-term therapy with procainamide may cause lupus erythematosus in 25-30% of patients. Interestingly, procainamide does not induce lupus erythematosus in mouse models. To explore the differences in this side-effect of procainamide between humans and mouse models, metabolomic analysis using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) was conducted on urine samples from procainamide-treated humans, CYP2D6-humanized mice, and wild-type mice. Thirteen urinary procainamide metabolites, including nine novel metabolites, derived from P450-dependent, FMO-dependent oxidations and acylation reactions, were identified and structurally elucidated. In vivo metabolism of procainamide in CYP2D6-humanized mice as well as in vitro incubations with microsomes and recombinant P450s suggested that human CYP2D6 plays a major role in procainamide metabolism. Significant differences in N-acylation and N-oxidation of the drug between humans and mice largely account for the interspecies differences in procainamide metabolism. Significant levels of the novel N-oxide metabolites produced by FMO1 and FMO3 in humans might be associated with the development of procainamide-induced systemic lupus erythematosus. Observations based on this metabolomic study offer clues to understanding procainamide-induced lupus in humans and the effect of P450s and FMOs on procainamide N-oxidation.

Oxygen isotope ratios (18O/16O) of hemicellulose-derived sugar biomarkers in plants, soils and sediments as paleoclimate proxy II: insight from a climate transect study

The oxygen isotopic composition of precipitation (δ18Oprec) is well known to be a valuable (paleo-)climate proxy. Paleosols and sediments and hemicelluloses therein have the potential to serve as archives recording the isotopic composition of paleoprecipitation. In a companion paper (Zech et al., 2014) we investigated δ18Ohemicellulose values of plants grown under different climatic conditions in a climate chamber experiment. Here we present results of compound-specific δ18O analyses of arabinose, fucose and xylose extracted from modern topsoils (n = 56) along a large humid-arid climate transect in Argentina in order to answer the question whether hemicellulose biomarkers in soils reflect δ18Oprec. The results from the field replications indicate that the homogeneity of topsoils with regard to δ18Ohemicellulose is very high for most of the 20 sampling sites. Standard deviations for the field replications are 1.5‰, 2.2‰ and 1.7‰, for arabinose, fucose and xylose, respectively. Furthermore, all three hemicellulose biomarkers reveal systematic and similar trends along the climate gradient. However, the δ18Ohemicellulose values (mean of the three sugars) do not correlate positively with δ18Oprec (r = −0.54, p < 0.014, n = 20). By using a Péclet-modified Craig-Gordon (PMCG) model it can be shown that the δ18Ohemicellulose values correlate highly significantly with modeled δ18Oleaf water values (r = 0.81, p < 0.001, n = 20). This finding suggests that hemicellulose biomarkers in (paleo-)soils do not simply reflect δ18Oprec but rather δ18Oprec altered by evaporative 18O enrichment of leaf water due to evapotranspiration. According to the modeling results, evaporative 18O enrichment of leaf water is relatively low (∼10‰) in the humid northern part of the Argentinian transect and much higher (up to 19‰) in the arid middle and southern part of the transect. Model sensitivity tests corroborate that changes in relative air humidity exert a dominant control on evaporative 18O enrichment of leaf water and thus δ18Ohemicellulose, whereas the effect of temperature changes is of minor importance. While oxygen exchange and degradation effects seem to be negligible, further factors needing consideration when interpreting δ18Ohemicellulose values obtained from (paleo-)soils are evaporative 18O enrichment of soil water, seasonality effects, wind effects and in case of abundant stem/root-derived organic matter input a partial loss of the evaporative 18O enrichment of leaf water. Overall, our results prove that compound-specific δ18O analyses of hemicellulose biomarkers in soils and sediments are a promising tool for paleoclimate research. However, disentangling the two major factors influencing δ18Ohemicellulose, namely δ18Oprec and relative air humidity controlled evaporative 18O enrichment of leaf water, is challenging based on δ18O analyses alone.

Metabolism of sulphonated anthraquinones in rhubarb, maize and celery: the role of cytochromes P450 and peroxidases

Sulphonated anthraquinones are precursors of many synthetic dyes and pigments, recalcitrant to biodegradation, and thus contaminating many industrial effluents and rivers. In the development of a phytotreatment to remove sulphonated aromatic compounds, rhubarb (Rheum rhaponticum), a plant producing natural anthraquinones, as well as maize (Zea mays) and celery (Apium graveolens), plants not producing anthraquinones, were tested for their ability to metabolise these xenobiotics. Plants were cultivated under hydroponic conditions, with or without sulphonated anthraquinones, and were harvested at different times. Either microsomal or cytosolic fractions were prepared. The monooxygenase activity of cytochromes P450 towards several sulphonated anthraquinones was tested using a new method based on the fluorimetric detection of oxygen consumed during cytochromes P450-catalysed reactions. The activity of cytosolic peroxidases was measured by spectrophotometry, using guaiacol as a substrate. Results indicated that the activity of cytochromes P450 and peroxidases significantly increased in rhubarb plants cultivated in the presence of sulphonated anthraquinones. A higher activity of cytochromes P450 was also detected in maize and celery exposed to the pollutants. In these two plants, a peroxidase activity was also detected, but without a clear difference between the control plants and the plants exposed to the organic contaminants. This research demonstrated the existence in rhubarb, maize and celery of biochemical mechanisms involved in the metabolism and detoxification of sulphonated anthraquinones. Taken together, results confirmed that rhubarb might be the most appropriate plant for the phytotreatment of these organic pollutants.

Comparative metabolism of cyclophosphamide and ifosfamide in the mouse using UPLC-ESI-QTOFMS-based metabolomics

Ifosfamide (IF) and cyclophosphamide (CP) are common chemotherapeutic agents. Interestingly, while the two drugs are isomers, only IF treatment is known to cause nephrotoxicity and neurotoxicity. Therefore, it was anticipated that a comparison of IF and CP drug metabolites in the mouse would reveal reasons for this selective toxicity. Drug metabolites were profiled by ultra-performance liquid chromatography-linked electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS), and the results analyzed by multivariate data analysis. Of the total 23 drug metabolites identified by UPLC-ESI-QTOFMS for both IF and CP, five were found to be novel. Ifosfamide preferentially underwent N-dechloroethylation, the pathway yielding 2-chloroacetaldehyde, while cyclophosphamide preferentially underwent ring-opening, the pathway yielding acrolein (AC). Additionally, S-carboxymethylcysteine and thiodiglycolic acid, two downstream IF and CP metabolites, were produced similarly in both IF- and CP-treated mice. This may suggest that other metabolites, perhaps precursors of thiodiglycolic acid, may be responsible for IF encephalopathy and nephropathy.

In vivo evaluation in cynomolgus monkey brain and metabolism of [¹⁸F]fluorodeprenyl: a new MAO-B pet radioligand

In this study, we evaluated the in vivo characteristics of a new monoamine oxidase type B (MAO-B) radioligand, [¹⁸F]fluorodeprenyl, by positron emission tomography (PET) in two cynomolgus monkeys. The brain uptake of [¹⁸F]fluorodeprenyl was more than 7% (600% SUV) of the total injected radioactivity and similar to that of [¹¹C]deprenyl, an established MAO-B radioligand. The highest uptake was observed in the striatum, one of the MAO-B-rich regions, with a peak at approximately 2-3 min after injection, followed by lower uptake in the thalamus and the cortex and lowest uptake in the cerebellum. Brain uptake of [¹⁸F]fluorodeprenyl was largely inhibited by preadministration of the MAO-B inhibitor, L-deprenyl, whereas clorgyline, a MAO Type A blocker, had no significant inhibitory effect, thus demonstrating selectivity for MAO-B. [¹⁸F]Fluorodeprenyl showed relatively slow metabolism with the presence of two radiometabolite peaks with similar retention time as the labeled metabolites of [¹¹C]deprenyl. These results suggest that [¹⁸F]fluorodeprenyl is a potential PET radioligand for visualization of MAO-B activity.

A comprehensive understanding of thioTEPA metabolism in the mouse using UPLC-ESI-QTOFMS-based metabolomics

ThioTEPA, an alkylating agent with anti-tumor activity, has been used as an effective anticancer drug since the 1950s. However, a complete understanding of how its alkylating activity relates to clinical efficacy has not been achieved, the total urinary excretion of thioTEPA and its metabolites is not resolved, and the mechanism of formation of the potentially toxic metabolites S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA) remains unclear. In this study, the metabolism of thioTEPA in a mouse model was comprehensively investigated using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based-metabolomics. The nine metabolites identified in mouse urine suggest that thioTEPA underwent ring-opening, N-dechloroethylation, and conjugation reactions in vivo. SCMC and TDGA, two downstream thioTEPA metabolites, were produced from thioTEPA from two novel metabolites 1,2,3-trichloroTEPA (VII) and dechloroethyltrichloroTEPA (VIII). SCMC and TDGA excretion were increased about 4-fold and 2-fold, respectively, in urine following the thioTEPA treatment. The main mouse metabolites of thioTEPA in vivo were TEPA (II), monochloroTEPA (III) and thioTEPA-mercapturate (IV). In addition, five thioTEPA metabolites were detected in serum and all shared similar disposition. Although thioTEPA has a unique chemical structure which is not maintained in the majority of its metabolites, metabolomic analysis of its biotransformation greatly contributed to the investigation of thioTEPA metabolism in vivo, and provides useful information to understand comprehensively the pharmacological activity and potential toxicity of thioTEPA in the clinic.

Antinociceptive effects, metabolism and disposition of ketamine in ponies under target-controlled drug infusion

Ketamine is widely used as an anesthetic in a variety of drug combinations in human and veterinary medicine. Recently, it gained new interest for use in long-term pain therapy administered in sub-anesthetic doses in humans and animals. The purpose of this study was to develop a physiologically based pharmacokinetic (PBPk) model for ketamine in ponies and to investigate the effect of low-dose ketamine infusion on the amplitude and the duration of the nociceptive withdrawal reflex (NWR). A target-controlled infusion (TCI) of ketamine with a target plasma level of 1 microg/ml S-ketamine over 120 min under isoflurane anesthesia was performed in Shetland ponies. A quantitative electromyographic assessment of the NWR was done before, during and after the TCI. Plasma levels of R-/S-ketamine and R-/S-norketamine were determined by enantioselective capillary electrophoresis. These data and two additional data sets from bolus studies were used to build a PBPk model for ketamine in ponies. The peak-to-peak amplitude and the duration of the NWR decreased significantly during TCI and returned slowly toward baseline values after the end of TCI. The PBPk model provides reliable prediction of plasma and tissue levels of R- and S-ketamine and R- and S-norketamine. Furthermore, biotransformation of ketamine takes place in the liver and in the lung via first-pass metabolism. Plasma concentrations of S-norketamine were higher compared to R-norketamine during TCI at all time points. Analysis of the data suggested identical biotransformation rates from the parent compounds to the principle metabolites (R- and S-norketamine) but different downstream metabolism to further metabolites. The PBPk model can provide predictions of R- and S-ketamine and norketamine concentrations in other clinical settings (e.g. horses).

A metabolomic approach to the metabolism of the areca nut alkaloids arecoline and arecaidine in the mouse

The areca alkaloids comprise arecoline, arecaidine, guvacoline, and guvacine. Approximately 600 million users of areca nut products, for example, betel quid chewers, are exposed to these alkaloids, principally arecoline and arecaidine. Metabolism of arecoline (20 mg/kg p.o. and i.p.) and arecaidine (20 mg/kg p.o. and i.p.) was investigated in the mouse using a metabolomic approach employing ultra-performance liquid chromatography-time-of-flight mass spectrometric analysis of urines. Eleven metabolites of arecoline were identified, including arecaidine, arecoline N-oxide, arecaidine N-oxide, N-methylnipecotic acid, N-methylnipecotylglycine, arecaidinylglycine, arecaidinylglycerol, arecaidine mercapturic acid, arecoline mercapturic acid, and arecoline N-oxide mercapturic acid, together with nine unidentified metabolites. Arecaidine shared six of these metabolites with arecoline. Unchanged arecoline comprised 0.3-0.4%, arecaidine 7.1-13.1%, arecoline N-oxide 7.4-19.0%, and N-methylnipecotic acid 13.5-30.3% of the dose excreted in 0-12 h urine after arecoline administration. Unchanged arecaidine comprised 15.1-23.0%, and N-methylnipecotic acid 14.8%-37.7% of the dose excreted in 0-12 h urine after arecaidine administration. The major metabolite of both arecoline and arecaidine, N-methylnipecotic acid, is a novel metabolite arising from carbon-carbon double-bond reduction. Another unusual metabolite found was the monoacylglyceride of arecaidine. What role, if any, that is played by these uncommon metabolites in the toxicology of arecoline and arecaidine is not known. However, the enhanced understanding of the metabolic transformation of arecoline and arecaidine should contribute to further research into the clinical toxicology of the areca alkaloids.

The influence of local and systemic preconditioning on oxygenation, metabolism and survival in critically ischaemic skin flaps in pigs

Stress proteins represent a group of highly conserved intracellular proteins that provide adaptation against cellular stress. The present study aims to elucidate the stress protein-mediated effects of local hyperthermia and systemic administration of monophosphoryl lipid A (MPL) on oxygenation, metabolism and survival in bilateral porcine random pattern buttock flaps. Preconditioning was achieved 24h prior to surgery by applying a heating blanket on the operative site (n = 5), by intravenous administration of MPL at a dosage of 35 microg/kg body weight (n = 5) or by combining the two (n = 5). The flaps were monitored with laser Doppler flowmetry, polarographic microprobes and microdialysis until 5h postoperatively. Semiquantitative immunohistochemistry was performed for heat shock protein 70 (HSP70), heat shock protein 32 (also termed haem oxygenase-1, HO-1), and inducible nitrc oxide synthase (iNOS). The administration of MPL increased the impaired microcirculatory blood flow in the proximal part of the flap and partial oxygen tension in the the distal part by approximately 100% each (both P<0.05), whereas both variables remained virtually unaffected by local heat preconditioning. Lactate/pyruvate (L/P) ratio and glycerol concentration (representing cell membrane disintegration) in the distal part of the flap gradually increased to values of approximately 500 mmol/l and approximately 350 micromol/l, respectively (both P<0.01), which was substantially attenuated by heat application (P<0.01 for L/P ratio and P<0.05 for glycerol) and combined preconditioning (P<0.01 for both variables), whereas the effect of MPL was less marked (not significant). Flap survival was increased from 56% (untreated animals) to 65% after MPL (not significant), 71% after heat application (P<0.05) and 78% after both methods of preconditioning (P<0.01). iNOS and HO-1 were upregulated after each method of preconditioning (P<0.05), whereas augmented HSP70 staining was only observed after heat application (P<0.05). We conclude that local hyperthermia is more effective in preventing flap necrosis than systemic MPL administration because of enhancing the cellular tolerance to hypoxic stress, which is possibly mediated by HSP70, whereas some benefit may be obtained with MPL due to iNOS and HO-1-mediated improvement in tissue oxygenation.