Marco H. Blokland

Screening for modulatory effects on steroidogenesis using the human H295R adrenocortical cell line: a metabolomics approach.

The recently OECD validated H295R steroidogenesis assay provides an in vitro alternative to evaluate the potential interference of exogenous compounds with endogenous steroid hormone synthesis. Currently, this assay is used for a simple negative-positive screening of compounds using testosterone and estradiol levels as end points, measured with specific enzyme immunoassays (EIAs) or targeted liquid chromatography (LC) and gas chromatography (GC)-mass spectrometry (MS) methods. However, recent developments in LC-MS and bioinformatics allow for more comprehensive approaches to evaluate changes in steroid profiles. In the current work, the H295R cell model was combined with a metabolomics approach to monitor changes in metabolite profiles in both a targeted and untargeted way. H295R cells were exposed for 48 h to model compounds, i.e., forskolin, abiraterone, prochloraz, ketoconazole, trilostane, formestane, aminoglutethimide, fadrozole, etomidate, and metyrapone, known to affect steroidogenesis. After exposure, the levels of 9 natural steroids were determined by a quantitative targeted GC-MS/MS method and compared to a metabolomics method using Ultra Performance Liquid Chromatography-Time-of-Flight-Mass Spectrometry (UPLC-ToF-MS). Like the EIAs, both methods were suited for negative-positive screening, but the MS methods also generated specific fingerprints, allowing chemical class prediction of the compound under investigation. Although the targeted GC-MS/MS was more sensitive, which was an advantage regarding analysis of the estrogens 17β-estradiol and estrone, the untargeted UPLC-ToF-MS was able to evaluate effects on the synthesis of the corticosteroids. Moreover, untargeted comparison of the aligned chemical profiles allowed identification of all m/z-values that are differential between exposed and nonexposed H295R cells. In conclusion, application of a comprehensive metabolite profiling methodology not only provides a tool to screen compounds for steroidogenic modulating properties, but also allows chemical class prediction. As such, steroid profiling methodologies in conjunction with the H295R assay can contribute to the prioritization of chemicals for additional safety testing.

Microbial Removal of the Pharmaceutical Compounds Ibuprofen and Diclofenac from Wastewater

Studies on the occurrence of pharmaceuticals show that the widely used pharmaceuticals ibuprofen and diclofenac are present in relevant concentrations in the environment. A pilot plant treating hospital wastewater with relevant concentrations of these pharmaceuticals was evaluated for its performance to reduce the concentration of the pharmaceuticals. Ibuprofen was completely removed, whereas diclofenac yielded a residual concentration, showing the necessity of posttreatment to remove diclofenac, for example, activated carbon. Successively, detailed laboratory experiments with activated sludge from the same wastewater treatment plant showed bioremediation potential in the treatment plant. The biological degradation pathway was studied and showed a mineralisation of ibuprofen and degradation of diclofenac. The present microbes were further studied in laboratory experiments, and DGGE analyses showed the enrichment and isolation of highly purified cultures that degraded either ibuprofen or diclofenac. This research illuminates the importance of the involved bacteria for the effectiveness of the removal of pharmaceuticals in a wastewater treatment plant. A complete removal of pharmaceuticals from wastewater will stimulate water reuse, addressing the worldwide increasing demand for clean and safe fresh water.

Fate in digestion in vitro of several food components, including some toxic compounds coming from omega-3 and omega-6 lipids

In this study it was proved the formation of oxygenated alpha,beta-unsaturated aldehydes (OαβUAs) of 6, 7, 9 and 10 carbon atoms during the thermal treatment (190°C with aeration) of a commercial vegetable oil rich in omega-3 and omega-6 acyl groups, which also contained small amounts of added proteins and carbohydrates to produce barbecue aroma when heated. The OαβUAs detected by Solid Phase Microextraction (SPME) followed by Gas Chromatography/Mass Spectrometry (GC/MS) were: 4-hydroxy-2-hexenal, 4-oxo-2-hexenal and 4,5-epoxy-2-heptenals, coming from omega-3 acyl groups; and 4-hydroxy-2-nonenal, 4-oxo-2-nonenal and 4,5-epoxy-2-decenals, coming from omega-6 acyl groups. Mixtures of this oil, either thermodegraded or not, with standard food were submitted to an in vitro digestion model. The study of the digestion products obtained revealed that OαβUAs remained unaltered, being bioaccessible in the gastrointestinal tract and so able to reach the systemic circulation. Besides, it was evidenced that during digestion Maillard, esterification and oxidation reactions take place.

Use of an in Vitro Digestion Model To Study the Bioaccessibility of 4-Hydroxy-2-nonenal and Related Aldehydes Present in Oxidized Oils Rich in Omega-6 Acyl Groups

Mixtures of either sunflower oil or thermodegraded sunflower oil and a standard meal were submitted to an in vitro digestion model. The same experiment was carried out with fluid deep-frying fat and thermodegraded fluid deep-frying fat. The thermodegradation of the oil and fat was provoked by submitting them to 190 degrees C with aeration in a convection oven, and the presence in the headspace of the thermodegraded oil and fat of oxygenated alpha,beta-unsaturated aldehydes (OalphabetaUAs), such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and 4,5-epoxy-2-decenal (EDE), was monitored by solid phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS). The digestion products were separated by centrifugation in a lipidic phase, an aqueous phase, and a pellet phase. The headspace of these three phases was also studied by SPME/GC-MS to check if the toxic and very reactive OalphabetaUAs above-mentioned remained unaltered after the in vitro digestion process or if they had reacted with the various compounds present in the digestion products, so disappearing from the samples. With the same aim the extract in ethyl acetate of the aqueous and pellet phases, and of the lipidic phase after dilution, were studied by GC-MS. All results obtained showed that a certain proportion of the toxic OalphabetaUAs remains unaltered after digestion, dispersed in the three phases above-mentioned, and thus are bioaccessible in the gastrointestinal tract and so could reach the systemic circulation. Compounds that may originate in Maillard type reactions (2-pentylpyridine) are found among digestion products, proving that these reactions are possible in this process if adequate substrates are present. In addition, it has been shown that toxic metabolites from the synthetic antioxidant BHT, present in fat before digestion, remain unaltered after this process and could reach the systemic circulation.

Sex hormone-binding globulin regulation of androgen bioactivity in vivo: validation of the free hormone hypothesis

Sex hormone-binding globulin (SHBG) is the high-affinity binding protein for androgens and estrogens. According to the free hormone hypothesis, SHBG modulates the bioactivity of sex steroids by limiting their diffusion into target tissues. Still, the in vivo physiological role of circulating SHBG remains unclear, especially since mice and rats lack circulating SHBG post-natally. To test the free hormone hypothesis in vivo, we examined total and free sex steroid concentrations and bioactivity on target organs in mice expressing a human SHBG transgene. SHBG increased total androgen and estrogen concentrations via hypothalamic-pituitary feedback regulation and prolonged ligand half-life. Despite markedly raised total sex steroid concentrations, free testosterone was unaffected while sex steroid bioactivity on male and female reproductive organs was attenuated. This occurred via a ligand-dependent, genotype-independent mechanism according to in vitro seminal vesicle organ cultures. These results provide compelling support for the determination of free or bioavailable sex steroid concentrations in medicine, and clarify important comparative differences between translational mouse models and human endocrinology.

Nortestosterone: endogenous in urine of goats, sheep and mares?†

For a number of species it is known that nortestosterone, either the alpha- or beta-epimer, can be of endogenous origin. For goats and mares similar results have not yet been published. As a follow-up on the experiments with cattle, a large number of urine samples per animal were collected from pregnant goats, sheep and mares. These samples were analysed for the presence of alpha- and beta-nortestosterone and alpha-estradiol using GC-MS. The results show that in the goats and mares studied alpha-nortestosterone is present during pregnancy. In this study no alpha-nortestosterone could be demonstrated in sheep. From our study and recently published data, however, it is proven that alpha-nortestosterone can occur endogenously.

Confirmatory analysis of Trenbolone using accurate mass measurement with LC/TOF-MS.

The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS). Confirmation of the identity of a substance using mass-spectrometric detection has been described. However, the use of accurate mass measurement for confirmatory analysis has not been taken into account. In this study, criteria for confirmation with accurate mass are proposed and feasibility is demonstrated. Mass accuracy better than 3ppm of the quasi-molecular ion and a fragment and their relative ratios determined with LC/TOF-MS are compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results show that these criteria can be met for Trenbolone in samples of bovine urine and that single MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation. The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which exact masses can be measured, make it important to formulate criteria for this type of instrumentation. It is shown in this study that accurate mass measurement can be used for confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis.

Metabolism of Ibuprofen by Phragmites australis: Uptake and Phytodegradation

This study explores ibuprofen (IBP) uptake and transformation in the wetland plant species Phragmites australis and the underlying mechanisms. We grew P. australis in perlite under greenhouse conditions and treated plants with 60 μg/L of IBP. Roots and rhizomes (RR), stems and leaves (SL), and liquid samples were collected during 21 days of exposure. Results show that P. australis can take up, translocate, and degrade IBP. IBP was completely removed from the liquid medium after 21 days with a half-life of 2.1 days. IBP accumulated in RR and was partly translocated to SL. Meanwhile, four intermediates were detected in the plant tissues: hydroxy-IBP, 1,2-dihydroxy-IBP, carboxy-IBP and glucopyranosyloxy-hydroxy-IBP. Cytochrome P450 monooxygenase was involved in the production of the two hydroxy intermediates. We hypothesize that transformation of IBP was first catalyzed by P450, and then by glycosyltransferase, followed by further storage or metabolism in vacuoles or cell walls. No significant phytotoxicity was observed based on relative growth of plants and stress enzyme activities. In conclusion, we demonstrated for the first time that P. australis degrades IBP from water and is therefore a suitable species for application in constructed wetlands to clean wastewater effluents containing IBP and possibly also other micropollutants.

Effect of ammonia in cigarette tobacco on nicotine absorption in human smokers.

The function of ammonia as tobacco additive is subject of scientific debate. It is argued that ammonia, by increasing the proportion of free nicotine, increases the absorption of nicotine in smokers. As a result of the addition of ammonia to cigarettes, smokers get exposed to higher internal nicotine doses and become more addicted to the product. On two occasions, the nicotine absorption in blood was measured after smoking a commercial cigarette of either brand 1 or brand 2, which differed 3.8-fold in ammonium salt content. Using a standardized smoking regime (six puffs, 30 s puff interval, 7 s breath hold before exhalation), 51 regular smokers smoked brand 1 (Caballero Smooth Flavor; 0.89 mg ammonium per gram tobacco) and brand 2 (Gauloise Brunes; 3.43 mg ammonium per gram tobacco). Puff volumes and cardiovascular parameters were monitored during and following smoking, respectively. Measurement of serum nicotine level in the blood samples collected over time following smoking of the two brands, showed that total amount of nicotine absorbed did not differ between the two brands. Present results demonstrate that smoking tobacco containing a higher amount of the tobacco additive ammonium does not increase the absorption of nicotine in the smokers body.

Distinction of clenbuterol intake from drug or contaminated food of animal origin in a controlled administration trial – the potential of enantiomeric separation for doping control analysis

ABSTRACT The differentiation of clenbuterol abuse and unintentional ingestion from contaminated meat is crucial with respect to the valuation of an adverse analytical finding in human sports doping control. The proportion of the two enantiomers of clenbuterol may serve as potential discriminating parameter. For the determination of the individual enantiomers, specific methods were developed and validated for the different matrices under investigation based on chiral chromatography coupled to tandem mass spectrometry. Data are presented from the administration to humans of clenbuterol from a pharmaceutical preparation, and from cattle meat and liver containing residues. A shift in the proportion of the enantiomers in cattle meat is detected and this signature is also found in human urine after ingestion. Thus, an altered enantiomeric composition of clenbuterol may be used to substantiate athletes’ claims following adverse analytical findings in doping control. However, in meat, the enantiomeric composition was found to be highly variable. Species as well as tissue dependent variances need to be considered in interpreting enantiomer discrimination. Analysis of post administration urines from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue-containing meat and liver (nonracemic mixture) from treated animals is reported. Furthermore doping control samples from Mexican U17 World Championship 2011 of the Fédération Internationale de Football Association (FIFA), with adverse analytical findings for clenbuterol, were re-analysed. GRAPHICAL ABSTRACT