Bovine liver slices combined with an androgen transcriptional activation assay: an in-vitro model to study the metabolism and bioactivity of steroids

Previously we described the properties of a rapid and robust yeast androgen bioassay for detection of androgenic anabolic compounds, validated it, and showed its added value for several practical applications. However, biotransformation of potent steroids into inactive metabolites, or vice versa, is not included in this screening assay. Within this context, animal-friendly in-vitro cellular systems resembling species-specific metabolism can be of value. We therefore investigated the metabolic capacity of precision-cut slices of bovine liver using 17β-testosterone (T) as a model compound, because this is an established standard compound for assessing the metabolic capacity of such cellular systems. However, this is the first time that slice metabolism has been combined with bioactivity measurements. Moreover, this study also involves bioactivation of inactive prohormones, for example dehydroepiandrosterone (DHEA) and esters of T, and although medium extracts are normally analyzed by HPLC, here the metabolites formed were identified with more certainty by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC–TOFMS) with accurate mass measurement. Metabolism of T resulted mainly in the formation of the less potent phase I metabolites 4-androstene-3,17-dione (4-AD), the hydroxy-T metabolites 6α, 6β, 15β, and 16α-OH-T, and the phase II metabolite T-glucuronide. As a consequence the overall androgenic activity, as determined by the yeast androgen bioassay, decreased. In order to address the usefulness of bovine liver slices for activation of inactive steroids, liver slices were exposed to DHEA and two esters of T. This resulted in an increase of androgenic activity, because of the formation of 4-AD and T

Recombinant cell bioassays for the detection of (gluco)corticosteroids and endocrine-disrupting potencies of several environmental PCB contaminants

Sensitive and robust bioassays for glucocorticoids are very useful for the pharmaceutical industry, environmental scientists and veterinary control. Here, a recombinant yeast cell was constructed that expresses the human glucocorticoid receptor alpha and a green fluorescent reporter protein in response to glucocorticoids. Both the receptor construct and the reporter construct were stably integrated into the yeast genome. The correct and specific functioning of this yeast glucocorticoid bioassay was studied by exposures to cortisol and other related compounds and critically compared to a GR-CALUX bioassay based on a human bone cell. Although less sensitive, the new yeast glucocorticoid bioassay showed sensitivity towards all (gluco)corticoids tested, with the following order in relative potencies: budesonide >> corticosterone > dexamethasone > cortisol = betamethasone > prednisolone > aldosterone. Hormone representatives for other hormone nuclear receptors, like 17β-estradiol for the oestrogen receptor, 5α-dihydrotestosterone for the androgen receptor and progesterone for the progesterone receptor, showed no clear agonistic responses, whilst some polychlorinated biphenyls were clearly able to interfere with the GR activity

Intestinal barrier function in response to abundant or depleted mucosal glutathione in Salmonella-infected rats

ABSTRACT: BACKGROUND: Glutathione, the main antioxidant of intestinal epithelial cells, is suggested to play an important role in gut barrier function and prevention of inflammation-related oxidative damage as induced by acute bacterial infection. Most studies on intestinal glutathione focus on oxidative stress reduction without considering functional disease outcome. Our aim was to determine whether depletion or maintenance of intestinal glutathione changes susceptibility of rats to Salmonella infection and associated inflammation. Rats were fed a control diet or the same diet supplemented with buthionine sulfoximine (BSO; glutathione depletion) or cystine (glutathione maintenance). Inert chromium ethylenediamine-tetraacetic acid (CrEDTA) was added to the diets to quantify intestinal permeability. At day 4 after oral gavage with Salmonella enteritidis (or saline for non-infected controls), Salmonella translocation was determined by culturing extra-intestinal organs. Liver and ileal mucosa were collected for analyses of glutathione, inflammation markers and oxidative damage. Faeces was collected to quantify diarrhoea. RESULTS: Glutathione depletion aggravated ileal inflammation after infection as indicated by increased levels of mucosal myeloperoxidase and interleukin-1beta. Remarkably, intestinal permeability and Salmonella translocation were not increased. Cystine supplementation maintained glutathione in the intestinal mucosa but inflammation and oxidative damage were not diminished. Nevertheless, cystine reduced intestinal permeability and Salmonella translocation. CONCLUSIONS: Despite increased infection-induced mucosal inflammation upon glutathione depletion, this tripeptide does not play a role in intestinal permeability, bacterial translocation and diarrhoea. On the other hand, cystine enhances gut barrier function by a mechanism unlikely to be related to glutathione

Proton gradient formation in early endosomes from proximal tubules

AbstractHeavy endosomes were isolated from proximal tubules using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. Two small GTPases (Rab4 and Rab5) known to be specifically present in early endosomes were identified in our preparations. Endosomal acidification was followed fluorimetrically using acridine orange. In presence of chloride ions and ATP, the formation of a proton gradient (ΔpH) was observed. This process is due to the activity of an electrogenic V-type ATPase present in the endosomal membrane since specific inhibitors bafilomycin and folimycin effectively prevented or eliminated endosomal acidification. In presence of chloride ions (Km = 30 mM) the formation of the proton gradient was optimal. Inhibitors of chloride channel activity such as DIDS and NPPB reduced acidification. The presence of sodium ions stimulated the dissipation of the proton gradient. This effect of sodium was abolished by amiloride derivative (MIA) but only when loaded into endosomes, indicating the presence of a physiologically oriented Na+/H+-exchanger in the endosomal membrane. Monensin restored the gradient dissipation. Thus three proteins (V-type ATPase, Cl−-channel, Na+/H+-exchanger) present in early endosomas isolated from proximal tubules may regulate the formation, maintenance and dissipation of the proton gradient

Microhabitat competition between Iberian fish species and the endangered Júcar nase (Parachondrostoma arrigonis; Steindachner, 1866)

"This is an Accepted Manuscript of an article published by Taylor & Francis in Journal of Ecohydraulics on 24-01-2017, available online: https://www.tandfonline.com/doi/full/10.1080/24705357.2016.1276417"[EN] Competition with invasive species is recognized as having a major impact on biodiversity conservation. The upper part of the Cabriel River (Eastern Iberian Peninsula) harbours the most important population of the Júcar nase (Parachondrostoma arrigonis; Steindachner, 1866), a fish species in imminent danger of extinction. Currently, this species cohabits with several non-native species, such as the Iberian nase (Pseudochondrostoma polylepis; Steindachner, 1864) and the bermejuela (Achondrostoma arcasii; Steindachner, 1866). The potential habitat competition with these species was studied by analysing the spatial and temporal overlapping of suitable microhabitats. Generalized Additive Mixed Models (GAMMs) were developed to model microhabitat selection and these GAMMs were used to assess the habitat suitability (i.e. probability of presence) under several flows simulated with River2D. The Júcar nase will compete, spatially and temporally, for the few suitable microhabitats with bermejuela and, to a lesser extent, with small Iberian nase; conversely, large Iberian nase was of minor concern, due to increased differences in habitat preferences. This study represents an important assessment of potential competition and, therefore, these results might assist to better define future management practices in the upper part of the Cabriel River.This study was funded by the Spanish Ministry of Economy and Competitiveness through the SCARCE project (Consolider Ingenio 2010 CSD2009 00065); the Universitat Politècnica de València, through the project UPPTE/2012/294 [PAID 06 12]; it was also partially funded by the IMPADAPT project (CGL2013-48424-C2-1-R) with Spanish MINECO (Ministerio de Economía y Competitividad) and FEDER funds. The authors would like to thank the help of the Conselleria de Territori i Vivenda (Generalitat Valenciana) and the Confederación Hidrográfica del Júcar (Spanish government), which provided environmental data to Alfredo Ollero, and the two anonymous reviewers who first suggested the submission of the paper to a regular journal. Finally, we would like to thank TECNOMA S.A. for the development of the hydraulic model.Muñoz Mas, R.; Soares Costa, RM.; Alcaraz-Hernández, JD.; Martinez-Capel, F. (2017). Microhabitat competition between Iberian fish species and the endangered Júcar nase (Parachondrostoma arrigonis; Steindachner, 1866). Journal of Ecohydraulics. 2(1):3-15. https://doi.org/10.1080/24705357.2016.1276417S31521Alcaraz, C., Carmona-Catot, G., Risueño, P., Perea, S., Pérez, C., Doadrio, I., & Aparicio, E. (2014). Assessing population status of Parachondrostoma arrigonis (Steindachner, 1866), threats and conservation perspectives. Environmental Biology of Fishes, 98(1), 443-455. doi:10.1007/s10641-014-0274-3ALMEIDA, D., & GROSSMAN, G. D. (2012). Utility of direct observational methods for assessing competitive interactions between non-native and native freshwater fishes. 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M., Alcaraz-Hernández, J. D., De Baets, B., Goethals, P. L. M., & Martínez-Capel, F. (2011). Data-driven fuzzy habitat suitability models for brown trout in Spanish Mediterranean rivers. Environmental Modelling & Software, 26(5), 615-622. doi:10.1016/j.envsoft.2010.12.001Mouton, A. M., De Baets, B., & Goethals, P. L. M. (2010). Ecological relevance of performance criteria for species distribution models. Ecological Modelling, 221(16), 1995-2002. doi:10.1016/j.ecolmodel.2010.04.017Muñoz-Mas, R., Fukuda, S., Vezza, P., & Martínez-Capel, F. (2016). Comparing four methods for decision-tree induction: A case study on the invasive Iberian gudgeon ( Gobio lozanoi ; Doadrio and Madeira, 2004). Ecological Informatics, 34, 22-34. doi:10.1016/j.ecoinf.2016.04.011Muñoz-Mas, R., Lopez-Nicolas, A., Martínez-Capel, F., & Pulido-Velazquez, M. (2016). Shifts in the suitable habitat available for brown trout (Salmo trutta L.) under short-term climate change scenarios. 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Application and testing of a procedure to evaluate transferability of habitat suitability criteria. Regulated Rivers: Research & Management, 8(3), 285-294. doi:10.1002/rrr.3450080307Vezza, P., Muñoz-Mas, R., Martinez-Capel, F., & Mouton, A. (2015). Random forests to evaluate biotic interactions in fish distribution models. Environmental Modelling & Software, 67, 173-183. doi:10.1016/j.envsoft.2015.01.005Vilizzi, L., Copp, G. H., & Roussel, J.-M. (2004). Assessing variation in suitability curves and electivity profiles in temporal studies of fish habitat use. River Research and Applications, 20(5), 605-618. doi:10.1002/rra.767Wood, S. N. (2004). Stable and Efficient Multiple Smoothing Parameter Estimation for Generalized Additive Models. Journal of the American Statistical Association, 99(467), 673-686. doi:10.1198/016214504000000980Wood, S. N. (2006). Generalized Additive Models. doi:10.1201/9781420010404Zuur, A. F., Ieno, E. N., Walker, N., Saveliev, A. A., & Smith, G. M. (2009). 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A large geometric distortion in the first photointermediate of rhodopsin, determined by double-quantum solid-state NMR

Double-quantum magic-angle-spinning NMR experiments were performed on 11,12-C-13(2)-retinylidene-rhodopsin under illumination at low temperature, in order to characterize torsional angle changes at the C11-C12 photoisomerization site. The sample was illuminated in the NMR rotor at low temperature (similar to 120 K) in order to trap the primary photointermediate, bathorhodopsin. The NMR data are consistent with a strong torsional twist of the HCCH moiety at the isomerization site. Although the HCCH torsional twist was determined to be at least 40A degrees, it was not possible to quantify it more closely. The presence of a strong twist is in agreement with previous Raman observations. The energetic implications of this geometric distortion are discussed

Effect of sodium bicarbonate supplementation on the renin-angiotensin system in patients with chronic kidney disease and acidosis:a randomized clinical trial

Background Acidosis-induced kidney injury is mediated by the intrarenal renin-angiotensin system, for which urinary renin is a potential marker. Therefore, we hypothesized that sodium bicarbonate supplementation reduces urinary renin excretion in patients with chronic kidney disease (CKD) and metabolic acidosis. Methods Patients with CKD stage G4 and plasma bicarbonate 15-24 mmol/l were randomized to receive sodium bicarbonate (3 x 1000 mg/day, similar to 0.5 mEq/kg), sodium chloride (2 x 1,00 mg/day), or no treatment for 4 weeks (n = 15/arm). The effects on urinary renin excretion (primary outcome), other plasma and urine parameters of the renin-angiotensin system, endothelin-1, and proteinuria were analyzed. Results Forty-five patients were included (62 +/- 15 years, eGFR 21 +/- 5 ml/min/1.73m(2), plasma bicarbonate 21.7 +/- 3.3 mmol/l). Sodium bicarbonate supplementation increased plasma bicarbonate (20.8 to 23.8 mmol/l) and reduced urinary ammonium excretion (15 to 8 mmol/day, both P <0.05). Furthermore, a trend towards lower plasma aldosterone (291 to 204 ng/L, P = 0.07) and potassium (5.1 to 4.8 mmol/l, P = 0.06) was observed in patients receiving sodium bicarbonate. Sodium bicarbonate did not significantly change the urinary excretion of renin, angiotensinogen, aldosterone, endothelin-1, albumin, or alpha 1-microglobulin. Sodium chloride supplementation reduced plasma renin (166 to 122 ng/L), and increased the urinary excretions of angiotensinogen, albumin, and alpha 1-microglobulin (all P <0.05). Conclusions Despite correction of acidosis and reduction in urinary ammonium excretion, sodium bicarbonate supplementation did not improve urinary markers of the renin-angiotensin system, endothelin-1, or proteinuria. Possible explanations include bicarbonate dose, short treatment time, or the inability of urinary renin to reflect intrarenal renin-angiotensin system activity

Microhabitat use by the white-clawed crayfish in a Tuscan stream.

Habitat modification, pollution, overfishing, poaching, competition from nonindigenous species, and diseases have led to the extinction in Europe of many populations of indigenous crayfish. Under the rationale that any programme of reintroduction should be preceded by a thorough understanding of habitat requirements of the species of concern, we studied the microhabitat use of an Austropotamobius pallipes population in Tuscany, central Italy. Microhabitat use was assessed for water depth, current velocity, substrate, percentages of boulders, underwater tree roots, and in-stream vegetation cover. Results show that A. pallipes’ habitat use is size-partitioned. Smaller individuals mostly occupy stream edges in shallow waters with submerged roots, whereas larger individuals use deeper waters, often associated with boulders. Crayfish spatial distribution is restricted mostly to the microhabitats characterised by extensive cover and slow current velocity. The study highlights the importance of habitat heterogeneity and cover elements for the protection of this indigenous species