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  • 1.
    Bechshoft, Thea
    et al.
    Danmark.
    Wright, Andrew J.
    Danmark.
    Weisser, Johan J.
    Danmark.
    Teilmann, Jonas
    Danmark.
    Dietz, Rune
    Danmark.
    Hansen, Martin
    Danmark.
    Björklund, Erland
    Danmark.
    Styrishave, Bjarne
    Danmark.
    Developing a new research tool for use in free-ranging cetaceans: recovering cortisol from harbour porpoise skin2015Inngår i: Conservation Physiology, E-ISSN 2051-1434, Vol. 3Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We developed a chemical analytical procedure for sampling, extracting and determining epidermal skin cortisol concentrations (SCCs) in the harbour porpoise (Phocoena phocoena) using gas chromatography–tandem mass spectrometry. In brief, this involved a pressurized liquid extraction with a two-step solid-phase clean-up. A derivatization step was conducted prior to detection. To evaluate the new assay, cortisol was analysed in three different sample types obtained from four harbour porpoises: skin plates, dorsal fin skin plugs (with and without lidocaine) and epidermal scrapes. Skin cortisol concentrations could be measured using the new assay in the majority of the tested skin samples down to a minimal sample size of 49 mg dry weight (dw). Water content ranged from 10 to 46% in the plug samples, which had SCCs from 2.1 to 77.7 ng/g dw. Epidermal scrape samples had the highest water content (83–87%) and lower SCCs (0.6–15 ng/g dw), while the skin plates had intermediate water contents (60–66%) and SCCs of 2.6–13.0 ng/g dw. SCC was slightly higher in plugs with lidocaine than without (average values of 41 and 33 ng/g dw, respectively). Substantial within-individual variations in cortisol concentrations are also common in other matrices such as blood and hair. Some important factors behind this variation could be e.g. the animal's sex, age, body condition, reproductive stage, and the body region sampled, as well as season, moulting cycles and water temperature. Clearly, more research into SCCs is required. The findings described here represent the first critical steps towards using epidermal skin cell samples to assess chronic stress levels in cetaceans and the development of a widely applicable health-assessment tool in these species.

  • 2.
    Hallgren, Pär
    et al.
    Högskolan Kristianstad, Sektionen för Lärarutbildning.
    Mårtensson, Lennart
    Högskolan Kristianstad, Sektionen för Lärarutbildning.
    Mathiasson, Lennart
    Division of Analytical Chemistry, Lund University.
    Improved spectrophotometric vitellogenin determination via alkali-labile phosphate in fish plasma: a cost effective approach for assessment of endocrine2009Inngår i: International Journal of Environmental Analytical Chemistry, ISSN 0306-7319, E-ISSN 1029-0397, Vol. 89, nr 14, 1023-1042 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Vitellogenin (VTG) is a well known protein biomarker for exposure to environmental estrogens and possible endocrine disruption in fish. VTG is very dominant in plasma after the onset of vitellogenesis and the protein is heavily phosphorylated. This enables indirect quantification through measurement of alkali-labile protein bound phosphate (ALP) as an alternative to the more expensive enzyme linked immunosorbent assay. Good correlation has previously been shown between ALP and actual VTG levels but little effort has been made to investigate the method in an analytical way e.g., to assure the origin of the measured phosphate. During this method development care has been taken to rule out non-VTG sources of phosphate such as phospholipids and free phosphate in the blood plasma. Sample preparation has been simplified and unnecessary steps have been omitted. The common spectrophotometric measurement for ALP involves measurement at two wavelengths and calculation of corrected absorbance values. With a quick phase separation step the spectrophotometric phosphate determination using molybdic acid and ascorbic acid has been improved and all matrix interference has been eliminated. The final ALP method presented here has a detection limit of 3.2 µg PO43-/ml plasma which is six times lower than similar methods and it also has less variability. A high sample throughput in comparison to previous ALP methods is possible after scaling down sample and reagent volumes to fit in a 96 well microtiter plate. The cost for buying all chemicals and plastic consumer goods for setting up the indirect protocol for the analysis of 1000 samples is only circa 350 euro. This is only 1% of the material cost for buying commercially available test kit for direct quantification of VTG in the same number of samples. The ALP method should thus be of interest also for applied scientists outside advanced research laboratories.

  • 3.
    Hansen, M.
    et al.
    University of California Berkeley.
    Popovic, O.
    University of Copenhagen.
    Björklund, Erland
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap.
    Krogh, K.
    University of Copenhagen.
    Stoumann, L.
    University of Copenhagen.
    Jacobsen, C.S.
    Geological Survey of Denmark and Greenland.
    Halling-Sørensen, B.
    University of Copenhagen.
    Impact of animal manure separation technologies on steriod hormone distribution: consequences for agricultural practices2014Konferansepaper (Fagfellevurdert)
    Abstract [en]

    When steroid hormones are emitted into the environment, they may have harmful effects on the reproduction system of aquatic life. Until now, research has primarily focused on human excretion, demonstrating that steroid hormones reach the aquatic environment due to insufficient removal in waste water treatment processes. However more recently, it has been revealed that agricultural practices also may add to the environmental burden of steroid hormones. So far, research activities have mainly focused on steroid estrogens, but also androgens, progestagens and glucocorticoids, expressed in the vertebrate steroidogenesis, may occur at substantial levels in animal manure and should be addressed. In agricultural practices the animal manure can be applied to the soil as raw manure, but also as a solid or liquid manure fraction, since current livestock production facilities utilizes a recently developed technology, which separates raw animal manure into a solid and a liquid fraction.This technology offers an improved handling and refined distribution of the manure nutrients to the farmlands and the possibility to reduce the environmental impact of manure nutrients, especially avoiding the surplus load of phosphorous. In the present work we investigated the distribution of 9 steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol and 17β-estradiol) in raw manure and manure separates from 10 to 15 different pig farms in Denmark utilizing 4 different separation technologies. Furthermore, we investigated a possible relationship between the steroid hormone concentration and the different manure fractions and separation technologies. The chemical steroid hormone analysis was done by inverse and integrated clean-up pressurized liquid extraction, and further cleaned by a two step solid-phase extraction before derivatization and finally analyzed by GC-MS/MS.It was found that the steroid hormones were predominant in the solid manure separate calling for manure management strategies to reduce the content of steroid hormones in separated manure solid fraction. This could potentially be achieved through composting or anaerobic digestion for biogas production of the solid fraction; however, the effects of these technologies on steroid hormones need to be verified.

  • 4.
    Hansen, Martin
    et al.
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Björklund, Erland
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Popovic, Olga
    Faculty of Science, University of Copenhagen.
    Jensen, Lars S.
    Faculty of Science, University of Copenhagen.
    Jacobsen, Carsten S.
    Geological Survey of Denmark and Greenland (GEUS).
    Sedlak, David L.
    Department of Civil and Environmental Engineering, University of California, Berkeley.
    Halling-Sørensen, Bent
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Animal manure separation technologies diminish the environmental burden of steroid hormones2015Inngår i: Environmental Science & Technology Letters, ISSN 2328-8930, Vol. 2, nr 4, 133-137 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Newly developed treatment technologies are capable of separating livestock manure into a liquid fraction and a solid fraction using sedimentation, mechanical, and/or chemical methods. These technologies offer a potential means of distributing nutrients to agricultural lands without the unwanted environmental risks associated with the release of steroid hormones to adjacent waterways. To assess the potential benefit of these technologies in reducing the level of release of steroid hormones to adjacent waterways, distribution profiles of nine steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol, and 17β-estradiol) were determined in raw swine manure, and in solid and liquid fractions separated from ten full-scale manure separation systems. Steroid hormone concentrations, normalized for nitrogen content, were significantly higher in separated solids than in liquids. If separated liquids are applied instead of raw manure, steroid hormone loading can be reduced by a factor of 2 at a constant nitrogen fertilization level.

  • 5.
    Hansen, Martin
    et al.
    Institut for Farmaci, Københavns Universitet.
    Rodríguez-Navas, Carlos
    University of Texas Southwestern Medical Center.
    Björklund, Erland
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap.
    Lægemidler i vandmiljøet på Mallorca2013Inngår i: Dansk kemi, ISSN 0011-6335, Vol. 94, nr 8, 24-27 s.Artikkel i tidsskrift (Annet (populærvitenskap, debatt, mm))
  • 6.
    Nielsen, Frederik Knud
    et al.
    Danmark.
    Hansen, Cecilie Hurup
    Danmark.
    Fey, Jennifer Anna
    Danmark.
    Hansen, Martin
    USA.
    Halling-Sørensen, Bent
    Danmark.
    Björklund, Erland
    Högskolan Kristianstad, Sektionen för lärande och miljö, Avdelningen för Naturvetenskap.
    Styrishave, Bjarne
    Danmark.
    Mixture effects of 3 mechanistically different steroidogenic disruptors (prochloraz, genistein, and ketoconazole) in the H295R cell assay2015Inngår i: International journal of toxicology, ISSN 1091-5818, E-ISSN 1092-874X, Vol. 34, nr 6, 534-542 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Mixture effects of 3 model endocrine disruptors, prochloraz, ketoconazole, and genistein, on steroidogenesis were tested in the adrenocortical H295R cell line. Seven key steroid hormones (pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone, and 17β-estradiol) were analyzed using gas chromatography and tandem mass spectrometry (GC-MS/MS) to investigate the effects throughout the steroidogenic pathway. Current modeling approaches often rely on models assuming compounds acting independently and that the individual effects in some way can be summarized to predict a mixture effect. In H295R cells with an intact steroidogenic pathway, such assumptions may not be feasible. The purpose of this study was therefore to evaluate whether effects of a mixture with differing modes of action followed or deviated from additivity (concentration addition) and whether the H295R cell line was suitable for evaluating mixture toxicity of endocrine disruptors with different modes of action. The compounds were chosen because they interfere with steroidogenesis in different ways. They all individually decrease the concentrations of the main sex steroids downstream but exert different effects upstream in the steroidogenic pathway. Throughout the study, we observed lowest observed effect concentrations of mixtures at levels 2 to 10 times higher than the predicted EC50, strongly indicating antagonistic effects. The results demonstrate that chemical analysis combined with the H295R cell assay is a useful tool also for studying how mixtures of endocrine disruptors with differing modes of action interfere with the steroidogenic pathway and that existing models like concentration addition are insufficient in such cases. Furthermore, for end points where compounds exert opposite effects, no relevant models are available.

  • 7.
    Oppong Bekoe, Samuel
    et al.
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Bak, Søren Alex
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Björklund, Erland
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Krogh, Kristine A.
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Okine, Nathaniel N. N. A.
    Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
    Adosraku, Reimmiel K.
    Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
    Styrishave, Bjarne
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Hansen, Martin
    Faculty of Health and Medical Sciences, University of Copenhagen.
    Determination of thirteen antibiotics in drug products: a new LC-MS/MS tool for screening drug product quality2014Inngår i: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 6, 5847-5855 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Poor quality antibiotic medicines in circulation in Sub-Saharan Africa continue to be a burden. Pharmaceutical trade in substandard and counterfeit medicines is on the rise. The chemical quality of antibiotics dispensed in health facilities and recognised drug outlets in Ghana, when compromised, could be a major drawback to efforts made in fighting antibiotic resistance globally. To improve on antibiotic drug quality monitoring, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology, which is capable of quantifying thirteen antibiotics in drug products, was developed and validated in present work. The methodology was applied to various drug products including tablets, capsules, suspensions, syrups, intravenous and injection solutions as well as ear and eye droplets used as essential medicines in a Sub-Saharan country, Ghana.

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