Untersuchungen zum Humanmetabolismus des Bismuts mittels massenspektrometrischer Kopplungstechniken

Biological methylation and hydride formation of metals and metalloids are ubiquitous environmental processes that can lead to the formation of chemical species with significantly increased mobility and toxicity. While much is known about the interaction of metal(loid)s with microorganisms in environmental settings, little information has been gathered on respective processes inside the human body as yet. Here, the biotransformation and excrDie Biomethylierung und die Hydridbildung von Metall(oid)en sind ubiquitäre Prozesse in der Umwelt, die zur Bildung von chemischen Spezies mit erhöhter Mobilität und Toxizität führen können. Während viel über die Wechselwirkung von Metall(oid)en mit Mikroorganismen unter Umweltbedingungen ermittelt wurde, sind bisher nur wenige Informationen über vergleichbare Prozesse im Menschen vorhanden. Diese Arbeit stellt einen Beitrag zur Biotransformation und Eliminierung von Bismut im Menschen dar, die in zwei Probandenstudien mit massenspektrometrischen Kopplungstechniken (GC/ICP-MS, GC/EI-MS/ICP-MS, GC/MS und ICP-MS) untersucht wurden. Weiterhin wurden humane Hepatozyten (HepG2) mit Bismut-haltigen Reagenzien (CBS, Bismutcystein und Bismutglutathion) inkubiert, um durch die Detektion der Metaboliten in vitro Hinweise auf die Beteiligung der humanen Leber an der Biomethylierung des Bismuts zu erhalten. Bei der ersten Probandenstudie wurde 20 männlichen Probanden eine Dosis von 215 mg Bismut in Form von Bismutsubcitrat oral verabreicht und die Biotransformation und Eliminierung des Bismut beobachtet. Die sehr geringe Bismut-Resorption im Magen uetion of bismuth following ingestion of bismuth in two studies were investigated with hyphenated mass spectrometric techniques (GC/ICP-MS, GC/EI-MS/ICP-MS, GC/MS and ICP-MS). In addition, human hepatoma cells (HepG2) were incubated with bismuth containing compounds (CBS, bismuth cysteine, bismuth glutathione) in order to investigate the involvement of the human liver in the biomethylation of bismuth in vitro. In the first study the biotransformation and excretion of bismuth following ingestion of bismuth subcitrate (215 mg bismuth) to 20 male human volunteers were investigated. Bismuth absorption in the stomach and upper intestine was very low, as evidenced by the small quantity of bismuth eliminated via the renal route. Total bismuth concentrations in blood increased rapidly in the first hour following ingestion. Most of the ingested bismuth was excreted via feces during the study period. Trace levels of the metabolite trimethylbismuth ((CH3)3Bi) were detected via low temperature-gas chromatography/inductively coupled plasma-mass spectrometry (LT-GC/ICP-MS) in blood samples and in exhaled air samples. Concentrations were in the range of up to 2.50 pg/ml (blood) and 0.8-458 ng/m3 (exhaled air), with high inter-individual variation. Elimination routes of bismuth were exhaled air (up to 0.03 ‰), urine (0.03-1.2 %), and feces. In the second study the biotransformation and excretion were observed with a higher time resolution. Here, in three experiments 215 mg bismuth (twice) and 430 mg bismuth (single) were given to the same male volunteer. The results of the first study were basically confirmed and, moreover, a few characteristic (CH3)3Bi concentration maxima were detected in exhaled air – each time after food intake. This observation indicates the involvement of enzymes in the biotransformation of bismuth, which were activated by food intake. In addition to this result (CH3)3Bi was detected in feces samples in a range from 1738 ng/kg to 5679 ng/kg. The headspace analysis of human hepatoma cells (HepG2) which were incubated with bismuth containing compounds (CBS, bismuth cysteine, bismuth glutathione) indicated that (CH3)3Bi was not detectable in any of the samples. After ethylation with sodium tetraethylborate at pH 7 monomethyl diethyl bismuth was found in cell lysates of cell cultures which were incubated with CBS or bismuth cysteine. In contrast, no monomethyl diethyl bismuth was not detectable after incubation with bismuth glutathione. These results show that human hepatoma cells have the potential to methylate bismuth and that the permethylated species is not generated within the observed time period. If methylation was not observed as in the case of bismuth glutathione, this might result from the low uptake of this compound into the hepatoma cells. In conclusion, it was shown in these studies that bismuth is methylated in the human body. It appears from the results that both the intestinal microflora and the liver are involved in this biotransformation.Die Biomethylierung und die Hydridbildung von Metall(oid)en sind ubiquitäre Prozesse in der Umwelt, die zur Bildung von chemischen Spezies mit erhöhter Mobilität und Toxizität führen können. Während viel über die Wechselwirkung von Metall(oid)en mit Mikroorganismen unter Umweltbedingungen ermittelt wurde, sind bisher nur wenige Informationen über vergleichbare Prozesse im Menschen vorhanden. Diese Arbeit stellt einen Beitrag zur Biotransformation und Eliminierung von Bismut im Menschen dar, die in zwei Probandenstudien mit massenspektrometrischen Kopplungstechniken (GC/ICP-MS, GC/EI-MS/ICP-MS, GC/MS und ICP-MS) untersucht wurden. Weiterhin wurden humane Hepatozyten (HepG2) mit Bismut-haltigen Reagenzien (CBS, Bismutcystein und Bismutglutathion) inkubiert, um durch die Detektion der Metaboliten in vitro Hinweise auf die Beteiligung der humanen Leber an der Biomethylierung des Bismuts zu erhalten. Bei der ersten Probandenstudie wurde 20 männlichen Probanden eine Dosis von 215 mg Bismut in Form von Bismutsubcitrat oral verabreicht und die Biotransformation und Eliminierung des Bismut beobachtet. Die sehr geringe Bismut-Resorption im Magen und im oberen Darmsegment wurde über die geringe renal eliminierte Bismutmenge nachgewiesen. Nach der Bismutgabe stiegen die Bismut-Konzentrationen im Blut sehr schnell innerhalb der ersten Stunde bis zu einem Maximum an. Der überwiedende Teil des Bismuts wurde fäkal eliminiert. Zudem wurden Spuren des Metaboliten Trimethylbismut ((CH3)3Bi) via LT-GC/ICPMS in Blut- und Atemproben detektiert. Bis zu 2,50 pg/ml (Blut) und 458 ng/m3 (Atemluft) wurden mit großer interindividueller Variation bestimmt. Die Eliminationspfade waren die Atemluft (bis zu 0,03 ‰), der Urin (0,03-1,2 %) und die Fäzes. In einer weiteren Probandenstudie wurde die Biotransformation und Eliminierung des Bismuts mit einer größeren Zeitauflösung beobachtet. Hierzu wurde in drei Teilstudien dem jeweils gleichen männlichen Probanden zweimal je 215 mg und einmal 430 mg Bismut in Form von Bismutsubcitrat verabreicht. Neben der grundsätzlichen Bestätigung der Ergebnisse aus der ersten Studie wurden durch das modifizierte Studiendesign mehrere charakteristische (CH3)3Bi-Konzentrationsmaxima in der Atemluft detektiert, welche stets nach der Nahrungsaufnahme beobachtet wurden. Diese Beobachtung deutet auf die Beteiligung von Enzymen bei der Methylierung des Bismuts hin, die durch die Nahrungsaufnahme aktiviert werden. Weiterhin wurde (CH3)3Bi in Fäzesproben nachgewiesen und (CH3)3Bi- Maximalkonzentrationen von 1738 ng/kg bis 5679 ng/kg bestimmt. Bei den In-vitro-Experimenten mit HepG2-Zellkulturen wurde nach der 24-stündigen Inkubation mit Bismut-haltigen Reagenzien kein (CH3)3Bi im Headspace der Zellkultur nachgewiesen. Nach der Ethylierung des Zelllysats mit Natriumtetraethylborat bei pH 7 wurde Monomethyldiethylbismut nur in den Zellkulturen nachgewiesen, die mit Bismutsubcitrat und Bismutcystein inkubiert wurden; der gleiche Versuch mit Bismutglutathion war negativ. Diese Ergebnisse zeigen, dass humane Leberzellen das Potenzial zur Bismutmethylierung haben und das die permethylierte Spezies nicht im Beobachtungszeitraum gebildet wird. Die Methylierung des Bismutgluthations wurde möglicherweise aufgrund der geringen Aufnahme des Bismutglutathions durch die humanen Leberzellen nicht nachgewiesen. Letztendlich wurde in dieser Arbeit gezeigt, dass Bismut im humanen Organismus methyliert wird. Durch die hier präsentierten Ergebnisse erscheint es so, dass die intestinale Mikroflora und die humane Leber an dieser Biotransformation beteiligt sind

CERTIFICATION REPORT The certification of the mass fraction of arsenobetaine in water: ERM®-AC626

This report describes the production of ERM-AC626, an aqueous solution material certified for the mass fraction of arsenobetaine (AB). The material was produced following ISO Guide 34:2009. The starting material was 10 g of solid AB monohydrate. The purity of AB was assessed through measurements carried out by laboratories of demonstrated competence and within the scope of accreditation to ISO/IEC 17025:2005. An aqueous solution was prepared and ampouled into 2 mL amber glass ampoules under argon atmosphere. Between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. Due to the inherent homogeneity of water solutions, determination of minimum sample intake (within-unit homogeneity) was not required. The sample intake used in the homogeneity study was adopted as the recommended minimum sample intake. The minimum sample intake is 50 µg. The certified value was obtained from the gravimetric preparation of the solution, taking into account the purity of the starting material. The certified value was confirmed by independent analyses carried out by laboratories of demonstrated competence and adhering to ISO/IEC 17025. Uncertainties of the certified values were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties related to possible inhomogeneity and instability and to characterisation. The material is intended to be used as a calibrant and quality control sample. The CRM is available in amber glass ampoules containing 1 mL of AB aqueous solution closed under argon atmosphere. The minimum amount of sample to be used is 50 µg. The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.F.6-Reference Material

The certification of the mass fractions of total arsenic, dimethylarsinic acid and the sum of arsenite and arsenate in rice: ERM-BC211

This report describes the production of ERM-BC211, a powdered rice material certified for the mass fractions of total arsenic, dimethylarsinic acid and the sum of arsenite and arsenate. The material has been produced following ISO Guide 34:2009. The starting material, which had been checked for its arsenic species was purchased and supplied by the University of Aberdeen. The rice was milled, sieved, dried, homogenised, filled in vials and sterilised. Between unit-inhomogeneity was quantified and stability during dispatch and storage was assessed in accordance with ISO Guide 35:2006 . Within-unit inhomogeneity was quantified to determine the minimum sample intake. The material was characterised by an intercomparison among laboratories of demonstrated competence and adhering to ISO/IEC 17025. Technically invalid results were removed but no outlier was eliminated on statistical grounds only. Uncertainties of the certified values were estimated in compliance with ISO/IEC Guide 98-3, Guide to the Expression of Uncertainty in Measurement (GUM) and they include contributions from possible between-unit inhomogeneity, instability and characterisation. The material is intended for quality control and assessment of method performance. Moreover, it can be used for validation purposes and trueness determination. The CRM is available in glass bottles containing 10 g of dried rice powder closed under argon atmosphere. The minimum amount of sample to be used for total arsenic and dimethylarsinic acid is 50 mg. The minimum amount of sample to be used for the sum of arsenite and arsenate is 100 mg. The CRM has been accepted as European Reference Material (ERM) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Standards for Innovation and sustainable Developmen

Titanium dioxide nanoparticles induce oxidative stress and DNA-adduct formation but not DNA-breakage in human lung cells

Titanium dioxide (TiO2), also known as titanium (IV) oxide or anatase, is the naturally occurring oxide of titanium. It is also one of the most commercially used form. To date, no parameter has been set for the average ambient air concentration of TiO2 nanoparticles (NP) by any regulatory agency. Previously conducted studies had established these nanoparticles to be mainly non-cyto- and -genotoxic, although they had been found to generate free radicals both acellularly (specially through photocatalytic activity) and intracellularly. The present study determines the role of TiO2-NP (anatase, ∅ < 100 nm) using several parameters such as cyto- and genotoxicity, DNA-adduct formation and generation of free radicals following its uptake by human lung cells in vitro. For comparison, iron containing nanoparticles (hematite, Fe2O3, ∅ < 100 nm) were used. The results of this study showed that both types of NP were located in the cytosol near the nucleus. No particles were found inside the nucleus, in mitochondria or ribosomes. Human lung fibroblasts (IMR-90) were more sensitive regarding cyto- and genotoxic effects caused by the NP than human bronchial epithelial cells (BEAS-2B). In contrast to hematite NP, TiO2-NP did not induce DNA-breakage measured by the Comet-assay in both cell types. Generation of reactive oxygen species (ROS) was measured acellularly (without any photocatalytic activity) as well as intracellularly for both types of particles, however, the iron-containing NP needed special reducing conditions before pronounced radical generation. A high level of DNA adduct formation (8-OHdG) was observed in IMR-90 cells exposed to TiO2-NP, but not in cells exposed to hematite NP. Our study demonstrates different modes of action for TiO2- and Fe2O3-NP. Whereas TiO2-NP were able to generate elevated amounts of free radicals, which induced indirect genotoxicity mainly by DNA-adduct formation, Fe2O3-NP were clastogenic (induction of DNA-breakage) and required reducing conditions for radical formation

Role of Intestinal Microbiota in Transformation of Bismuth and Other Metals and Metalloids into Volatile Methyl and Hydride Derivatives in Humans and Mice▿

The present study shows that feces samples of 14 human volunteers and isolated gut segments of mice (small intestine, cecum, and large intestine) are able to transform metals and metalloids into volatile derivatives ex situ during anaerobic incubation at 37°C and neutral pH. Human feces and the gut of mice exhibit highly productive mechanisms for the formation of the toxic volatile derivative trimethylbismuth [(CH3)3Bi] at rather low concentrations of bismuth (0.2 to 1 μmol kg−1 [dry weight]). An increase of bismuth up to 2 to 14 mmol kg−1 (dry weight) upon a single (human volunteers) or continuous (mouse study) administration of colloidal bismuth subcitrate resulted in an average increase of the derivatization rate from approximately 4 pmol h−1 kg−1 (dry weight) to 2,100 pmol h−1 kg−1 (dry weight) in human feces samples and from approximately 5 pmol h−1 kg−1 (dry weight) to 120 pmol h−1 kg−1 (dry weight) in mouse gut samples, respectively. The upshift of the bismuth content also led to an increase of derivatives of other elements (such as arsenic, antimony, and lead in human feces or tellurium and lead in the murine large intestine). The assumption that the gut microbiota plays a dominant role for these transformation processes, as indicated by the production of volatile derivatives of various elements in feces samples, is supported by the observation that the gut segments of germfree mice are unable to transform administered bismuth to (CH3)3Bi