Effects of arsenolipids on in vitro blood-brain barrier model.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids (AsLs) occurring in fish and edible algae, possess a substantial neurotoxic potential in fully differentiated human brain cells. Previous in vivo studies indicating that AsHCs cross the blood-brain barrier of the fruit fly Drosophila melanogaster raised the question whether AsLs could also cross the vertebrate blood-brain barrier (BBB). In the present study, we investigated the impact of several representatives of AsLs (AsHC 332, AsHC 360, AsHC 444, and two arsenic-containing fatty acids, AsFA 362 and AsFA 388) as well as of their metabolites (thio/oxo-dimethylpropionic acid, dimethylarsinic acid) on porcine brain capillary endothelial cells (PBCECs, in vitro model for the blood-brain barrier). AsHCs exerted the strongest cytotoxic effects of all investigated arsenicals as they were up to fivefold more potent than the toxic reference species arsenite (iAsIII). In our in vitro BBB-model, we observed a slight transfer of AsHC 332 across the BBB after 6 h at concentrations that do not affect the barrier integrity. Furthermore, incubation with AsHCs for 72 h led to a disruption of the barrier at sub-cytotoxic concentrations. The subsequent immunocytochemical staining of three tight junction proteins revealed a significant impact on the cell membrane. Because AsHCs enhance the permeability of the in vitro blood-brain barrier, a similar behavior in an in vivo system cannot be excluded. Consequently, AsHCs might facilitate the transfer of accompanying foodborne toxicants into the brain.

Arsenic-containing hydrocarbons: effects on gene expression, epigenetics, and biotransformation in HepG2 cells.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids found in fish and algae, elicit substantial toxic effects in various human cell lines and have a considerable impact on cellular energy levels. The underlying mode of action, however, is still unknown. The present study analyzes the effects of two AsHCs (AsHC 332 and AsHC 360) on the expression of 44 genes covering DNA repair, stress response, cell death, autophagy, and epigenetics via RT-qPCR in human liver (HepG2) cells. Both AsHCs affected the gene expression, but to different extents. After treatment with AsHC 360, flap structure-specific endonuclease 1 (FEN1) as well as xeroderma pigmentosum group A complementing protein (XPA) and (cytosine-5)-methyltransferase 3A (DNMT3A) showed time- and concentration-dependent alterations in gene expression, thereby indicating an impact on genomic stability. In the subsequent analysis of epigenetic markers, within 72 h, neither AsHC 332 nor AsHC 360 showed an impact on the global DNA methylation level, whereas incubation with AsHC 360 increased the global DNA hydroxymethylation level. Analysis of cell extracts and cell media by HPLC-mass spectrometry revealed that both AsHCs were considerably biotransformed. The identified metabolites include not only the respective thioxo-analogs of the two AsHCs, but also several arsenic-containing fatty acids and fatty alcohols, contributing to our knowledge of biotransformation mechanisms of arsenolipids.

Is it possible to agree on a value for inorganic arsenic in food? The outcome of IMEP-112.

Two of the core tasks of the European Union Reference Laboratory for Heavy Metals in Feed and Food (EU-RL-HM) are to provide advice to the Directorate General for Health and Consumers (DG SANCO) on scientific matters and to organise proficiency tests among appointed National Reference Laboratories. This article presents the results of the 12th proficiency test organised by the EU-RL-HM (IMEP-112) that focused on the determination of total and inorganic arsenic in wheat, vegetable food and algae. The test items used in this exercise were: wheat sampled in a field with a high concentration of arsenic in the soil, spinach (SRM 1570a from NIST) and an algae candidate reference material. Participation in this exercise was open to laboratories from all around the world to be able to judge the state of the art of the determination of total and, more in particular, inorganic arsenic in several food commodities. Seventy-four laboratories from 31 countries registered to the exercise; 30 of them were European National Reference Laboratories. The assigned values for IMEP-112 were provided by a group of seven laboratories expert in the field of arsenic speciation analysis in food. Laboratory results were rated with z and ζ scores (zeta scores) in accordance with ISO 13528. Around 85 % of the participants performed satisfactorily for inorganic arsenic in vegetable food and 60 % did for inorganic arsenic in wheat, but only 20 % of the laboratories taking part in the exercise were able to report satisfactory results in the algae test material.

Accuracy of a method based on atomic absorption spectrometry to determine inorganic arsenic in food: Outcome of the collaborative trial IMEP-41.

A collaborative trial was conducted to determine the performance characteristics of an analytical method for the quantification of inorganic arsenic (iAs) in food. The method is based on (i) solubilisation of the protein matrix with concentrated hydrochloric acid to denature proteins and allow the release of all arsenic species into solution, and (ii) subsequent extraction of the inorganic arsenic present in the acid medium using chloroform followed by back-extraction to acidic medium. The final detection and quantification is done by flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS). The seven test items used in this exercise were reference materials covering a broad range of matrices: mussels, cabbage, seaweed (hijiki), fish protein, rice, wheat, mushrooms, with concentrations ranging from 0.074 to 7.55mgkg(-1). The relative standard deviation for repeatability (RSDr) ranged from 4.1 to 10.3%, while the relative standard deviation for reproducibility (RSDR) ranged from 6.1 to 22.8%.

In vitro toxicological characterisation of arsenic-containing fatty acids and three of their metabolites.

Arsenic-containing fatty acids are a group of fat-soluble arsenic species (arsenolipids) which are present in marine fish and other seafood. Recently, it has been shown that arsenic-containing hydrocarbons, another group of arsenolipids, exert toxicity in similar concentrations comparable to arsenite although the toxic modes of action differ. Hence, a risk assessment of arsenolipids is urgently needed. In this study the cellular toxicity of a saturated (AsFA 362) and an unsaturated (AsFA 388) arsenic-containing fatty acid and three of their proposed metabolites (DMAV, DMAPr and thio-DMAPr) were investigated in human liver cells (HepG2). Even though both arsenic-containing fatty acids were less toxic as compared to arsenic-containing hydrocarbons and arsenite, significant effects were observable at µM concentrations. DMAV causes effects in a similar concentration range and it could be seen that it is metabolised to its highly toxic thio analogue thio-DMAV in HepG2 cells. Nevertheless, DMAPr and thio-DMAPr did not exert any cytotoxicity. In summary, our data indicate that risks to human health related to the presence of arsenic-containing fatty acids in marine food cannot be excluded. This stresses the need for a full in vitro and in vivo toxicological characterisation of these arsenolipids.

Determination of total cadmium, lead, arsenic, mercury and inorganic arsenic in mushrooms: outcome of IMEP-116 and IMEP-39.

The Institute for Reference Materials and Measurements (IRMM) of the Joint Research Centre (JRC), a Directorate General of the European Commission, operates the International Measurement Evaluation Program (IMEP). IMEP organises inter-laboratory comparisons in support of European Union policies. This paper presents the results of two proficiency tests (PTs): IMEP-116 and IMEP-39, organised for the determination of total Cd, Pb, As, Hg and inorganic As (iAs) in mushrooms. Participation in IMEP-116 was restricted to National Reference Laboratories (NRLs) officially appointed by national authorities in European Union member states. IMEP-39 was open to all other laboratories wishing to participate. Thirty-seven participants from 25 countries reported results in IMEP-116, and 62 laboratories from 36 countries reported for the IMEP-39 study. Both PTs were organised in support to Regulation (EC) No. 1881/2006, which sets the maximum levels for certain contaminants in food. The test item used in both PTs was a blend of mushrooms of the variety shiitake (Lentinula edodes). Five laboratories, with demonstrated measurement capability in the field, provided results to establish the assigned values (Xref). The standard uncertainties associated to the assigned values (uref) were calculated by combining the uncertainty of the characterisation (uchar) with a contribution for homogeneity (ubb) and for stability (ust), whilst uchar was calculated following ISO 13528. Laboratory results were rated with z- and zeta (ζ)-scores in accordance with ISO 13528. The standard deviation for proficiency assessment, σp, ranged from 10% to 20% depending on the analyte. The percentage of satisfactory z-scores ranged from 81% (iAs) to 97% (total Cd) in IMEP-116 and from 64% (iAs) to 84% (total Hg) in IMEP-39.

Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system.

Transcellular Ca2+ transport in the distal nephron involves passive Ca2+ influx at the apical membrane, diffusion through the cytosol and active extrusion across the opposing basolateral membrane. The molecular identity of the apical Ca2+ entry step is still elusive, but its regulatory aspects have been analyzed in the present study. To this end, rabbit connecting and cortical collecting tubular cells were cultured on permeable and transparent supports and the apical Ca2+ influx was deduced from Mn2+ quenching of Ca2+ independent Fura-2 fluorescence, while the intracellular Ca2+ concentration ([Ca2+]i) was measured simultaneously. In parallel experiments, transcellular Ca2+ transport was determined isotopically as 45Ca2+ flux from the apical to basolateral compartment. Decreasing the apical pH from 7.4 to 5.9 inhibited transcellular Ca2+ transport by 53 +/- 1%, whereas apical Ca2+ influx was reduced by 39 +/- 7% and [Ca2+]i decreased by 18 +/- 3%. Reversal of the Na+/Ca2+ exchanger by iso-osmotic replacement of Na+ by N-methyl-D-glucamine in the basolateral compartment resulted in 50 +/- 5% inhibition of Ca2+ transport, 46 +/- 3% reduction of apical Ca2+ influx and 60 +/- 3% increase in [Ca2+]i. In the absence of basolateral Ca2+, however, this manoeuvre decreased [Ca2+]i by 21 +/- 8%, while Ca2+ transport and apical Ca2+ influx were reduced by the same magnitude as in the presence of Ca2+, that is by 53 +/- 6% and 45 +/- 4%, respectively. Stimulation of adenylyl cyclase with forskolin (10(-5) M) increased transcellular Ca2+ transport by 108 +/- 40%, stimulated apical Ca2+ influx by 120 +/- 17% and increased [Ca2+]i by 110 +/- 2%. In conclusion, the apical Ca2+ influx is regulated by apical pH, intracellular cAMP and basolateral Na+/Ca2+ exchanger activity, and is coupled in an 1:1 fashion to the rate of transepithelial Ca2+ transport.

Effect of stobadine on Cu(++)-mediated oxidation of low-density lipoprotein.

The pyridoindole derivative stobadine [(-)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido(4,3b) indole] has been described as a drug with antihypoxic and antiarrhythmic cardioprotective properties. The antioxidative properties of this compound were studied during Cu(++)-mediated low-density lipoprotein (LDL) oxidation. Stobadine (concentration 0-5 microM) prolonged the lag phase (in min produced by one molecule antioxidant per LDL particle) as measured by diene formation more effectively than did ascorbate, trolox, or alpha-tocopherol. It also has the ability to decrease the rate of diene formation during the propagation phase very efficiently. Diene formation, Trp destruction, and alpha-tocopherol consumption were measured in the presence and absence of stobadine. Stobadine (10 microM) did not influence tocopherol consumption during oxidation and the Trp fluorescence quenching of Cu++ was not influenced by this compound. From these results, as well as polarographic measurements, we conclude that the antioxidative effect of stobadine is not simply a result of Cu(++)-ion complexation. In contrast to ascorbate, this compound is stable in the presence of Cu++. Stobadine inhibits the oxidation of LDL-Trp residues very efficiently via its radical scavenging properties, and may even have the ability to reduce Trp radicals to tryptophan. The concentration of stobadine used for LDL oxidation was in the range found in plasma (stobadine given p.o. in human and rats results in plasma concentrations between 0.2-3.9 microM.

New preconditioning strategy for the determination of inorganic anions with capillary zone electrophoresis using indirect UV detection.

It is widely accepted that preconditioning procedures are indispensable in capillary electrophoresis in order to achieve reproducibility of migration times and peak areas. Several preconditioning strategies have been employed for electrophoretic determinations of inorganic anions using indirect UV detection including simple flushing with buffer or alkaline or acid pre-rinsing followed by flushing with electrolyte. We investigated the influence of various preconditioning strategies on the reproducibility of migration times and peak areas of inorganic anions. The electrolyte systems for indirect UV detection were based on pyromellitic acid and chromic acid respectively as UV absorbing probes and hexamethonium hydroxide as electroosmatic flow modifier. Preconditioning agents under investigation were electrolyte buffer, NaOH, HCl and the free acids of the UV absorbing probes. Investigations showed that reproducibility of migration times and peak areas can be significantly improved by acid pre-rinsing using the corresponding acid of the UV absorbing probes compared to preconditioning by flushing the capillary with buffer. In contrast to acid pre-rinsing using hydrochloric acid no interfering signals within the migration time window of inorganic anions under investigation can be observed. The optimized preconditioning procedure yields relative standard deviations of migration times less than 0.25% (n = 10). Relative standard deviations of corrected peak areas were below 5% applying acid preconditioning using pyromellitic acid.

Anodic stripping voltammetric determination of titanium(IV) using a carbon paste electrode modified with cetyltrimethylammonium bromide.

A method is described for the voltammetric determination of titanium(IV) using a carbon paste electrode modified in situ with cetyltrimethylammonium bromide. The cationic micellar surfactant adsorbs onto the electrode particularly at negative potentials, simultaneously preconcentrating titanium(IV) as the oxalate complex with reduction to titanium(III). Anodic stripping voltammetry exploiting reoxidation can be used for the determination of trace levels of titanium(IV). Linearity between current and concentration exists between 5 and 160 mug l(-1) Ti(IV) (preconcentration time 2 min). The limit of detection (calculated as 3sigma) is 0.1 mug l(-1), with a preconcentration time of 10 min.

[Value of computerized tomography for the assessment of the parametrium in cervical cancer]. / Wert der Computertomographie zur Parametrienbeurteilung bei Zervixkarzinomen.

The clinical and CT appearances of the parametrium in 79 patients with carcinoma of the cervix were compared with the histological findings obtained at surgery. The accuracy of CT in stages T1/T2a was 46.6%, in category T2b 64.9% and in category T3b 66.6%. Clinical examination showed fewer errors. CT does not contribute to the evaluation of infiltration of the parametrium from carcinoma of the cervix, nor can it correct the findings on palpation. Tumour infiltration and operability are not demonstrated with sufficient accuracy by CT.

[Breast feeding, lactation disorders and inflammatory diseases of the female breast]. / Stillen, Stillschwierigkeiten und entzündliche Erkrankungen der weiblichen Brust.

Approximately 98% of our female population are capable of breast-feeding. Optimal success in breast-feeding is achieved when the first suckling stimulus occurs no later than 2 h following delivery. Obstetrical surgery impairs breast-feeding. Lactation is considered to be optimal with 7 to 10 min of milk uptake and 7 to 10 min of suckling for galactopoesis and discontinuation. The individual pattern of suckling is critical for initiation and duration of lactation. Due to environmental contamination of the milk, a critical assessment of advantages and disadvantages of breast-feeding needs to be performed if it is considered for more than four months. Early diagnosis and immediate initiation of treatment of mastitis helps avoiding abscess formation with potential surgical sequelae. Medical--dopamine agonists--and physical therapy permit continuation of breast-feeding.

[Nipple discharge: differential diagnostic considerations]. / Die sezernierende Brust: differentialdiagnostische Uberlegungen.

Each uni- or bilateral secretion of the nipple--spontaneous or easy to trigger--has to be examined thoroughly, independent of the colour or the amount of the secretion. Colours of the secretions do not refer to a specific breast disease. Main causes are functional disease with and without endogenous increase of prolactin secretion and benign and malignant breast diseases. Mastopathia, papilloma, fibroadenoma, nonpuerperal mastitis, and carcinoma of the breast are the most common pathological findings associated with secretions of the mammary gland.

Arsenic species in poultry feather meal.

Organoarsenical drugs are widely used in the production of broiler chickens in the United States. Feathers from these chickens are processed into a meal product that is used as an animal feed additive and as an organic fertilizer. Research conducted to date suggests that arsenical drugs, specifically roxarsone, used in poultry production result in the accumulation of arsenic in the keratinous material of poultry feathers. The use of feather meal product in the human food system and in other settings may result in human exposures to arsenic. Consequently, the presence and nature of arsenic in twelve samples of feather meal product from six US states and China were examined. Since arsenic toxicity is highly species-dependent, speciation analysis using HPLC/ICPMS was performed to determine the biological relevance of detected arsenic. Arsenic was detected in all samples (44-4100 µg kg(-1)) and speciation analyses revealed that inorganic forms of arsenic dominated, representing 37 - 83% of total arsenic. Roxarsone was not detected in the samples (<20 µg As kg(-1)). Feather meal products represent a previously unrecognized source of arsenic in the food system, and may pose additional risks to humans as a result of its use as an organic fertilizer and when animal waste is managed.

[Computerized tomography in the diagnosis of space-occupying gynecological processes. 2: examination technic and normal anatomy of the female pelvis in CT scans]. / Computertomographie zur Diagnostik raumfordernder gynäkologischer Prozesse. Teil 2: Untersuchungstechnik und normale Anatomie des weiblichen Beckens im CT-Schnittbild.

Differentiation of the organs in the small pelvis by computed tomography is often difficult, because of the small differences of density. General contrasting the bowel, intravenous injection of opaque matter and tamponing the vagina can facilitate the identification and estimation of pathological processes of female genitals. Presumption of any findings is the exact knowledge of X-ray topography. Following the explanation of patient preparation and of examination technique the normal anatomy of female pelvis is demonstrated by seven scans.

[The status of computerized tomography in the diagnosis of primary cervix cancer]. / Stellenwert der Computertomographie bei der Diagnostik des primären Zervixkarzinoms.

172 patients with a primary cervical carcinoma have been examined using computerized axial tomography. CT-diagnosis was then analysed and compared with the results of the gynecological examination, cystoscopy, rectoscopy, lymphography, conventional radiological examination procedures and histology. CT is unsuitable for the primary diagnosis of cervical carcinoma and anyway unnecessary. In the case, where the carcinoma expands to the vagina, the diagnosis is quicker and more exact through inspection, palpation and biopsy rather than through CT. Moreover, to exclude a possible expansion of the carcinoma to the bladder and rectum, it is necessary that cystoscopy, rectoscopy as well as colon enema be performed. These examination procedures are in this case more effective than CT. Using CT, it is not feasible either to diagnose correctly the degree of infiltration of the parametria or to make definite predictions on the siting and operability. It is not necessary to use CT as a primary means of judging lymph node status, since other findings relevant to therapy (infiltration of the parametria, bladder, rectum) cannot be undertaken simultaneously with adequate safety. The use of CT in the diagnosis of the primary cervical carcinoma is therefore only of minimal importance.