Marijn Van Hulle

The glycerol channel Fps1p mediates the uptake of arsenite and antimonite in Saccharomyces cerevisiae

The Saccharomyces cerevisiae FPS1 gene encodes a glycerol channel protein involved in osmoregulation. We present evidence that Fps1p mediates influx of the trivalent metalloids arsenite and antimonite in yeast. Deletion of FPS1 improves tolerance to arsenite and potassium antimonyl tartrate. Under high osmolarity conditions, when the Fps1p channel is closed, wild‐type cells show the same degree of As(III) and Sb(III) tolerance as the fps1Δ mutant. Additional deletion of FPS1 in mutants defective in arsenite and antimonite detoxification partially suppresses their hypersensitivity to metalloid salts. Cells expressing a constitutively open form of the Fps1p channel are highly sensitive to both arsenite and antimonite. We also show by direct transport assays that arsenite uptake is mediated by Fps1p. Yeast cells appear to control the Fps1p‐mediated pathway of metalloid uptake, as expression of the FPS1 gene is repressed upon As(III) and Sb(III) addition. To our knowledge, this is the first report describing a eukaryotic uptake mechanism for arsenite and antimonite and its involvement in metalloid tolerance.

A survey of arsenic species in chinese seafood

In the present report, thirty different types of Chinese edible seafood, including brown algae, red algae, fish, crab, shrimp, mussels, oysters, and clams, which are very popular foodstuffs in the Chinese kitchen, were examined for their total content of As as well as its different species. Total arsenic concentration in algae samples was 1.7-38.7 microg/g (dry weight), and 0.086-7.54 microg/g in fish and shellfish (wet weight), respectively. The arsenic species in seafood extracts were determined by using anion and cation exchange high performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICPMS). Arsenosugars were detected in all of the extracted algae samples (1.5-33.8 microg/g dry weight) and fish samples (0.018-0.78 microg/g wet weight). Arsenobetaine was detected in all of the extracted fish and shellfish samples (0.025-6.604 microg/g wet weight). In contrast, inorganic arsenic in fish and shellfish samples occurred at levels below 2% of the total arsenic. No inorganic arsenic was detected in the algae samples. This study provides information about the distribution pattern of arsenic species in seafood products. Since the major share of arsenic components in seafood is organic arsenic with a low toxicity, we can conclude that arsenic in seafood does not pose any risk to human health.

Arsenic speciation in Chinese seaweeds using HPLC-ICP-MS and HPLC-ES-MS

Three common Chinese edible seaweeds, one brown (Laminaria japonica) and two red (Porphyra crispata and Eucheuma denticulatum), were examined for their total arsenic content. The As species were extracted with yields of 76.4, 69.8 and 25.0%, respectively. Anion-exchange and cation-exchange high-performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS) were used for the separation of the different arsenic species in two of the three seaweed extracts (Laminaria and Porphyra). The main arsenic species in the algal extracts are arseno sugars, although it has been shown that the Laminaria seaweed contains significant amounts of dimethylarsinic acid (DMA). HPLC was coupled with electrospray mass spectrometry (ES-MS) for structural confirmation of the arsenic species. The mass spectrometer settings for the arseno sugars were optimised using standards. The conclusions drawn on the basis of HPLC-ICP-MS were confirmed by the HPLC-ES-MS data. The HPLC-ES-MS method is capable of determining both arseno sugars and DMA in the seaweeds. The unknown compounds seen in the HPLC-ICP-MS chromatogram of Laminaria could not be ascribed to trimethylarsenic oxide or tetramethylarsonium ion.

Identification of some arsenic species in human urine and blood after ingestion of Chinese seaweed Laminaria

Algae contain high amounts of arsenic in the form of arsenosugars. The metabolism and toxicology of these arsenic species are not yet fully understood. Three sets of experiments have been conducted in which the alga Laminaria was ingested by 2 to 5 healthy volunteers. Total arsenic concentrations in urine and in blood, packed blood cells and serum have been determined using ICP-MS and HGAFS, respectively. Neutron activation analysis was used for the determination of the total arsenic content in algae samples. Speciation analysis of urine and serum samples has been carried out using HPLC-ICP-MS. HPLC-ES-MS/MS has been used for structural confirmation. The stability of the arsenosugars in simulated gastric fluid was studied for both boiled and unboiled seaweed. A maximum level of arsenic in urine appears within 15 to 25 h after ingestion. Total arsenic and speciation analysis revealed no marked increase in arsenic blood, serum and packed cells levels up to 7 h after ingestion. Dimethylarsinic acid (DMA), methylarsonic acid (MA) and dimethylarsinoylethanol (DMAE) have been positively identified in urine sampled after algae intake. Another 5 species remain unknown. In simulated gastric fluid incubated with algae, the larger share of the arsenosugars degrade within a short time span into a compound with a mass of 254 Da.

Separation and detection of Se-compounds by ion pairing liquid chromatography-microwave assisted hydride generation-atomic fluorescence spectrometry

Liquid chromatography coupled to a hydride generation atomic fluorescence spectrometer has been applied for the speciation of Se in extracts of Saccharomyces cerevisiae. In order to develop a method which allows the separation of the compounds and detection of the element, seven Se standards were used: Se-methionine (Se-Met), Se-cystine (Se-(Cys)2), Se-cystamine (Se-Cya), Se-methylselenocysteine (Se-MeSeCys), Se-ethionine (Se-Et), selenate (SeVI), selenite (SeIV). Optimal chromatographic results were obtained with reversed-phase chromatography on an XTerra C18 column using a positively charged ion-pairing agent. It was observed that for these standards precise control of the pH was of utmost importance. Attention was devoted to the compatibility of the mobile phase with hydride generation. Efficient formation of the hydrides was obtained by optimisation of different parameters. The redox mixture which allowed optimum conversion of all different species was HBr–KBrO3. To assist in the conversion of the compounds, on-line microwave digestion was applied. The detection limits obtained for the standards were: 0.8 µg Se l−1 for selenite(IV); 1.3 µg Se l−1 for selenate(VI); 1.2 µg Se l−1 for Se-methionine; 1.2 µg Se l−1 for Se-cystine; 1.3 µg Se l−1 for Se-cystamine; and 1.1 µg Se l−1 for Se-methylselenocysteine, respectively. Se-compounds in Saccharomyces cerevisiae were extracted by hot water (50 °C) or proteolytic digestion with protease XIV (37 °C). The method developed for separation and elemental detection was applied to these extracts in order to distinguish between the different species extracted from the yeast matrix. Total Se concentration in the extracts was measured with pneumatic nebulization-inductively coupled plasma-mass spectrometry (PN-ICP-MS). Species transformation was investigated by analysing extracts preserved at 2 different temperatures (−20 °C and 4 °C). Only those extracts kept at −20 °C proved to be unchanged.

In vivo distribution and fractionation of indium in rats after subcutaneous and oral administration of [114mIn]InAs

Two in vivo experiments were carried out in this study. In the first experiment five rats were given two subcutaneous injections of [(114m)In]InAs. Major sites of accumulation were spleen, liver and kidney. The intracellular distribution of indium was examined by differential centrifugation. The cytoplasmic fraction contained most of the indium activity followed by the mitochondrial fraction. Both outcomes are in close agreement with the results obtained in previous studies. Chromatographic separations on a preparative size exclusion column were carried out. It was shown that indium was mostly bound to high molecular mass compounds in serum and in the cytoplasmic fraction of spleen, liver and kidney. In a second experiment five rats were given four oral doses of [(114m)In]InAs over a short period. Prior to this experiment the in vitro solubility of cold InAs in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was determined using graphite furnace atomic absorption spectroscopy. In the case of the SGF only 1.3% of an InAs suspension dissolved after 48 hours incubation at 37 degrees C. InAs was not soluble in SIF. Uptake of InAs after oral administration was minimal (<1%). Due to incomplete removal of traces of [(114m)In]InAs from the gastrointestinal tract, it was impossible to calculate accurately the in vivo distribution over the different organs. As the uptake and consequently the activity in the organs were very low, no further chromatographic separations could be carried out. Considering this very low uptake, it can be concluded that InAs will not accumulate in the body after oral exposure.

In vivo distribution and speciationof [114mIn]InCl3 in the Wistar rat

Five Wistar rats were given an intraperitoneal injection of [114mIn]InCl3 during four consecutive days. One hour after the last injection the rats were sacrificed. The in vivo distribution of 114mIn was studied in the blood and in different organs. Differential centrifugation was used to study the distribution in liver, kidney and spleen homogenate. Rat serum, packed cell lysate, urine and the cytosol of liver, kidney and spleen homogenate were examined by size exclusion fast protein liquid chromatography. The results showed that serum accounts for 90% of the indium activity in whole blood. Indium is preferentially accumulated within the liver, spleen and kidney, the highest amount of 114mIn being localised in the cytosolic fraction followed by the mitochondria. Size exclusion experiments showed that, in rat serum, indium is exclusively bound to transferrin. These results differed from earlier in vitro incubation experiments of human serum with 114mIn. It was not possible, from the experiments described herein, to conclude unequivocally whether indium is bound to haemoglobin of packed cell lysate or to another high molecular mass compound. Indium is associated with the high molecular mass fraction in liver, kidney and spleen cytosol; only in kidney are small amounts of 114mIn found in the low molecular mass fraction. The in vivo inhibitory effect of indium on the delta-aminolaevulinic acid dehydratase (ALAD) enzymatic activity in red blood cells and kidney tissue, well documented by other researchers, could not be attributed to direct binding of indium with this enzyme.