Determination of ions in Caenorhabditis elegans by ion chromatography.

The Caenorhabditis elegans (C. elegans) is a model organism that has been increasingly used in health and environmental toxicity assessments. The quantification of such elements in vivo can assist in studies that seek to relate the exposure concentration to possible biological effects. Therefore, this study is the first to propose a method of quantitative analysis of 21 ions by ion chromatography (IC), which can be applied in different toxicity studies in C. elegans. The developed method was validated for 12 anionic species (fluoride, acetate, chloride, nitrite, bromide, nitrate, sulfate, oxalate, molybdate, dichromate, phosphate, and perchlorate), and 9 cationic species (lithium, sodium, ammonium, thallium, potassium, magnesium, manganese, calcium, and barium). The method did not present the presence of interfering species, with R2 varying between 0.9991 and 0.9999, with a linear range from 1 to 100 µg L-1. Limits of detection (LOD) and limits of quantification (LOQ) values ranged from 0.2319 µg L-1 to 1.7160 µg L-1 and 0.7028 µg L-1 to 5.1999 µg L-1, respectively. The intraday and interday precision tests showed an Relative Standard Deviation (RSD) below 10.0 % and recovery ranging from 71.0 % to 118.0 % with a maximum RSD of 5.5 %. The method was applied to real samples of C. elegans treated with 200 uM of thallium acetate solution, determining the uptake and bioaccumulated Tl+ content during acute exposure.

Arsenolipids in salmon are partly converted to thioxo analogs during cooking.

BACKGROUND: Arsenic hydrocarbons, major arsenolipids occurring naturally in marine fish, have substantial cytotoxicity leading to human health-related studies of their distribution and abundance in foods. These studies have all investigated fresh foods; because most fish are cooked before being consumed, it is both food- and health-relevant to determine the arsenolipids present in cooked fish. METHODS: We used HPLC/mass spectrometry to investigate the arsenolipids present in salmon (Salmo salar) before and after cooking by either baking or steaming. RESULTS: In raw salmon (total As 2.74 mg kg-1 dry mass, of which 6% was lipid-soluble), major arsenolipids were three arsenic hydrocarbons (oxo-AsHC 332, oxo-AsHC 360, and oxo-AsHC 404, ca 55% of total arsenolipids) and a band of unidentified less-polar arsenolipids (ca 40%), trace amounts of another four arsenic hydrocarbons and two thioxo analogs were also detected. During the cooking process, 28% of the oxo-AsHCs were converted to their thioxo analogs. CONCLUSION: Our study shows that arsenic hydrocarbons naturally present in fresh fish are partly converted to their thioxo analogs during cooking by either baking or steaming. The greater lipophilicity of the thioxo analogs could alter the mode of toxicity of arsenic hydrocarbons, and hence future food regulations for arsenic should consider the influence of cooking on the precise type of arsenolipid in fish.

Nutritive Manganese and Zinc Overdosing in Aging C. elegans Result in a Metallothionein-Mediated Alteration in Metal Homeostasis.

SCOPE: Manganese (Mn) and zinc (Zn) are not only essential trace elements, but also potential exogenous risk factors for various diseases. Since the disturbed homeostasis of single metals can result in detrimental health effects, concerns have emerged regarding the consequences of excessive exposures to multiple metals, either via nutritional supplementation or parenteral nutrition. This study focuses on Mn-Zn-interactions in the nematode Caenorhabditis elegans (C. elegans) model, taking into account aspects related to aging and age-dependent neurodegeneration. METHODS AND RESULTS: Chronic co-exposure of C. elegans to Mn and Zn increases metal uptake, exceeding levels of single metal exposures. Supplementation with Mn and/or Zn also leads to an age-dependent increase in metal content, a decline in overall mRNA expression, and metal co-supplementation induced expression of target genes involved in Mn and Zn homeostasis, in particular metallothionein 1 (mtl-1). Studies in transgenic worms reveal that mtl-1 played a prominent role in mediating age- and diet-dependent alterations in metal homeostasis. Metal dyshomeostasis is further induced in parkin-deficient nematodes (Parkinson's disease (PD) model), but this did not accelerate the age-dependent dopaminergic neurodegeneration. CONCLUSIONS: A nutritive overdose of Mn and Zn can alter interactions between essential metals in an aging organism, and metallothionein 1 acts as a potential protective modulator in regulating homeostasis.

Characterizing effects of excess copper levels in a human astrocytic cell line with focus on oxidative stress markers.

BACKGROUND: Being an essential trace element, copper is involved in diverse physiological processes. However, excess levels might lead to adverse effects. Disrupted copper homeostasis, particularly in the brain, has been associated with human diseases including the neurodegenerative disorders Wilson and Alzheimer's disease. In this context, astrocytes play an important role in the regulation of the copper homeostasis in the brain and likely in the prevention against neuronal toxicity, consequently pointing them out as a potential target for the neurotoxicity of copper. Major toxic mechanisms are discussed to be directed against mitochondria probably via oxidative stress. However, the toxic potential and mode of action of copper in astrocytes is poorly understood, so far. METHODS: In this study, excess copper levels affecting human astrocytic cell model and their involvement in the neurotoxic mode of action of copper, as well as, effects on the homeostasis of other trace elements (Mn, Fe, Ca and Mg) were investigated. RESULTS: Copper induced substantial cytotoxic effects in the human astrocytic cell line following 48 h incubation (EC30: 250 µM) and affected mitochondrial function, as observed via reduction of mitochondrial membrane potential and increased ROS production, likely originating from mitochondria. Moreover, cellular GSH metabolism was altered as well. Interestingly, not only cellular copper levels were affected, but also the homeostasis of other elements (Ca, Fe and Mn) were disrupted. CONCLUSION: One potential toxic mode of action of copper seems to be effects on the mitochondria along with induction of oxidative stress in the human astrocytic cell model. Moreover, excess copper levels seem to interact with the homeostasis of other essential elements such as Ca, Fe and Mn. Disrupted element homeostasis might also contribute to the induction of oxidative stress, likely involved in the onset and progression of neurodegenerative disorders. These insights in the toxic mechanisms will help to develop ideas and approaches for therapeutic strategies against copper-mediated diseases.

Astrocyte-specific deletion of the transcription factor Yin Yang 1 in murine substantia nigra mitigates manganese-induced dopaminergic neurotoxicity.

Manganese (Mn)-induced neurotoxicity resembles Parkinson's disease (PD), but the mechanisms underpinning its effects remain unknown. Mn dysregulates astrocytic glutamate transporters, GLT-1 and GLAST, and dopaminergic function, including tyrosine hydroxylase (TH). Our previous in vitro studies have shown that Mn repressed GLAST and GLT-1 via activation of transcription factor Yin Yang 1 (YY1). Here, we investigated if in vivo astrocytic YY1 deletion mitigates Mn-induced dopaminergic neurotoxicity, attenuating Mn-induced reduction in GLAST/GLT-1 expression in murine substantia nigra (SN). AAV5-GFAP-Cre-GFP particles were infused into the SN of 8-week-old YY1 flox/flox mice to generate a region-specific astrocytic YY1 conditional knockout (cKO) mouse model. 3 weeks after adeno-associated viral (AAV) infusion, mice were exposed to 330 µg of Mn (MnCl2 30 mg/kg, intranasal instillation, daily) for 3 weeks. After Mn exposure, motor functions were determined in open-field and rotarod tests, followed by Western blotting, quantitative PCR, and immunohistochemistry to assess YY1, TH, GLAST, and GLT-1 levels. Infusion of AAV5-GFAP-Cre-GFP vectors into the SN resulted in region-specific astrocytic YY1 deletion and attenuation of Mn-induced impairment of motor functions, reduction of TH-expressing cells in SN, and TH mRNA/protein levels in midbrain/striatum. Astrocytic YY1 deletion also attenuated the Mn-induced decrease in GLAST/GLT-1 mRNA/protein levels in midbrain. Moreover, YY1 deletion abrogated its interaction with histone deacetylases in astrocytes. These results indicate that astrocytic YY1 plays a critical role in Mn-induced neurotoxicity in vivo, at least in part, by reducing astrocytic GLAST/GLT-1. Thus, YY1 might be a potential target for treatment of Mn toxicity and other neurological disorders associated with dysregulation of GLAST/GLT-1.

Toxicological assessment of arsenic-containing phosphatidylcholines in HepG2 cells.

Arsenolipids include a wide range of organic arsenic species that occur naturally in seafood and thereby contribute to human arsenic exposure. Recently arsenic-containing phosphatidylcholines (AsPCs) were identified in caviar, fish, and algae. In this first toxicological assessment of AsPCs, we investigated the stability of both the oxo- and thioxo-form of an AsPC under experimental conditions, and analyzed cell viability, indicators of genotoxicity and biotransformation in human liver cancer cells (HepG2). Precise toxicity data could not be obtained owing to the low solubility in the cell culture medium of the thioxo-form, and the ease of hydrolysis of the oxo-form, and to a lesser degree the thioxo-form. Hydrolysis resulted amongst others in the respective constituent arsenic-containing fatty acid (AsFA). Incubation of the cells with oxo-AsPC resulted in a toxicity similar to that determined for the hydrolysis product oxo-AsFA alone, and there were no indices for genotoxicity. Furthermore, the oxo-AsPC was readily taken up by the cells resulting in high cellular arsenic concentrations (50 µM incubation: 1112 ± 146 µM As cellular), whereas the thioxo-AsPC was substantially less bioavailable (50 µM incubation: 293 ± 115 µM As cellular). Speciation analysis revealed biotransformation of the AsPCs to a series of AsFAs in the culture medium, and, in the case of the oxo-AsPC, to as yet unidentified arsenic species in cell pellets. The results reveal the difficulty of toxicity studies of AsPCs in vitro, indicate that their toxicity might be largely governed by their arsenic fatty acid content and suggest a multifaceted human metabolism of food derived complex arsenolipids.

Transport of arsenolipids to the milk of a nursing mother after consuming salmon fish.

OBJECTIVE: We address two questions relevant to infants' exposure to potentially toxic arsenolipids, namely, are the arsenolipids naturally present in fish transported intact to a mother's milk, and what is the efficiency of this transport. METHODS: We investigated the transport of arsenolipids and other arsenic species present in fish to mother's milk by analyzing the milk of a single nursing mother at 15 sampling times over a 3-day period after she had consumed a meal of salmon. Total arsenic values were obtained by elemental mass spectrometry, and arsenic species were measured by HPLC coupled to both elemental and molecular mass spectrometry. RESULTS: Total arsenic increased from background levels (0.1 µg As kg-1) to a peak value of 1.72 µg As kg-1 eight hours after the fish meal. The pattern for arsenolipids was similar to that of total arsenic, increasing from undetectable background levels (< 0.01 µg As kg-1) to a peak after eight hours of 0.45 µg As kg-1. Most of the remaining total arsenic in the milk was accounted for by arsenobetaine. The major arsenolipids in the salmon were arsenic hydrocarbons (AsHCs; 55 % of total arsenolipids), and these compounds were also the dominant arsenolipids in the milk where they contributed over 90 % of the total arsenolipids. CONCLUSIONS: Our study has shown that ca 2-3 % of arsenic hydrocarbons, natural constituents of fish, can be directly transferred unchanged to the milk of a nursing mother. In view of the potential neurotoxicity of AsHCs, the effects of these compounds on the brain developmental stage of infants need to be investigated.

Mono-acyl arsenosugar phospholipids in the edible brown alga Kombu (Saccharina japonica).

Twenty one arsenolipids, including eight new compounds (AsSugPL 692, AsSugPL 706, AsSugPL 720, AsSugPL 734, AsSugPL 742, AsSugPL 746, AsSugPL 748, and AsSugPL 776) were identified in the edible brown alga Kombu, Saccharina japonica, by means of HPLC coupled with elemental and molecular mass spectrometry. The hitherto undescribed compounds are all mono-acyl arsenosugar phospholipids, differing from previously reported natural arsenic-containing phospholipids by containing only one fatty acid on the glycerol group. Collectively, this new group of mono-acyl compounds constituted about 30% of total lipid arsenic; other significant groups were the di-acyl arsenosugar phospholipids (50%) and arsenic hydrocarbons (20%). The origin and relevance of the mono-acyl arsenosugar phospholipids in Kombu, a commercial seafood product, is briefly discussed.

Quantifying Inorganic Arsenic and Other Water-Soluble Arsenic Species in Human Milk by HPLC/ICPMS.

Because the toxicity of arsenic depends on its chemical form, risk assessments of arsenic exposure must consider the type of arsenic compound, and hence they require sensitive and robust methods for their determination. Furthermore, the assessment should include studies on the most vulnerable people within a population, such as newborns and infants, and thus there is a need to quantify arsenic species in human milk. Herein we report a method for the determination of arsenic species at low concentrations in human milk by HPLC/ICPMS. Comparison of single and triple quadrupole mass analysers showed comparable performance, although the triple quadrupole instrument more efficiently overcame the problem of ArCl+ interference, from the natural chloride present in milk, without the need for gradient elution HPLC conditions. The method incorporates a protein precipitation step with trifluoroacetic acid followed by addition of dichloromethane or dibromomethane to remove the lipids. The aqueous phase was subjected to anion-exchange and cation-exchange/mixed mode chromatography with aqueous ammonium bicarbonate and pyridine buffer solutions as mobile phases, respectively. For method validation, a human milk sample was spiked with defined amounts of dimethylarsinate, arsenobetaine, and arsenate. The method showed good recoveries (99-103%) with detection limits (in milk) in the range of 10 ng As kg-1. The method was further tested by analyzing two Norwegian human milk samples where arsenobetaine, dimethylarsinate, and a currently unknown As species were found, but iAs was not detected.

Arsenic and arsenic species in shellfish and finfish from the western Arabian Gulf and consumer health risk assessment.

This study reports the levels of total arsenic and arsenic species in marine biota such as clams (Meretrix meretrix; N=21) and pearl oyster (Pinctada radiata; N=5) collected from nine costal sites in Jan 2014, and cuttlefish (Sepia pharaonis; N=8), shrimp (Penaeus semisulcatus; N=1), and seven commercially important finfish species (N=23) collected during Apr-May 2013 from seven offshore sites in the western Arabian Gulf. Total As and As species such as dimethylarsinic acid (DMA), arsenobetaine (AB), trimethylarsine oxide (TMAO), arsenocholine (AC), tetramethylarsonium ion (Tetra), arsenosugar-glycerol (As-Gly) and inorganic As (iAs) were determined by using ICPMS and HPLC/ICPMS. In bivalves, the total As concentrations ranged from 16 to 118mg/kg dry mass; the toxic iAs fraction contributed on average less than 0.8% of the total As, while the nontoxic AB fraction formed around 58%. Total As concentrations for the remaining seafood (cuttlefish, shrimp and finfish) ranged from 11 to 134mg/kg dry mass and the iAs and AB fractions contributed on average 0.03% and 81% respectively of the total As. There was no significant relationship between the tissue concentrations of total As and iAs in the samples. There was also no significant relationship between As levels in seafood and geographical location or salinity of the waters from which samples were collected. Based on our results, we recommend introducing a maximum permissible level of arsenic in seafood from the Gulf based on iAs content rather than based on total As. Our analyses of cancer risks and non-cancer hazards identified non-negligible risks and the potential for hazards; the greatest risks were identified for expatriate consumers of bivalves and high-end consumers of seafood. Despite this, many uncertainties remain that would be best addressed by further analyses.

Quantitative determination of small selenium species in human serum by HPLC/ICPMS following a protein-removal, pre-concentration procedure.

Protein precipitation was incorporated into a sample preparation method for the quantitative determination of small "non-protein" selenium species in human serum by high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC/ICPMS). The advantages of cleaner matrix and concomitant concentration of the small compounds result in quantification limits in the native serum at the sub-micrograms Se per litre level. Spiking experiments with methyl 2-acetamido-2-deoxy-1-seleno-ß-D-galactopyranoside (selenosugar 1), trimethylselenonium ion, selenomethionine, methylselenocysteine (MeSeCys) and selenate yielded recoveries from 73% to 103%. Selenite had a low recovery (44%), possibly owing to protein binding. The validated method was applied to serum samples from two volunteers before and after ingestion of a selenium food supplement. HPLC/ICPMS analysis showed, besides ingested selenate, the presence of selenosugar 1 and trace amounts of MeSeCys and methyl 2-amino-2-deoxy-1-seleno-ß-D-galactopyranoside, which have not been reported in human serum before.

A miniaturized microtiter plate protocol for the determination of in selenized yeast via enzymatic hydrolysis of protein-bound.

This paper describes a simple/low volume enzymatic extraction method for selenomethionine (SeMet) determination in selenized yeast samples. In contrast to traditional methods which generally utilize large sample volumes consuming significant amounts of costly enzymes, the modified protocol employs a microtiter plate format allowing a reduction of the required sample volumes to 1 mL per extract. The extraction is performed in a parallel (5 × 4 = 20 position microtiter plate) reaction platform made out of sintered silicon carbide, fitted with standard disposable glass HPLC/GC vials. Due to the high thermal conductivity of silicon carbide, this set-up can be placed on a standard hotplate to accurately maintain the desired extraction conditions (37 °C, 20 h) for all positions of the microtiter plate. Hydrolysis of selenium-enriched yeast with a combination of protease XIV and lipase VII (ratio 2 : 1, w/w) using these low-volume conditions provided identical results to the more traditional high-volume method. The amount of SeMet was determined by HPLC/ICPMS and confirmed a high recovery rate for SeMet (93 ± 2%, n = 3) for the certified reference material SELM-1.