Inorganic arsenic alters the development of dopaminergic neurons but not serotonergic neurons and induces motor neuron development via Sonic hedgehog pathway in zebrafish.

The mechanism of inorganic arsenic-induced neurotoxicity at the cellular level is not known. In zebrafish, teratological effects of inorganic arsenic have been shown at various concentrations. Here, we used similar concentrations of inorganic arsenic to evaluate the effects on specific neuron types. Exposure of zebrafish embryos at 5 h post fertilization (hpf) to sodium arsenite induced developmental toxicity (reduced body length) in 72 hpf larvae, beginning at a concentration of 300 mg/L concentration. Mortality or overt morphological deformity was detected at 500 mg/L sodium arsenite. While 200 mg/L sodium arsenite induced development of tyrosine hydroxylase-positive (dopaminergic) neurons, there was no significant effect on the development of 5-hydroxytryptamine (serotonergic) neurons. Sodium arsenite reduced acetylcholinesterase activity. In the hb9-GFP transgenic larvae, both 200 and 400 mg/L sodium arsenite produced supernumerary motor neurons in the spinal cord. Inhibition of the Sonic hedgehog (Shh) pathway that is essential for motor neuron development, by Gant61, prevented sodium arsenite-induced supernumerary motor neuron development. Inductively coupled plasma mass spectrometry (ICP-MS) revealed that with 200 mg/L and 400 mg/L sodium arsenite treatment, each larva had an average of 387.8 pg and 847.5 pg arsenic, respectively. The data show for the first time that inorganic arsenic alters the development of dopaminergic and motor neurons in the zebrafish larvae and the latter occurs through the Shh pathway. These results may help understand why arsenic-exposed populations suffer from psychiatric disorders and motor neuron disease and Shh may, potentially, serve as a plasma biomarker of arsenic toxicity.

Distribution of 26 major and trace elements in edible seaweeds from the US market.

In this study we present an elemental profile of 46 edible seaweed samples purchased in the United States. The seaweeds were grouped in 13 subgroups/species based on DNA barcoding analysis. The seaweeds were decomposed by microwave accelerated acid digestion followed by quantification of 26 elements by ICP-MS. Elements were grouped into macronutrient (Na, K, Ca, S, Mg and P), essential (Fe, Zn, Mn, V, Cu, Cr, Ni, Mo and Se), non-essential including toxic elements (Sr, Ba, Th, Sn and Sb As, Cd, Pb, U, W and Hg). The highest levels were found for Na and the lowest were for Hg. The elemental profiles depended on the taxonomy of the species and several elements (Fe, Ba, Cr, Pb, W and Th) also exhibited high intraspecies variations, likely due to geographic origin or food processing conditions. Higher Cd and Pb accumulation was observed in wakame, hijiki and nori, with Cd as high 4.05 mg/kg and Pb as high as 2.85 mg/kg in kombu. A study of correlation between the elements using Pearson's coefficients revealed multiple pairs of highly correlated elements in seaweed, as well as triple and quintuple correlations of certain elements.

Connecting inorganic mercury and lead measurements in blood to dietary sources of exposure that may impact child development.

Pre-natal and post-natal chemical exposures and co-exposures from a variety of sources including contaminated air, water, soil, and food are common and associated with poorer birth and child health outcomes. Poor diet is a contributing factor in the development of child behavioral disorders. Child behavior and learning can be adversely impacted when gene expression is altered by dietary transcription factors such as zinc insufficiency or deficiency or by exposure to toxic substances permitted in our food supply such as mercury, lead, or organophosphate pesticide residue. Children with autism spectrum disorder and attention deficit hyperactivity disorders exhibit decreased or impaired PON1 gene activity which is needed by the body to metabolize and excrete neurotoxic organophosphate pesticides. In this current review we present an updated macroepigenetic model that explains how dietary inorganic mercury and lead exposures from unhealthy diet may lead to elevated blood mercury and/or lead levels and the development of symptoms associated with the autism and attention deficit-hyperactivity disorders. PON1 gene activity may be suppressed by inadequate dietary calcium, selenium, and fatty acid intake or exposures to lead or mercury. The model may assist clinicians in diagnosing and treating the symptoms associated with these childhood neurodevelopmental disorders. Recommendations for future research are provided based on the updated model and review of recently published literature.

Suitability of DNA Sequencing Tools for Identifying Edible Seaweeds Sold in the United States.

Seaweeds have been consumed by billions of people around the world and are increasingly popular in United States (US) diets. Some seaweed species have been associated with adverse health effects-such as heavy metal toxicity-and higher priced seaweeds may be more prone to adulteration. Knowing which species of seaweeds are being marketed in the US is important for protecting human health and preventing economic adulteration. Therefore, the United States Food and Drug Administration is developing new DNA-based species identification tools to complement established chemical methods for verifying the accurate labeling of products. Here, seaweed products available in the United States were surveyed using a tiered approach to evaluate a variety of DNA extraction techniques followed by traditional DNA barcoding via Sanger sequencing; if needed, genome skimming of total extracted nuclear DNA via next-generation sequencing was performed. This two-tiered approach of DNA barcoding and genome skimming could identify most seaweed samples (41/46), even those in blends (2/2, 1 out of 3 labeled species in each). Only two commercial samples appeared to be mislabeled or to contain unintended algal species. Five samples, labeled as "hijiki" or "arame", could not be confirmed by these DNA-based identification methods.

Market Basket Survey of Arsenic Species in the Top Ten Most Consumed Seafoods in the United States.

The study focused on the determination of arsenic species in the top ten most consumed seafoods in the United States. Fifty-four samples were collected from local supermarkets, and their species identities were confirmed by DNA barcoding. The total arsenic in the samples varied greatly in the range of 8-22200 ng/g (wet mass). Speciation analysis based on extraction of water-soluble and nonpolar arsenic showed that inorganic arsenic (iAs) was found only in clams and crabs, while arsenobetaine (AsB) predominates in most samples. Among the other arsenicals, trimethylarsoniopropionate (TMAP) was found in most matrices with higher concentrations in crabs, and arsenosugars existed in most clams and crabs. Nonpolar arsenic accounted for 1-46% of the total arsenic in the samples. The accuracy of the analytical results was evaluated using standard reference materials and spike recovery tests. The survey showed that the iAs concentrations in America's most consumed seafood products are much lower than the tolerable intake set by the Joint FAO/WHO Expert Committee, even at the highest levels found in this study.

Matrix-induced transformation of arsenic species in seafoods.

The paper presents a study on the matrix-induced transformation of arsenic (As) species spiked into seafoods. Sixteen arsenicals were individually spiked into samples of finfish, crustaceans and molluscs. The spiked samples were subjected to hot water extraction at 90 °C, and extracts were analyzed by high pressure liquid chromatography (HPLC) interfaced with inductively coupled plasma mass spectrometry (ICP-MS). Arsenate (As5+), arsenobetaine (AsB), arsenocholine (AsC), dimethylarsinate (DMA), monomethylarsonate (MMA), tetramethylarsonium ion (TMA) and trimethylarsoniopropionate (TMAP) remained intact in all the matrices. Whereas arsenite (As3+), dimethylarsinoyl acetate (DMAA), dimethylarsinoyl ethanol (DMAE), dimethylarsinoyl propionate (DMAP), trimethylarsine oxide (TMAO) and glycerol-, sulfonate-, sulfate- and phosphate-arsinoylribosides (arsenosugars 328, 392, 408 and 482, respectively) were transformed to other forms in most finfish and crustaceans. The transformation of the arsenicals was discovered to be induced by matrix thiols. While As3+ was bound to sulfhydryl groups, DMAA, DMAE, DMAP, TMAO and arsenosugars 392, 408 and 482 were thiolated through conversion of their arsinoyl (As=O) functionalities to arsinothioyl (As=S). The newly formed arsinothioyl compounds were characterized by HPLC-ICP-MS and electrospray ionization high-resolution tandem mass spectrometry (ESI-HR-MS/MS) paired with HPLC. The observed matrix-induced transformation of the arsenic species could be prevented by treating the samples (prior to spiking) with a thiol-selective blocking agent, N-ethylmaleimide (NEM).

Chronic exposure to xenobiotic pollution leads to significantly higher total glutathione and lower reduced to oxidized glutathione ratio in red blood cells of children with autism.

Analyses of reduced glutathione (GSH), oxidized glutathione (GSSG), and total glutathione (tGSH) in red blood cell samples from 30 children diagnosed with autism and 30 age, gender, and socioeconomic status matched controls were undertaken. The children's ages ranged from 2 to 9. Samples were obtained from subjects residing in Western Pennsylvania, an area of the United States greatly affected by high levels of mercury deposition and airborne PM 2.5 particulates. Liquid chromatography - mass spectrometry was utilized by following EPA Method 6800 for sample analyses. The children with autism had a significantly lower mean red blood cell (RBC) reduced to oxidized glutathione ratio (GSH/GSSG) compared to the control children (p = 0.025). In addition, compared to the controls, the children with autism had significantly higher RBC tGSH values (p = 0.0076) and GSH values (p = 0.022). These results suggest that exposure to toxic elements may prompt compensatory increases in production of GSH in children with autism in environments higher in toxins. The compensation did not fully correct the anti-oxidant properties of exposure to xenobiotics as demonstrated by the significantly lower GSH/GSSG in children with autism compared to controls. Out of a set of glutathione biomarkers, GSH/GSSG may best determine the degree of compensation for oxidative stress in children with autism.

Speciation analysis of arsenic in seafood and seaweed: Part I-evaluation and optimization of methods.

Several extraction and chromatographic methods were evaluated to identify optimum conditions for arsenic speciation analysis in seafood and seaweed. The extraction systems, which include aqueous, aqueous-organic, acidic, basic, and enzymatic solutions, were examined for their efficiency in extracting arsenic from finfish, crustaceans, molluscs, and seaweed keeping the chemical forms of the native arsenicals intact. While dilute solutions of nitric acid, hydrochloric acid, and tetramethylammonium hydroxide (TMAH) extract high fractions of arsenic from most of the matrices, the extractants oxidized arsenite (As3+) to arsenate (As5+) and converted some arsenosugars and non-polar arsenicals to known and/or unknown forms. Hot water (90 °C) effectively maintained the integrity of the native arsenic species and enabled analysis of the extracts with no further manipulation than filtration and dilution. Stepwise extraction of water-soluble and non-polar arsenic with hot water and a mixture of dichloromethane and methanol, respectively, resulted in sufficiently quantitative (> 75%) arsenic extraction from seafood and seaweed. Anion and cation exchange chromatographic methods were optimized for separation and quantitation of the arsenicals extracted into hot water. The non-polar arsenicals were collectively determined after digesting the extract in acid. The application of the optimum extraction and chromatographic conditions was demonstrated by analyzing certified reference materials of tuna fish tissue (BCR 627), lobster hepatopancreas (TORT-2) and oyster tissue (SRM 1566b), and a sample of hijiki seaweed. For all the matrices, good agreement (80-92%) was found between the total water-soluble arsenic and the sum of the concentrations of the chromatographed species. Limits of quantification (LOQ) were in the range 4-11 ng g-1 for 16 arsenicals.

Speciation analysis of arsenic in seafood and seaweed: Part II-single laboratory validation of method.

Single laboratory validation of a method for arsenic speciation analysis in seafood and seaweed is presented. The method is based on stepwise extraction of water-soluble and non-polar arsenic with hot water and a mixture of dichloromethane and methanol, respectively. While the water-soluble arsenicals were speciated by anion and cation exchange liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS), the non-polar arsenicals were collectively determined by ICP-MS after digestion in acid. The performance characteristics and broad application of the method were evaluated by analyzing eight commercial samples (cod, haddock, mackerel, crab, shrimp, geoduck clam, oyster, and kombu) and four reference materials (fish protein (DORM-4), lobster hepatopancreas (TORT-3), mussel tissue (SRM 2976), and hijiki seaweed (CRM 7405-a)) representing finfish, crustaceans, molluscs, and seaweed. Matrices spiked at three levels in duplicates were also analyzed. The stepwise extraction provided 76-106% extraction of the total arsenic from the test materials. The method demonstrated satisfactory repeatability for analysis of replicate extracts prepared over several days. The accuracy of the method was evaluated by analyzing reference materials certified for both total arsenic and a few arsenicals; the experimental results were 90-105% of the certified values. Comparison between the total water-soluble arsenic and the sum of the concentrations of the chromatographed species gave 80-92% mass balance. While spike recoveries of most arsenicals were in the acceptance range set by CODEX, a few species spiked into cod, haddock, and shrimp were poorly recovered due to transformation to other forms. After thorough investigations, strategies were devised to improve the recoveries of these species by averting their transformations. Limits of quantification (LOQ) for the extraction and quantification of 16 arsenicals using the current method were in the range 6-16 ng g-1 arsenic.

Evaluation of Dietary Supplement Contamination by Xenobiotic and Essential Elements Using Microwave-Enhanced Sample Digestion and Inductively Coupled Plasma-Mass Spectrometry.

Dietary supplements were analyzed by evaluating the elemental content in six widely consumed products manufactured by four well-known companies. The elements included the neurotoxic and carcinogenic elements cadmium, mercury, aluminum, lead, arsenic, and antimony, as well as the essential elements zinc, selenium, chromium, iron, and copper, which were often not listed as ingredients on the product labels. Contamination from either xenobiotic or essential elements was found in all samples analyzed. The samples were prepared using US Environmental Protection Agency (EPA) Method 3052, microwave-enhanced digestion. The resulting digests were analyzed by Inductively Coupled Plasma-Mass Spectrometry based on EPA Method 6020B. The analytical protocols were validated by analyzing a multivitamin standard reference material, the National Institute of Standards and Technology Standard Reference Material 3280. The application of EPA standard methods demonstrated their utility in making accurate and precise measurements in complex matrices with multiple ingredients and excipients. In the future, the use of these methods could provide a uniform quality assurance protocol that can be implemented along with other industry guidelines to improve the production of dietary supplements.

Blood inorganic mercury is directly associated with glucose levels in the human population and may be linked to processed food intake.

BACKGROUND: The goals of the study were (1) to determine the impact of inorganic mercury exposure on glucose homeostasis; and (2) to evaluate the effectiveness of two community-based interventions in promoting dietary changes among American Indian college students to reduce risk factors for Type-2 Diabetes including fasting glucose, insulin, and mercury levels, weight, and body mass index. METHODS: To accomplish goal one, the National Health and Nutrition Examination Survey (NHANES) dataset was analyzed using a previously published method to determine if there is a relationship between inorganic blood mercury and fasting glucose. To accomplish goal two, ten college students were recruited and randomly assigned to a group receiving the online macroepigenetics nutrition course and the support group for eliminating corn sweeteners. Participants in both groups were assessed for diet patterns, weight, body mass index (BMI), fasting glucose, insulin, and mercury levels. The interventions were implemented over a 10-week period. RESULTS: Analysis of the NHANES data (n=16,232) determined a direct relationship between inorganic mercury in blood and fasting glucose levels (p<0.001). The participants who took the online macroepigenetics nutrition intervention course significantly improved their diets (p<0.01), and fasting blood glucose levels (p<0.01) while having lower levels of inorganic mercury in their blood compared to the subjects in the group who eliminated corn sweeteners from their diet and participated in the support group. The trend in lower blood inorganic mercury was strong with p=0.052. The participants in the support group who eliminated corn sweeteners from their diet achieved significant weight loss (p<0.01) and reduced their body mass index (p<0.01). CONCLUSION: Total blood mercury levels may be influenced by dietary intake of highly processed foods and lower inorganic mercury levels are associated with lower fasting glucose levels. Alternative community-based interventions emphasizing the role food ingredients and toxic substances play in gene modulation and the development of diseases can result in significant dietary improvements and reductions in risk factors associated with type-2 diabetes. A healthier diet can be promoted among community members using a novel online nutrition course. Consumption of corn sweeteners may be a risk factor in the development of obesity.

Molecular speciated isotope dilution mass spectrometric methods for accurate, reproducible and direct quantification of reduced, oxidized and total glutathione in biological samples.

Novel protocols were developed to accurately quantify reduced (GSH), oxidized (GSSG) and total (tGSH) glutathione in biological samples using molecular speciated isotope dilution mass spectrometry (SIDMS). For GSH and GSSG measurement, the sample was spiked with isotopically enriched analogues of the analytes ((310)GSH and (616)GSSG), along with N-ethylmaleimide (NEM), and treated with acetonitrile to solubilize the endogenous analytes via protein precipitation and equilibrate them with the spikes. The supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the analytes were quantified with simultaneous tracking and correction for auto-oxidation of GSH to GSSG. For tGSH assay, a (310)GSH-spiked sample was treated with dithiothreitol (DTT) to convert disulfide-bonded glutathione to GSH. After removing the protein, the supernatant was analyzed by LC-MS/MS and the analyte was quantified by single-spiking isotope dilution mass spectrometry (IDMS). The mathematical relationships in IDMS and SIDMS quantifications are based on isotopic ratios and do not involve calibration curves. The protocols were validated using spike recovery tests and by analyzing synthetic standard solutions. Red blood cell (RBC) and saliva samples obtained from healthy subjects, and whole blood samples collected and shipped from a remote location were analyzed. The concentrations of tGSH in the RBC and whole blood samples were 2 orders of magnitude higher than those found in saliva. The fractions of GSSG were 0.2-2.2% (RBC and blood) and 15-47% (saliva) of the free glutathione (GSH + 2xGSSG) in the corresponding samples. Up to 3% GSH was auto-oxidized to GSSG during sample workup; the highest oxidations (>1%) were in the saliva samples.

Measurement of mercury species in whole blood using speciated isotope dilution methodology integrated with microwave-enhanced solubilization and spike equilibration, headspace-solid-phase microextraction, and GC-ICP-MS analysis.

A biomonitoring method was developed for the determination of inorganic-, methyl-, and ethylmercury (Hg(2+), CH3Hg(+), and C2H5Hg(+), respectively) in whole blood by triple-spiking speciated isotope dilution mass spectrometry (SIDMS) using headspace (HS) solid-phase microextraction (SPME) in combination with gas chromatographic (GC) separation and inductively coupled plasma mass spectrometric (ICP-MS) detection. After spiking the blood sample with isotopically enriched analogues of the analytes ((199)Hg(2+), CH3(200)Hg(+) and C2H5(201)Hg(+)), the endogenous Hg species were solubilized in 2.0 mol L(-1) HNO3 and equilibrated with the spikes using a microwave-enhanced protocol. The microwaved sample was treated with a 1% (w/v) aqueous solution of sodium tetrapropylborate (buffered to pH 5.2), and the propylated Hg species were sampled in the HS using a Carboxen/polydimethylsiloxane-coated SPME fiber. The extracted species were thermally desorbed from the fiber in the GC injection port and determined by GC-ICP-MS. The analytes were quantified, with simultaneous correction for their method-induced transformation, on the basis of the mathematical relationship in triple-spiking SIDMS. The method was validated using a bovine blood standard reference material (SRM 966, Level 2). Analysis of human blood samples demonstrated the accuracy and reproducibility of the method, which can detect the Hg species down to 30 pg g(-1) in blood. The validity of the analytical results found for the blood samples was demonstrated using mass balance by comparing the sum of the concentrations of the individual Hg species with the total Hg in the corresponding samples; the latter was determined by isotope dilution mass spectrometry (IDMS) after decomposing the blood using EPA Method 3052 with single-spiking.

Method development for the redox speciation analysis of iron by ion chromatography-inductively coupled plasma mass spectrometry and carryover assessment using isotopically labeled analyte analogues.

An ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS) method was developed for the redox speciation analysis of iron (Fe) based on in-column complexation of Fe(2+) and Fe(3+) by dipicolinic acid (DPA). The effects of column type, mobile phase composition and molecular ion interference were studied in the method optimization. The carryover of the target species in the IC-ICP-MS method was uniquely and effectively evaluated using isotopically enriched analogues of the analytes ((54)Fe(2+) and (57)Fe(3+)). Standard solutions of the enriched standards were injected into the system following analysis of a sample, and the ratios of the isotopes of iron in the enriched standards were calculated based on the chromatographic peak areas. The concentrations of the analytes carried over from the sample to the enriched standards were determined using the quantitative relationship in isotope dilution mass spectrometry (IDMS). In contrast to the routine way of evaluating carryover effect by injecting a blank solution after sample analysis, the use of isotopically enriched standards identified significant analyte carryover in the present method. Extensive experiments were carried out to systematically identify the source of the carryover and to eliminate the problem; the separation column was found to be the exclusive source. More than 95% of the analyte carryover was eliminated by reducing the length of the column. The detection limit of the IC-ICP-MS method (MDL) for the iron species was 2ngg(-1). The method was used to determine Fe(2+) and Fe(3+) in synthetic aqueous standard solutions and a beverage sample.

Speciation analysis of arsenic in prenatal and children's dietary supplements using microwave-enhanced extraction and ion chromatography-inductively coupled plasma mass spectrometry.

A study was conducted to develop a microwave-enhanced extraction method for the determination of arsenic species in prenatal and children's dietary supplements prepared from plant materials. The method was optimized by evaluating the efficiency of various solutions previously used to extract arsenic from the types of plant materials used in the dietary supplement formulations. A multivitamin standard reference material (NIST SRM 3280) and a prenatal supplement sample were analyzed in the method optimization. The identified optimum conditions were 0.25 g of sample, 5 mL of 0.3 mol L(-1) orthophosphoric acid (H3PO4) and microwave heating at 90 °C for 30 min. The extracted arsenic was speciated by cation exchange ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS). The method detection limit (MDL) for the arsenic species was in the range 2-8 ng g(-1). Ten widely consumed prenatal and children's dietary supplements were analyzed using the optimized protocol. The supplements were found to have total arsenic in the concentration range 59-531 ng g(-1). The extraction procedure recovered 61-92% of the arsenic from the supplements. All the supplementary products were found to contain arsenite (As(3+)) and dimethylarsinic acid (DMA). Arsenate (As(5+)) was found in two of the supplements, and an unknown specie of arsenic was detected in one product. The results of the analysis were validated using mass balance by comparing the sum of the extracted and non-extracted arsenic with the total concentration of the element in the corresponding samples.