Methylated and thiolated arsenic species for environmental and health research - A review on synthesis and characterization.

Hundreds of millions of people around the world are exposed to elevated concentrations of inorganic and organic arsenic compounds, increasing the risk of a wide range of health effects. Studies of the environmental fate and human health effects of arsenic require authentic arsenic compounds. We summarize here the synthesis and characterization of more than a dozen methylated and thiolated arsenic compounds that are not commercially available. We discuss the methods of synthesis for the following 14 trivalent (III) and pentavalent (V) arsenic compounds: monomethylarsonous acid (MMAIII), dicysteinylmethyldithioarsenite (MMAIII(Cys)2), monomethylarsonic acid (MMAV), monomethylmonothioarsonic acid (MMMTAV) or monothio-MMAV, monomethyldithioarsonic acid (MMDTAV) or dithio-MMAV, monomethyltrithioarsonate (MMTTAV) or trithio-MMAV, dimethylarsinous acid (DMAIII), dimethylarsino-glutathione (DMAIII(SG)), dimethylarsinic acid (DMAV), dimethylmonothioarsinic acid (DMMTAV) or monothio-DMAV, dimethyldithioarsinic acid (DMDTAV) or dithio-DMAV, trimethylarsine oxide (TMAOV), arsenobetaine (AsB), and an arsenicin-A model compound. We have reviewed and compared the available methods, synthesized the arsenic compounds in our laboratories, and provided characterization information. On the basis of reaction yield, ease of synthesis and purification of product, safety considerations, and our experience, we recommend a method for the synthesis of each of these arsenic compounds.

Life cycle exposure of the frog Silurana tropicalis to arsenate: Steroid- and thyroid hormone-related genes are differently altered throughout development.

Arsenic contaminates water surface and groundwater worldwide. Several studies have suggested that arsenic acts as an endocrine disruptor in mammalian and non-mammalian species, although its chronic effect during development remains largely unknown. To address this question, life cycle exposures to 0, 0.3 and 0.8ppm of arsenate (pentavalent arsenic; As(V)) were performed in the Western clawed frog (Silurana tropicalis) from the gastrulae stage (developmental stage Nieuwkoop-Faber; NF12) until metamorphosis (NF66). Tissue samples were collected at the beginning of feeding (NF46; whole body), sexual development (NF56; liver), and at metamorphosis completion (NF66; liver and gonadal mesonephros complex). Real-time RT-PCR analysis quantified decreases in mRNA levels of genes related to estrogen- (estrogen receptor alpha and aromatase), androgen- (androgen receptor and steroid 5-alpha-reductase type 2), and cholesterol metabolism- (steroidogenic acute regulatory protein) at stage NF46. Similarly, arsenate decreased steroid 5-alpha-reductase type 2 expression in stage NF56 livers, but transcript increases were observed for both estrogen receptor alpha and steroidogenic acute regulatory protein at this stage. Given the changes observed in the expression of genes essential for proper sexual development, gonadal histological analysis was carried out in stage NF66 animals. Arsenate treatments did not alter sex ratio or produce testicular oocytes. On the other hand, arsenate interfered with thyroid hormone-related transcripts at NF66. Specifically, thyroid hormone receptor beta and deiodinase type 2 mRNA levels were significantly reduced after arsenate treatment in the gonadal mesonephros complex. This reduction in thyroid hormone-related gene expression, however, was not accompanied by any morphological changes measured. In summary, environmentally relevant concentrations of As(V) altered steroidogenesis-, sex steroid signaling- and thyroid hormone-related gene expression, although transcriptional changes varied among tissues and developmental stages.

Transcriptomic Responses During Early Development Following Arsenic Exposure in Western Clawed Frogs, Silurana tropicalis.

Arsenic compounds are widespread environmental contaminants and exposure elicits serious health issues, including early developmental anomalies. Depending on the oxidation state, the intermediates of arsenic metabolism interfere with a range of subcellular events, but the fundamental molecular events that lead to speciation-dependent arsenic toxicity are not fully elucidated. This study therefore assesses the impact of arsenic exposure on early development by measuring speciation and gene expression profiles in the developing Western clawed frog (Silurana tropicalis) larvae following the environmental relevant 0.5 and 1 ppm arsenate exposure. Using HPLC-ICP-MS, arsenate, dimethylarsenic acid, arsenobetaine, arsenocholine, and tetramethylarsonium ion were detected. Microarray and pathway analyses were utilized to characterize the comprehensive transcriptomic responses to arsenic exposure. Clustering analysis of expression data showed distinct gene expression patterns in arsenate treated groups when compared with the control. Pathway enrichment revealed common biological themes enriched in both treatments, including cell signal transduction, cell survival, and developmental pathways. Moreover, the 0.5 ppm exposure led to the enrichment of pathways and biological processes involved in arsenic intake or efflux, as well as histone remodeling. These compensatory responses are hypothesized to be responsible for maintaining an in-body arsenic level comparable to control animals. With no appreciable changes observed in malformation and mortality between control and exposed larvae, this is the first study to suggest that the underlying transcriptomic regulations related to signal transduction, cell survival, developmental pathways, and histone remodeling may contribute to maintaining ongoing development while coping with the potential arsenic toxicity in S. tropicalis during early development.

Arsenic(+3) and DNA methyltransferases, and arsenic speciation in tadpole and frog life stages of western clawed frogs (Silurana tropicalis) exposed to arsenate.

Western clawed frog (Silurana tropicalis) embryos were exposed to control, low (nominally 0.5 mg L(-1)) and high (nominally 1 mg L(-1)) arsenate (As(V)) culture water concentrations to investigate the effects of arsenic (As) on different life stages, namely tadpole (Nieuwkoop and Faber stage 56, NF56) and frog stages (NF66). The effects were assessed by measuring arsenic(+3) and DNA methyltransferases (AS3MT and DNMT1), as well as As speciation in the tissues. The As content in frog tissues increased with water As concentration. The As species observed by high performance liquid chromatography - inductively coupled plasma mass spectrometry (HPLC-ICPMS) were mostly inorganic, dimethylarsinic acid (DMA) and trimethylarsine oxide (TMAO). With solid state X-ray absorption near edge structure (XANES) analysis, arsenobetaine/tetramethylarsonium ion were also seen. AS3MT levels decreased upon low As exposure in NF56, rising again to control levels at the high As exposure. In NF66 tissues, on the other hand, AS3MT decreased only with NF66 high As exposure. DNMT1 increased with exposure, and this was statistically significant only for the high As exposure at both life stages. Thus these enzymes seem to be affected by the As exposure. Methylation of As to form monomethylarsonate (MMA), DMA and TMAO in the frogs appeared to be inversely related to AS3MT levels. A possible interpretation of this finding is that when AS3MT is higher, excretion of MMA + DMA + TMAO is more efficient, leaving lower concentrations in the tissues, with the opposite effect (less excretion) when AS3MT is lower; alternatively, other enzymes or linked genes may affect the methylation of As.

Therapeutic and analytical applications of arsenic binding to proteins.

Arsenic binding to proteins plays a pivotal role in the health effects of arsenic. Further knowledge of arsenic binding to proteins will advance the development of bioanalytical techniques and therapeutic drugs. This review summarizes recent work on arsenic-based drugs, imaging of cellular events, capture and purification of arsenic-binding proteins, and biosensing of arsenic. Binding of arsenic to the promyelocytic leukemia fusion oncoprotein (PML-RARα) is a plausible mode of action leading to the successful treatment of acute promyelocytic leukemia (APL). Identification of other oncoproteins critical to other cancers and the development of various arsenicals and targeted delivery systems are promising approaches to the treatment of other types of cancers. Techniques for capture, purification, and identification of arsenic-binding proteins make use of specific binding between trivalent arsenicals and the thiols in proteins. Biarsenical probes, such as FlAsH-EDT2 and ReAsH-EDT2, coupled with tetracysteine tags that are genetically incorporated into the target proteins, are used for site-specific fluorescence labelling and imaging of the target proteins in living cells. These allow protein dynamics and protein-protein interactions to be studied. Arsenic affinity chromatography is useful for purification of thiol-containing proteins, and its combination with mass spectrometry provides a targeted proteomic approach for studying the interactions between arsenicals and proteins in cells. Arsenic biosensors evolved from the knowledge of arsenic resistance and arsenic binding to proteins in bacteria, and have now been developed into analytical techniques that are suitable for the detection of arsenic in the field. Examples in the four areas, arsenic-based drugs, imaging of cellular events, purification of specific proteins, and arsenic biosensors, demonstrate important therapeutic and analytical applications of arsenic protein binding.

Chemical mechanism of arsenic biomethylation.

The bioconversion of inorganic arsenic to methylated metabolites affects the tissue distribution and retention of arsenic and its actions as a toxicant or carcinogen. Although enzymes that catalyze the methylation of arsenicals have been identified in all branches of the tree of life, fundamental questions persist about the chemical processes that underlie reactions that methylate this metalloid. Here, several reaction schemes for arsenic methylation are considered to encourage careful consideration of the chemical plausibility of these schemes.

Speciation and toxicity of arsenic in mining-affected lake sediments in the Quinsam watershed, British Columbia.

Anthropogenic arsenic inputs into fresh water lakes in the Quinsam watershed, British Columbia, were probed by using multiple methods of inquiry including sediment coring combined with (210)Pb dating, a principal components analysis of elemental composition of sediments, arsenic speciation, bioaccessibility, and toxicity testing. The quantification of arsenic inputs from anthropogenic sources was not trivial because a variety of processes redistribute the element throughout lakes. However, elevated arsenic and sulfate concentrations in Long Lake, a lake that receives arsenic from a seep, suggest that this lake is influenced by mine operations. X-ray absorption near edge structure (XANES) spectra reveal similar arsenic speciation for all sediments within the studied lakes. Bioaccessibility tests, which in this study were used to approximate the solubility and availability of arsenic to benthic organisms, indicate moderate bioaccessibility of arsenic in sediments (7.9-35%). Toxicity testing indicates that not all benthic organisms should be used for evaluating arsenic toxicity, and suggests that the amphipod, Corophium volutator, shows promise as a candidate for widespread use for arsenic sediment toxicity testing.

Arsenic speciation in freshwater snails and its life cycle variation.

Terrestrial snails are consumed by humans occasionally and they are an important food source for many creatures including fish and birds. Little is known about arsenic speciation in these gastropods, let alone life cycle variations. Here we report on the arsenic speciation in freshwater snails from Pender Island and Vancouver Island, B.C., Canada, which was determined on methanol/water extracts (43-59% extraction efficiency) by using high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) and HPLC-electrospray tandem mass spectrometry. The tetramethylarsonium ion, oxo-arsenosugars and thio-arsenosugars are the main arsenic species encountered. Arsenobetaine, which is commonly found in the marine environment, is minor. Live bearing snails Viviparidae sp. from Pender Island were maintained in aquaria and the arsenic speciation in the unborn, newly born, and adult animals was monitored. Oxo-arsenosugars predominate in the adults, whereas thio-arsenosugars seem to predominate in juveniles, suggesting that these arsenicals are snail metabolites.

Bioaccessibility of lead and arsenic in traditional Indian medicines.

Arsenic and lead have been found in a number of traditional Ayurvedic medicines, and the practice of Rasa Shastra (combining herbs with metals, minerals and gems), or plant ingredients that contain these elements, may be possible sources. To obtain an estimate of arsenic and lead solubility in the human gastrointestinal tract, bioaccessibility of the two elements was measured in 42 medicines, using a physiologically-based extraction test. The test consisted of a gastric phase at pH 1.8 containing organic acids, pepsin and salt, followed by an intestinal phase, at pH 7 and containing bile and pancreatin. Arsenic speciation was measured in a subset of samples that had sufficiently high arsenic concentrations for the X-ray absorption near edge structure analysis used. Bioaccessible lead was found in 76% of samples, with a large range of bioaccessibility results, but only 29% of samples had bioaccessible arsenic. Lead bioaccessibility was high (close to 100%) in a medicine (Mahayograj Guggulu) that had been compounded with bhasmas (calcined minerals), including naga (lead) bhasma. For the samples in which arsenic speciation was measured, bioaccessible arsenic was correlated with the sum of As(V)-O and As(III)-O and negatively correlated with As-S. These results suggest that the bioaccessible species in the samples had been oxidized from assumed As-S raw medicinal ingredients (realgar, As(4)S(4), added to naga (lead) bhasma and As(III)-S species in plants). Consumption at recommended doses of all medicines with bioaccessibile lead or arsenic would lead to the exceedance of at least one standard for acceptable daily intake of toxic elements.

Organoarsenicals. Distribution and transformation in the environment.

The widespread distribution of organoarsenic compounds has been reviewed in terms of the five kingdoms of life. Over 50 organoarsenicals are described. Pathways for their formation are discussed and significant data gaps have been identified.

Identification of arsenic-binding proteins in human cells by affinity chromatography and mass spectrometry.

Exposure to high levels of arsenic can cause a wide range of health effects, including cancers of the bladder, lung, skin, and kidney. However, the mechanism(s) of action underlying these deleterious effects of arsenic remains unclear. Arsenic binding to cellular proteins is a possible mechanism of toxicity, and identifying such binding is analytically challenging because of the large concentration range and variety of proteins. We describe here an affinity selection technique, coupled with mass spectrometry, to select and identify specific arsenic-binding proteins from a large pool of cellular proteins. Controlled experiments using proteins either containing free cysteine(s) or having cysteine blocked showed that the arsenic affinity column specifically captured the proteins containing free cysteine(s) available to bind to arsenic. The technique was able to capture and identify trace amounts of bovine biliverdin reductase B present as a minor impurity in the commercial preparation of carbonic anhydrase II, demonstrating the ability to identify arsenic-binding proteins in the presence of a large excess of non-specific proteins. Application of the technique to the analysis of subcellular fractions of A549 human lung carcinoma cells identified 50 proteins in the nuclear fraction, and 24 proteins in the membrane/organelle fraction that could bind to arsenic, adding to the current list of only a few known arsenic-binding proteins.

Arsenite and its mono- and dimethylated trivalent metabolites enhance the formation of benzo[a]pyrene diol epoxide-DNA adducts in Xeroderma pigmentosum complementation group A cells.

Recently, inorganic arsenite (iAs(III)) and its mono- and dimethylated metabolites have been examined for their interference with the formation and repair of benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts in human cells (Schwerdtle, ., Walter, I., and Hartwig, A. (2003) DNA Repair 2, 1449 - 1463). iAs(III) and monomethylarsonous acid (MMA(III)) were found to be able to enhance the formation of BPDE-DNA adducts, whereas dimethylarsinous acid (DMA(III)) had no enhancing effect at all. The anomaly manifested by DMA(III) prompted us to further investigate the effects of the three trivalent arsenic species on the formation of BPDE-DNA adducts. Use of a nucleotide excision repair (NER)-deficient Xeroderma pigmentosum complementation group A cell line (GM04312C) allowed us to dissect DNA damage induction from DNA repair and to examine the effects of arsenic on the formation of BPDE-DNA adducts only. At concentrations comparable to those used in the study by Schwerdtle et al., we found that each of the three trivalent arsenic species was able to enhance the formation of BPDE-DNA adducts with the potency in a descending order of MMA(III) > DMA(III) > iAs(III), which correlates well with their cytotoxicities. Similar to iAs(III), DMA(III) modulation of reduced glutathione (GSH) or total glutathione S-transferase (GST) activity could not account for its enhancing effect on DNA adduct formation. Additionally, the enhancing effects elicited by the trivalent arsenic species were demonstrated to be highly time-dependent. Thus, although our study made use of short-term assays with relatively high doses, our data may have meaningful implications for carcinogenesis induced by chronic exposure to arsenic at low doses encountered environmentally.

Tissue uptake, mortality, and sublethal effects of monomethylarsonic acid (MMA(V)) in nestling zebra finches (Taeniopygia guttata).

Monosodium methanearsonate (MSMA), an arsenic-based pesticide, has been used since the mid 1980s in attempts to suppress mountain pine beetle (Dendroctonus ponderosae) outbreaks in British Columbia, Canada. It was previously shown that cavity nesting forest birds forage and breed in MSMA-treated pine stands. The present study was designed to investigate the effects of ecologically relevant oral exposure to MSMA, including tissue distribution, growth parameters, and general health, including survival and immune function, of a model passerine, the zebra finch (Taeniopygia guttata). Nestling finches were orally dosed for 20 d from hatching to fledging with 4, 8, 12, 24, 36, or 72 microg/g bw/d of monomethylarsonic acid (MMA(V), which corresponds to MSMA at physiological pH). Preliminary trials showed complete mortality at 36 and 72 microg/g bw/d, and repeat trials also resulted in high mortality at 24 microg/g bw/d. Surviving nestlings showed dose-dependent trends in accumulation of arsenic in blood and specific tissues, and decreased tarsi and wing cord length upon fledging. There were no observed effects of dosing on measured immune function (phytohemagglutinin [PHA], hematocrit, and leukocrit). The data obtained suggest that passerine nestlings may be at risk of mortality and reduced growth due to exposure to MSMA under current environmental conditions.

Dose-dependent uptake, elimination, and toxicity of monosodium methanearsonate in adult zebra finches (Taeniopygia guttata).

Monosodium methanearsonate (MSMA), an arsenic-based pesticide, has been used for the past 10 years in attempts to suppress mountain pine beetle (Dendroctonus ponderosae) outbreaks in British Columbia, Canada. Previous studies have shown that cavity nesting forest birds such as woodpeckers forage and breed in MSMA treated pine stands. Here we examined the effects of MSMA in the laboratory using the zebra finch (Taeniopygia guttata), with the objective to examine tissue distribution and sublethal toxic effects in a model avian species. Zebra finches were exposed to this pesticide at doses similar to those found in bark beetle samples from MSMA stands of trees treated in the southern interior of British Columbia (8, 24, and 72 microg/g/d and a control group). Results showed high excretion (>90%) of arsenic in all dose groups, as well as dose-dependent trends in accumulation of arsenic in the blood (p < 0.001) and specific tissues. Monomethylarsonic acid, MMA (V), was the predominant form of arsenic in the blood plasma. Dimethylarsinic acid was the major form of arsenic found in the liver (83%) and kidney (61%) tissues. The brain tissue contained primarily the MMA (V) form (57%). Significant weight loss occurred in the two highest dose groups (p < 0.05). Birds in the highest dose group lost up to 15% of initial body mass.

Arsenic accumulation in bark beetles and forest birds occupying mountain pine beetle infested stands treated with monosodium methanearsonate.

The arsenic-based pesticide, monosodium methanearsonate (MSMA), is presently being evaluated for re-registration in Canada and the United States and has been widely used in British Columbia to help suppress Mountain Pine Beetle (MPB) outbreaks. We assessed the availability and exposure of MSMA to woodpeckers and other forest birds that may prey directly on contaminated bark beetles. Total arsenic residues in MPB from MSMA treated trees ranged from 1.3-700.2 microg g(-1) dw (geometric mean 42.0 microg g(-1)) with the metabolite monomethyl arsonic acid (MMAA) contributing 90-97% to the total arsenic extracted. Live adult and larval beetles were collected from treated trees and reached concentrations up to 327 microg g(-1) dw. MPBs from reference trees had significantly lower arsenic concentrations averaging 0.19 microg g(-1) dw. Woodpeckers foraged more heavily on MSMAtreesthat contained beetles with lower arsenic residues, suggesting those trees had reduced MSMAtranslocation and possibly greater live beetle broods. Blood samples from five species of woodpeckers and other forest passerines breeding within 1 km of MSMA stands contained elevated levels of total arsenic but with large individual variability (geometric mean = 0.18 microg g(-1) dw, range 0.02-2.20 microg g(-1). The results indicate that there is significant accumulation and transfer of organic arsenic within the food chain at levels that may present a toxicity risk to avian wildlife.

Bioaccessibility and excretion of arsenic in Niu Huang Jie Du Pian pills.

Traditional Chinese medicines (TCMs) often contain significant levels of potentially toxic elements, including arsenic. Niu Huang Jie Du Pian pills were analyzed to determine the concentration, bioaccessibility (arsenic fraction soluble in the human gastrointestinal system) and chemical form (speciation) of arsenic. Arsenic excretion in urine (including speciation) and facial hair were studied after a one-time ingestion. The pills contained arsenic in the form of realgar, and although the total arsenic that was present in a single pill was high (28 mg), the low bioaccessibility of this form of arsenic predicted that only 4% of it was available for absorption into the bloodstream (1 mg of arsenic per pill). The species of arsenic that were solubilized were inorganic arsenate (As(V)) and arsenite (As(III)) but DMAA and MMAA were detected in urine. Two urinary arsenic excretion peaks were observed: an initial peak several (4-8) hours after ingestion corresponding to the excretion of predominantly As(III), and a larger peak at 14 h corresponding predominantly to DMAA and MMAA. No methylated As(III) species were observed. Facial hair analysis revealed that arsenic concentrations did not increase significantly as a result of the ingestion. Arsenic is incompletely soluble under human gastrointestinal conditions, and is metabolized from the inorganic to organic forms found in urine. Bioaccessible arsenic is comparable to the quantity excreted. Facial hair as a bio-indicator should be further tested.

Chromatographic separation and identification of products from the reaction of dimethylarsinic acid with hydrogen sulfide.

The reaction of dimethylarsinic acid (DMAV) with hydrogen sulfide (H2S) is of biological significance and may be implicated in the overall toxicity and carcinogenicity of arsenic. The course of the reaction in aqueous phase was monitored, and an initial product, dimethylthioarsinic acid, was observed by using LC-ICP-MS and LC-ESI-MS. Dimethylarsinous acid was observed as a minor product. A second slower-forming product was identified, and the electrospray mass chromatograms for this species produced ions at m/z 275, 171, and 137 in positive mode. To aid in the identification of this slower-forming product, crystalline standards of sodium dimethyldithioarsinate and dimethylarsino dimethyldithioarsinate were prepared and re-characterized by using improved spectroscopic and structural analysis techniques. An aqueous solution of sodium dimethyldithioarsinate produced a single major chromatographic peak that matched the retention time (7.6 min) of the slower-forming product and contained similar molecular ions at m/z 275, 171, and 137 via LC-ESI-MS. The dimethylarsino dimethyldithioarsinate standard produced four aqueous phase species one of which coeluted with the slower forming product. This coeluting peak also produced the identical ESI-MS ions as the slower-forming product of DMAV + H2S. ESI-MS/MS experiments conducted on sodium dimethyldithioarsinate in deuterated water produced molecular ions at m/z 276, 173, and 137. Subsequent collisionally activated dissociation (CAD) experiments on m/z 276 did not produce a product ion at m/z 173. These data indicate that two different species are present in solution, while NMR data indicate that only dimethyldithioarsinic acid exists in aqueous solutions. This discrepancy was investigated by conducting NMR studies on the acidic solution of sodium dimethyldithioarsinate after taking this solution to dryness. The resolubilized solution produced a proton NMR signal characteristic of dimethylarsino dimethyldithioarsinate. Therefore, it was concluded that the ESI-MS ion at m/z 275 associated with the slowly forming second reaction product and the sodium dimethyldithioarsinate compound is a product of the ESI desolvation process.

Trace element distribution during the reproductive cycle of female and male spiny and Pacific scallops, with implications for biomonitoring.

Trace element concentrations and contents in gills, gonad, kidneys, mantle, muscle and remainder during the reproductive cycle of female and male spiny and Pacific scallops, from the Strait of Georgia, BC, Canada, were quantified by using ICPMS. The elements investigated were chromium, manganese, iron, cobalt, nickel, selenium, molybdenum, cadmium, tin and mercury. For all ten elements, the tissue distribution was to some extent influenced by species, sex and reproductive status. The implications of the present study in relation to the design of biomonitoring programmes are: (1) care should be taken to ensure an equal/constant sex composition when making interannual comparisons of pooled samples. Preferably the sexes should be monitored separately. (2) the practice of obtaining pooled samples in the interspawn phase is applicable only to monitoring long-term trends in contaminant levels, while the reproductive status should be heeded when studying short-term changes. (3) the present study confirms that direct temporal or spatial comparisons of absolute accumulated element concentrations are only valid intraspecifically.