William R. Cullen

Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells

Abstract. Biomethylation is considered a major detoxification pathway for inorganic arsenicals (iAs). According to the postulated metabolic scheme, the methylation of iAs yields methylated metabolites in which arsenic is present in both pentavalent and trivalent forms. Pentavalent mono- and dimethylated arsenicals are less acutely toxic than iAs. However, little is known about the toxicity of trivalent methylated species. In the work reported here the toxicities of iAs and trivalent and pentavalent methylated arsenicals were examined in cultured human cells derived from tissues that are considered a major site for iAs methylation (liver) or targets for carcinogenic effects associated with exposure to iAs (skin, urinary bladder, and lung). To characterize the role of methylation in the protection against toxicity of arsenicals, the capacities of cells to produce methylated metabolites were also examined. In addition to human cells, primary rat hepatocytes were used as methylating controls. Among the arsenicals examined, trivalent monomethylated species were the most cytotoxic in all cell types. Trivalent dimethylated arsenicals were at least as cytotoxic as trivalent iAs (arsenite) for most cell types. Pentavalent arsenicals were significantly less cytotoxic than their trivalent analogs. Among the cell types examined, primary rat hepatocytes exhibited the greatest methylation capacity for iAs followed by primary human hepatocytes, epidermal keratinocytes, and bronchial epithelial cells. Cells derived from human bladder did not methylate iAs. There was no apparent correlation between susceptibility of cells to arsenic toxicity and their capacity to methylate iAs. These results suggest that (1) trivalent methylated arsenicals, intermediary products of arsenic methylation, may significantly contribute to the adverse effects associated with exposure to iAs, and (2) high methylation capacity does not protect cells from the acute toxicity of trivalent arsenicals.

Arsenic binding to proteins.

Arsenic is a trace element found in the earth’s crust at an average concentration of ∼5 μg/g (ppm). Although its relative abundance in the earth’s crust is about 54th, arsenic can become concentrated in some parts of the world because of natural mineralization. Arsenic is a component of 245 minerals, associated most frequently with other metals such as copper, gold, lead, and zinc in sulfidic ores.1−3 When disturbed by natural processes, such as weathering, biological activity, and volcanic eruption, arsenic may be released into the environment. Anthropogenic activities, such as combustion of fossil fuels, mining, ore smelting, and well drilling, also mobilize and introduce arsenic into the environment. Chronic exposure to arsenic from groundwater has been recognized to cause the largest environmental health disaster in the world, putting more than 100 million people at risk of cancer and other arsenic-related diseases.4,5 Because of its prevalence in the environment, potential for human exposure, and the magnitude and severity of health problems it causes, the United States Agency for Toxic Substances and Disease Registry (ATSDR) has ranked arsenic as No. 1 on its Priority List of Hazardous Substances for many years. The recent priority list, posted in 2011 (http://www.atsdr.cdc.gov/SPL/index.html), shows arsenic as No. 1, ahead of lead, mercury, and polychlorinated biphenyls (PCBs). Epidemiological studies of populations exposed to high levels of arsenic due to ingestion from water, including those from Taiwan,6−8 Argentina,9,10 Chile,11,12 West Bengal, India,13,14 Bangladesh,15−17 and Inner Mongolia, China,18,19 have repeatedly shown strong associations between the exposure to high concentrations of arsenic and the prevalence of several cancers,20−23 most severely bladder, lung, and skin cancers. Arsenic is classified as a human carcinogen by the International Agency for Research on Cancer (IARC) and the U.S. Environmental Protection Agency (EPA). Chronic exposure to elevated concentrations of arsenic has also been associated with the increased risk of a number of noncancerous effects.24−27 Although the adverse health effects arising from exposure to arsenic have been well-recognized, the mechanism(s) of action responsible for the diverse range of health effects are complicated and poorly understood.26−30 It is believed that inorganic arsenate (HAsO42-), which is a molecular analogue of phosphate (HPO42-), can compete for phosphate anion transporters and replace phosphate in some biochemical reactions.28 For example, generation of adenosine-5′-triphosphate (ATP) during oxidative phosphorylation can be inhibited by the replacement of phosphate with arsenate. Depletion of ATP by arsenate has been observed in cellular systems.28 However, the replacement of phosphate in DNA by arsenic is not firmly established.31−35 The toxicity of trivalent arsenicals likely occurs through the interaction of trivalent arsenic species with sulfhydryl groups in proteins. Arsenic binding to a specific protein could alter the conformation and function of the protein as well as its recruitment of and interaction with other functional proteins. Therefore, there has been much emphasis on studies of arsenic binding to proteins, for the purpose of understanding arsenic toxicity and developing arsenic-based therapeutics. This review summarizes various aspects of arsenic binding to proteins. It discusses the chemical basis and biological implications and consequences of arsenic binding to proteins. It also describes analytical techniques and the characterization of arsenic binding, including the binding affinity, kinetics, and speciation.

Composition and distribution of polycyclic aromatic hydrocarbon contamination in surficial marine sediments from Kitimat Harbor, Canada

Surficial marine sediments from 20 sites within the Kitimat fjord system were analyzed for polycylic aromatic hydrocarbons (PAHs). Levels of the sum of the 16 USEPA priority pollutant PAHs varied from below detection limits (ca. 1 microg x g-1) to over 10 000 microg x g-1. Sediment PAH levels were highest in the immediate vicinity of a large aluminum smelter at the head of Kitimat Arm, and declined rapidly with increasing distance from the smelter. However, even at some of the more distant sites which are geographically isolated from the smelter PAH levels were elevated. The PAH distribution in the fjord system is consistent with a combination of aeolian and fluvial transport of PAHs emitted by the aluminum smelter at the head of Kitimat Arm. The mixture of PAHs present was qualitatively similar in all samples analyzed, including those from the distant sites. All aspects of the PAH composition are consistent with combustion generated PAHs. A correlation between PAH levels and sediment organic carbon was observed; however, this was only significant for highly contaminated sites in the harbor. This probably reflects the high organic content of particulate emissions from the smelter, rather than equilibrium partitioning of PAHs to sediment organic carbon within the harbor itself.

The reaction of methylarsenicals with thiols: Some biological implications

Abstract Address reprint requests to Dimethylarsinic acid and disodium methylarsonate are easily reduced by thiols in neutral solution to give the organosulfur derivatives of arsenic(III), (CH 3 ) 2 AsSR, CH 3 As(SR′) 2 , or CH 3 AsSR″S (RSH = R′SH = cysteine, glutathione, HSCH 2 CH 2 OH; RSH = HSCH 2 COOH; HSR″SH = dithiothreitol, 6.8-dithiooctanoic acid. The reactions are usually stoichiometric and quantitative: HSCH 2 COOH is an exception, apparently for kinetric reasons. The mechanism appears to be Me x As V O(OH) 3−x + 2RSH → Me x As V (SR) 2 (OH) 3−x + H 2 O Me x As V (SR) 2 (OH) 3−x 4− → Me x As III (OH) 3−x + RSSR Me x As III (OH) 3−x + (3−x)RSH → Me x As(SR) 3−x The possible importance of these reductions in the biological methylation of arsenic is discussed.

The reduction of trimethylarsine oxide to trimethylarsine by thiols: a mechanistic model for the biological reduction of arsenicals

Trimethylarsine oxide is reduced to trimethylarsine in aqueous solution by a variety of thiols and dithiols including cysteine, glutathione, and lipoic acid. Kinetic results and other observations suggest that the rate-determining step is the production of [Me3AsSR]+ from an initially formed Me3As(SR)OH species, and that the reduction occurs via a two-electron transfer from Me3As(SR)2 affording Me3As and RS-SR. A simple model for the biological methylation of arsenic is proposed based on oxidative methylation of arsenic(III) by S-adenosylmethionine and reduction by a thiol such as lipoic acid.

Arsenic in the Meager Creek hot springs environment, British Columbia, Canada

Levels of arsenic in water from Meager Creek hot springs, British Columbia, Canada, were found to be naturally elevated. Biota including microbial mats, green algae, sedge, cedar, fleabane, monkey flower, moss, mushrooms and lichens, that were expected to be impacted by the water, were analyzed for total levels of arsenic and for arsenic species. The major arsenic species extracted from all samples were arsenate and arsenite, which are toxic forms of arsenic. Additionally, small amounts of arsenosugars X and XI were detected in microbial mats and green algae, implying that cyanobacteria/bacteria, and possibly green algae are capable of synthesizing arsenosugars from arsenate. Low to trace amounts of arsenosugars X and XI were detected in lichens and the fungus Tarzetta cupularis. A large fraction (on average, greater than 50%) of arsenic was not extracted by using methanol/water (1:1) and the chemical and toxicological significance of this arsenic remains unknown.

SPECIATION OF ARSENIC COMPOUNDS BY HPLC WITH HYDRIDE GENERATION ATOMIC ABSORPTION SPECTROMETRY AND INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY DETECTION

An arsenic specific detection system utilizing on-line microwave digestion and hydride generation atomic absorption spectrometry (MD/HGAAS) is described for arsenic speciation by using high performance liquid chromatography (HPLC). Both ion exchange chromatography and ion pair chromatography have been studied for the separation of arsenite, arsenate, monomethylarsonic acid (MMAA), dimethylarsinic acid (DMAA), and arsenobetaine (AB). When the commonly used mobile phases, phosphate and carbonate buffers at pH 7.5, are used on an anion exchange column, arsenite and AB co-elute. However, selective determination of these two arsenic compounds can be achieved by using the new detection system. Partial separation between arsenite and AB can be achieved by increasing the mobile phase pH to 10.3 and by using a polymer based anion exchange column. The detection limit obtained by using anion exchange chromatography with MD/HGAAS detection is approximately 10 ng/ml (or 200 pg for a 20-mul sample injection) for arsenite, DMAA and AB, 15 ng/ml (or 300 pg) for MMAA, and 20 ng/ml (or 400 pg) for arsenate. Complete separation of the five arsenic compounds is achieved on a reversed phase C18 column by using sodium heptanesulfonate as ion pair reagent. Comparable resolution between chromatographic peaks is obtained by using MD/HGAAS detection and inductively coupled plasma mass spectrometry (ICPMS) detection.

The characterization of arsenosugars in commercially available algal products including a Nostoc species of terrestrial origin

The arsenic species present in a range of commercially available dried-algal food products were characterized by HPLC–ICP–MS. The products of marine origin contain up to four dimethylarsinylribosides (1) in the 8–49 ppm range and some also contain dimethylarsinic acid (DMAA). These species are easily extracted and account for most of the arsenic burden. One sample of a freshwater alga Nostoc sp. was found to contain a lower concentration of arsenic, 3 ppm, and only 34% of this was extractable. The extract representing 1 ppm of arsenic contained one of the arsenosugars 1 found in the marine samples (93%), the rest being DMAA. This is the first report of the identification of an arsenosugar from an organism of terrestrial origin. The implications of this result in connection with the global arsenic cycle are discussed.

Speciation of arsenic compounds in some marine organisms.

Speciation of arsenic compounds in some marine algae, bivalves, and crustaceans was studied by using two techniques: (i) high-performance liquid chromatography with inductively coupled plasma mass spectrometry detection and (ii) hydride generation atomic adsorption spectrometry following microwave-assisted digestion. Arsenosugars were identified as the major arsenic compounds present in marine algae, whereas arsenobetaine was the dominant arsenic species present in crab and skimp. In contrast to most previous reports, which claimed arsenobetaine as the only major arsenic species present in marine bivalves, tits study revealed the presence of arsenosugars in addition to arsenobetaine in the bivalves

Highly efficient catalysis of the Kemp elimination in the cavity of a cubic coordination cage

The hollow cavities of coordination cages can provide an environment for enzyme-like catalytic reactions of small-molecule guests. Here, we report a new example (catalysis of the Kemp elimination reaction of benzisoxazole with hydroxide to form 2-cyanophenolate) in the cavity of a water-soluble M8L12 coordination cage, with two features of particular interest. First, the rate enhancement is among the largest observed to date: at pD 8.5, the value of kcat/kuncat is 2 × 10(5), due to the accumulation of a high concentration of partially desolvated hydroxide ions around the bound guest arising from ion-pairing with the 16+ cage. Second, the catalysis is based on two orthogonal interactions: (1) hydrophobic binding of benzisoxazole in the cavity and (2) polar binding of hydroxide ions to sites on the cage surface, both of which were established by competition experiments.