Preparation and purification of 74As-labeled arsenate and arsenite for use in biological experiments

doi:10.1016/0003-2697(77)90117-8

Analytical Biochemistry

Volume 78, Issue 2, April 1977, Pages 557-560

https://doi.org/10.1016/0003-2697(77)90117-8 Get rights and content

Abstract

Inorganic arsenic may occur in biological systems as arsenite or arsenate, these two forms of arsenic differing markedly in both their chemical and biological properties (1). Preparations of arsenic-74 sometimes contain arsenic in both oxidation states. Lunde (2) reported that a sample of ⁷⁴As-labeled sodium arsenate contained 60% of the arsenic-74 as arsenite, while Chan et al. (3) found that labeled arsenate samples contained 0.1 to 1% of an impurity which did not migrate with authentic arsenate during paper electrophoresis.

The present paper describes simple procedures for purifying labeled samples of arsenic III and V, and for interconverting these two forms of arsenic.

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MAs(V) was obtained from ThermoFisher Acros Organics Division, Waltham, MA, and Chem Service, Inc., West Chester, PA, respectively. Pentavalent arsenicals were reduced as described (Reay and Asher, 1977). The reduced species were simultaneously analyzed for both arsenic and sulfur by high pressure liquid chromatography (HPLC) coupled with inductively coupled mass spectroscopy (ICP-MS) (ELAN DRC-e; PerkinElmer, Waltham, MA) (Qin et al., 2006), which showed that the trivalent organoarsenicals were not thiol adducts.
Arsenic can be methylated by arsenite (As(III)) S-adenosylmethionine methyltransferases (ArsMs) among various kingdoms of life. The intermediate product methylarsenite (MAs(III)) is highly toxic and can be utilized as an antibiotic by some microbes. ArsM gene is widely distributed in the members of every kingdom from bacteria to humans and displays a high diversity of sequence. Based on arsenic methylating capacity, ArsM proteins can be divided into two phylogenetically distinct clades (Groups 1 and 2). In this study, we show that Arcticibacter tournemirensis R1 isolated from arsenic contaminated paddy soil is resistant to both As(III) and MAs(III), but exhibits different methylation activities for As(III) and MAs(III). The A. tournemirensis R1 shows low As(III) methylation activity and produces an unknown arsenic compound. In contrast, it shows high methylation activity with MAs(III), with the main product of dimethylarsenate (DMAs(V)). An AtarsM gene is found in ars operon of A. tournemirensis R1 genome and is regulated by an atypical transcriptional repressor ArsR. Expressed in Escherichia coli AtArsM confers resistance to As(III) and MAs(III). Both in vivo and in vitro assays show that AtArsM methylates As(III) and MAs(III) to dimethyl- and trimethyl‑arsenicals. AtArsM has four conserved cysteine residues, which are present in most ArsMs and can be classified into phylogenetic group 2 family, producing trimethylated arsenic metabolites. The high arsenic methylation and volatilization activity of AtArsM provides a potential strategy for arsenic bioremediation. The methylation activity differs with As(III) and MAs(III) in A. tournemirensis R1 indicates that there may have different detoxification mechanisms for As(III) and MAs(III), which are worth investigating in the future.
Arsenic
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Arsenic in the environment occurs in both organic and inorganic compounds in their trivalent or pentavalent state. Certain fish and crustaceans contain very high levels of organic arsenic, often as arsenobetaine. In most other foodstuffs, levels of arsenic are low and the form is not known. The total daily intake of arsenic in the general population is reported to be approximately a few tenths of a milligram but varies to a great extent depending on the amount of fish consumed.
Both organic arsenic in seafood and inorganic arsenic in water, beverages, and drugs have been shown to be readily absorbed (70%–90%) by the gastrointestinal tract. Some reports also indicate a fairly high degree of absorption after the inhalation of arsenic.
Absorbed arsenic, irrespective of the form, is widely distributed in the body. After exposure to inorganic arsenic, clearance of arsenic from the skin, upper gastrointestinal tract, epididymis, thyroid, and skeleton is slower than from other organs. The highest levels of arsenic in humans are normally found in the hair, nails, and skin. The main route of excretion is through the kidneys. After ingestion of arsenite or arsenate, approximately 35% of the dose is excreted within 2 days. From animal experiments, it seems that insoluble inorganic arsenic inhaled through the airway is deposited and retained in lung tissue for a relatively long time. Animal data indicate arsenobetaine accumulation in cartilage, testes, epididymis, and muscle. Of ingested arsenobetaine, 50%–80% is excreted in the urine within 2 days.
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Inorganic arsenic-induced skin lesions such as dermatoses, which may include eruption, pigmentation, or leukodermal hyperkeratosis, may ultimately lead to the development of skin cancer and Bowen disease. Effects on the nervous system (e.g., peripheral nervous disturbance), as well as on the heart and circulatory system (e.g., abnormal electrocardiograms, peripheral vascular disturbances with gangrene of the extremities, ischemic heart disease, cerebral infarction, and erectile dysfunction), have also been reported after chronic exposure to inorganic arsenic. Hematological changes after inorganic arsenic exposure are characterized by anemia and leukopenia. Chronic oral ingestion of inorganic arsenic in drinking water has also been reported to cause internal cancers (of the lung, bladder, kidney, and liver), diabetes, hypertension, cataract, pterygium, and developmental retardation.
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Millions of people worldwide are exposed to unacceptable levels of arsenic, a proven human carcinogen, in drinking water. In animal models, arsenic and selenium are mutually protective through formation and biliary excretion of seleno-bis (S-glutathionyl) arsinium ion [(GS)₂AsSe]⁻. Selenium-deficient humans living in arsenic-endemic regions are at increased risk of arsenic-induced diseases, and may benefit from selenium supplementation. The influence of selenium on human arsenic hepatobiliary transport has not been studied using optimal human models. HepaRG cells, a surrogate for primary human hepatocytes, were used to investigate selenium (selenite, selenide, selenomethionine, and methylselenocysteine) effects on arsenic hepatobiliary transport. Arsenite + selenite and arsenite + selenide at different molar ratios revealed mutual toxicity antagonism, with the latter being higher. Significant levels of arsenic biliary excretion were detected with a biliary excretion index (BEI) of 14 ± 8%, which was stimulated to 32 ± 7% by selenide. Consistent with the formation and biliary efflux of [(GS)₂AsSe]⁻, arsenite increased the BEI of selenide from 0% to 24 ± 5%. Arsenic biliary excretion was lost in the presence of selenite, selenomethionine, and methylselenocysteine. Sinusoidal export of arsenic was stimulated ∼1.6-fold by methylselenocysteine, but unchanged by other selenium forms. Arsenic canalicular and sinusoidal transport (±selenide) was temperature- and GSH-dependent and inhibited by MK571. Knockdown experiments revealed that multidrug resistance protein 2 (MRP2/ABCC2) accounted for all detectable biliary efflux of arsenic (±selenide). Overall, the chemical form of selenium and human MRP2 strongly influenced arsenic hepatobiliary transport, information critical for human selenium supplementation in arsenic-endemic regions.
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Arsenic is a toxic metalloid that enters cells adventitiously via uptake systems for phosphate transporters, aquaglyceroporins (AQPs) or sugar permeases. However, transport of highly toxic methylarsenite (MAs(III)) and relatively nontoxic methylarsenate (MAs(V)) by bacterial AQPs has not been characterized. MAs(V) has a history of use as an herbicide. Here we used whole genome sequence analysis of AQPs in arsenic resistance (ars) operons. The aqp genes are frequently located next to MAs(III) resistance genes such as arsH, which suggests that they could be involved in MAs(III) uptake. Bacterial AQPs encoded by ars operons can be classified into two subgroups. One subgroup includes AqpS from the plant symbiont Sinorhizobium meliloti 1021. Our data suggests that AqpS has a substrate selectivity filter different from that of other bacterial AQPs. Both Escherichia coli GlpF and AqpS conduct MAs(III) efficiently, but GlpF conducts the MAs(V) anion poorly, so E. coli takes up MAs(V) inefficiently. In contrast, AqpS conducts MAs(V) under physiological conditions. A homology model of AqpS indicates that it has a substrate channel with a selectivity filter containing the nonpolar residue Val177 instead of the charged arginine residue found in other AQPs. While the selectivity filter in most AQPs prevents movement of anions, Val177 is predicted to allow movement of the MAs(V) anion through the channel. We propose that AqpS is a component of an MAs(III) resistance pathway in which MAs(III) enters cells of S. meliloti via AqpS, is oxidized by ArsH to MAs(V), which exits the cells via AqpS.
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Citation Excerpt :
Transport activity was normalized to total protein of cell lysates, determined using a bicinchoninic acid protein assay. Reduction of 73AsV into 73AsIII was performed as described previously [46]. Formation of 73AsIII from 73AsV was confirmed using thin layer chromatography (SigmaCell type 100 cellulose plates) developed with an isopropanol:acetic acid: water (10:1:5) solvent system, as described [47].
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^∗: Permanent address: Department of Agriculture, University of Queensland, Australia.

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Short communicationPreparation and purification of 74As-labeled arsenate and arsenite for use in biological experiments

Abstract

J. Biol. Chem

Anal Biochem

Anal. Biochem

Anal. Chim. Acta

Anal. Chim. Acta

CRC Critical Review in Environmental Control

Short communication
Preparation and purification of ⁷⁴As-labeled arsenate and arsenite for use in biological experiments