Occurrence and formation of thioarsenates in biological systems

For a long time, arsenite and arsenate were assessed to be the two main inorganic species that are present in arsenic-rich waters (Cullen & Reimer 1989). By now, it is known that in sulfidic environments, occurrence of arsenic is predominated by inorganic thioarsenic species which crucially influence arsenic chemistry (Hollibaugh et al. 2005, Planer-Friedrich et al. 2007, Stauder et al. 2005, Wilkin et al. 2003). These species were shown to form under abiotic and reducing conditions by mixing arsenite and sulfide (Wilkin et al. 2003). It was controversially discussed whether these species are trivalent thioarsenites (As^IIISnO3-n with n

= 1-3) or pentavalent thioarsenates (As^VSnO4-n with n = 1-4). Based on geochemically expectations, trivalent thioarsenites were assumed to form as the species were detected under anoxic conditions (Beak et al. 2008, Bostick et al. 2005, Helz et al. 1995, Wood et al.

2002). But chromatographic analyses using anion-exchange chromatography-inductively coupled plasma-mass spectrometry (AEC-ICP-MS) and electrospray ionization tandem mass spectrometry (ESI-MS-MS) identified these species as pentavalent thioarsenates. X-ray absorption spectroscopy (XAS) data confirmed the presence of thioarsenates in these solutions and identified thioarsenites as precursors of thioarsenate formation

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Friedrich et al. 2010). Mixing sulfide and arsenite at high SH^-/OH^- ratio under anoxic conditions leads to formation of mono-, di-, and trithioarsenite (MTA^III, DTA^III, and TTA^III) which are immediately oxidized either directly by addition of elemental sulfur (S0) or indirectly by O2 addition which oxidizes sulfide to S0, both resulting in the formation of di-, tri-, and tetrathioarsenate (DTA^V, TTA^V, and TetraTA^V). Monothioarsenate (MTA^V) is spontaneously formed from arsenite by addition of elemental sulfur (low SH^-/OH^- ratio) (Figure 3).

Figure 3: Reaction pathway of thioarsenite and thioarsenate formation according to Planer-Friedrich et al.

(2015); the blue arrows indicate spontaneous reactions, the green dashed arrows indicate slow reactions that need an additional reactant like O2, H2O2, or H⁺; monothioarsenate is spontaneously formed from arsenite by addition of zerovalent sulfur (low SH^-/OH^- ratio) and is kinetically stable; the occurrence of mono- and dithioarsenite during this metabolism pathway is only postulated, whereas trithioarsenite was detected by XAS and all thioarsenates were detected by AEC-ICP-MS.

Acidification of DTA^V, TTA^V, and TetraTA^V leads to arsenite formation, whereas MTA^V is stable under acidic conditions (Planer-Friedrich et al. 2010). Basically, thioarsenites cannot be detected by chromatographic methods to date as they are extremely sensitive towards the presence of oxygen. Even smallest amounts of oxygen are sufficient for immediate oxidation and high concentrations of OH^- groups in the eluent lead to immediate transformation of thioarsenites into arsenite (Planer-Friedrich et al. 2010).

Formation of thioarsenates is not only important in respect to arsenic behavior in the environment, but can crucially influence the behavior of arsenic in the body after ingestion.

Reduction and methylation during arsenic metabolism are long-known processes and it is estimated that 50-70 % of ingested inorganic arsenic - depending on the studied organism - is rapidly reduced to arsenite and subsequently methylated to dimethylated arsenic compounds which are detected in urine (Vahter 1999). High amounts of free sulfide in the

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human gut and high pH (Jorgensen & Mortensen 2001) facilitate the formation of thioarsenates after arsenite intake. The presence of aerobic microbiota - even if they present the minority group of all microbiota present in the human intestine (Ramakrishna 2007) - reveals that thioarsenites do not have to be considered in terms of arsenic-sulfur speciation in the intestine due to immediate oxidation. The presence of thiolated arsenic compounds in biological samples was proven in several studies implying the need to consider these compounds in pre-systemic arsenic metabolism and arsenic risk assessments:

Yoshida and co-workers detected an unknown sulfur-containing arsenic metabolite – presumably the same metabolite that was already reported by Hughes and Kenyon (1998) - in rat urine and feces after exposure to DMA^V (Yoshida et al. 2003). This metabolite was formed by Escherichia coli strain A3-6 that was isolated from the ceca of DMA^V exposed rats (Yoshida et al. 2003). The mechanism of the arsenic-sulfur-metabolite formation was reported to be reduction of DMA^V to DMA^III followed by thiolation. Oxidation of the unknown metabolite resulted in DMA^V formation. Therefore, Yoshida and co-workers deduced it to be a trivalent DMA^III derivative. By analyses with HPLC-ICP-MS coupled simultaneously to electrospray mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry, Hansen and co-workers could show later that this metabolite – formed as main product during DMA^V reduction with sodium-metabisulfite (Na2S2O5)/sodium thiosulfate (Na2S2O3) reagent – was not a trivalent, but a pentavalent sulfur-containing DMA^V derivate, namely dimethylmonothioarsenate (DMMTA^V). The structure was additionally confirmed by proton nuclear magnetic resonance analyses. In the same study (Hansen et al. 2004), DMMTA^V could be detected in urine and in wool extract from sheep that were naturally exposed to high concentrations of arsenosugars in their food. This study was the first that could distinctly identify thioarsenates in a biological sample.

Subsequently, several studies detected DMMTA^V after arsenic exposure in urine of hamsters (Naranmandura et al. 2007b), rats (Adair et al. 2007, Naranmandura et al. 2007b), and mice (Hughes et al. 2008), in urine of DMA^V-exposed hamsters, monomethylmonothioarsenate (MMMTA^V)was also detected (Naranmandura et al. 2007b). Furthermore, DMMTA^V was also shown to be a common arsenic metabolite in urine of women in Bangladesh who were exposed to arsenic (Raml et al. 2007).

Microbial thiolation of DMA^V leading to DMMTA^V and DMDTA^V formation was observed by Kubachka and co-workers (Kubachka et al. 2009): During incubation with microbiota from mouse cecum DMA^V was metabolized to DMMTA^V and dimethyldithioarsenate (DMDTA^V), and also to monothiotrimethylarsenate (MTTMA^V). Based on their results, Kubachka and co-workers proposed a scheme for DMA^V biotransformation leading to sulfur-containing metabolites. Either DMA^V is directly thiolated leading to formation of DMMTA^V and DMDTA^V

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or DMA^V is first reduced to DMA^III which is subsequently methylated (formation of trimethylarsenate) and finally thiolated (formation of MTTMA^V) (Figure 4).

Figure 4: Formation of methylated thioarsenicals by microbiota of mouse cecum incubated with DMA^V. DMMTA^V and DMDTA^V are formed by thiolation of DMA^V, MTTMA^V is formed via DMA^V methylation followed by thiolation; scheme proposed by Kubachka et al. (2009).

Incubating human gut microbiota with arsenate resulted in the formation of methylated arsenic compounds (MMA^V and MMA^III), methylated thioarsenic compounds (MMMTA^V) and interestingly also in one inorganic thioarsenic compound (MTA^V) (Van de Wiele et al. 2010).

Formation of inorganic thioarsenates by gut microbiota was confirmed in another study with microbiota from mouse ceca that were incubated with arsenate. The formation of seven different metabolites was observed: Besides the original substrate arsenate, methylated and inorganic thioarsenic species were detected in the reaction mixtures (Pinyayev et al. 2011).

Pinyayev and co-workers proposed a scheme for the metabolism of arsenate: Alternating steps of reduction and oxidative methylation lead to the formation of arsenite, MMA^V, MMA^III, DMA^V, DMA^III, and trimethylarsenate (TMA^V). Each pentavalent species present can subsequently be reversibly thiolated leading to the formation of Mono-, Di-, Tri-, and TetraTA^V (formed by thiolation of arsenate), MMMTA^V, MMDTA^V, and MMTTA^V (formed by thiolation of MMA^V), and DMMTA^V, DMDTA^V (formed by thiolation of DMA^V), and TMMTA^V (formed by thiolation of TMA^V) (Figure 5). Pinyayev and co-workers also suggest the presence of trivalent intermediates (inorganic and methylated thioarsenites) in terms of their arsenate metabolism scheme. It is not clear why Pinyayev and co-workers did not detect any arsenite or DMMTA^V in their experiments as it was shown in the mentioned study from

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Kubachka et al. (2009). Other studies disagree with the presented scheme as direct thiolation of pentavalent arsenic species was shown to occur only at pH < 4 for both arsenate (Planer-Friedrich et al. 2015) and methylated pentavalent arsenic species (Rochette et al.

2000) and support the hypothesis that pentavalent arsenic species have to be reduced prior to thiolation. However, in contrast to these observations from abiotic systems, it cannot be excluded that gut microbiota are able to directly thiolate pentavalent arsenic compounds also at near-neutral pH.

In summary, the conditions in the human gut – free sulfide and high pH - definitely facilitate thioarsenate formation after arsenite or arsenate ingestion, but the exact mechanisms of their formation remain to be completely elucidated. Both methylated and inorganic thioarsenates were already detected during pre-systemic metabolism. What is primarily missing, is any information about the ability of thioarsenates to pass the gastrointestinal barrier without changing their speciation and about the subsequent relevance of their toxicity for organs.

Figure 5: Arsenate metabolism by microbiota of mouse cecum; by alternating steps of reduction and oxidative methylation arsenate is metabolized firstly into arsenite and then into methylated pentavalent and trivalent arsenic compounds; each pentavalent compound can subsequently be thiolated leading to the formation of inorganic and methylated thioarsenic species. Scheme proposed by Pinyayev and co-workers (2008). The suggested presence of trivalent intermediates (inorganic and methylated thioarsenites) is not considered in this figure.

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1.2.2 Bioavailability of arsenic species determined by transport through a