In vitro toxicological characterization of two arsenosugars and their metabolites

Abstract

Scope: In their recently published Scientific Opinion on Arsenic in Food, the European Food Safety Authority concluded that a risk assessment for arsenosugars is currently not possible, largely because of the lack of relevant toxicological data. To address this issue, we carried out a toxicological in vitro characterization of two arsenosugars and six arsenosugar metabolites.

Methods and results: The highly pure synthesized arsenosugars, DMA(V) -sugar-glycerol and DMA(V) -sugar-sulfate, investigated in this study, as well as four metabolites, oxo-dimethylarsenoacetic acid (oxo-DMAA(V) ), oxo-dimethylarsenoethanol (oxo-DMAE(V) ), thio-DMAA(V) and thio-DMAE(V) , exerted neither cytotoxicity nor genotoxicity up to 500 μM exposure in cultured human bladder cells. However, two arsenosugar metabolites, namely dimethyl-arsinic acid (DMA(V) ) and thio-dimethylarsinic acid (thio-DMA(V) ), were toxic to the cells; thio-DMA(V) was even slightly more cytotoxic than arsenite. Additionally, intestinal bioavailability of the arsenosugars was assessed applying the Caco-2 intestinal barrier model. The observed low, but significant transfer rates of the arsenosugars across the barrier model provide further evidence that arsenosugars are intestinally bioavailable.

Conclusion: In a cellular system that metabolizes arsenosugars, cellular toxicity likely arises. Thus, in strong contrast to arsenobetaine, arsenosugars cannot be categorized as nontoxic for humans and a risk to human health cannot be excluded.

Keywords: Arsenosugars; Cellular and intestinal bioavailability; Cellular toxicity; Genotoxicity; Marine food.

Figure 1

Chemical structures, names and abbreviations of ten arsenic species investigated in this study.

Figure 2

Cytotoxicity of DMA^V-sugar-glycerol, DMA^V-sugar-sulfate, arsenobetaine, iAs^III (A–D) as well as DMA^V, thio-DMA^V, oxo-DMAA^V, thio-DMAA^V, oxo-DMAE^V and thio-DMAE^V (E–H) in UROtsa cells after 48-h incubation. Cytotoxicity was determined by impact on cell number (A, E), colony-forming ability (B, F), dehydrogenase activity as measured by CCK-8® (C, G), and lysosomal integrity as measured by neutral red uptake (D, H). The data represent mean values of at least three determinations ± SD. Colony-forming ability of untreated control cells was approximately 80%.

Figure 3

Cellular bioavailability of the arsenicals after 48-h incubation of UROtsa cells (A). Concentration-dependent bioavailability after 48-h incubation with iAs^III, DMA^V, and thio-DMA^V (B). The figure represents mean values of at least three independent determinations + SD; n.d. below LOD.

Figure 4

Formation of micronuclei in UROtsa cells after 48-h incubation with DMA^V-sugar-glycerol, DMA^V-sugar-sulfate, oxo-DMAA^V, thio-DMAA^V, oxo-DMAE^V, thio-DMAE^V or arsenobetaine (A). Increase in the number of micronuclei, bi- and multinucleated UROtsa cells after 48-h incubation with arsenite (B), DMA^V (C), or thio-DMA^V (D). Displayed are mean values of at least three independent determinations + SD. Representative fluorescence microscopic images (E). Asterisks mark statistically significant differences as compared to untreated cells (*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001).

Figure 5

Effects of the arsenicals on and transfer across the intestinal Caco-2 barrier model after apical incubation. Transepithelial electrical resistance (TEER) (A) and capacitance (B) were used as tracers for barrier integrity; shown are representative measurements. Arsenic concentration in the acceptor (basolateral) compartment (C), percent arsenic permeability (normalized to the respective incubation concentration) (D). Shown are mean values of at least three independent determinations +SD.