Cellular toxicological characterization of a thioxolated arsenic-containing hydrocarbon

doi:10.1016/j.jtemb.2020.126563

Journal of Trace Elements in Medicine and Biology

Volume 61, September 2020, 126563

https://doi.org/10.1016/j.jtemb.2020.126563 Get rights and content

Highlights

•
Thioxo-AsHC 348 is substantially cytotoxic in cultured human liver cells.
•
Cellular bioavailability of thioxo-AsHC 348 was 10 times lower than for oxo-AsHC 332.
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Thioxo-AsHC 348 neither increased cellular oxidative stress nor caused DNA lesions.

Abstract

Arsenolipids, especially arsenic-containing hydrocarbons (AsHC), are an emerging class of seafood originating contaminants. Here we toxicologically characterize a recently identified oxo-AsHC 332 metabolite, thioxo-AsHC 348 in cultured human liver (HepG2) cells. Compared to results of previous studies of the parent compound oxo-AsHC 332, thioxo-AsHC 348 substantially affected cell viability in the same concentration range but exerted about 10-fold lower cellular bioavailability. Similar to oxo-AsHC 332, thioxo-AsHC 348 did not substantially induce oxidative stress nor DNA damage. Moreover, in contrast to oxo-AsHC 332 mitochondria seem not to be a primary subcellular toxicity target for thioxo-AsHC 348. This study indicates that thioxo-AsHC 348 is at least as toxic as its parent compound oxo-AsHC 332 but very likely acts via a different mode of toxic action, which still needs to be identified.

Introduction

Water- and lipid-soluble organoarsenicals predominantly occur in seafood with concentrations up to 100 mg/kg wet weight [1]. The latter are also referred to as arsenolipids and are an ever-growing and emerging group of arsenicals not least because of recent advances in analytical techniques [[2], [3], [4], [5]]. When humans ingest arsenolipids, present in cod liver for example, they are metabolized to various arsenic-containing compounds including dimethylarsinic acid (DMA^V) and arsenic-containing fatty acids (AsFAs) [6,7].

Among the several classes of arsenolipids, arsenic-containing hydrocarbons (AsHCs) have been shown to be toxic in various in vitro systems. These studies indicate that AsHCs efficiently transfer across the intestinal barrier [8] and exert substantial toxicity in the same concentration range as compared to arsenite in several human cell lines, such as urothelial (UROtsa), hepatoblastoma (HepG2), astrocytoma (CCF-STTG1) cells as well as pre-differentiated and fully differentiated neurons (LUHMES) [[9], [10], [11], [12], [13]]. Recently, thioxo-AsHC 348 has been observed as a new discovered metabolite of oxo-AsHC 332 in human HepG2 cells [10]. This study characterizes the cellular toxicity of thioxo-AsHC 348 and directly compares it to the corresponding parent compound oxo-AsHC 332 (Fig. 1).

Section snippets

Materials

Sodium(meta)-arsenite (≥ 99% purity) and alcian blue were purchased from Fluka Biochemika, Munich, Germany. 7-Amino-4-trifluoromethyl coumarin (AFC) standard and staurosporine were bought from Enzo Life Sciences GmbH, Lörrach, Germany. Cell Counting Kit-8 was acquired from Dojindo molecular technologies, Munich, Germany. MitoTracker™ Orange CMTMRos and 5(&6)-carboxy-2′,7′-dichloro-dihydrofluorescein-diacetate (Carboxy-DCFH-DA) were obtained from Life Technologies GmbH, Darmstadt, Germany.

Cell viability and bioavailability

A 48 h incubation with thioxo-AsHC 348 comparably (to non-thiolated arsenic-containing hydrocarbons) decreased both viability markers as well as cell number in a concentration dependent manner (Fig. 2A). Thus, 15 μM thioxo-AsHC 348 caused a 40% decrease in both lysosomal integrity and dehydrogenase activity and a 50% decrease in cell number. The respective effective concentration (EC₃₀) values for thioxo-AsHC 348 of 11 μM for cell number, 12 μM for dehydrogenase activity, 13 μM for lysosomal

Conclusion

The aim of this study was to characterize toxic effects of thioxo-AsHC 348 in cultured human liver cells. Thioxo-AsHC 348 exerted a comparable cytotoxic potential like inorganic iAs^III and the arsenolipid oxo-AsHC 332. Whereas the accumulation was comparable to iAs^III, it was 10 times lower than for AsHC 332, probably due to differences in the hydrophilicity of the head group. Mechanistic studies showed that apoptosis and necrosis were not triggered. In contrast to oxo-AsHCs, thioxo-AsHC 348

Declaration of Competing Interest

None.

Acknowledgements

This work was supported by the German Research Foundation (DFG), grant number SCHW 903/10-1 and the Austrian Science Fund (FWF), project number I2412-B21.

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But limited toxicity studies were found for the arsenolipid-derived metabolites. There has been only one study on thioxo-AsHC 332; Ebert et al. (Ebert et al., 2020) found that the oxo- and thioxo- analogs of AsHC 332 affected the cell viability in cultured human liver cells in the same concentration range. For the metabolites of thioxo-AsFA 362 and AsFEs, no equivalent toxicity studies have been done so far.
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The same cannot be said, however, of arsenolipids. These compounds are abundant in marine fish, in particular in fatty fish where they can contribute up to 50% of the total arsenic [6], and in vitro toxicity testing with human liver or bladder cells has revealed that one group of arsenolipids, namely the arsenic-containing hydrocarbons (AsHCs) in both oxo- and thioxo-forms, has cytotoxicity comparable to that of inorganic arsenic [7,8]. Furthermore, arsenic hydrocarbons have been shown to accumulate in the brain of both the fruit fly [9] and tuna fish [10], and they can also disturb the neuronal network of human brain cells [11,12].
Arsenic hydrocarbons, major arsenolipids occurring naturally in marine fish, have substantial cytotoxicity leading to human health-related studies of their distribution and abundance in foods. These studies have all investigated fresh foods; because most fish are cooked before being consumed, it is both food- and health-relevant to determine the arsenolipids present in cooked fish.
We used HPLC/mass spectrometry to investigate the arsenolipids present in salmon (Salmo salar) before and after cooking by either baking or steaming.
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Our study shows that arsenic hydrocarbons naturally present in fresh fish are partly converted to their thioxo analogs during cooking by either baking or steaming. The greater lipophilicity of the thioxo analogs could alter the mode of toxicity of arsenic hydrocarbons, and hence future food regulations for arsenic should consider the influence of cooking on the precise type of arsenolipid in fish.
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Being an essential trace element, copper is involved in diverse physiological processes. However, excess levels might lead to adverse effects. Disrupted copper homeostasis, particularly in the brain, has been associated with human diseases including the neurodegenerative disorders Wilson and Alzheimer’s disease. In this context, astrocytes play an important role in the regulation of the copper homeostasis in the brain and likely in the prevention against neuronal toxicity, consequently pointing them out as a potential target for the neurotoxicity of copper. Major toxic mechanisms are discussed to be directed against mitochondria probably via oxidative stress. However, the toxic potential and mode of action of copper in astrocytes is poorly understood, so far.
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ToxicologyCellular toxicological characterization of a thioxolated arsenic-containing hydrocarbon

Highlights

Abstract

Introduction

Section snippets

Materials

Cell viability and bioavailability

Conclusion

Declaration of Competing Interest

Acknowledgements

Food Chem.

J. Trace Elem. Med. Biol.

Talanta

Toxicology

Biotechnol. Annu. Rev.

J. Trace Elem. Med. Biol.

Fundam. Appl. Toxicol.

Anal. Biochem.

Toxicology

Mutat. Res. Mol. Mech. Mutagen.

J. Environ. Sci.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Toxicol. Lett.

J. Environ. Sci.

Mutat. Res. Toxicol. Environ. Mutagen.

Free Radic. Biol. Med.

A review of human carinogens. Part C: arsenic, metals, fibres, and dusts

IARC Monogr.

Synthesis and characterization of arsenolipids: naturally occurring arsenic compounds in fish and algae

Organometallics

Quantification of arsenolipids in the certified reference material NMIJ 7405-a (Hijiki) using HPLC/Mass spectrometry after chemical derivatization

Anal. Chem.

Arsenolipid biosynthesis by the unicellular alga Dunaliella tertiolecta is influenced by As/P ratio in culture experiments

Metallomics

Arsenic-containing hydrocarbons: natural compounds in oil from the fish capelin, Mallotus villosus

Chem. Commun.

Arsenolipids exert less toxicity in a human neuron astrocyte co-culture as compared to the respective monocultures

Metallomics

Toxicity of two classes of arsenolipids and their water-soluble metabolites in human differentiated neurons

Arch. Toxicol.

Assessing neurodevelopmental effects of arsenolipids in pre-differentiated human neurons

Mol. Nutr. Food Res.

In vitro toxicological characterisation of three arsenic-containing hydrocarbons

Metallomics

Arsenic-containing hydrocarbons are toxic in the in vivo model Drosophila melanogaster

Metallomics

Arsenic-containing hydrocarbons: effects on gene expression, epigenetics, and biotransformation in HepG2 cells

Arch. Toxicol.

Toxicology
Cellular toxicological characterization of a thioxolated arsenic-containing hydrocarbon