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

Abstract

Arsenolipids are lipid-soluble organoarsenic compounds, mainly occurring in marine organisms, with arsenic-containing hydrocarbons (AsHCs) and arsenic-containing fatty acids (AsFAs) representing two major subgroups. Recently, toxicity studies of several arsenolipids showed a high cytotoxic potential of those arsenolipids in human liver and bladder cells. Furthermore, feeding studies with Drosophila melanogaster indicated an accumulation of arsenolipids in the fruit fly's brain. In this study, the neurotoxic potential of three AsHCs, two AsFAs and three metabolites (dimethylarsinic acid, thio/oxo-dimethylarsenopropanoic acid) was investigated in comparison to the toxic reference arsenite (iAs) in fully differentiated human brain cells (LUHMES cells). Thereby, in the case of AsHCs both the cell number and cell viability were reduced in a low micromolar concentration range comparable to iAs, while AsFAs and the applied metabolites were less toxic. Mechanistic studies revealed that AsHCs reduced the mitochondrial membrane potential, whereas neither iAs nor AsFAs had an impact. Furthermore, neurotoxic mechanisms were investigated by examining the neuronal network. Here, AsHCs massively disturbed the neuronal network and induced apoptotic effects, while iAs and AsFAs showed comparatively lesser effects. Taking into account the substantial in vitro neurotoxic potential of the AsHCs and the fact that they could transfer across the physiological barriers of the brain, a neurotoxic potential in vivo for the AsHCs cannot be excluded and needs to be urgently characterized.

Assessing neurodevelopmental effects of arsenolipids in pre‐differentiated human neurons

“…A comparison of the cytotoxic effects of the applied arsenolipids and iAs III in differentiating LUHMES cells and in fully differentiated LUHMES cells (the same but mature neuronal test system ) shows that all arsenicals exert about two fold higher toxicity in the differentiating neurons. Here for the first time, significant toxic effects for AsFA 388 were observed in a neuronal test system.…”

“…Purity of the compounds (>99%) was checked by NMR spectroscopy and HPLC/ICP‐MS. Stock solutions of the respective AsHCs and AsFAs were prepared in 100% ethanol (Roth, Karlsruhe, Germany) to a final concentration of 10 mM and stored at 4°C . For all experiments with lipid‐soluble arsenic compounds, the ethanol concentration was set to a constant level of 1%, which did not induce any cytotoxic effects (data not shown).…”

“…To assess the cell number via Hoechst staining of nuclei cells were washed, fixed with formaldehyde (Roth) and permeabilized with Triton TM X‐100 solution (Sigma‐Aldrich). Afterwards Hoechst 33258 (Merck, Darmstadt, Germany) was added and fluorescence was measured using a plate reader (Ex: 355 nm, Em: 460 nm; BMG FLUOstar Omega, Ortenberg, Germany).…”

“…Cell viability was assessed by the Resazurin reduction assay . The redox dye Resazurin is metabolized by viable cells to its fluorescent form Resorufin, which correlates with the cell viability .…”

“…The first and only in vivo study, carried out on the fruit fly Drosophila melanogaster , indicated an accumulation of arsenic‐containing hydrocarbons (AsHC), one important class of arsenolipids, in the fruit fly's brain . Recently, in vitro studies with fully differentiated human neurons revealed a substantial neurotoxic potential of AsHCs, which occurred in a concentration range similar to that observed for arsenite (iAs III ) . Upon elevated uptake, inorganic arsenic can induce neurotoxic effects in humans and has been considered as a potential neurotoxicant leading to brain‐related disorders such as declining cognitive functions .…”