During the metabolism of different arsenic-containing compounds in human, a variety of metabolites are produced with significantly varying toxicities. Currently available analytical methods can only detect a limited number of human metabolites in biological samples during one run due to their diverse characteristics. In addition, co-elution of species is often unnoticeable with most detection techniques leading to inaccurate metabolic profiles and assessment of toxicity. A high performance liquid chromatography inductively coupled mass spectrometry (HPLC-ICP-MS) method was developed that can identify thirteen common arsenic metabolites possibly present in human with special attention dedicated to thiolated or thiol conjugated arsenicals. The thirteen species included in this study are arsenite (AsIII), arsino-glutathione (As(GS)3), arsenate (AsV), monomethylarsonous acid (MMAIII), monomethylarsino-glutathione (MMAIII(GS)2), monomethylarsonic acid (MMAV), dimethylarsinous acid (DMAIII (from DMAIIII)), S-(dimethylarsinic)cysteine (DMAIII(Cys)), dimethylarsino-glutathione (DMAIII(GS)), dimethylarsinic acid (DMAV), dimethylmonothioarsinic acid (DMMTAV), dimethyldithioarsinic acid (DMDTAV), dimethylarsinothioyl glutathione (DMMTAV(GS)). The developed method was applied for the analysis of cancer cells that were incubated with Darinaparsin (DMAIII(GS)), a novel chemotherapeutic agent for refractory malignancies, and the arsenic metabolic profile obtained was compared to results using a previously developed method. This method provides a useful analytical tool which is much needed in unequivocally identifying the arsenicals formed during the metabolism of environmental arsenic exposure or therapeutic arsenic administration.
Here, we report the identification
of dimethylarsinothioyl glutathione
(DMMTAV(GS)) as a metabolite in cellular extracts of dimethyarsinous
glutathione (Darinaparsin, DMAIII(GS)) treated human multiple
myeloma (MM) cell lines. Co-elution of sulfur and arsenic on the inductively
coupled plasma mass spectrometer (ICP-MS) indicated the presence of
sulfur along with arsenic in the newly observed unidentified molecule
on the speciation chromatograms of cell lines treated with DMAIII(GS). Liquid chromatography–electrospray ionization–mass
spectrometry of the unknown peak in the MS and tandem MS modes revealed
molecular ion peaks at m/z = 443.9
and 466.0, corresponding to [DMMTAV(GS) + H]+ and [DMMTAV(GS) + Na]+, as well as peaks at
314.8 for the loss of glutamic acid and 231.1 for the loss of glycine.
In addition, peaks were observed at 176.9 corresponding to cysteine
and glycine adducts and at 137.1 for the [C2H6AsS]+ ion. An increase in the peak area of the unidentified
peak was observed upon spiking the cell extracts with a standard of
DMMTAV(GS). Heat deactivation of MM cells prevented the
formation of DMMTAV(GS) raising the possibility of its
formation via an enzymatic reaction. Formation studies in DMAIII(GS) treated MM cells revealed the dependence of DMMTAV(GS) formation on the depletion of DMAIII(GS).
The presence of 5 mM glutathione prevented its formation, indicating
that DMAIII, a dissociation product of DMAIII(GS), is likely a precursor for the formation of DMMTAV(GS). DMMTAV(GS) was observed to form under acidic and
neutral pH conditions (pH 3.0–7.4). In addition, DMMTAV(GS) was found to be stable in cell extracts at both acidic
and neutral pH conditions. When assessing the toxicity by exposing
multiple myeloma cells to arsenicals externally, DMMTAV(GS) was found to be much less toxic than DMAIII(GS) and
DMMTAV, potentially due to its limited uptake in the cells
(10 and 16% of the uptakes of DMAIII(GS) and DMMTAV, respectively).
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