Dimethylmonothioarsinic acid (DMMTAV) differentially modulates the expression of AHR-regulated cytochrome P450 1A enzymes in vivo and in vitro.

Dimethylmonothioarsinic acid (DMMTAV), a pentavalent thio-arsenic derivative, has been found in bodily fluids and tissues including urine, liver, kidney homogenates, plasma, and red blood cells. Although DMMTAV is a minor metabolite in humans and animals, its substantial toxicity raises concerns about potential carcinogenic effects. This toxicity could be attributed to arsenicals' ability to regulate cytochrome P450 1 A (CYP1A) enzymes, pivotal in procarcinogen activation or detoxification. The current study investigates DMMTAV's impact on CYP1A1/2 expression, individually and in conjunction with its inducer, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). C57BL/6 mice were intraperitoneally injected with 6 mg/kg DMMTAV, alone or with 15 µg/kg TCDD, for 6 and 24 h. Similarly, Hepa-1c1c7 cells were exposed to DMMTAV (0.5, 1, and 2 µM) with or without 1 nM TCDD for 6 and 24 h. DMMTAV hindered TCDD-induced elevation of Cyp1a1 mRNA, both in vivo (at 6 h) and in vitro, associated with reduced CYP1A regulatory element activation. Interestingly, in C57BL/6 mice, DMMTAV boosted TCDD-induced CYP1A1/2 protein and activity, unlike Hepa-1c1c7 cells where it suppressed both. DMMTAV co-exposure increased TCDD-induced Cyp1a2 mRNA. While Cyp1a1 mRNA stability remained unchanged, DMMTAV negatively affected protein stability, indicated by shortened half-life. Baseline levels of CYP1A1/2 mRNA, protein, and catalytic activities showed no significant alterations in DMMTAV-treated C57BL/6 mice and Hepa-1c1c7 cells. Taken together, these findings indicate, for the first time, that DMMTAV differentially modulates the TCDD-mediated induction of AHR-regulated enzymes in both liver of C57BL/6 mice and murine Hepa-1c1c7 cells suggesting that thio-arsenic pentavalent metabolites are extremely reactive and could play a role in the toxicity of arsenic.

Dimethylmonothioarsinic acid and dimethyldithioarsinic acid in the environment: Sorption characteristics on 2-line ferrihydrite and acute toxicity to Daphnia magna.

Dimethylmonothioarsinic acid (DMMTA(V)) and dimethyldithioarsinic acid (DMDTA(V)), which are commonly found in landfill leachate and pore water of rice paddy soil, have attracted considerable attention for their high toxicity. This study aims to evaluate the behavior and potential risks of DMMTA(V) and DMDTA(V) in the environment by conducting an equilibrium sorption test using 2-line ferrihydrite and acute toxicity (immobilization) test using Daphnia magna. The overall maximum sorption capacity (qm) derived by the Langmuir isotherm model showed an increase in the order of inorganic arsenate (As(V)) > dimethylarsinic acid (DMA(V)) > DMMTA(V) > DMDTA(V), which was likely due to steric hindrance due to the presence of methyl and thiol groups. Moreover, DMMTA(V) and DMDTA(V) showed no or negligible change in qm with pH change, while qm decreased rapidly with increasing pH in As(V) and DMA(V). The 50% inhibition concentrations (IC50) for As(V), DMA(V), DMMTA(V), and DMDTA(V), which were measured after 48 h exposure to D. magna, were 9.5, > 30, 1.7, and 6.5 mg/L, respectively. Overall, the results demonstrated that DMMTA(V) and DMDTA(V) have high mobility and eco-toxicity in the environment and that methylated thioarsenicals should be accurately monitored and controlled.

Origins, fate, and actions of methylated trivalent metabolites of inorganic arsenic: progress and prospects.

The toxic metalloid inorganic arsenic (iAs) is widely distributed in the environment. Chronic exposure to iAs from environmental sources has been linked to a variety of human diseases. Methylation of iAs is the primary pathway for metabolism of iAs. In humans, methylation of iAs is catalyzed by arsenic (+ 3 oxidation state) methyltransferase (AS3MT). Conversion of iAs to mono- and di-methylated species (MAs and DMAs) detoxifies iAs by increasing the rate of whole body clearance of arsenic. Interindividual differences in iAs metabolism play key roles in pathogenesis of and susceptibility to a range of disease outcomes associated with iAs exposure. These adverse health effects are in part associated with the production of methylated trivalent arsenic species, methylarsonous acid (MAsIII) and dimethylarsinous acid (DMAsIII), during AS3MT-catalyzed methylation of iAs. The formation of these metabolites activates iAs to unique forms that cause disease initiation and progression. Taken together, the current evidence suggests that methylation of iAs is a pathway for detoxification and for activation of the metalloid. Beyond this general understanding of the consequences of iAs methylation, many questions remain unanswered. Our knowledge of metabolic targets for MAsIII and DMAsIII in human cells and mechanisms for interactions between these arsenicals and targets is incomplete. Development of novel analytical methods for quantitation of MAsIII and DMAsIII in biological samples promises to address some of these gaps. Here, we summarize current knowledge of the enzymatic basis of MAsIII and DMAsIII formation, the toxic actions of these metabolites, and methods available for their detection and quantification in biomatrices. Major knowledge gaps and future research directions are also discussed.

Absorption and speciation of arsenic by microalgae under arsenic-copper Co-exposure.

Combined pollutions of arsenic (As) and copper (Cu) are common in water bodies near mines, non-ferrous metal smelting and power plants. This study investigated the effect of Cu(II) on the absorption and speciation of As(V) by microalgae. We compared the absorption and speciation of arsenic by microalgae (mainly Cyanophyta and Chlorophyta) when exposed to single As(V) with that exposed to As-Cu co-exposure in laboratory. The results showed that in the case of single As(V) exposure, the inhibitory effect of As(V) on microalgae was primarily affected by the exposure time, instead of the concentration of As(V) in the water solution. Compared with single As(V) exposure, the presence of Cu(II) under As-Cu co-exposure promoted the absorption and accumulation of As(V) by algae. The combination effect of As and Cu on algae was antagonistic instead of synergistic within the tolerance range of algae to them. In the presence of Cu(II), more monomethylarsonous acid (MMA) and dimethylarsinous acid (DMA), which are volatile organic arsenic compounds, were produced in algae compared with the control. The finding that Cu(II) can mediate the absorption and speciation processes of arsenic in algae has significance in possible bioremediation of arsenic pollution in aquatic environment.

Exposure to inorganic arsenic and its methylated metabolites alters metabolomics profiles in INS-1 832/13 insulinoma cells and isolated pancreatic islets.

Inorganic arsenic (iAs) is an environmental diabetogen, but mechanisms underlying its diabetogenic effects are poorly understood. Exposures to arsenite (iAsIII) and its methylated metabolites, methylarsonite (MAsIII) and dimethylarsinite (DMAsIII), have been shown to inhibit glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cells and isolated pancreatic islets. GSIS is regulated by complex mechanisms. Increase in ATP production through metabolism of glucose and other substrates is the ultimate trigger for GSIS in ß-cells. In the present study, we used metabolomics to identify metabolites and pathways perturbed in cultured INS-1 832/13 rat insulinoma cells and isolated murine pancreatic islets by exposures to iAsIII, MAsIII and DMAsIII. We found that the exposures perturbed multiple metabolites, which were enriched primarily in the pathways of amino acid, carbohydrate, phospholipid and carnitine metabolism. However, the effects of arsenicals in INS-1 832/13 cells differed from those in the islets and were exposure specific with very few overlaps between the three arsenicals. In INS-1 832/13 cells, all three arsenicals decreased succinate, a metabolite of Krebs cycle, which provides substrates for ATP synthesis in mitochondria. Acetylcarnitine was decreased consistently by exposures to arsenicals in both the cells and the islets. Acetylcarnitine is usually found in equilibrium with acetyl-CoA, which is the central metabolite in the catabolism of macronutrients and the key substrate for Krebs cycle. It is also thought to play an antioxidant function in mitochondria. Thus, while each of the three trivalent arsenicals perturbed specific metabolic pathways, which may or may not be associated with GSIS, all three arsenicals appeared to impair mechanisms that support ATP production or antioxidant defense in mitochondria. These results suggest that impaired ATP production and/or mitochondrial dysfunction caused by oxidative stress may be the mechanisms underlying the inhibition of GSIS in ß-cells exposed to trivalent arsenicals.

Arsenite and its trivalent methylated metabolites inhibit glucose-stimulated calcium influx and insulin secretion in murine pancreatic islets.

Chronic exposure to inorganic arsenic (iAs), a common drinking water and food contaminant, has been associated with an increased risk of type 2 diabetes in population studies worldwide. Several mechanisms underlying the diabetogenic effects of iAs have been proposed through laboratory investigations. We have previously shown that exposure to arsenite (iAs(III)) or its methylated trivalent metabolites, methylarsonite (MAs(III)) and dimethylarsinite (DMAs(III)), inhibits glucose-stimulated insulin secretion (GSIS) in pancreatic islets, without significant effects on insulin expression or insulin content. The goal of the present study was to determine if iAs(III) and/or its metabolites inhibit Ca2+ influx, an essential mechanism that regulates the release of insulin from ß cells in response to glucose. We found that in vitro exposures for 48 h to non-cytotoxic concentrations of iAs(III), MAs(III), and DMAs(III) impaired Ca2+ influx in isolated murine pancreatic islets stimulated with glucose. MAs(III) and DMAs(III) were more potent inhibitors of Ca2+ influx than iAs(III). These arsenicals also inhibited Ca2+ influx and GSIS in islets treated with depolarizing levels of potassium chloride in the absence of glucose. Treatment with Bay K8644, a Cav1.2 channel agonist, did not restore insulin secretion in arsenical-exposed islets. Tolbutamide, a KATP channel blocker, prevented inhibition of insulin secretion in MAs(III)- and DMAs(III)-exposed islets, but only marginally in islets exposed to iAs(III). Our findings suggest that iAs(III), MAs(III), and DMAs(III) inhibit glucose-stimulated Ca2+ influx in pancreatic islets, possibly by interfering with KATP and/or Cav1.2 channel function. Notably, the mechanisms underlying inhibition of GSIS by iAs(III) may differ from those of its trivalent methylated metabolites.

Using mathematical modeling to infer the valence state of arsenicals in tissues: A PBPK model for dimethylarsinic acid (DMAV) and dimethylarsinous acid (DMAIII) in mice.

Chronic exposure to inorganic arsenic (iAs), a contaminant of water and food supplies, is associated with many adverse health effects. A notable feature of iAs metabolism is sequential methylation reactions which produce mono- and di-methylated arsenicals that can contain arsenic in either the trivalent (III) or pentavalent (V) valence states. Because methylated arsenicals containing trivalent arsenic are more potent toxicants than their pentavalent counterparts, the ability to distinguish between the +3 and +5 valence states is a crucial property for physiologically based pharmacokinetic (PBPK) models of arsenicals to possess if they are to be of use in risk assessment. Unfortunately, current analytic techniques for quantifying arsenicals in tissues disrupt the valence state; hence, pharmacokinetic studies in animals, used for model calibration, only reliably provide data on the sum of the +3 and +5 valence forms of a given metabolite. In this paper we show how mathematical modeling can be used to overcome this obstacle and present a PBPK model for the dimethylated metabolite of iAs, which exists as either dimethylarsinous acid, (CH3)2AsIIIOH (abbreviated DMAIII) or dimethylarsinic acid, (CH3)2AsV(O)OH (abbreviated DMAV). The model distinguishes these two forms and sets a lower bound on how much of an organ's DMA burden is present in the more reactive and toxic trivalent valence state. We conjoin the PBPK model to a simple model for DMAIII-induced oxidative stress in liver and use this extended model to predict cytotoxicity in liver in response to the high oral dose of DMAV. The model incorporates mechanistic details derived from in vitro studies and is iteratively calibrated with lumped-valence-state PK data for intravenous or oral dosing with DMAV. Model formulation leads us to predict that orally administered DMAV undergoes extensive reduction in the gastrointestinal (GI) tract to the more toxic trivalent DMAIII.

Differences in apoptotic signaling and toxicity between dimethylmonothioarsinic acid (DMMTAV) and its active metabolite, dimethylarsinous acid (DMAIII), in HepaRG cells: Possibility of apoptosis cascade based on diversity of active metabolites of DMMTAV.

Dimethylmonothioarsinical acid (DMMTAV), a metabolite of arsenosugars (AsSug) and arsenolipids (AsLP), which are major organoarsenicals contained in seafoods, has been a focus of our attention due to its toxicity. It has been reported that the toxicity of DMMTAV differs according to the host cell type and that dimethylarsinous acid (DMAIII), which is a higher active metabolite of inorganic and organo arsenic compounds, may be the ultimate substance. To further elucidate the details of the mechanisms of DMMTAV, we carried out toxicological characterization by comparing DMMTAV and DMAIII using HepaRG cells, which are terminally differentiated hepatic cells derived from a human hepatic progenitor cell line that retains many characteristics, e.g, primary human hepatocytes including the morphology and expression of key metabolic enzymes (P450 s and GSTs, etc.) and complete expression of all nuclear receptors. HepaRG cells were induced to undergo differentiation by DMSO, which result red in increased levels of metabolic enzymes such as P450 and GST, in non-differentiated cells the cellular toxicities of DMMTAV and DMAIII were reduced and the induction of toxicity by DMMTAV was increased by GSH but not by DMAIII. Both DMAIII and DMMTAV induce apoptosis and increase caspase 3/7 activity. DMAIII exposure increased the activity of caspase-9. On the contrary, DMMTAV exposure resulted in markedly elevated activity of caspase-8 as well as caspase-9. These results suggest there are differences between the signaling pathways of apoptosis in DMAIII and DMMTAV and that between their active metabolites. Consequently, the ultimate metabolic substance of toxicity induction of DMMTAV may not only be DMAIII, but may also be partly due to other metabolic substances produced through the activation mechanism by GSH.

Multidrug Resistance Protein 1 (MRP1/ABCC1)-Mediated Cellular Protection and Transport of Methylated Arsenic Metabolites Differs between Human Cell Lines.

The ATP-binding cassette (ABC) transporter multidrug resistance protein 1 (MRP1/ABCC1) protects cells from arsenic (a proven human carcinogen) through the cellular efflux of arsenic triglutathione [As(GS)3] and the diglutathione conjugate of monomethylarsonous acid [MMA(GS)2]. Previously, differences in MRP1 phosphorylation (at Y920/S921) and N-glycosylation (at N19/N23) were associated with marked differences in As(GS)3 transport kinetics between HEK293 and HeLa cell lines. In the current study, cell line differences in MRP1-mediated cellular protection and transport of other arsenic metabolites were explored. MRP1 expressed in HEK293 cells reduced the toxicity of the major urinary arsenic metabolite dimethylarsinic acid (DMAV), and HEK-WT-MRP1-enriched vesicles transported DMAV with high apparent affinity and capacity (Km 0.19 µM, Vmax 342 pmol⋅mg-1protein⋅min-1). This is the first report that MRP1 is capable of exporting DMAV, critical for preventing highly toxic dimethylarsinous acid formation. In contrast, DMAV transport was not detected using HeLa-WT-MRP1 membrane vesicles. MMA(GS)2 transport by HeLa-WT-MRP1 vesicles had a greater than threefold higher Vmax compared with HEK-WT-MRP1 vesicles. Cell line differences in DMAV and MMA(GS)2 transport were not explained by differences in phosphorylation at Y920/S921. DMAV did not inhibit, whereas MMA(GS)2 was an uncompetitive inhibitor of As(GS)3 transport, suggesting that DMAV and MMA(GS)2 have nonidentical binding sites to As(GS)3 on MRP1. Efflux of different arsenic metabolites by MRP1 is likely influenced by multiple factors, including cell and tissue type. This could have implications for the impact of MRP1 on both tissue-specific susceptibility to arsenic-induced disease and tumor sensitivity to arsenic-based therapeutics.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Raman spectra of thiolated arsenicals with biological importance.

Surface enhanced Raman scattering (SERS) has great potential as an alternative tool for arsenic speciation in biological matrices. SERS measurements have advantages over other techniques due to its ability to maintain the integrity of arsenic species and its minimal requirements for sample preparation. Up to now, very few Raman spectra of arsenic compounds have been reported. This is particularly true for thiolated arsenicals, which have recently been found to be widely present in humans. The lack of data for Raman spectra in arsenic speciation hampers the development of new tools using SERS. Herein, we report the results of a study combining the analysis of experimental Raman spectra with that obtained from density functional calculations for some important arsenic metabolites. The results were obtained with a hybrid functional B3LYP approach using different basis sets to calculate Raman spectra of the selected arsenicals. By comparing experimental and calculated spectra of dimethylarsinic acid (DMAV), the basis set 6-311++G** was found to provide computational efficiency and precision in vibrational frequency prediction. The Raman frequencies for the rest of organoarsenicals were studied using this basis set, including monomethylarsonous acid (MMAIII), dimethylarsinous acid (DMAIII), dimethylmonothioarinic acid (DMMTAV), dimethyldithioarsinic acid (DMDTAV), S-(Dimethylarsenic) cysteine (DMAIII(Cys)) and dimethylarsinous glutathione (DMAIIIGS). The results were compared with fingerprint Raman frequencies from As─O, As─C, and As─S obtained under different chemical environments. These fingerprint vibrational frequencies should prove useful in future measurements of different species of arsenic using SERS.

Arsenite and methylarsonite inhibit mitochondrial metabolism and glucose-stimulated insulin secretion in INS-1 832/13 ß cells.

Growing evidence suggests that exposure to environmental contaminants contributes to the current diabetes epidemic. Inorganic arsenic (iAs), a drinking water and food contaminant, is one of the most widespread environmental diabetogens according to epidemiological studies. Several schemes have been proposed to explain the diabetogenic effects of iAs exposure; however, the exact mechanism remains unknown. We have shown that in vitro exposure to low concentrations of arsenite (iAsIII) or its trivalent methylated metabolites, methylarsonite (MAsIII) and dimethylarsinite (DMAsIII), inhibits glucose-stimulated insulin secretion (GSIS) from isolated pancreatic islets, with little effect on insulin transcription or total insulin content. The goal of this study was to determine if exposure to trivalent arsenicals impairs mitochondrial metabolism, which plays a key role in the regulation of GSIS in ß cells. We used a Seahorse extracellular flux analyzer to measure oxygen consumption rate (OCR), a proxy for mitochondrial metabolism, in cultured INS-1 832/13 ß cells exposed to iAsIII, MAsIII, or DMAsIII and stimulated with either glucose or pyruvate, a final product of glycolysis and a substrate for the Krebs cycle. We found that 24-h exposure to 2 µM iAsIII or 0.375-0.5 µM MAsIII inhibited OCR in both glucose- and pyruvate-stimulated ß cells in a manner that closely paralleled GSIS inhibition. In contrast, 24-h exposure to DMAsIII (up to 2 µM) had no effects on either OCR or GSIS. These results suggest that iAsIII and MAsIII may impair GSIS in ß cells by inhibiting mitochondrial metabolism, and that at least one target of these arsenicals is pyruvate decarboxylation or downstream reactions.

Exposures to arsenite and methylarsonite produce insulin resistance and impair insulin-dependent glycogen metabolism in hepatocytes.

Environmental exposure to inorganic arsenic (iAs) has been shown to disturb glucose homeostasis, leading to diabetes. Previous laboratory studies have suggested several mechanisms that may underlie the diabetogenic effects of iAs exposure, including (i) inhibition of insulin signaling (leading to insulin resistance) in glucose metabolizing peripheral tissues, (ii) inhibition of insulin secretion by pancreatic ß cells, and (iii) dysregulation of the methylation or expression of genes involved in maintenance of glucose or insulin metabolism and function. Published studies have also shown that acute or chronic iAs exposures may result in depletion of hepatic glycogen stores. However, effects of iAs on pathways and mechanisms that regulate glycogen metabolism in the liver have never been studied. The present study examined glycogen metabolism in primary murine hepatocytes exposed in vitro to arsenite (iAs3+) or its methylated metabolite, methylarsonite (MAs3+). The results show that 4-h exposures to iAs3+ and MAs3+ at concentrations as low as 0.5 and 0.2 µM, respectively, decreased glycogen content in insulin-stimulated hepatocytes by inhibiting insulin-dependent activation of glycogen synthase (GS) and by inducing activity of glycogen phosphorylase (GP). Further investigation revealed that both iAs3+ and MAs3+ inhibit insulin-dependent phosphorylation of protein kinase B/Akt, one of the mechanisms involved in the regulation of GS and GP by insulin. Thus, inhibition of insulin signaling (i.e., insulin resistance) is likely responsible for the dysregulation of glycogen metabolism in hepatocytes exposed to iAs3+ and MAs3+. This study provides novel information about the mechanisms by which iAs exposure impairs glucose homeostasis, pointing to hepatic metabolism of glycogen as one of the targets.

A novel MAs(III)-selective ArsR transcriptional repressor.

Microbial expression of genes for resistance to heavy metals and metalloids is usually transcriptionally regulated by the toxic ions themselves. Arsenic is a ubiquitous, naturally occurring toxic metalloid widely distributed in soil and groundwater. Microbes biotransform both arsenate (As(V)) and arsenite (As(III)) into more toxic methylated metabolites methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)). Environmental arsenic is sensed by members of the ArsR/SmtB family. The arsR gene is autoregulated and is typically part of an operon that contains other ars genes involved in arsenic detoxification. To date every identified ArsR is regulated by inorganic As(III). Here we described a novel ArsR from Shewanella putrefaciens selective for MAs(III). SpArsR orthologs control expression of two MAs(III) resistance genes, arsP that encodes the ArsP MAs(III) efflux permease, and arsH encoding the ArsH MAs(III) oxidase. SpArsR has two conserved cysteine residues, Cys101 and Cys102. Mutation of either resulted in loss of MAs(III) binding, indicating that they form an MAs(III) binding site. SpArsR can be converted into an As(III)-responsive repressor by introduction of an additional cysteine that allows for three-coordinate As(III) binding. Our results indicate that SpArsR evolved selectivity for MAs(III) over As(III) in order to control expression of genes for MAs(III) detoxification.

The relationships between arsenic methylation and both skin lesions and hypertension caused by chronic exposure to arsenic in drinking water.

The associations between arsenic exposure, arsenic methylation, and the prevalence of skin lesions and hypertension are investigated. The results indicate that the HS (hypertension and skin lesions) group and the S (skin lesions) group have higher urinary concentrations of iAs (inorganic arsenic), MMA (monomethylarsonic acid), DMA (dimethylarsinous acid) and%MMA, and lower SMI (secondary arsenic methylation index) compared to the H (hypertension) and N (without both hypertension and skin lesions) groups. The arsenic content in water which caused H may be lower than that which caused HS and S. In addition, the odds ratios suggest that higher urinary concentrations of iAs and MMA, %iAs, %MMA and PMI elevate the prevalence of only hypertension and skin lesions, and both hypertension and skin lesions. However, higher%DMA and SMI, and lower%MMA increase the prevalence of both hypertension and skin lesions compared to that of only skin lesions. It can be concluded that skin lesions subjects have higher prevalence of hypertension. Hypertension subjects may have higher prevalence of skin lesions. Lower%DMA and SMI, higher%iAs, %MMA and PMI enhance the prevalence of only hypertension and skin lesions, and both hypertension and skin lesions. Moreover, iAs and MMA may have higher toxicity and lead to both hypertension and skin lesions than to only hypertension.

Comparative cytotoxicity of fourteen trivalent and pentavalent arsenic species determined using real-time cell sensing.

The occurrence of a large number of diverse arsenic species in the environment and in biological systems makes it important to compare their relative toxicity. The toxicity of arsenic species has been examined in various cell lines using different assays, making comparison difficult. We report real-time cell sensing of two human cell lines to examine the cytotoxicity of fourteen arsenic species: arsenite (AsIII), monomethylarsonous acid (MMAIII) originating from the oxide and iodide forms, dimethylarsinous acid (DMAIII), dimethylarsinic glutathione (DMAGIII), phenylarsine oxide (PAOIII), arsenate (AsV), monomethylarsonic acid (MMAV), dimethylarsinic acid (DMAV), monomethyltrithioarsonate (MMTTAV), dimethylmonothioarsinate (DMMTAV), dimethyldithioarsinate (DMDTAV), 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone, Rox), and 4-aminobenzenearsenic acid (p-arsanilic acid, p-ASA). Cellular responses were measured in real time for 72hr in human lung (A549) and bladder (T24) cells. IC50 values for the arsenicals were determined continuously over the exposure time, giving rise to IC50 histograms and unique cell response profiles. Arsenic accumulation and speciation were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS). On the basis of the 24-hr IC50 values, the relative cytotoxicity of the tested arsenicals was in the following decreasing order: PAOIII≫MMAIII≥DMAIII≥DMAGIII≈DMMTAV≥AsIII≫MMTTAV>AsV>DMDTAV>DMAV>MMAV≥Rox≥p-ASA. Stepwise shapes of cell response profiles for DMAIII, DMAGIII, and DMMTAV coincided with the conversion of these arsenicals to the less toxic pentavalent DMAV. Dynamic monitoring of real-time cellular responses to fourteen arsenicals provided useful information for comparison of their relative cytotoxicity.

Thiolated arsenicals in arsenic metabolism: Occurrence, formation, and biological implications.

Arsenic (As) is a notoriously toxic pollutant of health concern worldwide with potential risk of cancer induction, but meanwhile it is used as medicines for the treatment of different conditions including hematological cancers. Arsenic can undergo extensive metabolism in biological systems, and both toxicological and therapeutic effects of arsenic compounds are closely related to their metabolism. Recent studies have identified methylated thioarsenicals as a new class of arsenic metabolites in biological systems after exposure of inorganic and organic arsenicals, including arsenite, dimethylarsinic acid (DMAV), dimethylarsinous glutathione (DMAIIIGS), and arsenosugars. The increasing detection of thiolated arsenicals, including monomethylmonothioarsonic acid (MMMTAV), dimethylmonothioarsinic acid (DMMTAV) and its glutathione conjugate (DMMTAVGS), and dimethyldithioarsinic acid (DMDTAV) suggests that thioarsenicals may be important metabolites and play important roles in arsenic toxicity and therapeutic effects. Here we summarized the reported occurrence of thioarsenicals in biological systems, the possible formation pathways of thioarsenicals, and their toxicity, and discussed the biological implications of thioarsenicals on arsenic metabolism, toxicity, and therapeutic effects.

Kinetics of Dimethylated Thioarsenicals and the Formation of Highly Toxic Dimethylmonothioarsinic Acid in Environment.

Dimethylmonothioarsinic acid (DMMTAV) is a highly toxic, thiolated analogue of dimethylarsinic acid (DMAV). In comparison, a further thiolated analogue, dimethyldithioarsinic acid (DMDTAV), and DMAV both exhibit lower toxicity. To understand the environmental conditions responsible for forming DMMTAV, the kinetics of DMAV thiolation are examined. The thiolation of DMAV is pH-dependent and consists of two consecutive first-order reactions under excess sulfide conditions. The first thiolation of DMAV to form DMMTAV is faster than the second one to DMDTAV. DMMTAV is therefore an intermediate. The first reaction is first-order in H2S at pH 6.0 and 20 °C; therefore, the overall reaction is second-order and the rate coefficient in this condition is 0.0780 M-1 s-1. The rate coefficient significantly decreases at pH 8.0, indicating that H2S(aq) triggers the thiolation of DMAV. The second reaction rate is significantly decreased at pH 2.5; therefore, reaction under strongly acidic conditions leads to accumulation of highly toxic DMMTAV in the early stages of thiolation. The transformation of DMDTAV to DMMTAV is catalyzed in the presence of ferric iron. Formation of DMMTAV should be considered when assessing risk posed by arsenic under sulfidic or sulfate reducing conditions.

Evaluating long-term cellular effects of the arsenic species thio-DMA(V): qPCR-based gene expression as screening tool.

Thio-dimethylarsinic acid (thio-DMA(V)) is a human urinary metabolite of the class 1 human carcinogen inorganic arsenic as well as of arsenosugars. Thio-DMA(V) exerts strong cellular toxicity, whereas its toxic modes of action are not fully understood. For the first time, this study characterises the impact of a long-term (21days) in vitro incubation of thio-DMA(V) on the expression of selected genes related to cell death, stress response, epigenetics and DNA repair. The observed upregulation of DNMT1 might be a cellular compensation to counterregulate the in a very recent study observed massive global DNA hypomethylation after chronic thio-DMA(V) incubation. Moreover, our data suggest that chronic exposure towards subcytotoxic, pico- to nanomolar concentrations of thio-DMA(V) causes a stress response in human urothelial cells. The upregulation of genes encoding for proteins of DNA repair (Apex1, Lig1, XRCC1, DDB2, XPG, ATR) as well as damage response (GADD45A, GADD45G, Trp53) indicate a potential genotoxic risk emanating from thio-DMA(V) after long-term incubation.

A novel metabolic activation associated with glutathione in dimethylmonothioarsinic acid (DMMTA(V))-induced toxicity obtained from in vitro reaction of DMMTA(V) with glutathione.

The purpose of the present study was to elucidate the metabolic processing of dimethylmonothioarsinic acid (DMMTA(V)), which is a metabolite of inorganic arsenic and has received a great deal of attention recently due to its high toxicity. The metabolites produced from an in vitro reaction with GSH were analyzed by high performance liquid chromatography-time of flight mass spectrometer (HPLC-TOFMS), HPLC with a photodiode array detector (PDA), and also gas chromatography-mass spectrometry (GC-MS) and GC with a flame photometric detector (FPD). The reaction of dimethylarsinic acid (DMA(V)) with GSH did not generate DMA(V)-SG but did generate dimethylarsinous acid (DMA(III)) or DMA(III)-SG. On the contrary, we confirmed that the reaction of DMMTA(V) with GSH directly produced the stable complex of DMMTA(V)-SG without reduction through a trivalent dimethylated arsenic such as DMA(III) and DMA(III)-SG. Furthermore, the present study suggests the production of hydrogen sulfide (H2S) and dimethylmercaptoarsine (DMA(III)-SH), a trivalent dimethylated arsenic, as well as DMA(III) and DMA(III)-SG in the decomposition process of DMMTA(V)-SG. These results indicate that the toxicity of DMMTA(V) depends not only on the formation of DMA(III) but also on at least those of H2S and DMA(III)-SH.