Dimethylarsinic acid induces bladder carcinogenesis via the amphiregulin pathway.

Dimethylarsinic acid (DMA) is a major metabolite in the urine of humans and rats exposed to inorganic arsenicals, and is reported to induce rat bladder carcinogenesis. In the present study, we focused on early pathways of carcinogenesis triggered by DMA that were also active in tumors. RNA expression in the bladder urothelium of rats treated with 0 and 200 ppm DMA in the drinking water for 4 weeks and in bladder tumors of rats treated with 200 ppm DMA for 2 years was initially examined using microarray analysis and Ingenuity Pathway Analysis (IPA). Expression of 160 genes was altered in both the urothelium of rats treated for 4 weeks with DMA and in DMA-induced tumors. IPA associated 36 of these genes with liver tumor diseases. IPA identified the amphiregulin (Areg)-regulated pathway as a Top Regulator Effects Network. Therefore, we focused on Areg and 6 of its target genes: cyclin A2, centromere protein F, marker of proliferation Ki-67, protein regulator of cytokinesis 1, ribonucleotide reductase M2, and topoisomerase II alpha. We confirmed high mRNA expression of Areg and its 6 target genes in both the urothelium of rats treated for 4 weeks with DMA and in DMA-induced tumors. RNA interference of human amphiregulin (AREG) expression in human urinary bladder cell lines T24 and UMUC3 decreased expression of AREG and its 6 target genes and decreased cell proliferation. These data suggest that Areg has an important role in DMA-induced rat bladder carcinogenesis.

Multigenerational effects of co-exposure to dimethylarsinic acid and polystyrene microplastics on the nematode Caenorhabditis elegans.

In the current study, we assessed the impact of DMA (dimethylarsinic acid) and MPs (microplastics) interactions in C. elegans over the course of five generations. We found that the redox state of the organisms changed over generations as a result of exposure to both pollutants. From the third generation onward, exposure to MPs reduced GST activity, indicating reduced detoxifying abilities of these organisms. Additionally, dimethylarsinic exposure decreased the growth of organisms in the second, fourth, and fifth generations. In comparison to isolated pollutants, the cumulative effects of co-exposure to DMA and MPs seem to have been more harmful to the organisms, as demonstrated by correlation analysis. These findings demonstrate that DMA, despite being considered less hazardous than its inorganic equivalents, can still have toxic effects on species at low concentrations and the presence of MPs, can worsen these effects.

New insights into surface behavior of dimethylated arsenicals on montmorillonite using X-ray absorption spectroscopy.

Although recent studies have revealed the occurrence of dimethylated arsenicals, little is known about their behavior in environment. This study investigates the adsorption behavior of dimethylarsinic acid (DMAV), dimethyldithioarsinic acid (DMDTAV), and dimethylmonothioarsinic acid (DMMTAV) on montmorillonite. Complicated transformations among arsenicals under normal environmental conditions were also considered. Our results clearly demonstrate that DMDTAV was oxidized to DMMTAV, which was relatively stable but partially transformed to DMAV when exposed to air during adsorption. The transformed DMAV exhibited high adsorption affinities for montmorillonite, while DMMTAV and DMDTAV were not appreciably retained by montmorillonite for 48 h. This is the first study to provide insights into DMDTAV oxidation under environmental conditions. X-ray absorption near edge structure and extended X-ray absorption fine structure studies confirmed that most of the adsorbed arsenicals on montmorillonite were DMAV. The significantly different bonding characteristics of each adsorbed DMAV provide direct evidence for the transformation of DMAV from DMDTAV and DMMTAV. Our study suggests the importance of incorporating the DMMTAV in the realistic risk management for soil environments because it is highly toxic, easily transformed from DMDTAV, and stable in the environment.

Dimethylated Thioarsenates: A Potentially Dangerous Blind Spot in Current Worldwide Regulatory Limits for Arsenic in Rice.

Arsenic (As) occurrence in rice is a serious human health threat. Worldwide, regulations typically limit only carcinogenic inorganic As, but not possibly carcinogenic dimethylated oxyarsenate (DMA). However, there is emerging evidence that "DMA", determined by routine acid-based extraction and analysis, hides a substantial share of dimethylated thioarsenates that have similar or higher cytotoxicities than arsenite. Risk assessments characterizing the in vivo toxicity of rice-derived dimethylated thioarsenates are urgently needed. In the meantime, either more sophisticated methods based on enzymatic extraction and separation of dimethylated oxy- and thioarsenates have to become mandatory or total As should be regulated.

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.

Mendelian randomization analysis of arsenic metabolism and pulmonary function within the Hispanic Community Health Study/Study of Latinos.

Arsenic exposure has been linked to poor pulmonary function, and inefficient arsenic metabolizers may be at increased risk. Dietary rice has recently been identified as a possible substantial route of exposure to arsenic, and it remains unknown whether it can provide a sufficient level of exposure to affect pulmonary function in inefficient metabolizers. Within 12,609 participants of HCHS/SOL, asthma diagnoses and spirometry-based measures of pulmonary function were assessed, and rice consumption was inferred from grain intake via a food frequency questionnaire. After stratifying by smoking history, the relationship between arsenic metabolism efficiency [percentages of inorganic arsenic (%iAs), monomethylarsenate (%MMA), and dimethylarsinate (%DMA) species in urine] and the measures of pulmonary function were estimated in a two-sample Mendelian randomization approach (genotype information from an Illumina HumanOmni2.5-8v1-1 array), focusing on participants with high inferred rice consumption. Among never-smoking high inferred consumers of rice (n = 1395), inefficient metabolism was associated with past asthma diagnosis and forced vital capacity below the lower limit of normal (LLN) (OR 1.40, p = 0.0212 and OR 1.42, p = 0.0072, respectively, for each percentage-point increase in %iAs; OR 1.26, p = 0.0240 and OR 1.24, p = 0.0193 for %MMA; OR 0.87, p = 0.0209 and OR 0.87, p = 0.0123 for the marker of efficient metabolism, %DMA). Among ever-smoking high inferred consumers of rice (n = 1127), inefficient metabolism was associated with peak expiratory flow below LLN (OR 1.54, p = 0.0108/percentage-point increase in %iAs, OR 1.37, p = 0.0097 for %MMA, and OR 0.83, p = 0.0093 for %DMA). Less efficient arsenic metabolism was associated with indicators of pulmonary dysfunction among those with high inferred rice consumption, suggesting that reductions in dietary arsenic could improve respiratory health.

Differential effects of arsenic species on Nrf2 and Bach1 nuclear localization in cultured hepatocytes.

Arsenic is a ubiquitous metalloid element present in both inorganic and organic forms in the environment. The liver is considered to be a primary organ of arsenic biotransformation and methylation, as well as the main target of arsenic toxicity. Studies have confirmed that Chang human hepatocytes have an efficient arsenic methylating capacity. Our previous studies have proven that arsenite activates nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in hepatocytes. This study aimed to explore the activation of the Nrf2 pathway upon treatment of arsenic in various forms, including inorganic and organic arsenic. Our results showed that inorganic arsenic-both As2O3 and Na2HAsO4 significantly induced the expression of Nrf2 protein and mRNA, enhanced the transcription activity of Nrf2, and induced the expression of downstream target genes. These results confirmed the inorganic arsenic-induced Nrf2 pathway activation in hepatocytes. Although all arsenic chemicals used in the study induced Nrf2 protein accumulation, the organic arsenic C2H7AsO2 did not affect the expression of Nrf2 downstream genes which were elevated by inorganic arsenic exposures. Through qRT-PCR and Nrf2 luciferase reporter assays, we further confirmed that C2H7AsO2 neither increased Nrf2 mRNA level nor activated the Nrf2 transcription activity. Mechanistically, our results confirmed inorganic arsenic-induced both the nuclear import of Nrf2 and export of Bach1 (BTB and CNC homology 1), which is an Nrf2 transcriptional repressor, while organic arsenic only induced Nrf2 translocation. The unique pattern of Nrf2 regulation by organic arsenic underlines the critical role of Nrf2 and Bach1 in the arsenic toxicology.

Inorganic arsenic influences cell apoptosis by regulating the expression of MEG3 gene.

Arsenic is a wildly distributed carcinogen in the environment. Arsenic-induced apoptosis has been extensively studied in therapeutics and toxicology. LncRNA MEG3 has been extensively studied as apoptosis regulatory gene in recent years. However, it stays unclear regarding how the mechanism of MEG3 regulates arsenic-induced apoptosis. Our focus was to explore the effects of MEG3 on arsenic-induced apoptosis. MTS assay was used to test cell viability, and qRT-PCR was for the examination of gene expressions. The effect of the apoptosis and necrosis after knockdown MEG3 was detected with double staining. Our results demonstrated that MEG3 expression was positively correlated with the concentration of three arsenic species (inorganic arsenic (iAs), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) (p < 0.05). The ability of iAs to induce MEG3 expression was much higher compared with that induced by MMA and DMA. In addition, our experiments confirmed that MEG3 knockdown increased cell viability and arsenic-induced apoptosis, but cell viability decreased after iAs treatment. Moreover, LncRNA MEG3 regulated apoptosis via down-regulate API5 while up-regulate CASP7, CCND3 and APAF1. It is further proved that arsenic-induced apoptosis increased after the knockdown of MEG3, which regulates these genes. These findings provide experimental evidence and possible mechanisms for subsequent research on the effects of arsenic on health.

Reduction by, ligand exchange among, and covalent binding to glutathione and cellular thiols link metabolism and disposition of dietary arsenic species with toxicity.

Arsenic (As) is a common contaminant in the earth's crust and widely distributed in food and drinking water. As exposures have been associated with human disease, including cancer, diabetes, lung and cardiovascular disorders, and there is accumulating evidence that early life exposures are important in the etiology. Mode-of-action analysis includes a critical role for metabolic activation of As species to reactive trivalent intermediates that disrupt cellular regulatory systems by covalent binding to thiol groups. The central role of glutathione (GSH) in the chemical reactions of metabolism and disposition of arsenic species was investigated here. The chemical kinetics were measured for reactions in which GSH is a ligand for trivalent As complex formation, a reductant for pentavalent As species, and a participant in ligand exchange reactions with other biological As-thiol complexes. The diverse reactions of GSH with As species demonstrate prominent roles in: (1) metabolic activation via reduction; (2) transport from tissues that are the primary sources of reactive trivalent As intermediates following ingestion (intestine and liver) to downstream target organs (e.g., lung, kidney, and bladder); and (3) oxidation to the terminal metabolite, dimethylarsinic acid (DMAV), which is excreted. Studies of As metabolism and disposition emphasize the link between metabolic activation vs. excretion of As (i.e., internal dosimetry of reactive species) and the disruption of critical cellular thiol-based regulatory processes that define the dose-response characteristics of disease in human epidemiological studies and animal models and underpin risk assessment.

Determination of arsenicals in mouse tissues after simulated exposure to arsenic from rice for sixteen weeks and the effects on histopathological features.

The accumulation of arsenic in rice has become a worldwide concern. In this study, dose-dependency in tissues (intestine, liver and kidney) and blood distribution of inorganic arsenicals and their methylated metabolites were investigated in male C57BL/6 mice exposed to four arsenic species (arsenite [iAs]III, arsenate [iAs]V, monomethylarsonate [MMA]V, and dimethylarsinate [DMA]V) at four doses (control [C]: 0 µg/g, simulation [S]: 0.91 µg/g, medium [M]: 9.1 µg/g and high [H]: 30 µg/g) according to the arsenical composition in rice for 8 and 16 weeks. No adverse effects were observed, while body weight gain decreased in group H. Increases in total arsenic concentrations (CtAs) and histopathological changes in the tissues occurred in all of the test groups. CtAs presented a tendency of kidney > intestine > liver > blood and were time-/dose-dependent in the liver and kidney in groups M and H. In the intestine and blood, abundant iAs (23%-28% in blood and 36%-49% in intestine) was detected in groups M and H, and CtAs decreased in group H from the 8th week to the 16th week. PMI decreased in the liver and SMI decreased in the kidney. These results indicate that the three tissues are injured through food arsenic. The intestine can also accumulate food arsenic, and the high arsenic dose will cause a deficiency in the absorbing function of the intestine. Thus, long-term exposure to arsenic-contaminated rice should be taken seriously attention.

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.

Dimethylarsinic acid (DMA) enhanced lung carcinogenesis via histone H3K9 modification in a transplacental mouse model.

Pregnant CD-1 mice received 200 ppm dimethylarsinic acid (DMA) in the drinking water from gestation day 8-18, and tumor formation was assessed in offspring at the age of 84 weeks. DMA elevated the incidence of lung adenocarcinoma (10.0%) and total tumors (33.3%) in male offspring compared to male control offspring (1.9 and 15.1%, respectively). DMA also elevated the incidence of hepatocellular carcinoma (10.0%) in male offspring compared to male control offspring (0.0%). DMA and its metabolites were detected in the lungs of transplacental DMA-treated neonatal mice. Transplacental DMA exposure increased cell proliferation in the epithelium in the lungs of both neonatal and 6-week-old male mice. Microarray and real-time PCR analyses detected high expression of keratin 8 (Krt8) in the lungs of both neonatal and 6-week-old DMA-treated mice. Western blot analysis indicated that DMA elevated methylation of histone H3K9, but not H3K27, in the lungs of male mice. Importantly, chromatin immunoprecipitation sequencing (ChIP-seq) analysis using an H3K9me3 antibody found differences in heterochromatin formation between mice exposed to DMA and the controls. Notably, ChIP-seq analysis also found regions of lower heterochromatin formation in DMA-treated mice, and one of these regions contained the Krt8 gene, agreeing with the results obtained by microarray analysis. High expression of Krt8 was also detected in adenoma and adenocarcinoma of the lung in male offspring. Overall, these data indicate that transplacental DMA treatment enhanced lung and liver carcinogenesis in male mice. In the lung, DMA caused aberrant methylation of histone H3K9, increased Krt8 expression, and enhanced cell proliferation.

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.

Genotoxic effect of dimethylarsinic acid and the influence of co-exposure to titanium nanodioxide (nTiO2) in Laeonereis culveri (Annelida, Polychaeta).

Few data are available about the effect of dimethylated forms (DMA) on aquatic organisms. As rarely a contaminant occurs alone, studies evaluating the combined effect of different contaminants in aquatic organisms are needed. In fact, the presence of nanomaterials, such as titanium dioxide nanoparticles (nTiO2), in the aquatic environment is now a reality due to its intensive production and use. So, this study evaluated the toxicological effects of DMA in an acute exposure condition and considered the potential influence of nTiO2 on the effects induced by DMA in the polychaete, Laeonereis culveri. The animals were exposed over 48 h to DMA (50 and 500 µg/l) alone or in combination with nTiO2 (1 mg/l). Biochemical parameters such as concentration of reactive oxygen species (ROS), glutathione-S-transferase (GST) activity, levels of reduced glutathione levels (GSH) and macromolecular (lipid and DNA) damage were evaluated, as well the DNA repair system. In addition, the accumulation of total As and the chemical speciation of the metalloid in the organisms was determined. The results showed that: (1) only the group exposed to 500 µg of DMA/l accumulated As and when co-exposed to nTiO2, this accumulation was not observed. (2) The levels of ROS increased in the group exposed to 50 µg/l of DMA alone and the effect was reversed when this group was co-exposed to nTiO2 (3) None of the treatments showed altered GST activity or GSH levels. (4) All groups that received nTiO2 (alone or in combination with DMA) showed lipid peroxidation. (5) The exposure to DMA (both concentrations) alone or in combination with nTiO2 induced DNA damage in L. culveri. These results showed that DMA exhibits a genotoxic effect and that co-exposure to nTiO2 had an influence on its toxicity. So the occurrence of both contaminants simultaneously can represent a threat to aquatic biota.

The effects of arsenic speciation on accumulation and toxicity of dietborne arsenic exposures to rainbow trout.

The effects on juvenile rainbow trout survival, growth, food consumption, and food conversion efficiency from dietborne exposures to inorganic arsenic (arsenite, arsenate) and to the organoarsenicals monomethylarsonate (MMA), dimethylarsinate (DMA), and arsenobetaine (AsB) were investigated in two experiments: (1) a 28-d exposure using live diets of oligochaete worms separately exposed via water to these five arsenic compounds and (2) a 56-d exposure using pellet diets prepared from commercial fish food to which arsenite, MMA, or DMA were added. In the live diet experiment, the degree to which worms could be contaminated with the organoarsenicals was limited by toxicity to the worms and other experimental constraints, so that their toxicity relative to inorganic arsenic could not be fully established, but AsB was concluded to have low toxicity, consistent with published results for mammals. For the pellet diet experiment, MMA and DMA were found to be at least an order of magnitude less toxic than inorganic As on the basis of concentration in the diet, as well as much less toxic on the basis of accumulation in the fish. The need to consider speciation in aquatic risk assessments for arsenic was further demonstrated by tissue analyses of three macroinvertebrate species from a mining-impacted stream, which showed large variations in both total arsenic and the relative amounts of inorganic and organic arsenic. Additionally, although effects of arsenic on trout appear to be well correlated with inorganic arsenic, worms were found to be more sensitive to waterborne DMA than to inorganic arsenic, showing that low toxicity of organoarsenicals cannot be assumed for all aquatic organisms. Various difficulties in evaluating and applying studies on dietborne exposures and fish growth are also discussed.

Arsenic content in two-year-old Acer platanoides L. and Tilia cordata Miller seedlings growing under dimethylarsinic acid exposure-model experiment.

The presence of cacodylic acid (dimethylarsinic acid, DMA) can be an important factor in limiting the abilities of young tree seedlings to adapt to unfavorable environmental conditions. For this reason, the aim of the study was to estimate the influence of different DMA additions (from 0.01 to 0.6 mM) to modified Knop solution to arsenic (As) and selected forms of this metalloid (As(III), As(V), DMA) phytoextraction by two-year-old Acer platanoides L. and Tilia cordata Miller seedlings. Additionally, the biomass and other elements important in As transport in plants were analyzed. Seedlings of both tree species were able to grow in all experimental systems except the one with the highest DMA concentration (0.6 mM). Exposure of tree seedlings was related to a general decrease in plant biomass. Phytoextraction of As in roots, stems, and leaves increased with a rise of DMA concentration in solution to the highest content of As in A. platanoides and T. cordata roots growing under 0.3 mM (135 ± 13 and 116 ± 14 mg kg-1 dry weight). Arsenic was accumulated mainly in roots, thereby confirming bioconcentration factor values BCF > 1 for all tree seedlings treated with DMA. Exposure of plants to low DMA concentrations (0.01 and 0.03 mM) was related to the transport of this element to aboveground parts, while increased DMA concentration in other experimental systems led to the limitation of As transport to stems, as confirmed by translocation factor values TF < 1. Changes in many other elements such as boron, silicon, phosphorus, or sulfur concentration indicated the possible influence of DMA on the transport of As from roots to leaves. The obtained results show that DMA can be an important factor in modulating As phytoextraction in the studied tree species.

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.

Inorganic arsenic exposure increased expression of Fas and Bax gene in vivo and vitro.

Accumulating evidences have shown that apoptosis plays an important role in mediating the therapeutic effects and toxicity of arsenic. Fas and Bax genes are critical regulatory genes for apoptosis. In this study, we investigated the association between levels of Fas and Bax expression and the three arsenic species (inorganic arsenic (iAs), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) in vivo and vitro. Three arsenic species in urine were measured and levels of Fas and Bax expression were examined by the quantitative real-time PCR (qPCR) for all subjects. We found that Fas and Bax mRNA expression in the exposed group were significantly higher than that in the control group. The levels of gene expression were positively correlated with the concentrations of urinary iAs, MMA and DMA in all subjects. Sodium arsenite induced Fas and Bax mRNA expression, then MMA and DMA did not induce mRNA expression in MDA-MB-231 and XWLC-05 cells. The findings of the present study indicated that iAs, MMA, and DMA had different effects on expression of Bax and Fas gene.

A comparative study on subacute toxicity of arsenic trioxide and dimethylarsinic acid on antioxidant status in Crandell Rees feline kidney (CRFK), human hepatocellular carcinoma (PLC/PRF/5), and epithelioma papulosum cyprini (EPC) cell lines.

Arsenic (As) is a global contaminant of terrestrial and aquatic environments posing concern for environmental and human health. The effects of subacute concentrations of arsenic trioxide (AsIII) and dimethylarsinic acid (DMAV) were examined using Crandell Rees feline kidney (CRFK), human hepatocellular carcinoma (PLC/PRF/5), and epithelioma papulosum cyprini (EPC). Whole monolayer with suffering cells (confluence 100%, pyknosis and refractive cells; value scale = 2) led to identification of subacute As concentrations for the three cell lines. The selected AsIII concentrations were 1.33 µM for CRFK and 33.37 µM for PLC/PRF/5 and EPC, at 48 hr time point. The selected DMAV concentrations were 0.67 mM for PLC/PRF/5, 1.33 mM for CRFK, and 2.67 mM for EPC for 48 hr. Unlike the AsIII test, the three cell lines did not exhibit marked susceptibility to DMAV-mediated toxicity. Several oxidative stress biomarker levels, directly or indirectly associated with reactive oxygen species (ROS) elimination including superoxide dismutase, catalase, glutathione peroxidases, glutathione reductase, glutathione S-transferase, glyoxalase I, glyoxalase II, and total glutathione, were determined in the three cell lines at 24 and 48 hr. Antioxidant responses in metal-treated cells were significantly altered compared to controls, suggesting a perturbation of redox state. The weakening of antioxidant pathway in either healthy or tumoral cells was greater using AsIII than DMAV. Differences in level of several oxidative stress biomarkers suggest that the oxidative stress mechanism induced by AsIII is distinctly different from DMAV. Multifaceted mechanisms of action underlying ROS generation in tumor and nontumor cells versus AsIII and DMAV exposure are thus involved. Since As-mediated toxicity is quite complex, more data regarding both oxidant-enhancement and oxidant-lowering strategies may be useful to improve knowledge regarding the influence of As on human and animal cells.