Uptake of inorganic and organic derivatives of arsenic associated with induced cytotoxic and genotoxic effects in Chinese hamster ovary (CHO) cells

Humans are exposed to arsenic and their organic derivatives, which are widely distributed in the environment, via food, water, and to a lesser extent, via air. Following uptake, inorganic arsenic undergoes biotransformation to mono- and dimethylated metabolites. Recent findings suggest that the methylation reactions represent a toxification rather than a detoxification pathway. In the present study, the genotoxic effects and the cellular uptake of inorganic arsenic [arsenate, As(i)(V); arsenite, As(i)(III)] and the methylated arsenic species monomethylarsonic acid [MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], dimethylarsinous acid [DMA(III)], trimethylarsenic oxide [TMAO(V)] were investigated in Chinese hamster ovary (CHO-9) cells. The chemicals were applied at different concentrations (0.1 microM to 10 mM) for 30 min and 1 h, respectively. Cytotoxic effects were investigated by the trypan blue extrusion test and genotoxic effects by the assessment of micronucleus (MN) induction, chromosome aberrations (CA), and sister chromatid exchanges (SCE). Intracellular arsenic concentrations were determined by ICP-MS techniques. Our results show that MMA(III) and DMA(III) induce cytotoxic and genotoxic effects to a greater extent than MMA(V) or DMA(V). Viability was significantly decreased after incubation (1 h) of the cells with > or = 1 microM As(i)(III), > or = 1 microM As(i)(V), > or = 500 microM MMA(III), > or = 100 microM MMA(V), and 500 microM DMA(V) and > or = 0.1 microM DMA(III). TMAO(V) was not cytotoxic at concentrations up to 10 mM. A significant increase of the number of MN, CA and SCE was found for DMA(III) and MMA(III). As(i)(III + V) induced CA and SCE but no MN. TMAO(V), MMA(V) and DMA(V) were not genotoxic in the concentration range tested (up to 5 mM). The nuclear division index (NDI) was not affected by any of the tested arsenic compounds after a recovery period of 14 to 35 h. When the uptake of the chemicals was measured by ICP-MS analysis, it was found that only 0.03% MMA(V) and DMA(V), and 2% MMA(III), As(i)(III) and (V) were taken up by the cells. In comparison, 10% of the DMA(III) dose was taken up. The total intracellular concentration of all arsenic compounds increased with increasing arsenic concentrations in the culture medium. Taken together, these data demonstrate that arsenic compounds in the trivalent oxidation state exhibit the strongest genotoxic effects. Trivalent organoarsenic compounds are more membrane permeable than the pentavalent species. The potency of the DNA damage decreases in the order DMA(III) > MMA(III) > As(i)(III and V) > MMA(V) > DMA(V) > TMAO(V). We postulate that the induction of genotoxic effects caused by the methylated arsenic species is primarily dependent upon their ability to penetrate cell membranes.     

Cytotoxic effects of S-(dimethylarsino)-glutathione: a putative intermediate metabolite of inorganic arsenicals

Glutathione (GSH) plays an important role in the metabolism of arsenite and arsenate by generating arsenic-glutathione complexes. Although dimethylarsinic acid (DMA(V)) is the major metabolite of inorganic arsenicals (iAs) in urine, it is not clear how DMA(V) is produced from iAs. In the present study we report that S-(dimethylarsino)-glutathione (DMA(III)(SG)), a putative precursor of dimethylarsinic acid DMA(V), was unstable in the culture medium without excess GSH and generated volatile substances which were highly cytotoxic for both rat heart microvascular endothelial cells and HL60, a human leukemia cell line. Cytotoxicity of DMA(III)(SG) was higher than that of iAs and its LC(50) value was calculated to be 7.8 microM in the endothelial cells. To our surprise DMA(III)(SG) effectively killed cells in the neighbor wells of the same multi-well dish, indicating that volatile toxic compounds generated from DMA(III)(SG) in the culture medium. High performance lipid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) analyses suggested that the freshly generated volatile compounds dissolved into aqueous solution and formed an unstable arsenic compound and the unstable compound was further converted to DMA(V). These results suggested that DMA(III)(SG) exerts its cytotoxicity by generating volatile arsenicals and is implicated in the metabolic conversion of inorganic arsenicals into DMA(V), a major final metabolite of inorganic arsenicals in most mammals.     

The accumulation and toxicity of methylated arsenicals in endothelial cells: important roles of thiol compounds

Excess intake of arsenic is known to cause vascular diseases as well as skin lesions and cancer in humans. Recent reports suggest that trivalent methylated arsenicals, which are intermediate metabolites in the methylation process of inorganic arsenic, are responsible for the toxicity and carcinogenicity of environmental arsenic. We investigated acute toxicity and accumulation of monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), trimethylarsine oxide (TMAO), and monomethylarsonous acid diglutathione (MMA(III) (GS)(2)) in rat heart microvessel endothelial (RHMVE) cells. MMA(V) (LC(50) = 36.6 mM) and DMA(V) (LC(50) = 2.54 mM) were less toxic than inorganic arsenicals (cf. LC(50) values for inorganic arsenite (iAs(III)), and inorganic arsenate (iAs(V)) was reported to be 36 and 220 microM, respectively, in RHMVE cells. TMAO was essentially not toxic. However, MMA(III) (GS)(2) was highly toxic (LC(50) = 4.1 microM). The order of cellular arsenic accumulation of those four organic arsenic compounds was MMA(III) (GS)(2) > MMA(V) > DMA(V) > TMAO. MMA(III) (GS)(2) was efficiently taken up by the cells and cellular arsenic content increased with the concentration of MMA(III) (GS)(2) in culture medium. N-acetyl-l-cysteine (NAC) reduced cellular arsenic content in DMA(V)-exposed cells and also decreased the cytotoxicity of DMA(V), whereas it changed neither cellular arsenic content nor the viability in MMA(V)-exposed cells. mRNA levels of heme oxygenase-1 (HO-1) were decreased by NAC in DMA(V)-exposed, but MMA(V)-exposed cells. Buthionine sulfoximine (BSO), a cellular glutathione (GSH) depleting agent, enhanced the cytotoxicity of MMA(V). However, BSO reduced, rather than enhanced, the cytotoxicity of DMA(V). These results suggest that intracellular GSH modulated the toxic effects of arsenic in opposite ways for MMA(V) and DMA(V). Even though intracellular GSH decreased the cytotoxicity of MMA(V), extracellularly added GSH enhanced the cytotoxicity of MMA(V). The use of high-performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometric analyses suggested that a small amount of MMA(V) was converted to MMA(III) (GS)(2) in the presence of GSH. These results suggest that MMA(III) (GS)(2) is highly toxic compared to other arsenic compounds because of faster accumulation of this species by cells, in addition to having the toxic nature of methylated trivalent organic arsenics.     

Methylated arsenicals: the implications of metabolism and carcinogenicity studies in rodents to human risk assessment

Monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)) are active ingredients in pesticidal products used mainly for weed control. MMA(V) and DMA(V) are also metabolites of inorganic arsenic, formed intracellularly, primarily in liver cells in a metabolic process of repeated reductions and oxidative methylations. Inorganic arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder, and lung. However, a good animal model has not yet been found. Although the metabolic process of inorganic arsenic appears to enhance the excretion of arsenic from the body, it also involves formation of methylated compounds of trivalent arsenic as intermediates. Trivalent arsenicals (whether inorganic or organic) are highly reactive compounds that can cause cytotoxicity and indirect genotoxicity in vitro. DMA(V) was found to be a bladder carcinogen only in rats and only when administered in the diet or drinking water at high doses. It was negative in a two-year bioassay in mice. MMA(V) was negative in 2-year bioassays in rats and mice. The mode of action for DMA(V)-induced bladder cancer in rats appears to not involve DNA reactivity, but rather involves cytotoxicity with consequent regenerative proliferation, ultimately leading to the formation of carcinoma. This critical review responds to the question of whether DMA(V)-induced bladder cancer in rats can be extrapolated to humans, based on detailed comparisons between inorganic and organic arsenicals, including their metabolism and disposition in various animal species. The further metabolism and disposition of MMA(V) and DMA(V) formed endogenously during the metabolism of inorganic arsenic is different from the metabolism and disposition of MMA(V) and DMA(V) from exogenous exposure. The trivalent arsenicals that are cytotoxic and indirectly genotoxic in vitro are hardly formed in an organism exposed to MMA(V) or DMA(V) because of poor cellular uptake and limited metabolism of the ingested compounds. Furthermore, the evidence strongly supports a nonlinear dose-response relationship for the biologic processes involved in the carcinogenicity of arsenicals. Based on an overall review of the evidence, using a margin-of-exposure approach for MMA(V) and DMA(V) risk assessment is appropriate. At anticipated environmental exposures to MMA(V) and DMA(V), there is not likely to be a carcinogenic risk to humans.     

Differential toxicity and gene expression in Caco-2 cells exposed to arsenic species

Inorganic arsenic [As(V) + As(III)] and its metabolites, especially the trivalent forms [monomethylarsonous acid, MMA(III), and dimethylarsinous acid, DMA(III)], are considered the forms of arsenic with the highest degree of toxicity, linked to certain types of cancer and other pathologies. The gastrointestinal mucosa is exposed to these forms of arsenic, but it is not known what toxic effect these species may have on it. The aim of the present work was to evaluate the toxicity and some mechanisms of action of inorganic arsenic and its metabolites [monomethylarsonic acid, MMA(V), dimethylarsinic acid, DMA(V), MMA(III) and DMA(III)] in intestinal epithelial cells, using the Caco-2 human cell line as a model.     

Arsenic uptake,cytotoxicity and detoxification studied in mammalian cells in culture

The cytotoxicity of trivalent and pentavalent inorganic arsenic salts was determined in mouse fibroblasts in vitro. Concentrations of As (III) in the M range led to a reduction of proliferation and viability with a concomitant increase in LDH release and stimulation of lactic acid production. Similar effects were noted with approximately 10-fold greater molar concentrations of As(V). Cells pretreated with a low As(III) concentration are less sensitive to toxic doses of As(III) or As(V).Uptake of As(III) by the fibroblasts is greater than that of As(V). Both forms of inorganic arsenic are converted intracellularly to monomethylarsonic (MMA) and dimethylarsinic (DMA) acids, which are then released into the culture medium. In As-pretreated cells, which are more resistant to As toxicity, biotransformation of inorganic arsenic to MMA and DMA is increased.     

Glutathione-conjugated arsenics in the potential hepato-enteric circulation in rats.

The metabolic pathways for arsenic were precisely studied by determining the metabolic balance and chemical species of arsenic to gain an insight into the mechanisms underlying the animal species difference in the metabolism and preferential accumulation of arsenic in red blood cells (RBCs) in rats. Male Wistar rats were injected intravenously with a single dose of arsenite (iAs(III)) at 2.0 mg of As/kg of body weight, and then the time-dependent changes in the concentrations of arsenic in organs and body fluids were determined. Furthermore, arsenic in the bile was analyzed on anion and cation exchange columns by high-performance liquid chromatography-inductively coupled argon plasma mass spectrometry (HPLC-ICP MS). The metabolic balance and speciation studies revealed that arsenic is potentially transferred to the hepato-enteric circulation through excretion from the liver in a form conjugated with glutathione (GSH). iAs(III) is methylated to mono (MMA)- and dimethylated (DMA) arsenics in the liver during circulation in the conjugated form [iAs(III)(GS)(3)], and a part of MMA is excreted into the bile in the forms of MMA(III) and MMA(V), the former being mostly in the conjugated form [CH(3)As(III)(GS)(2)], and the latter being in the nonconjugated free form. DMA(III) and DMA(V) were not detected in the bile. In the urine, arsenic was detected in the forms of iAs(III), arsenate, MMA(V), and DMA(V), iAs(III) being the major arsenic in the first 6-h-urine, and DMA(V) being increased in the second 6-h-urine. The present metabolic balance and speciation study suggests that iAs(III) is methylated in the liver during its hepato-enteric circulation through the formation of the GSH-cojugated form [iAs(III)(GS)(3)], and MMA(III) and MMA(V) are partly excreted into the bile, the former being in the conjugated form [CH(3)As(III)(GS)(2)]. DMA is not excreted into the bile but into the bloodstream, accumulating in RBCs, and then excreted into the urine mostly in the form of DMA(V) in rats.     

Cytotoxicity of combinations of arsenicals on rat urinary bladder urothelial cells in vitro

Based on epidemiological data, chronic exposure to high levels of inorganic arsenic in the drinking water is carcinogenic to the urinary bladder of humans. The highly reactive trivalent organic arsenicals dimethylarsinous acid (DMA(III)) and monomethylarsonous acid (MMA(III)) are formed during the metabolism of inorganic arsenic in vivo in addition to the corresponding mono-, di- and trimethylated pentavalent arsenicals. The objective of this study was to determine if combining arsenicals was additive or synergistic toward inducing cytotoxicity in a rat urothelial cell line. The MYP3 cell line, an immortalized but not transformed rat urinary bladder epithelial cell line, was seeded into appropriate culture wells. Treatment with the arsenicals was begun 24 h after seeding and continued for 3 days. Combinations of arsenicals used were DMA(III) with arsenite, dimethylarsinic acid (DMA(V)) or trimethylarsine oxide (TMAO). Combinations of concentrations used were the LC50, one-quarter or one-half the LC50 of one arsenical with one-half or one-quarter the LC50 of the other arsenical. To determine if MYP3 cells metabolize arsenicals, cells were treated with arsenate, arsenite and MMA(V) as described above and the medium was analyzed by HPLC-ICPMS to determine species and quantity of arsenicals present. When cells were treated with one-quarter or one-half the LC50 concentration of both arsenicals, the cytotoxicity was approximately the same as when cells were treated with half the LC50 concentration or the LC50 concentration, respectively, of either arsenical. Treatment with one-quarter the LC50 concentration of one arsenical plus the LC50 concentration of a second arsenical had similar cytotoxicity as treatment with the LC50 concentration of either of the arsenicals. Quantitation and speciation of arsenicals in the cell culture medium showed that MYP3 cells have some reductase activity but the cells do not methylate arsenicals. The effect on the cytotoxicity of arsenicals in combination was additive rather than synergistic toward a rat urothelial cell line.     

Monomethylarsenic diglutathione transport by the human multidrug resistance protein 1 (MRP1/ABCC1)

The ATP-binding cassette (ABC) transporter protein multidrug resistance protein 1 (MRP1; ABCC1) plays an important role in the cellular efflux of the high-priority environmental carcinogen arsenic as a triglutathione conjugate [As(GS)(3)]. Most mammalian cells can methylate arsenic to monomethylarsonous acid (MMA(III)), monomethylarsonic acid (MMA(V)), dimethylarsinous acid (DMA(III)), and dimethylarsinic acid (DMA(V)). The trivalent forms MMA(III) and DMA(III) are more reactive and toxic than their inorganic precursors, arsenite (As(III)) and arsenate (As(V)). The ability of MRP1 to transport methylated arsenicals is unknown and was the focus of the current study. HeLa cells expressing MRP1 (HeLa-MRP1) were found to confer a 2.6-fold higher level of resistance to MMA(III) than empty vector control (HeLa-vector) cells, and this resistance was dependent on GSH. In contrast, MRP1 did not confer resistance to DMA(III), MMA(V), or DMA(V). HeLa-MRP1 cells accumulated 4.5-fold less MMA(III) than HeLa-vector cells. Experiments using MRP1-enriched membrane vesicles showed that transport of MMA(III) was GSH-dependent but not supported by the nonreducing GSH analog, ophthalmic acid, suggesting that MMA(III)(GS)(2) was the transported form. MMA(III)(GS)(2) was a high-affinity, high-capacity substrate for MRP1 with apparent K(m) and V(max) values of 11 μM and 11 nmol mg(-1)min(-1), respectively. MMA(III)(GS)(2) transport was osmotically sensitive and inhibited by several MRP1 substrates, including 17β-estradiol 17-(β-D-glucuronide) (E(2)17βG). MMA(III)(GS)(2) competitively inhibited the transport of E(2)17βG with a K(i) value of 16 μM, indicating that these two substrates have overlapping binding sites. These results suggest that MRP1 is an important cellular protective pathway for the highly toxic MMA(III) and have implications for environmental and clinical exposure to arsenic.     

Toxicity of dimethylmonothioarsinic acid toward human epidermoid carcinoma A431 cells

Chronic ingestion of arsenic-contaminated drinking water induces skin lesions and urinary bladder cancer in humans. It is now recognized that thioarsenicals such as dimethylmonothioarsinic acid (DMMTA (V)) are commonly excreted in the urine of humans and animals and that the production of DMMTA (V) may be a risk factor for the development of the diseases caused by arsenic. The toxicity of DMMTA (V) was compared with that of related nonthiolated arsenicals with respect to cell viability, uptake ability, generation of reactive oxygen species (ROS), and cell cycle progression of human epidermoid carcinoma A431 cells, arsenate (iAs (V)), arsenite (iAs (III)), dimethylarsinic acid (DMA (V)), and dimethylarsinous acid (DMA (III)) being used as reference nonthiolated arsenicals. DMMTA (V) (LC 50 = 10.7 microM) was shown to be much more cytotoxic than iAs (V) (LC 50 = 571 microM) and DMA (V) (LC 50 = 843 microM), and its potency was shown to be close to that of trivalent arsenicals iAs (III) (LC 50 = 5.49 microM) and DMA (III) (LC 50 = 2.16 microM). The greater cytotoxicity of DMMTA (V) was associated with greater cellular uptake and distribution, and the level of intracellular ROS remarkably increased in A431 cells upon exposure to DMMTA (V) compared to that after exposure to other trivalent arsenicals at the respective LC 50. Exposure of DMMTA (V) to cells for 24 h induced cell cycle perturbation. Namely, the percentage of cells residing in S and G2/M phases increased from 10.2 and 15.6% to 46.5 and 20.8%, respectively. These results suggest that although DMMTA (V) is a pentavalent arsenical, it is taken up efficiently by cells and causes various levels of toxicity, in a manner different from that of nonthiolated pentavalent arsenicals, demonstrating that DMMTA (V) is one of the most toxic arsenic metabolites. The high cytotoxicity of DMMTA (V) was explained and/or proposed by (1) efficient uptake by cells followed by (2) its transformation to DMA (V), (3) producing ROS in the redox equilibrium between DMA (V) and DMA (III) in the presence of glutathione.     

Development of a human physiologically based pharmacokinetic (PBPK) model for inorganic arsenic and its mono- and di-methylated metabolites

A physiologically-based pharmacokinetic (PBPK) model was developed to estimate levels of arsenic and its metabolites in human tissues and urine after oral exposure to arsenate (As(V)), arsenite (As(III)) or organoarsenical pesticides. The model consists of interconnected individual PBPK models for inorganic arsenic (As(V) and As(III)), monomethylarsenic acid (MMA(V)), and, dimethylarsenic acid (DMA(V)). Reduction of MMA(V) and DMA(V) to their respective trivalent forms also occurs in the lung, liver, and kidney including excretion in urine. Each submodel was constructed using flow limited compartments describing the mass balance of the chemicals in GI tract (lumen and tissue), lung, liver, kidney, muscle, skin, heart, and brain. The choice of tissues was based on physiochemical properties of the arsenicals (solubility), exposure routes, target tissues, and sites for metabolism. Metabolism of inorganic arsenic in liver was described as a series of reduction and oxidative methylation steps incorporating the inhibitory influence of metabolites on methylation. The inhibitory effects of As(III) on the methylation of MMA(III) to DMA, and MMA(III) on the methylation of As(III) to MMA were modeled as noncompetitive. To avoid the uncertainty inherent in estimation of many parameters from limited human data, a priori independent parameter estimates were derived using data from diverse experimental systems with priority given to data derived using human cells and tissues. This allowed the limited data for human excretion of arsenicals in urine to be used to estimate only parameters that were most sensitive to this type of data. Recently published urinary excretion data, not previously used in model development, are also used to evaluate model predictions.     

Effects of Nickel Chloride on Human Platelets: Enhancement of Lipid Peroxidation,Inhibition of Aggregation and Interaction with Ascorbic Acid

In order to elucidate whether urinary levels of inorganic and organic arsenic metabolites are associated with previous exposure to high-arsenic artesian well water, a total of 302 residents of age 30 yr or older were recruited from three arseniasis-hyperendemic villages in Taiwan. Most study subjects had stopped consuming high-arsenic artesian well water for more than 20 yr. The mean total arsenic (Ast) determined by inductively cout pled plasma mass spectrometer (ICPMS) was 267.05 +/- 20.95 mug/L, and the mean level of inorganic arsenic and its metabolites (Ast) was 86.08 +/- 3.43 mug/L. In the multivariate analysis, urinary dimethylarsinic acid (DMA) levels were significantly inversely associated with age, with women exhibiting significantly lower urinary amounts of arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMA), organic arsenic (Aso), and Ast compared to men. After adjustment for age and sex, previous cumulative arsenic exposure through consumption of artesian well water was significantly associated with elevated urinary levels of MMA and DMA, but not As(III) + As(V), Asot, and Ast. In the multivariate analysis, the percentage of As in As was significantly higher in men than women, but this was not significantly associated with age. The percentage of As(III) + As(V) in As increased significantly with age, while the reverse was noted with DMA in Asi. Women had a significantly higher DMA percentage but lower As(III) + As(V) and MMA percentages in Asi than men. After adjustment for age and sex, the percentages of As(III) + As(V) in Asi were significantly inversely associated with previous arsenic exposure through consumption of artesian well water. Data suggested that women seem to possess a more efficient arsenic methylation capability than men, and aging diminishes this methylation capability; furthermore, the higher the cumulative arsenic exposure, the greater is the body burden of inorganic arsenic, mainly in the form of MMA and DMA.     

A comparative study of the sub-chronic toxic effects of three organic arsenical compounds on the urothelium in F344 rats; gender-based differences in response

Epidemiological studies indicated that human arsenic exposure can induce urinary bladder cancer. Methylation of inorganic arsenic can generate more reactive and toxic organic arsenical species. In this regard, it was recently reported that the methylated arsenical metabolite, dimethylarsinic acid [DMA(V)], induced urinary bladder tumors in rats. However, other methylated metabolites, like monomethylarsonic acid [MMA(V)] and trimethylarsine oxide (TMAO) were not carcinogenic to the urinary bladder. In order to compare the early effects of DMA(V), MMA(V), and TMAO on the urinary bladder transitional cell epithelium at the scanning electron microscope (SEM) level, we investigated the sub-chronic (13 weeks) toxicological effects of MMA(V) (187 ppm), DMA(V) (184 ppm), TMAO (182 ppm) given in the drinking water to male and female F344 rats with a focus on the urinary bladder in this study. Obvious pathological changes, including ropy microridges, pitting, increased separation of epithelial cells, exfoliation, and necrosis, were found in the urinary bladders of both sexes, but particularly in females receiving carcinogenic doses of DMA(V). Urine arsenical metabolic differences were found between males and females, with levels of MMA(III), a potential genotoxic form, higher in females treated with DMA(V) than in males. Thus, this study provides clear evidence that DMA(V) is more toxic to the female urinary bladder, in accord with sensitivity to carcinogenesis. Important gender-related metabolic differences including enhanced presentation of MMA(III) to the urothelial cells might possibly account for heightened sensitivity in females. However, the potential carcinogenic effects of MMA(III) need to be further elucidated.     

Further evidence against a direct genotoxic mode of action for arsenic-induced cancer

Arsenic in drinking water, a mixture of arsenite and arsenate, is associated with increased skin and other cancers in Asia and Latin America, but not the United States. Arsenite alone in drinking water does not cause skin cancers in experimental animals; therefore, it is not a complete carcinogen in skin. We recently showed that low concentrations of arsenite enhanced the tumorigenicity of solar UV irradiation in hairless mice, suggesting arsenic cocarcinogenesis with sunlight in skin cancer and perhaps with different carcinogenic partners for lung and bladder tumors. Cocarcinogenic mechanisms could include blocking DNA repair, stimulating angiogenesis, altering DNA methylation patterns, dysregulating cell cycle control, induction of aneuploidy and blocking apoptosis. Arsenicals are documented clastogens but not strong mutagens, with weak mutagenic activity reported at highly toxic concentrations of inorganic arsenic. Previously, we showed that arsenite, but not monomethylarsonous acid (MMA[III]), induced delayed mutagenesis in HOS cells. Here, we report new data on the mutagenicity of the trivalent methylated arsenic metabolites MMA(III) and dimethylarsinous acid [DMA(III)] at the gpt locus in Chinese hamster G12 cells. Both methylated arsenicals seemed mutagenic with apparent sublinear dose responses. However, significant mutagenesis occurred only at highly toxic concentrations of MMA(III). Most mutants induced by MMA(III) and DMA(III) exhibited transgene deletions. Some non-deletion mutants exhibited altered DNA methylation. A critical discussion of cell survival leads us to conclude that clastogenesis occurs primarily at highly cytotoxic arsenic concentrations, casting further doubt as to whether a genotoxic mode of action (MOA) for arsenicals is supportable.     

Tissue dosimetry, metabolism and excretion of pentavalent and trivalent dimethylated arsenic in mice after oral administration

Dimethylarsinic acid (DMA(V)) is a rat bladder carcinogen and the major urinary metabolite of administered inorganic arsenic in most mammals. This study examined the disposition of pentavalent and trivalent dimethylated arsenic in mice after acute oral administration. Adult female mice were administered [(14)C]-DMA(V) (0.6 or 60 mg As/kg) and sacrificed serially over 24 h. Tissues and excreta were collected for analysis of radioactivity. Other mice were administered unlabeled DMA(V) (0.6 or 60 mg As/kg) or dimethylarsinous acid (DMA(III)) (0.6 mg As/kg) and sacrificed at 2 or 24 h. Tissues (2 h) and urine (24 h) were collected and analyzed for arsenicals. Absorption, distribution and excretion of [(14)C]-DMA(V) were rapid, as radioactivity was detected in tissues and urine at 0.25 h. For low dose DMA(V) mice, there was a greater fractional absorption of DMA(V) and significantly greater tissue concentrations of radioactivity at several time points. Radioactivity distributed greatest to the liver (1-2% of dose) and declined to less than 0.05% in all tissues examined at 24 h. Urinary excretion of radioactivity was significantly greater in the 0.6 mg As/kg DMA(V) group. Conversely, fecal excretion of radioactivity was significantly greater in the high dose group. Urinary metabolites of DMA(V) included DMA(III), trimethylarsine oxide (TMAO), dimethylthioarsinic acid and trimethylarsine sulfide. Urinary metabolites of DMA(III) included TMAO, dimethylthioarsinic acid and trimethylarsine sulfide. DMA(V) was also excreted by DMA(III)-treated mice, showing its sensitivity to oxidation. TMAO was detected in tissues of the high dose DMA(V) group. The low acute toxicity of DMA(V) in the mouse appears to be due in part to its minimal retention and rapid elimination.     

Fast determination of arsenic species and total arsenic in urine by HPLC-ICP-MS: concentration ranges for unexposed german inhabitants and clinical case studies

A fast and reliable high-pressure liquid chromatography (HPLC)-inductively coupled plasma-mass spectrometry (ICP-MS) routine method was developed for the determination of inorganic arsenic [As(III) and As(V)], organic monomethylarsonate [MMA(V)], dimethylarsinate [DMA(V)], and arsenobetaine (As-B) in human urine. The complete method validation is described, including internal and external quality assurance. Limits of quantification for the As species are 0.1 microg/L, which is sufficient to determine background concentrations of the arsenic species in human urine. Additionally, total As in all urine samples was determined by conventional ICP-MS. Mean concentrations for 82 non-exposed inhabitants from northern Germany are 12.7, 5.9, 4.0, 0.23, 0.52, and 0.17 microg/L for total As, As-B, DMA(V), As(III), MMA(V), and As(V), respectively. Approximately 15% of the total As was not identified by the anion exchange HPLC-ICP-MS method, and could be other As metabolites in urine. Two case studies underline the need for As speciation, especially when total urinary arsenic concentrations are elevated. In the first case, we investigated the effect of seafood consumption on the concentration of different arsenic species in urine for different persons. A maximum enhancement of total As from 1 up to 2,200 microg/L (2,000 microg/L for As-B) was observed after a normal fish meal. The second case describes the exposure of a 7-year-old child to As(III) by inhalation of calcium arsenite powder. Five hours after exposure, the concentrations in the child's urine for As-B, DMA(V), As(III), MMA(V), and As(V) were < 0.1, 189, 304, 229, and 27 microg/L, respectively, and these concentrations were reduced to normal background values after 4 days.     

Identification of dimethylarsinous and monomethylarsonous acids in human urine of the arsenic-affected areas in West Bengal, India

A speciation technique for arsenic has been developed using an anion-exchange high-performance liquid chromatography/inductively coupled argon plasma mass spectrometer (HPLC/ICP MS). Under optimized conditions, eight arsenic species [arsenocholine, arsenobetaine, dimethylarsinic acid (DMA(V)), dimethylarsinous acid (DMA(III)), monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), arsenite (As(III)), and arsenate (As(V))] can be separated with isocratic elution within 10 min. The detection limit of arsenic compounds was 0.14-0.33 microg/L. To validate the method, Standard Reference Material in freeze-dried urine, SRM-2670, containing both normal and elevated levels of arsenic was analyzed. The method was applied to determine arsenic species in urine samples from three arsenic-affected districts of West Bengal, India. Both DMA(III) and MMA(III) were detected directly (i.e., without any prechemical treatment) for the first time in the urine of some humans exposed to inorganic arsenic through their drinking water. Of 428 subjects, MMA(III) was found in 48% and DMA(III) in 72%. Our results indicate the following. (1) Since MMA(III) and DMA(III) are more toxic than inorganic arsenic, it is essential to re-evaluate the hypothesis that methylation is the detoxification pathway for inorganic arsenic. (2) Since MMA(V) reductase with glutathione (GSH) is responsible for conversion of MMA(V) to MMA(III) in vivo, is DMA(V) reductase with GSH responsible for conversion of DMA(V) to DMA(III) in vivo? (3) Since DMA(III) forms iron-dependent reactive oxygen species (ROS) which causes DNA damage in vivo, DMA(III) may be responsible for arsenic carcinogenesis in human.     

Differential effects of trivalent and pentavalent arsenicals on cell proliferation and cytokine secretion in normal human epidermal keratinocytes

There is strong evidence from epidemiologic studies of an association between chronic exposure to inorganic arsenic (iAs) and hyperpigmentation, hyperkeratosis, and neoplasia in the skin. Although it is generally accepted that methylation is a mechanism of arsenic detoxification, recent studies have suggested that methylated arsenicals also have deleterious biological effects. In these studies we compare the effects of inorganic arsenicals (arsenite (iAs(III)) and arsenate (iAs(V))) and trivalent and pentavalent methylated arsenicals (methylarsine oxide (MAs(III)O), complex of dimethylarsinous acid with glutathione (DMAs(III)GS), methylarsonic acid (MAs(V)), and dimethylarsinic acid (DMAs(V))) in human keratinocyte cultures. Viability testing showed that the relative toxicities of the arsenicals were as follows: iAs(III) > MAs(III)O > DMAs(III)GS > DMAs(V) > MAs(V) > iAs(V). Trivalent arsenicals induced an increase in cell proliferation at concentrations in the 0.001 to 0.01 microM range, while at high concentrations (>0.5 microM) cell proliferation was inhibited. Pentavalent arsenicals did not stimulate cell proliferation. As seen in the viability studies, the methylated forms of As(V) were more cytotoxic than iAs(V). Exposure to low doses of trivalent arsenicals stimulated secretion of the growth-promoting cytokines, granulocyte macrophage colony stimulating factor and tumor necrosis factor-alpha. DMAs(V) reduced cytokine secretion at concentrations at which proliferation and viability were not affected. These data suggest that methylated arsenicals, products of the metabolic conversion of inorganic arsenic, can significantly affect viability and proliferation of human keratinocytes and modify their secretion of inflammatory and growth-promoting cytokines.     

Metabolic differences between two dimethylthioarsenicals in rats

Thioarsenicals are newly found arsenic metabolites in man and animals, and also in marine organisms. Dimethylmonothioarsinic acid (DMMTA(V)) and dimethyldithioarsinic acid (DMDTA(V)) are the only two thioarsenic metabolites detected in man and/or animals. However, their toxicological and biological significance is not known yet. The present study was performed to gain an insight into the significance of DMMTA(V) and DMDTA(V) in the metabolism of arsenic. The two thioarsenicals were synthesized chemically and injected intravenously into rats at the dose of 0.5 mg As/kg body weight. The distributions of arsenic in organs/tissues and body fluids were determined at 10 min and 12 h after the injection, and arsenic in liver and kidney supernatants, urine, plasma and red blood cell (RBC) lysates was subjected to speciation analysis by HPLC-ICP MS on a gel filtration GS 220 HQ column. Although both thioarsenicals are pentavalent arsenicals, they were distributed in organs/tissues and body fluids differently from the corresponding non-thiolated pentavalent arsenicals, and also from each other. Namely, DMMTA(V) was first found in organs/tissues at 10 min, and then redistributed and retained mostly in RBCs at 12 h, as in the case of trivalent dimethylarsinous acid (DMA(III)). On the other hand, although DMDTA(V) was also found in organs/tissues at 10 min, it had been efficiently excreted in urine in its intact form at 12 h. Thus, DMMTA(V) was unexpectedly distributed in and taken up by organs/tissues in a manner similar to DMA(III) rather than DMA(V), whereas DMDTA(V) was distributed similarly to DMA(V) as expected, but was much more efficiently excreted in urine.