Monomethylarsonous acid (MMA(III)) and arsenite: LD(50) in hamsters and in vitro inhibition of pyruvate dehydrogenase.

Monomethylarsonous acid (MMA(III)), a metabolite of inorganic arsenic, has received very little attention from investigators of arsenic metabolism in humans. MMA(III), like sodium arsenite, contains arsenic in the +3 oxidation state. Although we have previously demonstrated that it is more toxic than arsenite in cultured Chang human hepatocytes, there are no data showing in vivo toxicity of MMA(III). When MMA(III) or sodium arsenite was administered intraperitoneally to hamsters, the LD(50)s were 29.3 and 112.0 micromol/kg of body wt, respectively. In addition, inhibition of hamster kidney or purified porcine heart pyruvate dehydrogenase (PDH) activity by MMA(III) or arsenite was determined. To inhibit hamster kidney PDH activity by 50%, the concentrations (mean +/- SE) of MMA(III) as methylarsine oxide, MMA(III) as diiodomethylarsine, and arsenite were 59.9 +/- 6.5, 62.0 +/- 1.8, and 115.7 +/- 2.3 microM, respectively. To inhibit activity of purified porcine heart PDH activity by 50%, the concentrations (mean +/- SE) of MMA(III) as methylarsine oxide and arsenite were 17.6 +/- 4.1 and 106.1 +/- 19.8 microM, respectively. These data demonstrate that MMA(III) is more toxic than inorganic arsenite, both in vivo and in vitro, and call into question the hypothesis that methylation of inorganic arsenic is a detoxication process.     

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.     

Monomethylarsonic acid reductase and monomethylarsonous acid in hamster tissue

The formation of monomethylarsonous acid (MMA(III)) by tissue homogenates of brain, bladder, spleen, liver, lung, heart, skin, kidney, or testis of male Golden Syrian hamsters was assessed using [(14)C]monomethylarsonic acid (MMA(V)) as the substrate for MMA(V) reductase. The mean +/- SEM of MMA(V) reductase specific activities (nanomoles of MMA(III) per milligram of protein per hour) were as follows: brain, 91.4 +/- 3.0; bladder, 61.8 +/- 3.7; spleen, 30.2 +/- 5.4; liver, 29.8 +/- 1.4; lung, 21.5 +/- 0.8; heart, 19.4 +/- 1.5; skin, 14.7 +/- 1.6; kidney, 10.6 +/- 0.4; and testis, 9.8 +/- 0.6. The concentrations of MMA(III) in male Golden Syrian hamster livers were determined 15 h after administration of a single intraperitoneal dose of 145 microCi of [(73)As]arsenate (2 mg of As/kg of body weight). Trivalent arsenic species (arsenite, MMA(III), and dimethylarsinous acid, DMA(III)) were extracted from liver homogenates using carbon tetrachloride (CCl(4)) and 20 mM diethylammonium salt of diethyldithiocarbamic acid (DDDC). Pentavalent arsenicals (arsenate, MMA(V), and dimethylarsinic acid, DMA(V)) remained in the aqueous phase. The organic and the aqueous phases then were analyzed by HPLC. Metabolites of inorganic arsenate present in hamster liver after 15 h were observed in the following concentrations (nanograms per gram of liver +/- SEM): MMA(III), 38.5 +/- 2.9; DMA(III), 49.9 +/- 10.2; arsenite, 35.5 +/- 3.0; arsenate, 118.2 +/- 8.7; MMA(V), 31.4 +/- 2.8; and DMA(V), 83.5 +/- 6.7. This first-time identification of MMA(III) and DMA(III) in liver after arsenate exposure indicates that the significance of arsenic species in mammalian tissue needs to be re-examined and re-evaluated with respect to their role in the toxicity and carcinogenicity of inorganic arsenic.     

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.     

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.     

Arsenite uptake and metabolism by rat hepatocyte primary cultures in comparison with kidney- and hepatocyte-derived rat cell lines.

Biotransformation by methylation to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) influences inorganic arsenical toxicity, which is often investigated in cultured cells. Arsenic (III) uptake and methylation was assessed in rat hepatocytes in primary culture and in three established rat cell lines (hepatoma-derived McA-RH 7777 cells and H4-II-EC-3 cells, and kidney epithelium-derived NRK-52E cells) to compare their use as model systems for arsenite metabolism. Incubation of all cell types with 0.27, 0.67, 1.33, 2.67, or 6.67 microM As(III) concentrations resulted in concentration-dependent arsenic uptake and biomethylation. Arsenic uptake by the NRK-52E cells was initially slower than that of the other cells, but by 8 h, total uptake was similar in all cell types. At the lowest arsenite concentration, the percentages of total arsenic methylated to MMA and DMA by the hepatocytes and the McA-RH 7777 cells were similar (67 and 66%); methylation by the H4-II-EC-3 cells was somewhat lower (52%), and methylation by the kidney-derived NRK-52E cells was much lower (15%). Total arsenic methylation was inhibited in the cell lines, but not in the hepatocytes, at the highest arsenite concentrations. In all cases, exposure to increased arsenite concentrations inhibited conversion of MMA to DMA much more than it affected the initial methylation step (inorganic arsenite to MMA). These results indicate that rat hepatocytes in primary culture and established rat hepatoma-derived cell lines are similar in their abilities to accumulate and methylate arsenic to MMA and DMA at environmentally relevant arsenic concentrations in the medium. They differed from the kidney epithelium-derived cells, which exhibited substantially lower biomethylation activity.     

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.     

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.     

A review of the enzymology of arsenic metabolism and a new potential role of hydrogen peroxide in the detoxication of the trivalent arsenic species

This laboratory has studied the enzymology involved in the biotransformation of inorganic arsenic to dimethylarsinous acid (DMA(III)) and in human studies established that monomethylarsonous acid (MMA(III)) and DMA(III) appear in urine of people chronically exposed to arsenic. It appears that only two proteins are required for inorganic arsenic biotransformation in the human, namely, monomethylarsonic acid (MMA(V)) reductase and arsenic methyltransferase. MMA(V) reductase and the unique glutathione transferase omega (hGST-O) are identical proteins. Arsenicals with a +3 oxidation state are more toxic than the +5 species. While methylation of arsenite, MMA(III), and DMA(III) produces less toxic +5 oxidation arsenic species containing an additional methyl group such as MMA(V), dimethylarsinic acid (DMA(V)), and TMAO, a new mechanism involving hydrogen peroxide for detoxifying arsenite, MMA(III), and DMA(III) is proposed based on in vitro experiments.     

Toxicity of inorganic arsenic and its metabolites on haematopoietic progenitors "in vitro": comparison between species and sexes

Inorganic arsenic (iAs) and its metabolites are transferred to the foetus through the placental barrier and this exposure can compromise the normal development of the unborn. For this reason, we assessed the toxicity of sodium arsenite (iAs(III)) and its metabolites dimethylarsinic acid (DMA(V)), monomethylarsonic acid (MMA(V)) and monomethylarsonous acid (MMA(III)) on human haematopoietic cord blood cells and murine bone marrow progenitors in vitro, looking at the effects induced at different concentrations in the two genders. The expression of two enzymes responsible for arsenic biotransformation arsenic methyltranferase (AS3MT) and glutathione S-transferase omega 1 (GSTO1) was evaluated in human cord blood cells. Cord blood and bone marrow cells were exposed in vitro to iAs(III) at a wide range of concentrations: from 0.0001 microM to 10 microM. The methylated arsenic metabolites were tested only on human cord blood cells at concentrations ranging from 0.00064 microM to 50 microM. The results showed that iAs(III) was toxic on male and female colony forming units to about the same extent both in human and in mouse. Surprisingly, very low concentrations of iAs(III) increased the proliferation rate of both human and murine female cells, while male cells showed no significant modulation. MMA(V) and DMA(V) did not exert detectable toxicity on the cord blood cells, while MMA(III) had a marked toxic effect both in male and female human progenitors. AS3MT mRNA expression was not induced in human cord blood cells after iAs(III) exposure. GSTO1 expression decreased after MMA(III) treatment. This study provides evidence that exposure to iAs(III) and MMA(III) at muM concentrations is associated with immunosuppression in vitro.     

Occurrence of monomethylarsonous acid in urine of humans exposed to inorganic arsenic

Monomethylarsonous acid (MMA(III)) has been detected for the first time in the urine of some humans exposed to inorganic arsenic in their drinking water. Our experiments have dealt with subjects in Romania who have been exposed to 2.8, 29, 84, or 161 microg of As/L in their drinking water. In the latter two groups, MMA(III) was 11 and 7% of the urinary arsenic while the monomethylarsonic acid (MMA(V)) was 14 and 13%, respectively. Of our 58 subjects, 17% had MMA(III) in their urine. MMA(III) was not found in urine of any members of the group with the lowest level of As exposure. If the lowest-level As exposure group is excluded, 23% of our subjects had MMA(III) in their urine. Our results indicate that (a) future studies concerning urinary arsenic profiles of arsenic-exposed humans must determine MMA(III) concentrations, (b) previous studies of urinary profiles dealing with humans exposed to arsenic need to be re-examined and re-evaluated, and (c) since MMA(III) is more toxic than inorganic arsenite, a re-examination is needed of the two hypotheses which hold that methylation is a detoxication process for inorganic arsenite and that inorganic arsenite is the major cause of the toxicity and carcinogenicity of inorganic arsenic.     

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.     

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.     

Arsenic speciation in urine from acute promyelocytic leukemia patients undergoing arsenic trioxide treatment

Arsenic has been used successfully in clinical trials for treating acute promyelocytic leukemia (APL). Although sublethal doses of inorganic arsenic are used, little is known about the pharmacokinetics and metabolism of the high levels of arsenic in APL patients. To fill this important gap, this study describes the speciation of arsenic in urine from four APL patients treated with arsenic. Each patient was injected daily with an arsenite (As(III)) solution that contained 10 mg of As(2)O(3) precursor. Speciation analysis of the patient urine samples collected consecutively for 48 h, encompassing two intravenous injections of arsenic, revealed the presence of monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), monomethylarsonic acid (MMA(V)), and dimethylarsinic acid (DMA(V)). The intermediate methyl arsenic metabolites, MMA(III) and DMA(III), were detected in most urine samples from all of the patients when a preservative, diethyldithiocarbomate, was added to the urine samples to stabilize these trivalent arsenic species. The major arsenic species detected in the urine samples from the patients were As(III), MMA(V), and DMA(V), accounting for >95% of the total arsenic excreted. The relative proportions of As(III), As(V), MMA(V), and DMA(V) in urine samples collected 24 h after the injections of As(III) were 27.6 +/- 6.1, 2.8 +/- 2.0, 22.8 +/- 8.1, and 43.7 +/- 13.3%, respectively. The relatively lower fraction of the methylated arsenic species in these APL patients under arsenic treatment as compared with that from the general population exposed to much lower levels of arsenic suggests that the high levels of As(III) inhibit the methylation of arsenic (inhibits the formation of methyl arsenic metabolites). The arsenic species excreted into the urine accounted for 32-65% of the total arsenic injected. These results suggest that other pathways of excretion, such as through the bile, may play an important role in eliminating (removing) arsenic from the human body when challenged by high levels of As(III).     

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.     

The biouptake and toxicity of arsenic species on the green microalga Chlorella salina in seawater

The present study focuses on the biouptake, biotransformations, and toxicity of arsenic species on the marine green alga Chlorella salina in seawater from the Bulgarian Black Sea coast. Exposure to equal concentrations of As(III) or As(V) led to equivalent levels of toxicity and total intracellular arsenic content. Biouptake and toxicity of methylated arsenic species, monomethylarsonate (MMA) and dimethylarsinate (DMA), were approximately three orders of magnitude lower than those for inorganic arsenic species. Seawater enrichment with phosphate (up to 1.3 mg P L(-1)) resulted in a significant reduction of both intracellular As content and toxicity due to As(III) and As(V). In contrast, the toxicity and intracellular content of MMA and DMA were unaffected by the presence of phosphate. We measured the distribution and excretion of intracellular arsenic species, and demonstrated that the release of As(V) and/or As(III), together with the bio-reduction of As(V) and the subsequent methylation of As(III) may be a detoxification mechanism for these algae. The implications of the results with respect to arsenic species bioavailability and toxicity in marine water are further discussed.     

Arsenic species contents at aquaculture farm and in farmed mouthbreeder (Oreochromis mossambicus) in blackfoot disease hyperendemic areas.

A study was conducted to measure the arsenic species in farmed mouthbreeder (Oreochromis mossambicus) and culture ponds in water in blackfoot disease (BFD) hyperendemic areas in Taiwan. The relationships between arsenic species of aquaculture ponds and farmed fish were also explored. Biota samples were extracted with methanol/water (1/1, v/v) using a Soxhlet extraction apparatus. The concentrations of arsenite As (III), arsenate As (V), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) of extracts were measured by high-performance liquid chromatography (HPLC) linked to a hydride generator and atomic absorption spectrometry (HG-AAS). Moreover, arsenobetaine (AB) was analyzed by HPLC linked to ultra violet (UV) and HG-AAS. Concentrations of arsenic species were determined in 68 mouthbreeder (O. mossambicus) samples and 21 culture ponds from Putai and Yichu Townships of Chiayi County and Hsuehchia and Peimen Townships of Tainan County. The mean arsenic levels of culture ponds in Putai, Yichu, Hsuehchia, and Peimen were 75.8, 15.1, 14.4, and 221.0 microg/l, respectively. The water of culture ponds was dominated by As (V). The inorganic arsenic percentage of fish (7.4%) was higher than that reported by other seafood surveys. Except for the MMA and As (III) levels, As (V), DMA, AB, and total arsenic levels in fish significantly increased with inorganic and total arsenic concentrations of the pond water. Inorganic arsenic species are more toxic than methyl arsenic species. Therefore the effect of inorganic arsenic species might result in a greater number of adverse health effects to the general public. It is of importance to evaluate the inorganic arsenic levels of farmed seafood in arsenic-contaminated areas.     

Forced uptake of trivalent and pentavalent methylated and inorganic arsenic and its cyto-/genotoxicity in fibroblasts and hepatoma cells.

Mammals are able to convert inorganic arsenic to mono-, di-, and trimethylated metabolites. In previous studies we have shown that the trivalent organoarsenic compounds are more toxic than their inorganic counterparts and that the toxicity is associated with the cellular uptake of the arsenicals. In the present study, we investigated cyto-/genotoxic effects of the arsenic compounds arsenate [As(i)(V)], arsenite [As(i)(III)], monomethylarsonic acid [MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], dimethylarsinous acid [DMA(III)], and trimethylarsine oxide [TMAO(V)] after an extended exposure time (24 h) and compared the uptake capabilities of fibroblasts (CHO-9 cells: Chinese hamster ovary) used for genotoxicity studies, with those of hepatic cells (Hep G2: hepatoma cell-line). To find out whether the arsenic compounds are bound to membranes or if they are present in the cytosol, the amount of arsenic was measured in whole-cell extracts and in membrane-removed cell extracts by inductively coupled plasma-mass spectrometry (ICP-MS). In addition, we forced the cellular uptake of the arsenic compounds into CHO-9 cells by electroporation and measured the intracellular arsenic concentrations before and after this procedure. Our results show that organic and inorganic arsenicals are taken up to a higher degree by fibroblasts compared to hepatoma cells. The arsenic metabolite DMA(III) was the most membrane permeable species in both cell lines and induced strong genotoxic effects in CHO-9 cells after an exposure time of 24 h. The uptake of all other arsenic species was relatively low (<1% by Hep G2 and <4% by CHO cells), but was dose-dependent. Electroporation increased the intracellular arsenic levels as well as the number of induced MN in CHO-9 cells. With the exception of As(i)(III) and DMA(III) in CHO-9 cells, the tested arsenic compounds were not bound to cell membranes, but were present in the cytosol. This may indicate the existence of DMA(III)-specific exporter proteins as are known for As(i)(III). Our results indicate that the uptake capabilities of arsenic compounds are highly dependent upon the cell type. It may be hypothesized that the arsenic-induced genotoxic effects observed in fibroblasts are due to the high uptake of arsenicals into this cell type. This may explain the high susceptibility of skin fibroblasts to arsenic exposure.