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.     

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).     

Distributions and chemical forms of arsenic after intravenous administration of dimethylarsinic and monomethylarsonic acids to rats

The observed toxicity of arsenic is highly dependent on animal species and differences in metabolism. Rats are one of the most tolerant species, and the metabolic pathway is quite different in some aspects from those of other mammals. The distinct metabolic pathway including the preferential accumulation in red blood cells (RBCs) has been explained, whereby allowing an effective use of rats as an animal model for the arsenic metabolism. In the present study, distributions of arsenic among organs/tissues/body fluids and their chemical forms were studied after intravenous injection of arsenic in the forms of dimethylarsinic (DMA(V)) and monomethylarsonic acids (MMA(V)) to rats. DMA(V) and MMA(V) were mostly excreted into urine immediately after the injection as the intact forms, and both forms were taken up less effectively by organs/tissues than arsenite. The methylated arsenics distributed in organs/tissues were excreted directly into urine and excreted before being redistributed in RBCs. DMA(V) and MMA(V) taken up by the liver were transformed to metabolites not yet identified, accumulated transiently in the liver, and then they disappeared from the liver. The unidentified metabolites were assumed to be transformed from dimethylarsinic acid (DMA(III)) following the consecutive metabolic reactions [MMA(V) --> monomethylarsonous acid (MMA(III)) --> DMA(V) --> DMA(III)]. The unidentified metabolites were excreted not into the bile but into the bloodstream. Injections of DMA(V) and MMA(V) induced a biliary excretion of arsenic but only at 0.2-0.3% of the dose, the arsenic in the bile being their intact free forms.     

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.     

Biotransformation of dimethylarsinic acid in mouse, hamster and man

The metabolism of dimethylarsinic acid (DMA) a common pesticide and the main metabolite of inorganic arsenic in mammals, has been studied in mice, hamsters and man. Mice and hamsters were administered a single dose of 74As-DMA (40 mg As/kg body weight) orally, while a human subject ingested DMA corresponding to 0.1 mg As/kg body weight. Ion exchange chromatography, paper electrophoresis, thin layer chromatography as well as arsine generation--gas chromatography combined with atomic absorption spectrophotometry or mass spectrometry were used to characterize the arsenic metabolites in urine and feces collected over 48 hours after treatment. In mice and hamsters 3.5% and 6.4% of the dose, respectively, were excreted in urine in the form of trimethylarsine oxide (TMAO). No TMAO was found in feces. A DMA-complex was detected in urine and feces. It amounted to about 13% of the dose in mice and 15% in hamsters. About 80-85% of the dose was eliminated in urine and feces in the form of unmetabolized DMA. No demethylation of DMA to inorganic arsenic was observed. In man, about 4% of the dose was excreted in urine as TMAO and about 80% as DMA.     

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.     

Determination of arsenic metabolic complex excreted in human urine after administration of sodium 2,3-dimercapto-1-propane sulfonate

Sodium 2,3-dimercapto-1-propane sulfonate (DMPS) has been used to treat acute arsenic poisoning. Presumably DMPS functions by chelating some arsenic species to increase the excretion of arsenic from the body. However, the excreted complex of DMPS with arsenic has not been detected. Here we describe a DMPS complex with monomethylarsonous acid (MMA(III)), a key trivalent arsenic in the arsenic methylation process, and show the presence of the DMPS-MMA(III) complex in human urine after the administration of DMPS. The DMPS-MMA(III) complex was characterized using electrospray tandem mass spectrometry and determined by using HPLC separation with hydride generation atomic fluorescence detection (HGAFD). The DMPS-MMA(III) complex did not form a volatile hydride with borohydride treatment. On-line digestion with 0.1 M sodium hydroxide following HPLC separation decomposed the DMPS-MMA(III) complex and allowed for the subsequent quantification by hydride generation atomic fluorescence. Arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), MMA(III), and DMPS-MMA(III) complex were analyzed in urine samples from human subjects collected after the ingestion of 300 mg of DMPS. The administration of DMPS resulted in a decrease of the DMA(V) concentration and an increase of the MMA(V) concentration excreted in the urine, confirming the previous results. The finding of the DMPS-MMA(III) complex in human urine after DMPS treatment provides an explanation for the inhibition of arsenic methylation by DMPS. Because MMA(III) is the substrate for the biomethylation of arsenic from MMA(V) to DMA(V), the formation of DMPS-MMA(III) complex would reduce the availability of MMA(III) for the subsequent biomethylation.     

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.     

DMPS-arsenic challenge test. II. Modulation of arsenic species, including monomethylarsonous acid (MMA(III)), excreted in human urine

The administration of sodium 2,3-dimercapto-1-propane sulfonate (DMPS) to humans chronically exposed to inorganic arsenic in their drinking water resulted in the increased urinary excretion of arsenic, the appearance and identification of monomethylarsonous acid (MMA(III)) in their urine, and a large decrease in the concentration and percentage of urinary dimethylarsinic acid (DMA). This is the first time that MMA(III) has been detected in the urine. In vitro biochemical experiments were then designed and performed to understand the urinary appearance of MMA(III) and decrease of DMA. The DMPS-MMA(III) complex was not active as a substrate for the MMA(III) methyltransferase. The experimental results support the hypothesis that DMPS competes with endogenous ligands for MMA(III), forming a DMPS-MMA complex that is readily excreted in the urine and points out the need for studying the biochemical toxicology of MMA(III). It should be emphasized that MMA(III) was excreted in the urine only after DMPS administration. The results of these studies raise many questions about the potential central role of MMA(III) in the toxicity of inorganic arsenic and to the potential involvement of MMA(III) in the little-understood etiology of hyperkeratosis, hyperpigmentation, and cancer that can result from chronic inorganic arsenic exposure.     

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.     

Arsenic metabolites in urine and feces of hamsters pretreated with PCB

High pressure liquid chromatography and graphite tube atomizing atomic absorption spectrometry were used to quantify monomethyl arsonic acid (MMA), dimethyl arsinic acid (DMA), and inorganic arsenic (IA: arsenite plus arsenate) in the urine and feces of male and female hamsters pretreated with a single ip injection of PCB (100 mg/kg) and 4 days later given a single po dose of arsenite (10 mg As/kg). Approximately 17 to 23% and 35 to 63% of the arsenic given was eliminated in the urine and feces, respectively, during the 5 days after the administration of arsenic. Both DMA and MMA were found in the urine but only MMA was detected in the feces, as methylated metabolite. Fecal excretion of arsenic was significantly larger in female than in males. PCB influenced the metabolism of arsenic by significantly increasing the proportion of DMA excreted into the urine of female hamsters during the 5 days after the arsenic administration, but did not alter the total amount of arsenic metabolites in any group of male or female hamsters. PCB did not affect the cumulative amounts of fecal arsenic in any group, although the excretion in the PCB-treated group of females reached the maximum level 1 day earlier than in the controls. These results suggest that the metabolism of arsenic may be regulated by certain sex-relating factors which are influenced by PCB.     

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.     

DOSE-DEPENDENT EFFECTS ON THE DISPOSITION OF MONOMETHYLARSONIC ACID AND DIMETHYLARSINIC ACID IN THE MOUSE AFTER INTRAVENOUS ADMINISTRATION

The organic arsenicals monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) are the primary metabolites of inorganic arsenic, a known human carcinogen. The objective of this study was to examine if dose would affect the excretion and terminal tissue disposition of MMA and DMA in the mouse. 14C-MMA (4.84 and 484 mumol/ kg) and-DMA ( 8.04 and 804 mumol/kg) were administered to female micevia the tail vein. The mice were placed in metabolism cages for collection of urine (1, 2, 4, 8, 12, and 24 h) and feces ( 24 h) . The animals were then sacrificed at 24 h and tissues were removed and analyzed for radioactivity. The urine was also analyzed for parent compound and metabolites. Urinary excretion of MMA- and DMA-derived radioactivity predominated over fecal excretion. Dose did not affect the overall urinary excretion of both compounds. However, fecal excretion was significantly lower in the low-dose MMA-treated animals as opposed to in the high-dose group, whereas in the high-dose DMA-treated group excretion was lower than in the low-dose DMA group. The retention of radioactivity was low ( &lt;2% of dose) and the distribution pattern similar for both compounds, with carcass &gt; liver &gt;kidney &gt; lung. The concentration of radioactivity (% dose/ g tissue) was greater in kidney than in liver, lung, and blood for both compounds. The distribution and concentration of MMA-derived radioactivity was significantly greater in the liver and lung of the high-dose group. The MMA-treated animals excreted predominantly MMA in urine and lower amounts of DMA (&lt;10% of the dose). The percentage excreted as DMA was significantly higher in the low-dose MM A group. In the urine of DMA-treated anim als, an unstable metabolite and the parent compound were detected. Overall, it appears the dose of organic arsenical administered has a minimal effect on its excretion and terminal tissue disposition in the mouse. The rapid elimination and low retention of MMA and DMA explain in part their low acute toxicity.     

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.     

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.     

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.     

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.     

Role of metabolism in arsenic toxicity

In humans, as in most mammalian species, inorganic arsenic is methylated to methylarsonic acid (MMA) and dimethylarsinic acid (DMA) by alternating reduction of pentavalent arsenic to trivalent and addition of a methyl group from S-adenosylmethionine. The methylation of inorganic arsenic may be considered a detoxification mechanism, as the end metabolites, MMA and DMA, are less reactive with tissue constituents, less toxic, and more readily excreted in the urine than is inorganic arsenic, especially the trivalent form (AsIII, arsenite). The latter is highly reactive with tissue components, due to its strong affinity for sulfhydryl groups. Thus, following exposure to AsV the first step in the biotransformation, i.e. the reduction to AsIII, may be considered a bioactivation. Also, reactive intermediate metabolites of high toxicity, mainly MMAIII, may be formed and distributed to tissues. Low levels of MMAIII and DMAIII have been detected in urine of individuals chronically exposed to inorganic arsenic via drinking water. However, the contribution of MMAIIIand DMAIII to the toxicity observed after intake of inorganic arsenic by humans remains to be elucidated. The major route of excretion of arsenic is via the kidneys. Evaluation of the methylation of arsenic is mainly based on the relative amounts of the different metabolites in urine. On average human urine contains 10-30% inorganic arsenic, 10-20% MMA and 60-80% DMA.     

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.