Methylated arsenic species in plants originate from soil microorganisms

? Inorganic arsenic (iAs) is a ubiquitous human carcinogen, and rice (Oryza sativa) is the main contributor to iAs in the diet. Methylated pentavalent As species are less toxic and are routinely found in plants; however, it is currently unknown whether plants are able to methylate As. ? Rice, tomato (Solanum lycopersicum) and red clover (Trifolium pratense) were exposed to iAs, monomethylarsonic acid (MMA(V)), or dimethylarsinic acid (DMA(V)), under axenic conditions. Rice seedlings were also grown in two soils under nonsterile flooded conditions, and rice plants exposed to arsenite or DMA(V) were grown to maturity in nonsterile hydroponic culture. Arsenic speciation in samples was determined by HPLC-ICP-MS. ? Methylated arsenicals were not found in the three plant species exposed to iAs under axenic conditions. Axenically grown rice was able to take up MMA(V) or DMA(V), and reduce MMA(V) to MMA(III) but not convert it to DMA(V). Methylated As was detected in the shoots of soil-grown rice, and in rice grain from nonsterile hydroponic culture. GeoChip analysis of microbial genes in a Bangladeshi paddy soil showed the presence of the microbial As methyltransferase gene arsM. ? Our results suggest that plants are unable to methylate iAs, and instead take up methylated As produced by microorganisms.     

Determination of trivalent methylated arsenicals in rat urine by liquid chromatography-inductively coupled plasma mass spectrometry after solvent extraction

A method for the determination of trivalent arsenicals in urine was examined. Trivalent arsenicals, extracted as complexes with diethylammonium diethyldithiocarbamate (DDDC) into carbon tetrachloride, were determined by liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS). The trivalent methylated arsenicals monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), and trimethylarsine (TMA) were detected in urine of rats that had received dimethylarsinic acid (DMA(V)) or monomethylarsonic acid (MMA(V)) at concentration of 200 microg ml(-1) in drinking water for 24 weeks. This method is the first to permit quantification of trivalent methylated arsenicals in urine without significant changes in concentration during storage or pretreatment.     

Generation of thioarsenicals is dependent on the enterohepatic circulation in rats

Three minor sulfur-containing arsenic metabolites: monomethylmonothioarsonic acid (MMMTA(V)), dimethylmonothioarsinic acid (DMMTA(V)), and dimethyldithioarsinic acid (DMDTA(V)) were recently found in human and animal urine after exposure to inorganic arsenic. However, it remains unclear how the thioarsenicals are formed in the body and then excreted into the urine. It is hypothesized that the generation of thioarsenicals occurs during enterohepatic circulation. To address this hypothesis, male Sprague Dawley (SD) rats and Eisai hyperbilirubinuric (EHB) rats (with deficiency of multidrug resistance-associated protein 2) were orally administered a single dose of inorganic arsenite (iAs(III)) at 3.0 mg kg(-1) of body weight. Five hours after dosing, less than 1.0% of the dose was recovered in the bile of EHB rats, while more than 27% of the dose was recovered in the bile of SD rats, with the majority being monomethylarsinodiglutathione [MMA(SG)(2)] with a small amount of arsenic triglutathione [iAs(SG)(3)]. During the early time periods (3 h and 6 h) the arsenic levels in the liver, red blood cells (RBCs) and plasma of EHB rats were higher than those of SD rats, and approximately 76% and 87% of the dose was recovered in the RBCs of SD and EHB rats, respectively, at day 5 after dosing. However, there were no significant differences in arsenic concentration in urine between the two types of animal. Regarding the arsenic species in the urine of both types of rat, significant levels of thiolated arsenicals MMMTA(V) and DMMTA(V) were detected in SD rat urine, however in EHB rat urine only low levels of DMMTA(V) were detected. The present result of the metabolic balance and speciation study suggests that the formation of MMMTA(V) and DMMTA(V) in rats is dependent on enterohepatic circulation. In addition, in vitro experiments indicated that arsenicals excreted from bile may be transformed by gastrointestinal microbiota into MMMTA(V) and DMMTA(V), which are then absorbed into the bloodstream and finally excreted into the urine.     

Impact of arsenite and its methylated metabolites on PARP-1 activity, PARP-1 gene expression and poly(ADP-ribosyl)ation in cultured human cells

The underlying mechanisms of arsenic carcinogenicity are still not fully understood. Mechanisms currently discussed include the induction of oxidative DNA damage and the interference with DNA repair pathways. Still unclear is the role of biomethylation, which has long been considered to be one major detoxification process. Methylated arsenicals have recently been shown to interfere with DNA repair in cellular and subcellular systems, but up to now no DNA repair protein has been identified being particular sensitive towards methylated arsenicals in cultured cells. Here we report that the trivalent methylated metabolites MMA(III) and DMA(III) inhibit poly(ADP-ribosyl)ation in cultured human HeLa S3 cells at concentrations as low as 1nM, thereby showing for the first time an inactivation of an enzymatic reaction related to DNA repair by the trivalent methylated arsenicals at very low environmentally relevant concentrations. In contrast the pentavalent metabolites MMA(V) and DMA(V) showed no such effects up to high micromolar concentrations. All investigated arsenicals did not alter gene expression of PARP-1. However, all trivalent arsenicals were able to inhibit the activity of isolated PARP-1, indicating that the observed decrease in poly(ADP-ribosyl)ation in cultures human cells, predominantly mediated by PARP-1, is likely due to changes in the activity of PARP-1. Since poly(ADP-ribosyl)ation plays a major role in DNA repair, cell cycle control and thus in the maintenance of genomic stability, these findings could in part explain DNA repair inhibition and the genotoxic and carcinogenic effects of arsenic.     

Arsenic in the human food chain,biotransformation and toxicology – Review focusing on seafood arsenic

Fish and seafood are main contributors of arsenic (As) in the diet. The dominating arsenical is the organoarsenical arsenobetaine (AB), found particularly in finfish. Algae, blue mussels and other filter feeders contain less AB, but more arsenosugars and relatively more inorganic arsenic (iAs), whereas fatty fish contain more arsenolipids. Other compounds present in smaller amounts in seafood include trimethylarsine oxide (TMAO), trimethylarsoniopropionate (TMAP), dimethylarsenate (DMA), methylarsenate (MA) and sulfur-containing arsenicals. The toxic and carcinogenic arsenical iAs is biotransformed in humans and excreted in urine as the carcinogens dimethylarsinate (DMA) and methylarsonate (MA), producing reactive intermediates in the process. Less is known about the biotransformation of organoarsenicals, but new insight indicates that bioconversion of arsenosugars and arsenolipids in seafood results in urinary excretion of DMA, possibly also producing reactive trivalent arsenic intermediates. Recent findings also indicate that the pre-systematic metabolism by colon microbiota play an important role for human metabolism of arsenicals. Processing of seafood may also result in transformation of arsenicals.     

Method for the determination of five toxicologically relevant arsenic species in human urine by liquid chromatography-hydride generation atomic absorption spectrometry

An analytical method for the simultaneous quantitation of arseneous acid (As(III)), arsenic acid (As(V)), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and trimethylarsine oxide (TMAO) in human urine by coupling of high-performance liquid chromatography with hydride generation atomic absorption spectrometry (HPLC/HG-AAS) via a flow-injection interface is presented. After arsenic species separation by anion-exchange displacement chromatography the compounds are on-line reduced to their corresponding hydrides and detected by atomic absorption spectrometry. Detection limits range from 1.1 (TMAO) to 2.6 microg/L (As(V)). The method has been applied to determine arsenic species in the urine of a volunteer before and after consumption of seafood as well as to analyse certified reference urine samples for their arsenic species content.     

Effects of arsenic exposure among semiconductor workers: a cautionary note on urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine

Arsenic is a notorious environmental toxicant and was found to cause oxidative stress in cultured cells and animals. However, little work has been done in human studies, especially for the population occupationally exposed to arsenic. In order to investigate the effect of occupational exposure to arsenic in oxidative stress, we measured urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) from 90 semiconductor workers including 50 exposed and 40 nonexposed subjects. A highly sensitive and specific isotope dilution LC-MS/MS method was used for quantification of 8-oxodGuo. The levels of inorganic arsenic (iAs3+, iAs5+), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) in urine were determined by high-performance liquid chromatography-flow injection atomic absorption spectrometry (HPLC-FIAAS). Results showed that the mean urinary concentrations of total arsenic and 8-oxodGuo were significantly higher for exposed workers compared with the nonexposed workers. In addition, elevated urinary 8-oxodGuo concentrations of exposed workers were correlated with urinary levels of MMA (r = 0.44, P < 0.005) and the extent of primary methylation (the ratio of MMA to inorganic arsenic) (r = 0.40, P < 0.005). These findings suggested that occupational exposure to arsenic could result in the induction of oxidative stress. The presence and/or formation of MMA could play an important role in arsenic-involved injuries.     

Arsenic speciation in turnip as affected by application of chicken manure bearing roxarsone and its metabolites

Roxarsone (ROX) is widely used as a feed additive in intensive animal production. While animals are fed with ROX, the most commonly detectable As forms in fresh manures include ROX and small quantities of its metabolites such as arsenate (As(V)), arsenite (As(III)), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). A pot experiment was conducted to investigate the uptake, translocation and distribution of ROX, As(V), As(III), MMA and DMA in turnips, with the soil amended with 2% and 3% (w/w) chicken manure (CM) bearing ROX and its metabolites. Soil without any fertilizer was the control. The results show that only As(V) and As(III) were detected in turnip control samples. As(V), As(III) and DMA were found in all CM applied samples, but not ROX or MMA. This implies that turnip cannot take up ROX directly and accumulate MMA at detectable levels. The contents of DMA in tubers and the three As species in shoots increased with the CM rate in contrast to reduced levels of As(V) and As(III) in tubers. Increased CM rate enhanced the translocation of the three As species, especially for DMA, from tubers to shoots. DMA was the major form (42.9–61.4% in tubers and 38.1–76.3% in shoots), followed by As(III), in turnip plants fertilized with CM. The results indicate that ROX and its metabolites in animal manures can be introduced into human food chain by the way ROX → animal → manure → soil → crop.     

Alternative splicing variants of human arsenic (+3 oxidation state) methyltransferase

Arsenic (+3 oxidation state) methyltransferase (As3MT) catalyzes the methylation of trivalent arsenic (As(III)) to monomethylarsonate (MMA(V)) and dimethylarsinic acid (DMA(V)), and plays an important role in the detoxification of arsenicals. Here, we report the identification of two splicing variants of the human As3MT gene. One splicing variant was an exon-3 skipping (Δ3) form which produced a premature stop codon, and the other was an exon-4 and -5 skipping (Δ4,5) form which produced a 31.1 kDa As3MT protein. In addition to the full-length mRNA of As3MT, Δ4,5 mRNAs were detected in HepG2, A549, HL60, K562, and HEK293 cells. The methyltransferase activity of the recombinant Δ4,5 As3MT and wild-type As3MT proteins purified from Escherichia coli was determined. Speciation analysis by HPLC–ICP-MS showed a clear peak of MMA(V) after incubation of As(III) with the wild-type As3MT protein, but not with the Δ4,5 As3MT protein. In addition, COS-7 cells transfected with Δ4,5 As3MT cDNA did not convert As(III) to MMA(V) or DMA(V). The lack of methyltransferase activity of Δ4,5 As3MT seems to be related to the deletion of an S-adenosylmethionine-binding site and a critical cysteine residue. These data suggest that the expression pattern of splicing variants of the As3MT gene may affect the capacity for arsenic methylation in cells.     

Individual variations in arsenic metabolism in Vietnamese: the association with arsenic exposure and GSTP1 genetic polymorphism

We investigated the association of As exposure and genetic polymorphism in glutathione S-transferase π1 (GSTP1) with As metabolism in 190 local residents from the As contaminated groundwater areas in the Red River Delta, Vietnam. Total As concentrations in groundwater ranged from <0.1 to 502 μg l(-1). Concentrations of dimethylarsinic acid (DMA(V)), monomethylarsonic acid (MMA(V)), and arsenite (As(III)) in human urine were positively correlated with total As levels in the groundwater, suggesting that people in these areas may be exposed to As through the groundwater. The concentration ratios of urinary As(III)/arsenate (As(V)) and MMA(V)/inorganic As (IA; As(III) + As(V))(M/I), which are indicators of As metabolism, increased with the urinary As level. Concentration and proportion of As(III) were high in the wild type of GSTP1 Ile105Val compared with the hetero type, and these trends were more pronounced in the higher As exposure group (>56 μg l(-1) creatinine in urine), but not in the lower exposure group. In the high As exposure group, As(III)/As(V) ratios in the urine of wild type of GSTP1 Ile105Val were significantly higher than those of the hetero type, while the opposite trend was observed for M/I. These results suggest that the excretion and metabolism of IA may depend on both the As exposure level and the GSTP1 Ile105Val genotype.     

A simple and fast detection technique for arsenic speciation based on high‐efficiency photooxidation and gas‐phase chemiluminescence detection

High‐efficiency photooxidation (HEPO) and gas phase chemiluminescence detection (CL) combined with high‐performance liquid chromatography (HPLC) and hydride generation were developed for speciation of As(III), As(V), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). After chromatography separation, the arsenic species were passed through HEPO which performed efficient photooxidation and converted MMA and DMA to As(V) in several seconds. Then the reaction of ozone and arsine upon hydride generation produced a CL signal as the analytical parameter. The total analytical process was completed within 10 min. The effects of operational parameters such as the concentrations of hydrochloric acid and NaBH₄ solution, carrier gas flow and air gas flow for ozone generation were investigated. Detection limits were 3.7, 10.3, 10.2 and 10.0 µg/L for As(III), As(V), MMA and DMA, respectively. The recoveries of the four arsenic species in human urine sample ranged from 87 to 94%. Copyright © 2009 John Wiley & Sons, Ltd.     

Arsenite and its biomethylated metabolites interfere with the formation and repair of stable BPDE-induced DNA adducts in human cells and impair XPAzf and Fpg

The underlying mechanisms of arsenic carcinogenicity are only poorly understood and especially the role of biomethylation is still a matter of debate. Besides the induction of oxidative DNA damage the interference with DNA repair processes have been proposed to contribute to arsenic-induced carcinogenicity. Within the present study the effects of arsenite and its mono- and dimethylated trivalent and pentavalent metabolites on BPDE-induced DNA adduct formation and repair has been investigated and compared in cultured human lung cells. Whereas only arsenite and MMA(III) increased BPDE-DNA adduct formation, arsenite (>/=5 microM), the trivalent (>/=2.5 microM) and the pentavalent (>/=250 microM) metabolites diminished their repair at non-cytotoxic concentrations. As potential molecular targets, interactions with the zinc finger domain of the human XPA protein (XPAzf) and the Escherichia coli zinc finger protein Fpg, involved in NER and BER, respectively, have been investigated. All trivalent arsenicals were able to release zinc from XPAzf; furthermore, MMA(III) and DMA(III) inhibited the activity of isolated Fpg. Altogether the results suggest that besides arsenite, especially the trivalent methylated metabolites may contribute to diminished NER at low concentrations.     

Induction of structural and numerical changes of chromosome, centrosome abnormality, multipolar spindles and multipolar division in cultured Chinese hamster V79 cells by exposure to a trivalent dimethylarsenic compound

Dimethylarsine iodide (DMI) was used as a model compound of trivalent dimethylarsenicals [DMA(III)], and the biological effects were extensively investigated in cultured Chinese hamster V79 cells. When the cytotoxic effects of DMA(III) were compared with those of inorganic arsenite and dimethylarsinic acid [DMA(V)], DMA(III) was about 10,000 times more potent than DMA(V), and it was even 10 times more toxic than arsenite. Depletion of cell glutathione (GSH) did not influence the cytotoxic effects of DMA(III), whereas it enhanced the cytotoxicity of arsenite. Chromosome structural aberrations, such as gaps, breaks and pulverizations, and numerical changes, such as aneuploidy, hyper- and hypo-tetraploidy, were induced by DMA(III) in a concentration-dependent manner. Mitotic index increased 9-12h after the addition of DMA(III), and then declined. By contrast, the incidence of multinucleated cells increased conversely with the decrease in mitotic index at and after 24h of exposure. The mitotic cell-specific abnormality of centrosome integrity and multipolar spindles were induced by DMA(III) in a time- and concentration-dependent manner. Moreover, DMA(III) caused abnormal cytokinesis (multipolar division) at concentrations that were effective in causing centrosome abnormality, multipolar spindles and aneuploidy. These results showed that DMA(III) was genotoxic on cultured mammalian cells. Results also suggest that DMA(III)-induced multipolar spindles and multipolar division may be associated with the induction of aneuploidy. In addition, the centrosome may be a primary target for cell death via multinucleated cells.     

Exceptions in patterns of arsenic compounds in urine of acute promyelocytic leukaemia patients treated with As<Subscript>2</Subscript>O<Subscript>3</Subscript>

Arsenic trioxide (As(III) in solution) has been shown to be the most active single agent in combating acute promyelocytic leukemia (APL). It is metabolized and excreted via urine as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and As(V), along with excess As(III). In our study eight APL patients were treated (intravenously) with 0.15 mg As₂O₃/kg/day. During the therapy As(III) and its metabolites were followed in pre- and post-infusion urine using HPLC for separation followed by on-line detection using hydride generation-atomic fluorescence spectrometry. Five patients had a normal excretion pattern of residual arsenic compounds in morning pre-infusion urine, with 15–25 % of As(III), 35–55 % of DMA, 25–30 % of MMA and 1–5 % of As(V), while three patients showed unexpected exceptions from typical excretion patterns of arsenic compounds (i) a high DMA/MMA ratio (factor 5.3), (ii) severe As(III) oxidation (10.2 % As(III) converted to As(V)) or (iii) the presence of an excessive amount of As(III) (average 30.4 % of total arsenic). Intriguing was the occurrence of post-infusion oxidation of As(III) to As(V) observed in almost all patients and being especially high (>40 %) in patient with increased residual As(V). Results indicate that arsenic metabolites patterns can be unpredictable. Observed high levels of un-metabolised As(III) are a warning signal for side effects and for routine determination of arsenic metabolites during first days of treatment. High or low percentages of MMA or DMA did not show any observable effect on treatment results, while clear presence of post-infusion As(V) supports theoretical claims of in vivo oxidation (detoxification) of As(III) to As(V) associated with various metabolic processes.     

淹水条件对土壤砷形态转化的影响

通过淹水条件下的培养试验, 探讨了外源二甲基砷酸（DMA）、一甲基砷酸（MMA）、砷酸盐［As(V)］在土壤中的动态转化规律. 结果表明: 随着培养时间的推移, 加入土壤中的DMA和MMA均主要转化为As(V), 且土壤中As(V)含量均呈增加趋势, 培养到150 d时土壤中As(V)含量均显著高于1 d时的含量(P<0.01). 外源DMA通过脱甲基化作用, 在30 d内即基本转化为As(V), 且有少量的亚砷酸盐［As(Ⅲ)］生成; 而外源MMA的转化速度相对较慢, 培养60 d后才基本完成向As(V)的转化, 同时伴随少量DMA和As(Ⅲ)的生成; 在淹水条件下外源As(V)含量随培养时间的增加而逐渐降低,该过程中除有少量As(Ⅲ)生成外,其形态基本未发生改变.     

Arsenicals affect base excision repair by several mechanisms

Inorganic arsenic is a strong, widespread human carcinogen. How exactly inorganic arsenic exerts carcinogenicity in humans is as yet unclear, but it is thought to be closely related to its metabolism. At exposure-relevant concentrations arsenic is neither directly DNA reactive nor mutagenic. Thus, more likely epigenetic and indirect genotoxic effects, among others a modulation of the cellular DNA damage response and DNA repair, are important molecular mechanisms contributing to its carcinogenicity. In the present study, we investigated the impact of arsenic on several base excision repair (BER) key players in cultured human lung cells. For the first time gene expression, protein level and in case of human 8-oxoguanine DNA glycosylase 1 (hOGG1) protein function was examined in one study, comparing inorganic arsenite and its trivalent and pentavalent mono- and dimethylated metabolites, also taking into account their cellular bioavailability. Our data clearly show that arsenite and its metabolites can affect several cellular endpoints related to DNA repair. Thus, cellular OGG activity was most sensitively affected by dimethylarsinic acid (DMA(V)), DNA ligase IIIα (LIGIIIα) protein level by arsenite and X-ray cross complementing protein 1 (XRCC1 protein) content by monomethylarsonic acid (MMA(V)), with significant effects starting at ≥3.2μM cellular arsenic. With respect to MMA(V), to our knowledge these effects are the most sensitive endpoints, related to DNA damage response, that have been identified so far. In contrast to earlier nucleotide excision repair related studies, the trivalent methylated metabolites exerted strong effects on the investigated BER key players only at cytotoxic concentrations. In summary, our data point out that after mixed arsenic species exposure, a realistic scenario after oral inorganic arsenic intake in humans, DNA repair might be affected by different mechanisms and therefore very effectively, which might facilitate the carcinogenic process of inorganic arsenic.     

Kinetics of arsenic methylation by freshly isolated B6C3F1 mouse hepatocytes

The toxic and carcinogenic effects of arsenic may be mediated by both inorganic and methylated arsenic species. The methylation of arsenic(III) is thought to take place via sequential oxidative methylation and reduction steps to form monomethylarsenic (MMA) and dimethylarsenic (DMA) species, but recent evidence indicates that glutathione complexes of arsenic(III) can be methylated without oxidation. The kinetics of arsenic methylation were determined in freshly isolated hepatocytes from male B6C3F1 mice. Hepatocytes (>90% viability) were isolated by collagenase perfusion and suspended in Williams' Medium E with various concentrations of arsenic(III) (sodium m-arsenite). Aliquots of the lysed cell suspension were analyzed for arsenic species by hydride generation-atomic absorption spectrometry. The formation of MMA(III) from sodium arsenite (1 microM) was linear with respect to time for >90 min. DMA(III) formation did not become significant until 60 min. MMA(V) and DMA(V) were not consistently observed in the incubations. These results suggest that the glutathione complex mechanism of methylation plays an important role in arsenic biotransformation in mouse hepatocytes. Metabolism of arsenic(V) was not observed in mouse hepatocytes, consistent with inhibition of arsenic(V) active cellular uptake by phosphate in the medium. The formation of MMA(III) increased with increasing arsenic(III) concentrations up to approximately 2 microM and declined thereafter. The concentration dependence is consistent with a saturable methylation reaction accompanied by uncompetitive substrate inhibition of the reaction by arsenic(III). Kinetic analysis of the data suggested an apparent K(M) of approximately 3.6 microM arsenic(III), an apparent V(max) of approximately 38.9 microg MMA(III) formed/L/h/million cells, and an apparent K(I) of approximately 1.3 microM arsenic(III). The results of this study can be used in the physiologically based pharmacokinetic model for arsenic disposition in mice to predict the concentration of MMA(III) in liver and other tissues.     

In Vitro Evaluation of the Protective Role of Lactobacillus StrainsAgainst Inorganic Arsenic Toxicity

Inorganic arsenic [iAs, As(III) + As(V)] is considered a human carcinogen. Recent studies show that it has also toxic effects on the intestinal epithelium which might partly explain its systemic toxicity. The aim of this study is to evaluate the protective role of lactic acid bacteria (LAB) against the toxic effects of iAs on the intestinal epithelium. For this purpose, the human colonic cells Caco-2 were exposed to As(III) in the presence of various LAB strains or their conditioned medium. Results showed that some strains and their conditioned media partially revert the oxidative stress, the production of pro-inflammatory cytokines, the alterations of the distribution of tight junction proteins, and the cell permeability increases caused by As(III). These results show that both soluble factors secreted or resulting from LAB metabolism and cell-cell interactions are possibly involved in the beneficial effects. Therefore, some LAB strains have potential as protective agents against iAs intestinal barrier disruption.

     

Dose dependent changes in 74As-arsenate metabolism of Flemish Giant rabbits.

The metabolic handling of 74As-arsenate (As(V)) was studied in rabbits injected intraperitoneally (i.p.) with increasing doses of As(V) (0.00 to 1.00 mg As(V)/kg/day) over a period of 10 days. Plasma, packed cells, urine from the bladder and several tissues were analyzed for their 74As content and presence of 74As-As(V) metabolites 4 h after administration of 74As-As(V). 74As showed strong increases with increasing As(V) dose in nails and bone whereas in fat, thyroid and kidneys it decreased. Also with increasing As(V) dose, arsenate was less efficiently methylated to dimethylarsenic acid (DMA) and became more bound to insoluble tissue constituents. As a result 74As-DMA levels in tissues were systematically lower in the groups of rabbits receiving the higher doses, be it with a wide variation from one type of tissue to the other. The behaviour of 74As-monomethylarsonic acid (MMA) was different. The levels did not decrease significantly, occasionally even increased compared to the control group, indicating that especially the second methylation step is sensitive towards increasing doses of As(V). 74As-arsenite (As(III)), formed by in vivo reduction of As(V), reached maximal levels in the 0.25 mg As(V)/kg/day group as a result of the inhibited methylation. At doses > 0.25 mg As(V)/kg/day the amount of 74As-As(V) increased especially in plasma, packed cells and the urine in the bladder, indicative for a less efficient reduction of As(V).