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

Exposure to monomethylarsonic acid (MMA(V)) and monomethylarsonous acid (MMA(III)) can result from their formation as metabolites of inorganic arsenic and by the use of the sodium salts of MMA(V) as herbicides. This study compared the disposition of MMA(V) and MMA(III) in adult female B6C3F1 mice. Mice were gavaged po with MMA(V), either unlabeled or labeled with 14C at two dose levels (0.4 or 40 mg As/kg). Other mice were dosed po with unlabeled MMA(III) at one dose level (0.4 mg As/kg). Mice were housed in metabolism cages for collection of excreta and sacrificed serially over 24 h for collection of tissues. MMA(V)-derived radioactivity was rapidly absorbed, distributed and excreted. By 8 h post-exposure, 80% of both doses of MMA(V) were eliminated in urine and feces. Absorption of MMA(V) was dose dependent; that is, there was less than a 100-fold difference between the two dose levels in the area under the curves for the concentration-time profiles of arsenic in blood and major organs. In addition, urinary excretion of MMA(V)-derived radioactivity in the low dose group was significantly greater (P < 0.05) than in the high dose group. Conversely, fecal excretion of MMA(V)-derived radioactivity was significantly greater (P < 0.05) in the high dose group than in the low dose group. Speciation of arsenic by hydride generation-atomic absorption spectrometry in urine and tissues of mice administered MMA(V) or MMA(III) found that methylation of MMA(V) was limited while the methylation of MMA(III) was extensive. Less than 10% of the dose excreted in urine of MMA(V)-treated mice was in the form of methylated products, whereas it was greater than 90% for MMA(III)-treated mice. In MMA(V)-treated mice, 25% or less of the tissue arsenic was in the form of dimethylarsenic, whereas in MMA(III)-treated mice, 75% or more of the tissue arsenic was in the form of dimethylarsenic. Based on urinary analysis, administered dose of MMA(V) did not affect the level of its metabolites excreted. In the tested range, dose affects the absorption, distribution and route of excretion of MMA(V) but not its metabolism

Arsenicals in maternal and fetal mouse tissues after gestational exposure to arsenite

Exposure of pregnant C3H/HeNCR mice to 42.5- or 85-ppm of arsenic as sodium arsenite in drinking water between days 8 and 18 of gestation markedly increases tumor incidence in their offspring. In the work reported here, distribution of inorganic arsenic and its metabolites, methyl arsenic and dimethyl arsenic, were determined in maternal and fetal tissues collected on gestational day 18 of these exposure regimens. Tissues were collected from three females and from associated fetuses exposed to each dosage level. Concentrations of total speciated arsenic (sum of inorganic, methyl, and dimethyl arsenic) were higher in maternal tissues than in placenta and fetal tissues; total speciated arsenic concentration in placenta exceeded those in fetal tissues. Significant dosage-dependent (42.5 ppm versus 85 ppm of arsenite in drinking water) differences were found in total speciated arsenic concentrations in maternal lung (p < 0.01) and liver (p < 0.001). Total speciated arsenic concentrations did not differ significantly between dosage levels for maternal blood or for fetal lung, liver, and blood, or for placenta. Percentages of inorganic, methyl, or dimethyl arsenic in maternal or fetal tissues were not dosage-dependent. Over the range of total speciated arsenic concentrations in most maternal and fetal tissues, dimethyl arsenic was the most abundant arsenical. However, in maternal liver at the highest total speciated arsenic concentration, inorganic arsenic was the most abundant arsenical, suggesting that a high tissue burden of arsenic affected formation or retention of methylated species in this organ. Tissue concentration-dependent processes could affect kinetics of transfer of inorganic arsenic or its metabolites from mother to fetus

Examination of the effects of arsenic on glucose homeostasis in cell culture and animal studies: Development of a mouse model for arsenic-induced diabetes

Previous epidemiologic studies found increased prevalences of type 2 diabetes mellitus in populations exposed to high levels of inorganic arsenic (iAs) in drinking water. Although results of epidemiologic studies in low-exposure areas or occupational settings have been inconclusive, laboratory research has shown that exposures to iAs can produce effects that are consistent with type 2 diabetes. The current paper reviews the results of laboratory studies that examined the effects of iAs on glucose metabolism and describes new experiments in which the diabetogenic effects of iAs exposure were reproduced in a mouse model. Here, weanling male C57BL/6 mice drank deionized water with or without the addition of arsenite (25 or 50 ppm As) for 8 weeks. Intraperitoneal glucose tolerance tests revealed impaired glucose tolerance in mice exposed to 50 ppm As, but not to 25 ppm As. Exposure to 25 and 50 ppm As in drinking-water resulted in proportional increases in the concentration of iAs and its metabolites in the liver and in organs targeted by type 2 diabetes, including pancreas, skeletal muscle and adipose tissue. Dimethylarsenic was the predominant form of As in the tissues of mice in both 25 and 50 ppm groups. Notably, the average concentration of total speciated arsenic in livers from mice in the 50 ppm group was comparable to the highest concentration of total arsenic reported in the livers of Bangladeshi residents who had consumed water with an order of magnitude lower level of iAs. These data suggest that mice are less susceptible than humans to the diabetogenic effects of chronic exposure to iAs due to a more efficient clearance of iAs or its metabolites from target tissues

Arsenic (+ 3 oxidation state) methyltransferase and the methylation of arsenicals in the invertebrate chordate Ciona intestinalis

Metabolic conversion of inorganic arsenic into methylated products is a multistep process that yields mono-, di-, and trimethylated arsenicals. In recent years, it has become apparent that formation of methylated metabolites of inorganic arsenic is not necessarily a detoxification process. Intermediates and products formed in this pathway may be more reactive and toxic than inorganic arsenic. Like all metabolic pathways, understanding the pathway for arsenic methylation involves identification of each individual step in the process and the characterization of the molecules which participate in each step. Among several arsenic methyltransferases that have been identified, arsenic (+3 oxidation state) methyltransferase is the one best characterized at the genetic and functional levels. This review focuses on phylogenetic relationships in the deuterostomal lineage for this enzyme and on the relation between genotype for arsenic (+3 oxidation state) methyltransferase and phenotype for conversion of inorganic arsenic to methylated metabolites. Two conceptual models for function of arsenic (+3 oxidation state) methyltransferase which posit different roles for cellular reductants in the conversion of inorganic arsenic to methylated metabolites are compared. Although each model accurately represents some aspects of enzyme’s role in the pathway for arsenic methylation, neither model is a fully satisfactory representation of all the steps in this metabolic pathway. Additional information on the structure and function of the enzyme will be needed to develop a more comprehensive model for this pathway

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 [14C]-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 [14C]-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

Oxidation state specific generation of arsines from methylated arsenicals based on l-cysteine treatment in buffered media for speciation analysis by hydride generation-automated cryotrapping-gas chromatography-atomic absorption spectrometry with the multiatomizer

An automated system for hydride generation - cryotrapping- gas chromatography - atomic absorption spectrometry with the multiatomizer is described. Arsines are preconcentrated and separated in a Chromosorb filled U-tube. An automated cryotrapping unit, employing nitrogen gas formed upon heating in the detection phase for the displacement of the cooling liquid nitrogen, has been developed. The conditions for separation of arsines in a Chromosorb filled U-tube have been optimized. A complete separation of signals from arsine, methylarsine, dimethylarsine, and trimethylarsine has been achieved within a 60 s reading window. The limits of detection for methylated arsenicals tested were 4 ng l−1. Selective hydride generation is applied for the oxidation state specific speciation analysis of inorganic and methylated arsenicals. The arsines are generated either exclusively from trivalent or from both tri- and pentavalent inorganic and methylated arsenicals depending on the presence of L-cysteine as a prereductant and/or reaction modifier. A TRIS buffer reaction medium is proposed to overcome narrow optimum concentration range observed for the L-cysteine modified reaction in HCl medium. The system provides uniform peak area sensitivity for all As species. Consequently, the calibration with a single form of As is possible. This method permits a high-throughput speciation analysis of metabolites of inorganic arsenic in relatively complex biological matrices such as cell culture systems without sample pretreatment, thus preserving the distribution of tri- and pentavalent species

Speciation analysis of arsenic in biological matrices by automated hydride generation-cryotrapping-atomic absorption spectrometry with multiple microflame quartz tube atomizer (multiatomizer)

Analyses of arsenic (As) species in tissues and body fluids of individuals chronically exposed to inorganic arsenic (iAs) provide essential information about the exposure level and pattern of iAs metabolism. We have previously described an oxidation state-specific analysis of As species in biological matrices by hydride-generation atomic absorption spectrometry (HG-AAS), using cryotrapping (CT) for preconcentration and separation of arsines. To improve performance and detection limits of the method, HG and CT steps are automated and a conventional flame-in-tube atomizer replaced with a recently developed multiple microflame quartz tube atomizer (multiatomizer). In this system, arsines from AsIII-species are generated in a mixture of Tris-HCl (pH 6) and sodium borohydride. For generation of arsines from both AsIII- and AsV-species, samples are pretreated with L-cysteine. Under these conditions, dimethylthioarsinic acid, a newly described metabolite of iAs, does not interfere significantly with detection and quantification of methylated trivalent arsenicals. Analytical performance of the automated HG-CT-AAS was characterized by analyses of cultured cells and mouse tissues that contained mono- and dimethylated metabolites of iAs. The capacity to detect methylated AsIII- and AsV-species was verified, using an in vitro methylation system containing recombinant rat arsenic (+3 oxidation state) methyltransferase and cultured rat hepatocytes treated with iAs. Compared with the previous HG-CT-AAS design, detection limits for iAs and its metabolites have improved significantly with the current system, ranging from 8 to 20 pg. Recoveries of As were between 78 and 117%. The precision of the method was better than 5% for all biological matrices examined. Thus, the automated HG-CT-AAS system provides an effective and sensitive tool for analysis of all major human metabolites of iAs in complex biological matrices

Impact of life stage and duration of exposure on arsenic-induced proliferative lesions and neoplasia in C3H mice

Epidemiological studies suggest that chronic exposure to inorganic arsenic is associated with cancer of the skin, urinary bladder and lung as well as the kidney and liver. Previous experimental studies have demonstrated increased incidence of liver, lung, ovary, and uterine tumors in mice exposed to 85 ppm (∼8 mg/kg) inorganic arsenic during gestation. To further characterize age susceptibility to arsenic carcinogenesis we administered 85 ppm inorganic arsenic in drinking water to C3H mice during gestation, prior to pubescence and post-pubescence to compare proliferative lesion and tumor outcomes over a one-year exposure period. Inorganic arsenic significantly increased the incidence of hyperplasia in urinary bladder (48%) and oviduct (36%) in female mice exposed prior to pubescence (beginning on postnatal day 21 and extending through one year) compared to control mice (19 and 5%, respectively). Arsenic also increased the incidence of hyperplasia in urinary bladder (28%) of female mice continuously exposed to arsenic (beginning on gestation day 8 and extending though one year) compared to gestation only exposed mice (0%). In contrast, inorganic arsenic significantly decreased the incidence of tumors in liver (0%) and adrenal glands (0%) of male mice continuously exposed from gestation through one year, as compared to levels in control (30 and 65%, respectively) and gestation only (33 and 55%, respectively) exposed mice. Together, these results suggest that continuous inorganic arsenic exposure at 85 ppm from gestation through one year increases the incidence and severity of urogenital proliferative lesions in female mice and decreases the incidence of liver and adrenal tumors in male mice. The paradoxical nature of these effects may be related to altered lipid metabolism, the effective dose in each target organ, and/or the shorter one-year observational period

High-Throughput Identification of Catalytic Redox-Active Cysteine Residues

Cysteine (Cys) residues often play critical roles in proteins; however, identification of their specific functions has been limited to case-by-case experimental approaches. We developed a procedure for high-throughput identification of catalytic redox-active Cys in proteins by searching for sporadic selenocysteine-Cys pairs in sequence databases. This method is independent of protein family, structure, and taxon. We used it to selectively detect the majority of known proteins with redoxactive Cys and to make additional predictions, one of which was verified. Rapid accumulation of sequence information from genomic and metagenomic projects should allow detection of many additional oxidoreductase families as well as identification of redox-active Cys in these proteins

Supporting Online Material for “High-Throughput Identification of Catalytic Redox-Active Cysteine Residues”

Identification of redox-active Cys by homology to selenoproteins Identification of Cys/Sec pairsStatistical analysis of redox-active Cys neighborhoodsAdoMet-dependent methyltransferaseActivity of AS3MT and its Cys-to-Ser mutant