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 highthroughput 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.

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 As(III)-species are generated in a mixture of Tris-HCl (pH 6) and sodium borohydride. For generation of arsines from both As(III)- and As(V)-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 As(III)- and As(V)-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.

Biotransformation of inorganic arsenic (iAs) involves methylation catalyzed by arsenic (+ 3 oxidation state) methyltransferase (As3mt) yielding mono-, di-, and trimethylated arsenicals. To investigate the evolution of molecular mechanisms that mediate arsenic biotransformation, a comparative genomic approach focusing on the invertebrate chordate Ciona intestinalis was used. Bioinformatic analyses identified an As3mt gene in the C. intestinalis genome. Constitutive As3mt RNA expression was observed in heart, branchial sac, and gastrointestinal tract. Adult animals were exposed to 0 or 1 ppm of iAs for 1 or 5 days. Steady-state As3mt RNA expression in the gastrointestinal tract was not modulated significantly by 5 days of exposure to iAs. Tissue levels of iAs and its methylated metabolites were determined by hydride generation-cryotrapping-gas chromatography-atomic absorption spectrometry. At either time point, exposure to iAs significantly increased concentrations of iAs and its methylated metabolites in tissues. After 5 days of exposure, total speciated arsenic concentrations were highest in branchial sac (3705 ng/g), followed by heart (1019 ng/g) and gastrointestinal tract (835 ng/g). At this time point, the sum of the speciated arsenical concentrations in gastrointestinal tract and heart equaled or exceeded that of iAs; in branchial sac, iAs was the predominant species present. Ciona intestinalis metabolizes iAs to its methylated metabolites, which are retained in tissues. This metabolic pattern is consistent with the presence of an As3mt ortholog in its genome and constitutive expression of the gene in prominent organs, making this basal chordate a useful model to examine the evolution of arsenic detoxification.

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Trivalent arsenic [As(III)] is a well-known environmental toxicant that causes a wide range of organ-specific diseases and cancers. In the human liver, As(III) promotes vascular remodeling, portal fibrosis, and hypertension, but the pathogenesis of these As(III)-induced vascular changes is unknown. To investigate the hypothesis that As(III) targets the hepatic endothelium to initiate pathogenic change, mice were exposed to 0 or 250 parts per billion (ppb) of As(III) in their drinking water for 5 weeks. Arsenic(III) exposure did not affect the overall health of the animals, the general structure of the liver, or hepatocyte morphology. There was no change in the total tissue arsenic levels, indicating that arsenic does not accumulate in the liver at this level of exposure. However, there was significant vascular remodeling with increased sinusoidal endothelial cell (SEC) capillarization, vascularization of the peribiliary vascular plexus (PBVP), and constriction of hepatic arterioles in As(III)-exposed mice. In addition to ultrastructural demonstration of SEC defenestration and capillarization, quantitative immunofluorescence analysis revealed increased sinusoidal PECAM-1 and laminin-1 protein expression, suggesting gain of adherens junctions and a basement membrane. Conversion of SECs to a capillarized, dedifferentiated endothelium was confirmed at the cellular level with demonstration of increased caveolin-1 expression and SEC caveolae, as well as increased membrane-bound Rac1-GTPase. Conclusion: These data demonstrate that exposure to As(III) causes functional changes in SEC signaling for sinusoidal capillarization that may be initial events in pathogenic changes in the liver. T he vascular effects of arsenic are a global public health concern that contribute to disease in tens of millions of people worldwide. 1 Whereas the role of environmental contaminants in the etiology of vascular diseases and in the vascular contributions to organ dysfunction remains poorly defined, epidemiological studies have associated As(III) exposures to increased risk of cardiovascular diseases 1 and vascular contributions to liver disease. 2 Liver effects associated with arsenic in drinking water include noncirrhotic portal fibrosis and, to a lesser extent, portal hypertension. 2,3 These pathologic conditions involve increased vascular channels in the portal regions of the liver. Higher levels of chronic As(III) consumption increase urinary levels of porphyrins, a biomarker for liver injury, which are more pronounced in people under 20 years of age. 4 In addition, cardiac and liver disorders are the major side effects of therapeutic As(III) regimes that treat leukemias. 5 Despite epidemiological evidence that the liver vasculature is a pathogenic target of chronic As(III) ingestion, 2 the direct effects of As(III) on liver vascular cells are unknown.In other vascular beds and isolated cell cultures, As(III) affects both endothelial and smooth muscle cell physiology. Arsenic(III) stimulates angiogenic processes in cultured endotheli...