Biliary Excretion of [(GS)2AsSe]

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Biliary Excretion of [(GS)2AsSe]- after Intravenous Injection of Rabbits with Arsenite and Selenate Ju¨rgen Gailer,* Graham N. George,† Ingrid J. Pickering,† Roger C. Prince,‡ Husam S. Younis,§ and J. J. Winzerling| GSF National Research Center for Environment and Health, Institute for Ecological Chemistry, Ingolsta¨ dter Landstrasse 1, 85764 Neuherberg, Germany, Stanford Synchrotron Radiation Laboratory, SLAC, MS 69, 2575 Sand Hill Road, Menlo Park, California 94025, ExxonMobil Research and Engineering Co., Annandale, New Jersey 08801, Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, and Department of Nutritional Sciences, The University of Arizona, Tucson, Arizona 85721 Received April 3, 2002

It has been shown that the seleno-bis (S-glutathionyl) arsinium ion, [(GS)2AsSe]-, is the major arsenic and selenium excretory product in bile of rabbits treated with arsenite and selenite [Gailer, J., Madden, S., Buttigieg, G. A., Denton, M. B., and Younis, H. S. (2002) Appl. Organomet. Chem. 16, 72-75]. To investigate the in vivo interaction between the other environmentally common oxy-anions of arsenic and selenium in mammals, we have intravenously injected rabbits with different combinations of the arsenic and selenium oxo-anions (arsenite + selenate, arsenate + selenite, and arsenate + selenate) and analyzed the collected bile and whole blood samples by X-ray absorption spectroscopy. Only the injection of arsenite and selenate led to the biliary excretion of [(GS)2AsSe]- within 25 min. Whole blood collected from these animals (25 min postinjection) contained predominantly unchanged selenate, which suggests the presence of a mammalian selenate reductase in the liver. The lack of any significant biliary excretion of [(GS)2AsSe]- in the other treatment groups implies that arsenate was not reduced in the liver on the time scale of our experiments. The relevance of these results for the human toxicology of arsenic and selenium is discussed.

Introduction The interaction of aqueous solutions with mineral surfaces is one of the most important chemical reactions occurring in nature (1, 2). Such reactions play a major role in the dissolution of arsenic and selenium-containing minerals, rocks, and deposits (3, 4) and mobilize the water-soluble oxo-anions arsenite, selenite, arsenate, and selenate to natural waters. Life on earth therefore is, and always has been, exposed to background concentrations of numerous inorganic and organic arsenic and selenium compounds. At some point during the evolution of life selenium was recruited to actively participate in the biochemistry of the cell (5), and it is now an essential trace element for all higher forms of life (6). Since anthropogenic activities release quantities of numerous metals and metalloids into the environment that rival or exceed the natural inputs (7), however, humans are also exposed to elevated concentrations of these toxins compared with the preindustrial age (8). Much effort has been devoted to studying the metabolism of inorganic arsenic (arsenite + arsenate) in mammals. Despite this, the molecular basis for its carcinogenic effect in humans remains unknown (9). An understand* To whom correspondence should be addressed at the GSF National Research Center for Environment and Health. E-mail: [email protected]. † Stanford Synchrotron Radiation Laboratory. ‡ ExxonMobil Research and Engineering Co. § Department of Pharmacology and Toxicology. | Department of Nutritional Sciences.

ing of the molecular interactions between inorganic arsenic and micronutrients (such as selenium) may be important in elucidating the mechanism of carcinogenesis (10). This approach appears promising since it is well established that toxic compounds can interact with other compounds in vivo in antagonistic, additive, or synergistic modes (11). The antagonism between aqueous arsenite and selenite has its molecular basis in the formation of a novel arsenic-selenium compound, the seleno-bis (S-glutathionyl) arsinium ion, [(GS)2AsSe]-. We have shown that this species is formed in vivo and subsequently excreted in bile (12-14). [(GS)2AsSe]- therefore represents a fundamental molecular link between the metabolism of arsenite and selenite in mammals and thus is of considerable toxicological importance (12). Previous work on the individual metabolism of arsenate and selenate in mammals revealed that these compounds are individually reduced in vivo to arsenite (15, 16) and selenite (17). In the present work we address the question of whether [(GS)2AsSe]- will be excreted in bile after the combined iv injection of rabbits with selenite and arsenate, selenate and arsenite, or selenate and arsenate using X-ray fluorescence emission and X-ray absorption spectroscopy.

Experimental Procedures Caution: Inorganic arsenic compounds are established human carcinogens (18). Ingestion of inorganic arsenic may cause cancer of the skin, urinary bladder, kidneys, lungs, and liver, as well as disorders of the circulatory and nervous systems.

10.1021/tx025538s CCC: $22.00 © 2002 American Chemical Society Published on Web 10/24/2002

Biliary Excretion of [(GS)2AsSe]Chemicals. Na2SeO3‚5H2O (>97%) was purchased from Fluka (Buchs, Switzerland), and NaAsO2 (>99%) from GFS Chemicals (Columbus, OH), and Na2HAsO4‚7H2O and Na2SeO4 were from Sigma Chemicals (St. Louis, MO). Sodium hydroxide was purchased from MCB Reagents (Cincinnati, OH), and concentrated hydrochloric acid was obtained from Fischer Chemicals (Pittsburgh, PA). PBS-buffer (pH 7.4) was prepared from dry powder pouches (Sigma) and triply distilled water. Solutions at 80 mM of NaAsO2, Na2SeO3, Na2SeO4, and Na2HAsO4 each were prepared in PBS-buffer and were subsequently adjusted to pH 7.5 with hydrochloric acid. Test Tube Experiment. To study the chemical reaction between GSH and different oxidation states of arsenic and selenium oxo-anions, equimolar mixtures of selenite and arsenate, selenate and arsenite, and selenate and arsenate were reacted with between 1 and 16 M equiv of GSH (in PBS-buffer and adjusted to pH 7.4) at 37 °C. After the addition of the solution containing the metalloid compounds, the test tubes were sealed, mixed, and kept at 37 °C for 24 h. Finally, 100 µL of concentrated HCl was added in order to decompose and therefore visualize a potentially formed arsenic-selenium compound. All experiments were carried out in duplicate. Animal Experiment. The actual animal experiments were conducted on the 14th and 15th of December 2000. Male New Zealand white rabbits (1.35-1.65 kg) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and maintained for 1 week on a “high-fiber” rabbit diet (7015 Harlaw Tekland, Madison, WI). The animals were prepared for the animal experiment as previously reported (12). After a constant bile flow had been established, the metalloid solution(s) corresponding to 2.52 mg of Se/kg of body wt and/or 2.40 mg of As/kg of body wt was/were injected through the marginal ear vein and bile was collected for 25 min into ice-cold polypropylene tubes. Control animals were injected with aqueous solutions of sodium arsenite (one animal), disodium selenite (one animal), disodium selenate (one animal), and disodium hydrogen arsenate (one animal), and only bile samples were collected. The treatment groups were injected with disodium selenite followed 3 min later by disodium hydrogen arsenate (two animals; treatment 1), disodium selenate followed 3 min later by sodium arsenite (two animals; treatment two), and disodium selenate followed 3 min later by disodium hydrogen arsenate (two animals; treatment 3). In addition to bile samples, whole blood samples were collected immediately thereafter into heparinized Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ). Bile and whole blood samples were immediately mixed with glycerol (6:4, v/v) and frozen in liquid nitrogen for subsequent analysis by X-ray absorption spectroscopy. X-ray Absorption and Fluorescence Emission Spectroscopy. The X-ray absorption spectra (XAS) were measured at the Stanford Synchrotron Radiation Laboratory (SSRL) on beamline 7-3 using a Si(220) double-crystal monochromator with an upstream aperture of 1 mm. Harmonic rejection was accomplished by detuning one monochromator crystal to approximately 50% off-peak, and no specular optics were present. X-ray absorption spectra were measured as the X-ray fluorescence excitation spectra using a Canberra 13-element Gedetector. The incident X-ray intensity was monitored using a N2-filled ionization chamber, and energy calibration was performed by simultaneous measurement of a hexagonal Se and an As foil, the lowest energy inflection point of which was assumed to be 12658.0 and 11867.0 eV, respectively. X-ray fluorescence emission spectra for elemental analysis were measured with an incident X-ray energy of 13 400 eV, employing an amplifier Gaussian shaping time of 2 µs for greater resolution, while measurements of X-ray absorption spectra were taken using a shaping time of 0.125 µs. The latter allowed higher count rates, which were always nonsaturating. Samples were maintained at 10 K during data collection using an Oxford Instruments helium cryostat. X-ray absorption spectroscopic data were analyzed using the EXAFSPAK suite of computer programs (http://ssrl.slac.stanford.edu/exafspak.html).

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Figure 1. X-ray absorption fluorescence emission spectra of representative rabbit bile samples. Spectra were from animals treated with arsenite and selenate (a), arsenite alone (b), and selenate alone (c). The rising background on the high energy side of the plot is due to elastic and inelastic X-ray scattering from the sample. Ab initio theoretical extended phase and amplitude functions were calculated using FEFF, version 8.03 (19).

Results Test Tube Experiments. The aqueous chemistry of the interaction between the various oxy-anions of arsenic and selenium was investigated in test tube experiments. When equimolar amounts of selenite and arsenate were reacted with GSH at various stoichiometries, a red-brown precipitate invariably formed immediately. After 45 min the reactions carried out with 5-fold and greater molar equivalents of GSH contained a gray precipitate, and the color of the supernatant turned progressively more orange with increasing amounts of GSH. After 1 h the precipitate of the reaction mixture containing the most GSH (16 M eqiuv) had entirely dissolved, yielding an orange solution. The addition of concentrated HCl to the supernatant produced a red precipitate (presumably R-Se) and a garlic-like odor characteristic of low-valent selenium species (most likely selenides). Preliminary X-ray absorption spectroscopy of the orange solution species (not illustrated) showed pentavalent arsenic and reduced selenium, suggesting the presence of selenoarsenate. In contrast to these results, the addition of equimolar selenate and arsenite to aqueous solutions of GSH yielded colorless solutions, and the addition of concentrated HCl produced neither a color change nor the formation of a precipitate. Equimolar selenate and arsenate behaved similarly when reacted with increasing amounts of GSH. Thus, solutions containing selenate showed no obvious chemistry in the test tube, while those containing selenite did. Quantitation of As and Se in Bile. X-ray fluorescence spectroscopy was used to quantify As and Se in bile collected from rabbits that had been injected with arsenite, arsenate, selenite, selenate, and various combinations of these. Figure 1 shows the results for arsenite, selenate, or both in combination. Bile from rabbits injected with arsenite contained 2.8 ppm As, while bile from those injected with the same dose of arsenate contained approximately 0.3 ppm As. Bile from animals injected with selenite or selenate contained approximately 0.4 and 0.5 ppm Se. Animals treated with both selenite and arsenate and those treated with selenate and arsenate had negligible concentrations of As and Se in their bile (Table 1). Conversely, when selenate and arsenite were injected, substantial concentrations of both metalloids were detected in bile (Figure 1). The first

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Gailer et al. Table 1 bilea

As treatment 1 [As(V) + Se(IV)] treatment 2 [As(III) + Se(VI)] treatment 3 [As(V) + Se(VI)]

speciesc

[(GS)2AsSe]-

bloodb Se

speciesc

[(GS)2AsSe]-

speciesd

Se speciesd

unchanged changed unchanged

changed unchanged unchanged

As

a Collected for 25 min. b Collected 25 min after injection. c Predominant species according to the As and Se K near edge spectra. the basis of comparison of the As and Se K near edge spectra with the injected metalloid species.

Figure 2. As and Se K X-ray absorption near-edge spectra of bile from a representative arsenite- and selenate-treated animal, compared with selected model compounds. The abscissa scale at the top of the plot shows the energy scale for the As spectra, and the scale at the bottom shows the energy scale for the Se spectra. Arsenic K near-edges: (a) bile, (b) [(GS)2AsSe]-, (c) arsenite (‚‚‚), and (d) arsenate (- - -). Selenium K near-edges: (e) bile, (f) [(GS)2AsSe]-, (g) selenite (‚‚‚), and (h) selenate (- - -).

animal had 12.2 ppm As and 11.4 ppm Se, and the second had 6.9 ppm As and 4.9 ppm Se. The As/Se molar ratios in bile collected from these animals were 1.1 and 1.4, respectively, consistent with the arsenic observed when treatment was with arsenite alone, plus an additional quantity of both As and Se from a species containing As and Se in a molar ratio of 1:1. Speciation of As and Se in Bile. Figure 2 compares the As and Se K near-edge spectra of bile from a selenateand arsenite-treated animal with spectra of [(GS)2AsSe]and the oxy-anions of both metalloids. The data unambiguously indicate biotransformation of the injected arsenite and selenate. The As and Se spectra of the bile and of [(GS)2AsSe]- are almost identical, although the As near-edge peak in the bile is slightly broader than that of [(GS)2AsSe]-, suggesting the presence of a small quantity of an additional reduced arsenic form, e.g., (GS)3As (12). Speciation of As and Se in Whole Blood. Figure 3 shows As and Se K near-edge spectra of whole blood from all treatment groups. With selenate and arsenite treatment, the As spectrum indicates biotransformation (Figure 3, curves a and b) while the Se spectrum indicates that mainly unchanged selenate is present (Figure 3, curves c and d). Conversely, with selenite and arsenate treatment, the selenium is biotransformed (Figure 3c) while the arsenate remains predominantly unchanged (Figure 3a). For the selenate and arsenate treatment, both oxy-anions remain unchanged (Figure 3, curves a and c). These results are summarized in Table 1.

d

On

Figure 3. As and Se K X-ray absorption near-edge spectra of blood from representative animals, compared with selected model compounds. The top abscissa scale shows the energy scale for the As spectra, the bottom that for the Se spectra. Arsenic K near-edges: (a) blood [(s) arsenite/selenate treatment, (- - -) arsenate/selenite treatment, (‚‚‚) arsenate/selenate treatment], (b) models [(s) [(GS)2AsSe]-, (‚‚‚) arsenite, and (- - -) arsenate]. Selenium K near-edges: (c) blood [(s) arsenite/selenate treatment, (- - -) arsenate/selenite treatment, and (‚‚‚) arsenate/ selenate treatment], (d) models [(s) [(GS)2AsSe]-, (‚‚‚) selenite, and (- - -) selenate].

Discussion The general population is exposed to background concentrations of all four common oxy-anions of arsenic and selenium (arsenite, arsenate, selenite, and selenate) through the ingestion of food and drinking water (20, 21). A chronic daily intake of more than 200-250 µg of inorganic arsenic, however, will eventually result in cancer in humans (22). On the other hand, a daily intake between 50 and 200 µg of the essential trace element selenium is recommended to provide humans with an adequate supply of this metalloid for the synthesis of vital selenoproteins (e.g., glutathione peroxidases, selenoprotein P, and iodothyronine 5′-deiodinase) (23). Even though selenium exposure from burning selenium-rich cabonaceous shales can pose health problems (24, 25), the chronic exposure to inorganic arsenic via drinking water poses a considerably greater public health problem (26). Although substantial progress has been made toward understanding the metabolism of inorganic arsenic in mammals (27), little is known about the molecular form of arsenic inside cells and about the mechanism of arsenite-induced carcinogenesis in humans in particular (9). The first experimental evidence indicating a link between the metabolism of inorganic arsenic and selenium was reported more than 60 years ago (28). The discovered antagonism between arsenite and selenite was recently placed upon a molecular footing by the discovery

Biliary Excretion of [(GS)2AsSe]-

of the previously unknown [(GS)2AsSe]- in rabbit bile after the administration of arsenite and selenite (1214). Since arsenate and selenate are also frequently encountered in natural waters (20, 21), it was important to determine if [(GS)2AsSe]- is also excreted in bile after the combined exposure of rabbits to selenite and arsenate, selenate and arsenite, or selenate and arsenate. As or Se Alone in Bile. Injection of rabbits with arsenite (2.40 mg As/kg) resulted in bile containing 2.8 ppm As, approximately 2-fold higher than that previously reported with a quarter of the dose (12). Negligible As was detected in rabbit bile following a similar treatment with the same dose of arsenic in the form of arsenate. This trend is in agreement with previous results on rats (29). Rabbits injected with selenite or selenate (2.52 mg of Se/kg each) had approximately similar levels of selenium in their bile (0.4 and 0.5 ppm), which is also in overall agreement with previous results using rats (30). Whole Blood from As- and Se-Treated Animals. Near-edge spectra are very sensitive to the chemical environment around the absorber atom, and they can often be used to identify oxidation state, and even the chemical identity of a species under investigation (provided that model compound spectra are available) (31). Analysis of the near-edge spectra of the arsenate whole blood samples (treatments 1 and 3) by fitting with linear combinations of model spectra indicated only about 4% reduction of administered arsenate to arsenite after 25 min. (Figure 3a). This compares with previous work using one-sixth of our dose that indicated 10% of the total arsenic in plasma was present as arsenite 15 min after the injection (32). Another study reported that 30 min after the ip injection of Flemish Giant rabbits with carrier free 74As in form of arsenate, 23% of the total arsenic in plasma was present as arsenite (33). These quantitative differences may reflect the different doses of arsenic or the fact that we used an in situ probe, while previous work relied on ex situ methods, with the potential for altering the physiological partitioning between arsenite and arsenate. However, all three studies are in qualitative agreement that the bulk of the arsenate is not reduced on the time scales of the experiments. In a similar fashion, all selenate-treated animals (treatments 2 and 3) showed only a small amount (∼5-10%) of reduced Se forms in blood, with the balance being selenate (Figure 3c). The small amount of reduction of selenate is in reasonable agreement with literature data, which suggested that selenate (0.3 mg of Se/kg) was neither taken up nor reduced by red blood cells in rat and bovine blood (17, 34, 35). By contrast, for the selenite and arsenate treatment (treatment 1), no significant untransformed selenite remained in whole blood (Figure 3, curve c vs curve d), and the spectrum appears to be a mixture of reduced Se species (Figure 3c, Table 1) (12) which is expected (36). Likewise, after selenate and arsenite treatment, the As near edge spectrum suggests arsenic biotransformation, most likely to a (GS)3As species (Figure 3, curves a and b) (12). Bile from As- and Se-Treated Animals. Negligible amounts of either arsenic or selenium were detected in bile after the injection of rabbits with selenite and arsenate (data not shown). Thus, arsenate did not promote the biliary excretion of selenium. Previous work employing the sc injection of rats with arsenate and selenite indicated an interesting trend (37). Arsenate was given 10 min before selenite, at levels of 1:2, 3:2, and 5:2

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(As:Se, in mg of metalloid/kg), and while no increased selenium was present in bile at the 1:2 level (compared to the selenite only group), substantially elevated levels were found at the 3:2 ratio (2.5-fold, compared to control) and at the 5:2 ratio (4-fold, compared to control) (37). These experiments are in reasonable agreement with our findings using an in situ technique, where the iv injection of rabbits with essentially equimolar selenite and arsenate led to only negligible quantities of the metalloids being detected in bile by X-ray fluorescence emission spectroscopy. The detection of significant quantities of arsenic and selenium in bile after the combined selenate and arsenite treatment (Figures 1 and 2) is in good agreement with results obtained after similar treatment of rats (30, 38). The As and Se near-edge spectra of the bile were very similar to those previously reported for synthetic [(GS)2AsSe]- (Figure 2). On the basis of these data, and on the previous chromatographic identification of [(GS)2AsSe]- in rabbit bile (14), [(GS)2AsSe]- is identified as the major As/Se excretory species in bile after selenate and arsenite treatment (Table 1). Curve-fitting analysis indicated that close to 100% of the selenium in the bile, and approximately 75% of the arsenic was present in the form of this metabolite (the balance being reduced arsenic species), which is in agreement with the stoichiometry observed by X-ray fluorescence emission (Figure 1). The selenium near-edge spectra of the corresponding blood samples indicated predominantly unchanged selenate (Figure 3c). Since [(GS)2AsSe]- could not be synthesized from arsenite, selenate, and excess GSH in test tube experiments [which is in accord with the observation that selenate is not reduced by GSH (34)], the detection of [(GS)2AsSe]- in rabbit bile after the injection of rabbits with selenate and arsenite implies biochemical reduction of selenate in the liver. Previous studies by others have demonstrated reduction of selenate in rat liver in vivo (17) and in vitro (34). Selenate reductases and functionally equivalent biochemical systems are now well-known in a variety of organisms (39, 40, 41) and our results indicate the existence of a mammalian selenate reductase, probably in liver. Formation of [(GS)2AsSe]-. While the detailed mechanism of the formation of [(GS)2AsSe]- in mammals is unknown, some speculation may be in order. The detection of [(GS)2AsSe]- in bile after the iv injection of rabbits with selenate and arsenite suggests that both metalloids were first translocated to the liver. Selenate could then be reduced to selenite by a putative selenate reductase, and then further reduced to selenide (either enzymatically or by GSH) (36). At the high intracellular concentration of GSH in hepatocytes, the OH groups of arsenite are expected to be substituted by glutathionyl moieties (42). The known arsenite metabolite (GS)2As-OH (42, 43) may then react with selenide to form [(GS)2AsSe]-. The latter could then be exported from the hepatocytes to bile by ATP-driven glutathione S-conjugate export pumps (44). The fact that [(GS)2AsSe]- was not excreted in bile following selenite and arsenate treatment and selenate and arsenate treatment indicates that arsenate reduction in liver was negligible. This is in apparent disagreement with the existence of a mammalian arsenate reductase, which recently has been isolated from human liver (45). It is possible, however, that bile has to be collected for a longer period after the exposure to arsenate and selenite

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(or selenate) to observe the effects of liver arsenate reductase, and we note that a small quantity of reduced As was indeed present in blood. Further work is needed to clarify the role of arsenate reductase in these types of experiments. Despite extensive work, controversy surrounds the levels of various metals and metalloids that can safely be ingested by humans (46). The general population is simultaneously exposed to many toxic metals and metalloids, and synergistic, antagonistic, or additive effects that include essential elements such as Se potentially complicate the situation. The observed biliary excretion of [(GS)2AsSe]- after the iv injection of rabbits with selenate followed by arsenite emphasizes the need to further investigate the biochemical interaction between arsenic and selenium oxy-anions in mammals. It must always be kept in mind that the chemical form and the dose of a metal or metalloid determine its biological activity in vivo (47) and that the human health risk cannot be adequately examined by only studying the individual components. It seems likely that a much deeper knowledge of the metabolism of inorganic arsenic will be required to understand the molecular basis of its carcinogenicity.

Acknowledgment. This research was supported by the Alexander von Humboldt Foundation (J.G.). Donald W. DeYoung from University Animal Care of the University of Arizona is greatly acknowledged for help with the animal experiments (Protocol no. 98-052). The Stanford Synchrotron Radiation Laboratory (SSRL) is funded by the Department of Energy, Office of Basic Energy Sciences. The Structural Molecular Biology Program is supported by the National Institutes of Health, Biomedical Research Technology Program, Division of Research Resources. Further support is provided by the Department of Energy, Office of Biological and Environmental Research.

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