Chem. Res. Toxicol. 2000, 13, 1135-1142
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Structural Basis of the Antagonism between Inorganic Mercury and Selenium in Mammals Ju¨rgen Gailer,†,‡ Graham N. George,*,§ Ingrid J. Pickering,§ Sean Madden,| Roger C. Prince,⊥ Eileen Y. Yu,§ M. Bonner Denton,| Husam S. Younis,@ and H. Vasken Aposhian*,† Department of Molecular and Cellular Biology, The University of Arizona, Life Sciences South Building, Tucson, Arizona 85721, Stanford Synchrotron Radiation Laboratory, SLAC, P.O. Box 20450, MS 69, Stanford, California 94309, Department of Chemistry, The University of Arizona, Tucson, Arizona 85721, ExxonMobil Research and Engineering Company, Route 22 East, Annandale, New Jersey 08801, and Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721 Received March 2, 2000
Mercuric chloride toxicity in mammals can be overcome by co-administration of sodium selenite. We report a study of the mutual detoxification product in rabbit plasma, and of a Hg-Se-S-containing species synthesized by addition of equimolar mercuric chloride and sodium selenite to aqueous, buffered glutathione. Chromatographic purification of this Hg-Se-S species and subsequent structural analysis by Se and Hg extended X-ray absorption fine structure (EXAFS) spectroscopy revealed the presence of four-coordinate Se and Hg entities separated by 2.61 Å. Hg and Se near-edge X-ray absorption spectroscopy of erythrocytes, plasma, and bile of rabbits that had been injected with solutions of sodium selenite and mercuric chloride showed that Hg and Se in plasma samples exhibited X-ray absorption spectra that were essentially identical to those of the synthetic Hg-Se-S species. Thus, the molecular detoxification product of sodium selenite and mercuric chloride in rabbits exhibits similarities to the synthetic Hg-Se-S species. The underlying molecular mechanism for the formation of the Hg-Se-S species is discussed.
Introduction The protective effect of sodium selenite against the toxicity of mercuric chloride in mammals has been known for three decades (1). Simultaneous sc injection of sodium selenite with mercuric chloride (at a different site to avoid the formation of precipitates) completely protects rats from the characteristic histological changes associated with mercuric chloride toxicity (1). Further studies have substantiated this protective effect in mice (2), pigs (3), chicken (4), and rabbits (5), suggesting that it is a widespread phenomenon. When sodium selenite was given 1-2 h before the mercuric chloride, the toxicity was much greater and the animals died (6). This effect may arise from the in vivo production of the selenite metabolite dimethylselenide, which is highly toxic when coadministered with mercuric chloride (7). Additional studies revealed that the mutual detoxification of sodium selenite and mercuric chloride was most effective when the compounds were administered simultaneously (2, 8, 9). This caused a marked decrease in the level of urinary excretion of Hg (7) and a significant increase in the level * To whom correspondence should be addressed. † Department of Molecular and Cellular Biology, The University of Arizona. ‡ Present address: Department of Nutritional Sciences, Shantz Building 309, The University of Arizona, Tucson, AZ 85721. § Stanford Synchrotron Radiation Laboratory. | Department of Chemistry, The University of Arizona. ⊥ ExxonMobil Research and Engineering Co. @ Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona.
of body retention of Hg, which was still observable after 28 days in mice (10) and rats (11). In particular, the total Hg concentrations were increased several-fold in the blood of rats (11, 12), mice (10), and rabbits (5, 13), but the accumulation of Hg in the kidneys was prevented (3, 11, 14). Mercuric chloride strikingly changed the tissue distribution of 75Se in mice when various heavy metal salts were co-administered with Na275SeO3, resulting in an increased level of 75Se retention in erythrocytes, liver, spleen, and kidney (15). In addition, the level of pulmonary excretion of the volatile selenite metabolite dimethylselenide was decreased when mercuric chloride and sodium selenite were co-administered in rats (16). On the basis of these studies, it seems probable that the mutual detoxification of sodium selenite and mercuric chloride occurs predominantly in blood (2, 5, 8). Burk et al. (17) administered sodium selenite and mercuric chloride solutions to male rats and 20 h later detected a plasma protein that contained equimolar levels of Se and Hg. Even when the doses of injected sodium selenite and mercuric chloride were varied, the Se/Hg molar ratio in the protein remained close to unity (17). Naganuma et al. (18) showed that GSH is essential for the formation of this Hg-Se-protein complex, and more recently, the plasma protein to which the Hg-Se species specifically binds was identified by Yoneda et al. (19) as selenoprotein P. These authors postulated that a reduced form of selenite, possibly selenide, was effluxed from red blood cells and reacted with mercuric chloride in plasma to form a Hg-Se complex containing equimolar amounts
10.1021/tx000050h CCC: $19.00 © 2000 American Chemical Society Published on Web 09/29/2000
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of the two elements, which subsequently bound to selenoprotein P (19-21). Yoneda et al. (19, 20) reported that the Hg-Se species contained approximately 100 atoms each of Hg and Se. The structural details of this species, however, remained unknown. In this paper, we report the chemical identity of the Hg-Se-S species in blood using X-ray absorption spectroscopy.
Experimental Procedures Synthesis of the Hg-Se-S Species. GSH (231.0 mg, 0.75 mmol) was dissolved in 1.2 mL of PBS buffer and the pH adjusted to 7.5 by dropwise addition of 4.0 M NaOH. After dissolution of 0.47 mmol each of Na2SeO3‚5H2O and HgCl2 in PBS buffer to a final volume of 0.5 mL, 160 µL of this solution was added to the GSH solution (corresponding to 5 molar equiv) which had been previously incubated at 37 °C for 15 min. Immediately after the addition, a black solution (pH 7.1-7.2) was obtained (final volume of 1.80 mL). Size-Exclusion Chromatography. Samples were chromatographed in PBS buffer (pH 7.4) on Sephadex G-25 (Superfine). The void volume and the inclusion volume of the column were determined with rat hemoglobin and Mg2+ standards, and an inductively coupled plasma atomic emission spectrometer (see below) was used as the detector. Selenite and mercuric chloride eluted after Mg2+ ions, so an as yet unspecified chemical interaction must occur between these molecules and the Sephadex G-25 matrix. Se, S, and Hg in the Hg-Se-S species were quantified in fractions collected from the column. The recoveries of Hg, Se, and S from the column (fraction I and II) were 88.2% ((3.8), 86.7% ((3.3), and 90.1% ((0.7), respectively. Hg, S, Se, Fe, and Mg specific detection was accomplised using inductively coupled plasma-atomic emission spectroscopy (ICP-AES) using a Thermo Jarrel Ash IRIS HR radial view spectrometer (Franklin, MA). ThermoSPEC/CID software (version 2.10.04) was used to provide the necessary time scan functions, and the multitasking controller allowed the processing of one atomic emission line every 0.02 s. The nebulization gas flow was maintained at a rate of 2 L/min, the plasma forward power at 1150 W, and the CID temperature at -85 °C. Mg and Fe were monitored on-line by ICP-AES at the 285.213 nm (order 118) and 239.924 nm (order 140) lines, respectively. S and Se were monitored on-line with ICP-AES detection at the 182.034 nm (order 185) or the 196.090 nm (order 172) line. Hg was assessed at the 184.950 nm line (order 181). All quantitative measurements were performed at these S, Se, and Hg emission wavelengths. Custom-made software allowed off-line manipulation of the time scan files. To obtain the chromatograms, online monitoring by ICP-AES was started 6 min after the injection of the samples. Chromatograms were constructed by taking 1000 time slices (1 s) of the accumulating charge at the specified locations on the CID. CID knockdown events were detected and removed prior to a discreet interval derivative with a dt of 2. Finally, smoothing was performed with a five-point median filter. For the determination of the retention time of mercuric chloride, on-line monitoring by ICP-AES was started after 25 min. Animal Experiments. Male New Zealand white rabbits (2-3 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 fasted overnight and (following halothane anesthesia, midline abdominal incision, and gallbladder ligation), the common bile duct was cannulated with polyethylene tubing. Throughout the experiment, the rabbits were treated with an intravenous drip (0.5 mL/min) of lactated Ringer’s solution (Baxter Healthcare Corp., Deerfield, IL) through the marginal ear vein. In addition, a tracheal tube was inserted to ensure free airways during the experiment. After a constant bile flow had been established, a solution of HgCl2 or Na2SeO3‚5H2O (20 mM in PBS adjusted to pH 7.4) was injected as mercuric chloride (1.57 mg of Hg/kg of
Gailer et al. body weight), sodium selenite (0.63 mg of Se/kg of body weight), or both (selenite followed 3 min later by mercuric chloride). Injections were via the marginal ear vein; two rabbits were used for each experiment. Animal bile was collected for a total of 20 min into ice-cooled polypropylene vials. Experiments were started between 9:00 and 11:30 am to exclude the effects of diurnal variations in hepatic GSH levels. The bile samples were subsequently mixed with 40% (v/v) glycerol (to prevent ice crystal diffraction), transferred to Lucite sample cuvettes, and frozen in liquid nitrogen for later X-ray absorption spectroscopy (XAS) and X-ray fluorescence measurements. Preparation of Samples for XAS. An aliquot (200 µL) of the Hg-Se-S species was chromatographed on the same sizeexclusion column (Sephadex G-25, 29.0 cm × 1.0 cm) with 50 mM HEPES buffer (pH 7.3) serving as the mobile phase at a flow rate of 1.0 mL/min. The black band containing the HgSe-S species was collected (13.0-14.5 min). This solution (0.6 mL) was mixed with 0.4 mL of glycerol, transferred to a Lucite sample holder, and frozen in liquid nitrogen for subsequent XAS analysis. Commercial mercuric selenide was mixed with a calculated amount of boron nitride (used as an inert diluent) to give a maximum X-ray absorbance of 2 and packed into 0.5 mm thick aluminum sample holders. Mercury diglutathione [Hg(GS)2] was prepared by dissolving GSH (132 mmol) in 1.0 mL of HEPES buffer (50 mM, pH 7.3), adjusting to pH 7.7 and adding additional HEPES buffer to a final volume of 5.0 mL. Then 25 mmol of HgCl2 was dissolved in 1.0 mL of HEPES (50 mM, pH 7.3), and 40 µL of this solution was added to 560 µL of the GSH solution and mixed. Finally, 400 µL of glycerol was added; the mixture was transferred to Lucite sample holders and frozen in liquid nitrogen until it was analyzed by XAS. X-ray Absorption Spectroscopy (XAS). X-ray absorption spectra were measured at the Stanford Synchrotron Radiation Laboratory (SSRL) on Beamline 7-3 using a Si(220) doublecrystal monochromator with an upstream vertical 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 by monitoring the Hg Lβ1 or Se KR intensity using a Canberra 13element Ge detector. 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 foil, the lowest energy inflection point of which was assumed to be 12 658.0 eV. X-ray fluorescence emission spectra for elemental analysis were measured with an amplifier 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. During data collection, samples were maintained at a temperature of 10 K, 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). Ab initio theoretical extended phase and amplitude functions were calculated using FEFF, version 8.03 (22).
Results The concentrations of Hg and Se were high in plasma and erythrocytes, and virtually zero in bile (Table 1). When Hg and Se were co-administered, their concentrations were generally higher both in erythrocytes and in plasma than when administered individually (Table 1). In contrast to the Hg/Se molar ratio of 1.1 in plasma, the ratio was 0.7 in erythrocytes. To study the aqueous interaction of sodium selenite, mercuric chloride, and GSH, a solution equimolar in sodium selenite and mercuric chloride was added to
Mutual Detoxification of Hg and Se in Mammals
Chem. Res. Toxicol., Vol. 13, No. 11, 2000 1137
Table 1. X-ray Atomic Fluorescence-Derived Hg and Se Concentrationsa with Hg
with Se
with Hg and Se
[Hg] (mM) [Se] (mM) [Hg] (mM) [Se] (mM) Hg/Se red blood cells plasma bile
16(2) 58(15) 0
46(15) 25(16) 0
33(4) 88(10) 0
45(3) 79(13) 0
0.72(5) 1.12(9) -
a Hg and Se concentrations (millimolar) in body fluids of rabbits after sodium selenite, mercuric chloride, or sodium selenite and mercuric chloride injection. The values in parentheses are the estimated standard deviations which were obtained from four independent measurements (three for Hg-only plasma) from two animals per group.
Figure 2. X-ray absorption spectrum of solid HgSe, showing the three L-absorption edges of mercury and the K edge of selenium. The inset shows an expansion of the Hg LII edge, including the EXAFS oscillations.
Figure 1. Simultaneous on-line mercury, selenium, and sulfur specific chromatogram of the mixture prepared by reacting mercuric chloride and sodium selenite with 5 molar equiv of GSH in buffered, aqueous solution on a Sephadex G-25 (SF) column (29.5 cm × 1.0 cm) at a flow rate of 1 mL/min. The mobile phase was PBS buffer (pH 7.4); the injection volume was 200 µL.
solutions of between 1 and 10 molar equiv of GSH (buffered at pH 7.4). With
