Chem.-BioL Interactions, 16 (1977) 155--167 © Elsevier/North-Holland Scientific Publishers, Ltd.
155
INTERACTIONS BETWEEN SELENIUM AND METHYLMERCURY IN R A T B R A I N *
JOSEPH R. PROHASKA and HOWARD E. GANTHER
Department of Nutritional Sciences, University of Wisconsin, Madison, Wisc. 53706 (U.S.A.) (Received June 3rd, 1976) (Revision received September 27th, 1976) (Accepted October 8th, 1976}
SUMMARY
The interaction of selenium with methylmercury was investigated in brain of animals labeled with 7SSeO~- and CH32°3Hg÷. Brains were fractionated into subcellular components and the cytosol was further fractionated by chromatography on Sephadex G-150 and G-200. The main result of these studies was evidence suggesting a shift of 7SSe from the cytosol to the mitochondrial fraction in brain when CH3Hg÷ was given. Concurrent equimolar (10 ~moles/ kg) selenite injections increased the uptake of Hg b u t did n o t alter 2°3Hg distribution in brain. Changing the dose of CH3Hg ÷ from 1 to 38 ~moles/kg had little effect on Hg uptake (% of dose per g). Gel filtrations on Sephadex G-150 and G-200 revealed that 2°aHg in cytosol followed a pattern more closely related to protein (A280) than to 7SSe, although a considerable portion of both isotopes eluted with proteins in the void volume. Assays of whole brain homogenates revealed a slight reduction in glutathione peroxidase activity in CH3Hg*-treated rats which was n o t seen when equimolar selenite was injected with the CH3 Hg ÷ .
INTRODUCTION
The toxic effects of mercury on the brain have been known for centuries, y e t the mechanism of its action on this target organ is unknown. Because of mercury's high affinity for mercaptans, it is often stated that mercury is toxic because it binds to sulfur ligands in proteins, b u t this has n o t been established. * Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and the U.S. Public Health Service (AM 14184-07). Abbreviation: GSH-Px, glutathione peroxidase.
156 The essential trace element selenium has a well-established role in modifying mercury toxicity, but little is known about the mechanisms involved. Selenium fed in the diet [1] or given by injection [2] reduces mortality and growth depression caused by methylmercury. Mercury also reduces selenium toxicity [3]. Protection by selenium does not seem to result from accelerated elimination of mercury [4] or reduced uptake into tissues [5]. Indeed, selenium-treatment often causes an increase in mercury content of tissues and vice versa [5]. CH3Hg÷ has a high affinity for selenium, surpassing that for sulfur [6]. Thus mercury : selenium complexing is probably the basis for the biological interactions between mercury and selenium whereby the toxicity of each element is decreased in the presence of the other. Indeed, it is not unlikely that a biologically active form of selenium, not sulfur, may be the critical target for mercury. There is evidence that mercury and other heavy metals induce a conditioned nutritional deficiency of selenium [7,8]. The fact that selenium reduces mercury toxicity without decreasing mercury levels in brain raises the question of whether selenium may alter the form of mercury or its distribution within the tissue, preventing mercury from binding to critical sites. Studies with 2°3Hg-labeled mercuric chloride have demonstrated effects of selenium on the distribution of mercury in subcellular fractions of various tissues [9] and in plasma proteins [10]. The distribution of methylmercury (the form most commonly involved in brain damage) in rat brain has been described [11,12], as has the distribution of selenium [13] but they have not been studied concurrently. In this study we have examined the distribution of 2°3Hg-labeled methylmercury in brain subcellular fractions and in cytosolic proteins fractionated by gel filtration on Sephadex G-150 and G-200, in comparison to the distribution of 7SSe and the selenoenzyme, giutathione peroxidase. MATERIALS AND METHODS Glutathione, glutathione reductase (EC 1.6.4.2), NADPH and Triton X-100 were purchased from Sigma Chemical Co (St Louis, MO). Na2SeO3 and CH3HgC1 were obtained from Ventron-Alfa Products (Danvers, MA). New England Nuclear (Boston, MA) supplied H27SSeO3 and CH3 :°3HgC1. Cumene hydroperoxide was purchased from ICN K and K Chemicals (Cleveland, OH). Sephadex G-150 was obtained from Pharmacia (Piscataway, NJ). Rats were purchased from Holtzman Co. (Madison, WI) and were fed a commercial pellet diet (Lab Blox, Allied Mills Inc., Chicago, IL) containing 0.4 pg Se/g; Hg was not detectable (
155
INTERACTIONS BETWEEN SELENIUM AND METHYLMERCURY IN R A T B R A I N *
JOSEPH R. PROHASKA and HOWARD E. GANTHER
Department of Nutritional Sciences, University of Wisconsin, Madison, Wisc. 53706 (U.S.A.) (Received June 3rd, 1976) (Revision received September 27th, 1976) (Accepted October 8th, 1976}
SUMMARY
The interaction of selenium with methylmercury was investigated in brain of animals labeled with 7SSeO~- and CH32°3Hg÷. Brains were fractionated into subcellular components and the cytosol was further fractionated by chromatography on Sephadex G-150 and G-200. The main result of these studies was evidence suggesting a shift of 7SSe from the cytosol to the mitochondrial fraction in brain when CH3Hg÷ was given. Concurrent equimolar (10 ~moles/ kg) selenite injections increased the uptake of Hg b u t did n o t alter 2°3Hg distribution in brain. Changing the dose of CH3Hg ÷ from 1 to 38 ~moles/kg had little effect on Hg uptake (% of dose per g). Gel filtrations on Sephadex G-150 and G-200 revealed that 2°aHg in cytosol followed a pattern more closely related to protein (A280) than to 7SSe, although a considerable portion of both isotopes eluted with proteins in the void volume. Assays of whole brain homogenates revealed a slight reduction in glutathione peroxidase activity in CH3Hg*-treated rats which was n o t seen when equimolar selenite was injected with the CH3 Hg ÷ .
INTRODUCTION
The toxic effects of mercury on the brain have been known for centuries, y e t the mechanism of its action on this target organ is unknown. Because of mercury's high affinity for mercaptans, it is often stated that mercury is toxic because it binds to sulfur ligands in proteins, b u t this has n o t been established. * Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and the U.S. Public Health Service (AM 14184-07). Abbreviation: GSH-Px, glutathione peroxidase.
156 The essential trace element selenium has a well-established role in modifying mercury toxicity, but little is known about the mechanisms involved. Selenium fed in the diet [1] or given by injection [2] reduces mortality and growth depression caused by methylmercury. Mercury also reduces selenium toxicity [3]. Protection by selenium does not seem to result from accelerated elimination of mercury [4] or reduced uptake into tissues [5]. Indeed, selenium-treatment often causes an increase in mercury content of tissues and vice versa [5]. CH3Hg÷ has a high affinity for selenium, surpassing that for sulfur [6]. Thus mercury : selenium complexing is probably the basis for the biological interactions between mercury and selenium whereby the toxicity of each element is decreased in the presence of the other. Indeed, it is not unlikely that a biologically active form of selenium, not sulfur, may be the critical target for mercury. There is evidence that mercury and other heavy metals induce a conditioned nutritional deficiency of selenium [7,8]. The fact that selenium reduces mercury toxicity without decreasing mercury levels in brain raises the question of whether selenium may alter the form of mercury or its distribution within the tissue, preventing mercury from binding to critical sites. Studies with 2°3Hg-labeled mercuric chloride have demonstrated effects of selenium on the distribution of mercury in subcellular fractions of various tissues [9] and in plasma proteins [10]. The distribution of methylmercury (the form most commonly involved in brain damage) in rat brain has been described [11,12], as has the distribution of selenium [13] but they have not been studied concurrently. In this study we have examined the distribution of 2°3Hg-labeled methylmercury in brain subcellular fractions and in cytosolic proteins fractionated by gel filtration on Sephadex G-150 and G-200, in comparison to the distribution of 7SSe and the selenoenzyme, giutathione peroxidase. MATERIALS AND METHODS Glutathione, glutathione reductase (EC 1.6.4.2), NADPH and Triton X-100 were purchased from Sigma Chemical Co (St Louis, MO). Na2SeO3 and CH3HgC1 were obtained from Ventron-Alfa Products (Danvers, MA). New England Nuclear (Boston, MA) supplied H27SSeO3 and CH3 :°3HgC1. Cumene hydroperoxide was purchased from ICN K and K Chemicals (Cleveland, OH). Sephadex G-150 was obtained from Pharmacia (Piscataway, NJ). Rats were purchased from Holtzman Co. (Madison, WI) and were fed a commercial pellet diet (Lab Blox, Allied Mills Inc., Chicago, IL) containing 0.4 pg Se/g; Hg was not detectable (
