113
Biochimica et Biophysica Acta, 428 (1976) 113--122 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27844
INTERACTIONS OF CADMIUM AND SELENIUM IN R A T PLASMA IN VIVO AND IN VITRO
THOMAS A. GASIEWICZ and JOHN C. SMITH *
Department of Pharmacology and Toxicology, University of Rochester School of Medicine and Dentistry, Rochester, N.Y. 14642 (U.S.A.) (Received September 8th, 1975)
Summary 7SSe and t°gCd tracers were used to study the binding of Se and Cd to plasma proteins at various SeO32- doses and times up to 24 h after the simultaneous subcutaneous administration of SeO3 ~- and CdC12 to adult male rats. The simultaneous injection of CdC12 and SeO32- markedly increased both Se and Cd plasma levels over that in control animals. Gel permeation chromatography of plasma indicated that at all times up to 24 h Cd and Se were bound in an atomic ratio of approx. 1 : 1 in 330 000 and 130 000 dalton fractions. From 4 to 24 h, Cd and Se appeared in the 420 000 dalton fraction, also with an atomic ratio of approx. 1 : 1. The 330 000 dalton molecules appeared to have a maximal binding capacity for the Cd-Se complex at a concentration of approx. 30 pmol/ml of plasma, while the 130 000 and 420 000 dalton molecules show a higher binding capacity. Studies in vitro revealed that SeO32- does n o t interact directly with Cd and plasma proteins. It is metabolized b y erythrocytes to a form that interacts in an atomic ratio of 1 : 1 with Cd to form a protein-bound complex of 130 000 daltons.
Introduction Previous studies have noted that simultaneously administered selenite (SeO32-) markedly reduces cadmium toxicity [1--4]. Gunn et al. [3] found that in comparison to other protectors against Cd toxicity (cysteine, 2,3dimercaptopropanol), selenium as SeO32- is the most effective, completely preventing testicular degeneration in a molar dosing ratio of 1 : 1. Selenium, however, increases Cd uptake b y testicular tissue [4]. The mechanism of protection may be related to the change in distribution of Cd within the soluble protein fractions of the testes [ 5]. * To
whom reprint requests should be sent.
114 Parizek et al. [4,6] and Gunn et al. [3] have demonstrated that Se administration significantly alters the distribution of Cd in tissues within the first 5 h after Cd injection. In these studies blood Cd levels were found to be significantly increased by simultaneous SeO32- injection. Although a direct interaction between Cd and Se has been postulated [6,7], the mechanism by which Se decreases the toxicity and alters the tissue distribution of Cd is not understood. This report deals with the nature of Cd and Se in blood plasma within the first 24 h after CdCI: and SeO32- administration to adult rats. Analysis of the plasma components showing the largest initial change in both Cd and Se concentrations after the simultaneous injection of both elements might give some indication as to the nature of this interaction. Studies in vitro utilizing rat plasma and erythrocytes are also presented. Materials and Methods Male Sprague-Dawley rats (average weight 230 g), obtained from Holtzmann (Madison, Wisc.), were housed at least 2 weeks before experimentation under constant laboratory conditions with a 12-h light cycle. Charles River rat chow diet (Agway, Inc., Syracuse, N.Y.) and distilled water were provided ad libitum. 7SSe and 1°9Cd were obtained as H2SeO3 and CdC12 from New England Nuclear (Boston, Mass.). Carrier dosing solutions were prepared from Na2SeO3 and CdC12 dissolved in 0.9% NaC1. All Cd and Se injections were made subcutaneously in opposite thighs. Animals were exsanguinated by cardiac puncture under ether anesthesia. Blood was collected in heparinized syringes to prevent coagulation. Plasma was separated from red cells by centrifugation. For ~°gCd and ~SSe analysis, tissues were placed in 12 X 75 m m tubes and counted in a Packard Auto-Gamma Scintillation Spectrometer complete with Model 9601 Multi-Channel Analyzer System using photopeaks 0.16 and 0.465 MeV, respectively. Aliquots of the dosing solution were counted as standards. Calculations of Cd and Se concentrations are based upon the specific activity of the dosing solutions. Gel permeation chromatography was employed for the separation of plasma components. Sephadex G-200 (particle size 40--120 #m) and chromatographic columns (2.0 X 70 cm) were obtained from Pharmacia Fine Chemicals (Piscataway, N.J.). Sample volumes ranged between 0.8 and 1.7 ml of plasma. Void volume was 113 ml and total bed volume was 325 ml. A constant flow (15.5 ml/h) of buffer (1.0 M NaC1, 0.1 M Tris • HC1, pH 8.0) was maintained by a peristaltic pump. The eluate was monitored for absorbance at 280 nm. Fractions of 3.1 ml were collected and counted for radioactivity as described above for tissue samples. Recoveries on columns, for both ~°9Cd and 7SSe, ranged between 91 and 106%. All data were adjusted for the recovery of the particular experiment. For molecular weight determination, columns were calibrated by the method of Andrews [8], utilizing bovine albumin, ovalbumin, myoglobin, and cytochrome c obtained from Sigma Chemical (St. Louis, Mo.). To determine the effects of varying the SeO32- dose on the plasma protein distribution of Cd and Se (Experiment I), experimental animals were given 20.0 pmol CdC12/kg and SeO32- doses ranging between 1.0 and 20.0 #mol/kg. Control animals received either 20.0 #mol CdCl:/kg or the particular Se32- dose.
115 Injection volumes ranged between 0.15 and 0.20 ml. All animals were sacrificed 5 h after the injections. To determine the change in plasma protein distribution of Cd and Se with time (Experiment II), experimental animals were given 20.0 pmol CdC12/kg and 20.0 pmol SeO32-/kg, and were sacrificed 0.25--24 h after treatment. Control animals received either CdC12 or SeO32- . Injection volumes ranged from 0.4 to 0.5 ml. A modification of the method of Watkinson [9] was used for total Se analysis. Tissue samples were digested in a mixture of HNO3 and HC104 followed by complexing of Se with 2,3-diaminonaphthalene. The Se
Biochimica et Biophysica Acta, 428 (1976) 113--122 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27844
INTERACTIONS OF CADMIUM AND SELENIUM IN R A T PLASMA IN VIVO AND IN VITRO
THOMAS A. GASIEWICZ and JOHN C. SMITH *
Department of Pharmacology and Toxicology, University of Rochester School of Medicine and Dentistry, Rochester, N.Y. 14642 (U.S.A.) (Received September 8th, 1975)
Summary 7SSe and t°gCd tracers were used to study the binding of Se and Cd to plasma proteins at various SeO32- doses and times up to 24 h after the simultaneous subcutaneous administration of SeO3 ~- and CdC12 to adult male rats. The simultaneous injection of CdC12 and SeO32- markedly increased both Se and Cd plasma levels over that in control animals. Gel permeation chromatography of plasma indicated that at all times up to 24 h Cd and Se were bound in an atomic ratio of approx. 1 : 1 in 330 000 and 130 000 dalton fractions. From 4 to 24 h, Cd and Se appeared in the 420 000 dalton fraction, also with an atomic ratio of approx. 1 : 1. The 330 000 dalton molecules appeared to have a maximal binding capacity for the Cd-Se complex at a concentration of approx. 30 pmol/ml of plasma, while the 130 000 and 420 000 dalton molecules show a higher binding capacity. Studies in vitro revealed that SeO32- does n o t interact directly with Cd and plasma proteins. It is metabolized b y erythrocytes to a form that interacts in an atomic ratio of 1 : 1 with Cd to form a protein-bound complex of 130 000 daltons.
Introduction Previous studies have noted that simultaneously administered selenite (SeO32-) markedly reduces cadmium toxicity [1--4]. Gunn et al. [3] found that in comparison to other protectors against Cd toxicity (cysteine, 2,3dimercaptopropanol), selenium as SeO32- is the most effective, completely preventing testicular degeneration in a molar dosing ratio of 1 : 1. Selenium, however, increases Cd uptake b y testicular tissue [4]. The mechanism of protection may be related to the change in distribution of Cd within the soluble protein fractions of the testes [ 5]. * To
whom reprint requests should be sent.
114 Parizek et al. [4,6] and Gunn et al. [3] have demonstrated that Se administration significantly alters the distribution of Cd in tissues within the first 5 h after Cd injection. In these studies blood Cd levels were found to be significantly increased by simultaneous SeO32- injection. Although a direct interaction between Cd and Se has been postulated [6,7], the mechanism by which Se decreases the toxicity and alters the tissue distribution of Cd is not understood. This report deals with the nature of Cd and Se in blood plasma within the first 24 h after CdCI: and SeO32- administration to adult rats. Analysis of the plasma components showing the largest initial change in both Cd and Se concentrations after the simultaneous injection of both elements might give some indication as to the nature of this interaction. Studies in vitro utilizing rat plasma and erythrocytes are also presented. Materials and Methods Male Sprague-Dawley rats (average weight 230 g), obtained from Holtzmann (Madison, Wisc.), were housed at least 2 weeks before experimentation under constant laboratory conditions with a 12-h light cycle. Charles River rat chow diet (Agway, Inc., Syracuse, N.Y.) and distilled water were provided ad libitum. 7SSe and 1°9Cd were obtained as H2SeO3 and CdC12 from New England Nuclear (Boston, Mass.). Carrier dosing solutions were prepared from Na2SeO3 and CdC12 dissolved in 0.9% NaC1. All Cd and Se injections were made subcutaneously in opposite thighs. Animals were exsanguinated by cardiac puncture under ether anesthesia. Blood was collected in heparinized syringes to prevent coagulation. Plasma was separated from red cells by centrifugation. For ~°gCd and ~SSe analysis, tissues were placed in 12 X 75 m m tubes and counted in a Packard Auto-Gamma Scintillation Spectrometer complete with Model 9601 Multi-Channel Analyzer System using photopeaks 0.16 and 0.465 MeV, respectively. Aliquots of the dosing solution were counted as standards. Calculations of Cd and Se concentrations are based upon the specific activity of the dosing solutions. Gel permeation chromatography was employed for the separation of plasma components. Sephadex G-200 (particle size 40--120 #m) and chromatographic columns (2.0 X 70 cm) were obtained from Pharmacia Fine Chemicals (Piscataway, N.J.). Sample volumes ranged between 0.8 and 1.7 ml of plasma. Void volume was 113 ml and total bed volume was 325 ml. A constant flow (15.5 ml/h) of buffer (1.0 M NaC1, 0.1 M Tris • HC1, pH 8.0) was maintained by a peristaltic pump. The eluate was monitored for absorbance at 280 nm. Fractions of 3.1 ml were collected and counted for radioactivity as described above for tissue samples. Recoveries on columns, for both ~°9Cd and 7SSe, ranged between 91 and 106%. All data were adjusted for the recovery of the particular experiment. For molecular weight determination, columns were calibrated by the method of Andrews [8], utilizing bovine albumin, ovalbumin, myoglobin, and cytochrome c obtained from Sigma Chemical (St. Louis, Mo.). To determine the effects of varying the SeO32- dose on the plasma protein distribution of Cd and Se (Experiment I), experimental animals were given 20.0 pmol CdC12/kg and SeO32- doses ranging between 1.0 and 20.0 #mol/kg. Control animals received either 20.0 #mol CdCl:/kg or the particular Se32- dose.
115 Injection volumes ranged between 0.15 and 0.20 ml. All animals were sacrificed 5 h after the injections. To determine the change in plasma protein distribution of Cd and Se with time (Experiment II), experimental animals were given 20.0 pmol CdC12/kg and 20.0 pmol SeO32-/kg, and were sacrificed 0.25--24 h after treatment. Control animals received either CdC12 or SeO32- . Injection volumes ranged from 0.4 to 0.5 ml. A modification of the method of Watkinson [9] was used for total Se analysis. Tissue samples were digested in a mixture of HNO3 and HC104 followed by complexing of Se with 2,3-diaminonaphthalene. The Se
