Toxicology, 5 (1975) 243--254 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
THE ERYTHROCYTE TRANSPORT AND T R A N S F E R OF METHYLMERCURY TO THE TISSUES OF THE RAINBOW TROUT ( $ A L M O GAIRDNERI) *
F.J. GIBLIN and EDWARD J. MASSARO** Department of Biochemistry, State University of New York at Buffalo, Buffalo, N.Y. 14214 (U.S.A.)
(Received May 14th, 1975) (Revision received July 17th, 1975) (Accepted August 4th, 1975)
SUMMARY Methylmercury (MeHg) was found to be taken up rapidly and almost completely by t r o u t red blood cells (RBC) both in vitro and in vivo. The binding of MeHg within the RBC was freely reversible both in vitro, as shown by the efflux of MeHg from RBCs suspended in protein solutions, and in vivo following intracardial (i.c.) injection of RBC-bound MeHg. Hemoglobin (Hb) appeared to be the main MeHg transport protein in t r o u t blood since it bound 90% of whole blood Hg following an intragastric dose of Me2°aHgC1. MeHg, injected i.c. as MeHgS-cysteine, was f o u n d to be present in blood bound almost completely to hemoglobin 10 days postinjection. This suggests an ability of hemoglobin to compete for and bind MeHg bound to other sulfhydryl (--SH) compounds. The number of reactive --SH groups per molecule of t r o u t Hb was determined to be 4 by amperometric titration with MeHgC1. The concentration of Hb reactive--SH groups in the t r o u t RBC was calculated to be at least 20 mM. This accounts for the high affinity of the RBC for MeHg.
* A preliminary report of this work was presented at the 58th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.J., April 7--12, 1974. ** To whom all correspondence should be addressed. Abbreviations: RBC, red blood cells;--SH, sulfhydryl.
243
INTRODUCTION It has been firmly established that fishes, shellfishes and other aquatic organisms take up MeHg from their environment and sequester and concentrate it in their tissues [1--3]. Methylmercury is a potent neurotoxin and consumption of contaminated fishes and shellfishes has resulted in massive incidents of human intoxication [3]. We have investigated the pharmacodynamics of MeHg in a model piscine system, the rainbow trout (Salmo gairdneri) [1]. The blood of the rainbow trout was observed to concentrate methylmercury (MeHg) to a greater extent than any other tissue. A high affinity of MeHg for the blood has been reported to occur in a wide variety of other species and, apparently, is a general phenomenon that may relate to the unique toxic effects exhibited by this compound [2,3]. The blood element responsible for concentrating MeHg has been determined to be the RBC [3]. Relatively little is known concerning the mechanism of uptake of MeHg by the RBC, the binding of MeHg within the cell or the transport of MeHg from the RBC to tissues. Considerably more information is available concerning the interactions of inorganic Hg and the RBC. The status of this knowledge has been reviewed recently by Passow [4]. MeHg binds specifically to protein --SH groups as is expected from its high association constant with the - S H group of cysteine [5,6]. The - S H groups of hemoglobin have been shown to bind organic Hg strongly [7]; but little is known of the extent of MeHg binding to hemoglobin or of the reversibility of the MeHg-hemoglobin bond within the RBC. Work by Clarkson et al. [8] and White and Rothstein [9] has suggested that M e H g can be released from whole blood and RBCs. This work was u n d e ~ e n to elucidate the extent of binding of M e H g to hemoglobin in the rainbow trout, the reversibility of the MeHg-hemoglobin bond in vivo and in vitro and the role of hemoglobin in the transportation of M e H g to the tissues. METHODS
Animals, tissue sampling, Hg analysis. Hatchery.reared rainbow trout, averaging 400 g in weight, were o b ~ n e d from the New York state Hatchery at Caledonia, New York and maintained as previously described [1]. The collection of tissue samples and the determination of their Hg content by gamma-scintillation spectrometry have also been described [1]. Reagents. 2°3Hg-labeled MeHgCl was obtained from N e w England Nuclear, Boston, Mass. Its radiochemical purity was > 9 5 % as determined by paper electrophoresis [10]. 3sS-labeled L.cysteine, 96% radiochemical purity, was obtained from Amersham/Searle, Arlington Heights, Ill. M e H g O H , 100.8% by titration, was obtained from Alfa Inorganicsi Beverly, Mass. Collection and fractionation of whole blood. The fish were anesthetized 244
with ethyl m-aminobenzoate methanesulfonate (MS-222; Sigma Chemical Co., St. Louis, Mo.), the caudal fin was severed and a volume of blood was collected in an equal volume of modified Alsever's solution [11]. The RBCs, isolated by centrifugation for 10 min at 2000 g, were washed once by resuspension in 2 vol. of the Alsever's solution and lysed in 2 vol. of distilled water. The lysate was incubated for 6 h at 5 ° with gentle stirring and the stroma removed b y centrifugation at 40 000 g for 10 min. The stromal pellet was discarded except in one study in which its Hg content was measured. In this case, it was freed of residual hemoglobin by washing 4 times in 5 vol. 0 . 0 0 1 M N a H 2 P O 4 / N a 2 H P O 4 buffer, pH 7.4 containing 0.1% NaC1. Protein concentration measurements. Total soluble protein concentration was measured by the colorimetric procedure of L o w r y et al. [12]. Total nitrogen was determined by the Kjeldahl method [13]. The concentration of hemoglobin was determined spectrophotometrically employing the absorbance of cyanmethemoglobin at 540 mp [14]. The latter m e t h o d assumes a molecular weight for hemoglobin of 66 000. MeHg administration. MeHg was administered intragastrically through a stomach tube [1]. The dose solution (Me2°SHgC1 plus carrier MeHgOH in 0.14 M NaC1) contained 4.0 mg Hg/kg b o d y weight (bw) and 3.3 pCi/kg bw. Me 20 a Hg b o u n d to 35 S-labeled L-cysteine was administered intracardially. The injection solution contained a 9 molar excess of cysteine in 0 . 0 2 M N a H 2 P O 4 / N a 2 H P O a , pH 7.4, containing 0 . 0 1 M NaC1. The dose per kg bw was 0.01 mg Hg, 0.045 mg cysteine, 2.5 pCi 2 ° a H g and 24.2 uCi aSS. Tissue preparation. Livers were homogenized in a Virtis Model 60K Homogenizer (Virtis Co., Inc., Gardiner, N.Y.) at 45 000 rpm for 2 min. The tissue : buffer ratio was 1 : 1.5 (w/v). The buffers employed were 0.1 M Tris--citrate, pH 6.8, in preparation for gel filtration chromatography and 0 . 0 2 M N a H 2 P O a / N a 2 H P O 4 , pH 7.3, containing 0 . 1 4 M NaC1, for study of MeHg efflux from RBCs. Soluble protein extracts were prepared b y incubating the homogenates at 5 ° for 10 min and centrifuging for 20 min at 40 000 g. Measurement of beta-radioactivity. Beta-radioactivity was measured in a Beckman Model LS-230 Liquid Scintillation System. Column chromatographic fractions were dissolved in Scintisol-Complete (Isolab Inc., Akron, Ohio). Fractions containing hemoglobin were bleached with H 2 0 u before analysis. Cysteine levels were determined by measurement of the beta-radioactivity emitted by 35 S. The problem arising from the fact that both 203 Hg and a 5 S emit beta-radiation at approximately the same energy level was overcome by: (i) making the relative concentration of a 5 S in the injection solution very high with respect to 2°SHg and (ii) subtracting the betaradioactivity emitted b y 2°31-Ig from the total beta-radioactivity. The latter was accomplished with the data obtained by analyzing a standard 245
solution of Me 20 s HgCI for beta- and gamma-activity at the time of tissue analysis. The cpm of beta-activity per cpm of gamma-activity for 203Hg was used to calculate the beta-cpm contributed by 2 o s Hg in a tissue sample based on the gamma-cpm of the sample. The quench characteristics of the standard solution and tissue sample solution were identical. Amperometric titration of hemoglobin. The number of --SH groups per molecule of rainbow trout hemoglobin was determined by amperometric titration with MeHgC1 at the dropping mercury electrode. The method has been described in detail by Leach [15]. A conventional 3-electrode polarograph with operational ampl~iers was employed [16]. Amperage was recorded on a Model 1130 XY Variplotter (Electronics Associates Inc., West Long Branch, N.J.). A stream of purified nitrogen was used to remove oxygen from the solution and to mix MeHgC1 after each addition. RESULTS
Uptake of MeHg by trout blood components. The uptake of Me2°3Hg by trout RBCs was studied in vitro following the addition of 5 ppm Hg as MeHgC1 to whole blood in an equal volume of Aisever's solution and incubation at 3 ° . RBCs and plasma of sample aliquots were isolated by centrifugation and analyzed for 2V3Hg as described in METHODS. Approx. 3 min after addition, 84% of the Hg was found in the washed RBCs. Uptake reached 89% after 1 h and remained constant for the following 2 h. In vivo, almost 95% of whole blood Hg was contained in the soluble contents of the RBCs 2 weeks after an intragastric dose of Me2°3HgCl (Table I). The plasma contained ~2% of the whole blood Hg and
THE ERYTHROCYTE TRANSPORT AND T R A N S F E R OF METHYLMERCURY TO THE TISSUES OF THE RAINBOW TROUT ( $ A L M O GAIRDNERI) *
F.J. GIBLIN and EDWARD J. MASSARO** Department of Biochemistry, State University of New York at Buffalo, Buffalo, N.Y. 14214 (U.S.A.)
(Received May 14th, 1975) (Revision received July 17th, 1975) (Accepted August 4th, 1975)
SUMMARY Methylmercury (MeHg) was found to be taken up rapidly and almost completely by t r o u t red blood cells (RBC) both in vitro and in vivo. The binding of MeHg within the RBC was freely reversible both in vitro, as shown by the efflux of MeHg from RBCs suspended in protein solutions, and in vivo following intracardial (i.c.) injection of RBC-bound MeHg. Hemoglobin (Hb) appeared to be the main MeHg transport protein in t r o u t blood since it bound 90% of whole blood Hg following an intragastric dose of Me2°aHgC1. MeHg, injected i.c. as MeHgS-cysteine, was f o u n d to be present in blood bound almost completely to hemoglobin 10 days postinjection. This suggests an ability of hemoglobin to compete for and bind MeHg bound to other sulfhydryl (--SH) compounds. The number of reactive --SH groups per molecule of t r o u t Hb was determined to be 4 by amperometric titration with MeHgC1. The concentration of Hb reactive--SH groups in the t r o u t RBC was calculated to be at least 20 mM. This accounts for the high affinity of the RBC for MeHg.
* A preliminary report of this work was presented at the 58th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.J., April 7--12, 1974. ** To whom all correspondence should be addressed. Abbreviations: RBC, red blood cells;--SH, sulfhydryl.
243
INTRODUCTION It has been firmly established that fishes, shellfishes and other aquatic organisms take up MeHg from their environment and sequester and concentrate it in their tissues [1--3]. Methylmercury is a potent neurotoxin and consumption of contaminated fishes and shellfishes has resulted in massive incidents of human intoxication [3]. We have investigated the pharmacodynamics of MeHg in a model piscine system, the rainbow trout (Salmo gairdneri) [1]. The blood of the rainbow trout was observed to concentrate methylmercury (MeHg) to a greater extent than any other tissue. A high affinity of MeHg for the blood has been reported to occur in a wide variety of other species and, apparently, is a general phenomenon that may relate to the unique toxic effects exhibited by this compound [2,3]. The blood element responsible for concentrating MeHg has been determined to be the RBC [3]. Relatively little is known concerning the mechanism of uptake of MeHg by the RBC, the binding of MeHg within the cell or the transport of MeHg from the RBC to tissues. Considerably more information is available concerning the interactions of inorganic Hg and the RBC. The status of this knowledge has been reviewed recently by Passow [4]. MeHg binds specifically to protein --SH groups as is expected from its high association constant with the - S H group of cysteine [5,6]. The - S H groups of hemoglobin have been shown to bind organic Hg strongly [7]; but little is known of the extent of MeHg binding to hemoglobin or of the reversibility of the MeHg-hemoglobin bond within the RBC. Work by Clarkson et al. [8] and White and Rothstein [9] has suggested that M e H g can be released from whole blood and RBCs. This work was u n d e ~ e n to elucidate the extent of binding of M e H g to hemoglobin in the rainbow trout, the reversibility of the MeHg-hemoglobin bond in vivo and in vitro and the role of hemoglobin in the transportation of M e H g to the tissues. METHODS
Animals, tissue sampling, Hg analysis. Hatchery.reared rainbow trout, averaging 400 g in weight, were o b ~ n e d from the New York state Hatchery at Caledonia, New York and maintained as previously described [1]. The collection of tissue samples and the determination of their Hg content by gamma-scintillation spectrometry have also been described [1]. Reagents. 2°3Hg-labeled MeHgCl was obtained from N e w England Nuclear, Boston, Mass. Its radiochemical purity was > 9 5 % as determined by paper electrophoresis [10]. 3sS-labeled L.cysteine, 96% radiochemical purity, was obtained from Amersham/Searle, Arlington Heights, Ill. M e H g O H , 100.8% by titration, was obtained from Alfa Inorganicsi Beverly, Mass. Collection and fractionation of whole blood. The fish were anesthetized 244
with ethyl m-aminobenzoate methanesulfonate (MS-222; Sigma Chemical Co., St. Louis, Mo.), the caudal fin was severed and a volume of blood was collected in an equal volume of modified Alsever's solution [11]. The RBCs, isolated by centrifugation for 10 min at 2000 g, were washed once by resuspension in 2 vol. of the Alsever's solution and lysed in 2 vol. of distilled water. The lysate was incubated for 6 h at 5 ° with gentle stirring and the stroma removed b y centrifugation at 40 000 g for 10 min. The stromal pellet was discarded except in one study in which its Hg content was measured. In this case, it was freed of residual hemoglobin by washing 4 times in 5 vol. 0 . 0 0 1 M N a H 2 P O 4 / N a 2 H P O 4 buffer, pH 7.4 containing 0.1% NaC1. Protein concentration measurements. Total soluble protein concentration was measured by the colorimetric procedure of L o w r y et al. [12]. Total nitrogen was determined by the Kjeldahl method [13]. The concentration of hemoglobin was determined spectrophotometrically employing the absorbance of cyanmethemoglobin at 540 mp [14]. The latter m e t h o d assumes a molecular weight for hemoglobin of 66 000. MeHg administration. MeHg was administered intragastrically through a stomach tube [1]. The dose solution (Me2°SHgC1 plus carrier MeHgOH in 0.14 M NaC1) contained 4.0 mg Hg/kg b o d y weight (bw) and 3.3 pCi/kg bw. Me 20 a Hg b o u n d to 35 S-labeled L-cysteine was administered intracardially. The injection solution contained a 9 molar excess of cysteine in 0 . 0 2 M N a H 2 P O 4 / N a 2 H P O a , pH 7.4, containing 0 . 0 1 M NaC1. The dose per kg bw was 0.01 mg Hg, 0.045 mg cysteine, 2.5 pCi 2 ° a H g and 24.2 uCi aSS. Tissue preparation. Livers were homogenized in a Virtis Model 60K Homogenizer (Virtis Co., Inc., Gardiner, N.Y.) at 45 000 rpm for 2 min. The tissue : buffer ratio was 1 : 1.5 (w/v). The buffers employed were 0.1 M Tris--citrate, pH 6.8, in preparation for gel filtration chromatography and 0 . 0 2 M N a H 2 P O a / N a 2 H P O 4 , pH 7.3, containing 0 . 1 4 M NaC1, for study of MeHg efflux from RBCs. Soluble protein extracts were prepared b y incubating the homogenates at 5 ° for 10 min and centrifuging for 20 min at 40 000 g. Measurement of beta-radioactivity. Beta-radioactivity was measured in a Beckman Model LS-230 Liquid Scintillation System. Column chromatographic fractions were dissolved in Scintisol-Complete (Isolab Inc., Akron, Ohio). Fractions containing hemoglobin were bleached with H 2 0 u before analysis. Cysteine levels were determined by measurement of the beta-radioactivity emitted by 35 S. The problem arising from the fact that both 203 Hg and a 5 S emit beta-radiation at approximately the same energy level was overcome by: (i) making the relative concentration of a 5 S in the injection solution very high with respect to 2°SHg and (ii) subtracting the betaradioactivity emitted b y 2°31-Ig from the total beta-radioactivity. The latter was accomplished with the data obtained by analyzing a standard 245
solution of Me 20 s HgCI for beta- and gamma-activity at the time of tissue analysis. The cpm of beta-activity per cpm of gamma-activity for 203Hg was used to calculate the beta-cpm contributed by 2 o s Hg in a tissue sample based on the gamma-cpm of the sample. The quench characteristics of the standard solution and tissue sample solution were identical. Amperometric titration of hemoglobin. The number of --SH groups per molecule of rainbow trout hemoglobin was determined by amperometric titration with MeHgC1 at the dropping mercury electrode. The method has been described in detail by Leach [15]. A conventional 3-electrode polarograph with operational ampl~iers was employed [16]. Amperage was recorded on a Model 1130 XY Variplotter (Electronics Associates Inc., West Long Branch, N.J.). A stream of purified nitrogen was used to remove oxygen from the solution and to mix MeHgC1 after each addition. RESULTS
Uptake of MeHg by trout blood components. The uptake of Me2°3Hg by trout RBCs was studied in vitro following the addition of 5 ppm Hg as MeHgC1 to whole blood in an equal volume of Aisever's solution and incubation at 3 ° . RBCs and plasma of sample aliquots were isolated by centrifugation and analyzed for 2V3Hg as described in METHODS. Approx. 3 min after addition, 84% of the Hg was found in the washed RBCs. Uptake reached 89% after 1 h and remained constant for the following 2 h. In vivo, almost 95% of whole blood Hg was contained in the soluble contents of the RBCs 2 weeks after an intragastric dose of Me2°3HgCl (Table I). The plasma contained ~2% of the whole blood Hg and
