Properties of the cadmium and selenium complex formed in rat plasma in vivo and in vitro

Chem.-Biol. Interactions, 23 (1978) 171--183 © Elsevier/North-Holland Scientific Publishers Ltd.

171

PROPERTIES OF THE CADMIUM AND SELENIUM COMPLEX FORMED IN RAT PLASMA IN VIVO AND IN VITRO T.A. GASIEWICZ* and J.C. SMITH** Environmental Health Sciences Center and the Department of Pharmacology and Toxicology, University of Rochester School o f Medicine and Dentistry, Rochester, N.Y. 14642 (U.S.A.) (Received February 3rd, 1978) (Revision received May 12th, 1978) (Accepted May 21st, 1978)

SUMMARY

Following the simultaneous subcutaneous administration of CdC12 and Na~SeO~ to rats, evidence of a Cd-Se complex was detected in plasma by gel filtration chromatography. A similar complex was found in plasma after incubation of selenite, Cd, rat erythrocytes, and plasma in vitro, and after incubation of H:Se, Cd, and plasma in vitro. No interaction of selenite, selenate, or selenodiglutathione with Cd and plasma in the absence o f erythrocytes in. vitro was noted. Characterization by gel filtration, ionexchange chromatography, affinity chromatography, and ammonium sulfate fractionation showed that these Cd~e complexes are similar. The results support the hypothesis that H:Se or a similarly reduced selenide is the product of selenite metabolism by rat erythrocytes. Hydrogen selenide also altered the distribution of inorganic mercury in rat plasma in vitro in such a way that the apparent molecular weights of the Se-Hg and Cd-Se complexes associated with protein were similar. Hydrogen selenide had no effect upon the distribution of methylmercury in plasma. The stability of the Cd-Se complex in plasma depended upon the integrity of the native protein components, as shown by incubation with Proteinase K. The properties of the complex suggested that it existed in a single form associated with different plasma components under various conditions.

INTRODUCTION

Following the observation of Kar et al. [1] that testicular lesions in rodents produced by the acute administration of cadmium could be preven* Present address: Center in Toxicology, Department of Biochemistry, Vanderbilt University, Nashville, Tenn. (U.S.A.) ** To whom reprint requests should be addressed. Abbreviations: GSSeSG, selenodiglutathione; MeHg, methy mercury.

172 ted by the administration of selenium compounds, a number of workers [2--5] demonstrated the sparing action, especially of selenite, on many of the biological effects of Cd. The reversal of Se toxicity by Cd has also been demonstrated [6]. Although Se completely protected against Cd-induced testicular damage in rats, the uptake of Cd by testicular tissue was increased [7]. This suggested that Se diverted Cd from its usual target in the testes. A Se-provoked alteration in the distribution of Cd within the soluble protein fractions of the testes, liver, and kidney has been demonstrated [8,9]. Cadmium concentrations in other tissues were also altered, but the greatest change was in the plasma, in which Cd increased 30- to 50-fold over control [4,10]. Gasiewicz and Smith [11] presented evidence that the increase in plasma Cd concentration was due to the formation of the Cd-Se complex having an atomic ratio of approx. 1 : 1. A similar complex was formed in plasma following the incubation of selenite and Cd with rat erythrocytes and plasma [11], suggesting that the metabolism of selenite by erythrocytes had occurred. Hydrogen selenide or a similarly reduced selenide appears to be the ultimate product of selenite metabolism by rat erythrocytes [ 12]. The present paper reports on some of the properties of the Cd-Se complex formed under various conditions in vitro and in vivo. Evidence is presented that H:Se reacts with Cd in plasma to produce this complex. An abstract describing a portion of these findings has been published elsewhere [13]. MATERIALS AND METHODS Male Sprague--Dawley rats (250--300 g) were obtained from Holtzmann (Madison, Wisc.). Charles River rat chow and distilled water were provided ad libitum. 7SSe, 1°9Cd, and 2°3Hg were obtained as H2SeO3, CdC12, HgCl:, and CH3HgC1 from New England Nuclear. 7SSe-selenite was purified by the method described previously [12]. Carrier solutions were prepared by the addition of ultrapure Na:SeO3, CdCl2, HgCI~ or CH3HgC1 obtained from Ventron Corporation. Animals were injected with Cd and Se solutions simultaneously and subcutaneously in opposite thighs. The animals were exsanguinated by cardiac puncture under, light ether anesthesia. Erythrocytes were collected, washed in isotonic phosphate-NaC1 (pH 7.4) buffer and prepared for incubation in vitro as described previously [12]. Sephadex G-150, G-200, QAE Sephadex A-50, Blue Sepharose CL-6B resins and chromatographic columns were obtained from Pharmacia Fine Chemicals. Sample volumes ranged between 0.8 and 2.5 ml of plasma. The eluate was monitored for absorbance at 280 nm. Fractions were collected and radiochemical determinations of Se, Cd, and Hg were made as described previously [11]. Recoveries on all columns for l°gCd, ~SSe and :°~Hg ranged between 91 and 106%. All data were adjusted for the recovery of the particular experiment. In determining molecular weight, Columns were calibrated by the method of Andrews [ 14]. Selenodiglutathione (GSSeSG) was synthesized and isolated by the

173 method of Ganther [15]. Hydrogen selenide was generated by a modification of the method of Diplock et al. [16], as described previously [12]. Proteinase K from Tritirachium album was purchased from Boehringer Mannheim. Conductivity of eluate fractions from Sephadex QAE ion exchange chromatography was measured with a Yellow Springs Instrument conductivity bridge. All measurements were standardized against known concer~trations of the same buffer. Ammonium sulfate fractionation of diluted plasma samples or of pooled chromatography eluates were performed by a modified method of Cohn et al. [17]. RESULTS

Formation o f a Cd-Se complex in vitro As previously reported [11], selenite did not produce a complex with Cd when incubated with plasma. However, when washed rat erythrocytes were added, subsequent chromatography of plasma showed a single peak of Cd and Se at approximately 130 000, similar to that formed in vivo [11]. The atomic ratio of Cd and Se in these fractions was approximately 1 : 1. The interaction of Cd and Se in vitro occurred only when CdC12 was added to the rat erythrocyte and plasma preparations simultaneously or before the addition of selenite. When selenite (10.0 nmol/ml plasma) was added 15--60 min before.CdC12 (10.0 nmol/ml plasma), no interaction was noted, although Se was bound to plasma proteins. These results suggested that selenite was metabolized by rat erythrocytes to a compound which when bound to plasma components becomes unavailable to interact with Cd. This apparently was not the case for Cd. Further studies were performed to investigate the alteration in the formation of the complex with the change in Cd concentration. For these experiments a selenite concentration of 30.0 nmol/ml plasma was used. Cadmium concentrations varied between 0.7 and 30.0 nmol/ml plasma. At concentrations lower than approximately 1.0 nmol/ml plasma, Cd apparently became bound to sites in plasma which made it unavailable to interact with Se (Fig. 1). When 75.0 nmol of H~Se were bubbled into 2.5 ml of plasma, subsequent chromatography [12] showed Se to be distributed in a pattern similar to that bound in plasma in vivo or in the presence of plasma, erythrocytes, and selenite in ~itro [11]. When CdC12 was added to the preparations before the addition of H~Se, the distribution of both Se and Cd in plasma was altered from fractions of lower mol wt. to those of 130 000 (Fig. 2). The atomic ratio of Cd to Se in these fractions was approximately 1 : 1, just as when Se and Cd interact in vivo or in the presence of erythrocytes in vitro. No such interaction was noted when GSSeSG (60.0 nmol/ml plasma) or selenate (60.0 nmol/ml plasma) was incubated with CdCI~ and plasma alone or in the presence of erythrocytes. Selenium from GSSeSG, however, was bound to plasma components in a pattern similar to that following the incubation of H2Se with plasma [12].

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Fig. 1. Plasma protein distribution of Cd and Se after incubation of 2.0 ml plasma, 2.0 ml washed erythrocytes, and 30.0 nmol selenite/ml plasma with (a) 30.0 nmol CdCl~/ml plasma; (b) 7.0 nmol CdCl~/ml plasma; (c) 3.0 nmol CdCl~/ml plasma; and (d) 0.7 nmol CdCl2/ml plasma for 1 h at 37°C. Sephadex G-200 was used. Column dimensions were 2.6 × 70 cm. V o = 113 ml; V t = 325 ml. Flow of 0.1 M Tris--HC1, pH 8.0, was 15.5 ml/h.

The e[feet o f buffer pH on the distribution o f Cd and Se in plasma proteins When H2Se was bubbled into plasma containing CdC12 and applied to a Sephadex G-150 column equilibrated with a 0.1 M Tris--HC1, pH 6.5 buffer, the resulting Cd-Se complex appeared in fractions with an approximate mol. wt. of 330 000 (Fig. 3}. The Cd : Se atomic ratio of approximately 1 : 1 was

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maintained. A similar pattern of Cd and Se distribution in plasma was noted using the following buffers: 1.0 M NaC1, 0.1 M Tris--HC1, pH 6.5; 0.1 M Tris--HC1, pH 7.2 or 7.4; 0.1 M ammonium formate, pH 7.4; or 0.145 M NaC1, 0.01 M potassium phosphate, pH 7.4. These results were in contrast to that in which only the 130 000 peak appeared when plasma was fractionated on columns equilibrated with a pH 8.0 buffer (Fig. 2). There was no apparent alteration in the pattern of absorbance at 280 nm. When selenite (10.0 nmol/ml plasma) and CdC12 (10.0 nmol/ml plasma) were incubated with erythrocytes (1.5 ml) and plasma (0.5 ml) for 60 min at 37°C, chromatography of the plasma using a 0.1 M Tris--HC1, pH 6.5, buffer revealed results similar to that in Fig. 3. Plasma from rats injected E

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Fig. 3. P l a s m a p r o t e i n d i s t r i b u t i o n o f Cd a n d Se a f t e r b u b b l i n g 120 n m o l H=Se i n t o 3.0 m l p l a s m a c o n t a i n i n g 75.0 n m o l CdCI=. A S e p h a d e x G - 1 5 0 c o l u m n e q u i l i b r a t e d w i t h 0.1 M Tris--HCl, p H 6.5, w a s used. C o n d i t i o n s were similar t o t h o s e d e s c r i b e d in t h e legend for Fig. 2.

176

subcutaneously with 20.0 nmol selenite/kg and 20.0 pmol CdC12/kg, 1, 12, or 24 h before killing, showed a single Cd-Se peak at 330 000, using a pH 6.5 buffer for chromatography. Using a pH 8.0 buffer, peaks of 330 000 and 130 000 have been noted previously in vivo [11].

Ion-exchange chromatography of the Cd-Se complex in plasma Ion exchange chromatography of plasma was carried out using Sephadex QAE A-50 ion exchange resin equilibrated with 0.1 M Tris--HC1, pH 6.5. Following the application of whole plasma or a diluted sample, the column ~.

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177 was eluted with a linear gradient of 0.1--0.4 M Tris--HC1, pH 6.5. Fig. 4a depicts the ion exchange properties of the C d ~ e complex produced in plasma under conditions in vivo and in vitro. In all cases, a single Cd-Se peak eluted at approximately 0.12--0.13 M Tris--HC1. These results support the proposal that the complexes formed under conditions in vivo and in vitro were similar. The large protein peak at fractions 55--73 was tentatively identified as albumin b y the chromatography of bovine serum albumin under the same conditions. Figs. 4b and 4c show the distribution o f Se and Cd after ion exchange chromatography of plasma from erythrocyte and plasma preparations incubated with Cd or selenite. Both Cd and Se were found mainly in the albumin fractions.

Other characteristics o f the Cd~e complex in plasma In addition to the properties of the Cd-Se complex under conditions o f varying buffer pH and ion exchange chromatography, other characteristics were noted. These properties were c o m m o n among the Cd-Se complexes in plasma formed in vivo or in vitro. These results can be found in greater detail elsewhere [18]. The Cd-Se complex could be concentrated and nearly quantitatively recovered b y the use of ammonium sulfate precipitation. At 2.60--2.80 M ammonium sulfate, precipitation of both Cd and Se was nearly complete (Table I). R e ~ h r o m a t o g r a p h y o f the precipitate dissolved in 0ol M Tris--HC1, pH 6.5, showed the complex to maintain its molecular weight and Cd : Se ratio characteristics. It was of interest to determine whether the stability of the Cd-Se complex was dependent upon the integrity o f protein in plasma. For these experiments plasma containing the complex was incubated in a 0.1 M Tris--HC1, pH 7.0, buffer for 2 h at 37°C with or without 1.5 mg of proteinase K. The preparations without proteinase K showed a chromatographic pattern of Cd, Se, and protein similar to that in Fig. 3. With proteinase K, a marked alteration in the protein, Cd, and Se patterns was observed (Fig. 5). TABLE I P R E C I P I T A T I O N O F T H E Cd-Se C O M P L E X

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Sepharose covalently linked with Blue Dextran has been used for the removal of albumin from plasma [19]. Whole plasma containing the Cd~e complex was applied to a Blue Sepharose CL-6B column equilibrated with 0.05 M Tris--HC1, 0.1 M KC1, pH 7.0. Over 95% of both Cd and Se remained on the column. The radioactivity and an associated protein peak was removed by eluting with 0.05 M Tris--HC1, 1.5 M KC1, pH 7.0. The protein was tentatively identified as albumin by disc gel electrophoresis and gel chromatography. Both Cd and Se migrated with the albumin peak after Sephadex G-150 chromatography, but not with a 330 000 peak as noted using whole plasma (Fig. 3). However, when fractions from gel filtration corresponding to the 330 000 C d ~ e peak were pooled, concentrated with ammonium sulfate, dissolved in 0.1 M Tris--HC1, pH 6.5, buffer and applied to a Blue Sepharose column, over 90% of the Cd and Se eluted at the void volume of the column. Only a small amount of radioactivity was associated with a small albumin p e a k a f t e r elution of:the column with 0.05 M Tris-HC1, 1.5 M KC1, pH 7.0. These results suggested that while the Cd-Se complex was not normally associated with albumin or a polymer of albumin in whole plasma, the conditions of Blue Sepharose chromatography altered the binding properties so that the complex became associated with albumin.

Reaction o f H:Se with inorganic and organic mercury compounds in plasma in vitro Experiments were performed to determine if H~Se interacts with Hg or MeHg and plasma in vitro in a manner similar to the interaction with Cd. For the control experiments, nitrogen was bubbled for 20 min into plasma containing only MeHgC1 or HgCI~. Hydrogen selenide produced a marked alteration in the distribution of Hg in plasma (Fig. 6). An Hg-Se peak,

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similar to the Cd-Se peak previously noted, was found at approximately 330 000. The Hg : Se atomic ratio in this peak was approximately 1 : 1. There was no apparent interaction of H~Se with MeHg under the same conditions (results not shown}. DISCUSSION

These studies show that the Cd-Se complex produced in plasma in vivo following the injection of Cd and selenite into rats has gel filtration and ion exchange properties similar to those of the complex formed in plasma by the incubation of selenite with erythrocytes, plasma, and Cd in vitro or after the incubation o f H2Se, Cd, and plasma. The results support the previous hypothesis [12] that H2Se or a similarly reduced compound is the product of selenite metabolism by rat erythrocytes in vitro. Following intravenous [25] or subcutaneous [18] injections, previous studies have found that at initial times the largest percentage of the selenite dose was associated with the erythrocyte volume. It seems likely that the metabolism of selenite by rat erythrocytes in vivo plays a significant role in the formation of the Cd-Se complex in plasma. A similar pathway of selenite metabolism leading to the interaction with Cd may also occur in other tissues [21,22].

180 The concentrations of selenite, .CdCI~ or H~Se used in the experiments in vitro (10--100 nmol/ml plasma) are comparable to those found in plasma after Cd and Se administration in vivo [11]. The formation of the complex in vivo was directly related to the dose of selenite and was noted in plasma 15 min--24 h after treatment [11]. A mechanism whereby Se protects against the acute toxic effect of Cd may involve the formation of a Cd-Se complex subsequently altering its tissue distribution. Of the Se compounds tested, only Ht~Se produced this complex with Cd. However, the chemical reactivity of H~Se and its further metabolism to products which are excreted or utilized for essential biological functions limits the availability of H~Se to react with Cd in vivo. One would predict that Se would be effective in preventing the acute toxicity of Cd only when administered at or near the time of Cd exposure. Mason and Young [3] have obtained such results, using selenite. In the present studies, the Cd~e complex did not form when selenite was incubated with erythrocytes and plasma prior to the addition of Cd. The effects of selenite on Cd toxicity and tissue distribution have been shown to be similar to its effects on inorganic mercury (Hg) toxicity and distribution [10,20,21]. Although selenite appeared to alter the toxicity of methyimercury (MeHg), the effect on tissue distribution was unlike that with inorganic Hg or Cd [22--24]. While selenite increased the plasma levels of Cd or Hg, it had little effect on the plasma concentration of MeHg. In a reaction similar to that with Cd, HI~Se produced a marked alteration in the distribution of Hg, but not MeHg, in plasma. Burk et al. [25] have shown that the distribution of Hg and Se in plasma is altered when both were administered to rats. The ratio of Hg to Se in these plasma fractions was also 1 : 1. The authors did not report the mol. wt. of this peak; however, the chromatographic distribution was similar to that in the present study. Fang [26] has shown that the distribution, of Hg in rat tissue is altered by selenomethionine, selenocysteine, selenate, and selenite. Both selenate and selenite are metabolized to methylated selenides via an ~acid-volatile Se moiety, presumed to be H~Se [27--29]. Similarly, selenomethionine, selenocysteine, and S-methylselenocysteine are metabolized to the trimethylselenonium ion in vivo [30]. It is suggested that metabolism of these Se compounds takes place through similar mechanisms via the selenide intermediate. The ability of these compounds to alter Cd or Hg distribution may depend upon the difference in pharmacokinetics of the parent Se compound. It is apparent from the experiments with proteinase K that the stability of the Cd-Se complex in plasma was dependent upon the integrity of the protein components. The properties of the complex under various chromatographic conditions also suggest that it becomes reversibly associated with different proteins. Using a pH 8.0 buffer for chromatography, Cd-Se peaks at 130 000 and 330 000 appeared in plasma following the administration of Cd and selenite to rats. The Cd-Se peak at 330 000 appeared to be saturated at a concentration in plasma of approximately 30.0 nmol of Cd and Se per ml of plasma [11]. When a pH 6.5 buffer was used, a higher concentration

181

o f Cd and Se (up to 0.13 pmol/ml of plasma) was associated with this peak. It seems unlikely that sites on the 330 000 species enough to accommodate 100 ~mol of the complex became available through a pH change of less than 1.5 units. Although polymerization is an alternative explanation, the complete polymerization of a molecule by a similar pH change also appears unlikely. Experiments with Blue Sepharose showed that the distribution of the complex could be altered by changing conditions other than pH. In this case, the formation of a stationary albumin phase resulted in the altered distribution and the subsequent association with albumin. These results coupled with the ammonium sulfate precipitation data imply that this complex exists in a single form. The character of the complex, rather than the protein(s) it may be associated with, may play an important role not only in its properties in vitro b u t in its ultimate fate in intact biological systems. Like cadmium sulfide, cadmium selenide (CdSe) is virtually insoluble in water [31]. When 7SSe-labeled H~Se was bubbled into a buffer solution containing l°gCd-labeled CdCl~, over 90% of the radio-activity was associated with a precipitate following centrifugation at 2000 g for 10 min. The high affinity of Se -2 for Cd +2 and the stability of CdSe suggest that the complex forms even in the presence of plasma proteins. Many substances, including proteins, carbohydrates, and lipids, have been used to stabilize colloidal metal preparations [32]. The formation of a colloidal Cd-Se complex, the stability of which is dependent upon proteins, seems to be a reasonable possibility. ACKNOWLEDGEMENTS

This research was supported in part by grants GM-01781, ES-01247, and ES-01248 from the National Institutes of Health. REFERENCES 1 A.B. Kar, R.P. Das and F.N.I. Mukerji, Prevention of cadmium-induced changes in the gonads o~" rats by zinc and selenium -- a study in antagonism between metals in the biological system, Proc. Nat. Inst. Sci. India, 26B (1960) 40. 2 J. Parizek, I. Ostadalova, I. Benes and A. Babicky, Pregnancy and trace elements: the protective effect of compounds of an essential trace element-selenium against the peculiar toxic effects of cadmium during pregnancy, J. Reprod. Fert., 16 (1968) 507. 3 K.E. Mason and J.O. Young, Effectiveness of selenium and zinc in protecting against cadmium-induced injury of the rat testes, in Selenium in Biomedicine, O.H. Muth (Ed.), The AVI Publishing Company, Inc., Westport, Conn., 1967, p. 383. 4 S.A. Gunn, T.C. Gould and W.A.D. Anderson, The selectivity of response to cadmium injury and various protective measures, J. Path. Bact., 96 (1968) 89. 5 J. Parizek, I. Ostadalova, I. Benes and J. Pitha, The effect of subcutaneous injection of cadmium salts on the ovaries of adult rats in persistent oestrus, J. Reprod. Fert., 17 (1968) 559. 6 C.H. Hill, Reversal of selenium toxicity in chicks by mercury, copper, and cadmium, J. Nutr., 104 (1974) 593.

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