Archives of
Arch. Toxicol. 38, 217--228 (1977)
TOXICOLOGY 9 by Springer-Verlag 1977
Binding of Mercury and Selenium in Subcellular Fractions of Rat Liver and Kidneys Following Separate and Joint Administration* ElSbieta Komsta-Szumska and Jadwiga Chmielnicka** Department of Toxicology Chemistry, Institute of Environmental Research and Bioanalysis, Medical Academy of L6d$, Narutowicza 120 A, 90-145 Ltd$, Poland
Binding of Mercury and Selenium in Kidneys and Liver Abstract. The distribution of mercury and selenium has been examined in subcellular fractions of rat liver and kidneys in prolonged exposure to HgC12 and Na2SeO 3 administered separately and simultaneously. The molar ratio of mercury and selenium concentrations in subcellular fractions of the organs examined varied considerably. Selenium displaced mercury from the soluble kidney fraction bound mainly with metaUothionein to the nonhistone protein fraction of liver nuclei. The Hg-stimulated biosynthesis of metallothionein has been eliminated under the influence of selenium.
Key words: Interaction -- Mercury -- Selenium - Inorganic - Subcellular distribution - Mercury - Selenium -- Biocomplexes.
Zusammenfassung. Die Verteilung von Quecksilber und Selen wurde in zellfreien Fraktionen von Rattenleber und -nieren untersucht, nachdem getrennt oder zugleich HgC12 bzw. Na2SeO a zwei Wochen lang (0,5 mg Hg/kg jeden zweiten Tag i.v., 0,5 mg Se/kg jeden Tag per os, molares Verh~iltnis 1 : 5) verabreicht worden waren. Das molare Verh/iltnis der Hg- und Se-Konzentrationen schwankte in den zellfreien Fraktionen der untersuchten Organe betr/ichtlich. Selen verdr/ingte Quecksilber aus der 16slichen Nierenfraktion. Quecksilber war haupts/ichlich mit Metallothionein an die histonfreie Eiweil3fraktion der Leberzellkerne gebunden. Unter dem Einflu/3 yon Selen wurde die Hg-stimulierte Biosynthese yon Metallothionein unterbunden.
Introduction In the recent decade attention has been drawn to the interaction of selenium and mercury in vivo because of the significant role of these elements in the environmental * This work was supported by the Polish-Americanagreement No 05-009-2, with National Institute for Occupational Safety and Health, PHS, USA ** To whom offprint requests should be sent
218
El~bieta Komsta-Szumska and Jadwiga Chmielnicka
toxicology. Post morten examination of some organs in humans occupationally exposed to mercury compounds have shown that an increased level of mercury in these organs was accompanied by an increased level of selenium (Kosta et al., 1975). Investigations by Parizek and Ostadalova (1967), Parizek et al. (1974) and G r o t h et al. (1973) indicated that selenium counteracts the toxic action of inorganic mercury decreasing the nephrotoxic effect and diminishing proteinuria. Diet enriched by selenium prevented toxic effects caused by organic and inorganic mercury compounds (Potter and Matrone, 1974; Ohi et al., 1975). Several authors (Parizek et al., 1969a, 1969b, 1971; Eybl et al., 1968; Moffit and Clary, 1974) drew attention to the redistribution in the organs and to an increased retention of mercury in animals under the influence of selenium. A single dose of selenium and mercury administered simultaneously increased retention of both elements in the organism, caused a redistribution of mercury in the organs (Chen et al., 1974), and in the blood plasma (Burk et al., 1974) as well as their diversion from low mol. weight to high molecular weight proteins. Similar findings were reported in our previous investigations ( K o m s t a - S z u m s k a et al., 1976). It is well k n o w n that mercury increases the retention of selenium in the tissues of animals, both after a single dose (Parizek et al., 1973) and in prolonged exposure to selenium (Levander and Argrett, 1969; Johnson and Pond, 1974). There are but few investigations on the subcellular distribution of this element, limited to liver (Burk et al., 1974). Our investigations aim at learning more about the mechanism of mercury and selenium interaction. In the present paper we are concerned with subcellular distribution of both these elements administered to animals separately and simultaneously in the course of repeated exposure.
Methods The experiment was performed on white female rats of Wistar strain, average body weight 200 g, fed standard LSM diet. The animals were divided into 4 groups, 6 rats each: the first group received 2~ the second group - 2~ and Na2SeO3, the third group 75Sel as Na2SeO3, the fourth group - - 7 5 S e and HgC12. Mercuric chloride, 0.5 mg Hg/kg, was administered to the tail vein every other day for 2 weeks, activity 40 ~Ci/dose. Sodium selenite was supplied per os every day in a daily dose of 0.5 mg/Se/kg, activity 20 ~Ci per dose. The animals were killed in ether narcosis 24 h after the last administration, and kidneys and liver were removed for examination. The fractioning of organs was made by the method of Schnaitman and Greenawalt (1968). The scissed organs were suspended in 2 volumes of solution A (220 mM-D-mannitol, 70 mM sucrose, 2 mM HEPES-N-2-hydroxyethylpiperazine-N-2-ethanosulphonicacid, 0.5 mg crystalline bovine serum albumin; solution pH was 7.4). The organs were homogenized, 10% homogenates were centrifuged at 600 x g for 15 min. In the sediment were obtained proteins of the nuclear fraction (N) which were separated by the technique of Teng et al. (1971) into proteins solublein 0.14 M NaC1 (No), histones (Nn) and nonhistone proteins (NN). The post-nuclear supernatant was centrifugedat 7000 x g for 15 min. The supernatant was drawn off, the sediment was suspended in solution A (1/4in proportion to the original volume of the homogenate), and centrifuged again at 7000 x g for 15 min. The sediment contained the mitochondrial fraction (M-heavy mitochondria). Post-mitochondrial supernatants were pooled and centrifuged for 15 min, at Product of Instytut Badafi J~drowych, Swierk, Poland. Activity 770 mCi/g
Binding of Mercury and Selenium in Kidneys and Liver
219
12,000 • g. The sediment consisted of lysosomes and light mitochondria (L). The remaining solution was centrifuged at 144,000 • g for 1 h. The sediment contained the microsomal fraction (P) and the supernatant (soluble fraction - S). In the homogenates and individual subcellular fractions mercury and selenium were determined by gamma counting in a scintillation counter USB-2. The time of counting was 100 or 60 s; precision + 4%. Protein was determined by the method of Lowry et ai. (1951). Proteins binding mercury and selenium in soluble fractions of rat kidneys and liver were analysed by gel f'dtration. The columns filled with Sephadex G-75 gel were calibrated with Dextran Blue and Cytochrome C; 0.1 M formate buffer of pH 8 was used as eluent. Eluate fractions of 5 cm3 each were collected at the speed - 1 cm3/min. Metallothionein in the homogenates of liver and kidneys was determined by the radiochemical method of Piotrowski et al. (1973) as modified by Zelazowski and Piotrowski (in press). The metallothionein standard was obtained in our laboratory Zelazowski et al., in press) from horse kidneys by means of a technique similar to that described by Pullido et al. (1967). The sample contained about 3 ~xM groups SH/mg proteins: in the conditions of analysis about 200 txg Hg was bound by 1 mg of protein.
Results Selenium administered simultaneously with m er cu r y in repeated exposure significantly altered the level o f m e r c u r y in the examined organs o f rats (Table 1). M e r c u r y supplied without selenium was deposited mainly in the kidneys -- 32%, with only 3.5% in the liver. In simultaneous exposure to m er cu r y and selenium the situation was reversed. A higher cumulation o f mer cu r y was found in the liver, 29% o f cumulative dose, the kidneys retained only 8.5%. Selenium alone reached a higher level in the liver than in the kidneys. I f administered simultaneously with m e r c u r y its level increased several times both in the kidneys and in the liver. In the absence o f selenium m o r e than 50% o f the total a m o u n t o f m e r c u r y retained in the kidneys was located in the soluble fraction (Table 2). U n d e r the influence o f selenium the level o f m e r c u r y in this fraction was diminished m o r e than 15 times. The a m o u n t o f m e r c u r y decreased also in the remaining fractions o f the kidneys except o f the nuclear fraction. In the absence o f m er cu r y nearly half o f selenium contained in the kidneys was located in the nuclear fraction. This lowest a m o u n t o f selenium was retained in microsomes while in other fractions it was
Table 1. The levels of mercury and selenite in % of the dose in the kidneys and liver of rats in the course of 2 weeks of application of mercury, selenite alone and of mercury and selenite administered simultaneously. Groups 6 rats each 2~ contents in % of the cumulative dose
75Se contents in % of the cumulative dose
Hg (alone)
Hg + Se
Se (alone)
Se + Hg
Kidney
32.0 (29.0--33.0)
8.5 (6.5--9.5)
0.5 (0.4--0.6)
2.0 (1.8--2.5)
Liver
3.5 (2.7--4.3)
29.0 (28.0--30.0)
1.7 (1.5--2.0)
5.5 (4.3--6.5)
220
El~bieta Komsta-Szumska and Jadwiga Chmielnicka
Table 2. Mercury and selenite content in the homogenate and subcellular fractions of rat kidneysa Fractions
2~
(alone)
2~
+ Se
75Se (alone)
75Se + Hg
~xg/g tissue Homogenate
140.0 (122.0--165.0)
41.0 (32.0--51.0)
3.8 (3.3-5.6)
18.0 (16.9--24.0)
Nuclear
21.0 (17.0--23.0)
22.4 (19.7--27.0)
1.8 (1.5--2.6)
11.0 (9.5-15.0)
Mitochondrial (heavy)
12.5 (10.0--18.0)
4.0 (3.0--5.0)
0.6 (0.5-0.7)
2.7 (2.3--4.0)
Light mitochondrial and lysosomal
15.0 (13.0-16.0)
3.0 (2.2--3.7)
0.6 (0.5-0.7)
0.5 (0.4-0.6)
Microsomal
11.0 (8.4--9.4)
6.0 (5.0--7.0)
O.1 (0.1--0.2)
1.0 (0.9--1.2)
Soluble
80.5 (72.0-87.0)
5.6 (4.3-6.1)
0.7 (0.6-0.9)
2.8 (2.7-2.9)
a
Values are the means of 6 determinations
Table 3. Mercury and selenite content in the homogenate and subcellular fractions of rat livera Fractions
2~
(alone)
2~
+ Se
75Se (alone)
75Se + Hg
~g/g tissue Homogenate
3.2 (2.9--3.4)
22.0 (18.0--26.0)
2.6 (1.8-3.4)
9.0 (8.0--10.0)
Nuclear
1.0 (0.9--1.4)
13.3 (11.5-17.0)
1.4 (1.3--1.7)
5.1 (4.4--5.6)
Mitochondrial (heavy)
0.3 (0.2--0.4)
4.5 (3.8--5.8)
0.2 (0.1--0.2)
1.7 (1.4--2.0)
Light mitochondrial and lysosomal
0.7 (0.5-0.9)
1.5 (1.4-1.9)
0.1 (0.1-0.2)
0.4 (0.3-0.5)
Microsomal
0.2 (0.1--0.2)
2.0 (1.8--2.6)
0.1 (0.1--0.2)
0.4 (0.4--0.5)
Soluble
1.0 (0.8--1.2)
0.7 (0.6--0.8)
0.8 (0.7-0.9)
1.4 (1.0--1.6)
a Values are the means of 6 determinations
evenly distributed. I n the presence of m e r c u r y the level o f selenium rose c o n s i d e r a b l y in all fractions: a b o u t 10 fold in the m i c r o s o m a l fraction a n d 4 - 6 times in other fractions. Nevertheless, the highest level o f selenium r e m a i n e d characteristic of the n u c l e a r fraction (11 pxg Se/g tissue). I n the liver (Table 3), m e r c u r y alone was b o u n d m a i n l y b y the n u c l e a r a n d soluble fractions. U n d e r the influence of selenium the level of m e r c u r y increased
221
Binding of Mercury and Selenium in Kidneys and Liver Table 4. Mercury and selenite content in the nuclear fractions of rat kidneysa Nuclear fraction
2~
(alone)
2~
+ Se
75Se (alone)
75Se + Hg
~g/mg protein Protein soluble in 0.14 N NaCI
0.09 (0.08--0.10)
0.24 (0.22--0.26)
0.03 (0.02--0.04)
0.05 (0.04--0.06)
Histone
0.20 (0.18--0.21) 0.17 (0.14--0.19)
0.06 (0.05--0.07) 0.19 (0.18--0.21)
0.02 (0.02--0.03) 0.01 (0.01--0.02)
0.06 (0.05--0.06) 0.10 (0.08--0.11)
Nonhistone
a Values are the means of 6 determinations Table 5. Mercury and selenite content in the nuclear fractions of rat livera Nuclear fraction
2~
(alone)
2~
+ Se
75Se (alone)
75Se + Hg
0.03 (0.02--0.03) 0.02 (0.01-0.02) 0.01 (0.01--0.02)
0.06 (0.05--0.07) 0.07 (0.05--0.08) 0.07 (0.06--0.08)
~g/mg protein Protein soluble in 0.14 N NaC1 Historic Nonhistone
0.02 (0.01--0.02) 0.01 (0.01--0102) 0.01 (0.01--0.02)
0.10 (0.09--0.11) 0.12 (0.11--0.13) 0.24 (0.22--0.26)
a Values are the means of 6 determinations
1 0 - 1 5 times in the nuclear, mitochondrial and microsomal fractions. In the absence of mercury selenium was located in the liver mainly in the nuclear and soluble fractions which is in agreement with the results obtained by Burk et al. (1974). Under the influence of mercury the level of selenium rose in all examined liver fractions, particularly in the nuclear and mitochondrial fractions. In the renal nuclei (Table 4) in the absence of selenium mercury was retained mainly by nonhistone proteins (71%). Selenium administered with mercury slightly hightened the content of mercury in proteins soluble in NaC1 and lowered level in histones. In general the coefficient ~g H g / m g of protein in the nuclear fraction of the kidneys did not change under the influence of selenium. In the presence of mercury also selenium contained in the kidney nuclei was bound mainly by nonhistone proteins. In liver nuclei (Table 5), mercury was also bound mainly by nonhistone proteins (55%) and under the influence of selenium the share of this subfraction rose to 77%. A very high coefficient, ~g H g / m g protein, was characteristic for this fraction of the liver, much exceeding that of nonhistone proteins of the kidney (Table 5). Figures 1 and 2 illustrate the distribution of mercury and selenium in the kidneys and liver calculated per weight of the organ. In Table 6 molar rations Hg/Se were listed for the homogenates and single cellular fractions of the kidneys and liver as well as for high molecular weight proteins in postmicrosomal fraction.
El~bieta Komsta-Szumska and Jadwiga Chmielnicka
222
~ 16o
~5 300
14o
~ 250
~120
200
100
150
80
B Hg alone B Hg+Se
60
I00 50 0
Kidney
2O
H
160[
N
M
L
l
P
S
300 Liver
250 20Q
100
150
80 6O
100 4O
5O 0
2O H
N
M
L
P
S
Fig. 1. The distribution of mercury (bLg Hg per organ) in the cellular fractions of rat kidneys and liver. Fraction symbols: H homogenate, N - nuclear, M -- heavy mitochondrial, L -- light mitochondrial and lysosomal, P -- microsomal, S -- soluble
Kidney 30
c5 6~
Se alone B Se+Hg
~2o
0
N
H
100
50
80
40
6O
30
40
20
20
10
.B t4
L
P
A] S
Liver
0
H
N
M
L
P
S
Fig. 2. The distribution of selenite (~g Se per organ) in the cellular fractions of rat kidneys and liver. Symbols see Figure 1
Binding of Mercury and Selenium in Kidneys and Liver
223
Table 6. Molar ratio Hg/Sea
Homogenate Fractions: Nuclear
Kidneys
Liver
0.84 (0.70--0.88)
0.90 (0.78--1.00)
0.73 (0.66-0.76) 1.83 (1.40--1.85) 0.46 (0.29--0.49) 0.66 (0.64-0.70) 0.56 (0.47--0.58) 2.54 (2.20--2.57) 2.20 (2.10--2.42) 0.76 (0.58--0.77) 2.50
Soluble protein Histone Nonhistone Heavy mitochondrial Light mitochondrial and lysosomal Microsomal Soluble High molecular weight proteins
1.00
(0.91--1.10) 0.54 (0.52-0.60) 0.50 (0.50-0.70) 1.66 (1.60--1.80) 1.00
(1.00--1.00) 1.50 (1.40--1.60) 1.80 (1.72--1.84) 0.20 (0.18--0.22) 1.00
a Values are the means of 6 determinations
60
,01' '00fj
u
~2o 0
I
1.06~t~--, ~,
20
30 ~I
r
20
50
~0 (el
1.0 .--.
203Hg
~
Protein
,E o_
30
40 E
IOOl
-~v
(b)
50 (d)
I0
~o_40 x
E Q. u,
6ol
220
s~
2 401
o_
2O
0
20
30 40 Number of fractions
50
0
0
2O
30 40 Number of fractions
50 0
Fig. 3. Chromatographic separation of the soluble fractions: upper level, kidneys of the rat exposed to HgCI2 (a), HgC12 and Na2SeO 3 (b). Lower level, liver of the rat exposed to HgCI2 (c), HgC12 and Na2SeO 3 (d)
El~bieta Komsta-Szumska and Jadwiga Chmielnicka
224 & r~
1
x3
~
.--. 755e
cq
o
141
~'~. 0
I0
20
30
il
50'0
&O
0
I J I
IO
I
30
&O
~I
(c)
I 0 50
(d) 2~
C)
&
8
2&
E
is
m
2
o/
03
*6 i 0-
J 20 30 &O Number of fractions
50
I0
20 30 &O Number of fractions
5[
Fig. 4. Chromatographic separation of the soluble fractions: upper level, kidneys of the rat exposed to Na2SeO3 (a), Na2SeOa and HgCI2 (b). Lower level, liver of the rat exposed to Na2SeO3 (c), Na2SeO3 and HgCIE(d).
Table 7. The level of metallothionein in the kidneys and liver following separate and joint administrationa Organ
Metallothionein mg/g tissue Control
+Se
+Hg
Hg + Se
Kidneys
0.25 (0.18--0.31)
0.27 (0.26--0.28)
0.58 (0.50--0.65)
0.23 (0.21--0.24)
Liver
0.10 (0.10-0.1 I)
0.19 (0.18-0.20)
0.08 (0.08-0.09)
0.18 (0.17-0.19)
"Values are the means of 3 determinations
The c h r o m a t o g r a p h i c analysis o f the soluble fraction of the kidneys shows that m e r c u r y administered alone is bound mainly (Fig. 3a) b y metallothionein-like proteins, but if administered with selenium (Fig. 3b) almost all mercury is bound by high molecular weight proteins and only a negligible amount of mercury is retained in metallothionein-like proteins. The analysis of proteins o f the soluble fraction of the liver (Fig. 3c) shows that proteins of highly varying molecular weight were involved in the binding of mercury: about 60% o f mercury was bound by high molecular weight proteins. Selenium supplied simultaneously with mercury diverted this metal to a very narrow range of the highest molecular weight proteins (Fig. 3d).
Binding of Mercury and Selenium in Kidneys and Liver
225
The chromatography of the soluble kidney fraction (Fig. 4a and b) shows that selenium administered both alone and simultaneously with mercury was bound in about 80% by proteins of various mol. weight the participation of low molecular weight compounds being relatively small. In the liver selenium (Fig. 4c and b) was bound mainly with proteins of high molecular weight but it also appeared in the form of a low molecular weight fraction, presumably nonbound selenium. The presence of this fraction was more pronounced if mercury was administered simultaneously. Table 7 shows the level of metallothionein in the homogenates of rat liver and kidneys. Prolonged exposure to mercury alone doubled the level of metallothionein in the kidneys while in the presence of selenium the physiological level of this protein was maintained. In the liver mercury administered alone did not enhance the level of metallothionein while in the presence of selenium increased levels of this protein were found.
Discussion
The effect of mercury and selenium interaction in vivo depends on the molar ratios of these elements supplied to animals (Groth et al., 1973, 1976; Moffit and Clary, 1974; Burk et al., 1974; Hill, 1974). In our experiment the mercury dose was sufficiently high to provide saturation of the organism with this metal (Piotrowski et al., 1974) while the dose of selenium was selected so as to cause the assumed effect, i.e. diversion of mercury from the kidneys to the liver (Komsta-Szumska et al., 1976). The administration of mercury and selenium to rats in the molar ratio 1 : 5 contributed to the global diversion of about 200 ~g of mercury from the kidneys to the liver (Fig. 1). The liver has become the critical organ for mercury since in these conditions it cumulated nearly three times more of this metal than the kidneys. Under the influence of selenium disappeared from the kidneys mainly mercury contained in the postmicrosomal fraction while in the liver mercury was almost entirely bound by nonhistone proteins of the nuclear fraction and in the mitochondria. The altered picture of the redistribution of mercury under the influence of selenium refers not only to the organs examined and their subcellular fractions but also to the proteins binding mercury in the soluble fractions of both the kidneys and the liver. Selenium prevented the binding of mercury by metallothionein and it eliminated the stimulating effect of mercury on the biosynthesis of this protein in the kidneys. From the above it follows that in the presence of selenium the protective role of metallothionein (Wigniewska-Knypl et al., 1970) against mercury is eliminated. From the present data no alternative mechanism of protection involving selenium can be suggested as yet. Our investigations, however, provide a basis for further research: it seems necessary to examine more closely nonhistone nuclear proteins participating in the binding of mercury and selenium in the liver. This fraction plays an essential role in the mechanism of transferring mercury from the kidneys to the liver. It seems also legitimate to make a closer investigation of high molecular weight proteins of the postmicrosomal liver fraction since the sharp chromatographic separation of this fraction may indicate the appearance of a specific protein binding mercury with the participation of selenium. The values of molar ratio Hg/Se for the homogenate and the subcellular frac-
226
El~bieta Komsta-Szumska and Jadwiga Chmielnicka
tions of the organs examined are presented in Table 6. Approximately equimolar concentration of the elements was found in the homogenates. After fractioning their ratio approaching one had been found in the nuclear fractions of the organs examined and in the soluble fraction of kidney and in the mitochondria of the liver, while in other fractions the molar ratio Hg/Se varied within the range of 0.2-2.5. Further separation of the nuclear fraction into proteins soluble in NaC1, histones and nonhistones indicates that in nuclei there are two or three kinds of protein complexes differing in the molar ratio of these elements Ratio Hg/Se higher than one was found in the microsomal, mitochondrial and lysosomal fractions, as well as in high molecular weight proteins of the postmicrosomal fraction of the kidneys. The latter is particularly interesting since in the absence of selenium these proteins do not cumulate mercury (Fig 3a and b). Ratio lower than i was found in the soluble fraction of liver and in the mitochondrial fraction of the kidney. According to the opinion of several authors the molar ratio of mercury and selenium cumulated in the organs equals 1 (Kosta et al., 1975). By means of the electron microscopy Groth et al. (1976) showed the presence of inclusion bodies binding mercury and selenium in equimolar ratio in phagocytic liver cells and in other organs of rats exposed for 20 months to HgC12 and Na2SeO4. In our experiments, histopathological exminations of animals which received mercury and selenium did not reveal these bodies probably, because of a much shorter period of exposure, hence our results cannot be discussed with the results of the above mentioned authors. From our investigations it may be inferred however that in the organs of the animals examined various protein complexes appeared in which mercury and selenium were contained in non equimolar ratios. Thus we postulate, that the molar ratio approaching 1 considered characteristic of mercury and selenium in the organs is a resultant value which is stratified in the analysis of different subcellular as of chromatographically examined fractions. Though our main object was to examine the distribution of mercury under the influence of selenium in subceUular fractions of the kidneys and liver additional information was yielded on the distribution of selenium and on the role of mercury in this phenomenon. In the presence of mercury the level of selenium increased several times in the liver and in the kidneys mainly due to binding of this element by nonhistone proteins of the nuclear fractions of both organs. In the fractions of soluble proteins of the liver and kidneys high molecular weight proteins were involved in the binding of selenium, but selenium unbound with proteins was also detected. The presence of mercury slightly altered the picture of the distribution of selenium in the soluble fraction emphasizing the involvement of high molecular weight proteins in the binding of selenium. The localization of selenium does not change drastically under the influence of mercury (Fig. 2). The content of selenium in the examined organs as well as in the individual subcellular fractions and the participation of proteins of various molecular weights in binding selenium did not change so characteristically as it was the case with mercury. Liver remained the critical organ for selenium. Acknowledgements. We are deeply indebted to Prof. J. K. Piotrowski for the critical discussions and are also grateful to Mrs. Danuta Kujawifiskaand Honorata Andrzejczak for their useful technical assistance.
Binding of Mercury and Selenium in Kidneys and Liver
227
References Burk, R. F., Foster, K. A., Greenfield, P. M., Kiker, K. W.: Binding of simultaneously administered inorganic selenium and mercury to a rat plasma protein. Proc. Soc. exp. Biol. (N.Y.) 145,782--785 (1974a) Burk, R. F., Maekinnon, A. M., Simon, F. R.: Selenium and hepatic microsomal hemoproteins. Biochem. biophys. Res. Commun. 56, 431--436 (1974b) Chen, R. W., Whanger, P. D., Fang, S. C.: Diversion of mercury binding in rat tissues by selenium a possible mechanism of protection. Pharmacol. Res. Comm. 6, 571--579 (1974) Eybl, V., Sykora, J., Mertl, F.: Einflul3 yon Natriumselenit, Natriumtellurit und Natriumsulflt auf Retention und Verteilung von Quecksilber bei M,;iusen. Arch. Toxikol. 25, 296-305 (1969) Groth, D. H., Vignati, L., Lowry, L., Mackay, G., Stokinger, H. E.: Mutual antagonistic and synergistic effects of inorganic selenium and mercury salts in chronic experiments. In: Trace substances in environmental health (D. D. Hemphill, Ed.), pp. 187-189. Columbia: University of Missouri 1973 Groth, D. H., Stettler, L., Mackay, G.: In: Effects and dose response relationships of toxic metals (G. F. Nordberg, Ed.), pp. 527--543. Amsterdam: Elsevier 1976 Hill, C. H.: Reversal of selenium toxicity in chicks by mercury, cooper and cadmium. J. Nutr. 104, 593--598 (1974) Johnson, S. L., Pond, W. G.: Inorganic v.s., organic mercury toxicity in growing rats: Protection by dietary Se but not Zn. Nutr. Repts. Internat. 9, 135--147 (1974) Komsta-Szumska, E., Chmielnicka, J., Piotrowski, J. K.: The influence of selenium on binding of inorganic mercury by metallothionein in the kidney and liver of the rat. Biochem. Pharmacol. 25, 2539-2540 (1976) Kosta, L., Byrne, A. R., Zeienko, V.: Correlation between selenium and mercury in main following exposure to inorganic mercury. Nature (Lond.) 254, 238-239 (1975) Levander, O. A., Argrett, L. C.: Effects of arsenic, mercury, thalium and lead on selenium metabolism in rats. Toxicol. appl. Pharmacol. 14, 308--314 (1969) Lowry, O. H., Rosenbrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the Folin phenolreagent. J. biol. Chem. 193, 265--275 (1951) Moffit, A. E., Clary, J. J.: Selenite-induced binding of inorganic mercury in blood and other tissues in the rat. Res. Comm. Chem. Path. Pharmacol. 7, 593-603 (1974) Ohi, G., Nishigaki, S., Seki, H., Tamura, Y., Maki, T., Maeda, H. Ochial, S., Yamada, H., Shimamura, Y., Yagyu, H.: Interaction of dietary methylmercury and selenium on accumulation and retention of these substances in rat organs. Toxicol. appl. Pharmacol. 32, 527--533 (1975) Parizek, J., Ostadalova, I.: The protective effect of small amounts of selenite in sublimate intoxication. Experientia (Basel) 23, 142--143 (1967) Parizek, J., Babicky, A., Ostadalova, I., Kalouskova, J., Pavlik, L.: In: Radiation biology of fetal and juvenile mammal (M. R. Sikov, D. D. Mahium, Eds.), pp. 137--143. Washington: Proceedings of the Ninth Annual Hartford Biology Symposium at Richland 1969a Parizek, J., Benes, I., Ostadalova, I., Babicky, A., Benes, J., Lener, J.: Metabolic interrelations of trace elements the effect of some inorganic compounds of selenium on the metabolism of cadmium and mercury in the rat. Physiologia Bohemoslovaca 18, 95--103 (1969b) Parizek, J., Ostadalova, I., Kalouskova, J., Babicky, A., Benes, J.: The detoxiflng effects of selenium. Interrelation between compounds of selenium and certain metals. In: Newer trace element metabolism in nutrition (W. Mertz, W. E. Cornatzer, Eds.), pp. 85--122. New York: Marcel Dekker, Inc. 1971 Parizek, J., Ostadalova, I., Kalouskova, J., Babicky, A., Pavlik, L.: The effect of selenium compounds on the cross-placental passage of 2~ J. Reprod. Fertil. 25, 137-143 (1973) Parizek, J., Kalouskova, J., Babicky, A., Benes, J., Pavllk, L.: Interaction of selenium with mercury, cadmium and other toxic metals. In: Trace element metabolism in animals-2 (W. G. Hoekstra, J. W. Suttie, H. E. Ganther, W. Mertz, Eds.), pp. 119--131. London: Butterworth 1974 Piotrowski, J. K., Bolanowska, W., Sapota, A.: Evaluation of metallothionein content in animals tissue. Acta biochim, pol. 20, 207--215 (1973) Piotrowski, J. K., Trojanowska, B., Sapota, A.: Binding of cadmium and mercury by metallothionein in the kidneys and liver of rats following repeated administration. Arch. Toxicol. 32, 251--360 (1974)
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El~bieta Komsta-Szumska and Jadwiga Chmielnicka
Potter, S., Matrone, G.: Effect of selenite on the toxicity of dietay methyl mercury and mercuric chloride in the rat. J. Nutr. 104~ 638--647 (1974) Pullido, P., K~igi, J. H. R., Vallee, B. L.: Isolation and some properties of human metallothionein. Biochemistry 5, 1768-1777 (1966) Schnaitman, C., Greenawalt, J. W.: Enzymatic properties of the inner and other membranes of rat liver mitochondria. J. cell. Biol. 38, 158-175 (1968) Teng, C. S., Teng, C. T., Allfrey, V. G.: Tissues of nuclear acidic proteins. J. biol. Chem. 10, 3597--3609 (1971) Wi~niewska, J. M., Trojanowska, B., Piotrowski, J. K., Jakubowski, M.: Binding of mercury in the rat kidney by metallothionein. Toxieol. appl. Pharmacol. 16, 754-763 (1970) Zelazowski, A. J., Piotrowski, J. K.: A modified procedure for the determination of metallothioneinlike proteins in the animal tissue. Acta Biochim. Pol. 24, 97-104 (1977) 7.elezowski, A. J., Piotrowski, J. K., Mogilnicka, E. M., Szymafiska, J. A., Kaszper, W. B. Bromat. chem. toks. (in press) Received March 3, 1977
Arch. Toxicol. 38, 217--228 (1977)
TOXICOLOGY 9 by Springer-Verlag 1977
Binding of Mercury and Selenium in Subcellular Fractions of Rat Liver and Kidneys Following Separate and Joint Administration* ElSbieta Komsta-Szumska and Jadwiga Chmielnicka** Department of Toxicology Chemistry, Institute of Environmental Research and Bioanalysis, Medical Academy of L6d$, Narutowicza 120 A, 90-145 Ltd$, Poland
Binding of Mercury and Selenium in Kidneys and Liver Abstract. The distribution of mercury and selenium has been examined in subcellular fractions of rat liver and kidneys in prolonged exposure to HgC12 and Na2SeO 3 administered separately and simultaneously. The molar ratio of mercury and selenium concentrations in subcellular fractions of the organs examined varied considerably. Selenium displaced mercury from the soluble kidney fraction bound mainly with metaUothionein to the nonhistone protein fraction of liver nuclei. The Hg-stimulated biosynthesis of metallothionein has been eliminated under the influence of selenium.
Key words: Interaction -- Mercury -- Selenium - Inorganic - Subcellular distribution - Mercury - Selenium -- Biocomplexes.
Zusammenfassung. Die Verteilung von Quecksilber und Selen wurde in zellfreien Fraktionen von Rattenleber und -nieren untersucht, nachdem getrennt oder zugleich HgC12 bzw. Na2SeO a zwei Wochen lang (0,5 mg Hg/kg jeden zweiten Tag i.v., 0,5 mg Se/kg jeden Tag per os, molares Verh~iltnis 1 : 5) verabreicht worden waren. Das molare Verh/iltnis der Hg- und Se-Konzentrationen schwankte in den zellfreien Fraktionen der untersuchten Organe betr/ichtlich. Selen verdr/ingte Quecksilber aus der 16slichen Nierenfraktion. Quecksilber war haupts/ichlich mit Metallothionein an die histonfreie Eiweil3fraktion der Leberzellkerne gebunden. Unter dem Einflu/3 yon Selen wurde die Hg-stimulierte Biosynthese yon Metallothionein unterbunden.
Introduction In the recent decade attention has been drawn to the interaction of selenium and mercury in vivo because of the significant role of these elements in the environmental * This work was supported by the Polish-Americanagreement No 05-009-2, with National Institute for Occupational Safety and Health, PHS, USA ** To whom offprint requests should be sent
218
El~bieta Komsta-Szumska and Jadwiga Chmielnicka
toxicology. Post morten examination of some organs in humans occupationally exposed to mercury compounds have shown that an increased level of mercury in these organs was accompanied by an increased level of selenium (Kosta et al., 1975). Investigations by Parizek and Ostadalova (1967), Parizek et al. (1974) and G r o t h et al. (1973) indicated that selenium counteracts the toxic action of inorganic mercury decreasing the nephrotoxic effect and diminishing proteinuria. Diet enriched by selenium prevented toxic effects caused by organic and inorganic mercury compounds (Potter and Matrone, 1974; Ohi et al., 1975). Several authors (Parizek et al., 1969a, 1969b, 1971; Eybl et al., 1968; Moffit and Clary, 1974) drew attention to the redistribution in the organs and to an increased retention of mercury in animals under the influence of selenium. A single dose of selenium and mercury administered simultaneously increased retention of both elements in the organism, caused a redistribution of mercury in the organs (Chen et al., 1974), and in the blood plasma (Burk et al., 1974) as well as their diversion from low mol. weight to high molecular weight proteins. Similar findings were reported in our previous investigations ( K o m s t a - S z u m s k a et al., 1976). It is well k n o w n that mercury increases the retention of selenium in the tissues of animals, both after a single dose (Parizek et al., 1973) and in prolonged exposure to selenium (Levander and Argrett, 1969; Johnson and Pond, 1974). There are but few investigations on the subcellular distribution of this element, limited to liver (Burk et al., 1974). Our investigations aim at learning more about the mechanism of mercury and selenium interaction. In the present paper we are concerned with subcellular distribution of both these elements administered to animals separately and simultaneously in the course of repeated exposure.
Methods The experiment was performed on white female rats of Wistar strain, average body weight 200 g, fed standard LSM diet. The animals were divided into 4 groups, 6 rats each: the first group received 2~ the second group - 2~ and Na2SeO3, the third group 75Sel as Na2SeO3, the fourth group - - 7 5 S e and HgC12. Mercuric chloride, 0.5 mg Hg/kg, was administered to the tail vein every other day for 2 weeks, activity 40 ~Ci/dose. Sodium selenite was supplied per os every day in a daily dose of 0.5 mg/Se/kg, activity 20 ~Ci per dose. The animals were killed in ether narcosis 24 h after the last administration, and kidneys and liver were removed for examination. The fractioning of organs was made by the method of Schnaitman and Greenawalt (1968). The scissed organs were suspended in 2 volumes of solution A (220 mM-D-mannitol, 70 mM sucrose, 2 mM HEPES-N-2-hydroxyethylpiperazine-N-2-ethanosulphonicacid, 0.5 mg crystalline bovine serum albumin; solution pH was 7.4). The organs were homogenized, 10% homogenates were centrifuged at 600 x g for 15 min. In the sediment were obtained proteins of the nuclear fraction (N) which were separated by the technique of Teng et al. (1971) into proteins solublein 0.14 M NaC1 (No), histones (Nn) and nonhistone proteins (NN). The post-nuclear supernatant was centrifugedat 7000 x g for 15 min. The supernatant was drawn off, the sediment was suspended in solution A (1/4in proportion to the original volume of the homogenate), and centrifuged again at 7000 x g for 15 min. The sediment contained the mitochondrial fraction (M-heavy mitochondria). Post-mitochondrial supernatants were pooled and centrifuged for 15 min, at Product of Instytut Badafi J~drowych, Swierk, Poland. Activity 770 mCi/g
Binding of Mercury and Selenium in Kidneys and Liver
219
12,000 • g. The sediment consisted of lysosomes and light mitochondria (L). The remaining solution was centrifuged at 144,000 • g for 1 h. The sediment contained the microsomal fraction (P) and the supernatant (soluble fraction - S). In the homogenates and individual subcellular fractions mercury and selenium were determined by gamma counting in a scintillation counter USB-2. The time of counting was 100 or 60 s; precision + 4%. Protein was determined by the method of Lowry et ai. (1951). Proteins binding mercury and selenium in soluble fractions of rat kidneys and liver were analysed by gel f'dtration. The columns filled with Sephadex G-75 gel were calibrated with Dextran Blue and Cytochrome C; 0.1 M formate buffer of pH 8 was used as eluent. Eluate fractions of 5 cm3 each were collected at the speed - 1 cm3/min. Metallothionein in the homogenates of liver and kidneys was determined by the radiochemical method of Piotrowski et al. (1973) as modified by Zelazowski and Piotrowski (in press). The metallothionein standard was obtained in our laboratory Zelazowski et al., in press) from horse kidneys by means of a technique similar to that described by Pullido et al. (1967). The sample contained about 3 ~xM groups SH/mg proteins: in the conditions of analysis about 200 txg Hg was bound by 1 mg of protein.
Results Selenium administered simultaneously with m er cu r y in repeated exposure significantly altered the level o f m e r c u r y in the examined organs o f rats (Table 1). M e r c u r y supplied without selenium was deposited mainly in the kidneys -- 32%, with only 3.5% in the liver. In simultaneous exposure to m er cu r y and selenium the situation was reversed. A higher cumulation o f mer cu r y was found in the liver, 29% o f cumulative dose, the kidneys retained only 8.5%. Selenium alone reached a higher level in the liver than in the kidneys. I f administered simultaneously with m e r c u r y its level increased several times both in the kidneys and in the liver. In the absence o f selenium m o r e than 50% o f the total a m o u n t o f m e r c u r y retained in the kidneys was located in the soluble fraction (Table 2). U n d e r the influence o f selenium the level o f m e r c u r y in this fraction was diminished m o r e than 15 times. The a m o u n t o f m e r c u r y decreased also in the remaining fractions o f the kidneys except o f the nuclear fraction. In the absence o f m er cu r y nearly half o f selenium contained in the kidneys was located in the nuclear fraction. This lowest a m o u n t o f selenium was retained in microsomes while in other fractions it was
Table 1. The levels of mercury and selenite in % of the dose in the kidneys and liver of rats in the course of 2 weeks of application of mercury, selenite alone and of mercury and selenite administered simultaneously. Groups 6 rats each 2~ contents in % of the cumulative dose
75Se contents in % of the cumulative dose
Hg (alone)
Hg + Se
Se (alone)
Se + Hg
Kidney
32.0 (29.0--33.0)
8.5 (6.5--9.5)
0.5 (0.4--0.6)
2.0 (1.8--2.5)
Liver
3.5 (2.7--4.3)
29.0 (28.0--30.0)
1.7 (1.5--2.0)
5.5 (4.3--6.5)
220
El~bieta Komsta-Szumska and Jadwiga Chmielnicka
Table 2. Mercury and selenite content in the homogenate and subcellular fractions of rat kidneysa Fractions
2~
(alone)
2~
+ Se
75Se (alone)
75Se + Hg
~xg/g tissue Homogenate
140.0 (122.0--165.0)
41.0 (32.0--51.0)
3.8 (3.3-5.6)
18.0 (16.9--24.0)
Nuclear
21.0 (17.0--23.0)
22.4 (19.7--27.0)
1.8 (1.5--2.6)
11.0 (9.5-15.0)
Mitochondrial (heavy)
12.5 (10.0--18.0)
4.0 (3.0--5.0)
0.6 (0.5-0.7)
2.7 (2.3--4.0)
Light mitochondrial and lysosomal
15.0 (13.0-16.0)
3.0 (2.2--3.7)
0.6 (0.5-0.7)
0.5 (0.4-0.6)
Microsomal
11.0 (8.4--9.4)
6.0 (5.0--7.0)
O.1 (0.1--0.2)
1.0 (0.9--1.2)
Soluble
80.5 (72.0-87.0)
5.6 (4.3-6.1)
0.7 (0.6-0.9)
2.8 (2.7-2.9)
a
Values are the means of 6 determinations
Table 3. Mercury and selenite content in the homogenate and subcellular fractions of rat livera Fractions
2~
(alone)
2~
+ Se
75Se (alone)
75Se + Hg
~g/g tissue Homogenate
3.2 (2.9--3.4)
22.0 (18.0--26.0)
2.6 (1.8-3.4)
9.0 (8.0--10.0)
Nuclear
1.0 (0.9--1.4)
13.3 (11.5-17.0)
1.4 (1.3--1.7)
5.1 (4.4--5.6)
Mitochondrial (heavy)
0.3 (0.2--0.4)
4.5 (3.8--5.8)
0.2 (0.1--0.2)
1.7 (1.4--2.0)
Light mitochondrial and lysosomal
0.7 (0.5-0.9)
1.5 (1.4-1.9)
0.1 (0.1-0.2)
0.4 (0.3-0.5)
Microsomal
0.2 (0.1--0.2)
2.0 (1.8--2.6)
0.1 (0.1--0.2)
0.4 (0.4--0.5)
Soluble
1.0 (0.8--1.2)
0.7 (0.6--0.8)
0.8 (0.7-0.9)
1.4 (1.0--1.6)
a Values are the means of 6 determinations
evenly distributed. I n the presence of m e r c u r y the level o f selenium rose c o n s i d e r a b l y in all fractions: a b o u t 10 fold in the m i c r o s o m a l fraction a n d 4 - 6 times in other fractions. Nevertheless, the highest level o f selenium r e m a i n e d characteristic of the n u c l e a r fraction (11 pxg Se/g tissue). I n the liver (Table 3), m e r c u r y alone was b o u n d m a i n l y b y the n u c l e a r a n d soluble fractions. U n d e r the influence of selenium the level of m e r c u r y increased
221
Binding of Mercury and Selenium in Kidneys and Liver Table 4. Mercury and selenite content in the nuclear fractions of rat kidneysa Nuclear fraction
2~
(alone)
2~
+ Se
75Se (alone)
75Se + Hg
~g/mg protein Protein soluble in 0.14 N NaCI
0.09 (0.08--0.10)
0.24 (0.22--0.26)
0.03 (0.02--0.04)
0.05 (0.04--0.06)
Histone
0.20 (0.18--0.21) 0.17 (0.14--0.19)
0.06 (0.05--0.07) 0.19 (0.18--0.21)
0.02 (0.02--0.03) 0.01 (0.01--0.02)
0.06 (0.05--0.06) 0.10 (0.08--0.11)
Nonhistone
a Values are the means of 6 determinations Table 5. Mercury and selenite content in the nuclear fractions of rat livera Nuclear fraction
2~
(alone)
2~
+ Se
75Se (alone)
75Se + Hg
0.03 (0.02--0.03) 0.02 (0.01-0.02) 0.01 (0.01--0.02)
0.06 (0.05--0.07) 0.07 (0.05--0.08) 0.07 (0.06--0.08)
~g/mg protein Protein soluble in 0.14 N NaC1 Historic Nonhistone
0.02 (0.01--0.02) 0.01 (0.01--0102) 0.01 (0.01--0.02)
0.10 (0.09--0.11) 0.12 (0.11--0.13) 0.24 (0.22--0.26)
a Values are the means of 6 determinations
1 0 - 1 5 times in the nuclear, mitochondrial and microsomal fractions. In the absence of mercury selenium was located in the liver mainly in the nuclear and soluble fractions which is in agreement with the results obtained by Burk et al. (1974). Under the influence of mercury the level of selenium rose in all examined liver fractions, particularly in the nuclear and mitochondrial fractions. In the renal nuclei (Table 4) in the absence of selenium mercury was retained mainly by nonhistone proteins (71%). Selenium administered with mercury slightly hightened the content of mercury in proteins soluble in NaC1 and lowered level in histones. In general the coefficient ~g H g / m g of protein in the nuclear fraction of the kidneys did not change under the influence of selenium. In the presence of mercury also selenium contained in the kidney nuclei was bound mainly by nonhistone proteins. In liver nuclei (Table 5), mercury was also bound mainly by nonhistone proteins (55%) and under the influence of selenium the share of this subfraction rose to 77%. A very high coefficient, ~g H g / m g protein, was characteristic for this fraction of the liver, much exceeding that of nonhistone proteins of the kidney (Table 5). Figures 1 and 2 illustrate the distribution of mercury and selenium in the kidneys and liver calculated per weight of the organ. In Table 6 molar rations Hg/Se were listed for the homogenates and single cellular fractions of the kidneys and liver as well as for high molecular weight proteins in postmicrosomal fraction.
El~bieta Komsta-Szumska and Jadwiga Chmielnicka
222
~ 16o
~5 300
14o
~ 250
~120
200
100
150
80
B Hg alone B Hg+Se
60
I00 50 0
Kidney
2O
H
160[
N
M
L
l
P
S
300 Liver
250 20Q
100
150
80 6O
100 4O
5O 0
2O H
N
M
L
P
S
Fig. 1. The distribution of mercury (bLg Hg per organ) in the cellular fractions of rat kidneys and liver. Fraction symbols: H homogenate, N - nuclear, M -- heavy mitochondrial, L -- light mitochondrial and lysosomal, P -- microsomal, S -- soluble
Kidney 30
c5 6~
Se alone B Se+Hg
~2o
0
N
H
100
50
80
40
6O
30
40
20
20
10
.B t4
L
P
A] S
Liver
0
H
N
M
L
P
S
Fig. 2. The distribution of selenite (~g Se per organ) in the cellular fractions of rat kidneys and liver. Symbols see Figure 1
Binding of Mercury and Selenium in Kidneys and Liver
223
Table 6. Molar ratio Hg/Sea
Homogenate Fractions: Nuclear
Kidneys
Liver
0.84 (0.70--0.88)
0.90 (0.78--1.00)
0.73 (0.66-0.76) 1.83 (1.40--1.85) 0.46 (0.29--0.49) 0.66 (0.64-0.70) 0.56 (0.47--0.58) 2.54 (2.20--2.57) 2.20 (2.10--2.42) 0.76 (0.58--0.77) 2.50
Soluble protein Histone Nonhistone Heavy mitochondrial Light mitochondrial and lysosomal Microsomal Soluble High molecular weight proteins
1.00
(0.91--1.10) 0.54 (0.52-0.60) 0.50 (0.50-0.70) 1.66 (1.60--1.80) 1.00
(1.00--1.00) 1.50 (1.40--1.60) 1.80 (1.72--1.84) 0.20 (0.18--0.22) 1.00
a Values are the means of 6 determinations
60
,01' '00fj
u
~2o 0
I
1.06~t~--, ~,
20
30 ~I
r
20
50
~0 (el
1.0 .--.
203Hg
~
Protein
,E o_
30
40 E
IOOl
-~v
(b)
50 (d)
I0
~o_40 x
E Q. u,
6ol
220
s~
2 401
o_
2O
0
20
30 40 Number of fractions
50
0
0
2O
30 40 Number of fractions
50 0
Fig. 3. Chromatographic separation of the soluble fractions: upper level, kidneys of the rat exposed to HgCI2 (a), HgC12 and Na2SeO 3 (b). Lower level, liver of the rat exposed to HgCI2 (c), HgC12 and Na2SeO 3 (d)
El~bieta Komsta-Szumska and Jadwiga Chmielnicka
224 & r~
1
x3
~
.--. 755e
cq
o
141
~'~. 0
I0
20
30
il
50'0
&O
0
I J I
IO
I
30
&O
~I
(c)
I 0 50
(d) 2~
C)
&
8
2&
E
is
m
2
o/
03
*6 i 0-
J 20 30 &O Number of fractions
50
I0
20 30 &O Number of fractions
5[
Fig. 4. Chromatographic separation of the soluble fractions: upper level, kidneys of the rat exposed to Na2SeO3 (a), Na2SeOa and HgCI2 (b). Lower level, liver of the rat exposed to Na2SeO3 (c), Na2SeO3 and HgCIE(d).
Table 7. The level of metallothionein in the kidneys and liver following separate and joint administrationa Organ
Metallothionein mg/g tissue Control
+Se
+Hg
Hg + Se
Kidneys
0.25 (0.18--0.31)
0.27 (0.26--0.28)
0.58 (0.50--0.65)
0.23 (0.21--0.24)
Liver
0.10 (0.10-0.1 I)
0.19 (0.18-0.20)
0.08 (0.08-0.09)
0.18 (0.17-0.19)
"Values are the means of 3 determinations
The c h r o m a t o g r a p h i c analysis o f the soluble fraction of the kidneys shows that m e r c u r y administered alone is bound mainly (Fig. 3a) b y metallothionein-like proteins, but if administered with selenium (Fig. 3b) almost all mercury is bound by high molecular weight proteins and only a negligible amount of mercury is retained in metallothionein-like proteins. The analysis of proteins o f the soluble fraction of the liver (Fig. 3c) shows that proteins of highly varying molecular weight were involved in the binding of mercury: about 60% o f mercury was bound by high molecular weight proteins. Selenium supplied simultaneously with mercury diverted this metal to a very narrow range of the highest molecular weight proteins (Fig. 3d).
Binding of Mercury and Selenium in Kidneys and Liver
225
The chromatography of the soluble kidney fraction (Fig. 4a and b) shows that selenium administered both alone and simultaneously with mercury was bound in about 80% by proteins of various mol. weight the participation of low molecular weight compounds being relatively small. In the liver selenium (Fig. 4c and b) was bound mainly with proteins of high molecular weight but it also appeared in the form of a low molecular weight fraction, presumably nonbound selenium. The presence of this fraction was more pronounced if mercury was administered simultaneously. Table 7 shows the level of metallothionein in the homogenates of rat liver and kidneys. Prolonged exposure to mercury alone doubled the level of metallothionein in the kidneys while in the presence of selenium the physiological level of this protein was maintained. In the liver mercury administered alone did not enhance the level of metallothionein while in the presence of selenium increased levels of this protein were found.
Discussion
The effect of mercury and selenium interaction in vivo depends on the molar ratios of these elements supplied to animals (Groth et al., 1973, 1976; Moffit and Clary, 1974; Burk et al., 1974; Hill, 1974). In our experiment the mercury dose was sufficiently high to provide saturation of the organism with this metal (Piotrowski et al., 1974) while the dose of selenium was selected so as to cause the assumed effect, i.e. diversion of mercury from the kidneys to the liver (Komsta-Szumska et al., 1976). The administration of mercury and selenium to rats in the molar ratio 1 : 5 contributed to the global diversion of about 200 ~g of mercury from the kidneys to the liver (Fig. 1). The liver has become the critical organ for mercury since in these conditions it cumulated nearly three times more of this metal than the kidneys. Under the influence of selenium disappeared from the kidneys mainly mercury contained in the postmicrosomal fraction while in the liver mercury was almost entirely bound by nonhistone proteins of the nuclear fraction and in the mitochondria. The altered picture of the redistribution of mercury under the influence of selenium refers not only to the organs examined and their subcellular fractions but also to the proteins binding mercury in the soluble fractions of both the kidneys and the liver. Selenium prevented the binding of mercury by metallothionein and it eliminated the stimulating effect of mercury on the biosynthesis of this protein in the kidneys. From the above it follows that in the presence of selenium the protective role of metallothionein (Wigniewska-Knypl et al., 1970) against mercury is eliminated. From the present data no alternative mechanism of protection involving selenium can be suggested as yet. Our investigations, however, provide a basis for further research: it seems necessary to examine more closely nonhistone nuclear proteins participating in the binding of mercury and selenium in the liver. This fraction plays an essential role in the mechanism of transferring mercury from the kidneys to the liver. It seems also legitimate to make a closer investigation of high molecular weight proteins of the postmicrosomal liver fraction since the sharp chromatographic separation of this fraction may indicate the appearance of a specific protein binding mercury with the participation of selenium. The values of molar ratio Hg/Se for the homogenate and the subcellular frac-
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El~bieta Komsta-Szumska and Jadwiga Chmielnicka
tions of the organs examined are presented in Table 6. Approximately equimolar concentration of the elements was found in the homogenates. After fractioning their ratio approaching one had been found in the nuclear fractions of the organs examined and in the soluble fraction of kidney and in the mitochondria of the liver, while in other fractions the molar ratio Hg/Se varied within the range of 0.2-2.5. Further separation of the nuclear fraction into proteins soluble in NaC1, histones and nonhistones indicates that in nuclei there are two or three kinds of protein complexes differing in the molar ratio of these elements Ratio Hg/Se higher than one was found in the microsomal, mitochondrial and lysosomal fractions, as well as in high molecular weight proteins of the postmicrosomal fraction of the kidneys. The latter is particularly interesting since in the absence of selenium these proteins do not cumulate mercury (Fig 3a and b). Ratio lower than i was found in the soluble fraction of liver and in the mitochondrial fraction of the kidney. According to the opinion of several authors the molar ratio of mercury and selenium cumulated in the organs equals 1 (Kosta et al., 1975). By means of the electron microscopy Groth et al. (1976) showed the presence of inclusion bodies binding mercury and selenium in equimolar ratio in phagocytic liver cells and in other organs of rats exposed for 20 months to HgC12 and Na2SeO4. In our experiments, histopathological exminations of animals which received mercury and selenium did not reveal these bodies probably, because of a much shorter period of exposure, hence our results cannot be discussed with the results of the above mentioned authors. From our investigations it may be inferred however that in the organs of the animals examined various protein complexes appeared in which mercury and selenium were contained in non equimolar ratios. Thus we postulate, that the molar ratio approaching 1 considered characteristic of mercury and selenium in the organs is a resultant value which is stratified in the analysis of different subcellular as of chromatographically examined fractions. Though our main object was to examine the distribution of mercury under the influence of selenium in subceUular fractions of the kidneys and liver additional information was yielded on the distribution of selenium and on the role of mercury in this phenomenon. In the presence of mercury the level of selenium increased several times in the liver and in the kidneys mainly due to binding of this element by nonhistone proteins of the nuclear fractions of both organs. In the fractions of soluble proteins of the liver and kidneys high molecular weight proteins were involved in the binding of selenium, but selenium unbound with proteins was also detected. The presence of mercury slightly altered the picture of the distribution of selenium in the soluble fraction emphasizing the involvement of high molecular weight proteins in the binding of selenium. The localization of selenium does not change drastically under the influence of mercury (Fig. 2). The content of selenium in the examined organs as well as in the individual subcellular fractions and the participation of proteins of various molecular weights in binding selenium did not change so characteristically as it was the case with mercury. Liver remained the critical organ for selenium. Acknowledgements. We are deeply indebted to Prof. J. K. Piotrowski for the critical discussions and are also grateful to Mrs. Danuta Kujawifiskaand Honorata Andrzejczak for their useful technical assistance.
Binding of Mercury and Selenium in Kidneys and Liver
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References Burk, R. F., Foster, K. A., Greenfield, P. M., Kiker, K. W.: Binding of simultaneously administered inorganic selenium and mercury to a rat plasma protein. Proc. Soc. exp. Biol. (N.Y.) 145,782--785 (1974a) Burk, R. F., Maekinnon, A. M., Simon, F. R.: Selenium and hepatic microsomal hemoproteins. Biochem. biophys. Res. Commun. 56, 431--436 (1974b) Chen, R. W., Whanger, P. D., Fang, S. C.: Diversion of mercury binding in rat tissues by selenium a possible mechanism of protection. Pharmacol. Res. Comm. 6, 571--579 (1974) Eybl, V., Sykora, J., Mertl, F.: Einflul3 yon Natriumselenit, Natriumtellurit und Natriumsulflt auf Retention und Verteilung von Quecksilber bei M,;iusen. Arch. Toxikol. 25, 296-305 (1969) Groth, D. H., Vignati, L., Lowry, L., Mackay, G., Stokinger, H. E.: Mutual antagonistic and synergistic effects of inorganic selenium and mercury salts in chronic experiments. In: Trace substances in environmental health (D. D. Hemphill, Ed.), pp. 187-189. Columbia: University of Missouri 1973 Groth, D. H., Stettler, L., Mackay, G.: In: Effects and dose response relationships of toxic metals (G. F. Nordberg, Ed.), pp. 527--543. Amsterdam: Elsevier 1976 Hill, C. H.: Reversal of selenium toxicity in chicks by mercury, cooper and cadmium. J. Nutr. 104, 593--598 (1974) Johnson, S. L., Pond, W. G.: Inorganic v.s., organic mercury toxicity in growing rats: Protection by dietary Se but not Zn. Nutr. Repts. Internat. 9, 135--147 (1974) Komsta-Szumska, E., Chmielnicka, J., Piotrowski, J. K.: The influence of selenium on binding of inorganic mercury by metallothionein in the kidney and liver of the rat. Biochem. Pharmacol. 25, 2539-2540 (1976) Kosta, L., Byrne, A. R., Zeienko, V.: Correlation between selenium and mercury in main following exposure to inorganic mercury. Nature (Lond.) 254, 238-239 (1975) Levander, O. A., Argrett, L. C.: Effects of arsenic, mercury, thalium and lead on selenium metabolism in rats. Toxicol. appl. Pharmacol. 14, 308--314 (1969) Lowry, O. H., Rosenbrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the Folin phenolreagent. J. biol. Chem. 193, 265--275 (1951) Moffit, A. E., Clary, J. J.: Selenite-induced binding of inorganic mercury in blood and other tissues in the rat. Res. Comm. Chem. Path. Pharmacol. 7, 593-603 (1974) Ohi, G., Nishigaki, S., Seki, H., Tamura, Y., Maki, T., Maeda, H. Ochial, S., Yamada, H., Shimamura, Y., Yagyu, H.: Interaction of dietary methylmercury and selenium on accumulation and retention of these substances in rat organs. Toxicol. appl. Pharmacol. 32, 527--533 (1975) Parizek, J., Ostadalova, I.: The protective effect of small amounts of selenite in sublimate intoxication. Experientia (Basel) 23, 142--143 (1967) Parizek, J., Babicky, A., Ostadalova, I., Kalouskova, J., Pavlik, L.: In: Radiation biology of fetal and juvenile mammal (M. R. Sikov, D. D. Mahium, Eds.), pp. 137--143. Washington: Proceedings of the Ninth Annual Hartford Biology Symposium at Richland 1969a Parizek, J., Benes, I., Ostadalova, I., Babicky, A., Benes, J., Lener, J.: Metabolic interrelations of trace elements the effect of some inorganic compounds of selenium on the metabolism of cadmium and mercury in the rat. Physiologia Bohemoslovaca 18, 95--103 (1969b) Parizek, J., Ostadalova, I., Kalouskova, J., Babicky, A., Benes, J.: The detoxiflng effects of selenium. Interrelation between compounds of selenium and certain metals. In: Newer trace element metabolism in nutrition (W. Mertz, W. E. Cornatzer, Eds.), pp. 85--122. New York: Marcel Dekker, Inc. 1971 Parizek, J., Ostadalova, I., Kalouskova, J., Babicky, A., Pavlik, L.: The effect of selenium compounds on the cross-placental passage of 2~ J. Reprod. Fertil. 25, 137-143 (1973) Parizek, J., Kalouskova, J., Babicky, A., Benes, J., Pavllk, L.: Interaction of selenium with mercury, cadmium and other toxic metals. In: Trace element metabolism in animals-2 (W. G. Hoekstra, J. W. Suttie, H. E. Ganther, W. Mertz, Eds.), pp. 119--131. London: Butterworth 1974 Piotrowski, J. K., Bolanowska, W., Sapota, A.: Evaluation of metallothionein content in animals tissue. Acta biochim, pol. 20, 207--215 (1973) Piotrowski, J. K., Trojanowska, B., Sapota, A.: Binding of cadmium and mercury by metallothionein in the kidneys and liver of rats following repeated administration. Arch. Toxicol. 32, 251--360 (1974)
228
El~bieta Komsta-Szumska and Jadwiga Chmielnicka
Potter, S., Matrone, G.: Effect of selenite on the toxicity of dietay methyl mercury and mercuric chloride in the rat. J. Nutr. 104~ 638--647 (1974) Pullido, P., K~igi, J. H. R., Vallee, B. L.: Isolation and some properties of human metallothionein. Biochemistry 5, 1768-1777 (1966) Schnaitman, C., Greenawalt, J. W.: Enzymatic properties of the inner and other membranes of rat liver mitochondria. J. cell. Biol. 38, 158-175 (1968) Teng, C. S., Teng, C. T., Allfrey, V. G.: Tissues of nuclear acidic proteins. J. biol. Chem. 10, 3597--3609 (1971) Wi~niewska, J. M., Trojanowska, B., Piotrowski, J. K., Jakubowski, M.: Binding of mercury in the rat kidney by metallothionein. Toxieol. appl. Pharmacol. 16, 754-763 (1970) Zelazowski, A. J., Piotrowski, J. K.: A modified procedure for the determination of metallothioneinlike proteins in the animal tissue. Acta Biochim. Pol. 24, 97-104 (1977) 7.elezowski, A. J., Piotrowski, J. K., Mogilnicka, E. M., Szymafiska, J. A., Kaszper, W. B. Bromat. chem. toks. (in press) Received March 3, 1977
