ANALYTICAL
98, 287-292
BIOCHEMISTRY
(1979)
Effect of Selenium on Determination Mercury in Animal Tissues AKIRA NAGANUMA,"
HIROSHI SATOH,+ REIKO YAMAMoTO;t AND NOBUMASA IMURA*,~
Received
October
of TSUGUYOSHI SUZUK1.t
24. 1978
Selenium in animal tissues was found to influence the reactivity of mercury in the tissues with stannous chloride or with stannous chloride plus cadmium chloride added as reducing agents for the determination of mercury by the method developed by L. Magos (1971 .Ana/yst . 96, 847-853) and L. Magos and T. W. Clarkson (1972. J. AS.WC. Offic. And. Chern.. 55. 966-971). The recovery of mercury in the tissues of animals to which inorganic mercury and selenite were simultaneously administered was low compared to the case in which inorganic mercury alone was administered. Of the in t’itrr) interactions of inorganic mercury and selenite examined in tissue homogenates and blood samples, only those interactions in blood samples caused the difficulty in mercury analysis mentioned above. i.e., there was a marked decrease in ret overy of mercury when an equimolar amount of each compound was added to the blood. These facts suggest that selenium and inorganic mercury in the animal tissues are likely to interact with each other and might form a chemically stable state of inorganic mercury which resists reduction with stannous chloride in the procedure for mercury determination.
Knowledge concerning the mechanism of mutual modification of toxicities between selenium and mercurial compounds has accumulated in recent years. Selenium compounds reduce the excretion of inorganic mercury, ac’celerate its accumulation in the liver, spleen, and blood (3-5), and alter its distribution in the blood by increasing markedly its uptake by red blood cells (6.7). Gel filtration discloses that, when selenite and inorganic mercury are simultaneously administered, most of the mercury is present in the high molecular weight fraction of rat plasma(S), the soluble fractions of rat organs (9) and the erythrocytes of rabbits (6.7). These high molecular weight fractions are different from those in which mercury is found when inorganic mercury alone is administered. In the high molecular weight substance in plasma of rats treated with selenite and inorganic mercury, selenium ’ To whom
all inquiries
should
be addressed. 2x7
and mercury exist at a molar ratio of approximately one (8). These data indicate that the existing state of inorganic mercury changes when selenite is simultaneously administered. Using Magos’ selective determination method for inorganic and total mercury (1,2), we measured the mercurial content in the organs of animals to which inorganic mercury and selenite were simultaneously administered, and found that the mercury accumulated in the tissues reacted differently with SnCl, or SnCl,-CdCl, than when Hg’+ alone was administered. This also suggests that administration of selenite alters the chemical state of inorganic mercury in the animal tissues. MATERIALS
AND
METHODS
Preprrrutiorz o.f’ scmplrs. Adult IVCS strain female mice (weighing about 20 g) were administered HgCI, (3 mg Hg/kg) alone 0003.2697/79/140287-06$02.00/O CopyrIght ,979 by AcndemlL Precs. Inc. All right< ~4 rsproductmn !n any form reserved.
288
NAGANUMA
subcutaneously in the gluteal region with or without Na,SeO,, (1 mg Se/kg) in the neck. Blood was collected in a test tube containing 20 ~1 of heparin sodium salt (1000 units/ml) and various organs were also excised under ether anesthesia. To the organ homogenate (40 mg tissue/ml saline) and the blood taken from control mice was added HgCI, alone or with Na,SeO, to obtain 1 x lo-” M concentrations, respectively. The resulting mixtures were then incubated at 37°C for 1 h with continual shaking. A rabbit (male, weighing about 3 kg) was anesthetized with 30 mgikg of sodium pentobarbital (Nembutal, Abbot Laboratories, Chicago, Ill.). After administering 1.0 ml of heparin sodium salt intravenously in the rabbit’s ear, the blood was collected from its carotid and incubated at 37°C for 1 h with I x lo-” M HgCI, and various concentrations of Na,SeOB. Deterrninatim of mercury. Inorganic and organic mercury contents in the organ homogenates and the blood were determined by the method reported by Magos (1) and Magos and Clarkson (2). which is described as “Magos’ method” in this paper for convenience. Inorganic mercury was measured by “method 2” of Magos (I), and total mercury was measured by “method 1” of Magos’ text (I), which was more precisely described by Magos and Clarkson (2). The original procedure by Magos defines organic mercury concentration as the difference between total and inorganic mercury concentrations. In some experiments. however, we also employed his “method 3” (1): organic mercury can be determined from the same aliquot used for inorganic mercury determination. Magos had pointed out the difficulties in a quantitative analysis of organic mercury using this procedure: the second reading for organic mercury content may be influenced by the timing of disconnecting the airflow following the first reading for inorganic mercury content. Calibration curves for inorganic mercury
ET AL
and methylmercury in “method 3” were obtained using standard solutions of these mercurials in place of tissue homogenates. The method was modified in part by circulating the air for 1 min in the reductor before feeding mercury vapor into the cells for atomic absorption spectrometry using a three-way stopper in the apparatus.” The reagents for the second step of “method 3” (SnCl,-CdCl, solution, H,S04, and NaOH solution) were added to the mixture after the recorder returned to the baseline from the peak of the first step. In addition to mercury determination by Magos’ method, the total mercury was determined using the same sample by the wet incineration method. To the sample was added 3.5 vol of concentrated HNO, and the resulting mixture was placed in a Uniseal (Uniseal Decomposition Vessels, Ltd., Israel) and heated at 140°C for 90 min, and the amount of mercury was measured by the reductive vaporization-atomic absorption method (10). Organic mercury in the tissue homogenate (100 mg tissue/6 ml distilled water) was determined by gas-liquid chromatography with an electron capture detector after extraction and concentration by the cysteine-benzene extraction method (11). RESULTS In this report, the procedure to determine inorganic mercury using SnCl, as a reducing agent in Magos ’ “method 3” (1) is called the first step, whereas the subsequent determination of organic mercury in the presence of both CdCI, and SnCl, is called the second step. Table 1 shows the mercurial concentration in the organs of mice administered HgCIZ and Na,SeO, simultaneously. In various organs a portion of mercury was detected in the second step. This portion was particularly high in the liver and the spleen. When the ’ This improved method shows a sharper peak than that shown by Magos’ original method. Thus, the errors in measurement using peak height can be reduced.
EFFECT
OF SELENIUM
ON MERCURY TABLE
MERCURY
CONCENTRATION
Method for determination 1st step 2nd step 1st and 2nd step Total mercury: by wet incineration method Organic mercury: by glc”
tissue
OF MOUSE
IN TISSUES
1
INJECTED
WITH
CHLORIDE
CONCENTRATION
Method fol determination 1st step 2nd step 1st and 2nd step mercury:
incineration
SODIUM
SELENITE”
Brain
Spleen
3.53 2 1.12 1.72 IT 0.65 5.25 t- 1.57
Il.02 k 4.34 1.24 -+ 0.28 12.27 + 4.49
0.07 2 0.01 0.03 2 0.03 0.10 i 0.04
2.10 + 1.22 0.89 f 0.41 2.99 k 1.62
1.82 k 0.52 0.48 f 0.22 2.29 -+ 0.6.5
8.63 i- 1.15
13.58
0.11 ? 0.03
5.38 -t 2.12
2.56 ? 0.43
ND
-t 3.27
IN TISSUES
Blood
-
ND
’ Mice were sacrificed 24 h after the injection. Values in the table are mean (average of six mice). ’ Gas-liquid chromatography with electron capture detector.
MERCURY
AND
Kidney
-+ SD in micrograms
Hgigram
lower by 39% in the liver and 44% in the spleen. Table 2 shows the mercurial levels in rat organs to which HgC& was administered alone. No mercury was detected in the second step except for a trace amount in the kidney. There are no significant differences between the sum values of the first and the second steps and the total mercurial contents determined by the wet incineration methods. Figure 1 shows the effect of the amount of SnCl, used as a reducing agent in the first step in determining the mercury content in the liver. When HgCl, is administered alone, the sum values of mercury concentration were approximately the same as those determined
TABLE
tissue
MERCURIC
Liver
same samples were analyzed by gas chromatography, methylmercury and other organic mercurials were not detected. The detectable limit of methylmercury in the present experiment was 120 ng Hg/g tissue. Thus, methylmercury would have been detected, had it been the mercury determined in the second step. Accordingly, the mercury detected in tlhe second step of the present experiment was not organic. It is assumed that part of the inorganic mercury existing in the organs changed its form under the influence of simultaneously administered selenite. When the sum of mecurial content determined in the first and the second steps is compared to the value determined by the wet incineration method, the sum values are
Total
189
ANALYSIS
OF MOUSE
2 INJECTED
WITH
MERCURIC
CHLORIDE
AI ONE”
Liver
Kidney
Brain
Spleen
Blood
1.49 f 0.20 ND 1.49 -t 0.20
21.92 2 3.67 1.33 i- 0.46 23.31 f 3.69
0.09 2 0.02 ND 0.09 2 0.02
0.72 ? 0.07 ND 0.72 + 0.07
0.38 f 0.10 ND 0.38 2 0.10
1.43 k 0.19
19.47 -+ 1.70
0.09 f 0.01
0.78 k 0.06
0.35
by wet method
” Mice were sacrificed 24 h after (average of six mice).
the injection.
Values
in the table
are mean
-t SD in micrograms
-c 0.06 Hgigram
290
NAGANUMA
ET AL.
.
When the total mercury contents in the organs and tissues were measured by Magos’ “method 1” about the same values as those determined by the wet incineration method were obtained among the groups to which 1 HgCl, was administered alone, but approxi.P k WC-----* .--__ *-----------.* ;i; mately 50 and 70% of the values determined E + OlrrrS by the wet incineration method were re0 loo 200 300 400 500 covered as the total mercury in the spleen S&l2 added ( mg) and liver, respectively, when HgCl, and Na,SeO, were simultaneously administered FIG. I. Effect of the amount of SnCI, used as a reducing agent in the first step of Magos’ method (Table 3). (“method 3”; see text) in determining mercury content The influences of coexisting Na,SeO, over in liver of mouse injected with mercuric chloride the recovery rate of inorganic mercury alone (--) or with sodium selenite (- - -). Mice were added in rlitro and incubated for 1 h were sacrificed 24 h after the injection. Indicated values then studied using the organ homogenates are percentage of mercurial contents determined by and the blood of control mice (Table 4). Magos’ method to those by the wet incineration method. Results from the first step (0) and the second When HgCl, alone was added, the first step step (m) of Magos’ method, and the sum of these two could vaporize most of the added mercury in values (0). A: Hg determined by Magos’ method. all the organs, and hardly any mercury was B: Hg determined by wet incineration method. detected in the second step. When Na,SeO, was added simultaneously, the influence of by the wet incineration method irrespective coexisting selenite was demonstrated only of the amount of SnCl, used. When HgCl, in the blood-merely 72% of mercury and Na,SeO, were administered simulwas determined in the first step and about taneously, the amount of mercury deter- 14% in the second step. The total yield mined in the first step with less than 100 mg was as low as 86% of the amount added. of &Cl, was relatively small. However, No influence of coexisting Na,SeO,, added with more than 100 mg of SnCl,, the amount in rho was apparent in other organs. of mercury determined in the first step as The influences of selenium concentration well as that in the second step was approxion the recovery of mercury in Magos’ mately constant irrespective of the amount method from rabbit’s blood, to which of SnCl, used. 1 x lo-” M HgC12 and various concen-
s ‘O” I-=
TABLE TOTAL
MERCURY
CONCENTRATION
CHLORIDE
Method
for determination
3
IN TISSUES
OR MERCURIC
OF MOUSE
CHLORIDE
AND
INJECTED
SODIUM
WITH
MERCURIC
SELENITE”
Liver
Kidney
Brain
Spleen
Blood
W#W Magos ’ “method Wet incineration
1” method
3.19 2 0.68 3.57 + 0.83
31.41 + 11.06 32.57 2 7.44
0.14 ? 0.06 0.13 2 0.05
1.18 2 0.33 1.21 + 0.38
0.87 i- 0.36 0.80 2 0.21
[HgCI, + Na,SeO,]” Magos ’ “method Wet incineration
I” method
6.05 2 1.00 8.49 f 1.61
21.92 21.27
0.18 k 0.06 0.18 -+ 0.03
4.45 8.98
4.53 i- 0.66 5.44 k 0.76
tissue
(1 Mice were sacrificed (average of six mice). ’ Injected compound.
12 h after
the injection.
? 8.99 k 3.37 Values
in the table
are mean
k 1.88 r 3.71
? SD in micrograms
Hg/g
EFFECT
OF SELENIUM
ON TABLE
RECOVERY
OF MERCURY CHLORIDE
MERCURY
291
ANALYSIS
4
IN MAGOS’ METHOD FROM MOUSE TISSUES WITH ADDED IN THE PRESENCE OR ABSENCE OF SODIUM SELENITE”
MERCURIC
Kidney
Brain
Spleen
Blood
98.8 ND
98.2 ND
97.4 ND
98.4 ND
98.9 ND
97.2 ND
97.8 ND
96.8 ND
98.3 ND
71.5 14.1
Liver
WKLI 1st step 2nd step [HgCl, + Na,SeO:,] 1st step 2nd step ” Values
in the table
are percentage
recovered
of the added
trations of Na,SeO, were added, were studied and the results are plotted in Fig. 2. The figure reveals that when a nearly equimolar ratio of selenium to mercury was present, the amount of mercury determined in the first step was low whereas that in the second step was high. The sum of mercurial contents measured in the first and second steps, that is, the recovery rate of the added mercury, becomes smallest near the equimolar point. DISCUSSION In the organs, especially spleen, of mice administered
Concentration
of Na$e(+
the liver Na,SeO,,
and and
(MI
FIG. 2. Influence of sodium selenite concentration on recovery of mercury in Magos’ method from rabbit blood. To blood samples was added 1 x IO-” M HgCI, and various concentrations of Na,SeO,. Mercury was determined by the first (0) or the second step (m) of Magos’ method (“method 3”; see text). Closed circles indicate the sum of these two values. Indicated values are percentage recovered of the added amount (average of five samples).
amount;
average
of samples
from
six mice.
HgC& simultaneously, we found a part of the mercury accumulated was resistant to vaporization under the conditions used by Magos for determination of mercury (1,2). Absence of any detectable amount of organic mercury in the samples was confirmed by gas-liquid chromatography. The total mercury concentrations in the liver and the spleen, determined by Magos’ “method 1,” were lower by about 30 and 50%, respectively, than those determined by the wet incineration method (Table 3). In both the liver and the spleen the accumulation of inorganic mercury is known to be accelerated when selenite is administered simultaneously (3-5). These phenomena can reasonably be explained by the assumption that the existing state of inorganic mercury in the organs was modified by the simultaneous administration of selenite. An increased amount of SnCI,, a reducing agent added in the first step in “method 3,” did not vaporize all of the mercury. This also suggests that a portion of the inorganic mercury was made chemically stable by the simultaneously administered selenium. In the i/z \~ifro experiment a phenomenon such as that mentioned above was observed only in the case of blood (Table 4). Imura and Naganuma (7) studied the behavior of inorganic mercury in rabbit blood in the presence of selenite and showed the possibility that the blood might be a primary
292
NAGANUMA
site for the interaction of inorganic mercury and selenite. The results of the present study also show that the change in the existing state of inorganic mercury caused by the presence of selenium compound occurs first in the blood. This interaction of inorganic mercury and selenite in the blood was most marked when the molar ratio of mercury to selenium was 1: 1 (Fig. 2). This fact coincides well with the previous experimental results which show that the extent of the reaction between inorganic mercury and selenium is greatest when the molar ratio is one (4,5,12). Complete digestion of the samples in relatively high concentrations of alkaline solution (13,14) before the reducing procedure might improve the interference of selenium in mercury determination by Magos’ method. On this possibility a series of experiments are in progress. ACKNOWLEDGMENTS The authors wish to thank Dr. L. Magos for his enlightening and helpful discussion. They are also indebted to Dr. G. Ohi for valuable advice on the preparation of the manuscript.
ET AL
REFERENCES I. Magos, L. t 1971) Anal~&t 96, 847-853. 2. Magos, L., and Clarkson. T. W. (1972) ./. A.c.\oc,. Ojjic. And. Chem. 55, 966-971. 3. Eybl. V., Sykora, J., and Met-B, F. (1969) Arch. To.riXo/. 25, 296-305. 4. Moffitt, A. E., and Clary. J. J. t 1974) Res. Comrmm. Chem. Purhol. Phurtmm~l. 7. 593-603. 5. Fang, S. C. (1977) C1wnr. Biol. Interuc~riwr.~ 17, 25-40. 6. Naganuma, A., Pan, S. K., and Imura. N. (1978) Rcs. Conmun. Chrm. Pnthol. Phamucol. 20. 139-156. 7. Imura, N., and Naganuma, A. (1978) .I. Pharnz. Dw. 1, 67-73. 8. Burk. R. F.. Foster, K. A.. Greenfiels. P. M., and Kiker. K. W. (1974) Proc. Sm. E.rp. Bid. Med. 145, 782-785. 9. Chen, R. W..Whanger. P. D., and Fang. S. C. (1974) Pharmcrcol. Re;\. Conmun. 6, 571-579. 10. Imura. N., Pan, S. K., Shimizu, M.. Ukita, T.. and Tonomurd. K. (1977) Ecoro.uico/. Environ. Suj: 1, 255-261. Il. Pan. S. K.. Imura. N., and Ukita. T. (1973) Chemosphere. No. 6. 247-252. 12. Hill. C. H. (1974) J. Nlctr. 104, 593-598. 13. Gliovanoli-Jakubczak. T., Greenwood, M. R.. Smith. J. C.. and Clarkson. T. W. (1974) Chin. Chem. 20, 222-229. 14. Greenwood. M. R.. Dhahir. P.. Clarkson. T. W., Farant. J. P., Chartrand, A.. and Khayat. A. (1977) J. And. Toxicol. 1, 265-269.
98, 287-292
BIOCHEMISTRY
(1979)
Effect of Selenium on Determination Mercury in Animal Tissues AKIRA NAGANUMA,"
HIROSHI SATOH,+ REIKO YAMAMoTO;t AND NOBUMASA IMURA*,~
Received
October
of TSUGUYOSHI SUZUK1.t
24. 1978
Selenium in animal tissues was found to influence the reactivity of mercury in the tissues with stannous chloride or with stannous chloride plus cadmium chloride added as reducing agents for the determination of mercury by the method developed by L. Magos (1971 .Ana/yst . 96, 847-853) and L. Magos and T. W. Clarkson (1972. J. AS.WC. Offic. And. Chern.. 55. 966-971). The recovery of mercury in the tissues of animals to which inorganic mercury and selenite were simultaneously administered was low compared to the case in which inorganic mercury alone was administered. Of the in t’itrr) interactions of inorganic mercury and selenite examined in tissue homogenates and blood samples, only those interactions in blood samples caused the difficulty in mercury analysis mentioned above. i.e., there was a marked decrease in ret overy of mercury when an equimolar amount of each compound was added to the blood. These facts suggest that selenium and inorganic mercury in the animal tissues are likely to interact with each other and might form a chemically stable state of inorganic mercury which resists reduction with stannous chloride in the procedure for mercury determination.
Knowledge concerning the mechanism of mutual modification of toxicities between selenium and mercurial compounds has accumulated in recent years. Selenium compounds reduce the excretion of inorganic mercury, ac’celerate its accumulation in the liver, spleen, and blood (3-5), and alter its distribution in the blood by increasing markedly its uptake by red blood cells (6.7). Gel filtration discloses that, when selenite and inorganic mercury are simultaneously administered, most of the mercury is present in the high molecular weight fraction of rat plasma(S), the soluble fractions of rat organs (9) and the erythrocytes of rabbits (6.7). These high molecular weight fractions are different from those in which mercury is found when inorganic mercury alone is administered. In the high molecular weight substance in plasma of rats treated with selenite and inorganic mercury, selenium ’ To whom
all inquiries
should
be addressed. 2x7
and mercury exist at a molar ratio of approximately one (8). These data indicate that the existing state of inorganic mercury changes when selenite is simultaneously administered. Using Magos’ selective determination method for inorganic and total mercury (1,2), we measured the mercurial content in the organs of animals to which inorganic mercury and selenite were simultaneously administered, and found that the mercury accumulated in the tissues reacted differently with SnCl, or SnCl,-CdCl, than when Hg’+ alone was administered. This also suggests that administration of selenite alters the chemical state of inorganic mercury in the animal tissues. MATERIALS
AND
METHODS
Preprrrutiorz o.f’ scmplrs. Adult IVCS strain female mice (weighing about 20 g) were administered HgCI, (3 mg Hg/kg) alone 0003.2697/79/140287-06$02.00/O CopyrIght ,979 by AcndemlL Precs. Inc. All right< ~4 rsproductmn !n any form reserved.
288
NAGANUMA
subcutaneously in the gluteal region with or without Na,SeO,, (1 mg Se/kg) in the neck. Blood was collected in a test tube containing 20 ~1 of heparin sodium salt (1000 units/ml) and various organs were also excised under ether anesthesia. To the organ homogenate (40 mg tissue/ml saline) and the blood taken from control mice was added HgCI, alone or with Na,SeO, to obtain 1 x lo-” M concentrations, respectively. The resulting mixtures were then incubated at 37°C for 1 h with continual shaking. A rabbit (male, weighing about 3 kg) was anesthetized with 30 mgikg of sodium pentobarbital (Nembutal, Abbot Laboratories, Chicago, Ill.). After administering 1.0 ml of heparin sodium salt intravenously in the rabbit’s ear, the blood was collected from its carotid and incubated at 37°C for 1 h with I x lo-” M HgCI, and various concentrations of Na,SeOB. Deterrninatim of mercury. Inorganic and organic mercury contents in the organ homogenates and the blood were determined by the method reported by Magos (1) and Magos and Clarkson (2). which is described as “Magos’ method” in this paper for convenience. Inorganic mercury was measured by “method 2” of Magos (I), and total mercury was measured by “method 1” of Magos’ text (I), which was more precisely described by Magos and Clarkson (2). The original procedure by Magos defines organic mercury concentration as the difference between total and inorganic mercury concentrations. In some experiments. however, we also employed his “method 3” (1): organic mercury can be determined from the same aliquot used for inorganic mercury determination. Magos had pointed out the difficulties in a quantitative analysis of organic mercury using this procedure: the second reading for organic mercury content may be influenced by the timing of disconnecting the airflow following the first reading for inorganic mercury content. Calibration curves for inorganic mercury
ET AL
and methylmercury in “method 3” were obtained using standard solutions of these mercurials in place of tissue homogenates. The method was modified in part by circulating the air for 1 min in the reductor before feeding mercury vapor into the cells for atomic absorption spectrometry using a three-way stopper in the apparatus.” The reagents for the second step of “method 3” (SnCl,-CdCl, solution, H,S04, and NaOH solution) were added to the mixture after the recorder returned to the baseline from the peak of the first step. In addition to mercury determination by Magos’ method, the total mercury was determined using the same sample by the wet incineration method. To the sample was added 3.5 vol of concentrated HNO, and the resulting mixture was placed in a Uniseal (Uniseal Decomposition Vessels, Ltd., Israel) and heated at 140°C for 90 min, and the amount of mercury was measured by the reductive vaporization-atomic absorption method (10). Organic mercury in the tissue homogenate (100 mg tissue/6 ml distilled water) was determined by gas-liquid chromatography with an electron capture detector after extraction and concentration by the cysteine-benzene extraction method (11). RESULTS In this report, the procedure to determine inorganic mercury using SnCl, as a reducing agent in Magos ’ “method 3” (1) is called the first step, whereas the subsequent determination of organic mercury in the presence of both CdCI, and SnCl, is called the second step. Table 1 shows the mercurial concentration in the organs of mice administered HgCIZ and Na,SeO, simultaneously. In various organs a portion of mercury was detected in the second step. This portion was particularly high in the liver and the spleen. When the ’ This improved method shows a sharper peak than that shown by Magos’ original method. Thus, the errors in measurement using peak height can be reduced.
EFFECT
OF SELENIUM
ON MERCURY TABLE
MERCURY
CONCENTRATION
Method for determination 1st step 2nd step 1st and 2nd step Total mercury: by wet incineration method Organic mercury: by glc”
tissue
OF MOUSE
IN TISSUES
1
INJECTED
WITH
CHLORIDE
CONCENTRATION
Method fol determination 1st step 2nd step 1st and 2nd step mercury:
incineration
SODIUM
SELENITE”
Brain
Spleen
3.53 2 1.12 1.72 IT 0.65 5.25 t- 1.57
Il.02 k 4.34 1.24 -+ 0.28 12.27 + 4.49
0.07 2 0.01 0.03 2 0.03 0.10 i 0.04
2.10 + 1.22 0.89 f 0.41 2.99 k 1.62
1.82 k 0.52 0.48 f 0.22 2.29 -+ 0.6.5
8.63 i- 1.15
13.58
0.11 ? 0.03
5.38 -t 2.12
2.56 ? 0.43
ND
-t 3.27
IN TISSUES
Blood
-
ND
’ Mice were sacrificed 24 h after the injection. Values in the table are mean (average of six mice). ’ Gas-liquid chromatography with electron capture detector.
MERCURY
AND
Kidney
-+ SD in micrograms
Hgigram
lower by 39% in the liver and 44% in the spleen. Table 2 shows the mercurial levels in rat organs to which HgC& was administered alone. No mercury was detected in the second step except for a trace amount in the kidney. There are no significant differences between the sum values of the first and the second steps and the total mercurial contents determined by the wet incineration methods. Figure 1 shows the effect of the amount of SnCl, used as a reducing agent in the first step in determining the mercury content in the liver. When HgCl, is administered alone, the sum values of mercury concentration were approximately the same as those determined
TABLE
tissue
MERCURIC
Liver
same samples were analyzed by gas chromatography, methylmercury and other organic mercurials were not detected. The detectable limit of methylmercury in the present experiment was 120 ng Hg/g tissue. Thus, methylmercury would have been detected, had it been the mercury determined in the second step. Accordingly, the mercury detected in tlhe second step of the present experiment was not organic. It is assumed that part of the inorganic mercury existing in the organs changed its form under the influence of simultaneously administered selenite. When the sum of mecurial content determined in the first and the second steps is compared to the value determined by the wet incineration method, the sum values are
Total
189
ANALYSIS
OF MOUSE
2 INJECTED
WITH
MERCURIC
CHLORIDE
AI ONE”
Liver
Kidney
Brain
Spleen
Blood
1.49 f 0.20 ND 1.49 -t 0.20
21.92 2 3.67 1.33 i- 0.46 23.31 f 3.69
0.09 2 0.02 ND 0.09 2 0.02
0.72 ? 0.07 ND 0.72 + 0.07
0.38 f 0.10 ND 0.38 2 0.10
1.43 k 0.19
19.47 -+ 1.70
0.09 f 0.01
0.78 k 0.06
0.35
by wet method
” Mice were sacrificed 24 h after (average of six mice).
the injection.
Values
in the table
are mean
-t SD in micrograms
-c 0.06 Hgigram
290
NAGANUMA
ET AL.
.
When the total mercury contents in the organs and tissues were measured by Magos’ “method 1” about the same values as those determined by the wet incineration method were obtained among the groups to which 1 HgCl, was administered alone, but approxi.P k WC-----* .--__ *-----------.* ;i; mately 50 and 70% of the values determined E + OlrrrS by the wet incineration method were re0 loo 200 300 400 500 covered as the total mercury in the spleen S&l2 added ( mg) and liver, respectively, when HgCl, and Na,SeO, were simultaneously administered FIG. I. Effect of the amount of SnCI, used as a reducing agent in the first step of Magos’ method (Table 3). (“method 3”; see text) in determining mercury content The influences of coexisting Na,SeO, over in liver of mouse injected with mercuric chloride the recovery rate of inorganic mercury alone (--) or with sodium selenite (- - -). Mice were added in rlitro and incubated for 1 h were sacrificed 24 h after the injection. Indicated values then studied using the organ homogenates are percentage of mercurial contents determined by and the blood of control mice (Table 4). Magos’ method to those by the wet incineration method. Results from the first step (0) and the second When HgCl, alone was added, the first step step (m) of Magos’ method, and the sum of these two could vaporize most of the added mercury in values (0). A: Hg determined by Magos’ method. all the organs, and hardly any mercury was B: Hg determined by wet incineration method. detected in the second step. When Na,SeO, was added simultaneously, the influence of by the wet incineration method irrespective coexisting selenite was demonstrated only of the amount of SnCl, used. When HgCl, in the blood-merely 72% of mercury and Na,SeO, were administered simulwas determined in the first step and about taneously, the amount of mercury deter- 14% in the second step. The total yield mined in the first step with less than 100 mg was as low as 86% of the amount added. of &Cl, was relatively small. However, No influence of coexisting Na,SeO,, added with more than 100 mg of SnCl,, the amount in rho was apparent in other organs. of mercury determined in the first step as The influences of selenium concentration well as that in the second step was approxion the recovery of mercury in Magos’ mately constant irrespective of the amount method from rabbit’s blood, to which of SnCl, used. 1 x lo-” M HgC12 and various concen-
s ‘O” I-=
TABLE TOTAL
MERCURY
CONCENTRATION
CHLORIDE
Method
for determination
3
IN TISSUES
OR MERCURIC
OF MOUSE
CHLORIDE
AND
INJECTED
SODIUM
WITH
MERCURIC
SELENITE”
Liver
Kidney
Brain
Spleen
Blood
W#W Magos ’ “method Wet incineration
1” method
3.19 2 0.68 3.57 + 0.83
31.41 + 11.06 32.57 2 7.44
0.14 ? 0.06 0.13 2 0.05
1.18 2 0.33 1.21 + 0.38
0.87 i- 0.36 0.80 2 0.21
[HgCI, + Na,SeO,]” Magos ’ “method Wet incineration
I” method
6.05 2 1.00 8.49 f 1.61
21.92 21.27
0.18 k 0.06 0.18 -+ 0.03
4.45 8.98
4.53 i- 0.66 5.44 k 0.76
tissue
(1 Mice were sacrificed (average of six mice). ’ Injected compound.
12 h after
the injection.
? 8.99 k 3.37 Values
in the table
are mean
k 1.88 r 3.71
? SD in micrograms
Hg/g
EFFECT
OF SELENIUM
ON TABLE
RECOVERY
OF MERCURY CHLORIDE
MERCURY
291
ANALYSIS
4
IN MAGOS’ METHOD FROM MOUSE TISSUES WITH ADDED IN THE PRESENCE OR ABSENCE OF SODIUM SELENITE”
MERCURIC
Kidney
Brain
Spleen
Blood
98.8 ND
98.2 ND
97.4 ND
98.4 ND
98.9 ND
97.2 ND
97.8 ND
96.8 ND
98.3 ND
71.5 14.1
Liver
WKLI 1st step 2nd step [HgCl, + Na,SeO:,] 1st step 2nd step ” Values
in the table
are percentage
recovered
of the added
trations of Na,SeO, were added, were studied and the results are plotted in Fig. 2. The figure reveals that when a nearly equimolar ratio of selenium to mercury was present, the amount of mercury determined in the first step was low whereas that in the second step was high. The sum of mercurial contents measured in the first and second steps, that is, the recovery rate of the added mercury, becomes smallest near the equimolar point. DISCUSSION In the organs, especially spleen, of mice administered
Concentration
of Na$e(+
the liver Na,SeO,,
and and
(MI
FIG. 2. Influence of sodium selenite concentration on recovery of mercury in Magos’ method from rabbit blood. To blood samples was added 1 x IO-” M HgCI, and various concentrations of Na,SeO,. Mercury was determined by the first (0) or the second step (m) of Magos’ method (“method 3”; see text). Closed circles indicate the sum of these two values. Indicated values are percentage recovered of the added amount (average of five samples).
amount;
average
of samples
from
six mice.
HgC& simultaneously, we found a part of the mercury accumulated was resistant to vaporization under the conditions used by Magos for determination of mercury (1,2). Absence of any detectable amount of organic mercury in the samples was confirmed by gas-liquid chromatography. The total mercury concentrations in the liver and the spleen, determined by Magos’ “method 1,” were lower by about 30 and 50%, respectively, than those determined by the wet incineration method (Table 3). In both the liver and the spleen the accumulation of inorganic mercury is known to be accelerated when selenite is administered simultaneously (3-5). These phenomena can reasonably be explained by the assumption that the existing state of inorganic mercury in the organs was modified by the simultaneous administration of selenite. An increased amount of SnCI,, a reducing agent added in the first step in “method 3,” did not vaporize all of the mercury. This also suggests that a portion of the inorganic mercury was made chemically stable by the simultaneously administered selenium. In the i/z \~ifro experiment a phenomenon such as that mentioned above was observed only in the case of blood (Table 4). Imura and Naganuma (7) studied the behavior of inorganic mercury in rabbit blood in the presence of selenite and showed the possibility that the blood might be a primary
292
NAGANUMA
site for the interaction of inorganic mercury and selenite. The results of the present study also show that the change in the existing state of inorganic mercury caused by the presence of selenium compound occurs first in the blood. This interaction of inorganic mercury and selenite in the blood was most marked when the molar ratio of mercury to selenium was 1: 1 (Fig. 2). This fact coincides well with the previous experimental results which show that the extent of the reaction between inorganic mercury and selenium is greatest when the molar ratio is one (4,5,12). Complete digestion of the samples in relatively high concentrations of alkaline solution (13,14) before the reducing procedure might improve the interference of selenium in mercury determination by Magos’ method. On this possibility a series of experiments are in progress. ACKNOWLEDGMENTS The authors wish to thank Dr. L. Magos for his enlightening and helpful discussion. They are also indebted to Dr. G. Ohi for valuable advice on the preparation of the manuscript.
ET AL
REFERENCES I. Magos, L. t 1971) Anal~&t 96, 847-853. 2. Magos, L., and Clarkson. T. W. (1972) ./. A.c.\oc,. Ojjic. And. Chem. 55, 966-971. 3. Eybl. V., Sykora, J., and Met-B, F. (1969) Arch. To.riXo/. 25, 296-305. 4. Moffitt, A. E., and Clary. J. J. t 1974) Res. Comrmm. Chem. Purhol. Phurtmm~l. 7. 593-603. 5. Fang, S. C. (1977) C1wnr. Biol. Interuc~riwr.~ 17, 25-40. 6. Naganuma, A., Pan, S. K., and Imura. N. (1978) Rcs. Conmun. Chrm. Pnthol. Phamucol. 20. 139-156. 7. Imura, N., and Naganuma, A. (1978) .I. Pharnz. Dw. 1, 67-73. 8. Burk. R. F.. Foster, K. A.. Greenfiels. P. M., and Kiker. K. W. (1974) Proc. Sm. E.rp. Bid. Med. 145, 782-785. 9. Chen, R. W..Whanger. P. D., and Fang. S. C. (1974) Pharmcrcol. Re;\. Conmun. 6, 571-579. 10. Imura. N., Pan, S. K., Shimizu, M.. Ukita, T.. and Tonomurd. K. (1977) Ecoro.uico/. Environ. Suj: 1, 255-261. Il. Pan. S. K.. Imura. N., and Ukita. T. (1973) Chemosphere. No. 6. 247-252. 12. Hill. C. H. (1974) J. Nlctr. 104, 593-598. 13. Gliovanoli-Jakubczak. T., Greenwood, M. R.. Smith. J. C.. and Clarkson. T. W. (1974) Chin. Chem. 20, 222-229. 14. Greenwood. M. R.. Dhahir. P.. Clarkson. T. W., Farant. J. P., Chartrand, A.. and Khayat. A. (1977) J. And. Toxicol. 1, 265-269.
