Maternal Selenium Deficiency Enhances the Fetolethal Toxicity of Methyl Mercury NORIKONISHIKIDO,*KUMIKOFURUYASHIKI,*AKIRA TSUGUYOSHI
*Department ofPublic Health, School ofPharmaceutical Sciences, Kitasato University: Y-I, Shirokane 5 chornc’, Minato-ku. Tokyo 108, Japan and TDepartment of Human Ecology. School ofHealth Sciences. Facult) ofMedicine, University sf TolcJu. 3-l. Hongo 7 chome, Bunk~&x. Tokyo 113, Japan
ReceivedAugust .?I. 1986: accepted Januar!, 1.5.1987 Maternal Selenium Deficiency Enhances the Fetolethal Toxicity of Methyl Mercury. NIK., NAGANUMA. A..SUZUKI. T., ANDIMURA.N.(I~S~). Tolicol. Appl. Pharmacol. 88, 322-328. The effect of maternal selenium deficiency on methyl mercury fetotoxicity was examined in the ICR strain of mice. Pregnant mice were fed either seleniumdeficient diets based on torula yeast or selenium-supplemented diets which were identical to the former except that 0.1, 0.2, or 0.4 mg of selenium per kilogram of diet was added as sodium selenite. Fetolethality ofmethyl mercury wasexacerbated by maternal selenium deficiency when mothers were administered SC 15. 25, or 35 pmol/kg/day of methylmercuric chloride (MMC) on the 13. 14. and 15th days of pregnancy. One-tenth part per million of selenium in the diet was sufficient to protect the fetuses against MMC fetolethality when dams were administered 25 pmol/kg/day of MMC. Mercury concentrations in maternal and fetal tissues were independent of the dietary selenium level. Selenium concentration and glutathione peroxidase (GSH-Px) activity in maternal tissues were unaffected by MMC administration. In fetal liver. on the other hand. selenium concentration was increased and GSH-Px activity was decreased concurrently by maternal MMC administration in the selenium-supplemented groups. Therefore. as far as GSH-Px activity was concerned, the bioavailability of selenium was markedly decreased in fetal liver by maternal injection of MMC. The increase in selenium content in fetal liver. which was observed only in the selenium-supplemented groups. may play an important role in protection against fetolethal toxicity of MMC. IC 1987 Acadsmtc press. IIIC. SHIKIDO,N..FLJRUYASHIKI,
Selenium. an essential trace element (Schwarz and Foltz, 1957) is known to protect experimental animals from methyl mercury intoxication by simultaneous administration (Iwata et al., 1973; Ohi et al., 1975b) or by excessive supplementation in diets (Ganther et al., 1972; Potter and Matrone, 1974; Ohi et al., 1975a; Sell and Horani, 1976: El-Begearmi et al., 1977; Stoewsand et al., 1977). The nutritional level of selenium. which is contained in ordinary commercial diet at a level of approximately 0.4-0.6 ppm. ’ To whom all correspondence should be addressed. 004 1-008X/87 $3.00 Copyright :s 1987 by Academxc Press. Inc. All rights ofreproductmn in any form reserved.
was also found to alleviate methyl mercury toxicity in rats (Ganther et al., 1972; Stillings et al.. 1974) Japanese quail (Welsh and Soares, 1976) and mice (Oda et al., 1985). The mechanism of protection by selenium against methyl mercury intoxication has been discussed (Ganther, 1978; Magos and Webb, 1980: Naganuma et al., 1980) but has not yet been clarified. Methyl mercury is highly toxic to fetuses and causesdeath, malformations, behavioral disorders, and growth retardation (Spyker and Smithberg, 1972; Su and Okita, 1976: Fuyuta et al., 1979; Shimai and Satoh, 1985).
The effect of selenium simultaneously administered (Lee et al., 1979) or excessively supplemented in the diet (Nobunaga et al., 1979) on fetotoxicity of methyl mercury has been investigated in mice. Both studies revealed that excess selenium administered could not always prevent the fetotoxic manifestations of relatively high doses of methyl mercury. However, it is not known if the nutritional level of selenium ingested daily with food offers protection against methyl mercury fetotoxicity. In the present study, effects of the nutritional level of selenium on the fetotoxicity of methyl mercury were examined, and selenium-dependent changes in the distribution of methyl mercury, selenium, and glutathione peroxidase (GSH-Px) activity were investigated. METHODS A~~i~nu/s and d&s. Male and female ICR mice were obtained from Charles River, Japan. Selenium-deficient diet based on torula yeast (0 ppm selenium (Se) diet; Table 1) was purchased from Oriental Yeast Co. Ltd., Japan. Fluorometric analysis revealed that the torula yeast diet contained 0.009 ppm of selenium. Chmicals. ‘“3HgClZ was purchased from Amersham Int. Corp. Methylcobalamin was obtained from Sigma Chemical Co. ‘03Hg-labeled methylmercuric chloride (MMC. CH3’03HgCI. 3.5 @X/S rmol/ml) was prepared from ‘03HgC1z and methylcobalamin by the method of Naganuma et al. (I 985). Treamenr. From 4 weeks of age, female mice were fed diets containing varying amounts ofselenium, i.e.. the 0. 0.1.0.2. or 0.4 ppm Se diet. which was prepared by adding an appropriate amount of selenium as sodium selenite to the 0 ppm Se diet. At 9- 11 weeks of age, females in preestrus were mated overnight with males which had always been fed commercial stock diet containing approximately 0.4 ppm of selenium. When a vaginal plug or sperm was found the following morning, the day was designated as Day 0 of gestation. On Days 13. 14. and 15 ofgestation. dams were injected SC with CH3’03HgCI at a dose of0. 15.25. or 35 rmol/kg/day. On Day 17 ofgestation. dams were anesthetized with ether and blood was collected from the jugular vein with heparin. The uterus was exposed, and the numbers of surviving fetuses. dead fetuses. and early resorptions were recorded. Litter size was defined as the sum of surviving and dead fetuses, and the survival rate of fetuses in each treatment group was defined as the ratio of the total number of surviving fe-
COMPOSITIONOFSELENIUM-DEFICIENT DIEI % Torula yeast Sucrose Lard Cod-liver oil Mineral mixture” Vitamin mixture” Methionine
30.0 56.7 5.0 2.0 5.0 1.0 0.3
a Mineral mixture (%): CaHP04.2Hz0. 14.56: KH,PO,, 25.72; NaH2P04, 9.35: NaCl, 4.66; Ca-lactate. 35.09; Fe-citrate. 3.18: MgSO,, 7.17; ZnCO,, 0.11; MnS04 .4-6HzO. 0.12; CuSOI. 5HZ0, 0.03; Kl, 0.0 1: cc13. 6HZ0, 0.02. ’ Vitamin mixture (per 100 g of mixture): vitamin A acetate. 46,600 IU; vitamin D,, 23.300 IU; vitamin E acetate, 1200 mg; vitamin KA. 6 mg; vitamin B, HCI. 59 mg: vitamin B2, 59 mg; vitamin B6. HCI, 29 mg: vitamin B,?, 0.2 mg: vitamin C, 588 mg: D-biotin, 1 mg; folic acid. 2 mg; Ca-D-pantothenate, 235 mg; nicotinic acid, 294 mg: inositol. 1 176 mg.
tuses to the sum of litter sizes. Fetuses were rinsed in physiological saIine, blotted on filter paper, and weighed. Fetal livers were collected from fetuses and pooled per litter for the following analyses. Maternal and fetal tissues were then stored frozen at -90°C pending the analyses. An&es. Radioactive mercury contents in tissues were determined by the Aloka Autowell gamma system. Selenium concentrations in tissues were determined fluorometrically (Watkinson. 1966). For measurement ofselenium-dependent GSH-Px activity. each tissue was homogenized in 4 vol of0.25 M sucrose and the homogenate was centrifuged for 60 min at lOS,OOOg. The supernatant and lysed red blood cells (RBC) were analyzed for GSH-Px activity using H202 as substrate by the method of Lawrence and Burk (1976). Statistics. The significance of variances among the experimental groups was analyzed by two-way analysis of variance (ANOVA). Comparison of individual means was according to the Student I test.
RESULTS Eflect of selenium in diet on fetotouxicity of MA4C. The data in Table 2 show survival rates and mean body weights of fetuses after
maternal administration of MMC in groups fed diets with various selenium levels. Dams continued to gain weight after administration of MMC and there was no significant difference in maternal weight gain between the selenium-deficient dams and the seleniumsufficient ones. There were no significant variances in litter sizes or number of early resorptions among the groups tested. In the 0.4 ppm Se diet group, MMC had no effect on the survival rate of fetuses up to a dose of 25 pmol/kg/day. In the 0 ppm Se diet group, in contrast, only 30% of the fetuses survived at the dose of 15 pmol/kg/day of MMC. At 35 pmol/kg/day of MMC all fetuses were dead. These marked decreases in survival rates of fetuses observed in the 0 ppm Se diet group showed that the exacerbated dose-dependent fetolethal effect of MMC was caused by maternal selenium deficiency. When dams in four different selenium level groups were administered 25 pmol/kg/day of MMC, only 11.9% of the fetuses in the 0 ppm Se diet group survived, while all of the fetuses were alive in the 0.1,0.2, and 0.4 ppm Se diet groups. The degree of fetal growth suppression by maternal MMC administration in the 0.1 ppm Se diet group was about the same as that in the 0.4 ppm Se diet group. These results suggest that 0.1 ppm of dietary selenium is as equally protective as 0.4 ppm against adverse effects of maternally administered MMC. Mercury distribution. Approximately 20% of the dose of *03Hg was accumulated in the fetuses in all groups fed diets with various selenium levels. Mercury concentration in fetal liver was slightly higher than that in maternal liver. There were no remarkable differences in mercury concentrations in maternal tissues and fetal liver among the four groups fed diets containing different levels of selenium. Selenium concentration. Table 3 shows selenium concentrations in maternal tissues in the groups fed diets with various selenium levels, with or without MMC treatment (25 pmol/kg/day). Among the tissues analyzed the highest selenium concentration was
4.13 (1.22) 12.42 (1.61) 4.62 (0.38) 0.71 (0.26) 3.57 (0.48)
4.06 (1.35) 12.98 (2.08) 4.07 (0.23) 0.62 (0.24) 3.06 (0.39)
7.12 (1.62) 17.09 ( 1.90) 5.65 (0.43) 1.91 (0.47) 5.04 (0.55)
8.68 (2.53) 17.30 (3.11) 5.29 ( 1.OO) 1.38 (0.78) 3.84 (0.98)
.&‘flle. Dietary selenium effect was significant (p < 0.001) for all tissues but MMC effect was significant placenta (p < 0.0 1) by ANOVA. ’ Mice were injected with 25 pmol/kg/day of MMC on Days 13. 14, and 15 of gestation. h Mean (SD), nmol/g tissue.
only for the
Kidney RBC Plasma Placenta
0.18 (O.ll)h 4.19 (0.56) 1.31 (0.34) 0.09 (0.01) I .35 (0.23)
(0.06) 5.25 (0.18) 1.36 (0.20) 0.14 (0.02) 1.19 (0.06)
2.31 (1.04) 10.23 (1.34) 3.31 (0.49) 0.37 (0.07) 2.92 (0.44)
found in the kidneys. The selenium concentrations in maternal tissues decreased in proportion to those in the diets (ANOVA, dietary selenium effect, p < 0.001). MMC administration caused no remarkable change in selenium concentrations in maternal liver, kidneys, RBC, and plasma (ANOVA, MMC effect, p > 0.05). In the placenta, selenium levels were slightly decreased by MMC injection (ANOVA, MMC effect, a < 0.0 1). In fetal liver, however, selenium levels were markedly increased by 25 pmol/kg/day of maternal MMC administration (Fig. 1; ANOVA, MMC effect, F = 329.7,~ < 0.001). Depending on the selenium levels in diets, the effects caused by MMC injection were amplified (ANOVA, interaction, F = 44.4, p < 0.001). In the 0 ppm Se diet group, no significant change in fetal liver selenium concentration was caused by maternal MMC injection (t test, p > 0.05). On the other hand, in the 0.1, 0.2, and 0.4 ppm Se diet groups, striking increases in fetal liver selenium levels were observed by maternal administration of MMC (t test, p < 0.001).
in diet (ppm)
IN MATERNAL TISSUES IN GROUPS FED DIETS WITH VARIOUS WITH OR WITHOUT MMC TREATMENT
1.99 (0.67) 9.11 (1.37) 2.98 (0.46) 0.36 (0.12) 2.44 (0.23)
GSH-Px activity. Administration of MMC (25 pmol/kg/day) did not cause any significant change in GSH-Px activities in maternal liver, kidneys, placenta, and RBC (ANOVA,
FIG. 1. Effect of maternal injection of MMC on selenium concentration in fetal liver in groups fed diets with various selenium levels. (0) untreated; ( jetted (25 rmol/kg/day). Each value is the .i?* SD of four or five litters. Dietary selenium effect (p i O.OOl), MMC effect (p < O.OOl), and interaction (p < 0.001) were significant by ANOVA. *Significantly different from untreated group fed the same diet, p < 0.00 I by t test.
& .? -
m-30 Tic - ‘yj .-c- 0 2 ‘5
.- E z533 E lo:I o-
0.2 in diet
0.4 ( ppm
FIG. 2. Effect of maternal injection of MMC on GSHPx activity in fetal liver in groups fed diets with various selenium levels. GSH-Px activity was measured by the coupled assay using HtOz as substrate. (0) untreated; (BZ#J) MMC injected (25 rmol/kg/day). Each value is the S + SD of four or five litters. Dietary selenium effect (p < 0.001). MMC effect (p < 0.001). and interaction (p < 0.05) were significant by ANOVA. *. **, ***Significantly different from untreated group fed the same diet. p i 0.05. p < 0.0 1, and p < 0.00 1, respectively. by t test. n.d.. Not detected.
MMC effect, p > 0.05 for all tissues). In fetal liver, in contrast, the enzyme activity was markedly decreased by maternal MMC injection in all groups fed diets with various selenium levels (Fig. 2; ANOVA, MMC effect, p < 0.001). Correlation between selenium concentration and GSH-Px activity in fetal liver. There were significant correlations between selenium concentrations and GSH-Px activities in fetal liver both in the untreated group and in the MMC-treated (25 pmol/kg/day) group. The correlation coefficient was 0.89 (p < 0.001) in the untreated group and 0.71 (p < 0.001) in the MMC-treated group. However, the ratio of GSH-Px activities to selenium concentrations was markedly smaller in the MMC-treated group than in the untreated group.
DISCUSSION The present study demonstrated for the first time the protective effect of the nutri-
tional level of selenium on the fetolethal toxicity of methyl mercury. In the 0 ppm Se diet group, fetal death occurred at lower dose levels of MMC than in the 0.4 ppm Se diet group (Table 2), indicating that 0.4 ppm of selenium in the diet, which was almost the same level as that in commercial stock diet, has a protective effect on methyl mercury fetotoxicity. In addition, as also described in Table 2.0.1 ppm of selenium in the diet is sufficient to protect fetuses against fetolethal toxicity of 25 pmol/kg/day of MMC injected for 3 consecutive days in the late gestational period. Lee et al. (1979) and Nobunaga et cl/. ( 1979) reported that coadministration of relatively high doses of selenium with methyl mercury afforded only slight protection against the fetolethal and teratogenic toxicities of methyl mercury, depending on the dose combination of the two chemicals. The discrepancy between their findings and ours may be due to the different experimental design. The amount of MMC administered to dams was larger in their studies than that in this study. Also their dams were exposed to MMC for a longer period, i.e., during the precoital and the complete gestational period (Nobunaga et al., 1979) or in the middle gestational period when fetuses were more sensitive to induction of cleft palate by methyl mercury (Lee ez al.. 1979). In the present study dams were administered MMC for only 3 days in the late gestational period. Accumulation of mercury in the fetal liver does not appear to be correlated with selenium levels in the diets. Satoh and Suzuki (1979) also reported no change in mercury accumulation in fetal liver when it was coadministered with selenite. It was suggested, therefore, that the exacerbated fetolethality of MMC by maternal selenium deficiency might not be due to an increase of mercury content in the fetuses. Maternal injection of MMC markedly increased the selenium concentration in fetal liver in the groups of mice fed the seleniumsupplemented diets (Fig. 1). Iijima of ~1. (1978) previously found increased selenium
accumulation in fetuses 24 hr after simultaneous SCinjection of sodium selenite (0.20 mg Se/kg) and MMC (0.67 mg Hg/kg) into dams. However, Yonemoto et al. (1983) reported that there was no significant increase in selenium accumulation in fetuses 60 min after simultaneous iv injection of sodium selenite (0.75 pmol/kg) and MMC (1.5 pmol/ kg). This discrepancy may reflect the different times of analyses (24 hr vs 1 hr). Although the mechanism of the increase in the endogenous selenium level in fetal liver found in the present study is not clear, it may not be unreasonable to assume that the increased level of selenium has some role in protecting the fetuses against methyl mercury toxicity. To elucidate the mechanism of increased selenium content in fetal liver caused by maternal MMC injection, the chemical form of increased selenium in the fetus has to be investigated. Figure 2 shows that GSH-Px activity in fetal liver was decreased by maternal injection of MMC in all experimental groups. The cause of this decreased activity is not known. It is unlikely that it results from a direct effect of MMC on the enzyme assay, since we found MMC concentrations of more than lO-3 M were needed to inhibit GSH-Px activity in vifro. The final concentration of mercury derived from fetal liver cytosol of MMC-injetted mice was approximately 10m6M in the assay medium for GSH-Px activity. This seems too low to affect the enzyme activity. Kling and Soares (1978) reported that addition of MMC to the diet increased selenium concentration but not GSH-Px activity in the blood of the Japanese quail when selenium was supplemented in the diet at a concentration of 1 ppm. This result suggested that methyl mercury might reduce selenium bioavailability in the blood. In our study, however. neither selenium level (Table 3) nor GSH-Px activity was affected in the maternal blood and other tissues, except for placenta. The slight decrease in placental selenium content by MMC administration could result from the accelerated transport of selenium to fetuses.
On the other hand, in fetal liver, maternal MMC administration caused both the increase in selenium concentration (Fig. 1) and the decrease in GSH-Px activity (Fig. 2) in the selenium-supplemented groups. Consequently, the ratio of GSH-Px activities to selenium concentrations in fetal liver was dramatically reduced by maternal MMC administration. Thus, as far as GSH-Px activity is concerned, bioavailability of selenium was remarkably decreased in fetal liver after maternal injection of MMC, indicating that the fetuses are more sensitive to methyl mercury than the mother. Regarding fetal growth suppression by MMC administration, the nutritional level of selenium does not seem to be completely protective since body weights of fetuses on Day 17 of gestation were significantly decreased by maternal injection of 25 wmol/kg/day of MMC even in the 0.4 ppm Se diet group (Table 2). A lower dose level of MMC, which does not cause fetal death, even in the 0 ppm Se diet group, should be used to study the influence of the nutritional level of selenium on fetal growth suppression by MMC. In addition, appropriate selection of dosage and period of MMC administration should enable us to examine the effect of the nutritional level of selenium on the teratogenicity of methyl mercury.
H. E. (1977). and selenium _ A&. 13-J FUYUTA,
A mutual protective effect of mercury in Japanese quail. Poult. Sci. 56, 313FUJIMOTO,
Teratogenic effects of a single oral administration of methylmercuric chloride in mice. iicta .4nat. 104, 356-362. GANTHER. H. E. ( 1978). Modification of methylmercury toxicity and metabolism by selenium and vitamin E: Possible mechanisms. Environ. Health Perspect. 25, 11-76. GANTHER, M. J.,
H. E., GOUDIE, WAGNER, P.,
W. G. (1972).
C., SUNDE, OH. S-H.,
M. L., KOPUYKY. AND HOEKSTRA.
of methylmercury added to diets containing tuna. Srience 175,1122- 1124. IIJIMA,S.,TOHYAMA,C., Lu,C.-C., ANDMATSUMOTO, N. (1978). Placental transfer and body distribution of methylmercury and selenium in pregnant mice. To.xicol. Appl. Pharmacol.
44, 143- 146.
IWATA, H., OKAMOTO, H., AND OHSAWA, Y. ( 1973). Effect of selenium on methylmercury poisoning. Res. Commun.
KLING, L. J., AND SOARES, JR., J. H. (1978). Mercury metabolism in Japanese quail. II. The effects ofdietary mercury and selenium on blood and liver glutathione peroxidase activity and selenium concentration. Poult. Sri. 57, 1286-1292. LAWRENCE, R. A.. AND BURK, R. F. (1976). Glutathione peroxidase activity in selenium-deficient rat liver. Biothem.
LEE, M., CHAN, K. K.-S., SAIRENJI, E.. ANDNIIKUNI, T. (1979). Effect of sodium selenite on methylmercuryinduced cleft palate in the mouse. Environ. Res. 19, 39-48. MAGOS, L.. AND WEBB, M. (1980). The interactions of selenium with cadmium and mercury. CRCCrit. Rev. Toxicol.
NAGANUMA, A., KOJIMA, Y., AND IMURA, N. (1980). Interaction of methylmercury and selenium in mouse: Formation and decomposition of bis(methylmercuric) selenide. Res. Commun. Chem. Pathol. Pharmacol. 30,301-316. NAGANUMA, A., URANO, T., AND IMURA, N. (1985). A simple and rapid method for preparation of “3Hg-labeled methylmercury from 203HgC12and methylcobalamin. J. Pharmacobio-Dyn. 8,69-72. NOBUNAGA, T., SATOH, H., AND SUZUKI, T. (1979). Effects of sodium selenite on methylmercury embryotoxicity and teratogenicity in mice. Toxicol. .4ppl. Pharmacol.
ODA. N., NAGANUMA, A., AND IMURA. N. (1985). Enhancement of methylmercury toxicity by selenium deficiency. J. Pharmacobio-Dyn. 8, S22. OHI, G., NISHIGAKI, S., SEKI, H., TAMURA, Y., MAKI, T., MAEDA, H.. OCHIAI, S., YAMADA, H.. SHIMAMURA, Y.. AND YAGYU. H. (1975a). Interaction of dietary methylmercury and selenium on accumulation and retention of these substances in rat organs. Toxicol. Appl. Pharmacol.
ET AL. OHI,G., SEKI, H.,MAEDA,H.,ANDYAGYU, H.(l975b). Protective effect of selenite against methylmercury toxicity: Observations concerning time, dose, and route factors in the development of selenium attenuation. Ind. Health 13,93-99. POTTER, S., AND MATRONE. G. (1974). Effect of selenite on the toxicity of dietary methyl mercury and mercuric chloride in the rat. J. Nutr. 104,638-647. SATOH, H., AND SUZUKI, T. (1979). Effects of sodium selenite on methylmercury distribution in mice of late gestational period. Arch. Toxicol. 42,275-279. SCHWARZ,K., ANDFOLTZ, C. M. ( 1957). Selenium as an integral part of factor 3 against dietary necrotic liver degeneration. J. Amer. Chem. Sot. 79,3292-3293. SELL, J. L., AND HORANI. F. G. (1976). Influence of selenium on toxicity and metabolism of methylmercury in chicks and quail. Nutr. Rep. Int. 14,439-447. SHIMAI, S., ANDSATOH. H. (I 985). Behavioral teratology of methylmercury. J. Toxicol. Sci. 10, 199-216. SPYKER, J. M.. AND SMITHBERG, M. (1972). Effects of methylmercury on prenatal development in mice. Teratology& 181-187. STILLINGS, B. R., LAGALLY, H., BAUERSFELD. P., AND SOARES.J. ( 1974). Effect of cystine. selenium, and fish protein on the toxicity and metabolism of methylmercury in rats. Toxicol. Appl. Pharmacol. 30,243-254. STOEWSAND. G. S., ANDERSON. J. L., GUTENMANN. W. H., AND LISK. D. J. (I 977). Form of dietary selenium on mercury and selenium tissue retention and egg production in Japanese quail. Nutr. Rep. Int. 15, 81-87. Su. M.-Q., AND OKITA, G. T. (1976). Embryocidal and teratogenic effects of methylmercury in mice. To.~rsol. Appl. Pharmacol. 38.207-2 16. WATKINSON. J. H. (1966). Fluorometric determination of selenium in biological material with 2,3-diaminonaphthalene. .4nal. Chem. 38,92-97. WELSH, S. O., AND SOARES.JR.. J. H. (I 976). The protective effect ofvitamin E and selenium against methyl mercury toxicity in the Japanese quail. Nutr. Rep. Inr 13,43-5 1. YONEMOTO, J., NAGANUMA, A.. SUZUKI, T., AND IMURA, N. (1983). Effects of vitamin E, glutathione and methylmercury on distribution and placental transfer of selenium in mice. Chemosphere 12, 102 I 1029.