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Maternal-to-fetus transfer of mercury in metallothionein-null pregnant mice after exposure to mercury vapor
Toxicology 175 (2002) 215– 222 www.elsevier.com/locate/toxicol
Maternal-to-fetus transfer of mercury in metallothionein-null pregnant mice after exposure to mercury vapor Minoru Yoshida a,*, Masahiko Satoh b, Akinori Shimada c, Emi Yamamoto c, Akira Yasutake d, Chiharu Tohyama b a b
Department of Chemistry, St. Marianna Uni6ersity School of Medicine, 2 -16 -1 Sugao, Miyamae-ku, Kawasaki 261 -8511, Japan En6ironmental Health Sciences Di6ision, National Institute for En6ironmental Studies, 16 -2 Onogawa, Tsukuba 305 -8506, Japan c Department of Veterinary Pathology, Tottori Uni6ersity, Koyamachyo Minami 4 -101, Tottori-shi, Tottori 680 -0945, Japan d Biochemistry Section, National Institute for Minamata Disease, Minamata, Kumamoto 867 -0008, Japan Received 17 December 2001; received in revised form 1 March 2002; accepted 1 March 2002
Abstract This study examined the role of placenta metallothionein (MT) in maternal-to-fetal mercury transfer in MT-null and wild-type mice after exposure to elemental mercury (Hg0) vapor. Both strains were exposed to Hg0 vapor at 5.5–6.7 mg/m3 for 3 h during late gestation. Twenty-four hours after exposure to Hg0 vapor, accumulation of mercury in the major organs, except the brain, of MT-null maternal mice was significantly lower than that in organs of wild-type mice. In contrast to mercury levels in maternal organs, fetal mercury levels were significantly higher in MT-null mice than in wild-type mice. In placenta, mercury concentrations were not significantly different between the two strains. Although MT levels in major organs, except the brain, of wild type mice were markedly elevated after the exposure to Hg0 vapor, the placental MT levels were not elevated. However, endogenous MT level in the placenta is significantly higher than that in other organs, except the liver. Gel filtration profile of the placental cytosol in the wild-type mice revealed that a large amount of placental mercury was associated with MT. In MT-null mice, mercury in placental cytosol appeared mainly in the high-molecular-weight protein fractions. Mercury in the placenta was localized mainly in the yolk sac and decidual cells in the deep layer of the decidua in both mouse strains. The similar localization of MT was found in the placenta of wild type mice. These results suggest that MT in the placenta has a defensive role in preventing maternal-to-fetal mercury transfer. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Mercury vapor; Metallothionein; Fetus; Null mice; Placenta
1. Introduction Exposure to toxic metals, such as mercury (Hg), lead (Pb) and cadmium (Cd), during gestation * Corresponding author. Fax: + 81-44-977-8133. E-mail address: [email protected] (M. Yoshida).
potentially causes adverse effects on fetal development. Since the outbreak of fetal Minamata disease in Japan was found to be caused by in utero exposure to methyl mercury, numerous investigations on the placental transfer of various mercury compounds and the uptake of mercury by fetal tissues have been carried out (Suzuki et al., 1968;
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M. Yoshida et al. / Toxicology 175 (2002) 215–222
Grennwood et al., 1972; Satoh et al., 1981). Experimental exposure of pregnant animals to Hg0 vapor exposure has shown that although Hg0 is rapidly oxidized to ionic mercury (Hg++), some Hg0 penetrates the placental barrier and enters the fetus (Clarkson et al., 1972; Khayat and Dencker, 1982; Yoshida et al., 1986). Furthermore, our previous study of late-term pregnant guinea pigs showed that Hg0 transferred through the placental barrier is oxidized in fetal liver and is bound to fetal hepatic metallothionein (MT), which indicates that protein has a role in protecting the fetus from mercury toxicity (Yoshida et al., 1987). MT is a low-molecular-weight metal-binding protein with high cysteine content. In mammals, this protein is believed to play an important role in the metabolism, transport, and storage of essential metals, such as zinc and copper, and in the detoxification of heavy metals, especially Cd and Hg (Cherian and Goyer, 1978; Webb and Cain, 1982). MT synthesis is induced in various tissues by these metals, food restriction, physical and chemical stress, and glucocorticoid hormones. MT is also reported as present in human and rodent placenta during pregnancy and is induced by exposure to essential and non-essential metals (Lau et al., 1998; Goyer and Cherian, 1992). Goyer and Cherian (1992) described placental MT as playing a role in modulating maternal-to-fetal transfer of essential and non-essential metals. Lucis et al. (1972) and Boadi et al. (1991) reported that after exposure to Cd, most Cd in placenta is associated with MT, which may provide the fetus some protection against the toxic effects of heavy metals. Although some Hg0 persisting in blood penetrates the placenta barrier, this barrier prevents Hg++ from entering into the fetus. However, the role of placental MT in maternal-to-fetal mercury transfer after exposure to Hg0 vapor remains unclear. Recently, MT gene knockout mice (MT-null mice) that do not express MT-I and MT-II genes have been established (Palmiter et al., 1993; Michalska and Choo, 1993). The purpose of the present study was to clarify the role of placental MT in maternal-to-fetal mercury transfer in MTnull and wild-type mice after exposure to Hg0 vapor.
2. Materials and methods
2.1. Animals and exposure to mercury (Hg 0) 6apor MT-null mice and wild-type OLA129/C57BL6 control mice were provided by Dr A. Choo. Mating was achieved by placing a male and two females in the same cage overnight. The animal facility was maintained under a light/dark cycle of 12-h, temperature of 249 1 °C, relative humidity of 55 9 10%, and negative atmospheric pressure. The mice received mouse chow and filtered tap water ad libitum. When they reached day 16 of pregnancy, the animals were used in the experiment. Throughout the experiment, animals received humane care according to the National Institute for Environmental Studies’ guidelines for animal welfare. Four MT-null and 3 wild-type mice were exposed to mercury vapor as a group for 3 h as described previously (Yoshida et al., 1999b). Mercury concentration in the exposure chamber (20 l) was measured hourly by the air sampling method (Lindstedt and Skerfving, 1972) and was controlled at 5.5–6.7 mg/m3. Twenty-four hours after exposure to mercury vapor, the dams were killed with diethyl ether overdose, and placenta and fetuses were removed immediately from the uterus. Brain, lung, liver, and kidney of pregnant mice and non-pregnant controls were removed and stored at − 80 °C until analysis. The tissues for histological analysis were fixed in 10% neutral buffered formalin.
2.2. Gel filtration of tissue supernatant MT-null and wild type mouse tissues were homogenized in ice-cold 1.15% KCl under N2 atmosphere to yield a 20% (w/v) homogenate. The homogenate was centrifuged at 105 000× g for 60 min, and the supernatant was filtered through a 0.22-mm pore diameter membrane (Ultrafree C3, Millipore) at 5000× g for 1 min. A portion of the supernatant was applied to a Superdex 75 HR 10/30 column (Pharmacia Biotech, Tokyo, Japan) equilibrated with phosphate-buffered saline (PBS). The sample was eluted with the same
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buffer at 4 °C, and 1 ml fractions were collected at a flow rate of 0.5 ml/min (Yasutake et al., 1998).
2.3. Analysis of MT and mercury concentrations in tissue Tissue MT concentrations were measured with a radioimmunoassay (Tohyama and Shikh, 1981) as modified by Nishimura et al. (1990). The detection limit of this method was 0.2 mgMT/g of tissue. Mercury concentrations were measured with a cold atomic absorption spectrophotometer (RA-2A Mercury Analyzer; Nippon Instruments, Tokyo, Japan) after digestion with a concentrated acid mixture [HNO3/HClO4 3:1 (v/v)] (Satoh et al., 1997). After the fetuses were homogenized with 10 mM Tris– HCl buffer (pH 7.4), an aliquot was used for mercury analysis. The detection limit of this method was 0.5 ngHg with an intra-assay coefficient of variation of 4% (n =10).
2.4. Double staining: immunohistochemistry and autometallography The placental tissues fixed in 10% neutral buffered formalin were embedded in paraffin. Immunochemistry of MT was performed on 3 mm paraffin sections. The primary antibody used in this study was MT monoclonal antibody (E9, DAKO, Glostrup, Denmark) against horse MT-I and –II (1:200 in M.O.M.™). After treatment with 0.1% trypsin solution at 37 °C for 15 min and blockade of endogenous peroxidase activity with 3% H2O2 in PBS, sections were treated with M.O.M.™ Mouse IgG Blocking Reagent for 4 h, incubated with primary antibodies overnight at 4 °C, and then sequentially incubated with M.O.M.™ Biotinylated Anti-Mouse IgG Reagent for monoclonal antibody for 1 h at room temperature and with VECTASTAIN Elite ABC Reagent for 1 h. The sections were then washed 3 times for 10 min each in PBS and developed with 0.02% 3,3,-diaminobenzidine tetrahydrochloride and H2O2. After being immunostained, sections were then stained for mercury by autometallography (Danscher and M8ller-Madsen, 1985). The sections were pretreated with 1% potassium cya-
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nide for 2 h to eliminate non-specific staining from silver sulfides or selenides (Pamphlett and Waley, 1996), placed in a physical developer containing 50% gum arabic, citrate buffer, hydroquinone and silver nitrate at 26 °C for 40 min in the dark. Excess silver was dissolved in by 5% sodium thiosulfate. The sections were counterstained with haematoxylin. The reaction product was seen as small black grains of silver surrounding an invisible mercury core within the tissue.
2.5. Statistical analysis Data were analyzed statistically by Student’s t-test for comparison between MT-null and wildtype mice and one-way ANOVA for time-dependent changes with a preset probability level of PB 0.05 or PB 0.01.
3. Results When mercury concentrations in the maternal organs of MT-null and wild-type mice were checked 24 h after exposure to Hg0 vapor, both mouse strains showed the highest concentration in the kidney, followed by those in the lung, liver, and brain (Fig. 1). Mercury concentrations were significantly lower in the lung and liver of MTnull mice than of wild-type mice (PB 0.05). In the kidney, the mercury concentration in MT-null
Fig. 1. Mercury concentrations in major organs of pregnant MT-null and wild-type mice at 24 h after exposure to mercury vapor. Values are represented as mean 9standard deviation (n = 3 – 4). *Significant difference from wild-type mice at P B 0.05. **Significant difference from wild-type mice at PB 0.01.
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Table 1 Mercury concentrations in the placenta and fetus of pregnant MT-null and wild-type mice after exposure to mercury vapor Wild-type mice
MT-null mice
Control
Exposure
Control
Exposure
Fetus ngHg/g tissue ngHg/body weight
9 92 (n=9) 591 (n=9)
85 97 (n = 9) 579 9 (n = 9)
7 9 1 (n =9) 5 9 2 (n =9)
122 919* (n = 13) 90 9 10* (n =13)
Placenta ngHg/g tissue
19 9 3 (n= 9)
1702 9 327 (n = 9)
17 91 (n =9)
1866 9513 (n =13)
Ratio of placenta/fetus
–
209 4
–
15 9 3
Values are mean 9 standard deviation. The number of animals are given in parentheses. * Significant difference from wild-type mice at PB0.05.
mice was approximately one-third that in wildtype mice (PB0.01). As for the brain mercury levels, no significant difference was found between MT-null mice and wild-type mice. The concentration of mercury in the placenta in MT-null mice was also not significantly different from that in wild-type mice (Table 1). In contrast, fetal mercury concentrations, expressed as per gram or per whole body, were markedly higher in MT-null mice than in wild-type mice (P B0.01) after exposure to mercury vapor (Table 1). When placental transfer of mercury was investigated by determining the mercury concentration ratio of placenta to fetus, the ratio was about 20 in wild-type mice and 15 in MT-null mice. Table 2 shows MT-I and -II concentrations in the organs of maternal and non-pregnant wildtype mice before exposed and after exposure to Hg0 vapor. MT-I and -II was not detected in any tissues from MT-null mice even after exposure to Hg0 vapor (B0.2 mg/g tissue) (data not shown). In pregnant wild-type mice, 24 h after exposure to Hg0 vapor, MT-I and -II concentrations were 4-fold higher in the lung, 2-fold higher in the liver, and 10-fold higher in the kidney than the concentration were before exposure. The MT-I and -II concentrations in the brain and placenta of exposed mice did not differ significantly from those in the brain and placenta of non-exposed mice. MT showed the highest levels in the liver, placenta, and brain of non-exposed mice. Hepatic MT level in pregnant mice were approxi-
mately 25-fold higher than that in non-pregnant mice. Typical gel chromatograms of mercury distribution in the placental cytosol from MT-null and wild-type mice 24 h after exposure to mercury vapor are shown in Fig. 2. In wild-type mice, more than 60% of the eluted mercury was detected in the MT fraction (fraction numbers 13– Table 2 MT-I and -II concentrations in organs and placenta of wildtype mice at 24 h after exposure to mercury vapor Organ
Metallothionein (mg/g tissue) Non-exposed control mice
Brain Lung Liver Kidney Placenta
Non-pregnant
Pregnant
39.6 9 6.9 (n = 5) 1.9 90.6 (n =5)** 4.0 92.0 (n= 5)** 4.8 91.1 (n =5)** –
47.0 95.9 (n =3) 3.4 90.6 (n =3) 127 9 7.0 (n =3) 9.5 90.7 (n = 3) 94.7 9 14.7 (n =6)
Exposed pregnant mice
60.1 96.9 (n = 3) 14.9 90.6** (n =3) 240 954* (n = 3) 96.0 934** (n =3) 121 9 27 (n = 9)
Values are mean 9standard deviation. The number of samples are given in parentheses. * Significant difference from control pregnant mice at PB 0.05. ** Significant difference from control pregnant mice at PB 0.01.
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Fig. 2. Gel filtration profile of mercury in the placental cytosol from MT-null and wild-type mice at 24 h after exposure to Hg0 vapor. The mercury eluted in fractions 13 –16 is considered to be bound to MT.
15), and only residual mercury was eluted in the fractions of high-molecular-weight protein. In contrast, mercury was not found in MT fractions from MT-null mice, and most of the mercury was associated with the high-molecular-weight protein fraction. Thus, the elution profiles of the placenta cytosol fractions of MT-null mice differed from those of wild-type mice. Co-localization of mercury and MT-I in placenta of wild-type mice at 24 h after exposure to Hg0 vapor is shown in Fig. 3. Both Hg granules and MT-I immunoreactivity were detected in the yolk sac and decidual cells in the deep layer of the deciduas. MT immunoreactivity was observed in both the nucleolus and cytoplasm of decidual cells and yolk sac. Although MT immunoreactivity was not observed in placenta from MT-null animals, no appreciable difference was detected between MT-null and wild-type animals in either intensity or distribution of mercury positive granules in placenta.
4. Discussion MT is known not only to play an important role in the protection against mercury toxicity but also participate closely in the retention of mercury in various organs (Yoshida et al., 1999a; Satoh et al., 1997; Yoshida et al., 1999b). In the present study of MT-null mice and wild type mice, 24 h
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after exposure to Hg0 vapor the levels of mercury in the maternal lung, liver and kidney of MT-null mice were markedly lower than those in wild-type mice. Satoh et al. (1997) and Yoshida et al. (1999b) reported that although there are no differences in mercury uptake in both strains mice, the elimination rate of mercury from the kidney, liver, and lung was remarkably faster in MT-null mice than in wild-type mice. They also noted that mercury ions had a high affinity for MT and that the large amount of mercury present in organs was associated with MT after Hg exposure. The difference in accumulation of mercury in maternal organs between MT-null mice and wild type mice is apparently due to differences in elimination rate of mercury from organs, which suggests that MT plays a role in the retention of mercury in the kidney, liver, and lung. There is no difference in the placental mercury concentration between MTnull and wild-type mice although accumulation of Hg of MT-null fetuses was significantly higher than in wild-type fetuses. Chan and Cherian (1993) reported that hepatic Cd in pregnant rats after Cd exposure was transferred to the kidney and placenta through the plasma and consequently caused higher placental accumulation levels than those seen in normal rats. The accumulated mercury in the liver and kidney of MT-null mice could not be retained because of the relative lack MT in these organs compared with MT levels in the same organs of wild-type mice. Furthermore, maternal hepatic MT and mRNA levels are elevated from late-term pregnancy until birth in mice (Chan and Cherian, 1993; Quaife et al., 1986). Elevated maternal hepatic MT levels of wild-type mice during pregnancy may serve to retain mercury in this organ. The higher concentration of Hg in placenta and fetus of MT-null mice, therefore, may be due to redistribution of Hg from other organs to the placenta. Recently, Lau et al. (1998) reported that exposure of pregnant MT-null mice to cadmium resulted in a higher Cd accumulation in MT-null fetuses comparing to control fetuses. Our results also show that fetal mercury accumulation is greater in MT-null mice than in wild-type mice and suggest that MT influences maternal-to-fetal transfer of mercury.
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In our present study, induction of MT-I and -II in the placenta of wild-type mice did not occur following Hg0 exposure (Table 2), but endogenous placental MT level was nearly 10 times that in the kidney and 2 times that in the brain. In contrast, placental MT was undetectable in MT-null mice. The gel filtration elution pattern of placental cytosol in wild-type mice showed that over 60% of the mercury was retained in the MT fraction. In the cytosol of placenta from MT-null mice, mercury appeared only in the high-molecular-weight protein fractions. The results imply that the higher levels of endogenous MT in the placenta is associated with the accumulation of mercury.
Previous experimental studies on animals have shown that the placental membrane constitutes an important barrier against the penetration of mercury into the fetus (Clarkson et al., 1972; Khayat and Dencker, 1982; Yoshida et al., 1986). In our present study, the mercury concentration in the fetus was low in both mouse strains compared with concentrations in maternal brain, liver, kidney, and placenta, which indicates that only a small amount of mercury was transferred to the fetus. We did not detect, however, a significant difference in mercury uptake between the fetuses of MT-null mice and those of wild-type mice. Further, the placenta/fetus ratio of mercury con-
Fig. 3. Co-localization of mercury and MT-I in yolk sac (a) and decidua (b) in the placenta of wild-type mice at 24 h after exposure to Hg0 vapor. Determinations were made with an autometallographic technique and immunoperoxidase method. Note the co-localized mercury positive granules and MT immunoreactive findings in the cytoplasm (arrows) of yolk sac epithelial cells and decidual cells. ×450.
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centrations in wild-type mice was clearly higher than that in MT-null mice. These findings suggest that endogenous placental MT may play an important role in preventing maternal-to-fetal transfer of mercury. Hazelhoff Roelfzema et al. (1989) reported that MT in rat placenta is present mainly in trophoblastic labyrinth, spongiotrophoblast and yolk sac. In the present study, MT was intensely stained in the yolk sac and decidual cells in the deep layer of the decidua and only slightly stained in the labyrinth of the unexposed control and Hg0 vapor-exposed mice. MT immunoreactivity in the yolk sac and decidual cells did not differ between unexposed control and Hg0 vapor-exposed animals (data not shown), and no significant increase in MT was observed after Hg0 exposure. After exposure to Hg0 vapor, the mercury granules and MT were located mainly in the yolk sac and decidual cells in the placenta of wild type mice. In MT-null mice, the localization of mercury granules in placenta was similar to that of wild type mice. Co-localization of mercury granules and MT in the yolk sac and the deep layer of the decidua implies that the MT in placenta may be involved in the prevention of mercury transfer from the dam to the fetus. In conclusion, the exposure of pregnant mice to Hg showed that accumulation of mercury in the major organs of mother was significantly lower in MT-null mice than in wild-type but that fetal mercury levels were significantly higher in MTnull mice than in wild-type mice. Investigation of localization of Hg and MT in the placenta by gel chromatography and immunohistochemistry indicated that a large amount of mercury accumulated in wild-type placenta was associated with MT. In previous reports, we noted that a higher hepatic MT level in the fetus plays a defensive role against mercury crossing the placenta and is involved in regulating mercury distribution in the fetus (Yoshida et al., 1987, 1997). The findings of the present study suggest that MT in the placenta and maternal organs protects against maternal-to-fetal mercury transfer and may play a significant defensive role against mercury toxicity in fetuses.
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Acknowledgements We thank Mr H. Takimoto and Mr K. Hayashi at the Animal Care Company (Tokyo, Japan) for their excellent assistance in the maintenance of transgenic mice at National Institute for Environmental Studies.
References Boadi, W.Y., Yannai, S., Urbach, J., Brandes, J.M., Summer, K.H., 1991. Transfer and accumulation of cadmium, and the level of metallothionein in perfused human placentae. Arch. Toxicol. 65, 318 – 323. Chan, H.M., Cherian, M.G., 1993. Mobilization of hepatic cadmium in pregnant rats. Toxicol. App. Pharmacol. 120, 308 – 314. Cherian, M.G., Goyer, R.A., 1978. Metallothionein and their role in the metabolism and toxicity. Life Sci. 23, 1 – 10. Clarkson, T.W., Magos, L., Greenwood, M.R., 1972. The transport of elemental mercury into fetal tissue. Biol. Neonate. 21, 239 – 244. Danscher, G., M8ller-Madsen, B., 1985. Silver amplification of mercury sulfide and selenide: a histochemical method for light and electronmicroscopic localization of mercury in tissue. J. Histochem. Cytochem. 33, 219 – 228. Goyer, R.A., Cherian, M.G., 1992. Role of metallothionein in human placenta and rats exposed to cadmium. In: Nordberg, G.F., Herber, R.F.M., Alessio, L. (Eds.), Cadmium in the Human Environment: Toxicity and Carcinogenicity. IARC, New York, pp. 239– 247. Grennwood, M.R., Clarkson, T.W., Magos, L., 1972. Transfer of metallic into fetus. Experientia 28, 1455 – 1456. Khayat, A., Dencker, L., 1982. Fetal uptake and distribution of metallic mercury vapor in the mouse: influence of ethanol and aminotriazole. Int. J. Biol. Res. Pregnancy 3, 38 – 46. Lau, J.C., Joseph, M.G., Cherian, M.G., 1998. Role of placental metallothionein in maternal to fetal transfer of cadmium in genetically altered mice. Toxicology 127, 167 – 178. Lindstedt, G., Skerfving, S., 1972. Method of analysis. In: Friberg, L., Vostal, J. (Eds.), Mercury in the Environment. CRC Press, Cleveland, OH, pp. 3 – 13. Lucis, O.J., Lucis, R., Shikh, Z.A., 1972. Cadmium and zinc in pregnancy and lactation. Arch. Environ. Health 14, 419 – 432. Michalska, A.E., Choo, K.H., 1993. Targeting and germ line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc. Natl. Acad. Sci. USA 90, 8088 – 8092. Nishimura, H., Nishimura, N., Tohyama, C., 1990. Localization of metallothionein in the genital organs of male rat. J. Histochem. Cytochem. 38, 927 – 933.
222
M. Yoshida et al. / Toxicology 175 (2002) 215–222
Palmiter, R.D., Sandgren, E.R., Koeller, D.M., Brinster, R.L., 1993. Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mouse. Mol. Cell Biol. 13, 5266 –5275. Pamphlett, R., Waley, P., 1996. Uptake of inorganic mercury by the human brain. Acta Neuropathol. 92, 525 –527. Quaife, C.J., Hammer, R.E., Mottet, N.K., Palmiter, R.D., 1986. Glucocorticoid regulation of metallothionein during murine development. Dev. Biol. 118, 549 –555. Hazelhoff Roelfzema, W., Tohyama, C., Nishimura, H., Nishimura, N., Morselt, A.F.W., 1989. Quantitative immunohistochemistry of metallothonein in rat placenta. Histochemistry 90, 36 –369. Satoh, H., Suzuki, T., Nobunaga, T., Naganuma, A., Imura, N., 1981. Effects of sodium selenite on distribution and placental transfer of mercuric mercury in mice of late gestational period. J. Pharmacobiodyn. 4, 191 –196. Satoh, M., Nishimura, N., Kanauama, Y., Naganuma, A., Suzuki, T., Tohyama, C., 1997. Enhanced renal toxicity by inorganic mercury in metallothionein-null mice. J. Pharmacol. Exp. Ther. 283, 1529 –1533. Suzuki, T., Miyama, T., Katsunuma, H., 1968. Placenta transfer of mercury chloride, phenyl mercury acetate and methyl mercury acetate in mice. Ind. Health 5, 149 –155. Tohyama, C., Shikh, Z.A., 1981. Metallothionein in plasma and urine of cadmium-exposed rats determined by a signal antibody radioimmunoassay. Fund. App. Toxicol. 1, 1 –7.
Yasutake, A., Nakano, A., Hirayama, K., 1998. Induction by mercury compounds of brain metallothionein in rats: Hg exposure induces long-lived brain metallothionein. Arch. Toxicol. 72, 187 – 191. Yoshida, M., Yamamura, Y., Satoh, H., 1986. Distribution of mercury in guinea pig offspring after in utero exposure to mercury vapor during late gestation. Arch. Toxicol. 58, 225 – 228. Yoshida, M., Aoyama, H., Satoh, H., Yamamura, Y., 1987. Binding of mercury to metallothionein-like protein in fetal liver of the guinea pig following in utero exposure to mercury vapor. Toxicol. Lett. 34, 1 – 6. Yoshida, M., Satoh, H., Sumi, Y., 1997. Effect of ethanol pretreatment on mercury distribution in organs of guinea pigs following in utero exposure to mercury vapor. Toxicology 119, 193 – 201. Yoshida, M., Satoh, M., Shimada, A., Yasutake, A., Sumi, Y., Tohyama, C., 1999a. Pulmonary toxicity caused by acute exposure to mercury vapor is enhanced in metallothioneinnull mice. Life Sci. 64, 1861 – 1867. Yoshida, M., Satoh, M., Yasutake, A., Shimada, A., Sumi, Y., Tohyama, C., 1999b. Distribution and retention of mercury in metallothionein-null mice after exposure to mercury vapor. Toxicology 139, 129 – 136. Webb, M., Cain, K., 1982. Function of metallothionein. Biochem. Pharmacol. 31, 137 – 142.
Maternal-to-fetus transfer of mercury in metallothionein-null pregnant mice after exposure to mercury vapor Minoru Yoshida a,*, Masahiko Satoh b, Akinori Shimada c, Emi Yamamoto c, Akira Yasutake d, Chiharu Tohyama b a b
Department of Chemistry, St. Marianna Uni6ersity School of Medicine, 2 -16 -1 Sugao, Miyamae-ku, Kawasaki 261 -8511, Japan En6ironmental Health Sciences Di6ision, National Institute for En6ironmental Studies, 16 -2 Onogawa, Tsukuba 305 -8506, Japan c Department of Veterinary Pathology, Tottori Uni6ersity, Koyamachyo Minami 4 -101, Tottori-shi, Tottori 680 -0945, Japan d Biochemistry Section, National Institute for Minamata Disease, Minamata, Kumamoto 867 -0008, Japan Received 17 December 2001; received in revised form 1 March 2002; accepted 1 March 2002
Abstract This study examined the role of placenta metallothionein (MT) in maternal-to-fetal mercury transfer in MT-null and wild-type mice after exposure to elemental mercury (Hg0) vapor. Both strains were exposed to Hg0 vapor at 5.5–6.7 mg/m3 for 3 h during late gestation. Twenty-four hours after exposure to Hg0 vapor, accumulation of mercury in the major organs, except the brain, of MT-null maternal mice was significantly lower than that in organs of wild-type mice. In contrast to mercury levels in maternal organs, fetal mercury levels were significantly higher in MT-null mice than in wild-type mice. In placenta, mercury concentrations were not significantly different between the two strains. Although MT levels in major organs, except the brain, of wild type mice were markedly elevated after the exposure to Hg0 vapor, the placental MT levels were not elevated. However, endogenous MT level in the placenta is significantly higher than that in other organs, except the liver. Gel filtration profile of the placental cytosol in the wild-type mice revealed that a large amount of placental mercury was associated with MT. In MT-null mice, mercury in placental cytosol appeared mainly in the high-molecular-weight protein fractions. Mercury in the placenta was localized mainly in the yolk sac and decidual cells in the deep layer of the decidua in both mouse strains. The similar localization of MT was found in the placenta of wild type mice. These results suggest that MT in the placenta has a defensive role in preventing maternal-to-fetal mercury transfer. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Mercury vapor; Metallothionein; Fetus; Null mice; Placenta
1. Introduction Exposure to toxic metals, such as mercury (Hg), lead (Pb) and cadmium (Cd), during gestation * Corresponding author. Fax: + 81-44-977-8133. E-mail address: [email protected] (M. Yoshida).
potentially causes adverse effects on fetal development. Since the outbreak of fetal Minamata disease in Japan was found to be caused by in utero exposure to methyl mercury, numerous investigations on the placental transfer of various mercury compounds and the uptake of mercury by fetal tissues have been carried out (Suzuki et al., 1968;
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M. Yoshida et al. / Toxicology 175 (2002) 215–222
Grennwood et al., 1972; Satoh et al., 1981). Experimental exposure of pregnant animals to Hg0 vapor exposure has shown that although Hg0 is rapidly oxidized to ionic mercury (Hg++), some Hg0 penetrates the placental barrier and enters the fetus (Clarkson et al., 1972; Khayat and Dencker, 1982; Yoshida et al., 1986). Furthermore, our previous study of late-term pregnant guinea pigs showed that Hg0 transferred through the placental barrier is oxidized in fetal liver and is bound to fetal hepatic metallothionein (MT), which indicates that protein has a role in protecting the fetus from mercury toxicity (Yoshida et al., 1987). MT is a low-molecular-weight metal-binding protein with high cysteine content. In mammals, this protein is believed to play an important role in the metabolism, transport, and storage of essential metals, such as zinc and copper, and in the detoxification of heavy metals, especially Cd and Hg (Cherian and Goyer, 1978; Webb and Cain, 1982). MT synthesis is induced in various tissues by these metals, food restriction, physical and chemical stress, and glucocorticoid hormones. MT is also reported as present in human and rodent placenta during pregnancy and is induced by exposure to essential and non-essential metals (Lau et al., 1998; Goyer and Cherian, 1992). Goyer and Cherian (1992) described placental MT as playing a role in modulating maternal-to-fetal transfer of essential and non-essential metals. Lucis et al. (1972) and Boadi et al. (1991) reported that after exposure to Cd, most Cd in placenta is associated with MT, which may provide the fetus some protection against the toxic effects of heavy metals. Although some Hg0 persisting in blood penetrates the placenta barrier, this barrier prevents Hg++ from entering into the fetus. However, the role of placental MT in maternal-to-fetal mercury transfer after exposure to Hg0 vapor remains unclear. Recently, MT gene knockout mice (MT-null mice) that do not express MT-I and MT-II genes have been established (Palmiter et al., 1993; Michalska and Choo, 1993). The purpose of the present study was to clarify the role of placental MT in maternal-to-fetal mercury transfer in MTnull and wild-type mice after exposure to Hg0 vapor.
2. Materials and methods
2.1. Animals and exposure to mercury (Hg 0) 6apor MT-null mice and wild-type OLA129/C57BL6 control mice were provided by Dr A. Choo. Mating was achieved by placing a male and two females in the same cage overnight. The animal facility was maintained under a light/dark cycle of 12-h, temperature of 249 1 °C, relative humidity of 55 9 10%, and negative atmospheric pressure. The mice received mouse chow and filtered tap water ad libitum. When they reached day 16 of pregnancy, the animals were used in the experiment. Throughout the experiment, animals received humane care according to the National Institute for Environmental Studies’ guidelines for animal welfare. Four MT-null and 3 wild-type mice were exposed to mercury vapor as a group for 3 h as described previously (Yoshida et al., 1999b). Mercury concentration in the exposure chamber (20 l) was measured hourly by the air sampling method (Lindstedt and Skerfving, 1972) and was controlled at 5.5–6.7 mg/m3. Twenty-four hours after exposure to mercury vapor, the dams were killed with diethyl ether overdose, and placenta and fetuses were removed immediately from the uterus. Brain, lung, liver, and kidney of pregnant mice and non-pregnant controls were removed and stored at − 80 °C until analysis. The tissues for histological analysis were fixed in 10% neutral buffered formalin.
2.2. Gel filtration of tissue supernatant MT-null and wild type mouse tissues were homogenized in ice-cold 1.15% KCl under N2 atmosphere to yield a 20% (w/v) homogenate. The homogenate was centrifuged at 105 000× g for 60 min, and the supernatant was filtered through a 0.22-mm pore diameter membrane (Ultrafree C3, Millipore) at 5000× g for 1 min. A portion of the supernatant was applied to a Superdex 75 HR 10/30 column (Pharmacia Biotech, Tokyo, Japan) equilibrated with phosphate-buffered saline (PBS). The sample was eluted with the same
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buffer at 4 °C, and 1 ml fractions were collected at a flow rate of 0.5 ml/min (Yasutake et al., 1998).
2.3. Analysis of MT and mercury concentrations in tissue Tissue MT concentrations were measured with a radioimmunoassay (Tohyama and Shikh, 1981) as modified by Nishimura et al. (1990). The detection limit of this method was 0.2 mgMT/g of tissue. Mercury concentrations were measured with a cold atomic absorption spectrophotometer (RA-2A Mercury Analyzer; Nippon Instruments, Tokyo, Japan) after digestion with a concentrated acid mixture [HNO3/HClO4 3:1 (v/v)] (Satoh et al., 1997). After the fetuses were homogenized with 10 mM Tris– HCl buffer (pH 7.4), an aliquot was used for mercury analysis. The detection limit of this method was 0.5 ngHg with an intra-assay coefficient of variation of 4% (n =10).
2.4. Double staining: immunohistochemistry and autometallography The placental tissues fixed in 10% neutral buffered formalin were embedded in paraffin. Immunochemistry of MT was performed on 3 mm paraffin sections. The primary antibody used in this study was MT monoclonal antibody (E9, DAKO, Glostrup, Denmark) against horse MT-I and –II (1:200 in M.O.M.™). After treatment with 0.1% trypsin solution at 37 °C for 15 min and blockade of endogenous peroxidase activity with 3% H2O2 in PBS, sections were treated with M.O.M.™ Mouse IgG Blocking Reagent for 4 h, incubated with primary antibodies overnight at 4 °C, and then sequentially incubated with M.O.M.™ Biotinylated Anti-Mouse IgG Reagent for monoclonal antibody for 1 h at room temperature and with VECTASTAIN Elite ABC Reagent for 1 h. The sections were then washed 3 times for 10 min each in PBS and developed with 0.02% 3,3,-diaminobenzidine tetrahydrochloride and H2O2. After being immunostained, sections were then stained for mercury by autometallography (Danscher and M8ller-Madsen, 1985). The sections were pretreated with 1% potassium cya-
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nide for 2 h to eliminate non-specific staining from silver sulfides or selenides (Pamphlett and Waley, 1996), placed in a physical developer containing 50% gum arabic, citrate buffer, hydroquinone and silver nitrate at 26 °C for 40 min in the dark. Excess silver was dissolved in by 5% sodium thiosulfate. The sections were counterstained with haematoxylin. The reaction product was seen as small black grains of silver surrounding an invisible mercury core within the tissue.
2.5. Statistical analysis Data were analyzed statistically by Student’s t-test for comparison between MT-null and wildtype mice and one-way ANOVA for time-dependent changes with a preset probability level of PB 0.05 or PB 0.01.
3. Results When mercury concentrations in the maternal organs of MT-null and wild-type mice were checked 24 h after exposure to Hg0 vapor, both mouse strains showed the highest concentration in the kidney, followed by those in the lung, liver, and brain (Fig. 1). Mercury concentrations were significantly lower in the lung and liver of MTnull mice than of wild-type mice (PB 0.05). In the kidney, the mercury concentration in MT-null
Fig. 1. Mercury concentrations in major organs of pregnant MT-null and wild-type mice at 24 h after exposure to mercury vapor. Values are represented as mean 9standard deviation (n = 3 – 4). *Significant difference from wild-type mice at P B 0.05. **Significant difference from wild-type mice at PB 0.01.
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Table 1 Mercury concentrations in the placenta and fetus of pregnant MT-null and wild-type mice after exposure to mercury vapor Wild-type mice
MT-null mice
Control
Exposure
Control
Exposure
Fetus ngHg/g tissue ngHg/body weight
9 92 (n=9) 591 (n=9)
85 97 (n = 9) 579 9 (n = 9)
7 9 1 (n =9) 5 9 2 (n =9)
122 919* (n = 13) 90 9 10* (n =13)
Placenta ngHg/g tissue
19 9 3 (n= 9)
1702 9 327 (n = 9)
17 91 (n =9)
1866 9513 (n =13)
Ratio of placenta/fetus
–
209 4
–
15 9 3
Values are mean 9 standard deviation. The number of animals are given in parentheses. * Significant difference from wild-type mice at PB0.05.
mice was approximately one-third that in wildtype mice (PB0.01). As for the brain mercury levels, no significant difference was found between MT-null mice and wild-type mice. The concentration of mercury in the placenta in MT-null mice was also not significantly different from that in wild-type mice (Table 1). In contrast, fetal mercury concentrations, expressed as per gram or per whole body, were markedly higher in MT-null mice than in wild-type mice (P B0.01) after exposure to mercury vapor (Table 1). When placental transfer of mercury was investigated by determining the mercury concentration ratio of placenta to fetus, the ratio was about 20 in wild-type mice and 15 in MT-null mice. Table 2 shows MT-I and -II concentrations in the organs of maternal and non-pregnant wildtype mice before exposed and after exposure to Hg0 vapor. MT-I and -II was not detected in any tissues from MT-null mice even after exposure to Hg0 vapor (B0.2 mg/g tissue) (data not shown). In pregnant wild-type mice, 24 h after exposure to Hg0 vapor, MT-I and -II concentrations were 4-fold higher in the lung, 2-fold higher in the liver, and 10-fold higher in the kidney than the concentration were before exposure. The MT-I and -II concentrations in the brain and placenta of exposed mice did not differ significantly from those in the brain and placenta of non-exposed mice. MT showed the highest levels in the liver, placenta, and brain of non-exposed mice. Hepatic MT level in pregnant mice were approxi-
mately 25-fold higher than that in non-pregnant mice. Typical gel chromatograms of mercury distribution in the placental cytosol from MT-null and wild-type mice 24 h after exposure to mercury vapor are shown in Fig. 2. In wild-type mice, more than 60% of the eluted mercury was detected in the MT fraction (fraction numbers 13– Table 2 MT-I and -II concentrations in organs and placenta of wildtype mice at 24 h after exposure to mercury vapor Organ
Metallothionein (mg/g tissue) Non-exposed control mice
Brain Lung Liver Kidney Placenta
Non-pregnant
Pregnant
39.6 9 6.9 (n = 5) 1.9 90.6 (n =5)** 4.0 92.0 (n= 5)** 4.8 91.1 (n =5)** –
47.0 95.9 (n =3) 3.4 90.6 (n =3) 127 9 7.0 (n =3) 9.5 90.7 (n = 3) 94.7 9 14.7 (n =6)
Exposed pregnant mice
60.1 96.9 (n = 3) 14.9 90.6** (n =3) 240 954* (n = 3) 96.0 934** (n =3) 121 9 27 (n = 9)
Values are mean 9standard deviation. The number of samples are given in parentheses. * Significant difference from control pregnant mice at PB 0.05. ** Significant difference from control pregnant mice at PB 0.01.
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Fig. 2. Gel filtration profile of mercury in the placental cytosol from MT-null and wild-type mice at 24 h after exposure to Hg0 vapor. The mercury eluted in fractions 13 –16 is considered to be bound to MT.
15), and only residual mercury was eluted in the fractions of high-molecular-weight protein. In contrast, mercury was not found in MT fractions from MT-null mice, and most of the mercury was associated with the high-molecular-weight protein fraction. Thus, the elution profiles of the placenta cytosol fractions of MT-null mice differed from those of wild-type mice. Co-localization of mercury and MT-I in placenta of wild-type mice at 24 h after exposure to Hg0 vapor is shown in Fig. 3. Both Hg granules and MT-I immunoreactivity were detected in the yolk sac and decidual cells in the deep layer of the deciduas. MT immunoreactivity was observed in both the nucleolus and cytoplasm of decidual cells and yolk sac. Although MT immunoreactivity was not observed in placenta from MT-null animals, no appreciable difference was detected between MT-null and wild-type animals in either intensity or distribution of mercury positive granules in placenta.
4. Discussion MT is known not only to play an important role in the protection against mercury toxicity but also participate closely in the retention of mercury in various organs (Yoshida et al., 1999a; Satoh et al., 1997; Yoshida et al., 1999b). In the present study of MT-null mice and wild type mice, 24 h
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after exposure to Hg0 vapor the levels of mercury in the maternal lung, liver and kidney of MT-null mice were markedly lower than those in wild-type mice. Satoh et al. (1997) and Yoshida et al. (1999b) reported that although there are no differences in mercury uptake in both strains mice, the elimination rate of mercury from the kidney, liver, and lung was remarkably faster in MT-null mice than in wild-type mice. They also noted that mercury ions had a high affinity for MT and that the large amount of mercury present in organs was associated with MT after Hg exposure. The difference in accumulation of mercury in maternal organs between MT-null mice and wild type mice is apparently due to differences in elimination rate of mercury from organs, which suggests that MT plays a role in the retention of mercury in the kidney, liver, and lung. There is no difference in the placental mercury concentration between MTnull and wild-type mice although accumulation of Hg of MT-null fetuses was significantly higher than in wild-type fetuses. Chan and Cherian (1993) reported that hepatic Cd in pregnant rats after Cd exposure was transferred to the kidney and placenta through the plasma and consequently caused higher placental accumulation levels than those seen in normal rats. The accumulated mercury in the liver and kidney of MT-null mice could not be retained because of the relative lack MT in these organs compared with MT levels in the same organs of wild-type mice. Furthermore, maternal hepatic MT and mRNA levels are elevated from late-term pregnancy until birth in mice (Chan and Cherian, 1993; Quaife et al., 1986). Elevated maternal hepatic MT levels of wild-type mice during pregnancy may serve to retain mercury in this organ. The higher concentration of Hg in placenta and fetus of MT-null mice, therefore, may be due to redistribution of Hg from other organs to the placenta. Recently, Lau et al. (1998) reported that exposure of pregnant MT-null mice to cadmium resulted in a higher Cd accumulation in MT-null fetuses comparing to control fetuses. Our results also show that fetal mercury accumulation is greater in MT-null mice than in wild-type mice and suggest that MT influences maternal-to-fetal transfer of mercury.
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In our present study, induction of MT-I and -II in the placenta of wild-type mice did not occur following Hg0 exposure (Table 2), but endogenous placental MT level was nearly 10 times that in the kidney and 2 times that in the brain. In contrast, placental MT was undetectable in MT-null mice. The gel filtration elution pattern of placental cytosol in wild-type mice showed that over 60% of the mercury was retained in the MT fraction. In the cytosol of placenta from MT-null mice, mercury appeared only in the high-molecular-weight protein fractions. The results imply that the higher levels of endogenous MT in the placenta is associated with the accumulation of mercury.
Previous experimental studies on animals have shown that the placental membrane constitutes an important barrier against the penetration of mercury into the fetus (Clarkson et al., 1972; Khayat and Dencker, 1982; Yoshida et al., 1986). In our present study, the mercury concentration in the fetus was low in both mouse strains compared with concentrations in maternal brain, liver, kidney, and placenta, which indicates that only a small amount of mercury was transferred to the fetus. We did not detect, however, a significant difference in mercury uptake between the fetuses of MT-null mice and those of wild-type mice. Further, the placenta/fetus ratio of mercury con-
Fig. 3. Co-localization of mercury and MT-I in yolk sac (a) and decidua (b) in the placenta of wild-type mice at 24 h after exposure to Hg0 vapor. Determinations were made with an autometallographic technique and immunoperoxidase method. Note the co-localized mercury positive granules and MT immunoreactive findings in the cytoplasm (arrows) of yolk sac epithelial cells and decidual cells. ×450.
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centrations in wild-type mice was clearly higher than that in MT-null mice. These findings suggest that endogenous placental MT may play an important role in preventing maternal-to-fetal transfer of mercury. Hazelhoff Roelfzema et al. (1989) reported that MT in rat placenta is present mainly in trophoblastic labyrinth, spongiotrophoblast and yolk sac. In the present study, MT was intensely stained in the yolk sac and decidual cells in the deep layer of the decidua and only slightly stained in the labyrinth of the unexposed control and Hg0 vapor-exposed mice. MT immunoreactivity in the yolk sac and decidual cells did not differ between unexposed control and Hg0 vapor-exposed animals (data not shown), and no significant increase in MT was observed after Hg0 exposure. After exposure to Hg0 vapor, the mercury granules and MT were located mainly in the yolk sac and decidual cells in the placenta of wild type mice. In MT-null mice, the localization of mercury granules in placenta was similar to that of wild type mice. Co-localization of mercury granules and MT in the yolk sac and the deep layer of the decidua implies that the MT in placenta may be involved in the prevention of mercury transfer from the dam to the fetus. In conclusion, the exposure of pregnant mice to Hg showed that accumulation of mercury in the major organs of mother was significantly lower in MT-null mice than in wild-type but that fetal mercury levels were significantly higher in MTnull mice than in wild-type mice. Investigation of localization of Hg and MT in the placenta by gel chromatography and immunohistochemistry indicated that a large amount of mercury accumulated in wild-type placenta was associated with MT. In previous reports, we noted that a higher hepatic MT level in the fetus plays a defensive role against mercury crossing the placenta and is involved in regulating mercury distribution in the fetus (Yoshida et al., 1987, 1997). The findings of the present study suggest that MT in the placenta and maternal organs protects against maternal-to-fetal mercury transfer and may play a significant defensive role against mercury toxicity in fetuses.
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Acknowledgements We thank Mr H. Takimoto and Mr K. Hayashi at the Animal Care Company (Tokyo, Japan) for their excellent assistance in the maintenance of transgenic mice at National Institute for Environmental Studies.
References Boadi, W.Y., Yannai, S., Urbach, J., Brandes, J.M., Summer, K.H., 1991. Transfer and accumulation of cadmium, and the level of metallothionein in perfused human placentae. Arch. Toxicol. 65, 318 – 323. Chan, H.M., Cherian, M.G., 1993. Mobilization of hepatic cadmium in pregnant rats. Toxicol. App. Pharmacol. 120, 308 – 314. Cherian, M.G., Goyer, R.A., 1978. Metallothionein and their role in the metabolism and toxicity. Life Sci. 23, 1 – 10. Clarkson, T.W., Magos, L., Greenwood, M.R., 1972. The transport of elemental mercury into fetal tissue. Biol. Neonate. 21, 239 – 244. Danscher, G., M8ller-Madsen, B., 1985. Silver amplification of mercury sulfide and selenide: a histochemical method for light and electronmicroscopic localization of mercury in tissue. J. Histochem. Cytochem. 33, 219 – 228. Goyer, R.A., Cherian, M.G., 1992. Role of metallothionein in human placenta and rats exposed to cadmium. In: Nordberg, G.F., Herber, R.F.M., Alessio, L. (Eds.), Cadmium in the Human Environment: Toxicity and Carcinogenicity. IARC, New York, pp. 239– 247. Grennwood, M.R., Clarkson, T.W., Magos, L., 1972. Transfer of metallic into fetus. Experientia 28, 1455 – 1456. Khayat, A., Dencker, L., 1982. Fetal uptake and distribution of metallic mercury vapor in the mouse: influence of ethanol and aminotriazole. Int. J. Biol. Res. Pregnancy 3, 38 – 46. Lau, J.C., Joseph, M.G., Cherian, M.G., 1998. Role of placental metallothionein in maternal to fetal transfer of cadmium in genetically altered mice. Toxicology 127, 167 – 178. Lindstedt, G., Skerfving, S., 1972. Method of analysis. In: Friberg, L., Vostal, J. (Eds.), Mercury in the Environment. CRC Press, Cleveland, OH, pp. 3 – 13. Lucis, O.J., Lucis, R., Shikh, Z.A., 1972. Cadmium and zinc in pregnancy and lactation. Arch. Environ. Health 14, 419 – 432. Michalska, A.E., Choo, K.H., 1993. Targeting and germ line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc. Natl. Acad. Sci. USA 90, 8088 – 8092. Nishimura, H., Nishimura, N., Tohyama, C., 1990. Localization of metallothionein in the genital organs of male rat. J. Histochem. Cytochem. 38, 927 – 933.
222
M. Yoshida et al. / Toxicology 175 (2002) 215–222
Palmiter, R.D., Sandgren, E.R., Koeller, D.M., Brinster, R.L., 1993. Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mouse. Mol. Cell Biol. 13, 5266 –5275. Pamphlett, R., Waley, P., 1996. Uptake of inorganic mercury by the human brain. Acta Neuropathol. 92, 525 –527. Quaife, C.J., Hammer, R.E., Mottet, N.K., Palmiter, R.D., 1986. Glucocorticoid regulation of metallothionein during murine development. Dev. Biol. 118, 549 –555. Hazelhoff Roelfzema, W., Tohyama, C., Nishimura, H., Nishimura, N., Morselt, A.F.W., 1989. Quantitative immunohistochemistry of metallothonein in rat placenta. Histochemistry 90, 36 –369. Satoh, H., Suzuki, T., Nobunaga, T., Naganuma, A., Imura, N., 1981. Effects of sodium selenite on distribution and placental transfer of mercuric mercury in mice of late gestational period. J. Pharmacobiodyn. 4, 191 –196. Satoh, M., Nishimura, N., Kanauama, Y., Naganuma, A., Suzuki, T., Tohyama, C., 1997. Enhanced renal toxicity by inorganic mercury in metallothionein-null mice. J. Pharmacol. Exp. Ther. 283, 1529 –1533. Suzuki, T., Miyama, T., Katsunuma, H., 1968. Placenta transfer of mercury chloride, phenyl mercury acetate and methyl mercury acetate in mice. Ind. Health 5, 149 –155. Tohyama, C., Shikh, Z.A., 1981. Metallothionein in plasma and urine of cadmium-exposed rats determined by a signal antibody radioimmunoassay. Fund. App. Toxicol. 1, 1 –7.
Yasutake, A., Nakano, A., Hirayama, K., 1998. Induction by mercury compounds of brain metallothionein in rats: Hg exposure induces long-lived brain metallothionein. Arch. Toxicol. 72, 187 – 191. Yoshida, M., Yamamura, Y., Satoh, H., 1986. Distribution of mercury in guinea pig offspring after in utero exposure to mercury vapor during late gestation. Arch. Toxicol. 58, 225 – 228. Yoshida, M., Aoyama, H., Satoh, H., Yamamura, Y., 1987. Binding of mercury to metallothionein-like protein in fetal liver of the guinea pig following in utero exposure to mercury vapor. Toxicol. Lett. 34, 1 – 6. Yoshida, M., Satoh, H., Sumi, Y., 1997. Effect of ethanol pretreatment on mercury distribution in organs of guinea pigs following in utero exposure to mercury vapor. Toxicology 119, 193 – 201. Yoshida, M., Satoh, M., Shimada, A., Yasutake, A., Sumi, Y., Tohyama, C., 1999a. Pulmonary toxicity caused by acute exposure to mercury vapor is enhanced in metallothioneinnull mice. Life Sci. 64, 1861 – 1867. Yoshida, M., Satoh, M., Yasutake, A., Shimada, A., Sumi, Y., Tohyama, C., 1999b. Distribution and retention of mercury in metallothionein-null mice after exposure to mercury vapor. Toxicology 139, 129 – 136. Webb, M., Cain, K., 1982. Function of metallothionein. Biochem. Pharmacol. 31, 137 – 142.