* Department of Biochemistry, Division of Chemistry, St. Marianna University School of Medicine, 2161 Sugao, Miyamae-ku, Kawasaki 261-8511, Japan; Department of Human Ecology, School of International Health, Graduate School of Medicine, The University of Tokyo, 731 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan;
Department of Hygienics, Gifu Pharmaceutical University, 561, Mitahora-higashi,Gifu 502-8585, Japan;
Biochemistry Section, National Institute for Minamata Disease, Minamata, Kumamoto 867-0008, Japan; ¶ Department of Veterinary Pathology, Tottori University, Minami 4101, Tottori-shi, Tottori 680-0945, Japan; || Department of Chemistry, Meisei University, Hino, Tokyo 191-0041, Japan; and ||| Environmental Health Sciences Division, National Institute for Environmental Studies, 162 Onogawa, Tsukuba, Ibaraki 305-0053, Japan
Received January 18, 2004; accepted March 18, 2004
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ABSTRACT |
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Key Words: mercury (elemental); low-level exposure; metallothionein; knock-out mouse; behavior; learning.
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INTRODUCTION |
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Prolonged, lower-level exposures occur among dental personnel handling Hg-containing amalgams or among those who have dental amalgam fillings, for which health consequences have not been well documented. While recent human studies suggested that such a low-level exposure might be related with adverse neurobehavioral outcomes (Bittner et al., 1998; Echeverria et al., 1998
), animal experiments only dealt with exposure at higher mercury concentrations, except for those evaluating perinatal exposure (Danielsson et al., 1993
; Fredriksson et al., 1992
). Thus, experiments using adult rats or rabbits employed Hg0 concentrations far exceeding 1 mg/m3, resulting in brain mercury levels more than 1 µg/g (wet weight), while the LOAEL (Lowest-Observed-Adverse-Effect Level) and RfC (Reference Concentration) adopted by EPA (U.S. EPA, 2001
) were 0.025 and 0.0003 mg/m3, respectively, and the brain mercury concentrations found among the dental personnel or among those with dental fillings only reach up to 0.3 µg/g (Nylander and Weiner, 1991
). Therefore, experimental data corresponding for the low-level exposures found among adult human population is apparently lacking.
The present study has two objectives. The first was to examine the effects of prolonged low-level Hg0 exposure on neurobehavioral functions of mice. Two behavioral paradigms, open-field test and passive avoidance test, were used for the evaluation. These two behavioral tests were chosen primarily because they are easy to conduct and often very sensitive to many environmental manipulations, and also because the functions examined by these tests (i.e., response to a novel environmental stimuli and retention of a learned response, respectively) have some resemblances to those affected by low-level exposure to mercury vapor in human. That is, long-term, low-level exposure to mercury vapor is associated with symptoms of erethism, including irritability, excitability, and loss of memory (WHO, 1991).
The second objective was to test the hypothesis that mice lacking metallothionein-I,II (MT-I,II), the metal-binding proteins (MT-null), show enhanced susceptibility to the neurobehavioral effects of the Hg0 exposure, if any. Alleviation of heavy-metal toxicity by MT-I,II is well established, and we and others have shown that MT-null mice exhibited enhanced renal (Satoh et al., 1997) or pulmonary (Yoshida et al., 1999
) toxicity of inorganic mercury or cytotoxicity of methylmercury (Yao et al., 2000
). Since brain is a unique organ, in that it expresses a brain-specific MT, MT-III (Hidalgo et al., 2001
; Satoa and Kondoh, 2002
), such a protective role of MT-I,II might be obscured in case of neurotoxicity.
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MATERIALS AND METHODS |
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Gel filtration of tissue supernatant. Details of the procedure are given elsewhere (Yoshida et al., 1999). Briefly, tissues were homogenized in ice-cold KCl under a N2 atmosphere and centrifuged (105,000 x g for 60 min). The obtained supernatant was filtered, and an aliquot 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 buffer at 4°C, and 1-ml fractions were collected at a flow rate of 0.5 ml/min.
Analysis of mercury concentrations in tissue. Mercury concentrations in the tissues 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 1:3 [v/v]). The detection limit was 0.5 ng Hg with an intra-assay coefficient of variation (n = 10) of 4%.
Analysis of MT in tissue. Metallothionein levels in the maternal and fetal liver were determined using the mercury-binding method (Naganuma et al., 1987). Briefly, tissue homogenate in Tris-HCl (pH 7.6) was mixed with diethylmaleate and incubated at room temperature for 20 min. After the addition of CdCl2, the samples were heated to 100°C for 3 min and centrifuged (3000 x g for 5 min). HgCl2 was added to the supernatant, and metallothionein-unbound mercury was removed by adding ovalbumin. Afterward the ovalbumin was removed with trichloroacetic acid and centrifugation. After filtration, the amount of metallothionein-bound mercury was measured as above.
Open-field test. Both of the behavioral experiments were conducted by a person who was blind to the treatment allocation. The locomotor activity was assessed using the open-field apparatus after Tanaka et al. (2004) with slight modifications. The apparatus consisted of the floor (50 x 50 cm) surrounded by a 50-cm-high opaque wall. A CCD camera fixed above the apparatus was connected to a Macintosh computer, and the movement of the [image of] mouse was analyzed using Image OF (O'Hara & Co., Ltd., Tokyo, Japan), a modified NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). In this software, the position of the animal was defined as the position of the gravity center of the image of the animal, which was calculated in every 0.5 s. The total distance moved by the gravity center was calculated and converted into centimeters. Also, the area of floor was divided into 25 squares (10 x 10 cm), and the location of the animal was classified either as central (nine areas that did not have direct contact with the wall) or peripheral (other sixteen areas). Behavior was monitored for 10 min after placing the mice on the center of the floor. Between each trial, the floor and wall were cleaned with 70% alcohol followed by wet cotton. The test was conducted in 2 days; the wild type was tested on the first day, and the MT-nulls on the second.
Passive avoidance. The apparatus (model PA-2010A, O'Hara & Co., Ltd, Tokyo, Japan) consisted of a dark and an illuminated compartment, which were separated by a sliding door. On the first day (training trial), the mouse was placed in the illuminated compartment for 30 s, and then the door was opened. When the mouse entered the dark compartment, it received an unavoidable scrambled electric shock to its foot (4 mA x 2 s). Latency was defined as the interval between the opening of the door and the entry of the mouse to the dark chamber. Immediately after the mouse escaped to the illuminated compartment, the door was closed and the mouse was removed from the apparatus. The animal was placed once again in the illuminated compartment, 24 h after the training trial (retention trial), and the latency (avoidance latency) was recorded. The retention session lasted a maximum ("cut-off") of 300 s; when the latency of an animal exceeded 300 s, the latency was recorded as 300 s for later analyses. The test was conducted in 2 days immediately following the open field test; the wild type was tested on the first day, and the MT-nulls on the second.
Statistical analysis. Student's t-test was used to compare the nonexposed control with the exposed group, except for the passive avoidance test, in which Wilcoxon's nonparametric test was used because the distribution was skewed due to the existence of cut-off time. In either of these tests, the significance level was set to p < 0.05.
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RESULTS |
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Possible induction of brain MTs by mercury exposure was evaluated. As expected, brain total MT level of unexposed mice was significantly higher in wild-type mice than in MT-null mice (119 ± 17 and 91 ± 6 nmol/g tissue, respectively). The Hg0 exposure significantly increased the total MT both in wild type and, to a lesser extent, in MT-null (167 ± 11, 106 ± 5 nmol/g tissue, respectively).
As shown in Table 1, a significantly larger portion of total Hg was found in supernatant of the wild type than that of MT-null mice. The proportion of MT-bound Hg against total supernatant Hg, however, was similar between the strains, leaving more "free" (MT-unbound) Hg in wild-type than in MT-null mice.
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DISCUSSION |
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In the earlier study on rats (Kishi et al., 1978), the behavioral abnormality induced by mercury vapor exposure (17 mg/m3 x 2 h/day for 30 days) was associated with the brain Hg exceeding 10 µg/g. When the behavioral effects disappeared after the cessation of the exposure, the brain still contained approximately 5 µg/g of Hg, which is almost ten times the brain Hg in the Hg-exposed MT-null mice exhibiting behavioral abnormality. In rabbits, two of six animals exposed to 4 mg/m3 of Hg0 (6 hr/day, 4 days/week) for 13 weeks developed tremor; unfortunately, this study lacked the control group, and no correlation was found between the effects and the brain Hg levels (Fukuda, 1971
). The brain mercury level in humans could reach as high as 33 µg/g in retired mercury miners (Takahata et al., 1970
), 0.3 µg/g in a group of dentists exposed to low levels of Hg0 (while median is only 0.03 µg/g) (Nylander and Weiner, 1991
), or more than 0.1 µg/g resulting from exposure through dental amalgam fillings, but less than 0.01 µg/g is expected in the absence of any known exposure source (WHO, 1991
). Therefore, the present study denotes the fact that behavioral effects in mice were associated with the brain Hg levels found in humans occupationally exposed to low-levels of Hg0. Since it is unlikely that brain Hg level at 12 weeks of exposure was higher than the level at 23 weeks, the enhanced open-field locomotion observed at 12 weeks must be associated with further low level, especially for MT-null mice.
Perinatal exposure of rats to mercury vapor leads to behavioral alterations with brain Hg concentrations much lower than the present study. Thus, neonatal exposure of newborn rats to Hg0 at 0.05 mg/m3, 4 h/day between postnatal day 11 to 17 increased locomotion activity and depressed spatial learning (Fredriksson et al., 1992), whereas prenatal exposure (gestation day 1114 and 1720) at 1.8 mg/ m3 for 1 or 3 h decreased locomotion activity and depressed spatial learning (Danielsson et al., 1993
). In these studies, brain mercury concentrations well below 0.1 µg/g were reported, suggesting that fetus and neonates are more susceptible to Hg0 than adults are, as they are in case of methylmercury (WHO, 1990
). These concentrations, however, were measured several days after the end of exposure, and the peak concentrations were not determined.
The higher susceptibility of the MT-null mice compared to the wild-type mice indicated that MT-I,II had a protective role in mercury-induced neurobehavioral toxicity, which apparently could not be explained by brain Hg level. Alleviation of metal-induced toxicity by MTs is widely recognized and often ascribed to their binding to (sequestering of) toxic metals; thereby the metals could not interact with other important biological molecules (Morgan et al., 2002; Waalkes, 2002
). In the present study, since the MT determination method did not distinguish different MT species, the brain MT levels in MT-null mice presumably reflected the presence of MT-III, the brain-specific MT (Hidalgo et al., 2001
; Palmiter et al., 1993
). Therefore, the increase of total MT in wild type and the less pronounced increase in the MT-null are consistent with previous studies showing induction of MT-I and II and less pronounced induction of MT-III in the brain of Hg-exposed rats (Palmiter et al., 1993
; Yasutake et al., 1998
). The induction of MT-I,II, the cytosol-rich proteins, influenced the subcellular distribution of Hg by providing its binding site, resulting in similar Hg levels in the pellet fraction (despite the different brain Hg levels) of both the strains. The concentrations of "free" Hg in the cytosol, which is unbound to MT and thus can interact with HMW proteins, however, was calculated to be higher in the wild-type than MT-null mice. Similar observations were obtained in our previous studies in kidney (after HgCl2 injection) (Satoh et al., 1997
) or in lung (after Hg0 exposure) (Yoshida et al., 1999
) of MT-null mice. Therefore, the observed protective effect of MT-I,II are not entirely ascribed to kinetic mechanism (sequestering), suggesting an involvement of nonkinetic mechanisms, such as elimination of radicals (Hidalgo et al., 2001
; Satoh, 2002
).
The enhanced susceptibility of MT-null mice regarding the behavioral effects of Hg0 has dual implications for mercury neurotoxicology. First, as mentioned above, although there have been several reports showing the higher susceptibility of MT-null to lethal, renal, pulmonary, or cytotoxicity of metals including cadmium, mercury, or arsenic (Satoh and Kondoh, 2002; Yao, 2000
; Yoshida, 1999
), the present results appear to be the first demonstration of enhanced sensitivity of MT-null animal to metal-induced neurotoxicity. The findings indicated that the MT-I,II also play some roles in alleviating the brain mercury toxicity, where MT-III exists and potentially plays a similar role.
Second, it has a practical implication in human risk assessment. In the autopsy kidney samples of Japanese adults, MT concentrations increased with age, which is associated with accumulation of Cd with age; however, a subgroup did not show such induction of MT, although their Cd levels were high enough to trigger the induction (Yoshida et al., 1998). The MT-null mice in the present study, showing genetically determined susceptibility, may be regarded as a model of these "slow-responder" to low-level (nonoccupational) metal exposure, although no genetic analyses have been done for these autopsy samples.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Department of Human Ecology, School of International Health, Graduate School of Medicine, The University of Tokyo, 731 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Fax: +81-358413395. E-mail: chiho{at}humeco.m.u-tokyo.ac.jp.
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