Susceptibility of Metallothionein-Null Mice to the Behavioral Alterations Caused by Exposure to Mercury Vapor at Human-Relevant Concentration

Minoru Yoshida*, Chiho Watanabe{dagger},1, Masahiko Satoh{ddagger}, Akira Yasutake§, Masumi Sawada, Yuko Ohtsuka||, Yoshifumi Akama|| and Chiharu Tohyama|||

* Department of Biochemistry, Division of Chemistry, St. Marianna University School of Medicine, 2–16–1 Sugao, Miyamae-ku, Kawasaki 261-8511, Japan; {dagger} Department of Human Ecology, School of International Health, Graduate School of Medicine, The University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; {ddagger} Department of Hygienics, Gifu Pharmaceutical University, 5–6–1, 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 4–101, 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, 16–2 Onogawa, Tsukuba, Ibaraki 305-0053, Japan

Received January 18, 2004; accepted March 18, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
While recent human studies suggested adverse neurobehavioral outcomes of low-level exposure to mercury vapor (Hg0) as found among those having dental amalgam fillings and dental personnel, past animal experiments only dealt with exposure at much higher mercury concentrations. The present study aimed to examine neurobehavioral effects of prolonged, low-level Hg0 exposure in mice and to evaluate the protective role of metallothionein-I,II (MT-I,II) against Hg0-induced neurotoxicity, using a knock-out strain of mice. Adult female metallothionein-I,II-null (MT-null) and wild-type OLA129/C57BL6 mice were exposed to 0.06 mg/m3 of Hg0 for 8 h per day for 23 weeks. Neurobehavioral effects were evaluated at 12 and 23 weeks of exposure using open-field test and passive avoidance test. Subcellular distribution of mercury and the induction of MT were also assessed. The Hg0 exposure resulted in significantly enhanced locomotion in the open-field test and poorer performance in the passive avoidance test at a brain Hg concentration less than 1 ppm. These effects were slightly exaggerated in MT-null mice, which showed less induction of MT, lower brain Hg concentration, and lower calculated concentration of MT-unbound cytosolic Hg. The results showed, for the first time, that a concentration of Hg0 relevant to human exposure level could cause neurobehavioral effects in adult mice. The higher susceptibility of MT-null mice suggested that MT-I,II have protective roles in the metal-induced neurobehavioral toxicity, which cannot be entirely explained by kinetic mechanisms, thus suggesting an involvement of nonkinetic mechanisms.

Key Words: mercury (elemental); low-level exposure; metallothionein; knock-out mouse; behavior; learning.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two types of mercurials could potentially exert neurotoxic effects that are relevant to the general public: methylmercury and metallic mercury (Hg0). Of these, exposure to the latter occurs in occupational and nonoccupational settings, and the exposure at higher levels, as found among mercury miners, results in neurotoxicity such as tremor, ataxia, and psychic disturbances (WHO, 1991Go).

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., 1998Go; Echeverria et al., 1998Go), animal experiments only dealt with exposure at higher mercury concentrations, except for those evaluating perinatal exposure (Danielsson et al., 1993Go; Fredriksson et al., 1992Go). 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, 2001Go) 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, 1991Go). 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, 1991Go).

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., 1997Go) or pulmonary (Yoshida et al., 1999Go) toxicity of inorganic mercury or cytotoxicity of methylmercury (Yao et al., 2000Go). Since brain is a unique organ, in that it expresses a brain-specific MT, MT-III (Hidalgo et al., 2001Go; Satoa and Kondoh, 2002Go), such a protective role of MT-I,II might be obscured in case of neurotoxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and exposure to mercury (Hg0) vapor (Yoshida et al., 1986Go). Eight-week-old female MT-null mice and wild-type OLA129/C57BL6 control mice were kindly provided by Dr. A. Choo. The animal facility was maintained under a light/dark cycle of 12-h, temperature of 24 ± 1°C, relative humidity of 55 ± 10%. The mice received laboratory chow (CE-2, Japan Crea, Tokyo, Japan) and filtered tap water ad libitum with humane care according to the National Institute for Environmental Studies' guidelines for animal welfare. The total mercury content of the laboratory chow was routinely analyzed by the manufacturer and reported to be less than their detection limit (i.e., <10 ppb). Sixteen MT-null and fourteen wild-type female mice were exposed to mercury vapor for 8 h/day for 23 weeks starting at 8-weeks old. Briefly, mercury vapor was generated by passing air through a tandem flask containing Hg0, mixed with fresh air, and introduced into a plastic exposure chamber (50 x 50 x 70 cm). The median concentration of mercury vapor in the exposure chamber was 0.06 mg/m3 (range: 0.031–0.119 mg/m3), which was comparable to the LOAEL of 0.025 mg/m3 (USEPA; as described above). The control animals were treated identically but with no mercury in the flask. Animals were subject to two behavioral tests, at both 12 and 23 weeks of exposure period. After completion of all the behavioral evaluations, all animals were killed under diethyl ether anesthesia. Brain, lung, liver, and kidney were removed immediately and stored at –80°C until analysis.

Gel filtration of tissue supernatant. Details of the procedure are given elsewhere (Yoshida et al., 1999Go). 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., 1987Go). 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)Go 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
At 12 weeks of exposure, the mice were placed in the open-field apparatus for a 10-min observation to evaluate general activity levels in a novel environment (Fig. 1A). Regardless of the mice strain, the Hg-exposed mice showed enhanced locomotor activity when compared with the unexposed (control) mice. At the completion of the exposure (23 weeks), the enhanced activity was observed again, although the effect of the mercury was significant only for MT-null groups, presumably due to the small variation in the nonexposed MT-null group (Fig. 1B).




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FIG. 1. Locomotion activity of metallothionein-null (MT-null) and wild-type mice at 12 (A) or 23 (B) weeks of exposure to mercury vapor in the open-field task. MT(+/+) and MT(–/–) indicate the wild-type and MT-null mice, respectively. White bars and black bars stand for unexposed and mercury-exposed groups, respectively. Values are means ± standard deviations. Results of t-test are shown, where the difference is significant.

 
Performance in the passive avoidance test, a learning task motivated by strong aversive stimuli, was not affected at 12 weeks of exposure (Fig. 2A). When these mice were tested again at 23 weeks, the step-through latency at the "training" trial (i.e., the first session) was significantly shorter in exposed MT-null than in the unexposed MT-null mice. Such an effect of Hg exposure was not observed in the wild-type mice (Fig. 2B). In both the strains, these step-through latencies at 23 weeks were much longer than those in the training trial at 12 weeks, indicating the memory of learning experience at 12 weeks was retained at 23 weeks. In the retention trial (the second session), virtually all the mice stayed in the starting (light) chamber up to 300 s, the cut-off time, and no between-group difference was detected.




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FIG. 2. Avoidance latency of metallothionein-null (MT-null) and wild-type mice at the "trial" (1st day) and the "retention" (2nd day) session of 12 (A) or 23 (B) weeks of exposure to mercury vapor in the passive avoidance task. MT(+/+) and MT(–/–) indicate wild-type and MT-null mice, respectively. White bars and black bars stand for unexposed and mercury-exposed groups, respectively. Values are means ± standard deviations. Results of Wilcoxon test are shown, where the difference is significant.

 
Tissue Hg levels in the exposed groups evaluated at 23 weeks of exposure showed MT-null accumulated significantly less mercury in all the examined organs (lung, heart, and kidney) except for liver (data not shown) than the wild type, which were consistent with previous observations in lung (Yoshida et al., 1999Go) or in kidney (Satoh et al., 1997Go). In the brain, the mean Hg concentrations were 0.97 ± 0.07 and 0.66 ± 0.08 µg/g (wet weight) for the wild type and MT-null, respectively. Only the trace amounts (<0.01 µg/g) could be detected in the brains of unexposed groups.

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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TABLE 1 Mercury Concentration in the Brain of MT-Null and Wild-type Mice

 
Typical gel filtration elution patterns of mercury in the brain cytosol of the MT-null and wild-type mice are shown in Figure 3. A major portion of the mercury eluted was detected in the MT fraction (fraction numbers 13 to 15), both in MT-null and the wild-type mice. The retention time for the MT-null peak was slightly shorter than that for the wild-type, presumably reflecting the binding to different MT isoforms (MT-I,II vs. MT-III). A small amount was also eluted in the high-molecular-weight (HMW) protein fractions (fraction numbers 9–10).



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FIG. 3. Gel filtration profile of mercury in brain cytosol from MT-null and wild-type mice at 23 weeks of exposure to mercury vapor. The mercury eluted in fractions 13 to 16 is considered to be bound to MT.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To our knowledge, this is the first study to show that prolonged, low-level exposure to mercury vapor, which resulted in brain Hg level less than 1 µg/g (ppm), caused behavioral alterations in adult rodents. It is also the first in providing in vivo evidence suggesting that MT-I,II null mice might be more susceptible than the wild-type mice to metal-induced neurotoxicity, although this differential susceptibility was not so distinct as we have reported before for renal or pulmonary toxicity of mercury (Satoh et al., 1997Go; Yoshida et al., 1999Go).

In the earlier study on rats (Kishi et al., 1978Go), 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, 1971Go). The brain mercury level in humans could reach as high as 33 µg/g in retired mercury miners (Takahata et al., 1970Go), 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, 1991Go), 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, 1991Go). 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., 1992Go), whereas prenatal exposure (gestation day 11–14 and 17–20) at 1.8 mg/ m3 for 1 or 3 h decreased locomotion activity and depressed spatial learning (Danielsson et al., 1993Go). 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, 1990Go). 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., 2002Go; Waalkes, 2002Go). 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., 2001Go; Palmiter et al., 1993Go). 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., 1993Go; Yasutake et al., 1998Go). 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., 1997Go) or in lung (after Hg0 exposure) (Yoshida et al., 1999Go) 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., 2001Go; Satoh, 2002Go).

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, 2002Go; Yao, 2000Go; Yoshida, 1999Go), 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., 1998Go). 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.


    ACKNOWLEDGMENTS
 
The work was supported by Health and Labor Sciences Research Grants.


    NOTES
 

1 To whom correspondence should be addressed at Department of Human Ecology, School of International Health, Graduate School of Medicine, The University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Fax: +81-3–5841–3395. E-mail: chiho{at}humeco.m.u-tokyo.ac.jp.


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