Cadmium-Metallothionein Interactions in the Olfactory Pathways of Rats and Pikes

Jonas Tallkvist,1, Eva Persson, Jörgen Henriksson and Hans Tjälve

Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 573, SE-751 23 Uppsala, Sweden

Received October 22, 2001; accepted January 14, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Deposition of cadmium onto the olfactory epithelium results in transport of the metal along the primary olfactory neurons to the olfactory bulbs of the brain. The present investigation was undertaken to determine the intracellular ligand binding of cadmium during this process. 109Cd2+ was applied on the olfactory epithelium of rats and pikes, and the subcellular distribution of the metal in the olfactory pathways was then examined. Two groups of rats were used: one pretreated with intranasal instillations of nonlabeled cadmium and the other given physiological saline (controls). Cellular fractionations showed that the 109Cd2+ was predominantly present in the cytosol of all samples, both in the rats and the pikes. Gel filtrations of the olfactory epithelium of control rats killed 2 h after the 109Cd2+ instillation showed that the metal was recovered in two peaks with elution volumes corresponding to metallothionein (MT) and glutathione (GSH)—the latter peak being the predominant one. However, in the epithelium of the cadmium-pretreated rats killed at 2 h, 109Cd2+ was recovered in one peak corresponding to MT. In the olfactory epithelium and bulbs of both groups of rats killed at 48 h, as well as in the olfactory epithelium, nerves, and bulbs of pikes killed at this interval, 109Cd2+ was recovered in one peak corresponding to MT. Immunohistochemistry of the olfactory system of rats given cadmium in the right nasal cavity showed induction of MT in the neuronal, sustentacular, and basal cells of the right olfactory epithelium, in the nerve fascicles in the lamina propria of the right olfactory mucosa, and in the olfactory nerve layer of the right olfactory bulb. On the left side, the immunoreactivity was low in these structures. MT immunoreactivity was observed in the glomeruli of both the right and the left olfactory bulbs. However, the staining was homogeneously distributed within the entire glomeruli of the right bulb, whereas it showed a mesh-like pattern corresponding to the localization of astrocytes in the glomeruli of the left bulb. We conclude that exposure of the olfactory epithelium to cadmium results in induction of MT in the primary olfactory neurons and a transport of the metal in these neurons as a cadmium-metallothionein (CdMT) complex. Our results further indicate that GSH is a ligand that can interact with cadmium before the metal binds to MT.

Key Words: cadmium; olfactory; nerve; metallothionein; axonal transport; rat; pike.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous investigations in rats and fish have shown that exposure of the olfactory epithelium to cadmium results in an uptake of the metal in the primary olfactory neurons and transport along the axons of these neurons to the glomeruli of the olfactory bulbs (Evans and Hastings, 1992Go; Gottofrey and Tjälve, 1991Go; Hastings and Evans, 1991Go; Tjälve et al., 1986Go, 1996Go).

In the olfactory epithelium, the dendrites of the primary olfactory neurons are in contact with the environment, and via the axons, these neurons are also connected with the olfactory bulbs of the brain. Thus, the olfactory route provides a pathway by which metals and other toxicants that come into contact with the olfactory epithelium can enter the central nervous system (CNS) without interference from the blood-brain barrier.

It is not known to which subcellular constituent(s) cadmium is bound during its transport along the olfactory pathways. However, metallothionein (MT) constitutes a potential ligand for cadmium in these tissues. MT comprises a group of low molecular weight, cysteine-rich, metal-binding proteins that normally are expressed at relatively low levels in most cells but are highly inducible by cadmium (Aschner et al., 1997Go; Nordberg and Nordberg, 2000Go).

The present investigation was undertaken to examine whether MT plays a role in the transport of cadmium in the olfactory pathways. Cellular fractionations and gel filtrations were used to examine the subcellular distribution of the metal in the olfactory systems of rats and pikes. In addition, we performed immunohistochemistry on rat tissues to examine whether intranasal cadmium administration results in induction of MT in the olfactory pathways.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
109CdCl2 (specific activity 2.07 µCi/µg) was obtained from NEN Life Science Products (Vilvoorde, Belgium). For the in vivo experiments, the 109CdCl2 radioisotope was evaporated by N2-gas and physiological saline was added to obtain 500 µg 109Cd2+ (1 µCi)/µl. Superdex 30 prep-grade, dextran blue, aprotinin, neurotensin, cytidine, rabbit kidney MT, glutathione (GSH), L-cystein, phenyl-methyl-sulphonyl-fluoride (PMSF), and Na-azide were obtained from Sigma (St. Louis, MO).

Animals.
Thirty-four male Sprague-Dawley rats, weighing about 150 g and obtained from Bantin and Kingman (Sollentuna, Sweden), were housed in macrolon cages at 22°C with a 12-h light/dark cycle, free access to tap water, and a standard pellet diet (R36, Lactamin AB, Vadstena, Sweden). Two pikes (Esox lucius), weighing about 3 kg, were caught by net fishing in Lake Mälaren (Enköping, Sweden). They were kept at our laboratory in 200-l glass aquaria in aerated tap water at 10 ± 0.5°C. The Local Ethics Committee for Animal Research approved all experiments.

Experimental Procedures
Intranasal administration.
Twelve rats were pretreated with intranasal administration of 5 µg of nonlabeled Cd2+ in 10 µl saline and 12 control rats with 10 µl saline (controls). After 48 h, eight cadmium pretreated animals and eight controls were given intranasal administration of 5 µg 109Cd2+ in 10 µl saline as previously described (Tjälve et al., 1996Go). Four rats from each of these groups were killed by CO2-asphyxiation 2 h after the 109Cd2+ administrations, and four from each group were killed after 48 h. The olfactory epithelium and the olfactory bulbs were removed for {gamma}-spectrometry, cellular fractionation, and gel filtration. For MT immunohistochemistry, two groups of four rats each were treated in the same way as described above with the exception that these groups were dosed with nonlabeled Cd2+ the second time. All rats were killed 48 h after the second Cd2+ administration. For the purpose of immunohistochemistry, two additional rats were killed without any treatment.

For the experiments in pikes, 10 µl of the 109Cd2+-solution (10 µCi, 5 µg) was applied with a micropipette in the olfactory chambers as described (Gottofrey and Tjälve, 1991Go). The pikes were killed after 48 h and the olfactory epithelia, nerves, and bulbs were dissected as described (Gottofrey and Tjälve, 1991Go). The subcellular distribution of 109Cd2+ in these tissues was then examined.

Cell fractionation.
The tissues were homogenized on ice in 1.5-ml Eppendorf vials, using an Eppendorf micropistille, in 5–10 volumes of 50 mM Tris–HCl (pH 7.4) containing 0.4 mM PMSF; they were then centrifuged at 105,000 x g at 4°C for 60 min. Pellets and aliquots of the cytosols were taken for {gamma}-spectrometry. Cytosols not used for {gamma}-spectrometry were stored at –80°C until used for gel filtration.

Gel filtration.
Two-hundred microliter aliquots of the cytosols or protein standards were applied under the eluent to a Superdex 30 column (Column K9, Pharmacia Fine Chemicals AB, Uppsala, Sweden) equilibrated at 4°C with 50 mM Tris–HCl (pH 7.4) containing 0.02% Na-azide and 0.4 mM PMSF as described (Henriksson et al., 1999Go; Tallkvist et al., 1998Go; ). This gel filtration system has the capacity to separate molecules between 100 Da and 10 kDa. The protein standards were dissolved in the Tris buffer (5 mg/ml). The void volume of the column was determined with dextran blue (MW 2 MD) and the column calibrated with aprotinin (MW 6.5 kDa), neurotensin (MW 1.673 kDa), and cytidine (MW 243 Da). Fractions of 1 ml were collected at a flow rate of 0.8 ml/min. Absorbance at 280 nm was continuously recorded and the amount of 109Cd2+ in each fraction was determined by {gamma}-spectrometry.

Immunohistochemistry.
The expression of MT in the olfactory system was examined using a commercially available monoclonal antibody, Dako-MT (E9, Dako). All reactions were performed according to the avidin-biotin technique. Deparaffinized 5-mm horizontal sections were treated in a microwave oven 2 x 5 min in 0.01 M citrate buffer, pH 6.0. Endogenous peroxidase activity was blocked with 1% H2O2 in phosphate-buffered saline containing 0.3% Triton X-100 (PBS-T). After one rinse in PBS, nonspecific binding was blocked by incubating the sections for 30 min in 4% BSA (PBS). The Dako-MT antibody was diluted 1:50 in 4% BSA (PBS) and exposure of the sections was performed at 4°C overnight. Following three rinses in PBS-T and one rinse in PBS, the sections were incubated for 30 min at room temperature with a secondary antibody (rabbit antimouse, E464, Dako) diluted 1:500 as described above. After the incubation with the secondary antibody, the sections were rinsed as described above and then incubated with the avidin-biotin-peroxidase complex for 30 min at room temperature (according to the manufacturer's recommendations), rinsed as described above, and finally treated with 3,3`-diaminobenzidine (DAB)-H2O2 for color development. For histopathological evaluation of the tissues and also to identify MT-immunoreactive cells on the slides, parallel sections were taken and stained with hematoxylin/eosin (H&E). Periodic acid-Schiff (PAS) staining was performed to localize the mucopolysaccharide containing Bowman's glands.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular Fractionation
Cellular fractionations showed that in all examined tissues of rats and pikes, the major part of the 109Cd2+ (70–85%) was present in the cytosol at both 2 and 48 h.

Gel Filtration
Gel filtration of the olfactory epithelium of the control rats killed at 2 h showed that the 109Cd2+ was eluted in two peaks (Fig. 1Go). Thus, in these gel filtrations the 109Cd2+-peaks were eluted in fractions 15 and 22. It can be noted that the 109Cd2+ peak in fraction 22 was higher than the one in fraction 15 (Fig. 1Go). When 109Cd2+, mixed with either MT or GSH in vitro, was run on the column, the radioactivity and UV-peaks were eluted in fractions 15 and 22, respectively (Fig. 1Go). In the olfactory epithelium of the Cd2+-pretreated rats killed at 2 h, the 109Cd2+ was recovered in fraction 15 (Fig. 1Go). According to the equation of the standard curve obtained in the calibration of the Superdex 30 column, the molecular weight corresponding to fraction 15 is 6–7 kDa, whereas fraction 22 corresponds to about 1 kDa. There was not enough radioactivity to perform gel filtrations in the cytosol of the olfactory bulbs prepared from rats killed 2 h after the 109Cd2+ administrations.



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FIG. 1. Representative Superdex 30 filtration profiles of cytosol of control rat olfactory epithelium (top left), cadmium pretreated rat olfactory epithelium (top right), 109Cd2+ mixed with GSH in vitro (bottom left), and 109Cd2+ mixed with MT in vitro (bottom right). The cytosolic samples are from animals killed 2 h after intranasal administration of 109Cd2+ (5 µg). The 109Cd2+ in the cytosol of the control epithelium was mainly recovered in fractions 15 and 22, whereas the 109Cd2+ in the cytosol of the cadmium-pretreated epithelium was eluted in fraction 15. 109Cd2+ mixed with MT or GSH in vitro was eluted in fractions 15 and 22, respectively. Solid line, 109Cd2+; dotted line, UV absorbance (280 nm).

 
At 48 h, the 109Cd2+ in the olfactory epithelia and bulbs of both control and Cd2+-pretreated rats was eluted in fraction 15 (Fig. 2Go). The same pattern of cytosolic 109Cd2+ distribution was observed in the olfactory epithelium, olfactory nerve, and olfactory bulb of pikes examined 48 h after intranasal administration of the 109Cd2+ (Fig. 2Go).



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FIG. 2. Representative Superdex 30 filtration profiles of cytosol of rat olfactory epithelium, rat olfactory bulb, and pike olfactory nerve. The rat samples are from controls killed 48 h after intranasal administration of 109Cd2+ (5 µg). At this time interval the 109Cd2+ was recovered mainly in fraction 15 in all samples of both groups of rats (cadmium-pretreated and controls) as well as in all samples of pikes. As described in Figure 1Go, fraction 15 corresponds to the elution volume of CdMT. Solid line, 109Cd2+; dotted line, UV-absorbance (280 nm).

 
MT Immunohistochemistry
Right nasal cavity.
The results of the MT immunohistochemistry are shown in Figure 3Go. Similar staining was observed in all rats given Cd2+. Intranasal instillation of Cd2+ in the right olfactory cavity resulted in a strong induction of MT immunoreactivity in the cells of the right olfactory epithelium (A and C), in nerve fascicles in the lamina propria of the right olfactory mucosa (C), and in the olfactory nerve layer of the right olfactory bulb (A and B).



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FIG. 3. Immunohistochemical staining for MT in sections of the olfactory system of rats. Cadmium was instilled in the right olfactory mucosa and killed after 48 h as described in Materials and Methods. (A) Section showing the left and right olfactory mucosae, turbinates, and bulbs. The immunohistochemical staining is much higher on the right than on the left side. (B) Section showing the right and left olfactory bulb. Higher levels of MT are present in the olfactory nerve layer of the right bulb than of the left bulb. A homogeneously distributed staining is present in the glomeruli of the right olfactory bulb, whereas in the left bulb the glomerular staining shows a mesh-like pattern. (C) Detail of the right olfactory mucosa. MT staining is present in neuronal, sustentacular, and basal cells. Nerve fascicles in the lamina propria also show a marked immunoreactivity. The staining of Bowman's glands is low. Legends: b, basal cell; Bg, Bowman's glands; g, glomeruli; lb, left olfactory bulb; lm, left olfactory mucosa; n, olfactory nerve cell; nf, nerve fascicles; ns, nasal septum; onl, olfactory nerve layer; rb, right olfactory bulb; rm, right olfactory mucosa; s, sustentacular cell. Magnification: (A) x12, (B) x60, (C) x180.

 
Thus, in the olfactory surface epithelium, the neuronal, sustentacular, and basal cells showed a marked staining. Nerve fascicles in the lamina propria were distinctly stained. In most areas of the olfactory mucosa, Bowman's glands were weakly stained. However, strong staining in some Bowman's glands localized in the deep part of the lamina propria was observed in a few scattered areas. In the olfactory nerve layer of the right olfactory bulb there was a marked immunoreactivity. The immunoreactivity in the glomeruli of the right olfactory bulb was stronger than in the olfactory nerve layer. Within the glomeruli the staining was homogeneously distributed.

We also observed immunoreactivity in a few cells that we tentatively identified as representing osteoblasts of the periosteum, as well as in osteocytes of the bone tissue of the nasal turbinates and the nasal septum.

Left nasal cavity.
In the rats given cadmium in the right nasal cavity, the immunoreactivity was generally very weak in the cells of the olfactory mucosa on the non-cadmium-treated side (left side). This applied to the cells of the olfactory epithelium, the olfactory nerve fascicles, and the olfactory nerve layer of the olfactory bulb. In the glomeruli of the left olfactory bulb there was a marked immunoreactivity. This labeling showed a mesh-like pattern, and thus differed from the more homogeneous pattern seen in the right bulb. A few Bowman's glands, as well as osteocytes and cells identified as osteoblasts, were stained on the left side, a pattern similar to the one observed on the right.

In the untreated rats, the MT staining of the olfactory system showed a similar pattern to that observed on the nontreated side of the rats given cadmium by intranasal instillation. No staining for MT was detected when the primary antibody was omitted.

Histopathological evaluation of the slides showed that there were no visual differences between the cadmium- and saline-treated olfactory mucosae. However, it appears that the procedure of intranasal instillation in itself resulted in slight mechanical damage on the olfactory mucosa. Thus, the right olfactory mucosa was thinner in some areas as compared to the mucosa in the left nasal cavity (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of the present study provide strong evidence that cadmium administered by intranasal instillation induces MT in the primary olfactory neurons, and that the metal is transported as a CdMT complex along the axons of these cells from the olfactory epithelium to the olfactory bulbs of the brain.

The rationale for using pikes in the present study was to examine cytosolic localization of cadmium in the primary olfactory neurons by gel filtration, which is not possible in rats due to the short length of these neurons. Our results from these experiments indicate that the metal interacts with MT during the axonal transport in the primary olfactory neurons. In addition, the immunohistochemistry of the rats showed that intranasal administration of cadmium resulted in an induction of MT in the primary olfactory nerve fascicles in the olfactory epithelium as well as in the olfactory nerve layer in the olfactory bulb.

It should be noted that the synthesis of MT (as well as other proteins) occurs in the perikaryal region of the primary olfactory neurons. This implies that MT in the nerve fascicles, as well as in the olfactory nerve layers, stems from MT, which has been synthesized in the perikaryal region of the primary olfactory neurons.

Previous studies in pikes have shown that cadmium is transported along the primary olfactory neurons at a rate that falls into the category of fast anterograde axonal transport (Gottofrey and Tjälve, 1991Go). The results of the present study imply that MT is transported at the same rate as cadmium. The driving force for fast axonal transport is considered to occur via the action of kinesin (Brady and Lasek, 1982Go). This process is believed to mainly involve particulate materials and membrane-enclosed vesicles. However, it is not known whether MT is present in such vesicles in the neuroplasm. A potential alternative is that MT is transported in a free state in the neuroplasm, but there is at present no established molecular mechanism describing such a putative process.

The immunohistochemistry in the rat showed that intranasal instillation of cadmium resulted in induction of MT not only in the nerve cells, but also in the sustentacular and basal cells.

The reason for the nonspecific upregulation of MT in the entire surface epithelium is not known, but it may be related to a general role of the MT in the protection of the epithelial cells from cadmium-induced cytotoxicity. Previous studies in rodents have shown that the DAKO-MT (E9) antibody used in immunohistochemistry in the present study reacts both with MT-I and MT-II, but not with MT-III (Jasani and Elmes, 1991Go; Skabo et al., 1997Go). Thus, our results probably reflect an upregulation of the MT-I and/or MT-II isoforms.

To our knowledge there have been no previous reports of MT induction in the olfactory epithelium, nor has it been reported that the olfactory neurons can be induced to contain relatively large amounts of MT. The presence of constitutive MT in the olfactory mucosa has been examined and found to be mainly localized in a few sustentacular cells and some deeply localized Bowman's glands in certain areas of the olfactory mucosa. MT immunoreactivity was also observed in a few scattered olfactory nerve fibers in the olfactory mucosa (Chuah and Getchell, 1999Go; Shimada et al., 1996Go; Skabo et al., 1997Go). The MT distribution described in these studies corresponds to the pattern observed in untreated rats and in the nontreated sides in the nasal cavities of rats given intranasal cadmium instillations in the present study.

The glomeruli of the rat olfactory bulbs showed immunoreactivity on both the left and right sides. However, in the contralateral bulb, in relation to the side of the cadmium application, a mesh-like pattern of MT staining was observed that probably reflects a staining of astrocytes. Thus, astrocytes are localized to the outer regions of the glomeruli with extensions projecting into their interior (Bailey and Shipley, 1993Go; Chao et al., 1997Go). Our data showed a homogeneously distributed MT staining in the glomeruli ipsilateral to the side of the cadmium application. This may reflect an accumulation of MT in the terminal arborizations of the primary olfactory neurons following transport of protein in the olfactory axons.

Our data indicate that, following application of cadmium on the olfactory epithelium, the metal will initially interact with GSH. This is in line with previous investigations, which have demonstrated that GSH provides an initial cellular defense against cadmium toxicity prior to the induction of MT synthesis (Shimizu and Morita, 1990Go; Singhal et al., 1987Go; Zaroogian and Jackim, 2000Go).

In conclusion, our results show that cadmium is transported as a CdMT complex in the primary olfactory neurons to accumulate in the glomeruli of the olfactory bulbs. In addition, our results indicate that cadmium initially interacts with GSH before the onset of MT synthesis. The physiological role of MT in the olfactory pathways is elusive, but it is possible that one key function of the protein is to protect against cadmium-induced neurotoxicity.


    ACKNOWLEDGMENTS
 
We acknowledge Ms. Agneta Boström for excellent technical assistance. This study was supported by the Swedish Council for Working Life and Social Research and by the Foundation for Strategic Environmental Research (MISTRA).


    NOTES
 
1 To whom correspondence should be addressed. Fax: 46-18-50-41-44. E-mail: jonas.tallkvist{at}farmtox.slu.se. Back


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 ABSTRACT
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 MATERIALS AND METHODS
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 DISCUSSION
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