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
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ABSTRACT |
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Key Words: cadmium; olfactory; nerve; metallothionein; axonal transport; rat; pike.
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INTRODUCTION |
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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., 1997; Nordberg and Nordberg, 2000
).
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.
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MATERIALS AND METHODS |
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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., 1996). 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
-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, 1991). The pikes were killed after 48 h and the olfactory epithelia, nerves, and bulbs were dissected as described (Gottofrey and Tjälve, 1991
). 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 510 volumes of 50 mM TrisHCl (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 -spectrometry. Cytosols not used for
-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 TrisHCl (pH 7.4) containing 0.02% Na-azide and 0.4 mM PMSF as described (Henriksson et al., 1999; Tallkvist et al., 1998
; ). 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
-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.
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RESULTS |
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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. 1). 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. 1
). 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. 1
). In the olfactory epithelium of the Cd2+-pretreated rats killed at 2 h, the 109Cd2+ was recovered in fraction 15 (Fig. 1
). 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 67 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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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).
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DISCUSSION |
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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, 1991). 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, 1982
). 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, 1991; Skabo et al., 1997
). 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, 1999; Shimada et al., 1996
; Skabo et al., 1997
). 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, 1993; Chao et al., 1997
). 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, 1990; Singhal et al., 1987
; Zaroogian and Jackim, 2000
).
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.
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ACKNOWLEDGMENTS |
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NOTES |
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