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Investigation of Initial Changes in the Mouse Olfactory Epithelium Following a Single Intravenous Injection of Vincristine Sulphate
1 Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan Correspondence: Address correspondence to: Kiyonoir Kai, Daiichi Pharmaceutical Co., Ltd., Drug Safety Research Laboratory, 1-16-13, Kita-Kasai, Edogawaku Tokyo 134-8630, Japan; e-mail:kaikitrx{at}daiichipharm.co.jp
To investigate initial changes in the olfactory epithelium, vincristine sulphate (VCR) was administered intravenously once to male BALB/c mice on day 1 in comparison with unilateral bulbectomy (UBT). The light and electron microscopy of the olfactory epithelium, nerve and/or bulb with BrdU-morphometry was performed sequentially. Further, whole-body radioluminography was conducted at 1 and 24 hours postdose. Apoptosis and an increased number of mitotic cells with a tendency toward decreasing BrdU-positive olfactory epithelial cell counts were observed in olfactory epithelial cells at 6 hours postdose of VCR and became more pronounced at 24 hours postdose. These changes disappeared on days 4 or 15, but minimal axonal degeneration was seen in the olfactory nerve from day 4 onward. Semiquantitative measurement of VCR levels in the ethmoturbinals elicited high drug retention even 24 hours after administration. In contrast, UBT showed no effect on mitosis and BrdU-positive cell counts at 6 hours postdose, but severe lesions in the olfactory epithelium and nerve were seen on days 2, 4, and/or 15. The above results suggest that the initial event of VCR-induced apoptosis in the mouse olfactory epithelium would be mitotic arrest with high drug retention, unlike that evoked by UBT.
Key Words: Apoptosis • initial change • mice • olfactory epithelium • vincristine sulphate
Vincristine sulphate (VCR), a vinca alkaloid derivative, is used widely as part of multiagent chemotherapy for the treatment of malignant tumors in humans (Grindey, 1989; Gidding et al., 1999). VCR exerts its effect on microtubules by binding to tubulin or microtubules, and consequently leads to the inhibition of polymerization of mitotic spindle microtubules. The binding to microtubules damages spindle structures in a concentration-dependent manner, resulting in microtubule disruption (Bender et al., 1987; Grindey, 1989; Marcaigh and Betcher, 1995). Furthermore, VCR blocks the progress of cells from the metaphase to anaphase (Jordan et al., 1991; Gidding et al., 1999). Drug-induced disruption of the cell cycle could trigger apoptosis, but the relationship between VCR-associated metaphase arrest and apoptosis is poorly understood (Gidding et al., 1999). Although the toxic targets of cancer chemotherapeutic drugs are cells or tissues having a high mitotic activity (e.g., lymphohematopoietic tissue, intestine, skin, and testis), a dose-limiting toxicity for VCR in humans is neurotoxicity, which includes peripheral, symmetric mixed sensory-motor, and autonomic neuropathy with histopathological lesions (axonopathy and secondary demyelination). Hence, VCR-induced neurotoxicity is characterized by microtubule dysfunctions resulting in blockage of axonal transport and then axonal degeneration (Gidding et al., 1999; Schumburg, 2000). In laboratory animals such as mice, rats, and monkeys, neurotoxicities including abnormal clinical observations, and histopathological lesions in the peripheral nerve have been reported to be evoked by repeated or intermittent administration of VCR (Todd et al., 1976, 1979; Zhou and Rahmani, 1992; Aley et al., 1996; Nakamura et al., 2001; Ogawa et al., 2001; Borzan et al., 2004; Higueram and Luo, 2004). The globose basal cell is a precursor of the sensory cells, and possesses rapid proliferative activities (Schwartz et al., 1991; Huard and Schwob, 1995). The olfactory epithelium is a unique neural tissue, which is maintained by replacing dying neurons with new ones formed by cell proliferation of the globose basal cells (stem cells) in the basal region of the epithelium (Graziadei and Graziadei, 1978; Graziadei, 1980; Farbman et al., 1992). The sensory cell, a bipolar neuron, extends its axon through the cribriform plate into nerve cells (mitral, tufted and periglomerular cells) in the olfactory bulb glomeruli (Farbman et al., 1992; Graziadei and Graziadei, 1979a, 1979b, 1980; Von Bartheld et al., 2001). Apoptosis of the sensory cells has been recognized to be brought about by axotomy or bulbectomy that elicits the interruption of the axonal transport (Yaku and Saruta, 1986; Suzuki and Takeda, 1991; Suzuki et al., 1996). We have previously found that the class of tubulin-targeting anticancer drugs including VCR commonly induces apoptosis in the globose basal and sensory cells in the olfactory epithelium in mice (Kai et al., 2002, 2004). In the current paper, we investigated VCR-induced initial changes in olfactory epithelial cells, and compared them with those induced by the complete interruption of the axonal transport due to unilateral bulbectomy (UBT). Additionally, the implication of the toxicity and drug distribution to the target tissue were examined by measuring semiquantitative VCR concentrations in the ethmoturbinals with a whole-body radioluminography.
Animals One hundred and eight male BALB/c mice purchased from Charles River Japan, Inc. (Atsugi, Japan) were used at 8 weeks of age in the present study. This strain was chosen because of its high susceptibility to VCR-induced olfactory lesions (Kai et al., 2004). They were housed 5 animals or less per wire-mesh cage in an air-conditioned room (temperature, 23 ± 2°C; relative humidity, 55 ± 15%) with a 12-hour light/dark cycle. Basal diet (F-2, Funabashi Farm, Chiba, Japan) and tap water were available ad libitum. All experimental procedures were performed in accordance with the Guidelines for Animal Experimentation issued by the Japanese Association for Laboratory Animal Science (Japanese Association for Laboratory Animal Science, 1987).
Chemicals
Study1: Antimitotic Changes of the Olfactory Epithelium in Transverse Sections
VCR or saline was administrated once into the tail vein in an attempt to mimic a bolus intravenous injection used clinically. UBT was performed by the modified Herding’s method (Harding and Wright, 1979): the animals were anesthetized with inhalation of a mixture of N2O and O2 gases (700 ml/min N2O and 300 mL/min O2) containing 1.5% isoflurane (Dainippon Pharmaceutical, Osaka, Japan); a 0.8 mm diameter hole was drilled into the dorsal skull at a 2mm distance retrolaterally to the bregma; the right olfactory bulb was aspirated though the hole with a sterilized plastic pipette (100 µL, Eppendorf, Tokyo, Japan) connected to a vacuum pump; the opening was closed with a gel foam (Gelfoam Powder, Pfizer Pharmaceuticals, Tokyo, Japan) and the skin was sutured. Immediately after VCR injection or UBT, an osmotic pump MODEL 2001D (ALZET, Cupertino, CA) containing bromodeoxyuridine (BrdU, SIGMA, Tokyo, Japan) dissolved in sterilized phosphate buffered saline (PBS) at 15 mg/mL was subcutaneously implanted in the dorsal thoraco-lumbar area. BrdU solution was then automatically released at a rate of 8 µL/hr until euthanasia of animals. At necropsy, all mice were euthanized under ether anesthesia. Immediately after that, 4% paraformaldehyde was infused into the nasal cavity from the trachea, and the head of each mouse was carefully removed and fixed in the same fixative for 24 hours at 4°C. Subsequently, the nasal tissues were decalcified with 5% EDTA in 0.05 M TRIS buffer (pH 7.5) for 14 days. The specimens, trimmed transversely at the oblique olfactory level, were embedded in paraffin wax, cut at 5 µm thickness and stained with hematoxylin and eosin (H&E) for light microscopic examination. The nomenclature of the ethmoturbinals was partitioned according to the previous classification (Mery et al., 1994). The olfactory epithelium and nerve and olfactory bulb (except for the UBT groups) were examined for all animals. Histological findings were classified and scored as follows: minimal degree (score 0.5), local distribution and not detected by a low magnification; slight (score 1), sporadic distribution and not detected by a low magnification; moderate (score 2), easily found by a low magnification; severe (score 3), easily found by a low magnification and extensive distribution of the lesions. The total scores were summed and divided by the number of animals for each group to obtain the group mean score. For terminal TdT-mediated dUTP-nick-end labelling (TUNEL) assay, transverse sections obtained were treated with 20 mg/ml proteinase K (SIGMA, Tokyo, Japan) for 15 minutes at room temperature, and apoptotic DNA fragmentation was marked with an Apop TagTM peroxidase In Situ apoptosis detection kit (Intergen Company, Purchase, NY). For bromodeoxyuridine (BrdU) immunohistochemistry, the tissue sections were treated with 2N HCl for 10 minutes at 37°C to denature double-stranded DNA, since the anti-BrdU antibody binds only to single-strand DNA. Further, in order to expose the antigenic sites, the tissues were digested with 20 mg/ml proteinase K (SIGMA) for 10 minutes at room temperature, and stained with BrdU antibody (1: 180, Abcam Limited, Cambridgeshire, UK), followed by BioStain super ABC peroxidase sheep IgG (Biomeda, Foster City, CA). The numbers of BrdU-positive cells in the olfactory epithelium of the 3rd and 6th ethmoturbinals and the septum were counted for each animal on digital pictures taken under 10-fold magnification. The length of the basement membrane was measured first by tracing it with a computer system (Win-Roof version 5.02, MITANI Corporation, Chiba, Japan), and at the same time BrdU-positive cells were also counted. For the septum, the membrane length and cell counts were determined in the dorsal and ventral areas, and they were summed for each animal. The BrdU-positive cell counts per 500 µm length of the basement membrane were calculated for each area in the respective animals. The actually measured length of the basement membrane in the 3rd and 6th ethmoturbinals and the septum was in ranges of 1884 to 4672 µm, 1201 to 4521 µm, and 1880 to 3590 µm, respectively.
Study 2: Electron Microscopic Changes of the Olfactory Epithelium, Nerve, and Bulb in Sagittal Sections After necropsy, the abdominal aorta was clipped with a plastic clamp, and the right cardiac auricle was opened immediately. Then, 50 mL of 4% paraformaldehyde for light microscopy or 3% glutaraldehyde in 0.1 M phosphate buffer for electron microscopy was infused from the left cardiac ventricle into the vasculature via a 25G needle at a rate of 2 mL/hr with an infusion pump (STC-525, Terumo, Tokyo, Japan), preceded by infusion of 5 mL of physiological saline containing 0.4% heparin sodium to completely exclude blood. Sequentially, the head was fixed in the same fixative, decalcified and trimmed sagittally at the midline. Three animals each euthanized on days 2, 4, and 15 were examined by light microscopy. The decalcified tissues of 3 or 4 animals at all sampling points were postfixed with 1% OsO4 in 0.1 M phosphate buffer and embedded in epoxy resin 812. Afterward, the semi-thin sections were stained with toluidine blue for light microscopy. The microscopic findings observed in the olfactory epithelium, nerve and bulb were classified according to the criteria as described in study 1. Ultrathin sections of nasal tissue including the olfactory epithelium and nerve were stained with uranyl acetate and lead citrate, and examined under a transmission electron microscope.
Whole-Body Radioluminography The plates were exposed during a defined period in lead-shield boxes at room temperature, and images of the radioactivity were analyzed under the following conditions with a Bio-Imaging Analyzer (FUJIX BAS2500, Fuji Photo Film, Tokyo, Japan): resolution, 50 µm; gradation, 256; sensitivity, 30,000; latitude, 5. Photo-stimulated luminescence per unit area (PSL/mm2) was determined in each tissue area and background area on the radioluminograms. Radioactivity concentrations in tissues were determined from the PSL values by subtracting the background value. The PSL values in blood were measured at the abdominal aorta. The quantitative concentration was calculated from the calibration curve prepared from the PSL values of the standard samples.
Statistics
Study1: Antimitotic Changes of the Olfactory Epithelium in Transverse Sections Light Microscopy Histopathological findings in the transverse sections are presented in Table 2. In the VCR 1.17 mg/kg group, no changes were observed at 6 hour postdose, but single cell death in the olfactory epithelium was seen in the 2nd, 5th, and 6th ethmoturbinals at 24 hours postdose. In the VCR 1.95 mg/kg group, the basal olfactory epithelium contained single cell death, and an increased number of mitosis in the 6th ethmoturbinal was noted at 6 hours post-dose (Figures 1a, b). Single cell death became more prominent in the 6th ethmoturbinal (Figure 1c) and was additionally observed in the other ethmoturbinals at 24 hours postdose, but not in the septum. A severity order of the cell death in the ethmoturbinals (from highest to lowest) was 6th = 5th > 4th > 2nd > 1st > 3rd ethmoturbinals. No changes, however, were detected in the olfactory nerve and bulb at any dose level and on any sampling point. In the UBT group, no changes were observed at 6 hours postdose, but single cell death in the olfactory epithelium was noted in the septum and the 1st to 6th ethmoturbinals at 24 hours postdose (Figure 1d). No changes were seen in the olfactory nerves. In the both VCR and UBT groups, single cell death in the olfactory epithelium was severe in the basal to middle epithelial layers adjacent to the respiratory epithelium in the 5th and 6th ethmoturbinals, and the cell death was confirmed to be apoptosis by the TUNEL assay as mentioned below. Olfactory apoptosis in the UBT group was more severe than that in the VCR group, and apoptosis in the septum was only seen in the UBT group. The severity order of apoptosis in the UBT group, 5th > 6th = septum > 4th > 2nd > 1st > 3rd ethmoturbinals, was also different from that in the VCR groups.
In the TUNEL assay, nuclei of a few olfactory epithelial cells, which were observed as single cell death in the VCR 1.95 mg/kg group, revealed positive staining at 6 hours postdose. At 24 hours, numerous condensed and fragmented nuclei in the cells showed the positive staining in the VCR 1.17 and 1.95 mg/kg groups as well as in the UBT (Figures 2a, b, c).
BrdU-Positive Cell Morphometry BrdU-positive cell counts of the olfactory epithelium in the 3rd and 6th ethmoturbinals and the septum are presented in Table 2 and Figure 2. In the control group, BrdU-positive cells were mostly seen on the basement membrane of the olfactory epithelium adjacent to the respiratory epithelium (Figure 2d). The BrdU-positive cell counts per 500 µm length of basement membrane were highest in the 6th ethmoturbinal, followed, in decreasing order, by the septum and 3rd ethmoturbinal at 6 and 24 hours postdose. In the VCR 1.17 mg/kg group, no changes were seen at 6 hours postdose, but a significant decrease was observed in the 6th ethmoturbinal at 24 hours postdose. In the VCR 1.95 mg/kg group, the BrdU-positive cell counts exhibited a decrease in the 6th ethmoturbinal at 6 and 24 hours postdose, and tended to a decrease in the 3rd ethmoturbinal at 24 hours postdose (Figure 2e). In the septum cells, however, there were no changes in the BrdU-positive cell counts at any dose level and any sampling point. In the UBT group, although no changes were noted at 6 hours postdose, a significant decrease was observed only in the 6th ethmoturbinal at 24 hours postdose (Figure 2f). The severity order of olfactory apoptosis was the UBT > VCR 1.95 mg/kg > VCR 1.17 mg/kg groups, but for the decreased BrdU-positive cell counts, the order was VCR 1.95 mg/kg > UBT = VCR 1.17 mg/kg groups at 24 hours postdose. Taking into account the severity of olfactory apoptosis, the VCR groups showed stronger decreases in BrdU-positive cell counts than did the UBT group at 24 hours postdose, and the VCR 1.95 mg/kg group began revealing early signs of the decrease at 6 hours postdose.
Study 2: Electron Microscopic Changes of the Olfactory Epithelium, Nerve and Bulb in Sagittal Sections
Light Microscopy In the VCR group, single cell death and an increased number of mitotic cells in the olfactory epithelium were seen at 6 hours postdose; single cell death was moderately observed in the basal to middle layers on day 2; the cell death was decreased to minimal, and this was replaced by increased mitosis on day 4; they were recovered on day 15. There were no changes in the olfactory nerve throughout the observation period. In the UBT group, no changes were seen at 6 hours postdose; moderate single cell death was observed in the olfactory epithelium on day 2; in addition, moderate cell loss (basal to middle layers), and atrophy and hemorrhage in the olfactory epithelium were seen on day 4; slight cell death in the atrophied epithelium was evident on day 15. In the olfactory nerves of lamima propria, no changes were seen on day 2; severe degeneration was observed on day 4; additionally, macrophage infiltration was present on day 15.
Electron Microscopy
Metaphase chromatin was observed in the VCR group only at 6 hours postdose prior to the appearance of severe olfactory epithelial apoptosis (day 2). The onset and recovery of apoptosis in the olfactory epithelium occurred earlier in the VCR group than in the UBT group. The time course of the axonal lesions in the VCR group was essentially identical to, but somewhat milder than that in the UBT group. Neither horizontal basal nor supporting cells showed apoptosis in the VCR or UBT groups on any sampling point.
Whole-Body Radioluminography
VCR was intravenously injected once to male mice to elucidate initial changes in the olfactory epithelium, and to compare them with those induced by axonopathy using UBT. Six hours after treatment with VCR at 1.95 mg/kg, minimal apoptosis and an increased number of mitosis in basal layer cells were observed first in the olfactory epithelium of the 6th ethmoturbinal in which BrdU-positive cell counts were highest. Apoptosis was increased in number and in degree from the 1st to 6th ethmoturbinals at 24 hours postdose. The increased numbers of mitotic figures were associated with a tendency toward decreasing BrdU-positive cell counts in the 6th ethmoturbinal. Thereafter, the degree of apoptosis gradually decreased and disappeared on day 15. Thus, the increased number of cells in various stages of mitosis seen on day 4 was considered as a compensatory reaction. However, there was minimal axonal degeneration observed only in the olfactory nerve on days 4 and 15. Since VCR exerts its antimitotic effect on cells by interfering with mitotic spindle microtubules (O’ Marcaigh and Betcher, 1995; Gidding et al., 1999), it is conceivable that increased mitotic figures of olfactory epithelial cells implied mitotic arrest, which could precede the apoptosis. In contrast, UBT induced apoptosis of olfactory epithelial cells in the 1st to 6th ethmoturbinals more frequently than those due to VCR 1.95 mg/kg at 24 hours postdose, and apoptosis was additionally seen in the septum. The olfactory epithelial changes developed to a moderate degree including apoptosis, cell loss, atrophy and hemorrhage in the olfactory epithelium on day 4, but these were mostly resolved on day 15. On the other hand, moderate degeneration of the olfactory nerve started to appear from day 4 and became more severe on day 15 with macrophage infiltration. The severity order of apoptosis was UBT > VCR 1.95 mg/kg > VCR 1.17 mg/kg groups, but decreases in BrdU-positive cells were ordered as the VCR 1.95 mg/kg > UBT = VCR 1.17 mg/kg groups. Concerning the severity of olfactory apoptosis, antimitotic effects in the VCR groups were more severe than those in the UBT group. Although the mechanism of the apoptosis induced by axotomy or bulbectomy has still not been clarified, a transport inhibition of neurotrophic factors released from neural cells in axons (soma to axon/terminal) was considered to cause the olfactory apoptosis (Astic and Saucier, 2001; Yasuno et al., 2000). The decreased BrdU-positive cell counts seen in the 6th ethmoturbinal 24 hour after UBT were also thought due to the interrupted transport of the neurotrophic factors. The completely interrupted axonal transport evoked by UBT was shown to take time to produce the morphological alterations in the olfactory nerve. The UBT-induced changes were characterized as follows, inasmuch as the initial change in the present study was very frequent apoptosis of the olfactory epithelial cells; UBT exhibited marked atrophy of the epithelium, at which time profound degeneration of the olfactory nerve appeared and was then exacerbated. Hence, remarkable differences were shown in the development of the histological changes between UBT and VCR treatment. Moreover the apoptosis was seen in the septal olfactory epithelium in the former, but not in the latter. Semiquantitative VCR levels in the tissue revealed that the values of luminal contents in the intestine or gall bladder, a main excretion route, were higher than those in the other tissues, and the luminal contents in the urinary bladder, the other excretion route, also showed a high value. According to a previous paper (Krishna et al., 2001), the half-life of VCR following an intravenous injection was 1.36 hr, and VCR was mainly excreted into bile (El Dareer et al., 1977). The high values of the luminal contents in the intestine, gallbladder, and urinary bladder were also suggestive of rapid excretion after an intravenous injection of VCR. The bone marrow and lymphoid organs (spleen and thymus), the most preferable toxicity target tissues, elicited high values both 1 hour and 24 hours after administration. The values in the nasal tissue (ethmoturbinals) were approximately 2 times higher than those in the blood at either 1 hour or 24 hours postdose, but not higher than those in the other tissues. However, the VCR level in the nasal tissue was almost consistent with that in blood. Certain antimicrotubule drugs caused apoptosis in the olfactory epithelium, and high drug distribution and retention in the nasal tissue were also seen (Kai et al., 2004). In the present work, VCR was also shown to provoke apoptosis with the high drug retention in the nasal tissue even 24 hours after administration. The time course of morphological changes in the olfactory epithelium, nerve, and bulb by VCR were shown to be markedly different from those by UBT that had been reported to evoke apoptosis of the epithelial cells through the interruption of axonal transport (Michel et al., 1994; Pasterkamp et al., 1998). On the contrary, apoptosis in various tissues caused by antimicrotubule drugs including VCR has extremely complex signal pathways, and results in the rapid alteration of protein kinase activities in conjunction with the induction of bax. Overexpression of bax causes G2-M arrest, tubulin polymerization and bcl-phosphorylation, and the phosphorylations of bcl-2, as well as the elevations of p53 and p21, are supposed to contribute to apoptosis (Wang et al., 1999). Therefore, VCR is considered to initially induce mitotic arrest by its antimicrotubule action in the olfactory epithelial cells under the high drug retention, subsequently leading to apoptosis, without implication of the interrupted axonal transport in the olfactory nerve. The relevance of these mouse single-dose data to patients that received VCR as part of cancer chemotherapy regimen remained unclear, because VCR-induced olfactory toxicity in humans has never been reported so far, despite a long usage in the clinical field. However, based on the fact that the dose-limiting toxicity in humans is peripheral neurotoxicity, which is slowly reversed and enhanced by intermittent administration of VCR (Gidding et al., 1999), and the mouse olfactory lesion was noted at a relatively high dose, the absence of olfactory toxicity in humans may be due to a consequence of the lowered feasible dosage level of VCR. In conclusion, VCR-induced initial changes were shown to be apoptosis along with an increased number of mitosis and a tendency toward decreasing BrdU-positive cell counts in the olfactory epithelium. UBT-induced initial changes were quite different from the VCR lesions: apoptosis of the epithelial cells was observed in the early stage as well, but this developed into markedly atrophied epithelium, followed by profound degeneration of the olfactory nerve. Semiquantitative measurement of VCR levels in the ethmoturbinals revealed high drug retention at 24 hours postdose. Therefore, the results obtained suggest that the initial event of VCR-induced apoptosis in olfactory epithelial cells would be mitotic arrest, unlike that evoked by UBT.
The authors wish to thank Mr. Y. Ozaki, Mr. Y. Ishii, and Ms. K. Okado at the Technology Research Center, Daiichi Pharmaceutical Co., Ltd., for their technical assistance of tissue preparation.
Toxicologic Pathology, Vol. 33, No. 7,
752-761 (2005)
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Abstract
Introduction
