Abstract
Species and sex differences in susceptibility to vincristine sulphate (VCR)-induced olfactory epithelial lesions were investigated among the BALB/c mice, Crj: CD(SD) IGS rats and common marmoset monkeys following a single intravenous administration on day 1. As dosage levels, the 0.17-fold LD10, 0.6-fold LD10 and LD10 were used for mice and rats, and a maximum tolerated dose (MTD) was chosen only for monkeys. The order of strength of VCR action on peripheral neuropathic signs, body weight gain, and hematological parameters was mice > rats > monkeys, without clear sex differences. Histopathologically, on day 2, single cell death in the olfactory epithelium and vomeronasal organ was observed only in male mice at LD10, and in female mice at 0.6-fold LD10 or more. On day 5, the olfactory epithelium in these mice showed regenerative proliferation suggesting a sign of recovery. On day 10, axonopathy and demyelination in the sciatic and trigeminal nerves were noted in mice of both sexes at 0.6-fold LD10 or more. In rats and monkeys of either sex, however, no morphological changes were observed at any dose level. In conclusion, mice, particularly females, were shown to be more susceptible to VCR-induced apoptosis in the olfactory epithelium than rats and monkeys.
Introduction
Vincristine sulphate (VCR), a vinca alkaloid derivative, is widely used as chemotherapy for the treatment of malignant tumors in humans (Gidding et al., 1999). Although the toxic targets of cancer chemotherapeutic drugs are ordinarily cells or tissues having a high mitotic activity (e.g., lymphohematopoietic tissue, intestine, skin and testis), the dose-limiting toxicity for VCR in humans is neurotoxicity, which includes peripheral, symmetric mixed sensory-motor, and autonomic neuropathy with morphological lesions (axonopathy and secondary demyelination). VCR-induced neurotoxicity has been recognized to be caused by interference with microtubule function resulting in the blockage of axonal transport and thus axonal degeneration (Gidding et al., 1999). In laboratory animals such as mice, rats, and monkeys, neurotoxicity including abnormal behavior and histopathological lesions in the peripheral nerves has been reported to be evoked by repeated or intermittent administration of VCR (Gottschalk et al., 1968; Todd et al., 1976, 1979; Aley et al., 1996; Authier et al., 1999; Topp et al., 2000; Nakamura et al., 2001; Borzan et al., 2004). There had been no reports, however, on VCR-induced olfactory epithelial lesions in experimental animals prior to our description (Kai et al., 2002).
We have previously reported about VCR-induced apoptosis in the olfactory epithelium of male mice on days 2 to 5 after a single intravenous injection at a 10% lethal dose (LD10, Kai et al., 2002). In the present investigation, to clarify species and sex differences in susceptibility to the toxicity in the olfactory epithelium, VCR was intravenously injected once on day 1 at 0.17-fold LD10, 0.6-fold LD10 (corresponding to the maximum tolerated dose: MTD) and LD10 to mice and rats of both sexes, and only at an MTD to monkeys of both sexes. Afterward, these animals were serially euthanized on days 2, 5, and 10 for histological examinations.
Materials and Methods
Animals
Sixty-six males and 66 females of BALB/c mice and Crj:CD(SD) IGS rats were purchased from Charles River Japan, Inc. (Atsugi, Japan), and used at 8-week-old (body weight: 20 to 29 g) and 7-week-old (body weight: 173 to 295 g), respectively. Eight male and 8 female common marmoset monkeys purchased from Yaotsu Breeding Laboratory Japan, Inc. (Gifu, Japan) were used at 3- to 5-year-old (body weight: 310 to 470 g). The rodents were housed 3 to 5 animals per wire-mesh cage in air-conditioned rooms (temperature, 23 ± 2°C; relative humidity, 55 ± 15%). The monkeys were individually housed in stainless steel cages in an air-conditioned room (temperature, 24 ± 2°C; relative humidity, 60 ± 20%). A light/dark cycle was 12 h in the animal rooms. Basal diet (F-2 for mice and rats, Funabashi Farm, Chiba, Japan; CMS-1M for monkeys, CLEA Japan, Inc., Tokyo, 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
VCR was purchased from Shionogi & Co., Ltd. (Osaka, Japan). Lyophilized formulations of VCR were dissolved in physiological saline (saline) to make a constant dosing volume of 20 mL/kg for mice and rats, and 5 mL/kg for monkeys. Saline was also used for the concurrent control group.
Experimental Design
Group composition and experimental design are shown in Table 1. In the mouse and rat studies, four groups consisting of 1 control and 3 VCR-treated groups of 5 to 8 animals each were used to assess the time course of histological changes. In the monkey study, 2 groups consisting of 1 control and 1 VCR-treated group of 2 animals each were also set for changes in the time course. From the results of preliminary dose-finding studies in mice (1.0–2.5 mg/kg, n = 5), rats (0.3–1.5 mg/kg, n = 4) and monkeys (0.3–1.5 mg/kg, n = 1), the mouse LD10 was estimated as 1.95 mg/kg for males and 2.46 mg/kg for females; the rat LD10 was as 0.35 mg/kg for males and 0.40 mg/kg for females; the monkey MTD was as 0.35 mg/kg for combinated males and females because of the small sample size without sex difference. Additionally, as olfactory lesions are observed at the LD10 in the male mice (Kai et al., 2002), the LD10, 0.6-fold LD10 (middle dose, MTD) and ca. 0.17-fold LD10 (low dose) were set in both rodents; namely, 1.95, 1.17, and 0.35 mg/kg for mice and 0.35, 0.21, and 0.06 mg/kg for rats. For monkeys, only an MTD (0.35 mg/kg) was chosen to examine species-differences in the onset of VCR-induced olfactory lesions. VCR dissolved in saline was intravenously administrated once into the tail vein at an injection rate of 1 mL/min and the day of dosing was regarded as day 1 in the respective studies. As VCR-induced apoptosis in the olfactory epithelium was noted from days 2 to 5 in the previous study (Kai et al., 2002), the days of euthanasia were set on days 2, 5, and 10.
Clinical Investigation
Clinical signs were observed immediately after administration and once each on days 2, 5, and 10, body weight was measured on days 1, 2, 5, and 10, and hematology on days 2, 5, and 10. On days 2, 5, and 10, the blood sample was collected from the abdominal vein of mice and monkeys or the jugular vein of rats under ether (mice and rats) or pentobarbital (monkeys) anesthesia. The whole blood was analyzed for reticulocyte (RET), white blood cell (WBC) and platelet (PLT) counts by ADVIA 120 (Bayer Medical, Tokyo, Japan), a total hematology system. The animals of each group were subsequently euthanized by exsanguination for the following examinations.
Light Microscopy
Immediately after euthanasia, 10% neutral buffered formalin (for light microscopy) or 3% glutaraldehyde in 0.1 M phosphate buffer (for electron microscopy) was perfused into the nasal cavity from the trachea, and the head and sciatic nerve were carefully removed and fixed in the same fixatives. The rodent nasal tissues were decalcified with 5% EDTA in 0.05 M TRIS buffer (pH 7.5) for 2 weeks, and the heads of monkeys were with 25% buffered formic acid for 3 weeks. The rodent tissues were trimmed transversely at the upper incisor (level 1), incisive papilla (level 2), and oblique olfactory (level 3) as previously described (Kai et al., 2005). The whole monkey heads were cut at 5 levels at 5- to 8-mm intervals as shown in Figure 1, containing the squamous, respiratory, transitional and olfactory epithelium of the nasal mucosa, vomeronasal organ and trigeminal nerve. The respective nasal tissues and sciatic nerves were embedded in paraffin wax, cut at 5 μm in thickness and stained with hematoxylin and eosin (H&E) for light microscopic examination. Histopathological findings of the lesions were classified and scored for several areas in each mouse as follows: slight degree (score 1); sporadic distribution and little detected at a low magnification, moderate (score 2); easily found by a low magnification, severe (score 3); extensive distribution and easily found by a low magnification. Microscopic lesion were scored by a pathologist and reviewed by the other pathologists. The total scores were summed and divided by the number of animals for each group to get the group mean score.
Terminal TdT-Mediated dUTP-Nick-End Labelling (TUNEL) Assay
Sections at the level 3 made from mice of the control and VCR 1.95 mg/kg groups, which were euthanized on day 2, were subjected to TUNEL assay. Briefly, the sections were treated with 20 μg/ml proteinase K (SIGMA, Tokyo, Japan) for 15 min at room temperature and then apoptotic DNA fragmentation was marked by using an Apop Tag TM peroxidase In Situ apoptosis detection kit (Intergen Company, Purchase, NY).
Keratin Immunohistochemistry
In order to identify the horizontal basal cells (Suzuki et al., 1998, 2000), additional sections at the level 3 made from mice of the control and VCR 1.95 mg/kg groups (on day 2) were immunohistochemically stained with keratin antibody (1: 250, DAKO Japan Co., Ltd., Tokyo, Japan), followed by rabbit Envision/HRP (DAKO Japan Co., Ltd., Tokyo, Japan).
Electron Microscopy
Electron microscopic sections at the level 3 made from mice of the control and VCR 1.95 mg/kg groups (on day 2) were decalcificated, postfixed with 1% OsO4 in 0.1 M phosphate buffer and embedded in epoxy resin 812. Ultrathin sections of the nasal tissue were stained with uranyl acetate and lead citrate, and examined under a transmission electron microscope.
Statistics
Body weight and hematology data for mice and rats, which are expressed as the group mean and standard derivation (SD), were statistically analyzed between the control and VCR-treated groups by Dunnett’s multiple comparison test (2 tailed). The data for monkeys shown as the group mean values only were not statistically analyzed because of a small group size.
Results
Clinical Investigation
Clinical signs, body weights and hematology data are summarized in Table 2. In mice given VCR at a high dose (1.95 mg/kg), decreased locomotor activity, loss of extension reflex in the posterior limbs, and unkempt fur were observed in males and/or females from days 8 to 10, and one male died on day 8. No abnormal signs, however, were seen in rats and monkeys of either sex even at high doses. Body weights were decreased in mice, rats or monkeys of both sexes at the middle dose or more on days 5 and/or 10. This decrease was most severe in mice. Hematological data were shown in Figure 2 (data only from males in each species were represented, since a difference between both sexes was trivial as mentioned next). RET counts were decreased in mice and rats of both sexes at all doses on days 2 and 5, and in monkeys of both sexes on days 5 and 10. These decreases were common at similar degrees in three species. WBC counts were decreased in mice of both sexes and in male rats at all doses on days 2 and/or 5, and in female rats only at the high dose; however, no change was seen in monkeys of either sex. PLT counts were also decreased in mice of both sexes at all doses on days 2, 5, or 10, in male rats at the middle dose or more on days 2 and/or 5 and in female rats only at the high dose on day 5, and in female monkeys on day 10 only. All fluctuations in WBC and PLT counts were seen at similar degrees in mice and male rats, but were weaker in female rats. In monkeys, the decreased PLT was noted only in females. On later sampling day(s), decreases in RET, WBC and PLT counts observed in mice and rats were recovered to the control level.
Macroscopy and Light Microscopy
No macroscopic abnormalities were observed around the nostril or nasal cavity after trimming in any of the groups during the experimental period. Histopathological findings are presented in Table 3. On day 2, the olfactory epithelium in the basal layer (Figures 3a, 3b) and sensory cells of the vomeronasal organ (Figures 3c, 3d) revealed slight to moderate single cell death with condensation and fragmentation of nuclei in male mice at 1.95 mg/kg and in female mice at 1.17 mg/kg or more. Only female mice showed increased numbers of mitotic cells at 1.95 mg/kg. Single cell death was mostly seen in the basal to middle layers of the olfactory epithelium adjacent to the respiratory epithelium in the 5th and 6th ethmoturbinals. The olfactory lesion in female mice at 1.95 mg/kg was more severe than that in male mice. On day 5, the olfactory epithelium in male and female mice at 1.95 mg/kg showed a decrease in grade up to “slight” single cell death and increased numbers of mitotic cells. On day 10, axonopathy and/or demyelination in the trigeminal and sciatic nerves were observed in male and female mice at 1.95 and 1.17 mg/kg or more, respectively (Figures 3e, 3f). In rats and monkeys of either sex, no changes were observed in the olfactory epithelium, vomeronasal organs, and trigeminal and sciatic nerves.
TUNEL Assay
In mice of both sexes given 1.95 mg/kg, olfactory epithelial cells in the basal to middle layers revealed positive staining for TUNEL on day 2, but fragmented nuclei scarcely show the positive reaction (Figure 4a). No positive reaction was seen in the control group or negative control staining.
Keratin Immunohistochemistry
In mice of both sexes control and VCR group receiving 1.95 mg/kg, flattened basal cells located on the basement membrane of the olfactory epithelium and respiratory epithelium exhibited positive reaction to keratin staining (Figure 4b), but not to TUNEL on day 2. Therefore, the flattened basal cells of the epithelium were identified as being horizontal basal cells. No positive reaction was seen in negative control staining.
Electron Microscopy
In mice of both sexes given 1.95 mg/kg, the nuclei of globose basal cells, and sensory cells in the middle to basal layers of the olfactory epithelium elicited condensation, clumping, fragmentation or heterogeneity of their chromatin on day 2 (Figures 5a, 5b). Clusters of apoptotic bodies were also seen in globose basal and sensory cells; however, horizontal basal cells remained intact (Figure 5c).
Discussion
Species and sex differences in susceptibility to VCR-induced olfactory lesions were investigated among the mice, rats and common marmoset monkeys. Histopathologically, apoptosis was seen in the basal to middle layers of the olfactory epithelium adjacent to the respiratory epithelium only in mice. This apoptosis was limited to sensory cells and their precursor cells (globose basal cells), but it was not seen in keratin-positive horizontal basal cells of the olfactory epithelium. Single cell death was also observed in the vomeronasal organs. In addition, apoptosis was observed in male mice at 1.95 mg/kg and in female mice at 1.17 mg/kg or more, demonstrating higher susceptibility in females than in males; however, no such sex difference was noted for axonopathy and/or demyelination in the trigerminal and sciatic nerves. None of the changes seen in mice were detected in rats and monkeys of either sex. Thus, mice were shown to have high susceptibility to VCR-induced apoptosis in the olfactory epithelial cells and axonopathy in the peripheral nerves or rats and monkeys were resistant to olfactory epithelial lesions including apoptosis.
In clinical observations, loss of extension reflex in the posterior limbs was observed on day 8 or later only in mice reflecting the aforementioned axonopathy and demyelination of the sciatic nerves. In humans given weekly intermittent administration of VCR, neurotoxicity was also recognized to be the dose-limiting toxicity (Gidding et al., 1999). A decrease in body weight was strongest in mice among the species employed. Decreases in RET counts were common at similar degrees in mice, rats and monkeys. Decreased WBC and PLT counts were seen at similar degrees in mice and male rats but were stronger in female rats. In monkeys, the decreased PLT was noted only in females. Based on these data, the order (from highest to lowest) of strength of VCR action on peripheral neuropathic signs, body weight gain and hematological parameters was considered to be mice > rats > monkeys without clear sex differences. The most severe days for the appearance of olfactory epithelial lesions, hematotoxicity, and peripheral neuropathy were on day 2, 5, and 10, respectively. The dose for inducing hematotoxicity was lower than that for the peripheral neuropathy under the conditions of this study.
In our previous works (Kai et al., 2004, 2005), the tubulin-targeting antitumor agent VCR or paclitaxel, which caused severe apoptosis and/or atrophy in the olfactory epithelium of male mice, had high drug distribution and retention to the lesion site ethmoturbinates, whereas 5-fluorouracil, which showed no lesions in the olfactory epithelium of male mice, scarcely distributed and retained to these sites. This phenomenon was suggestive of evidently being a close implication of olfactory epithelial lesions and drug disposition. Accordingly, one possibility is raised that only a species showing a high drug disposition to the target site would exhibit olfactory lesions. Conversely, according to some reports from other authors (Castle et al., 1976; El Dareer et al., 1977; Krishna et al., 2001), a maximum concentration and half-life of VCR in blood have been proposed to be 0.3 μg/mL and 1.36 h, respectively for female BDF1 mice (body weight: 20 to 23 g) given a single administration of 2 mg/kg; 0.43 μg/mL and 1.5 h for SD rats (body weight: 200 to 250 g) receiving 1 mg/kg; and 0.77 μg/mL and 3.3 h for female rhesus monkeys (body weight: 3.3 to 6.7 kg) dosed with 1 mg/kg. Thus, the blood VCR concentration in rodents was likely to be low rather than that in monkeys. The discrepancy between our data (the magnitude of olfactory epithelial lesions: mice > rats and monkeys) and those of others (the height of blood drug levels: monkeys > rats and mice) may be explained by differences in sites (target tissue versus blood) where the drug level was measured, and experimental conditions such as dosage levels, strain, body weights and injection procedures used. The convert doses (Freireich et al., 1966) in mg/kg to mg/m2 at MTD in each species calculated were 3.51 mg/m2 for mice, 1.26 mg/m2 for rats and 2.1 mg/m2 for monkeys (marmosets). As another possibility, a difference in susceptibility to olfactory epithelial lesions was considered to be due to a difference in mortality. However, overt toxicities were observed in body weights and hematology in all species as mentioned above. Therefore, the species differences in olfactory lesions can not be explained only by the MTD. Alternatively, the further study is necessary to delineate various factors that elicit species differences.
Regarding sex difference, only in female mice given the high dose 1.95 mg/kg, increased numbers of mitotic cells were observed in conjunction with severe apoptosis on day 2, and regenerative proliferation of the olfactory epithelium was seen on day 5. The increased mitotic figures in an early time-point (day 2) are thought to imply mitotic arrests, which have been recognized to be an initial event of VCR-induced changes in olfactory epithelial lesions (Kai et al., 2005). On a cellular level, estrogen controls neural proliferation/survival and differentiation, stimulates axonal extension and synapse formation, and influences the physiological function of neural cells (Harlan 1988; Weeks and Levine, 1995; Arai et al., 1996; Beyer and karolczak, 2000). Neurotrophic factors including nerve growth factor, brain-derived neurotrophic factor, glial cell-line derived neutrophic factor and neurotrophin-3 are released from neural cells, and retrogradely transported (soma to axon/terminal) to axons as well as anterograde transport (van Bartheld et al., 2001). In the basal forebrain, estrogen receptors are localized within neurotrophin-sensitive neurons, and estrogen increases the expression of neurotrophins in the cerebral cortex, olfactory bulb and hippocampus (Guthrie and Gall, 1991; Toran-Allerand et al., 1992; Sohrabji et al., 1995; Jezierski and Sohrabji, 2001). Therefore, the higher susceptibility of female mice to the VCR-induced olfactory epithelial apoptosis may be due to higher levels of neurotrophins in the epithelial cells.
In conclusion, mice, particularly females, were shown to be more susceptible to VCR-induced apoptosis in the olfactory epithelium than rats and monkeys.
Footnotes
Acknowledgment
The authors appreciate Dr. M. Kato and Dr. T. Sugawara at the Drug Safety Research Laboratory, Daiichi Pharmaceutical Co., Ltd., for helpful discussion and review of the manuscript. 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.