Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box 573, S-751 23 Uppsala, Sweden
Received June 24, 1999; accepted January 25, 2000
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ABSTRACT |
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Key Words: aflatoxin B1; intranasal instillation; olfactory mucosa; nasal respiratory mucosa; olfactory bulb; neuronal transport; bioactivation.
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INTRODUCTION |
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AFB1 may contaminate various foods and feeds and may be present in high concentrations in respirable grain-dust particles (Burg and Shotwell, 1984; Sørensen et al., 1981
; Selim et al., 1998
). Therefore, inhalation of moldy dust may result in high local exposure of the nasal mucosa. The bioactivation of AFB1 in the nasal mucosa in vivo after local exposure of the nasal epithelium has not yet been examined. One objective of the present study was to evaluate bioactivation and toxicity of AFB1 in the nasal mucosa after intranasal administration of the mycotoxin in rats. For comparison, some rats were also given AFB1 orally. Tracing of cells with high bioactivating capacity was performed with a microautoradiographic technique, which involved extensive extractions during the fixation and embedding procedures and ensured that all unbound radioactivity would be removed (Larsson and Tjälve, 1992
). We also examined the covalent tissue-binding of radioactivity by ß-spectrometry. Our previous studies in several animal species have shown that accumulation of firmly bound metabolites of AFB1 in various tissues in vivo is due to local bioactivation of the mycotoxin in these tissues. (Larsson et al., 1989
, 1994
; Larsson and Tjälve, 1992
, 1993
, 1995
, 1996
).
In addition, we examined whether application of AFB1 on the olfactory epithelium will result in translocation of the mycotoxin to the brain along the olfactory route. This study was based on the observation that the olfactory mucosa poses a possible site of entry of foreign materials into the central nervous system (CNS). Thus, in the olfactory epithelium, the dendrites of the primary olfactory neurons are in contact with the environment in the nasal cavity and, via axonal projections, are also connected with the olfactory bulbs of the brain. It has been shown that some metals and organic xenobiotics can be transported via olfactory neurons (Holl, 1980; Tjälve et al., 1986
; Ghantous et al., 1990
; Gottofrey and Tjälve, 1991
; Hastings and Evans, 1991
; Tjälve et al., 1996
; Henriksson and Tjälve, 1998
; Tallkvist et al., 1998
).
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MATERIALS AND METHODS |
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Animals.
Female Sprague-Dawley rats, body weight about 180 g, were obtained from Bantin & Kingman Universal (Sollentuna, Sweden). The animals were housed at 22°C with 12 h light/dark cycle and given a standard pelleted diet (Lactamin AB, Vadstena, Sweden) and tap water ad libitum. The animals were kept for two weeks before the start of the experiments. The studies were approved by the Local Ethics Committee for Animal Research.
Experimental procedures.
Rats were given 3H-AFB1 intranasally. As a means of comparison, some rats were also given the mycotoxin orally. The rats given intranasal instillations were anaesthetized with pentobarbital sodium (40 mg/kg body weight intraperitoneally), and a polyethylene tubing coupled to a Hamilton syringe was inserted into the right nostril until the tubing stopped advancing (about 2 cm). The tubing was then retracted a few millimeters and 10 µl 3H-AFB1 solution was instilled into the naris. Control rats were given 10 µl physiological saline. The rats dosed orally were given 100 µl 3H-AFB1 solution into the stomach via a plastic tube attached to a syringe. After varying survival intervals, the rats were killed by CO2 asphyxiation and used as described below.
Autoradiography with freeze-dried sections of the head was performed in rats injected intranasally with 3H-AFB1 (50 µCi, 1 µg AFB1) and killed after 6 h and 24 h (two animals per interval). Autoradiography was then performed according to Ullberg et al. (1982), with horizontal tape-sections of the heads. Localization of bound 3H-AFB1 metabolites in respiratory mucosa, olfactory mucosa, and olfactory bulb was examined by high-resolution microautoradiography according to Larsson & Tjälve (1992) in rats killed 1 h, 6 h, 24 h, 2 d, 5 d, 10 d, 15 d, and 20 d after intranasal administration of 3H-AFB1 (10 µCi, 0.2 or 20 µg AFB1). Three animals were used for each treatment and survival interval. The extensive extractions during the fixation and embedding procedures removed all unbound radioactivity and the microautoradiograms therefore showed only tissue-bound labeling (Larson and Tjälve, 1992). Histopathology was performed in rats given the same doses of AFB1 and killed after 6 h, 24 h, and 5 d (three rats per survival interval). Tissues were fixed in 4% phosphate-buffered formaldehyde solution. Following fixation, the tissues were decalcified in 5.5% EDTA for 2 weeks and embedded in methacrylate (Technovit 7100, Heraus Kulzer, Wehrheim, Germany). Transversal sections 2 µm thick were taken and stained with either hematoxylin-eosin (HE), periodic acid-Schiff (PAS), or toluidine blue.
Quantitative data for the microautoradiography of the olfactory mucosa were obtained by counting silver grains over nuclei and cytoplasms. A scale was used in the ocular of the microscope, which permitted examination of cytoplasmic areas identical to nucleic areas. For each cell type, 10 nuclei and cytoplasms were counted at each survival interval. In the respiratory epithelium, the number of labeled mucous cells were determined by counting labeled cells over a length of 0.3 mm, using a scale in the ocular. For each survival interval, 5 areas were counted.
The amounts of 3H-labeled material in the olfactory nasal mucosa, the olfactory bulb, and the liver in rats killed 6 h and 24 h after intranasal or oral exposure to 3H-AFB1 (10 µ Ci, 0.2 µg or 20 µg) were determined by ß-spectrometry. Total amounts of radioactivity were measured in tissue pieces solubilized in Soluene 350TM (Packard). The amount of tissue-bound radioactivity was determined after extractions of homogenized tissue pieces with 1% SDS and acetone, using the method of Baker and Van Dyke (1984). Extracted protein pellets were dissolved in 1 M NaOH and aliquots were taken for scintillation counting and protein determination according to Lowry et al. (1951).
Statistical analysis.
Statistical significance was judged with the two-tailed Student's t-test for differences between mean values. Statistical analysis of ratios of silver grains over cell nuclei versus cytoplasms or over cell nuclei of darkly stained neuronal cells versus cell nuclei over other cell types were conducted on the logarithms of the data, using the theory of normal distribution. The obtained ratios and their 95% confidence intervals were then anti-logarithmated. A difference between cell nuclei and cytoplasms or darkly stained neuronal nuclei and other cell nuclei was considered significant if 1.0 is not included in the 95 % confidence interval.
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RESULTS |
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Microautoradiography of Nasal Respiratory Mucosa
At all survival intervals (from 1 h to 20 d), there was selective labeling of some mucous cells in the nasal respiratory mucosa (Fig. 3). Labeling was strongest at the shortest intervals. Both cytoplasms and nuclei were labeled. There was a successive decrease in the number of labeled cells from the shortest to the longest survival interval (Table 3
). Only a few silver grains were present over ciliated and basal cells in the respiratory epithelium.
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Twenty-four h after intranasal instillation of 20 µg AFB1, the olfactory epithelium was disorganized and had an undulating appearance (Fig. 4B). Several cells with pyknotic nuclei and shrinked cytoplasms were present. Both sustentacular and neuronal cells were injured, whereas cells of Bowman's glands appeared morphologically intact. Although less marked, similar injury was seen in rats given 0.2 µg mycotoxin. The extension of injured areas was usually most marked at dorsal parts of the nasal septum and medial parts of endoturbinates. In control rats (given saline), there were no injuries in the olfactory epithelium at the 24 h interval (Fig. 4A
). However, there was still decreased staining of glycoproteins in Bowman's glands at the site of intranasal instillation. Respiratory epithelium was largely intact 24 h after instillation of 0.2 µg AFB1. However, after instillation of 20 µg AFB1, mucous cells in some areas of the epithelium were severely damaged with pyknotic nuclei and vacuolized cytoplasms. Ciliated and basal cells appeared largely intact (Fig. 5
).
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ß-Spectrometry
The amount of covalently bound 3H-AFB1 metabolites in the nasal olfactory mucosa was much higher than in the liver, both at 6 h and 24 h after intranasal instillation of the mycotoxin (Table 4). In the two groups of animals exposed orally to 3H-AFB1 and killed after 6 h, higher levels of covalently bound 3H-AFB1 metabolites were found in the liver than in the olfactory mucosa. Similar amounts of bound 3H-AFB1 metabolites were found in liver and olfactory mucosa in the group of animals killed 24 h after oral administration.
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DISCUSSION |
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The neuronal cell population which accumulated bound 3H-AFB1 labeling were present superficially in the epithelium and showed darkly stained nuclei. It can be assumed that these neurons are the more mature ones, since they are characterized by a more condensed chromatin than basally localized immature neurons (Schwob et al. 1992).
In all cell types of the olfactory epithelium, there was preferential localization of silver grains over nuclei. We have previously shown nuclear localization of bound AFB1 metabolites in cells with a capacity to bioactivate the mycotoxin (Larsson et al., 1989, 1994
; Larsson and Tjälve, 1992
, 1995
, 1996
). It has been reported that the AFB1 epoxide readily binds to double-stranded DNA (Yu et al., 1990
), and labeling of nuclei probably reflects a specific affinity to bioactivated AFB1 for DNA.
It appeared that at long survival intervals, there was retention of bound 3H-AFB1 labeling in neuronal nuclei, which was more apparent than in other mucosal cells. The reason for this is not known, but one possibility is that repair of adducted DNA occurs at slower rate in neuronal cells than in other cells. The AFB1-8,9-epoxide binds to DNA primarily at the N7 position of guanine (Essigman et al., 1977; Lin et al., 1977). Altered guanine bases are either removed enzymatically or spontaneously at the glycosidic bond (Croy and Wogan, 1981
; Loeb, 1985
). It is conceivable that enzymatic adduct removal occurs at a slow rate in neuronal cells.
The finding that there was high labeling of some Bowman's glands, whereas others were virtually devoid of radioactivity, may be related to variations in contents of different cytochrome P450 enzymes in these glands. Bowman's glands present in the olfactory mucosa of the dorsal medial meatus of the nasal cavity contain higher levels of some cytochrome P450 enzymes than Bowman's glands at other sites (Genter et al., 1995). Our observation indicated that there may also be variations in cytochrome P450 contents in Bowman's glands within the same areas of the olfactory mucosa.
Our results indicated potent bioactivation of AFB1 in mucous cells in the nasal respiratory mucosa. The successive decrease in number of labeled mucous cells at increasing survival intervals may reflect the life span of these cells. The mucous cells have previously been shown to bioactivate other xenobiotics, such as N-nitrosodiethylamine and ipomeanol (Reznik-Schüller, 1982; Larsson and Tjälve, 1988
). We have previously shown high bioactivation of AFB1 in respiratory mucosa of several animal species (Larsson and Tjälve, 1992
, 1993
, 1996
; Larsson et al., 1994
).
We assume that the toxic effect of AFB1 in the nasal mucosa is related to bioactivation of the mycotoxin. Bioactivation-related toxicity of AFB1 in tracheal mucosa has been observed following intratracheal instillation of AFB1 in rabbits (Coulombe et al., 1986).
A part of the intranasally administered AFB1 can be expected to be absorbed into the blood either locally in the nasal area or after dislocation to the respiratory and alimentary tract followed by uptake to circulation. Indeed, bound AFB1 metabolites were found in the liver at similar levels after intranasal and oral administration of the mycotoxin.
Our data showed that 3H-AFB1 radioactivity was localized in the olfactory bulb ipsilateral to the site of administration. Since labeling was observed in olfactory nerve fascicles beneath the olfactory mucosa and in the olfactory nerve layer of the olfactory bulb at the side of application, and since labeling and was low in the contralateral bulb, we may assume that the radioactive material is transported along the axons of the olfactory receptor cells. Since our data indicated metabolism of AFB1 in a population of olfactory neurons, we may presume that the transported material is formed, at least in part, within the neuronal cells.
The observation that labeling reached the glomeruli of the olfactory bulb, in which synapses between primary and secondary olfactory neurons are localized, indicated that the transported material reached the terminals of the axons of primary olfactory neurons but was unable to pass to secondary olfactory neurons. The identity of the transported material is not known. However, the microautoradiography, which in the present study included fixation and embedding procedures and therefore will show only tissue-bound radioactivity (Larsson and Tjälve, 1992), demonstrated lack of labeling of olfactory nerve fascicles beneath the olfactory mucosa and of the nerve layer of the olfactory bulb. Thus, the material in the olfactory nerves should represent AFB1 and/or some of its nonreactive metabolites. The latter constitute various hydroxylated AFB1 metabolites and AFB1 conjugates. It is possible that the labeled material within the olfactory nerves will adhere to and move with some cellular components undergoing axoplasmic transport. Alternatively, movement of the labeled material may be due to diffusion along the olfactory axons.
Local exposure of nasal mucosa to AFB1 may induce tumorigenesis of this tissue. As mentioned, high levels of AFB1 can be present in respiratory grain-dust particles (Sørensen et al., 1981; Burg and Shotwell, 1984
; Selim et al., 1998
), which may lead to high local exposure of the nasal mucosa, and may conceivably increase risk for tumorigenesis of this tissue. Tumors originating from nasal mucosa are rare in humans (Silva et al., 1983
). However, in an epidemiological study in Dutch oil press workers industrially exposed to aflatoxins by inhalation, one case of nasal cancer was observed among 11 cancers in a group of 55 workers (Hayes et al., 1984
). Domestic animals, such as cattle, sheep, and swine, which are exposed to AFB1 via heavily contaminated feed and thus may inhale high amounts of the mycotoxin, have shown increased incidence of tumors originating from the nasal mucosa (Rajan et al., 1972
; Pospischil et al., 1979
; Sreekumaran and Rajan, 1983
). Nasal tumors have also been observed in sheep experimentally exposed to AFB1-contaminated groundnut feed (Lewis et al., 1967
).
Our observation that AFB1 appears to be bioactivated in some neuronal cells in the olfactory epithelium indicates that these cells may undergo tumorigenesis. It can be mentioned that in a carcinogenicity study in monkeys given AFB1 intraperitoneally or orally, one case of olfactory neuro-epithelioma was observed among nine cancers in a group of 45 animals (Adamson and Sieber, 1979).
To our knowledge, there is no evidence that AFB1 may induce tumors in olfactory bulbs. Thus, while tumors originating from nasal mucosa are frequently found in livestock exposed to moldy feed, no such evidence exists for forebrain neoplasms. The lack of CNS carcinogenesis is likely due to the inability of AFB1 to pass from primary olfactory neurons to secondary or other neuronal connections in the olfactory system.
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ACKNOWLEDGMENTS |
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NOTES |
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