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TOXICOLOGY
Departments of Anatomy, Physiology, and Cell Biology (M.G.L., A.P., C.P.) and Molecular Biosciences (D.M., A.B.), School of Veterinary Medicine, University of California, Davis, California
Received for publication
February 3, 2005
Accepted
April 13, 2005.
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Abstract |
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). Therefore, the olfactory mucosa has the potential to metabolize protoxicants that require in situ bioactivation. Toxic metabolites generated in the nasal passage could pass into the systemic circulation due to the rich vasculature and high perfusion rate of the nasal region (Ding and Dahl, 2003). Likewise, the nasal passage could be an injury target for xenobiotics undergoing systemic circulation (Placke et al., 1987; Gu et al., 1997).
Previous studies have shown that the olfactory mucosa of the rat is a major target of NA toxicity by intraperitoneal administration (
200 mg/kg NA, both the lowest observable adverse effect level and the lowest dose of their trial), and NA injury occurred uniformly across the olfactory region (Plopper et al., 1992). Bioactivation of NA by P450s to NA-1,2-epoxide is a key event in a series of steps leading to cytotoxicity (Buckpitt et al., 1995). For a number of species, including nonhuman primates, nasal tissue, especially olfactory mucosa, contains high concentrations of P450s. In humans, significant expression/activity of P450s in the nasal mucosa have been reported. For example, in the olfactory mucosa of humans, CYP2A6 and CYP2A13 are highly expressed and are catalytically active toward a number of toxicants, including tobacco-specific nitrosamines (Su et al., 2000; Chen et al., 2003).
NA is volatile (0.082 mm Hg at 25°C) and insoluble in water (31.7 mg of NA in 1 liter of water at 20°C) (Agency for Toxic Substances and Disease Registry, 2003). A major route of NA exposure in humans is via inhalation. A concentration-dependent increased incidence of nasal neoplasms was noted after inhalation exposure of rats to NA for 2 years, even at the lowest concentration tested (10 ppm; National Toxicology Program, 2000). Recently, the International Agency for Research on Cancer (IARC, 2002) categorized NA as a category 2B carcinogen (possibly carcinogenic to humans).
This study was designed to examine mucosal injury to the rat nasal passage from acute exposure to NA by inhalation. Our preliminary inhalation data indicated that olfactory mucosal injury was not uniform within the mucosa after inhalation exposure to NA, and this contrasts with the more uniform distribution of injury observed after systemic administration of NA. The hypothesis tested in this study was that the nonuniform pattern of olfactory mucosal injury from acute NA exposure by inhalation is due to both nasal airflow patterns and regional differences in NA bioactivation. To test the hypothesis, the goals were 1) to define the nasal pattern of injury from inhaled NA at doses that are substantially lower and higher than the current Occupational Safety and Health Administration standard for NA, 2) to compare the nasal injury pattern from inhaled NA with the results of previous studies on nasal airflow patterns by examining transverse sections of the rat nasal passage, 3) to compare the nasal injury pattern from inhaled NA with that from NA administered intraperitoneally, and 4) to determine whether the patterns of nasal injury from inhaled NA reflect the potential for regional bioactivation of NA in nasal tissue microsomes.
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Materials and Methods |
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-nicotinamide adenine dinucleotide phosphate hydrate (-NADP); L-glutathione, glucose 6-phosphate, glucose-6-phosphate dehydrogenase (Sigma-Aldrich, St. Louis, MO); paraformaldehyde (Fisher Scientific); formic acid (Merck, Darmstadt, Germany); and toluidine blue (Polysciences, Warrington, PA). Animals. Male Sprague-Dawley rats were purchased from Harlan (San Diego, CA). Animals were provided with food and water ad libitum and were housed in an Association for Assessment and Accreditation of Laboratory Animal Care accredited facility in HEPA-filtered cages at the University of California, Davis, for at least 7 days before use in an experiment. Rats weighed 180 to 200 g at the time of necropsy.
Inhalation Exposures. Three treatment groups of six rats each were exposed to filtered air, 3.4 ± 0.5 ppm NA, and 23.8 ± 1.7 ppm NA. All rats were exposed for 4 h. Exposures were done from 10:00 AM to 2:00 PM to minimize the effect of diurnal fluctuation of glutathione (Jaeschke and Wendel, 1985) on the extent of NA injury. Glass metabolism cages (Bioserve, Laurel, MD) housed two rats during the inhalation period. Rats were allowed free access to water while in the chamber. The desired NA concentrations inside the chamber were achieved by adjusting the flow rates of fresh air and the air flowing through a glass column (2.5 x 60 cm) packed with NA crystals. The air supply (medical air; Puritan Medical Products, Overland Park, KS) passed through an activated carbon filter before use in the chamber. Both fresh air and NA-containing air were mixed in a separate glass flask before entering the chamber. Total airflow inside the chamber was maintained at 1 liter min-1 throughout the exposure period, which resulted in a complete air exchange every 5 min. The temperature of the chamber was maintained at 25°C. Ten milliliters of air inside the chamber was removed using a gastight syringe (Hamilton, Reno, NV) and then dissolved in 3 ml of acetonitrile. Extraction efficiency of NA into acetonitrile was 80%. The concentrations of NA dissolved in acetonitrile were monitored by UV absorbance at 219.2 nm using an extinction coefficient of 89,100 M-1 cm-1. A conversion factor (1 ppm = 5.24 mg/m3 at 25°C) (IARC, 2002) was applied to obtain a concentration of NA in the gas phase as parts per million (ppm).
Intraperitoneal Administration of NA. Rats received a dose of 0, 25, 50, 100, or 200 mg NA/kg body weight in corn oil (Mazola, Cordova, TN) by intraperitoneal injection. Each group consisted of three rats. All rats were treated between 11:00 AM to 12:00 PM to minimize the influence of diurnal fluctuation of glutathione on the extent of NA injury.
Necropsy and Sample Preparation for High-Resolution Histopathology. Rats were euthanized with an overdose of sodium pentobarbital 24 h after inhalation or intraperitoneal NA exposures. The animals were exsanguinated by cutting the abdominal aorta. After the lower jaw was removed, the head was separated from the cadaver. The nasal passage was fixed in 40 ml of 4% paraformaldehyde (0.1 M sodium phosphate buffer, pH 7.4) at 4°C for at least 24 h after being flushed with 5 to 6 ml of ice-cold 4% paraformaldehyde through the nasopharyngeal duct. The fixed nasal passage was decalcified in 40 ml of 13% formic acid with gentle shaking for 5 days at room temperature. The decalcified nasal passage was washed with running tap water for 4 h and was grossly sectioned perpendicular to both the nose bridge and the nasal septum at the following four specific anatomic locations (Young, 1981; Mery et al., 1994): 1) immediately posterior to the upper incisive teeth, 2) through the incisive papilla, 3) through the second palatal ridge, and 4) in the middle of the first upper molar teeth. The sections were embedded in glycol methacrylate. Two- to 3-µm sections were generated from each embedded section using a Microme HM340E microtome (Carl Zeiss, Thornwood, NY). Sections were stained with 1% toluidine blue for examination by high-resolution light microscopy. For the purposes of this study, level 7 (L7) and level 23 (L23) were chosen. The levels were based on a diagram of the cross sections of the entire nasal passage of the rat by Mery et al. (1994) that has 30 cross sections. L7 and L23 mimic the cross sections where the airflow studies (Kimbell et al., 1997) have provided detailed information of nasal airflow, and the National Toxicity Program bioassay reported incidence of neoplasm (adenoma and neuroblastoma).
High-Resolution Light Microscopic Quantification of Injury. To quantify NA injury within the olfactory region along the basement membrane, the length of the basement membrane lined with injured and uninjured olfactory epithelium was measured with Scion Image (Scion Corporation, Frederick, MD). Results are expressed as a percentage of basement membrane lined with injured olfactory epithelium.
Microsomal Incubations. To determine whether the characteristic injury pattern from inhaled NA in the rat nasal passage reflected the differences in regional formation of NA-1,2-epoxide from NA, microsomes were prepared from nasal tissue isolates. Sampling sites were chosen based on nasal airflow patterns and the injury patterns from inhaled NA (see Results for more details of sampling sites.) Each sample was pooled from 14 unexposed rats. Samples were washed with ice-cold buffer (200 mM Tris/1.15% KCl, pH 7.4 at 4°C) after removal. All samples were homogenized similarly using a Polytron homogenizer (Glen Mills, Clifton, NJ). Microsomes were prepared by differential centrifugation at 9000g for 30 min at 4°C followed by centrifugation of the supernatant at 100,000g for 65 min at 4°C. Measurement of active microsomal P450s was performed as described by Schenkman and Jansson (1999). Microsomal protein concentration was determined according to Bradford (1976), with bovine serum albumin as the standard.
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). Incubations were prepared on ice in a final volume of 200 µl. Each incubation consisted of 100 mM sodium phosphate buffer at pH 7.4, 15.4 pmol of active P450s, 0 to 160 µM NA, 1 mM reduced glutathione, 6 1-chloro-2,4-dinitrobenzene units of glutathione S-transferases, and a NADPH-generating system. The NADPH-generating system was added to give a final concentration of 0.22 mM NADP, 6.2 mM glucose 6-phosphate, 0.1 unit of glucose 6-phosphate dehydrogenase, and 15 mM MgCl2. The NADPH-generating system was preincubated for 2 min at 37°C to ensure the presence of NADPH at the onset of incubation, and then it was added into the incubations on ice. NA solutions were prepared in methanol and added to the incubation mixture at 0.5% (v/v). Incubations were allowed to proceed for 10 min at 37°C in 12 x 35-mm capped glass vials (with Teflon polytetrafluoroethylene-lined closures; Fisher Scientific) in a shaking water bath. The reactions were terminated by adding 400 µl of ice-cold methanol, and samples were stored at -20°C overnight or longer. All incubations were prepared in triplicate or quadruplicate.
HPLC Analysis of NA Metabolites. This analysis was done as in Buckpitt et al. (1987) except that the acetonitrile gradient was modified. Terminated incubation mixtures were centrifuged at 13,000g for 30 min at -4°C to pellet the precipitated protein. The supernatant was collected and evaporated under a vacuum to complete dryness. Dried samples were stored at -80°C until high-performance liquid chromatography analysis. Samples were reconstituted in 100 µl of water and centrifuged to remove particulates. NA glutathione conjugates and diol were separated on a Phase Sep C18 ODS2 column (250 x 4.6 mm i.d.; 5-µm particle) (Waters, Milford, MA). The eluates were monitored by UV absorbance at 260 nm. A mobile phase of 0.06% triethylamine phosphate in water at pH 3.1, and acetonitrile was used at a flow rate of 1.0 ml/min with a linear increase from 10 to 14% acetonitrile over 60 min. Metabolites of NA were identified by comparing retention times with synthesized NA metabolite standards. NA metabolites were quantified against a standard curve of synthetic NA metabolite standards. Synthesized NA metabolites were made by conjugating NA-1,2-epoxide with glutathione and were purified by high-performance liquid chromatography. They were stored at -80°C until use.
Statistics. All data were imported into Prism 3.0 (GraphPad Software Inc., San Diego, CA) for statistical analysis. Differences in percentage of injured area among the groups of filtered air, 3.4 ppm NA, and 23.8 ppm NA were determined by one-way analysis of variance using the Bonferroni's post hoc test. Differences in Vmax for the formation of NA metabolites from the microsomal incubation of the septal olfactory mucosa versus ethmoturbinates were determined by unpaired t test.
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Results |
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). The olfactory epithelium of rats exposed to filtered air was not injured (Figs. 2a and 3a). The top of the olfactory epithelium was covered with fine cilia extending from the olfactory receptor cells, and most of the nuclei in the epithelial layer were from pseudostratified olfactory receptor cells. Cytoplasm from sustentacular cells was situated between the cilia and nuclei of the olfactory epithelium. After NA inhalation, injury was found only in the olfactory mucosa. At 3.4 ppm NA, the continuity of the olfactory mucosa in L7 and L23 was broken by areas of necrotic olfactory receptor cells, the volume of cytoplasm from sustentacular cells above the nuclei was reduced, and vacuoles were present in the olfactory epithelial layer (Figs. 2b and 3b). At this dose, L7 also had patches of exfoliated cells. This other type of cellular damage indicated that the injury was more severe at L7 compared with at L23 (Fig. 2c versus 3b). The frequency for the incidence of both types of injury at L7 from 3.4 ppm NA seemed to be the same. After exposure to 23.8 ppm NA, numerous exfoliated cells and cell debris were trapped in the nasal passage at both levels (Figs. 2d and 3c). However, the specific area of the DMM at L23 showed less injury (Fig. 3d) compared with the rest (Fig. 3c) of the olfactory region of L23. Despite the presence of numerous intraepithelial vacuoles and the reduced volume of cytoplasm from sustentacular cells of the DMM at L23 after 23.8 ppm NA exposure, the height of the epithelium was still preserved, and the nuclei from epithelial cells were visible within the epithelial layer of the DMM at L23. Basal cells of the olfactory mucosa seemed to remain unaffected in all of the NA-treated rats. In the underlying lamina propria of injured olfactory mucosa at both levels, the toluidine blue staining for seromucous materials in the Bowman's glands seemed to be depleted by NA treatment. However, both nerve bundles and blood vessels looked the same in NA-treated rats as in filtered air-treated rats.
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In general, it seemed that the pattern of injury from inhaled NA correlated well with the computational fluid dynamics-based model of nasal airflow patterns by Kimbell et al. (1997). At L23, olfactory mucosal injury from inhaled NA primarily occurred within the medial meatus of the nasal passage where the majority of the nasal airflow is allocated (approximately 81-92% of total airflow passing through L23 above the nasopharyngeal duct) after exposure to 23.8 ppm NA. At L23, olfactory mucosal injury from the lower dose (3.4 ppm) of NA was confined to the very first pathway of inspiratory nasal airflow at L23, the dorsal half of the medial meatus. Injury from either dose of NA by inhalation exposure was not observed in the low airflow area, mostly along the ethmoturbinates. Kimbell et al. (1997) reported that not only the airflow allocation but also the speed decreases along the stream of nasal air as inhaled air moves through the nasal passage. The degree of injury from inhaled NA was more severe at L7 than at L23 when we examined the DMM, where nasal air flows from L7 toward L23 direction.
Comparison of Nasal Injury after Inhalation and Systemic Exposure to NA. Cross sections of L7 and 23 from the nasal passage of rats treated intraperitoneally with varying doses of NA were prepared and examined in the same manner as the NA inhalation study. No mucosal injury was observed in the rats injected with 0, 25, or 50 mg/kg NA, and their mucosa looked similar to that in rats exposed to filtered air by inhalation at L7 and L23 (Figs. 2a and 3a). However, at 100 and 200 mg/kg NA, cellular injury was apparent. Systemic exposure to NA generated widespread injury in the olfactory mucosa at L7 (not shown) and L23 (Fig. 4g). In contrast to the pattern of injury observed after NA inhalation, injury occurred not only along the medial meatus but also throughout the ethmoturbinates at L23. The degree of olfactory mucosal injury decreased in a posterior to anterior direction through the nasal passage (not shown). Similar to what was seen with inhalation exposure, at L23, injury in the DMM was milder compared with the rest of the olfactory region of rats treated with 100 mg/kg NA; the only signs of injury were occasional degeneration of sustentacular cell cytoplasm and the presence of intraepithelial vacuoles (Fig. 4a). In contrast, the rest of the olfactory epithelium in L23 had extensive exfoliation (Fig. 4b). At 200 mg/kg NA, however, severe cellular exfoliation was uniform throughout L23 (Fig. 4, c and d). Basal cells were included in the population of exfoliated epithelium from systemic NA (Fig. 4, b and d), which was not apparent after inhalation exposure. Unlike inhaled NA, there was not a tendency for preferential distribution of injury in half of the nasal passage over the other. Another prominent difference between inhalation and systemic exposure was the presence of large vacuoles and loss of cilia in ciliated columnar cells, nonolfactory epithelium, along the lateral walls of L23 at 100 mg/kg NA (Fig. 4, e versus f). Similar to NA inhalation exposure, seromucous materials in the Bowman's glands were depleted by systemic NA treatment, and both nerve bundles and blood vessels seemed unaffected by NA.
NA Metabolism from Regional Nasal Microsomal Incubations. Three sampling sites for microsomal incubation of NA were chosen based on nasal airflow patterns and the injury patterns from inhaled NA: 1) the olfactory mucosa of the nasal septum (high airflow area), 2) the ethmoturbinates from both sides of the nasal passage (mostly lined with olfactory mucosa; mostly low airflow area), and 3) the nonolfactory region of the nasal septum (mostly lined with nonolfactory mucosa; high airflow area). The most dorsal ethmoturbinate was not used to avoid the chance of including any mucosa from the dorsal medial meatus. The levels of P450 by nasal region were 267 ± 11, 278 ± 17, and 74 ± 9 pmol/mg of microsomal protein for the septal olfactory region, the ethmoturbinates, and the septal nonolfactory region, respectively. Figure 5 summarizes the regional differences in rates of metabolism of 160 µM NA in microsomes isolated from rat nasal mucosa. The majority of NA metabolites generated in microsomal incubations were glutathione conjugates of NA-1,2-epoxide in all three nasal regions. A small portion of NA-1,2-epoxide was metabolized to 1,2-dihydroxy-1,2-dihydronaphthalene by microsomal epoxide hydrolase (4.1, 6.9, and 2.8%) for the septal olfactory region, the ethmoturbinates, and the septal nonolfactory region, respectively) instead of being conjugated with glutathione. Four diastereomeric glutathione conjugates were generated, and 1R-hydroxy-2R-glutathionyl-1,2-dihydronaphthalene, derived from 1R,2S-naphthalene oxide, was the primary metabolite for all three nasal regions. The percentage of total metabolites accounted for by 1R-hydroxy-2R-glutathionyl-1,2-dihydronaphthalene was 77.7% for the septal olfactory region and 72.6% for the ethmoturbinates and the septal nonolfactory region. The average rate of microsomal metabolism (the combined rate for the formation of both glutathione conjugates and dihydrodiol) of NA (0-160 µM) by nasal region is shown in Fig. 6.
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Discussion |
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; Gervasi et al., 1991; Gu et al., 2000; Su et al., 2000; Chen et al., 2003). The National Toxicology Program's bioassay (2-year inhalation of NA) has reported an exposure-related incidence of neuroblastoma in the olfactory region and adenoma in the respiratory and transitional epithelia of the anterior part of the nasal cavity along the medial or lateral aspects or tips of the nasoturbinates in the nasal passage of Fisher F344/N rats. In the current study, we were unable to observe cellular injury in the nonolfactory mucosa of the nasoturbinates. However, a significant degree of olfactory mucosal injury occurred below the lowest observable adverse effect level (10 ppm, female and 30 ppm, male) for nasal carcinoma (neuroblastoma) in the 2-year bioassay conducted by the National Toxicology Program.
One of the goals of this study was to determine whether nasal injury from inhaled NA is related to simulated airflow patterns. The studies of computational fluid dynamics on nasal airflow patterns in rats have shown that 15 to 19% of the total inspiratory airflow reaches the olfactory region at L7, whereas at L23 this decreases to 3 to 8% of the total inspiratory airflow (Kimbell et al., 1997). Of the airflow reaching the olfactory region at L23, approximately 90% moves through the dorsal half of the medial meatus. Once this air reaches the back of the nasal passage, it changes direction and approximately 85% flows toward the anterior region through a narrow passage, the ventral half of the medial meatus at L23. Shortly thereafter, the air changes direction again and joins the major inspiratory airflow from the nostril directly toward the nasopharyngeal duct. Therefore, the allocation of nasal airflow in the wide area of the lateral parts at L23, mostly along the ethmoturbinates, is very small.
Previous studies examining the susceptibility of the nasal mucosa of rats have shown that the formaldehyde mass flux predicted by computer fluid dynamics techniques correlates with both the patterns of nonolfactory mucosal injury from inhaled formaldehyde (Kimbell et al., 1993) and the sites of DNA-protein cross-linking induced by inhaled formaldehyde (Hubal et al., 1997). Hardisty et al. (1999) have found that eight toxicants (propylene glycol monomethyl ether acetate, n-butyl propionate, ethyl acetate, n-butyl acetate, dibasic esters, primary amyl acetate, vinyl acetate, and methyl methacrylate) result in a consistent pattern of olfactory mucosal injury after inhalation exposure, regardless of concentration and duration of exposure. The olfactory mucosal injury was confined to the medial meatus (high airflow area). In addition, Moulin et al. (2002) found that after 70 days of exposure of rats to hydrogen sulfide (30 or 80 ppm), nasal injury occurred only within the olfactory mucosa. The patterns of olfactory mucosal injury correlated with the high-deposition sites of inhaled hydrogen sulfide predicted by computational fluid dynamics techniques. However, the remaining nasal regions did not show such correlations. Olfactory mucosal injury from inhaled hydrogen sulfide was also confined within the medial meatus, which is similar to what was observed from inhaled NA. However, mechanisms involved in toxification and/or detoxification for most of the aforementioned compounds, including hydrogen sulfide, are unknown.
Since the balance between generation and detoxification of reactive metabolites is likely to be an important determinant in site-selective toxicity, another goal of this study was to examine regional differences in NA bioactivation as a possible reason for the specific injury patterns observed after exposure to naphthalene vapors. Buckpitt et al. (1995) have previously correlated the formation of NA-1,2-epoxide by P450s and its consequent cytotoxicity in lung tissues. Current data indicated that the average velocity (V) (the combined rate for the formation of both glutathione conjugates and 1,2-dihydroxy-1,2-dihydronaphthalene from NA-1,2-epoxide per milligram of microsomal protein) at the highest substrate concentration tested (160 µM) was not significantly different between the septal olfactory region and the ethmoturbinates (p < 0.05). The rates of microsomal naphthalene metabolism were similar at the remaining NA concentrations (Fig. 6, a and b). Similar rates of bioactivation may explain why the degree of injury was indistinguishable between the septal olfactory mucosa and ethmoturbinates when NA (100 mg/kg) was administered intraperitoneally since it should distribute uniformly within the olfactory area when delivered via the vasculature. However, the dependence on airflow patterns would explain why injury from inhaled NA primarily occurred in the septal olfactory mucosa. The presence of multiple P450 isoforms that are able to bioactivate NA in the nasal mucosa precluded classic kinetic analyses.
Injury from NA in the DMM at L23 was more severe with inhalation (23.8 ppm NA) than with systemic exposure (100 mg/kg NA) (Fig. 3d versus 4a), indicating that more NA reached the olfactory mucosa in the DMM at L23 via the inhalation route (23.8 ppm NA) compared with the systemic route (100 mg/kg NA). Nevertheless, olfactory mucosal injury in the low airflow area (mostly ethmoturbinates) at L23 was observed only from systemic NA (100 mg/kg NA) not from inhaled NA (23.8 ppm NA). This suggests that in the olfactory area at L23, a clear difference in the injury patterns from NA depends on the amount of NA delivered by the exposure route. Therefore, within the olfactory area (septal olfactory region and ethmoturbinates), differences in airflow (as predicted in simulations) seemed to govern the injury patterns from inhaled NA rather than just in situ formation of NA-1,2-epoxide.
NA was metabolized at a higher rate by the septal olfactory region and ethmoturbinates than the septal nonolfactory region (Figs. 5 and 6). In the septal nonolfactory region (high airflow region), it is likely that most of the NA metabolism came from the small isolated patch of olfactory mucosa situated in the septal nonolfactory mucosa (the organ of Masera; Weiler and Farbman, 2003; covering approximately 3% of the septal nonolfactory region by surface area). Severe injury of this small olfactory patch from inhaled NA was apparent at either concentration of NA when the nasal septum was examined by scanning electron microscopy (not shown). Therefore, virtually no NA bioactivation occurs in the septal nonolfactory mucosa, which is consistent with the finding that no cellular injury was observed in the septal nonolfactory mucosa regardless of the route of exposure or concentration of NA.
Less severe injury was noted in the DMM of the olfactory region at L23, regardless of the route of exposure (Figs. 3d and 4a). The small sample size precluded isolation of sufficient tissue for analysis of metabolic rates.
In conclusion, the current study found that 1) nasal injury in rats from inhaled and systemically administered NA was cell type-specific and occurred primarily in the olfactory mucosa; 2) olfactory mucosal injury was region-specific, and the regional patterns of olfactory mucosal injury were significantly different depending on exposure routes, i.e., inhalation versus intraperitoneal injection; 3) the incidence of acute nasal injury from systemic and inhaled NA correlates with regional microsomal formation of NA-1,2-epoxide; and 4) regional patterns of olfactory mucosal injury from inhaled NA correlate with the regions of highest airflow based on previous simulation studies in the rat nasal passages.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NA, naphthalene; L, level; P450, cytochrome P450; DMM, dorsal medial meatus; MMM, middle medial meatus.
Address correspondence to: Dr. Myong Gyong Lee, One Shields Ave., Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616. E-mail: mglee{at}ucdavis.edu
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References |
|---|
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Agency for Toxic Substances and Disease Registry (2003) Chemical and physical information, in Toxicological Profile for Naphthalene, 1-Methylnaphthalene, and 2-Methylnaphthalene.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.[CrossRef][Medline]
Buckpitt A, Buonarati M, Avey LB, Chang AM, Morin D, and Plopper CG (1992) Relationship of cytochrome P450 activity to Clara cell cytotoxicity. II. Comparison of stereoselectivity of naphthalene epoxidation in lung and nasal mucosa of mouse, hamster, rat and rhesus monkey. J Pharmacol Exp Ther 261: 364-372.
Buckpitt AR, Castagnoli N Jr, Nelson SD, Jones AD, and Bahnson LS (1987) Stereoselectivity of naphthalene epoxidation by mouse, rat, and hamster pulmonary, hepatic and renal microsomal enzymes. Drug Metab Dispos 15: 491-498.[Abstract]
Buckpitt A, Chang AM, Weir A, Van Winkle L, Duan X, Philpot R, and Plopper C (1995) Relationship of cytochrome P450 activity to Clara cell cytotoxicity. IV. Metabolism of NA and NA oxide in microdissected airways from mice, rats and hamsters. Mol Pharmacol 47: 74-81.[Abstract]
Chen Y, Liu Y, Su T, Ren X, Shi L, Liu D, Gu J, Zhang Q, and Ding X (2003) Immunoblot analysis and immunohistochemical characterization of CYP2A expression in human olfactory mucosa. Biochem Pharmacol 66: 1245-1251.[CrossRef][Medline]
Ding X and Dahl A (2003) Olfactory mucosa: composition, enzymatic localization and metabolism, in Handbook of Olfaction and Gustation, 2nd ed (Doty RL ed) pp 51-73, Marcel Dekker, New York.
Gervasi PG, Longo V, Naldi F, Panattoni G, and Ursino F (1991) Xenobiotic-metabolizing enzymes in human respiratory nasal mucosa. Biochem Pharmacol 41: 177-184.[CrossRef][Medline]
Gu J, Su T, Chen Y, Zhang QY, and Ding X (2000) Expression of biotransformation enzymes in human fetal olfactory mucosa: potential roles in developmental toxicity. Toxicol Appl Pharmacol 165: 158-162.[CrossRef][Medline]
Gu J, Walker VE, Lipinskas TW, Walker DM, and Ding X (1997) Intraperitoneal administration of coumarin causes tissue-selective depletion of cytochromes P450 and cytotoxicity in the olfactory mucosa. Toxicol Appl Pharmacol 146: 134-143.[CrossRef][Medline]
Hardisty JF, Garman RH, Harkema JR, Lomax LG, and Morgan KT (1999) Histopathology of nasal olfactory mucosa from selected inhalation toxicity studies conducted with volatile chemicals. Toxicol Pathol 27: 618-627.
Harkema JR, Hotchkiss JA, Barr EB, Bennett CB, Gallup M, Lee JK, and Basbaum C (1999) Long-lasting effects of chronic ozone exposure on rat nasal epithelium. Am J Respir Cell Mol Biol 20: 517-529.
Hubal EA, Schlosser PM, Conolly RB, and Kimbell JS (1997) Comparison of inhaled formaldehyde dosimetry predictions with DNA-protein cross-link measurements in the rat nasal passages. Toxicol Appl Pharmacol 143: 47-55.[CrossRef][Medline]
International Agency for Research on Cancer (IARC) (2002) Monographs on the evaluation of carcinogenic risks to humans: some traditional herbal medicines, some mycotoxins, NA and styrene. Vol 82, IARC, Lyon, France.
Jaeschke H and Wendel A (1985) Diurnal fluctuation and pharmacological alteration of mouse organ glutathione content. Biochem Pharmacol 34: 1029-1033.[CrossRef][Medline]
Kimbell JS, Godo MN, Gross EA, Joyner DR, Richardson RB, and Morgan KT (1997) Computer simulation of inspiratory airflow in all regions of the F344 rat nasal passages. Toxicol Appl Pharmacol 145: 388-398.[CrossRef][Medline]
Kimbell JS, Gross EA, Joyner DR, Godo MN, and Morgan KT (1993) Application of computational fluid dynamics to regional dosimetry of inhaled chemicals in the upper respiratory tract of the rat. Toxicol Appl Pharmacol 121: 253-263.[CrossRef][Medline]
Longo V, Pacifici GM, Panattoni G, Ursino F, and Gervasi PG (1989) Metabolism of diethylnitrosamine by microsomes of human respiratory nasal mucosa and liver. Biochem Pharmacol 38: 1867-1869.[CrossRef][Medline]
Mery S, Gross EA, Joyner DR, Godo N, and Morgan KT (1994) Nasal diagram: a tool for recording the distribution of nasal lesions in rats and mice. Toxicol Pathol 22: 353-372.
Moulin FJ, Brenneman KA, Kimbell JS, and Dorman DC (2002) Predicted regional flux of hydrogen sulfide correlates with distribution of nasal olfactory lesions in rats. Toxicol Sci 66: 7-15.
National Toxicology Program (NTP) (2000) Toxicology and carcinogenesis studies of NA (CAS no. 91-20-3) in F344/N rats (inhalation studies), in NTP Technical Report No. 500; National Institutes of Health Publication No. 01-4434, Research Triangle Park, NC.
Placke ME, Wyand DS, and Cohen SD (1987) Extrahepatic lesions induced by acetaminophen in the mouse. Toxicol Pathol 15: 381-387.[Medline]
Plopper CG, Suverkropp C, Morin D, Nishio S, and Buckpitt A (1992) Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after parenteral administration of NA. J Pharmacol Exp Ther 261: 353-363.
Schenkman JB and Jansson I (1999) Measurement of cytochrome P-450, in Current Protocols in Toxicology (Maines MD ed) pp 4.1.1-4.1.14, John Wiley & Sons, Hoboken, NJ.
Su T, Bao Z, Zhang QY, Smith TJ, Hong JY, and Ding X (2000) Human cytochrome P450 CYP2A13: predominant expression in the respiratory tract and its high efficiency metabolic activation of a tobacco-specific carcinogen, 4-(methylnitros-amino)-1-(3-pyridyl)-1-butanone. Cancer Res 60: 5074-5079.
Thornton-Manning JR and Dahl AR (1997) Metabolic capacity of nasal tissue inter-species comparisons of xenobiotic-metabolizing enzymes. Mutat Res 380: 43-59.[Medline]
Weiler E and Farbman AI (2003) The septal organ of the rat during postnatal development. Chem Senses 28: 581-593.
Young JT (1981) Histopathologic examination of the rat nasal cavity. Fundam Appl Toxicol 1: 309-312.[Medline]
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