Rat Nasal Epithelium Research Articles

Mucous cell metaplasia in rat nasal epithelium after a 20-month exposure to ozone: a morphometric study of epithelial differentiation.

The present study was designed to examine the effects of long-term ozone exposure on nasal epithelia and intraepithelial mucosubstances (IM) throughout the nasal airways of F344/N rats. Animals were exposed to 0 (controls), 0.12, 0.5, or 1.0 ppm ozone, 6 h/day, 5 days/wk, for 20 mo. Rats were killed 1 wk after the end of the exposure, and nasal tissues were processed for light and electron microscopy. Standard morphometric techniques were used to determine epithelial cell densities and the amounts of IM in the surface epithelium lining the nasal airways. No mucous cells or IM were present in the epithelia lining the nasal lateral meatus and maxillary sinus of rats exposed to 0 or 0.12 ppm ozone. In contrast, rats exposed to 0.5 or 1.0 ppm ozone had marked mucous cell metaplasia (MCM) with numerous mucous cells and conspicuous amounts of IM in the surface epithelium lining these upper airways. Ozone-induced increases in total epithelial cells (i.e., epithelial hyperplasia) were present only in rats exposed to 1.0 ppm. The results of this study indicate that rats chronically exposed to 1.0 or 0.5 ppm, but not 0.12 ppm, ozone can develop marked MCM with significant increases in IM in both proximal and distal nasal airways. The epithelial changes observed throughout the nasal passages of ozone-exposed rats may be adaptive responses in an attempt to protect the upper and lower respiratory tract from further ozone-induced injury.

Responses of rat nasal epithelium to short- and long-term exposures of ozone: image analysis of epithelial injury, adaptation and repair.

This article reviews the use of computerized image analysis and standard morphologic techniques to characterize the responses of nasal epithelium in laboratory rats to single or repeated exposures to a common urban air-pollutant, ozone. Alterations in the number and composition of the epithelial cell populations after either short- or long-term exposures are described. The principal nasal epithelial alteration induced by repeated exposures to this irritating, oxidant pollutant is mucous cell metaplasia (i.e., transformation of airway epithelium, normally devoid of mucous cells, to a secretory epithelium containing numerous mucus-secreting cells). This metaplastic change, induced by acute or chronic ozone exposures, has been morphometrically examined at various times post-exposure. In this article, we describe our current understanding of the pathogenesis and persistence of ozone-induced mucous cell metaplasia in nasal epithelium based on the results of these morphometric studies.

Confocal laser scanning microscopic visualization of the transport of dextrans after nasal administration to rats: effects of absorption enhancers.

To visualize the transport pathway(s) of high molecular weight model compounds across rat nasal epithelium in vivo using confocal laser scanning microscopy. Furthermore, the influence of nasal absorption enhancers (randomly methylated beta-cyclodextrin and sodium taurodihydrofusidate) on this transport was studied. Fluorescein isothiocyanate (FITC)-labelled dextrans with a molecular weight of 3,000 or 10,000 Da were administered intranasally to rats. Fifteen minutes after administration the tissue was fixed with Bouin. The nasal septum was surgically removed and stained with Evans Blue protein stain or DiIC18(5) lipid stain prior to visualization with the confocal laser scanning microscope. Transport of FITC-dextran 3,000 across nasal epithelium occurred via the paracellular pathway. Endocytosis of FITC-dextran 3,000 was also shown. In the presence of randomly methylated beta-cyclodextrin 2% (w/v) similar transport pathways for FITC-dextran 3,000 were observed. With sodium taurodihydrofusidate 1% (w/v) the transport route was also paracellular with endocytosis, but cells were swollen and mucus was extruded into the nasal cavity. For FITC-dextran 10,000 hardly any transport was observed without enhancer, or after co-administration with randomly methylated beta-cyclodextrin 2% (w/v). Co-administration with sodium taurodihydrofusidate 1% (w/v) resulted in paracellular transport of FITC-dextran 10,000, but morphological changes, i.e. swelling of cells and mucus extrusion, were observed. Confocal laser scanning microscopy is a suitable approach to visualize the transport pathways of high molecular weight hydrophilic compounds across nasal epithelium, and to study the effects of absorption enhancers on drug transport and cell morphology.

Acute histopathological effects of benzalkonium chloride and absorption enhancers on rat nasal epithelium in vivo

The aim of the study was to determine the acute effects of nasal absorption enhancers on the morphology of the rat nasal epithelium in vivo. Examples of three classes of enhancers were studied: the cyclodextrins dimethyl-β-cyclodextrin 2% (w/v) and randomly methylated-β-cyclodextrin 2% (w/v), the bile salt sodium glycocholate 1% (w/v), and the phospholipid L-α-lysophosphatidylcholine 1% (w/v). The preservative benzalkonium chloride 0.01% (w/v) was investigated for reasons of comparison. The compounds were dissolved in physiological saline (0.9% NaC1) and administered intranasally to anaesthetized rats. After 15 min the nasal cavity was fixated with Bouin and the tissue was processed for light microscopic examination. Morphological changes induced by physiological saline were graded as negligible or minor, and those of benzalkonium chloride as minor to major. The effects were mainly located in the respiratory epithelium. The effects of randomly methylated β-cyclodextrin and dimethyl-β-cyclodextrin were also minor and comparable to those of physiological saline. After administration of sodium glycocholate, large amounts of mucus were discharged from goblet cells and pyknosis occurred. l-α-lysophosphatidylcholine resulted in disruption of the epithelium, and damage was found not only in the respiratory epithelium but also in the olfactory epithelium. In conclusion, the acute effects of methylated β-cyclodextrins on the nasal epithelium morphology are relatively mild, and comparable to or less than those of the preservative benzalkonium chloride. Sodium glycocholate shows irreversible damage, and l-α-lysophosphatidylcholine is the most damaging absorption enhancer.

P 13 Effects of absorption enhancers on rat nasal epithelium in vivo: Release of marker compounds in the nasal cavity

P 13 Effects of absorption enhancers on rat nasal epithelium in vivo: Release of marker compounds in the nasal cavity

Hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium induced by endotoxin.

To elucidate the mechanisms of epithelial mucus hypersecretion in upper respiratory airway inflammation, we produced hypertrophic and metaplastic changes of goblet cells in rat nasal respiratory epithelium by intranasal instillation of endotoxin. Significant increase of hypertrophic goblet cells was induced in the septal epithelium transversely sectioned at the level of incisive papilla at 24 h after the intranasal instillation of 0.1 mg of endotoxin. This change was completed after 3 d of endotoxin instillations and recovered by normal epithelium 7 d after the last instillation. Total cell number and the number of basal and ciliated cells counted over 2 mm of basal lamina did not change; however, the number of goblet cells increased and that of nongranulated secretory cells decreased time-dependently after endotoxin instillations. Mitotic rates examined after a 6-h colchicine metaphase blockade were very low at any time point studied, and cell division did not play a major role in this process. These results indicate that endotoxin induces hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium rather than a hyperplastic change, and this metaplasia is produced by direct conversion of nongranulated secretory cells into the goblet cells. Histochemical examination of this epithelium revealed that most of the mucus produced by these goblet cells was sulfomucin. Intraperitoneal injection of antirat neutrophil antiserum or cyclophosphamide depleted circulating blood neutrophils. Endotoxin-induced changes of goblet cells were significantly inhibited in these neutrophil-depleted rats, and intranasal instillation of elastase also induced hypertrophic and metaplastic changes of goblet cells.

マクロライドと気道防御機能 マクロライドと粘液分泌

In the present study, we examined the in vivo and in vitro effects of erythromycin (EM) on epithelial mucus production. We produced hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium by the intranasal instillation of endotoxin. The in vivo effects of EM on epithelial mucus production were studied using this animal model. Intraperitoneal injection of EM (100 mg/kg body weight × 8 days), ampicillin (ABPC, 200 mg/kg body weight × 8 days), and cefalotin (CET, 200 mg/kg body weight × 8 days) inhibited epithelial mucus production induced after 7 days of endotoxin instillation. EM had a greater inhibitory effect than ABPC or CET.The in vivo effects of EM on epithelial mucus secretion were studied using cultured human nasal epithelial cells. Mucus secretion was quantitatively measured by the monoclonal antibody HCS18, which specifically reacts with mucin-like materials in human epithelial goblet cells. The spontaneous mucus secretion from cultured epithelial cells did not change after incubation with 10-4 to 10-6 M EM, 10-4 M ABPC, or 10-4 M CET.These results indicate that EM has a greater inhibitory effect on endotoxin-induced epithelial mucus production than ABPC or CET, and that this effect is not caused by the direct action of EM on epithelial secretory cells.

Changes in the Nasal Epithelium of Rats Exposed by Inhalation to Mixtures of Formaldehyde, Acetaldehyde, and Acrolein

Changes in the Nasal Epithelium of Rats Exposed by Inhalation to Mixtures of Formaldehyde, Acetaldehyde, and Acrolein

Cytochrome P450 2A of nasal epithelium: regulation and role in carcinogen metabolism.

In this study, we found that rat nasal coumarin-7-hydroxylase (COH) activity was two orders of magnitude higher than rat hepatic COH activity and could be induced by adding coumarin to the rats' drinking water. In western blot analysis, an anti-cytochrome P450 (Cyp) 2a-5 (mouse liver COH) antibody recognized a sharp band in the microsomal fraction of rat nasal epithelium but not of the liver; the band comigrated with Cyp2a-5. The intensity of the band was increased by the coumarin treatment. Similarly, in northern blot analysis, a cDNA probe specific for Cyp2a-5 recognized an mRNA in the nasal epithelium having the same size as mouse liver Cyp2a-5 mRNA; however, no hybridizable mRNA was recognized in liver preparations. Unlike the protein level, the level of the mRNA was not increased by coumarin. When northern blot analyses were performed with two oligoprobes specific for rat lung CYP2A3, an mRNA of similar size to Cyp2a-5 mRNA was recognized. In immunoinhibition analysis, anti-Cyp2a-5 antibody inhibited rat nasal COH activity and aflatoxin B1 (AFB1) metabolism completely. It inhibited N-nitrosodiethylamine (NDEA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism by 80-90%. In contrast, the hepatic metabolism of the four compounds was not affected by the antibody. When coumarin instead of anti-Cyp2a-5 antibody was used, a strong but variable inhibition of the nasal metabolism of AFB1, NDEA, and NNK was seen. The results suggest that an enzyme or enzymes similar to mouse liver Cyp2a-5, one of which may be CYP2A3, is expressed at high levels in rat nasal epithelium but not in the liver and that its expression is increased by coumarin, an odorant and a substrate of Cyp2a-5. The increase probably occurs by protein stabilization or stimulation of translation. The results also show that the enzyme has a key role in the nasal metabolism of three well-known carcinogens, AFB1, NDEA, and NNK and may therefore be an important contributing factor in nasal carcinogenesis.

Effects of Propylene Oxide on Nasal Epithelial Cell Proliferation in F344 Rats

Effects of Propylene Oxide on Nasal Epithelial Cell Proliferation in F344 Rats. Eldridge, S. R., Bogdanffy, M. S., Jokinen, M.P., and Andrews, L. S. (1995). Fundam. Appl. Toxicol. 27, 25-32. In chronic inhalation studies, propylene oxide (PO), widely used in the chemical and food industries, induced nasal tumors in F344 rats. Nonneoplastic findings of the chronic studies suggest a strong cytotoxic and proliferative component in the mechanism of PO carcinogenicity. A 4-week cell proliferation study was conducted to establish a no-observed-adverse-effect level (NOAEL) for nonneoplastic changes in the nasal epithelium of rats. Male F344 rats were exposed to 0, 10, 20, 50, 150, or 525 ppm PO vapor for up to 4 weeks with up to 4 weeks of recovery. Histopathology showed that the incidence and severity of respiratory epithelial hyperplasia increased with exposure time and regressed after termination of exposure with complete recovery after 4 weeks. Similarly, cell proliferation, as determined by bromodeoxyuridine incorporation into replicating cells, was elevated following 1 and 4 weeks of exposure, but decreased to control values after 1 week of recovery. Degeneration of the olfactory epithelium was found after 4 weeks of exposure with a decrease in incidence and severity after termination of exposure. Cell proliferation at this site was elevated during the 4-week exposure period and 1 week postexposure with return to control values after 4 weeks of recovery. Based on the cytotoxic and proliferative findings, the NOAEL for PO in nasal epithelium is 50 ppm.

Regional Differences in the Effects of Mainstream Cigarette Smoke on Stored Mucosubstances and DNA Synthesis in F344 Rat Nasal Respiratory Epithelium

This study was designed to determine the effects of mainstream cigarette smoke (MCS) on stored intraepithelial mucosubstances (IM) and DNA synthesis within the nasal respiratory epithelium of F344 rats and whether such effects persist after cessation of exposure. Rats were exposed to filtered air or diluted MCS for 9 days over a 2-week period. Two hours prior to termination rats were injected with bromodeoxyuridine (BrdU) to label cells synthesizing DNA 1 or 14 days after the last exposure. Sections of nasal tissue blocks taken from a region immediately posterior to the incisor teeth were processed for light microscopy and histochemically stained to detect acidic and neutral IM and immunochemically stained to detect BrdU-labeled cells. The nasal septum was divided into three measurement zones (dorsal, mid-, and ventral) for morphometric quantitation of the volume density of IM and the unit length labeling index (ULLI). MCS-exposed rats terminated 1 day after the last exposure had 270% more IM in the dorsal septum, 58% less IM in the midseptum (due to regions of squamous metaplasia), and amounts of IM in the ventral septum similar to controls. MCS-exposed rats sacrificed 14 days after exposure still had increased amounts of IM in the dorsal septal region, but no regions of squamous metaplasia or amounts of IM in the mid- and ventral septal regions that were different from air-exposed controls. MCS exposure resulted in a significant increase in the ULLI 1 day but not 14 days after exposure in the ventral and midseptal regions only. The results of this study indicate that MCS exposure induces transient alterations in the mucous-producing apparatus in the rat anterior nasal cavity that are resolved following 2 weeks of recovery. However, the type and magnitude of the initial epithelial responses are dependent on the intranasal location of the airway epithelium examined.

Effects of absorption enhancers on rat nasal epithelium in vivo: release of marker compounds in the nasal cavity.

The assessment of the effects of nasal absorption enhancers on the rat nasal epithelium and membrane permeability in vivo after a single nasal dose of the enhancers. The release of marker compounds (protein, cholesterol and acid phosphatase) from the nasal epithelium was measured using a lavage technique. The nasal membrane permeability was determined after intravenous administration of a systemic tracer (FITC-albumin). The effects of the absorption enhancers could be classified into four categories. The first consisted of HP beta CD (5%), DM beta CD (2%) and RAMEB (2%) and was not different from the control (physiological saline). For the second category, DM beta CD (5%), effects were significantly higher than for the control. The third category, SGC (1%), was more active than DM beta CD (5%) but less active than the last group. The fourth, most membrane damaging, category consisted of STDHF (1%), laureth-9 (1%) and LPC (1%). Administration of these three enhancers also resulted in release of acid phosphatase, indicating that severe membrane damage occurred. The release of cholesterol from nasal epithelium was largely dependent on the cholesterol solubilisation of the absorption enhancers. The amount of cholesterol released by laureth-9 and LPC was the largest. The results of this in vivo study are in agreement (i.e. similarity in rank order) with morphological and ciliotoxicity studies of nasal absorption enhancers, demonstrating that this in vivo model is a valuable tool to classify nasal absorption enhancers according to their effects on the rat nasal epithelium.

Strain-Related Differences in Ozone-Induced Secretory Metaplasia in The Nasal Epithelium of Rats

Strain-Related Differences in Ozone-Induced Secretory Metaplasia in The Nasal Epithelium of Rats

Effect of Type of Fixative on Labeling Rate of Rat Nasal Epithelium with a Monoclonal Antibody to Proliferating Cell Nuclear Antigen

Effect of Type of Fixative on Labeling Rate of Rat Nasal Epithelium with a Monoclonal Antibody to Proliferating Cell Nuclear Antigen

Biochemical and histopathological changes in nasal epithelium of rats after 3-day intermittent exposure to formaldehyde and ozone alone or in combination

To get a better insight into the pathophysiology of the nasal changes induced by formaldehyde-ozone mixtures, a 3-day inhalation study was carried out in rats, using intermittent exposure to formaldehyde (3.6 ppm) and ozone (0.4 ppm) alone or in combination and focusing on biochemical and histopathological changes in rat nasal respiratory epithelium. Formaldehyde dehydrogenase, glutathione S-transferase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities in this epithelium were not affected by the individual compounds. However, combined exposure to formaldehyde and ozone resulted in slightly decreased activities of these enzymes. Formaldehyde was found to induce rhinitis, degeneration, frank necrosis, hyperplasia and squamous metaplasia of the ciliated and non-ciliated nasal respiratory epithelium, while ozone induced disarrangement, flattening and slight basal cell hyperplasia of the non-ciliated cuboidal epithelium accompanied by influx of neutrophils. Proliferating cell nuclear antigen (PCNA) expression was elevated not only in nasal areas showing ozone-induced histopathological changes but also in the otherwise normal-appearing epithelium of the nasal septum. No interactive effects were found with respect to proliferative response of the nasal respiratory epithelium after exposure to the formaldehyde-ozone mixture. The present study did not provide evidence of a major role of glutathione and glutathione-dependent enzymes in the pathogenesis of nasal lesions induced by formaldehyde and/or ozone, demonstrated the potential of ozone to affect the mucociliary epithelium lining the nasal septum, and suggested that PCNA expression is a sensitive tool for detection of early effects of respiratory irritants.

Cyclodextrins as protection agents against enhancer damage in nasal delivery systems II. Effect on in vivo absorption of insulin and histopathology of nasal membrane

An in vivo rat model was used to study the nasal absorption of insulin in the presence of selected enhancers (Laureth 9, glycodeoxycholate and L-α-lysophosphatidylcholine) either alone or in combination with 2-hydroxypropyl-β-cyclodextrin or γ-cyclodextrin. All the enhancers when administered alone with insulin produced about 50% decrease in the blood glucose concentrations, an indirect measure of the absorption of insulin across the rat nasal mucosa. In the presence of cyclodextrins the enhancing effect of L9 was maintained, whereas that of GDC and LPC was considerably reduced, but the duration of action of insulin was prolonged. Concomitantly, the histological effect of these agents on the rat nasal epithelium was studied using a perfusion fixation technique. The absorption of insulin did not consistently correlate with the histological observations and the results obtained in previous haemolysis studies. However, the histological and haemolysis observations complemented each other in that the formulations [L9:HPβC (1:4), GDC:γ-CD (1:2) and LPC:HPβC (1:12)] which caused the least damage to the spithelial membrane had been shown to completely prevent haemolysis. In conclusion, the combination of L9 and possibly LPC with cyclodextrins may provide formulations which have almost the required balance between activity and safety, for nasal delivery of insulin and could possibly be used as an adjunct to subcutaneous therapy.

The potential of cyclodextrins as absorption enhancers in nasal delivery of peptide drugs

The potential of cyclodextrins, in particular dimethyl-β-cyclodextrin (DMβCD), as absorption enhancers was investigated for the nasal absorption of two peptide drugs: an ACTH (4–9) hexapeptide analogue and the polypeptide hormone, insulin. Studies in rats showed that DMβCD largely improved the absorption of both peptide drugs, resulting in bioavailabilities of 70 to 100%. The effective DMβCD concentration was in the order of 2% (w/v), and the increased permeability of the rat nasal epithelium as caused by DMβCD returned to normal physiological conditions in time. On the contrary, insulin/DMβCD solutions were ineffective in enhancing nasal insulin absorption in rabbits and men, whereas a small absorption enhancement was observed for the small ACTH (4–9) analogue in rabbits. Thus substantial interspecies differences appear to exist in the absorption of peptide drugs with DMβCD as enhancer. Nonetheless, studies in rabbits demonstrated that insulin/DMβCD powder formulations were much more effective than liquid preparations. The nasal insulin bioavailability was in the order of 13% for the insulin/DMβCD powder, and negligible for both a control insulin/lactose powder and insulin/DMβCD liquid formulations. In conclusion, the cyclodextrin derivative DMβCD is a potent enhancer of nasal peptide absorption. The efficacy of DMβCD, however, differs largely between species and is also greatly dependent upon the nasal dosage form.

In vivo effects of endotoxin on DNA synthesis in rat nasal epithelium.

Airway inflammation in bacterial infections is characterized by the presence of neutrophils and often epithelial injury and repair. Release of endotoxin from bacteria may contribute to these processes. The purpose of this study was to determine the in vivo effects of repeated endotoxin exposure on DNA synthesis in rat nasal epithelium in the presence and absence of neutrophilic influx. Rats were intranasally instilled, once a day for 3 days, with endotoxin or saline (controls). Before the first and third instillations, half of the saline and endotoxin-instilled animals were depleted of circulating blood neutrophils by administering a rabbit anti-rat neutrophil antiserum. Rats were sacrificed 6 or 24 h after the last instillation. Two hours prior to sacrifice, rats were intraperitoneally injected with bromodeoxyuridine (BrdU), an analog of thymidine that is incorporated in the nucleus of cells in the S-phase of the cell cycle. Nasal tissues were processed for light microscopy and immunohistochemical detection of BrdU in nasal epithelial cells. The numbers of nasal epithelial cells, BrdU-labeled epithelial nuclei, and neutrophils per millimeter of basal lamina in the epithelium lining the nasal turbinates in the proximal nasal passages were determined by morphometric analysis. We did not observe a neutrophilic influx in the nasal tissues of neutrophil-depleted rats at 6 or 24 h after the last endotoxin instillation; however, the numbers of nasal epithelial cells and the BrdU-labeling index were significantly increased compared to saline-instilled controls.(ABSTRACT TRUNCATED AT 250 WORDS)

An approach to mechanism-based cancer risk assessment for formaldehyde.

The established carcinogenicity of formaldehyde in the rat and suggestive epidemiological evidence that formaldehyde may be a human carcinogen have led to its regulation by U.S. Federal agencies as a probable human carcinogen. These risk assessments have typically been based on tumor data in F344 rats exposed chronically to formaldehyde by inhalation and used the inhaled concentration as a measure of dose and the linearized multistage model (LMS) for dose-response characterization. Low-dose risks estimated with the LMS are thought to be conservative but are also generally acknowledged to be highly uncertain. In this manuscript, we first consider in generic terms how use of chemical-specific data on mechanisms of target tissue dosimetry and the series of tissue responses to the chemical that culminate in tumor formation can lead to more accurate dose-response characterization. A planned mechanism-based risk assessment for formaldehyde is then described. This risk assessment uses data on target tissue dosimetry, size of the target cell population in the rat nasal epithelium, number and size of putative preneoplastic lesions, and tumor incidence. These data establish parameter values for a biologically based, multistage cancer model that is then used to predict cancer risk at low exposure levels. Such work provides insights into the relative roles of formaldehyde-stimulated cell replication and procarcinogenic mutation in tumor formation. Finally, future directions are outlined for research on tissue dosimetry and scaling of the mechanism-based formaldehyde risk model from rats to people.

Genotoxic effects of subacute treatments with wood dust extracts on the nasal epithelium of rats: assessment by the micronucleus and 32P-postlabelling.

Human exposure to wood dust has been epidemiologically linked to a number of enhanced incidences of various neoplasias, including those of the nose. Among different suspected woods, long-term exposure to the dust of beech (Fagus silvatica) is strongly associated with development of these tumors. Experimentally, it has been shown that a simple alcoholic extract of beech wood dust is mutagenic toward some bacteria and tumorigenic on mouse skin. For the present study, different groups of male Wistar rats were treated with aqueous, ethanol, or methanol extract of untreated beech wood dust via nasal drip at three concentrations for 48 h. Animals were killed 24 h after the last treatment and the nasal tissues were isolated to be examined for possible induction of micronuclei and DNA adducts. A clear dose-dependent increase in the number of micronuclei (P < 0.01) was observed after treatment of rats with each alcoholic extract of wood dust. In contrast, no DNA adducts could be detected with these extracts using the 32P-postlabelling technique. No increased number of micronucleated cells was found with solvents alone or with aqueous extract of dust. These data might support the early hypothesis that wood dust per se contains some in vivo genotoxic and thus possibly carcinogenic components extractable by an alcohol.