Age-, gender-, and species-dependent mutagenicity in T cells of mice and rats exposed by inhalation to 1,3-butadiene

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.     

Analysis of micronuclei, histopathological changes and cell proliferation in nasal epithelium cells of rats after exposure to formaldehyde by inhalation

The frequencies of micronuclei (MN), histopathological changes and cell proliferation were determined in the nasal epithelium of male Fischer-344 rats after exposure to formaldehyde (FA) by whole-body inhalation for four weeks (6h/day, 5 days/week). Groups of 12 rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15ppm. The micronucleus test (MNT) was performed with nasal epithelial cells prepared from six animals per group. Two thousand cells per animal were analysed for the presence of MN. The other six rats per group were subcutaneously implanted with osmotic pumps containing 5-bromo-2'-deoxyuridine (BrdUrd), three days prior to necropsy. Paraffin sections were made from the nasal cavity (four levels) of these animals for histopathology and cell-proliferation measurements. The frequency of cells with MN was not increased in any of the groups. However, there was also no induction of MN in nasal cells of rats exposed to a single dose of cyclophosphamide (CP, 20mg/kg) by gavage and analysed 3, 7, 14 or 28 days after the treatment. In contrast, nasal epithelial cells from rats exposed to 10 or 15ppm FA vapour showed clear site-specific pathological changes (focal epithelial degeneration, inflammation and squamous metaplasia) in a decreasing gradient (anterior to posterior). Analysis of slides after anti-BrdUrd antibody staining clearly indicated increased cell proliferation (unit length labelling indices, ULLI) after exposure to 6ppm and higher. No treatment-related effects were measured after exposure to 0.5, 1 and 2ppm. When comparing the cell-proliferation rate of normal epithelium with that of directly adjacent metaplastic epithelium, no consistent pattern was found: depending on the location, cell proliferation of normal epithelia was either higher or lower. Our results support previous findings demonstrating local cytotoxic effects in the nose of rats after inhalation of FA. However, induction of MN in the nasal epithelium as an indicator of a mutagenic effect was not seen. Because only limited experience exists for the MNT with rat nasal epithelial cells, this result has to be interpreted with great care. The contribution of mutagenicity to FA's carcinogenicity in rat nasal epithelium remains unclear.     

Effects of Localized Hydrophilic Mannitol and Hydrophobic Nelfinavir Administration Targeted to Olfactory Epithelium on Brain Distribution

Many nasally applied compounds gain access to the brain and the central nervous system (CNS) with varying degree. Direct nose-to-brain access is believed to be achieved through nervous connections which travel from the CNS across the cribriform plate into the olfactory region of the nasal cavity. However, current delivery strategies are not targeted to preferentially deposit drugs to the olfactory at cribriform. Therefore, we have developed a pressurized olfactory delivery (POD) device which consistently and non-invasively deposited a majority of drug to the olfactory region of the nasal cavity in rats. Using both a hydrophobic drug, mannitol (log P = -3.1), and a hydrophobic drug, nelfinavir (log P = 6.0), and POD device, we compared brain and blood levels after nasal deposition primarily on the olfactory region with POD or nose drops which deposited primarily on the respiratory region in rats. POD administration of mannitol in rats provided a 3.6-fold (p < 0.05) increase in cortex-to-blood ratio, compared to respiratory epithelium deposition with nose drop. Administration of nelfinavir provided a 13.6-fold (p < 0.05) advantage in cortex-to-blood ratio with POD administration, compared to nose drops. These results suggest that increasing the fraction of drug deposited on the olfactory region of the nasal cavity will result in increased direct nose-to-brain transport.     

Mitogenic responses of rat nasal epithelium to hexamethylphosphoramide inhalation exposure

Hexamethylphosphoramide (HMPA) is a rat nasal carcinogen that induces squamous cell carcinomas in the anterior portions of the nasal cavity following chronic inhalation exposures as low as 50 ppb. These tumors may arise as a result of P-450-mediated release of formaldehyde (HCHO), a known rat nasal carcinogen. The goal of this research was to investigate early responses of the nasal epithelium to inhaled HMPA. Rats were exposed nose-only to approximately 3 ppm HMPA for 6 h, and killed 18, 48, 96 or 144 h post-exposure. In a separate study, rats were exposed nose-only for 6 h for 1, 2, 3, or 5 consecutive days and killed 18 or 96 h post-exposure. With both single and repeated doses of HMPA, there was no evidence of cytotoxicity in the anterior nose. Olfactory degeneration and necrosis of the dorsal meatus, Bowman's glands and tips of the ethmoid turbinates increased in severity with repeated exposures to HMPA. Cell proliferation was assessed in levels of nasal tissue that included regions of squamous, respiratory, transitional and olfactory epithelium. Regional induction of cell proliferation was measured by BrdU incorporation, and reported as the number of labeled cells/mm basement membrane. At 18 h after a single exposure, there was an increase in cell proliferation in squamous epithelium, which returned to control levels within 48 h. A transitory increase in cell proliferation was observed regions of respiratory and transitional epithelium, although the response of each tissue, in terms of magnitude and peak time of response post-exposure, also differed. Along the dorsal meatus in Level 9, olfactory labeling initially decreased, returned to control levels by 96 h, but again declined at 144 h post-exposure. In repeat dose studies, the squamous epithelium response was variable 18 h post-exposure. For respiratory and transitional epithelium, increased cell proliferation 18 h post-exposure was correlated with increased dose (exposure) of HMPA. Cell proliferation responses following two or more exposures returned to near control levels within 96 h post-exposure. In conclusion, HMPA induced cell proliferation, but not cytotoxicity, in the anterior nose at approximately 3 ppm. These data suggest that HMPA induces proliferative, perhaps mitogenic, responses in the nasal epithelium, and this response may facilitate the fixation of low level genetic damage induced by liberated HCHO.     

Alterations in the proteome of the respiratory tract in response to single and multiple exposures to naphthalene

Protein adduction is considered to be critical to the loss of cellular homeostasis associated with environmental chemicals undergoing metabolic activation. Despite considerable effort, our understanding of the key proteins mediating the pathologic consequences from protein modification by electrophiles is incomplete. This work focused on naphthalene (NA) induced acute injury of respiratory epithelial cells and tolerance which arises after multiple toxicant doses to define the initial cellular proteomic response and later protective actions related to tolerance. Airways and nasal olfactory epithelium from mice exposed to 15 ppm NA either for 4 h (acute) or for 4 h/day × 7 days (tolerant) were used for label‐free protein quantitation by LC/MS/MS. Cytochrome P450 2F2 and secretoglobin 1A1 are decreased dramatically in airways of mice exposed for 4 h, a finding consistent with the fact that CYPs are localized primarily in Clara cells. A number of heat shock proteins and protein disulfide isomerases, which had previously been identified as adduct targets for reactive metabolites from several lung toxicants, were upregulated in airways but not olfactory epithelium of tolerant mice. Protein targets that are upregulated in tolerance may be key players in the pathophysiology associated with reactive metabolite protein adduction. All MS data have been deposited in the ProteomeXchange with identifier PXD000846 ( http://proteomecentral.proteomexchange.org/dataset/PXD000846 ).     

扬子鳄鼻腔上皮光学显微镜及扫描电镜观察

本文通过光镜和扫描电镜研究了爬行动物扬子鳄鼻腔上皮的组织学。结果表明:其嗅觉上皮的组成细胞类型与两栖类、鸟类和哺乳类基本相似,但嗅细胞纤毛形状则有所不同;扬子鳄与两栖类、鸟类嗅纤毛相似,呈丝状,而哺乳类嗅觉纤毛则呈棍棒状;据外,扬子鳄鼻腔不同部位可发现不同类型嗅纤毛,鸟兽则无此现象,扬子鳄嗅觉上皮的分布仅局限于鼻腔中部前甲区和鼻甲区狭小范围,而兽类嗅觉上皮一般分布较广;扬子鳄呼吸上皮下未见兽类具有的混合型粘液腺,也未见兽类用以温暖空气的静脉丛,这和扬子鳄属外温动物而兽类为恒温动物密切相关。     

The toxicity of styrene to the nasal epithelium of mice and rats: studies on the mode of action and relevance to humans

Inhaled styrene is known to be toxic to the nasal olfactory epithelium of both mice and rats, although mice are markedly more sensitive. In this study, the nasal tissues of mice exposed to 40 and 160 ppm styrene 6 h/day for 3 days had a number of degenerative changes including atrophy of the olfactory mucosa and loss of normal cellular organisation. Pretreatment of mice with 5-phenyl-1-pentyne, an inhibitor of both CYP2F2 and CYP2E1 completely prevented the development of a nasal lesion on exposure to styrene establishing that a metabolite of styrene, probably styrene oxide, is responsible for the observed nasal toxicity. Comparisons of the cytochrome P-450 mediated metabolism of styrene to its oxide, and subsequent metabolism of the oxide by epoxide hydrolases and glutathione S-transferases in nasal tissues in vitro, have provided an explanation for the increased sensitivity of the mouse to styrene. Whereas cytochrome P-450 metabolism of styrene is similar in rats and mice, the rat is able to metabolise styrene oxide at higher rates than the mouse thus rapidly detoxifying this electrophilic metabolite. Metabolism of styrene to its oxide could not be detected in human nasal tissues in vitro, but the same tissues did have epoxide hydrolase and glutathione S-transferase activities, and were able to metabolise styrene oxide efficiently, indicating that styrene is unlikely to be toxic to the human nasal epithelium.     

Identification and biochemical analysis of novel olfactory-specific cytochrome P-450IIA and UDP-glucuronosyl transferase

Two major transmembranal polypeptides of bovine olfactory epithelium were identified by SDS electrophoretic analysis of Triton X-114 solubilized membranes. Both polypeptides were present in large amounts in membranes of the olfactory epithelium but were barely detectable in membranes of the nasal respiratory epithelium. Both polypeptides are enriched in the deciliated epithelium as compared with isolated cilia. One of them is a glycoprotein with an apparent molecular mass of 56 kDa (gp56); the other is an unglycosylated protein with an apparent molecular mass of 52 kDa (p52). Sequence analysis of peptides obtained by CNBr cleavage of purified gp56 indicates that it is highly homologous to UDP-glucuronosyl transferase (UDPGT). Parallel analysis shows that p52 is highly homologous to cytochrome P-450 sequences of the IIA subfamily. This protein is assigned the name P-450olf2. Polyclonal antibodies were raised against synthetic peptides corresponding to gp56 and p52 peptide sequences. Immunoblots with these antibodies reveal the following properties of gp56 and p52: (1) they are enriched in the microsomal fraction of the bovine olfactory epithelium; (2) they are possibly specific to the olfactory epithelium, as we could not detect reactivity in microsomes derived from respiratory epithelium or lung, and only a very small amount of basal reactivity was seen with liver microsomes; (3) cross-reacting proteins exist in microsomes derived from the rat olfactory epithelium. These results are consistent with a mechanism whereby the microsomal enzymes are involved in odorant modification and clearance from the nasal tissue.     

Measurement of plasma or urinary metabolites and Hprt mutant frequencies following inhalation exposure of mice and rats to 3-butene-1,2-diol

Studies were performed to determine if the detoxification pathway of 1,3-butadiene (BD) through 3-butene-1,2-diol (BD-diol) is a major contributor to mutagenicity in BD-exposed mice and rats. First, female and male mice and rats (4-5 weeks old) were exposed by nose-only for 6h to 0, 62.5, 200, 625, or 1250 ppm BD or to 0, 6, 18, 24, or 36 ppm BD-diol primarily to establish BD and BD-diol exposure concentrations that yielded similar plasma levels of BD-diol, and then animals were exposed in inhalation chambers for 4 weeks to BD-diol to determine the mutagenic potency estimates for the same exposure levels and to compare these estimates to those reported for BD-exposed female mice and rats where comparable blood levels of BD-diol were achieved. Measurements of plasma levels of BD-diol (via GC/MS methodology) showed that (i) BD-diol accumulated in a sub-linear fashion during single 6-h exposures to >200 ppm BD; (ii) BD-diol accumulated in a linear fashion during single or repeated exposures to 6-18 ppm BD and then in a sub-linear fashion with increasing levels of BD-diol exposure; and (iii) exposures of mice and rats to 18 ppm BD-diol were equivalent to those produced by 200 ppm BD exposures (with exposures to 36 ppm BD-diol yielding plasma levels approximately 25% of those produced by 625 ppm BD exposures). Measurements of Hprt mutant frequencies (via the T cell cloning assay) showed that repeated exposures to 18 and 36 ppm BD-diol were significantly mutagenic in mice and rats. The resulting data indicated that BD-diol derived metabolites (especially, 1,2-dihydroxy-3,4-epoxybutane) have a narrow range of mutagenic effects confined to high-level BD (>or=200 ppm) exposures, and are responsible for nearly all of the mutagenic response in the rat and for a substantial portion of the mutagenic response in the mouse following high-level BD exposures.     

Measurement of HPRT mutations in splenic lymphocytes and haemoglobin adducts in erythrocytes of Lewis rats exposed to ethylene oxide

Young adult male Lewis rats were exposed to ethylene oxide (EO) via single intraperitoneal (i.p.) injections (10-80 mg kg-1) or drinking water (4 weeks at concentrations of 2, 5, and 10 mM) or inhalation (50, 100 or 200 ppm for 4 weeks, 5 days week-1, 6 h day-1) to measure induction of HPRT mutations in lymphocytes from spleen by means of a cloning assay. N-ethyl-N-nitrosourea (ENU) and N-(2-hydroxyethyl)-N-nitrosourea (HOENU) were used as positive controls. Levels of N-(2-hydroxyethyl)valine (HOEtVal) adducts in haemoglobin (expressed in nmol g-1 globin) were measured to determine blood doses of EO (mmol kg-1 h, mM h). Blood doses were used as a common denominator for comparison of mutagenic effects of EO administered via the three routes. The mean HPRT mutant frequency (MF) of the historical control was 4.3 x 10(-6). Maximal mean MFs for ENU (100 mg kg-1) and HOENU (75 mg kg-1) were 243 x 10(-6) and 93 x 10(-6), respectively. In two independent experiments, EO injections led to a statistically significant dose-dependent induction of mutations, with a maximal increase in MF by 2.3-fold over the background. Administration of EO via drinking water gave statistically significant increases of MFs in two independent experiments. Effects were, at most, 2.5-fold above the concurrent control. Finally, inhalation exposure also caused a statistically significant maximal increase in MF by 1.4-fold over the background. Plotting of mutagenicity data (i.e., selected data pertaining to expression times where maximal mutagenic effects were found) for the three exposure routes against blood dose as common denominator indicated that, at equal blood doses, acute i.p. exposure led to higher observed MFs than drinking water treatment, which was more mutagenic than exposure via inhalation. In the injection experiments, there was evidence for a saturation of detoxification processes at the highest doses. This was not seen after subchronic administration of EO. The resulting HPRT mutagenicity data suggest that EO is a relatively weak mutagen in T-lymphocytes of rats following exposure(s) by i.p. injection, in drinking water or by inhalation.     

Binding of the pheromonal steroid, 5alpha-androst-16-en-3-one, to membrane-enriched fractions of boar and rat olfactory epithelium; preliminary evidence for binding protein being a glycoprotein

A high degree of binding of 5alpha-[3H]-androstenone was recorded in membrane-enriched fractions of porcine olfactory tissue. The specific (i.e. high affinity, low capacity) binding had a mean Ka approximately 2x10(8)M(-1). A Hill plot of the data showed a Hill coefficient of approximately 2, possibly suggesting co-operativity of binding, with binding constants increasing from 8x10(7) to 1.6x10(9)M(-1) with increasing substrate concentration. The level of specific binding of 5alpha-[3H]-androstenone was nearly 10-fold higher than in corresponding respiratory tissue preparations and was markedly reduced in the presence of excess (approximately 1 microM) unlabelled 5alpha-androstenone. Corresponding fractions derived from rat olfactory tissue showed only 25% of the binding recorded for the pig. After incubation of 5alpha-[3H]-androstenone with solubilised olfactory cilial tissue (porcine), gel filtration and chromatography on a typical "glycoprotein" column (Concanavalin A-Sepharose B) were performed. Specific binding was recorded only in fractions corresponding to glycoproteins with Mr of approximately 70-90 kDa. In a third series of experiments, fractions containing high concentrations of cilia, some still attached to the dendritic endings (as shown by electron microscopy) were obtained by a novel method involving stripping them off the nasal epithelium. The basal adenylate cyclase (AC) activity was very significantly (P<0.01) higher in olfactory, compared with respiratory, cilia; storage at -70 degrees C for 3 weeks greatly reduced AC activity. When fresh male and female porcine olfactory cilia preparations were incubated with 5alpha-androstenone plus GTP, AC activity was increased fourfold (P<0.01). However, responses of porcine respiratory cilia were not significant statistically, neither were changes in basal levels of AC activities in rat olfactory cilia.     

Propylene oxide: mutagenesis, carcinogenesis and molecular dose

The results from mutagenic and carcinogenic studies of propylene oxide (PO) and the current efforts to develop molecular dosimetry methods for PO–DNA adducts are reviewed. PO has been shown to be active in several bacterial and mammalian mutagenicity tests and induces site of contact tumors in rodents after long-term administration. Quantitation of N7-(2-hydroxypropyl)guanine (7-HPG) in nasal and hepatic tissues of male F344 rats exposed to 500 ppm PO (6 h/day; 5 days/week for 4 weeks) by inhalation was performed to evaluate the potential of high concentrations of PO to produce adducts in the DNA of rodent tissues and to obtain information necessary for the design of molecular dosimetry studies. The persistence of 7-HPG in nasal and hepatic tissues was studied in rats killed three days after cessation of a 4-week exposure period. DNA samples from exposed and untreated animals were analyzed for 7-HPG by two different methods. The first method consisted of separation of the adduct from DNA by neutral thermal hydrolysis, followed by electrophoretic derivatization of the adduct and gas chromatography-high resolution mass spectrometry (GC–HRMS) analysis. The second method utilized ³²P-postlabeling to quantitate the amount of this adduct in rat tissues. Adducts present in tissues from rats killed immediately after cessation of exposure were 835.4±80.1 (respiratory), 396.8±53.1 (olfactory) and 34.6±3.0 (liver) pmol adduct/μmol guanine using GC–HRMS. Lower values, 592.7±53.3, 296.5±32.6 and 23.2±0.6 pmol adduct/μmol guanine were found in respiratory, olfactory and hepatic tissues of rats killed after three days of recovery. Analysis of the tissues by ³²P-postlabeling yielded the following values: 445.7±8.0 (respiratory), 301.6±49.2 (olfactory) and 20.6±1.8 (liver) pmol adduct/μmol guanine in DNA of rats killed immediately after exposure cessation and 327.1±21.7 (respiratory), 185.3±29.2 (olfactory) and 15.7±0.9 (liver) pmol adduct/μmol guanine after recovery. Current methods of quantitation did not provide evidence for the endogenous formation of this adduct in control animals. These studies demonstrated that the target tissue for carcinogenesis has much greater alkylation of DNA than liver, a tissue that did not exhibit a carcinogenic response.     

Distribution of epithelia and glands of the nasal septum mucosa in the rat.

A histotopographic study of the nasal septum mucosa in rats was made using semi-serial sections stained with PAS-hematoxylin, reconstructed in form of maps representing the structure in a sagittal plane. The stratified squamous, respiratory and olfactory epithelia and Masera's organ cover 14.8, 43.6, 41.6 and 1.8%, respectively, of the septal surface (117.1 mm2). In the vestibular region, only ducts of PAS-negative glands of the respiratory region are found, and below the septum there is the infraseptal gland with PAS-negative acini. In the respiratory region, PAS-negative acinous glands form two groups: the superior and the inferior one occupying 10.5 and 1.5%, respectively, of the septal area. PAS-positive acinous glands are in the inferior half of the respiratory region and in a small anteroinferior portion of the olfactory region. Besides goblet cells broadly distributed, the respiratory epithelium presents scattered intraepithelial PAS-positive glands which are concentrated in the anterior portion and close to the nasopharyngeal duct. In the olfactory region prevail Bowman's PAS-positive glands which are also present in the mucosa of Masera's organ, but are not seen in the olfactory mucosa of Jacobson's organ. In the latter, PAS-positive glands are found in the respiratory mucosa. Globular leukocytes, cells of connective tissue origin, are constantly infiltrating the superior regions of the respiratory and olfactory epithelia, being more numerous in female rats.     

Effect of conjugated linoleic acid on the formation of spontaneous and PhIP-induced mutation in the colon and cecum of rats

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid, has been reported to inhibit chemically induced mammary and colon carcinogenesis in rodents. In a preliminary experiment, we found that CLA significantly reduced the induction of mutations by the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the distal colon in male rats. Here, the chemopreventive properties of CLA were further evaluated by assessing its effect on PhIP-induced mutation and aberrant crypt foci (ACF) in both male and female rats. CLA (1%, w/w) was added to the diet (1) from weaning to 50-day-old, or (2) starting 1 week prior to exposure to PhIP. The 50-day-old Big Blue^® and F344 rats were then exposed to 100 ppm PhIP for 47 days. No sex differences were observed in mutagenic response to the various treatments in either the distal colon or cecum. The mutation frequency (MF) in the cecum and the distal colon from control animals is 4.3±1.3 and 5.3±1.4×10⁻⁵, respectively showing no statistically significant difference. Administration of PhIP induced a four-fold increase in the MF in the cecum and a seven-fold increase in the distal colon compared to the corresponding controls. Supplementation of the diet with CLA lowered the PhIP-induced MF in the distal colon by 23% (P<0.03), but had no effect in the cecum. The PhIP-induced ACF, determined 9 weeks after the termination of treatment with PhIP, were 0.75 ACF/rat, with 1.7 aberrant crypts /ACF in the colon of male rats, all located in the distal colon. This induction was completely inhibited by the addition of CLA.     

Hormonal influence on the olfactory response to a female-attracting pheromone, sodefrin, in the newt, Cynops pyrrhogaster

Sodefrin is a female-attracting pheromone isolated from the abdominal glands of male newts, Cynops pyrrhogaster. Previously, the preference of conspecific female newts for sodefrin was shown to be completely abolished by plugging the bilateral nostrils, indicating that it acts on the olfactory organ. To determine the sensitivity of the olfactory receptor cells to sodefrin, electro-olfactograms (EOGs) in response to sodefrin solution were recorded from the ventral nasal epithelium of sexually developed female newts. Sodefrin elicited marked EOG responses in a dose-dependent manner on the epithelium of the lateral nasal sinus (LNS) region, a putative vomeronasal organ. In ovariectomized females, treatment with prolactin (PRL) and estrogen markedly enhanced the EOG response to sodefrin. The EOG response to the pheromone was also enhanced considerably by treatment with either PRL or estrogen alone. A slight but significant elevation was observed in castrated males receiving PRL plus estrogen or estrogen alone. It was concluded that the main site of action of sodefrin resides in the lateral sinus region and that sensitivity to sodefrin is under the control of PRL and estrogen. The presence of a sex difference in olfactory sensitivity to the hormones and/or pheromone was also suggested.     

A study on specific behavioral effects of formaldehyde in the rat

It has been reported that single exposure of rats of low-level formaldehyde vapor concentrations causes significant alteration in their motor activity in the inhalation chamber. In this study, we determined the effects of formaldehyde on the locomotor activity and behavior of adult male and female Lew. 1K rats in an open field two hours after termination of a single two hours lasting inhalative exposure to approximately 0.1, 0.5, or 5 ppm. Following behavioral parameters were quantitatively examined: numbers of crossed floor squares, occurrence frequencies of air and floor sniffing, grooming, rearing, and wall climbing, as well as the incidence of fecal boli. In the open field situation, the males of all formaldehyde groups crossed significantly lower numbers of floor squares. Furthermore, significant decrease in the occurrence frequencies of floor sniffing, rearing, and wall climbing were observed. Within the female rat groups exposed to 0.5 or 5 ppm formaldehyde, a significantly decreased numbers of crossed squares were registered, while this parameter remained unchanged in the 0.1 ppm group. Other parameters were also affected by the formaldehyde inhalation (e.g. significant increase in the occurrence frequencies of air sniffing in the 0.1 and 0.5 ppm groups and significant decrease in the numbers of floor sniffing in the 0.5 and 5 ppm groups, respectively). The incidence of fecal boli was not affected in any exposure group neither in males nor in females. It is concluded from the results obtained that formaldehyde significantly affects the locomotor behavior of adult male and female rats in the open field after a single inhalative exposure to the above mentioned concentrations.     

Murine nasal septa for respiratory epithelial air-liquid interface cultures

Air-liquid interface models using murine tracheal respiratory epithelium have revolutionized the in vitro study of pulmonary diseases. This model is often impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. We describe a simple technique to harvest the murine nasal septum and grow the epithelial cells in an air-liquid interface. The degree of ciliation of mouse trachea, nasal septum, and their respective cultured epithelium at an air-liquid interface were compared by scanning electron microscopy (SEM). Immunocytochemistry for type IV beta-tubulin and zona occludens-1 (Zo-1) are performed to determine differentiation and confluence, respectively. To rule out contamination with olfactory epithelium (OE), immunocytochemistry for olfactory marker protein (OMP) was performed. Transepithelial resistance and potential measurements were determined using a modified vertical Ussing chamber SEM reveals approximately 90% ciliated respiratory epithelium in the nasal septum as compared with 35% in the mouse trachea. The septal air-liquid interface culture demonstrates comparable ciliated respiratory epithelium to the nasal septum. Immunocytochemistry demonstrates an intact monolayer and diffuse differentiated ciliated epithelium. These cultures exhibit a transepithelial resistance and potential confirming a confluent monolayer with electrically active airway epitheliumn containing both a sodium-absorptive pathway and a chloride-secretory pathway. To increase the yield of respiratory epithelial cells harvested from mice, we have found the nasal septum is a superior source when compared with the trachea. The nasal septum increases the yield of respiratory epithelial cells up to 8-fold.     

Cell-specific expression of CYP2A5 in the mouse respiratory tract: effects of olfactory toxicants.

We performed a detailed analysis of mouse cytochrome P450 2A5 (CYP2A5) expression by in situ hybridization (ISH) and immunohistochemistry (IHC) in the respiratory tissues of mice. The CYP2A5 mRNA and the corresponding protein co-localized at most sites and were predominantly detected in the olfactory region, with an expression in sustentacular cells, Bowman's gland, and duct cells. In the respiratory and transitional epithelium there was no or only weak expression. The nasolacrimal duct and the excretory ducts of nasal and salivary glands displayed expression, whereas no expression occurred in the acini. There was decreasing expression along the epithelial linings of the trachea and lower respiratory tract, whereas no expression occurred in the alveoli. The hepatic CYP2A5 inducers pyrazole and phenobarbital neither changed the CYP2A5 expression pattern nor damaged the olfactory mucosa. In contrast, the olfactory toxicants dichlobenil and methimazole induced characteristic changes. The damaged Bowman's glands displayed no expression, whereas the damaged epithelium expressed the enzyme. The CYP2A5 expression pattern is in accordance with previously reported localization of protein and DNA adducts and the toxicity of some CYP2A5 substrates. This suggests that CYP2A5 is an important determinant for the susceptibility of the nasal and respiratory epithelia to protoxicants and procarcinogens.     

Destruction of the main olfactory epithelium reduces female sexual behavior and olfactory investigation in female mice

We studied the contribution of the main olfactory system to mate recognition and sexual behavior in female mice. Female mice received an intranasal irrigation of either a zinc sulfate (ZnSO4) solution to destroy the main olfactory epithelium (MOE) or saline (SAL) to serve as control. ZnSO4-treated female mice were no longer able to reliably distinguish between volatile as well as nonvolatile odors from an intact versus a castrated male. Furthermore, sexual behavior in mating tests with a sexually experienced male was significantly reduced in ZnSO4-treated female mice. Vomeronasal function did not seem to be affected by ZnSO4 treatment: nasal application of male urine induced similar levels of Fos protein in the mitral and granule cells of the accessory olfactory bulb (AOB) of ZnSO4 as well as SAL-treated female mice. Likewise, soybean agglutinin staining, which stains the axons of vomeronasal neurons projecting to the glomerular layer of the AOB was similar in ZnSO4-treated female mice compared to SAL-treated female mice. By contrast, a significant reduction of Fos in the main olfactory bulb was observed in ZnSO4-treated females in comparison to SAL-treated animals, confirming a substantial destruction of the MOE. These results show that the MOE is primarily involved in the detection and processing of odors that are used to localize and identify the sex and endocrine status of conspecifics. By contrast, both the main and accessory olfactory systems contribute to female sexual receptivity in female mice.