Biochemical properties of the sensitivity to GABAAergic ligands, Cl-/HCO3--ATPase isolated from fish (Cyprinus carpio) olfactory mucosa and brain.

This paper presents a comparative study of the roles of Cl- and HCO3- in the functioning of the GABAAR-associated Cl-/HCO3--ATPase of the plasma membranes of the olfactory sensory neurons (OSNs) and mature brain neurons (MBNs) of fish. The ATPase activity of OSNs and its dephosphorylation were increased twofold by Cl-(15-30 mmol l-1), whereas the enzyme from MBNs was not significantly affected by Cl-. By contrast, HCO3-(15-30 mmol l-1) significantly activated the MBN enzyme and its dephosphorylation, but had no effect on the OSN ATPase. The maximum ATPase activity and protein dephosphorylation was observed in the presence of both Cl-(15 mmol l-1)/HCO3-(27 mmol l-1) and these activities were inhibited in the presence of picrotoxin (100 µmol l-1), bumetanide (150 µmol l-1), and DIDS (1000 µmol l-1). SDS-PAGE revealed that ATPases purified from the neuronal membrane have a subunit with molecular mass of ~ 56 kDa that binds [3H]muscimol and [3H]flunitrazepam. Direct phosphorylation of the enzymes in the presence of ATP-γ-32P and Mg2+, as well as Cl-/HCO3- sensitive dephosphorylation, is also associated with this 56 kDa peptide. Both preparations also showed one subunit with molecular mass 56 kDa that was immunoreactive with GABAAR ß3 subunit. The use of a fluorescent dye for Cl- demonstrated that HCO3-(27 mmol l-1) causes a twofold increase in Cl- influx into proteoliposomes containing reconstituted ATPases from MBNs, but HCO3- had no effect on the reconstituted enzyme from OSNs. These data are the first to demonstrate a differential effect of Cl- and HCO3- in the regulation of the Cl-/HCO3--ATPases functioning in neurons with different specializations.

Odorant-odorant metabolic interaction, a novel actor in olfactory perception and behavioral responsiveness.

In the nasal olfactory epithelium, olfactory metabolic enzymes ensure odorant clearance from the olfactory receptor environment. This biotransformation of odorants into deactivated polar metabolites is critical to maintaining peripheral sensitivity and perception. Olfactory stimuli consist of complex mixtures of odorants, so binding interactions likely occur at the enzyme level and may impact odor processing. Here, we used the well-described model of mammary pheromone-induced sucking-related behavior in rabbit neonates. It allowed to demonstrate how the presence of different aldehydic odorants efficiently affects the olfactory metabolism of this pheromone (an aldehyde too: 2-methylbut-2-enal). Indeed, according to in vitro and ex vivo measures, this metabolic interaction enhances the pheromone availability in the epithelium. Furthermore, in vivo presentation of the mammary pheromone at subthreshold concentrations efficiently triggers behavioral responsiveness in neonates when the pheromone is in mixture with a metabolic challenger odorant. These findings reveal that the periphery of the olfactory system is the place of metabolic interaction between odorants that may lead, in the context of odor mixture processing, to pertinent signal detection and corresponding behavioral effect.

Presence of Sex Steroid-Metabolizing Enzymes in the Olfactory Mucosa of Rats.

Although several lines of evidence have suggested that sex steroids influence olfaction, little is known about the cellular basis of steroid-metabolizing enzymes in the olfactory system. Thus, we aimed to examine gene expression and immunolocalization of four sex steroid-metabolizing enzymes in the olfactory mucosa (OM) of albino rats; steroid side chain-cleaving enzyme (P450scc), 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD-1), 17ß-HSD type 2 (17ß-HSD-2), and aromatase. P450scc is known to catalyze conversion from cholesterol to pregnenolone. 17ß-HSD-1 catalyzes conversion from estrone to estradiol, and 17ß-HSD-2 does the reverse. Aromatase catalyzes the conversion from testosterone to estradiol-17ß. Messenger (m) RNAs of all four enzymes mentioned above were detected in the OM. Western blot analysis demonstrated that P450scc, 17ß-HSD-1, and 17ß-HSD-2 were detected in the OM. Immunoreactivity for these three enzymes was observed in sustentacular cells of the olfactory epithelium and acinar cells of Bowman's glands. Immunoelectron microscopy analysis demonstrated immunoreactivity for P450scc in mitochondria, and for 17ß-HSD-1 and 17ß-HSD-2 in the well-developed smooth endoplasmic reticulum and myeloid bodies of the sustentacular cells. The present study suggests that sustentacular cells and acinar cells of the Bowman's glands in the rat OM express at least three of the steroid-metabolizing enzymes, that is, P450scc 17ß-HSD-1, and 17ß-HSD-2, and de novo synthesis of estradiol takes place in the OM. Anat Rec, 300:402-414, 2017. © 2016 Wiley Periodicals, Inc.

Canine olfactory ensheathing cells from the olfactory mucosa can be engineered to produce active chondroitinase ABC.

A multitude of factors must be overcome following spinal cord injury (SCI) in order to achieve clinical improvement in patients. It is thought that by combining promising therapies these diverse factors could be combatted with the aim of producing an overall improvement in function. Chondroitin sulphate proteoglycans (CSPGs) present in the glial scar that forms following SCI present a significant block to axon regeneration. Digestion of CSPGs by chondroitinase ABC (ChABC) leads to axon regeneration, neuronal plasticity and functional improvement in preclinical models of SCI. However, the enzyme activity decays at body temperature within 24-72h, limiting the translational potential of ChABC as a therapy. Olfactory ensheathing cells (OECs) have shown huge promise as a cell transplant therapy in SCI. Their beneficial effects have been demonstrated in multiple small animal SCI models as well as in naturally occurring SCI in canine patients. In the present study, we have genetically modified canine OECs from the mucosa to constitutively produce enzymatically active ChABC. We have developed a lentiviral vector that can deliver a mammalian modified version of the ChABC gene to mammalian cells, including OECs. Enzyme production was quantified using the Morgan-Elson assay that detects the breakdown products of CSPG digestion in cell supernatants. We confirmed our findings by immunolabelling cell supernatant samples using Western blotting. OECs normal cell function was unaffected by genetic modification as demonstrated by normal microscopic morphology and the presence of the low affinity neurotrophin receptor (p75(NGF)) following viral transduction. We have developed the means to allow production of active ChABC in combination with a promising cell transplant therapy for SCI repair.

Odorant Metabolism Analysis by an Automated Ex Vivo Headspace Gas-Chromatography Method.

In the olfactory epithelium (OE), odorant metabolizing enzymes have the dual function of volatile component detoxification and active clearance of odorants from the perireceptor environment to respectively maintain the integrity of the tissues and the sensitivity of the detection. Although emphasized by recent studies, this enzymatic mechanism is poorly documented in mammals. Thus, olfactory metabolism has been characterized mainly in vitro and for a limited number of odorants. The automated ex vivo headspace gas-chromatography method that was developed here was validated to account for odorant olfactory metabolism. This method easily permits the measurement of the fate of an odorant in the OE environment, taking into account the odorant gaseous state and the cellular structure of the tissue, under experimental conditions close to physiological conditions and with a high reproducibility. We confirmed here our previous results showing that a high olfactory metabolizing activity of the mammary pheromone may be necessary to maintain a high level of sensitivity toward this molecule, which is critical for newborn rabbit survival. More generally, the method that is presented here may permit the screening of odorants metabolism alone or in mixture or studying the impact of aging, pathology, polymorphism or inhibitors on odorant metabolism.

Essential role of the cytochrome P450 enzyme CYP2A5 in olfactory mucosal toxicity of naphthalene.

Naphthalene (NA), a ubiquitous environmental pollutant that can cause pulmonary and nasal toxicity in laboratory animals, requires cytochrome P450 (P450)-mediated metabolic activation to cause toxicity. Our recent study using a Cyp2f2-null mouse showed that CYP2F2 plays an essential role in NA-induced lung toxicity, but not in NA-induced nasal toxicity. The aim of this study was to determine whether mouse CYP2A5, abundantly expressed in nasal olfactory mucosa (OM) and the liver, but less in the lung, plays a major role in the bioactivation and toxicity of NA in the OM. We found, by comparing Cyp2a5-null and wild-type (WT) mice, that the loss of CYP2A5 expression led to substantial decreases in rates of NA metabolic activation by OM microsomes. The loss of CYP2A5 did not cause changes in systemic clearance of NA (at 200 mg/kg, i.p.). However, the Cyp2a5-null mice were much more resistant than were WT mice to NA-induced nasal toxicity (although not lung toxicity), when examined at 24 hours after NA dosing (at 200 mg/kg, i.p.), or to NA-induced depletion of total nonprotein sulfhydryl in the OM (although not in the lung), examined at 2 hours after dosing. Thus, mouse CYP2A5 plays an essential role in the bioactivation and toxicity of NA in the OM, but not in the lung. Our findings further illustrate the tissue-specific nature of the role of individual P450 enzymes in xenobiotic toxicity, and provide the basis for a more reliable assessment of the potential risks of NA nasal toxicity in humans.

Activation of ERK1/2 is required for normal response of isosexual social interactions in male rats.

Previous studies have indicated involvement of the mitogen-activated protein kinase (MAPK) pathway in heterosexual interactions among rats. Very few studies, however, have focused its role in isosexual social interactions. We studied the male rat's isosexual social interactional behavior using (i) the three-chambered social interaction box and (ii) phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) to localize the brain regions that are activated during isosexual behavior. When faced with the social target side of the box versus the inanimate side, all rats preferred the social target side. Within 10min, isosexual social interactions induced a rapid increase in pERK1/2 expression in the brain, especially the main olfactory epithelial (MOE)-related brain regions. After ZnSO4-induced olfactory deprivation, rats showed no preference for either the social target or inanimate side, with a concomitant decrease in pERK1/2 expression in MOE-related brain regions. Additionally, to determine the role of pERK1/2 in isosexual social interactional behavior, rats were injected intraperitoneally with SL327 (30mg/kg, a MAPK kinase inhibitor). Although SL327 dramatically down-regulated expression of brain pERK1/2, experimental animals also spent significantly more time in the social target side. These results indicate that (i) A brief interacting with a male partner induced rapidly phosphorylated ERK1/2 in the rat's brain. (ii) Destroy the function of MOE abolished the rats' isosexual social interactional behavior. (iii) Suppressed the phosphorylated ERK1/2 in the rats' brain disrupt their normal social behaviour.

Odorant metabolism catalyzed by olfactory mucosal enzymes influences peripheral olfactory responses in rats.

A large set of xenobiotic-metabolizing enzymes (XMEs), such as the cytochrome P450 monooxygenases (CYPs), esterases and transferases, are highly expressed in mammalian olfactory mucosa (OM). These enzymes are known to catalyze the biotransformation of exogenous compounds to facilitate elimination. However, the functions of these enzymes in the olfactory epithelium are not clearly understood. In addition to protecting against inhaled toxic compounds, these enzymes could also metabolize odorant molecules, and thus modify their stimulating properties or inactivate them. In the present study, we investigated the in vitro biotransformation of odorant molecules in the rat OM and assessed the impact of this metabolism on peripheral olfactory responses. Rat OM was found to efficiently metabolize quinoline, coumarin and isoamyl acetate. Quinoline and coumarin are metabolized by CYPs whereas isoamyl acetate is hydrolyzed by carboxylesterases. Electro-olfactogram (EOG) recordings revealed that the hydroxylated metabolites derived from these odorants elicited lower olfactory response amplitudes than the parent molecules. We also observed that glucurono-conjugated derivatives induced no olfactory signal. Furthermore, we demonstrated that the local application of a CYP inhibitor on rat olfactory epithelium increased EOG responses elicited by quinoline and coumarin. Similarly, the application of a carboxylesterase inhibitor increased the EOG response elicited by isoamyl acetate. This increase in EOG amplitude provoked by XME inhibitors is likely due to enhanced olfactory sensory neuron activation in response to odorant accumulation. Taken together, these findings strongly suggest that biotransformation of odorant molecules by enzymes localized to the olfactory mucosa may change the odorant's stimulating properties and may facilitate the clearance of odorants to avoid receptor saturation.

Antenatal insults modify newborn olfactory function by nitric oxide produced from neuronal nitric oxide synthase.

Newborn feeding, maternal, bonding, growth and wellbeing depend upon intact odor recognition in the early postnatal period. Antenatal stress may affect postnatal odor recognition. We investigated the exact role of a neurotransmitter, nitric oxide (NO), in newborn olfactory function. We hypothesized that olfactory neuron activity depended on NO generated by neuronal NO synthase (NOS). Utilizing in vivo functional manganese enhanced MRI (MEMRI) in a rabbit model of cerebral palsy we had shown previously that in utero hypoxia-ischemia (H-I) at E22 (70% gestation) resulted in impaired postnatal response to odorants and poor feeding. With the same antenatal insult, we manipulated NO levels in the olfactory neuron in postnatal day 1 (P1) kits by administration of intranasal NO donors or a highly selective nNOS inhibitor. Olfactory function was quantitatively measured by the response to amyl acetate stimulation by MEMRI. The relevance of nNOS to normal olfactory development was confirmed by the increase of nNOS gene expression from fetal ages to P1 in olfactory epithelium and bulbs. In control kits, nNOS inhibition decreased NO production in the olfactory system and increased MEMRI slope enhancement. In H-I kits the MEMRI slope did not increase, implicating modification of endogenous NO-mediated olfactory function by the antenatal insult. NO donors as a source of exogenous NO did not significantly change function in either group. In conclusion, olfactory epithelium nNOS in newborn rabbits probably modulates olfactory signal transduction. Antenatal H-I injury remote from delivery may affect early functional development of the olfactory system by decreasing NO-dependent signal transduction.

Neuronal nitric oxide synthase (nNOS) immunoreactivity in the olfactory system of a cartilaginous fish.

Nitric oxide is a regulative molecule with important roles in the olfactory system of vertebrates. Chondrichtyans have a key position in vertebrate evolution and nothing is known about nitric oxide in their olfactory system. Aim of this work was to investigate the neuronal nitric oxide synthase (nNOS) immunoreactivity in the olfactory system of the shark Scyliorhinus canicula. Because nitric oxide is often related to GABA in the olfactory system, also the distribution of GABA and its synthesis enzyme GAD has been investigated. In the olfactory epithelium scattered cells in the basal and medial zone of the epithelium thickness presented nNOS-like immunoreactivity. In the olfactory bulb the nNOS-like immunoreactivity has been highlighted in nerve fibers around some blood vessels and in scattered GABAergic granule cells. The presence of nNOS in the olfactory system of S. canicula is overall lesser than that described in other vertebrates, even if nitric oxide probably keeps some essential functions.

Differential expression of components of the retinoic acid signaling pathway in the adult mouse olfactory epithelium.

Position within a tissue often correlates with cellular phenotype, for example, differential expression of odorant receptors and cell adhesion molecules across the olfactory mucosa (OM). The association between position and phenotype is often paralleled by gradations in the concentration of retinoic acid (RA), caused by differential expression of the RA synthetic enzymes, the retinaldehyde dehydrogenases (RALDH). We show here that RALDH-1, -2, and -3 are enriched in the sustentacular cells, deep fibroblasts of the lamina propria, and the superficial fibroblasts, respectively, of the ventral and lateral OM as compared to the dorsomedial OM. The shift from high to low expression of the RALDHs matches the boundary defined by the differential expression of OCAM/mamFasII. Further, we found that RA-binding proteins are expressed in the epithelium overlying the RALDH-3 expressing fibroblasts of the lamina propria. Both findings suggest that local alterations in RA concentration may be more important than a gradient of RA across the epithelial plane, per se. In addition, RALDH-3 is found in a small population of basal cells in the ventral and lateral epithelium, which expand and contribute to the neuronal lineage following MeBr lesion. Indeed, transduction with a retrovirus expressing a dominant negative form of retinoic acid receptor type alpha blocks the reappearance of mature, olfactory marker protein (OMP) (+) olfactory neurons as compared to empty vector. These results support the notion of a potential role for RA, both in maintaining the spatial organization of the normal olfactory epithelium and in reestablishing the neuronal population during regeneration after injury.

Characterization of phospholipid hydroperoxide glutathione metabolizing peroxidase (gpx4) isoforms in Coho salmon olfactory and liver tissues and their modulation by cadmium.

Exposure to environmental contaminants, including various pesticides and trace metals, can disrupt critical olfactory-driven behaviors of fish such as homing to natal streams, mate selection, and an ability to detect predators and prey. These neurobehavioral injuries have been linked to reduced survival and population declines. Despite the importance of maintaining proper olfactory signaling processes in the presence of chemical exposures, little is known regarding chemical detoxification in the salmon olfactory system, and in particular, the antioxidant defenses that maintain olfactory function. An understudied, yet critical component of cellular antioxidant defense is phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4), an isoform within the family of selenium-dependent glutathione peroxidase (GPx) enzymes that can directly reduce lipid peroxides and other membrane-bound complex hydroperoxides. In this study, we cloned two gpx4 isoforms (gpx4a and gpx4b) from Coho salmon olfactory tissues and compared their modulation in olfactory and liver tissues by cadmium, an environmental pollutant and olfactory toxicant that cause oxidative damage as a mechanism of toxicity. Amino acid sequence comparisons of the two gpx4 isoforms shared 71% identity, and also relatively high sequence identities when compared with other fish GPx4 isoforms. Sequence comparisons with human GPx4 indicated conservation of three important active sites at selenocysteine (U46), glutamine (Q81), and tryptophan (W136), suggesting similar catalytic activity between fish and mammalian GPx4 isoforms. Tissue profiling confirmed the expression of gpx4a and gpx4b in all ten Coho tissues examined. The expression of gpx4 mRNAs in the Coho olfactory system was accompanied by comparably high initial rates of GPx4 enzymatic activity in mitochondrial and cytosolic fractions. Exposure to low (3.7 ppb) and high (347 ppb) environmental Cd concentrations for 24-48 h significantly decreased gpx4a expression in Coho olfactory rosettes, whereas olfactory gpx4b mRNA expression was not modulated by exposures at these concentrations. In summary, Coho salmon express two paralogs of gpx4, a key enzyme in the maintenance of signal transduction processes that protect against cellular oxidative damage. The Cd-associated downregulation of salmon olfactory gpx4a expression in particular, may be associated with the loss of olfactory signal transduction that accompanies metal-associated loss of olfaction in salmonids.

Immunohistochemical localization and biochemical changes in catalase and superoxide dismutase during metamorphosis in the olfactory system of frog Microhyla ornata.

Amphibian metamorphosis is characterized by rapid tissue remodeling and drastic changes in the body structure and function. Like other organs, olfactory system also undergoes a dramatic rearrangement as the animal experiences transition from aquatic to terrestrial habitat. Reactive oxygen species (ROS) are known to play an important role during anuran metamorphosis and role of antioxidant enzymes like catalase and superoxide dismutase (SOD) are believed to play a major role in these processes. Therefore, we hypothesize that antioxidant enzymes in the olfactory system may undergo changes that reflect metamorphic processes. Immunohistochemical study revealed the presence of catalase and SOD in the olfactory receptor neurons and also granular reaction in olfactory epithelium of medial diverticulum during metamorphosis. Catalase and SOD immunoreactivity were seen in the epithelium of lateral diverticulum, vomeronasal organ as metamorphosis proceeds and in the apical lining of olfactory epithelium of adult frog. Biochemical study showed that catalase activity gradually increases in the olfactory system from metamorphic stage 40-46 and adult, while SOD activity decreases from stage 40 to 46 and increases in adult. Thus, the localization and relative levels of catalase and SOD during metamorphosis in the olfactory system suggests that these enzymes may be involved in protection from oxidative damage.

Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors.

The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

The tissue-specific toxicity of methimazole in the mouse olfactory mucosa is partly mediated through target-tissue metabolic activation by CYP2A5.

The antithyroid drug methimazole (MMZ) can cause severe, tissue-specific toxicity in mouse olfactory mucosa (OM), presumably through a sequential metabolic activation of MMZ by cytochrome P450 (P450) and flavin monooxygenases (FMO). The aims of this study were to determine whether CYP2A5, one of the most abundant P450 enzymes in the mouse OM, is involved in MMZ metabolic activation, by comparing Cyp2a5-null with wild-type (WT) mice, and whether hepatic microsomal P450 enzymes, including CYP2A5, are essential for MMZ-induced OM toxicity, by comparing liver-Cpr-null (LCN) mice, which have little P450 activity in hepatocytes, with WT mice. We showed that the loss of CYP2A5 expression did not alter systemic clearance of MMZ (at 50 mg/kg, i.p.); but it did significantly decrease the rates of MMZ metabolism in the OM, whereas FMO expression in the OM was not reduced. MMZ induced depletion of nonprotein thiols, as well as pathological changes, in the OM of WT mice; the extent of these changes was much reduced in the Cyp2a5-null mice. Thus, CYP2A5 plays an important role in mediating MMZ toxicity in the OM. In contrast, the rate of systemic clearance of MMZ was significantly reduced in the LCN mice, compared to WT mice, whereas the MMZ-induced OM toxicity was not prevented. Therefore, hepatic P450 enzymes are essential for systemic MMZ clearance, but they are not required for MMZ-induced OM toxicity. We conclude that the tissue-specific toxicity of MMZ is mediated by target tissue metabolic activation, and the reaction is partly catalyzed by CYP2A5 in the OM.

Occurrence of neuropeptides and tyrosine hydroxylase in the olfactory epithelium of the lesser-spotted catshark (Scyliorhinus canicula Linnaeus, 1758).

Immunohistochemical studies using antisera against various neuropeptides (Substance P, vasoactive intestinal polypeptide, and cholecystokinin octapeptide) and tyrosine hydroxylase revealed both olfactory sensory neuron (OSN) polymorphisms and transepithelial-subepithelial nerves in the olfactory epithelium of the cartilaginous fish, Scyliorhinus canicula. This study provides the first evidence of three morphological types of OSNs within the olfactory epithelium of cartilaginous fish that are similar to those found in the teleosts. In fishes there is evidence that OSNs differ functionally, including their differential olfactory bulb projections and molecular properties. The Substance P positive olfactory neurons in S. canicula may have a separate bulbar projection site that is not known, but may indicate a characteristic found in olfactory neuron subtypes in both lampreys and teleost fish. Numerous Substance P immunopositive nerves are found at the base of and in the olfactory epithelium. Some of them were observed to extend outwards almost reaching the epithelial surface. Their presumptive origin from the trigeminal nerve and their interrelationship with chemosensory cells in the nasal passages of vertebrates are discussed.

Localization of eNOS in the olfactory epithelium of the rat.

Nitric oxide (NO) is a free radical and produced from L-arginine by nitric oxide synthase (NOS). Since NO is recently suggested to be involved in olfactory perception, the expression of eNOS, an isoform of NOS, was examined in the rat olfactory epithelium. The activity of NADPH-diaphorase was also examined as a marker of NOS. In the dorsomedial region of the nasal cavity, intensely positive reactions for NADPH-diaphorase were observed in the entire cytoplasm of sensory cells (olfactory cells). By immunohistochemistry, intensely positive reactions for eNOS were also found in the dorsomedial region of the nasal cavity. These reactions were observed on the free border of the olfactory epithelium. By immunoelectron microscopy, positive reactions for eNOS were found in the cilia of olfactory cells. In addition, in situ hybridization analysis of the olfactory epithelium revealed the expression of eNOS mRNA in the olfactory cells. These results indicate the presence of eNOS in the olfactory cells of the rat, and differential expression of eNOS in the olfactory epithelium depending on the regions of the nasal cavity. In addition, NO produced by eNOS may be involved in olfactory perception in the cilia of olfactory cells.

[Identification in the rat olfactory epithelium new subgroup YM-1 chitinase-like protein].

Novel protein with a molecular mass of ~43 kDa from rat olfactory epithelium in pathophysiological conditions was discovered. Its amino acid sequence and affiliation with the family 18 glycohydrolase subgroup of chitinase-like proteins YM-1 were determined.

Mechanisms of olfactory toxicity of the herbicide 2,6-dichlorobenzonitrile: essential roles of CYP2A5 and target-tissue metabolic activation.

The herbicide 2,6-dichlorobenzonitril (DCBN) is a potent and tissue-specific toxicant to the olfactory mucosa (OM). The toxicity of DCBN is mediated by cytochrome P450 (P450)-catalyzed bioactivation; however, it is not known whether target-tissue metabolic activation is essential for toxicity. CYP2A5, expressed abundantly in both liver and OM, was previously found to be one of the P450 enzymes active in DCBN bioactivation in vitro. The aims of this study were to determine the role of CYP2A5 in DCBN toxicity in vivo, by comparing the extents of DCBN toxicity between Cyp2a5-null and wild-type (WT) mice, and to determine whether hepatic microsomal P450 enzymes (including CYP2A5) are essential for the DCBN toxicity, by comparing the extents of DCBN toxicity between liver-Cpr-null (LCN) mice, which have little P450 activity in hepatocytes, and WT mice. We show that the loss of CYP2A5 expression did not alter systemic clearance of DCBN (at 25 mg/kg); but it did inhibit DCBN-induced non-protein thiol depletion and cytotoxicity in the OM. Thus, CYP2A5 plays an essential role in mediating DCBN toxicity in the OM. In contrast to the results seen in the Cyp2a5-null mice, the rates of systemic DCBN clearance were substantially reduced, while the extents of DCBN-induced nasal toxicity were increased, rather than decreased, in the LCN mice, compared to WT mice. Therefore, hepatic P450 enzymes, although essential for DCBN clearance, are not necessary for DCBN-induced OM toxicity. Our findings form the basis for a mechanism-based approach to assessing the potential risks of DCBN nasal toxicity in humans.

Effects of typical inducers on olfactory xenobiotic-metabolizing enzyme, transporter, and transcription factor expression in rats.

Several xenobiotic-metabolizing enzymes (XMEs) have been identified in the olfactory mucosa (OM) of mammals. However, the molecular mechanisms underlying the regulation of these enzymes have been little explored. In particular, information on the expression of the transcriptional factors in this tissue is quite limited. The aim of the present study was to examine the impact of five typical inducers, Aroclor 1254, 3-methylcholanthrene, dexamethasone, phenobarbital, and ethoxyquin, on the activities and mRNA expression of several XMEs in the OM and in the liver of rats. We also evaluated the effects of these treatments on the mRNA expression of transcription factors and transporters. On the whole, the intensities of the effects were lower in the OM than in the liver. Dexamethasone was found to be the most efficient treatment in the OM. Dexamethasone induced the transcription of several olfactory phase I, II, and III genes [such as cytochromes P450 2A3 and 3A9, UDP-glucuronosyltransferase (UGT) 2A1, and multidrug resistance-related protein type 1] and increased UGT activities. We observed that dexamethasone up-regulated sulfotransferase 1C1 expression in the OM but down-regulated it in the liver. Aroclor and ethoxyquin induced the gene expression of CYP1A and quinone reductase, respectively, in the OM. The transcription factors aryl hydrocarbon receptor, nuclear factor E2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor α, pregnane X receptor, and glucocorticoid receptor were detected in the OM, but no constitutive androstane receptor expression was observed. Dexamethasone and Aroclor enhanced olfactory Nrf2 expression. These results demonstrate that olfactory XME can be modulated by chemicals and that the mechanisms involved in the regulation of these enzymes are tissue-specific.