B-ENT , 2009, 5, Suppl. 13, 21-37 Defence mechanisms of olfactory neuro-epithelium: mucosa regeneration, metabolising enzymes and transporters

J.-B. Watelet *, M. Strolin-Benedetti ** and R. Whomsley *** *Department of Otorhinolaryngology, Ghent University, Belgium; **UCB sa, Colombes, France; ***Shire Pharmaceuticals, Basingstoke, Hampshire, UK

Key-words. Olfactory; regeneration; cytochrome; transporters

Abstract. Defence mechanisms of olfactory neuro-epithelium: mucosa regeneration, metabolising enzymes and trans - porters. The olfactory neuro-epithelium is highly sensitive to chemicals and its direct microbiological environment. It also plays a role as an interface between the airways and the nervous system, and so it has developed several defence instruments for rapid regeneration or for the detoxification of the immediate environment. This review illustrates three of these defence mechanisms: regeneration of the epithelium, local production of metabolising enzymes and xenobiotic transporters. Toxicants can inflict damage by a direct toxic response. Alternatively, they may require metabolic activation to produce the proximate toxicant. In addition to detoxifying inhaled and systemically derived xenobiotics, the local olfactory metabolism may fulfil multiple functions such as the modification of inhaled odorant, the modulation of endogenous signalling molecules and the protection of other tissues such as the CNS and lungs from inhaled toxicants. Finally, the permeability of nasal and olfactory mucosa is an important efficacy parameter for some anti-allergic drugs delivered by intranasal administration or inhalation. Efflux or update transporters expressed in these tissues may therefore significant - ly influence the pharmacokinetics of drugs administered topically.

Introduction matic control or transporter acti - affect this highly differentiated vation, have been the subject of mucosa. Most of what we know Inspired air is brought high into several recent publications. about the toxic action of inhaled the nasal cavity to come in contact This review will provide an compounds is derived from experi - with the olfactory nerves, trig- overview of three major defence mental animal studies. gering the , which is mechanisms for toxic or chemical intimately associated with taste. 1 insults to the olfactory mucosa. 1.1. Epidemiology The neuro-epithelium has several The inhalation of a number of instruments for ensuring optimal 1. Chemical injury of the olfac - environmental and industrial olfactory function. The olfactory tory epithelium chemicals can lead to olfactory neuro-epithelium is highly sensi - dysfunction. Even if the list of tive to the chemical or microbio - This section does not look at physi - theoretically toxic chemicals is logical environment and it acts as cal (e.g. irradiation...) or pharma - impressive, they are thought to be an interface between the airways cological injuries to the olfactory responsible for smell disorders in and the nervous system. As a mucosa. Many of the toxicity only 2% of patients. 5 The degree result, it is suspected that it facili - mechanisms are still under inves - of olfactory damage seems to be tates the direct transfection of bac - tigation and the literature is main - related to the timing and duration teria or viruses 2 or the transporta - ly based on case reports, making of the exposure, the concentration tion of chemicals 3,4 into the dura any generalisation hazardous. of the agent, and the intrinsic and nervous system. On the other The limited accessibility to biop - toxicity of the agent. It therefore hand, the defence mechanisms sies of human olfactory epitheli - remains difficult to establish a deployed by the olfactory neuro- um, in conjunction with their complete list of environmental epithelium for rapid regeneration small size, has resulted in an toxicants. Furthermore, the multi - or for detoxification of the imme - almost complete lack of informa - plicity of cofactors makes this diate environment, such as enzy - tion about how toxic compounds analysis even more complex. 22 J.-B. Watelet et al.

Table 1 Many dusts produced in industry Major toxicants and industrial agents that may induce smell disorders have also been associated with Classes Agents Reported effect olfactory dysfunction, including grain, silicone, cotton, paper, Major toxic chemicals Benzene or benzol Hyposmia/anosmia cement, lead, coal, chromium, and Butyl acetate Hyposmia/anosmia nickel. Carbon disulphide Hyposmia/anosmia Chronic metal toxicity was Chlorine Hyposmia clearly illustrated in the case of Ethyl acetate Hyposmia/anosmia cadmium during the 1950s. Up Formaldehyde Hyposmia to 60% of people exposed to Hydrogen Hyposmia/anosmia cadmium for more than 10 years 6 Selenide Hyposmia presented an olfactory disorder. Paint solvents : Hyposmia/anosmia The olfactory toxicity of solvents acetone (e.g. acetone, acetophenone, ben - mineral turpentine (turps) zenes...) is mainly due to their true turpentine high lipophilicity. Olfactory naphtha toluene deficit increases with cumulative white spirit exposure. Finally, chronic expo - xylene sure to gases (such as carbon methyl ethyl ketone disulphide, carbon monoxide, Sulphuric acid Hyposmia sulphur dioxide, formaldehyde...) Trichloroethylene Hyposmia/anosmia can also directly affect the Major industrial agents Ashes Hyposmia olfactory mucosa. For example, Cadmium Hyposmia/anosmia exposure to ozone induces an Chalk Hyposmia increase in olfactory thresholds in 7 Chromium Hyposmia healthy controls but there is an adaptation phase after the initial Iron carboxyl Hyposmia experiments. Lead Hyposmia Nickel Hyposmia/anosmia 1.3. Principles underlying Ozone Temporary hyposmia damage to the olfactory mucosa Silicone dioxide Hyposmia Smell disorders secondary to toxic exposure can be due to multiple pathogenesis mechanisms: inflam - mation of the nasal mucosa leading to obstruction, neuro - Table 1 lists the major environ - 1.2.2. Chronic toxicity sensorial lesions, central disorders mental toxicants and their clinical On the other hand, when doses are or a combination of the above. consequences. lower and presented for a long This review will focus mainly period of time, the diagnosis can 1.2. Clinical presentation on peripheral neurosensorial be more difficult to establish and, disorders related to toxic injuries 1.2.1. Acute toxicity because of the long course of to the neuro-epithelium. However, The relationship between toxi - events, many confounding factors it is possible that some toxicants cants and accidental smell disor - also need to be taken into account: could also have a central impact ders is easier to identify when the e.g. age, viral infections, trau - on the smell function, even though exposure is massive and sudden. mas... the supporting data is very frag - The smell disorder can occur Chronic exposure to low levels mentary. within the first seconds or the first of benzene, butyl acetate, for- hours after exposure and can lead maldehyde, and paint solvent has 1.3.1. Histological damage to hyposmia or a transitory or per - been commonly reported with Attempts to link clinical anosmia manent anosmia. associated olfactory dysfunction. to olfactory membrane abnor - Defence mechanisms of olfactory mucosa 23 malities have provided relevant information about the clinical consequences of olfactory tissue damage. 8 After exposure to olfactotoxins, cytotoxicity at the level of the olfactory mucosa is seen almost immediately (in less than 24 h). The tissue damage due to perchloroethylene gas is more persistent in the nasal mucosa of the olfactory region than in the respiratory region. Two weeks after exposure, ciliated epithelial cells, as well as a pseudostratified nonciliated columnar epithelium, begin to appear in the area Figure 1 previously covered by olfactory Chemical structure of 2,6-methylsulphonyl-substituted dichlorobenzene (a) and 3- epithelium and remain for up to methylindole (b), regularly used for anosmia models. 3 months after exposure. A base - ment membrane is also present under the ciliated epithelium, sug - gesting a possible persistence of 1.3.2. Direct or indirect toxicity glands, and an apparent redistribu - basal cells. The olfactory epitheli - tion of CYP immunoreactivity um may therefore be replaced by Toxicants can exert their damaging within sustentacular cells. The ciliated respiratory epithelium. properties in different ways. Some treatment of mice with metyra - The lamina propria of the olfacto - agents, such as methylbromide pone, a CYP inhibitor, at 10 min ry mucosa, however, loses its nor - and 3-methylindole (Figure 1b), prior to and 2, 4, 6, and 8 h after a mal structure, with atrophy of the produce a direct toxic response. 12,13 single dichlobenil injection, pro - olfactory nerves and Bowman’s Other compounds, such as vided some protection against the glands. 9 Interestingly, whereas dimethylamine and 3-methyl- damaging effects of dichlobenil. 18 2,5-methylsulfonyl-substituted sulphate, require metabolic activa - Complementary results suggested dichlorobenzene (diCl-MeSO2-B) tion to produce the proximate that, in mice, 3-aminobenzamide, induces no signs of toxicity in the toxicant . 14-17 another inhibitor of CYP, can olfactory mucosa at doses as high Finally, in addition their direct also reduce the local toxicity as 130 mg/kg (i.p. injection), toxic effects, some toxicants can of dichlobenil in the olfactory necrosis of Bowman’s glands also have a strong impact on the mucosa, probably because of a must be considered as the first enzymatic cascade. Cytochrome reduction in the metabolic activa - sign of 2,6-(diCl-MeSO2-B)- P450 (CYP) is a very large and tion of dichlobenil at this site. 19 induced toxicity at higher doses diverse super-family of haemopro - Human data are more incom - (Figure 1a), followed by degenera - teins using a plethora of both plete but it appears that individu - tion of the neuro-epithelium. This exogenous and endogenous com - als with olfactory loss caused by implies that, for some toxicants pounds as substrates. Twenty- epithelial damage, as in chronic with 2,6-positioned chlorine eight hours after a single dose rhinosinusitis, display evidence of atoms and an electron-with - (12 mg/kg) of dichlobenil, charac - nerve fascicle degeneration and drawing substituent in the primary teristic regions of the olfactory intra-epithelial neuromas. 20 position at least, Bowman’s mucosa showed signs of necrosis glands may be the primary site in haematoxyllin eosin-stained of toxicity and degeneration of sections. This was accompanied 1.4. Defence mechanisms the neuro-epithelium may be a by a dramatic reduction in CYP The olfactory mucosa has mecha - secondary effect. 10,11 immunoreactivity in Bowman’s nisms that can limit tissue lesions 24 J.-B. Watelet et al. secondary to inhaled toxicants. Bowman’s glands should be seen 2.2. Growth factors and olfactory The olfactory epithelium can as a key element in the develop - marker in olfactory secrete many proteins or enzymes ment of olfactory deficits in mucosa repair that act directly against the humans. Some biological factors involved pathogens or irritants. Further - In studies exploring the long- in the repair process have been more, the trigeminal reflex has a term response in the olfactory identified. As in other repair direct influence on the local respi - mucosa of mice after exposure to processes, the role played by ratory and secretory physiology the olfactory toxicants dichlobenil growth factors and enzymes is of the nasal cavities, reducing (a herbicide) or methimazole (an determinant. The development of the risk of olfactory damage. antithyroid drug), it appeared that, human olfactory mucosa shows However, chronic exposure to three and six months after expo - that epidermal growth factor- irritants such as trichloro - sure to dichlobenil (2 × or receptor, transforming growth fac - ethylenes, 21,22 tobacco smoke, 1 × 25 mg/kg i.p.), the dorsomedi - tor-alpha and - carbon monoxide and chloro- al part of the olfactory region beta proteins are reliable markers methanes can reduce the trigemi - showed a respiratory metaplasia for developing or regenerating nal defence reflex. with abundant invaginations and a olfactory epithelium. 27 Mucus This review looks at three com - fibrotic lamina propria. By con - covering the human olfactory plementary defence mechanisms trast, 3 months after exposure to a epithelium contains insulin engaged in the protection of the toxic dose of methimazole growth factor-I and insulin highly sensitive olfactory neuro- (2 × 50 mg/kg i.p.), the olfactory growth-factor-binding proteins, epithelium: regeneration of the neuro-epithelium and lamina pro - suggesting that these factors have epithelium and the roles played by pria had been restored. An intact a role in the activity of the olfac - metabolising enzymes and trans - lamina propria is thought to be a tory mucosa. The amounts are porters. prerequisite for the repopulation reduced in the mucus of patients of the neuro-epithelium after toxi - with neurodegenerative diseases, 2. Regeneration of olfactory cant-induced injury. 25 possibly reflecting a dysfunction epithelium Recently, round or oval of the mucosa itself. 28 On the other openings with a diameter of 50 The regeneration of the olfactory hand, a lack of beta 2-microglobu - to 500 microns were observed neuro-epithelium has also been a lin, and presumably class I, may on the surface of the olfactory subject of debate. 23 The precise be a general phenotype of neu - epithelium. 26 These olfactory pits events in the repair process are ronal cells regardless of their are blind pouches lined with still largely unknown and the mitotic state or exposure to envi - olfactory epithelium, presenting 29 existing data about the inducing ronmental antigens. as invaginations of the neuro- signals is sparse. Recently, in mice, another epithelium into the connective potential differentiation marker 2.1. Structural recovery of olfac - tissue to depths varying between has been identified: the olfactory tory tissue 150 and 200 microns. The func - marker (OMP). Cells After exposure to many olfacto - tion of the pit specialisation is expressing OMP have been toxins, at least in rodents, dead unclear, but it appears to be a fea - located in the anterior/ dorsal cells slough off, and during the ture of normal, young epithelium. region of the nose quite distant next 30 days, the olfactory epithe - The configuration of the blind from the regio olfactoria. The cells lium is restored. With other chem - pouches may prolong odorant are arranged in ganglion-like clus - icals, the return to a mature olfac - association with the olfactory ters during perinatal stages and tory epithelium is less rapid, espe - receptor neurons, or they may appear to persist in adult animals. cially when the lamina propria and contain specialised neurons that Although OMP is present in cells Bowman’s glands appear to be have not yet been recognised. of the central nervous system and severely damaged. 24 Recovery of These findings could also serve to the cribriform mesenchyme, 30-39 it Bowman’s glands is accompanied identify fully functional epitheli - is considered a valuable general by a return of olfactory function, um or newly regenerated epitheli - marker for mature olfactory suggesting that damage to um with restitutio ad integrum . sensory neurons in nasal neuro- Defence mechanisms of olfactory mucosa 25 epithelia, and growing evidence neurons may markedly affect CYP 3. Xenobiotic-metabolising en- has been accumulated that it is expression in non-neuronal cells zymes and enzymes involved in the indeed a participant in olfactory of the olfactory mucosa. 47 Finally, metabolism of reactive oxygen function. 37,40-45 Based on the con - the precursor nature of the highest species cept that cells expressing OMP reductase-expressing cells sug - typically operate as chemosen - gests that differentiation-specific Drug-metabolising enzymes, sors, it seems conceivable that the mechanisms regulate cytochrome which are theoretically supposed newly discovered OMP cells may P450 reductase transcription to participate in detoxication phe - have a chemosensory function as during organogenesis. 48 nomena, can also activate some well, but could also serve as indi - toxicants (Table 2). cators of fully functional neuro- Drug-metabolising enzymes 2.4. Metal chelators during the epithelium. have been grouped into phase I repair of olfactory mucosa reactions, in which enzymes con - 2.3. Metabolising enzymes during Finally, other biological factors trol oxidation, reduction, or repair of olfactory mucosa involved in repair are an important hydrolytic reactions, and phase II Several metabolising enzymes source of information about the reactions, which involve the have been considered as indicators local defence and repair mecha - introduction of a hydrophilic of the degree of olfactory regenera - nisms of the olfactory mucosa. endogenous species into the drug tion. In the peripheral olfactory Metallothionein (MT) is a family molecule. 50 organ, continual olfactory receptor of cysteine-rich, low molecular The phase I enzymes lead to the neuron (ORN) turnover exposes weight (MW ranging from 3500 to introduction of functional groups, neighbouring cells to potentially 14000 Da) proteins. MTs have the such as -OH, -COOH, -SH, -O- or damaging cellular debris such as capacity to bind both physiologi - NH2 groups, resulting in a modifi - free radicals. These, in turn, may cal (Zn, Cu, Se...) and xenobiotic cation of the drug. be inactivated by binding directly (Cd, Hg, Ag...) heavy metals The phase II reactions may to glutathione (GSH) or by enzy - through the thiol group of its cys - directly affect the parent com - matic conjugation with gluta - teine residues, which represents pounds that contain appropriate thione S-transferase (GST). The nearly 30% of its amino acid structural motifs, or functional recovery of GST activity and residues. MT function is not clear, groups added or exposed by phase widespread GST immunoreac- but experimental data suggest it I oxidation. 51 Sulphation, glu - tivity during regeneration indi - may provide protection against curonidation, and glutathione con - cates the modulation of neuro - metal toxicity, be involved in the jugation are the three most preva - protective, developmental, and/or regulation of physiological metals lent classes of phase II metabo - physiological processes by GST. 46 (Zn and Cu) and provide protec - lism. These conjugation reactions In mice with unilateral naris clo - tion against oxidative stress. There enhance the water solubility and sure for 3, 4, or 5 months, CYP are four main isoforms expressed molecular weight of the metabo - immuno reactivity was clearly in humans. When exploring the lite, and also add a negative charge reduced in rostral regions of the expression of the MT1 and MT2 to the molecule. Phase II reactions open-side olfactory mucosa, isoforms of metallothionein in the are generally cytosolic, with the where losses of receptor neurons mouse olfactory mucosa, it exception of glucuronidation, resulted after 3 to 5 months of clo - appeared that, in untreated mice, which is microsomal. Conjugation sure. Closed-side immunoreactivi - both were strongly expressed in reactions generally, although not ty was similar to controls. After 4 supporting cells, acinar cells of always, terminate the biological months of closure in animals that Bowman’s glands, and olfactory activity of the drug. The catalytic had regrown their receptor neu - neurons. Irrigation with irritative rates of phase II reactions are rons, open-side immunoreactivity solution caused exfoliation of the generally significantly faster than for CYP was comparable to olfactory epithelium and, during the rates of the CYPs 50 and so the controls . Furthermore, olfactory the resultant regeneration, metal - initial (phase I) oxidation reaction bulbectomy also depressed CYP lothionein immunoreactivity was is normally rate-limiting. immunoreactivity in mice. The associated with the proliferating Superoxide dismutases, metallo - presence or absence of receptor basal cells. 49 zymes that catalyse the dismuta - 26 J.-B. Watelet et al. tion of superoxide anion radicals 3.1.1. In animals (VNO), although their expression into hydrogen peroxide, are the levels were much lower than those cell’s major enzymatic defence The mammalian olfactory mucosa in the main olfactory epithelium. against cytotoxic reactive oxygen (OM) is unique among extra- These findings reinforce the species and oxidative stress. There hepatic tissues in having high hypothesis that olfactory mucosal are two main forms of superoxide levels , and tissue-selective forms, and VNO microsomal CYP dismutases: the superoxide dis- of CYP enzymes. These enzymes enzymes are actively involved in mutase containing manganese, may have important toxicological maintaining cellular hormonal which is a mitochondrial enzyme, implications, as well as biological homeostasis and other perirecep - and the superoxide dismutase functions, in this chemosensory tor processes associated with containing copper-zinc, which organ. In addition to tissue- olfactory chemosensory func - is located primarily in the cyto - selective, abundant expression of tion. 55 These high levels of CYP sol and secondarily in the CYP1A2, CYP2A, and CYP2G1, present in the olfactory mucosa in nucleus. 52 some of the OM CYPs are also mammalian animals may con - known to evince early develop - tribute to the known tissue-selec - 3.1. Enzymes present in olfactory mental expression, a resistance to tive toxicity of numerous chemi - mucosa xenobiotic inducers, and a lack cal compounds. Despite recent progress in the of responsiveness to circadian In rats, inspired acetaldehyde is 53 identification and characterisation rhythm. The respiratory and metabolised inside the olfactory of numerous nasal biotrans- olfactory tissues are also the first mucosa by aldehyde dehydroge - formation enzymes in laboratory line of contact with hazardous air - nase (ALDH). However, concen - animals, the expression of bio - borne chemicals. tration dependence upon uptake transformation in human In mice, CYP immunoreactivity suggests that a saturable process is nasal mucosa remains difficult to in olfactory mucosa is observed involved: at exposure concentra - study. Given the potential role of only in Bowman’s glands and sup - tions of 300 ppm or greater, the 47 nasal biotransformation enzymes porting cells, but not in receptor delivered dosage rate may equal in the metabolism of airborne neurons or their progenitor basal or exceed the capacity of this chemicals, including fragrance cells. This localisation of CYP in enzyme. 56 compounds and therapeutic the non-neuronal cells of the In bullfrogs, carbonic anhy - agents, as well as the potential olfactory mucosa supports the drase could play a role in the interspecies differences between view that one of these cells‘ major detection of CO 2 thanks to its laboratory animals and humans, roles is the biotransformation of location in olfactory neurons. several studies have attempted to inhaled compounds. However, only a small population identify the enzymatic content of In newborn rats, ultrastructural of olfactory receptor neurons are human nasal mucosa. studies have shown that mito - 57 CO 2-sensitive. Olfactory xenobiotic metabo - chondria are present in the apices lism may be responsible for multi - portions of olfactory receptor 3.1.2. In humans ple functions: neurons dendrites and of sup- The xenobiotic-metabolising 1°/ the modification of inhaled porting cell apices, suggesting that enzymes present in nasal mucosa, odorant, these regions near the surfaces are especially the olfactory neuro- 2°/ the modulation of endoge - metabolically the most active in epithelium, and in the lungs play nous signalling molecules, odorant detection, signal pro- important roles in the first-pass 3°/ the detoxification of inhaled cessing and detoxification, the metabolism of anti-allergic drugs and systematically derived xeno - latter for supporting cells. 54 that are administered through biotics, with some drugs having Several P450 isoforms, including nasal sprays, aerosols or puffs. a potential influence on the CYP1A2, CYP2A, CYP2B, Zhang et al .58 have found tran - development of smell disorders CYP2C, CYP2G1, and CYP3A, scripts for nine drug-metabolising (Table 3), NADPH cytochrome P450-reduc - enzymes [ALDH6, ALDH7, 4°/ the protection of other tis - tase, and microsomal epoxide CYP1B1, CYP2E1, CYP2F1, sues such as CNS and lungs from hydrolase have been detected in CYP4B1, flavin-containing mono- inhaled toxicants. the mouse vomeronasal organ oxygenase 1 (FMO1), GSTP1, Defence mechanisms of olfactory mucosa 27

UGT2A1] in human foetal nasal responsible, at least in part, for the dihydro-2,6-dimethylpyridine (4- mucosa. These observations, com - olfactory deficits in subjects with ethyl-DDC) to hamsters results in bined with the previous detection AD. 52 a marked loss of CYP-dependent of other enzymes in human nasal reactions (peroxidase, 7-ethoxy - mucosa (CYP2A6, CYP2A13, 3.2. Inhibition/induction of en- coumarin O-deethylase, and 7- CYP2B6, CYP2C, CYP2J2, zymes present in animal and/or ethoxyresorufin O-deethylase) in CYP3A, NADPH-cytochrome human olfactory mucosa by xeno - both the liver and the olfactory P450 reductase, microsomal epox - biotics, and the biological conse - epithelium. 66 quences ide hydrolase, GSTA, GSTP1, and Hydrogen sulphide (H 2S) is an UGT2A1), 59,60 provide strong sup - In rats, low-level m-xylene expo - important brain, lung, and nose port for the idea that both human sure results in the organ-selective toxicant. Inhibition of cytochrome foetal and adult olfactory mucosa alteration of CYP isozyme activi - oxidase is the primary biochemi - play an active role in the biotrans - ties and subsequent 1-nitro - cal effect associated with lethal 62 formation of numerous xenobi - naphthalene-induced toxicity. H2S exposure. In adult male rats, otics. Olfactory toxicity in the Furthermore, coumarin (50 µM) immediately after 3 hours of expo - perinatal period may have a inhibits the metabolism of three sure to H 2S (10, 30, 80, 200, and greater impact on behaviour, widely used gasoline oxygenates 400 ppm), decreased cytochrome growth, and development than in (ethyl tert-butyl ether and tert- oxidase activity is observed in the adults. 61 Prenatal expression of amyl methyl ether) by approxi - respiratory and olfactory epitheli - xenobiotic-bioactivating CYP mately 87%, these ethers being um following exposure to > or = enzymes in human OM suggests predominately metabolised through 30 ppm H 2S. Increased olfactory that the human foetal OM may be CYP of the rat olfactory mucosa. 63 epithelial sulphide concentrations a preferred target tissue for the Inhibition of CYP activities with are observed following exposure toxicity of maternally derived either metyrapone or carbon tetra - to 400 ppm H 2S. Cytochrome oxi - chemical compounds that are acti - chloride eliminates or significantly dase inhibition may be considered 61 vated by the CYP enzymes. reduces the olfactory toxicity of to be a sensitive biomarker of H 2S In the foetus, the level of beta,beta’-imino dipropionitrile in exposure in target tissues like the CYP2A13 mRNA is much higher the rat. 64 The dichlobenil-induced nose. 67 than that of CYP2A6 mRNA, as olfactory damage is accompanied When investigating the role of has been found previously in adult by a dramatic reduction in CYP metabolic activation in the olfac - nasal mucosa. Immuno histo- immunoreactivity in Bowman’s tory toxicity of methyl iodide chemical studies confirm that, in glands of the olfactory mucosa. (MeI), adult male rats were the foetus, the CYP2A proteins Finally, treatment of mice with exposed via nose-only inhalation are expressed in the supporting metyrapone or 3-aminobenzamide to 100 ppm MeI for 0-6 h, and cells in the olfactory epithelium (two CYP inhibitors) after a single non-protein sulphydryl (NP-SH) and in Bowman’s glands in the dichlobenil injection provides concentrations determined in lamina propria. some protection against the selected tissues. Depletion of NP- damaging effects of dichlo- SH occurred in all tissues, but was 3.1.3. In human diseases benil. 18,19 most marked and rapid in the res - Reactive oxygen species, which In vitro, with nasal and hepatic piratory epithelium of the nasal induce the expression of super - microsomes from rats and rabbits, cavity and the kidney. Olfactory, oxide dismutases, have been carbon monoxide binding and lung and liver NP-SH levels were implicated in the neurodegenera - hexamethylphosphoramide N- affected to a lesser extent. In order tion associated with Alzheimer’s demethylase activity have been to inhibit CYPs, animals were pre- disease (AD). 52 Individuals with found to be most sensitive to treated with cobalt protoporphyrin AD exhibit early, severe deficits in alkyl-substituted dioxolanes. IX. This reduced hepatic CYP olfactory ability. The pronounced Mono-oxygenase activity in the concentrations by > 90%, but increase in superoxide dismutase nasal mucosa is inhibited more when animals were then exposed immunoreactivity in the olfactory readily than that in the liver. 65 to 100 ppm MeI for four hours epithelium of AD subjects sug - The administration of 3,5- there was no effect on the severity gests that oxidative stress may be diethoxycarbonyl-4-ethyl-1,4- of the olfactory lesion. The 28 J.-B. Watelet et al.

Table 2 Instances in which nasal metabolism probably results in (a) detoxication or in (b) activation. Modified from Ding 59 Substrates Chemical structures Enzyme systems or enzymatic reactions A. Instances in which nasal metabolism probably results in detoxication Nitropyrenes Oxidases and hydroxylases

2,6-dichlorobenzonitrile Hydroxylases

Coumarin 7-hydroxylase

Cocaine Demethylation

Alkoxycoumarins Dealkylation

Lactones Carbooxylesterases

Styrene oxide Epoxide hydrolases

Naphthol Transferases

Chlorodinitrobenzene Transferases

Cyanide S-transferases (rhodanese)

Nicotine Demethylases

Formaldehyde Aldehyde deshydrogenases Defence mechanisms of olfactory mucosa 29

Table 2 Continuation B. Instances in which nasal metabolism probably results in activation 2,6-dichlorobenzonitrile Epoxygenase

Coumarin Epoxygenase Ferrocene

Oxidases

Benzo(a)pyrene Oxidases

Hexamethylphosphoramide Demethylases Ferrocene

Diethylnitrosamine Deethylases

Organonitriles Oxidases

Phenacetin Oxidases

Esters Carboxylesterases

Acetaminophen Oxidases

Trifluoromethylpyridine N-oxidases 30 J.-B. Watelet et al.

Table 3 authors’ conclusion was that GSH Major therapeutic agents susceptible to induce smell and taste disturbances conjugation of MeI is a detoxifica - Groups Agents tion pathway, and that there is no role for CYP in the development Antibiotics Ampicillin Azithromycin of toxicity, at least within the nasal Ciprofloxacin cavity. They suggest that, as MeI Clarithromycin is extremely effective at depleting Griseofulvin GSH, it is possible that those tis - Metronidazole Ofloxacin sues which suffer extensive GSH Tetracycline depletion and have slow rates of GSH turnover may become Anticonvulsants Carbamazepine Phenytoin vulnerable to oxidative insult due to prolonged GSH depletion. 68 Antidepressants Amitriptyline Clomipramine The prenatal human expression Desipramine of the CYP2A proteins in the Doxepin olfactory mucosa provides evi - Imipramine dence for the potential risks of Nortriptyline developmental toxicity associated Antihistamines and decongestants Chlorpheniramine with maternally derived xenobi - Loratadine otics, since both CYP2A6 and Pseudoephedrine CYP2A13 are known to be effi - Antihypertensives and cardiac medications Acetazolamide cient in the metabolic activation of Amiloride tobacco-specific nitrosamines and Betaxolol 69 Captopril other respiratory toxicants. Diltiazem Furthermore, recent kinetic, Enalapril immune-inhibition, and immuno - Hydrochlorothiazide and combinations blot data have confirmed that Nifedipine Nitroglycerin CYP2A13 is a functional enzyme Propranolol and the catalyst of 4-(methyl nitro - Spironolactone samino)-1-(3-pyridyl)-1-butanone Anti-inflammatory agents Auranofin (NNK) alpha-hydroxylation in Colchicine human foetal nasal mucosa. This Dexamethasone reaction is a key bio-activation Gold pathway in NNK-induced carcino - Hydrocortisone Penicillamine genesis. The results are also the first demonstration of high-effi - Antimanic drugs Lithium ciency NNK alpha-hydroxylation Antineoplastics Cisplatin in human tissue. 70 Reports indicate Doxorubicin Methotrexate that a significant level of GST Vincristine activity is located in the cytosol of olfactory and respiratory nasal Antiparkinsonian agents Levodopa mucosa in humans. The specific Antipsychotics Clozapine activity of this enzyme system in Trifluoperazine human nasal mucosa appears to be Antithyroid agents Methimazole higher (77 ± 21 nmol/min/mg, 1- Propylthiouracil chloro-2,4-dinitrobenzene used as Lipid-lowering agents Fluvastatin substrate) 71 than that reported for a Lovastatin Pravastatin number of extrahepatic tissues, suggesting the potential role of Muscle relaxants Baclofen nasal mucosa in the protection of Dantrolene the body against the toxic effect of Defence mechanisms of olfactory mucosa 31 compounds present in the inhaled air. On the other hand, by initially giving low doses of compounds that induce CYP metabolic enzymes, a protective status is induced and, subsequently, higher exposures produce little or no toxicity . For example, in humans, it has been shown that smokers are less susceptible to solvent-induced olfactory deficits than people who have never smoked. 72-74 3.3. Drugs used in the treatment of allergic rhinitis and adminis - tered by nasal route as subtrates/inhibitors/inducers of xenobiotic-metabolising enzymes present in the olfactory mucosa Figure 2 Suspected mechanisms for substrate transport by efflux transporters The reason for the absence of an extensive first-pass effect in the nasal mucosa for some drugs (e.g . and cell numbers of olfactory transport xenobiotics from bio- propranolol) is probably the fact neuro-epithelium and the expres - logical fluids into the cells). that only a few specific isozymes sion of OMP were all reduced Permeability-glycoprotein (P-gly - (phase I or phase II) are present more significantly in the group coprotein, P-gp, ABCB1 or in the nasal mucosa and that treated with dexamethasone. 77 MDR1) is an efflux pump, where - the others are not present or not This characteristic of dexametha - as organic anion transport poly - developed. sone treatment was associated peptides (OATPs), organic anion Through an increased with further deterioration in olfac - transporters (OATs) and organic Na(+)/K(+) ATPase expression in tory injury by 3-MI and recovery cation transporters (OCTs) are the regenerated olfactory mucosa, was achieved using a combination uptake transporters 78 (Figure 2). dexamethasone contributes to the treatment of dexamethasone and With some drugs, the affinity recovery of function after the mor - ginkgo biloba, probably explain - for transporters can confer posi - phological regeneration. This able by anti-oxidant effects. tive aspects. For example, a high mechanism involves, at least in affinity for the P-gp efflux pump part, its receptor by regulating the (which is also present at the 4. Efflux and uptake transporters ionic concentration in the olfac - blood-brain barrier (BBB)) might tory mucosal micro-environ - Transporters are membrane pro - explain the absence of central ment. 75 However, there is data to teins that can translocate endoge - nervous system side-effects asso - suggest that dexamethasone nous compounds (such as bile ciated with some H1 antihista - potentiates 3-methylindole (3-MI) acids, sugars, amino acids and mines. Differences in the ability of olfactotoxicity during the first hormones) and xenobiotics (such classical and modern antihista - 2 weeks after insult. This effect as drugs or toxicants) across bio - mines to interact with P-gp and may be due, at least in part, to the logical membranes to maintain other transport proteins at the inducing action of dexamethasone homeostasis and to detoxify any BBB may determine their CNS on the CYP responsible for the potentially harmful foreign sub - penetration and as a consequence metabolic bio-activation of 3- stances. Transporters are either the presence or absence of central methylindole. 76 Furthermore, in an efflux pumps ( i.e. they expel side-effects. 79-84 On the other hand, anosmia mouse model induced by xenobiotics out of the cells) or a high affinity for efflux trans - injection of 3-MI, the thickness uptake transporters ( i.e. they porters at the BBB may limit the 32 J.-B. Watelet et al.

Recently, both OCT1 and OCT2 have been localised in nasal mucosa. They may provide a route for the systemic absorption of cationic drugs. 89 A novel putative transporter, mouse OAT6, is expressed predominantly in the olfactory mucosa, but not in the kidney or brain. Sequence com - parisons and intron phasing analy - sis indicate that OAT6 is closely related to OAT1 and OAT3. In rats, the glucose transporter GLUT1, which mediates the spe - cific transfer of glucose across blood-brain, blood-cerebrospinal fluid and blood-nerve barriers, is abundant in occludin-positive Figure 3 cells. Both GLUT1 and tight junc - Localisation of transporters in the olfactory mucosa (a) in animals and (b) in humans tion protein occludin may serve as part of the machinery for the spe - cific transfer of glucose in the effectiveness of a drug when the porters in the nose is increasingly olfactory system while preventing intended site of action is the cen - available (Figure 3). the non-specific entry of sub - 90 tral nervous system. 4.1. Transporters in olfactory stances. The nasal mucosa and respira - mucosa Vomeromodulin, a glycoprotein tory tract may be an important synthesised by the lateral nasal point of entry for some anti-aller - 4.1.1. Animals glands, has been proposed as a gic drugs delivered by intranasal P-gp is present both in bovine pheromone transporter and func - administration or inhalation. olfactory and nasal respiratory tions as a chemosensory stimulus Transporters expressed in these mucosa, but expression seems to transporter associated with perire - tissues may therefore influence be greater in the olfactory epithe - ceptor processes in vomeronasal the pharmacokinetics of drugs lium than in the nasal respiratory and olfactory transduction. 91 In the administered in this way. epithelium. 85 It has also been rat, vomeromodulin mRNA and Drugs can be absorbed in the demonstrated in other species. 87 protein have been localised in nasal mucosa and throughout the P-gp was localised in the abundance in the glandular acini conducting airway from the tra - epithelial cells, nasal glands, and of the maxillary sinus component chea down to the bronchioles and the vascular endothelium of both of the lateral nasal glands. In addi - ultimately in the distal lung across the bovine olfactory and nasal tion, the vomeronasal and posteri - the alveolar epithelium. However, respiratory mucosae, and the or glands of the nasal septum, the most agents of pharmacologic expressed P-gp was capable of mucus of the sensory and non-sen - interest probably access the brain effluxing etoposide. Rates of sory epithelia of the vomeronasal via the olfactory epithelium, etoposide efflux were higher in organ, and the mucociliary com - which represents a more direct the olfactory mucosa than in the plex of the olfactory, respiratory, route of uptake. 85 nasal respiratory mucosa and the and associated rat nasal epithelia In the last 5-10 years, extensive staining density observed using also express vomeromodulin literature has been published immunohistochemistry suggests mRNA and protein. 92 Finally, in about hepatic, renal, intestinal that the expression of P-gp is rats, the expression of vomero - and brain transporters, 78,86 but greater in the olfactory epithelium modulin is dramatically upregu - interesting information con- than in the nasal respiratory lated by alachlor and butachlor, cerning the presence of trans - epithelium. 88 two chloracetanilide herbicides Defence mechanisms of olfactory mucosa 33 that can induce olfactory tumours Despite the presence of a number the roles played by metabolising in rats. 93 of protective barriers such as enzymes and transporters are only efflux transporters and metabo- partially understood and much 4.1.2. Humans lising enzymes in the olfactory fundamental data is missing, espe - P-gp is present in human nasal system, lipophilic compounds cially in humans. Unfortunately, respiratory mucosa, together with such as hydroxyzine and triproli - the animal models can only reflect 94,95 other transporters. The high- dine can access the CNS primarily some of the processes engaged by affinity transporter PEPT2 has by passive diffusion when admin - human olfactory mucosa because been found in the respiratory tract istered via the nasal cavity. 99 It has of the high inter-species variabili - and is expressed in the bronchial been reported that the lipophilicity ty in the content of metabolising epithelium and in alveolar type II of compounds such as hydroxy - enzymes and xenobiotic trans - 96 pneumocytes in human airways. zine and triprolidine, coupled with porters. However, a better under - It is possible, therefore, that this their ability to inhibit P-gp, enable standing of olfactory toxification/ may represent a target for the them to permeate freely across detoxification or the activation delivery of peptidomimetic drugs bovine olfactory mucosa. 99 It has of membrane transporters could and prodrugs. Its expression at the also been demonstrated that chlor - serve as a basis for the improve - level of upper airways and olfac - pheniramine and chlorcyclizine ment of existing treatment such as tory mucosa is still under investi - are effluxed from the olfactory intranasally administered drugs or gation. mucosa by efflux transporters for the development of novel ther - In human nasal mucosa, such as P-gp. 100 apeutic approaches. vomeromodulin immunoreactivity Furthermore, it has already is localised in the mucociliary been shown that topical steroids References complex of the vomeronasal and (e.g . budesonide) may increase the respiratory epithelia. 92 expression of P-gp in human nasal 1. Drettner B. The role of the nose in Finally, although monoamine mucosa. 95 the functional unit of the respiratory oxidase (MAO) has been reported It would be important to collect system. Rhinology . 1979;17:3-11. to be present in human olfactory additional information, in humans 2. Doty RL. The olfactory vector 97 hypothesis of neurodegenerative mucosa, the limited extent of in particular, about whether the dopamine metabolism should be disease: is it viable? Ann Neurol. drugs used in allergic rhinitis and 2008;63:7-15. considered in conjunction with administered by nasal routes are 3. Hastings L, Evans JE. Olfactory the strong activity of transporters substrates/inhibitors/inducers of primary neurons as a route of entry 98 observed at this level. Con se - the transporters found in the nose, for toxic agents into the CNS. Neuro toxicology quently, when administered intra - both to understand their nasal per - . 1991;12:707-714. nasally, dopamine can be trans - 4. Hastings L. Sensory neurotoxicolo - meability and to predict possible gy: use of the olfactory system in the ported in a nearly intact form 101,102 drug interactions. assessment of toxicity. Neurotoxicol through the BBB and then be Finally, in addition to trans - Teratol. 1990;12:455-459. metabolised exclusively in the porters, other substances such as 5. Deems DA, Doty RL, Settle RG, et central nervous system, which can ionic surfactants (sodium cholate, al . Smell and taste disorders, a study of 750 patients from the University be seen as the target organ here. sodium taurocholate, Tween 80 These findings should encourage of Pennsylvania Smell and Taste and Poloxamer F68) are potential - Center. Arch Otolaryngol Head Neck the researchers not to make an ly useful permeation enhancers for Surg . 1991;117:519-528. over-drastic distinction between the nasal delivery of hydrophilic 6. Potts CL. Cadmium Proteinuria – the their research on local metabo - compounds such as fexofenadine health of battery workers exposed to cadmium oxide dust. Ann Occup lising enzymes and research HCl. 103 looking at local transporter activity. Hyg . 1965;8:55-61. 7. Prah JD, Benignus VA. Decrements 4.2. Drugs used in the treatment Conclusions in olfactory sensitivity due to ozone of allergic rhinitis and adminis - exposure. Percept Mot Skills . 1979; tered by nasal route as Olfactory mucosa has different 48:317-318. 8. Yamagishi M, Hasegawa S, substrates/inhibitors/inducers of strategies for defending itself Nakano Y. Examination and classifi - transporters present in the olfac - against toxic environments. The cation of human olfactory mucosa in tory mucosa regeneration of the epithelium and patients with clinical olfactory dis - 34 J.-B. Watelet et al.

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