in vitro models of nasal cavity toxicity

Ž .Mutation Research 380 1997 97–111

In vitro models of nasal cavity toxicity

Celia J. ReedSchool of Biomolecular Sciences, LiÕerpool John Moores UniÕersity, Byrom Street, LiÕerpool L3 3AF, UK

Received 23 June 1996

1. Introduction

The effects of a xenobiotic on cellular integrityand function can be investigated at several differentlevels of complexity, and the particular model sys-tem chosen depends on a number of factors includ-ing what is known about the chemical of interest, itsmetabolism and mode of action, and also the type ofinformation that is required. Historically, the major-ity of toxicological studies, both regulatory and aca-demic, involved the use of whole animals. Today,we are influenced by the concept of the three R’s of

Žhumane animal use in research – replacement utili-.sation of models which do not involve live animals ,

Ž .reduction use of fewer animals and refinementŽ .minimisation of animal suffering . Thus in vitrosystems have found widespread use, not only be-cause of such ethical considerations, but also becauseof decreased costs and the absence of complexphysiological interactions which are encountered invivo and which may confound experimental interpre-tation.

A variety of different types of in vitro systems areavailable to the toxicologist, ranging from isolated,perfused organs, through to subcellular fractions andpurified enzymes. Each of these systems has its ownadvantages and disadvantages, and its own range ofuseful applications. Perfused organs are closest to thein vivo situation and thus easiest to extrapolate fromw x1,2 . However, an animal generally produces a sin-gle tissue preparation of limited viability. Tissue

slicesrexplants which maintain cell–cell interactionsare generally viable for longer than perfused organs,and many preparations may be obtained from one

w x Žanimal 3–5 . Cultured cells either primary or trans-.formed are convenient to work with, yield repro-

ducible results, are viable for considerable periods oftime and lead to significant reduction in animal

w xusage 6–8 . However, tissue architecture and cell–cell interactions are lost and dedifferentiation, result-ing in the loss of specific functions, may occurw x9,10 . Subcellular fractions and purified enzymesare ideal for studies of xenobiotic metabolism andmay be useful for detection of the toxicity of a

Ž .compound s whose mechanism of action is clearlyunderstood, but they have little general, predictivevalue.

In the last 15 years, a wide variety of chemicalsof both industrial and environmental importance havebeen shown to cause lesions and tumours in the nasalcavities of experimental animals. In vitro studieshave provided considerable mechanistic information,but at present there is no in vitro model that is of usefor prediction of effects in vivo. This is partly due tothe short time that has elapsed since the discovery ofthe nose as a target organ, but more importantly tothe morphological and physiological complexities ofthe nasal cavity which are discussed below.

Ž .1 Within the nasal passages, the convoluted,cartilaginous turbinates are lined with three differenttypes of epithelium – respiratory, transitional andolfactory, and frequently chemicals induce lesions in

0027-5107r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0027-5107 97 00129-2

( )C.J. ReedrMutation Research 380 1997 97–11198

specific regions of the nasal cavity. Thus a range ofŽwater-soluble, gaseous irritants including chlorine,

.hydrogen chloride, dimethylamine and acroleindamage the olfactory epithelium of the dorsal meatusand the respiratory epithelium adjacent to the nasalvestibule and lining the naso- and maxilloturbinatesw x11 . In contrast, ozone is principally toxic to transi-

w xtional epithelium 12,13 , whilst the lesions inducedw xby dichlobenil 14 , glycol ether acetate or acrylate

w x w xesters 15,16 , 3-trifluoromethylpyridine 17 , methylw x Ž . w x w xiodide 18 , nickel II 19 , methimazole 20,21 and

w xpyridine 22 are generally restricted to areas ofolfactory epithelium.

Ž .2 Within a single tissue type there may bemarked cellular heterogeneity. Thus the olfactoryepithelium consists of four major cell types – olfac-tory neurones, sustentacular cells, basal cells andsubepithelial or Bowman’s glands. Compounds such

Ž .as methyl iodide and nickel II appear to damagew xsustentacular cells in the first instance 18,19 , whilst

the Bowman’s glands are the targets for chloroformw x23 . Maintenance in vitro of the morphology andfunction of the olfactory epithelium is particularlychallenging since the olfactory neurones die follow-

w xing olfactory nerve section 24 .Ž .3 The nasal epithelia have been shown to be

active in the majority of both phase 1 and phase 2Žxenobiotic biotransformation reactions for recent re-

w xviews see 25–27 , and the olfactory epithelium con-tains novel olfactory-specific forms of cytochrome

w x w xP450 28,29 and UDP-glucuronosyltransferase 30 .Nasal drug metabolising enzymes play a critical rolein the in situ activation and detoxification of xenobi-otics, and thus their activities must be maintained inany predictive in vitro system.

Ž .4 Lesion distribution within the nasal cavity isdependent not only on the epithelial type and locali-sation of biotransformation enzymes, but also oncompound distribution which is determined by thephysicochemical properties of the compound, bynasal airflow patterns during inhalational exposurew x31 , and by blood flow during parenteral exposure.

Thus any in vitro model which is to be trulypredictive will have to overcome the problems posedby the epithelial and cellular heterogeneity of thenasal cavity and the regional distribution of com-pounds within this region. This review will focus onin vitro studies of the nasal cavity which are of

toxicological relevance, and will discuss the progressthat has been made to date in the development of apredictive nasal model.

2. Studies with subcellular fractions and purifiedenzymes

2.1. Xenobiotic biotransformation and its role innasal toxicity

Over the last 15 years, significant progress hasbeen made in the identification and characterisationof nasal xenobiotic biotransformation enzymes. Thus

w xthe glutathione S-transferases 32,33 and cy-w xtochromes P450 34–38 have been extensively char-

acterised, and there is considerable information re-garding other enzymes, such as the car-boxylesterases, UDP-glucuronosyltransferases, epox-ide hydrolase, formaldehyde dehydrogenase and rho-danese. For recent reviews in this area, the reader is

w x w xreferred to Dahl and Hadley 25 , Reed 26 and Dahlw x27 . These studies have, in general, used subcellularfractions prepared from discrete regions of the nasalcavity. There is relatively little information on puri-fied enzymes due to the inherent difficulties associ-ated with purifying proteins from the nasal cavityŽ .low levels and paucity of tissue . However, two

Ž .cytochromes P450 NMa and NMb have been puri-fied from rabbit olfactory epithelium and theirmetabolism of several known nasal toxins demon-

w xstrated 28,34,38–41 . NMa represents two geneproducts, CYP2A10 and CYP2A11, which have beenheterologously expressed and the metabolic activity

w xof the proteins investigated 35,42 . Furthermore, theŽ .rat ortholog of NMb CYP2G1 has been cloned

w x w x29,43 and transiently expressed in COS cells 43 .Work is currently underway to express this protein ina bacterial expression system, and once this has beenachieved significant advances in our understandingof this olfactory-specific cytochrome P450 and itsrole in nasal toxicity will become possible.

A knowledge of the capability of a tissue tometaboliseractivate chemicals is critical with regardto understanding mechanisms of toxicity. Table 1lists some of the xenobiotics which have been shownto be metabolised within the nasal epithelia andwhich are also nasal toxinsrcarcinogens. This is not

( )C.J. ReedrMutation Research 380 1997 97–111 99

meant to be a comprehensive review of such com-pounds, but rather a selection to illustrate the role ofin situ activation in nasal toxicity. In many cases, therole of a particular enzyme system andror isoen-zyme in this metabolism has been confirmed throughcofactor requirements, the use of specific

w xinhibitorsrinducing agents etc. Smith et al. 44Ždemonstrated that rat nasal metabolism of 4- methyl-

. Ž . Ž .nitrosamino -1- 3-pyridyl -1-butanone NNK wasNADPH-dependent and inhibited by carbon monox-ide, metyrapone and a-naphthoflavone, indicating arole for cytochromes P450. Furthermore, the use ofinhibitory antibodies to a variety of cytochrome P450isoenzymes implicated CYP1A2 in the metabolismof NNK. Similarly, we have recently demonstratedthat conjugation of methyl iodide with glutathioneoccurs within rat olfactory epithelium, and appears tobe catalysed by members of the theta class of glu-

w xtathione S-transferases 45 .If one is investigating a compound whose

metabolism and mechanism of toxicity in anotherorgan is well characterised, then demonstration ofthe formation within the nasal cavity of the putativetoxic metabolite may be sufficient to confirm themechanism of nasal toxicity. However, with com-pounds whose toxicity is less well understood or isrestricted to the nasal cavity, studies using subcellu-

lar fractionsrpurified enzymes are less informative.If metabolic activation of a chemical results in cova-lent binding of a metabolite to cellular macro-molecules, and if it is assumed that such covalentbinding is an initial event in the cascade of effectsleading to cytotoxicity, then routes andror rates ofnasal metabolism may be manipulated in vitro and

Ž .the effects on covalent binding toxicity determined.Thus preincubation of olfactory microsomes withsodium diethyldithiocarbamate or metyrapone, bothinhibitors of cytochromes P450, reduced the covalent

w xbinding of dichlobenil to the microsomes 46,47 ,suggesting cytochrome P450-dependent activation ofthis herbicide within the nasal cavity. However, co-valent binding is not necessarily causally related to

w xtoxicity 48 , and the major drawbacks of the use ofsubcellular fractionsrpurified enzymes in mechanis-

Ž .tic work are that: 1 studies tend to focus on the roleof a single enzyme system, whilst the majority ofchemicals are metabolised via multiple, and often

Ž .competing, pathways; and 2 no pathology is avail-able to confirm the relevance of the findings to themechanism of toxicity.

2.2. Mutagenicity assays

ŽBiotransformation of many promutagens for ex-ample, polycyclic aromatic hydrocarbons, ni-

Table 1Some of the xenobiotics which induce a carcinogenic or toxic response within the nasal cavity and which undergo biotransformationcatalysed by nasal drug metabolising enzymes

Enzyme system Compound Species Ref.

w xCytochromes P450 NNK Rat 44w xDiethylnitrosamine Rat, Syrian hamster 101w xN-Nitrosonornicotine Rat 81w xAflatoxin B Cow 921w x3-Trifluoromethylpyridine Rat 86

w x w xBenzo a pyrene Syrian hamster, rat 102,103w xHexamethylphosphoramide Rat 104w xPhenacetin Rat, rabbit 82w xDichlobenil Rat 46

X w xb,b -Iminodipropionitrile Rat 105

w xGlutathione S-transferases Alachlor Rat, squirrel monkey 106w xMethyl iodide Rat 45

w xCarboxylesterases Dibasic esters Rat 107w xVinyl acetate Rat, mouse 108

w xFlavin-containing monooxygenase Methimazole Rat 21

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Table 2Nasal cell isolation and culture methodologies

Species Age Cell isolation procedure Culture conditions Comments Ref.

w xHuman Adult Pronaserprotease digestion and mechanical Seeded on irradiated feeder cells Allows multiple passages of respiratory 109,54Ž .disaggregation of nasal polyps. in DMEMrHam’s F12 1:1 supplemented epithelial cells.

with FCS, EGF, HC, I, TF, sodiumselenite and antibiotics.

w xHuman Adult Pronase digestion and mechanical Plated on rat tail collagen gel Monolayer or suspension cultures, 110,65Ž .disaggregation of nasal polyps. in DMEMrHam’s F12 1:1 supplemented ciliogenesis occurred in suspension.

with cholera toxin, retinoic acid,Ultroser or NUSerum and antibiotics.

Human Adult Dispase digestion of excised and chopped Cultured at 378C in DMEMrHam’sturbinates. Then trypsinrEDTA digestionof sheets of epithelial cells.

w xConfluent cultures of respiratory 43Ž .F12 1:1 supplemented with glutamine, cells exposed to ozone.

HEPES buffer, NuDerum, antibiotics andantimycotics.

w xHuman Adult Walls of nasal turbinates brushed. Cell clusters cultured at 308C in Ham’s Cells formed multi-cellular spheroids 111F-12 I, HC, triiodothyronine, EGF, with synchronous ciliary beat.endothelial cell growth supplement, Non-mitotic.and cholera toxin.

6 w xHuman Adult Turbinates excised and sliced Slices cultured at 378C in DMEMrHam’s 2–3=10 cells obtained from 662Ž .F12 1:1 supplemented with NuSerum, 0.8–1.2 cm of tissue. Cells exposed

HEPES buffer, glutamine, antibiotics to sulphur dioxide.and antimycotics. Once confluence reachedŽ .14 days cells passaged.

Human Post- Turbinates excised and sliced. Immobilisedmortem in reconstituted basement membrane.Ž .-14 h

w xCultured in Coon’s modified Ham’s Cells grew out after 1–4 weeks, only 55F12 medium, neuroblast formulation, neuroblasts proliferated after 2–3 weeks.plus FCS, bovine hypothalamus extract, Clonal selection of neuroblasts.bovine pituitary extract, I, TF, HC,thyroxine, selenious acid and antibiotics

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w xRat Newborn Endoturbinates excised and minced. Cultured at 378C in minimal medium Cultures consist of basal cells and 59Trypsin and collagenase digestion followed supplemented with amino acids and neurones. EGF promotes basalby mechanical disaggregation. other essential nutrients plus I, TF, cell proliferation, TGF-b2

BSA, EGF, TGF-bs, sodium selenate promotes neurogenesis.and antibiotics.

Rat Newborn Turbinates and septum excised. Newborn: Cultured at 378C in Ham’s F10 supplemented Basal cells and neurones. Basal cellsand adult tissue minced. Adult: mucosa stripped. with glutamine, heat inactivated horse

Trypsin and collagenase digestion, serum, FCS, Nystatin, HC, dibutyrylŽand mechanical disaggregation. cyclic AMP, antibiotics and GH rat3

.pituitary somatomammotroph tumour cellsconditioned medium.

w x57maintained for months but could notbe passaged. Few sustentacular cells,lost after 1 week.

w xRat Adult Protease, collagenase and hyaluronidase Cultured in serum-free Ham’s In situ digestion gives high yields 112–114digestion – either excised turbinates F12 plus I, EGF, bovine hypothalamus with minimal fibroblast contamination,or in situ. extract, cholera toxin, HC and TF. but contains mixed populations nasal

epithelial cells.

w xRat Adult Ethmoturbinates excised. trypsinrDNase Cultured for 24 h at 378C in DCCM 1 Enrichment of metabolically competent 61digestion and mechanical disaggregation. plus Ultroser G in ECM-coated plates. sustentacular cells.Low-speed centrifugation to enrichsustentacular cells.

DMEM, Dulbecco’s modified Eagle’s medium; FCS, foetal calf serum; EGF, epidermal growth factor; HC, hydrocortisone; I, insulin; TF, transferrin; BSA, bovine serumalbumin; TGF-b, transforming growth factor-b; SMEM, suspension-minimum Eagle’s medium; ICM, extracellular matrix.


.trosamines and aromatic amines is essential for themutagenicrcarcinogenic responses of tissues to thesexenobiotics. A number of in vitro studies to assessthe capability of subcellular fractions prepared fromnasal tissue to bioactivate known carcinogens tomutagenic species have been documented. Bond usedrat nasal S9 in the Ames Salmonellarmammalianmicrosome assay for the detection of mutagens, and

w x w xshowed that 1-nitropyrene 49 , benzo a pyrene andw x2-aminoanthracene 50 could all be activated to

mutagenic species by nasal S9, and at rates that werecomparable to those found with liver S9. Similarly,

w xDahl 51 demonstrated the bioactivation of a num-ber of nitrosamines, most of which are rat nasalcarcinogens, using rat and rabbit nasal S9. Thesestudies concluded that carcinogenicity within thenasal cavity may not be reliably predicted frommutagenicity assays using hepatic tissue, and thatsuspected nasal carcinogens and compounds admin-istered via inhalation should be tested with nasal S9in the Ames test.

Nasal tissue has also been used in mutagenicityw xassays involving mammalian cells. Lawson 52 used

V79 cells to investigate the mutagenicity of the ratŽ .nasal carcinogen N-nitrosobis 2-oxopropyl amine,

w xwhile Tjalve et al. 53 showed that bovine olfactoryepithelium could activate aflatoxin B to species that:1Ž . Ž .1 covalently bind to DNA and protein; 2 are

Ž .mutagenic in Salmonella typhimurium; and 3 in-duce sister chromatid exchange in Chinese hamsterovary cells. Nasal tumours which develop from theolfactory epithelium are endemic in cattle of severaldeveloping countries, including southern India, and ithas been suggested that aflatoxins may be the

w xcausative agent. Tjalve et al. 53 demonstrated thatbovine liver had a much lower capacity to bioacti-vate aflatoxin B , and their studies support the role1

of aflatoxins in the aetiology of bovine nasal tu-mours.

3. Culture of nasal epithelial cells

3.1. Methodology for isolation and culture of nasalcells

A variety of different methodologies for the isola-tion and culture of nasal epithelial cells of both

human and animal origin have been described, andsome of these are summarised in Table 2. In general,enzyme digestion and mechanical disaggregation areused to prepare single-cell suspensions from excisedtissue, and these cells are then cultured in mediumsupplemented with antibiotics, antimycotics and vari-ous other components including insulin, hydrocorti-sone, transferrin, cholera toxin and epidermal growthfactor. The length of time that the cells remain viableappears to depend upon the origin of the tissue andthe cell types involved. Thus human nasal epithelialcells isolated from polyps could be maintained in

w xculture for up to 4 months 54 and neuronal cellcultures prepared from human cadavers maintained

w xgrowth for 3–6 months 55 . In contrast, in cellcultures derived from embryonic mouse olfactoryepithelium, all cells except the basal cells died after

w x w xabout 7 days 56 , and Pixley 57 found that, incultures derived from rat olfactory epithelium, veryfew neurones survived for more than 4 days. Neuro-genesis can apparently be prolonged by fibroblast

w xgrowth factors 58 and transforming growth factor-w xbs 59 , and recent work has demonstrated that zinc

sulphate-induced chemical trauma of the olfactoryepithelium in vivo increases the growth of olfactoryneurones when the tissue is subsequently maintained

w xin vitro 60 . Sustentacular cells appear to be particu-larly sensitive to the culture conditions, being rarely

w xseen and viable for less than a week 57 .w xEvans et al. 61 have described methodology for

the isolation of rat olfactory cells enriched withsustentacular cells, which they maintained for up to24 h in culture. The viability and metabolic potentialof these cells was not determined, but similar cul-tures had previously been found to biotransform the

w xolfactory toxin 3-trifluoromethylpyridine 62 . Unfor-tunately, relatively low yields were obtained, namely8–19=106 cells from 6 rats. Since toxicityrmetabolism studies generally use 0.5–1=106 cellsper incubation, such cultures are unlikely to be ofsignificant use to the toxicologist, unless the cellscan be induced to multiply and divide. In addition,further work is required to examine the long-termviability, maintenance of biotransformation, dediffer-entiation etc. of such cultures.

As is the case with many long-term cell culturesystems, nasal epithelial cells tend to dedifferentiate

w xin culture, and Wu et al. 63 reported that within a


week, human nasal epithelial cells lost their cilia andmucous granules. However, using denuded rabbittracheal graft as a vehicle, these cells reexpressedtheir mucociliary functions in nude mice, indicatingthat they retained the potential to differentiate, or tobecome the precursors for fully differentiated cellsw x63 . In contrast, the human nasal epithelial cells

w xcultured by Werner and Kissel 64 expressed mi-crovilli and actively beating cilia and Jorissen et al.w x65 described a culture system in which ciliogenesisoccurs. Cells were first plated on rat tail collagen andthen, after all cilia had disappeared, they were treatedwith collagenase to release sheaths of deciliated cellswhich were subsequently maintained in suspensionculture.

3.2. Nasal cells and toxicology

Relatively little toxicological investigation of nasalcell cultures has been carried out. McManus et al.w x66 have demonstrated the effects of sulphur dioxideon human nasal epithelial cells grown to confluence.

Ž .Short-term exposure 30–60 min to sulphur dioxideat concentrations known to produce physiological

Ž .effects in normal subjects 1–5 ppm significantlyw3 ximpaired cellular H leucine incorporation, whilst

there was no evidence of cytotoxicity as judgedmorphologically or by release of soluble 51chro-mium. In a similar system, ozone has been shown to

w xbe cytotoxic 67,68 , increasing the release of lactatedehydrogenase and 51chromium from cultured hu-man nasal epithelial cells. Recently, Pool-Zobel et al.w x69,70 used the ‘comet’ assay for detection of DNAdamage to assess the genotoxic effects of lindaneusing human and rat nasal mucosal cells, and foundthat it was indeed DNA damaging. Kuykendall et al.w x71 demonstrated cytochrome P450-dependent for-mation of DNA–protein crosslinks in rat nasal ep-ithelial cells as a result of exposure to the nasalcarcinogen hexamethylphosphoramide.

In addition, a number of studies have investigatedthe effects of xenobiotics on the ciliary beat fre-quency of human nasal cells in vitro. Thus naphazo-

Žline, oxymethazoline and xylometazoline nasal de-. w x Žcongestants 72,73 , cotitine a major metabolite of

. w x w xnicotine 74 , cocaine and lidocaine 75 have allbeen shown to be ciliotoxins.

4. Studies with intact nasal epithelium

Intact nasal epithelium can be obtained either byexcising the turbinates and using them whole orsliced, or by carefully strippingrscraping the mucosaaway from the underlying tissue. Which approach ischosen will depend, to a large extent, upon theanimal species being studied – for small rodents,such as rats and mice, it is easy to dissect out awhole turbinate and little damage is done to theepithelium, for larger animals, such as rabbits anddogs, stripping the epithelium may be the simplestoption.

4.1. Mechanistic studies

A number of groups have used excised turbinates,either whole or sliced, or nasal septa in studiesdesigned to elucidate mechanisms of toxicity withinthe nasal cavity. Thus Brittebo and coworkers haveexamined the metabolism andror covalent bindingof a variety of toxins including N-nitrosodiethyla-

w x w xmine 76,77 , N-nitrosopyrrolidine 78 , N-nitroso-w x w xdibutylamine 79 , N-nitrosonornicotine 80,81 ,

w x w xphenacetin 82,83 , carbon tetrachloride 84 andw xchlorobenzene 85 . For example, Brittebo and

w xAhlman 82 demonstrated that slices of rat or rabbitnasal mucosa were able to metabolise the nasalcarcinogen phenacetin more rapidly than any of theother tissues examined, and that this metabolism wasinhibited by both metyrapone and SKF525A, indicat-ing a role for cytochromes P450. In similar studies,excised ethmoturbinates from rats have been shownto accumulate the olfactory toxin 3-trifluoromethyl-pyridine and to metabolise it to its toxic N-oxidederivative. Both processes were decreased by in-

w xhibitors of cytochromes P450 86 .w xTrela and Bogdanffy 87–89 used a rat nasal

Ž .explant system excised turbinates to investigate thetoxicity of dibasic esters, a solvent mixture ofdimethyl adipate, dimethyl glutarate and dimethylsuccinate, which are all substrates for nasal car-boxylesterases. Dibasic esters induce degeneration ofolfactory, but not respiratory, epithelium in the rat,and female animals are more sensitive that malesw x w x90 . Trela and Bogdanffy 87 demonstrated concen-tration-dependent release of acid phosphatase from


the nasal explants following incubation with all threeesters, and this was more marked in incubationscontaining olfactory compared to respiratory epithe-

Ž .lium Fig. 1a . Metabolism to the monomethyl esterand diacid metabolites also occurred to a greater

Ž .extent in olfactory epithelium Fig. 1b , and there

Fig. 1. Toxicity and metabolism of dimethyl adipate in nasal explants. a,b: rat nasal explants were incubated for 2 h in William’s E-mediumŽ . Ž .containing 0–100 mM dimethyl adipate DMA . Media aliquots were assayed for acid phosphatase AcP release or production of

Ž . Ž . Ž . Ž .monomethyl adipate MMA and adipic acid AA . c,d: nasal explants from rats pretreated with bis p-nitrophenyl phosphate BNPP wereincubated as above with 50 mM DMA. Values are means"SE for at least four tissues prepared from four animals. MT, maxilloturbinate,

w xET1, endoturbinate-1. Adapted from Trela and Bogdanffy 87 .


was good correlation between acid phosphatase re-lease and metabolism, indicating a role formetabolism in the observed toxicity. Finally, inclu-

Ž .sion of bis p-nitrophenyl phosphate, a car-boxylesterase inhibitor, in the incubation system notonly inhibited metabolism, but also protected against

Ž .toxicity Fig. 1c,d . In further studies, they demon-strated the cytotoxicity of metabolites of dibasic

w xesters in rat nasal explants 88 . Thus the in vitronasal explant system was not only able to model thetoxicity seen in vivo, but also provided significantmechanistic information. More recently Bogdanffyand coworkers have used a similar in vitro system todemonstrate the carboxylesterase-dependentmetabolism and toxicity of the nasal carcinogen vinyl

w xacetate 91 .Since rodents are the most commonly used species

in toxicology, it is hardly surprising that little workusing stripped mucosa has been carried out. In one

w xstudy, Larsson et al. 92 investigated the metabolismof aflatoxin B in bovine liver slices and olfactory1

epithelium that had been scraped away from thecollagenous submucosa. They demonstrated differ-ences in the metabolic profiles of the two tissues and

the avid formation of tissue-bound metabolites withinolfactory epithelium, results which support the hy-pothesis that exposure to aflatoxin contaminated feedmay be an important factor in the development ofethmoid carcinoma in cattle. However, stripped ep-ithelium is ideal for investigation of transport acrossnasal mucosa. Several groups have mounted strippedovine or rabbit nasal epithelium in Ussing chambersand studied the absorption of insulin and other modelcompounds, and the effects of enhancers on this

w xprocess 93–95 .

4.2. DeÕelopment and characterisation of an in Õitronasal model for prediction of upper respiratory tracttoxicity

The studies described above have all provideduseful mechanistic information, but none of them hasattempted to validate their in vitro system. We haverecently started a program of work aimed at thedevelopment, characterisation and validation of an invitro nasal model. Excised turbinates were chosenfor this work since tissue architecture, cell–cell com-munication and cellular heterogeneity are all main-

Ž .Fig. 2. Characterisation of rat nasal turbinates maintained in culture for 24 h. ATP, potassium and non-protein sulphydryl NPSHŽ . Ž .concentrations, and rates of protein synthesis were monitored in olfactory ??? and respiratory ??? epithelium. Values are means"SE of

six turbinates.


tained in such a system, which is therefore closer tothe in vivo situation than disaggregated cells.

Initial studies aimed to optimise culture condi-Žtions for rat nasal ethmoturbinates predominantly

.olfactory epithelium and naso- and maxillo-Ž .turbinates predominantly respiratory epithelium , us-

ing simple equipment which is readily available inŽall laboratories a shaking waterbath with a gassing

.hood . For both epithelia, the optimum conditions ofthose tested were to maintain the tissue at 318Cunder an atmosphere of 95% O :5% CO , in 6-well2 2

plates containing 2 ml of William’s E-medium sup-Ž .plemented with glutamine 2 mM and gentamycin

Ž . Ž100 mgrml , with shaking at 60 strokesrmin am-. w xplitude 3.5 cm 96,97 . Lactate dehydrogenase re-

lease over a 24-h period under these conditions was40 and 30% for olfactory and respiratory epithelium,respectively. The culture system was then furthercharacterised with respect to a number of viabilityparameters which reflect different aspects of cellularintegrity and function, namely ATP, potassium andnon-protein sulphydryl concentrations and rates of

Ž .protein synthesis Fig. 2a–d . The results indicatethat rat nasal tissue is viable in this culture systemfor up to 24 h.

As discussed earlier, a critical feature of any invitro system for toxicological studies is the mainte-nance of xenobiotic biotransformation capability.Thus we have also characterised our explant systemwith respect to the activity of a number of importantdrug metabolising enzymes over 24 h in culture.Whereas carboxylesterase, glutathione S-transferaseand UDP-glucuronosyltransferase activity remainedessentially constant over this culture period, NADPHcytochrome c reductase activity declined steadily

Žand cytochrome P450 activity with 7-.ethoxycoumarin as substrate decreased dramatically,

w xbeing virtually undetectable by 8 h 98 . The reasonsfor this decline in cytochromes P450 are currentlyunder investigation, as are ways of maintaining activ-ity in culture. Although cytochrome P450-dependentmetabolism may only occur during the first fewhours of exposure of the tissue to a potential toxin,this may be sufficient for either activation or detoxi-fication and thus correct prediction of in vivo toxic-ity.

The next stage in the development of this modelis validation using compounds that are selectively

toxic to different regions of the nasal cavity andwhich exert their toxicity either directly or followingbioactivation. This work is ongoing and will corre-late in vitro and in vivo data in order to test thepredictive accuracy of the model. It is anticipatedthat this system will be of use not only in basicmechanistic research, but also as a screen for poten-tial upper respiratory tract toxins.

5. The way forward?

If it is accepted that there is a need for in vitromethodology to study toxicity within the nasal cavityof experimental animals, then there are a number ofquestions that need to be considered during thedevelopment of such methodology.

Firstly, what system will fulfil the criteria ofphysiological relevance, reduction in animal usage,decreased costs and simple application? In the near

Žfuture, intact tissue either excised turbinates or.stripped epithelium may be the system of choice as

tissue architecture and cell–cell communication ismaintained and relatively large amounts of tissue canbe obtained from a single animal. For example, one

Žrat will provide 12 turbinates 8 covered with olfac-.tory epithelium, 4 with respiratory which can be

cultured individually, and may even be cut intopieces to give a greater yield of tissue. Furthermore,significant advances have been made in the develop-ment and characterisation of a model using intact

Žtissue from the rat, and its usefulness is proven see.Section 4 . However, if long-term cell culture sys-

tems which maintain the cells in a sufficiently differ-entiated state were to be developed, even greaterreduction in animal numbers could be achieved, thedifferent cell types could be investigated individu-ally, and large numbers of replicate incubations couldbe carried out, thus minimising the variability inresults. Therefore it is important that efforts to suc-cessfully culture nasal cells continue.

The majority of the work aiming to develop nasalepithelial cell cultures has been motivated by a de-sire to understand the processes of neurogenesis, iontransport and inflammation in this tissue, and cultureconditions have been specifically tailored to produceuseful model systems. It may be inappropriate tomerely adopt one of these models for use in toxicol-


ogy, and the question of the most relevant cellularmodel for predictivermechanistic toxicity studies inexperimental animals must be addressed.

As discussed earlier, there is remarkable site-specificity in lesion distribution within the nasalcavity, some agents damaging respiratory epitheliumwhilst others are toxic to olfactory epithelium. How-ever, lesions restricted to respiratory epithelium aregenerally caused by direct-acting toxins, and therespiratory epithelium is the target because it isexposed to the highest concentrations of compound.Olfactory epithelium may prove to be equally sensi-tive to such agents in vitro. In contrast, olfactoryepithelium is usually targeted by those compoundsthat require metabolic activation in order to exerttheir toxic effects and, since there is relatively littlebioactivation capacity within respiratory epithelium,this tissue would not be appropriate for detection ofsuch protoxins. Therefore, cells derived from olfac-tory, rather that respiratory, epithelium may providethe most widely relevant model.

There are four major cell types that are importantin olfactory epithelium, namely sensory, sustentacu-lar and basal cells, and the subepithelial Bowman’sglands. It is likely that all four types will be sensitiveto direct-acting toxins, but olfactory biotransforma-tion is predominantly localised to the sustentacularcells and Bowman’s glands and thus only these cellsmay be damaged by protoxins. Thus it is logical toconclude that the most useful nasal cell culture willbe one composed of cells derived from sustentacularcells or Bowman’s glands. However, such a culturecannot be expected to provide 100% predictive accu-racy – although the sustentacular cells are frequentlythe primary cellular targets, there are compounds thatspecifically damage the sensory cells, possibly be-cause the latter are particularly sensitive to toxicinsult.

Having developed an appropriate culture system,what indices of toxicity should be monitored? Aswith the majority of in vitro work, investigationswith nasal tissue have to date focused primarily ongeneral parameters reflecting cytotoxicity, for in-stance release of intracellular enzymes, or rates ofprotein synthesis. The heterogeneity of the nasalepithelia and the existence of cell-specific macro-moleculesrbiochemical pathways open up possibili-ties for the study of markers which will indicate the

primary cellular target. For instance, carnosine andcarnosine synthetase are preferentially localised

w xwithin olfactory neurones 99 , whereas carnosinasew xis found within the sustentacular cells 99 and acidic

sulphomucins are markers for the Bowman’s glandsw x100 . Furthermore, it would be beneficial to monitorparameters which reflect the functional integrity ofthe tissuercell type, for example ciliary beat, secre-tion of mucous or inflammatory mediators, and re-sponsiveness to odorants.

6. Conclusion

Over the last decade, investigations using in vitromodels of the nasal cavity have contributed signifi-cantly to our understanding of mechanisms of toxic-ity within this target tissue. Subcellular fractions andpurified enzymes have proved invaluable formetabolic studies, but more physiologically relevant

Ž .systems intact tissue andror cells are required forthe prediction of toxic effects. Much work remains tobe done before such a predictive model is achievedand validated. Considering the structural complexityof the nasal cavity, and the number of factors whichcontribute to nasal toxicity, but which may provedifficult to model in vitro, for example compounddistribution, it is unlikely that we will ever be able toreplace in vivo inhalation studies with in vitro exper-iments.

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