(CANCER RESEARCH 46. 5438-5443. October 1986]

Xenobiotic Metabolism in Human Alveolar Type II Cells Isolated by Centrifugal

Elutriation and Density Gradient CentrifugationTheodora R. Devereux,1 Thomas E. Massey, Michael R. Van Scott, James Yankaskas, and James R. Fouts

laboratory of Pharmacology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709 [T. R. £>.,T. E. M., J. R. F.],and Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514 [M. R. V., J. Y.]

ABSTRACT

Alveolar type II cells were isolated from five human lung specimensobtained during resection or lobectomy and enriched to 63-85% purity.Digestion with Sigma protease type XIV followed by centrifugal elutria-tion and Percoli density gradient centrifugation yielded 1.2 ±0.4 x IO1'

cells/g lung in the type II cell fractions. The activities of some enzymesinvolved in the metabolism of \enobiolics were determined in thesefreshly isolated type II cells and compared with activities in alveolarmacrophages and fractions of unseparated cells from the same tissuesamples. Reduced nicotinamide adenine dinucleotide phosphate-cyto-chrome c reducíaseactivity was similar in the three cell fractions fromall five patients (18-29 nmol/mg protein/min). An antibody to rabbitreduced nicotinamide adcnine dinucleotide phosphate-cytochrome P-450reducíaseinhibited reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reduction as much as 70% in microsomal preparations ofthe isolated human pulmonary cells, although this same antibody barelyreacted with microsomes of the human cells in a Western blot assay.Kpoxidc hydrolase activity was highest in the alveolar type II cells (1.08±0.17 nmol/mg protein/min). This activity was 6 times higher than inthe alveolar macrophage or unseparated cell fractions. 7-Ethoxycoumarindeethylase activity, a cytochrome P-450-dependent pathway, was low orundctectable in the three cell fractions. Trace amounts of 7-ethoxyreso-rufin O-deethylase activity (0.5-1.5 pmol/mg protein/min) were detectedin microsomes of the isolated human cells, even though a polycyclichydrocarbon-inducible cytochrome P-450 which metabolizes 7-ethoxy-resorufin (form 6 in rabbits) was not detected immunochemically.

INTRODUCTION

An important component of chemical carcinogenesis is themetabolic activation of procarcinogens, a process often involving multiple steps. Because of the structural and functionaldiversity of the many pulmonary cell types, studies of xenobioticmetabolism in isolated pulmonary cells may help us to understand the relationships between metabolic activation of procarcinogens and carcinogenesis at specific sites in lung.

Results of recent studies with cells isolated from rabbit andrat lungs have demonstrated differential cellular localization ofseveral enzymes involved in the biotransformation of xenobiot-ics (1-7). In both rat and rabbit the nonciliated bronchiolarepithelial (Clara) cell catalyzes the metabolism of many compounds. In the rabbit, alveolar type II cells also have the abilityto metabolize compounds, such as 7-EC,2 benzo(a)pyrene, andbenzo(a)pyrene 4,5-oxide (2, 3). In contrast, alveolar type IIcells from rats exhibit only traces of 7-EC deethylase andbenzo(a)pyrene hydroxylase activities, and epoxide hydrolase isapparently not detected in these cells (7).

In general, the human lung possesses low levels of enzymes

Received 3/14/86; revised 6/3/86; accepted 7/10/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 D.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Laboratory of Pharmacology (MD D4-04). NIEHS/NIH, P. O. Box 12233, Research Triangle Park.NC 27709.

J The abbreviations used are: 7-EC. 7-elhoxycoumarin: HPBS, jV-2-hydroxy-ethylpiperazine-/V'-2-ethanesulfonic acid-buffered balanced salt solution; cyt creducíase. NADPH-cytochrome c reducíase; P-450 reducíase. NADPH-cylo-chromc P-450 reducíase;7-ERF. 7-elhoxyresorufm.

that metabolize xenobiotics (8, 9). Cytochrome P-450 has notbeen detected spectrally, and cytochrome P-450 monooxygen-ase activities in human lung microsomes are less than 3% ofthose in human liver microsomes (8). Human pulmonary epoxide hydrolase activity is about one-tenth that of liver (8). However, one or more of the many lung cell types might have highactivities of some of these enzymes. Studies of these enzymesystems in individual cell types of human lung have been limitedto alveolar macrophages and lymphocytes, which are easilyisolated but are not epithelial and exhibit low levels of xenobiotic metabolism. Attempts to correlate pulmonary carcinogenesis or cancer risk with elevated metabolism of xenobiotics,such as benzo(a)pyrene, in a single human cell type such asalveolar macrophages or lymphocytes have been unsuccessful(10, 11). Individual populations of human pulmonary epithelialcells, the stem cells of most pulmonary carcinomas, are moredifficult to isolate and have not been used for these types ofstudies. Carcinomas of type II pneumocytes do occur in humans(12, 13). Therefore, study of xenobiotic metabolism by thesecells is relevant to pulmonary carcinogenesis.

A procedure for the isolation of human alveolar type II cells,purified by differential attachment of cells to tissue cultureplates and subsequent culture of the cells for 1 day, has beendescribed recently (14). Studies of hepatocytes suggest thatcultured cells rapidly lose cytochrome P-450 and exhibitchanges in metabolism of xenobiotics (15). Preliminary experiments in our laboratory using rabbit trachéalcells supportthese findings.3 To avoid this potential problem, we developed

a method of cell isolation from surgically obtained human lungtissue which yields a large fraction of freshly isolated pulmonarycells containing a high percentage of alveolar type II cells. Thisprocedure uses a protease for cell dispersal, and elutriationfollowed by Percoli density gradient centrifugation for cellseparation. Several reactions important in xenobiotic metabolism have been assessed in this fraction, and the activities werecompared with those found in isolated alveolar macrophagesand in mixed pulmonary cells from five individuals.

MATERIALS AND METHODS

Chemicals. Protease type XIV (lot 45F-0545) was obtained fromSigma Chemical Company (St. Louis, MO). The HPBS and the cellisolation buffer have been described previously (4).

Lung Tissue. Excess lung tissue was obtained from five patientsundergoing clinically indicated lobectomy or pneumonectomy. Clinicaldetails, occupational exposures, and drugs administered within 2 weeksof surgery are shown in Table 1. The tissue used was remote from thearea of primary pathology and no macroscopic tumors were detectable.However, all the lung tissue contained abundant carbon particles. Afterlung cell fractionation, these were localized almost entirely in themacrophage fraction. Little or no carbon was observed in the type IIcell fractions by light or electron microscopy. The lung tissue was keptin minimal essential medium on ice until cell isolation was begun,within 2 h of resection. Permission to perform this study was obtainedfrom local committees responsible for protection of the rights of human

3 Unpublished data.

5438

on May 21, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

XENOBIOTIC METABOLISM IN HUMAN TYPE II CELLS

Table 1 Patient characteristics and type II cell purity for each specimen

Case12345Age(yr)4875753247SmokingRaceB'WWBWSexMMMMFPackyears"266040

(pipeonly)1520CurrentYesNoNoYesYesDrugsINH(1

wk)Pyri-doxineMethylclothiazideInsulinOccupational

exposuresIntermittent

sandblasting,coaldust

for 8 yrPathologyBronchiectasisLarge

cellcancerSquamous

cellcancerLargecellcancerSquamous

cell cancerType

II cellpurity(%)8585636571

" Number of years during which at least one pack of cigarettes was smoked/day.* B, black; W. white; INH, isoniazid.

subjects (at both University of North Carolina and National Instituteof Environmental Health Sciences).

Cell Isolation. Wedge-shaped sections of lung tissue weighing 26 to109 g were placed in a 12-cm2-polystyrene dish and kept on ice during

perfusion and instillation of protease. The cut surfaces of the tissuesection were examined to locate airways and blood vessels large enoughto insert a perfusion cannula (a blunted 20-gauge stainless steel needleattached to a repeating syringe). Airways were instilled with HPBS at4"C and allowed to drain in order to remove alveolar macrophages.

Blood was removed from the lung section by perfusing the vasculaturewith HPBS until the perfusate recovered was colorless. These washingswere discarded. Protease solution (1.0% Sigma protease type XIV inHPBS) was instilled via both the airways and the vasculature, and theends of the vessels were tied off with No. 00 silk sutures to preventleakage. The protease-filled tissue was then transferred to a beaker andincubated at 37°Cwith gentle shaking for 20 min. This incubation time

maximized yield of viable, dispersed cells, as shown by pilot studieswith rabbit and dog lungs. The digested tissue was chopped, degassedfor 30 s, and filtered as described previously for rabbit lung tissue (4).The filtered cells were centrifuged (800 x g for 10 min), resuspendedin 10 ml cell isolation buffer containing 0.5% DNase to 10 ml, andsubjected to centrifugal elutriation. A sample of unseparated cells,referred to as cell digest, was reserved to compare with separated cellfractions.

The details of the centrifugal elutriation system have been publishedpreviously (3, 4, 16). For preparation of human type II cells, threeelutriator fractions were collected. The first fraction (150 ml) collectedat 2200 rpm and 13 ml/min contained primarily blood cells and debrisand was discarded. The second fraction (100 ml at 2200 rpm and 21ml/min) contained 20-60% type II cells and was saved for furtherenrichment. The third fraction (100 ml at 1200 rpm and 18 ml/min)contained primarily macrophages (60-80% with less than 10% type IIcells) and was also saved.

Cells from the second elutriator fraction were layered on a 35%solution of Percoli in HPBS and centrifuged at 1000 x g for 15 min.The cell fraction obtained from the top of the Percoli solution wasenriched in type II cells. Following isolation and characterization, thedifferent cell fractions were frozen in liquid N2 and stored at -70°C

until used for assays.Cell Counts and Identification. Cells were counted with the Electro-

zone/Celloscope (Particle Data, Inc., Elmhurst, IL). Viability of cellswas estimated by trypan blue (0.04%) dye exclusion (17). Alveolar typeII cell and macrophage identification and enumeration were made usingthe modified Papanicolaou stain without acid alcohol (18) or by usingphosphine 3R fluorescent dye (19). Type II cells were identified by thepresence of stained cytoplasmic granules while macrophages were identified by their large size, round shape, and lack of stained granules.Clara cells were identified by electron microscopy (20). For electronmicroscopy, pellets of alveolar type II cells were fixed and processed bythe method of Williams (21). Thin sections of cell preparations wereexamined in a Phillips model 300 electron microscope.

Enzyme Assays. All samples were sonicated (5 s at 60 W output witha Sonifier-Cell Disruptor; Ultrasonics, Inc., Plainview, NY) prior toenzyme assay. Microsomal preparations of isolated human pulmonarycells were used in some assays as indicated. Microsomes were obtainedby centrifugation of sonicated cells at 10,000 x g for 20 min followed

by centrifugation of the 10,000 x g supernatant at 100,000 x g for 45min.

cyt c reducíaseactivity was measured by the method of Masters etal. (22) as modified by Peters and Fouts (23). The reduction of cyto-chrome c was measured at 550 nm using an extinction coefficient of18.5 cm"1 IHM"'. In experiments to inhibit cyt c reducíase,anti-rabbit

cytochrome P-450 reducíase or nonimmune goal IgG (for conlrolincubalions) was incubaled for 15 min on ice wilh concenlrated (>3mg/ml) microsomal preparations before substrale and cofactor wereadded.

Epoxide hydrolase aciivity was determined radiometrically using[3H]benzo(a)pyrene 4,5-oxide as the subslrate. Details of this method

have been published previously (24, 25).7-EC deethylase activily was assayed by fluoromelric measuremenl

of umbelliferone produciion. A modificalion of ihe melhod of Ullrichand Weber (26) was used. Sonicaied cells were incubaled wilh 0.4 mM7-EC in HPBS with 3 mM MgCl2 and in the presence of 1 mM NADPHfor 15 min at 37°C.The reaction was stopped with trichloroacelic acid,

and Ihe prolein was removed by centrifugation. The supernatanls weremade basic with Tris-glycine buffer, pH 9.0, and the unmelabolized 7-EC was removed by one exlraclion wilh heplane. The fluorescence ofihe umbelliferone formed was measured in the aqueous phase at anexcilalion wavelenglh of 375 nm and an emission wavelenglh of 458nm.

The O-deelhylalion of 7-ERF was assayed in microsomal prepara-lions of isolated human pulmonary cells by ihe melhod of Burke andMayer (27) as modified by Normal et al. (28). Since microsomes werenoi washed, 10 i/\t dicumarol was added lo ihe incubations to inhibitquinone reducíase(29). The limil of delection in our system was 0.3pinol resorufin produced/mg protein/min.

Enzyme activities were normalized to cell prolein conlenl [Bradfordtechnique (30)] to faciliiale comparison wilh published dala on enzymeaclivities of human lung microsomes (8) and of cells from other species(2, 3, 6). In separate calculations, epoxide hydrolase and cylochrome creducíaseactivities were expressed per cell number because of sizedifferences between macrophages (0.17 mg protein/106 cells) and alveolar lype II cells (0.08 mg proiein/106 cells).

Western blot analysis for ¡mmunochemicaldeteclion of proteins wasperformed on both sonicated cells and microsomal fractions by thetechnique of Domin et al. (31) with antibodies prepared in goat torabbit P-450 reducíaseand cytochrome P-450 form 6. These antibodieswere kindly supplied by Dr. Richard M. Philpot, National Institute ofEnvironmental Health Sciences. Pulmonary microsomes from rabbitor rat and purified reducíaseor cytochrome P-450 form 6 were analyzedin the same Western blot assays as positive conlrols for immunoreac-tivity of the antibodies. These same antibodies also showed ¡mimmoreactivity with microsomes from human placentas of smokers (32).

RESULTS

The yield of cells in the cell digest following proteolyticdigestion was 9.1 x 10" ±2.5 x 10" (SE)/g lung tissue, andthis fraction contained 10-30% alveolar type II cells. Similarnumbers of alveolar macrophages and some polymorphonuclearleukocytes were observed, but other cell types in the mixed cellfraction were not identified. The alveolar type II cell fractions

5439

on May 21, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

XENOBIOTIC METABOLISM IN HUMAN TYPE II CELLS

isolated from these cells by the procedure described abovecontained 1.2 x 10A±0.4 x IO6cells/g of lung tissue. Of thesecells, 74 ±5% (range, 63-85%) were identified as type II cells(Table 1) and demonstrated a viability of 90% or greater bytrypan blue dye exclusion. The major contaminants were alveolar macrophages and polymorphonuclear leukocytes. No Claracells were identified in the type II cell preparations.

The type II cells were examined both by light microscopy(Fig. 1) and by transmission electron microscopy (Fig. 2). Theseisolated cells displayed the characteristic lamellar bodies andmicrovilli observed in human type II cells (14) and in type IIcells isolated from other species (3, 6). The lamellar bodiesfrom type II cells of human have concentric lamellae (Ref. 14;Fig. 2), whereas those of rats and rabbits contain cross-barred

lamellae (3, 6).The three different cell fractions from all five patients exhib

ited similar cyt c reducíaseactivities (Table 2). In contrast, thecpoxide hydrolase activities indicated a large and consistentdifference between the cell types of human lung (Table 2).Epoxide hydrolation was detected in all the cell fractions,although the type II cells had about 6 times as much activity asthe alveolar macrophages on a per mg protein basis. Thisactivity varied only 2-fold in the type II cell fractions (0.7-1.4nmol/mg protein/min) from the five patients.

Cytochrome P-450-dependent 7-EC deethylation reflects theactivity of several cytochrome P-450 isozymes. 7-EC deethylaseactivities were low or undetectable in the cell fractions from thedifferent patients. Only the mixed cell fraction from one individual had significant activity (169 pmol/mg protein/min). Inall other fractions, the activity was less than 30 pmol/mgprotein/min. In an assay to ensure that low 7-EC deethylase

activity was not due to further metabolism of the productumbelliferone, no disappearance of umbelliferone (0.25 MM)was detected (n = 5 different cell preparations).

Western blot analysis was performed on the individual cellfractions and on microsomal preparations (for greater sensitivity) of the pooled type II cell or macrophage fractions in anattempt to detect immunochemically P-450 reducíase or apolycyclic hydrocarbon-inducible isozyme of cytochrome P-450(homologous to isozyme 6 in rabbit lung) in the isolated humancells. In rabbil lung ihis cytochrome P-450 isozyme is responsible for melabolism of both benzo(a)pyrene and 7-ERF (33).Antibodies made in goals lo rabbil pulmonary P-450 reducíaseor hepalic cytochrome P-450 form 6, both of which react wilhhuman placenta! microsomes (32), were used for the analyses.Under condilions which were oplimal for deteclion of ihe rabbitpulmonary proteins, neither anlibodies lo rabbit P-450 reducíasenor lo cylochrome P-450 form 6 formed idenlifiable bandswilh any of the human lung cell or microsomal preparations.(Maximum sensitivily of Ihe assay delecls 0.1 pmol/mg protein.)

Since a homologue of cytochrome P-450 form 6 was notobserved immunochemically in ihe Weslern blol analyses, assays for 7-ERF deethylation were carried out with microsomalfractions of isolaled human cells. Trace amounls of 7-ERFdeelhylase activily (0.5-1.5 pmol/mg prolein/min) were de-lecled in ihe microsomal preparalions of macrophages, lype IIcells, and unseparaled cells when 10 MMdicumarol was presenlin ihe incubations to inhibit quinone reducíaseactivily.

Cyt c reducíaseadivines in ihe human pulmonary cells (Table2) were similar lo reducíaseaclivilies measured in rabbil pulmonary cells (3), but P-450 reducíase (activily measured by

Fig. I. Modified Papanicolaou slain of a human lung cell fraction containing 85% human alveolar typ«II cells, x 725. Bar, 10 ^m.

5440

on May 21, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

XENOBIOTIC METABOLISM IN HUMAN TYPE II CELLS

fë

Fig. 2. Electron micrograph of isolated human alveolar type II cells showing characteristic lamellar bodies, x 15,000. Bar, 1 ion.

Table 2 NADPH-cytochrome c redactase and epoxide hydrolase activities inhuman pulmonary cells

Type II cellsMacrophagesCell digestCytochrome

c reducíase"(nmol cytochrome c re-duced/mgprotein/min)22

±3°(1.7)*

29 ±7 (4.8)18 ±2 (1.7)Epoxide

hydrolase'

[nmol benzolo )pyrene4.5-diol formed/mgprotein/min]1.08

±0.17 (0.082)0.18 ±0.05 (0.028)0.23 ±0.03 (0.022)

" Mean ±SE for 4-5 individual samples.'Numbers in parentheses, nmol metabolite formed/106cells/min.

cytochrome c reduction) was not detected by the Western blottechnique in microsomal fractions of the human cells. Therefore, we assayed cyt c reducíasein the microsomes from humanlung in the presence of the anti-rabbit P-450 reducíaseantibody(Fig. 3). Inhibition of Ihe reducíaseenzyme in ihe human lungpreparalions was observed, bui al a much higher concenlralionof Ihe anlibody lhan was required lo inhibil Ihe rabbit pulmonary enzyme.

Because inhibition of Ihe hman cytochrome c reducíaseacliv-ily by the anti-reductase antibody required a high anlibody

concenlration, ihe Weslern blol analysis for delection of bolhIhe reducíaseand cytochrome P-450 form 6 was repeated withmicrosomes of Ihe human cells and higher anlibody concenlra-tions (5 times greater lhan in the firsl experimenls). Wilh iheanli-reduclase anlibody, fainl bands were presenl in ihe ex-peeled molecular weighl range wilh microsomes of bolh Ihemacrophages and lype II cells (dala noi shown). However,because of the higher level of nonspecific binding due to theincreased concentralion of anlibody, these levels of slainingwere judged lo be al best al Ihe limit of deleclion and notquanlilalable. Wilh Ihe cylochrome P-450 form 6 anlibody, Ihefaint bands did not sland oui above Ihe high level of backgroundslaining, and Iherefore deleclion under ihese condilions wasnoi possible.

DISCUSSION

We have described a procedure for ihe isolalion of humanalveolar lype II cells in sufficienl number and purily lo charac-lerize several paramelers of xenobiolic melabolism in ihe

5441

on May 21, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

XENOBIOTIC METABOLISM IN HUMAN TYPE II CELLS

100

80

60

40

20

20 775155 465)m anti-Rd/unit Rd

Fig. 3. Effect of anti-rabbit cytochrome P-450 reducíaseon NADPH cyto-chrome c reducíase activity in rabbit lung microsomes (•),in human lungmicrosomes (O), and in microsomal preparations of human alveolar type II cells(D) and alveolar macrophages (A). Control incubation mixtures contained non-immune goat IgG at same concentrations as anti-P-450 reducíase(Rd} in thetreated incubation mixtures.

freshly isolated cells from small specimens (26-109g) of excisedlung. An important finding was the high epoxide hydrolaseactivity in type II cells relative to the other isolated pulmonarycells, indicating that human pulmonary cell types differ in theirability to metabolize chemicals.

The alveolar type II cell is a major pulmonary cell typerepresenting about 16% of the cells of human lung (34). Recently, Robinson et al. (14) described a technique for isolationof human alveolar type II cells in which the tissue was digestedwith elastase. In our hands, elastase (0.05%) did not release asmany human type II cells as did Sigma protease XIV (data notshown). Also, we were concerned that the isolation of cells fromhuman lung, which contains little cytochrome P-450, be carriedout as quickly as possible without a step involving differentialattachment and culture of the cells. Therefore, in our procedurethe cells were separated first using an elutriator centrifugefollowed by Percoli density gradient centrifugation, a techniquerequiring a relatively short time (2-3 h).

For several years, our laboratory has been interested in isolation of nonciliated bronchiolar epithelial (Clara) cells fromlung since in some species, they exhibit high rates of xenobioticmetabolism (2, 6). However, it was not surprising to us thatfew Clara cells were isolated in any of the cell fractions fromthe human lung tissue. Protease XIV does not yield many Claracells from either rabbit or dog lung,3 and may selectively remove

type II cells from human lung. Also, there may be a reductionin the number of Clara cells present in the bronchioles inresponse to smoking (35).

Epoxide hydrolase activity has been measured in microsomalpreparations from human lung (8, 36). This very stable (ascompared with cytochrome P-450) microsomal enzyme is important in the hydration of reactive epoxides. Epoxide hydrolasecan play a dual role of activation and deactivation of reactiveintermediates of polycyclic hydrocarbons depending on substrate concentrations and cytochrome P-450 isozymes present(37). Jones et al. (1) showed that type II cells from bothuntreated and ß-naphthoflavone-lrealed rats exhibited no detectable epoxide hydrolase activity. However, our results suggest that human type II cells, unlike type II cells of rats, mayfunction in metabolizing epoxides.

Cyt c reducíaseactivity was similar in all the pulmonary cellfractions, approximately 20-30 nmol cyt c reduced/mg protein/min. In comparison, activity measured in sonicated type II cellsof rabbits was 44 nmol/mg protein/min (3). The activity observed in the isolated human pulmonary cells was high enoughto suggest that immunoreactivity might occur in protein blots

with a goat antibody to rabbit P-450 reducíase.In Western blolanalysis to delecl a homologue of Ihe rabbil reducíase in themicrosomal preparations of isolated human pulmonary cells,the protein reacted minimally with a high concentration of theantibody. However, the same anlibody was used lo inhibitcylochrome c reduclion as much as 70% in microsomal prepa-ralions of Ihe human lung cells. These data provide evidencefor the presence of cytochrome P-450 reducíasein Ihe humanmacrophages and lype II cells.

7-EC deelhylase aclivily was assayed in Ihe isolated cellsbecause il is a very sensilive measure of cylochrome P-450-dependenl monooxygenase aclivity. Furlhermore, several isozymes of cylochrome P-450 metabolize Ihis compound. Sincemosl of Ihe patienls in Ihis sludy were long lerm smokers, wealso expecled lo delect a homologue of cytochrome P-450 form6, which is presenl in plácenla! microsomes of smokers (32).The lack of deleclion of Ihis isozyme in Ihe Weslern blol assaywilh human pulmonary cells of smokers could indicale a lackof immunological homology belween Ihe rabbil cylochromeprotein and similar prolein in human lung lissue. Indeed, Iracesof 7-ERF deelhylase aclivily, which has been correlaled wilhIhe presence of Ihis isozyme in human placenlal lissue ofsmokers (32), were found in microsomal preparalions of allIhree human pulmonary cell fraclions. However, the low orundeteclable 7-EC deethylase activilies as well as the Weslernblot data with an antibody to cytochrome P-450 form 6 and thelow 7-ERF deelhylase aclivilies measured in Ihe isolated humanpulmonary cell preparalions are consislenl wilh previous resultsshowing Hule or no cylochrome P-450 or cylochrome P-450monooxygenase aclivity in human lung microsomes (8).

The lissue oblained for these studies, allhough lacking grosslydeleclable tumors, came from cancerous lungs. Similarly, olhersludies on xenobiolic metabolism in human lung have beenperformed wilh "normal" lissue from diseased lungs (8, 9). In

rat livers conlaining 4-dimelhylaminoazobenzene-induced lu-rnors, lissue adjaceni lo i lie lumors had normal monooxygenaseaclivily whereas aclivily in Ihe lumors was significanlly decreased (38). This suggesls lhal cells from normal lung lissueof patients with lung cancer may have the same enzyme activilies as those from people wilhoul disease. Indeed, the cells fromIhe subject with bronchieclasis bul not cancer had enzymeactivilies comparable lo Ihe four palients with lung cancer.Further sludies wilh human lung epithelial cells, such as alveolar type II or Clara cells, may reveal differences in metabolismbetween cells from Ihe normal populalion and normal or lumorcells from lung cancer patients.

In Ihis report, we have shown thai lype II cells can be isolatedfrom pieces of excised human lung lissue in sufficient quantityto measure the aclivilies of several enzymes in Ihe freshlyisolated cells of one individual. The epoxide hydrolase dalademonslrated lhal marked differences in ihe aclivily of al leaslone enzyme involved in xenobiolic metabolism exist betweendifferenl human lung cell lypes. These isolated cell preparalionsmay be useful for ihe localizalion and sludy of endogenoussubstrate metabolism, such as biotransformation of arachidonicacid and prostaglandins, since many of ihese palhways do notinvolve cytochrome P-450.

ACKNOWLEDGMENTS

The authors thank Fred Talley for his asssistance in preparation ofthe electron micrographs, Drs. Barbara Domin and Richard Philpotfor performing the Western blot analysis, and Janet Diliberto and BlairHoyle for their excellent technical assistance.

5442

on May 21, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

XENOBIOTIC METABOLISM IN HUMAN TYPE II CELLS

REFERENCES

1. Devereux, T. R. Alveolar type II and Clara cells: isolation and xenobioticmetabolism. Environ. Health Perspect., 56: 95-101, 1984.

2. Devereux, T. R., Diliberto, J. J.. and Fouts, J. R. Cytochrome P-450monooxygenase, epoxide hydrolase and flavin monooxygenase activities inClara cells and alveolar type II cells isolated from rabbit. Cell Biol. Toxicol.,1: 57-65, 1985.

3. Devereux, T. R., and Fouts, J. R. Xenobiotic metabolism by alveolar type IIcells isolated from rabbit lung. Biochem. Pharmacol., 30:1231-1237, 1981.

4. Devereux, T. R., and Fouts, J. R. Isolation of pulmonary cells and use instudies of xenobiotic metabolism. In: W. Jakoby (ed.). Methods of Enzymol-ogy, p. 147-154. New York: Academic Press, 1981.

5. Devereux, T. R., Serabjit-Singh, C. J., Slaughter, S. R., Wolf, C. R., Philpot,R. M., and Fouts, J. R. Identification of cytochrome P-450 isozymes innonciliated bronchiolar epithelial (Clara) and alveolar type II cells isolatedfrom rabbit lung. Exp. Lung Res., 2: 221-230, 1981.

6. Jones, J. G., Holland, J. F.. and Fouts, J. R. Benzo(o)pyrene hydroxylaseactivity in enriched populations of Clara cells and alveolar type II cells fromcontrol and tf-naphthoflavone pretreated rats. Cancer Res., 42: 4658-4663,

1982.7. Jones, K. G., Holland, J. F., Foureman, G. L., Bend, J. R., and Fouts, J. R.

Xenobiotic metabolism in Clara cells and alveolar type II cells isolated fromlungs of rats treated with í¡-naphthoflavone.J. Pharmacol. Exp. Then, 225:316-319, 1983.

8. McManus, M. E., Boobis, A. R., Pacifici, G. M., Frempong, R. Y., Brodie,M. J., Kahn, G. C.. Whyte, C.. and Davies, D. S. Xenobiotic metabolism inthe human lung. Life Sci., 26:481-487, 1980.

9. Prough. R. A., Sipal, Z., and Jakobsson, S. W. Metabolism of benzo(a)pyreneby human lung microsomal fractions. Life Sci., 21: 1629-1636, 1977.

10. McLemore, T. L., Martin, R. R.. Pickard, L. R., Springer, R. R., Wray, N.P., Toppell, K. L., Mattox, K. L., Guinn, G. A., Cantrell, E. T., and Busbee,D. L. Analysis of aryl hydrocarbon hydroxylase activity in human lung tissue,pulmonary macrophages, and blood lymphocytes. Cancer (Phila.), 41: 2292-2300, 1978.

11. McLemore, T. L., Martin, R. R., Busbee, D. L., Richie, R. C., Springer, R.R., Toppell, K. L., and Cantrell, E. T. Aryl hydrocarbon activity in pulmonarymacrophages and lymphocytes from lung cancer and noncancer patients.Cancer Res., 3 7: 1175-1181. 1977.

12. Singh, G.. Katyal, S. L., and Torikata, C. Carcinoma of type II cells.Immunodiagnosis of a subtype of "bronchioalveolar carcinomas." Am. J.Pathol.. 102: 195-208, 1981.

13. Singh, G., Katayla. S.. Ordonez. N. G., Dail, D. H.. Negishi, Y., Weedn, V.W.. Marcus, P. B., Weldon-Linne, C. M., Axiotis, C. A., Alvarez-Fernandez,E., and Smith, W. I. Type II pneumocytes in pulmonary tumors. Arch.Pathol. Lab. Med., 108: 44-48, 1984.

14. Robinson, P. C., Voelker, D. R., and Mason, R. J. Isolation and culture ofhuman alveolar type II epithelial cells. Am. Rev. Resp. Dis., 130: 1156-1160,1984.

15. Guzelian, P. S., Bissell, D. M., and Meyer, U. A. Drug metabolism in adultrat heptocytes in primary monolayer culture. Gastroenterology, 72: 1232-1239, 1977.

16. Devereux, T. R., and Fouts, J. R. Isolation and identification of Clara cellsfrom rabbit lung. In Vitro (Rockville), 16: 958-968, 1980.

17. Phillips, H. J. Dye exclusion tests for viability. In: P. F. Kruse, Jr., and M.K. Patterson, Jr. (eds.), pp. 406-408. New York: Academic Press, 1973.

18. Kikkawa, Y., and Yoneda, K. The type II epithelial cell of the lung. I. Methodof isolation. Lab. Invest., 30: 76-84, 1974.

19. Mason, R. J., Williams, M. C., Greenleaf. R. D., and Clement. J. A. Isolation

of type II alveolar cells from rat lung. Am. Rev. Resp. Dis., 115:1015-1025,1977.

20. Plopper, C. G. Comparative morphologic features of bronchiolar epithelialcells. Am. Rev. Resp. Dis., 128: S37-S41. 1983.

21. Williams, M. C. Conversion of lamellar body membranes into tubular myelinin alveoli of fetal rat lungs. J. Cell Biol., 72: 260-277, 1977.

. 22. Master, B. S. S., Williams, C. H., Jr., and Kamin, H. The preparation andproperties of microsomal TPNH-cytochrome c reducíase from pig liver.Methods Enzymol., 10: 565-573, 1967.

23. Peters, M. A., and Fouts, J. R. A study of some possible mechanisms bywhich magnesium activates hepatic microsomal drug metabolism /;; vitro. J.Pharmacol. Exp. Ther., 775: 233-241, 1970.

24. Jerina, D. M., Dansette, P. M., Lu, A. Y. H., and Levin, W. Hepaticmicrosomal epoxide hydrase: a sensitive radiometrie assay for hydration ofarene oxides of carcinogenic aromatic hydrocarbons. Mol. Pharmacol., 13:342-351, 1977.

25. Smith, B. R., and Bend, J. R. Prediction of pulmonary benzo(a)pyrene 4,5-oxide clearance: a pharmacokinetic analysis of epoxide-metabolizing enzymesin rabbit lung. J. Pharmacol. Exp. Ther., 214:478-482, 1980.

26. Ullrich, V., and Weber, P. The O-dealkylation of 7-ethoxycoumarin by livermicrosomes. A direct fluorimetrie test. Hoppe Seyler's Z. Physiol. Chem.,35.J: 1173-1177, 1972.

27. Burke, M. M., and Mayer, R. T. Ethoxyresorufin: direct fluorimetrie assayof microsomal O-dealkylation which is preferentially inducible by 3-methyl-cholanthrene. Drug Metab. Disp., 2: 583-588, 1974.

28. Norman, R. L., Johnson, E. F., and Muller-Eberhard, U. Identification ofthe major cytochrome P-450 form transplacentally induced in neonatalrabbits by 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Biol. Chem., 253: 8640-8647, 1978.

29. Nims, R. W., Prough, R. A., and Lubet, R. A. Cytosol-mediated reductionof resorufin: a method for measuring quinone oxidoreductase. Arch.Biochem. Biophys., 229: 459-465, 1984.

30. Bradford, M. M. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem., 72:248-254, 1976.

31. Domin, B. A., Serabjit-Singh, C. J., and Philpot, R. M. Quantitation ofrabbit cytochrome P-450, form 2, in microsomal preparations bound directlyto nitrocellulose paper using a modified peroxidase-immunostaining procedure. Anal. Biochem., 136: 390-396, 1984.

32. Wong, T. K., Domin, B. A., Bent, P. E.. Blanton, T. E., Anderson, M. W.,and Philpot, R. M. Correlation of placenta! microsomal activities withprotein detected by antibodies to rabbit cytochrome P-450 isozyme 6 inpreparations from humans exposed to polychlorinated biphenyls, quater-phenyls and dibenzofurans. Cancer Res., 46: 999-1004, 1986.

33. Domin, B. A., and Philpot, R. M. The effect of substrate on the expressionof activity catalyzed by cytochrome P-450: metabolism mediated by rabbitisozyme 6 in pulmonary microsomal and reconstituted monooxygenase systems. Arch. Biochem. Biophys., 246: 128-142, 1986.

34. Crapo, J. D., Barry, B. E., Gehr, P., Bachofen, M., and Weibel, E. R. Cellnumber and cell characteristics of the normal human lung. Am. Rev. Resp.Dis., 126: 332-337, 1982.

35. Lumsden, A. B., McLean, A., and Lamb, D. Goblet and Clara cells of humandistal airways: evidence for smoking induced changes in their numbers.Thorax, 39: 844-849, 1984.

36. Greene, F. E., and Jernstrom, B. Epoxide hydratase and glutathione S-transferase activities in human lungs. Cancer Lett., 8: 235-239, 1980.

37. Bentley, P., Oesch, F., and Glatt, H. Dual role of epoxide hydratase in bothactivation and inactivation of benzo(a)pyrene. Arch. Toxicol., 39: 65-75,1977.

38. Hart, L. G., Adamson, R. H., Morris, H. P., and Fouts, J. R. The stimulationof drug metabolism in various rat hepatomas. J. Pharmacol. Exp. Ther., 149:7-15, 1965.

5443

on May 21, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1986;46:5438-5443. Cancer Res   Theodora R. Devereux, Thomas E. Massey, Michael R. Van Scott, et al.   by Centrifugal Elutriation and Density Gradient CentrifugationXenobiotic Metabolism in Human Alveolar Type II Cells Isolated

  Updated version

  http://cancerres.aacrjournals.org/content/46/10/5438

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/46/10/5438To request permission to re-use all or part of this article, use this link

on May 21, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from