(CANCER RESEARCH 46. 5438-5443. October 1986]
Xenobiotic Metabolism in Human Alveolar Type II Cells Isolated by Centrifugal
Elutriation and Density Gradient Centrifugation
Theodora 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.]
that metabolize xenobiotics (8, 9). Cytochrome P-450 has not
been detected spectrally, and cytochrome P-450 monooxygenAlveolar type II cells were isolated from five human lung specimens
ase activities in human lung microsomes are less than 3% of
obtained during resection or lobectomy and enriched to 63-85% purity.
those in human liver microsomes (8). Human pulmonary epox
Digestion with Sigma protease type XIV followed by centrifugal elutriation and Percoli density gradient centrifugation yielded 1.2 ±0.4 x IO1' ide hydrolase activity is about one-tenth that of liver (8). How
ever, one or more of the many lung cell types might have high
cells/g lung in the type II cell fractions. The activities of some enzymes
activities of some of these enzymes. Studies of these enzyme
involved in the metabolism of \enobiolics were determined in these
systems in individual cell types of human lung have been limited
freshly isolated type II cells and compared with activities in alveolar
to alveolar macrophages and lymphocytes, which are easily
macrophages and fractions of unseparated cells from the same tissue
samples. Reduced nicotinamide adenine dinucleotide phosphate-cytoisolated but are not epithelial and exhibit low levels of xeno
chrome c reducÃ-aseactivity was similar in the three cell fractions from
biotic metabolism. Attempts to correlate pulmonary carcino
all five patients (18-29 nmol/mg protein/min). An antibody to rabbit
genesis or cancer risk with elevated metabolism of xenobiotics,
reduced nicotinamide adcnine dinucleotide phosphate-cytochrome P-450
such as benzo(a)pyrene, in a single human cell type such as
reducÃ-aseinhibited reduced nicotinamide adenine dinucleotide phosphatealveolar macrophages or lymphocytes have been unsuccessful
cytochrome c reduction as much as 70% in microsomal preparations of
(10, 11). Individual populations of human pulmonary epithelial
the isolated human pulmonary cells, although this same antibody barely
cells,
the stem cells of most pulmonary carcinomas, are more
reacted with microsomes of the human cells in a Western blot assay.
difficult to isolate and have not been used for these types of
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 in studies. Carcinomas of type II pneumocytes do occur in humans
the alveolar macrophage or unseparated cell fractions. 7-Ethoxycoumarin
(12, 13). Therefore, study of xenobiotic metabolism by these
deethylase activity, a cytochrome P-450-dependent pathway, was low or cells is relevant to pulmonary carcinogenesis.
undctectable in the three cell fractions. Trace amounts of 7-ethoxyresoA procedure for the isolation of human alveolar type II cells,
rufin O-deethylase activity (0.5-1.5 pmol/mg protein/min) were detected
purified by differential attachment of cells to tissue culture
in microsomes of the isolated human cells, even though a polycyclic plates and subsequent culture of the cells for 1 day, has been
hydrocarbon-inducible cytochrome P-450 which metabolizes 7-ethoxydescribed recently (14). Studies of hepatocytes suggest that
resorufin (form 6 in rabbits) was not detected immunochemically.
cultured cells rapidly lose cytochrome P-450 and exhibit
changes in metabolism of xenobiotics (15). Preliminary exper
iments in our laboratory using rabbit trachéalcells support
INTRODUCTION
these findings.3 To avoid this potential problem, we developed
An important component of chemical carcinogenesis is the a method of cell isolation from surgically obtained human lung
metabolic activation of procarcinogens, a process often involv
tissue which yields a large fraction of freshly isolated pulmonary
ing multiple steps. Because of the structural and functional
cells containing a high percentage of alveolar type II cells. This
diversity of the many pulmonary cell types, studies of xenobiotic
procedure uses a protease for cell dispersal, and elutriation
metabolism in isolated pulmonary cells may help us to under
followed by Percoli density gradient centrifugation for cell
stand the relationships between metabolic activation of procar
separation. Several reactions important in xenobiotic metabo
cinogens and carcinogenesis at specific sites in lung.
lism have been assessed in this fraction, and the activities were
Results of recent studies with cells isolated from rabbit and compared with those found in isolated alveolar macrophages
rat lungs have demonstrated differential cellular localization of and in mixed pulmonary cells from five individuals.
several enzymes involved in the biotransformation of xenobiotics (1-7). In both rat and rabbit the nonciliated bronchiolar
MATERIALS AND METHODS
epithelial (Clara) cell catalyzes the metabolism of many com
pounds. In the rabbit, alveolar type II cells also have the ability
Chemicals. Protease type XIV (lot 45F-0545) was obtained from
to metabolize compounds, such as 7-EC,2 benzo(a)pyrene, and
Sigma Chemical Company (St. Louis, MO). The HPBS and the cell
benzo(a)pyrene 4,5-oxide (2, 3). In contrast, alveolar type II
isolation buffer have been described previously (4).
cells from rats exhibit only traces of 7-EC deethylase and
Lung Tissue. Excess lung tissue was obtained from five patients
benzo(a)pyrene hydroxylase activities, and epoxide hydrolase is undergoing clinically indicated lobectomy or pneumonectomy. Clinical
apparently not detected in these cells (7).
details, occupational exposures, and drugs administered within 2 weeks
of surgery are shown in Table 1. The tissue used was remote from the
In general, the human lung possesses low levels of enzymes
ABSTRACT
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 in
accordance with 18 D.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed, at Laboratory of Phar
macology (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-hydroxyethylpiperazine-/V'-2-ethanesulfonic
acid-buffered balanced salt solution; cyt c
reducÃ-ase. NADPH-cytochrome c reducÃ-ase; P-450 reducÃ-ase. NADPH-cylochromc P-450 reducÃ-ase;7-ERF. 7-elhoxyresorufm.
area of primary pathology and no macroscopic tumors were detectable.
However, all the lung tissue contained abundant carbon particles. After
lung cell fractionation, these were localized almost entirely in the
macrophage fraction. Little or no carbon was observed in the type II
cell fractions by light or electron microscopy. The lung tissue was kept
in minimal essential medium on ice until cell isolation was begun,
within 2 h of resection. Permission to perform this study was obtained
from local committees responsible for protection of the rights of human
3 Unpublished data.
5438
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1986 American Association for Cancer Research.
XENOBIOTIC
METABOLISM
IN HUMAN TYPE II CELLS
Table 1 Patient characteristics and type II cell purity for each specimen
years"266040
Case12345Age(yr)4875753247SmokingRaceB'WWBWSexMMMMFPack
exposuresIntermittent
II cell
purity
(%)8585636571
wk)Pyri-doxineMethylclothiazideInsulinOccupational
cancerSquamous
cell
only)1520CurrentYesNoNoYesYesDrugsINH(1
(pipe
cancerLargecell
sandblasting,
cancerSquamous
cell
coaldust
for 8 yrPathologyBronchiectasisLarge
cell cancerType
" 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 Institute
of Environmental Health Sciences).
Cell Isolation. Wedge-shaped sections of lung tissue weighing 26 to
109 g were placed in a 12-cm2-polystyrene dish and kept on ice during
perfusion and instillation of protease. The cut surfaces of the tissue
section were examined to locate airways and blood vessels large enough
to insert a perfusion cannula (a blunted 20-gauge stainless steel needle
attached to a repeating syringe). Airways were instilled with HPBS at
4"C and allowed to drain in order to remove alveolar macrophages.
Blood was removed from the lung section by perfusing the vasculature
with HPBS until the perfusate recovered was colorless. These washings
were discarded. Protease solution (1.0% Sigma protease type XIV in
HPBS) was instilled via both the airways and the vasculature, and the
ends of the vessels were tied off with No. 00 silk sutures to prevent
leakage. The protease-filled tissue was then transferred to a beaker and
incubated at 37°Cwith gentle shaking for 20 min. This incubation time
maximized yield of viable, dispersed cells, as shown by pilot studies
with rabbit and dog lungs. The digested tissue was chopped, degassed
for 30 s, and filtered as described previously for rabbit lung tissue (4).
The filtered cells were centrifuged (800 x g for 10 min), resuspended
in 10 ml cell isolation buffer containing 0.5% DNase to 10 ml, and
subjected to centrifugal elutriation. A sample of unseparated cells,
referred to as cell digest, was reserved to compare with separated cell
fractions.
The details of the centrifugal elutriation system have been published
previously (3, 4, 16). For preparation of human type II cells, three
elutriator fractions were collected. The first fraction (150 ml) collected
at 2200 rpm and 13 ml/min contained primarily blood cells and debris
and was discarded. The second fraction (100 ml at 2200 rpm and 21
ml/min) contained 20-60% type II cells and was saved for further
enrichment. The third fraction (100 ml at 1200 rpm and 18 ml/min)
contained primarily macrophages (60-80% with less than 10% type II
cells) 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 was
enriched in type II cells. Following isolation and characterization, the
different cell fractions were frozen in liquid N2 and stored at -70°C
by centrifugation of the 10,000 x g supernatant at 100,000 x g for 45
min.
cyt c reducÃ-aseactivity was measured by the method of Masters et
al. (22) as modified by Peters and Fouts (23). The reduction of cytochrome c was measured at 550 nm using an extinction coefficient of
18.5 cm"1 IHM"'. In experiments to inhibit cyt c reducÃ-ase,anti-rabbit
cytochrome P-450 reducÃ-ase or nonimmune goal IgG (for conlrol
incubalions) was incubaled for 15 min on ice wilh concenlrated (>3
mg/ml) microsomal preparations before substrale and cofactor were
added.
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 Ullrich
and Weber (26) was used. Sonicaied cells were incubaled wilh 0.4 mM
7-EC in HPBS with 3 mM MgCl2 and in the presence of 1 mM NADPH
for 15 min at 37°C.The reaction was stopped with trichloroacelic acid,
and Ihe prolein was removed by centrifugation. The supernatanls were
made basic with Tris-glycine buffer, pH 9.0, and the unmelabolized 7EC was removed by one exlraclion wilh heplane. The fluorescence of
ihe umbelliferone formed was measured in the aqueous phase at an
excilalion wavelenglh of 375 nm and an emission wavelenglh of 458
nm.
The O-deelhylalion of 7-ERF was assayed in microsomal preparalions of isolated human pulmonary cells by ihe melhod of Burke and
Mayer (27) as modified by Normal et al. (28). Since microsomes were
noi washed, 10 i/\t dicumarol was added lo ihe incubations to inhibit
quinone reducÃ-ase(29). The limil of delection in our system was 0.3
pinol resorufin produced/mg protein/min.
Enzyme activities were normalized to cell prolein conlenl [Bradford
technique (30)] to faciliiale comparison wilh published dala on enzyme
aclivities of human lung microsomes (8) and of cells from other species
(2, 3, 6). In separate calculations, epoxide hydrolase and cylochrome c
reducÃ-aseactivities were expressed per cell number because of size
differences between macrophages (0.17 mg protein/106 cells) and alveo
lar lype II cells (0.08 mg proiein/106 cells).
Western blot analysis for ¡mmunochemicaldeteclion of proteins was
performed on both sonicated cells and microsomal fractions by the
technique of Domin et al. (31) with antibodies prepared in goat to
rabbit P-450 reducÃ-aseand cytochrome P-450 form 6. These antibodies
were kindly supplied by Dr. Richard M. Philpot, National Institute of
Environmental Health Sciences. Pulmonary microsomes from rabbit
or rat and purified reducÃ-aseor cytochrome P-450 form 6 were analyzed
in the same Western blot assays as positive conlrols for immunoreactivity of the antibodies. These same antibodies also showed ¡mimmo
reactivity with microsomes from human placentas of smokers (32).
until used for assays.
Cell Counts and Identification. Cells were counted with the Electrozone/Celloscope (Particle Data, Inc., Elmhurst, IL). Viability of cells
was estimated by trypan blue (0.04%) dye exclusion (17). Alveolar type
II cell and macrophage identification and enumeration were made using
the modified Papanicolaou stain without acid alcohol (18) or by using
phosphine 3R fluorescent dye (19). Type II cells were identified by the
presence of stained cytoplasmic granules while macrophages were iden
tified by their large size, round shape, and lack of stained granules.
Clara cells were identified by electron microscopy (20). For electron
microscopy, pellets of alveolar type II cells were fixed and processed by RESULTS
the method of Williams (21). Thin sections of cell preparations were
The yield of cells in the cell digest following proteolytic
examined in a Phillips model 300 electron microscope.
digestion was 9.1 x 10" ±2.5 x 10" (SE)/g lung tissue, and
Enzyme Assays. All samples were sonicated (5 s at 60 W output with
this fraction contained 10-30% alveolar type II cells. Similar
a Sonifier-Cell Disruptor; Ultrasonics, Inc., Plainview, NY) prior to
numbers of alveolar macrophages and some polymorphonuclear
enzyme assay. Microsomal preparations of isolated human pulmonary
leukocytes were observed, but other cell types in the mixed cell
cells were used in some assays as indicated. Microsomes were obtained
by centrifugation of sonicated cells at 10,000 x g for 20 min followed
fraction were not identified. The alveolar type II cell fractions
5439
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1986 American Association for Cancer Research.
XENOBIOTIC
METABOLISM
IN HUMAN TYPE II CELLS
isolated from these cells by the procedure described above
contained 1.2 x 10A±0.4 x IO6cells/g of lung tissue. Of these
cells, 74 ±5% (range, 63-85%) were identified as type II cells
(Table 1) and demonstrated a viability of 90% or greater by
trypan blue dye exclusion. The major contaminants were alveo
lar macrophages and polymorphonuclear leukocytes. No Clara
cells 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). These
isolated cells displayed the characteristic lamellar bodies and
microvilli observed in human type II cells (14) and in type II
cells isolated from other species (3, 6). The lamellar bodies
from 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 reducÃ-aseactivities (Table 2). In contrast, the
cpoxide hydrolase activities indicated a large and consistent
difference 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 as
the alveolar macrophages on a per mg protein basis. This
activity varied only 2-fold in the type II cell fractions (0.7-1.4
nmol/mg protein/min) from the five patients.
Cytochrome P-450-dependent 7-EC deethylation reflects the
activity of several cytochrome P-450 isozymes. 7-EC deethylase
activities were low or undetectable in the cell fractions from the
different patients. Only the mixed cell fraction from one indi
vidual had significant activity (169 pmol/mg protein/min). In
all other fractions, the activity was less than 30 pmol/mg
protein/min. In an assay to ensure that low 7-EC deethylase
activity was not due to further metabolism of the product
umbelliferone, no disappearance of umbelliferone (0.25 MM)
was detected (n = 5 different cell preparations).
Western blot analysis was performed on the individual cell
fractions and on microsomal preparations (for greater sensitiv
ity) of the pooled type II cell or macrophage fractions in an
attempt to detect immunochemically P-450 reducÃ-ase or a
polycyclic hydrocarbon-inducible isozyme of cytochrome P-450
(homologous to isozyme 6 in rabbit lung) in the isolated human
cells. In rabbil lung ihis cytochrome P-450 isozyme is respon
sible for melabolism of both benzo(a)pyrene and 7-ERF (33).
Antibodies made in goals lo rabbil pulmonary P-450 reducÃ-ase
or hepalic cytochrome P-450 form 6, both of which react wilh
human placenta! microsomes (32), were used for the analyses.
Under condilions which were oplimal for deteclion of ihe rabbit
pulmonary proteins, neither anlibodies lo rabbit P-450 reduc
Ã-asenor lo cylochrome P-450 form 6 formed idenlifiable bands
wilh any of the human lung cell or microsomal preparations.
(Maximum sensitivily of Ihe assay delecls 0.1 pmol/mg pro
tein.)
Since a homologue of cytochrome P-450 form 6 was not
observed immunochemically in ihe Weslern blol analyses, as
says for 7-ERF deethylation were carried out with microsomal
fractions of isolaled human cells. Trace amounls of 7-ERF
deelhylase activily (0.5-1.5 pmol/mg prolein/min) were delecled in ihe microsomal preparalions of macrophages, lype II
cells, and unseparaled cells when 10 MMdicumarol was presenl
in ihe incubations to inhibit quinone reducÃ-aseactivily.
Cyt c reducÃ-aseadivines in ihe human pulmonary cells (Table
2) were similar lo reducÃ-aseaclivilies measured in rabbil pul
monary cells (3), but P-450 reducÃ-ase (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
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1986 American Association for Cancer Research.
XENOBIOTIC
METABOLISM
IN HUMAN TYPE II CELLS
fë
Fig. 2. Electron micrograph of isolated hu
man alveolar type II cells showing character
istic lamellar bodies, x 15,000. Bar, 1 ion.
Table 2 NADPH-cytochrome c redactase and epoxide hydrolase activities in
human pulmonary cells
hydrolase'
c reducÃ-ase"
[nmol benzolo )pyrene
(nmol cytochrome c re4.5-diol formed/mg
duced/mg
protein/min)22
protein/min]1.08
±3°
(1.7)*
Type II cells
±0.17 (0.082)
Macrophages
29 ±7 (4.8)
0.18 ±0.05 (0.028)
Cell digestCytochrome
18 ±2 (1.7)Epoxide
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 blot
technique in microsomal fractions of the human cells. There
fore, we assayed cyt c reducÃ-asein the microsomes from human
lung in the presence of the anti-rabbit P-450 reducÃ-aseantibody
(Fig. 3). Inhibition of Ihe reducÃ-aseenzyme in ihe human lung
preparalions was observed, bui al a much higher concenlralion
of Ihe anlibody lhan was required lo inhibil Ihe rabbit pulmo
nary enzyme.
Because inhibition of Ihe hman cytochrome c reducÃ-aseaclivily by the anti-reductase antibody required a high anlibody
concenlration, ihe Weslern blol analysis for delection of bolh
Ihe reducÃ-aseand cytochrome P-450 form 6 was repeated with
microsomes of Ihe human cells and higher anlibody concenlrations (5 times greater lhan in the firsl experimenls). Wilh ihe
anli-reduclase anlibody, fainl bands were presenl in ihe expeeled molecular weighl range wilh microsomes of bolh Ihe
macrophages and lype II cells (dala noi shown). However,
because of the higher level of nonspecific binding due to the
increased concentralion of anlibody, these levels of slaining
were judged lo be al best al Ihe limit of deleclion and not
quanlilalable. Wilh Ihe cylochrome P-450 form 6 anlibody, Ihe
faint bands did not sland oui above Ihe high level of background
slaining, and Iherefore deleclion under ihese condilions was
noi possible.
DISCUSSION
We have described a procedure for ihe isolalion of human
alveolar lype II cells in sufficienl number and purily lo characlerize several paramelers of xenobiolic melabolism in ihe
5441
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1986 American Association for Cancer Research.
XENOBIOTIC METABOLISM IN HUMAN TYPE II CELLS
100
80
60
40
20
20
155
465
)m anti-Rd/unit Rd
775
Fig. 3. Effect of anti-rabbit cytochrome P-450 reducÃ-aseon NADPH cytochrome c reducÃ-ase activity in rabbit lung microsomes (•),in human lung
microsomes (O), and in microsomal preparations of human alveolar type II cells
(D) and alveolar macrophages (A). Control incubation mixtures contained nonimmune goat IgG at same concentrations as anti-P-450 reducÃ-ase(Rd} in the
treated incubation mixtures.
freshly isolated cells from small specimens (26-109g) of excised
lung. An important finding was the high epoxide hydrolase
activity in type II cells relative to the other isolated pulmonary
cells, indicating that human pulmonary cell types differ in their
ability to metabolize chemicals.
The alveolar type II cell is a major pulmonary cell type
representing about 16% of the cells of human lung (34). Re
cently, Robinson et al. (14) described a technique for isolation
of human alveolar type II cells in which the tissue was digested
with elastase. In our hands, elastase (0.05%) did not release as
many human type II cells as did Sigma protease XIV (data not
shown). Also, we were concerned that the isolation of cells from
human lung, which contains little cytochrome P-450, be carried
out as quickly as possible without a step involving differential
attachment and culture of the cells. Therefore, in our procedure
the cells were separated first using an elutriator centrifuge
followed by Percoli density gradient centrifugation, a technique
requiring a relatively short time (2-3 h).
For several years, our laboratory has been interested in iso
lation of nonciliated bronchiolar epithelial (Clara) cells from
lung since in some species, they exhibit high rates of xenobiotic
metabolism (2, 6). However, it was not surprising to us that
few Clara cells were isolated in any of the cell fractions from
the human lung tissue. Protease XIV does not yield many Clara
cells from either rabbit or dog lung,3 and may selectively remove
type II cells from human lung. Also, there may be a reduction
in the number of Clara cells present in the bronchioles in
response to smoking (35).
Epoxide hydrolase activity has been measured in microsomal
preparations from human lung (8, 36). This very stable (as
compared with cytochrome P-450) microsomal enzyme is im
portant in the hydration of reactive epoxides. Epoxide hydrolase
can play a dual role of activation and deactivation of reactive
intermediates of polycyclic hydrocarbons depending on sub
strate concentrations and cytochrome P-450 isozymes present
(37). Jones et al. (1) showed that type II cells from both
untreated and ß-naphthoflavone-lrealed rats exhibited no de
tectable epoxide hydrolase activity. However, our results sug
gest that human type II cells, unlike type II cells of rats, may
function in metabolizing epoxides.
Cyt c reducÃ-aseactivity was similar in all the pulmonary cell
fractions, approximately 20-30 nmol cyt c reduced/mg protein/
min. In comparison, activity measured in sonicated type II cells
of rabbits was 44 nmol/mg protein/min (3). The activity ob
served in the isolated human pulmonary cells was high enough
to suggest that immunoreactivity might occur in protein blots
with a goat antibody to rabbit P-450 reducÃ-ase.In Western blol
analysis to delecl a homologue of Ihe rabbil reducÃ-ase in the
microsomal preparations of isolated human pulmonary cells,
the protein reacted minimally with a high concentration of the
antibody. However, the same anlibody was used lo inhibit
cylochrome c reduclion as much as 70% in microsomal preparalions of Ihe human lung cells. These data provide evidence
for the presence of cytochrome P-450 reducÃ-asein Ihe human
macrophages and lype II cells.
7-EC deelhylase aclivily was assayed in Ihe isolated cells
because il is a very sensilive measure of cylochrome P-450dependenl monooxygenase aclivity. Furlhermore, several iso
zymes of cylochrome P-450 metabolize Ihis compound. Since
mosl of Ihe patienls in Ihis sludy were long lerm smokers, we
also expecled lo delect a homologue of cytochrome P-450 form
6, which is presenl in plácenla! microsomes of smokers (32).
The lack of deleclion of Ihis isozyme in Ihe Weslern blol assay
wilh human pulmonary cells of smokers could indicale a lack
of immunological homology belween Ihe rabbil cylochrome
protein and similar prolein in human lung lissue. Indeed, Iraces
of 7-ERF deelhylase aclivily, which has been correlaled wilh
Ihe presence of Ihis isozyme in human placenlal lissue of
smokers (32), were found in microsomal preparalions of all
Ihree human pulmonary cell fraclions. However, the low or
undeteclable 7-EC deethylase activilies as well as the Weslern
blot data with an antibody to cytochrome P-450 form 6 and the
low 7-ERF deelhylase aclivilies measured in Ihe isolated human
pulmonary cell preparalions are consislenl wilh previous results
showing Hule or no cylochrome P-450 or cylochrome P-450
monooxygenase aclivity in human lung microsomes (8).
The lissue oblained for these studies, allhough lacking grossly
deleclable tumors, came from cancerous lungs. Similarly, olher
sludies on xenobiolic metabolism in human lung have been
performed wilh "normal" lissue from diseased lungs (8, 9). In
rat livers conlaining 4-dimelhylaminoazobenzene-induced
lurnors, lissue adjaceni lo ilie lumors had normal monooxygenase
aclivily whereas aclivily in Ihe lumors was significanlly de
creased (38). This suggesls lhal cells from normal lung lissue
of patients with lung cancer may have the same enzyme activi
lies as those from people wilhoul disease. Indeed, the cells from
Ihe subject with bronchieclasis bul not cancer had enzyme
activilies comparable lo Ihe four palients with lung cancer.
Further sludies wilh human lung epithelial cells, such as alveo
lar type II or Clara cells, may reveal differences in metabolism
between cells from Ihe normal populalion and normal or lumor
cells from lung cancer patients.
In Ihis report, we have shown thai lype II cells can be isolated
from pieces of excised human lung lissue in sufficient quantity
to measure the aclivilies of several enzymes in Ihe freshly
isolated cells of one individual. The epoxide hydrolase dala
demonslrated lhal marked differences in ihe aclivily of al leasl
one enzyme involved in xenobiolic metabolism exist between
differenl human lung cell lypes. These isolated cell preparalions
may be useful for ihe localizalion and sludy of endogenous
substrate metabolism, such as biotransformation of arachidonic
acid and prostaglandins, since many of ihese palhways do not
involve cytochrome P-450.
ACKNOWLEDGMENTS
The authors thank Fred Talley for his asssistance in preparation of
the electron micrographs, Drs. Barbara Domin and Richard Philpot
for performing the Western blot analysis, and Janet Diliberto and Blair
Hoyle for their excellent technical assistance.
5442
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1986 American Association for Cancer Research.
XENOBIOTIC
METABOLISM
IN HUMAN TYPE II CELLS
REFERENCES
1. Devereux, T. R. Alveolar type II and Clara cells: isolation and xenobiotic
metabolism. Environ. Health Perspect., 56: 95-101, 1984.
2. Devereux, T. R., Diliberto, J. J.. and Fouts, J. R. Cytochrome P-450
monooxygenase, epoxide hydrolase and flavin monooxygenase activities in
Clara 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 II
cells isolated from rabbit lung. Biochem. Pharmacol., 30:1231-1237, 1981.
4. Devereux, T. R., and Fouts, J. R. Isolation of pulmonary cells and use in
studies of xenobiotic metabolism. In: W. Jakoby (ed.). Methods of Enzymology, 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 in
nonciliated bronchiolar epithelial (Clara) and alveolar type II cells isolated
from rabbit lung. Exp. Lung Res., 2: 221-230, 1981.
6. Jones, J. G., Holland, J. F.. and Fouts, J. R. Benzo(o)pyrene hydroxylase
activity in enriched populations of Clara cells and alveolar type II cells from
control 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 from
lungs of rats treated with Ã-¡-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 in
the human lung. Life Sci., 26:481-487, 1980.
9. Prough. R. A., Sipal, Z., and Jakobsson, S. W. Metabolism of benzo(a)pyrene
by 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: 22922300, 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 pulmonary
macrophages 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 of
human alveolar type II epithelial cells. Am. Rev. Resp. Dis., 130: 11561160,1984.
15. Guzelian, P. S., Bissell, D. M., and Meyer, U. A. Drug metabolism in adult
rat heptocytes in primary monolayer culture. Gastroenterology, 72: 12321239, 1977.
16. Devereux, T. R., and Fouts, J. R. Isolation and identification of Clara cells
from 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. Method
of isolation. Lab. Invest., 30: 76-84, 1974.
19. Mason, R. J., Williams, M. C., Greenleaf. R. D., and Clement. J. A. Isolation
20.
21.
. 22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
of type II alveolar cells from rat lung. Am. Rev. Resp. Dis., 115:1015-1025,
1977.
Plopper, C. G. Comparative morphologic features of bronchiolar epithelial
cells. Am. Rev. Resp. Dis., 128: S37-S41. 1983.
Williams, M. C. Conversion of lamellar body membranes into tubular myelin
in alveoli of fetal rat lungs. J. Cell Biol., 72: 260-277, 1977.
Master, B. S. S., Williams, C. H., Jr., and Kamin, H. The preparation and
properties of microsomal TPNH-cytochrome c reducÃ-ase from pig liver.
Methods Enzymol., 10: 565-573, 1967.
Peters, M. A., and Fouts, J. R. A study of some possible mechanisms by
which magnesium activates hepatic microsomal drug metabolism /;; vitro. J.
Pharmacol. Exp. Ther., 775: 233-241, 1970.
Jerina, D. M., Dansette, P. M., Lu, A. Y. H., and Levin, W. Hepatic
microsomal epoxide hydrase: a sensitive radiometrie assay for hydration of
arene oxides of carcinogenic aromatic hydrocarbons. Mol. Pharmacol., 13:
342-351, 1977.
Smith, B. R., and Bend, J. R. Prediction of pulmonary benzo(a)pyrene 4,5oxide clearance: a pharmacokinetic analysis of epoxide-metabolizing enzymes
in rabbit lung. J. Pharmacol. Exp. Ther., 214:478-482, 1980.
Ullrich, V., and Weber, P. The O-dealkylation of 7-ethoxycoumarin by liver
microsomes. A direct fluorimetrie test. Hoppe Seyler's Z. Physiol. Chem.,
35.J: 1173-1177, 1972.
Burke, M. M., and Mayer, R. T. Ethoxyresorufin: direct fluorimetrie assay
of microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug Metab. Disp., 2: 583-588, 1974.
Norman, R. L., Johnson, E. F., and Muller-Eberhard, U. Identification of
the major cytochrome P-450 form transplacentally induced in neonatal
rabbits by 2,3,7,8-tetrachlorodibenzo-p-dioxin.
J. Biol. Chem., 253: 86408647, 1978.
Nims, R. W., Prough, R. A., and Lubet, R. A. Cytosol-mediated reduction
of resorufin: a method for measuring quinone oxidoreductase. Arch.
Biochem. Biophys., 229: 459-465, 1984.
Bradford, M. M. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem., 72:248-254, 1976.
Domin, B. A., Serabjit-Singh, C. J., and Philpot, R. M. Quantitation of
rabbit cytochrome P-450, form 2, in microsomal preparations bound directly
to nitrocellulose paper using a modified peroxidase-immunostaining proce
dure. Anal. Biochem., 136: 390-396, 1984.
Wong, T. K., Domin, B. A., Bent, P. E.. Blanton, T. E., Anderson, M. W.,
and Philpot, R. M. Correlation of placenta! microsomal activities with
protein detected by antibodies to rabbit cytochrome P-450 isozyme 6 in
preparations from humans exposed to polychlorinated biphenyls, quaterphenyls and dibenzofurans. Cancer Res., 46: 999-1004, 1986.
Domin, B. A., and Philpot, R. M. The effect of substrate on the expression
of activity catalyzed by cytochrome P-450: metabolism mediated by rabbit
isozyme 6 in pulmonary microsomal and reconstituted monooxygenase sys
tems. Arch. Biochem. Biophys., 246: 128-142, 1986.
Crapo, J. D., Barry, B. E., Gehr, P., Bachofen, M., and Weibel, E. R. Cell
number and cell characteristics of the normal human lung. Am. Rev. Resp.
Dis., 126: 332-337, 1982.
Lumsden, A. B., McLean, A., and Lamb, D. Goblet and Clara cells of human
distal airways: evidence for smoking induced changes in their numbers.
Thorax, 39: 844-849, 1984.
Greene, F. E., and Jernstrom, B. Epoxide hydratase and glutathione Stransferase activities in human lungs. Cancer Lett., 8: 235-239, 1980.
Bentley, P., Oesch, F., and Glatt, H. Dual role of epoxide hydratase in both
activation and inactivation of benzo(a)pyrene. Arch. Toxicol., 39: 65-75,
1977.
Hart, L. G., Adamson, R. H., Morris, H. P., and Fouts, J. R. The stimulation
of drug metabolism in various rat hepatomas. J. Pharmacol. Exp. Ther., 149:
7-15, 1965.
5443
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1986 American Association for Cancer Research.
Xenobiotic Metabolism in Human Alveolar Type II Cells Isolated
by Centrifugal Elutriation and Density Gradient Centrifugation
Theodora R. Devereux, Thomas E. Massey, Michael R. Van Scott, et al.
Cancer Res 1986;46:5438-5443.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/46/10/5438
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1986 American Association for Cancer Research.