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Annual report : National Institute of Environmental Health Sciences (Volume 1984 pt.1) online

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substrates. This conclusion, combined with measurements of individual isozymes
in microsomal fractions, results in calculations that divide activities between
minor isozymes with high activities and major isozymes with low activities. Our
results, in several cases, suggest that as little as 1% contamination of
purified preparations with a second isozyme can have significant effects on
substrate specificity. Methods for the detection of minor contamination are now
available. These methods also make it possible to evaluate the profile of P-450
isozymes in various tissue fractions and to make immunochemical comparisons
across species lines.

Objectives in near term :

The ability to detect and quantitate P-450 isozymes of interest is required if
they are to be studied in detail. The development of immunochemical methods of
high specificity and sensitivity is now underway. These methods are useful for
the determination of contamination in purified preparations, detection and quan-
titation of P-450 isozymes in tissue preparations, and assessment of the extent
to which tissues differences and responses to various inducers are similar in
different species.

Experimental design :

The primary technique employed for these studies is "Western blotting". This
method allows for the immunochemical detection of specific proteins that have
been separated on the basis of monomeric molecular weight. With Western
blotting it is possible to take advantage of high titer polyclonal antibodies
even if they cross- react with proteins other than the primary antigen. It is
also advantageous to use antibodies that recognize more than one form of P-450
with Western blotting. For studies that involve more than one species, antibo-
dies to both rat and rabbit isozymes of cytochrome P-450 are used.

Recent accomplishments :

We are now able to detect rabbit isozymes 2, 4, 5, and 6 and rat isozymes b, c,
d, e, and f using immunochemical methods. By employing antibodies to the pro-
teins of both species we have shown that a number of species exhibit common pro-
perties with respect to the P-450 monooxygenase systems of liver and lung. For
example, the major pulmonary isozyme in all species examined is the isozyme
found in liver that is induced to the greatest extent by phenobarbital . Also,
analogous isozymes are induced in the liver of all species by treatment with



440



ZOl ES 80041-01 LP

polycyclic aromatic hydrocarbons. Of the two isozymes induced in the liver,
only one is found in the lung. The immunochemical similarities between species
are such that the same proteins are detected by rat or rabbit antibodies in all
cases.

The ability to detect minute quantities of P-450 isozymes has allowed for the
measurement of less than 1% contamination of purified preparations by other
P-450 isozymes. Contamination at this level cannot be assessed by protein
staining, the method commonly used to evaluate purity. The consequences of low
levels of contamination can be highly significant. We reported previously that
isozyme 5 from rabbit metabolizes the pulmonary carcinogen, benzo(a)pyrene. We
can now show that this metabolism is due to contamination of the "5" prepara-
tions with small amounts of isozyme 6 which can only be detected by immunochemi-
cal methods. This finding has resulted in a complete reevaluation of the
metabolism of benzo{a)pyrene by the rabbit lung. In addition, we have found
that most of the "purified" isozymes of P-450 available to us (both from our
lab and others) have detectable levels of more than one form of P-450.

B. Plans

With the immunochemical methods available we will continue to assess the extent
to which the P-450 systems of different species are similar. This work will
include an examination of P-450 systems in human tissue samples. Also, we will
attempt to establish the substrate specificities of P-450 isozymes in prepara-
tions that are free from cross-contamination. Preliminary results suggest that
such preparations can be obtained with improved purification procedures.

C. Publications

Domin, B.A., Serabj it-Singh, C.J., and Philpot, R.M.: Quantitation of rabbit
cytochrome P-450, form 2, in microsomal preparations bound directly to nitro-
cellulose paper using a modified peroxidase-immunostaining procedure. Anal .
Biochem. 136: 390-396, 1984.



441



LABORATORY OF PULMONARY FUNCTION AND TOXICOLOGY



443



THE LABORATORY OF PULMONARY FUNCTION AND TOXICOLOGY
Summary Statement



The Laboratory of Pulmonary Function and Toxicology (LPFT) studies basic biolo-
gical mechanisms of pulmonary cell biology and biochemistry as well as mecha-
nisms of injury leading to pulmonary disease. The work of LPFT is focused on a
few select research topics and is divided accordingly into five groups each
having its own central research theme. The groups also collaborate on many pro-
jects thus sharing expertise and resources. The five groups are (1) the Cell
Biology Group which studies cellular and biochemical mechanisms of differen-
tiation of airway epithelial cells, (2) the Epithelial Carcinogenesis Group
which studies mechanisms of neoplastic transformation of epithelial cells from
the conducting airways, (3) the Environmental Carcinogenesis Group which
investigates the relationships between mutagenesis and carcinogenesis and the
molecular basis of multistage neoplastic transformation, (4) the Pulmonary
Pathology Group which investigate the cellular and biochemical mechanisms
underlying particle and fiber-induced pulmonary fibrosis, and (5) the
Biochemical Pathology Group which studies the regulation of secretions in the
distal air spaces and its disruption by environmental agents.

Cell Biology Group . One of the long-term research objectives of the Cell
Biology Group is to elucidate the regulation of mucin biosynthesis and secre-
tion. Towards this end two important steps were made during the past year:
(1) Methods were firmly established to grow, in primary culture, epithelial
cells from the tracheas of hamsters and rabbits under conditions which allow
expression of the normal differentiated phenotype. (2) Under appropriate con-
ditions primary hamster and rabbit tracheal epithelial cell cultures synthesize
and secrete mucins of large molecular size (>10^ daltons). The high molecular
weight materials are completely resistant to proteoglycan degrading enzymes,
they contain N_-acetylgalactosamine, N_-acetyl glucosamine, galactose and fucose as
well as sialic acid, but they do not contain mannose. This together with evi-
dence for 0-glycosidic linkage of N_-acetylgalactosamine to protein provides
strong evidence that the secreted materials are indeed mucins.

Further studies provided evidence for the heterogeneity of the mucins produced
by the cultures in terms of degree of sulfation, size of the oligosaccharide
chains (ranging from 5,000 to 15,000 MW) and binding affinity of the mucins for
various lectins. As soon as the studies on the characterization of mucin pro-
duced by the tracheal cell cultures are complete this model system will be used
to investigate the regulation of mucin biosynthesis and secretion.

Another major research topic pursued by the Cell Biology Group is to elucidate
the mechanisms regulating differentiation of the epithelial cells of the
conducting airways. It is known from in vivo as well as cell culture studies
that this epithelium has the potential~for mucociliary as well as epidermoid
(keratinocytic) differentiation. Using rabbit cell cultures several factors were
established which profoundly influenced tracheal epithelial cell differen-
tiation. Serum promotes keratinocyte differentiation so does Ca''"'" at high con-
centrations but only when the cultures have reached a state of confluency. On
the other hand, mesenchymal factors obtained from 3T3 fibroblasts and retinoids
inhibit keratinocyte differentiation and promote mucociliary differentiation.
Several important biochemical observations were made. Undi fferentiated-

445



proliferative cells made large amounts of hyaluronic acid. When stimulated to
undergo terminal differentiation hyaluronic acid production was drastically
reduced. When the rabbit tracheal cells underwent keratinocyte differentiation,
their keratin profile changed. The production of 56 kd keratin increased and 54
and 48 kd keratins appeared. Vitamin A inhibited the synthesis of these kera-
tins. The 48 kd keratin appeared to be a particularly good marker for epider-
moid differentiation and studies are currently underway to elucidate the
molecular mechanisms by which retinoids regulate its expression.

The mechanism of action of retinoid is one of the major research topics of the
Cell Biology Group. Retionids have been shown to effectively block the induc-
tion of the enzyme ornithine decarboxylase (ODC) by the phorbolester 12-0-
tetradecanoyl-phorbol-13-acetate (TPA). This enzyme is the rate limiting step
in polyamine biosynthesis and appears to play an important role in biochemical
events involved in tumor promotion. The question being asked how and at what
level of molecular organization do retinoids inhibit the induction of ODC by
TPA. It was shown that retinoids do not interfere with TPA binding nor do they
interfere with protein kinase C (the presumed TPA receptor) activity. This
raises the possibility that retinoids might interfere with signal transduction
from the TPA receptor to the nucleus or directly interfere with transcription.
These and other possible mechanisms of the retinoid effects will be examined in
the future.

Epithelial Carcinogenesis Group . The Epithelial Carcinogenesis Group has deve-
l(Dped a cell culture model of multistage carcinogenesis using tracheal epithe-
lial cells from rats (RTE cells). Several aspects of the work of this group are
closely related to research pursued in the Cell Biology Group. The major goals
of this work are: (1) To identify and characterize distinct steps in neoplastic
transformation of epithelial cells, (2) to study the rates of progression from
one preneoplastic stage to the subsequent stage, (3) to identify factors which
are important determinates for the rate of progression, and (4) to elucidate
cellular and molecular mechanisms involved. This work is being performed in
collaboration with several members of the Environmental Carcinogensis Group.
One of the problems which was addressed during the past year concerns the propa-
gation and growth kinetics of transformed epithelial cells. The question was
asked: what are the characteristics of the cells composing the transformed
colony in the RTE cell system? Do all the cells in such colonies, which are
presumably of clonal origin, exhibits transformed characteristics? The major
feature of transformed RTE cells, which distinguishes them from normal RTE
cells, is their capacity to grow under conditions which are nonpermissive for
normal tracheal cells and to become "immortal." This transformed cell variant
is therefore called enhanced growth (EG) variant. Studies showed that only a
small fraction of the cells in the transformed colony has transformed growth
characteristics. The majority of the cells in such colonies are terminal,
nonrepli eating cells. However it was also found that the proportion of
"transformed stem cells" increases with time after carcinogen exposure from a
frequency which was initially in the order of 10"^ to a frequency of 10'^.
These and other studies suggest that important qualitative as well as quan-
titative changes are occurring in the early transformed cell populations.

The early post-initiation phase of transformation also seems to be particularly
sensitive to modulators of neoplastic progression. This conclusion was reached
because it was found that repeated exposures during this phase of transformation
to the tumor promoter TPA enchances the development of the anchorage independent



446



phenotype (a cellular phenotype which is closely associated with late pre-
neoplastic and possibly neoplastic cell variants) during later stages of
neoplastic transformation. On the other hand the exposure to the anti-promoting
agent retinoic acid (RA) markedly inhibited the formation of transformed colo-
nies even when the exposure to the retinoic acid was limited to only 1 week (the
expression-selection time of the transformation studies is 5 weeks). Studies on
the sensitivity and response pattern of cells at various stages of transfor-
mation may help to define the stages in biological as well as biochemical terms.
Interesting differences between normal and stably transformed RTE cells in
response to the tumor promoter TPA as well as to retinoic acid are beginning to
emerge. Since these agents are known to have profound influences on cell proli-
feration as well as cell differentiation, these studies may lead us to the rele-
vant dysfunctioning biochemical control mechanisms in various stages of
neoplastic transformation. Studies have been initiated which are aimed at iden-
tifying important cellular genes which are responsible for the abnormal growth
behavior of the transformed RTE cells.

Environmental Carcinogenesis Group . One of the topics of investigation of the
Environmental Carcinogenesis Group is the elucidation of the relationship bet-
ween mutagenesis and carcinogenesis. The majority of carcinogens tested to date
have been found to be mutagenes. This has lead to the widely accepted hypothe-
sis that at least one of the key steps in carcinogenesis involves structural
damage to DNA and gene mutation. However a number of carcinogens do not appear
to cause point mutations (and thus appear not to be "mutagenic" in the narrow
sense of the word). The Environmental Carcinogenesis Group has selected a
number of such carcinogenic compounds to determine their mechanism of action.
Two particularly fascinating carcinogens which have been extensively studied are
di ethyl stilbestrol (DES) and asbestos. Both are important human carcinogens and
were tested in the Syrian hamster embryo (SHE) cell system for their ability to
cause point mutations, unscheduled DNA synthesis, sister chromatid exchanges,
chromosomal abnormalities and morphological transformation. The studies showed
that DES (without addition of a metabolic activation system) causes morphologi-
cal transformation in a dose-dependent fashion but neither causes mutations
at two specific genetic loci (HPRT and Na'^'/K"'' ATPase), unscheduled DNA synthesis
nor sister chromatid exchange. However DES, under the same experimental con-
ditions caused numerical chromosomal abnormalities by affecting spindle micro-
tubule organization and behaved in many respects similar to the well-known
aneuploidy-inducer, colcemid. Studies with asbestos fibers, glass fibers and
non-fibrous particulates carried out in collaboration with the Pulmonary
Pathology Group showed that, similar to in vivo tumori genesis, long and thin
fibers, regardless of chemical composition, caused morphological transformation
while thick and short fibers are very ineffective in this respect. It was also
found that neither the asbestos nor the fiberglass fibers induce measurable
point mutations or unscheduled DNA synthesis. However fibers of the appropriate
physical dimensions (average length approximately 10 microns and average
thickness approximately 1 micron) caused structural and in particular numerical
chromosomal abnormalities. Thus these studies suggest that aneuploidy induction
may be an important mechanism of transformation and carcinogenesis at least with
carcinogens which do not cause measurable structural DNA damage. These studies
are been extended to include compounds such as benzene and dioxins and to
further explore the possible role of aneuploidy in carcinogenesis.

A newly initiated area of investigation deals with exploration of the molecular
basis of the multiphasic development of neoplastic transformation using the SHE



447



cell system which has been well defined in terms of progression of preneoplastic
to neoplastic stages. It was found that normal SHE cells can be neoplastically
transformed with polyoma virus DNA but not with Ha-MSV DNA. RSV-DNA was occa-
sionally tumorigenic but the tumor latency was long. In contrast transfection
with combinations of viral DNAs (Ha-MSV plus pSVv-myc and RSV plus pSVv-myc)
resulted in efficient neoplastic transformation with short tumor latency. When
preneoplastic SHE cell lines were transfected with either polyoma Ha-MSV or
RSV-DNA rapid conversion to the neoplastic state occurred. These studies thus
provide strong support for the role of several oncogenes in the multistep pro-
cess of carcinogenesis. Future studies will more extensively explore the
cooperation of oncogenes and the expression of cellular oncogenes during
neoplastic progression in the SHE cell model.

Pulmonary Pathology Group . The overall research objectives of this group are to
elucidate pathogenetic mechanisms involved in the development of particle and
fiber induced pulmonary fibrosis specifically asbestosis and silicosis. The
pulmonary macrophage appears to play a key role in the early phases of tissue
damage caused by inhaled fibers. After the deposition of fibers in the air spa-
ces macrophages migrate to the site of deposition and within 48 hrs microscopic
lesions develop which are presumed to be the early manifestations of the deve-
loping fibrotic reaction. The questions being addressed by the Pulmonary
Pathology Group are: what is the nature of the signal responsible for
macrophage migration to the site of particle deposition, what factors govern the
particle macrophage interaction at the level of the cell membrane and what
characterizes, at the tissue level, the epithelial and the mesenchymal response?'

A number of in vivo and in vitro studies support the hypothesis that the signal
which attracts macrophages to the site of asbestos deposition is a complement
derived chemotactic factor (C5A) activated by interaction of asbestos fibers
with proteins in the alveolar lining layer. The evidence is as follows: gene-
tically complement deficient mice as well as pharmacologically complement
deprived rats have a markedly reduced macrophage response upon inhalation of
asbestos. Pulmonary lavage fluid obtained from rats shortly after a brief inha-
lation exposure to asbestos contains a factor which is chemotactic for pulmonary
macrophages. Based on molecular size this factor is similar to activated
complement which is a strong chemotactic agent. Chemotactic activity is removed
by decomplementation and treatment with EDTA. The chemotactic factor can be
generated in vitro by incubation of serum as well as cell free alveolar lavage
fluid obtaTnied from normal unexposed rats with asbestos. Studies are planned to
further examine this hypothesis, e.g., different particles and fibers will be
examined and compared in terms of their capacity (a) to activate complement in
vitro and (b) to stimulate marophage migration in vivo . Studies are also
planned to examine the effects of complement depTet i on on the development of a
interstitial lung disease.

If macrophages play a key role in the induction of interstitial lung disease by
fibers as is currently being assumed, then it is essential that we obtain a
better understanding of the factors controlling the interaction between
macrophages and fibers. Initial experiments, using erythrocytes as a model to
study fiber-membrane interactions, showed that the hemolysis caused by chryso-
tile involved the binding of the positively charged chrysotile to negatively
charge sialic acid residues on the erythrocyte membrane. More recently studies
have been carried out with pulmonary macrophages. These have shown, using the
lectin wheat germ agglutinin (WGA), that sialic acid (SA) binding sites are



448



evenly distributed over the macrophage membrane, that chrysotile asbestos as
well as carbonyl iron particles bind to the macrophage membrane upon incubation
at 40°C (no phagocytosis) and that this binding can be blocked by prior incuba-
tion of the macrophages with WGA which occupies the sialic acid groups. It was
further shown that wheat germ agglutinin also blocks phagocytosis at 37°C.
These data strongly suggest that the sialic acid residues on pulmonary
macrophage membranes are essential for binding as well as phagocytosis of posi-
tively charged fibers and particles. Current studies are concerned with the
pathophysiological and biochemical consequences of particle binding and particle
phagocytosis for macrophages themselves as well as for cells exposed to the
macrophage products resulting from the particle membrane interaction.

Other studies of the Pulmonary Pathology Group are concerned with deposition and
translocation of asbestos fibers in various tissue compartments of the lungs and
with the development of early tissue lesions believed to be the "precursors" of
the chronic fibrotic reaction characterizing asbestosis. These studies have
identified the alveolar duct bifurcations as the most important deposition site
with respect to development of fibrotic lung disease. They have further shown
rapid translocation of fibers through the epithelium into the interstitium.
Within 1 month after a short inhalation exposure to asbestos, increased cellu-
larity and deposition of extracellular matrix materials was observed in the
interstitium of the alveolar duct bifurcations. These findings strongly suggest
that the alveolar duct region is most likely the area in which pulmonary
asbestosis first develops. Other studies indicate that approximately 20% of the
fibers initially deposited in the lungs are retained 1 month after a brief
exposure and that the longer fibers appear to be selectively retained. It is
these remaining fibers which are suspected to play the key role in the intersti-
tial fibrosis, typical of asbestosis, by mechanisms which are unknown at the
moment .

Pulmonary Biochemical Group . The Pulmonary Biochemical Group has been
interested for many years in the phospholipids of the lung, the so-called pulmo-
nary surfactants, which are essential for pulmonary lung function, preventing
collapse of alveoli and distal airways at expiration. It is firmly established
that the source of the surfactant are the so-called Type II alveolar cells,
epithelial cells which also serve as "reserve cells" for the alveolar epithe-
lium. However the mechanisms regulating surfactant biosynthesis, secretion
and removal from the alveolar spaces are presently not known.

Silica dust which is a material of considerable environmental and occupational
concern since it can cause chronic obstructive lung disease (silicosis) was found
to cause a marked increase in surfactant levels in the lungs of exposed rabbits.
This effect of silica provides an opportunity to study the interrelationships
between surfactant synthesis, release and removal and as a means to disturb the
normal equilibrium and clarify some of the pathogenetic mechanisms involved in
silica induced pulmonary disease. The initial research efforts devoted to this
project were designed (1) to develop methodologies to quantitative intra- and
extracellular surfactant pools in the lungs of rabbits, and (2) to characterize
and quantitate the changes in intra- and extracellular surfactant resulting fr-
a large exposure to silica. Time and dose-response relationships were
established 1 month after a single dose of silica, administered by intratracheal
instillation. The intracellular pool was increased 80-fold and the extracellu-
lar surfactant pool was increased 29-fold above normal. Using ^H-palmitate as a
phospholipid precursor, measurements of the rates of surfactant synthesis.



449



om



transfer to the extracellular pool and turnover in the extracellular pool, were
conducted. These studies showed that biosynthesis was increased about 10-fold,
transfer to the extracellular pool about 4-fold and extracellular turnover
approximately 2-fold. This suggests that the primary (though not the only)
disruption of the surfactant system by silica may be resulting from a massive
stimulation of surfactant synthesis which neither the secretory mechanisms nor
the extracellular removal mechanism are able to cope with. Further studies willi
be conducted with isolated Type II cells from normal and silica exposed animals
to analyze the observed phenomena further.

In collaboration with the members of the Cell Biology Group, another project has;
been pursued, which is aimed at the elucidation of the function of the so-calledi
"Clara cell", an epithelial cell prominent in the distal airways. This cell is
believed to be a secretory cell based on ultrastructural evidence. It is a eel II



Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1984 pt.1) → online text (page 40 of 60)