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From the Department of Anatomy, Physiology and CellBiology*
and the Department of MolecularBiosciences,
School of Veterinary Medicine,University of California Davis, Davis, California; and the Departmentof Environmental Health Sciences,
School ofPublic Health, University of Alabama at Birmingham,Birmingham, Alabama
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-glutamylcysteine synthetase (-GCS) was induced in tolerant Clara
cells by repeated exposures to NA. Treating tolerant mice with
buthionine sulfoximine, a -GCS inhibitor, eliminates
resistance acquired by repeated exposures to NA. Broad phenotypic
shifts were not present. Marker proteins of differentiation declined
over the first 3 days in the development of tolerance, but
returned to control levels at 14 and 21 days. Epithelial organizational
structure and internal organelle composition in Clara cells from
tolerant mice were similar compared to corn oil-treated
controls, while subtle shifts in organelle distribution were
present. We conclude that induction of -GCS expression is
coordinated with the development of NA tolerance, but induction
of NA tolerance does not markedly alter Clara cell
differentiation, epithelial organization, or organelle
composition in bronchiolar epithelium.
The ubiquitous antioxidant glutathione (GSH) has various intracellular
roles, including the maintenance of enzyme structure, activity, and
intracellular redox state. Acting as a cycle, enzymes involved in the
synthesis, conjugation, and transport of GSH, collectively called the
-glutamyl cycle, are important in detoxification.9,10
Of critical importance is the role of GSH in the detoxification of
xenobiotics such as naphthalene. Mouse lung microsomes metabolize
naphthalene to glutathione conjugates11
which deplete
Clara cell glutathione in a dose dependent fashion.12,13
Toxicity is augmented by the treatment of mice with diethyl maleate, an
agent which depletes cellular glutathione.12
In addition,
it is becoming clear that the -glutamyl cycle is a dynamic system
with the ability to change under stress to protect cells from
additional toxic exposures.14,15
We have demonstrated that
the reduced susceptibility to NA injury by repeated exposure is
directly related to increased airway glutathione
resynthesis.16
However, it is possible that the changes in
susceptibility to naphthalene injury result from temporal shifts in the
repair process or the differentiation state of the cells in the target
site of injury, the bronchiolar epithelium.
Clara cell injury from bioactivated cytotoxicants such as naphthalene and dichloroethylene is followed by a repair phase that spans up to 30 days.2,3 During this time frame, distinct periods of proliferation are followed by maturation of undifferentiated cells back to a steady population containing both mature Clara and ciliated cells.17 In parallel to this differentiation process fluctuations in the expression of enzymes involved in the bioactivation of NA occur.17 Protein levels of cytochrome P-4502f2 protein drop dramatically after a single injurious administration of NA, but return to control levels after 14 days of repair.17 With respect to tolerance, the balance between bioactivation and detoxification appears to be critical. While after repeated exposures to NA protein levels of cytochrome P-450 appear to be decreased,18 our previous studies indicate that increased airway GSH was critical in the development of tolerance.16 Determining if the differentiation state and the ability of airways to repair while under constant stress from daily toxicant exposure is an important step in understanding how cells adapt to a state of tolerance to bioactivated xenobiotics, especially if the compounds are known carcinogens. This prompted us to ask the question whether changes in the differentiation state of Clara cells contributed to the development of NA tolerance.
This study was designed to test the hypothesis that changes in the
detoxification potential, in response to repeated exposures to NA,
result from a stable adaptation that favors maintenance of the GSH pool
without broad changes in cellular phenotype. To test this hypothesis we
addressed three questions: is induction of -glutamylcysteine
synthetase (-GCS) coordinated with the development of Clara cell
tolerance to long term exposures (21 days) of NA; do these cells in
tolerant mice express differentiation marker proteins characteristic of
normal Clara cells from naive mice and, do Clara cells from tolerant
mice retain the morphological and organelle structural characteristics
of the controls after repeated exposures of NA. Determining whether
Clara cells undergo specific alterations to maintain increased GSH
pools without drastic phenotypic shifts will provide insight into the
in vivo mechanism by which cells adapt to resist injury from
daily exposures to environmental pollutants.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Naphthalene was purchased from Fischer, Fairlawn, NJ. DL-buthionine-[L,R]-sulfoximine (99.0% purity) (BSO) was purchased from Sigma Biochemical, St. Louis, MO. All fixatives and embedding reagents were purchased from Electron Microscopy Sciences (Fort Washington, PA.) All other solvents were reagent grade or better.
Experimental Protocol
Male Swiss Webster mice (6 to 7 weeks) were purchased from Charles River (Wilmington, MA.) Animals were allowed free access to food and water and were housed in an AAALAC accredited facility in HEPA filtered cage racks at the University of California, Davis for at least 5 days before use in an experiment. Mice were administered NA (0 or 200 mg/kg) daily for 21 days. To determine whether GSH is critical in the development NA tolerance, mice receiving repeated exposures (4, 7, 14, and 21 days) were treated with BSO (0 or 800 mg/kg), and challenged with an additional NA dose then processed for high-resolution histopathology. To determine whether Clara cells retain normal phenotypic characteristics, mice were administered (i.p.) NA (0 or 200 mg/kg) daily for 21 days. Mice were killed and lungs fixed by tracheal infusion at 12 and 24 hours after a single exposure and 2, 3, 4, 7, 14, and 21 days after repeated daily exposures and processed for immunohistochemistry, scanning electron microscopy (SEM), or transmission electron microscopy (TEM).
Buthionine Sulfoximine Treatment
Previous studies have demonstrated that BSO can cause a
rebound19
or increases in GSH levels due to the feedback
inhibition of -GCS. We conducted several preliminary experiments to
determine the optimal timing of the BSO and NA doses and have
previously reported this data.16
The following protocol
was used for all mice in the results presented here. Mice receiving
repeated exposures of NA were treated with BSO (0 or 800 mg/kg), a GSH
resynthesis inhibitor, 24 hours after doses 4, 7, 14, and 21. One hour
later mice were challenged with an additional NA dose (0 or 200 mg/kg),
killed 3 hours after the NA challenge, and then processed for
histopathological assessment.
High-Resolution Histopathology
All mouse lungs for histopathological assessment were prepared by inflation via tracheal cannula with 1% glutaraldehyde/1% paraformaldehyde in 0.1 mol/L cacodylate buffer 335 mOsm for 1 hour at 30 cm H2O pressure. The entire fixed middle (cardiac) lobe was postfixed with osmium tetraoxide and incubated overnight in uranyl acetate.20 The postfixed tissue was embedded in Araldite-502 (Electron Microscopy Sciences), and embedded tissue was then grossly sectioned parallel to the long axis of the mainstem bronchi. Sections (0.5 µmol/L) were cut with glass knives using a Zeiss Microm HM340E microtome and stained with 1% toluidine blue (Electron Microscopy Sciences, Fort Washington, PA). Slides were imaged with a 330 CCD Dage camera on a Zeiss Axiakop MC80 microscope using Scion 1.59 imaging software.
Immunohistochemistry
Lung tissue from mice was fixed with 4% paraformaldehyde for 2
hours then placed in phosphate-buffered saline (PBS) until processing
for embedding. Tissue were embedded in paraffin and sectioned at 6-µm
thickness. The presence of the marker proteins for differentiated Clara
cells was detected using specific antibodies: rabbit anti-rat Clara
cell secretory protein (CC10) and rabbit anti-mouse cytochrome P4502f2
(CYP2f2). 21,22
Antigenic proteins were identified by the
avidin-biotin horseradish-peroxidase method as outlined by Plopper et
al.23
Controls for non-specific binding were performed by
substituting primary antibody with PBS. The antibody for CC10 was a
generous gift for Dr. Gurmukh Singh (Veterans Affairs Medical Center,
Pittsburgh, PA) and the antibody for cytochrome P-4502f was a generous
gift from Dr. Henry Sasame (National Institutes of Health, Bethesda,
MD). A 19-amino-acid peptide was synthesized according to the published
rat -GCS heavy subunit amino acid sequence (at position 295–313:
NH2-CRWGVISASVDDRTREERG-COOH).24
This peptide was conjugated to carrier keyhole limpet hemocyanin (KLH)
and used as an antigen to raise rabbit antisera against rat -GCS-HS.
The polyclonal antibodies against -GCS-HS were used in the
subsequent Western blot analysis.
Western Blot Analysis
To determine the relative expression of Cyp4502f2 and -GCS, the
distal lung region was a isolated via microdissection of agarose
inflated lungs. Briefly, tolerant mice were killed with a overdose of
pentobarbital 24 hours after the last dose of naphthalene. The trachea
of these animals was cannulated and lungs were infused with a 1%
agarose solution containing Waymouths media deficient of
sulfur-containing amino acids. Appropriate regions of lung were
microdissected as previously described.25
Microdissected
airways were placed in an ice cold lysis buffer (Tris 0.1 mol/L (pH
8.2), KCl 150 mmol/L, MgCl2 20 mmol/L, and EDTA 2 mmol/L) containing
protease inhibitors and homogenized. Homogenized proteins were
centrifuged at 9000 x g for 20 minutes. Soluble
protein content was determined by the microBradford
method26
and samples were frozen at -80°C until
analysis. Soluble protein were diluted in a 10% sodium dodecyl sulfate
(SDS) sample buffer containing ß-mercaptoethanol and separated by gel
electrophoresis using a Bio-Rad Tris-HCl-buffered 10% polyacrylamide
Minigel (Hercules, CA). Gels were transferred on to a
polyvinylydifluoroacetate membrane (NEN, Inc., Boston, MA) and probed
with the rabbit antibody produced against the cytochrome P4502f
(1:10,000) or the catalytic subunit of -glutamylcysteine synthetase
(1:5000). Bands were detected by chemilumenescence using a horseradish
peroxidase-linked secondary goat antibody produced against rabbit IgG.
Scanning Electron Microscopy
Briefly, the airway tree of the cranial lobe was exposed by microdissection, dehydrated through a graded ethanol series, immersed in hexamethyldisilazine for 5 minutes, and air dried overnight. Desiccated lungs were then gold-coated with a Polaron II E5100 sputter coater at 2.5 kV acceleration voltage in argon with a 10 mA current for 2 minutes. Airways were imaged using a Phillips SEM 501 microscope.
Transmission Electron Microscopy
Terminal airways from mice receiving repeated daily injections of NA (0 or 200 mg/kg) were selected from araldite sections used for high-resolution histopathology. Using the original blocks these airways were isolated and sectioned at 70 nm with a Sorvall MT 5000 utlramicrotome. These sections were stained with uranyl acetate and lead citrate then examined using a Zeiss EM-10 electron microscope at 60 kV. Complete profiles (including basal lamina, nucleus, and apical projections) of a minimum of 10 Clara cells per animal were categorized blindly by one investigator for differences in size, mitochondrial conformation, and secretory granule distribution and abundance.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Terminal airways of mice receiving repeated corn oil injections
were lined by a simple cuboidal epithelium (Figure 1A)
. The majority of the cells were
non-ciliated. Many of these non-ciliated, or Clara cells, had apical
projections into the airway lumen. Three hours after control mice were
administered 200 mg/kg NA, Clara cells were swollen (Figure 1B)
, formed
large clear cytoplasmic vacuoles, and had discrete apical blebs (**).
No signs of toxicity were detected in control mice treated with BSO
alone without NA (data not shown).
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. In contrast, 3 hours after administration of the NA
challenge dose, tolerant animals from the same time points treated with
BSO had severe airway injury similar to challenged non-tolerant mice
(Figure 1, D, F, H, J)
. After four repeated exposures to NA, the
resulting injury from pretreatment with BSO before NA challenge failed
to injure all of the cells in the distal airways (Figure 1D)
, there was
consistent sparing of the most terminal population of Clara cells. The
injured cells appeared to be clustered together, while large areas of
cells remained unaffected by the combined treatment. After 7, 14, and
21 days of repeated injections, pretreatment with BSO caused
significant NA-induced injury. The response in the distal airways of
these mice was less variable than mice receiving four repeated
injections of NA. Injury extended down to involve the most terminal
Clara cells, in contrast to those spared after four repeated injections
of NA. Occasional Clara cells were exfoliated and most had discrete
swollen apical membrane blebs (**), similar to NA-challenged controls.
Overall, the airway epithelium from the tolerant mice was similar to
NA-challenged non-tolerant mice. No signs of toxicity were detected in
tolerant mice treated with BSO alone without NA (data not shown).
Cellular Expression of -GCS
In control mice, detectable -GCS was present throughout the
airways and lung parenchyma as diffuse lightly positive staining within
the cells (Figure 2B)
compared to control
sections with PBS substituted for primary antibody (Figure 2A)
. This
corresponded to a faint band detectable by Western blot analysis
(Figure 3)
. Clara cells in control mice
appeared to have protein localized to the cytoplasm of the basal half
of the cell (Figure 2B
, inset). Twenty-four hours after a single
injection of NA, immunoreactive protein appeared to be induced in
bronchioles isolated by microdissection as assessed by Western blot
(Figure 3)
, and appeared in cells exfoliated from the basal lamina in
terminal bronchioles (Figure 2C)
. After two repeated injections,
-GCS protein was clearly elevated in microdissected bronchioles from
tolerant mice (Figure 3)
and immunohistochemically evident in both the
apical and basal regions of Clara cells (Figure 2D)
. A similar
expression pattern was present after 7, 14, and 21 repeated daily
injections of NA (Figure 2, G–I)
. After 14 and 21 repeated daily
injections, Western blot analysis revealed that -GCS expression
declined but remained elevated from control (Figure 3)
, while the
epithelium in terminal airways retained a strong immunoreactive signal
(Figure 2, H and I)
.
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Immunoreactive CC10 was only detected in Clara cells, and was
expressed throughout the airways of control mice (Figure 4A)
. Twelve hours after receiving one NA
exposure (200 mg/kg) CC10 positive cells were exfoliating into the
airway lumen (Figure 4B)
. Twenty-four hours after a single NA injection
immunoreactive CC10 was detected, but reduced in the terminal airways,
and was not prominent in the squamated cells after exfoliation (Figure 4C)
. After two daily injections of NA the most distal airways were
largely devoid of immunoreactive CC10 (Figure 4D)
and this pattern of
expression remained after 4 repeated daily exposures (Figure 4E)
. After
7 daily exposures to NA, (Figure 4F)
the number of CC10 immunoreactive
cells increased, but the overall intensity was less than controls.
After 14 and 21 daily injections, the expression of CC10 became
indistinguishable from control animals.
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The expression of immnunoreactive Cyp2f2 protein in control
animals was only detected in Clara cells (Figure 5A)
and was detected as a single band by
Western blot analysis (Figure 6)
in mice
receiving repeated corn oil injections. The overall pattern of changes
in CYP2f2 parallels that of CC10. Twelve hours after a single dose of
NA (200 mg/kg) cells with immunoreactive Cyp2f2 were exfoliated into
the airway lumen (Figure 5B)
. After 24 hours, immunoreactive Cyp450 was
not detectable by Western blot (Figure 6)
and, like CC10, at this time
was absent from the most terminal airway Clara cells (Figure 5C)
.
Terminal airways of mice receiving two or three daily exposures of NA
also expressed greatly reduced Cyp2f2 based on Western blot analysis
(Figure 6)
or immunohistochemistry (Figure 5D)
. After four daily
injections of NA, a faint, but detectable band was present in Western
blots (Figure 6)
, which was coordinated with focal expression of
immunoreactive protein in bronchiolar epithelial cells (Figure 5E)
.
After 1 week of repeated daily exposures to NA, Cyp2f2 expression
continued to increase (Figure 6)
; most Clara cells in the terminal
airways now expressed a diffuse signal for immunoreactive for CYP2f2
(Figure 5F)
. While these marker proteins differed at 7 days, after 14
and 21 repeated exposures of NA the expression of Cyp2f2 appeared
similar to control animals when assessed by both Western blot analysis
(Figure 6)
or immunohistochemistry (Figure 5, G and H)
.
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Terminal airways from control mice were lined by two predominant
cell types, non-ciliated (Clara) cells and ciliated cells (Figure 7A)
. The non-ciliated cells had apical
projections that extended into the lumen and were organized in linear
rows down the main axial path of the airways. Ciliated cells were less
numerous and interspersed among these rows. The apical portion of the
Clara cells was a defined dome-like region in the center of the cells.
The cilia covered the entire lumenal surface of the ciliated cells
(Figure 7A)
. Twenty-four hours after an intraperitoneal injection of
NA, the terminal airways were devoid of intact Clara cells (Figure 7B)
which had exfoliated and overlaid the subadjacent layer of attenuated
ciliated cells (Figure 7B)
. The cilia of these cells was now restricted
to the center of the cell as a discrete tuft (Figure 7B)
. After two or
three repeated injections of NA, cilia appear shorter and less
prominent and non-ciliated cells again are apparent but are poorly
differentiated and unorganized (Figure 7, C and D)
. The cilia are
difficult to distinguish and often absent after mice have received
three daily NA exposures (Figure 7D)
. After four repeated exposures
clusters of cells with small apical projections begin to appear in the
distal airways (Figure 7E)
. After seven daily injections of NA these
clusters of Clara cells have coalesced to the majority of the terminal
airway surface (Figure 7F)
and infrequently appeared as nodules. The
presence of these nodules as seen with high-resolution histopathology
has been previously reported elsewhere.16
Ciliated cells
were now more easily identifiable (Figure 7F)
, but few appeared to have
cilia comparable in length to those in control mice (Figure 7A)
. After
14 days of daily injections of NA, the epithelium of terminal airways
appeared more organized, the cells were more evenly dispersed and
morphologically similar to control animals (Figure 7G)
. After 21 daily
exposures to NA, the individual cell types of the epithelium of the
terminal airways are clearly recognizable, although both Clara and
ciliated cells appear to be larger with more distinct cell borders,
such that fewer cells are seen per field (Figure 7H)
.
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When compared to Clara cells of carrier-treated control animals,
there were considerable variations in the ultrastructure of Clara cells
in terminal bronchioles of animals exposed to NA for 21 days (Figure 8)
. Mitochondria in control animals were
a mixture of large circular profiles with electron dense matrix and few
cristae (Figure 8B)
and smaller mitochondria with elongated profiles
and more abundant cristae. All Clara cells evaluated from control
animals had an abundance of large mitochondria (Figure 8A)
. By
contrast, most of the mitochondria present in treated animals had long,
narrow profiles with large abundant cristae (Figure 8D)
. In very few of
the Clara cells evaluated in tolerant animals (7%) were large
mitochondria present. In comparison to control animals, smooth
endoplasmic reticulum (SER) in tolerant mice appeared to be more
abundant, very tightly packed, and distributed throughout the cytoplasm
(Figure 8, C and F)
. The Golgi apparatus also appeared to be more
abundant. In control animals, Golgi was not apparent in every
ultrastructural profile (Figure 8A)
, while in profiles of Clara cells
from tolerant mice, there were more (minimum of two) Golgi profiles
(Figure 8C)
, and each Golgi profile had more lamellae whose cisternal
content was more dense. In tolerant animals, secretory granules were
less abundant, more varied in size, and more widely dispersed around
all aspects of the nucleus (Figure 8C)
. In Clara cells from control
mice the granules were restricted to the apical portion of the
cytoplasm, generally in the lumenal projection (Figure 8A)
. However,
when compared quantitatively, fewer cells in tolerant animals (21%)
had a wide dispersion of granules than was the case for the controls
(36%). Rough endoplasmic reticulum (RER) was much more abundant in
Clara cells of tolerant mice, and was widely distributed dorsal, basal,
and lateral to the nucleus (Figure 8G)
. In Clara cells of control
animals, RER was restricted to a few cisternae on the basal-lateral
side of the nucleus (Figure 8A)
. In none of the Clara cells evaluated
was there any sign of injury or other cytopathology. In Clara cells of
animals exposed to NA for a shorter time (4 to 14 days), there was a
wide range of ultrastructural features varying between that of the
21-day exposed and carrier-treated controls. This mixture was found
within the Clara cell population of the lining the individual
bronchiole. All of the treated animals exhibited this range to varying
degrees. In animals exposed for 7 days, some of the Clara cells had SER
more tightly packed and more abundant than was observed in control
animals and also had evidence of the large mitochondria with few
cristae. A small population was not discernibly different from that
which lined the bronchioles of 21-day exposed animals and another which
was essentially indistinguishable from Clara cells from control
animals. In 21-day exposed mice most of the Clara cell profiles (73%)
were larger in size and had broader apical projections (compare Figure 8, A and C
). None of the Clara cells evaluated in control animals
exhibited profile sizes within the range found in tolerant animals. At
the apical surface of Clara cells in 21-day exposed mice (Figure 8F)
,
SER was packed closer to the plasmalemma and reduced the subplasmalemma
zone of organelle-free cytoplasm compared to Clara cells in controls
(Figure 8E)
.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-GCS protein, the rate limiting step of
glutathione synthesis; 3) differences in expression of proteins
considered markers of differentiated Clara cells were not discernable
between tolerant or control mice after 21 days of repeated exposures;
and 4) Clara cells of tolerant mice retained three-dimensional
conformation and mixtures of organelles similar to control mice, but
appear to have increased cell size and increased content of organelles.
Previous studies have indicated that the development of tolerance to NA
results from shifts in the balance between NA
bioactivation4,18
and detoxification.16
In
this study we found even longer exposure regimens result in a subtle
alterations that favor increased detoxification of naphthalene in the
resistant or tolerant cells of target airways. Our studies indicate
that mice receiving up to 14 and 21 repeated injections, have CYP2f2
expression that is no different from control, while -glutamyl
cysteine synthetase remains induced during continued exposure. These
data indicate that increases in -GCS expression plays a critical
role in the maintenance of NA tolerance even as the exposure duration
continues over a period of weeks.
Our previous studies demonstrated that the development of NA tolerance
was critically dependent on the thiol status.16
In those
studies we determined that repeated exposures of NA result in Clara
cell populations that had elevated the steady state GSH level to become
tolerant to further injury. We also found in tolerant animals the
maximal activity of -GCS, the rate limiting step of GSH
biosynthesis, had increased significantly above control. Further, when
we inhibited the activity of this enzyme, using BSO, in tolerant mice,
the induced resistance was eliminated and Clara cells again became
susceptible to injury.16
While important in addressing
the actual mechanism of NA tolerance, the previous studies did not
determine whether the GSH dependence of NA tolerance was merely a
result of temporal shifts in the repair process. In the current study,
the fact that we were able to eliminate tolerance with BSO at 4, 7, 14,
or 21 days indicates that tolerance is dependent on maintenance of the
thiol status from at least day 4 and continues for long-term exposure
periods. Further, using an antibody for -GCS we found protein
expression of this enzyme, which catalyzes the rate-limiting step of
glutathione synthesis is induced early and remains elevated in
tolerance. Overall, the present studies indicate the ability to induce
and maintain an elevated GSH pool to protect Clara cells from NA
cytotoxicity was critical in mice that had become tolerant, whether
they had received as few as 4 or as many as 21 repeated injections of
naphthalene.
Of particular interest, was the variable pattern of injury that
occurred when tolerant mice from different time points (4, 7, 14, and
21 days) were administered BSO and NA in combination. When mice
receiving 4 daily repeated NA injections were challenged with the
combination of BSO and NA, not all cells were injured. This was also
the case for tolerant mice receiving 7 repeated NA injections. Mice
receiving up to 14 and 21 injections appeared to have a more uniform
response to the combined challenge with BSO and NA. These differences
are highlighted by shifts in the cellular content of cytochrome CYP2f2.
Mice receiving 4 repeated daily injections had localized expression of
CYP2f2, which appeared to be expressed in a group of four or five cells
on a section. The changes were mirrored by expression of CC10, as well
as the formation of morphologically distinct clusters of Clara cells as
seen by SEM. After receiving 7 repeated exposures, these clusters of
cells appeared to be more abundant in the distal airways as did the
expression of CC10 and CYP2f2. This was also coordinated with a more
consistent injury pattern. After 2 and 3 weeks of repeated NA
exposures, Clara cells appeared morphologically similar to control both
surface structure and expression of CC10 and CYP2f2. The injury pattern
in these tolerant mice from combined treatment with BSO and NA appeared
similar in severity and extent to control mice. These results taken
together indicate that, as the bronchiolar epithelium returns to a
normal phenotypic state, the balance between phase I (cyp2f2) and phase
II (GSH conjugation) metabolism appears to be shifted to favor
detoxification of NA during prolonged exposure. Moreover, this data
combined with our previous studies16
indicate that the
induced expression of -glutamyl cysteine synthetase plays an
increasing role in the defense of cellular cytotoxicity from
bioactivated cytotoxicants as the exposure protocol extends from days
to weeks.
Shifts in the thiol status have been seen in other models of tolerance. Similar to NA tolerance in mice, rats exposed to ozone for prolonged periods have increased levels of reduced GSH.27 However, in contrast to NA tolerance, the development of tolerance to ozone includes phenotypic shifts in cells of the centriacinar region,28 including elevation of CC10, a cytoprotective protein considered a marker of differentiated Clara cells.29 This elevation of CC10 is in part due to the bronchiolarization of the alvealor ducts, where Clara cells appear to replace type II cells in the centriacinar region. The present study of NA tolerance, indicates that broad phenotypic shifts do not appear to play a role in NA-induced Clara cell tolerance. Using both SEM and TEM we found that cell size and organelle distribution had shifted in tolerant Clara cells. However, in contrast to ozone tolerance, Clara cells appeared to maintain overall structural and differentiation characteristics associated with Clara cells from carrier-treated animals, including normal expression of CC10 of mice receiving 14 and 21 days of repeated NA exposures.
Our hypothesis was that the development of tolerance to bioactivated cytotoxicants was based on a specific set of phenotypic changes. Using phenotypic markers of differentiation in Clara cells, we found that cell phenotype, instead of altering to a less metabolically active state, appeared to regain characteristics of the controls. Based on the relatively long clearance time for inhaled bioactivated carcinogenic compounds30 it seems reasonable to suggest that during repeated exposures to lipophilic volatile compounds, such as naphthalene, the return of P450 metabolism would favor the clearance of these compounds from the respiratory tract. Because phase I metabolism of these compounds, like naphthalene, is often the obligate step in the formation of the toxic intermediate, the system has evolved in two parts, bioactivation and detoxification. The second step, detoxification, in this case uses a ubiquitous tripeptide, GSH, to alleviate the toxicity of these often inhaled compounds while allowing for them to be removed from the body.
Because inhalation is the major route of exposure for NA in humans the next issues to be addressed by our studies will be whether Clara cells become tolerant by inhalation exposures and whether the mechanism for tolerance for repeated systemic exposures is shared by tolerance to repeat inhalation exposures. Additionally, while we saw no broad shifts in the phenotype of Clara cell in our long-term studies, the importance of other cytoprotective mechanisms such as heat shock proteins cannot be excluded. Combining techniques such tissue microdissection with cDNA microarray to identify changes in expression of genes will lead to more focused studies of the mechanisms of resistance to bioactivated xenobiotics. Of paramount importance is the link between the development of tolerance and the formation of tumors. While electrophilic intermediates of naphthalene do not appear to form DNA adducts (unpublished data), recent studies indicate that the compound is carcinogenic in mice and rats.7,8 It is possible that the development of tolerance, while conferring the ability of the cellular microenvironment to avoid acute injury, predisposes these cells to neoplastic transformation. The continuation of long-term studies and the characterization of changes in gene expression identified by microarray and applied to such techniques as in situ hybridization, with immunohistochemistry using reliable reagents, to identify changes in single cells will be required to identify the mechanism of tumor formation.
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Footnotes |
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Supported by National Institute of Health grants ES04311, ES06700, ES04699, ES05511 and NHLBI WL07013. The University of California at Davis is a NIEHS Center for Environmental Health Sciences (ES05707) and support for core facilities used in this work is gratefully acknowledged.
Accepted for publication December 13, 2001.
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  J. B. Morris and A. R. Buckpitt Upper Respiratory Tract Uptake of Naphthalene Toxicol. Sci., October 1, 2009; 111(2): 383 - 391. [Abstract] [Full Text] [PDF]
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J. A. A. West, L. S. Van Winkle, D. Morin, C. A. Fleschner, H. J. Forman, and C. G. Plopper Repeated Inhalation Exposures to the Bioactivated Cytotoxicant Naphthalene (NA) Produce Airway-Specific Clara Cell Tolerance in Mice Toxicol. Sci., September 1, 2003; 75(1): 161 - 168. [Abstract] [Full Text] [PDF]
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) of CC10 positive
cells (C) .
After 2 repeated exposures CC10 expression remained low in the terminal
airways (D).
Mice administered 4 repeated injections had multifocal expression of
CC10 (E),
which after 7 repeated exposures had become a diffuse signal throughout
most Clara cells
(F). Mice
receiving 14
(G), and 21
repeated injections
(H) had CC10
expression which was comparable to control mice. Scale bar
in A, 30 µm.
