Potential involvement of 4-hydroxynonenal in the response of
human lung cells to ozone
Raymond F.
Hamilton Jr.1,
Li
Li1,
William L.
Eschenbacher2,
Luke
Szweda3, and
Andrij
Holian1
1 Division of Pulmonary and
Critical Care Medicine, Department of Internal Medicine and
Pharmacology Toxicology Program, University of Texas Medical School,
and 2 Section of Pulmonary and
Critical Care Medicine, Baylor College of Medicine, Houston, Texas
77030; and 3 Department of
Physiology and Biophysics, Case Western Reserve University, Cleveland,
Ohio 44106-4970
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ABSTRACT |
Ozone is a photochemically generated pollutant that
can cause acute pulmonary inflammation and induce cellular injury and may contribute to the development or exacerbation of chronic lung diseases. Despite much research, the mechanisms of ozone- and oxidant-induced cellular injury are still uncertain. Ozone and secondary free radicals have been reported to cause the formation of
aldehydes in biological fluids. One of the most toxic aldehydes formed
during oxidant-induced lipid peroxidation is 4-hydroxynonenal (HNE).
HNE reacts primarily with Cys, Lys, and His amino acids, altering
protein function and forming protein adducts. The purpose of this study
was to determine whether HNE could account for the acute effects of
ozone on lung cells. Human subjects were exposed to 0.4 parts/million ozone or air for 1 h with exercise (each subject
served as his/her own control). Six hours after ozone exposure, cells
obtained by airway lavage were examined for apoptotic cell injury, and
cells from bronchoalveolar lavage were examined for apoptosis, presence
of HNE adducts, and expression of stress proteins. Significant
apoptosis was evident in airway lung cells after ozone exposure.
Western analysis demonstrated an increase in a 32-kDa HNE protein
adduct and a number of stress proteins, viz., 72-kDa heat shock protein
and ferritin, in alveolar macrophages (AM) after ozone exposure. All of
these effects could be replicated by in vitro exposure of AM to HNE.
Consequently, the in vitro results and demonstration of HNE protein
adducts after ozone exposure are consistent with a potential role for
HNE in the cellular toxic effects of ozone.
aldehydes; apoptosis; protein adducts; stress proteins; alveolar
macrophages
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INTRODUCTION |
OZONE is a photochemically generated pollutant that has
been demonstrated to produce acute pulmonary inflammation and cellular injury. It has been proposed to contribute to the development or
exacerbation of chronic lung diseases (20, 32). However, despite
extensive investigation, the mechanism(s) of ozone-induced lung injury
is not totally understood. Furthermore, not all of the effects of ozone
on the lung cells have been clearly defined.
Aldehyde production as a result of ozone inhalation could be an
important mediator of ozone toxicity (23). Furthermore, secondary
radicals formed after ozone exposure could react with polyunsaturated
fatty acids present in the lung lining fluid and cell membrane lipids
to form hydrogen peroxide and aldehydes, of which the latter is
considered to be more toxic (23). Although a large number of aldehydes
can be formed during polyunsaturated fatty acid peroxidation, three
aldehydes have been extensively studied as physiologically relevant
lipid peroxidation products [4-hydroxynonenal (HNE),
4-hydroxyhexenal, and malonaldehyde]. Among the three, HNE is
considered to be the most toxic aldehyde at the cellular level (7).
HNE has been shown to be formed in various models of inflammation and
oxidative stress (24). In addition, we have recently shown that HNE is
formed after ozone exposure, using murine and human models (10, 17). In
human studies, ozone was shown to decrease alveolar macrophage (AM)
function and to increase expression of 72-kDa heat shock protein
(HSP72) in AM (10). Results from ozone exposure in murine models also
demonstrated that ozone could induce apoptosis of lung cells (17).
These results were consistent with studies indicating that HNE could
induce HSP72 expression (3). Stress proteins, highly conserved between
species, are a family of proteins synthesized when the cell is
subjected to a variety of environmental assaults (e.g., heat, chemical,
etc.), and they perform various functions to protect and adapt the cell to the stress condition (26). Stress protein induction could explain or
contribute to the mechanism of adaptation that has been observed after
repeated ozone exposures (9). Furthermore, in vitro studies with HNE
using murine AM demonstrated that the effects of HNE could mimic those
of ozone exposure and were dose dependent (17, 19). Low concentrations
(5-25 µM) of HNE induced the stress protein heme oxygenase-1
(HO-1), 50-100 µM HNE induced apoptosis of AM, and higher
concentrations of HNE caused cellular necrosis.
The purpose of the present study was to further test the hypothesis
that HNE contributes to the effects of ozone injury. Human subjects
were exposed to 0.4 parts/million (ppm) ozone or air for 1 h with
exercise and then underwent both airway lavage (AL) and bronchoalveolar
lavage (BAL) 6 h later. BAL cells were examined for evidence of stress
protein expression, apoptosis, and formation of HNE-protein adducts.
Due to limited cell yields, AL cells were examined for apoptosis only.
In vitro studies were also conducted with human AM to
determine whether HNE could mimic the effects of ozone.
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METHODS |
Study design.
Detailed subject and ozone-exposure method descriptions are available
elsewhere (12). All subjects were healthy, nonsmoking volunteers who
participated in this approved study after giving informed consent that
was approved by the Institutional Review Boards of The Methodist
Hospital, Baylor College of Medicine, and The University of Texas
Houston Health Science Center. Each individual was evaluated with a
complete history and physical examination and was screened for
coagulation abnormalities. No subject had an upper respiratory tract
infection within 6 wk before either exposure. Briefly, four human
subjects received identical 1-h sham (air exposure) and 0.4 ppm ozone
exposures with exercise 14 to 44 days apart. Subjects wore a noseclip
and exercised continuously for 1 h on a cycle ergometer (type KEM-3;
Mijnhardt, Odijk, The Netherlands) at a workload sufficient to achieve
a minute ventilation of 30 l · min
1 · m2 body surface area
1. The order of exposure was
randomized. Bronchoscopies, including BAL of the right middle lobe
segment, were performed on all subjects after both air and ozone
exposure 6 h after the termination of exposure. Concomitant with
bronchoscopies, ALs were also performed on all subjects.
BAL and segmental AL.
Six hours after exposure, subjects underwent bronchoscopy in a room
adjacent to the exposure chamber in which
O2 and suction were available. No
oral or parenteral premedication was used. The subjects were
continuously monitored by pulse oximetry (OxyShuttle; Sensormedics,
Anaheim, CA). Both nares were anesthetized with 2% Xylocaine jelly.
Bronchoscopy was performed in the usual fashion without complications.
Vocal cords were anesthetized using topical 1% Xylocaine in small
boluses, with no more than 15 ml being used during the procedure. The
bronchoscope was wedged in a right middle lobe segment, and lavage was
performed with 35-ml aliquots of normal saline to a maximum of 140 ml.
Subsequently, we performed the segmental AL with a custom-designed
balloon catheter (no. 792015; Baxter, Santa Ana, CA) with two proximal
infusion and withdrawal ports. The catheter was introduced through the
nares not occupied by the bronchoscope and, under bronchoscopic
visualization, was advanced to the left main stem bronchus. At this
location, the balloon was inflated with adequate seal with no more than 2 ml of air. The bronchus was lavaged with 5-ml aliquots of normal saline until 5 ml of fluid were retrieved. The samples were immediately placed on ice and were transferred to the laboratory for analysis.
Cells were isolated from both AL and BAL and were examined for
apoptosis, stress proteins, and HNE-protein adducts as described below.
Cell counts were determined using a ZBI Coulter Counter (Coulter
Electronics, Hialeah, FL). BALs from the study groups yielded an
average of 6 × 106 cells
that were >90% viable by trypan blue exclusion. ALs yielded between
0.1 × 106 and 1.0 × 106 cells.
Lung lavage and cell isolation.
Lavage fluid was kept at 4°C until cells were isolated from the
lavage fluid by a 4°C centrifugation at 1,500 revolutions/min (rpm;
IEC centrifuge; Needham Heights, MA). The saline supernatant was
aspirated, and the cell pellet was resuspended in a small volume
(1-5 ml) of
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered medium 199 (GIBCO BRL, Gaithersburg, MD) with 10% heat-inactivated fetal bovine serum (Sigma Chemical, St. Louis, MO) and
added antibiotics (50 U/ml penicillin, 50 µg/ml gentamicin, and 50 µg/ml streptomycin). All culture media were pretreated with polymyxin
B beads, removed before use, to ensure an endotoxin-free media. For in
vitro cultures with HNE, 1 × 106 cells/ml were cultured at
37°C in the media described above for 4, 6, or 24 h in the presence
and absence of variable concentrations of HNE. The HNE was
pipetted directly into the cell culture from a 50 mM stock solution in
absolute ethanol stored at
80°C. Cell cultures were
maintained in suspension by slow end-over-end tumbling (Labquake
Shakers; Labindustries, Berkeley, CA) in sterile polypropylene tubes
(PGC Scientific, Gaithersburg, MD) at 37°C in a water-jacketed CO2 incubator (Queue, Parkersburg,
WV; see Ref. 25). Suspension cultures were selected for use in these
studies rather than adherent cultures for the following reasons:
1) they better model freshly isolated human AMs that were obtained by lung lavage because they were
not adherent; 2) adherence
downregulates macrophage function (A. Holian, unpublished
results) and may alter the response in these studies; and
3) suspension cultures allow for a
more uniform response and reliability with the assays in this study.
Cell viability assays (trypan blue exclusion).
Cells in culture suspension were exposed to trypan blue dye
[0.04% in phosphate-buffered saline (PBS)], placed on a
hemocytometer, and examined under light microscopy. Two hundred random
cells were counted after each treatment, and the percentage of blue cells was expressed as the percentage of nonviable cells for any given
condition.
Morphological differentials.
Immediately after cell isolation, 30 × 103 cells were placed in sterile
disposable cytofunnels (Shandon, Cheshire, UK) and then were
centrifuged at 1,500 rpm for 5 min onto positively charged glass slides
(Probe On Plus; Fisher Scientific, Pittsburgh, PA) using a Shandon
Cytospin 2 centrifuge. The cells were stained with Leukostat staining
protocol as described below (Fisher Scientific). Differentials were
performed at ×630 (dry objective) using a Zeiss microscope
(Zeiss). Two hundred random cells were counted and were characterized
as either macrophages, lymphocytes, neutrophils, eosinophils, or other.
Cells obtained from all air-exposed and ozone-exposed subjects were
>90% macrophages.
Apoptosis assays.
Macrophage apoptosis was examined by a combination of Leukostat
staining and detection of DNA fragmentation [Cell Death
enzyme-linked immunosorbent assay (ELISA) and DNA ladder] in the
cells. For Leukostat staining, cells were suspended in PBS (pH 7.2) at
room temperature, cytocentrifuged onto positively charged microscope slides (Fisher Scientific) at 1,500 rpm for 5 min, fixed in cold methyl
alcohol for 5 min, stained in Leukostat eosin stain for 2 min, and then
stained in Leukostat methylene blue stain for 4 s. The slides were
air-dried and were examined by light microscopy at ×630. For the
detection of oligonucleosomes in cytoplasmic fractions (present during
apoptosis) of the cells, the samples were processed and analyzed using
the Cell Death Detection ELISA kit (Boehringer Mannheim, Indianapolis,
IN) according to the manufacturer's protocol. The assay is based on
the quantitative sandwich-enzyme-immunoassay principle using monoclonal
antibodies directed against DNA and histone. Cells (1 × 105) from each sample
were processed, 5,000 cells were used for each reaction, and triplicate
reactions were performed for each sample.
To obtain DNA ladders, human AM were cultured with and without 50 µM
HNE for 6 and 24 h as described above and were washed one time with PBS
before DNA isolation. Genomic DNA was isolated by using the DNA
ISOLATOR (Genosys, Woodlands, TX) according to the manufacturer's
protocol. The isolated genomic DNA was dissolved in 10 mM
tris(hydroxymethyl)aminomethane (Tris)-1 mM EDTA buffer (pH 8.0) and
was 3'-end labeled with
[
-32P]dCTP (ICN
Pharmaceuticals, Costa Mesa, CA) by incubation of 1 µg of DNA in 50 µl of reaction buffer (50 mM Tris · HCl, pH 7.6, 10 mM MgCl2, 200 µM dATP, 200 µM
dGTP, 200 µM dTTP, 2 µl of
[
-32P]dCTP, and 2 U
Klenow) at 37°C for 30 min. The
[
-32P]dCTP-labeled
DNA was mixed with 10 µl of loading buffer (0.25% bromphenol blue,
100 mM EDTA, and 30% glycerol). The same amount of
[
-32P]dCTP-labeled
DNA (50 ng) was loaded onto a 2% agarose gel and was run at 5 V/cm for
5 h in 40 mM Tris-acetate buffer (pH 8.0) with 1 mM EDTA. The gel was
dried at 60°C under vacuum conditions in a gel dryer and was
exposed to X-ray film for detection of resolved labeled DNA fragments.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
Western blotting for stress proteins and HNE-protein adducts.
Immediately after cell isolation, 5 × 105 cells were collected in a
microcentrifuge tube (Eppendorf, Westbury, NY), and the pellet was
treated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis
denaturing buffer (10 µl/1 × 105 cells) and placed in a boiling
water bath for 5 min. The denatured sample was then vortexed,
sonicated, and stored at
20°C until use. After being thawed,
denatured cellular protein samples equivalent to 1 × 105 cells/lane were separated on
12% Ready Gels (Bio-Rad, Hercules, CA) using a minigel apparatus
(Bio-Rad). Resolved proteins were transferred to a nitrocellulose
membrane (Amersham, Arlington Heights, IL) using a wet mini-transfer
unit (Bio-Rad). The transferred blots were stained with 0.1% Ponceau S
to determine uniform transfer and equal loading of the gels. The
transferred blots were then incubated 16 h at 4°C in blocking
buffer (5% Blotto in 10 mM Tris and 150 mM NaCl, pH 7.2). Membranes
were then incubated with stress protein antibodies [HSP72, 65-kDa
heat shock protein (HSP65), and HO-1 from StressGen, Victoria, BC,
Canada, and ferritin from ICN Flow, Costa Mesa, CA] at a 1:2,000
dilution in blocking buffer for 18 h at 4°C or incubated with HNE
adduct antibody (provided by L. I. Szweda and shown to recognize
HNE-Cys, HNE-Lys, and HNE-His adducts; see Ref. 24) at a 1:500 dilution
(24, 30) in blocking buffer for 18 h at 4°C and were washed
extensively with Tris-buffered saline with 0.05% Tween 20 (TBST; pH
8.0). The membranes were then incubated with peroxidase-linked
anti-mouse immunoglobulin (Ig; Amersham) at a 1:20,000 dilution for
HSP72 (or peroxidase-linked anti-rabbit Ig at a 1:10,000 dilution for
HO-1, HSP65, ferritin, and HNE-protein adducts) in TBST for 1 h at room
temperature followed by extensive washing with TBST. The membranes were
incubated in enhanced chemiluminescence (ECL) reagents (Amersham) for 1 min and were exposed to ECL film (Amersham) for 2 min that was
developed by an automated film processor (Kodak, Rochester, NY). For
all Western blots, the resulting exposed areas on the film were
quantified by a scanning densitometer (Bioimage; MilliGen/Biosearch,
Division of Millipore, Bedford, MA), and the relative amounts of
protein are expressed as integrated optical density (IOD).
The polyclonal antibody raised to HNE-modified keyhole-limpet
hemocyanin was tested for cross-reactivity toward
malondialdehyde (MDA) and sodium acetylcysteine (NAC) derivatives of
acrolein, trans-2-pentenal
(t2P), and
trans-2-nonenal
(t2N), which are compounds structurally similar to HNE. Competitive Western blot experiments and
ELISA with these potential competitors were performed. Our results
indicate that the anti-HNE antibody is highly specific to HNE-derived
modifications to protein, exhibiting no binding to the Michael adducts
of NAC-acrolein, -t2P, and
-t2N or to MDA. In addition, antibody
binding requires the presence of the 4-hydroxyl group (29), is
sensitive to the chain length of the modifying 4-hydroxy-2-alkenal
(29), and, as judged by competitive ELISA experiments, recognizes Cys-,
His-, and Lys-HNE adducts (30). The epitope recognized by the antibody
appears to be the hemiacetal form of the HNE-derived portion of
protein-HNE adducts.
Statistical analysis.
Due to subject variability, stress protein Western blot analysis IOD
values were normalized by dividing each subject's ozone-exposed value
by his/her air-exposed value, and these values were averaged. A
one-tailed Student's t-test was then
applied for each stress protein using one-sample hypothesis testing,
assuming the population mean equaled one. For HNE adduct Western blots,
there was no need to normalize the IOD values, and these data were
analyzed by a paired one-tailed Student's
t-test (air vs. ozone). Because of high background optical density (OD) values, Cell Death
ELISA OD values were normalized by subtracting each subject's
ozone-exposed value from his/her air-exposed value, and these values
were averaged. Again, a one-tailed Student's
t-test was then applied using
one-sample hypothesis testing, assuming the population mean equaled
zero.
 |
RESULTS |
Evidence for HNE formation after ozone exposure.
To determine whether HNE was formed during in vivo ozone exposure in
humans, AM obtained by BAL were examined for HNE protein adducts by
Western analysis as described in
METHODS. Figure
1 shows the Western blot obtained for
HNE-protein adducts. The appearance of various HNE-adducted proteins
was common in controls and probably represents constitutive HNE adducts
due to the normal oxidizing environment of the lung. Similar to earlier
studies with human and murine ozone exposures, the dominant feature
after ozone exposure was a consistent increase in a 32-kDa protein
adduct in each subject. The results from densitometry scans for the
32-kDa protein adduct are presented in Fig.
2. Although the increase in HNE-protein adducts did not achieve statistical significance (0.1
P
0.5), this result in combination
with previous findings in the human (10) and murine (17) systems
provides strong evidence for ozone-induced formation of HNE and its
reaction with cell proteins.

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Fig. 1.
4-Hydroxynonenal (HNE)-protein adducts in alveolar macrophages (AMs) 6 h after exposure to 0.40 parts/million (ppm) ozone in 4 subjects. AMs
were isolated from each of 4 subjects, and Western blot analysis for
HNE-protein adducts in the cells was conducted as described in
METHODS. Most clearly evident was an
increase in an adducted protein at ~32 kDa. Conditions for each
subject are labeled air and
O3 exposure.
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Fig. 2.
Integrated optical density (IOD) values for the HNE-adducted 32-kDa
protein shown in Fig. 1. Intensity of the 32-kDa protein was measured
using a densitometer for each subject. Data are presented as means ± SE for 4 subjects.
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AM stress protein expression after ozone exposure.
We have previously shown that ozone could induce HSP72 levels in human
AM after ozone exposure (10). To determine whether ozone exposure
induced a stress response characteristic of oxidative stress, HSP72,
HSP65, HO-1, and ferritin levels in AM were quantitated after air and
ozone exposures. The results of in vivo stress protein induction are
illustrated in Fig. 3, and densitometry
results for these Western blots are shown in Fig.
4. Consistent with previous studies, there
was a variable fourfold increase in HSP72 after ozone exposure (10).
Expression of HO-1 increased in two subjects and remained unchanged in
two subjects after ozone exposure (Western blots not shown). In
contrast, there was no apparent net change in HSP65 protein after ozone
exposure. However, the stress protein most effected by ozone exposure
was ferritin, which showed a variable but consistent increase for all
subjects exposed to ozone. These results extend earlier studies by
demonstrating that a stress protein traditionally associated with
oxidative stress (i.e., ferritin) was also induced after ozone
exposure.

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Fig. 3.
Expression of 72-kDa heat shock protein (HSP72), 65-kDa heat shock
protein (HSP65), and ferritin in AMs 6 h after 0.4 ppm ozone exposure
compared with air exposure. Western blot analysis for each of the
stress proteins and -actin (representing protein loading in each
lane) was conducted as described in
METHODS. Each Western blot shows the
results from the 4 subjects in the study. Although not statistically
significant due to variance, ozone exposure induced the expression of
HSP72 and ferritin in every subject.
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Fig. 4.
Relative stress protein levels in bronchoalveolar lavage (BAL) cells
from Fig. 3. Data are expressed as a ratio of IOD obtained from Western
blot results. For each subject, ozone-exposed values were divided by
the air-exposed values, and resulting ratios were averaged. Results are
expressed as means ± SE for 4 subjects. HO-1, heme oxygenase-1.
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AM stress protein expression after in vitro HNE exposure.
HSP72, HSP65, HO-1, and ferritin levels in AM were quantitated to
determine whether HNE exposure could induce a stress response in human
AM in vitro in a similar manner to in vivo ozone exposure. Human AM
were incubated with 0, 5, or 10 µM HNE for 4 h at 37°C. Representative results of in vitro stress protein induction by HNE from
Western blot analysis are shown in Fig. 5.
Results for HSP72 have been presented elsewhere (10). These results
illustrate that a number of stress proteins can be induced by HNE in
vitro. Furthermore, stress proteins could be induced at relatively low concentrations of HNE, i.e.,
5 µM HNE. The response at low
concentrations of HNE was maximal because higher concentrations
(20-50 µM) did not further enhance stress protein expression
(data not shown). Concentrations of HNE >50 µM produced no increase
in stress proteins compared with control (data not shown).

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Fig. 5.
Dose response for induction of stress proteins in bronchoalveolar cells
in vitro. Results are representative Western blots illustrating HNE
induction of HSP65, HO-1, and ferritin. Human AM cells were incubated 4 h at 37°C with different amounts (0, 5, 10 µM) of HNE, and then
the cell samples were analyzed for stress protein expression in a
similar manner as in Fig. 3. Results are representative of 2 experiments.
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Lung cell apoptosis after ozone exposure.
We have previously demonstrated that HNE could induce AM apoptosis in
vitro in a murine model (19). Therefore, the effects of ozone exposure
on human lung cell apoptosis were examined. Figure
6 is a representative morphological
illustration of apoptosis in airway lung cells after ozone exposure.
Figure 6A is a photomicrograph of
cells obtained by AL after air (sham) exposure. Figure
6B is a photomicrograph of AL cells
from the same subject after 0.4 ppm ozone exposure. In contrast to Fig.
6A, airway cells exposed to ozone
showed a general shrinkage with a darkened, condensed nucleus,
suggesting apoptotic cell injury. Some cells remained unaffected and
have the appearance of control, air-exposed cells. In contrast, there
was no significant morphological evidence of apoptosis in BAL cells
after ozone exposure (data not shown).

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Fig. 6.
Photomicrographs (×630) of airway lung cells isolated 6 h after
air (sham) exposure (A) or 0.40 ppm
ozone exposure (B). Airway cells
were isolated and then were stained with Leukostat as described in
METHODS. Results are representative of
4 subjects. Cells displaying nuclear condensation and cell shrinkage
(consistent with morphological evidence of apoptosis) are indicated by
arrows.
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To further examine whether airway lung cells were undergoing apoptosis
after ozone exposure, the cells were assayed for apoptosis by Cell
Death ELISA. Figure 7 shows the results of
AL and BAL cells as quantified by Cell Death ELISA. Data are expressed
as the average of ozone-exposed values with the air-exposed value subtracted. Consistent with the morphological evidence, there was a
significant increase in cytosolic DNA fragments in AL cells after
ozone. Similar to the results from morphology, BAL cells exhibited only
a marginal but nonsignificant increase in DNA fragmentation.

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Fig. 7.
Cell Death enzyme-linked immunosorbent assay (ELISA) results for airway
lavage (AL) and BAL cells after 0.4 ppm ozone exposure. Both airway
lung cells and AMs were assayed for apoptosis 6 h after air and ozone
exposure by Cell Death ELISA as described in
METHODS. Data are expressed
[optical density (OD)] as means ± SE of 4 subjects'
ozone minus air values. * P < 0.05 compared with a hypothetical mean of 0.
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AM apoptosis after in vitro HNE exposure.
To test whether HNE could induce apoptosis, we exposed human AM to HNE
in vitro and measured apoptosis by several methods. Cell Death ELISA is
a very sensitive and early measure of apoptosis. Morphological changes
can also appear relatively early, and these changes are the hallmark of
apoptotic cells. DNA ladder formation is a late step in apoptosis and
is an excellent confirmatory method when enough cells are available.
Figure 8,
A and
B, is a photomicrograph of control and
HNE-treated AM incubated for 24 h, respectively. AM shown in Fig.
8A have normal cell and nuclear
appearance. HNE-exposed AM shown in Fig.
8B display nuclear condensation
characteristic of apoptosis. Figure 9 shows
the results from Cell Death ELISA from cells incubated for 6 h with
HNE. The results clearly indicate that apoptosis in human AM is HNE
dose dependent. Apoptosis was maximal at 50 µM HNE, whereas the
highest concentration of HNE (100 µM) produced less DNA
fragmentation. The results at 100 µM HNE suggested a necrotic process
rather than an apoptotic process due to excessive HNE toxicity. The
results shown in Fig. 10 further demonstrate that HNE induced apoptosis because characteristic DNA
ladder formation was evident after a 24-h culture with 50 µM HNE.

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Fig. 8.
Photomicrographs (×630) of AM cells after a 24-h control culture
(A) or 50 µM HNE culture
(B). Cells were stained in a similar
manner as in Fig. 6. Apoptotic cells are indicated by arrows. Results
are representative of 3 experiments.
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Fig. 9.
Dose response of HNE-induced apoptosis of human AMs. Cell Death ELISA
was used to quantitate apoptosis in a similar manner as in Fig. 7. AMs
were incubated with increasing concentrations of HNE for 6 h at
37°C. Results indicate a dose-dependent increase in cytosolic DNA
fragments in cultured AMs with increasing concentrations of HNE
(n = 1).
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Fig. 10.
Confirmation of HNE-induced apoptosis in human AMs. AMs were cultured
for 6 and 24 h at 37°C with 50 µM HNE. Genomic DNA was isolated
and analyzed as described in METHODS. Results are
representative of 2 experiments. MW, molecular weight; Ctr,
control.
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DISCUSSION |
This study examined two effects of acute ozone exposure (stress
response and apoptosis) on human AM and apoptosis on airway lung cells
to determine whether these effects could have been mediated by HNE. The
results demonstrated that the effects of ozone on lung cells are
consistent with the action of HNE. In addition, the results support the
hypothesis that the cell injury and inflammation that occur after ozone
exposure can be explained by initiation of lipid peroxidation and
formation of toxic aldehydes such as HNE. The source of the oxidants
that initiate HNE formation from polyunsaturated fatty acids such as
arachidonic and linolenic acids cannot be distinguished at this time.
It is possible that HNE formation may be secondary to the formation of
free radicals by ozone in the lung lining layer. It is also possible
that initiation of inflammation could trigger lipid peroxidation. We
suspect that the latter suggestion is less likely because HNE was
formed preceding recruitment of neutrophils in human exposures and in a
murine model (17). Nevertheless, it is evident that HNE is formed after ozone exposure and that HNE induces cellular effects very similar to
those caused by ozone.
The cytotoxic effects of HNE most likely occur because of the high
reactivity of HNE with certain amino acids (viz., Cys, Lys, and His),
resulting in the formation of Michael's adducts (8). When these
adducts occur in active sites or regulatory regions of proteins, then
the function of those proteins can be altered (4-6, 13). If this
occurs in one or more key cellular proteins, then alteration of cell
function, induction of stress proteins, and/or cell injury
could occur.
Evidence that HNE may be inhibiting specific and key proteins comes
from recent studies of enzyme inhibition by HNE (21). In addition, we
have recently demonstrated that HNE inhibits the cysteine protease
interleukin (IL)-converting enzyme in an in vitro model (4). HNE
inhibits the release of IL-1
from human monocytes (4) and
macrophages (10). This specific effect of HNE could explain the
inhibition of IL-1
release from AMs isolated from human subjects
exposed to ozone (10). Consequently, in vitro and in vivo studies
suggest that HNE is formed as a result of oxidant exposures
and that key proteins can be targets for HNE.
In this study, we observed the consistent increase of a 32-kDa HNE
protein adduct in AMs 6 h after ozone exposure. This finding extends
preliminary results obtained in another study (10). In addition, the
principal HNE-protein adduct detected after ozone exposure in a murine
model was also a 32-kDa protein (17). The half-life of HNE-protein
adducts can be estimated from the murine studies (17) to be on the
order of 4-12 h. Therefore, the adducts could have been formed
early during or after ozone exposure from secondary lipid peroxidation
and could still be present at the 6-h time point in this study. The
identity of this protein is unknown, and consequently it is not clear
whether it is associated with the effects of HNE on AMs. However, it
does appear to be a key target for HNE from in vivo ozone exposures and
has the potential to serve as a biomarker for ozone exposure. However, because the formation of HNE is not specific to ozone, it is not certain how specific this 32-kDa HNE-protein adduct would be to ozone.
A number of key enzymes have molecular masses of ~32 kDa, including
HO-1. Identification of this protein may help explain the mechanism of
oxidative stress on macrophages.
Oxidative stress can result in the induction of a number of proteins,
collectively referred to as a stress response (3, 15, 16, 32).
Induction of these proteins causes cells to become more resistant to
subsequent environmental stressors (1, 18, 27, 28). We had previously
reported that HSP72 was induced after ozone exposure (10). In this
study, those findings were extended to include induction of ferritin, a
protein known to be induced after oxidative stress (31). HO-1 and HSP65
were not consistently induced (Figs. 3 and 4). In
vitro, HNE induced all four of these stress proteins
(Fig. 5). There are a number of explanations for the difference between
the in vitro HNE and in vivo ozone results. HO-1 is induced at low
doses of HNE, whereas at higher doses of HNE, HO-1 is no longer induced
(19). It appears that at higher concentrations of HNE, in which
cytotoxic effects begin, HO-1 is no longer induced. Preliminary studies
in C57BL/6 mice exposed to ozone demonstrated that HO-1 was induced at
low ozone exposures (
0.4 ppm for 3 h), but no HO-1 induction was observed at higher ozone exposures (data not shown). Therefore, HO-1
induction may be very susceptible to excess oxidative stress, and the
dose of ozone in the present study may have been too high. Alternatively, other reactive aldehydes are also formed from lipid peroxidation and may have somewhat different effects on stress protein
expression or may have acted in an additive manner, blocking HO-1 and
HSP65 expression. Differences between the two models may also be due to
the time course of exposure to HNE. In the in vivo model, HNE was
probably formed over an extended time period (even after the cessation
of ozone exposure), whereas in the in vitro model, HNE was added as a
bolus. Consequently, the effects on specific stress protein expression
may be altered. However, it is clear that both ozone and HNE induce the
formation of multiple members of the stress protein family.
The implications of stress protein expression in human lung cells after
ozone exposure are that they are a clear and quantifiable indicator of
the presence of a significant environmental stress. In addition, the
stress response is a protective mechanism against subsequent
environmental stress. Although low levels of ozone exposure have
distinct effects within cells, they may also protect the cells against
subsequent ozone exposure. This protection may contribute to the
adaptation observed after multiple ozone exposures (2, 9, 22).
A significant new finding from these studies is that modest ozone
exposures can induce apoptosis in human lung cells. The apoptosis was
evident from morphological appearance and from Cell Death ELISA
results. Previous studies have demonstrated that the Cell Death ELISA
assay is a sensitive and reliable indicator of apoptosis (11, 14, 19).
Apoptosis was clearly evident in airway cells but not in peripheral
lung cells obtained by BAL. It should be noted that although the
increase in the Cell Death ELISA results was not significant in AMs,
there was an increase, suggesting that even at this ozone exposure,
minor cytotoxic events are beginning. In addition, in vitro studies
confirmed that HNE could induce apoptosis of AMs. HNE induced
morphological changes consistent with apoptosis, a dose-dependent
increase in Cell Death ELISA results, and a DNA ladder typical of cells
undergoing apoptosis. Therefore, both ozone in vivo and HNE in vitro
can induce apoptosis of human lung cells, demonstrating the adverse
health effects of ozone exposure.
The finding that only airway cells underwent significant apoptosis is
consistent with the prediction that upper airway cells would be exposed
to a higher concentration of ozone than the peripheral lung cells would
receive. Furthermore, these results along with our previous in vitro
and in vivo studies using human monocytes (4) and macrophages (10) can
provide some estimates on the levels of HNE that would have been
required to induce the biological effects of stress protein expression,
inhibition of IL-1
and induction of apoptosis. The fact that stress
proteins were induced and IL-1
release was inhibited in AMs but no
significant apoptosis was observed suggests that levels of HNE of ~5
µM could have been formed and could have generated the observed
effects. In contrast, ~10 times higher levels of HNE (50 µM) would
have to have been formed in the airways to induce apoptosis. These
upper values are based on the assumption that the primary active
component causing the biological effects was HNE. To the extent that
other agents formed as a result of ozone exposure contributed to the observed effects, then the amount of HNE formed and its relative overall contribution would correspondingly decrease.
In summary, we have established that acute ozone exposure in humans can
induce a stress response and apoptosis in lung cells. Airway cells
demonstrated evidence of undergoing apoptosis that may be due to the
higher concentrations of ozone in the airways versus lower
concentrations of ozone reaching the more distal alveolar spaces where
ozone produced a stress response. HNE-protein adducts were detected
after ozone exposure, primarily of a yet-to-be-identified 32-kDa
protein, confirming that HNE was formed as a result of ozone exposure.
Finally, all of the responses observed after ozone exposure were
mimicked by HNE in vitro, providing additional evidence for the
potential role reactive aldehydes may play in mediating the biological
effects of ozone exposure.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by a grant from the Methodist
Foundation and National Center for Research Resources Grant M01-RR-02558.
 |
FOOTNOTES |
Address for reprint requests: A. Holian, Dept. of Internal Medicine,
Univ. of Texas Medical School, 6431 Fannin, Rm. 1.276, Houston, TX
77030.
Received 8 October 1996; accepted in final form 12 September 1997.
 |
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