Cigarette Smoke Induces Apoptosis by Activation of Caspase-3 in Isolated Fetal Rat Lung Type II Alveolar Ep-ithelial Cells <i>in Vitro</i>

doi:10.4236/ojrd.2013.31002

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Open Journal of Respiratory Diseases, 2013, 3, 4-12

http://dx.doi.org/10.4236/ojrd.2013.31002 Published Online February 2013 (http://www.scirp.org/journal/ojrd)

Cigarette Smoke Induces Apoptosis by Activation of

Caspase-3 in Isolated Fetal Rat Lung Type II Alveolar

Epithelial Cells in Vitro

Asra Ahmed1, James A. Thliveris2, Anthony Shaw1,3, Michael Sowa1,3,

James Gilchrist1, J. Elliott Scott1,2,4*

1Departments of Oral Biology, Manitoba Institute for Child Health, University of Manitoba, Winnipeg, Canada

2Human Anatomy and Cell Science, Manitoba Institute for Child Health, University of Manitoba, Winnipeg, Canada

3Manitoba Institute for Child Health, The National Research Council Biodiagnostics Institute,

University of Manitoba, Winnipeg, Canada

4The Biology of Breathing Group, Manitoba Institute for Child Health, University of Manitoba, Winnipeg, Canada

Email: *mailto:[email protected]

Received December 6, 2012; revised January 8, 2013; accepted January 20, 2013

ABSTRACT

Smoking during pregnancy is a major source of fetal exposure to numerous harmful agents present in tobacco smoke.

Lung development involves complex biochemical processes resulting in dramatic changes which continue even after

birth. In addition to type I cells which form the blood-air barrier, type II alveolar epithelial (AE) cells have important

and diverse functions related to immunological protection and stabilization of the alveolus through synthesis and secre-

tion of the pulmonary surfactant. Apoptosis or programmed cells death is an important physiological process during

lung embryogenesis and for the proper maintenance of homeostasis. Caspases are proteases that play important roles in

regulating apoptosis. Caspase-3 is the key executioner caspase in the cascade of events leading to cell death by apop-

tosis. We explored the hypothesis that cigarette smoke extract (CSE) induces apoptosis in fetal rat lung type II AE cells

by activation of caspase-3. To analyze these factors, isolated fetal rat lung type II AE cells were used. The cells were

exposed to different concentrations of CSE (5%, 10% or 15%) (v/v) for 60 min. The results of the present study showed

that CSE induced apoptosis in fetal rat lung type II AE cells with a significant increase (p < 0.05) in caspase-3 activity

and decrease in cell proliferation at CSE concentrations of 10% and 15% (v/v). These observations indicate that ciga-

rette smoke extract induces apoptosis by activation of caspase-3 in fetal rat lung type II AE cells in a dose-dependent

manner and may potentially alter the regulated development of the lung and the appearance of the surfactant-producing

type II alveolar cells which are critical for the establishment of adequate gas exchange at birth.

Keywords: Cigarette Smoke Toxicity; Fetal Rat Lung Type II Alveolar Cells; Apoptosis; Protease; Caspase-3;

Lung Development; Developmental Toxicity; Maternal Smoking

1. Introduction

Cigarette smoke contains more than 4000 chemicals in-

cluding addictive and carcinogenic agents which signifi-

cantly contribute to the progression of pulmonary disease.

Cigarette smoking during pregnancy is a major source of

prenatal exposure to harmful agents in tobacco smoke to

the developing fetus. Despite the consequences, 30% -

40% of women smoke during pregnancy worldwide [1].

Exposure of tobacco smoke and second-hand smoke in

utero has been associated with neonatal mortality, growth

retardation [2], low birth weight, sudden infant death

syndrome, preterm delivery and higher incidence of still-

birth [3].

The respiratory system is one of the most structurally

complex and critical systems of the body and its deve-

lopment begins during the fifth week of gestation [4].

The adult lungs are comprised of forty different types of

cells. Among these the major cell types include fibro-

blasts, type I alveolar epithelial (AE) cells, type II alveo-

lar epithelial (AE) cells and macrophages. The alveolar

epithelium is formed of type I AE cells and type II AE

cells. The type II AE cells cover only ~5% of the total

alveolar epithelial surface comprising 16% of the total

lung cell population [5]. They are interspersed between

type I AE cells and are thought to be progenitor cells of

type I AE cells during lung development [6] and injury

[7]. Furthermore, these cells release growth factors which

help regulate cell growth following injury. Type II AE

*Corresponding author.

A. AHMED ET AL. 5

cells are characterized morphologically by the presence

of large intracellular concentric membrane bounded sto-

rage units of surfactant, known as lamellar bodies (LB).

The major functions of type II AE cells include synthesis,

secretion and regulation of surfactant which essential for

normal biophysical and immuno-modulatory functions of

the lung. It is the first site of defense against inhaled com-

ponents of air such as cigarette smoke (CS). There is pre-

liminary evidence that intrauterine fetal smoke exposure

alters lung development [3] and the infants born to mo-

thers who smoke during pregnancy are small for their

gestational age.

Typically, in normal lung development apoptosis or

programmed cell death plays an important role to main-

tain equilibrium between cell death and cell proliferation.

A defect in apoptotic processes during embryogenesis

may lead to developmental abnormalities [8]. Apoptosis

is an irreversible, timely regulated form of cell death es-

sential for proper maintenance of homeostasis, during em-

bryogenesis and for functional regulation of the immune

system [9]. Cells undergoing apoptosis show characteris-

tic well defined morphologic changes differentiating it

from necrosis. Apoptotic stimuli can be initiated extra-

cellularly or intracellularly. The process of apoptosis is not

associated with any inflammatory response as the cellular

contents of dying cells are not released in the surround-

ing interstitial tissues. Furthermore, apoptosis is a com-

plex, multi-step process which involves biochemical events

including activation of an intracellular proteolytic cas-

pase cascade, which is important in the regulation and

execution of apoptotic cell death. Knowledge of the im-

portance of caspases in apoptosis has been made possible

through studies on knockout animals deficient of parti-

cular caspases which confirmed profound defects in apo-

ptosis [10]. Moreover, studies involving use of inhibitors

of caspases which effectively inhibit apoptosis also help

understand the important role of caspase activation in

apoptosis [11].

Caspases, are proteases which belong to the family of

cysteine-aspartic acid endopeptidases. Caspases are pri-

marily localized in the cytoplasm and are synthesized as

inactive enzyme precursors or zymogens [12]. Recent stu-

dies report the existence of caspases in the mitochondrial

intra-membrane space (pro-caspase-2, -3, -8 and -9), en-

doplasmic reticulum (pro-caspase-12) nucleus and Golgi

apparatus (pro-caspase-2) [13].

Caspases are broadly classified into two groups; one

which is thought to play a central role in apoptosis (cas-

pases-2, -3, -6, -7, -8, -9, -10, and -12) and a second

group which is primarily involved in cytokine processing

during inflammation (caspases-1, -4, and -5) [14].

Of these enzymes caspase-3 also known as CPP32,

YAMA or apopain is considered as the major execution-

ers of apoptosis. It is the first of all the effector caspases

to be activated for amplifying downstream apoptotic

processes. Caspase-3 can be activated through caspase-8

and caspase-9 by extrinsic or intrinsic signaling, respec-

tively [15], suggesting, that the apoptotic signals from ei-

ther extrinsic or intrinsic pathways converge for active-

tion of caspase-3. Activated caspase-3 has been reported

to contribute mainly to the morphologic changes in apo-

ptotic cells including membrane blebbing, chromatin con-

densation and DNA fragmentation [10] and has been

reported in developing lung in the pseudoglandular bran-

ching morphogenesis phase [16].

The aim of the present study was to examine the effect

of cigarette smoke extract on fetal lung type II AE cells.

We hypothesized that cigarette smoke extract induces

apoptosis in type II AE cells through activation of cas-

pase-3.

2. Materials and Methods

2.1. Preparation of Cigarette Smoke Extract

Cigarette smoke extract (CSE) was prepared according to

method designed by Janoff and Carp [17]. Unfiltered

research cigarettes from University of Kentucky, each

containing 2.45 mg nicotine/cigarette [18] were used.

Cigarette smoke was drawn from each cigarette into a 50

ml syringe for two seconds maintaining a gap of 20 sec-

onds between each draw with the syringe and bubbled

through 50 ml of minimum essential medium (MEM) at

room temperature. This cycle was repeated until the end

of the cigarette. 50 ml of fresh MEM was used for the

next cigarette. The resulting smoke extracted MEM was

considered to be 100% CSE. It was filtered using 0.22

µm pore filters (Millipore) to sterile and stored at −80˚C.

Further dilutions (5%, 10% and 15%) were made in se-

rum-free media containing antibiotics and fungizone.

Before treating cells with conditioned media, the pH was

adjusted to 7.2.

2.2. Isolation and Culture of Type II AE Cells

Use of animals was approved according to the Canadian

Council on Animal Care guidelines. Timed pregnant

Sprague-Dawley rats purchased from Central Animal

Services, University of Manitoba were used to isolate fe-

tal lung type II alveolar cells. Rats were euthanized with

an intra-peritoneal injection of 1 ml Euthanyl (240 mg/ml

sodium pentobarbital) on gestational day 21 (day 22.5 is

term gestation). Fetuses were removed by hysterotomy,

decapitated and placed in cold, sterile Hanks Balanced

Salt Solution (HBSS, Gibco, ON Canada). Lungs were

dissected from fetuses by making an incision in the mid-

sternal region. Lungs were minced using a Sorval tissue

chopper (Sorval Instruments, Newton, CT) in a laminar

flow hood. The minced lung tissue was dissociated by

incubating with trypsin-EDTA (0.05%/0.02%) in HBSS

at 37˚C for 45 minutes in a water-jacketed trypsinization

flask which was placed on a magnetic stirrer. Minimal

A. AHMED ET AL.

essential medium (MEM) (Gibco, ON Canada) contain-

ing 10% of newborn calf serum (NCS), antibiotics/anti-

mycotic (1%) and fungizone (1%) (Gibco, ON Canada)

was added to stop further enzymatic disaggregating. The

dissociated cells were filtered through three layers of 150

μm Nitex gauze to remove tissue fragments and centri-

fuged for 10 min at 1000 rpm at 4˚C. The cell pellet was

re-suspended in 10 ml of MEM/NCS and cells were pla-

ted in five 75 cm2 tissue culture flasks in a humidified in-

cubator (95% air/5% CO2 and 37˚C) and allowed to ad-

here for one hour. Fibroblasts have the ability to attach

faster when compared to type II cells [19]. Fetal type II

AE cells were separated by differential adherence [20].

After one hour of incubation and adherence of fibroblasts,

the media with unattached cells was collected. Cells were

counted and re-plated at a density of 1.5 × 105 cells/flask.

Type II cells were cultured in media supplemented with

10% carbon stripped serum (sNCS) in MEM and 1% an-

tibiotics, antimycotic, fungizone and cultured in a humi-

dified incubator (95% air/5% CO2 and 37˚C). Medium

was changed after 24 hours and 48 hours thereafter.

2.3. Detection of Caspase-3 Activity in Adherent

Cells Exposed to CSE

Once the cultures reached 70% - 80% confluence, they

were washed twice with HBSS and treated for 60 min-

utes with different concentrations of CSE diluted in se-

rum-free media. Cells were washed three times with

HBSS to ensure complete removal of traces of CSE. Ca-

spase-3 activation in cells was determined using the cas-

pase-3 fluorometric assay kit, purchased from BioVision

(MountainView, CA). The cells were lysed using the ly-

sis buffer provided (50 ul per well), incubated on ice for

ten minutes, and incubated with 5 ul of fluorogenic sub-

strate 1mM DEVD-AFC (caspase-3 cleaves this substrate)

in a reaction buffer (containing 10 mM DTT) in the in-

cubator at 37˚C for two hours. The enzymatic activity

was measured using a fluorescence microplate reader

with 400 nm excitation and 505 nm emission filters. Cas-

pase-3 cleaves the AFC substrate (Figure 1) and releases

a fluorogenic signal; this signal is directly proportional to

the level of enzymatic activity of caspase-3 in cells. Ca-

spase-3 activity was calculated in samples of cells ex-

posed to CSE and compared to untreated controls.

2.4. Effect of N-benzyloxycarbonyl-Val-Ala-Asp-

fluoromethylketone (Z-VAD-fmk)

Z-VAD-fmk, is a broad spectrum caspase inhibitor which

was used in the present study to examine the involvement

of caspases in cell death due to CSE. The cells were in-

cubated with 80 mM Z-VAD-fmk in serum-free medium

at the time of exposure of cells to CSE for 60 min. After

which the cells were washed and the caspase-3 activity

was measured as described above.

(a) Caspase-3 enzyme (b) DEVD-AFC substrate

(d) Cleavage of substrate

(DEVD-AFC), Free AFC

emits yellow-green

fluorescence after cleavage

Figure 1. Schematic illustration of DEVD dependent detec-

tion of caspase-3 activity. Activation of caspase-3 is a key

event of apoptosis. Caspase-3 fluorometric assay kit (BioVi-

sion, Mountain View) is based on detection of cleavage of

substrate DEVD-AFC by caspase-3. DEVD-AFC substrate

emits blue light (400 nm). After cleavage of the substrate by

caspase-3, free AFC emits yellow-green fluorescence (505

nm). The amount of fluorescence can be quantified using a

fluorometric plate reader.

2.5. Determination of Cellular Viability

The cells were treated with different concentrations of

CSE as described above. After incubation with different

concentrations of CSE the cells were washed with HBSS

three times to ensure complete removal of CSE and fur-

ther incubated with MTT solution for three hours. The

MTT-based cell proliferation assay (Sigma Aldrich, St.

Louis, MO, USA), is a calorimetric assay used to meas-

ure the ability of mitochondrial dehydrogenase of viable

cells to reduce the key component, MTT or 3-[4,5-di-

methylthiazol-2-yl]-2,5d iphenyl tetrazolium bromide, a

yellow tetrazole to insoluble purple formazan crystals.

Viable cells cleave the tetrazolium ring of MTT and the

yellow water-soluble dye is converted to insoluble purple

crystals of formazan. After three hours of incubation with

MTT solution the crystals were dissolved in MTT sol-

vent. The plates were read spectrophotometrically at an

absorbance of 570 nm. The intensity of purple color in

the solution is indicative of the number of living cells.

2.6. Western Blot Analysis

At the end of treatment with CSE, cells were washed

three times with HBSS to ensure complete removal of

any remnants of CSE. The cells were lysed by adding

one ml of 2XRIPA buffer with protease inhibitor tablet

[20 mM Tris-HCL pH 7.6, 316 mM NaCl, 2 mM EDTA,

2% triton ×100, 0.2% SDS, 2% sodium deoxycholate, 1

mM PMSF, 1 mM Na3VO4, 1 protease inhibitor tablet]

and stored at −80 until processed. Protein samples were

quantified using Bradford protein determination method.

Equal amounts of protein extracts were subjected to elec-

trophoresis on 12% sodium dodecyl sulfate-polyacryla-

mide pre-cast gels (BIO-RAD, Mississauga, ON), elec-

trophoresed at 180 V and later transferred to nitrocellu-

lose membranes. The blots were probed with primary

antibody (rabbit polyclonal) purchased from Santa Cruz

Biotechnology (Santa Cruz, CA, USA) and diluted (1:500)

A. AHMED ET AL. 7

in blocking buffer overnight at 4˚C. This antibody recog-

nizes the p17 fragment of an activated form of caspase-3

(Santa Cruz, CA, USA). After three washes with TBS-T

blots were incubated with goat anti rabbit IgG-HRP se-

condary antibody (Santa Cruz, CA, USA) at a dilution of

1:1000 in blocking buffer for two hours at room tem-

perature and detected using ECL-plus (GE Healthcare,

NJ, USA) and exposed on Kodak films. The densities of

cleaved caspase-3 bands were quantified using Quantis-

can.

2.7. Subcellular Localization of Caspase-3 Using

Immunofluorescence

Subcellular localization of caspase-3 in cells exposed to

CSE was observed using immunofluorescence microcopy.

Cells were plated in four well glass chamber slides and

left overnight in incubator at 37˚C for attachment on

glass slides. The cells were exposed to 10% or 15% CSE

in serum-free medium and left in the incubator for 60

min at 37˚C. After which the cells were washed with

PBS three times and fixed with cold methanol (−10˚C)

for 5 min followed by three washes with PBS, suction

was used between each wash to completely remove the

reagents. The cells were blocked in 2% BSA/1×PBS for

one hour in a humidified chamber. The primary antibody

rabbit-polyclonal IgG, which recognizes active caspase-3

was diluted (1:800 dilution) in blocking solution was

incubated overnight at 4˚C with the cells in a humidified

chamber. After four washes with PBS cells were incu-

bated with secondary anti body, FITC conjugated donkey

anti-rabbit IgG which recognizes rabbit IgG (Santa Cruz).

The secondary antibody was diluted 1:80 in 2%BSA/1x

PBS and cells were incubated in a humidified chamber

for one hour in the dark. All steps after this were done in

the dark. After four washes with PBS the cells were

stained with Hoescht 33,342 (1:1000) for 15 seconds.

Following washing the slides were air-dried and mounted

with coverslips using 40 μl Prolong Anti-fade Gold. The

slides were observed under an inverted fluorescence mi-

croscope (BX61 Olympus microscope) using Image Pro.

Software.

2.8. Statistical Analysis

Statistical differences between group means were carri-

ed out using post ho c Duncan’s Multiple Range Test [21].

A value of p < 0.05 was considered for statistically sig-

nificant differences between the treated and untreated

groups.

3. Results

3.1. Detection of Caspase-3 Activity in Adherent

Cells Exposed to CSE

Exposure to CSE at concentrations of 10% or 15% (v/v)

produced significantly elevated activity (p < 0.05) of

caspase-3 compared to the non-exposed cells (Figure 2).

No significant differences were observed in the caspase-3

activity in cells exposed to 5% CSE compared to the non-

exposed cells.

3.2. Effect of N-benzyloxycarbonyl-Val-Ala-Asp-

fluoromethylketone (Z-VAD-fmk)

For further confirmation, apoptotic cell death was evalu-

ated after treatment with Z-VAD-fmk (Figure 3), a broad

spectrum caspase inhibitor. Isolated fetal rat lung type II

AE cells were exposed to different concentrations of

CSE (5%, 10% or 15%) and Z-VAD-fmk (80 uM con-

centration) was added for 60 minutes. Caspase-3 activity

in was measured using the fluorometric assay kit at 400

nm excitation and 505 nm emission. Z-VAD-fmk inhi-

bited caspase-3 activity in all samples exposed to CSE

when compared to the caspase-3 activity in cells without

Z-VAD-fmk (p < 0.05).

3.3. Determination of Cell Viability

MTT formazan assay (F igure 4) was used to measure cel-

lular mitochondrial dehydrogenase activity within a cell

and is based on the conversion of mitochondrial-depen-

dent MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-

tetrazolium bromide) to purple formazan crystals. There

was a significant decrease (p < 0.05) in the mitochondrial

activity of cells exposed to 10% or 15% CSE compared

to the cells not exposed to CSE.

Figure 2. Effect of CSE on caspase-3 activity in isolated fe-

tal rat lung type II alveolar epithelial cells. Fluorometric

assay to assess the activity of caspase-3 in fetal rat lung type

II AE cells exposed to different concentrations of CSE (5%,

10% or 15%) (v/v) for 60 minutes in 37˚C incubator. Cells

not exposed to CSE were considered as controls. Each bar

represents the mean ±SEM of three experiments of 16 sam-

ples in each. *(p < 0.05) significantly different from the cor-

responding controls.

A. AHMED ET AL.

Figure 3. Effect of Z-VAD-fmk and caspase-3 activity in iso-

lated fetal rat lung type II AE cells exposed to CSE. Cas-

pase-3 activity and effect of Z-VAD-fmk was measured us-

ing caspase-3 fluorometric assay in fetal rat lung type II

cells exposed to 5%, 10% or 15% (v/v) CSE for 60 minutes.

Each bar represents the mean of ±SEM of three experi-

ments of 16 samples each. *significantly (p < 0.05) different

from the corresponding controls.

Figure 4. Effect of CSE on cell viability in isolated fetal rat

lung type II AE cells. MTT activity was measured in fetal

rat lung type II AE cells expose d to different concentrations

of CSE (5%, 10% or 15%) (v/v) for 60 minutes. Cells not

exposed to CSE were considered as controls. Level of ab-

sorbance was measured at 540 nm. Each bar represents the

mean ±SEM of three experiments of 16 samples in each. *(p

< 0.05) significantly different from the corresponding con-

trols.

3.4. Western Blot Analysis

The expression of caspase-3 in isolated fetal rat type II

AE cells was analyzed by SDS-PAGE (Figure 5). Lys-

ates of cells not exposed to CSE were considered as con-

trols. The results of fetal rat lung type II AE cells shows

that an antibody specific for detecting active form of ca-

spase-3 bond to the protein band with relative molecular

mass of 17 kDa, which is the accepted molecular mass of

active caspase-3 [22]. The densitometric analysis of cas-

pase-3 expression in these cells was significantly in-

Controls 5% CSE 10% CSE 15% CSE

Figure 5. Effect of CSE on caspase-3 expression in fetal ra

reased (p < 0.05) in the samples exposed to 10% or 15%

3.5. Sub-Cellular Localization of Caspase-3

The sub-3 expressed in

we demonstrated that CSE induces

lung type II AE cells. (A) Representative Western bolt

showing caspase-3 expression in fetal rat lung type II AE

cells treated with 5%, 10% or 15% (v/v) CSE for 60 min-

utes. Cells not exposed to CSE were considered as controls.

(B) Densitometric analysis of band intensity shows each bar

representing the mean ±SEM of three experiments. *(p <

0.05) significantly different from the corresponding con-

trols.

CSE.

Using Immunofluorescence

-cellular localization of caspase

isolated fetal rat lung type II AE cells after exposure to

CSE was determined by immunofluorescence using fluo-

rescence microscopy (Figure 6). Controls consisted of

samples in which the primary antibody against caspase -3

was omitted. No green fluorescence was observed in

these samples. Caspase-3 activity was localized largely

within the cytoplasm of the isolated type II cells. A gra-

dient of expression appeared to be present in that rela-

tively little fluorescence was present at the periphery of

the cell immediately adjacent to the cell membrane but

fluorescence increased towards the nucleus. Caspase-3

activity appeared to be the most intense near the nuclear

membrane.

4. Discussion

In the present study

apoptosis through activation of caspase-3 in isolated fetal

rat lung type II AE cells in vitro. As previously reported

type II AE cells are regarded as the “defenders” of the

alveolus due to their diverse functions [22]. Any damage

A. AHMED ET AL.

(a)

(c)

Figure 6. Immunofluorype II AE cells. Iso-

type II AE cells may result in 1) alveolar collapse due phase contains less stable free radicals, which are more

escence staining for detection of caspase-3 expression in isolated fetal rat lung t

lated fetal rat lung type II AE cells were exposed to CSE (15% v/v). Immunofluorescence was performed using caspase-3

rabbit polyclonal IGg antibody. Caspase-3 was visualized using donkey anti-rabbit IGg-FITC (green fluorescence) and

counter stained with Hoescht 33,342 (nuclear staining—blue). Image (a) shows controls (without primary anti-body); Image

(b) shows expression of caspase-3 (green) primarily localized in the cytoplasm; (c) Shows an enlarged image of a positive

stained cell. FITC—Fluorescein isothiocyanate; DAPI—4,6-diamidino-2-phenylindole; Merge—merged image of FITC and

DAPI.

to decreased surfactant production; 2) defects in remod-

eling of alveolar structure due to decrease in cytokines

and growth factors; 3) increase in epithelial permeability

[23]. It has been reported that exposure of type II AE

cells to CS leads to oxidative damage of the respiratory

epithelium and induces DNA damage [24], which is an

important feature of cellular apoptotic death. Cigarette

smoke can be divided into two phases, the tar phase/par-

ticulate phase and gas phase, both of which are rich

sources of free radicals [25]. The tar phase contains free

radicals which are stable and are retained on the filter

when a cigarette is smoked. In contrast, the gas smoke

reactive and can penetrate through the filter [26]. The gas

phase can be further divided into mainstream smoke and

side stream smoke. The mainstream smoke is directly

inhaled through the cigarette and contains 8% tar and

92% gaseous components [26]. Side stream smoke is the

smoke emitted from the burning end of the cigarette.

Environmental smoke is a combination of side stream

and exhaled mainstream smoke. In the present study CSE

was prepared using unfiltered research cigarettes, which

included both phases of smoke components. Water solu-

ble components remain in the media during exposure to

cells. The solution which is considered as 100% CSE

A. AHMED ET AL.

was further diluted for all assays. Previous studies in our

laboratory have found these high levels of CSE to be

cytotoxic. Fetal growth and development is influenced by

intrauterine environment and governed by physical, en-

vironmental and hormonal factors [27]. This environment

is carefully regulated and consists of various hormones

including vascular endothelial growth factor (VEGF) and

fibroblast growth factor (FGF) [27]. Both of these hor-

mones contribute to type II alveolar cell differentiation as

well as production and secretion of surfactant phospho-

lipids and proteins [27]. Moreover, alterations in the in-

tra-uterine environment due to exposure to cigarette smoke

may affect lung function [28]. It may result in decreased

alveolarization [1] and altered formation of pulmonary

surfactant, which is the first line of defense against pol-

lutants and critical for proper lung expansion at birth [29].

Furthermore, intra-uterine smoke exposure affects lung

development adversely with a significant reduction in

lung growth [30], with an increased risk of asthma in

childhood [31]. The mechanisms underlying the pulmo-

nary effects due to CSE exposure are still not clearly elu-

cidated. It is widely accepted that apoptosis or program-

med cell death plays an important role during all stages

of lung development (pre and post-natal) and repair of

lungs following injury [16,32]. The deregulation of apop-

tosis may lead to development of lung disease. Hypero-

xia which inhibits distal airway branching within fetal

mouse pulmonary mesenchyme and is associated with

bronchopulmonary dysplagia activates caspase-3 [33]. In

addition acute respiratory syndrome induced by corona-

virus results in apoptotic activation through a caspase-

dependent mechanism [34]. Interestingly differential apo-

ptosis in type II alveolar cells and interstitial fibroblasts

may be involved with pulmonary fibrosis and post injury

inadequate re-epithelialization [35].

Caspases, which are responsible for initiation and exe-

cution of apoptosis, upon receiving apoptotic stimu

proliferation

y the Natural Science and

il of Canada (NSERC) and

[1] J. Stocks and C of Parental Smok-

ing on Lung Fnt during Infancy,”

li be-

me activated and the process of cell death is carried

out through proteolytic cleavage. Caspase-3 is the central

caspase in the caspase cascade that mediates the execu-

tion of the apoptotic process of cell death [36]; however,

little is known about the ability of CSE to activate cas-

pase-3 in fetal lung cells. Both the intrinsic and extrinsic

pathways of apoptosis converge to the activation of cas-

pase-3 [37]. Once activated caspase-3 cleaves nuclear

protein substrates leading to DNA fragmentation [38]

eventually leading to cell death.

In conclusion, our study showed that CSE induced an

increase in apoptosis and a reduction of cell

fetal rat lung type II AE cells following exposure to

CSE. As both these processes are critical for maturation

and acquisition of the adequate pulmonary surface of the

lung during development, observations that CSE disrupts

this program can account for the onset of respiratory in-

adequacies when the fetus is exposed in utero to chemi-

cals found in cigarette smoke. Since type II AE cells se-

crete surfactant which is the first line of defense against

pathogens and toxic substances, any damage to these

cells could mean an overall deterioration of lung function.

To our knowledge this is the first study which demon-

strates the increase in apoptosis through activation of ca-

spase-3 and decrease in cell growth in fetal lung type II

AE cells exposed to CS in vitro. However, the underlying

cellular mechanisms and signaling pathways that explain

the theory behind these changes remain to be resolved.

Further studies are required to investigate the specific fac-

tors inducing apoptosis in tobacco exposed fetal lungs.

5. Acknowledgements

This work was supported b

Engineering Research Counc

the Manitoba Institute for Child Health.

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