1 Center for Craniofacial
Molecular Biology, Although the effects of maternal smoking on
fetal growth and viability are overwhelmingly negative, there is a
paradoxical enhancement of lung maturation as evidenced, in part, by a
lower incidence of respiratory distress syndrome in infants of smoking mothers. Other epidemiologic and experimental evidence further support
the view that a tobacco smoke constituent, possibly nicotine, affects
the development of the lung in utero. We are studying the direct
effects of nicotine on murine lung development using a serumless organ
culture system. We have found that embryonic lungs explanted at 11 days
gestation showed a 32% increase in branching after 4 days in culture
in the presence of 1 µM nicotine and 7- to 15-fold increases in mRNAs
encoding surfactant proteins A and C after 11 days. The effect of
nicotine exposure on surfactant gene expression is apparently mediated
by nicotinic acetylcholine receptors because it was blocked by
D-tubocurarine. The
nicotine-induced stimulation of surfactant gene expression could, in
part, account for the effect of maternal smoking on the incidence of
respiratory distress syndrome.
surfactant protein A; surfactant protein C; messenger ribonucleic
acid; respiratory distress syndrome; nicotinic acetylcholine receptors; branching morphogenesis; lung development
THE NEGATIVE EFFECTS of maternal smoking on pregnancy
outcome are well known. Maternal smoking during pregnancy is associated with a higher incidence of spontaneous abortion, low birth weight, and
neonatal mortality (for reviews, see Refs. 1, 26). Immediate and
long-term deleterious effects on postnatal lung development have also
been associated with prenatal exposure to maternal smoking (8, 12).
This latter observation indicates that maternal smoking affects the
developmental program of the lung in utero. Nicotine is a possible
causative agent for these effects because it is a major pharmacological
constituent of tobacco smoke that crosses the placenta and is
concentrated in the fetal compartment (14). Animal studies show that
both nicotine and tobacco smoke have similar adverse effects on fetal
lung growth (7, 16). In addition, nicotine exposure in rabbits leads to
hyperplasia of pulmonary neuroendocrine cells (4), and a similar result has been observed in the lungs of human fetuses exposed to maternal smoking during pregnancy (5).
Although the overall effects of tobacco smoke exposure on fetal growth
and viability are negative, evidence suggests that prenatal exposure to
maternal smoking paradoxically enhances lung maturation. It is well
known that infants born to smoking mothers have a lower incidence of
respiratory distress syndrome (RDS) (9, 28), a condition resulting from
lung immaturity primarily due to inadequate production of pulmonary
surfactant (23). In addition, smoking-exposed fetuses have been found
to have saturated phosphatidylcholine levels and
lecithin-to-sphingomyelin ratios consistent with an increase in
functional lung maturity of ~1.5 wk relative to unexposed fetuses
(13).
We are examining the hypothesis that nicotine from maternal smoking
might perturb the developmental program of the lung, initially accelerating growth but eventually causing premature differentiational changes that could result in impaired growth and function after birth.
In these studies, the use of embryonic mouse lung buds grown in
serumless culture allows us to isolate direct, local effects of
nicotine from effects due to systemic mechanisms. Here we report that
nicotine exposure stimulates lung branching morphogenesis and increases
expression of mRNAs encoding the surfactant-associated protein (SP) A
and SP-C.
Organ culture. The serumless organ
culture system has been previously described (22). Briefly, timed
pregnant Swiss-Webster mice were obtained from Simonsen (Gilroy, CA).
Embryos were dissected on embryonic day
11 or 12. Lung buds
were removed from the embryos in sterile Hanks' balanced salt
solution, placed on Millipore filters resting on stainless steel wire
mesh, and cultured at the air-medium interface in chemically defined
medium (Fitton-Jackson modified BGJb, GIBCO, Grand Island, NY)
containing 0.2 mg/ml of ascorbic acid. The cultures were incubated
under 5% CO2 at 37°C for up
to 11 days, with the medium changed every second or third day. Drugs
[( Quantification of branching
morphogenesis. Quantification was done on lungs by
counting the number of terminal branches present in each explant after
4 days in culture. The terminal branches were visualized in whole
mounts, and the number of terminal branches for all the lobes of each
lung pair was counted with transillumination on a dissecting
microscope. Three to five lungs were counted for each data point. The
means ± SD were calculated, and the significance of differences
between means was evaluated by t-test
(criterion for significance, P < 0.05). Changes in the complexity of branching in response to nicotine
are expressed as a percent difference in the number of terminal
branches in treated versus control lungs.
Competitive reverse transcription-polymerase chain
reaction. Total RNA was extracted from cultured lungs
in guanidinium thiocyanate with the total RNA isolation kit from 5 Prime-3 Prime (West Chester, PA) and was reverse transcribed with
Moloney leukemia virus reverse transcriptase (RT) and oligo
deoxythymidine primers. Primers for SP-A, SP-C, Clara cell 10-kDa
protein (CC10), and Effects of nicotine on branching
morphogenesis. We initially observed that lung buds
from 11-day embryos cultured for 4 days in the presence of 1 µM
nicotine appeared to be more highly branched than lung buds grown in
control medium (Fig. 1,
A and
B). At the time of explant, lung
buds were at an early stage of branching, exhibiting primary bronchi
and, in some cases, the beginnings of the next generation of branches.
Lower concentrations of nicotine yielded inconsistent results (100 nM
nicotine: stimulation in one experiment, no change in two experiments)
or had no effect on branching (10 nM nicotine in three experiments).
Nicotine concentrations > 1 µM were not tested out of concern that
this would clearly exceed the physiologically relevant level of fetal
exposure from maternal smoking, although direct comparison of nicotine
exposure in vivo with exposure in the culture system is not possible.
Luck et al. (14) measured levels of nicotine up to 23 ng/ml in amniotic fluid at 12-16 wk gestation and up to 25 ng/ml in umbilical vein serum at delivery (slightly in excess of 0.1 µM). The level of exposure of a fetus in vivo would be expected to vary depending on how
recently the mother had smoked because the rate of transfer to the
fetal compartment is rapid relative to the half-life of nicotine in the
body (14). The concentration of nicotine in our cultures also probably
varied over time but in a different way because nicotine was introduced
only at 2-day intervals when the medium was changed and was presumably
lost at an unknown rate during the intervening periods due to such
processes as chemical breakdown, volatilization, and metabolism in the
lung tissue. Future studies will be needed to address the stability of
nicotine in the culture system.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
)-nicotine di-tartrate and/or
D-tubocurarine chloride (D-TC); Sigma Chemical, St.
Louis, MO] at the indicated concentrations were added to the
medium at the time the cultures were established and at each subsequent
medium change.
-actin were synthesized at the University of
Southern California Health Science Campus Microchemical Core Facility
on the basis of published mouse sequences (GenBank accession nos. are
S48768, M38314, S56696, and X03672, respectively). Amplification was
done in a Robocycler (Stratagene, La Jolla, CA) for 35 cycles at
appropriate denaturation, annealing, and extension temperatures.
Reaction products were analyzed by electrophoresis on 3% agarose gels,
stained with ethidium bromide, and photographed. Polymerase chain
reactions (PCRs) with RNA in place of the RT product were run to
control for DNA contamination. Validity of the PCR products was checked
by sequence determination. Quantitation by competitive RT-PCR was
performed as previously described (30). Briefly, the method involves
comparison of the intensity of the band amplified from the target cDNA
with the intensity of a band amplified from a known mass of competitor template DNA added to the same tube and amplified by the same primers.
A competitor template with appropriate specific primer sequences
flanking a stretch of heterologous DNA sequence was constructed for
each target mRNA. The competitor template was designed in each case to
be slightly longer than the intended target sequence to allow
resolution by gel electrophoresis of the PCR products generated from
the competitor template and from the target sequence. Yields of RNA
obtained from each lung were determined by ultraviolet
spectrophotometry. Equal masses of RNA were reverse transcribed for
each sample to be compared, and equal volumes of the RT reaction
product were loaded into each PCR tube together with a known amount of
competitor template DNA. After PCR amplification and gel
electrophoresis, quantification was accomplished by densitometric
scanning of the bands on photographs of the electrophoretic gels. A
standard curve was generated for each sequence under study by PCR
amplification of serial dilutions of a known concentration of the
specific cDNA sequence together with a constant amount of the
appropriate competitor template DNA. A straight-line standard curve for
each sequence was generated by plotting log (densitometric value of the
target band divided by the densitometric value of competitor band)
versus the amount of specific cDNA for each tube. From the log of the
ratio of the two bands for each sample lane, the amount of target cDNA
in each sample was obtained by using the computer-generated linear
regression equation. This approach controls for differences in priming
efficiency in the PCRs because the amount of target PCR product is
always determined relative to the amount of product generated from a known amount of a competitor template in the same tube with the same
pair of primers. To control for possible inaccuracies in spectrophotometric RNA determinations as well as for differences in the
efficiencies of the RT reactions, values for each sample studied were
normalized to values for
-actin obtained from matched samples by the
same competitive PCR technique. Graphed results are presented as means ± SD of values from three replicate lungs for each condition
graphed as a percentage of the control value. Significance of
differences between means was evaluated by
t-test, with the criterion for
significance being P < 0.05.
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
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Fig. 1.
Effect of nicotine exposure on branching morphogenesis. Lung buds were
grown for 4 days in presence or absence of 1 µM nicotine.
A: representative control lung.
B: nicotine-treated lung. Bars, 250 µm. C: quantitation of terminal
branches. Values are means ± SD; n = 4 lungs. Nicotine exposure significantly stimulates branching,
P < 0.05.
The stimulation of branching by 1 µM nicotine was quantified by counting terminal branches and found to be significant in three separate experiments. The mean increase in the number of terminal branches ranged from 20 to 53% and averaged 32% over all three experiments. Results from one experiment are shown in Fig. 1C.
Effects of nicotine on specific gene expression. To assess the effect of nicotine exposure on differentiation of the lung epithelium, we decided to examine the expression of genes, the products of which are characteristic of specific differentiated epithelial cell types. To date, we have looked at CC10, a specific marker for Clara cells of the bronchiolar epithelium (21), and at two of the four known surfactant-associated proteins, SP-A and SP-C. Increasing expression of the SP genes is known to correlate with increasing functional maturity of the fetal lung (for reviews, see Refs. 3, 20, 27). SP-C was chosen because it is the only SP that is a specific marker for type II pneumocytes, the surfactant-secreting cells of the alveolar epithelium. SP-A is expressed by both Clara and type II cells (see references in Ref. 27) and was chosen because in humans it increases later in gestation than SP-B or SP-C and might yield more information about changes in the timing of differentiation.
We have used a sensitive competitive RT-PCR assay to measure the relative levels of SP-A, SP-C, and CC10 mRNAs in control and nicotine-treated lung buds dissected from day 11-12 mouse embryos and maintained for 11 days in culture. Previously, Wuenschell et al. (29) and Slavkin et al. (22) showed that differentiation of various epithelial cell types begins within this culture period. We found that exposure to 1 µM nicotine significantly increased the levels of mRNA encoding the two SPs but had no effect on the level of CC10 mRNA (Fig. 2). A preliminary quantification of SP-B mRNA using recently designed primers and competitor template showed no change in the level of this mRNA in nicotine-treated versus control lungs in one experiment (data not shown). More work will be required to confirm this result. Interestingly, we noted that 10-fold less cDNA was required for optimal quantitation of SP-B mRNA than for SP-C mRNA and 25-fold less than for SP-A mRNA. Although some of this difference could be due to differences in priming efficiency of the different primer pairs, this observation strongly suggests that the control lungs contain substantially more SP-B than SP-C or SP-A mRNA.
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Because an increase in the levels of SP-A and SP-C mRNAs could reflect either an increase in the amount of message per cell or an increase in the fraction of cells expressing these genes, we used immunocytochemical staining with a polyclonal antibody directed against SP-C precursor protein (25) to obtain a preliminary assessment of the relative numbers of SP-C-positive cells. We did not observe any apparent difference in the number of stained cells in control versus nicotine-treated lungs in a small number of lungs taken from two separate experiments (data not shown). Although more extensive morphometric analysis will be required to determine whether there might be a small change in the number of type II cell precursors, we conclude that the majority of the effect of nicotine on SP-A and SP-C mRNA levels probably represents a change in gene expression (via either transcription or message stability) rather than an effect on type II cell differentiation.
To determine whether the effect of nicotine on expression of SP-A and SP-C mRNA was mediated by a member of the family of nicotinic acetylcholine receptors (nAChRs), we attempted to block the effect by coadministration of an equimolar concentration of the nAChR antagonist D-TC. As shown in Fig. 3, D-TC significantly blocked the nicotine-induced increase in SP-A and SP-C mRNA levels, demonstrating the involvement of nAChRs.
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DISCUSSION |
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The finding that nicotine exposure stimulates branching morphogenesis is consistent with the idea that nicotine may accelerate the developmental program of the lung. In contrast, the fact that expression of SP-A and SP-C mRNAs is stimulated by nicotine, whereas the expression of CC10 mRNA is unaffected, indicates that the effect on the SP-A and SP-C genes is not the result of a global stimulation of differentiation-associated genes or of differentiation in general. Thus, although we have demonstrated a direct stimulatory effect of nicotine on some indexes of lung development, it appears that the overall effect of nicotine exposure is more complicated than we had originally hypothesized.
We would like to know the identity of the cells bearing the nAChRs that we have detected. We do not know whether the precursors of parasympathetic ganglionic neurons are present in the embryonic lungs at the time of explant. Abundant evidence suggests that mature pulmonary neuroendocrine cells possess nAChRs (2, 18, 19). However, an earlier study by Wuenschell et al. (29) showed that these cells do not become overtly differentiated until 11 days in culture, whereas we saw an effect of nicotine on branching after just 4 days. The possibility that there could be nAChRs on undifferentiated epithelial cells is of great interest because a number of lung epithelial tumor lines have been shown to express nAChR subunits (6, 24) and because nicotine is known to be mitogenic for some of these cell lines (15).
The negative effects of smoking during pregnancy on fetal growth and viability are thought to be primarily due to fetal hypoxia (1). The mechanisms underlying the positive effect of maternal smoking on fetal lung maturation are less well understood. It has been proposed that chronic fetal stress, from a variety of causes, can lead to accelerated maturation of the lung and other organ systems as some sort of compensatory response (11). This has been the prevailing hypothesis to explain enhanced lung maturation in the smoking-exposed fetus (9, 28), although the exact mechanism is not clear (13). It is possible that the effect is mediated, at least in part, by glucocorticoids because these hormones are a major factor in normal fetal lung maturation (reviewed in Ref. 3) and because cortisol levels are elevated in the amniotic fluid of smokers (10). Increased glucocorticoid secretion due to stress-related release of adrenocorticotropic hormone could be a general mechanism for the stimulation of lung maturation in chronically stressed fetuses, and nicotine could play a direct role in the specific case of tobacco smoke exposure because it has been shown that nicotine acts centrally to increase secretion of adrenocorticotropic hormone (17).
Our new findings suggest that, in addition to the probable hormonal mechanisms acting to enhance lung maturation, there may be a direct, local effect of nicotine on the developing lungs of tobacco smoke-exposed fetuses. We hypothesize that the effect of nicotine on SP-A and SP-C mRNA expression could at least partially explain the effect of maternal smoking on the incidence of RDS even in the absence of a corresponding stimulation of SP-B mRNA production. SP-A, SP-B, and SP-C all contribute to the functional properties of pulmonary surfactant (for reviews, see Refs. 3, 20, 27), and identification of mutations in the human SP-B gene has shown that normal SP-B gene function is essential for viability (25). It has also been shown, however, that normal fetuses at 24 wk gestation already have SP-B mRNA levels that are 50% of the adult level, whereas SP-A mRNA is still barely detectable at this stage (3). Because both SP-A and SP-B are thought to be necessary for the formation of tubular myelin, the presumed structural intermediate between the lamellar bodies and the functional phospholipid monolayer (20, 27), it appears at least possible that a premature infant could develop RDS specifically due to a lack of SP-A and the consequent inability to form tubular myelin. Such an infant might be capable of producing enough SP-B to avoid developing RDS if SP-A expression were stimulated in some way.
Additional studies are required to further explore the effect of nicotine on lung maturation as well as to determine the nature and location of the nAChRs in the developing lung.
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ACKNOWLEDGEMENTS |
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This work was supported in part by funds from the Cigarette and Tobacco Surtax Fund of the State of California; by Grant 4KT-0339 from the Tobacco-Related Disease Research Program of the University of California (to C. W. Wuenschell); and by National Heart, Lung, and Blood Institute Grants HL-44977 and HL-44060 (to D. Warburton).
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FOOTNOTES |
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Address for reprint requests: C. W. Wuenschell, Center for Craniofacial Molecular Biology, Univ. of Southern California, 2250 Alcazar St., CSA 1st Floor, Los Angeles, CA 90033.
Received 25 April 1997; accepted in final form 26 September 1997.
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