Preparation stained with Leishman stain.
Arrowheads indicate the localization of gaps (fetuses S19 and S5) and
a triradial (S2).
de la Chica RA, Ribas I, Giraldo J, Egozcue J, Fuster C. Chromosomal Instability in Amniocytes From Fetuses of Mothers Who Smoke. JAMA. 2005;293(10):1212–1222. doi:10.1001/jama.293.10.1212
Author Affiliations: Departament de Biologia
Cel·lular, Fisiologia i Immunologia, Facultat de Medicina, Universitat
Autònoma de Barcelona, Bellaterra, Spain (Ms de la Chica and Drs Egozcue
and Fuster); Centro de Patología Celular, Barcelona, Spain (Dr Ribas);
and Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma
de Barcelona, Bellaterra (Dr Giraldo).
Context Tobacco increases the risk of systemic diseases, and it has adverse
effects on pregnancy. However, only indirect data have been published on a
possible genotoxic effect on pregnancy in humans.
Objectives To determine whether maternal smoking has a genotoxic effect on amniotic
cells, expressed as an increased chromosomal instability, and to analyze whether
any chromosomal regions are especially affected by exposure to tobacco.
Design, Setting, and Patients In this prospective study, amniocytes were obtained by routine amniocentesis
for prenatal diagnosis from 25 controls and 25 women who smoke (≥10 cigarettes/d
for ≥10 years), who were asked to fill out a smoking questionnaire concerning
their smoking habits. Chromosomal instability was analyzed in blinded fashion
by 2 independent observers in routine chromosome spreads. Breakpoints implicated
in chromosomal abnormalities were identified by G-banding.
Main Outcome Measures Association between maternal smoking and increased chromosomal instability
in amniotic fluid cells, expressed as chromosomal lesions (gaps and breaks)
and structural chromosomal abnormalities.
Results Comparison of cytogenetic data between smokers and nonsmokers (controls)
showed important differences for the proportion of structural chromosomal
abnormalities (smokers: 12.1% [96/793]; controls: 3.5% [26/752]; P = .002)
and to a lesser degree for the proportion of metaphases with chromosomal instability
(smokers: 10.5% [262/2492]; controls: 8.0% [210/2637]; P = .04),
and for the proportion of chromosomal lesions (smokers: 15.7% [391/2492];
controls: 10.1% [267/2637]; P = .045). Statistical
analysis of the 689 breakpoints detected showed that band 11q23, which is
a band commonly implicated in hematopoietic malignancies, was the chromosomal
region most affected by tobacco.
Conclusions Our findings show that smoking 10 or more cigarettes per day for at
least 10 years and during pregnancy is associated with increased chromosomal
instability in amniocytes. Band 11q23, known to be involved in leukemogenesis,
seems especially sensitive to genotoxic compounds contained in tobacco.
The long-term public health consequences of regular tobacco consumption
include an increased risk of coagulation problems, cancer, cardiovascular
disease, chronic obstructive pulmonary disease, and adverse effects on pregnancy.
Maternal smoking during pregnancy has many consequences both during and after
pregnancy, such as infertility, coagulation problems, obstetric accidents
such as extrauterine pregnancy or placenta previa, and intrauterine growth
retardation.1 A relationship between postnatal
exposure to tobacco and childhood cancer, especially leukemia and lymphomas,
has also been suggested.2
Tobacco contains a high number of mutagenic compounds.3 Recently,
the presence of tobacco-specific metabolites has been described in fetal blood
and cell-free amniotic fluid (transferred from the mother via placenta) and
in newborns from women who smoke,4- 6 suggesting
a possible genotoxic effect of smoking during pregnancy. However, although
many cytogenetic studies have demonstrated the existence of an increased incidence
of chromosomal aberrations, sister chromatid exchanges (SCEs), micronuclei,
and fragile-site expression in peripheral blood lymphocytes of adult smokers,7- 10 no data
regarding a possible genotoxic effect of tobacco on the embryo and fetus are
available. Only indirect data using chorionic villi have been published11,12; in one case, an increase in SCEs
was found in direct preparations,11 while in
the other, chromosomal lesions were not increased.12
In this study we assess the possible genotoxic effect of maternal smoking
on amniotic fluid cells, based on the presence of an increased chromosomal
instability expressed as chromosomal lesions (gaps and breaks) and structural
chromosomal abnormalities. We also analyze whether any chromosomal regions
are especially affected by exposure to tobacco in the fetus.
In this prospective study, amniocytes were obtained by amniocentesis
for prenatal diagnosis. The study group consisted of 25 women smokers and
25 nonsmoking women between the 13th and 26th postmenstrual week. Women were
first personally interviewed at length by one author (I.R.) regarding their
consumption of alcohol, coffee, and tea. Only if the answers were negative
were women asked to fill out the smoking questionnaire concerning their current
and previous smoking habits, those of their husbands, and smoking in their
occupational setting. Smokers had smoked 10 or more cigarettes per day for
at least 10 years. Nonsmokers (controls) were not exposed to tobacco at home
or at work (ie, no passive smoking). In the smokers group, 5 fathers smoked
5 to 20 cigarettes per day (S2, S5, S7, S8, and S17), 10 fathers were nonsmokers
(S1, S3, S12, S13, S15, S16, S18, S20, S23, and S25), and the smoking habits
of the rest of the fathers was unknown. The first 25 women who fulfilled all
of these conditions and were in good health were included in each group. In
total, 800 interviews were carried out. Four hundred ninety-six interviews
were required to find the 25 nonsmokers who fulfilled the strict criteria
set up in our protocol; 175 interviews were required to find the 25 mothers
who had smoked 10 or more cigarettes daily for at least 10 years and who continued
smoking during pregnancy. The 129 remaining interviews correspond either to
women who smoked fewer than 10 cigarettes per day, those who had smoked for
less than 10 years, or those who had quit smoking when they knew they were
Table 1 and Table 2 present data for maternal age, paternal age, number of previous
pregnancies, years of maternal smoking before present pregnancy, number of
cigarettes smoked per day, weeks of gestation, and the indications for prenatal
diagnosis for smokers and controls, respectively. The study was approved by
the Universitat Autònoma de Barcelona institutional ethics committee.
Informed consent was given in writing by all participants.
The amniotic fluid was centrifuged in 2 different tubes at 800 rpm for
5 minutes at room temperature. The supernatant was removed under sterile conditions,
leaving a pellet in 0.5 mL of amniotic fluid. Cells were resuspended with
fresh culture medium. Four cultures were set up: two 35-mm plastic petri dishes
containing a 22-mm–square coverslip and 2 flat plastic tubes. The culture
medium used for petri dishes was Chang (Irvine Scientific, Santa Ana, Calif)
with 1% penicillin-streptomycin (Invitrogen Corp, Carlsbad, Calif). The media
used for tubes were RPMI:HAM-F10 (1:1) (Invitrogen) with 5.5% fetal calf serum
(Invitrogen); 2.5% ultroser G, which is a substitute for calf bovine serum
(Ciphergen Biosystems Inc, Fremont, Calif); 2% L-glutamine
(Invitrogen); and 1% penicillin-streptomycin (Invitrogen). Cultures were placed
in an incubator with 5% carbon dioxide in ambient air at 37°C, monitored
visually, and the medium changed every 2 to 3 days. Petri dishes were used
only for prenatal diagnosis. For the present study, cultures from smokers
and controls were both first grown in an RPMI:HAM-F10 medium. When cultures
in a flat tube showed sufficient growth (≥5 colonies), the cells were distributed
into 2 plastic petri dishes containing Chang medium and harvested 24 hours
later using an in situ fixation technique; colcemid was added for the last
45 minutes. The medium containing colcemid was replaced by 0.8% sodium citrate
at room temperature for 12 to 15 minutes. A few drops of 3:1 methanol/acetic
acid fixative were added to the hypotonic solution for 5 minutes. The fixative
was replaced with fresh fixative for 20 minutes. One additional fixative change
was made. Following removal of the final fixative, the coverslips were allowed
to dry under specific humidity conditions (48%-52%).
Preparations were stained with Leishman stain (1:4 in Leishman buffer),
coded, and evaluated for the presence of gaps and breaks by 2 authors (R.A.C.,
C.F.) blinded to participant smoking status. Differences were resolved by
discussion and consensus. Location and types of anomaly were recorded by each
evaluator and compared at the end of the study. Cytogenetic evaluation was
performed according to standard procedures. Only high-quality metaphases were
analyzed. About 100 randomly selected metaphases uniformly stained were analyzed
in each case. Later, preparations were destained for 1 minute in 3:1 methanol/acetic
acid and immediately incubated for 10 to 30 minutes in 2xSSC at 65°C,
washed with distilled water, air dried, and stained for 3 minutes with Wright
Giemsa stain to identify the bands where the lesions were located. To characterize
structural chromosomal abnormalities (deletions, acentric fragments, duplications,
translocations, inversions, and marker chromosomes), only high-quality banded
metaphases were used; at least 25 banded metaphases per patient were karyotyped.
A generalized estimating equation (GEE)13 was
used for assessing the differences between the smoker and control groups for
the different types of chromosomal instability. The GEE approach is an extension
of generalized linear models designed to account for repeated within-individual
measurements. This technique is particularly indicated when the normality
assumption is not reasonable as, for instance, for discrete data. The GEE
model was used instead of the classic Fisher exact test because the former
takes into account the possible within-fetus correlation, whereas the latter
assumes that all observations are independent. Since several metaphases were
analyzed per fetus, the GEE model is more appropriate. In addition, this method
allows for the inclusion in the model of additional explanatory variables
as covariates. In our analyses, the variance function for the binomial distribution
and the logit link function were specified for the model. The response variable
was defined as the number of chromosomal anomalies/number of metaphases tested
for each fetus.
To identify which chromosome bands could be considered especially affected
by the genotoxic effect of tobacco, the fragile site multinomial method (version
995) was used.14,15 This multinomial
statistical method is specifically designed to identify chromosomal fragile
sites at loci where chromosome breaks are found. The fragile site multinomial
method can be used for a maximum of 30 individuals, and the program performs
the analyses for each individual separately and for the data pooled over all
individuals. Because the number of chromosomal abnormalities per individual
was much lower than the minimum (200 at the 400-band resolution level) required
by the program to perform reliable estimates, only results from data pooled
over the smoker and control groups were considered. The standardized χ2 and G2 tests were used for assessing
the statistical significance of the chromosome bands with breaks, gaps, or
rearrangements in each group.
To identify the bands with a greater sensitivity (implicated in structural
chromosomal abnormalities or in chromosomal lesions) in smokers relative to
controls, a variable was computed, defined for each band as the number of
gaps and breaks (including those involved in structural abnormalities) in
smokers minus their number in controls (difference). Bands with positive values
in the computed variable indicated a greater tendency to break in smokers,
while bands with negative values suggested the opposite. In addition, those
bands with a computed difference value more than 3 SDs from the mean difference
were considered extreme values and selected for further analysis with the
GEE model. In the particular case of a band presenting a zero value for each
of the individuals belonging to one group, alternative analyses such as the
Fisher exact test and the nonparametric Wilcoxon rank-sum test (for which
exact P value computation was requested) were applied.
Statistical significance was set at P<.05.
Statistical analyses were carried out with SAS/STAT release 8.01 (SAS Institute
Inc, Cary, NC). The GEE model was fitted using the REPEATED statement in the
GENMOD procedure. The conservative type 3 score statistics were used for the
analysis of the model effects.16
The number of metaphases with chromosomal instability, the frequency
and type of chromosomal lesions, and the frequency of structural abnormalities
in amniocytes from fetuses of the smoker and control groups are shown in Table 3. The clinical data of the patients (Table 1 and Table
2) revealed that the mean maternal age in the smoker group was significantly
higher than in the control group. However, the difference (3 years) found
in the mean values should not influence a study based on the analysis of lesions
and structural abnormalities, because maternal age influences numerical but
not structural abnormalities. In this regard, no significant correlation was
obtained in our data between any of the above cytogenetic variables and maternal
age, within either the smoker or control groups. Nevertheless, because the
GEE method used for the analysis allows for the inclusion of continuous explanatory
variables as covariates, the contribution of age was considered. Moreover,
the results obtained for the whole sample were consistent with those from
a particular subset (all women except those who underwent in vitro fertilization
or intracytoplasmic sperm injection) in which no significant difference in
maternal age between smokers and controls was present. Finally, no differences
were found between smokers and controls for the number of weeks of gestation.
First, we used a reduced model in which age was not considered. In all
analyses, the smoking effect was significant for chromosomal instability (smokers:
10.5% [262/2492]; controls: 8.0% [210/2637]; P = .04),
chromosomal lesions (smokers: 15.7% [391/2492]; controls: 10.1% [267/2637]; P = .045), and to a higher degree for structural
chromosomal abnormalities (smokers: 12.1% [96/793]; controls: 3.5% [26/752]; P = .002). In both groups, the most frequent
structural chromosomal abnormalities were deletions and translocations (Table 3). Deletions (smokers: 7.2% [57/793];
controls: 2.5% [19/752]) and translocations (smokers: 2.1% [17/793]; controls:
0.5% [4/752]) were both also significant (P = .006
and P = .01, respectively).
Next, a model in which age was included as a covariate was considered.
The age effect was not significant for any of the analyses performed (for
chromosomal instability, P = .40; chromosomal
lesions, P = .16; structural chromosomal
abnormalities, P = .64; deletions, P = .40; and translocations, P = .10). The high P values obtained
for maternal age indicate that this factor does not influence the chromosomal
anomalies observed and suggest that it could be removed from the model. Nevertheless,
the model incorporating maternal age was evaluated. The inclusion of this
covariate increased the P values of the smoking factor
for all chromosomal anomalies analyzed. A nearly significant increase was
observed in the percentage of metaphases with chromosomal instability in amniocytes
from smokers compared with those from controls (P = .05).
The proportion of chromosomal lesions was marginally influential in amniocytes
from smokers compared with those from controls (P = .10).
In the smoker group, 2 cases (S9 and S11) had metaphases with multiple chromosomal
lesions or pulverized cells; these metaphases were not included in the estimation
of the number of lesions. The much higher incidence of structural chromosomal
abnormalities in karyotyped metaphases in the smoker group than in the control
group remained significant (P = .01). The
incidence of deletions was higher in the smoker group than in the control
group (P = .01), while the incidence of
translocations became nonsignificant (P = .12).
More than one third of the fetuses from mothers who smoke (36% [9/25]) had
triradial or quadriradial figures in their metaphases (S2, S5, S9, S10, S11,
S13, S15, S19, and S20) (Figure 1);
in controls, only 1 quadriradial was found (C24).
Finally, 5 smokers and 6 controls had become pregnant by in vitro fertilization
or intracytoplasmic sperm injection. To discard a possible effect of the hormonal
treatment on the evaluation of the genotoxic effects of tobacco, the statistical
analyses were repeated excluding these individuals. It is worth noting that
in this subset of women excluding those who had undergone in vitro fertilization
or intracytoplasmic sperm injection, the maternal ages of smokers and controls
were not statistically different. However, for consistency with the previous
analyses, an extended model including maternal age as a covariate and a reduced
model not including this factor were considered. Similar results were obtained
for both models. In the extended model and as in the results obtained for
the whole sample, maternal age showed no significant association with observed
chromosomal anomalies. In this extended model, the results for the smoking
factor reached statistical significance for both the proportion of metaphases
with chromosomal instability (smokers: 10.3% [200/1951]; controls: 7.2% [145/2023]; P = .03) and the proportion of structural chromosomal
abnormalities (smokers: 13.3% [83/624]; controls: 3.0% [17/570]; P = .01) and showed a marginal influence for the proportion
of chromosomal lesions (smokers: 15.3% [298/1951]; controls: 9.0% [182/2023]; P = .08). The results obtained for the reduced
model reached statistical significance for both the proportion of metaphases
with chromosomal instability (P = .02)
and the proportion of structural chromosomal abnormalities (P = .002) and showed a nearly significant association for
the proportion of chromosomal lesions (smokers: 15.3% [298/1951]; controls:
9.0% [182/2023]; P = .05).
Aneuploid metaphases were found in smokers and controls (smokers: 12.5%
[99/793]; controls: 10.8% [81/752]) without showing statistical significance
between them (P = .52 for the reduced model; P = .36 for the extended model).
In sum, our results suggest that smoking during pregnancy has a genotoxic
effect that is not influenced by maternal age.
Cytogenetic results for each individual are shown in Table 4 and Table 5. All fetuses
had normal constitutional karyotypes (46,XX or 46,XY). A pseudomosaicism (46,XY,83%/46,XY,t[X;1][p22.2;q25]17%)
was detected in S16 but not confirmed after birth.
The breakpoint distribution of the 430 breakpoints clearly identified
by G-banding in structural abnormalities and in chromosomal lesions in the
smoker group and of the 259 breakpoints in the control group was not uniform
(Figure 2). With the exception of chromosome
22 in the smoker group and of chromosomes 21, 22, and Y in the control group,
all other chromosomes were involved in structural abnormalities or in chromosomal
lesions. To determine the possible existence of an association between the
breakpoints found (at the 400-band resolution level) and those chromosome
bands containing fragile sites, the data on fragile sites accepted by the
Committee on Human Gene Mapping 11 were used.19 The t test showed a preferential location of breakpoints in
chromosome bands containing fragile sites, both in smokers and in controls
(P<.001 and P = .002,
The fragile site multinomial method was used to identify those chromosome
bands that significantly expressed breakpoints in the 2 groups. In both groups,
the number of breaks required to consider a band to be nonrandomly affected
was 4 or more. The results in the control group indicated that 12 bands were
nonrandomly affected: 2q35, 7p15, 10q22, 11q13, and 14q24 (4 times each);
1p34, 1p22, 4q31, 6q21, and 12q13 (5 times each); and 1q32 and 17q21 (6 times
each). In the smokers group, 30 bands were nonrandomly affected: 1p34, 1q42,
2p13, 2p16, 2p23, 2q21, 3q21, 5q15, 6q22, 7p15, 15q24, 16q22, 16q23, and 17q23
(4 times each); 1q23, 2p21, 4q31, 6p21, 11q13, and 12q15 (5 times each); 1p36,
1q11.2, 1q32, 3p14, 7q11.2, 7q32, and 9q22 (6 times each); 11q23 (9 times,
but only in smokers) (Figure 3); 5q31
(10 times); and 17q21 (13 times) (Table 6).
To identify the bands with a greater propensity to break in smokers
relative to controls, the differences in the number of breaks for the bands
listed above were calculated as described in the “Methods” section.
The mean of these differences was 0.72 (SD, 1.78), with –3 and 9 the
most negative and positive values. Applying the criterion of 3 SDs of the
computed differences from their mean value as a classifying distance, no bands
with extreme negative values were detected, whereas 3 bands with extreme positive
values were found: 17q21 (difference, 7), 5q31 (difference, 7), and 11q23
(difference, 9). The Fisher exact test and the nonparametric Wilcoxon rank-sum
test reached statistical significance only for 11q23 (P = .02, both tests).
In this study, the main difficulty was to find heavy smokers (≥10
cigarettes/d for ≥10 years) who also smoked during pregnancy, and control
women not exposed to tobacco at home or at work (total of 800 interviews required).
Moreover, smokers and controls had to be free of exposure to other clastogenic
agents and not consume alcohol, coffee, or tea. In the present study it was
found that, under these conditions, fetuses from pregnant women who smoked
had an increased frequency of chromosomal instability, evaluated by the presence
of structural chromosomal abnormalities and chromosomal lesions.
Chromosomal instability and analyses of micronuclei in lymphocytes from
peripheral blood have been successfully used as biomarkers of genotoxicity
both for assessing DNA damage at the chromosomal level and for quantifying
early adverse human health effects, in particular cancer.20,21 Peripheral
blood lymphocytes from heavy smokers (>30 cigarettes/d) or from children born
to smokers show increases in structural chromosomal abnormalities, SCEs, micronuclei,
or fragile-site expression.7- 10 In
utero, only indirect data using chorionic villi have been published11,12; one study showed an increase in
SCEs while the other found no increase in chromosomal lesions.
In our study, comparison of cytogenetic data between groups of smokers
and controls showed important differences for the proportion of structural
chromosomal abnormalities and to a lesser degree for the proportion of metaphases
with chromosomal instability and for the proportion of chromosomal lesions.
This propensity for a strong genotoxic effect in mothers who smoke (highest
incidence of the most severe anomaly) is also observed for the chromosomal
lesions, where the differences are more marked for breaks than for gaps (Table 3).
Taking into account the way in which both groups had to be completed,
maternal age was by chance significantly higher in the smoker than in the
control group. It is well known that maternal age is related to an increase
in numerical chromosomal abnormalities (especially trisomies and, among them,
trisomy 21), but no study has related increasing maternal age to an increase
in chromosomal lesions and structural abnormalities. Nevertheless, 2 GEE models
were considered, either including or not including age as a covariate. In
all the analyses performed, inclusion of age as a covariate led to an increase
in the P value of the smoking factor relative to
that in the reduced model. As a result, 2 of the analyses that showed significance
in the reduced model, those for chromosomal instability (P = .04) and chromosomal lesions (P = .045),
became nearly significant (P = .05) and
marginally influential (P = .10), respectively,
in the extended model. The third chromosomal anomaly studied, structural chromosomal
abnormalities, remained significant in the extended model (P = .01). Finally, the analyses corresponding to a subset
in which women who had become pregnant by in vitro fertilization or intracytoplasmic
sperm injection were excluded showed similar significance values for the smoking
factor in the extended model compared with the reduced model for all 3 chromosomal
It is worth noting that the maternal age factor was not significant
in any of the analyses performed, suggesting that the reduced model in which
this factor was omitted could be more appropriate for the description of our
data. Keeping a nonsignificant covariate in an extended model can be considered
adequate when this factor belongs to the design configuration of the study
or its association with the response variable is widely accepted in the research
field. Neither of these circumstances applies in the present case. As indicated
above, maternal age was an observational variable and it is numerical chromosomal
abnormalities, not the anomalies studied in the present work, that are known
to be associated with maternal age. Because of these reasons, a reduced model
can be more suitable than the extended model including maternal age.
Our results show that fetuses exposed to tobacco smoke in utero have
increased chromosomal instability in amniocytes, expressed as an increase
of structural chromosomal abnormalities and chromosomal lesions, which is
not influenced by maternal age. In the present study, no direct relationship
between the level of genotoxic tobacco compounds and chromosomal instability
has been demonstrated because the levels of tobacco-specific compounds (eg,
cotinine) were not measured in amniotic fluid or maternal serum. However,
the fact that several studies have described the presence of these compounds
in the blood of fetuses from women who smoke4- 6 seems
to support our findings, suggesting a possible genotoxic effect of smoking
To determine if some chromosomal regions were especially affected by
exposure of the fetus to tobacco, we localized the breakpoints implicated
in chromosomal lesions and in structural abnormalities. An apparently nonrandom
distribution of breakpoints and a coincidence with fragile-site bands in the
smoker and control groups was observed. The preferential location of breakpoints
in fragile-site bands in chromosomal preparations from chorionic villi has
been previously described.22,23 This
coincidence has also been observed in lymphocyte chromosomes from cigarette
smokers.7,24 Recently, Stein et
al24 and Spitz et al25 have
stated that tobacco exposure increases chromosomal fragility due to an adaptation
of DNA repair mechanisms to smoking, which in turn leads to an accumulation
of genetic damage. It is worth noting that, according to these authors, this
ineffective repair is transient and reversible. Several data sets suggest
that tobacco exposure induces in vivo fragile-site expression, which contributes
to tumor formation.26,27
Our results show, in agreement with these studies, that tobacco exposure
increases chromosomal instability due to late or incomplete DNA replication
or to errors in repair mechanisms (inefficient response or poor inducible
repair response). Both mechanisms may affect the integrity of chromosomal
structure in these regions, leading to the appearance of structural chromosomal
abnormalities, gaps, and breaks. Therefore, the chromosome breakpoints could
produce deletions or disruptions of functional genes, producing developmental
defects or genetic disorders, including cancer.
By comparing the breakpoint distribution in both groups using the fragile
site multinomial method, 3 specific chromosome bands affected by exposure
to tobacco have been detected: 5q31, 17q21, and, especially, 11q23. Breaks
on 11q23, however, were only observed in smokers. Two of these bands, 5q31.1
and 11q23, correspond to regions where fragile sites FRA5C, FRA11B, and FRA11G
are located. According to the Committee on Human Gene Therapy,19 FRA5C
and FRA11G are considered “fragile sites, aphidicolin-type, common”
and FRA11B a “fragile site, folic acid-type, rare.” In this sense,
it should be noted that smokers have reduced concentrations of folic acid
in serum,28 a fact that could explain the high
incidence of breakpoints at 11q23.
It is worthwhile to note that chromosome breaks at 3p14.2, where the
most common fragile site (FRA3B) is located, were only found in mothers who
smoke (6 times). Although in the present study this site was not among the
3 breakpoints most expressed in amniocytes from fetuses of mothers who smoke
(more than 8 lesions each), this finding is consistent with that of a previous
study24 in which FRA3B expression is directly
correlated with cigarette smoking.
It has been suggested that the increase of chromosomal lesions and structural
abnormalities or the very existence of an increased chromosomal instability
resulting from the genotoxic effect of tobacco could be indicative of an increased
cancer risk and that fragile sites could be responsible for the chromosomal
instability observed in cancer cells.27 Moreover,
an increase of chromosomal instability is associated with an increase in the
risk of cancer, especially childhood malignancies.29
For the last 30 years, consumption of tobacco by parents has been related
to leukemia in infancy.2,30 It
is known that a high proportion of infants (40%-60%), children (18%), and
adults (3%-7%) with leukemia have molecular rearrangements in chromosome band
11q23, but these rearrangements are not always detectable by cytogenetic analysis.31- 34 According
to some authors,31- 35 there
is strong evidence that 11q23 rearrangements occur in utero. These findings
show the importance of the involvement of band 11q23 in events leading to
leukemogenesis in infants. The other 2 bands most affected by tobacco in our
study (5q31 and 17q21), although not affected in statistically significant
proportions, are also involved in childhood leukemia.34
In conclusion, maternal smoking of 10 or more cigarettes per day for
10 or more years, including during pregnancy, is associated with increased
chromosomal instability in amniocytes. Band 11q23, which seems to be especially
sensitive to compounds contained in tobacco, is known to be involved in leukemogenesis.
This band contains the genes ATM (cell prolymphocytic
leukemia), PLZF (leukemia acute, promyelocytic; PLZF/RARA type), and MLL (leukemia, myeloid/lymphoid, or mixed lineage). Thus, the transplacental
exposure to tobacco could be associated with an increased risk of pediatric
hematopoietic malignancies. Epidemiologic studies will be needed to determine
whether the offspring of parents who smoke have an increased lifetime risk
Corresponding Author: Josep Egozcue, MD,
Unitat de Biologia Cel·lular, Departament de Biologia Cel·lular,
Fisiologia i Immunologia, Edifici CS, Universitat Autònoma de Barcelona,
E-08193 Bellaterra, Spain (firstname.lastname@example.org).
Author Contributions: Dr Egozcue had full access
to all of the data in the study and takes responsibility for the integrity
of the data and the accuracy of the data analysis.
Study concept and design; analysis and interpretation
of data: de la Chica, Giraldo, Egozcue, Fuster.
Acquisition of data: Ribas.
Drafting of the manuscript: de la Chica, Ribas,
Giraldo, Egozcue, Fuster.
Critical revision of the manuscript for important
intellectual content; study supervision: Giraldo, Egozcue, Fuster.
Statistical analysis: Giraldo.
Obtained funding: Fuster.
Administrative, technical, or material support:
de la Chica, Ribas.
Financial Disclosures: None reported.
Funding/Support: Financial support for this
study was provided by the Comissionat per a Universitats i Recerca (2001,
Role of the Sponsor: The Comissionat per a
Universitats i Recerca was not involved in the design and conduct of the study;
in the collection, analysis, and interpretation of the data; or in the preparation,
review, or approval of the manuscript.
Previous Presentation: Preliminary results
of this study were presented at the Third European Cytogenetics Conference;
July 7-10, 2001; Paris, France.
Acknowledgment: We thank the Department of
Obstetrics and Gynecology, Institut Universitari Dexeus and the Centro de
Patología Celular for their kind collaboration in providing the samples.