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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Rev. Int. Contam. Ambient. 26 (1) 47-63, 2010
Centro de Ciencias de la Atmósfera, Universidad Nacional Autónoma de México (UNAM). Circuito Exterior,
Ciudad Universitaria, C.P. 04510, México D.F.
Autor responsable:
Instituto Nacional de Enfermedades Respiratorias. Calzada de Tlalpan 4502, Col. Sección XVI, Delegación
de Tlalpan. Ciudad de México. C.P. 14710
(Recibido octubre 2008, aceptado julio 2009)
Key words: micronuclei, human lymphocytes, nuclear division index (NDI), cytokinesis proliferation block
index (CPBI), nicotine, air pollution
The genotoxic damage that tobacco smoke produces in active smokers was evaluated
using cytokinesis-blocked micronucleus assay. The effect of tobacco smoke on the
cellular cycle was analyzed by means of nuclear division index (NDI) and cytokinesis
proliferation block index (CPBI). The results indicated a signi±cantly lower frequency
of micronuclei (MN) in the smoker group than in the control group. The nuclear divi-
sion and cytokinesis proliferation block indexes indicated a delay in the cellular cycle
of smokers and controls. The delay was greater in the controls (non-smokers) com-
pared to smokers. Nicotine and cotinine contents in the urine samples of the subjects
of both groups were also measured using gas chromatography/mass spectrometry;
signi±cantly higher levels were found in smokers, while values for controls could not
be established accurately due to the fact that they fell below the limits of resolution
accepted by the mass spectrometer. In general, no association was established between
evaluated cytogenetic variables –binucleated (BN) cells with MN, total MN, NDI and
CPBI– and nicotine and cotinine contents in smokers. However, when the informa-
tion was analyzed according to subgroups –light, moderate and heavy–, an increase in
correlation coef±cients was found. The same strategy was used to analyze the rest of
the cytogenetic variables and nicotine and cotinine. The results indicated that only the
light-smoker subgroup exhibited a signi±cant correlation coef±cient between nicotine
and the number of BN cells with MN.
Palabras clave: micronúcleos, linfocitos humanos, índice de división nuclear (IDN), índice de bloqueo de
proliferación de citocinesis (IBPC), nicotina, contaminación del aire
Se evaluó el daño genotóxico que produce el humo de tabaco en fumadores activos, a
través de la prueba de micronúcleos por bloqueo de la citocinesis. El efecto del humo
de tabaco sobre el ciclo celular también fue analizado mediante el empleo de los índi-
ces de división nuclear (IDN) y de bloqueo de proliferación de la citocinesis (IBPC).
C. Calderón-Ezquerro
et al.
Los resultados mostraron una frecuencia signiFcativamente menor de micronúcleos
(MN) en el grupo de fumadores, con respecto al grupo testigo. Los valores de los índices
IDN e IBPC mostraron que hubo retraso en el ciclo celular de los fumadores y de los tes-
tigos, aunque este retraso fue mayor en los testigos. También se midieron los contenidos
de nicotina y cotinina en la orina de los sujetos fumadores y testigos evaluados, mediante
cromatografía de gases/espectrometría de masas; en los fumadores se registraron los
niveles signiFcativamente más altos de estos compuestos. Los valores de los testigos
no pudieron establecerse con exactitud debido a que cayeron por debajo de los límites
de resolución aceptados por el espectrómetro de masas. En general, entre las variables
citogenéticas evaluadas no se estableció ninguna asociación –células binucleadas con
mincronúcleos, micronúcleos totales, índice de división nuclear, e índice de bloqueo
de proliferación de la citocinesis– y el contenido de nicotina y cotinina en fumadores.
Sin embargo, cuando la información fue analizada de acuerdo a subgrupos –ligeros,
moderados e intensos–, se encontró un incremento en los coeFcientes de correlación.
En consecuencia, la misma estrategia se aplicó para el análisis del resto de las variables
citogenéticas y los niveles de nicotina y cotinina. Como resultado, sólo el subgrupo de
fumadores ligeros mostró un coeFciente de correlación signiFcativo entre la nicotina
y el número de células binucleadas.
Genotoxicity studies on smokers have mainly
been based on the utilization of peripheral blood
lymphocytes to search for micronuclei (MN). These
cells come into contact with constitutive elements
of tobacco smoke as well as their by-products while
entering the body. MN assessment enables the estab-
lishment of DNA damage and determines the possible
affectation of the cellular cycle. Besides, it is an
easier and faster method than the conventional test
for chromosomal aberrations (Pardell 1996, ±enech
et al.
1999, ±enech 2000).
The relation between MN appearance and tobacco
smoking has been studied since mid-1980’s, not
only to evaluate the direct effect but also to discard
a confusing effect in studies with other genotoxic
agents. However, results have been contradictory
and controversial.
Among the studies assessing the effect of to-
bacco smoke on MN induction in healthy subjects
using the cytokinesis proliferation block technique
is the research carried out by Au
et al.
et al.
(1991) and Holmen
et al.
These authors reported a signiFcant increment in
MN, even though the last study describes that this
enhancement was restricted to the lymphocyte T8
cellular subpopulation.
On the other hand, Cheng
et al.
(1996) and Duf-
et al.
(1999) described a confusing effect for
tobacco smoke and showed a positive effect. Both
studies were focused on persons with cancer (pul-
monary and neck, respectively). Their results show a
signiFcant MN increment in sick patients that smoked
(Frst case) and in healthy subjects that smoked (sec-
Humans require clean environments to protect
themselves from diseases. However, such environ-
ments are frequently pervaded by different sub-
stances noxious to health (Bardana and Montanaro
1997). Mexico City’s inhabitants are exposed to an
atmosphere containing a large variety of gases and
contaminant particles such as ozone, carbon mon-
oxide and dioxide, aromatic hydrocarbons, alco-
hols, inorganic gases, volatile organic compounds,
aldehydes, metals, nitrogen oxide, particulate
pollutants, pesticides, biologic contaminants and
tobacco smoke, among others (Raga
et al.
et al.
It is well known that tobacco smoke is the cause
of various diseases and is responsible for numerous
deaths worldwide. Different types of cancer have
been associated with smoking. Moreover, it has been
shown that persons involuntarily exposed to environ-
mental tobacco smoke (ETS) are more susceptible to
these illnesses than unexposed persons (ASH 2006).
Noxious effects of tobacco smoke have been
assessed for a long time by various bioassays that
show alterations in the genetic material (Albertini
et al.
2000). As a consequence, a damaging effect
is caused to the integrity of the genetic material,
mitotic spindle, or centromere. This kind of damage
may be responsible for cellular death or the onset
of neoplastic processes (±enech
et al.
1999). The
cytokinesis-blocked micronucleus assay has been
employed to evaluate possible alterations in chro-
mosomes during interphase.
ond case). Zhao
et al
. (1998) described the effect of
atmospheric contamination, and Lohani
et al
. (2002)
that of asbestos. Both studies reported a higher MN
frequency, regardless of whether the subjetcs had
been exposed to contaminants or asbestos or not.
Conversely, studies reporting no difference be-
tween MN frequency in smokers and nonsmokers are
more numerous. Among the researches assessing the
possible genotoxic effect of tobacco is the work by
et al
. (1997) and Barale
et al
. (1998).
former found no signi±cant differences, whereas the
latter reported a signi±cant decrement.
Other studies evaluating the genotoxic effect
caused by various agents also included the possible
confusing effect of tobacco smoke. Among them
are Buckvic
et al
. (1998), Calvert
et al
. (1998),
et al
. (1999), Pitarque
et al
. (1999), Lucero
et al
. (2000), Maluf
et al
. (2000) and Palus
et al
(2003). The results of their researches conclude that
study subjects exposed to tobacco smoke (smokers)
showed no signi±cant differences in MN frequency
of lymphocytes when compared with their respective
controls. Finally, Falck
et al
. (1999), working with
pesticides, found that the control group that smoked
exhibited a decrement in MN frequency regarding
nonsmoker controls, even though the former also
showed an increase in the frequency of chromosomal
aberrations originating MN.
A possible explanation for these contradictory
results comes from Bonassi
et al
. (2003). They evalu-
ated and re-analyzed (meta-analysis) the results relat-
ing MN frequency to lymphocytes in smoking habits.
Overall, these studies recruited 5710 persons, of
whom 1409 were active smokers and 800 ex-smokers.
The main ±nding of this meta-analysis was that the
only subpopulation expressing a signi±cant increase
in MN frequency was that of heavy smokers (30
cigarettes or more daily), but only when these persons
had not been previously exposed to genotoxic agents.
Otherwise this increment was not found.
Studies evaluating nuclear division or cytokinesis
proliferation block index are scarce. Palus
et al
. (2003),
using the nuclear division index (NDI), reported no
differences between a smoker group and its control
group. Pitarque
et al
. (1999) employed the cytokine-
sis proliferation block index (CPBI) to ±nd a slight
non-signi±cant decrement in the smoker group. These
authors also evaluated the smoking effect in conjunc-
tion with the exposure to other toxic agents (cadmium,
lead, and contamination produced in airports). They
reported no differences between the considered groups
and their respective controls, whereas Pastor
et al
(2002) did ±nd a CPBI decrement caused by tobacco.
The joint analysis of MN in lymphocytes from
peripheral blood and nicotine determination has not
been undertaken yet, though some preliminary results
are available. Lee
et al
(1990) studied the genotoxic
effect of tobacco smoke in rats exposed for 90 days.
They measured nicotine levels in plasma to verify
smoke ingestion. No genotoxic effect was found in
bone marrow cells (micronuclei, sister chromatid
exchanges or chromosomal aberrations), so they did
not attempt to correlate both values.
Nersessian and Arutyunyan (1994) carried out
a similar study in mice; they analyzed genotoxic
effects of ten different types of cigarettes from East-
ern Europe. They found a strong increase in MN in
polychromatic erythrocytes from bone marrow. Their
results indicated not only that American cigarettes are
less clastogenic, but also that there is a strong cor-
relation between nicotine and tar contents in blood
plasma and MN frequency.
The following evidence has been determined
regarding to whether this correlation could be due
to a genotoxic effect of nicotine
per se
or is a con-
sequence of the amount of tobacco smoke one has
been exposed to.
et al
. (1997) found that nicotine values rang-
ing from 80 to 500 mg (per kilogram of body weight)
administered orally increase MN frequency in bone
marrow cells in mice infected with
as well as in uninfected mice. Likewise, in
ovary cells of the Chinese hamster (quantities similar
to those ingested with nicotine chewing gum), high
nicotine concentration caused an increment in the
frequency of both sister chromatid exchange and
chromosomal aberrations (Trivedi 1993). Lower
nicotine doses (1-2 mg per kilogram of body weight)
were not clastogenic as indicated by the MN test in
lymphocytes (Adler and Attia 2003).
et al
(1995) evaluated genotoxic activity of nicotine and
cotinine using Ames and SCE assays in Chinese
hamster cells, with negative results.
In México, studies on tobacco smoking are mainly
epidemiologic. The results indicate that health dam-
age due to tobacco is clearly associated with an
increase in morbidity and mortality (SSA 2002).
There have been few studies assessing DNA damage
in smokers and the applied method has been alkaline
single cell gel electrophoresis (comet assay) (Rojas
et al
. 1996). More recently, two methods have been
employed for the same purpose: the determination of
cell proliferation kinetics and genotoxicity in lym-
phocytes (Calderón-Ezquerro
et al
. 2007).
Therefore, the main objective of this study is to
assess the possible genotoxic effect in smokers living
C. Calderón-Ezquerro
et al.
in México City by micronucleus determination in
peripheral blood lymphocytes, using the proliferation
cytokinesis block assay, as well as its correlation with
exposure markers: nicotine and cotinine.
Sixty four subjects participated in this study; they
were divided into two groups. The Frst was made of
32 smokers recruited from the Anti-Tobacco Clinic in
the Instituto Nacional de Enfermedades Respiratorias
(INER, National Institute of Respiratory Diseases)
in México City while they still smoked. Each vol-
unteer attended the clinic in search of treatment to
quit smoking. The second group was formed by 32
healthy nonsmokers (control group) and was matched
in age and gender to the smoker group.
The smoker group consisted of 18 women and 14
men. Smoker age was 47.38 ± 12.97 years old (mean
± standard deviation), with an interval from 23 to 74
years of age. Number of cigarettes smoked per day
was 21.97 ± 10.73, ranging from 5 to 60. The mean
number of years smoking was 27.91 ± 13.10, with
a range from 4 to 60 years. In order to participate
in the study, smokers were required to have had a
daily minimum consumption of Fve cigarettes for
more than a year.
The control group included 18 women and 14
men. Mean age was 47.68 ± 13.19 years old (mean
± standard deviation), in an age range from 17 to 75
years. Participants in this group were nonsmokers,
unexposed to tobacco smoke (ETS)
(Albertini 2000)
at home or in their workplace. The age and gender
of the control group were matched to the smoking
group. In addition, it was veriFed that none of the
participants were on medication, were alcoholics, or
regularly exposed to genotoxic substances.
The classiFcation of subjects within the smoking
group was deFned according to the number of ciga-
rettes smoked per day: light smokers (<19 cigarettes
a day), moderate smokers (20 to 29 cigarettes), and
heavy smokers (> 30 cigarettes).
Samples of peripheral blood and urine were taken
from all participants.
Cytokinesis-blocked micronucleus (CBMN) assay
Venous blood samples from all volunteers of
both groups were drawn with heparinized vacutainer
tubes and transferred to the laboratory within a few
hours of taking the sample. Blood (400 µL) from
each sample was added to 4.5 mL RPMI medium
1640 containing L-glutamine (Gibco) plus 0.2 mL
phytohemagglutinin (4 % or 5 µg/mL) (Gibco) previ-
ously sterilized using a 0.22 µm nitrocellulose Flter.
This procedure was done by triplicate for each of the
subjects in the study.
Subsequently, cultures were incubated at 37 °C. Af-
ter 44-h incubation, cytochalasin B (Sigma) previously
dissolved in DMSO (6 µg/mL Fnal concentration)
was added to the cultures and stored at
20 °C for an
additional 28 hours; total culture time was 72 hours.
Afterwards, cells from each culture were harvested by
centrifugation, given a hypotonic shock (0.075 M KCl)
at 4 °C for 3 minutes followed by a pre-Fxed procedure
that slowly added four drops of a methanol-acetic acid
mixture (3:1) at 4 °C. Each tube was gently shaken and
let stand for 10 minutes at room temperature.
After a 10 minute centrifugation (1500 rpm),
a Fxation step was carried out by adding 10 mL
methanol-acetic acid mixture (3:1) at 4 °C. Tubes
were again centrifuged and the former procedure was
carried out until the pellet looked clean.
Cells were smeared on microscope slides and
air-dried. Slides were stained with the ²ulgen reac-
tion technique described by Stich and Rosin (1984)
and Stich (1987) and modiFed as follows (Gómez-
et al
. 2000): smeared cells were pretreated
in 1N HCl for 10 minutes at room temperature, then
placed in 1N HCl at 60 °C for another 10 minutes,
rinsed in distilled water, placed in Schiff’s reagent
for 90 minutes and washed with running tap water.
Afterwards, slides were immersed in a 1 % fast green
solution for 30 seconds, then submitted to four con-
secutive washes in ethanol to eliminate excess dye,
and Fnally air-dried. In order to avoid bias, all slides
were coded (smoker/nonsmoker) before counting
micronuclei (MN).
Using a light microscope (Olympus BX51 model),
MN were scored for every 1000 binucleated cells
counted per individual in accordance with accepted
criteria (²enech
et al
. 2003).
Micronuclei were reported in two different man-
ners: total micronuclei (TMN) and binucleated cells
with micronuclei (BN cells with MN). To obtain total
MN, the number of all MN was counted on 1000
cells. ²or binucleated cells with micronuclei, every
binucleated cell was counted as one unit, no matter
the number of micronuclei inside it.
The quantiFcation of TMN and BN cells with MN
was carried out following both procedures so that a
comparison of the results with those of other studies
could be established, since not all reports employ the
same way of counting micronuclei.
In addition, 500 more cells per individual were
analyzed to count the number of cells containing 1,
2, 3 and 4 nuclei, respectively. This was carried out
to derive the nuclear division and cytokinesis prolif-
eration block indexes with the following formulas:
Nuclear division index, NDI (provides the average
number of nuclei per cell):
NDI = [M1+2(M2) +3(M3) + 4(M4)/ total number of cells]
Cytokinesis proliferation block index, CPBI (pro-
vides the average number of complete cellular
divisions per cell):
CPBI = [M1+2(M2) + 3(M3+M4)/ total number of cells]
where M1 is the number of mononuclear cells,
M2 binuclear, M3 trinuclear, and M4 tetranuclear
et al
. 2003).
The value of these indexes in healthy subjects,
unexposed to toxic substances, is very near to 2 or
slightly higher. Values less than 2 indicate a delay
in the cellular cycle; values higher than 2 mean its
acceleration (Surallés
et al
. 1995, Albertini 2000).
Detection and quantifcation oF nicotine and its
metabolite cotinine in urine samples From the
subjects evaluated
For each individual, urine samples were collected
in the morning. From each sample, nine aliquots
were drawn, placed in 1.5 mL Eppendorf tubes, and
kept at
70 °C until needed to perform nicotine and
cotinine analyses.
Nicotine and cotinine in urine were quanti±ed
using gas chromatography-mass spectrometry.
The method of Hutchinson
et al
. (1998) was fol-
lowed taking into account their respective deuter-
ated internal standards. Analyses were isolated by
liquid-liquid extraction coupled to centrifugation
and evaporation. The sensitivity test indicated 10
and 100 ng/mL nicotine and cotinine, respectively.
Calibration curves were made ranging from 1 to
3000 and from 1 to 10,000 ng/mL for nicotine and
cotinine, respectively. All data were corrected for
recovery ef±ciencies.
Statistical analysis
Results did not follow a normal distribution func-
tion; therefore, the non-parametric Mann-Whitney U
test and Kruskal-Wallis test were used to determine
signi±cant differences. Pearson’s correlation coef-
±cient analysis was calculated to establish possible
relationships between TMN frequency, BN cells with
MN, NDI and CPBI parameters and nicotine and co-
tinine levels. Data were analyzed with the MINITAB
statistics program, version 13.2.
Intergroup variations in MN Frequency and cel-
lular cycle alterations
The genotoxic effect in smokers represented by
the number of binuclear cells with micronuclei (BN
cells with MN) and total micronuclei (TMN), as well
as the alteration of the cellular cycle measured with
nuclear division (NDI) and cytokinesis proliferation
block (CPBI) indexes, is shown in
Table I
. Smokers
exhibited mean values for BN cells with MN and
TMN signi±cantly smaller when compared with
the control group, as well as a faster cellular cycle
expressed as an increment in NDI and CPBI. Control
values showed a delay in cellular cycle regarding
smokers as well as in relation to the value equal to
2 reported for both indexes in healthy persons unex-
posed to genotoxic agents (Surallés 1995).
Possible DNA damage in smokers was analyzed
in the subgroups according to age and gender. Results
are shown in
Table II
. Male and female smokers ex-
hibited lower frequencies in BN cells with MN and
TMN compared to the control group. Regarding the
cellular cycle, female smokers showed higher mean
values and signi±cant differences for both indexes,
BN with MN
8.81 ² 6.59*** 9.94 ² 7.44*** 1.69 ² 0.27*** 1.63 ² 0.22**
16.25 ² 7.85
17.31 ² 8.76
1.48 ² 0.25
1.44 ² 0.21
Values are expressed as mean ² standard deviation
For every 1000 cells counted
The Mann-Whitney U test was employed; signi±cance was set at p < 0.01 (**) and
p < 0.001 (***)
C. Calderón-Ezquerro
et al.
NDI and CPBI, than controls. On the other hand, male
active smokers showed no signifcant diFFerences For
these indexes when compared with their controls.
Considering that the age interval in the smoker
group was wide (23 to 74 years old), it was divided into
three subgroups. Individuals From 23 to 40 years oF age
(smokers and controls) showed no signifcant diFFer-
ences among them in any oF the assessed parameters.
Smokers From 41 to 57 years old showed signifcant
diFFerences in the number oF BN cells with MN and
TMN, as well as signifcant increases in NDI and CPBI
with regard to the controls, whereas signifcant diFFer-
ences were only Found between smokers and controls
in both indexes in the 58 to 73 year-old subgroup.
Table III
shows the cytogenetic analysis For
smokers according to nicotine and cotinine concen-
trations Found in urine samples. Light and moderate
smokers exhibited signifcantly smaller values in
the number oF BN cells with MN and TMN, unlike
heavy smokers. Only moderate smokers showed a
signifcant diFFerence in CPBI in regard to controls,
though in the three smokers subgroups a higher mean
value was observed in NDI and CPBI variables when
compared with the corresponding controls.
NDI and CPBI were correlated to BN
cells with MN and TMN. ±or heavy smokers a cor-
relation coeFfcient equal to
0.758 (p = 0.049) was
Found between NDI and BN cells with MN; simi-
larly, a correlation coeFfcient equal to
0.764 (p =
0.046) was Found between NDI and TMN. ±or the
second index (CPBI), correlation coeFfcients equal
0.764 (p = 0.046) and
0.799 (p = 0.031) were
Found between this index and BN cells with MN and
TMN, respectively. ±or light and heavy smokers the
BN with MN
Total MN
10.44 ² 7.92*
12.18 ² 8.72*
1.66 ² 0.23*** 1.61 ² 0.19***
16.50 ² 8.56
18.06 ² 9.73
1.41 ² 0.20
1.37 ² 0.17
6.71 ² 3.60*** 7.07 ² 4.10*** 1.73 ² 0.32
1.65 ² 0.25
15.93 ² 7.13
16.36 ² 7.59
1.58 ² 0.27
1.53 ² 0.23
23-40 years old
7.70 ² 5.06
8.70 ² 5.77
1.66 ² 0.33
1.60 ² 0.27
14.90 ² 10.65
16.60 ² 12.27
1.55 ² 0.27
1.50 ² 0.22
41-57 years old
8.75 ² 5.79*** 10.13 ² 6.74**
1.71 ² 0.24*
1.65 ² 0.20*
18.25 ² 6.80
19.19 ² 7.40
1.52 ² 0.21
1.48 ² 0.18
58-73 years old
10.83 ² 10.72
11.50 ² 11.86
1.68 ² 0.24*
1.62 ² 0.19*
10.83 ² 3.92
11.33 ² 4.37
1.37 ² 0.27
1.34 ² 0.23
Values are shown as mean ² standard deviation
For every 1000 cells counted
The Mann-Whitney U test was employed setting the signifcance at p< 0.05 (*), p<0.01 (**), and p < 0.001 (***)
BN cells with MN
6.38 ² 2.88+*** 7.25 ² 3.33*** 1.74 ² 0.22 1.67 ² 0.17
16.13 ² 10.63
17.13 ² 11.80
1.49 ² 0.25 1.45 ² 0.21
7.75 ² 6.18***
8.63 ² 7.78*** 1.76 ² 0.30 1.69 ² 0.24***
15.88 ² 5.87
17.63 ² 6.89
1.49 ² 0.23 1.46 ² 0.18
7.63 ² 5.21
9.13 ² 6.24
1.78 ² 0.27 1.69 ² 0.22
13.38 ² 8.99
14.00 ² 9.64
1.56 ² 0.23 1.51 ² 0.19
Values are shown as mean ² standard deviation
±or every 1000 cells counted
The Mann-Whitney U test was used with signifcance set at p < 0.001 (***)
correlation coef±cients between the indexes and MN
frequencies were not signi±cant.
Intragroup variations in MN frequency and cel-
lular cycle alteration in smokers
The frequency of BN cells with MN and TMN,
as well as cellular cycle alterations evaluated by the
indexes (NDI and CPBI), was also assessed in an
intragroup manner. That is, the possible in²uence
of gender, age, number of consumed cigarettes (ac-
cording to nicotine levels) and time of exposure to
tobacco smoke was evaluated among the subjects in
each group (smokers and controls).
The outcome of the smoker group (
Table IV
showed that the only statistical difference encoun-
tered according to gender was that women presented
a signi±cant (p < 0.05; Kruskal-Wallis test) increase
in the frequency of total MN cells (12.17 ³ 8.72) in
regard to men (7.07 ³ 4.10).
Control groups in this study were classi±ed by age
and gender. The analysis of these variables showed
no signi±cant differences in any of the evaluated
Smokers were also divided into groups according
to gender, age, time with smoking habit and daily
cigarette intake to determine the differences in nico-
tine and cotinine levels in light, moderate and heavy
smokers (
Table V
). No signi±cant differences were
found between genders, subjects younger or older
than 45 years old, and time of smoking habit (more
or less than 25 years). However, when smokers were
divided into light, moderate and heavy, signi±cant
differences (p < 0.001) were found, considering that
recorded concentrations of these exposure biomarkers
re²ected the real number of cigarettes smoked daily.
Due to the small size of the sample and the resolu-
tion limits of the test employed, the analysis in control
group could not be undertaken.
Table VI
show the Pearson’s correlation coef-
±cients and signi±cance values for nicotine and
cotinine levels in smokers regarding other assessed
parameters. No relationship was established between
any of the assessed variables.
Likewise, in separating the group of smokers in
light, moderate and intense it was found that Pear-
son’s correlation coef±cients obtained comparing
nicotine and cotinine levels regarding the parameters
assessed (BN cells with MN, TMN, NDI, CPBI) and
some general characteristics (age, years of smoking
habit, daily cigarette intake), no relationship was
established between any of the assessed variables.
Nicotine and cotinine levels in evaluated subjects
From the 32 smokers participating in the study,
only 24 urine samples were collected. These samples
were used for the nicotine and cotinine quanti±cation.
Five urine samples were collected from controls.
Results are shown in
Table VII
Nicotine and cotinine values for controls were
below the mass spectrometer limits of resolution
(10 ng/mL and 100 ng/mL for nicotine and cotinine,
respectively). Hence, the exact minimum value could
not be determined for either of them. Signi±cant dif-
ferences were found (p < 0.05) between smokers and
nonsmokers. Smokers exhibited high concentrations
of both exposure biomarkers.
Nicotine and cotinine levels in urine of smokers
are shown in
Table VIII
BN cells with MN
10.44 ³ 7.92
12.17 ³ 8.72* 1.66 ³ 0.23
1.61 ³ 0.19
6.71 ³ 3.60
7.07 ³ 4.10
1.73 ³ 0.31
1.65 ³ 0.25
Age (years old)
7.70 ³ 5.06
8.70 ³ 5.77
1.66 ³ 0.33
1.60 ³ 0.27
8.75 ³ 5.79
10.13 ³ 6.74
1.71 ³ 0.24
1.65 ³ 0.20
10.83 ³ 10.72
11.50 ³ 11.86
1.68 ³ 0.24
1.62 ³ 0.19
Daily cigarette intake
< 30
8.15 ³ 5.18
8.85 ³ 5.44
1.66 ³ 0.29
1.60 ³ 0.24
> 30
9.92 ³ 8.59
11.75 ³ 9.96
1.75 ³ 0.22
1.68 ³ 0.18
Time with smoking habit
< 25
8.67 ³ 5.56
9.47 ³ 5.94
1.64 ³ 0.30
1.58 ³ 0.24
> 25
8.94 ³ 7.55
10.35 ³ 8.72
1.74 ³ 0.24
1.67 ³ 0.20
Values are mean ³ standard deviation
For every 1000 cells counted; Mann-Whitney U test was employed
Kruskal-Wallis test was used with signi±cance set at p < 0.05 (*)
C. Calderón-Ezquerro
et al.
MN frequency and cellular cycle alterations
Binuclear cells with micronuclei and total micro-
. The amount of MN found in smokers (
) was signiFcantly smaller than that in controls.
Moreover, this difference became more specific
when smokers were divided into ranges according
to age and gender, particularly when comparisons
were made between subjects 41-57 years old, unlike
comparisons involving smokers older than 58 years
of age (
Table III
). These results agree with Barale
. (1998) and Bonassi
et al
. (2003) in the sense that
tobacco smoking exerts a decreasing effect on MN
frequency; this study’s result is close to the historic
mean value of binuclear cells with micronuclei 7.8 ±
5.2 for every 1000 cells counted (Surallés and Nata-
rajan 1997). However, this marked reduction (almost
50 %) has not been previously reported. Barale
. (1998) and Bonassi
et al
. (2003) found 16 % and
from 3 to 10 % reductions, respectively.
By assessing genotoxicity in smokers through
nicotine in urine (
Table I
) and classifying them as
Nicotine (ng/mL)
Cotinine (ng/mL)
1088 ± 1084
1269 ± 581
1103 ± 581
1728 ± 1215
Age (years old)
< 45
1229 ± 1219
1120 ± 774
> 45
1014 ± 722
1696 ± 979
Smoking time (years)
< 25
1405 ± 1406
1338 ± 605
> 25
832 ± 739
1600 ± 1155
Daily cigarette intake
1-19 (light)
212 ± 159***
726 ± 458*
20-29 (moderate)
1010 ± 246***
1405 ± 200*
>30 (heavy)
2062 ± 851***
2309 ± 1086*
Values are mean ± standard deviation
²or every 1000 cell counted, using Mann-Whitney U test
Kruskal-Wallis test was employed with signiFcance set at
p <0.001 (*)
BN with
Years of
smoking habit
cigarette intake
SigniFcance values
SigniFcance values
Nicotine (ng/mL)
Cotinine (ng/mL)
983.00 ± 873.00***
1442.00 ± 905.00***
2.00 ±
0.00 ±
Values are expressed as mean ± standard deviation
*** The Mann-Whitney U test was employed; signiFcance was
set at p< 0.001
Nicotine level
Cotinine level
<10 - 475
<100 - 1187
476 - 1450
118 - 1743
1451 - 4110
1744 - 4946
light, moderate and heavy smokers, signifcant di±-
±erences were ±ound in the case o± BN cells with
MN and TMN ±or light and moderate smokers, but
not ±or heavy smokers (
Table IV
). The general trend
was a lower ±requency in these kind o± cells regard-
ing controls. These results are partly in accordance
with the fndings by Bonassi
et al
. (2003) in which
smokers with a daily intake lower than 20 cigarettes
showed a decrease in MN ±requency, whereas a non-
signifcant increase ±or subjects smoking between 20
and 29 cigarettes was reported. In smokers beyond
30 cigarettes, a signifcant increase was ±ound in BN
cells with MN and TMN, as long as these subjects
were not exposed to mutagenic or carcinogenic
agents; when exposed to such agents this increment
was not present.
All subjects in this study live in México City,
where they are exposed to an atmosphere containing
a large variety o± gaseous and particulate pollutants
et al
. 1999, Raga
et al
. 2001,
et al
. 2002). Thus, smokers living here are not
only a±±ected by their own tobacco smoke, but also
by environmental tobacco smoke (ETS) and a mix-
ture o± air-suspended chemical compounds. Several
authors have demonstrated various genotoxic e±±ects
and health damage. For example, Rubio
et al
. (1990)
suggest a possible synergistic e±±ect between tobacco
smoke and atmospheric pollution resulting in a pro-
cess harm±ul to respiratory ±unction. Studies o± the
e±±ects o± air pollutants on
Drosophila melanogaster
in México City have demonstrated the genotoxicity
o± polycyclic aromatic hydrocarbons (PAHs) and
their nitro derivatives (Delgado-Rodríguez
et al
1995), as well as that o± organic extracts o± airborne
particles (Delgado-Rodríguez
et al
. 1999). Similarly,
et al
. (2000),
in a genetic monitor-
ing study o± airborne particles using the Ames test
Salmonella typhimurium
), demonstrated that com-
pounds such as PAHs contained in urban atmospheres
exhibit mutagenic activity. Another study about
the genotoxic, cytokinetic and cytotoxic e±±ects o±
extracts ±rom airborne particles collected in México
City suggested that the extent o± the changes caused
by pollutants was partly dependent on the seasonal
weather. The highest genotoxicity rates (SCE) were
±ound in November (cool and dry); at this time o± the
year, all 15 polycyclic aromatic hydrocarbons studied
were present in the organic material extracted ±rom
air samples (Calderón-Segura
et al
. 2004). Recently,
a study by Roubicek
et al
. (2007) investigated the
ability o± chemically-characterized water and PM
organic/soluble extracts ±rom two areas o± México
City to induce micronuclei in a human epithelial cell
line; the association between PM chemical charac-
teristics and genotoxicity was also evaluated. The
authors reported that both industrial and residential
extracts induce a signifcant concentration-related
increase in micronucleus ±requency. Comparative
analyses between micronucleus induction and chemi-
cal compounds showed that cadmium and PAHs
exhibit a signifcant correlation with micronucleus
induction. E±±ects observed in this study emphasize
the risk o± PM exposure in México City.
On the other hand, Calderón-Ezquerro
et al
(2007) assessed the genotoxicity in tobacco smok-
ers living in México City and ±ound a delay in cell
proli±eration kinetics (CPK), as well as a decrease in
the replication index (RI). Cytokinetic e±±ects were
mainly detected in heavy and moderate smokers.
These results, common in all “healthy” smokers in
this study, are similar to those o± other authors who
studied diverse xenobiotic agents and concluded
that synergistic and/or potential e±±ects may be
involved in producing cytokinetic and genotoxic
e±±ects (Grandjean
et al
. 1983, Hedner
et al
. 1983,
et al
. 2000, Duydu
et al
. 2001, Palus
. 2003). These fndings suggest that smokers liv-
ing in places with high atmospheric contamination
levels are subject to a greater risk ±rom smoking due
to genotoxic, mutagenic and/or carcinogenic agents.
Hence, smokers evaluated in this study were not
only exposed to inhaled tobacco smoke, but also to
a complex mixture o± chemical compounds. Accord-
ing to Bonassi
et al
. (2003), the association between
the increase in MN ±requency and heavy smoking
in subjects unexposed to carcinogenic or mutagenic
environments may account ±or the outcome o± lower
MN ±requency in smokers than in controls. It could
also explain why heavy smokers exposed to various
pollutants were incapable o± expressing a higher MN
±requency compared with controls.
Studies in several countries reported lower ±re-
quencies o± MN in the control groups than those
reported in this study (Barale
et al
. 1998, Karahalil
. 1998, Zao
et al
. 1998, Burgaz
et al
. 1999, Pitarque
et al
. 1999, Baier
et al
. 2002, Palus
et al
. 2003).
et al
. (2003) state that “exposure to
genotoxins may stimulate the expression o± DNA
repair genes or detoxifcation mechanisms that are
also important in attenuating the genotoxic e±±ects o±
chemicals in cigarette smoke.” Similarly, the results
obtained in light and heavy smokers compared with
those o± nonsmokers, in whom a decrease in MN
±requency was ±ound, coincide with other reports.
These studies consider that a ±ew cigarettes per day
may stimulate an adaptive response (cell protective)
C. Calderón-Ezquerro
et al.
causing a decrease in MN frequency, whereas con-
tinued exposure to mutagens/carcinogens may induce
resistance to further DNA damage (Benner
et al
. 1992,
Gourabi and Mozdarani 1998, Rothfub
et al
. 1998,
et al
Control subjects exhibited no differences regard-
ing gender or age when compared with smokers.
However, they showed higher frequencies in BN cells
with MN and TMN; in some cases twice the values
(16.25 ± 7.85 and 17.31 ± 8.76, respectively) of
smokers (8.81 ± 6.59 and 9.94 ± 7.44, respectively).
It has been reported that factors such as gender and
age may account for 32 % (male) to 48 % (female)
variations in MN frequency (Fenech 1998a). Howev-
er, this is not the case in this study for age and gender
distributions were identical for both study groups. By
looking at the results summarized in
Tables II
subgroups of older ages did not present a signi²cant
increase in MN frequency, which may re³ect the
small sample size. The comparison of this study’s
results with those of other reports (Barale
et al
. 1998,
et al
. 1998, Zhao
et al
. 1998, Pitarque
et al
1999, Baier
et al
. 2002, Bonassi
et al.
2003, Palus
. 2003) indicates that MN values found in this study
are only surpassed by those in Lohani
et al
. (2002)
for the nonsmoker group (62.48 ± 5.96).
Similarly, when comparing MN frequencies in
this study with those reported in research on tobacco
smoking associated with genotoxic agents such as
PAHs (Karahalil
et al
. 1998), contaminants from
road traf²c (Zhao
et al
. 1998), airports (Pitarque
et al
1999), gas stations (Buckvic
et al
. 1998), leather fac-
tories (Somorovska
et al
. 1999), greenhouses (Lucero
et al
. 2000) and contaminated cities (Romanova and
Bezdrobna 2001),
it was found that MN frequency in
controls was similar to that in nonsmokers exposed
to diverse genotoxic agents. The same results were
obtained in studies where subjects had been exposed
to asbestos (Lohani
et al
. 2002), cadmium and lead
et al
. 2003), epichlorhidrine (Hindsă┐landin
. 1997), radiation (Chang
et al
. 1997) and low fre-
quency magnetic ²elds (Scar²
et al
. 1997). In cases
where individuals were subjected to intense exercise
et al
. 1997)
or suffered diseases such as cancer
et al
. 1999), leprosy (Kalaiselvi
et al
or alcoholism (Maffei
et al
. 2002), it was also found
that MN frequency in controls was similar to that in
nonsmokers exposed to genotoxic agents.
The increase in MN frequency in controls,
which coincides more with nonsmokers exposed
to genotoxic agents than with those living in clean
environments –with a minimum amount of pollut-
ants–, suggests a possible genotoxic effect, given
that a large amount of these pollutants have known
oxidative properties which may induce stress in cells
in contact with them. Effects of oxidative stress on
cells are varied; these include induction of sister
chromatid exchange (SCE), chromosomal aberra-
tions and reduction of cellular proliferation (Lioi
. 1998, Burgaz
et al
. 2002).
Furthermore, oxidative
stress causes decrease in the lymphocyte response
to mitogenic agents (Bechoua
et al
. 1999), rupture
of a strand of DNA (Chen
et al
. 2003), formation of
MN cells in the buccal mucosa in children (Lahiri
et al
. 2000) and splenocysts and ²broblasts in mice
et al
. 1999).
In addition, it has been found
that elder people are more sensitive to oxidative stress
than young persons (López-Hellin
et al
. 1998).
Several studies have shown that exposure to envi-
ronmental pollutants present in urban environments
contribute to the genotoxicity and carcinogenicity in
humans mainly exposed to PAHs. There is evidence
linking PAH metabolism with the generation of reac-
tive oxygen species (ROS) following oxidative stress,
leading to DNA damage (Flowers
et al
. 1997, Bolton
et al
. 2000, Singh
et al
. 2007).
Likewise, exposure to
breathable particulate matter can result in the in³ux of
alveolar macrophages, which in turn can generate free
radicals leading to oxidative stress (Li
et al
. 1997). Stud-
ies carried in México have shown that environmental
pollution causes oxidative stress (Sánchez
et al
. 2004)
leading to genotoxic damage in the nasal mucosa of
children (Calderón-Garcidueñas
et al
. 1999) which
in turn may reduce cellular proliferation (Calderón-
et al
. 2004) and enzymatic activity associated
with combating oxidative stress. This is the case with
superoxide dismutase which is reduced to half its normal
concentration after just 16 weeks of exposure (Medina-
et al
. 1997).
The former suggests that living in
contaminated cities can lead to an elevated frequency of
micronuclei, as happens in Kiev, where Romanova
. (2001)
reported a value of 10.5 in TMN frequency
in a nonsmoker sample with an average age of 42 years.
Similarly, Roubicek (2007) reported that in México
City exposition to particle extracts (PM
) present in
the air induced a signi²cant increase in micronucleus
frequency in a human epithelial cell line.
On the other hand, and in spite the fact that smok-
ers in the latter study live in México City and are
exposed to contamination, their low MN frequency
may be due to their erythrocyte protective properties.
These cells have elevated concentrations of reduced
glutathione (GSH) and catalase (Sierra
et al
. 2004).
Erythrocytes possess large amounts of antioxidizing
agents; it has been found that these kind of cells pres-
ent more reduced glutathione and catalase in smok-
ers than in nonsmokers. Considering that these cells
are constantly exposed to oxidative stress (caused
by tobacco smoke) and that cells are endowed with
adaptive mechanisms, a higher yield of these protec-
tive enzymes is produced as a consequence (Sierra
et al
. 2004).
This adaptive process has been previously de-
scribed by Rothfub
et al
. (1998). They discovered
that hyperbaric exposure with 100 % oxygen during
three 20-minute periods induces MN formation in
lymphocytes that is gradually reduced to levels lower
than baseline values at the beginning of the treatment.
There is evidence that enzymes such as 8-oxo-7,8-
dihydrodeoxyguanosine are involved in this process
and capable of repairing DNA adducts originated by
oxidizing agents. These enzymes have been found in
mammals and their function is to remove oxidized
bases in DNA. The amount and ef±ciency of these
enzymes are enhanced after exposure to reactive
oxygen species (Klaunig and Kamendulis 2004). Be-
sides, exposure to tobacco smoke increases reduced
glutathione levels in the bloodstream (Oesch
et al
1994) and even though tobacco smoke has oxidative
properties, it causes no reduction in the concentra-
tions of the main enzymes (superoxide dismutase,
catalase and glutathione peroxidase) involved in
the protection against this type of stress; however,
a reduction is present with age (Bolzan
et al
. 1997).
Some components in tobacco smoke, such as hy-
droquinone, are capable of reducing MN formation.
This compound, found in large quantities in tobacco
smoke, reduces MN frequency signi±cantly in bone
marrow of mice when they are exposed to a direct
antioxidizing effect (O’Donoghue
et al
. 1999).
Yildiz (2004) pointed out that a low nicotine
concentration can prevent oxidative stress, but high
concentrations induce it. An adequate adaptive re-
sponse in erythrocytes of smokers combined with the
presence in tobacco smoke of compounds capable of
reducing MN frequency makes viable the hypothesis
that tobacco smoke can in fact reduce MN frequency
in light and moderate smokers.
In addition, MN frequency is markedly sensitive
to other factors such as diet. Inclusion of vitamins C
and E, β-carotenes and zinc in food may signi±cantly
reduce MN frequency (Konopacka and Rzeszowska-
Wolny 2001); even amounts as small as 700 µg of
folic acid and 2.5 µg of vitamin B
may reduce MN
frequency in 25 % (Fenech 1998b). Polymorphisms
in gene GSTT1 are another variable affecting MN
frequency. Individuals lacking this gene or having
altered versions are more susceptible to have higher
MN frequencies, whether they have been exposed
to genotoxic agents or not; this is due to a de±cient
production of glutathione-S-transferase (Vlachodimi-
et al
. 1997). These factors are dif±cult to
control; hence their effect in this study is unknown,
though it is highly improbable that they would have
preferentially acted on a particular group. Moreover,
the participants in this study have diverse occupation-
al activities, educational levels and socioeconomic
backgrounds; the only common characteristic that
enabled their classi±cation into groups –smokers
and subjects in contact with tobacco smoke only oc-
casionally– is that all are inhabitants of México City.
In summary, results related to MN induction
indicate that subject controls have been exposed to
the action of one or more MN inducing agents that
elicit high MN frequencies. Tobacco smoke has the
capacity of: a) avoiding an increment in MN fre-
quency in active smokers (light and moderate) by the
activation of an adaptive response in erythrocytes that
enhances their antioxidant properties, and b) inducing
genotoxic and cytotoxic responses which reduce the
dividing rate of lymphocytes and therefore hinder
MN expression and observation.
Nuclear division and cytokinesis proliferation
block indexes
According to
Table I
, results associated with the
progression of the cellular cycle showed signi±cant
differences between smokers and controls consider-
ing NDI and CPBI. The corresponding control group
(N = 32) for smokers presented the following val-
ues: 1.48 ² 0.25 and 1.44 ² 0.21 for NDI and CPBI,
respectively. These results indicate that the cellular
cycle was faster for smokers than their controls.
These results suggest that tobacco smoke exerts an
accelerating effect on the cellular cycle in smokers.
This ±nding agrees with that of Woggner and Wong
(1994) and Argentin and Cicchetti (2004), who report
that nicotine stimulates cellular proliferation in some
cases (gingival ±broblasts and cervical epithelium,
respectively). However, other studies support the
opposite hypothesis, that is, tobacco smoke does
not accelerate the cellular cycle, but can even cause
a delay. Pastor
et al
. (2002) speci±cally report a
decrease in CPBI associated with tobacco smoking.
et al
. (1999) and Palus
et al
. (2003) show
NDI and CPBI values close to 2 in smokers as well
as in controls; no signi±cant differences resulted in
any of the cases. As mentioned before, a value equal
to 2 occurs when there are no external factors capable
of altering the cellular cycle. Scar±
et al
. (1997),
et al
. (2000) and Pastor
et al
. (2002), when
evaluating genotoxic agents, found CPBI values for
C. Calderón-Ezquerro
et al.
controls higher than those found in this study (1.79
± 0.15, 1.82 ± 0.15 and 1.52 ± 0.02, respectively).
On the other hand, Maffei
et al
. (2002) obtained a
lower mean value (1.37 ± 0.05), which might show
a cellular delay in the control group of that study.
There is evidence demonstrating that exposure
to atmospheric pollutants decreases the lymphocyte
response to phytohemagglutinin (Tomei
et al
. 2004)
and that tobacco smoking as well as chronic exposure
to nicotine reduce cellular proliferation (Sopori
et al
1993, Sánchez 2004, Yildiz 2004, Calderón-Ezquerro
These results suggest the existence of a delay
in the cellular cycle of smokers and controls; the lat-
ter might occur due to the environmental pollution
in México City.
Subjects in this study were exposed to high levels
of environmental pollution. Therefore, the Fnding
that NDI and CPBI values are signiFcantly lower for
control groups when compared with those of smokers
indicates that tobacco smoke may interact with pol-
lutants or agents responsible for the cellular delay in
such a way that its effect is reduced in smokers. The
mechanism of action is not known but perhaps among
the more than 400 chemical compounds comprising to-
bacco smoke, one or more exhibit antagonistic effects
et al
. 2007) toward components
borne in the atmosphere and/or may have genotoxic
effects, delay the cellular cycle, or even establish a
competition among components from both sources
once they are inside the body. Moreover, there is a
possibility that components coming from tobacco
smoke present a higher afFnity toward cells, thus ren-
dering less harmful. This hypothesis is supported by
the strong negative correlation between both indexes
(NDI and CPBI) and MN frequency. The correlation
coefFcient between NDI and BN cells with MN was
0.758 (p = 0.049), whereas for NDI and TMN it
0.764 (p = 0.046). As for the CPBI, it showed
a correlation coefFcient of
0.764 (p = 0.046) with
BN cells with MN and
0.799 (p = 0.031) with TMN.
These values were derived from the heavy smok-
ing group only. The former suggests that subjects
consuming more than 30 cigarettes a day present a
higher frequency of MN, and concurrently a higher
cellular delay. Although, a possible reason for the
lower MN frequency in smokers could be that heavy
damaged cells might have failed to divide or died
during their
culture, so it was not possible
the induction of MN.
This situation may not be provoked by tobacco
smoke, since no correlation was established among
nicotine and cotinine concentrations with BN cells
with MN and TMN in this type of smokers. In light and
moderate smokers, correlation coefFcients between
indexes and MN frequencies were not signiFcant.
Nicotine and cotinine
QuantiFcation of nicotine and cotinine was simi-
lar to that reported by the Environmental Protection
Agency (EPA 1997), who issued values of 1749 and
1391 ng/mL for nicotine and cotinine in urine of ac-
tive smokers. In this study the corresponding values
are 983 and 1442 ng/mL, respectively (
Table I
Our results indicate that gender, age and time
of smoking (
Table VI
) are not factors modifying
nicotine and cotinine concentrations. This is not in
agreement with Prather
et al
. (1993), who report
higher excretion levels of nicotine and cotinine for
women; Swan
et al
. (1993), who specify a positive
correlation of cotinine levels with age, and Pardell
(1996), who establishes a higher nicotine excretion
in chronic smokers.
There is evidence showing that nicotine me-
tabolism is affected by various factors such as food
regimes, gender, ethnic group, tar content, type of
cigarette and even genetic polymorphisms (Patterson
et al
. 2003). These aspects were not assessed in this
study and may participate in the outcome of this re-
search. Similarly, analyses were carried out with urine
samples collected early in the morning; this means that
nicotine and cotinine concentrations were indicative of
the number of cigarettes smoked before bedtime rather
than the number smoked throughout the entire day.
Nevertheless, the daily consumption of cigarettes
is not a good indicator of the nicotine content en-
tering the body. ²actors such as type of cigarette,
number, quality of inhalations, presence of additives
(menthol favors deeper inhalations, for example),
tar content, whole or partial consumption of ciga-
rettes, all contribute signiFcantly to the amount of
absorbed nicotine (Patterson
et al
. 2003). In a study
undertaken in Germany in 1998 (Heinrich
et al
2004), the contents of nicotine and cotinine were
assayed in 5000 participants. The authors concluded
that the number of cigarettes consumed accounted
for only 42 % of the differences found for nicotine
and 51 % for cotinine. Therefore, other factors are
involved in nicotine metabolism.
In this study, correlation values (
Table VII
between the number of cigarettes per day and nico-
tine and cotinine concentrations were low and non
signiFcant (0.90 and
0.44 for nicotine and cotinine,
respectively). This could mean that there is no cor-
relation between the previously mentioned vari-
ables. On the other hand, several reports show the
following correlation values for cotinine in urine:
0.24 (Wall
et al
. 1988), 0.54 (Suter
et al
. 1995),
0.62 (Jacob
et al
. 1988) and 0.67 (Fried
et al
. 1995).
However, Swan
et al
. (1993) found that cotinine
values in the saliva of smokers followed a non-linear
behavior, mainly in persons with moderate cigarette
consumption. As a result, the experimental popula-
tion in this study was divided in subgroups –light,
moderate and heavy– to render a better correlation
coef±cient for light and heavy smokers; for heavy
smokers this coef±cient was reduced. The same
scheme was followed in this study for nicotine and
cotinine values in urine.
The lack of a signi±cant correlation between daily
cigarette consumption and nicotine and cotinine lev-
els may be due to unreliable information provided by
smokers, with the possibility of being underestimated
in some cases and overestimated in others.
In general, no association was established among
evaluated cytogenetic variables (BN cells with MN,
TMN, NDI and CPBI) and nicotine and cotinine
contents in smokers. However, when the information
was analyzed according to subgroups –light, moder-
ate and heavy–, an increase in correlation coef±cients
was found.
The authors thank the Anti-tobacco Clinic of the
Instituto Nacional de Enfermedades Respiratorias
for their collaboration in allowing us to work with
smoker patients. We are also grateful to Miguel
Ángel Meneses for his technical assistance, and
Marcela Sánchez for revising the English version
of this manuscript.
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