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Mutagenesis vol. 26 no. 3 pp. 385–391, 2011 doi:10.

Advance Access Publication 30 December 2010

Seasonal variations in the levels of PAH–DNA adducts in young adults living

in Mexico City

W. A. Garcı́a-Suástegui, A. Huerta-Chagoya, Introduction

K. L. Carrasco-Colı́n, M. M. Pratt1, K. John1, P. Petrosyan,
One of the most densely populated cities in the world, the
J. Rubio, M. C. Poirier1 and M. E. Gonsebatt*
Mexico City Metropolitan Area (MCMA), has 20 million
Departamento de Medicina Genómica y Toxicologı́a Ambiental, Instituto de inhabitants, representing 18.6% of the national total according
Investigaciones Biomédicas, Universidad Nacional Autónoma de México, A.P. to the 2nd Count of Population and Housing (1). The MCMA
70-228, Ciudad Universitaria 04510, Mexico City, México and 1Carcinogen-
DNA Interactions Section, LCBG, Bldg.37 Rm 4032, National Cancer is an elevated basin 2240 m above sea level. At this altitude,
Institute, National Institutes of Health, 37 Convent Dr. MSC-4255, Bethesda, 23% less oxygen is available than at sea level, which makes
MD 20892, USA combustion less efficient and produces more polycyclic
* aromatic hydrocarbon (PAH) pollutants (2). Additionally, the
To whom correspondence should be addressed. Tel: þ52 55 56229179; Fax:
þ52 55 56229182; Email MCMA is surrounded on the south, west and east by mountains
that inhibit the dispersion of pollutants. Climate conditions
Received on May 10, 2010; revised on October 29, 2010; vary during the year, with precipitation occurring mainly
accepted on November 4, 2010
between the months of May and October (rainy season) and
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous very scarcely from November until May (dry season). Thus,
components of polluted air. The Mexico City Metropolitan critical air pollutants, such as volatile organic compounds

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Area (MCMA), one of the most densely populated areas in (VOC), which include PAHs and particulate matter (PM)
the world, is 2240 m above sea level. At this altitude, less emissions, increase during the dry season and decrease during
oxygen is available, making combustion less efficient and the rainy season (2).
therefore producing more PAH pollutants. According to the Studies designed to investigate the health risks associated
Automatic Monitoring Network in Mexico City (RAMA, for with PM emissions in the MCMA have reported that an increase
its Spanish initials; of 10 lg/m3 of PM caused an increment in mortality of 1.83 or
maciontecnica/index.php?opcion=5&opciondifusion_bd=90), 1.48% for particles with aerodynamic diameters of 10 lm
which performs environmental monitoring, the critical air (PM10) or 2.5 lm (PM2.5), respectively (3,4). In addition,
pollutants in Mexico City are ozone and particulate matter epidemiological studies performed in US cities suggest an
(PM). PM emissions increase during the dry season (winter increase in lung cancer risk in association with exposure to urban
to spring) and decrease during the rainy season (summer to air pollutants, particularly PM10 or PM2.5 (5).
autumn). The bioactivation of some PAHs produces reactive Particulate emissions are by-products of fuel combustion,
metabolites that bind to DNA, and the presence of elevated motor vehicle use and industrial processing. Organic extraction
levels of PAH–DNA adducts in tissues such as blood and analysis of PM in the southwest (SW) region of Mexico
lymphocytes represents an elevated risk for the development City have shown that benzo[ghi]perylene, coronene and
of cancer. We have compared the levels of PAH–DNA indeno[1,2,3-cd]pyrene are the most abundant PAHs of the
adducts and the percentage of cells with chromosomal 17 analysed (6). These observations and those from samples
aberrations (CWAs) using a matched set of peripheral blood obtained 4 years later (7) indicate that the main emission
lymphocytes obtained on two separate occasions from young sources for PAHs in the airborne particle phase in the MCMA
non-smoking inhabitants of the MCMA (n 5 92) during are vehicles utilising the combustion of gasoline and diesel and
the 2006 dry season and the following rainy season. PAH– not the combustion of wood.
DNA adducts were analysed using the r7, t8-dihydroxy-t-9, PAHs include a wide variety of genotoxic agents, result from
10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)–DNA the incomplete combustion of organic matter, such as wood,
chemiluminescence immunoassay (CIA). The percentages diesel or tobacco, and are ubiquitous components of environ-
of CWA were determined in cultured lymphocytes from the mental air pollution. Some PAHs have been classified as
same individuals. Both DNA adduct levels and chromosomal carcinogenic agents by the US Environmental Protection
aberrations were tested for correlation with lifestyle and the Agency (8) and the International Agency for Research on
polymorphisms of cytochromes P450 CYP1A1 and CYP1B1 Cancer (9). The weight of experimental evidence indicates that
as well as glutathione-S-transferases GSTM1 and GSTT1. binding of carcinogenic PAHs to DNA is a critical initiating
The levels of PAH–DNA adducts were significantly higher (P event in the process of tumour formation (10). PAH–DNA
< 0.001) in the dry season (10.66 6 3.05 per 109 nt, n 5 92) adducts formation in white blood cells is considered to be
than during the rainy season (9.50 6 2.85 per 109 nt, n 5 92) a biomarker of exposure to these compounds as well as an
and correlated with the seasonal levels of particulate matter indicator of cancer risk (11–15).
with a diameter of £10 mm (PM10). The percentage of CWA In previous studies, PAHs present in airborne PM collected
was not seasonally related; however, significant associations from industrial and residential regions of the MCMA were
between the number of risk alleles and adduct levels in the dry significantly associated with concentration-related induction of
(R 5 0.298, P 5 0.048) and in the wet seasons (R 5 0.473, P 5 micronuclei in A549 human alveolar epithelial cells (16). In
0.001) were observed. addition, the direct-acting mutagenicity observed in Salmonella
Ó The Author 2010. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
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W. A. Garcı́a-Suástegui et al.

typhimurium strains TA98 and YG1021 correlated with the Extraction of DNA
monthly concentrations of PM10 in SW Mexico City. Emission DNA was extracted from buffy coat cells as described by Daly et al. (29). DNA
of direct-acting mutagens occurred mainly in the coldest was dissolved in TE (10 mM Tris–HCl and 1 mM EDTA, pH 7.4) and stored at
20° C until use for the quantification of PAH–DNA adducts and genotyping.
months of the year, and December showed the highest
mutagenicity (6). Detection of DNA adducts
PAHs require metabolic activation to interact with DNA and PAH–DNA adducts were analysed using the highly sensitive 10-oxy-7,8,9,10-
form adducts. This activation is mainly accomplished by tetrahydrobenzo[a]pyrene (BPDE)–DNA chemiluminescence immunoassay
(CIA) (30), which has a limit of detection of 1.5 adducts/109 nt, using 20 lg
members of the cytochrome P450 (CYP) superfamily like the DNA. Samples collected from the same subjects in different seasons were
products of the CYP1A1 and CYP1B1 genes. Alternatively, assayed on the same enzyme-linked immunosorbent assay plate to minimise
elimination of PAHs occurs by interaction with phase II batch effects.
enzymes, such as the glutathione-S-transferases. There is
Lymphocyte cultures and analysis of chromosomal aberrations
evidence from a number of studies that polymorphic variants
Chromosomal aberrations were scored in the metaphase of whole-blood
in these genes confer altered capacity to activate or detoxify lymphocyte cultures. Cells were grown in RPMI 1600 culture media
genotoxic compounds (17). Indeed, exposed individuals have supplemented with L-glutamine (Sigma, St. Louis, MO, USA), nonessential
shown significant associations between the allelic variants amino acids (Sigma) and 0.2 ml phytohaemagglutinin (Invitrogen, Carlsbad,
CYP1A1*2A, CYP1A1*2C, CYP1B1*3, GSTM1 null and CA, USA) for 72 h, as described elsewhere (31). The cultures were arrested in
metaphase by the addition of 2 lg/ml demecolcine (Sigma) and further
GSTT1 null and increased risk for chromosome aberrations incubated for 90 min. The cells were centrifuged, treated with 7.4 mM KCl
and/or PAH–DNA adducts (14,18–27). hypotonic solution for 20 min and washed three times with methanol:glacial
Although the levels of VOC have been determined daily in acetic acid (3:1) fixing solution. The pellets were finally re-suspended in 400 ll
the MCMA (2), there have been no estimations of the levels of the fixing solution, and drops of the cell suspensions were placed on glass
of PAHs present in these emissions. Estimations of slides and air dried. After staining with phosphate-buffered 2% Giemsa
solution, pH 7.2, the metaphases were observed under a microscope. In
PAH–PM10 in the northern area of the MCMA demonstrate successful cultures, 100 well-defined metaphases with .45 chromosomes were
seasonal variations (28). On the other hand, high levels of analysed from each individual. The results of the analysis were expressed as the
particulate PAHs were found on the roadways of the MCMA percentage of cells containing chromatid and chromosomal gaps and breaks per
due to vehicle traffic (7). To estimate PAH exposures to individual (CWA).

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inhabitants of the MCMA, we performed a longitudinal Genotyping
study in young non-smoking adults, determining the levels of GSTM1 and GSTT1 deletion polymorphisms were determined simultaneously
PAH–DNA adducts and the frequency of chromosomal after multiplex polymerase chain reaction, as described by Abdel-Rahman et al.
aberrations in peripheral blood lymphocytes during the dry (32). Polymerase chain reaction–restriction fragment length polymorphism
and rainy seasons, as well as their association with the risk assay analysis was used to identify CYP1A1*2A (33), CYP1A1*2C (34),
CYP1A1*4 (35) and CYP1B1*3 (36) polymorphisms. All primers were
allelic variants of the cytochromes CYP1A1 (*2A, *2C and obtained from Invitrogen.
*4) and CYP1B1 (*3) and the phase II enzymes GSTM1 null
and GSTT1 null. Environmental monitoring
Overall, we observed significant differences between the Data on PM10 and PM2.5 concentrations recorded at five monitoring stations,
representing the northwest (NW, Tlalnepantla), northeast (NE, Xalostoc), central
average levels of PAH–DNA adducts, but not cells with (C, Merced), southwest (SW, PM10 Pedregal and PM2.5 Coyoacan) and southeast
chromosomal aberrations (CWAs), during the dry and rainy (SE, PM10 Cerro de la Estrella and PM2.5 UAM Iztapalapa) regions of the
seasons. Correlation analysis revealed that some allelic variants MCMA, were collected from the RAMA database (37) to estimate 24-h average
showed no seasonal differences. Also, the levels of PAH–DNA mass concentrations in these regions during the 2006 dry and rainy seasons.
adducts correlated with the seasonal levels of PM and with risk Statistical analysis
alleles in some of the areas. These results are consistent with Allele and genotype frequencies among the study samples were calculated, and
the fact that inhabitants of the MCMA are exposed to higher deviation from Hardy–Weinberg equilibrium was examined with the v2 test.
levels of PAHs and other xenobiotic compounds in the dry The paired t test and the non-parametric Wilcoxon paired-sample test were
season and lower levels in the rainy season. applied to compare the differences between the dry and the rainy seasons for
PAH–DNA adduct levels and the %CWA in subjects grouped by genotype,
respectively. To compare PAH–DNA adduct levels and %CWA between
genotypes during both seasons, the Mann–Whitney U test was used. Multiple
Materials and methods linear regression analyses were performed to evaluate the relationship between
Study subjects the PAH–DNA adduct levels from the 92 paired samples (both in dry and rainy
seasons) and the PM emissions. To investigate the relationship between PAH–
One hundred thirteen unrelated apparently healthy non-smoking 19- to
DNA adduct levels, the different allelic variants were grouped by genotype and
40-year-old volunteers, who remained in the MCMA for at least 6 months
place of residence (NW, NE, etc) and coded as follows: 0 (wild type
prior to the study and through the next rainy season, were recruited for this
homozygous), 1 (heterozygous, one variant allele) and 2 (mutant homozygous).
study. After a signed informed consent was obtained, the volunteers were
In the case of GSTM1 and GSTT1, the presence of allele was coded 0 and the
asked to complete a questionnaire concerning their age, residence area,
null genotype was coded 2 since the presence of GSTM1 or GSTT1 in
general health conditions, medications, smoking habits and exposure to X-
homozygous or heterozygous condition has not been associated with increased
rays and other genotoxic agents in the preceding 6 months. Peripheral venous
adduct levels (19,21,38–40). To explore the additive effect of risk alleles and
blood and urine samples were collected from each volunteer during the dry
adduct levels, the sum of risk alleles was computed for each individual and
season (March 1–31, 2006) and during the rainy season (August 22 to
a linear regression was performed. In all statistical tests, P , 0.05 was
September 27, 2006). Questionnaires, blood and urine samples were coded on
considered statistically significant. The analysis was performed using Stata for
site for an unbiased analysis.
Windows (version 10.1).
Determination of cotinine
Exposure of study participants to tobacco products within the past 48 h was
assessed by semi-quantitative determination of cotinine and hydroxycotinine Results
levels in urine using the Accutest NicAlert strip test (Jant Pharmaceutical Corp.,
Encino, CA, USA), according to the manufacturer’s protocol. Individuals Study population
showing levels of cotinine and hydroxycotinine .100 ng/ml were considered Of the initial 113 volunteers, 12 were absent during the second
smokers and excluded from the analysis. sampling period and 9 who showed levels of cotinine in

PAH-DNA adducts in young adults living in Mexico City

urine .100 ng/ml indicative of smoking were excluded from season). This amounted to 50 of 92 subjects, representing 54%
the study. The total number of paired samples analysed in the of the studied population. Those showing 9.90 adducts per
longitudinal study was 92. The volunteers were unrelated 42 109 nt during the dry season showed the smallest variation in
males and 50 females, between 19 and 40 years of age, with the level of adducts for dry and rainy seasons (8.64 versus 8.31
a mean weight of 65.02  12.82 kg, a median height of 1.66  per 109 nt, respectively), which was not statistically significant.
0.09 m and a body mass index of 23.32  3.22 kg/m2 (Table I). When individuals were grouped according to their region of
No significant effects on the levels of adducts or the % of residence (NE, NW, etc), the levels of PAH–DNA adducts
CWAs were observed due to residence location, age, body were not statistically different during the same season, but
mass index, consumption of vitamins or medical drugs (data adduct levels were higher during the dry season (Figure 2) for
not shown). all regions of residence except for those volunteers living in the
central region of the MCMA.
Particles and air quality data The mean values for PAH–DNA adduct levels observed
The data collected from RAMA (37) were used to estimate the among different genotypes (wild type homozygotes versus
24-h average PM2.5 and PM10 mass concentrations. The 24-h mutant homozygotes) are shown in Table II. No significant
averages at five representative residential locations (northwest, seasonal differences were observed in the case of the allelic
northeast, central, southwest and southeast) of the MCMA variants CYP1A1*4 þ/ (n 5 6), GSTT1 null (n 5 6) or the
were analysed during the dry and rainy seasons and the results combination of GSTM1 null and CYP1A1*4 þ/ (n 5 5),
are shown in Figure 1. During the dry season, higher PM10 and probably due to the small sample size (six or five subjects in
PM2.5 concentration measurements were reported (see median each group). However, the carriers of CYP 1B1*3 þ/ (n 5
values in Figure 1). 42), CYP1A1*2A (n 5 18) and *2C (n 5 18) also did not show
The highest median values (P , 0.05) for PM10 were significant seasonal differences, despite the large sample size.
observed at Xalostoc in the NE portion: PM10 5 101 (47–224) A multiple linear regression analysis of the 92 paired samples
lg/m3 in the dry season and PM10 5 51 (20–123) lg/m3 in the showed a significant correlation between the PAH–DNA
rainy season. At this monitoring station, the levels of PM10 adduct levels and the level of PM10 during the dry season
during the dry season sometimes exceeded the 120 lg/m3 but not with the %CWA (Table III). When risk alleles were

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standard of the Official Mexican Norm (NOM). This is taken into consideration to explain PAH–DNA adduct
consistent with the heaviest density of industrial activity and formation, we observed significant associations between
mobile sources being in the NE portion of the Mexico City PM10 and PM2.5 levels and GSTM1 null individuals living in
basin. Central, SE and NW portions of the basin showed more the SE region portion. In addition, there was a significant
mid-range median concentrations. The variability in daily association between to PM2.5 and CYP1B1*3 (n 5 9) in
PM2.5 mass concentrations observed was low compared with individuals living in the NW during the dry season.
daily PM10 concentrations. Central and NW portions showed When the additive effect of risk alleles was investigated, we
the highest values (P , 0.05) and variability. observed a relationship between the sum of the risk alleles and
PAH–DNA adduct levels in those individuals with four or
PAH–DNA adducts more risk variants in the dry (R 5 0.298, P 5 0.048) and wet
Significant differences were observed when the mean levels of seasons (R 5 0.473, P 5 0.001; Figure 3).
PAH–DNA adducts in both seasons were compared (10.66 
3.05 dry season versus 9.50  2.85 rainy season, P 5 0.0001, Chromosomal aberrations
Table I and Figure 2). The larger and significant seasonal Using conventional cytogenetic analysis, we analysed the
changes in adduct levels were observed in those volunteers percentage of cells with chromatid and chromosome aberra-
who showed .9.9 adducts per 109 nt during the winter (dry tions in the metaphase of peripheral blood lymphocyte cultures.
The analysis was performed in 79 duplicate successful cultures
Table I. Demographic variables stratified by sex from samples obtained in the dry season and in 89 from the
Number of volunteers
rainy season. Although recorded during the analysis, chromatid
and chromosomal gaps were excluded from the %CWA. We
Male Female did not observe significant seasonal differences in the %CWA
(Table I).
Dry season 53 60
Rainy season 42 50
Chromosomal damage was also correlated with genotypes.
Smokers (excluded) 7 2 The mean values for chromosomal aberrations observed among
Non-smokers, both seasons 42 50 groups with different genotypes are shown in Table II. No in-
Age 24.10  4.24 25.65  5.37 teractions were observed between polymorphisms in xenobiotic-
Weight (kg) 72.29  12.95 58.72  8.95 metabolising enzymes and the frequency of chromosomal
Height (m) 1.72  0.08 1.61  0.07
BMI 23.34  3.25 22.48  2.86 aberrations among seasons. In the rainy season, the combination
PAH–DNA GSTM1 homozygotes or heterozygotes plus CYP1B1*3 hetero-
Dry season 10.61 3.22* 10.93  2.85** zygotes showed a higher marginally significant %CWA than
Rainy season 9.29  3.03 9.9  2.58 GSTM1 null/CYP1B1*3 heterozygotic individuals (P 5 0.06;
%CWA (without gaps)
Dry season 0.47  0.71 (N 5 40) 0.48  0.73 (N 5 39)
Table II).
Rainy season 0.62  0.80 (N 5 41) 0.67  0.82 (N 5 48)

Average  SD of age, weight, height and body mass index (BMI), PAH–DNA Discussion
adducts and %CWA (excluding gaps). Paired Student t-test.
The types of chromosomal aberrations included in this analysis were
In this study, we observed a seasonal variation in the levels of
chromatid and isochromatid breaks and exchanges (21). PAH–DNA adducts in white blood cells from non-smokers
*P , 0.001; **P 5 0.012. living in the MCMA. Higher levels of adducts were found in

W. A. Garcı́a-Suástegui et al.

Fig. 1. Box plot distribution of PM2.5 and PM10 mass concentration average 24 h during the sampling period, at five monitoring stations representatives of the five
regions of the MCMA [northwest (NW), northeast (NE), central (C), southwest (SW) and southeast (SE)]. Panels correspond to (A) PM10 dry season; (B) PM10
rainy season; (C) PM2.5 dry season; (D) PM2.5 rainy season. The plot represents the minimum and maximum values (whiskers), the first and third quartiles (box) and

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the median value (midline). For PM10, the NW monitoring station reported higher (P , 0.05) median levels of emissions than the other four monitoring stations
while the SW region had the lowest levels. For PM2.5, the NW and C monitoring stations reported the highest (P , 0.05) levels of emissions. Initials above each box
indicate the regions that are significantly different from the data for that box.

damage (Table I). Similar negative results were observed when

chromosomal aberrations were compared, using conventional
cytogenetic analysis, between a group of policemen working
.8 h outdoors in the downtown area of Prague and age- and
sex-matched healthy volunteers spending .90% of each day
indoors (42). Nevertheless, previous in vitro studies have
documented the presence of clastogenic and mutagenic PAHs
in airborne PM from industrial and residential areas in the
MCMA (6,16), which emphasises the potential health risk
associated with exposure to air pollution in Mexico City.
Fig. 2. Box plot distribution of PAH–DNA adducts between subjects residing Furthermore, respiratory and systemic effects of chronic air
in the five regions of the MCMA. The plot shows the minimum and maximum pollution exposure have been reported in children living in
values (whiskers), the first and third quartiles (box) and the median values Mexico City (43), and long-term exposure to ozone, PM10 and
(midline). The levels of adducts were higher in dry season for individuals with NO2 have been associated with significant deficits in lung
residence in NW, NE, SW and SE regions (P , 0.05).
function growth (forced vital capacity and forced expiratory
volume in 1 second) among school-age children living in
blood samples obtained during the dry season (winter, Table I; Mexico City (44).
Figure 2), and this coincided with the presence of higher levels Besides the seasonal differences, we also observed a wide
of air pollutants (2) and PM (Figure 1). Moreover, the presence range of variation in the levels of PAH–DNA adducts and
of adducts correlated significantly with the estimated levels of %CWA in the study population (Table I; Figure 2). In-
PM10 (Table III), suggesting that the adduct levels show a close terindividual variations in DNA adduct levels have been
relationship with the intensity of environmental pollution reported in other studies (17,18,45). The range of individual
exposure. Because the antiserum used in the CIA recognises exposure biomarker levels in study populations is affected by
DNA modified with several different PAHs (10,41), our results factors such as accumulative exposure that could be related to
suggest that during the dry season (November to May) the region of residence in the MCMA, and to polymorphisms
residents of the MCMA are exposed to higher levels of PAHs in metabolic enzymes that convert PAHs to electrophilic
than during the rainy season (June to October). This is in metabolites able to damage DNA or polymorphisms in
agreement with PAH concentrations measured in PM10 and in enzymes that conjugate these compounds to render them
the gas phase in Mexico City’s atmosphere during 2005 (28). hydrophilic and therefore able to be excreted.
The benzo[a]pyrene in PM10 was almost three times higher Age was also investigated as a possible confounding factor
during the dry season (winter) than during the rainy season because positive correlations between age and levels of PAH–
(0.81 versus 0.29 ng/m3, respectively). DNA adducts in experimental animals (46) and in human brain
On the other hand, the increased exposure to PAH during the tissue from individuals with ages ranging from birth to 100
winter was not associated with chromatid or chromosome years (47) have been reported. The age range among donors in

PAH-DNA adducts in young adults living in Mexico City

Table II. Distribution of polymorphisms on metabolic PAHs enzymes in the studied population and their effect on the PAH–DNA adduct levels and %CWA in
each season

Gene Genotype N Dry season N Rainy season

Adductsa (SD) Pb N %CWAa (SD) Pb Adductsa (SD) Pb N %CWAa (SD) Pb

CYP1A1*2A, Msp I þ/þ 18 11.04 (3.56) 0.42 17 0.33 (0.59) 0.16 18 9.69 (3.10) 0.39 18 0.42 (0.76) 0.41
þ/ 49 10.45 (2.76) 41 0.63 (0.79) 49 9.31 (2.69) 49 0.65 (0.77)
/ 25 10.80 (3.28) 22 0.31 (0.64) 25 9.75 (3.05) 23 0.65 (0.88)
CYP1A1*2C, Ile462Val þ/þ 18 11.02 (3.57) 0.41 17 0.35 (0.60) 0.38 18 9.66 (3.12) 0.45 19 0.47 (0.77) 0.34
þ/ 57 10.48 (2.97) 49 0.57 (0.76) 57 9.47 (2.83) 56 0.71 (0.84)
/ 17 10.87 (2.84) 14 0.35 (0.74) 17 9.45 (2.67) 16 0.43 (0.62)
CYP1A1*4, Thr461Asn þ/þ 87 10.59 (2.94) 0.24 75 0.40 (0.54) 0.92 87 9.51 (2.89) 0.47 84 0.57 (0.78) 0.95
þ/ 7 11.74 (4.47) 5 0.49 (0.74) 7 9.38 (2.38) 7 0.61 (0.80)
CYP1B1*3, Leu432Val þ/þ 44 10.72 (2.90) 0.22 38 0.63 (0.81) 0.28 44 9.28 (2.89) 0.08 44 0.72 (0.92) 0.59
þ/ 42 10.51 (3.30) 37 0.37 (0.63) 42 10.02 (2.70) 40 0.52 (0.64)
/ 6 11.28 (2.62) 5 0.20 (0.44) 6 7.48 (2.91) 7 0.42 (0.78)
GSTM1 Active 51 10.46 (2.87) 0.31 46 0.54 (0.80) 0.74 51 9.29 (2.93) 0.11 51 0.61 (0.75) 0.78
Null 41 10.91 (3.27) 34 0.41 (0.60) 41 9.76 (2.76) 40 0.61 (0.86)
GSTT1 Active 86 10.69 (2.94) 0.33 76 0.51 (0.73) 0.28 86 9.49 (2.84) 0.39 85 0.63 (0.81) 0.41
Null 6 10.27 (4.65) 4 0.00 (0.00) 4 9.65 (3.18) 4 0.25 (0.50)
GSTM1/CYP1B1*3 Active (þ/þ, þ/) 48 10.43 (2.95) 0.22 44 0.38 (0.69) 0.93 48 9.48 (2.90) 0.22 47 0.77 (0.73) 0.06
Null (þ/, /) 25 11.22 (3.55) 22 0.36 (0.59) 25 9.72 (2.49) 25 0.31 (0.47)
GSTM1/CYP1A1 *4 Active/(þ/þ, þ/) 51 10.46 (2.87) 0.44 46 2.22 (1.87) 0.16 51 9.29 (2.93) 0.38 51 1.61 (1.55) 0.24
Null/(þ/) 5 10.50 (3.64) 4 1.25 (1.26) 5 8.50 (1.09) 5 2.00 (1.58)

þ, wild type alleles;, variant type. Marginally significant differences are shown in bold values.
Arithmetic mean per 109 nt or mean percentage for aberrant cells.
Derived from Mann–Whitney U test between the two homozygote groups (e.g. þ/þ and /).

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region as compared with a similar number of individuals living
Table III. Multiple regression analysis between the reported levels of PM, in the SW portion who were exposed to significantly lower
PAH–DNA adducts, %CWA, MCMA regions (NW, NE, C, SW and SE) and levels of PM. This was previously observed in individuals
allelic variants
occupationally exposed (49), smokers (50) and in a population
Variables R P exposed to urban air pollution (14). We found that volunteers
living in the NW region showed a significant correlation
PM10 PM2.5
between PM2.5, PAH–DNA adduct levels and the CYP1B1*3
Dry season genotype. Although the sample size in this group was small, this
PAH–DNA adducts (n 5 92) 0.209 0.048 0.216 association probably reflects the impact of exposure due to
SE, GSTM1 null (n 5 23) 0.533 0.035 0.013 commuting since this group of volunteers, according to the data
NW, CYP1B1*3 (n 5 9) 0.845 0.116 0.023
Rainy season
collected from questionnaires, travelled through a traffic corridor
PAH–DNA adducts (n 5 92) 0.169 0.528 0.128 where very high levels of PAHs have been reported (7). These
SE, GSTM1 null (n 5 23) 0.311 0.362 0.215 observations suggest that GSTM1 null and CYP1B1*3 are
NW, CYP1B1*3 (n 5 9) 0.285 0.780 0.775 relevant polymorphisms for leucocyte PAH–DNA adduct levels.
%CWA GSTM1 is involved in the conjugation of PAH diols to
Dry season (n 5 80) 0.132 0.669 0.399
Rainy season (n 5 91) 0.043 0.709 0.843 glutathione (41); thus, the existence of a null allele is associated
with the lack of expression of a functional protein (42–53) that
Significant associations are shown in bold values. could result in increased concentrations of epoxide intermediates
and hence higher DNA adduct levels. Indeed, the presence of
GSTM1 in homozygous or heterozygous condition has been
this study was relatively small (19–43 years), and we did not associated with lower levels of DNA adducts when compared
find any correlation between adduct levels and age. On the with GSTM1 null phenotype (19,21,38–40).
other hand, gender-based differences in adduct formation or CYP1B1 enzyme plays a significant role in the oxidation of
removal have been reported (48). We were not able to observe a variety of carcinogens, such as PAHs and arylamines (54).
gender differences, possibly due to the relatively low exposure An amino acid change from leucine to valine at Codon 432
levels in both groups. For this study, samples were collected (CYP1B1*3) has been associated with elevated levels of DNA
from the same individuals in the two seasons so that each adducts in white blood cells (49).
individual acted as their own control. Thus, we eliminated or Similar to reports by Matullo et al. (55) and Ketelslegers
minimised some potential confounders, such as lifestyles, et al. (50), we also observed an additive effect of risk alleles on
genetic polymorphisms, diets and other confounding factors PAH–DNA adduct formation when the levels of adducts were
that arise when different cohorts are analysed. correlated with the sum of risk alleles (Figure 3). These results
When the presence of risk alleles was investigated, we emphasise the importance of studying the simultaneous
observed that GSTM1 null individuals living in the SE portion contribution of many genotypes.
showed an association between PM and PAH–DNA adduct Several reports (14,17,19, 49,50,55) suggest that a single
levels during the dry season (Table III). This association is gene (or polymorphism) will never completely explain the
significant probably because of the large number of volunteer interindividual variations in DNA adduct levels. Therefore, we
living in the SE region and the higher levels of PM in the same focused on a combination of CYP1A1*2A, CYP1A1*2C,

W. A. Garcı́a-Suástegui et al.

Fig. 3. Relationship between PAH–DNA adduct levels and the sum of risk alleles in inhabitants of MCMA during the dry and the rainy seasons. Points represent
median values, the minimum and maximum values (whiskers), N 5 number of individuals. The sum of risk alleles per individual was estimated by adding the
number of polymorphisms that putatively increase the risk for formation of PAH–DNA adducts. We found an effect of the sum of risk alleles on adduct levels
among the carriers of more than four risk alleles both in the dry (R 5 0.298, P 5 0.048) and the wet (R 5 0.473, P 5 0.001) seasons.

CYP1A1*4, CYP1B1*3, GSTM1 and GSTT1 polymorphisms, compilation of particular matter emissions for the two analysed seasons. In
which were found in the majority of cases to be non-significant addition, we owe a great deal to our study subjects.
in the univariate analysis, but they were significant when Conflict of interest statement: None declared.
investigating multiple polymorphisms simultaneously in the
multivariate analysis, as a consequence of interactions. Our References
data indicate that assessing multiple genetic polymorphisms

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