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1093/mutage/geq104
Advance Access Publication 30 December 2010
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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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
Average SD of age, weight, height and body mass index (BMI), PAH–DNA Discussion
adducts and %CWA (excluding gaps). Paired Student t-test.
a
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
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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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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
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.
a
Arithmetic mean per 109 nt or mean percentage for aberrant cells.
b
Derived from Mann–Whitney U test between the two homozygote groups (e.g. þ/þ and /).
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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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