Urban air pollution induces micronuclei in peripheral erythrocytes of mice in vivo

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Environmental Research 92 (2003) 191–196

Urban air pollution induces micronuclei in peripheral erythrocytes of mice in vivo S.R.C. Soares, H.M. Bueno-Guimara˜es, C.M. Ferreira, D.H.R.F. Rivero, I. De Castro, M.L.B. Garcia, and P.H.N. Saldiva* Laboratory of Experimental Air Pollution, Faculty of Medicine, Department of Medicine and Department of Pathology, School of Medicine, University of Sa˜o Paulo, Av. Dr. Arnaldo 455, SP CEP 01246-903, Sa˜o Paulo, Brazil Received 22 February 2002; received in revised form 22 October 2002; accepted 4 November 2002

Abstract In this study, we explored the role of chronic exposure to urban air pollution in causing DNA damage (micronuclei frequency in peripheral erythrocytes) in rodents in vivo. Mice (n ¼ 20) were exposed to the urban atmosphere of Sa˜o Paulo for 120 days (February to June 1999) and compared to animals (n ¼ 20) maintained in the countryside (Atibaia) for the same period. Daily levels of inhalable particles (PM10), CO, NO2, and SO2, were available for Sa˜o Paulo. Occasional measurements of CO and O3 were made in Atibaia, showing negligible levels of pollution in the area. The frequency of micronuclei (repeated-measures ANOVA) increased with aging, the highest values obtained for the 90th day of experiment (Po0:001). The exposure to urban air pollution elicited a significant (P ¼ 0:016) increase of micronuclei frequency, with no significant interaction with time of study. Associations (Spearman’s correlation) between pollution levels of the week that precede blood sampling and micronuclei counts were observed in Sa˜o Paulo. The associations between micronuclei counts and air pollution were particularly strong for pollutants associated with automotive emissions, such as CO (P ¼ 0:037), NO2 (Po0:001), and PM10 (Po0:001). Our results support the concept that urban levels of air pollution may cause somatic mutations. r 2002 Elsevier Science (USA). All rights reserved. Keywords: Air pollution; Micronuclei test; Mutagenesis; Mice; Peripheral erythrocytes

1. Introduction The relationship between air pollution and cancer has been repeatedly reported in epidemiological studies (State of the Art, American Thoracic Society, 1996; Health Effects Institute (HEI), 2001). Chemical species present in polluted air or extracted from particulate matter sampled from urban centers exhibit mutagenic properties (Tokiwa and Ohnishi, 1986; Massad et al., 1987; Crebelli, 1989; Green et al., 1991; Lewtas, 1993; Sato et al., 1995; Farmer et al., 1996; Zhou and Ye, 1998; Kubiak et al., 1999; Batalha et al., 1999). However, the demonstration of carcinogenic and/or mutagenic potential of air pollution, using in vivo models exposed to real world air pollution is less evident. Such information would represent a link between the epidemiological and the in vitro laboratory *Corresponding author. Fax: +55-11-3064-2744. E-mail address: [email protected] (P.H.N. Saldiva).

studies, helping to determine the role of air pollution in the pathogenesis of cancer. Our group performed a series of studies exposing either rodents or higher plants to the polluted atmosphere of downtown Sa˜o Paulo (Saldiva et al., 1992; Lemos et al., 1994; Reyma˜o et al., 1997; Saldiva and Bo¨hm, 1998). Briefly, such studies reported a cancer promoter effect of air pollution in mice (Reyma˜o et al., 1997; Cury et al., 2000) and the presence of an increased frequency of mutations in plants chronically exposed to air pollution (Guimara˜es et al., 2000; Ferreira et al., 2000). These findings are supportive of the concept that low levels of air pollution are able to favor the development of tumors. In the present study, we aimed to move further on this topic, by studying the effects of air pollution on the development of micronuclei (MN) in peripheral erythrocytes of mice chronically exposed to urban levels of air pollution in downtown Sa˜o Paulo. More specifically, the objectives of the present study were (a) to determine

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whether chronic exposure to air pollution causes an increase of MN frequency, (b) to explore the dose– response relationship between the exposure level and the MN frequency, and (c) to assess the time lag between exposure and detection of MN in the peripheral blood.

2. Materials and methods 2.1. Exposure protocol Male Balb/c mice, 8–10 weeks old, obtained from the School of Medicine, University of Sa˜o Paulo, Brazil, were randomly divided into two groups: (1) Sa˜o Paulo group (SP) (n ¼ 20) exposed to one of the most polluted regions of Sa˜o Paulo city urban area, exhibiting an intense transit of vehicles (48,000 vehicles/day), and (2) Atibaia group (AT) (n ¼ 20), maintained in the rural area of Atibaia city, 65 km away from downtown Sa˜o Paulo. Atibaia was used as the control spot in several of our previous studies (Bo¨hm et al., 1989; Saldiva et al., 1992; Lemos et al., 1994; Cedon et al., 1997; Reyma˜o et al., 1997; Cury et al., 2000). Both groups were continuously exposed for up to 120 days, from February until June 1999. During the exposure period, all animals were kept in conventional cages installed in a wired jail to avoid insects, foreign animals, and direct sun and rain action. The animals were checked every 2 days and fed with commercial pellet food and water ad libitum. Daily levels of SO2, NO2, and particulate material (PM10) (24-h means) and of CO (highest 8-h moving average) were available for Sa˜o Paulo, provided by the stationary monitoring station of the Sa˜o Paulo State Environmental Sanitation Technology Company (CETESB) located in the same area where the animals were maintained. No continuous air pollution measurements were available for Atibaia. Occasional measurements of CO and O3 were made in Atibaia using colorimetric techniques in test tubes coupled to an aspiration pump (Dra¨ger, Lubeck, Germany). 2.2. Micronucleus test Blood samples were taken after 15, 30, 60, 90, and 120 days of exposure. The peripheral blood (5 mL) was collected from the tail vein of all experimental animals, and diluted in 45 mL of phosphate-buffered saline (pH 7.0). A drop of the solution was smeared on glass slides, fixed with absolute methanol (Merck) for 10 min, dried in air, and stained with Feulgen and Fast green. The slides were randomized and coded to blind score within an optical microscope at a magnification of 100  . For each animal, 3000 peripheral blood normochromatic erythrocytes/slides were examined for the presence of MN. The criteria for identifying MN was

based on Davies et al. (1998), with all slides being scored by one person to avoid interobserver variability. 2.3. Statistical analysis Data analysis was done by ANOVA for repeated measures, using either the absolute frequency of MN or its square root as the dependent variable in the models. The correlation coefficient (Pearson and Spearman) between MN frequency and air pollution measurements was explored within a window of 15 preceding days. The level of significance was set at 5%. The statistical software employed in this study was the SPSS v8.0.

3. Results Table 1 presents the mean levels of measured pollutants in Sa˜o Paulo observed during the study. The results of MN frequency measured at the different experimental times are presented in Table 2 and Fig. 1. The ANOVA models indicated a significant increase in MN within animals evaluated at different experimental times, the highest values being achieved after 90 days of experiment (Po0:001). A significant between-groups (Atibaia versus Sa˜o Paulo) effect was detected (P ¼ 0:016), with no significant interaction with time. Since we observed a wide variation in the MN frequency during the experiment, we reasoned that some degree of this variability could be dependent on changes of the levels of pollution. To test such a hypothesis, we computed the means of MN frequency for each group and each experimental time. We subtracted the mean of Sa˜o Paulo from the Atibaia group for each period. Thus, we considered the temporal variation of MN in Atibaia as a consequence of seasonality and/or aging processes (Sato et al., 1995b; Dass et al., 1997), whereas the difference between groups was considered a product of the pollution effect. In a second step, we explored the cross correlation between the differences of the means of Sa˜o Paulo and in Atibaia with the daily measures of air pollution, in a

Table 1 Monthly means of airborne pollutants measured in Sa˜o Paulo during the experimental period Period (month)

PM10 (mg/m3)

SO2 (mg/m3)

CO 8 h (ppm)

NO2 (mg/m3)

Feb Mar Apr May Jun

31 36 37 40 47

13 12 14 16 20

2.8 2.6 2.4 2.8 3.2

104 97 103 108 NMa

a

NM, not measured.

ARTICLE IN PRESS S.R.C. Soares et al. / Environmental Research 92 (2003) 191–196 Table 2 Descriptive statistics of micronuclei frequency measured in the different experimental periods Std. deviation

N

1.6250 3.8889

1.9621 3.5295

16 18

Atibaia S. Paulo

1.9375 1.6111

1.6520 1.7197

16 18

60

Atibaia S. Paulo

3.1875 5.2222

2.6387 3.2277

16 18

90

Atibaia S. Paulo

7.8125 8.0000

5.4064 5.3026

16 18

120

Atibaia S. Paulo

3.1875 4.7778

2.1360 4.0520

16 18

Period (days)

Groups

15

Atibaia S. Paulo

30

Mean

193

Table 3 Spearman correlation coefficient between the mean difference in micronuclei of Sa˜o Paulo and Atibaia groups and the mean levels of pollution of the previous week of blood sampling Pollutant PM10

CO

NO2

SO2

r¼1 P ¼ 000

r ¼ 0:9 P ¼ 0:037

r ¼ 0:27 P ¼ 0:005

r¼1 P ¼ 0:873

The corresponding levels of significance are also provided; r ¼ 1; Po0.05.

Fig. 2. Dose–response relationships between the mean differences in MN (Sa˜o Paulo–Atibaia), plotted against the mean daily levels of CO of the previous week.

Fig. 1. Estimated marginal means of MN (%) in the different experimental periods (days) as determined by generalized estimation models.

time window up to 15 days. In other words, we aimed to verify whether the difference between MN frequency measured in Sa˜o Paulo and in Atibaia was influenced by the previous pollution exposure, within a 15-day window of time. As a general rule, a positive association between all measures of air pollution and the MN frequency difference was observed, beginning with the exposure of the 8th day before blood sampling, becoming even more significant toward the previous 14th day. Thus, we considered these findings as suggestive that the mean difference in MN frequency between Sa˜o Paulo and

Atibaia is dependent on the pollution levels of the previous week of exposure. Table 3 shows the coefficients of Spearman’s cross correlation between the mean difference in MN between Sa˜o Paulo/Atibaia and the mean levels of pollution of the previous week of blood sampling. In fact, a dose–response pattern was observed when the mean difference in MN (Sa˜o Paulo–Atibaia) was plotted against the mean levels of pollution of the previous week of sampling, mainly for CO, NO2, and PM10, as exemplified in Fig. 2 for CO. Considering that, these gases are good markers of ‘‘fresh’’ automotive emissions, these results are suggestive for the air pollution derived from exhaust pipes of mobile sources.

4. Discussion The purpose of this study was to evaluate, under real world conditions, the potential of air pollution to cause mutations in mice, as detected by MN in the peripheral blood, by doing repeated blood samplings during a prolonged exposure.

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The exposure sites of the present study––downtown Sa˜o Paulo and the countryside of Atibaia––were the same previously used by our research group in several experiments, which disclosed positive air pollution effects in animals, in terms of both airway inflammation (Bo¨hm et al., 1989; Saldiva et al., 1992; Lemos et al., 1994) and lung tumor genesis (Reyma˜o et al., 1997; Cury et al., 2000). We moved further on the topic of the possible mutagenic effects of air pollution, by doing repeated measures in the same group of animals and following the time course as an indicator of DNA damage in two places with different pollution backgrounds after prolonged exposure in vivo. Using such an approach, we could not only detect cross-sectional differences but also observe the possible variations within groups and relate them to temporal changes in air pollution. After defining the exposure protocol and the study design, we chose a simple method of detection of DNA damage that allowed performing repeated measures within the same animal. Using such an approach, we could control for seasonal variations or age-dependent phenomena, which were accounted for by the changes observed in the control group. Due to its simplicity and practical nature, the micronucleus assay has become the test of choice for screening clastogenic and aneugenic activity of potential genotoxic agents (Heddle et al., 1991; Hayashi et al., 2000). It is very applicable to rodents, permitting the MN presence to be scored in blood samples with peripheral polychromatic or normochromatic erythrocytes, collected without killing the animals (Heddle et al., 1991; Torous et al., 1998; Hayashi et al., 2000). Hence, chronic (or long-lasting) in vivo assessment of chromosomal breakage can be performed using the population under study as its own control (Schlegel and MacGregor, 1982). The micronucleus assay proved to be easy to score and sensitive to detect DNA damage after hours of exposure (Massad et al., 1987) or days of exposure (Ong et al., 1985) to air pollution. The long-standing hypothesis that air pollution causes lung cancer has been based on geographic variation in cancer risk and the recognition that carcinogens, such as some of the air toxins, are emitted into the air (State of the Art, 1996; Farmer et al., 1996). According to Rothfuss et al. (2000) the MN frequency is a biomarker for cancer predisposition. We observed a significant association between MN frequency and air pollution levels of the previous days of exposure. The time lag between exposure and MN formation was relatively short, at about 1 week. The MN induction was correlated to CO, NO2, and PM10. The CO and NOx are formed in combustion processes and are major pollutants in urban air. Relatively few studies on the genotoxicity of NO2 and NO have been performed. These studies indicate that NO2 is genotoxic

in vitro, but the effect of NO seems to be very slight (Victorian, 1994). Nitrogen oxides are key components in atmospheric smog formation, which may lead to secondary effects. Strongly mutagenic nitro-polycyclic aromatic hydrocarbon compounds are easily formed, and mutagenic reaction products may be formed photochemically from alkenes. On the other hand, specific studies on the genotoxicity of CO are nonexistent, but this gas acts like a donor for new components in atmospheric formation. The mutagenic activities associated with inhalable airborne particulate matter (PM10) is due to the presence of irritating and genotoxic substances in both the gas phase and the particulate phase. Constituents are considered to have significant health implications, due to its chemical deposits in an organ that may lead to in vivo induction of a somatic cell mutation and carcinogenesis. Diesel exhaust particles have been studied in animal inhalation tests, indicating an association between diesel particle exposure and either lung or other types of cancers and expressing mutagenic activity, which is largely dependent on the aromatic content of the fuel (Torres-Bugarin et al., 1999). Traffic has been implicated as the major determinant for the concentration of PAHs and, therefore, for the genotoxic activity of urban air (Sato et al., 1995a; Torres-Bugarin et al., 1999; Karahalil et al., 1999). As we could not control the background exposure conditions, we could not identify what is (are) the pollutant(s) responsible for the observed effects. In fact, we could not rule out the possibility that the observed effects were not due to the measured pollutants, since important mutagenic compounds (such as nitroarenes and benzo(a)pyrene derivate from the mono- or polycyclic aromatic hydrocarbons) were not evaluated in our study. So, we think that our results are indicative that urban air pollution induces MN in a dose–response pattern, but have the limitation of not properly identifying the causal agent. It is more probable that the pollutants, which exhibited significant associations with MN in our study (Fig. 2), were acting as proxy variables for the complex mixture of substances present in automotive emissions (which are the dominant pollution source in Sa˜o Paulo) and their corresponding atmospheric reactions.

5. Conclusions Our study demonstrates the ease of evaluating the mutagenic action of urban air pollution of Sa˜o Paulo associated with ‘‘natural experiments’’ in mice, by performing an in vivo long-lasting exposure. The pattern of micronucleus frequency in mice peripheral blood evidence of mutagenicity in animals exposed to air

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pollution chronically. The micronuclei frequency in the Sa˜o Paulo group was also dependent on the levels of pollutants from the previous week. This dependency was related to the gases CO, NO2, and inhalable airborne particulate matter (PM10). This study shows the feasibility of simple biological assays such as the micronuclei test to better characterize mutagenicity of urban air pollution.

Acknowledgments The authors thank and acknowledge the assistance of the researchers from the Experimental Air Pollution and Experimental Therapeutic Laboratories who contributed to the realization of this project. This study was supported by LIM-HCFMUSP and was developed in the Experimental Air Pollution Laboratory, School of Medicine of Sa˜o Paulo University, Brazil. This research was conducted according to the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy of Sciences, Washington, DC (1996).

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