Detection of the genotoxicity of air pollutants in and
around the city of Sa˜o Paulo (Brazil) with the
Tradescantia
-micronucleus (Trad-MCN) assay
E.T. Guimara˜es
a, M. Domingos
b,*, E.S. Alves
b, N. Caldini Jr
a,
D.J.A. Lobo
a, A.J.F.C. Lichtenfels
a, P.H.N. Saldiva
aaDepartamento de Patologia,Faculdade de Medicina da USP,Laborato´rio de Poluic¸a˜o Atmosfe´rica Experimental,
A6enida Dr. Arnaldo 455,01246-903Sa˜o Paulo, Brazil bInstituto de Botaˆnica,Caixa Postal 4005,01061-970 Sa˜o Paulo, Brazil
Received 15 June 1999; received in revised form 19 December 1999; accepted 21 December 1999
Abstract
Tradescantia pallida cv. purpurea, a popular garden plant in Brazil, was used for the Tradescantia micronucleus (Trad-MCN) assay. In situ monitoring of the genotoxicity of air pollutants was carried out by sentinel approach, using the plant grown in the field or using the plants in pots which were carried to the monitoring sites. Two highly polluted sites, in Sa˜o Paulo city (Cerqueira Cesar and Congonhas) and two rural sites (the cities of Pirassununga, 200 km and Caucaia do Alto, 50 km from Sa˜o Paulo, respectively) were chosen for this study, in order to determine the gradient difference of the air pollution levels. Sentinel plants in Congonhas site presented the highest frequency of micronuclei (4.4%), in comparison with 2.2 and 2.3% found in plants from Pirassununga and Cerqueira Cesar sites, respectively (Kruskal – Wallis;PB0.020). Significant increases (F test; PB0.0001) in the frequency of micronuclei were observed in plants exposed in the polluted urban sites (Cerqueira Cesar: 5.7%; Congonhas: 7.1% and Caucaia do Alto: 2.3%). The increase in the frequency of micronuclei observed indicates the potential risk of mutagenicity in presence of high concentrations of pollutants. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Tradescantia pallida; Urban air pollution; Genotoxicity; Trad-MCN assay
www.elsevier.com/locate/envexpbot
1. Introduction
The air in large cities is usually contaminated by a variety of pollutants originated from
station-ary and mobile sources, mainly from the combus-tion of fossil fuel. The most important pollutants emitted into the air are particulate materials with varying composition, including heavy metals, and gases such as carbon, nitrogen and sulfur oxides and hydrocarbons. These gases are the primary sources of secondary pollutants, such as ozone and peroxy acetyl nitrate (PAN), formed by * Corresponding author. Tel.:+55-11-55846300; fax:+
55-11-5773678.
E-mail address: mdomingos@smtp-gw.ibot.sp.gov.br (M. Domingos).
photochemical reactions that take place in the atmosphere (Freedman, 1995). The effects of ur-ban air pollution on the human health have long been studied. Based on several studies, it is possi-ble to affirm that lung cancer is more common in urban areas than in the countryside. However, other features of the urban dwellers, such as differences in smoking (active and/or passive), drinking and eating habits, a higher risk of infec-tion, working conditions, and density-dependent factors may contribute to the urban increase in lung cancer as well (Cederlo¨f et al., 1978).
Although animal assays are well suited for lab-oratory studies of pathogenic mechanisms (Sal-diva and Bo¨hm, 1998), no effective animal assay for in situ monitoring is available so far. Higher plants, such asTradescantia, which are multi-cell-ular and eukaryotic organisms, have been the effective monitor for genotoxicity of air pollutants since the early 1960s (Sparrow et al., 1972; Ma et al., 1982; Arutyunyan et al., 1999; Batalha et al., 1999; Monarca, et al., 1999). According to Grant (1998) and Gopalan (1999), the contributions of plant bioassays will be their use in long-term ambient air monitoring studies and testing for genotoxicity on a global scale. The Tradescantia micronucleus (Trad-MCN) bioassay with several clones of Tradescantia has been used extensively for this purposes (Ma, 1981; Rodrigues et al., 1997; Grant, 1998). Recently, T. pallida cv. pur
-purea was employed for these purposes (Batalha
et al., 1999).
Although the genotoxic effects observed in plants can not be extrapolated directly to human populations, the finding of plant bioassays may be taken into account for this purposes. Some plants are known to be sensitive bioindicators of genotoxicity, disclosing positive results with very low levels of contamination. It is reasonable to assume that if a pollutant does not cause any detectable harm to the most sensitive species, it would not affect any other species, including hu-man beings. Besides this, epidemiological evi-dence, which reveals long-term changes in the rates of cancer in the exposed population, is not only difficult to characterize with clarity but also not acceptable on precautionary grounds. Consid-ering these aspects, studies in this area represents a key point in ecotoxicology.
Sa˜o Paulo, one of the largest cities in the world, with a population of 18 millions, represents an important industrialized and economic center of Brazil and present serious environmental prob-lems. According to the Environmental Protection Agency of the State of Sa˜o Paulo (CETESB), emissions from 4 million motor vehicles daily are the main sources of air pollution in the city (CETESB, 1998). Similarly to what happened in other urban environments, some studies in this big city have demonstrated that the air composi-tion is bad enough to produce inflammatory alter-ations in the pulmonary parenchyma in rats exposed for prolonged periods to downtown air (Saldiva et al., 1992) and to cause adverse health effects on the exposed human population (Saldiva et al., 1994, 1995). Reyma˜o et al. (1997), using mice, also predicted a gradient risk on lung malig-nancies between urban and rural populations.
We presume that the urban air pollution in Sa˜o Paulo can be evaluated by Trad-MCN assay to establish the urban – rural gradient, as observed by Reyma˜o et al. (1997), and demonstrate the genotoxicity of air pollutants. The aim of this study was to test this hypothesis, by performing in situ monitoring in highly polluted urban sites and in less polluted rural areas, using the MCN assay. Chemical data of gaseous contents of air samples were presented to confirm the high pollutant lev-els of the urban sites.
2. Materials and methods
2.1. Study sites
type, temperate, without dry season (mean tem-peratures of the hottest month below than 22°C and of the coldest month below to 18°C) and with annual rainfall 1500 mm (Setzer, 1966). Sa˜o Paulo city is situated in a lower region of South America Atlantic Planalt, occupying an area of 5000 km2, with an altitude of 715 – 900 m above sea level. According to the Ko¨ppen system, its climate is Cwa type, as described before, with
annual rainfall 1300 mm (Setzer, 1966). The city is subjected to frequent thermal inversions (CETESB, 1998).
Two highly polluted sites in Sa˜o Paulo city were chosen, both situated in avenues submitted to air pollutants from heavy traffic of passenger cars, buses and trucks: Bandeirantes Avenue, near to one of the air quality monitoring stations of CETESB (the governmental agency for air quality
Table 1
Annual mean concentrations, obtained in 1998, of the principal air pollutants and their maximum value ranges measured in Congonhas and Cerqueira Cesar stations, in Sa˜o Paulo citya
PM10(mg/m3)b
Stations CO (ppm)c SO
2(mg/m3)b NO2(mg/m3)c O3(mg/m3)c
Daily
Mean Mean Hourly Mean Daily Mean Hourly Mean Hourly
max max max
Congonhas 54 63 138 392 43 214
aData from CETESB (the governmental agency air quality control). bMean based on daily values.
cMean based on maximum hourly event in each day. dc, not measured.
control), denominated Congonhas, near an ele-mentary school; and Dr Arnaldo Avenue, near to the monitoring station Cerqueira Cesar, by Public Health School of Sa˜o Paulo University (Fig. 1b). The air pollutant concentrations in both urban sites, hereafter referred as Congonhas and Cerqueira Cesar, is shown in Table 1. Air quality data is not available for the rural sites, but CETESB considers the air as clean and non-pol-luted. Additionally, the better air quality in these sites in comparison with urban ones was confi-rmed by instantaneous measurements of CO and O3 concentrations, performed by colorimetric
techniques in test tubes coupled to an aspiration pump (Dra¨ger-Lubeck, Germany) at midday, in sunny days of November, 1999 (Table 2).
2.2. In situ monitoring
Two types of in situ monitoring were per-formed, using plants of Tradescantia pallida (Rose) Hunt. cv. purpurea Boom, the sentinel type, which included plants growing in the sites, and portable type, composed by plants cultivated in pots, exposed at the same sites.
Sentinel approach was applied to Pirassununga, Congonhas and Cerqueira Cesar. In one sampling period (June, 1998), 30 young inflorescences from plants of each site were sampled and immediately fixed in 1:3 acetic acid/ethanol solution for at least 24 h, and kept in the same solution from then on.
One hundred and eighty portable plants of T.
pallida were obtained from Viveiro Manequinho
Lopes (a public institution for plant cultivation) and cultivated in 60 pots (20 cm in diameter) containing a standardized substrate (Eucatex Plantmax) mixed with vermiculite (3:1), watered twice a week and supplemented with 100 ml of full strength Hoagland’s solution (Hoagland and Arnon, 1938) every 15 days. Twenty pots were maintained in open air and without shade, during August – December, 1998, near Congonhas and Cerqueira Cesar stations and in Caucaia do Alto. Around 30 young inflorescences from the portable plants were taken once, in the last month of exposure, and were treated in the same way, as described above.
2.3. Protocol of the Trad-MCN assay
To perform the Trad-MCN assay, we have followed the protocols established by Ma (1981).
Table 2
General characterization of air quality in the rural and urban sites, based on instantaneous measurements of CO and O3
Table 3
Mean MCN frequencies (%) and their standard errors derived from sentinel and portable plant monitoringa
MCN frequency
Sites N Maximum Minimum Standard error
Sentinel plant monitoring(June,1998)
2.2 a
Pirassununga 12 3.7 1.0 0.2
2.3 a 4.5 0.7 0.3
Cerqueira Cesar 15
4.4 b 8.0 1.7
10 0.7
Congonhas
Portable plant monitoring(January,1999)
2.3 a 6.0
15 0.6
Caucaia do Alto 0.4
Cerqueira Cesar 13 5.6 b 9.7 2.0 0.7
7.1 b 13.0 3.0
12 1.0
Congonhas
aIn each monitoring type, values indicated by different letters are significantly different (PB0.05).
Some 15 – 20 young inflorescences from each ex-periment were dissected and early tetrads of the meiotic microspore mother cells were squashed in aceto-carmine stain on a microslide. Only prepa-rations containing early tetrads were used. Scor-ing was performed by countScor-ing, under 400×
magnification, the number of micronuclei present in a random set of 300 early tetrads in each of ten to 15 slides per monitoring site. Slides were coded before scoring, the code being revealed only after all experimental groups had been evaluated.
2.4. Statistics and data analysis
The results obtained in the sentinel groups were statistically compared through a non-parametric
analysis of variance (ANOVA on Ranks –
Kruskal – Wallis), in function of the failure of the equal variance test. In this case, a multiple pair-wise Dunn’s method (PB0.05) was used to locate the differences among the sites. On the other hand, the results from the portable groups were compared through a parametric analysis of vari-ance (ANOVA; F test) and multiple comparison procedure of Student – Newman – Keuls (PB0.05) was employed to establish the differences among the sites.
3. Results
Both kinds of monitoring with T. pallida proved that the genotoxicity was increased from the rural to the urban environment (Table 3). The
samples of sentinel groups near Congonhas moni-toring station, at Bandeirantes Avenue, presented the highest frequency of micronuclei, in compari-son with plants from the other sites (Kruskal – Wallis; PB0.020). Although expected, the frequency of micronuclei in plants growing near the Medical School, next to Cerqueira Cesar mon-itoring station, was not significantly increased in comparison with the rural site (Pirassununga). In the portable group, significant increases (F test; PB0.0001) in the frequency of micronuclei were observed in plants exposed to the urban sites, in comparison with those exposed to the rural one, Caucaia do Alto. Although Caucaia is nearer to the metropolitan center of Sa˜o Paulo than Piras-sununga, the frequency of micronuclei in tetrads detected for plants in these rural sites is similar among them.
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Table 4
Mean concentrations of the principal air pollutants and their maximum values measured in Congonhas and Cerqueira Cesar and the number of micronucleus/100 tetrads counted during both in situ experimentsa
SO2(mg/m3)b
MCN PM10(mg/m3)b CO (ppm)c NO2 (mg/m3)c O3 (mg/m3)c Sites
frequency (%)
Mean Hourly max. Mean Daily max. Mean Hourly max. Mean Hourly max. Mean Daily max.
Sentinel plant monitoring(June,1998)
– – – –
– – – –
2.2
Pirassunungad – –
11 15 21 119 175 ce
Cerqueira 2.3 43 77 5 ce
Cesar
23 37 137 193 36 87
7 4.4
Congonhas 64 132 21
Portable plant monitoring(January,1999)
– – – – – – – – –
Caucaia do 2.3 –
Altod
Cerqueira 5.6 37 60 3 6 11 30 103 203 ce ce
Cesar
14 26 129 232 65
5 130
53
31 7
Congonhas 7.1
aData from CETESB (the governmental agency air quality control). bMean based on daily values.
4. Discussion
The frequency of micronuclei established in sentinel and portable plants in the rural sites is similar to the figures observed in negative control groups by Ma (1981), Ma et al. (1982), Rodrigues et al. (1998), Monarca et al. (1999) usingTrades -cantia clones and Batalha et al. (1999) using T. pallida. These results reinforce that there exists an urban-gradient of genotoxicity determined by the urban air, as mentioned in the literature, through epidemiological or animal monitoring studies (Cederlo¨f et al., 1978; Reyma˜o et al., 1997).
By fumigating plants ofTradescantia, Ma et al. (1982) found that NO2, SO2 and O3 were
genotoxic agents. Rodrigues et al. (1996), also performing fumigation experiments, observed that 100mg/m3of O3was enough to cause a significant
increase in the frequency of micronuclei. NOx
enhanced mutation significantly inSalmonellaand Saccharomycesassays (Arroyo et al., 1992; Poli et al., 1992). They also mentioned a direct correla-tion between other components of air pollucorrela-tion, such us CO or Pb and genotoxicity. Based on these findings, it is possible to confirm that the increase in the MCN frequency observed in the present study should have been caused by high concentrations of pollutants. Since we performed field experiments in real conditions, it is not possi-ble to relate our results with some specific pollu-tant (Table 4) or if they represent the consequences of the complex mixture of gases present in the atmosphere.
The observed differences between control and exposed groups were not very striking, compar-ing, for example, with results of Ma et al. (1982), who found a greater variation between control plants of Tradescantia, clone 4430 (8% of mi-cronucleus) and those exposed to the atmosphere of public parking garages (maximum values 28%). This may indicate a relative lower sensitiv-ity of T. pallida to air pollutants, in comparison with clones classically employed for in situ mea-surements, such as clone 4430. The portable plants seemed to be more sensitive than the sen-tinel ones, since the portable plants were exposed to a less polluted air in both urban sites, during the summer season, when the rainfall is higher
(Setzer, 1966) and, even though, a much clearer urban-rural gradient in MCN frequency was ob-served (Table 4). Other advantages of using portable plants include the possibility to eliminate certain factors, such as differences in soil charac-teristics, water supply, stage of development and plant exposure, that may modify the results.
In conclusion, exposure of portable plants ofT. pallida, a popular garden plant in Brazil, to air pollutants may serve as an important tool for detecting the potential for genotoxicity of urban air pollution. Since the genotoxic risk assessment is a time-dependent value strictly correlated with the evaluation system being tested (Poli et al., 1992), it is helpful to develop long-term active experiments using T. pallida in the future, as suggested by Grant (1998), to further evaluate the relationship between air pollutants and genotoxic risks.
Acknowledgements
This study was supported by grants from PIE/
CNPq. We are gratefully acknowledged to M.Sc. Yuri Tavares Rocha, for designing Fig. 1, to CETESB (the governmental agency air quality control), for furnishing data of the air quality of Sa˜o Paulo and to the Elementary School E.M.E.F. Joa˜o Carlos da Silva Borges, for using its area.
References
Arroyo, P.L., Hatchpigott, V., Mower, H.F., Cooney, R.V., 1992. Mutagenicity of nitric-oxide and its inhibition by antioxidants. Mutat. Res. 281, 193 – 202.
Arutyunyan, R.M., Pogosyan, V.S., Simonyan, E.H., Atoy-ants, A.L., Djigardjian, E.M., 1999. In situ monitoring of the ambient air around the chloroprene rubber industrial plant using theTradescantia-stamen – hair mutation assay. Mutat. Res. 426, 117 – 120.
methodology and epidemiological evidence. Environ. Health Perspect. 22, 1 – 12.
CETESB. 1998. Relato´rio de qualidade do ar no Estado de Sa˜o Paulo, 1997. Se´rie Relato´rios.
Freedman, B., 1995. Environmental Ecology, 2nd edn., Aca-demic Press, San Diego, pp. 606.
Gopalan, H.N.B., 1999. Ecosystem health and human well being: the mission of the international programme on plant bioassays. Mutat. Res. 426, 99 – 102.
Grant, W.F., 1998. Higher plants assays for detection of genotoxicity in air polluted environments. Ecosyst. Health 4, 210 – 229.
Hoagland, D.R., Arnon, D.I., 1938. The water-culture method for growing plants without soil. Circ. 347, College of Agric. Univ. of Calif., Berkerley, pp. 39.
Ma, T.H., 1981.Tradescantiamicronucleus bioassay and pol-len tube chromatid aberration test for in situ monitoring and mutagen screening. Environ. Health Perspect. 37, 85 – 90.
Ma, T.H., Anderson, V.A., Ahmed, I., 1982. Environmental clastogens detected by meiotic pollen mother cells of Tradescantia. In: Tice, R.R., Costa, D.L., Schaich, K.M. (Eds.), Genotoxic Effects of Airborne Agents. Environ-mental Science Research, vol. 25. Plenum Press, New York, pp. 141 – 157.
Monarca, S., Feretti, D., Zanardini, A., Falistocco, E., Nardi, G., 1999. Monitoring of mutagens in urban air samples. Mutat. Res. 426, 189 – 192.
Poli, P., Buschini, A., Campanini, N., Vettori, M.V., Cassoni, F., Cattani, S., Rossi, C., 1992. Urban air-pollution-use of different mutagenicity assays to evaluate environmental genetic hazard. Mutat. Res. 292, 113 – 123.
Reyma˜o, M.S.F., Cury, P.M., Lichtenfels, A.J.F.C., Lemos, M., Battlehner, C.N., Conceic¸a˜o, G.M.S., Capelozzi, V.L., Montes, G.S., Ju´nior, M.F., Martins, M.A., Bo¨hm, G.M., Saldiva, P.H.N., 1997. Urban air pollution enhances the formation of urethane-induced lung tumors in mice. Envi-ron. Res. 74, 150 – 158.
Rodrigues, G.S., Madkour, S.A., Weinstein, L.H., 1996. Genotoxic activity of ozone inTradescantia. Envrion. Exp. Bot. 36, 45 – 50.
Rodrigues, G.S., Ma, T.H., Pimentel, D., Weinstein, L.H., 1997. Tradescantia bioassays as monitoring systems for environmental mutagenesis — a review. Crit. Rev. Plant Sci. 16, 325 – 359.
Rodrigues, G.S., Ma, T.H., Pimentel, D., Weinstein, L.H., 1998. In situ assessment of pesticide genotoxity in an integrated pest management program I — Tradescantia micronucleus assay. Mutat. Res. 412, 235 – 244.
Saldiva, P.H.N., Bo¨hm, G.M., 1998. Animal indicator of adverse effects associated with air pollution. Ecosyst. Health 4, 230 – 235.
Saldiva, P.H.N., King, M., Delmonte, V.L.C., Macchione, M., Parada, M.A.C., Daliberto, M.L., Sakae, R.S., Criado, P.M.P., Silveira, P.L.P., Silveira, Z., Bo¨hm, G.M., 1992. Respiratory alterations due to urban air pollution: an experimental study in rats. Environ. Res. 57, 19 – 33. Saldiva, P.H.N., Lichtenfels, A.J.F.C., Paiva, P.S.O., Barone,
I.A., Martins, M.A., Massad, E., Pereira, J.C.R., Xavier, V.P., Singer, J.M., Bo¨hn, G.M., 1994. Association between air pollution and mortality due to respiratory diseases in children in Sa˜o Paulo, Brazil: a preliminary report. Envi-ron. Res. 65, 218 – 225.
Saldiva, P.H.N., Pope, C.A. III, Schwartz, J., Dockery, D., Lichtenfels, A.J., Salge, J.M., Barone, I., Bo¨hm, G.M., 1995. Air pollution and mortality in elderly people: a time-series study in Sa˜o Paulo, Brazil. Arch. Environ. Health 50, 159 – 163.
Setzer, J., 1966. Atlas clima´tico e ecolo´gico do Estado de Sa˜o Paulo. Comissa˜o Interestadual da Bacia do Parana´ — Uruguai/Centrais Ele´tricas de Sa˜o Paulo, Sa˜o Paulo, Brazil, pp. 41.
Sparrow, A.H., Underbrink, A.G., Rossi, H.H., 1972. Muta-tion induced inTradescantiaby small doses of X-rays and neutrons: analysis of dose – response curves. Science 176, 916 – 918.
