Mutation Research 544 (2003) 313–319 Mutagenic activity as a parameter to assess ambient air quality for protection of the environment and human health Vera Maria Ferrão Vargas∗ Programa de Pesquisas Ambientais, Fundação Estadual de Proteção Ambiental Henrique Luis Roessler (FEPAM), Avenida Dr. Salvador França, 1707 CEP: 90690-000 Porto Alegre, RS, Brazil Received 5 May 2003; received in revised form 27 June 2003; accepted 30 June 2003 Abstract Atmospheric pollution has significant effects on maintaining the integrity of ecosystems and on the population’s quality of life. Epidemiological studies have clearly associated related health problems, especially respiratory diseases, with exposure to air pollution. Organic compounds adsorbed to the airborne particulate matter are mutagenic in the Salmonella/microsome assay, and a considerable number of them are known to be carcinogenic to rodents. Studies performed at four sites within the urban area of Porto Alegre, capital of the state of Rio Grande do Sul, identified higher mutagenic activity at the sites with heavier vehicle traffic in assays without and with metabolic activation. The responses varied at different seasons of the year, and the highest revertants per cubic meter (rev/m3 ) values were observed in spring for moderately polar compounds, and in summer for non-polar ones. A pilot study was also performed in the region under the influence of a industrial petrochemical area. Most of the sites studied within the industrial area, as compared to others sampled in the nearby environment, presented higher levels of mutagenic activity independent of total suspended particulates (TSP) concentration in the sample. In the urban and industrial regions, the observed mutagenic activities were strongly associated with the presence of polycyclic aromatic hydrocarbons (PAHs). The responses observed in the TA98NR and TA98/1,8-DNP6 strains suggest the activity of nitrocompounds in both studies. The Salmonella/microsome assay is a sensitive method to define areas contaminated by these compounds, even in samples with TSP values that are consistent with the legal environmental quality standards. © 2003 Elsevier B.V. All rights reserved. Keywords: Salmonella/microsome; Microsuspension assay; Nitrocompounds; Airborne particulate matter 1. Introduction Air pollution is considered by the World Health Organization as one of the environmental exposure situations that can affect human health, leading to acute respiratory infections, cancer, chronic respiratory and cardiovascular diseases. Studies show that nearly 30% of the total mortality in various industrialized countries are due to cancer. Although the ∗ Tel.: +55-51-333-46765; fax: +55-51-333-46765. E-mail address: [email protected] (V.M.F. Vargas). major causes for the increases of cancer incidence include lifestyle, personal habits and diet, the role of environmental changes caused by the industrial activities favoring the release of chemicals into the environment, polluting air, water, soil and potentially contaminating the food should be considered [1,2]. Among the malignant types of neoplasm, lung cancer is most prevalent around the world, with 12.3% of all new cases of cancer. In Brazil, lung cancer is the main cause of death from cancer, with rates of mortality of 13.00/100,000 in males and 5.45/100,000 in females are expected for 2003 . The rates of 1383-5742/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2003.06.020 314 V.M.F. Vargas / Mutation Research 544 (2003) 313–319 occurrence per region for malignant neoplasm of the trachea, bronchi and lungs indicate major variations in the different Brazilian states. For 2003, the highest rates are estimated for Rio Grande do Sul both for males (46.90/100,000) and for females (16.80/100,000) . 1.1. Risk factors From most estimates, about 80% of cancers have causal relationship to the environment, ranging from the environment in general (water, land and air), to occupational (chemicals and related industries), consumption (foods, medications) and social and cultural (lifestyle and habits). Smoking is considered the main risk factor for cancer in the respiratory system. Other risk factors include certain chemicals that are mainly found in the occupational environment—arsenic, asbestos, beryllium, chromium, radon, nickel, cadmium and vinyl chloride; dietary factors, such as the low consumption of fruits and vegetables; chronic obstructive pulmonary disease; genetic factors that predispose to the carcinogenic action of carcinogens; and family history of lung cancer . Epidemiological studies have reported associations between health problems, especially respiratory diseases, and exposure to fine and ultrafine airborne particles. The size and composition of these particles determine their behavior in the respiratory system and the time of residence in the environment [4–6]. A recent study , using data from the American Cancer Society’s Cancer Prevention Study II, related risk factors for approximately 500,000 adults to data concerning air pollution, living conditions and cause of death. The study identified that an increase in pollution related to the fine airborne particulate matter and sulfur oxide was associated with mortality in general, lung cancer and cardiopulmonary mortality. 1.2. Air pollution, mutagenicity and cancer Studies of urban air in big cities have clearly shown that the presence of substances with genotoxic potential might increase the risk of cancer in the populations . In Brazil, the total suspended particulates (TSP) are regulated by the National Council of the Environment  as a measure of environmental pollution for environmental surveillance in health defining primary and secondary air quality standards. Organic components with mutagenic activity, including polycyclic aromatic hydrocarbons (PAHs), particularly nitro-PAHs, are adsorbed to the airborne particulate matter [8,9]. A considerable number of them are known to be carcinogenic to rodents. Extracts collected in different cities of the world, including studies performed in Brazil [10–14] have shown variable levels of mutagenic activity in the Salmonella/microsome assay, from total or fractionated size particulate matter associated with vehicle emissions, industrial activities and waste incineration [5,10–17]. The diesel particulates belong to the class of respiratory-size particles which has been implicated as responsible for increased daily mortality and morbidity rates under ambient conditions of exposure [4,6,18]. Several studies assessed the mutagenic activity of atmospheric particulate matter, concluding that the mutagenic compounds are almost exclusively located on particles less than 2.0–3.3 m in diameter [5,17]. Pagano et al. , studying the mutagenic activity of particle-sized fractions, concluded that PM2.5 monitoring (respirable particulate matter) seems to combine a better air quality concept with effective health risk, at least with regard to the mutagenicity. 1.3. Salmonella/microsome assay The basic mechanism of the test is to determine changes in the DNA molecule, caused by reverse mutations for prototrophy, using Salmonella typhimurium mutants to assess the mutagenic activity and the carcinogenic potential of chemicals. The indicator strains present different mutations in the histidine operon, selected to detect substances that are able to generate base pair substitution or frameshift mutation. Strains derived from the classical TA98 and TA100 were developed bearing a deficiency in a classical nitroreductase (NR strains) or in an O-acetyltransferase (1,8-DNP6 strains) [19,20], enabling the diagnosis of the presence of mononitro and dinitroarenes, respectively. Watanabe et al. [21,22] developed a series of YGs strains with similar properties— YG1021(pYG216), 1024(pYG219), 1026(pYG216), 1029(pYG219)—but presenting high enzymatic production. The genes of classical nitroreductase (pYG216) and of O-acetyltransferase (pYG219) are inserted in the plasmids, conferring high enzymatic V.M.F. Vargas / Mutation Research 544 (2003) 313–319 activity and greater sensitivity for nitrocompounds, such as nitroarenes or aromatic amines, respectively. The assay may be performed by various procedures, the most widely used being plate-incorporation, pre-incubation and microsuspension, which presents a 5–10 times higher sensitivity than the traditional Ames test [23–25]. The test is performed in the presence and absence of an in vitro system of metabolic activation, the most commonly used being the microsomal fraction—S9. The S9 was composed of a homogenate from cells of the livers of Sprague–Dawley rats that were pre-treated with a polychlorinated biphenyl mixture (Aroclor 1254) to induce an increase in the P-450 enzymes in this organ. 1.4. Studies in Rio Grande do Sul Studies in Rio Grande do Sul, Brazil, have been performed in our research group to assess the mutagenic activity of air samples in regions under the influence of different anthropic contributions. These studies have made it possible to investigate the presence of mutagenic activity in atmospheric samples in an urban area [12,14] and an industrial area under petrochemical influence . The research studies assess the contribution of moderately polar and non-polar compounds using sample fractionation techniques according to their chemical characteristic, based on total particulate matter. By using the Salmonella/microsome assay with specific conditions and strains, it was possible to identify compounds with direct action or after hepatic metabolism, as well as the presence of reactive nitrocompounds with a high carcinogenic potential. 1.5. Mutagenic activity of particulate matter in the urban area of Porto Alegre The study was performed at two different periods: summer (December) of 1994 ; summer (December/February) of 1997/1998 and spring (October) of 1997 . The mutagenic activity of organic extracts of atmospheric particulate matter was assessed at four sites within the urban area of Porto Alegre: site 1—Jardim Botˆanico District—a site of reference for the urban area of Porto Alegre; site 2—Anchieta District—a mixed residential/industrial neighborhood, with industries ranging from small to medium-sized; site 3—São João District—a site with heavy traffic 315 (about 7400 vehicles/h), at the intersection of two avenues; site 4—Centro District—a site with heavy traffic (about 6800 vehicles/h) at the intersection of avenues next to the main intermunicipal and interstate bus station. Samples of TSP matter, collected on fiber glass filters using a high-volume sampler, were extracted by sonication, sequentially, with cyclohexane (CX) and dichloromethane (DCM) for rough fractionation by polarity. The different fractions were tested for mutagenicity using the S. typhimurium strains TA98 and the nitroreductase-deficient strains TA98NR and TA98/1,8-DNP6 to detect the presence of mononitro- and dinitroarenes [18,19,23,24]. During the first period sites 1–3 were investigated using the Salmonella/microsome assay [12,23]. Mutagenic frameshift responses were observed for sites 2 and 3, suggesting risk from environmental contamination by non-polar (CX) and/or moderately polar (DCM) compounds. The values of revertants per cubic meter (rev/m3 ) were highest at the site 3 with heavy vehicle traffic, in assays without (9.56 rev/m3 ) and with S9 mix (5.08 rev/m3 ). The highest mutagenicity was observed in moderately polar fractions, decreasing after metabolic activation. The responses observed for TA98NR and TA98/1,8-DNP6 strains suggest the activity of nitrocompounds. The similarity in the behavior of the moderately polar compounds at sites 2 and 3 suggests the contribution of similar sources of pollution in these fractions, resulting in a mixture of mono and dinitroarenes. However, the differences in non-polar compounds suggest major differences with the presence of nitrocompounds at site 2 and absence of nitrocompounds at site 3. During the second period, four sites were investigated during the summer and spring periods using the Salmonella/microsuspension method without S9 activation . The mutagenic frameshift responses observed varied at different seasons of the year and the highest rev/m3 values were observed at site 3 with heavy traffic in spring (17.1 rev/m3) in DCM-fractions, and in summer (13.9 rev/m3) in CX-fractions. A similar relationship is observed at site 4, which possesses anthropic contribution characteristics similar to 3. As to site 1, previously characterized as negative for mutagenicity , positive responses could be observed for moderately polar compounds and indicative for non-polar ones during the same season of the year. 316 V.M.F. Vargas / Mutation Research 544 (2003) 313–319 The mutagenic activity of most of the samples indicates the presence of a mixture of mononitroarenes and dinitroarenes in different proportions, or even of substances from another group of chemicals, suggesting various sources of pollution. The similarity in the behavior of the fractions at sites 2 and 3 during spring, suggesting the contribution of similar sources of pollution with the predominance of dinitroarenes with a moderately polar behavior should be highlighted. During summer, differences occur, suggesting different sources of contamination. Air quality studies performed at site 4, from March 1996 to September 1997 , identified 65 volatile organic compounds (VOC). These data add evidence for the varied responses of the organic fractions from site 4, suggesting the presence of substances with mutagenic activity characteristic of vehicle emissions associated to compounds from different anthropic sources. Although the data in this study point to an indicative association between the increased mass per unit volume of air (TSP) and the mutagenicity of organic extracts of airborne particulate matter (Table 1), the mutagenic activity assay was a sensitive method to define areas contaminated by these compounds, even in samples that present TSP values according to the legal environmental quality standards. 1.6. Mutagenic activity of particulate matter in a petrochemical industrial area A pilot study  was performed to investigate the mutagenic activity of airborne particulate matter collected at different sites in areas under the influence of a petrochemical complex, with an area of 14,600 ha, located 30 km upstream from Porto Alegre, the capital of Rio Grande do Sul, in a mixed rural, urban and industrial area. The kilometers show the distance (radius) to the main industry responsible for the production and distribution of the raw materials benzene, toluene, xylene, ethene, propane and butadiene. The samplings were performed monthly during the period from January to April 2000. Extracts of moderately polar compounds have been tested for frameshift mutagenicity using the microsuspension assay in S. typhimurium TA98 strain, with/without S9 mix. The results show that due to preferential wind directions, samples from the industrial (AI) reference area (AI01) showed lower levels measured in rev/g of organic material extracted (0.99) and rev/m3 (0.93) as detected by direct assay. The site inside the complex (AI02), chosen because it is located in the area where there is the highest deposition of particulates (TSP: 129.15 g/m3 ) 1.5 km from the main smokestack, is among the highest levels measured in rev/g (9.19) and rev/m3 (18.17). However, at two other locations in the environmental area outside (AA) the plant 7 km (AA01) and 9 km (AA02) from the complex, considering the same preferential wind directions, a 83 and 89% decrease of rev/m3 (3.17; 2.02) with TSP values of 53.60 and 75.73 g/m3 , respectively, were observed. Under metabolism conditions the relationship was similar but with lower absolute values. New investigations performed during the second period of the study (May–August), 3 km from the main smokestack, and in an area for the treatment of liquid wastes (AI03), presented much lower TSP values (23.30 g/m3 ) showing 19.15 rev/g and 21.18 rev/m3 in the mutagenicity test. Ranking the mutagenic activity values in rev/m3 as compared to the TSP values, the absence of a relationship between the largest TSP magnitudes and the levels of induced mutagenesis can be observed. The sites sampled in the industrial area, except for AI01, presented the highest levels of mutagenic activity independent of TSP concentration in the sample (Table 2). These results agree with studies that indicate that the mutagenic activity of material is more highly concentrated in the fine particulate matter . Continuing this study, there are ongoing evaluations of the contribution of nitrocompounds, as well as studies at different periods of the year, increasing the number of sampling sites with a view to providing further information for analyses of population risks. 1.7. Comparative studies Claxton et al.  presented a list of different studies performed in urban and industrial areas of different cities in Europe, Asia and North America, specifying the differences in methodologies, as to assay conditions and strains used. For purposes of comparison (Table 3) it can be seen that the values of the first period of analysis in the urban area of Porto Alegre are the lowest observed among the studies evaluated by the classic Ames test. Relating the results obtained in the studies that used the microsuspension assay, First perioda Sample TSP (g/m3 )c CX (rev/m3 ) DCM (rev/m3 ) CX + DCM (rev/m3 ) a Ames Second periodb December 1994 Site 1 December 1994 Site 2 December 1994 Site 3 October 1997 Site 1 October 1997 Site 2 February 1998 Site 4b December 1997 Site 1 October 1997 Site 3 December 1997 Site 2 December 1997 Site 4a December 1997 Site 3 41 – – – 57 1.16 1.71 2.87 101 0.46 9.56 10.02 17 – – – 19 – 0.82 0.82 56 2.83 3.86 6.69 60 2.35 4.23 6.58 71 – 17.13 17.13 99 6.76 – 6.76 99 6.19 5.81 12.00 161 13.01 2.81 15.82 test . b Microsuspension assay . of TSP (g/m3 ) for 24 h: primary standards 240 g/m3 . Negative controls spectrophotometry grade dimethyl sulfoxide (DMSO 5 l/pl): first period 30.25 ± 11.266, second period 39.65 ± 14.097; positive controls: first period 4-oxide-1-nitroquinoline (4NQO–0.5 g/pl) 255.0 ± 5.60, second period 2-nitrofluorene (2-NF 0.15 g/pl) 268.70 ± 56.526 [12,14]. Secondary standards 150 g/m3 ; CX: non-polar compounds; DCM: moderately polar compounds. c Concentration V.M.F. Vargas / Mutation Research 544 (2003) 313–319 Table 1 Relationship between mutagenicity (rev/m3 ) and particulate matter concentration (TSP (g/m3 )) in urban areas 317 318 V.M.F. Vargas / Mutation Research 544 (2003) 313–319 Table 2 Relationship between mutagenicity (rev/m3 ) and particulate matter concentration (TSP (g/m3 )) in industrial areas by microsuspension assay Sample (g/m3 )a TSP DCM (rev/m3 ) AI03 AI01 AA01 AA02 AI02 23.30 21.18 24.30 0.93 53.60 3.17 75.73 2.02 129.15 18.17 Sample: AI inside AA outside the industrial area. a Concentration of TSP (g/m3 ) for 24 h: primary standards 240 g/m3 , secondary standards 150 g/m3 ; DCM: moderately polar compounds; negative control (DMSO 5 l/pl): 37.50 ± 7.746; positive control (2-NF 0.15 g/pl): 255.75 ± 7.632. Table 3 Comparative studies using Salmonella mutagenicity to evaluate organic extracts of airborne particulate matter Sites [references] Porto Alegre, Brazil  Oslo, Norway (traffic)  Delft, The Netherlands  Oslo, Norway (street)  Chiba, Japan  Rio de Janeiro, Brazil  São Paulo, Brazil  Porto Alegre, Brazil  Brescia, Italy  Martinez, CA, USA  RS, Brazil  Oslo, Norway  Norway  Revertants (m3 ) Other informations TA98 − S9 TA98 + S9 0–9.6 0.8–12 5–60 8–45 3.6–16.5 18.0 11.6–34.9 0.8–17.3 2.0 85–296 0.9–21 1.5–10 2–16 0–5.1 0.5–7.3 3–65 15–18 10.1–35.6 7.6 304–727 0.3–5.5 1.5–10 2–13 Urban, pre-incubation Urban, plate Urban, plate Urban, plate Urban, pre-incubation Urban, plate Urban, plate Urban, microsuspension Urban, microsuspension Urban, microsuspension Industrial, microsuspension Industrial, plate Industrial, plate Sites: urban or industrial areas; assays: plate-incorporation , pre-incubation , microsuspension . the values observed in Porto Alegre are higher than those obtained in Brescia, Italy, and much lower than those reported in Martinez, USA. Comparing studies performed in Brazil using the Samonella/microssoma assay with pre-incubation or plate-incorporation methods [10–12,14], it can be seen that the values in rev/m3 observed in Porto Alegre are lower than those obtained in the cities of Rio de Janeiro and São Paulo. Comparing the evaluations in the industrial areas, higher responses are observed in the Rio Grande do Sul study using the microsuspension assay, than those performed in Norway using the classic Ames assay. The evaluation of mutagenic activity, associated with comparable methodologies and the expression of standardized results, allows the use of this parameter to characterize air quality in urban and industrial centers, estimating the potential risks to health and the environment, as a result of exposure to these complex mixtures. The study of sample fractionation and chemical identification of the main compounds present, are advances define corrective measures and safe parameters for environmental control. 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