Mutagenic activity of airborne particulate matter from the urban area of Porto Alegre, Brazil

Mutagenic activity of airborne particulate matter from the urban area of Porto Alegre, Brazil

Vera Maria Ferrão Vargas, Rubem Cesar Horn, Régis Rolim Guidobono,

Ana Beatriz Mittelstaedt and Irascema Girardi de Azevedo

Programa de Pesquisas Ambientais, Divisão de Biologia, Departamento de Laboratórios, Fundação Estadual de Proteção Ambiental Henrique Luis Roessler (FEPAM), Avenida Dr. Salvador França, 1707, 90690-000 Porto Alegre, RS, Brasil. Send correspondence to V.M.F.V.

ABSTRACT

The mutagenic activity of airborne particulate matter collected from three different sites within the urban area of Porto Alegre, Brazil, was investigated using a Salmonella/microsome assay. Samples were extracted by sonication, sequentially, with cyclohexane (CX), and dichloromethane (DCM), for a rough fractionation by polarity. The different fractions were tested for mutagenicity using Salmonella typhimurium strains TA98, with and without metabolic activation (S9 mix fraction), and TA98NR and TA98/1,8-DNP₆, without metabolic activation. Mutagenic response was observed for frameshift strain TA98 in assays with and without metabolization for two sites (sites 2 and 3), which had considerable risk of environmental contamination by nonpolar (CX) and/or moderately polar (DCM) compounds. However, the values of revertants/m³ (rev/m³) were highest on the site subject to automobile exhaust (site 3) in assays without (9.56 rev/m³) and with metabolization (5.08 rev/m³). Maximum mutagenic activity was detected in the moderately polar fraction, decreasing after metabolization. Nevertheless, the nonpolar fractions (CX) gave higher mutagenic activity in the presence of metabolization than in the absence of the S9 mix fraction. The responses observed for TA98NR and TA98/1,8-DNP₆ strains suggest the activity of nitrocompounds.

INTRODUCTION

Air pollution has been related to an increase in the incidence of respiratory diseases such as bronchitis and pulmonary emphysema. Studies of urban air in big cities have clearly shown the presence of substances with genotoxic potential, indicating that air pollution might increase the risk of cancer in the populations (Menck et al., 1974; Walker et al., 1982). Extracts of airborne particulate matter, collected in different cities, have shown high levels of mutagenic activity in the Salmonella/microsome assay, associated with vehicle emissions, industrial activities and waste incineration (Hughes et al., 1980; Chrisp and Fisher, 1980; Claxton, 1983; Tokiwa et al., 1983; Barale et al., 1989; Miguel et al. 1990; Sato et al., 1995; Crebelli et al., 1995; DeMarini et al., 1996). Mutagenic activity of particulate matter has been associated with the presence of polycyclic aromatic hydrocarbons (PAH) derivatives. Among these, the nitro-PAHs have often been implicated in mutagenic and genotoxic activity. A considerable number of them are known to be carcinogenic to rodents. The diesel particulates belong to the class of "PM-10" material (respiratory size particles) which has been implicated as responsible for increased daily mortality and morbidity rates under ambient conditions of exposure (Rosenkranz and Mermelstein, 1983; Pope et al., 1995; Rosenkranz, 1996). In Brazil, genotoxic evaluation studies of particulate material in Rio de Janeiro (Miguel et al., 1990) and São Paulo (Sato et al., 1991, 1995) confirmed mutagenic activity related to industrial activity and vehicle emissions.

This paper investigates the mutagenic activity of urban air samples from Porto Alegre, in three different sites subject to different pollution sources, using the Salmonella/microsome assay. Porto Alegre is the seventh Brazilian city in population, and is the capital of Rio Grande do Sul State, having in its urban area 1,258,160 inhabitants and approximately 575,000 vehicles circulating daily.

MATERIAL AND METHODS

Sample collection

Samples of suspended particulate matter (corresponding to about 2,000 m³ air) were gathered using a high-volume sampler (General Metal Works Inc.) operated at a flow rate of 1.3-1.5 m³/min for 24 h, equipped with glass fiber filters (AP40-810, 20 x 25-cm Millipore). The filters were weighed before and after sampling (45% humidity) to calculate the total suspended particles (TSP) expressed in units of µg/m³ of sampled air (Associação Brasileira de Normas Técnicas, 1988).

Sampling sites

Particulate samples were taken from three different locations in Porto Alegre. This city is located 110 km from the coast, and comprises an area of 502.5 km² with an approximate altitude of 10 m. It has a temperate climate and average annual temperatures of 20°C. The sites chosen for this study were sampled during summer (average temperature of 28°C, with temperatures varying from 23°C to 36°C) with the following characteristics:

Site 1: located in a meteorological station about 1 km from an avenue with relatively moderate traffic (about 3,000 vehicles/h), far from big industrial areas and near the Botanical Garden;

Site 2: a mixed residential/industrial neighborhood, industries ranging from small to medium size (mainly: clothing, food, metallurgy, chemical products, paper goods, processing of latex and plastic), with the influence of about 600 vehicles per hour, and 2 km from a federal highway;

Site 3: a site of heavy traffic (about 6,300 vehicles/h), located at an intersection of two avenues.

Sample preparation

Half of each filter was extracted sequentially by sonication with cyclohexane (CX) and dichloromethane (DCM), to separate nonpolar and moderately polar fractions, respectively. The samples were sonicated in three rounds of 10 min each using 1 ml of solvent for each mg of TSP, at 40°C. The extracts were filtered in a Teflon membrane of 0.5-µm porosity and later concentrated in a rotating evaporator to 15 ml. The extractable organic matter (EOM) concentration in µg/m³ was determined from the total EOM quantity per filter divided by the total sampled air volume. The concentration of EOM in % represents the percentage of EOM in the TSP. The concentrated extracts were stored at -20°C. When mutagenicity tests were to be made, appropriate volumes of extracts were reduced until dryness with gaseous nitrogen and resuspended in spectrophotometry grade dimethyl sulfoxide (DMSO).

Mutagenic assay

Mutagenicity tests were made with the Salmonella/microsome assay, through the preincubation procedure according to Maron and Ames (1983), using Salmonella thyphimurium strains TA98 with and without metabolization (S9 mix fraction), kindly provided by Dr. Bruce Ames, University of California, Berkeley, CA, USA, and nitroreductase-deficient strains TA98NR and TA98/1,8-DNP₆ (kindly provided by Dr. Gisela Valent, CETESB, São Paulo, SP, Brasil) without S9 mix fraction. TA98NR is a derivative of the TA98 strain and is considered to be deficient in classical nitroreductase, whereas TA98/1,8-DNP₆ is likely to be deficient in a transacetylase (Rosenkranz and Mermelstein, 1980). The presence of mononitro and dinitroarenes was examined using TA98NR and TA98/1,8-DNP_6. The S9 mix fraction was prepared from livers of Sprague-Dawley rats pretreated with a polychlorinated biphenyl mixture (Aroclor 1254), as described by Garner et al. (1972) and Maron and Ames (1983). Five doses of each fraction (13-200 µg/plate) were tested and all assays were carried out in duplicate. Plates were incubated in the dark at 37°C for 48 h, after which revertants and surviving colonies were counted. A negative control (5 µl DMSO solvent) and positive controls (0.5 µg 4-oxide-1-nitroquinoline (4NQO) and 0.2 µg 2-nitrofluorene (2NF) per plate for strains TA98, TA98NR and TA98/1,8-DNP₆) were added to each test. For the metabolic activation assay 2-aminofluorene (2AF- 5 µg/plate) was added to TA98, as a positive control.

In the cytotoxicity test, the solution containing the sample and the bacterial culture (100-200 cells) were plated on nutrient agar plates and incubated at 37°C. A reading of the surviving colonies was taken 48 h later (Vargas et al., 1988).

Statistical analysis

The final criterion used to interpret the results of the Salmonella mutagenicity assay was similar to that described by Vargas et al. (1993), i.e., a number of revertants double that of the spontaneous yields, accompanied by a reproducible dose-response curve, were considered a significant effect. The significance of linear regressions from the linear portions of the dose-response curves was evaluated by the SALMONEL software (Myers et al., 1991) as described by Vargas et al. (1993). When one of these two criteria was satisfied, the sample was considered to present signs of mutagenicity. Samples were considered to be cytotoxic when less than 60% cell survival was obtained in the cell viability test, and tests were negative when a mutagenicity of less than twice the spontaneous yields was detected, with more than 60% cell viability.

RESULTS

Particulate and extractable organic matter concentrations

Site 1 presented the smallest values of TSP and EOM in µg/m³ (Table I); the particles collected at site 2 had the highest percentage of EOM of the total particle mass (22.32%), presenting also the largest quantity of substances with nonpolar characteristics (9.30%) and substances with moderately polar characteristics (13.02%) (Table I). Site 3 had the highest concentration of TSP and the smallest percentage of EOM. The DCM solvent proved to be the best extraction agent for the particulate matter. There was a larger concentration of moderately polar substances in relation to the concentration of nonpolar ones (extracted by CX).

Table I
- Concentrations of total suspended particles (TSP) and extractable organic matter (EOM) in air sampled from three sites in Porto Alegre, Rio Grande do Sul State.

		EOM (%)			EOM (µg/m³)
Samples	TSP (µg/m³)	CX	DCM	Total	CX	DCM	Total
Site 1	41.00	8.11	9.84	17.95	3.32	4.04	7.36
Site 2	57.00	9.30	13.02	22.32	5.30	7.42	12.72
Site 3	101.00	5.00	6.70	11.70	5.04	6.76	11.80

EOM (%) - Percentage of extractable organic matter in total suspended particles.

EOM (µg/m³) - Extractable organic matter, calculated from the total quantity of EOM per filter/total volume of sampled air.

CX - Cyclohexane, a nonpolar solvent; DCM - dichloromethane, a moderately polar solvent. Site 1 - Near the Botanical Garden; Site 2 - mixed residential/industrial neighborhood; Site 3 - site of heavy traffic.

Mutagenic activity

Mutagenic activity was observed in the two sites with high risk of environmental contamination (sites 2 and 3), in assays with and without metabolization (Table II). All dose-response curves were linear, although at higher doses the cytotoxicity test presented a decrease in cellular viability. The direct mutagenic activity was higher in moderately polar fractions, while the nonpolar fractions presented a higher induction in assays made after metabolization. Direct mutagenic induction was equal for nonpolar and moderately polar substances, for site 2. However, the mutagenic activity of moderately polar compounds was reduced by metabolization, resulting in an index (ratio between the number of revertants induced per plate and the number observed in the negative control) 1.8 times the negative control at the highest dose tested. This result, however, indicated signs of mutagenicity in the statistical analysis. At site 3, which presented higher mutagenic values (indexes of 8.9 and 5.9 times the negative control with or without S9 mix fraction for 200 µg/plate), the induction was higher for compounds extracted by DCM, mainly in the assay without metabolization (1.42 rev/µg). Metabolization reduced the mutagenicity of the existing compounds in this fraction (0.75 rev/µg) by 47%. However, the metabolites produced by S9 mix fraction in the compounds present in the nonpolar fraction gave 4.5 times the induction found without metabolic activation. Analysis for site 1 showed values lower than twice the spontaneous mutagenic activity values (Table II).

Table II
- Mutagenic activity of extractable organic matter fractions of airborne particulate matter in Porto Alegre city by TA98 strain Salmonella/microsome assay.

		Mutagenic indexes (a)						Mutagenicity
								Revertants/µg(b)	Revertants/m3(c)
Concentration µg/plate			13	25	50	100	200
		- S9 (d)	1.1	1.1	0.9	1.2	1.3	—	—
	DCM
		+ S9	0.9	1.0	1.3	1.2	0.5	—	—
SITE 1
		- S9	0.8	1.0	1.2	1.6	1.1	—	—
	CX
		+ S9	0.9	1.2	1.2	1.1	1.2	—	—
		- S9	0.7	0.7	1.2	1.2	2.0	0.23 ± 0.038	1.71
	DCM
		+ S9	0.6	0.7	1.2	1.1	1.8	0.15 ± 0.071	1.12
SITE 2
		- S9	0.9	0.9	1.4	1.6	2.2	0.22 ± 0.043	1.16
	CX
		+ S9	1.0	1.0	1.2	2.0	2.3	0.28 ± 0.081	1.50
		- S9	0.8	3.0	3.8	5.8	8.9	1.42 ± 0.094	9.56
	DCM
		+ S9	1.9	1.7	1.7	2.8	5.9	0.75 ± 0.112	5.08
SITE 3
		- S9	0.9	1.1	0.8	1.1	2.3	0.09 ± 0.036	0.46
	CX
		+ S9	1.7	1.7	1.7	3.2	4.3	0.41 ± 0.085	2.04

(a) Mutagenic index: No. of HIS+ (revertant colonies) induced in the sample/No. of spontaneous HIS+ in the control. Mutagenic index in bold type: number of revertants double the spontaneous yields.

(b) Number of revertants per unit mass of particles: data obtained from the linear regressions (slope ± SD, b £ 0.05) of the dose-response curve linear portions, calculated by the SALMONEL software (Myers et al., 1991).

(c) Number of revertants per volume unit of air.

(d) +S9, -S9: with or without metabolic activation.

Test controls _ average number of HIS+ revertants/plate: negative control DMSO (5 µl/plate) -S9mix 30.25 ± 11.266, +S9mix 32.58 ± 6.515; positive control: -S9mix 4NQO (0.5 µg/plate) 255 ± 5.60, +S9mix 2AF (5 µg/plate) 1028 ± 89.10.

Abbreviations and identification of sites - see Table I.

The EOM fractions, with mutagenic activity, were analyzed in the classical nitroreductase and O-acetyltrans-ferase-deficient derivative strains TA98NR and TA98/1,8-DNP₆. Various extracts had lower mutagenic activity in the nitroreductase-deficient strains than with the TA98 strain, except for the nonpolar fraction obtained from the site 3 sample, which did not show a reduction in mutagenic activity (Table III and Figure 1). There was a decrease of 61 to 91% in the mutagenic activity of the organic extracts from sites 2 and 3, highlighting the action of mono and dinitroarene compounds. An inhibition of 91% was observed in the TA98/1,8-DNP₆ strain for the sample from site 2 extracted with CX, highlighting the action of dinitroarene compounds.

Table III
- Mutagenic potencies (revertants/µg) of extracts from the airborne particulate sample fractions.

Salmonella typhimurium strains (a)
Site	TA98 - S9	TA98 + S9	TA98/NR - S9	TA98/1,8-DNP6-S9
Site 2
DCM	0.23 ± 0.038	0.15 ± 0.071	0.03 ± 0.013	0.09 ± 0.016
CX	0.22 ± 0.043	0.27 ± 0.081	0.08 ± 0.030	0.02 ± 0.022
Site 3
DCM	1.42 ± 0.094	0.75 ± 0.112	0.36 ± 0.041	0.49 ± 0.056
CX	0.09 ± 0.036	0.41 ± 0.085	0.13 ± 0.059	0.30 ± 0.132

^(a)TA98detects frameshift mutagens. The derivative strains TA98NR (classical nitroreductase deficient) and TA98/1,8-DNP₆ (transacetylase deficient) are useful in determining direct-acting nitrocompounds.

For abbreviations see Tables I and II.

Figure 1
- Dose-response curves of the extractable organic matter from different sites in TA98, TA98NR and TA98/1,8-DNP₆ strains, without metabolization. For abbreviations see Table I.

DISCUSSION

Evaluation of mutagenic activity in a Salmonella/microsome assay of compounds found in airborne particulate matter was used here, as a biological model to study urban air contamination. The determination of particulate concentration in the air is one of the quality evaluation measures that FEPAM (State Foundation for Environmental Protection) monitors in the State of Rio Grande do Sul. The concentration of particles present in the investigated samples (Table I) is in agreement with the annual average expected by the institutional studies for the different sites analyzed (site 1: 37, site 2: 66 and site 3: 99 µg/m³ of TSP).

It was possible to observe some important differences in the three sample sites. The predominant mutagenic activity in site 2 was associated with a nonpolar fraction, when subjected to metabolization. It was also present in moderately polar compounds, without the S9 mix fraction. Though these fractions (from site 2) presented the highest EOM values, the mutagenic activity observed in the majority of assays (except for the fractions extracted with cyclohexane, direct assay) was smaller than that observed for site 3. The mutagenic activity from site 3 was mostly due to moderately polar compounds, and its action was reduced by metabolization. The nonpolar fraction presented, in both sites (2 and 3), the highest mutagenic activity associated with the presence of metabolic activation. Miguel et al. (1990), in a study of fractions of airborne particulate matter collected in the city of Rio de Janeiro, found that the predominant mutagenic activity resides in the nonpolar fraction, which contains direct frameshift mutagens, with a decrease of the mutagenic activity after metabolic activation. The same study also emphasized that the activity observed for the moderately polar fractions was higher after metabolization. These results differ from ours, as a higher mutagenic activity was found in the DCM-fraction, without metabolization. In a comparison of airborne particulate matter studies in Brazil, Porto Alegre had less mutagenic activity (0 to 9.56 rev/m³ without S9 mix fraction and 0 to 5.08 rev/m³ with S9 mix fraction, in summer) than found by Miguel et al. (1990) in the city of Rio de Janeiro (18 rev/m³ without S9 mix fraction, in winter) and by Sato et al. (1995) in the city of São Paulo (34.92 rev/m³ without S9 mix fraction and 35.60 rev/m³ with S9 mix fraction, in summer). Considering all seasons, Sato et al. (1995) found higher values in spring (55.20 without S9 mix fraction and 68.28 rev/m³ with S9 mix fraction) and lower in winter (1.72 without S9 mix fraction and 1.34 rev/m³ with S9 mix fraction).

The values observed for urban and industrial sites of different cities throughout the world (see Sato et al., 1995) present a variation of 1 to 288 rev/m³ for the TA98 without metabolization and 1 to 690 rev/m³ for TA98 with metabolization. The methodologies applied in the different trials and the necessity of standardizing the procedures in order to obtain comparative data have to be considered (Claxton et al., 1992).

Contribution of nitrocompounds to mutagenic activity

The contribution of the nitrocompounds to the mutagenic activity of air samples from urban areas is associated with the presence of polycyclic aromatic hydrocarbon (PAH) derivatives. Among those, the mono and dinitro-PAHs associated with oxygenated PAH have been correlated with direct frameshift mutagenic activity (Miguel et al., 1990; Adonis and Gil, 1993; Crebelli et al., 1995; Sato et al., 1995; DeMarini et al., 1996). There have been investigations of mutagenic activity using TA98NR (nitroreductase deficient) and TA98/1,8-DNP₆ (transacetylase deficient) strains of S. typhimurium which have proved useful in determining direct-acting nitrocompounds (Mermelstein et al., 1981; Rosenkrantz, 1996).

Site 2 samples showed reduced mutagenic activity, pointing to the presence of mononitroarenes and dinitroarenes in the two fractions (DCM-fraction and CX-fraction). In fractions of site 3, only the moderately polar compounds presented a reduction in the mutagenic activity, indicating that nitroarenes have a significant contribution to the mutagenicity of this fraction. The CX-fraction compounds of site 3 did not present reduction in mutagenicity, suggesting the absence of the contribution of nitrocompounds in the frameshift mutation detected (Figure 1 and Table III).

The similarity in the behavior of the moderately polar compound fractions in the two sites (2 and 3) evaluated suggests the contribution of similar polluting sources in these fractions. The differences in the behavior of the nonpolar compound fractions were as follows: presence of nitrocompounds in site 2 (mononitro and dinitroarenes) and absence of nitrocompounds in site 3. This suggests important differences in the composition of these fractions. Additionally, there was a 4.5 times higher mutagenic induction, with metabolization. The composition of nonpolar fractions might include aliphatic hydrocarbons, PAH and nitro-PAHs. However, even after the fractionating by solvents, the extracts obtained are complex mixtures subject to synergistic and antagonistic interactions among different compounds (Butler et al., 1987; Miguel et al., 1990).

We can conclude that the different polluting sources resulted in different mutagenic activity patterns. The greatest mutagenesis induction came from air samples where there were the most vehicles and the highest concentration of total particulate matter in suspension. The moderately polar compounds gave the highest indexes of mutagenic activity. The assays conducted with the nitroreductase-deficient strains and fractionation of the sample allowed us to suggest the most important kind of contamination from each site studied.

ACKNOWLEDGMENTS

We are grateful to FEPAM's air collection team and to A. Pacheco from FEPAM's Quality Department, for the sampling performed and for the information on the history of the particulate material concentration. We also thank the official institutes for geographic (IBGE), meteorological (8°DISME), automobile traffic and industrial activity information (SMIC). This research was supported by CIAMB/PADCT/FINEP and CNPq.

RESUMO

Foi investigada a atividade mutagênica de material particulado de amostras de ar coletadas em três diferentes locais dentro da área urbana da cidade de Porto Alegre, Brasil, através do ensaio Salmonella/microssoma. As amostras foram extraídas, em ultra-som, por fracionamento seqüencial de acordo com a polaridade, utilizando os solventes ciclohexano (CX) e diclorometano (DCM). As diferentes frações foram testadas para mutagenicidade com as linhagens de Salmonella typhimurium TA98, em presença e ausência de ativação metabólica, e TA98NR e TA98/1,8-DNP₆ em ausência de metabolização. Observou-se resposta mutagênica positiva, do tipo erro no quadro de leitura, na linhagem TA98 (em ensaios em presença e ausência de metabolização) para compostos não polares (CX) e/ou moderadamente polares (DCM) nos locais com considerável risco de contaminação ambiental (locais 2 e 3). No entanto, os valores de revertentes por m³ (rev/m³) foram mais elevados no local sujeito a maior influência de veículos automotores (local 3), tanto em ensaios em ausência (9,56 rev/m³) como em presença de metabolização (5,08 rev/m³). A atividade mutagênica máxima foi detectada na fração moderadamente polar, decrescendo após metabolização. No entanto, as frações não polares (CX) apresentaram atividade mutagênica mais elevada na presença do que na ausência de fração S9mix. As respostas observadas para as linhagens TA98NR e TA98/1,8-DNP₆ sugerem a contribuição de nitrocompostos na atividade mutagênica observada.

(Received August 28, 1996)

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Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  June 1998

History

• Received
  28 Aug 1996