ALL NFL AND NCAA SPREADS AND O/U`s ARE -110

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 [3]. The rates of
1383-5742/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrrev.2003.06.020
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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) [3].
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 [3].
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 [6], 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 [2]. In Brazil, the total suspended particulates (TSP) are regulated by the National Council
of the Environment [7] 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. [5], 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 [13]. 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 [12]; summer (December/February) of 1997/1998 and spring (October) of
1997 [14]. 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 [14]. 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 [12], positive responses
could be observed for moderately polar compounds
and indicative for non-polar ones during the same
season of the year.
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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
[26], 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 [13] 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 [5].
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. [17] 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 [12].
b Microsuspension
assay [14].
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 [7]; 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 [7]; 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 [12]
Oslo, Norway (traffic) [17]
Delft, The Netherlands [17]
Oslo, Norway (street) [17]
Chiba, Japan [17]
Rio de Janeiro, Brazil [10]
São Paulo, Brazil [11]
Porto Alegre, Brazil [14]
Brescia, Italy [17]
Martinez, CA, USA [17]
RS, Brazil [13]
Oslo, Norway [17]
Norway [17]
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 [23], pre-incubation [23], microsuspension [24].
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.
Acknowledgements
We are grateful to Adriana Ducatti,1 Rubem C.
Horn, Jocelita A.V. Rocha,1 Regis R. Guidobono,1
Irascema Girardi,1 Ana B. Mittelstäedt,1 Lizandra
Bringueti,1 Carlos A.N. dos Santos, Ana Carolina
Costa,1 Andrea da Silva Lopes, for their great support
during the development and finalization of the study.
This research was supported by the Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnológico
(CNPq) and Financiadora de Estudos e Projetos
(CIAMB/PADCT/FINEP).
1 CNPq and FINEP also supplied the scholarships for graduate
students that enabled their participation in different phases of this
study.
V.M.F. Vargas / Mutation Research 544 (2003) 313–319
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