Mutagenic properties of PM2.5 urban pollution in the Northern Italy: The nitro-compounds contribution

Environment International 35 (2009) 905–910

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Environment International

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Mutagenic properties of PM2.5 urban pollution in the Northern Italy:The nitro-compounds contribution

Deborah Traversi a, Raffaella Degan a, Roberto De Marco c, Giorgio Gilli a, Cristina Pignata a,Simona Villani b, Roberto Bono a,⁎a Department of Public Health and Microbiology, University of Torino, Italyb Department of Health Sciences, Unit of Medical Statistics and Epidemiology, University of Pavia, Italyc Unit of Epidemiology and Medical Statistics, Department of Medicine and Public Health, University of Verona, Italy

⁎ Corresponding author. Department of Public Healthof Torino, via Santena 5 bis, 10126, Torino, Italy. Tel.0112365818.

E-mail address: [email protected] (R. Bono).

0160-4120/$ – see front matter © 2009 Elsevier Ltd. Aldoi:10.1016/j.envint.2009.03.010

a b s t r a c t

Article history:Received 15 December 2008Accepted 25 March 2009Available online 29 April 2009

Keywords:PM2.5MutagenicityUrban air pollutionNitroreductase activityNitro-compounds

PM2.5 is the breathable fraction of the particulate matter and some adverse health effects, such as respiratoryfunctionality, cardiological diseases and cancer, can be in some measure attributable to this risk factorexposure. Some of the most carcinogen compounds transported by PM2.5 are nitro-compounds. In this study,a strengthened in vitro bioassay — able to predict the mutagenic/carcinogenic activity of the environmentalmixtures — was conducted on PM2.5 organic extracts to define the nitro-compounds burden. PM2.5 airpollution was daily monitored, during 2006, in three cities located in the Northern part of Italy (Torino, Paviaand Verona) and themutagenic properties of the PM2.5 organic extractswere assessedwith the Ames test. Thebacterial used in this study were three Salmonella typhimurium strains: TA98, nitroreductase-less mutantTA98NR and YG1021 carrying a nitroreductase-producing plasmid. The annual PM2.5 mean level measured inTorino was 46.5 (±31.6) μg/m3, in Pavia 34.8 (±25.1) μg/m3, and in Verona 37.3 (±27.8) μg/m3, while themutagenicity expressed as TA98 net reverants/m3 was 28.0 (±22.1), 28.3 (±24.9), and 34.2 (±30.9)respectively. Monthly pool bioassays, conducted with the three different strains, showed a greater mutagenicresponse of the YG1021 in each city. The relationship among themutagenic answers for YG1021:TA98:TA98NRwas about 6:3:1 (pb0.001). Over nitroreductase activity enhanced the response of 2.2, 2.0 and 1.7 times forTorino, Pavia, and Verona (ANOVA Torino pb0.05) respectively. Without nitroreductase activity thegenotoxicity was limited. These biological findings are able to describe a relevant role played by the nitrocompounds in the mutagenic properties of the urban PM2.5 in the Padana plain; moreover the bacterialnitroreductase plays a predominant role in DNA interaction primarily for Torino PM2.5 extracts.

© 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Ambient air particulate matter (PM), a heterogeneous and com-plex mixture constituted by liquid and solid particles, is able to carrya large variety of chemical compounds among which hazardousmolecules for humanheath. PMoriginates as primary particles emitteddirectly into the atmosphere as well as secondary particles producedfrom atmospheric chemical reactions between gases precursors orbetween these and primary particles (WHO, 2005). The fine mode ofambient PM, named PM2.5, is defined as particles having aerodynamicdiameters below 2.5 μm. This PM fraction is part of PM10 and is able toovertake the alveolar lung region where blood exchange takes place.This physic property in association with the chemical characteristics

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underlines and explains hazardous properties of PM2.5 demonstratedfor human health (Areskoug, 2000; Brugge et al., 2007; Ciencewickiet al., 2008). Furthermore, fine particulate pollution is one of the mostimportant environmental issue not solved yet in Europe, especially intheMediterranean area (Hazenkamp-von Arx et al., 2004; Vineis et al.,2006; Rodriguez et al., 2007). PM2.5 origins from both natural andanthropogenic sources (resulting a complex chemical mix). Naturalsources are largely due to soil dust, sea salt, geological disturbances,biological debris, forest fires, and oxidation of biogenic reactive gaseswhile anthropogenic particles and reactive precursor gas sourcesinclude fossil fuel combustion from stationary and mobile sources,fugitive emissions, and various industrial, commercial, and residentialactivities (Chan andYao, 2008; Viana et al., 2008). Secondary inorganiccompounds (non-sea salt-SO4

2−, NO3−, NH4

+) are around 40–50% oftotal PM2.5, organic matter and elemental carbon around the 30–40%,mineral dust about 15%, then there are sea salt and trace elements.PM2.5 composition can vary in order to anthropic density and humanactivity in the area, season, meteorological and orographic condition(Querol et al., 2004; Viana et al., 2008).

906 D. Traversi et al. / Environment International 35 (2009) 905–910

Fine PM is involved in chronic adverse health effects such as thedecrease of respiratory functions and cancer (Sioutas et al., 2005).Exposition to PM has been linked to cancer effects in humans and otheranimals since more than 50 years (and to mutagenic effects inprokaryotic and eukaryotic systems since more than 30 years) (Claxtonand Woodall, 2007). Exposition to fine PM is correlated to lung cancermorbidity (Beeson et al., 1998; Nyberg et al., 2000; Gallus et al., 2008).These effects can mainly be due both to the quality of pollutants carriedon the particles surface and to the chemical nature of PM mixture.Pathogenesis pathway are proposed by several authors, especially forfine PM (Karlsson et al., 2008; Ciencewicki et al., 2008; Møller et al.,2008). Chemicals such ashydrocarbons (aliphatics, cycloalkanes,mono-and bicyclic arenes, and polycyclic arenes), oxygen-containing, sulfur-containing, nitrogen-containing, halogen-containing and organometal-lic compounds are responsible of the major mutagenicity and/orcarcinogenicity (Claxton et al., 2004; Claxton and Woodall, 2007).Among this wide set of molecules a relevant toxic role is attributed tonitro-compounds suchasnitro PolycyclicAromaticHydrocarbons, nitro-PAHs (Morales et al., 2006). Nitro-substituted compounds such assimple nitro aromatic and nitro heterocyclic chemicals are usuallyassociated with synthetically manufactured drugs, dyes, explosives andother highly energetic compounds. Their occurrence in the atmosphereis due both to direct emissions as primary pollutants, especially fromdiesel exhaust combustion, and to photochemical pollution (Cecinato,2003; Winkler and Hertweck, 2007). The concentration of thesepollutants in the urban air is around few ng/m3 and they are moreabundant in the PM fine fraction (Cecinato et al., 1999). Many of these

Fig. 1. Sampling cities are placed in the south part

compounds are potent bacterial mutagens, mammalian cell mutagensand clastogens, and some are also rodent carcinogens (Cecinato, 2003;Coronas et al., 2008).

Mutagenic activity of nitro aromatic compounds, in bacteria andmammals, generally depends on the biological reduction of NO2 func-tional groupbyenzymesknownasnitroreductases (Haynes et al., 2002).In bacteria and mammals, the mutagenicity of nitro-aromatic com-pounds is due to the biological reduction of the NO2 functional groupby enzymes named nitroreductases. This enzymatic activity can beassociated to ring-oxidation reactions producing the formation ofmolecules more reactive than the original ones. The possible biochem-ical activity of enzymatic pathway includes: 1) only nitroreduction,2) nitroreduction and esterification, 3) only ring oxidation by P450cytocrome, 4) ring oxidation and nitroreduction, 5) ring oxidation,nitroreduction and esterification (Hrelia et al., 1999; Salamanca-Pinzónet al., 2006). The intermediates of nitroreduction may yield ionelectrophiles that can interact with DNA to form adducts and otherkind of pro-lesions (Møller et al., 2008). The genotoxic activity of nitro-substituted compounds in both E. coli and Salmonella typhimuriumwasdemonstrated to be markedly influenced by cellular nitroreductaseactivities (Carroll et al., 2002). This evidence was observed testingextracts of diesel and gasoline emissions, fly ash particles, cigarettesmoke condensates and other environmental mixtures (Josephy et al.,1997). The role of the nitroreductases to metabolize into more muta-genic products fine PM nitro compounds is well known (Salamanca-Pinzòn et al., 2008); While lack of data are published on the nitrocompounds responsibility to produce mutagenic effects testing

of Europe, in the Padana Plain, under the Alps.

907D. Traversi et al. / Environment International 35 (2009) 905–910

European PM2.5 urban mixture. In this study, PM2.5 air levelswere monitored, during the 2006 in Torino, Pavia and Verona, threeNorthern Italian cities, known as heavy polluted cities among Europeancountries (Hazenkamp-von Arx et al., 2004). Salmonella assays wereconducted on the PM2.5 organic extracts by means of bacterial strainsmodified to detect nitro-compounds role on the observedmutagenicity.

2. Materials and methods

2.1. Sampling

The sampling activity of ECRHS II (Hazenkamp-von Arx et al.,2004, Götschi et al., 2005) was continued by the Italian group toevaluate the mutagenic properties of the PM2.5 organic extracts (Gilliet al., 2007a; Traversi et al., 2008). Sampling lasted 12 months con-tinuously from January to December 2006 including February 2006,the month of Torino Winter Olympic Games, in the three southernEuropean cities located in the Padana Plain: Torino, Pavia and Verona(Fig.1). In each city, the instrument for PM2.5measurementswas a BGIIncorporated Basel Sampler and the detailed procedures of samplingare illustrated in a previous paper. For each city, nine samples permonth were collected, representing 11 days (264 h) of measurementdistributed over themonth. Samplerswere located in a high traffic area(more than 10,000 vehicles/day) of each centre city. (Traversi et al.,2008). This kind of sampler works at a low flow rate of 16.7 l/min(Gilli et al., 2007a,b,c) that mimics the human respiratory functions innormal condition, equal to less than 20m3/day (Kim and Jaques, 2005;Nakayama et al., 2009).

2.2. Gravimetric analysis

The Torino Lab prepared and analyzed the filters for all centres.The filters were pre and post conditioned in a dry and dark envi-ronment for 24 h, and pre and post weighed in a roomwith controlled

Fig. 2. Daily concentration of PM2.5 over the sampl

temperature and humidity. Preconditioned filters were shipped byTorino Lab to Pavia (10 filters) and to Verona (10 filters). The filterswere shipped back to Torino at the end of monthly samplings for thepost conditioning and the post weighing. For eachmonthly samplings,one of the ten filters was used as blank control. The time storageand setting was respected in accordance with US EPA guidelines.Overall, up to 420 daily filters were pre and post conditioned andanalysed. Daily and monthly mean concentrations of PM2.5 werecalculated taking into account the effective sampling time of eachpump and subtracting the blank filter value (Gilli et al., 2007a,Hazenkamp-von Arx et al., 2004). The concentration of PM2.5 (μg/m3)was calculate by the application of a specific formula publishedelsewhere (Gilli et al., 2007a).

2.3. Extractions and biological assays

Daily filters were pooled to obtain one monthly sample in each ofthe three cities. Extractions of each pooled sample were carried outwith a Soxhlet apparatus for at least 80 cycles with acetone, sub-sequent evaporation induced by a Rotavapor instrument, and the re-suspension of the samplewith dimethyl-3-sulfoxide (DMSO) to obtainan equivalent concentration of 0.1 m3/μl. The mutagenicity assay wasexecuted according to Maron and Ames (1983). Definite concentra-tions of PM2.5 organic extractwere tested to generate a dose–responsecurve (20, 50, 100 μl of the DMSO 0.1 m3/μl suspension). The slope ofthe dose–response curve (revertants/m3) was calculated by the leastsquares linear regression from the first linear portion of the dose–response curve (Gilli et al. 2007c). All experiments were done intriplicate with at least three doses. Results were expressed as totalrevertants minus spontaneous revertants, to obtain net revertants percubic meters (rev/m3), and were calculated by the dose–responsecurve (Buschini et al., 2001; Cassoni et al., 2004; Claxton et al., 2004).The mutagenic activity of airborne particulate extracts was studiedusing S. typhimurium strains TA98, TA98NR and YG1021, both with

ing period (2006) in Torino, Pavia, and Verona.

Table 1Descriptive analysis of the daily PM2.5 levels in Pavia, Verona and Torino.

PM 2.5 μg/m3 N Minimum Maximum Mean Std. deviation

Pavia 107 4.0 107.8 34.8 25.1Verona 108 6.2 125.0 37.3 27.7Torino 91 7.5 153.3 46.5 31.6

Table 2Spearman's rho correlation coefficients (at pb0.01 significant level) between gravimetricanalyses (μg/m3) and Salmonella assays (net revertants/m3) for each cities.

TA98NR TA98 YG1021 TA98NR+S9 TA98+S9 YG1021+S9

Torino PM2.5 0.883 0.800 0.950 0.850 0.873 0.917Pavia PM2.5 0.841 0.853 0.806 0.890 0.839 0.794Verona PM2.5 0.948 0.867 0.891 0.939 0.951 0.952

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and without Aroclor-induced rat-liver homogenate activation (S9).YG1021 is a ‘classical’ nitroreductase-overproducing strain, obtainedby cloning the nitroreductase gene of S. typhimurium TA1538 into pBR322 and introducing the recombinant plasmid intoTA98 (Josephyet al.,1997). YG1021 strain has a nitrofurazone reductase activity more than50 times higher with respect to the original TA98 strain permitting anefficient detection of mutagenic nitroarenes. TA98NR lacks of the‘classical’ nitroreductase, so bioassays describe a reducedmutagenicityproportionally to the nitroarene amounts (Hrelia et al., 1999). Thesegenetically modified strains are supplied from Dr. T. Nohmi of theNational Institute of Hygienic Sciences, Tokyo (Einisto et al., 1991).

The spontaneous revertants obtained during 12 bioassay sessions,one for each sampling month, ranged from 15 to 24 (17±3) for TA98,from 11 to 14 (13±3) for TA98NR, from 23 to 40 (29±8) for YG1021,from 19 to 34 (24±5) for TA98+S9, from 11 to 25 (23±4) forTA98NR+S9, and from 20 to 35 (27±5) for YG1021+S9. Genotype ofeach tester strain was routinely confirmed and in each assay sessionpositive and negative controls were included. 2-nitrofluorene and2-aminofluorene (1 μg/plate) was tested in each assay as knownmutagen positive control in absence or presence of S9 mix metabolicactivation.

2.4. Statistics

The four seasons were priory subgrouped as follows: winter(January–March), spring (April–June), summer (July–September),autumn (October–December). Statistical analyses were performed

Fig. 3. Error bars of the results of the Salmonella assay performed on the whole samples (netthe Padana Plain.

using the SPSS Package, version 14.0. In particular, 1) a log-transformation of non-normally distributed data was carried out,2) the Spearman rank order correlation coefficient was calculated toassess relationship between variables; 3) Wilcoxon test was adoptedto compare means, 4) ANOVA was used for multivariate analysis inwhich an equal variance was assumed and Tukey test as post hocmultiple comparisons was applied. The mean differences and correla-tions were considered significant at pb0.05.

3. Results and discussion

The tendency of the daily PM2.5 levels in the three Italian cities is shown in Fig. 2while the annual descriptive analysis is reported in Table 1. The lower values wererecorded during summer in each city while highest values were recorded duringNovember in Verona, February in Pavia and January and February in Torino. WinterOlympic Games took place in Torino during February 2006, but the impact on the PM2.5concentration is neither somarked nor statistically significant (Traversi et al., 2008). Theannual level of the Padana Plain urban PM2.5 pollution, calculated among the threecities, was 38.3±21.1 μg/m3. This value is hotly above the proposed no human effectobserved level of 5 μg/m3 (WHO, 2006). Torino and Verona showed higher PM2.5contaminations but the air pollution trends in all the three cities are similar andsignificantly correlated (pb0.001) one to each other. The seasonal trend is confirmedand a significant higher concentration in PM2.5 was found in winter in the three cities(summer vs winter pb0.001).

Themutagenic properties of PM2.5 were summarized in Fig. 3 by each S. typhimuriumstrain (+ and−S9 activation). On the whole, an important mutagenic load of PM2.5 wasrecorded. The different mutagenic responses are positively correlated one to each other(Spearman's rho from 0.874 to 0.988 pb0.01) and to PM2.5 levels too (Table 2). Asignificant correlation among the mutagenicity recorded in the tree cities is also proved(pb0.01). Taking into account the spontaneous mutations, YG1021 strain showed thehighest sensitivity to urban airbornepollutants— expressed as awide enhance frame-shift

revertants/m3). The last error bar shows the PM2.5 annual level (μg/m3) calculated for

Table 3Variation of the net revertants modulated by the presence of the nitroreductase plasmidof by the lack of the major nitroreductase activity expressed as ratio between the netrevertants observed with YG1021 or TA98NR and TA98 strains.

Ratio Without S9 activation With S9 activation

Residualmutagenicity

Enhancedmutagenicity

Residualmutagenicity

Enhancedmutagenicity

Torino 0–0.52 1.67–2.97 0–0.16 1.76–5.22Pavia 0–0.45 1.59–4.13 0–0.58 1.65–3.90Verona 0–0.41 0.77–2.81 0–0.85 1.84–5.55

Residual mutagenicity linked to nitroreductase deficient strain calculated as [YG1021net revertants/m3/TA98 net revertants/m3] and the enhanced mutagenicity is linkedto amplified nitroreductase strain calculated as {1− [(TA98NR net revertants/m3−TA98 net revertants/m3)/TA98 net revertants/m3]}.

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mutagenicity— followedby the TA98 and in sequence by TA98NR strains. Furthermore, forthe three strains, the lowest values were recorded during the summer period in all thecities (summer vswinter pb0.001), while the highest valueswere observed inWinter, justduring the winter Olympic Games. Table 3 shows the influence of the nitroreductaseactivity on the Salmonella assay response. Two ratios between the net revertants obtainedduringall the biological assays are introduced. The role of thenitroreductase activity on thetotal measured mutagenicity is summarized by this ratio calculations. The ratio betweenthe over producing nitroreductase strain, YG1021, and the reference TA98 strain iscalculated as: [YG1021 net revertants/m3/TA98 net revertants/m3] and shows aquantification of the mutagenicity linked to the amplified nitroreductase activity. Therewas a clear increase of the response due to this augmentation. This increase is computedmeanly 2.2, 2 and 1.7 times for Torino, Pavia and Verona, respectively. The multiplecomparison analysis shows a significant difference only for Torino, comparing the threestrains (ANOVATorinop=0.012, Pavia p=0.073, Verona p=0.062) (Fig. 4). This differentbiological answer in Torino suggests a different chemical composition of PM2.5 bycomparison to the two other cities and in particular a higher contribution of the nitro-compounds on the total mutagenicity. The second ratio, used to quantify the residualmutagenicity linked to nitroreductase deficiency, is calculated as follows: {1−[(TA98NRnet revertants/m3−TA98 net revertants/m3)/TA98 net revertants/m3]}. This findingshows the residual mutagenic load after removal of the major nitroreductase activity.Withoutmain nitroreductase activity, the genotoxicity on S. typhimurium is reducedbelow30% (Table 3).

4. Conclusion

The annual level of urban PM2.5 pollution for the Padana Plain,recorded in this study during 2006, is about four times higher than10 μg/m3, the air quality guide value recently proposed by EuropeWHO. Furthermore daily air quality guide value for PM2.5 is ex-ceeded in the 58% of the sampling days. Several daily hot spots (over100 μg/m3) were also measured. A seasonal trend is confirmed, with

Fig. 4. Means of the net revertants obtained with the different Salmon

a higher PM2.5 pollution in winter. On the whole, the PadanaPlain represents an unlucky area where air exchanges are scarce,the dominant winds weak, and air pollutants can easily accumulateparticularly in Torino and Verona (Lonati et al., 2007). This pollution iswidely known andworrying especially for NOx and fine PM high levels(Chen et al., 2008). Moreover PM2.5 air pollution trends show a verysimilar behaviour in the three cities defining awide air pollution on thewhole area. Nitro compounds are not chemicals usually monitoring,therefore the new air quality European Directive introduce a chemicalcharacterization mandatory in the background rural site that includesNH4

+ and NO3− (2008/50/CE, attachment IV). IARC has classified

several nitro substituted compounds as possible human carcinogens(2B) (IARC,1997) but a deeper scientific attention to these pollutants isauspicial, also in consequence of the considerable amounts of nitricoxide contained in diesel exhaust (Heeb et al., 2008). Powerfulnitrating species may support nitration reactions that promote theconversion of PAHs to nitro-PAHs (Heeb et al., 2008). Moreover, abiological approach is desirable, too in the monitoring and toxicolo-gical characterization of the air pollution. Amutagenic load, associableto PM pollution, is well demonstrated (Ducatti and Vargas, 2003;Claxton et al. 2004; Claxton and Woodall, 2007), also in the presentstudy. In addition, in this study we showed a central role of the nitroderivates on urban PM2.5 mutagenicity with Salmonella assay inrelation to nitroreductase influence. The proportionality among themutagenic responses is 1:3:6, this crucial contribution is evident in allthe 3 included cities, especially in Torino. The nitro-derivatesresponsibility to increase DNA potential damage (Watanabe et al.,1998; Blackburn et al., 2008) emphasizes the need of an accuratehuman health risk evaluation and of the introduction of a newevaluation approach focused on the real toxicological properties of thedifferent environmental compartments considering the quality ofpollution in addition to the quantity. In this contest biological in vitromodels are suitable also to determine the specific class compoundscontribution to total observed effect of PM2.5. Thus, further investiga-tion have to integrate chemical characterizations with descriptions ofbiological effect for the emerging environmental aspects connected topublic health.

Acknowledgements

This study was partially financed by a 2006–2008 University ofTurin grant. The authors kindly thank Mr. Andrea Salomoni of Envi-ronmental Protection Regional Agency of Veneto for the collaboration

ella strains in each cities, the bars show the standard deviations.

910 D. Traversi et al. / Environment International 35 (2009) 905–910

in the Verona's sampling. At the end, an affectionate remembrance of afriend and colleague, Enzo Scursatone; his intelligence and hishumanity will be cherished in our hearts.

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