Mutageoeds vol.7 no.6 pp.421-425, 1992

Mutagens in urban air particulate

Barbara Nardini and Erminio Clonfero1 Institute of Occupational Health, University of Padova, Via Facciolati 71, 1-35127 Padova, Italy 'To whom correspondence should be addressed

Introduction Urban air, particularly that of industrialized areas, contains mutagenic and carcinogenic substances (IARC, 1983), many of which derive from combustion processes involving the emission of complex mixtures into the air. Single components may be transformed into compounds with different physical and biological properties and, for example, following the reaction of nonmutagenic compounds with oxidizing substances, may form airborne mutagens (Pitts etal., 1978; Pitts, 1979). Aromatic polycyclic hydrocarbons and their derivatives, detectable in air particulate, are among the main pollution markers from sources of combustion (central heating plants, industries, incinerators, combustion of mineral oils and coal, exhaust gases from diesel and petrol engines) and contribute most towards air mutagenicity (Pitts et al., 1978). The poly nuclear organic matter in the air of some cities mainly (80%) originates from motor vehicle emissions (IARC, 1983). Extracts of urban air particulate have long been known to cause skin cancer in experimental animals (Leiter etal., 1942; Heuper et al. ,1962; Epstein et al., 1979) and are mutagenic in the Salmonella/microsome test (Tokiwa et al., 1977; 1983; Pitts et al., 1979; Moller and Alfheim, 1980; Alfheim and Moller, 1981; Reali etal., 1984; Crebelli etal., 1988; Barale etal., 1989). Some in vitro genotoxicity studies on air particulate carried out on mammalian cells have shown the induction of SCE in human peripheral blood lymphocytes © Oxford University Press

Materials and methods Padova is a mainly commercial city, with a population of —250,000. Its urban air pollution is mostly due to intense traffic. Particulate material was collected on fiberglass filters using high-volume samplers (Sierra-Andersen mod. Tripod GMWT 2200) with an average flow rate of 1.95 m3/min, placed at street level. Eight city streets of various width and traffic density were sampled between June 1990 and February 1991. The sampling period was about three consecutive hours daily. All samples were obtained during the morning. The winter measurement survey began on 28/01/91 and ended on 02/03/91, with 13 days of good weather and nine overcast. The summer measurement survey began on 09/07/90 and ended on 04/08/90 (13 cloudless days, seven cloudy and two with slight rain). In both seasons, days of heavy rain were excluded from the survey. After having been conditioned in a desiccator for gravimetric determination of dusts, the filters were weighed before and after sampling, and then extracted in a Soxhlet apparatus with dichloromethane for 16 h and with methanol for another 16 h. The solvent was removed with a rotating evaporator and the extracts, resuspended in dimethylsulphoxide (DMSO), were mixed and placed in the dark at -20°C. The mutagenic activity of these extracts was evaluated in 22 winter samples and 23 summer samples on strains TA98 and TA100 of Salmonella typhimurium with and without S9 (50 /il/plate) obtained from rat liver induced with Aroclor 1254, and on strains TA98NR and TA100NR without metabolic activation. The plate incorporation technique was used (Maron and Ames, 1983). At least three doses different from zero were assayed in duplicate, ranging from 0.125 to 2 mg/plate, according to the availability of the extract of the particulate. A blank sample was extracted as described above and assayed on plate up to 200 mg equivalent, with negative results. Methylmethanesulphonate (TA100) and hycanthone (TA98) were used as direct positive controls. Benzo(a]pyrene was the indirect control mutagen for both strains. Controls for TA98NR and TA100NR were 1-nitropyrene and nitrofurantoin, respectively. Mean spontaneous revertants and standard deviations in all experiments (n = 21) were 16 ± 3 and 28 ± 7 for strain TA98, and 114 ± 27 and 135 ± 28 for strain TA100, in the absence and presence of S9 mix, respectively. For TA98NR (n = 21) and TA100NR (n •= 15), the values were 15 ± 4 and 125 ± 12, respectively. A sample was considered to be mutagenic if, at the maximum dose assayed, it was capable of producing at least double the number of spontaneous revertants of one of the strains used in our experimental conditions. Sample mutagenic activity was the slope of the linear portion of the dose-response curve calculated by linear regression analysis. Mutagenic activity was expressed as number of induced revertants per mg of particulate (specific mutagenic activity of urban air particulate) and as airborne mutagens in urban air (particulate fraction) expressed as revertants/m3. The latter value was obtained by multiplying the specific mutagenic activity of dusts (revertants/1 mg of dust) by the total airborne dust levels of the various samples collected. Statistical comparisons were carried out using a non-parametric test (Mann-Whitney test).

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Extracts of airborne particulate matter were demonstrated to be mutagenic in the Salmonella/microsome test. Urban airborne particulate was collected with high-volume samplers in an Italian town mainly polluted by traffic exhaust fumes. After being weighed for determination of total dust, the particulate was extracted with CH2CI2/methanol and assayed by Salmonella/microsome assay on strains TA98, TA100 and TA98NR. All samples were mutagenic on strain TA98, with a mutagenic potency of 50 ± 14(-S9), 128 ± 63(+S9)and 104 ± 51 ( - S 9 ) , 211 ± 97 (+S9) revertants/mg of particulate for summer (n = 23) and winter (n = 22) determinations, respectively. The mutagenic activity on strain TA98NR was about one-half that on strain TA98, indicating a large contribution of nitroaromatk mutagenic compounds. Mutagens from airborne particulate were less active on strain TA100. The summer and winter mean values of urban total dust were 0.15 ± 0.07 and 0.35 ± 0.18 mg/m3 respectively, and the mutagenkity of urban air on strain TA98 was 8 ± 5 (-S9), 22 ± 17 (+S9) and 30 ± 11 (-S9), 61 ± 21 (+S9) revertants/m3 in the two seasons, respectively. In winter, besides an increase in urban air mutagenkity, there also was a change in direct particulate activity per milligram, which was double that of summer.

(Lockard et al., 1981; Krishna et al., 1984), in V79 Chinese hamster cells (Alink et al., 1983) and in mouse bone marrow and spleen cells (Krishna et al., 1986). Diesel engine combustion and the reaction of aromatic polycyclic hydrocarbons with nitrogen oxides give rise to mutagenic and carcinogenic nitroderivatives in the air. These compounds are potent direct-acting bacterial mutagens, and dinitropyrenes are among the most highly mutagenic substances assayed by the Ames test (Rosenkranz et al., 1980; Mermelstein et al., 1981; Tokiwa et al., 1981; Rosenkranz, 1982; Rosenkranz and Mermelstein, 1983; Tokiwa and Onhishi, 1986). Their direct activity in the Salmonella/microsome test is related to the presence of bacterial nitroreductases which activate them to genotoxic agents (Rosenkranz and Mermelstein, 1983). This study evaluates the mutagenic activity on Salmonella typhimurium of extracts from the urban air particulate in the city of Padova (north-eastern Italy) in two different seasons.

B.Nardini and E.CIoafero

Results The mean values ( ± SD) of morning particulate pollution in the urban air of Padova were 0.15 (0.07) in summer and 0.35 (0.18) mg/m3 in winter. Dust pollution ranged from a minimum of 0.06 or 0.12 to a maximum of 0.33 or 0.72 in warm and cold seasons, respectively. The mutagenic activity of the particulate extracts and urban air mutagenicity (particulate fraction) are shown in Tables I and II. All samples turned out to be mutagenic on strain TA98, in both the presence and absence of metabolic activation at doses between 0.125 and 2 mg/plate. Strain TAIOO showed less mutagenic activity with respect to strain TA98,

mainly in the absence of S9 with summer filters, in which, at lower doses, it often did not reach double the number of revertants with respect to spontaneous ones. The mutagenic activity of the samples ranged from 26 to 76 and from 24 to 237 revertants/mg of particulate (summer), and from 41 to 244 and from 83 to 447 revertants/mg (winter), respectively, without and with S9. Figure 1 shows the mean values of specific particulate mutagenic activity on TA98: 50 ± 14(-S9), 128 ± 63 (+S9) and 104 ± 51 (-S9), 211 ± 9 7 (+S9) revertants/mg, respectively for summer and winter. The values of airborne mutagenic particulate varied from 2 to 25 (-S9) and from 1 to 68 ( + S9)

TaWe I. Mutaeenic activity on strains TA98 and TA98NR of Salmonella typhimurium of urban air paniculate extracts

Table D. Mutagenic activity on strains TA100 and TA100NR of Salmonella typhimurium of urban air particulate extracts

Sample

TA98NR

Net revertants/m TA98 10%

Sample TA98NR -

Summer Al A2 Bl B2 B3 Cl C2 C3 Dl D2 D3 El E2 E3 Fl F2 F3 Gl G2 G3 HI H2 H3 Winter A3 A4 A5 B4 B5 B6 C4 C5 D4 D5 E4 E5 E6 F4 F5 F6 G4 G5 G6 H4 H5 H6

422

63 51 53 36 52 41 ,60 40 34 56 48 37 26 70 28 38 40 76 70 54 54 63 63

110 77 53 109 80 109 76 72 56 45 140 68 164 202 140 244 151 % 41 87 88 81

184 130 151 82 64 117 193 148 74 98 102 162 24 120 74 51 30 204 204 185 237 224 97

272 147 99 290 189 117 254 145 175 83 351 150 291 447 145 340 322 221 114 136 158 185

13 22 13 0 28 14 20 17 13 19 24 29 5 10 14 20 20 23 35 19 23 26 26

52 32 27 69 51 51 37 39 32 16 61 38 87 87 68 85 63 51 14 43 64 63

12 10 6 4 3 4 10 6 3 13 6 4 2 8 4 4 3 25 15 14 11 9 13

21 51 38 20 14 39 20 29 29 20 22 27 20 41 28 30 25 32 16 49 51 39

34 24 17 9 4 11 32 23 8 22 13 17 1 14 11

5 2 68 44 49 48 33 20

53 98 70 54 33 42 68 58 90 36 56 59 35 90 29 42 52 73 44 76 91 89

2 4 1

0 2 1

3 3 1 4 3

3 0.3 1 2 2 1 8 8

5 5 4 5

10 21 19 13 9 18 10 16 16 7 10 15 10 18 14 11 10 17 5 24 37 30

Net revertants/mg TAIOO — 10%

Net revertants/m TA100NR TAIOO — 10%

TA100NR —

Summer Al A2 Bl B2 B3 Cl C2 C3 Dl D2 D3 El E2 E3 Fl F2 F3 Gl G2 G3 HI H2 H3

11 32 22 34 29 12 39 47 18 39 27 55 19 1 28 35 25 87 104 67 57 68 61

119 25 64 26 68 47 74 221 111 204 138 96

4 21 16 9 NT 11 0 24 11 9 NT 15 NT 6 4 NT NT 34 44 19 20 36 NT

2 6 2 4 2 1 6 7 2 9 4 6 1 0.1 4 4 2 29 23 18 12 10 13

16 15 11 10 4 8 16 18 8 18 13 13 1 7 4 7 4 25 48 29 41 20 20

1 4 2 1 NT 1 0 4 1 2 NT 2 NT 1 1 NT NT 11 10 5 4 5 NT

Winter A3 A4 A5 B4 B5 B6 C4 C5 D4 D5 E4 E5 E6 F4 F5 F6 G4 G5 G6 H4 H5 H6

92 45 72 97 73 78 62 51 70 33 112 53 73 160 152 102 106 101 39 72 58 88

128 94 114 149 176 153 163 161 123 78 220 131 178 206 230 245 211 175 124 88 149 215

28 18 NT 90 NT NT 28 16 60 NT NT 30 NT 84 46 NT 50 43 NT 50 61 NT

18 30 51 18 13 28 16 21 36 14 18 21 9 32 30 13 17 34 15 40 33 43

25 63 81 28 31 55 43 65 63 34 35 51 21 41 46 30 34 58 48 49 86 104

5 12 NT 17 NT NT 7 6 31 NT NT 12 NT 17 9 NT 8 14 NT 28 35 NT

S9

NT, not t ested.

86 82 100 92 68 90 97 115 76 81

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S9

Net revertants/mg TA98 10%

Mutagens in urban air partkulate 350 300 2 260 .2 o 7 200 3 Q.

CC 100 TA98+S9 TA98-S9

60

EZ3 TA98NR

0

SUMMER

WINTER

300

£ 260 cd

u 3= 200 150 CC 100 •

TA100+S9 TA100-S0

60

TA100NR SUMMER

WINTER

Fig. 1. Specific mutagenic activity (mean and standard deviation) of urban air paniculate.

revertants/m3 in summer, and from 14 to 51 (-S9) and from 29 to 98 (+S9) revertants/m3 in winter. The mean values of mutagenic activity in urban air (paniculate fraction) were 8 ± 5, 22 ± 17 (summer) and 30 ± 11,61 ± 2 1 (winter) revertants/ m3, respectively, without and with metabolic activation (Figure 2). The mean values of direct mutagenicity determined on TA98NR for all summer and winter samples were lower (38% and 49% respectively) than those obtained using strain TA98. All samples showed only very weak mutagenic activity on TA100NR. Statistical comparisons of mutagenic activity values on strain TA98 showed the greater presence of both direct and indirect mutagens in winter urban air (paniculate fraction) (z = 5.51 and 4.86 respectively, P < 0.01). Winter urban dusts also have greater specific activity (revertants/mg) both direct and indirect (z = 4.58 and 2.74, P < 0.01). The percentual reduction in the average values of direct mutagenic activity using strain TA98NR with respect to the original strain is greater in summer (61.8% versus 49.1%, z = 2.91, P < 0.01, data and calculations not shown). Discussion The summer dust levels measured respected the maxima laid down by Italian legislation of 28/3/1983, according to the 95

percentile peak value (0.3 mg/m3). The 95 percentile guideline value was exceeded by 55% of winter samples. The average dust levels found were double in winter and showed values similar to those found in another Italian city (Barale et al., 1989). The mutagens of the urban paniculate were more active on strain TA98 of Salmonella typhimurium, confirming results obtained in other studies (Tokiwa et al., 1983; Reali et al., 1984; De Flora et al., 1989). As already reported by other authors (De Flora et al., 1989; Reali et al., 1984; Alink et al., 1983), mutagenic activity is higher with S9, indicating the main contribution of indirect mutagen classes. But mutagenicity also occurs without metabolic activation because of the presence in urban air dust of another class of mutagens, acting directly or activatable to the ultimate mutagenic metabolites by the metabolic system of bacteria (i.e. nitroarenes). The mutagenic activity of the air of Padova is higher than that found in other Italian cities such as Pisa (Barale et al., 1989) and also with respect to three different seasonal sampling surveys in Rome, in which levels of paniculate mutagenic activity ranging from 3 to 13 (-S9) and from 4 to 23 (+S9) revertants/m? were found (Crebelli et al., 1992). The lower values reported by Crebelli and coworkers is partly due to the different sampling period, which was 24 h daily, with respect to a 3-h morning period in our study. The highest values found in Padova are mainly due to the stagnation of air which occurs in the Po Plain mainly in winter, and which favours the 423

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350

B.Nardini and E.CIonfero 100

80

CO

I

60

40

TA98+S9

20

TA98-S9 CD SUMMER

TA98NR

WINTER

80

CO

60

40

20

1

C D TA100+S9 IZH TA100-S9 H

SUMMER

TA100NR

WINTER

Fig. 2. Mutagenicity (mean and standard deviation) in urban air (paniculate fraction).

accumulation of pollutants in the low atmosphere (phenomenon of thermic inversion). Nonetheless, the mean values of mutagenic activity of paniculate in the urban air of Padova, determined in both winter and summer, fall within the large range of values found in 17 Italian towns by the Italian Railways national survey (Giromini et al., 1991), since the highest values, above all in winter and in the cities of North Italy, are subject to the same phenomenon (Turin, Milan, Aosta and Bolzano). According to a proposed classification (Tokiwa et al., 1983) based on the number of revertants/m3 obtained in the presence of S9 on TA98, the mutagenic values of our winter samples are similar to those commonly found in industrial areas. As well as an increase in mutagenicity per cubic meter of air in winter (3.8 times without S9; 2.8 times with S9), as opposed to summer, due to greater production of mutagenic pollutants (traffic, heating) and seasonal atmospheric phenomena (thermal inversion), increased mutagenicity per mg of dust (specific dust mutagenic activity) was also observed (2.1 times without S9 and 1.6 times with S9), as already reported by other authors (Barale et al., 1989; Reali et al.,. 1984). This may depend on more rapid photochemical destruction and increased volatilization of mutagens in summer, as against increased settling of mutagens 424

in the vapor phase on air particulate in winter (Atherholt et al., 1985), when traffic (Tokiwa et al., 1983) and heating plants (Reali et al., 1984; Alink et al., 1983) also favour the increase in mutagenic substances in urban air particulate. Nitroaromatic compounds contributed greatly to the total direct mutagenicity of urban air particulate in Padova, as shown by reduced activity values on nitroreductase deficient strain TA98NR. Instead, other studies revealed the limited role of nitroarenes on the mutagenic activity of urban dust (Crebelli etal., 1988; Crebelli et al., 1991; Giromini etal., 1991). As already reported (Crebelli et al., 1988; De Flora et al., 1989), the fraction of mutagenicity due to nitroderivatives is slightly greater in summer, probably due to increased photoactivation of aromatic polynuclear compounds and formation of nitroderivatives (De Flora etal., 1989). References Alfheim.I. and Moller.M. (1981) Mutagenicity of airborne particulate matter in relation to traffic and meteorological conditions. In Waters.M.D., Sandhu.S.S., HuisinghJ.L., Claxton,L. and Nesnow.S. (eds), Application of Short-term Bioassay in the Analysis of Complex Environmental Matures II, Environmental Science Research, Plenum, New York, pp. 85—89.

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Mutagens in urban air paniculate and their sources in the environment, CRC Crit. Rev., Toxicol, 17, 2 3 - 6 0 . Tokiwa.H., Morita.K., Takeyoshi.H., Takahashi,K. and Ohnishi.Y. (1977) Detection of mutagenic activity in paniculate air pollutants, Mutat. Res., 48, 237-248. Tokiwa.H., Nakagawa.R., Morita.K. and Ohnishi.Y. (1981) Mutagenicity of nitro derivatives induced by exposure of aromatic compounds to nitrogen dioxide, Mutat. Res., 85, 195-205. Tokiwa.H., Kitamore,S., Horikawa.K. and Nakagawa.R. (1983) Some findings on mutagenicity in airborne paniculate pollutants, Environ, Mutagenesis, S, 87-100. Received on February 3, 1992; accepted on July 16, 1992

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Alink,G.M., Smit.H.A., Van HoudtJ.J., KolkmanJ.R. and Boleij J.S.M. (1983) Mutagenic activity of airborne particulates at non-industnal locations, Mutal. Res., 116, 21-34. Aterhholt.T.B., McGarrity.G.J., LouisJ.B., McGeorge.L.J., Lioy.P.J., DaiseyJ.M., Greenberg.A. and Darack.F. (1985) Mutagenicity studies of New Jersey ambient air paniculate extracts. In Waters.M.D., Sandhu.S.S., LewtasJ., Claxton,L., Strauss,G. and Nesnow.S. (eds), Shon-Term Biosassay in the Analysis of Complex Environmental Mixtures IV, Plenum Press, New York and London, pp. 211-231. Barale,R., Zucconi.D., GiorgeUi.F., Carducci.A.L., Tonelli.M. and Loprieno.N. (1989) Mutagenicity of airborne particles from a non-industrial town in Italy, Environ. Mol. Mutagenesis, 13, 227-233. Crebelli.R., Fuselli.S., Meneguz.A., Aquilina.G., Conti.L., Leopardi.P., Zijno,A., Baris.F. and Carere.A. (1988) Ai vitro and in vivo mutagenicity studies with airborne paniculate extracts. Mutat. Res., 204, 565-575. Crebelli.R., Fuselli.S., Conti.G., Conti.L. and Carere.A. (1991) Mutagenicity spectra in bacterial strains of airborne and engine exhaust paniculate extracts. Mutat. Res., 261, 237-248. Crebelli.R., Fuselli.S., Benigni.R. and Carere.A. (1992) Mutagenkita e microinquinanti dell'aerosol urbano. In Frigerio,A. (ed.), Atti del Convegno Giomate di Studio del GSISR 'Inqwnamento atmosferico: modelli prediaivi, monitoraggio e interventi di disinquinamento', Milano, 18-19 Febbraio, 1992, CSI publ., in press. De Flora.S., Bagnasco.M., Izzotti.A., D'Agostini.F., Pala.M. and Valerio.F. (1989) Mutagenicity of polycyclic aromatic hydrocarbon fractions extracted from urban air particulates, Mutat. Res., 224, 305-318. Epstein.S.S., Fryi.K. and Asahina.S. (1979) Carcinogenicity of a composite organic extract of urban paniculate atmospheric pollutants following subcutaneous injection in infant mice, Environ. Res., 19, 163-176. Giromini.L., Bulleri,M., Bamini.B., Del Ry,S., Pala.M., Valerio.F., Barale.R. andBarrai.I. (1991) Assessment of mutagenic, chemical and physical parameters of some Italian towns' air. In Atti Associazione Genetica Italiana, Vol. 37, Congresso AGI, Porto Conte - Alghero, 2 - 5 Ottobre, 1991, Abstract Book, p. 73. Hueper.W.C, Kotin.P., Tabor.E.C, Payne.W.W., Falk.H. and Sawicki.E. (1962) Carcinogenic bioassay on air pollutants, Arch. Pathol., 74, 89-116. IARC (1983) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Vol. 32, IARC, Lyon. Krishna.G., NathJ. and Ong.T. (1984) Correlative genotoxicity studies of airborne particles in Salmonella typhimurium and cultured human lymphocytes, Environ, Mutagenesis, 6, 485-492. Krishna.G., NathJ., Soler.L. and Ong.T. (1986) Comparative in vivo and in vitro genotoxicity studies of airborne particle extract in mice, Mutat. Res., 171, 157-163. Leiter.J., Shimkin.M.B. and Shear.M.J. (1942) Production of subcutaneous sarcomas in mice with tars extracted from atmospheric dusts. /. Natl. Cancer lnst., 3, 155-165. LockardJ.M., Viau.CJ., Lee-Stephens.C, CakJwelU.C., WojciechowslciJ.P., Enoch.H.G. and Sabharwal.P.S. (1981) Induction of sister chromatid exchanges and bacterial revertants by organic extracts of airborne particles. Environ. Mutagenesis, 3, 671-681. Maron,D.M. and Ames.B.N. (1983) Revised methods for the Salmonella mutagenicity test, Mutat. Res., 113, 173-215. Mermelstein.R., KiriazkJes.D.K., Butler.M., McCoy.E.C. and Rosenkranz.H.S. (1981) The extraordinary mutagenicity of nitropyrenes in bacteria. Mutat. Res., 89, 187-196. Moller.M. and Alfheim.J. (1980) Mutagenicity and PAH-analysis of airborne paniculate matter. Atmosph. Environ., 14, 83-88. PittsJ.N., Jr (1979) Photochemical and biological implications of the atmospheric reactions of amines and benzo(a)pyrene. Phil Trans. R. Soc (Lon.) A, 290, 551-576. PittsJ.N.Jr, Cauwenberghe.K.A.V., Grosjean.D., SchmidJ.P., Fitz.D.R., Belser.W.L., Knudson.G.B. and Hynds.P.M. (1978) Atmospheric reactions of polycyclic aromatic hydrocarbons: facile formation of mutagenic nitro derivatives. Science, 202, 515-519. Reali.D., Schlitt.H., Lohse.C, Barale.R. and Loprieno.N. (1984) Mutagenicity and chemical analysis of airborne particulates from a rural area in Italy, Environ, Mutagen, 6, 813-823. Rosenkranz.H.S. (1982) Direct-acting mutagens in diesel exhausts: magnitude of the problem, Mutat. Res., 101, 1-10. Rosenkranz.H.S. and Mermelstein.R. (1983) Mutagenicity and genotoxicity of nitroarenes: all nitro-containing chemicals were not created equal. Mutat. Res., 114, 217-267. Rosenkranz.H.S., McCoy.E.C, Sanders.D.R., Butler.M., Kiriazides.D.K. and Mermelstein.R. (1980) Nitropyrenes: isolation, identification and reduction of mutagenic impurities in carbon black and toners. Science, 209, 1039—1043. Tokiwa.H. and Ohnishi.Y. (1986) Mutagenicity and carcinogenicity of nitroarenes

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