Correlations between 15 polyclclic aromatic hydrocarbons (PAH) and the mutagenicity of the total PAH fraction in ambient air particles in La Spezia (Italy)

227

Mutation Research, 249 (1991) 227-241 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 A D O N I S 0027510791001458

MUT 04997

Correlations between 15 polycyclic aromatic hydrocarbons (PAH) and the mutagenicity of the total PAH fraction in ambient air particles in La Spezia (Italy) R. Barale a, L. G i r o m i n i

2, G .

G h e l a r d i n i 2, C. S c a p o l i a, N . L o p r i e n o 2, M. P a l a F. V a l e r i o 3 a n d I. B a r r a i 1

3,

1 Istituto di Zoologia, Universit& di Ferrara, 1-44100 Ferrara, 2 Dipartimento di Scienze dell'Ambiente e del Territorio, Universitg~ di Pisa, 1-56100 Pisa and 3 Istituto Nazionale per la Ricerca sul Cancro, 1-16132 Genoa (Italy)

(Received 4 July 1990) (Revision received 12 November 1990) (Accepted 25 January 1991)

Keywords: Polycyclic aromatic hydrocarbons; Ambient air; Ames assay; Italy

Summary Airborne particulate matter has been monitored 4 times a month for 1 year (1988) in the city of La Spezia (Italy). The polycyclic aromatic hydrocarbon (PAH) fractions were extracted, purified and characterized for the content of 15 individual PAH. In general when concentrations of individual PAH were compared statistical correlation was obtained. Mutagenicity studies were performed by the use of the Ames plate test with the Salmonella strains TA98, TA100, TA98NR and TA98DNP6 with and without metabolic activation ($9 mix). The TA98 strain was by far the most responsive and the $9 mix was absolutely required as expected when PAH are assayed. Besides mutagenicity, toxicity was also considered and it proved to be correlated with mutagenicity in TA98, + $9. The TA98NR and TA98DNP6 strains showed no appreciable differences from the parental strain TA98 indicating the absence of significant amounts of direct-acting nitro derivatives in our PAH samples. Of the 15 PAH considered in this study the amounts of cyclopenta[c,d]pyrene (CPP) correlated best with mutagenicity. The role of CPP in contributing to the indirect mutagenicity of urban air PAH samples is discussed.

Organic fractions of particulate air matter are carcinogenic in animals (Hueper et al., 1962; Epstein et al., 1966; Asahina et al., 1972), mutagenic in bacteria (Pitts et al., 1977; Alflaeim and Moiler, 1981; Fukino et al., 1982; Moller and Alfheim,

Correspondence: Dr. R. Barale, Istituto di Zoologia, via L. Borsari 46, Universit~t di Ferrara, 1-44100 Ferrara (Italy).

1982; de Raat, 1983; Tokiwa et al., 1983; Takeda et al., 1984; Atherholt et al., 1985; Butler et al., 1985; de Raat et al., 1985; Crebelli et al., 1988; Barale et al., 1989; Miguel et al., 1990) and in mammalian cells (Lokard et al., 1981; Krishna et al., 1984, 1986; Hadnagy et al., 1986, 1989; Pyysalo et al., 1987; Barale et al., 1990). Among the variety of chemical classes adsorbed onto airborne particles, polycyclic aromatic hydro-

228 carbons (PAH) represent a very important group of carcinogenic and mutagenic compounds (IARC, 1983) and have received attention as a major class of atmospheric pollutants (U.S, National Academy of Sciences, 1983). Even if they account only for a part of the carcinogenicity and mutagenicity of organic extracts from airborne particles (Moiler et al., 1982; Reali et al., 1984; Lewtas, 1988), unsubstituted PAH have been studied as possible indicators of air quality in terms of mutagenicity (Moiler and Alfheim, 1980; Tokiwa et al., 1980, 1983; Garner et al., 1986; de Raat et al., 1987; de Raat, 1988). Recently, some studies have focused on possible correlations between the relative concentrations of specific PAH and the mutagenicity of organic extracts of ambient air samples. Concentrations of 15 PAH from 5 sites and of 7 PAH (overlapping with the previous 15) from 10 sites were determined from air samples and analyzed for possible correlations with mutagenicity on TA98 and TA100 Salmonella strains with and without metabolic activation ($9 mix) (de Raat et al., 1987; de Raat, 1988; De Flora et al., 1989). No clear indications of the presence of one or more PAH responsible for the major part of the observed mutagenicity was obtained. The aim of the present paper is to investigate further the possible presence of one or more descriptors of ambient air PAH mutagenicity by studying in detail the mutagenicity and the chemical composition of purified PAH samples collected throughout 1 year (1988) in the city of La Spezia, an important civil and military harbor on the east coast of Liguria, bordering on the Tirrenian sea, northern Italy. Materials and methods

Sites and sampling techniques Air particulate samples were collected mainly in one site (M) in the center of La Spezia, near a cross-roads characterized by intense, day-long vehicle traffic. Samples were collected at 1.70 m above street level, 3 - 4 m from the edge of the roadway. Sampling (24 h) was performed 4 times a month for 1 year excluding August and September 1988 for a total of 40 samples. Two additional sites, one along a harbor street in La Spezia (P), and the other at a distance of 7 km outside the

town in the countryside (N), were monitored for 3 months each in parallel with site M. Several meteorological data were recorded during the sampling periods. Two high-volume samplers (CMWL 2000, Metal Work, Cleveland, OH, U.S.A.) were used for the collection of particulate matter on fiberglass filters (Gelman type A / E , 23 x 25 cm) pretreated at 4 0 0 ° C for 60 min to eliminate organic contaminants and weighed after 24 h drying in a glass jar. The 2 samplers operated at constant air flow and due to normalization for temperature and pressure the normalized volumes of sampled air ranged from 1520 to 1900 m 3. The particulate matter ranged from 44.5 to 138 ~ g / m 3. Since only 2 samplers were available, one was kept fixed at one site (M) and the other was used at the other sites (N and F) for various lengths of time. The loaded filters were re-dried in a jar for 24 h at ambient temperature before weighing. Prolonging the drying time to 72 h did not appreciably change the weight of samples.

Extraction After sampling, 2 disks with surface areas of 10.4 cm 2 were cut from each filter for the analysis of metals. The remainder was extracted with 80 ml of cyclohexane in an ultrasonic bath for 15 min adding to the solution 2,3-dimethyl[2,b]fluoranthene, as internal standard. Extraction was repeated 3 times. Extracts were concentrated in a rotavapor apparatus, warmed to 45 ° C at reduced pressure. Concentration was stopped at a volume of about 0.5 ml, and the solution was applied with a capillary tube on a preparative TLC plate (PSC Fertigplatten Kieselgel 60, Merck). Elution of TLC plate was carried out with a mixture of nhexane : benzene (1 : 1, v/v). Band containing PAH were detected by their fluorescence under UV light (365 nm), scraped and extracted by 5 + 5 + 2 ml of toluene and the eluate was concentrated to dryness with nitrogen. The solute was resuspended in 600/~1 of benzene (pesticide grade). Analyses of PAH were performed by capillary column G C on a 25 x 0.2 mm I.D. SE-54 column (Supelchem) using a Perkin Elmer gas chromatograph, model Sigma 2. The injection of the extracts (1 ~1) was made by split-less mode with the injector at 300°C; FID detector at 300 ° C. Initial column temperature: 50 ° C for 1 min, 10 ° C / m i n to

229 145°C, 5 ° C / m i n to 290°C, 290°C for 15 min. PAH were identified according to their retention times. The minimum detectable concentration of each PAH in these experimental conditions was 0.1 n g / m 3. PAH standards were provided by the National Cancer Institute (NCI), Bethesda (MD, U.S.A). From the gas chromatographic profiles it was found that the 15 identified PAH constituted 50-90% of the total PAH fraction. However, when in the following we refer to 'total PAH value', we mean the sum of the 15 quantified PAHs.

Mutagenicity assays The mutagenicity of the dried PAH fractions dissolved in DMSO was tested in the Salmonella/ microsome assay using the standard plate incorporation test as described by Maron and Ames (1983). The TA98 and TA100 strains were routinely used. During the study, the TA100 strain was withdrawn because of its low responsiveness. It was replaced by the nitroreductase- or Oacetyltransferase-deficient strains TA98NR and TA98-1,8DNP6, in order to assess the possible contribution of residual nitro-PAH compounds to the total direct mutagenicity of PAH samples, since we could not a priori exclude contamination of the unsubstituted PAH fraction with such direct mutagens. The metabolic system was provided by $9 mix containing 4% of liver $9 fraction prepared from male Sprague-Dawley rats pretreated with sodium phenobarbital and 5,6-benzoflavone (Matsushima et al., 1976; Gatehouse and Delow, 1979; Ong et al., 1980). Each sample was tested at 5 doses of DMSO solution corresponding to 0, 5, 10, 20, 40 and 80 m3 eq of air. 2-Aminofluorene (Sigma, St. Louis, MO, U.S.A.), 2 #g/plate with $9, and hycanthone (Winthrop, Spain), 20/~g/plate without $9, were used as positive controls for checking both TA98 and TA100 sensitivity and $9 efficiency. The nitroreductase-deficient strains TA98NR and TA100NR were also used and their resistances to 8 ng/plate of 1-nitropyrene (Aldrich, purified by dr. M.A. Belisario according to Belisario et al., 1986) and to 4 ng/plate of 1,8-dinitropyrene (Sigma) were routinely confirmed (McCoy et al., 1983). The bacterial lawn was constantly monitored for the presence of gross toxicity by dissection

microscope examination of the plates ( x 30 magnification). However, when toxicity was not evident in the lawn, but indicated by a flattening of the response, higher magnification ( × 400) showed changes of microcolonies. In some cases we found (1) a decrease in microcolony number, or (2) a change of microcolony size, or (3) changes of individual cells which became enlarged or swollen to filamentous. All these features are considered signals of toxicity (Zeiger and Pagano, 1984). Typical examples of flattening responses are given in Fig. 1. These features were quite usual when cold month samples were assayed in the presence of metabolic activation.

Statistical analysis When the presence of gross toxicity is excluded by microscopic inspection of the bacterial lawn, there still remains the problem of assessing weak toxicity effects of the mixture on the mutagenic response, i.e., when revertants continue to increase with the dose, but not linearly. I

I

'

,'

I

1

I

150 TA90+S9

-~+

120

+,

II

Toxic

/

III

"I \

90

/ t

m ÷ II .4 1"

TAg@

I

60

I

/

30

I

0

,

q

)

,

20

m3 A I R

,

I

,

40

I

6e

]

80

EQUIUALENT

Fig. 1. F l a t t e n i n g r e s p o n s e s of his + r e v e r t a n t s i n d u c e d b y a P A H fraction f r o m a n o r g a n i c a i r b o r n e extract ( F e b r u a r y 2, 1988). D o s e s axe e x p r e s s e d as m 3 of air ¢quivaients. T A 9 8 + $9 response: n o t y p i c a l his + r c v e r t a n t s were further o b s e r v e d at the highest d o s e c o r r e s p o n d i n g to 80 n ~ ¢quivaic-nts.

230

The problem of jointly assessing mutagenicity and toxicity of compounds has been addressed, among others, by Margolin et al. (1981) and Bernstein et al. (1982). The simplest method of estimating simultaneously the 2 effects in the Ames test is the linear mutagenicity modified by exponential toxicity which results in the model: r=(a+b×X)

exp(-~'XX)

(1)

where a and b are the linear regression parameters, and ¢ is the toxicity parameter. In particular, b estimates the linear induction of revertants, and r the loss of survivors due to toxicity (Margolin et al., 1981). One consequence of this model is that any value of r > 0 at high doses leads to extinction of the reversion phenomenon. From a practical viewpoint, however, this equation can fit most types of curve, and can represent the behavior of most responses, including those experimental points which deviate from linearity and which are commonly excluded from analysis in mutagenicity assessment. Using the same model in all cases makes it possible to study the correlations between the values of b and ¢ in different test conditions. In the presence of toxicity, it is not correct to subtract the spontaneous revertants from the expected value at a given dose; in fact, toxicity of a given dose affects both the induced and the spontaneous revertants in the same way. Therefore, for the expected number of net revertants we use the equation: Y = (b × X) e x p ( - z x X)

(2)

where the spontaneous revertants are excluded from the equation, and only those revertants which are induced and survive are estimated. We are well aware that other factors, beside true toxicity, might influence the shape of the response curves, for example variation in solubility a n d / o r competition for $9 with increasing dose. An example of the type of response, with the experimental points and the fitted model, is given in Fig. 1.

Results

We shall first describe the results obtained from the data collected at site M (town), then those from sites P (harbor) and N (country). Site M (town) In Fig. 2, we give the variation, during the year, of particulate matter, PAH, and temperature, with the box and whisker (BW) plot method, and the regression of PAH over temperature. There is no significant difference between months for particulate matter amounts, but there is a strong temporal trend for PAH extractable from the particulate. The trend is in inverse correlation with that of temperature, and it is more precisely illustrated in the scatterplot and the exponential regression of PAH over temperature. The highly significant correlation between the model and the data is r = -0.824. The linear model is also significant, but there is not the same degree of correlation as in the case of the exponential model, it being only r = - 0.73. For the description of mutagenicity, we use the coefficient of linear regression over the dose obtained from the fitting of equation (1). This coefficient is the statistical unit we use. In Fig. 3, we illustrate the correlations between mutagenic activities (per m 3) and toxicity coefficients, with PAH amounts. Although to a lesser extent, under metabolic activation, there is a strong and significant linear correlation with mutagenicity while with toxicity it is less. Without metabolic activation, only weak responses are observed, indicating that these PAH samples contain prevalently indirect-acting mutagens. The strong correlation between the bs and total PAH and between these latter and temperature prompted the analysis of the mutagenicity responses as a function of temperature. The resulting scatterplot is given in Fig. 4, left, where the inverse and exponential correlation between mutagenicity of PAH samples and temperature is illustrated. Similar trends are in agreement with those obtained by other authors and ourselves showing that total organic extracts collected during cold months contain more mutagens than others (Alfheim, 1982; Alfheim et al., 1983; Alink et al., 1983; Atherholt et al., 1985; Van Houdt et

231 I 158

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

i

I

1@0

A

1.20

8@

\E I] 3

t

160

I.ti: J U

t

6@

E

\ rl r" v 'I" ,or.

o.

48

2@

O: O.

I

I

1

I

I

I

1.

2

4

S

S

7

I

I

I

I

1.8 1.1. 12

I

I

I

I

I

I

I

I

1

2

3

4

S

6

1'

1.0

I

I

I

I

I

I

MONTH

MONTH

t

I

1.1 1 2

I

I

i

I t

I

1.08

2S

5

1

I

I '\"

I

'

'

'

i

'

'

i

'

]

'

I

'

]

T

I

'

80 F

2@

'

,

'~ \0

X\

A

0 "15 @ k @ L n

E m P

"'"

40

. 2@

'"

[]

b.~'o

o

"'"'""

"-.

""

0 [] "'" o

"-.

"0

"'...

@

"'.

[]

"'.0

"

@ ~l

I

I

I

I

I

I

I

I

I

I

1.

:~

3

4

5

6

q'

10

11.

12

I

MONTH

i , , ,

i

4

,

i

i

I

8

,,,

i

X2

,,,

I

t

1.6

i

a

I

20

L i

i

24

TEMPERATURE ( O ' )

Fig. 2. Site M. (A) Top left, BW plot of the monthly variation of particulate matter (t~g/m3); right, BW plot of 15 PAH (ng/m3). Bottom left, BW of the yearly trend of temperature (o C); right, regression of 15 PAH amounts over temperature during sampling. Coefficient regression r = -0.824. (B) Top left, scatterplot of the particulate matter ( # g / m 3) of 40 air samples; right, amounts of 15 PAH ( n g / m 3) of the same samples. Bottom left, scatterplot of the average temperature ( ° C ) during the sampling days; right, regression of 15 PAH amounts over temperature during sampling. Coefficient regression r = -0.824.

al., 1987; Barale et al., 1989). This can be explained by several reasons such as more rapid photochemical destruction and increased volatili-

zation of mutagens during the summer and atmospheric inversion and increased deposition rates of vapor-phase mutagens onto airborne particulate

oql

qlIn~ pau.relqo

aql

ueql

sasuodsaa

o.raa~elntu aq. L •saldtues aatutuns oTJToads JarlS.itI e

o.ma~elnm

£1~!loe

aql u! , ( o u r l s u o o l~!ltrelsqns e Saleo!pu! qo.rq,~ 's[s/~

-IeUe I'eO.n.uoqo aql Kq lyalaoddns s! ~u!ptn.j s.[q~L •S~Olletu ale|rlo~.l

ssassod Ki.uessaoau lou op qa.rq~ H V d jo slunottre

-aed uo paqaospe H V d u]. sa~treqo aA!lel!ltrenb

aaleaa~

sqluotu

glU.retu ~u!lsa~',~rls 'qluotu aad atues aql /~[l~[.1Lrels

ploo oql ~u.unp }eql s! uo!lev!pu I ~UTllnsoa aqj~ •aeo~ aql ao^o iie H'v'd Ienp.~!PU! 5I jo uo!l!sodtuoo a^pelaa

-qns s[. slunome H V d lel O1 Iio poza..paeptrels slua!3 -l.jjaoo uolssaa~aa aq) £q paanseatu ,q~Tloe aql

u~luoo

SOlO!lied

auaoqa.re

aql

"IaAOA'~OH"(~861 "1e la l I o q a a q l v ) .IOlU1.Axu! aalletu

"(porlul.luo:)) ~ "~.ld (

~nz~d~z

O)

@ i

'

I

I

'

I

i

I

I

I

~

l

P

'

'

l

'

i

'

~

~

i

'

I t l l l t ~ ' ' l

. . . .

]

i

. . . .

. . . .

i

. . . .

I

0 :19~. . . . . . . . . . . "--.

-~1~. o

% o

0--.

o o

"

~, \ 0

"

"-0

8

o

.,1m. T

@'1:"

"..

o o

%

cPo o

'

0

o

tO \

@9

3

co

3>

{3

2T

[]

o%

I:lGo

9T

o

\

-4

G ~o nl

% 0

m --¢

"0 m ~0

[]

g

~o

08

o%

\ 0 o

@aT r

L

,

,i

. . . .

@S

8~'

. . . .

i

. . . .

@E

8~'

8l"

I ' ~ ' l l l ' ' ' l

. . . .

i

. . . .

i

. . . .

o O

D° D~

. . . .

I

@

% o

i ' l l , I

o o

O

O

t

~33M

>t33FI

89

i

o ~O

o

"D

o []

o

o

1:,9

~% 3000 o

o

O

[]

~,8

D

O O

\

O

tO

oo

09

D °

0

"0

o

D O

0

120 O

N r.i

17@T

c F D --4 m 1> .--i m

E

(3 O

-4

~NT

0~

O

o

o ~

o

8

@@T . . . .

i

. . . .

t

. . . .

I

. . . .

1 . . . .

I

. . . .

[

E~E

233 ''

|

I ' ' ' I ' ' ' I ' ' ' I ' ' ' 1

'

'

'

i

,

,

i

,

'

'

'

~

,

,

,

I

'

,

16

16 !

/"

,'o

le /o

• 0

[]

," 6"1

(~ +

O

""

.

i

12

12

\E ~ I L

9

•r v

6

[]

/'

Q .""

E

\

:;

s

II L

O/"

4. II •"( "1" v,

7"

n

6

II 3

X## i

i

[] i

@

i

i

i

i

28

'

'

I

'

'

i i i

4@

60

PRH

(mg/m3)

'

I

'

'

'

,

I

I

I

I

I

I

60

i

i

i

I

,

,

,

2e

1@@

i

,

I

'

•

'

I

'

'

'

i

i

I

i

,

i

68

4@ PRH

i

i

r

i

68

J

18,

(rig/m3)

' ' ' I ~ ' , I , , , i , , , i , , ,

I

I

8.08

8.86

8.

.... ~_ ........ _t:l:l.................................

8.e4

e4

,,

I -'~ ~r'l

° +-'~

.,°.o

o--°

rl

,.o o - - - ° " "

.... iiiii ......... - ........ " o~..o I 8

i

i

i

I 20

,

i

,

I

,

,

,

I

40

6e

PRH

(ng/m3)

,

,

,

I ~@

,

,

i

I 180

l

0

,

,

I

2e

o% "'"~ ..... ,

i

i

I

,

,,

I

I

48

68

PRH

(nCl/m3)

[] l

l

l

I

88

t

,

,

I-

.toe

Fig. 3. Site M, Top, correlations between the regression coefficient b of the dose-response curves and 15 PAH amounts. Left, in the presence of $9 mix (r = 0.72); right, without $9 mix (r = NS). Bottom: the toxicity coefficient obtained by equation (1) is reported on the left in the presence of $9 mix (r = 0.59) and on the right in the absence of $9 mix ( r = NS). The filled square is the anomalous point described in the text.

TA98NR and TA98DNP 6 strains are practically superimposible on those obtained with the parental strain TA98 ( - $9), suggesting that PAH sam-

pies have been adequately purified and so do not contain significant amounts of nitrated compounds. Moreover this finding is in agreement

234

18

0.4

\ 0

I

I

I

I

I

I

I

I

I

I

\\ \

IS

[3

\ \

D

~, "I-

D []

~.1.2 l= \

'

0 . 3

Q.

D

E \

",

II

k

9

2

0.2

L -.4

1" v n

"f3 ".D

6

[]

". D

"'-..

I

0

t

,

t

I

i

4

i

I

i

i

8

,

I

v

[]

"-.

i

7

"'-.

i

-D 0 . 1

0 D

i

12

TEMPEI~ATURE

i

I

i

i

i

16

I

i

i

20

24

1

2

3

4

(C " )

5

6

'7

10

11

1-'

MONTH

Fig. 4. Site M. Left, scatterplot of b values of mutagenicity of PAH samples in the presence of $9 mix as a function of temperature (r = - 0 . 5 5 ) . Right, the yearly trend of the mutagenic potency (b) of PAH mixtures (in the presence of $9 mix) per ng of 15 PAH is illustrated by a BW plot.

with the poor mutagenic activity of samples when the $9 was omitted. Correlations among P A H In order to study in greater detail the mutagenic effect of PAH, the concentrations of 15

different P A H were determined in each mixture. It was then possible to study the correlation between concentrations of individual PAH; the results are given in Table 1. It is apparent that several species of PAH are higly correlated among themselves and a few others are not correlated at all. To the

TABLE 1 C O R R E L A T I O N S (r) A M O N G T H E C O N C E N T R A T I O N S OF 15 PAH D E T E C T E D IN U R B A N A I R

CPP BeP FLE FEN ANT FLA PYR BaA CRY BbF BkF BaP IND DBA BgP

BeP

FLE

FEN

ANT

FLA

PYR

BaA

CRY

BbF

BkF

BaP

IND

DBA

BgP

0.52 1

-0.09 -0.03

0.74 0.51 -0.13 1

0.31 0.22 0.29 0.27 l

0.81 0.69 -0.11 0.88 0.39 1

0.88 0.68 -0.09 0.88 0.35 0.96 1

0.78 0.80 -0.07 0.86 0.33 0.94 0.92 1

0.63 0.84 -0.15 0.68 0.10 0.73 0.74 0.89 1

0.55 0.87 -0.12 0.57 -0.10 0.68 0.69 0.82 0.90 1

-0.16 -0.17 0.42 - 0.21 0.14 - 0.24 - 0.20 - 0.19 - 0.20 - 0.25

0.50 0.69 -0.13 0.71 -0.04 0.75 0.71 0.80 0.77 0.82 -0.15 1

0.30 0.61 -0.17 0.36 -0.16 0.42 0.42 0.58 0.75 0.78 -0.14 0.70 1

-0.09 0.19 0.12 - 0.07 0.17 - 0.08 - 0.10 - 0.07 0.01 0.02 0.02 - 0.05 -0.06 1

0.41 0.86 -0.13 0.36 -0.10 0.55 0.52 0.70 0.82 0.89 -0.13 0.75 0.75 0.06

1

1

235 first group belong cyclopenta[c,d]pyrene (CPP), benzo[elpyrene (BeP), fenanthrene (FEN), fluoranthene (FLA), pyrene (PYR), benzo[a]pyrene (BaP), chrysene (CRY), benzo[b]fluoranthene (BbF), benzo[a]anthracene (BaA), benzo[g,h,i] perylene (BgP) and possibly indeno[1,2,3-c,d]pyrene (IND). To the second group belong fluorene (FLE), anthracene (ANT), benzo[k]fluoranthene (BkF), dibenzo[a,h]anthracene (DBA). It has to be underlined that these 4 compounds often had concentrations below the detection level of the analytical instruments so that their correlations might have been particularly affected by this factor. We interpreted these results as indicating that the composition of the mixture tends to remain relatively constant over the period of time considered. Since what we are measuring are prevalently exhausts of internal combustion engines, these findings are not surprising. This assumption is supported by the results of analyses of BaP/BgP and P Y R / B a P ratios which are 0.57 (0.21-1.11) and 2.89 (1.25-4.59) respectively, without any apparent seasonal dependence. These ratio values fall within the ranges (0.2-0.6 and 2-6) typical for car exhausts, whereas those characteristic of domestic fuels show a ratio for BaP/BgP greater than 0.8 and for P Y R / B a P of 0.6-0.8 (Moiler and Alfheim, 1980). Moreover, woodsmoke as a cause of air pollution can be safely excluded since a methane network has been operating in the town and in the surrounding rural area for several years.

Correlation between P A H and mutagenicity and toxicity (TA98 + $9) In the following considerations, we have to underline that all compounds are simultaneously present in the mixture, so that when we estimate individual effects, we do so under this restriction. Several of the PAH which did not show significant correlation with mutagenicity often had concentrations below the detection level of the instruments, such as DBA, BkF, FLE and ANT. IND, although found in a measurable concentration, showed only a weak correlation with mutagenicity. However, after deleting a single aberrant point, the correlation became highly significant. Ten of the compounds show a significant individual correlation with the mutagenicity of the

PAH samples. Again, the group of correlating PAH may be further subdivided. Five out of 10, namely CRY, BbF, BaP, BeP, and BgP, although significantly correlated with mutagenicity, show a very wide dispersion about the regression. Three PAH, namely FEN, BaA and FLA, are better correlated and the dispersion is not as wide as in the previous case. Finally, 2 PAH, PYR and particularly CPP, show very narrow fiducial bands about the regression. In Fig. 5 we give the regression of the mutagenicity of PAH samples on the concentration of CPP (M and P sites). At both sites, CPP correlates more intensely than other PAH with mutagenicity and there is no effect of experimental scatter because even when the extreme points are excluded the correlation remains higly significant. If each individual PAH correlation is analyzed in detail, one point is almost constantly outside the fiducial bands, destroying some correlations, as we have pointed out for IND. The point is always the same observation. This sample is characterized by a high PAH value and an unusually low amount of CPP, i.e., 3.7%, while during the study its average value was 14.8%. This low mutagenicity was also observed for other samples characterized by a low CPP content. The relative amount of other PAH did not show any relevant deviation. We have no indication of the possible causes which resulted in such a low concentration of CPP in this particular sample. Deleting it, a new rank of correlations is obtained, also given in Table 2. It practically confirms the previous ordering, and it is in accordance with the known mutagenicity of IND (IARC, 1983), which is now included in the class correlated with mutagenicity of PAH samples. In our opinion the detailed analysis of this point underlines the relevance of CPP as a determinant of the mutagenicity of PAH in our samples. In Fig. 3 this point is identified by a filled square. If it were removed the correlations given would increase by about 15%. The correlation with toxicity is also given in Table 2. It is apparent that the PAH which correlate with mutagenicity also correlate with toxicity. Since we observe that toxicity is correlated with the PAH only in the presence of $9, we believe that it is mainly due to the activation of toxic compounds. Of course this does not mean that

236 i

I

i

15

15

E

/i/

12

W

9

L

," 6

,"

,

,

i

,~" ,;, -, .~-

[3

," ,"

~"

9 i

..4 I.

D,','o o ,' " £

n

/,'/,"

//

\

I

I1

i

-

~12 \E

/

,

i/ J/ lI ii t/

18'

o

I.

'

i

18

J

/,"

6

,'

'

[3"

,"

~'~ ," i

i

0

i

~

I

I

L

i

I

12

8

4

1

I

i

i

i

I

16

i

4

C~jclopemta(c~d)pgrene ( m g / m 3 )

i

,

I

,

i

12

8

16

Cyclopenta(c~d)pgrene (ng/m3)

Fig. 5. C o r r e l a t i o n s b e t w e e n C P P c o n c e n t r a t i o n s ( n g / m 3) a n d m u t a g e n i c i t y c o e f f i c i e n t ( b ) o f P A H s a m p l e s collected. Left, at site M ( r = 0.90); right, a t site P ( r = 0.93).

toxicity is due to the activated mutagens themselves. Fig. 6 clearly shows the relation existing between the 2 effects for the 15 species of PAH.

Site P (harbor) Site P was sampled 4 times a month for 3 months (in October, N o v e m b e r and December)

TABLE 2 CORRELATIONS (r) BETWEEN THE LINEAR MODEL AND THE OBSERVED + $9), S T U D E N T ' S t A N D L E V E L O F S I G N I F I C A N C E ( p ) O F 15 P A H

MUTAGENICITY

AND TOXICITY (TA98,

PAH

r

t

p

r *

t *

p *

r(r) *

t(~') *

CPP PYR FLA BaA FEN CRY BbF BaP BeP BgP IND ANT DBA BkF FEE

0.90 0.83 0.76 0.70 0.70 0.54 0.53 0.49 0.46 0.35 0.28 0.21 0.03 - 0.16 - 0.16

12.60 8.89 7.19 5.95 5.88 3.94 3.81 3.45 3.14 2.29 1.74 1.30 0.16 - 0.97 - 1.01

0.00 0.00 0.00 0.00 0.00 0.00 0.00 < 0.01 < 0.01 < 0.05 NS NS NS NS NS

0.90 0.84 0.79 0.78 0.78 0.73 0.72 0.72 0.70 0.69 0.58 0.21 0.09 - 0.16 - 0.16

12.40 9.50 7.76 7.45 7.38 6.45 6.19 6.19 5.93 5.79 4.32 1.30 0.53 - 0.97 - 1.01

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NS NS NS NS

0.66 0.66 0.65 0.64 0.63 0.62 0.58 0.56 0.53 0.44 0.41 0.13 0.06 0.03 - 0.08

5.39 5.26 5.13 5.02 4.91 4.77 4.31 4.08 3.71 2.96 2.66 0.78 0.39 0.23 - 0.52

* V a l u e s o b t a i n e d a f t e r d e l e t i n g the a b e r r a n t p o i n t , d i s c u s s e d in the text.

237

'

'

'

'

I

'

'

'

'

I

'

'

'

'

'

'

'

'

I

'

'

'

'

I

'

'

'

13

'

I

tween the total PAH at site M and at site P. It was r = 0.91, indicating substantially proportional concentrations of the PAH in the 2 sites, except for CPP which is halved (7.1%, Table 3). Mutagenicity, measured by the response of TA98 + $9, correlates with PAH concentrations (r = 0.62), but to a lesser extent than at site M (r = 0.72). All other correlations show the same trends as at site M, but their significance is limited by the small sample size. Moreover, at site P there is also a very strong and significant correlation between the mutagenicity of PAH samples and CPP concentrations, and the regression is practically superimposable on that observed at site P (Fig. 5).

I

12

1 t)

Pyg-[

.-

o ..-Tz

g4

...

Site N (country) Site N was sampled 4 times a month in parallel with site M in March, April, May, June, and July. The level of PAH was on average one fifth of that found at site M and many individual PAH were often non-measurable. All of the correlations studied at the other sites were non-significant. This can be interpreted as meaning that in the country, about 7 km from town, there is much less pollution than at the other sites. In Table 3 we give the summary of the main

-2 I

-

i

~

i

.6

i

I

i

0.4

i

p

i

i

i

,

1.4

I

,

;~.4

J

,

,

,

I

,

,

,

;3,4

,

I

4.4

i

t

i

r

]

5.4

tau Fig. 6. Simple regression analysis of mutagenicity coefficient (b) and toxicity values (1-) of PAH samples (site M) as a function of their content of 15 individual PAH (r = 0.93).

parallel to site M, so we have only 12 points to analyze for this site. The first correlation we studied was that beTABLE3

SUMMARY OF PAH AND GENOTOXICITYDATA FROM SITES M,P, AND N Site N

PAH

Site M

(ng/m3)

Average

%

(Min-Max)

Average

%

(Min-Max)

Average

%

(Min-Max)

15 PAH

26.6

100.0

(4.8 -92.2)

24.8

100.0

(6.6 -61.2)

4.5

100.0

(2.4

-9.8)

3.7 3.5 3.1 2.9 2.2 2.1 1.6 1.6 1.4 1.3 0.5 0.4 0.3 0.2 0.2

14.8 13.9 12.2 11.7 8.6 8.4 6.6 6.0 5.7 5.3 2.2 1.5 1.2 0.8 0.7

(0.20-15.4) (0.56-16.8) (0.18-17.3) (0.12-14.9) (0.16- 7.7) (0.16- 5.9) (0.04- 8.9) (0.16- 6.8) (0.16- 5.8) (0.19- 6.2) (0.10- 3.6) (0.17- 4.3) (0.09- 0.7) (0.09- 0.7) (0.11- 2.5)

1.8 2.2 4.7 2.9 1.7 2.3 2.2 2.2 1.6 1.9 0.3 0.3 0.1 0.2 0.2

7.1 9.1 19.2 11.7 6.8 9.4 8.9 8.8 6.7 7.8 1.4 1.0 0.5 0.8 0.8

(0.18- 5.4) (0.45- 7.1) (0.66-10.5) (0.67- 5.7) (0.28- 4.8) (0.46- 4.7) (0.13- 5.1) (0.28- 4.8) (0.27- 4.2) (0.45- 3.5) (0.15- 1.2) (0.24- 0.3) (0.12- 0.1) (0.12- 0.6) (0.15- 0.3)

0.7 0.2 0.4 0.3 0.2 0.7 0.3 0.4 0.2 0.4 0.1 0.2 0.2 0.2 0.1

14.8 3.9 9.7 5.8 3.9 15.7 6.0 8.8 3.6 8.3 2.7 5.5 4.6 3.8 2.7

(0.18 (0.08 (0.16 (0.10 (0.10 (0.17 (0.13 (0.13 (0.12 (0.16 (0.08 (0.16 (0.12 (0.10 (0.08

-1.3) -0.37) -2.2) -1.4) -0.39) -1.5) -0.64) -1.87) -0.37) -1.9) -0.18) -1.2) -0.69) -0.47) -0.32)

(0.84-16.97) (0.86-40.5)

3.2 14.0

(1.06- 9.7) (0.78-45.0)

0.8 4.4

CPP PYR BbF BgP FLA BeP CRY BaP BaA IND FEN FLE ANT BkF DBA b (×1000)

4.9 14.0

Site P

(0.0081-2.7) (0.05 -1.6)

238

chemical and genotoxic parameters for each site. The country is sharply differentiated from the harbor and the town. Conclusions Following De Raat (1988) and De Flora et al. (1989), we have investigated the effect of ambient air PAH on mutagenicity, with the aim of specifying one or more tracer substances correlated with reversion in the Ames test. No single or clear-cut indicator was found in the previous attempts. In the present work, we believe that we succeeded in detecting a strong candidate for mutagenicity of PAH samples on TA98 + $9 when urban air at street level is concerned. The molecule is CPP. The simplest explanation of this apparent discrepancy is that in the former studies CPP was not included among the PAH searched for and studied. It is also possible that having assayed many samples from the same location in the present study, instead of a few samples from several sites, has increased the homogeneity of PAH composition helping us in finding a possible mutagenicity indicator, A general review of the literature indicates that increasing attention is devoted to CPP because of its widespread environmental distribution, adverse biological activities and, unlike other PAH, the fact that a single metabolic step is required for its activation to a biologically reactive form. It was first separated and identified as a possible carcinogenic compound of carbon black by Gold (1975) and Wallcave et al. (1975). G r i m m e r et al., (1977) found that CPP was one of the most highly represented P A H (up to 1 rag/1 of fuel burned) in gasoline exhaust and Wallcave et al. (1975) found it to be 60-fold more abundant than BaP in carbon black. CPP exhibits much more mutagenicity than BaP on the TA98 Salmonella strain, requiring 10 times less $9 (Eisenstadt and Gold, 1978; Kaden et al., 1979; Gold and Eisenstadt, 1980; Wood et al., 1980), but it was less mutagenic than BaP in human diploid lymphoblasts (Skopek et al., 1979) and in inducing sister-chromatid exchanges in C 3 H / 1 0 T 1 / 2 cells (Krolewski et al., 1986). CPP was also positive in the mouse l y m p h o m a mutagen assay and in the morphological transformation assay (Gold et al., 1980). It has also been shown to

be an initiating tumor agent in mouse skin (Wood et al., 1980; Cavalieri et al., 1981) and mouse lung (Busby et al., 1988) and to interact synergistically with BaP in inducing tumors in mice, so accounting for most of the carcinogenicity of automobile exhaust emission (60-150%) (Cavalieri et al., 1983). Notwithstanding this widespread attention, CPP has not been adequately explored as a PAH air pollutant, possibly because it was only included in the list of standard PAH by the N C I in 1984. There are two inferences which support the hypothesis that CPP can play a relevant role in the mutagenic response of TA98 to urban air PAH in the presence of metabolic activation. The first is a general one, which comes from the literature, namely that CPP is much more active on TA98 than on TA100; practically, all works on urban ambient air mutagenicity report a higher response with metabolic activation in TA98 than TA100 and it is reasonable to suppose that CPP might give a contribution to that. Further, CPP is a typical product of gasoline engine combustion because it is present in considerable amounts among PAH from this source. The second originates from this study, which shows a very strict regression of the indicators of mutagenic response on CPP, as represented in Fig. 5. Such regression, with such narrow confidence limits, is rarely seen, at least in our experience. The finding is supported by two further observations, namely that CCP is the most highly represented PAH in all our samples (14.8%), and that it is by far the most mutagenic on TA98 among the 15 P A H we have tested in the laboratory (paper in preparation). It is about 5 times more powerful than BaP, which is the second in rank of potency, and about 30 times more so than I N D and BgP, the other 2 mutagens that were positive in our tests (TA98 + $9) on separate components of P A H mixtures in La Spezia. The reported data from the literature and the present results give reasonable grounds for proposing CPP as a valid candidate for genotoxicity assessment of PAH from urban ambient air particulate. Since CPP is also 8-15 times more concentrated than BaP ( G r i m m e r et al., 1977) it can be proposed as a good indicator of gasoline exhaust pollution. This point is of great concern since in the U.S.A., and

239 r e c e n t l y in E u r o p e , lead, a classical t r a c e r o f c a r p o l l u t i o n , has b e e n d r a s t i c a l l y r e d u c e d o r e v e n r e m o v e d f r o m gasoline.

Acknowledgements T h e a u t h o r s are g r a t e f u l to F r a n c o B e r t e l l i for his t e c h n i c a l a s s i s t a n c e a n d to D r . E l i z a b e t h P h i l p o t t for the r e v i s i o n o f the E n g l i s h . T h i s w o r k w a s s u p p o r t e d b y M U R S T 40% a n d 60% f u n d s , a n d b y C N R G r a n t s 88.03586 a n d 89.00853.

References Alfheim, I. (1982) Contribution from motor vehicle exhaust to the mutagenic activity of airborne particles, in: M. Sorsa and H. Vaino (Eds.), Mutagens in Our Environment, Alan R. Liss, New York, pp. 235-248. Alfheim, I., and M. Meller (1981) Mutagenicity of airborne particulate matter in relation to traffic and meteorological conditions, in: M.D. Waters, S.S. Shandu, J. LewtasHuisingh, L. Claxton and S. Nesnow (Eds.), Short-Term Bioassays in the Analysis of Complex Environmental Mixtures II, Plenum, New York, pp. 319-336. Alfheim, I., G. Lofroth and M. Moller (1983) Bioassay of extracts of ambient particulate matter, Environ. Health Perspect., 47, 227-238. Alink, G.M., H.A. Smit, J.J. van Houdt, J.R. Kolkman and J.S.M. Boleij (1983) Mutagenic activity of airborne particulates at non-industrial locations, Mutation Res., 116, 21-34. Asahina, S., J. Andrea, A. Carmel, E. Arnold, Y. Bishop, S. Joshi, D. Coffin and S.S. Epstein (1972) Carcinogenicity of organic fractions of particulate pollutants collected in New York city and administered subcutaneously to infant mice. Cancer Res., 32, 2263-2268. Atherholt, T.B., G.J. MacGerrity, J.B. Louis, L.J. McGeorge, P.J. Lioy, J.M. Daisey, A. Greenberg and F. Darack (1985) Mutagenicity studies of New Jersey ambient air particulate extract, in: M.D. Waters, S.S. Shandu, J. Lewtas-Huisingh, L. Claxton, G. Strauss and S. Nesnow (Eds.), Short-Term Bioassay in the Analysis of Complex Environmental Mixtures IV, Plenum, New York, pp. 211-231. Barale, R., D. Zucconi, F. Giorgelli, A.L. Carducci, M. Tonelli and N. Loprieno (1989) Mutagenicity of airborne particles from a non industrial town in Italy, Environ. Mol. Mutagen. 13: 227-233. Barale, R., L. Migliore, B. Cellini, L. Francioni, F. Giorgelli, I. Barrai and N. Loprieno (1990) Genetic toxicology of airborne particulate matter using cytogenetic assays and microbial mutagenicity assays, in: M.D. Waters, S.S. Sandhu, J. Lewtas, L. Claxton, N. Chernoff and S. Nesnow (Eds.), Short-Term Bioassays in the Analysis of Complex Environmental Mixtures VI, Plenum, New York, in press. Belisario, M.A., L. Carrano, A. Giulio amd V. Buonocore

(1986) Role of liver inducible enzymes in in vitro metabolic tranforrnation of 1-nitropyrene, Toxicol. Lett., 32, 89-96. Bernstein, L., J. Kaldor, J. McCann and M.C. Pike (1982) An empirical approach to the statistical analysis of mutagenesis data from the Salmonella test, Mutation Res., 97, 267-281. Busby, Jr., W.F., E.K. Stevens, E.R. Kellenbach, J. Cornelisse and J. Lugtenburg (1988) Dose-response relationships of the tumorogenicity of cyclopenta[c,d]pyrene, benzo[a]pyrene and 6-nitrochrysene in a newborn mouse lung adenoma bioassay, Carcinogenesis, 9, 741-746. Butler, J.P., T.J. Kneip, F. Mukai and J.M. Daisey (1985) Interurban variations in the mutagenic activity of the ambient aerosol and their relations to fuel use patterns, in: M.D. Waters, S.S. Shandu, J. Lewtas-Huising, L. Claxton, G. Strauss and S. Nesnow (Eds.), Short-Term Bioassays in the Analysis of Complex Environmental Mixtures IV, Plenum, New York, pp. 233-246. Cavalieri, E., E. Rogan, B. Toth and A. Munhall (1981) Carcinogenicity of the environmental pollutants cyclopenteno[cd]pyrene and cyclopentano[ed]pyrene in mouse skin, Carcinogenesis, 2, 277-281. Cavalieri, E., A. Munhall, E. Rogan, S. Salmasi and K. Patil (1983) Syncarcinogenic effect of the environmental pollutants cyclopenteno[cd]pyrene and benzo[a]pyrene in mouse skin, Carcinogenesis, 4, 393-397. Crebelli, R., S. Fuselli, A. Meneguz, G. Aquilina, L. Conti, P. Leopardi, A. Zijno, F. Baris and A. Carare (1988) In vitro and in vivo mutagenicity studies with airborne particulate extracts, Mutation Res., 204, 565-575. De Flora, S., M. Bagnasco, A. Izzotti, F. D'Agostini, M. Pala and F. Valerio (1989) Mutagenicity of polycyclic hydrocarbon fractions extracted from urban air particles. Mutation Res., 224, 305-318. de Raat, W.K. (1983) Genotoxicity of aerosol extracts. Some methodological aspects and the contribution of urban and industrial locations, Mutation Res., 116, 47-63. de Raat, W.K. (1988) Polycyclic aromatic hydrocarbons and mutagens in ambient air particles. Toxicol. Environ. Chem., 16, 259-279. de Raat, W.K., F.A. de Meijere and S.A.L.M. Kooijman (1985) Mutagenicity of ambient aerosol collected in an urban and industrial area of the Netherlands, Sci. Total Environ., 44, 17-33. de Raat, W.K., S.A.L.M. Kooijman and J.W.J. Gielen (1987) Concentration of polycyclic hydrocarbons in airborne particles in the Netherlands and their correlation with mutagenicity, Sci. Total Environ., 66, 95-114. Dipple, A. (1976) Polynuclear aromatic carcinogens, in: C.E. Searle (Ed.), Chemical Carcinogens, ACS Monograph 173, American Chemical Society, Washington, DC, pp. 245-314. Eisenstadt, E., and A. Gold (1978) Cyclopenta[c,d]pyrene: a highly mutagenic polycyclic aromatic hydrocarbon, Proc. Natl. Acad. Sci. (U.S.A.), 75, 1667-1669. Epstein, S.S., S. Joshi, J. Andrea, N. Mantel, E. Sawicki and T. Stanley (1966) Carcinogenicity of organic particulate pollutants in urban air after administration of trace quantities to neonatal mice, Nature (London), 212, 1305-1307. Fukino, H., S. Mimura, K. Inoue and Y. Yamane (1982)

240 Mutagenicity of airborne particles, Mutation Res., 102, 237-247. Garner, R.C., C.A. Stanton, C.N. Martin, F.L. Chow, W. Thomas, D. Hubner and R. Herrmann (1986) Bacterial mutagenicity and chemical analysis of polycyclic aromatic hydrocarbons and some nitro derivatives in environmental samples collected in West Germany, Environ. Mutagen. 8, 109-117. Gatehouse, D.G., and G.F. Delow (1979) The development of a 'microtitre' fluctuation test for the detection of indirect mutagens and its use in the evaluation of mixed enzyme induction of the liver, Mutation Res., 60, 239-252. Gold, A. (1975) Carbon black adsorbates: separation and identification of a carcinogen and some oxygenated polyaromatics, Anal. Chem., 47, 1469-1472. Gold, A., and E. Eisenstadt (1980) Metabolic activation of cyclopenta[c,d]pyrene to 3,4-epoxycyclopenta[cd]-pyrene by rat liver microsomes, Cancer Res., 40, 3940-3944. Grimmer, G., H. Bohnke and A. Glaser (1977) Investigation on the carcinogenic burden by air pollution in man XV. Polycyclic aromatic hydrocarbons in automobile exhaust gas - an inventory. Zbl. Bakt. Hyg., I. Abt. Orig. B, 164, 218-234. Hadnagy, W., N.H. Seemayer and R. Tomingas (1986) Cytogenetic effects of airborne particulate matter in human lymphocytes in vitro, Mutation Res., 175, 97-101. Hadnagy, W., N.H. Seemayer, R. Tomingas and K. Ivanfy (1989) Comparative study of sister-chromatid exchanges and chromosomal aberrations induced by airborne particulates from an urban and a highly industrialized location in human lymphocytes cultures, Environ. Mutagen., 6, 585-592. Hueper, W.C., P. Kotin, E.C. Tabor, W.W. Payne, H. Falk and E. Sawicki (1962) Carcinogenic bioassay on air pollutants, Arch. Pathol., 74, 89-116. IARC (1983) Polynuclear Aromatic Compounds, Part 1, Chemical Environmental and Experimental Data, IARC Monographs, Vol. 32, International Agency for Research on Cancer, Lyon. Kaden, D.A., R.A. Hites and W.G. Thilly (1979) Mutagenicity of soot and associated polycyclic aromatic hydrocarbons to Salmonella typhimurium, Cancer Res., 39, 4152-4159. Krishna, G., J. Nath and T. Ong (1984) Correlative genotoxicity studies of airborne particles in Salmonella typhimurium and cultured human lymphocytes, Environ. Mutagen., 6, 485-492. Krishna, G., J. Nath, L. Soler and T. Ong (1986) Comparative in vivo and in vitro genotoxicity studies of airborne particle extract in mice. Mutation Res., 171, 157-163. Krolewski, B., H. Nagasawa and J.B. Little (1986) Effect of aliphatic amides on oncogenic transformation, sister chromatid exchanges and mutations induced by cyclopenta [c,d]pyrene and benzo[a]pyrene, Carcinogenesis, 7, 16471650. Lewtas, J. (1988) Genotoxicity of complex mixtures: strategies for the identification and comparative assessment of airborne mutagens and carcinogens from combustion sources, Fund. Appl. Toxicol., 10, 571-589.

Lokard, J.M., C.J. Viau, C. Lee-Stephens, J.F. Wojciechowski, H.G. Enoch and P.S. Sabharwal (1981) Induction of sister chromatid exchanges and bacterial revertants by organic extracts of airborne particles. Environ. Mutagen., 3, 671681. Margolin, H.B., N. Kaplan and E. Zeiger (1981) Statistical analysis of the Ames Salmonella/microsome test, Proc. Natl. Acad. Sci. (U.S.A.), 78, 3779-3783. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Matsushima, H., K. Arashidani, M. Koyano and T. Handa (1976) A safe substitute for polychlorinated biphenyls as an inducer of metabolic activation system, in: F.J. de Serres, J.F. Fouts, J.R. Bend and R.M. Philpot (Eds.), In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier/ North-Holland, Amsterdam, pp. 85-88. McCoy, E.C., M. Anders and H.S. Rosenkranz (1983) The basis of the insensitivity of Salmonella typhimurium strain TA98/1,8-DNP6 to the mutagenic action of nitroarenes, Mutation Res., 121, 17-23. Miguel, A.G., J.M. Daisey and J.A. Sousa (1990) Comparative study of the mutagenic and genotoxic activity associated with inhalable particulate matter in Rio de Janeiro air. Environ. Mol. Mutagen., 15, 36-43. Moiler, M. and I. Alfheim (1980) Mutagenicity and PAH analysis of airborne particulate matter, Atmosph. Environ., 14, 83-88. Moiler, M., I. Alfheim, S. Larssen and A. Mikalsen (1982) Mutagenicity of airborne particles in relation to traffic and air pollution parameters, Environ. Sci. Technol., 16, 221225. National Academy of Sciences (1983) Polycyclic Aromatic Hydrocarbons: Evaluation of Sources and Effects, National Academy Press, Washington, DC. Ong, T., M. Muktar, C.R. Wolf and E. Zeiger (1980) Differential effects of cytochrome P-450 inducers on promutagen activation capabilities and enzymatic activities of $9 from rat liver, J. Environ. Path. Toxicol., 4, 55-65. Pitts, J.N., D. Grosjean and T.M. Mischke (1977) Mutagenic activity of airborne particulate organic pollutants, Toxicol. Lett., 1, 65-70. Pyysalo, H., J. Tuominen, K. Wickstrom, E. Skytta and L. Tikkanen (1987) Polycyclic organic material (POM) in urban air. Fractionation, chemical analysis and genotoxicity of particulate and vapor phases in an industrial town in Finland, Atmosph. Environ., 21, 1167-1180. Reali, D., H. Schlitt, C. Lohse, R. Barale and N. Loprieno (1984) Mutagenicity and chemical analysis of airborne particulates from a rural area in Italy, Environ. Mutagen., 6, 813-823. Skopek, T.R., H.L. Liber, D.A. Kaden, R.A. Hites and W.G. Thilly (1979) Mutation of human cells by kerosene soot, J. Natl. Cancer Inst., 63, 309-312. Takeda, N., K. Teranishi and K. Hamada (1984) Mutagenicity of air pollutants collected at industrial, urban-residential and rural areas, Bull. Environ. Contam. Toxicol., 32, 688692. Tokiwa, H., S. Kitamori, K. Takahashi and Y. Ohnishi (1980)

241 Mutagenic and chemical assay of extracts of airborne particulates, Mutation Res., 77, 99-108. Tokiwa, H., S. Kitamori, K. Horikawa and R. Nagakawa (1983) Some findings on mutagenicity in airborne particulate pollutants, Environ. Mutagen., 5, 87-100. Van Houdt, J.J., G.M. Alink and J.S.M. Boleij (1987) Mutagenicity of airborne particles related to meteorological and air pollution parameters, Sci. Total Environ., 61, 23-36. Wallcave, L., D.L. Nagel, J.W. Smith and R.D. Waniska (1975) Two pyrene derivatives of widespread environmental distribution: cyclopenta[c, d]pyrene and acepyrene, Environ. Sci. Technol., 9, 143-145.

Wood, A.W., W. Levin, R.L. Chang, M. Huang, D.E. Ryan, P.E. Thomas, R.E. Lehr, S. Kumar, M. Koreeda, H. Akagi, Y. Ittah, P. Dansette, H. Yagi, D.M. Jerina and A.H. Conney (1980) Mutagenicity and tumor-initiating activity of cyclopenta[c,d]pyrene and structurally related compounds, Cancer Res., 40, 642-649. Zeiger, E., and D.A. Pagano (1984) Suppressive effects of chemicals in mixture on the Salmonella plate test in the absence of apparent toxicity, Environ. Mutagen., 6, 683694.