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In vitro and in vivo mutagenicity studies with airborne particulate extracts
Mutation Research, 204 (1988) 565-575
565
Elsevier MTR01272
In vitro and in vivo mutagenicity studies with airborne particulate extracts R. Crebelli, S. Fuselli, A. Meneguz, G. Aquilina, L. Conti, P. Leopardi, A. Zijno, F. Baris and A. Carere Istituto Superiore di Sanit3, VialeRegina Elena 299, 00161 Rome (Italy)
(Received 18 June 1987) (Revision received12 October 1987) (Accepted 15 October 1987)
Keywords: Air particulate; Ames test; Micronucleus; Monooxygenases.
Summary The contribution of nitro compounds to airbome particulate mutagenicity was studied with Salmonella typhimurium strains TA98, TA98NR, T A 9 8 / 1 , 8 D N P 6. The results obtained indicate that nitropyrenes play a minor role in air particulate mutagenicity. Seasonal variations indicate a relatively greater contribution of nitro compounds to the mutagenicity of spring and summer samples. Fractionation of extracts into acidic, neutral and basic components shows that neutral compounds account for about two-thirds of the total mutagenic activity. Attempts to extract mutagens adsorbed onto particulate matter with aqueous media were almost completely negative. No significant mutagenicity was detected in urine and faecal extracts and in plasma samples of Sprague-Dawley rats treated with air particulate extracts at 80 m g / k g either per os or by i.p. injection. Negative results were obtained in the micronucleus test with Swiss mice treated at 200 and 400 m g / k g (twice by i.p. injection). A significant decrease in liver aminopyrine-N-demethylase was observed in Swiss mice injected with air particulate extracts or its basic and neutral fractions. In vitro experiments suggest a direct interaction of test materials with microsomal cytochrome P-450.
Several epidemiological studies have demonstrated a higher incidence of lung cancer in urban than in rural areas. This " u r b a n factor" was explained by the general exposure of the population to air pollutants in industrialized areas, beyond that by different smoking habits and occupational exposures (Higginson and Jensen, 1977). Several known carcinogenic polycyclic aromatic hydrocarbons and aromatic amines have been identified among the hundreds of chemical compounds adCorrespondence: Dr. R. Crebelli, Laboratorio di Tossicologia Applicata, Istituto Superioredi Sanith, Viale Regina Elena 299, 00161 Roma (Italia).
sorbed onto airborne particulate matter collected in polluted areas (Pellizzari, 1978; Helmes et al., 1982). Moreover, benzene extracts of urban air particulate have been known for decades to be carcinogenic in mammals by subcutaneous injection or skin application (Leiter and Shear, 1942; Hueper et al., 1962). However, to what extent exposure to air pollutants may pose a significant human hazard is still debated. Some estimates have indicated that air pollution could account for 5-10% of all cancers of the respiratory tract (Carnew, 1978; Doll, 1978), whereas a more recent reassessment of epidemiological data has suggested that the risk related to general air pollution
0165-1218/88/$03.50 © 1988 ElsevierSciencePubfishers B.V. (BiomedicalDivision)
566 ticulate matter collected in winter, spring and summer. In vivo experiments were carried out to evaluate both the mutagenic activity in body fluids of treated rats and the induction of micronuclei in mouse polychromatic erythrocytes. Furthermore, the possible in vitro and in vivo activity of air particulate extracts on mouse-liver microsomal enzymes was studied.
could be very much lower (Speizer, 1983). The biological evaluation of air pollution exposure is a hard task: in the case of such mixed concurrent exposures in fact synergistic or antagonistic interactions among carcinogens, promoters, cocarcinogens and inhibitors may occur in an unpredictable way (Saffiotti, 1983). Furthermore, in the evaluation of the genotoxic risks related to airborne particulate exposure some additional problems are encountered: (i) the undefined bioavailability of airbome genotoxic compounds adsorbed onto the particulate matter and (ii) the possible overestimation of the mutagenic potencies, due to the extraordinary mutagenic activity exerted in bacteria by some nitro compounds present as minor components in air particulate extracts (Mermelstein et al., 1981; Tokiwa et al., 1983). As a contribution to the study of the toxicological hazards related to urban air pollution in the city of Rome, the mutagenic activity of airborne particulate matter was evaluated in in vitro and in vivo experiments. Bacterial strains deficient in nitroreductase activities were used to characterize the mutagenic components of both unfractionated and fractionated extracts of par-
Materials and methods
Sampling and samples preparation Airborne particulate matter (particle size less than 10 /~) was collected at street level near a heavily used 4-lane street by a Hi-Vol cascade impactor operating at a flow rate of about 50 m3/h. Glass fibre filters used for the collection of particles were removed every 24 (48) h and stored in the dark. The organic phase of particulate matter was Soxhlet-extracted for 24 h with 150 ml dichloromethane (Carlo Erba, 99.5%) which was evaporated in a rotary evaporator at 40 o C. Fractionation of the organic extract into acidic, basic and neutral components was performed according to the scheme shown in Fig. 1. All samples were
ORGANIC EXTI~ACT(CH2Cl2)
NaOH
0.1
N
I
I
NaOH 0.I N
/\ CH2Cl 2
÷HCI IN pH=1 +CH2CI 2
H20
Acid Fraction
CH2Cl 2
I + HCl
0.1
N
/\ CH2CI 2
HCl 0.1 N ÷NaOH 1N ÷CH2Cl 2
Neutral Fraction
pH=13
/\ CH2Cl2
H20
Basic Fraction Fig. 1. Procedure followed for the fractionation of dichloromethane extracts of airborne particles into acidic, basic and neutral components.
567 dissolved in dimethyl sulphoxide (DSMO, Carlo Erba, analytical grade) just before use. Serum extraction of mutagenic compounds from the particulate matter was performed by incubating glass fibre filters in 250-ml bottles with 50 ml calf serum at 37 °C with shaking for 24-96 h. Afterwards serum was centrifuged for 20 min at 4000 g to remove particulate matter, filtered and passed over XAD-2 resin (Servachrome, analytical grade) to concentrate mutagenic compounds as described below for urine samples.
Mutagenicity assays The Salmonella/microsome plate-incorporation assay was performed as described by Maron and Ames (1983). Strain TA98 was kindly provided by Prof. B.N. Ames, University of Berkeley, CA; the nitropyrenes resistant strains TA98NR and TA98/1,8DNP6, (deficient in nitroreduction and hydroxylamine esterification, respectively), were a gift of Prof. H.S. Rosenkranz, Case Western Reserve University, Cleveland, OH. Exogenous metabolic activation was provided by liver postmitochondrial fractions ($9) obtained by Aroclor 1254 (Monsanto) induced Spra~ue-Dawley rats. Protein concentration, determined using the Lowry method (Lowry et al., 1951) with bovine serum albumin as standard, was 40 mg/ml. The $9 level routinely used was 50/~l/plate. For the assay of body fluids, 50 units of E. coli fl-glucuronidase (type VII, Sigma Chem. Co.) and 10 units of Aerobacter aryl sulphatase (type VII, Sigma) were added to each plate. All samples were tested over a wide range of concentrations in order to provide a dose-effect curve. The mutagenic potencies, i.e. the number of induced his + revertants/mg or m3 of filtered air were calculated from the linear portion of the curve. All determinations were made in triplicate in at least two independent experiments. Positive controls were always included to check both strain sensitivity and enzymic activities as well as routine controls of samples and $9 sterility. The micronucleus test was performed according to the recommended EPA protocol (Heddle et al., 1983). Air particulate extract dissolved in DMSO was administered to groups of 4 male and 4 female Swiss mice weighing 25-30 g in volumes of 5 ml/kg body weight by two intraperitoneal (i.p.)
injections with a 24-h interval. Two dosages (200 and 400 mg/kg) were chosen on the basis of preliminary toxicity tests. Animals were killed by cervical dislocation 24 and 48 h after the second injection. Negative controls were untreated or received the vehicle alone. Male positive controls received 1 m l / kg of benzene twice by gavage and were sacrificed 6 h after the second treatment; female positive controls were treated with 20 mg/kg of cyclophosphamide (Endoxan Asta) by i.p. injection and were sacrificed 24 h after the second injection. Bone marrow was sampled from both femurs following the procedure of Schmid (1975). Slides were prepared and stained according to Heddle et al. (1984). Two slides for each animal were scored by two different readers; 1000 polychromatic erythrocytes (PCE) per animal were examined for the presence of micronuclei and the ratio of polychromatic to normochromatic (NCE) erythrocytes determined.
Collection and processing of body fluids Male Sprague-Dawley rats (Charles River) weighing 200 + 10 g were used for the study of body-fluid mutagenicity. Airborne particulate extracts were dissolved in a small amount of DMSO, diluted in olive oil (highly refined, Sigma) and administered by gastric intubation or i.p. injection to pairs of rats which were housed in metabolic cages over 24 to 48 h after treatment. Controls received the vehicle alone by the same route. Excreta were collected in the dark on solid CO 2 and stored frozen until tested. Blood samples were obtained by ventricular puncture with sterile syringes containing preservative-free heparin (10 I U / m l of blood). Plasma was separated by centrifugation (500 g, 10 min) and assayed for mutagenicity as such. Urine and faecal samples were concentrated as described. Briefly, urine samples were thawed at room temperature, centrifuged for 10 rain at 3000 g and passed over 1 g of XAD-2 resin in 0.7 x 10 cm glass Bio-Rad Econo Colunms, the adsorbed material eluted with 10 ml acetone (Merck), the eluate dried under a flow of nitrogen and the residue dissolved in DMSO. Faecal samples were thawed, homogenized in 3 vol. 0.9% NaC1 and extracted with 10 vol. diethyl ether (Carlo Erba). The extract was
568
evaporated in a rotary evaporator and the residue dissolved in DMSO. Experiments on fiver microsomal monooxygenases Adult male albino Swiss mice weighing 24-27 g and Wistar rats weighing 200-250 g, purchased from Charles-River Italia (Calco, Como) with free access to water and standard pellet chow were used. In in vivo assays dichloromethane extract of airborne particulate matter dissolved in D M S O was administered to mice by single i.p. injection 24 h before sacrifice. Livers were immediately removed and 12.5% homogenates prepared in 0.01 M phosphate buffer containing 1.15% KC1, p H 7.4, using a Potter homogenizer with teflon pestle. The homogenates were centrifuged at 9000 g for 20 min at 4 ° C and the 9000 g supernatants used for enzyme assays. Aniline hydroxylase (AH) was measured by formation of p-aminophenol (PAP) and aminopyrine-N-demethylase (APND) by production of formaldehyde, according to the methods described by Mazel (1971). In in vitro assays, the 9000 g liver supernatants of untreated mice and rats were centrifuged at 100 000 g for 60 min to obtain the microsomal fraction. Cytochrome P-450 content after incubation with test materials with or without N A D P H was determined according to Mazel (1971) by the carbon monoxide difference spectrum of dithionite-reduced microsomes. Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. Statistical analyses were performed by the Student's t test. Results
Sampling of air particulates was performed over 2 - 3 weeks in winter, spring and summer 1986. D a t a on weight values and air concentration of
particulate matter and organic extracts are shown in Table 1. Such data demonstrate that the concentration of airborne particles was not strongly dependent on seasonal variations, although a 10% reduction was observed in the rainy spring months (Table 1, column " a i r p a r t i c u l a t e / m 3 "). Rather, a significant decrease in the amount of dichloromethane extractable matter was obtained, falling from 20% of the particulate weight in winter to 15 and 9% in spring and summer, respectively (Table 1, columns "organic f r a c t i o n / m 3" and "% organic matter"). This trend was paralleled by a reduction in the air concentration of mutagens (Fig. 2), due to the concurrent decrease in the mutagenicity of airborne matter (Fig. 3). N o major seasonal variation in the mutagenic properties of air particulate extracts was detected in assays carded out in strains TA98 and TA98NR, whereas a decreasing trend was observed in T A 9 8 / 1 , 8 D N P 6 (Fig. 4). Fractionation of air particulate extracts into acidic, basic and neutral components did not show large seasonal variations in weight values: the acidic fraction accounted for 21% of the whole extractable matter (range 16.8-25.2%), the neutral one for 75.1% (range 73.0-77.3) and the basic one for 3.9% (range 1.8-5.9). The mutagenic activities of acidic, basic and neutral fractions in S. typhimurium TA98, T A 9 8 N R and T A 9 8 / 1 , 8 D N P 6 are shown in Table 2. Mutagenic agents present in the basic fraction were shown to be proximate mutagens requiring exogenous metabolic activation by liver $9 and were unable to revert T A 9 8 / 1 , 8 D N P 6. Conversely, mutagens of the acidic and neutral fractions were similarly active with and without $9 in all tester strains, although acidic fractions were significantly less mutagenic in T A 9 8 N R and T A 9 8 / 1 , 8 D N P 6 with respect to TA98. N o definite time trend is evident in the data of Table 2, apart
TABLE 1 COLLECTION OF A I R PARTICULATE MATTER IN T H E URBAN AREA OF ROME: W E I G H T VALUES
Period covered by sampling
Duration of sampling (days)
Air volumes (m3)
P a r t i c u l a t e Organic collected fraction (g) (g)
% organic matter
Particulate/ m3 ( / ~ g )
Organic fraction/m3 (/tg)
01/23/86-02/14/86 04/01/86-05/12/86 06/23/86-07/19/86
14 20 22
18790 23240 25400
2.790 3.158 3.977
20 15 9
152 136 156
31 20 14
0.560 0.460 0.364
569
o)
o) TA98
TA98NR
~50
TA 98/1.8 DNP 6
TA98NR
TA98
~. IOO_ Q:
?,
50_
to
o
tll 2~
•~
TA98NR
b)
"~
t~ Q: 15o
TA 98 TA98/1.8DNP 6
c~ t.u
£3 lO
b)
TA98NR TA 98
lOO
TA98/1.8 DNP6
50_
£3 o
[] WINTER
[] SPRING
[] SUMMER
[]WINTER
[] SPRING
[]SUMMER
Fig. 2. Mutagenic activity of airborne particulate in $. typhimurium strains. Number of induced his + revertants/m3 of sampled air. (a) Without $9; (b) with $9.
Fig. 3. Mutagenic activity of airborne particulate in S. typhirnurium strains. N u m b e r of induced his + revertants/mg of particulate matter. (a) Without $9; Co) with $9.
from the decrease in the mutagenic activity of neutral fractions of summer samples in strain TA98/1,8DNP 6.
Reconstruction experiments demonstrated a recovery of mutagenic compounds greater than 80% in the fractionation procedure. Taking into account
TABLE 2 M U T A G E N I C ACTIVITY OF ACIDIC, BASIC A N D N E U T R A L COMPONENTS O F A I R PARTICULATE EXTRACTS IN S. typhimurium STRAINS Induced his + revertants/plate a Strain:
TA98
$9 mix:
-
+
TA98NR -
+
TA98/1,8DNP 6 .-
+
Winter samples
acidic fraction neutral fraction basic fraction
433 511 ns
455 589 1584
248 719 ns
317 820 1892
113 474 ns
99 366 ns
Spring samples
acidic fraction neutral fraction basic fraction
788 662 ns
809 713 462
358 769 ns
551 966 333
123 578 ns
93 358 ns
Summer samples
acidic fraction neutral fraction basic fraction
324 557 ns
267 624 450
129 345 ns
143 431 385
110 !88 ns
67 123 ns
a Calculated from the linear portion of the dose-effect relationship. ns N o significant mutagenic activity detected.
570 TABLE 3
o) TA 98 700.
EXTRACTION OF MUTAGENIC COMPOUNDS FROM AIRBORNE PARTICULATE MATTER
TA98NR
Extraction procedure a
I~1 500. (") 300,
Phosphate buffer, 96 h at 37 ° C
L) 1001
Calf serum, 96 h at 37 o C
E
Dichloromethane, 24 h in Soxhlet
b)
Q:: Lu
TA98NR TA 98
? ,ooj1 [ ] WINTER
-1I [ ] SPRING
TA98/1.SDNP 6
Induced his + revertants b
% recovery
780
3.1
1740
7.0
24780
100
a 3 identical samples of airborne particulate matter adsorbed on glass fibre filters (465 m g each) were submitted to different extraction procedures. b The a m o u n t of mutagens extracted from particulate matter was quantitated as the n u m b e r of induced his + revertants. Values shown in the Table were calculated from the linear portion of the dose-effect relationship in experiments with S. typhimurium TA98 without $9. Mutagens dissolved in aqueous media were concentrated on XAD-2 resin as described in Materials and Methods.
[~1 SUMMER
Fig. 4. Mutagenic activity of airborne particulate in S. typhimurium strains. N u m b e r of induced his + r e v e r t a n t s / m g of dichloromethane extract. (a) Without $9; (b) with $9.
the percent composition of air particulate extracts in acidic, basic and neutral fractions, the contribution of mutagens from each fraction to the in vitro mutagenicity of unfractionated extracts can be calculated. Roughly, neutral compounds accounted for two-thirds of the observed activities, whereas basic and, at a higher degree, acidic fractions contributed the remainder. To assess the biological availability of the mutagenic components of airborne particulate matter, the mutagenic activity of serum extracts was determined. Mutagens solubilized by serum were concentrated on XAD-2 resin to rule out the interference of serum with the performance of the mutagenicity assays (Ohsawa et al., 1983). Reconstruction experiments showed a recovery greater than 90% by such procedure. Data in Table 3 show that mutagens recovered in serum extracts accounted only for a minor fraction (about 7%) of the mutagenic activity exerted by dichloromethane extracts. Serum extracts, however, were significantly more mutagenic than saline extracts.
Useful information on the biological availability of materials adsorbed onto particulate matter can also be provided by the study of body-fluid mutagenicity in experimental animals (King et al., 1981; Belisario et al., 1984). To assess the suitability of this approach with airborne particulates, rats were exposed to dichloromethane extracts. Data in Tables 4 and 5 show that urine concentrates, faecal extracts and plasma samples of Sprague-Dawley rats treated at 80 mg/kg either by i.p. injection or per os, were unable to induce a 2-fold increase in the spontaneous number of revertants in Salmonella typhimurium TA98, although a positive trend was observed with urine concentrates. Taking into account the doses administered, the amounts assayed and the mutagenic activity of the extract in the test system used (lowest effective concentration 25 /~g/plate), it can be concluded that the concentration of active/ activable mutagens in the body fluids studied was below 2% of the administered dose in urines and faeces and below 5% in plasma. Obviously the absence of detectable activities in body fluids even after organic extract administration, although providing some information on in vivo biotransformation, made this approach unsuitable to assess
571 TABLE 4 M U T A G E N I C ACTIVITY OF U R I N E CONCENTRATES A N D FAECAL EXTRACTS F R O M S P R A G U E - D A W L E Y RATS A D M I N I S T E R E D W I T H AIR PARTICULATE EXTRACTS Treatment
Route
Administered
#l/plate a
dose (mg)
his + revertants/plate b Urine
Faeces
- $9
+ $9
- $9
+ $9
None
-
-
0
34+ 5
37+ 5
25+9
32+ 7
Vehicle
gavage
0
100
30+ 4
39+ 5
17+1
27+ 6
80 m g / k g
gavage
28.8
10 30 100
30 + 9 36+13 26+ 6
46 + 5 47+ 2 60+ 2
21 + 2 20+4 29+9
34 + 1 35+ 1 33+ 5
Vehicle
i.p.
0
100
33 + 2
37 + 1
21 + 2
30 + 3
80 m g / k g
i.p.
30.0
10 30 100
26 + 5 31+ 1 25+ 8
37 + 10 48+ 8 64+ 1
20 + 7 26+6 18+5
35 + 16 29+ 4 27± 1
a Urines and faeces collected over 48 h after treatment were concentrated as described in Materials and Methods. Concentrates were dissolved in 2 ml DMSO. b Means and SD from 3 plates; all experiments were carried out with S. typhimurium TA98 in the presence of deconjugating enzymes (fl-glucuronidase and aryl sulphatase).
the bioavailability of adsorbed materials. To gather further data on the biological consequences of the in vivo exposure to airborne particulate matter, the induction of micronuclei in mouse polychroTABLE 5 M U T A G E N I C ACTIVITY OF PLASMA SAMPLES COLLECTED F R O M S P R A G U E - D A W L E Y RATS A D M I N I S T E R E D W I T H AIR PARTICULATE EXTRACTS Treatment Route
Collection Volume of his + revertants/ time a plasma plate b (/~l/plate)
None
-
-
0
37 + 5
Vehicle
i.p.
2
200
38 ± 5
80mg/kg
i.p.
1 2 4
200 200 200
38+ 8 35 + 12 33+ 5
80mg/kg
gavage 1 2 4
200 200 200
36+ 7 44± 4 33+ 2
a Hours after treatment. b All data are based on single animals of 200 g body weight. Results are means + SD from three plates. Assays were carried out with S. typhimurium TA98 in the presence of $9 mix and deconjugating enzymes.
matic erythrocytes and the effects on liver microsomal enzymes were studied. The results of the micronucleus test are presented in Table 6. The frequency of micronucleated PCEs did not increase over control values in Swiss mice of both sexes after i.p. administration of dichloromethane extracts of air particulate matter at 200 and 400 mg/kg, although a highly significant ( P < 0.001) increase was observed in male and female positive control groups administered with benzene and cyclophosphamide, respectively. Toxicity to bone marrow, as shown by the decreased frequency of PCEs, was observed in vehicle controls and in animals treated with the highest dose of particulate extract 48 h after administration. Data in Table 7 show that i.p. administration of unfractionated dichloromethane extracts of both winter and spring samples of airborne matter caused a significant ( P < 0.01) decrease in aminopyrine-Ndemethylase activity without affecting aniline hydroxylase activity. The summer sample was assayed after fractionation into acidic, neutral and basic components. A significant decrease in both A P N D and A H activity was observed after administration of neutral components, whereas the basic fraction only affected the A P N D activity and the
572 TABLE 6 INCIDENCE OF MICRONUCLEI IN THE BONE-MARROW ERYTHROCYTES F R O M SWISS MICE A F T E R i.p. ADMINISTRATION OF AIRBORNE PARTICULATE EXTRACTS Treatment
Micronucleated PCE (%0 ± S.E.) Males
% PCE ± S.E.
Females
Total
Untreated
7 (1.7 + 0.2)
6 (1.5 + 0.6)
13 (1.6 + 0.3)
43.3 + 3.3
24 h vehicle (DMSO) 200 m g / k g b.w. 400 m g / k g b.w.
3 (0.7±0.2) 4 (1.0 ± 0.4) 8 (2.0 ± 0.4)
4 (1.0±0.7) 4 (1.0 ± 0.6) 5 (1.2 ± 0.5)
7 (0.9+0.3) 8 (1.0 ± 0.3) 13 (1.6 + 0.3)
30.4±2.2 ** 26.8 + 3.1 23.5 + 5.2
48 h vehicle (DMSO) 200 mg,/kg b.w. 400 m g / k g b.w.
2 (0.5 + 0.5) 5 (1.2±0.5) 3 (0.7 ± 0.5)
6 (1.5 + 0.6) 6 (1.5±0.3) 5 (1.2 ± 0.7)
8 (1.0 ± 0.6) 11 (1.4±0.3) 8 (1.0 ± 0.4)
32.2 + 4.2 * * 33.6±5.7 20.2 ± 4.1 * * *
Benzene (1 m l / k g b.w.) Cyclophosphamide (20 m g / k g b.w.)
94 (23.5 ± 1.5) *
-
-
31 (7.7 ± 1.5) *
-
24.2 ± 1.1 * *
-
36.6 ± 4.4
* Significantly greater than untreated controls ( p < 0.001, X2 test). * * Significantly lower than untreated controls ( p < 0.05, M a n n - W h i t n e y U-test). * * * Significantly lower than vehicle treated (48 h) ( p < 0.05, M a n n - W h i t n e y U-test).
acidic one was ineffective. Table 8 summarizes results
of
in
vitro
experiments
mouse-liver microsomes. cytochrome
P-450
with
rat-
the and
A significant decrease in
concentration
was
observed
a f t e r 15 m i n
incubation
with basic components This
effect was
NADPH
not
of microsomal of air particulate
related
in the incubation
to the
mixture,
fractions extract.
presence
of
thus indicat-
TABLE 7 INHIBITION OF HEPATIC MIXED F U N C T I O N OXIDASE SYSTEM IN MICE A F T E R i.p. ADMINISTRATION OF AIRBORNE PARTICULATE EXTRACTS Treatment
Dose b (mg/kg)
Aminopyrine-N-demethylase (nmoles H C H O / m i n / g liver)
Aniline hydroxylase (nmoles P A P / m i n / g liver)
322 + 24 c 317 + 18
48 + 7 50 + 6
Untreated Vehicle (DMSO)
-
Winter sample a Spring sample a
28 25
231 + 37 * * 251 +29 * *
44 + 7 47+5
16
305 + 16
50 + 8
13
192+12 * *
27+3 **
56
292 + 14 *
51 + 6
Summer sample (acidic fraction) (neutral fraction) (basic fraction)
a Unfractionated samples. b Doses were chosen on the basis of preliminary toxicity tests. c All values are means + SE from 4 animals. * p < 0.05; * * p < 0.01 (t test).
573 TABLE 8 IN VITRO EFFECTS OF ACIDIC, BASIC AND NEUTRAL COMPONENTS OF AIR PARTICULATE EXTRACTS ON CYTOCHROME P-450 CONCENTRATION IN RAT AND MOUSE HEPATIC MICROSOMES Dose (mg/ml)
NADPH (0.5 mM)
Control
0 0
+ -
Rat 1.28 -1-0.1 a 1.25 +0.1
Mouse 0.60 + 0.03 0.62+0.04
Acidic fraction
0.8 0.8
+ -
1.32 + 0.2 1.29 + 0.3
0.61 + 0.02 0.59 + 0.04
Neutral fraction
0.015 0.015
+ -
1.27+ 0.2 1.30 + 0.3
0.62 + 0.03 0.63 + 0.04
Basic fraction
2.8 2.8
+ -
0.69 + 0.07 * * 0.76+0.06 * *
0.35 + 0.04 * * 0.41 +0.01 * *
Cytochrome P-450 (nmoles/mg of protein)
a All values are means + SD of 5 determinations. * * p < 0.01 (t test).
ing a direct interaction of test material(s) with microsomal cytochrome P-450. Discussion
The occurrence of directly-acting bacterial mutagens absorbed onto airborne particulate matter is well established ( D e h n e n et al., 1977; Pitts et al., 1977; Talcott and Wei, 1977; Tokiwa et al., 1977). O u r data demonstrate a similar or lower concentration of airborne mutagens in the city of R o m e with respect to other E u r o p e a n (Lofroth, 1981) and American (Pitts et al., 1985) densely inhabited areas. The chemical identity of the mutagenic substances adsorbed o n t o air particulates is not fully elucidated. Airborne particulate matter is k n o w n to contain m o r e than 500 chemical c o m p o u n d s and chromatographic separation showed that the mutagenic activity in particulate extracts eluted over a wide range, with no major single peak (Lofroth, 1981). This suggests that m a n y different c o m p o u n d s contribute to the air particulate mutagenicity. This is not the case for particulates of other origin, such as diesel exhaust, in which a large fraction of the in vitro mutagenicity is due to the contribution of powerful mutagenic nitropyrenes (Rosenkranz, 1982). The results obtained in this study with S. typhimurium strain T A 9 8 N R (resistant to 1-nitropyrene) and T A 9 8 / 1 , 8 D N P 6 (resistant to 1,6- and 1 , 8 - d i -
nitropyrenes) suggest that nitropyrenes play only a limited role in air particulate mutagenicity. Similar conclusions were reached by Tokiwa et al. (1977) and by Siak et al. (1985). The seasonal variation observed in the strains' sensitivity (Fig. 4) suggests, however, a greater contribution of nitro c o m p o u n d s in spring and summer samples, possibly related to the greater contribution of m o t o r vehicle exhausts to u r b a n air pollution. After chemical fractionation of air particulate extracts, m u t a g e n s active in strain TA98 were detected in all acidic, neutral and basic c o m p o nents which exerted roughly similar mutagenic activities. Directly-acting mutagens were detected in acidic and neutral fractions, where aliphatic and polycyclic nitro c o m p o u n d s are expected. Proximate mutagens accounted for the mutagenicity of basic fractions (Table 2). Although no effort was made to identify these promutagens by chemical means, their inactivity in strain T A 9 8 / 1 , 8 D N P 6 is strongly suggestive of the involvement of aromatic amines or related c o m p o u n d s ( M c C o y et al., 1982). Several factors are to be taken into account in the evaluation of mutagenicity data of particulate extracts for the assessment of h u m a n risks related to exposure to air pollutants. F o r example, it was previously stressed that trace amounts of nitro c o m p o u n d s such as nitropyrenes could account for most of the mutagenic activity exerted by
574
particulate extracts in bacteria, due to the extraordinary activity of bacterial nitroreductases (Mermelstein et al., 1981). Another factor to be taken into account is the bioavailability of the mutagenic components adsorbed onto airborne particulate matter. Our data show that adsorbed mutagens are scarcely extractable by serum and therefore probably of limited bioavailability. Similar conclusions were reached by Ohsawa and coworkers (1983) and by Belisario et al. (1984) in a study on diesel exhaust. Also possible divergences between in vivo and in vitro metabolic systems are to be considered in the evaluation of in vitro data. In this connection it is noteworthy that, although liver $9 was unable to reduce significantly the in vitro mutagenicity of air particulate extracts, the study of body fluids' mutagenicity demonstrated the rapid and total disappearance of active/ activable mutagens in body fluids of treated animals, suggesting an extensive in vivo inactivation. These findings may be related to the negative outcome of in vivo cytogenetic assays obtained by us and by others even after positive in vitro results (Krishna et al., 1986). Due to the high chemical complexity of airborne materials, other biological properties beyond mutagenicity are also to be considered, for example the possible synergistic or antagonistic interaction with other chemical exposures. Air particulate extracts were previously shown to contain chemical compounds, different from polycyclic aromatic hydrocarbons and direct-acting mutagens, which were able to bind the TCDD-receptor protein in rat liver (Totfgard et al., 1983). Accordingly, we have shown that a single administration of air particulate extract is sufficient to modify the xenobiotic metabolizing system of mice. In vitro experiments with mouse and rat microsomes suggest a direct interaction with active sites of cytochrome P-450; however, other mechanisms, such as the decreased biosynthesis or increased breakdown of enzymes or their cofactors could also work in vivo. Moreover, it is conceivable that the air particulate matter, as with many other xenobiotics, could exert a biphasic effect working as an inhibitor in the acute phase and as an inducer after chronic exposure (Testa and Jenner, 1981). Taking into account the undefined bioavail-
ability of airborne material and possible unpredictable interactions among chemicals adsorbed onto particulate matter, it is suggested that only experimental models more closely related to the real human exposure could provide useful information on genotoxic risks. This goal is at present urgently needed due to the increasing levels of air pollution in industrialized areas and the wide human exposure to airborne materials.
Acknowledgements This work was partially supported by the E.E.C. (contract No. 530 ENV l-s). The technical assistance of Ms. C. D'Ascoli, Ms. L. Gargano and Mr. U. Cervelli is gratefully acknowledged.
References Belisario, M.A., V. Buonocore, E. De Marinis and F. De Lorenzo (1984) Biological availability of mutagenic compounds adsorbed onto diesel exhaust particulate, Mutation Res., 135, 1-9. Carnow, B.W. (1978) The "urban factor" and lung cancer: Cigarette smoking or air pollution, Environ. Health Perspect., 22, 17-21. Dehnen, W., N. Pitz and R. Tomingas (1977) The mutagenicity of airborne particulate pollutants, Cancer Lett., 4, 5-6. Doll, R. (1978) Atmospheric pollution and lung cancer, Environ. Health Perspect., 22, 23-31. Heddle, J.A., M. Hite, B. Kirkart, K. Mavourning, J.T. McGregor, G.W. Newell and M.F. Salamone (1983) The induction of micronuclei as a measure of genotoxicity, A Report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 123, 61-118. Heddle, J.A., E. Stuart and M.F. Salamone (1984) The bone marrow micronucleus test, in: B.J. Kilbey, M.S. Legator, W. Nichols and C. Ramel (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 441-475. Helmes, C.T., D.L. Atkinson, J. Jaffer, C.C. Sigman, K.L. Thompson, M.I. Kelsey, H.F. Kraybill and J.I. Munn (1982) Evaluation and classification of the potential carcinogenicity of organic air pollutants, J. Environ. Sci. Health, A17, 321-389. Higginson, J., and O.M. Jensen (1977) Epidemiological review on lung cancer, in: U. Mohr, D. Schmahl and L. Tomatis (Eds.), Air Pollution and Cancer in Man, IARC Sci. Publ. No. 16, IARC, Lyon, pp. 169-189. 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. King, L.C., M.J. Kohan, A.C. Austin, L.D. Claxton and J. Lewtas Huising (1981) Evaluation of the release of mutagens from diesel particles in the presence of physiological fluids, Environ. Mutagen., 3, 109-123.
575 Kotin, P., H.L. Falk, P. Mader and M. Thomas (1954) Aromatic hydrocarbons, 1. Presence in the Los Angeles atmosphere and the carcinogenicity of atmospheric extracts, Arch. Ind. Hyg. Occup. Med., 9, 153-177. 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. Leiter, J., M.B. Shimkin and M.J. Shear (1942) Production of subcutaneous sarcomas in mice with tars extracted from atmospheric dusts, J. Natl. Cancer Inst., 3, 155-165. Lofroth, G. (1981) Comparison of the mutagenic activity in carbon particulate matter and in diesel and gasoline engine exhaust, in: M.D. Waters, S.S. Shandu, J. Lewtas-Huisingh, L. Claxton and S. Nesnow (Eds.), Short-Term Bioassay in the Analysis of Complex Environmental Mixtures II, Plenum, New York, pp. 319-336. Lowry, O.H., N.J. Rosenbrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193, 265-275. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mazel, P. (1971) Experiments illustrating drug metabolism in vitro, in: B.N. La Du, M.G. Mandel and E.L. Way (Eds.), Fundamentals of Drug Metabolism and Drug Disposition, Williams and Wilkins, Baltimore, pp. 546-550. McCoy, E.C., G.D. McCoy and H.S. Rosenkranz (1982) Esterification of arylhydroxylamine, Biochem. Biophys. Res. Commun., 108, 1362-1367. Mermelstein, R., D.K. Kiriazides, M. Butler, E.C. McCoy and H.S. Rosenkranz (1981) The extraordinary mutagenicity of nitropyrenes in bacteria, Mutation Res., 89, 187-196.
Ohsawa, M., T. Ochi and H. Hayashi (1983) Mutagenicity in Salmonella typhimurium mutants of serum extracts from airborne particulates, Mutation Res., 116, 83-90. Pellizzari, E.D. (1978) State-of-the-art analytical techniques for ambient vapor phase organics and volatile organics in aqueous samples from energy-related activities, in: Application of Short-term Bioassays in the Fractionation and Analysis of Complex Environmental Mixtures, EPA Publ. 600/ 9-78-027, pp. 195-227. Pitts Jr., J.N., D. Grosjean, T.M. Mischke, V.F. Simmon and D. Poole (1977) Mutagenic activity of airborn particulate organic pollutants, Toxicol. Lett., 1, 65-70. Pitts Jr., J.N., J.A. Sweetman, W. Harger and D.R. Ditz (1985) Diurnal mutagenicity of airborne particulate organic matter adjacent to a heavily traveled west Los Angeles freeway, J. Air Pollut. Control Assoc., 35, 638-643. Rosenkranz, H.S. (1982) Direct-acting mutagens in diesel exhausts: magnitude of the problem, Mutation Res., 101, 1-10. Saffiotti, U. (1983) Evaluation of mixed exposure to carcinogens and correlations of in vivo and in vitro systems, Environ. Health Perspect., 47, 319-324. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Siak, J., T.L. Chan, T.L. Gibson and G.T. Wolff (1985) Contribution to bacterial mutagenicity from nitro-PAH compounds in ambient aereosols, Atm. Environ., 19, No. 2, 369-376. Testa, B., and P. Jenner (1981) Inhibitor of cytochrome P-450s and their mechanism of action, Drug Metab. Rev., 12, 1-118.
565
Elsevier MTR01272
In vitro and in vivo mutagenicity studies with airborne particulate extracts R. Crebelli, S. Fuselli, A. Meneguz, G. Aquilina, L. Conti, P. Leopardi, A. Zijno, F. Baris and A. Carere Istituto Superiore di Sanit3, VialeRegina Elena 299, 00161 Rome (Italy)
(Received 18 June 1987) (Revision received12 October 1987) (Accepted 15 October 1987)
Keywords: Air particulate; Ames test; Micronucleus; Monooxygenases.
Summary The contribution of nitro compounds to airbome particulate mutagenicity was studied with Salmonella typhimurium strains TA98, TA98NR, T A 9 8 / 1 , 8 D N P 6. The results obtained indicate that nitropyrenes play a minor role in air particulate mutagenicity. Seasonal variations indicate a relatively greater contribution of nitro compounds to the mutagenicity of spring and summer samples. Fractionation of extracts into acidic, neutral and basic components shows that neutral compounds account for about two-thirds of the total mutagenic activity. Attempts to extract mutagens adsorbed onto particulate matter with aqueous media were almost completely negative. No significant mutagenicity was detected in urine and faecal extracts and in plasma samples of Sprague-Dawley rats treated with air particulate extracts at 80 m g / k g either per os or by i.p. injection. Negative results were obtained in the micronucleus test with Swiss mice treated at 200 and 400 m g / k g (twice by i.p. injection). A significant decrease in liver aminopyrine-N-demethylase was observed in Swiss mice injected with air particulate extracts or its basic and neutral fractions. In vitro experiments suggest a direct interaction of test materials with microsomal cytochrome P-450.
Several epidemiological studies have demonstrated a higher incidence of lung cancer in urban than in rural areas. This " u r b a n factor" was explained by the general exposure of the population to air pollutants in industrialized areas, beyond that by different smoking habits and occupational exposures (Higginson and Jensen, 1977). Several known carcinogenic polycyclic aromatic hydrocarbons and aromatic amines have been identified among the hundreds of chemical compounds adCorrespondence: Dr. R. Crebelli, Laboratorio di Tossicologia Applicata, Istituto Superioredi Sanith, Viale Regina Elena 299, 00161 Roma (Italia).
sorbed onto airborne particulate matter collected in polluted areas (Pellizzari, 1978; Helmes et al., 1982). Moreover, benzene extracts of urban air particulate have been known for decades to be carcinogenic in mammals by subcutaneous injection or skin application (Leiter and Shear, 1942; Hueper et al., 1962). However, to what extent exposure to air pollutants may pose a significant human hazard is still debated. Some estimates have indicated that air pollution could account for 5-10% of all cancers of the respiratory tract (Carnew, 1978; Doll, 1978), whereas a more recent reassessment of epidemiological data has suggested that the risk related to general air pollution
0165-1218/88/$03.50 © 1988 ElsevierSciencePubfishers B.V. (BiomedicalDivision)
566 ticulate matter collected in winter, spring and summer. In vivo experiments were carried out to evaluate both the mutagenic activity in body fluids of treated rats and the induction of micronuclei in mouse polychromatic erythrocytes. Furthermore, the possible in vitro and in vivo activity of air particulate extracts on mouse-liver microsomal enzymes was studied.
could be very much lower (Speizer, 1983). The biological evaluation of air pollution exposure is a hard task: in the case of such mixed concurrent exposures in fact synergistic or antagonistic interactions among carcinogens, promoters, cocarcinogens and inhibitors may occur in an unpredictable way (Saffiotti, 1983). Furthermore, in the evaluation of the genotoxic risks related to airborne particulate exposure some additional problems are encountered: (i) the undefined bioavailability of airbome genotoxic compounds adsorbed onto the particulate matter and (ii) the possible overestimation of the mutagenic potencies, due to the extraordinary mutagenic activity exerted in bacteria by some nitro compounds present as minor components in air particulate extracts (Mermelstein et al., 1981; Tokiwa et al., 1983). As a contribution to the study of the toxicological hazards related to urban air pollution in the city of Rome, the mutagenic activity of airborne particulate matter was evaluated in in vitro and in vivo experiments. Bacterial strains deficient in nitroreductase activities were used to characterize the mutagenic components of both unfractionated and fractionated extracts of par-
Materials and methods
Sampling and samples preparation Airborne particulate matter (particle size less than 10 /~) was collected at street level near a heavily used 4-lane street by a Hi-Vol cascade impactor operating at a flow rate of about 50 m3/h. Glass fibre filters used for the collection of particles were removed every 24 (48) h and stored in the dark. The organic phase of particulate matter was Soxhlet-extracted for 24 h with 150 ml dichloromethane (Carlo Erba, 99.5%) which was evaporated in a rotary evaporator at 40 o C. Fractionation of the organic extract into acidic, basic and neutral components was performed according to the scheme shown in Fig. 1. All samples were
ORGANIC EXTI~ACT(CH2Cl2)
NaOH
0.1
N
I
I
NaOH 0.I N
/\ CH2Cl 2
÷HCI IN pH=1 +CH2CI 2
H20
Acid Fraction
CH2Cl 2
I + HCl
0.1
N
/\ CH2CI 2
HCl 0.1 N ÷NaOH 1N ÷CH2Cl 2
Neutral Fraction
pH=13
/\ CH2Cl2
H20
Basic Fraction Fig. 1. Procedure followed for the fractionation of dichloromethane extracts of airborne particles into acidic, basic and neutral components.
567 dissolved in dimethyl sulphoxide (DSMO, Carlo Erba, analytical grade) just before use. Serum extraction of mutagenic compounds from the particulate matter was performed by incubating glass fibre filters in 250-ml bottles with 50 ml calf serum at 37 °C with shaking for 24-96 h. Afterwards serum was centrifuged for 20 min at 4000 g to remove particulate matter, filtered and passed over XAD-2 resin (Servachrome, analytical grade) to concentrate mutagenic compounds as described below for urine samples.
Mutagenicity assays The Salmonella/microsome plate-incorporation assay was performed as described by Maron and Ames (1983). Strain TA98 was kindly provided by Prof. B.N. Ames, University of Berkeley, CA; the nitropyrenes resistant strains TA98NR and TA98/1,8DNP6, (deficient in nitroreduction and hydroxylamine esterification, respectively), were a gift of Prof. H.S. Rosenkranz, Case Western Reserve University, Cleveland, OH. Exogenous metabolic activation was provided by liver postmitochondrial fractions ($9) obtained by Aroclor 1254 (Monsanto) induced Spra~ue-Dawley rats. Protein concentration, determined using the Lowry method (Lowry et al., 1951) with bovine serum albumin as standard, was 40 mg/ml. The $9 level routinely used was 50/~l/plate. For the assay of body fluids, 50 units of E. coli fl-glucuronidase (type VII, Sigma Chem. Co.) and 10 units of Aerobacter aryl sulphatase (type VII, Sigma) were added to each plate. All samples were tested over a wide range of concentrations in order to provide a dose-effect curve. The mutagenic potencies, i.e. the number of induced his + revertants/mg or m3 of filtered air were calculated from the linear portion of the curve. All determinations were made in triplicate in at least two independent experiments. Positive controls were always included to check both strain sensitivity and enzymic activities as well as routine controls of samples and $9 sterility. The micronucleus test was performed according to the recommended EPA protocol (Heddle et al., 1983). Air particulate extract dissolved in DMSO was administered to groups of 4 male and 4 female Swiss mice weighing 25-30 g in volumes of 5 ml/kg body weight by two intraperitoneal (i.p.)
injections with a 24-h interval. Two dosages (200 and 400 mg/kg) were chosen on the basis of preliminary toxicity tests. Animals were killed by cervical dislocation 24 and 48 h after the second injection. Negative controls were untreated or received the vehicle alone. Male positive controls received 1 m l / kg of benzene twice by gavage and were sacrificed 6 h after the second treatment; female positive controls were treated with 20 mg/kg of cyclophosphamide (Endoxan Asta) by i.p. injection and were sacrificed 24 h after the second injection. Bone marrow was sampled from both femurs following the procedure of Schmid (1975). Slides were prepared and stained according to Heddle et al. (1984). Two slides for each animal were scored by two different readers; 1000 polychromatic erythrocytes (PCE) per animal were examined for the presence of micronuclei and the ratio of polychromatic to normochromatic (NCE) erythrocytes determined.
Collection and processing of body fluids Male Sprague-Dawley rats (Charles River) weighing 200 + 10 g were used for the study of body-fluid mutagenicity. Airborne particulate extracts were dissolved in a small amount of DMSO, diluted in olive oil (highly refined, Sigma) and administered by gastric intubation or i.p. injection to pairs of rats which were housed in metabolic cages over 24 to 48 h after treatment. Controls received the vehicle alone by the same route. Excreta were collected in the dark on solid CO 2 and stored frozen until tested. Blood samples were obtained by ventricular puncture with sterile syringes containing preservative-free heparin (10 I U / m l of blood). Plasma was separated by centrifugation (500 g, 10 min) and assayed for mutagenicity as such. Urine and faecal samples were concentrated as described. Briefly, urine samples were thawed at room temperature, centrifuged for 10 rain at 3000 g and passed over 1 g of XAD-2 resin in 0.7 x 10 cm glass Bio-Rad Econo Colunms, the adsorbed material eluted with 10 ml acetone (Merck), the eluate dried under a flow of nitrogen and the residue dissolved in DMSO. Faecal samples were thawed, homogenized in 3 vol. 0.9% NaC1 and extracted with 10 vol. diethyl ether (Carlo Erba). The extract was
568
evaporated in a rotary evaporator and the residue dissolved in DMSO. Experiments on fiver microsomal monooxygenases Adult male albino Swiss mice weighing 24-27 g and Wistar rats weighing 200-250 g, purchased from Charles-River Italia (Calco, Como) with free access to water and standard pellet chow were used. In in vivo assays dichloromethane extract of airborne particulate matter dissolved in D M S O was administered to mice by single i.p. injection 24 h before sacrifice. Livers were immediately removed and 12.5% homogenates prepared in 0.01 M phosphate buffer containing 1.15% KC1, p H 7.4, using a Potter homogenizer with teflon pestle. The homogenates were centrifuged at 9000 g for 20 min at 4 ° C and the 9000 g supernatants used for enzyme assays. Aniline hydroxylase (AH) was measured by formation of p-aminophenol (PAP) and aminopyrine-N-demethylase (APND) by production of formaldehyde, according to the methods described by Mazel (1971). In in vitro assays, the 9000 g liver supernatants of untreated mice and rats were centrifuged at 100 000 g for 60 min to obtain the microsomal fraction. Cytochrome P-450 content after incubation with test materials with or without N A D P H was determined according to Mazel (1971) by the carbon monoxide difference spectrum of dithionite-reduced microsomes. Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. Statistical analyses were performed by the Student's t test. Results
Sampling of air particulates was performed over 2 - 3 weeks in winter, spring and summer 1986. D a t a on weight values and air concentration of
particulate matter and organic extracts are shown in Table 1. Such data demonstrate that the concentration of airborne particles was not strongly dependent on seasonal variations, although a 10% reduction was observed in the rainy spring months (Table 1, column " a i r p a r t i c u l a t e / m 3 "). Rather, a significant decrease in the amount of dichloromethane extractable matter was obtained, falling from 20% of the particulate weight in winter to 15 and 9% in spring and summer, respectively (Table 1, columns "organic f r a c t i o n / m 3" and "% organic matter"). This trend was paralleled by a reduction in the air concentration of mutagens (Fig. 2), due to the concurrent decrease in the mutagenicity of airborne matter (Fig. 3). N o major seasonal variation in the mutagenic properties of air particulate extracts was detected in assays carded out in strains TA98 and TA98NR, whereas a decreasing trend was observed in T A 9 8 / 1 , 8 D N P 6 (Fig. 4). Fractionation of air particulate extracts into acidic, basic and neutral components did not show large seasonal variations in weight values: the acidic fraction accounted for 21% of the whole extractable matter (range 16.8-25.2%), the neutral one for 75.1% (range 73.0-77.3) and the basic one for 3.9% (range 1.8-5.9). The mutagenic activities of acidic, basic and neutral fractions in S. typhimurium TA98, T A 9 8 N R and T A 9 8 / 1 , 8 D N P 6 are shown in Table 2. Mutagenic agents present in the basic fraction were shown to be proximate mutagens requiring exogenous metabolic activation by liver $9 and were unable to revert T A 9 8 / 1 , 8 D N P 6. Conversely, mutagens of the acidic and neutral fractions were similarly active with and without $9 in all tester strains, although acidic fractions were significantly less mutagenic in T A 9 8 N R and T A 9 8 / 1 , 8 D N P 6 with respect to TA98. N o definite time trend is evident in the data of Table 2, apart
TABLE 1 COLLECTION OF A I R PARTICULATE MATTER IN T H E URBAN AREA OF ROME: W E I G H T VALUES
Period covered by sampling
Duration of sampling (days)
Air volumes (m3)
P a r t i c u l a t e Organic collected fraction (g) (g)
% organic matter
Particulate/ m3 ( / ~ g )
Organic fraction/m3 (/tg)
01/23/86-02/14/86 04/01/86-05/12/86 06/23/86-07/19/86
14 20 22
18790 23240 25400
2.790 3.158 3.977
20 15 9
152 136 156
31 20 14
0.560 0.460 0.364
569
o)
o) TA98
TA98NR
~50
TA 98/1.8 DNP 6
TA98NR
TA98
~. IOO_ Q:
?,
50_
to
o
tll 2~
•~
TA98NR
b)
"~
t~ Q: 15o
TA 98 TA98/1.8DNP 6
c~ t.u
£3 lO
b)
TA98NR TA 98
lOO
TA98/1.8 DNP6
50_
£3 o
[] WINTER
[] SPRING
[] SUMMER
[]WINTER
[] SPRING
[]SUMMER
Fig. 2. Mutagenic activity of airborne particulate in $. typhimurium strains. Number of induced his + revertants/m3 of sampled air. (a) Without $9; (b) with $9.
Fig. 3. Mutagenic activity of airborne particulate in S. typhirnurium strains. N u m b e r of induced his + revertants/mg of particulate matter. (a) Without $9; Co) with $9.
from the decrease in the mutagenic activity of neutral fractions of summer samples in strain TA98/1,8DNP 6.
Reconstruction experiments demonstrated a recovery of mutagenic compounds greater than 80% in the fractionation procedure. Taking into account
TABLE 2 M U T A G E N I C ACTIVITY OF ACIDIC, BASIC A N D N E U T R A L COMPONENTS O F A I R PARTICULATE EXTRACTS IN S. typhimurium STRAINS Induced his + revertants/plate a Strain:
TA98
$9 mix:
-
+
TA98NR -
+
TA98/1,8DNP 6 .-
+
Winter samples
acidic fraction neutral fraction basic fraction
433 511 ns
455 589 1584
248 719 ns
317 820 1892
113 474 ns
99 366 ns
Spring samples
acidic fraction neutral fraction basic fraction
788 662 ns
809 713 462
358 769 ns
551 966 333
123 578 ns
93 358 ns
Summer samples
acidic fraction neutral fraction basic fraction
324 557 ns
267 624 450
129 345 ns
143 431 385
110 !88 ns
67 123 ns
a Calculated from the linear portion of the dose-effect relationship. ns N o significant mutagenic activity detected.
570 TABLE 3
o) TA 98 700.
EXTRACTION OF MUTAGENIC COMPOUNDS FROM AIRBORNE PARTICULATE MATTER
TA98NR
Extraction procedure a
I~1 500. (") 300,
Phosphate buffer, 96 h at 37 ° C
L) 1001
Calf serum, 96 h at 37 o C
E
Dichloromethane, 24 h in Soxhlet
b)
Q:: Lu
TA98NR TA 98
? ,ooj1 [ ] WINTER
-1I [ ] SPRING
TA98/1.SDNP 6
Induced his + revertants b
% recovery
780
3.1
1740
7.0
24780
100
a 3 identical samples of airborne particulate matter adsorbed on glass fibre filters (465 m g each) were submitted to different extraction procedures. b The a m o u n t of mutagens extracted from particulate matter was quantitated as the n u m b e r of induced his + revertants. Values shown in the Table were calculated from the linear portion of the dose-effect relationship in experiments with S. typhimurium TA98 without $9. Mutagens dissolved in aqueous media were concentrated on XAD-2 resin as described in Materials and Methods.
[~1 SUMMER
Fig. 4. Mutagenic activity of airborne particulate in S. typhimurium strains. N u m b e r of induced his + r e v e r t a n t s / m g of dichloromethane extract. (a) Without $9; (b) with $9.
the percent composition of air particulate extracts in acidic, basic and neutral fractions, the contribution of mutagens from each fraction to the in vitro mutagenicity of unfractionated extracts can be calculated. Roughly, neutral compounds accounted for two-thirds of the observed activities, whereas basic and, at a higher degree, acidic fractions contributed the remainder. To assess the biological availability of the mutagenic components of airborne particulate matter, the mutagenic activity of serum extracts was determined. Mutagens solubilized by serum were concentrated on XAD-2 resin to rule out the interference of serum with the performance of the mutagenicity assays (Ohsawa et al., 1983). Reconstruction experiments showed a recovery greater than 90% by such procedure. Data in Table 3 show that mutagens recovered in serum extracts accounted only for a minor fraction (about 7%) of the mutagenic activity exerted by dichloromethane extracts. Serum extracts, however, were significantly more mutagenic than saline extracts.
Useful information on the biological availability of materials adsorbed onto particulate matter can also be provided by the study of body-fluid mutagenicity in experimental animals (King et al., 1981; Belisario et al., 1984). To assess the suitability of this approach with airborne particulates, rats were exposed to dichloromethane extracts. Data in Tables 4 and 5 show that urine concentrates, faecal extracts and plasma samples of Sprague-Dawley rats treated at 80 mg/kg either by i.p. injection or per os, were unable to induce a 2-fold increase in the spontaneous number of revertants in Salmonella typhimurium TA98, although a positive trend was observed with urine concentrates. Taking into account the doses administered, the amounts assayed and the mutagenic activity of the extract in the test system used (lowest effective concentration 25 /~g/plate), it can be concluded that the concentration of active/ activable mutagens in the body fluids studied was below 2% of the administered dose in urines and faeces and below 5% in plasma. Obviously the absence of detectable activities in body fluids even after organic extract administration, although providing some information on in vivo biotransformation, made this approach unsuitable to assess
571 TABLE 4 M U T A G E N I C ACTIVITY OF U R I N E CONCENTRATES A N D FAECAL EXTRACTS F R O M S P R A G U E - D A W L E Y RATS A D M I N I S T E R E D W I T H AIR PARTICULATE EXTRACTS Treatment
Route
Administered
#l/plate a
dose (mg)
his + revertants/plate b Urine
Faeces
- $9
+ $9
- $9
+ $9
None
-
-
0
34+ 5
37+ 5
25+9
32+ 7
Vehicle
gavage
0
100
30+ 4
39+ 5
17+1
27+ 6
80 m g / k g
gavage
28.8
10 30 100
30 + 9 36+13 26+ 6
46 + 5 47+ 2 60+ 2
21 + 2 20+4 29+9
34 + 1 35+ 1 33+ 5
Vehicle
i.p.
0
100
33 + 2
37 + 1
21 + 2
30 + 3
80 m g / k g
i.p.
30.0
10 30 100
26 + 5 31+ 1 25+ 8
37 + 10 48+ 8 64+ 1
20 + 7 26+6 18+5
35 + 16 29+ 4 27± 1
a Urines and faeces collected over 48 h after treatment were concentrated as described in Materials and Methods. Concentrates were dissolved in 2 ml DMSO. b Means and SD from 3 plates; all experiments were carried out with S. typhimurium TA98 in the presence of deconjugating enzymes (fl-glucuronidase and aryl sulphatase).
the bioavailability of adsorbed materials. To gather further data on the biological consequences of the in vivo exposure to airborne particulate matter, the induction of micronuclei in mouse polychroTABLE 5 M U T A G E N I C ACTIVITY OF PLASMA SAMPLES COLLECTED F R O M S P R A G U E - D A W L E Y RATS A D M I N I S T E R E D W I T H AIR PARTICULATE EXTRACTS Treatment Route
Collection Volume of his + revertants/ time a plasma plate b (/~l/plate)
None
-
-
0
37 + 5
Vehicle
i.p.
2
200
38 ± 5
80mg/kg
i.p.
1 2 4
200 200 200
38+ 8 35 + 12 33+ 5
80mg/kg
gavage 1 2 4
200 200 200
36+ 7 44± 4 33+ 2
a Hours after treatment. b All data are based on single animals of 200 g body weight. Results are means + SD from three plates. Assays were carried out with S. typhimurium TA98 in the presence of $9 mix and deconjugating enzymes.
matic erythrocytes and the effects on liver microsomal enzymes were studied. The results of the micronucleus test are presented in Table 6. The frequency of micronucleated PCEs did not increase over control values in Swiss mice of both sexes after i.p. administration of dichloromethane extracts of air particulate matter at 200 and 400 mg/kg, although a highly significant ( P < 0.001) increase was observed in male and female positive control groups administered with benzene and cyclophosphamide, respectively. Toxicity to bone marrow, as shown by the decreased frequency of PCEs, was observed in vehicle controls and in animals treated with the highest dose of particulate extract 48 h after administration. Data in Table 7 show that i.p. administration of unfractionated dichloromethane extracts of both winter and spring samples of airborne matter caused a significant ( P < 0.01) decrease in aminopyrine-Ndemethylase activity without affecting aniline hydroxylase activity. The summer sample was assayed after fractionation into acidic, neutral and basic components. A significant decrease in both A P N D and A H activity was observed after administration of neutral components, whereas the basic fraction only affected the A P N D activity and the
572 TABLE 6 INCIDENCE OF MICRONUCLEI IN THE BONE-MARROW ERYTHROCYTES F R O M SWISS MICE A F T E R i.p. ADMINISTRATION OF AIRBORNE PARTICULATE EXTRACTS Treatment
Micronucleated PCE (%0 ± S.E.) Males
% PCE ± S.E.
Females
Total
Untreated
7 (1.7 + 0.2)
6 (1.5 + 0.6)
13 (1.6 + 0.3)
43.3 + 3.3
24 h vehicle (DMSO) 200 m g / k g b.w. 400 m g / k g b.w.
3 (0.7±0.2) 4 (1.0 ± 0.4) 8 (2.0 ± 0.4)
4 (1.0±0.7) 4 (1.0 ± 0.6) 5 (1.2 ± 0.5)
7 (0.9+0.3) 8 (1.0 ± 0.3) 13 (1.6 + 0.3)
30.4±2.2 ** 26.8 + 3.1 23.5 + 5.2
48 h vehicle (DMSO) 200 mg,/kg b.w. 400 m g / k g b.w.
2 (0.5 + 0.5) 5 (1.2±0.5) 3 (0.7 ± 0.5)
6 (1.5 + 0.6) 6 (1.5±0.3) 5 (1.2 ± 0.7)
8 (1.0 ± 0.6) 11 (1.4±0.3) 8 (1.0 ± 0.4)
32.2 + 4.2 * * 33.6±5.7 20.2 ± 4.1 * * *
Benzene (1 m l / k g b.w.) Cyclophosphamide (20 m g / k g b.w.)
94 (23.5 ± 1.5) *
-
-
31 (7.7 ± 1.5) *
-
24.2 ± 1.1 * *
-
36.6 ± 4.4
* Significantly greater than untreated controls ( p < 0.001, X2 test). * * Significantly lower than untreated controls ( p < 0.05, M a n n - W h i t n e y U-test). * * * Significantly lower than vehicle treated (48 h) ( p < 0.05, M a n n - W h i t n e y U-test).
acidic one was ineffective. Table 8 summarizes results
of
in
vitro
experiments
mouse-liver microsomes. cytochrome
P-450
with
rat-
the and
A significant decrease in
concentration
was
observed
a f t e r 15 m i n
incubation
with basic components This
effect was
NADPH
not
of microsomal of air particulate
related
in the incubation
to the
mixture,
fractions extract.
presence
of
thus indicat-
TABLE 7 INHIBITION OF HEPATIC MIXED F U N C T I O N OXIDASE SYSTEM IN MICE A F T E R i.p. ADMINISTRATION OF AIRBORNE PARTICULATE EXTRACTS Treatment
Dose b (mg/kg)
Aminopyrine-N-demethylase (nmoles H C H O / m i n / g liver)
Aniline hydroxylase (nmoles P A P / m i n / g liver)
322 + 24 c 317 + 18
48 + 7 50 + 6
Untreated Vehicle (DMSO)
-
Winter sample a Spring sample a
28 25
231 + 37 * * 251 +29 * *
44 + 7 47+5
16
305 + 16
50 + 8
13
192+12 * *
27+3 **
56
292 + 14 *
51 + 6
Summer sample (acidic fraction) (neutral fraction) (basic fraction)
a Unfractionated samples. b Doses were chosen on the basis of preliminary toxicity tests. c All values are means + SE from 4 animals. * p < 0.05; * * p < 0.01 (t test).
573 TABLE 8 IN VITRO EFFECTS OF ACIDIC, BASIC AND NEUTRAL COMPONENTS OF AIR PARTICULATE EXTRACTS ON CYTOCHROME P-450 CONCENTRATION IN RAT AND MOUSE HEPATIC MICROSOMES Dose (mg/ml)
NADPH (0.5 mM)
Control
0 0
+ -
Rat 1.28 -1-0.1 a 1.25 +0.1
Mouse 0.60 + 0.03 0.62+0.04
Acidic fraction
0.8 0.8
+ -
1.32 + 0.2 1.29 + 0.3
0.61 + 0.02 0.59 + 0.04
Neutral fraction
0.015 0.015
+ -
1.27+ 0.2 1.30 + 0.3
0.62 + 0.03 0.63 + 0.04
Basic fraction
2.8 2.8
+ -
0.69 + 0.07 * * 0.76+0.06 * *
0.35 + 0.04 * * 0.41 +0.01 * *
Cytochrome P-450 (nmoles/mg of protein)
a All values are means + SD of 5 determinations. * * p < 0.01 (t test).
ing a direct interaction of test material(s) with microsomal cytochrome P-450. Discussion
The occurrence of directly-acting bacterial mutagens absorbed onto airborne particulate matter is well established ( D e h n e n et al., 1977; Pitts et al., 1977; Talcott and Wei, 1977; Tokiwa et al., 1977). O u r data demonstrate a similar or lower concentration of airborne mutagens in the city of R o m e with respect to other E u r o p e a n (Lofroth, 1981) and American (Pitts et al., 1985) densely inhabited areas. The chemical identity of the mutagenic substances adsorbed o n t o air particulates is not fully elucidated. Airborne particulate matter is k n o w n to contain m o r e than 500 chemical c o m p o u n d s and chromatographic separation showed that the mutagenic activity in particulate extracts eluted over a wide range, with no major single peak (Lofroth, 1981). This suggests that m a n y different c o m p o u n d s contribute to the air particulate mutagenicity. This is not the case for particulates of other origin, such as diesel exhaust, in which a large fraction of the in vitro mutagenicity is due to the contribution of powerful mutagenic nitropyrenes (Rosenkranz, 1982). The results obtained in this study with S. typhimurium strain T A 9 8 N R (resistant to 1-nitropyrene) and T A 9 8 / 1 , 8 D N P 6 (resistant to 1,6- and 1 , 8 - d i -
nitropyrenes) suggest that nitropyrenes play only a limited role in air particulate mutagenicity. Similar conclusions were reached by Tokiwa et al. (1977) and by Siak et al. (1985). The seasonal variation observed in the strains' sensitivity (Fig. 4) suggests, however, a greater contribution of nitro c o m p o u n d s in spring and summer samples, possibly related to the greater contribution of m o t o r vehicle exhausts to u r b a n air pollution. After chemical fractionation of air particulate extracts, m u t a g e n s active in strain TA98 were detected in all acidic, neutral and basic c o m p o nents which exerted roughly similar mutagenic activities. Directly-acting mutagens were detected in acidic and neutral fractions, where aliphatic and polycyclic nitro c o m p o u n d s are expected. Proximate mutagens accounted for the mutagenicity of basic fractions (Table 2). Although no effort was made to identify these promutagens by chemical means, their inactivity in strain T A 9 8 / 1 , 8 D N P 6 is strongly suggestive of the involvement of aromatic amines or related c o m p o u n d s ( M c C o y et al., 1982). Several factors are to be taken into account in the evaluation of mutagenicity data of particulate extracts for the assessment of h u m a n risks related to exposure to air pollutants. F o r example, it was previously stressed that trace amounts of nitro c o m p o u n d s such as nitropyrenes could account for most of the mutagenic activity exerted by
574
particulate extracts in bacteria, due to the extraordinary activity of bacterial nitroreductases (Mermelstein et al., 1981). Another factor to be taken into account is the bioavailability of the mutagenic components adsorbed onto airborne particulate matter. Our data show that adsorbed mutagens are scarcely extractable by serum and therefore probably of limited bioavailability. Similar conclusions were reached by Ohsawa and coworkers (1983) and by Belisario et al. (1984) in a study on diesel exhaust. Also possible divergences between in vivo and in vitro metabolic systems are to be considered in the evaluation of in vitro data. In this connection it is noteworthy that, although liver $9 was unable to reduce significantly the in vitro mutagenicity of air particulate extracts, the study of body fluids' mutagenicity demonstrated the rapid and total disappearance of active/ activable mutagens in body fluids of treated animals, suggesting an extensive in vivo inactivation. These findings may be related to the negative outcome of in vivo cytogenetic assays obtained by us and by others even after positive in vitro results (Krishna et al., 1986). Due to the high chemical complexity of airborne materials, other biological properties beyond mutagenicity are also to be considered, for example the possible synergistic or antagonistic interaction with other chemical exposures. Air particulate extracts were previously shown to contain chemical compounds, different from polycyclic aromatic hydrocarbons and direct-acting mutagens, which were able to bind the TCDD-receptor protein in rat liver (Totfgard et al., 1983). Accordingly, we have shown that a single administration of air particulate extract is sufficient to modify the xenobiotic metabolizing system of mice. In vitro experiments with mouse and rat microsomes suggest a direct interaction with active sites of cytochrome P-450; however, other mechanisms, such as the decreased biosynthesis or increased breakdown of enzymes or their cofactors could also work in vivo. Moreover, it is conceivable that the air particulate matter, as with many other xenobiotics, could exert a biphasic effect working as an inhibitor in the acute phase and as an inducer after chronic exposure (Testa and Jenner, 1981). Taking into account the undefined bioavail-
ability of airborne material and possible unpredictable interactions among chemicals adsorbed onto particulate matter, it is suggested that only experimental models more closely related to the real human exposure could provide useful information on genotoxic risks. This goal is at present urgently needed due to the increasing levels of air pollution in industrialized areas and the wide human exposure to airborne materials.
Acknowledgements This work was partially supported by the E.E.C. (contract No. 530 ENV l-s). The technical assistance of Ms. C. D'Ascoli, Ms. L. Gargano and Mr. U. Cervelli is gratefully acknowledged.
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