Mutation Research, 298 (1992) 113-123
113
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00
MUTGEN 01834
U r b a n air pollution: U s e of different m u t a g e n i c i t y assays to evaluate e n v i r o n m e n t a l genetic hazard P. Poli a, A. Buschini a, N. Campanini a, M.V. Vettori a, F. Cassoni b, S. Cattani b and C. Rossi a
a Istituto di Genetica, Universitk di Parma and b P.M.P. USL 4, 43100 Parma, Italy
(Received 20 February 1992) (Revision received 7 July 1992) (Accepted 8 July 1992)
Keywords: Airborne particulate genotoxicity; Mitochondrial mutagenesis; Nuclear mutagenesis; Urban air pollution
Summary The genotoxic activities associated with airborne particulate matter collected in Parma (northern Italy) have been determined. The airborne particle extracts were tested for mutagenicity using Salmonella frameshift (TA98) and base-substitution (TA100) tester strains with and without $9 microsomal activation and Saccharomyces cerevisiae strain D7 in order to determine the frequency of mitotic gene conversion and ilvl-92 mutant reversion in cells harvested at stationary and logarithmic growth phase. The relationship between mitochondrial DNA mutations and ageing, degenerative diseases and cancer prompted us to take into account the mitochondrial informational target, i.e., the respiratory-deficient (RD) mutants. The results obtained show a variability in the response for the different test systems during different months. The Salmonella mutagenicity trend was directly correlated with carbon monoxide, nitrogen oxides (NO x) and Pb concentration in airborne particulates and inversely correlated with temperature, whereas the mitochondrial genotoxic effect was higher during spring and late summer. These data suggest that the genotoxic risk assessment is a time-dependent value strictly correlated with the evaluation system being tested.
There is growing interest in the evaluation of urban air as a potential genotoxic system leading to harmful health effects. Health risks, associated with urban airborne particulates, include respiratory diseases such as bronchitis and an increase
Correspondence: Prof. C. Rossi, Istituto di Genetica, Universit5 di Parma, Viale delle Scienze, 43100 Parma, Italy. Tel. 521-580608; Fax 521-580665.
in lung cancer incidence (Walker et al., 1982). In addition there is an increase in a new class of disease that could be defined as an 'informational disease', i.e., ageing (Richter, 1988; Linnane et al., 1989). This has stimulated the search for mutagenicity tests able to detect, quickly and economically, the genotoxic activity of airborne particulate matter (Hughes et al., 1980). The Ames Salmonella typhimurium assay system (Maron and Ames, 1983) is the most exten-
114
sively used test in evaluating genotoxic potential (De Flora et al., 1989; Barale et al., 1989; Miguel et al., 1990). In particular strain TA98 is most often used for monitoring urban air. A quantitative/qualitative evaluation of the 'environmental genetic hazard' requires the building up of a screening system which is able to detect different mutagenic events at the molecular level. In this work the S. typhimurium assay is used. The diploid D7 strain of Saccharomyces cerevisiae (Zimmermann et al., 1975) was also used since, in addition to nuclear events such as gene conversion and point mutation, it is useful to verify whether a given 'potentially genotoxic system' induces mutation in the mitochondrial DNA, i.e., mutation from respiratory sufficiency (RS) to respiratory deficiency (RD). In spite of the fact that S. typhimurium appears more sensitive than S. cerevisiae, the eukaryotic organism highlights the mutational effects depending on the repair system and damage involving extranuclear DNA. Recent reports (Shay and Werbin, 1987; Richter, 1988; Wallace et al., 1988; Linnane et al., 1989; Zeviani et al., 1989; Morgan-Hughes et al., 1990) have underlined the relationship between mitochondrial genomes and ageing, degenerative diseases and cancer (for a recent review see Ferguson and von Borstel, 1992). The role of extrachromosomal factors suggests the need for evaluating the environmental mutational load (EML) not only on the nuclear compartment but also on the mitochondrial information. To verify whether there is seasonal variation of the E M L over a long period, samples of urban airborne particulates were collected from April to December 1990 in the centre of Parma (northern Italy). This variation could be of some importance in particular areas such as Parma, which is characterised by the season-dependent production of high-quality agro-industrial products. Materials and methods
Sample collection and extracts The sampling site was approximately 1.5 m above ground at the intersection of Via Spalato and Viale Martiri della Liberth, a busy road in a
mixed residential-commercial area in the centre of town. In order to simulate relatively well the real conditions of transfer of the potential EML from the air to the living system, i.e., man, a low-volume sampler was used. NO x, SO 2, CO concentrations were measured continuously by monitors that detect characteristic light emission. N O 2 concentration measurement is based o n N O 2 ~ NO catalytic conversion and the subsequent reaction NO + 0 3 ~ NO~' + O 2 where the excited NO~ decays in N O 2 with emission of characteristic wavelength light (chemiluminescence). Carbon monoxide is measured via non-dispersive infra-red emission and SO 2 with the pulsed fluorescence technique. The data, collected on a personal computer, were stored every hour on magnetic support. Particulate matter was collected using an air sampling apparatus consisting of: a cellulose acetate membrane filter, 47 ram, 0.45 /zm pore diameter; a filter holder; a low-volume sampler (Tecora Bravo H) with flow and sampled volume check. Sampling flow was 1 m3/h. The particulate measurement was gravimetric. Lead concentration was measured on the same filters by atomic absorption spectrometry. Whole particulate matter for the mutagenicity assays was collected on glass fibre filters (by the same sampling apparatus) during a 9-month period (April-December 1990), the sampling being continuous during a 24-h period. The daily filters were pooled to obtain the monthly samples. Each sample was extracted in a Soxhlet extraction apparatus for 24 h in 200-300 ml of toluene. The solvent was evaporated with a rotary evaporator. The residual dry matter from each extraction was redissolved in different volumes of dimethyl sulfoxide (DMSO RPE-ACS, Farmitalia Carlo Erba, Milan), to obtain a constant ratio (about 64 m3/ml) of air volume to extract volume.
Bioassay The samples were tested on strains TA98 and TA100 of Salmonella typhimurium according to the standard methods, with and without external metabolic activation ($9 hepatic fraction). The protein concentration of the $9 fraction (32 m g / m l ) was determined according to Lowry
115
testing samples, and incubated in an alternating shaker (110 rpm) at 37°C for 2 h. Mitochondrial DNA mutation induction was evaluated in the same D7 strain determining the frequency of respiration-deficient 'petite' colonies (Marmiroli et al., 1980). In complete medium 10 4 cells/ml were inoculated in the presence of different concentrations of the airborne particulate extracts. The cultures were incubated in an alternating shaker (110 rpm) at 28°C for 27 h. The cells were harvested, counted and plated at the scheduled concentration, on complete medium with glucose as sole carbon source. To detect 'petite' mutants, after 5 days, the plates were overlaid with agar containing tetrazolium (Ogur
et al. (1951). The $9 mix concentration used in the Salmonella plate test corresponded to 1.6 mg total protein/plate. The diploid strain D7 of Saccharomyces ceret:isiae obtained from F.K. Zimmerman, was used to determine the frequency of mitotic gene conversion of the trp5 locus and reversion of the ilvl-92 mutant, with and without metabolic activation. As an alternative system to the microsoreal assay, yeast cells were harvested during the logarithmic phase of growth (about 5 × 107 cells/ml) in 20% glucose at maximum activation of cytochrome P-450. In both systems, the cells were inoculated in phosphate buffer 0.1 M, pH 7.4 in the presence of different concentrations of
E
8 o - m ~o-
~.
4()3O-
10~. ;~
O
Apt May Jun Jul Allg 6ep Oct Nov Dec "~-q
:)5
25!
--i
20-
.............
3~
75 2-
. . . . . . . . . . . . . . . . . . . . . . . . ....
--o
O3 5O
Ap~ May J~l Jul Aug 6ep Ocl Nov
......
Dec
1
Apl May Jim Jul Aug 8ep 0~1 Nov
Dec
O.35-
J ~
-
o.,,
INO2 J
0.~
Z~
0.1-
I
Apt M~ Jun Jut Au0
Fig. 1. Airborne particulate,
SO2,
CO,
Sep
Od
Nov Dec
Apr
May
Jun
Jul
Aug Sep Ocl
Nov
Pb, NO x concentrations in urban air during the examined period (April-December).
116 TABLE 1 N U M B E R OF His + R E V E R T A N T S IN S. typhimurium TA98 AND TA100, W I T H O U T A N D WITH $9 MIX, I N D U C E D BY A I R B O R N E P A R T I C U L A T E SAMPLES The ratio between treated and control counted colonies is given in parentheses, T O X means a toxic effect on cell survival. Samples
Doses (~l/plate)
TA98 revertants/plate - $9 + $9
TA100 revertants/plate - $9 + $9
April
0 25 50 100 150
59 (1.0) 207 (3.5) 215 (3.6) 177 (3.0) TOX
36 (1.0) 72 (2.0) 79 (2.2) 104 (2.9) 148 (4.1)
199 (1.0) 279 (1.4) 245 (1.2) 299 (1.5) 179 (0.9)
236 283 307 401 309
May
0 25 50 100 150
59 (1.0) 130 (2.2) 36 (0.6) 94 (1.6) TOX
36 (1.0) 61 (1.7) 69 (1.9) 79 (2.2) 149 (4.1)
199 (1.0) 259 (1.3) 234 (1.2) 259 (1.3) TOX
236 (1.0) 283 (1.2) 294 (1.2) 425 (1.8) 242 (1.0)
June
0 50 100 150
40 (1.0) 122 (3.0) TOX TOX
47 (1.0) 78 (1.7) 100 (2.1) 105 (2.2)
329 (1.0) 401 (1.2) 577 (1.7) TOX
352 405 448 467
(1.0) (1.2) (1.3) (1.3)
July
0 50 100 150
40 (1.0) 143 (3.6) TOX TOX
47 (1.0) 76 (1.6) 87 (1.8) 108 (2.3)
329 411 467 552
352 437 453 495
(1.0) (1.2) (1.3) (1.4)
August
0 25 50 100 150
30 (1.0) TOX TOX TOX TOX
37 (1.0) 73 (1.9) 80 (2.2) 77 (2.1) TOX
277 (1.0) 468 (1.7) 505 (1.8) TOX TOX
242 (1.0) 371 (1.5) 396 (1.6) 420 (1.7) 558 (2.3)
September
0 25 50 100 150
30 (1.0) TOX TOX TOX TOX
37 (1.0) 83 (2.2) 113 (3.0) TOX TOX
277 (1.0) 580 (2.1) TOX TOX TOX
242 (1.0) 385 (1.6) 437 (1.8) 511 (2.1) TOX
October
0 25 50 100 150
42 (1.0) 69 (1.6) 99 (2.4) 132 (3.1) 136 (3.2)
43 (1.0) 69 (1.6) 79 (1.8) 120 (2.8) 164 (3.8)
266 268 317 365 401
(1.0) (1.0) (1.2) (1.4) (1.5)
280 338 371 427 478
(1.0) (1.2) (1.3) (1.5) (1.7)
November
0 25 50 100 150
42 (1.0) 80 (1.9) 97 (2.3) 139 (3.3) 178 (4.2)
43 (1.0) 52 (1.2) 92 (2.1) 114 (2.6) 164 (3.8)
266 (1.0) 321 (1.2) 362 (1.4) 386 (1.4) 400 (1.5)
280 366 423 457 393
(1.0) (1.3) (1.5) (1.6) (1.4)
December
0 50 100 150
55 289 357 405
55 234 391 491
299 (1.0) 412 (1.4) 490 (1.6) 550 (1.8)
311 (1.0) 525 (1.7) 608 (1.9) 732 (2.3)
(1.0) (5.2) (6.5) (7.4)
(1.0) (4.2) (7.1) (8.9)
(1.0) (1.2) (1.4) (1.7)
(1.0) (1.2) (1.3) (1.7) (1.3)
117 Table 1 (continued) The total volumes of monthly sampled air were: Month
Air volume (m 3)
Extract final volume (DMSO ml)
April May June July August September October November December
510439 599 434 638 669 674 058 669 402 645120 649 347 673 799 666 804
8.0 9.4 10.0 10.6 10.5 10.1 10.2 10.6 10.5
e t al., 1957). C e l l s d e r i v e d f r o m w h i t e c o l o n i e s were plated on complete medium with ethanol, a non-fermentable carbon source, to confirm the RD phenotype.
The data were analysed using the modified 2 - f o l d r u l e ( C h u e t al., 1981) in w h i c h a r e s p o n s e is c o n s i d e r e d p o s i t i v e if t h e a v e r a g e r e s p o n s e f o r a t l e a s t 2 c o n s e c u t i v e d o s e l e v e l s was: (i) m o r e
b °. [ ~ / , m , um~;u/rrv
0
J . c,~,r-~,~.,-b'a,i,a,e, b
Gene Conversion
= 10
m
"5
4
E
8
g
6
=
"c
4
~
2
g,
0
~
3,5 E
3
o 2,5
+ •
+
.$ 4_ +
÷
t
~ 0,5 0
10
20
30
40
50
60
70
80
90
o
0
10
20
air particulate ( pg/m3 )
b °. c,o_~Q,c,-/~/~ d
m 4 3,5 3
=
35
"5 E
30
o
o 2,5
50
60
70
80
90
RS ~
RD
25 20
~2 ,~1,5
~-
~-° +
4
+
E
o
-t-
~n
+
"c
+
15 10 5
~ 0,5 o
40
J . c_~e~/,~,~;c~
Point Mutation
"5 E
30
air particulate ( ug/m3 )
o
10
20
ao
40
50
60
air particulate ( ug/m3 )
70
80
90
0
,t 0
10
20
30
40
50
60
70
80
90
air particulate (.l/g/rn3)
Fig. 2. Ratio between treatment and control mutants at different airborne particulate concentrations: (a) S. typhimurium TA98 * and TA100 • with metabolic activation ($9); (b) S. cerevisiae locus trp5 convertants with + and without • metabolic activation; (c) S. cerevisiae locus ilvl-92 revertants with + and without • metabolic activation; (d) S. cerevisiae RD 'petite' mutants.
118 TABLE 2 I N D U C T I O N OF M I T O T I C G E N E C O N V E R S I O N AND POINT R E V E R S E M U T A T I O N IN B O R N E P A R T I C U L A T E SAMPLES
S. cerevisiae D7 BY AIR-
Incubation was performed with cells harvested at stationary (star) or logarithmic growth phase (log). Total number of counted colonies is given in parentheses. T O X indicates a toxic effect on cell survival. Samples
Titre
Locus trp 5 convertants/105 surv.
Locus ilv 1 revertants/106 surv.
(pA/ml)
star
log
stat
log
star
3924
4428 415l 3504 3 100 2 851
0.61 (240) 0.94 (398) 1.36 (456) * 1.59 (526) * *
1.31 (582) 1.23 (511) 2.02 (708) 3.19 (988) ** 3.82 (1089) * * *
0.18 0.27 0.19 0.18
April 0 25.0 37.5 50.0 62.5
4232 3344 3 312
log (70) (114) (64) (60)
0.46 (205) 0.45(185) 0.48 (168) 0.41 (128) 0.72 (204)
May 0 25.0 37.5 50.0 62.5
3372 1996 1 568 TOX
2843 2586 1606 964
0.53 (180) 0.84(168) 1.79 (280) ** TOX
1.45 3.09 3.41 3.07
(412) (800) * (548) ** (296) **
0.15 (50) 0.39 (78) 0.51 (80) ** TOX
0.43 0.44 0.41 0.76
(122) (114) (66) (73)
June 0 25.0 50.0 62.5
2841 2583 3006 2831
3757 3406 2416 2459
0.94 (266) 0.93 (240) 2.00 (600) * 2.06(582) *
0.86 0.85 1.09 1.16
(324) (288) (264) (286)
0.17 0.17 0.35 0.28
(47) (43) (104) (80)
0.35 0.35 0.34 0.37
(133) (120) (90) (92)
July 0 25.0 50.0 62.5
2841 2382 2671 2380
3757 3264 2582 2215
0.94 (266) 1.02 (243) 0.82 (220) 0.95(225)
0.86 (324) 0.94 (308) 1.04 (268) 1.42 (315)
0.17 0.26 0.19 0.18
(47) (61) (50) (43)
0.35 0.34 0.64 0.45
(133) (112) (164) (99)
August 0 5.0 10.0 15.0 25.0 50.0
4460 2692 1416 TOX
4946 5043 2595 1014
1.00 (448) 1.93 (520) * 2.46 (348) ** TOX
1.21 1.52 1.94 2.51
0.28 (124) 0.54 (146) 0.37 (53) TOX
0.52 0.65 0.71 0.60
(258) (327) (186) (61)
4038
4946 4350 2277 TOX
0.87 (352) 1.30 (456) 1.13 (248) 1.78 (208) * TOX
1.21 (597) 1.56 (678) 1.48 (336) TOX
0.29 (118) 0.62 (218) 0.71 (156) 0.53 (62) TOX
0.52 (258) 0.50 (219) 0.54 (123) TOX
0.40 (153) 0.55 (204) 0.40 (180)
0.65 (319) 0.85 (378) 0.75 (189)
0.55 (237) 0.66 (282) 0.43 (288)
0.65 (319) 0.91 (417) 0.52 (213)
September 0 5.0 10.0 15.0 20.0 25.0
3516 2204 1 166 TOX
(597) (768) (504) (255) *
October 0 25.0 50.0
3819 3714 4533
4932 4434 2508
0.82 (315) 1.18 (438) 0.93 (420)
1.13 2.06 2.46
November 0 25.0 50.0
4305 4284 5310
4932 4569 4068
0.72 (309) 0.62 (264) 0.64 (342)
1.13 (558) 1.77 (810) 1.45 (588)
(558) (912) (618) *
119 TABLE 2 (continued) Samples (~l/ml) December 0 25.0 50.0
Locus trp 5 convertants/10 s surv.
Locus ilv 1 revertants/106 surv.
stat
log
stat
log
star
log
4 508 4 660 3 664
4 932 4 683 1 638
0.52 (232) 0.69 (320) 1.08 (396) *
1.13 (558) 1.83 (858) 3.44 (564) **
0.45 121/4) 11.38 1176) 0.33 (120)
I).65 (319) 0.66 (310) 1.15 (1891
Titre
For sampled air volumes see Table 1. * p < 0 . 0 1 ; * * p
113
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00
MUTGEN 01834
U r b a n air pollution: U s e of different m u t a g e n i c i t y assays to evaluate e n v i r o n m e n t a l genetic hazard P. Poli a, A. Buschini a, N. Campanini a, M.V. Vettori a, F. Cassoni b, S. Cattani b and C. Rossi a
a Istituto di Genetica, Universitk di Parma and b P.M.P. USL 4, 43100 Parma, Italy
(Received 20 February 1992) (Revision received 7 July 1992) (Accepted 8 July 1992)
Keywords: Airborne particulate genotoxicity; Mitochondrial mutagenesis; Nuclear mutagenesis; Urban air pollution
Summary The genotoxic activities associated with airborne particulate matter collected in Parma (northern Italy) have been determined. The airborne particle extracts were tested for mutagenicity using Salmonella frameshift (TA98) and base-substitution (TA100) tester strains with and without $9 microsomal activation and Saccharomyces cerevisiae strain D7 in order to determine the frequency of mitotic gene conversion and ilvl-92 mutant reversion in cells harvested at stationary and logarithmic growth phase. The relationship between mitochondrial DNA mutations and ageing, degenerative diseases and cancer prompted us to take into account the mitochondrial informational target, i.e., the respiratory-deficient (RD) mutants. The results obtained show a variability in the response for the different test systems during different months. The Salmonella mutagenicity trend was directly correlated with carbon monoxide, nitrogen oxides (NO x) and Pb concentration in airborne particulates and inversely correlated with temperature, whereas the mitochondrial genotoxic effect was higher during spring and late summer. These data suggest that the genotoxic risk assessment is a time-dependent value strictly correlated with the evaluation system being tested.
There is growing interest in the evaluation of urban air as a potential genotoxic system leading to harmful health effects. Health risks, associated with urban airborne particulates, include respiratory diseases such as bronchitis and an increase
Correspondence: Prof. C. Rossi, Istituto di Genetica, Universit5 di Parma, Viale delle Scienze, 43100 Parma, Italy. Tel. 521-580608; Fax 521-580665.
in lung cancer incidence (Walker et al., 1982). In addition there is an increase in a new class of disease that could be defined as an 'informational disease', i.e., ageing (Richter, 1988; Linnane et al., 1989). This has stimulated the search for mutagenicity tests able to detect, quickly and economically, the genotoxic activity of airborne particulate matter (Hughes et al., 1980). The Ames Salmonella typhimurium assay system (Maron and Ames, 1983) is the most exten-
114
sively used test in evaluating genotoxic potential (De Flora et al., 1989; Barale et al., 1989; Miguel et al., 1990). In particular strain TA98 is most often used for monitoring urban air. A quantitative/qualitative evaluation of the 'environmental genetic hazard' requires the building up of a screening system which is able to detect different mutagenic events at the molecular level. In this work the S. typhimurium assay is used. The diploid D7 strain of Saccharomyces cerevisiae (Zimmermann et al., 1975) was also used since, in addition to nuclear events such as gene conversion and point mutation, it is useful to verify whether a given 'potentially genotoxic system' induces mutation in the mitochondrial DNA, i.e., mutation from respiratory sufficiency (RS) to respiratory deficiency (RD). In spite of the fact that S. typhimurium appears more sensitive than S. cerevisiae, the eukaryotic organism highlights the mutational effects depending on the repair system and damage involving extranuclear DNA. Recent reports (Shay and Werbin, 1987; Richter, 1988; Wallace et al., 1988; Linnane et al., 1989; Zeviani et al., 1989; Morgan-Hughes et al., 1990) have underlined the relationship between mitochondrial genomes and ageing, degenerative diseases and cancer (for a recent review see Ferguson and von Borstel, 1992). The role of extrachromosomal factors suggests the need for evaluating the environmental mutational load (EML) not only on the nuclear compartment but also on the mitochondrial information. To verify whether there is seasonal variation of the E M L over a long period, samples of urban airborne particulates were collected from April to December 1990 in the centre of Parma (northern Italy). This variation could be of some importance in particular areas such as Parma, which is characterised by the season-dependent production of high-quality agro-industrial products. Materials and methods
Sample collection and extracts The sampling site was approximately 1.5 m above ground at the intersection of Via Spalato and Viale Martiri della Liberth, a busy road in a
mixed residential-commercial area in the centre of town. In order to simulate relatively well the real conditions of transfer of the potential EML from the air to the living system, i.e., man, a low-volume sampler was used. NO x, SO 2, CO concentrations were measured continuously by monitors that detect characteristic light emission. N O 2 concentration measurement is based o n N O 2 ~ NO catalytic conversion and the subsequent reaction NO + 0 3 ~ NO~' + O 2 where the excited NO~ decays in N O 2 with emission of characteristic wavelength light (chemiluminescence). Carbon monoxide is measured via non-dispersive infra-red emission and SO 2 with the pulsed fluorescence technique. The data, collected on a personal computer, were stored every hour on magnetic support. Particulate matter was collected using an air sampling apparatus consisting of: a cellulose acetate membrane filter, 47 ram, 0.45 /zm pore diameter; a filter holder; a low-volume sampler (Tecora Bravo H) with flow and sampled volume check. Sampling flow was 1 m3/h. The particulate measurement was gravimetric. Lead concentration was measured on the same filters by atomic absorption spectrometry. Whole particulate matter for the mutagenicity assays was collected on glass fibre filters (by the same sampling apparatus) during a 9-month period (April-December 1990), the sampling being continuous during a 24-h period. The daily filters were pooled to obtain the monthly samples. Each sample was extracted in a Soxhlet extraction apparatus for 24 h in 200-300 ml of toluene. The solvent was evaporated with a rotary evaporator. The residual dry matter from each extraction was redissolved in different volumes of dimethyl sulfoxide (DMSO RPE-ACS, Farmitalia Carlo Erba, Milan), to obtain a constant ratio (about 64 m3/ml) of air volume to extract volume.
Bioassay The samples were tested on strains TA98 and TA100 of Salmonella typhimurium according to the standard methods, with and without external metabolic activation ($9 hepatic fraction). The protein concentration of the $9 fraction (32 m g / m l ) was determined according to Lowry
115
testing samples, and incubated in an alternating shaker (110 rpm) at 37°C for 2 h. Mitochondrial DNA mutation induction was evaluated in the same D7 strain determining the frequency of respiration-deficient 'petite' colonies (Marmiroli et al., 1980). In complete medium 10 4 cells/ml were inoculated in the presence of different concentrations of the airborne particulate extracts. The cultures were incubated in an alternating shaker (110 rpm) at 28°C for 27 h. The cells were harvested, counted and plated at the scheduled concentration, on complete medium with glucose as sole carbon source. To detect 'petite' mutants, after 5 days, the plates were overlaid with agar containing tetrazolium (Ogur
et al. (1951). The $9 mix concentration used in the Salmonella plate test corresponded to 1.6 mg total protein/plate. The diploid strain D7 of Saccharomyces ceret:isiae obtained from F.K. Zimmerman, was used to determine the frequency of mitotic gene conversion of the trp5 locus and reversion of the ilvl-92 mutant, with and without metabolic activation. As an alternative system to the microsoreal assay, yeast cells were harvested during the logarithmic phase of growth (about 5 × 107 cells/ml) in 20% glucose at maximum activation of cytochrome P-450. In both systems, the cells were inoculated in phosphate buffer 0.1 M, pH 7.4 in the presence of different concentrations of
E
8 o - m ~o-
~.
4()3O-
10~. ;~
O
Apt May Jun Jul Allg 6ep Oct Nov Dec "~-q
:)5
25!
--i
20-
.............
3~
75 2-
. . . . . . . . . . . . . . . . . . . . . . . . ....
--o
O3 5O
Ap~ May J~l Jul Aug 6ep Ocl Nov
......
Dec
1
Apl May Jim Jul Aug 8ep 0~1 Nov
Dec
O.35-
J ~
-
o.,,
INO2 J
0.~
Z~
0.1-
I
Apt M~ Jun Jut Au0
Fig. 1. Airborne particulate,
SO2,
CO,
Sep
Od
Nov Dec
Apr
May
Jun
Jul
Aug Sep Ocl
Nov
Pb, NO x concentrations in urban air during the examined period (April-December).
116 TABLE 1 N U M B E R OF His + R E V E R T A N T S IN S. typhimurium TA98 AND TA100, W I T H O U T A N D WITH $9 MIX, I N D U C E D BY A I R B O R N E P A R T I C U L A T E SAMPLES The ratio between treated and control counted colonies is given in parentheses, T O X means a toxic effect on cell survival. Samples
Doses (~l/plate)
TA98 revertants/plate - $9 + $9
TA100 revertants/plate - $9 + $9
April
0 25 50 100 150
59 (1.0) 207 (3.5) 215 (3.6) 177 (3.0) TOX
36 (1.0) 72 (2.0) 79 (2.2) 104 (2.9) 148 (4.1)
199 (1.0) 279 (1.4) 245 (1.2) 299 (1.5) 179 (0.9)
236 283 307 401 309
May
0 25 50 100 150
59 (1.0) 130 (2.2) 36 (0.6) 94 (1.6) TOX
36 (1.0) 61 (1.7) 69 (1.9) 79 (2.2) 149 (4.1)
199 (1.0) 259 (1.3) 234 (1.2) 259 (1.3) TOX
236 (1.0) 283 (1.2) 294 (1.2) 425 (1.8) 242 (1.0)
June
0 50 100 150
40 (1.0) 122 (3.0) TOX TOX
47 (1.0) 78 (1.7) 100 (2.1) 105 (2.2)
329 (1.0) 401 (1.2) 577 (1.7) TOX
352 405 448 467
(1.0) (1.2) (1.3) (1.3)
July
0 50 100 150
40 (1.0) 143 (3.6) TOX TOX
47 (1.0) 76 (1.6) 87 (1.8) 108 (2.3)
329 411 467 552
352 437 453 495
(1.0) (1.2) (1.3) (1.4)
August
0 25 50 100 150
30 (1.0) TOX TOX TOX TOX
37 (1.0) 73 (1.9) 80 (2.2) 77 (2.1) TOX
277 (1.0) 468 (1.7) 505 (1.8) TOX TOX
242 (1.0) 371 (1.5) 396 (1.6) 420 (1.7) 558 (2.3)
September
0 25 50 100 150
30 (1.0) TOX TOX TOX TOX
37 (1.0) 83 (2.2) 113 (3.0) TOX TOX
277 (1.0) 580 (2.1) TOX TOX TOX
242 (1.0) 385 (1.6) 437 (1.8) 511 (2.1) TOX
October
0 25 50 100 150
42 (1.0) 69 (1.6) 99 (2.4) 132 (3.1) 136 (3.2)
43 (1.0) 69 (1.6) 79 (1.8) 120 (2.8) 164 (3.8)
266 268 317 365 401
(1.0) (1.0) (1.2) (1.4) (1.5)
280 338 371 427 478
(1.0) (1.2) (1.3) (1.5) (1.7)
November
0 25 50 100 150
42 (1.0) 80 (1.9) 97 (2.3) 139 (3.3) 178 (4.2)
43 (1.0) 52 (1.2) 92 (2.1) 114 (2.6) 164 (3.8)
266 (1.0) 321 (1.2) 362 (1.4) 386 (1.4) 400 (1.5)
280 366 423 457 393
(1.0) (1.3) (1.5) (1.6) (1.4)
December
0 50 100 150
55 289 357 405
55 234 391 491
299 (1.0) 412 (1.4) 490 (1.6) 550 (1.8)
311 (1.0) 525 (1.7) 608 (1.9) 732 (2.3)
(1.0) (5.2) (6.5) (7.4)
(1.0) (4.2) (7.1) (8.9)
(1.0) (1.2) (1.4) (1.7)
(1.0) (1.2) (1.3) (1.7) (1.3)
117 Table 1 (continued) The total volumes of monthly sampled air were: Month
Air volume (m 3)
Extract final volume (DMSO ml)
April May June July August September October November December
510439 599 434 638 669 674 058 669 402 645120 649 347 673 799 666 804
8.0 9.4 10.0 10.6 10.5 10.1 10.2 10.6 10.5
e t al., 1957). C e l l s d e r i v e d f r o m w h i t e c o l o n i e s were plated on complete medium with ethanol, a non-fermentable carbon source, to confirm the RD phenotype.
The data were analysed using the modified 2 - f o l d r u l e ( C h u e t al., 1981) in w h i c h a r e s p o n s e is c o n s i d e r e d p o s i t i v e if t h e a v e r a g e r e s p o n s e f o r a t l e a s t 2 c o n s e c u t i v e d o s e l e v e l s was: (i) m o r e
b °. [ ~ / , m , um~;u/rrv
0
J . c,~,r-~,~.,-b'a,i,a,e, b
Gene Conversion
= 10
m
"5
4
E
8
g
6
=
"c
4
~
2
g,
0
~
3,5 E
3
o 2,5
+ •
+
.$ 4_ +
÷
t
~ 0,5 0
10
20
30
40
50
60
70
80
90
o
0
10
20
air particulate ( pg/m3 )
b °. c,o_~Q,c,-/~/~ d
m 4 3,5 3
=
35
"5 E
30
o
o 2,5
50
60
70
80
90
RS ~
RD
25 20
~2 ,~1,5
~-
~-° +
4
+
E
o
-t-
~n
+
"c
+
15 10 5
~ 0,5 o
40
J . c_~e~/,~,~;c~
Point Mutation
"5 E
30
air particulate ( ug/m3 )
o
10
20
ao
40
50
60
air particulate ( ug/m3 )
70
80
90
0
,t 0
10
20
30
40
50
60
70
80
90
air particulate (.l/g/rn3)
Fig. 2. Ratio between treatment and control mutants at different airborne particulate concentrations: (a) S. typhimurium TA98 * and TA100 • with metabolic activation ($9); (b) S. cerevisiae locus trp5 convertants with + and without • metabolic activation; (c) S. cerevisiae locus ilvl-92 revertants with + and without • metabolic activation; (d) S. cerevisiae RD 'petite' mutants.
118 TABLE 2 I N D U C T I O N OF M I T O T I C G E N E C O N V E R S I O N AND POINT R E V E R S E M U T A T I O N IN B O R N E P A R T I C U L A T E SAMPLES
S. cerevisiae D7 BY AIR-
Incubation was performed with cells harvested at stationary (star) or logarithmic growth phase (log). Total number of counted colonies is given in parentheses. T O X indicates a toxic effect on cell survival. Samples
Titre
Locus trp 5 convertants/105 surv.
Locus ilv 1 revertants/106 surv.
(pA/ml)
star
log
stat
log
star
3924
4428 415l 3504 3 100 2 851
0.61 (240) 0.94 (398) 1.36 (456) * 1.59 (526) * *
1.31 (582) 1.23 (511) 2.02 (708) 3.19 (988) ** 3.82 (1089) * * *
0.18 0.27 0.19 0.18
April 0 25.0 37.5 50.0 62.5
4232 3344 3 312
log (70) (114) (64) (60)
0.46 (205) 0.45(185) 0.48 (168) 0.41 (128) 0.72 (204)
May 0 25.0 37.5 50.0 62.5
3372 1996 1 568 TOX
2843 2586 1606 964
0.53 (180) 0.84(168) 1.79 (280) ** TOX
1.45 3.09 3.41 3.07
(412) (800) * (548) ** (296) **
0.15 (50) 0.39 (78) 0.51 (80) ** TOX
0.43 0.44 0.41 0.76
(122) (114) (66) (73)
June 0 25.0 50.0 62.5
2841 2583 3006 2831
3757 3406 2416 2459
0.94 (266) 0.93 (240) 2.00 (600) * 2.06(582) *
0.86 0.85 1.09 1.16
(324) (288) (264) (286)
0.17 0.17 0.35 0.28
(47) (43) (104) (80)
0.35 0.35 0.34 0.37
(133) (120) (90) (92)
July 0 25.0 50.0 62.5
2841 2382 2671 2380
3757 3264 2582 2215
0.94 (266) 1.02 (243) 0.82 (220) 0.95(225)
0.86 (324) 0.94 (308) 1.04 (268) 1.42 (315)
0.17 0.26 0.19 0.18
(47) (61) (50) (43)
0.35 0.34 0.64 0.45
(133) (112) (164) (99)
August 0 5.0 10.0 15.0 25.0 50.0
4460 2692 1416 TOX
4946 5043 2595 1014
1.00 (448) 1.93 (520) * 2.46 (348) ** TOX
1.21 1.52 1.94 2.51
0.28 (124) 0.54 (146) 0.37 (53) TOX
0.52 0.65 0.71 0.60
(258) (327) (186) (61)
4038
4946 4350 2277 TOX
0.87 (352) 1.30 (456) 1.13 (248) 1.78 (208) * TOX
1.21 (597) 1.56 (678) 1.48 (336) TOX
0.29 (118) 0.62 (218) 0.71 (156) 0.53 (62) TOX
0.52 (258) 0.50 (219) 0.54 (123) TOX
0.40 (153) 0.55 (204) 0.40 (180)
0.65 (319) 0.85 (378) 0.75 (189)
0.55 (237) 0.66 (282) 0.43 (288)
0.65 (319) 0.91 (417) 0.52 (213)
September 0 5.0 10.0 15.0 20.0 25.0
3516 2204 1 166 TOX
(597) (768) (504) (255) *
October 0 25.0 50.0
3819 3714 4533
4932 4434 2508
0.82 (315) 1.18 (438) 0.93 (420)
1.13 2.06 2.46
November 0 25.0 50.0
4305 4284 5310
4932 4569 4068
0.72 (309) 0.62 (264) 0.64 (342)
1.13 (558) 1.77 (810) 1.45 (588)
(558) (912) (618) *
119 TABLE 2 (continued) Samples (~l/ml) December 0 25.0 50.0
Locus trp 5 convertants/10 s surv.
Locus ilv 1 revertants/106 surv.
stat
log
stat
log
star
log
4 508 4 660 3 664
4 932 4 683 1 638
0.52 (232) 0.69 (320) 1.08 (396) *
1.13 (558) 1.83 (858) 3.44 (564) **
0.45 121/4) 11.38 1176) 0.33 (120)
I).65 (319) 0.66 (310) 1.15 (1891
Titre
For sampled air volumes see Table 1. * p < 0 . 0 1 ; * * p
