Mutagenesis vol.14 no.6 pp.547–555, 1999
Comet assay application in environmental monitoring: DNA
damage in human leukocytes and plant cells in comparison with
bacterial and yeast tests
Paola Poli2, Annamaria Buschini, Francesco Maria
Restivo, Antonella Ficarelli, Francesca Cassoni1, Iliana
Ferrero and Carlo Rossi
Istituto di Genetica, Università degli Studi di Parma, Parco Area delle
Scienze 11/a, 43100 Parma and 1ARPA Emilia Romagna, Sezione di Parma,
Parma, Italy
Urban airborne particulate is a complex mixture of air
pollutants, many of which have not been identified.
However, short-term mutagenesis tests together with chemicophysical parameter analysis are able to better assess air
quality and genotoxic load. The findings of continuous
monitoring (January 1991–August 1998) of urban air
genotoxicity of a Po Valley town (Italy) on Salmonella
typhimurium and Saccharomyces cerevisiae are reported.
During this period, various measures (catalytic devices,
unleaded fuels, annual vehicle overhaul, etc.) to improve
air-dispersed pollutant control were enforced. However, a
continuous presence of genotoxic compounds is shown and
more qualitative than quantitative changes are evident. We
also demonstrate the ability of the Comet assay to detect
DNA-damaging agents in airborne particulate samples. We
applied the test to human leukocytes and, with major
improvements, to plant cells (Allium cepa roots and epigean
tissues of Impatiens balsamina). The first findings on human
leukocytes confirm the sensitivity of this assay, its peculiarity and its applicability in assessing genotoxicity in environmental samples. The capability of plants to show the
response of multicellular organisms to environmental pollutants largely counterbalances a probable lowering in
sensitivity. Moreover, application of the Comet test to
epigean tissues could be useful in estimating the bioavailability of and genotoxic damage by air pollutants, including
volatile compounds (ozone, benzene, nitrogen oxides, etc.)
to higher plants.
Introduction
The ability of chemical substances, extracted from samples of
outdoor and indoor airborne particulates, to induce mutagenicity in prokaryotic cells such as Salmonella typhimurium
has been widely investigated (most recently by Azuma et al.,
1997; Chiang et al., 1997; Kuo et al., 1998; Monarca et al.,
1998; Wasserkort et al., 1998; Zhou and Ye, 1998). Some
studies have used eukaryotic cell systems, such as the yeast
Saccharomyces cerevisiae (Rossi et al., 1995a; Bronzetti et al.,
1997). However, human health risk determination associated
with air pollutants is not easily extrapolated from these findings.
On the other hand, direct monitoring, normally used on
professionally exposed workers, cannot be applied to the
general population. Furthermore, the international scientific
community is encouraging scientists to develop alternative
(non-animal) tests for DNA-damaging agents. Particularly
2To
appealing is the use of in vitro human cells for better extrapolation to human health (Sasaki et al., 1997; Holian et al., 1998)
and of plant material for a more accurate assessment of
environmental health (Fomin and Hafner, 1998; Monarca
et al., 1998). A good correlation between the data from
plant bioassays and those from mammalian systems has been
demonstrated. Furthermore, higher plants represent a stable
sensor in an ecosystem and hence allow us to follow the
evolution of the genotoxic impact.
In this contest the availability of possible alternative/integrative solutions could be of some relevance for environmental
genotoxicity assessment.
Our previous reports (Poli et al., 1992; Rossi et al., 1995b)
confirmed the complexity of environmental mixtures and the
difficulty of assessing the real amount of such genotoxic
compound emission in the environment.
The aim of the present research was to compare the genotoxic
effects induced by urban airborne particulate mixtures in
prokaryotic (Salmonella) and lower eukaryotic (yeast) cells
and the DNA damage in human leukocytes and plant cells.
Airborne particulate samples from a mixed residentialcommercial area of Parma, a Po valley town, were collected
over a long period (April 1990–September 1998) and tested
with different assays. The Ames test (Maron and Ames,
1983) and Saccharomyces cerevisiae diploid strain D7 assay
(Zimmermann et al., 1975) were performed on all the samples,
the latter being able to demonstrate nuclear DNA effects, such
as gene conversion and point mutation, and to assess mutation
induction in the mitochondrial genome, i.e. respiratory proficiency to respiratory deficiency. Alkaline single-cell gel electrophoresis (SCGE) on in vitro human leukocytes (Fairbairn et al.,
1995; Singh et al., 1988) and a modified and improved SCGE
on plant cells, Allium cepa roots (Navarrete et al., 1997) and
Impatiens balsamina tissues, were performed on samples
selected for their different effectiveness on bacterial and yeast
DNA. These last two assays involve detection, under alkaline
conditions, of cell DNA fragments which, on electrophoresis,
migrate from the nuclear core, resulting in a ‘comet’ formation.
The SCGE, or Comet assay, is a method for DNA alkali-labile
site and strand break detection in individual cells and is
becoming a major tool in environmental pollutant biomonitoring, both in vivo and in vitro (Fairbairn et al., 1994; Speit
et al., 1996; Calderón-Garcidueñas et al., 1997; Malyapa et al.,
1997a,b; Mitchelmore and Chipman, 1998; Steinert et al.,
1998; Wilson et al., 1998), which can be applied to virtually
any eukaryotic cell.
Materials and methods
Chemicals
Ethidium bromide, L-tryptophan, L-isoleucine and adenine were from Fluka
(RdH Laborchemika GmbH & Co. KG); Tris from ICN Biochemicals (Milan,
Italy); yeast extract, bacto peptone and agar from Difco (Detroit, MI); 2,3,5triphenyl-tetrazolium chloride, hycanthone, EDTA and all other laboratory
whom correspondence should be addressed. Tel: 139 521 905608; Fax: 139 521 905604; Email: [email protected]
© UK Environmental Mutagen Society/Oxford University Press 1999
547
P.Poli et al.
chemicals from Sigma (Milan, Italy); S9 from Laboratory Life Science
Research (Pomezia, Rome, Italy).
Airborne particulates: sample collection and extraction
Particulate matter was collected using an air sampling apparatus consisting of
a filter (Ø 5 47 mm), a filter holder and a low volume sampler (Tecora Bravo
H). The sampling was continuous during a 24 h period (sample flow 1 m3/h)
and the daily filters were pooled to obtain the monthly samples. The samples
were extracted in a Soxlet apparatus, evaporated with a rotary evaporator and
redissolved (50 m3/ml) in dimethyl sulfoxide (DMSO).
Tests on microorganisms
Ames test. The mutagenic activity of airborne particulate extract was studied
using S.typhimurium, strains TA98 and TA100, with or without in vitro
microsomial activation (Maron and Ames, 1983). The data obtained are
presented as revertants/m3 of air sampled, calculated from the linear portion
of the dose–response curves (six doses) by linear regression analysis. DMSO
(160 µl/plate) was used as a negative control, whereas hycanthone (50 µg/
plate) and 2-aminofluorene (2.5 µg/plate) were used as positive controls
without and with S9 mix, respectively (Gatehouse et al., 1994; OECD, 1994).
Yeast assays. The diploid strain D7 of S.cerevisiae (Zimmermann et al., 1975)
was used to determine the revertion frequencies of the ilv1-92 mutant and of
mitotic gene conversion at the trp5 locus with or without endogenous
activation. As an alternative system to the microsomal assay, yeast cells were
harvested during the logarithmic phase of growth in 20% glucose at maximum
activation of the cytochrome P450 complex (Rossi et al., 1997; Poli et al.,
1999). Both with and without endogenous metabolic activation, the cells (108
cells/ml) were inoculated in 0.1 M phosphate buffer, pH 7.4, in the presence
of different (at least five) concentrations of test samples and kept in an
alternating shaker (110 r.p.m.) at 37°C for 2 h. A least squares linear
regression analysis was used to calculate specific genotoxic activity (mutants
or convertants/m3 of air sampled, with reference to the treated population of
108 cells). DMSO (62.5 µl/ml) was used as a negative control; 2-aminofluorene
(50 µg/ml) and hycanthone (100 µg/ml) and ethyl methanesulfonate (100
mM) were used as positive controls when cytochrome 450 was induced or
not, respectively.
Mitochondrial DNA mutation induction was evaluated in the same strain
by determining the frequencies of respiratory-deficient colonies (Poli et al.,
1992; Rossi et al., 1995a,b). The cells (104 cells/ml) were cultured (28°C for
27 h) in the presence of different (at least five) sample concentrations and
then plated on a solid complete medium with glucose as the sole carbon
source. To detect petite mutants, the plates were overlayed with agar containing
tetrazolium after 5 days incubation at 28°C (Ogur et al., 1957). After ~1 h,
the respiratory-proficient colonies turned red, while the respiratory-deficient
colonies remained white. The data are reported as respiratory deficiency
increase/m3 of air, with reference to 104 cells. DMSO (12.5 µl/ml) and
ethidium bromide (1 µg/ml) were used as negative and positive controls,
respectively.
For all the assays the data were analysed using the modified two-fold rule
(Chu et al., 1981) in which a response is considered positive if the average
response for at least two consecutive dose levels was: more than double for
the Ames test and for the gene conversion test in strain D7; more than 3-fold
for the ilv1-92 revertion and respiratory deficiency mutant tests. The data
obtained were subjected to Student’s t-test.
Single cell gel electrophoresis
Human leukocytes. For leukocyte isolation, whole blood of healthy donors
was centrifuged twice in lysis buffer (155 mM NH4Cl, 5 mM KHCO3, 0.005
mM Na2EDTA, pH 7.4), washed with phosphate-buffered saline (PBS) and
resuspended (~106 cells/ml) in RPMI-1640 medium. Medium containing cells,
appropriate volumes of particulate sample and 0.1 M phosphate buffer (pH
7.4) or S9 mix were added to an Eppendorf tube. S9 was prepared as described
by Maron and Ames (1983). S9 was diluted 1:10 in 0.1 M phosphate buffer
(pH 7.4) containing NADP (4 mM), glucose 6-phosphate (5 mM), MgCl2 (8
mM) and KCl (33 mM) to constitute S9 mix, 250 µl of which was added to
each tube to make a total 1 ml cell suspension. Treatments were for 1 h at
37°C. Negative (DMSO, 50 µl/ml) and positive (Melphalan, 10 µg/ml) controls
were also performed.
Aliquots of the cell suspension were used to check viability by the Trypan
blue exclusion method and the percentage of apoptotic cells by Tunel assay
(Gorczyca et al., 1993). Another aliquot was used for SCGE, basically
performed according to Singh et al. (1988). Degreased slides were previously
dipped in 1% normal melting point agarose for the first layer. The cells
(~23105 cells) were mixed with low melting point agarose (LMA) and placed
on the first layer. Finally, LMA was added as the top layer. The cells were
lysed at 4°C in the dark (2.5 M NaCl, 10 mM Na2EDTA, 10 mM Tris–HCl,
1% Triton X-100 and 10% DMSO, pH 10) for at least 1 h. The DNA was
allowed to unwind for 20 min in alkaline electrophoresis buffer (1 mM
548
Na2EDTA, 300 mM NaOH, pH 13) and subjected to electrophoresis in the
same buffer for 20 min at 0.78 V/cm and 300 mA.
Plant cells. Growth of A.cepa roots was obtained by placing the bulbs at
25°C on a tray in the dark so that only their bases were in contact with tap
water. For the experiments, roots larger than 2 cm in length were excised
from the bulb and dipped (with the apex resting at the bottom) for 3 h at
26°C in Eppendorf tubes containing H2O and different concentrations of the
substances to be tested.
Seed derived plantlets (3–4 weeks old) of I.balsamina were used throughout.
Preliminary experiments showed that nuclear suspensions suitable for the
SCGE assay may be obtained from root, stem and leaf cuttings and processed
essentially as described for A.cepa roots. For the experiments, the plantlets
were maintained in the appropriate solution of the tested compounds for 3 h
at 26°C.
The procedure described by Navarrete et al. (1997) for nuclei isolation
from root cells of A.cepa was utilized with major improvements. Roots were
cut and gently squeezed, using forceps, directly into a drop of LMA (0.5%
in PBS) resting on the top of the first agarose layer. The slides were placed
on a warm surface (37°C) during this stage. The described modifications of
the procedure resulted in an increased yield of nuclei and a more uniform
distribution of nuclei in the agarose layer. Optimal pre-electrophoresis and
electrophoresis conditions were determined testing different voltage and time
schedules: 30 and 20 min of pre-electrophoresis and 10 and 20 min of
electrophoresis for A.cepa and I.balsamina, respectively, at 230 mA and 0.66
V/cm were selected for the experiments.
All the steps for slide preparation were performed under a yellow light to
prevent additional DNA damage. Once electrophoresis had been carried out,
the slides were washed in neutralization buffer (0.4 M Tris–HCl, pH 7.5).
Immediately before examination, the DNA was stained with 100 µl ethidium
bromide (2 µl/ml). The samples were examined under a fluorescent microscope
(Leitz Dialux 20) equipped with excitation filter BP 515–560 nm and barrier
filter LP 580 nm, using an automatic image analysis system (Cometa Release
2.1; Sarin, Florence, Italy). One hundred cells per sample, selected at random,
were analysed under constant sensitivity.
The comet length, measured as the distance between the leading edge of
the comet head and the end of the tail, was chosen to estimate DNA damage.
Results are presented as frequency distributions of single cell DNA damage
or as box and whisker plots. In this case measured values at the tested
concentrations are shown as boxes that include 50% of the data. The top and
bottom of the boxes mark the 25th and 75th centiles and the inner line marks
the median value; 25% of the data above the 75th centile and 25% below the
25th centile are marked as ‘whiskers’ limited by the maximum or minimum
values. Outliers are displayed as points.
The relationship between comet length and dose was calculated as mean
comet length increase/m3 air equivalent from the dose–response curves by
linear regression analysis.
A SPSS 8 statistical package was used. Variance analysis was by one or
multiple pairwise comparisons; the data obtained were also subjected to
Student’s t-test.
The samples were coded and evaluated blind. All the tests were usually
performed once, since the amounts of our environmental samples were
insufficient to repeat the tests in independent trials.
Results
Assays on microorganisms
The mutagenicity data on the two strains of S.typhimurium
(Table I), with or without S9, show a seasonal trend with
negligible values during the summer. The highest induction of
revertants occurred during the year 1992. A decrease during
the following years is evident, with special regard to the annual
mean values. This fact could be explained by the decreased
concentration of some compounds in the air due to the
enforcement of catalytic converters in new vehicles since the
year 1993. However, this hypothesis is not confirmed by the
data on S.cerevisiae (Table I). In this organism, with endogenous metabolic activation (i.e. a high cytochrome P450 cellular
content), induction of both convertants and revertants is
increased by the particulates collected after the introduction
of the catalytic device. Respiratory deficiency induction does
not appear to correlate with any measure against air pollution
or exogenous factors such as seasonal meteoclimatic conditions. The disagreement between the tests could be ascribed
Comet assay in environmental monitoring
Table I. Urban air particulate genotoxicity effects
Salmonella typhimurium
1991
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Mean
1992
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Mean
1993
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Mean
1994
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Mean
1995
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Mean
Saccharomyces cerevisiae
TA98
TA981
TA100
TA1001
Total
PM
PM1
GC
GC1
RD
Total
75
91
81
8
6
4
1
nd
8
22
15
22
30
64
58
63
6
5
3
1
nd
7
18
15
27
24
88
98
120
7
4
2
0
nd
11
35
19
35
38
71
60
84
7
9
5
4
nd
15
27
21
27
30
298
306
347
28
24
14
6
nd
41
103
70
112
123
2
10
0
0
5
1
1
1
6
4
0
3
3
17
5
7
2
0
16
4
8
2
7
27
10
9
84
61
0
46
0
9
0
11
249
120
3
36
52
56
340
34
8
67
63
6
441
241
205
179
40
140
38
8
56
9
0
0
7
1
0
9
21
5
13
197
424
97
65
72
89
18
462
498
345
230
94
216
33
42
1
nd
12
6
11
2
8
95
126
87
38
32
44
4
nd
12
5
12
3
14
102
160
149
49
38
45
1
nd
14
4
10
5
14
135
141
135
49
66
51
11
nd
15
12
11
6
21
88
191
88
51
168
182
18
0
53
27
44
16
56
421
618
460
188
0
0
0
nd
6
3
7
15
8
22
7
2
6
0
0
7
nd
0
24
24
0
1
131
6
2
18
2
0
0
nd
101
76
86
72
43
147
71
12
55
16
28
94
nd
0
30
2625
1012
428
289
59
0
416
213
41
10
nd
25
46
1306
325
144
142
12
7
206
231
69
111
0
132
179
4048
1424
624
731
155
23
702
105
44
24
9
5
5
2
3
6
22
44
24
24
139
57
25
11
4
2
2
3
3
11
47
27
28
130
22
15
9
4
6
2
2
1
10
23
21
20
129
50
18
14
6
3
5
4
4
14
35
52
28
503
173
82
42
18
16
10
12
13
58
148
125
100
0
2
1
2
1
4
2
2
1
3
8
2
2
11
10
6
12
15
11
1
3
6
8
32
28
12
21
39
12
10
113
13
80
85
93
107
430
78
90
87
191
3
188
142
128
29
12
27
1
664
343
151
25
2
9
17
18
16
14
3
8
35
62
4
18
144
244
31
229
289
172
126
105
135
154
1196
455
273
45
55
24
12
7
4
5
5
9
21
27
47
22
45
52
28
4
9
4
3
5
9
26
23
57
22
36
37
17
20
3
2
2
5
6
17
9
16
14
76
77
40
20
11
8
5
6
17
27
38
26
29
201
221
109
56
29
19
14
20
41
91
96
147
87
49
2
40
0
5
8
0
4
2
1
1
7
10
141
15
27
10
28
27
16
17
18
9
31
6
29
848
13
1472
21
69
0
483
4
79
62
113
155
277
1698
190
1702
60
161
316
695
342
314
142
296
462
532
400
4
2333
35
263
0
500
0
875
81
700
413
467
3136
224
5574
126
526
351
1694
367
1288
295
1141
1043
1314
32
41
12
8
9
6
4
4
10
16
12
18
14
50
46
15
8
8
5
4
3
11
16
13
19
17
29
14
108
13
13
0
0
2
13
12
8
9
18
68
23
85
20
16
11
9
1
17
12
10
13
24
180
124
220
50
47
23
16
10
50
56
44
59
73
13
2
13
17
6
13
0
0
0
2
2
2
6
304
1
116
115
24
2
3
10
10
8
1
12
51
250
0
263
492
156
49
0
43
26
12
6
246
129
2444
14
1006
431
608
34
4
0
35
750
0
0
444
47
3
88
500
0
0
0
0
1
0
0
12
54
3058
20
1486
1555
794
98
7
53
72
772
9
272
683
549
P.Poli et al.
Table I. Continued
Salmonella typhimurium
1996
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Mean
1997
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Mean
1998
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Mean
Saccharomyces cerevisiae
TA98
TA981
TA100
TA1001
Total
PM
PM1
GC
GC1
RD
Total
26
14
11
9
6
1
4
1
7
16
36
65
16
26
12
10
7
5
2
3
1
7
15
42
52
15
16
19
7
3
0
1
0
0
4
9
18
27
9
19
23
9
5
7
2
1
1
6
18
29
39
13
88
67
37
24
17
6
8
3
24
59
125
184
53
0
0
0
0
0
0
31
8
0
0
36
1
6
0
0
2
0
0
45
22
69
110
99
172
67
49
3
0
5
0
1
80
48
136
37
52
838
17
101
0
0
0
0
0
1244
591
634
5726
1264
1268
1484
1018
5
9
0
0
6
32
306
219
0
0
0
0
48
8
9
7
0
7
1401
998
1066
5873
1415
2314
1569
1222
38
30
21
11
3
5
0
2
5
23
15
21
15
33
34
22
8
3
3
1
1
4
31
17
23
15
21
20
18
9
3
5
8
5
6
15
7
9
10
31
35
25
13
7
10
4
12
10
10
18
11
15
123
118
86
40
15
23
13
20
25
79
57
64
55
3
3
0
2
0
3
0
0
3
4
0
0
2
41
336
209
93
47
41
0
0
12
378
24
39
102
54
97
119
1299
123
32
35
7
15
90
0
0
156
1220
17513
13358
1338
913
782
0
0
797
4192
53
429
3383
142
131
315
25
423
243
8
15
248
50
0
23
135
1460
18080
14001
2757
1506
1101
43
22
1075
4714
77
491
3777
13
68
15
4
10
1
3
5
3
3
11
33
14
23
83
16
5
2
0
2
2
3
2
12
33
15
9
62
5
6
6
0
0
0
3
9
7
35
12
22
43
13
4
4
3
4
3
2
1
22
59
15
67
256
49
19
22
4
9
10
11
15
52
160
56
0
0
0
11
4
3
0
0
139
0
2
2
13
440
13
0
2046
3
45
116
201
180
0
70
46
263
55
19
0
872
30
58
43
60
629
72
24
27
157
4251
86
0
29580
0
761
3280
2538
2260
161
887
1471
3773
200
72
0
668
79
53
250
145
2179
0
38
0
307
4946
190
0
33177
116
920
3689
2944
5387
233
1021
1546
4514
Monthly mean value per m3 air equivalent in different genetic targets: reversion in Salmonella strains TA98 and TA100 with (1) and without S9; gene
conversion (GC) and point mutation (PM), with (1) and without cytochrome P450 induction; respiratory deficiency (RD) in S.cerevisiae strain D7. Total 5
sum of effects. Annual means are reported. Italics represent the four months tested with the Comet assay.
to the different sensitivity of the two organisms toward different
classes of chemicals.
SCGE on human leukocytes
To evaluate the sensitivity of the Comet assay on human
leukocytes in vitro for detecting genotoxicity in urban airborne
particulates we tested samples from four different months,
collected in the centre of Parma. The four samples, previously
assayed on strains TA98 and TA100 of S.typhimurium (Figure
1) and on the diploid strain D7 of S.cerevisiae (Figure 2),
were selected since they showed different effectiveness on the
various targets.
July 1996. Very low revertant induction in Salmonella (ø4
revertants/m3 for the two strains); a low increase in convertant
(1- to 2-fold) and revertant (1- to 2.5-fold) frequencies; an
increase (~8 times) in respiratory deficiency (Table I).
February 1997. Very high effectiveness on TA100 (~20 and
35 revertants/m3 with and without S9, respectively), on TA98
(~30 and 34 revertants/m3 with and without S9, respectively)
550
and on strain D7 when induced for cytochrome P450 (~50
and 15 times the spontaneous frequencies of gene conversion
and reversion, respectively). A toxic effect was observed at
3.2 m3 air equivalent. Effects on mitochondria are not evident.
April 1997. Medium effects on Salmonella (~13 and 9
revertants/m3 in TA100, with or without S9 and ~8 and 11
revertants/m3 in TA98, with or without S9); convertant induction is high both with and without endogenous metabolic
activation (~10 versus ~5 times) whereas ilv1 revertants are
increased only in the presence of cytochrome P450 (~8 versus
~1 times). A toxic effect is induced at 4 m3 air equivalent.
There is no effect on respiratory-deficient induction.
November 1997. Medium-high effects on Salmonella (~18 and
7 revertants/m3 in TA100, with or without S9 and ~17 and 15
revertants/m3 in TA98, with or without S9); the effects on
S.cerevisiae are low or negligible.
These differences could be explained not only by the
different genotoxic load in the four samples but also by the
Comet assay in environmental monitoring
Fig. 1. Dose–effect relationships between four airborne particulate monthly
samples and revertants in Salmonella TA98 and TA100 strains, with (1S9)
and without (–S9) exogenous metabolic activation.
effectiveness on different targets of compounds present in the
four mixtures. The different cellular organization of Salmonella
and S.cerevisiae can affect the response to xenobiotic compounds. Furthermore, in yeast recombinational events are
evaluated together with reverse mutation. Recombinational
events are increased by exposure to genetically active chemicals
in a non-specific manner while the responses of the two
Salmonella strains are specific enough towards some classes
of compounds.
In order to determine the presence in these environmental
mixtures of chemical classes able to induce DNA strand breaks
and alkali-labile sites and also to compare the DNA effects
on prokaryotic or eukaryotic microorganisms and on human
cells, an SCGE assay on human leukocytes was performed,
with and without addition of the hepatic microsomal fraction
(S9) of induced rats.
The samples were tested at different concentrations. Neither
cell viability (95–100%) nor apoptosis (0–2%) were affected.
An analysis of the genotoxic effects, without (Figure 3) and
with S9 (Figure 4), shows the different effectiveness of the
four samples on the induction of DNA damage (P , 0.001,
univariate analysis of variance). Without S9, February is the
more genotoxic sample with respect to the negative control (P
, 0.001, Student’s t-test), with effects at the highest dose
comparable with the positive control effects. The addition of
S9 mix appears to partially detoxify some compounds in the
February sample (P , 0.01, univariate analysis of variance).
The differential genotoxic action of the four monthly extracts
is further confirmed by the mean comet length increase/m3 air
equivalent (Figure 5). Without S9 addition, February (P ,
0.001, Student’s t-test) and April (P , 0.01, Student’s t-test)
Fig. 2. Dose–effect relationships between four airborne particulate monthly
samples and gene conversion and ilv1-92 mutant reversion in S.cerevisiae
strain D7, with (1P450) or without (–P450) induction of cytochrome P450.
The arrows show values .5000 convertants/108 survivors.
are more genotoxic than July and November. In the presence
of S9 mix, February and April appear more effective on
DNA than July, although the differences are not significant.
November does not show a significant effect on DNA.
SCGE on A.cepa root cells
Preliminary experiments were performed to test the reliability
and sensitivity of the overall procedure. The Comet assay in
A.cepa was originally used (Navarrete et al., 1997) to test
DNA damage induced by irradiation of the bulbs, before root
growth; no data were reported by the authors on the effect of
chemical mutagens on the roots.
At first, for the sake of convenience, we wanted to ascertain
the possibility of using roots detached from the bulbs; this
would have provided us with the opportunity to use a single
bulb (or the least possible number of bulbs) for all the
treatments required for an experiment. To perform this test,
H2O2, a DNA-damaging agent, was used on roots detached
from or connected to the bulb. High concentrations (100 mM)
of H2O2 (30 min at 26°C) were used in order to obtain
significant effects on the nuclei, since a considerable amount
of the H2O2 may be inactivated by a specific enzymatic
apparatus or by the presence of protective agents such as
flavonoids (see for example Duthie et al., 1997) in A.cepa
root cells. The observation that comparable results were
obtained under both conditions (data not shown) prompted us
to adopt the detached root system for further experiments.
We then assayed the effect on this system of Cr61, a heavy
551
P.Poli et al.
Fig. 3. Genotoxic effects of four airborne particulate monthly samples on
human leukocytes. DNA breaks as measured by the Comet assay. Cells
were treated without S9. Negative (DMSO, 50 µl/ml) and positive
(Melphalan, 10 µg/ml) controls are reported. The data are displayed as
frequency distribution of comet length values. Values .70 µm are not
reported.
metal for which genotoxic effects have been assessed in Vicia
faba with the use of SCGE (Koppen and Verschaeve, 1996).
K2Cr2O7 at concentrations ranging from 10 to 80 mM was
effective in increasing the comet length of A.cepa root cells
(Figure 6) as compared with the negative control (P , 0.001,
Student’s t-test). Thus, the responsiveness of our modified
plant SCGE assay to Cr61 treatment as a positive control
prompted us to analyse the genotoxic potential of the previously
described four samples of airborne particulates from Parma.
Three concentrations corresponding to 1.25, 2.50 and 5.00
m3 air equivalent/ml were tested for their genotoxic effect
(Figure 7). A concentration of DMSO equivalent to that present
at the highest dose of particulate was used as a control. No
effects were observed in the samples treated with the July
1996, April 1997 and November 1997 samples, whereas a
significant dose-dependent increase in DNA damage was
observed in those treated with the February 1997 airborne
particulate (P , 0.001, Student’s t-test). The DNA damage at
the highest dose was comparable with that observed when the
roots were treated with 40 mM Cr61.
SCGE on I.balsamina
We wanted to verify the possibility of extending the plant
Comet assay to different plant systems that may prove more
suitable for in situ monitoring of the genotoxic potential of
air pollutants. Impatiens balsamina was chosen as the result
of a preliminary screening within ornamental plant species.
552
Fig. 4. Genotoxic effects of four airborne particulate monthly samples on
human leukocytes. DNA breaks as measured by the Comet assay. Cells
were treated in the presence of S9. Negative (DMSO, 50 µl/ml) and positive
(Melphalan, 10 µg/ml) controls are reported. The data are displayed as
frequency distribution of comet length values. Values .70 µm are not
reported.
Plant roots, stems and leaves can represent different targets
for the various environmental pollutants (in the soil, water or
air). With a view to improving our understanding of the
different bioavailability of a pollutant to roots, stems and
leaves depending on the means of uptake, we evaluated the
DNA damage in these different parts of I.balsamina treated
with Cr61 through a radical apparatus dipping. Whole plantlet
roots were exposed to different Cr61 concentrations and then
washed with water. The cuts were performed on the main root,
on the stem and on the first leaf. The data (Table II) show an
increase in DNA migration in all the treated samples with
respect to the controls (P , 0.001, Student’s t-test). Increasing
Cr61 concentrations resulted in a decrease in the comet length
values. This can be related to the simultaneous increase in the
number of ‘clouds’ of DNA fragments, with disintegration of
the head region, identified as ‘ghost’ cells (e.g. at 50 mM,
ghost cells were only 3% for root, whereas they were 44% for
stem and 28% for leaf). These cells were not considered in
the analysis.
Three concentrations of a different airborne particulate
sample from Parma, January 1998, were tested for their
genotoxic effect on stem cuttings of Impatiens (Figure 8). The
particulate induces a significant increase in DNA migration
with respect to the control (Student’s t-test, P , 0.001). The
2.5 m3 sample is different from the 1.25 m3 sample (P ,
Comet assay in environmental monitoring
Fig. 5. Dose–effect relationships between four airborne particulate monthly samples and mean values of comet length in human leukocytes, with (1S9) or
without (–S9) exogenous metabolic activation. Regression lines are reported.
Fig. 6. Genotoxic effects on A.cepa roots of K2Cr2O7. DNA breaks as
measured by the Comet assay. Results are reported as comet length and
displayed as box plots (see Materials and methods).
0.002, t test) whereas the 5.0 m3 sample does not show any
significant differences from the 2.5 m3 sample. This could be
due to the simultaneously increased induction of ‘ghost cells’
(not considered in the analysis) at the highest dose.
Discussion
Improvements in air quality is one of the main goals of The
European Community and, in this context, airborne particulate
genotoxicity appears to be one of the more promising indices
of human genotoxic/cancerogenic risk assessment.
The time series of the data allowed us to describe the Parma
urban air genotoxic load over a long period of time (1990–
1998), in the course of which various measures (catalytic
devices, unleaded fuels, annual vehicle overhaul, etc.) to
improve air-dispersed pollutant control were enforced. Some
chemicophysical parameters, such as lead and sulphur dioxide,
decreased with increased unleaded petrol use (data not
reported). However, the findings on airborne particulate genotoxicity, assessed over the period by short-term mutagenicity
Fig. 7. DNA damage induced by four different airborne particulate extracts
on A.cepa root cells. DNA breaks as measured by the Comet assay. Results
are reported as comet length and displayed as box plots.
553
P.Poli et al.
Table II. Effects of Cr61 on DNA migration of I.balsamina root, stem and
leaf derived nuclei (means 6 SD)
Dose (mM)
Comet length (µm)
Root
0
20
50
80
aGhost
25.8
34.2
43.8
30.1
Stem
6
6
6
6
6.7
6.4
9.6
9.2a
23.3
42.1
45.0
46.1
Leaf
6
6
6
6
5.9
6.7
8.8
14.2a
28.5
39.7
34.1
29.8
6
6
6
6
11.3
5.0
10.1
8.6a
cells .80%.
Fig. 8. DNA damage induced by the airborne particulate extract from
January 1998 on cells of I.balsamina stems. DNA breaks as measured by
the Comet assay. The data are displayed as frequency distribution of comet
length values.
tests on S.typhimurium and S.cerevisiae, showed the presence of
high levels of genotoxic compounds in the airborne particulates.
The differences between prokaryotic and eukaryotic cells can
affect sensitivity to a variety of substances. Gene conversion
frequencies were also evaluated in yeast. Recombinational
events were increased by exposure to genetically active chemicals in a non-specific manner, reflecting the response of the
cell to a wide spectrum of genetic damage. The composition
of airborne particulate mixtures is affected by the use of
different fuels (e.g. leaded and unleaded gasoline). Unleaded
gasoline by-products can be more effective on S.cerevisiae
than on Salmonella.
The approach proves to be very helpful in environmental
monitoring since these biological assays appear more sensitive
and effective than the chemicophysical parameter measure
alone. Nevertheless, further investigations are necessary for
more complete genotoxic compound detection in the urban
environment.
In this context, in order to evaluate the sensitivity of the
Comet assay for environmental mixture genotoxicity detection,
we applied the test to human leukocytes and to plant cells,
554
previously treated with some urban airborne particulate samples
presenting not only a different quantitative mutagenic load but
also a differential action on the considered DNA targets in
microorganisms. The Comet assay was used on human and
plant cells in comparison with results on Salmonella and
S.cerevisiae.
The first data on human leukocytes by SCGE confirm the
sensitivity of the assay and its applicability in assessing
genotoxicity in environmental samples such as airborne particulates. A peculiarity of the Comet assay is evident. Our
knowledge concerning the xenobiotic metabolizing capacity
of human blood cells is rather limited, however, blood monocytes have been shown to constitutively express P450 isoenzymes (Baron et al., 1998). On the other hand, addition of the
rat hepatic microsomal fraction allowed partial detoxification
of the February 1997 sample. A comparison of the data shows
a different response of human cells with respect to the tests
on Salmonella: leukocytes treated in the presence of S9 mix
show a behaviour similar to gene conversion in S.cerevisiae
when not induced for cytochrome P450 complex (P 5 0.05,
Spearman r); on the other hand, leukocytes without S9 correlate
with gene conversion in yeast induced for cytochrome P450
(see Table I and Figures 1, 2 and 5). Nevertheless, when the
sum of the two (6S9) mean comet length increases/m3 in
human cells (July, 1.9; February, 4.9; April, 2.8; November,
1.1) were compared with the sum of the effects in Salmonella
and in S.cerevisiae (see Table I), respectively, a similar trend
(P 5 0.01, Spearman r) was obtained between the effects on
leukocytes and on the eukaryotic microorganism.
These preliminary findings suggest a good correlation
between genotoxic effects in yeast and DNA damage in
human cells.
The measurement of DNA damage in the nuclei of higher
plant tissues is a new area of study with SCGE (Koppen and
Verschaeve, 1996; Gichner and Plewa, 1988; Koppen and
Angelis, 1998). This assay could be incorporated into in situ
plant monitoring of the atmosphere, water and soil: the Comet
assay allows fast detection of DNA damage without any need
to wait for progression into mitosis. However, the presence of
a cell wall causes technical difficulties for SCGE. This cell
wall proved to be a resistant barrier to cell lysis when using
a lysis buffer sufficient to degrade animal cells. In this context,
we improved previously described protocols (Navarrete et al.,
1997) in A.cepa, increasing the nuclear yield and widening its
application to different experimental conditions.
The protocol was validated by using a genotoxic compound
(Cr61): a dose–effect response was observed. The first findings
on airborne particulate extracts confirm the high genotoxic
load of the February 1997 sample.
Plant systems represent a complex multicellular environment
where the efficiency of different protection or repair mechanisms can be modulated by cellular homeostasis. However, the
capability of the system to show a multicellular organism
response against environmental pollutants widely counterbalances a probable lowering in sensitivity, for example, to
compounds such as free radicals (Duthie et al., 1997).
Furthermore, our revised Comet assay was applied to
different tissues (root, stem and leaf) of I.balsamina to verify
whether plant SCGE could be used as a suitable tool for
monitoring primary DNA damage induced by environmental
pollutants.
In order to improve our understanding of the different
bioavailability of a pollutant to roots, stems and leaves
Comet assay in environmental monitoring
depending on uptake, we evaluated the DNA damage in these
different parts of I.balsamina treated with Cr61 through a
radical apparatus dipping. Cr61 was able to induce DNA
damage in all the tissues. The different sensitivity (root , leaf
, stem) could be ascribed to the different cell types but also
to the different genotoxic action (i.e. Cr61 and/or generated
free radicals). DNA damage was also induced by airborne
particulate samples (January 1998). These preliminary findings
suggest that application of the test to different plant tissues
could be useful in estimation of the bioavailability and genotoxic damage due to environmental pollutants, including volatile compounds such as ozone, benzene, nitrogen oxides, etc.,
in higher plants.
In conclusion, the results of this study confirm that SCGE
is applicable to genotoxic load assessment of environmental
mixtures such as urban airborne particulates in comparison
with classical short-term mutagenesis tests. The use of human
cells could give further information regarding environmental
genotoxic load. Higher plants have a long tradition of use in
mutation research and the inclusion of SCGE in plant systems
(roots and epigean tissues) could extend their utility in in situ
environmental mutagenesis.
Acknowledgements
Financial support for this study was provided by the A.M.N.U. of Parma and
by the Ministero dell’Ambiente, Italy.
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Received on March 1, 1999; accepted on July 12, 1999
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