Mutagenesis vol.17 no.2 pp.
127–134, 2002
Indoor and outdoor genotoxic load detected by the Comet assay in
leaves of Nicotiana tabacum cultivars Bel B and Bel W3
Francesco Maria Restivo1, Maria Concetta Laccone,
Annamaria Buschini, Carlo Rossi and Paola Poli
Istituto di Genetica, Università degli Studi di Parma, Parco Area delle
Scienze 11/A, I-43100 Parma, Italy
Environmental pollution assessment and control are
priority issues for both developed and developing countries
of the world. The use of plant material for a more complete
picture of environmental health appears to be particularly
appealing. Here we validate a previous plant-adapted
Comet assay on leaf tissues of Nicotiana tabacum cultivars
Bel B and Bel W3. The effects of H2O2 on DNA damage
in Bel B and Bel W3 agree with the hypothesis that some
component of the machinery that protects DNA integrity
from oxidative stress may be impaired in cv. Bel W3.
Exposure in the field on sunny summer days (peak ozone
concentration >80 p.p.b.) showed significantly higher DNA
damage in cv. Bel W3 if plants were collected and subjected
to the Comet assay when the air ozone concentration was
reaching its peak value, but not when plants were sampled
early in the morning and hence after a period of low ozone
concentration. The different results suggest that Bel W3
possesses a less efficient recovery apparatus that requires
a longer period of activity to be effective and/or is less
protected against reactive oxygen species production during
exposure to ozone. However, it cannot be excluded that the
increase in mean DNA damage is the result of the presence
of a genotoxic agent(s) other than ozone. Interestingly, Bel
W3 also appears to be more responsive, compared with
Bel B, when exposed to ambient indoor pollutants. The use
of cv. Bel W3 increases the sensitivity of the assay under
both indoor and field conditions. However, different classes
of mutagens should be tested to define the range of
profitable utilization of this tobacco cultivar for environ-
mental genotoxicity detection.
Introduction
Improvements in air quality are an important goal for human
health protection. Airborne particulate genotoxicity appears
to be one of the more promising indices for genotoxic/
carcinogenic risk assessment and in many industrialized cities
throughout the world oxidizing air pollutants have become an
important public health concern.
Ozone (O3) is a secondary air pollutant, being formed in
the troposphere from primary precursor pollutants (e.g. in
motor vehicle engine exhaust) such as NOx and hydrocarbons.
In the presence of light, NO2 is cleaved to NO ⫹ O and allows
the formation of O3 (O2 ⫹ O). By reconversion of NO to NO2 in
complex reactions involving hydrocarbons, and photochemical
recycling of the NO2, O3 accumulates in the ambient air and
may reach high levels in an urban basin with heavy vehicular
traffic and adequate sunlight.
1To whom correspondence should be addressed. Tel: ⫹39 0521 905603; Fax: ⫹39 0521 905604; Email: restivo@unipr.it
© UK Environmental Mutagen Society/Oxford University Press 2002
O3 is a ubiquitous pollutant with a range of established
effects following acute and chronic exposure. Acute O3
exposure causes decrements in pulmonary function, alters
ventilation, induces inflammation and may alter host immune
defences (Bromberg and Koren, 1995; Kleeberger, 1995;
Miller, 1995). Ozone is genotoxic (Victorin, 1992; Boorman
et al., 1995; Sills et al., 1995) and has carcinogenic potential,
since it causes DNA damage by oxidative stress due to
hydroxyl radicals, superoxide, singlet oxygen and hydrogen
peroxide. Thus, an understanding of the molecular events that
underlie ozone toxicity is an important goal.
Exposure of plants to ozone results in the expression of a
number of defence-related genes that are also induced during
a hypersensitive response. A potential common link between
the activation of defence gene expression during a hyper-
sensitive response and by ozone treatment is the production
of reactive oxygen species (ROS) and the accumulation of
hydrogen peroxide (Baker and Orlandi, 1995). Ozone is
converted to ROS after entering the leaf intercellular spaces.
ROS react with membrane lipids to generate lipid peroxides
and, through a cascade reaction, can react with other macro-
molecules (DNA, proteins and lipids), resulting in photo-
synthesis reduction, electrolyte dispersion and accelerated
senescence (Mehlhorn and Wellburn, 1994; Kanofsky and
Sima, 1995; Miller et al., 1999).
O3-induced oxidative stress induces a large number of
antioxidant defence responses, such as ad hoc enzymatic
apparatus and the presence of protective agents (Kangasjärvi
et al., 1994; Sandermann, 1996; Sharma and Davis, 1997;
Turcsanyi et al., 2000). However, considerable variation
between taxa in the potential degree of protection afforded by
protective agents has been suggested (Moldau, 1999; Plochl
et al., 2000). Two tobacco cultivars, O3-tolerant cv. Bel B and
O3-sensitive cv. Bel W3, with different ozone sensitivities
have been widely used over the last 30 years (Heggestad, 1991).
A general scheme for the different sensitivities of Bel B
and Bel W3 is not well characterized. Small necrotic areas
seen as spotted patterns in Bel W3 are comparable with
hypersensitive response-like viral lesions of tobacco leaves,
both in terms of cell death and defence response (Morel and
Dangl, 1997; Schraudner et al., 1998). The oxidative stress
caused by ozone may serve as the initial signal for programmed
cell death and a hypersensitive response. This signalling may
involve ethylene, which is specifically induced in the ozone-
sensitive tobacco line (Sandermann et al., 1998).
Pasqualini et al. (1999) reported that ozone fumigation
produced visual injury in mature leaves of both cultivars,
particularly in Bel W3. After O3 treatment the leaf content of
abscissic acid, ascorbic acid and glutathione and the activity
of glutathione reductase were differently affected in the two
cultivars. After ozone exposure both cultivars showed a first
transient maximum of ROS in the apoplast, with subsequent
127
F.M.Restivo et al.
induction of comparable levels of glutathione peroxidase, but
a second oxidative burst was found only in Bel W3 (Schraudner
et al., 1998). A burst of ROS is known to be involved in local
cell death and therefore the effects of O3 could be amplified
in Bel W3 (Schraudner et al., 1998).
Previous reports have presented contrasting data on the
genotoxic activity of ozone. O3-induced chromosomal damage,
both physiological (chromosome stickiness) and physical
(bridges, fragments and micronuclei), was found in Vicia faba
(Janakiraman and Harney, 1976), with a higher susceptibility
to ozone during early than later stages of meiosis. On the
other hand, Gichner et al. (1992) did not find any increase in
the frequency of somatic mutations above the spontaneous
levels in Tradescantia and tobacco mutagenicity assays, while
distinct physiological damage to plant tissues was observed.
The single cell gel electrophoresis (SCGE) or Comet assay
(Singh et al., 1988) is a very sensitive method for detecting
DNA alkali-labile sites and strand breaks in individual cells.
It is becoming a major tool in environmental pollutant bio-
monitoring, both in vivo and in vitro (Fairbairn et al., 1994;
Calderón-Garcidueñas et al., 1997; Malyapa et al., 1997a,b;
Mitchelmore and Chipman, 1998; Wilson et al., 1998; Cotelle
and Ferard, 1999; Gluck and Gebbers, 2000; Kassie et al.,
2000; Moller et al., 2000), and can be applied to virtually any
eukaryotic cell. The measurement of DNA damage in the
nuclei of higher plant tissues is a new area of study by SCGE
and has been applied to various plant tissues: seeds (Cerda
et al., 1997), roots of V.faba (Koppen and Verschaeve, 1996;
Koppen and Angelis, 1998; Angelis et al., 2000; Gichner
et al., 2000a; Menke et al., 2000) and Allium cepa (Navarrete
et al., 1997) and leaves of Nicotiana tabacum (Gichner and
Plewa, 1998).
A previous study (Poli et al., 1999) showed that SCGE
assay of A.cepa roots and Impatiens balsamina tissues is
sensitive to environmental pollutants. Therefore, the Comet
assay seems particularly useful for monitoring the atmosphere,
water and soil in that it allows fast detection of DNA damage
without any need to wait for progression into mitosis.
The aim of the present research was to see if Bel W3
could be useful in the assessment of environmental pollutant
genotoxicity since, together with ozone hypersensitivity, many
other mechanisms are involved that are also induced during a
plant response to other exogenous attack. In other words, we
tried to verify whether the high sensitivity to ozone of Bel
W3 could also be useful to evaluate the genotoxicity of both
oxidizing agents and environmental mixtures using a short-
term genotoxicity test such as the Comet assay.
Materials and methods
Chemicals, media, cells and seeds
Ethidium bromide, L-tryptophan, L-isoleucine and adenine were obtained from
Fluka, Tris from ICN Biochemicals, yeast extract, BactoPeptone and agar
from Difco, reagents for electrophoresis, normal melting point (NMA) and
low melting point agarose (LMA), plant growth medium (MS; catalog
no. M5519) and general laboratory chemicals from Sigma. Saccharomyces
cerevisiae strain D7 (Zimmermann et al., 1975), I.balsamina common commer-
cial seeds and N.tabacum cultivars Bel B and Bel W3 (Heggestad, 1991),
kindly provided by Prof. G.Lorenzini (University of Pisa, Italy), were used in
the experiments.
Yeast assay
The diploid strain D7 of S.cerevisiae was used as a proven short-term
mutagenesis test to determine the induction of reversion (ilv1-92 mutant) and
mitotic gene conversion (trp5 locus) by ambient air (Poli et al., 1999; Buschini
et al., 2001). The cells (108 cells/ml) were inoculated into flasks containing
128
0.1 M phosphate buffer, pH 7.4, and maintained in an alternating shaker
(110 r.p.m.) at 20°C for 14 h. The flasks were stopped with a cork provided
with two outlets. The first was connected to a vacuum pump (Vacuum Gene
Pump; Pharmacia LKB); the second was fitted with a millipore filtration unit
(HVLP type, Ø ⫽ 0.45 µm; Millipore) to ensure sterile conditions inside the
flask. Using this system, the atmospheric mixture was directly introduced into
the flask without passing through the pump. Treated cells were then plated
on solid complete medium and trp⫹ and ile⫹ selection media. Plates were
scored for the number of survivors and revertant and convertant colonies.
Gene conversion and point mutation frequencies induced by hycanthone were
assessed in the cells to confirm the strain sensitivity (Poli et al., 1999).
Plant growth conditions
Seed germination was carried out in plastic pots (∅ ⫽ 10 cm) containing
vermiculite irrigated with tap water. After germination, seedlings were
transferred to new plastic pots (3 seedlings/pot). Both germination and growth
were performed in a plant growth chamber at 25°C with a dark/light
photoperiod of 8/16 h and irrigated with diluted (1/10) MS medium. If not
otherwise stated, intact plantlets (~45 days after germination) were used for
the different experiments described below.
Plant treatments
Hydrogen peroxide. Young seedlings (3 weeks old) were used for these
experiments. Plantlets were removed from the pots and the vermiculite was
carefully removed from the roots by several tap water washes. The roots of
whole plantlets were dipped in microcentrifuge tubes containing 1 ml of
different concentrations of H2O2 in distilled water and incubated at 26°C for
20 min in daylight conditions.
Ozone. The experiments were performed at the Experimental Station for
Environmental Monitoring (Department of Environmental Science, University
Campus, Parma), where O3 concentration in the air is routinely measured
(Philips Model-400 Ozone Analyser). The plantlets were placed on top of a
flat-roofed building (~2 m above ground), where ozone sensor inlets are
positioned. Plants were partially shadowed with a knitted net and watered
when necessary.
Indoor environment. The experiments were performed inside a microbiology
laboratory equipped with germicide lamps (two Philips TUV 36W/G36T8
lamps). Atmospheric ozone concentration was determined (Philips Model-400
Ozone Analyser) during the various treatments. Plantlets were exposed for
14 h either (i) directly on top of a bench or (ii) inside a plexiglas chamber
(57⫻43⫻52 cm) aerated (3 l/min) by means of the same type of system
described for the yeast assay.
To verify the persistence of genotoxicity in the environment, plantlets were
placed in the laboratory for 2 h, immediately after switching off the UV
lamps, but after a long (62 h) sterilization period. In all experiments plantlets
were protected from direct UV radiation. Control experiments were performed
either at the same time in an adjacent room or later, in the same room but
with the UV lamps switched off.
Single cell gel electrophoresis
Preliminary experiments showed that nuclei suspensions suitable for the SCGE
assay may be obtained from leaf cuttings of plantlets and processed essentially
as previously described (Poli et al., 1999). The procedure described by
Navarrete et al. (1997) for nuclei isolation from root cells of A.cepa was
utilized with major improvements. Degreased slides were previously dipped
in 1% NMA for the first layer. Leaf samples were sliced perpendicular to the
main rib and the various cut surfaces were dipped (10 times/cut) 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. Finally,
LMA was added as the top layer. The described modifications of the procedure
resulted in an increased yield of nuclei and a more uniform distribution of
nuclei in the agarose layer. If necessary, prepared slides can be incubated at
4°C in the dark in lysis buffer (2.5 M NaCl, 10 mM Na2EDTA, 10 mM Tris–
HCl, 1% Triton X-100 and 10% DMSO, pH 10) for at least 7 days without
any significant increase in DNA damage. The DNA was allowed to unwind
for 15 min in an electrophoretic alkaline buffer (1 mM Na2EDTA, 300 mM
NaOH, pH 艌 13) and subjected to electrophoresis in the same buffer for
10 min at 0.66 V/cm and 230 mA.
All the steps for slide preparation were performed under 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). To
obtain permanent samples, drained slides were than exposed briefly to cold
95% ethanol and allowed to dry.
Immediately before examination, the DNA was stained with 100 µl of
ethidium bromide (10 µg/ml). The samples were examined under a fluorescent
microscope (Leitz Dialux 20), equipped with an excitation filter (BP 515–560
nm) and a barrier filter (LP 580 nm) and linked to a CCD camera, using an

Fig. 1. (A) Comet images with increasing DNA damage in the plant
systems considered. Mean control nucleus diameter: I.balsamina,
6.59 ⫾ 1.79 µm; N.tabacum Bel B, 8.99 ⫾ 2.73 µm; N.tabacum Bel W3,
8.91 ⫾ 2.17 µm. (B) Average mean values (⫾ SD) of control TL.
automatic image analysis system (Cometa Release 2,1; Sarin, Florence, Italy).
One hundred cells per sample (50 cells/duplicate slide), selected at random,
were analysed. The samples were coded and evaluated blind.
The image analysis system allows us to make a quantitative description of
the comet using various parameters such as: (i) comet length (measured as
the distance between the leading edge of the comet head and the end of
the tail); (ii) head diameter; (iii) percentage DNA fluorescence intensity of
the total DNA in head and tail of the comet; (iv) tail moment (an integrated
value considering both the distance and the amount of migrated DNA, i.e.
tail length⫻per cent DNA fluorescence intensity in tail). However, a better
evaluation of the genotoxic effect could be obtained if the diameter of the
comet head (measured perpendicular to the direction of the electric field) was
subtracted from the comet length value in order to normalize for the different
cellular types in the tissue. Actually, this measurement resulted in lower
control values and hence increased sensitivity of the assay (Poli et al., 1999).
This parameter was defined as tail length (TL).
Results are presented as frequency distributions of single cell DNA damage
or as boxes with bars. In the latter case measured values are shown as boxes
that include 50% of the data. The top and the bottom of the boxes mark the
25th and 75th percentiles and the inner line marks the median value; quartiles
above the 75th and below the 25th percentile are marked as bars, limited by
the maximum or minimum values. Outliers are displayed as points. In addition,
we have analysed the number of cells which exhibit values greater than the
95 or 99% confidence limits (Superior Reference Group Limit, SRGL) for
the distribution of the control data (i.e. the frequency of damaged versus
undamaged cells).
The SPSS 10 statistical package was used. Analysis of variance was made
by one or multiple pairwise comparisons.
Results
The first step in our study was the evaluation of migration of
DNA from leaves of untreated plantlets of I.balsamina, as a
sensitive species previously used for environmental mixture
monitoring (Poli et al., 1999), and two tobacco cultivars, the
O3-tolerant cv. Bel-B and the O3-sensitive cv. Bel-W3, widely
used world wide for ozone biomonitoring (Heggestad, 1991).
Figure 1 shows the comet images of nuclei with different
amounts of DNA damage (Figure 1A) and the average mean
TL in untreated plantlets (Figure 1B). Impatiens balsamina
shows smaller nuclei and a shorter tail length than N.tabacum.
Since both Bel B and Bel W3 showed heterogeneous DNA
migration, we analysed the DNA integrity of leaves of different
size (L, length 4.9 cm, width 3.4–4.0 cm; S, length 2.5 cm,
width 1.5–2.0 cm), i.e. of different age, in the two N.tabacum
cultivars. Box plots of one experiment and average mean TLs
in large and small Bel B and Bel W3 leaves (at least three
independent experiments) are reported in Figure 2. Both
cultivars showed leaf age-dependent increases in cellular DNA
mobility. This observation has been previously reported by
Koppen et al. (1999). It can be concluded that with ageing of
leaves there is a decrease in DNA integrity, which could be
the result of increasing amounts of DNA single-strand breaks
Detection of genotoxic load
Fig. 2. Comparison of DNA damage distributions in small (S) and large (L)
leaves of untreated Bel B and Bel W3 plants. Data from each experiment
are reported as boxes with bars of TL values. The measured values are
shown as boxes that include 50% of the data. The top and the bottom of the
boxes mark the 25th and 75th percentiles and the inner line marks the
median value; quartiles above the 75th and below the 25th percentile are
marked as bars, limited by the maximum or minimum values. Average
mean values (at least three experiments) of TL (⫾ SD) are also reported.
and alkali-labile sites. Small leaves expressed a lower level of
DNA damage, possibly due to more efficient repair and/or
dilution of initial DNA lesions during cell division (Gichner
et al., 2000b).
For each of the subsequent experiments care was taken to
use leaves of similar dimensions (whenever possible small
leaves) from treated and control samples.
Hydrogen peroxide
To verify that the higher ozone sensitivity of Bel W3 resulted
in increased DNA sensitivity to oxidizing agents, plantlets were
treated with H2O2, whose accumulation has been demonstrated
after both ozone exposure and environmental stress (Baker
and Orlandi, 1995; Sharma and Davis, 1997). Furthermore,
H2O2 is able to induce DNA damage (Sharma and Davis,
1997), detected by SCGE analysis (Poli et al., 1999).
A significant increase (Dunnet’s C, P ⬍ 0.001, treated
versus control) in DNA damage was detected in all the tested
plant systems (I.balsamina and N.tabacum cultivars Bel B
and Bel W3) after H2O2 treatment (100 mM) in repeated
experiments (at least three) and in both large and small leaves.
The data from a representative experiment (small leaves) are
reported in Figure 3. The data for I.balsamina confirm the
previous found sensitivity to xenobiotics (Poli et al., 1999):
all the parameters considered (mean, median, percentiles and
number of damaged cells) were greatly increased after treat-
ment; ~50% of cells were damaged (see table in Figure 3).
The difference between treated and control Bel B plants was
less apparent, even if significantly different, especially when
considering damaged cells (compare percentiles and cell num-
129
F.M.Restivo et al.

ber with TL > SRGL), however, these data are affected by the
high dispersion of TL values in the control. The greatest
difference between control and treated samples was found for
cultivar Bel W3: the number of damaged cells was dramatically
increased (74 times control values).
Environmental exposure to ozone
Exposure at an O3 concentration 艌50 p.p.b. for 2–3 h is able
to induce necrotic areas on the leaf surface of cv. Bel W3
(Menser et al., 1976). These ozone levels are higher than natural
background and are indicative of photochemical reactions from
primary precursor pollutants such as NOx and hydrocarbons.
In summer, due to high solar radiation, O3 production is
generally increased. So, the first field exposures (72 h) of Bel
W3, and of Bel B as a control, were performed in June 2000.
As shown in Figure 4, ozone concentrations 艌50 p.p.b. (but
always ⬍80 p.p.b.) were observed for 4.6–8.1 h/day. However,
DNA damage detected in the sensitive cultivar was not
significantly different from that detected in the control cultivar
Bel B. A representative example (data from small leaf nuclei)
is reported in Table I. A second experiment was performed in
July 2000 (Figure 5) for 120 h. In this experiment also the
ozone concentration (艌50 p.p.b. for 0.07–7.8 h/day) did not
exceed the 80 p.p.b. limit; this relatively low level of ozone
in the atmosphere could be attributed to a period of un-

Table I. Comet tail length (TL) parameters in Bel B and Bel W3 plants
exposed in the field (June experiment)

Bel B Bel W3

Mean 7.79 7.77

Median 5.18 6.41
Standard deviation 7.83 6.68
Percentiles
95th 27.09 18.59
99th 34.45 34.31
TL ⬎ SRGL (cell no.)
95th 5 2
99th 1 0
Fig. 3. DNA damage distributions, reported as boxes with bars of TL
values, in untreated (C) and treated (T) (100 mM H2O2) I.balsamina, SRGL (Superior Reference Group Limit) refers to the Bel B distribution.
N.tabacum Bel B and N.tabacum Bel W3 plants. Other distribution
parameters are reported in the table. SRGL refers to the respective
control (C).

Fig. 4. (A) O3 concentration in the air during the test period (June 2000). Fig. 5. (A) O3 concentration in the air during the test period (July 2000).
(B) Exposure time and period (hours) with an ozone concentration (B) Exposure time and period (hours) with an ozone concentration 艌50
艌50 p.p.b. p.p.b.

130
 Detection of genotoxic load

characteristically low temperature and windy days. However, TL values between exposed and control plants (Dunnett’s C,
in this case a significant difference (Fisher’s F, P ⬍ 0.001) in P ⬍ 0.001 in all cases) were observed. The results are similar
DNA damage between ozone-sensitive cv. Bel W3 and ozone- to those found after H2O2 treatment: Bel B shows the lowest
tolerant cv. Bel B was found, with a peculiar frequency sensitivity, I.balsamina intermediate sensitivity and Bel W3 is
distribution in Bel W3. As an example, the effect on small the most sensitive. The two N.tabacum cultivar controls
leaf nuclei is reported in Figure 6. Box plots, frequency were not significantly different, but exposed Bel B showed
distributions, percentile values and cell number with TL ⬎ significantly less DNA migration than exposed Bel W3
SRGL showed a large sub-population of damaged cells in Bel (Fisher’s F, P ⬍ 0.001).
W3. The greatest mean DNA damage observed in the first In order to obtain confirmation of the possible presence of
experiment (Table I), in both Bel B and Bel W3, could be genotoxic agents in the UV laboratory atmosphere, we per-
ascribed to a greater mean exposure (Figures 4 and 5). The formed a different assay (Zimmerman et al., 1975) using
higher sensitivity of Bel W3 is only evident in the presence S.cerevisiae D7 mutation and gene conversion frequencies. As
of the genotoxic agent; indeed, the difference in DNA damage reported in Table II, neither of the parameters was significantly
between the two cultivars was not significant (one-way different between control and exposed yeasts. However, it is
ANOVA) when Bel B and Bel W3 were maintained inside the worth noting that S.cerevisiae was exposed to filtered air
plant growth room, as controls (Figure 1B). (0.45 µm pore size) to maintain sterility of the culture (see
Indoor genotoxicity experiment Materials and methods), preventing the cells from coming into
Germicidal lamps are currently used to maintain sterile contact with airborne particulate matter. These findings suggest
environments such as microbiology laboratories. Sterilization a lack of volatile genotoxic compounds and possible adsorption
is provided by UV emissions in a wavelength range that
minimizes or avoids ozone production. However, in our
Table II. Convertant and revertant frequencies in S.cerevisiae strain D7
laboratory a strong pungent smell had been noticed in the before (0 h) and after (15 h) exposure to filtered air withdrawn from the UV
morning, after overnight sterilization. To assess the potential laboratorya
genotoxicity of UV photoproducts in this indoor working
environment, the three plant systems were exposed to a routine Time (h) Control Treated
sterilization process (during the night, for 15 h) in the laboratory
Survival (%) Trp 5b Ilv 1-92c Survival (%) Trp 5b Ilv 1-92c
(UV laboratory). Ozone concentration was monitored during
the treatment, confirming the insignificant concentration 0 100 1.25 1.15 100 1.06 1.23
(艋10 p.p.b.) of this gas produced by the UV lamps (data 15 110 1.40 1.06 117 1.17 1.01
not shown). aThe germicidal lamps were either switched off (Control) or switched on
However, the presence of DNA-damaging compounds in (Treated) during the experiment.
the UV laboratory is clearly evident from the Comet test results bConvertants/105 survivors.
in all three plant systems (Figure 7). Significant differences in cRevertants/106 survivors.

Fig. 6. DNA damage distributions, reported as boxes with bars and as frequency of TL values, in Bel B and Bel W3 plants exposed in the field (July). Other
distribution parameters are reported in the table. SRGL refers to the Bel B distribution.

131
F.M.Restivo et al.
Table III. Comet tail length (TL) parameters in Bel W3 plants after
exposure to filtered air withdrawn from the UV laboratorya
Control
Mean 10.53
Median 10.90
Standard deviation 6.00
Percentiles
95th 21.75
99th 26.65
TL ⬎ SRGL (cell no.)
95th 5 5
99th 1 0
SRGL (Superior Reference Group Limit) is referred to the control.
aThe germicidal lamps were either switched off (Control) or switched on
(Treated) during the experiment.
of active compounds on particulate matter, blocked by filters.
To verify this hypothesis, Bel W3, the most sensitive plant
system, was exposed under the same experimental condition
as the yeast cells. The plants were put inside a plexiglass
chamber ventilated through the filtration apparatus used for the
yeast assay (see Materials and methods). Repeated experiments
confirmed the absence of DNA-damaging agents in filtered
air. An example is reported in Table III (large leaves).
The question of professional exposure is the persistence of
genotoxic compounds in the environment during working
hours. For this reason, we simulated the situation of maximum
risk by exposing Bel B and Bel W3 plants in the UV laboratory
for 2 h after switching off the UV lamps, but in this case the
UV laboratory was subjected to prolonged sterilization (a
weekend, 62 h). A potential professional risk, even if low, was
demonstrated: an increase in DNA mobility was found in both
systems (Dunnett’s C, P ⬍ 0.01), as reported in Figure 8.
Discussion
Environmental pollution assessment and control are priority
issues for both developed and developing countries of the
world. Monitoring the production and release into the environ-
ment of potentially hazardous compounds has been undertaken
by physico-chemical methods and biomonitoring. The latter
approach has the advantage of providing a prediction of the
biological consequences of interactions between the relevant
pollutant and several other fortuitous factors, such as other
pollutants, environmental conditions, physiological conditions
of the recipient, etc. For this reason the use of living organisms
is required in order to obtain a complete picture of potential
ecotoxic and genotoxic risks. In this respect plants seem to be
particularly attractive for the assessment of environmental
pollutants.
In a previous paper (Poli et al., 1999) we described the use
of a plant-adapted SCGE assay to assess the presence of
possible genotoxic compounds in the airborne particulates
of an Italian town. The data presented here confirm the
effectiveness of the procedure and extend its application to
leaf tissues of N.tabacum cultivars Bel B and Bel W3.
As reported by Koppen et al. (1999), we too have observed
a leaf age-dependent decrease in nuclear DNA integrity in
N.tabacum. Even if this inherent variability in leaf material
may be supposed to complicate data analysis and/or lower the
sensitivity of the assay, we have reported the results of different
experiments that clearly demonstrate its effectiveness. The use
of young leaves, with similar small dimensions, significantly
132
Treated
9.85
9.82
6.18
22.21
24.51
Fig. 7. Impatiens balsamina, Bel B and Bel W3 plants exposed indoors
overnight (C, control; T, UV lamp switched on). DNA damage distributions
are reported as boxes with bars of TL values. Other distribution parameters
are reported in the table. SRGL refers to the respective control (C).
decreases the range of variability in tail length in both
N.tabacum cultivars.
The preliminary data we report on the effect of H2O2 on
DNA damage in I.balsamina and N.tabacum Bel B and Bel
W3 agree with the hypothesis that some component of the
machinery that protects DNA integrity from oxidative stress
may be impaired in cv. Bel W3. The damage induced by H2O2
is significantly higher in Bel W3 compared with that observed
in cv. Bel B and I.balsamina. The concentration of H2O2 used
in this experiment was rather high, but, as reported (see Poli
et al., 1999, and references therein), plants have a particularly
efficient apparatus for H2O2 scavenging. In this respect, we
believe that the use of mutants or ‘knock-out’ transgenic plants
that are defective in one or more additional components of the
scavenging machinery or DNA repair apparatus could increase
the sensitivity of the Comet assay.
If the observed increase in H2O2 sensitivity of Bel W3 is
due to impairment of some steps of a molecular pathway
not specifically involved in nuclear DNA protection from
H2O2, one may expect that Bel W3 nuclei would be less
protected from the effect of other ROS generators, such as
ozone. We thus exposed the plants in the field under conditions
(sunny summer days) that were expected to favour an increase
in atmospheric ozone concentrations over the threshold of
50 p.p.b. for several hours during the day. Actually, exposure
of plantlets at concentrations of 50–60 p.p.b. for >3 h/day
have been reported to induce the appearance of necrotic areas
on Bel W3 leaves (see Heggestad, 1991, and reference therein).
Although the two reported experiments were performed during
June and July 2000, respectively, ozone concentration did not
increase as observed in the corresponding period of previous
years (peak ozone concentrations never exceeded 80 p.p.b.).

Fig. 8. Bel B and Bel W3 plants exposed indoor (C, control; T, UV lamp
switched off after a long sterilization period). DNA damage distributions are
reported as boxes with bars of TL values. Other distribution parameters are
reported in the table. SRGL refers to the respective control (C).
However, a significant difference in DNA damage parameters
between Bel B and Bel W3 was observed in the second
experiment.
The differences in the results of the two experiments cannot
be attributed to different mean exposures (exposure was higher
in the first experiment), but this higher mean exposure may
be responsible for the wide scattering of data observed in the
first experiment. It seems more probable that the observed
differences may be attributed to the different sampling periods
in the two experiments. In the second experiment plants were
collected and subjected to the Comet assay when the ozone
concentration was reaching its peak value. In the first experi-
ment, in contrast, plants were sampled early in the morning
and hence after a period of low ozone concentration. Do the
different results of the two experiments indicate that Bel W3
(i) possesses a less efficient recovery apparatus that requires
a longer period of activity to be effective or (ii) is less protected
from ROS production during exposure to ozone? We do not
have molecular evidence to confirm either of these hypotheses;
moreover, we cannot exclude the possibility that both defence
mechanisms are impaired in Bel W3.
Many different parameters may vary in the atmosphere in
parallel, or not, with ozone concentration, so it cannot be
excluded that the increase in mean DNA damage observed in
nuclei of plants exposed in the field is the result of the presence
of a genotoxic agent(s) other than ozone. Exposure of plants
Detection of genotoxic load
under controlled environmental conditions (i.e. monitored
ozone fumigations in cabinets) is required to assess specific
ozone effects. In any case, it is notable that whatever the
potential genotoxic agent(s) present in the atmosphere,
comparison of the Comet test data from Bel B and Bel W3
allows detection or assessment of potential genotoxic risk.
The idea of further testing the N.tabacum biomonitoring
system was suggested by an incidental observation: the
presence of an unknown strong pungent smelling compound
in the atmosphere of microbiological laboratories subject to
sterilization using UV germicidal lamps. Separation of the
compounds released into the laboratory atmosphere by physico-
chemical methods and individual risk assessment for each
of these compounds is not feasible from either a practical or
economic point of view.
We exposed both I.balsamina and the two N.tabacum
cultivars to the atmosphere of the laboratory overnight (i.e.
UV lamps switched on). The results demonstrate the production
of a compound(s) capable of inducing DNA damage in all the
tested plant systems. Interestingly, Bel W3 appears to be most
responsive (compared with Bel B and I.balsamina) to the
presumptive genotoxic agent(s). A yeast assay, performed in
parallel, suggested adsorption of the genotoxic compound onto
particulate matter. This hypothesis was confirmed by exposing
Bel W3 plants under the same conditions as used for the yeast
assay. Nevertheless, further investigations are necessary for a
more complete characterization of the nature of the potential
genotoxic compound. The possibility of absorption and
concentration of this compound on different pore size filters
should be tested. Collected and solubilized material could then
be re-tested for its genotoxic effect using different assays,
such as those previously reported (Poli et al., 1999).
The next question was does the genotoxic risk persist in the
laboratory during working hours? To investigate this we
simulated the situation of maximum risk. The laboratory was
subjected to a weekend sterilization (62 h) and, after switching
off the lamps, the plants (Bel B and Bel W3 cultivars) were
introduced into the laboratory. Two hours in the laboratory
significantly affected DNA migration from the nuclei as
compared with that observed in control plants.
In conclusion, the results reported in this paper confirm that
the Comet assay is suitable for genotoxic risk assessment in
indoor and field atmospheres. The use of cultivar Bel W3
increases the sensitivity of the assay under the conditions we
tested, but different classes of mutagens should be tested to
define the range of profitable utilization of this tobacco cultivar
in the Comet assay. Several plants and/or mutants may differ
in their responsiveness to different genotoxic agents, as a
consequence of morphological and physiological properties
and of environmental habitus. Since at present the Comet
assay protocols seem to have been adapted or are easily
adaptable to an increasing number of plant species and plant
tissues, it would be worth testing different plant genetic
materials for their capacity to assess genotoxic risk in different
geographical and environmental situations.
Acknowledgements
We would like to thank Prof. G.Lorenzini (University of Pisa) for the generous
gift of N.tabacum Bel B and Bel W3 seeds and Prof. F.Giusiano and Dr
G.Tamborino (University of Parma) for hospitality and help with field
experiments and for easy access to ozone and atmospheric parameter detectors.
We would also like to thank Elen Jones-Evans for English revision of the
manuscript. This work was supported in part by a grant from the Ministry of
133
F.M.Restivo et al.
University and Scientific and Technological Research of Italy (MURST),
Progetto giovani ricercatori e ricercatori singoli.
References
Angelis,K.J., McGuffie,M., Menke,M. and Schubert,I. (2000) Adaptation to
alkylation damage in DNA measured by the comet assay. Environ. Mol.
Mutagen., 36, 146–150.
Baker,C.J. and Orlandi,E.W. (1995) Active oxygen in plant pathogenesis.
Annu. Rev. Phytopathol., 33, 299–321.
Boorman,G.A., Sills,R.C., Grumbein,S., Hiley,R., Miller,R.A. and
Herbert,R.A. (1995) Long-term toxicity studies of ozone in F344/N rats
and B6C3F1 mice. Toxicol. Lett., 82/83, 301–306.
Buschini,A., Anceschi,E., Pasini,L., Cassoni,F., Poli,P. and Rossi,C. (2001)
Urban airborne particulate, genotoxicity evaluation of different size fractions
by mutagenesis tests on microorganisms and comet assay. Chemosphere,
44, 1723–1736.
Bromberg,P.A. and Koren,H.S. (1995) Ozone-induced human respiratory
dysfunction and disease. Toxicol. Lett., 82/83, 307–316.
Calderón-Garcidueñas,L., Osnaya,N., Rodrı́guez-Alcaraz,A. and Villarreal-
Calderón,A. (1997) DNA damage in nasal respiratory epithelium from
children exposed to urban pollution. Environ. Mol. Mutagen., 30, 11–20.
Cerda,H., Delincée,H., Haine,H. and Rupp,H. (1997) The DNA ‘comet assay’
as a rapid screening technique to control irradiated food. Mutat. Res., 375,
167–181.
Cotelle,S. and Ferard,J.F. (1999) Comet assay in genetic ecotoxicology: a
review. Environ. Mol. Mutagen., 34, 246–255.
Fairbairn,D.W., Meyers,D. and O’Neill,K.L. (1994) Detection of DNA
damaging agents in environmental water samples. Bull. Environ. Contam.
Toxicol., 52, 687–690.
Gichner,T. and Plewa,M.J. (1998) Induction of somatic DNA damage as
measured by single cell gel electrophoresis and point mutation in leaves of
tobacco plants. Mutat. Res., 401, 143–152.
Gichner,T., Langebartels,C. and Sandermann,H.,Jr (1992) Ozone is not
mutagenic in the Tradescantia and tobacco mutagenicity assays. Mutat.
Res., 281, 203–206.
Gichner,T., Menke,M., Stavreva,D.A. and Schubert,I. (2000a) Maleic
hydrazide induces genotoxic effects but no DNA damage detectable by the
comet assay in tobacco and field beans. Mutagenesis, 15, 385–389.
Gichner,T., Ptacek,O., Stavreva,D.A., Wagner,E.D. and Plewa,M.J. (2000b) A
comparison of DNA repair using the comet assay in tobacco seedlings after
exposure to alkylating agents or ionizing radiation. Mutat. Res., 470, 1–9.
Gluck,U. and Gebbers,J.O. (2000) The comet assay of nasal epithelia:
measurement of DNA damage for the assessment of genotoxic air pollution.
Laryngoscope, 110, 123–125.
Heggestad,H.E. (1991) Origin of Bel-W3, Bel-C and Bel-B tobacco varieties
and their use as indicators of ozone. Environ. Pollut., 74, 264–291.
Janakiraman,R. and Harney,P.M. (1976) Effects of ozone on meiotic
chromosomes of Vicia faba. Can. J. Genet. Cytol., 18, 727–730.
Kangasjärvi,J., Talvinen,J., Utriainen,M. and Karjalainen,R. (1994) Plant
defense systems induced by ozone. Plant Cell Environ., 17, 783–794.
Kanofsky,J.R. and Sima,P.D. (1995) Singlet oxygen generation from the
reaction of ozone with plant leaves. J. Biol. Chem., 270, 7850–7852.
Kassie,F., Parzefall,W. and Knasmuller,S. (2000) Single cell gel electrophoresis
assay: a new technique for human biomonitoring studies. Mutat. Res., 463,
13–31.
Kleeberger,S.R. (1995) Genetic susceptibility to ozone exposure. Toxicol.
Lett., 82/83, 295–300.
Koppen,G. and Angelis,K.J. (1998) Repair of X-ray induced DNA damage
by comet assay in roots of Vicia faba. Environ. Mol. Mutagen., 32, 281–285.
Koppen,G. and Verschaeve,L. (1996) The alkaline comet test on plant cells:
a new genotoxicity test for DNA strand breaks in Vicia faba root cells.
Mutat. Res., 360, 193–200.
Koppen,G., Toncelli,L.M., Triest,L. and Verschaeve,L. (1999) The comet
assay: a tool to study alteration of DNA integrity in developing plant leaves.
Mech. Ageing Dev., 110, 13–24.
Malyapa,R.S., Abern,E.W., Straube,W.L., Moros,E.G., Pickard,W.F. and
Roti,J.L.R (1997a) Measurement of DNA damage after exposure to 2450
MHz electromagnetic radiation. Radiat. Res., 148, 608–617.
Malyapa,R.S., Abern,E.W., Straube,W.L., Moros,E.G., Pickard,W.F. and
Roti,J.L.R. (1997b) Measurement of DNA damage after exposure to
electromagnetic radiation in the cellular phone communication frequency
band (835.62 and 847.74 MHz). Radiat. Res., 148, 618–627.
Mehlhorn,H. and Wellburn,A.R. (1994) Man-induced causes of free radical
damage to plants: O3 and other gaseous pollulants. In Foyer,C.H. and
Mullineaux,P.M. (eds), Causes of Photooxidative Stress and Amelioration
of Defence Systems in Plants. CRC Press, Boca Raton, FL, pp. 155–175.
134
Menke,M., Meister,A. and Schubert,I. (2000) N-methyl-N-nitrosourea-induced
DNA damage detected by the Comet assay in Vicia faba nuclei during all
interphase stages is not restricted to chromatid aberration hot spots.
Mutagenesis, 15, 503–506.
Menser,H.A.,Heggestad,H.E and Grosso,J.J. (1976) Registration of Bel W3,
an ozone susceptible tobacco germplasm. Reg. No G.P. 14. Crop Sci., 16, 606.
Miller,F.J. (1995) Uptake and fate of ozone in the respiratory tract. Toxicol.
Lett., 82/83, 277–285.
Miller,J.D., Arteca,R.N. and Pell,E.J. (1999) Senescence-associated gene
expression during ozone-induced leaf senescence in Arabidopsis. Plant
Physiol., 120, 1015–1024.
Mitchelmore,C.L. and Chipman,J.K. (1998) DNA strand breakage in aquatic
organisms and the potential value of the comet assay in environmental
monitoring. Mutat. Res., 399, 135–147.
Moldau,H. (1999) Ozone detoxification in the mesophyll cell wall during a
simulated oxidative burst. Free Radic. Res., 31S, 19–24.
Moller,P., Knudsen,L.E., Loft,S. and Wallin,H. (2000) The comet assay as a
rapid test in biomonitoring occupational exposure to DNA-damaging agents
and effect of confounding factors. Cancer Epidemiol. Biomarkers Prev., 9,
1005–1015.
Morel,J.-B. and Dangl,J.L. (1997) The hypersensitive response and the
induction of cell death in plants. Cell Death Differ., 4, 671–683.
Navarrete,M.H., Carrera,P., de Miguel,M. and de la Torre,C. (1997) A fast
comet assay variant for solid tissue cells. The assessment of DNA damage
in higher plants. Mutat. Res., 389, 271–277.
Pasqualini,S., Batini,P., Ederli,L. and Antonielli,M. (1999) Responses of the
xanthophyll cycle pool and ascorbate-glutathione cycle to ozone stress in
two tobacco cultivars. Free Radic. Res., 31S, 67–73.
Plochl,M., Lyons,T., Ollerenshaw,J. and Barnes,J. (2000) Simulating ozone
detoxification in the leaf apoplast through the direct reaction with ascorbate.
Planta, 210, 454–467.
Poli,P., Buschini,A., Restivo,F.M., Ficarelli,A., Cassoni,F., Ferrero,I. and
Rossi,C. (1999) Comet assay application in environmental monitoring:
DNA damage in human leukocytes and plant cells in comparison with
bacterial and yeast tests. Mutagenesis, 14, 547–555.
Sandermann,H.,Jr (1996) Ozone and plant health. Annu. Rev. Phytopathol.,
34, 347–366.
Sandermann,H.,Jr, Ernst,D., Heller,W. and Langebartels,C. (1998) Ozone: an
abiotic elicitor of plant defence reaction. Trends Plant Sci., 3, 47–50.
Schraudner,M., Moeder,W., Wiese,C., van Camp,W., Inzé,D., Langebartels,C.
and Sandermann,H.,Jr (1998) Ozone-induced oxidative burst in the ozone
biomonitor plant, tobacco Bel W3. Plant J., 16, 235–246.
Sharma,Y.K. and Davis,K.R. (1997) The effects of ozone on antioxidant
responses in plants. Free Radic. Biol. Med., 23, 480–488.
Sills,R.C., Hong,H.L., Greenwell,A., Herbert,R.A., Boorman,G.A. and
Devereux,T.R. (1995) Increased frequency of K-ras mutations in lung
neoplasms from female B6C3F1 mice exposed to ozone for 24 or 30
months. Carcinogenesis, 16, 1623–1628.
Singh,N.P., McCoy,M.T., Tice,R.R. and Schneider,E.L. (1988) A simple
technique for quantitation of low levels of DNA damage in individual cells.
Exp. Cell Res., 175, 184–191.
Turcsanyi,E., Lyons,T., Plochl,M. and Barnes,J. (2000) Does ascorbate in the
mesophyll cell walls form the first line of defence against ozone? Testing
the concept using broad bean (Vicia faba L.). J. Exp. Bot., 51, 901–910.
Victorin,K. (1992) Review of the genotoxicity of ozone. Mutat. Res., 277,
221–238.
Wilson,J.T., Pascoe,P.L., Parry,J.M. and Dixon,D.R. (1998) Evaluation of the
comet assay as a method for the detection of DNA damage in the cells of
a marine invertebrate, Mytilus edulis L. (Mollusca: Pelecypoda). Mutat.
Res., 399, 87–95.
Zimmermann,F.K., Kern,R. and Rosenberg,H. (1975) A yeast for simultaneous
detection of induced mitotic crossing-over, mitotic gene conversion and
reverse mutation. Mutat. Res., 28, 381–388.
Received on July 18, 2001; accepted on October 12, 2001