Mutagenesis, Vol. 17, No. 2, 127-134,
March 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Indoor and outdoor genotoxic load detected by the Comet assay in leaves of Nicotiana tabacum cultivars Bel B and Bel W3
Istituto di Genetica, Università degli Studi di Parma, Parco Area delle Scienze 11/A, I-43100 Parma, Italy
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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 environmental genotoxicity detection.
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Introduction |
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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.
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 hypersensitive 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 macromolecules (DNA, proteins and lipids), resulting in photosynthesis 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 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 biomonitoring, 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.
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Materials and methods |
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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 commercial 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 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 (Ø = 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 (57x43x52 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 
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 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 lengthxper 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.
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Results |
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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 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).
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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 treatment; ~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 number 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).
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Environmental exposure to ozone
Exposure at an O3 concentration
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 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 (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 uncharacteristically low temperature and windy days. However, in this case a significant difference (Fisher's F, P < 0.001) in DNA damage between ozone-sensitive cv. Bel W3 and ozone-tolerant cv. Bel B was found, with a peculiar frequency distribution in Bel W3. As an example, the effect on small leaf nuclei is reported in Figure 6. Box plots, frequency distributions, percentile values and cell number with TL > SRGL showed a large sub-population of damaged cells in Bel W3. The greatest mean DNA damage observed in the first experiment (Table I), in both Bel B and Bel W3, could be ascribed to a greater mean exposure (Figures 4 and 5). The higher sensitivity of Bel W3 is only evident in the presence of the genotoxic agent; indeed, the difference in DNA damage between the two cultivars was not significant (one-way ANOVA) when Bel B and Bel W3 were maintained inside the plant growth room, as controls (Figure 1B
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Indoor genotoxicity experiment
Germicidal lamps are currently used to maintain sterile environments such as microbiology laboratories. Sterilization is provided by UV emissions in a wavelength range that minimizes or avoids ozone production. However, in our laboratory a strong pungent smell had been noticed in the morning, after overnight sterilization. To assess the potential genotoxicity of UV photoproducts in this indoor working environment, the three plant systems were exposed to a routine sterilization process (during the night, for 15 h) in the laboratory (UV laboratory). Ozone concentration was monitored during the treatment, confirming the insignificant concentration (
10 p.p.b.) of this gas produced by the UV lamps (data not shown).
However, the presence of DNA-damaging compounds in the UV laboratory is clearly evident from the Comet test results in all three plant systems (Figure 7). Significant differences in TL values between exposed and control plants (Dunnett's C, P < 0.001 in all cases) were observed. The results are similar to those found after H2O2 treatment: Bel B shows the lowest sensitivity, I.balsamina intermediate sensitivity and Bel W3 is the most sensitive. The two N.tabacum cultivar controls were not significantly different, but exposed Bel B showed significantly less DNA migration than exposed Bel W3 (Fisher's F, P < 0.001).
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In order to obtain confirmation of the possible presence of genotoxic agents in the UV laboratory atmosphere, we performed a different assay (Zimmerman et al., 1975
) using S.cerevisiae D7 mutation and gene conversion frequencies. As reported in Table II, neither of the parameters was significantly different between control and exposed yeasts. However, it is worth noting that S.cerevisiae was exposed to filtered air (0.45 µm pore size) to maintain sterility of the culture (see Materials and methods), preventing the cells from coming into contact with airborne particulate matter. These findings suggest a lack of volatile genotoxic compounds and possible adsorption 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).
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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
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Discussion |
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Environmental pollution assessment and control are priority issues for both developed and developing countries of the world. Monitoring the production and release into the environment 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 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.). 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 experiment, 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 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.
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Acknowledgments |
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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 University and Scientific and Technological Research of Italy (MURST), Progetto giovani ricercatori e ricercatori singoli.
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Notes |
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1 To whom correspondence should be addressed. Tel: +39 0521 905603; Fax: +39 0521 905604; Email: restivo{at}unipr.it
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References |
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Received on July 18, 2001; accepted on October 12, 2001.
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