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Rapid detection of mutagens accumulated in plant tissues using a novel Vibrio harveyi mutagenicity assay
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Ecotoxicology and Environmental Safety 70 (2008) 231–235 www.elsevier.com/locate/ecoenv
Highlighted article
Rapid detection of mutagens accumulated in plant tissues using a novel Vibrio harveyi mutagenicity assay Beata Podgo´rskaa, Aleksandra Kro´lickab, Ewa Łojkowskab, Grzegorz W˛egrzyna,c, Department of Genetics and Marine Biotechnology, Institute of Oceanology, Polish Academy of Sciences, S´w. Wojciecha 5, 81-347 Gdynia, Poland b Intercollegiate Faculty of Biotechnology, University of Gdan´sk and Medical University of Gdan´sk, K!adki 24, 80-822 Gdan´sk, Poland c Department of Molecular Biology, University of Gdan´sk, K!adki 24, 80-822 Gdan´sk, Poland
a
Received 14 October 2006; received in revised form 11 March 2007; accepted 8 April 2007 Available online 18 May 2007
Abstract Detection of mutagenic pollution in natural environment is difficult as there are thousands of known chemical mutagens, and they have mutagenic effects usually at very low concentrations. Plants are able to accumulate various substances, including mutagens, in their tissues. Here, we demonstrate that rapid detection of mutagenic activities of compounds accumulated in plant tissues is possible using a recently developed microbiological mutagenicity assay, based on induction of bioluminescence of a dim mutant of a marine bacterium Vibrio harveyi. Using this assay, it was possible to detect mutagenic activity in extracts from relatively small amounts of tissues (1.5 g) collected from plants, which were cultured in vitro for 30 days in the presence of nano-molar concentrations of various chemical mutagens. Moreover, contrary to control samples, cultured in vitro without any mutagens, significant mutagenicity was detected in several extracts of plant tissues collected from natural environment. The whole procedure was as short as 4 h or less. r 2007 Elsevier Inc. All rights reserved. Keywords: Mutagenic pollution of environment; Detection of mutagens; Mutagenicity assay; Accumulation of mutagens in plants
1. Introduction Currently, mutagenic pollutants appear in environment mostly as side effects of industrial processes (Heddle et al., 1999; Goldman and Shields, 2003; Vargas, 2003; Jha, 2004). These chemicals occur in various habitats and can induce serious diseases, including cancer, due to their genotoxic (mutagenic) activities (El-Bayoumy, 1992; Depledge, 1998; Au et al., 2001; Martin, 2001; Tornqvist and Ehrenberg, 2001; Barton et al., 2005). The germ line of higher organisms may be also damaged by these compounds, which may lead to fertility problems and to negative genetic changes in future generations (Shelby et al., 1993). Although detection of mutagenic pollutants in the environment appears very important (because of the reasons mentioned above), there are no simple chemical Corresponding author. Department of Molecular Biology, University of Gdan´sk, K"adki 24, 80-822 Gdan´sk, Poland, Fax: +48 58 523 5501. E-mail address: [email protected] (G. W˛egrzyn).
0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.04.004
procedures which might be employed as universal methods for testing the presence of mutagens. This is because there are thousands of known mutagens that occur in natural habitats in very low amounts, while having mutagenic effects at very low concentrations. Therefore, it appears that for preliminary and rapid detection of mutagenic activities in environmental samples, biological assays are more useful than chemical analyses. Among them, microbiological tests are commonly used as they are relatively simple and rapid (W˛egrzyn and Czyz˙ , 2003). Nevertheless, even using such tests, to obtain results of measurements one must usually wait as long as two days from sample withdrawal, like in the commonly used Ames test (Mortelmans and Zeiger, 2000) or its analogue, based on the use of bacteria which are more likely to survive in samples of natural waters (Czyz˙ et al., 2000, 2002b, 2003; Podgo´rska et al., 2005). More rapid tests, based on bacterial bioluminescence have been developed, including commercially available Mutatox (in which a strain of Vibrio fischeri is used; Ulitzur et al., 1980, Ulitzur and Weiser, 1981), which requires
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16–24 h to obtain results. Recently, a novel bioluminescence mutagenicity assay has been reported, which allows to obtain results in 2–4 h from sample withdrawal (Podgo´rska and W˛egrzyn, 2006). In this assay, a dim luxE mutant of Vibrio harveyi is employed, and cultures of this strain (named A16) are induced for light emission upon contact with mutagenic compounds. The luxE gene codes for the synthetase of the fatty acid reductase complex, a holoenzyme (containing also reductase and transferase) responsible for production of a long-chain aliphatic aldehyde, which is a substrate in the oxidation reaction performed by luciferase (for a review see Meighen, 1988). Since this luciferase-mediated reaction results in light emission, dysfunction of the luxE gene causes impairment of V. harveyi bioluminescence. However, restoration of the luxE function due to reverse mutation(s) results in restoration of light emission. Therefore, the A16 strain responds to contact with various mutagens by increasing the level of luminescence (Podgo´rska and W˛egrzyn, 2006). This may arise from induced reversion to the wild-type luxE allele or a pseudoreversion that allows proper functions of the gene product and restoration of luminescence (Podgo´rska and W˛egrzyn, 2006). Light emission by the A16 strain after its incubation with mutagenic compounds may be further enhanced, as stimulation of this process by both physical and chemical mutagens has been reported (Czyz˙ et al., 2002a). Although mutagenic compounds occur in the environment usually at very low concentrations, they can accumulate in various organisms, which makes them even more dangerous for humans. It appears that plants are especially capable of accumulation of environmental xenobiotics, including mutagens and promutagens (Ockenden et al., 1998; Stepanova et al., 1999). Moreover, it was demonstrated that some promutagens can be activated in plant tissues (Plewa, 1991; Plewa and Wagner, 1993), which may be considered as an additional hazardous factor. The aim of this work was to test an applicability of the V. harveyi bioluminesce mutagenicity assay in assessment of accumulation of mutagenic compounds in tissues of plants, both cultured in vitro and occurring in natural environment.
(plant tissue extract) and 5 mL of 4% S9 mix (consisting of rat liver microsomal enzymes and cofactors, as described previously by Maron and Ames, 1983) were added to 4.5 mL of the bacterial culture at absorbance (A575) ¼ 0.1, and incubation was continued for 3 h. Before addition of the tested sample, and 3 h after its addition, 1 mL of the bacterial culture was withdrawn and its luminescene was measured using a Sirius luminometer (Berthold), and expressed as relative light units (RLU). At the same times, A575 of cultures was measured to estimate number of bacterial cells (a correlation between cell number, determined by plating on BOSS plates, and absorbance of the culture was established according to Podgo´rska and W˛egrzyn (2006); A575 ¼ 0.1 corresponded to 3 107 colony forming units per 1 mL). Results were calculated as relative luminescence (RLU) per cell or per absorbance unit.
2. Materials and methods
Plants were collected from natural habitats, and species or genera were determined at Department of Plant Taxonomy and Nature Protection, University of Gdan´sk, Poland. A 1.5 g of plant tissue was homogenized and filtered as described in the preceding paragraph, and 0.5 mL of such an extract was used for the mutagenicity assay (performed as described above). Each measurement was repeated three times, and the mean value7SD was calculated. The result was considered positive (indicating mutagen accumulation) when at least two-fold increase of luminescence, relative to a control experiment, was observed, according to the previously published recommendation (Podgo´rska and W˛egrzyn, 2006).
2.1. Chemical mutagens Following mutagens (purchased from Sigma) were used: 2-methoxy-6chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine 2HCl (ICR-191), benzo(a)pyrene (B[a]P), and sodium azide (SA).
2.2. V. harveyi bioluminescence mutagenicity assay The assay was performed according to Podgo´rska and W˛egrzyn (2006), with the V. harveyi A16 strain (a dim luxE mutant) as an indicator strain. Bacteria were grown in the liquid BOSS nutrient medium (bacto-peptone, 1%; beef extract, 0.3%; glycerol, 0.011 mol L1; NaCl, 0.51 mol L1), described by Klein et al. (1995), at 30 1C. 0.5 mL of the tested sample
2.3. Plant cultures The plantlets of Petunia hybrida, Nicotiana tabacum and Solanum brevidens were grown on the Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 3% sucrose and 0.75% agar. Cultures were grown at a temperature of 20–22 1C under white fluorescent light with a 16 h photoperiod (white cool fluorescent light, Philips, TLD 58W/84o, 20 mmol m2 s1) and kept under 16/8 h light/dark cycles. The pH of the medium was adjusted to 5.8 prior to autoclaving. A voucher specimen of the plant is deposited at the Department of Plant Protection and Biotechnology, University of Gdan´sk and Medical University of Gdan´sk, Poland.
2.4. Estimation of mutagens’ accumulation in in vitro cultured plants Different concentrations of applied mutagens (ICR-191: 40, 400, 4000 nM; B[a]P: 80, 800, 8000 nM; and SA: 30, 300, 3000 nM) were tested by adding them to the MS medium after autoclaving (in control experiments no mutagen was added). Explants containing apical and one secondary meristem were cultured in mutagen-containing or mutagenfree (control experiments) MS medium for 30 days. After sample withdrawal, 1.5 g of plant tissue were homogenized (using Heidolph homogenizator) with 3 mL of artificial marine water (prepared according to MacLeod et al., 1954), and the extract was filtered using Whatmann 3 mm paper filters. A 0.5 mL of such an extract was used for the V. harveyi bioluminescence mutagenicity assay (performed as described above). Each experiment was repeated three times, and the mean value7SD was calculated. The result was considered positive (indicating mutagen accumulation) when at least two-fold increase of luminescence, relative to a control experiment, was observed, according to the previously published recommendation (Podgo´rska and W˛egrzyn, 2006).
2.5. Estimation of mutagens’ accumulation in plants from natural environment
3. Results To estimate accumulation of various mutagens in plant tissues in in vitro cultures with the use of the V. harveyi
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bioluminescence mutagenicity assay, plantlets of P. hybrida, N. tabacum and S. brevidens were cultured in the presence (or absence) of different concentrations of mutagenic compounds for 30 days. Usually, after this time plantlets have 4–5 internodes (from 8 to 10 cm height) and did not indicate any morphological disorder irrespective of an applied mutagen and its concentration (data not shown). Extracts obtained from 1.5 g fresh weight of plant tissue were tested for mutagenicity. Mutagenicity of extracts from tissues of all plants cultured in the presence of mutagens (ICR-191, B[a]P and SA) could be detected (Fig. 1). Therefore, accumulation of mutagens in these tissues was assumed, though different chemicals apparently accumulated in different plants to various extends. In P. hybrida, accumulation of ICR-191 present in the culture medium at final concentration of 40 nM was undetectable, while an unambiguous signal was observed at 400 nM. Ten times higher concentration of this mutagen resulted in a decrease of the signal to the level of the control experiment. Such a phenomenon was also observed previously, when chemical mutagens were added directly to V. harveyi A16 (a dim luxE mutant) cultures, and the effect of decreased luminescence was ascribed to toxicity of high concentrations of mutagens (Podgo´rska and W˛egrzyn, 2006). The extracts of S. brevidens, cultured
30
300
Relative luminescence (RLU)
(RLU)
3000
C
sodium azide (nM)
300
Nicotiana tabacum
4000
C
3000
14 12 10 8 6 4 2 0 C
40 400 ICR 191 (nM)
80 800 8000 benzo (a) pyrene (nM) Solanum brevidens
Relative luminescence (RLU)
(RLU)
Relative luminescence
40 400 ICR 191 (nM)
80 800 8000 benzo (a) pyrene(nM)
14 12 10 8 6 4 2 0
Solanum brevidens Relative luminescence (RLU)
30
C
Relative luminescence
(RLU)
C
Solanum brevidens
sodium azide (nM)
4000
14 12 10 8 6 4 2 0
30 300 3000 sodium azide (nM)
14 12 10 8 6 4 2 0 C
40 400 ICR 191 (nM)
14 12 10 8 6 4 2 0
Nicotiana tabacum Relative luminescence
Relative luminescence (RLU)
Nicotiana tabacum 14 12 10 8 6 4 2 0 C
Petunia hybrida
14 12 10 8 6 4 2 0
(RLU)
C
in the presence of ICR-191, were mutagenic to similar extent as those of P. hybrida, while a strong signal could be detected in N. tabacum extracts even when the mutagen occurred at final concentration of 4000 nM (Fig. 1). Quite similar results were obtained when accumulation of B[a]P was tested (Fig. 1). The strongest signal was observed, however, in N. tabacum tissue extracts at the highest tested concentration of this mutagen (8000 nM). P. hybrida extracts were mutagenic at all tested concentrations of B[a]P. Results of experiments with S. brevidens in the presence of B[a]P were similar to those obtained for the same species in the presence of ICR-191 (Fig. 1). A direct dose–response correlation could be observed when accumulation of SA was tested in N. tabacum and S. brevidens (Fig. 1). In fact, these samples gave strong signals in the mutagenicity assay. Accumulation of SA could also be detected in P. hybrida, but the presence of the highest tested concentration of this compound (3000 nM) in the medium resulted in a decrease rather then an increase in the mutagenicity signal. Since results of the experiments described above indicated that accumulation of mutagenic chemicals in plant tissues can be detected using the V. harveyi bioluminescence mutagenicity assay, we aimed to use this assay in testing environmental samples. Extracts of plants’
Petunia hybrida Relative luminescence
(RLU)
Relative luminescence
Petunia hybrida 14 12 10 8 6 4 2 0
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4000
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80 800 8000 benzo (a) pyrene (nM)
Fig. 1. Accumulation of various mutagens in tissues of plants cultured in vitro. Particular compounds were added to the culture medium to indicated concentrations, and cultivations were performed for 30 days. Analogous control experiments (C), with no mutagen added, were performed. Mutagenicity was estimated as described in Materials and methods. Average values from three measurements, with bars representing SD, are shown. Relative luminescence of 1 corresponds to values obtained for particular species cultured with no mutagen added, and is equal to: 0.011370.0015 RLU/cell for P. hybrida, 0.010870.0009 for N. tabacum, and 0.009970.0003 for S. brevidens.
ARTICLE IN PRESS B. Podgo´rska et al. / Ecotoxicology and Environmental Safety 70 (2008) 231–235
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Relative luminescence (RLU)
tissues (the same weight as in case of in vitro experiments) occurring in different water (mostly marine) habitats were tested for their mutagenicity. These tests indicated that mutagenicity could be detected, to various extent, in all tested samples (Fig. 2), suggesting that accumulation of mutagenic compounds in plants occurred in all tested habitats. Nevertheless, significantly stronger signals were detected in extracts of plants collected from habitats expected to contain relatively high concentrations of mutagens (samples 6, 7 and 8 in Fig. 2) than in analogous samples from definitely less polluted regions (samples 1, 2 and 3 in Fig. 2). In these experiments, to be sure that control plants had no contact with mutagens, we used extracts from P. hybrida, N. tabacum and S. brevidens cultured in vitro as without mutagens. Although different plant species were used in these controls than in environmental samples, very similar background signals with no mutagens were obtained for each species (Figs. 1 and 2). This suggests that the source of tested control tissue has little, if any, effect on the background signal detected in the mutagenicity assay with plant tissue extracts.
9 8 7 6 5 4 3 2 1 0 C
Sample C
1
2
3
5 4 sample
Genus/species
6
7
8
9
Source of the sample/habitat
Petunia hybrida, Nicotiana In vitro cultures grown in the MS medium in tabacum and Solanum the absence of mutagens brevidens
1
Enteromorpha sp.
Hel Peninsula, beach (open sea side)
2
Enteromorpha sp.
Gdynia, city boulevard
3
Enteromorpha sp.
Gdynia, Kolibkowski Potok river
4
Enteromorpha sp.
Sopot, mouth of the Kamienny Potok river
5
Ceratophylum sp.
Gdansk (Górki Wschodnie), estuary lake
6
Ceratophylum sp. Myriophyllum spicatum
8
Myriophyllum spicatum
Gdansk (Sobieszewo), estuary of Vistula river – a region polluted with phosphorgypsum compounds
Potamogeton sp.
5. Conclusions
Gdansk (Sobieszewo), estuary of Vistula river
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In this report, we demonstrate that accumulation of mutagenic compounds in plant tissues can be assessed using the recently developed V. harveyi bioluminescence mutagenicity assay. Unambiguous signals indicating mutagenic activities in tested samples have been detected both in samples from experiments with plantlets cultured in in vitro conditions and in samples from plants growing in natural environment. The extent of the accumulation of mutagenic chemicals could be assessed preliminarily. However, due to apparent toxic effects of higher concentrations of mutagens to the V. harveyi A16 tester strain (described by Podgo´rska and W˛egrzyn, 2006, and noted also in this study), conclusions about amounts of accumulated mutagens should not be definitive. On the other hand, the presence of mutagenic compounds in natural habitats in amounts that could cause toxic effects to V. harveyi appears rather unlikely, or at least very rare. It was demonstrated previously that various organic pollutants accumulate differentially in various plant species (Ockenden et al., 1998). This is true also for mutagenic pollutants, as demonstrated in this report (Fig. 1). Therefore, according to the suggestion by Ockenden et al. (1998), intraspecies and not interspecies comparisons should be performed when using plants as biomonitors of pollutant accumulation, including accumulation of mutagenic compounds (as demonstrated in this report). Although it appears that other microbiological mutagenicity assays, like the Ames test or Mutatox, may give more quantitative results in assessments of accumulation of mutagenic pollutants in plants than the V. harveyi bioluminescence mutagenicity test, the advantage of the latter assay is its simplicity and rapidity. While one needs 48 h to obtain results in the Ames test and 16–24 h in Mutatox, optimal results in the V. harveyi bioluminescence mutagenicity test are obtained within 2–4 h from addition of the tested sample (Podgo´rska and W˛egrzyn, 2006). Therefore, this assay can be recommended when rapid (while not necessary very precise) assessment of environmental safety is required.
Gdansk (Górki Wschodnie), estuary of Vistula river
7
4. Discussion
Gdansk (Swibno), fishery port
Fig. 2. Accumulation of mutagens in tissues of plants collected from natural habitats. Mutagenicity of samples was tested as described in Materials and methods. Average values from three measurements, with bars representing SD, are shown in the upper panel (relative luminescence of 1 corresponds to 0.010570.0019 RLU/cell). In the control experiment (column C), the mean luminescence obtained from three plants species, growing in vitro without mutagens, are shown. The lower panel presents characteristics of the samples (all environmental samples were collected from the Baltic Sea region in Northern Poland).
Accumulation of mutagenic pollutants in plant tissues can be assessed using the V. harveyi bioluminescence mutagenicity test. This assay may be especially useful for rapid testing of and/or preliminary studies on environmental genotoxicity (mutagenicity). Funding sources This work was supported by the Ministry of Science and Higher Education (project Grant no. 2 P04G 011 26 to B.P.) and by the Institute of Oceanology of the Polish Academy of Sciences (task Grant no. IV.3. to G.W.).
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Disclaimer Experiments involving humans or animals were not conducted in the studies described in this paper. Since carcinogenic chemicals were used in this work, safety precautions, described in the Safety of Work Code of Practice of the University of Gdan´sk (Regulation no. 26/R/ 04, Part XII) and the Regulation of the Safety of Work of the Institute of Oceanology of Polish Academy of Sciences, were adopted, according to regulations 91/155/EEC and Dz.U. 2002/140/1171. Acknowledgments We thank Dr. Ryszard Markowski for determination of genera and species of plants from natural habitats. References Au, W.W., Oberheitmann, B., Heo, M.Y., Hoffmann, W., Oh, H.Y., 2001. Biomarker monitoring for health risk based on sensitivity to environmental mutagens. Rev. Environ. Health 16, 41–64. Barton, H.A., Cogliano, V.J., Flowers, L., Valcovic, L., Setzer, R.W., Woodruff, T.J., 2005. Assessing susceptibility from early-life exposure to carcinogens. Environ. Health. Perspect. 113, 1125–1133. Czyz˙ , A., Jasiecki, J., Bogdan, A., Szpilewska, H., W˛egrzyn, G., 2000. Genetically modified Vibrio harveyi strains as potential bioindicators of mutagenic pollution of marine environments. Appl. Environ. Microbiol. 66, 599–605. Czyz˙ , A., Plata, K., Wegrzyn, G., 2002a. Induction of light emission by luminescent bacteria treated with UV light and chemical mutagens. J. Appl. Genet. 43, 377–389. Czyz˙ , A., Szpilewska, H., Dutkiewicz, R., Kowalska, W., BiniewskaGodlewska, A., W˛egrzyn, G., 2002b. Comparison of the Ames test and a newly developed assay for detection of mutagenic pollution of marine environments. Mutat. Res. 519, 67–74. Czyz˙ , A., Kowalska, W., W˛egrzyn, G., 2003. Vibrio harveyi mutagenicity assay as a preliminary test for detection of mutagenic pollution of marine water. Bull. Environ. Contamin. Toxicol. 70, 1065–1070. Depledge, M.H., 1998. The ecotoxicological significance of genotoxicity in marine invertebrates. Mutat. Res. 399, 109–122. El-Bayoumy, K., 1992. Environmental carcinogens that may be involved in human breast cancer etiology. Chem. Res. Toxicol. 5, 585–590. Goldman, R., Shields, P.G., 2003. Food mutagens. J. Nutr. 133 (Suppl 3), 965S–973S. Heddle, J.A., Moody, J., Thompson, L.U., Torous, D.K., Trentin, G., 1999. New approaches to antimutagenesis. J. Environ. Pathol. Toxicol. Oncol. 18, 95–101. Jha, A.N., 2004. Genotoxicological studies in aquatic organisms: an overview. Mutat. Res. 552, 1–17.
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Ecotoxicology and Environmental Safety 70 (2008) 231–235 www.elsevier.com/locate/ecoenv
Highlighted article
Rapid detection of mutagens accumulated in plant tissues using a novel Vibrio harveyi mutagenicity assay Beata Podgo´rskaa, Aleksandra Kro´lickab, Ewa Łojkowskab, Grzegorz W˛egrzyna,c, Department of Genetics and Marine Biotechnology, Institute of Oceanology, Polish Academy of Sciences, S´w. Wojciecha 5, 81-347 Gdynia, Poland b Intercollegiate Faculty of Biotechnology, University of Gdan´sk and Medical University of Gdan´sk, K!adki 24, 80-822 Gdan´sk, Poland c Department of Molecular Biology, University of Gdan´sk, K!adki 24, 80-822 Gdan´sk, Poland
a
Received 14 October 2006; received in revised form 11 March 2007; accepted 8 April 2007 Available online 18 May 2007
Abstract Detection of mutagenic pollution in natural environment is difficult as there are thousands of known chemical mutagens, and they have mutagenic effects usually at very low concentrations. Plants are able to accumulate various substances, including mutagens, in their tissues. Here, we demonstrate that rapid detection of mutagenic activities of compounds accumulated in plant tissues is possible using a recently developed microbiological mutagenicity assay, based on induction of bioluminescence of a dim mutant of a marine bacterium Vibrio harveyi. Using this assay, it was possible to detect mutagenic activity in extracts from relatively small amounts of tissues (1.5 g) collected from plants, which were cultured in vitro for 30 days in the presence of nano-molar concentrations of various chemical mutagens. Moreover, contrary to control samples, cultured in vitro without any mutagens, significant mutagenicity was detected in several extracts of plant tissues collected from natural environment. The whole procedure was as short as 4 h or less. r 2007 Elsevier Inc. All rights reserved. Keywords: Mutagenic pollution of environment; Detection of mutagens; Mutagenicity assay; Accumulation of mutagens in plants
1. Introduction Currently, mutagenic pollutants appear in environment mostly as side effects of industrial processes (Heddle et al., 1999; Goldman and Shields, 2003; Vargas, 2003; Jha, 2004). These chemicals occur in various habitats and can induce serious diseases, including cancer, due to their genotoxic (mutagenic) activities (El-Bayoumy, 1992; Depledge, 1998; Au et al., 2001; Martin, 2001; Tornqvist and Ehrenberg, 2001; Barton et al., 2005). The germ line of higher organisms may be also damaged by these compounds, which may lead to fertility problems and to negative genetic changes in future generations (Shelby et al., 1993). Although detection of mutagenic pollutants in the environment appears very important (because of the reasons mentioned above), there are no simple chemical Corresponding author. Department of Molecular Biology, University of Gdan´sk, K"adki 24, 80-822 Gdan´sk, Poland, Fax: +48 58 523 5501. E-mail address: [email protected] (G. W˛egrzyn).
0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.04.004
procedures which might be employed as universal methods for testing the presence of mutagens. This is because there are thousands of known mutagens that occur in natural habitats in very low amounts, while having mutagenic effects at very low concentrations. Therefore, it appears that for preliminary and rapid detection of mutagenic activities in environmental samples, biological assays are more useful than chemical analyses. Among them, microbiological tests are commonly used as they are relatively simple and rapid (W˛egrzyn and Czyz˙ , 2003). Nevertheless, even using such tests, to obtain results of measurements one must usually wait as long as two days from sample withdrawal, like in the commonly used Ames test (Mortelmans and Zeiger, 2000) or its analogue, based on the use of bacteria which are more likely to survive in samples of natural waters (Czyz˙ et al., 2000, 2002b, 2003; Podgo´rska et al., 2005). More rapid tests, based on bacterial bioluminescence have been developed, including commercially available Mutatox (in which a strain of Vibrio fischeri is used; Ulitzur et al., 1980, Ulitzur and Weiser, 1981), which requires
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16–24 h to obtain results. Recently, a novel bioluminescence mutagenicity assay has been reported, which allows to obtain results in 2–4 h from sample withdrawal (Podgo´rska and W˛egrzyn, 2006). In this assay, a dim luxE mutant of Vibrio harveyi is employed, and cultures of this strain (named A16) are induced for light emission upon contact with mutagenic compounds. The luxE gene codes for the synthetase of the fatty acid reductase complex, a holoenzyme (containing also reductase and transferase) responsible for production of a long-chain aliphatic aldehyde, which is a substrate in the oxidation reaction performed by luciferase (for a review see Meighen, 1988). Since this luciferase-mediated reaction results in light emission, dysfunction of the luxE gene causes impairment of V. harveyi bioluminescence. However, restoration of the luxE function due to reverse mutation(s) results in restoration of light emission. Therefore, the A16 strain responds to contact with various mutagens by increasing the level of luminescence (Podgo´rska and W˛egrzyn, 2006). This may arise from induced reversion to the wild-type luxE allele or a pseudoreversion that allows proper functions of the gene product and restoration of luminescence (Podgo´rska and W˛egrzyn, 2006). Light emission by the A16 strain after its incubation with mutagenic compounds may be further enhanced, as stimulation of this process by both physical and chemical mutagens has been reported (Czyz˙ et al., 2002a). Although mutagenic compounds occur in the environment usually at very low concentrations, they can accumulate in various organisms, which makes them even more dangerous for humans. It appears that plants are especially capable of accumulation of environmental xenobiotics, including mutagens and promutagens (Ockenden et al., 1998; Stepanova et al., 1999). Moreover, it was demonstrated that some promutagens can be activated in plant tissues (Plewa, 1991; Plewa and Wagner, 1993), which may be considered as an additional hazardous factor. The aim of this work was to test an applicability of the V. harveyi bioluminesce mutagenicity assay in assessment of accumulation of mutagenic compounds in tissues of plants, both cultured in vitro and occurring in natural environment.
(plant tissue extract) and 5 mL of 4% S9 mix (consisting of rat liver microsomal enzymes and cofactors, as described previously by Maron and Ames, 1983) were added to 4.5 mL of the bacterial culture at absorbance (A575) ¼ 0.1, and incubation was continued for 3 h. Before addition of the tested sample, and 3 h after its addition, 1 mL of the bacterial culture was withdrawn and its luminescene was measured using a Sirius luminometer (Berthold), and expressed as relative light units (RLU). At the same times, A575 of cultures was measured to estimate number of bacterial cells (a correlation between cell number, determined by plating on BOSS plates, and absorbance of the culture was established according to Podgo´rska and W˛egrzyn (2006); A575 ¼ 0.1 corresponded to 3 107 colony forming units per 1 mL). Results were calculated as relative luminescence (RLU) per cell or per absorbance unit.
2. Materials and methods
Plants were collected from natural habitats, and species or genera were determined at Department of Plant Taxonomy and Nature Protection, University of Gdan´sk, Poland. A 1.5 g of plant tissue was homogenized and filtered as described in the preceding paragraph, and 0.5 mL of such an extract was used for the mutagenicity assay (performed as described above). Each measurement was repeated three times, and the mean value7SD was calculated. The result was considered positive (indicating mutagen accumulation) when at least two-fold increase of luminescence, relative to a control experiment, was observed, according to the previously published recommendation (Podgo´rska and W˛egrzyn, 2006).
2.1. Chemical mutagens Following mutagens (purchased from Sigma) were used: 2-methoxy-6chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine 2HCl (ICR-191), benzo(a)pyrene (B[a]P), and sodium azide (SA).
2.2. V. harveyi bioluminescence mutagenicity assay The assay was performed according to Podgo´rska and W˛egrzyn (2006), with the V. harveyi A16 strain (a dim luxE mutant) as an indicator strain. Bacteria were grown in the liquid BOSS nutrient medium (bacto-peptone, 1%; beef extract, 0.3%; glycerol, 0.011 mol L1; NaCl, 0.51 mol L1), described by Klein et al. (1995), at 30 1C. 0.5 mL of the tested sample
2.3. Plant cultures The plantlets of Petunia hybrida, Nicotiana tabacum and Solanum brevidens were grown on the Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 3% sucrose and 0.75% agar. Cultures were grown at a temperature of 20–22 1C under white fluorescent light with a 16 h photoperiod (white cool fluorescent light, Philips, TLD 58W/84o, 20 mmol m2 s1) and kept under 16/8 h light/dark cycles. The pH of the medium was adjusted to 5.8 prior to autoclaving. A voucher specimen of the plant is deposited at the Department of Plant Protection and Biotechnology, University of Gdan´sk and Medical University of Gdan´sk, Poland.
2.4. Estimation of mutagens’ accumulation in in vitro cultured plants Different concentrations of applied mutagens (ICR-191: 40, 400, 4000 nM; B[a]P: 80, 800, 8000 nM; and SA: 30, 300, 3000 nM) were tested by adding them to the MS medium after autoclaving (in control experiments no mutagen was added). Explants containing apical and one secondary meristem were cultured in mutagen-containing or mutagenfree (control experiments) MS medium for 30 days. After sample withdrawal, 1.5 g of plant tissue were homogenized (using Heidolph homogenizator) with 3 mL of artificial marine water (prepared according to MacLeod et al., 1954), and the extract was filtered using Whatmann 3 mm paper filters. A 0.5 mL of such an extract was used for the V. harveyi bioluminescence mutagenicity assay (performed as described above). Each experiment was repeated three times, and the mean value7SD was calculated. The result was considered positive (indicating mutagen accumulation) when at least two-fold increase of luminescence, relative to a control experiment, was observed, according to the previously published recommendation (Podgo´rska and W˛egrzyn, 2006).
2.5. Estimation of mutagens’ accumulation in plants from natural environment
3. Results To estimate accumulation of various mutagens in plant tissues in in vitro cultures with the use of the V. harveyi
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bioluminescence mutagenicity assay, plantlets of P. hybrida, N. tabacum and S. brevidens were cultured in the presence (or absence) of different concentrations of mutagenic compounds for 30 days. Usually, after this time plantlets have 4–5 internodes (from 8 to 10 cm height) and did not indicate any morphological disorder irrespective of an applied mutagen and its concentration (data not shown). Extracts obtained from 1.5 g fresh weight of plant tissue were tested for mutagenicity. Mutagenicity of extracts from tissues of all plants cultured in the presence of mutagens (ICR-191, B[a]P and SA) could be detected (Fig. 1). Therefore, accumulation of mutagens in these tissues was assumed, though different chemicals apparently accumulated in different plants to various extends. In P. hybrida, accumulation of ICR-191 present in the culture medium at final concentration of 40 nM was undetectable, while an unambiguous signal was observed at 400 nM. Ten times higher concentration of this mutagen resulted in a decrease of the signal to the level of the control experiment. Such a phenomenon was also observed previously, when chemical mutagens were added directly to V. harveyi A16 (a dim luxE mutant) cultures, and the effect of decreased luminescence was ascribed to toxicity of high concentrations of mutagens (Podgo´rska and W˛egrzyn, 2006). The extracts of S. brevidens, cultured
30
300
Relative luminescence (RLU)
(RLU)
3000
C
sodium azide (nM)
300
Nicotiana tabacum
4000
C
3000
14 12 10 8 6 4 2 0 C
40 400 ICR 191 (nM)
80 800 8000 benzo (a) pyrene (nM) Solanum brevidens
Relative luminescence (RLU)
(RLU)
Relative luminescence
40 400 ICR 191 (nM)
80 800 8000 benzo (a) pyrene(nM)
14 12 10 8 6 4 2 0
Solanum brevidens Relative luminescence (RLU)
30
C
Relative luminescence
(RLU)
C
Solanum brevidens
sodium azide (nM)
4000
14 12 10 8 6 4 2 0
30 300 3000 sodium azide (nM)
14 12 10 8 6 4 2 0 C
40 400 ICR 191 (nM)
14 12 10 8 6 4 2 0
Nicotiana tabacum Relative luminescence
Relative luminescence (RLU)
Nicotiana tabacum 14 12 10 8 6 4 2 0 C
Petunia hybrida
14 12 10 8 6 4 2 0
(RLU)
C
in the presence of ICR-191, were mutagenic to similar extent as those of P. hybrida, while a strong signal could be detected in N. tabacum extracts even when the mutagen occurred at final concentration of 4000 nM (Fig. 1). Quite similar results were obtained when accumulation of B[a]P was tested (Fig. 1). The strongest signal was observed, however, in N. tabacum tissue extracts at the highest tested concentration of this mutagen (8000 nM). P. hybrida extracts were mutagenic at all tested concentrations of B[a]P. Results of experiments with S. brevidens in the presence of B[a]P were similar to those obtained for the same species in the presence of ICR-191 (Fig. 1). A direct dose–response correlation could be observed when accumulation of SA was tested in N. tabacum and S. brevidens (Fig. 1). In fact, these samples gave strong signals in the mutagenicity assay. Accumulation of SA could also be detected in P. hybrida, but the presence of the highest tested concentration of this compound (3000 nM) in the medium resulted in a decrease rather then an increase in the mutagenicity signal. Since results of the experiments described above indicated that accumulation of mutagenic chemicals in plant tissues can be detected using the V. harveyi bioluminescence mutagenicity assay, we aimed to use this assay in testing environmental samples. Extracts of plants’
Petunia hybrida Relative luminescence
(RLU)
Relative luminescence
Petunia hybrida 14 12 10 8 6 4 2 0
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4000
14 12 10 8 6 4 2 0 C
80 800 8000 benzo (a) pyrene (nM)
Fig. 1. Accumulation of various mutagens in tissues of plants cultured in vitro. Particular compounds were added to the culture medium to indicated concentrations, and cultivations were performed for 30 days. Analogous control experiments (C), with no mutagen added, were performed. Mutagenicity was estimated as described in Materials and methods. Average values from three measurements, with bars representing SD, are shown. Relative luminescence of 1 corresponds to values obtained for particular species cultured with no mutagen added, and is equal to: 0.011370.0015 RLU/cell for P. hybrida, 0.010870.0009 for N. tabacum, and 0.009970.0003 for S. brevidens.
ARTICLE IN PRESS B. Podgo´rska et al. / Ecotoxicology and Environmental Safety 70 (2008) 231–235
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Relative luminescence (RLU)
tissues (the same weight as in case of in vitro experiments) occurring in different water (mostly marine) habitats were tested for their mutagenicity. These tests indicated that mutagenicity could be detected, to various extent, in all tested samples (Fig. 2), suggesting that accumulation of mutagenic compounds in plants occurred in all tested habitats. Nevertheless, significantly stronger signals were detected in extracts of plants collected from habitats expected to contain relatively high concentrations of mutagens (samples 6, 7 and 8 in Fig. 2) than in analogous samples from definitely less polluted regions (samples 1, 2 and 3 in Fig. 2). In these experiments, to be sure that control plants had no contact with mutagens, we used extracts from P. hybrida, N. tabacum and S. brevidens cultured in vitro as without mutagens. Although different plant species were used in these controls than in environmental samples, very similar background signals with no mutagens were obtained for each species (Figs. 1 and 2). This suggests that the source of tested control tissue has little, if any, effect on the background signal detected in the mutagenicity assay with plant tissue extracts.
9 8 7 6 5 4 3 2 1 0 C
Sample C
1
2
3
5 4 sample
Genus/species
6
7
8
9
Source of the sample/habitat
Petunia hybrida, Nicotiana In vitro cultures grown in the MS medium in tabacum and Solanum the absence of mutagens brevidens
1
Enteromorpha sp.
Hel Peninsula, beach (open sea side)
2
Enteromorpha sp.
Gdynia, city boulevard
3
Enteromorpha sp.
Gdynia, Kolibkowski Potok river
4
Enteromorpha sp.
Sopot, mouth of the Kamienny Potok river
5
Ceratophylum sp.
Gdansk (Górki Wschodnie), estuary lake
6
Ceratophylum sp. Myriophyllum spicatum
8
Myriophyllum spicatum
Gdansk (Sobieszewo), estuary of Vistula river – a region polluted with phosphorgypsum compounds
Potamogeton sp.
5. Conclusions
Gdansk (Sobieszewo), estuary of Vistula river
9
In this report, we demonstrate that accumulation of mutagenic compounds in plant tissues can be assessed using the recently developed V. harveyi bioluminescence mutagenicity assay. Unambiguous signals indicating mutagenic activities in tested samples have been detected both in samples from experiments with plantlets cultured in in vitro conditions and in samples from plants growing in natural environment. The extent of the accumulation of mutagenic chemicals could be assessed preliminarily. However, due to apparent toxic effects of higher concentrations of mutagens to the V. harveyi A16 tester strain (described by Podgo´rska and W˛egrzyn, 2006, and noted also in this study), conclusions about amounts of accumulated mutagens should not be definitive. On the other hand, the presence of mutagenic compounds in natural habitats in amounts that could cause toxic effects to V. harveyi appears rather unlikely, or at least very rare. It was demonstrated previously that various organic pollutants accumulate differentially in various plant species (Ockenden et al., 1998). This is true also for mutagenic pollutants, as demonstrated in this report (Fig. 1). Therefore, according to the suggestion by Ockenden et al. (1998), intraspecies and not interspecies comparisons should be performed when using plants as biomonitors of pollutant accumulation, including accumulation of mutagenic compounds (as demonstrated in this report). Although it appears that other microbiological mutagenicity assays, like the Ames test or Mutatox, may give more quantitative results in assessments of accumulation of mutagenic pollutants in plants than the V. harveyi bioluminescence mutagenicity test, the advantage of the latter assay is its simplicity and rapidity. While one needs 48 h to obtain results in the Ames test and 16–24 h in Mutatox, optimal results in the V. harveyi bioluminescence mutagenicity test are obtained within 2–4 h from addition of the tested sample (Podgo´rska and W˛egrzyn, 2006). Therefore, this assay can be recommended when rapid (while not necessary very precise) assessment of environmental safety is required.
Gdansk (Górki Wschodnie), estuary of Vistula river
7
4. Discussion
Gdansk (Swibno), fishery port
Fig. 2. Accumulation of mutagens in tissues of plants collected from natural habitats. Mutagenicity of samples was tested as described in Materials and methods. Average values from three measurements, with bars representing SD, are shown in the upper panel (relative luminescence of 1 corresponds to 0.010570.0019 RLU/cell). In the control experiment (column C), the mean luminescence obtained from three plants species, growing in vitro without mutagens, are shown. The lower panel presents characteristics of the samples (all environmental samples were collected from the Baltic Sea region in Northern Poland).
Accumulation of mutagenic pollutants in plant tissues can be assessed using the V. harveyi bioluminescence mutagenicity test. This assay may be especially useful for rapid testing of and/or preliminary studies on environmental genotoxicity (mutagenicity). Funding sources This work was supported by the Ministry of Science and Higher Education (project Grant no. 2 P04G 011 26 to B.P.) and by the Institute of Oceanology of the Polish Academy of Sciences (task Grant no. IV.3. to G.W.).
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