Environ. Sci. Technol. 2008, 42, 5746–5751
Hydroxy-PCBs, Methoxy-PCBs and Hydroxy-Methoxy-PCBs: Metabolites of Polychlorinated Biphenyls Formed In Vitro by Tobacco Cells J A N R E Z E K , †,‡ T O M A S M A C E K , * ,† MARTINA MACKOVA,‡ JAN TRISKA,§ AND KAMILA RUZICKOVA§ Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo namesti 2, 166 10 Prague 6, Czech Republic, Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6, Czech Republic, Department of Analytical Chemistry, Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic.
Received February 13, 2008. Revised manuscript received May 13, 2008. Accepted May 15, 2008.
While the metabolism of polychlorinated biphenyls (PCBs) in plant cells is a rarely studied field, hydroxy-PCBs have been detected in several studies involving the use of various plant species. The ability of the tobacco (Nicotiana tabacum) callus culture WSC-38 to metabolize six dichlorobiphenyls under aseptic conditions was studied, and the resulting PCB metabolites were analyzed. WSC-38 cultures were cultivated with individual dichlorinated PCB congeners. The metabolites were identified based on mass spectra characteristics after gas chromatography separation. In addition, metabolites of PCB 9 (2,5dichlorobiphenyl) were identified by comparing their retention characteristics with the available standards. In most cases at least two hydroxy-PCBs were produced from each parent PCB. Methoxy-PCBs and hydroxy-methoxy-PCBs were other groups of metabolites produced. To the best of our knowledge, ours is the first report to determine the presence of methoxy- and hydroxy-methoxy-metabolites of PCBs in plants. The role of the O-methyltransferases (OMTs) in the methylation of hydroxy-PCBs is discussed. As methoxy-metabolites of acetophenone were found among our samples, we posit that the OMTs responsible for the methylation of these compounds are also involved in the metabolism of PCBs in cultures of WSC38.
Introduction Although their production was banned in the 1970s (a ban which took effect in the former Czechoslovakia in 1984), polychlorinated biphenyls (PCBs) remain the subject of great environmental concern. There are 209 PCB congeners, all of which are unique in terms of the number and position of * Corresponding author phone: +420-220-183-340; fax: +420-220183-582; e-mail:
[email protected]. † Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic. ‡ Institute of Chemical Technology Prague. § Department of Analytical Chemistry, Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic. 5746
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chlorine atoms attached to the biphenyl molecule. Historically, PCBs were commercially produced as complex mixtures of different congeners (e.g., Aroclor, Clophen, Delor, Kanechlor), and, due to their excellent physical and chemical properties (nonflammability, high stability, lipophilicity, resistance to degradation), were used throughout the world in a wide range of industrial applications (e.g., as coolants for industrial transformers, hydraulic fluids, fire retardants, etc). Conversely, these properties also mark PCBs as recalcitrant compounds that accumulate in the environment. Such lipophilic compounds are able to spread over large distances (1), affecting the whole environment as they do so. In particular, their ability to enter the food chain, and accumulate in fat tissues, means that their most significant impact is on end consumers, including human beings (2, 3). The toxicity of PCBs has been well documented in recent decades, and it now seems clear that the toxic properties they possess may have a number of harmful effects. Apart from possibly possessing carcinogenic properties (4), PCBs are known to act as endocrine disrupters (5) and as thyroid hormone analogues (6). Of the approximately 1.3 million metric tons of PCBs that were produced worldwide (7), it has been estimated that about 30% were released into the environment (8, 9), making the need for techniques capable of cleaning polluted sites very clear. Due to the high cost of, as well as public opposition to, the physical methods traditionally used to remove PCBs from contaminated soil, bioremediation appears to be a promising technology (10). Apart from traditional bioremediation technologies that use natural bacterial or fungal degraders, another technique for PCB removal involves the use of plants in phytoremediation processes (11, 12). The potential of this technique makes it important to study the plant metabolites of PCBs. Several studies have shown that PCB metabolism in plants exhibits apparent similarities to the same mechanism in animals and humans (“green liver” model). The plant detoxification of lipophilic compounds, including PCBs, consists of three phases. Phase I (activation) involves the oxidation or hydroxylation of the toxic compound. Phase II (conjugation) consists of the covalent binding of the formed metabolite to endogenous hydrophilic molecules, such as glucose, glutathione, or malonate; this step increases the hydrophilic character of the parent compounds. Then, in phase III (compartmentation), inactive conjugated watersoluble xenobiotics are relocated from cytosol into either vacuole or apoplast (13). Moza et al. (14, 15) described the production of monohydroxylated metabolites (chlorobiphenylols) of PCB 4 (2,2′dichlorobiphenyl) in carrot and sugar beet, and later, also identified mono- and dihydroxy-metabolites of PCB 31 (2,4′,5trichlorobiphenyl) in carrot. Fletcher et al. (16), studying the metabolism of 2-chlorobiphenyl with a tissue culture of Paul’s Scarlet Rose, showed that plant cells are able to metabolize PCBs under axenic conditions. Butler et al. (17) partially identified their metabolites as monohydroxylated PCBs that were subsequently glycosylated. Tissue cultures, thus, became popular for the study of plant metabolism because they result in faster biomass development and are easier to maintain under sterile cultivation conditions. More importantly, tissue cultures enable metabolites to be unambiguously assigned to plant cells with the certainty that contaminating microorganisms play no role in plant metabolism. More recently, Wilken et al. (18) reported hydroxylated metabolites of PCB 1 (2-chlorobiphenyl) and PCB 52 (2,2′,5,5′tetrachlorobiphenyl) in soybean and wheat culture, respec10.1021/es800445h CCC: $40.75
2008 American Chemical Society
Published on Web 06/25/2008
tively. Metabolites of PCB 77 (3,3′,4,4′-tetrachlorobiphenyl), hydroxylated in positions 2-, 5-, and 6-, have been identified in cultures of rose, tomato, sunflower, and lettuce (19). The commercial mixture Delor 103, which consists of 59 PCB congeners with an average of 3 chlorine atoms per biphenyl, has also been used to study PCB metabolism (20–23). Approximately 40 in vitro tissue cultures of different plant species were tested, with a hairy root culture of black nightshade (Solanum nigrum), SNC-9O, shown to have the highest ability to degrade PCBs. Using the same culture, Kucerova et al. (24) identified mono- and dihydroxychlorobiphenyls as metabolites of some mono- and dichlorobiphenyls. Monohydroxylated metabolites of monochlorinated PCBs were later also found in cultures of Armoracia rusticana, N. tabacum, S. nigrum, and Medicago sativa (25). In our previous study (26) with the promising culture S. nigrum cultivated with 25 di-, tri-, tetra-, and pentachlorobiphenyl congeners only monohydroxylated PCBs were detected as biodegradation products. The identification of PCB metabolites in plants is particularly important in terms of the environmental safety of both the bioremediation process itself, and the independent formation of such metabolites in nature. Compounds such as PCBs are globally spread; their effects not limited to their original site of usage, production, and contamination. The widespread occurrence of PCBs in the environment, together with the ubiquitous presence of plants, inevitably leads to their interaction. The use of plants and plant organs cultivated aseptically makes it possible to distinguish the remediation steps that can be performed by plant cells alone, without the involvement of microorganisms. Our present study is focused on determining the phase I detoxification products of dichlorinated PCB congeners formed in vitro by the tobacco callus culture, WSC-38. We chose tobacco because field experiments have demonstrated its high potential to accumulate PCBs in its tissues (27).
Materials and Methods Plant Cultures and Cultivation Conditions. WSC-38 callus cultures of tobacco (Nicotiana tabacum cv. Wisconsin 38) were used to identify metabolites of PCB biodegradation. Cultivation was performed in the media described by Linsmaier and Skoog (28), to which we added 0.1075 mg/L of kinetin and 0.225 mg/L of dichlorophenoxyacetic acid. In vitro callus cultures were cultivated in 100 mL of liquid media in 250 mL Erlenmeyer flasks. Aluminum-lined caps were used to minimize evaporation. The metabolites were analyzed in both the media and biomass. Cultivation started with 5 g of fresh weight tissue culture, in the dark, at 24 °C, with shaking at 110 RPM. After 7 days precultivation, PCB 4 (2,2′dichlorobiphenyl), PCB 5 (2,3-dichlorobiphenyl), PCB 6 (2,3′dichlorobiphenyl), PCB 7 (2,4-dichlorobiphenyl), PCB 8 (2,4′dichlorobiphenyl) and PCB 9 (2,5-dichlorobiphenyl) congeners (manufacturer: Dr. Ehrenstorfer GmbH, Augsburg, Germany) were added to reach a final PCB concentration of 1 mg/L media in all cases. Cultivation continued for 14 days under the same conditions. Two controls were cultivated in the same way, but without the addition of a PCB. However, we added the same amount of methanol (as used to dilute the PCB congeners in the real samples) to the second control. Metabolite Extraction and Derivatization. At the end of the cultivation period, the media and biomass were separated by filtration. Forty grams of biomass was homogenized in liquid nitrogen, and liquid-liquid extracted with dichloromethane (2 × 30 mL). Fifty mL of medium was extracted with dichloromethane (2 × 30 mL). The extracts were dried with natrium sulfate (anhydrous), filtered, evaporated to dryness in a vacuum evaporator (40 °C, 150 RPM, 7 Torr), and redisolved in 1 mL of methanol. Acetylation was then performed using a slightly modified version of the procedure
TABLE 1. Dichlorobiphenylols Formed from PCBs by Nicotiana tabacum (WSC-38 Culture) hydroxy-PCB metabolites in biomass PCB
Cl position
4
2,2′
5
2,3
6
2,3′
7 8
2,4 2,4′
9
2,5 a
retention time (min:s) 29:40 30:46a 31:56 33:35a 31:23 31:40 32:23a 32:37 31:45 32:46a 31:51 32:32a
identification dichlorobiphenylol dichlorobiphenylol dichlorobiphenylol dichlorobiphenylol dichlorobiphenylol dichlorobiphenylol dichlorobiphenylol dichlorobiphenylol dichlorobiphenylol dichlorobiphenylol 2′,5′-dichloro-3-biphenylol 2′,5′-dichloro-4-biphenylol
Main metabolite.
described by Triska et al. (29). Methanolic extracts were dissolved in 50 mL of 0.1 M K2CO3 in a separation funnel. Then 1 mL of acetic anhydride was added and the mixture was shaken until no more gas evolved (typically 5-10 min). The reaction mixture was extracted with hexane (2 × 6 mL) and evaporated to dryness using a vacuum evaporator. The postevaporation residues were dissolved in 100 µL of hexane for GC-MS analysis. The available biphenylol standards were also acetylated. Analysis of PCB Metabolites. A Finnigan Mat GCQTM (gas chromatograph equipped with a quadrupole ion trap mass spectrometer) was used to perform the analysis. One µL of extract plus 1 µL of hexane were injected splitless into a GCMS system (Zebron Capillary GC Column ZB-5: 30 m; internal diameter 0.25 mm; 5% phenylpolysiloxane stationary phase; thickness 0.1 µm). Helium was used as the carrier gas at a flow rate of 0.4 m/s. The injector temperature was 250 °C. The initial temperature of 60 °C was linearly increased until it reached 283 °C (4 °C/min), at which point a constant temperature of 283 °C was maintained for 4.25 min. MS detection was performed in “full scan” mode: EI ionization potential 70 eV; ion source at 175 °C; transfer line at 275 °C; one spectra measured every second. Basic identification was performed by comparing the mass spectra of the metabolites with the NIST library (National Institute of Standards and Technology, Gaithersburg, MD). Standards of 2′,5′-dichloro2-biphenylol, 2′,5′-dichloro-3-biphenylol and 2′,5′-dichloro4-biphenylol (J.T.Baker B.V., Deventer, The Netherlands), were available and, based on a comparison of retention characteristics, used to identify metabolites of PCB 9.
Results and Discussion Hydroxy-PCBs. Based on our earlier findings of PCB metabolites in an SNC-9O black nightshade (S. nigrum) hairy root tissue culture, we expected to find hydroxylated PCB metabolites in the tobacco callus culture WSC-38, because the ability of tobacco plants to accumulate PCBs from contaminated soil in their tissues has already been demonstrated (27, 30). WSC-38 cultures, each cultivated with one of six different dichlorinated PCB congeners, yielded the expected monohydroxylated PCB metabolites. Identification, based on comparing their mass spectra to the NIST library, provided basic information about the metabolites formed. As can be seen from Table 1, all of the tested dichlorinated PCB congeners yielded at least one monohydroxylated metabolite, and in most cases two were detected. The mass spectra obtained for these metabolites revealed a visible molecular ion [M+] at m/z 280. The typical fragmentation cluster surrounding ion at m/z 238 [M+-CH2CO] was also VOL. 42, NO. 15, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Mass spectrum of methoxy-PCB 4 found in biomass of WSC-38 cultivated with PCB 4.
TABLE 2. Methoxy-Metabolites Formed from PCBs by Nicotiana tabacum (WSC-38 Culture) methoxy-PCB metabolites in biomass Cl PCB position 4 5 6
2,2′ 2,3 2,3′
7 8 9
2,4 2,4′ 2,5
retention time (min:s) 28:04 29:34 29:29 29:52 29:07 not detected 28:29
identification ortho- or meta-methoxy-PCB 4 ortho- or meta-methoxy-PCB 5 para-methoxy-PCB 6 ortho- or meta-methoxy-PCB 6 para-methoxy-PCB 7 ortho- or meta-methoxy-PCB 9
present in all spectra. In addition, fragments of m/z 168 [M+CH2CO-2Cl] and m/z 139 [M+-CH2CO-2Cl-HCO] were also detected. Due to limited availability, we were unable (with the exception of PCB 9) to compare the retention times of the metabolites formed with those of the standards. We did, however, compare their retention times to those of the metabolites detected in the SNC-9O cultures used in our earlier experiment. In general, the number of metabolites identified in WSC-38 cells was lower than the number detected in the SNC-9O cultures (26). All metabolites determined in the WSC-38 culture were also found in the SNC-9O samples (with corresponding retention times). Some additional metabolites (e.g., 2′,5′-dichloro-2-biphenylol for PCB 9) were detected in the SNC-9O culture. As free hydroxy-PCBs were found neither in the cultivation media extracts nor in the PCB-untreated control extracts, we conclude that they were maintained in the plant cells and not exuded in their unbound form from living cells to the cultivation media. Our findings confirm that phase I in the plant metabolism of PCBs is the hydroxylation of the parent compounds. Various authors have shown that this phase can largely be attributed to the work of the cytochrome P450 enzyme system (31). Indeed, this system also seems to be responsible for the production of hydroxy-PCBs as metabolites in yeasts (32), animals (33), and humans (34). The uptake of PCBs by plants, together with their metabolism in plants, depends on the plant species, tissue culture and PCB congener used. Although, due to the unavailability of 14C-labeled PCBs, the mass balance of PCB uptake and metabolism was not estimated in our work, differences among plant species can be demonstrated by reference to examples from the literature. For instance, previous studies (14, 15) have demonstrated that carrot has 5748
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a higher uptake and metabolism of PCB 4 (2,2′-dichlorobiphenyl) than sugar beet; the concentration of unchanged PCB 4 in carrot root being 0.240 ppm compared to less than 0.001 ppm in sugar beet root. Similarly, the concentration of PCB metabolites in carrot root was 0.012 ppm (5% of applied radioactivity) and just 0.004 ppm in sugar beet root. The uptake of radioactivity from PCB 31 (2,4′,5-trichlorobiphenyl-14C) reached 3.1% in carrot root and 0.2% in sugar beet root, with metabolites only being detected in the carrot. With PCB 100 (2,2′,4,4′,6-pentachlorobiphenyl-14C), the uptake of radioactivity was higher in carrot (1.4%) than in sugar beet (0.1%), but no metabolites were detected in either species. Later, Fletcher et al. (35) applied PCB 47 (2,2′,4,4′tetrachlorobiphenyl-14C) to a rose suspension culture. After 8 days of cultivation, the authors recovered 5.5% of radioactivity in the form of metabolites of PCB 47; 90% of the total radioactivity applied being recovered. Furthermore, in an experiment in which 86% of PCB-1 (2-chlorobiphenyl-14C) and 3.4% of PCB 47 (2,2′,4,4′-tetrachlorobiphenyl-14C) were metabolized by rose culture within 4 days of cultivation, Groeger and Fletcher (36) showed that the rate of PCB metabolism decreases as the chlorination level increases. In this experiment, PCB 153 (2,2′,4,4′,5,5′-hexachlorobiphenyl14C) remained intact. Ryslava et al. (27) demonstrated that the concentration of PCBs in tissues of tobacco plant (0.244 mg PCB/kg fresh biomass) cultivated in PCB-contaminated soil was 5-9 times higher compared to that in black nightshade (S. nigrum) and alfalfa (M. sativa). After 6 months, the residual concentration of PCBs in the same soil had declined to 4.3 mg PCB/kg of dry soil (66% of the PCB concentration in unplanted control soil). In spite of their generally low uptake and metabolism of PCBs, plants possess various advantageous properties that aid phytoremediation. In addition to phytoextraction alone, certain plants also support degrading microbes in the soil in a variety of ways, which include the release of root exudates that serve as extra nutrients for degraders and the development of rhizosphere that enhances soil aeration. It is likely that PCB-contaminated soils will need to be repeatedly cultivated with the most beneficial species. With this approach, we anticipate that, even taking into account the low metabolic rates of PCBs in plant tissues, after several years of such cultivation the total amount of catechol metabolites in the plant biomass will reach significant levels. As the toxicity of hydroxylated PCBs is known to be higher than that of their parent PCBs (37), PCB metabolism by plants could lead to catechol metabolites of PCBs entering food chains, thereby creating a new environmental problem. Consequently, with respect to the developing phytoremediation technologies, special attention must be paid to the
FIGURE 2. Mass Spectrum of Hydroxy-Methoxy-PCB 4 Found in Biomass of WSC-38 Cultivated with PCB 4.
TABLE 3. Hydroxy-Methoxy-Metabolites Formed from PCBs by Nicotiana tabacum (WSC-38 culture) hydroxy-methoxy-PCB metabolites in biomass PCB
Cl position
4
2,2′
5 6
2,3 2,3′
7 8
2,4 2,4′
9
2,5
retention time (min:s) 33:48 34:40 37:17 35:35 36:20 36:22 35:58 36:46 36:10
identification hydroxy-methoxy-PCB hydroxy-methoxy-PCB hydroxy-methoxy-PCB hydroxy-methoxy-PCB hydroxy-methoxy-PCB hydroxy-methoxy-PCB hydroxy-methoxy-PCB hydroxy-methoxy-PCB hydroxy-methoxy-PCB
4 4 5 6 6 7 8 8 9
establishment of procedures that ensure the careful handling of plant biomass cultivated in contaminated soil to avoid the consequent dissemination of toxic compounds. Without such procedures, contamination by hydroxy-PCBs, recently monitored in Canada (38), could become as serious a problem as PCB contamination itself. Methoxy-PCBs. While checking the mass spectra of PCB metabolites, in one sample we observed a typical chlorination cluster surrounding the ion at m/z 252. This fragmentation pattern did not correspond to the typical pattern for hydroxylated PCBs, suggesting that this molecule could be a methoxy-metabolite of the parent PCB. Therefore, we compared the mass spectra obtained for this metabolite with the MS results obtained by other authors (39–43). In all cases, the spectra included a molecular ion [M+] at m/z 252 surrounded by a cluster of chlorinated compounds, as well as ions at m/z 217 [M+-Cl], m/z 209 [M+-CH3CO], m/z 202 [M+-CH3Cl], m/z 173 [M+-79], and m/z 139 [M+-CH3COCl2], thereby confirming that the compound was indeed a methoxy-derivative of its parent PCB. The spectrum of methoxy-PCB 4 is shown in Figure 1. With the exception of PCB 8, methoxy-PCBs were also found in the extracts of biomass cultivated with the other PCBs (see Table 2). As shown in previous studies (39, 40), in each case it is likely that these metabolites were substituted by a methoxy group in either the meta or ortho positions. Both methoxy-PCB 6 and methoxy-PCB 7 showed the addition, at m/z 237 [M+CH3], of another ion typical for methoxylated PCBs, thereby suggesting that both compounds were para-substituted by a methoxy group. These compounds were found neither in the cultivation media samples nor in the PCB-untreated control extracts. Although previously identified in fungi (40), rats (44), and rabbits (45), as far as we are aware, the presence
of PCB methoxy-metabolites in plants has not previously been reported in the literature. Hydroxy-Methoxy-PCBs. Typical chlorination fragmentation patterns were also found in the mass spectra of other compounds. Figure 2 shows the mass spectrum of these compounds, in which a molecular ion [M+] appears at m/z 268 surrounded by the cluster typical for chlorinated aromatic compounds, together with ions at m/z 253 [M+-CH3] and m/z 225 [M+-COCH3]. Present in each extract of biomass cultivated with a PCB, Kamei et al. (40) have shown that these compounds are almost certainly hydroxy-methoxyPCBs (see Table 3). These hydroxy-methoxy-PCBs were found neither in the cultivation media extracts nor in the PCBuntreated control extracts. To the best of our knowledge, neither hydroxy-methoxy- nor methoxy-PCBs have previously been reported in plant cells as intermediates of PCB transformation. The Methylation of Hydroxy-PCBs. Our findings raise an important question. Namely, are specialized enzymes induced to perform the methylation of hydroxy-PCBs, or do hydroxylated PCBs, as alternative substrates, enter a biosynthetic pathway in which the hydroxy-substituted aromatic ring naturally undergoes subsequent methylation? Several groups of plant metabolites exhibiting remarkable structural similarities to the methoxy- and hydroxy-methoxyPCBs described above have been reported for some plant species, e.g., for aucuparin and magnolol and their related biphenyl derivatives (46, 47). None, however, were detected in our samples. Other plant metabolites that exhibit close structural similarities to the PCB metabolites identified in our samples act as lignin precursors in plants (48). In the case of tobacco, several isoforms of O-methyltransferase (OMT), the enzyme that controls the methylation of monolignols, have been identified (49). Compounds that share partial structural similarity with methoxy- and hydroxy-methoxy-PCBs were found among our PCB-treated cell extracts, including hydroxyacetophenone, dihydroxyacetophenone, hydroxy-methoxyacetophenone, hydroxy-dimethoxyacetophenone, dimethoxyacetophenone, trimethoxyacetophenone, and vanillin (data not shown). Substances of this type have previously been reported in various plant species, frequently having been found conjugated with a sugar moiety (50). It has even been shown that a hairy root culture of Pharbitis nil can metabolize, although only to a minor extent, hydroxy-methoxyacetophenone and, thereby, yield a biphenyl metabolite (51). Frick and Kutchan (52) suggested that certain plant OMTs might have a broad substrate range, and may be common to various metabolic pathways. Later, Chen et al. (53) showed VOL. 42, NO. 15, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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that OMTs of the monolignol biosynthetic pathways of five plant species, including tobacco, have a wider range of substrates than previously suspected. Such phenomena support our hypothesis that hydroxy-PCBs in a WSC-38 culture may undergo subsequent methylation by OMTs belonging to another metabolic pathway, thereby yielding the methoxy- and hydroxy-methoxy-PCBs found in our samples. In this case it is most likely that these OMTs are responsible for the methylation of hydroxyacetophenone metabolites. Similarly, methoxy-PCBs have been identified in rats, with catechol PCB metabolites acting as substrates of catechol-O-methyltransferase (COMT), the enzyme responsible for the methylation of catechol estrogen (54). Our discovery of methoxy-metabolites of PCBs in plant cells makes the whole process of the detoxification of PCBs in plants considerably more complicated than previously realized. While enzymes responsible for the oxidation of PCBs, such as the cytochromes P450 and the peroxidases, have already been well studied (19, 31, 55), enzymes responsible for the subsequent methylation of PCB catechol metabolites, most likely the OMTs, should be the focus of further research. With increasing understanding of the metabolism of PCBs in plant cell cultures, we plan to next focus on the in vivo determination of PCB metabolites present in those plant species naturally able to colonize PCB dump sites, some species of which have recently been analyzed for PCB accumulation (56).
Acknowledgments This work was supported by grant 203/06/0563 of the Grant Agency of the Czech Republic, as well as by research projects Z 40550506 and MSM 6046137305.
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