Hydroxy-PCBs, Methoxy-PCBs andHydroxy-Methoxy-PCBs: Metabolitesof Polychlorinated Biphenyls FormedIn Vitro by Tobacco CellsJ A N R E Z E K , † , ‡ T O M A S M A C E K , * , †

M A R T I N A M A C K O V A , ‡ J A N T R I S K A , § A N DK A M I L A R U Z I C K O V A §

Institute of Organic Chemistry and Biochemistry, Academy ofSciences of the Czech Republic, Flemingovo namesti 2, 166 10Prague 6, Czech Republic, Department of Biochemistry andMicrobiology, Faculty of Food and Biochemical Technology,Institute of Chemical Technology Prague, Technicka 3, 166 28Prague 6, Czech Republic, Department of AnalyticalChemistry, Institute of Systems Biology and Ecology, Academyof Sciences of the Czech Republic, Branisovska 31, 370 05Ceske Budejovice, Czech Republic.

Received February 13, 2008. Revised manuscript receivedMay 13, 2008. Accepted May 15, 2008.

While the metabolism of polychlorinated biphenyls (PCBs) inplant cells is a rarely studied field, hydroxy-PCBs have beendetected in several studies involving the use of various plantspecies. The ability of the tobacco (Nicotiana tabacum) callusculture WSC-38 to metabolize six dichlorobiphenyls underaseptic conditions was studied, and the resulting PCB metaboliteswere analyzed. WSC-38 cultures were cultivated withindividual dichlorinated PCB congeners. The metabolites wereidentified based on mass spectra characteristics after gaschromatography separation. In addition, metabolites of PCB 9 (2,5-dichlorobiphenyl) were identified by comparing their retentioncharacteristics with the available standards. In most casesat least two hydroxy-PCBs were produced from each parentPCB. Methoxy-PCBs and hydroxy-methoxy-PCBs wereother groups of metabolites produced. To the best of ourknowledge, ours is the first report to determine the presenceof methoxy- and hydroxy-methoxy-metabolites of PCBs in plants.The role of the O-methyltransferases (OMTs) in the methylationof hydroxy-PCBs is discussed. As methoxy-metabolites ofacetophenone were found among our samples, we posit thatthe OMTs responsible for the methylation of these compoundsare also involved in the metabolism of PCBs in cultures of WSC-38.

IntroductionAlthough their production was banned in the 1970s (a banwhich took effect in the former Czechoslovakia in 1984),polychlorinated biphenyls (PCBs) remain the subject of greatenvironmental concern. There are 209 PCB congeners, all ofwhich are unique in terms of the number and position of

chlorine atoms attached to the biphenyl molecule. Histori-cally, PCBs were commercially produced as complex mixturesof different congeners (e.g., Aroclor, Clophen, Delor,Kanechlor), and, due to their excellent physical and chemicalproperties (nonflammability, high stability, lipophilicity,resistance to degradation), were used throughout the worldin a wide range of industrial applications (e.g., as coolantsfor industrial transformers, hydraulic fluids, fire retardants,etc). Conversely, these properties also mark PCBs as recal-citrant compounds that accumulate in the environment. Suchlipophilic compounds are able to spread over large distances(1), affecting the whole environment as they do so. Inparticular, their ability to enter the food chain, and ac-cumulate in fat tissues, means that their most significantimpact is on end consumers, including human beings (2, 3).The toxicity of PCBs has been well documented in recentdecades, and it now seems clear that the toxic propertiesthey possess may have a number of harmful effects. Apartfrom possibly possessing carcinogenic properties (4), PCBsare known to act as endocrine disrupters (5) and as thyroidhormone analogues (6). Of the approximately 1.3 millionmetric tons of PCBs that were produced worldwide (7), it hasbeen estimated that about 30% were released into theenvironment (8, 9), making the need for techniques capableof 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 fromcontaminated soil, bioremediation appears to be a promisingtechnology (10). Apart from traditional bioremediationtechnologies that use natural bacterial or fungal degraders,another technique for PCB removal involves the use of plantsin phytoremediation processes (11, 12). The potential of thistechnique makes it important to study the plant metabolitesof PCBs.

Several studies have shown that PCB metabolism in plantsexhibits apparent similarities to the same mechanism inanimals and humans (“green liver” model). The plantdetoxification of lipophilic compounds, including PCBs,consists of three phases. Phase I (activation) involves theoxidation or hydroxylation of the toxic compound. Phase II(conjugation) consists of the covalent binding of the formedmetabolite to endogenous hydrophilic molecules, such asglucose, glutathione, or malonate; this step increases thehydrophilic character of the parent compounds. Then, inphase III (compartmentation), inactive conjugated water-soluble xenobiotics are relocated from cytosol into eithervacuole or apoplast (13).

Moza et al. (14, 15) described the production of mono-hydroxylated metabolites (chlorobiphenylols) of PCB 4 (2,2′-dichlorobiphenyl) in carrot and sugar beet, and later, alsoidentified mono- and dihydroxy-metabolites of PCB 31 (2,4′,5-trichlorobiphenyl) in carrot. Fletcher et al. (16), studying themetabolism of 2-chlorobiphenyl with a tissue culture of Paul’sScarlet Rose, showed that plant cells are able to metabolizePCBs under axenic conditions. Butler et al. (17) partiallyidentified their metabolites as monohydroxylated PCBs thatwere subsequently glycosylated. Tissue cultures, thus, be-came popular for the study of plant metabolism becausethey result in faster biomass development and are easier tomaintain under sterile cultivation conditions. More impor-tantly, tissue cultures enable metabolites to be unambigu-ously assigned to plant cells with the certainty that con-taminating microorganisms play no role in plant metabolism.

More recently, Wilken et al. (18) reported hydroxylatedmetabolites of PCB 1 (2-chlorobiphenyl) and PCB 52 (2,2′,5,5′-tetrachlorobiphenyl) in soybean and wheat culture, respec-

* Corresponding author phone:+420-220-183-340; fax:+420-220-183-582; e-mail: tom.macek@uochb.cas.cz.

† Institute of Organic Chemistry and Biochemistry, Academy ofSciences 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.

Environ. Sci. Technol. 2008, 42, 5746–5751

5746 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 15, 2008 10.1021/es800445h CCC: $40.75 2008 American Chemical SocietyPublished on Web 06/25/2008

tively. Metabolites of PCB 77 (3,3′,4,4′-tetrachlorobiphenyl),hydroxylated in positions 2-, 5-, and 6-, have been identifiedin cultures of rose, tomato, sunflower, and lettuce (19).

The commercial mixture Delor 103, which consists of 59PCB congeners with an average of 3 chlorine atoms perbiphenyl, has also been used to study PCB metabolism(20–23). Approximately 40 in vitro tissue cultures of differentplant species were tested, with a hairy root culture of blacknightshade (Solanum nigrum), SNC-9O, shown to have thehighest ability to degrade PCBs. Using the same culture,Kucerova et al. (24) identified mono- and dihydroxychlo-robiphenyls as metabolites of some mono- and dichloro-biphenyls. Monohydroxylated metabolites of monochlori-nated PCBs were later also found in cultures of Armoraciarusticana, 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 penta-chlorobiphenyl congeners only monohydroxylated PCBs weredetected as biodegradation products.

The identification of PCB metabolites in plants is par-ticularly important in terms of the environmental safety ofboth the bioremediation process itself, and the independentformation of such metabolites in nature. Compounds suchas PCBs are globally spread; their effects not limited to theiroriginal site of usage, production, and contamination. Thewidespread occurrence of PCBs in the environment, togetherwith the ubiquitous presence of plants, inevitably leads totheir interaction. The use of plants and plant organs cultivatedaseptically makes it possible to distinguish the remediationsteps that can be performed by plant cells alone, without theinvolvement of microorganisms. Our present study is focusedon determining the phase I detoxification products ofdichlorinated PCB congeners formed in vitro by the tobaccocallus culture, WSC-38. We chose tobacco because fieldexperiments have demonstrated its high potential to ac-cumulate PCBs in its tissues (27).

Materials and MethodsPlant Cultures and Cultivation Conditions. WSC-38 calluscultures of tobacco (Nicotiana tabacum cv. Wisconsin 38)were used to identify metabolites of PCB biodegradation.Cultivation was performed in the media described byLinsmaier and Skoog (28), to which we added 0.1075 mg/Lof kinetin and 0.225 mg/L of dichlorophenoxyacetic acid. Invitro callus cultures were cultivated in 100 mL of liquid mediain 250 mL Erlenmeyer flasks. Aluminum-lined caps were usedto minimize evaporation. The metabolites were analyzed inboth the media and biomass. Cultivation started with 5 g offresh weight tissue culture, in the dark, at 24 °C, with shakingat 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/Lmedia in all cases. Cultivation continued for 14 days underthe same conditions. Two controls were cultivated in thesame way, but without the addition of a PCB. However, weadded the same amount of methanol (as used to dilute thePCB congeners in the real samples) to the second control.

Metabolite Extraction and Derivatization. At the end ofthe cultivation period, the media and biomass were separatedby filtration. Forty grams of biomass was homogenized inliquid nitrogen, and liquid-liquid extracted with dichlo-romethane (2 × 30 mL). Fifty mL of medium was extractedwith dichloromethane (2 × 30 mL). The extracts were driedwith natrium sulfate (anhydrous), filtered, evaporated todryness in a vacuum evaporator (40 °C, 150 RPM, 7 Torr),and redisolved in 1 mL of methanol. Acetylation was thenperformed using a slightly modified version of the procedure

described by Triska et al. (29). Methanolic extracts weredissolved in 50 mL of 0.1 M K2CO3 in a separation funnel.Then 1 mL of acetic anhydride was added and the mixturewas 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. Thepostevaporation residues were dissolved in 100 µL of hexanefor GC-MS analysis. The available biphenylol standards werealso acetylated.

Analysis of PCB Metabolites. A Finnigan Mat GCQTM (gaschromatograph equipped with a quadrupole ion trap massspectrometer) was used to perform the analysis. One µL ofextract plus 1 µL of hexane were injected splitless into a GC-MS system (Zebron Capillary GC Column ZB-5: 30 m; internaldiameter 0.25 mm; 5% phenylpolysiloxane stationary phase;thickness 0.1 µm). Helium was used as the carrier gas at aflow rate of 0.4 m/s. The injector temperature was 250 °C.The initial temperature of 60 °C was linearly increased untilit reached 283 °C (4 °C/min), at which point a constanttemperature of 283 °C was maintained for 4.25 min. MSdetection was performed in “full scan” mode: EI ionizationpotential 70 eV; ion source at 175 °C; transfer line at 275 °C;one spectra measured every second. Basic identification wasperformed by comparing the mass spectra of the metaboliteswith the NIST library (National Institute of Standards andTechnology, Gaithersburg, MD). Standards of 2′,5′-dichloro-2-biphenylol, 2′,5′-dichloro-3-biphenylol and 2′,5′-dichloro-4-biphenylol (J.T.Baker B.V., Deventer, The Netherlands),were available and, based on a comparison of retentioncharacteristics, used to identify metabolites of PCB 9.

Results and DiscussionHydroxy-PCBs. Based on our earlier findings of PCBmetabolites in an SNC-9O black nightshade (S. nigrum) hairyroot tissue culture, we expected to find hydroxylated PCBmetabolites in the tobacco callus culture WSC-38, becausethe ability of tobacco plants to accumulate PCBs fromcontaminated soil in their tissues has already been dem-onstrated (27, 30). WSC-38 cultures, each cultivated with oneof six different dichlorinated PCB congeners, yielded theexpected 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 dichlorinatedPCB congeners yielded at least one monohydroxylatedmetabolite, and in most cases two were detected. The massspectra obtained for these metabolites revealed a visiblemolecular ion [M+] at m/z 280. The typical fragmentationcluster surrounding ion at m/z 238 [M+-CH2CO] was also

TABLE 1. Dichlorobiphenylols Formed from PCBs by Nicotianatabacum (WSC-38 Culture)

hydroxy-PCB metabolites in biomass

PCB Cl positionretention

time (min:s) identification

4 2,2′ 29:40 dichlorobiphenylol30:46a dichlorobiphenylol

5 2,3 31:56 dichlorobiphenylol33:35a dichlorobiphenylol

6 2,3′ 31:23 dichlorobiphenylol31:40 dichlorobiphenylol32:23a dichlorobiphenylol

7 2,4 32:37 dichlorobiphenylol8 2,4′ 31:45 dichlorobiphenylol

32:46a dichlorobiphenylol9 2,5 31:51 2′,5′-dichloro-3-biphenylol

32:32a 2′,5′-dichloro-4-biphenylola Main metabolite.

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present in all spectra. In addition, fragments of m/z 168 [M+-CH2CO-2Cl] and m/z 139 [M+-CH2CO-2Cl-HCO] were alsodetected.

Due to limited availability, we were unable (with theexception of PCB 9) to compare the retention times of themetabolites formed with those of the standards. We did,however, compare their retention times to those of themetabolites detected in the SNC-9O cultures used in ourearlier experiment. In general, the number of metabolitesidentified in WSC-38 cells was lower than the numberdetected in the SNC-9O cultures (26). All metabolitesdetermined in the WSC-38 culture were also found in theSNC-9O samples (with corresponding retention times). Someadditional metabolites (e.g., 2′,5′-dichloro-2-biphenylol forPCB 9) were detected in the SNC-9O culture.

As free hydroxy-PCBs were found neither in the cultivationmedia extracts nor in the PCB-untreated control extracts, weconclude that they were maintained in the plant cells andnot exuded in their unbound form from living cells to thecultivation media.

Our findings confirm that phase I in the plant metabolismof PCBs is the hydroxylation of the parent compounds.Various authors have shown that this phase can largely beattributed to the work of the cytochrome P450 enzyme system(31). Indeed, this system also seems to be responsible for theproduction of hydroxy-PCBs as metabolites in yeasts (32),animals (33), and humans (34).

The uptake of PCBs by plants, together with theirmetabolism in plants, depends on the plant species, tissueculture and PCB congener used. Although, due to theunavailability of 14C-labeled PCBs, the mass balance of PCBuptake and metabolism was not estimated in our work,differences among plant species can be demonstrated byreference to examples from the literature. For instance,previous studies (14, 15) have demonstrated that carrot has

a higher uptake and metabolism of PCB 4 (2,2′-dichlorobi-phenyl) than sugar beet; the concentration of unchangedPCB 4 in carrot root being 0.240 ppm compared to less than0.001 ppm in sugar beet root. Similarly, the concentrationof PCB metabolites in carrot root was 0.012 ppm (5% ofapplied radioactivity) and just 0.004 ppm in sugar beet root.The uptake of radioactivity from PCB 31 (2,4′,5-trichloro-biphenyl-14C) reached 3.1% in carrot root and 0.2% in sugarbeet root, with metabolites only being detected in the carrot.With PCB 100 (2,2′,4,4′,6-pentachlorobiphenyl-14C), theuptake of radioactivity was higher in carrot (1.4%) than insugar beet (0.1%), but no metabolites were detected in eitherspecies. Later, Fletcher et al. (35) applied PCB 47 (2,2′,4,4′-tetrachlorobiphenyl-14C) to a rose suspension culture. After8 days of cultivation, the authors recovered 5.5% of radio-activity in the form of metabolites of PCB 47; 90% of the totalradioactivity applied being recovered. Furthermore, in anexperiment in which 86% of PCB-1 (2-chlorobiphenyl-14C)and 3.4% of PCB 47 (2,2′,4,4′-tetrachlorobiphenyl-14C) weremetabolized by rose culture within 4 days of cultivation,Groeger and Fletcher (36) showed that the rate of PCBmetabolism decreases as the chlorination level increases. Inthis experiment, PCB 153 (2,2′,4,4′,5,5′-hexachlorobiphenyl-14C) remained intact. Ryslava et al. (27) demonstrated thatthe concentration of PCBs in tissues of tobacco plant (0.244mg PCB/kg fresh biomass) cultivated in PCB-contaminatedsoil was 5-9 times higher compared to that in blacknightshade (S. nigrum) and alfalfa (M. sativa). After 6 months,the residual concentration of PCBs in the same soil haddeclined to 4.3 mg PCB/kg of dry soil (66% of the PCBconcentration in unplanted control soil).

In spite of their generally low uptake and metabolism ofPCBs, plants possess various advantageous properties thataid phytoremediation. In addition to phytoextraction alone,certain plants also support degrading microbes in the soil ina variety of ways, which include the release of root exudatesthat serve as extra nutrients for degraders and the develop-ment of rhizosphere that enhances soil aeration. It is likelythat PCB-contaminated soils will need to be repeatedlycultivated with the most beneficial species. With this ap-proach, we anticipate that, even taking into account the lowmetabolic rates of PCBs in plant tissues, after several yearsof such cultivation the total amount of catechol metabolitesin the plant biomass will reach significant levels.

As the toxicity of hydroxylated PCBs is known to be higherthan that of their parent PCBs (37), PCB metabolism by plantscould lead to catechol metabolites of PCBs entering foodchains, thereby creating a new environmental problem.Consequently, with respect to the developing phytoreme-diation technologies, special attention must be paid to the

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 Nico-tiana tabacum (WSC-38 Culture)

methoxy-PCB metabolites in biomass

PCBCl

positionretention

time (min:s) identification

4 2,2′ 28:04 ortho- or meta-methoxy-PCB 45 2,3 29:34 ortho- or meta-methoxy-PCB 56 2,3′ 29:29 para-methoxy-PCB 6

29:52 ortho- or meta-methoxy-PCB 67 2,4 29:07 para-methoxy-PCB 78 2,4′ not detected9 2,5 28:29 ortho- or meta-methoxy-PCB 9

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establishment of procedures that ensure the careful handlingof plant biomass cultivated in contaminated soil to avoid theconsequent dissemination of toxic compounds. Without suchprocedures, contamination by hydroxy-PCBs, recently moni-tored in Canada (38), could become as serious a problem asPCB contamination itself.

Methoxy-PCBs. While checking the mass spectra of PCBmetabolites, in one sample we observed a typical chlorinationcluster surrounding the ion at m/z 252. This fragmentationpattern did not correspond to the typical pattern forhydroxylated PCBs, suggesting that this molecule could bea methoxy-metabolite of the parent PCB. Therefore, wecompared the mass spectra obtained for this metabolite withthe MS results obtained by other authors (39–43). In all cases,the spectra included a molecular ion [M+] at m/z 252surrounded by a cluster of chlorinated compounds, as wellas 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 amethoxy-derivative of its parent PCB. The spectrum ofmethoxy-PCB 4 is shown in Figure 1. With the exception ofPCB 8, methoxy-PCBs were also found in the extracts ofbiomass cultivated with the other PCBs (see Table 2). Asshown in previous studies (39, 40), in each case it is likelythat these metabolites were substituted by a methoxy groupin either the meta or ortho positions. Both methoxy-PCB 6and methoxy-PCB 7 showed the addition, at m/z 237 [M+-CH3], of another ion typical for methoxylated PCBs, therebysuggesting that both compounds were para-substituted bya methoxy group. These compounds were found neither inthe cultivation media samples nor in the PCB-untreatedcontrol 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 previouslybeen reported in the literature.

Hydroxy-Methoxy-PCBs. Typical chlorination fragmen-tation patterns were also found in the mass spectra of othercompounds. Figure 2 shows the mass spectrum of thesecompounds, in which a molecular ion [M+] appears at m/z268 surrounded by the cluster typical for chlorinated aromaticcompounds, together with ions at m/z 253 [M+-CH3] andm/z 225 [M+-COCH3]. Present in each extract of biomasscultivated with a PCB, Kamei et al. (40) have shown thatthese compounds are almost certainly hydroxy-methoxy-PCBs (see Table 3). These hydroxy-methoxy-PCBs were foundneither in the cultivation media extracts nor in the PCB-untreated control extracts. To the best of our knowledge,neither hydroxy-methoxy- nor methoxy-PCBs have previouslybeen reported in plant cells as intermediates of PCBtransformation.

The Methylation of Hydroxy-PCBs. Our findings raisean important question. Namely, are specialized enzymesinduced to perform the methylation of hydroxy-PCBs, or dohydroxylated PCBs, as alternative substrates, enter a bio-synthetic pathway in which the hydroxy-substituted aromaticring naturally undergoes subsequent methylation?

Several groups of plant metabolites exhibiting remarkablestructural similarities to the methoxy- and hydroxy-methoxy-PCBs described above have been reported for some plantspecies, e.g., for aucuparin and magnolol and their relatedbiphenyl derivatives (46, 47). None, however, were detectedin our samples.

Other plant metabolites that exhibit close structuralsimilarities to the PCB metabolites identified in our samplesact as lignin precursors in plants (48). In the case of tobacco,several isoforms of O-methyltransferase (OMT), the enzymethat controls the methylation of monolignols, have beenidentified (49).

Compounds that share partial structural similarity withmethoxy- and hydroxy-methoxy-PCBs were found amongour PCB-treated cell extracts, including hydroxyacetophe-none, dihydroxyacetophenone, hydroxy-methoxyacetophe-none, hydroxy-dimethoxyacetophenone, dimethoxyace-tophenone, trimethoxyacetophenone, and vanillin (data notshown). Substances of this type have previously been reportedin various plant species, frequently having been foundconjugated with a sugar moiety (50). It has even been shownthat a hairy root culture of Pharbitis nil can metabolize,although only to a minor extent, hydroxy-methoxyacetophe-none and, thereby, yield a biphenyl metabolite (51).

Frick and Kutchan (52) suggested that certain plant OMTsmight have a broad substrate range, and may be commonto various metabolic pathways. Later, Chen et al. (53) showed

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 byNicotiana tabacum (WSC-38 culture)

hydroxy-methoxy-PCB metabolites in biomass

PCB Cl positionretention

time (min:s) identification

4 2,2′ 33:48 hydroxy-methoxy-PCB 434:40 hydroxy-methoxy-PCB 4

5 2,3 37:17 hydroxy-methoxy-PCB 56 2,3′ 35:35 hydroxy-methoxy-PCB 6

36:20 hydroxy-methoxy-PCB 67 2,4 36:22 hydroxy-methoxy-PCB 78 2,4′ 35:58 hydroxy-methoxy-PCB 8

36:46 hydroxy-methoxy-PCB 89 2,5 36:10 hydroxy-methoxy-PCB 9

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that OMTs of the monolignol biosynthetic pathways of fiveplant species, including tobacco, have a wider range ofsubstrates than previously suspected. Such phenomenasupport our hypothesis that hydroxy-PCBs in a WSC-38culture may undergo subsequent methylation by OMTsbelonging to another metabolic pathway, thereby yieldingthe methoxy- and hydroxy-methoxy-PCBs found in oursamples. In this case it is most likely that these OMTs areresponsible for the methylation of hydroxyacetophenonemetabolites. Similarly, methoxy-PCBs have been identifiedin rats, with catechol PCB metabolites acting as substratesof catechol-O-methyltransferase (COMT), the enzyme re-sponsible for the methylation of catechol estrogen (54).

Our discovery of methoxy-metabolites of PCBs in plantcells makes the whole process of the detoxification of PCBsin plants considerably more complicated than previouslyrealized. While enzymes responsible for the oxidation of PCBs,such as the cytochromes P450 and the peroxidases, havealready been well studied (19, 31, 55), enzymes responsiblefor 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 PCBsin plant cell cultures, we plan to next focus on the in vivodetermination of PCB metabolites present in those plantspecies naturally able to colonize PCB dump sites, somespecies of which have recently been analyzed for PCBaccumulation (56).

AcknowledgmentsThis work was supported by grant 203/06/0563 of the GrantAgency of the Czech Republic, as well as by research projectsZ 40550506 and MSM 6046137305.

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