their general metabolic pathway in mammals involves cytochrome p-450 ( cyp ) enzyme - mediated oxidation to form hydroxylated metabolites and epoxides and further conjugation with glucuronide or glutathione to create corresponding conjugates , leading further to methylsulfonyl metabolites catalyzed by glutathione s - transferase ( gst ) ( 1 ) .
pcbs with vicinal hydrogens at the 3 and 4 positions of the aromatic ring as well as 2,5- or 2,3,6-substituted chlorines are most likely to be metabolized to methyl sulfones ( 2,3 ) .
it is important to study the fate of individual pcb congeners because the properties , toxicities , and behavior of pcbs vary widely in the environment .
the coplanar 3,3,4,4-tetrachlorobiphenyl ( cb77 , iupac number ) is a dioxin - like congener , with a relatively high toxicity compared to most tetrachlorobiphenyls .
the main monohydroxy metabolites of cb77 in mammals includes 4-hydroxy-3,3,4,5-tetrachlorobiphenyl ( 4oh - cb79 ) , 5-hydroxy-3,3,4,4- tetrachlorobiphenyl ( 5oh - cb77 ) , a trace of 6-hydroxy-3,3,4,4-tetrachlorobiphenyl ( 6oh - cb77 ) , and 2-hydroxy-3,3,4,4-tetrachlorobiphenyl ( 2oh - cb77 ) .
the methylsulfonyl metabolites of cb77 were documented in rats , mice , and fish , although cb77 is not one of the preferred structures for methyl sulfone metabolism ( 58 ) . according to the  green liver  model , metabolism in plants
is similar to the detoxification mechanism in animal livers because of similar enzyme systems ( 9 ) . to date ,
metabolism of pcbs in plants is generally reported in in vitro studies using cell or tissue cultures , which have the advantages of rapidly growing biomass and axenic conditions where microorganisms play no role in plant metabolism ( 1012 ) .
different plant species , including paul s scarlet rose , tobacco , solanum spp . ,
horseradish , and alfalfa have been screened for their ability to biotransform pcbs using cell cultures .
mono- and dihydroxy - chlorobiphenyls were identified as the metabolites of mono- and dichlorobiphenyls ( 13 ) .
other research showed only monohydroxy - pcbs as the metabolites of di- , tri- , tetra- , and penta - chlorobiphenyls ( 14 ) .
metabolism depends on the plant species and specific pcb congeners to which the plant tissue cultures are exposed . in general , lower chlorinated congeners are metabolized more rapidly than those with higher chlorine substitutions .
however the position of chlorine atoms and molecular structures are also important factors affecting pcb metabolism ( 10 ) . in the study of wilken et al .
, only three in 12 cell cultures , tomato ( lycopersicum esculentum ) , lettuce ( lactuca sativa ) , and paul s scarlet rose ( rosa spp . ) , were able to hydroxylate cb77 , and the main identified metabolites were 2-oh - cb77 , 6-oh - cb77 , and 5-oh - cb77 ( 15 ) .
more recently , methoxy- and hydroxy - methoxy - pcbs were reported as the metabolites of tobacco cells biodegrading dichlorobiphenyls ( 16 ) . despite the advantage of cell and tissue cultures over whole plants as a fast screening tool ,
the extrapolation of metabolism from in vitro to in vivo is tenuous . to better understand the biodegradation of pcbs in ubiquitous plants in nature , we find in vivo studies are necessary . in our previous studies ( 17 )
, it was found that whole hybrid poplars take up and bind cb77 strongly in root tissues , and metabolism was suggested but not confirmed . to study the biotransformation of cb77 in whole plants and the applicability of phytoremediation for such pcb congeners , we hydroponically exposed intact plants to cb77 , and the presence of metabolites in hydroponic solution and plant tissues were tracked through time . to our knowledge
, this is the first in vivo study on hydroxylation of pcb within intact plants .
the standards of cb77 , hydroxy metabolites ( 4-oh - cb79 , 5-oh - cb77 , 6-oh - cb77 ) , and the derivatization reagent ( diazomethane ) were all synthesized by the national institute of environmental health sciences ( niehs ) superfund basic research program center at the university of iowa .
cb77 was synthesized from 3,4-dichlorobenzene boronic acid and 3,4-dichlorobromobenzene using the suzuki coupling reaction ( 18 ) .
the 4-oh - cb79 metabolite was synthesized using the suzuki coupling of 3,4-dichlorobenzene boronic acid and 2,6-dichloro-4-bromoanisole , followed by demethylation of the methoxy pcb with boron tribromide ( 19 ) .
the 5-oh - cb77 and 6-oh - cb77 metabolites were prepared using the cadogan coupling of 3,4-dichloroaniline with 2,3-dichloroanisole and subsequent demethylation with boron tribromide ( 20 ) .
all four compounds were > 99% pure according to gas chromatographic analysis ( based on relative peak area ) . a solution of diazomethane in diethylether was synthesized from n - methyl - n - nitroso - p - toluenesulfonamide ( diazald ) using an aldrich mini diazald apparatus ( milwaukee , wi ) following manufacturer instructions .
the synthesization and use of diazomethane must be completed in a fume hood because of high toxicity .
stock solutions of cb77 and hydroxy - pcbs at 1 mg ml as compounds were prepared in acetone .
working solutions were prepared by gradual dilution of the stock solution . because of no commercial methylsulfonyl - cb77 standards available , 3-methylsulfonyl-2,2,5,5-tetrachlorobiphenyl ( 3-meso2-cb52 ) and 4-methylsulfonyl-2,2,5,5-tetrachlorobiphenyl ( 4-meso2-cb52 ) with the same amounts of chlorine substitutions as cb77 were purchased from accustandard as references .
internal standard 2,2,3,4,4,5,6,6-octachlorobiphenyl ( cb204 , iupac number ) was purchased from cambridge isotope laboratories , inc .
all standards and solutions were stored hermetically in amber glass vials at room temperature in the dark . silica gel ( 70230 mesh , fisher scientific , inc . )
two kinds of cleanup silica gel ( concentrated and 90% sulfuric acid silica gel ) were prepared by blending 100 g of activated silica gel with 50 g of concentrated h2so4 and 50 g of 90% ( w / w ) h2so4 , respectively .
acetone ( hplc grade ) , methyl tert - butyl ether ( mtbe ) ( hplc grade ) , and hexane ( pesticide grade ) were purchased from fisher scientific .
all other chemicals and reagents used in this experiment were of analytical reagent grade or higher purity .
cuttings from male clones of the adult imperial carolina hybrid poplar tree ( populus deltoides  nigra , dn34 ) and switchgrass ( panicum vigratum , alamo ) were used in this study .
each cutting was fit snugly with a predrilled screw cap and predrilled ptfe - faced septum .
then , the cuttings were grown hydroponically in half strength hoagland nutrient solution ( 21 ) .
after 25 days , healthy whole poplar plants were selected to carry out the experiment .
seeds of switchgrass were germinated on a bed of perlite with 1/10 strength hoagland solution .
the grasses were transferred into half strength hoagland solution ( without perlite ) when they grew 3 cm high . after enough biomass developed , bunches of grass ( five to six individual grasses in a bunch ) were grown in 500 ml glass jars for 1 week and then used for exposure .
the exposure reactors were 500 ml glass screw top conical flasks for poplars , 500 ml glass screw top jars for switchgrass , which have a sampling port with a ptfe - faced silicon septum and predrilled screw cap .
hoagland nutrient solution was made by using autoclaved deionized water saturated with oxygen by bubbling with compressed air flowing through a sterilized 0.20 m filter to prevent microbial contamination .
then , each autoclaved reactor was filled with 400 g of nutrient solution and a suitable amount of cb77 .
each reactor was planted with 56 switchgrass plants or a single 8 in . individual poplar plant .
three of nine poplar plants with shoots cutoff acted as the excised poplar controls , three of them were air - dried to act as dead poplar controls , and the remaining three were autoclaved before exposure to create autoclaved poplar controls . an untreated whole poplar control and an untreated switchgrass control , without cb77 in the hydroponic solutions , were used to detect any pcb contamination emanating from laboratory air during the experiment .
all reactors were wrapped with aluminum foil , which supported roots in the dark environment and eliminated photolysis of cb77 .
the temperature during plant growth was maintained at 23  1 c , and the photoperiod was 16 h a day under fluorescent lighting with a light intensity between 120 and 180 mol m s. transpiration of whole plants was determined daily by weighing the reactors .
oxygen - saturated , autoclaved deionized water was injected into the reactors to replace transpiration losses .
roots and hydroponic solutions of whole plants and controls were sampled and analyzed after 1 and 5 day exposures .
the extraction , separation , and cleanup procedures for pcbs , hydroxy - pcbs , and methylsulfonyl - pcbs were modified from the method for plasma ( 22 ) . in brief , the hydroponic solutions were amended with 10 drops of 37% hcl and 5 ml of 2-propanol , and then extracted with hexane / mtbe ( 1:1 , v / v ) for 30 min twice .
roots samples were mixed with 2 ml of 37% hcl and 5 ml of 2-propanol , homogenized , and extracted with 3 ml of hexane / mtbe ( 1:1 , v / v ) per gram of sample under vigorous shaking overnight .
the hexane phase was partitioned with 500 l of naoh solution ( 0.5 m in 50% ethanol ) and transferred to a clean vial after phase separation .
the combined hexane extract contained pcbs , methylsulfonyl - pcbs , and any other neutral metabolites ( neutral fraction ) , while hydroxy - pcbs were left in alkaline solution .
the alkaline solution was acidified with 125 l hcl ( 2 m ) and extracted with 1 ml of hx / mtbe ( 9:1 , v / v ) twice .
the extract was amended with a few drops of methanol and 0.5 ml of diazomethane to derivatize the oh - pcb to meo - pcb .
the derivatization reaction was performed in a refridgerator at 48 c for 3 h. excess diazomethane was evaporated under a gentle nitrogen stream .
then , the derivatization products , methyoxy pcb , were cleaned with acid silica gel column a ( 1 g of concentrated sulfuric acid silica gel with 0.1 g activated silica gel at the bottom ) and eluted by 10 ml of dichloromethane .
the neutral fraction was partitioned with 0.5 ml of anhydrous dimethyl sulphoxide ( dmso ) for 10 min .
the dmso phase containing methylsulfonyl - pcbs was then mixed with 1 ml of water and extracted with 3 ml of hexane .
the pcb fraction was cleaned by silica gel column a with 10 ml of dichloromethane for elution .
the methylsulfonyl - pcbs were cleaned by silica gel column b ( 0.5 g anhydrous na2so4 on top , 0.5 g of 90% sulfuric acid silica gel in the middle , and 0.1 g of activated silica gel at the bottom ) and eluted by 15 ml of dichloromethane .
eluates were evaporated to dryness and dissolved in hexane for gas chromatograph ( gc ) analysis .
qualitative and quantitative analysis of hydroxylated and methylsulfonyl metabolites was performed on an agilent 6890 gas chromatograph equipped with split detector system , gc / ms / ecd .
analytes were separated in a hp-5 fused silica capillary column ( 5% phenyl methyl siloxane , 60.0 m  250 m  0.25 m ) , and then split to a mass spectrometry and electron capture detector .
the gc was set as follows : injection port , 270 c with splitless mode ; high purity helium carrier gas at a constant flow rate of 1.9 ml min ; ecd , 360 c ; 95% argon and 5% methane makeup gas flow at 30 ml min ; electron impact ( ei ) ionization ms source , 250 c , scan from m / z 50 to 500 .
the oven program started at 75 c and was held for 3 min , then was increased at 5 c min to 150 c and held for 1 min , and then heated at 2 c min to 300 c and held for 5 min .
identification and quantification of daughter pcbs derived from dechlorination were performed by an agilent 6890n gas chromatograph coupled to a waters micromass quattro micro gc mass spectrometer ( gc / ms / ms ) ( milford , ma ) operating under electron impact ( ei ) positive mode at 70 ev and multiple reaction monitoring ( mrm ) , and the trap current was 200 a . the gc was equipped with an agilent 7683 series autosampler ( 23 ) .
analytes were separated in a supelco spb - octyl capillary column ( 30 m  250 m  0.25 m ) with helium at a constant flow rate of 0.8 ml min .
the oven temperature was programmed from 75 c and held for 5 min , heated to 150 at 15 c min and held for 1 min , and then heated to 280 at 2.5 c min and held for 3 min .
the mass recoveries and reproducibility of cb77 and 6-oh - cb77 were investigated by measuring the blank aqueous concentrations and root samples spiked with standards .
results in table 1 show that the analytical method worked well without the occurrence of chemical or biological transformations during sample analysis .
identification of the hydroxy metabolites of cb77 in plant roots and nutrient solutions was based on a comparison of the retention times and mass spectra of derivatives of analytes ( in samples ) to those of the standards .
the derivatives of hydroxy - cb77 , 6-meo - cb77 , were coeluted with 4-meo - cb79 , but they were separated from 5-meo - cb77 on the hp-5 capillary column . according to their mass spectra , they all have the molecular ion [ m ] at m / z 322 but have different fragmentation patterns due to the different positions of methyoxy substitutions .
the 6-oh - cb77 metabolite was identified in roots and nutrient solutions of exposed whole poplar plants and excised poplar controls , but it was not found in autoclaved poplar controls , dead poplar controls , or untreated whole poplar controls ( table 2 ) .
the derivative of 6-oh - cb77 , 6-meo - cb77 ( figure 1 ) , has a visible molecular ion and typical fragmentation clusters surrounding ions at m / z 267 [ m - ch3-cl ] and 207 [ m - coch3 - 2cl ] in the mass spectrum , and the ions around 207 possess the highest abundance .
there were no hydroxy - pcb metabolites detected in whole switchgrass , indicating that switchgrass was not able to hydroxylate cb77 during the experimental period .
all metabolites were identified and quantitatively analyzed by gc / ms / ecd and gc / ms / ms .
. coeluted pcb congeners that were not able to be separated by the used gc column .
chromatogram of 6-meo - cb77 after derivitization from 6-oh - cb77 in a root sample , detected by the split detector system ( gc / ms / ecd ) .
compound corresponds to the peaks highlighted in panel a and b. the autoclaved poplar controls were dead plants , minimizing the action of microbes and eliminating the role of plant enzymes . in the unautoclaved
the metabolite 6-oh - cb77 was not detected in either control , but it was detectable in the live poplars , suggesting that hydroxylation in poplars was caused by the plant itself , rather than by microorganisms in the root zone . excised poplar controls had no significant evapotranspiration , but the plant tissue was viable during the exposure period .
the 6-oh - cb77 metabolite was detected in the excised controls , and the concentration was similar to that in exposed whole poplars , indicating that transpiration was not required for metabolic transformation of cb77 in plant tissues .
hydroxylation occurred very rapidly in poplars with 6-oh - cb77 detected in roots and solutions after 1 day exposure .
the concentrations of 6-oh - cb77 increased slightly over time but without a significant difference between 1 and 5 day exposures , indicating that further transformation in poplars is possible if the exposure period was lengthened .
there was no significant difference between the concentrations of hydroxy metabolites under different dosages , although the lower dosage of cb77 resulted in slightly higher concentrations of metabolites .
other researchers reported that cb77 at a low dose was metabolized more rapidly than that at high dose in scup ( 7 ) .
the mass and concentrations of hydroxylated metabolites in roots were far higher than those in nutrient solution .
it is concluded that small amounts of 6-oh - cb77 exuded from roots into the rhizosphere compartment during the exposure period .
pcbs can be biotransformed to a multitude of sulfur - containing pcb metabolites , including thiol , methylthio- , and methylsulfonyl - pcbs under the function of cyp and gst enzymes in animals via forming glutathione conjugates of the pcbs ( 1,2426 ) .
most studies have focused on methylsulfonyl - pcbs ( 2527 ) , which have been found in various tissues of mammals and fish ( 28 ) .
methylsulfonyl - cb77 and two possible sulfur - containing intermediates ( figure 2 ) are of concern in this study . because the methylsulfonyl metabolites of cb77 are not commercially available , two standards of methylsulfonyl - cb52 with the same degree of chlorine substitutions as cb77 were selected as references .
similar mass spectra were detected in which typical molecular ion [ m ] at m / z 370 and fragmentation clusters surrounding at m / z 307 [ m - ch3-ccl ] , 279 [ m - cso2ch3 ] , and 184 [ m - so2ch3 - 3cl ] were displayed . taking account of the possibility of various mass spectrum patterns of methylsulfonyl - cb77 , molecular ions were powerful fragments to identify meso2-cb77 .
however , no similar mass spectra were found in the exposed poplar and switchgrass samples .
there were also no molecular ions of cb77-thiol and methylthio - cb77 in any mass spectra of plant samples .
it appears that neither poplar trees nor switchgrass are able to metabolize cb77 to methyl sulfones in 5 days .
according to the literature ( 2,3,29 ) , the structure of cb77 , without not only vicinal 3,4 hydrogen substitutions but also 2,5-dichloro- or 2,3,6 trichloro - phenyl rings , makes it difficult to biotransform to methyl sulfones .
various microbial dechlorination products of pcbs have been observed in anaerobic sediments ( 10 ) .
magee et al . found that the crude extract of nitrate reductase from leaves of the plant , medicago sativa , was capable of dechlorination of cb153 by observing the decrease of cb153 after its incubation with the plant crude extract ( 30 ) .
anaerobic microbial dechlorination was avoided in the experiments of this study by autoclaving the reactors and solution , saturating the hydroponic solution with oxygen at the beginning of the exposure and replacing the transpiration loss with sterilized and oxygen saturated water .
thus , the hydroponic solution was aerobic during the short period of exposure ( 1 and 5 days ) .
a large proportion of the exposed whole plants were observed to contain significant concentrations of the metabolites of 2,3,5,6-cb ( cb73 ) , 2,2,5,5-cb ( cb52 ) , 3,3,4-cb ( cb35 ) , 2,3,3-cb/2,4,4-cb ( cb20/28 ) , 4,4-cb ( cb15 ) , and 4-cb ( cb3 ) ( cb20 and 28 are coeluted congeners on the supelco spb - octyl capillary column .
both of them have the possibility of occurrence . ) . for various controls , cb3 and 35 were the congeners detected most frequently .
the concentrations of cb35 in autoclaved poplar control roots ( with minimized roles of microbes and without the role of poplars ) were far lower than that in dead poplar roots ( with the presence of microbes but without viable poplar tissue ) indicating that the symbiotic association of microbes in the root zone of the plant aid in the formation of cb35 .
the concentrations of cb35 in dead poplar roots were similar to that in excised poplar roots and exposed poplar roots ( all with functioning root - associated microbes and poplar roots ) at a lower dosage for 5 days , indicating that viable poplar tissue is not necessary for the formation of cb35 .
however , by comparison of the exposed whole poplars to the excised , dead , and autoclaved poplar controls , the formation of cb3 , 15 , 20/28 , 52 , and 73 were due to the role of the viable poplar ( plant ) tissues on dechlorination of the parent compound cb77 . according to the daughter pcbs detected in the reactors , the dechlorination of cb77 progressed from the tetra - chlorine ( 3,3,44-cb ) congener to the di-(4,4-cb ) congener to the mono - chlorine ( 4-cb ) congener .
in addition , tetra - chlorine congeners , cb73 , cb52 , and trichlorine congeners cb20/cb28 were detected by gc / ms / ms , which were presumed to be the products of chlorine rearrangement of cb77 during metabolism by plants .
the chlorine shift was suggested by our experiment in which a relatively high concentration of cb73 was detected after a whole poplar plant was exposed to cb52 for 5 days .
the rearrangement of chlorines on the biphenyl rings was clearly observed in this study , although the mechanism is not understood .
the concentrations of the dechlorination products and chlorine - shift metabolites in switchgrass roots were lower than those in exposed poplar roots .
this suggests that the enzymatic dechlorination and rearrangement capability of poplar is better than that of switchgrass .
generally , meta- and para - chlorines in pcb congeners are more susceptible to dechlorination ( 31 ) .
the 2,3,4,3,4-cb congener would biodegrade following the sequence as 2,3,4,3,4-cb , 2,4,3,4-cb , 2,4,3-cb , 2,3-cb , and 2-cb ( 32,33 ) .
intermediates following dechlorination of cb77 by a palladium / iron nanoparticles system were determined to be ( a ) from 3,3,4,4-cb ( cb77 ) to 3,3,4-cb ( cb35 ) , 3,3-cb ( cb11 ) , 3-cb ( cb2 ) , and biphenyl ; ( b ) from 3,3,4,4-cb to 3,4,4-cb ( cb37 ) , 4,4-cb ( cb15 ) , 3,4-cb ( cb12 ) , 3,4-cb ( cb13 ) , 4-cb ( cb3 ) , and biphenyl ( 34 ) .
although the metabolism of pollutants in vivo is very complex and requires a wide range of enzymatic capability , a proposed mechanistic scheme ( figure 3 ) is shown for how poplars and associated microbes might interact to affect the observed transformation products from this study .
all of the compounds shown in figure 3 were confirmed by gc / ms / ms and gc / ms / ecd .
the rearrangement of chlorines during metabolism of cb77 in poplar was suggested by the metabolites detected , cb52 and cb73 ( figure 3 , compounds in the dotted square ) , during our experiments .
the para / meta and meta / ortho chlorine shifts are hypothesized in the transformation from cb77 to cb52 , from cb77 to cb73 , from cb52 to cb73 , and from cb35 to cb20 .
the meta / ortho shift was reported in the formation of pcdfs from pcb pyrolysis ( 35,36 ) . however , these chlorine rearrangements have not been previously reported in microbial , mammal , or plant degradation studies of pcbs .
metabolites in the dotted square were also positively confirmed by gc / ms / ms in the poplar roots .
our experiment showed that relatively high concentrations of cb15 and cb3 were detected after a whole poplar plant was exposed to cb28 for 5 days . taking the daughter polychlorinated biphenyls in the reactors into account ,
we established the stepwise dechlorination pathway from cb28 to cb15 to cb3 . however , the key intermediate , cb37 , which should have occurred in the dechlorination from cb77 to cb15 , was not detected .
possible hypotheses for the observed lack of cb37 are the following : ( i ) cb37 disappeared very quickly due to further transformation , including the formation of cb15 by dechlorination .
( ii ) two meta chlorines on coplanar cb77 are of the same bond length , bond energy , and charge , which makes these two chlorines depart easily and form cb15 directly .
( iii ) cb77 was metabolized to cb28 directly under an enzymatic mechanism by poplar , which might involve dechlorination and rearrangement in one step .
there are two reported hydroxylation pathways for introducing hydroxyl substitution to the aromatic ring of pcbs ( 1 ) .
for example , cb77 could be metabolized to a 5,6-epoxide , which would form both 5-oh - cb77 and 6-oh - cb77 .
the second way is via an enzymatic hydroxyl substitution to a carbon on the aromatic ring directly to form the corresponding hydroxylated metabolite .
these two hydroxylation pathways could be involved in poplars , but only the 6-oh - cb77 was detected in this paper .
 polychlorinated biphenyls ( pcbs ) are widely distributed persistent organic pollutants . in vitro research 
 has shown that plant cell cultures might transform lower chlorinated congeners to hydroxylated pcbs , but there are few studies on in vivo metabolism of pcbs by intact whole plants . in this research , poplar plants ( populus deltoides  nigra , dn34 ) and switchgrass ( panicum vigratum , alamo ) were hydroponically exposed to 3,3,4,4-tetrachlorobiphenyl ( cb77 ) . 
 metabolism in plants occurred rapidly , and metabolites were detected after only a 24 h exposure . 
 rearrangement of chlorine atoms and dechlorination of cb77 by plants was unexpectedly observed . in addition , poplars were able to hydroxylate cb77 and the metabolite 6-hydroxy-3,3,4,4-tetrachlorobiphenyl ( 6-oh - cb77 ) was identified and quantified . 
 hybrid poplar was able to hydroxylate cb77 , but switchgrass was not , suggesting that enzymatic transformations are plant specific . 
 sulfur - containing metabolites ( from the action of sulfotransferases ) were investigated in this study , but they were not detected in either poplar or switchgrass . 
Introduction
Experimental Section
Results and Discussion