although the industrial 
 production of polychlorinated biphenyls 
 ( pcbs ) has been banned worldwide under the stockholm convention , pcbs still represent a significant environmental 
 and human health concern .
specifically , pcbs continue to be released 
 into the environment from building materials and other sites of pcb 
 use .
electronic waste processing has resulted 
 in considerable environmental and occupational pcb exposure at electronic 
 waste sites around the world .
several recent studies revealed 
 the presence of pcbs in certain paint pigments , highlighting 
 the fact that the inadvertent production of pcbs by industrial processes 
 represents a current , environmental source of pcbs .
there is increasing 
 evidence that , in particular , pcb congeners with multiple ortho chlorine substituents predominate in outdoor and indoor air , environmental and human samples .
these ortho - substituted pcb congeners 
 and their hydroxylated metabolites are endocrine disruptors and have been implicated as developmental neurotoxicants 
 by mechanisms involving altered ca signaling , interference 
 with thyroid hormone signaling , and decreased dopamine content . in vitro metabolism studies
have shown that 
 pcbs 
 are oxidized by p450 enzymes to ho - pcbs .
the p450 isoforms involved 
 in the metabolism of a particular pcb congener depend on its chlorine 
 substitution pattern
. pcb congeners with ortho substituents 
 are typically metabolized by phenobarbital - inducible cyp2b enzymes , whereas pcb congeners without ortho substituents 
 are metabolized by cyp1a enzymes .
several 
 environmentally relevant pcb congeners ( e.g. , pcb 136 ) are chiral 
 because they exist as rotational isomers , or atropisomers , that are 
 nonsuperimposable mirror images of each other .
atropselective metabolism 
 of these chiral pcb congeners by p450 enzymes is thought to result 
 in their atropisomeric enrichment in wildlife , laboratory animals , 
 and humans .
ho - pcbs may be further metabolized 
 in the liver to dihydroxylated metabolites or glucuronide and sulfate 
 conjugates .
there is experimental 
 evidence from in vitro studies using recombinant 
 p450 enzymes that the oxidation of ho - pcbs to dihydroxylated metabolites 
 is also atropselective .
it is likely 
 that glucuronide or sulfate conjugates of ho - pcbs are also formed 
 atropselectively ; however , this has not been shown experimentally . despite a large number of in vitro metabolism 
 studies , the importance of hepatic vs extrahepatic p450 enzymes for the atropselective 
 metabolism and excretion of pcb 136 and
its ho - pcb metabolites remains 
 unproven in vivo . to address this knowledge gap , 

the present study uses a genetic mouse model with defective hepatic 
 metabolism due to the liver - specific deletion of the cytochrome p450 
 reductase ( cpr ) gene to investigate the role of hepatic 
 metabolism in the atropselective disposition of pcb 136 and its metabolites 
 in mice . in this mouse model
all p450 isoforms involved in the hepatic 
 metabolism of pcbs are inactivated or inhibited because cpr , the required 
 electron donor of all microsomal p450 enzymes , is not expressed in 
 the liver .
pcb 136 was selected for this disposition study because 
 it is a chiral pcb congener of environmental relevance and atropspecifically alters neuronal connectivity 
 in rat hippocampal neurons by mechanisms involving ryanodine receptors .
analytical 
 standards of 2,3,4,5,6-pentachlorobiphenyl 
 ( pcb 117 ) , 2,2,3,4,4,5,6,6-octachlorobiphenyl 
 ( pcb 204 ) , and 2,3,3,4,5,5-hexachlorobiphenyl-4-ol 
 ( 4-159 ) were purchased from accustandard ( new haven , ct , usa ) . 

2,2,3,3,6,6-hexachlorobiphenyl ( pcb 136 ) and 
 the corresponding ho - pcb metabolites ( 2,2,3,4,6,6-hexachlorobiphenyl-3-ol , 
 3 - 150 ; 2,2,3,3,6,6-hexachlorobiphenyl-4,5-diol , 
 4,5 - 136 ; 2,2,3,3,6,6-hexachlorobiphenyl-4-ol , 
 4 - 136 ; 2,2,3,3,6,6-hexachlorobiphenyl-5-ol , 
 5 - 136 ) were synthesized as described earlier .
diazomethane for the derivatization of ho - pcbs 
 to methoxylated pcbs was synthesized from n - methyl - n - nitroso - p - toluenesulfonamide ( diazald ) 
 using an aldrich mini diazald apparatus ( milwaukee , wi , usa ) . simplified metabolism scheme of pcb 136 atropisomers showing 
 major 
 ho - pcb metabolites investigated and the corresponding abbreviations . 

only one atropisomer of pcb 136 and the corresponding metabolites 
 are shown for clarity reasons .
p450 , cytochrome p450 enzyme ; ugt , 
 uridine 5-diphospho - glucuronosyltransferase ; sult , sulfotransferase ; 
 glc , glucuronide .
alb - cre / cpr mice with liver - specific deletion 
 of the cytochrome p450 oxidoreductase ( ec 1.6.2.4 ) gene ( ko ) and congenic 
 alb - cre / cpr mice ( wild 
 type , wt ) were obtained from dr .
xinxin ding ( school of public health , 
 state university of new york , albany , ny ) to establish a breeding 
 colony at the university of iowa .
breeding pairs 
 were set up between ko and wt mice , and littermates were genotyped 
 by pcr in the university of iowa transgenic animal facility as described 
 previously .
animals were housed in standard 
 plastic cages in a controlled environment maintained at 22 c 
 with a 12 h light  dark cycle .
the mice were fed a basal diet 
 ( harlan 7913 with 18% protein , 6% fat , and 5% fiber ) and water ad 
 libitum .
housing of breeding pairs and animal experiments were approved 
 by the institutional animal care and use committee at the university 
 of iowa ( protocol # 1206120 ) .
liver microsomes were prepared 
 from eight week old , nave ( i.e. , untreated ) female ko ( n = 6 ) and wt mice ( n = 5 ) using established 
 procedures to characterize the effects 
 of the liver - specific deletion of cpr on total p450 
 levels and hepatic cpr , cyp1a , and cyp2b activities ( for additional 
 experimental details , see table s1 ) .
the 
 effects of the liver - specific deletion on these markers were consistent 
 with published findings .
in particular , 
 a drastically reduced cpr activity was observed in the liver of ko 
 compared to wt mice .
in contrast , liver - specific deletion of cpr did not significantly alter the expression of synaptic 
 plasticity - associated genes ( i.e. , activity - regulated cytoskeleton - associated 
 protein , myelin basic protein , neurogranin , and spinophilin ) in the 
 cortex , hippocampus , or cerebellum of ko compared to wt mice ( table s2 ) .
ko and congenic wt mice ( 8 
 week old females ) were obtained from the breeding colony described 
 above and randomly divided into the following treatment and control 
 groups ( table s3 ) : 1 ) wt mice ( n = 4 ) received a single oral dose of pcb 136 ( 30 mg / kg 
 b.w . ) on a vanilla wafer cookie ( 7.5 g / kg b.w . ) ; 2 ) ko mice ( n = 7 ) received a single oral dose of pcb 136 ( 30 mg / kg 
 b.w . ) on a vanilla wafer cookie ( 7.5 g / kg b.w . ) ; 3 ) wt control mice 
 ( n = 5 ) received the vehicle ( cookie ; 7.5 g / kg b.w . ) 

alone ; and 4 ) ko control mice ( n = 5 ) received the 
 vehicle ( cookie ; 7.5 g / kg b.w . )
the dosing of all mice using 
 a cookie was performed as described previously . after eating the entire cookie ,
animals were housed individually 
 in metabolic cages , and urine and feces were collected daily from 
 each animal for 3 days .
tissues ( brain , liver , 
 and adipose tissue ) were excised en bloc , and their 
 wet weight was determined .
pcb treatment had no significant effect 
 on body or organ weights ( table s3 ) .
lipids were extracted 
 from tissues ( liver , brain , and adipose tissue ) and feces of pcb or 
 vehicle - treated animals by pressurized liquid extraction using a dionex 
 ase200 system ( dionex , sunnyvale , ca ) as described earlier .
briefly , the samples were mixed with 2 g of 
 diatomaceous earth ( fisher scientific ) and placed in ase extraction 
 cells ( 11 ml ) .
the cells were extracted using a chloroform / methanol 
 mixture ( 2:1 , v / v ) at 120 c and 1500 psi .
the extractable lipid content of each tissue or feces 
 are summarized in table s4 .
pcb 136 
 and its hydroxylated metabolites were extracted by pressurized liquid 
 extraction from liver ( 0.300.74 g ) , brain ( 0.190.30 
 g ) , adipose ( 0.040.29 g ) , and feces samples ( 0.610.75 
 g ) from individual mice using a dionex ase200 system following a published 
 method with modifications .
briefly , the 
 tissues were mixed with diatomaceous earth ( 2 g ) and placed in the 
 extraction cell ( 33 ml ) containing florisil ( 60100 mesh , 12 
 g ; fisher scientific ) . pcb 117 ( 250 ng ) and 4-159 ( 137 ng ) 
 were added to each sample as surrogate standards , and the cells were 
 extracted with hexane - dichloromethane - methanol ( 48:43:9 , v / v / v ) at 
 100 c and 1500 psi ( 10 mpa ) with preheat equilibration for 6 
 min , 60% of cell flush volume , and 1 static cycle of 5 min .
sample 
 blanks containing only florisil and diatomaceous earth were extracted 
 in parallel with each sample set .
the extracts were concentrated to 
 approximately 1 ml using a turbo vap ii ( biotage , llc , nc , usa ) and 
 transferred with 1 ml of hexane to glass tubes .
the solvent was evaporated 
 to dryness under a gentle stream of nitrogen and redissolved in 1 
 ml of hexane .
after derivatization of the ho - pcbs with diazomethane , 
 the organic extracts were subjected to a sulfur cleanup step as described 
 earlier , followed by treatment with concentrated sulfuric acid and 
 addition of the internal standard ( pcb 204 ) .
pcb 136 and its hydroxylated metabolites were extracted from 
 blood samples ( 0.490.87 g ) from individual mice by liquid 
briefly , 
 blood samples were diluted with aqueous kcl ( 1% ; 3 ml ) .
the surrogate 
 standards ( pcb 117 and 4-159 , 50 ng each ) were added to each 
 sample .
samples were acidified with hcl ( 6 m ; 1 ml ) , followed by addition 
 of 2-propanol ( 3 ml ) . samples were extracted with hexane : mtbe ( 1:1 , v / v ; 5 ml ) and hexane ( 3 ml ) .
the combined organic extracts 
 were washed with aqueous kcl ( 1% ; 3 ml ) , evaporated to dryness , derivatized 
 with diazomethane , and further treated as described above for tissue 
 and feces samples .
two aliquots of each urine sample ( 0.2 ml ) were diluted 
 with an equal 
 volume of 0.2 m sodium acetate buffer ( ph = 5 ) . to determine if glucuronide 
 and/or sulfate conjugates of pcb 136 were present in urine samples 
 both aliquots were incubated in parallel with or without -glucuronidase / sulfatase 
 mixture ( 20 l ; type h-2 from helix pomatia , 100,000 units / ml ; sigma - aldrich co. st .
subsequently , pcb 136 
 and its hydroxylated metabolites were extracted from urine samples 
 as described above for blood .
analysis of pcb 136 and the 
 methylated derivatives of hydroxylated 
 pcb 136 metabolites was performed on a db1-ms ( 60 m  0.25 mm 
 i d  0.25 m film thickness ; agilent , santa clara , ca ) 
 or an equity-1 capillary column ( 60 m  0.25 mm i d  0.25 
 m film thickness ; supelco , bellefonte , pa ) using an agilent 
 7890a gas chromatograph equipped with two ni-ecds 
 as described previously .
the levels of 
 pcbs were calculated using pcb 204 as internal standard , adjusted 
 for surrogate recoveries , and are presented adjusted for tissue wet 
 weight , lipid content , and as percent of the total pcb 136 dose ( tables s7s11 ) .
enantiomeric fractions , 
 a measure of the atropisomeric enrichment of pcb 136 and its metabolites , 
 were determined on the same instrument described above .
if not stated 
 otherwise ( see table s12 ) , enantioselective 
 determinations were performed with extracts from tissues or excreta 
 collected from individual animals .
briefly , pcb 136 and 5 - 136 atropisomers 
 were separated using a chiraldex bdm column ( bdm column , 30 m  
 250 m  0.12 m ; supelco , analytical , st .
the temperature program was as follows : 
 10 c / min from 100 to 148 c , hold for 400 min , 10 c / min 
 to 200 c , and hold for 13 min .
pcb 136 and 4 - 136 atropisomers 
 were separated with a cyclosil - b column ( cb column , 30 m  0.25 
 mm i d  0.25 m film thickness ; agilent , santa clara , ca , 
 us ) using a previously published temperature program .
to allow a comparison with previously published data , the ef values were determined as ef = area e2/(area e1 + area e2 ) , were area e1 and area e2 denote the peak area of the first and second eluting atropisomer . 

on both enantioselective columns , e1-pcb 136 and e2-pcb 136 correspond to ( )-pcb 136 and ( + ) -pcb 136 , 
 respectively .
the e1- and e2-atropisomers of 5 - 136 ( bdm column ) and 4 - 136 ( cb column ) 
 are formed from ( )- and ( + ) -pcb 136 , respectively .
the ef values of pcb 136 , 5 - 136 , and 4 - 136 atropisomers 
 from different tissues are summarized in table 
 s12 .
the ni-ecds used for the pcb and ho - pcb analysis 
 were linear up 
 to concentrations of 1,000 ng / ml for all analytes investigated ( r > 0.999 ) .
a detailed summary of the limits 
 of detection , limits of quantification , and background levels of pcb 
 136 and its metabolites is presented in tables s5 and s6 , supporting information .
the recovery of pcb 117 
 and 4-159 was 94  7% ( range : 84102 ) and 95  
 6% ( range : 83105 ) , respectively .
the average resolution of pcb 136 , 5 - 136 , and 4 - 136 atropisomers on 
 the enantioselective columns were 0.92 ( bdm column or 0.87 ( cb column ) , 
 0.69 , and 0.75 , respectively .
the ef values for the racemic standards 
 of pcb 136 on the bdm column and the cb column , 5 - 136 on the bdm column , 
 and 4 - 136 on the cb column were 0.502  0.001 , 0.505  0.004 , 
 0.513  0.016 , and 0.499  0.011 , respectively .
the ef values 
 of pcb 136 determined on both enantioselective columns were not significantly 
 different from each other ( t test , p > 0.05 ) .
unless otherwise 
 stated , all data 
 are reported as mean  one standard deviation .
differences in 
 biological measurements , tissue pcb and ho - pcb levels , and ef values 
 between different treatment groups as well as differences of ef values 
 from the respective racemic standard were tested with two - sample , 
 two - tailed student s t test .
ko mice had consistently 
 higher pcb levels in tissues and excreta than wt mice , presumably 
 due to defective hepatic pcb 136 metabolism . pcb
136 levels , expressed 
 as percent of the total dose , decreased in the rank order adipose 
 > liver > brain > blood in both wt and ko mice ( figure 2 ) .
the same rank order was observed when the pcb 
 levels were 
 adjusted for wet weight or extractable lipid content ( tables s7 and s9 ) .
the pcb 136 tissue distribution 
 in wt mice was comparable to similar studies in c57bl/6 mice .
pcb 136 levels in feces and urine decreased from day 1 to day 3 , which 
 is also consistent with earlier pcb 136 disposition studies in rodents .
female mice with defective hepatic metabolism 
 due to liver - specific 
 deletion of cytochrome p450 oxidoreductase ( ko ) have significantly 
 higher levels of pcb 136 in tissues and feces compared to age - matched 
 congenic wild type mice ( wt ) .
data represent the mean  standard 
 deviation of the pcb 136 levels determined in the tissues and excreta 
 of individual pcb l36-treated ko ( n = 7 ) and wt mice 
 ( n = 5 ) and are expressed on a logarithmic scale 
 as percent of the total pcb 136 dose ( see table 
 s11 for additional details ) . * significantly different from 
 wt ( t test , p < 0.05 ) ;   different from wt ( t test , 0.05  p < 0.1 ) ; nd , not detected .
phenotypic differences in liver metabolism also contributed 
 to 
 higher pcb 136 levels in the liver and feces from ko versus congenic 
 wt mice , thus resulting in a modest distribution of pcb 136 away from 
 other organs , such as the brain ; however , the overall effect of the 
 higher liver and feces levels was small and , at most , slightly decreased 
 systemic pcb 136 levels in ko mice .
consistent with previous studies , 
 ko mice have larger livers as well as higher fat and p450 protein 
 levels in the liver compared to congenic wt mice ( tables s1 , s3 , and s4 ) .
the differences in the liver size and composition likely contribute 
 to the retention of a considerable percentage of the total pcb 136 
 dose in the liver of ko versus wt mice ( 4.2% in ko versus 0.07% in 
 wt mice , respectively ) .
we also observed higher fecal lipid levels 
 in ko compared to wt mice , which is likely due to a decreased absorption 
 of fats caused by a lack of bile acid synthesis in ko mice . because systemic uptake , excretion , and reuptake 
 of pcbs are driven by their lumen - to - tissue concentration gradient , the higher gastrointestinal fat content in ko mice increased the 
 fecal excretion pcb 136 levels in ko compared to wt mice .
specifically , 
 4.5% vs 1.0% of the total pcb 136 dose are excreted in the feces of 
 ko and wt mice , respectively .
an increased gastrointestinal fat content 
 due to non - absorbable fats , such as olestra , has also been shown to 
 increase the fecal excretion of pcbs and other lipophilic pollutants 
 in mice and humans . based on in vitro metabolism studies in different species ,
pcb 136 
 is metabolized 
 by hepatic p450 enzymes to several hydroxylated metabolites , including 
 5 - 136 , 4 - 136 , and 3 - 150 , a 1,2-shift metabolite of pcb 136 ( figure 1 ) . both 5 - 136 and 4 - 136 can be further metabolized by p450 enzymes 
 to a dihydroxylated metabolite , 4,5 - 136 . in agreement with these earlier metabolism studies , 5 - 136 ,
4 - 136 , 
 and 4,5 - 136 were detected in the liver , blood , and excreta from both 
 ko and wt mice ( figure 3 ) .
3 - 150 was a minor 
 metabolite in the liver but was not detected in blood from ko and 
 wt mice .
all ho - pcb metabolites were below the detection limit in 
 adipose and brain tissue . in an earlier disposition study ,
we were 
 also unable to detect ho - pcb metabolites of racemic pcb 95 in the 
 brain of adult c57bl/6 mice ; however , 
 other studies have detected ho - pcbs in the brain of cetaceans , polar bears , and 
 rats .
other metabolites , including methylsulfone 
 pcbs , pcb sulfates , and glucuronides were not analyzed in tissues 
 because suitable analytical methods and standards are not available .
comparison 
 of the levels of ( a ) 5 - 136 , ( b ) 4 - 136 , and ( c ) 4,5 - 136 
 in tissues and excreta from female mice with defective hepatic metabolism 
 ( ko mice ) and their age - matched congenic wild type mice ( wt ) .
pcb 
 136 is primarily eliminated as 5 - 136 , and to a lesser extent as ( b ) 
 4 - 136 and ( c ) 4,5 - 136 , in the feces of ko and wt mice ; however , the 
 time course of fecal ho - pcb metabolite and/or conjugate levels differ 
 significantly between ko and wt mice .
data represent the mean  
 standard deviation of the ho - pcb metabolites levels determined in 
 the tissues and excreta of individual , pcb l36-treated ko ( n = 7 ) and wt mice ( n = 5 ) and are expressed 
 on a logarithmic scale as percent of the total pcb 136 dose ( see table s11 for additional details ) . * significantly 
 different from wt ( t test , p < 
 0.05 ) ;   different from wt ( t test , 0.05 
  p < 0.1 ) ; nd , not detected .
5 - 136 , which is formed by rat , dog , and , likely , 
 mouse cyp2b enzymes , was the major metabolite in the liver from both ko and wt mice , 
 with 5 - 136 levels being 3-fold higher in the liver of ko mice ( figure 3a ) .
livers from ko mice also contained higher levels 
 of 4 - 136 and 4,5 - 136 compared to wt mice ( figures 3b and 3c ) , and a higher percentage 
 of the total pcb 136 dose were recovered as ho - pcbs from the liver 
 of ko compared to wt mice ( 0.045% vs 0.018% of the total pcb 136 dose , 
 respectively ) .
in contrast to a pcb 95 disposition study , ho - pcb levels in blood were typically lower 
 compared to liver levels .
blood levels of 5 - 136 and 4,5 - 136 were higher 
 in ko compared to wt mice ( figures 3a and 3c ) , whereas 4 - 136 levels were higher in wt compared 
 to ko mice ( figure 3b ) .
these differences between 
 ko and wt mice were observed both for ho - pcb levels adjusted for wet 
 weight or expressed as percent of the total pcb 136 dose ( tables s8 and s11 ) . the higher ho - pcb levels 
 in ko mice on day 3 are due to higher pcb 136 levels in ko mice and , 
 consequently , result in the formation of more ho - pcb metabolites in 
 ko than wt mice on day 3 .
similarly , a toxicokinetics study with cyclophosphamide 
 showed higher metabolite levels at certain time points due to the 
 altered toxicokinetics in ko vs wt mice .
the levels of all ho - pcbs detected 
 in feces decreased from day 1 to day 3 ( figure 3 ) .
since the intestinal flora has glucuronidase and sulfatase activity , it is unclear whether free ho - pcbs or the corresponding conjugates 
 were eliminated via the bile into the feces .
a recent study with 4-chlorobiphenyl 
 ( pcb 3 ) in rats suggests that ho - pcb conjugates were most likely eliminated 
 into the feces , followed by their efficient deconjugation by the intestinal 
 flora .
interestingly , the time course 
 of ho - pcb excretion differed between ko and wt mice , with fecal ho - pcb 
 levels decreasing more rapidly in wt compared to ko mice ( figure 3 ) .
these differences in the time course of fecal 
 ho - pcb excretion are consistent with a decreased hepatic pcb metabolism 
 and , as a result , a higher internal pcb dose in ko mice . over 
 the entire three - day study period
, 32% and 27% of the total pcb 136 
 dose was recovered as ho - pcbs from the feces of wt and ko mice , respectively 
 ( table s11 ) .
similarly , a large percentage 
 of pcb 136 was excreted as metabolites in the feces of senescent rats .
5 - 136 was the major metabolite recovered from 
 feces , with 26% and 24% of the total pcb 136 dose being excreted as 
 5 - 136 in the feces from wt and ko mice , respectively ( table s11 ) .
the high levels of 5 - 136 recovered 
 from feces indicate a major role of cyp2b enzymes in the elimination 
 of pcb 136 in mice .
a smaller percentage of the total pcb 136 dose 
 was recovered from feces as 4 - 136 ( 5.9% and 2.3% for wt and ko mice , 
 respectively , table s11 ) .
the large percentage 
 of ho - pcbs recovered over the three day study period from feces of 
 ko mice is surprising because , as we ( see table 
 s1 ) and others have shown , ko 
 mice have essentially no hepatic cpr activity and , therefore , should 
 not be able to metabolize pcb 136 in the liver ; however , other xenobiotics , 
 such as cyclophosphamide , also undergo 
 considerable biotransformation in ko mice .
extrahepatic metabolism 
 of pcb 136 is the most likely explanation for the high fecal levels 
 of ho - pcbs or their conjugates observed in our study . indeed , p450 
 enzymes are expressed and functional in extrahepatic tissues in many 
 organisms , including fish and mammals .
. however , we can not completely rule 
 out that liver cells other than hepatocytes metabolize pcbs in ko 
 mice or that the cytochrome b5 electron 
 transfer pathway may support low levels of p450 enzyme activity , thus 
 resulting in the formation of ho - pcbs .
moreover , 
 we can not exclude the possibility that pcb 136 is differentially metabolized 
 in ko vs wt by the gut microbiome . an earlier study in rats reported 
 that urine was only a minor route of pcb 136 excretion , with most 
 of the pcb 136 excreted as metabolites ( > 99% ) .
recent studies of 4-chlorobiphenyl metabolism in rats suggest 
 that these urinary metabolites are likely pcb conjugates .
levels of ho - pcbs were , therefore , quantified in mouse urine with 
 or without -glucuronidase / sulfatase deconjugation to investigate 
 if pcb conjugates are also excreted with the urine in mice .
urine 
 was a minor route of excretion of ho - pcbs ( figure 3 ) and the corresponding sulfate and/or glucuronide conjugates 
 ( figure 4 ) , accounting for < 0.2% of the 
 total pcb 136 dose ( table s11 ) .
after deconjugation , 
 5 - 136 , 4 - 136 , and 4,5 - 136 , but not 3 - 150 , were detected in day 1 and , 
 to some extent , day 2 urine samples ( figure 3 ) .
-glucuronidase / sulfatase 
 treatment significantly increased urinary 5 - 136 and 4,5 - 136 but not 
 4 - 136 levels in urine samples ( figure 4 ) , which 
 demonstrates that some ho - pcb 136 metabolites , such as 5 - 136 and 4,5 - 136 , 
 are further metabolized to the corresponding sulfate and/or glucuronide 
 conjugates in mice .
there is growing evidence that sulfate conjugates 
 of ho - pcbs in particular are important metabolites formed by plants , wildlife , or laboratory animals .
further studies are needed to further characterize 
 the conjugates , their chiral signatures , and assess their role in 
 pcb developmental neurotoxicity in wildlife and laboratory animals .
treatment 
 with -glucuronidase / sulfatase significantly increased 
 the levels of 5 - 136 and 4,5 - 136 in day 1 urine from ( a ) female wild 
 type ( wt ) mice and ( b ) female mice with a liver - specific deletion 
 of the cpr gene ( ko ) , suggesting the presence of glucuronide and/or 
 sulfate conjugates of both ho - pcbs in urine .
data represent the mean 
  standard error of the mean of the ho - pcb metabolites levels 
 determined in urine from individual , pcb l36-treated mice .
* significantly 
 higher ho - pcb levels following deconjugation ( t test , p < 0.05 ) ;   significantly higher 4,5 - 136 
 levels following deconjugation in wt mice compared to ko mice ( t test , p < 0.05 ) ; nd , not detected .
the enantiomeric fractions 
 of pcb 136 were determined to assess whether 
 decreased hepatic metabolism alters the atropselective disposition 
 and excretion of pcb 136 . in both ko and wt mice ,
the ef values of 
 pcb 136 were > 0.5 in all tissues and excreta investigated , with 
 ef 
 values significantly different from the racemic standards ( t test , p < 0.05 ) ( figure 5 ) .
this direction of atropisomeric enrichment is consistent 
 with the enrichment of ( + ) -pcb 136 reported previously for mice .
the most pronounced atropisomeric enrichment was observed in liver 
 samples , with ef values of 0.70 and 0.74 in ko and wt mice , respectively . 

ef values decreased in the order liver > blood  brain > 
 adipose . 

several other disposition studies have also reported the most pronounced 
 atropisomeric enrichment of pcb 136 in liver and a less pronounced 
 enrichment in adipose tissue .
interestingly , the 
 ef values of pcb 136 in different tissues were by and large not significantly 
 different between ko and wt mice ( table s12 ) , i.e. , reduced hepatic pcb metabolism did not alter the chiral 
 signature of pcb 136 in the mouse , at least at the time point investigated . 

vice versa , increased hepatic pcb metabolism due to the induction 
 of cyp2b enzymes with phenobarbital also had no significant effect 
 on chiral pcb 136 signatures in mice . because extrahepatic p450 enzyme activities are probably not affected 
 by the liver - specific deletion of the cpr gene ( this 
 study ) or phenobarbital treatment , a 
 contribution of extrahepatic metabolism to the biotransformation of 
 pcb 136 may explain why changes in the metabolic capacity of the liver 
 have no clear effect on chiral signatures in mice ; however , additional 
 studies are needed to further explore this hypothesis .
comparison of the enantiomeric 
 fractions ( efs ) of pcb 136 ( a ) , 
 5 - 136 ( b ) , and 4 - 136 ( c ) in tissues and feces reveals significant 
 differences in the atropisomeric enrichment between female mice with 
 a liver - specific deletion of the cpr gene ( ko ) and 
 congenic wild type ( wt ) mice .
the ef values are presented as the mean 
  standard deviation and were determined in the tissues of individual 
 pcb l36-treated ko ( n = 7 ) and wt mice ( n = 5 ) .
the only exceptions are the ef determination for 5 - 136 in 
 blood , which were performed with a single sample pooled by genotype . 

a cb column was used to separated pcb 136 and 4 - 136 , whereas a bdm 
 column was used for separation of 5 - 136 ( see table 
 s12 for additional details ) . * significantly different from 
 wt ( t test , p < 0.05 ) ;   different from wt ( t test , 0.05  p < 0.1 ) ; nd , not detected ; the dotted line indicates 
 the ef values of the respective racemic standard .
several recent studies have reported that hydroxylated metabolites 
 of chiral pcbs are formed atropselectively in mice . in agreement with these earlier studies ,
5 - 136 and 4 - 136 displayed 
 atropisomeric enrichment in tissues and excreta from both ko and wt 
 mice .
remarkably , the extent and direction of the enrichment was different 
 between ko and wt mice ( figures 5b and 5c ) .
e2 - 5 - 136 was enriched in the liver 
 of ko mice , whereas 5 - 136 was near racemic in liver samples from wt 
 mice ( figure 5b ) ; however , the difference in 
 the ef values between ko and wt mice in the liver did not reach statistical 
 significance ( t test , p = 0.058 ) .
in contrast , analysis 
 of pooled blood samples revealed an atropisomeric enrichment of e1 - 5 - 136 in the blood from ko mice .
a much less pronounced enantiomeric 
 enrichment of e1 - 5 - 136 was present in pooled blood samples 
 from wt animals .
e2 - 4 - 136 was enriched in blood and liver 
 samples of ko and wt mice ( figure 5c ) , with 
 wt mice displaying a more pronounced atropisomeric enrichment in blood 
 and liver compared to ko mice .
interestingly , the direction 
 of the atropisomeric enrichment of ho - pcbs in the liver was different 
 compared to the enrichment observed in in vitro metabolism 
 studies .
while e2 - 5 - 136 and e2 - 4 - 136 were typically 
 enriched in the liver from ko and wt mice , e1 - 5 - 136 and 
 e1 - 4 - 136 were preferentially formed in studies using mouse 
 liver microsomes and tissue slices .
in contrast , in vitro models 
 generally predict the direction of the atropisomeric enrichment of 
 the parent pcb in vivo , which is consistent with 
 a major role of p450 enzymes in the atropisomeric enrichment of pcbs in vivo .
it is therefore likely that , in addition to their 
 atropselective formation by p450 enzymes , other processes play a role 
 in the atropselective disposition of ho - pcb metabolites .
the ef values 
 of 5 - 136 and 4 - 136 in feces samples changed over 
 time ( figures 5b and 5c ) .
e1 - 5 - 136 and e1 - 4 - 136 were enriched in 
 day 1 feces samples from both ko and wt mice , with ko mice displaying 
 significantly more pronounced atropisomeric enrichment compared to 
 wt mice .
ef values of both 5 - 136 and 4 - 136 changed from day 1 to day 
 3 feces samples , with ef values in ko mice always being significantly 
 smaller compared to wt mice . in ko mice , e1 - 5 - 136 and e1 - 4 - 136 were enriched in day 2 feces samples , whereas both 
 metabolites were near racemic in day 3 feces samples .
in contrast , 
 5 - 136 was near racemic and e2 - 4 - 136 was enriched in day 
 2 and day 3 feces samples from wt mice .
ef values of ho - pcb could 
 only be determined in day 1 urine samples .
consistent with day 1 feces 
 samples , pooled day 1 urine samples also displayed an enrichment of 
 e1 - 5 - 136 and e1 - 4 - 136 ( table 
 s12 ) .
differences in the subsequent atropselective phase 
 ii metabolism of ho - pcbs as well as the active transport of ho - pcbs 
 and their glucuronide or sulfate conjugates are one possible explanation 
 for these differences .
for example , we have shown that ho - pcbs are 
 atropselectively oxidized to dihydroxylated metabolites .
moreover , differences in the atropselective 
 biotransformation of ho - pcb 136 metabolites to sulfate and/or glucuronide 
 conjugates may contribute to the differences in the chiral ho - pcb 
 signatures between wt and ko mice . indeed
, earlier studies have demonstrated 
 the compensatory induction of various drug metabolizing enzymes ( e.g. , 
 glucuronosyltransferases ) in ko mice .
in addition 
 to phase ii metabolism , differences in the atropselective active transport 
 of the ho - pcb and/or their conjugates may explain why the extent and 
 direction of the atropisomeric enrichment of the ho - pcb metabolites 
 in feces samples changed over time and differed between ko and wt 
 mice .
further studies to understand the atropselective disposition 
 of pcb metabolites are therefore needed in terrestrial and aquatic 
 organisms , especially considering the fact that ho - pcb and , possibly 
 their conjugates , are also toxic . to date
, most pcb metabolism 
 studies have focused on mammalian p450 enzymes ; however , there is growing evidence 
 that p450 enzymes contribute to the biotransformation of pcbs in plants , fish , birds , and marine mammals .
as suggested by our results , it is likely that 
 hepatic and possibly extrahepatic metabolism by p450 enzymes plays 
 a major role in the biotransformation of pcbs and structurally related 
 compounds in wildlife , laboratory animals , and humans and contributes 
 to the atropisomeric enrichment of pcbs observed in wildlife .
moreover , ho - pcbs can undergo phase ii biotransformation 
 to glucuronides and sulfates in laboratory animal models and in environmentally relevant species , such 
 as polar bear .
our findings in mice suggest 
 that , in addition to p450 enzymes , phase ii biotransformation represents 
 a currently unexplored factor in the atropselective disposition of 
 chiral ho - pcb .
further understanding these biotransformation processes 
 is important because ho - pcbs are potentially toxic and contribute 
 to the adverse effects of pcbs in the environment and in humans .
since 
 animal models with a deletion or knockdown of genes of key enzymes 
 involved in pcb biotransformation ( e.g. , cpr or p450 
 enzymes ) can be generated for various species , including fish and mice , such models are powerful tools 
 to explore the importance of hepatic vs extrahepatic metabolism in 
 pcb disposition in wildlife and laboratory animals .
furthermore , these 
 types of models may be useful for studying how metabolism modulates 
 the developmental neurotoxicity of pcbs and other environmental contaminants . 

the feasibility of such studies is indicated by our observation that 
 genetic deletion of cpr in the liver does not significantly 
 alter neurodevelopment as determined by an assessment of synaptic 
 plasticity genes in the brains of ko vs wt mice ( table s2 ) .
 to understand the role of hepatic 
 vs extrahepatic metabolism in 
 the disposition of chiral pcbs , we studied the disposition of 2,2,3,3,6,6-hexachlorobiphenyl 
 ( pcb 136 ) and its hydroxylated metabolites ( ho - pcbs ) in mice with 
 defective hepatic metabolism due to the liver - specific deletion of 
 cytochrome p450 oxidoreductase ( ko mice ) . 
 female ko and congenic wild 
 type ( wt ) mice were treated with racemic pcb 136 , and levels and chiral 
 signatures of pcb 136 and ho - pcbs were determined in tissues and excreta 
 3 days after pcb administration . 
 pcb 136 tissue levels were higher 
 in ko compared to wt mice . 
 feces was a major route of pcb metabolite 
 excretion , with 2,2,3,3,6,6-hexachlorobiphenyl-5-ol 
 being the major metabolite recovered from feces . 
 ( + ) -pcb 136 , the 
 second eluting pcb 136 atropisomers , was enriched in all tissues and 
 excreta . 
 the second eluting atropisomers of the ho - pcbs metabolites 
 were enriched in blood and liver ; 2,2,3,3,6,6-hexachlorobiphenyl-5-ol 
 in blood was an exception and displayed an enrichment of the first 
 eluting atropisomers . 
 fecal ho - pcb levels and chiral signatures changed 
 with time and differed between ko and wt mice , with larger ho - pcb 
 enantiomeric fractions in wt compared to ko mice . 
 our results demonstrate 
 that hepatic and , possibly , extrahepatic cytochrome p450 ( p450 ) enzymes 
 play a role in the disposition of pcbs . 
Introduction
Experimental Procedures
Results
and Discussion