polycyclic aromatic 
 hydrocarbons ( pahs ) are characterized by the 
 presence of two or more fused benzene rings arranged in various configurations .
pahs are ubiquitous airborne environmental pollutants 
 that arise from the incomplete combustion of fossil fuels and are 
 also present in automobile exhaust and first- and secondhand cigarette 
 smoke .
pahs are one of the major classes of carcinogens found in tobacco 
 smoke and are suspect lung carcinogens .
benzo[a]pyrene 
 ( b[a]p ) , the most studied pah , has been recently 
 upgraded to a group 1  known human carcinogen  by the 
 international agency for research on cancer .
pahs are not biological reactive , and the biotransformation of 
 pahs to electrophilic metabolites is required to elicit their tumorigenic 
 effects .
there are three major routes 
 for pah activation which include the formation of radical cations , diol epoxides , and electrophilic 
 and redox - active o - quinones . in the o - quinone pathway , akrs catalyze the oxidation 
 of proximate pah carcinogens , trans - dihydrodiols , 
 to yield ketols which spontaneously rearrange to catechols .
pah catechols 
 are not stable and undergo autooxidation to form pah o - quinones with the concomitant production of reactive oxygen species 
 ( ros ) ( figure 1 ) .
pah o - quinones 
 are electrophilic and highly reactive with endogenous nucleophiles 
 and yield l - cysteine , n - acetyl - l - cysteine , and gsh conjugates .
electrophilic 
 pah o - quinones can also form both depurinating dna 
 adducts in vitro(13 ) and 
 stable covalent dna adducts in vitro and in human 
 lung cells .
apart from their electrophilicity , pah o - quinones are also redox active and undergo nonenzymatic or enzymatic 
 reduction to reform catechols at the expense of consuming nadph .
enzymes 
 that contribute to this redox cycling include nad(p)(h):quinone oxidoreductase 
 ( nqo1 ) , carbonyl reductases ( cbr1 and cbr3 ) , and akrs themselves . 

ros produced by redox cycling of the pah o - quinones mediates dna damage and can lead to 7,8-dihydro-8-oxo-2deoxyguanosine 
 ( 8-oxo - dguo ) lesions which contribute to g - to - t transversions 
 in p53 .
it was found that even 
 nanomolar concentrations of pah o - quinones were able 
 to generate sufficient ros to cause a significant increase in 8-oxo - dguo .
metabolic 
 activation of b[a]p by akrs and detoxication 
 of b[a]p-7,8-dione by ugts . using b[a]p as a representative pah and 
 stable 
 isotope dilution liquid chromatography  tandem mass spectrometry
, 
 we found that all three pathways of pah activation were functional 
 in human bronchoalveolar h358 cells .
also , 
 using a549 cells which show high constitutive expression of akrs , 
 we found that b[a]p-7,8-trans - dihydrodiol 
 ( an akr substrate ) was converted to b[a]p-7,8-dione 
 and that the ros produced increased the level of 8-oxo - dguo in cellular 
 dna as measured by stable isotope dilution liquid chromatography 
importantly , the level 
 of 8-oxo - dguo produced from b[a]p-7,8-trans - dihydrodiol was elevated further in the presence of a comt inhibitor 
 suggesting that a redox cycle was occurring .
these observations led to a systematic study of the role of 
 phase 
 ii conjugating enzymes in intercepting pah - catechols to prevent redox 
 cycling .
human catechol - o - methyl transferase ( comt ) 
 and sulfotransferases ( sult ) 1a1 , 1a3 , and 1e1 were able to detoxify 
 b[a]p-7,8-dione by intercepting b[a]p-7,8-catechol through the formation of o - methylated - b[a]p-7,8-catechol and o - sulfated - b[a]p-7,8-catechol , respectively .
another superfamily of phase ii metabolic enzymes , uridine diphosphate 
 glucuronosyltransferases ( ugts ) , are microsomal enzymes which catalyze 
 the transfer of the glucuronosyl group from uridine 5-diphospho - glucuronic 
 acid ( udpga ) to substrates that contain alcohols , amines , or carboxylic 
 acids as functional groups .
ugts are 
 divided into two main subfamilies ugt1 and ugt2 based on amino acid 
 sequence identity and are responsible 
 for the glucuronidation of a variety of endogenous compounds and xenobiotics .
ugts are widely distributed in a variety of 
 tissues , including the liver , intestine , brain , and kidney , and the 
 aerodigestive tract , etc . ugt1a7 , ugt1a8 , 
 ugt1a9 , and ugt1a10 and ugt2b7 are active against several pah metabolites , while ugt1a10 > ugt1a9 > ugt1a1 > ugt1a7 are the preferred 
 isoforms 
 for catalyzing the glucurondiation of b[a]p-7,8-trans - dihydrodiol
. however , 
 the glucuronidation of pah catechols by ugts has not been previously 
 examined .
studies of b[a]p-7,8-dione metabolism 
 and disposition 
 in three human lung cells hbec - kt , h358 , and a549 cells showed rapid 
 disappearance of the quinone accompanied by the formation of phase 
 ii conjugates and a n1 or n3-adenine adduct originating from the nucleotide 
 pool . in a549 cells , one of the phase 
 ii conjugates was o - monoglucuronsyl - b[a]p-7,8-catechol indicating that ugts play a role in the interception / detoxication 
 of pah catechols . in the present study , we investigated whether glucuronidation 
 catalyzed by human ugts is a feasible detoxication pathway for b[a]p-7,8-dione and identified two major enzyme isoforms responsible 
 for the glucuronidation of b[a]p-7,8-catechol .
7-hydroxy-4-(trifluoromethyl)coumarin(hfc ) , 
 alamethicin , and uridine-5-diphosphoglucuronic acid ( udpga ) 
 were purchased from sigma - aldrich co. ( st . louis , mo ) .
[ c]-uridine-5diphosphoglucuronic acid ( 180 mci / mmol ) was purchased 
 from perkin elmer inc .
all solvents were of hplc 
 grade , and all other chemicals used were of the highest grade available . 

ugt1a1 , 1a3 , and 2b7 supersomes ( microsomes from baculovirus infected 
 insect cells expressing ugts ) were obtained from bd biosciences ( san 
 jose , ca ) and titered before use , using standard substrates .
a549 cells ( human 
 lung adenocarcinoma cells ) were from american type culture collection 
 ( atcc number ccl-185 ) and cultured in f-12k medium ( kaighn s 
 modification ) with supplementation of 10% heat - inactivated fbs , 2 
 mm l - glutamine , 100 units / ml penicillin , and 100 g / ml 
 streptomycin .
hepg2 cells ( hepatoma cells ) from atcc were cultured 
 in eagle s minimal essential medium supplemented with 10% heat - inactivated 
 fetal bovine serum , 1% l - glutamine , and 100 units / ml penicillin / streptomycin 
 solution .
h358 cells ( human bronchoalveolar cells ) were purchased 
 from american type culture collection ( atcc number crl-5807 ) and cultured 
 in rpmi 1640 medium containing 10% heat - inactivated fbs , 2 mm l - glutamine , 100 units / ml penicillin , and 100 g / ml streptomycin . 

hbec - kt cells ( immortalized human bronchial epithelial cells ) originating 
 from a patient without lung cancer were kindly provided as a gift 
 by dr .
john minna at university of texas southwestern medical center 
 and cultured in keratinocyte - serum - free medium with 0.10.2 
 ng / ml recombinant egf , 2030 g / ml bovine pituitary extract , 
 and 2 mm l - glutamine .
beas-2b cells ( normal human bronchial 
 epithelium cells ) were obtained from american type culture collection 
 ( atcc number crl-9609 ) and cultured in begm bronchial epithelium medium 
 ( cambrex cc-3170 ) .
cells were maintained at 37 c in a humidified 
 atmosphere containing 5% co2 and 95% o2 .
cultured 
 cells used in the experiments were confined to passage numbers of 
 1020 .
extraction of total rna from each cell line was 
 conducted using the 
 rneasy kit ( qiagen , valencia , ca ) .
reverse transcription ( rt ) was 
 conducted by using geneamp rna pcr core kit according to the manufacturer s 
 protocol ( applied biosystems , carlsbad , ca ) .
an aliquot of 1 l 
 of cdna synthesized in the above rt reaction was used for pcr .
the 
 pcr system ( 25 l ) contained 1 pcr buffer ( 10 mm tris - hcl 
 buffer , ph 8.3 , and 50 mm kcl ) , 2.5 mm mgcl2 , 250 m 
 dntps , 0.2 m primers , and 0.5 u of taq dna polymerase ( applied 
 biosystems , carlsbad , ca ) .
the sequences of the forward and reverse 
 primer pairs for the amplification of ugt1a1 , 1a3 , 1a7 , 1a8 , 1a9 , 
 1a10 , and 2b7 and -actin transcripts are shown in table 1 .
pcr amplification was performed with a mycycler thermal 
 cycler pcr system ( bio - rad , ca ) using the following protocol .
after 
 an initial denaturation step at 94 c for 3 min , amplification 
 was conducted by denaturation at 95 c for 30 s , annealing at 
 61 c ( for ugt1a1 ) , 63 c ( for ugt1a8 ) , or 57 c ( for 
 other ugt isoforms ) for 30 s , and extension at 72 c for 45 s 
 for 35 cycles . the final extension reaction was performed at 72 c 
 for 7 min .
the control 
 samples were amplified using the same conditions as those described 
 above .
an aliquot of 20 l of pcr products was analyzed by electrophoresis 
 in 2% agarose gel with ethidium bromide and visualized under uv light .
the reverse primer for the amplification 
 of ugt2b7 is located between 931 - 912 nts .
the same exon-3 derived antisense 
 primer was used for all rt - pcr amplification of family 1 ugts .
ugt enzyme assays were 
 conducted as recommended by bd biosciences ( san jose , ca ) .
briefly , 
 the reaction system was composed of 50 mm tris buffer of ph 7.4 , 10 
 mm mgcl2 , 1 mm udpga , 0.025 mg / ml alamethicin , 50 m 
 7-hydroxy-4-(trifluoromethyl)coumarin , and 10 g of ugt supersome 
 in a final volume of 100 l .
after incubation at 37 c for 15 min , the 
 reactions were terminated by the addition of 50 l of 94% acetonitrile/6% 
 glacial acetic acid and were centrifugated at 16,000 g for 10 min .
an aliquot of 100 l of supernatant was carefully 
 pipetted , and 50 l was injected into hplc / pda for the quantification 
 of 7-hydroxy-4-(trifluoromethyl)coumarin glucuronide .
the reversed - phase 
 column ( agilent zorbax - ods c18 , 5 m , 4.6  250 mm , ca ) 
 was used for the separation of substrate from product .
elution conditions 
 used a flow rate of 1 ml / min with 80%:20% water / methanol ( v / v ) containing 
 0.1% formic acid .
the methanol concentration was increased from 20% 
 to 80% over 3 min and kept at 80% methanol for 4 min , then changed 
 back to 20% methanol in 1 min followed by a re - equilibration phase 
 of 7 min at 20% methanol .
the experiments were conducted 
 in 1.5 ml amber glass vials with polytetrafluoroethylene / silicone 
 septa closures .
the reaction system was composed of 10 mm kpo4 buffer at ph 7.4 , 1.0 mm dithiothreitol , 5.0 mm mgcl2 , 0.025 mg / ml alamethicin , 1 mm [ c]-udpga ( 4 
 dpm / pmol ) , 020 m b[a]p-7,8-dione , 
 and 1530 g of microsomes containing human recombinant 
 ugts in a final volume of 0.2 ml .
the reactions were initiated by 
 the addition of udpga and incubated for 30 min ( for ugt 1a1 ) or 60 
 min ( for ugt 1a3 and 2b7 ) at 25 c . the incubation time and amount 
 of enzyme used were always in the linear range as determined by plots 
 of initial velocity versus incubation time or enzyme concentration . 

the reactions were terminated by the addition of 50 l of ice - cold 
 1% formic acid and were chilled on ice .
the reaction mixtures were 
 extracted with 0.5 ml aliquots of ethyl acetate twice by vortex mixing 
 and centrifuged at 16,000 g to help phase separation . 

the combined ethyl acetate layer was backwashed with 0.2 ml of 1% 
 formic acid by vigorous vortexing and centrifuged at 16,000 g .
the ethyl acetate was then dried by a speedvac concentrator 
 ( thermo scientific ) .
the residue was dissolved in 100 l of 
 methanol and analyzed by scintillation counting or by hplc analysis . 
 kinetic analyses using nonlinear regression
were performed by fitting 
 the michaelis  menten equation to the data with the program 
 grafit , where v is the initial velocity 
 of the reaction , [ s ] is the molar concentration of the substrate , 
 and km is the michaelis 
because of the iterative fits of the equations 
 to each data set , each fit provided estimates of the kinetic parameters 
 as a mean  se . because it is not possible to normalize vmax values to ugt protein expression , vmax values are apparent values only based on 
 total protein .
b[a]p-7,8-dione was prepared in hbss 
 buffer containing 1 mm sodium pyruvate at final concentration of 2 
 m with 0.2% dmso and then was used to treat a549 , hbec - kt , 
 and h358 cells ( 5  10 ) at confluency .
the media 
 were collected at 0 and 24 h and acidified subsequently with 0.1% 
 formic acid before extraction with 2  1.5-fold volume of cold 
 h2o - saturated ethyl acetate .
the organic phases of ethyl 
 acetate were combined and dried by a speedvac concentrator ( thermo 
 scientific ) .
the o - glucuronsyl - b[a]p-7,8-catechol conjugates formed in ugt reaction systems 
 were analyzed by a waters alliance 2695 chromatographic system ( waters 
 corp . ,
milford , ma ) in tandem with a waters 996 photodiode array detector 
 and a -ram inline radiometric detector ( in / us systems inc . , 
 tampa , fl ) or with a finnigan tsq quantum ultra spectrometer ( thermo 
 fisher , san jose , ca ) .
chromatographic separation was conducted on 
 a reverse - phase column ( agilent zorbax - ods c18 , 5 m , 4.6  
 250 mm , ca ) .
the c18 reverse - phase column was eluted with the following 
 linear gradient of h2o ( 0.1% formic acid ; solvent a)/meoh 
 ( solvent b ) at a flow rate of 0.5 ml / min .
solvent b was changed from 
 50 to 95% ( v / v ) over 15 min , kept at 95% over 10 min , changed from 
 95 to 50% over 1 min , and kept at 50% for equilibration for 4 min . 

the o - glucuronsyl - b[a]p-7,8-catechols 
 formed in reactions with [ c]-udpga were eluted from the 
 c18 reverse - phase column and introduced into the inline radiometric 
 detector using a mixture of the scintillant with the hplc effluent 
 at a flow rate 1.5 ml / min .
the o - glucuronsyl - b[a]p-7,8-catechols generated with unlabeled udpga were eluted 
 from the c18 reverse - phase column and analyzed by electrospray ionization 
 ms / ms .
the mass spectrometer analysis was carried out in the positive 
 or negative ion mode with the following parameters : spray voltage 
 ( 4500 v at positive ion mode or 2000 v at negative ion mode ) , 
 vaporizer temperature ( 400 c ) , sheath gas pressure ( 35 arbitrary 
 units ) , auxiliary gas pressure ( 10 arbitrary units ) , capillary temperature 
 ( 350 c ) , and collision energy ( 20 v ) .
the molecular masses of 
 the metabolites were acquired by detecting the molecular ion from 
 q1 full scan , and the corresponding mass spectrum of each metabolite 
 was obtained from a q3 full scan of the product ions of the molecular 
 ions .
we performed 
 rt - pcr to identify the ugt isoforms expressed in hepg2 cells and four 
 human lung cells .
the ugts selected were those that have been previously 
 characterized for the glucuronidation of b[a]p-7,8-trans - dihydrdodiol ( ugt1a10 > ugt1a9 > ugt1a1 > 
 ugt1a7 ) and the catechol estrogens ( ugt1a8 , 
 ugt1a9 , 
 and ugt2b7 ) . although rt - pcr is a semiquantitative 
 approach to analyze gene expression , we found that ugt1a1 , 1a3 , and 
 2b7 were expressed at the mrna level in hepg2 and a549 cells among 
 the seven different ugt isoforms examined ( figure 2 ) .
the current studies focused on the glucuronidation of b[a]p-7,8-catechol by recombinant human ugt1a1 , 1a3 , and 2b7 , 
 which were major ugt isoforms in a549 cells .
( hepg2 , hepatoma 
 cells ; a549 , human lung adenocarcinoma cells ; h358 , human bronchoalveolar 
 cells ; hbec - kt , immortalized human bronchial epithelial cells ; beas-2b , 
 normal human bronchial epithelial cells . ) supersomes ( bd - biosciences ) 
 containing overexpressed human recombinant ugts were tittered in standard 
 assays . the standard assay for ugt activity using 7-hydroxy-4-(trifluoromethyl)coumarin 
 as a substrate demonstrated that the specific activities of ugt1a1 , 
 1a3 , and 2b7 were 1.18 ( 0.8 ) , 0.96 ( 0.7 ) , and 1.74 ( 1.51 ) nmol of 
 7-hydroxy-4-(trifluoromethyl)coumarin glucuronide formed / min / mg , respectively , 
 which are comparable to those values previously reported as indicated 
 by the values in parentheses .
ugt1a1 , 
 1a3 , and 2b7 were all able to catalyze the glucuronidation of b[a]p-7,8-catechol in the presence of udpga ( figure 3a , b , c ) , while no conjugates were generated in the 
 absence of udpga ( figure 3d ) . ugt1a3 and
2b7 
 catalyzed the formation of one o - glucurosonyl - b[a]p-7,8-catechol ( m2 ) , while ugt1a1 catalyzed the formation 
 of two o - glucuronsyl - b[a]p-7,8-catechols 
 ( m1 , m2 ) .
the uv spectra of the two glucuronides of b[a]p-7,8-catechol ( m1 , m2 ) were quite different ( figure 4 ) . when they were compared with the uv spectrum of the o - glucuronsyl - b[a]p-7,8-catechol found 
 in a549 cell culture media ,
the formation 
 of glucuronides of b[a]p-7,8-catechol was confirmed 
 by conducting the glucuronidation studies in the presence of [ c]-udpga .
[ c]-o - glucuronsyl - b[a]p-7,8-catechol was detected with hplc - ram analysis ( figure 3 e ) .
hplc / uv / ram identification of b[a]p-7,8-catechol 
 glucuronide metabolites ( m1 and m2 ) .
b[a]p-7,8-catechol 
 ( 20 m ) was generated in situ under anaerobic 
 conditions in an incubation buffer containing 1 mm dithiothreitol . 

b[a]p-7,8-catechol was converted to the o - glucuronide(s ) by ugt supersomes supplemented with udpga ( a , ugt1a1 ; 
 b , ugt1a3 ; and c , ugt2b7 ) in the presence of udpga .
( e ) hplc - ram chromatogram 
 of o - monoglucuronsyl - b[a]p-7,8-catechol 
 formed by ugt1a3 in the presence of [ c]-udpga .
the delayed 
 retention time of the m2 metabolite is due to the fact that the radiometeric 
 detector is in - line and downstream from the absorbance ( pda ) detector .
b[a]p-7,8-catechol 
 ( 20 m ) was generated in situ under anaerobic 
 conditions in an incubation buffer containing 1 mm dithiothreitol . 

uv spectra of m1 and m2 were 
 collected by an in - line waters 996 photodiode array detector . to determine whether b[a]-7,8-catechol formed 
 mono- or bis - glucuronide conjugates ,
lc - ms / ms was used to further 
 characterize the structure the m1 and m2 metabolites .
the molecular 
 ions of both metabolites ( m1 , m2 ) were the same and showed a ( [ m + 
 h ] ) m / z = 461 in the 
 positive ion mode and a ( [ m  h ] ) m / z = 459 in the negative ion mode , respectively , 
 indicating that both m1 and m2 are o - monoglucuronsyl - b[a]p-7,8-catechols where the position of glucuronidation 
 is different .
ms / ms analysis provided further evidence to the identity 
 of the metabolites . in the positive ion mode , the product ions of 
 the two metabolites were identical , and characteristic scission under 
 collision - induced dissociation occurred at the c  o glycosidic 
 bond with the loss of 176 amu resulting from the loss of the monodehydrated 
 glucuronic acid moiety to yield a fragment ion m / z = 285 .
oh bonds resulted 
 in a daughter ion of m / z = 267 representing 
 the loss of h2o .
rearrangement resulting in a change of 
 the remaining phenolic group from a c  oh to c = o 
 bond is followed by the loss of c = o group which resulted 
 in a fragment ion at m / z = 239 . 
 in the negative ion mode
, product ion spectra of two metabolites also 
 demonstrated characteristic cleavage at the c  o glycosidic 
 bond with a loss of 176 amu resulting in daughter ions at m / z = 283 .
lc - ms / ms analyses in both the 
 positive and negative ion modes confirmed that the metabolites ( m1 
 and m2 ) formed in the reaction system were o - monoglucuronsyl - b[a]p-7,8-catechols .
since the mass spectra of m1 and m2 were 
 identical ( figure 5 ) , it was not possible to 
 distinguish the position of glucuronic acid conjugation in the two 
 isomers . however , our studies on the sulfonation of b[a]p-7,8-catechol catalyzed by sults also led to the formation of two 
 regioisomeric o - monosulfated catechols , where the 
 earlier eluting species was shown by 2d - nmr to be the o7-monosulfate .
thus , in this study the 
 later eluting m2 metabolite is tentatively assigned as the o8-monoglucuronsyl - b[a]p-7,8-catechol . 

lc - ms / ms in the selected reaction monitoring mode ( srm ) further confirmed 
 that ugt1a1 catalyzed the formation of isomeric o - monoglucuronsyl - b[a]p-7,8-catechols ( m1 , m2 ) and 
 ugt1a3 and that 2b7 catalyzed the formation of only one o - monoglucuronsyl - b[a]p-7,8-catechol ( m2 ) ( figure 6 ) .
lc / ms / ms identification of o - monoglucuronsyl - b[a]p-7,8-catechols ( m1 and m2 ) .
b[a]p-7,8-catechol 
 ( 20 m ) was generated in situ under anaerobic 
 conditions in the presence of 1 mm dithiothreitol .
b[a]p-7,8-catechol was converted to o - glucuronide(s ) 
 in the presence of udpga and ugts in the incubation buffer .
the 
 organic phase was then dried and dissolved in meoh for lc - ms / ms analysis 
 ( positive ion mode , ms spectrum of isomer m1 and ms spectrum of isomer m2 ) .
lc - ms / ms detection of o - monoglucuronsyl - b[a]p-7,8-catechols ( m1 and m2 ) formed by recombinant ugt1a1 
 ( a ) , 1a3 ( b ) , 2b7 ( c ) , and by a549 ( d ) at 24 h. b[a]p-7,8-catechol ( 20 m ) was generated in situ under anaerobic conditions in the presence of 1 mm dithiothreitol . 

b[a]p-7,8-catechol was converted to the o - glucuronide(s ) in the presence of udpga and ugts in the incubation 
 buffer ( a , ugt1a1 ; b , ugt1a3 ; and c , ugt2b7 ) .
( d ) b[a]p-7,8-dione ( 2 m , 0.2% dmso ) in hbss medium was incubated 
 with a549 cells and the culture media collected at 0 and 24 h , respectively . 

o - monoglucuronsyl - b[a]p-7,8-catechols were analyzed with lc - ms / ms in a negative ion mode 
 by monitoring the mass transition m / z 459  283 ( [ m  h ]
 [ m 
  h - glucuronic acid group ] ) . to elucidate the steady state 
 kinetic properties of the recombinant 
 ugts to glucuronidate b[a]p-7,8-catechol
, we performed 
 discontinuous assays to monitor the initial velocity of conjugate 
 formation using [ c]-udpga as the cofactor .
because of 
 substrate solubility , the substrate concentration was limited to 020 
 m .
apparent utilization ratios ( vmaxapp / km ) showed 
 that ugt1a1 supersomes were the most catalytically efficient for the 
 glucuronidation of b[a]p-7,8-catechol , followed by 
 ugt1a3 and 2b7 supersomes ( table 2 ) .
the km values of ugt1a1 and 1a3 were 9.6 and 8.5 
 m , respectively , which were slightly lower than that of ugt2b7 
 at 16.3 m indicating that the substrate concentration at which 
 half maximal velocity was observed was similar in each case . kinetic characterization 
 of glucuronidation of b[a]p-7,8-catechol by ugts . 

kinetic analyses were performed by fitting 
 the michaelis  menten equation to the data .
reactions contained 
 10 mm kpo4 buffer at ph 7.4 , 1.0 mm dithiothreitol , 5.0 
 mm mgcl2 , 1 mm [ c]udpga , 020 m 
 b[a]p-7,8-dione , and 1530 g of human 
 recombinant ugt supersomes at 25 c .
a , b , and c , velocity versus 
 [ s ] curve for the glucuronidation of b[a]p-7,8-catechol 
 by ugt1a1 , 1a3 , and 2b7 , respectively .
only one o8-monoglucuronsyl - b[a]p-7,8-catechol ( m2 ) was detected by lc - ms / ms in the srm mode in 
 a549 cells after incubation of the cells with 2 m b[a]p-7,8-dione for 24 h. by contrast , o - monoglucuronsyl - b[a]p-7,8-catechol was not detected in h358 and hbec - kt cell 
 lines , which was consistent with the low mrna level of ugts in these 
 cell lines ( figures 2 and 6d ) .
we have been conducting 
 a systematic study to elucidate the roles 
 of human enzymes in catalyzing the redox cycling of pah o - quinones and the roles of phase ii enzymes in conjugating pah catechols 
 that arise from the akr pathway of pah activation .
previous studies 
 have shown that akrs are efficient pah o - quinone 
 reductases and that the pah catechols 
 formed can be intercepted by comts and sults .
we now show that ugt1a1 , 1a3 , and 2b7 are all expressed in a549 
 cells . of these ,
ugt1a3 and 2b7 are the enzymes most likely involved 
 in b[a]p-7,8-catechol glucuronidation based on the 
 glucuronide product profile . while recombinant ugt1a1 expressed in 
 supersomes
produces two regioisomeric monoglucuronides with b[a]p-7,8-catechol , only one of the isomers is produced in 
 a549 cells and corresponds to the single isomer produced by ugt1a3 
 and ugt2b7 .
the monoglucuronide formed is tentatively assigned as o8-monoglucuronsyl - b[a]p-7,8-catechol based 
 on differences in retention time observed for the regioisomeric o - monosulfated b[a]p-7,8-catechols .
it is noteworthy that ugt2b7 is also the 
 dominant isoform involved 
 in the glucuronidation of the structurally related catechol estrogens .
ugt2b7 catalyzes the glucuronidation of 4-hydroxycatecholestrogens 
 ( hydroxylated estrone / estradiol ) , which are structurally similar to 
 b[a]p-7,8-catechol .
the 
 glucuronidation of 4-hydroxycatecholestrogens shows preference for 
 the c4-hydroxyl group , which would be equivalent to the c7 position 
 of b[a]p-7,8-catechol . although ugt1a1 was found 
 in the kinetic analysis to have the highest utilization ratio ( vmax / km ) and catalyzed 
 the formation of both regioisomeric b[a]p-7,8-catechol 
 glucuronides , no significant m1 was found in a549 cells indicating 
 that it did not contribute to the detoxication of this catechol in 
 these cells .
it is noteworthy that only a549 cells expressed 
 the ugt isoforms 
 studied to any extent .
the low expression of ugts might be anticipated 
 due to the low level of expression previously reported in human lung 
 specimens .
zheng at al . measured the expression of multiple ugts in 
 32 human lung tissues by duplex rt - pcr and found that ugt1a1 , ugt1a3 , 
 ugt1a4 , ugt1a6 , ugt1a7 , ugt1a8 , ugt1a9 , and ugt1a10 were not expressed .
similarly , ugt2b4 , ugt2b7 , ugt2b15 , and ugt2b17 
 were not detected . in later studies ,
dillinger et al . showed that 
 ugt1a10 was found in low amounts in human lung specimens . the difference in ugt
isoform expression observed 
 in our study versus that observed in the earlier work could reflect 
 differences in measuring ugt expression in human bronchial epithelial 
 cells versus whole lung tissue .
other ugts that could have been included 
 in our study would be ugt2b10 , but up until now , this has only been 
 shown in glucuronidate tobacco specific nitrosamines .
ugt1a1 , 
 ugt1a9 , and ugt2b7 , which are predominately expressed in the liver , 
 were found to form the 7s - monoglucuronide from ( )-b[a]p-7,8-trans - dihydrodiol , whereas the 
 extrahepatic ugt1a7 , ugt1a8 , and ugt1a10 could form either the 7r - monoglucuronide or the 8s - monoglucuronide , 
 i.e. , they produce two different enantiomers . by contrast ,
ugt1a1 formed two different regioisomeric b[a]p-7,8-catechol monoglucuronides , whereas ugt1a3 and ugt2b7 
 produced a single regioisomeric o - monoglucuronsyl - b[a]p-7,8-catechol .
the ability of ugts to form different 
 enantiomers from b[a]p-7,8-trans - dihydrodiol may be related to the fact that the dihydrodiol is nonplanar 
 and has both axial and equatorial alcohols . by contrast
no bis - glucuronide 
 has been observed with either b[a]p-7,8-trans - dihydrodiol or b[a]p-7,8-catechol possibly due 
 to steric hindrance .
we next compared the specific activities 
 for the redox cycling 
 of b[a]p-7,8-dione catalyzed by akrs , with the specific 
 activities observed for o - methylation , o - sulfation , and o - glucuronidation of b[a]p-7,8-catechol catalyzed by comts , sults , and ugts in table 3 .
these data reveal that the rates of redox cycling 
 are much greater than the rates of phase ii conjugating reactions . 

although some uncertainties exist in this comparison due to the differences 
 in expression levels of these enzymes in specific cells , it is unlikely 
 that these will account for the > 6,000-fold difference in the specific 
 activities of nqo1 and ugt1a3 to use b[a]p-7,8-dione 
 and b[a]p-7,8-catechol as substrates , respectively . 

thus , the ability to intercept the pah catechols and prevent redox 
 cycling is not an efficient process and could be easily overwhelmed 
 in a cellular environment .
this is supported by earlier work in which 
 we showed that b[a]p-7,8-trans - dihydrodiol 
 ( akr substrate ) and b[a]p-7,8-dione ( akr product ) 
 produced significant ros and 8-oxo - dguo formation in a549 cells .
although ugts were not detected 
 in hbec - kt , h358 , and beas-2b under 
 current culture conditions , the contribution of ugts in human lung 
 cells to b[a]p-7,8-catechol conjugation may be affected 
 by the induction of ugts .
it has been proposed that the expression level of ugt1a4 in the 
 human small airway epithelium was elevated as a result of nuclear 
 factor erythroid 2 p45-related factors ( nrf2 ) activation .
however , 
 ugt1a4 does not appear to be responsible for b[a]p-7,8-catechol 
 glucuronidation in our experiments to date .
it has also been demonstrated that ugt1a10 and ugt1a8 are coordinately 
 regulated by the aryl hydrocarbon receptor ( ahr ) and nrf2 and that 
 the nrf2 response requires the presence of ahr .
both pahs and pah o - quinones such as b[a]p-7,8-dione are the ligands of the ahr , which is required 
 for the induction of ugts by nrf2 . with the induction of 
 ugts ,
the akr1c genes involved in the formation and 
 redox cycling of pah o - quinones are also highly induced 
 by the nrf2-keap 1 system .
inducers that activate 
 nrf2 include electrophiles and ros and not surprisingly pah o - quinones .
thus , the induction of akr1c genes with the products of pah - trans - dihydrodiol 
 oxidation , namely , pah o - quinones , could lead to 
 an exacerbation of ros formation which may not be easily countered 
 by ugt induction .
 polycyclic 
 aromatic hydrocarbons ( pahs ) are environmental and tobacco 
 carcinogens . 
 proximate carcinogenic pah trans - dihydrodiols 
 are activated by human aldo - keto reductases ( akrs ) to yield electrophilic 
 and redox - active o - quinones . 
 interconversion among 
 benzo[a]pyrene ( b[a]p)-7,8-dione , 
 a representative pah o - quinone , and its corresponding 
 catechol generates a futile redox - cycle with the concomitant production 
 of reactive oxygen species ( ros ) . 
 we investigated whether glucuronidation 
 of b[a]p-7,8-catechol by human udp glucuronosyltransferases 
 ( ugts ) could intercept the catechol in three different human lung 
 cells . 
 rt - pcr showed that ugt1a1 , 1a3 , and 2b7 were only expressed 
 in human lung adenocarcinoma a549 cells . 
 the corresponding recombinant 
 ugts were examined for their kinetic constants and product profile 
 using b[a]p-7,8-catechol as a substrate . 
 b[a]p-7,8-dione was reduced to b[a]p-7,8-catechol 
 by dithiothreitol under anaerobic conditions and then further glucuronidated 
 by the ugts in the presence of uridine-5-diphosphoglucuronic 
 acid as a glucuronic acid group donor . 
 ugt1a1 catalyzed the glucuronidation 
 of b[a]p-7,8-catechol and generated two isomeric o - monoglucuronsyl - b[a]p-7,8-catechol products 
 that were identified by rp - hplc and by lc - ms / ms . 
 by contrast , ugt1a3 
 and 2b7 catalyzed the formation of only one monoglucuronide , which 
 was identical to that formed in a549 cells . 
 the kinetic profiles of 
 three ugts followed michaelis  menten kinetics . on the basis 
 of the expression levels of ugt1a3 and ugt2b7 and the observation 
 that a single monoglucuronide was produced in a549 cells 
 , we suggest 
 that the major ugt isoforms in a549 cells that can intercept b[a]p-7,8-catechol are ugt1a3 and 2b7 . 
Introduction
Materials and Methods
Results
Discussion