Detoxication of benzo[a]pyrene-7,8-dione by sulfotransferases (SULTS) in human lung cells

Article abstract of DOI:10.1074/jbc.M112.386052

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Human aldo-keto reductases catalyze the metabolic activation of proximate carcinogenic PAH transdihydrodiols to yield electrophilic and redox-active o-quinones. Benzo[a]pyrene-7,8-dione a representative PAH o-quinone is reduced back to the corresponding catechol to generate a futile redox-cycle. We investigated whether sulfonation of PAH catechols by human sulfotransferases (SULT) could intercept the catechol in human lung cells. RT-PCR identified SULT1A1, -1A3, and -1E1 as the isozymes expressed in four human lung cell lines. The corresponding recombinant SULTs were examined for their substrate specificity. Benzo[a]pyrene-7,8-dione was reduced to benzo[a]pyrene-7,8-catechol by dithiothreitol under anaerobic conditions and then further sulfonated by the SULTs in the presence of 3′-[³⁵S]phosphoadenosine 5′-phosphosulfate as the sulfonate group donor. The human SULTs catalyzed the sulfonation of benzo[a]pyrene-7,8-catechol and generated two isomeric benzo[a]pyrene-7,8-catechol O-monosulfate products that were identified by reversed phase HPLC and by LCMS/ MS. The various SULT isoforms produced the two isomers in different proportions. Two-dimensional ¹H and ¹³C NMR assigned the two regioisomers of benzo[a]pyrene-7,8-catechol monosulfate as 8-hydroxy-benzo[a]pyrene-7-O-sulfate (M1) and 7-hydroxy-benzo[a]pyrene-8-O-sulfate (M2), respectively. The kinetic profiles of three SULTs were different. SULT1A1 gave the highest catalytic efficiency (k_cat/K_m) and yielded a single isomeric product corresponding to M1. By contrast, SULT1E1 showed distinct substrate inhibition and formed both M1 and M2. Based on expression levels, catalytic efficiency, and the fact that the lung cells only produce M1, it is concluded that the major isoform that can intercept benzo[a]pyrene-7,8-catechol is SULT1A1.

Full text of DOI:10.1074/jbc.M112.386052

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 35, pp. 29909–29920, August 24, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Detoxication of Benzo[a]pyrene-7,8-dione by
□
S
Sulfotransferases (SULTs) in Human Lung Cells*
Received for publication, May 28, 2012, and in revised form, July 7, 2012 Published, JBC Papers in Press, July 9, 2012, DOI 10.1074/jbc.M112.386052
Li Zhang, Meng Huang, Ian A. Blair, and Trevor M. Penning1
From the Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Department of Pharmacology, Perelman
School of Medicine, University of Pennsylvania Philadelphia, Pennsylvania 19104-6084
Background: Benzo[a]pyrene-7,8-dione is a genotoxic metabolite produced by aldo-keto reductases.
Results: SULT1A1 is identified as the major SULT in lung cells involved in the detoxication of the corresponding catechol.
Conclusion: SULT1A1 may protect lung cells from the genotoxic benzo[a]pyrene-7,8-dione.
Significance: Polymorphisms in SULT1A1 may affect lung cancer susceptibility.
Polycyclic aromatic hydrocarbons (PAH)2 are ubiquitous
environmental pollutants that originate from fossil fuel com-
bustion and tobacco smoke and are suspect lung carcinogens
(1, 2). PAH are not biologically reactive and require metabolic
activation to elicit their deleterious effects; thus they are pro-
carcinogens (3). Benzo[a]pyrene (B[a]P) is a representative
PAH and is widely used to study the metabolic activation of
PAH (3, 4).
Polycyclic aromatic hydrocarbons (PAH) are environmental
and tobacco carcinogens. Human aldo-keto reductases catalyze
the metabolic activation of proximate carcinogenic PAH trans-
dihydrodiols to yield electrophilic and redox-active o-quinones.
Benzo[a]pyrene-7,8-dione a representative PAH o-quinone is
reduced back to the corresponding catechol to generate a futile
redox-cycle. We investigated whether sulfonation of PAH cat-
echols by human sulfotransferases (SULT) could intercept the
catechol in human lung cells. RT-PCR identified SULT1A1,
-1A3, and -1E1 as the isozymes expressed in four human lung
cell lines. The corresponding recombinant SULTs were exam-
ined for their substrate specificity. Benzo[a]pyrene-7,8-dione
was reduced to benzo[a]pyrene-7,8-catechol by dithiothreitol
under anaerobic conditions and then further sulfonated by the
SULTs in the presence of 3ꢀ-[35S]phosphoadenosine 5ꢀ-phos-
phosulfate as the sulfonate group donor. The human SULTs cat-
alyzed the sulfonation of benzo[a]pyrene-7,8-catechol and gener-
ated two isomeric benzo[a]pyrene-7,8-catechol O-monosulfate
products that were identified by reversed phase HPLC and by LC-
MS/MS. The various SULT isoforms produced the two isomers
There are three pathways for the activation of B[a]P. In the
first pathway, called the radical cation pathway, B[a]P is acti-
vated by one-electron oxidation catalyzed by either P450
monooxygenases or peroxidases to yield a radical cation at C6
of B[a]P, which leads to the formation of depurinating adducts
and results in G to T transversions (5, 6). The second pathway is
known as the diol-epoxide pathway catalyzed by cytochrome
P450s. In this pathway B[a]P is initially converted to (Ϫ)trans-
7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene (B[a]P-7,8-trans-
dihydrodiol), a proximate carcinogen intermediate by the
sequential actions of P4501A1/1B1 and epoxide hydrolase.
B[a]P-7,8-trans-dihydrodiol is then further monooxygenated
by P450 1A1/1B1 to yield the ultimate carcinogen (ϩ)-anti-7,8-
dihydroxy-9␣,10␣-epoxy-7,8,9,10-tetra-hydrobenzo[a]pyrene
(anti-B[a]PDE) (4, 7, 8). Anti-B[a]PDE is electrophilic and
reacts with dGuo to form (ϩ)-trans-anti-BPDE-N2-dGuo
adducts (9, 10), which are believed to be the cause of mutagen-
icity in bacterial and mammalian cells as well as tumorigenicity
in pulmonary adenomas of mice (11, 12). The third pathway is
termed the o-quinone pathway catalyzed by aldo-keto reducta-
ses (AKR). In this pathway, B[a]P-7,8-trans-dihydrodiol is oxi-
dized by AKR1A1 and AKR1C1–1C4 to yield a ketol that spon-
taneously rearranges to form B[a]P-7,8-catechol, which is not
stable and undergoes autooxidation to yield benzo[a]pyrene-
7,8-dione (B[a]P-7,8-dione) and the generation of reactive oxy-
gen species (ROS) (13–15) (Fig. 1).
1
in different proportions. Two-dimensional H and 13C NMR
assigned the two regioisomers of benzo[a]pyrene-7,8-catechol
monosulfate as 8-hydroxy-benzo[a]pyrene-7-O-sulfate (M1)
and 7-hydroxy-benzo[a]pyrene-8-O-sulfate (M2), respectively.
The kinetic profiles of three SULTs were different. SULT1A1
gave the highest catalytic efficiency (kcat/Km) and yielded a sin-
gle isomeric product corresponding to M1. By contrast,
SULT1E1 showed distinct substrate inhibition and formed both
M1 and M2. Based on expression levels, catalytic efficiency, and
the fact that the lung cells only produce M1, it is concluded that
the major isoform that can intercept benzo[a]pyrene-7,8-cate-
chol is SULT1A1.
B[a]P-7,8-dione is electrophilic and highly reactive with
endogenous nucleophiles. It readily forms thioether conjugates
* This work was supported, in whole or in part, by National Institutes of
Health Grants P30-ES-013508, R01-CA39504, and PA-DOH4100038714 2 The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; B[a]P,
(to T. M. P.).
This article contains supplemental Fig. S1.
benzo[a]pyrene; B[a]P-7,8-trans-dihydrodiol, (Ϫ)trans-7,8-dihydroxy-7,8-
dihydro-benzo[a]pyrene; AKR, aldo-keto reductase; B[a]P-7,8-catechol,
benzo[a]pyrene-7,8-catechol; B[a]P-7,8-dione, benzo[a]pyrene-7,8-dione;
COMT, catechol-O-methyl transferase; PAPS, 3Ј-phosphoadenosine 5Ј-phos-
phosulfate; SULT, sulfotransferase; ROS, reactive oxygen species; RAM, radio-
active monitor.
□S
1 To whom correspondence should be addressed: Dept. of Pharmacology,
University of Pennsylvania, School of Medicine, 3620 Hamilton Walk, Phil-
adelphia, PA 19104-6084. Tel.: 215-898-9445; Fax: 215-573-7188; E-mail:
penning@upenn.edu.
AUGUST 24, 2012•VOLUME 287•NUMBER 35
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Detoxication of PAH o-Quinones by SULT
FIGURE 1. Metabolic activation of B[a]P by AKRs and detoxication of B[a]P-7,8-dione by SULTs.
with L-cysteine, N-acetyl-L-cysteine, and GSH (16, 17). B[a]P- enzymes responsible for transfer of a sulfonate group from a
7,8-dione can also react with DNA to form both stable and 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) to either a
depurinating adducts (18–20). B[a]P-7,8-dione is also redox- hydroxyl moiety or an amine group (27, 28). On the basis of
active, and in the presence of NADPH it is reduced back to the sequence identity, human SULTs are divided into two major
catechol for subsequent rounds of autooxidation thereby families, SULT1 and SULT2, which are also termed the phenol
amplifying ROS in futile redox-cycles (21). Recently, we found sulfotransferase and the hydroxysteroid sulfotransferase fam-
that this process was exacerbated enzymatically by NQO1 and ily, respectively. The SULT1 family is further classified into
by AKRs themselves (21). Using a yeast-based p53 mutagenesis four subfamilies, phenol sulfotransferases (SULT1A), thy-
assay, we find that B[a]P-7,8-dione was more mutagenic than roid hormone sulfotransferases (SULT1B), hydroxyaryl-
diol-epoxides provided that the o-quinone was allowed to amine sulfotransferases (SULT1C), and estrogen sulfotrans-
redox cycle and that the mutagenic efficiency of B[a]P-7,8-di- ferases (SULT1E) (29). The SULT2 family consists of SULT2A
one showed a linear correlation with the presence of 8-oxo- and -2B subfamilies and catalyze sulfonation of the hydroxyl
dGuo in the p53 DNA (22–24). In human lung adenocarcinoma group of steroids including both 3␣- and 3␤-hydroxysteroids
(A549) cells with high constitutive expression of AKRs, the (30). Estrogen o-quinones are structurally related to the PAH
metabolic activation of B[a]P-7,8-trans-dihydrodiol to B[a]P- o-quinones, and multiple SULTs catalyze the sulfonation of
7,8-dione by AKRs led to the formation ROS and 8-oxo-dGuo estrogen catechols such as 2-hydroxyestradiol, 4-hydroxyestra-
adducts in cellular DNA, which were exacerbated by the pres- diol, 2-hydroxyestrone, and 4-hydroxyestrone (31–33). SULT
ence of a catechol-O-methyl transferase (COMT) inhibitor enzymes are also widely expressed and found in human lung
(25). The contribution of COMT to detoxication of PAH o-qui- (34). Therefore, it is imperative to investigate whether sulfo-
none was confirmed by demonstrating that human recombinant nation of B[a]P-7,8-catechol by SULTs is a detoxication path-
COMT detoxified structurally diverse PAH o-quinones, including way for B[a]P-7,8-dione that will limit its ability to redox cycle.
B[a]P-7,8-dione, via O-methylation of PAH catechols (26).
EXPERIMENTAL PROCEDURES
It is still unknown whether sulfonation of B[a]P-7,8-catechol
by human sulfotransferases (SULT) is a feasible detoxication
Chemicals and Reagents—p-Nitrophenol and PAPS, were
route for B[a]P-7,8-dione. SULTs are a group of cytosolic purchased from Sigma. 3Ј-[35S]Phosphoadenosine 5Ј-phos-
29910 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 35•AUGUST 24, 2012

Detoxication of PAH o-Quinones by SULT
phosulfate (1–3 Ci/mmol, 0.5 mCi/ml) was obtained from
PerkinElmer Life Sciences. B[a]P-7,8-dione was synthesized
according to published procedures (35). All solvents were
HPLC grade, and all other chemicals used were of the highest
grade available.
Preparation of pGEX-2TK-SULT1A1*1 Wild Type Expres-
sion Vectors—The pGEX-2TK-SULT1A1*3 (Met223Val) was
used as a template to prepare a wild type construct by conduct-
ing site-directed mutagenesis using the QuikChange method
following the manufacturer’s protocol. The following forward
and reverse primers were used (where the underlined nucleo-
tides indicate the mutation introduced): 5Ј-dGAGACCGTGGA-
CTTCATGGTTCAGCACACGTCG-3Ј and 5Ј-dCGACGTGT-
GCTGAACCATGAAGTCCACGGTCTC-3Ј. The introduction
of the wild type sequence of SULT1A1*1 into the construct was
verified by dideoxysequencing.
Cell Lines and Culture Conditions—A549 (human lung ade-
nocarcinoma cells) were obtained from American Type Culture
Collection (ATCC number CCL-185) and maintained in F-12K
nutrient mixture (Kaighn’s modification) with 10% heat-inacti-
vated FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100
␮g/ml streptomycin. H358 (human bronchoalveolar cells) were
obtained from American Type Culture Collection (ATCC
number CRL-5807) and maintained in RPMI 1640 nutrient
mixture with 10% heat-inactivated FBS, 2 mM L-glutamine, 100
units/ml penicillin, and 100 ␮g/ml streptomycin. HBEC-KT
(immortalized human bronchial epithelial cells) originated
from a patient without lung cancer were a gift from Dr. John
Minna at University of Texas Southwestern Medical Center
and maintained in keratinocyte-serum free medium with 0.1–
0.2 ng/ml recombinant EGF, 20–30 ␮g/ml bovine pituitary
extract, and 2 mM L-glutamine. BEAS-2B (normal human bron-
chial epithelium cells) cells from American Type Culture Col-
lection (ATCC number CRL-9609) were cultured in BEGM
Bronchial Epithelium Medium (Cambrex CC-3170). Cells were
incubated at 37 °C in a humidified atmosphere containing 5%
CO2 and were passaged every 3 days at a 1:6 dilution. Cultured
cells with a passage number of 10–20 were used in the experi-
ments to reduce variability during cell culture.
RT-PCR Analysis of SULT mRNA Expression in Human Lung
Cells—Total RNA from each cell line was extracted using
RNeasy Kits (Qiagen, Valencia, CA). cDNA was synthesized
with GeneAmpꢁ RNA PCR Core kit according to the manufac-
turer’s protocol (Applied Biosystems, Carlsbad, CA). An ali-
quot of 1 ␮l of the reverse transcriptase reaction mixture was
used for PCR. In 25 ␮l of PCR system there was 1ϫ PCR buffer
(10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl), 2.5 mM MgCl2,
250 ␮M dNTPs, 0.2 ␮M primers and 0.5 units of TaqDNA
polymerase (Applied Biosystems). The sequences of forward
and reverse primer pairs for SULT1A1, -1A3, -1B1, -1C2, -1E1,
and -2A1, ␤-actin were taken from previous studies (36, 37).
PCR amplification was carried out with PerkinElmer Life Sci-
ences GeneAmp PCR System 2400 (Waltham, MA) using the
following protocol. After an initial denaturation step at 95 °C
for 45 s, amplification was conducted by denaturation at 95 °C
for 15 s, annealing at 68 °C (for SULT1E1) or 66 °C (for other
SULT isoforms) for 30 s, and extension at 72 °C for 45 s for 30
cycles. The final extension reaction was performed at 72 °C for
7 min. Plasmids corresponding to pGEX-2TK-SULT1A1*3,
-1A3, -1B1, -1C2, -1E1, and -2A1 were kindly provided by Prof.
Ming-Cheh Liu (Department of Pharmacology, College of
Pharmacy, University of Toledo, OH) and were used as positive
controls, whereas H2O was used as negative control. The con-
trol samples were amplified using the same conditions as
described above. An aliquot of 10 ␮l of PCR products was ana-
lyzed by electrophoresis in a 2% agarose gel containing
ethidium bromide and visualized under UV light.
Standard Radiometric Assay for SULT Activity—SULT
enzyme assays were conducted as described by Foldes and
Meek (38). Briefly, the reaction system contained 10 mM KPO4
buffer of pH 7.4, 1.0 mM dithiothreitol, 5.0 mM MgCl2, 20 ␮M
[35S]PAPS (100 cpm/pmol), 10 ␮M (for SULT1A1) or 500 ␮M
(for SULT1A3 and -1E1) of p-nitrophenol, and human recom-
binant SULTs in a final volume of 100 ␮l. Reactions were initi-
ated by addition of [35S]PAPS. After incubation at 37 °C for 15
min, the reactions were terminated by the sequential addition
of 50 ␮l each of 0.1 M barium acetate, 0.1 M barium hydroxide,
and 0.1 M zinc sulfate. The precipitate was pelleted by centrif-
ugation at 16,000 ϫ g for 10 min. An aliquot of 50 ␮l superna-
tant was pipetted and mixed with scintillation fluid. The radio-
activity was measured on a Tri-Carb 2100TR scintillation
counter (machine efficiency for tritium is 65%). Control reac-
tions were performed in the absence of p-nitrophenol. The
trace amount of [35S]PAPS unprecipitated by the barium treat-
ment in the controls served as a background value that was
subtracted from the values obtained from reactions containing
p-nitrophenol. The volume-corrected cpm were converted to
pmol of p-nitrophenol sulfate formed using the specific radio-
activity of [35S]PAPS. It was also confirmed that a single prod-
uct of p-nitrophenol-sulfate was formed by comparing the
retention time of authentic p-nitrophenyl sulfate (Sigma) by
reversed phase HPLC.
Expression and Purification of SULT—The SULTs expressed
in human lung cells were purified as recombinant enzymes fol-
lowing the reported method with slight modification (39). The
pGEX-2TK-SULT1A1, -1A3, and -1E1 expression vectors were
transformed respectively into competent Escherichia coli
C41(DE3) cells. The cells were grown in 750-ml cultures of
Luria-Bertani medium at 37 °C (containing 100 ␮g/ml ampicil-
lin). Upon reaching A600 0.6, 1 mM isopropyl-1-thio-␤-D-galac-
topyranoside was added to induce enzyme expression, and cells
were cultured overnight. The culture was centrifuged for 10
min at 10,000 ϫ g at 4 °C. Harvested cells were washed with
saline, and the pellets were resuspended in buffer A (20 mM
Tris-HCl (pH 7.5) containing 10 mM NaCl and 1 mM EDTA).
Resuspensions were lysed by sonication and centrifuged twice
for 10 min at 10,000 ϫ g at 4 °C. The lysate was loaded onto a
glutathione-Sepharose column (GE Healthcare) equilibrated
with buffer A. GST-SULT fusion proteins were eluted with
buffer consisting 10 mM GSH, 20 mM Tris-HCl (pH 7.5), 10 mM
NaCl, 1 mM EDTA. The collected fractions were digested with
40 units of thrombin for 1 h to cleave the GST tag from the
fusion protein and release native SULTs, and the thrombin was
inactivated by adding 20 ␮g of aprotinin. The resulting cleavage
AUGUST 24, 2012•VOLUME 287•NUMBER 35
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Detoxication of PAH o-Quinones by SULT
products were dialyzed overnight in buffer. The cleaved GST kinetic parameters as mean Ϯ S.E. Dividing Vmax by the molar
tags were removed by loading the dialysate onto a glutathione- concentration of the enzyme gave kcat. Ki is the dissociation
Sepharose column equilibrated with buffer A. The eluent con- constant for the substrate to dissociate from the enzyme inhib-
taining native SULTs was concentrated into 2 ml using a Cen- itor complex.
tricon (Millipore, Billerica, MA) and further purified with
Metabolism of B[a]P-7,8-dione in Human Lung Cells—The
HiPrep 16/60 gel filtration column (GE Healthcare). The active A549, HBEC-KT, and H358 cells (5 ϫ 106) at confluency were
fractions containing SULTs were identified using the standard treated with B[a]P-7,8-dione (2 ␮M, 0.2% DMSO) in HBSS
SULT assay conditions, measurement of A280 nm, and visualiza- buffer containing 1 mM sodium pyruvate. The culture media
tion of the protein content of each fraction by SDS-PAGE. The were collected at 0 and 24 h, respectively, and subsequently
homogeneous human recombinant SULT1A1, -1A3, and -1E1 acidified with 0.1% formic acid before extraction with a 2ϫ
with an Mr 35 kDa were obtained by this procedure, as judged 1.5-fold volume of cold H2O-saturated ethyl acetate. The
by SDS-polyacrylamide gel electrophoresis. The specific activ- organic phases of culture media were combined and dried
ities of SULT1A1, -1A3, and -1E1 were 25, 33, and 27 nmol of under a vacuum. The residue was dissolved in 100 ␮l of meth-
p-nitrophenol sulfate formed/min/mg, respectively, and com- anol. A 20-␮l aliquot was analyzed by LC-MS/MS.
pare favorably with published values for these enzymes (39–
Identification of B[a]P-7,8-catechol Sulfates by HPLC-
41). The purified enzymes were stored in 20 mM Tris-HCl (pH RAM-UV and LC-MS/MS—The B[a]P-7,8-catechol sulfates
7.5) containing 1 mM EDTA, 10 mM DTT, 20% glycerol at generated with [35S]PAPS were analyzed by a Waters Alliance
Ϫ80 °C for future use.
2695 chromatographic system (Waters Corp., Milford, MA) in
Kinetic Studies on the Formation of B[a]P-7,8-Catechol tandem with a Waters 996 photodiode array detector and a
Sulfates—Experiments were conducted anaerobically in a glove ␤-RAM inline radiometric detector (IN/US Systems Inc.,
box purged with argon. All the solvent and aqueous solutions Tampa, FL). Chromatographic separation was achieved on a
were degassed by freeze-pump-thaw cycling five times and reversed phase column (Zorbax-ODS C18, 5 ␮m, 4.6 ϫ 250
stored in sealed containers filled with argon. The reactions were mm, DuPont) eluted with the following linear gradient of H2O
performed in 1.5-ml amber glass vials with polytetrafluoroeth- (0.1% trifluoroacetic acid and 5 mM ammonium acetate; solvent
ylene/silicone septa closures. The reaction system contained 10 A)/MeOH (0.1% trifluoroacetic acid and 5 mM ammonium ace-
mM KPO4 buffer of pH 7.4, 1.0 mM dithiothreitol, 5.0 mM tate; solvent B) at a flow rate of 0.5 ml/min. Solvent B was
MgCl2, 20 ␮M [35S]PAPS (100 cpm/pmol), 0–10 ␮M B[a]P-7,8- changed from 65 to 80% (v/v) over 45 min, kept at 80% over 5
dione in a final volume of 0.2 ml. The reactions were initiated by min, changed from 80 to 65% over 1 min, and kept at 65% for 9
the addition of 0.48–0.96 ␮g of human recombinant SULTs at min. Eluates from the column were introduced into the inline
25 °C. The amount of enzyme used was always in the linear radiometric detector following mixture of the scintillant with
range as determined by plots of initial velocity versus enzyme the HPLC effluent (IN/US Systems Inc.) at a flow rate 1.5
concentrations. The reactions were quenched by the addition ml/min.
of 50 ␮l of ice-cold 1% formic acid and were placed on ice. The
LC-MS/MS identification of the O-sulfated catechols was
reaction mixtures were then extracted twice with 0.5 ml ali- conducted with a Finnigan TSQ Quantum Ultra spectrometer
quots of ethyl acetate twice by vortex mixing and centrifuged at (Thermo Fisher, San Jose, CA) equipped with a heated electro-
16,000 ϫ g to help phase separation. The combined ethyl ace- spray ionization probe as ionization source. The mass spec-
tate layer was backwashed with 0.2 ml of 1% formic acid by trometer was operated in the positive ion mode or negative ion
vigorous vortexing and centrifuged at 16,000 ϫ g. The organic mode with the following parameters: spray voltage (4500 V at
phase was then dried by a SpeedVac concentrator (Thermo positive ion mode or Ϫ2000 V at negative ion mode), vaporizer
Scientific). The residue was dissolved in 100 ␮l methanol, and temperature (400 °C), sheath gas pressure (35 arbitrary units),
an aliquot of 50 ␮l was analyzed by scintillation counting or by auxiliary gas pressure (10 arbitrary units), capillary temperature
HPLC analysis. The initial velocity was estimated by the slope of (350 °C), and collision energy (20 V). The masses of the metab-
the linear portion of the progress curve over 10 min. Kinetic olites were obtained by detecting the molecular ion from Q1 full
analyses using nonlinear regression were performed by fitting scan, and the corresponding mass spectrum of each metabolite
the Michaelis-Menten equation to the data with the program was obtained from a Q3 full scan of the product ions of the
Grafit,
molecular ion. The chromatography conditions for the metab-
olites were identical to those used for HPLC-RAM-UV
detection.
v ϭ Vmax ϫ ͓S͔͑͞Km ϩ ͓S͔͒
(Eq. 1)
Identification of the B[a]P-7,8-catechol Sulfates by 1H and 13
C
NMR Analysis—To generate sufficient B[a]P-7,8-catechol-O-
monosulfates for characterization, the sulfonation reactions
were performed on a large scale. Metabolites were synthesized
in a 100-ml system containing 10 mM KPO4 buffer (pH 7.4), 1.0
mM dithiothreitol, 5.0 mM MgCl2, 20 ␮M PAPS, and 10 ␮M
When substrate inhibition was observed, the following equa-
tion was fitted to the initial velocity data in a similar manner,
v ϭ Vmax ϫ ͓S͔͑͞Km ϩ ͓S͔ ϩ ͓S͔2/Ki͒
(Eq. 2)
where v is the initial velocity of the reaction, [S] is the molar B[a]P-7,8-dione under anaerobic conditions. The reaction was
concentration of the substrate, and Km is the Michaelis-Menten started by the addition of 1.2 mg of human recombinant
constant for the substrate. Because of the iterative fits of the SULT1A3 and incubated for 90 min at 37 °C. The reaction was
equations to each data set, each fit provided estimates of the quenched by the addition of 250 ␮l of formic acid. Metabolites
29912 JOURNAL OF BIOLOGICAL CHEMISTRY
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Detoxication of PAH o-Quinones by SULT
FIGURE 2. Gene expression of SULTs in human lung cells is shown (A). A549, human lung adenocarcinoma cell; H358, human bronchoalveolar cell; HBEC-KT,
immortalized human bronchial epithelial cell; BEAS-2B, normal human bronchial epithelial cell. SDS-PAGE of purified human recombinant SULTs is shown (B).
were extracted with 1ϫ 100 ml of ethyl acetate followed by 1 ϫ [35S]PAPS as the sulfonate group donor, and the products were
50 ml of ethyl acetate. The pooled extracts were evaporated to
complete dryness. The residues were dissolved in 2 ml of meth-
anol and subjected to reversed phase HPLC (Zorbax-ODS C18,
5 ␮m, 4.6 ϫ 250 mm, DuPont). The column was eluted with the
linear gradient of H2O (0.1% trifluoroacetic acid solvent
A)/MeOH (0.1% trifluoroacetic acid; solvent B) at a flow rate of
0.5 ml/min. Solvent B started with 50% for 5 min and was
changed from 50 to 70% (v/v) in 3 min and changed afterward
from 70 to 73% over 27 min and then further changed from 73
to 80% in 5 min, kept at 80% for 5 min, changed from 80 to 50%
over 1 min, and kept at 50% for 14 min. The B[a]P-7,8-catechol-
O-monosulfates were collected on the basis of their character-
istic UV spectra. The pooled elutes were evaporated to com-
profiled by reversed phase HPLC with online radiometric
detection. Reactions were subsequently performed with unla-
beled PAPS for product identification by LC-MS/MS. Recom-
binant SULT1A1 catalyzed the formation of a single metabolite
(M1), whereas SULT1A3 and -1E1 catalyzed the formation of
two metabolites (M1, M2) of B[a]P-7,8-catechol, which were
detected by HPLC-UV-RAM (Fig. 3). The HPLC-UV-RAM
gave information on the ratios of the two [35S]B[a]P-7,8-cate-
chol sulfates generated with [35S]PAPS. SULT1A3 and -1E1
formed both M1 and M2 in the ratios of 82:18 and 37:63 (M1:
M2), respectively. The UV spectra of two metabolites were dif-
ferent. LC-MS analysis demonstrated that the molecular ions of
both metabolites were the same. In the positive ion mode, they
gave an m/z 365, and in the negative ion mode they gave an m/z
363. MS/MS analysis provided additional structural informa-
tion for the two metabolites. In the positive ion mode, the prod-
uct ion spectra of the two metabolites were identical and gave
the characteristic cleavage at -SO3H with the loss of 81 atomic
mass units from the molecular ion (Fig. 4), indicating that they
were sulfate conjugates. Cleavage at one of the C-OH bonds
resulted in a daughter ion of m/z 267 representing the loss of
-OH. Rearrangement at the remaining phenolic group likely
resulted in a change from a -C-OH to -CAO bond. The-CAO
group was then lost, resulting in fragment ion at m/z 239. In the
negative ion mode, product ion spectra of two metabolites also
1
plete dryness. One- and two-dimensional H and 13C NMR
data were collected on a Bruker AVIII (cryo500) NMR spec-
trometer operating at 500 MHz using methanol-D4 as the sol-
vent (Sigma).
RESULTS
Gene Expression of SULTs in Human Lung Cells—To deter-
mine the expression level of SULTs in four human lung cells,
RT-PCR was employed. Among the six different SULT iso-
forms, SULT1A1, -1A3, and -1E1 were expressed at the mRNA
level (Fig. 2). Although semiquantitative, this screen showed
that SULT1A3 was the most abundant at the mRNA level in
human lung cells followed by SULT1A1. By contrast. SULT1E1
mRNA showed only a low level of expression in human lung
cells. Therefore, the following studies focused on the sulfo-
nation of B[a]P-7,8-catechol by recombinant SULT1A1, -1A3,
and -1E1.
Ϫ
demonstrated characteristic cleavage at -SO3 resulting in
daughter ions at m/z 283. LC-MS/MS analyses in both the pos-
itive and negative ion modes confirmed that the metabolites
formed in the reaction system were B[a]P-7,8-catechol-O-
monosulfates. However, because the mass spectra of M1 and
M2 were identical, LC-MS/MS could not distinguish the posi-
tion of sulfate group in the two isomers.
Kinetic Studies on the Formation of B[a]P-7,8-catechol
Sulfates—To obtain steady state kinetic parameters for the for-
mation of the B[a]P-7,8-catechol-O-monosulfates, we con-
Identification of B[a]P-7,8-catechol-O-Monosulfates Pro-
duced by Human Recombinant SULT Isoforms—Recombinant
human SULT1A1, -1A3, and -1E1 were expressed as GST-fu-
sion proteins, and upon removal of the tag and subsequent
chromatography they were obtained in homogeneous form
with appropriate specific activities for the sulfonation of p-
nitrophenol. Discontinuous assays were performed with ducted discontinuous assays using the recombinant SULT iso-
AUGUST 24, 2012•VOLUME 287•NUMBER 35
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Detoxication of PAH o-Quinones by SULT
FIGURE 3. HPLC/UV/RAM identification of sulfated metabolites (M1 and M2) of the B[a]P-7,8-catechol. B[a]P-7,8-catechol (10 ␮M) was generated in situ
under anaerobic conditions in the presence of 1 mM dithiothreitol. The B[a]P-7,8-catechol was converted to the O-sulfate(s) in the presence of [35S]PAPS and
SULTs in the incubation buffer. A, B, and C, shown are HPLC-UV chromatograms of B[a]P-7,8-catechol sulfates formed by SULT1A1, -1A3, and -1E1, respectively;
D, E, and F, shown are HPLC-RAM chromatograms of B[a]P-7,8-catechol sulfates formed by SULT1A1, -1A3, and -1E1, respectively. The red triangles indicate the
beginning and end of peak integration in the chromatograms. AU, absorbance units.
forms. We used an assay based on ethyl acetate extraction of the
Formation of B[a]P-7,8-catechol Sulfates in Human Lung
products under acidic conditions, which was favorable for the Cells Treated with 2 ␮M B[a]P-7,8-dione—One B[a]P-7,8-
partition of metabolites in the organic phase. Reverse phase- catechol-O-monosulfate isomer was detected in A549,
HPLC analysis confirmed that when M1 and M2 were gener- H358, and HBEC-KT cell lines after incubation of the cells
ated, they were formed concurrently in reactions containing with 2 ␮M B[a]P-7,8-dione for 24 h and was detected by
SULT1A3 and -1E1. Only M1 was formed in reactions contain- LC-MS/MS (Fig. 6). Similar experiments were not per-
ing SULT 1A1. We find that the kinetic profiles for the forma- formed on BEAS-2B cells as they are AKR null by real-time
tion of B[a]P-7,8-catechol sulfates catalyzed by the three PCR.3 Using the negative ion mode by monitoring the reac-
SULTs were quite different (Fig. 5 and Table 1). Both the tion transition m/z 3633283, the metabolite in the human
Michaelis-Menten equation and the equation for substrate lung cells had the same retention time as M1. No significant
inhibition were fitted to kinetic data with nonlinear regression amount of the other isomeric B[a]P-7,8-catechol-O-mono-
analysis. Goodness of fit clearly distinguished between these sulfate was detected in the cells.
two kinetic patterns. B[a]P-7,8-catechol sulfonation catalyzed
NMR Analysis of B[a]P-7,8-catechol Monosulfates—To identify
bySULT1A3followednormalMichaelis-Mentenkinetics. Bycon- the M1 and M2 B[a]P-7,8-catechol-O-monosulfates unequivo-
trast, B[a]P-7,8-catecholsulfonationcatalyzedbySULT1A1*1and cally, they were enzymatically generated using SULT1A3 on a pre-
-1E1 showed modest to potent substrate inhibition yielding Ki parative scale and purified by reversed phase-HPLC. Two-dimen-
values of 30.0 and 0.08 ␮M, respectively. kcat/Km values showed sional H NMR and 14C NMR of M1 and one-dimensional H
that SULT1A1 was the most catalytically efficient isozyme fol- NMR of M2 were obtained. Two-dimensional 1H and 13C NMR
lowed by SULT1E1 and -1A3, respectively. The Km value for of M2 could not be obtained due to its poor yield. By comparing
SULT1A1*1 was the lowest, yielding a value in the submicro- previous published two-dimensional 1H NMR of B[a]P-7,8-cat-
1
1
echol, the protons in the spectra of B[a]P-7,8-catechol sulfates
could be assigned (17). Generally, the 1H NMR spectra of cate-
chol and catechol sulfates are quite similar. All the protons were
aromatic and were located between 7.5 and 9.1 ppm. H-6 in
both M1 and M2 was a singlet due to a lack of coupling with
other protons. The cross-peaks at two-dimensional 1H NMR of
M1 indicated the coupling between H-9 and H-10, H-11 and
molar range that was 63- and 15-fold lower than those seen with
SULT1A3 and -1E1. B[a]P-7,8-catechol sulfonation by the
thermostable SULT1A1*3 variant followed normal Michaelis-
Menten kinetics. The Km value of SULT1A1*3 was close to that
of SULT1A1*1, whereas the kcat/Km value of SULT1A1*3 was
about 50% that of the wild type enzyme (Table 1). Interestingly,
the catalytic efficiency of SULT1A1*1 and COMT to form
B[a]P-7,8-catechol monosulfate and O-methylated B[a]P-7,8-
catechol were similar.
3 M. E. Kushman and T. M. Penning, unpublished information.
29914 JOURNAL OF BIOLOGICAL CHEMISTRY
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Detoxication of PAH o-Quinones by SULT
FIGURE 4. LC/MS/MS identification of sulfated metabolites (M1 and M2) of the B[a]P-7,8-catechol. B[a]P-7,8-catechol (10 ␮M) was generated in situ
under anaerobic conditions in the presence of 1 mM dithiothreitol. The B[a]P-7,8-catechol was converted to the O-sulfate(s) in the presence of PAPS and
SULTs in the incubation buffer. The reactions were quenched by 1% formic acid and extracted with of ethyl acetate. The organic phase was then dried
and dissolved in MeOH for LC-MS/MS analysis. A, B, and C, shown are positive ion chromatograms of B[a]P-7,8-catechol sulfates by monitoring the
reaction transition m/z 3653284 ([MϩH]ϩ3 [MϩH-SO3H]ϩ). D, shown is a positive ion MS2 of isomer 1 and MS2 of isomer 2; E, shown is a negative ion
MS2 of isomer 1 and MS2 of isomer 2.
H-12, H-1 and H-2, and H-2 and H-3 as well as H-4 and H-5 ing of protons at these positions. When the -SO3 group is at C7,
(Fig. 7). A complete list of the connectivities obtained from the this would place H-10 at the para position with respect to the
cross-peaks is provided in Table 2. To determinate the position -SO3 group, and its resonance would be shifted to a more down-
of the sulfate group, it was necessary to examine the influence of field position. This is observed because H-10 and H-6 now
the -SO3 group on the chemical shift of the related protons. overlap in the spectrum of the M1 isomer. By contrast the
Based on diagnostic peaks of H-6, H-9, and H-10, M1 was chemical shift of H-9 would be unaltered because the -SO3
assigned as 8-hydroxy-B[a]P-7-O-sulfate, and M2 was 7-hy- group would be in a meta position relative to this proton, and
droxy-B[a]P-8-O-sulfate. 13C NMR spectrum of M1 was also this is observed in the spectrum of M1. When the SO3 group
acquired (see supplemental Fig. S1). The chemical shifts were as is at the C8 position, H-10 and H-6 are clearly separated, but
follow: ␦ ϭ 146.72, 131.36, 130.99, 130.07, 128.20, 127.94, H-9 would now be in the ortho position, and its resonance
127.46, 127.30, 127.18, 125.49, 125.15, 125.09, 124.52, 123.69, would be expected to be shifted downfield. For M2, the -SO3
121.97, 121.81, 121.36, 119.18, 118.50 ppm. The carbon at the group shifted the H-9 resonance downfield when it was com-
most downfield position was modified by -O-SO3H.
pared with the chemical shifts of H-9 in the spectra of either
It was found that the 1H NMR spectra of M1 and M2 were B[a]P-7,8-catechol or M1. This indicated that in M2, H-9
different. H-6, H-9, and H-10, are closest to the 7- and 8-hy- should be in the ortho position with respect to the -SO3
droxyl groups, and their chemical shift values are most influ- group. The chemical shift values of the other aromatic pro-
enced by conjugation of the -SO3 group. Chemical shift values tons in M1 and M2 were quite similar because they are far
were used as diagnostic peaks to assign the position of the -SO3 away for the -SO3 group and thus there was a minimal effect
group, which was accomplished by comparing their values in on their chemical environment. This analysis shows that M1
the spectra of M1 and M2 with those previously reported for is 8-hydroxy-B[a]P-7-O-sulfate and M2 is 7-hydroxy-B[a]P-
B[a]P-7,8-catechol (17). The -SO3 moiety is an electron with- 8-O-sulfate. Using these compounds as standards, the iso-
drawing group that makes carbons at the ortho and para posi- mer produced in human lung cells was found to be M1 or
tions carry a partial positive charge that results in the deshield- 8-hydroxy-B[a]P-7-O-sulfate.
AUGUST 24, 2012•VOLUME 287•NUMBER 35
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Detoxication of PAH o-Quinones by SULT
DISCUSSION
We are conducting a systematic study to identify the conju-
gating enzymes that can intercept PAH-catechols and prevent
their redox cycling to the corresponding PAH o-quinones.
These studies will document which enzymes attenuate the abil-
ity of AKR products to form ROS that can lead to the mutagenic
lesion 8-oxo-dGuo. Previously we have shown that PAH o-qui-
nones are enzymatically redox-cycled at a robust rate by recom-
binant NQO1 and by AKRs themselves and that human COMT
can O-methylate a series of PAH-catechols (21, 26). In this
study we identify SULT1A1 as the major isoform responsible
for the formation of B[a]P-7,8-catechol-O-monosulfate based
on catalytic efficiency and enzyme expression level in human
lung cells. We also identify the major metabolite formed as
8-hydroxy-benzo[a]pyrene-7-O-sulfate based on NMR data.
Three SULT isoforms, SULT1A1, -1A3, and -1E1, were
detected in human lung cells and were recombinantly
expressed for this study. SULT1A1 and -1A3 share 95.6% sim-
ilarity in amino acid sequence, whereas SULT1A1 and -1E1
share only 65.4% similarity. SULT1A1 formed only the M1
metabolite, 8-hydroxy-benzo[a]pyrene-7-O-sulfate, whereas
SULT1A3 showed a distinct preference for the formation of
M1. By contrast SULT1E1 formed M1 and M2 in approxi-
mately equal amounts. It is probable the high amino acid
sequence similarity and, therefore, similar three-dimensional
structureofSULT1A1and-1A3accountsfortheirsimilarregio-
selectivity of sulfonation of B[a]P-7,8-catechol. SULTs perform
sulfonation via a SN2 mechanism without going through a sul-
fonated enzyme intermediate. To determine the structural
basis for the formation of M1 and M2 at the same active site will
require an x-ray crystallographic approach.
SULT1A1 was the most catalytically efficient isoform among
the three SULTs in sulfonating B[a]P-7,8-catechol. The Km
value of SULT1A1 was in the nanomolar range, which indicates
that it can detoxify B[a]P-7,8-catechol at very low substrate
concentrations but would also be easily saturated. Even though
there is 95.6% similarity in amino acid sequence between
SULT1A1 and -1A3, their kinetic profiles were different.
Although the kcat value of SULT1A3 was higher than that of
SULT1A1, the Km of SULT1A3 is orders of magnitude greater
that of SULT1A1; thus, SULT1A3 is less efficient at detoxica-
tion of B[a]P-7,8-catehcol than SULT1A1. It was also found
that B[a]P-7,8-catechol caused significant substrate inhibition
of SULT1E1, which would compromise its detoxication ability.
Moreover, the mRNA level of SULT1E1 was relatively low in
human lung cells. When the kinetic profiles and expression
levels of the SULTs are considered, SULT1E1 was not as impor-
tant as SULT1A1 and -1A3 in the sulfonation of B[a]P-7,8-
catechol. Interestingly, SULT1E1 is the most efficient SULT
isoform for sulfonation of 17␤-estradiol and estrogen-3,4-cat-
echol, which prevents formation of estrogen o-quinones (31).
Estrogen o-quinones are regarded as endogenous tumor initia-
tors and are able to attack DNA to form potentially carcino-
genic stable or depurinating DNA adducts (42). It is possible
that inhibition to SULT1E1 by B[a]P-7,8-catechol and other
PAH catechols may increase the risk factor for estrogen-depen-
dent genotoxicity.
FIGURE 5. Kinetic characterization of sulfonation of B[a]P-7,8-catechol by
SULTs. Initial velocities were estimated by the slope of a linear portion of the
progress curve over 10 min. Kinetic analyses were performed by fitting the
Michaelis-Menten equation or the substrate inhibition equation to the data.
Reactions contained 10 mM KPO4 buffer of pH 7.4, 1.0 mM dithiothreitol, 5.0 mM
MgCl2, 20 ␮M [35S]PAPS, 0–10 ␮M B[a]P-7,8-dione, and 0.48–0.96 ␮g of human
recombinant SULTs at 25 °C. A, B, and C, shown is velocity versus S curve for sul-
fonation of B[a]P-7,8-catechol by SULT1A1, -1A3, and -1E1, respectively.
29916 JOURNAL OF BIOLOGICAL CHEMISTRY
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Detoxication of PAH o-Quinones by SULT
TABLE 1
Kinetic constants of o-sulfonation of B[a]P-7,8-catechol
Enzymes
Vmax
nmol/min/mg
kcat
min
Km
␮M
Ki
␮M
kcat/Km
min ␮M
Ϫ1
Ϫ1
Ϫ1
SULT1A1*1
SULT1A1*3
SULT1A3
SULT1E1
COMTa
1.68 Ϯ 0.07
0.84 Ϯ 0.02
9.2 Ϯ 0.9
15.4 Ϯ 3.7
54.2
0.059 Ϯ 0.002
0.030 Ϯ 0.001
0.32 Ϯ 0.03
0.54 Ϯ 0.13
1.48
0.061 Ϯ 0.004
0.08 Ϯ 0.009
3.8 Ϯ 0.8
29.9 Ϯ 3.5
NA
0.97
0.38
0.084
0.60
0.70
NA
0.90 Ϯ 0.26
2.10
0.08 Ϯ 0.02
NA
a Ref. 26.
FIGURE 6. LC-MS/MS detection of sulfated metabolites (M1 and M2) of the B[a]P-7,8-catechol formed by recombinant SULT1A3 (A) and by A549
(B), H358 (C), and HBEC-KT cells (D) at 24 h. B[a]P-7,8-dione (2 ␮M, 0.2% DMSO) in HBSS buffer were incubated with lung cells and collected at 0 and 24 h,
respectively. The culture media were extracted with ethyl acetate. The organic phases were dried under vacuum and redissolved in methanol. B[a]P-7,8-
catechol sulfates were analyzed with LC-MS/MS in the negative ion mode by monitoring the reaction transition m/z 3633283 ([M-H]Ϫ 3 [M-H-SO3]Ϫ). The
peak M1 had the same retention time and mass transition as authentic 8-hydroxy-B[a]P-7-O-sulfate.
We also demonstrated that B[a]P-7,8-dione was metabolized treated with B[a]P-7,8-dione, which supports the contention
to B[a]P-7,8-catechol sulfate in three human lung cell-based that O-methylation of B[a]P-7,8-catechol contributes to the
models. At 2 ␮M B[a]P-7,8-dione, only 8-hydroxy-B[a]P-7-O- detoxication of B[a]P-7,8-dione by preventing its ability to
sulfate (M1) was detected in the cells. In the metabolism study redox cycle (25). Because of the higher level of O-sulfonation in
using recombinant SULTs, the ratios of M1/M2 formation for these cells, we predict that SULTs will play a more important
SULT1A1, -1A3, and -1E1 were 100:0, 82:18, 36:63, respec- role than COMT in intercepting B[a]P-7,8-dione to prevent the
tively. This ratio could be used as an index to reflect in part the generation of reactive oxygen species. The expression of SULT
contribution of SULTs in cellular detoxication of B[a]P-7,8- and COMT in lung tissue has been established by others (34,
dione. The predominant metabolite found in the cells (M1) 44) and suggests that is reasonable to infer from our cell-based
implied that SULT1A1 makes the most significant contribution study that both enzymes will play a significant role in the detox-
to O-sulfonation. This is consistent with the analysis based on ication of B[a]P-7,8-dione.
the kinetic profiles and expression levels of each SULT.
SULT1A1 polymorphism has been associated with an
We found that in A549, H358, and HBEC-KT human lung increased lung cancer risk (45), and polymorphic variants of
cells, the O-monosulfate conjugate of B[a]P-7,8-catechol SULT1A1 may affect the detoxication of PAH o-quinones. The
accounts for 12.2, 15, and 3.5% of the metabolites derived from common SULT1A1 allozymes mainly include *1 (wild type), *2
B[a]P-7,8-dione (43). The amount of O-monosulfated B[a]P- variant (R213H), and *3 variant (M223V) (46). The allelic fre-
7,8-catechol formed was about twice that of the O-methylated quencies for SULT1A1*1, -*2, and *3 in Caucasian were 0.656,
B[a]P-7,8-catechol. This indicated that detoxication of B[a]P- 0.332, 0.012, respectively. Despite the low frequency of
7,8-dione through the sulfation pathway is preferred over SULT1A1*3 in Caucasians, it has an allelic frequency of 0.229 in
O-methylation. Inhibition of COMT in A549 cells increased African Americans (46). It has been reported that SULT1A1
DNA strand breaks and formation of 8-oxo-dGuo in cells recombinant allozymes have variable thermal stability and spe-
AUGUST 24, 2012•VOLUME 287•NUMBER 35
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Detoxication of PAH o-Quinones by SULT
FIGURE 7. 500 MHz 1H COSY 8-hydroxy-B[a]P-7-O-sulfate (M1) and 1H NMR spectra of 7-hydroxy-B[a]P-8-O-sulfate (M2).
TABLE 2
Proton assignments for 8-hydroxy-benzo[a]pyrene-7-O-sulfate
m, multiplet; d, doublet; and s, singlet.
Proton
Chemical shift, multiplicity
Coupling constants (Hz)
Connectivities
ppm
J
H-1 or H3
H-2
8.08, d; 8.22, d
7.95, m
7.92, m
8.05, d
7.7 or 7.8
H-2 with H-1/H-3
H-1/H-3 with H-2
H-4 with H-5
H-4
H-5
9.1
H-5 with H-4
H-6
8.88, s
H-9
7.52, d
9.3
9.1
9.1
9.1
H-9 with H-10
H-10 with H-9
H-11 with H-12
H-12 with H-11
H-10
H-11
H-12
8.88, d
9.02, d
8.31, d
cific activity toward p-nitrophenol, catechol estrogens, and die- efficiency of SULT1A1*3 for sulfonation of B[a]P-7,8-cate-
tary flavonoids (31, 47, 48). The SULT1A1*2 variant was asso- chol was about half that observed for the wild type
ciated with low enzyme activity and thermal stability (45, 47, SULT1A1. Because SULT1A1 plays an important role in
48). Although SULT1A1*3 had compatible thermal stability to detoxication of B[a]P-7,8-dione, this study provides some
wild type enzyme, its specific activities for SULT1A1 substrates evidence that the polymorphic variants that reduce stability
were usually lower than that of wild type (48).
or catalytic efficiency of SULT1A1 in sulfonation of PAH
Although the Km value of SULT1A1*3 was comparable to its catechols may increase susceptibility to lung cancer caused
wild type counterpart, its kcat was less. As a result the catalytic by smoking and air pollution. Unlike high allele frequencies
29918 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 35•AUGUST 24, 2012

Detoxication of PAH o-Quinones by SULT
ster cells. Mutat. Res. 44, 313–326
of SULT1A1 variants, SULT1A3 and SULT1E1 variants were
found to be very rare, which indicates that genetic polymor-
phism of these two enzymes may have less influence on
detoxication of B[a]P-7,8-dione (49, 50).
12. Nesnow, S., Ross, J. A., Stoner, G. D., and Mass, M. J. (1995) Mechanistic
linkage between DNA adducts, mutations in oncogenes, and tumorigen-
esis of carcinogenic environmental polycyclic aromatic hydrocarbons in
strain A/J mice. Toxicology 105, 403–413
Sulfonation of PAH catechols by SULTs described in this
study may be one important mechanism for detoxication of
PAH o-quinones. First, SULTs could terminate the futile redox
cycling of PAH o-quinones by intercepting o-quinones that
cause ROS generation and subsequent oxidative DNA damage.
Second SULTs could eliminate the electrophilicity of PAH
o-quinones as the formation of catechol O-sulfates prevents the
formation of covalent PAH o-quinone adducts with protein or
DNA. Third, sulfation usually results in more polar metabolites
and enhances renal or biliary excretion of xenobiotics or drugs;
thus, sulfate conjugation of PAH catechols may facilitate the
elimination of PAH o-quinones.
13. Burczynski, M. E., Harvey, R. G., and Penning, T. M. (1998) Expression
and characterization of four recombinant human dihydrodiol dehydro-
genase isoforms. Oxidation of trans-7, 8-dihydroxy-7,8-dihydrobenzo-
[a]pyrene to the activated o-quinone metabolite benzo[a]pyrene-7,8-di-
one. Biochemistry 37, 6781–6790
14. Palackal, N. T., Burczynski, M. E., Harvey, R. G., and Penning, T. M. (2001)
The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcin-
ogen trans-dihydrodiols to o-quinones. Potential role in polycyclic aro-
matic hydrocarbon activation. Biochemistry 40, 10901–10910
15. Palackal, N. T., Lee, S. H., Harvey, R. G., Blair, I. A., and Penning, T. M.
(2002) Activation of polycyclic aromatic hydrocarbon trans-dihydrodiol
proximate carcinogens by human aldo-keto reductase (AKR1C) enzymes
and their functional overexpression in human lung carcinoma (A549)
cells. J. Biol. Chem. 277, 24799–24808
16. Murty, V. S., and Penning, T. M. (1992) Polycyclic aromatic hydrocarbon
(PAH) ortho-quinone conjugate chemistry. Kinetics of thiol addition to
PAH ortho-quinones and structures of thioether adducts of naphthalene-
1,2-dione. Chem. Biol. Interact. 84, 169–188
Acknowledgments—We thank Dr. George Furst for conducting NMR
analysis, Drs. Adegoke Adeniji and Ding Lu for the advice on NMR
assignments, Dr. Mo Chen for help in recombinant SULT purifica-
tion, and Dr. Xiaojing Liu for advice on LC/MS method development.
17. Murty, V. S., and Penning, T. M. (1992) Characterization of mercapturic
acid and glutathionyl conjugates of benzo[a]pyrene-7,8-dione by two-di-
mensional NMR. Bioconjug. Chem. 3, 218–224
18. Shou, M., Harvey, R. G., and Penning, T. M. (1993) Reactivity of benzo-
[a]pyrene-7,8-dione with DNA. Evidence for the formation of deoxy-
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29920 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 35•AUGUST 24, 2012

Metabolism:
Detoxication of Benzo[a]pyrene-7,8-dione
by Sulfotransferases (SULTs) in Human
Lung Cells
Li Zhang, Meng Huang, Ian A. Blair and
Trevor M. Penning
J. Biol. Chem. 2012, 287:29909-29920.
doi: 10.1074/jbc.M112.386052 originally published online July 9, 2012
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