Effect of benzo-ring hydroxyl groups on site-specific mutagenesis by tetrahydrobenzo[a]pyrene adducts at N6 of deoxyadenosine

Article abstract of DOI:10.1021/tx010076o

We have previously investigated the mutations induced on replication in Escherichia coli of the M13mp7L2 genome containing each of the eight possible adducts derived from the four optically active 7,8-diol 9,10-epoxide metabolites of benzo[a]pyrene (B[a]P) by alkylation of a specific deoxyadenosine (dAdo) residue at N⁶. Observed mutational frequencies depended in part on the relative spatial orientations of the three hydroxyl groups in these adducts. To determine how the presence or absence of these hydroxyl groups affects mutational response, we have synthesized 16-mer oligonucleotides with the same sequence as one of those previously studied with the diol epoxide adducts, but containing B[a]P-dAdo adducts in which two or all three of the adduct hydroxyl groups were replaced by hydrogen. Transfection of the adducted M13 constructs into SOS-induced Escherichia coli consistently gave fewer infective centers than the control construct, with viabilities ranging from 8.4 to 44.9% relative to control. In general, decreasing the number of adduct hydroxyls decreased the total frequency of substitution mutations induced. For all but one of the present adducts, the total mutational frequency was lower than that for any of the previously reported diol epoxide adducts in the same sequence. Remarkably, this (9S,10R)-adduct with cis orientation of the dado residue and the 9-OH group gave the highest mutational frequency of all the B[a]P adducts studied in this sequence, including the diol epoxide adducts. With the present adducts, A → T transversions predominated, with smaller numbers of A → G transitions and even fewer A → C transversions.

Full text of DOI:10.1021/tx010076o

1082
Chem. Res. Toxicol. 2001, 14, 1082-1089
Effect of Ben zo-Rin g Hyd r oxyl Gr ou p s on Site-Sp ecific
Mu ta gen esis by Tetr a h yd r oben zo[a ]p yr en e Ad d u cts a t
N6 of Deoxya d en osin e
Leilani A. Ramos,† J ane M. Sayer,‡ Haruhiko Yagi,‡ J amshed H. Shah,‡
Anthony Dipple,†,§ and Donald M. J erina*,‡
Chemistry of Carcinogenesis Laboratory, National Cancer Institute at Frederick,
Frederick, Maryland 21702, and Laboratory of Bioorganic Chemistry, National Institute of Diabetes
and Digestive and Kidney Diseases, The National Institutes of Health, Bethesda, Maryland 20892
Received April 12, 2001
We have previously investigated the mutations induced on replication in Escherichia coli of
the M13mp7L2 genome containing each of the eight possible adducts derived from the four
optically active 7,8-diol 9,10-epoxide metabolites of benzo[a]pyrene (B[a]P) by alkylation of a
specific deoxyadenosine (dAdo) residue at N6. Observed mutational frequencies depended in
part on the relative spatial orientations of the three hydroxyl groups in these adducts. To
determine how the presence or absence of these hydroxyl groups affects mutational response,
we have synthesized 16-mer oligonucleotides with the same sequence as one of those previously
studied with the diol epoxide adducts, but containing B[a]P-dAdo adducts in which two or all
three of the adduct hydroxyl groups were replaced by hydrogen. Transfection of the adducted
M13 constructs into SOS-induced Escherichia coli consistently gave fewer infective centers
than the control construct, with viabilities ranging from 8.4 to 44.9% relative to control. In
general, decreasing the number of adduct hydroxyls decreased the total frequency of substitution
mutations induced. For all but one of the present adducts, the total mutational frequency was
lower than that for any of the previously reported diol epoxide adducts in the same sequence.
Remarkably, this (9S,10R)-adduct with cis orientation of the dAdo residue and the 9-OH group
gave the highest mutational frequency of all the B[a]P adducts studied in this sequence,
including the diol epoxide adducts. With the present adducts, A f T transversions predomi-
nated, with smaller numbers of A f G transitions and even fewer A f C transversions.
omers, four DEs are metabolically formed. Each of these
four DEs reacts with DNA via cis or trans opening of the
epoxide ring by the exocyclic N2 and N6 amino groups of
the purine nucleosides deoxyguanosine (dGuo) and deoxy-
adenosine (dAdo) (4, 5); thus a total of eight isomeric
adducts are possible from a given base. Our laboratory
had previously investigated the effect of these eight
isomeric B[a]P DE adducts at dAdo in two sequence
contexts on the mutagenic outcome of translesion DNA
replication in an Escherichia coli-M13 system (6, 7). For
all eight stereoisomers, base substitution mutations
occurred at a high rate, with frequencies ranging from 5
to 68%. Observed mutational frequencies depended to
some extent on the stereochemical relationships among
the hydroxyl groups on C7, C8 and C9 of the DEs.
However, chirality at the site of attachment of the amino
group also had an influence on mutagenicity. This effect
of chirality was much larger in the case of benzo[c]-
phenanthrene DE-dAdo adducts (8). In the present study
we sought to determine the effect of the tetrahydro benzo-
ring hydroxyl groups on the mutagenicity of B[a]P
derivatives by the use of adduct-containing inserts 1-6
(Figure 1A) in which some or all of these hydroxyl groups
in B[a]P DE adducts had been replaced by a hydrogen.
In tr od u ction
The occurrence of mutations, especially in genes that
influence cell cycle regulation or proto-oncogene activa-
tion, is recognized as the initial event in the multistage
process of carcinogenesis. Chemical carcinogens that
alkylate DNA produce lesions in the form of covalent
adducts (1). While most organisms are capable of repair-
ing damaged DNA, some lesions either escape repair or
are repaired inaccurately (2). Incorrect replication past
such unrepaired lesions will result in mutations that
could potentially lead to cell transformation.
The environmental pollutant benzo[a]pyrene (B[a]P)1
is metabolized in mammals to two diastereomeric benzo-
[a]pyrene 7,8-diol-9,10-epoxides (B[a]P DE-1, in which
the benzylic 7-hydroxyl group and the epoxide oxygen are
cis, and B[a]P DE-2, in which these groups are trans)
(3). Since each diastereomer exists as a pair of enanti-
* To whom correspondence should be addressed. Phone (301) 496-
4560. Fax: (301) 402-0008. E-mail: dmjerina@nih.gov.
† National Cancer Institute at Frederick.
‡ National Institutes of Health.
§ Deceased May 26, 1999.
1
Abbreviations: B[a]P, benzo[a]pyrene; B[a]P DE, benzo[a]pyrene
7,8-diol-9,10-epoxides (B[a]P DE-1, in which the benzylic 7-hydroxyl
group and the epoxide oxygen are cis, and B[a]P DE-2, in which these
groups are trans); B[a]P H4E, 7,8,9,10-tetrahydrobenzo[a]pyrene 9,-
10-epoxide; 10-amino H4 B[a]P, 10-amino-7,8,9,10-tetrahydrobenzo[a]-
pyrene; 6-FP, 6-fluoro-9-(2-deoxy-â-D-erythro-pentofuranosyl)purine;
UDG, uracil DNA glycosylase.
Exp er im en ta l P r oced u r es
T4 polynucleotide kinase, T4 DNA ligase, uracil DNA glyco-
sylase, X-gal, and IPTG were obtained from USB Corp. (Cleve-
10.1021/tx010076o CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/03/2001

Effect of Hydroxyl Groups on Benzo[a]pyrene Mutagenesis
Chem. Res. Toxicol., Vol. 14, No. 8, 2001 1083
Sch em e 1
(br d, 1NH, J 10,NH ) 6.9), 7.92-8.22 (m, 8 aromatic protons).
HRMS (FAB+, m/z) calcd for C22H18INO2 (M+): 455.0382. Found,
455.0384. Anal. calcd for
Found: C, 58.01; H, 3.89.
C22H18INO2: C, 58.04; H, 3.98.
Cyclic Carbamate of cis-9-Hydroxy-10-amino-7,8,9,10-tetrahy-
drobenzo[a]pyrene (8). A solution of iodocarbamate 7 (1.2 g, 2.6
mmol) in diglyme (12 mL) was refluxed under argon for 2.5 h.
The reaction mixture was concentrated in vacuo to give a
crystalline product (0.83 g, 99%). Recrystallization from THF-
n-hexane gave colorless scales, mp 314-315 °C (decomp); 1H
NMR (CDCl3): δ 2.08 (m, 1H8′), 2.60 (m, 1H8), 3.15 (dt, 1H7,
J 7,7′ ) 16.0, J 7,8 ) J 7,8′ ) 4.0), 3.46 (m, 1H7′), 5.33 (br s, 1NH),
5.45 (dt, 1H9, J 9,10 ) 8.0, J 9,8 ) J 9,8′ ) 4.0), 5.91 (d,1H10, J 9,10
)
8.0), 8.00-8.27 (m, 8 aromatic protons). HRMS (FAB+, m/z)
calcd for C21H15NO2 (M+): 313.1103. Found: 313.1112. Anal.
calcd for C21H15NO2: C, 80.49; H, 4.82; N, 4.47. Found: C, 80.35;
H, 4.85; N, 4.38.
F igu r e 1. (A) Structures of the six optically active tetrahydro
B[a]P dAdo adducts in the oligonucleotide 16-mers 1-6 prepared
for the present study. (B) Reaction of the racemic H4 B[a]P cis
and trans amino alcohols and 10-amino H4 B[a]P with the
fluorinated oligonucleotide 16-mer. For graphical purposes the
6-FP residue is represented as a fluoro derivative of dAdo. Note
that oligonucleotides 1 and 2 correspond to cis opening, and 3
and 4 to trans opening of the corresponding B[a]P tetrahydroe-
poxide (B[a]P H4E).
cis-9-Hydroxy-10-amino-7,8,9,10-tetrahydrobenzo[a]pyrene (cis
amino alcohol, 9). A mixture of cyclic carbamate 8 (370 mg, 1.2
mmol), Amberlite IRA-400 (OH--form, 4 g), H2O (30 mL), and
THF (200 mL) was refluxed with stirring for 3 days. The
reaction mixture was filtered, and the filtrate was evaporated
to leave colorless crystals (320 mg, 94%). Recrystallization from
THF-MeOH gave colorless needles, mp 227-228 °C (decomp);
1H NMR (DMSO - D2O): δ 1.85 (m, 1H8′), 2.14 (m, 1H8), 3.25
(m, 1H7 and 1H7′), 3.94 (dt, 1H9, J 9,8′ ) 12.4, J 9,10 ) J 9,8 ) 3.6),
4.73 (br d, 1H10, J 9,10 ) 3.6), 7.95-8.27 (m, 7 aromatic protons),
land, OH). [γ-32P]ATP and EcoRI restriction enzyme were from
Amersham Corp. (Piscataway, NJ ). E. coli strain SMH77 and
the bacteriophage M13mp7L2 were generous gifts from Dr. C.
W. Lawrence (University of Rochester, NY).
Syn th etic In ter m ed ia tes. Proton NMR spectra were ob-
tained at 300 MHz. Chemical shifts (δ) are reported in parts
per million, and coupling constants (J ) are reported in hertz.
Mass spectra were measured on a J EOL J MS-SX102 spectrom-
eter with a direct exposure probe. Melting points are uncor-
rected. For structures and relative stereochemistry, see Scheme
1. The known precursor compounds (9), 9,10-dihydrobenzo[a]-
pyrene-7-(8H)-one and 7,8,9,10-tetrahydrobenzo[a]pyrene, were
prepared using improved methods as described in the Support-
ing Information.
trans-9-Iodo-10-carbomethoxyamino-7,8,9,10-tetrahydrobenzo-
[a]pyrene (7). To a stirred mixture of 7,8-dihydrobenzo[a]pyrene
(10) (1.7 g, 6.8 mmol), silver isocyanate (6 g, 40 mmol) and dry
THF (40 mL) was added a solution of iodine (1.7 g, 6.8 mmol)
in dry THF (10 mL) at room temperature over a period of 5 min
in the dark. After being stirred for 30 min, the reaction mixture
was treated with decolorizing charcoal (1 g) and filtered. The
filtrate was evaporated to leave a reddish oil, which was
dissolved in a mixture of THF (50 mL) and MeOH (350 mL)
and refluxed for 2 h. After treatment with decolorizing charcoal
(2 g) and filtration, the filtrate was concentrated to give a
crystalline product (2.9 g, 94%). Recrystallization from MeOH
gave colorless minute needles, mp 123 °C (decomp); 1H NMR
(acetone-d6): δ 2.20-2.45 (m, 1H8 and 1H8′), 3.30-3.65 (m, 1H7
and 1H7′), 3.70 (s, MeCO), 5.10 (m, 1H9), 6.05 (m, 1H10), 7.32
8.53 (d, 1H11, J 11,12 ) 9.3). HRMS (FAB+, m/z) calcd for C20H18
NO (M+ + 1): 288.1388. Found: 288.1392. Anal. calcd for
20H17NO: C, 88.52; H, 5.96; N, 4.87. Found: C, 88.45; H, 5.88;
N, 4.90.
tr a n s-9-H yd r oxy-10-a m in o-7,8,9,10-tetr a h yd r oben zo[a ]-
-
C
pyrene (trans amino alcohol, 10). A mixture of 9,10-epoxy-7,8,9,-
10-tetrahydrobenzo[a]pyrene (10) (540 mg, 2.0 mmol) in liquid
ammonia (∼50 mL) was heated at 85-95 °C for 18 h in a Parr
high-pressure reactor. After cooling to dry ice temperature and
evaporation of the ammonia under N2, the crude product was
washed with cold ether. Chromatography on a silica column
packed in CHCl3 and eluted with 7% MeOH in CHCl3 provided
450 mg (85%) of off-white solid: mp 176-178 °C. 1H NMR
(CDCl3): δ 2.25 (m, 1H8′), 2.34 (m, 1H8), 3.24 and 3.47 (each m,
1H7 and 1H7′), 4.32 (ddd, 1H9, J 9,8′ ) 5.5, J 9,10 )3.3, J 9,8 ) 2.3),
4.88 (d, 1H10, J 9,10 ) 3.3), 7.50-8.04 (m, 4 aromatic protons),
8.17 (d, 1H12, J 11,12 ) 9.1 ), 8.19 (br d, 1H3) 8.21 (br d, 1H1), 8.37
(d, 1H11, J 11,12 ) 9.1 ). m/z: 288 (M+ + 1). Treatment with Ac2O/
pyridine gave the 9-acetoxy 10-acetamido derivative (mp 258-
260 °C). Anal. calcd for C24H21NO3: C, 77.62; H, 5.66; N, 3.77.
Found: C, 77.38; H, 5.77; N, 3.65.
10-Amino-7,8,9,10-tetrahydrobenzo[a]pyrene (10-amino H4
B[a]P, 11). 7,8-dihydrobenzo[a]pyrene-10-(9H)-one (11) (1 g, 3.7
mmol) was converted to its O-benzyloxime by treatment with
O-benzylhydroxylamine hydrochloride (2 g, 7.4 mmol) in

1084 Chem. Res. Toxicol., Vol. 14, No. 8, 2001
Ramos et al.
Ta ble 1. HP LC Reten tion Tim esa a n d Con figu r a tion a l
Assign m en tsb for Oligon u cleotid es Con ta in in g
Tetr a h yd r o B[a ]P -d Ad o Ad d u cts in Con text II(A)
4 mL pyridine for 16 h at room temperature. The resultant
product was directly reduced (12) to 10-amino-7,8,9,10-tetrahy-
drobenzo[a]pyrene 11 by treatment with borane-THF (26 mmol)
in 50 mL THF for 1 h at 0 °C followed by 16 h at room
temperature. After dilution with organic solvent and washing
with NaOH, the organic layer was concentrated to give an oil
(800 mg) that contained three major products (tR ) 1.8, 3.0 and
4.3 min) by HPLC on an Axxiom Sil column (9.5 × 250 mm)
eluted at a flow rate of 10 mL/min with 20% EtOAc and 0.1%
Et3N in n-hexane (detection at 270 nm). The mixture was
subjected to column chromatography on silica gel eluted with
20% EtOAc and 0.1% Et3N in hexane to give three fractions, in
order of elution, that were identified as 7,8,9,10-tetrahydro
B[a]P (250 mg), the desired amine (11, 28 mg) and benzyl alcohol
(420 mg). Although amine yields of 68-86% on reduction of
other O-benzyloximes by the described procedure have been
reported (12), the above reaction gave extremely poor yields of
the desired amine as a result of over-reduction at the 10-position
to give predominantly 7,8,9,10-tetrahydro B[a]P. The amine was
further purified by HPLC as above to give a colorless powder
(20 mg): 1H NMR (acetone-d6): δ 2.00 (m, 2H), 2.15 (m, 2H),
3.18 (m, 2H7), 4.80 (br s, NH2 and 1H10), 7.80-8.27 (7 aromatic
protons), 8.45 (d, 1H11, J 11,12 ) 9.3). HRMS (EI, m/z) calcd for
retention time
oligonucleotidec
configuration at C10
(min)
1
2
3
4
5
6
R
S
R
S
S
R
21.7
23.7
21.3
22.7
24.2
26.9d
a
On a 7 µm Hamilton PRP-1 column (10 × 250 mm) eluted at
3 mL/min with a linear gradient of acetonitrile in 0.1 M am-
monium carbonate buffer, pH ∼7.5, that increased the acetonitrile
concentration from zero to 17.5% over 20 min, followed by a 5 min
ramp to 50% acetonitrile and 5 min isocratic elution at this solvent
b
composition. Configurational assignments were based on CD
spectra of the oligonucleotides (see text). c For structures see
Figure 1. Note that for each pair of diastereomers the early and
late eluting oligonucleotides always have the dAdo substituent in
the R and â orientations, respectively, as shown in Figure 1A,
although the Cahn-Ingold-Prelog nomenclature changes for 5
d
and
6
on application of the sequence rules. An additional
chromatographic step (Beckman Ultrasphere C18, 10 × 250 mm,
eluted at 3 mL/with the above solvent system) was required to
separate this oligonucleotide (tR 23.3 min) from ammonolysis
product(s) of the organic protecting groups.
C
20H17N (M+): 271.1361. Found: 271.1364. A portion was
acetylated with Ac2O and pyridine to give the acetamido
derivative as a colorless powder. 1H NMR (CDCl3): δ 1.90-2.40
(m, 4H), 3.25 (m, 2H7), 2.00 (s, CH3CO), 5.85 (br d, 1H10, J 10,NH
) 8.0), 6.09 (br d, 1NH), 7.90-8.26 (8 aromatic protons). HRMS
(EI, m/z) calcd for C22H19NO (M+): 313.1466. Found: 313.1458.
Ad d u cted Oligon u cleotid es. Oligonucleotides containing
adducts corresponding to cis or trans opening of 9,10-epoxy-
7,8,9,10-tetrahydro B[a]P by N6 of dAdo (one hydroxyl group
on the tetrahydro benzo-ring) or containing a N6-10-(7,8,9,10-
tetrahydrobenzo[a]pyrenyl) dAdo residue (no hydroxyl groups)
were prepared by postoligomerization modification (13) of a
support-bound oligonucleotide 16-mer, 5′-CAG(6-FP)TTTA-
GAGTCTGC-3′ [Context II(A), corresponding to region 141-126
in the supF gene] containing a 6-fluoro-9-(2-deoxy-â-D-erythro-
pentofuranosyl)purine (6-FP) residue at the modification site.
The fluorinated oligonucleotide was prepared essentially as
described (14) on a 15 micromole scale using 150 mg of 170 Å
controlled pore glass (cpg) derivatized with N4-benzoyl-5′-O-(4,4′-
dimethoxytrityl)-2′-deoxycytidine-3′-succinic acid (102 µmol/g),
with the following modifications. 4,5-Dicyanoimidazole (0.5 M
in CH3CN, 250 µL) was used instead of 1H-tetrazole in the
manual coupling with a 6-fold molar excess of 6-FP phosphora-
midite. End capping following this step was omitted (15). Any
failure to couple the fluorinated residue would eventualy result
in a shorter oligonucleotide lacking the hydrocarbon, which is
easily separable from the desired, adducted oligonucleotides on
HPLC. The yield on manual coupling was essentially quantita-
tive. The 5′-DMT group was removed from the support-bound
oligonucleotide before use. Hydrocarbon adducted oligonucle-
otides were prepared on a 2 µmol scale. In a typical reaction,
20 mg of the above cpg-bound, fluorinated oligonucleotide, 12
mg of racemic, trans amino alcohol 10, and 13 µL of triethyl-
amine were heated at 40-45 °C for 65 h in 200 µL Me2SO.
Similar procedures were followed for postoligomerization syn-
thesis with the racemic cis amino alcohol 9 and racemic 10-
amino H4 B[a]P 11. Washing of the glass beads and cleavage
and deprotection of the modified oligonucleotides were as
described (14). The adducted oligonucleotides were purified by
reversed-phase HPLC (Table 1). Isolated yields of each diaster-
F igu r e 2. Polyacrylamide gel electrophoresis of the16-mer
oligonucleotides 1-6 after end-labeling following their purifica-
tion by HPLC and gel electrophoresis (see Experimental Pro-
cedures). Structures for 1-6 are shown in Figure 1. The lane
labeled C corresponds to the unadducted 16-mer control.
[γ-32P] ATP-labeling of aliquots (Figure 2).
All M13 minipreps were performed using QIAprep 8 M13 kits
from Qiagen (Valencia, CA). DNA sequencing reactions were
carried out using ABI PRISM Dye Terminator Cycle Sequencing
Ready Reaction kit from PE Applied Biosystems (Foster City,
CA). The sequencing runs were done on an ABI Prism 377 DNA
Sequencer.
Con str u ction of Site-Sp ecifica lly Mod ified M13 Ge-
n om es. The construction of the M13 vectors was based on the
procedure developed by Lawrence and co-workers (16, 17) and
is essentially the same as that used in previous studies in our
laboratory (6, 7). Briefly, bacteriophage M13mp7L2 (200 µg) was
digested with EcoRI (800 units) in 50 mM Tris-HCl, pH 8.0, 10
mM MgCl2, 100 mM NaCl, for 2.5 h at 30 °C. The enzyme was
inactivated by heating at 65 °C for 15 min, and the solution
was extracted with phenol/chloroform. The linearized DNA was
then purified by ethanol precipitation. Each oligonucleotide 16-
mer (50 pmol), with or without the adduct, was 5′-phosphory-
lated with kinase and annealed with a 56-mer uracil-containing
scaffold (2 pmol) by heating them together to 50 °C for 5 min
and slow cooling to room temperature overnight. The scaffold
contained a middle sequence complementary to the 16-mer
insert and 20-mer overhangs complementary to the ends of the
linearized M13 DNA. The 16-mer/scaffold duplex was annealed
with the linear M13 (1 pmol) by incubation at room temperature
overnight. Ligation was done at 16 °C overnight upon addition
of T4 DNA ligase (30 units). The ligation mixture was then
incubated with uracil DNA glycosylase (UDG) (1 unit) to remove
uracil residues in the scaffold. UDG produces abasic sites that
lead to degradation of the scaffold by apurinic endonucleases
eomeric oligonucleotide were typically 15-25 A260
.
All oligodeoxynucleotides were further purified by electro-
phoresis on a denaturing 20% polyacrylamide gel. The oligo-
nucleotides were detected by UV shadowing. The corresponding
gel areas were cut out and crushed, incubated overnight in
elution buffer [0.5 M NH4OAc, 10 mM Mg(OAc)2], and desalted
on Waters Sep-Pak cartridges (Milford, MA) by elution with 60%
methanol in water. The eluate was dried and then washed with
90% ethanol. Purity was confirmed by electrophoresis after

Effect of Hydroxyl Groups on Benzo[a]pyrene Mutagenesis
Chem. Res. Toxicol., Vol. 14, No. 8, 2001 1085
and exonucleases upon transfection into bacterial cells (6).
To estimate the apparent ligation efficiency (defined as the
ratio of circular DNA to total DNA), the components of the vector
construction mixture (∼100 ng of DNA) were separated by
agarose gel electrophoresis and transferred to nitrocellulose by
Southern blotting. A [γ-32P]ATP-labeled probe complementary
to a sequence in the M13 construct, 5′-GGCGAAAGGGGGAT-
GTGC (1 ng/mL hybridization solution), was used to detect
closed circular and linear DNA, the amounts of which were
quantified by PhosphorImager analysis (Storm 860, Molecular
Dynamics, Inc.).
Mu ta gen esis Assa y. E. coli strain SMH77 cells were SOS-
induced by UV irradiation (254 nm) at 40 J /m2 for 40 s (18)
before they were made competent by CaCl2 treatment. For each
transfection, an aliquot of the ligation mixture (20 ng) was added
to 100 µL of the competent cell suspension, heat shocked at 42
°C for 90 s, cooled on ice for 120 s and incubated at room
temperature for 10 min. The DNA/cell suspension was then
mixed with top agarose containing X-gal and IPTG, poured onto
2XYT agar plates, and grown at 37 °C overnight. Survival was
estimated by comparing the number of blue plaques (containing
the insert) obtained from equal amounts of total DNA with and
without adduct (with the survival of the control taken as 100%).
Either three or four separate ligations were performed followed
by transfection, and the survival results were averaged. Mu-
tagenicity data were generally obtained from three ligation-
transfection experiments, except for the two most mutagenic
adducted sequences 1 and 3, which gave 595 and 697 total
plaques, respectively, and good agreement between the data
from two replicate experiments.
Mutation analysis of progeny phage was done by differential
oligonucleotide hybridization as described (6). The DNA from
the plaques produced on each plate was transferred to four
Protran nitrocellulose membranes (Schleicher & Schuell). After
baking the membranes for 2 h at 80 °C in a vacuum oven, they
were washed with 3× SSC containing 0.1% SDS for 2 h at 37
°C. To eliminate nonspecific binding, they were then incubated
in 2× Prehybridization/Hybridization solution (Gibco/BRL) for
1.5 h at 37 °C. One of four [γ-32P]ATP-labeled 13-mers designed
to be complementary to either side of the adduct site, 5′-
TCTAAAXCTGCAC, where the adduct is opposite X (X)A, C,
G, or T), was added to each transfer membrane at 37 °C. The
solutions were cooled slowly to room temperature and incubated
overnight with constant agitation. The membranes were then
washed with 6× SSC at room temperature (4 × 30 min) and
finally at the stringent temperature 35.5 °C for another 30 min.
The membranes were subsequently exposed to X-ray film with
an intensifying screen at -70 °C overnight. The autoradiographs
were compared to the actual plates to identify single-base
mutations at the target site. The DNA from any plaque that
did not correspond to any signal in the autoradiograph was
extracted and sequenced.
F igu r e 3. Circular dichroism spectra of oligonucleotides in 0.02
M phosphate buffer, pH 7, with ionic strength adjusted to 0.1
M with NaCl. Spectra are normalized to 1.0 absorbance unit at
260 nm. Note that the early (5) and late (6) eluting adducted
oligonucleotides (Table 1) with no hydroxyl groups on the
saturated ring (10-amino H4 B[a]P adduct; partial structures
as indicated) exhibit major CD bands that are very similar to
those for the oligonucleotides with the same sequence (14)
containing B[a]P DE adducts (3 hydroxyl groups). Correspond-
ing similarities are observed with oligonucleotides containing
the cis opened DE adducts (7) as well as the cis and trans opened
B[a]P H4E adducts in oligonucleotides 1-4 (not shown).
pressure as previously described for the corresponding
amino triols from B[a]P DEs (19). The corresponding cis
amino alcohol was prepared by the general method of
Hassner et al. (20) from 7,8-dihydro B[a]P in three steps
(Scheme 1), each of which proceeded with g94% yield.
In contrast, preparation of 10-amino H4 B[a]P via reduc-
tion of the O-benzyloxime proved quite difficult, as the
major reaction pathway resulted in loss of the 10-amino
group to give 7,8,9,10-tetrahydro B[a]P.
The diastereomeric adducted oligonucleotides derived
from reaction of the support-bound, fluorinated 16-mer
with each racemic amino alcohol or with racemic 10-
amino H4 B[a]P were well separated on HPLC (Table 1).
Absolute configurations at C10, the point of attachment
of N6 of dAdo to the hydrocarbon, were assigned on the
basis of the circular dichroism (CD) spectra of the purified
oligonucleotides (Figure 3). In the monomeric nucleoside
adducts, the hydroxyl groups have a negligible effect on
the appearance of the CD spectra, as shown by compari-
son of the spectra of B[a]P H4E adducts (21, 22) at N6 of
dAdo with the corresponding DE adducts (23, 24).
Similarly, the present B[a]P adducted oligonucleotides
lacking two or three of the benzo-ring hydroxyl groups
exhibited CD spectra whose long-wavelength bands were
very similar to those reported (7, 14) for the correspond-
ing DE-adducted oligonucleotides. For example, the CD
spectra for the H4 B[a]P-modified oligonucleotides 5 and
6 (prepared from 10-amino H4 B[a]P in this study) are
compared with those for the previously assigned trans
DE-2 adducted analogues in Figure 3. Similar results
were obtained with the adducted oligonucleotides 1-4
Resu lts
Ad d u cted Oligon u cleotid es. The required oligo-
nucleotides were constructed by postoligomerization modi-
fication (13) of a 16-mer containing a 6-fluoropurine base
(Figure 1B), utilizing racemic 10-amino tetrahydro B[a]P
11 as well as the racemic cis and trans amino alcohols 9
and 10. The cis and trans amino alcohols are easily
distinguished from each other by the NMR coupling
constants between the two H8 protons and H9. Thus, for
the trans amino alcohol (diaxial amino and hydroxyl
substituents), H9 is equatorial, and J 9eq,8eq and J 9eq,8ax are
both relatively small (2.3 and 5.5 Hz). In contrast, for
the cis amino alcohol (axial amino and equatorial hy-
droxyl group), H9 is axial, and J 9ax,8ax is 12.4 Hz. The
trans amino alcohol was easily prepared (85%) from the
corresponding 9,10-epoxide of 7,8,9,10-tetrahydro B[a]P
(Scheme 1) by ammonolysis in liquid ammonia under

1086 Chem. Res. Toxicol., Vol. 14, No. 8, 2001
Ramos et al.
Ta ble 2. F r equ en cies of In d ivid u a l Rep lica tion Even ts
a n d Tota l Ba se Su bstitu tion Mu ta tion s Resu ltin g fr om
Rep lica tion P a st th e B[a ]P Ad d u cts in In ser ts 1-6 in
SOS-In d u ced Cellsa
(not shown). On the basis of their CD spectra, all three
early-eluting oligonucleotides (1, 3, and 5) had the same
spatial orientation at C10 of the hydrocarbon relative to
the dAdo substituent (see Figures 1 and 2), and all three
late-eluting eluting oligonucleotides (2, 4, and 6) had the
opposite orientation. Note that early- and late-eluting
adducts correspond to 10R and 10S, respectively, for the
amino alcohol derivatives, but the nomenclature (al-
though not the relative spatial orientation of the dAdo
substituent and the hydrocarbon) reverses for the 10-
amino B[a]P derivatives because of the application of the
sequence rule.
b
no. of
A f A
no. of
A f T
no. of
A f G
no. of
A f C
survival
(%)
MFtot
(%)
1c
2d
3e
4f
421
1918
649
2110
2380
3556
165
28
43
12
43
4
5
16
5
3
4
4
2
0
1
1
1
13
23
13
19
33
46
29.2
2.3
6.9
0.8
2.0
0.2
5g
6h
1
a
For the control sequence, 3668 plaques were screened and no
Con st r u ct ion of Sit e-Sp ecifica lly Mod ified M13
Gen om es. The M13 vectors were constructed based on
the method of Lawrence and co-workers (16, 17) with
minor modifications (6). After digestion with EcoRI, the
linear M13 DNA was recircularized by annealing and
ligating a duplex consisting of a 56-mer scaffold and a
16-mer oligonucleotide insert with or without the adduct.
Ligation efficiencies, defined as the amount of circular
DNA as a fraction of the sum of linear and circular DNA,
were determined by running an aliquot of the constructs
on an agarose gel, transferring the DNA onto a mem-
brane by Southern blotting and radioactive probing.
Ligation of the unadducted 16-mer into the M13 vector
occurred with an apparent efficiency of 36%, whereas the
oligonucleotides 1, 2, 3, 4, 5, and 6 containing the dAdo
adducts were ligated with somewhat lower efficiencies
of 26, 26, 20, 29, 27, and 28%, respectively, that were
quite similar to each other. These apparent ligation
efficiencies for the adducted 16-mers are comparable to
those observed wirh cis opened B[a]P DE-dAdo adducts
in the same sequence (7), and may result from distortion
of the DNA by the adducts.
mutation at the targeted site was detected (background MF <
b
0.027%). The percentage of total progeny that had undergone
base substitution mutations at the adduct site. c Nontargeted
mutations for the insert sequence 5′-C1A2G3A4T5T6T7A8G9A10
-
G
11T12C13T14G15C16, where the adduct is at A4, were as follows
through footnote h: 1 C1 f T; 4 A2 f G; 1 A2 f C; 1 G3 f T; 1 T5
f A; 1 A8 f G; 2 G9 f A; 2 A10 f C; 1 G insert between T14 and
d
G
15; 3 C16 deletions. 1 C1 f G; 3 A2 f C, 2 G3 f C; 1 T5 f A; 1
A8 f C; 3 G9 f A; 3 G9 f C; 4 A10 f C; 1 A10 f T; 1 G insert
between A10 and G11; 1 C16 deletion. e 1 G3 f T; 1 G9 f A; 4 C16
deletions. f 3 C1 f T; 4 A2 f T/G3 f T/T5 f G; 2 G3 f C; 4 G9
f
A; 1 A10 f C; 1 A10 f T; 1 G11 f A; 1 C16 deletion; 1 deletion of
g
first 6 nucleotides. 1 T5 f A; 1 T6 f A; 2 A8 f G; 1 G9 f A; 1 G9
h
f T; 1 G insert between A10 and G11; 6 C16 deletions. 1 A2 f C;
1 G3 f T; 1 T7 f A; 1 A8 f G; 3 G9 f A; 2 A10 f C; 1 A10 f T; 1
G11 f A; 4 C16 deletions.
When transfected into SOS-induced E. coli, the ad-
ducted M13 constructs gave fewer plaques than the
control M13 construct. The values for survival were
determined from the ratio of infective centers containing
the insert (blue plaques) obtained from a given amount
of adducted relative to control DNA. These values were
13, 23, 13, 19, 33, and 46%, for the constructs with dAdo
adducts 1, 2, 3, 4, 5, and 6, respectively. In the presence
of adducts, low apparent survival values reflect contribu-
tions from two factors: (1) a smaller fraction of the total
DNA that is circular and contains the insert, and (2)
reduced efficiency of DNA polymerases to replicate past
the adducts. Consequently, the survival data by them-
selves would overestimate the contribution of the latter
factor (25). In the present study, apparent ligation
efficiencies for all six adducted oligonucleotides were
quite large (∼60-70% of control). Observed survival
values (13-46%) that are much less than this suggest
that the present H4 B[a]P dAdo adducts are inefficiently
bypassed. Interestingly, the recoveries of progeny in the
present experiments are generally higher than those
(<10%) previously observed (6, 7) with the B[a]P DE
adducted oligonucleotides relative to the control.
F igu r e 4. Distribution of individual base substitution muta-
tions as a fraction of total mutations induced by B[a]P-dAdo
adducts in the 16-mer inserts 1-6. Structures are shown in
Figure 1.
mutational frequencies than their trans counterparts (3
and 4). With respect to the hydroxyl groups on the
tetrahydro benzo-ring, 5 and 6 (no hydroxyl groups) had
lower mutational frequencies than 1, 2, 3, and 4 (one
hydroxyl group).
The distribution of mutations is given in Figure 4. For
all six adducts investigated, the major base substitution
mutations observed were A f T transversions, with
frequencies ranging from 60.9 to 94.8% of total muta-
tions. The frequencies for A f G transitions were higher
than for A f C transversions in all cases except for 6,
where these two mutational events occurred with the
same frequency.
Mu ta tion a l F r equ en cies. Mutation data for the six
adducted oligonucleotide inserts are summarized in Table
2. Overall mutational frequencies ranged from 0.2 to
29.2%. Oligonucleotides 1 (10R), 3 (10R), and 5 (10S),
having the same spatial arrangement of the pyrene rings
with respect to dAdo, gave higher mutational frequencies
than their counterparts, 2 (10S), 4 (10S), and 6 (10R).
For the H4E adducts, those with the hydroxyl group and
the dAdo in a cis arrangement (1 and 2) showed higher
Discu ssion
An objective of our ongoing studies on site-specific
mutations induced by polycyclic aromatic hydrocarbon

Effect of Hydroxyl Groups on Benzo[a]pyrene Mutagenesis
Chem. Res. Toxicol., Vol. 14, No. 8, 2001 1087
F igu r e 5. Total frequencies of base substitution mutations induced by the B[a]P-dAdo adducts in 16-mers 1-6 in comparison with
those induced by the related B[a]P diol epoxide (DE-1 and DE-2) dAdo adducts in the same sequence context. Data for the DE
adducts are from Page et al. (6, 7). The top row of structures (DE adducts) correspond to the rear row of bars in each bar graph. All
of the adducts on side A have the same spatial orientation at C10, and the adducts on side B have the opposite orientation. The
structures are arranged such that those on the right-hand edge of side A and those along the left-hand edge of side B, etc., are
mirror images of each other.
DNA adducts has been to evaluate the effect of adduct
structure on mutagenicity. Previously, we reported data
for dAdo adducts derived from the cis and trans opening
of B[a]P DEs (6, 7) and benzo[c]phenanthrene DEs (8)
in two sequence contexts. In those studies, we observed
a dependence of the mutational frequencies on the
stereochemical relationships among the hydroxyl groups
as well as on the configuration at the site of attachment
of the DEs to the adenine base. The aim of the present
study was to investigate systematically the effect of the
hydroxyl groups on mutational response by examining
adducts in which some or all of these hydroxyl groups
were absent. The bay-region tetrahydroepoxide, 9,10-
epoxy-7,8,9,10-tetrahydro B[a]P (B[a]P H4E) (see Figure
1), which lacks hydroxyl groups at C7 and C8, has been
shown to form adducts with both dAdo and dGuo in DNA
in vitro (21). This tetrahydroepoxide is formed on mam-
malian metabolism of 7,8-dihydro B[a]P (26-28), with
the (+)-(9S,10R)-enantiomer predominating upon cyto-
chrome P450-mediated metabolism. This stereoselectivity
is analogous to that for the formation of the B[a]P DEs
(3). Racemic B[a]P H4E is generally more mutagenic than
the corresponding B[a]P DEs in several strains of Sal-
monella typhimurium, but is somewhat less mutagenic
than the B[a]P DEs in Chinese hamster V79 cells (29).
However, the optically active (+)-B[a]P H4E is about
three times more mutagenic in V79 cells than the
corresponding (+)-B[a]P DE-2 (R. L. Chang, personal
communication). In contrast, racemic B[a]P H4E was not
tumorigenic on mouse skin (30), presumably as a conse-
quence of its high chemical reactivity (31) and its
sensitivity to epoxide hydrolase (28) which likely result
in its rapid hydrolysis to inactive diols.
corresponding DE adducts. Thus, we chose context II(A)
for the present experiments, since the relatively low
mutational frequency observed with DE adducts in this
sequence would potentially allow a better assessment of
increases in mutational frequency than context I(A),
whose mutational frequency with the DE adducts was
already quite high (6, 7). Surprisingly, however, only one
of the H4E adducts (the cis 10R adduct in 1; see below)
was more mutagenic than the DEs, and all the other
adducts were much less so.
There is no clear effect of adding hydroxyl groups on
the mutational distribution (Figure 4). In all cases A f
T transversions predominated (g60%). The set of adducts
configurationally related to 5 (same orientation of dAdo
relative to the hydrocarbon) exhibited a higher preference
(g90%) for A f T mutations than those (2, 4, and 6) with
the opposite configuration at C10. Comparison with our
previous data (6, 7) for B[a]P DE adducts in this sequence
context indicated that the 10R DE adducts also exhibited
a higher preference for this mutation relative to their 10S
counterparts. Concomitant with lower proportions of A
f T mutations, 2, 4, and 6 gave relatively higher
percentages (17-35%) of A f G transitions. The highest
percentage of A f G transitions (35%) in the present
series of compounds was observed with the cis 10S B[a]P
H4E adduct in 2. Notably, in the same sequence II(A) (7),
the cis 10S B[a]P DE-1 adduct also gave the highest
percentage (65%) of this mutation. In all cases but one
(insert 6), A f C mutations, which were also relatively
infrequent with the B[a]P DE adducts, were uncommon.
Total mutational frequencies for adducts lacking two
or three of the hydroxyl groups, as well as those previ-
ously reported for the DE adducts, are summarized in
Figure 5. For this purpose, we divided the adducts into
two families, one formally derived from the S adducted
oligonucleotide 5 and the other from the R adducted
On the basis of the above considerations, we antici-
pated that the BaP H4E adducts would be at least as
mutagenic and might well be more mutagenic than the

1088 Chem. Res. Toxicol., Vol. 14, No. 8, 2001
Ramos et al.
(6) Page, J . E., Zajc, B., Oh-hara, T., Lakshman, M. K., Sayer, J . M.,
J erina, D. M., and Dipple, A. (1998) Sequence context profoundly
influences the mutagenic potency of trans-opened benzo[a]pyrene
7,8-diol 9,10-epoxide-purine nucleoside adducts in site-specific
mutation studies. Biochemistry 37, 9127-9137.
(7) Page, J . E., Pilcher, A. S., Yagi, H., Sayer, J . M., J erina, D. M.,
and Dipple, A. (1999) Mutational consequences of replication of
M13mp7L2 constructs containing cis-opened benzo[a]pyrene 7,8-
diol 9,10-epoxide-deoxyadenosine adducts. Chem. Res. Toxicol. 12,
258-263.
(8) Ponte´n, I., Sayer, J . M., Pilcher, A. S., Yagi, H., Kumar, S., J erina,
D. M., and Dipple, A. (2000) Factors determining mutagenic
potential for individual cis and trans opened benzo[c]phenan-
threne diol epoxide-deoxyadenosine adducts. Biochemistry 39,
4136-4144.
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A postoligomerization synthesis of oligonucleotides containing
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Cheh, A. M., J erina, D. M., Bohr, V. A. and Mazur, S. J . (1999)
Stereospecific differences in repair by human cell extracts of
synthesized oligonucleotides containing trans-opened 7,8,9,10-
tetrahydrobenzo[a]pyrene 7,8-diol 9,10-epoxide N2-dG adduct
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oligonucleotide 6 by successive additions of hydroxyl
groups on the tetrahydro ring. The figure is arranged so
that the left- and right-hand families of structures, A and
B, are mirror images of each other, e.g., enantiomeric
pairs of trans opened adducts are on the inside edges of
the graphs A (R) and B (S), and enantiomeric pairs of
cis adducts are on their outside edges. Pairs of adducts
occupying equivalent left-to-right positions in graphs A
and B differ only in their absolute configuration at C10.
In general, the 10-amino H4 derivative 5 and its related
10R H4E adducts 1 and 3 (A), as well as the related DE
adducts, gave higher mutational frequencies than the
corresponding adducts with the opposite spatial orienta-
tion at C10 (B). This trend is opposite to that observed
with the benzo[c]phenanthrene DE-dAdo aducts (8).
Interestingly, the cis 10R adduct in 1 was the most
mutagenic of all the adducts studied. With this one
outstanding exception, the mutational frequency de-
creased within each family as the number of hydroxyl
groups was decreased from three to one to none. Thus,
contrary to our original expectation, decreasing the
number of hydroxyl groups generally decreased the
mutagenicity of these B[a]P adducts.
Within a set of adducts (A or B) with the same spatial
orientation at C10, the H4E adducts in oligonucleotides
1 and 2, in which the dAdo substituent and the hydroxyl
group are cis, gave higher mutation frequencies than
their trans counterparts 3 and 4. For the 10S DE adducts
(B), a similar general pattern of higher mutagenicity for
adducts with cis orientation of the dAdo and 9-OH
substituents is similar, but this pattern is less pro-
nounced with the 10R DE adducts (A). It has previously
been observed that cis-opened B[a]P DE adducts in an
N-ras sequence (32) were severalfold more mutagenic
than the corresponding trans-opened adducts. In the
present work, the high mutagenicity of the cis 10R H4E
adduct 1 is particularly striking. Since dAdo adducts are
formed from racemic B[a]P H4E on reaction with DNA
in vitro (21), it is tempting to speculate that a cis dAdo
adduct or adducts may be in part responsible for the high
mutagenicity observed in previous studies with this
tetrahydroepoxide.
(18) Gibbs, P. E. M., and Lawrence, C. W. (1993) U-U and T-T
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6594.
Su p p or tin g In for m a tion Ava ila ble: Improved methods
for the preparation of 9,10-dihydrobenzo[a]pyrene-7-(8H)-one
and 7,8,9,10-tetrahydrobenzo[a]pyrene. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
(20) Hassner, A., Lorber, M. E., and Heathcock, C. (1967) Addition of
iodine isocyanate to olefins. Scope and synthetic utility. J . Org.
Chem. 32, 540-549.
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