Influence of Na+ on DNA reactions with aromatic epoxides and diol epoxides: Evidence that DNA catalyzes the formation of benzo[a]pyrene and benz[a]anthracene adducts at intercalation sites

Article abstract of DOI:10.1021/tx960134j

Reactions of the benzo[a]pyrene (BP) and benz[a]anthracene (BA) metabolites, (±)-trans-7, 8-dihydroxy-anti-9, 10-epoxy-7, 8, 9, 10- tetrahydro-BP (BPDE), (±)-trans-3, 4-dihydroxy-anti-1, 2-epoxy-1, 2, 3, 4- tetrahydro-BA (BADE), (±)-BP-4, 5-oxide (BPO), and (±)-BA-5, 6-oxide (BAO), were examined under pseudo-first-order conditions at varying Na⁺ (2.0-100 mM) and native, calf thymus DNA (ctDNA) concentrations. In 0.2 mM ctDNA and 2.0 mM Na⁺, at a pH of 7.3, most BPDE, BADE, BPO, and BAO (87-95%) undergo DNA catalyzed hydrolysis or rearrangement. For BPDE and BPO, overall, pseudo- first-order rate constants, k, in 2.0 mM Na⁺ and 0.2 mM ctDNA are 21-72 times larger than values obtained without DNA. For BADE and BAO, the rate constants are less strongly influenced by DNA; k values in 0.2 mM ctDNA are only 9-12 times larger than values obtained without DNA. Kinetic data for BPDE, BPO, BADE, and BAO and DNA intercalation association constants (K(A)) for BP and BA diols which are model compounds indicate that K(A) values for BPDE and BPO in 2.0 mM Na⁺ are 6.6-59 times larger than those of BADE and BAO. The greater DNA enhancement of rate constants for BPDE and BPO, versus BADE and BAO, correlates with the larger K(A) values of the BP metabolites. DNA adducts, which account for less than 10% of the yields, also form. For BPDE in 0.20 mM ctDNA, k decreases 5.1 times as the Na⁺ concentration increases from 2.0 to 100 mM. Nevertheless, the DNA adduct level remains constant over the range of Na⁺ concentrations examined. These results provide evidence that, for BPDE in 0.20 mM DNA and 2.0 mM Na⁺, ctDNA adduct formation follows a mechanism which is similar to that for DNA catalyzed hydrolysis. The pseudo-first-order rate constant for adduct formation, k(Ad), given approximately by k(Ad) ? (k(cat, Ad)K(A)[DNA])/(1 + K(A)[DNA]), where k(cat, Ad) is a catalytic rate constant. For BADE, BPO, and BAO, the influence of varying DNA and Na⁺ concentrations on k values is similar to that for BPDE, and provides evidence that the formation of adducts follows the same rate law.

Full text of DOI:10.1021/tx960134j

Chem. Res. Toxicol. 1996, 9, 1391-1402
1391
In flu en ce of Na + on DNA Rea ction s w ith Ar om a tic
Ep oxid es a n d Diol Ep oxid es: Evid en ce Th a t DNA
Ca ta lyzes th e F or m a tion of Ben zo[a ]p yr en e a n d
Ben z[a ]a n th r a cen e Ad d u cts a t In ter ca la tion Sites
Harshica Fernando, Chao-Ran Huang, Ann Milliman, Luchuan Shu, and
Pierre R. LeBreton*
Department of Chemistry, The University of Illinois at Chicago, Chicago, Illinois 60607-7061
Received J uly 30, 1996X
Reactions of the benzo[a]pyrene (BP) and benz[a]anthracene (BA) metabolites, (()-trans-
7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-BP (BPDE), (()-trans-3,4-dihydroxy-anti-
1,2-epoxy-1,2,3,4-tetrahydro-BA (BADE), (()-BP-4,5-oxide (BPO), and (()-BA-5,6-oxide (BAO),
were examined under pseudo-first-order conditions at varying Na+ (2.0-100 mM) and native,
calf thymus DNA (ctDNA) concentrations. In 0.2 mM ctDNA and 2.0 mM Na+, at a pH of 7.3,
most BPDE, BADE, BPO, and BAO (87-95%) undergo DNA catalyzed hydrolysis or rear-
rangement. For BPDE and BPO, overall, pseudo-first-order rate constants, k, in 2.0 mM Na+
and 0.2 mM ctDNA are 21-72 times larger than values obtained without DNA. For BADE
and BAO, the rate constants are less strongly influenced by DNA; k values in 0.2 mM ctDNA
are only 9-12 times larger than values obtained without DNA. Kinetic data for BPDE, BPO,
BADE, and BAO and DNA intercalation association constants (KA) for BP and BA diols which
are model compounds indicate that KA values for BPDE and BPO in 2.0 mM Na+ are 6.6-59
times larger than those of BADE and BAO. The greater DNA enhancement of rate constants
for BPDE and BPO, versus BADE and BAO, correlates with the larger KA values of the BP
metabolites. DNA adducts, which account for less than 10% of the yields, also form. For BPDE
in 0.20 mM ctDNA, k decreases 5.1 times as the Na+ concentration increases from 2.0 to 100
mM. Nevertheless, the DNA adduct level remains constant over the range of Na+ concentra-
tions examined. These results provide evidence that, for BPDE in 0.20 mM DNA and 2.0 mM
Na+, ctDNA adduct formation follows a mechanism which is similar to that for DNA catalyzed
hydrolysis. The pseudo-first-order rate constant for adduct formation, kAd, given approximately
by kAd ≈ (kcat,AdKA[DNA])/(1 + KA[DNA]), where kcat,Ad is a catalytic rate constant. For BADE,
BPO, and BAO, the influence of varying DNA and Na+ concentrations on k values is similar
to that for BPDE, and provides evidence that the formation of adducts follows the same rate
law.
than K region metabolites (3-7). Investigation of PAH
diol epoxide reactivities has led to the bay region theory.
The theory relates the π electronic structure of diol
epoxides to the reactivities of these metabolites toward
biochemical targets, such as DNA (3). In this way,
reactivity influences carcinogenic potency. A large num-
ber of factors affect genotoxic potency (1, 3, 8-12);
nevertheless, predictions based on chemical reactivity
often correlate with the mutagenic activities of PAH diol
epoxides (3).
In tr od u ction
Covalent DNA modification by epoxide containing
metabolites of polycyclic aromatic hydrocarbons (PAHs)1
is important to carcinogenic mechanisms involving these
molecules (1-3). Figure 1 shows structures of four
epoxides, (()-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,-
10-tetrahydrobenzo[a]pyrene (BPDE), (()-benzo[a]pyrene
4,5-oxide (BPO), (()-trans-3,4-dihydroxy-anti-1,2-epoxy-
1,2,3,4-tetrahydrobenz[a]anthracene (BADE), and (()-
benz[a]anthracene 5,6-oxide (BAO), which are derived
from benzo[a]pyrene (BP) or benz[a]anthracene (BA).
BPDE and BADE are bay region diol epoxides, while BPO
and BAO are K region epoxides. Generally, bay region
metabolites are more genotoxic and chemically reactive
In aqueous solution with DNA, BPDE hydrolyzes to
7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-BP (BP tetrol)
and forms DNA adducts. These reactions are shown in
Figure 2. Adduct formation occurs primarily at the
2-amino group of guanine (1). Hydrolysis is both spon-
taneous and catalyzed by DNA (6, 7, 13-17). The
hydrolysis and adduct reactions of BPDE proceed via
transition states with carbocationic character at the C-10
atom of BPDE (3).
X Abstract published in Advance ACS Abstracts, November 1, 1996.
Abbreviations: BA, benz[a]anthracene; BA34D, trans-3,4-dihy-
1
droxy-3,4-dihydro-BA; BA56D, cis-5,6-dihydroxy-5,6-dihydro-BA; BADE,
(()-trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydro-BA; BAO, (()-
BA-5,6-oxide; BA tetrol, 1,2,3,4-tetrahydroxy-1,2,3,4-tetrahydro-BA;
BP, benzo[a]pyrene; BP45D, trans-4,5-dihydroxy-4,5-dihydro-BP; BP78D,
trans-7,8-dihydroxy-7,8-dihydro-BP; BPDE, (()-trans-7,8-dihydroxy-
anti-9,10-epoxy-7,8,9,10-tetrahydro-BP; BPO, (()-BP-4,5-oxide; BP
tetrol, 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-BP; ctDNA, calf thy-
mus DNA; 5-OH-BA, 5-hydroxy-BA; 6-OH-BA, 6-hydroxy-BA; 4-OH-
BP, 4-hydroxy-BP; 5-OH-BP, 5-hydroxy-BP; PAH, polycyclic aromatic
hydrocarbon; poly(A), poly(riboadenylic acid); poly(C), poly(ribocytidylic
acid); poly(G), poly(riboguanylic acid); poly(I), poly(riboinosinic acid);
poly(U), poly(ribouridylic acid).
At low BPDE concentrations, under pseudo-first-order
conditions, the overall, pseudo-first order rate constant
(k) has contributions (see following equation) from hy-
drolysis (kHy) and from reaction leading to DNA adducts
(kAd) (6, 7, 13-16).
k ) kHy + kAd
(1)
S0893-228x(96)00134-8 CCC: $12.00 © 1996 American Chemical Society

1392 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Fernando et al.
F igu r e 1. Structures of the bay region diol epoxides, BPDE and BADE, of the K region epoxides, BPO and BAO, and of the model
compounds BP78D, BA34D, BP45D, and BA56D.
significant reaction occurs at guanine, and, for BPO, it
is likely that the 2-amino group of guanine is a major
target site (19, 21). For BAO, adduct yields in reactions
with poly(G) are more than 5 times greater than yields
with poly(U), poly(C), poly(A), or poly(I) (20). In addition
to adduct reactions, BPO and BAO undergo DNA cata-
lyzed hydrolysis to diols (4,5-dihydroxy-4,5-dihydro-BP
and 5,6-dihydroxy-5,6-dihydro-BA) and rearrangement to
phenols (6, 23). For BPO and BAO, the following
equation gives the overall, pseudo-first-order rate con-
stant, where kRe is the pseudo-first-order rate constant
for rearrangement (6).
k ) kHy + kAd + kRe
(2)
Without DNA, BPO and BAO have major reaction
pathways leading to hydrolysis and rearrangement (6,
24). While only one BPO rearrangement product, 4-hy-
droxy-BP (4-OH-BP), was reported in the DNA catalyzed
rearrangement of BPO (23), results from later reactivity
measurements (25) suggest that 5-hydroxy-BP is also
formed. Analysis of relative yields of 5-hydroxy-BA (5-
OH-BA) and 6-hydroxy-BA (6-OH-BA) formed via DNA
catalyzed rearrangement of BAO has not been reported.
However, in 1:9 dioxane-water with 0.1 M NaClO4 at a
pH of 4.3, BAO, without DNA, forms 5-OH-BA and 6-OH-
BA in the ratio 2:1 (24). At pH 4.3, the BAO hydrolysis
yield is 37% (24); however, at pH 7.1, almost all reaction
proceeds via hydrolysis (6). Figure 2 shows products of
BADE, BPO, and BAO reactions with DNA.
In buffer with DNA, BPDE hydrolysis accounts for
approximately 90% of the total reaction (6). The majority
of investigations indicate that the catalyzed hydrolysis
in double-stranded DNA relies on the formation of
intercalated complexes (6, 7, 14, 15, 26-28). Similarly,
for BPDE hydrolysis in closed-circular, single-stranded
DNA, current results provide evidence that catalysis
occurs at local regions of the single-stranded DNA where
base stacking occurs (7). Equation 3 gives the pseudo-
first-order rate constant (kHy) for BPDE hydrolysis with
DNA (15). Here, kcat,Hy is the rate constant for DNA
catalyzed hydrolysis; k0,Hy, the rate constant for sponta-
neous hydrolysis; kH,Hy, the rate constant for H+ catalyzed
hydrolysis without DNA; and KA, the intercalation as-
sociation constant.
F igu r e 2. Major pathways for reactions of BPDE, BADE, BPO,
and BAO in buffer with DNA. For BPO and BAO, adduct
structures are based on preliminary results indicating that
guanine is a major target site, and on the structures of the
primary BPDE and BADE adducts. For BPO and BAO, the C-5
and C-6 adducts are shown, respectively. However, the forma-
tion of C-4 adducts of BPO and C-5 adducts of BAO cannot be
ruled out. See refs 1, 19-21, 24, and 25.
BADE, like BPDE, forms DNA adducts and hydrolyzes
(18). The hydrolysis product is 1,2,3,4-tetrahydroxy-
1,2,3,4-tetrahydro-BA (BA tetrol). BPO and BAO, while
less reactive than BPDE and BADE, also form DNA
adducts (19-21). Like BPDE, BADE adducts form most
favorably at the 2-amino group of guanine (22). For BPO
and BAO, adduct structures have not been fully charac-
terized. However, preliminary evidence indicates that

Influence of Na+ on DNA Reactions with PAH Epoxides
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1393
k0,Hy + kH,Hy[H+] + kcat,HyKA[DNA]
concerning the relationship between reactions leading to
BPDE hydrolysis and to DNA modification, nor to agree-
ment concerning the role that intercalation plays in
adduct reactions. A great deal remains to be elucidated
regarding the reactions of hydrocarbon epoxides with
DNA (2).
The goal here is to examine the relationship between
BPDE, BADE, BPO, and BAO reactions leading to DNA
adduct formation and reactions leading to hydrolysis or
rearrangement. The BP and BA diol epoxides and
epoxides examined were chosen because they exhibit a
wide variation in reactivities, and in DNA intercalation
association constants. The relationships between reac-
tions leading to adduct formation, rearrangement, and
hydrolysis in DNA were examined over Na+ concentra-
tions where the overall, pseudo-first-order rate constants
of the diol epoxides and epoxides vary 2.7-15.4 times,
and the model compound intercalation association con-
stants vary 2.3-3.3 times.
kHy
)
(3)
1 + KA[DNA]
In eq 3, k0,Hy(1 + KA[DNA])-1 is the contribution to kHy
from spontaneous hydrolysis; kH,Hy[H+](1 + KA[DNA])-1
,
the contribution from specific acid catalyzed hydrolysis;
and kcat,HyKA[DNA](1 + KA[DNA])-1, the contribution from
catalysis at intercalation sites. Earlier investigations
indicate that DNA enhanced BPDE hydrolysis occurs via
general acid catalysis involving phosphate or exocyclic
amino groups on the bases (2, 13, 27).
Without DNA, at Na+ concentrations below 100 mM,
Na+ has negligible influence on BPDE hydrolysis (7).
However, with DNA, the pseudo-first-order rate constant
for hydrolysis decreases as Na+ concentrations increase
in the range 2.0-100 mM. This is due to Na+ induced
decreases in the association constant, KA, and catalytic
rate constant, kcat,Hy (7, 15). For BPDE at a pH of 7.3,
an increase in the Na+ concentration from 2.0 to 100 mM
causes KA and kcat,Hy to decrease 1.8-2.7 and 4.8-9.0
times, respectively (7).
Exp er im en ta l Section
Earlier investigations of intercalation association con-
stants employed the diols trans-7,8-dihydroxy-7,8-dihy-
dro-BP (BP78D), trans-4,5-dihydroxy-4,5-dihydro-BP
(BP45D), trans-3,4-dihydroxy-3,4-dihydro-BA (BA34D),
and cis-5,6-dihydroxy-5,6-dihydro-BA (BA56D) as model
compounds of BPDE, BPO, BADE, and BAO, respectively
(6, 7, 29, 30). The parallel between the intercalation
association constants of the models and of the corre-
sponding epoxide containing metabolites is supported by
the observation that the ratio of ctDNA association
constants for BPDE versus BPO is similar to the ratio of
association constants for BP78D versus BP45D (6).
Figure 1 shows structures of the model compounds. A
comparison of results from fluorescence quenching (7, 29,
31, 32), UV absorption (29, 32), linear dichroism (33), and
dialysis binding measurements (7, 29, 31) demonstrates
that, in the concentration range 10-6 to 10-7 M, virtually
all reversible binding between BP and BA derivatives
with native, double-stranded DNA occurs via intercala-
tion (7, 29, 31).
Because DNA adduct formation is a minor pathway,
the mechanism is not well understood (2, 13, 14, 16, 17).
For reaction of BPDE with DNA, a decrease in pH causes
an increase in the pseudo-first-order rate constant for
hydrolysis. A proportional increase in the rate of covalent
binding also occurs, with the net effect that covalent
binding levels remain constant (13, 34). This has led to
speculation (13) that DNA catalyzes covalent adduct
formation at intercalation sites, and, contrary to conclu-
sions from other experiments, that catalyzed hydrolysis
occurs at external sites. In contrast, it has also been
speculated that hydrolysis of BPDE is catalyzed at
intercalation sites, while covalent binding results from
reaction at external sites (17). A third description
invokes a mechanism in which hydrolysis and covalent
adducts form in the same reversibly bound domains of
DNA (14, 26, 34). The more recent development of a
general theory (16) for mechanisms of DNA reaction with
BPDE considers two possibilities. The first is that
catalysis of either hydrolysis or covalent binding occurs
in reversibly bound intercalation complexes. The second
is that these reactions occur without complex formation.
The results indicate that either possibility leads to
equations for overall, pseudo-first-order rate constants
and covalent binding fractions with the same DNA
concentration dependence. Earlier theoretical and ex-
perimental investigations have not led to consensus
Fluorescence emission spectra were measured with a Perkin-
Elmer 650-10 fluorescence spectrometer. Magic angle fluores-
cence lifetime measurements employed a Photochemical Re-
search Associates Model 2000 nanosecond fluorescence
spectrophotometer equipped with aberration-corrected optics.
A Glan polarizer was mounted on the excitation side and a film
polarizer on the emission side. Ultraviolet absorption spectra
were measured with a Cary 17 spectrometer. Samples used in
fluorescence experiments were contained in 1 cm cells.
Samples of BPDE, BADE, BPO, BAO, trans-7,8-dihydroxy-
7,8-dihydro-BP (BP78D), trans-4,5-dihydroxy-4,5-dihydro-BP
(BP45D), trans-3,4-dihydroxy-3,4-dihydro-BA (BA34D), and cis-
5,6-dihydroxy-5,6-dihydro-BA (BA56D) were purchased from
Chemsyn Science Laboratories (Lenexa, KS). 9,10-Dimethyl-
anthracene was obtained from Sigma Chemical Co. (St. Louis,
MO). Native calf thymus DNA (ctDNA) was purchased from
Worthington (Freehold, NJ ) and was found to have a hypochro-
micity of 35%. ctDNA concentrations are reported in terms of
-
PO4 molarity calculated from an average base-pair molecular
weight of 617.8. This is based on a calf thymus DNA composi-
tion which is 60% A-T base pairs (31). Concentrations have been
corrected for the amounts of H2O and Na+ reported by the
supplier for each DNA batch. From the DNA absorption
spectrum, it was found that one A260 unit equals 1.5 × 10-4
M
[PO4-] (31). In control experiments, ctDNA obtained from the
supplier was purified by using a phenol-chloroform procedure
(31) which yielded an A260/A280 ratio of 2.0. When Stern-Volmer
quenching constants for 9,10-dimethylanthracene were com-
pared in experiments using purified DNA and DNA directly
from the supplier, the difference was less than 10%. Subsequent
experiments were performed without repurification of the DNA.
All experiments were carried out in 10 mM Tris‚HCl and 1.0
mM EDTA buffer at pH 7.3. The temperature was maintained
at 23 ( 0.5 °C with an Isotemp Circulator Model 210. Ca u tion :
BPDE, BPO, BADE, BAO, BP78D, BP45D, BA34D, and BA56D
are hazardous chemicals which were handled in accordance with
NIH guidelines (35).
In order to introduce anions, as well as cations, which occur
under physiological conditions, Na+ concentrations were ad-
justed by addition of NaCl (36). While the present experiments
focus on the effects which Na+ has on DNA catalysis, earlier
investigations indicate that Cl- also influences reactions of diol
epoxides and epoxides (25, 36, 37, 38). For BPDE reactions with
ctDNA, Cl- enhances the formation of cis opened hydrolysis
products and adducts, at the expense of trans opened products,
through the formation of chlorohydrin intermediates (36, 37).
However, with ctDNA, a significant decrease in the formation
of trans products (36) has only only been observed at much
higher Cl- concentrations (1 M) than the concentrations em-
ployed here, which never exceed 100 mM. In reactions of the
(+) and (-) enantiomers of BPDE with poly(G) at
a
Cl-

1394 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Fernando et al.
concentration (250 mM) which is 2.5 or more times greater than
the Cl- concentrations employed in the present experiments,
the yield of trans opened BPDE adducts (36) is less than 15%
smaller than the yield without Cl-. Similarly, in 3 mM ctDNA
at a pH of 7.0, the percentage of BPDE hydrolysis resulting in
trans products decreases only slightly, from 93% to 91%, as the
NaCl concentration increases from 0 to 100 mM. With DNA,
the influence of Cl- on overall, pseudo-first-order rate constants,
k, has not been observed. Without DNA, significant effects of
NaCl on k values for PAH epoxide hydrolysis and rearrange-
ment reactions have, again, only been observed (37, 38) at high
Cl- concentrations (0.5-1 M). For BPDE and BPO, without
DNA, at the low buffer concentrations used in the present
experiments, k values measured in 100 mM Cl- are the same
as those measured without Cl- (7). These observations strongly
indicate that the effects of the secondary chlorohydrin mecha-
nism on the results presented here are negligible.
Over a ll, P seu d o-F ir st-Or d er Ra te Con sta n ts. All reac-
tions were carried out under pseudo-first-order conditions with
concentrations of diol epoxides and epoxides in the range 10-6
to 10-7 M. Overall, pseudo-first-order rate constants of BPDE,
BADE, BPO, and BAO were obtained by measuring the increase
in fluorescence intensity which occurs as products are formed.
The epoxides have negligible fluorescence intensities compared
to the DNA adducts or to the hydrolysis and rearrangement
products (6, 7, 18). Excitation wavelengths used to monitor
BPDE, BADE, BPO, and BAO reactions were 350, 365, 330, and
310 nm, respectively. The emission wavelengths were 404, 410,
388, and 365 nm. Each reaction was initiated by injecting 20
µL of a THF solution containing BPDE, BADE, BPO, or BAO
into 1.0 mL of buffer with or without ctDNA (6, 7). Overall,
pseudo-first-order rate constants are equal to minus the slopes
of least-squares linear fits of plots of ln([Imax - I(t)]/[Imax - I0])
versus t. Here, Imax is the maximum fluorescence intensity
measured after the reaction was completed, I(t) is the intensity
at time t, and I0 is the intensity when the reaction was initiated.
In the present experiments, the fluorescence emission intensi-
ties, extrapolated to the time at which the reactions were
initiated, were less than 8% of the intensities observed at the
completion of the reactions. For the experiments reported here,
the overall, pseudo-first-order rate constants for BPDE and
BPO, with and without ctDNA, differ by factors of 1.4-2.4 from
values reported earlier (7). These differences are due to the
improved temperature control which was employed in the
current experiments.
St er n -Volm er Qu en ch in g Con st a n t s. Stern-Volmer
plots were obtained from measurements of fluorescence quench-
ing caused by ctDNA. Quenching measurements were carried
out on BP78D, BA34D, BP45D, and BA56D which are model
compounds for BPDE, BADE, BPO, and BAO, respectively.
Hydrocarbon concentrations were in the range 10-6 to 10-7 M.
Excitation and emission wavelengths used for each model com-
pound were the same as those used to measure the overall,
pseudo-first-order rate constants of the corresponding diol
epoxide or epoxide. Quenching constants, KSV, were obtained
from Stern-Volmer plots and are equal to the slopes of least-
squares fits to plots of the ratio I0/I versus the ctDNA concen-
tration. Here, I0 corresponds to the fluorescence emission inten-
sity of the model compound without ctDNA, and I corresponds
to the intensity with DNA. For BP78D, BA34D, BP45D, and
BA56D, the slopes of the Stern-Volmer plots (KSV) are equal
to association constants (KA) for intercalation into DNA (6, 7, 30).
F lu or escen ce Lifetim es. Fluorescence lifetime measure-
ments of BP78D, BP45D, BA34D, and BA56D were carried out
at the same excitation and emission wavelengths used in the
fluorescence quenching measurements. The quenching of the
diol fluorescence by ctDNA required that, in measurements with
DNA, a subtraction procedure be employed to correct for
scattered light. In this procedure, the decay profile of a ctDNA
blank was subtracted from the profile measured for a diol with
DNA (7, 29). Lifetime measurements with ctDNA were carried
out at DNA concentrations of 0.10, 0.20, 0.50, and 0.50 mM for
BP78D, BP45D, BA34D, and BA56D, respectively. The more
weakly quenched diols (BP45D, BA34D, and BA56D) were
examined at higher ctDNA concentrations than BP78D, which
is strongly quenched. Analysis of the lifetime data was carried
out using a least-squares deconvolution method (6, 7).
Ad d u ct a n d Hyd r olysis Yield s. BPDE, BADE, BPO, and
BAO adduct yields were determined for reactions in 0.20 mM
ctDNA at varying Na+ concentrations. BPO and BAO hydroly-
sis yields were also determined under these conditions, and for
reactions in buffer without ctDNA. The reactions were run
under the same conditions as the kinetics experiments. Reac-
tions in ctDNA were initiated by adding 20 µL of BPDE, BADE,
or BPO in THF, or 20 µL of BAO in 95% ethanol, to a conical
tube containing 0.20 mM ctDNA in 1.0 mL of buffer. For the
analysis of BPO and BAO hydrolysis yields without DNA, the
buffer conditions were the same as for reactions with DNA. The
separation of ctDNA adducts employed a method similar to that
described previously (6). After the reactions were completed,
samples containing DNA adducts were extracted 3 times with
3.0, 2.0, and 2.0 mL of buffer saturated ethyl acetate.2 This
removed the hydrolysis and rearrangement products from the
adducts, which remained in the aqueous phase. Ethyl acetate
dissolved in the buffer was removed by placing the sample in
boiling water for 1 min. The covalently bound adducts were
released by HCl hydrolysis for 1.5 h in sealed tubes. For BPDE
and BADE adduct samples in buffer, the hydrolysis was carried
out by adding 50 µL of 1 M HCl and heating to 93 °C. For BPO
and BAO adduct analysis, 20 µL of HCl was added and the
samples were heated to 89 °C. By carrying out one set of control
experiments in which the reaction times were varied, and a
second set of control experiments in which standard samples of
BP tetrol, BA tetrol, BP45D, and BA56D were subjected to the
hydrolysis conditions, it was determined that the hydrolysis
reaction conditions, which were employed, resulted in complete
adduct hydrolysis without decomposition of the hydrolysis
products (6). After cooling, the pH of the solutions containing
the hydrolyzed adducts was adjusted to 4.5 by adding 2.0 M
NaOH. Adducts from BPDE and BADE were analyzed as BP
tetrol and BA tetrol; adducts from BPO and BAO were analyzed
as 4,5-dihydroxy-4,5-dihydro-BP and 5,6-dihydroxy-5,6-dihydro-
BA.
(A) Deter m in a tion of BP DE a n d BADE Ad d u ct Yield s.
The observation that reactions of BPDE and BADE, in buffer
without DNA, lead exclusively to BP tetrol and BA tetrol was
exploited in the preparation of standards for determining BPDE
and BADE adduct yields. The percentage of the initial BPDE
or BADE reactant which formed DNA adducts was quantified
with a standard BP tetrol or BA tetrol solution prepared from
the same solution of diol epoxide in THF which was employed
to prepare the adducts. The standards were obtained by
hydrolyzing 20 µL of the THF solution containing BPDE or
BADE in 1.0 mL of buffer without DNA. The standards were
treated in the same manner as the samples. To each standard,
0.5 mL of ethyl acetate was added and then removed by placing
in boiling water. Each standard was also acidified with HCl,
heated, and neutralized with NaOH in the same manner as the
sample. To measure the percentage of diol epoxide which
formed adducts in reactions with ctDNA, the fluorescence
intensity of the BP tetrol or BA tetrol obtained via hydrolysis
of the adducts was compared to that of the corresponding
standard at the same emission and excitation wavelengths
employed in the BPDE or BADE kinetics measurements.
(B) Deter m in a tion of BP O a n d BAO Ad d u ct a n d Hy-
d r olysis Yield s. The analysis of BPO and BAO adduct yields
was also carried out using standards of the products formed in
the adduct hydrolysis reactions. However, because BPO and
BAO, in buffer without DNA, not only hydrolyze to diols but
can also rearrange to phenols, the method used to prepare diol
standards for analysis of BPO and BAO adducts was different
from that used to prepare tetrol standards. The percentage of
the epoxide which formed ctDNA adducts was calculated from
the number of moles of reactant epoxide, which was determined
from UV absorption measurements of the initial epoxide con-
2
Products from reactions of BPO and BAO in buffer without ctDNA
were also extracted in this manner. The hydrolysis products were then
analyzed in ethyl acetate.

Influence of Na+ on DNA Reactions with PAH Epoxides
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1395
F igu r e 3. Overall, pseudo-first-order rate constants, k, of BPDE, BADE, BPO, and BAO versus ctDNA concentration. Solid lines
show least-squares fits to the rate constants obtained using eq 4. For BPDE and BPO, values of kcat and KA were optimized to obtain
the calculated curves. For BADE and BAO, values of KA were based on association constants for the model compounds BA34D and
BA56D, respectively, and values of kcat were optimized to obtain the calculated curves. See text.
centration,3 and from the number of moles of diol formed after
and BAO. As the ctDNA concentration increases, values
hydrolysis of the resultant DNA adducts. Determination of the
diol concentration resulting from hydrolysis of BPO or BAO
of k for all of the epoxides and diol epoxides increase.
However, the results in Figure 3 also point out small
adducts was carried out by comparing the fluorescence intensity
differences between the behavior of BPDE and BPO
of the hydrolysis product with that of a BP45D or BA56D
versus BADE and BAO. For example, the results for
standard solution in buffer, for which the concentration was
BPDE and BPO show evidence of saturation kinetics. At
the higher ctDNA concentrations, the increases in the
rate constants of BPDE and BPO, caused by increases
in ctDNA concentration, become small. In contrast, the
results for BADE and BAO do not exhibit evidence of
saturation over the range of ctDNA concentrations
examined. For BADE and BAO, the rates at which k
increases with increasing ctDNA concentration are nearly
constant. Furthermore, the results in Figure 3 demon-
strate that, when compared to rate constants without
DNA, ctDNA causes a greater increase in k values of
BPDE and BPO than of BADE and BAO. In 0.20 mM
ctDNA, the rate constants of BPDE and BPO are 21.2
and 71.9 times larger than rate constants without DNA.
For BADE and BAO in 0.20 mM ctDNA, the rate
constants are only 9.1 and 12.0 times larger than rate
constants without DNA. The results in Figure 3 also
demonstrate that the more effective way in which DNA
enhances the rate constants of the BP metabolites causes
the ordering of the overall, pseudo-first-order rate con-
stants obtained with ctDNA to differ from that obtained
without DNA. Without DNA, the rate constant of BAO
is larger than that of BPO; in 0.2 mM ctDNA, the rate
constant of BPO ((1.23 ( 0.1) × 10-4 s-1) is larger than
that of BAO ((5.00 ( 0.6) × 10-5 s-1).
determined by UV absorption.3 The fluorescence intensities of
the adduct hydrolysis products and the standard solutions were
compared at the same excitation and emission wavelengths used
in the epoxide kinetic measurements. Like the tetrol standard
solutions, the BP45D and BA56D standards were treated in a
manner identical to the diols formed by hydrolysis of the ctDNA
adducts.
The percentage of BPO or BAO which formed hydrolysis
products in reactions with or without ctDNA was determined
by analyzing the reaction products contained in the ethyl acetate
extract obtained after the reactions in buffer were completed.
The percentage of BPO or BAO which hydrolyzed was deter-
mined by comparing the fluorescence intensity of the hydrolysis
products with that of a BP45D or BA56D standard which was
prepared in ethyl acetate.3
Resu lts
Figure 3 shows plots of the overall, pseudo-first-order
rate constants (k) for BPDE, BADE, BPO, and BAO at
varying ctDNA concentrations. Without DNA, the order-
ing of the rate constants is BPO ((1.71 ( 0.2) × 10-6 s-1
)
< BAO ((4.15 ( 0.3) × 10-6 s-1) < BADE ((1.47 ( 0.2) ×
10-4 s-1) < BPDE ((1.44 ( 0.2) × 10-3 s-1). With ctDNA,
the data in Figure 3 are similar for BPDE, BPO, BADE,
3
Extinction coefficients, in M L-1 cm-1, for BPO (ꢀ276 ) 118 537) in
Results from earlier investigations (6, 7, 29, 31) have
THF, and for BAO (ꢀ270 ) 54 577), P45D (ꢀ273 ) 90 950), and BA56D
(ꢀ267 ) 43 284) in 95% ethanol, were obtained from Chemsyn Science
Laboratories product data sheets. To prepare standard solutions of
BP45D and BA56D in ethyl acetate or in buffer, 20 µL of a known
concentration of the diol in 95% ethanol was added to 1 mL of ethyl
acetate or buffer. In the quantification of the BPDE, BADE, BPO, and
BAO adducts, and of the BPO and BAO hydrolysis yields, the extinction
coefficients and the fluorescence quantum yields of corresponding cis
and trans tetrols, and cis and trans diols, were taken to be equal.
shown that Stern-Volmer quenching constants (KSV
)
obtained from measurements of the fluorescence quench-
ing of BP and BA derivatives by double-stranded DNA
are equal to intercalation association constants (KA) when
the fluorescence lifetimes of the metabolites measured,
with and without DNA, are equal. This occurs if the

1396 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Fernando et al.
F igu r e 4. Stern-Volmer plots for the ctDNA quenching of
BP78D (closed squares), BP45D (closed triangles), BA34D (open
squares), and BA56D (open triangles) in 2.0 mM Na+. Excitation
wavelengths were 350, 365, 330, and 310 nm, respectively.
Emission wavelengths were 404, 410, 388, and 365 nm. DNA
concentrations are given on the bottom scale for BP78D and
BP45D, and on the top scale for BA34D and BA56D. Stern-
Volmer quenching constants obtained from least-squares fits of
the data are given without parentheses. Stern-Volmer quench-
ing constants measured in 100 mM Na+ are given in parenthe-
ses.
F igu r e 5. (A) Overall, pseudo-first-order rate constants of
BPDE (open squares) and percentage of BPDE resulting in
ctDNA adducts (closed squares) at varying Na+ concentrations.
(B) Overall, pseudo-first-order rate constants of BADE (open
triangles) and percentage of BADE forming ctDNA adducts
(closed triangles) at varying Na+ concentrations. Adduct yields
are given on the left-hand scales. Overall, pseudo-first-order rate
constants are given on the right-hand scales. The ctDNA
concentration was 0.2 mM.
fluorescence quenching is static, and if the fluorescence
quantum yields of the intercalated BP or BA derivatives
are negligible compared to those of the unbound mol-
ecules (29, 31). Figure 4 shows Stern-Volmer plots,
measured in 2.0 mM Na+, for the fluorescence quenching
of the model compounds, BP78D, BP45D, BA34D, and
BA56D, by ctDNA. The figure shows the ratio of fluo-
rescence intensity measured without DNA (I0) over the
intensity measured with ctDNA (I) as a function of DNA
concentration. In addition to Stern-Volmer quenching
constants measured in 2.0 mM Na+, the figure also shows
Stern-Volmer quenching constants measured in 100 mM
Na+.
Measurements of the influence of ctDNA on the
fluorescence lifetimes of the diol epoxide and epoxide
model compounds were carried out in order to determine
whether, for these molecules, KSV equals KA under the
conditions at which the kinetic experiments were carried
out. Fluorescence decay profiles of the BADE and BPDE
model compounds, BA34D and BP78D, were measured
in 10 mM Tris‚HCl and 1.0 mM EDTA buffer at pH 7.3,
with and without ctDNA. For BP78D, BP45D, BA34D,
and BA56D, the ctDNA concentrations used were 0.1, 0.2,
0.5, and 0.5 mM, respectively. Fluorescence lifetimes
were obtained by fitting the decay profiles with a single-
exponential decay law. Within experimental uncertainty,
the fluorescence lifetimes are unchanged when ctDNA
is added. For BP78D, BP45D, BA34D, and BA56D,
fluorescence lifetimes without DNA are 25.1, 20.4, 7.37,
and 44.1 ns, respectively. The ø2 values are 1.01, 1.08,
1.02, and 1.11. With ctDNA, the fluorescence lifetimes
are 24.2, 19.8, 7.36, and 43.9 ns; and the ø2 values are
0.98, 1.10, 0.97, and 1.16. Results from fluorescence
lifetime measurements support the conclusion that the
fluorescence quenching shown in Figure 4 is static; that
the fluorescence quantum yields of the diols bound to
ctDNA are negligible; and that KSV equals KA.
Figure 3 indicates that the reactivities of BPDE and
BPO are more effectively enhanced by ctDNA than are
the reactivities of BADE and BAO. Figure 4 indicates
that the intercalation association constants of the BPDE
and BPO model compounds are larger than those of the
BADE and BAO model compounds. In 2.0 mM Na+, the
intercalation association constants of BP78D and BP45D
are 6.6-59 times larger than the association constants
of BA34D and BA56D. The data in Figure 4 also
indicates that an increase in the Na+ concentration from
2.0 to 100 mM is accompanied by a 2.3-3.3-fold decrease
in the KA values of the model compounds. These data
are consistent with previously reported results (7, 14, 15,
28, 39).
Figure 5 shows overall, pseudo-first-order rate con-
stants of BPDE and BADE in ctDNA, and the percent-
ages of these diol epoxides which form ctDNA adducts
at varying Na+ concentrations. Panel A of Figure 5
shows results for BPDE. In Panel A, k decreases 5.1

Influence of Na+ on DNA Reactions with PAH Epoxides
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1397
the rate constant decreases 15.4 times with an increase
in Na+ concentration from 2 to 100 mM, the adduct yield
varies no more than 1.1 times. For BPO, the hydrolysis
and rearrangement yields are also nearly constant. Both
vary by only 1.2 times. For BAO, the results are different
from BPO. Here the low adduct yields (∼2%), and the
large relative errors ((150%) make it impossible to
determine whether the adduct yield remains constant.
However, for BAO, quantitative analysis of the 5,6-
dihydroxy-5,6-dihydro-BA formed indicates that the hy-
drolysis and rearrangement yields change. As the Na+
concentration increases from 2.0 to 50 mM, the hydrolysis
yield increases from 18% to 56%, and the rearrangement
yield decreases from 80% to 42%.
Discu ssion
The results for BPDE in Figure 5, like results from an
earlier investigation of the pH dependence of BPDE
kinetics (13), demonstrate that the rate of adduct forma-
tion is changing in a manner which parallels changes in
the rate of hydrolysis. The observed parallel kinetics,
coupled with the finding that, under the conditions
employed, most of the BPDE reaction (82-88%)4 occurs
via ctDNA catalyzed hydrolysis, support the earlier
suggestion that BPDE adduct formation is autocatalyzed
(13). For BADE and BPO, the results in Figures 5 and
6 are similar to those for BPDE. Again, as the Na+
concentration increases, and the overall, pseudo-first-
order rate constants decrease 2.7 and 15.4 times, the
product distributions remain almost unchanged. For
BAO, the behavior of the adduct yield is uncertain;
however, the hydrolysis yield increases and the rear-
rangement yield decreases as the Na+ concentration
increases.
With ctDNA, the available reaction pathways are
different for BPDE and BADE, which form adducts and
hydrolyze, than for BPO and BAO, which form adducts,
hydrolyze, and also rearrange. However, in all cases
adduct formation is a minor pathway, and the most
important contribution to the overall, pseudo-first-order
rate constants (k), given in Figures 5 and 6, is due to
catalyzed hydrolysis or rearrangement. The results in
Figure 3 indicate that the k values of the BP metabolites,
BPDE and BPO, are more strongly influenced by ctDNA
than those of the BA metabolites, BADE and BAO. The
results in Figure 4 indicate that intercalation association
constants, KA, for the BPDE and BPO model compounds
(BP78D and BP45D, respectively) are significantly larger
than association constants for the corresponding BADE
and BAO model compounds (BA34D and BA56D). To-
gether, the results in Figures 3 and 4 provide qualitative
evidence that the catalytic contribution to k increases as
KA of the diol epoxides and epoxides increases.
In earlier investigations of reactions of BPDE, BPO,
and BAO in DNA, Na+ and Mg2+ induced decreases in k
have been reported (6, 7, 13-15, 28). For BPDE, these
decreases arise from reductions in both the association
constant, KA, and the catalytic rate constant for hydroly-
sis, which occur as the Na+ or Mg2+ concentration
increases (7, 15). The similar way in which Na+ reduces
the overall, pseudo-first-order rate constants of BPDE,
F igu r e 6. Overall, pseudo-first-order rate constants (closed
squares) and percentage yields of ctDNA adducts (closed circles),
and hydrolysis (open triangles) and rearrangement products
(open squares) in 0.2 mM ctDNA at varying Na+ concentrations.
Panels A and B show results for BPO and BAO, respectively.
Adduct, hydrolysis, and rearrangement yields are given on the
left-hand scales. Overall, pseudo-first-order rate constants are
given on the right-hand scales. Yields for rearrangement
products were obtained by subtraction using results from the
determination of adduct and hydrolysis yields.
times as the Na+ concentration increases from 2.0 to 100
mM. Earlier experiments indicated that varying the Na+
concentration, over the range examined in Figure 5, has
no influence on the values of k for BPDE and BPO
measured without DNA (7).
The decrease in the value of k for BPDE, observed in
Figure 5, is due to decreases in the intercalation associa-
tion constant, KA, and in the catalytic rate constant for
hydrolysis, kcat,Hy, which occur as the Na+ concentration
increases (7, 14, 15). In contrast to the strong depen-
dence of k on the Na+ concentration, the BPDE adduct
yield remains almost constant. The adduct yield changes
less than 1.2 times. Panel B shows similar results for
BADE, at Na+ concentrations between 2.0 and 50 mM.
Here, the value of k decreases 2.7 times as the Na+
concentration increases, while the adduct yield changes
less than 1.4 times.
Figure 6 contains overall, pseudo-first-order rate con-
stants for BPO and BAO at Na+ concentrations between
2.0 and 100 mM, and 2.0 and 50 mM, respectively.
Figure 6 also shows yields for adduct formation, hydroly-
sis, and rearrangement. For BPO, the results in panel
A are like those for BPDE and BADE in Figure 5. As
4
These values were obtained from calculations using eqs 2 and 3,
and earlier results from Tables I and IV of ref 7 indicating that as the
Na+ concentration increases from 2.0 to 100 mM, KA decreases 1.8-
2.7 times, and kcat,Hy decreases 4.8-9.0 times. In Table IV of ref 7 there
is a misprint in the heading of the third column. The correct heading
is (k0 + kH[H+])/(1 + KA[DNA]).

1398 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Fernando et al.
Ta ble 1. Ra te Con sta n ts for Hyd r olysis, Rea r r a n gem en t, a n d ctDNA Ad d u ct F or m a tion for Rea ction s of Ben zo[a ]p yr en e
a n d Ben z[a ]a n th r a cen e Diol Ep oxid es a n d Ep oxid esa
[DNA] ) 0.0 mMb
[DNA] ) 0.20 mM
kd,g
kcat,Hy
kcat,Re
kcat,Ad
c,d
d,e
f
d,h
d,i
d,j
ki,Hy
ki,Re
KA
BPDE (1.44 ( 0.2) × 10-3
BADE (1.47 ( 0.2) × 10-4
14500 ( 1500
(16000 ( 2000)
820 ( 100
(3.05 ( 0.1) × 10-2 (3.75 ( 0.3) × 10-2
(1.34 ( 0.2) × 10-3 (9.33 ( 0.7) × 10-3
(5.00 ( 0.6) × 10-5 (1.39 ( 0.1) × 10-4 (6.23 ( 0.6) × 10-4 (1.08 ( 0.1) × 10-5
(1.23 ( 0.1) × 10-4 (8.74 ( 0.9) × 10-5 (1.21 ( 0.1) × 10-4 (1.10 ( 0.1) × 10-5
(4.17 ( 0.3) × 10-3
(7.02 ( 0.5) × 10-4
(910 ( 240)
BAO
(3.53 ( 0.6) × 10-6 (6.2 ( 1) × 10-7 320 ( 64
(270 ( 30)
BPO
(9.6 ( 1.8) × 10-7 (7.5 ( 1) × 10-7 7100 ( 1400
(6000 ( 800)
a
In all experiments the Na+ concentration was 2.0 mM. Reactions were carried out in 10 mM Tris‚HCl and 1.0 mM EDTA buffer at
b
pH 7.3. For reactions without ctDNA, overall, pseudo-first-order rate constants are equal to the sum of rate constant for hydrolysis plus
rearrangement. c Pseudo-first-order rate constant for hydrolysis, measured in buffer without ctDNA. In units of s-1
.
e Pseudo-first-
d
order rate constant for rearrangement to phenols, measured in buffer without ctDNA. f Association constants of diol epoxides and epoxides
given without parentheses. Association constants of corresponding model compounds BP78D, BP45D, BA34D, and BA56D given in
parentheses. In units of M-1
.
Overall, pseudo-first-order rate constants measured in ctDNA. Rate constant for ctDNA catalyzed
g
h
i
j
hydrolysis. Rate constant for ctDNA catalyzed rearrangement. Rate constant for ctDNA catalyzed adduct formation.
BADE, BPO, and BAO provides evidence that these two
factors are also important for understanding the influ-
ence of Na+ on reactions of BADE, BPO, and BAO in
ctDNA.
Under conditions where most diol epoxide or epoxide
reacts via DNA catalysis (ki , kcatKA[DNA]), the overall,
pseudo-first-order rate constant in eq 4 is approximately
given by
The manner in which ctDNA enhances the overall,
pseudo-first-order rate constants, k, of all of the epoxides
and diol epoxides examined, and the manner in which
values of k, for all of these epoxides and diol epoxides,
decrease as the Na+ concentration increases, points to
basic similarities between the reactions of BPDE, BADE,
BPO, and BAO in ctDNA. For BPDE, BADE, and BPO,
the finding that the product distributions remain nearly
unchanged, as the Na+ concentration increases, also
provides evidence of similar mechanisms. These results
support the conclusion that, for BPDE, BADE, BPO, and
BAO, k, in eqs 1 and 2, is given by eq 4, which has the
same form (2) as that of the pseudo-first-order rate
constant for BPDE hydrolysis in eq 3. In eq 4, ki
represents the contribution to k from reactions which are
not DNA catalyzed.
kcatKA[DNA]
k ≈
(7)
1 + KA[DNA]
For BPDE and BADE, kcat can be written in the form
kcat ) kcat,Ad + kcat,Hy (8)
For BPO and BAO,
kcat ) kcat,Ad + kcat,Hy + kcat,Re
(9)
Under conditions where eq 7 is valid,
kcat,P ) kcatYP
(10)
Here, kcat,P is either kcat,Ad, kcat,Hy, or kcat,Re, the catalytic
rate constant for adduct formation, hydrolysis, or rear-
rangement, respectively, and YP is the corresponding
percent yield of adducts, or hydrolysis or rearrangement
products obtained from reaction in ctDNA.
ki + kcatKA[DNA]
k )
(4)
1 + KA[DNA]
For BPDE and BADE, ki has components associated with
adduct formation and hydrolysis.
Under conditions where eq 7 is valid, eqs 1 or 2, and 8
or 9, indicate that
ki ) ki,Ad + ki,Hy
(5)
kcat,PKA[DNA]
kP ≈
(11)
1 + KA[DNA]
For BPO and BAO, ki has components associated with
adduct formation, hydrolysis, and rearrangement.
where kP is either kAd, kHy, or kRe, the pseudo-first-order
rate constant for adduct formation, hydrolysis, or rear-
rangement. Equations 7, 8 or 9, and 10 are able to
describe the invariance in the product distributions
observed in Figures 5 and 6 for reactions of BPDE,
BADE, and BPO, even though kcat,P and KA are changing
significantly, if the relative contribution of kcat,P to kcat is
nearly constant over the range of Na+ concentrations
examined. This relationship is expressed by the equa-
tion:
ki ) ki,Ad + ki,Hy + ki,Re
(6)
For BPDE and BAO, ki,Hy equals k0,Hy + kH,Hy[H+] (15,
24). For BAO, ki,Re (equal5 to k0,Re + kH,Re[H+]) has the
same general form as ki,Hy (24). It is likely that ki,Hy for
BADE and BPO and ki,Re for BPO also have the same
form. In eqs 5 and 6, ki,Ad equals k2[DNA], where k2 is
the second-order rate constant for reaction which does
not proceed through formation of a reversibly bound DNA
complex. Table 1 lists values of ki,Hy and ki,Re obtained
from overall, pseudo-first-order rate constants and from
hydrolysis product yields measured without DNA.6
kcat,P/kcat ≈ constant
(12)
In Figures 5 and 6, the BPDE, BADE, and BPO product
distributions, which remain nearly constant as the rate
constants change, agree with product distributions that
are described by eqs 1 or 2, 7, 8 or 9, 11, and 12.
While these equations provide a simple description of
epoxide and diol epoxide kinetics that accounts for the
BPDE, BADE, and BPO results in Figures 5 and 6, it is
5
Here, k0,Re is a rate constant for spontaneous rearrangement and
kH,Re is a rate constant for H+ catalyzed rearrangement without DNA.
6
For BPDE and BADE, ki,Hy equals the overall, pseudo-first-order
rate constant measured without DNA. For BPO and BAO, values of
ki,Hy were obtained by multiplying k by the percent yield of hydrolysis
products measured without DNA. Values of ki,Re were obtained by
subtraction.

Influence of Na+ on DNA Reactions with PAH Epoxides
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1399
important to note the limitations. They apply only when
DNA catalysis dominates. The finding, in Figure 5, that
a measurable decrease occurs in the BADE adduct yield,
over the range of Na+ concentrations examined, suggests
that noncatalyzed hydrolysis is not entirely negligible at
high Na+ concentrations. As the Na+ concentration
increases and DNA catalyzed reactions account for a
smaller fraction of the total reaction, eqs 7 and 11 become
less accurate. This is especially apparent in the BAO
results of Figure 6. For BAO, which has the smallest
DNA intercalation association constant, the fraction of
total reaction that is DNA catalyzed in 50 mM Na+ is
smaller than the fractions of BADE, BPDE, and BPO
reactions that are DNA catalyzed at the highest Na+
concentrations examined in Figures 5 and 6. For BAO,
the relative increase in hydrolysis versus rearrangement
yields which occurs as the Na+ concentration increases
from 2.0 to 50 mM reflects a reduction in the fraction of
reaction which is DNA catalyzed. At 2.0 mM Na+, most
of the reaction undergoes general acid catalysis involving
DNA, and the product distribution reflects the favored
rearrangement pathway which is observed at low pH
(24). In 50 mM Na+, a significantly smaller fraction of
the reaction proceeds via ctDNA catalysis. This is
accompanied by an increase in the hydrolysis yield, which
in buffer without DNA at pH 7.3 accounts for 85% of the
total product.
This calculation was carried out using eq 10 and results
from measurements of product yields in 0.20 mM ctDNA
and 2.0 mM Na+.8
Va lu es of KA, kca t,Hy, kca t,Re, a n d kca t,Ad for BADE
a n d BAO. The small values of KA for the model
compounds BA34D and BA56D, and the results in Figure
3, indicating that the plots of the overall, pseudo-first-
order rate constants of BADE and BAO show no evidence
of saturation over the range of ctDNA concentrations
examined, support the conclusion that the association
constants of BADE and BAO are small. For this reason,
reliable values of KA for BADE and BAO cannot be
obtained from the kinetic data of Figure 3.9 Instead,
values of KA for these molecules were calculated using
eq 13, association constants of the model compounds
BA34D, BA56D, BP78D, and BP45D, and KA values of
BPDE and BPO listed in Table 1.
KA ) KA,mRm
(13)
In evaluating KA for BADE (BAO), KA,m is the association
constant of the model compound BA34D (BA56D), and
Rm is the ratio of the association constant of BPDE (BPO)
to that of BP78D (BP45D). A comparison of the KA values
listed in Table 1 indicates that the intercalation associa-
tion constant of BPDE is 18 times larger than that of
BADE, and the association constant of BPO is 22 times
larger than that of BAO. The KA values of the bay region
diol epoxides, BPDE and BADE, are 2.0 and 2.6 times
larger than those of the corresponding K region epoxides,
BPO and BAO.
ctDNA Associa tion Con sta n ts (KA) of BP DE a n d
BP O, a n d ctDNA Ca ta lytic Ra te Con sta n ts for
Hyd r olysis (kca t,Hy), Rea r r a n gem en t (kca t,Re), a n d
Ad d u ct F or m a tion (kca t,Ad ). For BPDE, BPO, BADE,
and BAO, Table 1 lists intercalation association constants
(KA) in 2.0 mM Na+, and overall, pseudo-first-order rate
constants, k, measured in 2.0 mM Na+ and 0.2 mM
ctDNA. The table gives catalytic rate constants (kcat,Hy
and kcat,Ad) for hydrolysis and adduct formation. For BPO
and BAO, the table also lists catalytic rate constants
(kcat,Re) for rearrangement.
For BADE and BAO, kcat was obtained from a least-
squares fit of the kinetic data in Figure 3. However, in
determining kcat for BADE and BAO, values of KA were
calculated from eq 13, and only kcat was varied during
the fitting process. Values of kcat,Ad, kcat,Hy, and kcat,Re
,
which are listed in Table 1, were calculated from eq 10
using values of kcat obtained from the least-squares fitting
procedure, and product distributions measured in 0.20
mM ctDNA and 2.0 mM Na+.
Values of KA, for BPO and BPDE, which are large
(7100-14 500 M-1), have been obtained using a different
procedure from that used to evaluate values of KA for
BAO and BADE, which are small (320-820 M-1). At
ctDNA and Na+ concentrations of 0.2 and 2.0 mM,
respectively, 58% of BPO and 74% of BPDE are reversibly
intercalated. For BPDE and BPO, KA and kcat were
determined using eq 4 and the results in Figure 3 which
show how the overall, pseudo-first-order rate constants,
k, change as the ctDNA concentration increases. For
BPDE and BPO, values of KA and kcat in Table 1 were
obtained from a least-squares optimized fit of the data
in Figure 3 with calculated values of k obtained from eq
4. In the fitting procedure, ki is equal to the overall,
pseudo-first-order rate constant, measured without DNA,
and to the sum of ki,Hy and ki,Re values listed in Table 1.
Values of KA and kcat are variable parameters.7 For
BPDE and BPO, values of kcat,Ad, kcat,Hy, and kcat,Re, listed
in Table 1, were calculated from values of kcat obtained
from the least-squares analysis of the data in Figure 3.
Com p a r ison of BP DE , BADE , BP O, a n d BAO
Rea ction s. As the product distributions in Figures 5
and 6 indicate, most reaction involves DNA catalyzed
hydrolysis or rearrangement which, in 0.20 mM ctDNA
and 2.0 mM Na+, accounts for 89%, 95%, 87%, and 90%
of the total reaction of BPDE, BPO, BADE, and BAO,
respectively.10 For BPDE and BPO in 100 mM Na+ and
0.2 mM ctDNA, DNA catalyzed hydrolysis or rearrange-
ment accounts for 82% and 77% of the total reaction; for
BADE and BAO in 50 mM Na+ and 0.20 mM ctDNA,
catalyzed hydrolysis or rearrangement accounts for 68%
and 55% of the total reaction.10
The results also demonstrate that the overall, pseudo-
first-order rate constants (k ) kHy,i + kRe,i) obtained
without DNA increase in the order BPDE > BADE >
BAO > BPO. In earlier work, values of k were employed
8
The validity of this approach depends on the accuracy of eq 7
which, in turn, relies on the relationship kcatKA[DNA] . ki. For BPO,
BPDE, BAO, and BADE in 0.2 mM ctDNA and 2.0 mM Na+, the results
in Table 1 indicate that (kcatKA[DNA])/ki equals 182, 84, 12, and 11.
9 For BADE and BAO, it is not currently possible to carry out kinetic
measurements at sufficiently high ctDNA concentrations to achieve
saturation kinetics. For BAO in 2.0 mM Na+, a ctDNA concentration
of 8.9 mM is required to achieve the 74% reversible binding level that
occurs for BPDE in 0.20 mM ctDNA. The smaller BADE and BAO
association constants give rise to large uncertainty in the KA values
obtained when the least-squares fitting procedure, which was used for
BPDE and BPO, is applied to the BADE and BAO kinetic results in
Figure 3. This procedure requires k values near saturation in order to
yield accurate KA values.
7
In the least-squares fitting procedure for BPDE and BPO, as well
as in a similar procedure used for BADE and BAO, ki,Ad, the rate
constant for adduct formation which does not proceed through a ctDNA
complex, was taken to be negligible when compared to other contribu-
tions to the overall, pseudo-first-order rate constant, k (see eqs 4-6).
This is supported by the finding that, for all of the BP and BA epoxides
and diol epoxides, the total adduct yield is less than 10%, coupled with
the observation that for the BPDE, BADE, and BAO reactions, which
have the greatest adduct yields, adduct formation parallels the
hydrolysis and rearrangement reactions which occur primarily via
ctDNA catalyzed pathways. See text and footnotes 10 and 11.

1400 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Fernando et al.
F igu r e 7. Mechanisms for major reaction pathways of BPDE, BPO, BADE, and BAO in buffer with ctDNA.
as markers of the inherent reactivities of diol epoxides
(40). The ordering of k values measured without ctDNA
agrees with earlier data indicating that BPDE is more
reactive than BADE, BAO, or BPO (2, 6, 7, 15, 18, 26,
40). Without DNA, the overall, pseudo-first-order rate
constants of the bay region diol epoxides, BPDE and
BADE, are 842 and 35 times larger than rate constants
for the corresponding K region epoxides, BPO and BAO.
More importantly, the results in Table 1 indicate that
the ordering of the values of k, measured without ctDNA,
is the same as the ordering of the values of kcat, the
catalytic rate constants for hydrolysis, rearrangement,
and adduct formation. The catalytic rate constants (kcat
) kcat,Hy + kcat,Ad) for BPDE ((4.17 ( 0.3) × 10-2) and for
BADE ((1.00 ( 0.1) × 10-2) are 190 and 13 times larger
for BAO because the intercalation association constant
of BPO (7100 M-1) is 22 times larger than that for BAO.
These observations point out that differences occurring
between the intercalation association constants of BP and
BA epoxides can significantly alter the relative rates of
reactions occurring with DNA.
For BPDE, BADE, BAO, and BPO, the similar influ-
ence that DNA and Na+ have on the overall, pseudo-first-
order rate constants, together with the finding that, for
BPDE, BADE, and BPO, the ratios of the pseudo-first-
order rate constants for adduct formation, hydrolysis, and
rearrangement are constant under conditions where the
absolute values of the rate constants are rapidly chang-
ing, provides evidence that a common catalytic mecha-
nism leads to all of the products which have been
examined. In ctDNA, the similarities between reactions
resulting in BPDE adduct formation and BPDE hydroly-
sis, and the similarity between the BPDE reactions and
the reactions of BADE, BPO, and BAO, provide evidence
that, for all of these epoxides and diol epoxides, the most
important reaction mechanisms are shown in Figure 7.
Of the two major reaction routes shown in the figure, one
involving direct reactions and the second involving
catalysis at ctDNA intercalation sites, the latter is, by
far, the most important for hydrolysis and rearrangement
reactions in 0.2 mM ctDNA and 2.0 mM Na+. For BPDE
in 0.2 mM ctDNA at Na+ between 100 and 2.0 mM,
ctDNA catalyzed hydrolysis accounts for 82-89% of all
reaction.10 Because of the manner in which the yield for
adduct formation tracks the yields for hydrolysis and
rearrangement, under conditions where the rate con-
stants and the intercalation association constants vary
significantly, it is likely that catalysis at intercalation
sites represents the most important route through which
adduct formation occurs. Interestingly, the important
role that the initial formation of intercalated complexes
plays is consistent with early speculation about the
mechanism of BPDE adduct formation (41, 42).
than the rate constants (kcat ) kcat,Hy + kcat,Re + kcat,Ad
)
for BPO ((2.19 ( 0.1) × 10-4) and BAO ((7.73 ( 0.3) ×
10-4). For BPDE and BADE, the catalytic rate constants
for adduct formation alone, kcat,Ad, are 379 and 65 times
larger than values for the corresponding K region ep-
oxides. This observation indicates that the ordering of
reactivities, at ctDNA catalytic sites where reaction
occurs 9-72 times faster than in buffer, is the same as
the ordering in buffer without DNA and supports the bay
region theory.
While the ordering of the catalytic rate constants, kcat
,
reflects inherent reactivity, as indicated by the ordering
of k values, measured without DNA, the results in Table
1 demonstrate that reversible intercalation significantly
influences the rate of epoxide and diol epoxide reactions
in ctDNA. This is illustrated by a comparison of BPO
and BAO overall, pseudo-first-order rate constants, for
which the ordering is reversed with and without DNA.
Although the value of kcat for BAO is larger than that for
BPO, the value of k for BPO in ctDNA is larger than that
10
In 2.0 mM Na+ and 0.2 mM ctDNA, values of the percent reaction
which occurs via ctDNA catalyzed hydrolysis and rearrangement were
calculated by using eqs 1 or 2, 4, 5 or 6, and 8 or 9; values of k, ki,Hy
,
ki,Re, kcat,Hy, kcat,Re, kcat,Ad, and KA from Table 1; and the simplifying
approximation that ki,Ad contributes negligibly to k (see footnote 7).
For BPDE and BPO in 100 mM Na+, and for BADE and BAO in 50
mM Na+, values of the percent reaction which occurs via ctDNA
catalyzed hydrolysis and rearrangement were obtained in the same
manner, using adduct and hydrolysis yields, k values, and model
compound KA values measured in 100 and 50 mM Na+. For BPDE and
BPO in 100 mM Na+, k values of (6.03 ( 0.6) × 10-3 and (6.00 ( 0.6)
× 10-6 s-1 and KA values of 6300 and 2400 M-1 were used in the
calculations. For BADE and BAO in 50 mM Na+, k values of (5.11 (
0.6) × 10-4 and (1.02 ( 0.1) × 10-5 s-1 and KA values of 300 and 177
M-1 were used. Values of kcat,Hy and kcat,Re in 100 and 50 mM Na+ were
calculated from eqs 4 and 10. For BPDE and BPO in 100 mM Na+,
and BADE and BAO in 50 mM Na+, the calculated kcat,Hy values were
(8.9 ( 1) × 10-3, (6.3 ( 0.3) × 10-6, (6.3 ( 0.5) × 10-3, and (1.0 ( 0.1)
× 10-4 s-1. For BPO in 100 mM Na+ and BAO in 50 mM Na+, the
For BADE and BAO, the smaller association constants
and the greater variation in product yields over the range
of Na+ concentrations examined indicate that at high Na+
concentrations noncatalyzed hydrolysis and rearrange-
ment become significant. However, for all of the epoxides
and diol epoxides, the data in Table 1 indicate that, in
0.2 mM ctDNA and 2.0 mM Na+, 90-99% of all hydroly-
sis and rearrangement occurs via DNA catalysis.11 These
11
The percentages of hydrolysis and rearrangement which are
ctDNA catalyzed were obtained using a method analogous to that used
in determining the percentage of total reaction which occurs via ctDNA
catalyzed hydrolysis and rearrangement. See footnote 10.
kcat,Re values were (8.0 ( 3) × 10-6 and (7.2 ( 3) × 10-5 s-1
.

Influence of Na+ on DNA Reactions with PAH Epoxides
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1401
results provide evidence that, under the same conditions,
similarly high percentages of adducts are also formed via
DNA catalysis.
epoxides. In 2.0 mM Na+, the association constants of
the BP metabolites, BPDE and BPO, are 18 and 22 times
larger than the association constants of the corresponding
BA metabolites, BADE and BAO.
While the present experiments provide a qualitative
description of the reaction mechanisms examined, the
need remains for a full characterization of the transition
states associated with the formation of DNA adducts from
BP and BA epoxides and diol epoxides. Of particular
interest will be information which provides clues relating
reversibly bound intercalated conformations, the impor-
tance of which is indicated by binding and kinetic data,
to the nonintercalated equilibrium conformations associ-
ated with covalently bound adducts (43, 44). One expla-
nation is that reactions occur via “loose” transition states
involving reactants which are approaching or have
recently exited a reversibly bound intercalation site.
Another possibility is that the conformational flexibility
of covalently modified DNA and the reaction exother-
micity result in a change in adduct conformation from
initial high energy intercalated geometries to more
favorable external geometries. Further investigation will
be required to test the validity of these or of other
possibilities which have not yet been considered.
Nevertheless, recognition that DNA catalysis and
reversible intercalation influence the covalent modifica-
tion of ctDNA by BPDE, BADE, BPO, and BAO leads to
an understanding of the complexity of factors influencing
the biochemical activity of aromatic DNA alkylating
agents. In biochemical environments which provide
additional reaction pathways, that have not been exam-
ined here, DNA modification, which relies on catalysis
at intercalation sites may influence adduct yields by
enhancing adduct formation at the expense of DNA
independent detoxification pathways. These include
glutathione S-transferase catalyzed formation of glu-
tathione conjugates (8) and epoxide hydrolase catalyzed
hydrolysis (9-11). In this way, the catalytic role which
DNA plays in enhancing adduct formation may be
important to mutagenic and carcinogenic mechanisms of
BP and BA metabolites which rely on covalent modifica-
tion of DNA.
3. In 2.0 mM Na+ and 0.2 mM ctDNA, the ordering of
catalytic rate constants (kcat) for reactions is BPDE >
BADE > BAO > BPO. This is the same ordering as that
of the inherent reactivities indicated by overall, pseudo-
first-order rate constants, k, measured without DNA.
However, for reactions in ctDNA, the ordering of the k
values is influenced not only by reactivity but also by
intercalation association constants. Comparisons of
pseudo-first-order rate constants for hydrolysis and rear-
rangement in buffer without DNA (ki,Hy and ki,Re) and of
catalytic rate constants (kcat) indicate that BPO is less
reactive than BAO. However, in ctDNA, the larger
intercalation association constant of BPO gives rise to
an overall, pseudo-first-order rate constant for reactions
leading to hydrolysis, rearrangement, and adduct forma-
tion which is 2.3 times larger than that of BAO.
4. The similar catalytic influence of ctDNA on the
hydrolysis of BPDE, BADE, BPO, and BAO, and on the
rearrangement reactions of BPO and BAO, coupled with
the invariance in the BPDE, BADE, and BPO adduct
yields, which occurs at varying Na+ concentrations,
provide evidence that, in DNA at low Na+ concentrations,
all reactions are dominated by DNA catalysis. For each
pathway, the contribution to the overall, pseudo-first-
order rate constant is given approximately by eq 11.
5. For BAO, which has the smallest DNA intercalation
association constant, and for which DNA catalysis is least
efficient, increasing the Na+ concentration from 2.0 to
50 mM, in reactions with DNA, causes an increase in the
hydrolysis yield and a decrease in the rearrangement
yield. This is consistent with the kinetic data in Table
1, which indicate that, for BAO at the highest Na+
concentration examined, the percentage of the total
reaction which proceeds via DNA catalysis is approxi-
mately 55%. The smaller role that DNA catalysis plays
at high Na+ concentrations results in product distribu-
tions which are more similar to that occurring in buffer
without DNA.
6. For BPDE, BADE, BPO, and BAO in 0.2 mM ctDNA
and 2.0 mM Na+, the percentages of the hydrolysis and
rearrangement reactions which undergo catalysis at DNA
intercalation sites are between 90% and 99%. Under the
same conditions, it is likely that similarly high percent-
ages of reactions resulting in adducts proceed in the same
manner.
Con clu sion s
In summary, this investigation leads to a description
of the relationship between reversible binding which
occurs at DNA intercalation sites and DNA catalyzed
reactions of BPDE, BADE, BPO, and BAO which result
in covalent DNA modification. The main conclusions are
as follows:
1. For BPDE in 0.2 mM ctDNA and at Na+ concentra-
tions between 2.0 and 100 mM, the kinetics for the minor
pathway resulting in adducts closely parallels the kinet-
ics for the most important pathway, which results in
hydrolysis, which accounts for 82-89% of all reaction,
and which occurs via catalysis at ctDNA intercalation
sites. The reactions of BPDE, BADE, and BPO resulting
in DNA adducts behave similarly. In all cases, Na+
induced decreases in the pseudo-first-order rate constant
for DNA adduct formation parallel decreases in pseudo-
first-order rate constants for major pathways leading to
hydrolysis and rearrangement. Over ranges of increasing
Na+ concentration, in which the overall, pseudo-first-
order rate constants of BPDE, BADE, and BPO decrease
2.7-15.4 times, the adduct yields vary only by factors of
1.2, 1.4, and 1.1, respectively.
Ack n ow led gm en t. Support of this work by the
American Cancer Society (Grant CN-37E) is gratefully
acknowledged.
Refer en ces
(1) Harvey, R. G. (1991) In Polycyclic Aromatic Hydrocarbons:
Chemistry and Carcinogenesis, Chapters 4, 9, 12, 13, and 14,
Cambridge University Press, Cambridge, England.
(2) J erina, M., Chadha, A., Cheh, A. M., Schurdak, M. E., Wood, A.
W., and Sayer, J . M. (1990) Covalent bonding of bay region diol
epoxides to nucleic acids. In Biological reactive Intermediates IV
(Witmer, C. M., Ed.) pp 533-553, Plenum Press, New York.
(3) Conney, A. H. (1982) Induction of microsomal enzymes by foreign
chemicals and carcinogenesis by polycyclic aromatic hydrocar-
bons: G. H. A. Clowes Memorial Lecture. Cancer Res. 42, 4875-
4917.
(4) Wood, A. W., Levin, W., Lu, A. Y. H., Ryan, D., West, S. B., Lehr,
R. E., Schaefer-Ridder, M., J erina, D. M., and Conney, A. H.
(1976) Mutagenicity of metabolically activated benzo[a]an-
thracene 3,4-dihydrodiol: evidence for bay region activation of
2. There is significant variation in the association
constants of different BP and BA epoxides and diol

1402 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Fernando et al.
(25) Hylarides, M. D., Lyle, T. A., Daub, G. H., and J agt, D. L. (1979)
Carbon-13 nuclear magnetic resonance study of the nucleophilic
additions to benzo[a]pyrene 4,5-oxide and of its acid catalyzed
rearrangement. J . Org. Chem. 44, 4652-4657.
(26) Geacintov, N. E., Yoshida, H., Ibanez, V., and Harvey, R. G. (1982)
Noncovalent binding of 7,8-dihydroxy-9,10-epoxy-tetrahydro-
benzo[a]pyrene to deoxyribonucleic acid and its catalytic effect
on the hydrolysis of the diol epoxide to tetrol. Biochemistry 21,
1864-1869.
(27) MacLeod, M. C., and Zachary, K. L. (1985) Involvement of
exocyclic amino group of deoxyguanosine in DNA-catalyzed
carcinogen detoxification. Carcinogenesis 6, 147-149.
(28) MacLeod, M. C., and Selkirk, J . K. (1982) Physical interactions
of isomeric benzo[a]pyrene diol epoxides with DNA. Carcinogen-
esis 3, 287-292.
(29) Abramovich, M., Prakash, A. S., Harvey, R. G., Zegar, I. S., and
LeBreton, P. R. (1985) A comparison of the intercalative binding
of nonreactive benzo[a]pyrene metabolites and metabolite model
compounds to DNA. Chem.-Biol. Interact. 55, 39-62.
(30) Urano, S., Fetzer, S., Harvey, R. G., Tasaki, K., and LeBreton, P.
R. (1988) Influences of 7-alkyl substitution on the reversible
binding of the proximate carcinogen trans-3,4-dihydroxy-3,4-
dihydrobenzo[a]anthracene to DNA. Biochem. Biophys. Res. Com-
mun. 154, 789-795.
carcinogenic polycyclic hydrocarbons. Biochem. Biophys. Res.
Commun. 72, 680-686.
(5) Slaga, T. J ., Bracken, W. M., Viaje, A., Levin, W., Yagi, H., J erina,
D. M., and Conney, A. H. (1977) Comparison of the tumor-
initiating activities of benzo(a)pyrene arene oxides and diol-
epoxides. Cancer Res. 37, 4130-4133.
(6) Urano, S., Price, H. L., Fetzer, S. M., Briedis, A. V., Milliman,
A., and LeBreton, P. R. (1991) Photoemission probes of hydro-
carbon-DNA interactions: a comparison of DNA influences on the
reactivities of (()-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-
tetrahydrobenzo[a]pyrene, benzo[a]pyrene 4,5-oxide, and benzo-
[a]anthracene 5,6-oxide. J . Am. Chem. Soc. 113, 3881-3893.
(7) Huang, C. R., Milliman, A., Price, H. L., Urano, S., Fetzer, S. M.,
and LeBreton, P. R. (1993) Photoemission probes of catalysis of
benzo[a]pyrene epoxide reactions in complexes with linear, double-
stranded and closed-circular, single-stranded DNA. J . Am. Chem.
Soc. 115, 7794-7805.
(8) Awasthi, Y. C., and Singh, S. V. (1985) Differential activities of
rat and human lung glutathione S-transferase isoenzymes to-
wards benzo(a)pyrene epoxides. Biochem. Biophys. Res. Commun.
133, 863-867.
(9) Oesch, F. (1980) Microsomal epoxide hydrolase. In Enzymatic
Basis of Detoxication (J akoby, W. B., Ed.) Vol. 2, pp 277-290,
Academic Press, New York.
(31) Zegar, I. S., Prakash, A. S., Harvey, R. G., and LeBreton, P. R.
(1985) Stereoelectronic aspects of the intercalative binding prop-
erties of 7,12-dimethylbenz[a]anthracence metabolites with DNA.
J . Am. Chem. Soc. 107, 7990-7995.
(32) Yang, N.-C., Hrinyo, T. P., Petrich, J . W., and Yang, D.-D. (1983)
Base sequence selectivity in binding of aromatic hydrocarbons
with synthetic polynucleotides. Biochem. Biophys. Res. Commun.
114, 8-13.
(33) Geacintov, N. E., Ibanez, V., Gagliano, A. G., Yoshida, H., and
Harvey, R. G. (1980) Kinetics of hydrolysis to tetrols and binding
of benzo(a)pyrene-7,8-dihydrodiol-9,10-oxide and its tetraol de-
rivatives to DNA. Conformation of adducts. Biochem. Biophys.
Res. Commun. 92, 1335-1342.
(34) Geacintov, N. E., Ibanez, V., Benjamin, M. J ., Hibshoosh, H., and
Harvey, R. G. (1983) Mechanism of reaction of benzo[a]pyrene
diol epoxide with DNA. In Polynuclear Aromatic Hydrocarbons;
Formation, Metabolism and Measurement (Cook, M., and Dennis,
A., Eds.) pp 554-570, Battelle, Columbus, OH.
(35) NIH guidelines for the laboratory use of chemical carcinogens
(1981) NIH Publication No. 81-2385, U.S. Government Printing
Office, Washington, DC.
(36) Wolfe, A. R., Yamamoto, J ., and Meehan, T. (1994) Chloride ions
catalyze the formation of cis adducts in the binding of anti-benzo-
[a]pyrene diol epoxide to nucleic acids. Proc. Natl. Acad. Sci.
U.S.A. 91, 1371-1375.
(37) Wolfe, A. R., and Meehan, T. (1994) Halide-catalyzed cis formation
in the hydrolysis of anti-benzo[a]pyrene diol epoxide and its
alkylation of poly(A). Chem. Res. Toxicol. 7, 110-119.
(38) Whalen, D. L., Ross, A. M., Dansette, P. M., and J erina, D. M.
(1977) Specific effects of chloride ion in the hydrolysis of a
K-region arene oxide. J . Am. Chem. Soc. 99, 5672-5676.
(39) Wolfe, A., Shimer, G. H., J r., and Meehan, T. (1987) Polycyclic
aromatic hydrocarbons physically intercalate into duplex regions
of denatured DNA. Biochemistry 26, 6392-6396.
(40) Sayer, J . M., Lehr, R. E., Whalen, D. L., Yagi, H., and J erina, D.
M. (1982) Structure reactivity indices for the hydrolysis of diol
epoxides of polycyclic aromatic hydrocarbons. Tetrahedron Lett.
23, 4431-4434.
(10) Wood, A. W., Levin, W., Lu, A. Y. H., Yagi, H., Hernandez, O.,
J erina, D. M., and Conney, A. H. (1976) Metabolism of benzo(a)-
pyrene and benzo(a)pyrene derivatives to mutagenic products by
highly purified hepatic microsomal enzymes. J . Biol. Chem. 251,
4882-4890.
(11) Bentley, P., Schmassmann, H., Sims, P., and Oesch, F. (1976)
Epoxides derived from various polycyclic hydrocarbons as sub-
strates of homogeneous and microsome bound epoxide hydratase.
Eur. J . Biochem. 69, 97-103.
(12) Loechler, E. L. (1995) How are potent bulky carcinogens able to
induce such a diverse array of mutations? Mol. Carcinog. 4, 213-
219.
(13) Meehan, T., and Bond, D. M. (1984) Hydrolysis of benzo[a]pyrene
diol epoxide and its covalent binding to DNA proceed through
similar rate-determined steps. Proc. Natl. Acad. Sci. U.S.A. 81,
2635-2639.
(14) Geacintov, N. E., Hibshoosh, H., Ibanez, V., Benjamin, M. J ., and
Harvey, R. G. (1984) Mechanisms of reaction of benzo[a]pyrene-
7,8-diol-epoxide with DNA. Biophys. Chem. 20, 121-133.
(15) Michaud, D. P., Gupta, S. C., Whalen, D. L., Sayer, J . M., and
J erina, D. M. (1983) Effects of pH and salt concentration on the
hydrolysis of a benzo[a]pyrene-7,8-diol-epoxide catalyzed by DNA
and polyadenylic acid. Chem.-Biol. Interact. 44, 41-52.
(16) MacLeod, M. C. (1990) The importance of intercalation in the
covalent binding of benzo[a]pyrene diol epoxide to DNA. J . Theor.
Biol. 142, 113-122.
(17) Chen, F. M. (1983) Binding of pyrene to DNA, base sequence
specificity and its implication. Nucleic Acids Res. 11, 7231-7250.
(18) Carberry, S. E., Shahbaz, M., Geacintov, N. E., and Harvey, R.
G. (1988) Reactions of stereoisomeric and structurally related bay
region diol epoxide derivatives of benz[a]anthracene with DNA.
Conformations of non-covalent complexes and covalent carcinogen-
DNA adducts. Chem.-Biol. Interact. 66, 121-145.
(19) Rojas, M., and Alexandrov, K. (1986) In vivo formation and
persistence of DNA and protein adducts in mouse and rat skin
exposed to (()benzo[a]pyrene-4,5-oxide. Carcinogenesis 7, 235-
240.
(20) Grover, P. L., and Sims, P. (1973) K-region epoxides of polycyclic
hydrocarbons: reactions with nucleic acids and polyribonucle-
otides. Biochem. Pharmacol. 22, 661-666.
(41) Meehan, T., and Straub, K. (1979) Double-stranded DNA stereo-
selectively binds benzo(a)pyrene diol epoxides. Nature 277, 410-
412.
(42) Lin, J . H., LeBreton, P. R., and Shipman, L. L. (1980) Reaction
of the 7,8-diol 9,10-epoxide of benzo[a]pyrene with guanine in
DNA. Ab initio calculations and proposed reaction mechanism.
J . Phys. Chem. 84, 642-649.
(21) J ennette, K. W., J effrey, A. M., Blobstein, S. H., Beland, F. A.,
Harvey, R. G., and Weinstein, I. B. (1977) Nucleoside adducts
from the in vitro reaction of benzo[a]pyrene-7,8-dihydrodiol 9,-
10-oxide or benzo[a]pyrene 4,5-oxide with nucleic acids. Biochem-
istry 16, 932-938.
(43) Cosman, M., de los Santos, C., Fiala, R., Hingerty, B. E., Singh,
S. B., Ibanez, V., Margulis, L. A., Live, D., Geacintov, N. E.,
Broyde, S., and Patel, D. J . (1992) Solution conformation of the
major adduct between the carcinogen (+)-anti-benzo[a]pyrene diol
epoxide and DNA. Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918.
(44) LeBreton, P. R., Huang, C.-R., Fernando, H., Zajc, B., Lakshman,
M. K., Sayer, J . M., and J erina, D. M. (1995) Multiple fluorescence
lifetimes for oligonucleotides containing single, site-specific modi-
fication at guanine and adenine corresponding to trans addition
of exocyclic amino groups to (+)-(7R,8S,9S,10R)- and (-)-
(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroben-
zo[a]pyrene. Chem. Res. Toxicol. 8, 338-348.
(22) Cheh, A. M., Chadha, A., Sayer, J . M., Yeh, H. J . C., Yagi, H.,
Pannell, L. K., and J erina, D. M. (1993) Structures of covalent
nucleoside adducts formed from adenine, guanine, and cytosine
bases of DNA and the optically active bay-region 3,4-diol 1,2-
epoxides of benz[a]anthracene. J . Org. Chem. 58, 4013-4022.
(23) Murray, A. W., Grover, P. L., and Sims, P. (1976) The conversion
of benzo[a]pyrene-4,5-oxide into 4-hydroxybenzo[a]pyrene in the
presence of polyriboguanylic acid. Chem-Biol. Interact. 13, 57-
66.
(24) Nashaad, N. T., Balani, S. K., Loncharich, R. J ., Sayer, J . M.,
Shipley, D. Y., Ram, M. S., Whalen, D. L., and J erina, D. M. (1991)
Solvolysis of K-region arene oxides: Substituent effects on
reactions of benz[a]anthracene 5,6-oxide. J . Am. Chem. Soc. 113,
3910-3919.
TX960134J