Benzene and dopamine catechol quinones could initiate cancer or neurogenic disease

Article abstract of DOI:10.1016/j.freeradbiomed.2009.11.002

Catechol quinones of estrogens react with DNA by 1,4-Michael addition to form depurinating N3Ade and N7Gua adducts. Loss of these adducts from DNA creates apurinic sites that can generate mutations leading to cancer initiation. We compared the reactions o

Full text of DOI:10.1016/j.freeradbiomed.2009.11.002

Free Radical Biology & Medicine 48 (2010) 318–324
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Original Contribution
Benzene and dopamine catechol quinones could initiate cancer or neurogenic disease
Muhammad Zahid a, Muhammad Saeed a, Eleanor G. Rogan a,b, Ercole L. Cavalieri a,
⁎
a
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA
b
Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Catechol quinones of estrogens react with DNA by 1,4-Michael addition to form depurinating N3Ade and
N7Gua adducts. Loss of these adducts from DNA creates apurinic sites that can generate mutations leading to
cancer initiation. We compared the reactions of the catechol quinones of the leukemogenic benzene (CAT-Q)
and N-acetyldopamine (NADA-Q) with 2′-deoxyguanosine (dG) or DNA. NADA was used to prevent
intramolecular cyclization of dopamine quinone. Reaction of CAT-Q or NADA-Q with dG at pH 4 afforded
CAT-4-N7dG or NADA-6-N7dG, which lost deoxyribose with a half-life of 3 h to form CAT-4-N7Gua or 4 h to
form NADA-6-N7Gua. When CAT-Q or NADA-Q was reacted with DNA, N3Ade adducts were formed and lost
from DNA instantaneously, whereas N7Gua adducts were lost over several hours. The maximum yield of
adducts in the reaction of CAT-Q or NADA-Q with DNA at pH 4 to 7 was at pH 4. When tyrosinase-activated
CAT or NADA was reacted with DNA at pH 5 to 8, adduct levels were much higher (10- to 15-fold), and the
highest yield was at pH 5. Reaction of catechol quinones of natural and synthetic estrogens, benzene,
naphthalene, and dopamine with DNA to form depurinating adducts is a common feature that may lead to
initiation of cancer or neurodegenerative disease.
Received 31 August 2009
Revised 22 October 2009
Accepted 4 November 2009
Available online 10 November 2009
Keywords:
1,4-Michael addition
Catechol quinones
Depurinating DNA adducts
Kinetics of depurination
© 2009 Elsevier Inc. All rights reserved.
Experiments on estrogen metabolism, formation of DNA adducts,
mutagenicity, cell transformation, and carcinogenicity led to and
support the hypothesis that reaction of specific metabolites, mostly
catechol estrogen-3,4-quinones, with DNA forms small amounts of
stable adducts (b1%) and predominant amounts of depurinating
adducts (N99%) [1,2]. This reaction occurs by a proton-assisted 1,4-
Michael addition [3]. The depurinating adducts obtained are
4-hydroxyestrone(estradiol)-1-N3Ade [4-OHE1(E2)-1-N3Ade]1 and
4-OHE1(E2)-1-N7Gua. The stable adducts remain in DNA unless
removed by repair, whereas the depurinating adducts detach from
DNA, leaving behind apurinic sites. Errors in the repair of these sites
can lead to the critical mutations initiating breast, prostate, and other
human cancers [1,2,4,5].
Strong evidence has also been obtained that the synthetic
estrogen hexestrol and the human carcinogen diethylstilbestrol are
metabolized predominantly to their respective catechols. When the
catechols are oxidized to quinones, they also react with DNA by 1,4-
Michael addition to form depurinating N3Ade and N7Gua adducts
that are analogous to those formed by the catechol estrogen-3,4-
quinones [6–8]. Similarly, the catechol quinone of naphthalene reacts
with DNA in vitro and in vivo to form analogous depurinating N3Ade
and N7Gua adducts [9,10].
We have evidence that the ortho-quinones of the leukemogenic
benzene and the neurotransmitter dopamine can also react with DNA
by 1,4-Michael addition to form depurinating N3Ade and N7Gua
adducts [11] analogous to those formed by the natural and synthetic
estrogens, as well as naphthalene. In this article, we report a further
study of the benzene catechol (1,2-dihydroxybenzene; CAT) and
N-acetyldopamine (NADA), which is itself a catechol (Fig. 1). Benzene
is metabolized to phenol in the liver by cytochrome P450 2E1 [12,13].
Other metabolites include CAT and hydroquinone (1,4-dihydroxy-
benzene) [14]. Oxidation of CAT and hydroquinone is catalyzed by
peroxidases, forming quinones that can exert myelotoxic effects [15]
and produce stable [16,17] and depurinating [11] DNA adducts. In fact,
higher levels of peroxidases and the lack of quinone reductase in the
bone marrow allow formation of toxic quinones without the
possibility of their being reduced [18].
One of the major metabolic pathways of the neurotransmitter
dopamine is oxidation to its catechol quinone, which at neutral pH
regularly undergoes intramolecular cyclization by 1,4-Michael addi-
tion, followed by oxidation to form leukochrome and then amino-
chrome. Polymerization of the aminochrome leads to neuromelanin
(Fig. 2, top). At lower pH, however, partial protonation of the amino
group of dopamine slows down the intramolecular cyclization of the
dopamine quinone, rendering competitive the reaction of the quinone
with DNA to form depurinating N3Ade and N7Gua adducts (Fig. 2,
bottom). Although the two depurinating adducts were obtained from
dopamine activated by tyrosinase [11], NADA was used in this study
Abbreviations: Ade, adenine; CAT, 1,2-dihydroxybenzene; CAT-Q, catechol-1,2-
quinone; DA, dopamine; dG, 2′-deoxyguanosine; DMF, dimethylformamide; ESI,
electrospray ionization; Gua, guanine; NADA, N-acetyldopamine; NADA-Q, N-acetyldo-
pamine-3,4-quinone; OHE1(E2), hydroxyestrone(estradiol); TFA, trifluoroacetic acid;
UPLC-MS/MS, ultraperformance liquid chromatography/tandem mass spectrometry.
⁎
Corresponding author. Fax: +1 402 559 8068.
E-mail address: ecavalie@unmc.edu (E.L. Cavalieri).
0891-5849/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2009.11.002

M. Zahid et al. / Free Radical Biology & Medicine 48 (2010) 318–324
319
to protect the amino group of dopamine and totally avoid the pathway
of intramolecular cyclization after formation of the quinone. In this
way we could compare the ability of the catechol quinone of benzene
and the dopamine quinone to react intermolecularly via 1,4-Michael
addition at various pH.
Materials and methods
Chemicals and reagents
CAT, Ag2O, potassium tetraborate, acetic anhydride, formic acid,
acetic acid, trifluoroacetic acid (TFA), dimethylformamide (DMF), and
dimethyl sulfoxide-d6 were purchased from Aldrich Chemical Co.
(Milwaukee, WI, USA) and used as such without further purification.
2′-Deoxyguanosine (dG) and calf thymus DNA were purchased from
USB (Cleveland, OH, USA). Dopamine (DA) hydrochloride and
mushroom tyrosinase were purchased from Sigma Chemical (St.
Louis, MO, USA). The standard depurinating adducts of CAT, i.e., CAT-
4-N7Gua and CAT-4-N3Ade, and those of NADA, NADA-6-N7Gua and
NADA-6-N3Ade, were synthesized according to the procedure
reported earlier [11].
Instrumentation
UV
UV spectra were obtained during HPLC by using a Waters (Milford,
MA, USA) 996 or 2996 photodiode array detector for all compounds
synthesized. HPLC separations were monitored at 290 nm for both
CAT and NADA.
HPLC
Analytical HPLC was conducted on a Waters 2690 separations
module equipped with a Waters 996 or 2996 photodiode array
detector and a reverse-phase Phenomenex Luna-2 C-18 column
(250×4.6 mm, 5 μm; 120 Å; Torrance, CA, USA). For CAT, the column
was eluted starting with 8% CH3CN in H2O (0.25% TFA) and continuing
Fig. 1. Oxidation CAT and NADA to their respective quinones and reaction of the
quinones with Ade or dG to form N3Ade and N7Gua adducts.
Fig. 2. Metabolism of dopamine to form neuromelanin and depurinating DNA adducts.

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M. Zahid et al. / Free Radical Biology & Medicine 48 (2010) 318–324
with a linear gradient to 10% CH3CN in 25 min. For NADA, the column
was eluted starting with 6% CH3CN in H2O (0.25% TFA) and then with a
linear gradient to 8% CH3CN in 20 min at a flow rate of 1 ml/min.
adduct, i.e., CAT-4-N7Gua and NADA-6-N7Gua. The rationale for using
the N7Gua adducts for quantitation of the N7dG adducts derives from
their similar UV spectra. The reactions were conducted and analyzed
in triplicate.
Mass spectrometry
Kinetics of depurinating adduct formation after reaction of DNA with
CAT-Q or NADA-Q
Electrospray ionization (ESI)-MS.
All experiments were performed
on a Waters QuattroMicro triple-quadrupole mass spectrometer by
using ESI in the positive ion mode, with an ESI-MS capillary voltage of
3.0 kV, an extractor cone voltage of 2 V, and a detector voltage of
650 V. Desolvation gas flow was maintained at 400 l/h. Cone gas flow
was set at 60 l/h. Desolvation temperature and source temperature
were set to 200 and 100°C, respectively. The acquisition range was
50–700 Da. The sample was introduced to the source at a flow rate of
10 μl/min by using a built-in pump.
CAT (19.2 mg, 174 μmol) or NADA (33.9 mg, 174 μmol) was
dissolved in 1 ml of acetone and stirred at 0 °C. Then Ag2O (242.2 mg,
6 equivalents) was slowly added. After 30 min, the solution was
filtered through a Gelman acrodisc. Five microliters of the freshly
prepared quinone (8.7 μmol of CAT-Q or NADA-Q) in acetone was
reacted with 3 mM DNA in a 10-ml reaction mixture at pH 4. At
various time points (1, 2, 3, 4, 5, 6, 7, and 10 h), aliquots of the
mixtures were treated quickly with 2 volumes of ethanol to remove
DNA and the supernatants were analyzed for the formation of
depurinating adducts, as described above. The reactions were
performed in triplicate and control experiments were conducted
with 5 μl of acetone in place of the quinone solution.
UPLC-MS/MS. Samples were analyzed on an Acquity UPLC system
attached to a MicroMass QuattroMicro triple-quadrupole mass
spectrometer. The 10-μl injections were made to a Waters Acquity
UPLC BEHC18 column (1.7 μm, 1×100 mm). The instrument was
operated in the positive ESI mode. All aspects of system operation,
data acquisition, and processing were controlled using QuanLynx
version 4.0 software (Waters). The column was eluted with 5% CH3CN
in H2O (0.1% formic acid) for 10 min at a flow rate of 150 μl/min.
Ionization was achieved using the following settings: capillary voltage
3 kV, cone voltage 15–60 V, source block temperature 100°C,
desolvation temperature 200°C, with a nitrogen flow of 400 l/h.
Nitrogen was used as both the desolvation and the auxiliary gas.
Argon was used as the collision gas. Three-point calibration curves
covering the range of analytes detected were run for each standard.
Covalent binding of CAT-Q or NADA-Q to DNA at various pH
Five microliters (87 μM final concentration) of a stock solution of
CAT-Q or NADA-Q was mixed with DNA in a total 10-ml reaction
mixture containing 3 mM calf thymus DNA in 0.1 M sodium acetate
(for pH 4 or 5) or 0.1 M sodium potassium phosphate (pH 6 or 7).
After incubation at 37 °C for 10 h, the DNA was precipitated with 2
volumes of ethanol; the supernatant was evaporated and dissolved in
methanol (1 ml), lyophilized, and redissolved in 50 μl of methanol:
water (1:1). The final solution was passed through a 5000 MW cut-off
filter (Millipore, Billerica, MA, USA) and 10 μl of each sample was
analyzed for depurinating adducts by UPLC-MS/MS. Control reactions
were carried out under identical conditions using 50 μl of acetone in
place of the quinone solution.
Synthesis of NADA
NADA was synthesized from DA with a slight modification of the
reported method [19]. DA hydrochloride (0.8 g) was dissolved in
40 ml of 10% potassium tetraborate that had been purged with
nitrogen. Then 0.44 ml of 99% acetic anhydride was slowly added over
a period of 30 min to the DA hydrochloride solution under a nitrogen
atmosphere. The reaction mixture was stirred at room temperature
for 8 h, and then 3 ml of concentrated formic acid was added to
precipitate boric acid. After filtration, the supernatant was extracted
with ethyl acetate (3×50 ml). The combined organic layers were
concentrated and purified by column chromatography on silica gel
using CHCl3:CH3OH (1:1) as the solvent system. Fractions containing
common thin-layer chromatography spots were pooled and concen-
trated to obtain pure NADA (540 mg, 66%). The NMR and MS data for
the compound matched exactly those already reported in the
literature [19].
Covalent bindingof tyrosinase-activated CAT orNADA to DNA at various pH
Stock solutions of CAT (0.96 mg) or NADA (1.69 mg) were made in
50 μl of acetone and 5 μl of each solution was mixed with DNA in a
total 10-ml reaction mixture containing 3 mM calf thymus DNA in
0.1 M sodium acetate (pH 5) or 0.1 M sodium potassium phosphate
(pH 6, 7, or 8) and 1 mg of mushroom tyrosinase (2577 units). The
reaction mixture was incubated at 37°C in the presence of air for 10 h.
After precipitation of DNA with ethanol, the supernatant was used for
analysis of depurinating adducts, as described above. Control reac-
tions were carried out under identical conditions using 50 μl of
acetone in place of the CAT or NADA solution.
Reaction of CAT-Q or NADA-Q with dG and loss of deoxyribose
Results
CAT (11 mg, 0.1 mmol) or NADA (20 mg, 0.1 mmol) was dissolved
in 2 ml of DMF and stirred at 0°C. To this solution was added Ag2O
(140 mg, 0.6 mmol) portion-wise and the reaction mixture was
stirred at 0 °C for 30 min. A dark-colored solution of CAT-Q or NADA-Q
was filtered quickly through a syringe filter into a solution of dG
(142 mg, 0.5 mmol) in H2O:CH3COOH (2 ml, 1:1) and the pH of the
solution was adjusted to 4. The reaction mixture was stirred at room
temperature and aliquots at various time intervals (0.33, 0.66, 1, 2, 3,
4, 5, 7, 8, 12, 16, 20, and 24 h) were analyzed by using analytical HPLC
monitored at 290 nm. The CAT-4-N7dG and CAT-4-N7Gua peaks were
identified and showed Rt at 9.5 and 10.2 min, respectively. The NADA-
6-N7dG and NADA-6-N7Gua peaks were eluted at 8.8 and 10.4 min,
respectively. The compounds were analyzed and quantified at 290 nm
using Empower software after comparing the response of samples
with the calibration curves made with the standard depurinating
Formation of CAT-4-N7dG and NADA-6-N7dG adducts
The reaction of CAT-Q with dG at pH 4.0 (Fig. 1) afforded initially a
labile adduct as observed by HPLC analysis (retention time 9.5 min,
data not shown). After 1 h an aliquot of the reaction mixture was
directly infused into the mass spectrometer (Fig. 3) to afford the
molecular ions [M]+ at m/z 376, 267, 260, and 152, corresponding to
CAT-4-N7dG (C16H18N5O+6 ), dG, CAT-4-N7Gua, and Gua, respectively.
MS/MS analysis of the peak at m/z 376 afforded a daughter ion at m/
z 260, corresponding to CAT-4-N7Gua. Attempts to isolate and record
the NMR spectrum of the CAT-4-N7dG adduct were not successful,
because the compound decomposed during the removal of the
solvent.
Analogously, the reaction of NADA-Q with dG at pH 4.0 yielded
initially a labile adduct, NADA-6-N7dG, which slowly converted to

M. Zahid et al. / Free Radical Biology & Medicine 48 (2010) 318–324
321
Fig. 3. Mass spectrum of the reaction mixture of CAT-Q with dG after 1 h at 22 °C. Top: MS/MS of m/z 376, CAT-4-N7dG, showing the daughter ion at m/z 260, CAT-4-N7Gua.
Fig. 4. Mass spectrum of the reaction mixture of NADA-Q with dG after 3 h at 22 °C. Top: Right, MS/MS of m/z 461, NADA-6-N7dG, showing the daughter ions at m/z 345, NADA-6-
N7Gua, and m/z 152, Gua. Left, MS/MS of m/z 345, NADA-6-N7Gua, showing the daughter ions at m/z 303, NADA-6-N7Gua minus the acetyl group, and m/z 152, Gua.

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M. Zahid et al. / Free Radical Biology & Medicine 48 (2010) 318–324
the depurinating NADA-6-N7Gua adduct. The reaction mixture was
analyzed after 3 h by direct infusion into the mass spectrometer. A
molecular ion [M]+ peak at m/z=461 (Fig. 4) showed the formation
of NADA-6-N7dG adduct that was selectively fragmented to its
daughter ion at m/z 345, corresponding to the depurinating NADA-
6-N7Gua adduct; m/z 303, corresponding to NADA-6-N7Gua minus an
acetyl group; and m/z 152, corresponding to the released Gua moiety.
Kinetics of loss of the deoxyribose moiety
Loss of deoxyribose from CAT-4-N7dG
The rate of loss of the deoxyribose moiety was studied by reacting
CAT-Q with dG and analyzing aliquots of the reaction mixture at
various time points. The time course of this reaction is represented in
Fig. 5 by plotting the concentrations of CAT-4-N7dG and CAT-4-
N7Gua over time. The maximum formation (6.4 1.1 mM) of the
CAT-4-N7dG adduct was obtained within 20 min, indicating that most
of the CAT-Q had reacted during this time. This adduct subsequently
released the deoxyribose moiety and was converted to CAT-4-N7Gua.
The concentration of CAT-4-N7Gua was minimal (1.6 0.3 mM) at
20 min, but slowly increased with time, indicating the slow
depurination of CAT-4-N7dG. The concentrations of the CAT-4-N7dG
and CAT-4-N7Gua were almost equal (4.4 0.6 and 4.3 0.8 mM,
respectively) at 2 h. By 12 h, almost complete conversion of CAT-4-
N7dG to CAT-4-N7Gua (9.5 1.4 mM) was observed, and after that
the amount of the depurinating adduct remained constant. The half-
life of CAT-4-N7dG conversion to CAT-4-N7Gua was approximately
3 h (Fig. 5).
Fig. 6. Time course of the reaction of NADA-Q with dG to form NADA-6-N7dG and its
conversion to NADA-6-N7Gua. The same reaction conditions as for Fig. 5 were used,
except that NADA was used instead of CAT.
Time course of the formation of depurinating adducts of CAT and NADA
in DNA
After finding the optimum conditions for the formation of
depurinating adducts, we conducted a time course study by reacting
CAT-Q or NADA-Q with DNA at pH 4. Aliquots of the reaction mixture
were analyzed at various time points. As shown in Fig. 7, depurination
of the N3Ade adduct (26.0 4.4 μmol/mol DNA-P) was almost
complete within 1 h of incubation time and its level remained
constant after that. On the other hand, depurination of the N7Gua
adduct was not instantaneous. A gradual increase in the level of the
N7Gua adduct formed was observed, starting with 3.4 0.1 μmol/mol
DNA-P at 1 h and reaching a maximum level of 12.1 1.4 μmol/mol
DNA-P at about 6 h.
In the reaction between NADA-Q and DNA, instantaneous
depurination of the N3Ade adduct was observed and its level of
14.8 0.7 μmol/mol DNA-P remained unchanged after 1 h (Fig. 8).
Depurination of the N7Gua adduct was slow. At 1 h, a minimal
amount, 1.2 0.1 μmol/mol DNA-P, of the N7Gua adduct was
observed in the reaction mixture, which continuously increased
until it reached its maximum level of 15.2 1.2 μmol/mol DNA-P at
5 h. The adduct level remained unchanged afterward (Fig. 8).
The half-life of CAT-4-N7dG and NADA-6-N7dG in DNA was
approximately 3 h.
Loss of deoxyribose from NADA-6-N7dG
In the reaction between NADA-Q and dG, the concentration of
NADA-Q decreased rapidly, with the concomitant formation of NADA-
6-N7dG. This adduct subsequently released the deoxyribose moiety
and was converted to the depurinating NADA-6-N7Gua adduct. The
concentration of the N7dG adduct increased initially and reached its
highest level of 1.8 0.6 mM at 2 h and then decreased continuously
until it reached a trace amount of b0.1 mM at 12 h (Fig. 6). On the
other hand, the amount of the N7Gua adduct was minimal (0.2
0.2 mM) at 30 min and then increased continuously until it reached
the maximum amount of 2.9 0.7 mM at 12 h. After 12 h, the adduct
concentrations remained constant as shown in Fig. 6. The half-life of
NADA-6-N7dG conversion to NADA-6-N7Gua was approximately 4 h
(Fig. 6).
Fig. 5. Time course of the reaction of CAT-Q with dG to form CAT-4-N7dG and its
conversion to CAT-4-N7Gua. CAT (0.05 M) was oxidized to CAT-Q with 3 M Ag2O in 2 ml
of DMF and filtered into 0.25 M dG in 2 ml of H2O:CH3COOH (1:1). The pH was adjusted
to 4 and the reaction continued at room temperature.
Fig. 7. Time course of the formation of CAT-4-N3Ade and CAT-4-N7Gua after the
reaction of CAT-Q with DNA at pH 4. CAT-Q (87 μM) was reacted with 3 mM DNA at pH
4 and room temperature.

M. Zahid et al. / Free Radical Biology & Medicine 48 (2010) 318–324
323
that of the N7Gua adduct to 3.3 0.3 μmol/mol DNA-P. At pH 6, the
level of CAT-4-N3Ade was 3 0.5 μmol/mol DNA-P, and CAT-4-
N7Gua, 1.5 0.4 μmol/mol DNA-P. At neutral pH, a sharp reduction
in the level of depurinating adducts was observed, giving a level of
1.1 0.3 μmol/mol DNA-P for the N3Ade adduct and 0.4 0.1 μmol/
mol DNA-P for N7Gua. Quite surprisingly, the N3Ade adduct was
formed in much higher amounts than the corresponding N7Gua
adduct (Fig. 9A). When tyrosinase-activated CAT was reacted with
DNA at various pH (5–8, Fig. 9B), the highest level of depurinating
adducts was observed at pH 5, 95 15 μmol/mol DNA-P for N3Ade
and 114 18 μmol/mol DNA-P for N7Gua. Again, at higher pH (6–8),
the depurinating adduct levels were reduced. However, slightly more
Gua than Ade adducts were detected at pH 5 and 6, which is reversed
for pH 7 and 8.
For NADA, the reaction between NADA-Q and DNA at pH 4 was
optimum with respect to the formation of depurinating adducts
(NADA-6-N7Gua and NADA-6-N3Ade, Fig. 10A). The N3Ade and
N7Gua adducts were detected in similar amounts, 16.4 3.2 and
13.8 2.7 μmol/mol DNA-P, respectively. By increasing the pH to 5,
the amounts of the depurinating adducts were lowered to half, with
the formation of 7.2 0.4 μmol/mol DNA-P for the N3Ade adduct and
7.7 0.3 μmol/mol DNA-P for the N7Gua adduct. At neutral pH, the
amount of the N3Ade adduct was 2.0 0.3 μmol/mol DNA-P and that
of the N7Gua adduct was 1.8 0.3 μmol/mol DNA-P. A similar trend
was seen when NADA was activated with tyrosinase in the presence
of DNA and the reaction was conducted at various pH (5–8, Fig. 10B).
Fig. 8. Time course of the formation of NADA-6-N3Ade and NADA-6-N7Gua after the
reaction of NADA-Q with DNA at pH 4. The same reaction conditions as for Fig. 7 were
used, except that NADA-Q was used instead of CAT-Q.
Formation of depurinating adducts of CAT and NADA at various pH
The effect of pH on the formation of depurinating adducts was
studied by reacting CAT-Q (or NADA-Q) or enzymatically activated
CAT (or NADA) with DNA. The level of the depurinating adducts was
measured by using UPLC-MS/MS. For CAT, the highest level (29.0
4.5 μmol/mol DNA-P for N3Ade and 15.4 2.6 μmol/mol DNA for
N7Gua) of depurinating adducts was detected at pH 4 (Fig. 9A).
When the pH of the reaction mixture was increased to 5, the level of
the N3Ade adduct was reduced to 10.1 2.1 μmol/mol DNA-P and
Discussion
Reaction of catechol quinones with nucleophiles occurs via a
proton-assisted 1,4-Michael addition [3]. In this article, we have
Fig. 9. Effect of pH on formation of the depurinating CAT-4-N3Ade and CAT-4-N7Gua
adducts by reaction of (A) CAT-Q with DNA for 10 h and (B) tyrosinase-activated CAT
with DNA for 10 h. (A) CAT-Q (87 μM) was reacted with 3 mM DNA at the indicated pH
and 37 °C or (B) 87 μM CAT was reacted with 3 mM DNA in the presence of 2577 units of
mushroom tyrosinase at the indicated pH and 37°C.
Fig. 10. Effect of pH on formation of the depurinating NADA-6-N3Ade and NADA-6-
N7Gua adducts by reaction of (A) NADA-Q with DNA for 10 h and (B) tyrosinase-
activated NADA with DNA for 10 h. The same reaction conditions as for Fig. 9 were used,
except that (A) NADA-Q or (B) NADA was used instead of CAT-Q or CAT.

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M. Zahid et al. / Free Radical Biology & Medicine 48 (2010) 318–324
compared the reaction of the catechol quinone of benzene, CAT-Q,
with dG or DNA to that of the catechol quinone of NADA, NADA-Q.
Reaction of the quinones with dG forms N7dG adducts that slowly
lose the deoxyribose moiety to form N7Gua adducts. We have
investigated the kinetics of this loss of the deoxyribose moiety for
both CAT-4-N7dG and NADA-6-N7dG at pH 4 (Figs. 5 and 6) and
observed the rate of disappearance of the N7dG adducts with
formation of the N7Gua adducts. The conversion of the CAT-4-N7dG
adduct to the N7Gua adduct had a half-life of approximately 3 h
(Fig. 5), whereas the conversion of the NADA-6-N7dG adduct had a
half-life of approximately 4 h (Fig. 6).
A further comparison of CAT-Q and NADA-Q was obtained by
reaction of the quinones with DNA at pH 4. With DNA, it was
observed that the CAT-4-N3Ade and NADA-6-N3Ade adducts were
formed and lost from DNA instantaneously, whereas the N7Gua
adducts were lost more slowly, with half-lives of approximately 3 h
(Figs. 7 and 8). With CAT-Q, the amount of the N3Ade adducts was
more than twice that of the N7Gua adduct after 10 h (Fig. 7). In
contrast, NADA-Q formed almost equal amounts of the N3Ade and
N7Gua adducts in 10 h (Fig. 8).
Finally, the formation of N3Ade and N7Gua adducts by reaction of
CAT-Q with DNA was studied at pH 4 to 7 (Fig. 9A). The maximum yield
of both adducts was observed at pH 4, whereas at pH 7 the yield was
negligible. In contrast, when tyrosinase-activated CAT was reacted with
DNA atpH 5 to 8 (Fig. 9B), the amounts of the N3Ade and N7Gua adducts
were about the same. The highest yield was at pH 5 and the yield
decreased as the pH increased. The amounts of the adducts were much
larger than those obtained from the reaction of the CAT-Q with DNA. For
example, at pH 5, the amounts of the adducts obtained from tyrosinase-
activated CAT were at least 10 times higher than those obtained with
CAT-Q (Figs. 9A and 9B). This is presumably due to the instability of the
quinone and the efficient reaction of the catechol after intercalation in
the DNA and activation to quinone by tyrosinase. This mechanism has
been demonstrated with the catechol of diethylstilbestrol [8].
When formation of N3Ade and N7Gua adducts by reaction of
NADA-Q with DNA was studied at pH 4 to 7 (Fig. 10A), the maximum
yield of both adducts was observed at pH 4 and decreased as the pH
rose from 5 to 7. Both adducts were observed at similar levels at all pH.
When NADA was activated by tyrosinase to react with DNA at pH 5 to
8, the amounts of adducts were at least 15 times higher than after the
direct reaction of NADA-Q with DNA (Fig. 10B). Again, this is
presumably due to the instability of NADA-Q and the efficient reaction
of NADA after intercalation into the DNA and activation to NADA-Q by
tyrosinase. The levels of the N3Ade and N7Gua adducts were not
significantly different from each other.
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Acknowledgments
This research was supported by Grants P01 CA49210 from the
National Cancer Institute and DAMD17-03-10229 from the Depart-
ment of Defense Breast Cancer Research Program and by Prevention
LLC. Core support at the Eppley Institute was provided by Grant P30
CA36727 from the National Cancer Institute.
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Toxicol. 19:164–172; 2006.