Synthesis of N2-(4-hydroxyphenyl)-2'-deoxyguanosine 3'-phosphate: Comparison by 32P-postlabeling with the DNA adduct formed in HL-60 cells treated with hydroquinone

Article abstract of DOI:10.1021/tx9500991

A new adduct has been isolated from the reaction of guanosine 3'- phosphate and p-benzoquinone. The structure of this adduct has been determined as N²-(4-hydroxyphenyl)guanosine 3'-phosphate. ³²P- Postlabeling showed that this adduct is similar to the DNA adduct formed in HL-60 cells treated with hydroquinone. For comparison with the corresponding deoxyribonucleotide, a synthetic procedure was developed for the preparation of N²-substituted derivatives of 2'-deoxyguanosine 3'-phosphate. 2-Bromo- 2'-deoxyinosine 3'-phosphate was synthesized with a combination of synthetic and enzymatic methods. Reaction of 2-bromo-2'-deoxyinosine 3'-phosphate with 4-hydroxyaniline gave N²-(4-hydroxyphenyl)-2'-deoxyguanosine 3'-phosphate. Using ³²P-postlabeling, we compared this product with the DNA adduct produced in HL-60 cells treated with hydroquinone. The results of these studies suggest that the DNA adduct formed in HL-60 cells treated with hydroquinone corresponds to N²-(4-hydroxyphenyl)2'-deoxyguanosine 3'- phosphate.

Full text of DOI:10.1021/tx9500991

Chem. Res. Toxicol. 1996, 9, 593-598
593
Syn th esis of N2-(4-Hyd r oxyp h en yl)-2′-d eoxygu a n osin e
3′-P h osp h a te: Com p a r ison by 32P -P ostla belin g w ith th e
DNA Ad d u ct F or m ed in HL-60 Cells Tr ea ted w ith
Hyd r oqu in on e
Krisztina Pongracz and William J . Bodell*
Brain Tumor Research Center, Department of Neurological Surgery, School of Medicine,
University of California, San Francisco, California 94143
Received J une 9, 1995X
A new adduct has been isolated from the reaction of guanosine 3′-phosphate and p-
benzoquinone. The structure of this adduct has been determined as N2-(4-hydroxyphenyl)-
guanosine 3′-phosphate. 32P-Postlabeling showed that this adduct is similar to the DNA adduct
formed in HL-60 cells treated with hydroquinone. For comparison with the corresponding
deoxyribonucleotide, a synthetic procedure was developed for the preparation of N2-substituted
derivatives of 2′-deoxyguanosine 3′-phosphate. 2-Bromo-2′-deoxyinosine 3′-phosphate was
synthesized with a combination of synthetic and enzymatic methods. Reaction of 2-bromo-2′-
deoxyinosine 3′-phosphate with 4-hydroxyaniline gave N2-(4-hydroxyphenyl)-2′-deoxyguanosine
3′-phosphate. Using 32P-postlabeling, we compared this product with the DNA adduct produced
in HL-60 cells treated with hydroquinone. The results of these studies suggest that the DNA
adduct formed in HL-60 cells treated with hydroquinone corresponds to N2-(4-hydroxyphenyl)-
2′-deoxyguanosine 3′-phosphate.
peroxidase activity and that treatment of cells with either
HQ or p-BQ results in the production of the same DNA
adduct. Recently, we have demonstrated that benzene
administration results in the formation of DNA adducts
in the bone marrow of male B6C3F1 mice (23). These
adducts were identical to those produced in HL-60 cells
or in mouse bone marrow treated in vitro with HQ or
p-BQ.
To characterize these DNA adducts, we have investi-
gated the DNA adducts formed by the reaction of p-BQ
with purified DNA. The DNA adducts formed were
identified as 3′-hydroxy-3,N4-benzetheno-2′-deoxycytidine
3′-phosphate, 3′-hydroxy-1,N6-benzetheno-2′-deoxyade-
nosine 3′-phosphate, and 3′-hydroxy-1,N2-benzetheno-2′-
deoxyguanosine 3′-phosphate (20, 24, 25). However,
these identified adducts were not the same as those
produced in HL-60 cells treated with HQ or p-BQ (20).
In this study, we further investigated the DNA adducts
produced in HL-60 cells treated with HQ or p-BQ.
In tr od u ction
Benzene is widely used in the chemical industry as an
intermediate in the production of other agents and as a
fuel additive; it is also found in automobile exhaust and
cigarette smoke (1). Thus, human exposure to benzene
occurs from both industrial and environmental sources
(1-3). Concern regarding this exposure arises from
evidence that acute exposure to benzene is leukemogenic
in humans (4, 5).
Benzene must be metabolized to exert its toxic and
leukemogenic effects (6-8). The principal metabolites
of benzenesphenol, hydroquinone (HQ),1 catechol, and
muconaldehyde (9-11)saccumulate in the bone marrow
(12, 13), where it is proposed that they are further
activated to produce the observed myelotoxic and leuke-
mogenic effects (8, 14). The enzymatic mechanisms
responsible for the activation of benzene metabolites in
bone marrow have not been elucidated; however, these
metabolites appear to be further activated by peroxidases
(15-17), including prostaglandin H-synthase (18, 19).
Our laboratory has been interested in the role of DNA
adduct formation by benzene metabolites in the myelo-
toxic effects of benzene exposure (19). In these studies
we have used HL-60 cells, a human promyelocytic cell
line, to investigate the activation of p-benzoquinone (p-
BQ), HQ, catechol, and 1,2,4-benzenetriol to form DNA
adducts (20-22). These studies have demonstrated that
activation of HQ to form adducts is dependent upon
Ma ter ia ls a n d Meth od s
1H NMR spectra were recorded on a QE-300 300 MHz
instrument (General Electric, Fremont, CA). Liquid secondary-
ionization mass spectra (LSIMS) were recorded on a Kratos MS-
50S mass spectrometer (Manchester, U.K.) equipped with a 23-
kg magnet and postaccelerator detector. A Cs+ primary electron
beam of 8 keV was used. Samples were run in a glycerol matrix
in the negative-ion mode. High resolution direct chemical
ionization (DCI) (NH4+) and electron impact (EI) mass spectra
were obtained on a VG 70-SE mass spectrometer (VG Analytical,
Ltd., Manchester, U.K.) at a source temperature of 180 °C
(Resolution 5000). Electrospray ionization (ESI) mass spectra
were recorded on a VG Biotech/Fisons Bio-Q instrument (VG
Analytical, Ltd., Manchester, U.K.) equipped with an electro-
spray ionization source operating in the negative-ion mode. The
ESI voltage and current were 5 kV and 20 mA, respectively,
and the capillary temperature was 250 °C. The UV absorption
spectra were recorded on a Beckman DU-7 spectrophotometer
(Beckman Instruments, Inc., Fullerton, CA).
* Correspondence and requests for reprints should be addressed to
this author at the University of California Brain Tumor Research
Center, Box 0806, San Francisco, CA 94143-0806. Tel: (415) 476-4899;
Fax: (415) 476-5799; e-mail: bodell@itsa.ucsf.edu.
X Abstract published in Advance ACS Abstracts, March 1, 1996.
1
Abbreviations: p-BQ, p-benzoquinone; HQ, hydroquinone; LSIMS,
liquid secondary ionization mass spectrometry; DCI MS, direct chemi-
cal ionization mass spectrometry; HR EI MS, high resolution electron
impact mass spectrometry; ESI, electrospray ionization; tR, retention
time; TSP, 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt; AU,
absorbance unit; PEI, poly(ethylenimine).
0893-228x/96/2709-0593$12.00/0 © 1996 American Chemical Society
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594 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
Pongracz and Bodell
HP LC An a lysis. Sample purifications were performed on
a Perkin-Elmer HPLC system equipped with an ISCO gradient
programmer and a 10 µm C-18 reversed-phase Econosil column
eluted with a linear gradient of 0-70% methanol for 30 min in
10 mM ammonium acetate (pH 5.1), at a flow rate of 1 mL/min.
UV absorption was monitored with a Perkin-Elmer LC-235
diode array detector.
ether. The ether extracts were dried with sodium sulfate and
evaporated. The product was dissolved in a small volume of
dichloromethane and slowly added to 200 mL of petroleum
ether. The precipitated product was further purified by column
chromatography using silica gel eluted with chloroform-
methanol (9:1). The collected fractions (Rf 0.4) were evaporated
and used for phosphorylation.
N2-(4-Hyd r oxyp h en yl)gu a n osin e 3′-P h osp h a te (1). Gua-
nosine 3′-phosphate (Sigma Chemical Co., St Louis, MO; 100
mg) and p-BQ (100 mg) were dissolved in 20 mL of 200 mM
HCl-KCl (pH 1) and incubated overnight at 37 °C. After
adjustment of pH to 6.5 with 1 N sodium hydroxide and addition
of 30 mL of water, the reaction mixture was loaded onto 5 BOND
ELUT cartridges (Analytichem International, Division of Vari-
an, Harbor City, CA), each eluted with 12 mL of 0.4 M sodium
phosphate (pH 6). The eluants were desalted on SEP PAK C-18
reversed-phase cartridges (Waters Division of Millipore, Milford,
MA). The products were eluted from the cartridges with 50%
methanol and evaporated. This prepurified material was
separated by HPLC as described above. The major product was
eluted at a retention time (tR) of 23 min. UV absorption
spectra: pH 6, UVmax 274 nm, UVmin 244 nm; pH 1, UVmax 275
nm, UVmin 245 nm; pH 12, UVmax 250, 272s nm, UVmin 233 nm.
1H NMR (recorded in D2O with 3-(trimethylsilyl)-1-propane-
sulfonic acid sodium salt (TSP) as internal standard) 8.011 (s,
1H, C8), 7.326 (dd, 2H, C-2, C-6, J 9.0 Hz), 6.952 (dd, 2H, C-3,
C-5), 5.907 (d, 1H, H-1′ J 5.9 Hz), 4.564 (m, 1H, H-2′), 4.282
(m, 1H, H-4′), 3.604 (m, 2H, H-5′) ppm. LSIMS m/ z [M - H]-
454.
Dep u r in a tion Stu d ies: N2-(4-Hyd r oxyp h en yl)gu a n in e
(2A). One absorbance unit (AU) of 1 was heated at 90 °C in 1
mL of 0.1 N HCl for 2 h. The product was purified by HPLC
and eluted at a tR of 27 min. The UV absorption spectra were
as follows: pH 6, UVmax 272 nm, UVmin 242 nm; pH 1, UVmax
268 nm, UVmin 242 nm. HR EI MS m/ z M+ 243.07553.
N2-(4-Hyd r oxyp h en yl)gu a n in e (2). 2-Bromo-6-hydroxy-
purine (216 mg, 1 mmol) and 4-hydroxyaniline (10 mmol) were
refluxed for 2 h in 5 mL of methoxyethanol-water (4:1) and
cooled. The resulting crystals were filtered and dried. With
HPLC, the product eluted at a tR of 27 min. 1H NMR (dimethyl
sulfoxide-d6) 10.365 (s, 1H, D2O exchangeable), 9.162 (s, 1H,
OH, D2O exchangeable), 8.270 (s, 2-NH, D2O exchangeable),
7.741 (s, 1H C8), 7.324 (dd, 2H, C-2, C-6 J 8.4 Hz), 6.707 (dd,
2H, C-3, C-5) ppm. DCI MS m/ z [M + H]+ 244. UVmax 271
(pH 6), 267 (pH 1), 244, 270 nm (pH 12).
2-Br om o-2′-d eoxyin osin e 3′-P h osp h a te (5). 4 was dried
by repeated coevaporations from dry pyridine. Ten milliliters
of phosphorylating agent (310 mg of triazole, 140 µL of phos-
phoryl chloride, 340 µL of triethylamine, 10 mL of dioxane, 5
°C, 1 h, filtered) was added, and the solution was stirred for 1
h (28). Water (5 mL) was added, and the solution was stirred
for 15 min. After the addition of 20 mL of phosphate buffer,
the product was extracted three times with 50 mL of chloroform,
and the collected organic phases were evaporated to dryness.
Twenty milliliters of 80% acetic acid was added; the solution
was kept at room temperature for 20 min and was then carefully
evaporated (30 °C). The dry residue was dissolved in 100 mL
of water, the pH of the solution was adjusted to 7 with diluted
ammonium hydroxide, and in a sepratory funnnel it was washed
twice with 50 mL of diethyl ether. The volume of the water
phase was reduced to 50 mL and analyzed by HPLC. The tR of
5
was 20.5 min. The concentration of this solution was
estimated by measurement at 260 nm using the relationship
25 µg/AU, and this value was used for reactions with the
aminophenols. Samples for mass spectrometry were lyophilized.
LSIMS m/ z [M - H]- 409, 411. UVmax 257 (pH 6), 254 (pH 1),
259 nm (pH 12).
N2-(4-Hyd r oxyp h en yl)-2′-d eoxygu a n osin e 3′-p h osp h a te
(6). 5 (40 µmol) was reacted with 400 µmol of 4-hydroxyaniline
in 2 mL of water at 37 °C for 24 h. The reaction mixture was
loaded onto four previously washed SEP PAK cartridges, and
the products were eluted with 50% methanol. The end product
was purified by HPLC as described above. N2-(4-Hydroxyphen-
1
yl)-2′-deoxyguanosine 3′-phosphate eluted at a tR of 23 min. H
NMR (D2O) 8.036 (s, 1H, C8), 7.434 (dd, 2H, C-2, C-6, J 8.7 Hz),
7.014 (dd, 2H, C-3, C-5), 6.382 (t, 1H, H-1′, J 7.0 Hz), 4.776 (m,
1H, H-3′), 4.333 (m, 1H, H-4′), 3.864 (m, 2H, H-5′), 3.171, 2.778
(m, m, 2H, H-2′) ppm. ESI MS m/ z [M - H]- 438.
One AU of 6 was depurinated in 0.1 N HCl at 90 °C for 20
min. The product eluted at a tR of 27 min, and the UV spectra
were identical with the UV spectra of 2.
In Vitr o Stu d ies. HL-60 cells were cultured as previously
described (20-22). The cultures were incubated at 37 °C in a
humidified 5% CO2/95% air atmosphere. HQ (Aldrich Chemical
Co., Milwaukee, WI) was dissolved in distilled water and added
directly to the culture medium to achieve the desired final
concentrations. After treatment, the HL-60 cells were centri-
fuged at 3000g for 10 min, washed with cold Hanks’ balanced
salt solution (Ca2+ -Mg2+-free), recentrifuged at 3000g for 10
min, and stored at -70 °C until the DNA was isolated.
32P -P ostla belin g. The DNA was isolated from treated cells
as previously described (29). The P1 nuclease enhanced 32P-
postlabeling technique was used to analyze the DNA adducts
formed in HL-60 cells (20-22). N2-(4-Hydroxyphenyl)guanosine
3′-phosphate, N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phos-
phate, and the HPLC fraction containing the cellular adduct
were labeled directly using T4 kinase. The radioactive materials
were applied to 10 × 10 cm poly(ethylenimine) (PEI)-cellulose
sheets. The PEI plates were developed in the following solvent
systems: D0, 0.4 M sodium phosphate buffer (pH 6.8); D1, 1.8
M lithium formate and 4.5 M urea (pH 3.5); D2, 0.36 M lithium
chloride, 0.22 M Tris, and 3.8 M urea (pH 8); D3, 1.7 M sodium
phosphate (pH 6). The adducts were located by autoradiography
using Kodak XAR-5 film (Kodak, Rochester, NY) and a DuPont
Chronex Lightning Plus intensifying screen (DuPont, Boston,
MA) The areas corresponding to adducts were scraped into
scintillation vials containing 5 mL of scintillation cocktail
(Safety Solve, Research Products Inc., Mount Prospect, IL), and
the radioactivity was determined by scintillation counting. The
relative adduct levels were calculated as previously described
(20-22).
2-Br om o-2′-d eoxyin osin e (3). 2-Bromo-6-hydroxypurine
(216 mg; 26, 27) and thymidine (242 mg) were dissolved in 100
mL of 20 mM potassium phosphate buffer (pH 7.4) and
incubated overnight at 37 °C with 60 units of thymidine
phosphorylase (EC 2.4.2.4, Sigma) and 90 units of purine
nucleotide phosphorylase (EC 2.4.2.1, Sigma). After evaporation
to dryness, the residue was dissolved in 50 mL of water and
filtered. The solution was placed in a 250 mL round-bottom
flask with 10 g of silica gel and evaporated to dryness. A silica
gel column (2.5 × 25 cm) was prepared with chloroform-
methanol-triethylamine (80:15:5) as the solvent, and the silica
gel containing the reaction products was placed on top of the
column. The same solvent system was used to elute the
products. 2-Bromo-2′-deoxyinosine eluted in the late fractions
of the chromatography (280 mg, 84%). The tR of this compound
1
was 23.7 min. LSIMS m/ z [M + H]+ 331, 333; H NMR (D2O)
8.066 (s, 1H, C8), 6.339 (t, 1H, H-1′), 4.638 (m, 1H, H-3′), 4.176
(m, 1H, H-4′), 3.828 (m, 2H, H-5′), 2,782 and 2.535 (m, 2H H-2′)
ppm. UVmax 252 nm (pH 1), 254 nm (pH 6), 258 nm (pH 12).
5′-DMT-2-br om o-2′-d eoxyin osin e (4). 3 (280 mg) was
dried with repeated evaporations from dry pyridine and dis-
solved in 20 mL of pyridine. Dimethoxytrityl chloride (400 mg),
(dimethylamino)pyridine (10 mg), and triethylamine (500 µL)
were added, and the reaction mixture was stirred at room
temperature overnight. The reaction was followed by thin-layer
chromatography using chloroform-methanol (9:1) as the solvent
system. After the addition of 20 mL of water to the reaction
mixture, the product was extracted three times with 80 mL of
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DNA Adduct Formed in HL-60 Cells Treated with HQ
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 595
F igu r e 1. UV spectra of N2-(4-hydroxyphenyl)guanosine 3′-
phosphate, recorded at pH 1, pH 6, and pH 12.
Rech r om a togr a p h y of 32P -P ostla beled Ad d u cts. For
rechromatography experiments, the postlabeled adducts were
excised from the PEI-cellulose plates, and the cellulose was
extracted at room tempreature with 200 µL of 1.5 M pyri-
dinium formate (pH 4.5), for 1 h. The solution was evapo-
rated, and the residue was dissolved in 20 µL of water and
spotted on another PEI-celluose sheet. The plates were sub-
sequently developed in 0.5 M sodium phosphate and 3 M urea
(pH 5).
HP LC Isola t ion of Cellu la r DNA Ad d u ct . HL-60 cells
were treated with HQ as described above. The DNA was
subsequently isolated and purified. DNA (50 µg) was digested
with 80 µg of spleen phosphodiesterase and 80 µg of micrococcal
nuclease in 60 µL of 20 mM sodium succinate and 8 mM CaCl2
(pH 6), for 2 h at 37 °C. The sample was diluted with water to
1 mL and heated at 100 °C for 90 s. After cooling, the sample
was filtered with Millex GSO 0.22 µm filters (Millipore, Bedford,
MA) before injection. The sample was separated by HLPC as
described above. The eluant between 21 and 25 min was
collected and lyophilized. The residue was dissolved in 10 µL
of water and 32P-postlabeled.
F igu r e 2. 1H NMR spectrum of N2-(4-hydroxyphenyl)guanosine
3′-phosphate, recorded in deuterium oxide with TSP as internal
standard.
N2-(4-Hydroxyphenyl)guanine was also prepared from
2-bromo-6-hydroxypurine and 4-hydroxyaniline. 1H NMR
analysis of this product confirmed the structure. Com-
parison of the UV spectrum and the HPLC tR of N2-(4-
hydroxyphenyl)guanine with those of the product derived
from depurination of N2-(4-hydroxyphenyl)guanosine 3′-
phosphate showed them to be the same. These results
further establish the structure of the p-BQ-guanosine
reaction product as N2-(4-hydroxyphenyl)guanosine 3′phos-
phate.
Previous studies have concluded that the DNA adducts
formed in HL-60 cells treated with either HQ or p-BQ
do not correspond to the benzetheno adducts previously
identified (19, 20). Figure 3A shows the results of 32P-
postlabeling analysis of DNA isolated from HL-60 cells
treated with 250 µM HQ for 16 h. Under these treatment
conditions, one principal (1) and two minor (2 and 3) DNA
adducts were detected; the relative adduct level was 4.4
Resu lts
From the reaction of guanosine 3′-phosphate with p-BQ
at pH 1, a new product was isolated. The UV absorption
spectra of this compound are shown in Figure 1. The
neutral and the acidic spectra are similar to those
reported for N2-(p-n-butylphenyl)-2′-deoxyguanosine (30).
The molecular weight of the compound was 455 (LSIMS
m/ z [M - H]- 454), indicating the presence of a hydroxy-
phenyl group as the substituent modifying guanosine 3′-
phosphate. The exact position of the hydroxyl group on
× 10-7
.
N2-(4-Hydroxyphenyl)guanosine 3′phosphate
was 32P-postlabeled and chromatographed in a similar
fashion (Figure 3B). Comparison of these results sug-
gested that the principal adduct formed in HL-60 cells
treated with HQ was similar to N2-(4-hydroxyphenyl)-
guanosine 3′-phosphate.
1
the phenyl ring was determined by H NMR (Figure 2).
The aromatic protons of the phenyl ring appeared as two
distorted doublets, characteristic of an AA′XX′ coupling
system. These results indicated that the position of the
hydroxyl group is para to the N2 amino group of gua-
nosine. Therefore, we assigned the structure of N2-(4-
hydroxyphenyl)guanosine 3′-phosphate to the product.
N2-(4-Hydroxyphenyl)guanosine 3′-phosphate was depu-
rinated by acid hydrolysis, and the product was purified
by HPLC. EI MS of the depurinated product determined
the molecular weight as 243.07553, which corresponds
to elemental composition C11H9N5O2. Two fragments
with exact masses of 134.04782 (C7H6N2O) and 110.03628
(C4H4N3O) resulted from fragmentation of the six-
membered purine ring. These results are consistent with
the structure of N2-(4-hydroxyphenyl)guanine for the
depurinated product.
For comparison with the cellular adduct, it was neces-
sary to synthesize the 2′-deoxyguanosine derivative.
Enzymatic coupling procedures have been described to
prepare both modified and unmodified deoxyribonucle-
otides (31, 32). We have developed a method for prepar-
ing N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phos-
phate; the synthetic scheme is shown in Figure 4.
2-Bromo-6-hydroxypurine was prepared using published
procedures (26, 27). Enzymatic coupling of this com-
pound to deoxyribose resulted in 2-bromo-2′-deoxyinosine;
the expected molecular weight of 331 was confirmed by
MS (LSIMS m/ z [M + H]+ 331, 333) (data not shown).
Dimethoxytritylation and phosphorylation followed by
mild acid treatment gave 2-bromo-2′-deoxyinosine 3′-
phosphate. Reaction of 2-bromo-2′-deoxyinosine 3′-
phosphate with 4-hydroxyaniline produced the expected
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596 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
Pongracz and Bodell
PEI plates. As shown in Figure 6, the DNA adduct
formed in HL-60 cells treated with HQ has the same
chromatographic properties as N2-(4-hydroxyphenyl)-
2′-deoxyguanosine 3′-phosphate when analyzed by a
combination of HPLC and PEI-cellulose chromatogra-
phy.
Discu ssion
In these studies we have investigated the reaction
of p-BQ with guanylic acid at different pH levels. Gua-
nylic acid was chosen due to its stability at acidic pHs.
At pH 1, a new product was detected. Spectroscopic
analysis showed that the structure of this product is
N2-(4-hydroxyphenyl)guanosine 3′-phosphate. The struc-
ture of the depurinated product was confirmed by com-
parison with an independently prepared sample of N2-
(4-hydroxyphenyl)guanine. This product may form from
the reaction of the N2 amino group of guanosine with
one of the carbonyls of p-BQ, as has been observed in
the reaction of primary amines with estrogen o-quinones
(33, 34).
In our previous studies of the reaction of p-BQ with
DNA at neutral pH, the principal products detected by
postlabeling were 3′-hydroxy-3,N4-benzetheno-2′-deoxy-
cytidine 3′-phosphate; 3′-hydroxy-1,N6-benzetheno-2′-
deoxyadenosine 3′-phosphate, and 3′-hydroxy-1,N2-ben-
zetheno-2′-deoxyguanosine 3′-phosphate (20, 24, 25). In
the present investigation, in which the pH of the reaction
mixture was raised above 3.5, the previously identified
cyclic guanosine adduct 3′-hydroxy-1,N2-benzethenogua-
nosine 3′-phosphate became dominant.2
In previous investigations several modified deoxyribo-
nucleotides and the corresponding ribonucleotides have
been prepared (24).2 Both sets of adducts were 32P-
postlabeled, and the chromatographic properties on PEI-
cellulose sheets were analyzed. These experiments dem-
onstrated that the bisphosphate derivative of a 2′-
deoxyribonucleotide or ribonucleotide with the same
modified base structure chromatographed similarly on
PEI-cellulose plates. We observed that 32P-postlabeled
N2-(4-hydroxyphenyl)guanosine 3′-phosphate was very
similar to the 32P-postlabeled adduct from HL-60 cells
treated with p-BQ.
Because of the general acid lability of deoxyguanylic
adducts, it was necessary to develop an alternative
method for preparing the corresponding N2-(4-hydroxy-
phenyl)-2′-deoxyguanosine 3′-phosphate. 2-Bromo- or
2-fluoro-2′-deoxyinosine is the most suitable starting
material for the syntheses. However, all published
methods for preparing these compounds involve several
steps and the use of protecting groups (35-37). 2-Fluoro-
2′-deoxyinosine was reported in the syntheses of N2-
modified guanosine derivatives, but this compound had
a low reactivity with 4-hydroxyaniline.2 The synthesis
of 2-bromo-2′-deoxyinosine via the O6-methyl derivative
has been described (38), and a stereoselective method
using 2-bromo-6-hydroxypurine as the starting material
has been developed (39). Recently, Chapeau and Marnett
(32) described an enzymatic method for preparing de-
oxyribonucleoside adducts from the modified base. We
developed a chemical-enzymatic strategy for the prepa-
ration of 2-bromo-2′-deoxyinosine 3′-phosphate. This
compound would be a useful intermediate not only in the
preparation of N2-(4-hydroxyphenyl)-2′-deoxyguanosine
F igu r e 3. (A) Autoradiogram from 32P-postlabeling analysis
of DNA isolated from HL-60 cells treated with 250 µM hydro-
quinone for 16 h. The film was exposed at -70 °C for 2 h. (B)
32P-Postlabeling analysis of N2-(4-hydroxyphenyl)guanosine 3′-
phosphate.
productsN2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phos-
phate, as determined by ESI MS m/ z [M - H]- 438. The
UV spectra of this product were identical to the UV
spectra of N2-(4-hydroxyphenyl)guanosine 3′-phosphate,
and depurination of the compound produced N2-(4-
hydroxyphenyl)guanine (results not shown).
N2-(4-Hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate
was 32P-postlabeled and chromatographed on PEI-cel-
luose sheets (Figure 5A). The chromatographic proper-
ties of the bisphospate derivative were similar to those
previously observed for the ribo derivative (Figure 3B).
DNA isolated from HL-60 cells treated with HQ was
postlabeled, the adducts were resolved on PEI-cellulose
sheets and located by autoradiography, and DNA ad-
duct 1 was eluted from the PEI-cellulose sheets. Simi-
lar procedures were applied to 32P-postlabeled N2-(4-
hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. Rechro-
matography experiments to compare the adducts showed
that DNA adduct 1 formed in HL-60 cells treated with
HQ has the same chromatographic properties as N2-
(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate (Fig-
ure 5B).
To confirm these observations, we performed an ex-
periment that combined HPLC separation with 32P-
postlabeling. DNA isolated from HL-60 cells treated with
HQ was enzymatically digested to 3′-monophosphates.
The mononucleotides were chromatographed using the
same HPLC procedures used to purify N2-(4-hydroxy-
phenyl)-2′-deoxyguanosine 3′-phosphate. The eluant be-
tween 21 and 25 min after injection was collected,
evaporated, 32P-postlabeled, and chromatographed on
2
K. Pongracz, unpublished results.
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DNA Adduct Formed in HL-60 Cells Treated with HQ
Chem. Res. Toxicol., Vol. 9, No. 3, 1996 597
F igu r e 4. Synthetic scheme for the preparation of N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. DMT ) dimethoxytrityl;
TPase ) thymidine phosphorylase; PNPase ) purine nucleotide phosphorylase.
F igu r e 6. Autoradiogram of 32P-postlabeling analysis of HPLC-
enriched fractions (21-25 min) from enzymatic digestion of HL-
60 cells treated with hydroquinone. The films were exposed for
30 min at -70 °C.
yl)-2′-deoxyguanosine 3′-phosphate with the DNA adduct
formed in HL-60 cells treated with either p-BQ or HQ.
In previous studies we have demonstrated that the
principal adduct formed in HL-60 cells treated with
either HQ or p-BQ was the same (20). The results of the
present study suggested that the adduct formed in HL-
60 cells treated with these agents was N2-(4-hydroxy-
phenyl)-2′-deoxyguanosine 3′-phosphate. Further evi-
dence for this conclusion was obtained by HPLC separa-
tion of deoxyribonucleotides produced by enzymatic
digestion of DNA isolated from treated HL-60 cells. 32P-
Postlabeling of the fraction with the same retention time
as N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate
led to the detection of the same adduct as with P1-
enhanced 32P-postlabeling. These results taken together
suggest that the adduct formed in HL-60 cells treated
with p-BQ or HQ corresponds to N2-(4-hydroxyphenyl)-
2′-deoxyguanosine 3′-phosphate. Additional proof of this
conclusion will require direct structural analysis of the
DNA adduct formed in these treated cells (40).
F igu r e 5. (A) Autoradiogram of 32P-postlabeling of N2-(4-
hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. (B) Rechro-
matography analysis of N2-(4-hydroxyphenyl)-2′-deoxyguanosine
3′-phosphate with adduct 1 formed in HL-60 cells treated with
HQ: Lane a, DNA adduct 1; lane b, 1:1 mixture of DNA adduct
1 and N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate;
lane c, N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate.
The exposure times for (A) and (B) were 10 min at room
temperature and overnight at -70 °C, respectively.
3′-phosphate but also in the preparation of a series of
N2-modified deoxyguanosine 3′-phosphate derivatives for
32P-postlabeling analysis.
Using this procedure, we successfully prepared N2-
(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. The
structure of the product was confirmed by ESI MS.
Using 32P-postlabeling, we compared N2-(4-hydroxyphen-
Ack n ow led gm en t. This work was supported by
Grant P42ES04705 from the National Institute of Envi-
ronmental Health Sciences. The authors thank Dr. G.
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598 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
Pongracz and Bodell
(20) Levay, G., Pongracz, K., and Bodell, W. J . (1991) Detection of DNA
adducts in HL-60 cells treated with hydroquinone and p-benzo-
quinone by 32P-postlabeling. Carcinogenesis 12, 1181-1186.
(21) Le´vay, G., and Bodell, W. J . (1992) Potentiation of DNA adduct
formation in HL-60 cells by combinations of benzene metabolites.
Proc. Natl. Acad. Sci. U.S.A. 89, 7105-7109.
(22) Le´vay, G., Ross, D., and Bodell, W. J . (1993) Peroxidase activation
of hydroquinone results in the formation of DNA adducts in HL-
60 cells, mouse bone marrow macrophages and human bone
marrow. Carcinogenesis 14, 2329-2334.
Le´vay for treatment of the HL-60 cells, Cheryl Chris-
tensen for manuscript preparation, and Stephen Ordway
for editorial assistance. Mass spectrometry was per-
formed at the Mass Spectrometry Facility, University of
California San Francisco, which is supported by Grant
NIH NCRR BRTP RRO1614.
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