higher than that of FapydGuo 4. This may be accounted for by
a lower ability of HPT than ascorbate or cysteine to reduce the
8-hydroxy-7,8-dihydroguanin-7-yl radical.
nium formate and methanol. The amount of NQS derivative of
guanidine (Rt: 11.3 min) was inferred from a calibration curve
established with derivatized authentic guanidine. FapydGuo 4
and free guanine 6 (Gua) were measured by GC-MS. An
aliquot of the solution of dGuo 1 (50 µL) was dried under
vacuum together with 10 µL of a 66 µM solution of [15N3]Fapy-
Gua. The resulting residue was solubilized in 150 µL of 88%
formic acid. The samples were left 15 min at room temperature
and then evaporated to dryness under vacuum. Water (50 µL)
was added and the solution dried under vacuum. The latter step
was resumed once. The dry residue was then silylated in 100 µL
of a [1:1] mixture of N-bis(trimethylsilyl)trifluoroacetamide ϩ
1% trimethylsilyl chloride (BSTFA, Aldrich, Milwaukee, WI)
and acetonitrile for 25 min at 110 ЊC. The resulting solution was
evaporated to dryness and silylated. Samples were analysed on
a GC-MS apparatus consisting of a HP 5890 chromatograph
and a MSD 5972 mass detector used in the single ion monitor-
ing mode. Samples (1 µL) were injected in the splitless mode
onto an Optima-5 column (25 m; 0.2 mm id; 0.1 µm film thick-
ness) from Macherey-Nagel (Düren, Germany). The tempera-
ture of the injector was 250 ЊC. The column was maintained at
110 ЊC for 1 min. Then, the temperature was linearly increased
to 280 ЊC at a rate of 25 ЊC minϪ1. The ions collected were the
following: FapyGua (retention time 5.95 min): m/z = 457
(FapyGua ϩ 4 TMS), 442 (FapyGua ϩ 4 TMS Ϫ methyl), 460
([15N3]FapyGua ϩ 4 TMS), 445 ([15N3]FapyGua ϩ 4 TMS Ϫ
methyl). For the measurement of guanine, 9 µL of a 57 µM
solution of [15N3,13C]Gua was added to 50 µL of the dGuo
solution. The sample was evaporated to dryness, silylated by
BSTFA and analysed by GC-MS. The ions collected were
(retention time 6.01 min): m/z = 367 (Gua ϩ 3 TMS), 352
(Gua ϩ 3 TMS Ϫ methyl), 371 ([15N3,13C]Gua ϩ 3 TMS), 356
([15N3,13C]Gua ϩ 3 TMS Ϫ methyl).
Experimental
Chemicals
2Ј-Deoxyguanosine (dGuo) was obtained from Pharma-
Waldhof. -Cysteine, ascorbic acid, N-hydroxypyridine-2-
thione (HPT) and 1,2-naphthoquinone-4-sulfonic acid (NQS)
were purchased from Sigma (St. Louis, MO). Analytical grade
formic acid (99%) was obtained from Merck (Darmstadt,
Germany).
Gamma irradiation and HPT-mediated photodegradation of
dGuo
Fresh stock solutions of cysteine and ascorbic acid (10 mM,
pH 7) were prepared before each series of irradiations. The
required volume of the stock solutions of reducing agents and
water were added to 2.7 mL of a 1 mM solution of dGuo (final
volume 3 mL) through which air was bubbled for 1 h. The final
concentrations in cysteine and ascorbate were 0, 0.01, 0.03, 0.1,
0.3 and 1 mM. Aqueous solutions of dGuo containing either 1
mM HPT, 10 mM HPT or 1 mM UVB-degraded HPT were
also prepared. The samples were then exposed under constant
air saturation to the gamma rays emitted by a 60Co source. The
dose rate was 20 Gy minϪ1. Aliquot fractions (1 mL) were col-
lected after 0, 15 and 30 min of irradiation. All experiments
were duplicated with new solutions of all reagents. A 1 mM
solution of dGuo containing 10 mM HPT was exposed for
increasing periods of time to the 300 nm light emitted by a
Rayonet photoreactor (The Southern New England Ultraviolet
Company, Hamden, CT) equipped with ten 15 W lamps.
Irradiation times were 0, 20, 32, 45 and 60 min. Air bubbling
was maintained during the experiment.
Determination of the radiolytic degradation yield of the
nucleosides
Aqueous solutions (5 mL, 1 mM) of either 2Ј-deoxycytidine,
thymidine or dGuo were exposed to the γ-rays emitted by the
60Co source under constant air bubbling. After increasing
periods of time (0, 15, 30 and 60 min) aliquot fractions (0.5 mL)
were collected. Each fraction was injected on the HPLC system
described above. Content of nucleoside was inferred from the
area of the peaks observed on the UV chromatogram. The deg-
radation was found to be linear over the dose range studied.
Radiolytic degradation yields were calculated from the slopes
obtained by linear regression. Each experiment was done in
triplicate.
Analysis of modified bases
8-OxodGuo 3 was measured by HPLC-EC. The analytical sys-
tem consisted of a model 2150 LKB pump (Pharmacia LKB
Biotechnology, Uppsala, Sweden) connected to a SIL-9A
autosampler (Shimadzu, Kyoto, Japan) equipped with an
Uptisphere ODSB (particle size 5 µm) octadecylsilyl silica gel
column (250 × 4.6 mm id) (Interchim, Montluçon, France). The
isocratic eluent was a 50 mM aqueous solution of potassium
phosphate (pH 4.7) containing 8% methanol. Coulometric
detection of 8-oxodGuo 3 was provided by a Coulochem II
detector equipped with a 5010 cell (ESA, Chelmsford, MA) with
the potentials of the two electrodes set at 200 and 400 mV. The
retention time of 8-oxodGuo 3 was 17 min. Elution of dGuo
was simultaneously monitored by a Waters 484 UV variable
wavelength spectrophotometer set at 280 nm. Both EC and UV
signals were collected on a D7500 Hitachi integrator (Tokyo,
Japan). Oxazolone 2 and its imidazolone precursor were meas-
ured by HPLC-fluorescence following decomposition under
alkaline conditions. Typically, the irradiated solution of dGuo 1
(50 µL) was placed in a polypropylene vial together with 50 µL
of 1 M sodium hydroxide. After homogenization, the solution
was held at 70 ЊC for 30 min in a water bath. Then, the sample
was removed and cooled down to room temperature. An aque-
ous solution of NQS (10 µL, 8 mg mLϪ1) was added and the
resulting solution incubated for 30 min in a water bath at 37 ЊC.
After cooling to room temperature, the sample was neutralised
by addition of 50 µL of 1 M hydrochloric acid. The solution
was then analysed with the HPLC system described previously,12
using the fluorescence detection provided by a F-1050 fluori-
meter (Hitachi, Tokyo, Japan) with the excitation and emission
wavelengths set at 355 and 405 nm, respectively. The eluent was
a [90:10] v/v mixture of a 25 mM aqueous solution of ammo-
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