Chemical Research in Toxicology
Article
HPLC-QTOF/MS analysis of aliquots taken from the reaction
mixtures.
N7-GHESEHG-N7. ESI+-MS/MS, m/z 421 [M + H]+, m/z 389 [M +
H − 2O]+, m/z 270 [M + H − Guo]+, m/z 211 [Guo + CH2CH2S]+,
l
2.6. Preparation of DVS-Adducts Reference Standards.
2.6.1. Reaction of DVS with Adenosine or Guanosine. Adenosine
(10.68 mg, 0.04 mmol) or guanosine (11.32 mg, 0.02 mmol) was
dissolved in 10 mL of deionized water containing 100 μL of formic
acid, and 112 mg of DVS (1.12 mmol) was added to the solution. The
resulting reaction mixture was adjusted to pH 7.4 using disodium
hydrogen phosphate and stirred at 37 °C for 12 h. After reacting, the
mixture was added into 2 M HCl aqueous solution (10 mL) to
undergo depurination, heated at 100 °C for 30 min, and then cooled
to room temperature. The reaction mixture was diluted and subjected
to analysis by HPLC-QTOF/MS.
2.6.2. Preparation of the Monoadducts. Adenosine or guanosine
(0.2 g, 0.7 mmol) was suspended in acetic acid (5 mL), and 0.4 mmol
DVS was added dropwise to the suspension. The resulting reaction
mixture was stirred at 60 °C for 24 h then cooled to room
temperature. The unreacted nucleoside was filtrated and discarded,
and the filtrate was evaporated to dryness. The residue was dissolved
of 1 M HCl aqueous solution (20 mL). After extraction of unreacted
DVS with dichloromethane (3 × 10 mL), the aqueous solution was
heated at 100 °C for 1.5 h, and a pale-yellow clear solution was
obtained, cooled, and neutralized with concentrated ammonia. A white
crude product was achieved. After the mixture was filtered, the
resultant precipitate was redissolved in a small amount of acidic water
and then purified by a preparative liquid chromatograph equipped with
an ultraviolet (UV) detector (Unisil 10-120 C18 column 21.2 mm ×
250 mm I.D. 10 μm; water as mobile phase A, methanol as mobile
phase B, using gradient elution program, flow rate of 15 mL/min). The
yield of 31% was obtained with more than 96% purity. All purities of
the synthesized adducts were checked by HPLC-UV detection with an
area normalization method.
2.6.3. Preparation of Cross-Linked Adducts. The cross-linked
adducts were prepared based on the monoadducts as the following:
DVS-A or DVS-G was first dissolved in PBS buffer (50 mM, pH 7.4),
and guanosine or adenosine was added at room temperature,
respectively. The reaction mixture was stirred at 55 °C for 1 h, and
the solution was dissolved using 1 M HCl aqueous solution to undergo
hydrolysis. The residue was purified using preparative liquid
chromatography as mentioned above. Molecular structures of all
synthesized DVS-DNA adducts are shown in Figure 1.
2.7. Identification and Characterization of Adducts by NMR
and LC-HRMS. All synthesized DVS-DNA adducts were characterized
by LC-HRMS or NMR analysis. The samples were directly delivered
to a QTOF (Waters, Xevo G2, USA) mass spectrometer for analysis.
In the QTOF/MS analysis, the equipment was operated in a positive
ion electrospray (ESI+) mode, and the scanning mode was MSE. The
source conditions were: capillary voltage, 3 kV; cone voltage, 15 V;
extraction voltage, 4 V; ion source temperature, 100 °C; desolvation
temperature, 300 °C; low collision energy, 6 eV; high collision energy,
10−40 eV; resolution, 10000 fwhm. In addition, 1HNMR spectra were
collected on a Bruker AV 400 MHz NMR spectrometer. The
characterization results are as follows:
m/z 151 [Guo]+. H NMR (DMSO-d6, 400 MHz) δ 8.21 (s, 2H,
−CH), 4.46(s, 4H, −NH2), 3.78 (m, 4H, −CH2). HRMS (Q-TOF),
obsd m/z 421.1154, calcd for C14H16N10O4S, m/z 421.1155 [M + H]+.
Retention time is 4.0 min.
N3-AHESEHG-N7. ESI+-MS/MS, m/z 405 [M + H]+, m/z 203 [Guo
l
+ CH2CH2 + Na]2+. H NMR (DMSO-d6, 400 MHz) δ 8.18, (s, 2H,
-NH2), 8.11 (s, 2H, −NH2), 4.46 (m, 2H, −CH), 3.97 (m, 2H, −CH),
3.76 (s, 2H, −CH), 3.52 (m, 2H, −CH2). HRMS (Q-TOF), obsd m/z
405.1165, calcd for C14H16N10O3S, m/z 405.1206 [M + H]+.
Retention time is 4.5 min.
2.8. Reaction of DVS with dsDNA. Incubations of DVS with
salmon sperm DNA were performed in a final volume of 5 mL
containing 112 mg of DVS, 10 mg salmon sperm DNA, and 50 mM
phosphate buffer at pH 7.4. Reactions were performed at 55 °C for 24
h in the water bath. The unreacted DVS was removed by repetitive
extraction with cold anhydrous ethanol (2.5 volumes) and washed
three times with 70% ethanol (v/v). Then, the DNA pellet was
precipitated from the aqueous layer and subsequently hydrolyzed by
15% formic acid at 75 °C for 30 min. The supernatant was diluted and
subjected to the analysis by HPLC-QTOF/MS.
2.9. Cell Culture. The human keratinocyte cell line HaCaT was
provided by the Shanghai Cell Bank of the Chinese Academy of
Sciences and all maintained in a standard culture medium. HaCaT
cells were maintained in complete DMEM culture medium with 4.5 g/
L glucose supplemented with 10% FBS (Gibco, NY, USA). All cultures
were maintained at 37 °C in a humidified incubator in 5% CO2.
HaCaT cells were individually plated at 3 × 105 cells per 10 cm dish
and incubated overnight. DVS was initially diluted with DMSO and
subsequently diluted to 30 μM with culture medium for cell exposure,
in which the final concentration of DMSO was strictly restricted to
<0.1%. After incubation for 24 h at 37 °C, the cells were washed three
times with 1 × PBS and harvested for extract DNA.
2.10. Identification of DNA Adducts of DVS in HaCaT Cells.
DVS was initially diluted with DMSO and subsequently diluted to 30
μM with medium for exposure. First, the medium was removed, and
the cells were washed twice with FBS-free culture medium. Then,
cultured cells in 6-well plates were exposed to 30 μM DVS for 1 h at
37 °C. The supernatant was removed, and the cells were washed three
times with medium. Subsequently, the cells were cultured in a
complete culture medium at 37 °C in the presence of 5% CO2. The
cells were lysed with 0.5 mL of lysis buffer per well at time points of 1,
3, 6, 12, and 24 h after the 1 h DVS exposure.
The lysate was collected to extract DNA as follows: The solution
containing cellular DNA (300 μL) was subjected to enzyme hydrolysis.
The proteinase K was added and incubated for 1 h at 55 °C. At the
end of the incubation, an aliquot of 0.5 mL chloroform and 0.5 mL
phenol were added into the lysis solution, and the sample was
incubated for 1.5 h at 37 °C. The supernatant was added with 2.5
times ethanol (V/V) to the digestion solution, and the sample was
chilled at 0 °C for 10 min. The sample solution was centrifuged at
14000 rpm for 10 min at 4 °C. The DNA precipitant was washed with
2.5 times ethanol (2 × 2 mL) and then evaporated under vacuum.
Lastly, the DNA was hydrolyzed using 15% formic acid at 100 °C for
30 min. The solution was evaporated to dryness, redissolved using 100
μL H2O, and then analyzed by a 5500 QTrap tandem mass
spectrometer (AB Sciex, Framingham, MA, USA). The conditions
for the quantification of DVS-DNA adducts were as follows:
chromatographic column, ACQUITY UPLC BEH C18 column (2.1
× 50 mm I.D, 1.7 μm); mobile phase A, water; mobile phase B,
methanol; gradient elution program: 0−4 min, 5−35% B; 4.1−4.5 min,
35−80% B at a flow rate of 0.35 mL/min; injection volume 3 μL;
column temperature 40 °C; MS conditions: positive ion electrospray
MS/MS in multiple reaction monitoring (MRM) mode; gas
atomization (GS1) 35 psi and the auxiliary heating dry gas (GS2) 60
psi; spray voltage, 5.5 kV; ion source temperature, 550 °C; the
transition of precursor to product ion, the optimized parameters
declustering potential (DP), collision energy (CE), collision cell exit
N3-HESVA. ESI+-MS/MS, m/z 254 [M + H]+. 1H NMR (DMSO-d6,
400 MHz) δ 8.14 (s, 2H, −NH2), 8.09 (s, H, −CH), 7.76 (s, H,
−CH), 7.06 (q, H, −CH), 6.26 (m, 2H, −CH2), 3.85 (m, 2H, −CH2),
3.49 (t, 2H, CH2). HRMS (Q-TOF), obsd m/z 254.0701, calcd for
C9H11N5O3S, m/z 254.0702 [M + H]+. Retention time is 5.0 min.
N7-HESVG. ESI+-MS/MS, m/z 270 [M + H]+, m/z 103 [M + H −
Ade]+. 1H NMR (DMSO-d6, 400 MHz) 7.88 (s, H, −CH), 6.86 (t, H,
−CH), 6.29 (s, 2H, −NH2), 6.13 (m, 2H, −CH2), 4.46 (m, 2H,
−CH2), 3.73 (m, 2H, −CH2). HRMS (Q-TOF), obsd m/z 270.0643,
calcd for C9H11N5O3S, m/z 270.0682 [M + H]+. Retention time is 4.4
min.
N3-AHESEHA-N3. ESI+-MS/MS, m/z 389 [M + H]+, m/z 254 [M +
l
H − Ade]+, m/z 195 [Ade + CH2CH2S]+, m/z 136 [Ade]+. H NMR
(DMSO-d6, 400 MHz) δ 8.11 (s, 2H, −CH), 3.96 (s, 4H, −NH2),
3.81 (m, 4H, −CH2). HRMS (Q-TOF), obsd m/z 389.1249, calcd for
C14H16N10O2S, m/z 389.1257 [M + H]+. Retention time is 1.2 min.
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Chem. Res. Toxicol. 2017, 30, 1874−1882