Article
pubs.acs.org/crt
Abundance of Four Sulfur Mustard-DNA Adducts ex Vivo and in Vivo
Revealed by Simultaneous Quantification in Stable Isotope Dilution−
Ultrahigh Performance Liquid Chromatography−Tandem Mass
Spectrometry
Lijun Yue,†,‡,§ Yuxia Wei,† Jia Chen,† Huiqin Shi,‡ Qin Liu,† Yajiao Zhang,† Jun He,‡ Lei Guo,*,†
Tingfen Zhang,‡ Jianwei Xie,*,† and Shuangqing Peng‡
†
State Key Laboratory of Antitoxic Drugs and Toxicology, and Laboratory of Toxicant Analysis, Institute of Pharmacology and
Toxicology, Academy of Military Medical Sciences, No. 27 Taiping Road, Haidian District 100850, Beijing, China
‡
Beijing Institute for Disease Control and Prevention, No. 20 Dongdajie Street, Fengtai District 100071, Beijing, China
S Supporting Information
*
ABSTRACT: Sulfur mustard (SM) is a highly reactive alkylating vesicant and causes blisters upon contact with skin, eyes, and
respiratory organs. It covalently links with DNAs by forming four mono- or cross-link adducts. In this article, the reference
standards of SM-DNA adducts and deuterated analogues were first synthesized with simplified procedures containing only one or
two steps and using less toxic chemical 2-(2-chloroethylthio)ethanol or nontoxic chemical thiodiglycol as starting materials. A
sensitive and high-throughput simultaneous quantification method of N7-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N7-HETEG),
O6-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (O6-HETEG), N3-[2-[(2-hydroxyethyl)thio]-ethyl]adenine (N3-HETEA), and
bis[2-(guanin-7-yl)ethyl]sulfide (Bis-G) in the Sprague−Dawley rat derma samples was developed by stable isotope dilution−
ultrahigh performance liquid chromatography−tandem mass spectrometry (ID-UPLC-MS/MS) with the aim of revealing the
real metabolic behaviors of four adducts. The method was validated, the limit of detection (S/N ratio greater than 10) was 0.01,
0.002, 0.04, and 0.11 fmol on column for N7-HETEG, O6-HETEG, Bis-G, and N3-HETEA, respectively, and the lower limit of
quantification (S/N ratio greater than 20) was 0.04, 0.01, 0.12, and 0.33 fmol on column for N7-HETEG, O6-HETEG, Bis-G, and
N3-HETEA, respectively. The accuracy of this method was determined to be 76% to 129% (n = 3), and both the interday (n = 6)
and intraday (n = 7) precisions were less than 10%. The method was further applied for the quantifications of four adducts in the
derma of adult male Sprague−Dawley rats exposed to SM ex vivo and in vivo, and all adducts had time− and dose−effect
relationships. To the best of our knowledge, this is the first time that the real presented status of four DNA adducts was
simultaneously revealed by the MS-based method, in which Bis-G showed much higher abundance than the result previously
reported and N3-HETEA showed much less. It should be noted that since the interstrand cross-linked adduct is believed to stall
DNA replication and finally induce a double-strand break, the higher abundance of Bis-G is a great indication of a more serious
DNA lesion by SM alkylation.
■
INTRODUCTION
Sulfur mustard (bis(2-chloroethyl)sulphide, SM) is a wellknown, highly reactive alkylating vesicant and can cause blisters
upon contact with skin, eyes, and respiratory organs. SM is also
a predominant agent found in the chemical weapons
abandoned in China by the Japanese, and has caused several
civilian casualty incidents and environmental contamination.1−3
© 2014 American Chemical Society
As a highly reactive electrophile, it readily reacts with a variety
of nucleophiles via the episulfonium ion.4−7 Previous studies
showed that SM reacts with DNA by forming mono- or crosslinked adducts.8,9 As a consequence, DNA replication is
Received: September 19, 2013
Published: January 27, 2014
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Figure 1. Chemical structures of four SM-DNA adducts and their corresponding deuterated analogues.
and accurate method is urgently required. In recent years, liquid
chromatography−tandem mass spectrometry (LC-MS/MS)
provides a powerful tool for DNA adduct quantification since
such a technique can provide ultrahigh sensitivity as well as
structural and unambiguous quantification information in an
analysis cycle.16−24 In LC-MS/MS, accurate quantification of
trace analytes in a complicated biological matrix requires an
appropriate internal standard (IS). An isotopomer of identical
structure to the analyte is an ideal IS because it has chemical
and physical properties identical to those of the analyte except
for the mass. A stable isotope-labeled IS can be used to
precisely identify the peak of analyte in a complicated
chromatogram based on almost the same retention times of
the analyte and IS, which provides the highest possible accuracy
and specificity for quantitative measurements in many
reports.21−25
In the present work, N7-HETEG, N3-HETEA, O6-HETEG,
Bis-G, and their corresponding deuterated analogues were
successfully synthesized and characterized, and a stable isotope
dilution−ultrahigh performance liquid chromatography−tandem mass spectrometry (ID-UPLC-MS/MS) method for
simultaneous quantification of four SM-DNA adducts was
developed and validated. The method was then applied to
analyze SM-DNA adducts in the derma of male Sprague−
Dawley rats by ex vivo dermal exposure of SM in three
concentrations and in vivo exposure at four dosages, so as to
further illustrate the dose and time dependence profiles with a
good relevance to the abundance of four SM-DNA adducts.
blocked, leading to DNA breakage and cell cycle arrest.
Eventually, the unrepaired lesions may lead to miscoding,
altered gene expression, mutation, cancer, and thus, death.
Therefore, identification and quantification of DNA adducts are
essential for establishing the relationship between DNA adduct
formation and other biological end points (mutations, DNA
double-strand break etc.), and the evaluation of genetic
damage.10−12
According to the multiple independent experiments ex vivo
or in vivo summarized in a review, 13 it has been presumed that
these adducts are formed preferentially at the N7 position of
guanine (61%), the N3 position of adenine (16%), two N7
positions of guanine as intra- or interstrand cross-links (nearly
17%), and the O6 position of guanine (0.1%). With regard to
the determination point of SM-DNA adducts, most research
only focused on the detection of the frequently formed SMDNA adduct, i.e., N7-[2-[(2-hydroxyethyl)thio]-ethyl]guanine
(N7-HETEG), as an excellent biomarker for internal exposure.
However, N7-guanine adducts are chemically unstable and do
not participate in Watson−Crick base pairing, indicating that
N7-guanine maintained limited biological relevance.14 Even of
comparatively much lower abundance, O 6 -[2-[(2hydroxyethyl)thio]-ethyl]guanine (O6-HETEG) is regarded as
a critical DNA lesion because the formation of O6-HETEG may
affect the hydrogen bonds between guanine and cytosine, and
the human DNA repair mechanism fails to remove such SM
adducts from this position.15 The interstrand cross-link, i.e.,
bis[2-(guanin-7-yl)ethyl]sulfide (Bis-G), is also believed to stall
replication and finally induce a double-strand break. It is
noteworthy that no simultaneous determination of four SMDNA adducts has been reported until now. Its development
would definitely be helpful for clarifying the presumption on
the presented status of four SM-DNA adducts and for a much
further understanding of the SM toxicological mechanism.
Considering the significant biological relevance and low
abundance of N3-[2-[(2-hydroxyethyl)thio]-ethyl]adenine (N3HETEA), O6-HETEG, and Bis-G, a highly sensitive, specific,
■
MATERIALS AND METHODS
Caution: SM is a highly reactive alkylating vesicant and cytotoxic
agent. This agent should be handled only in well-ventilated f ume hoods.
The use of gloves and stringent protective measures should be adopted.
Chemicals. SM was provided by China Institute of Chemical
Defense, with purity higher than 95%. Methanol (HPLC grade) was
obtained from J. T. Baker (New Jersey, USA). Guanosine and adenine
were the Amresco packaging products from Beijing Xinjingke
Biotechnology Limited Company (Beijing, China). Thiodiglycol
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Figure 2. Mass spectra of SM-DNA adducts and their corresponding deuterated analogues.
purity greater than 98%. Guanosine-5′-monophosphate disodium
(GMP) was from J&K Scientific LTD (Beijing, China), with a purity
of 99%. 1,2-Dichloroethane-d4 was from Cambridge Isotope
(GC grade) was from Fluka (Buchs SG, Switzerland), at purity higher
than 98.5%. 6-Chloroguanine was purchased from Beijing Coupling
Science and Technology Limited Company (Beijing, China), with a
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O6-[2-[(2-Hydroxyethyl)thio]ethyl] guanine (O6-HETEG) and O6HETEG-d4. TDG or TDG-d4 of 1.64 mmol was dissolved in 10 mL of
dry tetrahydrofuran. The resulting solution was added dropwise to a
60% oil suspension of sodium hydride (130 mg, 3.25 mmol) in 10 mL
of dry tetrahydrofuran. After 10 min of stirring under N2 at room
temperature, 6-chloroguanine (90 mg, 0.53 mmol) was added, and the
resultant mixture refluxed for 3 h. The solvent was evaporated, and the
residue was dissolved in 30 mL of water and extracted with ethyl
acetate (30 mL, three times). The layer of water was acidified to pH
5.0 with 20% acetic acid and evaporated to dryness, then the mixture
was placed in a silica gel column and eluted with dichloromethanemethanol-concentrated ammonia (10:1:0.05, v/v/v). The solvent was
evaporated to dryness, and 40 mg of target compound was obtained.
The yield was 29%, and the purity was more than 98%.
O6-HETEG: lH NMR (DMSO-d6, 300 MHz) δ 12.44 (br, s, 1H),
7.83 (s, 1H), 6.25 (s, 2H), 4.86 (s, 1H), 4.53 (t, J = 7.2 Hz, 2H), 3.58
(d, J = 4.8 Hz, 2H), 2.94 (t, J = 7.2 Hz, 2H), 2.70 (t, J = 6.9 Hz, 2H).
UPLC-MS/MS: m/z 256 [M+H]+, 210[M-HOCH2CH2]+, 105
[HOCH2CH2SCH2CH2]+, 87 [HOCH2CH2SCH2CH2−H2O]+.
O6-HETEG-d4: lH NMR (CD3OD, 300 MHz) δ 8.09 (s, 2H), 4.74
(t, J = 6.9 Hz, 2H), 3.77 (t, J = 6.9 Hz, 2H), 3.04 (t, J = 6.9 Hz, 2H),
2.81(t, J = 6.9 Hz, 2H). UPLC-MS/MS: m/z 260 [M+H]+, 109
[HOCH2CH2SCD2CD2]+, 91 [HOCH2CH2SCD2CD2-H2O]+.
Bis[2-(guanin-7-yl)ethyl] Sulfide (Bis-G) and Bis-G-d4. The
procedures reported by Fidder and co-workers were followed.9 Yield
was ca. 1%, and the purity was more than 97%.
Bis-G: lH NMR (DMSO-d6, 300 MHz) δ 8.41 (s, 2H), 6.81 (s, 4H),
4.46−4.44(m, 4H), 3.04−3.02(m, 4H). UPLC-MS/MS: m/z 389 [M
+H]+, 210 (for fragment ions, see Figure 2D).
Bis-G-d4: lH NMR (DMSO-d6, 300 MHz) δ 8.26 (s, 2H), 6.56 (s,
4H), 4.42(t, J = 6.9 Hz, 2H), 3.02(t, J = 6.9 Hz, 2H). UPLC-MS/MS:
m/z 393 [M+H]+, 214, 210 (for fragment ions, see Figure 2D).
Mass spectra of synthetic SM-DNA adducts and their deuterated
analogues are shown in Figure 2, and all 1H NMR spectra of these four
SM-DNA adducts and their deuterated analogues are shown in Figures
S1−S8 (Supporting Information).
Animals and Treatment. Adult male Sprague−Dawley rats of
specific pathogen free (SPF) grade were purchased from the
Laboratory Animal Center of Beijing. The animal experiment was
conducted in the Beijing Center for Toxicological Evaluation and
Research, in accordance with the protocol approved by the
Institutional Animal Care and Use Committee of the Center, which
is in compliance with the guidelines of the Association for Assessment
and Accreditation of Laboratory Animal Care International (AAALAC).
The animals were allowed to acclimate for at least 1 week prior to
experimental use. They ate food and drank water freely. After
acclimatization, Sprague−Dawley rats of 207 ± 8 g weight were
assigned randomly into 21 groups of five each. The Sprague−Dawley
rats were treated by dermal exposure, including a control and four
dosage groups. After closely clipping the hair (the hair was clipped 24
h before the application), a fresh dilution of SM in 1,2-propanediol
was uniformly applied by injecting 42 μL with a microsyringe onto the
back of the rats onto a square area of approximately 1 cm2. SM was
administrated as a single dose of 5.6, 11.2, 22.5, and 45.0 mg/kg body
weight, equivalent to 0.25, 0.5, 1.0, and 2.0 times the median lethal
dose (LD50), respectively (experimental determination of LD50, see
Table S1, Supporting Information). Relatively higher doses were used
so as to meet the detection needs of the lowest biomarker, i.e., O6HETEG. Control rats were only administered with the equivalent
volume of 1,2-propanediol. Considering the mortalities for animals,
the rats treated with a dosage of 45.0 mg/kg were euthanized at 1, 2, 4,
and 5 days after dosing. The other rats were euthanized with urethane
by intraperitoneal injection at 1, 2, 4, 7, and 14 days postdosing, and
the derma of exposure spots, blood, spleen, liver, lung, kidney, and
other tissues were immediately isolated and stored at −70 °C prior to
DNA extraction. Only derma samples were examined in this research.
Results of DNA adducts in other organ tissues samples will be
reported later.
Laboratories, Inc. (MA, USA) with a deuterated ratio of 99%. 2Mercaptoethanol (biotechnological grade) was obtained from SigmaAldrich (MO, USA), and the purity was better than 98%. All other
reagents were of analytical grade and were purchased from Beijing
Chemical Works and Sinopharm Chemical Reagents Co. Ltd. (Beijing,
China). Ultrapure water was produced in a Mill-Q water purification
system (Millipore, MA, USA).
Synthesis of Standards and Stable Isotope-Labeled Internal
Standards. Molecular structures of standards and stable isotopelabeled ISs are shown in Figure 1.
Chemicals of 2-(2-Chloroethylthio)ethanol (Semi-sulfur Mustard,
Semi-SM) and Semi-SM-d4. Semi-SM or semi-SM-d4 was synthesized
as the raw material for N7-HETEG and N3-HETEA. It was synthesized
from equimolar amounts of sodium methoxide and 2-mercaptoethanol
in the presence of 1,2-dichloroethane or 1,2-dichloroethane-d4
according to the procedures previously described (Rao et al.).26
Both of them were characterized by gas chromatography (GC)-MS.
Thiodiglycol-d4 (TDG-d4). A mixture of 0.5 g of semi-SM-d4 and 8
mL of hydrochloric acid solution of 1 M was heated to 90 °C for 2 h,
and the solvent was evaporated to dryness. The resultant compound
was characterized by GC-MS, and the deuterated ratio was over 99%.
Sulfur Mustard-d4 (SM-d4). Thionyl chloride of 2 mL was added to
0.2 g (1.4 mmol) of semi-SM-d4, refluxed at 80 °C for 2 h, and then
evaporated to dryness. The product was characterized by GC-MS, and
the deuterated ratio was over 99%.
N7-[2-[(2-Hydroxyethyl)thio]ethyl]-guanine (N7-HETEG) and N7HETEG-d4. Guanosine of 1 g (3.5 mmol) was suspended in 12.5 mL of
acetic acid, and 1.8 mmol semi-SM or semi-SM-d4 was added dropwise
to the suspension. The resulting reaction mixture was stirred at 100 °C
for 3 h, then cooled to room temperature. The unreacted guanosine
was filtrated and discarded, and the filtrate was evaporated to dryness.
The residue was dissolved in 19 mL of HCl solution of 1 M. After
extraction of unreacted semi-SM with dichloromethane (19 mL, three
times), the solution was heated at 100 °C for 1.5 h, and a pale-yellow
clear solution resulted, which was cooled and neutralized with
concentrated ammonia. A white crude product was achieved. After
the mixture was filtered, the resultant precipitate was redissolved in
acidic water, then purified by the preparative chromatography
equipped with an ultraviolet (UV) detector (mode, Gilson GX-281,
Gilson Inc., WI, USA; column, Phenomenex C18, 150 × 21.1 mm, 5
μm; mobile phase, 0.1% aqueous trifluoro acetic acid and acetonitrile;
flow rate, 1 mL/min). The yield was 22%, and the purity was more
than 96%. All purities of synthesized adducts and their deuterated
analogues were provided by HPLC-UV detection with an area
normalization method.
N7-HETEG: lH NMR (DMSO-d6, 300 MHz) δ 8.57 (s, lH), 6.96
(br, s, 2H), 4.44−4.42 (m, 2H), 3.54 (t, J = 6.3 Hz, 2H), 3.02−2.98
(m, 2H), 2.61 (t, J = 6.3 Hz, 2H). UPLC-MS/MS: m/z 256 [M+H]+,
239[M+H-NH3]+, 105 [HOCH2CH2SCH2CH2]+, 87
[HOCH2CH2SCH2CH2−H2O]+.
N7-HETEG-d4: lH NMR (CD3OD, 300 MHz) δ 8.71 (s, 1H), 3.70
(d, J = 1.2 Hz, 2H), 2.69 (d, J = 1.2 Hz, 2H). UPLC-MS/MS: m/z 260
[M+H]+, 109 [HOCH2CH2SCD2CD2]+, 91 [HOCH2CH2SCD2CD2H2O]+.
N3-[2-[(2-Hydroxyethyl)thio]ethyl]adenine (N3-HETEA) and N3HETEA-d4. Adenine of 345 mg (2.5 mmol) was dissolved in 20 mL of
dimethylacetamide, and 2.99 mmol semi-SM or semi-SM-d4 was added
dropwise to the solution. The reaction mixture was stirred at 110 °C
for 5 h and then cooled to room temperature. The crude product was
purified by preparative chromatography equipped with a UV detector
(the parameters are shown in the N7-HETEG purification section).
The yield was 26%, and the purity was more than 98%.
N3-HETEA: lH NMR (CD3OD, 300 MHz) δ 8.72 (s, 1H), 8.52 (s,
1H), 4.75−4.73(m, 2H), 3.74(t, J = 4.5 Hz, 2H), 3.30−3.20(m, 2H),
2.75(t, J = 4.5 Hz, 2H). UPLC-MS/MS: m/z 240 [M+H]+, 105
[HOCH2CH2SCH2CH2]+, 87 [HOCH2CH2SCH2CH2−H2O]+.
N3-HETEA-d4: lH NMR (CD3OD, 300 MHz) δ 8.72 (s, 1H), 8.53
(s, 1H), 3.74(t, J = 6.3 Hz, 2H), 2.75(t, J = 6.3 Hz, 2H). UPLC-MS/
MS: m/z 244 [M+H] + , 109 [HOCH 2 CH 2 SCD 2 CD 2 ] + , 91
[HOCH2CH2SCD2CD2-H2O]+.
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Table 1. ID-UPLC-MS/MS Parameters for the Analysis of Four SM-DNA Adducts
compound
N7-HETEG
declustering potential
(V)
collision energy
(V)
collision cell exit potential
(V)
entrance potential
(V)
qualitative ion
pairs
quantitative ion
pairs
80
20
16
10
256→105
256→87
260→109
260→91
256→105
256→87
260→109
260→91
389→210
389→238
393→214
393→210
393→242
240→105
240→87
244→109
244→91
256→105
N7-HETEG-d4
O6-HETEG
80
20
16
10
O6-HETEG-d4
Bis-G
71
31
16
10
Bis-G-d4
N3-HETEA
66
21
10
10
N3-HETEA-d4
260→109
256→105
260→109
389→210
393→214
240→105
244→109
voltage was 2350 V, and the source temperature was 650 °C. All other
mass spectrometric parameters were listed in Table 1. For qualitative
analysis, two or three ion pairs were chosen, and for quantitative
analysis, only the most abundant ion pair was chosen to achieve the
highest sensitivity.
In order to demonstrate the suitability of the developed method,
systematic validation was carried out, including linearity, intra- and
interassay accuracy and precision, limit of detection (LOD, S/N is
greater than 10) and the lower limit of quantification (LLOQ, S/N is
greater than 20), and the sensitivity, recovery, and selectivity were
provided.
SM Administrated Sprague−Dawley Derma Sample Preparation
ex Vivo. After closely clipping the hair, derma samples were taken from
the untreated adult male Sprague−Dawley rats after euthanization.
The frozen Sprague−Dawley rat derma samples were homogenized in
a proportion of 0.1 g tissue per milliliter of cold physiological saline
and stored at −70 °C prior to exposure ex vivo. Aliquots of 90 μL SM
solutions (dissolved in 1,2-propanediol) at the concentrations of 0.5
mM, 0.1 M, and 0.99 M were added to 1.8 mL blank Sprague−Dawley
rat derma (0.18g derma, DNA amounts is about 13 μg) homogenized
solutions (n = 3) and incubated for 2 h at 37 °C with continuous
mixing. The final low, medium, and high concentration levels are
0.024, 4.8, and 48 mM, respectively.
Extraction and Neutral Thermal Hydrolysis of Sprague−Dawley
Rat Derma DNA. The frozen Sprague−Dawley rat derma samples
were homogenized in a proportion of 0.1 g of derma tissue (DNA
amounts are about 7.3 μg) per milliliter of cold physiological saline,
and they were subjected to the same procedures as those for the blood
sample described previously.18 Amounts and purity of DNA were
determined by UV−vis spectrometry according to the equation
20A260 nm = 1 mg/mL DNA. The A260 nm/A280 nm ratios were found to
be between 1.7 and 1.9, ensuring minimal protein contamination. The
yields of DNA are 73 μg per gram of Sprague−Dawley rat derma. The
extracted DNA was dissolved in water, then the stable isotope ISs were
spiked, and the final volumes were 50 μL in vivo and 100 μL ex vivo.
The mixture was continuously shaken at 70 °C for 1 h. After
hydrolysis, the sample was cooled down to room temperature for IDUPLC-MS/MS analysis.
ID-UPLC-MS/MS Analysis. The qualitative and quantitative analysis
of four adducts were carried out with stable isotope dilution UPLCMS/MS in multiple reaction monitoring (MRM) mode. The stock
solutions of N7-HETEG, N3-HETEA, and Bis-G were prepared in
formic acid solution, while the stock solution of O6-HETEG was
prepared in methanol because it is unstable in acidic conditions.
All UPLC-MS/MS analyses were performed in a triple quadrupole
ion trap (QqQtrap) 5500 UPLC-MS/MS instrument (AB SCIEX,
Framingham, MA, USA) by using a Waters ACQUITY UPLC BEH
C18 column (2.1 mm ID × 50 mm, 1.7 μm, Waters Co., MA, USA).
Mobile phases consisted of ultrapure water (A) and HPLC-grade
methanol (B). The gradient elution started with 15% B, held for 1 min,
then linearly increased to 60% B over 1 min, held 0.5 min, then
decreased to 15% B in 0.1 min, and held for 1.4 min; thus, the total
run time was 4 min. The flow rate was 0.35 mL/min, and the injection
volume was 3 μL.
MS detection was initiated from a positive turbo ion spray source.
Curtain gas was 20 psi (1 psi = 6895 Pa), collision gas was 8 psi, ion
source gas 1 and gas 2 were 40 and 50 psi, respectively, ionspray
■
RESULTS AND DISCUSSION
Synthesis of DNA Adducts and the Deuterated
Analogues. To establish a rapid and highly selective method
for the simultaneous detection, identification, and characterization of four SM-DNA adducts, a stable ID method may be
the best choice. Since the reference standards of adducts and
the stable isotope ISs were not commercially available, four SMpurine alkylated products and their deuterated analogues were
synthesized and characterized as an important issue to develop
this method. The synthesis approaches are shown in Scheme S1
(Supporting Information). All obtained compounds were
identified with MS and 1H NMR, and the results of four SMDNA adducts were in high accordance with the references.9
Purity and the deuterated ratio of all compounds meet the
analysis demand well.27
In the synthesis of N7-HETEG and N3-HETEA, considering
the toxicity of two alkylating agents, SM and semi-SM, semi-SM
with much less toxicity was chosen as the original reactant.
Another reactant used in the preparation of N7-HETEG is
guanosine. Since both N7 and N9 of guanine were the most
reactive positions in the presence of acetic acid, guanosine with
a deoxyribose occupying its N9 position can make the alkylating
agent selectively bind to the N7 position and can efficiently
reduce byproduct. 9 Even though guanosine and semi-SM can
react in equal moles theoretically, considering the poor
solubility of guanosine in acetic acid, excessive guanosine was
mixed with semi-SM to ensure complete reaction. Pure N7HETEG was easily isolated from the solution since it is not
soluble in neutral pH. Both materials and the reaction
condition in this approach were different from those described
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Scheme 1. Procedures of Simultaneous Analysis for N7HETEG, O6-HETEG, N3-HETEA, and Bis-G in Rat Derma
Samples by ID-UPLC-MS/MS
but both phenomena did not affect the compounds acting as
ISs.
ID-UPLC-MS/MS Method Development and Validation. The whole procedure for the determination of N7HETEG, O6-HETEG, N3-HETEA, and Bis-G in derma samples
by ID-UPLC-MS/MS is shown in Scheme 1.
Here, we selected SM binding DNA nucleobases instead of
nucleosides as SM-DNA adducts for three reasons. First, the
altered molecular shape and hydrogen bonding characteristics
occurred in the level of structurally modified bases.25 Second,
MS-based analysis on nucleobases can provide better sensitivity
than nucleosides since nucleosides have larger hydrophilicity
due to the occurrence of glycosides and thus have a lower
ionization efficiency than nucleobases. In the high temperature
of the ion source in MS, some thermal labile nucleosides are
inclined to decompose as the nucleobases. Third, prior to
UPLC-MS/MS analysis, DNA is typically hydrolyzed to release
SM-bound nucleotides, nucleosides, or nucleobases. Here, we
used a mild neutral thermal hydrolysis condition, which causes
hydrolytic release of the alkylated purines at neutral pH,8 and
bis-G adducts can be quantitatively released,20,24 as well as
thermally labile N7-HETEG and N3-HETEA. Besides, even O6HETE-deoxyguansine (dG) has better stability than N7-HETEdG and N3-HETE-deoxyadesine (dA), the O6-HETEG adduct
is more thermal and acidic liable than N7-HETEG and N3HETEA during typical acidic and/or heating conditions after
the enzymatic hydrolysis procedure. We need to pursue a
balance between conditions to fully release O6-HETEG from
SM-DNA adduction and to maintain the stability of such a
compound. In our work, a relatively mild thermal hydrolysis
condition, i.e., 70 °C for 1 h was employed because a certain
amount of O6-HETEG can only be preserved in such a
condition in comparison with other acidic hydrolysis (70 °C, 1
h at pH 2) and stronger thermal hydrolysis (100 °C, 1 h)
conditions (Figure S9, Supporting Information).
In this research, biological samples spiked with the
deuterium-labeled ISs were subjected to UPLC-MS/MS. This
method achieved high sensitivity, fast data-collection speed, and
high efficiency. Several mobile phases were tested, such as
water, 0.1% formic acid (pH 2.7), ammonium formate/formic
acid buffer (pH 4.2), ammonium formate/formic acid buffer
(pH 5.2), ammonium formate solution (10 mM, pH 6.1),
ammonium acetate solution (10 mM, pH 6.8), etc. When the
mobile phase was acidic, N3-HETEA had a good peak shape,
while the other three adducts showed poor peak shape and
sensitivity. The best sensitivity for all four adducts was only
achieved with a gradient of water (solvent A) and methanol
(solvent B) without any acidic adjustment. Under such a
condition, the peak symmetry of N3-HETEA was slightly
poorer, but its detection sensitivity was the same as the
sensitivity in the acidic mobile phase.
Since the four SM-DNA adducts have high polarity and
similar properties, several different stationary phases and mobile
phases were tested. N3-HETEA was never separated with the
other three adducts, especially with N7-HETEG (Figure 3).
However, it did not affect their qualitative and quantitative
results in MRM mode. MRM mode offers a great advantage
here since only selected multiple pairs of precursor ion of
correct molecular weight and its product ion are detected,25
and it promises superior specificity from the point of view of
m/z values.
To validate this new method, blank DNAs were isolated from
Sprague−Dawley rat derma, digested, and spiked with certain
in previous literature;9 this preparation way is much simpler
and easier.
In the preparation of N3-HETEA, adenine was reacted
directly with semi-SM in the presence of N,N-dimethylacetamide since the N3 position of adenine was most reactive in
neutral conditions, which was found in 1979 by Fujii and his
co-workers.28 The optimized synthesis procedure was simpler
and safer than the earlier reported methods.9
Since the O6 position of guanine or guanosine is less reactive,
the synthesis of O6-HETEG was quite difficult. The only
available reference was reported by Fidder et al., which had
tedious and time-consuming procedures.9 However, Gundersen
et al. successfully synthesized 2-amino-6-(2-methoxyethoxy)
purine from 2-amino-6-chloropurine in strong basic conditions.29 Enlightened by the latter research, a facile method
from nontoxic raw material adenine was designed and
successfully achieved with only a one-step reaction.
In the synthesis of Bis-G, the main byproduct is N7-HETEG.
In order to decrease the output of this monoadduct, the
reactants were added according to the theoretic ratio, namely,
the ratio of GMP to SM was 2 to 1. The acidity of the Bis-G
dissolving solution utilized was slightly higher than that of N7HETEG; benefiting from this minor difference, pure Bis-G was
produced successfully in this approach.
Compared with stable isotope-labeled agents originated from
nitrogen, sulfur, oxygen, and carbon atoms, deuterium-labeled
agents were more cost-effective. Except for the fact that the
synthesis began from 1,2-dichloroethane-d4 instead of 1,2dichloroethane, the procedures were exactly same as those for
the four SM-DNA adducts. Because the reactant involves four
deuterium atoms, i.e., only occupying half of all eight hydrogen
atoms, O6-HETEG-d4 has two isomeric compounds (see Figure
1, compound H1 and H2) and Bis-G-d4 has two main fragment
ions (m/z 210 and 214) in the mass spectrum (see Figure 2D),
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Figure 3. Chromatograms of four SM-DNA adducts, N7-HETEG and O6‑HETEG (panel A), N3-HETEA (panel B), and Bis-G (panel C) in IDUPLC-MS/MS using MRM mode. The chromatograms of the control, the sample in vivo, and the sample spiked with IS are shown from top to
bottom. Transition m/z: N7-HETEG, 256→105; O6-HETEG, 256→105; N3-HETEA, 240→105; Bis-G, 389→210; N7-HETEG-d4, 260→109; O6HETEG-d4, 260→109; N3-HETEA-d4, 244→109; Bis-G-d4, 393→214.
amounts of N7-HETEG, N3-HETEA, O6-HETEG, Bis-G, and
corresponding deuterium-labeled ISs, and UPLC-MS/MS
analysis was followed. An excellent correlation was observed
between the expected and observed concentrations of adducts,
and the linearity was good in 5 to 7 orders of magnitude with
the value of correlation coefficient (r2) over 0.99, i.e., N7HETEG (0.003−198 ng/mL), N3-HETEA (0.026−260 ng/
mL), O6-HETEG (0.0007−149 ng/mL), and Bis-G (0.016−
1550 ng/mL). The LOD values (S/N ratio greater than 10)
were 1, 0.2, 5, and 9 pg/mL (0.01, 0.002, 0.04, and 0.11 fmol on
column) for N7-HETEG, O6-HETEG, Bis-G, and N3-HETEA,
respectively, and the LLOQ values (S/N ratio greater than 20)
were 3, 0.7, 16, and 26 pg/mL (0.04, 0.01, 0.12, and 0.33 fmol
on column) for N7-HETEG, O6-HETEG, Bis-G, and N3HETEA, respectively. The sensitivity was higher than that of
the previous method developed in our group18 and at the same
level with the quantification method of similar DNA
Table 2. Intra- and Interday Precisions and Recoveries of
Four SM-DNA Adducts
precision (RSD)
adduct
concentration
(ng/mL)
intraday
(n = 7)
interday
(n = 6)
recovery
n=3
0.009
9.90
180
0.002
2.93
133
0.16
17.1
1318
0.09
9.53
238
8.8%
2.1%
1.4%
5.6%
3.6%
2.2%
4.9%
2.8%
2.2%
6.2%
0.9%
0.4%
9.5%
2.0%
0.9%
4.4%
3.0%
2.4%
5.2%
2.7%
4.2%
5.9%
1.7%
1.0%
118 ± 0.6%
107 ± 0.2%
100 ± 2%
103 ± 2%
76 ± 3%
86 ± 2%
81 ± 12%
129 ± 2%
123 ± 4%
109 ± 3%
112 ± 1%
93 ± 2%
N7-HETEG
O6-HETEG
Bis-G
N3-HETEA
Table 3. Determination Results of Four SM-DNA Adducts ex Vivo (n = 3)
N7-HETEG
N3-HETEA
O6-HETEG
Bis-G
exposure concentration (mM)
ng/g
%
ng/g
%
ng/g
%
ng/g
%
0.024
4.8
48
6.9 ± 0.8
170 ± 40
6570 ± 820
51.6
63.8
83.2
0.3 ± 0.2
8.8 ± 2.8
158 ± 16
2.5
3.3
2.0
0.08 ± 0.01
0.41 ± 0.05
9.8 ± 6.9
0.56
0.15
0.12
6.1 ± 1.4
89 ± 42
1160 ± 123
45.3
32.7
14.7
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to be 76% to 129% (n = 3), and both the interday (n = 6) and
intraday (n = 7) precisions were less than 10% (Table 2).
Analysis of SM-DNA Adducts in Rat Derma ex Vivo. In
derma samples exposed to low, medium, and high SM
concentrations ex vivo, all four SM-DNA adducts were
positively detected in exposed derma samples (Table 3). The
results showed that the N7 position of guanine was the most
reactive nucleophilic site and can react with SM readily. The
next reactive position was the N3 position of adenine, and the
O6 of guanine was the least reactive. Only 0.01% to 0.1% (w/w)
of administrated SM reacted with DNA by an alkylating
reaction, and the amounts of total SM-DNA adducts were
about 0.002%, 0.360%, and 10.8% (w/w) of total DNA
(calculated according to the extracted amount of DNA).
The content of adduct was also transformed to the molar
concentration to investigate the molar contribution of each SMDNA adduct. The simultaneous quantification results showed
that the molar percentages of N7-HETEG, Bis-G, N3-HETEA,
and O6-HETEG were 61−88%, 10−35%, 2−4%, and 0.1−0.7%,
respectively. The molar percentages of Bis-G and N3-HETEA
were significantly different from previous published results,13
i.e., Bis-G has much higher abundance, 10−35% versus 17%,13
and N3-HETEA has much lower abundance, 2−4% versus 16%.
Meanwhile, our results were well supported by an in vitro
Figure 4. Amounts of four SM-DNA adducts in different derma
exposed concentrations ex vivo (n = 3). The value beyond the column
represents the mean value of each amount for SM-DNA adduct. The
error bar represents the standard deviation of results for three parallel
derma samples ex vivo.
adducts. 21−24 Three levels of low, medium, and high
concentration were tested, method accuracy was determined
Figure 5. Dose and time dependence profiles of four SM-DNA adducts in the derma of Sprague−Dawley rats (n = 5). Levels of the four adducts’
response positively with the exposed dosages and negatively with the elapsed time after dosing. Inserts show the time dependence profiles of four
SM-DNA adducts at the dosage of 45 mg/kg. Regarding the ultrahigh dose of 45.0 mg/kg group, the last data were collected on the 5th day
postexposure due to high mortality. The error bar represents the detection standard deviation of five derma samples of rats in the same group.
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Figure 6. Linearity between four SM-DNA adducts and derma exposed dosages in vivo (n = 5). The error bar represents the detection standard
deviation of five derma samples of rats in the same group.
obviously observed. All adducts decreased with time after
exposure and positively correlated with the exposure dosages.
The mean maximum contents of all four SM-DNA adducts
were achieved on day 1 postexposure, i.e., 112, 346, 628, and
1910 ng/g for dose 5.6, 11.2, 22.5, and 45.0 mg·kg−1,
respectively, and the contents of adducts in derma decreased
rapidly during postdosing days 1 to 4. The results indicate a
dose dependent relationship between content of adducts and
exposure dose. Regression analysis of mean maximum
concentration of adducts versus dose showed an r2 beyond
0.964 (Figure 6). The total amount of four adducts was ca.
from 0.001% to 0.01% (w/w) of the applied dose of SM.
The abundance of four adducts in the different exposure
dosage levels is shown in Figure 7. The molar percentages of
N7-HETEG, Bis-G, N3-HETEA, and O6-HETEG were ca. 64 to
81%, 18 to 42%, 1.3 to 4.6%, and 0.04 to 0.62%, respectively,
which correlates with the results ex vivo very well. SM
penetrated the rat derma from epidermis in vivo, while it
reacted directly with the derma homogenate in the ex vivo
experiment, and the molar percentage results in vivo and ex vivo
are quite similar. Therefore, it suggests that the formation rate
and ratio of four SM-DNA adducts were not significantly
influenced by skin structure, such as the epidermis and dermis.
Here, for the first time, the real status of four DNA adducts
presented in vivo was revealed, and a much higher abundance of
Bis-G and a lower abundance of N3-HETEA were found
compared with the previous results.13 It is important to note
that the higher abundance of Bis-G in all SM-DNA adducts
indicates a more serious DNA lesion by SM alkylation because
this interstrand cross-linked adduct is believed to stall DNA
replication and finally induce a double-strand break.13
Figure 7. Amounts of four SM-DNA adducts in different derma
exposed dosages in vivo (n = 5) at the first day postdosing. The value
beyond the column represents the mean value of each amount for the
SM-DNA adduct. The error bar represents the standard deviation of
results from five derma samples of rats in the same group.
investigation report published in 2013, in which N7-HETEG,
Bis-G, and N3-HETEA in cellular DNA were isolated and
determined by HPLC-MS/MS. Bis-G has the second
abundance, existing as less than half of the content of N7HETEG.30 All trends in the molar percentage values of four
SM-DNA adducts were the same at three different exposure
concentrations (Figure 4). It is indicated that SM primarily
reacts with the most reactive site, N7 of guanine, and then with
other but less reactive positions, N3 of adenine, and O6 of
guanine ex vivo.
Analysis of SM-DNA Adducts in Rat Derma in Vivo. In
derma samples exposed to low, medium, high, and ultrahigh
SM dosage levels in vivo, the dose and time dependence profiles
of four SM-DNA adducts are shown in Figure 5. Regarding the
dose of the 45.0 mg/kg group, the last data were collected on
the fifth day postexposure due to high mortality. The DNA
adduct was not detected in rat derma samples collected before
dosing or from the control group. For all dosage levels, both
time and dose dependent trends of four SM-DNA adducts were
■
CONCLUSIONS
To develop a highly sensitive and selective method, four SMDNA adducts and their stable isotope-labeled ISs were
synthesized. The stable ID-UPLC-MS/MS method for
simultaneous determination of four SM-DNA adducts was
developed, and high sensitivity, precision, and accuracy were
achieved. This method was successfully applied to determine
four SM-DNA adducts in the SM-exposed Sprague−Dawley rat
derma ex vivo and in vivo, and a dose or time dependent
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relationship was found. It is the first time that the real
presented status of four DNA adducts was revealed, in which
we found a much higher abundance for the cross-linked adduct
Bis-G and much less for N3-HETEA than previous published
results. We hope that our experimental proof provides a
quantitative view for further understanding DNA lesions which
are formed on exposure to SM.
■
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ASSOCIATED CONTENT
S Supporting Information
*
Synthesis route of four SM-DNA adducts and deuterated
analogues; 1H NMR spectra of N7-HETEG, N3-HETEA, O6HETEG, Bis-G, N7-HETEG-d4, N3-HETEA-d4, O6-HETEG-d4,
and Bis-G-d4; and the determined median lethal dose of
Sprague−Dawley rats with dermal exposure to SM. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Authors
*(L.G.) Tel: +86 10 66930621. Fax: +86 10 68225893. E-mail:
[email protected].
* (J.X.) Tel/Fax: +86 10 68225893. E-mail: [email protected].
Present Address
§
(L.Y.) College of Life and Environmental Sciences, Minzu
University of China, 27 South Zhongguancun Avenue, Haidian
District 100081, Beijing, China.
Funding
This work was supported by National Science and Technology
Major Project of the Ministry of Science and Technology of
China (Grant No. 2012ZX09301003-001-010), and China
Postdoctoral Science Foundation (No. 20090450198).
Notes
The authors declare no competing financial interest.
■
ABBREVIATIONS
SM, sulfur mustard; N7-HETEG, N7-[2-[(2-hydroxyethyl)thio]ethyl]guanine; O6-HETEG, O6-[2-[(2-hydroxyethyl)thio]ethyl]guanine; N3-HETEA, N3-[2-[(2-hydroxyethyl)thio]ethyl]adenine; Bis-G, bis[2-(guanin-7-yl)ethyl]sulfide; IDUPLC-MS/MS, stable isotope dilution−ultrahigh performance
liquid chromatography−tandem mass spectrometry; MRM,
multiple reaction monitoring; LOD, limit of detection; LLOQ,
lower limit of quantification; S/N ratio, the ratio of signal-tonoise; IS, internal standard; GC-MS, gas chromatography−
mass spectrometry; GMP, guanosine-5′-monophosphate disodium; TDG, thiodiglycol; NMR, nuclear magnetic resonance;
DMSO, dimethyl sulfoxide; UV−vis, ultraviolet visible; HPLC,
high performance liquid chromatography; MS, mass spectrometry; SPF, specific pathogen free; AAALAC, Association for
Assessment and Accreditation of Laboratory Animal Care
International; LD50, median lethal dose; QqQtrap MS, triple
quadrupole-linear ion trap mass spectrometry
■
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