Facile Formation of a DNA Adduct of Semicarbazide on Reaction with

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Facile Formation of DNA Adduct of Semicarbazide in Reaction with Apurinic/Apyrimidinic Sites in DNA Yinan Wang, Ho-Wai Chan, and Wan Chan Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00011 • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 14, 2016

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Chemical Research in Toxicology

Facile Formation of DNA Adduct of Semicarbazide in Reaction with Apurinic/Apyrimidinic Sites in DNA

Yinan Wang, Ho Wai Chan, and Wan Chan*

Department of Chemistry, The Hong Kong University of Science and Technology, Clear

Water Bay, Kowloon, Hong Kong

* Correspondence author. Phone: +852 2358-7370; Fax: +852 2358-1594; E-mail:

[email protected].

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Abstract Mutagenic semicarbazide (SEM) is a hydrazine-containing food contaminant found in

a wide variety of foods. Despite decades of research, the toxicity of SEM remains

incompletely understood. In this study, we demonstrated for the first time that SEM

reacts rapidly with apurinic/apyrimidinic sites in an endogenous DNA lesion to form

covalently bonded DNA adducts in vitro and in bacteria. Specifically, we performed

high-performance liquid chromatography with high accuracy and tandem mass

spectrometry to characterize the DNA adduct formed by reacting SEM with

2’-deoxyribose, and single-stranded and double-stranded oligonucleotides containing

abasic sites under physiological relevant conditions. By analyzing the reaction

mixture at different time points, the reaction kinetics of SEM with DNA was also

elucidated. Moreover, by using a highly sensitive and selective liquid

chromatography-tandem mass spectrometry method, we showed that SEM induced a

dose-dependent formation of DNA adduct in Escherichia coli. Results from our

studies provide the first direct evidence suggesting that SEM may exerts genotoxicity

by forming covalently bonded DNA adducts.


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Introduction Semicarbazide (SEM, Figure 1) is a protein-binding metabolite produced when livestock or aquatic products were treated with bactericidal nitrofurazone.1,2 Although

nitrofurazone has been banned worldwide from use in food production, illegal use of nitrofurazone exists.3-7 Emerging evidence has also suggested that SEM is a food contaminant produced in processes other than nitrofurazone metabolism.8 For

instance, SEM is produced when azodicarbonamide, a foaming agent used extensively in plastic production and bakery, is subjected to thermal treatments.9,10 Previous

research also reported that the combined use of hypochlorite and peroxyacetic acid for milk sterilization generates SEM.11

Although adverse reproductive effects have been confirmed in SEM-treated laboratory rodents, the carcinogenicity of SEM remains controversial.12-14 While in some studies multiple tumors were observed in SEM-treated mice,15 studies reporting no noticeable carcinogenic potential of SEM in rats also exist.2,16,17 The current study

aims to probe the genotoxicity of SEM by using a molecular biology approach to

characterize the SEM-induced DNA adducts. To the best of our knowledge, the



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formation of covalently bonded DNA adducts by SEM has not been reported in the

literature.

In this study, we explored the feasibility of SEM in forming covalently bonded

DNA adducts with apurinic/apyrimidinic (AP, Figure 1) sites in DNA. AP sites are

among the most prevalent DNA lesions formed in various physiological processes, 18,19

such as spontaneous hydrolysis and DNA damage/repair.20,21 The ring-opened

form of the 2’-deoxyribose (dR) in AP sites can process a reactive carbonyl moiety that can react with hydrazines and hydroxylamines.22,23 Hence, we hypothesized that

facial chemical reactions between AP sites in DNA and SEM have occurred in

biological systems. A similar phenomenon for covalent adduct formation between hydrazine-containing drugs and AP sites was observed in previous studies.24-26

In the current study, high-performance liquid chromatography (HPLC) and mass

spectrometry were used to characterize the SEM–DNA adducts formed in both in

vitro and in bacteria. We first characterized the adducts formed by reacting SEM with

AP site-containing DNA oligonucleotides and dR, which is the hydrolysis product of

the AP sites. We then performed a series of in vitro experiments to assess the


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reactivity of SEM with single- and double-stranded AP site-containing

oligonucleotides at physiologically relevant conditions. We conducted liquid

chromatography-tandem mass spectrometry (LC–MS/MS) coupled with a stable

isotope–isotope dilution method, and the frequencies of SEM–DNA adducts in DNA

samples isolated from SEM-treated Escherichia coli were accurately quantified.

Materials and Methods Chemicals. All chemicals used in this study were of the highest purity available and

used without purification unless noted otherwise. 2’-Deoxy-D-ribose, semicarbazide hydrochloride (SEM), semicarbazide-13C-15N2 hydrochloride (13C-1,2,-15N2-SEM),

methoxyamine, methylhydrazine, 1-aminohydantonin, 3-amino-2-oxaz-olidone,

alkaline phosphatase, DNase I, and nuclease P1 were obtained from Sigma-Aldrich

(St. Louis, MO). Snake venom phosphodiesterase was purchased from US Biological

(Swampscott, MA). Uracil glycosylase was acquired from New England Biolabs

(Ipswich, MA). A uracil-containing oligonucleotide 5′-GCCGT-U-AGGTA-3′ and its



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complementary strand 3′-CGGCAATCCAT-5′ were synthesized by Integrated DNA

Technologies (Coralville, IA). LC–MS grade methanol and acetonitrile were

purchased from JT-Baker (Philisburg, NJ). Deionized water was further purified in a

Milli-Q Ultrapure water system (Billerica, MA) and was used in all experiments.

Instrumentation. LC–MS/MS analyses were performed on an Agilent 1100 HPLC

system equipped with an API 4000 QTRAP tandem mass spectrometer (AB Sciex,

Foster City, CA). High-mass accuracy and collision-induced dissociation mass

spectrometric analyses were performed on a Waters Xevo G2 Q-TOF mass

spectrometer (Milford, MA) with a standard electrospray ionization source. HPLC

analysis and purification were performed on an Agilent 1100 HPLC system coupled with a diode-array detector. 1HMR spectra were collected on a Bruker AV 400 MHz

NMR spectrometer (Figure S1). UV absorbance spectrometric measurements were

recorded on a Varian UV–vis absorption spectrophotometer (Cary 50, Walnut Creek,

CA).

Synthesis, Purification, and Characterization of the SEM-dR Adducts. The dR adducts of SEM and 13C-15N2-SEM were synthesized in an overnight reaction of a


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10-fold molar excess of SEM in potassium phosphate buffer (50 mM, pH 7.4) at

37 °C. The reaction of the ring-opened, aldehyde-containing dR with SEM (or 13

C-15N2-SEM) led to the formation of a hydrazone, which was separated from the

starting materials by HPLC.

20 µL of the reaction mixture was injected onto a Alltech Prevail carbohydrate

ES column (250 mm × 4.6 mm, 5 µm) by using water (A) and acetonitrile (B) as the

mobile phase (60% to 40% B over 20 min, held at 40% B for 5 min). The eluate was

monitored by a UV detector (238 nm), and a single product eluted at 9.1 min was

collected. The products were characterized by UV absorbance spectrophotometry,

high-accuracy mass spectrometry, and collision-induced dissociation mass

spectrometry.

Synthesis of Single- and Double-Stranded Oligonucleotides. An AP site-containing

oligonucleotide (5′-GCCGT-X-AGGTA-3′) was synthesized by incubating 5′-GCCGT-U-AGGTA-3′ with uracil glycosylase as previously described.19,27 In accordance with standard procedures,28,29 the HPLC-purified AP site-containing

oligonucleotide was annealed with its complementary strand 3′-CGGCATTCCAT-5′



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to produce a double-stranded oligonucleotide DNA. Efficient formation of the AP

site-containing double-stranded oligonucleotide DNA was confirmed by gel

electrophoretic analysis (Figure S2).

Reaction of SEM with Single- and Double-Stranded Oligonucleotides. SEM (1

mM) was incubated with the AP site-containing single-stranded (60 µM) or

double-stranded (60 µM) DNA oligonucleotides in potassium phosphate buffer (50

mM, H 7.4) at 37 °C. Aliquots of the samples were sampled at different reaction times

and reaction kinetics followed by HPLC analysis. 10 µL of the sample was injected

onto a GraceSmart C18 column eluted with a binary solvent of (A) 20 mM

ammonium acetate and (B) methanol at 0.3 mL/min (1% B for 10 min, increased

linearly to 10% B in 20 min). The formation of the SEM-modified oligonucleotides

was monitored at 254 nm by a UV detector. Control experiments were conducted by

using single-stranded (5′-GCCGT-U-AGGTA-3′) and double-stranded

oligonucleotides that do not contain abasic sites.

Exposure of E. coli to SEM. After growing E. coli (DH5α, ATCC) to mid-log phase,

it was harvested by centrifugation at 4500 ×g for 15 min. The sample was washed


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thrice with phosphate buffer solution (PBS, 50 mM, pH 7.4), and cells were

resuspended in PBS for SEM exposure. Up to 0.5 g of E. coli in 5 mL of PBS was

added to SEM at final concentrations of 0.05, 0.1, 0.2, 0.5, and 1.0 mM. After 3 h of

exposure at 37 °C, cells were harvested by centrifugation, and the cellular DNA was isolated by salting out method.30 Cells were quantified by spectrophotometry, and the DNA samples were hydrolyzed enzymatically as previously described.27,31

Hydrolysates were analyzed by LC−MS/MS after passing through a Omega Nanosep

spin filter (MWCO 3K, PALL) to remove the enzymes.

LC−MS/MS Analysis. The frequency of SEM-dR adduct in the cell-isolated DNA

samples was quantified by LC−MS/MS analysis. Chromatographic separation of the

SEM-dR adduct from the canonical nucleobases was performed on a Waters XBridge

Amide column (50 mm × 3.0 mm, 3.5 µm). A 10 µL aliquot of the sample extract was

injected into the column eluted with the following gradients of acetonitrile in 0.1%

formic acid at a flow rate of 200 µL/min at 40 °C: 0−2 min, 100%; 2−4.0 min,

100−1%; 4.0−6.0 min, 1%; 6.1−15 min, 100%.

HPLC was coupled to a tandem mass spectrometer operated in multiple-reaction



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monitoring (MRM) mode. Parameters for positive electrospray ionization were

optimized as follows: declustering potential, 32 V; entrance potential, 9 V; and ion

spray voltage, 5000 V. The curtain gas (CUR), collision gas (CAD), ion source gas I

(GSI), ion source gas II (GSII), and temperature of GSII were set at 30, 5, 20, 40, and

400 °C, respectively. The collision energy for collision-induced dissociation was set at

12 V. MRM transitions of m/z 192 →76 and 192 →117 were set to monitor the

SEM-dR adduct, whereas MRM transitions of m/z 195 →79 and 195 →117 were set to monitor the 13C-15N2-SEM-dR internal standard. The dwell time for each transition

was set at 100 ms.

RESULTS AND DISCUSSION Characterization of the Adduct Formed by Reacting SEM with dR. An AP site is

produced by spontaneous hydrolysis of nucleobase and through base-excision repair mechanism.32,33 AP sites exist in an equilibrium of a ring-closed form and a ring-opened aldehyde-containing form (Figure 1),34 and the carbonyl moiety of the


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ring-open form is highly reactive toward nucleophilic attacks from hydrazine- and hydroxylamine-containing compounds.35 Thus, we investigated the reaction of

hydrazine-containing SEM with AP sites in DNA. First, we incubated SEM with dR,

which was the hydrolysis product of AP sites in DNA. The reaction of SEM with dR

produced a covalently bonded SEM-dR adduct. The adduct was purified by HPLC

and then characterized by UV absorption spectrometry, high-accuracy MS, and

MS/MS analyses (Figure 2).

High-accuracy MS analysis of the adduct revealed a close correlation between the measured (192.0982) and theoretical (192.0984) m/z values of the [M+H]+ ion,

with a mass error of 1.0 ppm. Collision-induced dissociation MS/MS analysis of the

pseudo-molecular ion at m/z 192.1 led to the formation of fragment ions at m/z 117.1

and 76.1, which originated from the cleavage at the imine bond linking SEM and dR.

The corresponding daughter ions at m/z 117.1 and 79.1 were also identified when the 13

C-15N2-SEM-dR internal standard (m/z 195.0958) was analyzed under identical

conditions.

UV absorption spectrometric analysis of the SEM-dR adduct in acetonitrile



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showed the maximum local absorption at 238 nm (Figure 2C), with an extinction coefficient of 4,300 M−1 cm−1. Moreover, the concentration of the internal standard 13

C-15N2-SEM-dR was determined using the molar extinction coefficient for the

SEM-dR adduct.

Characterization of the Adduct Formed by Reacting SEM with AP

Site-Containing DNA Oligonucleotide. After characterizing the SEM-dR adduct,

this study was extended to investigate the formation of DNA adducts in SEM with AP

site in DNA oligonucleotide. After incubating the AP site-containing oligonucleotide

under physiological conditions, a clear formation of the SEM-tagged oligonucleotide

was observed (Figure 3). The SEM-modified AP site-containing oligonucleotide was

well characterized by high-accuracy mass spectrometry. Figure 4 depicts the MS

spectra from the analysis of the AP site-containing oligonucleotides and their SEM

adducts. The close correlation of the measured (3319.5973) and theoretical (3319.6007) m/z values of the monoisotopic peak of the [M-3H]3- ion in the SEM

adduct indicated that the AP site-containing oligonucleotide was SEM-tagged. A

similar phenomenon of adduct formation was observed when the double-stranded


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oligonucleotide was incubated with SEM (Figure 4D).

The AP site-containing oligonucleotide and its SEM adduct were then analyzed

by MS/MS analysis. Upon subjecting to collision-induced dissociation, a

characteristic fragmentation pathway at the 3’- end of phosphate backbone adjacent to

the SEM-tagged AP site was observed for both the unmodified and SEM-modified

oligonucleotide (Figure S3), which demonstrated unambiguously that SEM adduct

formation at the AP site of the oligonucleotide.

In contrast to previous studies that demonstrated strand breaks at the AP site

when AP site-containing DNA was incubated with phenylhydrazine hydrochloride of similar chemical structure,36,37 both our HPLC and high-accuracy MS analyses of

SEM-treated DNA showed no detectable amounts of degradation products from

hydrolysis of the DNA oligonucleotide (Figures. 3 and 4). This significantly different

result may be ascribed to the varied acidity of the incubation buffers used. Previous

studies used reaction systems at which the pH was determined to ranged from pH 3.5

to 5.0. In the present study, we used a mildly buffered system at pH 7.4 for the

incubation. We have also demonstrated in our study that under the acidic environment,



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e.g. pH 3.5, AP site-containing DNA oligonucleotide was hydrolyzed to short chain

oligonucleotides (Figure S4).

A previous study has proposed that the genotoxicity of SEM is attributed to its reaction with cytosine.38 However, both our studies on AP site-containing single- and

double-stranded DNA oligonucleotides and on oligonucleotides that do not contain

abasic sites revealed no indication of adduct formation at the canonical nucleobases of

DNA (Figures 3, 4, and S5). Moreover, our findings indicating that SEM reacts

rapidly with AP sites in single- and double-stranded DNAs under physiologically

relevant conditions (pH 7.5, 37 °C) suggest that the covalently bonded semicarbazone

adducts of SEM with endogenous AP sites in DNA may have contributed to the

observed genotoxicity in SEM.

Reaction Kinetics of SEM with Single- and Double-Stranded DNAs. The reaction

kinetics of SEM with AP site-containing oligonucleotides was followed by HPLC

analyses. Accordingly, we incubated SEM (50 µM) with single- and double-stranded

DNAs (50 µM) in potassium phosphate at 37 °C. The reactions were monitored at

different time points by HPLC analysis (Figure 3), and the peak area of the SEM


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adduct was plotted against the reaction time. As shown in Figure 5, our study revealed

a time-dependent increase in detection response for the reactions with both single-

and double-stranded AP site-containing DNA. A complete reaction was observed at 30

min after the start of incubation, indicating that the reaction between AP

site-containing DNA oligonucleotides is highly efficient.

The kinetics data were also analyzed by plotting ln|Yt − Y∞| versus time as described previously (Figure S6).35 Linear regression analysis revealed pseudo-first-order rate constants of 1.2 ± 0.1 × 10−3 s−1 and 1.9 ± 0.3 × 10−3 s−1 for the

reaction of SEM with single- and double-stranded DNAs, respectively, in which a

higher reaction rate was observed for double-stranded oligonucleotide. These results correspond to second-order rate constants of 24 ± 2 M−1 s−1 and 38 ± 6 M−1 s−1 for

SEM, in which a higher reaction rare was observed for the double-strand DNA

oligonucleotide. Similarly, a higher reaction constant for double-strand DNA than that

of single-strand DNA was reported by Melton et al with the antihypertensive drug hydralazine.35 Furthermore, our findings are in reasonable agreement with the formation rate constants of other hydrazone-forming reactions (2 to 20 M–1 s–1),



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which were reported by Kool et al.,39 who tested different hydrazines and

oligonucleotides. Similar rate constants were also observed when the studies were performed on methoxyamine and methylhydrazine40,41 (Table 1).

Quantification of SEM-dR Adducts in DNA of SEM-Exposed E. coli.

Subsequently, we determined the effects of the cellular environment on SEM-dR

adduct formation by quantifying SEM-dR adducts in DNA samples isolated from

SEM-treated E. coli. As such, E. coli was treated with different amounts of SEM, and

the level of SEM-dR adduct in the genomic DNA was analyzed using the stable

isotope-dilution LC–MS/MS method, as described in the Materials and Methods.

The limit of detection (LOD) and quantitation (LOQ) of the optimized LC–

MS/MS method, defined as the amount of SEM-dR adduct generating a signal 3 and

10 times that of the noise level, respectively, was found to be 13 fmole and 43 fmole. These data correspond to LOD and LOQ of 0.6 adduct/106 nt and 2 adduct/106 nt,

respectively, in 25 µg of DNA. To evaluate the method accuracy, the combined effect

of the DNA isolation process and the hydrolysis efficiency on the level of SEM-dR

adduct measured in the isolated DNA was accessed by spiking a certain quantity of


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SEM-tagged DNA oligonucleotide to unexposed cells during the DNA isolation

process, and the levels of SEM-dR adduct were quantified in the isolated DNA. The

measured quantities of SEM-dR adducts were expressed relative to the quantity of

SEM-tagged DNA added to the cells. As shown in Figure S7, a close correlation of

the measured SEM-dR adduct level with that of the spike SEM-tagged DNA amount

was observed. These results correspond to 91.5% of the SEM-tagged DNA used to

spike the cell samples. Moreover, these findings reveal a tangible loss of SEM-dR

during the cell processing and enzymatic hydrolysis steps and provide a correction

factor of 1.1 for cellular yields of SEM-dR for the analytical method.

LC–MS/MS analysis revealed a clear identification of SEM in the E. coli-isolated

DNA samples. Figure 6 shows a typical chromatogram obtained from LC–MS/MS

analysis of SEM-dR adduct in a hydrolyzed E. coli DNA sample. The analysis results

of SEM-dR in SEM-exposed cells are illustrated in Figure 7. A dose-dependent

formation of SEM-dR adduct was observed in the SEM-treated cells. After correction

for adduct losses during DNA isolation and processing (8.5%), linear regression analysis of the results revealed a corrected damage frequency of 23 SEM-dR per 106



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nt per mM SEM. To our knowledge, this study is the first to report about the detection

of covalently bonded DNA adducts in SEM.

Conclusion In this study, we demonstrated for the first time that food-borne SEM formed

covalently bonded DNA adducts with AP sites in DNA under physiologically relevant

conditions. By using a combination of state-of-the-art analytical techniques, we

characterized the SEM–DNA adducts formed in vitro and in bacteria. Our study also

revealed a dose-dependent formation of SEM–DNA adducts in SEM-treated E. coli,

indicating the biological relevance of the DNA adducts. Our results suggest that the

unclear toxicity of SEM may be (or may possibly be) ascribed to its reaction with the

ring-opened, aldehyde-containing form of AP sites in DNA. Considering that SEM

can be found in a wide variety of foods, the current results also highlighted the need

for a thorough reinvestigation on SEM toxicity. Furthermore, the results can provide a

solid foundation for the use of SEM–DNA adducts as biomarkers in assessing the risk

of mutagenic SEM exposure.


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Conflict of interest statement The authors declare that there are no conflicts of interest.

Supporting Information Available 1

H NMR spectrum of SEM adduct of 2’-deoxyribose. Gel electrophoretic analysis of

the single- and double stranded oligonucleotides; The negative ESI-MS/MS analysis

of fragment ions of AP site-containing oligonucleotide and its SEM adduct; Analysis

of DNA oligonucleotide after overnight reaction with SEM in pH 3.5 at 37 °C; HPLC

and Q-TOF MS analysis of the reaction conducted by using single-stranded (5′

-GCCGT-U-AGGTA-3′) and double-stranded oligonucleotides that do not contain

abasic sites; Reaction kinetic of SEM with single- and double- stranded

oligonucleotide DNA; and Method validation with synthesized SEM-tagged, AP

site-containing oligodeoxynucleotides. This material is available free of charge via the

Internet at http://pubs.acs.org



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Acknowledgements We thank Drs. Yueru Sun and Zhihong Gou (Department of Chemistry, HKUST) for

their assistance in performing the gel electrophoretic analysis. We also thank AB

Sciex for providing the LC–MS/MS system for this research.

Funding Sources This work was supported by the Research Grant Council of Hong Kong (ECS

609913). W. Chan thank The Hong Kong University of Science and Technology for a

Startup Funding (Grant R9310).

Abbreviations AP site, apurinic/apyrimidinic site; dR, 2’-Deoxy-D-ribose; HPLC, high-performance

liquid chromatography; LC–MS/MS, liquid chromatography-tandem mass

spectrometry; SEM, semicarbazide.


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Table 1 Comparative Reactivity of Semicarbazide, Methoxyamine, and Methylhydrazine with Single-stranded and Double-stranded AP Site-containing DNA Oligonucleotides.

Second Order Rate Constant, M-1s-1 With Single-Stranded DNA

With Double-Stranded DNA

Semicarbazide

24±2

38±6

Methoxyamine

34±8

42±8

Methylhydrazine

18±4

28±6

1-aminohydantonin

ND a

ND a

3-amino-2-oxaz-olidone

ND a

ND a

a

No reaction. Rate constants not determined.



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FIGURE LEGENDS

Figure 1. Reaction of semicarbazide (SEM) with the ring-opened form of

apurinic/apyrimidinic sites in DNA to form a covalently bonded DNA adduct.

Figure 2. HPLC chromatogram for the analysis of the purified SEM-dR adduct on a

carbohydrate column (A); Shown in panels (B) and (C) are the positive ESI-MS and

UV absorbance spectra of the SEM-dR adduct, respectively.

Figure 3. HPLC chromatograms for the analyses of AP site-containing

oligonucleotide (A) before and (B) after reacting with SEM. Clear formation of

SEM-DNA adduct was observed.

Figure 4. Negative ESI-MS analyses of AP site-containing single- (A, B) and

double-stranded (C, D) DNA oligonucleotides before (A, C) and after (B, D) reacting

with SEM. The analyses revealed unambiguous identification of SEM-DNA adducts in their [M-3H]3- form.

Figure 5. Time course for the formation of the SEM-DNA adduct in single- and

double-stranded DNA. DNA oligonucleotides were allowed to react with SEM and

analyzed by HPLC as described in the Material and Methods section.


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Figure 6. Typical chromatograms obtained from LC–MS/MS analysis of (A)

unlabeled SEM-dR (B) and isotope-labeled SEM-dR internal standard in hydrolyzed E. coli DNA samples. Shown in the insets are the ESI-MS/MS spectra of the [M+H]+

ion of the (A) unlabeled SEM-dR and (B) labeled SEM-dR adduct at m/z 192 and m/z

195, respectively.

Figure 7. Dose-dependent formation of SEM-DNA (SEM-dR) adduct in DNA

samples isolated from SEM-treated E. Coli cells.



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Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5


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Figure 6



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Figure 7


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Graphic for manuscript


Facile Formation of a DNA Adduct of Semicarbazide on Reaction with

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