Identification of a Fused-Ring 2′-Deoxyadenosine Adduct Formed in

May 9, 2016 - Identification of a Fused-Ring 2′-Deoxyadenosine Adduct Formed in Human Cells Incubated with 1-Chloro-3-buten-2-one, a Potential React...
1 downloads 4 Views 498KB Size
Download PDF
Article pubs.acs.org/crt

Identification of a Fused-Ring 2′-Deoxyadenosine Adduct Formed in Human Cells Incubated with 1‑Chloro-3-buten-2-one, a Potential Reactive Metabolite of 1,3-Butadiene Fang-Mao Zeng,†,∥ Ling-Yan Liu,†,∥ Jin Zheng,† Cong Kong,‡ Jing An,† Ying-Xin Yu,† Xin-Yu Zhang,*,† and Adnan A. Elfarra*,§ †

Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, China ‡ East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China § Department of Comparative Biosciences and the Molecular and Environmental Toxicology Center, University of WisconsinMadison, Madison, Wisconsin 53706, United States S Supporting Information *

ABSTRACT: 1-Chloro-3-buten-2-one (CBO) is an in vitro metabolite of 1,3-butadiene (BD), a carcinogenic air pollutant. CBO exhibited potent cytotoxicity and genotoxicity that have been attributed in part to its reactivity toward DNA. Previously, we have characterized the CBO adducts with 2′-deoxycytidine and 2′deoxyguanosine. In the present study, we report on the reaction of CBO with 2′-deoxyadenosine (dA) under in vitro physiological conditions (pH 7.4, 37 °C). We used the synthesized standards and their decomposition and acid-hydrolysis products to characterize the CBO−DNA adducts formed in human cells. The fused-ring dA adducts (dA-1 and dA-2) were readily synthesized and were structurally characterized as 1,N6-(1-hydroxy-1-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine and 1,N6-(1-hydroxy-1-chloromethylpropan-1,3-diyl)-2′-deoxyadenosine, respectively. dA-1 exhibited a half-life of 16.0 ± 0.7 h and decomposed to dA at pH 7.4 and 37 °C. At similar conditions, dA-2 decomposed to dA-1 and dA, and had a half-life of 0.9 ± 0.1 h. These results provide strong evidence for dA-1 being a degradation product of dA-2. dA-1 is formed by replacement of the chlorine atom of dA-2 by a hydroxyl group. The slow decomposition of dA-1 to dA, along with the detection of hydroxymethyl vinyl ketone (HMVK) as another degradation product, suggested equilibrium between dA-1 and a ring-opened carbonyl-containing intermediate that undergoes a retro-Michael reaction to yield dA and HMVK. Acid hydrolysis of dA-1 and dA-2 yielded the corresponding deribosylated products A-1D and A-2D, respectively. In the acid-hydrolyzed reaction mixture of CBO with calf thymus DNA, both A-1D and A-2D could be detected; however, the amount of A-2D was significantly larger than that of A-1D. Interestingly, only A-2D could be detected by LC-MS analysis of acidhydrolyzed DNA from cells incubated with CBO, suggesting that dA-2 was stable in DNA and thus may play an important role in the genotoxicity and carcinogenicity of BD. In addition, A-2D could be developed as a biomarker of CBO formation in human cells.

■

INTRODUCTION 1-Chloro-3-buten-2-one (CBO), an α,β-unsaturated ketone, is a potential metabolite of 1,3-butadiene (BD), a known human carcinogen.1 It is formed from BD via a two-step process (Scheme 1). The first step is a myeloperoxidase (MPO)mediated reaction of BD with hydrogen peroxide in the presence of high concentrations of chloride ion (>50 mM) to form 1-chloro-2-hydroxy-3-butene (CHB).2 The second step is an alcohol dehydrogenase (ADH)-mediated oxidation of CHB to produce CBO.3 BD is an air pollutant with high cancer risks.4−7 It is considered the most carcinogenic agent in cigarette smoke.8 BD is ubiquitous in the environment because it is formed as a product of incomplete combustion of fossil fuels and biomass. © XXXX American Chemical Society

The major environmental sources include automobile exhaust, cigarette smoke, and exhaust from biomass burning.1 Carcinogenicity of BD originates from its reactive metabolites, including 3,4-epoxy-1-butene, 3,4-epoxy-1,2-butanediol, 1,2,3,4-diepoxybutane, and likely hydroxymethyl vinyl ketone (HMVK).9 The metabolites can form DNA adducts and cause DNA damage and mutation, which is probably one of the underlying mechanisms of carcinogenicity of these metabolites.10 Evidence for an alternative metabolic pathway, in which two different metabolites, i.e., CHB and CBO, are formed, has been Received: March 19, 2016

A

DOI: 10.1021/acs.chemrestox.6b00095 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology

All phosphate buffers (100 mM) contained 100 mM KCl. The HepG2 cell line was purchased from the Center of Cell Resources of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Grand Island, NY, U.S.). Biomiga EZgene Tissue gDNA Miniprep Kit was obtained from Biomiga, Inc. (San Diego, CA, U.S.). Instruments and Methods. Mass spectra were obtained on a Bruker microTOF II mass spectrometer with electrospray ionization (ESI). NMR spectra were recorded on a Bruker Instruments Avance AV500 spectrometer (500 MHz). Analysis of samples and isolation of products were performed on a Waters Alliance 2695 HPLC system (Milford, MA, U.S.) equipped with a Waters 2996 diode array detector. An Agilent ZORBAX 80 Å StableBond SB-C18 analytical column (4.6 × 250 mm, 5 μm) was used for analysis of samples. Different gradient programs and isocratic elution conditions were used in the analysis of samples. For analysis of CBO-dA reaction mixtures, the acid hydrolysis products of dA-1 and dA-2, and the decomposition products of dA-1 and dA-2 under in vitro physiological conditions, a linear gradient program was used, starting at 1 min from 0% pump B to 22% pump B over 11 min [pump A, 1% (v/v) methanol with the pH being adjusted to 2.5 with TFA; pump B, 10% (v/v) methanol at pH 2.5] at a flow rate of 1 mL/min and then at 12 min from 22% to 100% pump B over 2 min, at 22 min from 100% to 0% pump B over 1 min, and stopped at 23 min. The gradient for analyses of the CBO− DNA reaction mixtures started at 1 min from 0.1% pump B to 10% pump B over 19 min (pump A, water with the pH being adjusted to 2.5 with TFA; pump B, acetonitrile containing 0.1% TFA) at a flow rate of 1 mL/min and then at 20 min from 10% to 15.3% pump B over 9 min, at 29 min from 15.3% to 0.1% pump B over 1 min, and stopped at 30 min. The column temperature was set at 30 °C. For preparative isolation of the products, a Waters SunFire C18 preparative column (10 × 150 mm, 5 μm) was employed with the same mobile phases. dA-1 and its decomposition product dA were isolated through isocratic elution with 50% pump B at a flow rate of 3 mL/min. A-1D was purified through isocratic elution with 100% pump A at a flow rate of 2 mL/min, and A-2D was similarly isolated with 20% pump B at a flow rate of 3 mL/min. Fractions were collected manually and were lyophilized on a CHRIST ALPHA 1−4 LD-2 freeze-dryer (Osterode am Harz, Germany). For analysis of the HMVK standard, and HMVKGSH and dA-1-GSH reaction mixtures, the linear gradient program started at 1 min from 0% pump B to 10% pump B over 19 min (pump A, water with the pH being adjusted to 2.5 using TFA; pump B, acetonitrile containing 0.019% TFA) at a flow rate of 1 mL/min and then at 20 min from 10% to 0% pump B over 1 min, and stopped at 21 min. The column temperature was set at 30 °C except the preparative isolation of the products. LC-MS analysis was performed on an Agilent 1290 Infinity HPLC system connected to an Agilent 6224 TOF mass spectrometer with ESI. An Agilent Poroshell 120 PFP column (3.0 × 100 mm, 2.7 μm) was used with a linear gradient program, starting at 1 min from 0.1% pump B to 53.2% pump B over 10 min [pump A, water containing 0.1% (v/v) formic acid; pump B, acetonitrile containing 0.1% formic acid] at a flow rate of 0.3 mL/min, at 11 min from 53.2% to 0.1% pump B over 1 min, and stopped at 12 min. Synthesis of HMVK and the HMVK-Glutathione (GSH) Conjugate. HMVK was synthesized according to the procedure in the literature with some modifications.14 Briefly, 2-butyn-1,4-diol (1.72 g, 20 mmol) was dissolved in 15 mL of water. Mercury(II) sulfate (0.09 g, 0.30 mmol) and 0.11 mL of concentrated sulfuric acid were added, and the suspension was stirred at ambient temperature for 20 h. Barium carbonate (1.3 g, 6.6 mmol) was added to neutralize the mixture to pH 6−7. After filtration, a pale yellow solution was obtained, whose HPLC chromatogram exhibited only one peak at 7.4 min with the λmax at 212 nm. The solution was directly used to prepare the HMVK-GSH conjugate as described previously.15 The HMVKGSH conjugate was eluted at 10.1 min. Reactions of CBO with dA at in Vitro Physiological Conditions and at Other pH Values. Time-Course Experiments. dA (5.0 mg, 0.019 mmol) was dissolved in 2 mL of phosphate buffer

Scheme 1. Formation of CHB and CBO from BD

obtained.2,3 CHB is expected to be generated in immune and bone marrow cells due to the requirement for MPO,2 and BD has been known to cause lymphohematopoietic cancers in occupationally exposed workers,1 implying an association between the carcinogenicity of BD in humans and the alternative MPO-mediated metabolic pathway. However, the toxicological significance of CBO has not been well investigated in part because of the high reactivity of CBO which prevents the development of direct methods to assess its formation in vivo. Recently, we reported both CHB and CBO to be cytotoxic and genotoxic to human liver cells in culture with CBO being approximately 100-fold more potent than CHB.11 Results obtained with the Ames test indicated that CHB was a mutagen; however, the high toxicity of CBO prevented a reliable assessment of its mutagenicity.11 CBO is a Michael acceptor that is expected to react with nucleobases to form DNA adducts. Indeed, we have found that CBO reacted readily with 2′-deoxycytidine (dC) and 2′deoxyguanosine (dG) to produce multiple adducts under in vitro physiological conditions (pH 7.4, 37 °C).12,13 The reactivity of CBO toward nucleobases is probably the basis of CBO genotoxicity and implies that CBO may be a mutagen. In the present study, we investigated the reaction of CBO with 2′deoxyadenosine (dA) under in vitro physiological conditions and characterized the formed adducts, their stabilities, and the decomposition and acid-hydrolysis products to obtain standards that were used to characterize the major CBO-dA adducts formed in the reactions with DNA and in human cells in vitro.

■

EXPERIMENTAL PROCEDURES

Materials. BD (99%) was purchased from Dalian Da-Te Gas Ltd. (Dalian, China). Calcium hypochlorite and dA monohydrate were obtained from Alfa Aesar (Ward Hill, MA, U.S.). Chromium oxide was obtained from Adamas Reagent Company (Shanghai, China). LButhionine-sulfoximine (BSO), calf thymus DNA (CT DNA, Cat. No. D1501), and dimethyl sulfoxide-d6 (DMSO-d6) was obtained from Sigma-Aldrich (St. Louis, MO, U.S.). Trifluoroacetic acid (TFA, purity ≥99.0%), tris(hydroxymethyl)aminomethane (Tris), and other reagents (analytical reagent grade) were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). CBO was synthesized as reported previously and was usually used without further purification (purity ∼90%).3 Pure CBO (>98%) was obtained through purification on a silica gel column with dichloromethane as the eluent. B

DOI: 10.1021/acs.chemrestox.6b00095 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology with the specified pH value, and CBO (6.3 μL, purity 95%, 0.065 mmol, CBO/dA = 3.5:1) was added. The mixture was incubated at 37 °C for 7 h. Aliquots (50 μL) were withdrawn every hour and were subjected to HPLC analysis. Isolation of Products for Structural Characterization. To prepare the products for structural characterization, the reactions were performed on a larger scale. To prepare dA-1, dA (9.0 mg, 0.033 mmol) was dissolved in 3.6 mL of pH 7.4 phosphate buffer, and crude CBO (12 μL, purity 90%, 0.12 mmol, CBO/dA = 3.5:1) was added. The solution was incubated at 37 °C for 6 h, extracted with diethyl ether three times, and subjected to isolation of products on preparative HPLC. dA-2 was quite labile with a short half-life (0.9 h at pH 7.4 and 37 °C). Attempts to isolate pure dA-2 for structural characterization failed, even when the beakers to collect fractions were kept on ice. The problem was partly avoided by directly lyophilizing the reaction mixture at pH 4.0 without isolation of dA-2, although the dA-2 obtained was not highly pure (purity ∼80%). Nonetheless, structural characterization of dA-2 by NMR spectroscopy was achieved in this way. To prepare dA-2 for structural characterization, the reaction of CBO with dA was carried out in TFA-acidified water instead of phosphate buffer as in the preparation of other products. Specifically, dA (11.0 mg, 0.041 mmol) was dissolved in 4.4 mL of water with the pH being adjusted to 4.0 using TFA, and pure CBO (75 μL, 0.82 mmol, CBO/dA = 20:1) was added. The mixture was incubated at 37 °C for 6 h, extracted with diethyl ether three times, and lyophilized. The resulting solid, which predominantly contained dA-2 (accounted for ∼82% of all compounds except CBO) as estimated by HPLC, was directly dissolved in DMSO-d6 for NMR analysis or in acetonitrile− water (1:1, v/v) for ESI-MS analysis. To prepare dA formed from decomposition of dA-1, 15.0 mg of dA (0.056 mmol) was dissolved in 6.0 mL of pH 4.0 phosphate buffer, and 22 μL of crude CBO (purity 90%, 0.22 mmol, CBO:dA = 3.9:1) was added. Incubation was carried out at 37 °C for 2.75 h, and HPLC analysis indicated that the solution contained almost pure dA-2 (98% as estimated by peak areas at 260 nm). After being extracted with diethyl ether 5 times, the aqueous solution was divided into two equal parts (3.0 mL per part). One part was used to prepare the decomposition product of dA-1 (i.e., dA) and the other to prepare A-2D. To investigate the decomposition of dA-1, the pH of the solution was adjusted to 7.4 with KOH, and dA was isolated on preparative HPLC after incubation at 37 °C for 8 h. The other part of the solution was acidified by adding 61 μL of 5 M HCl and was left in a boiling water bath for 1 h. After cooling down, the pH was adjusted to ∼5, and then A-2D was isolated using preparative HPLC. A-1D was obtained by dissolving purified dA-1 in 0.1 M HCl and heating the acidic solution in a boiling water bath for 1 h. After cooling down and adjusting the pH to ∼6, A-1D was isolated on preparative HPLC. Stabilities of Products, Determination of the Half-Lives, and Decomposition of Products under in Vitro Physiological Conditions. General Procedure. A small quantity of purified product (1−2 mg) was dissolved in pH 7.4 phosphate buffer and incubated at 37 °C for different time points. The solution was then analyzed using HPLC. For the determination of the half-lives, aliquots were withdrawn every hour and were subjected to HPLC analysis. The peak areas of the compounds to be examined were determined and were regressed against time using exponential decay (y = a × e‑bx). The half-lives were calculated using the data obtained after regression. The experiments were carried out in triplicate, and the reported values were the means and standard deviations of three independent experiments. Detection of HMVK Generated during the Decomposition of dA-1 under in Vitro Physiological Conditions. To detect HMVK in the incubation mixture, GSH was used as the trapping agent. A pH 7.4 phosphate buffer (305 μL) containing ∼0.024 μmol of purified dA1 (estimated by the peak area) and 0.14 μmol of GSH was incubated at 37 °C for 29 h. The solution was then analyzed for the formation of the HMVK-GSH conjugate by HPLC as described above. Reaction of CBO with CT DNA under in Vitro Physiological Conditions. CT DNA (2 mg/mL) was rehydrated overnight at 4 °C

in 50 mM, pH 7.2 Tris buffer containing 1 mM MgCl2. CBO (2 μL, purity 80%, 17 μmol) was dissolved in 40 μL of isopropanol. To 0.5 mL of DNA solution, 2 μL of CBO solution in isopropanol was added. The reaction mixture was then incubated at 37 °C for 9 h. After hydrolysis in 0.1 M HCl at 75 °C for 20 min, the solution was cooled to room temperature, neutralized to pH ∼5, filtered with a 0.22 μm membrane, and then subjected to HPLC analysis. Detection of A-2D in Cells Incubated with CBO. HepG2 cells were cultured as monolayers in DMEM supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere with 5% CO2 and routinely passaged by trypsinization when nearly confluent. Cells at ∼60% confluence were incubated with 100 μM BSO in FBS-containing DMEM at 37 °C for 24 h, and then with 1 μM CBO in D-Hank’s balanced salt solution at 37 °C for 1 h. Cellular DNA was extracted with Biomiga EZgene Tissue gDNA Miniprep Kit. The DNA extracted was immediately hydrolyzed in 0.1 M HCl at 75 °C for 20 min. After cooling to room temperature, adjusting pH to ∼3, and filtering with a 0.22 μm membrane, the hydrolyzed solution was subjected to LC-MS analysis.

■

RESULTS Reaction of CBO with dA under in Vitro Physiological Conditions. The reaction of CBO with dA under in vitro physiological conditions proceeded readily. Two major products, which were designated as dA-1 and dA-2 in the order of the retention times (which were 10.6 and 20.6 min, respectively), were formed (Figure 1). Their UV/vis absorption

Figure 1. Typical chromatogram of the reaction mixture of CBO and dA under in vitro physiological conditions (monitored at 220 nm). The CBO/dA molar ratio was 3.5:1, and the reaction time was 1 h.

spectra were similar to that of dA with the absorption maxima being 212 and 263 nm for dA-1, and 212 and 264 nm for dA-2 (λmax for dA was 257 nm) (Figure S1). Thus, dA-1 and dA-2 were expected to be adducts of dA. Time- and pH-Dependence of the Formation of Products. The formation of dA-2 was rapid; in fact, it could be observed immediately after CBO and dA were mixed at room temperature. However, the formation of dA-1 could be observed only after starting the incubation of the reaction mixture at 37 °C. The two products showed different patterns of formation (Figure 2). The reaction of CBO with dA was examined at pH 4, 7.4, and 10 to determine the optimal conditions for preparation of the products. Under all three pH conditions, the amounts of dA-2 increased over time at the early stages of the reactions (within 1−2 h) and subsequently decreased, whereas those of dA-1 only showed increases up to 7 h (Figure 2), suggesting that dA-2 underwent an initial formation and subsequent C

DOI: 10.1021/acs.chemrestox.6b00095 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology

overlapped the signals of three protons at 3.52 and 3.59 ppm, rendering the integrals of these signals less accurate (Figure S3). The proton number suggested that dA-1 was a quaternary ammonium salt. In the spectrum, five broad single peaks appeared at 10.80, 6.75, 5.49, 5.35, and 5.07 ppm, and the corresponding signals were absent from the heteronuclear multiple-quantum correlation (HMQC) spectrum (Figure S4), indicating that these peaks were the signals of protons at hydroxyl or amino groups. The peak at 10.80 ppm apparently could only be assigned as the proton at the exocyclic amino group. The fact that it contained only one proton and appeared at low field (the signal of exocyclic amino group in dA in DMSO-d6 appears at 7.40 ppm) clearly indicated that both N1and N6-positions of dA were alkylated. Alkylation at the N1position led to the formation of a positive charge at this nitrogen atom. The positive charge can move to the nitrogen atom of the exocyclic amino group via tautomerization, explaining why the proton at the exocyclic amino group in dA-1 appeared at such a low field (the feature seems a reliable tool to identify adenine adducts containing a quaternary nitrogen at the N1 position of adenine, as similar proton shifts were also observed with dA-2 and A-2D; see Table 1). dA-1 contains four hydroxyl groups, consistent with cyclization by the addition of a nitrogen atom to the carbonyl carbon of CBO. The two protons on the purine ring (H2 and H8) exhibited very similar chemical shifts (8.69 and 8.74 ppm). The assignment of the proton and carbon signals at positions 2 and 8 could be established through the heteronuclear multiplebond correlation (HMBC) spectrum (see below). The signals of the deoxyribose protons of dA-1 shifted little in comparison to the deoxyribose protons of dA, thus being easily identified (Table 1). After these protons were assigned, the remaining six protons could only be assigned to those on three methylene groups in the CBO moiety. The HMQC spectrum shows corresponding 13C signals at 65.3, 43.5, and 27.5 ppm (Table 1), which are assigned to 1″-, 4″-, and 3″-CH2 (the numbering of the carbon atoms in dA-1 is shown in Scheme 2), respectively, based on the most downfield signal belonging to the carbon bearing a hydroxyl substituent (1″-CH2) and the highest field signal belonging to the carbon attached only to H and C (3″-CH2). In the HMBC spectrum (Figure S5), H1′ was observed to couple to two carbon atoms at 142.4 and 146.3 ppm, which were thus assigned as C8 and C4, respectively, because the carbon atom at 142.4 ppm carries a hydrogen atom as indicated by the HMQC spectrum. The assignment was further supported by a very weak coupling of H8 at 8.74 ppm with C1′. Therefore, the other hydrogen-carrying carbon atom on the purine ring, i.e., C2, which had a 13C chemical shift at 146.9 ppm, was identified. The determination of the H2 and C2 chemical shifts was critical to establish the structure of dA-1. The HMBC spectrum showed that H2 coupled with C4″, and in turn, H-4″ coupled with C2, indicating that C4″ was connected to N1. Thus, it was C2″ (i.e., the carbon atom at the original carbonyl group in the CBO moiety) that was connected to the exocyclic amino group. Other coupling information was consistent with such a structure. Therefore, dA-1 was characterized as 1,N6-(1-hydroxy-1-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (Scheme 2). Structural Characterization of dA-2. The first clue for the structure of dA-2 came from the fact that incubation of dA2 at pH 7.4 led to the formation of dA-1 as the predominant product (see below).

Figure 2. Time-dependent formation of dA-1 and dA-2 at different pH values. (A) pH 7.4; (B) pH 4.0; and (C) pH 10.0. The CBO/dA molar ratios were all 3.5:1, and the detection wavelength was 260 nm.

decomposition. The incubation pH significantly affected the yields and ratios of the products. At pH 7.4, the peak area ratio of dA-2 to dA-1 changed over time, from 1.7 at 1 h to 0.11 at 7 h. At pH 10, the yields and ratios of products were similar to those obtained at pH 7.4. However, different results were observed at pH 4. At this pH, the yield of dA-2 greatly increased and that of dA-1 significantly decreased, leading to a dA-2:dA-1 ratio of 135:1 at 1 h and 12:1 at 7 h. This indicated that the formation of dA-2 was greatly favored at pH 4. Meanwhile, decomposition of dA-2 was considerably decreased at this pH (Figure 2A). Structural Characterization of dA-1. The high-resolution ESI-mass spectrum of dA-1 (Figure S2A) showed the base peak at m/z 222.0978 and a strong signal at m/z 338.1453, which did not contain a chlorine atom based on the intensity of the isotopic peak at m/z 340.1503. The data matched a structure with one molecule of CBO being added to dA with the chlorine atom being replaced by a hydroxyl group (the calculated formula weight for such a structure with a formula of C14H20N5O5 is 338.1464). It was also consistent with the observed base peak, which was an ion formed after losing the deoxyribose moiety (the calculated mass for C9H12N5O2 was 222.0991). The 1H NMR spectrum of dA-1 in DMSO-d6 showed the presence of 20 protons, although the water peak at 3.42 ppm D

DOI: 10.1021/acs.chemrestox.6b00095 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology Table 1. NMR Data of the Products Characterized in the Present Studya dA-1 1

position 2 4 5 6 8 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ NH OH OH OH OH

H

8.69 (s, 1H)

8.74 6.39 2.66 2.38 4.43 3.90 3.61 3.52 3.75 3.58

(s, 1H) (t, 1H, J = 6.5 Hz) (m, 1H) (m, 1H) (s, 1H) (m, 1H) (m, 1H) (m, 1H) (d, 1H, J = 10.5 Hz) (1H)b

2.24 (m, 1H) 2.09 (m, 1H) 4.58 (m, 1H) 4.34 (m, 1H) 10.80 (br s, 1H) 6.75 (br s, 1H) 5.49 (br s, 1H) 5.35 (br s, 1H) 5.07 (br s, 1H)

dA-2 13

1

C

146.9 146.3 118.5 146.8 142.4 83.8 39.6 70.2 88.1 61.1 65.3 79.4 27.5 43.5

A-1D 13

H

8.77 (s, 1H)

8.80 6.40 2.67 2.40 4.43 3.91 3.59 3.52 4.07 3.94

146.9 146.5 118.3 146.6 142.7 83.9 39.5

(s, 1H) (t, 1H, J = 6.5 Hz) (m, 1H) (m, 1H) (m, 1H) (m, 1H) (m, 1H) (m, 1H) (d, 1H, J = 11.5 Hz) (dd, 1H, J = 11.5, 2 Hz)

1

C

H

8.60 (s, 1H)

8.50 (s, 1H)

A-2D 13

1

C

H

146.0 e e e 144.0

8.66 (s, 1H)

65.4

4.06 (d, 1H, J = 11 Hz) 3.94 (d, 1H, J = 11 Hz)

8.56 (s, 1H)

13

C

146.3 148.0 117.6 146.4 143.6

70.1 88.0 61.1 47.9 78.4 27.4

2.33 (m, 1H) 2.28 (m, 1H) 4.63 (m, 1H) 4.38 (m, 1H) 11.22 (br s, 1H) 7.35 (br s, 1H) 5.47 (br s, 1H) 5.41 (br s, 1H)

43.3

3.75 (d, 1H, J = 12 Hz) 3.58 (1H)d 2.23 2.09 4.58 4.34 c c c

(m, (m, (m, (m,

1H) 1H) 1H) 1H)

e 27.4 43.3

2.33 (m, 1H) 2.25 (m, 1H) 4.60 (m, 1H) 4.33 (m, 1H) 11.09 (br s, 1H) c

48.0 78.3 27.5 43.2

c

a

The 13C chemical shifts of the carbon atoms carrying hydrogen were obtained from the HMQC spectra, and those without hydrogen (i.e., C4, C5, C6, and C2″) were from the HMBC spectra. bThe signal should be a double peak. However, it overlapped with the signal of H5′ at 3.61 ppm; therefore, its multiplicity could not be identified. cThe signals were not observed. dThe signal was expected to be a double peak. However, its multiplicity was unable to be determined due to overlapping with the peaks of impurities. eThe data were not available because the HMBC spectrum of A-1D was not collected.

accounted for 10%, and unreacted dA for 1%). Therefore, CBO was reacted with dA under these conditions, the reaction mixture was lyophilized after extraction with diethyl ether, and the formed residue was dissolved in DMSO-d6 and was subjected to NMR data collection. Using this approach, the NMR and ESI-mass spectra were obtained. The high-resolution ESI-mass spectrum of dA-2 exhibited the base peak at m/z 240.0651 and a strong peak at m/z 356.1125 (Figure S2B) with a peak at m/z 358.1095 having approximately one-third the intensity, consistent with the presence of a chlorine atom. The formula weight was consistent with a structure with a CBO molecule being added to dA (the calculated formula weight for C14H19N5O4Cl is 356.1126). Obviously, the base peak was the signal of the daughter ion of the molecular ion after losing the deoxyribose moiety (the calculated formula weight for C9H11N5OCl is 240.0652). Although the 1H NMR spectrum exhibited the presence of significant amounts of impurities (Figure S6), the proton signals of dA-2 could be identified with assistance from the HMQC and correlation spectroscopy (COSY) spectra (Figures S7 and S8) and also the 1H NMR data of dA-1. Overall, the chemical shifts of protons in dA-2 were similar to those in dA-1 (Table 1). Similar to dA-1, the HMBC spectrum of dA-2 (Figure S9) exhibited the coupling between H2 and C4″, and between H4″ and C2, confirming that the vinyl terminus of CBO had added to N1. H1″, H3″, and H4″ were all observed to couple to a carbon atom at 78.4 ppm, which was assigned as C2″. The chemical shift precludes the possibility that C2″ is a carbonyl carbon and is consistent with addition of the exocyclic

To obtain the NMR data, a sufficient amount of dA-2 (at milligram level) needed to be isolated with HPLC. However, it was found that dA-2 was so labile that most of dA-2 decomposed during isolation; keeping the collection beakers on ice seemed not to decrease the decomposition. Considering that dA-2 was the overwhelmingly predominant product at pH 4, especially at the early stage of the reaction (2−3 h) (Figure 2B), the possibility to obtain the spectroscopic data directly using the reaction mixture was explored. Indeed, as estimated by peak areas, dA-2 accounted for ∼94% of all compounds in the reaction mixture at 2 h (the monitoring wavelength was 260 nm), which included dA-1, dA-2, and unreacted dA (unreacted CBO was not considered because it can easily be removed by extraction and lyophilization). Thus, in our first attempt, the reaction was performed in pH 4 phosphate buffer at 37 °C for 3 h, and the excess of CBO was removed by extraction with diethyl ether. The obtained solution was lyophilized, DMSO-d6 was directly added to the residual solid, and the 1H NMR spectrum was recorded. However, the spectrum exhibited broad peaks and a poor baseline, probably due to the presence of inorganic salts, such as phosphate and KCl. To avoid this problem, we examined the reaction of CBO with dA in water whose pH was adjusted to 4 with TFA. However, in this situation it was found that a higher CBO/dA molar ratio was needed to obtain a high yield of dA-2. Finally, the reaction conditions were optimized to a CBO/dA molar ratio of 20:1 and incubation time of 6 h at 37 °C. Under these conditions, 99% of dA was consumed/ and the peak area of dA-2 accounted for 89% of the total peak areas (dA-1 E

DOI: 10.1021/acs.chemrestox.6b00095 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology Scheme 2. Structures of the Reaction Products of CBO with dA under in Vitro Physiological Conditions and of the Decomposition/Acid Hydrolysis Products of the Reaction Productsa

Figure 3. Decomposition of dA-1 and dA-2 over time at pH 7.4 and 37 °C as monitored by HPLC at 260 nm.

a

The numbering of the positions in the compounds, which was used in the text and Table 1, was shown in dA-1 and A-1D. On the basis of the numbering rules, for the CBO moieties in A-1D and A-2D, 1′, 2′, 3′, and 4′ (rather than 1″, 2″, 3″, and 4″) should be used because the deoxyribose moieties are already lost. However, the numbering with double primes (i.e., 1″, 2″, 3″, and 4″) were still used in A-1D and A2D to facilitate comparison of data.

Figure 4. Decomposition of dA-1 and dA-2. (A) The HPLC chromatogram of dA-1 monitored at 260 nm after incubation at pH 7.4 and 37 °C for 9 h. (B) The HPLC chromatogram of dA-2 monitored at 260 nm after incubation at pH 7.4 and 37 °C for 1 h.

amino group. Therefore, CBO reacted with N1 and N6 of dA to form a fused ring, and dA-2 was characterized as 1,N6-(1hydroxy-1-chloromethylpropan-1,3-diyl)-2′-deoxyadenosine (Scheme 2). Stabilities of dA-1 and dA-2 under in Vitro Physiological Conditions. At pH 7.4 and 37 °C, dA-1 and dA-2 were both observed to undergo decomposition with dA-2 being much more labile than dA-1 (Figure 3). The half-lives of dA-1 and dA-2 were determined to be 16.0 ± 0.7 and 0.9 ± 0.1 h, respectively. Characterization of the Decomposition Products of dA-1 and dA-2. Incubation of dA-1 at pH 7.4 and 37 °C led to the formation of a product with the retention time at 16.9 min and λmax at 257 nm, which was designated as dA-1B (Figure 4A). dA-1B had the same retention time and λmax as dA (see Figure 1 for comparison). The product was isolated and subjected to ESI-MS and NMR analysis. The high-resolution ESI-mass spectrum of dA-1B exhibited the protonated molecular ion peak at m/z 252.1097 and a base

peak at m/z 136.0620, which was consistent with the data of dA. The 1H NMR spectrum of dA-1B in DMSO-d6 matched that of dA in DMSO-d6 well. In addition, dA-1B coeluted with the dA standard. Furthermore, acid hydrolysis of dA-1B yielded a product with the retention time at 6.3 min, which coeluted with the acid hydrolysis product of dA. The HPLC chromatogram of the reaction mixture of CBO with dA-1B matched that of CBO with dA well. Collectively, these results provided clear evidence that dA-1B was dA. Incubation of dA-2 at pH 7.4 and 37 °C yielded two products, which were identified as dA-1 and dA-1B (dA) based on the coelution experiments with the purified dA-1 and dA standard (Figure 4B). The decrease in the peak area of dA-2 (monitored at 260 nm) was equal to the combined increases in the peak areas of dA-1 and dA within the experimental errors (data not shown), indicating that decomposed dA-2 was completely converted to dA-1 and dA. F

DOI: 10.1021/acs.chemrestox.6b00095 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology

Scheme 3. Proposed Mechanism of Decomposition of dA-1 to dA-1B (i.e., dA) and HMVK under in Vitro Physiological Conditions

the absence of the deoxyribose moiety. The 1H NMR spectrum showed that purified A-1D still contained impurities (Figure S10); however, the impurities did not contain aromatic rings because only two peaks, which appeared at 8.50 and 8.60 ppm (Table 1), were observed at low field (>5.5 ppm). Apparently, the two peaks were the signals of the two protons (H2 and H8) on the purine ring of A-1D. The signals of H3″, which appeared at 2.09 and 2.23 ppm, were easily identified because their chemical shifts changed little in comparison with dA-1 (Table 1), and no other signals from 2.0 to 3.0 ppm could be assigned to H3″. However, it was somewhat difficult to identify the signals of H1″ and H4″ due to overlapping with the peaks of impurities (Figure S10). To overcome the problem, the HMQC spectrum of A-1D was collected (Figure S11). The signals of H1″ and H4″ were assigned on the basis of the 13C chemical shifts of the corresponding carbon atoms, which changed little in comparison with dA-1 (for example, the 13C chemical shift of C4″ in A-1D was 43.3 ppm, and this value in dA-1 was 43.5 ppm, indicating that the loss of the deoxyribose moiety virtually had no effect on the 13C chemical shifts of the carbon atoms in the CBO moiety; Table 1). In fact, the final results showed that, compared to its precursor dA-1, the signals of the protons in the CBO moiety in A-1D (i.e., H1″, H3″, and H4″) did not change in terms of chemical shifts and peak splitting. Thus, A-1D was characterized as 1,N6-(1-hydroxy-1hydroxymethylpropan-1,3-diyl)adenine (Scheme 2). Hydrolysis of dA-2 in 0.1 M HCl at 100 °C for 1 h gave one major product with a retention time of 15.9 min and λmax at 267 nm (see Figure S1 for the UV absorption spectrum), which was designated as A-2D. In the high-resolution ESI-mass spectrum (Figure S2D), its protonated molecular ion appeared at m/z 240.0653 and contained a chlorine atom, which was consistent with the expected structure of a deribosylated product (the calculated formula weight of protonated molecular ion was 240.0652). The 1H NMR spectrum indicated the absence of the deoxyribose moiety (Figure S12). Obviously, like A-1D, it was the hydrolysis product after dA-2 lost the deoxyribose moiety. The signals of the two protons on the purine ring (H2 and H8) appeared at 8.56 and 8.66 ppm (Table 1, Figure S12). The HMBC spectrum (Figure S13) indicated that the proton at 8.66 ppm coupled to carbon atoms at 43.2, 117.6, and 146.4 ppm, whereas the proton at 8.56 ppm coupled to carbon atoms at 117.6 and 148.0 ppm. Therefore, apparently the proton at 8.66 ppm was H2 (the 13C chemical shift of C2 was 146.3 ppm based on the HMQC spectrum) because the carbon atom at 43.2 ppm was one at the CBO moiety (see below). The HMQC spectrum (Figure S14) indicated that the signals at 4.60 and 4.33 ppm, 4.06 and 3.94 ppm, and 2.33 and 2.25 ppm

Further Confirmation of the Decomposition Mechanism of dA-1. To explain the decomposition of dA-1 to dA, a mechanism was proposed (Scheme 3). On the basis of the mechanism, dA-1 decomposed to dA and HMVK. To provide evidence for this mechanism, we used GSH to trap HMVK as the HMVK-GSH conjugate because HMVK is unstable at pH 7.4 and 37 °C.15 dA-1 was incubated with an excess of GSH (the molar ratio of GSH/dA-1 was about 5:1) at pH 7.4 and 37 °C for 29 h. As expected, a new peak at 10.1 min was observed in the HPLC chromatogram of the reaction mixture (Figure 5), which was determined to be the HMVK-GSH conjugate by matching its elution time and UV absorption spectrum with those of the HMVK-GSH standard.

Figure 5. HPLC chromatogram of the reaction mixture of dA-1 with GSH after incubation at pH 7.4 and 37 °C for 29 h to confirm the formation of HMVK during the incubation of dA-1 (monitoring wavelength: 210 nm). The retention times of GSH and oxidized GSH (GSSG) were 6.5 and 10.5/10.7 (a doublet peak), respectively.

Characterization of the Acid Hydrolysis Products of dA-1 and dA-2. To be used as potential biomarkers, the acid hydrolysis products of dA-1 and dA-2 were prepared and characterized. Hydrolysis of dA-1 in 0.1 M HCl at 100 °C for 1 h yielded a predominant product (which was designated as A1D) with a retention time of 4.5 min and λmax at 265 nm (see Figure S1 for the UV absorption spectrum). The formula weight of A-1D was 222.0992 (contained no chlorine atom) as determined by its high-resolution ESI-mass spectrum (Figure S2C), which was consistent with the expected formula C9H12N5O2 (the calculated formula weight is 222.0991). Thus, A-1D was the hydrolysis product of dA-1 following the loss of the deoxyribose moiety. This was further confirmed by the 1H NMR spectrum of A-1D, which indicated G

DOI: 10.1021/acs.chemrestox.6b00095 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology were those of three methylene groups with the corresponding 13 C chemical shifts at 43.2, 48.0, and 27.5 ppm, respectively. The signals at 4.60 and 4.33 ppm were identified as those of H4″ because the HMBC spectrum indicated that the two signals coupled to a carbon atom at 146.3 ppm, i.e., C2. The COSY spectrum (Figure S15) indicated that the signals at 4.60 and 4.33 ppm coupled with those at 2.33 and 2.25 ppm, which were thus assigned as the signals of H3″. Thus, the peaks at 4.06 and 3.94 ppm were the signals of H1″ (Table 1). In addition, A-2D was converted to A-1D when incubated at pH 7.4 and 37 °C. Therefore, A-2D was characterized as 1,N6-(1hydroxy-1-chloromethylpropan-1,3-diyl)adenine (Scheme 2). Detection of A-2D from the Reaction of CBO with CT DNA under in Vitro Physiological Conditions. To examine if CBO could react with dA residues in DNA to generate products corresponding to dA-1 and dA-2, CT DNA (2 mg/ mL) was incubated with CBO (1.7 mM) at pH 7.4 and 37 °C for 9 h and then was hydrolyzed in 0.1 M HCl at 75 °C for 20 min to release purines. The resulting solution was analyzed by HPLC. On the basis of identical retention times (confirmed by coelution experiments with the standards) and similar UV absorption spectra to those of the standards, A-1D and A-2D were identified (Figure 6). It should be noted that the amount

Figure 7. LC-MS chromatogram of acid-hydrolyzed cellular DNA under selected ion monitoring mode (the mass range was from 240.0649 to 240.0655). Cellular DNA was extracted from HepG2 cells incubated with 100 μM BSO at 37 °C for 24 h and then 1 μM CBO for 1 h.

cells to form dA adducts, which predominantly consisted of dA2, i.e., dA-2 was stable in DNA and retained its chlorine atom.

■

DISCUSSION CBO is a potential reactive metabolite of BD. We recently demonstrated that it was genotoxic in cultured human liver cells.11 CBO induced the formation of alkali-labile sites on DNA and was capable of directly generating DNA breaks as examined by the comet assay.11 Because CBO is expected to be reactive toward DNA, we characterized the reaction products of CBO with dC and dG under in vitro physiological conditions.12,13 In the present study, we report on the reactivity of CBO with dA and the chemical characterization of products, and their degradation and acid-hydrolyzed products. We also provide evidence for the formation of a fused-ring dA adduct in CT DNA and human cells incubated with CBO. The reaction of CBO with dA (Scheme 2) is similar to that of CBO with dC described previously. Both reactions proceeded through initial Michael addition of CBO to nucleoside ring nitrogen atoms and subsequent cyclization of the resulting intermediates via reaction of the exocyclic amino groups with the carbonyl groups of the CBO moieties, forming fused-ring adducts. These adducts underwent similar hydrolysis of the chloromethyl groups to yield the corresponding hydroxylated products (Scheme 2). A major difference between the two reaction profiles was, however, that there existed a minor product in the reaction of CBO with dC, which was formed through an initial Michael addition of CBO with the exocyclic amino group of dC. The corresponding product was not detected in the reaction of CBO with dA. This may be attributed to multiple factors, including the relative nucleophilic strength of the exocyclic amino groups in comparison with the ring nitrogen atoms, “hardness/softness” of the electrophiles and nucleophiles,16 and kinetic or thermodynamic control of the reactions.17,18 It was interesting to observe the decomposition of dA-1 to produce dA under the used incubation conditions. To explain this decomposition, a mechanism (Scheme 3), in which dA-1 is present in equilibrium with 1, a carbonyl-containing intermediate, and 1 undergoes a retro-Michael reaction to produce dA and HMVK, is proposed. The formation of HMVK was confirmed through its trapping with GSH, providing evidence for this mechanism. Similarly, the observed rapid equilibrium of the two diastereomers of the CBO-dC adduct with 3,N4-fused

Figure 6. HPLC chromatogram of the reaction mixture of CBO (1.7 mM) with CT DNA after incubation at pH 7.4 and 37 °C for 9 h, and acid-hydrolysis (monitoring wavelength: 260 nm). The peaks of A-1D and A-2D were indicated by arrows. Inset: the magnified chromatogram to clearly show the peaks of A-1D and A-2D.

of A-2D was significantly larger than that of A-1D (the peak area of A-2D was 3-fold as large as that of A-1D). In addition, no guanine adducts were detected. The results provide strong evidence that CBO reacted with the dA residues in DNA to form products corresponding to dA-1 and dA-2. Detection of A-2D in Cells Incubated with CBO. To examine if CBO could cause the formation of dA adducts in living cells, GSH-depleted HepG2 cells were incubated with 1 μM CBO at 37 °C for 1 h. DNA was extracted, acidhydrolyzed, and then subjected to LC-MS analysis. Indeed, as shown in Figure 7, a peak with the elution time at 2.26 min was observed under selected ion monitoring mode (the mass range was set at 240.0649−240.0655). The compound that the peak represents exhibited a signal at m/z 240.0650 with an isotope one at m/z 242.0609 (∼1/3 intensity) and had the same retention time as the A-2D standard. Apparently, this compound was A-2D. However, A-1D was not detected. Moreover, no guanine adducts were detected. Therefore, the results demonstrated that CBO could react with DNA in living H

DOI: 10.1021/acs.chemrestox.6b00095 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology

*(A.A.E.) University of WisconsinMadison, Department of Comparative Biosciences, 2015 Linden Dr., Madison, WI 53706, United States. Tel: + 1-608-262-6518. Fax: + 1-608-2623926. E-mail: [email protected].

rings and hydroxyl groups in place of the chlorine atoms suggested the formation of a carbonyl-containing intermediate.12 While dA-2 could also undergo retro-Michael reaction to regenerate dA and CBO, we were unable to obtain evidence for this hypothesis, possibly because of the rapid decomposition of dA-2 to yield dA-1. CBO can form fused-ring adducts with dA, dC, and dG. Similarly, other α,β-unsaturated aldehydes/ketones, such as acrolein,19 4-hydroxy-2-nonenal and crotonaldehyde,20 4-oxo-2nonenal,21 and lipid peroxidation products,22 also form fusedring adducts with the N1- and N6-positions of dA. Bifunctional alkylating agents, such as 1,2,3,4-diepoxybutane, can also lead to the formation of dA adducts with fused-rings.23 These adducts could be mutagenic because they have potential to significantly disrupt the Watson−Crick base pairing in DNA.24,25 Levine et al. demonstrated that 1,N6-ethenodeoxyadenosine, the simplest dA adduct with a fused-ring, was substantially mutagenic in human cells.26 The formation of 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine during stage-specific tumor development in mouse skin has been demonstrated.27 1,N6Ethenodeoxyadenosine was detectable in human urine and its levels were associated with lipid peroxidation-derived DNA damage in vivo, and these adducts have been implicated in the development of hepatocellular carcinoma.28,29 Therefore, the CBO-dA adducts could also be mutagenic and play an important role in the genotoxicity of CHB and BD. In addition, A-2D may be used as a biomarker of CBO formation in human cells. It was somewhat surprising that the amount of A-2D obtained in the CT DNA experiment was significantly greater than that of A-1D. The result suggested that, unlike the adduct at the free nucleoside level, hydrolysis of dA-2 at the DNA level was largely inhibited, probably due to the stacking of nucleobases in DNA. In addition, the fact that no guanine adducts were detected suggested that CBO predominantly reacted with the dA residues in DNA. The result obtained with HepG2 cells was qualitatively consistent with that obtained with CT DNA, supporting the observation that dA-2 was stable in DNA. A-1D was detected in CT DNA but not in HepG2 cells, probably due to the large difference in the CBO concentrations used in these experiments (1.7 mM in the DNA experiment vs 1 μM in the cell one). In addition, no guanine adducts were detected in both experiments, suggesting that, in terms of purines, CBO predominantly reacted with adenine in DNA. The basis for the selectivity of CBO toward dA residues in DNA is presently unclear. More studies with multiple time points and different CBO concentrations are being conducted to clarify the basis for the observed CBO selectivity toward dA residues in DNA.

■

Author Contributions ∥

This work was supported by the National Nature Science Foundation of China (Nos. 21077070 and 21377080), the Shanghai Municipal Education Commission (No. 11ZZ90), the Shanghai Leading Academic Discipline Project (No. S30109), and the National Institutes of Health of the U.S.A. (R01 DK044295 and R01 ES06841). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The NMR spectra of all samples were recorded on the Bruker Avance AV500 spectrometer in the Instrumental Analysis & Research Center of Shanghai University. We are grateful to Dr. Hong-Mei Deng of the Nuclear Magnetic Resonance Laboratory in this center for her assistance with the collection of the NMR spectra. We also thank Dr. Ying Wen and Professor Tao Yi of the Chemistry Department in Fudan University for their assistance in submitting samples for ESI-MS and LC-MS analysis, and Mr. Gang-Feng Tang in this department for the ESI-MS and LC-MS analysis of samples.

■

ABBREVIATIONS ADH, alcohol dehydrogenase; BD, 1,3-butadiene; BSO, Lbuthionine-sulfoximine; CBO, 1-chloro-3-buten-2-one; CHB, 1-chloro-2-hydroxy-3-butene; COSY, correlation spectroscopy; CT DNA, calf thymus DNA; dA, 2′-deoxyadenosine; dC, 2′deoxycytidine; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; ESI, electrospray ionization; FBS, fetal bovine serum; GSH, glutathione; HMBC, heteronuclear multiple-bond correlation; HMQC, heteronuclear multiplequantum correlation; HMVK, hydroxymethyl vinyl ketone; MPO, myeloperoxidase; TFA, trifluoroacetic acid; Tris, tris(hydroxymethyl)aminomethane

■

REFERENCES

(1) Koppikar, A. M., Wu, C., Leavens, T., Valcovic, L., Kimmel, C., Faust, R., and Jinot, J. (2002) Health Assessment of 1,3-Butadiene, EPA/600/P-98/001F, U.S. Environmental Protection Agency, Office of Research and Development, National Center for Environmental Assessment-Washington Office, U.S. Government Printing Office, Washington, DC, http://cfpub.epa.gov/ncea/cfm/recordisplay. cfm?deid=54499 (accessed Mar 19, 2016). (2) Duescher, R. J., and Elfarra, A. A. (1992) 1,3-Butadiene oxidation by human myeloperoxidase. J. Biol. Chem. 267, 19859−19865. (3) Elfarra, A. A., and Zhang, X.-Y. (2012) Alcohol dehydrogenaseand rat liver cytosol-dependent bioactivation of 1-chloro-2-hydroxy-3butene to 1-chloro-3-buten-2-one, a bifunctional alkylating agent. Chem. Res. Toxicol. 25, 2600−2607. (4) Loh, M. M., Levy, J. I., Spengler, J. D., Houseman, E. A., and Bennett, D. H. (2007) Ranking cancer risks of organic hazardous air pollutants in the United States. Environ. Health Perspect. 115, 1160− 1168. (5) McCarthy, M. C., O’Brien, T. E., Charrier, J. G., and Hafner, H. R. (2009) Characterization of the chronic risk and hazard of hazardous air pollutants in the United States using ambient monitoring data. Environ. Health Perspect. 117, 790−796.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.6b00095. UV absorption, NMR, and high-resolution ESI-mass spectra of dA-1, dA-2, A-1D, and A-2D (PDF)

■

F.-M.Z. and L.-Y.L. contributed to the work equally.

Funding

AUTHOR INFORMATION

Corresponding Authors

*(X.-Y.Z.) Tel: + 86-21-6613-7736. Fax: + 86-21-6613-6928. Email: [email protected]. I

DOI: 10.1021/acs.chemrestox.6b00095 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Article

Chemical Research in Toxicology

(2015) Polymerase bypass of N6-deoxyadenosine adducts derived from epoxide metabolites of 1,3-butadiene. Chem. Res. Toxicol. 28, 1496− 1507. (25) Kotapati, S., Maddukuri, L., Wickramaratne, S., Seneviratne, U., Goggin, M., Pence, M. G., Villalta, P., Guengerich, F. P., Marnett, L., and Tretyakova, N. (2012) Translesion synthesis across 1,N6-(2hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N6-γHMHP-dA) adducts by human and archebacterial DNA polymerases. J. Biol. Chem. 287, 38800−38811. (26) Levine, R. L., Yang, I.-Y., Hossain, M., Pandya, G. A., Grollman, A. P., and Moriya, M. (2000) Mutagenesis induced by a single 1,N6ethenodeoxyadenosine adduct in human cells. Cancer Res. 60, 4098− 4104. (27) Nair, J., Furstenberger, G., Burger, F., Marks, F., and Bartsch, H. (2000) Promutagenic etheno-DNA adducts in multistage mouse skin carcinogenesis: correlation with lipoxygenase-catalyzed arachidonic acid metabolism. Chem. Res. Toxicol. 13, 703−709. (28) Nair, J., Srivatanakul, P., Haas, C., Jedpiyawongse, A., Khuhaprema, T., Seitz, H. K., and Bartsch, H. (2010) High urinary excretion of lipid peroxidation-derived DNA damage in patients with cancer-prone liver diseases. Mutat. Res., Fundam. Mol. Mech. Mutagen. 683, 23−28. (29) Bartsch, H., and Nair, J. (2005) Accumulation of lipid peroxidation-derived DNA lesions: potential lead markers for chemoprevention of inflammation-driven malignancies. Mutat. Res., Fundam. Mol. Mech. Mutagen. 591, 34−44.

(6) Zhou, J., You, Y., Bai, Z., Hu, Y., Zhang, J., and Zhang, N. (2011) Health risk assessment of personal inhalation exposure to volatile organic compounds in Tianjin, China. Sci. Total Environ. 409, 452− 459. (7) Logue, J. M., McKone, T. E., Sherman, M. H., and Singer, B. C. (2011) Hazard assessment of chemical air contaminants measured in residences. Indoor Air 21, 92−109. (8) Fowles, J., and Dybing, E. (2003) Application of toxicological risk assessment principles to the chemical constituents of cigarette smoke. Tob. Control 12, 424−430. (9) Kirman, C. R., Albertini, R. J., Sweeney, L. M., and Gargas, M. L. (2010) 1,3-Butadiene: I. Review of metabolism and the implications to human health risk assessment. Crit. Rev. Toxicol. 40 (Suppl. 1), 1−11. (10) Albertini, R. J., Carson, M. L., Kirman, C. R., and Gargas, M. L. (2010) 1,3-Butadiene: II. Genotoxicity profile. Crit. Rev. Toxicol. 40 (Suppl. 1), 12−73. (11) Liu, X.-J., Zeng, F.-M., An, J., Yu, Y.-X., Zhang, X.-Y., and Elfarra, A. A. (2013) Cytotoxicity, genotoxicity, and mutagenicity of 1-chloro2-hydroxy-3-butene and 1-chloro-3-buten-2-one, two alternative metabolites of 1,3-butadiene. Toxicol. Appl. Pharmacol. 271, 13−19. (12) Sun, L., Pelah, A., Zhang, D.-P., Zhong, Y.-F., An, J., Yu, Y.-X., Zhang, X.-Y., and Elfarra, A. A. (2013) Formation of fused-ring 2′deoxycytidine adducts from 1-chloro-3-buten-2-one, an in vitro 1,3butadiene metabolite, under in vitro physiological conditions. Chem. Res. Toxicol. 26, 1545−1553. (13) Zheng, J., Li, Y., Yu, Y.-X., An, J., Zhang, X.-Y., and Elfarra, A. A. (2015) Novel adducts from the reaction of 1-chloro-3-buten-2-one with 2′-deoxyguanosine. Structural characterization and potential as tools to investigate 1,3-butadiene carcinogenicity. Chem.-Biol. Interact. 226, 40−48. (14) Reppe, W., and Mitarbeiter (1955) Ethynylation. IV. Reactions of α-alkynols and γ-alkynediols. Justus Liebigs Ann. Chem. 596, 38−79. (15) Krause, R. J., Kemper, R. A., and Elfarra, A. A. (2001) Hydroxymethylvinyl ketone: a reactive Michael acceptor formed by the oxidation of 3-butene-1,2-diol by cDNA-expressed human cytochrome P450s and mouse, rat, and human liver microsomes. Chem. Res. Toxicol. 14, 1590−1595. (16) LoPachin, R. M., Gavin, T., Petersen, D. R., and Barber, D. S. (2009) Molecular mechanisms of 4-hydroxy-2-nonenal and acrolein toxicity: nucleophilic targets and adduct formation. Chem. Res. Toxicol. 22, 1499−1508. (17) Freccero, M., Gandolfi, R., and Sarzi-Amade, M. (2003) Selectivity of purine alkylation by a quinone methide. Kinetic or thermodynamic control? J. Org. Chem. 68, 6411−6423. (18) Veldhuyzen, W. F., Shallop, A. J., Jones, R. A., and Rokita, S. E. (2001) Thermodynamic versus kinetic products of DNA alkylation as modeled by reaction of deoxyadenosine. J. Am. Chem. Soc. 123, 11126−11132. (19) Pawlowicz, A. J., Munter, T., Zhao, Y., and Kronberg, L. (2006) Formation of acrolein adducts with 2′-deoxyadenosine in calf thymus DNA. Chem. Res. Toxicol. 19, 571−576. (20) Chen, H.-J. C., and Chung, F.-L. (1994) Formation of etheno adducts in reactions of enals via autoxidation. Chem. Res. Toxicol. 7, 857−860. (21) Rindgen, D., Lee, S. H., Nakajima, M., and Blair, I. A. (2000) Formation of a substituted 1,N6-etheno-2′-deoxyadenosine adduct by lipid hydroperoxide-mediated generation of 4-oxo-2-nonenal. Chem. Res. Toxicol. 13, 846−852. (22) el Ghissassi, F., Barbin, A., Nair, J., and Bartsch, H. (1995) Formation of 1,N6-ethenoadenine and 3,N4-ethenocytosine by lipid peroxidation products and nucleic acid bases. Chem. Res. Toxicol. 8, 278−283. (23) Seneviratne, U., Antsypovich, S., Goggin, M., Dorr, D. Q., Guza, R., Moser, A., Thompson, C., York, D. M., and Tretyakova, N. (2010) Exocyclic deoxyadenosine adducts of 1,2,3,4-diepoxybutane: synthesis, structural elucidation, and mechanistic studies. Chem. Res. Toxicol. 23, 118−133. (24) Kotapati, S., Wickramaratne, S., Esades, A., Boldry, E. J., Quirk Dorr, D., Pence, M. G., Guengerich, F. P., and Tretyakova, N. Y. J

DOI: 10.1021/acs.chemrestox.6b00095 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX