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Analysis of Different Fates of DNA Adducts in Adipocytes Post-sulfur Mustard Exposure in Vitro and in Vivo Using a Simultaneous UPLCMS/MS Quantification Method Peng Wang,† Yajiao Zhang,† Jia Chen,† Lei Guo,† Bin Xu,† Lili Wang,‡ Hua Xu,*,† and Jianwei Xie*,† †

State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27 Taiping Road, Haidian District, 100850, Beijing, China ‡ State Key Laboratory of Toxicology and Medical Countermeasures, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 27 Taiping Road, Haidian District, 100850, Beijing, China S Supporting Information *

ABSTRACT: Sulfur mustard (SM) is a powerful alkylating vesicant that can rapidly penetrate skin, ocular, and lung bronchus mucous membranes and react with numerous nucleophiles in vivo. Although the lesion mechanisms of SM remain unclear, DNA damage is believed to be the most crucial factor in initiating SM-induced toxicity. Four major DNA adducts were identified for retrospective detection and DNA lesion evaluation, namely, N7-[2-[(2-hydroxyethyl)thio]-ethyl]guanine (N7-HETEG), bis(2ethyl-N7-guanine)thioether (Bis-G), N3-(2-hydroxyethylthioethyl)-2′-adenine (N3-HETEA), and O6-[2-[(2-hydroxyethyl)thio]ethyl]guanine (O6-HETEG). Because of previous observations that the levels of SM-DNA adducts were relatively higher in adipose-rich organs, such as the brain, we focused on the in vitro and in vivo fates of the DNA adducts in exposed adipocytes. A UPLC-MS/MS method developed in our laboratory was used to profile the N7-HETEG, Bis-G, and N3-HETEA levels in human mature adipocytes (HA-s) that had differentiated from human subcutaneous preadipocytes (HPA-s). This method was also used to profile three other cell lines related to the targeting of major tissues, including human keratinocytes (HaCaT), human hepatocytes (L-02), and human lung fibroblasts (HLF). Long-lasting adduct persistence and a high proportion of Bis-G were found in exposed adipocytes in vitro. The survival properties of exposed adipocytes were also tested. At the same time, the fate of SM-DNA adducts in vivo was characterized using a rat model exposed to 1 and 10 mg/kg doses of SM. The level of DNA adducts in the exposed adipose tissue (AT) was much lower than those in other organs studied in our previous work. The adduct persistence behavior was observed in AT with an extremely high proportion of Bis-G, which was higher than N7-HETEG. In light of these results, we suggest that an adipose-rich environment may promote the formation of Bis-G and that adipocyte-specific DNA repair mechanisms may result in adduct persistence and the survival of adipocytes after SM exposure. These conclusions should be further investigated.

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INTRODUCTION

Furthermore, residual SM contamination from war remains a continuous threat to civilians.6,7 For example, SM chemical weapons abandoned by the Japanese army after 1945 were scattered throughout China; exposure accidents occasionally occurred.8 Furthermore, because it is relatively simple to synthesize and maintain, the potential terrorist use of SM is a

Sulfur mustard (2,2′-dichloroethyl sulfide, SM) is one of the most utilized chemical warfare agents.1 Because of its highly reactive chemical properties, liquid or vapor SM can cause serious damage to exposed organisms.2 Since World War I in the early 20th century to the recent Iraq−Iran conflict, in addition to SM-inflicted death, a large number of victims have also suffered from SM-induced blistering, necrosis, painful wounds, metabolic disorders, and chronic diseases.3−5 © 2015 American Chemical Society

Received: February 2, 2015 Published: May 8, 2015 1224

DOI: 10.1021/acs.chemrestox.5b00055 Chem. Res. Toxicol. 2015, 28, 1224−1233

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Chemical Research in Toxicology present threat.9 Therefore, understanding the effects of SM requires urgent attention. As a powerful vesicant and a bifunctional alkylating agent, SM can rapidly penetrate the skin and ocular and lung bronchus mucous membranes to react with numerous nucleophiles, including most in vivo biomolecules.3,10 Soon after SM exposure, the hydrolysis and oxidation of metabolites and alkylates of nucleic acids and proteins can be detected in the body. These alkylation products and metabolites have been used as biomarkers to verify SM exposure.11−13 These products are associated with SM lesions; however, the exact mechanism through which SM lesions appear remains unclear. DNA damage is believed to be the most crucial factor in initiating SM-induced toxicity.14 With the formation of SMDNA mono and biadducts, nucleic acid and peptide syntheses are influenced. As a result, the cell cycle is interrupted and apoptosis often results.10 Moreover, high levels of DNA adducts can induce the consumption of ATP and the death of pro-inflammatory cells, with subsequent comprehensive damage to adjacent cells and tissues.15 Therefore, interest has grown in investigating SM-DNA adducts. Because of DNA sensitivity to alkylating agents, alkylation can occur on purine bases after DNA is exposed to SM. As a result, the reaction between SM and DNA is mainly at the N7 or O6 positions of guanine and the N3 position of adenine to form four major types of SM-DNA adducts (Figure 1). These

disparate results, including differences in exposure methods, time point selections, or the analytical method. There have also been instances in in vivo studies that were not anticipated or controlled, including the absorbance ratio of SM, the state of experimental animals, and the individual responses to SM exposure. To directly and objectively examine the DNA adduct formation profiles without the complexity of in vivo factors, an improved analytical method should be established for the detection of DNA adducts in different tissue-derived cell lines that have been exposed to SM in vitro. In our previous work, we observed that the SM-DNA adduct levels in adipose-rich tissues, including the brain, were peculiarly high.25 SM has lipophilic properties.26 It has been suggested that the accumulation of SM in adipose or hydrophobic environments (such as lipids) and the subsequent biological effects may be crucial for the distribution of and damage caused by SM. However, because of its chemical properties and rapid reaction rates, the in vivo accumulation of SM in adipose tissue is rarely considered, and its significance has been ignored. One study did report higher levels of SM in skin with subcutaneous adipose tissue (AT) and the brain from a dead Iranian victim 7 days postexposure, as detected by GCMS.27 Our recent experiment demonstrated that higher levels and persistent SM adduct formations occurred in AT in vivo, as detected by a novel UPLC-ESI-MS/MS derivatization method.28 This was an interesting and significant phenomenon that reflected the cumulative effects on AT by SM. Because SM accumulation in AT has historically been overlooked, research associated with SM-DNA adducts in adipocytes have been particularly rare. Therefore, studies on SM-DNA adduct profiles in adipocytes are urgently needed. Using an established isotope dilution UPLC-MS/MS method,29 a few changes were made to accommodate adduct acquisitions from cultured cells. After our method was validated, we focused on the fate of the SM-DNA adducts in adipocytes in vitro and in vivo. Adipocytes that had differentiated from human subcutaneous preadipocytes were selected for further investigation on the influence of high adipose content on DNA adduct formation and repair in vitro. The major target tissues of the adducts include the liver, skin, and lungs. Therefore, three types of human cell lines were used as controls. At the same time, an animal model was used to identify the fate of SM-DNA adducts in adipocytes in vivo. Adducts of N7-HETEG, Bis-G, and N3-HETEA were detected, and their time and dose-dependent profiles were recorded and are presented herein.

Figure 1. Chemical structure for SM-DNA adducts. (A) N7-HETEG, (B) N3-HETEA, (C) Bis-G, and (D) O6-HETEG.

include monoadducts, such as N7-[2-[(2-hydroxyethyl)thio]ethyl]guanine(N7-HETEG), N3-(2-hydroxyethylthioethyl)-2′adenine(N 3 -HETEA), and O 6 -[2-[(2-hydroxyethyl)thio]ethyl]guanine(O6-HETEG) adducts, and a biadduct, such as bis(2-ethyl-N7-guanine)thioether (Bis-G).16−18 These adducts have been identified as biomarkers that indicate internal exposure and can be used to characterize the DNA lesions caused by SM. Various analytical methods including immunoslot-blot assays, isotope labeling, and LC-MS have been used to detect DNA adducts.19−22 Because of the different mutagenic properties and repair rates, adduct-specific data even from the same source are significant. Recently, HPLC-ESI-MS/MS was utilized for the simultaneous detection of SM-DNA adducts after acidic or enzymatic DNA digestion.12,23 Using these methods, the fate of DNA adducts was investigated after SM exposure in vivo, and the distinct time and dose-dependent profiles of the adducts and their distribution in tissues were examined in mice, rats, and humans. Although significant progress has been achieved, the results have been difficult to compare. For example, the prevalence of Bis-G was observed over a very wide range, from 17% to 45%.12,15,23,24 Several factors may contribute to these

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MATERIALS AND METHODS

Caution: SM is a reactive alkylating and cytotoxic agent. All associated experiments were carefully carried out in a f ume hood with adequate protection by experienced personnel. Chemicals and Reagents. SM was supplied by the Institute of Chemical Defense of the Chinese People’s Liberation Army, with purity higher than 95%. HPLC grade methanol and chloroform were purchased from Dunsan Pure Chem. Co., Ltd. (Ansan-si, Korea). Analytical grade formic acid was obtained from Sinopharm Chemical Reagents Co., Ltd. (Beijing, China). Phenol was supplied by HaoYang Biological Co., Ltd. (Tianjin, China). Standards (N7-HETEG and BisG, N3-HETEA) and deuterated internal standards (ISs) (d4-N7HETEG, d4-Bis-G, d4-N3-HETEA) were synthesized with purity above 95%, and deuterated ratios were more than 99%.12 Protease K was purchased from Merck KGaA (Darmstadt, Germany). 3-Isobutyl-1methylxanthine (IBMX), dexamethasone (DEX), and insulin (INS) were purchased from Sigma-Aldrich Co. (MO, USA). Oil-red-O was 1225

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Chemical Research in Toxicology purchased from Solar Bio S&T Co., Ltd. (Beijing, China). Ultrapure water was produced by a Milli-Q A10 purification system from EMD Millipore Co. (MA, USA). Animals, Cell Lines, Cell Culture, and Differentiation. Adult male Sprague−Dawley rats of a specific pathogen-free grade were supplied by the Laboratory Animal Center of Beijing and maintained in the Beijing Center for Toxicological Evaluation and Research in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the center. All animal experiments were in compliance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International. The human keratinocyte cell line HaCaT, the human hepatocyte cell line L-02, and the human lung fibroblasts cell line HLF were provided by the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose supplemented with 10% fetal bovine serum (FBS, Gibco, NY, USA). The HPA-s cell line that was derived from human subcutaneous fat tissue was purchased from Sciencell Research Laboratories (CA, USA) and cultured in preadipocyte medium (PAM) supplemented with 5% FBS. All cultures were maintained at 37 °C in a humidified incubator in 5% CO2. For adipocyte differentiation, HPA-s cells were seeded onto poly-Llysine-coated 6-well plates at 2.5 × 105 cells per well until the cells reached 100% confluence. Then, the culture medium was replaced with a preadipocyte differentiation medium in DMEM containing 10% (v/v) FBS, 0.5 μM DEX, 0.5 mM IBMX, and 10 μg/mL INS. From day 3, the cells were cultured in DMEM supplemented with 10% (v/v) FBS and 10 μg/mL INS. The medium was changed every 2 days until approximately 80% of the cells had differentiated. The mature adipocytes are referred to as HA-s in this article. Oil-red-O staining was performed to monitor the differentiation of HPA-s according to the protocol provided by Sciencell Research Laboratories. Briefly, the medium was removed, and cells were washed with 0.01 M PBS (pH 7.4) followed by 10% (v/v) formaldehyde fixation for 1 h. After removing the fixative solution, the culture vessels were rinsed with water. Then, oil-red-O solution was used to visualize lipid droplets for 1 h, and the stained cells were observed on a phase contrast microscope (Olympus Co., Tokyo, Japan). All steps were carried out at room temperature. SM Exposure. For Cells. SM was initially diluted with DMSO and subsequently diluted to 100 μM with medium for exposure. First, the medium was removed, and the cells were washed twice with FBS-free culture medium. Then, cultured cells in 6-well plates were exposed to 100 μM SM for 1 h at 37 °C. The supernatant was removed, and the cells were washed three times with medium. Subsequently, cells were cultured at 37 °C in the presence of 5% CO2. The cells were lysed with 0.5 mL of lysis buffer (5 mM Tris-HCl, 100 mM ethylene diaminetetraacetic acid, and 0.75% w/v sodium dodecyl sulfonate) per well at time points of 5, 15, 30, and 45 min and 1, 3, 6, 12, 24, and 48 h after the 1 h SM-exposure. The lysate was collected to extract DNA. For Animals. Rats of 277 ± 15 g weight were randomly divided into eight groups of five each. Two groups were exposed to dosages at four time points. After several days of adaptable feeding, hair cleaning was performed by treatment with 8% Na2S for 24 h before SM treatment. Following anesthesia by intraperitoneal injection with 10% urethane, animals were dermally exposed to 10 μL of DMSO-diluted SM at dosages of 1 mg/kg or 10 mg/kg. After exposure, the treatment site of the skin was immediately covered by plastic wrap. Eventually, animals were euthanized with urethane by intraperitoneal injection at 1, 3, 9, and 12 h after exposure, and subcutaneous, epididymis, perirenal and brown AT samples were isolated. Samples (0.2 g) of the tissues were transferred into a cleaning tube with a sterile steel ball and were lysed by agitation. After the removal of the supernatant, lysis buffer was added to extract DNA. DNA Extraction and Acquisition of DNA Adducts. After cell lysis, the supernatant was collected into a clean tube with additional protease K and incubated at 55 °C for 1 h. Proteins were denatured by adding 0.5 mL of denaturation buffer (isoamylol/chloroform/phenol, 1:24:25 v/v/v) while vortexing. After centrifugation, the supernatant

aqueous phase was transferred into a new tube, and a DNA pellet was extracted by adding 2.5 volumes of chilled ethanol and incubated at −20 °C overnight. To extract the DNA, the sample was subjected to pelleting at 14,000g for 15 min. The resultant supernatant was discarded, and the pellet was washed with 2 mL of 75% (v/v) ethanol. After a second centrifugation, the ethanol was removed, and the ethanol was evaporated at room temperature. The DNA was redissolved in 0.75 mL of water. Then, the absorption of a 0.05 mL DNA sample was measured at 260 and 280 nm on a Cary 300 UV−vis analyzer (Varian Medical Systems Inc., CA, USA). A 0.7 mL DNA sample was mixed with 0.3 mL of formic acid for acidolysis at 110 °C for 30 min. After the addition of 0.05 mL of IS, the solvent was evaporated at 70 °C under vacuum. Samples were redissolved in 0.05 mL of water and analyzed by UPLC-ESI-MS/MS. UPLC-ESI-MS/MS Quantification of DNA Adducts. The UPLCESI-MS/MS method for the quantification of the DNA adducts was performed as previously described.29 Different DNA adducts were simultaneously quantified. Briefly, samples were separated by an ACQUITY UPLC system (Waters Co., Manchester, UK), which included a binary pump, an autosampler, and an ACQUITY UPLC BEH C-18 column (50 × 2.1 mm I.D., 1.7 μm particle size, Waters Co.). Water was used as mobile phase A; methanol was used as mobile phase B. After 3 μL of the sample was injected, the gradient elution was linearly increased from 5% to 35% B (v/v) over 4 min at 40 °C at a flow rate of 0.35 mL/min. The LC elute was then delivered into a 5500 tandem mass spectrometer (AB Sciex, MA, USA) for ESI-MS/ MS analysis. Analytes and ISs were eventually detected in positive mode at unit resolution for both Q1 and Q3 using multiple reaction monitoring (MRM) with a dwell time of 100 ms. The quantification precursor/product ion pairs were monitored for N7-HETEG, N3HETEA, and Bis-G and were m/z 256 → 105, m/z 240 → 105, and m/z 389→ 210, respectively (Figure S1, Supporting Information). The pairs for corresponding deuterated ISs were m/z 260 → 109, m/z 244 → 109, and m/z 393 → 214, respectively. Analytical Method Validation. The analytical method was validated as follows. The N7-HETEG, Bis-G, and N3-HETEA standards that were prepared in water and stored at −20 °C were diluted to 0.7 mL in a series of concentrations of 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, and 20 μg/L using a blank matrix, which was prepared using DNA extracted from unexposed HaCaT cells. An additional 0.3 mL of formic acid was added to 0.05 mL of ISs (d4-N7-HETEG, d4-BisG, and d4-N3-HETEA at concentrations of 21, 62, and 37 μg/L), and standard samples were evaporated and redissolved as described above. The detection results of UPLC-ESI-MS/MS were used to plot a calibration curve, and the lower limit of detection (LLOD) and the lower limit of quantification (LLOQ) for the DNA adducts were defined as the lowest concentration with a signal-to-noise ratio (S/N) greater than 5 and 10, respectively. Low, medium, and high quality control measures (LQC, MQC, and HQC) were accordingly defined and used to evaluate the within-run and between-run precision and accuracy. The IS-normalized matrix effect (ME), recovery, freeze and thaw stability, and short-term stock stability at 4 and 25 °C for 24 h were also evaluated. Data Analysis and Statistics. Both in vitro and in vivo results were expressed as the means ± standard errors of the mean (SE). Data were analyzed with IBM SPSS version 19.0 using single factor analysis of variance (ANOVA). Comparison analyses among the means of groups were performed by Fisher’s least significant difference (LSD) method, in which differences were considered significant at p < 0.05. The kinetic parameters were calculated with WinNonlin 6.3 software from Pharsight Company (NJ, USA).

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RESULTS AND DISCUSSION Validation of a UPLC- MS/MS Simultaneous Quantification Method for DNA Adducts. In this work, an established UPLC-MS/MS method was utilized to detect three major adducts within the same sample, i.e., N7-HETEG, Bis-G, and N3-HETEA. The method validation was carried out according to the European Medicines Agency guidelines.30 1226

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Chemical Research in Toxicology Table 1. Linearity Parameters of the UPLC-MS/MS Method for SM-DNA Adduct Simultaneous Quantification analyte 7

N -HETEG Bis-G N3-HETEA

linear range (μg/L)

calibration curve

r2

LLOD (μg/L)

LLOQ (μg/L)

0.02−20 0.05−20 0.10−20

Y = 0.894X + 0.003 Y = 3.142X + 0.035 Y = 2.940X − 0.002

0.995 0.995 0.999

0.01 0.02 0.05

0.02 0.05 0.1

Table 2. Matrix Effects, Recovery, within and between-Run Precision and Accuracy Validation Parameters for the Methoda between-runb n = 6

within-run n = 6 analyte N7-HETEG

Bis-G

N3-HETEA

conc. (μg/L) LLOQ LQC MQC HQC LLOQ LQC MQC HQC LLOQ LQC MQC HQC

0.02 0.05 10 15 0.05 0.1 10 15 0.1 0.2 10 15

RSD (%)

accuracy (%)

RSD (%)

accuracy (%)

3.3 3.3 3.3 3.7 3.4 5.0 2.2 5.4 3.2 4.6 6.2 5.4

104 102 91 88 92 95 101 94 98 94 105 92

6.8 7.0 3.7 2.8 6.9 5.6 3.3 4.2 6.7 6.3 6.8 5.7

102 100 91 88 95 95 99 95 95 95 99 91

I-MF (%) n = 6

I-R (%) n = 6

100 97 102

100 90 88

112 86 88

113 96 92

109 98 107

92 93 100

a

Conc.: Concentration. I-MF: IS-normalized matrix factor. I-R: IS-normalized recovery. bThree batches of QC samples were used for between-run precision and accuracy validation.

Table 3. Concentration and Percentage of SM-DNA Adducts in Different Cell Lines at 5 min after Exposurea analyte N7-HETEG Bis-G N3-HETEA

conc. percent of total adducts (%) conc. percent of total adducts (%) conc. percent of total adducts (%)

HaCaT

HLF

L-02

HPA-s

HA-s

225 ± 6 73.3 77.6 ± 8.2 25.2 5.1 ± 0.2 1.7

219 ± 8 79.4 51.9 ± 3.1 18.8 5.1 ± 0.2 1.8

166 ± 7 66.7 79.3 ± 3.5 31.7 4.1 ± 0.2 1.6

297 ± 10 73.8 99.8 ± 4.4 24.8 5.6 ± 0.3 1.4

200 ± 9 65.6 101 ± 2 33.2 3.9 ± 0.3 1.3

Conc.: Concentration was expressed as adducts per 106 bases (mean ± SE). HaCaT: A human keratinocyte cell line derived from skin. HLF: A human lung fibroblasts cell line derived from lung. L-02: A human hepatocyte cell line derived from liver. HPA-s: A human subcutaneous preadipocyte cell line derived from subcutaneous adipose tissue. HA-s: A human adipocyte cell differentiated from HPA-s.

a

precision of the method. For accuracy, all mean concentrations of the QC samples were within 15% of nominal values. For precision, all CV values of the QC samples were less than 15%. The accuracy and precision results satisfied the EMEA guidelines and are shown in Table 2. To evaluate the method matrix effects and recovery, three sets of QC samples were prepared. Samples in the first set (i.e., Set 1) were prepared by adding the standards and ISs into an acid hydrolyzed blank matrix from different individuals at low, medium, and high levels (n = 3). For Set 2, standards and ISs were spiked into water at the same corresponding concentrations. For Set 3, the mixtures of standards, ISs, and the blank matrix at the same corresponding concentrations were acid hydrolyzed. The matrix factor (MF) of the standards and ISs were calculated by dividing the peak areas of Set 1 by the peak areas of Set 2. The IS-normalized MF was calculated by dividing the MF of the standard by the MF of the IS; the ISnormalized MF ranged from 86% to 112% (Table 2). This indicated that there was no obvious influence of the matrix on MS detection. The CV values of the IS-normalized MF at the low, medium, and high levels for each adduct were all below 15%. The recovery was determined by comparing the peak areas in Set 3 to the peak areas in Set 1. The IS-normalized recovery was calculated by dividing the recovery of the standard

Three batches of adduct standards were diluted to nine concentrations by the blank matrix to verify the LLOD and linear detection range. The LLOQ, LQC, MQC, and HQC were determined to evaluate the precision, accuracy, matrix effects, recovery, and stability of the method. A calibration curve of the ratios of the adduct peak to the ISs versus the adduct concentrations was plotted, and analytical linear ranges were confirmed (Table 1). The LLOD was 0.01, 0.02, and 0.05 μg/L for N7-HETEG, Bis-G, and N3-HETEA, respectively. The ULOD was 20 μg/L for all three adducts, while the LLOQ was 0.02, 0.05, and 0.1 μg/L for N7-HETEG, Bis-G, and N3-HETEA, respectively. Compared with a similar report,23 the isotope dilution UPLC-MS/MS method accurately detected adducts even at lower concentrations with a wider linear range. Therefore, our method meets the requirements for evaluating SM-DNA adducts. After confirming the analytical range, the standards were diluted to different concentrations with a blank matrix to prepare the QC samples. The LLOQ (0.02, 0.05, and 0.1 μg/L for N7-HETEG, Bis-G, and N3-HETEA, respectively), LQC (0.05, 0.1, and 0.2 μg/L for N7-HETEG, Bis-G, and N3HETEA, respectively), MQC (10 μg/L for all adducts), and HQC (15 μg/L for all adducts) from three batches were utilized within and between runs to validate the accuracy and 1227

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Chemical Research in Toxicology to the recovery of the IS; the IS-normalized recovery ranged from 88% to 113%. The CV values of the IS-normalized MF at low, medium, and high levels for each adduct were all below 15%, indicating that acidolysis did not influence the recovery of the three adducts. To acquire reliable data, the samples were analyzed as quickly as possible, to reduce the exposure of the samples to freezing conditions. We investigated the short-term stability at 4 and 25 °C for 6, 12, and 24 h and one freeze−thaw cycle, using the LQC, MQC, and HQC samples. The subsequent storage of the analytical sample was also within these conditions. The relative error values between the poststorage and original QC samples were all within ±15% (Table S1, Supporting Information), showing good short-term stability. Analysis of SM-DNA Adducts by the End of Exposure in Vitro. As validated, the UPLC-MS/MS method was used to evaluate the levels of the three SM-DNA adducts in vitro. After a 1 h exposure to SM, cells were rinsed and collected at 5 min, and the SM-DNA adducts were analyzed for all five cell lines. The concentration of each adduct was calculated and expressed as adducts per 106 bases. As shown in Table 3, the concentration of each adduct reached a high level shortly after exposure. There were a few differences in adduct levels among the cell lines. However, previous reports indicated that the differences in adduct levels in exposed organs were so significant that there were multiplefold differences between exposed organs. For example, after a 10 mg/kg SM exposure, the maximum levels of N7-HETEG in the pancreas, liver, and lungs were 36, 84, and 137 ng/g tissue, respectively.25 By comparison, the differences in adduct levels among cell lines exposed in vitro were less pronounced. This indicated that the degree of DNA lesions in different tissues most likely resulted from differential SM distribution by the circulatory system. These observations could be attributed to the fact that vascular tissues are more likely exposed to large fractions of SM in the bloodstream.14 The in vitro results indicated that the order of concentration levels of the adducts was N7-HETEG > Bis-G > N3-HETEA, which agreed with previous reports.31 The N7-HETEG adduct was the most abundant; however, the concentration was slightly lower than that reported by Batal et al. In their experiment, the SM exposure time was as long as 4 h, and the utilized cells were cultured in suspension, which may have resulted in the contamination of the samples with dead cells that could not be removed.23 Here, the Bis-G proportions in cells were all above 18%, which were significantly higher than the N3HETEA levels (below 2%) and were similar to the results of our previous studies.12,29,32 However, these results contradicted previous studies that reported Bis-G proportions similar to N3HETEA proportions.4,33 We propose that different preparation and detection methods may influence the recovery and detection sensitivity of Bis-G. By using synthetic standards and deuterated ISs for the simultaneous detection of three adducts, our method may be a truer reflection of the profiles of each adduct in a single sample, especially for Bis-G. These results indicated that the abundance of Bis-G was higher than that previously expected and suggests that a serious DNA lesion caused by SM may arise in part due to the additional formation of Bis-G.34 Time-Dependent Profiles of SM-DNA Adducts in Vitro. The time-dependent concentration profiles of SM-DNA adducts were completely analyzed and are shown in Figure 2. The detected values of N3-HETEA after a 24 h exposure were

Figure 2. Concentration- and time-dependent profiles of adducts in different cell lines after 1 h of SM exposure at a dose of 100 μM. Results are expressed as the means ± SE (n = 3). (A) Time-dependent trend of the N7-HETEG level in five cell lines after exposure. (B) Time-dependent trend of the Bis-G level in five cell lines after exposure. (C) Time-dependent trend of the N3-HETEA level in five cell lines after exposure. The data for N3-HETEA level in cells after 24 h of SM exposure were below the limit of detection and are not presented.

mostly below the detection limits and are not presented. To visually describe the variation tendency, the elimination percentages of adduct concentrations were calculated and are plotted in Figure S2 (Supporting Information). The values of time-to-peak (Tmax), half-life time (T1/2), maximum concentration (Cmax), and area under the concentration−time curve (AUC) were calculated and are listed in Table 4. Except for HA-s, the level of SM-DNA adducts in the cells reached a maximum within 1 h of exposure, followed by a continual decline. The T1/2 values of N7-HETEG and Bis-G showed no significant differences among the liver, lungs, and skin-derived cells and preadipocytes. We also found that the T1/2 and Tmax 1228

DOI: 10.1021/acs.chemrestox.5b00055 Chem. Res. Toxicol. 2015, 28, 1224−1233

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Chemical Research in Toxicology Table 4. Tmax, T1/2, Cmax, and AUC Values for DNA Adducts in Each Cell Typea analyte N7-HETEG

Bis-G

N3-HETEG

Tmax T1/2 Cmax AUC Tmax T1/2 Cmax AUC Tmax T1/2 Cmax AUC

HaCaT

HLF

L-02

HPA-s

HA-s

0.08 46.6 ±12.0b 225 ± 6 8198 0.75 46.6 ± 9.1b 103 ± 9 2302 0.25 21.5 ± 5.7c 7.3 ± 0.6 62.2

0.08 46.0 ± 4.5b 219 ± 8 5949 0.50 40.4 ± 4.9b 74.9 ± 2.1 1346 0.08 11.0 ± 3.7b,c 5.1 ± 0.2 43.0

0.08 47.6 ± 5.5b 166 ± 7 4924 0.08 43.8 ± 12.2b 79.3 ± 3.5 1550 0.08 21.2 ± 2.2c 4.1 ± 0.2 37.1

0.08 56.2 ± 8.1b 297 ± 10 9587 0.75 74.5 ± 10.1b 123 ± 5 3319 0.08 57.1 ± 18.4 5.6 ± 0.4 48.2

3.00 195 ± 22 242 ± 9 10263. 3.00 323 ± 226 112 ± 5 3958 1.00 30.3 ± 11.6c 5.5 ± 0.3 55.5

Tmax: the time to maximum concentration was expressed as hours. T1/2: half-life time was expressed as h (mean ± SE). Cmax: the maximum concentration was expressed as adducts per 106 bases (mean ± SE). AUC: areas under concentration−time curve from 0.08 to 48 h (for N7-HETEG and Bis-G) or 12 h (for N3-HETEA) were expressed as adducts per 106 bases. bSignificantly different from data in HA-s (p < 0.01). cSignificantly different from data in HPA-s (p < 0.01). a

values for N7-HETEG and Bis-G in HA-s were significantly higher than those in other cells (Table 4). Fate of DNA Adducts from Exposed Adipocytes Both in Vitro and in Vivo. In the results described above, the different profiles of SM-DNA adducts in adipocytes were observed in vitro. However, we also observed that the differences between the DNA adduct levels by the end of SM exposure in exposed adipocytes versus the other target tissue cell lines were smaller than the differences observed between the exposed tissues in vivo.14,32 Therefore, additional in vivo experiments were used to characterize the profiles of the DNA adducts in the exposed AT to compare with the other tissues. Four types of AT were collected from exposed rats, and SMDNA adducts were analyzed. As shown in Figure 3, the results clearly show a dose-dependent profile. A high dose (10 mg/kg) of SM exposure resulted in more DNA adducts in subcutaneous, epididymis, perirenal, and brown adipose tissues, which were about three to 30 times greater than that caused by a low exposure dose (1 mg/kg) (Table S2, Supporting Information). The total level of adduct in exposed ATs was quite low, although animals were exposed to a high dose of SM. The level and dose-dependent trends of DNA adduct distributions were similar in these four types of ATs. For example, the maximum levels of adducts in the subcutaneous AT from the high dose exposure group were 284 ± 47, 250 ± 89, and 14 ± 4 per 109 bases for N7-HETEG, Bis-G, and N3HETEA, respectively. By comparison, our previous work reported that the maximum level of N7-HETEG in the liver from a 10 mg/kg dose exposed rat was approximately 98 ± 62 per 106 bases.25 There were multiple magnitude differences between the adduct levels in the ATs and other tissues. These results were different from the comparative results in vitro and may be a result of a more complex physiological environment in vivo. After characterizing the time-dependent profile of adducts in adipocytes, several interesting results were observed. As described above, persistent adducts were found in postexposure adipocytes in vitro. The time-related trends of adducts in the in vivo AT were different from those of the SM-exposed in vitro samples. In the low-dose exposure group, the level of adducts slowly decreased and surprisingly exhibited an upward trend at 12 h after exposure. This was very similar to the phenomenon observed in vitro and indicated that the DNA lesions in the low-

dose exposed AT were similar to those in vitro. This also indicated that in vitro research may somewhat contribute toward the elucidation of the mechanism of SM damage in lowdose exposed AT. The DNA adducts in AT declined over time after a high-dose SM exposure (Cmax). However, the rate and range of the decrease were slow and small. After reaching a maximum adduct level within 3 h, the residual adduct concentrations in the four types of AT 12 h after exposure to Cmax were approximately 16.67% to 59.71% for N7-HETEG, Bis-G, and N3-HETEA, respectively (Table S2, Supporting Information). In our previous work, the residual adducts in liver at 12 h post-exposure were only 2.2, 2.2, and 2.3% for N7HETEG, Bis-G, and N3-HETEA, respectively. This also indicated that the DNA adducts were persistently present in exposed AT in vivo. These results were in agreement with our experiments in vivo, which showed that a plateau or secondary peak and a long half-life time were detected in exposed rat brain, an adipose-rich organ.25 Consequently, we proposed that the time-dependent trend of DNA adduct levels was separate from the SM exposure dosage or the adipose content of the cell. With respect to the abundance of the adduct, the highest proportion of Bis-G was observed in adipocytes in vitro (Table 3). In the in vivo experiments, it was unexpectedly observed that the proportion of Bis-G in AT was also extremely high and reached or exceeded the proportion of N7-HETEG, as was the case in exposed subcutaneous AT (Figure 3). Taken together, we proposed that an adipose-rich environment in the cell may promote the formation of Bis-G. The formation of SM-DNA adducts is directly related to the DNA lesions caused by SM. The majority of SM-DNA adducts are monoadducts; however, it should be noted that interstrand cross-links (ICLs) are the primary causes of cytotoxic damage.35 It has been estimated that merely 20 ICLs in a mammalian genome can be fatal to cells because of a lack of ability to remove the cross-links.34 Research by Jowsey et al. found that homologous recombination (HR) in DNA repair could confer protection against acute SM toxicity and acted in the repair of DNA double strand breaks.35 In AT, although the proportion of Bis-G was high, the concentrations of adducts were extremely low. We assumed that the lesion degree of the exposed AT may not be severe. To verify this assumption, a half maximal inhibitory concentration (IC50) experiment was performed using the same five cell lines 1229

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Figure 3. DNA adduct levels in rat adipose tissues at 1, 3, 9, and 12 h postcutaneous exposure to a low (1 mg/kg) or high (10 mg/kg) dosage of SM. Results are expressed as the means ± SE (n = 5). Four types of adipose tissues were tested: subcutaneous, epididymis, perirenal, and brown adipose tissues. Left panel: time-related levels of adduct in DNA of low-dose SM-exposed rats. Right panel: time-related levels of adduct in the DNA of highdose SM-exposed rats.

to evaluate the effect of SM exposure on the cell survival rate. The results showed that the IC50 value of SM-exposed HA-s was as high as 510 μM and particularly higher than those of other cell lines, including preadipocytes, which had IC50 values of only approximately 70 μM. Microscope observations also

showed that large amounts of HA-s cells were alive until 48 h after the 1 h SM exposure at a dose of 1000 μM. This suggested that adipocytes are relatively resistant to lesions by SM. Despite the identification of a cell line with strong survival properties even after SM exposure, we had difficulties in 1230

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investigate the cellular damage in adipocytes caused by SM and the protection afforded by DNA repair mechanisms of adipocytes against SM exposure.

accounting for the persistence of adducts in the adipocytes while simultaneously expressing smaller lesions. Because of the relatively simple structure of adipocytes, i.e., an adipose vacuole occupies most of the space in the cytoplasm (Figure S3, Supporting Information), we believed that SM may be stored in lipids and thus lead to decreased cellular damage and higher survival rates. Given their specific differentiation and simple function, the DNA repair mechanisms in adipocytes may function abnormally. Additionally, there may be an adipocytespecific mechanism that delays the clearance of adducts. The determination of the specific mechanism responsible for these observations would shed light on DNA repair mechanisms and AT-related illnesses. Adipose tissues are one of the largest organs in the body and include a range of cell types that are related to several physiological functions, including metabolic regulation, energy storage, and endocrine functions.36 White adipose tissue is a major type of AT that contains a reservoir of lipophilic environmental pollutants, such as dioxins. Severe diseases, such as diabetes, cause an excessive accumulation of these pollutants in white adipose tissue.37 Furthermore, recent data demonstrated that AT is involved in inflammation and the immune response.38 Adipocytes and adipose-related immune cells can affect lymphocyte proliferation, differentiation, and activation to affect immune function.39 Therefore, AT is not only a target but also a regulator of lipophilic environmental pollutant toxicity.36 Given the lipophilicity of SM and the persistence of DNA adducts, the retention time of intact SM in AT should be longer than that in other tissues. The accumulated residual SM in AT may cause continuous damage because of AT inflammation and immune response alteration. Alternatively, SM may be released from adipocytes due to the consumption of AT cells induced by energy mobilization, leading to extensive damage to adjacent cells and tissues. AT consumption is suspected based on the observations of a clear reduction in AT cell population in SM-exposed rats. Therefore, in vivo, AT or hydrophobic environments (such as lipids) are key areas of focus for eliminating internal residual SM. The timely elimination of residual SM may be crucial for the treatment of SM-exposed individuals.

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ASSOCIATED CONTENT

* Supporting Information S

MS/MS fragmentation information for SM-DNA adduct detection, the elimination of SM-DNA adducts in different cells after 1 h of SM-exposure at a dosage of 100 μM, mature adipocyte cells (HA-s) differentiated from human subcutaneous preadipocytes (HPA-s), stability validation parameters of the method and the values of Tmax and Cmax, and a comparison of results of DNA adducts in four types of ATs exposed to low and high doses of SM. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00055.

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AUTHOR INFORMATION

Corresponding Authors

*(J.X.) Tel/Fax: +86 10 68225893. E-mail: xiejwbmi@163. com; [email protected]. *(H.X.) Tel: +86 10 66930621. E-mail: [email protected]. Funding

This study was supported by the Chinese National Scientific Research Special-Purpose Project in Public Health Profession (Grant No. 2015SQ00192) and the National Science and Technology Major Project of the Ministry of Science and Technology of China (Grant No. 2012ZX09301003-001-010). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS AT, adipose tissue; AUC, area under concentration−time curve; Bis-G, bis(2-ethyl-N7-guanine)thioether; Cmax, maximum concentration; CV, coefficient of variation; DEX, dexamethasone; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HaCaT, human keratinocyte cell line; HA-s, human subcutaneous adipocyte; HLF, human lung fibroblasts cell line; HPA-s, human subcutaneous preadipocyte; HQC, high quality control; IBMX, 3-isobutyl-1-methylxanthine; IC50, half-maximal inhibitory concentration; ICLs, interstrand crosslinks; INS, insulin; IS, internal standard; L-02, human hepatocyte cell line; LLOQ, lower limit of quantification; LOD, limit of detection; LQC, low quality control; ME, matrix effect; MF, matrix factor; MQC, medium quality control; O6HETEG, O6-[2-[(2-hydroxyethyl)thio]-ethyl]guanine; N3HETEA, N 3 -(2-hydroxyethylthioethyl)-2′-adenine; N 7 HETEG, N7-[2-[(2-hydroxyethyl)thio]-ethyl]guanine; QC, quality control; S/N, the ratio of signal-to-noise; SM, sulfur mustard; SPSS, Statistical Product and Service Solutions; T1/2, half-life time; Tmax, time to peak; UPLC-MS/MS, ultrahigh performance liquid chromatography tandem mass spectrometry

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CONCLUSIONS For the simultaneous quantification of three SM-DNA adducts, we established, applied, and validated a UPLC-MS/MS method. We selected five common cell types to investigate the formation, specificity, and time-dependent relationships of SM-DNA adducts in vitro after SM exposure. The fate of DNA adducts in AT were examined in rats with a low (1 mg/kg) or a high dose (10 mg/kg) of SM exposure. Contradictory to the results from in vivo studies, there were no differences for the T1/2 and Tmax values of adducts in the liver, lungs, skin, or preadipocyte-derived cells in vitro. The persistence of adducts and the high proportion of Bis-G in adipocytes was observed both in vitro and in vivo. To the best of our knowledge, this is the first study to definitively show that adipose-rich environments in cells influence the formation and repair of SM-DNA adducts and promote the formation of Bis-G. Abnormal or new DNA repair mechanisms are suggested to explain the high survival of adipocytes postexposure, despite the persistence of adducts; this may be a key topic for future research. AT was also important for SM distribution and toxicity. This may pioneer a new model for both the prevention and treatment of SM exposure. Further studies are required, especially to

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