Monoclonal antibodies for the detection of a specific cyclic DNA

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Article Cite This: Chem. Res. Toxicol. 2018, 31, 772−783

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Monoclonal Antibodies for the Detection of a Specific Cyclic DNA Adduct Derived from ω‑6 Polyunsaturated Fatty Acids Marcin Dyba,† Brandon da Silva,‡,§ Heidi Coia,† Yanqi Hou,† Sumire Noguchi,† Jishen Pan,† Deborah Berry,∥ Karen Creswell,∥ Jacek Krzeminski,⊥ Dhimant Desai,⊥ Shantu Amin,⊥ David Yang,‡ and Fung-Lung Chung*,†

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†

Department of Oncology, Department of Biochemistry and Molecular and Cellular Biology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States ‡ Department of Chemistry, Georgetown University, Washington, DC 20057, United States ∥ Histopathology and Tissue Shared Resource, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States ⊥ Department of Pharmacology, Pennsylvania State University, Hershey, Pennsylvania 17033, United States S Supporting Information *

ABSTRACT: Lipid peroxidation of polyunsaturated fatty acids (PUFAs) is an endogenous source of α,β-unsaturated aldehydes that react with DNA producing a variety of cyclic adducts. The mutagenic cyclic adducts, specifically those derived from oxidation of ω-6 PUFAs, may contribute to the cancer promoting activities associated with ω-6 PUFAs. (E)-4-Hydroxy2-nonenal (HNE) is a unique product of ω-6 PUFAs oxidation. HNE reacts with deoxyguanosine (dG) yielding mutagenic 1,N2-propanodeoxyguanosine adducts (HNE-dG). Earlier studies showed HNE can also be oxidized to its epoxide (EH), and EH can react with deoxyadenosine (dA) forming the wellstudied εdA and the substituted etheno adducts. Using a liquid chromatography-based tandem mass spectroscopic (LC-MS/MS) method, we previously reported the detection of EH-derived 7-(1′,2′-dihydroxyheptyl)-1,N6-ethenodeoxyadenosine (DHHεdA) as a novel endogenous background adduct in DNA from rodent and human tissues. The formation, repair, and mutagenicity of DHHεdA and its biological consequences in cells have not been investigated. To understand the roles of DHHεdA in carcinogenesis, it is important to develop an immuno-based assay to detect DHHεdA in cells and tissues. In this study we describe the development of monoclonal antibodies specifically against DHHεdA and its application to detect DHHεdA in human cells.

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fatty acid hydroperoxide, and possibly lipoxygenase.18−20 Compared to HNE, EH is more reactive and readily modifies nucleobases.18,19 It primarily reacts with deoxyadenosine (dA) and dG forming unsubstituted and substituted etheno purine adducts, such as 1,N6-ethenodeoxyadenosine (εdA), 1,N2ethenodeoxyguanosine (εdG), 7-(1′,2′-dihydroxyheptyl)-1,N2ethenodeoxyguanosine (DHHεdG), and 7-(1′,2′-dihydroxyheptyl)-1,N6-ethenodeoxyadenosine (DHHεdA) (Scheme 1). Two pairs of diastereomers (designated as DHHεdA 1 and 2) are formed from the reaction of EH with dA because of the two chiral carbons in the side chain. The mutagenicity of the LPO-derived cyclic adducts suggests that they may contribute to carcinogenesis. DHHεdA is a cyclic adduct in vivo detected only in recent years.21 Its biological consequences, such as repair and mutagenicity, have not yet been investigated. Although EH also modifies dG to yield DHHεdG, the dG adduct has not been detected in vivo, possibly due to its poor stability.18,19 Like its reactions in vitro,

INTRODUCTION In addition to exposure to environmental carcinogens, the endogenously formed DNA-reactive compounds are believed to play an important role in carcinogenesis.1 Lipid peroxidation (LPO) of polyunsaturated fatty acids (ω-3 and ω-6 PUFAs) is an endogenous source of protein and DNA damage.2,3 The oxidation of PUFAs produces α,β-unsaturated aldehydes (enals), such as (E)-4-hydroxy-2-nonenal (HNE) and acrolein (Acr), that can modify DNA bases forming a variety of cyclic DNA adducts (Scheme 1).4−10 HNE is an oxidation product of ω-6 PUFAs, such as arachidonic and linoleic acids,11 and it can also be formed from trans,trans-2,4-decadienal in food products as a breakdown product of LPO.12,13 HNE is believed to be a major cytotoxic product of LPO.11,14 In addition to its ability to modify proteins, HNE reacts with deoxyguanosine (dG) via Michael addition to form cyclic 1,N2-propanodeoxyguanosine adducts (HNE-dG).14 HNE-dG is shown to be mutagenic,15−17 however, its levels in vivo are relatively low and have hindered its consistent and quantitative detection by 32P-postlabeling and LC-MS/MS.14 HNE is readily epoxidized to 3,4-epoxy-4hydroxynonenal (EH) by hydrogen peroxide, organic peroxides, © 2018 American Chemical Society

Received: April 25, 2018 Published: July 12, 2018 772

DOI: 10.1021/acs.chemrestox.8b00111 Chem. Res. Toxicol. 2018, 31, 772−783

Article

Chemical Research in Toxicology

Scheme 1. Formation of Selected Cyclic DNA Adducts Derived from Enals As Secondary Products of Oxidized PUFAs via Hydroperoxy Fatty Acids (FAOOH): α-OH-1,N2-propano-2′- deoxyguanosine (Acr-dG 1,2), γ-OH-1,N2-propano-2′deoxyguanosine (Acr-dG 3), 1,N6-etheno-2′-deoxyadenosine (εdA), 7-(1′,2′-dihydroxyheptyl)-1,N6-etheno-2′-deoxyadenosine (DHHεdA), and 1,N2-deoxyguanosine adducts of (E)-trans-4-hydroxy-2-nonenal (HNE-dG)a

a

EH, 2,3-epoxy-4-hydroxynonanal, HNE, (E)-4-hydroxy-2-nonenal. internal standard used for quantitative mass spectrometry was described earlier.21 Water used in all experiments was from in house Milli-Q Biocel water polisher (EMD Millipore, Billerica, MA). All other chemicals, unless stated otherwise, were purchased from Sigma-Aldrich (St. Louis, MO) and were analytical or HPLC grade. Calf thymus (CT) DNA, normal nucleosides, and 8-oxo-dG are purchased from Sigma-Aldrich. Acr-dG and HNE-dG were synthesized as described previously.28,29 εdA was synthesized following a published method.30,31 Synthesis of Immunogens. Synthesis of 2′,3′-O-Isopropylidene-5′-carboxyadenosine (2). 2′,3′-O-Isopropylideneadenosine (5.00 g, 16.27 mmol), 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) (510 mg, 3.26 mmol), and (diacetoxyiodo)benzene (DIB) (11.53 g, 35.80 mmol) were placed in a 100 mL round-bottom flask equipped with a magnetic stir bar. Thirty mL of 1:1 acetonitrile (ACN):water was added, and the mixture was stirred for 3 h. After a few initial minutes of stirring, the reaction mixture showed an orangebrown color, and compounds began to dissolve. Shortly, compound 2 precipitated as a white solid (Scheme 2). Three h later, the product is filtered and triturated with acetone (3 × 15 mL) and diethyl ether (3 × 15 mL) and dried overnight by vacuum.32 No further purification was needed. The purity of compound 2 analyzed by HPLC system 1 was >98%. The reaction yield was 91%. High-resolution mass spectrometry: observed mass: 322.1149 m/z (expected mass: 322.1151 m/z). Synthesis of 5′-Carboxyadenosine (3). The isopropylidene protecting group in compound 2 was removed by reacting 1.013 g (3.16 mmol) with 50 mL of 4:1 mixture of formic acid and water for 43 h at room temperature. The reaction progress was monitored using HPLC system 1. After completion of the reaction, the reaction mixture was evaporated using a centrifugal vacuum evaporator (SpeedVac). Dry material was redissolved in dimethyl sulfoxide (DMSO) and then

DHHεdA in rodent and human tissues was detected as two pairs of diastereomers by two methods: 32P-postlabeling/HPLC and isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS).21 The levels of DHHεdA in vivo are considerably higher than that of HNE-dG, making it a potential biomarker of DNA damage by ω-6 PUFAs. Because DHHεdA is specifically derived from ω-6 PUFAs, we hypothesize that it may play a role in the tumor promoting activities associated with ω-6 PUFAs.22−25 This assumption is supported by the observations that EH is more mutagenic and reactive toward DNA than HNE and it is tumorigenic.26 To better understand its mutagenicity and repair and its activity toward apoptosis and cell cycle arrest and, ultimately, its role in cancer, monoclonal antibodies (mAbs) against DHHεdA need to be developed as tools for the studies in cells and tissues. In this study, we raised and characterized mAbs against DHHεdA. The specificity and reactivity of the mAbs were determined by direct and competitive ELISA assays. The applications of the mAbs for the detection of DHHεdA, confirmed by LC-MS/MS, in EH-modified DNA using highly sensitive ELISA assay and in human hepatocytes using flow cytometry-based fluorescenceactivated cell sorting (FACS) analysis and immunocytochemistry staining (ICC) are described.

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EXPERIMENTAL PROCEDURES

Chemicals. (E)-4-hydroxy-2-nonenal (HNE) and 2,3-epoxy-4hydroxynonanal (EH) were synthesized by previously described methods.18,27 Synthesis of DHHεdA standard and [15N5]-DHHεdA 773

DOI: 10.1021/acs.chemrestox.8b00111 Chem. Res. Toxicol. 2018, 31, 772−783

Article

Chemical Research in Toxicology Scheme 2. Synthesis of DHHεdA Protein Conjugates As Antigensa

a

(a) TEMPO, BIB, 1:1 ACN:water, 3 h; (b) 80% formic acid, room temperature, 43 h; (c) HNE in THF, 30% H2O2 and ACN mixed with 3 dissolved in 2:1 THF:100 mM phosphate buffer, 50 °C, 7 days; (d) EDAC, BSA or KLH, 0.1 M MES buffer; (e) raising mAbs against DHHεdA by immunization of mice with antigen 5. purified using HPLC system 2. The purity of the final product as compound 3 was >98%. Reaction yield was 78%. High-resolution mass spectrometry: observed mass: 282.0836 m/z (expected mass: 282.0838 m/z). Synthesis of the 5′-Carboxy derivative of 7-(1′,2′-Dihydroxyheptyl) Adenosine (4). HNE (469 mg, 3.00 mmol) was dissolved in 1.2 mL of tetrahydrofuran (THF), 1.6 mL ACN, and 3.25 mL (30 mmol) 30% hydrogen peroxide. The resulting mixture was added to compound 3 (527 mg, 1.88 mmol) dissolved in 50 mL of 2:1 THF:100 mM phosphate buffer (pH 7.4). The reaction was kept in a water bath at 50 °C for 7 days. One mL of 30% H2O2 was added 19 h after reaction began. The pH was checked periodically and adjusted by 1.0 M NaOH to pH 7.4. The reaction progress was followed using HPLC system 1. The excess of peroxides was removed using a saturated solution of sodium metabisulfite in water. The reaction mixture separated into two layers. Both layers were collected, evaporated on a SpeedVac, redissolved in water, and purified using HPLC system 2. The final hapten 5′-carboxy-DHHεA was separated on HPLC, as two peaks on HPLC as two pairs of isomers: 1 and 2 with ratio of 1:2, respectively. For conjugating with proteins (see below), an equimolar mixture of 5′-carboxy-DHHεA 1 and 2 was used. The purity of the final product was >98%. Reaction yield was 24%. High-resolution mass spectrometry: observed mass: 436.1851 m/z (expected mass, 436.1832 m/z). The high-resolution mass spectrometry for compounds 2, 3, and 4 was performed using a Waters ESI-Q-TOF Premiere mass spectrometer with Waters ACQUITY UPLC as the front end (Waters Corporation, Milford, MA). Conjugation of Hapten 4 to Bovine Serum Albumin (BSA). BSA (2 mg) was dissolved in 200 μL of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 4.7 buffer and mixed with compound 4 (0.5 mg, 0.0011 mmol) dissolved in 500 μL of MES buffer. 1-(3(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDAC) (22 mg, 0.11 mmol) was dissolved in 1 mL of water, and 100 μL (×10, 0.011 mmol) was added to the reaction mixture. The reaction mixture was stirred for 3 h at room temperature and then separated on 7K MWCO Zeba Spin Desalting columns (Thermo Scientific #89891, Fisher Scientific, Pittsburgh, PA) using water as eluent.

Protein concentration in the final eluent was determined by NanoDrop Microvolume spectrophotometry (Thermo Scientific #ND-2000, Fisher Scientific, Pittsburgh, PA). The conjugation was confirmed with MALDI-TOF/TOF mass spectrometry. Briefly 1 μL of protein with conjugated hapten was mixed with 1 μL of 10 mg/mL sinapinic acid in 30% (v/v) ACN containing 0.3% (v/v) trifluoroacidic acid (TFA) in water and then spotted on a MALDI plate. Mass spectra were acquired in linear high mass mode using a 4800 MALDI-TOF/ TOF mass spectrometer (AB SCIEX, Framingham, MA). Conjugation of Hapten 4 to Keyhole Limpet Hemocyanin (KLH). Lyophilized Imject mcKLH in MES buffer (6 mg) (Thermo Scientific #77653, Fisher Scientific, Pittsburgh, PA) was reconstituted in 600 μL of water and mixed with compound 4 (5.4 mg, 0.012 mmol) dissolved in 1.5 mL of 0.1 M MES, 0.9 M NaCl, pH = 4.7 buffer. A 150 μL (7.5 μmol) aliquot of EDAC (10 mg, 0.05 mmol) in water (1 mL) was added to solution of KLH containing compound 4. The reaction mixture was gently stirred for 2 h at room temperature and then separated on 7K MWCO Zeba Spin Desalting Columns using Imject Purification Buffer Salts as eluent (Thermo Scientific #77159, Fisher Scientific, Pittsburgh, PA). Protein concentration was confirmed as above. The complex structure of KLH, its high molecular weight, and stability issues for this protein prohibited MALDI-MS analysis. mAbs Development. Immunization, Cell Fusion, and Hybridoma Screening. Two separate immunizations (Im I and Im II) were carried out in an effort to raise mAbs. In Im I, the immunization and antibody isolation/purification was done using commercial service (GenScript USA Inc.; Piscataway, NJ). Briefly, five BALB/c mice were immunized with 100 μg of DHHεdA-conjugated to KLH in 0.1 mL of saline emulsified with an equal amount of Freund’s complete adjuvant, given in a split dose, intraperitoneal (i.p.) and subcutaneous (s.c). A second immunization was given 2 weeks after the first one in incomplete Freund’s adjuvant. Test bleeds were taken to check the antibody reactivity toward immunogens using ELISA and DHHεdAconjugated to BSA. Mice were boosted with 100 μg of the conjugate in saline given i.p. on days 1, 2, 3, and 4 on fourth week after the second injection. On day 5, mice were sacrificed, and spleens were removed for fusion. Satisfactory immune responses were seen in all mice, two of which were used for cell fusion and hybridoma production. 774

DOI: 10.1021/acs.chemrestox.8b00111 Chem. Res. Toxicol. 2018, 31, 772−783

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Chemical Research in Toxicology

in 100 μL of PBS containing 1% (w/v) BSA were added, and the plates were incubated at 37 °C for 30 min. The wells were washed twice with PBST and once with PBS and developed with 100 μL of working solution of SuperSignal ELISA Femto Maximimum Sensitivity Substrate kit (Thermo Scientific #37074, Fisher Scientific, Pittsburgh, PA). Plates were gently shaken for 3−4 min at room temperature protected from light, then luminescence intensity was measured on plate reader (GloMax-Multi Detection System, Promega, Madison, WI). Detection of DHHεdA in EH-Modified Calf Thymus DNA (CT-DNA) Samples by ELISA. EH-Modified CT-DNA. EH was generated by reacting HNE (20.6 mg) with 30% hydrogen peroxide (21.4 μL) in the mixture of 250 μL THF, 10 μL ACN, and 8 mg sodium carbonate over 1 h at room temperature. At the end of the reaction, 1 mL of chloroform and 1 mL of water were added, and after vigorous shaking the chloroform fraction was collected, washed twice with 1 mL of water, dried over anhydrous sodium sulfate, filtered, and evaporated using a SpeedVac. After evaporation, reaction mixture was dissolved in 100 μL of ethanol (EtOH), diluted with PBS buffer, and used directly to modify DNA. CT-DNA (0.5 mg in 0.5 mL PBS pH 7.4) was mixed with 5 μL of previously prepared EH solutions with 10× and 1000× dilution in ethanol and kept at 37 °C for 19 h. The solution was extracted three times by 0.5 mL of chloroform, then DNA was precipitated by adding 100 μL of cold 4 M sodium chloride solution and 1.5 mL of cold EtOH. Sample was kept for 30 min at −20 °C, centrifuged, washed twice by cold 80% EtOH, and air-dried. DHHεdA modification levels were checked by LC-MS/MS. ELISA Detection. The ELISA plate wells were coated with 50 μL of EH-modified CT-DNA (see below) in PBS (20 μg/mL to 2 ng/mL). The buffer was evaporated overnight at room temperature. After evaporation, plates were washed five times with 100 μL PBST. The wells were then blocked with 200 μL of 1% (m/v) BSA in PBS at room temperature with moderate shaking for 1 h and washed trice with 200 μL PBST. The purified mAb (3C9C9) solution (50 μL, 1:200,000 dilution) in 1% (w/v) BSA was added, and the plates were incubated at 37 °C for 1 h and washed trice with PBST. Horseradish peroxidase conjugated goat antimouse IgG secondary antibodies in 50 μL of PBS containing 1% (w/v) BSA were added, and the plates were incubated at 37 °C for 30 min. The wells were washed twice with PBST and once with PBS and developed with 50 μL of working solution of SuperSignal ELISA Femto Maximimum Sensitivity Substrate kit. Protected from light, plates were gently shaken for 3−4 min at room temperature. The luminescence intensity was measured on a plate reader. Cell Culture and Treatments. HepG2 cells (#HB-8065, ATCC, Manassas, VA) were grown in complete DMEM (#10−013, Corning Life Sciences, Tewksbury, MA) until 85−90% confluent. Cells were treated with a final concentration of 100 μM or 300 μM of either arachidonic acid (AA) or EH solubilized in 70% EtOH for 24 h. Cells were then scraped down in media, transferred to a centrifuge tube, and spun for 3000 rpm for 5 min. The pellet was washed 2× with 1× PBS, centrifuging between washes. DNA was then isolated from the cells and prepared for LC-MS/MS-MRM after enzymatic hydrolysis as described below. Primary human hepatocytes (#HUFS1M, lot #HUM4132, Triangle Research Laboratories, Durham, NC) were cultured in hepatocyte media (#5201, ScienCell Research Laboratories, San Diego, CA) until 85−90% confluent. Cells were then treated with a final concentration of 300 μM EH dissolved in 70% EtOH and added to the culture media. The cells were collected at the designated time points following EH treatment: 0, 4, 8, 12, and 24 h. The nuclei were extracted using Nuclei EZ Prep Nuclei Isolation Kit (#NUC-101, Sigma-Aldrich, St. Louis, MO) and fixed with 3.7% formaldehyde for 10 min. The nuclei were then prepared for FACS analysis using the method shown previously.33 Immunocytochemistry (ICC) Staining. Cells were cultured and treated as described above and then fixed with 3.7% formaldehyde on the cover glass (VWR #16004-302, Radnor, PA) for 10 min at room temperature and washed 3 times with 1 × PBS. HCl (2M) + 0.5% PBST was added, and cells were incubated for 5 min, then neutralized with 0.1 M Na2B4O7 (pH8.5), and washed 2 times with PBS. Cells were

A total of 20 hybridoma cell lines were produced. Splenocytes and myelomas were fused, plated into 96-well culture plates, and screened by ELISA to detect the positive clones. Four selected clones were then subcloned by limiting dilution until they were monoclonal and stable hybridomas. One subclonal line 8H6 lost its activity during cloning process. Two subclones from the three remaining parental clone (3C3B6, 3C3E12, 3C9C9, 3C9G2, 4E10B8, and 4E10F2) were produced, expanded into culture flasks, and 4−6 vials of cells for each subclonal cell line were cryopreserved. In Im II, a rapid immunization protocol and hybridoma development platform was carried out by Precision Antibody (Columbia, MD). Three SJL/J mice were immunized with KLH conjugated DHHεA. Over 21 days, multiple immunizations were performed. Per immunization, a total of 10 μg of antigen was injected subcutaneously per mouse in multiple sites. On days 17−20, serum test bleeds were taken to check the antibody reactivity toward the target using ELISA and DHHεdA conjugated to BSA. Target-specific Ab titer was determined by ELISA against immobilized antigen by screening serial dilutions of the serum. An additional round of boosting was performed to increase the titer followed by serum testing. Once a sufficient titer was detected, a high efficiency electrofusion was performed. Splenocytes and lymph nodes were harvested from immunized mice with high serum titer to the target antigen and fused with a murine myeloma partner. Direct cloning was performed where the fused cells were cloned in a semisolid, HAT selection media prior to screening utilizing an automated clone picking system. Supernatant from the resulting 24 clonal hybridomas were subsequently screened by ELISA against the target BSAconjugated DHHεA. Six positive clones (2E9, 2G7, 6A1, 3A11, 4D2, and 4B2) with mAbs specific to the target antigen were expanded and cryopreserved. mAbs Isolation and Purification. Im I. mAbs was produced using roller bottle cell culture technique. Briefly, selected hybridoma cells were cultured with Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C, 6% CO2 in humidified incubator. When hybridoma cells were at >70% confluence, they were transferred to T-25 tissue culture flask. After reaching >70% confluency, cells were suspended in roller bottle cell culture medium to make a concentration of 2.5−3.5 × 104 cells/mL and transferred to tissue culture roller bottle in electro-thermal incubator for around 10−14 days. Supernatant was collected when the cell density was below 1 × 105 cells/mL. Supernatant was centrifuged, filtered with 0.22 μm filter, and concentrated using centrifugal evaporator to final volume of 100 mL. mAbs was purified by affinity column chromatography using recombinant protein A resin (GenScript USA Inc.; Piscataway, NJ). Im II. Selected hybridoma cell lines were first expanded in complete RPMI medium containing 10% FBS. After cells reached >80% confluency, the concentration of FBS was reduced by using a high-density hybridoma cell culture in a medium with a gradient of decreasing serum concentration from 8%, 5%, and 2%. Supernatant was collected after 50% of hybridoma cells underwent apoptosis in complete RPMI medium with 2% FBS. The supernatant was centrifuged and collected in a clean conical tube. OD absorption was measured to estimate the presence of mAb in supernatant. Characterization of mAbs. ELISA and Competitive ELISA. The ELISA plate wells (white 96-well Nunc MaxiSorp, Thermo Scientific #436110, Fisher Scientific, Pittsburgh, PA) were coated with 100 μL of 0.1 μg/mL antigens of DHHεdA conjugated to BSA in PBS at 4 °C overnight and washed three times with PBS containing 0.1% (v/v) Tween 20 (PBST). The wells were then blocked with 100 μL of 1% (m/v) BSA in PBS at room temperature with moderate shaking for 1 h and washed three times with PBST. For ELISA, 100 μL of antibody solutions with different dilutions or test bleeds in PBS containing 1% (w/v) BSA were added. For competitive ELISA, first 50 μL of competitive reagent with different dilutions of tested competing agents in 1% (w/v) BSA were added, then antibody solutions or test bleeds in PBS containing 1% (w/v) BSA were added into the wells. The plates were incubated at 37 °C for 1 h and washed three times with PBST. Horseradish peroxidase conjugated goat antimouse IgG secondary antibodies (2 ng HRP IgG/ml in Im I and 33.3 ng HRP IgG/ml in Im II) 775

DOI: 10.1021/acs.chemrestox.8b00111 Chem. Res. Toxicol. 2018, 31, 772−783

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Chemical Research in Toxicology

DHHεdA on column for both peaks (1/× weighting; r2 = 0.9998 and r2 = 0.9984 for both LC resolved pairs of DHHεdA stereoisomers 1,2 and 3,4 respectively). Measured limit of quantification (LOQ) was 0.37 fmol/column, and limit of detection (LOD) was 0.1 fmol/ column for both peaks. The overall method detection limit (MDL) for DNA samples was calculated to be 1−5 fmol of each HPLC resolved isomers per sample. To express adduct levels as number of adducts per T, which were quantified in DNA hydrolysate using HPLC System 3 with detection at 258 nm, a standard curve (from 5 nmol to 5 pmol of T on column) was constructed using UV quantified T standard (ε267 = 9650 M−1 × cm−1 in water).34 Quantification of DHHεdA in DNA of Primary Human Hepatocytes Treated with EH by Competitive ELISA. The ELISA plate was coated, blocked, and washed as described above. The ELISA quantification was performed using the remaining portion of the samples for the LC-MS/MS. The samples were diluted with 1% (w/v) BSA (1:10 dilution) and used as competitive reagent. Solutions made from pure DHHεdA were used to construct standard curves. Purified mAb (3C9C9) solution (50 μL, 1:200,000 dilution) in 1% (w/v) BSA was added as antibody solution. Horseradish peroxidase conjugated goat antimouse IgG secondary antibodies 33.3 ng/mL were added after washing, and the plate was developed with 100 μL of

blocked in 10% normal goat serum in PBS for 10 min, then incubated with mAb solution (1:20 or 1:200 dilution) for 1 h. From this point on, cells were washed 3 times with PBS after each incubation process. Fluorescein-conjugated secondary antibody (Alexa Fluor Plus 488, A32723, Invitrogen, Carlsbad, CA) solution (1:2000 dilution) was added, and cells were incubated for 30 min in dark, incubated with 1:1000 DAPI in dark for 4 min, and then mounted onto microslide with mounting media (#17985-10, Electron Microscopy Sciences, Hatfield, PA). Cells were imaged under Olympus IX-71 Inverted Epifluorescence Microscope. FACS Analysis. The geometric mean of the fluorescence intensity of Alexa Fluor 488 labeled DHHεdA positive cells was measured using FACS. The output values of triplicate control and EH treatment groups (see above) were averaged, and the standard deviation was calculated using Excel. Additionally, a Student’s t-test was performed to determine significance by comparing the triplicate values for the control group to those of the EH-treated group for each individual time point of 4, 8, 12, and 24 h. Detection and Quantification of DHHεdA in DNA by LC-MS/ MS-MRM. DNA was isolated by a QIAGEN Blood and Cell Culture DNA Maxi Kit (#13362, QIAGEN Inc., Valencia, CA) using the protocol as recommended by the manufacturer. For hydrolysis, dry DNA (50 to 1000 μg) was dissolved in 5 mM magnesium chloride and 0.5 mM GSH solution (1 mL per 1 mg of DNA), and then 50 fmol of [15N5]-DHHεdA 1,2 and 50 fmol of [15N5]-DHHεdA 3,4 were added as internal standards. DNA was hydrolyzed by incubation with DNase I (1300 units per mg of DNA, #D4527-40KU, Sigma-Aldrich, St. Louis, MO) for 30 min at 37 °C, followed by a second addition of DNase I (1300 units per mg of DNA) and incubation for additional 10 min at 37 °C. Finally, phosphodiesterase I (0.06 units per mg DNA, #P32431VL, Sigma-Aldrich, St. Louis, MO), alkaline phosphatase (380 units per mg DNA, #10 108 146 001, Roche Diagnostic GmbH, Mannheim, Germany), and adenosine deaminase (0.5 units, #LS009043, Worthington Biochemical Corporation, Lakewood, NJ) were added, and the sample was incubated for 60 min at 37 °C. After hydrolysis small portion of hydrolysate was saved for further T quantification, and the remaining sample was purified using Phenomenex Strata-X 33 μm 30 mg/1 mL polymeric reversed-phase solid-phase extraction columns (#8B-S100-TAK, Phenomenex, Torrance, CA). Before loading samples, columns were washed by ACN (3 × 1 mL) and stabilized by 25 mM ammonium formate pH = 4.00 (3 × 1 mL). After loading DNA hydrolysate columns were washed by 2.5% ACN in 25 mM ammonium formate pH = 4.00 (1 × 1 mL), 5% ACN in 25 mM ammonium formate pH = 4.00 (1 × 1 mL), followed by DHHεdA collection by 30% ACN in 25 mM ammonium formate pH = 4.00 (1 × 1 mL). DHHεdA fraction was dried using SpeedVac rotary concentrator, redissolved in 400 μL 1:1 water:ACN, transferred to HPLC vials, dried, and kept at −20 °C. Before quantification, samples were dissolved in 60 μL of water, and 37 μL was injected on LC-MS. Quantification of DHHεdA in DNA was carried out on SCIEX 6500 QTRAP triple quadrupole mass spectrometer (AB Sciex LLC, Framingham, MA) interfaced with a Waters ACQUITY UPLC liquid chromatography system equipped with Waters ACQUITY UPLC BEH C18 50 × 2.1 mm, 1.7 μm particle size column (Waters Corporation, Milford, MA). The separation of adducts was performed isocratically by eluting with 13.5% ACN, 1 mM ammonium formate buffer over 6.5 min using 0.5 mL/min flow rate at 40 °C, followed by 100% ACN wash. The ESI source operated in positive mode. The MRM experiment was performed using ion transitions of 406.2 → 290.2 m/z (DHHεdA) and 411.2 → 295.1 m/z ([15N5]-DHHεdA) with CE of 28 eV for quantification, and those of 406.2 → 160.1 m/z (DHHεdA) and 411.2 → 165.0 m/z ([15N5]-DHHεdA) with a CE of 76 eV were used for structural confirmation. All other parameters were optimized to achieve maximum signal intensity. Calibration curves were constructed for two HPLC resolved peaks before each analysis using standard solutions of DHHεdA and [15N5]-DHHεdA. A constant concentration of [15N5]-DHHεdA (1 fmol/μL) was used with different concentrations of DHHεdA (3.3 amol/μL to 65 fmol/μL) and analyzed using 37 μL injections by LC-MS/MS-MRM. The standard curves were linear in the range from 0.37 to 800 fmol of

Figure 1. Reactivity of mAbs against DHHεA-BSA by ELISA. (a) Im I and (b) Im II. DHHεA-BSA conjugate (×2, 100 ng) was immobilized on plate and incubated with varying dilutions of mAbs from the six monoclonal cell lines. HRP IgG at 2 ng/mL and 33.3 ng/mL was used in Im I and Im II, respectively. 776

DOI: 10.1021/acs.chemrestox.8b00111 Chem. Res. Toxicol. 2018, 31, 772−783

Article

Chemical Research in Toxicology

Figure 2. Reactivity of mAbs from Im I against normal nucleosides by competitive ELISA for (a) dG, (b) dA, (c) dC, and (d) T. DHHεA-BSA conjugate (×2, 100 ng) was immobilized on plate and incubated with mAbs and varying concentration (0, 0.1, 1, 10, and 100 ng) of competing agents. None of six mAbs tested binds to normal nucleotides. HRP IgG at 2 ng/mL was used. working solution of SuperSignal ELISA Femto Maximimum Sensitivity Substrate kit. HPLC Systems. System 1. Agilent 1200 HPLC system consisting of a G1322A degasser, a G1311A quaternary pump, and a G1315D photodiode array detector (Agilent Technologies, Inc., Santa Clara, CA). The system was equipped with a Phenomenex Prodigy ODS3, 250 × 4.6 mm, 100 Å, and 5 μm column protected by a Phenomenex guard cartridge (Phenomenex, Inc., Torrance, CA). Solvent A: water with 0.1% TFA; solvent B: ACN with 0.1% TFA. A flow rate of 1 mL/min was established. The gradient program was 0−25 min from 0% B to 100% B, followed by 12 min washing by 100% B. Before each run, the column was equilibrated with 100% A for 12 min. The detector recorded chromatograms at 200, 210, 227, 254, and 280 nm. Spectra for all time points were recorded from 190 to 400 nm with a resolution of 1 nm. System 2. Shimadzu HPLC system was comprised of a SPD-M10A VP diode array detector, a SCL-10A VP controller, and two LC-10AD VP pumps (Shimadzu Scientific Instruments, Columbia, MD) equipped with Phenomenex Prodigy 250 × 21.2 mm, 5 μm particle size, 100 Å, and ODS3 (C18) columns (Phenomenex, Torrance, CA). Solvent A: water with 0.1% TFA; solvent B: ACN with 0.1% TFA. Flow rate was set to 9.999 mL/min. The gradient program was 0−6 min 100% A, 6−96 min from 0% B to 100% B, followed by 20 min washing with 100% B. Before each run the column was equilibrated with 100% A for 20 min. The detector recorded chromatograms from 200 to 320 nm with 1 nm resolution and 4.2 Hz sampling rate. System 3. Waters ACQUITY UPLC system with PDA detector and Waters ACQUITY UPLC BEH C18 50 × 2.1 mm, 1.7 μm particle size column (Waters Corporation, Milford, MA). Solvent A was water with 0.1% heptafluorobutyric acid (HFBA), solvent B was methanol (MeOH) with 0.1% HFBA. Column temperature was 40 °C, and the flow rate was 0.5 mL/min with linear gradient program: 0 to 4 min from 0 to 4% B. Between injections, the column was washed by 100% B for 2.6 min and stabilized with 100% A for 2.6 min.

by epoxidation of HNE as an exclusive oxidation product of ω-6 PUFAs, is likely responsible for the in vivo formation of DHHεdA. While both HNE-dG and DHHεdA may be considered as DNA damage biomarkers of ω-6 PUFAs,35 the levels of DHHεdA detected in some tissues are approximately 10−100-fold higher than HNE-dG (×107−8 vs 109, respectively).21 The biological consequences of DHHεdA formation in cells and its interactions with cellular proteins remain to be elucidated. The fact that EH is a more potent mutagen and tumorigen than HNE implicates DHHεdA as a more relevant biomarker for the study of tumor promoting activity of ω-6 PUFAs.26 In this study, two separate immunizations (Im I and Im II) using an antigen derived from DHHεdA were performed in an effort to raise mAbs as tools for detecting DHHεdA in cells and tissues. Producing mAbs against DHHεdA. The vicinal diols in the side chain of DHHεdA hapten prohibited its conjugation to carrier proteins using the standard periodate oxidation because it will result in side chain cleavage.36 We decided to adopt an alternative conjugation strategy, as shown in Scheme 2, involving conversion of guanosine 5′-hydroxy group into 5′-carboxy and conjugation of modified hapten to protein by water-soluble carbodiimide. Mass spectrometry confirmed that the molecular weight represents the modification levels of 3.7 (×2) and 10.0 (×10) adducts per BSA (with 2 × excess of EDEC and 10 × excess for the conjugation reaction of 5′-carboxy-DHHεdA with BSA, respectively). Levels of modifications for KLH conjugate were not determined by mass spectrometry due to high molecular mass, complex structure, and poor stability of carrier protein. Five mice were used in Im I. After immunization with DHHεA-KLH, all of them showed satisfactory immune responses. Based on ELISA results on specificity and selectivity, only two mice were chosen for further antibody development. After spleen fusion, a total of 20 hybridomas were produced from Im I. In Im II, three mice were used. Similarly, 24 hybridomas

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RESULTS AND DISCUSSION In recent years DHHεdA was reported to be detected in rodent and human tissues as an endogenous DNA lesion.21 EH, formed 777

DOI: 10.1021/acs.chemrestox.8b00111 Chem. Res. Toxicol. 2018, 31, 772−783

Article

Chemical Research in Toxicology

Figure 3. Reactivity of mAbs from Im I against selected commonly detected DNA adducts by competitive ELISA for (a) Acr-dG, (b) 8-oxo-dG, (c) HNE-dG, and (d) εdA. DHHεA-BSA conjugate (×2, 100 ng) was immobilized on plate and incubated with mAbs and varying concentration (0, 0.1, 1, 10, and 100 ng) of competing agents. None of six mAbs tested binds to Acr-dG and 8-oxo-dG. Moderate affinity to εdA was found for two mAbs. All six mAbs showed moderate binding to HNE-dG. HRP IgG at 2 ng/mL was used.

were produced. The supernatants of all fused cell lines were screened by direct ELISA for their activity toward DHHεABSA, from which three positive clones were produced from Im I (Figure S1a) and six from Im II (Figure S1b). The three clones from Im I were further grown into two subclones, each forming six stable monoclonal hybridomas (3C3B6, 3C3E12, 3C9C9, 3C9G2, 4E10B8, 4E10F2). They were characterized by direct and competitive ELISA for their activity and specificity against DHHεA-BSA. Among the 24 clones obtained from Im II, six showed relatively high reactivity against the antigen (3A11, 4B2, 2E8, 2G7, 6A1, 4D2). For all competitive ELISA experiments, mAbs concentrations were normalized by total protein content in the supernatants. To test activity, sequential dilutions of mAbs were incubated with 100 ng of DHHεABSA conjugate ((×2) with an average of 3.7 modifications per protein) deposited on ELISA plate. All six anti-DHHεdA monoclonal cell lines from Im I and Im II each displayed similar and strong binding activities (Figure 1). Determining Reactivity and Specificity of mAbs. The specificity of antibodies was determined using competitive ELISA by testing reactivity toward immunogen in the presence of normal nucleotides and other structurally related cyclic DNA adducts. All six mAbs from Im I showed no reactivity toward normal nucleotides, including dA (Figure 2a−d). Also, no reactivity toward Acr-dG (or γ-OHPdG) or 8-oxo-dG was observed for all mAbs (Figure 3a,b). All six mAbs showed weak reactivity toward HNE-dG only at concentrations above 10 ng/well (Figure 3c). The competing effects of HNE-dG at 100 ng/well were similar or weaker than competing effects for DHHεdA at one ng/well in that assay (data not shown), indicating that the mAbs has at least 100× stronger affinity toward DHHεdA than HNE-dG. Because of the relatively low endogenous levels of HNE-dG compared to DHHεdA in vivo,21 the weak crossreactivity is not expected to affect the specificity of the mAbs to

Figure 4. Reactivity of purified 3C9C9 mAb against (a) normal nucleotides and (b) selected DNA adducts by competitive ELISA. DHHεA-BSA conjugate (×2, 100 ng) was immobilized on plate and incubated with 3C9C9 and varying concentration (0, 0.1, 1, 10, 100, and 1000 ng) of competing agents. 3C9C9 does not bind to normal nucleotides, Acr-dG, 8-oxo-dG, and εdA. Moderate binding to HNE-dG adducts was observed. HRP IgG at 2 ng/mL was used. 778

DOI: 10.1021/acs.chemrestox.8b00111 Chem. Res. Toxicol. 2018, 31, 772−783

Article

Chemical Research in Toxicology

Figure 5. Reactivity of mAbs obtained Im II against normal nucleosides and related DNA adducts by competitive ELISA. DHHεdA-BSA conjugate (×2, 100 ng) was immobilized on plate and incubated with mAbs and varying concentration (0, 0.1, 1, 10, 100, and 1000 ng) of competing agents. None of six mAbs tested bind to Acr-dG, 8-oxo-dG, and εdA, and three mAbs (2G7, 4D2 and 4B2) showed moderate binding to HNE-dG adducts. HRP IgG at 33.3 ng/mL was used.

by quantitative mass spectrometry. In CT-DNA treated with EH diluted with ethanol (1:10), we detected a total of 2475.7 adducts per 106 unmodified nucleotides (in the absence of dA in DNA hydrolysate due to deaminase treatment T was quantified as dA complementary base) or 2158.8 fmol adduct per 1 μg of DNA (Figure 6a and Table S1). On reaction of CT-DNA with 1:1000 diluted EH solution, the combined modification of DHHedA 1 and 2 was detected at 40.4 adducts 106 unmodified nucleotides or 34.8 fmol of adduct per 1 μg of DNA. The background levels of endogenous DHHεdA detected in unmodified CT-DNA were 12.9 adducts per 109 unmodified nucleotides or 11.7 amol adduct per 1 μg of DNA. For ELISA, EH-modified CT-DNA and unmodified CT-DNA were coated on plates with amounts from 1 μg to 0.1 ng per well. The plates were incubated with 3C9C9, then HRP conjugated secondary antibody and finally ultrasensitive chemiluminescence HRP substrate were added to enhance signal. The signals increased with increasing amounts of 1:10 EH-modified DNA immobilized on plate, whereas the signals from 1:1000 EH-modified DNA or unmodified CT-DNA remain steady (Figure 6b). The data clearly showed that 3C9C9 recognizes DHHεdA in DNA,

detect DHHεdA. The two cell lines (3C3B6 and 3C3E12) showed moderate competitive effects against εdA (Figure 3d), and they were not further used in the study. All other mAbs showed similar affinity and specificity. Because 3C9C9 performed slightly better in the competitive ELISA for DHHεdA than HNE-dG (Figure 3 c), it was used to produce purified mAb. The purified 3C9C9 mAb was then evaluated against dA, dC, dG, T, Acr-dG, 8-oxo-dG, and εdA to confirm that it has no cross-reactivity, and similar results were obtained (Figure 4a,b). Similar, if not identical, results on specificity and reactivity against normal and related adducts were obtained with the mAbs from Im II (Figure 5), demonstrating the reproducibility of DHHεdABSA as an antigen for raising mAbs. It should be noted that although the dG counterpart of DHHεdA, 7-(1,2-dihydroxyheptyl)-εdG (DHHεdG), can also be formed by reaction with EH, it has not yet been detected in vivo, possibly due to its rapid conversion to 1,N2-εdG under neutral and basic conditions.18,37 Detection of DHHεdA in CT-DNA by LC-MS/MS and ELISA. The purified 3C9C9 was used to study the sensitivity of ELISA for detecting DHHεdA in DNA using EH-modified CT-DNA. The levels of DHHedA 1 and 2 were first determined 779

DOI: 10.1021/acs.chemrestox.8b00111 Chem. Res. Toxicol. 2018, 31, 772−783

Article

Chemical Research in Toxicology

Figure 6. (a) A representative chromatogram of LC-MS/MS determination of DHHεdA in CT DNA modified by EH (1:10). A combined levels of DHHedA 1 and 2 of 2475.7 adducts per 106 unmodified nucleotides were detected (Table 2) and (b) reactivity of purified 3C9C9 mAb against the EH (1:10 and 1:1000) modified and unmodified CT DNA by ELISA. DNA was immobilized on plate (from 1 μg to 0.1 ng DNA per well) and exposed to 3C9C9 mAb.

Figure 7. Detection of DHHεdA in EH-treated human hepatocyte using (a) LC-MS/MS, a representative chromatogram obtained from cells treated with 300 μM EH and (b) FACS analysis with S9C9 in extracted nuclei of human hepatocytes, a significant difference (t-test, p < 0.05) between treatment and control groups at the time points 4, 8, 12, and 24 h. The significant decrease of DHHεdA in cells treated with EH at 24 h may be attributed to repair as