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Quantitation of Lipid Peroxidation Product DNA Adducts in Human Prostate by Tandem Mass Spectrometry: A Method that Mitigates Artifacts Haoqing Chen, Sesha Krishnamachari, Jingshu Guo, Lihua Yao, Paari Murugan, Christopher J. Weight, and Robert J Turesky Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00181 • Publication Date (Web): 30 Jul 2019 Downloaded from pubs.acs.org on July 31, 2019

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Chemical Research in Toxicology

Quantitation of Lipid Peroxidation Product DNA Adducts in Human Prostate by Tandem Mass Spectrometry: A Method that Mitigates Artifacts Haoqing Chen†, Sesha Krishnamachari†, Jingshu Guo†, Lihua Yao†, Paari Murugan‡, Christopher J. Weight∥, Robert J. Turesky*†

†Masonic

Cancer Center and Department of Medicinal Chemistry, ‡Department of Laboratory

Medicine and Pathology, and ∥Department of Urology, University of Minnesota, Minneapolis, Minnesota 55455, United States Corresponding author: *Robert J. Turesky, Ph.D. Masonic Cancer Center and Department of Medicinal Chemistry, College of Pharmacy, 2231 6th St SE, University of Minnesota, Minneapolis, MN 55455. Tel: 612-626-0141; Fax: 612-624-3869; Email: [email protected]

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ABSTRACT

Reactive oxygen species (ROS) and chronic inflammation contribute to DNA damage of many organs, including the prostate. ROS cause oxidative damage to biomolecules, such as lipids, proteins, and nucleic acids, resulting in the formation of toxic and mutagenic intermediates. Lipid peroxidation (LPO) products covalently adduct to DNA and can lead to mutations. The levels of LPO DNA adducts reported in humans range widely. However, a large proportion of the DNA adducts may be attributed to artifact formation during the steps of isolation and nuclease digestion of DNA. We established a method that mitigates artifacts for most LPO adducts during the processing of DNA. We have applied this methodology to measure LPO DNA adducts in the genome of prostate cancer patients, employing ultra-high-performance liquid chromatography electrospray ionization ion trap multistage mass spectrometry. Our preliminary data show that DNA adducts of acrolein, 6-hydroxy-1,N2-propano-2′-deoxyguanosine (6-OH-PdG) and 8hydroxy-1,N2-propano-2′-deoxyguanosine (8-OH-PdG) (4 – 20 adducts per 107 nucleotides) are more prominent than etheno (ε) adducts (< 0.5 adducts per 108 nucleotides). This analytical methodology will be used to examine the correlation between oxidative stress, inflammation, and LPO adduct levels in patients with benign prostatic hyperplasia and prostate cancer.


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INTRODUCTION Endogenous reactive oxygen species (ROS) play an important role in cellular signaling and homeostasis. ROS are generated from normal metabolism or triggered by environmental factors, such as ultraviolet light, ionizing radiation, chemotherapeutic treatments, and inflammatory cytokines. Cellular antioxidant defense systems, including enzymatic scavengers and nonenzymatic antioxidants (e.g., ascorbate, vitamin E, and glutathione), reduce the intracellular burden of these oxidants.1 If the balance between ROS generation and antioxidant defense is shifted in favor of the former, the excessive ROS will attack cellular components and produce an oxidative stress that is associated with chronic and degenerative diseases, such as arthritis, autoimmune disorders, cardiovascular and neurological disorders, aging, and cancer.2 ROS cause direct damage on the DNA bases and deoxyribosyl backbone. However, free radicals are extremely short-lived species and mainly attack molecules in very close proximity. Cellular polyunsaturated fatty acids are a major target of ROS attack, which can generate a free radical chain reaction resulting in a series of lipid peroxidation (LPO) end products,3 such as 4-hydroxy2-nonenal (HNE), 4-oxo-(2E)-nonenal (ONE), malondialdehyde (MDA), and acrolein.3 Many LPO products conjugate with intracellular glutathione (GSH), which is present at millimolar concentrations in the cell and serves as a detoxification pathway.4, 5 However, a portion of the LPO products may escape detoxification due to their high local concentration (as much as 10 mM in microsomal and mitochondrial intramembranes).6 Aldehydes are more stable than free radicals. In the presence of GSH, LPO such as 4-ONE and 4-HNE have half-lives of roughly 1 s and 2 min, respectively.6 Thus, some LPO products can diffuse into the nucleus and form DNA adducts.7, 8 Multiple classes of exocyclic LPO DNA adducts have been characterized, including the HNE adduct HNE-dG; ONE adducts heptanone-etheno-2′-deoxycytidine (HεdC); heptanone-etheno-2′-


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deoxyguanosine (HεdG); heptanone-etheno-2′-deoxyadenosine (HεdA); MDA adduct 3-(2Deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG);

the

acrolein

adducts 6-hydroxy-1,N2-propano-2′-deoxyguanosine (6-OH-PdG) and 8-hydroxy-1,N2-propano2′-deoxyguanosine (8-OH-PdG); and the etheno adducts 3,N4-etheno-2′-deoxycytidine (εdC), 1,N6-etheno-2′-deoxyadenosine (εdA), 1,N2-etheno-2′-deoxyguanosine (εdG) generated from epoxides (Scheme 1).9-16 These adducts are potential biomarkers of oxidative stress-related diseases and cancer risk; some of the adducts induce high mutagenicity during cell replication.1719

Analytical methods have been developed for quantitating LPO adducts in human tissues, blood, and urine. The techniques include

32P-postlabeling,

immunoblot assay, immunohistochemistry,

gas chromatography-mass spectrometry, and liquid chromatography-tandem mass spectrometry (LC-MS/MS).20 However, the variation of levels of LPO adducts reported are large, ranging from one adduct in 1010 nucleotides (nts) to one adduct in 105 nts.20-24 This wide variation may be attributed to artifact formation during DNA workup, where free radicals, LPO end products, redoxactive transition metals (e.g. iron and copper),25,

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and environmental pollutants (e.g. vinyl

chloride and acrolein) can serve as sources of artifacts. Antioxidants (AO) are commonly used to minimize artifact formation, such as the metal chelators deferoxamine (DFO)27 and 8hydroxyquinoline (8-HQ),28 the free radical scavengers butylated hydroxytoluene (BHT)27 and 2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO),29 the aldehyde reducing agent [Na(CN)BH3],30 or thiol compounds such as glutathione (GSH).31 Although these antioxidants can mitigate artifact formation, most studies have not proven that AOs are fully effective, or focused on only a few LPO adducts with similar chemical properties.


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A reliable approach that mitigates artifactual formation of DNA adducts during the isolation and nuclease digestion of DNA is required to understand the true occurrence of LPO adduct formation in human tissues. We have conducted a systematic evaluation of artifact formation and the loss of LPO adducts during DNA isolation, enzymatic digestion, and storage steps with different tissue workup methods. We established an ultra-high-performance liquid chromatography electrospray ionization ion trap multistage mass spectrometric (UHPLC/ESI-IT-MS3) method to quantitate LPO adducts, covering most of the reported adduct types (Scheme 1). We demonstrate that metal chelators and free radical scavengers do not completely prevent artifact formation, while the thiol compound β-mercaptoethanol (BME) efficiently mitigates LPO-induced DNA damage during sample workup and digestion of DNA. Using our optimized method, the level of adducts measured is relatively free of artifact formation. Inflammation is a common pathology in the prostate of aging men, and plays a critical role in the development of benign prostatic hyperplasia (BPH) and prostate cancer (PC),32-35 which is the second leading cause of cancer death for men in the United States.36 BPH is the most common urologic disease in men at old age.37 PC and BPH usually develop from different zones of the prostate gland, the former from the peripheral zone (PZ) and the latter from the transitional zone (TZ).37 Both diseases are associated with inflammation, an oxidative-stress condition38 that occurs in prostate cancer and BPH patients,33, 39 with the level of oxidative stress often higher in PC patients.40 Determining the overall LPO adduct levels in human prostate specimens may shed light on the link between oxidative stress, inflammation, and PC. Although some LPO adducts have been detected in human lung, liver, pancreas, colon, breast, gastric mucosa, oral cells, blood and urine samples,20 to our knowledge, there are no previous reports on the detection of LPO adducts


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in human prostate. Thus, we applied our new methodology to examine the formation of LPO adducts in the peripheral and transition zones of prostate cancer patients.


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EXPERIMENTAL SECTION Caution! α,β-unsaturated aldehydes (enals) are toxic and potential human carcinogens. These chemicals must be handled in a well-ventilated fume hood with proper use of gloves and protective clothing. Instrumentation information are reported in Supporting Information (SI). Materials. Escherichia coli (ATCC® 15224™) was purchased from American Type Culture Collection (Manassas, VA). Calf thymus (CT) DNA, DNase I (type IV, bovine pancreas), alkaline phosphatase (Escherichia coli), nuclease P1 (Penicillium citrinum), RNase A (bovine pancreas), RNase T1 (Aspergillus oryzae), and proteinase K (Tritirachium album) were purchased from Sigma-Aldrich (St. Louis, MO). Phosphodiesterase I (Crotalus adamanteus venom) and adenosine deaminase were purchased from Worthington Biochemical Corp. (Lakewood, NJ). LC-MS grade solvents were purchased from Fisher Chemical Co. (Pittsburgh, PA). Puregene protein precipitation solution was purchased from Qiagen (Germantown, MD). LPO adduct standards and isotopic labeled internal standards were synthesized according to reported methods.10-16 M1dG and [15N5]-M1dG, HNE-dG, and [2H11]-HNE-dG were kindly provided by Dr. Carmelo Rizzo, Vanderbilt University. All other chemicals were ACS grade and purchased from Sigma-Aldrich unless stated. Bond Elut C18 solid phase extraction (SPE) cartridge (40 μm, 100 mg/1 mL) was purchased from Agilent (Santa Clara, CA). MicroLiter autosampler vials with silanized glass inserts were purchased from Wheaton (Millville, NJ). Animal studies. Protocols were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee. Male F344 rats at 43−62 days, body weight range 170-210 g (Charles River Laboratories, Wilmington, MA) were housed in the animal facility under


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standard conditions on AIN-76A diet. After one week of acclimatization, the rats were sacrificed, and the organs were immediately frozen on dry ice and stored at −80 ºC until sample workup. Human prostate tissue collection and treatments. The research protocol was reviewed and approved by the Institutional Review Board at University of Minnesota. The study population consisted of men from Minnesota, Wisconsin, South Dakota, North Dakota, and Iowa, who were diagnosed with PC and underwent radical prostatectomy (mean age: 64.2 ± 8.5). All patients consented to participate in this research. The de-identified tissues were flash-frozen in liquid nitrogen within 30 minutes of surgery. Tissue homogenization, DNA isolation, and DNA digestion methods with AO. Frozen tissues were thawed and cut into small pieces on ice. The minced tissue (30−50 mg) was homogenized on ice with a PRO 200 homogenizer (PRO Scientific, Oxford, CT) at medium speed (9000 to 17000 rpm) in TE buffer [50 mM Tris-HCl, 10 mM EDTA, pH 8.0, with or without AO] with a dilution factor of 5. The nuclei were pelleted at 3000g, 4 ºC for 10 min and resuspended in 500 μL TE buffer (with or without AO) for DNA isolation (PUR, PUR/CHCl3, or Phe/CHCl3 method). Subsequent nuclease digestion (method D-a or D-b) was performed in Bis-Tris or HEPES buffer to circumvent Tris-conjugation to M1dG.41 The DNA isolation and nuclease digestion conditions are reported in Table 1. Detailed information on AO efficacy, and the method for determining artifact formation during sample preparation are described in the following text and summarized in Table 2. Solid phase extraction (SPE) method. The digested DNA (50−100 µg, 100 μL solution) was diluted with 900 μL LC-MS grade water and loaded on a Bond Elut C18 SPE cartridges (100 mg bed mass) preconditioned with CH3CN and H2O. The cartridge was washed with H2O (4 mL), 1% CH3CN (1 mL), and eluted with 50% CH3CN (1 mL) to a 2 mL Eppendorf tube. The eluent was


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dried under vacuum centrifugation, resuspended in 90% CH3CN (90 μL) to recover the non-polar ONE adducts that adsorbed to the surface of the Eppendorf tubes, and centrifuged at 21,000g for 10 min. The supernatant was transferred to autosampler vials with silanized glass inserts and dried under vacuum centrifugation. Water (20 μL) was added to dissolve the residue for LC-MS3 analysis. Quantitation of LPO adducts by UHPLC/ESI-IT-MS3. Quantitation was conducted on a Velos Pro Dual-Pressure Linear Ion Trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an UltiMate™ 3000 RSLCnano System (Thermo Fisher Scientific, San Jose, CA) and an Advance CaptiveSpray source (Michrom Bioresource Inc., Auburn, CA). A Magic C18AQ reversed-phase column (Michrom Bioresources, Inc., 0.2 × 150 mm, 3 μm particle size, 200 Å pore size) was employed for chromatographic separation. The mobile phases were (A) 2 mM NH4OAc and (B) 2 mM NH4OAc in 95% CH3CN. SPE-enriched sample (9 μL) was injected on to the analytical column. Adducts in human prostate samples were separated with the following gradient: 0−9 min: 3 μL/min, 1% B; 9−15 min: 3 μL/min, 1−7 % B; 15−16 min: 3−1 μL/min, 7−8% B; 16−23 min: 1 μL/min, 8−14 % B; 23−24 min, 1 μL/min, 14−30 % B; 24−30 min: 1 μL/min, 30−65 % B; 30−31 min: 1 μL/min, 65−90 % B; 31−33 min, 1−3 μL/min, 90 % B; 33−36 min, 3 μL/min, 90−1 % B; 36−41 min, 3 μL/min, 1 % B. The sample loop was bypassed at 3.5 min. Adducts were analyzed in positive ion mode at the MS3 stage. The major product ions of the aglycones [M + H]+ > [M + H − 116]+ were extracted and employed for quantification. The adducts were analyzed in two segments. Segment one (0 to 26 min): dG (268.0 → 152.0 → 135.0); [15N5]-dG (273.0 → 157.0 → 139.0); M1dG (188.0 → 160.0 → 106.0); [15N5]-M1dG (193.0 → 165.0 → 109.0); 6-OH-PdG (324.2 → 208.1 → 152.1, 190.0, first peak); [15N5]-6-OH-PdG (329.2 → 213.1 → 157.1, 195.0, first peak); 8-OH-PdG (324.2 → 208.1 → 164.0); [15N5]-8-OH-PdG


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(329.2 → 213.1 → 169.0); εdC (252.1 → 136.1 → 81.1); [15N3]-εdC (255.1 → 139.1 → 83.0); εdA (276.1 → 160.0 → 106.0, 119.0, 133.0); [15N5]-εdA (281.1 → 165.0 → 109.0, 123.0, 137.0); εdG (292.1 → 176.0 → 121.0, 148.1); [15N5]-εdG (297.1 → 181.0 → 125.0, 153.0). Segment two (26 to 42 min): HεdC (364.2 → 248.2 → 150.0); [15N3]-HεdC (367.2 → 251.2 → 153.0); HεdA (388.2 → 272.1 → 174.0); [15N5]-HεdA (393.2 → 277.1 → 179.0); HεdG (404.2 → 288.1 → 190.0); [15N5]-HεdG (409.2 → 293.1 → 195.0); HNE-dG (424.2 → 308.2 → 152.1, 290.2); [15N5]HNE-dG (429.2 → 313.2 → 157.1, 295.2); [2H11]-HNE-dG (435.2 → 319.2 → 152.1, 301.2). Seven-point calibration curves (Figure S5) were constructed by spiking isotopically labeled internal standards (25 fmol/45 μg DNA, 1.83 adduct per 107 nts) and authentic standards (n = 3) using liver DNA from an untreated rat isolated with PUR/CHCl3 method with BME (10 mM) and digested with D-b with BME (2.5 mM) as the AO (Table 1). The amount of DNA was quantitated by UV spectroscopy, assuming an absorbance at 260 nm of 1.0 OD corresponds to 50 μg/mL double stranded DNA solution, and converted to nucleotides (nts) using an average molecular weight of 330 g/mol for unmodified nts. The adduct levels were then expressed per 107 or 108 nts. The background level of εdA, M1dG, HεdC, HεdA, and HNEdG (Table S2) were subtracted from each calibration level to ensure accuracy. The least-squares linear regression was used to fit the experimental data with a weighting factor of 1/y. Antioxidant efficacy in DNA isolation (Table 1, method PUR + D-a, Figure 1a). Freshly frozen rat kidney tissue was homogenized in TE buffer containing either freshly added BME (10 mM), BHT (0.1 mM), 8-HQ (0.35 mM), GSH (0.5 or 5 mM), DFO (0.1 or 1 mM), TEMPO (15 mM), or no AO (Table 2). After pelleting the nuclei, 15N-labeled bacterial DNA [see SI for bacteria growth, 15N-incorporation

efficiency, and bacterial DNA isolation method] was added to either the nuclear


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pellet or the supernatant (500 μL) of tissue homogenate. DNA was isolated by PUR method (Table 1) with or without the same AO used in homogenization. No AO was added during the DNA desalting step. Prior to DNA digestion, [2H11]-HNE-dG was added as a global ISTD. DNA (60 to 100 μg in total, a mixture of 14N- and 15N-DNA, quantitated by UV) was digested with method D-a (Table 1), pH 7.1 in the presence of the AO acetohydroxamic acid (AHA, 1 mM).42, 43 The concentration of 15N adducts was determined by comparing the peak area ratio of adducts to that of [2H11]-HNE-dG and calculated based on standards with known concentration employing a calibration curve. The 15N adduct levels were determined using the concentration of unmodified deoxyribonucleosides from 15N-DNA, which was calculated based on the total DNA determined by UV spectroscopy and the ratio of 15N-DNA to 14N-DNA using the peak area of [15N5]-dG and dG in diluted DNA by UHPLC/ESI-IT-MS3 (Table S1 and S2). Antioxidant efficacy in DNA digestion (Table 1, method PUR + D-a, Figure 1d). We chose spleen as our study model because it contains a much higher iron content than other organs,44 making it possible to create a high level of artifacts in the DNA digestion step. Rat spleen DNA was isolated by the PUR method with BME (10 mM). [2H11]-HNE-dG was spiked prior to digestion. HPLC-purified 15N-labeled 2′-deoxyribonucleosides (dNs) including [15N5]-dG, [15N5]dA, and [15N3]-dC (10 μg each) were added to the isolated rat DNA (30 μg) to measure artifact formation of the 15N-labeled dNs during enzyme digestion. The DNA was digested with method D-a in Bis-tris (5 mM, pH 7.1) or HEPES (5 mM, pH 8.0) buffer without AO, or with GSH (0.5, 1, 2.5 mM), BME (0.5, 1, 2.5 mM), or AHA (1 mM). For the negative control, 15N-dNs (10 μg each) and [2H11]-HNE-dG were treated with adenosine deaminase for 1.5 h and processed through SPE. The amount of artifactual 15N-LPO adducts was estimated using [2H11]-HNE-dG as the ISTD (vide supra) (Table S3).


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Enzymatic digestion efficiency of DNA (method D-b). Method D-b was found to be optimal for enzyme digestion and mitigation of artifacts (vide infra). To confirm the completeness of digestion of modified DNA bases, LPO-modified CT DNA (100 μg, see SI for synthesis protocols) was digested with one or two equivalents of enzyme using method D-b with BME (2.5 mM) as the AO. The concentration of each adduct was quantitated by comparing the peak area of the adduct to its corresponding stable isotopically labeled internal standard (Figure S1). The completeness of digestion for unmodified nucleosides was monitored by HPLC-UV showing four peaks of dC, dI, dG and dT.45 Effect of chloroform extraction (method PUR/CHCl3 or Phe/CHCl3 + D-b). Optimzations were made to the PUR method to remove lipid residues from DNA. Briefly, DNA was purified from tissue homogenate by Puregene protein precipitation (PUR/CHCl3) or phenol protein extraction (Phe/CHCl3), followed by lipid removal with chloroform extraction (Table 1). The effect of BME was determined as follows: Test 1 (BME in DNA isolation) (Figure 3a). Rat liver DNA was isolated by the PUR/CHCl3 or Phe/CHCl3 method with or without BME (10 mM) in the presence of

15N-DNA

and digested using method D-b with BME (2.5 mM).

15N-DNA

was

directly digested with the same method (without processing) and served as the control. Test 2 (BME in DNA digestion) (Figure 3b). Rat liver DNA (45 μg) was isolated by PUR/CHCl3 or Phe/CHCl3 method with BME (10 mM) and digested using method D-b with or without BME (2.5 mM) in presence of 15N-dNs. For control, 15N-dNs were spiked in the same digestion matrix of method D-b (same buffer and enzyme cocktail, with BME), treated with adenosine deaminase for 1.5 h, and processed through SPE. [2H11]-HNE-dG was used as a global ISTD. Artifact LPO adducts were estimated against the concentration of 15N-labeled unmodified deoxyribonucleosides (Tables S4 and S5).


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Determining adduct stability and artifact formation during DNA isolation using CT DNA (PUR/CHCl3 or Phe/CHCl3 + D-b). Commercial CT DNA (600 μg) was processed through DNA isolation steps using PUR/CHCl3 or Phe/CHCl3 method with BME (10 mM). Processed DNA and control DNA (that did not go through DNA isolation steps) were dissolved in HEPES buffer (5 mM, pH 8.0) containing freshly added BME (2.5 mM), quantitated by UV-Vis, and digested (400 μg) with method D-b (using 4 x enzymes and buffer) with BME. The LPO adducts were quantitated against their corresponding labeled ISTDs (spiked at 2.06 adducts per 108 nts). Method validation. The within-day and between-day reproducibility assays were performed using LPO-modified CT DNA (Table 4). DNA (45 μg, determined by UV spectroscopy) was digested with method D-b with BME (2.5 mM) in quadruplicate on three different days. The concentration of each adduct was quantitated using the corresponding stable isotopically labeled internal standard, and the DNA adduct level was calculated against the concentration of DNA. The percent coefficient of variation (%CV) was used to measure reproducibility. LPO adducts in human prostate tissue. Prostate tissue from the transitional and peripheral zones from six prostate cancer patients was isolated using the PUR/CHCl3 method with BME (10 mM) as the AO, and the DNA (45 μg) was digested using method D-b with BME (2.5 mM). The level of adduct was quantitated against corresponding labeled ISTDs using the validated method. Statistical analysis. DNA adduct levels were log-transformed and one-way ANOVA tests with Holm-Sidak's multiple comparisons test to show statistically significant differences between the processed groups and control group for each adduct, to determine the effect of antioxidants on mitigating artifact formaton. GraphPad Prism 7 (GraphPad, La Jolla, CA) was employed. For all tests, p

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