Reagents and chemicals
Cell lysis, Proteinase K, and RNase A solutions were purchased from QIAGEN. DNA purified from calf thymus (ctDNA) was purchased from Worthington Biochemical Corporation, C3H8O and CH3OH were purchased from Honeywell, and CH2O (37%), MgSO4, and CaCl2 were purchased from Thermo Fisher Scientific. All other chemicals, materials and enzymes were purchased from Millipore Sigma. All solvents used for chromatography and mass spectrometry analyses were of the purest commercially available grade.
Generation of isotope-labeled DNA from E.coli
15N-labeled bacterial DNA was generated by growing E.coli (MG1655 strain) in M9 minimal medium (standard) fortified with 15NH4Cl. 98% DNA labeling was achieved by growing the bacteria for at least three generations. Briefly, 10 μL of bacterial stock culture in 25% glycerol were inoculated in 5 mL M9 minimal media started culture and incubated overnight in a thermoshaker (37 °C, 200 rpm). Afterwards, 50 μL of cells from the starting culture were added to 1 L M9 minimal medium containing 15NH4Cl and further incubated in the thermoshaker (37 °C, 200 rpm) until an optical density (measured by absorbance at 600 nm) of 1.2 absorbance units was reached. The culture was then split in 50 mL volumes, and the cells were pelleted by centrifugation at 4,000 x g for 10 min. Cell pellets were stored at -80 °C. The same protocol was performed in parallel for generating bacterial DNA that did not contain the 15N-isotope.
Extraction of bacterial DNA
Cell pellets were vortexed and re-suspended in the remaining liquid. Three 50 mL Eppendorf tubes containing 15N-DNA were combined into one 50 mL Eppendorf tube and 25 mL of cell lysis solution was added. Next, 150 μL Proteinase K were added followed by overnight incubation in the shaker at room temperature. A total of 7.5 mL of protein precipitation solution was added and vortexed for 20 s followed by incubation on ice for 10 min. The solution was then centrifuged (4000 x g for 10 min) and the remaining supernatant was divided evenly into two parts (~16.25 mL) and each were poured into clean Eppendorf tubes containing 17 mL cold isopropanol (IPA) to allow the DNA to precipitate. The precipitated DNA pellet was transferred in a clean silanized glass vial and subsequently washed using 3 mL 70% IPA and 3 mL 100% IPA. Pellets were air-dried and subsequently combined into one 50 mL Eppendorf tube.
The DNA was re-suspended in 10 mL 10 mM PIPES/5 mM MgCl2. A total of 150 μL RNAse A was added followed by incubation at 37 °C for 2 h. A total of 5 mL protein precipitation solution was added followed by 20 s of vortexing, 5 min incubation on ice, and centrifugation for 10 min at 4000 x g. DNA precipitation was performed by addition of 2 mL cold IPA to each vial. The precipitated DNA was removed from the sample, placed in a clean, silanized glass vial, and washed twice with 1 mL 70% IPA and 1 mL 100% IPA. DNA pellets were air-dried and stored at -20 oC.
Reaction of calf thymus DNA (ctDNA) or isotope-labeled bacterial DNA with DOX
DOX (100 μL, 0.6 mg/mL) in Tris-HCl buffer (10 mM, pH 7.4) was added to a reaction mixture containing formaldehyde (500 μL, 300 μM) in water and either ctDNA (400 μL, 2.5 mg/mL), 14N-bacterial DNA (500 μL, 1 mg/mL) or 15N-bacterial DNA (500 μL, 0.8 mg/mL) in Tris-HCl buffer (10 mM, pH 7.4). The reaction mixtures were incubated at 37 °C for 24 h. The same reaction mixtures without DOX were used as negative controls. Isolation of DNA was performed by IPA precipitation. Briefly, 2 mL cold IPA were added to each vial. The precipitated DNA was removed from the sample, placed in a clean, silanized glass vial, and washed twice with 1 mL 70% IPA and 1 mL 100% IPA. The DNA pellet was dried under a nitrogen stream. All of the steps of this procedure were performed in silanized glass vials.
Animal Ethics
All procedures involving live vertebrates, including both mouse and canine patients, were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Minnesota and were carried out in accordance with relevant guidelines and regulations. Additionally, all animal studies, both murine and canine, were performed in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (42).
Mouse treatment
Single dose: Adult male C57BL/6J mice were administered with a 10 mg/kg intraperitoneal injection of DOX or sterile saline vehicle. This dose was selected upon literature evaluation of similar studies involving an acute administration of DOX (43-46). Mice were then sacrificed 24, 48, or 96 h following DOX injection (n=2/time point). Control mice (n=2) were sacrificed 48 h following vehicle injection. The liver and blood were harvested and stored at -80 °C.
Weekly dose: Five week old male C57BL/6N mice (n=3/group) were administered once a week with DOX 4 mg/kg/week or equivalent volume of sterile saline vehicle by intraperitoneal injection for 3 weeks as we previously reported (47). Animals were sacrificed at designated time points (1 or 3 weeks) after the last injection. Liver and blood samples were collected and stored at -80 °C.
Isolation of DNA from liver tissue samples
Genomic DNA from mice exposed to DOX was extracted with the QIAGEN Gentra Puregene Tissue Kit (Qiagen Sciences) following the manufacturer’s instructions with minor modifications. In brief, frozen liver tissues (270-390 mg) were minced with a razor blade while on dry ice. The minced tissues were lysed with 3 mL cell lysis solution and incubated for 5 min on ice to allow for degradation. The tissue was then homogenized using a tissue homogenizer set at low-medium speed for no more than 1 min. Additional 3 mL of cell lysis solution were added and mixed by inverting 25 times. Next, 30 µl of Puregene Proteinase K were added and tubes were mixed by inverting 25 times and incubated overnight in a shaker at room temperature. RNase A solution (30 µl) was added to each lysate and mixed before incubation for 2 h in a shaker at room temperature. Then, 2 mL of protein precipitation solution were added and tubes were vortexed vigorously for 20 s prior to centrifugation (2500 x g for 15 min). Supernatants were added to cold IPA, and DNA was precipitated and washed as previously described, with the only difference being the DNA pellets were air-dried. The DNA pellets were stored at -20 oC. The amounts described above were reduced by a factor of 4 when using 50 mg of liver tissue.
Recruitment and Sample Collection from Patients Undergoing Chemotherapy with Doxorubicin
Dogs with spontaneously arising tumors of various histologies undergoing treatment with a DOX-based chemotherapy protocol at the University of Minnesota Veterinary Medical Center were recruited. Dogs eligible for enrollment had a constitutional clinical signs score of 0 or 1 according to the Eastern Cooperative Oncology Group performance scale (48), body weight ≥10Kg, and adequate hematologic, renal, and hepatic function. Following written informed consent of each dog owner, blood (6-10 mL, depending on dog’s size) was collected via routine venipuncture into a potassium EDTA tube 7 days post-treatment with doxorubicin when dogs returned for their post-chemotherapy CBC per routine protocol at our institution.
Isolation of DNA from blood tissue samples
Genomic DNA was extracted with the QIAGEN Gentra Puregene Blood Kit following the manufacturer’s instructions for DNA Purification from Whole Blood with minor modifications. In brief, 3 mL of whole blood were lysed with 9 mL red blood cell (RBC) lysis solution and mixed by inverting 10 times followed by 5 min of incubation at room temperature. Next, the solution was centrifuged for 2 min at 2,000 x g to pellet the white blood cells. The supernatant was then discarded leaving approximately 200 µL of residual liquid. The pellet was resuspended in the residual liquid by vortexing vigorously. A total of 3 mL of cell lysis solution was added and tubes were vortexed. RNase A solution (30 µl) was added to each lysate and mixed by inverting 25 times followed by 15 min of incubation at 37 oC, which was followed by 3 min of incubation on ice. Then, 1 mL of protein precipitation solution was added and the tubes were vortexed vigorously for 20 s prior to centrifugation (2000 x g for 5 min). Supernatants were added to cold IPA, and DNA was precipitated and washed as previously described, with the only difference being the DNA pellets were air-dried. The dried pellets were stored at -20 oC. The amounts described above were reduced by a factor of 6 when using about 0.5 mL of whole blood.
DNA clean-up, hydrolysis and sample enrichment
Prior to hydrolysis and adduct enrichment, purified DNA samples and mouse liver DNA from the acute treatment study were dissolved in 2 mL 10 mM Tris + 1 mM EDTA (pH 7.0). Then, 2 mL of chloroform/isoamyl alcohol (24:1, purified DNA samples) or phenol/chloroform/isoamyl alcohol (25:24:1, mouse liver DNA samples) was added and the solution was vortexed vigorously for 60 s followed by centrifugation (2,000 x g for 10 min), and the upper layer was collected and transferred into a clean 5 mL Eppendorf tube. The extraction was performed twice. After the second extraction, 200 µl 5 M NaCl were added. DNA was precipitated using cold IPA as previously described. The dried pellets were stored at -20 oC until further use. The extraction was performed in an attempt to remove leftover drug from the samples.
Prior to DNA hydrolysis, DNA was re-dissolved in a 10 mM Tris-HCl/5 mM MgCl2 buffer (pH 7.4) solution. Initial digestion of DNA was performed overnight at room temperature by addition of 124 U/mg DNA (ctDNA and bacterial DNA) or 600 U/mg DNA (liver and blood DNA) DNase I (recombinant, from Pichia pastoris). Then, an additional 124 or 600 U/mg DNA, 6.6 mU/mg DNA (ctDNA and bacterial DNA) or 20 mU/mg DNA (liver and blood DNA) phosphodiesterase I (type II, from Crotalus adamanteus venom) and 46 U/mg DNA (ctDNA and bacterial DNA) or 240 U/mg DNA (liver and blood DNA) of alkaline phosphatase (recombinant, from Pichia pastoris) were added and samples were incubated at 37 °C for 70 min. and followed by overnight incubation at room temperature. Enzymes were removed by centrifugation using a Centrifree ultrafiltration device (MW cutoff of 30,000, Millipore Sigma) at 2000 x g for 45 min. A 10-15 µL aliquot was removed from each sample for dGuo quantitation.
Samples were desalted and enriched using a Strata-X solid phase extraction (SPE) cartridge (33 µm, 30 mg/1 ml, Phenomenex). Briefly, the cartridge was pre-conditioned and equilibrated with 3 mL CH3OH and 1 mL H2O. Samples were loaded, and the cartridge was washed with 3 mL H2O and 1 mL 10% CH3OH in H2O. The two eluting fractions collected were 1 mL 100% CH3OH and 1 mL CH3OH + 2% formic acid. The fractions were evaporated until dry and stored at -20 °C. Prior to LC-MS analysis, samples were reconstituted in 500 µL (ctDNA), 250 µL (bacterial DNA) or 10 µL (liver and blood DNA) 5% CH3OH in LC-MS grade water. For the DNA samples extracted from mouse liver and dog blood, the two SPE fractions were pooled together prior to LC-MS analysis.
dGuo quantitation by HPLC-UV analysis
Quantitation of dG was carried out on an UltiMate 3000 UHPLC System (Thermo Fisher Scientific) with a UV detector set at 254 nm. A 250 x 0.5 mm Luna C18 100A column (Phenomenex, Torrance, CA) at 40 °C was used with a flow rate of 15 µl/min and a gradient from 5% to 25% CH3OH in H2O over the course of 10 min followed by an increase to 95% CH3OH in 3 min and a hold at 95% CH3OH for 5 min. The column was re-equilibrated to initial conditions for 8 min.
LC-MS parameters
Samples were injected onto an UltiMate 3000 RSLCnano UPLC (Thermo Fisher Scientific) system equipped with a 5 μL injection loop. Liquid chromatography (LC) separation was performed on a capillary column (75 μm ID, 20 cm length, 10 μm orifice) created by hand packing a commercially available fused-silica emitter (New Objective) with 5 μm Luna C18 bonded separation media (Phenomenex). Gradient conditions were 1000 nL/min for 5.5 min at 5% CH3CN in 0.05% formic acid aqueous solution, then decreased to 300 nL/min followed by a linear gradient of 1%/min over 44 min for the untargeted screening and over 30 min for the targeted MS/MS analysis. Column wash was performed with a flow rate of 300 nL/min at 98% CH3CN for 5 min (untargeted screening) or at 95% CH3CN for 2 min (targeted MS/MS analysis). Re-equilibration was performed with a flow rate of 1000 nL/min at 5% CH3CN for 5 min (untargeted screening) or for 1 min (targeted MS/MS analysis). The injection valve was switched at 5.5 min to remove the sample loop from the flow path during the gradient. All MS data was acquired on an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific). Positive mode electrospray ionization and nanospray (300 nL/min) were used on a Thermo Scientific Nanoflex ion source with a source voltage of 2.2 kV, a capillary temperature of 300 °C, a S-Lens RF level set at 60%, and EASY-IC lock mass (m/z 202.0777) enabled.
Constant Neutral Loss (CNL)-MSn data-dependent acquisition (DDA)
CNL-MSn DDA was performed by repeated full scan detection followed by MS2 acquisition and constant neutral loss triggering of MS3 fragmentation. Full scan (range 200-2000 Da) detection was performed by setting the Orbitrap detector at 60,000 resolution with 1 microscan, automatic gain control (AGC) target settings of 2.0E5, and maximum ion injection time set at 50 ms. The most intense full scan ions were fragmented over a 2 s cycle. The MS2 fragmentation parameters were as follows: quadrupole isolation window of 1.6, HCD collision energy of 20% ± 10%, Orbitrap detection at a resolution of 7,500, AGC of 2.0E5, 1 microscan, maximum injection time of 50 ms, and EASY-IC lock mass (m/z 202.0777) enabled. Data-dependent conditions were as follows: triggering intensity threshold of 2.5E4, repeat count of 1, exclusion duration of 30 s, and exclusion mass width of ± 5 ppm. The MS3 fragmentation parameters were as follows: HCD fragmentation, 2 amu isolation window, collision energy of 20% ± 10%, Orbitrap detection at a resolution of 7,500 upon the observation of neutral losses (± 5 ppm) of 116.0474 (- dR), 151.0494 (- G), 135.0545 (- A), 126.0429 (- T), 111.0433 (- C), 156.0346 (- 15N-G), 140.0413 (- 15N-A), 128.037 (- 15N-T), or 114.0344 (- 15N-C) between the parent ion and one of the most intense product ions from the MS2 spectrum, provided minimum signal of 2.5E4, AGC of 2.0E5, maximum injection time of 50 s, and EASY-IC lock mass (m/z 202.0777) enabled.
Targeted data acquisition
Targeted MS2 acquisition was performed with a quadrupole isolation window of m/z 1.5 centered on m/z 609.2, 598.2, 735.2, 592.1, 619.2, 608.1, 743.1, 358.1, 376.1, 378.6, 470.2, 472.2, 363.1, 381.1, and 383.6 during the time span 0-14 min, m/z 541.2, 345.2, 356.2, 373.2, and 432.2 during the time span 12-20 min, m/z 415.2, 531.2, 340.2, and 425.1 during the time span 14-20 min, m/z 680.2, 809.3, 814.2, 685.2, 546.2, 544.2, and 791.9 during the time span 20-41 min, and m/z 823.3 during the time span 0-41 min. The other settings were: scan range 80-1000, HCD fragmentation of 20% ± 10%, Orbitrap detection at a resolution of 60,000, AGC of 5.0E4, 1 microscan, maximum injection time of 118 ms, RF lens set at 60% and EASY-IC lock mass (m/z 202.0777) enabled.