Simultaneous quantitative determination of 5-aza-2'-deoxycytidine genomic incorporation and DNA demethylation by liquid chromatography tandem mass spectrometry as exposure-response measures of nucleoside analog DNA methyltransferase inhibitors

Abstract

The epigenetic and anti-cancer activities of the nucleoside analog DNA methyltransferase (DNMT) inhibitors decitabine (5-aza-2'-deoxycytidine, DAC), azacitidine, and guadecitabine are thought to require cellular uptake, metabolism to 5-aza-2'-deoxycytidine triphosphate, and incorporation into DNA. This genomic incorporation can then lead to trapping and degradation of DNMT enzymes, and ultimately, passive loss of DNA methylation. To facilitate measurement of critical exposure-response relationships of nucleoside analog DNMT inhibitors, a sensitive and reliable method was developed to simultaneously quantitate 5-aza-2'-deoxycytidine genomic incorporation and genomic 5-methylcytosine content using LC-MS/MS. Genomic DNA was extracted and digested into single nucleosides. Chromatographic separation was achieved with a Thermo Hyperpcarb porous graphite column (100mm×2.1mm, 5μm) and isocratic elution with a 10mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-aza-2'-deoxycytidine, 2'-deoxycytidine, and 5-methyl-2'-deoxycytidine. The assay range was 2-400ng/mL for 5-aza-2'-deoxycytidine, 50-10,000ng/mL for 2'-deoxycytidine, and was 5-1000ng/mL for 5-methyl-2'-deoxycytidine. The assay proved to be accurate (93.0-102.2%) and precise (CV≤6.3%) across all analytes. All analytes exhibited long-term frozen digest matrix stability at -70°C for at least 117 days. The method was applied for the measurement of genomic 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine content following exposure of in vitro cell culture and in vivo animal models to decitabine.

Keywords: 5-Aza-2′-deoxycytidine; 5-Methyl-2′-deoxycytidine; 5-Methylcytosine; DNA methyltransferase inhibitor; Decitabine; Genomic DNA; Global DNA demethylation; LC/MS/MS.

Copyright © 2016 Elsevier B.V. All rights reserved.

Figures

Figure 1

Representative chromatograms of blank matrix monitoring DAC, 2dC, 5mC, 5AC-15N4 (I.S.), 2dC-13C15N2 (I.S.), and 5mC-d3 (I.S.).

Figure 2

Representative chromatograms of blank matrix spiked with internal standard monitoring DAC, 2dC, 5mC, 5AC-15N4 (I.S.), 2dC-13C15N2 (I.S.), and 5mC-d3 (I.S.).

Figure 3

Representative chromatograms of the lowest calibration standard spiked with 2 ng/mL DAC, 5 ng/mL 5mC and 50 ng/mL 2dC monitoring DAC, 2dC, 5mC, 5AC-15N4 (I.S.), 2dC-13C15N2 (I.S.), and 5mC-d3 (I.S.).

Figure 4

Incorporation of DAC into genomic DNA follows a linear correlation with dose of DAC exposure. DU145 human prostate cancer cells were treated with increasing doses of DAC for 48 hours. The amount of DAC per thousand 2dC in the genomic DNA showed a strong linear correlation with the initial treatment dose. Shown are the mean ± standard error of the mean (SEM) of three replicate measurements.

Figure 5

DAC and 5mC content in genomic DNA of colon and bone marrow cells from mice treated with a dose response of DAC. Mice were treated with a dose response of DAC in normal saline at various doses for two 1-week cycles consisting of every day administration for 5 days followed by two days of rest. Results for DAC and 5mC content per thousand 2dC are reported. The * indicates that there is no detectable DAC. Shown are the mean values from 3 mice in each drug treated group and the mean value of four replicate measurements for Control. Error bars represent standard deviations.