Direct Drug Metabolism Monitoring in a Live Single Hepatic Cell by Video Mass Spectrometry

Abstract

The monitoring of drug metabolism in a hepatic cell, or in a target cell is a very important trace analysis in drug discovery and development. However, these analyses have been mostly performed by liquid chromatography-mass spectrometry (LC-MS), which needs more than thousand cells. Cells were pretreated by sonication or homogeneization by which any information of drug localization within a cell was lost, in most cases.If the drug metabolism and the localization of drug metabolites within a live single cell can be directly observed under a video microscope, the drug discovery and development become speedy and precise. Furthermore, in personalized medicine, 1 a drug can be properly selected by single-cell drug metabolism analysis using only a few cells in biopsied tissues. We developed the live single-cell video mass spectrometry, 2-4 and applied to the analysis of live single-cell drug metabolism using a hepatic model cell, HepG2.HepG2 cells (human hepatocellular carcinoma cells) were cultured in 35 mm dishes at 37 C in 95% air/5% CO2 in Dulbecco's Modified Eagle's Medium with 4500 mg/L glucose, supplemented with 10% fetal bovine serum (FBS). FBS was inactivated by a heating treatment at 56 C for 30 min.Tamoxifen (TAM) was added to cultured HepG2 cells to be 5 mM final concentration, and incubated for 12 h. An organelle (cytoplasm, a vacuole or a nucleus) was sucked into a nanospray tip (HUMANIX) independently, as shown in Fig. 1. Then, 3 mL of a solvent (90% MeOH/8.0% H2O/2.0% formic acid) was added from the back-end of the tip. These samples were directly introduced by nano-electrospray ionization into a LTQ Orbitrap XL (Thermo Fisher Scientific) at a positive ion mode.Each metabolite was identified according to its exact mass m/z value of Δppm <±3.0. Tamoxifen (m/z 372.232) was detected in cytoplasm (Fig. 2).Its five metabolites, 1,[5][6][7][8][9][10] (N-desmethyl tamoxifen (N-DM-T, m/z 358,216), 4-hydroxy tamoxifen (4-OHT), tamoxifen-N-oxide (T-NO) (m/z 388.227), 3,4-dihydroxy tamoxifen (3,4-diOHT), 4-hydroxy tamoxifen N-oxide (4-OHT-NO) (m/z 404.222)) were also detected (Fig. 2). N-DM-T, which is mainly metabolized by CYP3A4, was preferably detected. This result is in accord with the finding that N-DM-T is a major metabolite of TAM, 5 and that CYP3A4 is the most expressed isozyme in P450 subfamilies of the HepG2 cell.11 Weak molecular peaks were also identified by LC-MS/MS with many cells. Besides the peaks of tamoxifen and its metabolites, many peaks were detected at the same time, most of which were found in the control (without tamoxifen). Most of these peaks were hard to identify, however, peak origin was told apart. The peak of m/z 372.186, for example, is solvent peak, and the peak of m/z 372.267 is from cytoplasm.Two metabolites, (4-OHT and T-NO, which have the same mass (m/z 388.227), and two metabolites, 3,4-diOHT and 4-OHT-NO, that have the same mass (m/z 404.222)) were identified using LC-MS/MS methods, however, endoxifen which is known as the most active metabolites, was not detected. Since HepG...

Time-resolved ICP-MS Measurement: a New Method for Elemental and Multiparametric Analysis of Single Cells

“…Due to cellular heterogeneity within an isogenic cell population, individual analysis of cells will lead to a more sensitive representation of cell-to-cell variations, instead of a stochastic average analysis by bulk measurements. [24][25][26][27][28][29] Classical ICP-MS analysis of cells is the bulk analysis of large amounts of cells with a lysis, extraction or digestion; 30 this is an approach that is hardly adapted for evaluating any cell-to-cell variance. Time-resolved measurement via a direct cell introduction method is a relatively recent method for individual analysis of cells by ICP-MS, [14][15][16][17][18][19][20][21][22][23] an approach that can detect the elements in individual cells with a high sensitivity, often called "single particle mode".…”

“…The instrument has been launched by DVS Science (CyTOF TM ). 39 Ho et al successfully performed single-cell analysis of algal cells (1 -6 μm) using quadrupole ICP-MS (ICP-QMS) at its lowest integration time (10 ms), focussing on the 25 Mg isotope. 15 They applied this method for tracking bismuth antiulcer drug uptake in Helicobacter pylori cells.…”

Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry

“…Indeed, whereas a laser/ion beam can be easily focused on a tiny spot, the utilization of organic chemical matrices could lead to an inability to maintain cell viability before MS measurement (24,25). Much effort has been devoted to the collection of raw cytoplasm from living cells, such as by directly drawing the cytoplasm from a cell using a micropipette (27)(28)(29). The application of patch clamp technique combined with capillary electrophoresis (CE) separation would provide enhanced dynamic range and high sample coverage (30).…”

Fluorescence Probing Live Single-cell Mass Spectrometry for Direct Analysis of Organelle Metabolism

“…[1][2][3][4]11 However, since organelles are extremely small and difficult to capture within cells, until now, targets that could be captured were limited. We have thus developed methods to capture single vacuoles and single granules.…”

“…We have thus developed methods to capture single vacuoles and single granules. [1][2][3]11 When we combined live fluorescence imaging techniques with live single-cell and organelle mass spectrometry, we found we can trap even mitochondria. Mitochondria are a very important target in biology because of the important roles they play.…”