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ANALYTICAL SCIENCES MARCH 2012, VOL. 28
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2012 © The Japan Society for Analytical Chemistry
Rapid Communications
Direct Drug Metabolism Monitoring in a Live Single Hepatic Cell by Video
Mass Spectrometry
Sachiko DATE,* Hajime MIZUNO,** Naohiro TSUYAMA,** Takanori HARADA,** and
Tsutomu MASUJIMA*,**†
*Quantitative Biology Center (QBiC), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565–0874, Japan
**Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami, Hiroshima 734–8551, Japan
The metabolism of anti-breast cancer drug, tamoxifen, in a single human hepatocellular carcinoma cell, HepG2, was
directly monitored by a video-mass spectroscope. The cytoplasm, a vacuole or nucleus of the cell was directly sucked by
a nano-spray tip under a video-microscope, and then was introduced into a mass spectrometer. Unchanged drug molecules
were found in cytoplasm and a vacuole, but the metabolites were only found in the cytoplasm. This direct detection of
drug metabolites in a live single cell is useful for speedy drug metabolism monitoring.
(Received February 4, 2012; Accepted February 13, 2012; Published March 10, 2012)
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 μL
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,1,5–10
(N-desmethyl tamoxifen (N-DM-T, m/z 358,216), 4-hydroxy
To whom correspondence should be addressed.
E-mail: [email protected]
†
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 HepG2 is reported to have less metabolizing activity,11
these secondary metabolites can be found in greater numbers in
primary cultured cells.
In vacuoles, tamoxifen was detected (Fig. 3), but no
metabolites were detected. On the other hand, neither tamoxifen
nor metabolites were detected in the nucleus. These data
indicate that trapped cytoplasm includes a metabolizing
organelle, such as endoplasmic reticulum. Since tamoxifen
metabolites were only found in cytoplasm, transport of
metabolites into a vacuole or a nucleus seems to be minimal.
The role of vacuole in the HepG2 cell is still unknown, however,
the vacuole appears to be vacant and having no granules in it.
Vacuole seems to be a reservoir or wasting envelope, and to
have a less viscous aqueous solution, because it can be sucked
easily and absorbed into the glass capillary inner wall when it is
trapped.
This study of live single-cell level drug metabolism analysis
will open the possibility of speedy, low-cost and site-specific
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MARCH 2012, VOL. 28
Fig. 1 Trapping of the cell contents of cytoplasm, a vacuole and a nucleus by a nanospray tip from a
single HepG2 cell under a video microscope.
monitoring of drug metabolism and a useful tool for single-cell
tailored medicine using biopsied cells.
Acknowledgements
This work was supported by the Grant-in-Aid (No. 20229003)
from the Ministry of Education, Culture, Sports, Science and
Technology of Japan.
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Fig. 3 Detected mass spectra for a vacucole from a single HepG2
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Fig. 2 Detected mass spectra for cytoplasm from a single HepG2
cell that shows TAM and its metabolites.