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In vitro primary human lymphocyte flow cytometry micronucleus assay: simultaneous assessment of cell proliferation, cell cycle changes, apoptosis and micronucleus induction

woensdag, 26 oktober, 2011 - 16:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculteit: Science and Bio-engineering Sciences
D
0.02
Magdalena Lukamowicz
doctoraatsverdediging

Exposure to DNA damaging agents and mutagens as well as genomic instability
may contribute to carcinogenesis. Therefore, determination of potential genotoxic hazard
became one of the goals of the preclinical phase of the drug development process. The
results obtained within the genotoxicity studies should help in the determination of a
potential cancer risk in humans. International Conference on Harmonization (ICH) and
Organization for Economic Co-operation and Development (OECD) as well as other
organizations provide guidance on the test selection and data interpretation using multiple
in vitro and in vivo approaches. In vitro tests, however, show, in some conditions,
positive results that find no confirmation in rodent in vivo tests. Such results are referred
to as false or irrelevant positive results and may be caused by the use of systems that are
p53 and DNA repair deficient or are based on transformed cells of non-human origin.
Moreover, the lack of fully metabolic competent test systems may as well contribute to
the inconsistency of in vitro and in vivo results.

The in vitro micronucleus test is currently widely used in determination of
genotoxic activity. The assay has the potential to detect both clastogenic and aneugenic
chemicals. It has been shown that the primary human lymphocyte micronucleus test gives
very promising results regarding reduction of irrelevant positive results if compared to
standard rodent cell lines. Therefore, the goal of the presented project was to develop a
human primary lymphocyte in vitro micronucleus test using flow cytometry, taking into
account cell division and other confounding factors related to apoptosis/necrosis or
excessive cytotoxicity. The technology was based on sequential staining with
carboxyfluorescein diacetate succinimidyl ester as a division control marker, ethidium
monoazide to determine the apoptosis/necrosis level and 4-6-diaminodino-2-phenyl
indole as a DNA marker. The assay was validated and miniaturized using genotoxic and
non-genotoxic compounds with various modes of action. Moreover, application of flow
cytometry technology provided the tool to analyze and understand the basic mechanisms
of the compounds modes of action. Our data showed that the flow cytometry based
micronucleus test is a sensitive and reliable tool for the determination of clastogenic and
aneugenic activity of the tested compound with an indication of mode of action.