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Image analysis of mechanistic protein biomarkers for the characterization of genotoxicants

Aneugens, clastogens, and reactive oxygen species inducers
: Wilde, S.; Queisser, N.; Sutter, A.

Volltext ()

Environmental and Molecular Mutagenesis 61 (2020), Nr.5, S.534-550
ISSN: 0893-6692
ISSN: 1098-2280
Zeitschriftenaufsatz, Elektronische Publikation
Fraunhofer ITEM ()

The early detection of genotoxicity contributes to cutting‐edge drug discovery and development, requiring effective identification of genotoxic hazards posed by drugs while providing mode of action (MoA) information in a high throughput manner. In other words, there is a need to complement standard genotoxicity testing according to the test battery given in ICH S2(R1) with new in vitro tools, thereby contributing to a more in‐depth analysis of genotoxic effects. Here, we report on a proof‐of‐concept MoA approach based on post‐translational modifications of proteins (PTMs) indicative of clastogenic and aneugenic effects in TK6 cells using imaging technology (with automated analysis). Cells were exposed in a 96‐well plate format with a panel of reference (geno)toxic compounds and subsequently analyzed at 4 and 24 hr to detect dose‐dependent changes in PTMs, relevant for mechanistic analysis. All tested compounds that interfere with the spindle apparatus yielded a BubR1 (S640) (3/3) and phospho‐histone H3 (S28) (7/9) positive dose–response reflecting aneugenicity, whereas compounds inducing DNA double‐strand‐breaks were associated with positive FANCD2 (S1404) and 53BP1 (S1778) responses pointing to clastogenicity (2/3). The biomarker p53 (K373) was able to distinguish genotoxicants from non‐genotoxicants (2/4), while the induction of reactive oxygen species (ROS), potentially causing DNA damage, was associated with a positive Nrf2 (S40) response (2/2). This work demonstrates that genotoxicants and non‐genotoxicants induce different biomarker responses in TK6 cells which can be used for reliable classification into MoA groups (aneugens/clastogens/non‐genotoxicants/ROS inducers), supporting a more in‐depth safety assessment of drug candidates.