Mapping and Quantification of Genome-wide DNA Damages by Catechol Estrogens Using Click probe-Seq and LC/MS2: Unraveling Endogenous Genotoxicity Beyond Receptor-mediated Signaling

Genotoxic estrogen metabolites generate various endogenous DNA lesions but their carcinogenesis mechanisms have been overlooked by well-known cell proliferation pathways through estrogen receptor (ER). Genome-wide sequencing using click probe enrichment coupled with liquid chromatography-mass spectrometry (click probe-Seq/LCMS2) was developed to identify damaged genes and characterize global generation profiles of depurinating and stable adducts induced by 4-hydrolyxyl estradiol (4OHE2) in MCF-7 chromatin. Both data were combined to show guanine nucleobase in GC-rich transcription-relevant domain are main target sites. The damage abundance exhibited positive correlation with DNase hypersensitive sites, indicating 4OHE2 attacks chromatin exposure region beyond ER binding. Cell-based comparability studies indicated accumulated 4OHE2 caused suppressed transcription of target genes, in-effective damage repair, and decreased cell viability, differing from uncontrolled cell growth by extensive ER-signaling. Click probe-Seq/LCMS2 approach revealed the first chromatin damage map by endogenous metabolites, exposing a previously unexplored landscape in cancer research and being applicable to other genotoxic species. To study the DNA sequence where catechol estrogen DNA adducts are located, we treated extracted chromatin from MCF-7 cells with either catechol estrogen (experimental group) or acetonitrile (control/mock group). After treatment, the DNA was purified, and a biotin-labeled probe was introduced. This was followed by streptavidin bead pulldown. The pulled-down DNA sequences were then submitted for sequencing.