Cellular levels and molecular dynamics simulations of estragole DNA adducts point at inefficient repair resulting from limited distortion of the double-stranded DNA helix

Estragole, naturally occurring in a variety of herbs and spices, can form DNA adducts after bioactivation. Estragole DNA adduct formation and repair was studied in in vitro liver cell models, and a molecular dynamics simulation was used to investigate the conformation dependent (in)efficiency of N2-(trans-isoestragol-3′-yl)-2′-deoxyguanosine (E-3′-N2-dG) DNA adduct repair. HepG2, HepaRG cells, primary rat hepatocytes and CHO cells (including CHO wild-type and three NER-deficient mutants) were exposed to 50 μM estragole or 1′-hydroxyestragole and DNA adduct formation was quantified by LC–MS immediately following exposure and after a period of repair. Results obtained from CHO cell lines indicated that NER plays a role in repair of E-3′-N2-dG adducts, however, with limited efficiency since in the CHO wt cells 80% DNA adducts remained upon 24 h repair. Inefficiency of DNA repair was also found in HepaRG cells and primary rat hepatocytes. Changes in DNA structure resulting from E-3′-N2-dG adduct formation were investigated by molecular dynamics simulations. Results from molecular dynamics simulations revealed that conformational changes in double-stranded DNA by E-3′-N2-dG adduct formation are small, providing a possible explanation for the restrained repair, which may require larger distortions in the DNA structure. NER-mediated enzymatic repair of E-3′-N2-dG DNA adducts upon exposure to estragole will be limited, providing opportunities for accumulation of damage upon repeated daily exposure. The inability of this enzymatic repair is likely due to a limited distortion of the DNA double-stranded helix resulting in inefficient activation of nucleotide excision repair.</p

Molecular Dynamics and In Vitro Quantification of Safrole DNA Adducts Reveal DNA Adduct Persistence Due to Limited DNA Distortion Resulting in Inefficient Repair

The formation and repair of N2-(trans-isosafrol-3'-yl)-2'-deoxyguanosine (S-3'-N2-dG) DNA adduct derived from the spice and herbal alkenylbenzene constituent safrole were investigated. DNA adduct formation and repair were studied in vitro and using molecular dynamics (MD) simulations. DNA adduct formation was quantified using liquid chromatography-mass spectrometry (LCMS) in wild type and NER (nucleotide excision repair) deficient CHO cells and also in HepaRG cells and primary rat hepatocytes after different periods of repair following exposure to safrole or 1'-hydroxysafrole (1'-OH safrole). The slower repair of the DNA adducts found in NER deficient cells compared to that in CHO wild type cells indicates a role for NER in repair of S-3'-N2-dG DNA adducts. However, DNA repair in liver cell models appeared to be limited, with over 90% of the adducts remaining even after 24 or 48 h recovery. In our further studies, MD simulations indicated that S-3'-N2-dG adduct formation causes only subtle changes in the DNA structure, potentially explaining inefficient activation of NER. Inefficiency of NER mediated repair of S-3'-N2-dG adducts points at persistence and potential bioaccumulation of safrole DNA adducts upon daily dietary exposure.</p

Rates of bone loss among women initiating antidepressant medication use in midlife

10.1210/jc.2013-1971Journal of Clinical Endocrinology and Metabolism98114355-436