title: Detection of DNA strand breaks and oxidized DNA bases at the single-cell level resulting from exposure to estradiol and hydroxylated metabolites.
abstract: Long-term exposure to steroidal estrogens is a key factor contributing to increases in the risk of developing breast cancer. Proposed mechanisms include receptor-activated increases in the rate of cell proliferation leading to the accumulation of genetic damage resulting from reading errors, and the production of DNA damage by species arising from metabolism of 17beta-estradiol (E2) resulting in mutations. In support of the second mechanism, catechol metabolites of E2 induce DNA damage in vitro. In the present study, utilizing the single-cell gel electrophoresis (Comet) assay, we observed increases in the number of single-strand breaks in estrogen receptor alpha-positive (MCF-7) and -negative (MDA-MB-231) breast cancer cells exposed to E2 (for 24 hr) or 4-hydroxy-17beta-estradiol (4-OH-E2; for 2 hr). The concentrations of 4-OH-E2 sufficient to induce these effects were approximately 100 nM, substantially lower than reported previously. The catechol 2-hydroxy-17beta-estradiol (2-OH-E2) also induced strand breaks. 2-OH-E2, often referred to as an improbable carcinogen in humans, is not a major metabolite of E2 in the breast; however, our findings show that it is as DNA-damaging as 4-OH-E2. Formamidopyrimidine glycosylase posttreatment of E2-, 4-OH-E2-, and 2-OH-E2-exposed MCF-7 cells led to an up to sixfold increase in mean tail moment, suggesting that oxidative DNA damage was formed. Comet formation could be partially attenuated by coincubation with dimethylsulfoxide, attributing a small DNA-damaging role to oxyradicals emanating from catechol redox cycling. Similar findings were obtained with MDA-MB-231 cells, indicating that estrogen receptor status is not relevant to these effects. Our observations show that exposure to E2 adds to the oxidative load of cells, and this may contribute to genomic instability.
authors.affiliation: Centre for Toxicology, School of Pharmacy, London, United Kingdom.
authors.forename: Nissanka
authors.initials: N
authors.lastname: Rajapakse
authors.forename: Michael
authors.initials: M
authors.lastname: Butterworth
authors.forename: Andreas
authors.initials: A
authors.lastname: Kortenkamp
mesh_terms: D045744:Cell Line, Tumor; D020552:Comet Assay; D004249:DNA Damage; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity*; D047628:Estrogen Receptor alpha / Q000378:metabolism; D002393:Estrogens, Catechol / Q000633:toxicity*; D006801:Humans; D010084:Oxidation-Reduction
chemical_list: D047628:Estrogen Receptor alpha; D002393:Estrogens, Catechol; D004958:Estradiol; C001390:2-hydroxyestradiol; C014036:4-hydroxyestradiol