Estrogens are major risk factors for the development of breast cancer; they can be metabolized to catechols, which are further oxidized to DNA-reactive quinones and semiquinones (SQs). These metabolites are mutagenic and may contribute to the carcinogenic activity of estrogens. Redox cycling of the SQs and subsequent generation of reactive oxygen species (ROS) is also an important mechanism leading to DNA damage. The SQs of exogenous estrogens have been shown to redox cycle, however, redox cycling and the generation of ROS by endogenous estrogens has never been characterized. In the present studies, we determined whether the catechol metabolites of endogenous estrogens, including 2-hydroxyestradiol, 4-hydroxyestradiol, 4-hydroxyestrone and 2-hydroxyestriol, can redox cycle in breast epithelial cells. These catechol estrogens, but not estradiol, estrone, estriol or 2-methoxyestradiol, were found to redox cycle and generate hydrogen peroxide (H ₂ O ₂ ) and hydroxyl radicals in lysates of three different breast epithelial cell lines: MCF-7, MDA-MB-231 and MCF-10A. The generation of ROS required reduced nicotinamide adenine dinucleotide phosphate as a reducing equivalent and was inhibited by diphenyleneiodonium, a flavoenzyme inhibitor, indicating that redox cycling is mediated by flavin-containing oxidoreductases. Using extracellular microsensors, catechol estrogen metabolites stimulated the release of H ₂ O ₂ by adherent cells, indicating that redox cycling occurs in viable intact cells. Taken together, these data demonstrate that catechol metabolites of endogenous estrogens undergo redox cycling in breast epithelial cells, resulting in ROS production. Depending on the localized concentrations of catechol estrogens and enzymes that mediate redox cycling, this may be an important mechanism contributing to the development of breast cancer.

Tox21 and ToxCast are high-throughput in vitro screening programs coordinated by the U.S. National Toxicology Program and the U.S. Environmental Protection Agency, respectively, with the goal of forecasting biological effects in vivo based on bioactivity profiling. The present study investigated whether mechanistic insights in the biological targets of food-relevant chemicals can be obtained from ToxCast results when the chemicals are grouped according to structural similarity. Starting from the 556 direct additives that have been identified in the ToxCast database by Karmaus et al. [Karmaus, A. L., Trautman, T. D., Krishan, M., Filer, D. L., and Fix, L. A. (2017). Curation of food-relevant chemicals in ToxCast. Food Chem. Toxicol. 103, 174–182.], the results showed that, despite the limited number of assays in which the chemical groups have been tested, sufficient results are available within so-called “DNA binding” and “nuclear receptor” target families to profile the biological activities of the defined chemical groups for these targets. The most obvious activity identified was the estrogen receptor-mediated actions of the chemical group containing parabens and structurally related gallates, as well the chemical group containing genistein and daidzein (the latter 2 being particularly active toward estrogen receptor β as a potential health benefit). These group effects, as well as the biological activities of other chemical groups, were evaluated in a series of case studies. Overall, the results of the present study suggest that high-throughput screening data could add to the evidence considered for regulatory risk assessment of food chemicals and to the evaluation of desirable effects of nutrients and phytonutrients. The data will be particularly useful for providing mechanistic information and to fill data gaps with read-across.

Open access science on alternatives to the use of animals for scientific purposes