Abstract P5-06-04: Synergistically elevated hallmarks of cancer induced in non-malignant human breast cells by mixtures of environmental chemicals
Authors: Dairkee SH , Luciani-Torres G , Moore DH , Goodson WH 
Published: May 2016

Abstract
BACKGROUND: Estrogenic overexposure is a well-accepted risk factor in the etiology of breast cancer. Synthetic chemicals that simulate natural estrogens, i.e., xenoestrogens (XEs), occur widely in the environment entering the human body through multiple routes. Consequently, mixtures of XEs are known to circulate in the blood at any given time. Current protocols to evaluate chemical safety rely primarily on testing chemicals individually, ignoring the fact that the general population is exposed daily to mixtures of XEs. We asked whether mixtures of low dose XEs would intensify the cellular effects of individual XEs at the same concentrations.
METHODS: We exposed non-malignant breast epithelial cell cultures (HRBECs), obtained from high-risk human donors by random periareolar fine needle aspiration (RPFNA), to the high volume chemicals - bisphenol-A (BPA), methylparaben (MP) and perflourooctanoic acid (PFOA). Cells were exposed to individual XEs or mixtures of all three at a range of concentrations encompassing environmentally relevant levels. Breast cancer associated phenotypes were quantitated as test endpoints using fluorescence-activated cell sorting (FACS) and/or Western blotting. The endpoints included: total and phosphorylated estrogen receptor (ER)α, ERβ, S-phase fraction, and 4-hydoxytamoxifen (OHT) induced apoptotic fraction. We fit a log-linear dose-response model to the data generated from 3 doses for each chemical, individually and as a mixture. Evidence of synergism was tested by comparing the observed data for mixtures with that predicted by an additive-in-dose model for single doses.
RESULTS: Our data demonstrates that the degree of perturbation induced by the chemical mixture tested here is not merely an additive effect of each chemical; instead it reflects considerable synergism in the mode of action. In 3/3 SNP-authenticated non-malignant HRBEC cell lines exposed to the chemical mixture at the lowest test dose, total and phosphorylated ERα levels were consistently elevated. Concurrently, ERβ level was reduced. A dose-dependent increase was observed in the S-phase fraction, together with a marked reduction of the OHT-induced apoptotic fraction in HRBECs exposed to BPA, MP, and PFOA individually (p&lt;0.001 for each exposure). A significantly greater effect, or synergism, was detected for these endpoints upon exposure to a mixture of the chemicals at the same concentrations (p&lt;0.001 for the combination vs. the predicted sum of individual doses). Primary HRBEC cultures from 7 additional human volunteers displayed a similar pattern of response.
CONCLUSION: Routine tests conducted to assess the carcinogenic potential of chemicals overlook possible synergism among different chemicals. We show that the combined effects of low doses of various chemicals can be much stronger than predicted by addition of their effects as single agents. Despite being cost, time, and labor-intensive, current testing of individual chemicals fails to inform regulators about the safe dose range for human exposure. It is thus imperative to expand present methods to include combinatorial approaches pertaining to test chemicals, endpoints, and target cells.
Citation Format: Dairkee SH, Luciani-Torres G, Moore DH, Goodson WH. Synergistically elevated hallmarks of cancer induced in non-malignant human breast cells by mixtures of environmental chemicals. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-06-04.