A Ternary Mixture of Common Chemicals Perturbs Benign Human Breast Epithelial Cells More Than the Same Chemicals Do Individually

Abstract

As a continuous source of hormonal stimulation, environmentally ubiquitous estrogenic chemicals, ie, xenoestrogens (XEs), are a potential risk factor for breast carcinogenesis. Given their wide distribution in the environment and the fact that bisphenol-A (BPA), methylparaben (MP), and perfluorooctanoic acid (PFOA) are uniformly detected in unselected body fluid samples, it must be assumed that humans are simultaneously exposed to these chemicals almost daily. We studied the effects of a ternary mixture of BPA, MP, and PFOA on benign breast epithelial cells at the range of concentrations observed for single chemicals in human samples. Measurements of exposure impact relevant to the breast were based on endpoints associated with “hallmarks” of cancer and “key characteristics” of carcinogens. These included modulation of total estrogen receptor (ER)α, phosphorylated ERα (pERα), total ERβ, S-phase induction, and apoptotic evasion. Data from live cell measurements were fit to a log-linear dose-response model. Concentration-dependent reduction of ERβ and apoptosis evasion was observed concurrently with the induction of ERα, pERα, and S-phase fraction, and an increased rate of cell proliferation. Beyond additive effects predicted by the sum of individual test XEs, mixture treatment demonstrated synergism for the ERβ and apoptosis suppression phenotypes (p > .001). Nonmalignant breast cells were more sensitive than commonly used breast cancer lines to XE treatment in 3 of 5 endpoints. All observations were validated with cells isolated from the normal breast tissue of 14 individuals. At relatively low concentrations, a chemical mixture has striking effects on normal cell function that are missed by evaluation of single components.

Carcinogenesis: Failure of resolution of inflammation?

“…These key characteristics may also lead to the development of human-based assays and biomarkers for assessing cancer risk ( Fielden et al, 2018 ). At low concentrations, a chemical mixture has synergistic pro-tumorigenic activity on benign and malignant cells at a significantly lower concentration than as single chemicals ( Dairkee, Luciani-Torres, Moore, Jaffee, & Goodson 3rd., 2018 ) . Carcinogens may not directly be genotoxic but cause DNA damage by stimulating inflammation.…”

The Key Characteristics of Carcinogens: Relationship to the Hallmarks of Cancer, Relevant Biomarkers, and Assays to Measure Them

“…Static measures of apoptosis include identification of apoptotic cells on histology (sometimes with TUNEL assay or Annexin-V), and expression of pro-and anti-apoptotic genes. Evasion of apoptosis is determined as a change in apoptosis in response to a known therapeutic agent, e.g., tamoxifen, compared to controls after exposure to a chemical of interest (108,109). The vascularity of a tumor can be assessed by visual observation of blood vessel density, sometimes employing stains for capillary basement membrane.…”

Application of the Key Characteristics of Carcinogens to Per and Polyfluoroalkyl Substances

“…PFOA and PFOS both increased proliferation, cell migration and invasion of normal human breast epithelial cells and altered the expression of proteins involved in cell cycle regulation, including cyclin dependent kinase 4 [202,203]. Similarly, in a mixture with two other chemicals found in consumer products, environmentally relevant concentrations of PFOA increased cell proliferation in non-malignant breast cancer cells through cell cycle disruption, reduced cell apoptosis and increased estrogen receptor alpha levels [204].…”

“…PFOA Association: [61] Association: [202]; [204]; [205]; [207]; [209] Long-chain PFAS a PFOS Association: [203]; [207]; [208]…”