Investigating phenotypic plasticity due to toxicants with exposure disparities in primary human breast cells in vitro.

Introduction: Breast cancer is the second most diagnosed cancer, as well as the primary cause of cancer death in women worldwide. Of the different breast cancer subtypes, triple-negative breast cancer (TNBC) is particularly aggressive and is associated with poor prognosis. Black women are two to three times more likely to be diagnosed with TNBCs than white women. Recent experimental evidence suggests that basal-like TNBCs may derive from luminal cells which acquire basal characteristics through phenotypic plasticity, a newly recognized hallmark of cancer. Whether chemical exposures can promote phenotypic plasticity in breast cells is poorly understood. Methods: To investigate further, we developed a high-content immunocytochemistry assay using normal human breast cells to test whether chemical exposures can impact luminal/basal plasticity by unbiased quantification of keratin 14 (KRT14), a basal-myoepithelial marker; keratin 8 (KRT8), a luminal-epithelial marker; and Hoechst 33342, a DNA marker. Six cell lines established from healthy tissue from donors to the Susan G. Komen Normal Tissue Bank were exposed for 48 hours to three different concentrations (0.1µM, 1µM, and 10µM) of eight ubiquitous chemicals (arsenic, BPA, BPS, cadmium, copper, DDE, lead, and PFNA), with documented exposure disparities in US Black women, in triplicate. Automated fluorescence image quantification was performed using Cell Profiler software, and a random-forest classifier was trained to classify individual cells as KRT8 positive, KRT14 positive, or hybrid (both KRT8 and KRT14 positive) using Cell Profiler Analyst. Results and discussion: Results demonstrated significant concentration-dependent increases in hybrid populations in response to BPA, BPS, DDE, and PFNA. The increase in hybrid populations expressing both KRT14 and KRT8 is indicative of a phenotypically plastic progenitor-like population in line with known theories of carcinogenesis. Furthermore, BPA, BPS, DDE, and copper produced significant increases in cell proliferation, which could be indicative of a more malignant phenotype. These results further elucidate the relationship between chemical exposure and breast phenotypic plasticity and highlight potential environmental factors that may impact TNBC risk.

Applying Cell Painting in Non-Tumorigenic Breast Cells to Understand Impacts of Common Chemical Exposures.

There are a substantial number of chemicals to which individuals in the general population are exposed which have putative, but still poorly understood, links to breast cancer. Cell Painting is a high-content imaging-based in vitro assay that allows for rapid and unbiased measurements of the concentration-dependent effects of chemical exposures on cellular morphology. We optimized the Cell Painting assay and measured the effect of exposure to 16 human exposure relevant chemicals, along with 21 small molecules with known mechanisms of action, for 48 hours in non-tumorigenic mammary epithelial cells, the MCF10A cell line. Through unbiased imaging analyses using CellProfiler, we quantified 3042 morphological features across approximately 1.2 million cells. We used benchmark concentration modeling to quantify significance and dose-dependent directionality to identify morphological features conserved across chemicals and find features that differentiate the effects of toxicants from one another. Benchmark concentrations were compared to chemical exposure biomarker concentration measurements from the National Health and Nutrition Examination Survey to assess which chemicals induce morphological alterations at human-relevant concentrations. Morphometric fingerprint analysis revealed similar phenotypes between small molecules and prioritized NHANES-toxicants guiding further investigation. A comparison of feature fingerprints via hypergeometric analysis revealed significant feature overlaps between chemicals when stratified by compartment and stain. One such example was the similarities between a metabolite of the organochlorine pesticide DDT (p,p'-DDE) and an activator of canonical Wnt signaling CHIR99201. As CHIR99201 is a known Wnt pathway activator and its role in ß-catenin translocation is well studied, we studied the translocation of ß-catenin following p'-p' DDE exposure in an orthogonal high-content imaging assay. Consistent with activation of Wnt signaling, low dose p',p'-DDE (25nM) significantly enhances the nuclear translocation of ß-catenin. Overall, these findings highlight the ability of Cell Painting to enhance mode-of-action studies for toxicants which are common exposures in our environment but have previously been incompletely characterized with respect to breast cancer risk.

High-Throughput Transcriptomics of Nontumorigenic Breast Cells Exposed to Environmentally Relevant Chemicals.

BACKGROUND: There is a suite of chemicals, including metals, pesticides, and personal care product compounds, which are commonly detected at high levels in US Center for Disease Control's National Health and Nutrition Examination Survey (NHANES) chemical biomarker screens. Whether these chemicals influence development of breast cancer is not well understood. OBJECTIVES: The objectives were to perform an unbiased concentration-dependent assessment of these chemicals, to quantify differences in cancer-specific genes and pathways, to describe if these differences occur at human population-relevant concentrations, and to specifically test for differences in markers of stemness and cellular plasticity. METHODS: We treated nontumorigenic mammary epithelial cells, MCF10A, with 21 chemicals at four concentrations (25 nM, 250 nM, 2.5µM, and 25µM) for 48 h. We conducted RNA-sequencing for these 408 samples, adapting the plexWell plate-based RNA-sequencing method to analyze differences in gene expression. We calculated gene and biological pathway-specific benchmark concentrations (BMCs) using BMDExpress3, identifying differentially expressed genes and generating the best fit benchmark concentration models for each chemical across all genes. We identified enriched biological processes and pathways for each chemical and tested whether chemical exposures change predicted cell type distributions. We contextualized benchmark concentrations relative to human population biomarker concentrations in NHANES. RESULTS: We detected chemical concentration-dependent differences in gene expression for thousands of genes. Enrichment and cell type distribution analyses showed benchmark concentration responses correlated with differences in breast cancer-related pathways, including induction of basal-like characteristics for some chemicals, including arsenic, lead, copper, and methyl paraben. Comparison of benchmark data to NHANES chemical biomarker (urine or blood) concentrations indicated an overlap between exposure levels and levels sufficient to cause a gene expression response. DISCUSSION: These analyses revealed that many of these 21 chemicals resulted in differences in genes and pathways involved in breast cancer in vitro at human exposure-relevant concentrations. https://doi.org/10.1289/EHP12886.

PIWI-Interacting RNA (piRNA) and Epigenetic Editing in Environmental Health Sciences.

PURPOSE OF REVIEW: The epigenome modulates gene expression in response to environmental stimuli. Modifications to the epigenome are potentially reversible, making them a promising therapeutic approach to mitigate environmental exposure effects on human health. This review details currently available genome and epigenome editing technologies and highlights ncRNA, including piRNA, as potential tools for targeted epigenome editing. RECENT FINDINGS: Zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease (CRISPR/Cas) research has significantly advanced genome editing technology, with broad promise in genetic research and targeted therapies. Initial epigenome-directed therapies relied on global modification and suffered from limited specificity. Adapted from current genome editing tools, zinc finger protein (ZFP), TALE, and CRISPR/nuclease-deactivated Cas (dCas) systems now confer locus-specific epigenome editing, with promising applicability in the field of environmental health sciences. However, high incidence of off-target effects and time taken for screening limit their use. FUTURE DEVELOPMENT: ncRNA serve as a versatile biomarker with well-characterized regulatory mechanisms that can easily be adapted to edit the epigenome. For instance, the transposon silencing mechanism of germline PIWI-interacting RNAs (piRNA) could be engineered to specifically methylate a given gene, overcoming pitfalls of current global modifiers. Future developments in epigenome editing technologies will inform risk assessment through mechanistic investigation and serve as potential modes of intervention to mitigate environmentally induced adverse health outcomes later in life.

Toxicoepigenetics and Environmental Health: Challenges and Opportunities.

The rapidly growing field of toxicoepigenetics seeks to understand how toxicant exposures interact with the epigenome to influence disease risk. Toxicoepigenetics is a promising field of environmental health research, as integrating epigenetics into the field of toxicology will enable a more thorough evaluation of toxicant-induced disease mechanisms as well as the elucidation of the role of the epigenome as a biomarker of exposure and disease and possible mediator of exposure effects. Likewise, toxicoepigenetics will enhance our knowledge of how environmental exposures, lifestyle factors, and diet interact to influence health. Ultimately, an understanding of how the environment impacts the epigenome to cause disease may inform risk assessment, permit noninvasive biomonitoring, and provide potential opportunities for therapeutic intervention. However, the translation of research from this exciting field into benefits for human and animal health presents several challenges and opportunities. Here, we describe four significant areas in which we see opportunity to transform the field and improve human health by reducing the disease burden caused by environmental exposures. These include (1) research into the mechanistic role for epigenetic change in environment-induced disease, (2) understanding key factors influencing vulnerability to the adverse effects of environmental exposures, (3) identifying appropriate biomarkers of environmental exposures and their associated diseases, and (4) determining whether the adverse effects of environment on the epigenome and human health are reversible through pharmacologic, dietary, or behavioral interventions. We then highlight several initiatives currently underway to address these challenges.

Identifying the link between chemical exposures and breast cancer in African American women via integrated in vitro and exposure biomarker data.

Among women, breast cancer is the most prevalent form of cancer worldwide and has the second highest mortality rate of any cancer in the United States. The breast cancer related death rate is 40 % higher in non-Hispanic Black women compared to non-Hispanic White women. The incidence of triple negative breast cancer (TNBC), an aggressive subtype of breast cancer for which there is no targeted therapy, is also approximately three times higher for Black, relative to, White women. The drivers of these differences are poorly understood. Here, we aimed to identify chemical exposures which play a role in breast cancer disparities. Using chemical biomonitoring data from the National Health and Nutrition Examination Survey (NHANES) and biological activity data from the EPA's ToxCast program, we assessed the toxicological profiles of chemicals to which US Black women are disproportionately exposed. We conducted a literature search to identify breast cancer targets in ToxCast to analyze the response of chemicals with exposure disparities in these assays. Forty-three chemical biomarkers are significantly higher in Black women. Investigation of these chemicals in ToxCast resulted in 32,683 assays for analysis, 5172 of which contained nonzero values for the concentration at which the dose-response fitted model reaches the cutoff considered "active". Of these chemicals BPA, PFOS, and thiram are most comprehensively assayed. 2,5-dichlorophenol, 1,4-dichlorobenzene, and methyl and propyl parabens had higher biomarker concentrations in Black women and moderate testing and activity in ToxCast. The distribution of active concentrations for these chemicals in ToxCast assays are comparable to biomarker concentrations in Black women NHANES participants. Through this integrated analysis, we identify that multiple chemicals, including thiram, propylparaben, and p,p' DDE, have disproportionate exposures in Black women and have breast cancer associated biological activity at human exposure relevant doses.

Pesticide exposure and adverse health effects associated with farmwork in Northern Thailand.

OBJECTIVES: To assess pesticide exposure and understand the resultant health effects of agricultural workers in Northern Thailand. METHODS: This was a cross-sectional study. We quantified exposure to pesticides, including chlorpyrifos, methomyl, and metalaxyl, by air sampling and liquid chromatography/mass spectrometry. We estimated differences in self-reported health outcomes, complete blood counts, cholinesterase activity, and serum/urine calcium and creatinine concentrations at baseline between farmworkers and comparison workers, and after pesticide spraying in farmworkers only. RESULTS: This study included 97 men between the ages of 22 and 76 years; 70 were conventional farmworkers; and 27 did not report any prior farmwork or pesticide spraying. None of the farmworkers wore standardized personal protective equipment (PPE) for the concentrated chemicals they were working with. Methomyl (8.4-13 481.9 ng/m3 ), ethyl chlorpyrifos (11.6-67 759 ng/m3 ), and metalaxyl (13.9-41 191.3 ng/m3 ) were detected via personal air sampling. When it came to reporting confidence in the ability to handle personal problems, only 43% of farmworkers reported feeling confident, which reflects higher stress levels in comparison to 78% of comparison workers (P = .028). Farmworkers also had significantly lower monocyte counts (P = .01), serum calcium (P = .01), red blood count (P = .01), white blood cell count (P = .04), and butyrylcholinesterase activity (P < .0001), relative to comparison workers. After adjusting for body mass index (BMI), age, and smoking, methomyl air concentrations were associated with a decrease in farmworker acetylcholinesterase activity (beta = -0.327, P = .016). CONCLUSIONS: This population of farmworkers had significant alterations in stress measures and clinical biomarkers, including decreased blood cell counts and cholinesterase activity, relative to matched controls. These changes are potentially linked to occupational pesticide exposures. Improving PPE use presents a likely route for preventive intervention in this population.

A comprehensive analysis of racial disparities in chemical biomarker concentrations in United States women, 1999-2014.

BACKGROUND: Stark racial disparities in disease incidence among American women remain a persistent public health challenge. These disparities likely result from complex interactions between genetic, social, lifestyle, and environmental risk factors. The influence of environmental risk factors, such as chemical exposure, however, may be substantial and is poorly understood. OBJECTIVES: We quantitatively evaluated chemical-exposure disparities by race/ethnicity, life stage, and time in United States (US) women (n = 38,080) by using biomarker data for 143 chemicals from the National Health and Nutrition Examination Survey (NHANES) 1999-2014. METHODS: We applied a series of survey-weighted, generalized linear models using data from the entire NHANES women population along with cycle and age-group stratified subpopulations. The outcome was chemical biomarker concentration, and the main predictor was race/ethnicity with adjustment for age, socioeconomic status, smoking habits, and NHANES cycle. RESULTS: Compared to non-Hispanic White women, the highest disparities were observed for non-Hispanic Black, Mexican American, Other Hispanic, and Other Race/Multi-Racial women with higher levels of pesticides and their metabolites, including 2,5-dichlorophenol, o,p'-DDE, beta-hexachlorocyclohexane, and 2,4-dichlorophenol, along with personal care and consumer product compounds, including parabens and monoethyl phthalate, as well as several metals, such as mercury and arsenic. Moreover, for Mexican American, Other Hispanic, and non-Hispanic black women, there were several exposure disparities that persisted across age groups, such as higher 2,4- and 2,5-dichlorophenol concentrations. Exposure levels for methyl and propyl parabens, however, were the highest in non-Hispanic black compared to non-Hispanic white children with average differences exceeding 4-fold. Exposure disparities for methyl and propyl parabens are increasing over time in Other Race/Multi-Racial women while fluctuating for non-Hispanic Black, Mexican American, and Other Hispanic. Cotinine levels are among the highest in Non-Hispanic White women compared to Mexican American and Other Hispanic women with disparities plateauing and increasing, respectively. DISCUSSION: We systematically evaluated differences in chemical exposures across women of various race/ethnic groups and across age groups and time. Our findings could help inform chemical prioritization in designing epidemiological and toxicological studies. In addition, they could help guide public health interventions to reduce environmental and health disparities across populations.