Application of an in Vitro Assay to Identify Chemicals That Increase Estradiol and Progesterone Synthesis and Are Potential Breast Cancer Risk Factors

Abstract

BACKGROUND: Established breast cancer risk factors, such as hormone replacement therapy and reproductive history, are thought to act by increasing estrogen and progesterone (P4) activity. OBJECTIVE: We aimed to use in vitro screening data to identify chemicals that increase the synthesis of estradiol (E2) or P4 and evaluate potential risks. METHOD: Using data from a high-throughput (HT) in vitro steroidogenesis assay developed for the U.S. Environmental Protection Agency (EPA) ToxCast program, we identified chemicals that increased estradiol (E2-up) or progesterone (P4-up) in human H295R adrenocortical carcinoma cells. We prioritized chemicals by their activity. We compiled in vivo studies and assessments about carcinogenicity and reproductive/developmental (repro/dev) toxicity. We identified exposure sources and predicted intakes from the U.S. EPA's ExpoCast. RESULTS: We found 296 chemicals increased E2 (182) or P4 (185), with 71 chemicals increasing both. In vivo data often showed effects consistent with this mechanism. Of the E2-and P4-up chemicals, about 30% were likely repro/dev toxicants or carcinogens, whereas only 5-13% were classified as unlikely. However, most of the chemicals had insufficient in vivo data to evaluate their effects. Of 45 chemicals associated with mammary gland effects, and also tested in the H294R assay, 29 increased E2 or P4, including the well-known mammary carcinogen 7,12-dimethylbenz(a)anthracene. E2-and P4-up chemicals include pesticides, consumer product ingredients, food additives, and drinking water contaminants. DISCUSSION: The U.S. EPA's in vitro screening data identified several hundred chemicals that should be considered as potential risk factors for breast cancer because they increased E2 or P4 synthesis. In vitro data is a helpful addition to current toxicity assessments, which are not sensitive to mammary gland effects. Relevant effects on the mammary gland are often not noticed or are dismissed, including for 2,4-dichlorophenol and cyfluthrin. Fifty-three active E2-up and 59 active P4-up chemicals that are in consumer products, food, pesticides, or drugs have not been evaluated for carcinogenic potential and are priorities for study and exposure reduction.

Comment on “Application of an in Vitro Assay to Identify Chemicals That Increase Estradiol and Progesterone Synthesis and Are Potential Breast Cancer Risk Factors”

“…Our reanalysis identified either 17 or 3 chemicals (depending upon stringency) that we allege may be stimulating the production of estrogen in vitro ; however, all of those are likely false positives due to small sample sizes and sampling bias. This is far less than the number identified by Cardona and Rudel (2021) , suggesting most of the chemicals they identified are false positives. Therefore, we suggest that these 17 or 3 chemicals would be good starting points for further research at human-relevant concentrations, with the understanding that they very well could be false positives due to the limited number of replicates.…”

“…The article by Cardona and Rudel (2021) analyzed ToxCast results for the H295R steroidogenesis assays and reported that “296 chemicals increased [estradiol] (182) or [progesterone] (185), with 71 chemicals increasing both.” This result is both fascinating and rather striking in suggesting that in vitro 32% of chemicals increase estrogen production, 32% increase progesterone production, and 12% increase both. Based on our reanalysis of the data (available on Github: https://github.com/DataSciBurgoon/toxcast_steroidogenesis/blob/main/Raptor_SSI_Steroidogenesis.ipynb ) and based on the fact that most ToxCast chemicals have small sample sizes (generally , atypically ) and therefore likely suffer from sampling bias, most if not all of the chemicals identified by Cardona and Rudel (2021) are false positives ( Christley 2010 ; Gelman and Carlin 2014 ; Lin 2018 ).…”

“…Peculiarly, Cardona and Rudel (2021) did not base their analysis directly on ToxCast data from the U.S. Environmental Protection Agency’s invitrodb database. Rather, they used data from Haggard et al.…”

“…2021 ). Thirty-seven and 42 of the chemicals that Cardona and Rudel (2021) identified as increasing estrogen or progesterone production, respectively, have quality flags that indicate that the chemical used may not be what was intended, or that the concentration may be 30% or less than the intended concentration.…”

Invited Perspective: Prioritizing Chemical Testing and Evaluation Using Validated in Vitro Assays Relevant to Key Characteristics

“…Refers to https://doi.org/10.1289/EHP8608 By interfering with hormone action, endocrine-disrupting chemicals (EDCs) can increase the risk of various adverse health outcomes, including cancer and reproductive impairment (La Merrill et al 2020). In their article, Cardona and Rudel (2021) have identified nearly 300 chemicals that increased estradiol, progesterone, or both in an in vitro steroidogenesis assay that is internationally validated for use in regulatory contexts. They screened publicly available testing data for more than 2,000 chemicals tested in the ToxCast™ high-throughput in vitro steroidogenesis assay in cultured human H295R adrenocarcinoma cells.…”