Per- or polyfluoroalkyl compounds (PFASs) are a class of completely or partially fluorinated chemicals used in industry and consumer applications such as in fire extinguishing agents and surfactants. Accumulation of PFASs in humans has been correlated to changes in total hormone levels, including decreased levels of testosterone and increased levels of estrogens. The effect of PFASs as endocrine disruptors is not fully understood, and the potential impact of novel PFASs has not been extensively studied. This study will develop a high throughput assay to measure the changes in 17 steroid hormones in H295R cells as a result of PFAS exposure at environmentally relevant concentrations. This study will culture H295R cells which express the enzymes necessary to produce 17 steroid hormones. These cells will be exposed to various PFAS conditions and any changes in hormone levels will be measured to assess endocrine disrupting potential. H295R cells will be grown in a 96-well plate so multiple conditions can be tested simultaneously. Hormones will be extracted from the cellular media using micro solid phase extraction (SPE) in a 96 well plate. Hormones will then be derivatized with a parallel methoxyamine and dansyl chloride reactions for low-level detection by liquid chromatography with tandem mass spectrometry (LC/MS/MS). Separation and mass analysis will be done using an AgilentTM 1200 series LC coupled to a Thermo ScientificTM TSQ Quantum Ultra MS, equipped with an InertsustainTM phenyl column. The development of this method will yield a highly sensitive hormone assay that will save time and material compared to other methods. This analysis will also facilitate the increased understanding of mechanisms of endocrine disruption by both novel and legacy PFASs.

Assessment of endocrine disrupting effects of per and poly fluorinated alkyl compounds using a high throughput derivatization-based LC-MS/MS assay

ACS Spring 2022

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