Development of a Liquid Chromatography–Mass Spectrometry-Based In Vitro Assay to Assess Changes in Steroid Hormones Due to Exposure to Per- and Polyfluoroalkyl Substances

Abstract

Per-and poly-fluorinated substances (PFASs) are organic pollutants that have been linked to numerous health effects, including diabetes, cancers, and dysregulation of the endocrine system. This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to measure changes in 17 hormones in H295R cell line (a steroid producing adrenocortical cells) upon exposure to PFASs. Due to the challenges in the analysis of steroid hormones using electrospray ionization MS, a chemical derivatization method was employed to achieve 0.07−2 μg/L detection limits in LC-MS/MS. Furthermore, a 10-fold concentration factor through solid-phase extraction (SPE) allows for consistent sub-parts per billion detections. Optimization of the derivatization conditions showed doubly-derivatized products in some hormone analytes, including progesterone, corticosterone, and cortisol, and gave improved ionization efficiency up to 20-fold higher signal than the singly-derivatized product. The use of SPE for sample cleanup to analyze hormones from cellular media using weak anion exchange sorbent yielded 80−100% recovery for the 17 targeted hormones. The method was validated by exposing H295R cells to two known endocrine disruptors, forskolin and prochloraz, which showed expected changes in hormones. An initial exposure of H295R cells with various PFAS standards and their mixtures at 1 μM showed significant increases in progestogens with some PFAS treatments, which include PFBS, PFHxA, PFOS, PFDA, and PFDS. In addition, modest changes in hormone levels were observed in cells treated with other sulfonated or carboxylated headgroup PFASs. This sensitive LC-MS/MS method for hormone analysis in H295R cells will allow for the investigations of the alterations in the hormone production caused by exposure to various environmental insults in cell-based assays and other in vitro models.

Transcriptomics effects of per- and polyfluorinated alkyl substances in differentiated neuronal cells

“…Many forms of PFAS toxicity are chronic, taking place over years. 20 PFAS are commonly detected in human serum at low part per billion levels (ng/mL) or nanomolar concentrations, 45, 46 but do not exhibit toxicity in cultured cells up to 3∼100 μM 14, 47, 48 , consistent with our results. PFAS can reversibly bind to proteins which decreases bioavailability for uptake in vitro .…”

“…13 PFAS are also routinely detected in human blood samples and in breast milk, resulting in early exposure of infants. 14 PFAS affect endocrine system 15,16 , disrupting menstrual cycle, decreasing sperm mobility, and lowering the age of onset puberty. 17 The liver accumulation of PFAS is common, and hepatoxicity has been observed in human studies.…”

“…PFAS affect many systems and processes, including the endocrine system 14, 15 , menstrual cycle, sperm mobility, and the age of onset puberty. 16 The liver accumulation of PFAS is common, and hepatoxicity has been observed in human studies.…”

“…21 PFAS are commonly detected in human serum at low part per billion levels (ng/mL) or nanomolar concentrations, 14, 41 but do not exhibit toxicity in cultured cells up to 3∼100 μM. 15, 42, 43 Another factor that should be considered is PFAS, specifically long-chain ionic PFAS, reversibly binds to proteins through complex interactions that decrease bioavailability non-linearly in protein-rich cellular media used for in vitro studies. 44 Based on these factors, exposure studies use much higher concentrations than environmentally relevant PFAS levels.…”

“…While certain professions are at an increased risk for PFAS exposure including firefighters, industrial workers and ski board waxers, general communities are also at risk from PFAS, the ones found in our water sources, food packing and cosmetics. 12 PFAS affect many systems and processes, including the endocrine system 14,15 , menstrual cycle, sperm mobility, and the age of onset puberty. 16 The liver accumulation of PFAS is common, and hepatoxicity has been observed in human studies.…”