Steroidomic Footprinting Based on Ultra-High Performance Liquid Chromatography Coupled with Qualitative and Quantitative High-Resolution Mass Spectrometry for the Evaluation of Endocrine Disrupting Chemicals in H295R Cells

Abstract

The screening of endocrine disrupting chemicals (EDCs) that may alter steroidogenesis represents a highly important field mainly due to the numerous pathologies, such as cancer, diabetes, obesity, osteoporosis, and infertility that have been related to impaired steroid-mediated regulation. The adrenal H295R cell model has been validated to study steroidogenesis by the Organization for Economic Co-operation and Development (OECD) guideline. However, this guideline focuses solely on testosterone and estradiol monitoring, hormones not typically produced by the adrenals, hence limiting possible in-depth mechanistic investigations. The present work proposes an untargeted steroidomic footprinting workflow based on ultra-high pressure liquid chromatography (UHPLC) coupled to high-resolution MS for the screening and mechanistic investigations of EDCs in H295R cell supernatants. A suspected EDC, triclocarban (TCC), used in detergents, cosmetics, and personal care products, was selected to demonstrate the efficiency of the reported methodology, allowing the simultaneous assessment of a steroidomic footprint and quantification of a selected subset of steroids in a single analysis. The effects of exposure to increasing TCC concentrations were assessed, and the selection of features with database matching followed by multivariate analysis has led to the selection of the most salient affected steroids. Using correlation analysis, 11 steroids were associated with a high, 18 with a medium, and 8 with a relatively low sensitivity behavior to TCC. Among the candidates, 13 identified steroids were simultaneously quantified, leading to the evaluation and localization of the disruption of steroidogenesis caused by TCC upstream of the formation of pregnenolone. The remaining candidates could be associated with a specific steroid class (progestogens and corticosteroids, or androgens) and represent a specific footprint of steroidogenesis disruption by TCC. This strategy was devised to be compatible with medium/high-throughput screening and could be useful for the mechanistic elucidation of EDCs.

In utero and lactational exposure to triclocarban: reproductive effects on female rat offspring

“…The decrease in progesterone levels in the offspring could suggest a decrease in steroidogenic enzyme expression, as some in vitro steroidogenesis studies showed down‐regulation of progesterone and 17α‐hydroxyprogesterone levels after TCC exposure, impairing an early step of steroidogenesis (Strajhar et al, ; Tonoli et al, ). Moreover, the steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis, transporting cholesterol from the outer to the inner mitochondrial membrane and a decreased StAR expression has been reported in fathead minnows (Zenobio, Sanchez, Archuleta, & Sepulveda, ).…”

“…The decrease in progesterone levels in the offspring could suggest a decrease in steroidogenic enzyme expression, as some in vitro steroidogenesis studies showed down-regulation of progesterone and 17α-hydroxyprogesterone levels after TCC exposure, impairing an early step of steroidogenesis (Strajhar et al, 2017;Tonoli et al, 2015).…”

“…Moreover, in vitro studies have also shown enhanced estrogen activity mediated via the estrogen receptor (ER) (Ahn et al, 2008;Tarnow, Tralau, Hunecke, & Luch, 2013) and weak estrogenic activity in the ER reporter gene assay and breast cancer cell line (MCF-7) proliferation assay (Huang et al, 2014). Tonoli et al (2015) reported that TCC exposure altered an early step in adrenal steroidogenesis in H295R cells, and human aromatase inhibition in vitro (Li et al, 2017).…”

Sex-dependent impact of Roundup on the rat gut microbiome

“…X-variables which combine high magnitude and high reliability are of relevance in the search for discriminating variables. The extraction of the discriminating variables from the S-plot could be combined with the jack-knifed confidence intervals seen in the loading plot [ 33 , 34 ].…”

Effects of the antimicrobial contaminant triclocarban, and co‐exposure with the androgen 17β‐trenbolone, on reproductive function and ovarian transcriptome of the fathead minnow (Pimephales promelas)

“…However, in contrast to the hypothesis that TCC can augment androgenic activity, these data were more suggestive of a potential antiandrogenic action than augmentation. At least 1 recent study suggests that TCC may impair steroid biosynthesis in vitro . However, the concentrations tested in H295R cells were in excess of 150 µg/L , so it is unclear whether those effects would be relevant in vivo given that similar concentrations cause overt toxicity in fish.…”