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.

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.

Impact of Tralopyril and Triazolyl Glycosylated Chalcone in Human Retinal Cells' Lipidome.

Antifouling (AF) coatings containing booster biocides are used worldwide as one of the most cost-effective ways to prevent the attachment of marine organisms to submerged structures. Nevertheless, many of the commercial biocides, such as Econea® (tralopyril), are toxic in marine environments. For that reason, it is of extreme importance that new efficient AF compounds that do not cause any harm to non-target organisms and humans are designed. In this study, we measured the half-maximal inhibitory concentration (IC50) of a promising nature-inspired AF compound, a triazolyl glycosylated chalcone (compound 1), in an immortalized human retinal pigment epithelial cell line (hTERT-RPE-1) and compared the results with the commercial biocide Econea®. We also investigated the effects of these biocides on the cellular lipidome following an acute (24 h) exposure using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS). Our results showed that compound 1 did not affect viability in hTERT-RPE-1 cells at low concentrations (1 µM), in contrast to Econea®, which caused a 40% reduction in cell viability. In total, 71 lipids were found to be regulated upon exposure to 10 µM of both compounds. Interestingly, both compounds induced changes in lipids involved in cell death, membrane modeling, lipid storage, and oxidative stress, but often in opposing directions. In general, Econea® exposure was associated with an increase in lipid concentrations, while compound 1 exposure resulted in lipid depletion. Our study showed that exposure to human cells at sublethal Econea® concentrations results in the modulation of several lipids that are linked to cell death and survival.

Polycyclic aromatic hydrocarbons in silicone wristbands of Uruguayan children: measurement and exposure source exploration.

Polycyclic aromatic hydrocarbons (PAHs) pose health risks to children, potentially resulting in stunted growth, obesity, and cognitive deficits, but lack of reliable and noninvasive means to measure PAHs results in poor understanding of exposure patterns and sources in this vulnerable population. In this study, 24 children aged ∼7 years (9 boys and 15 girls) from Montevideo, Uruguay wore silicone wristbands for 8 days to monitor the exposure of 27 PAHs. Wristbands were extracted using a modified ethyl acetate tandem solid phase extraction clean up and then analyzed via gas chromatography with tandem mass spectrometry. This analysis has reported LODs for 27 PAHs between 0.05 and 3.91 µg L-1. Eighteen PAHs were detected in >50% of the samples with concentration medians ranging 1.2-16.3 ng g-1 of wristband. Low molecular weight PAHs (2-3 rings) such as naphthalene and its alkyl derivatives were highly correlated (0.7-0.9) in the wristbands, suggesting exposure from related sources. Exposure source exploration focused on secondhand tobacco smoke, potentially through caregivers who reported on smoking habits in an associated survey. A principal components analysis (PCA) was conducted to examine patterns in PAH compounds detected in the wristbands; subsequently, the resulting components were compared according to current smoking among caregivers. The PCA analysis revealed a grouping of participants based on higher exposure of 1-methyl naphthalene, pyrene, fluoranthene, 1-methylphenanthrene, dibenzothiophene and 2-phenyl naphthalene. The derived components did relate with parental smoking, suggesting that some participants experienced exposure to a common source of certain PAHs outside of parental smoking. This is the first study to assess PAH exposure in young children from South America. Using wristbands, our study indicates exposure to multiple, potentially harmful chemicals. Wristbands could provide a comprehensive picture of PAH exposure in children, complementing other non-invasive biomonitoring approaches.

Perfluorooctane Sulfonate (PFOS) Negatively Impacts Prey Capture Capabilities in Larval Zebrafish.

Per- and polyfluoroalkyl substances (PFAS) are widely used in many industrial and domestic applications, which has resulted in unintentional human exposures and bioaccumulation in blood and other organs. Perfluorooctane sulfonate (PFOS) is among the most prevalent PFAS in the environment and has been postulated to affect brain functions in exposed organisms. However, the impacts of PFOS in early neural development have not been well described. We used zebrafish larvae to assess the effects of PFOS on two fundamental complex behaviors, prey capture and learning. Zebrafish exposed to PFOS concentrations ranging from 2 to 20 µM for differing 48-h periods were viable through early larval stages. In addition, PFOS uptake was unaffected by the presence of a chorion. We employed two different experimental paradigms; first we assessed the impacts of increasing organismal PFOS bioaccumulation on prey capture and learning, and second, we probed stage-specific sensitivity to PFOS by exposing zebrafish at different developmental stages (0-2 vs. 3-5 days post fertilization). Following both assays we measured the amount of PFOS present in each larva and found that PFOS levels varied in larvae from different groups within each experimental paradigm. Significant negative correlations were observed between larval PFOS accumulation and percentage of captured prey, whereas nonsignificant negative correlations were observed between PFOS accumulation and experienced-induced prey capture learning. These findings suggest that PFOS accumulation negatively affects larval zebrafish's ability to perform complicated multisensory behaviors and highlights the potential risks of PFOS exposure to animals in the wild, with implications for human health. Environ Toxicol Chem 2024;43:847-855. © 2023 SETAC.

Preliminary hazard assessment of a new nature-inspired antifouling (NIAF) agent.

A recently synthesized aminated 3,4-dioxygenated xanthone (Xantifoul2) was found to have promising antifouling (AF) effects against the settlement of the macrofouler Mytilus galloprovincialis larvae. Preliminary assessment indicated that Xantifoul2 has reduced ecotoxicological impacts: e.g., being non-toxic to the marine crustacea Artemia salina (<10 % mortality at 50 µM) and showing low bioconcentration factor in marine organisms. In order to meet the EU Biocidal Product Regulation, a preliminary hazard assessment of this new nature-inspired antifouling (NIAF) agent was conducted in this work. Xantifoul2 did not affect the swimming ability of the planktonic crustacean Daphnia magna, the growth of the diatom Phaeodactylum tricornutum, and the cellular respiration of luminescent Gram-negative bacteria Vibrio fischeri, supporting the low toxicity towards several non-target marine species. Regarding human cytotoxicity, Xantifoul2 did not affect the cell viability of retinal human cells (hTERT-RPE-1) and lipidomic studies revealed depletion of lipids involved in cell death, membrane modeling, lipid storage, and oxidative stress only at a high concentration (10 µM). Accelerated degradation studies in water were conducted under simulated sunlight to allow the understanding of putative transformation products (TPs) that could be generated in the aquatic ecosystems. Both Xantifoul2 and photolytic-treated Xantifoul2 in the aqueous matrix were therefore evaluated on several nuclear receptors (NRs). The results of this preliminary hazard assessment of Xantifoul2, combined with the high degradation rates in water, provide strong evidence of the safety of this AF agent under the evaluated conditions, and provide the support for future validation studies before this compound can be introduced in the market.

Ceramide-1-Phosphate Is Involved in Therapy-Induced Senescence.

Sphingolipids are key signaling lipids and their dysregulation has been associated with various cellular processes. We have previously shown significant changes in sphingolipids in therapy-induced senescence, a state of cell cycle arrest as a response to chemotherapy, including the accumulation of ceramides, and provided evidence suggesting that ceramide processing is important for this process. Herein, we conducted a focused small molecule inhibitor screen targeting the sphingolipid pathway, which highlighted a new lipid regulator of therapy-induced senescence. Among the inhibitors tested, the inhibition of ceramide kinase by NVP-231 reduced the levels of senescent cells. Ceramide kinase knockdown exhibited similar effects, strongly supporting the involvement of ceramide kinase during this process. We showed that ceramide-1-phosphate was upregulated in therapy-induced senescence and that NVP-231 reduced ceramide-1-phosphate levels in different cell line models of therapy-induced senescence. Finally, ceramide-1-phosphate addition to NVP-231-treated cells reversed the effects of NVP-231 during senescence. Overall, our results identify a previously unknown lipid player in therapy-induced senescence and highlight a potential targetable enzyme to reduce the levels of therapy-induced senescent cells.

Comprehensive assessment of chemical residues in surface and wastewater using passive sampling, chemical, biological, and fish behavioral assays.

Effluents from ten full-scale municipal wastewater treatment plants (WWTPs) that discharge into the Hudson River, surface waters, and wild-caught fish samples were analyzed using liquid chromatography with tandem mass spectrometry (LC/MS/MS) to examine the influence of wastewater discharge on the concentrations of contaminants of emerging concern (CECs) and their ecological impacts on fish. Analysis was based on targeted detection of 41 pharmaceuticals, and non-targeted analysis (suspect screening) of CECs. Biological effects of treated WWTP effluents were assessed using a larval zebrafish (Danio rerio) swimming behavior assay. Concentrations of residues in surface waters were determined in grab samples and polar organic chemical integrative samplers (POCIS). In addition, vitellogenin peptides, used as biomarkers of endocrine disruption, were quantified using LC/MS/MS in the wild-caught fish plasma samples. Overall, 94 chemical residues were identified, including 63 pharmaceuticals, 10 industrial chemicals, and 21 pesticides. Eight targeted pharmaceuticals were detected in 100% of effluent samples with median detections of: bupropion (194 ng/L), carbamazepine (91 ng/L), ciprofloxacin (190 ng/L), citalopram (172 ng/L), desvenlafaxine (667 ng/L), iopamidol (3790 ng/L), primidone (86 ng/L), and venlafaxine (231 ng/L). Over 30 chemical residues were detected in wild-caught fish tissues. Notably, zebrafish larvae exposed to chemical extracts of effluents from 9 of 10 WWTPs, in at least one season, were significantly hyperactive. Vitellogenin expression in male or immature fish occurred 2.8 times more frequently in fish collected from the Hudson River as compared to a reference site receiving no direct effluent input. Due to the low concentrations of pharmaceuticals detected in effluents, it is likely that chemicals other than pharmaceuticals measured are responsible for the behavioral changes observed. The combined use of POCIS and non-target analysis demonstrated significant increase in the chemical coverage for CEC detection, providing a better insight on the impacts of WWTP effluents and agricultural practices on surface water quality.

Endocrine Therapy-Resistant Breast Cancer Cells Are More Sensitive to Ceramide Kinase Inhibition and Elevated Ceramide Levels Than Therapy-Sensitive Breast Cancer Cells.

ET resistance is a critical problem for estrogen receptor-positive (ER+) breast cancer. In this study, we have investigated how alterations in sphingolipids promote cell survival in ET-resistant breast cancer. We have performed LC-MS-based targeted sphingolipidomics of tamoxifen-sensitive and -resistant MCF-7 breast cancer cell lines. Follow-up studies included treatments of cell lines and patient-derived xenograft organoids (PDxO) with small molecule inhibitors; cytometric analyses to measure cell death, proliferation, and apoptosis; siRNA-mediated knockdown; RT-qPCR and Western blot for gene and protein expression; targeted lipid analysis; and lipid addback experiments. We found that tamoxifen-resistant cells have lower levels of ceramides and hexosylceramides compared to their tamoxifen-sensitive counterpart. Upon perturbing the sphingolipid pathway with small molecule inhibitors of key enzymes, we identified that CERK is essential for tamoxifen-resistant breast cancer cell survival, as well as a fulvestrant-resistant PDxO. CERK inhibition induces ceramide-mediated cell death in tamoxifen-resistant cells. Ceramide-1-phosphate (C1P) partially reverses CERK inhibition-induced cell death in tamoxifen-resistant cells, likely through lowering endogenous ceramide levels. Our findings suggest that ET-resistant breast cancer cells maintain lower ceramide levels as an essential pro-survival mechanism. Consequently, ET-resistant breast cancer models have a unique dependence on CERK as its activity can inhibit de novo ceramide production.