Untargeted lipidomics reveals the toxicity of bisphenol A bis(3-chloro-2- hydroxypropyl) ether and bisphenols A and F in zebrafish liver cells.

Given the opposing responses reported for bisphenol A (BPA) in terms of induction of obesogenic effects and impaired lipid metabolism, the increasing use of bisphenol F (BPF), and the relatively low information available regarding the effects of bisphenol A bis(3-chloro-2- hydroxypropyl) ether (BADGE·2HCl) in aquatic organisms, this work aims to use the zebrafish liver cell line (ZFL) as an alternative model to characterize the toxicity and the lipid metabolism disruptive potential of the selected compounds in fish. All three bisphenols increased intracellular levels of dihydroceramides and ether-triacylglycerides (ether-TGs), suggestive of inhibited cell growth. However, while BPA and BADGE·2HCl caused an increase of saturated and lower unsaturated TGs, BPF caused oxidative stress and the decrease of TGs containing polyunsaturated fatty acids (PUFAs). Analysis by qPCR highlighted the up-regulation of the lipogenic genes scd and elovl6 by BPA and BPF in line with an increase of lipids containing saturated and monounsaturated FA and a decrease of lipids containing PUFAs. This study shows that BPA, BPF and BADGE·2HCl target lipid homeostasis in ZFL cells through different mechanisms, and highlights the higher lipotoxicity of BADGE·2HCl compared to BPA and BPF.

Dysregulation of lipid metabolism in PLHC-1 and ZFL cells exposed to tributyltin an all-trans retinoic acid.

There is increasing awareness that exposure to endocrine disrupters interferes with lipid homeostasis in vertebrates, including fish. Many of these compounds exert their action by binding to nuclear receptors, such as peroxisome proliferator-activated receptors and retinoid X receptor. This work investigates the use of fish liver cells (PLHC-1 and ZFL cells) for the screening of metabolic and lipid disrupters in the aquatic environment by assessing changes in the cell's lipidome after exposure to the model compounds, tributyltin chloride and all-trans retinoic acid. Lipid extracts, analyzed by FIA-ESI (+/-) Orbitrap, evidenced the intracellular accumulation of triglycerides and diglycerides in both cell models after exposure to 100 and 200 nM tributyltin chloride for 24 h. Exposure to 1 µM all-trans retinoic acid led to a significant accumulation of triglycerides in PLHC-1 cells, while few triglycerides were accumulated in ZFL cells. Retinoic acid (cyp26b1, cyp3a65, lrata) and lipid metabolism (fasn, scd, elovl6) related genes were up-regulated by tributyltin chloride and all-trans retinoic acid, while only all-trans retinoic acid down-regulated the expression of dgat1a. The two cell models show sensitivity and responses to tributyltin chloride and all-trans retinoic acid comparable to those previously reported in mammalian cells. These results support the use of fish liver cells as alternative models for the detection of contaminants that act as lipid disrupters in the aquatic environment.

Drospirenone induces the accumulation of triacylglycerides in the fish hepatoma cell line, PLHC-1.

Drospirenone (DRO) is one of the most commonly used progestins reaching the aquatic environment through wastewater treatment plant effluents. It is a progesterone receptor agonist, and as such, can act primarily in the brain and reproductive organs of fish. In order to better understand and predict its effects, this work evaluates the lipidomic changes induced in PLHC-1 cells after exposure to drospirenone at concentrations below the EC10 (1 and 10 µM) by direct injection of the lipid extracts into a ESI(+/-) Orbitrap mass spectrometer. A significant accumulation of triacylglycerides, particularly long chain ones with unsaturated fatty acid moieties (TGs 46:2, 56:4-7; 58:5-8) and a concomitant decrease of diacylglycerides (DGs 32:1, 34:1-2, 36:1-2, 38:2-4) was observed after 48 h exposure to 10 µM DRO, which corresponded to an intracellular concentration of 8.3 ng·mg-1 protein. No significant alteration of PLHC-1 cell lipids was observed following exposure to 1 µM DRO. EC50 for the cytotoxicity of DRO ranged from 105 to 119 µM (24 h exposure) to 51-58 µM (48 h exposure). The study evidences a dysregulation of neutral lipid metabolism and increased TG/DG ratio in fish hepatic cells exposed to DRO.

Skeletal Muscle Lipidomics as a New Tool to Determine Altered Lipid Homeostasis in Fish Exposed to Urban and Industrial Wastewaters.

This work applies ultrahigh performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS) to characterize for the first time the lipidome of the skeletal muscle of two fish species (Barbus meridionalis, Squalius laietanus) collected in a Mediterranean River affected by urban and industrial wastewater outflows. The untargeted analysis allowed a clear separation of the lipidome of fish from polluted and reference sites; phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and their lyso and ether-linked forms were among the distinctive features. The targeted analysis consistently detected a decrease in PC-plasmalogens (36:4, 36:6, 38:6) and highly unsaturated PCs (36:5, 36:6, 38:6, 40:6, 40:7) and an increase in plasmanyl-PCs (36:5, 38:5), lyso-PCs (16:1, 18:1, 22:4) and cholesteryl esters (CEs) (16:0, 18:0, 20:4) in fish from polluted sites. These lipid profiles were indicative of oxidative stress and dysregulation of cholesterol homeostasis in fish from polluted sites. This methodology represents a promising tool for the development of novel noninvasive diagnostic methods based on muscle tissue biopsies to assess the effects of water pollution in wildlife.

Toxic effects of bisphenol A diglycidyl ether and derivatives in human placental cells.

BADGE (bisphenol A diglycidyl ether) is a synthesis product of bisphenol A (BPA), which, like other plasticizers, can cross the human placenta and reach the foetus. However, compared to BPA, there is almost no toxicological information. This work investigates the toxicity, endocrine and lipid disruption potential of BADGE and its hydrolysed and chlorinated derivatives (BADGE·H2O and BADGE·2HCl) in human placental JEG-3 cells. The analysis of culture medium by HPLC-ESI(+)-QqQ evidenced a good bioavailability of BADGE·2HCl and BADGE·H2O, but low stability of BADGE. Regardless, BADGE·2HCl and BADGE showed higher cytotoxicity than BADGE·H2O, which was the only compound that significantly inhibited CYP19 activity (IC50 49 ±â€¯5 µM). JEG-3 cells lipidome analyzed by FIA-ESI(+/-)-Orbitrap was significantly altered by exposure to BADGE·2HCl and BADGE at concentrations at the low µM range. BADGE·2HCl lead to a strong decrease of diacyl- and triacyl-glycerides (DGs,TGs) together with some membrane lipids, while BADGE lead to an accumulation of TGs. The results evidence the ability of BADGE and derivatives to affect placental lipid handling and to modulate placental CYP19 activity (BADGE·H2O) and highlights the need to monitor human exposure to these compounds, at least as intensely as BPA is monitored.

Comparative toxicity and endocrine disruption potential of urban and rural atmospheric organic PM1 in JEG-3 human placental cells.

Outdoor ambient air particulate matter and air pollution are related to adverse effects on human health. The present study assesses the cytotoxicity and ability to disrupt aromatase activity of organic PM1 extracts from rural and urban areas at equivalent air volumes from 2 to 30 m3, in human placental JEG-3 cells. Samples were chemically analyzed for particle bounded organic compounds with endocrine disrupting potential, i.e. PAH, O-PAH, phthalate esters, but also for organic molecular tracer compounds for the emission source identification. Rural samples collected in winter were cytotoxic at the highest concentration tested and strongly inhibited aromatase activity in JEG-3 cells. No cytotoxicity was detected in summer samples from the rural site and the urban samples, while aromatase activity was moderately inhibited in these samples. In the urban area, the street site samples, collected close to intensive traffic, showed stronger inhibition of aromatase activity than the samples simultaneously collected at a roof site, 50 m above ground level. The cytotoxicity and endocrine disruption potential of the samples were linked to combustion products, i.e. PAH and O-PAH, especially from biomass burning in the rural site in winter.