Replacement Flame-Retardant 2-Ethylhexyldiphenyl Phosphate (EHDPP) Disrupts Hepatic Lipidome: Evidence from Human 3D Hepatospheroid Cell Culture

Abstract

The present study aims to evaluate the effects of repeated exposure to 2-ethylhexyldiphenyl phosphate (EHDPP) on human liver cells. In vitro three-dimensional (3D) hepatospheroid cell culture was utilized to explore the potential mechanisms of EHDPP-mediated metabolic disruption through morphological, transcriptional, and biochemical assays. Lipidomics analysis was performed on the individual hepatospheroids to investigate the effects on intracellular lipid profiles, followed by hepatospheroid morphology, growth, functional parameters, and cytotoxicity evaluation. The possible mechanisms were delineated using the gene-level analysis by assessing the expression of key genes encoding for hepatic lipid metabolism. We revealed that exposure to EHDPP at 1 and 10 μM for 7 days alters the lipid profile of human 3D hepatospheroids. Dysregulation in several lipid classes, including sterol lipids (cholesterol esters), sphingolipids (dihydroceramide, hexosylceramide, ceramide, sphingomyelin), glycerolipids (triglycerides), glycerophospholipids, and fatty acyls, was noted along with alteration in genes including ACAT1, ACAT2, CYP27A1, ABCA1, GPAT2, PNPLA2, PGC1α, and Nrf2. Our study brings a novel insight into the metabolic disrupting effects of EHDPP and demonstrates the utility of hepatospheroids as an in vitro cell culture model complemented with omics technology (e.g., lipidomics) for mechanistic toxicity studies.

Keywords: 3D spheroids; flame retardants; lipidomics; metabolic disrupting chemicals; repeat dose toxicity.

Figure 1

HepG2 spheroid culture and exposure scheme. One thousand viable cells were seeded into 1.5% agarose-coated 96-flat-bottom well plates and were cultivated at a 37 °C humidified incubator with 5% CO₂ and maintained for 14 days. The culture medium was refreshed every 3–4 days. Treatments started on D7 (mean diameter of spheroids: 250–300 μm). Treatment was performed every second to third day, and the hepatospheroids were repeatedly exposed for 7 days (three exposures). The concentration of DMSO was maintained at 0.01% at all experimental conditions. Spheroid growth was monitored using a phase-contrast microscope (BioTek) during every treatment duration.

Figure 2

Morphological assessment and growth kinetics of hepatospheroids. Quantitative evaluation of hepatospheroid morphology: (A) spheroid size (diameter), (B) spheroid area, and (C) circularity. (D) Representative photomicrograph showing cell viability of hepatospheroids using live/dead staining with calcein-AM (live cell indicator) and propidium iodide (dead cell indicator). Images were taken in BioTek Cytation 5 with fluorescence microscopy mode, using green fluorescence protein (GFP) and Texas red (TR) filter. Scale bar, 100 μm. (E) Representative photomicrographs (brightfield) showing morphological differences between hepatospheroids exposed repeatedly to EHDPP (1, 10, 50 μM) and SC on D0 before exposure and D7 after exposure. (F) Endpoint cell viability was determined by CellTiter-Glo luminescent and (G) LDH release assay after 7 days of exposure with EHDPP (1 and 10 μM). (H) Urea, (I) albumin, and (J) total protein (n = 10 spheroids) as hepatic function parameters after 7 days of exposure to EHDPP 1 and 10 μM or SC. Data presented as mean ± SEM of three to four independent experiments. Significance was determined by one-way ANOVA, **p < 0.01; ***p < 0.001.

Figure 3

PCA score plot of the lipid profile of 3D hepatospheroids treated with EHDPP 1 and 10 μM or SC for 7 days.

Figure 4

Targeted lipidomic analysis of hepatospheroids exposed to EHDPP (1 and 10 μM) or SC for 7 days. (A) Acylcarnitines (CAR), (B) diglyceride (DG), (C) triglyceride (TG), (D) TG:DG ratio, (E) phosphatidylcholine (PC), (F) phosphatidylethanolamines (PEs), (G) PC/PE ratio, (H) alkenylphosphatidylcholine (PC-P), (I) alkylphosphatidylcholine (PC-O), (J) lysophosphatidylethanolamines (LPEs), (K) lysophosphatidylcholine (LPC), (L) phosphatidylglycerol (PG), (M) phosphatidylinositol (PI), (N) phosphatidylserine (PS), (N) phosphatidylethanolamines (PEs), (O) dihydroceramides (dhCer), (P) ceramide (CER), (Q) hexosylceramide (HexCer), (R) hexosylceramide (Hex2Cer), (S) sphingomyelin (SM), (T) free cholesterol (FC), (U) cholesterol esters, (V) total cholesterol, and (W) CE/FC ratio. Statistical significance was evaluated by one-way ANOVA post-Dunnett’s multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001 (n = 10 spheroids/group).

Figure 5

mRNA expression of key genes associated with hepatic lipid metabolism. For all statistical plots, the data are presented as mean ± SEM of three to four independent experiments. Statistical significance was evaluated by ANOVA followed by Dunnett’s multiple comparison test. *p < 0.05; **p < 0.01.

Figure 6

Cellular ROS production and mRNA expression of Nrf2 transcription factor. (A) Elevated ROS production in hepatospheroids treated with EHDPP (1 and 10 μM) or SC for 7 days; mean ± SEM of three independent experiments (n = 3). (B) mRNA expression of Nrf2 and (C) PGC1α; mean ± SEM of four independent experiments. *p < 0.05; **p < 0.01; and ***p < 0.001.

Figure 7

Summary of proposed mechanisms for EHDPP-induced hepatic lipid metabolism dysregulation.