A wide range of novel replacement flame retardants (nFRs) is consistently detected in increasing concentrations in the environment and human matrices. Evidence suggests that nFRs exposure may be associated with disruption of the endocrine system, which has been linked with the etiology of various metabolic disorders, including nonalcoholic fatty liver disease (NAFLD). NAFLD is a multifactorial disease characterized by the uncontrolled accumulation of fats (lipids) in the hepatocytes and involves multiple-hit pathogenesis, including exposure to occupational and environmental chemicals such as organophosphate flame retardants (OPFRs). In the present study we aimed to investigate the potential mechanisms of the nFRs-induced hepatic steatosis in the human liver cells. In this study, we employed an in vitro bioassay toolbox to assess the key events (KEs) in the proposed adverse outcome pathways (AOP) (s) for hepatic steatosis. We examined nine nFRs using AOP- based in vitro assays measuring KEs such as lipid accumulation, mitochondrial dysfunction, gene expression, and in silico approach to identify the putative molecular initiating events (MIEs). Our findings suggest that several tested OPFRs induced lipid accumulation in human liver cell culture. Tricresyl phosphate (TMPP), triphenyl phosphate (TPHP), tris(1,3-dichloropropyl) phosphate (TDCIPP), and 2-ethylhexyl diphenyl phosphate (EHDPP) induced the highest lipid accumulation by altering the expression of genes encoding hepatic de novo lipogenesis and mitochondrial dysfunction depicted by decreased cellular ATP production. Available in vitro data from ToxCast and in silico molecular docking suggests that pregnane X receptor (PXR) and peroxisome proliferator-activated receptor gamma (PPARγ) could be the molecular targets for the tested nFRs. The study identifies several nFRs, such as TMPP and EHDPP, TPHP, and TDCIPP, as potential risk factor for NAFLD and advances our understanding of the mechanisms involved, demonstrating the utility of an AOP-based strategy for screening and prioritizing chemicals and elucidating the molecular mechanisms of toxicity.

Endocrine disrupting chemicals (EDCs) constitute a major public health concern because they can induce a large spectrum of adverse effects by interfering with the hormonal system. Rapid identification of potential EDCs using in vitro screenings is therefore critical, particularly for chemicals of emerging concerns such as replacement flame retardants (FRs). The review aimed at identifying (1) data gaps and research needs regarding endocrine disrupting (ED) properties of replacement FRs and (2) potential EDCs among these emerging chemicals. A systematic search was performed from open literature and ToxCast/Tox21 programs, and results from in vitro tests on the activities of 52 replacement FRs towards five hormone nuclear receptors (NRs) associated with reproductive outcomes (estrogen, androgen, glucocorticoid, progesterone, and aryl hydrocarbon receptors) were compiled and organized into tables. Findings were complemented with information from structure-based in silico model predictions and in vivo information when relevant. For the majority of the 52 replacement FRs, experimental in vitro data on activities towards these five NRs were either incomplete (15 FRs) or not found (24 FRs). Within the replacement FRs for which effect data were found, some appeared as candidate EDCs, such as triphenyl phosphate (TPhP) and tris(1,3-dichloropropyl)phosphate (TDCIPP). The search also revealed shared ED profiles. For example, anti-androgenic activity was reported for 19 FRs and predicted for another 21 FRs. This comprehensive review points to critical gaps in knowledge on ED potential for many replacement FRs, including chemicals to which the general population is likely exposed. Although this review does not cover all possible characteristics of ED, it allowed the identification of potential EDCs associated with reproductive outcomes, calling for deeper evaluation and possibly future regulation of these chemicals. By identifying shared ED profiles, this work also raises concerns for mixture effects since the population is co-exposed to several FRs and other chemicals.

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.

Nonalcoholic fatty liver disease (NAFLD) is a growing concern worldwide, affecting 25% of the global population. NAFLD is a multifactorial disease with a broad spectrum of pathology includes steatosis, which gradually progresses to a more severe condition such as nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and eventually leads to hepatic cancer. Several risk factors, including exposure to environmental toxicants, are involved in the development and progression of NAFLD. Environmental factors may promote the development and progression of NAFLD by various biological alterations, including mitochondrial dysfunction, reactive oxygen species production, nuclear receptors dysregulation, and interference in inflammatory and immune-mediated signaling. Moreover, environmental contaminants can influence immune responses by impairing the immune system’s components and, ultimately, disease susceptibility. Flame retardants (FRs) are anthropogenic chemicals or mixtures that are being used to inhibit or delay the spread of fire. FRs have been employed in several household and outdoor products; therefore, human exposure is unavoidable. In this review, we summarized the potential mechanisms of FRs-associated immune and inflammatory signaling and their possible contribution to the development and progression of NAFLD, with an emphasis on FRs-mediated interferon signaling. Knowledge gaps are identified, and emerging pharmacotherapeutic molecules targeting the immune and inflammatory signaling for NAFLD are also discussed.