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.

Mycotoxins are natural metabolites produced by fungi that contaminate food and feed worldwide. They can pose a threat to human and animal health, mainly causing chronic effects, e.g., immunotoxic and carcinogenic. Due to climate change, an increase in European population exposure to mycotoxins is expected to occur, raising public health concerns. This urges us to assess the current human exposure to mycotoxins in Europe to allow monitoring exposure and prevent future health impacts. The mycotoxins deoxynivalenol (DON) and fumonisin B₁ (FB₁) were considered as priority substances to be studied within the European Human Biomonitoring Initiative (HBM4EU) to generate knowledge on internal exposure and their potential health impacts. Several policy questions were addressed concerning hazard characterization, exposure and risk assessment. The present article presents the current advances attained under the HBM4EU, research needs and gaps. Overall, the knowledge on the European population risk from exposure to DON was improved by using new harmonised data and a newly derived reference value. In addition, mechanistic information on FB₁ was, for the first time, organized into an adverse outcome pathway for a congenital anomaly. It is expected that this knowledge will support policy making and contribute to driving new Human Biomonitoring (HBM) studies on mycotoxin exposure in Europe.

Humans are chronically exposed to the mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1), as indicated by their widespread presence in foods and occasional exposure in the workplace. This exposure is confirmed by human biomonitoring (HBM) studies on (metabolites of) these mycotoxins in human matrices. We evaluated the exposure–health relationship of the mycotoxins in humans by reviewing the available literature. Since human studies did not allow the identification of unequivocal chronic health effects upon exposure to DON and FB1, the adverse outcome pathway (AOP) framework was used to structure additional mechanistic evidence from in vitro and animal studies on the identified adverse effects. In addition to a preliminary AOP for DON resulting in the adverse outcome (AO) ‘reduced body weight gain’, we developed a more elaborated AOP for FB1, from the molecular initiating event (MIE) ‘inhibition of ceramide synthases’ leading to the AO ‘neural tube defects’. The mechanistic evidence from AOPs can be used to support the limited evidence from human studies, to focus FB1- and DON-related research in humans to identify related early biomarkers of effect. In order to establish additional human exposure–health relationships in the future, recommendations are given to maximize the information that can be obtained from HBM.

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.

Emerging environmental contaminants, organophosphate flame retardants (OPFRs), pose significant threats to ecosystems and human health. Despite numerous studies reporting the toxic effects of OPFRs, research on their epigenetic alterations remains limited. In this study, we investigated the effects of exposure to 2-ethylhexyl diphenyl phosphate (EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP) on DNA methylation patterns during zebrafish embryonic development. We assessed general toxicity and morphological changes, measured global DNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase (DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven translocation (TET) methylcytosine dioxygenase genes. Additionally, we analyzed genome-wide methylation patterns in zebrafish larvae using reduced-representation bisulfite sequencing. Our morphological assessment revealed no general toxicity, but a statistically significant yet subtle decrease in body length following exposure to TMPP and EHDPP, along with a reduction in head height after TPHP exposure, was observed. Eye diameter and head width were unaffected by any of the OPFRs. There were no significant changes in global DNA methylation levels in any exposure group, and TMPP showed no clear effect on DNMT expression. However, EHDPP significantly decreased only DNMT1 expression, while TPHP exposure reduced the expression of several DNMT orthologues and TETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation. Differential methylation occurred primarily in introns (43%) and intergenic regions (37%), with 9% and 10% occurring in exons and promoter regions, respectively. Pathway enrichment analysis of differentially methylated region-associated genes indicated that TPHP exposure enhanced several biological and molecular functions corresponding to metabolism and neurological development. KEGG enrichment analysis further revealed TPHP-mediated potential effects on several signaling pathways including TGFβ, cytokine, and insulin signaling. This study identifies specific changes in DNA methylation in zebrafish larvae after TPHP exposure and brings novel insights into the epigenetic mode of action of TPHP.

The ever-increasing use of chemicals and the rising incidence of adverse reproductive effects in the modern environment have become an emerging concern. Several studies have shown that environmental contaminants, such as organophosphate flame retardants (OPFRs), negatively impact reproductive health. To evaluate the potential endocrine-related adverse reproductive effects of widely used and priority-listed compound 2-Ethylhexyl diphenyl phosphate (EHDPP), we characterized its effects on adrenal steroidogenesis in human adrenocortical (H295R) cells. The cells were exposed to EHDPP (1 and 5 μM) for 48 h, and the production of hormones, including progesterone, androstenedione, testosterone, estradiol, cortisol, and aldosterone, was measured. In addition, LC-MS/MS-based lipidomics analysis was done to quantify intracellular lipid profiles, and transcriptional assays were performed to examine the expression of genes related to corticosteroidogenesis, lipid metabolism, and mitochondrial dynamics. Our findings indicate that EHDPP disrupts hormone regulation in vitro, as evidenced by increased estradiol, cortisol, and aldosterone secretion. The expression of key corticosteroidogenic genes (CYP11B2, CYP21A1, 3β-HSD2, and 17β-HSD1) was upregulated significantly upon EHDPP exposure. Intracellular lipidomics revealed EHDPP-mediated disruption, including reduced total cholesterol ester, sphingolipids, and increased phospholipids, triglyceride species, and saturated-monounsaturated lipids subspecies. These alterations were accompanied by decreased ACAT2 and SCD1 gene expression. Moreover, a shift in mitochondrial dynamics was indicated by increased MF1 expression and decreased FIS1 expression. These data suggest that EHDPP disrupts adrenal steroidogenesis and lipid homeostasis, emphasizing its potential endocrine-disrupting effects.