Tris(1,3-dichloro-2-propyl) phosphate disrupts cellular metabolism within human embryonic kidney (HEK293) cells

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TL;DR

This study found that low concentrations (<1 μM) of the flame retardant TDCIPP unexpectedly increased cell viability and glycolysis in HEK293 cells, rather than causing toxicity. The effect was linked to a Warburg-like metabolic shift, decreased reactive oxygen species, and increased carnosine levels. These findings suggest TDCIPP may disrupt cellular metabolism in human embryonic cells at environmentally relevant concentrations.

Full summary

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**Background:** Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is an additive organophosphate flame retardant widely used in polyurethane foam and other consumer products. It is detected in human serum, urine, breast milk, and placental tissues, indicating ubiquitous exposure. TDCIPP is a suspected carcinogen (California Prop 65) and has been linked to adverse pregnancy outcomes. Prior studies in human cell lines from pediatric and adult patients reported TDCIPP-induced oxidative stress, apoptosis, and cytotoxicity at concentrations ranging from 1–300 μM, depending on exposure duration. However, no studies had investigated TDCIPP effects in human embryonic cells. The objective of this study was to assess how TDCIPP exposure affects human embryonic kidney (HEK293) cell physiology, including cell viability, ATP production, reactive oxygen species (ROS) generation, and markers of DNA methylation, cell membranes, and mitochondria.

**Methods:** HEK293 cells were exposed to TDCIPP (0.015–31.25 μM) or vehicle (0.1% DMSO) for 24 or 48 hours. Cell viability was measured using the CellTiter-Glo (CTG) luminescence assay, which quantifies ATP. To explore mechanisms, cells were pre-treated with oligomycin (ATP synthase inhibitor) or compound 3K (pyruvate kinase M2 inhibitor) at 0.1 μM (maximum tolerated concentration) for 12 hours before TDCIPP exposure. Real-time ATP production rates (glycolysis vs. mitochondrial respiration) were measured using a Seahorse XFp Analyzer (ATP rate assay and glycolytic rate assay). ROS levels were assessed with CellROX Green fluorescence. Targeted metabolomics of central carbon metabolism was performed using UPLC-MS/MS, focusing on polar primary metabolites. Additionally, 5-methylcytosine (5-mC) immunocytochemistry, cell membrane labeling (WGA), and MitoTracker Orange staining were evaluated in situ. Statistical analyses used GLM ANOVA with Tukey post-hoc tests (α=0.05).

**Key Results:** - TDCIPP significantly increased cell viability at concentrations <1 μM after both 24 and 48 hours, with a peak effect at 0.98 μM. Higher concentrations (>1 μM) did not affect viability. Pre-treatment with oligomycin or compound 3K did not block this increase, suggesting the effect was not mediated by direct activation of ATP synthase or PKM2. - Seahorse assays showed no significant change in mitochondrial respiration (OCR) but a significant increase in extracellular acidification rate (ECAR) and proton efflux rate (PER) after rotenone/antimycin A injection, indicating enhanced glycolysis. The effect was most pronounced at 0.245 μM TDCIPP. - ROS levels were unchanged at 24 hours but significantly decreased at 48 hours from 0.245 to 15.63 μM TDCIPP, with a concentration-dependent reduction. - Metabolomics revealed significant alterations in central carbon metabolism at 0.245 μM TDCIPP, with carnosine (a dipeptide in the histidine metabolism pathway) being the most significantly affected metabolite. Carnosine levels increased approximately 2-fold relative to vehicle. - No significant changes were observed in 5-mC abundance, cell membrane integrity, or mitochondrial number at any TDCIPP concentration tested.

**Clinical Implications:** This study reveals a novel mechanism of TDCIPP toxicity at low, environmentally relevant concentrations (<1 μM) in human embryonic kidney cells. Contrary to prior studies in adult/pediatric cell lines, TDCIPP did not induce cytotoxicity but instead promoted cell viability and glycolysis, consistent with a Warburg-like metabolic shift. The decrease in ROS and increase in carnosine suggest adaptive antioxidant responses. These findings raise concerns about potential developmental effects of TDCIPP exposure, as metabolic disruption in embryonic cells could impact cell proliferation and differentiation. The study highlights the need for further research in human embryonic stem cells and in vivo models to assess the relevance of these metabolic changes for developmental toxicity and carcinogenicity.