Potential developmental neurotoxicity of short-chain di-n-alkyl phthalates and their mixtures to neurospheres derived from human neural progenitor cell lines

Short-chain phthalates (SCPs), are commonly found in personal care products (PCPs) as solvents, leading to complex human exposures. PCPs are primarily utilized by women, with heightened usage during pregnancy. While SCPs absorbed through the skin can traverse the placenta, their potential for developmental neurotoxicity (DNT) remains inadequately investigated. This study evaluated the impacts of single and combined SCP exposures to assess proliferation, migration, differentiation, and neurite outgrowth in human neural progenitor cell lines. Three individual SCPs—diethyl phthalate (DEP, 46.9% ± 7.55%), dipropyl phthalate (DPrP, 64.2% ± 7.16%), and dipentyl phthalate (DPP, 31.9% ± 9.10%)—significantly reduced only neurite outgrowth at 100uM100 μM compared to the vehicle control. The equimolar-mixture of these SCPs (Mix) also showed a similar reduction (57.5% ± 2.46%). In RNA-seq analysis, DEP, DPrP, DPP, and Mix treatments identified neurite outgrowth inhibition related 720 common DEGs showing the functional annotations most strongly enrichimentenrichment associated with oxidative phosphorylation. Additionally, individual SCPs and Mix significantly reduced mitochondrial ATP production and were able to bind to mitochondrial respiratory complexes I, III, IV, and V, as confirmed by docking analysis. Treatment with exogenous ATP and vitamin B complex could restore the inhibition of neurite outgrowth caused by SCP exposure. In conclusion, this study identified the possibility that SCPs induce DNT by interfering with mitochondrial energy metabolism, with similar effects observed even in mixtures with low individual concentrations. Future research is needed to evaluate the cumulative effects of SCPs. This study aimed to investigate the potential developmental neurotoxicity (DNT) of SCPs exposure in vitro. Among the various SCPs, we focused on DEP, which is commonly used in PCPs, and di-n-alkyl phthalate, a linear phthalate similar to DEP but differing only in the number of carbon atoms. In this study, we investigated the DNT of chemicals using a neurosphere Assay. This assay relies on human neural stem cells (hNPCs) and is an important part of an in vitro test battery recommended by the OECD to assess DNT hazards. The neurosphere assay helps assess potential hazards of chemicals to developing brains by evaluating key events (KE) in neural development, such as NPC proliferation (NPC1), NPC migration (NPC2), neuronal differentiation (NPC3), neurite outgrowth (NPC4), and oligodendrocyte differentiation (NPC5). Here, we replaced the original use of primary hNPCs with an ReNcell VM, an NPC line. This change was made to improve the ease of obtaining cells, ensure consistent results between experiments, and make the testing process more manageable. Given that humans are exposed to multiple phthalates simultaneously, individual SCPs and composite SCPs were assessed to understand the mechanisms involved in SCP-induced DNT.