Fig. 5.

Analysis of cell viability upon treatments with mixtures using CellTiter-Blue assay. (a) hiPSC-derived NSCs were differentiated for 7 DIV and treated with individual chemicals or three different types of mixtures for either 3 days (b-d) or 14 days (e-g) with different LOAECs specific for each DNT endpoint (i.e., synaptogenesis (b and e), neurite outgrowth (c and f), and BDNF levels (d and g)). After 3 days (b-d) or 14 days (e-g), resazurin test (with CellTiter Blue) was performed. All samples were normalised to control medium with solvent (0.1% DMSO, Ctr) at the respective time point. LOAECs (red curves) and their serial dilutions (respectively black (LOAEC/2), blue (LOAEC/4) and light blue (LOAEC/8) curves) were tested to assess whether mixed chemicals elicited cytotoxic effects. Mixture labelled as ‘3-Sim’ contained similar MoA chemicals (affecting BDNF levels i.e., BPA, CPF and Lead), whilst mixture with dissimilar MoA chemicals (i.e., Methyl-Hg, PCB138 and VA) is labelled as ‘3-Diss’. The ‘All’ mixture comprised all 6 chemicals together. Data are represented as mean ± S.E.M. of 3–4 biological replicates