BPA and its analogues are facing increasingly stringent regulations restricting their use due to the increasing knowledge of their harmful effects. It is therefore expected that novel BPA analogues and alternatives will replace them in plastic products, cans and thermal paper to circumvent restrictions imposed by legislation. This raises concerns about the safety of "BPA-free" products, as they contain BPA substitutes whose safety has not been sufficiently assessed prior to their market introduction. The regulatory agencies have recognised BPAP, BPBP, BPC2, BPE, BPFL, BPG, BPP, BPPH, BPS-MAE, BPS-MPE, BP-TMC, BPZ and the alternatives BTUM, D-90, UU and PF201 as compound with insufficient data regarding their safety. We demonstrate that the mentioned compounds are present in consumer products, food and the environment, thus exhibiting toxicological risk not only to humans, but also to other species where their toxic effects have already been described. Results of in silico, in vitro and in vivo studies examining the endocrine disruption and other effects of BPA analogues show that they disrupt the endocrine system by targeting various nuclear receptors, impairing reproductive function and causing toxic effects such as hepatotoxicity, altered behaviour and impaired reproductive function. In vitro and in vivo data on BPA alternatives are literally non-existent, although these compounds are already present in commonly used thermal papers. However, in silico studies predicted that they might cause adverse effects as well. The aim of this article is to comprehensively collate the information on selected BPA substitutes to illustrate their potential toxicity and identify safety gaps.

RT-qPCR is the gold standard and the most commonly used method for measuring gene expression. Selection of appropriate reference gene(s) for normalization is a crucial part of RT-qPCR experimental design, which allows accurate quantification and reliability of the results. Because there is no universal reference gene and even commonly used housekeeping genes’ expression can vary under certain conditions, careful selection of an appropriate internal control must be performed for each cell type or tissue and experimental design. The aim of this study was to identify the most stable reference genes during osteogenic differentiation of the human osteosarcoma cell lines MG-63, HOS, and SaOS-2 using the geNorm, NormFinder, and BestKeeper statistical algorithms. Our results show that TBP, PPIA, YWHAZ, and EF1A1 are the most stably expressed genes, while ACTB, and 18S rRNA expressions are most variable. These data provide a basis for future RT-qPCR normalizations when studying gene expression during osteogenic differentiation, for example, in studies of osteoporosis and other bone diseases.

This study investigates the immunomodulatory effects of BPA substitutes using in silico and in vitro approaches. A comprehensive in silico screening with Endocrine Disruptome of BPA and its 25 substitutes revealed that BPG, BPS-MAE, BPS-MPE, BPPH and PF201 have the highest potential to bind nuclear receptors. Based on the in silico ranking, 12 compounds were selected and tested in vitro to investigate their effects on THP-1 derived macrophages, Jurkat T cells and LCLs. The metabolic activity tests showed that BPA has IC₅₀ values of 99-182 µM, while the values for BPG, BPPH and BPP are 5-10 times lower. Comparison of the IC₅₀ values with the logP of the tested compounds showed that the BPA analogues affect cell viability in proportion to their lipophilicity (R² = 0.9185). The results of the cytokine release assays showed that the BPA substitutes generally stimulated the proinflammatory response in THP-1 macrophages in a statistically significant manner when exposed to the environmentally relevant concentrations and suppressed it in the micromolar range in all cell lines tested. Further analysis using RamosBlue reporter cells indicated that BPAP, BPG, BPP and BPPH alter the NF-κB/AP-1 signalling pathway. Moreover, BPAP, BPG, BPP, BPPH, BPS-MAE, BPS-MPE, BTUM and PF201 were predicted in silico to be TLR4-MyD88 inhibitors. The overall results of this study identify BPG, BPP, BPPH, BPZ and TCBPA as BPA analogues with the highest immunomodulatory potential. In addition, the immunomodulatory potential of the BPA alternatives Pergafast201 and BTUM was confirmed in vitro for the first time. This study emphasises the need for cautious evaluation of BPA substitutes due to their potential immunomodulatory effect, which could significantly affect public health.

The strong appeal to reduce animal testing calls for the development and validation of in vitro, in chemico and in silico models that would replace the need for in vivo testing and ex vivo materials. A category that requires such new approach methods is the assessment of immunosuppression that can be induced by chemicals including environmental pollutants. To assess the immunosuppressive action on monocytes and lymphocytes, we mimicked the whole-blood cytokine-release assay by preparing an in vitro coculture of THP-1 and Jurkat cell lines. We optimised its activation and investigated the effects of known immunosuppressive drugs with different mechanisms of action on the release of proinflammatory cytokines. Decreased secretion of IL-8 was achieved by several immunosuppressive mechanisms and was therefore selected as an appropriate marker of immunosuppression. A set of environmentally occurring bisphenols, BPA, BPAP, BPP, BPZ, BPE, TCBPA and BPS-MAE, were then applied to the model and BPP and BPZ were found to act as potent immunosuppressants at micromolar concentrations.

This study investigated whether the Endocrine Disruptome and VirtualToxLab in silico platforms are suitable for predicting the endocrine disrupting effects of per- and polyfluoroalkyl substances (PFASs)—in particular, for interactions with oestrogen receptors (ERs) and androgen receptor (AR). Compounds included in the U.S. Environmental Protection Agency’s PFAS working list were analysed with both models, and the results were compared with the available in vitro data regarding their modulation of nuclear receptors. Based on the identified prediction parameters, such as sensitivity, specificity, accuracy, and Mathews’ correlation coefficient, VirtualToxLab was found to be a reliable model for predicting the reactivity of PFASs with AR, while a positive consensus approach of both platforms provided reliable predictions of the PFAS reactivity with ERα and ERβ. This study provides the evidence that Endocrine Disruptome and VirtualToxLab can be used as a tier 1 screening tool for assessment of the endocrine disrupting effect of PFASs. Furthermore, it demonstrates that the likelihood of endocrine disrupting properties increases with the lipophilicity of PFASs and identifies the understudied PFHpS, PFNS, PFDS, 9-Cl, NMeFOSAA, NEtFOSAA, 4:2 FTS, 6:2 FTS, 8:2 FTS, 6:2 monoPAP, 8:2 monoPAP, and 5:3 acid as potential ligands of AR and/or ERs.