The development of reliable and cost-efficient methods to assess the toxicity of nanomaterials (NMs) is critical for the proper identification of their impact on human health and for ensuring a safe progress of nanotechnology. In this study, we investigated the reliability and applicability of label-free impedance flow cytometry (IFC) for in vitro nanotoxicity screening, which avoids time-consuming labelling steps and minimizes possible NM-induced interferences. U937 human lymphoma cells were exposed for 24 h to eight different nanomaterials at five concentrations (2, 10, 20, 50, and 100 μg/mL). The NMs’ effect on viability was measured using IFC and the results were compared to those obtained by trypan blue (TB) dye exclusion and conventional flow cytometry (FC). To discriminate viable from necrotic cells, the IFC measurement settings regarding signal trigger level and frequency, as well as the buffer composition, were optimised. A clear discrimination between viable and necrotic cells was obtained at 6 MHz in a sucrose-based measurement buffer. Nanomaterial-induced interferences were not detected for IFC. The IFC and TB assay results were in accordance for all NMs. The IFC was found to be robust, reliable and less prone to interferences due to the advantage of being label-free.

To adjust cell growth and proliferation to changing environmental conditions or developmental requirements, cells have evolved a remarkable network of signaling cascades that integrates cues from cellular metabolism, growth factor availability and a large variety of stresses. In these networks, cellular information flow is mostly mediated by posttranslational modifications, most notably phosphorylation, or signaling molecules such as GTPases. Yet, a large body of evidence also implicates cytosolic pH (pHc) as a highly conserved cellular signal driving cell growth and proliferation, suggesting that pH-dependent protonation of specific proteins also regulates cellular signaling. In mammalian cells, pHc is regulated by growth factor derived signals and responds to metabolic cues in response to glucose stimulation. Importantly, high pHc has also been identified as a hall mark of cancer, but mechanisms of pH regulation in cancer are only poorly understood. Here, we discuss potential mechanisms of pH regulation with emphasis on metabolic signals regulating pHc by Na + /H + -exchangers. We hypothesize that elevated NHE activity and pHc in cancer are a direct consequence of the metabolic adaptations in tumor cells including enhanced aerobic glycolysis, generally referred to as the Warburg effect. This hypothesis not only provides an explanation for the growth advantage conferred by a switch to aerobic glycolysis beyond providing precursors for accumulation of biomass, but also suggests that treatments targeting pH regulation as a potential anti-cancer therapy may effectively target the result of altered tumor cell metabolism.

BackgroundOral cavity is a doorway for a variety of products containing titanium dioxide (TiO2) nanoparticles (NPs) (nano‐TiO2) such as food additives, oral healthcare products and dental materials. Their potential to penetrate and affect normal human oral mucosa is not yet determined.ObjectivesTo evaluate the ability of nano‐TiO2 to penetrate the in vitro reconstructed normal human buccal mucosa (RNHBM).Methods RNHBM was generated from primary normal human oral keratinocytes and fibroblasts isolated from buccal oral mucosa of healthy patients (n = 6). The reconstructed tissues were exposed after 10 days to clinically relevant concentrations of spherical or spindle rutile nano‐TiO2 in suspension for short (20 min) and longer time (24 h). Ultrahigh‐resolution imaging (URI) microscopy (CytoViva™, Auburn, AL, USA) was used to assess the depth of penetration into reconstructed tissues.ResultsUltrahigh‐resolution imaging microscopy demonstrated the presence of nano‐TiO2 mostly in the epithelium of RNHBM at both 20 min and 24‐h exposure, and this was shape and doze dependent at 24 h of exposure. The depth of penetration diminished in time at higher concentrations. The exposed epithelium showed increased desquamation but preserved thickness.ConclusionNano‐TiO2 is able to penetrate RNHBM and to activate its barrier function in a doze‐ and time‐dependent manner.

Enhanced cell growth and proliferation are accompanied by profound changes in cellular metabolism. Originally identified as the Warburg effect in cancer, such metabolic changes are also common under physiological conditions and include increased fermentation and elevated cytosolic pH (pHc) 1,2 . However, how these changes contribute to enhanced cell growth and proliferation is unclear. Here, we demonstrate that elevated pHc specifically orchestrates an E2F-dependent transcriptional program to drive cell proliferation by promoting Cyclin D1 expression. pHc-dependent transcription of Cyclin D1 requires the transcription factors CREB1/ATF1 and ETS1 and the Histone Acetyltransferases p300/CBP. Interestingly, biochemical characterization revealed that the CREB1-p300/CBP interaction acts as a pH-sensor and coincidence detector linking different mitotic signals to Cyclin D1 transcription. We also show that elevated pHc contributes to increased Cyclin D1 expression in Malignant Pleural Mesotheliomas (MPMs) and renders them hypersensitive to pharmacological reduction of pHc. Taken together, these data demonstrate that elevated pHc is a critical cellular signal regulating G1 progression and provide a mechanism linking elevated pHc to oncogenic activation of Cyclin D1 in MPMs and possibly other Cyclin D1-dependent tumors. Thus, an increase of pHc may represent a functionally important, early event in the etiology of cancer amenable to therapeutic intervention..