Multiparametric In Vitro Toxicity Assessment of Consumer-relevant Nanomaterials in a Human Placental Model

The increasing number of consumer products containing nanomaterials (NM) has raised serious concerns regarding their environmental and human safety. Among the categories of NM of large market volume are metal nanoparticles (M-NP), inorganic non-metallic and carbon-based NM [1]. So far, little is known about the impact of NM exposure on placental development and function. Thus, our study aimed at evaluating the in vitro toxicity of NM of consumer relevance in human trophoblastic BeWo clone b30 epithelial cells, a widely used human placental model. BeWo b30 cells were exposed for 24 h to varied concentrations (0.8-48 µg/cm2) of NM of different chemical composition [Au, Ag, TiO2, SiO2 and graphene oxide (nano_GO)], primary size (10, 30 and 60 nm Au- and AgNP), capping (citrate- and PEGylated AuNP) and crystal structure (TiO2 NP rutile and anatase forms). In vitro cytotoxicity was assessed by determining changes in cell morphology (optical microscopy), metabolic activity (AlamarBlue assay), plasma membrane integrity (LDH release) and intracellular reactive oxygen species (ROS; DCFH-DA assay) and ATP levels (luminescent assay). In vitro genotoxicity (DNA strand breaks and oxidative damage) was assessed by the comet assay. No evident changes in cell morphology were observed after exposure to any tested NM. Overall, NM can be ranked for cytotoxicity as AgNP > nano_GO > AuNP ~TiO2 NP ~ SiO2 NP, being the effects more visible at higher concentrations. Regarding M-NP, the influence of the size in the cytotoxic-induced effects was more evident for AgNP than for AuNP, with the smaller NP causing more cytotoxicity in BeWo b30 cells. PEG capping was an effective protection, preventing the cytotoxic effects that were visible in cells exposed to the tested citrate-capped AuNP. No significant differences between rutile-anatase and anatase TiO2 NP-induced cytotoxicity were observed. Exposure to AgNP and nano_GO significantly increased ROS levels of the exposed cells suggesting that oxidative stress is a possible mechanism underlying their cytotoxicity in BeWo b30 cells. All tested NM significantly increased intracellular ATP levels compared to control cells, except for 10 nm AuNP. No significant changes in both DNA strand-breaks levels and DNA oxidative damage were detected for all tested NM. Our findings highlight the potential hazard associated with human placental exposure to NM, where the physicochemical properties are important determinants of their toxicity. Additional research is needed for a deeper understanding of NM impact on human placental barrier, to unravel their mechanisms of action and the properties responsible for NM-mediated toxicity, and thus support regulatory decisions that protect consumers and ultimately assist in the development of safer NM.Study carried out under the project NanoBioBarriers: Unravelling the mechanisms underlying the effects of metal-based nanoparticles on biological barriers - a contribution towards the implementation of the safe-by-design concept (PTDC/MED-TOX/31162/2017)N/