Multi-walled carbon nanotubes decrease neuronal NO synthase in 3D brain organoids

Abstract

Multi-walled carbon nanotubes (MWCNTs) might induce the dysfunction of neuronal NO synthase (nNOS) and impair the function of brains. But to the best of our knowledge, this conclusion was made by using laboratory animals or conventional nerve cell cultures; however, these models might not reflect the complex conditions of human brains. Recently, the development of 3D brain organoids (also known as organotypic cultures) derived from human induced pluripotent stem cells (iPSCs) provides a platform to investigate the behaviors of human brains in vitro. In this study, we investigated the toxicity of MWCNTs to 3D brain organoids which expressed the cortical layer markers. It was shown that MWCNTs induced cytotoxicity to 3D brain organoids but not in dose-dependent manner. Exposure to high level of MWCNTs (64 μg/mL) reduced the levels of intracellular NO but increased superoxide. As the mechanism, 64 μg/mL MWCNTs significantly reduced the protein level of nNOS. The nNOS regulators nuclear factor kappa-B (NF-κB) proteins were significantly induced by MWCNTs, whereas Kruppel-like factor 4 (KLF4) proteins were reduced particularly after exposure to low level of MWCNTs (16 μg/mL). The results from fluorescence micro-optical sectioning tomography (MOST) confirmed the decrease of nNOS proteins, not only at the out-layers that directly contacted MWCNTs, but also at the inner-layers. Combined, our results suggested that MWCNTs could decrease nNOS activity by inducing oxidative stress and modulating NF-κB-KLF4 pathway. This study also showed the potential of 3D brain organoids in mechanism-based toxicology studies.