Influences of repeated exposure to low levels of TiO nanoparticles on Kruppel-like factors in 3D Caco-2 spheroids and mouse intestines.

Kruppel-like factors (KLFs) are a set of transcription factors (TFs) involved in the regulation of many basic biological processes, and recent studies suggested that nanoparticles (NPs) were capable to change KLFs in different models even at non-cytotoxic concentrations. In this study, we repeatedly exposed 3D Caco-2 spheroids and mice to TiO NPs, one of the most frequently used metal oxide NPs, and investigated the changes of KLF-signaling pathways based on RNA-sequencing. Although the internalization of TiO NPs did not induce cytotoxicity in vitro, repeated exposure (three times within 7 days) to 15.7 ng/ml TiO NPs increased KLF4 but decreased KLF6. Consistently, KLF4/KLF6-regulated gene ontology terms were altered, including those involved in the regulation of gene expression. We further verified that repeated exposure to 15.7 ng/ml TiO NPs increased the expression of KLF4 and proto-oncogene, bHLH transcription factor (MYC), but decreased the expression of KLF6 and activating transcription factor 3 (ATF3). But with the increase of NP concentrations, the expression of these genes was decreased. In mice following intragastrical exposure to 4.39 and 43.9 mg/kg TiO NPs (once a day for 5 continuous days), we observed increased expression of klf4, klf6, myc, and atf3, along with morphological changes of intestines. We concluded that repeated exposure to low levels of TiO NPs altered KLF-signaling pathways in intestinal cells both in vitro and in vivo.