Influences of repeated exposure to low levels of TiO₂ nanoparticles on Kruppel‐like factors in 3D Caco‐2 spheroids and mouse intestines

Abstract

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 TiO2 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 TiO2 NPs did not induce cytotoxicity in vitro, repeated exposure (three times within 7 days) to 15.7 ng/ml TiO2 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 TiO2 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 TiO2 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 TiO2 NPs altered KLF‐signaling pathways in intestinal cells both in vitro and in vivo.

Exposure to MoS2 nanosheets or bulk activated Kruppel‐like factor 4 in 3D Caco‐2 spheroids in vitro and mouse intestines in vivo

“…For example, we recently found that titanate nanofibers activated KLF6‐ATF3‐CCL2 signaling pathway, but RNAi revealed that KLF6 only directly regulated ATF3 in endothelial cells 68 . Nevertheless, the results from this study indicated that MoS 2 materials could activate KLF4‐signaling pathways both in vitro and in vivo, consistent with our recent observations in TiO 2 nanoparticle‐exposed intestines 25 …”

“…68 Nevertheless, the results from this study indicated that MoS 2 materials could activate KLF4-signaling pathways both in vitro and in vivo, consistent with our recent observations in TiO 2 nanoparticle-exposed intestines. 25 In summary, the results from this study showed that MoS 2 NSs or bulk reduced bodyweights of mice and increased organ coefficients, indicating the in vivo toxicity of these materials. The oral toxicity of MoS 2 NSs and bulk could be explained by that these materials T A B L E 1 The levels of trace elements in mouse intestines.…”

“…22 However, although it has been suggested that KLFs play a pivotal role in the regulation of proper functions of intestines, 23,24 the influences of NMs on KLFs and KLF-mediated downstream events in intestines have gained little attention. Recently, we reported that TiO 2 nanoparticles affected KLF4-signaling pathways in 3D Caco-2 spheroids and mouse intestines, 25 but more studies are still needed to investigate the possible influences of NMs on KLFs in intestines.…”

Mechanisms of Nanoparticle Transport across Intestinal Tissue: An Oral Delivery Perspective

“…Several groups have developed 3D Caco-2 spheroids by seeding Caco-2 cells in ultralow attachment tissue culture materials. 82 Newer developments in 3D gut studies have emerged by implementing intestinal tissues from patients because they more closely mimic the human intestinal epithelium. 3D gut organoids are particularly advantageous and of interest because they encompass all intestinal cell types.…”

“…Intestinal epithelial cells can also be grown into a 3D, spheroid conformation to represent the morphological geometry of the gut. Several groups have developed 3D Caco-2 spheroids by seeding Caco-2 cells in ultralow attachment tissue culture materials . Newer developments in 3D gut studies have emerged by implementing intestinal tissues from patients because they more closely mimic the human intestinal epithelium.…”