An in vitro investigation of biochemical and molecular markers of nanomaterial toxicity to the intestine

Abstract

Application of nanomaterials (NMs) cuts across all facets of life, including agriculture, food, medicine, cosmetics, textiles and electronics, and can lead to ingestion by humans. Although researchers have investigated the toxicity of NMs, there is still a paucity of studies which have assessed NM toxicity to the gastrointestinal (GI) tract. The Caco-2 cell line is most commonly used to assess impacts of NMs on the GI tract, but in vitro coculture models that are more complex can be used to better mimic intestinal physiology. Therefore, the aim of this research was to identify the most appropriate in vitro model, and biochemical and molecular markers for assessing the toxicity of NMs to the intestine in vitro. Undifferentiated Caco-2 cells, differentiated Caco-2 cells, Caco-2/HT29-MTX (mucus secreting intestinal model) and Caco-2/Raji B (M cell) co-cultures were used for this study. Copper oxide (CuO) NMs were investigated and copper sulphate (CuO4) included as an ionic control. CuO NMs are useful due to their antimicrobial properties for wood preservation, inks, cosmetics, textiles and food contact materials and their incorporation in these products may enhance oral exposure. CuO NMs and CuSO4, impaired the function of the intestinal barrier (as indicated by a reduction in transepithelial electrical resistance (TEER) and Zonular occludens (ZO-1) staining intensity), and shortened microvilli in all differentiated in vitro intestinal models tested. An increase in interleukin (IL)-8 secretion, upregulation of haem oxygenase (HMOX)1, IL8, metallothionein (MT) 1A and 2A and mucin (MUC) 2 expression were also observed following exposure of all models to CuO NMs and CuSO4. CuO NMs and CuSO4 increased reactive oxygen species (ROS) production in acellular conditions, but only CuSO4 induced ROS production in the cell models. The Caco-2/Raji B co-culture demonstrated the highest level of NM translocation. Silica NMs and montmorillonite nanoclays did not stimulate a toxic response. The toxicity of CuO NMs and CuSO4 was typically 2 fold greater in undifferentiated Caco-2 cells, suggesting that undifferentiated cells may overestimate the toxicity of NMs. Therefore, it is suggested that differentiated Caco-2 cells, Caco-2/HT29-MTX and Caco-2/Raji B co-cultures are powerful in vitro intestinal models and can be used to assess NM toxicity to the intestine. In addition, HMOX1, IL8, MT2A and MUC2 expression, IL-8 protein, TEER measurement, ZO-1 staining and light microscopy to assess cell morphology and viability are presented as biomarkers for investigating the intestinal toxicity of NMs across in vitro models of varied complexity.