Model Complexity as Determining Factor for In Vitro Nanosafety Studies: Effects of Silver and Titanium Dioxide Nanomaterials in Intestinal Models

Abstract

With the rising interest in the effects of orally ingested engineered nanomaterials (ENMs), much effort is undertaken to develop and advance intestinal in vitro models. The cytotoxic, proinflammatory, and DNA damaging properties of polyvinylpyrrolidone‐capped silver (Ag‐PVP) and titanium dioxide (TiO2, P25) ENM in four in vitro models of increasing complexity—from proliferating Caco‐2 and HT29‐MTX‐E12 monocultures to long‐term transwell triple cultures including THP‐1 macrophages to reproduce the human intestine in healthy versus inflamed‐like state—are studied. Results are compared against in vivo effects of the same ENM through intestinal tissue analysis from 28‐day oral exposure studies in mice. Adverse responses are only observed in monocultures and suggest toxic potential for both ENM, typically showing stronger effects for Ag‐PVP than for TiO2. By contrast, no adverse effects are observed in either the transwell cultures or the analyzed murine tissues. The data provide further support that monoculture models represent a cost and time efficient tool for early‐phase hazard assessment. However, the observed similarities in morphology and ENM effects in murine intestinal tissue and the in vitro triple culture model suggest that advanced multifacetted research questions concerning oral ENM exposure are more adequately addressed by the more complex and time intensive models.

Grouping Hypotheses and an Integrated Approach to Testing and Assessment of Nanomaterials Following Oral Ingestion

“…The DNs and the relative TTS applied to the oral hazard endpoints should be addressed using appropriate doses and exposure regimes to reflect a realistic human exposure scenario, when possible. For instance, when direct oral exposure is expected by food grade NFs, doses mimicking daily intake values are preferred, in association with repeated exposure to simulate long-term toxicological outcomes (e.g., up to five days for the treatment of intestinal cells [88] as suggested by the PATROLS EU project). The use of physiologically relevant in vitro models to better resemble the characteristics of in vivo intestinal tissues or liver/kidney tissues (site of NF accumulation) is preferred.…”

Ingested Engineered Nanomaterials Affect the Expression of Mucin Genes—An In Vitro-In Vivo Comparison

“…The delivered ENM doses in E12 cell monoculture experiments, performed in 6-well plates, were estimated using the In Vitro Diffusion and Dosimetry model (ISDD) [51]. The input parameters described in Kämpfer et al [52] were applied and, where necessary, adapted to the 6-well format and complemented for CeO 2 and SiO 2 . The effective agglomerate diameters of CeO 2 and SiO 2 were measured as described in Kämpfer et al [52].…”

“…The input parameters described in Kämpfer et al [52] were applied and, where necessary, adapted to the 6-well format and complemented for CeO 2 and SiO 2 . The effective agglomerate diameters of CeO 2 and SiO 2 were measured as described in Kämpfer et al [52]. Briefly, ENM were suspended in exposure medium to 150 µg/mL and the Z-average and polydispersity index (PDI) were measured using a Zetasizer NanoZS (Malvern Pananalytics, Malvern, UK).…”

“…The cell numbers and volumes of medium were adjusted to the size of the well surfaces so that equal exposure concentrations in µg/mL and µg/cm 2 were obtained for both plate formats (Table 1). The triple cultures in stable/healthy and inflamed state were prepared as described by Kämpfer et al [52]. Briefly, 1.8*10 5 Caco-2 and E12 cells per cm 2 were seeded on 12-well transwell inserts (Falcon, 353103) at a ratio of 9:1 and cultivated for 21 days.…”

Stem Cells for Next Level Toxicity Testing in the 21st Century

“…Whereas strong agreement exists on the suitability of transwell cultures over undifferentiated monocultures, studies failed to demonstrate a clear advantage of more complex models. [ 416,417 ]…”