Investigation of twenty metal, metal oxide, and metal sulfide nanoparticles' impact on differentiated Caco-2 monolayer integrity
Graphical abstract
Introduction
As the field of nanotechnology evolves, there is a need for comprehensive biological response profiles to guide the development of safe and sustainable use of nanomaterials. In the past two decades, the number of consumer products containing engineered nanomaterials (ENMs) has increased substantially, including those within the food and agriculture sectors (Bergin and Witzmann, 2013, Bouwmeester et al., 2014, EFSA, 2015). According to several consumer product databases and inventories, there are now hundreds of commercially available food and beverage products that contain ENMs (Nanotechnologies, 2015; Nanodatabase, 2019; CFS, 2017). It has been estimated that the daily number of ingested metal or metal oxide particles amounts to 1012, comprised mainly of titanium dioxide (TiO2) and silicon dioxide (SiO2) (Lomer et al., 2002). Food grade TiO2 is a food additive used as a whitening agent that has been found in food at concentrations between 0.02 and 9.0 mg TiO2/g product (Peters et al., 2014), and the estimated daily consumption of TiO2 ranges from 2 mg/kg body weight (bw) for children between age 2.5–4.5 years to 0.2 mg/kg bw for adults (Weir et al., 2012). That ingested particles cross the intestinal barrier has been demonstrated by the detection of TiO2 particles in 7 out of 15 adult (age 56–104) human liver samples (collected post mortem) (Heringa et al., 2018). While TiO2 and SiO2 nanoparticles (NP) are the most prevalent in food, a range of other ENMs are also used as e.g. food supplements, antimicrobial agents, and colorant (Table 1). The level and diversity of ingested ENMs are therefore likely to keep rising, and more knowledge is needed to estimate the interaction between ENMs and the intestinal tract.
The gastrointestinal tract is a selective barrier between the lumen and the body, and responsible for nutrient absorption as well as providing protection against harmful pathogens and xenobiotics. Disruption of the gastrointestinal tract health and homeostasis can lead to many chronic diseases. Increased intestinal permeability, called “Leaky Gut Syndrome”, happens when the intestinal tight junctions (TJs) do not work properly (as reviewed (Kiefer and Ali-Akbarian, 2004, Liu et al., 2005, Hollander and Kaunitz, 2019)). TJs connect the intestinal epithelial cells and control passive diffusion of ions and other small solutes paracellularly, maintaining any gradient created by the active transport pathways associated with the transcellular route (as reviewed (Mitic and Anderson, 1998, Anderson and Van Itallie, 2009)). Disruption of TJs therefore leads to decrease in protection against xenobiotics and pathogens present in the lumen. Chronic disruption of gastrointestinal integrity has been associated with a predisposition to developing autoimmune diseases, liver dysfunction, arthritic and other degenerative diseases (Meletis, 1998; Arrieta et al., 2006; Fukui, 2016; Konig et al., 2016).
In vitro models of the intestinal tract have been used to investigate ENM translocation across cell monolayers and cytotoxic effects (Zha et al., 2008; Esch et al., 2012; Fisichella et al., 2012; Loo et al., 2012; Piret et al., 2012; Gerloff et al., 2013; Gitrowski et al., 2014; Ude et al., 2017; Ye et al., 2017; Vila et al., 2018a; Vila et al., 2018b). A well-established model for the intestinal barrier is the epithelial colorectal adenocarcinoma Caco-2 cells. Fully differentiated Caco-2 cells resemble mature enterocytes, expressing an organized brush border with a dense network of functional TJs (Hilgendorf et al., 2000; Carr et al., 2012). Differentiated Caco-2 transwell (TW) cultures are the model of choice for uptake studies into the small intestine (Christensen et al., 2012), and are considered a good model for studying intestinal barrier function (Alvarez-Hernandez et al., 1991; Jarc et al., 2019). It has been established that Caco-2 cells need to grow on a semipermeable surface, such as a TW membrane, in order to fully differentiate into polarized enterocytes, exhibiting well-developed microvilli and polarized distribution of brush border enzymes and transporters (Hidalgo et al., 1989; Sambuy et al., 2005; Natoli et al., 2012). The use of undifferentiated cells is quicker and cheaper than using the differentiated Caco-2 model; however, differentiated cells more accurately mimic in vivo conditions and allow for studies of monolayer integrity.
There is an increasing incentive to reduce animal testing and develop more physiological relevant in vitro models. With the large number of diverse ENMs implemented in food and consumer products, where safety needs to be assessed, it is important to align nanosafety and toxicology studies to the 3Rs principles (Replacement, Reduction and Refinement of animal testing) (Burden et al., 2017). Initiatives in developing and validating 2-dimensional (2D) and 3-dimensional (3D) mono- and co-culture in vitro models have been expanding rapidly during the past decade and are showing great promise. However, dependent on the biological mechanism in focus, i.e. studying enterocyte integrity, transwell mono-culture may be the most suitable in vitro model.
For testing ENM transport across the intestinal tract in vitro co-culture models including Caco-2, human mucus producing goblet cells (HT29-MTX) (Lesuffleur et al., 1993; Walter et al., 1996) and/or human B-lymphocytes (Raji B), which convert a portion of the Caco-2 cells to resemble Peyer's patch follicle-associated epithelium (FAE) microfold (M) cells (Lo et al., 2004; Beloqui et al., 2017), have been utilized. These cell models have been used to evaluate the role of the mucus layer in ENM interaction with the intestinal tract in vitro (Loo et al., 2012; Walczak et al., 2015). The M-cells playing a central role in the intestinal translocation and transport of ENMs have been included in studies focusing on ingested ENM uptake (Loo et al., 2012, Walczak et al., 2015). It is also worth noting that these co-culture models have lower transepithelial electrical resistance (TEER) measurements than the Caco-2 mono-culture, indicating that both HT29-MTX and Raji B cells impact the TJs of the monolayer (Araujo and Sarmento, 2013). While this has been argued to more closely resemble the human intestinal tract than the Caco-2 mono-culture, it may pose a challenge when investigating ENM interaction with enterocyte integrity. Additionally, the Caco-2 monolayer model resembles the enterocyte in the small intestinal tract where the mucus layer is considerably thinner than that of the large intestinal tract, and M-cells are located in Peyer's patches at the base of the epithelium and not in the microvilli. It is therefore important to recognize that the intestinal tract has a considerable variation in its anatomy which must be taken into consideration when selecting an in vitro model. Furthermore, these complex cell models are often expensive, with low through-put and might not aid in answering the biological function or mechanism in question. There is therefore still a need for established and well-characterized in vitro models, including the human enterocyte monolayer model, in evaluating the potential health risk of ingesting ENMs, and investigating a fundamental question such as enterocyte health and integrity.
Another aspect of in vitro evaluation of ENM exposure is simulated gastrointestinal digestion, first presented as an in vitro digestion model for bioaccessibility of toxins (Versantvoort et al., 2005), and later adopted in nanotoxicity studies (Peters et al., 2012; Lichtenstein et al., 2015; Bove et al., 2017; DeLoid et al., 2017b). While in vitro digestion provides valuable insight in the potential physiochemical changes of ENMs, it also raises questions of what food matrix to use. As illustrated in the literature, the presence of a food matrix, and the type of food matrix, dramatically changes the outcome of the ENM in vitro digestion (Peters et al., 2012; Lichtenstein et al., 2015; Kästner et al., 2017; McClements et al., 2017). Assumptions of orogastric transition time, medication, and underlying medical conditions, are all important aspects to address and need to be systematically investigated in future research of ENM ingestion studies.
This study was conducted as part of National Institute of Environmental Health Sciences (NIEHS) Nanomaterials Health Implications Research (NHIR) Consortium and the investigated ENMs were provided by the NIEHS-NHIR Consortium. Herein we compare the impact of 20 metal, metal oxide, and metal sulfide ENMs, selected by NIEHS NHIR, on intestinal integrity in vitro using the well-established Caco-2 model of fully differentiated cells resembling small intestinal enterocytes. In this study we tested a dose of 50 μg/mL ENM. Determination of the most realistic dosage is challenging since no actual data of ENM concentration in the intestinal tract is available. We based our dose selection on literature of what ENM concentrations are present in food. E.g. food grade TiO2 has been found in food at a concentration between 0.02 and 9.0 mg TiO2/g product (Peters et al., 2014). Therefore, 50 μg/mL for TiO2 E171 may be substantially lower than the concentration in food. We do acknowledge that this dose might not be as realistic for other tested ENMs (e.g. V2O5 flakes, ZnS NP, and WO3 NP). However, since the aim of this study is to provide a comparison between 20 ENMs, we have decided to test the same concentration for all ENMs, at the same time modeling the effective dose. We investigated the cell layer integrity following in vitro exposure, using fluorescence labeled dextran, and cytotoxic response by lactate dehydrogenase (LDH) release and mitochondrial activity assays.
Section snippets
Engineered nanomaterial formulation and characterization
Twenty metal, metal oxide, and metal sulfide ENMs were selected by NIEHS NHIR. The ENMs were synthesized or procured and comprehensively characterized by the Consortium Engineered Nanomaterials Resource and Coordination Core (ERCC) as part of the NIEHS NHIR Consortium (Table 1). Eighteen of the ENMs were provided as powders (20 nm Ag NP, 3.8 w/w% Ag-SiO2 NP, 15.9 w/w% Ag-SiO2 NP, 30 nm Al2O3 NP, 60 nm CdS NP, 10 nm CeO2 NP, 30 nm CeO2 NP, 50 nm CuO NP, 10 nm Fe2O3 NP, 100 nm Fe2O3 NP, 20 nm MgO
ENM characterization and stability
The DSEcr (J/mL) needed to achieve the most monodisperse and stable ENM suspension possible varied considerably between the 20 metal, metal oxide, and metal sulfide ENMs, with 30 nm Al2O3 NP (407 J/mL), 15 nm SiO2 NP (795 J/mL), and 25 nm TiO2 P25 (908 J/mL) requiring the lowest amount of energy, and 15 nm WO3 NP (2130 J/mL), 60 nm CdS NP (3446 J/mL), and 100 nm V2O5 flakes (3636 J/mL) requiring the highest amount (Table 2). The criteria for ENM formulation stability was <30% change of the
Discussion
Considerable effort has been made in the field of nanotoxicology and nanotechnology to generate hazard rankings, safety and risk assessments in order to provide stakeholders and policy makers with robust risk governance tools to ensure safe and sustainable use of ENMs in food and consumer products. However, the large number of available ENMs, in vitro and in vivo models, biological assays, and endpoints often make it challenging to compare the biological interactions between different ENMs for
Conclusion
This study provides a comparison between 20 metal, metal oxide, and metal sulfide ENMs' impact on differentiated Caco-2 monolayer integrity. While the majority of the investigated ENMs did not cause cytotoxicity or impacted cell monolayer integrity, we did find that some ENMs adversely impact the monolayer integrity. As the differentiated Caco-2 cells resemble the enterocytes of the small intestinal tract, our findings suggest that ingestion of ENMs may results in perturbation of intestinal
CRediT authorship contribution statement
Ninell P. Mortensen:Methodology, Validation, Formal analysis, Visualization, Writing - original draft.Maria Moreno Caffaro:Investigation, Data curation.Purvi R. Patel:Investigation, Software.Md Jamal Uddin:Investigation.Shyam Aravamudhan:Validation.Susan J. Sumner:Conceptualization, Funding acquisition.Timothy R. Fennell:Supervision, Funding acquisition, Writing - review & editing.
Acknowledgement
Research reported in this publication was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number (NIH grant # U01ES027254) as part of the Nanotechnology Health Implications Research (NHIR) Consortium. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The ENMs used in the research presented in this publication have been
Declaration of competing interest
The authors have no conflicts of interest and nothing to disclose. The National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH) and the Nanotechnology Health Implications Research (NHIR) reviewed the article prior to submission, however, the content is solely the responsibility of the authors and does not represent the official view of the NIH or the NHIR Consortium.
References (80)
- et al.
Caco-2 cell line: a system for studying intestinal iron transport across epithelial cell monolayers
Biochim. Biophys. Acta
(1991) - et al.
Transport and metabolism of opioid peptides across BeWo cells, an in vitro model of the placental barrier
Int. J. Pharm.
(2002) - et al.
Towards the characterization of an in vitro triple co-culture intestine cell model for permeability studies
Int. J. Pharm.
(2013) - et al.
Development of reference metal and metal oxide engineered nanomaterials for nanotoxicology research using high throughput and precision flame spray synthesis approaches
NanoImpact.
(2018) - et al.
State of the safety assessment and current use of nanomaterials in food and food production
Trends Food Sci. Technol.
(2014) - et al.
The 3Rs as a framework to support a 21st century approach for nanosafety assessment
Nano Today
(2017) - et al.
In vitro assessment of CeO2 nanoparticles effects on intestinal microvilli morphology
Toxicol. in Vitro
(2019) - et al.
Silver nanoparticles in polymeric matrices for fresh food packaging
Journal of King Saud University - Science.
(2016) - et al.
Morphological aspects of interactions between microparticles and mammalian cells: intestinal uptake and onward movement
Prog. Histochem. Cytochem.
(2012) - et al.
Effective delivery of sonication energy to fast settling and agglomerating nanomaterial suspensions for cellular studies: implications for stability, particle kinetics, dosimetry and toxicity
NanoImpact.
(2018)
Uptake of different crystal structures of TiO(2) nanoparticles by Caco-2 intestinal cells
Toxicol. Lett.
Validation of an LDH assay for assessing nanoparticle toxicity
Toxicology.
Characterization of the human-colon carcinoma cell-line (Caco-2) as a model system for intestinal epithelial permeability
Gastroenterology.
Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport
J Pharm Sci.
Monitoring the fate of small silver nanoparticles during artificial digestion
Colloids Surf. A Physicochem. Eng. Asp.
Comparative study of nanoparticle-mediated transfection in different GI epithelium co-culture models
J. Control. Release
Physicochemical and colloidal aspects of food matrix effects on gastrointestinal fate of ingested inorganic nanoparticles
Adv. Colloid Interf. Sci.
Good Caco-2 cell culture practices
Toxicol. in Vitro
Comprehensive study on regional human intestinal permeability and prediction of fraction absorbed of drugs using the Ussing chamber technique
Eur. J. Pharm. Sci.
Evaluation of tumorigenic potential of CeO2 and Fe2O3 engineered nanoparticles by a human cell in vitro screening model
NanoImpact.
Mechanical, optical and antibacterial properties of polylactic acid/polyethylene glycol films reinforced with MgO nanoparticles
Materials Today: Proceedings.
Applicability of an in vitro digestion model in assessing the bioaccessibility of mycotoxins from food
Food Chem Toxicol.
Assessing the effects of silver nanoparticles on monolayers of differentiated Caco-2 cells, as a model of intestinal barrier
Food Chem Toxicol.
Effects of cerium oxide nanoparticles on differentiated/undifferentiated human intestinal Caco-2cells
Chem. Biol. Interact.
HT29-MTX/Caco-2 cocultures as an in vitro model for the intestinal epithelium: in vitro−in vivo correlation with permeability data from rats and humans
J. Pharm. Sci.
Mussel-inspired 3D fiber scaffolds for heart-on-a-chip toxicity studies of engineered nanomaterials
Anal. Bioanal. Chem.
Physiology and function of the tight junction
Cold Spring Harb Perspect Biol.
Alterations in intestinal permeability
Gut.
A human intestinal M-cell-like model for investigating particle, antigen and microorganism translocation
Nat Protoc.
Nanoparticle toxicity by the gastrointestinal route: evidence and knowledge gaps
Int. J. Biomed. Nanosci. Nanotechnol.
In vitro human digestion test to monitor the dissolution of silver nanoparticles
Nanotechnology in food product inventory
Nanotechnology in Food Interactive Tool
Defining new criteria for selection of cell-based intestinal models using publicly available databases
BMC Genomics
Presence and risks of nanosilica in food products
Nanotoxicology.
Preparation, characterization, and in vitro dosimetry of dispersed, engineered nanomaterials
Nat. Protoc.
An integrated methodology for assessing the impact of food matrix and gastrointestinal effects on the biokinetics and cellular toxicity of ingested engineered nanomaterials
Part Fibre Toxicol.
Robust antibacterial activity of tungsten oxide (WO3-x) nanodots
Chem Res Toxicol.
Inventory of Nanotechnology Applications in the Agricultural, Feed and Food Sector. E. F. S. Authority. EN-621
Annual Report of the EFSA Scientific Network of Risk Assessment of Nanotechnologies in Food and Feed for 2014
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