Elsevier

Toxicology Letters

Volume 325, 1 June 2020, Pages 14-24
Toxicology Letters

Soluble silver ions from silver nanoparticles induce a polarised secretion of interleukin-8 in differentiated Caco-2 cells

https://doi.org/10.1016/j.toxlet.2020.02.004Get rights and content

Highlights

  • Silver nanoparticles induce the polarised secretion of interleukin-8 into the apical compartment of Caco-2 cells.

  • Interleukin-8 production is Nrf2 dependent.

  • This increased secretion of interleukin-8 is likely exerted by soluble Ag ions and not by nanoparticles per se.

Abstract

Because of their antimicrobial properties, silver nanoparticles are increasingly incorporated in food-related and hygiene products, which thereby could lead to their ingestion. Although their cytotoxicity mediated by oxidative stress has been largely studied, their effects on inflammation remain controversial. Moreover, the involvement of silver ions (originating from Ag0 oxidation) in their mode of action is still unclear. In this context, the present study aims at assessing the impact of silver nanoparticles on the secretion of the pro-inflammatory chemokine interleukin-8 by Caco-2 cells forming an in vitro model of the intestinal mucosal barrier. Silver nanoparticles induced a vectorized secretion of interleukin-8 towards the apical compartment, which is found in the medium 21 h after the incubation. This secretion seems mediated by Nrf2 signalling pathway that orchestrates cellular defense against oxidative stress. The soluble silver fraction of silver nanoparticles suspensions led to a similar amount of secreted interleukin-8 than silver nanoparticles, suggesting an involvement of silver ions in this interleukin-8 secretion.

Introduction

With the advances of nanotechnology, manufactured nanoparticles came on the market with promises of improved optical, catalytical, electronical or antimicrobial properties (Vigneshwaran et al., 2007; El-Nour et al., 2010; León-Silva et al., 2016; Syafiuddin et al., 2017). However, their smaller size and higher reactivity could also raise their toxicity. Among them, silver nanoparticles (AgNPs) are increasingly used in consumer products because of their antimicrobial properties (Wijnhoven et al., 2009). According to the Project of Emerging Nanotechnologies (The project on emerging nanotechnologies, 2019), AgNPs are found in the highest number of consumer products containing nanoparticles, with 50 % of the consumer products containing silver (The project on emerging nanotechnologies, 2019). In particular, "health and fitness" and "food and beverages" applications are the most represented categories for AgNPs, potentially resulting in their ingestion (Laloux et al., 2017). The presence of AgNPs has also been proven in the food additive E174 added in pastry decorations such as chocolates or silver pearls (Verleysen et al., 2015). Aside this example, AgNPs are not directly incorporated in food but can migrate into it from packaging materials, cooking instruments, cleaning sprays or storage boxes (Wijnhoven et al., 2009; Emamifar et al., 2012; Cushen et al., 2014; Echegoyen and Nerín, 2013; Hauri and Niece, 2011; Song et al., 2011; von Goetz et al., 2013). Even if the potential migrated amount remains low, the increasing use of AgNPs in consumer products could dramatically raise the consumer exposure to AgNPs. Moreover, AgNPs constitute some unauthorised food supplements whose consumption can lead to up to 0.02 mg/kg BW/day exposure (Larsen et al., 2015). After ingestion, these AgNPs will come in contact with the gut. Although the cytotoxicity of AgNPs has been largely studied, their effect on inflammation remains controversial.

Because of its major role in inflammation, we decided to focus on the production of interleukin-8 (IL-8) by intestinal epithelial cells (IECs), a chemokine involved in inflammatory processes. For this purpose, Caco-2 cells were used as an in vitro model of the gut mucosal barrier. Although isolated from a colonic adenocarcinoma (Fogh and Trempe, 1975), they differentiate spontaneously in cells presenting characteristics similar to small intestine enterocytes such as the presence of active tight junctions and efflux pumps (Artursson, 1990; Sambuy et al., 2005; Smetanová et al., 2011). These cells are thus commonly used as a simple in vitro model of the small intestine in pharmaco-toxicology studies. In particular, they are the most widely used for nanomaterial translocation assessment (Braakhuis et al., 2015; Georgantzopoulou et al., 2015).

Although toxicity of AgNPs has been extensively reported in the literature, the involvement of silver ions (Ag+) in their mode of action still remains unclear. These ions, as a subproduct of Ag0 oxidation (Reidy et al., 2013), are commonly found in AgNPs suspensions in variable proportions that can go even up to 69 % of total silver content (Beer et al., 2012), depending on the synthesis method (Beer et al., 2012), the AgNPs size (Bouwmeester et al., 2011) and concentration (Hadioui et al., 2013) or the chemical nature of their coating (van der Zande et al., 2012). Together with different chloride complexes (Behra et al., 2013), Ag + forms the "soluble Ag fraction" in AgNPs suspensions. Although silver salts are known to be toxic (Miura and Shinohara, 2009), only a few studies have addressed this issue. Most of the published reports concerning AgNPs have not distinguished the effect of soluble Ag from the global observed effects (Zhao and Wang, 2012). Papers addressing this issue have generally compared the effect of a silver salt such as acetate or nitrate as a source of Ag+ (Böhmert et al., 2015; Bilberg et al., 2011; Martirosyan et al., 2016; Navarro et al., 2008; Yin et al., 2011). Only a few tests have been performed with the soluble fraction separated from AgNPs suspensions although this was recommended by Beer et al. (2012) for all studies concerning metallic nanoparticles such as silver or copper (Beer et al., 2012). In addition, AgNPs toxicity was suppressed by cysteine, which inactivates soluble Ag by complexing the ions (Zhao and Wang, 2012). Another way to assess the involvement of this soluble Ag is to separate it from the suspension, either by ultracentrifugation (Beer et al., 2012) or by filtration through a membrane with an appropriate cut-off (Zhao and Wang, 2012). Silver ions could also be separated from the rest of the suspension by ion exchange resin (Hadioui et al., 2013) although this is less used in literature.

The gut is a major immune organ, with about 60 % of the total immunoglobulin content of the human body (Salminen et al., 1998). Within this organ, the gut-associated lymphoid tissue (GALT) containing the largest pool of immune cells is found (Bourlioux et al., 2003). However, due to the presence of overwhelming potentially immune-stimulatory bacterial and food antigens, this tissue should respond adequately to stimulations. A complex regulation takes place in the gut to allow pathogens recognition while avoiding any unwanted response to the "normal" gut microflora. IECs form the first barrier encountered by luminal antigens and should respond appropriately to have a role in the regulation of immune response (Blikslager et al., 2007). For this purpose, they can secrete chemokines, cytokines and eicosanoids (Mason et al., 2008) to communicate with immune cells and to direct them selectively towards antigens. In particular, IL-8 is an important mediator for these cells, being the major secreted product of infected epithelial cells (Eckmann et al., 1995), e.g. Caco-2 cells (Van De Walle et al., 2010; Sergent et al., 2010). This chemokine produced among others by IECs, has the ability to attract neutrophils to guide them to the site of inflammation and it is thus commonly classified as a pro-inflammatory cytokine (Andoh et al., 2000; Rossi et al., 2013).

As the involvement of oxidative stress in the toxicity of AgNPs has been extensively proven (Völker et al., 2013; Kim and Choi, 2012; Georgantzopoulou et al., 2015; Gaillet and Rouanet, 2015; Akter et al., 2018), we have decided to evaluate whether a transcription factor i.e. Nuclear factor-erythroid 2-related factor (Nrf2) could be involved in this crosstalk as it orchestrates the defence against oxidative stress. Under normal conditions, Nrf2 is sequestrated by the cytosolic protein Keap1, which leads to its proteosomal degradation. Keap1 contains a series of reactive cysteine residues, acting as a sensor through reaction with electrophiles or oxidants. This modification releases Nrf2 that then translocates in the nucleus and regulates the expression of its target genes, containing an antioxidant response element (ARE) in their promoters (Kobayashi and Yamamoto, 2005). They modulate the cellular response to stress such as phase 2 detoxifying enzymes, thiol molecule generating system, reactive oxygen species (ROS) removing enzymes or stress response proteins (Kwak et al., 2004; Kobayashi and Yamamoto, 2005; Kensler et al., 2007). Among these target genes, heme oxygenase-1 (HO-1) is one of the most used to assess the activation of Nrf2 as it has been probably the best characterised ARE (Simmons et al., 2011). This enzyme, also named "heat shock protein 32", catalyzes the heme degradation, releasing free iron, bilirubin and carbon monoxide (Gozzelino et al., 2010). Moderate levels of these three molecules could exert anti-inflammatory and antioxidant properties (Ryter et al., 2006). Indeed, HO-1 seems to have a major role in the protection against acute and chronic inflammation of the gut (Zhu et al., 2011), its induction being associated with a protective response that contributes to the preservation of the gastro-intestinal tract (Zhu et al., 2011). Activation of Nrf2 (Prasad et al., 2013; van der Zande et al., 2016; Mao et al., 2018; Aueviriyavit et al., 2014; Ambrožová et al., 2017; Böhmert et al., 2015) and/or induction of HO-1 (Stępkowski et al., 2014; Bouwmeester et al., 2011; Miura and Shinohara, 2009; Xin et al., 2015; Kang et al., 2012a; Sahu et al., 2015; Ambrožová et al., 2017; Aueviriyavit et al., 2014; Fizesan et al., 2019) have been largely observed as a response to AgNPs.

In this study, we investigated if this Nrf2 cascade could play a role in the secretion of IL-8 mediated by AgNPs as IL-8 displays an ARE in its promoter (Zhang et al., 2005). This study aims at evaluating if AgNPs could modulate the secretion of IL-8 in Caco-2 cells. Moreover, we investigated the involvement of soluble Ag and Nrf2 signalling pathway in this secretion.

Section snippets

Cell culture and exposure

Caco-2 cells from a human colon adenocarcinoma (clone 1 from Dr. M. Rescigno, University of Milano, IT) were cultivated between p + 10 and p + 30 at 37C under a water-saturated atmosphere with 10 % (v/v) CO2. Caco-2 cells were grown in tissue culture flasks (Corning incorporated, Corning, NY) in Dulbecco modified Eagle’s medium (DMEM) with 4.5 g/L glucose (Lonza, Basel, CH), supplemented with 10 % (v/v) fetal bovine serum (Biowest, Nuaillé, FR), 1% (v/v) non-essential amino acids 100X (Lonza),

Characterisation of the nanomaterial

The characterisation of dry particles from this batch was performed by Klein et al. (2011) (Klein et al., 2011). We also performed an additional characterisation of AgNPs in HBSS (67.5 μg/mL) by transmission electron microscopy (TEM) (Fig. 2), UV–vis spectrophotometry (Fig. 3A) and dynamic light scattering (DLS). The hydrodynamic diameter measured by DLS was 57.75 nm with a polydispersity index of 0.226. In addition, the absorbance of soluble silver fraction was measured between 350 and 750 nm

Discussion

With their increasing use in food-related consumer products, it is very likely that a certain proportion of these AgNPs are ingested and that they come in contact with IECs from the gut. Although the important role of these cells in immune regulation against luminal potential antigens has been well described, the effect of AgNPs on inflammation in IECs still remains largely unknown. This study analyses the implication of one of the key players of these mechanisms, i.e. IL-8 upon exposure of

Conclusion

In summary, Caco-2 cells produce IL-8 in response to AgNPs exposure. This secretion is polarized towards the luminal compartment and is activated, at least partially, by a Nrf2 dependent pathway suggesting the involvement of oxidative stress in this secretion. This IL-8 secretion seems to be mediated by the soluble fraction composed of Ag + ions present in AgNPs suspensions, suggesting that it is not specific to nanoparticles.

Funding information

The authors declare no conflict of interest. This work has received some financial support from Association Luxembourgeoise des Amis de la Fondation de l’Université Catholique de Louvain (ALAF) and from UCLouvain (FSR Grant).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We would like to thank the SMCS platform from UCLouvain and in particular Vincent Bremhorst and Catherine Rasse for the statistical support.

References (111)

  • Y. Echegoyen et al.

    Nanoparticle release from nano-silver antimicrobial food containers

    Food Chem. Toxicol.

    (2013)
  • S. Gaillet et al.

    Silver nanoparticles: their potential toxic effects after oral exposure and underlying mechanisms–a review

    Food Chem. Toxicol.

    (2015)
  • S. Gioria

    Proteomics study of silver nanoparticles on Caco-2 cells

    Toxicol. Vitr.

    (2018)
  • C. Greulich

    Uptake and intracellular distribution of silver nanoparticles in human mesenchymal stem cells

    Acta Biomater.

    (2011)
  • S. Hackenberg

    Silver nanoparticles: evaluation of DNA damage, toxicity and functional impairment in human mesenchymal stem cells

    Toxicol. Lett.

    (2011)
  • M. Hadioui et al.

    Multimethod quantification of Ag+ release from nanosilver

    Talanta

    (2013)
  • S.J. Kang

    Silver nanoparticles-mediated G2/M cycle arrest of renal epithelial cells is associated with Nrf2-GSH signaling

    Toxicol. Lett.

    (2012)
  • S.J. Kang

    Role of the Nrf2-heme oxygenase-1 pathway in silver nanoparticle-mediated cytotoxicity

    Toxicol. Appl. Pharmacol.

    (2012)
  • A. Keshavarzian

    Leaky gut in alcoholic cirrhosis: a possible mechanism for alcohol-induced liver damage

    Am. J. Gastroenterol.

    (1999)
  • M.K. Kwak et al.

    Chemoprevention through the Keap1-Nrf2 signaling pathway by phase 2 enzyme inducers

    Mutat. Res. Mol. Mech. Mutagen.

    (2004)
  • D.H. Lim

    The effects of sub-lethal concentrations of silver nanoparticles on inflammatory and stress genes in human macrophages using cDNA microarray analysis

    Biomaterials

    (2012)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCt method

    methods

    (2001)
  • F. Martínez-Gutierrez

    Antibacterial activity, inflammatory response, coagulation and cytotoxicity effects of silver nanoparticles

    Nanomed. Nanotechnol. Biol. Med.

    (2012)
  • A. Martirosyan

    Tuning the inflammatory response to silver nanoparticles via quercetin in Caco-2 (co-) cultures as model of the human intestinal mucosa

    Toxicol. Lett.

    (2016)
  • R. Miethling-Graff

    Exposure to silver nanoparticles induces sizeand dose-dependent oxidative stress and cytotoxicity in human colon carcinoma cells

    Toxicol. Vitro

    (2014)
  • N. Miura et al.

    Cytotoxic effect and apoptosis induction by silver nanoparticles in HeLa cells

    Biochem. Biophys. Res. Commun.

    (2009)
  • M.J. Piao

    Silver nanoparticles induce oxidative cell damage in human liver cells through inhibition of reduced glutathione and induction of mitochondria-involved apoptosis

    Toxicol. Lett.

    (2011)
  • T. Sergent

    Anti-inflammatory effects of dietary phenolic compounds in an in vitro model of inflamed human intestinal epithelium

    Chem. Biol. Interact.

    (2010)
  • D.I. Sonnier

    TNF-α induces vectorial secretion of IL-8 in Caco-2 cells

    J. Gastrointest. Surg.

    (2010)
  • T. Stępkowski et al.

    Silver nanoparticles induced changes in the expression of NF-κB related genes are cell type specific and related to the basal activity of NF-κB

    Toxicol. Vitr.

    (2014)
  • J. Van De Walle

    Inflammatory parameters in Caco-2 cells: effect of stimuli nature, concentration, combination and cell differentiation

    Toxicol. Vitro

    (2010)
  • A. Abdelkhaliq

    Impact of in vitro digestion on gastrointestinal fate and uptake of silver nanoparticles with different surface modifications

    Nanotoxicology

    (2019)
  • N. Ambrožová

    Low concentrations of silver nanoparticles have a beneficial effect on wound healing in vitro

    J. Nanopart. Res.

    (2017)
  • R. Behra

    Bioavailability of silver nanoparticles and ions: from a chemical and biochemical perspective

    J. R. Soc. Interface

    (2013)
  • A.T. Blikslager

    Restoration of barrier function in injured intestinal mucosa

    Physiol. Rev.

    (2007)
  • L. Böhmert

    Molecular mechanism of silver nanoparticles in human intestinal cells

    Nanotoxicology

    (2015)
  • H. Bouwmeester

    Characterization of translocation of silver nanoparticles and effects on whole-genome gene expression using an in vitro intestinal epithelium coculture model

    ACS Nano

    (2011)
  • H.M. Braakhuis

    Progress and future of in vitro models to study translocation of nanoparticles

    Arch. Toxicol.

    (2015)
  • C. Carlson

    Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species

    J. Phys. Chem. B

    (2008)
  • T.E.A. Chalew et al.

    Toxicity of commercially available engineered nanoparticles to Caco-2 and SW480 human intestinal epithelial cells

    Cell Biol. Toxicol.

    (2013)
  • M. Cushen

    Evaluation and simulation of silver and copper nanoparticle migration from polyethylene nanocomposites to food and an associated exposure assessment

    J. Agric. Food Chem.

    (2014)
  • S. Dodd

    N-acetylcysteine for antioxidant therapy: pharmacology and clinical utility

    Expert Opin. Biol. Ther.

    (2008)
  • L. Eckmann

    Entamoeba histolytica trophozoites induce an inflammatory cytokine response by cultured human cells through the paracrine action of cytolytically released interleukin-1 alpha

    J. Clin. Investig.

    (1995)
  • K.M.A. El-Nour

    Synthesis and applications of silver nanoparticles

    Arab. J. Chem.

    (2010)
  • A. Emamifar

    Effect of nanocomposite packaging containing Ag and ZnO on reducing pasteurization temperature of orange juice

    J. Food Process. Preserv.

    (2012)
  • I. Fizesan

    In vitro exposure of a 3d-tetraculture representative for the alveolar barrier at the air-liquid interface to silver particles and nanowires

    Part. Fibre Toxicol.

    (2019)
  • J. Fogh et al.

    New human tumor cell lines

    Human Tumor Cells in Vitro

    (1975)
  • R.D. Fusunyan

    Butyrate enhances interleukin (IL)-8 secretion by intestinal epithelial cells in response to IL-1β and lipopolysaccharide

    Pediatr. Res.

    (1998)
  • A. Georgantzopoulou

    Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium

    Part. Fibre Toxicol.

    (2015)
  • K. Gerloff

    Cytotoxicity and oxidative DNA damage by nanoparticles in human intestinal Caco-2 cells

    Nanotoxicology

    (2009)
  • Cited by (0)

    View full text