BOUWMEESTER ET AL. VOL. 5 ’ NO. 5 ’ 4091–4103 ’ 2011 www.acsnano.org 4091 April 11, 2011 C 2011 American Chemical Society Characterization of Translocation of Silver Nanoparticles and Effects on Whole-Genome Gene Expression Using an In Vitro Intestinal Epithelium Coculture Model Hans Bouwmeester, * Jenneke Poortman, Ruud J. Peters, Elly Wijma, Evelien Kramer, Sunday Makama, Kinarsashanti Puspitaninganindita, Hans J. P. Marvin, Ad A. C. M. Peijnenburg, and Peter J. M. Hendriksen RIKILT, Institute of Food Safety, Wageningen University and Research Center, Akkermaalsbos 2, P.O. Box 230, 6700 AE Wageningen, The Netherlands T he use of nano-based consumer prod- ucts is growing rapidly, and many of such products are available in the market. To date, more than 600 consumer products that are self-identified by the man- ufacturers as containing nanotechnology are included in a public database. 1 Appli- cations in the food sector are eminent, widely discussed, and cover many aspects such as improving food packaging materi- als, efficient nutrient delivery, formulations with improved bioavailability, and new tools for molecular and cellular detection of contaminants. 2À5 Nanoparticles, as a consequence of their small size, exhibit different physicochemical properties and biological effects compared to their respective bulk materials, even at the same mass dose. 6 A nanoparticle is generally defined as a discrete entity that has three dimensions on the order of 100 nm or less. 7 Various types of nanopar- ticles are used in the food sector. With respect to inorganic nanoparticles, various metal oxides like magnesium oxide, tita- nium dioxide, silicon dioxide, and silver are used in, for example, coatings of food packaging materials. Notably, the conven- tional forms of magnesium oxide, titanium dioxide, silicon dioxide, and silver are per- mitted food additives (e.g., E530, E171, E551, E174), 2 but without a definition of the sizes of the materials. For example, recently, we showed that silicon dioxide (E551) as used in food products contains particle sizes in the nanorange. 8 Silver (E174) is only limit- edly used as food additive (as colorant). If total use, including in food packaging mate- rials, is considered, silver nanoparticles are among the most frequently used nanopar- ticles, mainly because of its antimicrobial action. 9,10 In conclusion, consumer exposure to nanoparticles is inevitable, and it is there- fore necessary to assess the health impact of human exposure to nanoparticles. 4,11 Only a few oral repeated dose studies using silver nanoparticles are available (reviewed in refs 12 and 13). Kim and co- workers exposed rats orally for 28 consecu- tive days to high doses of silver nanoparti- cles (30, 300, or 1000 mg/kg/day), resulting in an increased silver content in various organs. The liver appeared to be the main accumulating organ for silver nanopar- ticles. 14 This was confirmed in a 90 day study from the same authors. 15 So far, these are the only oral in vivo studies using silver * Address correspondence to hans.bouwmeester@wur.nl. Received for review February 22, 2011 and accepted April 11, 2011. Published online 10.1021/nn2007145 ABSTRACT Applications of nanoparticles in the food sector are eminent. Silver nanoparticles are among the most frequently used, making consumer exposure to silver nanoparticles inevitable. Information about uptake through the intestines and possible toxic effects of silver nanoparticles is therefore very important but still lacking. In the present study, we used an in vitro model for the human intestinal epithelium consisting of Caco-2 and M-cells to study the passage of silver nanoparticles and their ionic equivalents and to assess their effects on whole-genome mRNA expression. This in vitro intestine model was exposed to four sizes of silver nanoparticles for 4 h. Exposure to silver ions was included as a control since 6À17% of the silver nanoparticles were found to be dissociated into silver ions. The amount of silver ions that passed the Caco-2 cell barrier was equal for the silver ion and nanoparticle exposures. The nanoparticles induced clear changes in gene expression in a range of stress responses including oxidative stress, endoplasmatic stress response, and apoptosis. The gene expression response to silver nanoparticles, however, was very similar to that of AgNO 3 . Therefore, the observed effects of the silver nanoparticles are likely exerted by the silver ions that are released from the nanoparticles. KEYWORDS: silver nanoparticles . in vitro . oral . translocation . Caco-2 . microarray ARTICLE