title: The common food additive carrageenan is not a ligand for Toll-Like- Receptor 4 (TLR4) in an HEK293-TLR4 reporter cell-line model.
abstract: Carrageenan (CGN) is widely used in the food manufacturing industry as an additive that stabilizes and thickens food products. Standard animal safety studies in which CGN was administered in diet showed no adverse effects. However, several in vitro studies have reported that intestinal inflammation is caused by CGN and that this effect is mediated through Toll-Like-Receptor 4 (TLR4). The purpose of this study was to evaluate the ability of different types of CGN to bind and activate TLR4 signaling. To accomplish this a TLR4/MD-2/CD14/NFκB/SEAP reporter construct in a HEK293 cell line was used. The reporter molecule, secretable alkaline phosphatase (SEAP), was measured as an indicator of TLR4 activation. The test compounds were exposed to this system at concentrations of 0.1, 1, 10, 50, 100, 500, 1000, and 5000 ng/mL for 24 h. Cytotoxicity was evaluated following the 24 h exposure period by LDH leakage and ATP. CGN binding to serum proteins was characterized by Toluidine Blue. The results show that CGN does not bind to TLR4 and is not cytotoxic to the HEK293 cells at the concentrations and experimental conditions tested and that CGN binds tightly to serum proteins.
authors: IONTOX, LLC, 4717 Campus Dr., Kalamazoo, MI 49008, USA. Electronic address: jmckim@iontox.com.
authors: James M
authors: JM
authors: McKim
authors: Cyprotex/CeeTox, Inc., 4717 Campus Dr., Kalamazoo, MI 49008, USA.
authors: Paul C
authors: PC
authors: Wilga
authors: Cyprotex/CeeTox, Inc., 4717 Campus Dr., Kalamazoo, MI 49008, USA.
authors: Jeffrey F
authors: JF
authors: Pregenzer
authors: Celtic Colloids, Inc., 13 Garnet Dr., Topsham, ME 04086, USA.
authors: William R
authors: WR
authors: Blakemore
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D002351:Carrageenan / Q000009:adverse effects*; D002470:Cell Survival / Q000187:drug effects; D005503:Food Additives / Q000009:adverse effects*; D057809:HEK293 Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008024:Ligands; D008070:Lipopolysaccharides / Q000009:adverse effects; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D019055:No-Observed-Adverse-Effect Level; D018570:Risk Assessment; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*
chemical_list: D005503:Food Additives; D008024:Ligands; D008070:Lipopolysaccharides; D016328:NF-kappa B; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D000255:Adenosine Triphosphate; D002351:Carrageenan; D007770:L-Lactate Dehydrogenase; D000469:Alkaline Phosphatase
keywords: Carrageenan; In vitro; Inflammation; Protein binding; TLR4