55th Annual Meeting of the Society of Toxicology
Effects of Food Grade Carrageenan on Inflammatory Signaling Pathways in a Human Hepatoma Cell Line (HepG2).
Abstract no. 2589. Click here to view poster.
H. M. Baas and J. M. McKim. IONTOX, Kalamazoo, MI
Carrageenan (CGN) is a common food additive used primarily as a thickener and stabilizing agent. Animal safety studies using high molecular weight CGN have demonstrated that CGN is safe for human consumption. Recently, it has been reported that CGN can bind to Toll-Like Receptor 4 (TLR4) and induce proinflammatory pathways in HepG2 cells. These results have not been repeated by other laboratories, which is important in order to understand potential human risk. Poligeenan (PGN) (formerly referred to as degraded CGN) with Mw 10-20 kDa has been associated with intestinal inflammation in animals, but is not used as a food additive. CGN is a natural polysaccharide (Mw of 200-800 kDa), is resistant to breakdown in the gut, and is not absorbed across intestinal epithelium. In addition, CGN and PGN bind tightly to proteins, which would reduce cellular exposure in vitro and in vivo. Therefore, the aim of this study was to carefully evaluate whether CGN can cross intestinal cells in vitro and induce an inflammatory response in HepG2 cells. Intestinal absorption was measured using a standard Caco-2 permeability model. HepG2 cells were seeded into 96-well plates at a density of 15,000 cells/ 100 µL/well in the presence and absence of 10% fetal bovine serum (FBS). The cells were exposed to lambda-CGN (0.1, 1.0, and 10 µg/mL) for 24 hr. Positive control groups included PGN (Mw = 19 kDa, 10 µg/mL) and lipopolysaccharide (LPS) (10 ng/mL and 100 ng/mL). Three (media/ exposure) conditions were tested; 1) compound plus media with 10% FBS for 24 hr, 2) compound plus media with 10% FBS for 20 hr followed by compound plus FBS free media for 4 hr, and 3) compound plus serum free media for 24 hr. Following the exposure times the cells were either evaluated for ATP or used to prepare total RNA. Cellular ATP was used to monitor cell viability while changes in the expression of interleukin-8 (IL-8), as determined by qRT-PCR, was used to assess TLR-4 signaling. Following the exposure period, LPS produced a concentration dependent increase in IL-8 expression and this was greatest in media 3 (>10- fold). PGN induced IL-8 by 8-fold in both media 2 and 3, but had no effect on IL-8 when FBS was present (media 1). Lambda-CGN had no effect on IL-8 induction in any of the media formulations. Cell viability was greater than or equal to 80% of untreated controls. CGN did not cross the Caco-2 cell barrier and did not cause cytotoxicity. In conclusion, CGN is not absorbed from the gut and does not induce a proinflammatory response mediated by TLR-4 binding in HepG2 cells.
H. M. Baas and J. M. McKim. Effects of Food Grade Carrageenan on Inflammatory Signaling Pathways in a Human Hepatoma Cell Line (HepG2). In: The Toxicologist: Supplement to Toxicological Sciences, 150 (1), Society of Toxicology, 2016. Abstract no. 2589.
Click here to view poster.