The CaCo-2 cell junction derangement exerted by the single addition of oxysterols commonly detected in foods is markedly quenched when they are in mixture

The selective permeability of the gut epithelial barrier is heavily reliant on the stability of cell junctions, often challenged by a variety of dietary stressors, including non-enzymatic cholesterol oxidation products (COPs). A marked decrease of the tight junctions claudin-1 and occludin, and of the adherens junction E-cadherin was previously detected in differentiated CaCo-2 monolayers challenged by a single addition of 7β-hydroxycholesterol (7βOHC) or 7-ketocholesterol (7KC) in the lowest micromolar range. However, in the diet, oxysterols are occurring in a mixture. Hence, the aim of the present study was to evaluate whether cell incubation with all the main dietary COPs together quench the intercellular junction derangement previously observed as exerted by 7βOHC and 7KC singularly added. Two chocolate prototypes, respectively made with fresh (oxy-Mix1) or six-months stored whole milk powder (oxy-Mix2), were compared. The second prototype showed an almost double content of total COPs (3.34 µM, approximately 1337 ng /g of chocolate) than the first one (1.69 µM, approximately 675 ng /g of chocolate). Importantly, even in the CaCo-2 cell monolayers treated with six-months stored mixture of COPs oxy-Mix2, no alterations were observed of those cell junctions markedly affected by identical concentration of 7βOHC or 7KC used alone. The junctions’ derangement started to be significantly evident when oxy-Mix2 was used at higher concentration (5 µM, approximately 2 µg oxysterols/g of product) or when treatments were carried out with repeated doses of oxy-Mix2 every 24 hours. Although achieved in a still widely adopted in vitro model system, these findings could orientate the definition of a safe shelf-life for dairy products, certainly for milk chocolate. • Two chocolate prototypes made with fresh or 6-months stored milk powder were tested. • CaCo-2 cell monolayers were exposed to 2 oxysterol mixtures from the 2 prototypes. • Oxysterol mixture-dependent gut damage can be monitored as cell junction loss. • Only oxysterols from high/repeated doses of 6-months chocolate induced gut damage. • Chocolate with longer shelf-life, i.e., oxysterol excessive intake, is harmful.