To assess the ability of the common food additive E407a (semi-refined carrageenan) to enter leukocytes in vitro and generate reactive oxygen species (ROS) in leukocytes as a whole and granulocytes in particular, both during incubation and in experimental animals.
ROS production was assessed in leukocytes incubated with E407a for 2 h at the final concentrations of 5 and 10 g/L using the dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), as well as in cells isolated from rats orally exposed to E407a (140 mg/kg of weight) during 2 weeks (n = 8) and control rats (n = 8), by flow cytometry. Carrageenan uptake by leukocytes was estimated by confocal microscopy using incubation of rhodamine B isothiocyanate-labelled carrageenan with leukocyte suspensions.
Uptake of carrageenan by viable neutrophils, monocytes, and lymphocytes was confirmed. Oral administration of the food additive E407a was associated with excessive ROS formation by viable leukocytes (CD45+, 7‑aminoactinomycin D− cells) and especially in granulocytes. Unexpectedly, a direct impact of semi-refined carrageenan during incubation for 2 h did not affect ROS production in leukocytes, evidenced by statistically insignificant differences in mean fluorescence intensity values of 2′,7′-dichlorofluorescein, which is a ROS-sensitive product of intracellular H2DCFDA conversion. Oral intake of E407a and direct exposure of leukocyte suspensions to it decreased the viability of leukocytes.
Food-grade carrageenan can enter leukocytes without affecting ROS generation as a result of incubation for 2 h with leukocyte suspensions. On the contrary, oral exposure to E407a is accompanied by ROS overproduction by white blood cells, suggesting an indirect mechanism for the stimulation of ROS synthesis in vivo. E407a promotes cell death of leukocytes both in vivo and in vitro.