Abstract
Background: Semi-refined carrageenan (food additive E407a) is a widely used thickener, which has been reported to exert toxic and pro-inflammatory effects. In particular, there is accumulating evidence that it induces eryptosis, i.e. a programmed cell death of eryptocytes, via ROS-mediated pathways. However, the role of Ca2+-dependent mechanisms in E407a-induced eryptosis is not elucidated. Material and methods: Semi-refined carrageenan at concentrations of 0 mg/ml, 1 mg/ml, 5 mg/ml and 10 mg/ml was incubated with blood of intact female WAG rats (n=9) for 24 h in RPMI and fetal bovine serum. After 24 h, the samples were used to obtain erythrocyte suspensions. The obtained suspensions were stained with a Ca2+-sensitive FLUO4 AM probe (30 min, 2.5 µM). The fluorescence of FLUO4 in erythrocytes was detected by a BD FACSCanto II flow cytometer. Results: The intracellular Ca2+ levels are proportional to the fluorescence of FLUO4. The mean fluorescence intensities (MFI) were compared. Low levels (1 mg/ml) of E407a had no impact on Ca2+ concentrations in erythrocytes (p>0.05). On the contrary, high concentrations (5 mg/ml and 10 mg/ml) of this food additive promoted an increase in the intracellular Ca2+ levels. The MFI values were 2.3- and 2.5-fold higher, respectively (p<0.0001). In addition, the exposure to E407a at concentrations of 5 mg/ml and 10 mg/ml (p<0.0001) increased the percentage of cells with high FLUO4 fluorescence. Conclusion: Food additive E407a induces eryptosis in a Ca2+-dependent manner.
Highlights
The lifespan of erythrocytes is approximately 120 days
Since calcium release into the cytosol of erythrocytes plays a pivotal role in induction of eryptosis, its concentration in red blood cells exposed to E407a was determined by FLUO4 staining
Intracellular Ca2+ levels were estimated by comparing mean fluorescence intensities (MFI) values of FLUO4, which depend on Ca2+ concentrations, and the percentage of erythrocytes with the increased FLUO4 fluorescence
Summary
The lifespan of erythrocytes is approximately 120 days. After that period, they are eliminated from the bloodstream by the fixed macrophages. In response to unfavorable factors, including lack of energy, hyperosmolarity, accumulation of reactive oxygen species (ROS), they undergo cell death referred to as eryptosis [1, 2]. In some ways, it resembles apoptosis of nucleuscontaining cells and is characterized by blebbing, shrinkage and plasma phospholipid membrane scrambling [3]. High concentrations (5 mg/ml and 10 mg/ml) of this food additive promoted an increase in the intracellular Ca2+ levels. The exposure to E407a at concentrations of 5 mg/ml and 10 mg/ml (p
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