Experimental Evaluation of Food-Grade Semi-Refined Carrageenan Toxicity

Abstract

The safety of food additives E407 and E407a has raised concerns in the scientific community. Thus, this study aims to assess the local and systemic toxic effects of the common food additive E407a in rats orally exposed to it for two weeks. Complex evaluations of the effects of semi-refined carrageenan (E407a) on rats upon oral exposure were performed. Local effects of E407a on the intestine were analyzed using routine histological stains and CD68 immunostaining. Furthermore, circulating levels of inflammatory markers were assessed. A fluorescent probe O1O (2- (2'-OH-phenyl)-5-phenyl-1,3-oxazole) was used for evaluating the state of leukocyte cell membranes. Cell death modes of leukocytes were analyzed by flow cytometry using Annexin V and 7-aminoactinomycin D staining. Oral administration of the common food additive E407a was found to be associated with altered small and large intestinal morphology, infiltration of the lamina propria in the small intestine with macrophages (CD68⁺ cells), high systemic levels of inflammation markers, and changes in the lipid order of the phospholipid bilayer in the cell membranes of leukocytes, alongside the activation of their apoptosis. Our findings suggest that oral exposure to E407a through rats results in the development of intestinal inflammation.

Keywords: E407a; animal model; carrageenan; fluorescent probe; inflammation; intestine; leukocytes; processed Eucheuma seaweed; toxicity.

Figure 1

Representative hematoxylin-eosin (H&E)-stained and periodic acid-Schiff (PAS)-stained sections of the small and large intestines and PAS stain. (A) Control group. Small intestine is not damaged. Villi are preserved. H&E stain, 100×. (B) Control group. Moderately PAS-positive mucin-containing goblet cells are visible. PAS stain, 400×. (C) Control group. No signs of inflammation in the large intestine. The mucosal layer is intact. H&E stain, 400×. (D) Control group. PAS stain reveals goblet cells with mucin inside. PAS stain, 400×. (E) Experimental group. Villi are absent. Significant leukocyte infiltration is observed. H&E stain, 100×. (F) Experimental group. Multi-row epithelia is noticed. Goblet cells contain less PAS-positive mucin. PAS stain, 400×. (G) Experimental group. Epithelium is desquamated. Leukocyte infiltration is observed. H&E stain, 100×. (H) Experimental group. PAS positivity of mucin in goblet cells is less pronounced compared with the control group. PAS stain, 400×.

Figure 2

Immunostaining for CD68 (brown stain) in the small intestine of control animals ((A), 100×; (B), 400×) and rats treated with E407a for two weeks ((C), 100×; (D), 400×). Moderate amounts of CD68⁺ cells (macrophages) are observed in control samples. Exposure to E407a results in an increase in the number of CD68⁺ cells in the lamina propria.

Figure 3

Quantitative analysis of CD68 expression in the small intestine reveals an infiltration of the small intestinal lamina propria with CD68⁺ cells in rats orally exposed to E407a.

Figure 4

Representative fluorescence spectra of probe O1O in leukocyte suspensions: (a) the control group of rats (black solid line); (b) the animals orally exposed to E407a for two weeks (red solid line). For a correct comparison, the spectra were normalized to the fluorescence intensity of the normal form.

Figure 5

Representative SSC/APC-CyTM 7 CD45 demonstrating the gating strategy for identifying CD45⁺ cells.

Figure 6

Representative Annexin V/7-AAD dotplots that demonstrate the percentage (Me [IQR]) of viable leukocytes (Annexin V⁻, 7-AAD⁺ cells); early apoptotic cells (Annexin V⁺, 7-AAD⁻ cells); late apoptotic/necrotic cells (Annexin V⁺, 7-AAD⁺); and dead necrotic CD45⁺ cells (Annexin V⁻, 7-AAD⁺) in the control (A) and experimental groups (B). A statistically significant increase in the percentage of early apoptotic cells is found in rats exposed to semi-refined carrageenan.

Figure 7

Scheme of excited state intramolecular proton transfer (ESIPT) reaction in 2 (2′-hydroxyphenyl)-5-phenyl-1,3-oxazole (probe O1O). The upwards arrow denotes the electronic excitation and the downwards arrow designates the emission of light (fluorescence). Corresponding maximum absorption and the ranges of emission are shown in nanometers. (Modified from Posokhov and Kyrychenko, 2018).

Figure 8

Localization and orientation of fluorescent probe O1O (2- (2′-OH-phenyl)-5-phenyl-1,3-oxazole) in phospholipid membranes. Two molecules of phosphatidylcholine from the outer leaflet are shown to denote the localization of the probe. (Adapted from Posokhov and Kyrychenko, 2018).