Sunset Yellow and Allura Red modulate Bcl2 and COX2 expression levels and confer oxidative stress-mediated renal and hepatic toxicity in male rats

Abstract

Studies on the adverse health effects caused by azo dyes are insufficient and quite contradictory. This work aims to investigate the possible toxic effect of two types of widely used food additives, Sunset Yellow and Allura Red, by assessing the physiological, histopathological and ultrastructural changes in the liver and kidney. Also, we investigated the genotoxic effect of both dyes on white blood cells. Thirty adult male albino rats were divided into three groups of 10 animals each: control (received water), Sunset Yellow-treated (2.5 mg/kg body weight) and Allura Red-treated (seven mg/kg body weight). The doses were orally applied for 4 weeks. Our results indicated an increase in the biochemical markers of hepatic and renal function (Aspartate aminotransferase, alanine aminotransferase, urea, uric acid and creatinine) in animals administered with the azo dyes. We also observed a noticeable increase in MDA and a marked decrease in total antioxidant levels in azo dye-treated animals compared to controls. Conversely, both dyes adversely affected the liver and kidney of albino rats and altered their histological and fine structure, with downregulation of Bcl2 and upregulation of COX2 expression. Our comet assay results showed a significant elevation in the fold change of tail moment in response to application of Sunset Yellow but not Allura Red. Collectively, we show that Sunset Yellow and Allura Red cause histopathological and physiological aberrations in the liver and kidney of male Wistar albino rats. Moreover, Sunset Yellow but not Allura Red induces a potential genotoxic effect.

Keywords: Allura Red; Bcl2; COX2; Genotoxicity; Histopathology; Serum biochemistry; Sunset Yellow.

Figure 1. Effect of Sunset Yellow and Allura Red on serum biochemistry and oxidative biomarkers in male rats.

(A) Bar graph showing the fold change in the level of measured parameters in response to administration of Sunset Yellow. (B) Bar graph showing the fold change in the level of measured parameters in response to administration of Allura Red. Control values are adopted for meta-analysis from Khayyat et al. (2017) and are normalized to 1 (Red line) for all measured parameters.

Figure 2. Sunset Yellow induces histological and ultrastructural alterations in liver tissue of rat.

(A) LM of liver section from rats treated with Sunset Yellow showing disorganized hepatic strands, necrosis of most hepatocytes (H), congestion of blood vessel (star). H&E, Scale bar: 50 µm. (B) LM of liver section from rats treated with Sunset Yellow showing irregular shaped nuclei (N), thickened blood sinusoid with erythrocytes (star), leucocytic infiltration (I), vacuoles (V) and Kupffer cell (K). H&E, Scale bar: 20 µm. (C) EM of liver rats treated with Sunset Yellow showing cytoplasmic degeneration, electron dense mitochondria (M), clustered rER and pyknotic nuclei (N). Scale bar: 500 nm. (D) EM of liver rats treated with Sunset Yellow showing numerous glycogen particles (g), myelin bodies (my), collagen fibers (star). M, mitochondria; N, nucleus; L, lipid droplets. Scale bar: 500 nm.

Figure 3. Adverse effect of Allura Red on the histology and ultrastructure of liver tissue of rat.

(A) LM of liver section from rats treated with Allura Red showing hydropic degeneration in hepatocytes (H), damage in central vein (star), leucocytic infiltration (I). H&E, Scale bar: 50 µm. (B) LM of liver section from rats treated with Allura Red showing vacuoles in hepatocytes (V), irregular nuclei (N), Kupffer cell (K), leucocytic infiltration (I). H&E, Scale bar: 20 µm. (C) EM of liver rats treated with Allura Red showing disrupted hepatocyte, degenerated mitochondria (M), packed rER, lipid droplets (L) and irregular nucleus (N). Scale bar: 500 nm. (D) EM of liver rats treated with Allura Red showing altered mitochondria (M), disintegration rER, lipid droplets (L), pyknotic nucleus (N) and Kupffer cell (K). Scale bar: 500 nm.

Figure 4. Sunset Yellow alters the normal architecture of kidney tissue of rat.

(A) LM of kidney section from rats treated with Sunset Yellow showing degenerated renal tubules (RT) and destructed glomeruli (G). H&E, Scale bar: 100 µm. (B) LM of kidney section from rats treated with Sunset Yellow showing vacuolar degenerated renal tubules (RT), vacuoles in glomerulus (G), leukocytes infiltration (I), large vacuole between the tubules (star), haemorrhage (h). H&E, Scale bar: 50 µm. (C) EM of kidney rat treated with Sunset Yellow showing proximal tubular cell with altered nucleus (N), condensed mitochondria (M), lysosomes (Ly), vacuoles (star), microvilli (mv). Scale bar: 500 nm. (D) EM of kidney rat treated with Sunset Yellow showing disrupted distal tubular cells with pyknotic nucleus (N), disordered mitochondria (M), vacuolated cytoplasm (star). Scale bar: 500 nm.

Figure 5. Allura Red induces histological and ultrastructural changes in kidney tissue of rat.

(A) LM of kidney section from rats treated with Allura Red showing necrosis of renal tubules (RT) and degenerated glomeruli (G). H&E, Scale bar: 100 µm. (B) LM of kidney section from rats treated with Allura Red showing vacuolated renal tubules (RT), dilated Bowman’s space (star), leukocytes infiltration (I), haemorrhage (h). H&E, Scale bar: 50 µm. (C) EM of kidney rat treated with Allura Red showing disrupted proximal tubular cells, irregular nucleus (N), disordered mitochondria (M), lysosomes (Ly), vacuoles (V), microvilli (mv). Scale bar: 500 nm. (D) EM of kidney rat treated with Allura Red showing disrupted distal tubular cells. N, nucleus; M, mitochondria; V, vacuoles. Scale bar: 500 nm.

Figure 6. Immunohistochemical microscopic images showing the expression of Bcl2 and COX2 in the kidney of rat.

(A–C) Bcl2 expression levels in control, Sunset Yellow-, and Allura Red-treated animals. Bcl2 expressions are decreased in Sunset Yellow and Allura Red as compared to control. (A–F) COX2 expression levels in control, Sunset Yellow-, and Allura Red-treated animals. Application of Sunset Yellow did not alter COX2 levels as compared to control. However, application of Allura Red elevated the expression levels of COX2 specially in the uriniferous tubules and Malpighian corpuscles. Scale bar: 20 µm.

Figure 7. The genetic toxicity of Sunset Yellow and Allura Red on white blood cells of rat.

Bar graph showing the tail moment in nuclei of leucocytes of control, Sunset Yellow-, and Allura Red-treated animals after Comet Assay. Data are Mean ± SD (n = 3, t-test, ∗p < 0.05).