Side Effects of In ovo Given Sunset Yellow FCF on the Embryonic Development of the Spleen by Means of Histological and Enzyme Histochemical Methods

Research Article

J Immun Res. 2019; 7(1): 1035.

Side Effects of In ovo Given Sunset Yellow FCF on the Embryonic Development of the Spleen by Means of Histological and Enzyme Histochemical Methods

Celik S, Celik I* and Berktay E

Department of Histology and Embryology, Selçuk University, Turkey

*Corresponding author: Ilhami Celik, Department of Histology and Embryology, Selcuk University, Veterinary Faculty, Selçuklu 42301, Konya, Turkey

Received: March 08, 2021; Accepted: March 29, 2021; Published: April 05, 2021

Abstract

Background: E110 is one of the food colorants which is widely used in dairy products, fast foods, jam and dry beverage powders, aqueous drug solutions, tablets, capsules, toothpastes mouthwashes, hair care products and cosmetics. Doubts have accumulated in recent years that food additives might cause allergic reactions in humans or increase these ailments. In this study, side effects of Sunset yellow FCF (E110) on the embryonic development of chicken spleen were evaluated by means of histological, histomorphometrical and enzyme histochemical methods.

Methods: In the study, 250 fertilized broiler eggs obtained from a commercial broodstock were used. Eggs were divided into 5 groups each having 50 eggs. Control eggs were either nontreated or distilled water injected via air sac. The eggs in the experimental groups were injected with 100ng/egg, 500ng/egg and 1.000ng/egg E110 prior to incubation. Blood and spleen samples were taken from randomly selected 10 eggs of each group at 11th, 15th, 18th and 21st days of incubation. Leukocyte formula, alpha-naphtyl acetate esterase-positive and acid phosphatase-positive lymphocyte percentages were determined in the blood samples and embriyonic development of the spleen was assessed in the tissue sections.

Results: In the 500ng/egg and 1.000ng/egg E110 injected experimental groups, embryonic development of the spleen retarded, alpha-naphtyl acetate esterase-positive and acid phosphatase-positive lymphocyte rates were significantly depressed.

Conclusions: Significant disturbances in the immune system functions of the affected animals might occure at post-natal period of their life.

Keywords: E110; Spleen; Immune system; Acid phosphatase; Alphanaphthyl acetate esterase

Abbreviations

ACPase: Acid Phosphatase; ADI: Acceptable Daily Intake; ADHD: Attention Deficit Hyper-Activity Disorder; ANAE: Alpha- Naphthyl Acetate Esterase; BW: Body Weight; BW/D: Body Weight/ Day; CAS: Chemical Abstracts Service; CHEST: Embryotoxicity Screening Test; ChEs: pseudo Cholinesterases; E110: Sunset Yellow FCF; E123, Amaranth; EFSA: European Food Safety Authority; FAO: Food and Agriculture Organization; GCs: Germinal Centers; IRDC: International Research and Development Corporation; LD50: Lethal Dose-50; LOAEL: Lowest Observed Adverse Effect Level; MN: Micronucleus; NOAEL: No-Observed-Adverse-Effect Level; NTP: National Toxicology Program; PALS: Periarteriolar Lymphoid Sheath; SCE: Sister Chromatid Exchange; UNESDA: Union of European Soft Drinks Associations.

Introduction

Sunset Yellow FCF is a very important food colorant azo dye, which is used to increase appealing of the foods. E110 is widely used in beverages, gel confectionery, cereal products, macaroni, desserts, snacks, ice cream and canned fish. It also participates as a colorant in the drugs [1]. E110 is often used in combination with E123 to achieve brown color in the chocolate and caramel and it is the most commonly used food colorant in soft beverages in many European countries and Turkey [2].

E110 was first introduced as a food colorant in 1929 and its reliability as food additive was evaluated in 1982. ADI level of E110 was determined as 0-2.5 mg/kg body weight/day [3]. The LD50 doses of E110 are >6.000mg/kg BW in the mouse and >10.000mg/kg BW in the rats [4].

Although E110 did not have significant side effects when the permitted safe limits were not exceeded, these limits are often exceeded in daily practice. Moreover, because of E110 is a sulfonated form of sudan I, which is considered a possible carcinogen, there is a certain amount of sudan I in the produced final E110 product, although it is not desired. E110 is suspected to be responsible for the health problems such as, allergic reactions, diarrhea, vomiting and urticaria [5], angioedema [6,7], rarely anaphylactic shock and headache in the children with aspirin intolerance [8]. These reactions are common symptoms of ailments caused by azo group food colorings, including E110. In recent years, it has been suggested that E110 is also associated with childhood hyperactivity and also might trigger aggression in children [9]. The affected children return to normal, when artificial food colorants including E110 are removed from the drinks of hyperactive children, but behavioral disorders reappear by reintroducing the beverages containing these substances [10].

Water solubility of E110 is 190.000mg/L at 25°C and 200.000mg/L at 60ºC [11]. Since E110 does not contain functional groups such as ester, amide, acetal, epoxide, lactone, which are hydrolyzed in water, its reaction potential in water depends on the desulfonation of aromatic sulfonic acid or its equivalent sulfonic acid salt. Since aromatic sulfonic acids are not desulfonated in the natural environment, E110 is not biodegradable in the natural conditions and tends to maintain its stable structure in water [12].

Metabolism studies showed that 3.6% of orally ingested E110 is absorbed through the digestive tract, and only 0.8% of 100mg single dose was observed in the feces. The main metabolic pathway in the breakdown of the dye is probably occurs via bacterial activity in the gut. This activity provides the breakdown of aromatic amines and aminosulfonic acids, and the resulting products are partially absorbed from the intestine [13]. In the rat, relatively important (20-30%) part of intravenously administered E110 is excreted via bile without destruction after 6 hours, and urine is also a significant excretion route of E110 and its metabolites [14].

Results of the cell metabolism experiments showed that E110 reversibly inhibits true and ChEs in a mixed manner in vitro; both types of inhibition occur, via competitive and non-competitive mechanisms [15]. In previous mutagenicity and clastogenicity studies [16-19] on E110, consistent and inconsistent results have been revealed. In the SCE assay with E110, concentrations up to 5.000μg/ ml gave incompatible results [20]. MN induction test results showed that E110 administered orally at a dose of 500 or 1.000 mg/kg BW, increased the MN frequency in the bone marrow of male rats [21]. E110 caused clastogenic effects [22]. And increased MN frequency in Chinese hamster fibroblasts [23], whereas similar effects were not observed in the in vivo studies on different laboratory animals [24]. Results of the previous genotoxicity studies on E110 are also contradictory. In a previous experiment, E110 administered orally at 500mg/kg BW did not change the timing of DNA synthesis [25]. E110 did not cause chromosomal disorders [26]. Similarly, Ishidate et al. [23] suggested that metabolically inactive E110 caused chromosomal damage at 6,000μg/mL concentration. However, in another study, 5.000μg/mL of both metabolically activated and inactive E110 was found to be ineffective [20]. NOAEL of E110 was determined as 6.000ppm for female and 12.500ppm for male rat [27]. In the mouse, 2.000mg/kg BW E110 given twice at 24 hours intervals increased mitosis frequency in the intestinal epithelial cells, whereas MN frequency did not change [28]. In the embryotoxicity trials, side effects were not observed in Charles River CD rats of 100, 300 or 1.000 mg/kg BW/D E110 administered via nasogastric gauge. Similarly, there were no negative effects on the reproductive system [29,30]. However, Mathur et al. [31] have observed significant effects on the testes of the rats received 250 and 1.500 mg/kg BW/D E110 for 90 days accepted LOAEL as 250mg/kg BW/D.

In the skin tests, the people with eczematous hypersensitivity to p-phenylenediamine gave cross-sensitivity to E110 [32]. This crossreaction is explained by the ability of the dye molecule to easily transform into compounds similar to quinone structure binding structural molecules [33].

Because the lack of a placental barrier in the avian species, chicken eggs have become a widely preferred test material in experimental studies to determine the negative effects of external factors on the embryonic development of the immune system. Spleen, which is the largest peripheral lymphoid organ in both the adult mammals and poultry species, mainly allows the removal of foreign organisms and aged red blood cells from the circulating blood [34].

The white pulp is mainly constituted of lymphoid follicles and lymphatic cords, and red pulp contains red pulp regions and venous sinuses all those form parenchym of the organ. The most abundant cells are lymphocytes in the white pulp of the spleen. Specific regions populated by T- and B-lymphocytes are distinguished in the spleen, as in the other secondary lymphoid organs. T-lymphocytes mainly populate adventitial layer of the central artery and form PALSs, while B-lymphocytes mostly occupy the GCs of the lymphoid follicles. Because that the lymph nodes are not well developed in the avails, the role of the spleen in the chicken immune system is vital and its embryonic development should not to be adversely affected by either external or internal factors. Therefore, disorders occurring during embryonic development of the spleen may result in significant deficiencies in both cellular and humoral immune functions in the post-hatch period of the chicken [35].

Precursors of T and B lymphocytes migrate to primordium of thymus and bursa of Fabricius via blood circulation and mature in these central lymphoid organs [36]. In the chicken embryo, the migration begins between 10th-14th days of day of incubation [37]. These cells form organ-specific lymphatic cords and lymphoid follicles in later periods of the embryonic development. In these follicles, B-lymphocytes populate GCs and T-lymphocytes mainly locate in cortical areas of the folicles and PALS [35,36].

In many animal species including chickens, ruminants, dogs and humans, mature T-lymphocytes give ANAE positivity with a very specific localized granular color reaction [38,39]. While the null cells give a fine granular staining [37], monocytes/macrophages display a strong and diffuse ANAE positivity [38-40].

ACPase is also a lysosomal enzyme of myelocytes, PMNLs, lymphocytes, plasma cells, megakaryocytes, blood platelets and mononuclear phagocytes. Lymphocytes give large granular positivity, whereas the reaction product is diffuse granular in monocytes [40]. In the avian species, the ACPase positivity has been suggested to be specific for B-lymphocytes [40-42].

In the present study, effects of E110 on the development of spleen, migration and localization of T- and B-lymphocytes were determined by means of histological, and enzyme histochemical methods during embryonic development of the chicken embryo.

Materials and Methods

From Ross 508 line, 250 fertilized eggs were used as egg material. The eggs were weighed and then disinfected by fumigating with 130ml of formaldehyde 37% and 80g of potassium permanganate vapor. Relative embryo weight was calculated with the following formula; (embryo weight/egg weight) X 100. The eggs were grouped as given in the Table 1, each group having 50 eggs. Sterile 20μl of test solution was injected via blunt end of each egg, immediately sealed with liquid paraffin and incubated in 1.000 egg-capacity incubator (Veyisoğlu, Istanbul, Turkey) under optimum conditions (37.8ºC and 65% relative humidity).