Open access peer-reviewed chapter - ONLINE FIRST
Body weight and relative kidney weight of experimental animals.
Abstract
The food azo dye tartrazine (Е102) is not metabolized in humans, and it is excreted unchanged through the kidneys. A study was carried out on the biochemical and histological features in the kidneys of rats, as well as the biochemical features in the blood and urine during long-term use of tartrazine E102 (1 ml of 0.1% solution per 100 g of animal weight daily). In the first experiment, animals received a solution of tartrazine from 1 month up to 3 months age per os; in the second experiment, individuals received tartrazine first transplacentally (in utero), then with mother’s milk, and during 1 month only with food. In the animals of the first experiment, the biochemical changes are not profound, and it is morphologically shown that the losses of the parenchyma and stroma of the kidney are compensated and regenerated. In the second experiment, oxidative stress and signs of chronic glomerulonephritis and chronic tubulointerstitial nephritis were detected against the background of disorders of intrauterine kidney development.
Keywords
- food additive tartrazine (E102)
- kidneys
- biochemical disorders
- histological damage
- rats
1. Introduction
In modern society, the incidence of various nephropathies in children has sharply increased [1]. Children’s chronic glomerulonephritis, which occurs latently and supposedly does not have an etiological factor, is especially difficult to treat and prognosis. The most common nephropathies in children are pyelonephritis and interstitial tubular nephritis [2].
According to Goyer [3], the kidneys, as the main excretory organ, are the target of many xenobiotics. Xenobiotics damage filtering membranes and disrupt the glomerular-tubular balance. The high level of blood supply and the long distance of the tubular apparatus determine the duration of contact of toxic substances and their metabolites with the renal endothelium, epithelium, and cell interstitium. Positive hydrostatic pressure necessary for ultrafiltration, other intrarenal processes aimed at preserving essential metabolites and eliminating toxins, and the work of the countercurrent multiplying system lead to recirculation of low molecular weight metabolites in the body, deposition of uncharged toxins in the renal interstitium and the development of the inflammatory process [4].
According to the literature, various toxic agents can play the role of an etiological factor in 5–20% of cases of interstitial nephritis, in 8% of pyelonephritis, and in 20% they could be the cause of acute renal failure [5].
The effect on the kidneys of children of a large number of various synthetic food additives that are present in the food of urban residents has not been sufficiently studied. A combined harmful effect of these impurities on the kidneys and liver can be expected.
Research in recent decades has proven that certain kidney diseases, which predominate in preschool and school age, originate in the antenatal and prenatal period [6]. The formation of renal pathology is associated with impaired renal embryogenesis. Dysembryogenesis of the urinary system organs can manifest itself as gross anomalies of the kidneys and urinary tract (often complicated by pyelonephritis and urolithiasis), and tissue dysembryogenesis. This can serve as the basis for the formation of immune glomerulopathies or dysplasia of renal tissue, the development of nephropathies, and tumor processes [7, 8]. It is known that the maternal and fetal organisms are closely interconnected (the structural and functional unity of the “mother-placenta-fetus” system), therefore it is clear that the functional state of the mother’s kidneys affects the state of the offspring’s kidneys [9]. In this regard, interest has arisen in studying the possible effect of women’s long-term use of foods containing nutritional supplements, before conception and during pregnancy, on the condition of the kidneys in their offspring.
The opinion about the hidden causes of nephropathies in children led to an attempt to evaluate the effect on the kidneys of a food additive—a synthetic yellow dye—tartrazine (E 102), widely used in the food industry in the production of soft drinks, confectionery, ice cream, caramel, and sweets [10]. From the literature, it is known that tartrazine is not involved in metabolic processes in the body but is excreted from the body unchanged. There is no information in the literature about its possible accumulation in the kidneys. At the same time, in the scientific literature, there is information about the toxic effect of tartrazine on the kidneys of experimental animals (article on turmeric).
The purpose of the study was to evaluate the possible role of tartrazine in the latent development of various nephropathies.
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2. Data literature
One of the most important organoleptic properties of food products that attracts the buyer’s attention is their color. Particularly popular is bright yellow, a sunny color that is loved by adults and children; everyone associates it with joyful feelings. There are many yellow plant pigments, but they are not resistant to environmental factors. Therefore, almost all manufacturers use synthetic dyes when preparing products. One of these dyes, most often used in the food industry, is tartrazine (food additive E 102). Tartrazine is used to color egg yolks, in the manufacture of chips, confectionery, drinks, spaghetti, fish pates, and to color smoked fish in all countries [11]. Currently, tartrazine is also used in the pharmaceutical industry in the manufacture of capsules [12].
A regulated concentration policy for various dyes in foods, cosmetics, and drugs has been emphasized and stated. The average daily intake of tartrazine was set as 7.5 mg/kgbw [13]. Synthetic dyes have shown controversial effects on the health of humans.
Tartrazine is a xenobiotic; it is not included in metabolic processes, is excreted from the body unchanged, and is used exclusively as a dye. That is why manufacturers and users consider it absolutely harmless. Doubts arose only after 8 years of using E102 in connection with the publication of its name and formula. It turned out that this is 5-hydroxy-1-(4-sulfonatophenyl)-4-(4-sulfonatophenylazo)-H-pyrazol-3-carboxylate – trisodium, because it is a compound with an azo group, and are prepared from aromatic amines, which contain an azo group of two nitrogen atoms linked together (-N〓N-) and linked to aromatic rings [14]. Compounds with such a group activate free radical oxidation in biological objects, which leads to damage to cellular structures. However, the toxic effect of tartrazine is not immediately detected; in the long term, it can only be revealed in experiments. However, experimental data carried out by different researchers gave conflicting data—non-toxic [15] and highly toxic [16, 17, 18]—and did not clarify the understanding of the issue. Later studies carried out using high-precision methods showed that E102 has the ability to modify genes and cause mutations [18].
Medical statistics have revealed that tartrazine is also suspected of triggering urticaria [19] and angioedema (the swelling of the lips, tongue, throat, and neck caused by the release of histamine in an allergic reaction) [20]. It further acts as a neurotoxin, at least in rats (namely inducing problems with spatial memory) [21].
Back in the 90s, it was established that tartrazine is excreted unchanged from the body, but it is not known at what speed it is excreted. This is important since tartrazine will necessarily pass through the liver and kidneys, that is, organs with loose parenchyma and a large contact surface. Given the structure and chemical properties of tartrazine, it can be expected that long-term use of tartrazine may lead to disruption of the structure and function of these organs. Therefore, studying the effect of prolonged administration of tartrazine on the kidneys is a pressing issue.
2.1 Materials and methods
The experimental material was studied using biochemical and morphological methods, which made it possible to elucidate the metabolic, functional, and structural links of the possible mechanism of nephropathy development.
The experiments were carried out on 50 rats (male and female) of the Wistar line. From the age of 2 months (“adolescent” age), rats received every day for 1 month, first for 10 days intragastrically (
The control group—intact animals of the same age—was given 1 ml of saline according to the same scheme. Some of the animals, 17 females and males of the main group and 17 females and males of the control group, were removed from the experiment by decapitation at the age of 3 months. The second part of the animals (five females of the main group and five females of the control group) were placed with males. After fertilization, pregnant females, and then mothers-rats while nursing continued to give the tartrazine solution.
The descendants of these females, who received tartrazine during feeding with mother’s milk and immediately after the end of feeding—as part of food, 1 ml of a 1% solution every day for 1 month, were withdrawn from the experiment at the age of 2 months (20 rats of the main group and 20 rats control group). Thus, two pairs of animals’ “control-main group” were formed, namely young adult animals that consumed tartrazine for 2 months at a “young” age from 1 month to 3 months (gr. K1 and gr. O1, respectively), and 2-month-old offspring animals that were born under conditions of tartrazine consumption by pregnant animals, while breastfeeding, and consumed tartrazine independently for 1 month (gr. K2 and gr. O2, respectively).
Objects of study: blood serum, kidney homogenates, urine, kidney tissue.
2.2 Research methods
After completion of the administration of tartrazine, the rats were removed from the experiment by decapitation using a guillotine. Cubital blood was collected, and at autopsy, the kidneys were isolated. A sample of kidneys (approximately 150 mg) was crushed with scissors in a mortar on ice, then saline was added (at the rate of 10 ml of saline per 1 g of tissue), homogenized in a Potter homogenizer (on ice) until a formation of homogeneous mass. The homogenate was centrifuged at 600 g for 10 minutes. The supernatant was used for biochemical studies. Serum was obtained from cubital blood (coagulation followed by centrifugation), which was used for biochemical studies.
All experimental procedures with animals were carried out in accordance with the ethical standards of the 1975 Declaration of Helsinki, as revised in 2008, and in accordance with national legislation.
To assess the activity of lipid peroxidation, the content of diene conjugates (DC) and TBA-active products in kidney homogenates was determined. Determination of the content of diene conjugates was carried out according to the method of I Kammanesky, V. Cruer [21]. The DC content in the samples was expressed in mmol/g of protein. Determination of the concentration of TBA-active products in liver homogenates was carried out according to method Botsoglow et al. [22]. The content of TBA-active products was expressed in μmol per gram of protein. Measurements were carried out on a Scolar PV-128 spectrophotometer.
As is known, the intensity of free radical processes in cells and tissues is controlled by antioxidants; the state of the antioxidant system was evaluated by the activity of superoxide dismutase (SOD) and catalase in kidney homogenates. Superoxide dismutase (SOD) activity was determined by enzyme immunoassay using SOD ELISA Kit reagents from Elabscience (USA) and expressed in pkg/ml. The measurements were carried out on a semiautomatic Stat Fax analyzer (USA). Catalase activity was determined by the spectrophotometric method [23]. Measurements were carried out on a Scolar-PV 128 spectrophotometer.
The protein composition of blood serum was determined by the turbodimetric method using reagents from the company “Filicit-diagnostics” (Dnepr, Ukraine).
The content of matrix metalloproteinase-2 (MMP-2) was determined by enzyme immunoassay using a reagent kit from Elabscience (USA), expressed in ng/mg tissue. The measurement was carried out on an enzyme immunoassay analyzer STAT-FAX (USA).
The content of total protein, urea, creatinine, and aminotransferase activity were determined using reagent kits from Filicit-diagnostics (Dnepr, Ukraine).
The content of total protein in homogenates of renal tissue and urine was studied using the Lowry spectrophotometric method [24].
ATP concentration [25] was determined by spectrophotometric methods. Measurements were made on a Scolar PV-128 spectrophotometer.
The concentration of glycosaminoglycans in the urine was determined using a spectrophotometric method [26].
The material for morphological research—the right kidney—after fixation in 10% formaldehyde, was embedded in paraffin, and sections 5–6 μm thick were stained with hematoxylin-eosin, picrofuchsin according to Van Gieson, halocyanin according to Einarson, and the PAS-reaction was used to clearly identify the brush border of the tubular epithelium. Light microscopy and photography were carried out using Olympus BX-41 (Japan) and Axiostar (Zeiss, Germany) microscopes.
Immunohistochemical examination of the kidneys was carried out on paraffin sections 5—6 μm thick of the kidney, thymus and spleen using the direct Koons method according to the method of M. Brosman. The localization and abundance of IgG and CD16 were determined using monoclonal antibodies (mAbs) with a luminescent label (Chemicon, USA and Novocastra Ltd.). The preparations were studied and photographed in a luminescent microscope Axioscop 40 (Zeiss, Germany), using light filters: FS-1-2, SZS-24, BS-8-2, UFS-6-3.
All digital data were processed by variation statistics methods on a personal computer using the Statgraph software package.
2.3 Biochemical examination of blood, urine, and kidney tissue
When studying a group of adult female rats aged 3 months, which received an adequate dose of tartrazine with food for 1 month, it was revealed that the rats of the main group had body weight and relative kidney weight less than females of the control group (Table 1).
| Animal groups | Body weight, g | Relative weight of kidneys (%) |
|---|---|---|
| Group К1: | ||
| Females, | 289,5 ± 12,6 | 0,55 ± 0,03 |
| Males, | 277,5 ± 14,02 | 0,61 ± 0,04 |
| Group O1: | ||
| Females, | 205,7 ± 10,8; p < 0,02 | 0,41 ± 0,02; p < 0,02 |
| Males, | 233,3 ± 11,4; p < 0,02 | 0,52 ± 0,04; p < 0,02 |
Table 1.
The daily diuresis of animals in the main group did not differ from those in the control group. However, in the animals of the main group, hyaline casts were found in the urine (from 2 to 4 in the preparation).
The data obtained indicate the toxic effect of tartrazine on animals, which contributes to kidney damage.
One of the important indicators for assessing renal function is the proteinogram. Analysis of the total protein content in blood serum showed that the level in males of the main group is practically no different from its content in the control group. In females of the main group, it is slightly lower than in the control group (Table 2).
| Animal groups | Protein, g per L, Females | Protein, g per L, Males |
|---|---|---|
| Group К1, | 60,55 ± 3,48 | 57,82 ± 0,37 |
| Group O1, | 58,13 ± 2,63*; p < 0,05 | 57,55 ± 0,42; p > 0,05 |
Table 2.
Content of total protein in blood serum of experimental rats.
When studying the protein spectrum of blood serum, it was found that the use of food coloring tartrazine with food leads to a probable decrease in the amount of albumin and an increase in the concentration of γ-globulins in male and female rats. At the same time, the amount of other protein fractions does not differ from the control (Table 3). These data indicate activation of the humoral immune system and possible toxic damage to the kidneys.
| Animal groups | Albumins | Globulins | |||
|---|---|---|---|---|---|
| α1 | α2 | β | γ | ||
| Group К1: | |||||
| Females, | 50,51 ± 2,16 | 3,72 ± 0,22 | 8,52 ± 0,65 | 12,24 ± 1,12 | 25,04 ± 1,38 |
| Males, | 51,32 ± 3,0 | 3,84 ± 0,32 | 8,15 ± 0,53 | 13,32 ± 1,23 | 23,47 ± 1,33 |
| Group O1: | |||||
| Females, | 45,38 ± 3,01; p < 0,02 | 3,60 ± 0,26 p > 0,05 | 8,44 ± 0,72 p > 0.05 | 11,86 ± 1,00 p > 0.05 | 31,14 ± 2,0 p < 0.05 |
| Males, | 48,22 ± 3,46 p < 0,05 | 3,78 ± 0,25 p > 0,05 | 7,98 ± 0,68 p > 0,05 | 12,83 ± 1,00 p > 0,05 | 27,26 ± 1,03 p < 0.05 |
Table 3.
Protein fractions of blood serum of 3-month-old animals (in %).
As can be seen from the Table 4, in females and males of rats (more in females) the content of MMP-2 is significantly increased—one of the proteases of the extracellular matrix. It is specifically active in relation to collagen IV—the main component of basement membranes and plays a significant role in tissue remodeling, proliferation, and apoptosis. An increase in the content of MMZ-2 is probably a consequence of kidney damage.
| Index | Females | Males | ||
|---|---|---|---|---|
| Group К1 n = 10 | Group O1 n = 10 | Group К1 n = 10 | Group O1 n = 10 | |
| MMP-2 μg/mg | 1,43 ± 0,09 | 4,85 ± 0,12* | 1,36 ± 0,11 | 3,55 ± 0,14* |
Table 4.
MMP-2 content in blood serum of 3-month-old animals.
It is known that tartrazine is excreted from the body through the kidneys. In this regard, it can be assumed that its accumulation in the kidneys can have a toxic effect on the kidneys, and contribute to the development of immune inflammation. To assess kidney function, the content of creatinine and urea in blood serum was determined. As shown by our results (Table 5), the content of creatinine and urea in the blood serum of females and males of the main group is significantly higher than in animals of the control group, which indicates a decrease in filtration processes in the kidneys and, in connection with this, an increase in concentration slags in the blood.
| Animal groups | Transamidinase activity, μM/hour ml | Creatinine, μmol/1 | Urea, mmol/1 |
|---|---|---|---|
| Group К1: | |||
| Females, | 3,02 ± 0,19 | 91,35 ± 5,17 | 6,37 ± 0,45 |
| Males, | 2,15 ± 0,12 | 111,5,61 ± 9,55 | 7,00 ± 0,55 |
| Group O1: | |||
| Females, | 5,66 ± 0,14; p < 0,02 | 122,13 ± 6,22; p < 0,02 | 8,96 ± 0,48; p < 0,02 |
| Males, | 3,81 ± 0,27; p < 0,02 | 139,91 ± 7,33*; p < 0,02 | 8,62 ± 0,63; p < 0,02 |
Table 5.
Transamidinase activity, the amount of creatinine and urea in the blood of 3-month-old animals.
As can be seen from the given data (Table 5), the activity of the organ-specific “renal” enzyme transamidinase (to a greater extent in females) was significantly increased in the blood serum of rats, which indicates a violation of the permeability of nephrocyte membranes, that is, kidney damage.
The study of the content of protein, urea, creatinine, and glycosaminoglycans (GAG) in the urine of female rats revealed that the animals of the main group probably increased all the indicators that were studied (Table 6).
| Animal groups | Protein, mg/day | Creatinine, mM/day | Urea, mM/day | GAG, mg/g creatinine |
|---|---|---|---|---|
| Group К1, | 9,95 ± 0,55 | 10,52 ± 0,82 | 368,58 ± 11,27 | 1,34 ± 0,18 |
| Group O1, | 12,48 ± 1,08*; p < 0,01 | 12,89 ± 1,03; p < 0,05 | 385,22 ± 20,3; p < 0,05 | 2,45 ± 0,25; p < 0,01 |
Table 6.
The content of protein, urea, creatinine, and glycosaminoglycans in the urine of experimental animals.
Glucosaminoglycans are a carbohydrate component of proteoglycans (mucopolysaccharides), which is part of the intercellular substance of connective tissue. An increase in their content in urine indicates the destruction of connective tissue.
An increase in the content of protein, creatinine, and urea in the urine with a simultaneous increase in GAG may indicate pathological changes in the kidneys of animals. In eight females of the main group, a decrease in creatinine clearance was noted (1.12 ± 0.11 mg/min versus 2.08 ± 0.09 in the control group), which may indicate a decrease in the glomerular filtration rate.
The activity of the mitochondrial enzyme succinate dehydrogenase was determined in the urine of four animals (activity 3.14–4.11 μM/l). The presence of this enzyme in the urine may indicate damage to the renal tubules.
In kidney tissue homogenates, we investigated protein content and aminotransferase activity. The protein content in kidney tissue homogenates of animals of the main group (males and females) does not differ from the level of animals of the control group (Table 7). The activity of aminotransferases (AST and ALT) in males and females of the main group is probably higher than the control (Table 8), which indicates the activation of protein catabolism in the kidneys. However, since the protein content has not changed, it can be assumed that protein synthesis is also stimulated in the kidney tissue.
| Index | Control group | Main group | ||
|---|---|---|---|---|
| Females | Males | Females | Males | |
| Protein (total), g/l | 2,65 ± 0,17 | 2,55 ± 0,19 | 2,73 ± 0,23 p > 0,05 | 2,43 ± 0,22 p > 0,05 |
Table 7.
Protein content in kidney homogenates of 3-month-old animals.
| Animal groups | AcAT, μmol/g protein hour | AlAT, μmol/g protein hour |
|---|---|---|
| Group К1: | ||
| Females, | 55,42 ± 3,12 | 40,45 ± 2,63 |
| Males, | 46,84 ± 2,55 | 33,28 ± 2,31 |
| Group O1: | ||
| Females, | 65,58 ± 3,18*; p < 0,02 | 54,43 ± 2,63; p < 0,01 |
| Males, | 64,42 ± 3,11*; p < 0,02 | 52,49 ± 3,37; p < 0,01 |
Table 8.
Aminotransferase activity in the kidneys of 3-month-old animals that consumed tartrazine for 1 month.
That is, in the animals of the main group, the rate of protein metabolism is increased and the regeneration of protein structures in the kidney tissue is enhanced, which may indicate a high tension of functioning and metabolic processes in general.
Our study of the state of the LPO and AOS system in kidney tissue homogenates revealed that the content of lipid peroxidation products (malondialdehyde and diene conjugates) in females of the main group is somewhat higher than in the control group. The activity of catalase and superoxide dismutase (AOS components) was significantly higher than in the control group (Table 9). That is, the presence of oxidative stress in the kidneys of females who used tartrazine for 1 month was not detected, although there was an increase in the activity of lipid peroxidation processes.
| Animal groups | TBA-active products, μmol/1 | DC, μmol/1 | Catalase, μcatal/g of protein | SOD, pkg./ml |
|---|---|---|---|---|
| Group К1, | 5,73 ± 0,47 | 20,64 ± 1,38 | 2,07 ± 0,11 | 342,17 ± 1,48 |
| Group O1, | 7,08 ± 0,32*, p < 0,02 | 27,45 ± 1,28*, p < 0,05 | 3,25 ± 0,23*, p < 0,02 | 405,23 ± 3,17*, p < 0,01 |
Table 9.
Content of lipid peroxidation products and activity of antioxidant enzymes in kidney homogenates of female rats that consumed tartrazine.
Almost all the main processes in the kidneys are energy-dependent. Therefore, in our opinion, it was necessary to determine the concentration of ATP in the homogenate of rat kidneys. The data we obtained are presented in Table 10.
| Animal groups | ATP, μmol/g tissue, males | ATP, μmol/g tissue, females |
|---|---|---|
| Group К1, | 3,34 ± 0,22 | 2,51 ± 0,11 |
| Group O1, | 2, 21 ± 0,17; p < 0,02 | 1,66 ± 0,13*; p < 0,02 |
Table 10.
ATP content in kidney homogenates of experimental animals.
The study of the content of ATP—the main macroergic compound in the kidneys—proved that the production of ATP is significantly reduced in both females and males, which may be due to the accumulation of tartrazine in the kidneys and, in connection with this, the activation of lipid and protein peroxidation processes.
The use of tartrazine, as a food additive, by adult rats for months leads to a clear violation of metabolic processes in the kidneys, which is evidenced by a decrease in the level of ATP in the tissue. An increase in the content of urea and creatinine in blood serum and urine, a decrease in creatinine clearance, and an increase in the content of GAG in the urine can be considered as criteria for functional disorders in the kidneys. Thus, the obtained results allow us to draw a conclusion about the toxicity of adequate doses of tartrazine with its long-term use. In 2-month-old offspring of rats, signs of kidney dysfunction are more pronounced—significantly reduced body weight and specific weight of kidneys, compared to the control group, more pronounced increase in creatinine, urea, GAG in urine, and the content of MMP-2 in the blood.
2.4 Histological features of the kidney tissue (1st experiment)
In animals of group K1, females and males, the glomeruli are compact and dense; the capillaries have no gaps. This can be explained by the softness of their walls and a slight decrease after the blood leaves the lumen at the time of killing.
In general, glomeruli are paucicellular, that is, they consist of a small number of capillaries, and the mesangium has single macrophages. This is confirmed by an immunomorphological reaction with CD 16. Due to the small number of capillaries in the glomeruli, some of them have the shape of a divided glomerulus. The nuclei of the endothelial cells of the glomerular capillaries are dark and strongly flattened. In some places, thin and clear PAS-positive basement membranes of the glomerular capillaries can be seen. The lumen of Bowman’s capsule is wide.
The tubular epithelium is well preserved, and has a high content of RNA in the cytoplasm and round, moderately euchromic nuclei. The microvillus border of the tubular epithelium is wide and intensely stained with fuchsin (PAS-reaction). The interstitium in both the cortex and the medulla of the kidney is narrow, with single macrophages.
Compared with the histological structure of the kidney tissue of group K1 animals, the kidney of group O1 animals predominantly has multicellular large glomeruli. They appear spread out, often with erythrocyte cells in the lumen of the capillaries.
The basement membrane of the glomerular capillaries is thickened in some places (PAS reaction), and when performing an immunohistochemical reaction with antibodies to IgG, in contrast to group K1, a significant presence of IgG was found both on the basement membrane of the glomerular and in the mesangium. The nuclei of endothelial cells are lighter than the control ones. The mesangium contains more macrophages than in the control, which was also proven by an immunohistochemical reaction with mAb CD16. The lumen of Bowman’s capsule is wider than control.
The epithelium of the tubules was significantly damaged: the apical parts with a microvillus border are absent in some epithelial cells. For others, on the contrary, it is wider than control. This is most likely a compensatory reaction. In such epithelial cells, there is an increased content of RNA in the cytoplasm and more euchromic nuclei.
It is necessary to pay attention to the presence of a large number of bodies in a state of apoptosis in the epithelial cell layer of the tubules and in the glomeruli. This is a sign of intense apoptosis of both tubular epithelial cells and capillary endothelial cells. The interstitium of the kidneys is enriched with macrophages and lymphocytes.
Thus, taking an adequate dose of tartrazine solution with food in young rats for 1 month caused the appearance of morphological signs of increased morphofunctional activity of the glomeruli. Intense apoptosis and compensatory hypertrophy of the epithelium of the proximal tubules with microvillus border hyperplasia are observed. The stroma of the renal tissue is enriched with macrophages, and IgG is found on the basement membrane of the glomerular capillaries and in the mesangium.
2.5 Biochemical examination of blood, urine, and kidney tissue
The study of the protein spectrum of blood serum revealed that in the blood serum of 2-month-old males and females of the main group, in comparison with the control group. The content of α-globulins and γ-globulins increases with a significant decrease in the content of albumins, which can be considered as a nephrotic type of proteinogram (Table 11).
| Animal groups | Albumins, % | Globulins, % | ||||
|---|---|---|---|---|---|---|
| α1 | α2 | β | γ | |||
| Group К2 | Females, | 58,84 ± 2,31 | 2,45 ± 0,12 | 8,34 ± 0,41 | 9,61 ± 0,54 | 21,37 ± 1,54 |
| Males, | 56,74 ± 3,06 | 3,42 ± 0,24 | 7,62 ± 0,51 | 10,43 ± 0,65 | 21,81 ± 1,45 | |
| Group O2 | Females, | 50,84 ± 1,84 p < 0,02 | 2,03 ± 0,17 p > 0,05 | 11,45 ± 0,55 p < 0,05 | 10,07 ± 0,84 p > 0,05 | 25,24 ± 1,85 p < 0,05 |
| Males, | 49,36 ± 2,05 p < 0,02 | 3,76 ± 0,18 p > 0,05 | 10,24 ± 0,45 p < 0,05 | 9,79 ± 0,43 p > 0,05 | 27,13 ± 1,38 p > 0,05 | |
Table 11.
Protein spectrum of blood serum of 2-month-old rats.
The study of the content of MMP-2 in the blood serum (Table 12) showed that in animals of the main group, the content of the enzyme increases (especially in males). This fact, together with an increase in the γ-globulin and α2 globulin fraction, can be considered as the presence of the process of immune inflammation and apoptosis. The α2-globulin fraction includes ceruloplasmin, haptoglobin, and α2-macroglobulin, the concentration of these proteins is known to increase in nephropathy (Table 13).
| Group К2 | Group O2 | |||
|---|---|---|---|---|
| Females, | Males, | Females, | Males, | |
| MMP-2, μg/ml | 0,75 ± 0,04 | 0,97 ± 0,04 | 2,17 ± 0,13; P < 0,01 | 2,89 ± 0,05; p < 0,01 |
Table 12.
Content of MMP-2 in blood serum of rats.
| Index | Гpyпa К2 | Гpyпa O2 | ||
|---|---|---|---|---|
| Females, | Males, | Females, | Males, | |
| Creatinine, μmol/L | 40,85 ± 3,11 | 48,14 ± 6,32 | 55,11 ± 4,55; p < 0,01 | 61,22 ± 8,15; p < 0,01 |
| Urea, mmol/L | 5,64 ± 0,37 | 6,75 ± 0,54 | 8,59 ± 0,28; p < 0,01 | 9,36 ± 0,37; p < 0,02 |
Table 13.
Content of creatinine and urea in blood serum.
The study of the content of creatinine and urea in blood serum showed that in males of the main group in 95% of cases and females in 100% of cases, the content of both substances in blood serum increases, which reflects a violation of the filtration function of the kidneys (Table 14).
| Animal groups | Protein, mg/day | Urea, mmol/day | Creatinine, mmol/day |
|---|---|---|---|
| Group К2, females, | 11,35 ± 1,08 | 305,67 ± 10,33 | 11,48 ± 0.94 |
| Group O2, females, | 40,65 ± 1,47; p < 0,001 | 346,47 ± 12,55; p < 0,01 | 19,22 ± 1,11*; p < 0,01 |
| Group К2, males, | 10,47 ± 1.11 | 310,37 ± 14,77 | 13,43 ± 1,07 |
| Group O2, males, | 34,14 ± 1,48*; p < 0,001 | 367,43 ± 2,34; p < 0,01 | 23,56 ± 1,22*; p < 0,001 |
Table 14.
Content of protein, urea, and creatinine in experimental animals.
Urinalysis revealed a significant increase in the concentration of urea and creatinine, and protein excretion (Table 14), which indicates a violation of glomerular filtration in both female and male rats of the main group. That is, the degree of kidney damage in the offspring of rats that used tartrazine is higher than in adults.
The assessment of daily diuresis revealed that there were no significant differences between females of the control and experimental groups on this indicator. In males, diuresis was reduced in 80% of cases (compared to the control group of animals), hyaline cylinders (4–6 in the preparation) were found in the urine of all males. At the end of the experiment, almost all males showed swelling in the neck and head, which, as can be seen, is associated with hypoproteinemia.
Analysis of biochemical indicators of kidney homogenates also revealed features that indicate impaired kidney function in 2-month-old rats that consumed tartrazine (Tables 15 and 16). As can be seen from Table 15, the activity of aminotransferases increased in males and females of the main group, which indicates an increase in protein catabolism. This assumption is confirmed by the decrease in the content of total protein in the homogenates of the kidneys of animals of the main group (Table 16).
| Animal groups | AcAt, μmol/h/l | AlAt, μmol/h/l | Total protein, g/l | |
|---|---|---|---|---|
| Group К2 | females, | 66,24 ± 2,18 | 47,85 ± 1,89 | 2,65 ± 0,17 |
| males, | 51,28 ± 1,89 | 36,43 ± 2,0 | l,89 ± 0,12;p < 0,05 | |
| Group O2 | females, | 71,35 ± 2,13; p < 0,05 | 51,67 ± 1,63; p < 0,02 | 1,82 ± 0,29 |
| males, | 59,75 ± 3,11; p < 0,02 | 42,61 ± 1,55; p < 0,02 | 1,28 ± 0,07; p < 0,05 | |
Table 15.
Aminotransferase activity and the presence of protein in blood homogenates.
| Animal groups | TBA-active products, μmol/L | DC, μmol/L | Кaтaлaзa, мккaт/л | SOD, y.oд./г бiлкy | |
|---|---|---|---|---|---|
| Group К2 | females, | 7,65 ± 0,53 | 23,11 ± 1,65 | 1,89 ± 0,12 | 54,81 ± 1,5 |
| males, | 8,39 ± 0,62 | 30,25 ± 2,08 | 1,23 ± 0,08 | 49,75 ± 1,22 | |
| Group O2 | females, | 9,33 ± 0,57; p < 0,02 | 27,13 ± 1,38; p < 0.02 | 2,03 ± 0,19; p > 0,05 | 52,45 ± 2,09; p < 0,05 |
| males, | 12,45 ± 1,13* | 41,55 ± 1,89* | 0,92 ± 0,06* | 40,79 ± 2,03* | |
Table 16.
Content of lipid peroxidation products and activity of antioxidant protection enzymes in kidney homogenates.
A study of the indicators of the process of lipid peroxidation (LPO) and the state of the antioxidant system in kidney homogenates revealed an increase in the concentration of diene conjugates and TBA-active products (more pronounced in males) and an insufficient response of the AOS system. As can be seen from the data presented in Table 15, in females the levels of TBA-active products (MDA) and diene conjugates are significantly increased, catalase activity does not differ from the level in rats of the control group, and the level of SOD in females of the main group is lower than in the control group. This is a manifestation of the presence of oxidative stress. In males of the main group, the increase in the concentration of lipid peroxidation products is higher than in females. At the same time, the state of the oxidative system is even worse; the activities of both Catalase and SOD are reduced compared to the control group of males. Thus, in 2-month-old rats that received tartrazine from the prenatal period, there is oxidative stress in the kidneys, which is more pronounced in males.
Urinalysis revealed a significant increase in the concentration of urea and creatinine, and protein excretion (Table 16), which indicates impaired glomerular filtration in both females and males of the main group. That is, the degree of kidney damage in the offspring of rats that used tartrazine is higher than in adults.
In 2-month-old offspring, signs of renal dysfunction are more pronounced—a higher degree of increase in the concentration of creatinine, urea, protein in the urine, the appearance of a significant number of hyaline casts, a decrease in the specific gravity of the kidneys, a more significant increase in the concentration of MMP-2 in the blood, the presence of oxidative stress in kidney homogenates, a marked decrease in the concentration of ATP in the kidneys, the appearance of enzymes in the urine. The features of biochemical parameters we identified in the offspring of rats receiving tartrazine in their diet indicate that kidney damage develops quickly, which is apparently associated with a violation of their structure during embryogenesis.
Thus, the use of tartrazine as a dietary supplement by adult rats for months leads to a clear violation of metabolic processes in the kidneys, which is evidenced by a decrease in the level of ATP in the tissue, an increase in the content of urea and creatinine in blood serum and urine, a decrease in creatinine clearance and an increase in the content of GAG in the urine can be considered as criteria for functional disorders in the kidneys. Thus, the obtained results allow us to draw a conclusion about the toxicity of adequate doses of tartrazine with its long-term use.
2.6 Histological features of the kidney tissue (2nd experiment)
In group K2, the features of the histological structure of the kidneys correspond to a state of low morphofunctional load. Most glomeruli look compact, the lumen of the capillaries cannot be seen, and the nuclei of all glomerular cells are hyperchromic and small in size. The epithelium of the proximal tubules is well preserved, the apical parts have a high PAS-positive border with a color of medium intensity. The tubules are located very densely, the interstitial layers are very thin. Immunohistochemical reactions revealed the episodic presence of macrophages in the interstitium of the kidneys, in the glomerular mesangium, as well as a small amount of IgG in the basement membrane and glomerular mesangium.
In gr. O2 (photo) glomeruli of the kidneys are unevenly located in the cortex. A significant part of them is reduced, represented by a small number of capillaries. In some glomeruli, when stained with picrofuchsin using the Van Gieson method, small foci of mesangium sclerosis can be seen. The remaining glomeruli, on the contrary, are multicellular and large in size, which also has a compensatory value. Many glomeruli showed large numbers of macrophages in the mesangium and IgG in the capillary wall. The basement membrane of the capillaries in the glomeruli is thickened and rigid, since the capillaries do not collapse due to their sclerosis. In some glomeruli, the presence of serous exudate is detected in the lumen of Bowman’s capsule.
The epithelium of te proximal tubules in the outer layer of the cortex has signs of hydropic lystrophy, the PIC-positive border is poorly preserved, the nuclei of epithelial cells are often pyknotic, some epithelial cells are without nuclei. In the inner layer of the cortex, the epithelium of the proximal tubules is better preserved, the nuclei are larger and lighter than the control ones, and the cytoplasm contains more RNA, but the PAS-positive apical border is discontinuous, that is, absent in some places, and preserved in other places, of different heights and varying degrees of staining intensity. Halocyanin staining for nucleic acids reveals a large number of apoptotic bodies at the level of the proximal tubule epithelium. In some places, desquamated epithelial cells are found in the lumen of the distal tubules. In the interstitium of the renal medulla, there are macrophage infiltrates and foci of sclerosis. In medium-sized vessels, perivascular sclerosis occurs, and the wall of the arterioles is sclerotic.
Thus, in the offspring of tartrazine-using mothers (group O2), a microscopic picture of a violation of kidney embryophetogenesis is observed—the glomeruli in the cortex are unevenly located, many glomeruli turned out to be reduced. Normally developed glomeruli function under increased load and are hypertrophied. The appearance of IgG and an increased number of macrophages in some glomeruli indicates the formation of immune damage and inflammation of the glomeruli, the process of sclerosis of both the capillary walls and the mesangium is intense, and there are histological signs of chronic mesangiocapillary glomurolephritis.
At the same time, the morphological picture of damage to the tubular epithelium, in places with the accumulation of macrophages in the stroma, can be called the beginning of the development of chronic tubulointerstitial nephritis.
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3. Discussion
There is very little information in the scientific literature available to us about the effect of tartrazine on the condition of internal organs in particular the kidneys. Along with the statement about the complete safety of tartrazine (no toxicity [27]), there are works that show that tartrazine is highly toxic—it contributes to an increase in the level of free radicals, which leads to oxidative stress and disorders of metabolism [28]. There is also information about the genotoxicity of tartrazine and its ability to activate carcinogenesis, oxidative stress, and the development of metabolic disorders [29].
Our experimental study in two parts showed that the use of tartrazine, as a grub supplement, in young rats over the course of 1 month leads to a significant disruption of metabolic processes in young rats. This leads to a decrease in the level of ATP in the tissue, which is very important since almost all processes in the kidneys are energy-dependent. The data we obtained on studying the state of the LPO/AOS system indicate an increase in the level of LPO with an adequate increase in AOS. Perhaps the reason for the difference in the results of the previously mentioned publication and our data is that the study [30] used a longer period of administration (50 days), which could contribute to the exhaustion of the AOS system.
A morphological study of renal tissue in the first experiment revealed facts confirming the activation of both dystrophic and hypertrophic processes. For example, the brush border of the proximal tubular epithelium is absent in places, lysed, and in places appears very wide and intensely PAS-positive. The studies [30 turmeric] revealed an increase in the activity of caspase-3, which is consistent with the increase in MMP-2 content that we detected, and histological examination made it possible to det Оect apoptotic bodies both in the glomeruli and at the level of the tubular epithelium. The increase in the concentration of urea and creatinine in blood serum and urine, a decrease in creatinine clearance, and an increase in the content of GAG in urine can be considered as criteria for functional disorders in the kidneys. Moreover, microscopic examination revealed a noticeably increased infiltration of the kidney stroma and mesangium by macrophages, which directly indicates damage to the stromal elements. Thus, the obtained results allow us to conclude about the toxicity of adequate doses of tartrazine with и удельной массы почек its long-term use, but for some time the lesions may be compensated.
In 2-month-old rat pups who were exposed to tartrazine in utero and during milk feeding, and then received it independently with food, that is, in offspring rats, renal dysfunction is more pronounced—a higher degree of increase in the concentration of creatinine, urea, and protein in the urine. The appearance of a significant number of hyaline casts in the urine sediment is observed, a more pronounced decrease in the body weight of rats and the specific gravity of the kidneys, a more significant increase in the concentration of MMP-2 in the blood, the presence of oxidative stress in kidney homogenates, a pronounced decrease in the concentration of ATP in the kidneys, and the appearance of enzymes in the urine.
Histological examination visualized this biochemical pattern. It was found that the glomeruli are distributed unevenly in the cortex, apparently as a result of the early intrauterine death of part of the glomeruli. Due to the presence of transplacental entry of tartrazine into the fetal body, a restructuring of the renal tissue apparently occurred, which can be called disembryofetogenetic. Analyzing frequent cases of renal hypoplasia and dysplasia in children, clinical researchers recognize that not only genetic changes can lead to this pathology but also the action of epigenetic factors and environmental factors [30, 31, 32]. This experimental study of the transplacental effect of the food coloring tartrazine E 102 on the kidney tissue of the offspring fully confirms this point of view. Further, the individual-descendant received this substance from the mother’s milk, and then independently caused damage in hypoplastic and dysplastic kidneys and the beginning of the formation of chronic masangio-capillary glomerulonephritis and tubulo-interstitial nephritis.
The experimental data we obtained, a decrease in body weight and specific gravity of the kidneys of rats of groups O1 and O2 compared with rats K1 and K2, respectively, as well as typical “kidney” proteinograms in rats of groups O1 and O2 indicate the nephrotoxicity of tartrazine.
The mechanism of the damaging effect on the kidneys can be presented as follows. Under the influence of the dye, which is slowly removed from the kidneys, accumulating in the arenchyma, lipid peroxidation processes are activated, and oxidative stress develops in O2 group rats. Activation of LPO is known to be accompanied by an increase in the concentration of proteins and lipids oxidized by peroxides. This leads to damage to membranes, including the basement membrane (as evidenced by an increase in the content of protein and GAGs in the urine); and other authors have found persistent microscopic hematuria in children [33]. Damage to the membranes leads to disruption of oxidative processes in the kidneys, a low-energy shift in the adenyl system, and, as a consequence, disruption of the processes of filtration, reabsorption, and secretion. This may not be the only way tartrazine can damage the kidneys. Available data in the literature on the toxic effects of tartrazine (with long-term use) on organs indicate that the dye contributes to memory impairment and behavioral reactions by activating the synthesis of neurotoxins by intestinal bacteria [34]. In addition, there is evidence that tartrazine activates the inflammatory process in the intestine, affecting the state of lymphocytes [29], has genotoxic and mutagenic properties, increases histamine secretion [17], and has a specifically damaging effect on the liver [35]. This information suggests that tartrazine is not immediately (and possibly not completely) eliminated from tissues and has the ability to accumulate. Also, it is the multiplicity of mechanisms of action that makes one wonder whether it has any specific surface properties. In medicine, there is no convincing evidence of the connection between diseases and the duration of tartrazine use; most likely, signs of pathology appear after a long period of time, that is, latent kidney pathology is formed, which makes it very difficult to identify a connection with the use of tartrazine. However, experimental studies prove this connection; moreover, it has been established that the development of pathological processes in mice under the influence of tartrazine depends on genetics, diet, and duration of use [36], which confirms the diversity of mechanisms of action and determines the complexity of preventive measures.
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4. Conclusion
The features of biochemical and histological parameters that we identified in offspring rats that received tartrazine in utero, then with milk feeding and independently with food, indicate that kidney damage develops quickly, which is apparently associated with a violation of their structure in embryfetogenesis, and occurs latently (if we consider the human).
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