Effect of food azo-dye tartrazine on physiological functions of pancreas and glucose homeostasis

Introduction

Currently, food additives including food azo-dyes are most commonly used to enhance the color and taste of the food items and are considered as an important item of the food industry [1]. More than 2500 food additives have been reported to be used as colorants and preservatives [2]. Generally, food additives are classified into five major types; among which, coloring agents are an essential class that are mostly obtained from the animal and/or plant sources [3]. Since 1995, natural and synthetic coloring agents have been widely used in majority of food and pharmaceutical industries [4]. Pharmaceutical products and medicines such as antacids, vitamins, shells of medicated capsules and other prescription drugs are known to contain coloring agents [5].

There are number of coloring agents that are commonly used in pharmaceutical and food industries among which tartrazine is most commonly used. Tartrazine is a yellow to orange colored synthetic azo-dye which is also known as FD&C Yellow 5 and/or E102. Tartrazine is a water soluble colorant that is used in many food products like cotton candy, corn flakes, cake, soft drinks, chips, sauces, jelly, ice creams, sauces, snacks, mustard, chees, foodstuffs, pickles, chewing gum and drugs [5], [6]. The chemical name of tartrazine is trisodium-5 hydroxy-1-(4-sulfonatophenyl)-4-(4-sulfonatophenylazo)-H-pyrazole-3-carboxylate (Figure 1) and contains azo linkage (–N=N–) in its structure that is known to be metabolized into sulfanilic acid and aminopyrazolone by intestinal bacteria. Whereas, aminopyrazolone is further cleaved into sulfanilic acid by the action of intestinal bacteria [7]. However, excess intake of tartrazine has shown various adverse effects such as urticaria, migraine, rashes and gastro-intestinal disturbances [8]. World Health Organization (WHO) expert committee on food additives has recommended 0–7.5 mg/kg body weight of tartrazine as a maximal daily acceptable intake [7], [9]. Several studies have reported variable results about deleterious effects of tartrazine. Tartrazine has known to show chromosomal aberration in somatic cells of rats [10]. Moreover, 10 mg/kg dose of tartrazine has depicted carcinogenic effects on colon which is a dose that is very close to the daily acceptable intake of tartrazine recommended by WHO [7]. Despite of such toxic effects observed on tartrazine-exposure, this azo-dye is still cheaply available worldwide and has been observed to be used as an alternative for β-carotene which is considered to be more expensive than tartrazine [11].

Figure 1:

Structural formula of tartrazine.

Pre-diabetes is a condition that has high risk for diabetes and is characterized by high blood sugar level as compared to that of the normal, but lower than the level found after onset and progression of diabetes. However, interestingly, 5–10% pre-diabetic people have been recognized to develop diabetes mellitus (DM) every year. Experts have estimated that more than 470 million people might be in the range of pre-diabetes by the year 2030 globally. Pre-diabetes is correlated with β-cell’s dysfunction and insulin resistance which is the inability of peripheral tissues to utilize insulin for metabolism of glucose [12], [13]. Studies have also shown that pre-diabetes can lead to the increased risk of neuropathy, nephropathy, retinopathy, kidney and macrovascular diseases. Thereby, it is essential to identify and eradicate unnecessary exposure to the risk factors that not only lead towards pre-diabetes but also increases the chances of prevalence of DM.

The growing literature provides a great deal of evidences regarding toxic compounds like tartrazine and their relation to human health particularly, has revealed the toxic effects of tartrazine on public health however, till now, a very limited work has been done on exploring the exposure of tartrazine on endocrine system and its association with endocrine dysfunction and metabolic disorders. Hence, we opted to evaluate the effect of tartrazine-exposure on the normal functioning of pancreas, an important organ of the body that regulates the glucose homeostasis using rats as an experimental animal model. Furthermore, we also investigated the effect of tartrazine on insulin sensitivity, glucose tolerance, status of antioxidant enzyme, serum levels of amylase and lipase and morphology of pancreas. We hypothesized that exposure to tartrazine might probably alter the normal morphology of pancreas and glucose homeostasis.

Materials and methods

Results

Effect of tartrazine-exposure on glycemic status

In present study, we investigated the exposure of tartrazine on glycemic status of experimental animal model. Normal glucose homeostasis is indicated by the serum and/or blood levels of glucose and insulin. The present study showed that tartrazine-exposure exhibited its effects on the glucose homeostasis by altering the levels of blood glucose and serum insulin. At the end of treatment, both low and high dose of tartrazine significantly increased the levels of glucose (118.5±1.68 mg/dL) and (130±2.59 mg/dL), respectively as compared to the control group (Figure 2A). Similarly, at the end of treatment, we also investigated the impact of tartrazine-exposure on serum level of insulin. We found that both low and high dose of tartrazine significantly decreased the levels of insulin (0.468±0.008 ng/mL) and (0.395±0.012 ng/mL), respectively as compared to the control group (Figure 2B).

Figure 2:

Effect of tartrazine-exposure on glycemic status: rats were divided into three groups having equal number of rats (n=6) in each group.

Group I was control group that only received normal saline, while group II and III received low dose (10 mg/kg body weight of rat) and high dose (50 mg/kg body weight of rat) of tartrazine respectively for 30 days. The time courses of serum levels of glucose (A) and insulin (B) at pre-treatment (0 day), during treatment (15^th day) and post-treatment (30^th day) have been expressed as mean±SD. *,**p<0.05 when compared to control group.

Effect of tartrazine-exposure on amylase activity

To investigate the effect of tartrazine-exposure on carbohydrate metabolism, we measured the serum level of amylase. The results mentioned in Figure 3 indicate that at the end of treatment, both low and high dose of tartrazine significantly increased the serum level of amylase (429.660±6.940 U/L) and (646.830±9.780 U/L) respectively as compared to that of control group. This indicate the disruption of normal function of pancreas by tartrazine due to which the production of carbohydrate metabolizing enzyme (amylase) was increased which in turn might have increased the production of glucose in GIT.

Figure 3:

Effect of tartrazine-exposure on amylase level: rats were divided into three groups having equal number of rats (n=6) in each group.

Group I was control group that only received normal saline, while group II and III received low dose (10 mg/kg body weight of rat) and high dose (50 mg/kg body weight of rat) of food azo-dye tartrazine respectively for 30 days. The time course of serum levels of amylase at pre-treatment (0 day), during treatment (15^th day) and post-treatment (30^th day) have been expressed as mean±SD. *,**p<0.05 when compared to control group.

Effect of tartrazine-exposure on lipase activity

We also assessed the exposure of tartrazine on fat metabolism by measuring the serum level of lipase (U/L). Lipase is an enzyme that plays active role to hydrolyze lipids and ester bonds in triglycerides, to form fatty acids and triglycerides. At the end of treatment, we found that tartrazine significantly increased the serum level of lipase enzyme in both tartrazine-exposed groups as compared to that control group (Figure 4).

Figure 4:

Effect of tartrazine-exposure on lipase level: rats were divided into three groups having equal number of rats (n=6) in each group.

Group I was control group that only received normal saline, while group II and III received low dose (10 mg/kg body weight of rat) and high dose (50 mg/kg body weight of rat) of food azo-dye tartrazine respectively for 30 days. The time course of serum levels of lipase at pre-treatment (0 day), during treatment (15^th day) and post-treatment (30^th day) have been expressed as mean±SD. *,**p<0.05 when compared to control group.

Effect of tartrazine-exposure on calcium and magnesium level

We also evaluated the effect of tartrazine-exposure on the serum levels of calcium (mg/dL) and magnesium (mg/dL). The results mentioned in Figure 5 indicate that the serum levels of calcium and magnesium were decreased in tartrazine-exposed groups as compared to that of control group.

Figure 5:

Effect of tartrazine-exposure on calcium and magnesium level: rats were divided into three groups having equal number of rats (n=6) in each group.

Group I was control group that only received normal saline, while group II and III received low dose (10 mg/kg body weight of rat) and high dose (50 mg/kg body weight of rat) of food azo-dye tartrazine respectively for 30 days. The time courses of serum levels of calcium (A) and magnesium (B) at pre-treatment (0 day), during treatment (15^th day) and post-treatment (30^th day) have been expressed as mean±SD. *,**p<0.05 when compared to control group.

Effect of tartrazine-exposure on antioxidant enzyme status

We also investigated the effect of tartrazine on antioxidant enzyme capacity by measuring the serum level of SOD. At the end of treatment, we found that tartrazine-exposure significantly decreased the antioxidant enzyme capacity as the serum levels of SOD were found to be significantly decreased in tartrazine-exposed rat groups as compared to that of control (Figure 6).

Figure 6:

Effect of tartrazine-exposure on antioxidant enzyme capacity of pancreas: rats were divided into three groups having equal number of rats (n=6) in each group.

Group I was control group that only received normal saline, while group II and III received low dose (10 mg/kg body weight of rat) and high dose (50 mg/kg body weight of rat) of food azo-dye tartrazine respectively for 30 days. The time course of serum levels of superoxide dismutase (SOD) at pre-treatment (0 day), during treatment (15^th day) and post-treatment (30^th day) have been expressed as mean±SD. *,**p<0.05 when compared to control group.

Effect of tartrazine-exposure on glucose tolerance

Before the end of treatment period, we performed OGTT to investigate the effect of tartrazine-exposure on glucose tolerance in experimental animal model. We administered glucose at the rate of 2 g/kg body weight of the rat after an overnight fasting. We collected the blood from the tail of rats before (0 min) and after the administration of glucose (30, 60, 90 and 120 min). From the results mentioned Figure 7, we found that the maximum concentration of blood glucose was achieved at 30 min. Among the three rat groups, the concentration of blood glucose in rat group exposed with high dose of tartrazine (50 mg/kg body weight) was significantly high (230.830±0.940 mg/dL) as compared to the control rat group treated with normal saline (195.830±1.240 mg/dL). Moreover, the blood glucose level of rats exposed to high and low dose of tartrazine were also found to be significantly high at all-time points when compared with control rat group (Figure 7). Tartrazine significantly impaired the glucose tolerance due to which the ability of peripheral tissues to utilize the exogenously administered glucose, was impaired. During OGTT, no significant difference (p<0.05) was observed in the blood glucose levels between the rat groups exposed to low dose (10 mg/kg body weight) and high dose (50 mg/kg body weight) of tartrazine at almost all time points after the administration of glucose. After 120 min of glucose administration, the blood glucose level in control group was 99.500±0.760 mg/dL having non-significant difference with its basal blood glucose level (88.660±2.450 mg/dL) whereas, the low and high dose of tartrazine exposed rat groups exhibited the higher levels of blood glucose (117.830±0.940 mg/dL, 131.160±1.440 mg/dL) as compared to their basal blood glucose levels (92.000±0.570 mg/dL, 90.160±0.600 mg/dL), respectively (Figure 7).

Figure 7:

Effect of tartrazine-exposure on glucose tolerance: before the end of treatment period, oral glucose tolerance test (OGTT) was performed by administering glucose (2 g/kg body weight) to all rat groups (control, low dose and high dose groups) after an overnight fasting.

The time course of serum levels of glucose at 0 min, 30, 60, 90 and 120 min has been expressed as mean±SD. *,**p<0.05 when compared to control group.

Effect of tartrazine-exposure on insulin sensitivity

During OGTT, we further elucidated the impact of tartrazine-exposure on insulin sensitivity and/or resistance using HOMA-IR model which was based on the fasting levels of insulin and glucose. While performing OGTT, we evaluated the levels of fasting insulin and glucose. Upon the evaluation of HOMA-IR, the values for low and high dose of tartrazine-exposed rat groups were found to be significantly high (1.370±0.120, 1.450±0.090, respectively) as compared to that of the control. These high values indicated that tartrazine decreased the insulin sensitivity by increasing insulin resistance (Figure 8).

Figure 8:

Effect of tartrazine-exposure insulin sensitivity: insulin sensitivity/resistance was assessed using HOMA-IR (homeostasis model assessment for insulin resistance).

HOMA-IR was calculated using the values of serum levels of fasting glucose and fasting insulin.

Effect of tartrazine-exposure on morphology of pancreas

H&E staining of pancreas was done at the end of treatment period. After histopathological examination, we found that control group treated with normal saline exhibited the normal endocrine cells that were properly arranged and lined up surrounded with plenty of capillaries (Figure 9A). Both low dose (10 mg/kg body weight) and high dose (50 mg/kg body weight) of tartrazine affected the pancreas massively. Tartrazine exhibited significant impact on the normal morphology of pancreas (Figure 9B and C, respectively). Moreover, it was observed that the endocrine cells were smaller in size due to their proliferation.

Figure 9:

Effect of tartrazine-exposure on pancreas morphology: histopathology of pancreas was performed at the end of treatment period.

(A) Control group showing normal ratio and structure of pancreatic beta islet cells (IC) and acinar cells (AC), (B) low dose exposed group showing reduced number of islet cells (IC) and sloughed acinar cells (AC) and (C) high dose exposed group depicting moderate to severe damage of islet cells (IC) and acinar cells (AC) along with vacuolated cells (arrows).

Discussion

In the last many years, considerable interest has been paid to evaluate the toxicological evaluation of food additives and colorants. Food colorant and/or additive is any pigment or dye that exhibits its color when added into the soft drink, food and/or any non-food item such as drugs or pharmaceuticals [14], [15]. Among the five classes of food colorants, tartrazine is considered as very toxic to the human beings if consumed in excess amount [16], [17], [18], [19]. Toxicological studies conducted on human beings exhibit that ingestion of tartrazine may cause several behavioral changes, sleep disturbance and endocrine disruptions in children [20] whereas, toxicological studies conducted on experimental animals indicate that tartrazine (both higher and lower doses) alters various biochemical markers of vital organs [5], [21]. More recent studies have found that tartrazine has the ability to bind with albumin and cease the normal physiological functions of this protein [22], [23], [24].

In present study, we investigated the impact of tartrazine-exposure on pancreatic function of experimental animal model. Pancreatitis is a condition in which the inflammation of pancreas occurs. Pancreas is one of the main organ of the endocrine system that consists of alpha-(α) and beta-(β) cells. The α-cells are known to secrete the hormone glucagon and β-cells are known to secrete insulin hormone. Insulin mainly responsible to regulate the glucose homeostasis within the body. When the level of glucose in blood exceeds the normal limit, it triggers the β-cells of pancreatic islets to secret insulin which in return metabolizes the excess glucose accordingly, but if the β-cells of pancreatic islets are not functioning properly, insulin secretion is impaired due to which the levels of glucose in blood becomes high [12], [13], [25]. In our study, we found that tartrazine significantly impaired the normal functioning of pancreas due to which the secretion of insulin in response of glucose was impaired (Figure 2B). Moreover, due to impaired insulin secretion, the levels of glucose in blood were also increased exponentially (Figure 2A). Production of oxidative stress in pancreatic islets disturbs the balance between antioxidative enzymes and reactive oxygen species (ROS) which leads to further increase in the production of oxidative stress and as results, normal functioning of the pancreatic islets is disrupted. Tartrazine exhibited its toxic effects in dose-dependent manner by potentiating the mechanism of oxidative stress [5], [26] which was further verified by measuring the levels of SOD (Figure 6). The suppressed expression of SOD indicated that tartrazine induced oxidative stress in β-cells of pancreatic islets due to which the impaired insulin secretion occurred (Figure 2B). Moreover, tartrazine also induced the oxidative stress in peripheral tissues due to which impaired glucose tolerance (Figure 7) and insulin resistance (Figure 8) occurred. Further, we verified our results by applying HOMA-IR model to assess the ability of peripheral tissues to utilize insulin for the metabolism of glucose. We found that insulin resistance in tartrazine exposed animal groups was very high (Figure 8) as compared to the control group which indicated that tartrazine induced the insulin resistance (Figure 8) by generating the mechanism of oxidative stress.

Carbohydrates and fats are the main sources of energy in our daily diet. Once ingested, these are metabolized into glucose and fatty acids by enzymes namely amylase and lipase, respectively. Acute pancreatitis is a condition in which the pancreas is inflamed. Pancreas is one of the main endocrine glands that secrets variety of enzymes and hormones in feed-back mechanism. In case of acute pancreatitis, the secretion of these enzymes in response to feed-back mechanism is affected. Likewise, amylase and lipase are metabolizing enzymes that are also secreted from the pancreas. In acute pancreatitis, the secretion of these enzymes is also altered. In our study, we also measured the levels of these two enzymes to assess the impact of tartrazine on pancreatic function. We found that the levels of amylase (Figure 3) and lipase (Figure 4) were significantly increased in the groups exposed to tartrazine. The increased levels of amylase and lipase in blood indicate that tartrazine exhibited its toxic effects on pancreas by inducing the oxidative stress in pancreas due to which the inflammation in pancreas occurred and altered the normal functioning of pancreas.

Calcium and magnesium are known to play their significant role in the proteins synthesis of pancreatic enzymes and in acute pancreatitis, the levels of calcium and magnesium are decreased due to which hypocalcemia and hypomagnesemia occur [27], [28], [29]. In our present study, we found that tartrazine significantly decreased the levels of calcium and magnesium (Figure 5) which indicated that tartrazine impaired the normal functioning of pancreas by inducing the oxidative stress in pancreas. From several studies, it has been reported that deficiency of calcium and magnesium is strongly associated with the development of diabetes mellitus [30], [31], [32]. Tartrazine is a food colorant that belongs to group of azo dye. It is metabolized into aromatic amine by intestinal bacteria and the resultant aromatic amines will generate ROS due to their metabolism by interaction of nitrite or nitrate containing foods with these amino groups [5]. ROS such as hydroxyl radical, superoxide anion and hydrogen peroxide could be produced due to the metabolism of nitrosamines and increase oxidative stress. Due to ROS formation, the anti-oxidant defensive mechanism of the cells starts to become activated and compromised to prevent the cell death by toxic radicals. Therefore, SOD levels in the serum are lowered [5]. In our study, we also assessed the anti-oxidant enzyme capacity of pancreas by measuring the serum levels of SOD. Tartrazine decreased the serum levels of SOD (Figure 6) in tartrazine exposed experimental rats. The results of our study were inconsistent with already published report [5]. Once oxidative stress is produced, it exhibits its drastic effects on surrounding cells and tissues. We also investigated the hazardous effects of tartrazine on the normal morphology of pancreas by performing the H&E staining of pancreas. The results exhibited in Figure 9 indicate that tartrazine affected the pancreas massively (Figure 9C) and endocrine cells were smaller in size due to their proliferation. Due to alteration in the morphology of pancreas, it could not secret sufficient amount of insulin to metabolize the glucose.

Our study may have some limitations as we only focused on the impact of tartrazine on pancreatic functions and glycemic status of experimental animal model. Therefore, we propose that further studies may be required to investigate the impact of tartrazine on endocrine systems and its association with the causative factors of DM. Another limitation of our study is that we only focused the impact of tartrazine-exposure on the pancreatic function and glycemic status of experimental animal model, therefore, based on significant impact of tartrazine-exposure on pancreatic function, further studies may also be required to compare the association of tartrazine-exposure and development of DM in diabetic patients.

Conclusion

This study provides scientific evidence that exposure of tartrazine even in small doses may exhibit its potential toxic effects on endocrine system due to which the normal functioning of endocrine system alters which may lead to interfere the glucose homeostasis, carbohydrate and fat metabolism, and glucose tolerance. Moreover, tartrazine also alters the normal physiology of pancreas due to which the impaired insulin secretion from β-cells of pancreatic islets occurred. The results of present study also highlight the unexpected toxic effects of tartrazine and urge the attention of regulatory authorities for use of colorant to ensure the public health. Furthermore, there should be proper labeling on tartrazine containing food items so that, the patients that are at high risk of diabetes should not consume these products.