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Effect of Artificial Food Coloring Agents on Health Biomarkers, Antioxidant Enzymes, Hepatic Functions, Metabolic Hormones and DNA Damage in Rabbits

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Background: Food coloring agent is any coloring chemical substance added in food or drinks to impart
color and to enhance the aesthetic sense of food. These coloring agents are used blindly and excessively
in everyday life without considering their potential impact on the health status.
Objective(s): The present study was conducted to find any harmful physiological effects aggravated by
the use of artificial food colors.
Methodology: A total of twenty (20) rabbits were procured from the local market and divided into two
assemblies, treated and control. The treated assembly was granted 30mg/kg body weight of a blend of
synthetic food colors (red (amaranth) E123, green E140 and sunset yellow E110) through gastric tube
daily for 30 days. The health biomarkers, antioxidant enzymes, liver enzymes, metabolic hormones and
DNA impairment of the lymphocytes were estimated in both assemblies.
Results: The outcomes revealed that the health biomarkers like serum total antioxidant capacity (TAC)
and total oxidant status (TOS) were radically higher (P≤0.05) in the treated rabbits contrast to control.
The serum malondialdehyde (MDA) concentrations were statistically significant (P≤0.05) on day 30, in
the treated rabbits mismatched to control group. The nitrite and nitrate concentrations showed significant
raise (P≤0.05) on days ‘0’ and 7 of the experimental period, respectively in the treated group as compared
to control. The antioxidant enzyme catalase was substantially higher (P≤0.05) on day 7 post treatment in
the treated rabbits than control assembly. Paraoxonase-1 activity (PON1) and ceruloplasmin (Cp)
concentrations were considerably higher (P≤0.05) on day 30, post treatment in the treated assembly. The
aspartate transaminase (AST) and alanine transaminase (ALT) concentrations of treated rabbits were
significantly higher (P≤0.05) on days 0 and 30, as compared to control. The serum metabolic hormones
T4 and T3 concentrations were significantly increased (P≤0.05) at days 0 and 30, in the treated rabbits
than in control ones. The extent of DNA damage was significantly higher (P≤0.05) in treated rabbits on
day 30 of the experiment than in the control group.
Significance: The findings of our study signify that synthetic food colors should be considered harmful
as they induce oxidative and cellular damage by altering the genetic make-up of the body through the
production of reactive oxygen species.
Keywords: health biomarkers, oxidative stress, synthetic food colors
Food additives are being incorporated into food products internationally for preservation, coloring and
sweetening of food items. They may be natural or synthetic (Harris, 1986) and principal classes of food
additives are coloring agents, preservatives, flavors, emulsifiers and stabilizers (Lindsay, 1985). Food
coloring agent is any coloring chemical substance added in food or drinks to impart color and to enhance
the aesthetic appearance of food (Vaclavik and Christian, 2003). Therefore from the organoleptic point of
view, the visual aspect is an important factor for the choice of the products by the consumer (Himri et al.,
2011). On the other side, some of the food additives and colorants however, have been omitted from use
because of their health hazard risks through experiment on some vital organ functions of the animals
(Hassan, 2009). Many azo compounds when used as colorants in foods proved to be genotoxic and
carcinogenic (Sasaki et al., 2002). Now a days interest in natural and hygenic foods has therefore raised
numerous questions regarding the safety of the many chemicals, both synthetic and naturally occurring
that are present in food. Keeping in view the above said issue, the present study has been designed to
investigate the physiological effects of food colors on the health status of rabbits.
Results and Discussion
1. Health biomarkers
Total antioxidant capacity in the serum of rabbits treated with synthetic food colors was significantly
(P≤0.05) higher on days 0 and 30 of treatment than control group (Table 1). The serum total oxidant
status of treated rabbits was increased significantly (P≤0.05) throughout the experimental trial compared
to control ones (Table 1). Under physiological conditions, the body usually has sufficient antioxidant
reserves to cope with the production of free radicals (Miller et al., 1993; Castillo et al., 2003), which are
produced continuously during metabolism and may increase as a result of pathological and other
circumstances (Roth, 2000). Therefore, free radical generation developed the oxidative stress (Castillo et
al., 2005). In the present study level of antioxidants was decreased due to generation of free radicals and
oxidative stress on day seven and thirty of experimental trial in rabbits fed with synthetic food colorants
as reported earlier (Himri et al., 2011). The serum malondialdehyde level was increased significantly
(P≤0.05) on day 30 post treatment as compared to control group (Table 1) and plasma MDA is considered
as biomarker for oxidative stress (Nielsen et al., 1997). The MDA level was increased as a result of
action of the ROS on lipids of cellular membrane (Bansal, 2005). Further, Rybczynska et al. (1996) found
that lipid peroxidation of cell membranes is associated with inactivation of membrane bound enzymes.
Nitric oxide production assay revealed significantly higher (P≤0.05) level of serum nitrite and nitrate
level on days 0 and 7 of treatment trail, respectively as compared to control group (Table 1). Moreover,
lowest level of serum nitrate and nitrite was observed on day 30 of treatment in treated rabbits than
control rabbits (Table 1). These results find good support from the study carried out by Hassan (2009)
who illustrated a marked increase in nitrate and nitrite rats after treatment with sunset yellow or carmine.
Moreover, the adverse effects of nitrate diet may occur in relation to peroxidation (Angelis et al., 1996).
2. Antioxidant enzymes
Intracellular antioxidant enzyme catalase activity was statistically higher (P≤0.05) on day 7 post
treatment in the serum of rabbits treated with synthetic food colors as compared to control group (Table
1). As a result of the reactive oxygen species (ROS) formation, the antioxidant defense mechanism of the
cells including catalase and reduced glutathione (GSH) began to consume to prevent the cell death by
these toxic radicals so their levels were decreased (Bansal, 2005). Therefore catalase is frequently used
by cells to rapidly catalyze the decomposition of hydrogen peroxide into less reactive gaseous oxygen
and water molecules (Gaetani et al., 1996). It was further noticed that activity of PON1 and
concentrations of serum ceruloplasmin were increased significantly (P≤0.05) on day 30 of treatment trial
compared to control rabbits (Table 1). Ceruloplasmin is important in the control of membrane lipid
oxidation, probably by direct oxidation of cations, thus preventing their catalysis of lipid peroxidation
(Taysi et al,. 2002).
3. Liver enzymes
The serum concentration of alanine aminotransferase in the rabbits treated with synthetic food colors
was significantly higher (P≤0.05) at day 30 than control group and lowest concentration of ALT was
recorded on day 7 post treatment when compared with control (Table 1). Moreover significantly higher
(P≤0.05) concentration of aspartate aminotransferase was noticed at day 0 than days 7 and 30 post
treatment in the serum of treated rabbits (Table 1). ROS plays an important role in pathological changes
in the liver (Poli and Parola, 1997). Increased generation of ROS or free radicals is therefore able to
cause auto-oxidation of the hepatic cells, resulting in marked hepatic lesions (Suzuki et al., 1998).
4. Metabolic hormones
Triiodothyronine concentration was significantly higher (P≤0.05) at day 30 as compared to control and
day 0 of treatment (Table 1). The serum thyroxine concentration was increased significantly (P≤0.05) at
day 0 of treatment and lowest level was recorded at day 30 post treatment in the serum of treated rabbits
(Table 1). In present work the combination of food colors may give a new chemical component, which
has a stimulatory effect on thyroid gland. This effect could be attributed to its chemical structure that can
compete with thyroxine binding globulin leading to its deficiency and to hyperthyrodism by feed
back mechanism (Gold and Vladutin, 1994).
5. DNA damage
The results of DNA damage revealed that extent of mean DNA damage after day 30 of treatment was
found to be significantly higher (P≤0.01) in the treated group compared to control rabbits (Figure 1).
The formation of comet (tail) of DNA at day 30 post treatment was shown in the microphotograph of
treated and control rabbits (Figure 2, 3). The percentage of DNA in the tail is the most appropriate
parameter to analyze induced DNA damage (De-Boeck et al., 2000) and considered a good biomarker of
toxicity of food additives (Himri et al., 2011). Therefore, the imbalance between free radical production
and body inability to scavenge them result in the oxidation of DNA and nucleic acids results in strand
breaks possibly through the production of free radicals (Zeiger, 1993).
Our findings revealed that use of synthetic food colorants should be considered harmful and should be
used cautiously to minimize the adverse effects of oxidative stress in terms of free radical production
ultimately resulting in DNA damage, liver damage and lipid peroxidation of cell membranes.
A) Study design
A total of twenty (20) rabbits viz treated and control comprised of 10 each; were procured from the
local market and kept under normal conditions in the animal room of the Department of Physiology and
Pharmacology, University of Agriculture, Faisalabad, Pakistan. The treated group was given a mixture of
3 synthetic food colors (red (amaranth) E123, green E140 and sunset yellow E110; Xi'an Day Natural
Tech Co., Ltd. Shaanxi, China) through gastric tube on daily basis at the dose rate of 30mg/kg body
weight for a period of 30 days. The first sampling of blood was carried out from all rabbits at day zero
and 2nd and 3rd sampling from treated rabbits was done on day 7 and 30 of the experimental trial
respectively. The blood was centrifuged at 1107×g for 15 minutes for 15 minutes for the separation of
serum and collected in small eppendorfs that were stored at -20 0C till further analysis. All the
experimental procedures were carried out in accordance with the guide for the humane use and care of
animals, approved by the Animal Care Committee of University of Agriculture-Faisalabad, Pakistan.
B) Health biomarkers
1. Total antioxidant capacity (TAC; mmol/L)
The total antioxidant capacity (TAC) was measured through spectrophotometer Biosystem BTS-330
Biosystems, S.A. Costa Brava, Barcelona, Spain) by adopting the methodology as described by Erel
2. Total oxidant status (TOS; μmol/L)
Total oxidant status was determined according to the method of Erel (2005) using Biosystem BTS-330
(Biosystems, S.A. Costa Brava, Barcelona, Spain) spectrophotometer.
3. Malondialdehyde (MDA; nmol/mL)
A spectrophotometric method described by Okhawa et al. (1979) was used for the determination of
MDA level in the collected samples.
4. Nitric oxide production assay (µmol/L)
The concentrations of nitrate and nitrite in the serum samples were determined using
spectrophotometer according to the method established by Kitrina et al. (2001).
C) Antioxidant enzymes
1. Catalase activity (Ku/L)
Serum catalase activity was determined spectrophotometerically by using the method established by
Goth (1991).
2. Paraoxonase-1 activity (PON1; U/L)
The enzymatic activity of paraoxonase-1 was measured through spectrophotometer by using the
method of Juretic et al. (2006).
3. Ceruloplasmin (Cp; U/L)
Serum ceruloplasmin concentration was determined by adopting the procedure as described by
Schoslnsky et al. (1974) using spectrophotometer (Biosystem BTS-330; Biosystems, S.A. Costa Brava,
Barcelona, Spain).
D) Liver enzymes and metabolic hormones
The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum samples
were measured using commercially available kits (Randox Laboratories, Ltd. United Kingdom).
For the quantitative determination of thyroxine (T4) in the serum, T3 and T4 EIA (enzyme
immunoassay) kit (JD Biotech, Via Scozia-Zona Ind.le-64026, Roseto degli Abruzzi (TE), Italy was
E) Comet assay (DNA damage; µm)
The comet assay (extent of DNA damage) in the present study was performed under alkaline
conditions using serrated microscopic slides according to Singh et al. (1988) and the extent of DNA
damage was assessed on the basis of flouricensing tail, making a comet on florescent microscope at X-
400 and X-1000. The tail length was measured by using computer operated automated image analysis
software (Image J- 1.44p; National Institute of Health, USA).
D) Statistical analysis
Data obtained was analyzed by one way analysis of variance to check the significance of results using
SPSS version 11.5 (SPSS Inc., Chicago, IL, USA). Tukey’s HSD test was applied to determine the
significant difference between treated and control rabbits at different days of treatment.
Table-1: Health biomarkers, antioxidant enzymes, liver enzymes, and serum metabolic hormones
(mean±SE) of treated and control rabbits (fed orally with synthetic food colors)
Figure: -1 DNA damage (mean±SE) of control and treated rabbits
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The authors are thankful to the Chairman Department of Physiology & Pharmacology, University of
Agriculture, Faisalabad, Pakistan for providing technical and financial assistance to complete the
research work.
For additional information contact:
Saima Sadaf
Govt. College of Home Economics, Gulberg, Lahore, Pakistan.
1Government College of Home Economics, Lahore, Pakistan
2Department of Rural Home-economics, University of Agriculture, Faisalabad, Pakistan
3Sub-campus Toba Tek Singh, University of Agriculture, Faisalabad, Pakistan
4Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan
Saima Sadaf*1, Naheed Abbas2, Zia ur Rahman3, Zafar Iqbal4 and Asma Lodhi2
0 day 7 day 30 day
Total antioxidant capacity
(TAC; mmol/L)*
Total oxidant status
(TOS; μmol/L)*
(MDA; nmol/mL)*
16.17±0.059dc 17.01±0.322cb 18.35±0.369b26.04±0.774a
Nitrate (µmol/L)*49.09±0.078b49.68±0.122b76.22±0.376a33.46±0.136c
Nitrite (µmol/L)*0.90±0.024b1.90±0.031a0.94±0.005b0.76±0.009c
Catalase activity (Ku/L)* 48.50±0.215d68.60±0.574c140.04±0.563a83.96±0.496b
Paraoxonase-1 activity
(PON1; U/L)*
(Cp; U/L)*
Alanine transaminase
(ALT; U/L)*
16.96±0.195cb 17.65±0.126ba 15.36±0.403d18.14±0.149a
Aspartate transaminase
(AST; U/L)*
16.59±0.150ba 17.47±0.225a15.66±0.353cb 15.26±0.421dc
(T3; ng/mL)*
0.89±0.008dc 0.94±0.003cb 1.01±0.021ba 1.080±0.037a
(T4; µg/dL)*
3.86±.05099ba 4.16±0.247a3.46±0.092cb 2.84±0.223dc
“*” indicates significance at (P≤0.05)
Similar alphabets do not differ significantly at (P≤0.05)
Control Treated
DNA damage (µm)
Figure:- 2 Microphotograph of DNA of control rabbit (not showing
any comet)
Figure:-3 Microphotograph of DNA (lymphocytes) of treated rabbit
fed with synthetic food colors showing comet (arrows)
There are various undesirable products generated from endogenous aerobic metabolism such as reactive oxygen species (ROS). Physiological and biochemical lesions are caused by ROS and which give rise to oxidative damages towards DNA, proteins and lipids which ultimately lead to cell death. This study was aimed to examine the effect of oral administration of food colorants (tartrazine and curcumin) on the oxidants and antioxidants level in blood and fecal of rats after 15, 30, and 45 days. Two doses were used based on the admissible daily intake (ADI) of 9.6 and 96 (high) mg/kg/body weight for tartrazine, 3.85 and 38.5 6 mg/kg/body weight for curcumin. The results showed that oral administration of tartrazine had significantly increased the total oxidant level, arylesterase, glutathione reductase, and MDA whereas there was significantly decreased the total antioxidants level, catalase, glutathione peroxidase in plasma and fecal after 30 and 45 days. Vitamin E and C were decreased in plasma. Fecal showed high level of vitamin A. High dose of tartrazine caused alteration in the aldehyde reactive probe (ARP) sites of DNA showing the DNA damage. After 45 day, significant increment was observed in the level of AST in low and high curcumin treated group. Whereas, elevations of arylestrase were seen in high curcumin group after 45 day. High dose of curcumin significantly (P≤ 0.05) decreased the concentration of vitamin C after 45 days of treatment and increased the level of vitamin E in plasma of treated groups after 30 and 45 days of treatment. The present study showed that the ADI and doses up to 10 times higher than ADI showed negative effects on antioxidant level and demonstrated the importance of using appropriate doses of food colorants such as tartrazine and curcumin in different processed food products.
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