Effect of food azo dyes tartrazine and carmoisine on biochemical parameters

related to renal, hepatic function and oxidative stress biomarkers in young male rats
K.A. Amin
a,
*
, H. Abdel Hameid II
b
, A.H. Abd Elsttar
b
a
Biochemistry Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, Egypt
b
Chemistry Department, Faculty of Science Beni-Suef, Beni-Suef University, Beni-Suef, Egypt
a r t i c l e i n f o
Article history:
Received 18 May 2010
Accepted 26 July 2010
Keywords:
Tartrazine carmoisine
Oxidative stress
Liver
Biochemical parameters
a b s t r a c t
Tartrazine and carmoisine are an organic azo dyes widely used in food products, drugs and cosmetics. The
present study conducted to evaluate the toxic effect of these coloring food additives; on renal, hepatic
function, lipid prole, blood glucose, body-weight gain and biomarkers of oxidative stress in tissue.
Tartrazine and carmoisine were administered orally in two doses, one low and the other high dose for
30 days followed by serum and tissue sample collection for determination of ALT, AST, ALP, urea, creat-
inine, total protein, albumin, lipid prole, fasting blood glucose in serum and estimation of GSH, catalase,
SOD and MDA in liver tissue in male albino rat.
Our data showed a signicant increase in ALT, AST, ALP, urea, creatinine total protein and albumin in
serum of rats dosed with tartrazine and carmoisine compared to control rats and these signicant change
were more apparent in high doses than low, GSH, SOD and Catalase were decreased and MDA increased in
tissue homogenate in rats consumed high tartrazine and both doses of carmoisine.
We concluded that tartrazine and carmoisine affect adversely and alter biochemical markers in vital
organs e.g. liver and kidney not only at higher doses but also at low doses.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Food additives play a vital role in todays bountiful and nutri-
tious food supply, they allow our growing population to enjoy a
variety of safe, wholesome and tasty foods year round, and they
make possible an array of convenience foods without the inconve-
nience of daily shopping.
The Egyptian famous food additives which are used as coloring
substances are tartrazine and carmisione.
Food colors are materials of natural origin have been used to
provide color in foods; drugs and cosmetics for thousands of years,
Ash from res mineral compounds and plants were probably
among the rst materials used for cosmetic purposes (Gaunt
et al., 1972).
By the early 1995, natural and synthetic color additives were
used extensively to color foods, drugs and cosmetics (Hallagan
et al., 1995). Color is an important characteristic and selection cri-
terion for food choice, recent studies have high lighted this impor-
tance and have shown how selection may change among certain,
populations, and over time (Clydesdale, 1993). Colorant plays a sig-
nicant role in enhancing the artistic appeals of foods that are aes-
thetically pleasing more likely to be consumed and to contribute to
a varied diet (Hallagan et al., 1995).
Tartrazine known as E102 or FD&C Yellow 5 or C.I. 19140 is a
synthetic lemon yellow azo dye used as a food coloring. It is de-
rived from coal tar. It is water soluble and principally is the triso-
dium 5-hydroxy-1-(4-sulfonatophenyl)-4-(4-sulfonatophenylazo)-
H-pyrazol-3-carboxylate.
Many products contain tartrazine like foods cotton candy, soft
drinks, avored chips (Doritos, Nachos, etc.), cereals (corn akes,
muesli, etc.), cake mixes, soups, sauces, some rice, ice cream, can-
dy, chewing gum, marzipan, jam and jelly, some of non food prod-
ucts include tartrazine such as soaps, cosmetics, shampoos and
other hair products, also some medical preparations contain tartra-
zine such as vitamins, antiacids, medicinal capsules and certain
prescription drugs. The ADI for tartrazine is 7.5 mg/kg/day (Walton
et al., 1999).
Carmoisine and its different names as Azorubine, Food Red 3,
Azorubin S, Brillant carmoisine O, Acid Red 14, or C.I. 14720 is a
synthetic red food dye from the azo dye group. It usually comes
as a disodium salt. It is a red to maroon powder; it has been used
for the purposes where the food is heat-treated after fermentation.
It has E number E122 carmoisine present in food like blancmange,
marzipan, Swiss roll, jams, preserves, yoghurts, jellies, bread-
crumbs, and cheesecake mixes. It is also present in oraldene
mouthwash.
0278-6915/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.07.039
* Corresponding author.
E-mail address: [email protected] (K.A. Amin).
Food and Chemical Toxicology 48 (2010) 29942999
Contents lists available at ScienceDirect
Food and Chemical Toxicology
j our nal homepage: www. el sevi er . com/ l ocat e/ f oodchemt ox
Because tartrazine and carmoisine are a nitrous derivatives (azo
class) they can be reduced in the organism to an aromatic amine
which is highly sensitizing. The main metabolite identied to date
is sulfanylic acid (Maekawa et al., 1987; Chung et al., 1992).
Two doses of synthetic dyes (low or high doses) mostly attrib-
utable to hepatocellular damage when the toxic effects of synthetic
dyes that tartrazine and carmoisine were among of them (ponceau,
carmoisine, erythrosine, sunset yellow, tartrazine, fast green,
indigotine, brilliant blue and brilliant black) were tested in rats
by the biochemical and histopathological examinations. Dyes exert
histopathological effects on the hepatic and renal tissues of the
rats, indicated by vacuolation, swelling, necrosis and pyknosis of
their cells (Mekkawy et al., 1998).
Histopathological studies showed brown pigment deposition in
the portal tracts and Van Kpffer cells of the liver and renal mainly
induced by colorant that include carmoisine (Aboel-Zahab et al.,
1997).
The individual response varies not only according to dose, age,
gender, nutritional status and genetic factors, but also according
to long term exposure to low doses (Sasaki et al., 2002).
Therefore the aim of this work is to study the effects of tartra-
zine and carmoisine as coloring agent widely used in food prod-
ucts, drugs and cosmetics on on some biochemical parameters in
serumof young male albino rats (as a model of children) associated
with liver and kidney functions, blood glucose and serum lipids,
also our study was extended to evaluate the effect of these addi-
tives on the biomarkers of the oxidative stress in tissue
homogenates.
2. Materials and methods
2.1. Chemicals utilized in the experiment
Tartrazine is a yellow color substance known as E102 or FD&C Yellow 5, dye
content in the substance is 87%, loss in drying and sodium chloride/sulphate is less
than 13%, water insoluble matter less than 0.2% lead less than 0.01 ppm Arsenic less
than 1 ppm, heavy metals less than 40 ppm, tartrazine was labeled as food colorant.
Carmoisine is a red color substance known as E122 or Food Red 3 Azorubin S,
Brillant carmoisine O, Acid Red 14, or C.I. 14720, dye content in the substance is
87%, loss in drying and sodium chloride/sulphate is less than 13%, water insoluble
matter less than 0.2% lead less than 0.01 ppm Arsenic less than 1 ppm, heavy metals
less than 40 ppm, carmoisine was labeled as food colorant. Tartrazine and carmoi-
sine were obtained from Star Aromatic Company for avors and fragrance food col-
ors (Egypt), which obtain these chemicals from Ajanta Chemical Industries (India).
2.2. Animals and treatments
A total of 36 young male (Rattus Norvegicus) albino rats weighting about 60
80 g were used in the present study. They were obtained from (National Research
Center, Cairo, Egypt). Animals were kept under observation for about 7 days before
the onset of the experiment to exclude any intercurrent infection. They were main-
tained in stainless steel cages at normal atmospheric temperature of 27 5 C as
well as under good ventilation. Our work was carried out in accordance with the
guidelines of Beni Suef University for animal use.
Tartrazine, and carmoisine were in a solid state so we prepared two solutions of
each substance (one low and the other high) by dissolving the solid in distilled
water, low doses of tartrazine and carmoisine were 15 and 8 mg/kg bw respectively
while high doses were were 500 and 100 mg/kg bw respectively.
Animals were fed on the standard basal diet and provided with tap water a
composition of experimental diets according to Kim et al. (2005) as follows: (fat
5%, carbohydrates 65%, protein 20.3%, ber 5%, salt mixture 3.7%, vitamins mixture
1%).
2.3. Sampling and tissue preparation
By the end of the experimental periods, venous blood samples were collected
from the orbital sinus of control, and food dyes treated rats via glass capillaries at
fasting state. The blood samples were collected in dry glass centrifuge tubes and al-
lowed to coagulate at room temperature and centrifuged at 3500 rpm for 15 min at
room temperature for separation of serum.
Homogenates of liver tissues were prepared by dissolving 0.25 g of liver tissue
in 5 ml of NaOH 0.9% in test tube then homogenized by the homogenizer for 15 min
then centrifuged by centrifuge for 10 min at 3000 rpm then the supernatants were
collected for determination of biomarkers of oxidative stress.
Activity of serum ALT and AST were determined by the method of Reitman and
Frankel (1957). Total protein was determined according to the method of Henry
(1964). Urea was determined in serum by the method of Patton and Crouch
(1977). Total cholesterol was estimated in serum by enzymatic colorimetric method
according the method of Allain et al. (1974) and Tamaoku et al. (1982). Triglycerides
in serum were estimated by enzymatic colorimetric method of Buccolo (1973).
HDL-cholesterol in serum was determined by enzymatic colorimetric method of
Fruchart et al. (1982). Glucose concentration in serum was estimated by enzymatic
colorimetric method according to Trinder (1969). Catalase activity was estimated in
tissue homogenate by method of Cohen et al. (1970). Liver reduced glutathione con-
tent (GSH) was determined according to the procedure of Beuther et al. (1963).
Determination of lipid peroxidation in tissue homogenate was determined accord-
ing to the method of Presuss (1998).
2.4. Statistical analysis
The statistical analysis was carried out using GraphPad Instat software (version
3, ISS-Rome, Italy). Unless differently specied, groups of data were compared with
un-paired t-test and one-way analysis of variance (ANOVA) followed by Tukeykra-
mer (TK) multiple comparisons post-test. Values of P < 0.05 were regarded as signif-
icant. The data, as clearly indicated are reported in tables and gures as
mean standard error (SE).
3. Results
The present study revealed that high dose of tartrazine and low
and high doses of carmoisine that were 15.4 0.80, 14.2 1.19, and
17.2 0.94 u/l respectively induced a signicant increase in serum
ALT activity in comparison to control group where being
8.91 0.27 u/l. Also high doses of tartrazine and carmoisine and
low and high doses of saccharin that their values are 55.38 1.00
and 52.91 2.54 u/l showed a signicant increase in serum AST
activity when compared to control rats where being
47.85 2.70 u/l (Table 1).
Both low and high doses of tartrazine, carmoisine that their val-
ues are 117.16 2.3, 119 2.93, 114.16 3.85, and 115.45
2.89 iu/l exhibited a signicant increase in serumALP activity when
compared to control group where being 87.56 2.79 iu/l (Table 1).
Table 1 showed that high dose of carmoisine and both low and
high doses of tartrazine that their values were 6.16 0.10,
6.66 0.18, and 6.70 0.18 g/dl respectively induced a signicant
increase in serum total protein concentration when compared to
control group where being 5.59 0.12 g/dl, also serum albumin le-
Table 1
Effect of food colorants both low and high doses on serum ALT, AST and ALP activities also serum level of T proteins, albumin and globulin in rats.
Groups Control Low tartrazine High tartrazine Low carmoisine High carmoisine
ALT (u/l) 8.91 0.27
a
10.78 0.65
a
15.4 0.80
b
14.2 1.19
b
17.2 0.94
c
AST (u/l) 47.85 2.70
a
48.96 0.96
a
55.38 1.0
b
47.68 2.41
a
52.91 2.54
b
ALP (iu/l) 87.56 2.79
a
117.16 2.3
b
119 2.93
b
114.16 3.85
b
115.45 2.89
b
T protein (g/dl) 5.59 0.12
a
6.66 0.18
c
6.70 0.18
c
5.71 0.26
a
6.16 0.10
b
Albumin (g/dl) 3.81 0.14
a
4.57 0.08
c
4.76 0.13
c
3.94 0.03
a
4.27 0.27
b
Globulin (g/dl) 1.77 0.05
a
1.88 0.08
a
2.13 0.04
b
1.8 0.07
a
1.89 0.05
a
Data expressed as means SE. Number of animals is eight for low dose groups and ten for high dose groups. Means which share the same letter are not signicantly different.
Means which have different letters are signicantly different (P < 0.05).
K.A. Amin et al. / Food and Chemical Toxicology 48 (2010) 29942999 2995
vel increased in these groups where their values are 4.27 0.27,
4.57 0.08, and 4.76 0.13 g/dl, respectively when compared to
control value where being 3.81 0.14 g/dl. High tartrazine dosed
group showed a signicant increase in serum globulin concentra-
tion when compared to control group.
Low and high doses of tartrazine, and carmoisine where their
values were 1.65 0.05, 1.80 0.05, 1.55 0.05, and
1.56 0.09 mg/dl induced a signicant increase in serum creati-
nine level when compared to control value that was
1.32 0.06 mg/dl (Table 2).
Low and high doses of tartrazine, and carmoisine where their
values were 37.41 1.59, 37.46 0.94, 37.76 2.05, and
42.15 1.03 mg/dl respectively, induced a signicant increase in
serum urea level when in comparison to control value where being
17.54 0.68 mg/dl.
Table 2 showed that serum total cholesterol levels were signif-
icantly reduced in groups of rats dosed with tartrazine or carmoi-
sine in both low and high doses and their values were
115.86 4.03, 104.1 2.009, 121.71 4.68, and 118.2 3.31 mg/
dl when compared to control value where being
143.41 4.32 mg/dl, as a result serum HDL-cholesterol and LDL-
cholesterol were signicantly reduced in azo dyes dosed rats.
Rats consumed low and high doses of carmoisine and that con-
sumed high dose of tartrazine where their values were
36.15 1.44, 27.25 1.86, and 34.2 1.16k 10
2
showed a signif-
icant decrease in liver catalase activity when compared to control
value where being 47.03 2.01k 10
2
(Table 3)
Low and high doses of tartrazine and high dose of carmoisine
where values were 64.8 0.68, 53.0 1.27, and 50.18 2.36 u/g
showed a signicant decrease in liver SOD activity in comparison
to control value that being 70.68 1.50 u/g (Table 3).
Rats consumed low and high doses of carmoisine and that con-
sumed high dose of tartrazine where their values were
66.25 1.83, 61.12 1.07, and 67.37 2.73 nmol/100 mg showed
a signicant decrease in liver GSH content in comparison to control
value where being 72.9 1.41 nmol/100 mg. Low and high doses of
carmoisine and high dose of tartrazine where their values were
6.27 0.44, 8.32 0.36, 6.63 0.16, and 7.64 0.62 nmol MDA/g/
h showed a signicant increase in liver MDA in comparison to con-
trol value where being 4.82 0.41 nmol MDA/g/h (Table 3).
In Table 3 all groups of rats dosed with food azo dyes showed
signicant reduction in body-weight gain when compared to con-
trol group.
4. Discussion
In this study some trials were adopted to throw a light on the
side toxic effects and biochemical changes in some constituents
in serum of experimental rats treated with 2 compounds (each of
low and high doses) that are commonly used in Egyptian eld of
food additives.
We considered low dose (double of ADI) because our young
children in Egypt can consume a double of ADI (or more) daily in
several products without control, in addition we used the high
dose (a much higher than ADI) to evaluate the toxicity and health
hazards of these additives on biochemical assay and oxidative
stress.
High dose of carmoisine showed a signicant decrease more
than low carmoisine dose in body-weight gain; also low dose of
tartrazine produced a signicant decrease more than low carmoi-
sine dose in body-weight gain (Fig. 1).
These data are in agreement with Ford et al. (1987) who re-
ported that high carmoisine dose groups had reduced body-weight
gain compared with that of the controls.
Body weight loss is considered by some authors to be a good
reliable sensitive toxicity indicator (Ezeuko et al., 2007). Thus the
body weight loss in the present study may represent the primary
marker of dye bad effect.
4.1. Effect of food azo dyes on liver enzymes
The present study revealed that rats consumed high dose of tar-
trazine (500 mg/kg bw) or high dose of carmoisine (100 mg/kg bw)
exhibited a signicant increase in serum ALT, AST and alkaline
phosphatase activities when compared to control rats, while low
dose of carmoisine (8 mg/kg bw) showed a signicant increase in
serum ALT and alkaline phosphatase activities when compared to
control rats in addition, low dose of tartrazine (15 mg/kg bw)
showed a signicant increase in serum alkaline phosphatase activ-
ity when compared to control rats (Table 1).
The present ndings are in a agreement with Mekkawy et al.
(1998) who indicated that two doses of synthetic dyes (low or high
doses) where tartrazine and carmoisine were among of them (pon-
ceau, carmoisine, erythrosine, sunset yellow, tartrazine, fast green,
indigotine, brilliant blue and brilliant black) showed a signicant in-
crease in serum AST, ALT, and alkaline phosphates activities.
Table 2
Effect of food colorants both low and high doses on serum level of creatinine and urea, TC, TG, HDL, LDL in rats.
Groups Control Low tartrazine High tartrazine Low carmoisine High carmoisine
Urea (mg/dl) 17.54 0.68
a
37.41 1.59
b
37.46 0.94
b
37.76 2.05
b
42.15 1.03
c
Creatinine (mg/dl) 1.32 0.06
a
1.65 0.05
b
1.80 0.05
c
1.55 0.05
b
1.56 0.09
b
TC (mg/dl) 143.41 4.32
a
115.86 4.03
b
104.1 2.009
b
121.71 4.68
b
118.2 3.31
b
TG (mg/dl) 130.6 3.98
a
143.5 1.50
a
144 3.24
a
132.95 2.8
a
144.6 3.01
a
HDL (mg/dl) 87.11 1.47
a
65.63 1.68
b
58.11 1.14
c
71.33 2.89
b
59.93 1.01
c
LDL (mg/dl) 34.18 1.52
a
17.45 0.91
b
15.78 1.43
b
28.18 1.91
a
17.83 1.30
b
Data expressed as means SE. Number of animals is eight for low dose groups and ten for high dose groups. Means which share the same letter are not signicantly different.
Means which have different letters are signicantly different (P < 0.05).
Table 3
Effect of food colorants both low and high doses on biomarkers of Oxidative stress {catalase, SOD, GSH, and malondialdehyde (MDA)} in liver homogenate.
Groups Control Low tartrazine High tartrazine Low carmoisine High carmoisine
SOD (u/g) 70.68 1.50
a
64.8 0.68
b
53.0 1.27
b
69.3 1.91
a
50.18 2.36
b
Catalase (k 10
2
) 47.03 2.01
a
46.01 1.83
a
34.2 1.16
b
36.15 1.44
b
27.25 1.86
c
GSH (nmol/100 mg) 72.9 1.41
a
70.21 3.85
a,b
67.37 2.73
b
66.25 1.83
b,d
61.12 1.07
c,d
MDA (nmol/g/h) 4.82 0.41
a
4.93 0.23
a
6.63 0.16
b
6.27 0.44
b
8.32 0.36
c
Data expressed as means SE. Number of animals is eight for low dose groups and ten for high dose groups. Means which share the same letter are not signicantly different.
Means which have different letters are signicantly different (P < 0.05).
2996 K.A. Amin et al. / Food and Chemical Toxicology 48 (2010) 29942999
Mekkawy et al. (1998) attributed these results to hepatocellular
damage caused by the toxic effects of these synthetic dyes which
indicatedby vacuolation, swelling, necrosis andpyknosis of the liver
cells.
Also these results are deal with Aboel-Zahab et al. (1997) who
found that liver enzymes ALT, AST, and Alkaline phosphatase were
elevated in rats whose diets were supplemented with chocolate
colors A and B (sunset yellow, tartrazine, carmoisine and brilliant
blue in varying concentrations), the histopathological studies
showed brown pigment deposition in the portal tracts and Van
Kpffer cells of the liver, in addition congested blood vessels and
areas of haemorrhage in both liver and renal sections were re-
vealed in those rats given colorants B and C (sunset yellow, tartra-
zine, carmoisine and brilliant blue in varying concentrations).
Furthermore, these results are in accordance with Sharma et al.
(2006) who found that the two doses of Tomato Red (blend of car-
moisine and ponceau 4R) showed a signicant increase in alkaline
phosphatase activity when swiss albino mice consumed these col-
orants for 21 days as short term or 42 days as long term.
Also, Sharma et al. (2005) observed a signicant increase in ser-
um transaminases in rats whose diets were supplemented with
chocolate colors A and B (sunset yellow, tartrazine, carmoisine
and brilliant blue in varying concentrations).
The present ndings are in agreement with Helal et al. (2000)
who found that oral administration of synthetic or natural colo-
rants induced a marked increase in the serum AST and ALT level
of all treated groups after 30 days of treatment.
Abdel-Rahim et al. (1987) found a signicant increase in both
serum AST and ALT of rats fed on brown food dye for three months,
he attributed these changes in liver function to hepatocellular
impairment which subsequently caused the release of greater than
normal levels of intracellular enzymes into the blood.
The elevation of aminotransferases activities in serum may be
due to tissue damage particularly in liver, kidney and heart (Varely
et al., 1988). And increased permeability of cell membrane or in-
creased synthesis or decreased catabolism of transaminases may
be involved (Malik et al., 1980), also Westlake et al. (1981) men-
tioned that the release of abnormally high levels of specic tissue
enzymes into blood stream is dependent on both the degree and
the type of damage exerted by the toxic compound administration.
In the same concern, Webner (2003) reported that the damaged
or diseased tissues release enzymes into the blood, so serum alka-
line phosphatase measurements can be abnormal in many condi-
tions including bone diseases and liver diseases.
The signicant elevation of serum aminotransferases may be
attributed to what mentioned by Shakoori et al. (1987) who re-
vealed that under pathological conditions the parenchymal cells
of hepatic lobules fail to carry out vital functions, which usually re-
sults in disturbed or imbalanced intermediatory metabolism, as a
result of cellular damage, several enzymes like ALT, AST, LDH and
ALP beach out into the serum and hence their level indicate the
type and extent of damage inicted.
4.2. Effect of food azo dyes on serum proteins
Our work revealed that rats consumed high dose of tartrazine
(500 mg/kg bw) or low dose of tartrazine (15 mg/kg bw) and that
consumed high dose of carmoisine (100 mg/kg bw) exhibited a sig-
nicant increase in serum total protein and serum albumin con-
centration when compared to control rats (Table 1).
These results are in accordance with Mekkawy et al. (1998) who
found a signicant increase in serum total protein also this is in
agreement with Aboel-Zahab et al. (1997) who found the same ef-
fect on serum total protein with rats whose diets were supple-
mented with chocolate colors A and B. Also Sharma et al. (2006)
found that total protein was signicantly elevated when Tomato
Red (blend of carmoisine and ponceau 4R) was consumed by swiss
albino mice.
Sharma et al. (2005) observed signicant increase in serumtotal
protein and globulin in rats whose diets were supplemented with
chocolate colors A and B.
Moreover, these results are in agreement with Ali et al. (1998)
who found that the nucleic acids and the total protein had marked
increase during the various periods of treatment when the rats
were treated with two doses of carmoisine limdose (0.011 mg/
100 g/day) and high dose (0.022 mg/100 g/day) during different
periods of treatment 30, 60, and 90 days.
Furthermore, our study demonstrated that high dose of tartra-
zine caused a signicant increase in serum globulin, which were
in accordance with Mekkawy et al. (1998) and Aboel-Zahab et al.
(1997).
Proteins are the fundamental components of all living cells and
include many substances such as enzymes, hormones and antibod-
ies that are necessary for the proper functioning of an organism, in-
creased release of enzymes by the damaged tissues and the
antibodies to counter act the dye might be the cause of increase
in serum protein (Sharma et al., 2005), so increase of release of li-
ver enzymes caused by toxic effect of synthetic food dyes tartra-
zine and carmoisine can result in increasing of serum protein
concentration.
Furthermore, Al-Shinnawy (2009) attributed the elevation in
serum total protein of rats whose supplemented with synthetic
food dye to the stimulation of protein biosynthesis to produce
the specic enzymes required for all processes.
In our opinion these hypersensitivity reactions caused by yel-
low food dye tartrazine can increase production of immunoglobu-
lin which lead to elevation of serum globulin concentration and
this phenomenon is more appreciable in the group of rats consum-
ing high dose of tartrazine due to increasing of immune hyperac-
tivity caused by this food colorant.
Our opinion is agreed with that reported by Al-Shinnawy (2009)
who mentioned that the specic elevation in globulin fraction
points towards increased immunoglobin synthesis, the defense
mechanism which aims to protect the body from the toxic effects
of this synthetic food dye.
C
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i
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e

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i
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h

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a
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a
z
i
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o
w

C
a
r
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i
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H
i
g
h

C
a
r
m
o
i
s
i
n
e
0
50
100
150
200 Initial Bwt
Final Bwt
Bwt gain
a
b
b
a
a
b
*
*
*
B
w
t

(
g
m
)
* Indicated significant changes (P <0.05) between control and both tartrazine
and carmoisine either low or high dose.
Fig. 1. Effect of food colorants both low and high doses on initial, nal body weight
and body-weight gain in rats.
K.A. Amin et al. / Food and Chemical Toxicology 48 (2010) 29942999 2997
4.3. Effect of food azo dyes on kidney function
Our study demonstratedthat the daily intake for 30 day of tartra-
zineor carmoisine either lowor highdoses exhibiteda signicant in-
crease in serum creatinine and urea concentration when compared
withcontrol rats, whilehighdoseof tartrazineexhibitedasignicant
increase more than low dose in serum creatinine level (Table 2).
Our results were parallel to our ndings those recorded by Helal
et al. (2000) who found a signicant elevation in serum creatinine
and urea in rats consumed a synthetic or natural food colorants
after 30 days of treatment.
Furthermore, the present ndings are in accordance with data
reported by Ashour and Abdelaziz (2009) who observed a signi-
cant elevation in serum creatinine and urea level of rats dosed with
organic azo dye (fast green) orally for 35 days.
We believe that the signicant elevation in urea and creatinine
levels is closely related to the impairment of renal function. These
results are in agreement with Varely (1987) who determined that
the blood urea can be increased in all forms of kidney diseases such
as hydronephrosis congenital cystic, kidney renal tuberculosis,
condition in which deposition of calcium occurs as hypervitamin-
osis D. Also plasma creatinine increases in renal diseases gave
prognostic signicance than those of other nitrogenous substances.
4.4. Effect of food azo dyes on lipid prole
The reduction in serum cholesterol levels obtained in this study
are in accordance with results recorded by Sharma et al. (2006)
who reported that two doses of Tomato Red (blend of carmoisine
and ponceau 4R) showed a signicant decrease in serum total cho-
lesterol and triglycerides when swiss albino mice consumed these
colorants for 21 days as short term or 42 days as long term.
Also, these results are correlate well with that reported by
Ashour and Abdelaziz (2009) who obtained a signicant reduction
in serum total cholesterol and triglycerides level when food color
azo dye (fast green) was consumed orally to male albino rats for
35 days. While our results are in a contrary with Aboel-Zahab
et al. (1997) who observed a signicant increases in serum total
lipids, cholesterol and triglycerides in rats whose diets were sup-
plemented with chocolate colors A and B that tartrazine and car-
moisine were among of them (sunset yellow, tartrazine,
carmoisine and brilliant blue) in varying concentrations. Choles-
terol is a soft waxy substance found among the lipids in the blood
stream and in the bodys cells. It is an important part of healthy
body because it is used to form cell membranes and to produce
certain hormones. The total body content of cholesterol depends
on the balance between the amount of cholesterol formed in the
body plus that absorbed from diet. Intestinal cholesterol absorp-
tion represents another major route for the entry of cholesterol
into the body, and, thus, this source can inuence the plasma
LDL-cholesterol concentration (Turley, 2004). The cholesterol pool
in the intestine comes from dietary cholesterol and the majority
from biliary excretion. Approximately 50% of the intestinal choles-
terol pool is reabsorbed by the intestines (Turley and Dietschy,
2003) and recirculated through the body (via the enterohepatic cir-
culation), with the remainder excreted in feces (Grigore et al.,
2007). The deviation from normal values of cholesterol, in the
blood serum is considered as symptoms of liver diseases (Singh
et al., 1988). In the present study the decreased cholesterol level
implies liver damage which is in accordance with increased alka-
line phosphatase level discussed earlier.
4.5. Effect of food colorants (azo dyes) on oxidative stress biomarkers
The present study revealed that rats consumed high and low
doses of carmoisine and that consumed high dose of tartrazine
showed a signicant decrease in liver GSH content and catalase
activity. Also high and low doses of tartrazine and high dose of car-
moisine showed a signicant decrease in liver SOD activity when
compared to control group, while high and low doses of carmoisine
and high dose of tartrazine showed a signicant increase in liver
MDA (Table 3).
There is a shortage in the literatures dealing with this subject so
we have not been able to nd a studies targeting oxidative stress
with a given food additives.
While our results may be in accordance with Sweeney et al.
(1994) who suggested that various azo dye products are genotoxic,
not through N-hydroxylation and esterication, which is charac-
teristic of many aromatic amines but rather through a mechanism
involving oxygen radicals and the superoxide free radical was pro-
duced by the azo dyes only after reduction by the intestinal bacte-
ria Enterococcus faecalis.
Moreover, Siraki et al. (2002) found that incubation of hepato-
cytes with aromatic amines caused a decrease in the mitochondrial
membrane potential before cytotoxicity ensued. Hepatocyte GSH
was also depleted by all arylamines tested and extensive GSH oxi-
dation occurred with o-anisidine and aminouorene.
Azo compounds contain an aromatic ring linked by an azo bond
to second naphthalene or benzene ring.
Many intestinal bacteria are able to reduce the azo bond (termed
azossion), which liberates the substituted naphthol compounds.
Coloring matter entering the intestinal tract is subjected to the ac-
tion of acid, digestive enzymes, and microora. Azo compounds
may reach the intestine directly after oral ingestion or through the
bile after parenteral administration. They are reduced by azo reduc-
tases from intestinal bacteria and, to a lesser extent, by enzymes of
the cytosolic andmicrosomal fractions of the liver. The rst catabolic
stepinthereductionof azodyes, whichis accompaniedbyadecrease
inthevisiblelight absorbanceandthendecolorationof thedye, is the
reduction of the azo bond to produce aromatic amines. Aromatic
amines, some of which are known carcinogens, have been found in
the urine of dyestuff workers andtest animals following administra-
tion of azo dyes (Cerniglia et al., 1986).
Tartrazine is transformed into aromatic amine sulfanilic acid
after being metabolized by the gastrointestinal microora (Mout-
inho et al., 2007).
Because the food dyes (tartrazine and carmoisine) are from the
group of azo dye food colorants, they are metabolized into aromatic
amine by intestinal ora andthe formedaromatic amines cangener-
ate reactive oxygen species as part of their metabolism (NOS) by
interaction of these amino groups with nitrite or nitrate containing
foods or in the stomach., The reactive oxygen species (ROS) such as
superoxide anion, hydroxyl radical and H
2
O
2
could be produced in
the metabolismof nitrosamines and increase oxidative stress (Ban-
sal, 2005). As a result of the ROS formationof the antioxidant defense
mechanism of the cells including catalase, SOD, and GSH began to
consumed to prevent the cell death by these toxic radicals so their
levels in the tissue homogenate were decreased specially at higher
doses when the need for them was increased, on the other hand
MDAlevel was increased as a product of lipid peroxidation occurred
by the ROS action on lipids of cellular membrane.
Reactive oxygen species play an important role in pathological
changes in the liver (Poli and Parola, 1997). Biological membranes
are particularly prone to the ROS effect, the peroxidation of unsatu-
rated fatty acids in biological membranes leads to a decrease of
membrane uidity and disruption of membrane integrity and func-
tion, which is implicated in serious pathological changes (Halliwell,
1987).
Increased generation of ROS or free radicals is able to cause
auto-oxidation of the hepatic cells, resulting in marked hepatic le-
sions (Suzuki et al., 1998). In the present study, the increased activ-
ities of serum enzymes (AST and ALT) have been detected in food
2998 K.A. Amin et al. / Food and Chemical Toxicology 48 (2010) 29942999
colorants administrated in rats specially at higher doses (Table 2),
implying the increased permeability, damage and injuries of hepa-
tocytes. Because the enzyme ALT is located in the cytoplasm and
the soluble enzyme AST is located mainly in organelles such as
mitochondria (Senthil et al., 2003). Increased levels of AST and
ALT suggested damage of both hepatic cellular and mitochondrial
membranes in food azo dyes administered rats.
This study suggests a potential role for commonly consumed
beverages in elevating the risk of pathophysiologies associated
with peroxyl radical-mediated events.
5. Conclusion
Food azo dyes like tartrazine and carmoisine can affect ad-
versely and alter biochemical markers in vital organs e.g. liver
and kidney not only at higher doses but also at lowdoses. Tartazine
and carmoisine not only cause changes in hepatic and renal param-
eters but also their effect become more risky at higher doses be-
cause they can induce oxidative stress by formation of free
radicals. Therefore, it is necessary to create consumer awareness
regarding the ill effects of these food azo dyes and mention the
type and concentration of each material added to food.
Based on our results, we believe that more extensive assess-
ment of food additives in current use is warranted.
Conict of Interest
The authors declare that there are no conicts of interest.
Acknowledgements
This study was supported by Beni Suef University. We appreci-
ate the assistance and advice of Prof. Dr. Bastawy M., Vice Dean of
College of Science, Beni Suef Univ.
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