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Effects of Synthetic Food Color (Carmoisine) on Expression of Some Fuel Metabolism Genes in Liver of Male Albino Rats


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Food additives were known since the old man from the ancient civilizations to introduce special color or taste in his food. Nowadays, there are over-use of synthetic chemicals as food additives to preserve foods, to improve characters and attract consumers especially children. Previous studies had reported many effects of overdoses from food additives, however, further scientific studies are needed in the molecular levels. The current work studied the effects of doses equivalent to the acceptable daily intake (ADI), 5 and 10 ADI folds of the synthetic food color (Carmoisine) on the experimental animals (male albino rats) for different periods. Gene expression of some fuel metabolism genes e.g. PPAR-alfa, ACo-A and CPT-1 were studied and supported by histological studies on rat liver. There were down-regulations of the studied genes which may leads to the conclusion that, carmoisine may decrease the fuel metabolism. The histological studies indicate also that high doses of carmoisine may affect the liver.
Content may be subject to copyright. 22013;10(Life Science Journal,
Effects of Synthetic Food Color (Carmoisine) on Expression of Some Fuel Metabolism Genes in Liver of Male
Albino Rats
Metwally M. Montaser
, Mohamed E. Alkafafy
Biotechnology Department, Faculty of Science, Taif University, P.O. Box 888, Taif 21974, KSA.
Zoology Department, Faculty of Science, Al-Azhar University, 11884 Nasr City, Cairo, Egypt.
Histology Department, Faculty of Veterinary Medicine, University of Sadat City, Egypt.
Abstract: Food additives were known since the old man from the ancient civilizations to introduce special color or
taste in his food. Nowadays, there are over-use of synthetic chemicals as food additives to preserve foods, to
improve characters and attract consumers especially children. Previous studies had reported many effects of
overdoses from food additives, however, further scientific studies are needed in the molecular levels. The current
work studied the effects of doses equivalent to the acceptable daily intake (ADI), 5 and 10 ADI folds of the synthetic
food color (Carmoisine) on the experimental animals (male albino rats) for different periods. Gene expression of
some fuel metabolism genes e.g. PPAR-alfa, ACo-A and CPT-1 were studied and supported by histological studies
on rat liver. There were down-regulations of the studied genes which may leads to the conclusion that, carmoisine
may decrease the fuel metabolism. The histological studies indicate also that high doses of carmoisine may affect
the liver.
[Metwally M. Montaser
, Mohamed E. Alkafafy. Effects of Synthetic Food Color (Carmoisine) on Expression of
Some Fuel Metabolism Genes in Liver of Male Albino Rats. Life Sci J 2013;10(2):2191-2198] (ISSN:1097-8135). 306
Keywords: Carmoisine, rat liver, Fuel metabolism, PPAR-alfa, ACo-A, CPT-1.
1. Introduction:
The addition of colorants to make food more
attractive is not a recent invention. Extracts from spices
and vegetables were used as early as 1500 BC, in India
and China for coloring skin. Wine was colored as early
as 400 BC, and spices and condiments were being
colored by AD 1400. Colorants derived from naturally
occurring minerals, plants, and animals were prepared
along with the spices that played such a prominent part
in the development of early civilizations Tannahill,
The advent of the use of food colorants in the late
1800S and early 1900S was unfortunately accompanied
by their misuse in food adulteration. Before the
development of synthetic colorants, dangerous mineral
extracts were often used to color foods, frequently to
disguise food of poor quality. Some of these deceptive
practices included coloring of pickles with copper
sulfate, cheese with vermilion and red lead, tea with
copper arsenate, lead chromate, and Indigo, and Candy
with lead chromate, red and white lead, and vermilion
(NAS/NRC 1971.
In 1956, the discovery of the first synthetic dye,
mauve, by Sir William Henry Perkins prompted a
search for other dyes. Further development of synthetic
colorants then became attractive to the food industry
because these colorants were superior to natural
extracts in tinctorial strength, hue, and stability and was
readily available in many hues. The addition of
synthetic colorants to food in the United States was
first legalized for butter in 1885, followed by the
authorization to add colorants to cheese in 1896
(Newsome, 1986 .
Historically, synthetic colorants, also known as
aniline dyes, were manufactured from coal tar
derivatives. Although the colorants were highly
purified before they were added to foods, the negative
connotation of their association with coal tar resulted in
much unfavorable publicity. As a result, synthetic
colorants are no longer manufactured from coal tar
derivatives but instead are developed form highly
purified petrochemicals. The U.S. Department of
Agriculture began to investigate the use of colorants in
foods to establish principles for their regulation in the
early 1900s. The first comprehensive regulation
regarding food colorants was the Federal Food and
Drug Act of 1906. Previously, 80 different colorants
had been used in foods. Following establishment of the
Federal food and Drugs Acts, the U.S. Department of
agriculture eliminated all but seven of these colorants,
on the basis of known composition and purity. This act
and a series of food inspection decisions lead to
establish of a voluntary certification program for food
colorants with the food inspection decision of 1907.
Subsequently, seven more colorants were added to the
approved list. In 1938, The Federal Food, Drug, and
Cosmetic Act established mandatory certification (21
CFR parts 73-74, 81-820) requiring submission of
samples from each batch of colorant for evaluation of
purity. Synthetic colorants that had previously been 22013;10(Life Science Journal,
known by common names were there given specific
names to ensure distinction of those for use in foods
from those for use in drugs or cosmetics. Three
categories for designating colorant names. FD & C
(Suitable for use in foods, drugs and cosmetics), D & C
(suitable for use in drugs and cosmetics), and external
D & C (Suitable for use in externally applied drugs and
cosmetics) were defined (Branen et al., 1990).
In 1960, the color additive amendments to the FD
& C act were established, defining the term “color
additive” as any dye, pigment or other substances made
or obtained form a vegetable, animal or mineral or
other source capable of coloring a food, drug or
cosmetic or any part of the human body. Included in
the two part amendments was a Delaney-type clause.
Part (1) of the clause prohibited addition to food of any
colorant found to induce cancer in human or animals.
Part (2) of the clause permitted the use of current color
additives under a provisional list in pending the
completion of scientific investigations needed to
determine their safety for permanence listing.
Synthetic colorants were thus required to undergo
premarketing safety clearance, and previously
authorized colorants were reevaluated (Newsome,
The development and use of food colors in
countries other than United States has been extremely
varied. Havel and Smith (1980) reviewed development
of regulations for use of food colorants in other
countries. Legalization for food colorants in the United
Kingdom was not established until the mid-1950s.
Some countries do not regulate the use of food
colorant, thus permitting the use of any colorants, while
others prohibit the use of all synthetic colorants. The
regulatory status of colorants in use outside as well as
within the U.S. is reported in the International Life
Science Institute Nutrition Foundations Catalog of
Food Colors (ILSI/NF 1981) and updated periodically
as new information becomes available
Specific food products are expected to appear in
certain color shades, and when deviations from these
expectations occur, flavor perception is attended. The
importance of color to the perception of the quality,
odor, flavor, and texture of food is well documented. In
studying how individuals react to sherbets of
mismatched flavor and color, Hall (1958) found that
white sherbets made with one of six test flavors
(Lemon, Lime, Orange, Grape, Pine apple, and
Almond) were confused, and flavor was difficult to
identifyAlso, when the sherbets were deceptively
colored, most individuals mistakenly identified flavors.
Color far outweighs flavor in the impression it makes
on the consumer, even when the flavors are pleasant
and the food is a popular one. It powerfully influences
not only the consumer’s ability to identify the flavor
but also her or his estimation of its strength and quality
The use of food colorants aids in the production of food
of preferred color values and provides significant
functional advantages in a variety of situations
colorants. It corrects for variation and irregularities
resulting from storage, processing packaging, and
distribution, assuming greater uniformity in appearance
and hence acceptability Color also, help preserve the
identity or character by which foods are recognized
(Hall, 1958)
The Certified Color manufacturer’s Association
(CCMA) has petitioned the FDA for approval of
Carmoisine, a colorant similar in shade to FD and C
Red No. 2. Carmoisine is widely used in food in
Central and South America and Europe. However,
colorants considered safe in one country may not be
considered safe in other parts of the world. In the U.S.
the toxicity of a colorant is tested under conditions
similar to those under which it will be used. Therefore,
since it is important that food colors be safe when
ingested, animal feeding studies play a key role in their
evaluation (Marmion, 1984). Present day toxicological
testing, as suggested by the FDA, is listed in the RDA
“Red Book” (FDA, 1982). Scientists in many other
parts of the world, however, place emphasis on the
effects of subcutaneous injection of the colorant.
Because of the different mechanisms involved, the
results of the studies conducted under these different
test conditions vary and have resulted in much
scientific and political debate
Gerd and Lennart (1973) reported that
“recurrences of urticaria could be prevented through
the avoidance of food and drugs containing azo dyes
and preservatives
Feingold (1979) had two case reports illustrate the
therapeutic response of congenital nystagmus to a diet
eliminating synthetic food colors. A brief discussion of
the hyperkinetic syndrome was offered with the
proposal that a variety of neurologic and
neuromuscular disturbances (grand mal, petit mal,
psychomotor seizures, la tourette syndrome, autism,
retardation, the behavioral component of Down’s
syndrome, and oculomotor disturbances) may be
induced by identical chemicals, depending upon the
individual’s genetic profile and the interaction with
other environmental factors. Juhlin (1981) and Twaroj
(1983) reported that artificial food additives
particularly, azo dyes and benzoate preservatives are a
common cause of chronic urticaria and angioedema in
both adult and children. Brozelleca et al. (1989)
reported a reduction in body weight in female rats by
high-dose of (FD and C red No, 40). Hong et al. (1989)
reported that aspirin and food additives are known to
induce bronchoconstriction, angioedema or urticaria in
susceptible patients
Van Bever et al.(1989) reported that children feed
on food colors shown to have an adverse effect on a 22013;10(Life Science Journal,
daily Conner’s rating of behavior. Osman et al. (1995)
reported that synthetic food colorants administration
increased the body weight gain until the fourth month
but after that forth month a decrease in body weight as
observed either in female or male mice, there were also
an increase in organ/body weight. Gaunt et al. (1967)
carried acute toxicity studies in rats and mice and a
short term feeding study in rat. They reported an
elevation in the relative kidney weight at the 1% level
in females. Gaunt et al. (1969) reported that pig is less
sensitive to Carmoisine than rat
Rowe (1988) reported that subjects (children)
were maintained on a diet free from synthetic additives
and were challenged daily for 18 weeks with placebo
(during lead-in and washout periods) or tartrazine or
Carmoisine 50), each for 2 separate weeks. Two
significant reactors were identified whose behavioral
pattern featured extreme irritability, restlessness and
sleep disturbance. One of the reactors did not have
inattention as a feature. The findings raise the issue of
whether the strict criteria for inclusion in studies
concerned with hyperactivity” based on “attention
deficit disorder” may miss children who indicate
behavioral changes associated with ingestion of food
colorings. Moreover, for further studies, the need to
construct a behavioral rating instrument specifically
validated for dye challenge is suggested. Booth
(1993) illustrated the effectiveness of dietary advice in
a young body with chronic idiopathic urticaria. An azo
dye and preservative-free diet was initially advised,
resulting in a total improvement in urticarial symptoms.
Double-blind challenges confirmed the boy was
intolerant to E127 (Erythrosine), E122 (Carmoisine),
E128 (red 2G), and E102 (tartrazine) but not to E211
(sodium benzoate)
Murdoch et al. (1987) reported that ten healthy
adults with a negative history of adverse reactions to
foods and food additives were asked to exclude foods
known to be high in histamine and food containing azo
dyes and other coloring agents. He found that 200 mg
tartrazine caused a significant increase in plasma and
urinary histamine concentrations 30 min to 3 hrs after
ingestion with a mean time of 100 min. Abdel-Rahim
(1988) and Ibrahim et al.[38] reported pronounced
increase in serum and liver transaminases activity of
rats by ingestion of synthetic colorants. The load and
species of food colorants ingested into animals for
assimilation at any time may alter the activity of GOT
and GPT followed by changes of overall protein
Abou El-Zahab et al. (1997) found that
administration of the synthetic colorants (Carmoisine,
sunset yellow, indigocarmine, brilliant blue and brown
chocolate HT) after 60 days induced damage to liver
tissue as evidenced by a significant increase in AST,
ALT and ALP in serum of rat groups. But, some
researchers, (Ford et al. 1987 and Brozelleca and
Hallagan 1988a, b) stated that Carmoisine and
tartrazine caused insignificant changes in rat serum
AST, ALT and ALP. Yet these contradictory results
were recorded after long term toxicity studies which
may indicate an adaptation mechanism on part of the
liver. He, also stated that serum total protein exhibited
significant increase in rats fed on colors containing
Carmoisine after 30 and 60 days of diet
supplementation, while albumin revealed significant
increase in rats fed with color B (sunset yellow,
tartrazine, Carmoisine and brilliant blue) after 60 days.
In agreement with these findings, El-Sadany whose rats
ingested the food colors indigocarmine and Carmoisine
as well as Lord who administered tartrazine dye to
the experimental rats. The accumulation of serum
protein can be attributed to the stimulation of protein
biosynthesis to produce the specific enzymes required
for all processes Amin et al. (2010) reported that
Tartrazine and Carmoisine in low and high doses affect
adversely and alter biochemical markers in vital
El-Saadany (1991) pointed out that the ingestion
of synthetic chocolate colorants (indigocarmine and
Carmoisine) specifically increased RNA (not DNA) in
rat liver cell homogenates. In connection with this, it is
noted that their hydrolytic enzymes (DNase) in
cytoplasmic as well as mitochondrial fractions were
also stimulated to provide the necessary enzymatic
machinery to cope with increased flow of
ribnucleotides. Abou El-Zahab et al. (1997) stated that,
the concentrations of ribonucleic acid (RNA) in liver
cell homogenates exhibited a highly significant
increase (P < 0.01) only on rats fed on diet
supplemented with color C (brown chocolate HT and
indigo carmine in addition to small fractions of
Carmoisine, tartrazine, sunset yellow and brilliant blue)
for 60 days, whereas DNA remained unchanged. Ali et
al. (1988) also reported expected mutagenic effects of
high doses from carmoisine and fast green
Gaunt et al. (1988b) reported that the types and
incidence of histological changes were comparable in
control and test groups. A no-effect level of 0.5%
Carmoisine was established in the diet of rats for 90
days, a level equivalent to 250 mg/kg/day. Shaker et al.
(1989) noted an increase in hemoglobin content of rat
administered chocolate brown color (0.1% w/w)
consisting of tartrazine, noval coccine, Carmoisine and
Abou El-Zahab et al. (1997) stated that, the
marked discrepancies observed between the various
research studies may be attributed to dose variations as
well as the duration of colorant intake, where small
doses for longer periods induced positive stimulatory
effect on erythropoiesis, moderate duration and dose
revealed inhibition and long term high dose 22013;10(Life Science Journal,
administrations produced no alterations. The total
count in his study remained unchanged in all his
experimental groups
Tartrazine and carmoisine are an organic azo dyes
widely used in food products, drugs and cosmetics.
Therefore, Amin and his coworkers (Amin et al., 2010)
evaluated the toxic effect of carmoisine on hepatic
function, lipid profile, blood glucose, body-weight gain
and biomarkers of oxidative stress in tissue. They
administered carmoisine orally in two doses, one low
(8 mg/kg bw) and the other high dose (100 mg/kg bw)
for 30 days and concluded that carmoisine affects
adversely and alter biochemical markers in liver at both
In the liver, PPARα is a critical transcription
factor for lipid metabolism, because several genes
coding for enzymes involved with oxidation (either in
peroxisomes or mitochondria) contain a functional
peroxisome proliferatorresponsive element in their
enhancer regions (e.g., acyl-CoA oxidase, liver fatty
acidbinding protein, cytochrome p 450A, hepatic
lipoprotein lipase, and others) (Schoonjans et al.,
The current study focus on the action of
carmoisine (synthetic food color) in different doses for
different periods on gene expression of the common
genes controlling for fuel metabolism. It also supports
the molecular studies with histological studies for the
2. Material and Methods:
Chemicals: carmoisine (C
) was
ordered from Lobachemie PVL Ltd company
(Mumbai, India).
Animals: A total of 30 Male Sprague-Dawley rats, 160
+ 10 g body weight (B Wt) were used in this study.
Rats were kept on standard diet (Degrace et al., 2003)
and randomly divided in to 4 groups according to
treatment. Control negative group (G0), group 1(ADI
or acceptable daily intake) was treated with a dose
equivalent to ADI (50mg/kg B Wt), group 2 (5xADI)
was treated with 5 folds of ADI, group 3(10xADI) was
treated with 10 folds of ADI. Three rats were taken
from each group every 15 days for a period of 45 days.
Histological analysis: Animals were sacrificed.
Removed liver slices were fixed in 10%
neutral-buffered formalin and embedded in paraffin.
Five μm thick sections were stained with
hematoxylin-eosin, Alcian blue or Crossmon’s
trichrome stain for histological examination.
Semi-quantitive PCR: Total RNA from about 40mg
liver was extracted using Biozol (Biolabs, USA) . The
final amount of RNA was estimated by determining the
optical density at 260 nm. First strand cDNA synthesis
with total RNA was performed using reverse
transcriptase (Jones et al., 1999). Subsequently,
PCR-amplification was performed using specific
primers and conditions (Table 1) for 30 of 1 min at
94 °C, 1 min at the annealing temperature indicated in
Table 1, and 1 min at 72 °C. The final extension step
was 5 min at 72 °C, PCR products were separated on
1.5% agarose gels visualized under UV light and
analyzed using Alfa Ease FC software.
Statistical analysis: Data were expressed as mean ±
standard deviation. Student’s t-test was used to
compare means. A level of p<0.05 was considered as
statistically significant.
3. Results and Discussion:
Histological effects: In the control group there were
normal hepatocytes with normal un-engorged hepatic
sinusoids or any blood vessels, normal portal tracts
(Figure 4), no alcianophilic hepatocytes (Figure
3A). The ADI treated groups didn't show deleterious
effects especially for short period of treatment.
However, the longer time of treating with ADI (for 30
and 45 days) resulted in swollen hepatocytes with fatty
changes and some with congested hepatic blood
sinusoids (Figure 6C). There were also, ballooned
swollen hepatocytes that stores fat droplets as also
was present mucoid degeneration of the hepatocytes
with alcianophilia (5C).
Increasing the doses of carmoisine more than ADI
(5xADI and 10xADI) for 15 days did not vary (Figure
6C) from those of 60 and 90 days fed of light doses.
There were more degenerative changes of hepatocytes
that revealed hepatic cells mucoid degeneration as
shown in figure 6C.
Gene expression effects:
The levels of PPAR alfa gene expression
fluctuated around that of the control group, because of
the different doses along the various periods of
treatments. After 15 days The expression levels were
elevated more than that of the control group. The ADI
at 15 days elevated the level of PPAR from 0.770
(control) into 0.910, the medium (5xADI) dose resulted
in elevation into 0.913, and the high (10xADI) dose
elevated the level from 0.770 (control) into 0.900.
However, all the increments were non-significant.
At the 30 day group, only the ADI and medium
doses were non-significantly increased from 0.772
(control) into 0.860 and 0.84 respectively. While, the
high dose resulted in significant decrease in the
expression level from 0.770 (control) into 0.670.
At the 45 day group, the ADI dose produced
non-significant increase in level of expression from
0.780 (control) into 0.810. The medium and high doses
resulted in significant decreases from 0.772 (control)
into 0.770 and 0.690 respectively. 22013;10(Life Science Journal,
The expression pattern of ACO-A gene was
similar to that of PPAR- alfa. After 15 days, there were
non-significant increases in expression level due to
treatments with ADI, medium and high doses from
0.630 (control) into 0.645, 0.640 and 0.637
respectively. However, at 30 day group there were
non-significant increases when treated with ADI and
medium from 0.670 (control) into 0.677 and 0.675
respectively. The high dose of carmoisine after 45 days
resulted in significant decrease into 0.660.
Carnetine palmetoyl transferase-1 was expressed
in heterogeneous pattern among the different periods of
treatment. At the 15 day period, there were
non-significant increases due to ADI, medium and high
doses from 0.392 (control) into 0.410, 0.451 and 0.444
respectively. After 30 days, there were non-significant
increases in response to ADI and high doses from
0.395 (control) into 0.511 and 0.472 respectively.
Meanwhile, the increase in CPT-1 gene expression due
to the medium dose at 30 day period was significant
into 0.470.
After 45 days of treatment, CPT-1 gene was
significantly decreased from 0.430 (control) into 0.400,
0.410 and 0.400 due to ADI, medium and high doses
The down regulation of PPAR-α due to the high
doses of carmoisine may lead to disturbance of fuel
metabolism, which is manifested by the disturbance in
some fuel metabolism genes e.g. ACo-A oxidase and
CPT-1. The interpretation of our results could be
supported by an explanation according to Videla and
Pettinelli (2012), who summarized the expected
consequences of disturbance in PPAR.
Videla and Pettinelli (2012) stated that, Liver
PPAR-α downregulation and substantial enhancement
in the hepatic sterol regulatory element binding
protein-1c (SREBP-1c)/PPAR-α mRNA content ratio
represent major metabolic disturbances between de
novo lipogenesis and FA oxidation favouring the
former, as a central issue triggering liver steatosis in
obesity-induced oxidative stress and insulin resistance.
The prosteatotic action of PPAR-α downregulation
may be reinforced by PPAR- γ upregulation favouring
hepatic FA uptake, binding, and transport, representing
a complementary lipogenic mechanism to SREBP-1c
induction leading to de novo FA synthesis and TAG
accumulation. In addition, PPAR-α downregulation
may play a significant role in enhancing the DNA
binding capacity of proinflammatory factors NF-κB
and AP- 1 in the liver of obese patients, thus
constituting one of the major mechanisms for the
progression of simple steatosis to steatohepatitis. In the
past, PPARs have been studied as drug targets for the
management of Nonalcoholic fatty liver disease
NAFLD in obesity and the broader metabolic
syndrome (MetS) (George and Liddle 2008).
However, PPAR-α agonists such as fibrates used to
diminish steatosis and inflammatory scores in human
steatohepatitis revealed poor effectiveness,
thiazolidinediones have weight gain limitations,
whereas that of dual PPAR-αγ agonists has safety
concerns. Hepatic lipid metabolism was regulated via
ligands of PPAR-α promoted the expression of genes
encoding for proteins involved in FA oxidation at
mitochondrial, peroxisomal, and microsomal levels, FA
binding in cells, and lipoprotein assembly and transport
and reduction in PPAR-α controlling FA oxidation
(carnetine palmitoyltransferase-1a; CPT-1a), with the
consequent enhancement in the hepatic
SREBP-1c/PPAR-α ratios denoting a prolipogenic
status. This condition may also involve diminution in
TAG export from the liver via very-low density
lipoprotein (VLDL) due to decreased production of
apolipoprotein B-100, which is upregulated by
LCPUFA n-3 and PPAR-α activation (Videla and
Pettinelli 2012).
Table (1): The primer sequences and PCR conditions.
Gene name
size (bp)
ACO oxidase
PPARα (Peroxisome proliferator-activated receptor alfa); CPT1 (carnetine palmetoyl transferase 1); ACo oxidase
(Acyl Co-A oxidase). 22013;10(Life Science Journal,
Figure 1: Histogram showing changes in expression level of mRNA for PPAR-α gene in relation to
GAPDH due to treatment with carmoisine. Relative mRNA levels expressed in relative IDV (integrated
density value) of PPAR-α/ GAPDH as measured by AlfaEaseFC software.
Figure 2: Histogram showing changes in expression level of mRNA for Acyl-COA gene in relation to
GAPDH due to treatment with carmoisine. Relative mRNA levels expressed in relative IDV (integrated
density value) of ACO A/ GAPDH as measured by AlfaEaseFC software.
Figure 3: Histogram showing changes in expression level of mRNA for CPT1 (carnetine palmetoyl
transferase 1) gene in relation to GAPDH due to treatment with carmoisine. Relative mRNA levels expressed
in relative IDV (integrated density value) of CPT1/ GAPDH as measured by AlfaEaseFC software. 22013;10(Life Science Journal,
Figure 4: Liver section stained with routine stain, showing normal histology of liver in the control rat liver. (Hx& E.
st., X 100 ).
Figure 5: Liver sections stained with Alcian blue stain X 400, showing: A) alcianophilia ; B) mucoid degeneration
of the hepatocytes; C) ballooned swollen hepatocytes that stores fat droplets as also was present mucoid
degeneration of the hepatocytes due to the effect of carmoisine on histology of liver.
Figure 6: Liver sections stained with Crossmon’ s trichrome stain showing: A) fibrinoid deposition around some
central veins and in the portal areas-X 100 ; B) swollen with fatty changes and some congestion of the hepatic blood
vessels -X 400; C) congested hepatic blood sinusoids-X 400, due to the effect of carmoisine on histology of liver.
4. Conclusion:
From our studies we can conclude that, the
expression levels of fuel metabolism proteins and
enzymes e.g. PPAR-alfa, Acyl Co-A and Carnetine
palmetoyl transferase-1 could be affected by the use of
carmoisine especially the high doses for long time.
The high doses of carmoisine could be harmful to
liver and lowers the expression level of some metabolic
enzymes in it.
1. Abdel-Rahim, G.A. (1988): Effect of some natural and
synthetic food colorants on protein nucleic acids and
nucleases in albino rats organs. Minia J. Agric. Res.
and Dev., 10(4)112-117.
2. Abou El-Zahab, H.S.H.; El-Khyat, Z.A.; Awadallah,
R. and Mahdy, K.A. (1997): Physiological effects of
some synthetic food coloring additives on rats. Boll.
Chim. Farm.,136(10):615-627.
3. Ali, M. O.; Al-Ghor, A.; Sharaf, A. K.; Mekkawy,
H.; Montaser, M. M.(1988) Genotoxic effects of food
color (Carmoisine) on the chromosome of bone marrow
cells of rat. Toxicology Letters, 95(1):44.
4. Amin, K.A.,; Abdel-Hameid, H.; Abd Elsattar, A. H.
(2010):Effect of food azo dyes tartrazine and
carmoisine on biochemical parameters related to renal,
hepatic function and oxidative stress biomarkers in
young male rats. Food and Chemical Toxicology,
5. Beverley, S.M., (2001). Current Protocols in Molecular
Biology 15.5.1-15.5.6, Copyright © 2001 by John
Wiley & Sons, Inc.
6. Booth, J. (1993): Food inolerance in a child with
urticaria. J. Human Nutrition and Dietetics,
6(4):377-380. 22013;10(Life Science Journal,
7. Branen, A.L.;Davidson, P.M. and Salminen,
S.(1990):Food additives ,Marcel,INC.,270 Madison
Avenue, New York 10016, Printed in the USA.
8. Brozelleca, J.F. and Hallagan, J.B. (1988a): A
chronic toxicity/carcinogenicity study of FD & C
yellow No. 5 (tartrazine) in mice. Fd. Chem. Toxic., 26
9. Brozelleca, J.F. and Hallagan, J.B. (1988b): Chronic
toxicity/carcinogenicity studies of FD & C yellow No.
5 (tartrazine) in rats. Fd. Chem. Toxic., 26 (3):179-187.
10. Brozelleca, J.F.; Olson, J.W. and Reno, F.E. (1989):
Life time toxicity/carcinogenicity study of FD & C Red
no. 40 (Allura Red) in Sprague-Dawley rats. Fd. Chem.
Toxic., 27 (11): 701-706.
11. Chromoczynski, P. and N. Sacchi, 1987. Single-step
method for RNA isolation by acid guanidium U.S.
thiocyanate-phenol-chloroform extraction. Anal
Biochem., 162: 156-9.
12. El-Saadany, S.S. (1991): Biochemical effect of
chocolate colouring and flavouring like substances on
thyroid function and protein biosynthesis. Die
Nahrung., 35 (4):335-343.
13. FDA (1982): Toxicological principles for safety
assessment of direct food additives used in foods. In :
Red book. Food and Drug Administration, Washington,
14. Feingold, B.F. (1979): Dietary management of
nystagmus. J. Neural. Transm., 45 (2):107-115.
15. Ford, G.P.; Stevenson, B.I. and Evans, J.G. (1987):
Long term toxicity study of carmoisine in rats using
animals exposed in utero. Fd. Chem., 25 (12):919-925.
16. Gaunt, I.F.; Carpanini, F.M.B.; Grasso, P.; Kis,
Idas, S. and Gangolli, S.D. (1972): Long term feeding
study on brilliant blak PN in rats. Fd Cosmet. Toxicol.,
10 (1):17-27.
17. Gaunt, I.F.; Grasso, P.; Kiss, Ida, S. and Gangolli,
S.D. (1969): Short-term toxicity study on carmoisine in
the miniature pig. Food Cosmet. Toxicol., 7(1):1-7.
18. Gaunt, I.F.;Madge Farmer;Grasso , P. and
Gangolli, S. D.(1967):Acute (mouse and rat ) and short
term (rat) toxicity studies on Carmoisine.Food Cosmet.
19. George, J. and Liddle, C. (2008) Nonalcoholic fatty
liver disease: pathogenesis and potential for nuclear
receptors as therapeutic targets. Molecular
Pharmaceutics. 5(1) 4959.
20. Gerd, M. and Lennart. J. (1973): Urticaria induced
by preservatives and dye additives in food and drugs.
Bull. JSME (Jap. Soc. Mech. Engl.), 88 (6):525-532.
21. Hall, R.L. (1958): Flavor study approach at
McCormick and Company, Inc. In : Flavor Research
and Food Acceptance, Reinhold, New York, p. 224.
22. Haveland-Smith, R.B. (1980): An evalauation of the
genetic effects of some food colors using microbial test
systems. Ph. D. Thesis, Council for National Academic
Awards, London.
23. Hong, S.P.; Park, H.S.; Lee, M.K. and Hong, C.S.
(1989): Oral provocation tests with aspiring and food
additives in asthmatic patients. Yonsei Medical J., 30
24. ILSI/NF (1981): Catalog of Food Colors. International
Life Sciences Institute/ Nutrition Foundation,
Washington, D.C.
25. Juhlin, L. (1981): Recurrent urticaria : Clinical
investigation of 330 patients. Br. J. Dermatol.,
26. Kim, J.H.; Hahm, D.H.; Yang, D.C.; Lee, H.J.;
Shim, I. (2005) Effect of crude saponin of Korean
red ginseng on high-fat diet-induced obesity in the rat.
J. Pharmacol. Sci. 97: 124131.
27. Marmion, D.M. (1984): Handbook of U.S colorants
for foods, drugs and cosmetics. Wiley, New York.
28. Murdoch, R.D.; Pollock, I. and naeem, S. (1987):
Tartrazine induced histamine release in vivo in normal
subjects. J. Royal College Physicans (London),
29. NAS/NRC (1971): Food colors. Nat. Acad. Sci./Nat.
Res. Council. Nat. acad. Press, Washington, D.C.
30. Newsome, R. L.(1986): Food colors. Food
31. Osman, M.A.; Afifi, A.; Hussien, R.M.; Kamilia, B.;
Abdel-Aziz and Salah, S.H. (1995): Long-term
biochemical and genotoxicity studies of four synthetic
food and drug colorants in mice. Bull. Fac. Pharm.
(Cairo Univ.), 33 (1):13-21.
32. Pollock, I. and Warner, J.O. (1990): Effect of
artificial food colors on childhood behaviour.
Archieves of Disease in Childhood, 65(1): 74-77.
33. Rowe, S.K. (1988): Synthetic food colourings and
hyperactivity : a double-blind crossover study. Aust.
Paediat. J., 24 (2):143-147.
34. Sato, H.; Tsai-Morris, C.; Dufau, M. L. (2010)
Relevance of gonadotropin-regulated testicular RNA
helicase (GRTH/DDX25) in the structural integrity of
the chromatoid body during spermatogenesis. Bioch et
Bioph Acta, 1803: 534-543.
35. Shaker, A.M.H.; Ismail, I.A. and Eilnemr, S.E.
(1989): Effect of different food stud colurants added to
case in diet on biological evaluation. Bull. Nutr. Inst.,
Cairo, Egypt, 9 (1):77-86.
36. Tannahill, R. (1973): Food in history. Stein and Day,
New York.
37. Twaroj, F.J. (1983): Urticaria in childhood:
pathogenesis and management. Paediat. Clin. North.
Am., 30:887-898.
38. Van Bever, H..P.; Doxx, M. and Stevens, W.J.
(1989): Food and food additives in severe atopic
dermatitis. Allergy (Copenhagen), 44 (8):588-594.
39. Videla, L.A. and Pettinelli, P. (2012) Misregulation of
PPAR Functioning and Its Pathogenic Consequences
Associated with Nonalcoholic Fatty Liver Disease in
Human Obesity. PPAR Research. Article ID
... According to the classification of the International Agency for Research on Cancer, azo dyes such as carmoisine are placed in category 3 of carcinogens. Carmoisine can reduce fuel metabolism in the liver (98). In brief, hydrophobic azo dyes are not safe for use due to their ability to induce tumors in the body systems (99). ...
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: Azo dyes, as a major group of the synthetic colorants, are added to food products not only to make them aesthetic but also to preserve their appearance. However, the use of azo dyes in food has been banned worldwide due to side effects on human health. The search was conducted using PubMed, Scopus, Web of Science, Europe PMC beta, Science Direct, and Springer database considering all articles published up to 9 July 2021. The inclusion criteria were double-blind, randomized, cohort studies, placebo-controlled trials, case reports, non-controlled trials, and case series. Several studies suggest that azo dyes induce oxidative stress, which subsequently increases the concentration of malondialdehyde and reduces superoxide dismutase (SOD) activity and glutathione (GSH) concentration in brain tissue. Also, results showed the adverse effects of azo dyes on the brain parts such as the prefrontal cortex, cerebellum, and cerebrum which, are accompanied by changes in brain function. It can be concluded that azo dyes with an increase in oxidative stress affect the most important parts of the brain and cause brain dysfunction. This study aimed to evaluate the effects of the food additive azo dyes on brain tissues.
... Gene expression is one of the molecular biomarkers representing the ultimate effect of cells and has become an important tool in ecotoxicology research (Dondero et al. 2011). Carmoisine has been reported to affect the normal expression of MAPK8, Bcl-X, PARP, p53 and ACo-A in mice and promote apoptosis and activation of inflammatory pathways in cells (Raposa et al. 2016;Reza et al. 2019;Montaser and Alkafafy 2013). However, few studies have reported that the toxicity of carmoisine affects insect gene expression. ...
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Carmoisine belongs to a water-soluble synthetic dye and is often used as a food additive. Previous research has shown that carmoisine is toxic to rats and zebrafish, but there have been few reports on the effect of carmoisine on soil-dwelling social insects. The present study evaluated carmoisine toxicity in Polyrhachis vicina Roger. We found that the effects of different concentrations of carmoisine on the mortality of workers were dose-dependent. The 10% lethal dose (LD10), 50% lethal dose (LD50) and 90% lethal dose (LD90) of carmoisine to workers at 96 h was calculated to be 0.504, 5.491 and 10.478 g/L, respectivily. LD10 of workers were selected to treat the fourth instar larvae, pupae and adults for 10 days. The results showed that the survival rate of all ants, except for females, was significantly reduced, especially larvae and workers. The body weight of larvae, pupae and males decreased significantly, while weight gain was observed in the females and workers. The appearance of larvae, pupae and workers changed after carmoisine treatment, such as body darkening and epidermis shrinking of larvae and pupae, as well as body segment expansion of workers. Furthermore, carmoisine altered the expression of the estrogen-related receptor, tailless and homothorax of P. vicina (Pv-ERR, Pv-tll and Pv-hth) to varying degrees in larvae and adults. We believe that variations in body weight can lead to a decrease in survival rate and appearance changes in the ants, which may be related to abnormal gene expressions caused by carmoisine treatment. Therefore, we confirm that carmoisine has negative effects on the growth and development of P. vicina.
... In recent times, the use of artificial coloring in food items is quite widespread. Since some synthetic colours have been found to have played a vital role in the causation of cancer in laboratory animals, the search for alternative, safe natural food colours has intensified and continues to top the priority lists of many research laboratories around the world [1]. ...
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The application of coloring to food is widespread around the globe. Some of the artificial food colorants are highly carcinogenic. Exploration of new natural colorants has therefore been under research. The present study was aimed to evaluate Hibiscus rosasinensis flower as a potential feedstock for natural colorant. Crude anthocyanin was first extracted from Hibiscus rosasinensis flower using a Soxhlet apparatus with ethanol as the solvent. Flower to solvent ratio was found to affect the extraction largely. A ratio of 1:20 was found to give the highest fraction of extract. The cytotoxicity of crude anthocyanin extract was subsequently determined by Brine Shrimp Lethality Test. The LC 50 value for crude anthocyanin extract was 2332 µg/ml, which is higher than the required minimum threshold value. Hence the findings of present study would be useful towards the application of H. rosasinensis flower as a feedstock for natural colorant to food.
... On the other hand, the liver sections of rats treated an oral dose of carmoisine showed mildly edematous portal tracts with mildly dilated congested portal veins and average hepatocytes in peri-portal area, and mildly dilated central veins with mildly congested blood sinusoids and average hepatocytes in peri-venular area (Figure 3). Montaser and Alkafafy (2013) found increasing the doses of carmoisine more than ADI led to more degenerative changes of hepatocytes that revealed hepatic cells mucoid degeneration. ...
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This study was carried out to evaluate and compare the possible toxic effect of some synthetic food colorants (carmoisine, raspberry, sunset-yellow and fast green) with some natural food colorants (anthocyanin, betalain, carotenoids and chlorophyll) on biochemical parameters as well as liver and kidney histological of experimental rats. 45 young male albino rats (weight about 100-120 gram) were used in this study. All rats were fed on a balanced diet for one week, when divided randomly into nine groups and fed on tested diets for four weeks The results showed significant increase in levels of serum alanine amino transferase, aspartate amino transferase, urea, creatinine, total protein and albumin, with decreased levels of immune-globulins were observed in all rats groups treated by synthetic colorants groups when compared to rats groups treated by natural food colorants and control group. Histological examinations revealed alterations in kidneys include: congestion and hemorrhage with infiltration and deformation of the glomeruli structure. Whereas, alterations in liver include: congestion, hemorrhage and dilated of sinusoids and central vein with micro vesicular steatosis. Therefore, it is advisable to limit the uses of synthetic food colorants or replacing them by natural ones especially for children foods.
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Aim: To evaluate chronic exposure of carmoisine at ADI doses on some hepatocellular and renal parameters of male and female albino rats as well as to determine sex-dependent toxicity. Study Design: The study involves treatment for 30, 60, and 90 days. Each phase consists of 40 rats, divided into treatment and control groups. The treated groups were orally administered with 4.0 mg/kg of carmoisine daily for the periods of 30, 60, and 90 days. Methodology: At the end of the treatment, the rats were allowed to fast for 18 hours followed by the collection of 5 ml of whole blood specimens by means of cardiac puncture into Lithium Heparin bottles and fluoride oxalate bottles (for glucose only). Plasma obtained was analyzed for glucose (GLU), AST, ALT, ALP, creatinine (CRT), and urea. Hepatic and Renal tissues collected were fixed in 10% formol saline and later examined histologically using H&E stain. Statistical data analysis was done using GraphPad Prism version 9.02. Results: Glucose indicated significant increases after 30, 60, and 90 days of chronic treatment at ADI doses. Urea, Creatinine, AST, ALT and ALP showed significantly higher values after 60 and 90 days of treatment (except creatinine in male rats and ALP in female rats after 60 and 90 days respectively). Hepatic distortions, vacuolation, compression of central vein were seen in the liver section while distortion of proximal and distal tubules, and inflammation of the glomerulus were observed in the renal tissue of the treated rats. Conclusion: The administration of camoisine over a period of 30 days at ADI dose did not indicate hepatocellullar and renal derangements as well histological distortions in liver, and kidneys. However, after 60 and 90 days, mild hepatocellular, and renal derangements were seen. No sex-dependent toxicity was observed.
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Food color additives are used to make food more appetizing. The United States Food and Drug Administration (FDA) permitted nine artificial colorings in foods, drugs, and cosmetics, whereas the European Union (EU) approved five artificial colors (E-104, 122, 124, 131, and 142) for food. However, these synthetic coloring materials raise various health hazards. The present review aimed to summarize the toxic effects of these coloring food additives on the brain, liver, kidney, lungs, urinary bladder, and thyroid gland. In this respect, we aimed to highlight the scientific evidence and the crucial need to assess potential health hazards of all colors used in food on human and nonhuman biota for better scrutiny. Blue 1 causes kidney tumor in mice, and there is evidence of death due to ingestion through a feeding tube. Blue 2 and Citrus Red 2 cause brain and urinary bladder tumors, respectively, whereas other coloring additives may cause different types of cancers and numerous adverse health effects. In light of this, this review focuses on the different possible adverse health effects caused by these food coloring additives, and possible ways to mitigate or avoid the damage they may cause. We hope that the data collected from in vitro or in vivo studies and from clinical investigations related to the possible health hazards of food color additives will be helpful to both researchers and the food industry in the future.
Human food is composed of loads of chemicals derived naturally as well as unintentionally through environmental sources. Food additives added purposefully, play an important role in the palatability of foods. Most additives are synthetic whose essentiality in food processing is well-known however their health risks are not overlooked. The palatability of food should not only stimulate our eating desire alone but, also assure sufficient quality and safety. Application of food additives varies from region to region due to cultural or ethnic differences and the local food availability. There are about more than ten thousand chemicals allowed in food whereas due to weak enforcement, it becomes onerous for regulatory bodies identifying chemicals that are inadequately or not tested at all for safety. The hiking population and urbanization in many industrialized and developing countries resulted in life-style changes including culinary and eating choices. Particularly, the modern way of this globalised life demands ready-to-cook or ready-made foods, snacks, sweets, soft drinks, desserts, confectionery and so on. These sorts of food would be most uninteresting unless processed with additives. This puts food industries under demand to robustly supply foods that are either partially, fully or ultra-processed using plenty of additives. Recent research warns consuming food additives may result in serious health risks, not only for children but also for adults. Growing body of studies on food additives in various experimental animals, cell cultures, and human population suggest elevation of number of obesity and diabetes risk factors i.e. adiposity, dyslipidemia, weight gain, hyperglycaemia, insulin resistance, glucose intolerance, energy imbalance, hormonal intervention etc. Hence, it is important to identify and explore food obesogens or obesogenic food additives posing potential impact. Based on the recent toxicological findings, the review aspires to establish the association between exposure of food obesogen and metabolic disruption which may help filling knowledge gaps and distributing more knowledge, awareness and effective measures to implement treatment and preventive strategies for metabolic syndrome.
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The objective of the present study was to assess the effects of various doses of Azorubine which is a food additive on the female reproductive organs in Sprague Dawley rats. Twenty four female Sprgue Dawley rats were divided randomly into 4 equal groups. Group 1(control group), group, 2, 3 and 4 were received Azorubine (5, 10 and 20 mg/kg) orally, daily for 30 days respectively. Blood samples were taken for estimation of white blood cells, red blood cells, hemoglobin and platelets, in addition Luteinizing, follicular stimulation, estrogen and progesterone hormones from the sera. The reproductive hormones levels affected drastically under the effects of different doses of treatment like Luteinizing hormone (0.69±0.25, 0.60±0.75 and 0.55±0.63), Follicular Stimulation hormone (0.17±0.11, 0.13±0.33 and 0.3±0.45), Progesterone hormone (0.50±0.77, 0.14±0.56 and 0.10±0.85), and estrogen hormone (0.45+0.43, 0.30±0.29 and 0.14±0.27) hormones were decreased significantly (P˂0.05) in groups of rats treated with each 5, 10 and 20 mg/kg doses of Azorubine respectively. Histopathologically, the ovaries treated with 5 mg/kg doses of Azorubine showing follicles at the beginning stages of growth with no Graffian follicle while the ovaries with 10 mg/kg doses of Azorubine contain fully grown Graffian follicles with no follicles at various stages as well as those with 20 mg/kg doses of treatment displaying no mature Graffian follicle with many atretic and shrunk follicles. The hematological outcomes are significantly affected by this food additive. The results of this work is concluded that Azorubine can be considered as one of the most important causes of infertility, hormonal disturbances and irregular estrus cycle in the female rat.
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Dyes in food products, drugs and cosmetics are used to maintain color and classified as artificial or natural with an estimated world production of ~8 million tons per year. The FDA and U.S. Department of Agriculture have identified safe colorants (GRAS) based on safe use in food and monitor GRAS continuously. If published evidence suggests that GRAS are mutagenic, they are delisted. For most additives, JECFA/FAO has allowed a classification of “Admissible Daily Intake Dose” (ADI), most frequently provisional but still requires additional evaluation, being genotoxic if dose is lower than ADI dose. Even certified colorants can elicit adverse reactions (WHO, 1991) and in foodstuffs can induce cancer. It appears that synthetic colorants are undesirable and efforts to use natural colorants due to consumer preference as well as legislative action have promoted delisting of registered synthetic dyes. Tartrazine (E120) is the 2nd most used food dye derived from coal tar and used in food, pharmaceuticals and cosmetics but can be mutagenic and affect cell viability. Curcumin (E100) from the minced root of the herb Curcuma longa Linn. Is a double-edged sword, like other antioxidants with anticancer, antioxidant and pro-oxidant with possible but doubtful mutagenic properties. We review conflicting toxicity of natural versus synthetic food colorants with special emphasis on curcumin and tartrazine.
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Nonalcoholic fatty liver disease in human obesity is characterized by the multifactorial nature of the underlying pathogenic mechanisms, which include misregulation of PPARs signaling. Liver PPAR-α downregulation with parallel PPAR-γ and SREBP-1c up-regulation may trigger major metabolic disturbances between de novo lipogenesis and fatty acid oxidation favouring the former, in association with the onset of steatosis in obesity-induced oxidative stress and related long-chain polyunsaturated fatty acid n-3 (LCPUFA n-3) depletion, insulin resistance, hypoadiponectinemia, and endoplasmic reticulum stress. Considering that antisteatotic strategies targeting PPAR-α revealed that fibrates have poor effectiveness, thiazolidinediones have weight gain limitations, and dual PPAR-α/γ agonists have safety concerns, supplementation with LCPUFA n-3 appears as a promising alternative, which achieves both significant reduction in liver steatosis scores and a positive anti-inflammatory outcome. This latter aspect is of importance as PPAR-α downregulation associated with LCPUFA n-3 depletion may play a role in increasing the DNA binding capacity of proinflammatory factors, NF-κB and AP-1, thus constituting one of the major mechanisms for the progression of steatosis to steatohepatitis.
Groups of 24 male and 24 female weanling rats were fed for 2 yr on diets containing 0 (control), 1000, 5000 or 10,000 ppm Black PN. No effects attributable to treatment were found in respect of mortality, body-weight gain, food intake, haematology, serum chemistry, renal concentration tests, organ weights or incidence of pathological findings, including tumours. No carcinogenic potential was detected in Black PN and the no-untoward-effect level was 10,000 ppm (approximately 500 mg/kg/day).RésuméDeux groupes de jeunes rats sevrés, 24 mâles et 24 femelles, ont reçu pendant deux ans du Noir PN à raison de 0 (animaux témoins), 1000, 5000 ou 10 000 ppm du régime. Aucun effet imputable au traitement n'a été observé sous le rapport de la mortalité, du gain de poids, de la consommation de nourriture, de l'hématologie, de la chimie du sérum, des tests de concentration rénale, du poids des organes et de la fréquence des phénomènes pathologiques, tumeurs comprises. Le Noir PN n'a manifesté aucun pouvoir cancérigène et son seuil d'indifférence est de 10 000 ppm (environ 500 mg/kg/jour).ZusammenfassungGruppen von 24 männlichen und 24 weiblichen abgesetzten Ratten wurden 2 Jahre lang mit Futter gefüttert, das 0 (Kontrolle), 1000, 5000 oder 10 000 ppm Black PN enthielt. Es wurden keine dieser Verabreichung zuzuschreibenden Erscheinungen bei der Mortalität, Körpergewichtszunahme, dem Futterverbrauch, der Hämatologie, Serumchemie, Nieren-Konzentrationstest, Organgewichten oder der Häufigkeit pathologischer Befunde, einschliesslich Tumoren, festgestellt. Eine carcinogene Wirkung wurde bei Black PN nicht gefunden, und die von schädlichen Wirkungen freie Konzentration war 10 000 ppm (etwa 500 mg/kg/Tag).
This case study illustrates the effectiveness of dietary advice in a young boy with chronic idiopathic urticaria. An azo dye and preservative-free diet was initially advised, resulting in a total improvement in urticarial symptoms. Double-blind challenges confirmed the boy was intolerant to E127 (erythrosine), E122 (carmoisine), 128 (red 2G), and E102 (tartrazine) but not to E211 (sodium benzoate).
Three different synthetic chocolate colourant agents (A, B and C) were administered to healthy adult male albino rats for 30 and 60 day periods to evaluate their effects on body weight, blood picture, liver and kidney functions, blood glucose, serum and liver lipids, liver nucleic acids (DNA and RNA), thyroid hormones (T3 and T4) and growth hormone. In addition, histopathological examinations of liver, kidney and stomach sections were studied. These parameters were also investigated 30 days after colourant stoppage (post effect). Ingestion of colourant C (brown HT and indigocarmine) significantly decreased rat body weight, serum cholesterol and HDL-cholesterol fraction, while, T4 hormone, liver RNA content, liver enzymes (S. GOT, S. GPT and alkaline phosphatase), total protein and globulin fractions were significantly elevated. Significant increases were observed in serum total lipids, cholesterol, triglycerides, total protein, globulin and serum transaminases in rats whose diets were supplemented with chocolate colours A and B (sunset yellow, tartrazine, carmoisine and brilliant blue in varying concentrations). Haematological investigations demonstrated selective neutropenia and lymphocytosis with no significant alterations of total white blood cell counts in all rat groups, while haemoglobin concentrations and red blood cell counts were significantly decreased in the rats who were administered food additives A and B. Eosinophilia was noted in rats fed on colourant A only. No changes were recorded for blood glucose, growth hormone and kidney function tests. Histopathological studies showed brown pigment deposition in the portal tracts and Van Küpffer cells of the liver as well as in the interstitial tissue and renal tubular cells of the kidney mainly induced by colourant A. Congested blood vessels and areas of haemorrhage in both liver and renal sections were revealed in those rats who were given colourants B and C. There were no-untoward-effects recorded in the stomach tissue.
Fifty-two patients with recurrent urticaria or angio-oedema and thirty-three controls have been provoked with five different food dyes and the preservatives sodium benzoate and 4-hydroxy-benzoic acid, as well as aspirin, sulphanilic acid and a placebo. The reaction was judged as positive in thirty-nine patients who developed urticaria within 14 h. Of these, thirty-five reacted to aspirin, twenty-seven to benzoic acid compounds and twenty-seven to azo dyes. The four patients who did not have urticaria after aspirin, reacted with urticaria to benzoic acid compounds, and three of them to azo dyes. No definite pattern for the reaction to the different azo dyes was seen. None had an urticarial reaction from sulphanilic acid, Patent Blue (a non-azo dye) or placebo. The doses of additives used in the provocation tests are easily exceeded in daily life by the consumption of foods and drugs. Recurrences of urticaria could be prevented through the avoidance of food and drugs containing azo dyes and preservatives.