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Study of the pathological effects to one of Food additives (Gelatin 441)in male rats

Authors:
  • Al Muthanna University/ Medicine college

Abstract and Figures

The amount of locally consumed gelatin gel 441 has increased in foodstuffs in the Iraqi market as a digestible substance or as food additives. Although the high eating of this substance, no study has been found for its side effects, So that come this study to determine the effect of high-concentration (375 mg / kg) of gelatin in some body tissues and blood tests for liver and kidney function and oxidative state tests. Materials and Methods: In the study, 12 laboratory rats were trained and studied at the Animal House of the College of Education \ University of Qadisiyah. The animals were divided into two groups (for each group 6 animals),The control group : was treated orally Normal saline(0,9) for one month (30 days), The treated group: was treated orally 375 mg / kg gelatin 441 of body weight for 30 Days. Results: The results showed significant changes(P <0.05)in all the tests that were used. There was a significant increase in liver function tests (ALT, AST) and significant increase in kidney function tests (Urea, creatinine).As for oxidative state tests, there was a significant decrease in (SOD, GST, GPx, T-AOC),while the rates of (MDA, NO) tests were significantly higher when compared to the control group. The histological sections of the tissue used in the study appeared different tissue changes by treatment with gelatin 441. In the heart sections there was damage in the branching structure of the fibers and relapse of some cells. WhileIn the spleen tissue sections, there was reduction of the white pulp area compared to the red pulp and Receding of the PALS sheath, Also there was also damage to the general structure of the kidney tissue and the disintegration of the glomerulus and the lining of twisted tubules. In addition, there appeared damage to the radial regulation of 2 hepatic cells, the expansion of the central vein and the relapse of some cells in the tissue of liver. In addition, the tissue of the stomach showed disintegration. Where observed that the dissociation in structure of the columnar epithelial tissue and the area of the chief cells was reduced. As for the testicular tissue, there was a disintegration of Tunica vasculosa tissue and a clear expansion between the tubules and the disappearance of their cells. Conclusions: It can be concluded that eating high concentrations of gelatin 441 leads to side effects and disease in the body may be dangerous to the health of consumers, especially children, so we recommend avoiding the eating of large amounts of gelatin directly and putting of laws by the government to regulate the amounts of food additives and follow-up side effects.
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Study of the pathological effects to one of Food additives
(Gelatin 441)in male rats
Nael Mohammed Sarheed*, Ali Noory Fajer**, Meaad Nasser Hussein***
* Dep. of Physiology \ Medicine College\Al-Muthanna University\ Nael.serheed@mu.edu.iq
**,***Dep. of Chemistry \ Education College\Al-Qadisiyah University\Ali.fajer@qu.edu.iq
Abstract
The amount of locally consumed gelatin gel 441 has increased in foodstuffs in the
Iraqi market as a digestible substance or as food additives. Although the high eating of
this substance, no study has been found for its side effects, So that come this study to
determine the effect of high-concentration (375 mg / kg) of gelatin in some body tissues
and blood tests for liver and kidney function and oxidative state tests. Materials and
Methods: In the study, 12 laboratory rats were trained and studied at the Animal House
of the College of Education \ University of Qadisiyah. The animals were divided into
two groups (for each group 6 animals),The control group : was treated orally Normal
saline(0,9) for one month (30 days), The treated group: was treated orally 375 mg / kg
gelatin 441 of body weight for 30 Days. Results: The results showed significant
changes(P <0.05)in all the tests that were used. There was a significant increase in liver
function tests (ALT, AST) and significant increase in kidney function tests (Urea,
creatinine).As for oxidative state tests, there was a significant decrease in (SOD, GST,
GPx, T-AOC),while the rates of (MDA, NO) tests were significantly higher when
compared to the control group. The histological sections of the tissue used in the study
appeared different tissue changes by treatment with gelatin 441. In the heart sections
there was damage in the branching structure of the fibers and relapse of some cells.
WhileIn the spleen tissue sections, there was reduction of the white pulp area compared
to the red pulp and Receding of the PALS sheath, Also there was also damage to the
general structure of the kidney tissue and the disintegration of the glomerulus and the
lining of twisted tubules. In addition, there appeared damage to the radial regulation of
2
hepatic cells, the expansion of the central vein and the relapse of some cells in the tissue
of liver. In addition, the tissue of the stomach showed disintegration. Where observed
that the dissociation in structure of the columnar epithelial tissue and the area of the
chief cells was reduced. As for the testicular tissue, there was a disintegration of Tunica
vasculosa tissue and a clear expansion between the tubules and the disappearance of
their cells. Conclusions: It can be concluded that eating high concentrations of gelatin
441 leads to side effects and disease in the body may be dangerous to the health of
consumers, especially children, so we recommend avoiding the eating of large amounts
of gelatin directly and putting of laws by the government to regulate the amounts of food
additives and follow-up side effects.
Keyword's \ Gelatin , Blood tests , Oxidation , Tissue , rate
Introduction
The interest recently increased on the importance of secondary industrial products
and improved conditions for extraction such as gelatin and collagen (1),Where the
attention of researchers in the last century on the importance of the sources of these
products, especially gelatin, which is extracted from many sources such as pigskin,
cattle , cattle bones , many mammals and fish (2)(3),This substance has been used in many
industries including cosmetics, pharmaceuticals and many food items such as ice-cream
and beer (1)(4), Gelatin was also used in practical fields such as scaffolding to create
tissue cell lines which showed great importance for cell proliferation and profusion in a
short time (5)(6), In addition, gelatin has been found to be an important nutrient for objects
(7), and to save food at high humidity through improved thermal stability (8).
Gelatin is a protein-rich peptide that has digestibility and water solubility and
contains all amino acids except tryptophan (9). It is obtained from the breakdown of
collagen fibers that form bones, skin and cartilage In the bodies of animals that has age
and the type of decomposed collagen are of fundamental importance in the
characteristics of gelatin type(10)(11), The synthesis of collagen consists of three
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interlocking alpha chains called triglyceride collagen, in which the chains are bound
together by hydrogen bonds that combine proline and hydroxyproline compounds (12),In
the process of extracting gelatin, the non-biodegradable collagen is processed before it is
converted into gelatin, which is heated by water at 45°C (13)(14), in which the collagen is
transformed from the helical form into the gelatin coil Soluble in water(15), and
measuredthe gelatin gel by a test called the Bloom test(16), where acids and alkalis are
usually used to extract gelatin of A and B type from fish skin and other sources in order
to obtain the required properties (17)(18) , while experts found this type of important
benefits related to prevention of mad cow disease ,inflammation and Foot-and-mouth
disease (19).
There are healthy effects in the human body that may be due to take of large amounts
of gelatin, and these effects depend on the source of extraction of this type of gel,
inaddition , researchers said that eating gelatin inhibits low bone mineral density, but
does not affect the main biochemical characteristics (2), As well as the eating of gelatin
to the body at high concentrations lead to high blood pressure , disturbance of the rates
of systolic, diastolic, heart beat and average of arterial pressure (20).
There is no previous study to know the side effects and pathogenic effects of high
concentrations of gelatin, although the widespread in the Iraqi market as a substance or
food additives, the study came to know these side effects and changes resulting from the
eating of gelatin in many tissues of the body and the changes in the biochemical tests
and the oxidative state within the body of the organism.
Materials and Methods
1.Chemicals used: GelatinE441 obtained from the local Iraqi market imported
from Aganata Chemical Industries (India)
2.Experimental animals: Experimental rats animals (12 rats) were obtained from
the Animal House of the Faculty of Science at the University of Babylon and for the
males in weights ranging from 180-200 grams. The animals were left for two weeks to
adapt to the new environment in the animal house of the Department of Life Sciences /
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Faculty of Education / Al Qadisiyah University. They were putting under standard
conditions (20-25 ° C) and sterile polypropylene cages (15 * 35 * 50 cm) With stainless
cedar husk of sterile conditions, supply them with water and standard diet pellet the
length of the experiment period. They were orally administered for one month (30 days)
with gelatin E441.
3.Design of the experiment: The animals are divided in two groups, each group has
6 animals as follows: The control group ,was given orally the saline solution (Normal
saline) for the duration of the experiment, The treated group, was given Gelatin E441 at
a concentration of 375 mg / kg body weight throughout the study period (30 days).
4. Blood sampling and tests: At the end of the trial period, blood samples were
collected directly from the heart to obtain the serum. Blood samples were used to
prepare serum after centrifugation at 1500 rpm. Biochemical tests were carried out as
follows: were measured ALT , AST tests according to (21 ) , Urea and Creatinine tests
according to (22 ) , SOD(23), GST(24), GPX(25), MDA(26), NO(27) and T-AOC by (28).
5. Histological study: The process of tissue cutting and staining of organs (heart,
liver, kidney, spleen, stomach and testis) was performed according to the method(29 ).
6. Statistical analysis: They were made between the control group and the
treatment group according to (SPSS) program, where aused T-test for marital
comparisons (30).
Results and discussion
1. Liver function tests
The statistical analysis of blood biochemical parameters showed clear changes in all
tests for animals treated with gelatin E441 during the trial period and the concentration
used. The liver function tests (ALT, AST) appear significant increase (P <0.05) for both
tests after treatment with gelatin E441when compared Control group, which was evident
through histological changes in the liver structure(Table 1).
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The liver is one of the most important organs to perform many functions in the body,
most importantly detoxification, which causes it to be exposure to damaged as a result of
these substances and what is generated from its metabolism(31). The measurement of
enzymes (AST, ALT) is a sensitive measure of the extent of damage in hepatocytes
(32),The increase in the efficiency of these enzymes for the group of animals treated with
gelatin E441 may be due to the negative role to the free radicals caused by oxidative
stress due to the effect of this substance confirmed by the abnormal oxidation tests in
this study, Where free radicals have an effect through histological changes in the liver
that have been evident in the tissue sections of the liver and led to the perfusion of large
amounts of these enzymes to the blood plasma, which led to a high level in the serum
(33). The increase may also be caused by membrane destruction of Hepatocytes as a
result of oxidative stress resulting from treatment with gelatin E441, which produces
free radicals that increase the proportion of these enzymes in the serum (34).
2. Kidney function tests
The results showed a significant increase (P <0.05)in the tests of the kidney function.
A significant increase was found in the level of blood urea for the animals treated with
gelatin E441 , also significant increase in serum creatinine in the same group when
compared with Non-treated group(control)(Table 2).
The measurement of urea and creatinine is a good indicator of kidney function
efficiency(35). The reason for the increase in the amount of these substances in the serum
of animals treated with gelatin E441 may be due to the pathological changes in the
structure of the kidney in particular the glomerular structure after exposure to this
substance, Which may lead to functional deficiencies of the kidney, and the cause may
be disorders of kidney function, which are caused by the effects of this food additives
leads to disruption of metabolism through by production of toxic compounds and then
affect the tissue of kidney causing damage(36),or the increase in the level of urea and
creatinine may be result from the decrease in the rate of passage of the urinary plasma
produced by the low glomerular filtration rate(37), as well as the increase in the resistance
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of glomerular arteries and the decrease in blood volume which leads to a decrease in the
release of urea and creatinine, They accumulate in the body and thus increase their level
in serum (33).
3. Oxidative state tests
In the oxidative condition, the results of the tests were completely different from the
normal proportions that appeared in the blood of animals were not treated with gelatin
E441, where there was a decrease in all the antioxidants that are manufactured by the
cells of the body through significant decrease (P <0.05) in the levels of SOD, GST, GPx
in the blood of animals treated with gelatin E441 when compared to the normal
proportions of the control group, which confirms the presence of an unstable oxidation
caused by spread of free radicals as a result of the treated with this substance(Table 3).
Such anti-oxidants act as protective agents of oxidative stress damage through the free
radicals equation, or reduce the toxicity of the influencing substances or their analysis
into simple non-oxidizing substances(38).The measurement of the antioxidant enzymes
(SOD, GST, GPx) is not only an important indicator of oxidative stress, but a good sign
of the non-regulation of antioxidants(39), and the decrease in SOD, GST, GPx and T-
AOC may be due to the oxidative stress of this metabolism, where there are many free
radicals that cause many health problems and lead to imbalance between oxidants and
their antioxidants (35), Because the free radicals resulting from eating industrial nutrients
lead to the consumption of large amounts of antioxidants, which play a role in protecting
cells from the damage of free radicals (40).
While the results were showing a significant increase (P <0.05) in the amount of
substances produced by the process of oxidation, which confirms the oxidative stress in
animal cells after treatment with gelatin E441, where the proportion of malondialdehyde
(MDA) significantly increase (P <0.05) during the trial period (Table 3) ,which is a
chemical evidence of lipid oxidation and is a major indicator of oxidative stress, which
consists of two types, ROS and RNS, causing the depletion of the cell membrane(41).
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There was also a significant increase (P <0.05) in the level of nitric oxide NO (Table
3)due to the expected amount of free radicals resulting from the metabolism of the
material, which led to this increase when compared with the group of control animals
were not treated with gelatin E441, or perhaps due to the increase in the oxidative state
resulting from the reduced amount of antioxidants and increase the amount of radicals
that generated from the metabolism of gelatin E441, which appeared clear in this
experiment, As may be the increase resulted from the presence of inflammation that
stimulate the cells to produce Cytoscines that stimulate Immune cells to release
superoxide O2 to interact with NO active oxygen component (42).
4. Histological study
4.1. Heart
The transverse tissue sections of untreated animals demonstrated that the
myocardial tissue consists of circular structures that contain clear nuclei called cardio
muscular fibers, Confine between them blood vessel. The longitudinal sections showed
that the heart muscle consists of parallel longitudinal fibers and branching at points
called Intercalated discs, Which are not clear when staining with H & E, and their
nucleus is almost oval in the center of the cell (Images1).While the treatment of gelatin
E441 had an effect on the tissue of the heart in exposed animals, it was found in the
cross sections of the expansion and decomposition of the internal lining of the blood
vessel and the relapse of the cells around it whose nuclei appeared scattered, while the
changes in longitudinal sections are damage the structure of the branching muscle fibers
and the breakdown of some of the walls, As well as the emergence of some cellular
death(Images 2).The changes in the tissue of the heart can be attributed to the role of
free radicals generated by gelatin E441 metabolism, where act the free oxygen radicals
(ROS) on damage the tissue of the heart muscle through the oxidation of fat, proteins
and DNA, As well as reducing the heart muscle (shrinking) by influencing the property
of ion transport and the role of calcium in the performance of function(35),As a result of
decrease of calcium (which may be due to gelatin)occurs changes in the tissue of the
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heart muscle and the emergence of large numbers of Mast cells known with secretion of
histamine and some substances that cause inflammation, which may affect the
metabolism of calcium and phosphorus and change their concentration in the blood,
Resulting in an increase in myocardial dysfunction (43).
4.2.Spleen
The histological spleen sections of non-treated rats have been shown to consist of
two regions, The first consists of lymphocytes, which are organized around the central
artery called white pulp region, which surrounds the PALS sheath that contain lymph
nodules. The second is with light color composed of chords, Splenic Sinusoids called
Red Pulp (Image3).As for the sections of the spleen tissue treated with gelatin E441,
they differed from the normal state. A change was found in the general structure of the
spleen and a large spread of the red pulp area compared to shorthand of the white pulp
area, disappearance of the Germinal center and a clear receding of the PALS sheath area.
As well as the expansion of splenic sinusoids and the appearance of some necrotic cells
(Image4).May be attributed the reason for this is that the spleen tissue is affected by the
high concentration of gelatin, which may impede the work of the mitochondria, causing
the collapse of the Crista structure affecting the production of energy and thus deficincy
of some enzymes necessary for the production of energy sources(44) which is consistent
with the results of the current study, Where there was a decrease in the level of
antioxidant enzymes and an increase in oxidative state. In addition, some food additives
act to increase apoptosis in lymphocytes and break down organelles within them,
leading to necrosis of cells due to high concentrations(45)(46).
4.3. Kidney
The kidney is consist of two primary layers as shown in the kidneys of the control
group animals, the cortex containsa glomeruli, distal convoluted tubules and proximal
convoluted tubules. The other pulp contains the convoluted tubules only (Image 5).But
the sections of animals treated with gelatin E441 were found to be different due to their
relapses and weakness in their functions, were the renal glomeruli decomposed and
9
disintegrated within Bowman's capsule, which appeared crash in its internalized lining.
It also analyzed the lining of distal convoluted tubules and proximal convoluted tubules,
as well as the emergence of hemorrhages in confined areas, which led to damage to the
structure of the kidney(Image 6).These changes in the kidney tissue may be attributed to
be the first members which exposed to toxins in the blood and most affected by, or may
result from the metabolism of toxic food and thus lead to the generation of large
amounts of ROS and RNS, which also play an important role in the occurrence of many
setbacks and damage Of the kidney and damage to some of its tissues (37).The
contraction of the glomerular structure may be due to the effect of the permeability of
the epithelial cells that forming the capillaries, causing an increase in the perfusion of
plasma elements, which leads to shrinkage of the glomerulus, as indicated by the
researcher (47).
4.4.Liver
In the histological sections of the non-gelatin-treated group, the histological structure
of the liver is composed of hexagonal-shaped structures in which the hepatic cells form
radially around a central vein and separated by hepatic Sinusoids. The lobes are
surrounded by barriers contain the portal vein(Image7).The microscobical sections of
group that treated with a concentration of 375 mg / kg of E441 gelatin have suffered
from significant changes as the irregularity of the Radial regulation of the hepatic cells
and apparent expansion of the central vein, which has appeared crash in the internal
lining, as well as inflammation and necrosis of the cells and the appearance of many Red
blood corpuscles in the vein and sinusoids regions (Image 8).The causes of these
pathological changes in the liver tissue of experimental animals may be due to the
formation of many free radicals that generate oxidative stress, Subsequently the death
and decomposition of hepatocytes and the growth of inflammatory cells. These roots act
on the oxidation of lipid membranes of the liver cells and thus their death and the
occurrence of necrosis and enlargement of bloody sinusoids Subsequently(48).As the loss
of radial regulation of hepatic cords may be the result of treatment with highly
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concentrated substances that attack cytoskeleton, causing the destruction of adjacent cell
Milestones, also the presence of necrosis and congestion in the tissue may be due to the
resulting changes in vascular permeability (49).
4.5.Stomach
In the transverse sections of the stomach tissue of the control group, its surface is
covered with a simple columnar epithelium, punctuated by a pits called Gastric pits,
Inside of the pits open What's called the gastric glands, which end in dark cells, are the
chief cells (Zymogenic cells) that are based on the lamina propria layer(Image 9).While
the histological sections of the animals treated with gelatin E441 showed a breakdown
of the layers constituent in the gastric tissue, where the cells of the columnar surface
tissue decomposed, Also the decomposition of the tissue led to spaces between the
gastric glandsand gradual atrophy of the chief cells region, as well as the cellular
necrosis that appeared in the composition of the gastric tissue that led to degenerative in
lamina propria layer (Image 10),Gastric tissue is the most affected tissue with food
substance because it is the first site of digestion and is most affected by toxic industrial
substances, Resulting in damage to the stomach cells and therefore death and damage to
stomach lining and bleeding in the tissue (50), which resulted from treated with high
concentration of gelatin in the current study.
4.6.Testis
Observed in the microscopic histological structure of the testis in the group of
untreated animals, It is surrounded by a thick connective tissue called Tunica albuginea,
It is lined with a connective tissue called Tunica vasculosa, which extends inside the
testicle to contain blood vessels and separate seminiferous tubules from each other, In
these tubules, sperm cells are generated and transformed into a sperm (Image11).While
treatment with Gelatin E441 appear damage in the testicular tissue sections of these
animals, Where deformation was found in Tunica albuginea and Tunica vasculosa and
high expansion between the tubules during decomposition and disappearance of some
connective tissue that surrounds them, as well as disappearance of some cells in the
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tubules (Image 12).The testicular tissue damage to animals given gelatin E441 may be
due to the free radicals generated by metabolism of this substance, which act on
oxidation of fat and proteins. These tissue can be well linked to the protein and lipid of
cells membrane affecting the cells of the body, including testicular cells(51).
(Table 1)The effect of Gelatin E441 on liver function tests
Test Control (Mean ± SE ) Treated (Mean ± SE) P-value
ALT (U/L)
41 ±1
6
4.6 ±4.5
S
AST (U/L)
5
9
.66 ± 2
S
M: the arithmetic mean, SE: standard errer, the number of animals per group (6), (S):significant difference (P
<0.05) when compared to the control group.
(Table 2)The effect of Gelatin E441 on kidney function tests
Test Control (Mean ± SE )
Treated (Mean ± SE) P-value
Urea (mg/dl)
17.3 ±
0.57
2
9
.8± 0.8
S
Creatinine
(mg/dl)
1.
0
6 ±0.15
S
M: the arithmetic mean, SE: standard errer, the number of animals per group (6), (S):significant difference (P
<0.05) when compared to the control group.
(Table 3)The effect of Gelatin E441 on Oxidative Stress tests
Test Control (Mean ± SE )
Treated (Mean ± SE) P-value
SOD (U/L)
1.53 ± 0.066
S
GST(U/L)
13.3± 0.23
6.4± 0.31
S
GPX(U/L)
72.86 ± 0.57
S
MDA (µmol/L)
4.6 ± 0.45
S
NO(µmol/L)
5.2 ± 0.24
14.05 ±0.4
S
T
-
AOC (U/ml)
2.1 ±0.11
0.99 ± 0..03
S
M: the arithmetic mean, SE: standard error, the number of animals per group (6), (S):significant difference (P
<0.05) when compared to the control group.
12
13
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