Curcumin Reduces Pyramid Cell Necrosis in Mice

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International Journal of Civil Engineering and Technology (IJCIET)
Volume 10, Issue 05, May 2019, pp. 430-437, Article ID: IJCIET_10_05_046
Available online at http://www.iaeme.com/ijciet/issues.asp?JType=IJCIET&VType=10&IType=05
ISSN Print: 0976-6308 and ISSN Online: 0976-6316
© IAEME Publication
POTENTIAL OF CURCUMIN IN PYRAMID CELL
NECROSIS OF MICE (MUS MUSCULUS) DUE TO
METHYLMERCURY EXPOSURE
Gibran Dewanda
Department of Anatomy and Histology, Faculty of Veterinary, Universitas Airlangga
Widjiati
Department of Anatomy and Histology, Faculty of Veterinary, Universitas Airlangga
Sri Pantja Madyawati
Department of Veterinary Reproduction, Faculty of Veterinary, Universitas Airlangga
ABSTRACT
Mercury pollution in the aquatic environment can cause intoxication of marine
organisms and the formation of free radicals in the human body if consumed. Curcumin
is a natural ingredient that contains extracellular antioxidants to overcome the
formation of free radicals in the body. The purpose of the study was to determine the
effect of administering curcumin to the number of pyramid cells that were necrotic in
mice (Mus musculus) exposed to methylmercury. This experiment used a completely
randomized design with 4 groups of mice, i.e. 0.5 ml distilled water, 0.056 mg/kg
methylmercury, 0.0056 mg/kg methylmercury + 150 mg/kg curcumin and 0.0056 mg/kg
methylmercury + 300 mg/kg curcumin. Examination of total pyramid cell necrosis was
observed with a 400x magnification light microscope. In this study, there was a
decrease in the number of pyramid cell necrosis in mice. The decrease in the number
of necrotic cells was directly proportional to the increase in dose given. The treatment
results as positive controls showed the highest value with the number of pyramid cells
experiencing necrosis of 23.28. The highest decreases in the number of each treatment
were 10.32 in 150 mg/kgBW curcumin and 5.80 in 300 mg/kgBW curcumin. Curcumin
can reduce the number of pyramid cells that experience necrosis due to exposure to
methylmercury.
Key words: Methylmercury, Curcumin, Pyramid cell, Necrosis
Cite this Article: Gibran Dewanda, Widjiati and Sri Pantja Madyawati, Potential of
Curcumin in Pyramid Cell Necrosis of Mice (Mus Musculus) Due to Methylmercury
Exposure. International Journal of Civil Engineering and Technology, 10(05), 2019,
pp. 430-437
http://www.iaeme.com/IJCIET/issues.asp?JType=IJCIET&VType=10&IType=05

Gibran Dewanda, Widjiati and Sri Pantja Madyawati
1. INTRODUCTION
Developing countries are very related to industrial growth. Industrial growth has a positive
impact on improving people’s living standards. However, the negative impact that results in
environmental pollution which disrupts the environment ecosystem. One form of pollution is
water pollution due to organic substances, pathogenic substances, dangerous heavy metals, and
pesticides. One of the heavy metals that cause pollution is mercury. Mercury pollution in the
aquatic environment, both in the sea and rivers, will cause intoxication of marine organisms.
Mercury poisoning is usually caused by the habit of eating food from the sea originating from
waters contaminated by mercury (Palar, 2008). The whole body can easily absorb food
contaminated by methylmercury after being absorbed by methylmercury carried by the blood
to all body tissues (Paterson M and A.P. Talcott, 2006).
Mercury in contaminated fish or marine organisms, if consumed, will be digested and
absorbed by the digestive tract. The mercury absorbed will enter the bloodstream and be
distributed to certain organs, such as the liver, kidneys, and brain. Mercury is neurotoxic which
shows changes in cells in the cerebral cortex. The accumulation of mercury in the cerebrum
cortex will cause damage to pyramid cells (Despopoulus, 2000) exposure to large amounts of
mercury which occurs continuously. There will be the formation of free radicals in the body
which will cause a decrease in the body’s antioxidant activity and can cause disease. To
overcome this problem, extracellular antioxidants are usually needed in foods, such as vitamin
C, vitamin E, and curcumin (Bhagya, 2013).
Curcumin is a yellow pigment found in the plant rhizome of the genus curcuma (wahyuni
et al., 2018). Curcumin is expressed as a phytochemical that is safe to use, not toxic, or
teratogenic even when used at high doses. Curcumin can be used as an antioxidant compound.
The body needs antioxidants that can reduce free radical (purba and martosupono, 2009). In
addition, curcumin inhibits brain damage due to the activity of chemical mediators and at the
time of the occurrence of foreign objects into the brain (jayaprakasha, 2009). In vitro and in
vivo trials prove that curcumin is efficacious as an anti-amyloidogenic, antioxidant, and anti-
inflammatory drug that can prevent alzheimer’s disease (ringman et al., 2005). The purpose of
this study was to determine the effect of administering curcumin to mice (mus musculus)
exposed to methylmercury to decrease the number of pyramid cells that experienced necrosis.
2. MATERIALS & EXPERIMENTAL PROCEDURES
2.1. Materials and Equipment
This research was conducted in an experimental animal cage, Faculty of Veterinary,
Universitas Airlangga. The performance of histopathological preparations of mice brains was
carried out at the Veterinary Pathology Laboratory of the Faculty of Veterinary, Universitas
Airlangga.
The research material used in this study were as follows: (i) The experimental animals used
in this study were 20 male mice that were determined using the federe formula. Mice receiving
treatment were controlled by the criteria of 16 weeks of age with a body weight of 30 grams
divided into four groups. The mice were obtained from the Veterinaria Farma Center in
Surabaya. (ii) The equipment used in this study was a scale to measure the body weight of
mice, microscopes to examine histopathological preparations of cerebrum mice, mice cages,
sonde, syringes, surgical scissors, scalpels, tubes, and Petri disks. (iii) The research material
used was methylmercury in the form of Methylmercury (II) chloride with the chemical formula
(CH3ClHg) made by Sigma-Aldrich, distilled water, formalin, tube, and curcumin (Merck).

Potential of Curcumin in Pyramid Cell Necrosis of Mice (Mus Musculus) Due to Methylmercury
Exposure
The administration of curcumin was carried out in multilevel dosages of 150 mg/KgBW and
300 mg/KgBW.
2.2. Methods
The research design used was a laboratory experimental method. The study design used was a
completely randomized design (crd) with a factor of the number of pyramid cells that were
damaged with an assumption:
K- : Group of controlled mice given only with distilled water
K+ : Group of mice with 0.1 methylmercury at a dose of 0.0056 mg/kgbw for 30 days
P1 : Group of mice with 0.0056 mg/kgbw methylmercury per day and 150 mg/kgbw
curcumin for 30 days
P2 : Group of mice with 0.0056 mg/kgbw methylmercury and 300 mg/kgbw curcumin for
30 days
Histopathological assessment was carried out microscopically on histological changes that
occurred in the cerebral cortex of the brain of mice and focused on the number of pyramid cells
that experience cell necrosis (picnosis, karyorrhexis, and karyolysis). Observations were made
in five different fields of view. Observations were carried out in each of the five fields of view,
starting from the right, bottom, right, up and right side of the preparation then viewed under a
microscope with 400x magnification. The signs of cell damage observed are: (Price, S. A. And
Wilson, 2006)
I. Cells that undergo karyopyknotic will give a profile of the small nucleus, cells are
more basophilic, darker and obscure boundaries.
II. Cells that undergo karyorrhexis will give a profile of fragmented cell nucleus by
leaving the remnants of chromatin in the cell.
III. Cells that undergo karyolysis will give a profile of the cell nucleus that disappears.
Data collection in this study was carried out by: (i) Mice were adapted to the environment
around 7 days. During the adaptation process, mice were fed and given with water ad libitium.
(ii) Preparation of the treatment using 0.04 mg/KgBW dose (Johansson et al., 2007) (9) and it
was converted to the dose of mice 0.04 x 0.14 = 0.0056mg/KgBW. Toxic doses given were
based on fractions that did not kill mice but had the potential to cause toxic effects (Hamid et
al., 2014). The doses of curcumin were 150mg/kgBW and 300mg/kgBW (Aggarwal, 2007).
(iii) Experimental animals were divided randomly into four treatment groups with each
treatment consisting of 5 replications. (iv) Sampling was carried out on the 31st day. The brain
organs were taken and then histopathological preparations with Hematoxylin-Eosin Staining
was made. (v) Histopathological preparation was performed. (vi) Examination of brain
histopathology preparations was done using a microscope with 400x magnification on five
different fields of view.
Data in this study were analyzed using the Analysis of Variance (ANOVA) test with p
<0.05 if significant differences were found to be followed by Duncan’s multiple distance test.
3. RESULTS AND DISCUSSION
3.1. Result
The results showed that there were significant comparisons of the number of cerebrum pyramid
cells of mice (Mus musculus) which were damaged after being given with curcumin due to the

exposure of methylmercury to which curcumin was administered and no curcumin was
administered.
Table 1. Analysis of the number of cerebrum pyramid cells of mice (Mus musculus) which have
necrosis due to exposure to methylmercury and given with curcumin.
Treatments
The mean number of pyramid cells necrosis ±
SD
K-
K+
P1
P2
2.56 a
± 0.83
23.28c
± 8.64
10.32b
± 4.93
5.80ab
± 2.97
Note: Different superscripts in the same column show significant differences (p<0.05)
K-: Negative control, only given with distilled water
K +: Positive control, exposed to methylmercury at a dose of 0.0056 mg/KgBW orally
P1: First treatment, exposed to methylmercury at a dose of 0.0056 mg/KgBW orally and
given with curcumin at a dose of 150 mg/KgBW orally
P2: Second treatment, exposed to methylmercury at a dose of 0.0056 mg/KgBW orally and
given with curcumin at a dose of 300mg/KgBW orally.
The results of the statistical analysis of variance (ANOVA) test showed a significant
difference of 0.000 (p<0.05). Furthermore, to find out the differences between treatments, a
further test was conducted with Duncan’s test with the results showing negative control
treatment which was only given aquadest (K-) significantly different (p<0.05) with P1 and K+.
Positive treatment which was only exposed to methylmercury (K+) was significantly different
from K-, P1, and P2 which were given with methylmercury and curcumin. The profile of
cerebrum pyramid cells in mice can be seen in Figure 1.
Figure 1. Pyramid cell cerebrum histopathology (Hematoxylin-Eosin staining; 1000 times
magnification. Blue arrow shows normal pyramid cells and red arrows show pyramid cells that
experience necrosis). Normal pyramid cells of the cell nucleus are clear, whereas necrotic pyramid
cells have a darker cell nucleus.

Exposure
This study also found a comparison between the number of decreases in pyramid cells that
experienced necrosis between the control group and the treatment group. The results of the
examination of pyramid cell histopathology cerebrum mice are shown in Figure 2 below:
Figure 2. Analysis of the number of mean cerebral pyramid cell necrosis of mice exposed to
methylmercury and given with curcumin.
The results of K+ treatment as a positive control showed the highest value with a large
number of pyramid cells experiencing necrosis. Treatments P1 and P2 showed a reduction in
the number of pyramid cells that experienced necrosis with the administration of curcumin.
The decrease in the number of cells experiencing necrosis was directly proportional to the
increase in the dose given. The decrease in each treatment was P1 of 150mg/kgBW curcumin
at 10.32 and P2 of 300mg/kgBW curcumin at 5.80.
3.2. Discussion
Methylmercury (Hg2+
) contained in the body induces apoptosis in neuron cells. In addition,
methylmercury is neurotoxic which shows changes in cells in the cerebral cortex.
Accumulation of mercury in the cerebral cortex will cause damage to pyramid cells
(Despopoulus, 2000).
The results of counting the number of necrotizing pyramid cells showed significant results
(p<0.05) among all treatments. In the positive control group which was only given with
methylmercury (K+) as much as 23.28 ± 8.642 showed an increase in the number of necrosis
cells when compared to negative control which was only given with distilled water (K-) as
much as 2.56 ± 0.832. This shows that methylmercury can increase pyramid cell necrosis due
to exposure to methylmercury. Longer exposure to methylmercury will reduce the number of
normal cells. This can be seen in the treatment of three weeks and six weeks, the number of
living cells decreases on exposure to mercury for six weeks. Cell necrosis was very large. In
this study, it was seen that most of the pyramid cells in the brain experienced necrosis due to
exposure to methylmercury (Bhagya, 2013).
Methylmercury exposure can produce very varied effects. All forms of the effects of
methylmercury depend on the magnitude and duration of exposure and also the age and health
status of the individual. Exposure to methylmercury at large levels can produce effects on the
nervous, respiratory, kidney, immune system, pregnancy, dermatology, and various other

effects. The nervous system is the main target of metal toxicity, especially organic metal
compounds. Methylmercury, which has fat soluble properties, easily passes through the blood-
brain barrier and enters the nervous system (Fouad et al., 2009).
As one of the strongest thiol binding agents, Hg2+
specifically binds to thiols in cells. The
direct chemical interactions between Hg2+
and thiol groups of proteins or nonprotein molecules
may play an important role in Hg-induced neurotoxicity, resulting in disorders of endogenous
antioxidants and non-enzymatic antioxidants. NPSH acts as a nucleophilic binder of many of
its compounds and metabolites. Binding of NPSH metabolites through enzymatic and chemical
mechanisms and processes plays an important role in protecting against oxidative stress caused
by reactive oxygen species (ROS). About 90% NPSH is glutathione (GSH), which is the most
intracellular low molecular weight sulfhydryl compound (Ayer et al., 2010). GSH can join
Hg2+
directly and form a series that prevents Hg2+
from binding to cellular proteins. In addition,
GSH can react with free radicals, eliminating excessive ROS produced by oxidants.
Free radicals contained in mercury can cause lipid peroxidation, protein oxidation, ROS,
and ultimately cause brain neuron cell death. This is not surprising because the brain is more
susceptible to oxidative damage than other organs or tissues because of the high levels of
oxygen consumption, high polyunsaturated fat content, and the relative lack of antioxidant
enzymes (Ayer et al., 2010). In the normal state, the antioxidant defense system can easily
overcome the free radicals formed. The time of increased use of oxygen free radical production
is believed to be very instrumental in causing cardiovascular disease, cancer, Alzheimer’s
disease and Parkinson’s (Musalmah, 2009).
Histopathological description of the group given with Curcumin (P1 and P2) showed a
difference with the positive control group which was only exposed to methylmercury. The
number of pyramid cell necrosis was significantly different compared to the group exposed to
only 0.0056 mg/kgBW (K+). The P1 group, which was given a dose of 150 mg/kg of curcumin,
showed a decrease in the amount of necrosis compared to the K+ group which was only
exposed to methylmercury. P2 group, which administered by the dose of 300 mg/kgBW
curcumin, showed a decrease in the number of pyramid cells. The positive control group (K+)
exposed only to methylmercury showed results of 23.28 ± 8.642, while the treatment given
with curcumin (P1) was 10.32 ± 4.937 and (P2) was 5.80 ± 2.9777. This indicates that curcumin
can reduce the number of pyramid cell necrosis.
Curcumin has antioxidant activity. Antioxidants based on how they work function as an
antidote to the effects of free radicals. Besides, antioxidants can also prevent the occurrence of
free radical ion bonds with cells, repair cells and tissues that have been damaged so that no
damage to cells (Capelli and Cysewski, 2010). Curcumin has the function of repairing damaged
cells and tissues caused by free radicals. Curcumin has the properties of Scavenger and
Chelators. The nature of Scavenger is to function to bind oxygen so as to prevent the occurrence
of oxidation reactions. Whereas, Chelators properties function to bind metals and are able to
catalyze the occurrence of oxidation reactions.
Curcumin has gone through clinical trials of its effectiveness as a therapy in phases I, II
and III, so that even at high doses (12g/day for three months) and it is still safe to use. The
study shows that this is related to the ability of curcumin as an antioxidant, anti-inflammatory,
anti-cholestatic, anti-fibrogenesis, and anti-carcinogenic (Oktaviana, 2010). The human body
naturally has an antioxidant system defense. Antioxidants in curcumin work by protecting
lipids from the process of peroxidation by free radicals. When free radicals get electrons from
antioxidants, the free radicals no longer need to attack cells and the oxidation chain reaction
will be broken. After giving electrons, antioxidants become free radicals. By definition,

Exposure
antioxidants in this state are dangerous because they have the ability to change electrons
without becoming reactive.
Curcumin is known to protect biomembrane from peroxidative damage. Lipid peroxidation
is known as a chain reaction mediated by free radicals, which causes damage to cell
membranes. The inhibition of peroxidation by curcumin is caused by the rinsing of reactive
free radicals involved in peroxidation. Most antioxidants have phenolic group or diketone
group functions. Curcumin is a unique antioxidant, which contains a variety of functional
groups, including the B-diketo group, double carbon-carbon bonds, and phenyl rings
containing varying amounts of hydroxyl and methoxy substituents (García-Niño and Pedraza-
Chaverrí, 2014).
Curcumin as an antioxidant performs extraordinary H-atom donors from the central
methylene group rather than from a phenolic group dissolved in acid and neutral acetonitrile
(S, 2002). Besides that, curcumin is a classic phenolic chain-breaking antioxidant, donating H-
atoms from phenolic groups (Jovanovic et al., 1999). This phenolic group is very important for
free radical scavenging activities by increasing the activity of the methoxy group (Barclay et
al., 2000).
The most common mechanism by which free radicals can fight antioxidant defenses is by
attacking the biochemical components in the body and forming hydroperoxide. In this
pathophysiological form, cells will start producing free radicals in large quantities, due to
exogenous stress (chemical, physical and biological elements) and or nine metabolic activities
(especially in the plasma membrane, mitochondria, endoplasmic reticulum, and cytosol).
Cytosols, including dangerous hydroxyl (HOH) radicals, are one of the most dangerous
reactive oxygen species (ROS). Hydroxyl radicals can attack every type of molecule (including
carbohydrates, fats, amino acids, peptides, proteins, nucleotides, nucleic acids, and others). As
a result of this process, each molecule will lose one electron and then become radical. After
that, a radical chain reaction will occur due to the presence of oxygen molecules (through
breathing), and the formation of hydroperoxide (ROOH), a type of Reactive Oxygen
Metabolites (ROMs). Although hydroperoxide is a relatively stable type of chemical, it has the
potential to form free radicals again and can oxidize other target molecules. Furthermore, the
cell will pull out hydroperoxide in the extracellular environment, including blood,
cerebrospinal fluid, pleural fluid and others (Priyadarsini et al., 2003).
4. CONCLUSION
Curcumin can reduce the number of pyramid cells that are damaged due to exposure to
methylmercury. The decrease in the number of necrotic pyramid cells is directly proportional
to the increase in the dose given. The highest decrease in the number of pyramid cell necrosis
is in the provision of curcumin at a dose of 300 mg/KgBW.
ACKNOWLEDGEMENTS
The authors would like to thank every lecture in Faculty of Veterinary, Universitas Airlangga,
Surabaya, Indonesia for the support to finish this paper
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