Crosstalk between Necrosis and Apoptosis of Embryonal Cerebral Cortex Neuron Mice (Mus musculus) Caused by Carbofuran Exposure
Epy Muhammad Luqman1,2*, Widjiati2, Maslichah Mafruchati2,
Bambang Poernomo Sunardi Rahardjo2, Eka Pramyrtha Hestianah2
1Post Graduate, School of Universitas Airlangga Surabaya, Indonesia
2Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Universitas Airlangga Surabaya, Indonesia
*Corresponding Author E-mail: epy-m-l@fkh.unair.ac.id
ABSTRACT:
The study aimed to explain the crosstalk of cerebral cortex neuron cell death due to the carbofuran exposure during the embryonal development. This experimental laboratory study used 81 fetuses from 27 mice mothers and carbofuran were exposed in gavage manner at the pregnancy age of 6-15 days old at a dose of 0.0417mg/Kg body weight and 0.0208mg/Kg body weight. On the 17th day of pregnancy, the mice mothers were terminated and its fetal cerebrums were measured for its ROS activity such as the malondialdehyde/MDA levels and superoxide dismutase/SOD activity and examined its cerebral cortex neuron cells which included the apoptosis (tunnel assay) and necrosis (HE staining). The examination of p53 and caspase 3 used the immunohistochemistry. This study concluded that there was moderate in these relationships between carbofuran doses and MDA and p53 levels; and between SOD activity and MDA and P53 levels; and between p53 and caspase 3, apoptotic and necrotic cells. Meanwhile, both relationships between carbofuran doses and SOD activity and between caspase 3 and apoptotic cells were strong. Furthermore, there was no any relationship between MDA levels and necrotic cells. The expression of p53, caspase 3 and apoptotic cells showed that carbofuran could increase the embryonal cerebral ROS activity and injure the core DNA which caused the apoptosis via intrinsic pathways through p53 or mitochondria. MDA levels did not increase any damage to the cell membrane system and cause necrosis. There was a relationship between necrotic cells and p53 due to a decrease in mitochondrial function for producing energy. Apoptotic and necrotic neuron cells occurred because p53 opened the opportunities for prevention and treatment of the ROS activities’ effects due to the carbofuran exposure during pregnancy.
KEYWORDS: Crosstalk, carbofuran, neuron, embryonal, mus musculus.
INTRODUCTION:
This increase in ROS can reduce the activity of superoxide dismutase (SOD) in the brain. The presence of ROS can trigger hydroxyl radicals (OH•) which can break the DNA chain and cause an apoptosis3. Furthermore, the presence of OH can also damage all cell membrane systems by increasing lipid peroxidation which will generate an injury and a necrotic cell death1,4.
DNA damage due to OH• will activate p53 and triggers Bax activity. Bax will suppress BCl-2 activity causing some changes in the permeability of mitochondrial membrane and lead to the release of cytochrome C to cytosol. Then, Cytochrome C will activate Apaf-1 and caspase cascade starting from pro-caspase 9 to caspase 3. Later, Caspase 3 will activate DNA-se and penetrate the core membrane and damage DNA, thus, the cell undergoes apoptosis5.
The mechanism and morphology of apoptosis and necrosis are different, but sometimes there is still an overlap between the two processes6. Using the conventional histology, it is not easy to distinguish apoptosis from necrosis, because both processes can occur simultaneously depending on the intensity and the duration of injury, the ATP depletion rates and the availability of caspase7,8. The type or degree of injury determines a cell death whether it is by apoptosis or necrosis. Various injuries such as heat, hypoxia, radiation, and low-dose anticancer drugs can induce an apoptosis, however, the same injuries with higher doses can lead to a necrosis6.
Carbofuran can pass trans-mammary and increase ROS activity by increasing the MDA levels and necrosis of Purkinje cells during lactation9. The two main metabolites of carbofuran which cause toxicity and can penetrate the placental barrier are 3-hydroxycarbofuran and 3-ketocarbofuran which catalyze a serious damage to maternal units of fetal placenta10.
Understanding the relationship of cell death processes - both through a necrosis and an apoptosis in fetal cerebral neuron cells due to the exposure of carbofuran during the pregnancy is needed to obtain a molecular basis of treatment and prevention of the carbofuran exposure during pregnancy. If the connection between the causes of cell death is mainly apoptosis, thus, there will be a chance to open some opportunities in prevention efforts by applicating the variants of antioxidant along with some treatment efforts to improve the effects of ROS by giving the antidotes such as N-acetylcysteine, vitamin E, atropine, memantine etc.
MATERIAL AND METHODS:
Ethics Approval:
The study was approved by the Faculty of Veterinary Medicine’s Animal Ethics Committee. All variables had beenconsidered in accordance with the Ethics Committee related to the animal handling to ensure no discomfort or pain caused to the animals during sampling (2011/111-KE).
Laboratory Animals:
The animals used in this study were 27 female mice (Mus musculus), 10 weeks old, with the weight range of 25-30 grams, and 12-weeks-old male mice. Environmental adaptation was done to female mice (Mus musculus) for 7 days. On the 8th day, pregnant mare serum gonadotropin (PMSG) with a dosage of 5 IU/mouse was injected into the female mice and followed by Human Chorionic Gonadotrophin (HCG) injections with a dosage of 5 IU/mouse which was performed on the 10th day. Afterwards, the female mice were mated with the 12-week-old male mice. On the 11th day, a gestation examination was carried out. The gestation of female mice was indicated by the visible mating plug covering the female mice vulva; and then the day was considered as the first day of gestation11.
Carbofuran Exposure:
The dosage of carbofuran insecticide using in this research was 0.0417mg/kg BW (1/12 LD50, where LD50 = 0.5mg/kg BW) and 0.0208mg/kg BW (1/24 LD50)11. At the 6th to 15th day of gestation, the mice were exposed by carbofuran in gavage manner using disposable syringe12. At the 17th day of gestation, the mice were sacrificed to collect fetuses’ cerebrum. Cerebrum ROS activity was measured based on its Malondialdehyde (MDA) level, Superoxide Dismutase (SOD) activity, p53 and caspase 3 expressions, cerebral cortex neuronal cells death (apoptosis and necrosis).
Measuring Malondialdehyde (MDA) Level:
Malondialdehyde (MDA) was determined by the method of Conti et al13. The measurement of malondialdehyde level on the six-day-old mice pups’ brains was performed using the MDA/Thiobarbituric Acid Reactive Substance (TBARS). This method was performed by weighing 1gram of the infant mice brain sample then putting it into a reaction tube, mixing it with 9mL cold PBS and then crushing it with a spatula. The liquid was then centrifuged at 3000rpm for 15 min. 4 mL of supernatant was collected and added to 1 mL of trichloroacetic acid (TCA) 15% solution. Then, 1 mL of0. 37% Thiobarbituric acid (TBA) solution was added into HCl 0.25 N and heated in a water bowl at 80ºC for 15 min. After cooling the solution at room temperature for 60 min,the solution was centrifuged at 3000 rpm for 15 min. Finally,the value of the absorbance was read against the red lines which were formed using a spectrophotometer at λ = 532 nm.
Superoxide Dismutase (SOD) Activity Examination:
The SOD activity was measured using SOD assay Kit-WST purchased from Sigma-Aldrich. Cerebral tissue was mashed using PBS or KCl 150mM to remove erythrocyte. The mashed tissue was homogenized with 0.1 M Tris/ HCl (pH 7.4) containing 5mM β-ME 0.5% Triton X-100 and 0.1mg/mL RMSF. The compound was centrifuged at 14000G and 4°C for 5 minutes. The debris of supernatant was removed to obtain cytolytic and mitochondrial SOD. The examination was conducted by putting 20mL sample into sample cuvette and cuvette blank 2 added by 20mL H2O on cuvette blank 1 and cuvette blank 3. 200mL WST Working Solution were added on each cuvette (sample cuvette, cuvette blank 1, cuvette blank 2, and cuvette blank 3). 20mL Dilution Buffer were added to cuvette blank 2 and cuvette blank 3. 20mL Enzyme Working Solution were added to sample cuvette and stirred. The cuvettes were incubated at 37°C for 20 minutes. Absorbance can be read on 450 nm wavelength using microplate reader. SOD activity (inhibition percentage) was measured based on formula:
SOD Activity (Inhibition Percentage)
(Ablank1 - Ablank3) – (A sample - Ablank2)
------------------------------------------------x100%
(Ablank1 - Ablank3)
Examination on p53 and Caspase 3 Expressions Using Immunohistochemistry Technique:
Examination on p53 and caspase 3 was conducted by applying streptavidin and biotin methods of immunohistochemistry technique. Brain tissue of 17-day-old gestated fetuses was fixated using buffer formalin 10% applying paraffin methods with 5mm thickness and being stuck on object glass using polylysine. The area which was being examined was motor cortex area obtained through coronal sectioning. Coronal sectioning was conducted at 11/1.56 mm from frontal lobe 14.
The object glasses were re-hydrated using gradual ethanol series (100%, 90%, 80%, 70%, and 30%) for 5 minutes every process. The object glasses were washed in pH 7.4 Phosphate Buffer Saline (PBS) and re-washed in citric buffer (pH 5.2) at 90° C for 30 minutes. Object glasses were cooled down and blocked with 1% Bovine Serum Albumin (BSA) for an hour and being washed in PBS (pH 7.4) for 5 minutes conducted in three repetitions. Slide was labeled with primary antibodies (p53 monoclonal antibody - Santa Cruz Biotechnology, Catalogue Number pab 240- lot 104p212 and caspase 3 monoclonal antibody Santa Cruz Biotechnology, Catalogue Number ab-1 lot 11144p201 (1:100)) inside BSA 1% overnight at 4° C. Object glasses were washed in PBS (pH 7.4) for 5 minutes in three repetitions. The object glasses were labeled with biotin secondary antibody label (Dako Cytomation-Denmark) in composition 1:500 for an hour at room temperature and being washed using PBS (pH 7.4). The object glasses were supplemented with Strepovidin Horserodish Peroxidase (SA-HRP) 1:500 for 40 minutes at room temperature and being washed with PBS (pH 7.4). Object glasses were applied with diaminobenzidine tetrahydrochloride (DAB) chromogen substrate for 20 minutes and being washed with PBS (pH 7.4) followed by aquadest for 5 minutes in three repetitions continued by counterstaining object glasses.
Apoptosis Examination Using Tunel Assay:
The examination of apoptotic cells was performed by Tunel assay. The paraffin blocks were cut using a microtome with a thickness of 5μm in a series and then glued to the object glass using polylysine. For counting the apoptotic cells, pieces of the tissue were processed with S7101 Apoptag Plus Peroxidase. Apoptotic cells were identified by the color absorbent (dark brown).
Data Analysis:
The relationship between variables using regression analysis, the value of r = 0.7 - less than 1 means that the level of relationship is high, the value of r = 0.4 - less than 0.7 means the level of the relationship is moderate, the value of r = 0.2 - less than 0.4 means a low level of relationship and a value of r = less than 0.2 means that the level of the relationship is very low/neglected15. The statistical analysis was performed using Statistical Product and Service Solutions (SPSS) version 17.0.
Results:
Fig 1 Relationship between SOD activity and the level of MDA with R value of 0,406.
Fig 2 Relationship between SOD activity and p53 expression with the r value of 0,535
The r value of 0.406 between SOD activity and MDA levels had a moderate relationship. It showed that the lower SOD activity occurred, the higher MDA level was displayed (see Figure 1). Meanwhile, the r value of 0.535 between SOD activity and p53 expression exhibited a moderate relationship. It meant that the lower SOD activity happened, the higher expression of p53 was shown. Furthermore, the r value of 0.158 between MDA levels and necrotic cells described no relationship which said that the higher MDA level was indicated, there was no any relationship with necrotic cells (see Figures 2 and 3).
Meanwhile, the r value of 0.578 between the p53 expression and caspase 3 had a moderate relationship, it showed that the higher dose of carbofuran was exposed, the higher p53 was expressed. Additionally, the r value of 0.748 between the caspase 3 expression and apoptotic cells exhibited a strong relationship which revealed that the higher the caspase 3 is expressed, the higher the apoptotic cells existed (see Figures 4 and 5).
Fig 4. Relationship between the p53 expressions and caspase 3 expressions with the r value of 0,578.
Fig 5. Relationship between the caspase 3 expressions with apoptotic cells with the r value of 0,748.
Fig 6 Relationship between the p53 expressions and apoptotic cells with the r value of 0,569.
Fig 7 Relationship between the p53 expressions and necrotic cells with the r value of 0,547.
Additionally, the R value of 0.569 between p53 expression and apoptotic cells revealed a moderate relationship, which showed that the higher caspase3 was expressed, the higher the apoptotic cells appeared. Lastly, the r value of 0.547 between p53 expression and necrotic cells had also a moderate relationship which presented the higher p53 was expressed, the higher necrotic cells were generated (see Figures 6 and 7).
Discussion:
Figure 8 shows a relationship between carbofuran exposure, SOD activity, MDA levels, p53 expression, caspase 3 expression, apoptosis and necrosis based on the regression analysis between variables.
Carbofuran exposure during an embryonal period could increase superoxide anion (O2•) marked by a decrease in SOD activity. This decrease was catalyzed by a continuous increase in O2• level due to the carbofuran exposure, as a result, it increased the use of SOD and ultimately decreases the SOD activity3. The strong relationship between carbofuran doses and SOD activity showed that the higher carbofuran dose was exposed, the lesser SOD activity occurred. The decreasing SOD activity would also increase MDA levels and p53 expression. SOD antioxidants had a function in neutralizing O2• into hydrogen peroxide (H2O2) and oxygen (O2). Then, H2O2 would react with O2• and transitional metal ions (Fe and Cu) through Haber Weiss and Fenton’s reaction to form OH• 5.
Fig 8. Relationship between carbofuran exposure, SOD activity, MDA, p53 expression, caspase 3 expression, apoptosis and necrosis levels
Note: There is a relationship is presented by this symbol ( ) and no relationship is presented by this symbol ( )
Later, the presence of OH caused a peroxidation reaction with a membrane system which is rich with polyunsaturated fatty acids and produces compounds such as MDA16. There was a moderate relationship between carbofuran doses and MDA levels, the higher carbofuran dose was exposed, the higher MDA level was generated. The moderate relationship between carbofuran exposure dose and MDA levels expressed that hydroxyl radical products (OH•) generated by carbofuran exposure would react with the cell membrane systems which were rich with PUFA and delivered the lipid peroxidation products including MDA2. Besides that, OH• also reacted with DNA and caused a DNA injury17,18.
The increased MDA levels indicated a damage in the membrane and caused the necrotic cells. Free radical attacked the membrane and caused the cell membranes lost its integrity and viability which led to the necrotic cells16. However, in this study, there was not any increasing relationship found between MDA levels with necrotic cells. Necrotic cells were expected as the result of DNA damage by OH• and the DNA damage would stimulate the protein p53 expression and then would activate PARP and create NAD and ATP depletion which then trigger the necrotic cells.
The molecular mechanism of necrosis has not been well defined, but it can occur as a result of apoptosis incomplete execution19. Apoptotic cell death requires an ATP energy, while necrotic cell death does not need any ATP energy. Therefore, one of the main factors that determines whether the cells follow an apoptotic or necrosis pathway is the intracellular ATP concentration 20. Furthermore, apoptosome formation requires ATP for proapoptotic activity. The absence of sufficient energy reserves can lead to a condition where a cell death shifts from apoptotic to necrotic forms. A progressive energy depletion drives a greater proportion of necrotic cell death than apoptotic cell death7. Poly (ADP-ribose) polymerase (PARP) is considered as one of the switch points that determines whether cells will experience an apoptosis, which is a condition when PARP is split and inactive and does not drain ATP bags; or undergo a necrosis, which is a condition when PARP is not split and remains active. The lack of PARP cleavage through caspase resistance or PARP activation can eventually lead to a necrosis by ADP-ribose depletion which leads to a decrease in ADP bag to form ATP20.
This increasingp53 expression also causes necrotic cells. An identification of p53’s new role in the activating necrotic cell death is found in the oxidative stress and ischemia. The p53 protein is accumulated in the mitochondrial matrix and triggers the opening of PTP, mPT and necrosis by physical interaction with the cyclophilin D critical regulator and PTP. The p53 protein is able to quickly open the PTP pores on the inner membrane of mitochondrial and p53 is also able to cause some structural changes in the PTP pores. The old paradigm stated that p53 only controls apoptosis and plays no role in necrosis. However, the new and first concepts stated that p53 does play a role in the necrosis 21.
A DNA damage by OH• will also increase p53 as an attempt to repair the damaged DNA or the cell will decide to perform programmed death by activating the proapoptotic proteins in the mitochondrial membrane. This increase in proapoptotic protein leads to a porous formation of mitochondrial membranes, thus, the cytochrome C is released from mitochondria into the cytoplasm and binds Apaf-1 to initiate the apoptosome formation, which then binds pro-caspase-9. Oligomerization of caspase-9 in apoptosomes activates proteases. The active Caspase-9 splits two executioner caspases, they are caspase-3 and caspase-722. In this study, there was a moderate correlation between p53 expression and caspase 3 expression which explained that an injured DNA told us that p53 was still trying to improve itself by not activating the proapoptotic and caspase 3 proteins. The classic role of p53 is to increase the resting cell cycle which follows the DNA damage and mediated by the transcription protein stabilization mechanism21. The Increase of p53 expression due to carbofuran induction can stimulate the expression of p21 inhibitors as an effort to repair the DNA damage. In addition, the presence of p53 will affect the mitochondrial membrane’s integrity, decrease the intracellular ATP concentration, and cause the necrotic cell 21, 22, 23.
During the brain development, most neuron cells will be eliminated in an apoptotic manner to optimize the neural tissue. Caspase 3 activation will execute the apoptosis of neuronal cells during brain development24. Caspase 3, which has been formed, will activate the cytoplasmic DNA-se, thus, the typical intra-nucleus DNA splitting occurs and then the cell undergoes apoptosis. In this study, the strong relationship between caspase 3 and the cells that were experiencing apoptosis showed that when an active caspase 3 had been formed, the cell had no chance to improve itself and would experience an apoptotic death25.
When caspase 3 is active, the cell is truly committed to death and this process of apoptosis can be considered as a condition which lead to the point of no return. The active form expression of caspase 3 in the cells occurs at the beginning of apoptosis process and precedes any changes in the morphological picture of classical cell death, thus, that the detection of caspase 3 expression increase in cells through immunohistochemical examination can be used as a basis for detecting cells which undergo an apoptosis26,27. Healthy neuron cells also need to be analyzed for neural network functions, because neural network also contributes the area of research on neurology and psychology28. Likewise the impact of apoptosis needs evaluation of the transcriptional control of the genes demage in vitro by mutational analysis and reporter assay studies29.
Embryonal necrotic neuron cell death occurs with the increasing MDA levels, but in this study, it was found that an increase in MDA levels was not associated with an increase in necrotic cells. The increased necrotic cells were created by the increased expression of p53 which affected the mitochondria in producing energy. Carbofuran exposure in pregnant mice generated a high apoptotic neuron cells and, in this study, there was a relationship between the expression of p53 and necrotic cells, which opened up the opportunities for prevention and strategies for treating the embryonal neuronal cell death due to carbofuran exposure during pregnancy such as by giving the antioxidant variations. Antioxidants include a group of vitamins, minerals, herbs and enzymes that help to protect the body from the formation of free radicals. Free radicals are substances or electromagnetic fields that cause damage to cells30,31. The antibiotic stops “cell suicide” (neuronal apoptosis). Apoptosis includes the activation of caspase enzyme and the possible treatment by herbal medicines32. Gongronema latifolia, Psidium guajava, Morinda tinctoria could serve an important role against the peroxidation of membrane lipids, alteration of redox balance, enzyme inactivation and DNA damage33,34,35.
This study can conclude that carbofuran exposure in the embryonal period can trigger the generation of intrinsic ROS and initiate the oxidative damage. The presence of ROS creates a decrease in SOD and increases the MDA levels. MDA levels are the products of membrane system peroxidation but, in this study, it is found that they do not launch any necrotic cells. The presence of ROS also drives the DNA damage of neuron cell characterized by a relationship between the expression of p53 and caspase 3 which then can initiate the apoptotic cells. The strong relationship of apoptotic and necrotic cells caused by p53 expression opens the opportunities for prevention and treatment of the ROS activity’s effects due to the carbofuran exposure during pregnancy.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
Acknowledgments:
The authors express sincere thanks to the Ministry of Research, Technology and Higher Education of the Republic of Indonesia for funding the research, and the Director of Post Graduate School Universitas Airlangga for providing all necessary facilities and funds in conducting this research work.
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Received on 11.12.2018 Modified on 18.04.2019
Accepted on 16.06.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(11):5492-5498.
DOI: 10.5958/0974-360X.2019.00953.3