Research code: IR.IAU.VARAMIN.REC.1399.003
Ethics code: IR.IAU.VARAMIN.REC.1399.003
Clinical trials code: IR.IAU.VARAMIN.REC.1399.003
islamic azad university , m.salehypur@gmail.com
Abstract: (526 Views)
Background: Mice models as in-vivo model for cancer research provide significant help to researchers about the mechanisms of tumorigenesis in dynamic systems which affected by many factors such as hormones, various cellular communications, diets, and chemicals. One of the most common cancer is hepatocarcinoma or liver cell cancer, which has different stages and caused by various environmental and genetical factors. Thioacetamide is a kind of toxin which destroys liver tissue through affects the mechanisms of cell proliferation, differentiation and apoptosis by the destruction or damage of cell structure induction pathways, increasing the risk of genetic errors and stimulating the evolution of cells into malignant neoplasia, and is used in many industries such as leather, textile, paper and rubber, but its carcinogenic mechanisms are not well defined. In order to diagnose liver cancer, there are various methods including liver function test such as ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), GGT (Gamma glutamyl transferase) and tumor markers such as CEA (Carcinoembryonic Antigen) and AFP (Alpha fetoprotein), which are most widely used prognosticators for determining cancer, but their specificity and sensitivity is not high. Therefore, the direct method of examining the tissue, pathology method is used for the final diagnosis of cancer. It shows the structural changes of the cells. In this study, by using the in-vivo modeling of cancer in rats exposed to a toxic substance, we have investigated different stages of hepatocarcinogenesis process, in order to help doctors to have in vivo samples at every stage of the occurrence of this cancer for treatment purposes and to make decisions about how to treat and test different drugs.
Material and methods: In this research, in order to determine the tolerance and lethal dose of thioacetamide, before starting the main experiment, 18 male mice in 3 groups of 6 were treated for 4 months with different doses of thioacetamide including 100, 200 and 300 mg/L respectively and after observing the process of mortality, causing shock, lethargy and investigating the cause of death after surgery in different groups, the best dose was determined. More than half of the mice in the high-dose group were shock and died in less than a month, while in the low-dose group, the tissues were still healthy after surgery and just in the middle dose receiver mice, the tissue changes was obtained, which was precisely determined during the main test by examining the pathology of the changes. After determining the carcinogenic concentration of thioacetamide, in this study, 200 mg/L (equivalent to 33 mg/l daily TAA) of thioacetamide was consumed as a solution in drinking water to make liver cancer in mice. Mice were euthanized by ketamine/xylazine after 2 and 4 months. Then the livers of mice were separate in formalin to investigate the cancer process. After staining with Hematoxylin/Eosin, the tissue samples were studied using a light microscope with different magnifications by a camera installed on the microscope and a computer system connected to the camera with DINO CAPTURE software.
Results: Liver samples were examined in terms of morphology, cytoplasmic staining, nuclear size and cellular atypia (abnormality and uniformity of cells). Also, for staging in case of cancer, the size of tumors, the number of lymph nodes containing tumor and the rate of spread (metastasis) were investigated. No histopathological lesion was observed in the samples taken from the liver of the control group and the liver had a normal structure. Among the tissue lesions created in the group that were euthanized after 2 months, we can mention mild to moderate dysplasia, which is associated with hypertrophy (enlargement) of liver cell nuclei, bordering of nuclei, degeneration of some cells, and swelling of cell walls. Also, in some areas, the expansion of disc space and sinusoid can be seen. The liver tissue of mice exposed to thioacetamide for 4 months is completely necrotic. Hepatic lobules and dysplastic hepatic portal space can be seen and focal structural changes are also observed. Many necrotic foci are seen in the liver, which indicates the destructive effect of thioacetamide in acute liver damage. Hepatic lobules and dysplastic hepatic portal space can be seen and focal structural changes are also observed. Many necrotic foci are seen in the liver, which indicates the destructive effect of thioacetamide in acute liver damage.
Conclusion: In vivo mice models are key tools for oncology, pharmaceutical, genetics and other research studies. Considering that nearly five hundred thousand patients are diagnosed with liver cancer every year and it is associated with a poor prognosis, therefore creating experimental models to define the pathogenesis of HCC that occurs in the early stages of liver cancer can be a solution in the direction of reduce the cost of cancer research. Our results showed that thioacetamide as an organosulfur can change the structure of liver cells and severely inflame the tissue during 4 months. Thioacetamide leading to apoptosis and necrosis of these cells by CYP2E1 enzymes present in liver microsomes through the mechanism of oxidation to thioacetamide S-oxide, which changes the permeability of cells, increases intracellular calcium concentration, increases nuclear volume and inhibits mitochondrial activity by reducing their membrane potential in liver cells. TAA has long been recognized as a hepatotoxin that requires biological activation of S oxidation to thioacetamide oxide (TASO) and then to the highly reactive S-Oxide (TASO2) and can be tautomerized to acyl species which capable of transforming cellular nucleophiles include phosphatidylethanolamine (PE) lipids and proteins with lysine side chains. Liver cells can effectively oxidize TA to TASO and TA make strongly covalent bonds with lipids and proteins. Exposure to this substance can activate the ROS system (reactive oxygen species) such as nitric oxide (NO), which causes cell death due to interaction with superoxidase ion and the formation of peroxynitrite, which causes a significant decrease in FRAP values, which indicates the capacity of antioxidant. It can also affect the pre-apoptotic signaling pathways through G protein-coupled receptors, as well as the effect on the phosphorylation of the transcription factor P53, the production of inflammatory cytokines such as NF-kB, the metabolism and apoptosis of cells. In this project, after 2 months of exposure the mice to thioacetamide, liver tissue cells gradually changed towards fibrosis and after 4 months, some cells changed to necrosis, which indicates the irreversible effects of this substance in the body. In conclusion, it seems that mice model has the potential to be used as accessible strategies replace human samples.
Material and methods: In this research, in order to determine the tolerance and lethal dose of thioacetamide, before starting the main experiment, 18 male mice in 3 groups of 6 were treated for 4 months with different doses of thioacetamide including 100, 200 and 300 mg/L respectively and after observing the process of mortality, causing shock, lethargy and investigating the cause of death after surgery in different groups, the best dose was determined. More than half of the mice in the high-dose group were shock and died in less than a month, while in the low-dose group, the tissues were still healthy after surgery and just in the middle dose receiver mice, the tissue changes was obtained, which was precisely determined during the main test by examining the pathology of the changes. After determining the carcinogenic concentration of thioacetamide, in this study, 200 mg/L (equivalent to 33 mg/l daily TAA) of thioacetamide was consumed as a solution in drinking water to make liver cancer in mice. Mice were euthanized by ketamine/xylazine after 2 and 4 months. Then the livers of mice were separate in formalin to investigate the cancer process. After staining with Hematoxylin/Eosin, the tissue samples were studied using a light microscope with different magnifications by a camera installed on the microscope and a computer system connected to the camera with DINO CAPTURE software.
Results: Liver samples were examined in terms of morphology, cytoplasmic staining, nuclear size and cellular atypia (abnormality and uniformity of cells). Also, for staging in case of cancer, the size of tumors, the number of lymph nodes containing tumor and the rate of spread (metastasis) were investigated. No histopathological lesion was observed in the samples taken from the liver of the control group and the liver had a normal structure. Among the tissue lesions created in the group that were euthanized after 2 months, we can mention mild to moderate dysplasia, which is associated with hypertrophy (enlargement) of liver cell nuclei, bordering of nuclei, degeneration of some cells, and swelling of cell walls. Also, in some areas, the expansion of disc space and sinusoid can be seen. The liver tissue of mice exposed to thioacetamide for 4 months is completely necrotic. Hepatic lobules and dysplastic hepatic portal space can be seen and focal structural changes are also observed. Many necrotic foci are seen in the liver, which indicates the destructive effect of thioacetamide in acute liver damage. Hepatic lobules and dysplastic hepatic portal space can be seen and focal structural changes are also observed. Many necrotic foci are seen in the liver, which indicates the destructive effect of thioacetamide in acute liver damage.
Conclusion: In vivo mice models are key tools for oncology, pharmaceutical, genetics and other research studies. Considering that nearly five hundred thousand patients are diagnosed with liver cancer every year and it is associated with a poor prognosis, therefore creating experimental models to define the pathogenesis of HCC that occurs in the early stages of liver cancer can be a solution in the direction of reduce the cost of cancer research. Our results showed that thioacetamide as an organosulfur can change the structure of liver cells and severely inflame the tissue during 4 months. Thioacetamide leading to apoptosis and necrosis of these cells by CYP2E1 enzymes present in liver microsomes through the mechanism of oxidation to thioacetamide S-oxide, which changes the permeability of cells, increases intracellular calcium concentration, increases nuclear volume and inhibits mitochondrial activity by reducing their membrane potential in liver cells. TAA has long been recognized as a hepatotoxin that requires biological activation of S oxidation to thioacetamide oxide (TASO) and then to the highly reactive S-Oxide (TASO2) and can be tautomerized to acyl species which capable of transforming cellular nucleophiles include phosphatidylethanolamine (PE) lipids and proteins with lysine side chains. Liver cells can effectively oxidize TA to TASO and TA make strongly covalent bonds with lipids and proteins. Exposure to this substance can activate the ROS system (reactive oxygen species) such as nitric oxide (NO), which causes cell death due to interaction with superoxidase ion and the formation of peroxynitrite, which causes a significant decrease in FRAP values, which indicates the capacity of antioxidant. It can also affect the pre-apoptotic signaling pathways through G protein-coupled receptors, as well as the effect on the phosphorylation of the transcription factor P53, the production of inflammatory cytokines such as NF-kB, the metabolism and apoptosis of cells. In this project, after 2 months of exposure the mice to thioacetamide, liver tissue cells gradually changed towards fibrosis and after 4 months, some cells changed to necrosis, which indicates the irreversible effects of this substance in the body. In conclusion, it seems that mice model has the potential to be used as accessible strategies replace human samples.
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