Defense of the dissertation of Ainagýlova Galija Sijundýkovna for the degree of Doctor of Philosophy (PhD) in the specialty «8D05107 - Biology»
L.N. Gumilyov Eurasian National University, a dissertation defense for the degree of Doctor of Philosophy (PhD) by Ainagýlova Galija Sijundýkovna on the topic «Study of the molecular-cellular effect of asbestos dust on mammals» to the educational program «8D05107 – Biology».
The dissertation was carried out at the «General Biology and Genomics» of L.N. Gumilyov Eurasian National University.
The language of defense is kazakh
Official reviewers:
Ережепов Даурен Адилович
Шалахметова Тамара Минажевна - Doctor of Science, Professor, professor
Temporary members of the Dissertation Committee:
Тулеуханов Султан Тулеуханұлы
Акильжанова Айнур Рахметуловна - Doctor of Science, Professor of practice
Мукантаев Канатбек Найзабекович - Professor of practice
Scientific advisors:
Bersimbaev Rakhmetkazhi Iskendirovich
Gasimov Eldar Kochari Оgly
The defense will take place on December 24, 2024, at 12:00 PM in the Dissertation Council for the training direction «8D051 – Biological and related sciences» in the specialty «8D05107 – Biology» of L.N. Gumilyov Eurasian National University. The defense meeting is planned to be held online.
Link: https://us06web.zoom.us/j/85675283893?pwd=7wpEA3bnCdbkaLKSWO6Zaxa24MN2Dr.1, идентификатор конференции 856 7528 3893, код доступа 234542.
Address: г. Астана, ул. Кажымукана, 13, корпус №3, ауд. №333
Abstract (English): General description of the study. The dissertation is devoted to the study of the molecular cellular effects of chrysotile asbestos dust on the lungs of rats. Relevance of the research topic. Environmental pollution is a global problem all over the world. Asbestos is one of the important environmental carcinogens. Asbestos is a naturally occurring silicate mineral fiber, which due to its structure has high fire resistance, electrical and thermal insulation, this material is widely used in various industrial fields. Asbestos was widely used in many industries in the 19th and 20th centuries. In the late 19th century, asbestos was used as a major component in construction and shipbuilding for fire protection and insulation, and as an anti-friction material for clutch and brake pads, vehicle supports and in the manufacture of filters. In addition, asbestos has been used as an additive to asphalt concrete to improve the stability of road surfaces. As a result, a large number of industrial workers and a significant portion of the urban population were exposed to asbestos dust during the processes of asbestos mining and processing, asbestos cement production and the use of asbestos for commercial purposes. According to the World Health Organization (WHO), approximately 125 million people worldwide are exposed to asbestos in their workplaces, which is responsible for approximately 230,000 deaths each year. Between 1970 and 2005 Most Western countries have banned the use of asbestos and by 2019 asbestos was banned in 66 countries. Bans on the use of chrysotile asbestos were adopted by the European Community based on research results and the consequences of the uncontrolled use of amphibole asbestos. These studies indicated a high risk of developing asbestosis, lung cancer and pleural mesothelioma among industrial workers and the population. Global asbestos production in 2020 amounted to more than 1.2 million tons. The main producing countries are Russia, China and Brazil. Kazakhstan currently produces about 250 thousand tons of chrysotile asbestos per year, of which 95% is exported. The Zhitikarinsk enterprise, which is located in the Kostanay region, for the extraction and processing of chrysotile-asbestos ores, produces about 10% of the world's volume of chrysotile fiber. Asbestos-related lung disease is a major health problem worldwide. All identified forms of asbestos have been classified as human carcinogens by the International Agency for Research on Cancer (IARC). Lung cancer is commonly associated with smoking and exposure to carcinogenic components of tobacco smoke. About 90% of lung cancer cases in men and 80% in women are caused by tobacco smoking. Smoking is responsible for about 25% of deaths among women and men. However, lung cancer in people who have never smoked accounts for 10 to 25% of all cases. Experts also estimate that about 110,000 people die each year from lung cancer, mesothelioma and asbestosis as a result of exposure to asbestos. Between 5% and 7% of all lung cancer cases worldwide are caused by high levels of asbestos as a cause of the disease, mainly due to occupational exposure. In addition, several thousand deaths annually may be caused by the use of asbestos and asbestos-containing materials in the home. Co-exposure to tobacco smoke and asbestos fibers has also been shown to significantly increase the risk of developing lung cancer. Asbestos may influence various immune cells, cytokines, cytokine binding proteins, and growth factors in regulating disease manifestations in fibrotic lung diseases, including asbestosis. Some of the important cytokines and growth factors involved in the pathogenesis of pulmonary fibrosis enhance cellular damage and activate fibroblast proliferation and collagen deposition. The cytokine TNF-α is a key cytokine involved in asbestos-induced pulmonary toxicity. Alveolar macrophages in asbestosis and idiopathic pulmonary fibrosis release increased levels of TNF-α. TNF-α may contribute to lung inflammation through its effects on epithelial cells. More research is needed to determine their mechanism of action in lung cells when exposed to chrysotile asbestos. Asbestos causes asbestosis and malignancy through molecular mechanisms that are not fully understood. Pathological processes at the cellular and molecular levels can be determined by a violation of the functional state of mitochondria. Mitochondrial dysfunction plays a critical role in the pathogenesis of many lung diseases. Mitochondria are the main source of reactive oxygen species (ROS) generated during the flow of the electron transport chain. ROS of mitochondrial origin are involved in mediating asbestos-induced apoptosis of alveolar epithelial cells. Chrysotile and crocidolite types of asbestos have been shown to cause oxidative stress and induce local inflammatory mediators, resulting in a reactive inflammatory microenvironment and cell proliferation. All these processes occurring in cells are carried out with the participation of ROS. Asbestos induces ROS production through multiple mechanisms, which in part include activation of inflammatory cells, the ionic state of iron in asbestos fibers, mitochondria, and other intracellular sources. Asbestos fibers affect mitochondrial DNA and functional electron transport, resulting in the production of mitochondrial-derived ROS. The connection between the functional state of mitochondria and changes in their ultrastructure is well known. Therefore, studies of morphological changes in mitochondria caused by asbestos are relevant and promising, since disturbances in the ultrastructure of mitochondria occur much earlier than the appearance of detectable clinical symptoms of diseases. At the same time, the characteristic ultrastructure of mitochondria indicates changes in the functional state of the tissue and, as a consequence, the onset of the development of pathological processes. In recent years, quite effective methods have been developed for morphometric analysis of mitochondrial ultrastructure, which make it possible to assess the degree of ultrastructural disorders of mitochondria, indicating the functional state of these cellular organelles and the tissue as a whole. Typical mitochondrial ultrastructure is a prerequisite for mitochondrial function, which is critical to the fitness of cells, tissues, and organisms. Discovered relatively recently, the mitochondrial contact site and cristae organizing system complex MICOS is an important protein complex that contributes to the formation, maintenance and stability of mitochondrial cristae and its proteins, including mitofilin, play a key role in organizing the architecture of mitochondrial membranes in various organisms, from yeast to humans. Since mitochondrial dysfunction can lead to the release of toxic levels of ROS that cause cell death, to combat this, a number of quality control systems are in place to repair damaged mitochondria and protect the overall integrity of the mitochondrial network. However, when mitochondrial damage is too severe to repair, mitochondria can be selectively destroyed through mitophagy, an intracellular process that removes damaged mitochondria, and can cause cell degeneration. During mitophagy, Parkin (PRKN) directs damaged mitochondria to the lysosome for degradation. Mitochondrial dysfunction involves impaired fission and fusion, changes in transport and morphology, as well as abnormal PRKN levels and mitochondrial protein activity. Mitochondria provide the most important cellular functions such as energy supply, apoptosis, and play a key role in other important regulatory physiological processes, including the formation of adaptive reactions of the body. A high level of apoptosis can provoke the development of inflammatory processes in the lung tissue due to freely circulating nucleic acids - freely circulating mitochondrial DNA (fc-mtDNA). The number of fc-mtDNA copies changes in various types of malignant neoplasia, including lung cancer. For the study of malignant neoplasia, fc-mtDNA is acquiring great importance as a biomarker for a number of diseases, which serves as a kind of trigger for the transduction of pro-inflammatory signals in animal cells. However, the role of fc-mtDNA in the pathogenesis of lung cancer induced by exposure to chrysotile asbestos remains unexplored. Considering the above, studying the molecular cellular effect of asbestos dust in lung tissue, which allows us to identify intracellular and molecular mechanisms leading to pathology in cells and understanding the changes that occur when exposed to chrysotile asbestos on the mammalian body, is a relevant topic of research. The purpose of the study is to study the molecular cellular effects of chrysotile asbestos on rat lung tissue. To achieve the goal, the following tasks have been set: 1. To study the morphological structure of rat lung tissue when exposed to chrysotile asbestos. 2. Determine the levels of cytokines (IL-6, IL-4 and TNF-α) and indicators of the immune system of B- and T-lymphocytes in the blood serum of rats exposed to chrysotile asbestos. 3. Conduct electron microscopic studies and study the morphometric parameters of the ultrastructure of mitochondria in the lung tissue of rats under the influence of chrysotile asbestos. 4. To study the content of parkin (PRKN) and mitofilin (IMMT - Inner mitochondrial membrane transmembrane) proteins in the lung tissue of rats exposed to chrysotile asbestos. 5. Determine the effect of chrysotile asbestos on the content of free-circulating mitochondrial DNA (fc-mtDNA) in the blood plasma of rats. The object of the study were sexually mature male white Wistar rats. To reproduce experimental asbestosis, chrysotile asbestos dust was injected intratracheally into the lungs of experimental animals, and saline solution was injected into control animals. The animals were divided into three groups: 1) control; 2) poisoned with asbestos at a dose of 25 mg; 3) poisoned with asbestos at a dose of 50 mg. Two months after inoculation with asbestos, the lungs of the rats were removed and the necessary parameters were studied. The dissertation work was carried out at the Department of General Biology and Genomics, Institute of Cell Biology and Biotechnology of the Eurasian National University named after L.N. Gumilev (Astana, Kazakhstan) and at the Department of Cytology, Embryology and Histology of the Azerbaijan Medical University (Baku, Azerbaijan). Research methods. The dissertation work used light and electron microscopy methods; histological, molecular and immunological research methods; Real-time PCR; immunofluorescence assay using FITC-conjugated monoclonal antibodies; morphometric analysis of mitochondrial ultrastructure; ELISA for parkin (PRKN) and mitofilin (IMMT) proteins; statistical data processing. Scientific research innovation: - the morphological structure of rat lung tissue was studied by light microscopy after intratracheal administration of chrysotile asbestos to animals; - the levels of pro- and anti-inflammatory cytokines IL-6, IL-4 and TNF-α in the blood serum of rats under the influence of chrysotile asbestos were determined; - the parameters of the immune system of T- and B-lymphocytes of the lungs of rats poisoned with asbestos dust were determined when exposed to different doses of chrysotile asbestos; - morphometric studies of the ultrastructure of mitochondria in the lungs of rats were carried out after intratracheal administration of chrysotile asbestos to animals and changes occurring in the morphometric characteristics of mitochondrial ultrastructure depending on the dose of asbestos exposure were shown; - the content of mitofilin proteins, which support the structure of mitochondrial cristae, and parkin, which restore damaged mitochondria and protect the integrity of the mitochondrial network through mitophagy, under the influence of chrysotile asbestos, was determined; - the levels of free-circulating mitochondrial DNA in the lungs of rats were determined when exposed to chrysotile asbestos. Theoretical and practical significance of the research. The conducted studies allow us to deepen knowledge about the molecular cellular mechanisms of the development of inflammatory processes in the lung tissues of rats caused by chrysotile asbestos. The results obtained make it possible to identify early symptoms of structural and molecular cellular disorders of the body. Identification of the mechanisms of the molecular cellular response to the action of asbestos makes it possible to prevent and reduce the risks of complications, reduce the number of cases of disease, optimize methods of personalized treatment, improve the well-being and quality of life of patients with asbestos-related diseases in Kazakhstan. The results of the dissertation work were tested and introduced into the educational process of the Department of General Biology and Genomics of ENU named after L.N. Gumilyov (Act of introduction into the discipline «Fundamentals of molecular genetic processes» of the educational program 7M05107 - Biology and «Molecular and Cellular Biology» of the educational program 7M01513 - Biology for 1st year undergraduates of the Department of General Biology and Genomics of the Eurasian National University named after L.N. Gumilyov). The main points put forward for the defense of the dissertation: 1. In the morphological structure of the lung tissue of rats, when exposed to chrysotile asbestos, chronic bronchitis, small-focal bronchopneumonia and small-focal perivascular sclerosis develop, and also at a higher dose, obstructive emphysema, respiratory failure and pulmonary hypertension develop. 2. Increased levels of pro- and anti-inflammatory cytokines IL-6, TNF-α, IL-4 and immune system parameters indicate the acute phase of the inflammatory process in the lung tissue of rats exposed to chrysotile asbestos. 3. An increase in mitochondrial area, perimeter, average diameter of external and internal membranes, cristae diameter and intermembrane space, as well as a decrease in the length of cristae in the ultrastructure of mitochondria in rat lung tissue are mitochondrial dysfunction when exposed to chrysotile asbestos. 4. Reduced levels of mitochondrial proteins mitofilin and parkin upon exposure to chrysotile asbestos are indicators of cristae destruction, accumulation of damage and mitochondrial dysfunction in the lung tissues of rats. 5. An increase in the level of fc-mtDNA in the blood plasma of rats is due to the development of inflammatory processes in the body when exposed to chrysotile asbestos. Connection of work with research programs. The dissertation work was carried out within the framework of the scientific project AR09259700 «Study of molecular changes induced by asbestos in the pathogenesis of lung cancer», state registration number No. 171/36-21-23 in 2020-2023. The main results of the study were reported at international and national conferences: 1. International scientific and practical conference «Occupational Medicine of the 21st Century: Issues of Protecting the Health of the Working Population» (Karaganda, 2022). 2. International scientific forum «Omarov Readings: Biology and Biotechnology of the 21st Century» (Astana, 2023). 3. International scientific and practical conference «Prospects for the development of occupational medicine and medical ecology» (Karaganda, 2023). 4. International scientific conference «Priority directions for the development of science and education» (Astana, 2023). 5. Proceedings of the international scientific conference of students and young scientists «Science and Education – 2024» (Astana, 2024). Publications. Based on the research materials of the dissertation work, 8 works were published, of which: 1 article in publications included in the Scopus database (Cytokine, IF 3.8, percentile 78); 4 articles in the journal of the Republic of Kazakhstan, included in the list recommended by the SHEQAC of the Ministry of Science and Higher Education of the Republic of Kazakhstan; 5 works in collections of articles of International scientific and practical conferences. Structure and scope of the dissertation. The dissertation is presented on 104 pages. The work consists of symbols and abbreviations, introduction, literature review, materials and research methods, results and discussion, conclusion, list of references, including 239 sources. The dissertation is illustrated with 4 tables, 32 figures and 1 application.
Conclusion of the Research Ethics Committee
Defense of the dissertation: https://youtu.be/PI3AfLlUddA?si=Aox4D_REHybmuVpr
