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The influence of in utero and early postnatal exposure to pesticides on the process of cells apoptosis and proliferation.
DOI: http://dx.doi.org/10.20534/ESR-17-3.4-57-59
Tukhtaev Nodirbek Kadirovich, Senior Researcher of Tashkent Medical Academy, E-mail: [email protected] Zokirova Nargiza Bakhodirovna, Senior Researcher of Tashkent Medical Academy
The influence of in utero and early postnatal exposure to pesticides on the process of cells apoptosis and proliferation in immune and endocrine organs of the offspring
Abstract
Objective: Immunohistochemical evaluation of apoptosis and cells proliferation in the thyroid and thymus of offspring in a prenatal and early postnatal exposure to pesticides.
Material and methods: Experiments were performed on white adult female rats, which were divided into 3 groups of 30 animals each. Two groups of animals for 30 days daily per os obtained respectively pesticides cyhalothrin (8 mg/kg), or fipronil (3.6 mg/kg). The third group receives only the same volume of sterile saline served as control. Apoptotic and proliferating cells of the thyroid gland and thymus were determined by immunohistochemistry method using monoclonal antibodies (Thermo Scientific, USA).
Results: It was found that both fipronil and cyhalothrin under the influence through the mother causes a marked induction of apoptosis in the offspring thyroid and thymus. Simultaneously, these pesticides cause inhibition of proliferative activity of cells in these organs.
Conclusions: Intrauterine and early postnatal exposure of pesticides to cause the induction of apoptosis, but the inhibition of proliferative activity of cells in the thyroid and thymus of offspring. In the mechanism of induction of apoptosis, besides the direct toxic effects of drugs are important hypothyroidism and oxidative stress observed in the offspring. These data contributes to the development of a new method of prevention and treatment of hidden toxic effects in pregnant women and their newborns.
Keywords: pesticides; postnatal ontogenesis; thyroid; thymus; cells apoptosis; cells proliferation; immunohistochemistry.
Introduction. Programmed cell death (PCD), or apoptosis, is the mechanism which is crucial to the body under the control of cell proliferation and the maintenance of tissue homeostasis [2; 3; 5]. Under physiological conditions, between the processes of apoptosis and cell proliferation there is a balance that allows you to maintain tissue homeostasis [2; 7]. Defects in apoptosis physiological mechanisms can lead to a variety of human diseases, including the development of malignancy and cancer process [2; 5]. This explains the great interest in the in-depth study of the mechanisms of apoptosis and effector proteins, as well as genes involved in apoptosis. The results of these studies have opened great possibilities to control the process of apoptosis and to develop new methods for treating various diseases, including cancer [2]. In recent years, it revealed that many environmental contaminants, including pesticides of new generations, act as apoptotic cells triggers [3; 4; 6; 7]. It is found that one of the most widespread and effective insecticide Fipronil (F) substantially induces apoptosis in line SH-SY5Y and Drosophila S2 cells under in vitro [12; 13]. Another, no less common, pesticide of pyrethroid class lambda-cyhalothrin (C) led to an increase in fragmentation of rat bone marrow of human lymphocyte cell nuclei, indicating the induction of apoptosis [1; 6]. Most authors believe that the induction of apoptosis under the influence of pesticides associated with the development of oxidative stress and the subsequent activation of the caspase family enzymes and apoptosis-stimulating p 53-family proteins [3; 4; 6; 12; 13]. However, it should be noted that all of these studies were conducted on cell lines or on adult individuals of experimental animals. We have previously shown that in utero and early postnatal exposure to pesticides through the mother's body has a negative effect on the organs of the endocrine and immune progeny systems, leading to
their structural and functional impairment of growth and formation in the dynamics of postnatal ontogenesis [10; 11]. However, the value of cells apoptosis and proliferation in the mechanism of these disorders remains unclear.
The purpose of the study was a immunohistochemical assessment of cells proliferation and apoptosis in the thymus and thyroid of offspring in conditions of in utero and early postnatal exposure to pesticides.
Material and methods. Experiments were performed on nul-liparous, white adult female rats, which were divided into 3 groups of 30 animals each. Two groups of animals for 30 days daily per os obtained respectively pesticides cyhalothrin (8 mg/kg), or fipronil (3.6 mg/kg). The third group receiving only the same volume of sterile saline served as a control. The following day, the females were coupled to healthy males for fertilization. Pregnancy was monitored for the presence of sperm in vaginal smears. Exposure of pesticides was continued incessantly during pregnancy and after delivery until the end of lactation. Offspring obtained from the experimental and control females were studied in dynamics on days 7, 14, 21 and 30 after birth. For immunohistochemical studies of proliferation and apoptotic cells paraffin sections of thymus and thyroid have been used. Apoptotic cells were detected using a rabbit monoclonal antibody to fragments of caspase - 3 proteins and the family of p - 53 (manufactured by Thermo Scientific, USA). Proliferation cells have been detected using a rabbit monoclonal antibody to protein Ki - 67 (manufactured by Thermo Scientific, USA). Further procedures for visualization of proliferation and apoptotic cells by labeled with horseradish peroxidase were carried out by the standard procedure using reagents Ultra-Vision complex (manufactured by Thermo Scientific, USA). Further sections have been stained
Section 5. Medical science
methylene blue and neutral red. Further the numbers of labeled proliferating and apoptotic cells counted on 1000-5000 total cells and calculated an index of proliferation and apoptosis, that is expressed in parts per thousand. All digital data were processed by the method of variation statistics. Statistical significance between control and experimental groups was compared using the Student's t test and P values < 0.05 were considered significant.
Results and discussion. Apoptotic and proliferating cells on sections after carrying out immunohistochemical reaction looked dark brown due to visualization of horseradish peroxidase-labeled secondary antibodies. They were easily identified from unlabeled cells. Differences between the levels of proliferation and apoptosis of the cells of the studied organs were most clearly manifested in the quantitative calculation of the indices of proliferating and apoptotic cells. The cell proliferation index of both thymus and thyroid gland was highest on day 7 after birth. In the future, it gradually decreased and on the 30 th day after birth was about 55-60% of the baseline. Similar dynamics was characteristic for both control and experimental groups of rats. The effect of pesticides led to inhibition of the proliferative activity of cells of both thymus and thyroid gland. On the 7th day after birth, the index of thymus cells proliferation under the action of cyhalothrin in 1,5 times, and when exposed to fipronil 1.8 times decreased in comparison with the control (P < 0.05). A similar decrease in the proliferation index was found in the thyroid gland. A significant decrease in the cell proliferation index in both organs persisted up to 21 days after birth. At 30 days after birth, the decrease in the cell proliferation index in both organs was statistically unreliable (P > 0.05). Thus, exposure to pesticides led to inhibition of proliferative activity of cells in both the immune and endocrine organs of the offspring. In this case, the negative effect of fipronil was more pronounced in comparison with cyhalothrin.
Data of a different nature were obtained when calculating the apoptosis index of cells in the thymus and thyroid gland. The level of apoptosis in the thyroid gland of control animals was very low and at the dynamic of postnatal development the apoptosis index gradually increased. In contrast, in the thymus, apoptosis was initially high and increased significantly in the dynamics of postnatal growth of animals. Intrauterine and early postnatal exposure to pesticides led to a significant increase in the degree of apoptosis of cells of the thyroid gland and thymus. In the thyroid gland of the offspring, under the influence of cyhalothrin, the apoptosis index was 3.5-4 times, and when fipronil was 4.5-5 times higher than the control group in all periods of the study (P < 0.05). A similar significant increase in the apoptosis index at all times of the study was observed in the thymus of the experimental animals. Thus, exposure to pesticides led to a significant increase in the degree of apoptosis of cells of both the thyroid gland and thymus. Un-
der the influence of fipronil, the induction of apoptosis of the cells was manifested to a higher degree as compared with cyhalothrin.
Thus, both fipronil and cyhalothrin when exposed through the mother's organism cause a marked induction of apoptosis in the thyroid and thymus glands of the offspring. Simultaneously, these pesticides cause inhibition of proliferative activity of cells in these organs. Thus, there is an imbalance between the processes of proliferation and apoptosis in organs and tissues. Our previous studies have shown that both pesticides in conditions of prenatal and early postnatal expose cause a slowdown in the growth and formation of the thyroid gland, a decrease in organ function in the offspring in the form of hypothyroidism [10; 11]. In this case, hypothyroidism was more pronounced when exposed to fipronil compared with cyhalothrin. It has long been known that the thyroid hormones, along with the growth hormone, regulate the proliferation of cells in virtually all tissues. Recent data show that thyroid hormones also have a high anti-apoptotic effect, which opens great prospects for the regulation of apoptosis in various diseases [5]. All this makes it possible to consider that the intensity of induction of apoptosis in our experiments is to a certain extent determined by the degree of thyroid dysfunction and the weakening of the proliferation-stimulating and anti-apoptotic effects of its hormones. This is confirmed by our data indicating a more pronounced induction of apoptosis when exposed to fipronil. On the other hand, an important role in the induction of apoptosis is played by oxidative stress, caused by an increase in the production of free radicals [3; 7]. Earlier, we showed that both pesticides in prenatal and early postnatal conditions cause pronounced oxidative stress in offspring [8; 9]. Consequently, the induction of apoptosis in the experimental progeny is caused not only by the direct toxic effect of pesticides, but also largely mediated by the weakening of the anti-apoptotic function of thyroid hormones due to hypothyroidism and the resulting oxidative stress in the form of an increase in the number of free radicals.
Conclusions:
1. Pesticides of the new generation of cyhalothrin and fipronil in conditions of in utero and early postnatal expose cause induction of apoptosis in the thyroid and thymus glands of the offspring.
2. In the mechanism of induction of apoptosis of cells, along with direct toxic effects of drugs, an important role is played by hypothyroidism and oxidative stress observed in offspring.
3. Induction of apoptosis and inhibition of cells proliferation are more pronounced when exposed to fipronil compared with cy-halothrin.
4. Disclosure of mechanisms of apoptosis induction under the influence of pesticides contributes to the development of a new methods for the prevention and treatment of latent toxic effects in pregnant women and their newborn children.
References:
1. Celik A., Mazmanci B., Camlica Y., Askin A., Comelekoglu U. Induction of micronuclei by lambda-cyhalothrin in Wistar rat bone marrow and gut epithelial cells.//Mutagenesis. - 2005. - Vol. 20 (2). - P. 125-129.
2. Goldar S., Khaniani M. S., Derakhshan S. M., Baradaran B. Molecular mechanisms of apoptosis and roles in cancer development and treatment.//Asian Pac J Cancer Prev. - 2015. - Vol. 16 (6). - P. 2129-2144.
3. Ki Y. W., Lee JE, Park J. H., Shin I. C., Koh H. C. Reactive oxygen species and mitogen-activated protein kinase induce apoptotic death of SH-SY5Y cells in response to fipronil.//Toxicol Lett. - 2012. - Vol. 211 (1). - P. 18-28.
4. Lee J. E., Kang J. S., Ki Y. W., Lee S. H., et al. Akt/GSK3p signaling is involved in fipronil-induced apoptotic cell death of human neuroblastoma SH-SY5Y cells.//Toxicol Lett. - 2011. - Vol. 202. (2). - P. 133-141.
5. Lin H. Y., Glinsky G. V., Mousa S. A., Davis P. J. Thyroid hormone and anti-apoptosis in tumor cells.//Oncotarget. - 2015. 6 (17): - P. 14735-14743.
6. Muranli F. D. Genotoxic and cytotoxic evaluation of pyrethroid insecticides X-cyhalothrin and a-cypermethrin on human blood lymphocyte culture.//Bull Environ Contam Toxicol. - 2013. - Vol. 90. (3) - P. 357-363.
The roleof adipokines, triglycerides and freefatty acids in the development of insulin resistance in the presence of metabolicsyndrome
7. Park J. H., Park Y. S., Lee J. B., Park K. H. et al., Meloxicam inhibits fipronil-induced apoptosis via modulation of the oxidative stress and inflammatory response in SH-SY5Y cells.//J Appl Toxicol. - 2015. Mar 13. - doi: 10.1002/jat.3136.
8. Tukhtaev K. R., Tulemetov S. K., Zokirova N. B., Tukhtaev N. K. Effect oflong term exposure low doses of lambda-cyhalothrin on the level of lipid peroxidation and antioxidant enzymes of the pregnant rats and their offspring.//Medical and Health Science Journal -2012. - Vol. 13. - P. 93-98. URL: http: www.pradec.en
9. Tukhtaev K. R., Tulemetov S. K., Zokirova N. B., Tukhtaev N. K. et al. Prolonged exposure of low doses of Fipronil causes oxidative stress in pregnant rats and their offspring.//The Internet Journal of Toxicology. - 2013. - Vol. 10. - No. 1. URL: http: www.ispub. com/IJT0/10/1/14550.
10. Tukhtaev K. R., Zokirova N. B., Tulemetov S. K., Tukhtaev N. K. Effect of prolonged exposure of low doses of lamda-cyhalothrin on the thyroid function of the pregnant rats and their offspring//Medical and Health Science Journal - 2012. - Vol. 13. - P. 86-92. URL: http: www.pradec.en
11. Tukhtaev K. R., Zokirova N. B., Tulemetov S. K., Tukhtaev N. K. et al. Effect of Prolonged exposure low doses of Fipronil on thyroid function of pregnant rats and their offspring.//The Internet Journal of Toxicology. - 2013. - Vol. 10. - No. 1. URL: http: www.ispub. com/IJT0/10/1/14550.
12. Vidau C., González-Polo R. A., Niso-Santano M., Gómez-Sánchez R. et al. Fipronil is a powerful uncoupler of oxidative phosphorylation that triggers apoptosis in human neuronal cell line SHSY5Y.//Neurotoxicology. -2011. - Vol. 32 (6). - P. 935-943.
13. Zhang B., Xu Z., Zhang Y., Shao X. et al. Fipronil induces apoptosis through caspase-dependent mitochondrial pathways in Drosophila S2 cells.//Pestic Biochem Physiol. - 2015. - Vol. 119. - P. 81-89.
DOI: http://dx.doi.org/10.20534/ESR-17-3.4-59-62
Uzbekova Nelly Rafikovna, Candidate of medical sciences, Associate Professor of the Department of Intermediate Level Therapy of Andijan State Medical Institute Vakhobov Bakhrom Mumindzhanovich, Candidate of medical sciences, Associate Professor of the Department of Intermediate Level Therapy of Andijan State Medical Institute Khuzhamberdiyev Mamazair Akhmedovich, Doctor of medical sciences, Professor, Head of the Department of Intermediate Level Therapy of Andijan State Medical Institute E-mail: [email protected]
The role of adipokines, triglycerides and free fatty acids in the development of insulin resistance in the presence of metabolic syndrome
Abstract: The objective of the study was to investigate the possible role of adipokines (leptin, adiponectin), triglycerides (TG) and free fatty acids (FFA) in the development of insulin resistance (IR) in patients with metabolic syndrome (MS).
The study included 170 patients with MS (67 men and 103 women) at the age of 51,5±3,93 years. It was determined that the increase of TG, FFA, leptin and the decrease of adiponectin were observed in the patients with MS with high HOMA index. However, the analysis of data in the groups of patients with different body mass demonstrated that the development of IR in patients with obesity may be influenced by the increase of TG, FFA and leptin concentrations, while in patients without significant obesity — by the increase of TG, FFA levels and the decrease of adiponectin.
Thus, it is possible to assume that leptin, adiponectin, TG and FFA may influence the development of IR, however, their role depends on the severity of obesity.
Keywords: adipokines, leptin, adiponectin, triglycerides, free fatty acids, insulin resistance, metabolic syndrome.
Insulin resistance is an important pathogenic factor in the number of diseases and disorders such as diabetes mellitus of the 2nd type, atherosclerosis, arterial hypertension (AH), atherogenic dislipoproteinemia [1; 2; 3; 14]. Insulin resistance (IR) means the reduction in the response of insulin-sensitive tissues to insulin in the case of its sufficient concentration [3; 12; 13; 17]. In this case, these disorders are combined into a single complex called "metabolic syndrome".
In the pathogenesis of metabolic syndrome (MS) the special role is assigned to obesity or abdominal fat distribution, which con-
tribute to the development of IR [1; 2; 8; 9; 13]. Therefore, with regard to the mechanisms of development of IR with MS a special attention is paid to the change of adipokine levels in the blood -biologically active proteins that are produced by fatty tissue (leptin, adiponectin, visfatin, resistin, interleukin-6, tumor necrosis factor alpha) [5; 7; 8; 15]. Moreover, an important role in the development of IR can be assigned to triglycerides (TG) and free fatty acids (FFA) that are produced in the course of lipolysis by fatty tissue and in the blood stream by lipoproteins, which contain a great amount of
