The goal of the dissertation was to investigate the involvement of four members of the amine oxidases family: FAD-containing polyamine oxidase and Cu-containing enzymes, diamine oxidase, soluble and membrane-bound forms of semicarbazide-sensitive amine oxidase, extracellular lysyl oxidase in the development of the kidney and lung pathology, as well as the potential impact of semicarbazide, a Cu-containing amine oxidases inhibitor, on disease manifestations.
The following methods were used in the work: modeling of acute and chronic kidney and lung diseases in laboratory animals (glycerol-induced rhabdomyolysis, streptozotocin-induced type 1 diabetes, and bleomycin C-induced pulmonary fibrosis in rats, ovalbumin-induced bronchial asthma in guinea pigs), cellular and molecular biology methods (a model of highly invasive Lewis lung carcinoma cells with stable knockdown of the adaptor protein Ruk/CIN85), Western-blot analysis, preparative biochemistry as well as differential centrifugation, spectrophotometry, spectrofluorometry, cytofluorometry, enzyme immunoassay, EPR spectroscopy, light microscopy, histological and morphometric studies, statistical analysis.
According to the results obtained during the work, it was shown for the first time that the activity levels of the studied amine oxidases, semicarbazide-sensitive amine oxidase, polyamine oxidase, and diamine oxidase, in the rat kidney, the target organ of rhabdomyolysis and diabetes; lysyl oxidase, semicarbazide-sensitive amine oxidase, diamine oxidase in the lung of guinea pigs and rats, the target organs of bronchial asthma and pulmonary fibrosis, were increased many times as compared to the control. The relationship between the functioning of amine oxidases and the metabolism of active forms of nitrogen was revealed. The original are the data regarding a potent increase in the activity of semicarbazide-sensitive amine oxidase in the kidney of diabetic rats is accompanied by an elevation in the level of nitrosative stress in the blood and liver, while under conditions of diabetes complicated by rhabdomyolysis, the binding of an excessive amount of reactive nitrogen species in the tissues of experimental animals by labile Fe3+ and Fe2+ of the iron-sulfur proteins leads to the normalization of a number of biochemical indicators. The direct involvement of copper-containing amine oxidases, lysyl oxidase, semicarbazide-sensitive amine oxidase, and diamine oxidase, in the development of lung pathology under bronchial asthma in guinea pigs and pulmonary fibrosis in rats was demonstrated by the use of semicarbazide, irreversible inhibitor of these enzymes. For the first time, it was established that knockdown of the adapter protein Ruk/CIN85 in highly invasive mice adenocarcinoma Lewis lung cells is accompanied by a corresponding decrease in the activity levels of the studied amine oxidases and enzymes/metabolites involved in the realization of the Warburg effect, which indicates direct involvement of these enzymes in carcinogenesis. In in vitro experiments has been shown that the metabolic transformation of semicarbazide in the body takes place with the participation of cytochromes P-450 of the liver microsomal fraction with the formation of formaldehyde, ammonium, and nitric oxide that determines the toxic effects of this inhibitor of Cu-containing amine oxidases depending on its concentration. 1.5 mM concentration of semicarbazide was the limit of the liver monooxygenase system sensitivity.
The results obtained during the dissertation research provide grounds for a general conclusion: all studied amine oxidases are a source of oxidative, carbonyl, and nitrosative stress, which makes them involved in the development of pathological conditions of the kidney and lungs, while the activity levels of these enzymes correspond to the degree of the course of the disease. Moreover, these indicators are markers of changes in the metabolism of reactive nitrogen species and are influenced in vivo by a specific inhibitor, semicarbazide, which can be used in the development of therapeutic approaches in the complex treatment of systemic diseases.
