ABSTRACT
Ethambutol (EMB) is conventionally used to treat tuberculosis and atypical Mycobacterium infections in combination with other antimycobacterial drugs. Eventually, EMB testicular toxicity has not been explored extensively yet. The aim of the study is to evaluate testicular toxicity of EMB. We explored the impact of EMB on male rats' fertility, testosterone level and germ cells state, testicular pro- and anti-oxidant status and DNA damage, as well as identified EMB effects on cytochrome P-450 2E1 (CYP2E1) both with computer simulation and in vivo. We demonstrated that EMB administration to male rats decreased in epididymal sperm count (19%) and fertility index (53%). These events were accompanied by reduction in serum testosterone content (1.6 times) and appearance of spermatogenic epithelium damages. It was also found in testes the intensification of lipid peroxidation, decrease in reduced glutathione content and changes in DNA fragmentation. Additionally, computer simulation showed direct interaction of EMB with CYP2E1 active site and heme. On the top of this, we demonstrated that level of testicular CYP2E1 messenger RNA in EMB-treated rats was increased 8.7 folds and p-nitrophenol hydroxylase activity in testes rose three folds. As this shows, EMB-caused CYP2E1 induction, oxidative stress, and apoptosis in the testes contribute to inhibition of steroidogenesis enzymes and spermatogenesis disruption.
Subject(s)
Antitubercular Agents/toxicity , Ethambutol/toxicity , Spermatogenesis/drug effects , Testis/drug effects , Animals , Cytochrome P-450 CYP2E1/genetics , Cytochrome P-450 CYP2E1/metabolism , DNA Fragmentation , Fertility/drug effects , Male , RNA, Messenger/metabolism , Rats, Wistar , Sperm Count , Testis/metabolism , Testis/pathology , Testosterone/bloodABSTRACT
Diabetes mellitus is one of the three most common modem diseases. A number of animal models is used in investigations of the mechanisms of development of the disease. Most of these models replicate the symptoms of type 1 diabetes mellitus. The development of type 2 diabetes is caused by the insulin resistance, hyperglycemia, structural and functional disorders of the pancreatic cells. Investigation of pathogenesis of type 2 diabetes is complicated by the lack of adequate models of this disease. In this work, based on existing hyperglycemia model, we propose the model of metabolic syndrome as a precursor of type 2 diabetes. The development of metabolic syndrome symptoms was caused by 28 days long intramuscular injection of protamine sulfate to guinea pigs at a dose of 15 mg/kg along with keeping of animals on a high glucose diet. Increased blood glucose and cholesterol levels, reduction of glycogen in liver, the structural and functional damage and reduce in the number of functionally active beta-cells in the pancreas of the experimental animals were observed. The results confirm the development of the metabolic syndrome symptoms in experimental animals, which makes it possible to use such methodical approach in creation of promising type 2 diabetes model.
Subject(s)
Diabetes Mellitus, Type 2/pathology , Disease Models, Animal , Glucose/administration & dosage , Hyperglycemia/pathology , Insulin-Secreting Cells/pathology , Metabolic Syndrome/pathology , Animals , Diabetes Mellitus, Type 2/chemically induced , Diabetes Mellitus, Type 2/metabolism , Diet , Glucose/metabolism , Glycogen/metabolism , Guinea Pigs , Hyperglycemia/chemically induced , Hyperglycemia/metabolism , Injections, Intramuscular , Insulin Resistance , Insulin-Secreting Cells/metabolism , Liver/metabolism , Male , Metabolic Syndrome/chemically induced , Metabolic Syndrome/metabolism , ProtaminesABSTRACT
A comparison has been made between spatial structures of human CYP2E1 obtained experimentally and those calculated using the computer methods. The structures were characterized by such parameters as total energy, protein pocket volume and total volume of molecules as well as the analysis of spatial geometry. The obtained results have proved that the model calculated and optimized by us can be used for studing the mechanisms of interaction of the active center of the enzyme with substrates and inhibitors. An assumption was made in the course of the research that one of the possible mechanisms of inactivation of the enzyme is the reduction of protein pocket volume, which prevents substrate access to the active center.
Subject(s)
Computer Simulation , Cytochrome P-450 Enzyme System/chemistry , Models, Chemical , Models, Molecular , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P450 Family 2 , Enzyme Inhibitors/chemistry , Humans , Ligands , Protein Binding , Protein Conformation , Substrate SpecificityABSTRACT
A computer model of human cytochrome P450 2E1 (CYP2E1) three-dimensional structure and active site was constructed based on homology with crystallographic coordinates of CYP2C5 and CYP2C9. A high degree of secondary structure homology for human, mouse, rat and rabbit CYP2E1 was demonstrated. The location of heme and the supporting alpha-helices was established. CYP2E1, CYP2C5 and CYP2C9 active sites are distinguished by pocket size and their amino acid residues composition. Key amino acid residues forming the active site channel and substrate-binding cavity are presented. Active site surface area and volume for CYP2E1, CYP2C5 and CYP2C9 were calculated.
