Visnyk of Dnipropetrovsk University. Biology, medicine

3-6

Authors: O. Kuzminov, D. Ostapiv, T. Alekhina

Antihistamines with active ingredients of loratadine and desloratadine are produced by Ukrainian pharmaceutical industry. According to the law, ther are assessed for their potential danger to human health and the environment, including alternative test objects. Evaluation has been carried out with regard to cytotoxic effect of pharmacological substances (loratadine and desloratadine) using the bull sperm suspension as test objects, standardized and highly sensitive to toxic substances. Sperm was divided into the control sample (dissolved by phosphate-buffered saline) and the investigated sample. Loratadine was added to phosphate-buffered saline in doses of 1/500 LD50 (12.3 mg), 1/100 LD50 (61.5 mg) and LD50 (6150 mg). Desloratadine doses were 1/500 LD50 (1.25 mg), 1/100 LD50 (6.25 mg) and LD50 (625 mg). Survival of spermatozoa was defined until termination of rectilinear forward movement in sperm intacted at +2…+5 °C. Respiratory activity (ex tempore) was defined in 1.0 ml thermostated cell (temperature of 38.5 °C) by polarography with the automatic registration of process flow by potentiometer; restorative activity was defined potentiometrically, using the open microelectrodes that were inserted in thermostated polarographic cell. Survival of spermatozoa in the sperm under the impact of loratadine in doses of 1/500 LD50 and 1/100 LD50 is respectively 136.0 ± 26.2 hours and 144.0 ± 19.6 hours. Adding LD50 dose of loratadine reduced survival to 112.0 ± 26.2 hours, which is lower than the control (160.0 ± 26.1 hours), respectively, by 10.0–15.0 and 30.0%. Loratadine reduces the respiratory activity of sperm: in the dose of 1/500 LD50 by 20.5%, in the dose of 1/100 LD50 – by 43.6%, and that of 100 LD50 – by 61.5% compared to the control. Restorative sperm activity under the impact of the loratadine reduced by 84.0% (dose of 1/500 LD50), 98.0% (dose of 1/100 LD50), 80.0% (dose LD50) compared to controls. The survival of spermatozoa in the sperm under desloratadine action in the dose of 1/500 LD50 is 144.0 ± 19.6 hours (10.0% reduction compared with the control – 152.0 ± 23.6 hours), in the dose of 1/100 LD50 – 136.0 ± 26.1 hours (15.0% reduction), in the dose of LD50 – 72.0 ± 19.6 hours (30.0% reduction). The intensity of oxygen consumption by sperm reduced by 16.2% when adding desloratadine in the dose of 1/500 LD50, by 38.8% in the dose of 1/100 LD50 and by 80.7% – in the dose of LD50, compared with the control. Inhibition of respiratory activity is accompanied by decreasing in the transport of protons and electrons from spermatozoa into the extracellular medium, that manifests decreasing of reconstructive activity when the dose of desloratadine is 1/500 LD50 (by 12.0%), 1/100 LD50 (by 40.0%) and LD50 (by 60,0%) compared with the control.

12-16

Authors: O. V. Ketsa, M. V. Zazulyk, M. V. Himchak

The effect of fat compositions with the varying ratio of polyunsaturated fatty acids (PUFAs) of families ω-3 and ω-6 on oxidation process intensity in microsomal fraction of rat liver has been investigated. The aim of the study was to investigate the level of markers of oxidative modification of lipids and proteins in microsomal fraction of rat liver. Fat components in the experiment diets were presented by sunflower oil, soybean oil and fish oil. Rats were fed using one of the fillowing 5 diets for the period of 4 weeks: 1) AIN-93 diet with 7% sunflower oil and fish oil, with the inclusion of linoleic acid, eicosapentaenoic acid and docosahexaenoic acid in the ratio of ω-6:ω-3 – 7:1 (control diet); 2) AIN-93 diet with 7% soybean oil, with the inclusion of linoleic acid and α-linolenic acid in the ratio of 7:1; 3) the diet containing only ω-6 PUFAs; 4) the diet containing only ω-3 PUFAs; 5) the diet without PUFAs. The fatty acid compositions of the diets were analysed by gas chromatography. We measured the primary and secondary lipoperoxidation products, proteins carbonyl derivatives and SH-groups of proteins. It was shown that inclusion of linoleic acid and α-linolenic acid in the ratio of 7:1 or ω-6 PUFAs into the animal diet increased lipid peroxidation in microsomal fraction of the rat liver as compared with the control group. Only ω-6 PUFAs increased the oxidative modification of proteins in microsomal fraction of the rat liver as compared with the control rat group. High dose of ω-3 PUFAs – eicosapentaenoic acid and docosahexaenoic acid had no influence on free radical oxidation of lipids and proteins. Using the diet without PUFAs increased oxidation process intensity in microsomal fraction of rat liver. According to our study, ω-6 PUFAs increased the oxidative modification of lipids and proteins in microsomal fraction of the rat liver. ω-3 PUFAs, in particular, eicosapentaenoic acid and docosahexaenoic acid, increased lipid and protein resistance to oxidative modification in microsomal fraction of the rat liver.

23-27

Authors: L. V. Yashchuk, N. V. Cherevach, A. I. Vinnikov

Cats and dogs kept at home, as well as other animals, are susceptible to infectious diseases caused by pathogenic microorganisms, including viruses. Viral infections in urban environments are extremely common and cause severe diseases in domestic animals which often lead to death, resulting in high material and moral damages to owners of animals. Therefore, investigation of the prevalence of pathogens of viral diseases is very important in our time. The aim of this work is to define the indicators of spreading viral diseases of animals in Dnipropetrovsk, to analyze the seasonal spread of viral infections in animals, and influence of sex and breed on the frequency of disease manifestations. Materials for research were obtained on the basis of three veterinary clinics in Dnipropetrovsk. During the study, we used serological methods of diagnostics of clinical materials, namely ELISA and IHA. Immunosorbent assay was performed using the thermostatic shaker ST-3 and strip immunoassay analyzer Stat Fax 303 Plus. Also we used commercial test systems VetExpert CAV Ag and Feline VacciCheck ImmunoComb®, based on the immuno-chromatographic method. During clinical studies of 491 animals (268 cats and 223 dogs) it was found that the most common respiratory viral infections in cats were calicivirus infection and rhinotracheitis, while in dogs there were viral diseases of gastrointestinal tract, i.e. enteritis and hepatitis. Using IHA method, we revealed the antibodies to respiratory viruses in the blood of deseased cats: to calicivirus – in 95 cats, to rhinotracheitis – in 60 cats; by ELISA method we identified antigens of parvovirus, enteritis pathogen, in biological material of 61 dogs, by IHA we found antibodies to virus of infectious hepatitis in 49 individuals. Based on these data, it has been revealed that during the winter months the animals suffered mostly the respiratory viral infections (60%), and in spring there were increased occur-rence of enteritis, observed in all age groups of animals. From May, hepatitis started to appear and prevailed for all summer months, mixed with enteritis (about 50% of total number of cases). Hepatitis was recorded both in cats and dogs. By the fall, recurrence was observed for calicivirus and herpes virus infections in cats, and the cases of enteritis decreased, while hepatitis was recorded until mid-October. Such statistics is kept relatively constant. The research results can be applied in the practice of veterinary laboratories for the development of diagnostic measures and prevention of viral diseases in domestic animals, as well as in the prediction of dissemination of viral infections in animals in the near future.

33-38

Authors: K. R. Kotsyuba, O. S. Voronkova, A. І. Vіnnіkov, T. M. Shevchenko

The paper deals with the basic medical scheme of antibiotics use for treatment of lesions caused by enterobacteria and mechanisms of resistance of Enterobacteriaceae to different classes of antibiotics. It is known that the main mechanisms of resistance to antibiotics are enzymatic inactivation, modification of the target, efflux, violation of conduct through the membrane and formation of metabolic shunt. The most common cases of resistance to beta-lactams among Enterobacteriaceae relate to production of plasmid and chromosomal beta-lactamases, violation of the permeability of the outer membrane, and modification of target penicillin binding proteins. Active release of antibiotics from the cell, or efflux, in Enterobacteriaceae is used for maintaining resistance to tetracyclines, macrolides, carbapenems. Genes of efflux system are localized on plasmids and contribute to rapid spreading among Enterobacteriaceae. Mutations are the basis of resistance to novobiocinum and rifampicinum. Enzymatic inactivation by modifying is typical for resistance to aminoglycosides. Three groups of enzymes are engaged in the process, by adding the molecule of acetic acid, phosphate or adenine. Joining of these groups is irreversible and leads to complete loss of biological activity of the antibiotic. Resistance to aminoglycosides appears also due to inhibition of drug penetration, that is associated with genetically determined mechanisms of electron transport through the membrane. Resistance to quinolones and fluoroquinolones is associated with the modification of topoisomerase II and IV which are targets of these groups of antibiotics. Resistance is possible as a result of changes in the structure of the target, breaching of penetration into the cell, and active release from the cell. The highest level of resistance is develope in the case of two- or three-stage mutations in one or the other, or both, subunits in different genes. At the same time, for breaching of the bacterial cell it is enough to suppress the activity of only one enzyme associated with different functions of both topoisomerases. Another mechanism of resistance to quinolones is the reduction of permeability through bacterial outer membrane, that it’s possible due to decreasing of permeability of porine channels. In this case, decreasing of permeability efficacy takes place not only for quinolones, but also for other classes of antibitics. In addition, reduced sensitivity to quinolones efflux may play the significant role. For clinical strains of microorganisms, cross-resistance to various drugs, associated with simultaneous production of various enzymes that inactivate antibiotics, is typical.

45-48

Authors: K. V. Bubalo, L. P. Golodok, A. I. Vinnikov

The frequency of urogenital mycoplasmas detection in women of different ages was studied in culture with the help of DUO test-system in order to determine their etiological significance in the development of inflammatory processes of women urogenital tract. We identified the researched cultures Mycoplasma hominis, Ureaplasma urealyticum in the diagnostic titer >104 TEM/ml indicating severe contamination by microorganisms, and in the titer <103 TEM/ml, the carrier state of the identified microorganisms. Of 120 studied isolates of women urogenital tract there have been identified 113 strains of genital mycoplasmas, among which 63% – U. urealyticum, 32% – M. hominis, 3% – microbial association of U. urealyticum – M. hominis. According to the study of frequency of detection of urogenital mycoplasma using DUO test-system culture method, it was found that the most frequently observed ones were U. urealyticum in 75 women (63%) of all individuals, M. hominis in 38 women (32%) in different diagnostic titers (>104 TEM/ml, <103 TEM/ml) in 4 women (3%) U. urealyticum – M. hominis was observed in microbial associations and mycoplasma were not found in 3 women (2%) of all surveyed patients. U. urealyticum and M. hominis in the diagnostic titer of >104 TEM/ml was observed in 55 women (46%) and 20 women (17%), respectively, and the titer of <103 CFU/ml U. urealyticum was observed in 20 women (17%), and M. hominis in 18 women (15%). Analysis of genital mycoplasmas distribution among women of different ages has shown that there was the certain correlation between the patient age and frequency of genital mycoplasmas detection: the highest detection rate was observed in women age of 24–29. The dominant pathogen of urogenital tract inflammatory processes in women in 24–29 age group is U. urealyticum. The comparison of DUO test-system and PCR data has shown that DUO test-system in culture allowed more sensitive quantitave characterization of mycoplasmas, however, for the more effective laboratory diagnostics it was necessary to use complex methods to increase the probability of pathogen detection. Incidence of mycoplasmas in women with the presence of inflammation was higher than in women having the inflammation in the genital tract. In this case, potential symptom-free carriers exist for the development of inflammation of urogenital tract of women. Scientists have proved that mycoplasma could cause vulvovaginitis, urethritis, paraurethritis, bartholinitis, adnexitis, salpingitis, endometritis, and ovaritis.

54-57

Authors: I. A. Gordiienko

MMP2 and MMP9 matrix metalloproteinases due to their ability to destroy basement membranes collagen and remodeling extracellular matrix (ECM) in the micro-environment of blood progenitor cells in the bone marrow play the important role in hematopoiesis. Displacement of normal hematopoiesis and dissemination of malignant cells in proliferative diseases of blood is also accompanied by catalytic ECM rearrangement. However, it is not known exactly how activity of MMP2 and MMP9 changes in various forms of leukemia and how it is affected by chemotherapeutic drugs. The aim of this study was to determine the influence of anthracycline antibiotics (daunorubicin and adriablastin) on MMP2 and MMP9 activity in blood plasma of patients with acute myeloid leukemia, chronic lymphocytic leukemia and multiple myeloma. It was established that proMMP9 activity was significantly reduced (0,03 ± 0,01 rel. u.) in patients with acute myeloid leukemia before the treatment, however, after chemotherapy, it increased approximately 7 times. Chronic lymphocytic leukemia and multiple myeloma were accompanied by significant increase of MMP9 activity. Application of daunorubicin led to decrease of proMMP9 activity (0,25 ± 0,10 rel. u.) in patients with chronic lymphocytic leukemia. ProMMP9 activity was significantly reduced (16 times) and that of MMP9 increased in case of multiple myeloma. In studying of MMP2 activity it did not significantly change. The conclusion is that the ratio of proMMP9/MMP9 can be used as the additional criterion for monitoring the effectiveness of chemotherapy of proliferative diseases of blood.

62-65

Authors: H. V. Sukharenko

The modern ecology situation in waters of the Kerch Strait requires assessment of disturbances in biotopes and monitoring of the degree of impact of industrial pollutants on ecosystem. Deposit of oil products after the 2007 year ships’ accidents might have considerable impact on the water biocenosis area. The investigation of cytoskeleton marker of astrocytes glial fibrillary acidic protein (GFAP) in brain of the bullhead (Neogobius fluviatilis), which is the typical representative of the commercial ground fish of the Kerch Strait, has been carried out. The results of comparative analysis of GFAP content in the brain of fish from the Kerch Bay near-shore waters and fish from conditionally clear area of Vorskla river shows the reliable (2.18 times) increasing of GFAP in the area of industrial pollution. Rising GFAP content indicates the astrogliosis development as a result of metabolic disturbances which can be induced by higher content of oil products in the near-bottom biotopes of the Kerch Bay. Increase in lipid peroxidation level was observed in the brain of fish from the Kerch Bay. The results provided with regard to violations of the state of astrocyte cytoskeleton and oxidative stress in the brain of bullhead from the Kerch Bay prove the sublethal biology effect of industrial pollutants in hydrobionts from this area. Results of this investigation also indicate the necessity of continuous ecology monitoring and comprehensive study of hydrobiont populations in the industrial regions and ecological disaster zones.

71-78

Authors: O. V. Severynovska, O. O. Galinskij, A. I. Rudenko, O. B. Mursin, V. V. Babicheva, L. D. Skubytska

Nitric oxide is the main inhibitory mediator of the gastrointestinal smooth muscles’ relaxation which stimulates duodenal mucus and bicarbonate secretion. More recent studies have demonstrated that NO also protected the gastrointestinal tract by inhibiting gastric acid secretion. In this study we investigated gastric secretory and motor activity considering the NO imbalance condition. The experiments were carried out on male white laboratory rats (200–230 g). The control group was treated with 0.9% NaCl solution. The injections of NO donator (1.5 mg/kg of 0.1% (Sigma-Aldrich) sodium nitroprusside solution) were made in the second and the third groups during 6 and 12 days. The fourth and the fifth group were treated with NO synthesis inhibitor (40 mg/kg of 1% solution (Sigma-Aldrich) Nω-nitro-L-arginine) during 6 and 12 days respectively. Recording of gastric myoelectric activity (GMA) and gastric juice collection were carried out under anesthesia (ketamine, 110 mg/kg). Next, its volume, pH, glycoprotein and pepsin were measured. Within 6-days stimulation of NO excess decreased gastric juice volume by 47% and increased pH compared to control samples. Pepsin level increased by 62% and glycoprotein level decreased by 68% compared to the checkpoint. After 6 days of L-NNA injections we observed the increase of gastric juice secretion volume (78%) and pH level, however, pepsin concentration remained unchanged. Glycoprotein level increased by 21% compared to control samples. After 12 day NO synthesis inhibitor injections gastric secretion volume increased by 85%. Gastric juice pH level was 200% higher than the control value and exceeded gastric juice pH level (62%) in the third group. In addition, pepsin level tended to decrease when NO deficiency simulation was prolonged. Glycoprotein level decreased by 41% compared to control samples and by 51% compared to the third group. Pepsin level decreased after 12 day NO-inhibitor injections as gastric juice pH level increased. After 12 day Na-nitroprusside treatment, gastric myoelectric index decreased by 42% compared to the checkpoint. The type of contractions is typical to the I phase of the basic electrical rhythm (BER). Also, retrograde entrainment of duodenal rhythm took place. After 6 day L-NNA injections, GMA was the same as the period between II and III phase of BER. Late second and early third phases predominated, while the I phase wasn’t detected during the recording. In case of long-lasting disorder of NO-system, motor index value significantly differed from the control group samples and depended on the duration of NO-synthase blocking. On the 6th day of L-NNA treatment, the level of motor index increased 1.5 times. GMA phases could be identified only on the basis of motor index values. Stomach’s own rhythms disappeared and duodenogastric reflux resulted in domination of duodenal rhythms. After 12 day L-NNA injections, duration of the II and III phases increased while the I phase of gastric BER disappeared; gastric motor index decreased by 27%. After considering the results of the current investigation, it can be stated that NO system imbalance leads to desynchronization of gastric active phases and, perhaps, reassigns the influence of different compensatory-adaptive mechanisms. The longer is the L-NNA treatment, the more gastric functions are imbalanced and the stronger is the process of destruction.