COMPARISON OF CELL CYCLE DURATION IN THE MONOLAYER CELL LINE OF HEPATOMA ZAJDELA AND ITS SUBLINES 3H AND 9C CULTIVATED IN VITRO.

We studied proliferative features of cells in monolayer line of rat hepatoma Zajdela (the original, parent line) and in the sublines 3H and 9C cloned from different types of the colonies of the parental line. These sublines also differed by cytomorphometric parameters, by the types of colonies formed at recloning of these cells in vitro, and by tumorogenicity at transplantation to a rat. Using a time-lapse video of native living cells, we analyzed the cell cycle duration (CCD) and its relationship to a cell shape. Direct measurement of the CCD (a time period from mitosis to mitosis) was performed in individual cells of non-synchronized cultures. Average value of CCD in the parent Zajdela line appeared to be 14.6 ± 0.2 hours, that was higher (at P < 0.05) than in 3H and 9C sublines (13.9 ± 0.2 and 13.5 ± 0.3 hours, respectively). The analysis of CCD distribution histogram showed that all three lines contained a common population of cells with CCD close to 14 hours. Besides, the parent cell line had about 1/3 of cells with a higher CCD (16.7 ± 0.2 hours) while the subline 9C had, on the contrary, 1/3 of cells with a lower CCD (12.6 ± 0.1 hours). In addition, the parameters of cell area, coefficient of cell spreading and coefficient of cell polarization showed the highest correlation to CCD in cells of subline 3H, which are primarily fibroblast-shaped cells (P < 0.01) : the larger the cell area, the longer the CCD; the more flattened or polarized the cell is, the shorter its CCD. In the parental cell line and the subline 9C, both consisting of preferably epithelium-shaped cells, the correlation between CCD and cell shape was less pronounced and showed the opposite direction, that may be explained by a difference in the origin of the cell lines. When considering the differences of CCD in the pairs of daughter cells, we introduce the concept of «the coefficient of symmetry of a cell division¼. The lower its value, the greater the similarity of CCD in a pair of daughter cells. Possible connection of the cell parameters studied in vitro to the tumorigenicity of these cells is discussed.

USING OF CELL LINE HepG2 FOR ASSESSMENT OF TOXIC AND METABOLIC EFECTS OF ANTIPSYCHOTIC DRUGS.

In order to study in vitro the toxic and metabolic effects of antipsychotic drugs (AP) on the cells of hepatic origin we used human hepatoblastoma cell line HepG2. We cultured HepG2 cells in the presence of two AP of the first and second generations (haloperidol and olanzapine, respectively) adding them to the culture medium in concentrations that may at the therapeutic use of AP take place in liver and other tissues of a high lipid content. In the process of cultivation, we detected several products of carbohydrate and lipid metabolism, measured activity of four hepatocellular enzymes in the culture medium, and estimated cell viability/proliferation in the MTS-test. We observed that both AP performed a toxic effect on HepG2 cells, the effect was manifested by a decrease in cell viability/proliferation and an increase in alkaline phosphatase activity in the culture medium. The toxic effect of olanzapine was less pronounced in comparison to haloperidol. According to the data from literature, AP upregulate the expression of lipogenesis genes in the cells of central nervous system, adipose tissue and liver, that might lead to hyperlipidemia. However, we observe in our experiments no increase in the levels of total cholesterol, of cholesterol in lipoproteins of high and low density, of triglycerides in the culture medium containing haloperidol or olanzapine. That observation may have been due to the fact that both AP, which are cationic amphiphiles, are capable to inhibit intracellular traffic of lipids. We also found no effects of haloperidol and olanzapine on the activity of aspartate aminotransferase and gamma-glutamyltransferase, while both AP did reduce the alanine aminotransferase activity. Our work proves that HepG2 cells can be helpful as an in vitro model to obtain new data on metabolic effects of drugs on the cells of hepatic origin and to assess the risk of a drug hepatotoxicity in preclinical studies.

[Retarded excision of pyrimidine dimers in human unstimulated lymphocytes].

Using immuno-labelling of cyclobutane pyrimidine dimers (CPDs) in nuclei of peripheral lymphocytes after their UVC-irradiation and cultivation, we have found that within the first four hours of cultivation the CPD-specific fluorescent signal from cell nuclei increased. Earlier, a similar increase in binding of antibody specific for pyrimidine (6-4) pyrimidone photoproducts to undenatured DNA isolated from UV-irradiated Chinese hamster ovary cells was reported (Mitchell et al., 1986). Our experiments showed that nucleotide excision repair enzyme might induce such of DNA modification in lymphocyte nuclei that increased specific antibody binding to DNA fragments with lesions. We suggest that enzymatic formation of open structures in DNA predominated qualitatively over dual-incision and excision of these fragments, and resulted in the enhanced exposure of the pyrimidine dimers in nuclei to specific antibodies. The results evidence that nucleotid excision repair in unstimualted human lymphocytes being deficient in dual incision and removal of UV-induced DNA lesions appear to be capable of performing chromatin relaxation and pre-incision uncoiling of DNA fragments with lesions.

Circulating leukocyte and cytokine responses to measles and poliovirus vaccination in children after ultraviolet radiation exposures.

Suberythemal ultraviolet radiation (UVR) exposures of children are used routinely in Russia to prevent rickets and to strengthen general health. The aim of the present study was to re-evaluate the effects of such a regime on immune responses as UVR is now recognised to suppress cell-mediated immunity in many animal models. Seventeen infants were immunised with attenuated measles and recall polio vaccines of whom 10 had been given a course of prophylactic UV exposures before the vaccinations. All the infants in the study developed an acute infectious conjunctivitis one week prior to the vaccinations and were convalescent at the time of the vaccination. They were bled on the day of the vaccinations and at several times thereafter to assess leukocyte percentages and plasma cytokine levels. On the day of the vaccinations, an active immune response was apparent. The UV-exposed children differed from the unexposed children by having a smaller percentage of natural killer cells and a higher percentage of CD25-positive cells. In the days following the vaccinations, the UV-exposed infants had a lowered percentage of total lymphocytes with increased percentages of monocytes, eosinophils, neutrophils and HLA-DR-positive cells as well as higher concentrations of plasma IL-1beta and IL-10 compared with the unexposed infants. There were no local or systemic clinical reactions to the vaccines in the UV-group while a moderate rise in temperature of three children in the unexposed group occurred. Thus the UV irradiations modulated leukocyte percentages and plasma cytokine levels following the vaccinations, perhaps through the activation of a T helper 2-like response.

[Stimulation of DNA repair in human cells damaged by UV and ionizing radiation with soluble growth factors of photomodified blood]. / Stimuliatsiia reparatsii DNK rastvorimymi faktorami fotomodifitsirovannoi krovi v kletkakh cheloveka, povrezhdennykh UF- i ioniziruiushchei radiatsiei.

Exposure of a small skin area of volunteers to UV light in 1 minimal erythemal dose is accompanied by rapid appearance in the circulating blood of soluble factors able to restore proliferation of X-ray-damaged autologous lymphocytes, to decrease frequency of chromosome breaks, and to stimulate unscheduled DNA synthesis. The appearance of such an activity in the blood can be also induced without skin irradiation. For this, one volume of a directly UV-irradiated blood is to be mixed in vitro with 10-fold volumes of intact blood, thus modeling the in vivo situation, when a small amount of transcutaneously UV-irradiated blood mixes with intact blood in the circulation. It has been found that the platelet-derived growth factor and epidermal growth factor, added at physiological concentrations to the culture medium, decrease chromosome break frequency in X-damaged cells.

Molecular dosimetry by flow cytometric detection of thymine dimers in mononuclear cells from extracorporally UV-irradiated blood.

UV-induced DNA damage in mononuclear leucocytes can be quantified by flow cytometry of fluorescence from a labelled monoclonal antibody that specifically binds to thymine dimers (T<-->T): specific fluorescence is already detectable after exposures of 1-2 J m-2 of 254 nm radiation and shows a linear relationship with dose. The distribution of UV fluences over an irradiated volume can thus be ascertained by measuring the UV-induced T<-->T loads of the individual cells from that volume. After irradiation of mononuclear cells in a phosphate buffer solution in a Petri dish, most cells showed a similar intensity of specific T<-->T fluorescence, forming a single sharp peak in the fluorescence histogram. This signifies an even distribution of fluences over the cells. It was noticed, however, that a variable minor fraction of mononuclear cells (usually less than 10%) could be resistant to immunostaining; this fraction was rejected from the calculation of the specific fluorescence. The flow cytometric technique was also applied to blood cells exposed in an ISOLDA device, which is in use in Russian clinics for UV irradiation of whole blood for therapeutical purposes. Only a small fraction of mononuclear cells in a sample of whole blood treated in ISOLDA acquired a detectable T<-->T load after exposure to lamps which emit predominantly either UVC or UVB light ((3.6 +/- 1.0)% and (1.8 +/- 0.4)% of all analysed cells respectively). This small fraction had received a large variation in fluences, resulting in differences in nuclear T<-->T loads by a factor of 200.(ABSTRACT TRUNCATED AT 250 WORDS)

[The relationship of the potassium transport function of human erythrocyte membranes to the structural state of the glycocalyx]. / O sviazi kaliitransportnoi funktsii membran éritrotsitov cheloveka so strukturnym sostoianiem glikokaliksa.

The two structural-functional state parameters of erythrocyte membrane in healthy donors and ischemic heart disease patients have been compared--the potassium exchange rate constant (x), and the quantity of Alcian blue sorption capacity of glycocalyx (y). The connection between the first parameter--x--and the other one--y--has been observed in cases, when the value of the latter experienced both the seasonal oscillations (the coefficient of lineal correlation was: r = -0.87, P less than 0.01) and the changes in the cause of ischemic heart disease (r = -0.72, P less than 0.01) and its treatment (the coefficients of correlation attitude of each of parameter changes were: eta xy = 0.83, P less than 0.01; eta yx = 0.52, P less than 0.05). The above connection may indicate the participation of membrane glycoproteins and glycolipids in a regulation of the cation transport function of the erythrocyte membrane.

[Trigger mechanisms of the therapeutic effects of autologous transfusion of UV-irradiated blood (membranotropic effect on erythrocytes and thrombocytes)]. / Puskovye mekhanizmy lechebnykh éffektov autotransfuzii UF-obluchennoi krovi (membranotropnoe deistvie na éritrotsity i trombotsity).

The article sums up numerous investigations of the authors on studying different parameters of membrane-dependent properties of erythrocytes and thrombocytes after UV irradiation of blood samples, mixing of irradiated and nonirradiated blood, transfusions of UV-irradiated auto blood. It was shown that membranotropic action of UV-irradiated blood and transfusion of UV-irradiated blood are responsible for rheological and hemostatic properties of blood.

[Functional and structural changes in the surface of human erythrocytes following irradiation with ultraviolet rays of various wave lengths. V. Modification of the glycocalyx in autotransfusions of UV-irradiated blood]. / Funktsionalnye i strukturnye izmeneniia poverkhnosti éritrotsitov cheloveka posle oblucheniia UF luchami raznoi dliny volny. V. Mofifikatsiia glikokaliksa pri autotransfuziiakh UF-obluchennoi krovi.

A study was made of the structural and functional state of the erythrocyte surface components of patients with ischemic heart disease, and of patients with ulcer disease during the treatment with UV-irradiated blood autotransfusions. The cytochemical and isoserological methods employed showed some structural disturbances in the state of erythrocyte, glycocalyx and its modification in the course of treatment. The clinical results of treatment correlated with these changes.

[Functional and structural changes in the surface of human erythrocytes after UV irradiation with rays of various wavelengths. IV. Changes in the physicochemical properties of autotransfused UV-irradiated blood]. / Funktsional'nye i strukturnye izmeneniia poverkhnosti éritrotsitov cheloveka posle oblucheniia UF luchami raznoi dliny volny. IV. Izmeneniia fiziko-khimicheskikh svoistv pri autotransfuziiakh UF-obluchennoi krovi.

Properties of erythrocyte surface were investigated for patients with ischemic heart disease in the course of treatment with the UV-irradiated blood autotransfusion (UVIBA). Application of methods of light-scattering, photometry and cytochemistry revealed rapid and significant changes in deformability and aggregation properties of the erythrocytes immediately following each UVIBA procedure, which was accompanied by considerable blood viscosity decrease.

[Functional and structural changes in the surface of human erythrocytes after irradiation by different wave lengths of UV rays. III. The immediate effect of the autotransfusion of UV-irradiated blood]. / Funktsional'nye i strukturnye izmeneniia poverkhnosti éritrotsitov cheloveka posle oblucheniia UF luchami raznoi dliny volny. III. Nemedlennyi éffekt autotransfuzii UF-obluchennoi krovi.

The autotransfusion of UV-irradiated blood (70-200 ml) results in the structural modification of cell surface in all the circulating erythrocytes of cardiological patients. The effect is registered within 1 hour after transfusion and involves some decrease in the distribution coefficient of erythrocytes registered in two-phase polymer system dextran-poly(ethylene glycol), which depends on membrane surface properties other than charge. This effect is suggested to be responsible for the main peculiarities of the therapeutic effect of UV-irradiated blood autotransfusion--high rate of appearance, prolongation and wide spectrum of the therapeutic action.