[Cyclosporine A fails to prevent thymocyte death induced by ionizing radiation and hydrocortisone]. / Tsiklosporin A ne predotvrashchaet gibel timotsitov, indutsirovannuiu oblucheniem i gidrokortizonom.

In experiments with albino mongrel mice the influence of cyclosporine A on thymocyte death in vivo under the effect of ionizing radiation and hydrocortisone has been investigated. The effect was assessed by the amount of soluble polydeoxynucleotides (PDN) that are formed in the cells. Dose dependence of the PDN yield and kinetics of their formation in thymocytes as well as electrophoretic mobility of PDN were studied after irradiation and the cyclosporine A treatment. Cyclosporine A was shown to have no effect on the thymocyte death induced by irradiation in vivo. The same results were obtained with hydrocortisone. Possible mechanisms of action of cyclosporine A on death of lymphocyte and proliferating cells are discussed.

The use of flow cytometry for the investigation of cell death.

Flow cytometry is more and more widely used for investigations of cell death, predominantly in the study of DNA degradation in cells dying by apoptosis. There are different interpretations of changes observed in DNA histograms of these cells. We describe an approach based on extraction of chromatin degradation products from fixed cells and subsequent staining with DNA specific dyes. Apoptotic cells containing fragmented DNA are observed in < 2C DNA region of DNA histograms. DNA histograms of irradiated thymocytes dying in vitro and stained without extraction of fragmented DNA do not differ from control. Under the same staining conditions DNA histograms of lymphocytes dying in thymus of irradiated animals reveal fluorescent material in < 2C DNA region, most likely due to formation of apoptotic bodies (cell fragments, some of them contain fragments of nuclei). Similar changes are observed in thymocytes dying upon glucocorticoid treatment. Our present results and other data indicate that reduced amount of DNA in dying cells is the main reason for changes of DNA histograms. Examples of application of the method described for the investigations of cell death modifiers are presented.

[D2O inhibition of interphase thymocyte death]. / Ingibirovanie D2O interfaznoi gibeli timotsitov.

Using the method of flow cytometry and biochemical analysis it was shown that D2O, an agent that stabilizes microtubules, prevented the internucleosome fragmentation of DNA in thymocytes exposed to gamma radiation and dexamethasone in vitro. It was also found that D2O is ineffective with respect to Ca2+/Mg2(+)-dependent nuclease. The transfer of irradiated cells from a medium containing 90% of D2O to a normal one caused rapid DNA degradation; the fragmentation process ceased with the irradiated cells being transferred from H2O to heavy water. The results obtained permit us to assume that the disturbance of microtubules is not a trigger mechanism of DNA degradation by apoptosis, but is some intermediate stage of cell death preceding the chromatin fragmentation proper.

[General similarities and differences in the postradiation death of various animal species and of man]. / Obshchie zakonomernosti i razlichiia postradiatsionnoi gibeli limfotsitov raznykh vidov zhivotnykh i cheloveka.

It was found in various animal species and man that an ordered internucleosome fragmentation of DNA is characteristic of lymphoid cells dying in the interphase. Both in vivo and in vitro, the postirradiation DNA degradation in thymocytes of rodents and piglets preceded the increase in the permeability of their plasma membrane. The in vivo kinetics of death of lymphoid cells from the thymus and spleen is similar in rodents and piglets. Rat thymocytes died in vitro earlier than thymocytes of piglets, calves and man which was evidently associated with a worse adaptive capacity of the latter to cultivation conditions.

[The patterns of colchicine-induced death of lymphoid cells]. / Zakonomernosti indutsirovannoi kolkhitsinom gibeli limfoidnykh kletok.

It is shown that colchicine injection at doses higher than 1 mg/kg of animal weight induces cell death in thymus, spleen, bone marrow and intestine mucosa. The cell death is accompanied by a regular internucleosomal cleavage of nuclear DNA and by the elimination of the formed fragments from cells. Both the processes begin after a 1.5 hour lag-period and proceed before the outer membrane permeability for supravital dyes increases. DNA degradation is prevented by the inhibitor of protein synthesis cycloheximide. Cytochalasin B does not induce chromatin degradation or cell death and has no effect on radiation death of lymphocytes. A possible role of microtubule destruction as a switch-on mechanism of DNA degradation and cell death is discussed.

The effects of prenatal exposure to methylmercury on aggressive behavior in the rat.

The sensitivity of isolation-induced aggressive behavior to prenatal treatment of 6.0 mg/kg methylmercuric chloride (MMCl) by gavage on gestation days 6-9 was assessed in a subset of animals from the Collaborative Behavioral Teratology Study (CBTS). CBTS behavioral measures consisted of negative geotaxis, olfactory discrimination, auditory startle habituation, 1-hr activity, 23-hr activity, activity following pharmacological challenge and visual discrimination learning. Auditory startle was the only CBTS behavioral measure that discriminated among prenatal treatment groups. MMCl animals also were reliably more aggressive than vehicle controls in dyadic encounters. The results suggest that tests of aggressive behavior, which rarely have been included in teratologic assessments, be considered in the formulation of behavioral screening paradigms. The advantages of including tests of aggression and the relationship between aggressive and startle responses are discussed.

[Determination of the radiosensitivity of interphase-dying cells in the thymus, spleen and bone marrow of rats by flow cytometry]. / Opredelenie radiochuvstvitel'nosti interfazno gibnushchikh kletok timusa, selezenki i kostnogo mozga krys metodom protochnoi tsitometrii.

The flow cytofluorometry of cells stained with a DNA-specific probe was used to determine the share of dying cells (containing less than 2C DNA) in thymus, spleen and bone marrow cells of irradiated rats. The cell death curves for spleen and bone marrow had a plateau by the 6th h, and for thymus, by the 10th h following irradiation with different doses. On the basis of the dose-response relationship the share of cells dying in the interphase was determined in each organ under study, and dose-response curves shaped. All the curves had no shoulder. Do was 3.0, 3.0 and 3.7 Gy for thymus, spleen, and bone marrow cells, respectively.

[Quantitative analysis of the effect of cysteamine on interphase cell death by flow cytometry]. / Kolichestvennyi analiz vliianiia tsisteamina na interfaznuiu gibel' kletok metodom protochnoi tsitometrii.

The method of flow cytofluorometry was used to study the radioprotective effect of cysteamine on cells dying in the interphase. DMF was 1.67, 1.67, and 1.76 for thymus, spleen, and bone marrow, respectively.

Flow cytometry analysis of DNA degradation in thymocytes of gamma-irradiated or hydrocortisone treated rats.

The pattern of DNA degradation in thymocytes of irradiated or hydrocortisone-treated rats has been studied by means of flow cytometry of the cells, treated with probes specifically bound to the AT or GC-pairs of DNA. It has been shown that the death of thymocytes is accompanied by a decrease in their DNA content. The main features of the occurrence and accumulation of cells with a DNA content less than the normal diploid level correspond with those of internucleosomal DNA fragmentation: such cells appear after a 1 hour lag-period, their accumulation is prevented by cycloheximide injection and is lower at 300 Gy than at doses of 10 to 30 Gy. At the same time, no increase in permeability of the cell membrane to ethidium bromide was observed up to the sixth hour after irradiation. Most of the thymocytes dying under the action of irradiation or hydrocortisone are in the G0 or G1 phases of the cell cycle. The method used allows detection of the cells with cleaved but not removed DNA.

Flow cytometry and biochemical analysis of DNA degradation characteristic of two types of cell death.

By flow cytometry it has been shown that in rat thymocytes dying upon gamma-irradiation the reduction in DNA content per cell occurs well before the increase in outer membrane permeability. In contrast, in irradiated Burkitt's lymphoma cells the disturbance of plasma membrane permeability precedes the decrease of DNA content. Genome degradation in dying thymocytes of irradiated or hydrocortisone-treated rats is accounted for by internucleosomal DNA fragmentation. Postirradiation DNA cleavage in the 3 investigated cell cultures is disorderly and is probably caused by the activation of hydrolases in dead cells.

Collaborative Behavioral Teratology Study: results.

Behavioral measures used in the Collaborative Behavioral Teratology Study (CBTS) were negative geotaxis (PNDs 7-10), olfactory discrimination (PNDs 9-11), auditory startle habituation (PNDs 18-19 and 57-58), 1-hr activity (PNDs 21, 60, 100 and 120), 23-hr activity (PND 100), activity following a pharmacological challenge (PND 120), and an operant, discrete trial visual discrimination task. Maternal and offspring body weights and the appearance of certain physical landmarks of development were also monitored. The design of the CBTS allowed evaluation of the reproducibility and detection sensitivity of these behavioral test methods, as well as the impact of early testing experience on later behavioral assessment, offspring sex differences in response levels and variability, and the contribution of litter-to-litter and animal-to-animal variation to behavioral measures in a standardized test protocol. The results obtained in this test system are discussed in relation to each of these factors and to the degree of overt toxicity obtained using prenatal treatment with 0, 0.5 or 2.0 mg/kg d-amphetamine sulfate, SC, on gestation days 12-15 (Study 1) or methylmercuric chloride, 0, 2.0 or 6.0 mg/kg by gavage, on gestation days 6-9 (Study 2).

[DNA degradation during radiation death of cultured cells]. / Zakonomernosti degradatsii DNK pri radiatsionnoi gibeli kletok v kul'ture.

The method of flow cytofluorometry was used to study postirradiation changes in plasma membrane permeability and DNA content of the Burkitt's lymphoma cells (Raji line). At a dose of 10 Gy, the increase in membrane permeability preceded the appearance of cells with diminished DNA content. The synchronization of cells in phase G2 was associated with a virtually complete radiation arrest of mitoses. The postirradiation electrophoretic analysis of DNA of the Burkitt's lymphoma cells and of Syrian and Chinese hamster fibroblasts showed that the DNA degradation is unordered and results perhaps from activation of hydrolases in the dead cells.

[Internucleosomal fragmentation of nuclear DNA in the mucosa of the small intestine and in bone marrow cells of irradiated rats]. / Mezhnukleosomnaia fragmentatsiia iadernoi DNK v slizistoi tonkogo kishechnika i kostnogo mozga obluchennykh krys.

DNA was isolated from thymus, bone marrow and small intestine epithelium of gamma-irradiated rats and analyzed by electrophoresis in 3% polyacrylamide gel - 0.5% agarose. The internucleosomal chromatin fragmentation was observed in all tissues investigated. In the thymus and bone marrow, DNA fragmentation increased to a maximum by the 6th hour, and in the small intestine, by the 4th hour following irradiation. No low molecular weight fragments were found in the organs under study by the 16th hour after irradiation.

[Analysis of DNA degradation in the thymocytes of irradiated rats by flow cytometry]. / Analiz degradatsii DNK v timotsitakh obluchennykh krys metodom protochnoi tsitometrii.

The method of flow cytofluorometry of cells treated with probes specifically bound to AT- or GC-pairs of DNA was used to study DNA degradation in thymocytes of irradiated and hydrocortisone-treated rats. Death of thymocytes was shown to be accompanied by the decrease in the DNA content. The main regularities in the formation and accumulation of cells, the DNA content of which being lower than that of diploid cells, were the same as those of the internucleosome DNA fragmentation.

[Densitometric determination of nucleic acids in gels after electrophoresis]. / Densitometricheskoe opredelenie nukleinovykh kislot v geliakh posle élektroforeza.

A technique for the quantitative determining of nucleic acids after electrophoresis by double-wave densitometry in UV spectrum by scanning agar gel along both axes was developed. Optical characteristics of agar and acrylamide-agar gels are given. The range of quantities measured is 6 divided by 30 micrograms of DNA per gel; the standard deviation adjusted to 1 microgram is less than +/- 3%.

[Comparison of chromatin degradation products formed in rat thymus cells under the effects of irradiation and hydrocortisone injection]. / Sravnitel'nyi analiz produktov degradatsii khromatina, obrazuiushchikhsia v kletkakh timusa krys pod vozdeistviem oblucheniia i vvedeniia gidrokortizona.

The death of rat thymocytes after irradiation or hydrocortisone injection is accompanied by degradation of DNA. DNA degradation in the thymocytes of irradiated animals starts after a 2-hr lag, reaches its maximum by the 6th hour and remains constant between the 6th and 10th hours. The accumulation of chromatin fragments after hydrocortisone injection starts without a pronounced lag period and reaches its maximum (approximately equal to 15%): by the 9th-10th hour. The amount of degraded DNA is correlated with the number of cells with psychotic nuclei. The chromatin degradation products in rat thymocytes after irradiation or hydrocortisone injection are nucleosomes and their oligomers. The number of intranucleosomal breaks in DNA is negligibly small. In vivo experiments with inhibitors of RNA and protein synthesis in the system described have demonstrated that chromatin degradation depends on protein synthesis and is almost independent of RNA synthesis. Thus, the molecular mechanisms of nuclear DNA degradation in dying thymocytes appear to be similar and independent of the nature of the agents used.

[DNA degradation in vivo in dying thymocytes, model for the study of chromatin structure]. / Degradatsiia DNK in vivo v gibnushchikh timotsitakh--model' dlia izucheniia struktury khromatina.

Properties of chromatin fragments which are formed as a result of in vivo DNA degradation in thymocytes of gamma-irradiated rats were studied. It is shown that under these conditions chromatin breakdown is accounted for by internucleosomal DNA scission without proteolysis and disturbance of nucleosome structure as judged from how they are split by DNAase I. Amount of mononucleosomes and their dimers, trimers and tetramers is in average about 10% of the fragments formed, and that of oligomers composed of about 8--10 nucleosomes equals about 80%. Core-particles and subnucleosomal fragments are never observed. Chromatin fragments of different size have been purified and their composition was studied. Electrophoresis in 3% PAAG--0.5% agarose divides mononucleosomes into two fractions. The less mobile fraction contains all histones, non-histone proteins and DNA fragments of 178 base pairs long. More mobile mononucleosomes do not contain at least fragments of 178 base pairs long. More mobile mononucleosomes do not contain at least one of the histone H1 subfraction and the size of their DNA is 168 base pairs. Chromatin fragments of different size contain both similar and different non-histone proteins. In mononucleosomes and their dimers, trimers and tetramers proteins are found which are absent in chromatin fragments of high molecular weight. It is assumed that mononucleosomes and their dimers, trimers and tetramers are products of active chromatin degradation and that high molecular weight oligomers are supernucleosomal structures of inactive chromatin.

In vivo DNA degradation in thymocytes of gamma-irradiated or hydrocortisone-treated rats.

Gamma-irradiation or introduction of hydrocortisone bring about degradation of nuclear DNA in rat thymocytes. The chromatin degradation products were extracted from purified nuclei by 0.7 mM EDTA. The quantity of low molecular weight chromatin fragments formed 6 h after irradiation increases up to the doses of 3 Gy, then remains constant up to 30 Gy and decreases at doses 100 to 300 Gy. Whatever the irradiation dose, DNA degradation starts after a 2-h lag, reaches a maximum by the 6th hour and remains constant between the 6th and 10th hours. The quantity of chromatin fragments formed coincides with the number of cells with pycnotic nuclei. The chromatin fragments present nucleosomes and their oligomers with a normal histone content and an intact structure, as judged from how they are split by DNAase I. The number of intranucleosomal breaks in DNA is negligible. DNA fragmentation is not accompanied by degradation of histones and nonhistone proteins of chromatin. Hence, DNAase I and proteases are not involved in degradation of chromatin. The ratio between mononucleosomes and oligomers of different lengths does not depend on the dose and the time after irradiation. The quantity of DNA degraded is determined by the number of dying cells in which all DNA is fragmented rather than the degree of chromatin degradation over the whole thymocyte population. Hydrocortisone-induced degradation of chromatin in rat thymocytes occurs similarly. A possible role of chromatin degradation in cell death is discussed.