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Biomedical subjects

G Speit

Publications and source records attributed to G Speit.

At least 91 records · Page 5Linked to original sources
PubMed1985

On the mechanism of differential Giemsa staining of bromodeoxyuridine-substituted chromosomes. II. Differences between the demonstration of sister chromatid differentiation and replication patterns.

Experiments were performed to find out whether different mechanisms are involved in FPG-(fluorescent plus Giemsa) staining for the demonstration of replication patterns and sister chromatid differentiation (SCD) after bromodeoxyuridine (BrdU)-substitution of V79 Chinese hamster chromosomes. The influence of variations of the staining procedure on the quality of both SCD and replication patterns was comparatively investigated and differences in the demonstration of these two phenomena within the same chromosome were studied using various BrdU-labeling protocols. The results show that at least graduated differences exist. For a good differentiation of replication patterns a stronger FPG-treatment is necessary than it is for SCD. Partial BrdU substitution only leads to replication patterns in the next mitosis. A further round of replication either in the presence or absence of BrdU causes a reduced staining of the complete chromatid and three-way differentiation is seen in third generation mitoses. These results support the view that alterations of chromosomal proteins during BrdU-incorporation and replication of BrdU-substituted DNA are decisive for differential staining.

Animals↗
PubMed1985

Do the frequencies of sister chromatid exchanges in endoreduplicated mitoses provide a measure for lesion persistence and repair?

Endoreduplication was induced in V 79 cells using Colcemid. The concentration of Colcemid necessary to induce endoreduplication is about 1000 times higher than that needed to arrest mitoses or to induce ordinary tetraploid cells. Diplochromosomes with sister chromatid differentiation were obtained by adding BrdU for the duration of one cell cycle prior to the induction of endoreduplication. The induction of endoreduplication with Colcemid had no influence on the frequency of sister chromatid exchanges (SCEs). Treating the cultures with mitomycin C (MMC) before adding BrdU increased the percentage of endoreduplicated mitoses and also led to marked SCE induction. In the diplochromosomes, the frequencies of both twin SCEs (first cycle) as well as single SCEs (second cycle) were increased. It was also found that the SCE frequencies in mitoses after endoreduplication were lower than the values found in diploid and ordinary tetraploid metaphases of the same preparation. The possible conclusions concerning the lifetime of SCE-inducing lesions and the influence of repair processes are discussed.

Animals↗
PubMed1985

Characterization of chromosome replication during S-phase with bromodeoxyuridine labelling in Chinese hamster ovary and HeLa cells.

Chinese hamster ovary (CHO) and HeLa cells were successively pulse labelled at 1-h intervals after the cultures were synchronized at the end of G1 (monitored by flow cytometry). The metaphases analysed afterwards showed R-type replication patterns after 1-h pulses during the early S-phase (SE; from h 1-5 after release) and replication of G- and C-bands in late S-phase (SL: from h 6-8 after release). The transition from SE to SL is abrupt, constituting a sudden switch of replication between different types of chromatin as has been described for human lymphocytes. The differences between these two cell lines and earlier results reported on a V79 Chinese hamster cell line and on normal diploid human and Chinese hamster fibroblasts are discussed.

Animals↗
PubMed1985

Characterization of mitoses with sister chromatid differentiation (SCD) and consequences for the analysis of proliferation kinetics and sister chromatid exchanges in asynchronously growing cells.

Labeling cells with bromodeoxyuridine (BrdU) permits the differentiation of mitoses of the first, second, and third generation after the addition of BrdU. The term "second mitoses" is used for those cells which have incorporated BrdU for two S-phases and which exhibit sister chromatid differentiation (SCD). However, SCD can also be obtained if the cell was in S-phase at the time of BrdU-addition and had already replicated part of its DNA. Such cells with incomplete BrdU-substitution in the first S-phase can only be differentiated from completely substituted ones by the quality of the SCD and are usually also grouped as "second mitoses" in the evaluation of experiments. Due to the heterogeneity of the evaluated "second mitoses", the determination of proliferation delay and the incidence of sister chromatid exchange-induction can depend on the time of chromosome preparation.

Animals↗
PubMed1985

Effect of diethylstilbestrol on the frequencies of sister chromatid exchange in vitro and in vivo.

The influence of diethylstilbestrol (DES) on the SCE frequency was studied in various cell systems (V79, HTC, DON, WI-38, human lymphocytes) differing in their ability to metabolize the two promutagens benzo(a)pyrene and cyclophosphamide as well as after incubation with exogenous metabolic systems (S9 mix and peroxidase/H2O2) and in vivo on cells from the bone marrow of the Chinese hamster. DES concentrations from 10(-6) to 10(-4) M were tested. Slight SCE induction was observed only at the highest concentration (10(-4) M) of DES in human lymphocytes treated with DES for the entire culture period of 72 h and in DON cells. No increase of the SCE rate was determined in the other cell lines and in vivo. The combination with S9 mix or peroxidase/H2O2 also had no influence on the SCE frequency. These findings cast doubt on the assumption that DES is metabolized to a DNA-damaging compound subsequently leading to SCE induction. The positive findings in the SCE test are more likely to be products of an indirect and rather unspecific effect.

Animals↗
PubMed1984

Different effects of mutagens on sister chromatid exchange induction in three Chinese hamster cell lines.

The sister chromatid exchange (SCE) induction of mutagens with different mechanisms of action was comparatively investigated on permanent cell lines of the Chinese hamster (CHO, V-79, and DON) with and without exogenous metabolic activation and with the use of various experimental protocols. CHO and V-79 cells were treated with ethylmethanesulfonate (EMS), a direct mutagen; with the two indirect mutagens cyclophosphamide (CP) and benzo[a]pyrene (BP); as well as with the radical former hydrogen peroxide (H2O2) and hydroxyurea (HU), an inhibitor of DNA synthesis. Aside from an increased basal SCE level and a higher bromodeoxyuridine (BrdUrd) sensitivity, there was no decisive difference between CHO and V-79 cells. However, there was a distinct relationship between SCE induction and the experimental protocol used, which was most pronounced after HU treatment. Neither cell line was able to metabolize the indirect mutagen BP. Only in CHO cells did CP lead to increased SCE frequencies. However, in all cases, the simultaneous application of S9 mix produced a distinct SCE induction. In contrast, BP caused SCE induction in DON cells, whereas CP was not metabolized. The reason for these findings must obviously be sought in the metabolization of CP and BP via different monooxygenase systems, whose activity can differ in these permanent cell lines. One notable finding was that the number of SCE induced by H2O2 could be distinctly reduced by the simultaneous application of S9 mix. This effect can be explained by the fact that S9 mix contains H2O2-degrading enzymes. The results indicate that closely related cell lines differ in their capability for inducing SCE and that investigations of SCE inductions performed on only one cell line do not necessarily produce a representative response.

Animals↗
PubMed1984

Sister chromatid exchange (SCE) induced by laser-UV-microirradiation: correlation between the distribution of photolesions and the distribution of SCEs.

Small, medium, and large nuclear areas comprising approximately 5, 30, or 80% of the total area of the interphase nuclei of Chinese hamster cells (M3-1) cultivated in vitro were irradiated with a laser-UV-microbeam of wavelength 257 nm. The DNA of the cells was substituted with 5-bromodeoxyuridine (BrdUrd) for 1 cell cycle in one set of experiments. After microirradiation the cells were grown for a second cycle in medium without BrdUrd (protocol A). In a second set, cells with nonsubstituted DNA were microirradiated and grown for 2 additional cycles, the first in the presence, the second in the absence of BrdUrd (protocol B). In situ chromosome preparation and differential chromatid staining was subsequently performed. The induction of sister chromatid exchanges (SCEs) was found to be dependent on both the ultraviolet (UV) dose and the spatial distribution of the UV energy within the cell nucleus. Following both protocols the average number of chromosomes with SCEs was significantly higher after microirradiation of a large nuclear area as compared to microirradiation of a small nuclear area. In the latter case, multiple SCEs were noted on individual chromosome arms at the first postirradiation mitosis (protocol A). In other cells, especially at higher doses, protocol A resulted in shattering of a few closely neighbored chromosomes which were surrounded by intact ones with normal SCE levels. Microirradiation of medium-sized nuclear areas produced high levels of SCEs over a number of chromosomes which still appeared spatially related in a part of the metaphase spread. Finally, high SCE levels could be observed over most or all chromosomes when a large nuclear area (up to 100%) was exposed to the microbeam. Following protocol B the increase of SCEs was much less pronounced. Microirradiation of a small part of the cytoplasm in addition to the nuclei did not induce SCEs. Our results support the concept (i) that interphase chromosomes occupy distinct nuclear domains and indicate (ii) that the induction of SCEs by UV light is restricted to microirradiated chromatin.

Animals↗
PubMed1984

The contribution of DNA single-strand breaks to the formation of chromosome aberrations and SCEs.

The induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) with bleomycin (BLM), hydrogen peroxide (H2O2), short-wave ultraviolet (UV)-irradiation, and long-wave UV-irradiation was investigated in V79 cells with BrdUrd-substituted DNA. The application of a Neurospora endonuclease (NE) which specifically cleaves single-stranded DNA after these treatments showed that DNA single-strand breaks (SSBs) are induced by these agents. The SSBs are converted to double-strand breaks (DSBs) by NE and become visible as CAs on metaphase chromosomes. H2O2 and both types of UV-irradiation also led to an induction of CAs and SCEs, whereas BLM only induced aberrations. Cysteine (Cys) reduced the frequency of the induced SSB-dependent CAs in all treatments, but had no influence on the SCE frequencies after BLM and H2O2 treatment and had only a slight effect on the UV-induced SCEs. The results confirm the opinion that directly induced SSBs can contribute to the induction of CAs in cells with BrdUrd-substituted DNA, but that these SSBs are not efficiently converted to SCEs. The more recent conceptions regarding the mechanism of SCE are in accordance with these findings and the conclusions derived therefrom.

Animals↗
PubMed1984

Considerations on the mechanism of differential Giemsa staining of BrdU-substituted chromosomes.

The staining properties of unifilarly bromodeoxyuridine (BrdU)-substituted chromatids were compared using fluorescent-plus-Giemsa (FPG) staining methods. It was found that the staining intensity of chromatids which had incorporated BrdU in the next to last S-phase is less than that of chromatids whose BrdU-containing strand came from the last cell cycle. Thus, FPG-staining is not a function of the number of BrdU-substituted DNA strands alone. These findings lead to the conclusion that the primary point of action of PFG staining leading to sister chromatid differentiation (SCD) are chromosomal proteins which have been altered in the replication of BrdU-substituted DNA and that the demonstration of the SCD and replication patterns with the same staining procedure is based on different mechanisms.

Azure Stains↗
PubMed1984

Induction of endoreduplication by hydrazine in Chinese hamster V 79 cells and reduced incidence of sister chromatid exchanges in endoreduplicated mitoses.

Hydrazine in high concentrations very effectively induces endoreduplication in Chinese hamster V79 cells. The addition of 5-bromodeoxyuridine (BrdU) for the duration of one cell cycle prior to the induction of endoreduplication produces diplochromosomes with sister chromatid differentiation (SCD) after differential chromatid staining. The fact that diplochromosomes with complete SCD are obtained shows that endoreduplication was induced in cells that were in G2-phase. The analysis of sister chromatid exchanges (SCEs) showed that hydrazine treatment rarely led to increased SCE frequencies in mitoses after endoreduplication, but that it caused a strong SCE induction in diploid second division metaphases in the same culture. Neither catalase nor cysteine had an effect on the induction of endoreduplication or the incidence of SCEs. Treatment of the cells with mitomycin C prior to addition of BrdU led to increased SCE frequencies. Compared with the normal mitoses from the same preparation, the mitoses after endoreduplication showed a significantly reduced induction of SCEs. In contrast to these findings, SCE induction was not reduced in the common tetraploid V 79 cells after colcemid-induced polyploidization.

Animals↗
PubMed1984

Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix.

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na2SeO3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H2O2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H2O2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.

Animals↗
PubMed1983

Maleic hydrazide induces sister-chromatid exchanges in mammalian cells in vitro.

Maleic hydrazide (MH) in high concentrations (from 8 X 10(-3)M upward) induces SCEs in human blood cultures if it is added either for the entire culture period (72 h) or for the last 24 h. Parallel to the induction of SCEs, there is a delay in the proliferation of the cultures, as can be seen by the distribution of the first, second and third mitoses following the addition of BrdUrd. MH leads to a pronounced induction of SCEs in V79 cells (quadrupeling the control value) if added for 24 h simultaneously with BrdUrd. Treating the cells for 1 or 3 h prior to the addition of BrdUrd has no or little effect on the frequencies of SCEs.

Animals↗
PubMed1982

Characterization of sister chromatid exchange induction by hydrogen peroxide.

Hydrogen peroxide (H2O2) induces SCEs in V-79 Chinese hamster cells. The incorporation of BrdUrd sensitizes the cells to the action of H2O2, but damage leading to the formation of SCEs can also be induced in DNA not substitued with BrdUrd. Catalase leads to a complete reduction of the SCE-producing as well as the toxic effect. The amino acid L-cysteine does not reduce the H2O2-induced SCE-frequencies. The results yield information about the relevance of SCE investigations in vitro for the determination of DNA-damaging substances and about the effects of antimutagenic SH-compounds.

Animals↗
PubMed1982

The effect of sulfhydryl compounds on sister-chromatid exchanges. II. The question of cell specificity and the role of H2O2.

In contrast with earlier reports on the induction of sister-chromatid exchanges (SCEs) by SH compounds in cell lines of the Chinese hamster, cysteine, cysteamine and cystamine did not cause an increase of the SCE frequency in human lymphocyte cultures. Differences in the treatment protocols or variations of the BrdUrd concentration had no effect on the induction of SCEs by these substances. The inclusion of H2O2 and comparative investigations with V79 cells of the Chinese hamster showed that the probable reason for the SCE induction by SH compounds is the inability of the cells to degrade H2O2. Furthermore, for cystamine it became clear that additional effects must exist besides the induction of SCEs through H2O2. The present study underlines the fact that the examination of a substance within one cell system does not necessarily permit a reliable statement about the DNA-damaging property of this substance.

Animals↗
PubMed1982

A comparative investigation of the antimitotic action of 2-mercaptoethanol and colcemid on V-79 Chinese hamster cells.

The current study was performed to characterize the antimitotic action of 2-mercaptoethanol (MET) on mammalian cells. At concentrations of 2.5 x 10(-2) m, MET arrests V-79 Chinese hamster cells in metaphase. Smaller concentrations (from 5 x 10(-3) m) only produce a mitotic block after several hours, only arresting those mitoses which have gone through one cell cycle in the presence of MET. The accumulation of mitoses by MET is smaller in comparison with colcemid, explained by an effect reducing the number of cells which enter mitosis. In contrast to colcemid, MET-concentrations which do not lead to a mitotic block cause a delay in proliferation. It was shown, by means of the BUdR-labelling method that cells in the presence of colcemid concentrations which arrest mitosis again enter interphase and become polyploid, whereas MET leads to an irreversible arrest of mitosis and does not produce polyploidy in V-79 cells.

Animals↗
PubMed1981

Demonstration of sister chromatid differentiation in human amniotic fluid cells after partial synchronization of BrdU or dT surplus.

Protocols are compared demonstrating sister chromatid differentiation (SCD) in human amniotic fluid (AF) cells with and without partial synchronization. Partial synchronization both with an excess of 5-bromodeoxyuridine (BrdU) and an excess of thymidine leads to an increase of metaphases with SCD. Compared with unsynchronized cells, the rate of sister chromatid exchanges (SCE) is not increased. Studies on the late replicating X chromosome of female cells showed that the addition of mitomycin C (MMC) after releasing the thymidine block preferentially induces SCEs in late replicating regions. The partial synchronization with thymidine surplus provided a good basis for SCE experiments with AF cells and facilitates the prenatal diagnosis of diseases characterized by changes in the SCE rate.

Amniotic Fluid↗