Biomedical subjects

G Speit

Publications and source records attributed to G Speit.

At least 73 records · Page 4Linked to original sources

PubMed1993

Molecular characterization of methyl methanesulphonate (MMS)-induced HPRT mutations in V79 cells.

Spontaneous and methyl methanesulphonate-induced HPRT-deficient mutants were analysed for changes in the hprt gene structure using multiplex polymerase chain reaction. The PCR amplification pattern of 21 MMS-induced mutations revealed one total deletion of the hprt coding exons and one small deletion within exon 5, while 19 mutants showed the V79 wild-type pattern. Molecular analysis of 30 spontaneous mutations revealed no mutants with amplification patterns which differed from those of wild-type cells. We further analysed MMS-induced mutants in a different V79 cell line with a high (40%) spontaneous deletion frequency. MMS caused a dose-dependent increase in the mutant frequency but the incidence of deletions was reduced to 6% at 2 x 10(-4) M and to 13% at 5 x 10(-4) M indicating that mainly point mutations were induced. The repair inhibitor cytosine arabinoside (araC) enhanced mutation induction by MMS but did not change the proportion of deletions in the mutation spectrum. The results indicate that different V79 cell lines spontaneously produce different amounts of deletion mutations. The frequency of MMS-induced deletions does not depend on the frequency of spontaneous deletions in a given cell line. The MMS-induced mutation spectrum seems to be unchanged even at high concentrations with a strong cytotoxic effect. Deletions are not increased as a consequence of araC-inhibited repair of MMS-induced lesions.

Animals↗

PubMed1993

Absence of genotoxic activity of beta-myrcene in the in vivo cytogenetic bone marrow assay.

beta-Myrcene (MYR, 7-methyl-3-methylene-1,6 octadiene) is a peripheral analgesic substance and one of the major constituents of lemongrass oil (Cymbopogon citratus, Stapf), a plant widely used in Brazilian folk medicine. In the present study the genotoxicity of MYR was evaluated in vivo using the rat bone marrow cytogenetic assay. Male and female Wistar rats weighing 250 g (223 to 286 g) and 178 g (168 to 186 g), respectively, were used. Two or four rats of either sex were treated orally with MYR (0.1, 0.5 and 1.0 g/kg po), corn oil (negative control) and cyclophosphamide 30 mg/kg ip (positive control). Animals were sacrificed and bone marrow cells were harvested 24 and 48 h after MYR administration. The mitotic index and the frequency of chromosome aberrations were evaluated. Fifty metaphase cells were examined per animal. A dose related increase in mitotic index was observed 24-h after MYR administration. No evidence of MYR-induced clastogenicity was observed under the experimental conditions of this in vivo assay. The present results and previous negative findings of in vitro mutagenicity tests strongly indicate that MYR is not a genotoxic substance.

Acyclic Monoterpenes↗

PubMed1992

Cytogenetic and molecular characterization of the mutagenicity of chlorambucil in V79 cells.

Chlorambucil (CBC) is used as a chemotherapeutic agent and immunosuppressant. Recently, it could be shown that CBC is considerably more effective than radiation or any chemical investigated to date in inducing high yields of germ-line mutations that appear to be multilocus deletions or other structural changes. We therefore reinvestigated the in vitro genotoxic effects of CBC in V79 cells and characterized induced sister-chromatid exchanges (SCEs), chromosome aberrations and gene mutations by means of cytogenetic and molecular methods. CBC effectively induced chromosome aberrations and SCEs in a dose-dependent manner. The chromosome aberrations found after a 14-h treatment were mainly chromatid-type aberrations. 3-Aminobenzamide (3AB) did not influence the incidence of CBC-induced SCEs and chromosome aberrations. Combined treatment with CBC and caffeine (CAF) strongly increased the frequency of aberrations, but had no effect on the yield of SCEs. CAF at lower concentrations enhanced the production of chromatid breaks and exchange figures while higher concentrations (10(-3) M) caused multiple breaks and pulverised mitoses. Mutations at the hprt locus were induced in a narrow range of CBC concentrations (10(-5) M-2 x 10(-5) M) and the mutagenic effect was accompanied by strong cytotoxicity. The CBC-induced gene mutation frequency was not increased after CAF treatment. The molecular analysis of CBC-induced mutations by Southern hybridization and PCR demonstrated that CBC predominantly produced small alterations but not deletions or gross structural alterations in the hprt gene of V79 cells. For the first time, these results reveal striking differences in the mutagenic action of an alkylating agent in cultivated cells compared to germ-line cells at the molecular level.

Animals↗

PubMed1991

Evaluation of the mutagenicity of beta-myrcene in mammalian cells in vitro.

The genotoxicity of the terpene beta-myrcene was evaluated in mammalian cells in vitro. Myrcene is the major constituent of oil of bay and hop which are used in the manufacture of alcoholic beverages. Myrcene is also present in lemon grass (Cymbopogon citratus), a plant widely used in Brazilian folk medicine. Recently, it was shown that myrcene is a very potent analgesic substance and might be an alternative to the already available analgesic drugs. Myrcene was tested up to 1,000 micrograms/ml (limit of solubility) in the presence and absence of S9-mix and did not induce chromosome aberrations and sister chromatid exchanges (SCEs) in human lymphocytes in vitro. Neither the mitotic index nor the proliferation index was influenced by the myrcene treatment. Myrcene did not cause increased mutation frequencies at the hprt-locus in V79-cells. Tests with and without S9-mix revealed negative results. There was no indication for induced cytotoxicity. However, myrcene reduced the SCE-inducing effect of cyclophosphamide in human lymphocytes in a dose dependent manner and also reduced the toxic and mutagenic effect of cyclophosphamide in V79-cells. Under the same test conditions, SCE induction by ethyl methanesulfonate (EMS) and benzo [a]pyrene (BP) was not significantly influenced by simultaneous myrcene treatment. The in vitro results show that myrcene is not mutagenic in mammalian cells, but has antimutagenic properties. The possibility that myrcene exerts its antimutagenic activity by inhibiting certain forms of the cytochrome P-450 isoenzymes required for activation of premutagens and precarcinogenes is discussed.

Acyclic Monoterpenes↗

PubMed1991

Molecular characterization of mutations at the hprt locus in V79 Chinese hamster cells induced by bleomycin in the presence of inhibitors of DNA repair.

The molecular basis of bleomycin (BLM)-induced mutations in the absence and presence of inhibitors of DNA repair was investigated in V79 cells with Southern hybridization techniques. 43% of the BLM-induced thioguanine-resistant mutants suffer from large alterations of hprt DNA sequences. To understand the role of DNA repair in the process of mutagenesis, the effect of inhibitors of DNA repair on the frequency and types of BLM-induced mutations was tested. The inhibitors used were arabinofuranosyl cytosine (araC), didesoxythymidine (ddThd) and 3-aminobenzamide (3AB), which inhibit different steps of excision repair. Only 3AB caused a comutagenic effect. The increased mutation frequency was mainly due to additionally induced gene deletions. In the presence of 3AB, 70% of the HPRT-deficient mutants revealed partial or total deletions of the hprt coding sequences. Thus, it could be shown that BLM induces a broad range of types of mutation and that inhibited repair of BLM-induced DNA damage leads to specific types of mutations.

Animals↗

PubMed1991

Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells.

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1991

Mutation screening of bleomycin-induced V79 Chinese hamster hprt mutants using multiplex polymerase chain reaction.

We have shown by the filter hybridization technique that bleomycin (BLM) induces different types of mutations at the hprt gene locus of V79 Chinese hamster cells. DNA of mutants identified by Southern blots as partial deletions was subjected to further analysis using multiplex polymerase chain reaction (PCR) to localize the endpoints of the deletions over the hprt gene. The PCR analysis revealed that deletions occur in all parts of the hprt gene but are distributed non-randomly. Deletions occurred most frequently at the 3'-end of the hprt gene suggesting a possible existence of a hot spot for deletions in this region; exons 1, 2 and 3 appeared to be less affected by deletions. As PCR can detect microdeletions which are below the limit of resolution of Southern blot hybridization we analysed 25 HPRT- mutants with Southern wild-type pattern to distinguish between point mutations and small deletions. Of these HPRT- mutants, all except five showed PCR amplification products identical to that of V79 wild-type cells. These results are consistent with previous Southern analyses indicating that a large portion of BLM-induced HPRT- mutants are real point mutations. Five mutants, however, showed differences in fragment sizes of single PCR products or did not yield one single exon fragment and thus are probably the result of deletions which were not to be detected by Southern analyses.

Animals↗

PubMed1990

The effect of glutathione depletion on sister-chromatid exchange induction by cytostatic drugs.

Using sister-chromatid exchanges (SCEs) as an indicator for DNA damage, we investigated the role of glutathione (GSH) as a determinant of cellular sensitivity to the DNA-damaging effects of the cytostatic drugs adriamycin (AM) and cyclophosphamide (CP). Exposure of V79 cells to buthionine sulfoximine (BSO) resulted in a complete depletion of cellular GSH content without toxicity and without increasing the SCE frequency. Subsequent 3-h treatment of GSH-depleted cells with AM or S9-mix-activated CP caused a potentiation of SCE induction. In Chinese hamster ovary (CHO) cells, which showed a higher GSH level compared to V79 cells, BSO treatment led to a depletion of GSH to about 5% of the control and increased SCE induction by AM and CP. Compared to V79 cells, the effect of AM on SCE frequencies was less distinct in CHO cells, while CP exerted a similar effect in both cell lines. Pretreatment of V79 cells with GSH increased the cellular GSH content, but had no effect on the induction of SCEs by AM, and pretreatment with cysteine influenced neither GSH levels nor SCE induction by AM. The study shows that SCEs are a suitable indicator for testing the modulation of of drug genotoxicity by GSH. The importance of different GSH contents of cell lines for their response to mutagens is discussed.

Animals↗

PubMed1990

Genetic effects of 2-aminopurine in mammalian cells.

The effects of 2-aminopurine (APur) on mutations, sister-chromatid exchanges (SCEs) and proliferation were investigated in V79 cells by means of cytogenetic and flowcytometric experiments. APur did not induce SCEs after a 3-h treatment before the addition of BrdUrd but SCE frequencies were increased after treatment for two cell cycles in the presence of BrdUrd. SCEs were mainly produced during the second cell cycle of the SCE experiment when BrdUrd substituted DNA is replicated. APur also caused a high percentage of polyploid cells. Compared on the basis of DNA content, SCE induction was the same in diploid and tetraploid metaphases. APur-induced SCEs are strongly influenced by nucleosides. The presence of deoxycytidine (dCyd) caused a reduction of AP-induced SCEs to about control level while addition of deoxythymidine (dThd) enhanced SCE induction. Flow cytometric measurements revealed a small increase in S-Phase cells and a strong accumulation in G2/M after APur treatment in the presence of BrdUrd. S-phase delay was strongly enhanced when BrdUrd substituted DNA is replicated. Addition of dCyd removed the APur-induced inhibition of S-phase in both protocols. Using the same treatment protocol, APur also induced mutations at the HPRT locus. In contrast to their effects on SCEs and proliferation neither BrdUrd nor dCyd had an effect on APur-induced mutations, and dThd reduced the mutation frequency. The results demonstrate that APur-induced SCEs and mutations occur independently from each other. APur-induced mutations obviously occur by a mispairing mechanism while SCEs are a consequence of pool imbalances during replication.

2-Aminopurine↗

PubMed1990

An improved procedure for the cultivation and in situ chromosome preparation of monolayer cell cultures.

A method for the cultivation of monolayer cell cultures on microslides in quadruple culture dishes together with a simple procedure for in situ chromosome preparation are described. The cells fixed to the slide can be stained according to standard procedures and analysed microscopically. The method is simple, rapid and reliable and provides many advantages especially for cytogenetic diagnostics with fibroblasts and amniotic fluid cells. It simplifies the performance of cytogenetic mutagenicity testing with primary cultures and permanent cell lines, e.g. the analysis of chromosome aberrations, sister chromatid exchanges (SCEs) and induced aneuploidy, as well as large-scale cytogenetic experiments.

Animals↗

PubMed1987

Cytogenetic effects of penicillamine.

Penicillamine (PA), a drug used for the treatment of rheumatoid arthritis induces sister-chromatid exchanges (SCEs) and chromosome aberrations in cultivated mammalian cells. PA in concentrations from 400 micrograms/ml upward induced SCEs and proliferative delay in human blood cultures when added for the last 24 h of the culture period. In V79 Chinese hamster cells SCE induction was found after acute exposure to PA before the addition of BrdUrd and after chronic exposure during one cell cycle in the presence of BrdUrd. The effect of PA on SCE frequencies occurred both after treatment in complete medium and in serum-free medium and was not influenced by the application of an S9 mix. The simultaneous addition of peroxidase reduced the PA-induced SCEs whereas catalase did not show any effect. Chromosome analysis in the first mitosis after PA treatment revealed a significant increase in the incidence of chromosome aberrations and endoreduplication. The results are discussed with respect to the cause and the significance of the observed effects in connection with mutagenicity testing.

Animals↗

PubMed1987

DHFR gene amplification in cultured skin fibroblasts of ataxia telangiectasia patients after methotrexate selection.

During selection for methotrexate resistance, SV40-transformed human skin fibroblasts from patients with ataxia telangiectasia (A-T) underwent amplification of the dihydrofolate reductase (DHFR) gene, experienced nearly complete loss of the integrated SV40 sequences and showed a 3.6-fold increase in Ki-ras gene copy number. Over a period of months methotrexate-resistant (MTXr) A-T subclones were obtained, which were able to grow in progressively increasing MTX concentrations up to 100 microM. The ED50 values determined as the effective dose of MTX causing 50% growth inhibition in comparison to control cells increased from 3 x 10(-2) microM for MTXs AT5BI-VA cells to 250 microM MTX for the MTXr AX100 subclone. In contrast, human skin fibroblasts of healthy individuals did not show DHFR gene amplification and loss of SV40 sequences under comparable conditions and were unable to grow in MTX concentrations greater than 1 microM. Gene amplification and loss of DNA sequences are features underlying the genomic instability known to be a characteristic property of A-T cells and being probably responsible for the high cancer incidence in these patients.

Ataxia Telangiectasia↗

PubMed1986

Cytogenetic replication studies with short thymidine pulses in bromodeoxyuridine-substituted chromosomes of different mouse cell lines.

In normal diploid fibroblasts of the mouse, 3T3-SV-3T3-, and Meth A-cells, the chromosome replication patterns were studied by a bromodeoxyuridine (BrdU)-labelling technique. SV-3T3 is a subline of 3T3 transformed by SV 40 and Meth A is a permanent cell line from Balb c transformed by methylcholanthrene. The use of 1 h thymidine pulses permits high resolution of the S-phase after partial synchronization of the cells at G1/S in an otherwise BrdU-substituted S-phase. It could be shown that the autosomal heterochromatin of the mouse (Mus musculus) starts replication during the early S-phase (R-band replication), continues while R-band chromatin finishes, and still replicates when G-band chromatin starts. The heterochromatin finishes before the majority of G-bands have been replicated. There is no fundamental difference in the course of chromosome replication between the different cell lines studied here. It is concluded that there are no obligate changes in the course of the S-phase linked to the process of transformation.

Animals↗

PubMed1986

Detection of bromodeoxyuridine-incorporation in mammalian chromosomes by a bromodeoxyuridine-antibody. I. Demonstration of replication patterns.

We have shown that a commercially available antibody allows the demonstration of bromodeoxyuridine (BrdU) incorporation into mammalian chromosomes by the typical immunologic staining techniques. The replication patterns obtained are comparable to those after BrdU-incorporation and FPG-staining, though with reversed staining specificity. The quality of the patterns equals that of commonly used differential staining but the sensitivity of BrdU-detection is superior. Thus the new technique may be very promising for all cytogenetic studies concerning any kind of chromosome replication.

Animals↗

PubMed1986

Variation in the frequency of sister chromatid exchanges in repeated human lymphocyte cultures.

Repeated blood samples from two healthy donors were taken over a period of about one year to determine the temporal variation in human lymphocyte baseline sister chromatid exchange (SCE)-frequencies. The investigations were performed on whole blood cultures and purified lymphocyte cultures using a standardized protocol for blood collection and cultures. Significant differences in the frequencies of SCEs were found between the two cultivation systems and the two blood donors but also between repeated cultures of the same individual. There was no systematic relationship between the proliferation of the cultures and the basal SCE values. The results indicate the necessity of concurrent controls and repeated blood samples whenever SCEs are used as a test for monitoring human exposure to potential mutagens. Temporal variation in human lymphocyte baseline SCE frequencies is a limiting factor for the detection of minor effects of genotoxic agents.

Cells, Cultured↗

PubMed1986

Detection of bromodeoxyuridine incorporation in mammalian chromosomes by a bromodeoxyuridine antibody. II. Demonstration of sister chromatid exchanges.

A commercially available bromodeoxyuridine (BrdUrd) antibody was used to demonstrate sister chromatid differentiation (SCD) and to evaluate sister chromatid exchanges (SCEs) in V79 Chinese hamster cells. V79 cells were cultivated for one cell cycle in the presence of BrdUrd, followed by a second cell cycle in the absence of BrdUrd. Chromosome preparations were stained by a common immunologic staining technique. The staining pattern observed is similar to that after FPG (fluorescent plus Giemsa) staining, though with reverse staining specificity. The sensitivity of BrdUrd detection is enhanced by a factor of 20 compared to the FPG technique and thus allows the evaluation of SCEs at very low BrdUrd concentrations. The application of the antibody technique gives information about the origin and localization of SCEs and produces further evidence for the spontaneous occurrence of SCEs.

Animals↗

PubMed1986

The relationship between the induction of SCEs and mutations in Chinese hamster cells. I. Experiments with hydrogen peroxide and caffeine.

Hydrogen peroxide (H2O2) and caffeine were examined for their capacity for inducing SCEs and mutations at the HPRT locus in V79 Chinese hamster cells. Although, under standard conditions, both substances induced SCEs neither caused gene mutations. The SCE induction by both H2O2 and caffeine is influenced by BrdUrd substitution. Whereas H2O2 also induces lesions leading to SCEs in normal DNA, the SCe induction by caffeine depends on the replication of BrdUrd-substituted DNA. In cells with BrdUrd-substituted DNA, H2O2 induces mutations at the HPRT locus parallel to its SCE induction, whereas caffeine in the presence of BrdUrd only has an influence on the SCE rate. It is shown that the experimental conditions of the two test systems can play a decisive role when contradictory results are obtained.

Animals↗

PubMed1985

"Unusual SCD" explained by differences in BrdU incorporation during subsequent cell cycles.

In the course of three consecutive cell cycles, V79 cells were labeled with BrdU by different labeling protocols. Cells treated for three cycles with bromodeoxyuridine (BrdU) showed third division metaphases, with the typical appearance after fluorescent plus Giemsa (FPG) staining (i.e., 75% of the chromatids showed light staining, 25% showed dark staining). The same staining pattern is achieved by the second labeling protocol, during which the cells have replicated for two cycles in the presence of BrdU and, during the last cycle, in the absence of BrdU. Cells that have replicated only for one cell cycle in BrdU-containing medium, and the following two cycles in normal medium, depict just the opposite staining pattern (i.e., 75% dark, 25% light). These experiments explain how the variation of BrdU substitution in the DNA leads to altered FPG staining. Unexpected staining patterns ("unusual SCD") are also observed after seemingly permanent BrdU substitution. This phenomenon, which has been found in cancer cells, is due to the decrease in BrdU concentration and not to a peculiarity of the cells investigated.

Animals↗