Comparative evaluation of the genotoxic properties of potassium bromate and potassium superoxide in V79 Chinese hamster cells.

The genotoxic potential of two oxidizing compounds, potassium bromate and potassium superoxide, was comparatively tested in various genotoxicity tests with V79 Chinese hamster cells. Both substances clearly induced cytotoxicity, chromosome aberrations and increased DNA migration in the alkaline comet assay. Using a modified comet assay protocol with FPG protein, a DNA repair enzyme which specifically nicks DNA at sites of 8-oxoguanines and formamidopyrimidines, we detected oxidative DNA base damage only after potassium bromate treatment. HPLC analysis also revealed significantly increased levels of 8-oxodeoxyguanosine after potassium bromate treatment but not after potassium superoxide treatment. Furthermore, potassium bromate clearly induced gene mutations at the HPRT locus while potassium superoxide only had a small effect on HPRT mutant frequencies. Molecular analysis of potassium bromate-induced mutations indicated a high portion of deletion mutations. Three out of four point mutations were G to T transversions which typically arise after replication of 8-oxoguanine. Our results suggest that the two oxidizing compounds induce specific patterns of genotoxic effects that reflect the types of DNA alterations induced by different reactive oxygen species (ROS).

Evaluation of the genotoxic properties of paraquat in V79 Chinese hamster cells.

The genotoxic potential of the herbicide paraquat (PQ), an intracellular generator of superoxide, was comparatively tested in various genotoxicity tests with V79 Chinese hamster cells. PQ clearly induced cytotoxicity and chromosome aberrations but did not induce gene mutations at the HPRT locus or increased DNA migration in the comet assay under the same treatment conditions. Using a modified comet assay protocol with formamidopyrimidine-DNA glycosylase (FPG) protein, a DNA repair enzyme which specifically nicks DNA at sites of 8-oxo-guanines and formamidopyrimidines, we could not detect oxidative DNA base damage after PQ treatment. When cells were treated directly on the slides after lysis (i.e, after the cell membrane barrier was eliminated), increased DNA migration was observed after treatment with high PQ-concentrations. Our results suggest that PQ does not significantly induce DNA lesions relevant for HPRT gene mutations in cultivated V79 cells. Since PQ-induced chromosome aberrations only occur after treatment with high concentrations which totally prevent cell survival and are not preceded by an induction of DNA strand breakage in intact cells, their biological significance has to be questioned.

Characterization of spontaneous and induced mutations in SV40-transformed normal and ataxia telangiectasia cell lines.

Spontaneous and induced mutations at the HPRT locus were analyzed in one normal (MRC5CV1) and one ataxia telangiectasia (AT5BIVA) SV40-transformed cell line derived from male donors. Multiplex PCR and Southern analyses revealed a high frequency of spontaneous deletion mutations that may be a consequence of the SV40 transformation. Four mutagens (ethyl methanesulfonate, bromodeoxyuridine, bleomycin, adriamycin), which differ in their types of primary DNA lesions, caused specific patterns of mutations. By using fluorescence in situ hybridization (FISH) techniques, we were able to show that more than 90% of the AT5BIVA cells contained two X chromosomes with HPRT alleles, while in more than 90% of the MRC5CV1 cells genomic hemizygosity for the HPRT gene was found. Taking into account these findings we found that the AT5BIVA cell line possesses spontaneous hypermutability as well as hypersensitivity and hypermutability to bleomycin (BLM) and adriamycin (AM). Both mutagens induced deletion mutations in both cell lines, but more complex mutations and larger deletions were found in AT5BIVA cells.

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.

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.

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.

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.

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.

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.