European HPRT workshop in collaboration with GUM Gatersleben-Quedlinburg, 4-7 May, 1995.
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Humans↗
Biomedical subjects
G Speit
Publications and source records attributed to G Speit.
Effect of arsenic and cadmium on the persistence of mutagen-induced DNA lesions in human cells.
The alkaline single cell gel electrophoresis (SCG test or comet assay) was used to characterize the influence of sodium arsenite (NaAsO2) and cadmium sulphate (CdSO4) on the persistence of mutagen-induced DNA lesions. Human blood and SV4O-transformed fibroblasts (MRC5CV1) were treated for 2 hr with methyl methanesulphonate (MMS) or benzo(a)pyrene (BaP). MMS induced concentration-related DNA damage in white Blood cells (WBC) and fibroblasts in similar concentrations. For the induction of DNA damage in white blood cells (WBC) and fibroblasts in similar concentrations. For the induction of DNA damage by BaP, higher concentrations had to be applied to WBC than to the fibroblast cell line. To study the influence of metal ions on the persistence of DNA lesions, treated cells were further incubated for 2 hr in the absence (postincubation) or presence (posttreatment) of NaAsO2 or CdSO4. After postincubation, MMS and BaP-induced DNA effects were reduced in both cell types, indicating that repair of DNA lesions had taken place. When the cells were posttreated with NaAsO2 or CdSO4, BaP- and MMS-induced DNA lesions persisted in both cell types, indicating an inhibition of DNA repair by these metals. The results suggest a strong interaction of arsenic and cadmium with BaP- and MMS-induced DNA repair processes.
Arsenites↗
Detection of DNA effects in human cells with the comet assay and their relevance for mutagenesis.
The single cell gel test (SCG-test or comet assay) is a rapid and sensitive method for measuring DNA damage and repair in individual cells. A wide variety of mutagens have been shown to cause DNA alterations detectable with the comet assay, but it is not yet clear whether a relationship exists between the DNA effects and the induction of mutations. We are therefore investigating in a cell culture system with human cells (MRC5CV1) the induction of DNA damage by environmental mutagens and the formation of mutations at the HPRT gene. In the present study we investigated benzo[a]pyrene (BP), an environmental mutagenic and carcinogenic polycyclic aromatic hydrocarbon, and its reactive metabolite (+)-anti-benzo[a]pyrene-7,8-diol 9, 10-oxide ((+)-anti-BPDE). S9 mix activated BP and the direct acting mutagen (+)-anti-BPDE caused a concentration-related increase in DNA migration in the comet assay. Postincubation experiments indicated that induced DNA effects are eliminated by DNA repair within 24 h. BP-treatment caused a strong genotoxic effect in the comet assay but had only a marginal effect on the frequency of gene mutations. When cells were treated with BP in the presence of cadmium sulphate, a clear increase in genotoxicity was observed while the effect on mutations was unchanged. Our results indicate that DNA alterations detected with the comet assay do not necessarily relate to mutagenesis. The absence of a close relationship between DNA migration in the comet assay and mutagenesis may be explained by the fact that some effects seen in the comet assay occur as a consequence of an error free DNA repair process.
Benzo(a)pyrene↗
Evaluation of the mutagenicity of the molluscicidal latex of Christ's Crown (Euphorbia milii var. hislopii) in mammalian cells in vitro and in vivo.
The latex of Christ's Crown (Euphorbia milii var. hislopii, syn. E. splendens var. hislopii) is a highly active plant molluscicide and could be used for snail control to reduce the prevalence of schistosomiasis in endemic areas. In the course of its toxicological evaluation, the mutagenicity of the latex of Euphorbia milii was tested in mammalian cells in vitro and in vivo. Latex was investigated for its capability of inducing gene mutations and chromosome aberrations in V79 cells in the absence and presence of S9-mix. Concentrations up to 800 micrograms/ml neither induced gene mutations at the HPRT locus nor chromosome aberrations. Latex had no effect on the frequencies of chromosome aberrations in the bone marrow of male and female rats at a dose of 1000 mg/kg. The results indicate that latex of E. milii is not mutagenic in mammalian cells in vitro and in vivo and its use as a molluscicide does not pose a mutagenic hazard for humans.
Animals↗
DNA damage after exhaustive treadmill running in trained and untrained men.
The single cell gel electrophoresis (SCG) assay was used to compare the occurrence of DNA damage in peripheral white blood cells in 6 trained (TR) and 5 untrained (UT) men after exhaustive exercise. The subjects completed an incremental treadmill test until exhaustion (maximal lactate: 12.9 +/- 1.7 in TR and 12.2 +/- 2.5 mmol.l-1 in UT). A clear and significant increase of DNA migration from 2.31 +/- 0.20 (TR) and 2.22 +/- 0.16 (UT) at rest to 2.65 +/- 0.30 (TR) and 3.00 +/- 0.41 tail moment (UT) was found 24 hours after exercise. Noteworthy is that the increase of DNA migration was significantly lower in TR (+ 18.7 +/- 6.8%) compared to UT (+ 35.7 +/- 8.9%). Plasma levels of malondialdehyde (MDA) were not significantly increased in TR and UT after exercise. At rest and 15 minutes after exercise MDA-values were significantly lower in TR compared to UT. In conclusion the present investigation demonstrates the occurrence of DNA damage in white blood cells following exhaustive exercise. This observation may be induced by oxidative stress. Our data suggest that adaptation to training seems to be capable of reducing free radical associated effects, such as DNA damage. Further investigations are needed to clarify the causal mechanisms and biological relevance of exercise-induced DNA damage.
Adaptation, Physiological↗
Detection of DNA damage after hyperbaric oxygen (HBO) therapy.
Hyperbaric oxygen (HBO) therapy is successfully used for the treatment of a variety of conditions. However, exposure to high concentrations of oxygen is known to induce damage to cells, possibly due to an increased oxygen radical production. As reactive oxygen species also cause DNA damage, we investigated the DNA-damaging effect of HBO with the alkaline version of the single cell gel test (comet assay). Oxidative DNA base modifications were determined by converting oxidized DNA bases to strand breaks using bacterial formamidopyrimidine-DNA glycosylase (FPG), a DNA repair enzyme, which specifically nicks DNA at sites of 8-oxo-guanines and formamidopyrimidines. HBO treatment under therapeutic conditions clearly and reproducibly induced DNA damage in leukocytes of all test subjects investigated. Increased DNA damage was found immediately at the end of the treatment, while 24 h later, no effect was found. Using FPG protein we detected significant oxidative base damage after HBO treatment. DNA damage was detected only after the first treatment and not after further treatments under the same conditions, indicating an increase in antioxidant defences. DNA damage did not occur when the HBO treatment was started with a reduced treatment time which was then increased stepwise.
Adult↗
DNA-damaging effect of cyclophosphamide on human blood cells in vivo and in vitro studied with the single-cell gel test (comet assay).
The single-cell gel (SCG) test was used to study the induction and persistence of DNA damage by cyclophosphamide (CP) in human blood cells after treatment in vitro and in vivo. S9-mix-activated CP (from 0.1 mM upward) induced DNA effects in a concentration-dependent manner in the in-vitro SCG test. Blood cells from various donors showed considerable intra- and interindividual variability. Incubation of CP-treated blood samples at 37 degrees C caused a rapid decrease in DNA effects, but DNA migration was still significantly increased 1 hr after the end of the CP treatment. Comparative studies with the in vitro sister chromatid exchange (SCE) test were performed that demonstrated that much lower CP concentrations (about 100 times) were required for a significant induction of SCEs. A group of 11 patients who suffered from vasculitis/collagen disease and were treated with low CP doses (50-200 mg/day) exhibited an elevated level of DNA damage in the SCG test with peripheral blood cells, compared with a group of 11 control persons or 5 patients without chemotherapy. Increases in DNA damage were variable and not clearly related to the CP dose. SCE tests could successfully be performed with 5 out of the 11 CP-treated patients; all showed significantly increased SCE frequencies. For six patients no result could be obtained with the SCE test due to a failure of lymphocyte proliferation. Three multiple sclerosis patients who received high doses of CP were investigated with the SCG test before, during, and after the treatment. The results indicate that CP-induced DNA effects that are detectable with the SCG test persist in vivo for a period of several days, but for less than 2 weeks. The results of our study provide information with regard to the use of the SCG test in human monitoring. The advantages and limitations of the test are discussed.
Adult↗
The pattern of adriamycin-induced mutations in V-E5 Chinese hamster cells with chromosomal instability.
The V-E5 cell line, a mutant V79 Chinese hamster cell line, was used to study the effect of chromosomal instability on the spectrum of gene mutations and chromosome aberrations induced by the anthracycline antibiotic adriamycin (AM). V-E5 cells showed hypersensitivity to the cytotoxic effects of AM when compared to the parental cell line. AM caused both, chromosome-type aberrations and chromatid-type aberrations in V-E5 cells. Under the same experimental conditions, gene mutations were induced at the hprt locus which mainly represented deletion mutations. The spectrum of AM-induced chromosomal aberrations and gene mutations did not show any peculiarities in comparison to normal V79 cells. It is concluded that the genomic instability in V-E5 cells does not influence the pathways leading to chromosome aberrations and gene mutations after AM treatment.
Animals↗
Vitamin E prevents exercise-induced DNA damage.
The single cell gel test (SCG test or comet assay) was used to study DNA damage in peripheral white blood cells (WBC) of humans after a single bout of exhaustive exercise and the effect of vitamin supplementation. Human subjects were asked to run on a treadmill until exhaustion and blood samples were taken before and 24 h after the run. A clear increase in DNA strand breakage was observed in the 24-h sample of all probands. A short-term application of multivitamin pills or vitamin E (3 x 800 mg) resulted in a significantly smaller increase of DNA effects in WBC of some probands. When the volunteers were given a supplement of vitamin E (1200 mg daily) for 14 days prior to a run, exercise-induced DNA damage was clearly reduced in all probands. In four out of five subjects, vitamin supplementation completely prevented the induction of DNA damage after exhaustive exercise. Intake of vitamin E for 14 days led to a clear increase in vitamin E serum concentrations. Malondialdehyde (MDA), a marker of lipid peroxidation, was measured in the serum of probands in tests with and without vitamin supplementation for 14 days. MDA concentrations were significantly decreased following vitamin E supplementation but not significantly changed 15 min and 24 h after a run. Our results demonstrate that vitamin E prevents exercise-induced DNA damage and indicate that DNA breakage occurs in WBC after exhaustive exercise as a consequence of oxidative stress.
Adult↗
Genotoxic effects of chemicals in the single cell gel (SCG) test with human blood cells in relation to the induction of sister-chromatid exchanges (SCE).
In a comparative study, benzo[a]pyrene (BaP), cyclophosphamide (CP), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and tetrachloroethylene (PER) were tested for their ability to induce genotoxic effects in the single cell gel (SCG) test and the sister-chromatid exchange (SCE) test with human blood cells. MNNG as well as S9 mix activated BaP- and CP-induced DNA effects in both tests in a dose-dependent manner. While the range of concentrations which induced DNA migration or SCE was the same for MNNG and for BaP, much higher CP concentrations were necessary for a positive response in the SCG test than in the SCE test. PER was tested in the absence and in the presence of S9 mix and neither induced DNA migration nor increased SCE frequencies. In these experiments, a clear cytotoxic effect of PER was observed. To investigate a possible influence of DNA repair on the effects in the SCG test, cells were treated for 2 h and further incubated for 1 h after removal of the test substance. This procedure caused a clear decrease in induced DNA migration in experiments with BaP and CP, whereas no reduction was found with MNNG. This modified protocol did not lead to the detection of DNA effects after treatment with PER. The results indicate that the SCG test responds to various DNA lesions and does not seem to be sensitive to non-genotoxic cell killing. Its sensitivity obviously depends on the type(s) of induced DNA lesions and the effects can be modified by DNA repair processes in a complex manner. For the detection of genotoxic properties of chemicals with the in vitro SCG test, a single evaluation at the end of the exposure period seems to be sufficient.
Benzo(a)pyrene↗
The pattern of mutations induced by neocarzinostatin and methyl methanesulfonate in the ataxia telangiectasia-like Chinese hamster cell line V-E5.
The Chinese hamster cell line V-E5 is a mutant cell line isolated from V79 cells. The phenotypic characteristics of V-E5 strongly resemble those of cells from patients suffering from the genomic instability syndrome ataxia telangiectasia. In order to further characterize the mutant cell line and to get insight into the underlying genetic defect we compared the clastogenic and mutagenic effects of neocarzinostatin (NCS) and methyl methanesulfonate (MMS) in V-E5 and V79 wild-type cells (V79-LE). V-E5 cells were 2-3 times more sensitive to the cytotoxic effect of NCS or MMS. The clastogenic action of NCS was characterized by the predominant induction of chromosome breaks and dicentrics in both cell lines, whereas MMS mainly induced chromatid-type aberrations. The frequency of mutations induced by NCS as well as MMS was slightly enhanced in V-E5 cells compared to V79 cells treated with the same dose. However, the mutant cell line was found to be hypomutable when considering the same survival level as in the parental cell line. Molecular analysis of mutants induced by NCS revealed a high frequency of total deletions of the hprt gene in both cell lines. In contrast, among MMS-induced mutations only 11% deletion mutations were found in V79-LE, whereas in V-E5 MMS-induced deletions were seen in 52% of the hprt-deficient mutants. These results are discussed with respect to a possible relation between genomic instability, cell cycle control and mutational spectra.
Animals↗
The contribution of excision repair to the DNA effects seen in the alkaline single cell gel test (comet assay).
The alkaline single cell gel test (SCG test or comet assay) was used to study the contribution of excision repair activity to the observed DNA effect after mutagen treatment. The cytotoxicity and genotoxicity of UV-irradiation and the chemical mutagens 4-nitroquinoline-1-oxide (4NQO), benzo[a]pyrene (BP) and 7,12-dimethyl-benz[a]anthracene (DMBA) were compared in a normal human cell line (MRC5CV1) and an excision-deficient xeroderma pigmentosum (XP) cell line (XP12ROSV). The XP cells showed increased cell killing after treatment with all mutagens tested, but did not show a clear increase in DNA migration in the comet assay. DNA effects in MRC5 cells were strongly enhanced by the repair inhibitor aphidicolin (APC), while under the same experimental conditions, APC had no effect on the XP cell line. The enhancing effect of APC on DNA migration in MRC5 cells and the lack of effects in XP cells indicate that the induced DNA effects of 4NQO, BP and DMBA in the comet assay mainly represent the activity of an excision repair process.
4-Nitroquinoline-1-oxide↗
The effect of defective DNA double-strand break repair on mutations and chromosome aberrations in the Chinese hamster cell mutant XR-V15B.
The radiosensitive Chinese hamster cell line XR-V15B was used to study the effect of decreased rejoining of DNA double-strand breaks (DSBs) on gene mutations and chromosome aberrations. XR-V15B cells are hypersensitive to the cytotoxic effects of neocarzinostatin (NCS) and methyl methanesulfonate (MMS). Both mutagens induced more chromosome aberrations in XR-V15B cells than in the parental cell strain. The clastogenic action of NCS was characterized by the induction of predominantly chromosome-type aberrations in cells of both strains, whereas MMS induced mainly chromatid aberrations. The frequency of induced gene mutations at the hprt locus was not increased compared to the parental V79 cells when considering the same survival level. Molecular analysis by multiplex polymerase chain reaction (PCR) of mutants induced by NCS revealed a high frequency of deletions in cells of both cell lines. Methyl methane-sulfonate induced mainly mutations without visible changes in the PCR pattern, which probably represent point mutations. Our findings suggest a link between a defect in DNA DSB repair and increased cytotoxic and clastogenic effects. However, a decreased ability to rejoin DNA DSBs does not seem to influence the incidence and types of gene mutations at the hprt locus induced by NCS and MMS.
Animals↗
Comparative investigations of the genotoxic effects of metals in the single cells gel (SCG) assay and the sister chromatid exchange (SCE) test.
Sodium arsenite (NaAsO2) and cadmium sulphate (CdSO4) were tested for their ability to induce genotoxic effects in the single cell gel (SCG) assay and the sister chromatid exchange (SCE) test in human blood cultures in vitro. Both metals induced DNA damage in white blood cells that was expressed and detected as DNA migration in the SCG assay. Dose dependent effects were seen for cadmium in concentrations from 5 x 10(-4)-5 x 10(-3) M and for arsenic in concentrations from 2 x 10(-4)-1.5 x 10(-3) M. The distribution of DNA migration among cells, a function of dose, revealed that the majority of exposed cells expressed more DNA damage than cells from control cultures and that with increasing length of DNA migration the variability in migration among cells increased as well. Treatment of cells for 2 hr or 24 hr beginning 48 hr after the start of the blood cultures did not increase the SCE frequency in the case of cadmium but caused a small but significant SCE induction with arsenic at the highest concentration. The metal concentrations which could be investigated in the SCE test were much lower due to a strong toxic effect. Metal concentrations which were toxic in the SCE test were without visible effect in the SCG assay. Thus the two endpoints for the determination of genotoxic effects in vitro differed markedly with respect to the detection of genotoxicity induced by metals. These differences and the biological significance of the findings are discussed.
Arsenites↗
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.
Ataxia Telangiectasia↗
Differences in the response to mutagens between two V79 sublines.
Two V79 Chinese hamster sublines (V79-UL and V79-MZ) which differed markedly with respect to their spontaneous pattern of mutations at the HPRT locus were comparatively investigated in genotoxicity tests with ethyl methanesulphonate (EMS). EMS-induced frequencies of HPRT mutations and sister chromatid exchanges (SCEs) were much higher in V79-MZ than in V79-UL. V79-MZ were not hypersensitive against EMS and had normal frequencies of spontaneous gene mutations and chromosome aberrations. Baseline SCE frequencies at various BrdUrd concentrations were slightly increased compared to V79-UL. EMS induced a similar amount of chromosome aberrations in both cell lines but exchange figures occurred with lower frequency in V79-MZ. The results indicate that specific and significant differences in the response to mutagens may exist between 'normal' Chinese hamster cell lines which might be relevant for genotoxicity testing.
Animals↗
The molecular nature of mutations induced by adriamycin at the hprt locus of V79 cells.
Adriamycin (AM), a widely used chemotherapeutic drug, induced a broad spectrum of gene mutations at the hprt locus of V79 cells. The frequency and distribution of AM-induced deletions was analyzed with multiplex polymerase chain reaction in two V79 cell lines, which differed considerably in their spontaneous deletion frequency. Among AM-induced mutants, deletions predominated in both cell lines. Apart from total deletions of the hprt gene, partial deletions were found which were distributed all over the hprt gene with breakpoints in nearly all introns. Under the same experimental conditions, chromosome aberrations were induced by AM which mainly represented chromatid-type aberrations. Neither the induction of gene mutations nor the induction of chromosome aberrations was enhanced by the repair inhibitor 3-aminobenzamide. These results are discussed in the context with our earlier findings on bleomycin-induced mutations and it is suggested that at least two mechanisms lead to the formation of gene deletions. One of them seems to be associated with a misrepair process of frank DNA double-strand breaks and related to chromosome aberrations while the other is not.
Animals↗
Does physical activity induce DNA damage?
The single cell gel electrophoresis (SCG) assay (comet assay) is a sensitive technique for detecting the presence of DNA strand-breaks and alkali-labile damage in individual cells. This technique was used to study peripheral blood cells from three volunteers after physical activity. The test subjects had to run on a treadmill and were checked for blood pressure and ECG, lactate concentration and creatine kinase activity. Blood was taken before and several times during and after the run. In a first multiple step test, the volunteers ran as long as possible with increasing speed. In a second test they had to run for 45 min with a fixed individual speed which was defined to ensure an aerobic metabolism. In the first test, the white blood cells of all subjects showed increased DNA migration in the SCG assay. The effect was seen 6 h after the end of the exercise and reached its maximum 24 h later. After 72 h, DNA migration decreased to about control level. The distribution of DNA migration among cells clearly demonstrated that the majority of white blood cells exhibited increased DNA migration and that the effect was not only due to a small fraction of damaged cells. From the same blood samples, blood cultures were set up to study a possible effect on the frequency of sister chromatid exchanges (SCE), another indicator for genotoxic effects. However, there was no significant increase in SCE in any of the cultures. In the second exercise, during aerobic metabolism, the effect on DNA migration was not seen.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗