Liquid-holding experiments with human lymphocytes: III. Experiments with G0 and G1 cells

Liquid holding (LH) experiments were performed with human peripheral lymphocytes treated in the GO (G0-LH) or the G1 (G1-LH) phase of the cell cycle with diepoxybutane (DEB) or methylnitrosourea (MNU). In the G0-LH system, treatment with DEB but not with MNU led to a lowering of the frequencies of sisterchromatid exchanges (SCE). In the G1-LH system treatment with both chemicals led to a lowering of the SCE frequencies during the LH. These results are concluded to mean that lesions induced by DEB but not by MNU can be repaired in GO cells and that G1 cells can repair both DEB and MNU induced lesions.     

Effects of 50-Hertz electromagnetic fields on proliferation and on chromosomal alterations in human peripheral lymphocytes untreated or pretreated with chemical mutagens

Cultivation of human peripheral lymphocytes (HPL) in the presence of 50Hz electromagnetic fields (EMFs) does not alter the spontaneous frequencies of sister-chromatid exchanges (SCE) and of chromosomal aberrations (CA), but leads to an enhancement of the cell cycle progression of HPLs in vitro. Pretreatment of HPLs with trenimon (TRN), diepoxybutane (DEB), or methylnitrosournea (MNU) in the G₀ phase of the cell cycle results in dose-dependent elevations of the SCE frequencies. In some cases culturing of HPLs pretreated with MNU or TRN in the presence of EMFs led to significantly higher frequencies of SCEs when compared to cells cultivated in the absence of EMDs. Since we did not use multiple fixation times these data may rather result from differential influences on HPL subsets than from EMF exposure.     

In vivo repair during G1 of DNA lesions eliciting sister chromatid exchanges induced by methylnitrosourea or ethylnitrosourea in BrdU substituted or unsubstituted DNA in murine salivary gland cells

The difference in efficiency of methylnitrosourea (MNU) and ethylnitrosourea (ENU) to induce SCE in early or late G1 was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation during G1. The repair during G1 was determined by treating the cells in early or late G1. Treatment was in the first cycle (G1 before incorporation of 5-bromodeoxyuridine (BrdU)) or in G1 of the second cycle (after a single round of BrdU incorporation). It was observed that 50% of the lesions induced by MNU that elicit SCE are repaired during G1. BrdU incorporation into DNA increases the sensitivity of the cell to SCE induction by MNU nearly 40%; however under this circumstance a slightly lower SCE frequency was observed in the cells exposed to MNU at early G1, indicating that during G1 only few lesions are repaired. The ENU-induced DNA-lesions involved in SCE production are nearly 100% persistent along G1; besides, a slight but significantly higher SCE frequency was observed in cells exposed at early G1, suggesting the formation of SCE-inducing lesions during G1. BrdU incorporation to DNA sensitizes the cell to SCE induction by ENU, increasing the SCE frequency to nearly to a 40%, although these additional lesions involved in SCE induction seem to be susceptible to repair during G1.     

Use of three-way differential staining and liquid holding for the assessment of individual repair capacity

Many studies on DNA repair mechanisms in mammalian cells have used liquid holding (LH) recovery to evaluate premutational damage repair. We used human peripheral lymphocytes (HPL) to assess damage reduction during the G0 phase. This technique was matched with the three-way differential (TWD) staining that allows identification of sister-chromatid exchanges (SCE) per cell cycle in third metaphases. By adopting this approach, the persistence of diepoxybutane (DEB)-induced lesions during subsequent cycles and individual repair capacity in LH conditions were measured. Our results show that most DEB-induced damage was repaired during the first cell cycle; a large part of lesions were removed during LH recovery, demonstrating G0 HPL repair capacity.     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.     

Comparison of cytogenetic effects of 3,4-epoxy-1-butene and 1,2:3, 4-diepoxybutane in mouse, rat and human lymphocytes following in vitro G0 exposures

To understand better the species differences in carcinogenicity caused by 1,3-butadiene (BD), we exposed G0 lymphocytes (either splenic or peripheral blood) from rats, mice and humans to 3, 4-epoxy-1-butene (EB) (20 to 931 microM) or 1,2:3,4-diepoxybutane (DEB) (2.5 to 320 uM), two of the suspected active metabolites of BD. Short EB exposures induced little measurable cytogenetic damage in either rat, mouse, or human G0 lymphocytes as measured by either sister chromatid exchange (SCE) or chromosome aberration (CA) analyses. However, DEB was a potent inducer of both SCEs and CAs in G0 splenic and peripheral blood lymphocytes. A comparison of the responses among species showed that the rat and mouse were approximately equisensitive to the cytogenetic damaging effects of DEB, but the situation for the human subjects was more complex. The presence of the GSTT1-1 gene (expressed in the erythrocytes) reduced the relative sensitivity of the lymphocytes to the SCE-inducing effects of DEB. However, additional factors also appear to influence the genotoxic response of humans to DEB. This study is the first direct comparison of the genotoxicity of EB and DEB in the cells from all three species.     

Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes. I. Correlation with chromosomal aberrations

We carried out a cross-sectional analysis of sister-chromatid exchanges (SCEs) and chromosomal aberrations induced by diepoxybutane (DEB) in lymphocyte cultures from 58 normal blood donors. DEB-induced SCE frequencies were measured in all subjects and chromosomal aberrations in 18. Analysis of variance was used to assess the contributions of exposure to organic solvents, age, smoking history, alcohol and coffee consumption, and red and white blood cell counts to variations in DEB-induced SCEs. In 10 individuals, the epoxide-detoxifying enzyme, glutathione (GSH)-S-transferase mu, was also measured. We observed a bimodal distribution of DEB-induced SCEs in the study population. Approx. 24% of the individuals were twice as sensitive to the induction of SCEs by DEB as the remaining 76%. Lymphocytes from persons sensitive to SCE induction by DEB contained a 4.4-fold increase in the number of DEB-induced chromatid deletions and exchanges. Within sensitive and resistant groups, significant interindividual variations in DEB-induced SCE frequencies were noted. Cigarette smoking was weakly associated with lower SCE frequencies within each group. Genetic deficiency in GSH-S-transferase mu was not correlated with increased sensitivity to SCE induction by DEB. Sensitivity to induction of SCEs by DEB can be rapidly determined and may be a marker of sensitivity to the induction of genotoxicity by certain classes of mutagens.     

Induction and rapid repair of sister-chromatid exchanges in multiple murine tissues in vivo by diepoxybutane

Sister-chromatid exchange (SCE) induction by the direct-acting bifunctional carcinogen, diepoxybutane (DEB), was investigated in multiple tissues in vivo. The log-log dose SCE response relationship was found to be parallel to that previously reported for DEB induction of lung adenomas. However, the SCE assay is approximately 20 times as sensitive in detecting genotoxic effects of DEB than indicated by the lung adenoma assay. Examination of second and third division cells following various treatment protocols revealed that regardless of the nature of initially induced lesions, they are rapidly repaired with no evidence of persistence beyond 1 cell cycle.     

Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes. II. Relationship to baseline SCE frequency

Environmental and genetic factors have been implicated as important sources of individual variation in baseline sister-chromatid exchange (SCE) frequency in humans. The current study was designed to test whether the frequency of baseline SCEs in 58 normal blood donors is associated with previously observed variations in SCE frequencies induced by diepoxybutane (DEB). Because 12 subjects were current cigarette smokers and smoking is known to be an in vivo inducer of baseline SCE frequencies, we specifically tested whether higher baseline SCE frequencies in smokers would be associated with in vitro sensitivity to SCE induction by DEB. Analysis of variance showed that DEB-induced SCE frequencies were significantly associated with baseline SCE frequencies; those who were sensitive to SCE induction by DEB were more likely to have higher baseline SCE frequencies. This effect, however, was independent of in vivo induction of SCE by smoking. Chromosomal sensitivity to the induction of SCE by DEB explained approx. 15-20% of the variation in baseline SCE. This was similar in magnitude to the effect of cigarette smoking. Because increased sensitivity to DEB-induced SCEs is common in normal blood donors (approx. 24%) and is associated with an increase in baseline SCEs, it should be investigated as a source of bias and/or a potential marker of sensitivity to environmental mutagens in future cytogenetic studies.     

Strand breaks arising from the repair of the 5-bromodeoxyuridine-substituted template and methyl methanesulphonate-induced lesions can explain the formation of sister chromatid exchanges

5-Bromodeoxyuridine (BrdU)-induced sister chromatid exchanges (SCEs) are mainly determined during replication on a BrdU-substituted template. The BrdU, once incorporated, is rapidly excised as uracil (U), and the gap is repaired with the incorporation of BrdU from the medium, which leads to further repair. During the second S period in BrdU medium, this process continues as the strand acts as template. Experiments suggest that 3-amino-benzamide (3AB) delays the ligation of the gaps formed after U excision, resulting in enhanced SCE levels during the second cycle of BrdU incorporation. When normal templates of G1 cells are treated before BrdU introduction with methyl methanesulphonate (MMS), 3AB in the first cycle doubles the MMS-induced SCEs but has no effect on them during the second cycle. When the BrdU-substituted template is treated with MMS in G1 of the second cycle, 3AB again doubles the SCEs due to MMS and also enhances the SCEs resulting from delays in ligation of the gaps following U excision in the BrdU-substituted template. The repair processes of MMS lesions that are sensitive to 3AB and lead to SCEs take place rapidly, while the repair process of late repairing lesions that lead to SCEs appear to be insensitive to 3AB. A model for SCE induction is proposed involving a single-strand break or gap as the initial requirement for SCE initiation at the replicating fork. Subsequent events represent natural stages in the repair process of a lesion, ensuring replication without loss of genetic information. G1 cells treated with methylnitrosourea (MNU) and grown immediately in BrdU medium rapidly lose the O⁶-methylguanine from their DNA and the rate of loss is BrdU-dose dependent. The rapid excision of the U lesions can explain the effect of BrdU concentration on SCE reduction following both MNU or MMS treatment.     

Reduction of diepoxybutane-induced sister chromatid exchanges by glutathione peroxidase and erythrocytes in transgenic Big Blue mouse and rat fibroblasts

We have investigated the effect of glutathione peroxidase (GSH-Px) and mammalian erythrocytes (RBCs) on spontaneous and diepoxybutane (DEB)-induced sister chromatid exchange (SCE) in primary Big Blue(R) mouse (BBM1) and Big Blue(R) rat (BBR1) fibroblasts. DEB is the putative carcinogenic metabolite of 1,3-butadiene (BD) for which inhalation exposure yields a high rate of malignancies in mice but not in rats. BD is metabolized differently in mice and rats, producing much higher levels of DEB in mice than in rats, which may partly explain the different carcinogenic responses. However, other factors may contribute to the observed differences in the rodent carcinogenic response to BD. DEB is a highly reactive compound. Upon epoxide hydrolysis, DEB can covalently bind to DNA bases. Likewise, DEB generates reactive oxygen species that, in turn, can either damage DNA or produce H(2)O(2). Reduced glutathione (GSH) is known to play a role in the metabolism and detoxification of DEB; and GSH is reduced by GSH-Px in the presence of H(2)O(2). GSH-Px is a constitutive enzyme that is found at high concentrations in mammalian RBCs. Therefore, we were interested in examining the role of RBCs and GSH-Px on DEB-induced SCE in rat and mouse cells for detection of possible differences in the species response. Transgenic BBM1 and BBR1 fibroblasts were treated with either 0, 2 or 4 microM DEB plus 0, 2 or 20 units of GSH-Px with and without 2x10(8) species-specific RBCs. DEB effectively induced SCEs in both rat and mouse cells. The relative induction of SCEs in both cell types was comparable. Both GSH-Px and RBCs alone and in combination were effective in significantly reducing DEB-induced SCEs in both mouse and rat fibroblasts, although there was more variability in the SCE response in rat cells. The present study suggests that GSH-Px may be important in the detoxification of DEB-induced DNA damage that results in the formation of SCEs.     

Effects of repair inhibition in the G1 phase of clastogen-treated human lymphocytes on the frequencies of chromosome-type and chromatid-type aberrations and sister-chromatid exchanges

It has been shown that certain types of DNA lesions induced by an S-dependent clastogen are converted to chromosome-type aberrations when their repair is inhibited in the G1 phase of the cell cycle. The purpose of the present study was to investigate which kinds of repair inhibitors have the ability to induce chromosome-type aberrations in cells having DNA lesions and which kinds of DNA lesions will be converted to chromosome-type aberrations when their repair is inhibited. For this purpose, human peripheral blood lymphocytes, which were treated with a clastogen in their G0 phase, were post-treated with one of several kinds of repair inhibitors in the G1 phase, and resulting frequencies of both chromosome-type and chromatid-type aberrations as well as of sister-chromatid exchanges (SCEs) were compared with those of the control cultures: chromatid-type aberrations and SCEs were adopted as cytogenetic indicators of lesions remaining in S and G2 phases. Chemicals used for the induction of DNA lesions were 4-nitroquinoline 1-oxide (4NQO), methyl methanesulfonate (MMS) and mitomycin C (MMC); inhibitors used were excess thymidine (dThd), caffeine, hydroxyurea (HU), 5-fluoro-2'-deoxyuridine (FdUrd), 1-beta-D-arabinofuranosylcytosine (ara C), 9-beta-D-arabinofuranosyladenine (ara A), 1-beta-D-arabinofuranosylthymine (ara T) and aphidicolin (APC). Induction of chromosome-type aberrations was observed in cells pretreated with 4NQO or MMS followed by ara C, ara A, ara T or APC, whereas other combinations of a clastogen and an inhibitor did not induce them. Among the inhibitors, ara C alone induced chromosome-type aberrations in cells without pretreatment. Chromatid-type aberrations were increased only in cells pretreated with MMC and their frequency was enhanced further by post-treatment with ara C. All of the clastogens used in the present experiments induced SCEs. Most inhibitors did not modify the SCE frequencies except for ara C which synergistically increased the frequency in MMC-treated cells. The present study offers further evidence that the lesions responsible for chromosome-type aberrations are those which are repaired quickly, and that they are converted to chromosome-type aberrations when repair by polymerase alpha is inhibited. The effects of ara C on MMC-induced lesions are considered residual effects of ara C treatment in the S or G2 phases rather than repair inhibition in the G1 phase.     

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 V 79 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.     

Survival and liquid holding recovery in UV-sensitive strains of Saccharomyces cerevisiae after treatment with chemical mutagens and radiation.

Two UV-sensitive mutants of Saccharomyces cerevisiae rad 3 and rad 6 were tested for sensitivity to X-rays, MMS, EMS, HNO2 and DEB. Rad 3 mutant is more sensitive than the wild type strain only to HNO2 and DEB, while rad 6 is cross sensitive both to X-rays and all chemicals tested. Liquid holding recovery (LHR) was studied by comparison of cell survival immediately after mutagen treatment and after 5 days of storage in phosphate buffer. LH greatly increases cell survival of rad 3 mutant after DEB and slightly after EMS, MMS and HNO2, while after UV treatment LH significantly decreases survival of this mutant. LH increases survival of rad 6 mutant after exposure to UV, MMS and HNO2, but decreases survival of DEB-treated cells. Exposure of wild type strain to LH results in an increase of survival after UV, and DEB but not after MMS and HNO2. The results suggest that LHR is a strain- and mutagen-specific phenomenon and cannot be explained within the present knowledge of repair processes in yeast.     

The influence of GSTM1 and GSTT1 genotypes on the induction of sister chromatid exchanges and chromosome aberrations by 1,2:3,4-diepoxybutane

Cytogenetic tests - chromosome aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) - are most often applied in biomonitoring of the genotoxicity of potentially carcinogenic chemicals in human cells. One of the extensively studied genotoxins is diepoxybutane (DEB) - reactive biometabolite of butadiene (BD). Several studies showed a high SCE induction in human lymphocytes exposed in vitro to various concentrations of DEB. DEB also proved to be a potent inducer of chromosome aberrations and micronuclei. A bimodal distribution of SCE frequency after in vitro DEB treatment was observed. The aim of the present study was to examine the ability of DEB to induce different individual cytogenetic response measured by SCE and CA frequency. The possible influence of genetic polymorphism has also been taken into account, by including donors representing positive or null GSTM1 and GSTT1 genotypes. Our study supported the earlier results showing that DEB is an effective inducer of SCEs and CAs, causing also the decrease in replication index (RI). DEB bioactivity measured by SCE induction - but not by CA test - was significantly higher in GSTT1 negative than in GSTT1 positive donors. GSTM1 polymorphism had no influence on these endpoints. The donors GSTT1-/GSTM1+ were shown to be slightly more sensitive to DEB than GSTT1-/GSTM1- individuals. There was also observed a unimodal distribution of DEB-induced SCEs and CAs in the group, despite the fact that the experiment was performed on the lymphocytes obtained from both GSTT1 positive and negative donors.     

Some insights into the mode of action of butadiene by examining the genotoxicity of its metabolites

1,3-Butadiene (BTD) is an important commodity chemical and air pollutant that has been shown to be a potent carcinogen in mice, and to a lesser extent, a carcinogen in rats. To better assess butadiene's carcinogenic risk to humans, it is important to understand its mode of action and how this relates to differences in responses among species. In a series of in vitro experiments, lymphocytes from rats, mice, and humans were exposed to 3,4-epoxy-1-butene (EB) or 1,2:3,4-diepoxybutane (DEB) for 1h at the G(0) stage of the cell cycle, stimulated to divide, and cultured to assess the ability of these metabolites to induce sister chromatid exchange (SCE) and chromosome aberrations (CAs). EB induced no increases in SCEs or CAs in the cells from the three species. DEB was a potent SCE- and CA-inducer, with the results being similar in each rodent species. The response for SCEs seen in the human cells was more complex, with genetic polymorphism for glutathione-S-transferases (GST) possibly modulating the response. The single cell gel electrophoresis assay was used on genetically engineered V79 cell lines to investigate a possible influence of GST status. Experiments were also conducted to investigate the reason for EB's failure to induce SCEs or CAs in G(0) cells. The results indicate that EB-induced DNA damage was repaired before DNA synthesis in unstimulated lymphocytes, but EB caused a large increase in SCEs if actively cycling cells were treated. Thus, the results indicate that DEB damage is persistent in G(0) cells, and DEB is a much more potent genotoxicant than EB. The carcinogenic effect of butadiene will most likely depend on the degree to which DEB is produced and reaches target tissues, and to a lesser extent on the ability of EB to reach actively dividing or repair deficient cells.     

The action of N-methyl-N-nitrosourea on non-established human cell lines in vitro. I. Cell cycle inhibition and aberration induction in diploid and Down's fibroblasts.

The effect of N-methyl-N-nitrosourea (MNU) on the cell cycle, DNA synthesis and chromosomal sensitivity of cultivated diploid fibroblasts and fibroblasts with trisomy 21 was investigated in vitro. With the exception of the inhibition of G2, Down's cells proved to be more sensitive than diploid cells with respect to the decrease of the mitotic and labelling index, the inhibition of the progression of cells through the early and middle S and the frequency of induced chromosomal aberrations. The chromosomal sensitivity was dependent on the position of cells in the cell cycle during treatment with MNU. If treated during late S no differences concerning the S block and aberration frequencies were found between diploid and Down's cells. However, if MNU treatment took place in the middle and early S, Down's cells were more sensitive. The higher aberration frequencies in Down's cells resulted from elevated levels of chromatid breaks, multiple fragmentations and chromatid translocations. Possible reasons for the increased sensitivity of Down's cells are discussed.     

Comparison of sensitivity and liquid holding recovery in rad mutants of Saccharomyces cerevisiae inactivated by UV and DEB.

Summary Twenty one UV-sensitive rad mutants were tested for their sensitivity towards DEB. All mutants were more sensitive to this treatment than the wild type. Seven mutants were classified as supersensitive to DEB (radl-1, 2, 3, 6, 15 and 18-2), while only rad2 and rad3 can be classified as supersensitive to UV. For all mutants ability for liquid holding recovery (LHR) after UV and DEB was compared. Mutants radl-1, 3, 5, 6, 9 and 11 differ in their response to LH afterr the two treatments. Survival of radl-1 and rad3 increases significantly during LH after DEB but not after UV exposure. In contrast rad5, 6, 11 and 22 show marked LHR after UV but no increase of survival after DEB treatment.     

Clastogenic effects of methylnitrosourea and ethylnitrosourea on chromosomes from human fibroblast cell lines.

Human fibroblast cell lines were pulse-treated for 1 h with either methylnitrosourea (MNU) or ethylnitrosourea (ENU) at various time intervals before harvesting for chromosome analysis. Cells treated with 1 X 10(-3) M, 5 X 10(-4) M, and 1 X 10(-4) M final concentrations of MNU and ENU during the G2 or M phases of the cell cycle showed a significant increase in chromatid-type abnormalities over controls. Cells exposed to MNU or ENU 23 h before harvest showed some chromosome-type abnormalities, reflecting probable damage induced during the G1 phase of the cell cycle or derived from chromatid damage induced during the previous cell cycle. The mitotic indices and incidences of abnormalities suggested a dose response effect when cells were treated with the two higher concentrations and the three concentrations, respectively, of MNU or ENU. Chromatid abnormalities were observed in MUN and ENU-treated cells from each of four cell lines. From this investigation, it was concluded that MNU and ENU treatment of human diploid cell lines in vitro induced both chromatid and chromosome aberrations. MNU and ENU, both of which had previously been shown to be mutagenic in experimental animals, are, therefore, also considered to be mutagenic at the chromosome level in human fibroblasts grown and treated in cell culture.