Higher frequency of chromosome aberrations in late-arising first-division metaphases than in early-arising metaphases after exposure of human lymphocytes to X-rays in G0.

PURPOSE: To determine whether metaphases arising at different times after mitogen stimulation of G0 lymphocytes differ in frequencies of X-ray-induced chromosome aberrations. MATERIALS AND METHODS: Human G0 lymphocytes from peripheral blood exposed to 0, 1.5 or 3.0 Gy X-rays were stimulated to divide with the mitogen phytohaemagglutinin (PHA). First-division metaphases were distinguished from second and third divisions by chromatid labelling with 5-bromodeoxvuridine (BUdR) and staining with Giemsa or DAPI. Cultures harvested 48, 70 and 94 h after mitogen stimulation were analysed for unstable aberrations on Giemsa-stained slides and for stable and unstable aberrations by fluorescence in situ hvbridization (FISH) with painting probes for chromosomes 1, 2 and 4. RESULTS: Frequencies of aberrations declined at the later culture periods, as expected on the basis of unstable aberrations being lost in mitotic division. Whe n scoring was restricted to firstdivision metaphases, however, aberration frequencies were higher in 94-h cultures than in 48-h cultures. CONCLUSIONS: Frequencies of radiation-induced chromosome aberrations in first-division metaphases increase with culture time after mitogen stimulation. Possible explanations for this finding are a delay of damaged cells in mitogenic response or progression through divisions and heterogeneity among lymphocytes in culture kinetics and radiosensitivity. The data argue against the common assumption that all first-division cells are equivalent as indicators of radiation-induced chromosome aberrations.

Induction of -2 frameshift mutations by 2-nitrofluorene, N-hydroxyacetylaminofluorene, and N-2-acetylaminofluorene in reversion assays in Escherichia coli strains differing in permeability and acetyltransferase activity.

The mutagenicity of 2-nitrofluorene (NF), N-hydroxyacetylaminofluorene (N-OH-AAF), and N-2-acetylaminofluorene (AAF) was measured in strains of Escherichia coli that contain a lacZ allele that reverts by -2 frameshift mutations from CG(5) to CG(4). Mutagenesis was compared in a strain having wild-type permeability and metabolism, a strain with increased permeability caused by a lipopolysaccharide-defective (LPS(d)) mutation, a strain with N- and O-acetyltransferase (NAT/OAT) activity conferred by the Salmonella nat gene on plasmid pYG219, and a strain carrying both an LPS(d) mutation and pYG219. The LPS(d) mutation facilitated the measurement of mutagenicity but was not absolutely required, in that lower levels of mutagenicity were detected in LPS(+) strains. The NAT/OAT activity conferred by pYG219 strongly potentiated the mutagenicity of NF and N-OH-AAF. Surprisingly, AAF was mutagenic in the NAT/OAT LPS(d) strain without an exogenous P450 metabolic activation system. Its activity may be ascribable to the detection of a directly mutagenic impurity by the highly sensitive strain or to a low level of metabolic activation by the bacteria under the assay conditions. The findings add to the evidence that the lacZ allele derived from E. coli strain CC109 is an effective indicator of -2 frameshift mutagenesis and that strains expressing high levels of NAT/OAT activity are highly sensitive in monitoring the mutagenicity of nitroarenes and aromatic amides.

Structure-activity analysis of the potentiation by aminothiols of the chromosome-damaging effect of bleomycin in G0 human lymphocytes.

The radioprotective aminothiols 2-[(aminopropyl)amino] ethanethiol (WR-1065) and cysteamine (CSM) potentiate the induction of chromosomal damage by the radiomimetic compound bleomycin (BLM) in G0 human lymphocytes. To investigate the mechanism of potentiation, we measured the clastogenic activity of BLM in the cytokinesis-block micronucleus assay in the presence and absence of amines, thiols, and aminothiols. The hydroxy analog of WR-1065, 2-(3-aminopropylamino) ethanol (WR-OH), potentiates BLM only slightly, indicating the critical nature of the thiol group. As thiols, WR-1065 and CSM may donate electrons for the activation of Fe(+2)-BLM or for the regeneration of Fe(+2)-BLM from inactive Fe(+3)-BLM. The amines putrescine, spermidine, and spermine all potentiate BLM, but they are weaker potentiators than the aminothiols, and they are effective only at high concentrations. Their activity, like that of WR-OH, is probably a consequence of conformational alteration of DNA. Dithioerythritol (DTE) and 2-mercaptoethanol (2-ME), thiols lacking an amino group, are less effective potentiators of BLM than are the aminothiols. The thiol group of WR-1065 and CSM is therefore essential, but insufficient, for explaining the strong enhancement of BLM activity. The cationic nature of CSM and WR-1065, conferred by the amino groups, evidently concentrates the active thiol function at the site of BLM action on DNA. As expected on this basis, the diamine WR-1065 is a more effective potentiator of BLM than is the monoamine CSM, whereas cysteine and N-acetylcysteine (NAC), which lack a net positive charge, potentiate BLM only weakly. These studies suggest that potentiation of the clastogenic action of BLM by aminothiols can be explained by the combination of a thiol-mediated redox mechanism and an amine-mediated targeting of the thiol function to DNA.

Analysis by FISH of the spectrum of chromosome aberrations induced by X-rays in G0 human lymphocytes and their fate through mitotic divisions in culture.

The induction, distribution, and persistence of chromosome aberrations in human lymphocytes exposed to X-rays in G0 were analyzed in 48-, 70-, and 94-hr cultures by conventional metaphase analysis and painting of chromosomes 1, 2, and 4 by FISH. All cells that had been scored by FISH were relocated to determine by differential staining of chromatids whether they had passed through 1, 2, or > or =3 divisions. FISH revealed a dose-dependent induction of stable and unstable aberrations, while chromatid labeling showed mitotic lag caused by irradiation in G0. Relative to their DNA contents, there was a small but significant overrepresentation of chromosome 4 and underrepresentation of chromosome 2 among the aberrations involving chromosomes 1, 2, and 4. FISH slightly underestimated the genomic frequency of unstable aberrations measured by conventional metaphase analysis. There was a slight excess of translocations relative to dicentrics, but the data are compatible with the 1:1 ratio expected from cytogenetic theory. Many of the translocations were apparently incomplete (i.e., nonreciprocal). Incomplete translocations were more frequent at higher X-ray dose and in first division, suggesting that they may be associated with complex damage and are more apt to be lost in mitosis than complete translocations. Among the incomplete translocations, t(Ab) outnumbered t(Ba) -- a difference ascribable to the FISH technique. Aberration frequencies declined as the cells divided in culture. The overall decline in the frequency of aberrant cells (approximately 29% per cell generation) reflects a rapid decline in dicentrics and fragments (approximately 60% per cell generation) and the relative stability of translocations. The frequency of translocation-bearing cells underwent a modest decline in culture (approximately 13% per cell generation).

Two mechanisms of antimutagenicity of the aminothiols cysteamine and WR-1065 in Saccharomycescerevisiae.

The aminothiols cysteamine (CSM) and 2-[(aminopropyl)amino] ethanethiol (WR-1065) are radioprotectors, in that they reduce the mutagenic and potentially lethal consequences of ionizing radiation. We have studied the effects of these compounds on the induction of gene conversion at the trp5 locus in Saccharomyces cerevisiae strain D7 by two chemical mutagens: bleomycin (BLM) and beta-propiolactone (beta-PL). Both mutagens are potent recombinagens in this assay, giving rise to trp5 convertant frequencies greater than 10(-3). CSM and WR-1065 are effective antimutagens, reducing the recombinagenic effect of beta-PL by about 95%. The maximum reduction in the recombinagenic activity of BLM was 91% with CSM and 89% with WR-1065. Although the antimutagenic effects of the aminothiols against beta-PL and BLM appear to be similar, they stem from different underlying mechanisms. Aminothiols protect against the recombinagenicity of beta-PL through direct inactivation of the electrophilic mutagen by the nucleophilic antimutagen. They protect against that of BLM through modification of physiological conditions, most notably by depletion of oxygen required for BLM activity.

Modulation of bleomycin-induced mitotic recombination in yeast by the aminothiols cysteamine and WR-1065.

The cancer chemotherapy drug bleomycin (BLM) is a potent inducer of genetic damage in a wide variety of assays. The radioprotectors cysteamine (CSM) and WR-1065 have been shown in previous studies to potentiate the induction of micronuclei and chromosome aberrations by BLM in Go human lymphocytes. By contrast, WR-1065 is reported to reduce the induction of hprt mutations by BLM in Chinese hamster cells. To elucidate the basis for these interactions, we examined the effects of CSM and WR-1065 on the induction of mitotic gene conversion by BLM in the yeast Saccharomyces cerevisiae. Treatment with BLM causes a dose-dependent increase in the frequency of mitotic gene conversion and gene mutations. Unlike its potentiation of BLM in Go lymphocytes, WR-1065 protected against the recombinagenicity of BLM in yeast. CSM was also strongly-antirecombinagenic under, some conditions, but the nature of the interaction depended strongly on the treatment conditions. Under hypoxic conditions, cysteamine protected against BLM, but under oxygen-rich conditions CSM potentiated the genetic activity of BLM. The protective effect of aminothiols against BLM may be ascribed to the depletion of oxygen required for the activation of BLM and the processing of BLM-induced damage. Aminothiols may potentiate the effect of BLM by acting as an electron source for the activation of BLM and/or by causing conformational alterations that make DNA more accessible to BLM. The results indicate that aminothiols have a strong modulating influence on the genotoxicity of BLM in yeast as they do in other genetic assays. Moreover, the modulation differs markedly depending on physiological conditions. Thus, yeast assays help to explain why aminothiols have been observed to potentiate BLM in some genetic systems and to protect against it in others.

Enhancement of the activity of bleomycin by cysteamine in a micronucleus assay in G0 human lymphocytes.

The aminothiol cysteamine enhances the induction of micronuclei by bleomycin in G0 human lymphocytes. The potentiation of bleomycin (12.5, 25, 50, or 100 micrograms/ml) increased with cysteamine concentration from 5 to 20 mM in a 2-h treatment before culturing the cells for the cytokinesis-block assay. The maximum clastogenic activity of bleomycin in the presence of cysteamine was more than 10-fold greater than that of the same dosage of bleomycin alone. Both the thiol and amine functions of aminothiols seem to contribute to the potentiation of bleomycin.

Potentiation of bleomycin by the aminothiol WR-1065 in assays for chromosomal damage in G0 human lymphocytes.

The aminothiol radioprotector WR-1065 potentiates the induction of chromosome aberrations and micronuclei by the chemotherapy drug bleomycin in G(0) human lymphocytes. Potentiation by 5 mM WR-1065 was observed at bleomycin concentrations from 0.1 to 100 micrograms/ml in a 2-h treatment. The frequencies of micronuclei induced by bleomycin in the presence of WR-1065 reached that of 500-fold higher concentrations of bleomycin alone. The potential therapeutic implications of these findings are discussed.

Induction of genetic recombination: consequences and model systems.

Radiation and many chemicals have been found to induce homologous genetic recombination. Experimental systems that allow the detection and characterization of recombinagens exist in organisms as diverse as bacteria, fungi, plants, insects, and mammals. Recombination plays an important role in many biological processes, and studies of recombinagens can provide insight into underlying mechanisms. Studies of recombinagens are also of applied interest in genetic toxicology, because recombinational events in somatic cells can contribute to human disease. Clear connections have been established between mitotic recombination and the etiology of some cancers. This article briefly reviews two aspects of the induction of genetic recombination by radiation and chemicals--the health implications of recombinagenic effects and assays for detecting recombinagens.

Induction of micronuclei by bleomycin in G0 human lymphocytes: I. Dose-response and distribution.

The cytokinesis-block micronucleus assay was used to investigate the induction of chromosomal damage by bleomycin in G0 human lymphocytes. A dose-dependent increase in the frequency of micronuclei was observed in binucleate cells, and the frequency approached 0.5 micronuclei per cell at the highest dosage tested. The distribution of micronuclei among cells was overdispersed, rather than fitting a Poisson distribution. Even at the highest dosage, more than two-thirds of the cells did not contain micronuclei, while some cells were highly damaged, containing more than 4 micronuclei per cell.

Induction of micronuclei by bleomycin in G0 human lymphocytes: II. Potentiation by radioprotectors.

Dimethylsulfoxide (DMSO) and WR-1065 are radioprotectors, in that they reduce the effectiveness with which ionizing radiation causes genetic damage. Unlike their protective effects with radiation, these agents potentiate the induction of micronuclei by bleomycin in the cytokinesis-block assay in G0 human lymphocytes. High concentrations of DMSO (1 M) are required to cause potentiation. In contrast, WR-1065 causes dose-dependent potentiation at relatively low concentrations (1.25 to 10 mM). Cytogenetic analysis supports the results from the micronucleus assay, showing higher levels of genetic damage induced by the combination of bleomycin with DMSO or WR-1065 than by bleomycin alone. Possible mechanisms of potentiation are proposed.

Bacterial assays for recombinagens.

Two principal strategies have been used for studying recombinagenic effects of chemicals and radiation in bacteria: (1) measurement of homologous recombination involving defined alleles in a partially diploid strain, and (2) measurement of the formation and loss of genetic duplications in the bacterial chromosome. In the former category, most methods involve one allele in the bacterial chromosome and another in a plasmid, but it is also possible to detect recombination between two chromosomal alleles or between two extrachromosomal alleles. This review summarizes methods that use each of these approaches for detecting recombination and tabulates data on agents that have been found to be recombinagenic in bacteria. The assays are discussed with respect to their effectiveness in testing for recombinagens and their potential for elucidating mechanisms underlying recombinagenic effects.

DNA sequence analysis of mutations induced by N-2-acetylamino-7-iodofluorene in plasmid pBR322 in Escherichia coli.

The spectrum of mutations induced by N-2-acetylamino-7-iodofluorene (AAIF) was analyzed in a forward mutation system based on mutagenesis directed to a small restriction fragment in the tetracycline resistance gene of plasmid pBR322. AAIF was found to induce frameshift mutations and base-pair substitutions at approximately equal frequencies. The frameshift mutations were mostly deletions of single base-pairs, but -2 frameshifts and +1 frameshifts were also detected. With one exception, the substitutions were transversions initiated at a G.C base-pair. Both frameshift mutations and transversions occurred preferentially at sites of repetitive guanine residues. Although AAIF and the related aromatic amines N-2-acetylaminofluorene (AAF) and N-2-aminofluorene (AF) all bind to the C-8 position of guanine, they have different effects on DNA conformation, and these differences are reflected in their mutation spectra. Previous studies have provided evidence that AAF adducts can trigger a B to Z conformational change in alternating GC sequences or displacement of the guanine by the fluorene ring in other sequences; the principal result is two classes of frameshift mutations. AF, whose DNA interaction involves outside binding rather than insertion and denaturation, primarily induces base-pair substitutions. AAIF adducts are chemically similar to AAF adducts, but the iodo group apparently hinders insertion of the fluorene ring into DNA. Consistent with this model, the mutation spectrum of AAIF combines properties of the mutation spectra of both AAF and AF.

Induction of genetic duplications and frameshift mutations in Salmonella typhimurium by acridines and acridine mustards: dependence on covalent binding of the mutagen to DNA.

The aroC321 allele permits positive selection for the detection of a large genetic duplication that arises in the Salmonella typhimurium chromosome by homologous recombination. Strains that contain both aroC321 and the hisC3076 allele were constructed so that the induction of genetic duplications and frameshift mutations in a run of GC base pairs could be studied simultaneously by selecting for tryptophan and histidine prototrophy, respectively. Using these strains, we examined the ability of 9-aminoacridine, quinacrine, four acridine mustards (ICR-170, ICR-191, ICR-372, and quinacrine mustard) and the nitroacridine Entozon to induce genetic duplications and frameshift mutations. Although all these compounds induce reversion of hisC3076, only the four mustards and Entozon are effective as inducers of genetic duplications under identical treatment conditions. The induction of genetic duplications by acridine mustards, like the toxic and mutagenic effects of these compounds, is enhanced by a deficiency for excision repair caused by a deletion through the uvr B gene. The ineffectiveness of 9-aminoacridine and quinacrine in the test for genetic duplications indicates that simple intercalation is sufficient for the mutagenic effect measured with the hisC3076 allele but that the induction of duplications by the acridine mustards and Entozon requires covalent binding of the chemical to DNA.