Effects of pre-treatment with sodium butyrate on the frequencies of X-ray-induced chromosomal aberrations in human peripheral blood lymphocytes

The effects of sodium butyrate-mediated alterations in chromatin structure on the yields of X-ray-induced chromosomal aberrations were studied in human peripheral blood lymphocytes. Unstimulated (G0) lymphocytes were pre-treated with sodium butyrate (5 mM) for 24 h, X-irradiated and then stimulated to pass through the cell cycle. Cells in their first post-radiation metaphase were scored for chromosomal aberrations. In parallel biochemical experiments nucleoid sedimentation technique was used to examine the induction and repair of DNA-strand breaks. The results show that sodium butyrate pre-treatment leads to a significant increase in the frequencies of dicentrics and rings, but not of fragments. The data from biochemical studies suggest that the numbers and rates of repair of X-ray-induced DNA-strand breaks are the same in butyrate-treated and untreated cells. We therefore suggest that the observed effect is probably a consequence of butyrate-induced conformational changes in the chromatin of G0 lymphocytes.     

The mouse splenocyte assay, an in vivo/in vitro system for biological monitoring: studies with X-rays, fission neutrons and bleomycin.

A modified mouse splenocyte culture system was standardized after testing different mitogens (i.e., phytohemagglutinin (PHA), concanavalin A (Con A)). The mitotic index was determined for comparison between different mitogens. Following selection of appropriate mitogen (PHA 16, Flow), a series of experiments were conducted to evaluate the application of a cytokinesis-block for scoring micronuclei and assays for chromosomal aberrations produced by treatment in G0 and G2 for the purposes of biological dosimetry following in vivo and/or in vitro exposure to X-rays, fission neutrons and bleomycin. In the X-irradiation studies, the frequencies of micronuclei and chromosomal aberrations (i.e., dicentrics and rings) increased in a dose-dependent manner. These data could be fitted to a linear-quadratic model. No difference was observed between irradiation in vivo and in vitro, suggesting that measurement of dicentrics and micronuclei in vitro after X-irradiation can be used as an in vivo dosimeter. Following in vivo irradiation with 1 MeV fission neutrons and in vitro culturing of mouse splenocytes, linear dose-response curves were obtained for induction of micronuclei and chromosomal aberrations. The lethal effects of neutrons were shown to be significantly greater than for a similar dose of X-rays. The relative biological effectiveness (RBE) was 6-8 in a dose range of 0.25-3 Gy for radiation-induced asymmetrical exchanges (dicentrics and rings), and about 8 for micronuclei in a dose range of 0.25-2 Gy. Furthermore, the induction of chromosomal aberrations by bleomycin was investigated in mouse G0 splenocytes (in vitro) and compared with X-ray data. Following bleomycin treatment (2 h) a similar pattern of dose-response curve was obtained as with X-rays. In this context a bleomycin rad equivalent of 20 micrograms/ml = 0.50 Gy was estimated.     

Influence of sodium butyrate on the induction of radiation-induced chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary (CHO) cells.

CHO cells were pre-treated with sodium butyrate (SB) for 24 h and then X-irradiated in G1. Metaphases were scored for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs). The data were compared with those obtained after irradiation of cells not pre-treated with SB and showed that SB has different effects on the endpoints examined. The frequencies of dicentric chromosomes were elevated and of small acentric rings (double minutes, DMs) reduced. These results are discussed to be a consequence of conformational changes in hyperacetylated chromatin which could lead to more interchromosomal and to less intrachromosomal exchanges. SB itself induces a few SCEs but suppresses the induction of SCEs by X-rays. We assume that a minor part of radiation induced SCEs are 'false' resulting from structural chromosomal aberrations, such as inversions, induced in G1. Inversions are the symmetrical counterparts of DMs. If inversions are suppressed by SB treatment to a similar extent as DMs a small reduction of SCEs by SB can be expected.     

Clastogenic effects of bleomycin, cyclophosphamide, and ethyl methanesulfonate on resting and proliferating human B- and T-lymphocytes

The effects of bleomycin (BM), cyclophosphamide (CP), and ethyl methanesulfonate (EMS) on the frequencies of chromosomal aberrations were tested in mitogen-stimulated highly purified human B- and T-lymphocytes. In unstimulated G0/G1 B- and T-lymphocytes the clastogen induction of chromosome fragments was investigated in prematurely condensed chromosomes (PCC) induced by cell fusion with xenogenic mitotic cells. BM, CP (with metabolic activation), and EMS induced a significant increase in chromosome aberrations in proliferating human B- and T-lymphocytes. There were no significant differences in the BM-induced aberration rates between the cell populations. CP and EMS induced more aberrations in T- than in B-lymphocytes. In the PCC tests, BM-exposed G0/G1 lymphocytes showed dose-dependent high yields of chromosome fragments. No significant differences between B- and T-lymphocytes were observed. CP and EMS induced no clear increase in fragments in either cell population.     

Lack of synergistic effect between X-ray and UV irradiation on the frequency of chromosome aberrations in PHA-stimulated human lymphocytes in the G1 stage.

PHA-stimulated human lymphocytes in the G1 stage were irradiated with UV radiation and X-rays, and the cells were analyzed for chromosomal aberrations in the first mitotic division. The frequency of dicentric chromosomes after single X-irradiation in the G1 stage was about twice the yield in the G0 stage. No increase in the yield of dicentrics was observed after combined irradiation with UV and X-rays. This is contrary to the finding for G0 lymphocytes, where a 2-fold increase of chromosome aberrations was observed. UV irradiation of G1 lymphocytes induced chromatid-type aberrations whereas no significant yield of dicentric chromosomes was observed. This is in agreement with previous findings in Chinese hamster cells in the G1 stage [7]. Irradiation of G0 lymphocytes with UV radiation induce a low frequency of dicentric chromosomes. Thus, the present data indicate that the ratio between chromosome-type and chromatid-type aberrations is different in the G1 and G0 stages in human lymphocytes irradiated with UV radiation.     

The relevance of caffeine post-treatment to SCE incidence induced in Chinese hamster cells

The influence of caffeine post-treatment on sister-chromatid exchanges (SCE) and chromosomal aberration frequencies on Chinese hamster cells exposed to a variety of chemical and physical agents followed by bromodeoxyuridine (BrdUrd) was determined. After 2 h treatment, N-methyl-N′-nitrosoguanidine (MNNG) and cis-platinum(II)diamine dichloride (cis-Pt(II)) induced a 7- and 6-fold increase in SCE, respectively, while 4-nitroquinoline-1-oxide (4NQO), methyl methanesulfonate (MMS), proflavine, and N-hydroxyfluorenylacetamide (OH-AAF) caused a 2–3-fold increase in SCE compared to controls treated with BrdUrd alone. Ultraviolet light doubled the number of SCE. The lowest increase of SCE was obtained with bleomycin and X-irradiation. Caffeine post-treatment caused a statistically significant increase in the frequency of SCE induced by UV- and X-irradiation as well as by 4NQO and MMS but did not alter the number of SCE induced by MNNG, cis-Pt(II), proflavine, OH-AAF, and bleomycin.

Caffeine post-treatment increased the number of cells with chromosomal aberrations induced by MNNG, cis-Pt(II), UV, 4NQO, MMS, and proflavine. With the exception of proflavine, these agents are dependent on DNA and chromosome replication for the expression of the chromosomal aberrations. Caffeine enhancement of cis-Pt(II) chromosomal aberrations occurred independently of the time interval between treatment and chromosome preparations. Chromosomal damage produced by bleomycin and X-irradiation, agents known to induce chromosomal aberrations independent of “S” phase of the cell cycle, as well as the damage induced with OH-AAF was not influenced by caffeine post-treatment.

The enhancement by caffeine, an inhibitor of the gap-filling process in post-replication repair, of chromosomal aberrations induced by “S” dependent agents, is consistent with the involvement of this type of repair in chromosomal aberration formation. The lack of inhibition of SCE frequency by caffeine indicates that post-replication repair is probably not important in SCE formation.     

Bleomycin-induced chromosomal aberrations in Down's syndrome lymphocytes

Blood samples from 4 Down's syndrome (DS) patients with a 47,XY,21 + karyotype and from 4 normal male probands were cultured for 72 h in the presence of BrdU and lymphocytes analysed at their first mitosis for chromosomal aberrations. The frequencies of spontaneous aberrations and the proportions of cells in the first or later mitoses in culture were not different between the groups. Treatment with various doses of bleomycin in vitro resulted in similar delays in cell development for both DS and normal lymphocytes and dose-dependent increases in the incidence of chromosome-type aberrations. However, the induction of both dicentric aberrations and acentric fragments was significantly enhanced in DS cells relative to cells of normal karyotype.     

Isolation and subregional mapping of a human cDNA clone detecting a common RELP on chromosome 12

Summary Peripheral blood lymphocytes from three patients with Down syndrome (DS; trisomy 21; aged 5–6 years) and three age-matched control children were studied for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs).Cells in G₀ were exposed to bleomycin (20–100 g/ml) for 3 h, and then cultured in medium containing 5-bromodeoxyuridine and phytohemagglutinin for 66 h. By the sister chromatid differential staining method, chromosome analyses were performed on metaphase cells that had divided one, two, or three or more times after treatment. The results indicate that DS cells exposed to bleomycin are hypersensitive to the production of dicentric and ring chromosomes compared to normal cells. Bleomycin also led to a dose-related increase in the frequency of SCEs, but no difference was found between the SCE frequencies in DS or normal lymphocytes exposed to bleomycin.     

The effects of post-treatments with caffeine during S and G2 on the frequencies of chromosomal aberrations induced by thiotepa in root tips of Vicia faba and in human lymphocytes in vitro

The effects of post-treatments with caffeine on the frequencies of chromosomal aberrations induced by the trifunctional alkylating agent thiotepa were studied in human lymphocytes and in root tips of Vicia faba. In lymphocytes the frequency of aberrations induced in G0 or G1 was most strongly increased when the caffeine post-treatments were given during G2. In Vicia faba, on the other hand, the frequency of aberrations induced in early interphase was unaffected by post-treatments with caffeine during G2, but strongly increased when the root tips were exposed to caffeine during the S phase.     

The effect of caffeine posttreatment of X-ray-induced chromosomal aberrations in human blood lymphocytes in vitro

Summary The potentiating effect of caffeine on X-ray-induced chromosomal aberrations in human blood lymphocytes has been investigated, with special reference to cell cycle stages (G0 and G2). Both quantitative and qualitative differences in the yield of chromosomal aberrations were detected in caffeine-posttreated cells, depending on the cell stage irradiated. The studies on caffeine potentiating effects on X-irradiated G0 lymphocytes from normal adults, newborns, Down syndrome patients, and an ataxia telangiectasia patient pointed to interindividual variations in the response to caffeine potentiation among normal probands and a very profound effect in ataxia cells.     

Effects of X-irradiation on cell-cycle progression, induction of chromosomal aberrations and cell killing in ataxia telangiectasia (AT) fibroblasts

Survival, cumulative labeling indices, chromosomal aberrations and cell-cycle distribution by flow microfluorometry (FMF) were studied in fibroblasts from normal and three ataxia telangiectasia (AT) families after X-irradiation during density-inhibition of growth and immediate release by subculture to low density. Homozygotic AT (proband) fibroblasts were very hypersensitive to cell killing by X-irradiation (D0 = 40-45 rad). Fibroblasts from AT heterozygotes (parents) were minimally hypersensitive, with D0's (100-110 rad) slightly lower than those for normal fibroblasts (D0 = 120-140 rad). There were three different response groups for a G1 phase block induced by 400 rad of X-rays: (1) minimal or no G1 block was observed in AT homozygote cell strains; (2) 10-20% of the cells were blocked in G1 in normal cell strains; and (3) 50% or more of the cells were blocked in AT heterozygote strains. FMF profiles and cumulative labeling indices showed that homozygotic AT cells irradiated in plateau phase moved into the S-phase following subculture with no additional delay over non-irradiated controls. Homozygotic AT cells showed not only a 4-5 times higher frequency of X-ray-induced chromosomal aberrations than normal strains, but approximately 30% of these were of the chromatid-type. There were no differences in the frequency or type of X-ray-induced chromosomal aberrations between normal and heterozygotic AT cells.     

3-Aminobenzamide does not affect X-ray-induced cytogenetic damage in G0 human lymphocytes

The inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide (3AB) has been reported to have very different effects on X-ray-induced chromosome aberrations in G0 human lymphocytes. One group of investigators observed a 2-3-fold increase in the yield of rings, dicentrics and chromosome breaks after X-irradiation and 3AB treatment, whereas another group found that 3AB had no effect on X-ray-induced chromosome aberrations. To resolve this discrepancy, we repeated the experiments as described by both groups and found no effect of 3 mM or 5 mM 3AB on the frequency of chromosome aberrations induced by either 1 Gy or 2 Gy of X-rays. Furthermore, we found no effect of 3AB on X-ray-induced aberration yields in C-banded prematurely condensed chromosome preparations from unstimulated human lymphocytes. These results indicate that poly(ADP-ribose) polymerase is not involved in the repair of cytogenetic damage in G0 human lymphocytes.     

Damage to lymphocytes by X-ray and bleomycin measured with the cytokinesis-block micronucleus technique.

Chromosome damage induced by X-irradiation or bleomycin was measured using the cytokinesis-block micronucleus assay in the peripheral blood lymphocytes of 6 newborn, 8 young and 10 elderly individuals. An increase in the frequency of spontaneous micronuclei with age was observed. There was no difference in the X-irradiation-induced micronucleus frequency between the 3 groups. There was a significant increase with age in the number of micronuclei induced by bleomycin. Kinetochore-labelling studies revealed that the percentage of kinetochore-positive induced micronuclei was higher for bleomycin (36.2-43.3%) than for X-irradiation (17.1-19.7%). The age-related increase in frequency of spontaneous or bleomycin-induced micronuclei was due to increases in both kinetochore-positive and kinetochore-negative micronuclei. The frequency of kinetochore-positive or -negative micronuclei induced by X-irradiation was not different between the 3 age groups. These results suggest that bleomycin is more potent in inducing whole-chromosome loss than X-rays, and that lymphocytes from aged individuals are more sensitive to bleomycin in terms of both chromosome breakage and whole chromosome loss.     

Influence of ataxia telangiectasia gene dosage on bleomycin-induced chromosome breakage and inhibition of replication in human lymphoblastoid cell lines

Long-term lymphoblastoid cell lines, obtained by E-B virus transformation of peripheral blood lymphocytes, retain many of the features of hypersensitivity to environmental agents found in primary cultures and fibroblast strains from patients with genetic diseases. Primary lymphocyte cultures from patients with ataxia telangiectasia, a cancer-prone genetic disease, have increased sensitivity to chromosomal damage induced by the radio-mimetic drug, bleomycin. In order to study the expression of ataxia telangiectasia gene dosage in lymphoblastoid cell lines, we examined chromosomal aberrations in lines containing two, one, or no alleles for ataxia telangiectasia. These were derived from ataxia telangiectasia homozygotes, from ataxia telangiectasia obligate heterozygotes, and from presumably normal donors, respectively. Chromosome preparations were made 46 h after a 2 h exposure to bleomycin and scored for chromosome breakage, for the relative rate of cell replication as assessed by sister chromatid differentiation patterns, and for the frequency of sister chromatid exchanges. Baseline frequencies of chromosome breakage and sister chromatid exchanges, and baseline rates of cell replication were similar in all nine lymphoblastoid cell lines. Following treatment with 25 or 250 mU/ml bleomycin, all the lymphoblastoid cell lines showed increased chromosome breakage and decreased cell replication. The lymphoblastoid cell lines from the ataxia telangiectasia homozygotes had significantly increased chromosome breakage and decreased rate of cell replication after either bleomycin dose in comparison with the normal or with the ataxia telangiectasia heterozygous lines. Sister chromatid exchange frequencies were not altered by bleomycin exposure.     

Analysis of spontaneous and bleomycin-induced chromosome damage in peripheral lymphocytes of long-haul aircrew members from Argentina

Spontaneous and bleomycin (BLM)-induced chromosomal aberrations in G0 and G2 stages of the cell cycle have been analyzed in peripheral lymphocytes of 21 long-haul aircrew members from Argentina in order to assess BLM-induced clastogenesis as a first approach to determine the DNA repair capacity and thereby the susceptibility to environmental cancers in aircrew. The possibility that occupational exposure of flight personnel to cosmic radiation can induce an adaptive response in their peripheral lymphocytes that can be detected by a subsequent in vitro treatment with BLM was also investigated. For comparison, aberrations were also scored in the lymphocytes of 15 healthy volunteers matched by age, health, sex, drinking and smoking habits to the flight personnel group. Aircrew exhibited a higher frequency of spontaneous dicentrics and ring chromosomes than the control population (p<0.05). BLM sensitivity test showed that aircrew and controls are equally sensitive to BLM G2 clastogenic effects, since both groups exhibited a similar frequency of chromatid breaks per cell (p>0.05). However, the aircrew sampled population was almost two times more sensitive to BLM G0 clastogenic effects than controls (p<0.05). Therefore, our data suggest that chronic exposure of aircrew to cosmic radiation increases the in vitro chromosomal sensitivity of their peripheral lymphocytes to BLM (at least in the G0 stage of the cell cycle), and that occupational exposure of flight personnel to cosmic radiation does not induce an adaptive response to this radiomimetic compound. Our results justify further studies aimed at determine if those aircrew members hypersensitive to BLM are more prone to develop environmental cancer than BLM-insensitive individuals.     

Clastogenic action of ellipticine over the cell cycle of human lymphocytes and influence of posttreatments with caffeine and ara-C at G2

The clastogenic potential of the intercalating compound ellipticine, an antitumor alkaloid, has been demonstrated in mammalian cells. To characterize the mechanism of action of this drug over the cell cycle, human lymphocyte cultures from 2 healthy donors were treated with 3 micrograms/ml ellipticine in 30-min pulses during different phases of the cell cycle and analyzed for chromosomal aberrations and sister-chromatid exchanges. The G2 phase was most sensitive in terms of induction of aberrations, followed by S and G1. Chromatid-type aberrations were the most common type of chromosomal damage. Induction of SCEs was significantly high only after treatment at G1, when the frequencies of SCEs doubled. The post-treatment effect of lymphocytes with inhibitors of DNA repair, 10(-3) M caffeine and 5 x 10(-6) M 1-beta-D-arabinofuranosylcytosine, was also tested by adding 3 micrograms/ml ellipticine at G2 in 30-min pulses and immediately followed by caffeine and/or ara-C during the last 3 h before harvesting. Three experiments performed on blood from 3 donors showed a moderate potentiation effect on the frequency of chromatid-type aberrations (about 2-3 times) by both inhibitors. Likewise, a 3-fold increase was observed in the frequencies of chromosomal aberrations when caffeine and ara-C were combined. The present data demonstrate that posttreatment with caffeine and ara-C at G2 can modify the response of human lymphocytes treated with ellipticine by increasing the clastogenic action of this compound or by changing the cell-cycle progression.     

Protective effect of cysteine against X-ray- and bleomycin-induced chromosomal aberrations and cell cycle delay

The protective effect of cysteine was studied in muntjac and human lymphocytes in vitro scoring chromosomal aberrations in harlequin stained first cycle metaphases, induced by X-irradiation at G₀. Its protective efficiency was also studied against the radiomimetic clastogen, bleomycin, in muntjac cells. 30 μg and 1 mg/ml of cysteine were given prior to 2, 3, and 4 Gy, and 2 mg/ml prior to only 4 Gy. 30 μg cysteine protected only against deletions in 4 Gy-treated cells while 1 mg protected against deletions by all three doses of X-rays. However, rearrangements were not reduced significantly in any of these, probably due to their low frequency. But when cysteine was increased to 2 mg, both types of aberrations were reduced significantly. This shows that a sufficient number of aberrations and an optimum concentration of the protector are essential for eliciting the best protective effect. This conclusion is further supported by the results of 2 mg cysteine treatment in human lymphocytes which yielded higher frequencies of rearrangements with 2 and 3 Gy X-rays than 4 Gy in muntjac, but had a relatively lower frequency of deletions. Thus the most abundant categories of aberration, i.e., deletions in muntjac and exchanges in humans, were reduced significantly by 2 mg cysteine, associated with a prominent reduction in the frequency of aberrant metaphases. Therefore, the differential protection observed with a low concentration of the protector and an insufficient yield of aberrations induced only indicates protection provided to the most frequent type of aberration by a protector when present in lower concentration.

Cysteine pretreatment yielded weak protection against the effects of bleomycin, but posttreatment caused a mild potentiation of the clastogenic effect of BLM without altering the cell cycle kinetics. In this context, an action of cysteine as a reducing agent on BLM is suggested. Although cysteine alone caused severe retardation of the cell cycle, when given prior to X-irradiation, not only its delaying effect was not observed, but also it reduced the X-ray induced cell cycle delay. This might be due to the oxidation of cysteine by its radical scavenging action.     

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or gamma-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5 mM caffeine plus 3 mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p < 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p < 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p < 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p < 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p < 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation.     

Hypersensitivity to ionizing radiation, in vitro, in a new chromosomal breakage disorder, the Nijmegen Breakage Syndrome

The Nijmegen Breakage Syndrome (NBS) is a new chromosomal instability disorder different from ataxia telangiectasia (AT) and other chromosome-breakage syndromes. Cells from an NBS patient appeared hypersensitive to X-irradiation. X-rays induced significantly more chromosomal damage in NBS lymphocytes and fibroblasts than in normal cells. The difference was most pronounced after irradiation in G2. Further, NBS fibroblasts were more readily killed by X-rays than normal fibroblasts. In addition, the DNA synthesis in NBS cells was more resistant to X-rays and bleomycin than that in normal cells. The reaction of NBS cells to X-rays and bleomycin was similar to that of cells from patients with ataxia telangiectasia. Our results indicate that NBS and AT, which also have similar chromosomal characteristics, must be closely related.     

Effects of pingyanymycin on chromosomes: a possible structural basis for chromosome aberration

Pingyanymycin (PYM), and antitumor-antibiotic complex which belongs to the bleomycin family can induce "G2-free chromatin" and "uncompleted-packing-mitotic figures" (UPM) at increased frequency after treatment of cultured human lymphocytes. PYM can also induce an extraordinarily high frequency of chromosomal breaks but few sister-chromatid exchanges (SCE) in the same experiment, which is similar to the action of bleomycin. To solve this remarkable contradiction we presume that the UPM is related to a basic mechanism for producing chromosomal aberrations. Our results also show that various steps of the chromosomal cycle can be affected by certain chemical agents, and these treatments lead to chromosomal aberrations. Thus, other testing systems should be used in addition to the SCE system.