Defective G2 repair in Down syndrome: effect of caffeine, adenosine and niacinamide in control and X-ray irradiated lymphocytes

Lymphocytes from both Down syndrome (DS) patients and age-matched control donors have been investigated to identify a possible disturbance in chromosomal G2 repair. Analyses of caffeine treatments during G2 have shown that the frequency of chromosomal aberrations is higher in DS lymphocytes than in normal lymphocytes. Likewise, G2 duration is longer in DS cells than in normal cells. In both control and DS lymphocytes, caffeine treatments increase the frequencies of chromatid breakages and decrease the average of G2 duration. The reversal of the caffeine potentiation effect by adenosine and niacinamide is higher in DS cells than in normal cells. Furthermore, ATP content per cell in DS lymphocytes is one third of that estimated in normal lymphocytes. The increase of ATP level produced by adenosine or niacinamide generally correlates with the reversal of the caffeine effect on chromosome aberrations. Under the experimental conditions tested, a good negative exponential correlation between ATP level and chromosome aberrations has been detected in both normal and DS lymphocytes which were or were not X-irradiated. Finally, we postulate a decrease in G2 repair capability of DS lymphocytes caused by a low availability of ATP and/or some other factor correlating with it.     

The implications of S-phase exchanges for the mechanisms of radiosensitivity in trisomy 21

Human lymphocytes obtained from four patients with Down syndrome and from two normal individuals were irradiated with X-rays during their S phase and examined for chromatid type aberrations. It is suggested that the significantly increased frequency of asymmetrical chromatid interchanges found in trisomic cells is related to an altered DNA repair system. This altered repair system is probably responsible for the increased frequency of chromosome aberrations that can be induced in these cells by x-rays and the increased tendency for leukemia observed in Down syndrome as well.     

Specificity of arsenite in potentiating cytogenetic damage induced by the DNA crosslinking agent diepoxybutane.

In the present study, the induction of sister chromatid exchanges (SCEs) and chromosomal aberrations were measured in normal human lymphocytes treated with low concentrations of arsenite alone (0.5-2.0 microM) and arsenite in combination with the potent DNA crosslinking agent diepoxybutane (DEB). Experiments were carried out with lymphocytes from blood donors with different sensitivities to SCE induction by DEB. Arsenite, beginning at concentrations as low as 1 microM, increased SCE frequencies; chromosomal aberration frequencies were increased at 2 microM of arsenite. DEB treatments alone increased SCE frequencies and chromosomal aberrations. The yields of chromatid deletions and exchanges in lymphocytes exposed to both arsenite and DEB were markedly increased above the levels expected if the effects of the two agents had been simply additive. The frequencies of chromatid deletions were 4- to 8-fold greater than expected and chromatid exchanges were increased 7- to 40-fold. Chromatid exchanges detected in cells treated with arsenite and DEB were predominately incomplete exchanges. The most dramatic increases in chromatid aberrations were observed in lymphocytes from an individual sensitive to SCE induction by DEB, indicating that individuals may vary in their sensitivity to the co-clastogenic effects of arsenite. At concentrations that dramatically affect aberrations, arsenite had no effect on the induction of SCEs by DEB. These studies suggest a specific interaction of arsenite with the induction or repair of DNA damage produced by DEB that leads to chromosomal aberrations but not to SCEs. Based on the selective chemical reactivity of low concentrations of arsenite with proteins containing vicinal dithiols and the occurrence of these groups within DNA repair proteins, it is proposed that the specific co-clastogenic effects of arsenite may be mediated by its interference with DNA repair activities.     

Chromosomal radiosensitivity of lymphocytes from Alzheimer''s disease patients.

We have examined chromosome aberrations in gamma irradiated (3 Gy) lymphocytes from five patients with Alzheimer's disease (AD). In each case, the number of dicentrics was significantly higher than the number in irradiated lymphocytes from five age matched normal subjects, the mean value for AD cells being about 25% higher. There was no significant difference in number of acentrics between AD and normal cells. Examination of the number of first, second, and third division metaphases, using fluorescence plus Giemsa staining, indicated that there was no difference in cycling time between AD and normal cells, and that after irradiation both groups showed the same mitotic delay. The similarity of our findings to those of others with irradiated Down's syndrome cells (from adult patients) is discussed.     

Interaction of radiation- and bleomycin-induced lesions and influence of glutathione level on the interaction

Radiation-induced exchange aberrations are thought to arise as a consequence of misrejoining of free ends of DNA double strand breaks (dsbs). In quiescent mammalian cells this process of misrejoining is prevalently taken up by the non-homologous end joining (NHEJ) process. In order to investigate the role of glutathione (GSH) in DNA dsb rejoining, the interaction of the lesions induced by bleomycin (Blem) and by radiation was studied since the lesions caused by both have similar and apparent rapid rates of repair. Endogenous GSH was depleted by buthionine sulfoximine (BSO) and chromosome aberrations (CAs) of human lymphocytes were scored from first cycle metaphases. Gamma radiation was administered 2 h after Blem treatment in combined studies. In the case of BSO, the treatment was given 3 h before Blem treatment. The BSO-treated samples showed higher sensitivity to radiation than BSO-untreated ones. Combined treatment of Blem and radiation induced higher frequency of CAs, in particular the exchange aberrations and interstitial deletions. However, such increased frequency of exchange aberrations was reduced drastically and the frequency of terminal deletions was increased significantly when combined treatment was given to BSO-pretreated cells. The consistent level of Ku70 protein in all the treated samples, with undetectable level of Rad51 in the G0-lymphocytes indicates the involvement of NHEJ pathway in misrejoining of DNA dsbs. It may be hypothesized that reduction in the frequency of exchange aberrations as induced by Blem + radiation combined treatment in BSO-treated samples could be because of reduced NHEJ pathway.     

Peripheral blood chromosome aberrations in MDS.

The frequency of non-clonal structural and numerical chromosome aberrations in peripheral blood lymphocytes of 51 patients with MDS and 37 age-matched hematologically normal subjects is assessed. The frequency of aneuploid cells (p less than 0.001) and of structural aberrations (p less than 0.005) was significantly higher in MDS patients than in normal subjects, but showed no relationship with FAB type or with the presence of clonal karyotype abnormalities in the bone marrow. Exchange configurations were only observed in MDS patients (27.5%). The data also suggest that there may be an association between high peripheral blood aberration levels and rapidly progressive disease. This may indicate increased mutagen sensitivity and have implications for treatment.     

Cell division,chromosomal damage and micronucleus formation in peripheral lymphocytes of healthy donors: related to donor's age

Spontaneous baseline frequencies of chromosome aberrations, micronucleus counts and cell division were analysed in peripheral lymphocytes of 127 normal healthy individuals in vitro. Cells were subjected to culture for 48 h in serum and PHA supplemented culture medium RPMI 1640. 100 metaphases were observed for chromosome aberrations and 1000 cells each for micronucleus counts and mitotic index. Regression analyses were carried out to see the effect of age on spontaneous abnormalities. The correlation of aberrations, micronucleus formation and mitotic index with donor's age is highly significant. The elevation of abnormalities and depression of mitotic index were linear to the increase of donor's age, with a higher frequency in males. Aged males and females from the age range of 40–70 years showed larger numbers of aberrations. Individuals with the smoking habit possessed higher frequencies of abnormalities than non-smokers.     

Enhancement of induced sister chromatid exchange and chromosomal aberrations by inhibitors of DNA repair processes

The effect of post-treatment with inhibitors of DNA synthesis (hydroxyurea, aphidicolin) and repair (caffeine, 3-aminobenzamide) on the frequencies of chromosomal aberrations and sister chromatid exchange (SCE) induced by mitomycin C and decarbamoyl mitomycin C both in Chinese hamster cells and in human lymphocytes in vitro has been studied. The data show that in the case of Chinese hamster and human lymphocytes mitomycin C-treated cells there is an increased frequency of both chromosomal aberrations and SCE after a G2 post-treatment with the inhibitors, while no increase is observed for decarbamoyl mitomycin C-treated cells. Since SCE are DNA synthesis-dependent phenomenon, an increase in the frequency of SCE also in the G2 phase might suggest that after mitomycin C treatment there is a residual DNA synthesis still going on very late in the cell cycle.     

Sensitivity to ultraviolet radiation in a dominantly inherited form of xeroderma pigmentosum.

An Australian family is described in which a mild form of xeroderma pigmentosum (XP) is inherited as an autosomal dominant trait. Studies of lymphoblastoid cells and fibroblasts from affected persons demonstrated cellular sensitivity to ultraviolet (UV) light as judged by diminished clonogenicity and higher frequencies of UV induced chromosome aberrations compared to normal controls. After UV irradiation of dominant XP cells, replicative DNA synthesis was depressed to a greater extent than normal and the level of UV induced DNA repair synthesis was lower than that in normal cells. The level of sister chromatid exchanges and the numbers of 6-thioguanine resistant mutants induced by UV irradiation were equal to those found in normal controls. Although two subjects in the family had skin cancers, this dominant form of XP is not apparently associated with high risk, or large numbers, of skin cancers in affected persons.     

Acid phosphatase and succinate and dehydroorotate dehydrogenase activity during interaction between allogeneic lymphocytes and target cells in tissue culture

Investigation of acid phosphatase activity during interaction between lymphocytes and target cells showed that its greatest increase is observed in lymphocytes in 1-h and sometimes 3-h cultures, whereas in the cells it was observed after 3–6 h. Initial acid phosphatase activity was higher in immune lymphocytes, but later, on their addition to a culture of L-cells, no significant difference was found between the change in enzyme activity in immune and normal lymphocytes. Acid phosphatase activity in L-cells was lower on the addition of normal than of immune lymphocytes. Activity of the dehydrogenases in the lymphocytes increased until 3 h of incubation and the increase was greater in immune than in normal lymphocytes. Activity of succinate and dehydroorotate dehydrogenases in the L-cells changed at virtually the same times as in the lymphocytes. Increased activity of oxidoreductases and acid phosphatase in the first few hours of contact is evidence of the rapid activation of effector lymphocytes.     

Inhibitory effect of ascorbic acid post-treatment on radiation-induced chromosomal damage in human lymphocytes in vitro

In the present study, the effect of exposure to ascorbic acid (vitamin C) after gamma-ray-induced chromosomal damage in cultured human lymphocytes was examined to explore the mechanism by which this antioxidant vitamin protects irradiated cells Non-irradiated lymphocytes were exposed to increasing concentrations of ascorbic acid (1-100 micro g/ml) and DNA damage was estimated using chromosomal aberration analysis and the comet assay. The results showed that ascorbic acid did not influence the frequency of chromosomal aberrations in non-irradiated cells, except at the highest concentration (20 micro g/ml), which induced breakage-type chromosomal aberrations. Vitamin C at the concentration of 50 micro g/ml caused DNA damage detected by the comet assay. A significant (34%) decrease in the frequency of chromosomal aberrations was observed in lymphocytes exposed to gamma-radiation and then cultured in the presence of ascorbic acid (1 micro g/ml). The removal of DNA breaks in cells exposed to 2 Gy of gamma-radiation was accelerated in the presence of ascorbic acid as determined by the comet assay, suggesting that it may stimulate DNA repair processes.     

Comparison of AluI-induced frequencies of dicentrics and translocations in human lymphocytes by chromosome painting.

It has been shown repeatedly that following irradiation of human lymphocytes in the G0 stage, more translocations are induced than dicentrics. To check the role of DNA double-strand breaks (DSB) alone for the induction of symmetrical and asymmetrical chromosome aberrations, the frequencies of induced exchange aberrations by the restriction enzyme AluI were analyzed. The enzyme was introduced into cells using the pellet pipetting technique. Frequencies of induced translocations and dicentrics were determined using a chromosome painting assay with chromosome-specific DNA libraries for chromosomes 1, 4 and X (representing 16.8% of the human genome). The number of translocations detected was approximately 3-fold higher than the number of dicentrics, indicating that the increased frequency of translocations compared with dicentrics found in irradiated human lymphocytes does not result from DNA lesions other than DSB but from differential processing of DSB.     

Specific chromosome aberrations in peripheral blood lymphocytes are associated with risk of bladder cancer

Specific chromosome aberrations in peripheral blood lymphocytes (PBLs) in chromosomes 9 and 11 may be associated with bladder cancer. To investigate this hypothesis, in this study, we used a whole-chromosome painting technique to detect chromosomal aberrations in PBLs from 100 patients with bladder cancer and 100 matched controls. We also used a locus-specific fluorescence in situ hybridization technique to study 9p21 and cyclin D1 gene (CCND1) aberrations in PBLs of 10 patients and 10 controls and in tumor tissues of 38 additional cases. The chromosome-painting analysis showed that there were more aberrations of chromosomes 9 and 11 in bladder cancer patients than in controls. When categorized by type, the number of deletions of 9p and of translocations of chromosome 11 was significantly higher in patients than in controls (P < 0.05). Stratified analysis showed a larger odds ratio (OR) for bladder cancer in individuals with either a 9p deletion or a chromosome 11 translocation/amplification and an even larger OR in individuals with both aberrations. Using locus-specific analysis, we found that 9p21 aberrations occurred more frequently in bladder cancer patients (12.1 per 1,000 interphase cells) than in controls (6.4 per 1,000 interphase cells, P < 0.05); CCND1 translocation and amplification were observed only in bladder cancer patients. Tumor tissue analysis showed that aberrations of 9p21 (40.0 per 100 interphase cells) and CCND1 (43.8 per 100 interphase cells) were very common. Thus, we concluded that 9p deletions and translocations of chromosome 11, especially at 9p21 and CCND1, are associated with bladder cancer.     

Chromosome abnormalities in a referred population for suspected chromosomal aberrations: a report of 4117 cases.

A cytogenetic study was performed on 4,117 Korean patients referred for suspected chromosomal abnormalities. Chromosome aberrations were identified in 17.5% of the referred cases. The most common autosomal abnormality was Down syndrome and Turner syndrome in abnormalities of sex chromosome. The proportions of different karyotypes in Down syndrome (trisomy 21 92.5%, translocation 5.1%, mosaic 2.4%) were similar to those reported in other countries. However, it was different in Turner syndrome (45, X 28.1%, mosaic 50.8%, 46, X, del (Xq) 4.4%, 46, X, i (Xq) 16.7%), in which proportions of mosaics and isochromosome, 46, X, i(Xq), were higher than those reported in other countries. In structural chromosome aberrations of autosome, translocation was the most common (43.6%), and duplication (21.3%), deletion (14.4%), marker chromosome (7.9%) and ring chromosome (4.0%) followed in order of frequency. Rates of several normal variant karyotypes were also described. Inversion of chromosome 9 was observed in 1.7% of total referred cases.     

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).     

Induction and repair of formaldehyde-induced DNA-protein crosslinks in repair-deficient human cell lines

We have previously shown that the alkaline Comet assay (single cell gel electrophoresis) in a modified version is a sensitive test for the detection of formaldehyde-induced DNA-protein crosslinks (DPC). Our results also indicated that formaldehyde-induced DPC are related to the formation of chromosomal effects such as micronuclei and sister chromatid exchanges. To better understand the genetic consequences of formaldehyde-induced DPC we have now investigated the induction and removal of DPC in relationship to the formation of micronuclei in normal and repair-deficient human cell lines. We did not find significant differences between normal cells, a xeroderma pigmentosum (XP) cell line and a Fanconi anaemia (FA) cell line with respect to the induction and removal of DPC. However, the induction of micronuclei was enhanced in both repair-deficient cell lines, particularly in XP cells, under the same treatment conditions. Comparative investigations with the DNA-DNA crosslinker mitomycin C (MMC) revealed a delayed removal of crosslinks and enhanced induction of micronuclei in both repair-deficient cell lines. FA cells were found to be particularly hypersensitive to micronucleus induction by MMC. In contrast to the results with formaldehyde, induction of micronuclei by MMC occurred at much lower concentrations than the effects in the Comet assay. Our results suggest that more than one repair pathway can be involved in the repair of crosslinks and that disturbed excision repair has more severe consequences with regard to the formation of chromosomal aberrations after formaldehyde treatment than has disturbed crosslink repair.     

Genetic effects of drug interaction in tuberculosis patients and their fate

In this paper we will discuss the genetic consequences of drug interaction in tuberculosis patients. Blood from tuberculosis patients was cultured before, during, and after withdrawal of therapy involving five different drug combinations of isoniazid (INH), thiacetazone (TAZ), para-aminosalicylic acid (PAS), and streptomycin (SM). The approaches used to detect DNA damage were chromosome aberrations and sister chromatid exchanges (SCEs). A total of 179 subjects were analyzed. In combination these drugs showed synergistic, additive, and antagonistic effects, though they were found to be nonclastogenic individually. Four of the drug combinations, INH + TAZ, INH + PAS, INH + TAZ + SM, and INH + PAS + SM, induced a significant increase in the frequency of aberrations, whereas INH + SM did not induce aberrations. In fact, SM appeared to reduce the frequency of aberrations. SCEs were increased in only two patients: one treated with INH + TAZ and the other with INH + PAS. The frequency of aberrations after withdrawal of therapy was decreased; it was slightly higher than the controls, though it was insignificant. The return to normalcy could be due to elimination of damaged cells or the repair of DNA in lymphocytes. Though the drug-induced aberrations do not persist after withdrawal of therapy, the chromosome damaging combinations of drugs should be used with caution, because the possibility of meiotic chromosome damage in germ cells (during therapy), which might be passed on to the next generation, cannot be ruled out.     

Studies of cytogenetic effects of sodium arsenicals on mammalian cells in vitro

The cytotoxic and cytogenetic effect of sodium arsenite and sodium arsenate on Chinese hamster ovary (CHO) cells is reported. Chromosome aberrations were induced with both arsenic compounds. Trivalent arsenic was more clastogenic than pentavalent arsenic. Sodium arsenite was also shown to produce increased sister chromatid exchange in CHO cells and increased chromosome breakage in human lymphocytes.     

The repair of gamma-radiation-induced DNA damage is inhibited by microcystin-LR, the PP1 and PP2A phosphatase inhibitor

The genotoxic activity of microcystin-LR (MC-LR) is a matter of debate. MC-LR is known to be a phosphatase inhibitor and it may be expected that it is involved in the regulation of the activity of DNA-dependent protein kinase (DNA-PK), the key enzyme involved in the repair of radiation-induced DNA damage. We studied the effect of MC-LR on the repair capacity of radiation-induced DNA damage in human lymphocytes and human glioblastoma cell lines MO59J and MO59K. A dose of 0.5 microg/ml of MC-LR was chosen because it induced very little early apoptosis which gives no false positive results in the comet assay. Human lymphocytes in G0-phase of the cell cycle were pre-treated with MC-LR for 3 h and irradiated with 2 Gy of gamma radiation. The kinetics of DNA repair was assessed by the comet assay. In addition the frequencies of chromosomal aberrations were analysed. The pre-treatment with MC-LR inhibited the repair of radiation-induced damage and lead to enhanced frequencies of chromosomal aberrations including dicentric chromosomes. The results of a split-dose experiment, where cells were exposed to two 1.5 Gy doses of radiation separated by 3 h with or without MC-LR, confirmed that the toxin increased the frequency of dicentric chromosomes. We also determined the effect of MC-LR and ionizing radiation on the frequency of gamma-H2AX foci. The pre-treatment with MC-LR resulted in reduced numbers of gamma-H2AX foci in irradiated cells. In order to elucidate the impact of MC-LR on DNA-PK we examined the kinetics of DNA repair in human glioblastoma MO59J and MO59K cells. Both cell lines were exposed to 10 Gy of X-rays and DNA repair was analysed by the comet assay. A strong inhibitory effect was observed in the MO59K but not in the MO59J cells. These results indicate that DNA-PK might be involved in DNA repair inhibition by MC-LR.     

Gender differences in age-related decline in DNA double-strand break damage and repair in lymphocytes

DNA repair capacity has been suggested to be a genetic mechanism which influences the rate of ageing and length of life. We recently reported an age-related decline in DNA double-strand break (DSB) repair in unstimulated human lymphocytes (Mayer, Lange, Bradley, and Nichols 1989, 1990). In that work peripheral lymphocytes isolated from whole blood donated by 20 normal, healthy subjects aged 23-78 years, were X-irradiated (30 Gy) on ice and incubated at 37 degrees C for repair times of 15, 30, and 120 min, and neutral filter elution was used to assay DSB induction and completeness of DSB rejoining. Further analysis reveals that the decrease in DSB rejoining appears to be more pronounced in older women than in older men (and may begin after age 65 years). Moreover the reported age-related decline in DSB induction occurs more rapidly (by a factor of ca. 2) in women than in men. We had previously demonstrated that, independently of in vivo age, cells with lower radiosensitivity (i.e. lesser DSB induction) appear to be less repair-proficient (Mayer et al. 1990). The present analysis reveals a gender-specific pattern in this correlation between extent of DSBs induced and percentage of DSBs rejoined: at comparable levels of DSB induction, cells from men rejoin a higher percentage of DSBs than do cells from women. This preliminary study suggests the following hypothesis: age-related DSB effects (i.e. induction and rejoining) are due to changes in chromatin structure and males are less sensitive than females to the influence which age-related alterations in chromatin exert on DSB effects.