The influence of culture medium composition on the incidence of chromosomal breakage

Summary In patients with chromosomal instability and in healthy subjects, significant differences were observed in the chromosomal breakage incidence in simultaneous lymphocyte cultures set up with TC Medium 199, Eagle's Minimum Essential Medium, or RPMI 1629. The importance of the choice of culture medium for mutagenicity testing and studied of so-called spontaneous breakage is shown. Cultures incubated with TC Medium 199 showed the highest chromosomal breakage incidence.     

Increased sensitivity to bleomycin in upper aerodigestive tract mucosa of head and neck squamous cell carcinoma patients

Previous studies on lymphocytes have suggested that patients with head and neck squamous cell carcinoma (HNSCC) have an increased susceptibility for chromosomal damage induced by bleomycin, a known radiomimetic mutagen. However, it has so far not been possible to study whether this genetic instability is present also in the epithelial component of the upper aerodigestive tract mucosa, the tissue from which HNSCC originates. In the present study, we have successfully cultured epithelial cells and fibroblasts isolated from non-neoplastic mucosa samples of 30 HNSCC patients and 56 controls. All cell cultures were exposed to bleomycin and chromosome instability was assessed by analysis of chromosome breakage in cells harvested after 2h of exposure and subsequent removal of bleomycin. Furthermore, the status of the fragile histidine triad gene (FHIT) in chromosome band 3p14.2 was studied by fluorescence in situ hybridization (FISH) in epithelial cells that had been cultured after removal of bleomycin. Chromosomal damage, in the form of chromosomal breaks and gaps, was seen in all cell cultures harvested 2h after exposure to bleomycin. In epithelial cells, the frequency of chromosome breakage was significantly higher among HNSCC patients than among controls [mean breaks per cell (b/c) 1.02 vs. 0.77, p=0.02]. When subdivided according to smoking status, age, and sex, a significantly higher frequency of chromosome breakage was still found in HNSCC patients (smokers, p=0.01, age相似文献   

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.     

The effect of aphidicolin on Fanconi's anemia lymphocyte chromosomes

The cytogenetic effect of the DNA polymerase alpha inhibitor aphidicolin (APC) at a dose which did not affect cell cycle progression was determined in normal and Fanconi's anemia (FA) lymphocytes. APC enhanced sister-chromatid exchange (SCE) levels by about twice both in control and FA cells, while the yields of chromosome breakage increased up to 20 times in normal lymphocytes and 4 times in FA cells. APC did not act synergistically with the bifunctional alkylating diepoxybutane in terms of SCE either in normal or in FA lymphocytes. However, chromosome aberrations in cultures from normal subjects were much more than expected by an additive mode of action.     

Effects of gamma-irradiation on chromosomes of cultured lymphocytes from rheumatoid arthritis patients and their family members

Increased rates of spontaneous or induced chromosome breakage are seen in many types of diseases, including the 'collagen-type' autoimmune diseases. Using a 60Co gamma cell, we irradiated lymphocyte cultures from three related rheumatoid arthritis patients, their immediate family members, and two unrelated rheumatoid arthritis patients. Although these individuals had not shown abnormally high levels of spontaneous chromosome breakage, they did show an abnormal sensitivity to irradiation, which was manifested in several ways. Two of the probands showed induced breakage rates that were twice as high as those seen in controls. In addition, the reduction of mitotic index, due either to increased cell death or to induction of a G2 lag period, was higher in the arthritis group (including non-symptomatic family members) than in the control group. Finally, we observed a high frequency of an unusual type of cell in the arthritis group. These unusual cells resembled c-anaphases seen with extended colcemid treatment, and may indicate that the mitotic apparatus in cells from this group is particularly sensitive to ionizing radiation.     

Role of haemoglobin in the protection of cultured lymphocytes against diepoxybutane (DEB), assessed by in vitro induced chromosome breakage

Diepoxybutane (DEB) is an alkylating agent that can be used to assess chromosome instability in repair-deficient subjects. Previous authors investigated the role of red blood cells (RBC) in determining individual susceptibility to DEB in normal healthy donors, and demonstrated that a polymorphic enzyme in RBC, Glutathione S-transferase T1 (GSTT1), is involved in DEB detoxification. In the present work we studied the influence of individual GSTM1 and GSTT1 genotypes and the presence of RBC on the frequency of DEB-induced chromosome breakage in lymphocyte cultures from normal individuals and, in particular, the influence of isolated components of RBC: RBC membranes, RBC lysate, and haemoglobin. Our results confirm that individual GSTT1 genotypes modulate the level of genetic lesions induced by DEB; however, this effect was not sufficient to explain the highly significant variation in chromosome breakage between whole blood and RBC-depleted cultures. We showed that RBC can protect cultured lymphocytes against chromosome breakage induced by DEB and we demonstrated the particular role of haemoglobin in the protective effect.     

Cytogenetic analyses utilizing various clastogens in two sibs with Fanconi anemia,their relatives,and control individuals

Summary Structural chromosome damage, sister chromatid exchange (SCE), and proliferation kinetics were studied on lymphocyte cultures from the peripheral blood of two sibs exhibiting signs of Fanconi anemia, their relatives, and control individuals. While the rate of spontaneous chromosome breakage was at the lower limit of that known for Fanconi anemia in our patients, a distinctly greater increase than in controls of breakage frequency could be induced by isoniazid (INH), 4-nitroquinoline-1-oxide (NQO), and diepoxybutane (DEB) in their lymphocytes. Increased aberration frequencies as compared with controls were also observed in the clastogen-exposed lymphocyte cultures of the parents of both sibs, but in some experiments (NQO, DEB 24h) only in the cells of the healthy brother. There was an increase in the breakage rate of bromodeoxyuridine (BrdU)-labeled consecutive mitoses under the action of NQO, but a decrease with INH as the test clastogen.No significantly higher SCE frequency was found throughout the study in untreated and clastogen-exposed FA lymphocytes as compared with the respective controls. Proliferation was clearly inhibited by INH and NQO as indicated by a distinct increase of the percentage of BrdU-labeled first and a drastic decrease of third metaphases. The present test clastogens were shown not only to be suitable for ensuring the diagnosis of FA in patients with a low incidence of spontaneous breakage but also for determining clastogen-sensitive heterozygotes. According to these results cross-link repair cannot be the only mechanism affected by the basic defect of Fanconi anemia.Dedicated to Professor Dr. A. Barthelmess on the occasion of his 75th birthdayThis paper contains parts of the M.D. theses of D.K., H.M., and M.N.     

Chromosome-breaking agent of low molecular weight in human systemic Lypus Erythematosus

Summary The serum of patients with systemic lupus erymathosus contains a chromosome-breaking agent of low molecular weight, which produces chromosomal breakage and rearrangement in lymphocyte cultures of healthy donors. This breakage factor is probably produced by or released from the cells of patients, since lymphocyte extracts and cocultivation of patients' lymphocytes with lymphocytes from healthy subjects results in an increased frequency of chromosome aberrations in the normal cells. The aberration rate induced by this clastogenic agent is reduced to normal values if the free radical scavenging enzyme superoxide dismutase is added to the culture medium at a final concentration of 0.05 mg/ml.     

In vitro chromosome damage due to PCB interactions

In order to study the possible mutagenic properties of polychlorinated biphenyls (PCBs), human lymphocyte cultures were examined for chromosome breakage, rearrangements, sister-chromatid exchange, and mitotic delay. The present study, which used cyclophosphamide as a positive control, shows that one planar PCB congener, 3,4,3',4'-tetrachlorobiphenyl, caused dose-related chromosome breakage in human lymphocytes exposed in vitro to 0.1-10(-4) micrograms/ml. In contrast, the non-planar PCB, 2,5,2',5', did not cause chromosome damage in comparable tests even at concentrations as high as 1 microgram/ml. However, when 3,4,3',4' at a concentration lower than that which causes chromosome breakage (10(-5) micrograms/ml) was combined with a non-clastogenic concentration of 2,5,2',5', the chromosomal damage observed was far in excess of what one would expect from higher doses of 3,4,3',4' alone. These results suggest that some PCB congeners may interact to cause synergistic genotoxic effects.     

Structural chromosomal rearrangements in HpaII-treated human lymphocytes.

Restriction endonucleases have been shown to induce chromosome damage in a variety of cultured cells. We recently reported the coincidence between MspI-induced breakage and the location of common fragile sites. We have extended our study to HpaII, which induced a 4.5-fold increase in total breakage compared to controls. It appeared that a major contribution was given by stable chromosome rearrangements, which were present at a 14-fold increased frequency in comparison to the spontaneous levels. Moreover, several chromosome bands were involved in rearrangements in different cultures from different donors. Notably, HpaII-induced breakage occurred in the same bands where breakpoints of constitutional and neoplastic rearrangements are located.     

A simple diagnostic test for Fanconi anemia by flow cytometry

A simple diagnostic test for Fanconi anemia (FA) by flow cytometry is proposed. It is based on the cell cycle disturbances of FA cells and their sensitisation by alkylating agents. Following PHA-stimulation of whole blood cell cultures in the presence or absence of nitrogen mustard, the accumulation of cells in G2/M phase was measured. A sharp increase of cells in G2/M was observed in cultures from FA patients when nitrogen mustard was added. This increase allows one to distinguish FA patients from patients with anemias of other origin, healthy controls, and FA heterozygotes, as effectively as chromosome breakage studies. The rapidity of the test and its reliability as demonstrated on the ten FA patients studied, will make the diagnosis of FA easier in centers without cytogenetic laboratory facilities.     

Monocyte-derived clastogenic factor in rheumatoid arthritis

Blood or lymphocyte cultures from patients with rheumatoid arthritis show increased chromosome breakage. This is due to the presence of a clastogenic factor (CF) inducing also chromosome damage in blood cultures of healthy persons. CF may be isolated not only from patients' plasma or synovial fluid, but also from the supernatant of blood or lymphocyte cultures. No CF was detectable, if the lymphocyte cultures were free of other contaminating blood cells. Addition of neutrophils did not considerably influence the production of CF, and platelets were without any effect. However, addition of increasing numbers of monocytes resulted in increasing clastogenic activity. Also monocytes in adherence, in absence of lymphocytes and without any chemical stimulant, produced CF. This indicates that monocytes are responsible for CF production. The protective effect of superoxide dismutase, as well against CF formation as against CF action on cells of normal subjects, suggests a role of the superoxide radical O2-. Inhibitors of arachidonic acid metabolism were only slightly anticlastogenic.     

Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli.

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.     

Mutagenic activity of anticancer agent cis-dichlorodiammine platinum-II.

cis-Dichlorodiamminoplatinum-II (cis-DDP) has been widely used as an anticancer chemotherapeutic agent. The mutagenicity of cis-DDP was investigated in vitro and in vivo using sister-chromatid exchange analysis and the analysis of chromosomal aberrations. Parallel human lymphocyte cultures were incubated with and without the addition of BrdU at 4 concentrations of cis-DDP. Significant increases in SCE rate were observed at 0.25 micrograms/ml and higher, showing a clear dose-response relation between SCE rate and cis-DDP concentration. A significant increase in chromosome breakage and tetraradial figures was observed in BrdU free cultures treated with cis-DDP again showing a dose dependency. Analysis of the distribution of cells in the first, second and third division in cis-DDP treated cultures demonstrated the depressing effect of the drug on mitotic activity. In vivo analysis of SCE and chromosome aberrations in mouse showed that 13.85 mg/kg i.p. of cis-DDP produces significant increases in the rate of SCE and chromosome aberrations in bone-marrow cells.     

Chromosome breakage in low birth weight newborns.

The frequency of chromosome aberrations in cells cultured from umbilical cord blood was determined for 50 low birth weight (LBW) and 50 normal birth weight (NBW) euploid newborns matched for sex, race, and maternal age. The metaphase spreads had been prepared in the course of an earlier study of frequency of aneuploidy and results are from 72-h cultures, i.e., presumably, at the second division in vitro. There were no significant differences between the two groups in the frequency of cells with chromosome breakage, chromosome gaps, or hyperdiploid cells. There was, however, a significantly higher frequency of hypodiploid cells in the LBW group. The present findings differ from those of others who have reported an increase in chromosome breakage in premature newborns.     

Chromosome studies on lymphocytes of patients under cytostatic therapy

Summary Lymphocyte cultures from the peripheral blood of 38 patients undergoing a cytostatic interval therapy with a regimen of methyl-CCNU (1-[2-chloroethyl-3-(4-methyl-cyclohexyl)]-1-nitrosourea), 5-fluorouracil, and vincristine (each 5-day course of therapy was followed by a therapy interval of 4 weeks) were supplied with 5-bromodeoxyuridine (BUDR) for the whole culture time to determine the sister chromatid labelling pattern. From a total of 92 individual blood samples sister chromatid exchange (SCE) studies were performed including analyses before the start of the therapy, and immediately and 4 weeks after each course of therapy. In addition, the frequency of first, second, and third metaphases in the 72-h cultures was estimated using the characteristic labelling patterns.A distinct increase of SCE frequency over the control level (i.e., lymphocyte cultures of patients before the start of therapy) was observed at all phases of therapy. It was clearly correlated with the number of courses of therapy up to course 7, later on the SCE rate remained more or less at the level reached. The influence of the composition of each drug regimen on the SCE rate was less pronounced than it was on the breakage rate. Moreover, although a clear correlation existed between the individual rates of breakage and SCE, the formation of the latter appeared to reflect a long-term effect of the therapy rather than did the formation of break aberrations. In addition, as the intercellular variability of the number of SCEs per cell was much higher than that of breaks, the interindividual variability (variation of the mean values for each patient) was small compared to the respective variability of breakage rates.The proportion of first, second, and third metaphases present in 72-h cultures evidently was influenced by single courses of therapy. The observed delay of proliferation was also reflected in different amounts of chromosome damage. Although the BUDR treatment enhanced the cytostatic effect of the therapy on the lymphocytes in culture rendering SCE analysis rather difficult in several cases, the other data of this study and in particular the experiences with the long-term effect make it imperative to include BUDR-labelling in further cytogenetic studies in subjects with exceptional exposure to chemicals. However, the SCE method can by no means, replace the classic cytogenetic analysis.     

Clastogenic factors in plasma of HIV-1 infected patients

The objective of this study was to investigate the clastogenic activity of plasma ultrafiltrates from HIV-I infected patients. Clastogenic factors are chromosome-damaging agents with low molecular weight (<10,000 daltons) which cause chromosome aberrations, sister chromatid exchanges, DNA strand breakage, and gene mutation. They have first been described in the plasma of irradiated persons, but they are also found in hereditary breakage syndromes and chronic inflammatory diseases with autoimmune reactions. Their formation and their clastogenic effects are modulated by superoxide anion radicals. We analyzed a total of 22 HIV-1 positive patients in comparison to 20 reference plasma samples from healthy HIV negative blood donors of similar age. The plasma ultrafiltrates (filter cutoff 10,000 daltons) from patients induced a statistically significant increase in chromosomal breakage in the cytogenetic test system (20.5 ± 6.8 aberrations per 100 cells), while no increase was observed in test cultures exposed to plasma ultrafiltrates from healthy blood donors (6.3 ± 2.9 aberrations per 100 cells). The breakage values were slightly, but not significantly, lower in the 10 patients with more than 200 T-helper cells/ml (18 ± 4 aberrations per 100 cells), than in the 12 patients with less than 200 T-helper cells/ml (22.3 ± 7.9 aberrations per 100 cells). HIV patients with high clastogenic activity (induction of more than 20 aberrations per 100 cells, range 20 to 39) showed higher plasma levels for malondialdehyde than those with lower clastogenic activity (less than 20 aberrations per 100 cells, range 12 to 18). However, the difference was statistically not significant. Another lipid peroxidation product, 4-hydroxynonenal, was increased equally in both groups. There were no significant differences in water- and lipid-soluble plasma antioxidants between the low- and high-breakage group. In agreement with previous findings, the clastogenic effects of plasma ultrafiltrates in the test cultures were reduced by the antioxidant enzyme superoxide dismutase. The presence of clastogenic factors in the plasma of HIV patients is further evidence for a prooxidant state in these persons. Since clastogenic factor formation appears to occur at an early stage of the disease, it may be significant for virus release or activation, because of the superoxide anion stimulating effects of clastogenic factors. From a practical standpoint, clastogenic factors may be useful for evaluation of promising drugs.     

Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures

Summary Investigations have been carried out on karyotype change in both callus and suspension cell cultures of Haplopappus gracilis (2n=4). It has been found that polyploidization arises directly in culture to give up to six times the normal diploid chromosome number in some cultures. In polyploid cultures, both chromosome loss and chromosome rearrangements occur to give rise to aneuploid karyotypes displaying chromosomes which differ in morphology from the diploid set. Whole or partial chromosome loss has been observed in the form of lagging chromosomes and chromosome bridges at anaphase, and micronuclei, ring chromosomes and chromosome fragments at other stages in mitosis. C-banded preparations have confirmed the occurrence of chromosomal rearrangements. Comparative investigations suggest that (i) more polyploidy occurs in callus cultures than in suspension cell cultures, and (ii) the presence of cytokinin (kinetin) in the culture medium may reduce the extent of karyotype change.     

Chromosome damage and aneuploidy detected by interphase multicolour FISH in benzene-exposed shale oil workers

A multicolour tandem-labelling fluorescence in situ hybridization (FISH) procedure was used to detect chromosome alterations in peripheral blood cells of a group of Estonian petrochemistry workers. Twelve workers employed in benzene production and five cokery workers, together with eight unexposed rural controls, were enrolled in the study. The methodology employed, based on the in situ hybridization of adjacent centromeric and pericentromeric regions, allowed the simultaneous detection of both chromosome breakage, involving damage-prone pericentromeric regions, and hyperploidy in interphase cells. Blood smears from all subjects were hybridized with chromosome 1 specific probes, in order to detect genotoxic damage in circulating lymphocytes and granulocytes. Moreover, lymphocyte cultures were established, harvested 48 h following mitogen stimulation and hybridized with the tandem chromosomes 1 and 9 probes. No significant difference in the incidence of breakage was detected in the nucleated cells of blood smears of exposed vs. control subjects. In contrast, modest but significantly increased frequencies of breakage affecting both chromosomes 1 and 9 were observed in the cultured lymphocytes of the benzene-exposed workers compared to the unexposed controls, suggesting an expression of premutagenic lesions during the S-phase in vitro. Across the entire study group, the frequencies of breakage affecting chromosomes 1 and 9 in the stimulated lymphocytes were highly intercorrelated (p < 0.001). No significant difference was found in the incidence of hyperploidy among the study groups, although a tendency to higher values was observed in benzene-exposed workers. Although the relatively small size of the study groups does not allow firm conclusions on the role of occupational exposure, the observed patterns are suggestive of effects in the benzene-exposed workers. This work also shows that tandem labelling FISH can be usefully applied in human biomonitoring, allowing the simultaneous detection of both hyperploidy and chromosome breakage at interphase in different cell types.     

Bands involved in primary chromosome rearrangements in sarcomas are not constitutionally liable to breakage in sarcoma patients

Summary The localization of breakpoints in spontaneous chromosome aberrations, i.e., chromatid and chromosome gaps, breaks, and exchanges, has been studied in cultured skin fibroblasts from 34 untreated patients with musculoskeletal sarcoma and 38 controls. A total of 325 aberrations in the sarcoma group and 251 in the control group could be assigned to particular bands. The distribution was non-random (P<0.001) in both groups. Twenty-one bands in the sarcoma group and 20 in the control group appeared as hot spots, with 11 represented in both groups. Only three hot spots, all of which were present among both patients and controls, coincided with bands involved in primary sarcoma-associated chromosome rearrangements. The results indicate that the chromosome breakage pattern of non-malignant cells is similar in sarcoma patients and controls. Hence, the occurrence of primary structural rearrangements in sarcomas cannot be accounted for by any constitutional proneness to chromosome breakage at these bands.