Cytogenetic effects of methotrexate on human cells in vivo: comparison between results obtained by chromosome studies on bone-marrow cells and blood lymphocytes and by the micronucleus test

Cytogenetic studies were performed in 22 patients treated with methotrexate (MTX). In some patients, metaphases from both bone-marrow cells and peripheral blood cells were studied. In the bone-marrow preparations an increased number of structural chromosomal aberrations was present, whereas abnormalities were not observed in the peripheral blood cells. An examination of the bone-marrow chromosomes must therefore be included in the study of the possible chromosome-breaking effect of chemical agents. The results obtained with the micronucleus test and chromosome studies were compared in 10 patients treated with MTX. The micronucleus test was more sensitive than the chromosome analysis as regards the clastogenic effect of MTX.     

Cytogenetic studies in patients treated with penicillamine.

Cytogenetic studies were performed on bone-marrow cells from 11 patients with rheumatoid arthritis treated with penicillamine. One of the patients was studied while developing a granulocytopenia and thrombocytopenia. The findings show that penicillamine had no chromosome-damaging effect as estimated by the micronucleus test and by the number of structural chromosomal aberrations.     

Clastogenic effects of biosynthetic human growth hormone in Snell dwarf mice and CHO cells in vitro

Treatment of Snell dwarf mice with high concentrations of human growth hormone from pituitaries as well as of bacterial origin, significantly increased the frequencies of chromosomal aberrations in bone-marrow cells, as measured by the micronucleus test. In vitro treatment of Chinese hamster ovary (CHO) cells with the two types of hormone likewise induced structural chromosomal aberrations.     

Bleomycin-induced structural chromosomal aberrations in spermatogonia and bone-marrow cells of mice

The induction of structural chromosomal aberrations by bleomycin (BLM) was studied in bone-marrow cells and spermatogonia of the mouse at doses of 10, 20, 40 and 80 mg/kg. BLM induced genetically important reciprocal translocations in stem-cell spermatogonia as measured with the spermatocyte test, and the response of bone-marrow cells to BLM was not markedly different from that of spermatogonia.     

Louis-Bar syndrome: spontaneous and induced chromosomal aberrations in lymphocytes and micronuclei in lymphocytes,oral mucosa and hair root cells

Summary Louis-Bar (L-B) syndrome, also called ataxia-telangiectasia, is cytogenetically characterized by an increased frequency of spontaneous and induced chromosomal aberrations (CA) in cultured lymphocytes and skin fibroblasts. However, it is not yet clear whether the chromosomal instability is also present in uncultured cells. The spontaneous and bleomycin-induced CA in peripheral lymphocytes of 8 L-B patients were evaluated. The micronucleus test was also performed, for the first time in lymphocytes by the cytokinesis-block method, and in uncultured cells of the oral cavity and hair root. The spontaneous frequency of CA and micronuclei in lymphocytes was about 3 times higher in L-B patients than in controls, these two cytogenetic parameters being highly correlated. Moreover, the induction by bleomycin of CA was higher in patients than in controls. The micronuclei in buccal and hair root cells of patients were normal. It remains to be determined whether the different responses obtained with cultured and uncultured cells are the result of the different L-B gene expression of chromosomal instability or whether they arise because of a particular cell sensitivity to culture conditions. The spontaneous and induced CA in lymphocytes of heterozygotes cultured in the presence of L-B serum were studied to evaluate a possible increased sensitivity of heterozygotes to a possible diffusible clastogenic factor present in the plasma of L-B patients. We could not demonstrate the presence of any factor that enhances CA in normal subjects or in heterozygote carriers.     

Effect of follicle-stimulating hormone (FSH) on radiation-induced chromosomal aberrations in mouse germ cells and Chinese hamster cells in vitro

Earlier observations on the induction by X-rays of reciprocal translocations in stem-cell spermatogonia of the rhesus monkey have established a correlation between the level of follicle-stimulating hormone (FSH) in blood at the moment of irradiation and the final recovery of translocations (van Buul, 1980). In the present study, FSH treatment of mice did not induce chromosomal aberrations in bone-marrow cells or stem-cell spermatogonia, nor did it change the radiosensitivity of stem-cell spermatogonia for the induction of chromosomal translocations. Experiments in vitro with Chinese hamster ovary cells (CHO), however, showed a clear radiosensitizing effect of FSH on the induction of structural chromosomal aberrations.     

Relative ranking of culture media based on proliferation kinetics and spontaneous chromosomal aberrations in PHA-responsive human peripheral blood lymphocytes.

Proliferation kinetics and spontaneous yield of chromosomal aberrations phytohemagglutinin (PHA)-responsive peripheral blood lymphocytes were studied from blood samples collected from 45 individuals in 4 different synthetic media. Except for a significant difference for Eagle's MEM and RPMI 1640, the other media did not show difference for the yield of chromatid or chromosome type of aberrations. Differences were however noticed in the proliferation kinetics (mitotic and proliferative rate indices) of cells among the media used. The study indicated that (i) the intrinsic properties of media which influence proliferation rate and yield of chromosomal aberrations are independent of each other as higher proportion of first division cells do not correspond with higher frequency of chromosomal aberrations, (ii) the amount of free-radical scavengers present in the medium, apart from the genetic make-up of the individuals, may contribute to the spontaneous yield of chromosomal aberrations and (iii) RPMI 1640 medium, which showed higher transformation and faster cycling rate for the lymphocytes, may be considered as medium of choice for analysing two main cytogenetic end-points, chromosomal aberrations and sister chromatid exchanges (SCEs).     

Spontaneous and radiation-induced chromosomal instability and persistence of chromosome aberrations after radiotherapy in lymphocytes from prostate cancer patients

The aim of the study was to compare the spontaneous and ex vivo radiation-induced chromosomal damage in lymphocytes of untreated prostate cancer patients and age-matched healthy donors, and to evaluate the chromosomal damage, induced by radiotherapy, and its persistence. Blood samples from 102 prostate cancer patients were obtained before radiotherapy to investigate the excess acentric fragments and dicentric chromosomes. In addition, in a subgroup of ten patients, simple exchanges in chromosomes 2 and 4 were evaluated by fluorescent in situ hybridization (FISH), before the onset of therapy, in the middle and at the end of therapy, and 1 year later. Data were compared to blood samples from ten age-matched healthy donors. We found that spontaneous yields of acentric chromosome fragments and simple exchanges were significantly increased in lymphocytes of patients before onset of therapy, indicating chromosomal instability in these patients. Ex vivo radiation-induced aberrations were not significantly increased, indicating proficient repair of radiation-induced DNA double-strand breaks in lymphocytes of these patients. As expected, the yields of dicentric and acentric chromosomes, and the partial yields of simple exchanges, were increased after the onset of therapy. Surprisingly, yields after 1 year were comparable to those directly after radiotherapy, indicating persistence of chromosomal instability over this time. Our results indicate that prostate cancer patients are characterized by increased spontaneous chromosomal instability. This instability seems to result from defects other than a deficient repair of radiation-induced DNA double-strand breaks. Radiotherapy-induced chromosomal damage persists 1 year after treatment.     

Chromosomal instability in homo- and heterocygocity for microcephalia vera )author's transl)]

In a family with microcephalia vera, not only the 3 microcephalic probands, but also their healthy mother showed a significantly increased rate of spontaneous structural chromosomal aberrations in cultures of lymphocytes of the peripheral blood. Thus an increased aberration rate in the lymphocytes of asymptomatic family members could serve as a sign of heterocygocity. The qualitative analysis revealed aberrations of the chromatid and chromosomal type; exchanges that are frequently found in Fanconi's and Bloom's syndrome could not be detected. Therefore it can be assumed that the chromosomal instability of microcephalia vera is caused by a specific mechanism.     

Chromosomal aberrations and sister-chromatid exchanges in tool and die workers

Because tool and die workers are exposed to a number of potentially genotoxic agents, including mutagenic metals, polyaromatic hydrocarbons, and nitrosamines, and may be at increased cancer risk, the present study was undertaken to test whether chromosomal damage in peripheral blood cells is associated with work in the tool and die industry. Lymphocyte cultures were established from 27 tool and die fabrication workers from one manufacturing plant who had worked in the trade for more than 15 years. 15 of these workers also had some form of malignancy at the time of the study, but had not been treated with radiation or chemotherapies that could themselves induce chromosomal damage. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCEs) were measured in workers and the data compared with those of a control group consisting of 7 non-fabrication workers from the same plant and 8 age-matched community controls. In addition, the relative rates of lymphocyte proliferation were estimated for each group by analyzing the percentages of first-, second- and third-division mitotic cells after 72 h of culture. The results of the chromosomal studies show that tool and die workers have significantly increased frequencies of aberrations whether engaged in fabrication work or not, compared to control subjects. The frequency of SCEs and the frequencies of 1st, 2nd and 3rd division figures are not different among the study groups. Among workers who are engaged in fabrication, including those who are cancer patients, the frequency of more complex aberrations (i.e., interstitial deletions and small acentric fragments) is increased. In a five-year follow-up of these workers, 2 of the 13 workers with these aberrations developed some form of colon cancer. Whether the presence of interstitial deletions and small acentric fragments is related to the occupation of these workers, or is tangentially related to the development of cancer, is presently under consideration.     

A sequential screening of the cytogenetic damage induced by triaziquone.

A sequence is described of test procedures for a screening in vivo of the clastogenic potential of the alkylating agent triaziquone (Trenimon). Two intraperitoneal injections of 0.125 mg/kg body weight caused a considerable increase in the number of aberrations in both the micronucleus test and the bone-marrow metaphase test, but not in the spermatocyte translocation test or the spermatogonial metaphase test. With the latter test a severe cell-killing effect was detected. An analysis of whole mounts of seminiferous tubules showed that 0.125 mg/kg was a lethal dose for all B- and intermediate-type spermatogonia and partly killed A-type spermatogonia. A single administration of the same dose caused stable chromosomal rearrangements in spermatids that could be demonstrated with the F1 translocation test, and gave rise to dominant lethality of fetuses originating from post-meiotic sperm. The comparative triaziquone study has provided arguments in favor of the micronucleus test as a reliable screening method for chromosomal aberrations. The analysis of seminiferous tubules is a recommendable method for studying lethal effects of a compound on germ cells, whereas the F1 translocation test gives important information about viable aberrations and their effect on the fertility of the progeny.     

Mutagenicity of polycyclic hydrocarbons. II. Monitoring genetical hazards of chrysene in vitro and vivo.

Mutagenicity tests were performed with chrysene in the Salmonella/microsome test, NMRI-mice oocytes, bone-marrow cells and spermatogonia of Chinese hamsters. Only in mice oocytes was a weak but significant increase of structural chromosome aberrations observed. Correlations were found between weak carcinogenic and observed weak mutagenic activities of chrysene in vitro and in vivo.     

Effects of hormone treatment on chromosomal radiosensitivity of somatic and germ cells of Snell's dwarf mice

The X-ray induction of micronuclei and structural chromosomal aberrations was studied in bone-marrow cells of normal and dwarf (dw) mice in combination with thyroxine and/or prolactin treatment or otherwise. Hormone treatment clearly increased micronuclei induction but not chromosome breakage, suggesting that indirect effects were involved. Since no clear differences in the timing of the final stage of erythropoiesis could be found, it is likely that the indirect effects are mediated via the formation-differentiation kinetics of erythroblasts. The induction of reciprocal translocations by X-rays in stem cell spermatogonia of dwarf mice was lower than in normals and treatment with prolactin, growth hormone and/or thyroxin, did not influence the chromosomal radiosensitivity of spermatogonial stem cells.     

Cytogenetic evaluation of arsenic trioxide toxicity in Sprague-Dawley rats

Acute exposure to arsenic trioxide has been reported to induce death and/or multiple organ damage with symptoms including nausea, vomiting, diarrhea, gastrointestinal hemorrhage, cerebral edema, tachycardia, dysrhythmias and hypovolemic shock. Its toxic effects are due to its ability to bind to sulfhydryl groups of proteins and to inhibit energy production. Although the chronic exposure to arsenic trioxide has been linked to various types of cancer, such as skin, liver, lung, bladder and kidney neoplasms, studies of its carcinogenic potential in animals have not been conclusive. In this study, we investigated the genotoxic potential of arsenic trioxide in bone-marrow cells obtained from Sprague-Dawley rats; using chromosomal aberrations (CA), mitotic index (MI) and micronuclei (MN) formation as the toxicological endpoints. Four groups of six male rats each, weighing approximately 60+/-2 g per rat, were injected intraperitoneally, once a day for 5 days with doses of 5, 10, 15 and 20 mg/kg body weight (BW) of arsenic trioxide dissolved in distilled water. A control group was also made of six animals injected with distilled water without chemical. All the animals were sacrificed at the end of the treatment period. Chromosome and micronuclei preparation was obtained from bone-marrow cells following standard protocols. Arsenic trioxide exposure significantly increased the number of structural chromosomal aberrations, the frequency of micronucleated cells and decreased the mitotic index in treated groups when compared with the control group. Our results demonstrate that arsenic trioxide has a clastogenic/genotoxic potential as measured by the bone-marrow CA and MN tests in Sprague-Dawley rats.     

Cytogenetic effect of the anticancer drug epirubicin on Chinese hamster cell line in vitro

The present study demonstrated the cytogenetic effect of the anticancer drug epirubicin on cultures of Chinese hamster cell line in vitro. The cultures were exposed to the drug for 24 h at three final concentrations; 10, 20 and 40 microg/ml. All treatments were carried out in the absence of any exogenous metabolic activation system.The different types of structural chromosomal aberrations, including gaps, breaks, deletions and fragments were increased in epirubicin-treated cultures. This increase was dose dependent where there was a positive correlation between increased drug concentration and induction of structural chromosomal aberrations. Also, the numerical chromosomal aberrations, including hypodiploidy and hyperdiploidy, were increased significantly in epirubicin-treated cultures. Like structural aberrations, the increase of numerical chromosomal aberrations was also dose-dependent.The frequency of sister chromatid exchanges in cultures treated with epirubicin increased significantly and this increase was dose-dependent. On the other hand, the epirubicin significantly decreased the mitotic index in treated cultures of Chinese hamster cell line.     

Chromosomal aberrations and micronuclei in lymphocytes of breast cancer patients after an accident during radiotherapy with 8 MeV electrons

In February 2001 a radiation accident occurred in a radiotherapy unit of an oncology hospital in Poland. Five breast cancer patients undergoing radiotherapy received a single high dose of 8 MeV electrons. The exact doses are not known, but they were heterogeneous and may have reached about 100 Gy. To assess whether such exposure would be detectable in peripheral blood lymphocytes, chromosomal aberrations and micronuclei were analyzed in lymphocytes from the accident patients and compared to values for lymphocytes from 10 control patients who were not involved in the accident but who received similar radiotherapy treatments. Lymphocytes were harvested for analysis of chromosomal aberrations at three different culture times to determine whether heavily damaged cells reached mitosis with a delay. There was no effect of harvest time on the frequencies of chromosomal aberrations, indicating that there was no delay of heavily damaged cells in entering mitosis. A good correlation was observed between micronuclei and chromosomal aberrations. In lymphocytes from three of the accident patients, significantly enhanced frequencies of both aberrations and micronuclei were found. The great individual variability observed in the frequency of cytogenetic damage in lymphocytes from both control and accident patients precluded the unambiguous identification of all accident patients.     

Cytogenetic effects of myleran in vivo on bone-marrow cells from male mice

The cytogenetic effects of Myleran were examined in bone-marrow cells from male mice. To study the dose-response relationship the male mice were injected with 5, 10, 20 or 40 mg of Myleran/kg. Bone-marrow samples were prepared 24 h later. The time response was investigated by examining bone-marrow samples 1, 2, 4 or 10 days after i.p. injection of 40 mg of Myleran. Most of the structural aberrations were of the chromatid type and the dose-response relationship was linear. The chromatid and chromosomal aberrations were maximal at 2 days and decreased sharply after longer intervals of time.     

Chromosomal abnormalities in maternal and fetal tissues of magnesium- or zinc-deficient rats.

The effect of dietary deficiency during pregnancy of zinc or magnesium on maternal and fetal chromosomes was studied. Pregnant rats were given a zinc-deficient or a magnesium-deficient diet from the beginning of pregnancy and maternal bone marrow and fetal liver were removed on day 19 of gestation. Chromosome spreads were prepared and metaphases examined for abnormalities. Both magnesium- and zinc-deficient maternal bone-marrow and fetal liver cells showed significantly more chromosomal abnormalities than did those of controls. The chromosomal aberrations occurring in highest incidence in magnesium-deficient animals were terminal deletions and fragments. A higher than normal incidence of "stickiness" was also observed in cells from magnesium-deficient animals. In zinc-deficient animals, on the other hand, the chromosomal aberrations with the highest incidence were gaps and terminal deletions.     

Induction of chromosomal aberrations in active oxygen-generating systems. I. Effects of paraquat in Chinese hamster cells in culture

A possible role for the superoxide anion radical (O2-) in the clastogenicity of paraquat (PQ) was investigated in cultured Chinese hamster cells. When cells were treated with 0.8 mg/ml of PQ for 3 h followed by 21 h of recovery time, structural chromosome aberrations were induced in about 50% of the metaphases examined. Almost all aberrations were of the chromatid-type and involved exclusively gaps and breaks. The induction of chromosomal aberrations by PQ was enhanced by a 1-h pretreatment with diethyldithiocarbamate, an inhibitor of superoxide dismutase. Diethyl maleate, a glutathione scavenger, also enhanced the induction of chromosomal aberrations, but 3-aminotriazole, an inhibitor of catalase, showed no such effects. Enhanced induction of chromosomal aberrations was also observed when PQ-treated cells were cultured at a high oxygen concentration (80%). The present results suggest that the production of chromosomal aberrations by PQ may be directly or indirectly related to the generation of O2-, but not to the formation of hydrogen peroxide by the dismutation reaction of O2- or of other active oxygen species including the hydroxyl radical and singlet oxygen.     

The rate of sister chromatid exchanges parallel to spontaneous chromosome breakage in Fanconi's anemia and to trenimon-induced aberrations in human lymphocytes and fibroblasts.

The incidence of structural chromosome aberrations and the rate of sister chromatid exchanges (SCE) was investigated in lymphocyte cultures from a patient with typical Fanconi's anemia and his parents. The rate of SCEs was found to be normal. In experiments with the alkylating agent Trenimon the SCE rates proved to be a sensitive indicator for the induction of structural aberrations: in presence of an induced aberration rate half as high as the spontaneous rate in the Fanconi's anemia case, the rate of SCEs was found to be quintupled. Dose-effect relationships for the induction of SCE rates by Trenimon were studied over a wide dose range in lymphocyte and fibroblast cultures. The results reflect the same difference in sensitivity earlier observed in the induction of structural chromosome aberrations, fibroblasts being far more sensitive.