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Thioridazine (Mellaril), a psychotropic drug belonging to the phenothiazine group of drugs, was evaluated for its in vivo clastogenic effect on human chromosomes in lymphocyte cultures. Lymphocyte cultures were set up with peripheral blood samples of psychiatric patients (diagnosis: schizophrenia, mania, manic depressive psychosis, acute psychotic episode) who were on therapeutic doses of thioridazine for over 4 weeks. Two sets of controls were used: (1) newly diagnosed psychiatric cases (same type as above) who were not yet under therapy and (2) healthy individuals from the general population. The chromosomal aberration frequency was found to be significantly increased in the patients exposed to this drug as compared to the two sets of controls. The results point to a clastogenic and mutagenic potential of thioridazine and warrent a more careful consideration of its use in medicine.

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 micrograms/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.

Fanconi anaemia (FA) lymphocytes were tested for their susceptibility to chromosomal breakage by cis-diamminedichloroplatinum (II) [cis-Pt(II)] and its stereoisomer trans-diamminedichloroplatinum (II) [trans-Pt(II)]. Unlike trans-Pt(II), which is a rather inefficient clastogen, cis-Pt(II) is very efficient in inducing chromosomal breakage in FA cells at concentrations that hardly affect control cells. As both cis-Pt(II) and trans-Pt(II) are capable of inducing DNA interstrand crosslinks but only cis-Pt(II) can induce DNA intrastrand crosslinks, this result suggests that FA cells may be specifically sensitive to the intrastrand type of DNA crosslink.

Post-treatment of mutagen-treated human peripheral lymphocytes with a single-strand specific endonuclease from Neurospora crassa leads to a significant elevation of the rate of structural chromosomal aberrations. Our results indicate that DNA double-strand breaks (DSB) are ultimate lesions for the formation of chromosomal aberrations in the G1 and G2 phase of the cell cycle and probably also in the S-phase. Post-treatment of X-irradiated G2 cells with polyethylene glycol (PEG) leads to an elevation of the frequencies of chromatid type aberrations. This result is taken as an indication that nucleases from PEG-damaged lysosomes transform lesions in X-ray damaged chromosomes to DSB. With respect to the origin of chromosomal aberrations, our results are in favour of the breakage and reunion hypothesis of K. Sax , and not of Revell 's exchange hypothesis.

Among human survivors following the methyl isocyanate (MIC) gas tragedy the major complaints have been related to deep-seated suffocation, terrible pain in breathing, and severe ocular irritations. In order to assess the possible genetic effects we have used lymphocyte cultures and screened chromosomes by two techniques; one by looking for chromosomal aberrations and the other by estimating sister-chromatid exchange (SCE) frequencies. Both these parameters are good indicators of genetic damage in chromosomal DNA. SCE frequencies in lymphocytes have been increased more than three times in MIC-exposed persons. The results were compared to two groups of controls (one group comprising persons present in the same house; the second group of persons were chosen from distant places, 20-50 km away from the incident). Chromosomal breaks have been observed in 10 out of 14 MIC-affected people (71.4%) studied while only 6 out of 28 (21.4%) controls had chromosomal breaks. Some MIC-exposed persons had chromatin bodies in addition to the normal 46 chromosomes. These observations suggest that chromosomal DNA has been damaged.

Posttreatment with sodium arsenite in log phase synergistically increases the chromosomal aberrations induced by ethyl methanesulfonate in Chinese hamster ovary cells, human fibroblasts, and human lymphocytes. However, posttreatment with sodium arsenite in stationary phase has no apparent effect on the clastogenicity of ethyl methanesulfonate. These results indicate that the cycling state of the cell plays a crucial role in the action of arsenite coclastogenicity. One prediction from this finding is that in combined treatment, posttreatment with sodium arsenite should preferentially kill cancer cells.

At least four folic acid sensitive fragile sites have been detected in the karyotype of the Persian vole Ellobius lutescens Th. (Rodentia, Microtinae). Two such "hot spots" are located on chromosome 1 flanking the pericentric segment which is inverted in many individuals. The fragile site on 1p was observed on both homologues in one cell. Our findings suggest that fragile sites are common properties of mammalian chromatin and might have played a role in karyotype evolution.

Bovine superoxide dismutase (SOD) (Peroxinorm, Grünenthal Stolberg) was injected intramuscularly or subcutaneously in a daily dose of 4 mg over a period of six weeks into two patients with Fanconi anaemia. The effects (measured by the decrease of chromosome aberrations in blood lymphocytes and the increase in blood cells in venous blood) were evident but temporary. The hypothetical mode of action of SOD and the failure of a prolonged therapeutic effect are discussed.

In man a common fragile site is known to occur at 3p14. We studied the expression of this fragility in a group of 70 normal healthy subjects. Chromosome breaks, chromatid breaks and gaps at 3p14 could be observed in every examined individual, and in a total of 7000 metaphases they were seen in a mean of 4% of cells. Fluorescence studies in ten persons with chromosome No. 3 polymorphism showed that in all cases both Nos. 3 were about equally liable to breakage. A considerable variation in the fra 3p14 expression was found between individuals as well as in repeated cultures from the same person. Neither sex nor age influences could be detected. Cultures with a high percentage of lesions at 3p14 tended to have also a high number of lesions at other sites. Methotrexate and fluorodeoxyuridine markedly enhanced the expression of fra 3p14 and other fragilities. It is concluded that the chromosomal region at 3p14 represents man's most common fragile site, the expression of which seems to be influenced by environmental and heritable factors.

A modified technique has been developed for the visualization of the chromosomes in human sperm. The cytogenetic analysis of 129 G-banded human sperm metaphases of 6 normal donors showed an incidence of structural and numerical chromosome abnormalities of 7.8%. Two out of 129 spermatozoa were aneuploid (1.6%). The frequency of sperms with chromatid-type aberrations was 2.3% (3/129). Chromosome-type aberrations were found in 5 out of 129 (3.9%) spermatozoa. X to Y ratio did not differ significantly from the expected one-to-one ratio. Twenty-six sperm complements from a patient 18-20 months after testes exposure to 30 Gy were examined. A significant increase of numerical and structural chromosome abnormalities was not observed. Chromatid-type aberrations were found in two sperm complements (7.7%) and chromosome-type aberrations in one sperm complement (3.9%). The cytogenetic analysis of 15 human sperms from a cancer patient 26 months after chemotherapy showed an increased frequency of aberrant sperm complements (33.4%). One chromatid-type (6.7%), three chromosome-type aberrations (20.0%) and one (6.7%) hyperploid sperm complement could be observed. The sample size is still too small to answer the question whether chemical mutagens may increase the frequency of chromosomal abnormalities in human sperm.

Using cultured trophoblast cells obtained by chorionic villus biopsy, we diagnosed Fanconi anemia (FA) in two pregnancies and excluded it in eight pregnancies at risk for the syndrome. Baseline chromosomal breakage and breakage induced by diepoxybutane (DEB) were analyzed. Increased breakage was used as a marker for the syndrome. Our results were unambiguous and provide a reliable method for prenatal detection of FA in the first trimester of pregnancy.

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.

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 24 h) 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.

Chromosomal aberrations were scored in normal human lymphocytes cultured for 3 to 7 days at 20, 40, 50, and 60% oxygen. Compared to normoxic cultures (20% O2), in hyperoxic cultures (40% to 60% O2) the frequency of aberrant cells was significantly increased, especially at the longer incubation times. The increase was mainly due to chromatid-type damage: chromatid gaps, breaks, and interchanges. In addition, long-term hyperoxic cultures had a remarkably high incidence of endoreduplications, up to 7.4%. It appears that prolonged hyperoxic incubation induces a pattern of cytogenetic abnormalities in normal human lymphocytes similar to that present "spontaneously" in 3-day normoxic cultures from patients with Fanconi anemia.

Human lymphocytes from 18 healthy adults were cultured in minimal essential medium without folic acid. The addition of 4 mM cytidine or 1 mM guanosine to cultures 24 h prior to harvest produced a statistically significant increase in hot point 3p14 breaks. An excess of 3 mM adenosine or thymidine had no such effect on the hot point. The mechanism of the effects of nucleosides on hot points was discussed.

The treatment of human lymphocytes in vitro with 5-methoxypsoralen (5-MOP) plus UV-A induces a dose-dependent increase in the SCE rate and in structural chromosome aberrations. We carried out tests to see whether the clastogenic effect of 5-MOP plus UV-A could be reduced by the anticlastogen beta-aminoethylisothiouronium (AET). The occurrence of a protective effect proved to be dependent upon the conditions of treatment. When AET was present over a long period of time (22 h) in cultures with 5-MOP, the number of breaks was reduced compared with such cultures without AET (reduction factor 0.5-0.6). On the other hand, a short period of action by AET (1.5 h) in the presence of 5-MOP produced no reduction of breaks. Posttreatment with AET (20 h) yielded an obvious protective effect (reduction factor 0.2-0.4). The possible mechanisms of the protective effect of AET are discussed.

Utilizing the phenomenon of premature chromosome condensation (PCC) studies were carried out on interphase chromatin of metastatic cells from 52 cancerous effusions obtained from 45 patients presenting with various solid carcinomas. A highly individual pattern of distribution of the various interphase stages was detected, reflecting the heterogeneity of human solid tumors in an advanced stage. Nevertheless a variety of clinical, biologic, and technical factors were examined for their possible influence on these PCC patterns. The duration in culture was one of the influencing factors, as were the time lapse between the first diagnosis and the sampling of the respective effusion, or the nature of cytostatic therapy. Cytogenetic equivalents of gene amplification, as represented by "double minutes", could be found in the prematurely interphase chromatin of 35 of the 52 effusions. G1-PCC proved to be most reliable with regard to screening of double minutes. In addition, an adequate quality of Giemsa banding was achieved in PCC of 21 out of 24 effusions yielding a sufficient number of well-spread PCC. In six of these 21 cases PCC was superior to metaphase analysis in obtaining karyotypes, while the same was true for 14 of the 52 effusions screened for double minutes. Thus the PCC technique was shown to be an indispensable additional source of cytogenetic information in cells of human solid tumors.

Eleven folic acid sensitive fragile sites (3p14, 7p13, 7q31.1, 7q32, 9q32, 11p13, 14q23, 15q22, 16q23, Xp22.2, Xq22) were detected in one individual, eight of them previously unknown. These sites seem to bear each its specific sensitivity to folic acid deficiency. Six of the sites were observed simultaneously on both homologous chromosomes in at least one cell. Each of these 11 sites was also found in at least one among 12 individuals further examined. Some of these individuals showed six of these 11 sites. The fragile site 3p14 was detected in all individuals examined. The homologous sites 3p14 of one individual differed from each other in their frequency of lesions induced by methotrexate as well as fluorodeoxyuridine. This observation suggests that folic acid sensitivity is a property inherent in the chromatin of an individual chromosome at the site involved in fragility. This property seems to be responsible for the nonrandom fragility at that site and also for the individual sensitivity of each chromosomal site.

In the present study we examined cells from several patients clinically diagnosed as having ataxia-telangiectasia (AT), for the capacity of their cells to inhibit DNA synthesis following exposure to gamma irradiation, and for the rate of spontaneous or bleomycin-induced chromosomal aberrations. Cells from two patients showed normal inhibition of DNA synthesis and levels of induced chromosomal aberrations intermediate between normal and AT cells. These two patients had only minimal immunologic impairment. These findings appear to define one distinct subset of AT.

Chromosome aberrations were studied in peripheral lymphocytes from 50 patients treated with melphalan against ovarian carcinoma. The chromosome analyses were carried out 4-132 months (mean 57 months) after the end of melphalan therapy. Most of the patients were studied several times during four years. The mean frequency of cells with chromosome and chromatid aberrations was 5.4% in the patients and 2.3% in an untreated control group. The highest aberration frequency (average 18%) was found in a patient who later developed gastric carcinoma. The dominating types of aberrations in the patients were chromosome exchanges occurring as single marker chromosomes or as multiple chromosome rearrangements. These types of aberrations were found in only 0.3% of the control cells as compared to 3.8% of the patient cells. Patients with a high total dose of melphalan (above 420 mg) and a long duration of the therapy (average 22.5 months) had a higher frequency of cells with aberrations (6.3%) than patients with a lower total dose (below 420 mg) and a shorter therapy (12 months) (4.2%). No additive effect of radiation therapy was observed on the aberration frequency.

Caffeine has been reported to enhance the expression of the fragile X [fra(X)] and common fragile sites in peripheral blood lymphocyte cultures (PBLC) treated with 5-fluorodeoxyuridine (FUdR). One of the effects of caffeine on replicating cells is inhibition of DNA repair suggesting that fragile sites may be regions of DNA with a high rate of misreplication under the conditions of thymidylate stress induced by FUdR. We have studied the effect of caffeine on the expression of the fra(X) and common folate-dependent fragile sites in PBLC from two fra(X) expressing individuals and in five lymphoblastoid cell lines (LCL) established from individuals in families in which the fra(X) is segregating. Caffeine did not enhance the expression of the fra(X) in the PBLC or in the three LCL from fra(X) expressing individuals nor did it elicit fra(X) expression in LCL from a non-expressing obligate-carrier female and a transmitting male. However, in all cultures there was a marked increase of common fragile site expression due to caffeine treatment. These data suggest that the mechanism of expression of the common fragile sites and the fra(X) may be quite different.