Adriamycin-induced chromosome aberrations in human fibroblasts.

Adriamycin (AM) induced chromosome lesions and cell division delay in human foreskin fibroblasts. Cells treated with 0.01, 0.03, and 0.05 mug AM/ml culture medium for 1 hour and evaluated 5-12 hours post treatment exhibited a wide spectrum of cytogenetic injuries, ranging from moderately damaged metaphases with predominantly simple chromatid-type lesions to heavily damaged metaphases with chromosome stickiness and fragmentation. In moderately damaged metaphases that could be scored for specific types of aberrations, we observed a paucity of chromatid exchanges and chromosome-type lesions even in cultures having a very high frequency of breakages. Further, the distribution of breaks among chromosomes within groups A-G appeared to be random, which suggested that the drug does not show breakage specificity in human fibroblasts; The number of heavily damaged metaphases increased with an increase in concentration of AM and with longer periods of recovery.

Establishment of two parental cell lines and three clonal cell strains from rat colonic carcinoma.

Two epithelial cell lines were established from separate pools of cells that were harvested by a sequential proteolytic treatment of a single specimen of rat transplantable colonic carcinoma. The cells were small (10-micrometer diameter) and cuboidal and displayed desmosomes and light junctions. Both cell lines grew rapidly with population-doubling times of 20 to 22 hr, were near diploid in chromosome number, contained A-2 and B-7 marker chromosomes, and were tumorigenic in rats. Each was distinguishable from the other by the shape of its cell clusters in monolayer culture, its serum requirement for growth, its modal chromosome number, and its karyotypic alterations. Undifferentiated cells, cystic cells, and vacuolated cells, but not mature mucous cells, were observed in monolayer cultures. Clonal cell strains mimic the morphological and growth properties of their respective parental cell line but display unique karyotypic alterations in addition to the A-2 and B-7 marker chromosomes.

Modification of lipid acyl groups by serum deprivation does not affect phorbol ester-induced differentiation of human leukemia cells.

12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, can induce differentiation of human promyelocytic leukemia cells grown in a serum-free environment. Removal of serum produces altered acyl group composition of cellular phospholipids, most notably, marked decreases in the polyunsaturated fatty acid content. Although higher amounts of TPA are required to induce differentiation in serum-free cell populations, induction assessed by morphological and enzymatic markers in similar to the "macrophage-like' differentiated state reported for cells grown in a serum-rich medium.

Statistical methods for the blood beryllium lymphocyte proliferation test.

The blood beryllium lymphocyte proliferation test (BeLPT) is a modification of the standard lymphocyte proliferation test that is used to identify persons who may have chronic beryllium disease. A major problem in the interpretation of BeLPT test results is outlying data values among the replicate well counts (approximately 7%). A long-linear regression model is used to describe the expected well counts for each set of Be exposure conditions, and the variance of the well counts is proportional to the square of the expected count. Two outlier-resistant regression methods are used to estimate stimulation indices (SIs) and the coefficient of variation. The first approach uses least absolute values (LAV) on the log of the well counts as a method for estimation; the second approach uses a resistant regression version of maximum quasi-likelihood estimation. A major advantage of these resistant methods is that they make it unnecessary to identify and delete outliers. These two new methods for the statistical analysis of the BeLPT data and the current outlier rejection method are applied to 173 BeLPT assays. We strongly recommend the LAV method for routine analysis of the BeLPT. Outliers are important when trying to identify individuals with beryllium hypersensitivity, since these individuals typically have large positive SI values. A new method for identifying large Sls using combined data from the nonexposed group and the beryllium workers is proposed. The log(SI)s are described with a Gaussian distribution with location and scale parameters estimated using resistant methods. This approach is applied to the test data and results are compared with those obtained from the current method.

Telomere staining of human chromosomes and the mechanism of radiation-induced dicentric formation.

The majority of models of radiation action developed over the past half century hold that the curvilinear dose responses exhibited by eukaryotic cells to sparsely ionizing radiations result from the interaction of pairs of lesions produced in sensitive targets of the cell. Within this conceptual framework, chromosomal exchange aberrations (e.g., interchanges) are believed to occur through the interaction of damaged sites on both chromosomes participating in the exchange. In contrast, the model proposed by Chadwick and Leenhouts (as well as some other models) suggests that such exchanges arise from initial radiation damage to only one chromosome, which then becomes associated with an undamaged chromosome. A particular aspect of this theory is that asymmetrical exchanges, such as dicentrics, may be formed from the rejoining of a broken end of one chromosome to the telomere of another. By using a DNA probe that specifically hybridizes to the telomeric region of human chromosomes, we were able to test this assertion directly. After scanning more than 200 dicentrics produced in normal human fibroblasts by 6 Gy of 60Co gamma rays, virtually none were found that contained telomeres located between the centromeres of this aberration type. Therefore, since the proposed telomere-break rejoining process, per se, is not necessarily a central element of the Chadwick-Leenhouts model, we suggest the theory be modified to exclude this mechanism.

Evaluations of cellular proliferation and chromosome breakages after in vitro exposure of human lymphocytes to calcium or zinc DTPA.

The analysis of mitotic indices (MI) and chromosome breakages in metaphases of 50-hr lymphocyte cultures exposed to the calcium or zinc chelates of diethylenetriamine pentaacetic acid (DTPA) demonstrated: (1) an 80% reduction in MI in cultures from three women but no reduction in those from two men after in vitro exposure to CaDTPA in concentrations as low as 10 micrograms/ml culture medium, and complete suppression of mitoses in cultures from men and women after exposure to 40 micrograms/ml CaDTPA; (2) minor suppression in MI in cultures from women and none in those from men after exposure to 40 or 80 micrograms/ml ZnDTPA; (3) no ring or dicentric chromosomes in 1700 metaphases from DTPA-treated cultures. Likewise, in other experiments we observed no differences in the frequency or distributions of rings and dicentrics in lymphocyte cultures from two persons after in vitro exposure to 250-R 60Co gamma radiation in the presence or absence of 10 micrograms/ml CaDTPA or 10 or 80 micrograms/ml ZnDTPA. These data indicate that while accurate estimates of the frequencies of radiation-induced rings and dicentrics in lymphocytes can be made in actinide-contaminated persons undergoing DTPA chelation therapy, blood samples for cytogenetic cultures should not be obtained from chelated patients until the compound has been cleared from the blood plasma.

Concentration-dependent protection against X-ray-induced chromosome aberrations in human lymphocytes by the aminothiol WR-1065.

WR-1065, the free-thiol form of WR-2721, has radioprotective effects in various biological systems. We measured the efficiency of WR-1065 in modifying the induction of chromosome aberrations by X rays in human lymphocytes. G0 lymphocytes were incubated for 30 min in medium containing 1-12 mM WR-1065, exposed to 0 or 3.1 Gy 220-kV X rays, washed, and cultured for evaluations of chromosome aberrations and micronuclei (MN). Neither proliferation kinetics nor baseline frequencies of aberrations or MN were affected in nonirradiated cultures incubated in WR-1065 for up to 45 min. Radiation-induced chromosome aberrations and MN varied inversely as a logarithmic function of thiol concentration. At extracellular concentrations of 8-12 mM, WR-1065 protected against > 85% of X-ray-induced chromosome damage as measured by either cytogenetic end point. WR-1065 is more efficient in modulating X-ray-induced chromosome aberrations than dimethyl sulfoxide, which provides protection by scavenging OH radicals. Our data suggest that mechanisms in addition to OH radical scavenging are involved in radioprotection by WR-1065.

Chromosome aberrations in peripheral lymphocytes and radiation dose to active bone marrow in patients treated for cancer of the cervix.

An international study of cervical cancer patients reported a doubling of the risk for leukemia following radiotherapy. To evaluate the extent of residual chromosome damage in circulating T-cell lymphocytes in this population, approximately 200 metaphases were examined from each of 96 irradiated and 26 nonirradiated cervical cancer patients treated more than 17 years ago (average 23 years). Radiation dose averaged over the total red bone marrow was estimated to be 8.1 Gy. The type and frequency of stable and unstable chromosome aberrations were quantified in 24,117 metaphases. Unstable aberrations did not differ significantly between irradiated and nonirradiated patients (P greater than 0.5). Stable aberrations (i.e., translocations, inversions, or chromosomes with deleted segments), however, were significantly higher among irradiated (2.8 per 100 cells) compared to nonirradiated (0.7 per 100 cells) women (P less than 10(4). The frequency of these stable aberrations was found to increase significantly with increasing dose to the bone marrow. These data indicate that a direct relationship between radiation dose and extent of damage to somatic cells persists in populations and can be detected many years after partial-body radiation exposure. The stable aberration rate in irradiated cervical cancer patients was 50 to 75% lower than those observed 25 years or more after radiation exposure in atomic bomb survivors and in ankylosing spondylitis patients treated with radiotherapy. The average marrow dose was only 1 Gy in the examined atomic bomb survivors and 3.5 Gy in the ankylosing spondylitis patients. It appears, then, that a very high dose delivered to the pelvic cavity in fractionated doses resulted in far fewer persistent stable aberrations than lower doses delivered either in acute whole-body exposure or in fractionated doses to the spinal column and sacroiliac joints. The higher radiation dose and the concentration of that dose in a smaller area of the body appear to be responsible for the lower rate of persistent aberrations observed in cervical cancer patients.

Chromosome aberrations in relation to radiation dose following partial-body exposures in three populations.

Structural chromosome aberrations were evaluated in peripheral blood samples obtained from three populations exposed to partial-body irradiation. These included 143 persons who received radiotherapy for enlarged thymus glands during infancy and 50 sibling controls; 79 persons irradiated for enlarged tonsils and 81 persons surgically treated for the same condition during childhood; and 77 women frequently exposed as young adults to fluoroscopic chest X rays during lung collapse treatment for tuberculosis (TB) and 66 women of similar ages treated for TB with other therapies. Radiation exposures occurred 30 and more years before blood was drawn. Doses to active bone marrow averaged over the entire body were 21, 6, and 14 cGy for the exposed thymic, tonsil, and TB subjects, respectively. Two hundred metaphases were scored for each subject, and the frequencies of symmetrical (stable) and asymmetrical (unstable) chromosome aberrations were quantified in 97,200 metaphases. Cells with stable aberrations were detected with greater frequency in the irradiated subjects compared with nonirradiated subjects in all three populations, and an overall test for an association between stable aberrations and partial-body ionizing radiation was highly significant (P less than 0.001). We found no evidence that radiation-induced aberrations varied by age at exposure. These data show that exposure of children or young adults to partial-body fractionated radiation can result in detectable increased frequencies of stable chromosome aberrations in circulating lymphocytes 30 years later, and that these aberrations appear to be informative as biological markers of population exposure.

Biological effectiveness of neutrons from Hiroshima bomb replica: results of a collaborative cytogenetic study.

The effectiveness of neutrons from a facsimile of the Hiroshima bomb was determined cytogenetically. The "Little-Boy" replica (LBR), assembled at Los Alamos as a controlled nuclear reactor for detailed physical dosimetry, was used. Of special interest, the neutron energy characteristics (including lineal energy) measured 0.74 m from the LBR were remarkably similar to those calculated for the 1945 Hiroshima bomb at 1 to 2 km from the hypocenter, as shown in a companion dosimetric paper (Straume, et al., Radiat. Res. 128, 133-142 (1991)). Thus we examine here the effectiveness of neutrons closely resembling those that the A-bomb survivors received at Hiroshima. Chromosome aberration frequencies were determined in human blood lymphocytes exposed in vitro to graded doses of LBR radiation (97% neutrons, 3% gamma rays). Vials of blood suspended in air at distances up to 2.10 m from the center of the LBR uranium core received doses ranging from 0.02 to 2.92 Gy. The LBR neutrons (E approximately 0.2 MeV) produced 1.18 dicentrics and rings per cell per Gy. They were more effective than the higher-energy fission neutrons (E approximately 1 MeV) commonly used in radiobiology. The maximum RBE (RBEM) of LBR neutrons at low doses is estimated to be 60 to 80 compared to 60Co gamma rays and 22 to 30 compared to 250-kVp X rays. These results provide a quantitative measurement of the biological effectiveness of Hiroshima-like neutrons.

Cumulative genetic damage in hematopoietic stem cells in a patient with a 40-year exposure to alpha particles emitted by thorium dioxide.

Thorotrast, a colloidal suspension of the long-lived radionuclide, thorium-232, was widely used as a radiographic contrast medium for several decades. Due to the poor excretion of the sol, however, Thorotrast would deposit in the liver, bone marrow and other tissue, and patients would receive alpha-particle irradiation for life. To gauge the cumulative genetic damage to hematopoietic stem cells due to chronic exposure to alpha particles, we conducted a multi-end-point evaluation in a 72-year-old man who had been administered a 32-ml bolus of Thorotrast during cerebral angiography performed over 40 years ago in 1950. Peripheral T lymphocytes were cultured to quantify the frequencies and cellular distributions of asymmetrical and symmetrical types of chromosome aberrations in first-division metaphases and micronuclei in cytokinesis-arrested interphase II cells. Aberrations were scored using classical chromosome group analysis methods and chromosome painting techniques. Assays of glycophorin-A (GPA) mutations in red blood cells were also performed to obtain a relative measurement of damage sustained by the erythroid stem cell population. Results revealed that approximately 30% of the lymphocytes in this patient contained one or more chromosome aberrations, the majority of which were of the "stable" type. About one-third of the lymphocytes with chromosome damage carried multiple aberrations, suggesting that significant numbers of stem cells survive exposures to alpha-particle radiation that induce complex genomic alterations. Increased frequencies of GPA mutations were observed, demonstrating that genomic damage is also induced in erythroid progenitors. The numbers of micronuclei in lymphocytes were only moderately increased compared to expected values for persons of comparable age, and thus this end point was not useful for quantifying exposure level. Despite the relatively severe burden of somatic cell damage induced by 40 years of internal alpha-particle irradiation, the patient remains surprisingly free of any serious illness.

Chromosome aberrations in lymphocytes from women irradiated for benign and malignant gynecological disease.

Excess leukemias have occurred after partial-body radiotherapy for cervical cancer and benign gynecological disease (BGD). However, the level of risk is nearly the same in both groups, about twofold, despite a tenfold difference in average dose to active bone marrow (8 Gy vs 0.7 Gy, respectively). High-dose cell killing has been postulated as one explanation for this apparent inconsistency. To examine whether chromosome aberration rates observed in lymphocytes many years after exposure might serve as population markers of cancer risk, blood samples were taken from 60 women treated for BGD (34 with radiation) and cytogenetic data compared with previous results from 96 women irradiated for cervical cancer. Remarkably, the rate of stable aberrations, which reflects nonlethal damage in surviving stem cells, was only slightly higher among the cancer patients. Thus the lower-dose regimens to treat benign disorders resulted in much higher aberration yields per unit dose than those for cervical cancer. Assuming that the fraction of cytogenetically aberrant stem cells that survive radiotherapy contributes to the leukemogenic process, these data are then consistent with the epidemiological observations of comparable overall leukemia risks seen in these two irradiated populations. Accordingly, for patient populations given partial-body radiotherapy, stable aberrations at a long time after exposure appear to serve as biomarkers of effective risk rather than as biomarkers of radiation dose received.

Thyroid nodularity and cancer among Chernobyl cleanup workers from Estonia.

Thyroid examinations, including palpation, ultrasound and, selectively, fine-needle aspiration biopsy, were conducted on nearly 2,000 Chernobyl cleanup workers from Estonia to evaluate the occurrence of thyroid cancer and nodular thyroid disease among men with protracted exposure to ionizing radiation. The examinations were conducted in four cities in Estonia during March-April 1995, 9 years after the reactor accident. The study population was selected from a predefined cohort of 4,833 cleanup workers from Estonia under surveillance for cancer incidence. These men had been sent to Chernobyl between 1986 and 1991 to entomb the damaged reactor, remove radioactive debris and perform related cleanup activities. A total of 2,997 men were invited for thyroid screening and 1,984 (66%) were examined. Estimates of radiation dose from external sources were obtained from military or other institutional records, and details about service dates and types of work performed while at Chernobyl were obtained from a self-administered questionnaire. Blood samples were collected for assay of chromosomal translocations in circulating lymphocytes and loss of expression of the glycophorin A (GPA) gene in erythrocytes. The primary outcome measure was the presence or absence of thyroid nodules as determined by the ultrasound examination. Of the screened workers, 1,247 (63%) were sent to Chernobyl in 1986, including 603 (30%) sent in April or May, soon after the accident. Workers served at Chernobyl for an average of 3 months. The average age was 32 years at the time of arrival at Chernobyl and 40 years at the time of thyroid examination. The mean documented radiation dose from external sources was 10.8 cGy. Biological indicators of exposure showed low correlations with documented dose, but did not indicate that the mean dose for the population was higher than the average documented dose. Ultrasound examinations revealed thyroid nodules in 201 individuals (10.2%). The prevalence of nodules increased with age at examination, but no significant associations were observed with recorded dose, date of first duty at Chernobyl, duration of service at Chernobyl, building the sarcophagus or working on the roof of neighboring buildings or close to the damaged reactor. Nodularity showed a nonsignificant (p(1) = 0.10) positive association with the proportion of lymphocytes with chromosome translocations, but associations with the frequency of variant erythrocytes in the GPA assay were weak and unstable (p(1) > or = 0.46). The majority of fine-needle biopsies taken on 77 study participants indicated benign nodular disease. However, two cases of papillary carcinoma and three benign follicular neoplasms were identified and referred for treatment. Both men with thyroid cancer had been sent to Chernobyl in May of 1986, when the potential for exposure to radioactive iodines was greatest. Chernobyl cleanup workers from Estonia did not experience a markedly increased risk of nodular thyroid disease associated with exposure to external radiation. Possible reasons for the apparent absence of effect include low radiation doses, the protracted nature of the exposure, errors in dose measurement, low sensitivity of the adult thyroid gland or the insufficient passage of time for a radiation effect to be expressed.

Do recorded doses overestimate true doses received by Chernobyl cleanup workers? Results of cytogenetic analyses of Estonian workers by fluorescence in situ hybridization.

Studies of workers who were sent to Chernobyl after the 1986 reactor accident are being conducted to provide a better understanding of the effects of chronic low-dose radiation exposures. A crucial component to these investigations is an accurate assessment of the radiation doses received during the cleanup activities. To provide information on biological measurements of dose, fluorescence in situ hybridization (FISH) with whole-chromosome painting probes has been applied to quantify stable chromosome aberrations (translocations and insertions) among a defined cohort of 4,833 cleanup workers from Estonia. Cytogenetic analysis of 48-h lymphocyte cultures from 118 Estonian cleanup workers (10.3 cGy mean recorded dose; 25 cGy maximum), 29 Estonian population controls and 21 American controls was conducted by three laboratories. More than 258,000 painted metaphases were evaluated. Overall, we observed lower translocation frequencies than has been reported in previous studies using FISH among Chernobyl cleanup workers. In our data, a clear association with increased levels of translocations was seen with increasing age at blood drawing. There was no correlation, however, between aberration frequency and recorded measurements of physical dose or any category of potential high-dose and high-dose-rate exposure such as being sent to Chernobyl in 1986, working on the roof near the damaged nuclear reactor, working in special zones or having multiple tours. In fact, the translocation frequency was lower among the exposed workers than the controls, though not significantly so. To estimate the level of effect that would have been expected in a population of men having an average dose of approximately 10 cGy, blood from six donors was exposed to low-LET radiation, and more than 32,000 metaphases were scored to estimate dose-response coefficients for radiation-induced translocations in chromosome pairs 1, 2 and 4. Based on these results, we estimate that had this group of 118 men received an average whole-body dose of 10-11 cGy, as chronic or acute exposures, an increase in the mean frequency of chromosome translocations of more than 40-65% would have been observed in their lymphocytes compared to findings in nonirradiated controls. In spite of evaluating more than a quarter of a million metaphases, we were unable to detect any increase in the mean, median or range in chromosome aberrations in lymphocyte cultures from a group of Estonian men who took part in the cleanup of the Chernobyl nuclear power site and those who did not. We conclude that it is likely that recorded doses for these cleanup workers overestimate their average bone marrow doses, perhaps substantially. These results are consistent with several negative studies of cancer incidence in Chernobyl cleanup workers and, if borne out, suggest that future studies may not be sufficiently powerful to detect increases in leukemia or cancer, much less distinguish differences between the effects of chronic compared to brief radiation exposures.

Premature separation of centromeres in marrow chromosomes from an untreated patient with acute myelogenous leukemia.

Cytogenetic studies in an untreated patient with acute myelogenous leukemia revealed that greater than 60% of his marrow metaphases had pronounced anomalies in the centromere region. In the least affected chromosomes, the centromere appeared as a nonstaining hole, whereas in the most affected chromosomes, it appeared that the centromere segment had prematurely migrated at right angles to the telomeric portions. The population of mitoses exhibiting premature separation of the centromeres was reduced to virtually none during periods of drug induced remission. Our findings suggest that cytogenetic changes in malignant myeloid cells may be characterized by defects in centromere separation, as well as by alternations in karyotype.

Analysis of mutagen-induced chromosome damage in a primate species (Saguinus oedipus oedipus) at risk for spontaneous adenocarcinoma of the colon.

A high incidence of adenocarcinoma of the colon (greater than 16%) has been observed at necropsy in the South American primate, Saguinus oedipus oedipus (S. oedipus), while the disease has not been found in tamarins of the closely related species, Saguinus fuscicollis spp, housed in the same research colony. Cytogenetic analyses in cultured lymphocytes from 10 S. oedipus and 10 S. fuscicollis illigeri (S. fuscicollis) demonstrated no differences in the average frequencies of spontaneous or mitomycin C (MMC)-induced sister chromatid exchanges (SCEs) between animals of the two species. However, highly significant variability in MMC-induced chromosome lesions was observed between the individual S. oedipus, with one animal exhibiting increased sensitivity for both SCEs and chromosome breakages. At present we do not know the relationship, if any, between increased sensitivity to mutagen-induced cytogenetic lesions in specific S. oedipus tamarins and the increased risk for colon cancer that has been documented in this primate species. However, our cytogenetic findings in this one S. oedipus are similar to data obtained in evaluations of persons with several autosomal recessive conditions in which there is a genetic predisposition for developing malignancies.

Induction of micronuclei by bleomycin in G0 human lymphocytes: I. Dose-response and distribution.

The cytokinesis-block micronucleus assay was used to investigate the induction of chromosomal damage by bleomycin in G0 human lymphocytes. A dose-dependent increase in the frequency of micronuclei was observed in binucleate cells, and the frequency approached 0.5 micronuclei per cell at the highest dosage tested. The distribution of micronuclei among cells was overdispersed, rather than fitting a Poisson distribution. Even at the highest dosage, more than two-thirds of the cells did not contain micronuclei, while some cells were highly damaged, containing more than 4 micronuclei per cell.

Induction of micronuclei by bleomycin in G0 human lymphocytes: II. Potentiation by radioprotectors.

Dimethylsulfoxide (DMSO) and WR-1065 are radioprotectors, in that they reduce the effectiveness with which ionizing radiation causes genetic damage. Unlike their protective effects with radiation, these agents potentiate the induction of micronuclei by bleomycin in the cytokinesis-block assay in G0 human lymphocytes. High concentrations of DMSO (1 M) are required to cause potentiation. In contrast, WR-1065 causes dose-dependent potentiation at relatively low concentrations (1.25 to 10 mM). Cytogenetic analysis supports the results from the micronucleus assay, showing higher levels of genetic damage induced by the combination of bleomycin with DMSO or WR-1065 than by bleomycin alone. Possible mechanisms of potentiation are proposed.

Structure-activity analysis of the potentiation by aminothiols of the chromosome-damaging effect of bleomycin in G0 human lymphocytes.

The radioprotective aminothiols 2-[(aminopropyl)amino] ethanethiol (WR-1065) and cysteamine (CSM) potentiate the induction of chromosomal damage by the radiomimetic compound bleomycin (BLM) in G0 human lymphocytes. To investigate the mechanism of potentiation, we measured the clastogenic activity of BLM in the cytokinesis-block micronucleus assay in the presence and absence of amines, thiols, and aminothiols. The hydroxy analog of WR-1065, 2-(3-aminopropylamino) ethanol (WR-OH), potentiates BLM only slightly, indicating the critical nature of the thiol group. As thiols, WR-1065 and CSM may donate electrons for the activation of Fe(+2)-BLM or for the regeneration of Fe(+2)-BLM from inactive Fe(+3)-BLM. The amines putrescine, spermidine, and spermine all potentiate BLM, but they are weaker potentiators than the aminothiols, and they are effective only at high concentrations. Their activity, like that of WR-OH, is probably a consequence of conformational alteration of DNA. Dithioerythritol (DTE) and 2-mercaptoethanol (2-ME), thiols lacking an amino group, are less effective potentiators of BLM than are the aminothiols. The thiol group of WR-1065 and CSM is therefore essential, but insufficient, for explaining the strong enhancement of BLM activity. The cationic nature of CSM and WR-1065, conferred by the amino groups, evidently concentrates the active thiol function at the site of BLM action on DNA. As expected on this basis, the diamine WR-1065 is a more effective potentiator of BLM than is the monoamine CSM, whereas cysteine and N-acetylcysteine (NAC), which lack a net positive charge, potentiate BLM only weakly. These studies suggest that potentiation of the clastogenic action of BLM by aminothiols can be explained by the combination of a thiol-mediated redox mechanism and an amine-mediated targeting of the thiol function to DNA.

Methods for improving the yield and quality of metaphase preparations for FISH probing of human lymphocyte chromosomes.

Procedures are described for the in vitro culture of human lymphocytes, which have been concentrated by density gradient centrifugation, and for a modified slide-making technique for the fixed cells. The method yields improved percentages of mitotic cells which are largely synchronized at harvest. Controlled placement of fixed cells on slides produces well-spread metaphase preparations with little background material to interfere with fluorescence in situ hybridization (FISH) probe procedures. The FISH reagents and microscope scanning time required are minimized by concentrating cells in a defined area of the slide.