Inhibition of heterochromatin condensation of human Y chromosome by distamycin-A

Distamycin-A, an oligopeptide antibiotic with a N-methylpyrrole ring system and propionamide side chain, preferentially forms stable bonds with AT rich double stranded DNA. When introduced to cell cultures, it inhibits condensation of the heterochromatic region of the Y chromosome. The frequency of metaphases showing inhibition of heterochromatin condensation of the Y chromosome was found to be dependent on the treatment time and concentration of distamycin-A in the culture medium. When distamycin-A was added to a concentration of 100 micrograms/ml at the start of the culture (72 hours), the frequency of Y heterochromatin decondensation was found to be 48%, 30% and 6% in amniotic fluid, lymphocyte and fibroblast cultures respectively. The highest frequency of metaphases with decondensed Y heterochromatin were observed when distamycin-A treatment was carried out for the last 24 hours prior to harvest, the frequencies being 94%, 72% and 59% in amniotic fluid, lymphocyte and fibroblast cultures respectively. Increase in the concentration of distamycin-A from 25 micrograms/ml to 50 micrograms/ml during the last 24 hours of culture increased the incidence of metaphases with Y heterochromatin decondensation from 51% to 69% in amniotic fluid, 40 to 49% in lymphocyte and 29% to 31% in fibroblast cultures. Highest frequency of metaphases with Y heterochromatin decondensation were observed when the cultures were exposed to distamycin-A at a concentration of 100 micrograms/ml for the last 24 hours of culture.     

Reducing the radiation dose from inhaled americium-241 using continuously administered DTPA therapy

Accelerating the removal of a radionuclide from the body of a contaminated individual is the only available approach to decreasing the radiation dose from such exposures. In this study, continuous infusion of a chelating agent, DTPA, was given to dogs that had inhaled a moderately soluble aerosol, 241 AmO2, not only to accelerate clearance of the radionuclide from the lung but also to prevent its deposition in liver and bone. Treatment was begun with an intravenous injection of CaDTPA 1 h after exposure, and was continued for 64 days after exposure by implanting subcutaneously osmotic pumps containing ZnDTPA at 1 day after exposure. The results showed that the infusion therapy was effective in blocking the translocation of 99.5 per cent of the 241Am that would have been deposited in liver, and 98.3 per cent of the 241Am that would have been deposited in bone. This result was significantly better than the result achieved using repeated intravenous injections of DTPA, the method of treatment in current use for actinide contamination cases.     

Nuclei with protrusions--"tailed" nuclei--and radiation cytogenetic markers in a lymphocyte culture after x-ray irradiation

Frequency of the appearance of binuclear cells with nuclei having outgrowth into the cytoplasmic space and arise after first mitosis in human lymphocyte culture is linear-square dependent on the X-irradiation at doses from 0.0 to 4.0 Gy. Positive correlation between frequency of cells with "tailed" nuclei and frequency of metaphases of first mitosis having dicentrics and rings was established. Apparently, formation such "tailed" nuclei is connected with dicentrics and rings.     

Quantitative analyses of the induction of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed to gamma-rays and mitomycin-C in combination

Most chemicals are S-dependent and are potent inducers of SCE, but do not produce chromosome-type aberrations in the first metaphases after exposure. Ionizing radiation, which is an S-independent agent, produces chromosome-type aberrations, especially dicentrics and rings, but inefficiently produces chromatid-type aberrations. A series of experiments has been performed to investigate whether cytogenetic damage induced by ionizing radiation (gamma-rays) might be assessed separately from that induced by the alkylating chemical, mitomycin C (MMC), when human lymphocytes were exposed to these 2 agents in combination. Whole-blood cultures of human lymphocytes in G0 phase were exposed to gamma-rays and MMC in combination or separately. Cytogenetic analyses were done for both chromosome aberrations (CA), analyzed in cultures incubated for 56 h without BrdUrd, and sister-chromatid exchanges (SCEs) in cultures incubated for 72 h with BrdUrd. The frequency of chromosome-type aberrations (dicentrics and rings) increased with increasing doses of gamma-rays from 0.5 to 4.0 Gy. The dose-response relationships were the same with or without concomitant treatment with MMC (10(-6) M). Although the SCE frequency increased with increasing doses of MMC, the increase was nearly the same as when cells were treated with both MMC and gamma-rays (2 Gy). There was no interaction between MMC and gamma-rays concerning these 2 endpoints.     

On the genotoxicity of the pesticides Endodan and Kilacar in 6 different test systems

Two pesticides, the fungicide Endodan (ethylene thiuram monosulphide) and the insecticide-acaricide Kilacar (bis(parachlorophenyl)cyclopropyl methanol), produced or used in the neighbouring countries of Bulgaria and Greece were investigated in a coordinated research programme for their genotoxic effects in a variety of test systems. This included the Ames test, Aspergillus nidulans for mitotic segregation, in vitro human lymphocyte cell cultures for SCE and chromosomal aberrations, in vivo bone marrow cells in hamsters and rats and the dominant lethal test in rats. The genotoxicity of Endodan was found to range from negative to slightly positive in different test systems. At concentrations of 7.5 and 12.0 micrograms/plate together with S9 mix it induced base-pair substitutions in the TA100 strain of Salmonella typhimurium at a rather low level. At a dose of 93 mg/kg b.w. it also caused chromosomal aberrations in acutely treated hamster bone marrow cells. A significant increase of SCE was also found in human lymphocyte cultures at a concentration of 20.0 micrograms/ml. Endodan was found to be negative in A. nidulans for somatic segregation, lymphocyte cultures for chromosomal aberrations and mitotic activity and in rats for dominant lethals and chromosomal aberrations. Kilacar was found to be a weak mutagen in the TA97 strain of S. typhimurium at concentrations of 2.5 and 5.0 micrograms/plate together with S9 mix. At concentrations of 1.0, 1.5 and 2 micrograms/ml Kilacar increased the number of mitotic segregants in A. nidulans by 160%, 220% and 156% respectively over the control. In Syrian hamster bone marrow cells after acute administration at concentrations of 0, 40, 80 and 160 mg/kg, the MI was 5.50, 4.30, 3.10 and 1.30 respectively, and an increase in chromosomal aberrations of about 300% over the control was observed with a concentration of 80 mg/kg. In human lymphocytes no significant changes were observed in either MI or SCE. In the dominant lethal test after chronic treatment of male rats at doses of 5.1, 10.2 and 102.0 mg/kg b.w. no significant mutagenic effect was found although a decrease was shown in the percentage of females with implants mated with treated males in the first week.     

Factors influencing the efficiency of chelation therapy.

The purpose of the present study was to obtain new data on the effect of age, route, dose and time of metal and chelating agent administration on the efficiency of chelation therapy. The experiments were performed on 1-2 and 6-week-old rats which received radioisotopes of metals--203Pb, 115 mCd, 203Hg and 141Ce intraperitoneally or orally. Chelating agents calcium ethylenediaminetetraacetate (CaEDTA), calcium and zinc diethylenetriaminepentaacetate (CaDTPA, ZnDTPA), 2,3-dimercapto-propane-sulfonate-1 (DMPS), dimercaptosuccinic acid (DMSA) and sodium N-(4-methoxybenzyl)-D-glucamine dithiocarbamate monohydrate (MeOBDCG) were administered twice by intraperitoneal or oral administration as early (immediately and 24 hr after metals) or delayed treatment (24 and 48 or 48 and 72 hr after metals). The animals were killed six days after metal administration and the retention was determined in the whole body, carcass and gut. After intraperitoneal administration of metals and chelating agents chelation therapy had much lower efficacy in younger than older animals. After ingestion of metals oral chelation therapy was more effective in younger than older animals. In suckling rats the treatment effectively reduced metal retention and this was mostly due to decrease in gut retention. This treatment in sucklings was also very effective in condition of late administration. In older rats early oral DMPS treatment after 203Hg ingestion is contraindicated since it increases significantly mercury retention while DMSA and ZnDTPA treatments reduced mercury retention. Delayed oral treatment with ZnDTPA and DMSA caused increased cadmium retention in older rats and decreased retention in sucklings. Opposite to results with CaDTPA, MeOBDCG was effective in reducing cadmium retention also when given as delayed treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of phytohemagglutinin-stimulated lymphocyte blastogenesis by ovine uterine milk protein

Two basic glycoproteins (UTM-P) with molecular weights of 57,000 and 59,000 were purified from ovine uterine milk collected on Days 125 and 130 of pregnancy. The UTM-P were evaluated for immunosuppressive activity in phytohemagglutinin (PHA)-treated, mixed lymphocyte (MLC) and resting lymphocyte (RLC) cultures. For PHA and RLC cultures, UTM-P (2.5 to 800 micrograms UTM-P/ml) were added to 1 X 10(6) lymphocytes and 0.8 micrograms of PHA (for PHA cultures only), while for the MLC, UTM-P (50 to 1600 micrograms UTM-P/ml) were added to 5 X 10(5) lymphocytes combined from each of two ewes. Following [3H] thymidine addition, cells were later harvested for determination of thymidine incorporation. Lymphocyte blastogenesis was suppressed by UTM-P in PHA (R2 = 0.32 to 0.92, P less than 0.01 to 0.001), MLC (R2 = 0.8, P less than 0.001) and RLC (R2 = 0.65, P les than 0.01) experiments. To determine reversibility, PHA-treated lymphocytes were incubated with UTM-P for 6, 12 or 24 h, then washed to remove surface UTM-P. Incubation was continued in the presence of PHA as with other experiments. Exposure of lymphocytes to UTM-P for 6 or 12 h did not result in suppression of blastogenesis, whereas exposure for 24 h was sufficient for suppression (P less than 0.01). In an additional experiment, UTM-P were added to PHA-treated cultures at 0, 6, 12 or 24 h. Suppression (P less than 0.01) of blastogenesis was observed for each time period. Immunosuppressive activity was not mediated by overall cytotoxicity and was not affected by routine handling and storage of UTM-P. Data from these experiments provide one explanation for tolerance of the conceptus allograft during defined stages of ovine pregnancy.     

Phenomenon of delayed disruption of telomeric links between chromosomes in polykaryocytes formed from human-Chinese hamster hybrid cells

The clones MOM-8-1 and MOM-8-3 of human--Chinese hamster cell hybrids were used for induction of the phenomenon of delayed disruption of the telomeric links between chromosomes. Colcemide (0.08 microgram/ml) and 5-bromodeoxyuridine (20 micrograms/ml) were present in cell culture for 30 hours. The dicentrics were observed in tetraploid, hypotetraploid and hypodiploid metaphases. No differences between the clones were found. The hybrid cells were the second object in which the phenomenon under discussion was reproduced.     

The fate of X-ray-induced chromosome aberrations in blood lymphocyte culture

The BrdU-Giemsa method which facilitates an unequivocal identification of metaphases at different cycles has been utilized to investigate the fate of X-ray-induced chromosome aberrations in the blood lymphocyte culture system of the Indian muntjac which has the lowest diploid number (2n = 6 female/7 male) and easily distinguishable large-sized chromosomes. The results demonstrate that about 50% of dicentrics and only 12% of rings were transmitted from the first cycle to the second. There were as high as 73% abnormal cells in the second cycle as against 94% in the first cycle following 4.0 Gy. However, the frequencies of dicentrics, rings and of abnormal cells were greatly reduced in the third+ cycles. The frequencies of acentric fragments per post-irradiated first, second and third+ division cell were 2.21, 0.64 and 0.24, respectively. In sharp contrast to all earlier reports, about 75% of them were retained as a single acentric fragment in the second cycle. Analysis of fragment segregation during anaphase separation supports this finding. The survival probability of dicentrics and rings was found to be more than 60% in the second and only 18% in the third+ cycle.     

Reduction of benzo(a)pyrene induced chromosomal aberrations by DL-alpha-tocopherol

The antioxidant, DL-alpha-tocopherol (vitamin E), has been demonstrated to significantly reduce the percentage of benzo(a)pyrene (BP) induced chromosomal aberrations in vitro. Chinese hamster lung (Don) and Chinese hamster ovary (CHO) cells were treated with either 1 microgram/ml or 5 micrograms/ml BP for 4 to 28 h; some cultures were treated with S9 mix activated BP. Additional cultures of Don and CHO were treated simultaneously with 100 micrograms/ml of DL-alpha-tocopherol and BP. In CHO cells 1 microgram/ml non-activated BP significantly increased the chromosomal aberration percentage above the control level. Aberrations observed included breaks, gaps, fusions, rings, dicentrics, and polyploids. Chinese hamster Don cells treated with 1 microgram/ml or 5 micrograms/ml S9 mix activated BP contained significant increases in aberration percentages above the control levels. When Don cells were treated simultaneously with activated BP and DL-alpha-tocopherol for 4 h, there was a slight decrease in the total aberration frequency to less than that of cells treated with activated BP only; however, when Don cells were treated with BP and DL-alpha-tocopherol for 28 h, there was a significant reduction in the aberration percentage below that of BP-treated cells alone. Similar results have been obtained with CHO cells treated with nonactivated BP and DL-alpha-tocopherol. The results reported here provide further evidence that antioxidants may prevent the potential mutagenic and carcinogenic effects of certain polycyclic compounds.     

The effect of staphylococcal alpha-toxin on DNA replication in human cells]

The influence of Staphylococcus alpha-toxin has been investigated on the duration of S-phase of lymphocyte mitotic cycle and on DNA replication in human fibroblasts in vitro. The duration of the S-phase of lymphocytes was measured by counting labeled metaphases and by making replication curves. Alpha-toxin in a dose of 3 micrograms/ml enhances the onset of S-phase, which is inhibited at a dose of 33 micrograms/ml of alpha-toxin. The action of alpha-toxin resulted in a decreased rate of replication fork and in a progressive activation of replicon groups. This effect was most prominent at 33 micrograms/ml of alpha-toxin. The data obtained allow to suggest that immunodeficiency of the second order, so characteristic of the staphylococcal sepsis, may be due, in many respects, to suppression of DNA replication.     

Identifying the fluorescent body (F-body) with quinacrine mustard staining of canine

The fluorescent body (F-body) was identified with quinacrine mustard (Q-M) staining in spermatozoon and lymphocyte of canine. Well washed sperm suspension was treated with protease (125 mg/ml) or dispase (2000p. u./ml) and staining with Q-M (final dilution 50 micrograms/ml) for 15 min to 24 hr at 37 degrees C. The lymphocyte cultures from whole blood were prepared as routine human investigation. The chromosomal preparation made by air dry method was stained with Q-M (final dilution 0.5 to 50 micrograms/ml) after pretreatment of enzyme digestion. The examination using a reflected fluorescent microscope revealed that the same F-body in human was present in both spermatozoon (20.1-39.7%) and interphase of lymphocyte (0.37.2%) of male origin.     

Quinacrine dihydrochloride, the non-surgical female sterilant induces dicentrics, rings, and marker chromosomes in human peripheral blood lymphocytes treated in vitro: a preliminary report

During the last decade, quinacrine dihydrochloride (QDH) has been promoted for clinical trials as a much needed non-surgical female sterilant, largely in the Third World. Recently, however, these human trials have come under severe criticism due to lack of adequate evidence of biological safety of QDH, particularly of its genotoxicity in mammalian systems. In the present study, the cytogenetic analysis of QDH-treated human lymphocytes, grown as whole blood cultures in vitro, surprisingly showed a wide range of chromosomal aberrations. At a concentration of 3.0 and 6.0 microg/ml in culture, QDH was cytotoxic, as shown by the very few analyzable metaphases that could be observed. G(0) lymphocytes, treated with 0. 6 microg/ml QDH, exhibited chromosome aberrations including dicentrics, ring configurations, translocations, inversions, and marker chromosomes. Near haploid, polyploid, and endoreduplicated cells were also observed. All the rings appeared to be formed as a result of telomere fusion/association. Twenty percent of the dicentrics observed also indicated telomere fusion/association in the D and G groups of chromosomes. Overall, a frequent involvement of chromosomes 1, 2, and 3 in both unstable and stable chromosome rearrangements was also observed. Exposure of 72-h cultures to 0.45 microg/ml QDH at 69 h resulted in an accumulation of C-metaphases, suggesting that probably QDH behaves as a mitotic spindle inhibitor. The G(2) lymphocytes from two donors exposed to 0.6, 1.5 or 3.0 microg/ml of QDH showed no increase in chromatid aberrations in two donors. However, QDH at 0.6 microg/ml increased the frequency of micronucleated binucleate cells. No increase in sister chromatid exchanges was observed at this concentration. Though preliminary, these observations demonstrate the chromosome damaging ability of QDH in human lymphocytes treated in vitro. Surprisingly, like ionizing radiation, QDH acted by an S-phase-independent mechanism, unlike most of the chemical mutagens. These results warrant detailed investigations on the cytogenetic effects of QDH in vitro, as well as among women exposed to this agent during clinical trials for non-surgical sterilization. The interesting cytogenetic profile of QDH deserves to be pursued and the underlying mechanisms, in particular, the DNA topoisomerase II inhibitory effect, if any, needs to be elucidated.     

A method of estimating the dose to and magnitude of an irradiated area during partial radiation damage based on the results of a cytogenetic analysis of a culture of peripheral blood lymphocytes

The curves, obtained in the in vitro experiments, that show the dependence of the frequency of dicentrics (per a cell with dicentrics) upon dose and percentage of cells with dicentrics are proposed to be used in estimating a dose and body volume affected by partial irradiation (an extreme case of nonuniform exposure) by the analysis of chromosome aberrations in peripheral blood lymphocyte cultures.     

Analyses of sister-chromatid exchange and cell-cycle kinetics in mouse T- and B-lymphocytes from peripheral blood cultures

A reliable mouse peripheral blood lymphocyte culture assay has been developed for sister-chromatid exchange analysis. Crucial aspects for optimal mitogenesis include: (1) the addition of 5 X 10(5) leucocytes/ml culture; (2) the use of animals with leucocyte counts from 5 to 7 X 10(6)/ml; and (3) the addition of 6% mouse plasma for the first 24 h of a total 54-h incubation. When 7 micrograms phytohemagglutinin/ml were used to stimulate T-lymphocytes, the mitotic index was 3.4 +/- 0.3%, 28 +/- 2.3% of the metaphases were in first-division, and the SCE frequency/metaphase was 7.3 +/- 0.2 (n = 14 mice). When B-lymphocytes were stimulated with 60 micrograms lipopolysaccharide/ml, the mitotic index was 4.5 +/- 0.3%, 64 +/- 3.3% of the metaphases were in first-division, and the SCE frequency/metaphase was 4.6 +/- 0.1 (n = 7 mice). This culture method consistently yields sufficient numbers of metaphases from both B- and T-lymphocytes for SCE and chromosome-breakage studies.     

Long-term follow-up cytogenetic survey and biological dosimetry in persons evacuated from 30-km Chernobyl NPP zone

The paper presents the results of the follow-up cytogenetic survey and biological dosimetry carried out in inhabitants of Pripiat' town and nearby villages, who were departured from the Chernobyl NPP 30-km exclusive zone during first days after the Chernobyl catastrophe. The unstable chromosome aberration level in inhabitants were significantly increased above control in terms up to 1 year after evacuation and declined gardually during next 14 years. In early period the cytogenetic damage frequency in evacuees showed no dependence on gender. The chromosome type aberration level appeared to be lower in young persons comparing with adults. The dicentrics plus centric rings yield had a positive correlation with duration of staying at Chernobyl zone. The average doses of protracted exposure were calculated from the dicentrics and centric rings yields; the dose estimations appeared to be 1.4 times higher in persons evacuated 3-11 days after the accident than that of in persons with shorter departure time. Uing the Bayesian analysis the probabilistic distribution of biological doses was constructed for the studied evacuees group. This distribution was characterized by a mean dose of 360 mGy, the modal doses of 200-450 mGy and 80% of probability density within the dose range 0-1000 mGy, that seems to be sufficient for considering the increased risk of late somatic radiation effects for this cohort.     

Analysis of chromosome aberrations in lymphocytes of long-term heavy smokers

We assessed the incidence of structural chromosome aberrations in 500 diploid first-division metaphases from 48-h lymphocyte cultures from each of 6 non-smokers and from 6 persons who had smoked a minimum of 1 pack of cigarettes per day for at least 20 years. Cytogenetic analyses of coded slides revealed a single dicentric chromosome with its accompanying fragment and two symmetrical chromatid exchanges in 3000 metaphases from the non-smokers. In contrast, 9 dicentric chromosomes, 8 translocations or inversions, and 7 chromatid exchanges were observed in 3000 metaphases from lymphocyte cultures from the 6 heavy smokers. A total of 13 metaphases having chromosome-type inter- or intra-changes was noted including 9 with a single aberration, and 4 with 2 or more. Our findings provide additional evidence of the in vivo clastogenicity of cigarette smoke in long-term heavy smokers, and further demonstrate that the distribution of chromosome-type exchange aberrations is overdispersed relative to that expected based on Poisson assumptions.     

The mouse splenocyte assay, an in vivo/in vitro system for biological monitoring: studies with X-rays, fission neutrons and bleomycin.

A modified mouse splenocyte culture system was standardized after testing different mitogens (i.e., phytohemagglutinin (PHA), concanavalin A (Con A)). The mitotic index was determined for comparison between different mitogens. Following selection of appropriate mitogen (PHA 16, Flow), a series of experiments were conducted to evaluate the application of a cytokinesis-block for scoring micronuclei and assays for chromosomal aberrations produced by treatment in G0 and G2 for the purposes of biological dosimetry following in vivo and/or in vitro exposure to X-rays, fission neutrons and bleomycin. In the X-irradiation studies, the frequencies of micronuclei and chromosomal aberrations (i.e., dicentrics and rings) increased in a dose-dependent manner. These data could be fitted to a linear-quadratic model. No difference was observed between irradiation in vivo and in vitro, suggesting that measurement of dicentrics and micronuclei in vitro after X-irradiation can be used as an in vivo dosimeter. Following in vivo irradiation with 1 MeV fission neutrons and in vitro culturing of mouse splenocytes, linear dose-response curves were obtained for induction of micronuclei and chromosomal aberrations. The lethal effects of neutrons were shown to be significantly greater than for a similar dose of X-rays. The relative biological effectiveness (RBE) was 6-8 in a dose range of 0.25-3 Gy for radiation-induced asymmetrical exchanges (dicentrics and rings), and about 8 for micronuclei in a dose range of 0.25-2 Gy. Furthermore, the induction of chromosomal aberrations by bleomycin was investigated in mouse G0 splenocytes (in vitro) and compared with X-ray data. Following bleomycin treatment (2 h) a similar pattern of dose-response curve was obtained as with X-rays. In this context a bleomycin rad equivalent of 20 micrograms/ml = 0.50 Gy was estimated.     

Mitosis of maternal lymphocytes in the presence of fetal cells: Possible implication in prenatal diagnosis from fetal blood samples

Summary The suppression of proliferation of maternal lymphocytes by the lymphocytes of their own male newborns have been tested in a PHA-induced two-way stimulation system. The mixed lymphocyte cultures of 6 out of 12 such mother/son pairs had 23–50% metaphases with 46,XX karyotype. In 2 more cases 10% maternal metaphases have been observed. Hence, it appears that fetal lymphocytes are unable to suppress the proliferation of maternal cells completely.     

Cytogenetic investigation in lymphocytes of people living in cadmium-polluted areas

Chromosome aberrations were analyzed from peripheral lymphocyte cultures of 21 men and 19 women who had been exposed to environmental cadmium, and 11 controls (9 men and 2 women). The average cadmium level in the urine of the Cd-polluted group was 3.32 micrograms/l for men and 3.83 micrograms/l for women. There were significant differences in chromosome aberration frequencies between the Cd-polluted and non-polluted groups. The number of individuals with relatively high aberration frequencies (greater than or equal to 5%) in the Cd-polluted group was greater than in the controls. Individuals with a high cadmium content in urine (greater than or equal to 3 micrograms/l) had higher aberration frequencies and more severe aberration types in comparison with the low-cadmium group (less than 3 micrograms/l). There were significant correlations between chromosome aberration frequencies and urinary cadmium content (r = 0.463). The linear regression equation was determined. Considering the conflicting results in other published reports, it is hard to say that the conclusion that cadmium only acts synergistically to enhance the mutagenicity of other compounds present in the environment is correct. According to our study, environmental cadmium cannot only induce chromosome aberrations but also increases the chromosomal aberration frequencies and the frequency of severe aberration types.