Exchanges In Human Lymphocytes Research Articles

Discrimination between complete and incomplete chromosome exchanges in X-irradiated human lymphocytes using FISH with pan-centromeric and chromosome specific DNA probes in combination with telomeric PNA probe

Purpose : To discriminate precisely between radiation-induced complete and incomplete chromosome exchanges using chromosome painting together with the detection of the centromeres and telomeres in one FISH assay. Materials and methods : Human lymphocytes were exposed in vitro to X-rays at a dose of 4 Gy. Chromosome aberrations were analysed using the FISH technique in combination with a whole chromosome-specific DNA probe for chromosome 8, human pan-centromeric DNA and telomeric PNA probes. Results : The combined FISH assay has improved the resolution of detecting chromosomal exchanges in human lymphocytes. Results indicate that the frequency of observed incomplete exchange patterns was 21% when telomeric signals were ignored during the analysis. When the telomeric signals were included in the analysis a large proportion of apparently incomplete exchange patterns appeared complete and should be re-classified. The percentage of true incomplete exchanges was found to be less than 5%. Conclusion : The combination of chromosome painting and the detection of centromeres and telomeres enable unequivocal discrimination between incomplete and complete exchanges. The fraction of true incomplete exchanges observed in X-irradiated human lymphocytes was found to be low in comparison with previous reports in the literature.

Genetic damage in operating room personnel exposed to isoflurane and nitrous oxide.

OBJECTIVES: To evaluate genetic damage as the frequency of sister chromatid exchanges and micronuclei in lymphocytes of peripheral blood of operating room personnel exposed to waste anaesthetic gases. METHODS: Occupational...

Na(+)/Ca(2+) exchange inhibitors modulate thapsigargin-induced Ca(2+) and Na(+) influx in human lymphocytes.

Thapsigargin has been shown the elevate intracellular Na(+) concentration in human lymphocytes, but mechanisms underlying thapsigargin-induced Na(+) entry are little understood. In the present study we investigated thapsigargin-induced changes in cytosolic free Na(+) and Ca(2+) concentration in human lymphocytes after inhibition of the Na(+)/Ca(2+) exchange with two structurally unrelated compounds, dimethylthiourea ad bepridil. The intracellular Na(+) increase induced by 5 microM thapsigargin was significantly enhanced in the presence of 5 mM dimethylthiourea or 40 microM bepridil. In contrast, both compounds significantly decreased the thapsigargin-induced intracellular Ca(2+) elevation. No effect of dimethylthiourea or bepridil on thapsigargin-induced Ca(2+) influx was observed in the absence of extracellular Na(+). These observations are consistent with the hypothesis that thapsigargin stimulates Na(+)/Ca(2+ )exchange in human lymphocytes. However, Na(+)/Ca(2+) exchange does not mediate Na(+) influx in human lymphocytes.

Sister chromatid exchange in human lymphocytes exposed to isoflurane and nitrous oxide in vitro

The question of whether or not inhalation anaesthetics are genotoxic remains controversial. Therefore, we have studied the in vitro genotoxic potential of isoflurane and nitrous oxide in human lymphocytes. Blood samples were obtained from eight healthy male, non-smoking volunteers, which were incubated and exposed to increasing concentrations of isoflurane (0.0, 0.3, 0.6 and 1.2 mmol litre-1) or 50% nitrous oxide in oxygen. Baseline sister chromatid exchange (SCE) rate per cell was mean 7.65 (SD 1.5) which increased to 9.15 (1.0), 9.55 (1.4) and 9.95 (1.8) SCE/cell during exposure to isoflurane 0.3, 0.6 and 1.2 mmol litre-1, respectively. During 50% nitrous oxide exposure, SCE rate was 9.26 (1.4). The difference between the control and exposed cells was statistically significant (P < or = 0.05). We conclude that exposure to nitrous oxide and subanaesthetic concentrations of isoflurane can produce genetic damage in peripheral lymphocytes in vitro.

Combined FISH with pan-telomeric PNA and whole chromosome-specific DNA probes to detect complete and incomplete chromosomal exchanges in human lymphocytes

Purpose: To combine FISH with pan-telomeric peptide nucleic acid (PNA) and whole chromosome-specific DNA probes to detect complete and incomplete chromosome exchanges in human lymphocytes. Materials and methods: Human lymphocytes were irradiated in vitro with 0.9Gy low dose-rate (0.019 Gy/h) tritium beta-rays. Metaphase spreads were treated with RNase, fixed in 1:3 acetic acid:methanol, and then further treated with KCl, proteinase K and fixed in 4% paraformaldehyde. Slides were denatured, hybridized for 1.5h with an FITC-labelled telomeric PNA probe, and rehybridized overnight with a spectrum-orange whole-chromosome probe specific for chromosomes 1, 2 and 4. Hybridized spreads were washed with 70% formamide/20 x SSC and counterstained with DAPI. Results: All three pairs of labelled chromosomes together with 92 telomeres were readily visible after hybridization. The whole chromosomes 1, 2 and 4 were painted orange, and all telomeres were stained green. Unpainted chromosomes were counterstained blue. In the observed 680 chromosome aberrations induced by tritium beta-rays in human lymphocytes after 52h of culture, no evidence of telomere addition was detected. Incomplete and hidden complete exchanges and terminal deletions were definitively discriminated. Conclusion: The simultaneous detection of telomeres and specific whole chromosomes allows for the first time accurate analysis of complete and incomplete chromosome exchanges involving painted chromosomes in human lymphocytes.

Estimate of the frequency of true incomplete exchanges in human lymphocytes exposed to 1GeV/u Fe ions in vitro

Purpose: To study the frequency of true incomplete exchanges induced by high-LET radiation. Materials and methods: Human lymphocytes were exposed to 1GeV/u Fe ions (LET 140keV/ mu m). Chromosome aberrations were analysed by a fluorescence in situ hybridization using a combination of whole-chromosome-specific probes and human telomere probes. Chromosomes 1, 3 and 4 were investigated. Results: The percentage of incomplete exchanges was between 23 and 29% if telomere signals were not considered. The percentage decreased to 10% after ruling out false incomplete exchanges containing telomere signals. The final estimation of true incomplete exchanges was 10%. Conclusion: Within a degree of uncertainty, the percentage of true incomplete exchanges in 1GeV/u Fe ion-irradiated human lymphocytes was similar to that induced by gamma rays.

Use of semipermeable membrane devices for studying effects of organic pollutants: Comparison of pesticide uptake by semipermeable membrane devices and mussels

Uptake of four pesticides—the organochlorines chlordane and endosulfan and the synthetic pyrethroids fenvalerate and allethrin—by triolein-containing semipermeable membrane devices (SPMD) and by the lake mussel Anodonta piscinalis was studied in a laboratory continuous-flow system. Uptake of the analytes by the SPMDs and mussels was linear during the exposure period of 20 d. These kinetic data were used to calculate the first-order uptake rate constants. On a SPMD-whole body basis, the uptake rates were 3.5 to 5.5 times higher in the membrane devices than in the organisms. The synthetic pyrethroids were sampled at lower rates than the organochlorines, and this difference may be attributed to the larger molecular dimensions of the pyrethroids rather than analyte molecular weight and lipophilicity, which were similar for all test compounds. Because of the disparate sampling rates, concentration factors of analytes differed between SPMDs and mussels. However, the percent composition (ratios) of analytes in SPMDs and in mussels was similar, which indicates that SPMDs may serve as good surrogates for aquatic organisms with respect to the discriminatory uptake of hydrophobic chemicals. Semipermeable membrane device dialysate, mussel extract, as well as two artificial mixtures of the four pesticides were tested with standard toxicity and genotoxicity tests, including Microtox® (inhibition of bacterial luminescence), Daphtoxkit™, and Rotoxkit™ (toxicity tests with freshwater invertebrates Daphnia pulex and Brachionus calyciflorus, respectively), and sister chromatid exchange in human lymphocytes in in vitro assay. Results of these tests suggest that integration of the SPMD technique and bioassays may be a valuable approach for the assessment of levels and effects of bioavailable hydrophobic pollutants.

Sister chromatid exchanges in human lymphocytes treated in vitro with cadmium in G o and S phase of their cell cycles

Sister chromatid exchanges (SCEs) were analyzed in human phytohemagglutinin-activated peripheral lymphocyte cultures exposed to varying concentrations (10 −7–10 −3 M) of cadmium chloride in vitro at two different stages of the cell cycle, G o and early S phase. When cadmium chloride was administered at the G o phase, no increase in the SCEs were observed for the doses 10 −6 and 10 −5 M. Concentrations equal to or larger than 10 −4 M cadmium chloride were lethal to human lymphocytes in our experimental conditions. A highly statistically significant increase was observed in the SCE frequency with increasing cadmium chloride concentration (10 −7–10 −4) when cadmium was administered at the early S phase, which was 24 h after culture initiation. The increase in SCE frequency was higher when the cultures were terminated at 54 h, compared to termination at 72 h. In order to examine the effects of cadmium administered at the S phase on SCE frequency in different individuals, 10 −5 M concentration was used and the cultures were terminated at 54 h after culture initiation. A 2- to 3-fold increase in the SCE frequency was observed in all six individuals examined. A progressive decrease in the proliferative index was also observed by increasing cadmium chloride concentration. These results demonstrate that the genotoxicity of cadmium chloride may be changed depending on the stage of the cell cycle in human lymphocytes. This may be one of the reasons of contradictory findings in the literature.

Rejoining and Misrejoining of Radiation-Induced Chromatin Breaks. III. Hypertonic Treatment

It has been shown that treatment in anisotonic medium modifies rejoining of radiation-induced breaks in interphase chromosomes. In previous work, we have demonstrated that formation of exchanges in human lymphocytes has a slow component (half-time of 1-2 h), but a fraction of exchanges are also observed in samples assayed soon after exposure. In this paper we studied the effect of hypertonic treatment on rejoining and misrejoining of radiation-induced breaks using fluorescence in situ hybridization of prematurely condensed chromosomes in human lymphocytes. Isolated lymphocytes were irradiated with 7 Gy gamma rays, fused to mitotic hamster cells and incubated in hypertonic solution (0.5 M NaCl) for the period normally allowed for interphase chromosome condensation to occur. The data from hypertonic treatment experiments indicate the presence of a class of interphase chromosome breaks that rejoin and misrejoin very quickly (half-time of 5-6 min). The fast misrejoining of these lesions is considered to be responsible for the initial level of exchanges which we reported previously. No significant effect of hypertonic treatment on the yield of chromosome aberrations scored at the first postirradiation mitosis was detected.

Variation in arsenic-induced sister chromatid exchange in human lymphocytes and lymphoblastoid cell lines

This study was undertaken to compare the genotoxic effects of arsenite in cultured human lymphocytes and lymphoblastoid cell lines from a group of normal human volunteers. The goal was to determine whether, as found with other genotoxins, subgroups might exist which showed relative high or low sensitivity to induction of sister chromatid exchanges (SCEs) by this metal. Primary lymphoblast cultures were established by treatment with phytohemagglutinin (PHA-L). Lymphoblastoid cell lines were established by transformation with Epstein-Barr virus. Cultures were exposed for 40 h to sodium arsenite (AsIII) and SCEs assayed by 5-bromo-2'-deoxyuridine incorporation and staining by fluorescence plus Giemsa. SCEs were increased by arsenite in a dose-dependent manner over the concentration range of 10(-7)-10(-5) M. SCEs could not be scored above 10(-5) M because of cytotoxicity. Comparison of SCE frequency in primary lymphocyte cultures among individuals showed substantial variation in sensitivity to arsenite, with some showing no significant effect while others showed a 2-3-fold increase in SCE frequency. In one lymphoblastoid cell line especially sensitive to arsenite, arsenic acid (AsV) or dimethylarsinic acid (DMA) at concentrations up to 10(-5) M did not increase the SCE frequency suggesting that AsIII is the active form of arsenic. When pooled data from the primary lymphocytes was compared to that obtained with the lymphoblastoid cells, the slopes of the dose-response curves for ASIII-induced SCEs were similar. The sensitivity of the majority of the individual primary lymphocyte cultures to SCE induction by arsenite was correlated with the sensitivity of the lymphoblastoid cultures established from the same individual. However, in three individuals no correlation was found. Individual lymphoblastoid cell lines retained their As sensitivity after cryopreservation and subsequent revival. Whether the genotoxic response to As is genetically controlled or the result of phenotypic selection is being explored in these stable lymphoblastoid cell lines.

K+/H+ exchange in human lymphocytes and its alteration during tumor growth

K+/H+ exchange in human lymphocytes and its alteration during tumor growth

K +/H + exchange in human lymphocytes and its alteration during tumor growth

K +/H + exchange in human lymphocytes and its alteration during tumor growth

The Influence of Track Structure on the Understanding of Relative Biological Effectiveness for Induction of Chromosomal Exchanges in Human Lymphocytes

A biophysical model has been applied to describe the production of exchange chromosomal aberrations (dicentrics) in human lymphocytes by radiations of different qualities. The model includes a detailed description of the energy deposition pattern in the form of computer-generated tracks. Energy deposition events are further converted to DNA double-strand breaks (DSBs). Formation of chromosomal exchanges is modeled in competition with repair in a distance-dependent manner with breaks in proximity being most likely to interact. We demonstrate that an assumption of an RBE > 1 for production of DSBs at higher LET leads to a significant increase with LET of both the linear and the quadratic coefficients of the dose response for exchange formation. The latter is not supported experimentally and argues against high RBE values for production of DSBs, at least for those breaks involved in chromosomal exchanges. Assuming that the RBE for production of DSBs is unity, the calculated dose-response curves conformed to experimental data for 60Co gamma rays, 250 kVp X rays and 8.7 MeV protons. The linear coefficient for 23.5 MeV 3He ions is underpredicted. The model predicts that a quadratic term in the dose response for exchange aberrations should be observed at LET values of 20-30 keV/microm. The curvature is not observed experimentally, and the contradiction is discussed.

Lack of effect of long-term fluoride ingestion on blood chemistry and frequency of sister chromatid exchange in human lymphocytes

Two studies were conducted to assess the potential for adverse physiologic and genotoxic effects of long-term fluoride ingestion in adults residing in three communities with varying water fluoride levels (0.2 ppm, 1.0 ppm, and 4.0 ppm). All were long-time (> or = 30 years) residents of their respective communities. Plasma and urine samples were collected for fluoride analyses. Additional plasma was collected to determine blood chemistry, and plasma lymphocytes were examined to determine the frequency of sister chromatid exchange. Significant differences in urine (P = 0.001) and plasma (P = 0.0001) fluoride levels were found in the three communities. Seven of the blood parameters were statistically different among the communities, although all were within the defined normal range of the pathology laboratory. Sister chromatid exchange frequency was statistically higher in the 4.0 ppm fluoride community as compared to the other communities. Because of the higher SCE frequency in the 4.0 ppm fluoride community, a second study was performed to determine if the increased frequency was potentially related to the fluoride level of the communal water supply. Of the 58 adults recruited; 30 had used city water and 28 had used well water (< or = 0.3 ppm fluoride) as their principal water source for 30 years. Data analyses showed that the sister chromatid exchange frequency did not differ between the groups, indicating that the increased sister chromatid exchange frequency was not related to the fluoride level of the communal water. The investigation provided evidence that the long-term ingestion of water containing 4.0 ppm fluoride did not have any clinically important physiologic or genotoxic effects in healthy adults.

Dose dependent of sister chromatid exchanges in humans lymphocytes induced by in vitro alpha-particle irradiation.

Exposure of human G0 lymphocytes to high-LET particles under different conditions has been seen, unlike low-LET radiations, to be substantially effective in the induction of sister chromatid exchanges (SCE). However, whereas for fast neutrons a linear dose response of SCE has been determined, there is no sign of a dose-response relationship for alpha-particles. A likely reason for this lack of dose dependence may be the irradiation procedure. Therefore, a technique developed in our laboratory to ensure uniformity of irradiation with alpha-particles was used in the present study. Monolayers of 3 h-stimulated lymphocytes were exposed with alpha-particles from 241Am. Underdispersion was found for the cell-to-cell variance of the number of SCE. The dose response of SCE was linear, with a yield of 3.4 SCE per cell and per Gray.

On the formation kinetics of radiation-induced chromosome exchanges in human lymphocytes.

On the formation kinetics of radiation-induced chromosome exchanges in human lymphocytes.

Genotoxicity of 4-chloro- o-toluidine in Salmonella typhimurium, human lymphocytes and V79 cells

In the absence of a metabolizing system (S9 mix) 4-chloro- o-toluidine (4-COT) was found to be ineffective in a combination of assays for gene mutations in Salmonella typhimurium, for chromosome aberrations and sister chromatide exchanges in human lymphocytes, and for the induction of spindle disturbances in V79 Chinese hamster cells. In the presence of S9, 4-COT was also ineffective in producing structural or numerical changes in mammalian cells, but the yields of 4-COT induced revertants in S. typhimurium strains TA 100 and TA 98 were about 2-fold higher than those in controls.

Interaction of nickel with mutagens in the induction of sister chromatid exchanges in human lymphocytes

In this study, individual treatments of human lymphocytes with Ni(II) [0.5–25 μM], Cr(VI) [0.65–1.30 μM], UV-light or X-rays induced SCEs in a dose-dependent fashion, and combined treatments of Ni(II) with Cr(VI), UV-light or X-rays interacted antagonistically. Nickel, at environmentaly relevant exposure levels, cna have the effect in complex mixtures of reducing an otherwise positive SCE response and could lead to underestimating human exposures to certain classes of chemicals or radiation. Furthermore, our data indicate that antagonism may occure when human lymphocytes are exposed simultaneously to Ni(II) and Cr(VI), suggesting an explanation for epidemiological studies reporting conflicting results for cytogenetic effects in lymphocytes of workers exposed to chromium and nickel.

Sister chromatid exchange in human lymphocytes induced by propoxur following plant activation by Vicia faba.

Because the carbamate insecticide propoxur induced sister chromatid exchanges (SCE) in Vicia faba but was ineffective in producing SCE in lymphocytes in culture, it was hardly suspected that plant metabolism was involved. Experiments were conducted in which metabolic activation was afforded by Vicia faba roots, and SCE in human lymphocytes in vitro was used to assess cytogenetic damage. Several concentrations of propoxur (250, 500, 1,000, 1,500, and 2,000 ppm) were applied for 4 hr to the roots of Vicia faba. Extracts prepared from these treatments were added to the lymphocyte cultures and a significant increase of SCE frequencies with a concentration-response relationship could be detected. The lymphocyte proliferation kinetics and the proliferation rate index (PRI) were not affected (except in the highest concentration, of 2,000 ppm). This general behavior was in agreement with the presence of an enzymatic system (S10 fraction) in Vicia roots capable of metabolizing or activating the propoxur. With 2,000 ppm, cell necrosis was produced in Vicia; therefore, this extract did not induce SCE in lymphocytes. However, lymphocyte proliferation kinetics were delayed and PRI was significantly decreased. Ethanol, a promutagen activated by this plant, was applied directly to the lymphocyte cultures as a positive control, and the response was negative. On the other hand, the extracts of roots treated with ethanol increased the SCE to more than twice that of the negative control, but the lymphocyte proliferation kinetics and PRI were not affected.

Effect of oxygen concentration in culture on the frequency of sister-chromatid exchanges in human lymphocytes and CHL cells

Effect of oxygen concentration in culture on the frequency of sister-chromatid exchanges in human lymphocytes and CHL cells