Assessment of DNA/Chromosome Damage in the Peripheral Blood Lymphocytes of Workers Exposed to Indium Compounds.

This study was conducted to investigate possible genotoxic effects resulting from occupational exposure to indium compounds. We performed a cytogenetic analysis of peripheral blood lymphocytes gathered from 57 individuals exposed to indium at an indium ingot production plant in Guangxi, China, and compared the results with those obtained from 63 control subjects. The lymphocytes from both groups were examined in the chromosomal aberrations (CAs) assay, cytokinesis-block micronucleus (CBMN) assay, and single-cell gel electrophoresis (comet) assay. Samples of personal breathing zone air were collected throughout the work shift of each subject. Blood and urine samples were collected before and after each work shift on the same day as the air samples were collected. Our assay results showed that workers in the indium production plant were exposed to significantly higher levels of indium (median exposure, 8.00 µg/m3) than the control subjects. Also, higher concentrations of urinary indium (U-In) were found in the exposed workers than the control subjects. When compared with the control subjects, the exposed workers showed higher levels of DNA damage as detected by the comet assay (tail length and TDNA%), significantly higher frequencies of CAs/100 cells, and increased CBMN frequencies. Moreover, the mean CBMN frequency in the non-smokers exposed to indium was significantly higher than that in the non-smoker control subjects (3.14‰ vs 1.00‰, respectively; P < .01). U-In levels, comet assay, CBMN, and CA test proved to be the most sensitive biological markers for detecting occupational exposure to indium compounds and can also be used to assess the health risks of the exposed workers.

[Assessment of hematopoiesis and cytogenetics changes in interventional radiologists].

Objective: To investigate hematopoiesis and cytogenetics changes in staff of interventional radiology. Methods: A total of 121 intervention radiation workers, 245 common radiation workers and 100 medical personnel (healthy control) without exposure to radiation were enrolled in the study. The peripheral lymphocyte chromosomal aberrations and micronucleus were detected, and the result of white blood cells examination was analyzed. Results: Compared with common radiation group and healthy control group, decreases in white blood cells count, neutrophil ratio, and increase in lymphocyte ratio were observed in intervention radiation group (all P<0.05). Intervention radiation group had higher chromosome aberration rate and micronuclear rate than common radiation group and healthy control group (all P<0.05). Most common chromosome aberrations were dicentric chromosome, acentric ring, fragments and minute chromosome. Abnormal rates in chromosome aberration and micronucleus rates were increased with the rise of length of service, but no statistically significant difference was observed (P>0.05). Conclusion: Long term exposure to ionizing radiation may lead to changes in the human hematopoietic system and cause human chromosome aberration, and the severity of such injuries may be associated with the dose of ionizing radiation.

Assessment of oxidative stress and chromosomal aberration inducing potential of three medical grade silicone polymer materials.

Medical expenditures for devices are increasing along with the ageing of human population and the synthesis of materials such as silicone polymers is on upsurge for manufacturing these devices. The International Organization for Standardization (ISO) emphasizes a battery of tests for preclinical assessment of biocompatibility of medical devices. Genotoxicity assays have become an integral component of these test procedures and it employs a set of in vitro and in vivo experiments to detect mutagens. Hence, this study was performed with an intention to investigate the genotoxic potential of the physiological saline extracts of three medical grade silicone polymer materials by the in vitro chromosomal aberration assay using human peripheral blood lymphocytes. Further, the oxidative stress inducing potential of the material extracts was investigated in vivo in mice liver homogenates using cyclophosphamide as positive control. The investigation revealed that none of the three materials were able to produce marked human lymphocyte chromosomal aberration, suggesting the absence of mutagens. The materials also showed negative results in their oxidative stress inducing potential, which was revealed by the normal levels of lipid peroxidation and unaltered levels of glutathione and its metabolizing enzymes in the mice liver tissue homogenates. It was interesting to observe a significant correlation between the genotoxic and antioxidant parameters investigated. Hence, it is suggested that the estimation of antioxidant status would serve as a better preliminary testing procedure prior to evaluating the genetic and molecular toxicity mechanisms of medical devices and/or materials intended for manufacture of such devices.

Genotoxicity assessment in patients with thalassemia minor.

Thalassemia is an inherited blood disorder that affects both genders and results in reduced synthesis of hemoglobin, and thus causing anemia. Previous studies have shown that the severe form of this disease, thalassemia major, is associated with genotoxicity. This includes increases in the level of sister chromatid exchange (SCEs), chromosomal aberrations (CAs) and micronuclei. In this study, we assessed genotoxicity in the lymphocytes of thalassemia minor subjects using sister chromatid exchange (SCE) and chromosomal aberration (CA) assays. In addition, we investigated the level of oxidative DNA damage by measuring 8-hydroxy-2'-deoxyguanosine (8OHdG) biomarker in urine samples. Eighteen thalassemia minor subjects and eighteen matched normal healthy controls were volunteered in the study. In addition, seven thalassemia major patients were recruited as positive controls. The results showed increases in the frequency of SCEs (P<0.05) in thalassemia minor compared to healthy controls. However, no difference in CAs frequency was detected between thalassemia minor and controls (P>0.05). Both SECs and CAs in thalassemia major patients were significantly higher compared to other groups (P<0.05). Regarding urine 8OHdG levels, the result showed a slight increase in thalassemia minor compared to healthy controls but the difference was not significant (P>0.05). In conclusion, our results showed that thalassemia minor is associated with genotoxicity to blood lymphocytes as indicated by SCEs assay.

Assessment of genetic damage in peripheral blood of human volunteers exposed (whole-body) to a 200 muT, 60 Hz magnetic field.

AIM: To investigate the extent of damage in nucleated cells in peripheral blood of healthy human volunteers exposed to a whole-body 60 Hz, 200 microT magnetic field. MATERIALS AND METHODS: In this study, 10 male and 10 female healthy human volunteers received a 4 h whole-body exposure to a 200 microT, 60 Hz magnetic field. In addition, five males and five females were treated in a similar fashion, but were exposed to sham conditions. For each subject, a blood sample was obtained prior to the exposure period and aliquots were used as negative- (pre-exposure) and positive- [1.5 Gray (Gy) (60)Cobalt ((60)Co) gamma-irradiation] controls. At the end of the 4 h exposure period, a second blood sample was obtained. The extent of DNA damage was assessed in peripheral human blood leukocytes from all samples using the alkaline comet assay. To detect possible clastogenic effects, the incidence of micronuclei was assessed in phytohemagglutinin (PHA)-stimulated lymphocytes using the cytokinesis-block micronucleus assay. RESULTS: There was no evidence of either increased DNA damage, as indicated by the alkaline comet assay, or increased incidence of micronuclei (MN) in the magnetic field exposed group. However, an in vitro exposure of 1.5 Gy gamma-irradiation caused a significant increase in both DNA damage and MN induction. CONCLUSIONS: This study found no evidence that an acute, whole-body exposure to a 200 microT, 60 Hz magnetic field for 4 hours could cause DNA damage in human blood.

Toxicological and cytogenetic assessment of a Salacia oblonga extract in a rat subchronic study.

Salacia oblonga holds potential as a natural method to mitigate the blood glucose response for people with diabetes by inhibiting the activity of intestinal alpha-glucosidases. As part of a safety evaluation of novel ingredients for use in blood glucose control, the toxicity of a S. oblonga root extract (SOE) was evaluated in a subchronic 90-day feeding study in rats. An in vivo-in vitro rat peripheral blood lymphocyte chromosomal aberrations assay was added at termination of the subchronic rat study to examine cultured lymphocytes for possible chromosomal aberration induction. This was conducted due to a previous weak; although reproducible, positive chromosomal aberrations response in cultured peripheral blood human lymphocytes after acute in vitro treatment with SOE. The present study results indicate that SOE was negative for the induction of chromosomal aberrations in cultured rat peripheral blood lymphocytes after 90 consecutive days of treatment with SOE. The no observable adverse effect level (NOAEL) was determined to be 2,500 mg/kg/day following daily subchronic oral gavage administrations to rats.

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model.

Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics+/-antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. 'Tail intensity' (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3+/-1.7% TI versus 10.2+/-4.1% TI, n=19), but not of non-smokers (8.6+/-2.8% TI versus 8.3+/-2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4+/-2.9% TI versus 18.9+/-13.1% TI, n=15) but not in smokers (15.5+/-10.7% TI versus 20.4+/-14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread+/-antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.

Assessment of DNA strand breakage by the alkaline COMET assay in dialysis patients and the role of Vitamin E supplementation.

Although the role of reactive oxygen species (ROS) in chronic renal failure (CRF) is not definitely demonstrated, a consistent number of observations has provided evidence for the presence of oxidative stress in uremic patients undergoing maintenance dialysis. In order to investigate this hypothesis further and to understand the role of antioxidant supplementation, peripheral blood lymphocytes were taken from 36 dialysis patients before and after Vitamin E supplementation in a dosage of 600 mg per day (2x300 mg) for 14 weeks and examined in the alkaline Comet assay for DNA strand breakage. The results were also compared with those of 36 controls with comparable age, sex, and smoking habits, and with no history of renal disease. The DNA breakage observed in the lymphocytes of patients before Vitamin E supplementation was significantly higher than in the controls (P<0.001) but a clear protective effect of Vitamin E supplementation were observed after 14 weeks of therapy.

DNA damage assessment by comet assay of human lymphocytes exposed to jet propulsion fuels.

Exposure to jet fuel damages DNA and results in a number of physiological changes in liver, lung, immune, and neurological tissue. In this study the single-cell gel electrophoresis assay or comet assay was used to compare the DNA damage in human peripheral lymphocytes produced by three jet propulsion fuels: JP-8, JP-5, and JP-8+100. These fuels consist of complex mixtures of aliphatic, aromatic, and substituted naphthalene hydrocarbons. Two exposure times were investigated which correspond to estimated occupational exposure times and concentrations of fuels were used that were based on previous fuel toxicity studies. Analysis of samples for the extent of DNA damage as determined by tail moment and percent tail DNA was performed on exposed cells following a brief recovery time. All fuels produced significant increases in DNA damage; however, only JP-8+100 was genotoxic at the lowest exposure concentration (1:500). At the highest exposure concentration (1:75), the mean tail moments for JP-8 and JP-8+100 (32.041 +/- 2.599 and 45.774 +/- 4.743, respectively) were significantly greater than for JP-5 (1.314 +/- 0.474). These results indicate that JP-8+100 is the most potent inducer of DNA damage in human peripheral lymphocytes and that both JP-8+100 and JP-8 are capable of damaging lymphocyte DNA to a greater extent than JP-5.

Computer analysis of mFISH chromosome aberration data uncovers an excess of very complicated metaphases.

PURPOSE: To analyse spectra of chromosome aberrations induced in vitro by low LET radiation, in order to characterize radiation damage mechanisms quantitatively. METHODS: Multiplex fluorescence in situ hybridization (mFISH) allows the simultaneous identification of each homologous chromosome pair by its own colour. mFISH data, specifying number distributions for colour junctions in metaphases of human peripheral blood lymphocytes 72 hours after exposure in vitro to a 3 Gy gamma-ray dose, were combined with similar, previously published results. Monte Carlo computer implementations of radiobiological models for chromosome aberration production guided quantitative analyses, which took into account distribution of cells among different metaphases and lethal effects or preferential elimination of some aberrations at cell division. RESULTS AND CONCLUSIONS: Standard models of DNA damage induction/repair/misrepair explain the main trends of the data as regards the fraction of metaphases having a particular number of colours involved in colour junctions. However, all standard models systematically under-predict the observed fraction of metaphases where a large number of different chromosomes participate in aberrations. An early appearance of chromosomal instability could explain most of the discrepancies.

[Assessment of single-stranded DNA breaks in spleen lymphocytes using the DNA comet assay in the long term after acute irradiation of mice at sublethal and moderately-lethal doses]. / Otsenka metodom DNK-komet odnonitevykh razryvov DNK limfotsitov selezenki v otdalennye sroki posle ostrogo oblucheniia myshei v subletal'nykh i sredneletal'nykh dozakh.

The single-strand DNA level in spleen lymphocytes of BALB/c male-mice after 11 month acute exposure to gamma-radiation at doses 1, 3 and 6 Gy has been investigated by comet assay. The results of our study showed that at 11 month after irradiation at different doses a significant increase in the level of DNA breaks in spleen lymphocytes and decrease in the total number of these cells in mice was registered. It is possible that the increase in the DNA breaks is due to the effort of the compensatory proliferation process in lymphoid system that can give the increase in the number of different genetic disturbances in lymphocytes.

Assessment of DNA strand breakage by comet assay in diabetic patients and the role of antioxidant supplementation.

Diabetes patients often show increased production of reactive oxidative species (ROS) together with vascular complications. The presence of these ROS may lead to increased DNA damage in peripheral blood lymphocytes that may be revealed by the comet assay. To test whether DNA is damaged in diabetes, peripheral blood samples were taken from 30 control individuals and 63 diabetic patients (15 insulin dependent (IDDM) and 48 non-insulin dependent (NIDDM)) and the alkaline comet assay was used to evaluate background levels of DNA damage. Significant differences were detected between control and diabetic patients in terms of frequencies of damaged cells. The extend of DNA migration was greater in NIDDM patients by comparison with IDDM patients which might indicate that IDDM patients are handling more oxidative damage on a regular basis. Smoker individuals had higher frequencies of cells with migration by comparison with the non-smokers in both groups. Also, clear differences between patients on placebo and on Vitamin E supplementation for 12 weeks were observed on the basis of the extend of DNA migration during single cell gel electrophoresis.

Assessment of the adaptive response induced by quercetin using the MNCB peripheral blood human lymphocytes assay.

Over more than two decades the existence of an adaptive response (AR) has been reported in several cell types and extensively studied with low doses of radiation. Besides radiation, some chemicals [alkylating compounds, mitomycin C (MMC), bleomycin, hydrogen peroxide and metals] may also induce an adaptive response. We have recently reported that the food mutagen quercetin can also induce an adaptive response in V79 Chinese hamster cells. In this work we have studied the effect of low doses of quercetin on the genotoxicity of MMC and bleomycin assessed by the formation of micronuclei in cytokinesis-blocked (MNCB) human peripheral blood lymphocytes. Our results suggest the existence of an AR induced by quercetin in human lymphocytes. Seven of the nine donors studied showed in at least one independent experiment a significant decrease in the frequency of MNCB induced by MMC. The range of these decreases varied between 31 and 58%. In addition, we observed an AR induced by quercetin towards challenging doses of bleomycin. In accordance with other studies with ionizing radiation in which heterogeneity of the AR in the population has been extensively observed, the response here reported also showed some degree of variability between the different donors studied. In view of the results obtained one cannot rule out a possible protective effect of low doses of quercetin leading to adaptation to further exposure to mutagens or carcinogens.

[The assessment of the course of an infectious process by the membrane status of the peripheral blood lymphocytes]. / Otsenka techeniia infektsionnogo protsessa po sostoianiiu membran limfotsitov perifericheskoi krovi.

Lipid peroxidation in lymphocyte membranes is studied in patients with acute viral hepatitis B and B + C and in chronic alcoholics with Flexner's dysentery and with uneventful premorbid history. The intensity of lipid peroxidation in lymphocytes was increased, corresponding to the severity and period of infection. The premorbid background and therapy influenced the lymphocyte membrane status, which can serve as an integral indicator of immunological reactivity of the organism.

Measurement accuracy in confocal microscopy.

Confocal laser scanning microscopy provides optical serial sections through thick biological samples, making it possible to perform both three-dimensional visualization and three-dimensional quantitative analysis. On human lymphocytes, we measured geometrical features, cell contents in DNA and in cyclin A and CDK1 proteins, localization and colocalization of these two proteins. Cells were acquired at a vertical sampling step of 0.5 micron, which gives sufficient information about cell labelling. For the purpose of obtaining fast and reliable data at a reduced time cost, we examined various possibilities to simplify acquisition. For example, it might be possible to increase the vertical sampling step to 2.0 microns while preserving an acceptable accuracy of measurements. Further limiting the acquisition to the central sections appeared to give only rough estimations about the whole cells. Finally, we compared confocal microscopy to conventional two-dimensional epifluorescence microscopy. Confocal microscopy appeared slightly less accurate as regards content estimation, but was an invaluable tool when investigating three-dimensional structures and, more especially, localization of proteins.

Sister chromatid exchange and micronucleus frequency in human lymphocytes of 1,650 subjects in an Italian population: I. Contribution of methodological factors.

The influence of several methodological factors on mean values of sister chromatid exchanges (SCEs) and micronuclei (MN) in peripheral lymphocytes of 1,650 subjects was analyzed. Donors belonged to a general healthy population living in Pisa and in two nearby small cities: Cascina and Navacchio (Ca-Na). Blood samples were collected over a period of 29 months and processed in three different laboratories of the some institute. Slides were analyzed by several scorers. Our data showed that lymphocyte proliferation indexes (PIs) and baseline mean values of SCEs were affected mainly by sampling period. This factor accounted for a percentage ranging from roughly 10% (Pisa) to 20% (Ca-Na) of total SCE variance and from roughly 10% (Pisa) to 13% (Ca-Na) of total PIs variance. A marginal effect was attributable to the different laboratories involved (maximum 3% for SCEs and 7% for PIs). The sampling period variable included many sources of variability such as culture media batches, fetal calf serum, PHA, BrdUrd, and seasonality. MN counts revealed a more marked dependence on processing laboratories. This factor accounted for a percentage of roughly 10% (Pisa and Ca-Na) of total variance, while the sampling period was marginally effective (about 1-4% of total variability). Because laboratories were equipped and supplied with the same materials and consumables and technicians were rotated constantly, the only variable ascertained was represented by the three different models of CO2 incubators used for lymphocyte culturing. When "month" and "incubator" variables were considered jointly, experimental variability accounted for 15-20% of total variance, both for PIs and mean values SCEs and MN. The variability due to slide scoring was reduced by assigning each slide to five different scorers and matching low with high scorers in each group. Present data show that when the study is performed under these controlled conditions, about 20% of total interdonor variability can be explained by experimental or seasonal factors.

Distribution of ABL and BCR genes in cell nuclei of normal and irradiated lymphocytes.

Using dual-color fluorescence in situ hybridization (FISH) combined with two-dimensional (2D) image analysis, the locations of ABL and BCR genes in cell nuclei were studied. The center of nucleus-to-gene and mutual distances of ABL and BCR genes in interphase nuclei of nonstimulated and stimulated lymphocytes as well as in lymphocytes stimulated after irradiation were determined. We found that, after stimulation, the ABL and BCR genes move towards the membrane, their mutual distances increase, and the shortest distance between heterologous ABL and BCR genes increases. The distribution of the shortest distances between ABL and BCR genes in the G0 phase of lymphocytes corresponds to the theoretical distribution calculated by the Monte-Carlo simulation. Interestingly, the shortest ABL-BCR distances in G1 and S(G2) nuclei are greater in experiment as compared with theory. This result suggests the existence of a certain regularity in the gene arrangement in the G1 and S(G2) nuclei that keeps ABL and BCR genes at longer than random distances. On the other hand, in about 2% to 8% of lymphocytes, the ABL and BCR genes are very close to each other (the distance is less than approximately 0.2 to 0.3 microm). For comparison, we studied another pair of genes, c-MYC and IgH, that are critical for the induction of t(8;14) translocation that occurs in the Burkitt's lymphoma. We found that in about 8% of lymphocytes, c-MYC and IgH are very close to each other. Similar results were obtained for human fibroblasts. gamma-Radiation leads to substantial changes in the chromatin structure of stimulated lymphocytes: ABL and BCR genes are shifted to the nuclear center, and mutual ABL-BCR distances become much shorter in the G1 and S(G2) nuclei. Therefore, we hypothesize that the changes of chromatin structure in the irradiated lymphocytes might increase the probability of a translocation during G1 and S(G2) stages of the cell cycle. The fact that the genes involved in the t(8;14) translocation are also located close together in a certain fraction of cells substantiates the hypothesis that physical distance plays an important role in the processes leading to the translocations that are responsible for oncogenic transformation of cells.

[The possibilities of using biological dosimetry methods for the retrospective assessment of dosages in relation to the sequelae of the accident at the Chernobyl Atomic Electric Power Station. An assessment of the dosages based on an analysis of unstable chromosome aberrations]. / Vozmozhnosti primeneniia metodov biologicheskoi dozimetrii dlia retrospektivnoi otsenki doz v sviazi s posledstviiami avarii na Chernobyl'skoi AES. Otsenka doz na osnove analiza nestabil'nykh khromosomnykh aberratsii.

The problem of retrospective dose assessment in connection with the Chernobyl accident is discussed. Existing methods of dose quantification based on the analysis of unstable aberrations in blood lymphocytes are discussed in details. Starting from the own data it is concluded that unstable aberration technique could not be applied to verify individual doses for ameliorators as well as for inhabitants of contaminated areas.

[The possibilities of using biological dosimetry methods for the retrospective assessment of dosages in relation to the sequelae of the accident at the Chernobyl Atomic Electric Power Station. An assessment of the dosages based on an analysis of stable chromosome aberrations]. / Vozmozhnosti primeneniia metodov biologicheskoi dozimetrii dlia retrospektivnoi otsenki doz v sviazi s posledstviiami avarii na Chernobyl'skoi AES. Otsenka doz na osnove analiza stabil'nykh khromosomnykh aberratsii.

The problem of retrospective dose assessment in connection with the Chernobyl accident is discussed. Reasons justifying the applicability and advantages of technique based on the analysis of stable aberrations in blood lymphocytes are given. Two modern methods for registration aberrations of these registration, namely fluorescence in situ hybridization and full karyotyping of G-banded chromosomes are considered in details.

Sister-chromatid exchanges and their distribution in human lymphocytes in relation to age, sex and smoking.

The effects of age, sex and smoking on sister-chromatid exchange (SCE) frequency and distribution in human lymphocytes were assessed by means of multiple linear regression. Differences in SCE scores were associated with all above variables: SCE increased with age and cigarette smoking intensity, and higher SCE frequencies were observed in females. Changes in SCE distribution were associated with age and smoking: the ratio of sample variance to sample mean (heterogeneity index) increased with age and smoking intensity. Cell proliferation kinetics, as measured by replication index, inversely correlated with age. Monte Carlo methods were used to show that in the occupational study, analysis of 20-50 persons per group and 25 cells per person may be recommended.