Gentiana lutea exerts anti-atherosclerotic effects by preventing endothelial inflammation and smooth muscle cell migration.

BACKGROUND AND AIMS: Studies suggest that Gentiana lutea (GL), and its component isovitexin, may exhibit anti-atherosclerotic properties. In this study we sought to investigate the protective mechanism of GL aqueous root extract and isovitexin on endothelial inflammation, smooth muscle cell migation, and on the onset and progression of atherosclerosis in streptozotocin (STZ)-induced diabetic rats. METHODS AND RESULTS: Our results show that both GL extract and isovitexin, block leukocyte adhesion and generation of reactive oxygen species in human umbilical vein endothelial cells (HUVECs) and rat aortic smooth muscle cells (RASMCs), following TNF-alpha and platelet derived growth factor-BB (PDGF-BB) challenges respectively. Both the extract and isovitexin blocked TNF-α induced expression of ICAM-1 and VCAM-1 in HUVECs. PDGF-BB induced migration of RASMCs and phospholipase C-γ activation, were also abrogated by GL extract and isovitexin. Fura-2 based ratiometric measurements demonstrated that, both the extact, and isovitexin, inhibit PDGF-BB mediated intracellular calcium rise in RASMCs. Supplementation of regular diet with 2% GL root powder for STZ rats, reduced total cholesterol in blood. Oil Red O staining demonstrated decreased lipid accumulation in aortic wall of diabetic animals upon treatment with GL. Medial thickness and deposition of collagen in the aortic segment of diabetic rats were also reduced upon supplementation. Immunohistochemistry demonstrated reduced expression of vascular cell adhesion molecule-1 (VCAM-1), inducible nitric oxide synthase (iNOS), and vascular endothelial cadherin (VE-cadherin) in aortic segments of diabetic rats following GL treatment. CONCLUSIONS: Thus, our results support that GL root extract/powder and isovitexin exhibit anti-atherosclerotic activities.

The radiorecovery potential of nutraceuticals in cellular defense after ionizing radiation in vitro.

BACKGROUND: The aim of this study was to evaluate the effects of two nutraceuticals Amazon Megamin and Immunarc forte in radiorecovery of human lymphocytes after exposure to ionizing radiation in vitro. MATERIAL AND METHODS: The incidences of micronuclei, cell proliferation, apoptosis and lipid peroxidation products were examined in cultured human peripheral blood lymphocytes before and after ionizing radiation in a present of nutraceuticals in vitro. RESULTS: Results showed that Amazon Megamin and Immunarc forte possess antioxidant properties; they act by eliminating the toxic metabolites, scavenging the free radicals and decreasing lipid peroxidation. CONCLUSION: The obtained results indicated that the studied nutraceuticals can help in prevention of the development of injurious caused by ionizing irradiation and, therefore, they encourage studies on their radioprotective properties.

Molecular analysis of ring y chromosome in a 10-year-old boy with mixed gonadal dysgenesis and growth hormone deficiency.

Ring Y chromosome is a very rare chromosomal aberration. The published mixed gonadal dysgenesis (MGD) patients with a ring Y chromosome are short in stature, but are not growth hormone (GH) deficient. We present the molecular cytogenetic and molecular characterization of ring Y chromosome mosaicism in a 10-year-old boy with MGD whose short stature could be explained by the high percentage of cells monosomic for the X-chromosome, but also by the presence of severe GH deficiency. The ring Y chromosome in our patient is a de novo structural aberration. The father's karyotype was normal.

Accelerated telomere shortening and telomere abnormalities in radiosensitive cell lines.

We examined telomere maintenance in cells of 11 primary fibroblast cell lines with differing genetic defects that confer sensitivity to ionizing radiation. These included cell lines derived from patients with ataxia telangiectasia, Nijmegen breakage syndrome, Fanconi anemia, defective Artemis, DNA ligase I and DNA ligase IV, an immunodeficient patient with a defect in DNA double-strand break repair, and a patient diagnosed with xeroderma pigmentosum who, in addition, showed severe clinical sensitivity to ionizing radiation. Our results, based on Southern blot, flow-FISH and Q-FISH (quantitative FISH) measurements, revealed an accelerated rate of telomere shortening in most cell lines derived from the above patients compared to cell lines from normal individuals or a cell line isolated from a heterozygotic parent of one radiosensitive patient. This accelerated telomere shortening was accompanied by the formation of chromatin bridges in anaphase cells, indicative of the early loss of telomere capping function and in some cases low levels of chromosome abnormalities in metaphase cells. We also analyzed telomere maintenance in mouse embryonic stem cells deficient in Brca1, another defect that confers radiosensitivity. Similarly, these cells showed accelerated telomere shortening and mild telomere dysfunction in comparison to control cells. Our results suggest that mechanisms that confer sensitivity to ionizing radiation may be linked with mechanisms that cause telomere dysfunction.

Role of antioxidant enzymes in radiosensitivity of human blood cells.

The marked variability in radiation response among individuals of the same age group prompted us to investigate the role of antioxidative enzyme activity. Micronuclei (MN) and enzyme assays were performed on blood samples of healthy male volunteers. The procedure consisted of micronucleus analysis and measurement of the superoxide dismutase (SOD) activity in harvested blood samples irradiated in vitro with 2 Gy gamma-rays and in unirradiated control samples for each individual. We found that the yield of radiation-induced micronuclei was in the range of 112 to 378 micronuclei per 1000 binucleated cells. The activity of cytosol superoxide dismutase (CuZnSOD) was reduced, whereas the activity of manganese superoxide dismutase (MnSOD) was markedly elevated in the blood samples harvested in lymphocyte cultures after irradiation. The analysis of our results showed that MnSOD plays the most important role in radiation-induced cellular damage. The results of this investigation showed that measurement of micronuclei and the activities of SOD in harvested human blood cells can serve as a rapid predictive assay of radiosensitivity in a clinical setting.

Radiosensitivity of different aged human lymphocytes following electron irradiation in vitro.

Cytochalasin B-blocking micronucleus test and chromosomal aberration analysis were used in this study to estimate the yield of individual variability in radiation response of different aged human lymphocytes. Both analyses were performed in three groups of adults, aged 18-65 years, on two sampling times, following irradiation by therapeutical dose of 2 Gy e- in vitro. No statistically significant difference in the induced yield of exchange aberrations between individuals under consideration was found. The yield of total aberration data showed greater variability and was statistically significant in the oldest group against two other adult groups. Regarding to fixation times no statistically significant differences in the induced yield of chromosomal aberrations (exchanges as well as total aberrations) were observed. The study has shown a slight increase in spontaneously occurring micronuclei with age. Almost equal mean number of radiation induced micronuclei was observed in the groups of adults aged 18-20 and 45-55 years. The highest mean number was observed in the oldest group. Evident variation in number of radiation induced micronuclei among individuals from the same age group was observed. The results of micronuclei assay for all individuals under consideration show statistically significant difference in the yield of radiation-induced micronuclei regarding the second fixation time. This study has shown that cytochalasin-B blocking micronucleus test is more sensitive assay than chromosomal aberration analysis for the estimation of individual radiosensitivity.

Influence of ribavirin on the micronucleus formation and in vitro proliferation of human lymphocytes.

Cytotoxic effects of the antiviremic drug ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) were evaluated in vitro measuring micronucleus formation and cell proliferation kinetics in whole blood cultures employing cytokinesis block (CB) micronucleus test. The cells were exposed to ribavirin doses ranging from 0.05 0.65 micromol/ml at three different incubation times. The frequency of micronuclei in treated samples demonstrated relatively low ability of ribavirin to induce micronuclei. However, the lowest concentration ofribavirin markedly changed the frequency of mononucleated and multinucleated cells, particularly binucleated ones, which declined significantly. The decline in the frequency ofbinucleate cells was followed with accumulation of greater number of mononucleate cells. Decreased proliferation potential of lymphocytes treated with ribavirin indicates that cells are arrested prior to metaphase. The present investigation showed that ribavirin is capable to induce a delay in cellular proliferation at all the doses assayed. The study demonstrated that CB micronucleus assay is simple and rapid method that can be used to assess toxic effect of drugs in vitro.

Radiosensitivity of human lymphocytes in vitro correlates more with proliferative ability of cells than with the incidence of radiation-induced damages of the genome.

The purpose of this study was the estimate the extent of individual variability in radioresponse of human lymphocytes in vitro and to establish the reasons of variability. Individual variability in radiation-response was evaluated using the cytochalasin B micronucleus test among 82 healthy individuals (36 men and 46 women), of mean age 38 (range 30-48). Blood samples were irradiated with gamma (60Co) rays at a dose of 2 Gy in vitro. The yield of radiation-induced micronuclei (MN), cytochalasin blocked proliferation index (CBPI), fraction of micronucleated binucleate (BN) cells and mean incidence of MN per micronucleated BN cells at a sampling time of 72 hours were scored. Our results brought out a significant effect of gender on the level of spontaneously occurring micronuclei, the lack of statistical differences between gender in the yield of radiation-induced micronuclei and marked variability in radiation response among individuals. Likelihood of expressing hypersensitivity was correlated with ability of cells to proliferate in vitro (beta = 0.41, p < 0.000) more than with the incidence of radiation-induced micronuclei per micronucleated cell (beta = 0.20, p < 0.000).

Age-related changes in radiation-induced micronuclei among healthy adults.

The aim of the present study was to establish the extent of in vitro radioresponse of lymphocytes among 62 healthy adults of both genders and to estimate the distribution of baseline micronuclei and radiosensitivity among individuals of the study population using the cytochalasin block micronucleus test. A younger study group consisted of 10 males (mean age, 22.4 years; range, 21-27) and 12 females (mean age, 24.8 years; range, 20-29), whereas an older study group consisted of 18 males (mean age, 35.1 years; range, 30-44) and 22 females (mean age, 38.5 years; range, 30-48). For evaluation of radiosensitivity blood samples were irradiated in vitro using 60Co gamma-ray source. The radiation dose employed was 2 Gy, the dose rate 0.45 Gy/min. The study revealed a significant gender effect on baseline micronuclei favoring females (Z = 3.25, P < 0.001), while yields of radiation-induced micronuclei did not differ significantly (Z = 0.56, P < 0.56) between genders. The distribution of baseline micronuclei among the individuals tested followed Poisson distribution in both study groups and in both genders, whereas the distribution of radiosensitivity among individuals of the older study group did not fulfill Poisson expectations (Kolmogorov-Smirnof test, P < 0.01). In contrast to a nonsignificant difference in radiosensitivity between males and females of the same age group (Z = 1.97, P < 0.56), a statistically significant difference in radiosensitivity between younger and older group for both genders was found (Z = 3.03, P < 0.03). Since the individuals tested were healthy, the observed variability in radiation response is considered to be an early effect of ageing.

[Cytogenetic changes in persons exposed to polychlorinated biphenyls]. / Citogenetske promene u osoba izlozenih polihlorovanim bifenilima.

A cytogenetic study performed in a group of workers exposed to polychlorinated biphenyls comprised analysis of structural chromosomal aberrations in the lymphocytes in the first division in vitro, analysis of micronuclei frequency in binuclear lymphoblasts induced by cytochalasin B and analysis of sister chromatid exchanges. The study included 48 exposed workers and 15 controls. In 16 exposed workers increased incidence of unstable chromosomal aberrations (dicentrics, rings and acentrics) was found, while in 19 no structural chromosomal aberrations could be detected. In 11 workers changes defined as chromosomal and chromatid breaks were noted. This type of change, however, can be evidenced in a non-exposed population as well. In two workers stable chromosomal aberrations were recorded (translocations and inversions). The incidence of micronuclei as well as exchange of sister chromatids were elevated in the exposed group of workers compared to controls.

In vitro evaluation of the genotoxicity of ritodrine and verapamil in human lymphocytes.

The aim of this study was to investigate the genotoxic effects of ritodrine and verapamil on human peripheral lymphocytes in vitro using micronucleus (MN) test. Also, fluorescence in situ hybridization (FISH) with a centromeric probe was performed to determine the origin of the induced MN. Cells were treated with 8.4 × 10(-6) M - 25.2 × 10(-4) M concentrations for ritodrine and 0.56 - 11 × 10(-5) M concentrations for verapamil, separately and combined. The MN frequencies showed increase after all treatments, but the difference between treated cells and untreated controls were found to be statistically significant only in the concentration range from 8.4 × 10(-5) M - 4.5 × 10(-4) M for ritodrine, 1.1 - 3.3 × 10(-5) M for verapamil, and in combined treatment with concentrations 8.4 × 10(-5) M + 1.1 × 10(-5) M for ritodrine and verapamil. The highest tested concentrations of both medicaments showed cytotoxic effect. Both medicaments decreased the nuclear division index (NDI) in tested concentrations. The results of FISH analysis suggest that verapamil, separately or combined with ritodrine, shows to a larger extent aneugenic than clastogenic effect.

Chromosome analysis of lymphocytes from radiation workers in tritium-applying industry.

OBJECTIVES: The frequency of chromosomal aberrations (CA) and micronuclei (MN) in peripheral blood lymphocytes was compared in two groups of persons occupationally exposed to tritium either through the use of luminous paints or in the weapons industry, with the aim of finding a correlation between the CA and MN assays, the extent of radiation hazard, and the duration of occupational exposure to chemical forms of tritium and the ability of the latter to induce hazards in human lymphocytes. METHODS: CA analysis and the cytochalasin B micronucleus test were performed. Urinary tritium in both groups was measured using a liquid scintillation method. For purposes of comparison, 2 control groups (40 persons) were examined; 24 exposed individuals were matched to their controls according to age, sex, and smoking habits. RESULTS: Values recorded for the frequency of CA in the group of workers handling tritium in luminous dial painting were 0.015 +/- 0.014 dicentrics/cell, 0.023 +/- 0.017 total unstable CA/cell, and 0.035 +/- 0.019 MN/binucleated cell. Urinary levels of tritium in this group of employees ranged from 1.35 to 9.43 MBq/l. As compared with their matched controls, in these workers the differences in the yield of CA as well as in MN were statistically highly significant (P < 0.005 and P < 0.003, respectively). The same parameters analyzed in workers employed in the armament industry gave values of 0.001 +/- 0.003 dicentrics/cell, 0.010 +/- 0.008 total unstable CA/cell, and 0.021 +/- 0.029 MN/binucleated cell. No detectable concentration of urinary tritium was found. As compared with their matched controls, in these workers the differences were also statistically significant (P < 0.027 and P < 0.012, respectively). The yield of CA, particularly the yield of dicentrics, was significantly higher (P < 0.005) in the group of luminous dial painters as compared with the weapons industry workers. The difference found in the yield of MN between the two groups of workers was also statistically significant (P < 0.084). Intercontrol differences in the yields of spontaneous CA as well as in MN were insignificant (P < 0.683 and P < 0.735, respectively). The results are discussed with respect to individual variations in the response to low doses of ionizing radiation, disorders in the handling of radionuclides, the duration of occupational exposure, and the chemical properties of the radionuclides used. CONCLUSION: Tritium in the chemical form of luminous paint is a more powerful clastogenic agent than tritium gas. Statistical analysis demonstrated that lymphocyte effects are due to the concentration of tritium in urine rather than to the duration of occupational exposure. Since the radiation hazard measured by the MN assay did not differ from that determined by the CA assay, the MN test can be recommended as a rapid assay for screening purposes in cases of occupational exposure to low radiation doses.

Cytogenetic monitoring of pesticide sprayers.

The induction of chromosome aberrations, micronuclei, and sister-chromatid exchanges (SCEs) was examined in cultured lymphocytes of 27 vineyard growers exposed to pesticides. Cytogenetic examinations were performed during the prespraying period, a month after spraying, and at the end of the spraying season. For comparison purposes, the same cytogenetic monitoring program was applied to two control groups. The first consisted of 15 individuals from a nearby town, and the second consisted of 20 volunteers living 200 km from the vine-growing area (reference control group). A positive, though low statistically significant (P < 0.017) difference in the yield of unstable chromosomal aberrations in exposed sprayers was observed compared with both control groups during the prespraying period. The mean group value of micronuclei in exposed workers averaged 5.41 per 1000 binucleated cells, with individual means ranging from 0 to 15. In both control groups, the yield of micronuclei averaged 5.09 per 1000 binucleated cells, with individual means ranging from 1 to 10. No statistically significant (P < 0.5) differences in yield of micronuclei were found in exposed subjects compared with both control groups. Significant individual variation (F = 14.09, P < 0.000) in SCE frequency was observed in exposed subjects, as well as in both control groups (F = 14.09, P < 0.000). A month after spraying, the average incidence of unstable aberrations in pesticide sprayers was 0.22%, and the yield of micronuclei averaged 17.78 per 1000 binucleated cells, with individual means ranging from 7 to 28. The incidence of micronuclei a month after spraying in exposed subjects was elevated (statistically significant at P < 0.01) in comparison with the prespraying period, while the difference in the yield of chromosomal aberrations in exposed subjects was insignificant (P < 0.5). At the end of the spraying season, the average incidence of unstable aberrations in exposed subjects was 0.79%, and the yield of micronuclei averaged 39.92 micronuclei per 1000 binucleated cells, with individual means ranging from 21 to 62. The appearance of more than one micronucleus per binucleated cell was related to the results on chromosome aberrations. The frequencies of chromosomal aberrations and micronuclei were significantly higher (P < 0.001, P < 0.000) in the exposed group than in their matched control groups. The yield of micronuclei in pesticide sprayers at the end of the season was higher than expected with respect to chromosomal aberration frequency, which provides some evidence that some of the micronuclei are induced by the spindle-inhibiting effects of pesticides. A statistically significant (P < 0.003) difference in micronuclei in the first control group was observed compared with the reference control group at the end of the spraying season. With respect to the incidence of micronuclei in the control group in the vine-growing area, a poor but positive correlation (r = 0.074, P < 0.104) with duration of the spraying season was found, which is probably due to airborne pesticides in the vine-growing area. SCE frequencies of the workers' lymphocytes were not significantly changed due to the exposure. The yield of aberrations as well as that of micronuclei in exposed subjects correlated positively (r = 16, P = 0.016) with duration of exposure.

Chromosome aberrations, micronuclei, and activity of superoxide dismutases in human lymphocytes after irradiation in vitro.

The goal of this study was to provide data on the dose-dependent production of dicentrics and micronuclei in human lymphocytes irradiated with 22.6 MeV protons and to estimate the possible contribution of intracellular superoxide dismutases (SOD) to the relative biological effectiveness (RBE) of protons. For the dose-response study, heparinized whole blood of a healthy volunteer was irradiated with protons and X-rays employing radiation doses of 0.5-4 Gy. Three biological endpoints were analyzed: chromosomal aberrations, micronuclei, and specific activity of cytosolic (CuZnSOD) and mitochondrial (MnSOD) superoxide dismutases in harvested human blood cells. Dicentric dose-response curves fit a linear-quadratic form (alpha = 0.094 +/- 0.006, beta = 0.032 +/- 0.001) induced with X-rays and (alpha = 0.119 +/- 0.057, beta = 0.029 +/- 0.014) for 22.6 MeV protons. Protons were more effective than X-rays in producing exchanges, particularly at 0.5 and 1 Gy. In contrast to X-ray irradiated samples where a significant increase in the specific activity of MnSOD was recorded (up to a radiation dose of 1 Gy), irradiation with protons markedly reduced its activity. As a consequence of the reduced activity of MnSOD, the chromosomal dose-response curve became quadratic. The RBE for dicentrics varies with dose (from 2.2 to 1.01) and reduced activity of MnSOD is an important contributor to the RBE of protons. SODs, particularly MnSOD, play an important role in defending DNA from reactive oxygen species. A reduced activity of SOD, particularly MnSOD, is an important contributor to the RBE of protons.

Age-related changes in radiation-induced micronuclei among healthy adults

The aim of the present study was to establish the extent of in vitro radioresponse of lymphocytes among 62 healthy adults of both genders and to estimate the distribution of baseline micronuclei and radiosensitivity among individuals of the study population using the cytochalasin block micronucleus test. A younger study group consisted of 10 males (mean age, 22.4 years; range, 21-27) and 12 females (mean age, 24.8 years; range, 20-29), whereas an older study group consisted of 18 males (mean age, 35.1 years; range, 30-44) and 22 females (mean age, 38.5 years; range, 30-48). For evaluation of radiosensitivity blood samples were irradiated in vitro using 60Co g-ray source. The radiation dose employed was 2 Gy, the dose rate 0.45 Gy/min. The study revealed a significant gender effect on baseline micronuclei favoring females (Z = 3.25, P < 0.001), while yields of radiation-induced micronuclei did not differ significantly (Z = 0.56, P < 0.56) between genders. The distribution of baseline micronuclei among the individuals tested followed Poisson distribution in both study groups and in both genders, whereas the distribution of radiosensitivity among individuals of the older study group did not fulfill Poisson expectations (Kolmogorov-Smirnof test, P < 0.01). In contrast to a nonsignificant difference in radiosensitivity between males and females of the same age group (Z = 1.97, P < 0.56), a statistically significant difference in radiosensitivity between younger and older group for both genders was found (Z = 3.03, P < 0.03). Since the individuals tested were healthy, the observed variability in radiation response is considered to be an early effect of ageing.