Response of thyroid follicular cells to gamma irradiation compared to proton irradiation: II. The role of connexin 32

The objective of this study was to determine whether connexin 32-type gap junctions contribute to the "contact effect" in follicular thyrocytes and whether the response is influenced by radiation quality. Our previous studies demonstrated that early-passage follicular cultures of Fischer rat thyroid cells express functional connexin 32 gap junctions, with later-passage cultures expressing a truncated nonfunctional form of the protein. This model allowed us to assess the role of connexin 32 in radiation responsiveness without relying solely on chemical manipulation of gap junctions. The survival curves generated after gamma irradiation revealed that early-passage follicular cultures had significantly lower values of alpha (0.04 Gy(-1)) than later-passage cultures (0.11 Gy(-1)) (P < 0.0001, n = 12). As an additional way to determine whether connexin 32 was contributing to the difference in survival, cultures were treated with heptanol, resulting in higher alpha values, with early-passage cultures (0.10 Gy(-1)) nearly equivalent to untreated late-passage cultures (0.11 Gy(-1)) (P > 0.1, n = 9). This strongly suggests that the presence of functional connexin 32-type gap junctions was contributing to radiation resistance in gamma-irradiated thyroid follicles. Survival curves from proton-irradiated cultures had alpha values that were not significantly different whether cells expressed functional connexin 32 (0.10 Gy(-1)), did not express connexin 32 (0.09 Gy(-1)), or were down-regulated (early-passage plus heptanol, 0.09 Gy(-1); late-passage plus heptanol, 0.12 Gy(-1)) (P > 0.1, n = 19). Thus, for proton irradiation, the presence of connexin 32-type gap junctional channels did not influence their radiosensitivity. Collectively, the data support the following conclusions. (1) The lower alpha values from the gamma-ray survival curves of the early-passage cultures suggest greater repair efficiency and/or enhanced resistance to radiation-induced damage, coincident with the expression of connexin 32-type gap junctions. (2) The increased sensitivity of FRTL-5 cells to proton irradiation was independent of their ability to communicate through connexin 32 gap junctions. (3) The fact that the beta components of the survival curves from both gamma rays and proton beams were similar (average 0.022 +/- 0.008 Gy(-2), P > 0.1, n = 39) suggests that at higher doses the loss of viability occurs at a relatively constant rate and is independent of radiation quality and the presence of functional gap junctions.     

The effect of 835.62 MHz FDMA or 847.74 MHz CDMA modulated radiofrequency radiation on the induction of micronuclei in C3H 10T(1/2) cells

To determine if radiofrequency (RF) radiation induces the formation of micronuclei, C3H 10T(1/2) cells were exposed to 835.62 MHz frequency division multiple access (FDMA) or 847.74 MHz code division multiple access (CDMA) modulated RF radiation. After the exposure to RF radiation, the micronucleus assay was performed by the cytokinesis block method using cytochalasin B treatment. The micronuclei appearing after mitosis were scored in binucleated cells using acridine orange staining. The frequency of micronuclei was scored both as the percentage of binucleated cells with micronuclei and as the number of micronuclei per 100 binucleated cells. Treatment of cells with cytochalasin B at a concentration of 2 microg/ml for 22 h was found to yield the maximum number of binucleated cells in C3H 10T(1/2) cells. The method used for the micronucleus assay in the present study detected a highly significant dose response for both indices of micronucleus production in the dose range of 0.1-1.2 Gy and it was sensitive enough to detect a significant (P > 0.05) increase in micronuclei after doses of 0.3 Gy in exponentially growing cells and after 0.9 Gy in plateau-phase cells. Exponentially growing cells or plateau-phase cells were exposed to CDMA (3.2 or 4.8 W/kg) or FDMA (3.2 or 5.1 W/kg) RF radiation for 3, 8, 16 or 24 h. In three repeat experiments, no exposure condition was found by analysis of variance to result in a significant increase relative to sham-exposed cells either in the percentage of binucleated cells with micronuclei or in the number of micronuclei per 100 binucleated cells. In this study, data from cells exposed to different RF signals at two SARs were compared to a common sham-exposed sample. We used the Dunnett's test, which is specifically designed for this purpose, and found no significant exposure-related differences for either plateau-phase cells or exponentially growing cells. Thus the results of this study are not consistent with the possibility that these RF radiations induce micronuclei.     

Regulation of collagenase and collagenase mRNA production in early- and late-passage human diploid fibroblasts

The levels of collagenase and collagenase mRNA produced by early-passage (less than 40% of lifespan completed) and late-passage (greater than 80% of lifespan completed) cultures of human fibroblasts were analyzed. The constitutive levels of collagenase and collagenase mRNA produced by the late-passage cultures were 10-30 x greater than the levels observed in similarly treated early-passage cultures. Immunofluorescence analysis established that the percentage of collagenase-positive cells was also greater (77% vs. 4%) in the late-passage cultures. To determine whether the difference in collagenase production resulted from cell-derived regulatory factors, collagenase production was examined in cultures plated onto substrates coated with fibroblast extracellular matrix (ECM). Collagenase and collagenase mRNA production was enhanced in both types of cultures, although amounts produced by ECM-induced early-passage cultures was significantly less than that produced by similarly treated late-passage cultures. Collagen-coated substrates also induced collagenase synthesis.     

DNA damage and micronucleus induction in human leukocytes after acute in vitro exposure to a 1.9 GHz continuous-wave radiofrequency field

Human blood cultures were exposed to a 1.9 GHz continuous-wave (CW) radiofrequency (RF) field for 2 h using a series of six circularly polarized, cylindrical waveguides. Mean specific absorption rates (SARs) of 0.0, 0.1, 0.26, 0.92, 2.4 and 10 W/kg were achieved, and the temperature within the cultures during a 2-h exposure was maintained at 37.0 +/- 0.5 degrees C. Concurrent negative (incubator) and positive (1.5 Gy (137)Cs gamma radiation) control cultures were run for each experiment. DNA damage was quantified immediately after RF-field exposure using the alkaline comet assay, and four parameters (tail ratio, tail moment, comet length and tail length) were used to assess DNA damage for each comet. No evidence of increased primary DNA damage was detected by any parameter for RF-field-exposed cultures at any SAR tested. The formation of micronuclei in the RF-field-exposed blood cell cultures was assessed using the cytokinesis-block micronucleus assay. There was no significant difference in the binucleated cell frequency, incidence of micronucleated binucleated cells, or total incidence of micronuclei between any of the RF-field-exposed cultures and the sham-exposed controls at any SAR tested. These results do not support the hypothesis that acute, nonthermalizing 1.9 GHz CW RF-field exposure causes DNA damage in cultured human leukocytes.     

The cell composition in the lymphocyte population and the radiation adaptive response

Using lymphocytes of 5 healthy individuals the ability to adaptive response (AR), cell composition of population after PHA stimulation, changes in cell composition population after irradiation in the dose of 1.0 Gy and after irradiation in adaptive (0.05 Gy) and challenge (1.0 Gy) doses have been studied. AR observed in 2 of the 5 individuals only. After PHA stimulation the persons with AR have the total amount of cells after mitosis or during mitosis (the number of binucleated cells + the number of multinucleated cells + the whole cells with micronuclei + the number of mitotic cells) on average is higher than in persons without AR. In individuals with AR the linear correlation between the number of binucleated cells with micronuclei (on the 1000 scored binucleated cells) and the part of binucleated cells in the population is observed with coefficients of correlation -0.89 and -0.91. In the humans without AR this correlation is absent. The correlation observed permits to suppose that AR may occur at the expense of not only the decrease in number of damaged lymphocytes, but also the increase in the share of not damaged binucleated cell with the stable number of damaged cells.     

Non-disjunction and chromosome loss in gamma-irradiated human lymphocytes: a fluorescence in situ hybridization analysis using centromere-specific probes

Centromere-specific DNA probes for chromosomes 4, 7 and 18 were used to simultaneously analyze chromosome loss, non-disjunction, breaks within the labeled region, and nucleoplasmic bridges induced by gamma rays in binucleated human lymphocytes. The doses used were 0, 1, 2 and 4 Gy, and approximately 1000 cells were scored per dose. Micronucleus frequency increased in a linear-quadratic fashion. For chromosome loss, significant increases were observed at 2 and 4 Gy, whereas for non-disjunction significant increases were observed at 1 Gy; thus non-disjunction allowed us to detect the effects of radiation at a lower dose than chromosome loss. The use of centromere-specific probes allowed discrimination between the clastogenic and aneugenic effects of ionizing radiation. The analysis of chromosome loss, not taking fragmented signals into account, ensures the detection of an aneugenic effect, which was not possible using pancentromeric probes. The frequency of chromosome breakage within the labeled regions was higher in nuclei than in micronuclei, suggesting an increase in the engulfment of chromosomal material by nuclei as a consequence of the presence of cytochalasin B in the cultures. Chromatin filaments connecting main nuclei, the so-called nucleoplasmic bridges, were observed in irradiated samples, and are a manifestation of rearranged chromosomes producing anaphase bridges.     

Enhanced proliferation and altered intracellular zinc levels in early- and late-passage mouse aorta smooth muscle cells

Cell growth and DNA synthesis were studied from a cultured early- and late- passage mouse aorta smooth muscle cell (MASMC) because the proliferation of vascular smooth muscle cell (VSMC) is a key factor in development of atherosclerosis. In this study, the cells were cultured in fetal bovine serum (FBS) and stimulated by growth factors such as thrombin and platelet-derived growth factor-BB (PDGF-BB). Compared to the number of early-passage MASMC (passage 3 to 9) the number of late-passage MASMC (passage 30 to 40) in a normal serum state was increased 2 fold at Day 1,3 and 6 in culture, respectively. Incorporation of [³H] thymidine into DNA induced by serum, PDGF and thrombin in late-passage MASMC was greater than those in early-passage MASMC. We also examined whether intracellular zinc levels would be an aging factor or not. The intracellular zinc level in, early- and late-passage MASMC was monitored by using the zinc probe dyeN-(6-methoxy-8-quinolyl)-p-toluenesulfonamide. It is interested that late-passage MASMC increased the intracellular fluorescence level of zinc, more than the early passage MASMC did. The alterations of intracellular zinc level occur concurrently with changes in MASMC proliferation rate during aging. This data suggest that the age-associated changes in zinc concentrations may provide a newin vitro model for the study of smooth muscle cell differentiation.     

Use of C-banding for identifying radiation induced micronuclei

Differentiation of micronuclei (MN) caused by ionizing radiation from those caused by chemicals is a crucial step for managing treatment of individuals exposed to radiation. MN in binucleated lymphocytes in peripheral blood are widely used as biomarkers for estimating dose of radiation, but they are not specific for ionizing radiation. MN induced by ionizing radiation originate predominantly as a result of chromosome breaks (clastogenic action), whereas MN caused by chemical agents are derived from the loss of entire chromosomes (aneugenic action). C-banding highlights centromeres, which might make it possible to distinguish radiation induced MN, i.e., as a byproduct of acentric fragments, from those caused by the loss of entire chromosomes. To test the use of C-banding for identifying radiation induced MN, a blood sample from a healthy donor was irradiated with 3 Gy of Co-60 gamma rays and cultured. Cells were harvested and dropped onto slides, divided into a group stained directly with Giemsa and another processed for C banding, then stained with Giemsa. The frequency of MN in 500 binucleated cells was scored for each method. In preparations stained with Giemsa directly, the MN appeared as uniformly stained structures, whereas after C banding, some MN exhibited darker regions corresponding to centromeres that indicated that they were not derived from acentric fragments. The C-banding technique enables differentiation of MN from acentric chromosomal material. This distinction is useful for improving the specificity of the MN assay as a biomarker for ionizing radiation.     

应用原位杂交技术检测X射线诱发的特定染色体的微核率

为了研究X射线与X染色体的微校率之间的关系．本实验利用原位杂交技术同时检测了经X射线诱发人双核淋巴细胞的7号和X染色体的微核率。结果发现：经2．5Gy的X射线照射后．X和7号染色体的微核率男性分别为3．4％和7．1％;女性分别为6．6％和6．0％。X和7号染色体微核率的实验观察值与理论预期值之间在统计学上无显著性差异。实验结果提示：X射线并不特异性引起X染色体的微核率增高。     

Involvement of DNA polymerase beta in protection against the cytotoxicity of oxidative DNA damage

We had shown previously that DNA polymerase beta (beta-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H2O2). This is surprising because beta-pol is thought to be involved in BER of oxidative as well as methylated DNA damage. We confirm these findings here in early-passage cells. However, with time in culture, beta-pol null cells become hypersensitive to H2O2 and other reactive oxygen species-generating agents. Analysis of in vitro BER reveals a strong deficiency in single-nucleotide BER of 8-oxoguanine (8-oxoG) by both early- and late-passage beta-pol null cell extracts. Therefore, in early-passage wild-type and beta-pol null cells, the capacity for single-nucleotide BER of 8-oxoG does not correlate with cellular sensitivity to H2O2. Expression of beta-pol protein in the late-passage null cells almost completely reverses the H2O2-hypersensitivity phenotype. Methoxyamine (MX) treatment sensitizes late-passage wild-type cells to H2O2 as expected for beta-pol-mediated single-nucleotide BER; however in beta-pol null cells, MX has no effect. The data indicate a role(s) of beta-pol-dependent repair in protection against the cytotoxicity of oxidative DNA damage in wild-type cells.     

Effects of 5-bromo-2-deoxy-uridine and 2-deoxy-D-glucose on the radiosensitivity of proliferating transformed mammalian cells.

Effects of 5-bromo-2-deoxy-uridine (BrdU) and 2-deoxy-D-glucose (2-DG) were studied in exponentially growing transformed mammalian (BHK-21) cells, grown as monolayer. Micronuclei formation as an index of radiation damage was studied by i) cytokinesis block technique from cytochalasin-B induced binucleated cells, and ii) conventional technique. Presence of BrdU (0.8 microM) for nearly 2 cell cycles before gamma-irradiation (2.5 Gy) significantly increased frequencies of cells with micronuclei. Post-irradiation incubation of cultures in liquid holding medium (HBSS) however, reduced micronuclei formation, especially in the BrdU treated cells. Presence of 2-DG (4 hr, equimolar with glucose) in growth as well as liquid holding medium further increased micronuclei frequencies. These observations suggest that radiation induced DNA lesions in BrdU substituted cells, leading to chromosome fragmentation are partly repairable. 2-DG increased cytogenetic damage, possibly by inhibiting the repair of such repairable lesions. Present studies suggest that combination of 2-DG could optimize BrdU-radiation therapy of brain tumors, by reducing the BrdU doses required for tumor radiosensitization.     

Frequency of radiation-induced micronuclei in neuronal cells does not correlate with clonogenic survival

It is generally assumed that radiation-induced micronuclei (MN) in cytokinesis-blocked cells are an expression of cellular radiosensitivity. Therefore, radiosensitive cells should have a high frequency of MN and radioresistant cells should show lower levels. We have irradiated cells of a panel of 13 neuronal cell lines of widely differing radiosensitivity [human neuroblastomas: N2alpha, SHSY5Y, SK-N-SH, KELLY and SK-N-BE(2c); murine neuroblastomas: OP-6 and OP-27; human glioblastomas: G120, G60, G28, G112, G44 and G62] and compared their radiation response using the micronucleus and standard clonogenic assays. It was found that micronucleus frequency was much higher in some of the radioresistant cell lines (N2alpha, G28, G120 and G44; SF2 >/= 0.60). These cell lines showed a high frequency of more than 0.32 MN per gray of (60)Co gamma radiation per binucleated cell. On the other hand, the more radiosensitive cell lines (OP-27 and SK-N-SH, SF2 相似文献   

Freeze-fracture study of the junctional complexes of human and rabbit thyroid follicles

Summary Zonulae occludentes, gap junctions and desmosomes have been demonstrated in replicas of freeze-fractured follicular cells of normal human and rabbit thyroid glands. The zonulae occludentes between the human follicular cells are composed of two to eight strands, which completely separate the intercellular space from the follicular lumen. Four to twelve or more strands are visible between the follicular cells of the rabbit thyroid gland.In the meshes of the zonulae occludentes as well as below them, gap junctions are present. They are numerous on the fracture faces of the human follicular cell membranes, but infrequent in those of the rabbit.Aggregates of particles related to desmosomes are found in the deeper meshes of the zonulae occludentes or close to them.     

Induction of micronuclei in human fibroblasts across the Bragg curve of energetic heavy ions

The space environment consists of a varying field of radiation particles including high-energy ions, with spacecraft shielding material providing the major protection to astronauts from harmful exposure. Unlike low-LEpsilonTau gamma or X rays, the presence of shielding does not always reduce the radiation risks for energetic charged-particle exposure. The dose delivered by the charged particle increases sharply as the particle approaches the end of its range, a position known as the Bragg peak. However, the Bragg curve does not necessarily represent the biological damage along the particle path since biological effects are influenced by the track structures of both primary and secondary particles. Therefore, the "biological Bragg curve" is dependent on the energy and the type of the primary particle and may vary for different biological end points. Here we report measurements of the biological response across the Bragg curve in human fibroblasts exposed to energetic silicon and iron ions in vitro at two different energies, 300 MeV/nucleon and 1 GeV/nucleon. A quantitative biological response curve generated for micronuclei per binucleated cell across the Bragg curve did not reveal an increased yield of micronuclei at the location of the Bragg peak. However, the ratio of mono- to binucleated cells, which indicates inhibition of cell progression, increased at the Bragg peak location. These results confirm the hypothesis that severely damaged cells at the Bragg peak are more likely to go through reproductive death and not be evaluated for micronuclei.     

Folic acid deficiency increases chromosomal instability, chromosome 21 aneuploidy and sensitivity to radiation-induced micronuclei

Folic acid deficiency can lead to uracil incorporation into DNA, hypomethylation of DNA, inefficient DNA repair and increase chromosome malsegregation and breakage. Because ionising radiation increases demand for efficient DNA repair and also causes chromosome breaks we hypothesised that folic acid deficiency may increase sensitivity to radiation-induced chromosome breakage. We tested this hypothesis by using the cytokinesis-block micronucleus assay in 10 day WIL2-NS cell cultures at four different folic acid concentrations (0.2, 2, 20, and 200 nM) that span the "normal" physiological range in humans. The study showed a significant dose-dependent increase in frequency of binucleated cells with micronuclei and/or nucleoplasmic bridges with decreasing folic acid concentration (P<0.0001, P=0.028, respectively). These biomarkers of chromosomal instability were also increased in cells irradiated (1.5 Gy gamma-rays) on day 9 relative to un-irradiated controls (P<0.05). Folic acid deficiency and gamma-irradiation were shown to have a significant interactive effect on frequency of cells containing micronuclei (two-way ANOVA, interaction P=0.0039) such that the frequency of radiation-induced micronucleated cells (i.e. after subtracting base-line frequency of un-irradiated controls) increased with decreasing folic acid concentration (P-trend<0.0001). Aneuploidy of chromosome 21, apoptosis and necrosis were increased by folic acid deficiency but not by ionising radiation. The results of this study show that folate status has an important impact on chromosomal stability and is an important modifying factor of cellular sensitivity to radiation-induced genome damage.     

No evidence for genotoxic effects from 24 h exposure of human leukocytes to 1.9 GHz radiofrequency fields

The current study extends our previous investigations of 2-h radiofrequency (RF)-field exposures on genotoxicity in human blood cell cultures by examining the effect of 24-h continuous-wave (CW) and pulsed-wave (PW) 1.9 GHz RF-field exposures on both primary DNA damage and micronucleus induction in human leukocyte cultures. Mean specific absorption rates (SARs) ranged from 0 to 10 W/kg, and the temperature within the cultures was maintained at 37.0 +/- 1.0 degrees C for the duration of the 24-h exposure period. No significant differences in primary DNA damage were observed between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures when processed immediately after the exposure period by the alkaline comet assay. Similarly, no significant differences were observed in the incidence of micronuclei, incidence of micronucleated binucleated cells, frequency of binucleated cells, or proliferation index between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures. In conclusion, the current study found no evidence of 1.9 GHz RF-field-induced genotoxicity in human blood cell cultures after a 24-h exposure period.     

Micronucleus induction in mammalian cell cultures treated with ionizing radiations

Summary Exponentially growing and plateau phase cultures of Ehrlich ascites tumor cells (suspension strain) were treated with either fast electrons, X-rays, fast neutrons or Am-241-alpha-particles in a dose range from about 0.02 Gy to 1 Gy and for comparison also at higher doses. After the first post-irradiation division, cells were scored for the presence of micronuclei and the micronucleus fraction as well as the number of micronuclei/cell was determined. Micronuclei were counted using the DNA specific stain H 33258 in a fluorescence microscope. A comparison with cytofluorometric measurements established that microscopic detection accounted for up to 90% of all micronuclei present within a sample, the rest probably being hidden in direct observation by the main nucleus.Dose response curves based on the micronucleus fraction as well as on the number of micronuclei/cell were found to be linear in the whole dose range tested at low and at high ionization density. Linearity was maintained also when repair of primary lesions was promoted or suppressed. The RBE of alpha-particles compared with X-rays was dependent on the time of fixation and was at a maximum immediately after the first division (RBE = 4.8 ± 0.5). Micronucleus distribution showed overdispersion relative to Poissonian statistics with every radiation quality used, in accordance with earlier observations on the distribution of acentric fragments in irradiated cultures.     

RBE of 10 kV X rays determined for the human mammary epithelial cell line MCF-12A

The dependence of relative biological effectiveness (RBE) on photon energy is a topic of extensive discussions. The increasing amount of in vitro data in the low-energy region indicates this to be a complex dependence that is influenced by the end point and cell line studied. In the present investigation, the RBE of 10 kV X rays (W anode) was determined relative to 200 kV X rays (W anode, 0.5 mm copper filter) for cell survival in the dose range 1-10 Gy and for induction of micronuclei in the range 0.5-3.6 Gy for MCF-12A human mammary epithelial cells. The RBE for cell survival was found to increase with decreasing dose, being 1.21+/-0.03 at 10% survival. Considerably higher values were obtained for micronucleus induction, where the RBE(M) obtained from the ratio of the linear coefficients of the dose-effect curves was 2.6+/-0.4 for the fraction of binucleated cells with micronuclei and 4.1+/-1.0 for the number of micronuclei per binucleated cell. These values, together with our previous data, support a monotonic increase in RBE with decreasing photon energy down to the mean energy of 7.3 keV used in the present study.     

Differential oxidation of deoxyribose in DNA by gamma and alpha-particle radiation

Emerging evidence points to the importance of deoxyribose oxidation in the toxicity of oxidative DNA damage, including the formation of protein-DNA crosslinks and base adducts. With the goal of understanding the differences in deoxyribose oxidation chemistry known to occur with different oxidants, we have compared the formation of one product of 3'-oxidation of deoxyribose in DNA, 3'-phosphoglycolaldehyde (PGA) residues, in isolated DNA and cells exposed to ionizing radiations. A recently developed gas chromatography/negative chemical ionization mass spectrometry method was used to quantify PGA residues in purified DNA and in human TK6 lymphoblastoid cells exposed to gamma radiation (60Co) and alpha particles (241Am). The level of PGA residues was then correlated with the total quantity of deoxyribose oxidation determined by plasmid topoisomer analysis. Alpha-particle irradiation (0-100 Gy) of purified DNA in 50 mM potassium phosphate (pH 7.4) produced a linear dose response of 0.13 PGA residues per 10(6) nucleotides per gray. When normalized to an estimate of the total number of deoxyribose oxidation events (2.0 per 10(6) nucleotides per gray), PGA formation occurred in 7% (+/-0.5) of deoxyribose oxidation events produced by alpha-particle radiation. In contrast, the efficiency of PGA formation in gamma-irradiated DNA was found to be 1% (+/-0.02), which indicates a shift in the chemistry of deoxyribose oxidation, possibly as a result of the different track structures of the two types of ionizing radiation. Studies with gamma radiation were extended to TK6 cells, in which it was observed that gamma radiation produced a linear dose response of 0.0019 PGA residues per 10(6) nucleotides per gray. This is consistent with an approximately 1000-fold quenching effect in cells, similar to the results of other published studies of oxidative DNA damage in vivo.