Dose response and repair kinetics of γ-H2AX foci induced by in vitro irradiation of whole blood and T-lymphocytes with X- and γ-radiation

Purpose:?Dose response and repair kinetics of phosphorylated histone H2A isoform X (γ-H2AX) foci in T-lymphocytes were investigated in the low-dose range after in vitro irradiation of whole blood and T-lymphocytes with 100 kVp X-rays and ⁶⁰Co γ-rays.

Materials and methods:?Whole blood or isolated T-lymphocytes were irradiated in vitro and γ-H2AX foci were scored. Dose response was determined in the 0–500?mGy dose range. Foci kinetics were studied at doses of 5 and 200?mGy up to 24?h post-irradiation.

Results:?After X-irradiation, the dose response for whole blood shows a biphasic behaviour with a low-dose hypersensitivity, which is less pronounced for isolated T-lymphocytes. In contrast, γ-radiation shows a linear dose response for both irradiation conditions. Concerning repair kinetics, delayed repair was found after X-ray whole blood irradiation (5 and 200?mGy) with 40% of the foci persisting 24?h post-irradiation. This number of foci is reduced to 10% after irradiation of isolated T-lymphocytes with 200?mGy X-rays. On the contrary, γ-H2AX foci are reduced to background levels 24?h post-irradiation with 200?mGy ⁶⁰Co γ-rays.

Conclusion:?γ-H2AX foci response and repair kinetics depend on irradiation conditions and radiation quality, possibly linked to Bystander response.     

Cell cycle arrest and aberration yield in normal human fibroblasts. II: Effects of 11 MeV u−1 C ions and 9.9 MeV u−1 Ni ions

Purpose: To investigate further the relationship between high linear energy transfer (LET) induced cell cycle arrests and the yield of chromosome aberrations observable in normal human fibroblasts at the first post-irradiation mitosis.

Materials and methods: Normal human fibroblasts (AG01522C) were exposed in G0/G1 to either 11 MeV u^?1 C ions (LET = 153.5 keV μm^?1) or 9.9 MeV u^?1 Ni ions (LET = 2455 keV μm^?1), subcultured in medium containing 5-Bromo-2'-deoxyuridine (BrdU) and at multiple time-points post-irradiation the yield of chromosomal damage, the mitotic index and the cumulative BrdU-labelling index were determined. Furthermore, a mathematical approach was used to analyse the entire cell population.

Results: Following high LET exposure normal fibroblasts suffer a transient delay into S-phase and into mitosis as well as a prolonged, probably permanent cell cycle arrest in the initial G0/G1-phase. Cells that reach the first mitosis at early times carried less aberrations than those collected at later times indicating a relationship between cell cycle delay and the number of aberrations. However, with respect to the whole cell population, only a few aberrant fibroblasts are able to progress to the first mitosis. For all endpoints studied the relative biological effectiveness (RBE) of C ions is in the range of 2 – 4, while for Ni ions RBE < 1 is estimated. In contrast, when compared on a per particle basis Ni ions with the higher ionization density were found to be more effective.

Conclusions: Detailed analysis of the data demonstrates that the number of fibroblasts at risk for neoplastic transformation is significantly reduced by a chronic cell cycle arrest in the initial G0/G1-phase and, for the first time, the LET-dependence of this effect has been shown.     

Dose–response relationship of locally applied nimodipine in an ex vivo model of cerebral vasospasm

Introduction

Cerebral vasospasm is a severe complication of subarachnoid hemorrhage (SAH). The calcium channel inhibitor nimodipine has been used for treatment of cerebral vasospasm. No evidence-based recommendations for local nimodipine administration at the site of vasospasm exist. The purpose of this study was to quantify nimodipine's local vasodilatory effect in an ex vivo model of SAH-induced vasospasm.

Methods

SAH-induced vasospasm was modeled by contracting isolated segments of rat superior cerebellar arteries with a combination of serotonin and a synthetic analog of prostaglandin A₂. A pressure myograph system was used to determine vessel reactivity of spastic as well as non-spastic arteries.

Results

Compared to the initial vessel diameter, a combination of serotonin and prostaglandin induced considerable vasospasm (55?±?2.5 % contraction; n?=?12; p?<?0.001). Locally applied nimodipine dilated the arteries in a concentration-dependent manner starting at concentrations as low as 1 nM (n?=?12; p?<?0.05). Concentrations higher than 100 nM did not relevantly increase the vasodilatory effect. Nimodipine's vasodilatory effect was smaller in spastic than in non-spastic vessels (n?=?12; p?<?0.05), which we assume to be due to structural changes in the vessel wall.

Conclusion

The described ex vivo model allows to investigate the dose-dependent efficacy of spasmolytic drugs prior to in vivo experiments. Low concentrations of locally applied nimodipine have a strong vasodilatory effect, which is of relevance when considering the local application of nimodipine in cerebral vasospasm.     

Role of DNA methylation in long-term low-dose γ-rays induced adaptive response in human B lymphoblast cells

Abstract

Purpose: With widespread use of ionizing radiation, more attention has been attracted to low-dose radiation (LDR); however, the mechanisms of long-term LDR-induced bio-effects are unclear. Here, we applied human B lymphoblast cell line HMy2.CIR to monitor the effects of long-term LDR and the potential involvement of DNA methylation.

Materials and methods: HMy2.CIR cells were irradiated with 0.032 Gy γ-rays three times per week for 1–4 weeks. Some of these primed cells were further challenged with 2 Gy γ-rays. Cell proliferation, micronuclei formation, gene expression of DNA methyltransferases (DNMT), levels of global genomic DNA methylation and protein expression of methyl CpG binding protein 2 (MeCP2) and heterochromatin protein-1 (HP1) were measured.

Results: Long-term LDR enhanced cell proliferation and clonogenicity and triggered a cellular adaptive response (AR). Furthermore, global genomic DNA methylation was increased in HMy2.CIR cells after long-term LDR, accompanied with an increase of gene expression of DNMT1 and protein expression of MeCP2 and HP1. After treatment with 5-aza-2′-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, the long-term LDR-induced global genomic DNA hypermethylation was decreased and the AR was eliminated.

Conclusion: Global genomic DNA hypermethylation accompanied with increases of DNMT1 and MeCP2 expression and heterochromatin formation might be involved in long-term LDR-induced adaptive response.     

Cell cycle arrest and aberration yield in normal human fibroblasts. I. Effects of X‐rays and 195 MeV u−1 C ions

Purpose: To examine the relationship between cell proliferation and the expression of chromosomal damage in normal human skin fibroblasts after X‐ray and particle irradiation.

Materials and methods: Confluent G0/G1 AG1522B cells were exposed to X‐rays or 195?MeV?u^?1 C ions with a linear energy transfer of 16.6?keV?µm^?1 in the dose range 1–4?Gy. Directly after irradiation, cells were reseeded at a low density in medium containing 5‐bromo‐2′‐deoxyuridine. At multiple time points post‐irradiation, the cumulative BrdU‐labelling index, mitotic index and aberration frequency were measured. Based on these data, the total amount of damage induced within the entire cell population was estimated by means of mathematical analysis.

Results: Both types of radiation exposure exert a pronounced effect on the cell cycle progression of fibroblasts. They result in delayed entry of cells into S‐phase and into the first mitosis, and cause a dramatic reduction in mitotic activity. Measurement of chromosomal damage in first‐cycle cells at multiple time points post‐irradiation shows that the frequencies of aberrant cells and aberrations increase with time up to twofold for the lower doses. However, for the higher doses, this effect is less pronounced or even disappears. When the data for the whole cell population are analysed, it becomes evident that only a few damaged fibroblasts can progress to the first mitosis, a response attributable at least in part to a long‐term arrest of injured cells in the initial G0/G1‐phase. As observed in other investigations, the effectiveness of 195?MeV?u^?1 C ions was similar or slightly higher than X‐rays for all endpoints studied leading to a relative biological effectiveness in the range 1.0–1.4.

Conclusions: Cell cycle arrests affect the aberration yield observable in normal human fibroblasts at mitosis. The data obtained for the cell population as a whole reveal that injured cells are rapidly removed from the mitotically active population through a chronic cell cycle arrest, which is consistent with other studies that indicate that this response is a specific strategy of fibroblasts to minimize the fixation and propagation of genetic alterations.     

Induction of single- and double-strand breaks in plasmid DNA by 100–1500 eV electrons

Purpose : To investigate the induction of DNA strand breaks by electrons with energies ranging from 0.1 to 1.5 keV. Materials and methods : Dry supercoiled plasmid DNA was irradiated with electrons of energies ranging from 0.1 to 1.5keV and the results were compared with those obtained by gamma-irradiation of the same plasmid in solution. For electron irradiation, the plasmid was deposited on a gold substrate under a controlled atmosphere to minimize contamination of the DNA film. Electron bombardments were performed under ultra-high vacuum conditions (UHV 10 9 torr). DNA damage was detected by gel electrophoresis followed by quantitation of the DNA bands by fluorescence or by hybridization with a radioactive probe. Results : Electrons with energies from 0.1 to 1.5keV induced single, double and multiple double-strand breaks in supercoiled plasmid DNA. For equal doses, we observed a marked increase in the efficiency of induction of double- and multiple-strand breaks in supercoiled DNA as a function of electron energy. In contrast to γ-irradiation, the formation of small DNA fragments by electrons did not seem to be related to the production of the linear form of the plasmid. Conclusions : Electrons within the energy range of the secondary electrons generated by high-energy ionizing radiation induce single, double and multiple double-strand breaks in DNA. Problems associated with low-energy electron irradiation experiments and dose calculations in thin films are also discussed.     

Distribution of radiation‐induced exchange aberrations in all human chromosomes

Purpose: To investigate the DNA‐proportional distribution of radiation‐induced chromosome aberrations for all chromosomes of a male and a female human karyotype.

Materials and methods: Metaphases were prepared from whole blood cultures obtained from two healthy donors and set up after irradiation with 3?Gy 220?kV X‐rays. Single whole‐chromosome FISH painting simultaneously with pancentromeric DNA painting were performed separately for each chromosome of the human karyotype. One thousand exclusively first‐division metaphases were analysed per chromosome and donor. After statistical analysis, the data obtained were compared with theoretically expected values.

Results: All aberration types (translocations, dicentrics) showed deviations from a DNA‐proportional distribution. For both donors, chromosomes 2 and 3 exhibited significantly less and chromosome 4 more symmetrical translocations than expected. Chromosomes 15 and 22 showed more symmetrical translocations than predicted for one of the two donors. Less dicentrics than expected became apparent for chromosomes 2, 3 and 18, while more dicentrics were seen for chromosomes 15, 16 and 17. Moreover, chromosomes 4, 14 and 22 showed a significant deviation from the theoretically expected 1:1 ratio of the yields of symmetrical translocations to the yields of dicentrics.

Conclusion: The results from the whole‐chromosome complement in two different donors confirmed published data from the analysis of single chromosomes that some human chromosomes were not involved in radiation‐induced dicentrics and symmetrical translocations proportional to their DNA content. This must be taken into account if chromosome subsets for dose reconstruction are selected or if whole genomic frequencies have to be calculated from partial genome analysis.     

Sensitive determination of midazolam and propofol in human plasma by GC–MS/MS

Forensic Toxicology - A sensitive method was developed for the simultaneous determination of midazolam and propofol in human plasma samples via modified quick, easy, cheap, effective, rugged, and...     

Dose–response effects of customised foot orthoses on lower limb muscle activity and plantar pressures in pronated foot type

Customised foot orthoses (FOs) featuring extrinsic rearfoot posting are commonly prescribed for individuals with a symptomatic pronated foot type. By altering the angle of the posting it is purported that a controlled dose–response effect during the stance phase of gait can be achieved, however these biomechanical changes have yet to be characterised. Customised FOs were administered to participant groups with symptomatic pronated foot types and asymptomatic normal foot types. The electromyographic (EMG) and plantar pressure effects of varying the dose were measured. Dose was varied by changing the angle of posting from 6° lateral to 10° medial in 2° steps on customised devices produced using computer aided orthoses design software. No effects due to posting level were found for EMG variables. Significant group effects were seen with customised FOs reducing above knee muscle activity in pronated foot types compared to normal foot types (biceps femoris p = 0.022; vastus lateralis p < 0.001; vastus medialis p = 0.001). Interaction effects were seen for gastrocnemius medialis and soleus. Significant linear effects of posting level were seen for plantar pressure at the lateral rearfoot (p = 0.001), midfoot (p < 0.001) and lateral forefoot (p = 0.002). A group effect was also seen for plantar pressure at the medial heel (p = 0.009). This study provides evidence that a customised FOs can provide a dose response effect for selected plantar pressure variables, but no such effect could be identified for muscle activity. Foot type may play an important role in the effect of customised orthoses on activity of muscles above the knee.     

Induction and disappearance of G2 chromatid breaks in lymphocytes after low doses of low-LET γ -rays and high-LET fast neutrons

Purpose : To determine by means of the G2 assay the number of chromatid breaks induced by low-LET γ-rays and high-LET neutrons, and to compare the kinetics of chromatid break rejoining for radiations of different quality. Materials and methods : The G2 assay was performed on blood samples of four healthy donors who were irradiated with low-LET γ-rays and high-LET neutrons. In a first set of experiments a dose-response curve for the formation of chromatid breaks was carried out for γ-rays and neutrons with doses ranging between 0.1 and 0.5 Gy. In a second set of experiments, the kinetics of chromatid break formation and disappearance were investigated after a dose of 0.5 Gy using post-irradiation times ranging between 0.5 and 3.5 h. For the highest dose of 0.5 Gy, the number of isochromatid breaks was also scored. Results : No significant differences in the number of chromatid breaks were observed between low-LET γ-rays and high-LET neutrons for the four donors at any of the doses given. The dose-response curves for the formation of chromatid breaks are linear for both radiation qualities and RBEs = 1 were obtained. Scoring of isochromatid breaks at the highest dose of 0.5 Gy revealed that high-LET neutrons were, however, more effective at inducing isochromatid breaks (RBE = 6.2). The rejoining experiments further showed that the kinetics of disappearance of chromatid breaks following irradiation with low-LET γ-rays or high-LET neutrons were not significantly different. Half-times of 0.92 h for γ-rays and 0.84 h for neutrons were obtained. Conclusions : Applying the G2 assay, the results demonstrate that at low doses of irradiation, the induction as well as the disappearance of chromatid breaks is independent of the LET of the radiation qualities used (0.24 keV μ m -1 60 Co γ-rays and 20 keV μ m -1 fast neutrons). As these radiation qualities produce the same initial number of double-strand breaks, the results support the signal model that proposes that chromatid breaks are the result of an exchange process which is triggered by a single double-strand break.     

Lovastatin causes sensitization of HeLa cells to ionizing radiation‐induced apoptosis by the abrogation of G2 blockage

Purpose: To investigate the effect of inhibition of Ras/Rho‐regulated signalling by 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors (statins) on radiation‐induced cell killing and apoptosis.

Materials and methods: Different human cell lines were pretreated or not with lovastatin before exposure to γ‐rays. Afterwards, radiation‐induced cell killing, formation and repair of double‐strand breaks, activation of radiation‐inducible signal mechanisms (i.e. p53, p21, extracellular‐signal‐related kinase (ERK), NF‐κB), changes in cell cycle progression and apoptosis were analysed.

Results: As shown by a colony formation assay, lovastatin sensitized HeLa cells to γ‐radiation‐induced cell killing. The lovastatin effect was cell‐type specific. Neither the level of γ‐ray‐induced double‐strand breaks nor its repair were affected by lovastatin. Sensitization was independent of p53/p21^Waf1‐ and NF‐κB‐related mechanisms. Radiation‐stimulated activation of ERKs was attenuated by lovastatin. Cell cycle analyses revealed that the level of γ‐ray‐induced G2 blockage was not affected by lovastatin. However, as analysed up to 72?h after irradiation, lovastatin pretreated cells showed an accelerated abrogation of G2 blockage as compared with the control. G2 abrogation is paralleled by an increase in the frequency of apoptotic and necrotic cells.

Conclusions: The data show that lovastatin can render human cells more sensitive to the cytotoxic effect of γ‐rays. This is related to abrogation of G2 blockage and a concomitant increase in apoptotic/necrotic cell death.     

Catalytic inhibition of topoisomerase II in Werner‚s syndrome cell lines enhances chromosomal damage induced by X-rays in the G2 phase of the cell cycle

Purpose : To investigate whether catalytic topoisomerase II activity by ICRF187, a compound that interferes with the catalytic cycle of topoisomerase II without causing DNA damage, could result in a modulation of X-ray-induced chromosomal damage in Werner's syndrome (WS) cell lines. Materials and methods : Two WS (KO375, DJG) and one normal lymphoblastoid cell line (SNW646) were exposed to X-rays, post-treated with ICRF187 and harvested after various recovery times. Cell progression to mitosis was monitored by 5-bromo-2'-deoxyuridine (BrdUrd) and fluorescent immmunodetection to analyse chromosomal damage in homogeneous treated cell populations in the G1, S or G2 phase of the cell cycle. Results : In WS cell lines, catalytic inhibition of topoisomerase II activity by ICRF187 resulted in potentiation of X-ray-induced chromosomal damage in the G2 phase of the cell cycle. This potentiation was not observed in the G1 or S phases of the cell cycle, neither in WS nor normal cells. Conclusion : These results point out the possibility that Werner's syndrome protein (WRNp) might play a role in a G2 recombinational pathway of double-strand break repair, cooperating with topoisomerase II and thus contributing to maintain genomic integrity.     

Studies on mechanism of treatment of granulocyte colony-stimulating factor,recombinant human interleukin-11 and recombinant human interleukin-2 on hematopoietic injuries induced by 4.5 Gy γ-rays irradiation in beagles

Objective To investigate the mechanism of treatment of granulocyte colony-stimulating factor(rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2 (rhIL-2)on hematopoietic injuries induced by 4.5 Gy60 Coγ-ray irradiation in beagles,and to provide experimental evidence for the clinical treatment of extremely severe myeloid acute radiation sickness (ARS).Methods Sixteen beagle dogs were given 4.5 Gy60 Co γ-ray total body irradiation(TBI),then randomly assigned into irradiation control group,supportive care group or cytokines+supportive care (abbreviated as cytokines)group.In addition to supportive care,rhG-CSF,rhlL-11 and rhIL-2 were administered subcutaneously to treat dogs in cytokines group.The percentage of CD34+cells,cell cycle and apoptosis of nucleated cells in peripheral blood were examined by Flow cytometry.Results After 4.5 Gy 60 Co γ-ray irradiation,the CD34+cells in peripheral blood declined obviously(61.3%and 52.1% of baseline for irradiation control and supportive care group separately).The cell proportion of nucleated cells in Go/G1 phase was increased notably(99.27% and 99.49% respectively).The rate of apoptosis(26.93% and 21.29% separately)and necrosis(3.27% and 4.14%,respectively)of nucleated cells were elevated significantly when compared with values before irradiation(P<0.05) 1 d post irradiation.When beagles were treated with cytokines and supportive care,the CD34+cells in peripheral blood were markedly increased(135.6% of baseline).The effect of G0/G1 phase blockage of nucleated cells became more serious(99.71%).The rate of apoptosis(5.66%)and necrosis(1.60%)of nucleated cells were significantly lower than that of irradiation control and supportive care groups 1 d after exposure.Conclusions Cytokines maybe mobilize CD34+cells in bone marrow to peripheral blood,indce cell cycle block at G0/G1 phase and reduce apoptosis,and eventually cure hematopoieticinjuries induced by irradiation.     

Studies on mechanism of treatment of granulocyte colony-stimulating factor,recombinant human interleukin-11 and recombinant human interleukin-2 on hematopoietic injuries induced by 4.5 Gy γ-rays irradiation in beagles

Objective To investigate the mechanism of treatment of granulocyte colony-stimulating factor(rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2 (rhIL-2)on hematopoietic injuries induced by 4.5 Gy60 Coγ-ray irradiation in beagles,and to provide experimental evidence for the clinical treatment of extremely severe myeloid acute radiation sickness (ARS).Methods Sixteen beagle dogs were given 4.5 Gy60 Co γ-ray total body irradiation(TBI),then randomly assigned into irradiation control group,supportive care group or cytokines+supportive care (abbreviated as cytokines)group.In addition to supportive care,rhG-CSF,rhlL-11 and rhIL-2 were administered subcutaneously to treat dogs in cytokines group.The percentage of CD34+cells,cell cycle and apoptosis of nucleated cells in peripheral blood were examined by Flow cytometry.Results After 4.5 Gy 60 Co γ-ray irradiation,the CD34+cells in peripheral blood declined obviously(61.3%and 52.1% of baseline for irradiation control and supportive care group separately).The cell proportion of nucleated cells in Go/G1 phase was increased notably(99.27% and 99.49% respectively).The rate of apoptosis(26.93% and 21.29% separately)and necrosis(3.27% and 4.14%,respectively)of nucleated cells were elevated significantly when compared with values before irradiation(P<0.05) 1 d post irradiation.When beagles were treated with cytokines and supportive care,the CD34+cells in peripheral blood were markedly increased(135.6% of baseline).The effect of G0/G1 phase blockage of nucleated cells became more serious(99.71%).The rate of apoptosis(5.66%)and necrosis(1.60%)of nucleated cells were significantly lower than that of irradiation control and supportive care groups 1 d after exposure.Conclusions Cytokines maybe mobilize CD34+cells in bone marrow to peripheral blood,indce cell cycle block at G0/G1 phase and reduce apoptosis,and eventually cure hematopoieticinjuries induced by irradiation.     

Chiral analysis of dextromethorphan and levomethorphan in human hair by liquid chromatography–tandem mass spectrometry

Forensic Toxicology - Methorphan exists in two enantiomeric forms including dextromethorphan and levomethorphan. Dextromethorphan is an over-the-counter antitussive drug, whereas levomethorphan is...