Excitatory cholinergic responses in mouse primary bronchial smooth muscle require both Ca2+ entry via l-type Ca2+ channels and store operated Ca2+ entry via Orai channels.

Malfunctions in airway smooth muscle Ca2+-signalling leads to airway hyperresponsiveness in asthma and chronic obstructive pulmonary disease. Ca2+-release from intracellular stores is important in mediating agonist-induced contractions, but the role of influx via l-type Ca2+ channels is controversial. We re-examined roles of the sarcoplasmic reticulum Ca2+ store, refilling of this store via store-operated Ca2+ entry (SOCE) and l-type Ca2+ channel pathways on carbachol (CCh, 0.1-10 µM)-induced contractions of mouse bronchial rings and intracellular Ca2+ signals of mouse bronchial myocytes. In tension experiments, the ryanodine receptor (RyR) blocker dantrolene (100 µM) reduced CCh-responses at all concentrations, with greater effects on sustained rather than initial components of contraction. 2-Aminoethoxydiphenyl borate (2-APB, 100 µM), in the presence of dantrolene, abolished CCh-responses, suggesting the sarcoplasmic reticulum Ca2+ store is essential for contraction. The SOCE blocker GSK-7975A (10 µM) reduced CCh-contractions, with greater effects at higher (e.g. 3 and 10 µM) CCh concentrations. Nifedipine (1 µM), abolished remaining contractions in GSK-7975A (10 µM). A similar pattern was observed on intracellular Ca2+-responses to 0.3 µM CCh, where GSK-7975A (10 µM) substantially reduced Ca2+ transients induced by CCh, and nifedipine (1 µM) abolished remaining responses. When nifedipine (1 µM) was applied alone it had less effect, reducing tension responses at all CCh concentrations by 25% - 50%, with greater effects at lower (e.g. 0.1 and 0.3 µM) CCh concentrations. When nifedipine (1 µM) was examined on the intracellular Ca2+-response to 0.3 µM CCh, it only modestly reduced Ca2+ signals, while GSK-7975A (10 µM) abolished remaining responses. In conclusion, Ca2+-influx from both SOCE and l-type Ca2+ channels contribute to excitatory cholinergic responses in mouse bronchi. The contribution of l-type Ca2+ channels was especially pronounced at lower doses of CCh, or when SOCE was blocked. This suggests l-type Ca2+ channels might be a potential target for bronchoconstriction under certain circumstances.

Chromosome aberration induction is dependent on the spatial distribution of energy deposition through a cell nucleus.

The importance of the spatial distribution of energy deposition through the nucleus in determining the resultant chromosome rearrangements was investigated using fluorescent in situ hybridisation technique following either uniform or partial irradiation of HF19 human fibroblast cells with low-LET 1.5 keV ultrasoft X-rays. Irradiations were performed with and without a copper irradiation mask with a Poisson distribution of micron-sized holes immediately below the irradiation dish and the results are compared with previous results obtained following exposure to a Poisson distribution of alpha particles. For the same radiation quality, the spatial distribution of energy deposition within the nucleus was found to be important in determining the ultimate biological response, with an increased ratio of complex-to-simple aberrations observed for partial compared to uniform irradiation. Comparisons between low-LET ultrasoft X-rays and high-LET alpha particles indicate that the sub-micron clustering of damage along the alpha particle track is more important than just the total number of double-strand breaks produced.

The role of homologous recombination repair in the formation of chromosome aberrations.

The repair of DNA double strand breaks by homologous recombination can occur by at least two pathways: a Rad51-dependent pathway that is predominantly error free, and a Rad51-independent pathway (single strand annealing, SSA) that is error prone. In theory, chromosome exchanges can result from (mis)repair by either pathway. Both repair pathways will involve a search for homologous sequence, leading to co-localization of chromatin. Genes involved in homologous recombination repair (HRR) have now been successfully knocked out in mice and the role of HRR in the formation of chromosome exchanges, particularly after ionising radiation, is discussed in the light of new evidence.

Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel.

PURPOSE: To investigate within the framework of a multilaboratory study the suitability of FISH chromosome painting to measure so-called stable translocations in peripheral lymphocytes of Mayak nuclear-industrial workers (from the Southern Urals) and their use for retrospective biodosimetry. MATERIALS AND METHODS: Chromosime analyses were carried out from 69 workers who had received protracted occupational radiation exposures (0.012-6.065 Gy) up to approximately 40 years before blood sampling. Twenty-one unexposed people living in the same area were controls. A multicolour FISH-painting protocol with the target chromosomes 1, 4 and 8 simultaneously with a pancentromeric probe was used to score potentially transmissible chromosome-type aberrations (reciprocal translocations 2B and related 'one-way' patterns I-III according to the S&S classification). RESULTS: Individual biodosimetry estimates were obtained in terms of these potentially long-term surviving aberration types based on the linear component of a low dose-rate gamma-ray calibration curve produced using identical staining and scoring protocols. For comparison, the workers personal and total background doses were converted to red bone marrow doses. The estimated doses were mainly lower than would be predicted by the calibration curve, particularly at accumulated higher dose levels. CONCLUSIONS: Owing to the limited life-time of circulating T-lymphocytes, the long-term persistence of translocations in vivo requires the assumption of a clonal repopulation of these naturally senescing cells from the haemopoietic stem cell compartments. Obviously such a replacement cannot be fully achieved, leading to a temporal decline even of the yield of transmissible aberrations types. Assuming further a highly selective capacity of stem cells against any type of chromosomal damage and the fact that one must rely on partial genome findings, the potential of FISH chromosome painting for retrospective dose reconstruction is probably limited to a decade or so after high-level protracted radiation exposure.

Xrcc2 is required for genetic stability, embryonic neurogenesis and viability in mice.

Repair of DNA damage by homologous recombination has only recently been established as an important mechanism in maintaining genetic stability in mammalian cells. The recently cloned Xrcc2 gene is a member of the mammalian Rad51 gene family, thought to be central to homologous recombination repair. To understand its function in mammals, we have disrupted Xrcc2 in mice. No Xrcc2(-/-) animals were found alive, with embryonic lethality occurring from mid-gestation. Xrcc2(-/-) embryos surviving until later stages of embryogenesis commonly showed developmental abnormalities and died at birth. Neonatal lethality, apparently due to respiratory failure, was associated with a high frequency of apoptotic death of post- mitotic neurons in the developing brain, leading to abnormal cortical structure. Embryonic cells showed genetic instability, revealed by a high level of chromosomal aberrations, and were sensitive to gamma-rays. Our findings demonstrate that homologous recombination has an important role in endogenous damage repair in the developing embryo. Xrcc2 disruption identifies a range of defects that arise from malfunction of this repair pathway, and establishes a previously unidentified role for homologous recombination repair in correct neuronal development.

Mammalian recombination-repair genes XRCC2 and XRCC3 promote correct chromosome segregation.

Growth and development are dependent on the faithful duplication of cells. Duplication requires accurate genome replication, the repair of any DNA damage, and the precise segregation of chromosomes at mitosis; molecular checkpoints ensure the proper progression and fidelity of each stage. Loss of any of these highly conserved functions may result in genetic instability and proneness to cancer. Here we show that highly significant increases in chromosome missegregation occur in cell lines lacking the RAD51-like genes XRCC2 and XRCC3. This increased missegregation is associated with fragmentation of the centrosome, a component of the mitotic spindle, and not with loss of the spindle checkpoint. Our results show that unresolved DNA damage triggers this instability, and that XRCC2 and XRCC3 are potential tumour-suppressor genes in mammals.

Absence of delayed chromosomal instability in a normal human fibroblast cell line after 125I iododeoxyuridine.

PURPOSE: To seek the delayed appearance of chromosomal abnormalities in human fibroblasts exposed to the Auger electron emitter 125I. MATERIALS AND METHODS: Normal untransformed human fibroblasts, HF19, were exposed to a concentration of [I125]IUdR, which allowed the survival of 37% of clonogens. Chromosomal analysis using both conventional Giemsa and fluorescence in situ hybridization (FISH) was undertaken on non-clonal bulk cultures from 2 to 39 days after treatment. RESULTS: The data show a declining level of unstable aberrations in the progeny of HF19 fibroblasts exposed to [I125]IUdR, eventually reaching control levels. CONCLUSIONS: The results provide evidence that [125I]IUdR does not induce ongoing chromosomal instability in long-term culture, and gives further support to the use of Auger-electron emitting radionuclides in the treatment and diagnosis of tumours.

Complex chromosome aberrations in peripheral blood lymphocytes as a potential biomarker of exposure to high-LET alpha-particles.

PURPOSE: To determine the induction and transmission, to second and third division cells, of complex chromosome aberrations in peripheral blood lymphocytes after exposure to high-LET alpha-particles in vitro. MATERIALS AND METHODS: Separated peripheral blood lymphocytes collected from four healthy donors were irradiated in vitro with either high-LET alpha-particles (121 keV/microm; 0.5 Gy) or low-LET X-rays (250kV constant potential; 3 Gy). Cells were harvested in first, second and third division post-irradiation and chromosome aberrations observed at each cell division were analysed by combining the techniques of FISH and DAPI/Hoechst 33258 harlequin staining. Whole chromosome probes were used for chromosomes 1, 2 and 5, together with a pan-centromeric probe and the resulting chromosome 'painting' patterns were classified according to the Savage and Simpson (S & S) scheme (Savage and Simpson 1994a, Savage and Tucker 1996). RESULTS: A greater proportion of complex chromosome aberrations was observed, defined as involving three or more breaks in two or more chromosomes, relative to total exchanges, after exposure to 0.5 Gy alpha-particles (mean 1 track/cell) than after the high reference dose of 3 Gy X-rays (49-56% and 20-22%, respectively). Qualitatively, alpha-particles induced chromosome aberrations of far greater complexity than those observed after X-rays. This was reflected by both the rapid reduction in the percentage of damaged cells between first and second division indicative of cell death, and the spectrum of aberration types observed in second and third division cells post-irradiation. Regardless of this complexity, 15% of the complexes induced by alpha-particles at first division were potentially transmissible and by third division, transmissible-type complexes, specifically insertions, represented the predominant complex type (65%). CONCLUSION: Transmissible-type complexes were observed, specifically insertions, in both second and third division cells after exposure to high-LET alpha-particles (0.5 Gy) in vitro. The authors predict these cells to be stable and to be capable of persisting through many cell generations. Considering that the induction of complex chromosome aberrations by low-LET radiation is strongly dependent on dose, so that they are expected to be undetectable at environmental exposures, insertions are much more likely to be a characteristic feature of high-LET radiation at all doses. From this the usefulness of insertions as biomarkers of past exposure to environmentally relevant doses of high-LET alpha-particles is supported.

Effectiveness of 0.28 keV carbon K ultrasoft X-rays at producing simple and complex chromosome exchanges in human fibroblasts in vitro detected using FISH.

PURPOSE: To study the effects of carbon K ultrasoft X-rays, which produce a single photoelectron with a track length of < 7 nm, on the production of structural chromosome-type changes. MATERIALS AND METHODS: Untransformed human fibroblasts (HF12) were irradiated in G1 phase. Aberrations were analysed using fluorescence in situ hybridization using multi-coloured chromosome specific DNA probes for chromosomes 1 and 2 and an alpha-satellite pan-centromeric probe. RESULTS: CK X-rays have a high efficiency per unit absorbed dose for producing simple and complex exchanges. Mean absorbed doses of 0.33-1.31 Gy produce simple exchanges with a predominantly linear dose dependency, and visibly complex exchanges increased by more than the power 2 of the dose, with no evidence of a linear component. The proportion of exchanges that are visibly complex ranged from 9% to 46%. CONCLUSIONS: The linear response for simple exchanges provides further support to the hypothesis that damaged DNA may be able to interact with undamaged DNA. The high proportion of complex exchanges may be due to the increased efficiency of double-strand break induction and to the high density of tracks per unit absorbed dose targeting pre-existing sites, some of which may be close to the incident nuclear membrane.

Computer simulation of data on chromosome aberrations produced by X rays or alpha particles and detected by fluorescence in situ hybridization.

With fluorescence in situ hybridization (FISH), many different categories of chromosome aberrations can be recognized-dicentrics, translocations, rings and various complex aberrations such as insertions or three-way interchanges. Relative frequencies for the various aberration categories indicate mechanisms of radiation-induced damage and reflect radiation quality. Data obtained with FISH support a proximity version of the classic random breakage-and-reunion model for the formation of aberrations. A Monte Carlo computer implementation of the model, called the CAS (chromosome aberration simulator), is generalized here to high linear energy transfer (LET) and compared to published data for human cells irradiated with X rays or 238Pu alpha particles. For each kind of radiation, the CAS has two adjustable parameters: the number of interaction sites per cell nucleus and the number of reactive double-strand breaks (DSBs) per gray. Aberration frequencies for various painted chromosomes, of varying lengths, and for 11 different categories of simple or complex aberrations were simulated and compared to the data. The optimal number of interaction sites was found to be approximately 13 for X irradiation and approximately 25 for alpha-particle irradiation. The relative biological effectiveness (RBE) of alpha particles for the induction of reactive DSBs (which are a minority of all DSBs) was found to be approximately 4. The two-parameter CAS model adequately matches data for many different categories of aberrations. It can use data obtained with FISH for any one painting pattern to predict results for any other kind of painting pattern or whole-genome staining, and to estimate a suggested overall numerical damage indicator for chromosome aberration studies, the total misrejoining number.

Analysis of lymphocytes from uranium mineworkers in Namibia for chromosomal damage using fluorescence in situ hybridization (FISH).

Workers in the open pit uranium mine in Namibia appear to suffer from health problems including malignant diseases at a much higher prevalence when compared with the general population. The objective of the present study was to determine whether long-term exposure to low-dose uranium increases the risk of biological radiation damage which could lead to malignant diseases. In order to investigate this risk, we measured the relative frequency of chromosome alterations using Fluorescence in situ hybridization (FISH). A representative cohort of 11 non-smoking miners, were compared to a control group of 9 individuals with no occupational history in mining. We determined a significant increase in chromosome aberrations in the circulating lymphocytes of miners versus the non-smoking controls (p = 0.0000096). Therefore, we concluded that these uranium exposed miners are at an increased risk to acquire genetic damage, which may be associated with an increased risk for malignant transformation.

Ultrasoft 1.5 keV aluminum K X rays are efficient producers of complex chromosome exchange aberrations as revealed by fluorescence in situ hybridization.

The electron pairs generated by ultrasoft 1.5 keV aluminum K X-ray photons deposit their energy in tracks of length < 70 nm and provide an ideal tool for analyzing the spatial distribution of breaks and misrepair processes. We have undertaken the analysis of changes in chromosome structure produced by aluminum K X rays in untransformed HF12 human fibroblasts in G1 phase using fluorescence in situ hybridization (FISH). Multicolored chromosome-specific DNA probes for chromosomes 1 and 2 and an alpha-satellite pan-centromeric probe were used to examine in vitro radiation-induced chromosome-type exchange aberrations. After mean doses of 0.37-2.93 Gy the relative frequencies of complex exchanges, derived from three or more breaks in two or more chromosomes, ranged from 15-35%. For the classic break-age-and-rejoining theory to hold, very large interaction distances are needed to account for this high frequency of multibreak interactions, unless many sites pre-exist where several different chromosomes come very close together. Alternatively, damaged DNA may be able to interact with adjacent undamaged DNA, obviating the need for large rejoining distances.

Frequencies of complex chromosome exchange aberrations induced by 238Pu alpha-particles and detected by fluorescence in situ hybridization using single chromosome-specific probes.

We undertook an analysis of chromosome-type exchange aberrations induced by alpha-particles using fluorescence in situ hybridization (FISH) with whole chromosome-specific probes for human chromosomes 1 or 4, together with a pan-centromeric probe. Contact-inhibited primary human fibroblasts (in G1) were irradiated with 0.41-1.00 Gy 238Pu alpha-particles and aberrations were analysed at the next mitosis following a single chromosome paint. Exchange and aberration painting patterns were classified according to Savage and Simpson (1994a). Of exchange aberrations, 38-47% were found to be complex derived, i.e. resulting from three or more breaks in two or more chromosomes, and the variation with dose was minimal. The class of complex aberrations most frequently observed were insertions, derived from a minimum of three breaks in two chromosomes. There was also an elevated frequency of rings. The high level of complex aberrations observed after alpha-particle irradiation indicates that, when chromosome domains are traversed by high linear energy transfer alpha-particle tracks, there is an enhanced probability of production of multiple localized double-strand breaks leading to more complicated interactions.

Chromatid damage induced by 238Pu alpha-particles in G2 and S phase Chinese hamster V79 cells.

The comparative induction of chromatid aberrations by 238Pu alpha-particles, or by 250 kVp X-rays was investigated in V79 Chinese hamster cells. Metaphases were sampled at hourly intervals postirradiation up to 8 h and BrdU/FPG staining methods were used to distinguish G2, S and G1 phase cells. Two experiments were performed. In the first, an alpha-particle dose of 0.41 Gy was compared with an X-ray dose of 1.5 Gy used in a previously published study. In the second, an X-ray dose of 1.2 Gy was used in parallel with 0.41 Gy of alpha-particles to produce a similar overall frequency of interchanges, and allow comparative ratios to be derived for other aberration types. At these isoexchange doses, alpha-particles produce relatively less gaps and breaks, particularly in late G2, and significantly more isochromatid deletions. A very high proportion of the isochromatid deletions were incomplete after alpha-particles compared with X-rays, but no difference in incompleteness was found for interchanges. With X-rays, about 6% of interchanges are complex intra-interchange forms. At similar exchange frequencies this increases to 26.7% for alpha-irradiation, suggesting increased multiple lesion interaction. Differences in dose distribution between alpha-particles and X-rays are discussed and mitotic delay is examined after separation of the analysed cells into damaged and undamaged classes.

Aberrant expression of the gene for lens major intrinsic protein in the CAT mouse.

Immunocytochemistry fails to detect expression of the lens major intrinsic protein (MIP) in 16-day embryos of the congenitally cataractous mouse, CAT, which inherits a dominant mutation assigned to the distal end of mouse chromosome 10. In situ hybridisation, however, detects MIP mRNA in CAT embryo lens fibre cells at a level approximating 60% of that detected in embryonic lens fibres of the non-cataractous MF1 mouse. Northern blot hybridisation reveals that the most abundant MIP mRNA transcript in the adult CAT lens is truncated when compared to that in the adult MF1 lens. The results are consistent with a cataractogenic mutation in the mouse gene for MIP (Mip) which has also been mapped to the distal end of mouse chromosome 10.

Localisation of the gene for the major intrinsic protein of eye-lens-fibre cell membranes to mouse chromosome 10 by in situ hybridisation.

The gene encoding the major intrinsic protein (Mip) of eye-lens-fibre cell membranes has been assigned to region D1 of mouse Chromosome 10 by in situ hybridisation of a cDNA for rat MIP to G-banded metaphase chromosomes. The mouse Mip gene maps within or near to a segment homoeologous with human chromosome 12q and may be linked to the Cat locus at the distal end of mouse Chromosome 10.

In situ hybridisation localises the gene for the major intrinsic protein of eye lens fibre cell membranes to human chromosome 12q14.

The gene encoding the major intrinsic protein (MIP) of eye lens fibre cell membranes has been localised to human chromosome 12q14 by in situ hybridisation of a cDNA for rat MIP to G-banded metaphase chromosomes. The human MIP gene maps within a conserved region of synteny with mouse Chromosome 10.

Localisation of the beta crystallin Cryb gene to mouse Chromosome 2B-->C1 by in situ hybridisation.

The beta crystallin gene Cryb (betaA3/A1) has been assigned to mouse Chromosome 2 region B-->Cl by in situ hybridisation to metaphase chromosomes from mouse foetal liver and bone marrow preparations of Rb(2.17)4H mice using a murine cDNA (pMbeta23Crl) probe.

Immunochemical comparison of the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts.

Expression of the major intrinsic protein (MIP) of eye-lens fibre cell membranes was compared in normal (DBA), cataractous (CAT, LOP, NCT) and chimaeric (CBA-LOP) mice at different stages of development using immunofluorescence microscopy and immunoblotting techniques. MIP of apparent molecular mass 26 kDa was detected in extracts of adult DBA, LOP and CBA-LOP lenses, but only low molecular mass (less than 26 kDa) immunoreactive proteins were detected in similar extracts from adult CAT and NCT lenses. The corresponding MIP distribution patterns confirmed the highly organised fibre-cell histology in embryonic DBA and adult CBA-LOP lenses and also highlighted the severe fibre-cell degeneration in the LOP lens. In contrast, however, no immunoreactive MIP was detected in situ in embryonic CAT and NCT lenses. These results suggest that a structural alteration of MIP occurs during embryonic lens development in the cataractous CAT (dominant) and NCT (recessive) mutant mice.