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.

Hepatocytes of double-transgenic mice expressing high levels of hepatitis B virus e antigen and interferon-gamma are not injured by HBeAg specific autoantibodies.

Seroconversion from HBeAg to alphaHBe of persons chronically infected by HBV is usually associated with a transient exacerbation of liver disease and subsequent normalization of liver histology. It is speculated that these clinico-pathological features may be due to the activation of cytodestructive mechanisms by alphaHBe antibodies. The aim of the present study was to investigate the pathogenic potential of alphaHBe antibodies in a transgenic mouse model. Therefore, alphaHBe autoantibodies were elicited in double-transgenic mice expressing high amounts of HBeAg and interferon-gamma in the liver. Interferon-gamma has reviously been shown to play an important role in the development of hepatic necroinflammation associated with hepadnaviral infection, probably via tumor-necrosis-factor-alpha secreted by activated macrophages. We found no evidence that alphaHBe antibodies have the potential to destroy HBeAg-secreting hepatocytes even if the cells were predisposed to injury due to high-level interferon-gamma expression. We conclude that seroconversion from HBeAg to alphaHBe of persons chronically infected with HBV seems to be an immunological epiphenomenon without pathogenic significance.

H-2(d) mice born to and reared by HBeAg-transgenic mothers do not develop T cell tolerance toward the hepatitis B virus core gene products.

The function of the secretory core gene product (HBeAg) of the human hepatitis B virus (HBV) is unknown. It has been proposed that this protein may be passed from the mother to her offspring at the perinatal stage where it might induce immune tolerance. In a previous study we have shown that the murine placenta presents an efficient barrier for the HBe protein and that H-2(b) mice born to HBeAg-positive transgenic mothers do not develop tolerance of specific cytotoxic T cells. In the present work we demonstrate that transgenic mice expressing high serum levels of HBeAg secrete only small amounts of this protein into their milk and excrete minute amounts of the viral gene product in their urine. Furthermore, it is shown that nontransgenic H-2(d) mice born to and reared by HBeAg-positive mothers exhibit a reactivity of HBc/eAg-specific CD4(+) Th cells and CD8(+) cytotoxic T cells comparable to that of normal isogenic control mice. In accordance with this observation the humoral immune responses directed against the HBeAg were comparable between these two groups of animals. This finding indicates that H-2(d) mice potentially exposed to small amounts of maternal HBeAg transferred by the transplacental, lactogenic, or renal route do not develop tolerance toward the HBV core gene products. These data challenge the hypothesis that a potential function of the HBeAg may be to operate as a tolerogen at the perinatal developmental stage.

Multicolour FISH painting for the analysis of chromosomal aberrations induced by 220 kV X-rays and fission neutrons.

PURPOSE: The quantification of radiation-induced chromosome aberrations identified by multicoloured FISH painting and classified according to different nomenclature systems (PAINT, S&S and a conventional method). MATERIAL AND METHODS: Blood samples were irradiated with five different doses of 220 kV X-rays or fission neutrons respectively. Cell cycle-controlled, multicoloured FISH painting was performed with a cocktail of chromosomes 1, 4, 12 and a pancentromeric probe. RESULTS: Ten aberration parameters or categories were selected according to the three nomenclature systems and dose-response curves were constructed for the observed yields. Fitted coefficients of the linear-quadratic dose-response function show the relative importance of the quadratic term for aberration parameters of the low-LET radiation (X-rays) data set and of the linear term for those of the high-LET radiation (fission neutrons) data set. The relative proportion of complex aberrations observed was larger for the densely ionizing fission neutrons than for sparsely ionizing X-rays. CONCLUSION: Compared with single-colour FISH painting, a multicolour approach provides extended information for a mechanistic and quantitative interpretation of radiation-induced chromosome aberrations. The choice of a nomenclature system and the selection of an appropriate aberration parameter or category depend on these specific aspects. Practical application requires a rapid and reproducible description of the observed painting patterns and should also throw light on the origin of aberrations.

DNA-proportional distribution of radiation-induced chromosome aberrations analysed by fluorescence in situ hybridization painting of all chromosomes of a human female karyotype.

PURPOSE: This is the extension of a previous study, showing deviations from a DNA-proportional involvement of 12 single chromosomes (1-4, 6-10, 12, 14 and X) in radiation-induced translocations and dicentrics measured by FISH-painting and classified by standard cytogenetic scoring criteria. By adding data on chromosomes 2, 4, 5, 9, 11-13, 15-22 and X the analysis now comprises all chromosomes of a human female karyotype evaluated with three nomenclature systems (PAINT, S & S and a conventional method). MATERIAL AND METHODS: Metaphase spreads were prepared from lymphocytes irradiated with 3 Gy 220 kV X-rays. FISH painting was performed with single chromosome-specific probes in combination with a pancentromeric probe. RESULTS: Deviations from a DNA-proportional distribution became apparent for all aberration parameters analysed with the three nomenclature systems. Chromosomes 2, 3 and 6 were less frequently involved and chromosomes 16, 17 and 20 were more frequently involved in exchange aberrations. Generally, smaller chromosomes (15-22, with the exception of chromosome 19) were more frequently involved in aberration formation than expected. CONCLUSION: The assumption that the probability of a chromosome being involved in an exchange aberration is proportional to its DNA content is not supported by the present data.

FISH-based analysis of radiation-induced chromosomal aberrations using different nomenclature systems.

PURPOSE: Comparison of three nomenclature systems for the description of chromosomal aberrations involving painted chromosomes (PAINT, S&S and a conventional method) by parallel application to one data set. MATERIALS AND METHODS: Radiation-induced (3 Gy 220 kV X-rays) chromosome aberrations in human lymphocytes were analysed by FISH-painting of seven single chromosomes (1, 3, 6, 7, 8, 10 and 14) simultaneously with a pancentromeric probe. RESULTS: Each system is based on different prerequisites and uses different criteria for the classification and quantification of structural chromosome aberrations. Due to the frequent occurrence of complex exchanges (resulting from > or = 3 breaks on > or = 2 chromosomes), standard cytogenetic scoring criteria used for solid-stained preparations are inadequate for a precise and reproducible classification of aberrant painting patterns. S&S is particularly suitable if a mechanistic interpretation of aberration origins is required. The descriptive terminology of PAINT enables a rapid, reproducible description, even of most extensively rearranged chromosomes by classifying each abnormal painting pattern individually. CONCLUSION: A modification of PAINT criteria, allowing also for mechanistic aspects, would be most advantageous for practical application.

Chromosome analysis by fluorescence in situ hybridization: further indications for a non-DNA-proportional involvement of single chromosomes in radiation-induced structural aberrations.

The frequencies of symmetrical complete and incomplete translocations and dicentrics induced in human lymphocytes after in vitro irradiation with 3Gy X-rays were analysed by the use of fluorescence in situ hybridization (FISH). Single whole chromosome painting (WCP) probes, specific for chromosomes 1-4, 6-10, 12, 14 and X were hybridized separately. A human pancentromeric DNA-probe was used simultaneously for unequivocal centromere detection. For both aberration types, symmetrical translocations and dicentrics, a significant deviation from a DNA-proportional distribution was found. In general, chromosomes with a higher DNA content (chromosomes 1-3, 6 and 7) were less frequently involved in the formation of symmetrical translocations and dicentrics than expected according to their DNA-content, whereas smaller chromosomes were more frequently involved. The only exception was chromosome 4, exhibiting the highest translocation frequency of all chromosomes analysed. Ratios of the yields of symmetrical translocations to the yields of dicentrics varied between 0.9 and 1.8 for the single chromosomes. The present results substantiate our previous data obtained with identical chromosomes but examined in four different triple combinations.

Combined FISH painting and harlequin staining for cell cycle-controlled chromosome analysis in human lymphocytes.

We present a simple method that allows scoring of FISH-painted chromosomes exclusively in the first division human lymphocytes. It consists of a combination of FISH with chromosome-specific libraries and a differential sister chromatid staining after BrdU treatment of lymphocyte cultures. The method allows a precise quantification of induced chromosome damage for human biodosimetry.

Analysis for DNA-proportional distribution of radiation-induced chromosome aberrations in various triple combinations of human chromosomes using fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with five cocktails of composite whole chromosome-specific DNA probes 1, 4, 12; 2, 7, 9; 2, 7, 9dig; 3, 6, 10dig and 8, 14 Xdig and a degenerate alpha-satellite pancentromeric DNA probe was used to examine in vitro radiation-induced symmetrical translocations and dicentrics in peripheral lymphocytes for a DNA-proportional distribution. For a discrimination between morphologically similar target chromosomes, chromosomes 9, 10 and X were labelled with digoxigenin (dig). Among the five combinations, significantly higher translocation frequencies than expected from the DNA content were found in 8, 14, Xdig, whereas for this combination no deviation became apparent for dicentrics. The chromosome-specific analysis showed that chromosome 2 was involved in fewer symmetrical translocations, whereas chromosomes 9, 10 and 12 were more frequently involved in dicentrics than predicted. Comparing the ratios of symmetrical translocations to dicentrics, an excess of symmetrical translocations was found for the combinations 1, 4, 12; 2, 7, 9dig and 8, 14, Xdig and for the chromosomes 1, 4, 6, 7, 8 and X. Provided the present data can be confirmed in further experiments, the formula Y = 2.05fi(1--fi)FG, relating the translocation or dicentric frequency measured by FISH to the respective genomic FG (fi is the labelled genomic fraction) cannot be used to scale up to equal the full genome unless appropriate weighting factors are included.