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Biomedical subjects

W U Müller

Publications and source records attributed to W U Müller.

At least 19 recordsLinked to original sources
PubMed2000

Chromosomal aberrations induced in mice bone marrow by treating with cisplatin and irradiation.

BACKGROUND: The aim of this study was to quantify the combined effect of cisplatin and radiation on chromosomal damage with emphasis on the time interval between cisplatin and radiation. METHODS AND MATERIALS: Bone marrow of female NMRI-nu(+) mice was taken as a model system for a highly proliferative tissue irradiated with cobalt-60 (1 to 4 Gy). Cisplatin was injected intraperitoneally (i.p.) at 1.1 to 36 mg/kg. Cisplatin was given at various time intervals before and after radiation. Bone marrow and metaphases were prepared according to standard procedures. RESULTS: The percentage of aberrant metaphases after radiation or cisplatin alone increased in a dose-dependent manner (sigmoidal dose-response curve). Combining both modalities led to additive values at all time points for the percentage of aberrant metaphases. Borderline significant (p < 0.05) supraadditive effects were found 2 hours before or 1 hour after irradiation. However, a supraadditive percentage of aberrant chromosomes was found only at 2 or 1.5 hours with cisplatin before irradiation indicating the dependence of supraadditivity on the chosen parameter. CONCLUSION: It is doubtful to expect a true supraadditive or "radiosensitizing" effect, e.g. in the clinical setting from combined treatment with cisplatin and radiation. Rather, cisplatin might act as an independent cytotoxic agent.

Animals↗
PubMed2000

DNA excision repair and DNA damage-induced apoptosis are linked to Poly(ADP-ribosyl)ation but have different requirements for p53.

Poly(ADP-ribose) polymerase (PARP) is a DNA binding zinc finger protein that catalyzes the transfer of ADP-ribose residues from NAD(+) to itself and different chromatin constituents, forming branched ADP-ribose polymers. The enzymatic activity of PARP is induced upon DNA damage and the PARP protein is cleaved during apoptosis, which suggested a role of PARP in DNA repair and DNA damage-induced cell death. We have generated transgenic mice that lack PARP activity in thymocytes owing to the targeted expression of a dominant negative form of PARP. In the presence of single-strand DNA breaks, the absence of PARP activity correlated with a strongly increased rate of apoptosis compared to cells with intact PARP activity. We found that blockage of PARP activity leads to a drastic increase of p53 expression and activity after DNA damage and correlates with an accelerated onset of Bax expression. DNA repair is almost completely blocked in PARP-deficient thymocytes regardless of p53 status. We found the same increased susceptibility to apoptosis in PARP null mice, a similar inhibition of DNA repair kinetics, and the same upregulation of p53 in response to DNA damage. Thus, based on two different experimental in vivo models, we identify a direct, p53-independent, functional connection between poly(ADP-ribosyl)ation and the DNA excision repair machinery. Furthermore, we propose a p53-dependent link between PARP activity and DNA damage-induced cell death.

Animals↗
PubMed1999

Lethal and teratogenic effects in two successive generations of the HLG mouse strain after radiation exposure of zygotes -- association with genomic instability?

We analysed the transmission of lethal and teratogenic events to the subsequent generation in HLG/Zte mice after exposure of the zygote stage to 1 Gy X-rays. As observed in previous studies, our results on teratogenic events occurring in the same generation, which was exposed during the zygote stage, reveal a significantly higher risk for the induction of gastroschisis. Interesting new insights came from the study of lethal and teratogenic effects in the generation obtained after mating female mice, which were exposed during their zygote stage, to unexposed males. An approximately 2-fold higher level of damage was manifest in this generation compared with controls, expressed mainly as a significant increase of prenatal mortality (P<0.01). Although there was an increase in the number of malformed fetuses on day 19 of gestation (6.5% cases of gastroschisis compared to 3.5% in the controls), the frequency of gastroschisis in the exposed group was just not statistically significant (P>0.05). These results are in line with the hypothesis that genomic instability is involved in the damage seen after radiation exposure of the zygote stage of HLG mice.

Animals↗
PubMed1999

Radiation-induced malformations after exposure of murine germ cells in various stages of spermatogenesis.

We studied radiation effects in day 19 fetuses of the mouse strain 'Heiligenberger' after exposure (2.8 Gy, 137 Cs gamma rays, dose rate 0.28 Gy/h) of their fathers. We observed an increased lethality (exclusively due to preimplantation death and early resorptions) after exposure of all stages of spermatogenesis with the exception of early spermatogonia. In addition, there was a significant increase in the frequency of malformed fetuses (gastroschises only); this increase was observed primarily after exposure of the meiotic stages.

Animals↗
PubMed1999

Automated comet assay analysis.

BACKGROUND: Recently the "comet assay" or "single-cell gel electrophoresis assay" has been established as a sensitive method for the detection of DNA damage and repair. Most of the software now available to quantify various parameters for DNA damage requires the interaction of a human observer. In this report, we describe an automated analysis system that is based on self-developed software and hardware and needs minimal human interaction. METHODS: The image analysis is divided into two parts: 1) automatic cell recognition and comet classification and 2) quantification of desired comet parameters. Image preprocessing, segmentation, and feature classification were developed with algorithms based on mathematical morphology. To enhance evaluation speed, we have introduced parallel processing of data under the Windows NT operating system (Microsoft Corporation, Redmond, WA). Use of an analogue real-time autofocus unit (Böcker et al.: Phys Med Biol 1997;42:1981-1992) allows for faster analysis. RESULTS: Our recognition software shows a sensitivity of 95.2% and a specificity of 92.7% when tested on test samples from routine work with DNA damage by low-dose radiation (0-2 Gy). The parallel hardware and software concept enables us to analyze 100 comets on one slide in less than 15 min. CONCLUSIONS: A comparison of measurements made on the same samples by manual and automated analysis systems revealed that there are no significant differences. The slope of the dose-response curves and the repair kinetics are very similar and demonstrate that automatic comet assay analysis is possible.

Algorithms↗
PubMed1999

Optimization and standardization of the "comet assay" for analyzing the repair of DNA damage in cells.

BACKGROUND: The "comet assay" has become an interesting and a very useful tool for the analysis of the induction and amount of DNA damage in single cells thus offering the opportunity to measure the effectiveness of DNA repair. On the basis of the Ostling and Johanson protocol we have developed a modified method with increased sensitivity and high reproducibility. MATERIAL AND METHODS: Human tumor cells or isolated human peripheral blood lymphocytes were analyzed in the experiments. The amount of DNA damage and the effectiveness of DNA repair was measured after X-irradiation using the "comet assay" technique. RESULTS: In this presentation the influences of different methodological factors like agarose concentration, buffer pH, electrophoresis time, electric field strength on the applicability of the "comet assay" are described in detail and optimum conditions for "comet assay" experiments have been evaluated. Additionally the authors will show a comparison of different fluorescent DNA dyes pointing out their advantages or disadvantages for "comet" analysis. The usefulness of this technique and its capabilities are exemplified by showing DNA repair kinetics of human lymphocytes of different healthy or radiosensitive donors after in-vitro irradiation with 2 Gy X-rays. CONCLUSIONS: This paper presents data on the optimization and standardization of the original "comet assay" leading to an extremely fast and practicable protocol in the field of single cell gel electrophoresis. After irradiation with 0.1 Gy an increase in the amount of DNA damage can be measured with high statistical significance and the DNA repair capacity of individual cells after X-ray doses of 2 Gy can be analyzed with high reproducibility. The results comparing DNA repair capacities of different donors point out that the "comet assay" may have the potential for the estimation of individual radiosensitivity.

Adult↗
PubMed1998

Micronuclei-biological indicator for retrospective dosimetry after exposure to ionizing radiation.

Micronuclei can be measured through a conventional method after staining with Giemsa or fluorescence dyes for DNA. However, a technique with cell proliferation control should be preferred. This is done by incubation with cytochalasin B and counting the micronuclei in binucleated cells. Satisfactory dose relationships are observed after irradiation of human lymphocytes in vitro. The RBE for fast neutrons is around three. An automatic analysis is possible by image analysis. The dose range in which significant increases can be observed is 0.3 to 5 Gy X-rays. The assay becomes more sensitive when the micronuclei are determined only in B-lymphocytes. Another possibility exists by determination of the number of micronuclei with centromeres. For this purpose the hybridization with pancentromeric DNA probes and fluorescence labelling is of advantage. By this technique a radiation dose of 0.1 Gy X-rays can be detected. It is apparently also possible under these conditions to detect radiation exposures which have taken place decades before the measurements.

Cell Division↗
PubMed1998

The sensitivity of the in vitro cytokinesis-blocked micronucleus assay in lymphocytes for different and combined radiation qualities.

PURPOSE: The dose-response relationship and the relative biological effectiveness (RBE) for the induction of micronuclei in lymphocytes was analyzed after irradiation in vitro with a 6-MeV neutron beam that was followed by 240-kV X-rays. The dose range of the combined exposure comprised 1 to 3 Gy. For reference, the dose-effect relationships found after X-ray (0.5 to 5 Gy)- and neutron (0.5 to 4 Gy) exposure applied separately are presented. The possibility of an interaction between the 2 radiation qualities is investigated by the method of isobole calculation termed "envelope of additivity". METHODS: Micronuclei were analyzed in PHA-stimulated, cytokinesis-blocked human lymphocytes. RESULTS: The dose-response relationships for the micronucleus frequencies induced by the neutron irradiation, as well as by the mixed exposure, were linear. A saturation effect was indicated after neutron doses higher than 3 Gy. After low LET exposure the dose-response curves were describable by a linear-quadratic model. For neutron-induced micronucleus frequencies, RBE-values of 2 to 3 and for the combined exposure RBE values of 1.5 to 2 were calculated for a range of effect of 0.5 to 1.5 micronuclei/binucleated lymphocyte. No indication was found for an interaction between the damage induced by X-rays and that produced by neutrons under our experimental conditions. CONCLUSIONS: These studies demonstrate a clear dependence of micronucleus induction on radiation quality and emphasize the usefulness of the micronucleus assay in biological dosimetry, also in cases in which high LET radiation or a mixed beam is involved as the radiation source.

Adult↗
PubMed1997

Image analysis of comet assay measurements.

In the last decade the 'comet assay' or 'single cell gel electrophoresis assay' has been established as a sensitive method for the detection of DNA damage and the measurement of its recovery. The results published in the literature have often been obtained with different methods for comet structure measurement. In most cases these data are not comparable with each other. Even when using similar systems for the analysis, it is difficult to obtain matching data. This presentation will describe some technical aspects of our measurement equipment and evaluation software. It focuses on necessary experimental conditions to minimize errors in obtaining such data. The software developed here allows the rapid analysis of the microscopic samples (< 2 s per image). The image analysis was designed with respect to the morphological shapes of comet cells, which were investigated with a confocal laser microscope. The system is built with standard components which are commercially available. As a measure of the amount of DNA damage the ratio of fluorescence intensity was used inside the comet tail and the fluorescence intensity of the comet head. Other parameters such as DNA content, comet area, head radius, tail length and tail moment are also determined. The reproducibility of the system has been evaluated in several experiments over a period of 5 years.

Ataxia Telangiectasia↗
PubMed1997

A fast autofocus unit for fluorescence microscopy.

In this paper a fast autofocus unit is introduced for fluorescence microscopy based on image content information. The module, an electronic board with several differentiators and integrators, is designed for high-speed autofocusing and is directly coupled to the output of the video camera. A Leitz MPV II fluorescence microscope with x, y, z stepping motors was used as basic equipment. The microscope images were focused by using an intensified target camera with a following analogue-digital converter and a PC. Thus one obtains a focus value for the current image within one video cycle. Furthermore, it is possible to process three different focus functions (weighted intensity, first derivative and second derivative) simultaneously. The flexibility to select a certain focus function or a combination of these functions allows the use of the analogue detector for various cell types and fluorescent dyes. In order to test the experimental set-up we use three different kinds of biological specimen (lymphocytes, fibroblasts and comet cells) which are distinguished by large differences in their morphological structure. Successful focusing is carried out in more than 95% of cases (investigation of several hundred different cells). The focusing procedure is almost finished after 1-2 s.

Algorithms↗
PubMed1997

Detection of chromosome 2 and chromosome 7 within X-ray- or colchicine-induced micronuclei by fluorescence in situ hybridization.

The occurrence of chromosome 2 and chromosome 7 within micronuclei of binucleated lymphocytes induced by X-rays or colchicine was scored using the whole chromosome painting technique. The observed frequency of involvement in micronucleus formation was compared with the yield that would be expected theoretically, when the random participation of each chromosome is assumed. No difference was observed between the expected and observed inclusion of chromosome 2 or chromosome 7 into micronuclei after X-ray exposure (2.5 Gy). This was also the case for chromosome 2, but not for chromosome 7 after colchicine treatment (0.04/0.06 microgram/ml); chromosome 7 was detected approximately 1.5 times more frequently in micronucleus formation than would be expected from the assumption of a random distribution.

Aneuploidy↗
PubMed1996

Genetic analysis of the cause of gastroschisis in the HLG mouse strain.

An inbred mouse strain HLG shows a high incidence of gastroschisis after X-ray exposure to the zygotes. About 11% of the fetuses display this malformation after irradiation with 1 Gy. The C57BL-strain does not show the increased frequency of gastroschisis after radiation-exposure to the zygotes. The genetic background of this malformation was investigated in a backcross of HLG x C57BL females to HLG males. The pregnant HLG x C57BL females were irradiated in a stage in which the (HLG x C57BL) x HLG [BC1] embryos were in the 1-cell stage. The frequency of gastroschisis in the BC1 generation was compared with a genetic model of a single recessive mutation with 11% penetrance. This frequency does not fit a single-locus inheritance. The number of loci involved was estimated to be about two or three. HLG mouse strain may be a valuable animal model in the study of polygenic traits.

Abdominal Muscles↗
PubMed1996

Effects of cell cycle specific exposure to 3H-thymidine or 3H-arginine on development and cell proliferation of mouse embryos.

One-cell mouse embryos were exposed to either 3H-thymidine (100 or 200 kBq/ml) or 3H-arginine (2.5 to 50 kBq/ml) for 2 h either in G1, S or G2 phase. 3H-Arginine affected embryonic development and cell proliferation in an activity-dependent way irrespective of the cell cycle stage exposed, whereas 3H-thymidine was effective only at higher activities and only after exposure during S phase.

Animals↗
PubMed1996

Automated scoring of micronuclei in binucleated human lymphocytes.

Manual and automatic scoring of micronuclei (MN) in binucleated human lymphocytes (BNC) were compared after irradiation of whole blood samples. The blood samples were irradiated with X-ray doses (1, 2 or 3 Gy) and stained with Giemsa. The preparation technique was optimized in such a way that acceptable conditions (cell density, contrast) were obtained for both scoring procedures. To estimate the quality of automatic micronucleus detection, two researchers who had different experience in scoring MN (6 months and 5 years) analysed the samples independently from each other. Automatic scoring was carried out with a digital image analysis system and the recognition procedure was divided into two parts. The BNC positions were detected with low microscope magnification (100x), and the recognition of micronuclei within the cytoplasm of the classified BNC was carried out at high magnification (630x). A fuzzy logic classification system as well as two different segmentation steps (preclassification and postclassification) made it possible that about 94% of all automatically recognized BNC were classified correctly). On the other hand, the classification system was optimized in such a way that false positive decisions were minimized (95% of automatically recognized micronuclei were classified correctly). Failure to recognize micronuclei (8.5%-25% false negatives) was mainly due to extremely small micronuclei, poor contrast with respect to the cytoplasm, and aggregation of micronuclei especially at higher doses.

Automation↗
PubMed1996

Micronuclei in lymphocytes of children from the vicinity of Chernobyl before and after 131I therapy for thyroid cancer.

The present study addresses the monitoring of children from the Belorussian and Ukrainian Republics exposed to the fall-out of the Chernobyl accident. Micronucleus analysis has been performed on 56 children from different areas. The micronucleus frequencies in individuals as well as in regional groups were comparable with controls, except for three donors. Such results had to be expected, taking into account that at least 7 years have passed since the accident. Most of the children whose micronucleus frequencies were determined are suffering from thyroid cancer and were treated by radioiodine (131I) therapy. We studied the effect of in vitro exposure with 131I on micronucleus induction and that proliferative ability of lymphocytes. The present investigation indicates that micronuclei can be usefully employed to detect individual exposures to the incorporated radionuclide within several days after the intake of the radionuclide in a dose range of around 65-390 mGy (effective dose).

Adolescent↗
PubMed1996

Automated cell cycle analysis with fluorescence microscopy and image analysis.

Automatic cell cycle analysis (DNA histograms) is usually performed with flow cytometry. In cases when only few cells are available, the DNA content has to be measured with a fluorescence microscope combined with sensitive camera systems (SIT, MCP, cooled CCDs) and a frame grabber for image analysis. The fluorescent cells are observed on the monitor of the image analyser. The DNA content of specific cells of interest is calculated after an interactive selection via mouse click on the monitor screen. It is desirable to automate this time-consuming procedure by a computer "cell-finding' and "cell-measurement' system, which is comparable in speed and measurement accuracy with manual scoring. A software program, based on image analysis, was designed for the automated cell finding, the cell measurement, and finally the interactive DNA data evaluation. The system has already been used to determine cell cycle stages in which specific manipulations of a lymphocyte culture were required. However, the system is also able to determine, automatically, DNA distributions of different kinds of cell (different leucocytes) within one sample, as long as the automatic recognition software can distinguish between them by morphological differences.

Algorithms↗
PubMed1996

Comet assay studies indicate that caffeine-mediated increase in radiation risk of embryos is due to inhibition of DNA repair.

It is well known that under specific conditions caffeine is able to enhance radiation risk of mammalian cells by a factor of approximately 1.5-2. Various mechanisms are discussed in the literature as possible explanations for this interaction. Inhibition of DNA repair plays a crucial role in the discussion, although direct evidence for this assumption is difficult to obtain. We used the "comet assay' in order to analyse the significance of repair inhibition by caffeine in the two-cell stage of mammalian gestation. Our data show that at the concentration necessary for increasing radiation risk (2 mM), caffeine effectively inhibits the restitution of radiation-damaged DNA.

Animals↗