Biomedical subjects

W U Müller

Publications and source records attributed to W U Müller.

At least 19 recordsLinked to original sources

PubMed1995

Image processing algorithms for the automated micronucleus assay in binucleated human lymphocytes.

The frequency of micronuclei in binucleated lymphocytes (cytochalasin B assay) may serve as a biological dosimeter after radiation exposure. The automation of the micronucleus assay in binucleated human lymphocytes has been considerably advanced in recent years. In our studies for this purpose the detection of binucleated cells (BNCs) and the scoring of micronuclei (MN) was divided into two parts. First, detection of BNCs was feasible with low microscopic magnification (x 100). The positions of classified BNCs were stored. Second, after an automatic change of microscope objective, the stored BNCs were automatically analyzed in sequence at high microscopic magnification (x 630) for occurrence of MN. For both phases of image analysis we used empirical methods based on mathematical morphology. The system is able to recognize nearly 65% of BNCs with false positive decisions of 6% and about 75% of the MN with false positive decisions of 7%.

Algorithms↗

PubMed1995

No indications of an enhanced UV-light-induced unscheduled DNA synthesis in splenocytes of mice following a low-dose irradiation in vivo or in vitro.

One of the open questions regarding the adaptive response to ionizing radiation is whether it can be induced in G0 lymphocytes. In the majority of experiments in which an adaptive response in G0 lymphocytes was observed, the adapting dose was applied in vivo. In order to investigate whether there is some in vivo component of adaptive response, mouse splenocytes of the C57BL/6 strain were irradiated with 0.1 Gy x-rays either in vivo or in vitro, and their UV-light-induced unscheduled DNA synthesis (UDS) levels were determined autoradiographically. An augmented UV-light-induced UDS following an adapting dose applied in vivo has previously been described by several authors in splenocytes of C57BL/6 mice, indicating that the adapting dose enhanced the DNA repair capacity of lymphocytes. In the present investigation, however, no evidence of an adaptive response could be seen regardless of whether the adapting dose was given in vivo or in vitro. Those results present a further indication for the fact that the adaptive response to ionizing radiation is not always inducible, even in lymphocytes of an inbred mouse strain in which its existence has been reported before.

Adaptation, Physiological↗

PubMed1995

Induction of malformations by X-ray exposure of various stages of the oogenesis of mice.

We studied the effects of ionizing radiation (X-rays, 0-3 Gy, dose rate 1 Gy/min) on various stages of oogenesis of a mouse strain (HLG/Zte) that had been shown previously to respond with an increased number of malformations after radiation exposure of preimplantation stages. Like the results obtained with preimplantation stages, we observed a statistically significant increase in the number of malformed fetuses (exclusively gastroschises) on day 19 of gestation, when oocytes within 1-4 weeks before ovulation were exposed to 2 or 3 Gy. Less mature oocytes were killed by radiation doses of 0.5 Gy and higher. In addition to the increased frequency of malformations, we found a dose-dependent increase of lethal effects (affecting either oocytes or specific gestational stages) and of runts ('dwarfs').

Abnormalities, Radiation-Induced↗

PubMed1995

Micronucleus determination as a means to assess radiation exposure.

Radiation accidents require an easy to perform and rapid determination of the radiation response of the victim(s) affected by the exposure. Micronuclei in cytochalasin B blocked lymphocytes are able to serve this purpose. Micronuclei are particularly useful when it comes to screening many exposed people after a more or less homogeneous whole body exposure with a high dose rate. There are problems, as is the case for most biological indicators, with partial body (especially when it is due to internal emitters), chronic and fractionated exposure.

Dose-Response Relationship, Radiation↗

PubMed1994

Comet assay studies of radiation-induced DNA damage and repair in various tumour cell lines.

We used the 'comet assay' to compare the amount of radiation-induced DNA damage in three tumour cell lines (MeWo, PECA 4451 and PECA 4197) and the extent of DNA repair in two of these lines (MeWo and PECA 4197). Tumour cells were irradiated with X-rays (0.1-10 Gy), embedded in agarose on slides, lysed with sodium dodecyl sulphate and exposed to an electric field. DNA migrated within the agarose and formed comets whose length depended on the amount of DNA damage. When the cells were incubated at 37 degrees C for various time intervals before electrophoresis started, the comets shrank in the course of time, indicating repair of DNA damage. All three cell lines showed the same extent of DNA damage after radiation exposure, despite the fact that in the colony-forming assay MeWo and PECA 4451 were much more sensitive to radiation exposure than PECA 4197. The repair characteristics, however, were markedly different for MeWo and PECA 4197 cells. PECA 4197 cells showed a much faster restoration of the original shape of the cell nucleus than MeWo cells.

DNA Damage↗

PubMed1993

Radiation induced micronuclei in subpopulations of human lymphocytes.

The micronucleus expression in T-helper, T-suppressor and B lymphocytes of the peripheral blood was studied after in vitro exposure to high (2.5 Gy and 5 Gy) and low (0.5 Gy and 1 Gy) doses of ionizing radiation. Investigations were carried out by combining the micronucleus assay with immunofluorescence staining using subpopulation specific antibodies. While in the higher dose range B cell proliferation was inhibited nearly completely-so that micronuclei could not be expressed-we found after exposure to lower doses that B cells were the lymphocyte subpopulation which was most sensitive to micronucleus induction. Among the T cell population, the T-suppressor subset revealed a higher yield of micronuclei than T-helper cells, whereas with regard to the effect of radiation on proliferative ability, T-helper cells reacted more sensitivity than the T-suppressor lymphocytes. Our studies provide insight into the effect of radiation exposure on the micronucleus expression of lymphocyte subpopulations and new information which may be useful for the further development of biological dosimetry.

Adult↗

PubMed1992

Adaptive response in mouse embryos?

Pre-implantation embryos of the mouse were studied for the occurrence of an adaptive response, i.e. induction of radio-resistance by a previous low dose. Various experimental designs were checked (initial doses between 3 and 10 cGy; second dose 2-6 Gy at 6-24 h after the first dose). Some of the experiments were carried out in exactly the same way that resulted in an adaptive response of human lymphocytes reported previously. However, when cell proliferation and differentiation of mouse embryos were examined, none of the conditions tested indicated the induction of an adaptive response.

Adaptation, Physiological↗

PubMed1992

Absence of adaptive response to low doses of X-rays in preimplantation embryos and spleen lymphocytes of an inbred mouse strain as compared to human peripheral lymphocytes: a cytogenetic study.

The adaptive response was studied in preimplantation embryos and spleen lymphocytes of a mouse inbred strain and in peripheral lymphocytes of three human donors, using chromosomal aberrations as the endpoint. Embryos were adapted to 0.05 Gy X-ray 50 h post-conception either in vitro or in vivo and challenged 6 h later. Chromosome aberrations of the 8----16 cell stage mitoses were scored. No adaptive response was seen in the embryos. Of 14 female mice studied, an adaptive response was seen in spleen lymphocytes of only one mouse. However, because variable chromosomal aberration levels were observed in lymphocytes of different donors, it is concluded that the adaptive response detected was merely a result of this heterogeneity. In human peripheral lymphocytes an adaptive response was seen in all three donors. It is speculated that the inbred mouse strain used is deficient in the adaptive response.

Adaptation, Physiological↗

PubMed1992

Micronucleus assay prediction and application optimized by cytochalasin B-induced binucleated tumor cells.

Improvement in the predictive assertion of the micronucleus assay was achieved by treating human malignant melanoma cells (Mewo) with cytochalasin B (CB), generating binucleated cells (BNC) representing cells after a single karyokinesis. Optimal cell binucleation was determined by testing several cytochalasin B concentrations and different incubation times. On average, 56% binucleated cells were found after incubation with 2 to 3 micrograms/ml cytochalasin B for 48 h. Cells with at least one micronucleus (Mn) were defined as fraction of cells with micronuclei and describes the degree of damaged cells. We found in binucleated cells 2.2 fold the fraction of cells with micronuclei than in mononucleated cells (MNC), as expected assuming that an induced micronucleus is associated with only one single daughter cell after mitosis. The mean of micronuclei per binucleated cells, however, was enhanced about 2.9 fold in relation to that of micronuclei per mononucleated cells and is related to the nuclear damage per cell. The application of cytochalasin B did not enhance the fraction of damaged cells although the degree of the injury per cell is intensified. A micronuclei promoting or inhibiting effect of the experimental design due to changes in cell proliferation was excluded by cytofluorometric investigations of DNA content and synthesis after cytochalasin B application. A comparison of the modified with the conventional micronucleus assay shows the superiority of the former.

Cell Line↗

PubMed1992

Preimplantation growth delay and micronucleus formation after in vivo exposure of mouse zygotes to fast neutrons.

Mouse zygotes were irradiated with fast neutrons (0.06 to 1.00 Gy) 1 h after conception and examined at various intervals (24 to 100 h after conception) for embryonic development and micronucleus formation. The frequency of micronuclei per cell increased linearly with dose in 2-cell embryos observed at 24 h after conception and in 4-cell and 8-cell embryos at 48 h after conception. Compared with X rays, the relative biological effectiveness of neutrons for the induction of micronuclei per embryo was 2.5 at 24 h after conception and 3.5 at 48 h after conception. Neutron-induced micronucleus formation was accompanied by morphological growth delay and a significant decrease in the number of cells in the embryos. An inverse relationship was found between the number of cells in embryos and the number of micronuclei when observed at 48 h after conception following irradiation with 0.12 to 1.00 Gy and at 78 h after conception following exposure to 0.50 Gy. The effect of neutron irradiation on embryonic development was likely to be mediated by cell death, as suggested by a significantly increased dead cell index in blastocysts following irradiation of zygotes.

Animals↗

PubMed1991

Cell proliferation and vascularization in human breast carcinomas.

Cell proliferation and vascularization were studied in 10 human breast carcinomas by an immunoenzyme technique. The monoclonal antibody (MAb) Ki-67 was used as a marker for proliferating cells and a polyclonal antibody directed against human von-Willebrand factor to identify blood vessels. The proportion of Ki-67-labelled cells varied from 1% to 20%, the number of small blood vessels from 4.4/mm2 to 57.6/mm2. Within single histological sections of individual tumours the percentage of proliferating cells was not related to the number of small blood vessels. However, after evaluation of 5 sections of each tumour, the average values showed that tumours with a high grade of vascularization had a higher percentage of Ki-67-positive cells than poorly vascularized samples. The influence of vascular density on cell proliferation was investigated in a selected area of one of the tumours (in 2-dimensions) and with regard to the over- and underlying sections (in 3-dimensions). After 2-dimensional evaluation, distances from proliferating cells to the closest blood vessel between 10 and 390 microns were observed, and after 3-dimensional evaluation none of the proliferating cells measured was located more than 130 microns away from the closest vessel.

Antibodies↗

PubMed1991

Direct evidence that radiation induced micronuclei of early embryos require a mitosis for expression.

The naturally synchronous development of early mouse embryos was exploited to address the question, whether micronuclei require a mitosis for expression or whether they can be expressed in the same cell cycle, in which exposure to X-rays or caffeine took place. Experiments with 2-cell and with 4-cell embryos showed that micronuclei are expressed only if a mitosis is completed. There was no indication, even after doses up to 20 Gy, that micronuclei can be expressed before the mitosis was reached, which followed exposure. Furthermore, no nuclear fragmentation pointing to apoptosis could be detected in the cycle, in which cells were exposed. The same results were obtained when caffeine (5 mM) was used as micronucleus inducing agent.

Animals↗

PubMed1991

Biological indicators for radiation damage.

Methods for estimating radiation dose using biological indicators have made rapid progress during recent years. Chromosome analysis in lymphocytes still plays a central role, but it is no longer the only quantitative system in biological dosimetry. The best approach seems to be to combine several of the assays exploiting their specific advantages: the high sensitivity in the case of dicentrics in lymphocytes (starting at about 0.05 Gy low-LET radiation), the broad dose range covered by the electron spin resonance technique (0.5-100 Gy), the possibility of identifying the localization of partial-body exposure when determining hair diameter, and the individual prognostic information obtained from changes in the frequency of blood cells after exposures exceeding about 1 Gy. In specific situations other methods may replace or supplement these indicators for radiation damage.

Biomarkers↗

PubMed1991

Micronuclei in human lymphocytes irradiated in vitro or in vivo.

Venous blood from healthy donors or from patients with various lympho- and myeloproliferative diseases was incubated in vitro in the presence of cytochalasin B for the induction of binucleated lymphocytes. The time at which cytochalasin B was added depended on the proliferation rate of the lymphocytes. Proliferation was monitored using a semiautomatic microscope photometer/computer system. The background level of micronuclei in binucleated lymphocytes of the patients before radiotherapy was statistically indistinguishable from that of healthy persons. Blood from both groups was irradiated in vitro for the study of the dose-response relationship. The dose-response curves were very similar up to 3.75 Gy, and a somewhat lower micronucleus frequency was found in lymphocytes of patients after a 5-Gy exposure. These in vitro results were compared with in vivo exposure after total-body irradiation of leukemic patients. Due to heavy medication that accompanied radiation therapy, only two doses (1.25 and 2.5 Gy) could be checked after in vivo exposure. There was no statistically significant difference between in vitro and in vivo results after 1.25 Gy, but a slightly lower number of micronuclei was observed after in vivo exposure to 2.5 Gy.

Adult↗

PubMed1990

Lethal and teratogenic effects after exposure to X-rays at various times of early murine gestation.

Various well-defined stages during completion of the second meiotic division and early organogenesis of mouse embryos were X-irradiated with doses of 1-4 Gy (100-400 rad). The major risk was prenatal mortality with radiation sensitivity changing markedly with dependence on the developmental stage irradiated; in the case of day 1 even within hours. The surviving fetuses did show a significantly enhanced frequency of malformations on day 19 of gestation (mostly gastroschisis and some exencephalies). This was true for all stages between days 1 and 8; only sensitivity again changed considerably. The radiation doses used in this study are markedly higher than doses that can be expected from radiation diagnostics, but exposure is in a range comparable to doses that can occur in radiation therapy (e.g., Morbus Hodgkin).

Abnormalities, Radiation-Induced↗

PubMed1990

Temperature dependence of combined exposure of preimplantation mouse embryos to X-rays and mercury.

Usually preimplantation mouse embryos are cultured in vitro at 37 degrees C. In this study, mouse embryos (starting with the 2-cell stage) were incubated in vitro throughout the preimplantation period at 34 degrees, 35 degrees, 36 degrees, 37 degrees, 38 degrees or 39 degrees C. Embryonic development, in particular hatching of blastocysts (144 h after conception), and cell numbers (at the same time) were analysed. Embryos were exposed to X-rays (1 Gy) or to mercuric chloride (3 microM, 10 microM) or to a combination of the two agents. Control embryos developed rather well in a temperature range of 35 degrees to 38 degrees C. A combined risk greater than expected from the sum of the individual effects was observed only at the optimum temperature (for a combination of 1 Gy + 3 microM) or in the range of the optimum temperature, that is between 36 degrees to 38 degrees C (for a combination of 1 Gy + 10 microM).

Animals↗

PubMed1989

Micronucleus formation in 2-cell embryos after in vitro X-irradiation of mouse spermatozoa.

Mouse spermatozoa were exposed in vitro to various X-ray doses and added to a medium containing superovulated oocytes. The percentage of fertilized oocytes was determined 24 h after sperm addition and 2-cell embryos were investigated for micronucleus formation. The fertilization rate was drastically decreased after exposure to 470 cGy whereas smaller doses had no effect. The dose-response relationship for micronucleus formation per embryo was linear-quadratic and the number of embryos with micronuclei increased linearly with dose. The distribution of micronuclei among 2-cell embryos showed a significant overdispersion relative to the Poisson distribution after sperm irradiation for doses above 188 cGy.

Animals↗

PubMed1989

Toxicity of various combinations of X-rays, caffeine, and mercury in mouse embryos.

Preimplantation mouse embryos in vitro were exposed to various doses of X-rays (0.25-2 Gy) and to different concentrations of two chemicals: caffeine (0.5-2 mM) and mercury (0.5-5 microM). X-irradiation was given first, followed immediately by exposure to the chemicals. The effects of the agents, applied either singly or in combination of two or of three, were studied using morphological, proliferative and cytogenetic endpoints (formation of blastocysts, hatching, trophoblast outgrowth, formation of inner cell mass, cell numbers, micro-nucleus frequency). The term 'enhancement in risk' was used whenever the effects observed after combined exposure (two or three agents) significantly exceeded the sum of the effects due to the component individual agents. The enhancement in risk observed after exposure to the three agents could be explained by the interactions already detected at the level of a combined exposure to only two agents. There was no increase in risk specific for the presence of all three agents.

Animals↗