[Agencies for radiation protection in Germany, Europe and worldwide. Responsibilities and relations between]. / Strahlenschutz-Gremien in Deutschland, Europa und weltweit. Aufgaben und Beziehungen untereinander.

The complex radiation protection system is affected by various organisations. The most important bodies worldwide, in Europe and in Germany will be introduced including a description of their relationships. Three levels of responsibility have to be discriminated: the scientific, the philosophical/methodical, and the level of jurisdiction and regulations.

Clinical, molecular- and cytogenetic analysis of a case of severe radio-sensitivity.

In radiotherapy the normal tissue reaction is often a limiting factor for radiation treatment. Still there is no screening method, which predicts normal tissue reaction on radiotherapy, especially in comparison to tumor tissue, and therefore allows tailoring of the radiation dose to each patient. Here, we present a case of severe radiation-related side effects. We applied classical cytogenetic techniques (Giemsa-banding and staining of centromeric regions), the comet assay as well as multicolor fluorescence in situ hybridization on peripheral blood lymphocytes of this patient in order to determine the radio-sensitivity on the DNA level and to correlate these findings with the clinical outcome. Our investigations revealed abnormalities on chromosome 9, deficiencies in the DNA-repair capacity after radiation exposure and a high number of radiation induced chromosomal aberrations. A detected high amount of residual damage two or three hours after radiation exposure and repair as well as the high number of chromosomal aberrations (ChAs) suggests a correlation between repair capacity and radiation induced ChAs. We concluded that the detected abnormalities might serve as a genetic basis for the radio-sensitive phenotype of this patient. Taken together this report strengthens the idea that intensive DNA genomic analysis of individual patients can serve as the basis for more favourable treatment of cancer patients.

Micronuclei in lymphocytes of uranium miners of the former Wismut SDAG.

We studied micronucleus frequencies in former German uranium miners of the Wismut SDAG (Sowjetisch-Deutsche Aktiengesellschaft). Various other groups were analyzed for comparison (individuals with lung tumors or lung fibrosis, controls). We had shown previously that micronucleus frequencies were not different among the various groups. Differences were observed, however, when centromere-positive and -negative micronuclei were distinguished. In the analyses presented here, we looked for the effects of smoking habits, alcohol consumption, vitamin uptake, chronic diseases, allergies, doing sports, gamma-GT (gamma-glutamyltranspeptidase), lymphocyte numbers, CEA (carcinoembryonic antigen), X-ray diagnostics, computer tomographies, and scintigraphies. With the exception of more than one scintigraphy carried out during the last four months before micronucleus analysis, none of the factors mentioned above significantly affected micronucleus numbers. One result deserves specific attention: individuals with low percentages of binucleated lymphocytes after in vitro cytochalasin B exposure showed higher micronucleus frequencies than those individuals with high percentages of binucleated cells. The same result was obtained for various other populations that we monitored in the past.

Micronucleus formation in lymphocytes of children from the vicinity of Chernobyl after (131)I therapy.

After the Chernobyl accident a statistically significant increase in the number of children with thyroid tumours was observed. In this study 166 children with and 75 without thyroid tumours were analysed for micronucleus formation in peripheral blood lymphocytes using the cytochalasin B approach. The following factors did not significantly affect micronucleus formation: gender, age at the time of the first (131)I treatment, tumour stage, tumour type, or metastases; a statistically significant increase in the number of micronuclei, however, was observed for the residents of Gomel compared to other locations, such as Brest, Grodno, and Minsk. The children with tumours received (131)I treatment after surgical resection of the tumours. This gave us the opportunity to systematically follow the effect of (131)I on micronucleus formation. A marked increase was observed 5 days after the (131)I treatment followed by a decrease within a 4-7 months interval up to the next application, but the pre-treatment levels were not achieved. Up to 10 therapy cycles were followed each including an analysis of micronucleus formation before and 5 days after (131)I application. The response of the children was characterised by clear individual differences and the increase/decrease pattern of micronucleus frequencies induced by iodine-131 was correlated with a decrease/increase pattern in the number of lymphocytes.

Effects of asbestos on initiation of DNA damage, induction of DNA-strand breaks, P53-expression and apoptosis in primary, SV40-transformed and malignant human mesothelial cells.

Human mesothelial cells (HMC), the progenitor cells of asbestos-induced mesothelioma, are particularly sensitive to the genotoxic effects of asbestos, although the molecular mechanisms by which asbestos induces injury in HMC are not well known. The high susceptibility of HMC to simian virus 40 (SV40)-mediated transformation is assumed to play a causative role in the pathogenesis of mesothelioma. The aim of this study was to investigate the asbestos-induced DNA damage in cultured HMC and SV40-transformed HMC (MeT-5A) compared with their malignant counterparts, i.e. human mesothelioma cells (MSTO). The time-dependent initiation of DNA-strand breaks as well as the induction of oxidative DNA base modifications were key factors for investigation. HMC, MeT-5A and MSTO cells were exposed to chrysotile and crocidolite asbestos (3 microg/cm2) during different time periods (1-72 h). DNA damage was investigated by use of the Comet assay and alkaline unwinding, the latter in combination with the Fpg protein. The P53 level was analyzed by immunofluorescence, and measurement of apoptosis was conducted by flow cytometry. We found a significant induction of DNA damage in asbestos-treated HMC already after an exposure time of 1.5 h. This effect could not be observed in treated MeT-5A and MSTO cells. Also, a time-dependent significant increase in DNA-strand breaks was observed by alkaline unwinding in asbestos-treated HMC, but not in treated MeT-5A and MSTO cells. In none of the three cell lines we could detect oxidative DNA damage recognized by the Fpg protein (e.g. 8-oxo-guanine), up to 24 h after exposure to asbestos. In contrast to what was found in HMC, P53 was over-expressed in untreated MeT-5A and MSTO. The induction of apoptosis by asbestos fibers was suppressed in MeT-5A and MSTO cells. Crocidolite fibers induced the higher genotoxic effects and chrysotile the more pronounced apoptotic effects. We conclude that asbestos induces DNA damage in HMC already after a very short exposure time in the absence of 8-oxo-guanine formation. The presence of SV40-Tag in MeT-5A and MSTO cells results in an increased expression of P53, but not in additive genotoxic effects after exposure to asbestos. The deregulation of the apoptotic pathway may lead to proliferation of genomically damaged cells and finally to the development of mesothelioma.

Does radiotherapy affect the outcome of the comet assay?

This study was designed to assess possible effects of fractionated radiotherapy (5 or 10 fractions at 2 Gy per fraction) on the DNA repair capacity of lymphocytes, as measured by the comet assay. 50 patients with various tumour types were chosen. They had received no chemotherapy during the 6 months prior to radiotherapy and did not receive cortisone. 10 ml of heparinized blood was collected before radiotherapy, after 5 fractions and after 10 fractions. Lymphocytes were isolated and analysed using the comet assay. On average, no effect on DNA repair capacity was observed that could be attributed to radiotherapy. On an individual basis, there were a few patients who showed a comparatively pronounced variability in their response to radiotherapy (three patients with a relative coefficient of variability of more than 30%). There was some indication of a weak correlation between poor repair capacity and severe side effects in normal tissue. We also found that alcohol in particular, and smoking to some extent, may impair repair capacity during radiotherapy. Age, gender, field size, medication and tumour entity showed no effect on repair capacity.

A cytogenetic analysis of the long-term effect of uranium mining on peripheral lymphocytes using the micronucleus-centromere assay.

PURPOSE: To assess the long-term effect of radiation exposure of uranium miners on a cytogenetic endpoint: micronuclei (Mn) with and without a centromere. MATERIALS AND METHODS: Mn were scored using the cytochalasin-B technique. It is known that Mn can comprise acentric fragments or/and whole chromosomes. Mn containing whole chromosomes were identified by means of fluorescence in situ hybridization (FISH) with a centromere-specific probe. The frequency and percentage of Mn were analysed with centromeres (MnC+) in lymphocytes of healthy donors and uranium miners with large radiation exposures several decades ago employed by the Wismut AG in the former German Democratic Republic. The miners were subdivided into those with and those without bronchial carcinoma. RESULTS: It was shown previously that the relative frequency of MnC+ decreased with dose; this means that the number of Mn originating from acentric fragments increases. In the study presented here, no statistically significant difference in the overall Mn frequency was seen between the analysed groups. The fraction of MnC+, however, was highest in lymphocytes of healthy male donors (mean: 74.6%) followed by healthy miners (mean: 62.1%) and those suffering from cancer (mean: 55.8%). CONCLUSION: The results indicate the occurrence of a genomic instability in lymphocytes of miners, especially those with cancer. It appears that the low percentage of MnC+ may be a marker of genomic instability and cancer predisposition.

Heterogeneity in 2-deoxy-D-glucose-induced modifications in energetics and radiation responses of human tumor cell lines.

PURPOSE: The glucose analog and glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), has been shown to differentially enhance the radiation damage in tumor cells by inhibiting the postirradiation repair processes. The present study was undertaken to examine the relationship between 2-DG-induced modification of energy metabolism and cellular radioresponses and to identify the most relevant parameter(s) for predicting the tumor response to the combined treatment of radiation + 2-DG. METHODS AND MATERIALS: Six human tumor cell lines (glioma: BMG-1 and U-87, squamous cell carcinoma: 4451 and 4197, and melanoma: MeWo and Be-11) were investigated. Cells were exposed to 2 Gy of Co-60 gamma-rays or 250 kVP X-rays and maintained under liquid-holding conditions 2-4 h to facilitate repair. 2-DG (5 mM, equimolar with glucose) that was added at the time of irradiation was present during the liquid holding. Glucose utilization, lactate production (enzymatic assays), and adenine nucleotides (high performance liquid chromatography and capillary isotachophoresis) were investigated as parameters of energy metabolism. Induction and repair of DNA damage (comet assay), cytogenetic damage (micronuclei formation), and cell death (macrocolony assay) were analyzed as parameters of radiation response. RESULTS: The glucose consumption and lactate production of glioma cell lines (BMG-1 and U-87) were nearly 2-fold higher than the squamous carcinoma cell lines (4197 and 4451). The ATP content varied from 3.0 to 6.5 femto moles/cell among these lines, whereas the energy charge (0.86-0.90) did not show much variation. Presence of 2-DG inhibited the rate of glucose usage and glycolysis by 30-40% in glioma cell lines and by 15-20% in squamous carcinoma lines, while ATP levels reduced by nearly 40% in all the four cell lines. ATP:ADP ratios decreased to a greater extent ( approximately 40%) in glioma cells than in squamous carcinoma 4451 and MeWo cells; in contrast, presence of 2-DG reduced ADP:AMP ratios by 3-fold in the squamous carcinoma 4451, whereas an increase was noted in the glioma cell line BMG-1. 2-DG significantly reduced the initial rates of DNA repair in all cells, resulting in an excess residual damage after 2 h of repair in BMG-1, U-87, and 4451 cell lines, whereas no significant differences could be observed in the other cell lines. Recovery from potentially lethal damage was also significantly inhibited in BMG-1 cells. 2-DG increased the radiation-induced micronuclei formation in the melanoma line (MeWo) by nearly 60%, while a moderate (25-40%) increase was observed in the glioma cell lines (BMG-1 and U-87). Presence of 2-DG during liquid holding (4 h) enhanced the radiation-induced cell death by nearly 40% in both the glioma cell lines, while significant effects were not observed in others. CONCLUSIONS: The modifications in energetics and radiation responses by 2-DG vary considerably among different human tumor cell lines, and the relationships between energy metabolism and various radiobiologic parameters are complex in nature. The 2-DG-induced modification of radiation response does not strictly correlate with changes in the levels of ATP. However, a significant enhancement of the radiation damage by 2-DG was observed in cells with high rates of glucose usage and glycolysis, which appear to be the two most important factors determining the tumor response to the combined treatment of 2-DG + radiation therapy.

Radiation sensitivity of lymphocytes from healthy individuals and cancer patients as measured by the comet assay.

Lymphocytes of healthy volunteers (n = 24) and of tumour patients (n = 30, 18 of whom had experienced severe side-effects) were irradiated with x-rays in vitro. DNA damage was analysed after 0.25-2 Gy and DNA repair after 2 Gy, and quantification of both endpoints was done by the comet assay. The individual differences in radiation-induced DNA damage as well as in the repair kinetics were observed to be striking for both healthy donors and tumour patients. After a repair time of 3 h, following 2 Gy x-irradiation, some of the healthy volunteers showed no residual DNA damage at all in their lymphocytes, whereas others revealed about 30%. There was no indication that our results were affected by either age, gender or smoking habits. Slow repair kinetics and high amounts of residual damage were characteristic for many but not all tumour patients who had experienced severe side-effects in their normal tissues during or after radiotherapy (n = 18). Our conclusion is that those individuals showing poor DNA repair characteristics in the lymphocytes following in vitro irradiation, have a high probability of being radiosensitive. The opposite conclusion is not necessarily true: if repair is effective, this does not mean that the individual is radioresistant, because factors other than impaired repair may cause radiosensitivity.

HUman MicroNucleus project: international database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei.

Micronucleus (MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis-block micronucleus (CBMN) assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of "normal" values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin-B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e., normal) subjects was 6.5 per thousand and the interquartile range was between 3 and 12 per thousand. An increase in MNC frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MNC frequency (95% confidence interval: 14-24%). Statistical analyses were performed using random-effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods.

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.

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.

Biomarkers of genetic damage and inflammation in blood and bronchoalveolar lavage fluid among former German uranium miners: a pilot study.

Former East German uranium miners who are known to have been exposed to radon are estimated to be at high risk for lung carcinogenesis. Among these miners over 200 occupationally caused lung cancer cases are expected to occur each year, resulting in a total of 7,000-24,000 excess lung cancer cases in the coming years. It is still unknown whether there is a correlation between biomarkers and the exposure of the uranium miners to ionizing radiation that might enable us to trace those miners with high lung cancer risk. The primary aim of this pilot study was to test the possibility of performing a biomarker study in this unique cohort of former uranium miners in spite of several limitations that had to be taken into consideration when comparing them with healthy controls, such as old age, age-dependent diseases and potential confounding artefacts from dissimilar smoking patterns. The second aim was to test a range of biomarkers for DNA damage and inflammation in leukocytes and bronchoalveolar fluid for their ability to detect biological effects. In this cohort of miners we found an increased frequency of chromosomal aberrations in blood lymphocytes and an increased prevalence of both fibronectin and tumour necrosis factor alpha in the bronchoalveolar fluid.

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.

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.

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.

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.

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.

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.

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.