A novel micronucleus in vitro assay utilizing human hematopoietic stem cells.

The induction of micronucleated reticulocytes in the bone marrow is a sensitive indicator of chromosomal damage. Therefore, the micronucleus assay in rodents is widely used in genotoxicity and carcinogenicity testing. A test system based on cultured human primary cells could potentially provide better prediction compared to animal tests, increasing patient safety while also implementing the 3Rs principle, i.e. replace, reduce and refine. Hereby, we describe the development of an in vitro micronucleus assay based on animal-free ex vivo culture of human red blood cells from hematopoietic stem cells. To validate the method, five clastogens with direct action, three clastogens requiring metabolic activation, four aneugenic and three non-genotoxic compounds have been tested. Also, different metabolic systems have been applied. Flow cytometry was used for detection and enumeration of micronuclei. Altogether, the results were in agreement with the published data and indicated that a sensitive and cost effective in vitro assay to assess genotoxicity with a potential to high-throughput screening has been developed.

[Impact of preoperative patient education on postoperative pain in consideration of the individual coping style]. / Einfluss präoperativer patienteninformationen auf postoperative schmerzen unter berücksichtigung individueller stressverarbeitung.

OBJECTIVE: the German guideline for the treatment of acute perioperative and post-traumatic pain (S3-Leitlinie zur Behandlung akuter perioperativer und posttraumatischer Schmerzen) recommends giving preoperative information about postoperative pain and how to influence it. It is expected that the effect of preoperative information is modified by psychological characteristics of the patient. One of these psychological characteristics is the individual coping style. The purpose of the study is to evaluate whether or not patients benefit from preoperative education in relation to their level of negative coping style. METHODS: the study is based on a 2×2 factorial experimental design with the experimental factor "treatment" (education vs control condition) and the factor "negative coping style" (high vs low). After informed consent 96 patients undergoing abdominal or vascular surgery were enrolled in the study. Outcomes were pain intensity, pain quality and psychic state. They were assessed by using numerical rating scales and psychometric methods of self-assessment. The data were collected preoperatively and on the first to third postoperative day. RESULTS: patients who received preoperative education experience a greater reduction in postoperative pain than patients without preoperative education do (ES=0.48). The risk for stronger pain (NRS>3) on the third postoperative day is decreased (2.1 vs 14.6%). The influence of negative coping style is altogether minimal. CONCLUSIONS: preoperative patient information has positive effects on the postoperative development of pain. Patient information is a valuable addition to the drug pain treatment. The application can be recommended regardless of the level of the patients' negative coping style.

Identification and characterization of a novel splicing variant of vesicular monoamine transporter 1.

Vesicular monoamine transporter 1 (VMAT1) is an integral protein in the membrane of secretory vesicles of neuroendocrine and endocrine cells that allows the transport of biogenic monoamines, such as serotonin, from the cytoplasm into the secretory vesicles. The full-length VMAT1 transcript is produced from 16 exons. We have identified and characterized an alternatively spliced form of VMAT1 that lacks exon 15, the next to last exon of VMAT1. The new form was therefore denoted VMAT1Delta15. Exon 15 does not contain an even multiple of three nucleotides. As a consequence, there is a shift of reading frame, and exon 16 is translated in an alternative reading frame, yielding a novel protein with a shorter and unrelated C-terminus compared with the native VMAT1 protein. VMAT1 and VMAT1Delta15 mRNAs are simultaneously expressed in normal and neoplastic neuroendocrine cells of the GI tract. However, VMAT1 expression is always higher than VMAT1Delta15 expression. We prove that VMAT1Delta15 is not localized in large, dense core vesicles as the native form but in the endoplasmic reticulum. Furthermore, while VMAT1 can take up serotonin, VMAT1Delta15 cannot, indicating different functions for the two forms of VMAT1.

Pilot study for comparison of reticulocyte-micronulei with lymphocyte-micronuclei in human biomonitoring.

Biomonitoring tries to determine the consequences for humans of exposures to environmental or pharmaceutical agents. Different end points have been employed to assess the burden of genomic damage. This is the first report comparing a recently introduced new end point, the reticulocyte-micronuclei analyzed by flow cytometry with the widely used lymphocyte-micronucleus assay, applied to two exposure scenarios leading to enhanced genomic damage. Radioiodine therapy was chosen to represent a short time exposure and hemodialysis treatment in end-stage renal failure was chosen to represent a chronic exposure. The results show that iodine radiation induced measurable genomic damage in the lymphocyte-micronucleus assay as well as in the reticulocyte-micronucleus test. Of two groups of patients under hemodialysis treatment, a reduced genomic damage was found with the lymphocyte-micronucleus test, but not with the reticulocyte-micronucleus test in the group undergoing daily hemodialysis, which removes uremic toxins more efficiently as compared to conventional hemodialysis, the treatment applied in the other group. The limited life-span of reticulocytes may make them less suitable for accumulation of chronic low level damage than lymphocytes. In conclusion, the lymphocyte-micronucleus test may be applicable to more exposure situations (including low chronic exposure), but the reticulocyte-micronucleus assay may be easier to perform in a clinical setting. The latter reflects a more rapid reduction of genomic damage after an acute exposure.

Extended exposure of adult and fetal mice to 50 Hz magnetic field does not increase the incidence of micronuclei in erythrocytes.

The flow cytometer-based micronucleus assay was used to study the effects on chromosomes in erythroid cells of CBA/Ca mice after extended exposure to 50 Hz magnetic field (MF), 14 microT, peak-to-peak (p-p). The study included two different experiments: (a) mice exposed in utero during 18 days of their prenatal stage, and (b) adult mice exposed for 18 days. In experiment (a) 35 days after exposure was terminated, peripheral blood was drawn from the mice exposed in utero to determine whether the exposure had a genotoxic effect on the pluripotent erythroid stem cells. About 200000 polychromatic erythrocytes (PCE) and 200000 normochromatic erythrocytes (NCE) were analysed from each of 20 exposed mice. The EMF exposure did not significantly change the frequency of micronucleated PCE or NCE in comparison with 20 sham-irradiated mice. There was no difference in the proportion of PCE between exposed and unexposed animals. Similarly, in experiment (b) no differences were seen between EMF exposed and unexposed adult mice when samples of peripheral blood were taken at the end of exposure and analyzed for micronuclei in PCE and NCE. The proportion of PCE was the same in both groups. The results indicate that exposure to EMF does not induce direct or indirect effects on chromosomes in erythroid cells expressed as increased levels of micronucleated erythrocytes of mice. No indications of delayed genetic effects were found.

The multiplicative model for cancer risk assessment: applicability to acrylamide.

According to a multiplicative model for prediction of cancer risk for genotoxic agents the incremental cancer risk is, for low-intermediate exposures, proportional to target doses of the genotoxic substance and to the background risk in control groups. This model has been applied to evaluate cancer tests of acrylamide in rodents. Because of its reactivity toward DNA, glycidamide is assumed to be the causative genotoxic metabolite of acrylamide. Evaluation of experimental data according to the multiplicative model shows that mice, compared with rats, are of the order of 10 times more sensitive per administered dose of acrylamide. The US EPA procedure would, however, generally predict rats to be about twice as sensitive as mice to carcinogenic chemicals, because their estimates are based on scaling of the dose per square meter body surface area, as a surrogate for metabolic differences between the species. The comparison of rats and mice with respect to observed cancer incidence is at a key position in the evaluation of the usefulness of risk models for extrapolation between species. In the present study mice and rats were compared, with respect to in vivo doses of acrylamide and the metabolite glycidamide, after exposure to acrylamide. The relative in vivo doses were inferred from levels of hemoglobin adducts. The adduct levels from glycidamide were, per administered dose of acrylamide, approximately 3-10 times higher in mice than in rats. In combination with the above mentioned higher sensitivity of mice than rats in cancer tests of acrylamide this is compatible with the concept that glycidamide is the key genotoxic factor in acrylamide exposure. Furthermore, it is shown that the multiplicative, i.e. relative, risk model and measurements of the dose of the genotoxic factor give good prediction of the observed risk from acrylamide in cancer tests with rats and mice.

The in vivo dose rate effect of chronic gamma radiation in mice: translocation and micronucleus analyses.

The in vivo effects of chronic, ultra low dose rates of gamma radiation in mice were evaluated using fluorescence in situ hybridization and the in vivo micronucleus test. SWRxC57BL/6 mice were divided into nine exposure groups and continuously exposed to 0.5, 2.0 or 4.0cGy 137Cs per day for 30, 60 or 90 days; unexposed control mice were also included. Following exposure, blood samples were taken from each animal and the frequencies of micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE) were determined using flow cytometry. Peripheral blood lymphocytes were cultured and analyzed by chromosome painting to determine translocation frequencies. A significant dose rate response was seen in translocations and both MPCE and MNCE. Comparisons were made between the three chronic dose rates and it was determined that there was no significant difference among translocation frequencies for each rate. However, a significant difference was found between the chronic exposures reported here and the fractionated daily exposures reported previously. Dose rate reduction effects, ranging from 3 at low doses to 14 at high doses, were found for chronic versus acute exposures. The possibility of gender effects was investigated in both micronucleus and translocation data. No gender effect was found in translocation induction, but a slight effect was suggested in micronucleus induction.

Absence of genomic instability in mice following prenatal low dose-rate gamma-irradiation.

PURPOSE: To determine whether mice exposed to an extended low dose of gamma-irradiation during most of their prenatal period express increased frequencies of micronucleated polychromatic erythrocytes (fMPCE) and/or micronucleated normochromatic erythrocytes (fMNCE) several weeks after the end of irradiation. METHODS: Female CBA/Ca mice were gamma-irradiated for an average of 16 days during their pregnancy. The mice were exposed to dose rates of 0, 44, 99 and 265 mGy/day. At 1-2 days prior to parturition the mice were removed from exposure. Then, 36 days after birth, peripheral blood was drawn from all offspring (74 mice). Using flow-cytometer-based analysis, the frequencies of MPCE and MNCE were determined. From each animal about 170,000 PCE were analysed. RESULTS: No delayed effects in terms of higher fMPCE or fMNCE were observed among the in utero exposed mice of either gender. On the contrary, a significant (p<0.001) reduction of fMPCE was found among the male offspring exposed at the highest dose rate. CONCLUSION: Gamma-irradiation of mice during their prenatal stage did not induce damage in erythroid stem cells that can be detected as persistent or delayed chromosome aberrations (i.e. micronucleated erythrocytes) at 35 days after the end of exposure.

Human cytogenetic biomonitoring using flow-cytometric analysis of micronuclei in transferrin-positive immature peripheral blood reticulocytes.

We have developed a method to isolate and analyze nascent human reticulocytes in peripheral blood for the presence of micronuclei (MN). For a very short time peripheral reticulocytes show residual expression of the transferrin receptor. Using immunomagnetic separation of cells expressing the transferrin receptor, a population of immature reticulocytes (Trf-Ret) was isolated from peripheral blood. In humans, the spleen actively removes micronucleated erythrocytes but during the short lifetime of the isolated Trf-Ret only a fraction (less than about 20%) of the MN-containing reticulocytes will have been eliminated. Cells were stained with the fluorescent dyes Thiazole Orange for RNA and Hoechst 33342 for DNA and analyzed by flow cytometry and fluorescence microscopy. Baseline frequencies of MN-Trf-Ret on a group of healthy donors were found to be 1.1% for males and 1.4% for females; however, the gender difference was not significant. The frequency of MN-Trf-Ret in the studied group increased with age, and was dependent on blood group. In three donors studied over 4 months, the baseline level remained stable. In cancer patients treated with radiation or chemotherapy, the frequency of MN-Trf-Ret increased 10- to 20-fold after 1-4 days, depending on the treatment. A high correlation between flow and manual analysis of MN-Trf-Ret was seen. We believe the method has a high potential as a sensitive and rapid method for biological monitoring in presumed exposed groups and individuals.

The micronucleus test in rat erythrocytes from bone marrow, spleen and peripheral blood: the response to low doses of ionizing radiation, cyclophosphamide and vincristine determined by flow cytometry.

The frequency of micronucleated polychromatic erythrocytes (fMPCE) was determined in samples from bone marrow, spleen and peripheral blood of rats exposed to low doses of X-rays, cyclophosphamide or vincristine. The fMPCE values were lower in the peripheral blood than in bone marrow or spleen. This is due to the elimination of MPCE from the circulating blood, which was confirmed by the results from prolonged exposure of rats to gamma-radiation. When the analysis was restricted to the youngest PCE in peripheral blood, the sensitivity of the assay was considerably improved. This can be reproducibly achieved with the flow cytometric analysis.

Low dose effects of chemicals as assessed by the flow cytometric in vivo micronucleus assay.

Using flow cytometric automation of the mouse in vivo, micronucleus assay increases the sensitivity of the test. This is achieved through a very large increase in the number of cells scored, by a factor of 100x, which in turn greatly reduces the sampling error. With this method, dose-response relationships of in vivo micronucleus induction for four model agents mitomycin C (MMC), diepoxybutane (DEB), cyclophosphamide (CPA), and colchicine (COL) were studied at low dose levels. For the three clastogens MMC, DEB and CPA, linear dose-response relationships were found over the dose ranges studied, even in the very low dose region (defined as the dose region where the frequency of micronucleated erythrocytes is less than twice the baseline frequency). This is consistent with the view that no threshold should exist for genotoxic agents which target DNA. For COL a dose range was found, in which the frequency of micronucleated erythrocytes did not increase with dose, possibly indicating an in vivo threshold. The flow cytometric in vivo micronucleus assay represents one possibility for in vivo low dose-response studies.

Spontaneous and radiation-induced micronuclei in erythrocytes from four species of wild rodents: a comparison with CBA mice.

Almost 100 animals of 4 different species of small wild rodents (bank vole, Clethrionomys glareolus; field vole, Microtus agrestis; yellow-necked mouse, Apodemus flavicollis; and wood mouse, Apodemus sylvaticus) were trapped in central Sweden and used in experiments to determine the spontaneous and radiation-induced frequencies of polychromatic (fMPCE) and normochromatic erythrocytes (fMNCE) from bone marrow (bm) and peripheral blood (pb) using flow cytometric analysis. The results were compared with those from similar experiments with CBA mice. The saving of time and labour by the use of the flow cytometer-based analysis was a prerequisite for this study in which about 135 million PCE were analysed. The two species of voles had a mean background fMPCE (bm) of about the same value as CBA mice, while the yellow-necked mice had about five times higher fMPCE (bm). Wood mice had more than twice the fMPCE (bm) compared to CBA mice. Between individual animals in each of the 4 species, the background fMPCE (bm) varied more than between individual CBA mice, and the elimination of micronucleated erythrocytes was considerable. When exposed to ionizing radiation, the voles did not show a significant response. The response of the two Apodemus species was similar to that of the CBA mice, although it varied between individual animals and was not correlated to their background fMPCE. This study indicates that bank voles and field voles are unsuitable testing objects in the in vivo micronucleus assay. On the other hand, yellow-necked mice and wood mice seem to be useful in this test. Since the variation between individuals is considerable in wild Apodemus mice, large groups will be needed for obtaining statistically significant results when exposure to a genotoxic agent is low. Alternatively, repeated samples can be taken from individual wild mice to study the effect of a decreased exposure after keeping the animals for a period of time in an uncontaminated environment.

Flow cytometric analysis of micronucleus induction in rat bone marrow polychromatic erythrocytes by 1,2;3,4-diepoxybutane, 3,4-epoxy-1-butene, and 1,2-epoxybutane-3,4-diol.

Automation of the analysis of micronucleus induction with flow cytometry was developed by using mouse bone marrow or peripheral blood. In the present study, we report the use of flow cytometry for the identification and quantification of micronuclei (MN) induced in rat bone marrow polychromatic erythrocytes. Three metabolites of the industrial chemical 1,3-butadiene, namely 1,2;3,4-diepoxybutane (DEB), 3,4-epoxy-1-butene (EB) and 1,2-epoxybutane-3,4-diol (diol-EB), were studied in addition to mitomycin C and cyclophosphamide, which served as positive controls. DEB showed a dose-dependent increase in the frequency of MN, whereas EB was completely negative and diol-EB only weakly positive at one dose level. The effect of the positive control compounds was observed 48 h after a single injection in a dose-dependent manner. Flow cytometry was an effective method to quantitate bone marrow MN induction in the rat when density gradient separation of polychromatic erythrocytes is used. The results are compatible with the theory that oxidation of EB to the mutagenic metabolite DEB occurs at a low rate in rat bone marrow and that EB is detoxified by epoxide hydrolase and by conjugation with glutathione by glutathione transferase yielding nonmutagenic metabolites. Thus, the reported lack of MN induction by 1,3-butadiene inhalation in rat bone marrow is explained.

Quantitative and qualitative studies of micronucleus induction in mouse erythrocytes using flow cytometry. I. Measurement of micronucleus induction in peripheral blood polychromatic erythrocytes by chemicals with known and suspected genotoxicity.

Quantitative and qualitative aspects of the in vivo micronucleus-inducing potential of five chemicals were studied using flow cytometric enumeration of micronucleated polychromatic peripheral blood erythrocytes in mice. The chemicals were hydroquinone, vinblastine sulphate, chloral hydrate (tested in two different mouse strains), 5-bromo-2-deoxyuridine and 2-chlorobenzylidene malonitrile. Repeat samplings of peripheral blood were made at 0, 24, 40, 48 and 72 h and for low doses of 5-bromo-2-deoxyuridine 96 h after i.p. treatment. The agents hydroquinone (lowest effective dose 25 mg/kg), vinblastine sulphate (lowest effective dose 0.05 mg/kg) and 5-bromo-2-deoxyuridine (lowest effective dose 200 mg/kg) gave rise to significant increases in the frequencies of micronucleated polychromatic erythrocytes. No significant induction of micronucleated polychromatic erythrocytes by 2-chlorobenzylidene malonitrile or chloral hydrate was found. The frequencies of induced micronucleated polychromatic erythrocytes peaked at 40 h after hydroquinone treatment, at 48 h after vinblastine treatment and at 72 h after 5-bromo-2-deoxyuridine treatment with evident dose-dependent differences in the kinetics of the induction of micronucleated polychromatic erythrocytes. The mean relative Hoechst 33342 fluorescence of the populations of induced micronucleated polychromatic erythrocytes was used as an indicator of the DNA content of induced micronuclei. These values were found to be in agreement with the presumed mechanisms of micronucleus induction for hydroquinone, vinblastine sulphate and 5-bromo-2-deoxyuridine. Flow cytometric enumeration of micronucleated polychromatic erythrocytes in peripheral blood is an efficient method for the study of in vivo micronucleus induction, combining rapid analysis and high sensitivity with information on possible mechanisms of micronucleus induction. The method also allows a substantial reduction in the number of animals needed.

Quantitative and qualitative studies of micronucleus induction in mouse erythrocytes using flow cytometry. II. Analysis of micronuclei of aneugenic and clastogenic origin by dual-colour FISH on populations of bone marrow PCEs flow sorted on the basis of their relative DNA content.

Simultaneous dual-colour fluorescent in situ hybridization (FISH) with the major and minor mouse satellite probes was performed on flow-sorted fractions of micronuclei with defined Hoeschst 33342 fluorescence intensities, reflecting their relative DNA content. The purpose of the study was to investigate the relationship between probe binding and the size of micronuclei induced by a set of chemicals with different mechanisms of micronucleus induction. The agents studied were vinblastine, cyclophosphamide, hydroquinone and 5-bromo-2-deoxyuridine. The Hoechst 33342 fluorescence distributions of induced micronuclei differ markedly between cyclophosphamide, vinblastine and hydroquinone. While the distributions for cyclophosphamide and vinblastine agree well with those obtained for other model aneugens and clastogens, hydroquinone shows a distribution with characteristics similar to both clastogens and aneugens. A comparison between the Hoechst 33342 fluorescence distributions of hydroquinone-induced micronuclei positive for both major and minor probes and those positive for the major probe only indicates that the former consist of whole chromosomes, while the latter originate from chromosome fragments. The absolute frequency and Hoechst 33342 fluorescence distribution of major-only-positive micronuclei induced by vinblastine indicates that this model aneugen also shows a certain in vivo clastogenic potential. Micronuclei induced by 5-bromo-2-deoxyuridine indicate that this agent is a clastogen, preferentially inducing pericentric breaks, but may also to some extent be aneugenic.

Erythropoiesis and the induction of micronuclei in mouse spleen determined by flow cytometry.

Erythrocytes from the spleen of CBA mice have been prepared for analyses by flow cytometry. About 80% of the polychromatic erythrocytes (PCE) in the spleen originate from erythropoiesis in the spleen, while the remaining 20% come from the peripheral blood. Analyses of the RNA content of PCE revealed that splenic PCE do not mature into normochromatic erythrocytes (NCE) in the spleen but leave the organ at a more immature stage. A considerable part of the PCE from bone marrow also mature into NCE in the bone marrow. The rate of RNA breakdown in PCE follows an exponential function. Time-courses for the appearance of micronucleated PCE (MPCE) from spleen and from bone marrow were determined by analysis of samples taken with short intervals after an acute dose of 0.1 Gy X-rays. The time-courses were identical for MPCE from the spleen and the bone marrow. The frequency of MPCE (fMPCE) starts to increase at about 10 h after irradiation and reaches its maximum after about another 20 h upon which fMPCE returns to control level. The first induced MPCE in peripheral blood appear at about 20 h after irradiation. The effects of the carcinogen DMBA, 9,10-dimethyl-1,2-benzanthracene, at low doses were determined in PCE from spleen and bone marrow. The sensitivity was found to be about the same for erythroblasts in the spleen and the bone marrow. Protracted exposure to gamma-irradiation at a very low dose rate (44 mGy/day) gave a similar increase of fMPCE in bone marrow and spleen. The suitability of using splenic erythrocytes in the micronucleus test is discussed.

Analysis of the DNA content distribution of micronuclei using flow sorting and fluorescent in situ hybridization with a centromeric DNA probe.

The DNA content distributions of micronuclei induced in mouse 3T3 cells by ionizing radiation and chemicals was measured by flow cytometry. For a quantitative understanding of these distributions, micronuclei with increasing DNA contents were sorted and analysed for the presence of centromeric signals using fluorescent in situ hybridization (FISH) with a mouse centromeric gamma satellite probe. Radiation-induced micronuclei were found to be produced mainly by chromosome fragments, whereas micronuclei induced by the tear gas chlorobenzylidene malonitrile (CS) were found to be produced mainly by whole chromatids. In contrast, micronuclei induced by vinblastine (VBL) were, according to the shape of their DNA content distributions, produced mainly by whole chromosomes and by combinations of two or more whole chromosomes. With increasing DNA content, micronuclei induced by ionizing radiation also contained one or more whole chromosomes, whereas micronuclei induced by CS or VBL were found to contain several whole chromatids or chromosomes respectively. Computerized random breakage of chromosomes and random combination of chromosome fragments, whole chromatids and whole chromosomes were used according to the FISH results to simulate the measured DNA content distributions of micronuclei. A good agreement was obtained between measured and simulated distributions of micronuclei as well as between results of the measured frequency of micronuclei showing centromeric signals as a function of their DNA content and those predicted by the simulations. These results demonstrate the usefulness of flow cytometry and sorting combined with the FISH technique and computer simulations for producing a more detailed analysis of mechanisms of micronucleus induction.

The time-course of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood.

The time-course of micronucleated polychromatic erythrocytes (MPCE) in mouse bone marrow and peripheral blood, induced by an acute 0.1 Gy dose of X-rays, was determined using flow cytometric analysis, which made frequent sampling possible and allowed use of a dose low enough not to affect erythroid cell proliferation. The frequency of MPCE (fMPCE) began to increase in the bone marrow at 10 h after irradiation and reached a maximum at 28 h after irradiation. In the peripheral blood fMPCE began to increase at 20 h after irradiation and peaked at about 40 h after irradiation. The time-course found is discussed on the basis of data on the differentiation of erythroid cells. The results indicate that the micronuclei registered in polychromatic erythrocytes may originate from lesions induced not only during the last cell cycle but also during earlier ones. After an acute dose of 1.0 Gy of X-rays the maximum fMPCE was delayed both in bone marrow and peripheral blood reflecting an effect on the cell cycle progression of erythroblasts.

Flow-cytometric identification and follow-up of mice exposed to x-irradiation: evaluation of a model system.

An experimental model system is presented that allows the identification and follow-up of mice exposed to ionizing radiation using flow-cytometric measurements of peripheral blood cells. In an experiment, properties of peripheral blood cells were analysed with flow cytometry for a rapid identification of individuals exposed to radiation. Individuals were then followed longitudinally in an attempt to identify those developing neoplasias. Male CBA mice, 25 days old, were subjected to fractionated x-irradiation (4 x 1.31 Gy) to induce haematopoietic malignancies. By repeated blood sampling followed by flow cytometry, frequencies of micronucleated erythrocytes and of proliferating nucleated cells were determined. Neoplasias were diagnosed by histopathology. Five days after the end of radiation exposure, increased frequencies of proliferating cells, polychromatic erythrocytes and micronucleated normochromatic erythrocytes clearly distinguished the exposed group from the control group. Increased cell proliferation in peripheral blood cells could be used to identify animals with manifest tumours, although these animals were at a late stage of tumour development. Animals with thymic lymphoma (not generalized) could not be identified with the flow-cytometric parameters used. We consider that this model system has a potential use when a small number of risk individuals need to be identified and monitored within a large population.

Flow cytometric analysis of micronucleus induction in mice by internal exposure to 137Cs at very low dose rates.

Internal radiation from 137Cs, intraperitoneally injected into mice, induced chromosome damage seen as micronuclei in erythrocytes of peripheral blood harvested 72 h after injection and analysed with flow cytometry. The retention of injected 137Cs activity was determined and the absorbed doses obtained from the beta-radiation of 137Cs were calculated for the whole bodies and bone marrow of the treated mice. The absorbed doses during the most relevant period for micronucleus induction were 2.7-18.3 mGy per day. The dose to the bone marrow during the same period was calculated to be 6-44 mGy per day. A linear dose response relationship was found.