Gösta Zetterberg

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.

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. Environ. Mol. Mutagen. 36:22–31, 2000.

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 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.

The influence of pH on the effects of 2,4-D (2,4-dichlorophenoxyacetic acid, Na salt) on Saccharomyces cerevisiae and Salmonella typhimurium

The genetic effects of 2,4-D (2,4-dichlorophenoxyacetic acid, Na salt) have been investigated in cells of the yeast Saccharomyces cerevisiae and of the bacterium Salmonella typhimurium in experiments in vitro and in vivo. Experiments in vitro showed that the killing of both yeast and bacteria is dependent on the pH in the treatment solution of 2,4-D. A dose-dependent increase of the frequency of mitotic gene conversion and mitotic recombination in yeast was observed at pH 4.50 and 4.30. In experiments in vitro with two strains of Salmonella no significant increase of the number of revertants to prototrophy was obtained. The positive correlation between survival of cells and dissociation of 2,4-D in the pH region 2.8-5.0 indicates that the cells are unable to take up dissociated 2,4-D. Therefore the survival is high at a high pH when most 2,4-D is in dissociated form, and the survival is low at a relatively low pH when more of the 2,4-D is in its undissociated form. No genetic effects were induced by oral administration of tolerable doses of 2,4-D in host-mediated assays using mice as hosts and yeast or Salmonella as indicator cells.

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.

Effects of Extended Low-dose-rate Exposure to 137Cs Detected by Flow-cytometric Enumeration of Micronucleated Erythrocytes in Mouse Peripheral Blood

An automated variant the of micronucleus assay for erythrocytes in mouse peripheral blood has recently been developed. The flow-cytometric technique used allows very large numbers of erythrocytes to be analysed with a relatively small effort. Here we report the potential of this method to detect a response to extended low-dose-rate exposure to gamma-irradiation. The mice were irradiated with a 137Cs source at a dose rate of 4.8 cGy/day for 26 days. Sampling was continued for another 39 days after irradiation. Elevated frequencies compared with the control group were found at days 2, 9 and 20 after the start of the irradiation for micronucleated polychromatic erythrocytes, and at days 9, 20, 29, 42, 51 and 65 for micronucleated normochromatic erythrocytes.

Mechanism of the lethal and mutagenic effects of phenoxyacetic acids in Saccharomyces cerevisiae

MCPA and salicylic acid, two compounds with similar structures and almost the same dissociation pattern, were tested for lethal and mutagenic effects on, and uptake by, cells of Saccharomyces cerevisiae strain rad18. The results obtained with the two compounds were similar, suggesting a common mechanism of action. It is proposed that they act by increasing the concentration of hydrogen ions within the cell, so that killing and mutation occur. Mutations were induced only when killing reached 95--99%. The compounds are considered weak mutagens for yeast cells. The methyl ester of MCPA also induced killing and reverse mutation, but only at concentrations about 100 times higher than for the undissociated acid. MCPA methyl ester did not increase the number of revertants in the Salmonella/liver microsome test. It is suggested that the effects of the methyl ester of MCPA depends on the ester being hydrolysed to the acid by yeast cells and the liver microsome preparation.

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.

THE LETHAL AND MUTAGENIC EFFECTS OF N-NITROSO-N-METHYL-URETHANE AND N-NITROSO-N-ETHYLURETHANE IN COLLETOTRICHUM COCCODES.

Abstract The lethal and mutagenic effects of N -nitro- N -methylurethane (NMU) and N -nitroso- N -ethylurethane (NEU) was investigated in the fungus Colletotrichum coccodes . NMU was found to be much more toxic than NEU. Studies on the breakdown of the compounds showed that their stability was high in water solutions at room temperature, and that the rate of breakdown was considerably increased in cell suspensions. The shapes of the survival curves obtained at different concentrations of NMU and NEU are interpreted on the basis of these findings. The mechanism of action is discussed. At the same survival level NMU was found to be more mutagenic than NEU. A total of 177 auxotrophic mutants were isolated. Indications were found that the mutation spectra induced by NMU and NEU in Colletotrichum are different.