Jan Grawé

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.

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.

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.Purpose : To determine whether mice exposed to an extended low dose of γ-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 γ-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 170000 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.

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.

Flow cytometric analysis of micronuclei in erythrocytes.

The in vivo micronucleus (MN) test in bone marrow or peripheral blood erythrocytes is widely used as a short-term assay for the detection of agents able to induce chromosome aberrations in somatic cells and has also been shown to have good predictive potential for the identification of carcinogens and germ cell mutagens. The endpoint used is the scoring of micronuclei (MN) in bone marrow or peripheral blood erythrocytes of mice or rats. In this chapter, a detailed description of the flow cytometric micronucleus test will be given, as well as a more general description of the manual micronucleus assay. The DNA of MN is identified using the DNA-specific fluorescent stain Hoechst 33342; discrimination between polychromatic and normochromatic erthrocytes is based on staining with thiazole orange, a fluorescent probe with high RNA affinity. The use of flow-cytometric quantification of micronucleated polychromatic and normochromatic erythrocytes (MNPCE and MNNCE), beyond replacing manual enumeration, provides substantial advantages in terms of speed of analysis, as well as sensitivity. The general description of the MN assay briefly covers choice of animal species and strains, treatment regime, sampling times, and data interpretation. The description of the flow cytometric assay covers in detail erythrocyte preparation and purification, fixation, staining, data acquisition, and data analysis.

Genotoxicity of alcohol is linked to DNA replication-associated damage and homologous recombination repair

Although alcohol consumption is related to increased cancer risk, its molecular mechanism remains unclear. Here, we demonstrate that an intake of 10% alcohol for 4 weeks in rats is genotoxic due to induction of micronuclei. Acetaldehyde (AA), the first product of ethanol metabolism, is believed to be responsible for DNA damage induced by alcohol. Here, we observe that AA effectively blocks DNA replication elongation in mammalian cells, resulting in DNA double-strand breaks associated with replication. AA-induced DNA damage sites colocalize with the homologous recombination (HR) repair protein RAD51. HR measured in the hypoxhantineguaninefosforibosyltransferase (HPRT) gene is effectively induced by AA and recombination defective mammalian cells are hypersensitive to AA, clearly demonstrating that HR is essential in the repair of AA-induced DNA damage. Altogether, our data indicate that alcohol genotoxicity related to AA produces replication lesions on DNA triggering HR repair.

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.

The Src Homology 2 Protein Shb Promotes Cell Cycle Progression In Murine Hematopoietic Stem Cells By Regulation Of Focal Adhesion Kinase Activity

The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shbs known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time.

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.