Micronuclei in the blood and bone marrow cells of mice exposed to specific complex time‐varying pulsed magnetic fields

For 8 weeks, adult CD‐1 male mice were continuously exposed to complex time‐varying pulsed magnetic fields (PMF) generated in the horizontal direction by a set of square Helmholtz coils. The PMF were <1000 Hz and delivered at a peak flux density of 1 mT. Sham‐exposed mice were kept in a similar exposure system without a PMF. Positive control animals exposed to 1 Gy gamma radiation were also included in the study. Blood samples were collected before (time 0) and at 2, 4, 6, and 8 weeks. All mice were euthanized at the end of 8 weeks and their bone marrow was collected. From each blood and bone marrow sample, smears were prepared on microscope slides, fixed in absolute methanol, air‐dried, and stained with acridine orange. All slides were coded and examined using a fluorescence microscope. The extent of genotoxicity and cytotoxicity was assessed from the incidence of micronuclei (MN) and percent polychromatic erythrocytes (PCE) in the blood and bone marrow, respectively. The data indicated that both indices in PMF‐exposed mice were not significantly different from those observed in sham‐exposed animals. In contrast, positive control mice exhibited significantly increased MN, and decreased percentages of PCE in both tissues. Thus, the overall data suggested that 8 weeks of continuous exposure to PMF did not induce significantly increased genotoxicity and cytotoxicity in experimental mice. Further investigations are underway using other genotoxicity assays (comet assay, γ‐H2AX foci, and chromosomal aberrations) to assess genotoxicity following PMF exposure. Bioelectromagnetics 31:445–453, 2010. © 2010 Wiley‐Liss, Inc.     

Micronuclei in peripheral blood lymphocytes of workers exposed to lead

Lead plays an important role in many industrial processes. Although highly useful to man, lead has various types of toxic effects. There is constantly growing evidence of a relationship between the induction of chromosome breaks and an increased risk of onset of cancer. However, available data about the possible genotoxic and carcinogenic action of lead are conflicting. In this report we present the results of studies on lead concentrations in blood and the respective micronucleus frequencies in peripheral blood lymphocytes from workers employed in the recycling of automotive batteries in the surroundings of Porto Alegre, Brazil. We observed that in the occupationally exposed group, both lead concentration in peripheral blood and micronucleus frequency in lymphocytes were significantly higher compared to control (Z=6.35, P<0.0001 and Z=4.47, P<0.0001). The nuclear division index (NDI) values were significantly higher in the control group than in the exposed group (Z=2.13, P=0.0330), indicating a possible effect of Pb on nuclear proliferation. We also detected a negative correlation between micronuclei and progression of nuclear division (tau=-0.312, P=0.0129). There were no changes in micronucleus frequency between smoking and non-smoking workers exposed to lead (Z=0.03, P=0.9790). The only difference found between the groups of smokers and non-smokers was with respect to NDI, whose values were significantly higher among non-smokers (Z=1.98, P=0.0481).     

Micronuclei in peripheral blood lymphocytes and buccal epithelial cells of Polish farmers exposed to pesticides.

In this biomonitoring study, we investigated whether an occupational exposure to a complex mixture of chemical pesticides produced a significant increase of micronuclei (MN) in both peripheral blood lymphocytes and buccal cells. Forty-nine male workers exposed to pesticides, from an agricultural area of Malopolska Region in Southern Poland, together with 50 men from the same area without indication of exposure to pesticides that served as controls, were used in this investigation.No statistically significant differences in the frequencies of cytogenetic damage were detected between exposed and control individuals, for either type of cells. The multiple linear regression analysis in the case of lymphocytes indicated that the studied cytogenetic endpoints were inversely influenced by alcohol; whilst a negative binomial regression, in the case of buccal cells, indicated that the MN values were directly influenced by the ingestion of red meat. An inverse negative relationship between the cytokinesis-block proliferation index and age, and a significant increase of miscarriages due to the exposure to pesticides were also observed.     

Micronuclei in human peripheral blood lymphocytes exposed to mixed beams of X-rays and alpha particles

The purpose of this study was to analyse the cytogenetic effect of exposing human peripheral blood lymphocytes (PBL) to a mixed beam of alpha particles and X-rays. Whole blood collected from one donor was exposed to different doses of alpha particles ((241)Am), X-rays and a combination of both. All exposures were carried out at 37 °C. Three independent experiments were performed. Micronuclei (MN) in binucleated PBL were scored as the endpoint. Moreover, the size of MN was measured. The results show that exposure of PBL to a mixed beam of high and low linear energy transfer radiation led to significantly higher than expected frequencies of MN. The measurement of MN size did not reveal any differences between the effect of alpha particles and mixed beam. In conclusion, a combined exposure of PBL to alpha particles and X-rays leads to a synergistic effect as measured by the frequency of MN. From the analysis of MN distributions, we conclude that the increase was due to an impaired repair of X-ray-induced DNA damage.     

Changes of bone marrow cell and peripheral blood cell counts in newborn mice

Differential bone marrow cell and peripheral blood cell counts in new-born mice from zero to twenty-one days of age were investigated. In the bone marrow, erythroblasts rapidly increased whereas granulocytic cells decreased with time. The G/E ratio was 6.7 on day zero and 0.5 on day four, and as the G/E ratio of adult mice is normally from 0.8 to 2.4, these values would be the highest and lowest respectively for the whole life span of a mouse. In the peripheral blood, MCV decreased each day, closely matching the changes in the Price-Jones curves. The results of erythrocyte area measurements indicate that the larger the erythrocyte area, the earlier in days of age it disappeared and the shorter its life span.     

Protein changes in macrophages induced by plasma from rats exposed to 35 GHz millimeter waves

A macrophage assay and proteomic screening were used to investigate the biological activity of soluble factors in the plasma of millimeter wave‐exposed rats. NR8383 rat macrophages were incubated for 24 h with 10% plasma from male Sprague–Dawley rats that had been exposed to sham conditions, or exposed to 42 °C environmental heat or 35 GHz millimeter waves at 75 mW/cm² until core temperature reached 41.0 °C. Two‐dimensional polyacrylamide gel electrophoresis, image analysis, and Western blotting were used to analyze approximately 600 protein spots in the cell lysates for changes in protein abundance and levels of 3‐nitrotyrosine, a marker of macrophage stimulation. Proteins of interest were identified using peptide mass fingerprinting. Compared to plasma from sham‐exposed rats, plasma from environmental heat‐ or millimeter wave‐exposed rats increased the expression of 11 proteins, and levels of 3‐nitrotyrosine in seven proteins, in the NR8383 cells. These altered proteins are associated with inflammation, oxidative stress, and energy metabolism. Findings of this study indicate both environmental heat and 35 GHz millimeter wave exposure elicit the release of macrophage‐activating mediators into the plasma of rats. Bioelectromagnetics 31:656–663, 2010. © 2010 Wiley‐Liss, Inc.     

Colony formation inhibition in human bone marrow stromal cells exposed to a factor formed in vitro by peripheral blood leukocytes]

Addition of human peripheral blood leukocytes or a medium in which the leukocytes were cultivated to the monolayer culture of human bone marrow produced an inhibitory action of the growth of the fibroblast colonies. This effect was not associated with the immunological incompatibility of the cells in a culture--autologous blood cells produced the same action as the heterologous ones. The factor inhibiting the fibroblast growth failed to depress the growth of other cells, of macrophages in particular.     

Gene expression profile in bone marrow and hematopoietic stem cells in mice exposed to inhaled benzene

Acute myeloid leukemia and chronic lymphocytic leukemia are associated with benzene exposure. In mice, benzene induces chromosomal breaks as a primary mode of genotoxicity in the bone marrow (BM). Benzene-induced DNA lesions can lead to changes in hematopoietic stem cells (HSC) that give rise to leukemic clones. To gain insight into the mechanism of benzene-induced leukemia, we investigated the DNA damage repair and response pathways in total bone marrow and bone marrow fractions enriched for HSC from male 129/SvJ mice exposed to benzene by inhalation. Mice exposed to 100 ppm benzene for 6h per day, 5 days per week for 2 week showed significant hematotoxicity and genotoxicity compared to air-exposed control mice. Benzene exposure did not alter the level of apoptosis in BM or the percentage of HSC in BM. RNA isolated from total BM cells and the enriched HSC fractions from benzene-exposed and air-exposed mice was used for microarray analysis and quantitative real-time RT-PCR. Interestingly, mRNA levels of DNA repair genes representing distinct repair pathways were largely unaffected by benzene exposure, whereas altered mRNA expression of various apoptosis, cell cycle, and growth control genes was observed in samples from benzene-exposed mice. Differences in gene expression profiles were observed between total BM and HSC. Notably, p21 mRNA was highly induced in BM but was not altered in HSC following benzene exposure. The gene expression pattern suggests that HSC isolated immediately following a 2 weeks exposure to 100 ppm benzene were not actively proliferating. Understanding the toxicogenomic profile of the specific target cell population involved in the development of benzene-associated diseases may lead to a better understanding of the mechanism of benzene-induced leukemia and may identify important interindividual and tissue susceptibility factors.     

Chromosomal aberrations in bone marrow and peripheral blood cells from the same rats

Spontaneous cytogenetic aberrations were analyzed in bone-marrow cells and cultured peripheral lymphocytes from the same animals. No significant differences in the total number of cells with aberrations or total aberrations were detected between the bone-marrow cells and cultured lymphocytes. It was concluded that short-term culture does not contribute significantly to in vivo aberration yield within the experimental conditions used.     

In vivo flow cytometry combined with intravital microscopy to monitor kinetics of transplanted bone marrow mononuclear cells in peripheral blood and bone marrow

Bone marrow mononuclear cells (BM-MNCs) transplantation has evolved as a promising experimental treatment in various regenerative therapy fields, especially in clinical hematopoietic stem cells transplantation (HSCT). In vitro methods have mainly been used to study the pre-clinical kinetics of BM-MNCs in mice after transplantation. And it is difficult to monitor the dynamic homing of BM-MNCs in living mice. The present study obtained the kinetics of transplanted BM-MNCs in the peripheral blood (PB) and the dynamic homing of BM-MNCs in the BM in living mice by a combination of in vivo flow cytometry (IVFC) and calvarium intravital microscopy. We found out that BM-MNCs were cleared rapidly from the PB and mainly localized to various hematopoietic tissues after transplantation. The number of BM-MNCs in the PB decreased over time accompanied by an increase in the BM indeed after transplantation. In addition, a lower number of BM-MNCs were found home to calvaria than long bone, probably indicating long bone marrow might also be an important hematopoietic organ. Clinical studies will benefit from non-invasive measurements to monitor the dynamic homing of transplanted cells. Our pre-clinical kinetics of BM-MNCs in living mice will have important clinical guiding significance in HSCT and other regenerative therapy fields.

     

Frequency dependence of heating of human skin exposed to millimeter waves

In this paper we studied experimentally the frequency dependence of heating of human skin exposed to millimeter waves. Theoretical modeling of obtained data was performed using the hybrid bio-heat equation. It was found that the skin heating and SAR increased with increasing the exposure frequency. The frequency dependence of heating was entirely resulted from that of reflection from the skin. Unlike temperature, the frequency dependence of the SAR was due to the increased absorption of millimeter wave energy within the thin surface layer of the skin.     

Frequency dependence of heating of human skin exposed to millimeter waves

In this paper we studied experimentally the frequency dependence of heating of human skin exposed to millimeter waves. Theoretical modeling of obtained data was performed using the hybrid bio-heat equation. It was found that the skin heating and SAR increased with increasing the exposure frequency. The frequency dependence of heating was entirely resulted from that of reflection from the skin. Unlike temperature, the frequency dependence of the SAR was due to the increased absorption of millimeter wave energy within the thin surface layer of the skin.     

Haemoglobin types in peripheral blood and bone marrow cells in rats after a single blood loss.

The authors studied quantitative changes in the synthesis of haemoglobin types in rat peripheral erythrocytes and in the cells of the bone marrow erythrocytic series after a single blood loss. They demonstrated that raised synthesis of given haemoglobin types could be found in the peripheral cells 5-7 days after blood loss. These changes could already be detected in the bone marrow cells 24 hours after blood loss. The question of the probable site and level of the origin of altered haemoglobin synthesis is discussed.     

Supernatant of bone marrow mesenchymal stromal cells induces peripheral blood mononuclear cells possessing mesenchymal features

Increasing evidence shows that some cells from peripheral blood fibroblast-like mononuclear cells have the capacity to differentiate into mesenchymal lineages. However, the insufficiency of these cells in the circulation challenges the cell isolation and subsequently limits the clinical application of these cells. In the present study, the peripheral blood mononuclear cells (pbMNCs) were isolated from wound animals and treated with the supernatant of bone marrow mesenchymal stromal cells (bmMSCs). Results showed these pbMNCs were fibroblast-like, had stromal morphology, were negative for CD34 and CD45, but positive for Vimentin and Collagen I, and had the multipotency to differentiate into adipocytes and osteoblasts. We named these induced peripheral blood-derived mesenchymal stromal cells (ipbMSCs). Skin grafts in combination with ipbMSCs and collagen I were applied for wound healing, and results revealed ipbMSC exhibited similar potency and effectiveness in the promotion of wound healing to the bmMSCs. Hereafter, we speculate that the mixture of growth factors and chemokines secreted by bmMSCs may play an important roles in the induction of the proliferation and mesenchymal differentiation of mononuclear cells. Our results are clinically relevant because it provide a new method for the acquisition of MSCs which can be used as a candidate for the wound repair.     

Rhythms in human bone marrow and blood cells

In 24h studies of bone marrow (BM), circadian stage-dependent variations were demonstrated in the proliferative activity of BM cells from subsets of 35 healthy diurnally active men. On an average, the percentage of total BM cells in deoxyribonucleic acid (DNA) synthesis phase was 188% greater at midday than at midnight (circadian rhythm: p = 0.018; acrophase or peak time of 13: 16h). Patients with malignant disease (n = 15) and a normal cortisol circadian rhythm showed higher fractions of BM cells in S-phase at midday. Colony-forming units--granulocyte/macrophage (CFU-GM), an indicator of myeloid progenitor cells, showed the same circadian variation as DNA S-phase (average range of change or ROC = 136%; circadian rhythm: p < 0.001; acrophase of 12:09h). Deoxyribonucleic acid S-phase and CFU-GM in BM both showed a circannual rhythm (p = 0.015 and 0.008) with an identical acrophase of August 12. The daily peak in BM glutathione content, a tripeptide involved in cellular defense against cytotoxic damage, preceded BM proliferative peaks by 4-5 h (ROC = 31-90%; circadian rhythm: p = 0.05; acrophase of 08:30h). Myeloid (ROC = 57%; circadian rhythm: p = 0.056; acrophase at 08:40h) and erythroid (ROC = 26%; circadian rhythm: p = 0.01; acrophase of 13:01h) precursor cells were positively correlated (r = 0.41; p < 0.001), indicating a circadian temporal relationship and equal influence on S-phase of total BM cells. Yield of positive selected CD34+ progenitor stem cells also showed significant circadian variation (ROC = 595%; circadian rhythm: p = 0.02; acrophase of 12:40h). Thus, the temporal synchrony in cell cycling renders BM cells more sensitive at specific times to hematopoietic growth factors and cell cycle-specific cytotoxic drugs. Moreover, proper timing of BM harvesting may improve progenitor cell yield. When using marker rhythms in the blood to allow for individualized timing of BM procedures, the times of low values in white blood corpuscles, neutrophils, and lymphocytes and high values in cortisol were predictive of the times of highest BM erythroid, myeloid, and total S-phase numbers occurring in the following 12 h.     

Absence of mutagenicity effects of Psidium cattleyanum Sabine (Myrtaceae) extract on peripheral blood and bone marrow cells of mice

Cattley guava (Psidium cattleyanum Sabine) is a native fruit of Brazil that is popular both as a sweet food and for its reputed therapeutic properties. We examined whether it could damage DNA using the alkaline single-cell gel electrophoresis (comet assay) and the micronucleus test in leukocytes and in bone marrow cells of mice. P. cattleyanum leaf extract was tested at concentrations of 1000, 1500 and 2000 mg/kg. N-nitroso-N-ethylurea was used as a positive control. Peripheral blood leukocytes were collected 4 and 24 h after the treatments for the comet assay, and bone marrow cells were collected after 24 and 48 h for the micronucleus test. Unlike N-nitroso-N-ethylurea, P. cattleyanum extract failed to induce a significant increase in cell DNA damage, in micronucleated cell frequency, and in bone marrow toxicity. The lack of mutagenicity and cytotoxicity with high doses of this plant extract means that it can be safely used in traditional medicine.