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1.
Int J Tuberc Lung Dis ; 14(10): 1272-9, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20843418

ABSTRACT

SETTING: Health care students in Sweden. OBJECTIVE: To analyse the distribution of tuberculin skin test (TST) reactions and epidemiological factors related to TST reactivity. DESIGN: TST reactivity was analysed in 1190 students. A linear regression model was created for the relative contribution of background factors of TST reactivity. A subgroup of 287 non-vaccinated subjects was comparatively skin-tested with Mycobacterium avium sensitin and tuberculin. RESULTS: Among non-bacille Calmette-Guérin (BCG) vaccinated students, 91% had no TST reaction (0 mm induration) and reactions of ≥ 10 mm were found in 2.9%, whereas 34% of BCG-vaccinated students had no TST reaction and 42% had reactions of ≥ 10 mm. The expected contribution to TST reactivity was 6.0 mm for a history of BCG vaccination, 3.0 mm for a country of birth with medium/high incidence of TB and 1.6 mm per 10 years of age. The sensitin reactions exceeded the TST reactions by ≥ 3 mm in 52% of the comparatively tested subjects with TST reactions of ≥ 1 mm. CONCLUSION: BCG vaccination, cross-reactivity with non-tuberculous mycobacteria, geographic origin and age had a decisive influence on TST reactivity. Most non-vaccinated health care students were non-reactive, which highlights the need to organise preventive measures in settings where TB exposure is expected.


Subject(s)
Health Personnel , Mycobacterium/immunology , Students , Tuberculin Test , Tuberculosis/diagnosis , Adolescent , Adult , Age Factors , BCG Vaccine , Female , Health Personnel/statistics & numerical data , Humans , Linear Models , Male , Middle Aged , Predictive Value of Tests , Prevalence , Sensitivity and Specificity , Sex Factors , Students/statistics & numerical data , Sweden/epidemiology , Tuberculosis/ethnology , Tuberculosis/immunology , Tuberculosis/prevention & control , Young Adult
2.
Hum Exp Toxicol ; 19(7): 402-11, 2000 Jul.
Article in English | MEDLINE | ID: mdl-11002390

ABSTRACT

Exposure to quartz induces pulmonary inflammation and development of fibrosis. In order to study the fibrosing process, we investigated morphology, function and phenotype of alveolar (AMs) and interstitial (IMs) macrophages at an early stage of fibrosis in rats. Rats were exposed by intratracheal instillations of 10 mg quartz (n=8) or saline (n=8) and studied 3 months later. AMs were obtained by bronchoalveolar lavage and IMs by mechanical fragmentation, followed by enzymatic digestion of lung tissue. Histology revealed subacute silicosis, with early focal fibrosis and alveolar lipoproteinosis. AM quartz exposure increased phagocytic activity and expression of major histocompatibility complex (MHC) Ia antigens, the latter being associated with cellular antigen presenting capacity. IM had an even more pronounced expression of MHC than AM after quartz exposure. Both macrophage fractions had a higher expression of OX-42 (complement receptor 3, CR3) than controls, but the increase in the IM fraction might be explained by the remaining AM in the IM fraction. Exposed AM adhered less to extracellular matrix components (vitronectin and fibronectin) than controls. In contrast, the adhesion of IM to vitronectin increased after exposure. Besides increased adhesion, the effects on IM were scarce. Our results therefore do not support the hypothesis that IM has a key role in the process of inflammation, including fibrosis.


Subject(s)
Macrophages, Alveolar/pathology , Macrophages/pathology , Pulmonary Alveoli/pathology , Pulmonary Fibrosis/pathology , Quartz/toxicity , Animals , Cell Adhesion , Cell Count , Intubation, Intratracheal , Macrophages/metabolism , Macrophages/ultrastructure , Macrophages, Alveolar/metabolism , Macrophages, Alveolar/ultrastructure , Male , Microscopy, Electron , Phagocytosis/drug effects , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/ultrastructure , Pulmonary Fibrosis/metabolism , Quartz/administration & dosage , Rats , Rats, Sprague-Dawley , Receptors, Cell Surface/metabolism
3.
Int J Radiat Biol ; 76(7): 971-7, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10923621

ABSTRACT

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.


Subject(s)
Chromosome Aberrations , Erythroid Precursor Cells/radiation effects , Fetus/radiation effects , Animals , DNA Damage , Female , Gamma Rays , Male , Mice , Mice, Inbred CBA , Pregnancy
4.
Environ Mol Mutagen ; 36(1): 22-31, 2000.
Article in English | MEDLINE | ID: mdl-10918356

ABSTRACT

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.


Subject(s)
Cytogenetics/methods , Flow Cytometry/methods , Micronucleus Tests/methods , Reticulocytes/physiology , Transferrin/metabolism , Adult , Age Factors , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Neoplasms/blood , Neoplasms/therapy , Reference Values
5.
Inflammation ; 23(2): 179-89, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10213273

ABSTRACT

The effects of pulmonary surfactant on granulocytes were studied by flow cytofluorometry. Cells from hemolyzed blood were first activated by N-formylmethionyl-leucyl-phenylalanine (fMLP) which mobilizes complement receptor 1 (CR1) from the intracellular pool to the cell surface. The reduced CR1 expression observed after quartz incubation was abolished by a porcine surfactant preparation containing phospholipids and the hydrophobic surfactant proteins. Phospholipids alone had no preservative capacity. However, addition of the surfactant proteins to the phospholipids did not restore the CR1 values to that of intact surfactant, probably due to changed protein structure during the purification procedure. Heating of surfactant at 37 degrees C up to 72 h reduced the preservative effect of surfactant on CR1 expression. Congruent results of CR1 expression were achieved when 1-10% lysophosphatidylcholine was added to the surfactant preparations. Our results imply that lysophosphatidylcholine formed during storage of surfactant at elevated temperatures influences CR1 expression on granulocytes.


Subject(s)
Granulocytes/drug effects , Lysophosphatidylcholines/pharmacology , Pulmonary Surfactants/antagonists & inhibitors , Quartz/pharmacology , Receptors, Complement 3b/metabolism , Hot Temperature/adverse effects , Humans , Lysophosphatidylcholines/metabolism , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Phosphatidylcholines/metabolism , Pulmonary Surfactants/pharmacology , Receptors, Complement 3b/biosynthesis
6.
Mutat Res ; 423(1-2): 113-24, 1999 Jan 25.
Article in English | MEDLINE | ID: mdl-10029688

ABSTRACT

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.


Subject(s)
Cyclophosphamide/toxicity , Erythrocytes/drug effects , Erythrocytes/radiation effects , Spleen/pathology , Vincristine/toxicity , Animals , Bone Marrow Cells/drug effects , Bone Marrow Cells/pathology , Bone Marrow Cells/radiation effects , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Erythrocytes/pathology , Erythroid Precursor Cells/drug effects , Erythroid Precursor Cells/pathology , Erythroid Precursor Cells/radiation effects , Flow Cytometry , Male , Micronucleus Tests/methods , Rats , Rats, Sprague-Dawley , Spleen/drug effects , Spleen/radiation effects , X-Rays
7.
Mutat Res ; 405(2): 199-208, 1998 Sep 20.
Article in English | MEDLINE | ID: mdl-9748577

ABSTRACT

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.


Subject(s)
Flow Cytometry/methods , Micronucleus Tests/methods , Mutagens/toxicity , Animals , Dose-Response Relationship, Drug , Male , Mice , Mice, Inbred CBA , Sensitivity and Specificity
8.
Respir Med ; 92(4): 653-8, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9659532

ABSTRACT

Lung volume reduction surgery for severe emphysema with removal of 20-30% of the most destroyed parts of the lung parenchyma has been reported to improve lung function substantially. Increased elastic recoil has been suggested as one underlying mechanism for the improvement. Fourteen patients, seven men and seven women with a mean age of 62 years, who underwent bilateral lung volume reduction surgery have been followed up for 3 months. We here report the data on quality of life, lung function and elastic recoil. FEV1.0 increased by a mean of 26% from 0.581 to 0.731 (P < 0.01). The mean TLC was reduced by 16% from 8.91 to 7.51 (P < 0.001). The level of hyperinflation decreased as implied by a reduction in the ratio of RV to TLC from 0.70 to 0.60 (P < 0.001). The pulmonary elastic recoil improved, with an increase in the transpulmonary pressure at maximal inspiration (PelTLC) from 0.95 kPa to 1.35 kPa (P < 0.05) and an average increase in the coefficient of retraction PelTLC/TLC) from 0.12 kPa l-1 to 0.19 kPa l-1 (P < 0.01). The resting PaO2 increased from a mean of 8.7 kPa to 9.8 kPa (P < 0.01). The patients reported a high degree of subjective improvement according to the St. George's Respiratory Questionnaire and the working capacity on a bicycle increased by 26% from a mean of 38 W to 48 W (P < 0.01). The promising short-term results of lung volume reduction surgery for severe emphysema appear to be related to improved pulmonary elastic recoil.


Subject(s)
Lung Compliance/physiology , Lung/physiology , Pneumonectomy , Pulmonary Emphysema/surgery , Quality of Life , Aged , Female , Forced Expiratory Volume , Humans , Male , Middle Aged , Pulmonary Emphysema/physiopathology , Total Lung Capacity
9.
Am J Physiol ; 274(2): L226-34, 1998 02.
Article in English | MEDLINE | ID: mdl-9486207

ABSTRACT

Macrophages play an essential role in pulmonary host defense. We investigated differences between rat alveolar (AM) and interstitial (IM) macrophages after exposure in vivo to quartz, an inducer of intensive alveolitis. Rats were exposed to 0.5 ml of saline without (n = 8) or with (n = 8) 10 mg of quartz by intratracheal instillation. In a third group (n = 8), 10 mg of surfactant were added to the quartz mixture. Five weeks later, AM were recovered by bronchoalveolar lavage and IM by mechanical fragmentation of the lung, followed by enzymatic treatment. Contamination of AM to the IM fraction was calculated to be 12-15%. After quartz exposure, the expression of major histocompatibility complex class Ia was increased in both AM and IM fractions. The receptor corresponding to human complement receptor 3 increased in AM after quartz exposure, and AM from quartz-exposed animals had a lower metabolic activation. Our findings indicate that IM are immunocompetent cells and that differences between AM and IM fractions occur upon quartz-induced inflammation. This response is not affected by addition of surfactant.


Subject(s)
Macrophages, Alveolar/drug effects , Macrophages/drug effects , Quartz/pharmacology , Animals , Bronchoalveolar Lavage Fluid/cytology , Cell Adhesion/drug effects , Humans , Lung/cytology , Macrophages/physiology , Macrophages, Alveolar/physiology , Male , Phagocytosis , Rats , Rats, Sprague-Dawley
10.
Mutat Res ; 393(1-2): 55-71, 1997 Sep 18.
Article in English | MEDLINE | ID: mdl-9357562

ABSTRACT

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.


Subject(s)
Animals, Wild/genetics , Arvicolinae/genetics , Erythrocytes/radiation effects , Erythrocytes/ultrastructure , Micronuclei, Chromosome-Defective/radiation effects , Micronucleus Tests/methods , Muridae/genetics , Animals , Animals, Wild/blood , Arvicolinae/blood , Bone Marrow/radiation effects , Bone Marrow/ultrastructure , Environmental Monitoring , Flow Cytometry , Gamma Rays , Mice , Mice, Inbred CBA , Muridae/blood , Species Specificity , X-Rays
11.
Inflammation ; 21(1): 83-92, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9179624

ABSTRACT

Quartz is known to induce an inflammatory response in the alveolar space by recruitment of different effector cells. We investigated the interaction between granulocytes and quartz with respect to expression of complement receptor type 1 (CR1) and CR3, with and without the presence of surfactant. Granulocytes from hemolyzed blood were stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), which mobilize the intracellular pool of CR1 to the surface, and the mean fluorescence intensity (MFI) measured by cytofluorometry was 47.4 (46-63.6) (median; interquartile range). Quartz exposure reduced the CR1 expression to 23.2 (22.8-30.6) MFI units (P < 0.01), a porcine surfactant preparation added during quartz exposure abolished the down-regulation completely, 47.7 (43.2-62.3) MFI units (P < 0.001). Similar results were obtained after preincubation of the cells with surfactant followed by quartz exposure. No significant influence on CR1 expression was found by a synthetic lipid mixture, nor was the CR3 expression affected. In conclusion, this study demonstrates that the presence of surfactant inhibits quartz induced down-regulation of CR1 on activated granulocytes.


Subject(s)
Down-Regulation/drug effects , Granulocytes/drug effects , Pulmonary Surfactants/pharmacology , Quartz/toxicity , Receptors, Complement/antagonists & inhibitors , Receptors, Complement/drug effects , Adolescent , Adult , Aged , Animals , Granulocytes/metabolism , Humans , Lipids/chemical synthesis , Lipids/pharmacology , Middle Aged , Receptors, Complement/biosynthesis , Swine
12.
Mutat Res ; 394(1-3): 17-28, 1997 Nov 27.
Article in English | MEDLINE | ID: mdl-9434839

ABSTRACT

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.


Subject(s)
Erythropoiesis , Micronuclei, Chromosome-Defective , Spleen/ultrastructure , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Bone Marrow Cells , Erythrocytes/metabolism , Erythrocytes/ultrastructure , Flow Cytometry , Male , Mice , Mice, Inbred CBA , RNA/metabolism , Spleen/drug effects , Spleen/physiology , Spleen/radiation effects
13.
Mutat Res ; 350(2): 349-58, 1996 Mar 09.
Article in English | MEDLINE | ID: mdl-8600364

ABSTRACT

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.


Subject(s)
Bone Marrow/radiation effects , Erythrocytes/physiology , Micronuclei, Chromosome-Defective/radiation effects , Animals , Bone Marrow/physiology , Bone Marrow Cells , Erythrocytes/cytology , Erythrocytes/radiation effects , Flow Cytometry , Kinetics , Male , Mice , Mice, Inbred CBA , Micronuclei, Chromosome-Defective/physiology , Time Factors , X-Rays
14.
Sarcoidosis ; 12(1): 46-50, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7617976

ABSTRACT

In disorders affecting the alveoli and lung interstitium and altered composition of the epithelial lining fluid, i.e. the surfactant, may affect the outcome of the disease. The phospholipid composition in bronchoalveolar lavage (BAL) fluid was determined in healthy non-smoking (n = 8) and smoking (n = 12) volunteers, and in non-smoking patients with clinically active sarcoidosis (n = 7). The total amount of phosphatidylcholines (median +/- SD) were in the non-smoking control group (21.8 +/- 5.7 mumol/L) and in the non-smoking sarcoidosis group (26.1 +/- 9.1 mumol/L), while healthy smokers had significantly (p < 0.05 for both) lower amounts (14.6 +/- 5.6 mumol/L). The composition of phosphatidylcholines was similar in all three groups with one exception. Palmitoylmyristoyl-phosphatidylcholine constituted a significantly higher fraction among the smokers (12.7 +/- 2.1 mol%) compared to the non-smoking control group (10.6 +/- 1.4 mol%; p < 0.05) and the sarcoidosis group (10.6 +/- 0.6 mol%; p < 0.01). In conclusion, no quantitative or qualitative differences in phosphatidylcholines were observed between non-smoking healthy volunteers and non-smoking patients with clinically active sarcoidosis. However, in smoking healthy volunteers the total amount of phosphatidylcholines was reduced and their composition altered. Earlier reported conflicting results may be due to the fact that the smoking habits have not been considered.


Subject(s)
Bronchoalveolar Lavage Fluid/chemistry , Phosphatidylcholines/metabolism , Phospholipids/metabolism , Pulmonary Surfactants/metabolism , Sarcoidosis, Pulmonary/metabolism , Smoking/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Chromatography, Gas , Female , Humans , Male , Middle Aged , Sarcoidosis, Pulmonary/diagnosis
15.
Int J Radiat Biol ; 67(1): 29-36, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7852814

ABSTRACT

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.


Subject(s)
Bone Marrow/radiation effects , Cesium Radioisotopes , Erythrocytes/radiation effects , Micronuclei, Chromosome-Defective/radiation effects , Micronucleus Tests/methods , Animals , Benzothiazoles , Cesium Radioisotopes/administration & dosage , Dose-Response Relationship, Radiation , Flow Cytometry/methods , Fluorescent Dyes , Injections, Intraperitoneal , Male , Mice , Mice, Inbred CBA , Quinolines , Thiazoles
16.
Mutagenesis ; 9(1): 31-8, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8208128

ABSTRACT

A fixation method for mouse bone marrow erythrocytes was developed which allows both flow cytometric enumeration of micronucleated polychromatic erythrocytes (MPCEs) and detection of centromeres using fluorescent in situ hybridization (FISH) with a mouse gamma satellite probe on flow-sorted MPCEs. Male CBA mice were treated with clastogens or spindle poisons: X-irradiation (XR; 0.5 Gy), cyclophosphamide (CPA; 80 mg/kg), vincristine sulphate (VCR; 0.125 mg/kg) and colchicine (COL; 1 mg/kg). At 30 and 50 h after treatment bone marrow suspensions were prepared and subsequently analysed by flow cytometry to enumerate and determine the DNA content of induced MPCEs. The mean DNA content in MPCEs was found to be higher after treatments with the two aneugens, VCR and COL, than with the two clastogens, CPA and XR. The mean DNA content of MPCEs was positively correlated with the mean proportion of micronuclei (MN) containing centromeres, indicating that determination of the mean DNA content alone can give information about the mechanism of MN induction. When the MPCEs induced with VCR were outsorted according to four classes of increasing DNA content and the presence or absence of centromeres was determined with FISH, it was found that only the class with the lowest DNA content (0.8-1.7% of the diploid DNA content) had a low proportion (< 20%) of centromere-containing micronuclei while in the three classes with higher DNA content (1.7-10.2% of the diploid DNA content) > 80% of the MN had centromeres. This was in contrast to the results from treatments with CPA where the proportion of centromere-containing MN did not increase with increasing DNA content.


Subject(s)
Bone Marrow/metabolism , Bone Marrow/ultrastructure , Centromere/ultrastructure , DNA/metabolism , Micronuclei, Chromosome-Defective/metabolism , Micronuclei, Chromosome-Defective/ultrastructure , Animals , Base Sequence , Bone Marrow/drug effects , Centromere/drug effects , Colchicine/toxicity , Cyclophosphamide/toxicity , DNA/genetics , DNA Primers/genetics , Flow Cytometry , In Situ Hybridization, Fluorescence , Male , Mice , Mice, Inbred CBA , Micronuclei, Chromosome-Defective/drug effects , Molecular Sequence Data , Mutagenicity Tests , Mutagens/toxicity , Vincristine/toxicity
17.
Int J Radiat Biol ; 64(5): 555-64, 1993 Nov.
Article in English | MEDLINE | ID: mdl-7902395

ABSTRACT

Male CBA-S mice were subjected to protracted gamma-irradiation. Two groups of five animals were each exposed to dose rates of 6 and 30 mGy/day for 56 days, respectively, upon which irradiation was terminated and the groups were followed for an additional 49 days. Frequencies of micronucleated poly- and normochromatic erythrocytes in peripheral blood samples were determined before (day 0), during (day 14, 28 and 56), and after (day 70 and 105) irradiation using flow cytometry. A second experiment was performed as above, but with exposure limited to 7 days. Frequencies of micronucleated polychromatic erythrocytes in bone marrow and peripheral blood were determined. Significantly elevated frequencies of micronuclei in peripheral blood polychromatic erythrocytes were found for the 30-mGy/day dose group on day 14, 28 and 56 and for the 6-mGy/day dose group on day 28 and 56. In normochromatic erythrocytes from peripheral blood significantly elevated frequencies were found on all sampling occasions with mouse given 30 mGy/day, while those given 6 mGy/day showed significantly elevated frequencies on day 28, 56 and 70. The frequencies of micronucleated polychromatic erythrocytes were found to be similar in bone marrow and peripheral blood, while the frequencies of micronucleated normochromatic erythrocytes were consistently lower than for micronucleated polychromatic erythrocytes at all samplings for all groups. On day 28 the frequencies (mean +/- SE) of micronucleated polychromatic erythrocytes in peripheral blood were 0.0016 +/- 0.0001 for the control group, 0.0019 +/- 0.0001 for the 6-mGy/day group and 0.0028 +/- 0.0003 for the 30-mGy/day group. The results show an elevated induction of micronuclei in erythroblasts at a dose rate of approximately 3 mGy per cell cycle.


Subject(s)
Erythrocytes/radiation effects , Micronucleus Tests , Animals , Cesium Radioisotopes , Dose-Response Relationship, Radiation , Flow Cytometry , Gamma Rays , Male , Mice , Mice, Inbred CBA , Radiation Genetics
18.
Mutagenesis ; 8(3): 249-55, 1993 May.
Article in English | MEDLINE | ID: mdl-8332088

ABSTRACT

The frequencies and DNA distributions of micronuclei (MN) in polychromatic erthrocytes (PCEs) from bone marrow (BM) and peripheral blood (PB) of mice after four different treatments were determined by flow cytometry. PCEs were differentiated from normochromatic erythrocytes (NCEs) using the fluorescent RNA stain Thiazole orange, while MN in erythrocytes were detected with the DNA stain Hoechst 33342. The treatments were X-irradiation (1 Gy), cyclophosphamide (CPA; 30 mg/kg), vincristine sulphate (VCR; 0.08 mg/kg), and cholchicine (COL; 1 mg/kg). All treatments showed increased frequencies of micronucleated PCEs at 30 h after treatment in BM and at 50 h in PB. The clastogens (X-irradiation and CPA) and the spindle poisons (VCR and COL) could be grouped according to the fluorescent characteristics of the induced MN as well as the relative frequency of small (0.5-2% of the diploid DNA content) and large (2-10%) MN. In PB the relative frequency of large MN was lower than in BM, indicating that they were partly eliminated before entrance into the peripheral circulation.


Subject(s)
Bone Marrow/drug effects , Colchicine/toxicity , Cyclophosphamide/toxicity , DNA/metabolism , Erythrocytes/drug effects , Micronucleus Tests , Mutagens/toxicity , Vincristine/toxicity , Animals , Bone Marrow/pathology , Bone Marrow/radiation effects , DNA/drug effects , DNA/radiation effects , Erythrocytes/pathology , Erythrocytes/radiation effects , Male , Mice , Mice, Inbred CBA , Time Factors , X-Rays
19.
Int J Radiat Biol ; 63(3): 339-47, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8095284

ABSTRACT

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.


Subject(s)
Erythrocytes/radiation effects , Radiation Injuries, Experimental/genetics , Animals , Cesium Radioisotopes , Flow Cytometry , Male , Mice , Mice, Inbred CBA , Micronucleus Tests , Radiation Dosage , Radiation Genetics , Time Factors
20.
Cell Biol Toxicol ; 8(4): 233-53, 1992.
Article in English | MEDLINE | ID: mdl-1493584

ABSTRACT

Attempts have been made to use the hypoxanthine-guanine-phospho-ribosyl-transferase-assay as a method for automated screening of agent-induced phenotypic variants of human peripheral lymphocytes reflecting 6-thioguanine resistance and assumed to indicate genotoxic action. Different protocols of the hypoxanthine-guanine-phospho-ribosyl-transferase-system were used in this study in order to investigate whether the system can be a candidate for a short-term test for a rapid and reliable identification of biological systems exposed to agents. The current protocols were based on: 1) fluoresceinated monoclonal antibodies against bromodeoxyuridine-DNA for labelling of 6-thioguanine-resistant human lymphocytes and direct flow-cytometric enumeration of bromodeoxyuridine-positive events and: 2) indirect flow-cytometric enrichment of 6-thioguanine-resistant cells labelled with 3H-thymidine followed by autoradiographic enumeration of positive events. Both the direct and the indirect enumeration method yielded similar results down to the range 10(-4) with respect to frequency of variants. For the less time-consuming direct enumeration method the resolution was limited due to non-specific binding of the antibody and false positives. It was, nevertheless, sufficient to score variants induced in vitro with the mutagens EMS, MMC and TT in the same range as e.g. that of cancer patients during and after chemotherapy or radiotherapy, or that of psoriasis patients during the after PUVA (8-methoxypsoralen and long range UV light)-therapy. We conclude that the direct enumeration protocol can be used for a rapid screening of so called outliers, but a more sensitive test, such as the more time-consuming enrichment protocol based on autoradiography, must be used in order to score variants in the range 10(-5)-10(-6).


Subject(s)
Lymphocytes/drug effects , Mutagenicity Tests , Mutagens/toxicity , Thioguanine/pharmacology , Autoradiography , Bromodeoxyuridine , Cell Cycle/drug effects , Cells, Cultured , DNA/analysis , Drug Resistance , Ethyl Methanesulfonate/toxicity , Flow Cytometry , Humans , Hypoxanthine Phosphoribosyltransferase/metabolism , Lymphocyte Activation , Male , Microscopy, Fluorescence , Mitomycin/toxicity , Thiotepa/toxicity
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