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1.
Biol. Res ; 47: 1-5, 2014. ilus, graf
Article in English | LILACS | ID: biblio-950771

ABSTRACT

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.


Subject(s)
Animals , Rats , Technology Assessment, Biomedical/methods , Tetrazolium Salts/pharmacology , Trachea/cytology , Bromodeoxyuridine/pharmacology , Myocytes, Smooth Muscle/physiology , Cell Proliferation/physiology , Reagent Kits, Diagnostic , Trachea/growth & development , Enzyme-Linked Immunosorbent Assay , Cell Survival/physiology , Calgranulin B/administration & dosage , Primary Cell Culture
2.
Braz. j. med. biol. res ; 43(12): 1143-1152, Dec. 2010. ilus
Article in English | LILACS | ID: lil-569006

ABSTRACT

5-Bromo-2’-deoxyuridine (BrdUrd) has long been known to interfere with cell differentiation. We found that treatment ofBradysia hygida larvae with BrdUrd during DNA puff anlage formation in the polytene chromosomes of the salivary gland S1 region noticeably affects anlage morphology. However, it does not affect subsequent metamorphosis to the adult stage. The chromatin of the chromosomal sites that would normally form DNA puffs remains very compact and DNA puff expansion does not occur with administration of 4 to 8 mM BrdUrd. Injection of BrdUrd at different ages provoked a gradient of compaction of the DNA puff chromatin, leading to the formation of very small to almost normal puffs. By immunodetection, we show that the analogue is preferentially incorporated into the DNA puff anlages. When BrdUrd is injected in a mixture with thymidine, it is not incorporated into the DNA, and normal DNA puffs form. Therefore, incorporation of this analogue into the amplified DNA seems to be the cause of this extreme compaction. Autoradiographic experiments and silver grains counting showed that this treatment decreases the efficiency of RNA synthesis at DNA puff anlages.


Subject(s)
Animals , Bromodeoxyuridine/pharmacology , DNA , Diptera/genetics , Insect Proteins/drug effects , Salivary Glands/chemistry , Salivary Proteins and Peptides/drug effects , Autoradiography , Cell Differentiation , Insect Proteins/genetics , Larva/drug effects , Salivary Glands/drug effects , Salivary Proteins and Peptides/genetics
3.
Journal of Korean Medical Science ; : 500-505, 2006.
Article in English | WPRIM | ID: wpr-47125

ABSTRACT

We investigated the effect of low dose radiation on diabetes induced suppression of neurogenesis in the hippocampal dentate gyrus of rat. After 0.01 Gy, 0.1 Gy, 1 Gy and 10 Gy radiation was delivered, the dentate gyrus of hippocampus of streptozotocin (STZ)-induced diabetic rats were evaluated using immunohistochemistry for 5-bromo-2-deoxyuridine (BrdU), caspase-3, and terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) staining. The number of BrdU positive cells in the non-diabetic rats, diabetic rats without radiation, diabetic rats with 0.01 Gy radiation, diabetic rats with 0.1 Gy radiation, diabetic rats with 1 Gy radiation and diabetic rats with 10 Gy radiation were 55.4+/-8.5/mm2, 33.3+/-6.4/mm2, 67.7+/-10.5/mm2, 66.6+/-10.0/mm2, 23.5+/-6.3/mm2 and 14.3+/-7.2/mm2, respectively. The number of caspase-3 positive cells was 132.6+/-37.4/mm2, 378.6+/-99.1/mm2, 15.0+/-2.8/mm2, 57.1+/-16.9/mm2, 191.8+/-44.8/mm2 and 450.4+/-58.3/mm2, respectively. The number of TUNEL-positive cells was 24.5+/-2.0/mm2, 21.7+/-4.0/mm2, 20.4+/-2.0/mm2, 18.96+/-2.1/mm2, 58.3+/-7.9/mm2, and 106.0+/-9.8/mm2, respectively. These results suggest low doses of radiation paradoxically improved diabetes induced neuronal cell suppression in the hippocampal dentate gyrus of rat.


Subject(s)
Rats , Male , Animals , Rats, Sprague-Dawley , Radiotherapy/methods , Neurons/metabolism , In Situ Nick-End Labeling , Hippocampus/cytology , Diabetes Mellitus, Experimental/radiotherapy , Dentate Gyrus/drug effects , Cell Proliferation , Caspase 3/metabolism , Bromodeoxyuridine/pharmacology , Apoptosis
4.
Indian J Exp Biol ; 2005 Nov; 43(11): 1068-79
Article in English | IMSEAR | ID: sea-61615

ABSTRACT

Earlier studies had shown that long term treatment with estradiol arrests spermatogenesis in adult male rats, at a dose of 0.1 mg/kg/day. The present study was therefore undertaken to ascertain the causes underlying the reduction in sperm counts by administering estradiol for a short term, at the dose of 0.1 mg/kg/day. Estradiol valerate was injected at a dose of 0.1 mg/kg/day, for a period of 10 days to one group of adult male rats, which were administered saline for 12 days prior to estradiol injection, and sacrificed after 22 days. The control group was administered saline for 22 days. The sera were analyzed for testosterone and FSH levels. One testis of each male was immersion fixed for histology, and for immunohistochemistry of two testicular cytoskeletal proteins, vimentin and vinculin. The contralateral testes were used for analysis of vimentin and vinculin gene expression by reverse transcriptase polymerase chain reaction (RTPCR) and western blotting. Another group exposed to estradiol for 10 days was injected with bromodeoxyuridine (BrdU), at a dose of 100 mg/kg/day, to ascertain the effect on germ cell proliferation, and sacrificed 12 days later, while estradiol treatment was continued till sacrifice. BrdU, at a dose of 100 mg/kg/day was injected i.p. to a group of control rats treated with saline for 10 days, and sacrificed 12 days later. The testes from both groups were immersion fixed for immunohistochemical detection of BrdU. Histology of estradiol treated testis showed predominance of tubules with round spermatids with accumulation of lipid droplets in Sertoli cell cytoplasm and decreased cell height, whereas controls showed elongating spermatids. BrdU immunolocalization in the testis, irrespective of treatment, indicated its incorporation in deoxyribonucleic acid (DNA) suggesting that estradiol sustained germ cell proliferation. Both vimentin and vinculin could be immunolocalized to the testis. The testicular levels of vimentin and vinculin, quantified after western blotting, were unaffected. The testicular expression of vimentin and vinculin seen by RTPCR was also unaffected. The study suggested that estradiol induced reduction in sperm counts was not due to adverse effects on proliferation. The observed predominance of seminiferous tubules showing round spermatids, accumulation of lipid droplets as compared to controls suggested that reduction in elongated spermatids occurred through reduced spermiation and phagocytosis. The study also suggested that reduction in Sertoli cell height after short-term estradiol treatment was not due to reduced expression of vimentin and vinculin, which could be maintained by estradiol. However, reduction in Sertoli cell height could have been due to suppression of FSH and testosterone, implicated in the polymerization of vimentin and organization of vinculin, two cytoskeletal proteins involved in inter-Sertoli or Sertoli-germ cell junctions. The study suggested that disorganization of Sertoli cell cytoskeleton and reduction in the volume of Sertoli cells could be an important factor for reduced efficiency of spermatogenesis after exposure to estrogenic molecules.


Subject(s)
Animals , Bromodeoxyuridine/pharmacology , Cell Lineage , Cytoskeleton/metabolism , Dose-Response Relationship, Drug , Estradiol/analogs & derivatives , Lipids/chemistry , Male , Polymerase Chain Reaction , RNA/chemistry , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sertoli Cells/drug effects , Spermatogenesis/drug effects , Spermatozoa/metabolism , Testis/metabolism , Testosterone/metabolism , Time Factors , Vimentin/metabolism , Vinculin/metabolism
5.
Indian J Exp Biol ; 1997 Nov; 35(11): 1156-60
Article in English | IMSEAR | ID: sea-61149

ABSTRACT

Certain qualitative criteria for primed lymphocytes in the expression of cytotoxic function have been studied. Unlike normal lymphocytes, primed lymphocytes expressed cytotoxicity even when DNA synthesis and new gene expression were inhibited by hydroxyurea (HU) and bromodeoxyuridine (BU) respectively. Such differential cytotoxic expression in presence of HU and BU by primed lymphocytes might have their basis in conformational change within the chromatin. Chromatin from primed lymphocytes was more susceptible to DNase I digestion than virgin lymphocytes indicating exposition of more DNase I sensitive sites in primed state. The result suggest the presence of more ready to act sites for the polymerases in the genomic material of primed lymphocytes even at quiescent state.


Subject(s)
Animals , Antimetabolites/pharmacology , Bromodeoxyuridine/pharmacology , Chromatin/metabolism , Deoxyribonuclease I/metabolism , Hydroxyurea/pharmacology , Lymphocytes/drug effects , Mice , Nucleic Acid Synthesis Inhibitors/pharmacology
6.
Rev. bras. genét ; 19(3): 501-9, set. 1996. ilus, tab
Article in English | LILACS | ID: lil-189668

ABSTRACT

Os efeitos da concentraçäo de bromodeoxiuridine (BUdR) e o comprimento do período de síntese (S) na freqüência basal de intercâmbios de cromátides irmäs (SCEs)e na taxa de proliferaçäo de culturas de sangue total (WBC) e de leucócitos plasmáticos (PLC) humanos foram estudados. Um método imunofluorescente foi usado para induzir a diferenciaçäo de cromátides irmäs. Este método emprega uma baixa concentraçäo de BUdR para a marcaçäo de cromossomos durante o primeiro ciclo celular in vitro (3,3µM), e o anticorpo monoclonal anti-BUdR conjugado com fluoresceína isotiocianada (FITC) para detecçäo. Permitiu-se que WBC e PLC crescessem durante o primeiro ciclo celular (48h) na presença de BUdR; o meio de cultura foi entäo substituído pelo meio de cultura sem BUdR e as células foram cultivadas por um ciclo celular adicional (24h) até a colheita (72h). Os cromossomos foram contrastados com iodeto de propidium DAPI. PLC mostrou no mínimo um aumento em dobro na freqüência de SCE sobre os valores de WBC e a paralela, independente da concentraçäo de BUdR no meio de cultura, pelo menos na faixa do análogo de base empregada (3,3µM-33,0µM). Além disso, uma estimativa do comprimento do período S, feita pela análise da proporçäo de diferentes tempos de colheira, revelou que PLC tem um período S marcadamente extenso, comparado a WBC. Conseqüentemente, a taxa de deslocamento de fork do DNA de linfócitos em PLC poderia ser mais lenta que em WBC, resultando em um aumento na freqüência de SCE destas culturas.


Subject(s)
Humans , Animals , Sister Chromatid Exchange , Bromodeoxycytidine , Bromodeoxyuridine/pharmacology , Fluorescent Antibody Technique/methods , Leukocytes/drug effects , Sister Chromatid Exchange
7.
Indian J Exp Biol ; 1994 Sep; 32(9): 637-42
Article in English | IMSEAR | ID: sea-59039

ABSTRACT

Effects of 5-bromo-2-deoxy-uridine (BrdU) and 2-deoxy-D-glucose (2-DG) on 60-Co-gamma-ray induced damage were studied in monolayer cultures of glioma (BMG-1) cells, and PHA-stimulated peripheral leukocytes from normal donors. Micronuclei formation was used as an index of cytogenetic damage. BrdU and 2-DG treatments did not induce micronuclei formation in unirradiated cultures. Presence of BrdU (0.8 microM) for more than one cell cycle (24 hr) significantly increased gamma-ray (1-4 Gy) induced micronuclei formation in exponentially growing BMG-1 cells. Incubation of irradiated cells under sub-optimal growth conditions (DMEM with 1% serum) for 3 hr, instead of growth medium, significantly decreased micronuclei formation. Post-irradiation presence of 2-DG (5 mM; 3 hr, in DMEM + 1% serum) significantly increased radiation damage. In BrdU sensitized cells also, 2-DG significantly increased radiation damage further. In PHA-stimulated leukocytes from normal donors, 2-DG (5mM, equimolar with glucose; for 2 hr) did not increase gamma-ray (2-Gy, 42 hr after PHA-stimulation) induced micronuclei formation. Pre-irradiation presence of BrdU (1.6 microM) significantly increased micronuclei. On the contrary, 2-DG treatment reduced radiation induced micronuclei formation in BrdU sensitized leukocyte cultures. These results suggest that (i) radiation induced lesions leading to micronuclei formation in proliferating tumour cells, are, at least, partly repairable; (ii) combination of 2-DG could reduce BrdU doses required for radiosensitization of proliferating tumour cells; and (iii) 2-DG could differentially increase radiation damage in BrdU sensitized proliferating tumour cells, while reducing manifestation of damage in normal proliferating cells.


Subject(s)
Adult , Bromodeoxyuridine/pharmacology , Cells, Cultured , Deoxyglucose/pharmacology , Glioma/pathology , Humans , Leukocytes/drug effects , Male , Phytohemagglutinins , Radiation-Sensitizing Agents/pharmacology , Tumor Cells, Cultured
8.
Indian J Exp Biol ; 1993 Mar; 31(3): 224-30
Article in English | IMSEAR | ID: sea-62571

ABSTRACT

The effects of 2-deoxy-D-glucose (2-DG) and 5-bromo-2-deoxy-uridine (BrdU) on gamma ray (60Co) induced damage were studied in monolayer cultures of transformed mammalian (BHK-21) cells. Micronuclei formation and changes in DNA content dispersion were used as indices of cytogenetic damage. Exposure of cells to BrdU (0.8 microM) for nearly two cell cycles before irradiation significantly increased micronuclei formation in exponentially growing cells. Incubation of irradiated cells under suboptimal growth conditions (in HBSS) for 4 hr, instead of growth medium, decreased the manifestation of damage. However, post-irradiation presence of 2-DG (5 mM, equimolar with glucose; 4 hr) in growth medium or HBSS significantly increased radiation damage. The effects of 2-DG treatment following irradiation in plateau phase were quantitatively less. These results suggest that: (i) radiation induced DNA lesions leading to micronuclei formation in BrdU incorporated cells are partly repairable; (ii) 2-DG could increase radiation induced cytogenetic damage in transformed mammalian cells, possibly by inhibiting the cellular repair processes; and (iii) combination of 2-DG treatment may decrease the BrdU doses required for radiosensitization of proliferating tumour cell populations.


Subject(s)
Animals , Bromodeoxyuridine/pharmacology , Cell Line, Transformed , Cricetinae , DNA Damage/drug effects , Deoxyglucose/pharmacology , Micronuclei, Chromosome-Defective/drug effects
9.
Indian J Exp Biol ; 1993 Feb; 31(2): 101-5
Article in English | IMSEAR | ID: sea-56975

ABSTRACT

Chromosomal DNA of the synchronously mitotic plasmodia of P. polycephalum was substituted with 5-bromo-2'-deoxyuridine, by growing the plasmodia during S phase, on a medium containing this nucleoside analog. A strong synergism was observed between bromodeoxyuridine and UV-irradiation, in late G2-irradiated plasmodia in that, the mitotic delay obtained in them was much more than a simple sum of the delays induced by these two agents individually. It was also observed that the mitotic delay in this system is reduced significantly by different concentrations of caffeine applied immediately after irradiation and there was a stage specificity in this effect. The reduction in mitotic delay was maximum (80%) in those plasmodia irradiated 20-30 min before control metaphase, when mitogenic factors also reach their maximum activity in this system. It is proposed that the mitotic delay reducing effect of caffeine is due to its ability to promote the activity of the mitogenic factors, largely independent of the system which is responsible for monitoring the state of the chromosomal DNA.


Subject(s)
Animals , Bromodeoxyuridine/pharmacology , Caffeine/pharmacology , DNA/drug effects , Mitosis/drug effects , Physarum polycephalum/drug effects , Ultraviolet Rays
10.
Indian J Exp Biol ; 1992 Aug; 30(8): 664-9
Article in English | IMSEAR | ID: sea-57053

ABSTRACT

Phytohaemagglutinin (PHA)-responsive lymphocytes from human peripheral blood samples, either irradiated or un-irradiated, showed increased frequency of first division metaphase cells (detected by fluorescence plus Giemsa (FPG) staining) as a function of duration of storage. Irradiated and subsequently stored samples showed small but significant increase for the yield of dicentrics. The yield of aberrant metaphases and deletions (excess acentrics) remained unchanged. Increasing Bromodeoxyuridine (BrdU) concentrations slowed down the cell cycle progression but did not influence the yield of aberrations including that of dicentrics.


Subject(s)
Analysis of Variance , Blood Preservation/adverse effects , Bromodeoxyuridine/pharmacology , Cell Cycle/drug effects , Chromosome Aberrations , Chromosomes/drug effects , Dose-Response Relationship, Drug , Humans , Lymphocytes/cytology , Phytohemagglutinins , Time Factors
11.
Indian J Exp Biol ; 1991 Sep; 29(9): 826-30
Article in English | IMSEAR | ID: sea-61034

ABSTRACT

Effects of 5-bromo-2-deoxy-uridine (BrdU) and 2-deoxy-D-glucose (2-DG) were studied in exponentially growing transformed mammalian (BHK-21) cells, grown as monolayer. Micronuclei formation as an index of radiation damage was studied by i) cytokinesis block technique from cytochalasin-B induced binucleated cells, and ii) conventional technique. Presence of BrdU (0.8 microM) for nearly 2 cell cycles before gamma-irradiation (2.5 Gy) significantly increased frequencies of cells with micronuclei. Post-irradiation incubation of cultures in liquid holding medium (HBSS) however, reduced micronuclei formation, especially in the BrdU treated cells. Presence of 2-DG (4 hr, equimolar with glucose) in growth as well as liquid holding medium further increased micronuclei frequencies. These observations suggest that radiation induced DNA lesions in BrdU substituted cells, leading to chromosome fragmentation are partly repairable. 2-DG increased cytogenetic damage, possibly by inhibiting the repair of such repairable lesions. Present studies suggest that combination of 2-DG could optimize BrdU-radiation therapy of brain tumors, by reducing the BrdU doses required for tumor radiosensitization.


Subject(s)
Animals , Bromodeoxyuridine/pharmacology , Cell Division/drug effects , Cell Line, Transformed , Cricetinae , Deoxyglucose/pharmacology , Radiation Tolerance , Radiotherapy
12.
Article in Spanish | LILACS | ID: lil-113724

ABSTRACT

En la actualidad se acepta que muchas neoplasias humanas están causadas por factores y muchos mutágenos químicos inducen aberraciones cormosómicas (AC), las caules se cree que se origina por roturas en zonas cromosómicas específicas o sitios frágiles (SF). Los SF se estudiaron en cultivos de linfocitos expuestos a diversos inductores químicos, pero todavia no se conoce como influyen los factores ambientales en su expresión. Hasta ahora no hay estudios de SF inducidos con rayos X, ni se conoce la interacción de este agente con los inductores quimicos. Este es el primer trabajo que analiza la expresión de SF inducida por rayos X y por 3 inductores de SF: BUDR, FUDR y anfidicolina. Se identificaron 17 bandas cromosómicas significtivamente afectadas (p<0.oo1), que se definieron como SF. Los SF más frecuentes estaban localizados en las bandas 3p14 y 16q23. Se observó un aumento significativo (p<0.01) de AC y SF en el cultivo expuesto a rayos X y en el tratado con FUDR más radiación, indicando la conveniencia de emplear otro agente radiosensibilizador. Los resultados observados sugerirían que muchos SF pueden ser causados por factores ambientales tales como sustancias químicas o radiación. La alta correlación establecida entre los SF y la ubicación de AC inducidos por radiación, AC en cáncer y oncogenes demostraría la importante interacción entre SF, AC y oncogenes en el proceso neoplásico


Subject(s)
Humans , Aphidicolin/pharmacology , Bromodeoxyuridine/pharmacology , Chromosome Fragility , Floxuridine/pharmacology , Mutation , Neoplasms, Radiation-Induced , Lymphocytes/drug effects , Lymphocytes/radiation effects , Mutation , Mutation/radiation effects
18.
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