BrdU Exposure Research Articles

Role of p53 in Growth Suppression by Bromodeoxyuridine in Human Gastric Cancer Cell Lines.

Bromodeoxyuridine (BrdU) is known to cause base mispairs and DNA strand breaks during DNA synthesis, culminating in growth suppression. To know whether P53 gene plays any role in the BrdU-induced growth suppression, we continuously gave BrdU (20 μM) to the synchronized culture of human gastric cancer cell lines, MKN-45 (P53-wild type) and MKN-28 (P53-mutant), shortly after release from the G1/S block by hydroxyurea. In comparison with the control culture, the growth of MKN-28 was not suppressed until 48 hr of BrdU exposure, while that of MKN-45 was already suppressed at the 24 hr point. Continuous exposure to BrdU caused a S-phase delay and G2 arrest of around 6 hr each in MKN-45 and a delay only of the second S phase in MKN-28 in comparison with synchronized control cultures of matched cell cycle phase. BrdU-exposed MKN-45 cells showed a significantly higher incidence of apoptosis after the cells passed through the first G2 phase and the second S/G2 phases, but MKN-28 did not. It thus appears that the delays of S/G2 phases and an increased incidence of apoptosis in the first cell cycle are p53-dependent. The growth suppression in the second S/G2 phase or later observed in both of the cell lines may be p53-independent.

Effects of prenatal exposure to 5-bromo-2′-deoxyuridine on reproductive function in male mouse offspring

The effect of the halogenated pyrimidine analog 5-bromo-2′-deoxyuridine (BrdU), on reproductive functions of male mouse offspring treated prenatally was studied. BrdU was administered intraperitoneally to pregnant ICR mice at 100 mg/kg/d on days 8 through 13 of gestation, and at 100 or 200 mg/kg/d on days 14 through 18 of gestation. Dams were allowed to deliver naturally. Male offspring were aged for 10 weeks and then cohabited with untreated female mice for assessment of reproductive performance. Histopathologic examination of the testes and pituitary, sperm analysis, and determination of plasma testosterone concentrations of offspring at 12 weeks of age were performed. In the subsequent study, pregnant ICR mice were treated with 200 mg BrdU/kg on day 10,13, or 15 of gestation. The embryos or fetuses were obtained from mothers from 6 to 48 h after treatment, and pyknotic cells in the ventricular zone of the telencephalon were counted. There was a significant decrease in body weight gain of offspring in all of the BrdU-treated groups. A marked decrease in copulation rate was noted in the male offspring of dams treated on days 8 through 13 of gestation, whereas no significant decreases in copulation and fertility rates were found in the male offspring of dams treated on days 14 through 18 of gestation. Neither histopathologic examination of testes nor sperm analyses revealed adverse effects of this compound, whereas cysts in the pars distalis of the pituitary were observed in the male offspring treated on days 8 through 13 of gestation. Dilatation of the lateral ventricles was also observed in male offspring at 12 weeks of age in the group treated on days 8 through 13 of gestation. The incidence of pyknotic cells in the ventricular zone of embryos was markedly increased 24 h after treatment on day 10 of gestation. These results indicate that the impaired fertility of the male offspring of dams treated with BrdU on days 8 through 13 of gestation may be due to the effects of BrdU exposure on central nervous system function that result in loss of libido rather than to the direct effects of this compound on the male reproductive organs.

Reproductive effects of prenatal exposure to 5-bromo-2′-deoxyuridine on male rats

5-Bromo-2′-deoxyuridine (BrdU) was administered intraperitoneally to Sprague-Dawley rats at doses of 12.5, 25, 50, or 100 mg/kg/d on days 9 through 15 of gestation, and at 50 or 100 mg/kg/d on days 16 through 20 of gestation. Dams were allowed to deliver naturally and the numbers of live and dead pups were recorded. Male offspring were allowed to mature and then cohabited with untreated female rats for assessment of reproductive performance. Dam body weight gain during pregnancy and lactation periods was not reduced by the treatment with BrdU. Dams treated with 50 and 100 mg BrdU/kg on days 9 through 15 of gestation had litters with decreased survival rates. The male offspring from dams treated with 25, 50, and 100 mg BrdU/kg on days 9 through 15 of gestation had reduced body weights over the course of the entire study. A dose-related decrease in copulation and fertility rates was found in the male offspring of dams treated on days 9 through 15 of gestation, while no significant decrease in those rates were found in the male offspring of dams treated on days 16 through 20 of gestation. Neither histopathologic examination of testes nor sperm examination indicated the cause of the impaired fertility in the male offspring from dams treated with BrdU on days 9 through 15 of gestation. All of the male offspring of dams treated with 100 mg BrdU/kg on days 9 through 15 of gestation failed to copulate, and some of the male offspring of dams treated with 50 mg BrdU/kg on days 9 through 15 of gestation did not form copulatory plugs or formed very small plugs. Dilatation of the lateral ventricles and cysts in the pars distalis of the pituitary were observed in all of the male offspring of dams treated with 100 mg BrdU/kg on days 9 through 15 of gestation. The impaired fertility of the male offspring of dams treated prenatally with BrdU may have resulted from BrdU exposure effects on central nervous system action such as loss of libido and from failure to form proper copulatory plugs, rather than the direct effects of BrdU on the male reproductive organs.

Activation of thyrotropin-releasing hormone gene expression in cultured fetal diencephalic neurons by differentiating agents.

The present studies were undertaken to investigate the effect of 5-bromo-2'-deoxyuridine (BrdU; 50 microM) or forskolin/3-isobutyl-1-methylxanthine (F/I; 10/500 microM) on TRH gene expression in cultured fetal diencephalic cells. BrdU as well as drugs such as F/I that raise intracellular cAMP levels had been previously termed differentiating agents because they cause morphological and functional differentiation of IMR-32 neuroblastoma cells. We postulated that neurons of fetal diencephalons may remain relatively undifferentiated in vitro and that this might be the reason for low or undetectable TRH production. We hypothesized that treatment with differentiating agents might increase neuropeptide expression. Both BrdU and F/I dramatically (P < 0.01) raised intracellular TRH and pro-TRH messenger RNA concentrations in cultured diencephalic neurons. Although a short BrdU exposure during the first 4 days of culture was sufficient to irreversibly change TRH neurons and to cause maintenance of high TRH levels after withdrawal of the drug, F/I had to be present continuously throughout the observation period of 16 days to significantly elevate TRH expression. This suggests that BrdU and F/I act at different intracellular sites to activate TRH expression in cultured diencephalic neurons. The reduction of glial cells that occurs concurrent with the BrdU treatment was not observed after F/I exposure, and therefore, this effect does not appear to be a key factor for the induction of TRH expression. As the intracellular accumulation of somatostatin and arginine vasopressin, which were determined in parallel, was similarly enhanced after treatment with BrdU or F/I, our culture system might provide a valuable tool for the study of these and possibly other neuropeptides in vitro.

Selective aflatoxin B1‐induced sister chromatid exchanges and cytotoxicity in differentiating B and T lymphocytes in vivo

The purpose of this study was to assess the genotoxic and cytotoxic effects of the fungal metabolite aflatoxin B1 (AfB1) on the developing immune system of the chick embryo, a model in vivo system. Of particular interest was the assessment of AfB1-mediated selective toxicity toward developing B lymphocytes as compared to T lymphocytes. In vivo bromodeoxyuridine (BrdU) labelling of DNA was used to detect the induction of sister chromatid exchanges (SCE) in lymphocytes and to assess the progression of these cells through successive cell cycles. Cytotoxicity was also assessed by studying the entrance and maintenance of cells in mitosis (mitotic index). Graded doses of AfB1 (1.09-17.4 micrograms/g embryo) were applied to chick embryos at 18 days of incubation (DI). Embryos also received two doses of BrdU at 3 mg/200 microliters (3 hr apart) to provide continuous labelling of B and T lymphocyte replicating DNA. B and T lymphocytes were harvested 20 hr post-AfB1/BrdU exposure from the bursa and thymus, respectively, and were processed for cytogenetic analyses. AfB1 induced dose-related increases in SCE in B lymphocytes; this induction was 6- to 8-fold that of controls at the higher doses tested. AfB1-mediated induction of SCE in T cells was just 2-fold that of controls at the highest dose tested. AfB1 reduced the progression of B cells and to a lesser extent T cells through successive rounds of replication. Furthermore, AfB1 dramatically reduced the mitotic index of B cells but not of T cells. These data indicate both selective genotoxicity and cytotoxicity of AfB1 toward B cells in the late stage embryo.

Identification of 'non-proliferating' B16 melanoma cells using monoclonal antibody (AD203) against the M1 subunit of ribonucleotide reductase.

Ribonucleotide reductase catalyses a critical reaction in DNA synthesis. Its M1 subunit is present during all proliferative phases of the cell cycle, but apparently not in the quiescent phase G0. We have used a monoclonal antibody (AD203) directed against the M1 subunit to distinguish immunocytochemically proliferating from non-proliferating cultured B16 mouse melanoma cells during exponential growth. The presumption that AD203 unstained cells constituted a non-proliferating fraction was tested by simultaneously counting the cells that failed to incorporate bromodeoxyuridine (BrdU) into DNA during a prolonged BrdU exposure. The proportion of cells which did not incorporate BrdU was found to correlate closely with the proportion not staining with AD203 and therefore presumably lacking the M1 subunit. The respective morphological features of AD203 stained and unstained cells were found not to differ significantly.

Alterations in the baseline sister-chromatid exchange frequency in human lymphocyte culture following a number of cell divisions

A significant decrease in the baseline of sister-chromatid exchanges (SCEs) was observed in cultured human lymphocytes, if 5-bromodeoxyuridine (BrdU) was added after 60 h of culture, and the cells were harvested at least 24–30 h after BrdU exposure. This decrease is supposed to occur if at least one cell division takes place before the addition of BrdU. For cytogenetic monitoring of mutagenic environmental factors, using human lymphocyte cultures, it is assumed that two time periods are sufficient for comparison.

Effect of 5-bromodeoxyuridine substitution on sister chromatid exchange induction by chemicals

The fluorescence-plus-Giemsa (FPG) technique for analysis of sister chromatid exchange (SCE) is widely used as an assay for mutagenic carcinogens. There is very little information, however, on whether incorporation of the bromodeoxyuridine (BrdU) necessary for visualization of SCEs affects the sensitivity of the SCE test system to different chemical agents. We have investigated the effect of BrdU incorporation on SCE induction by labeling cells with BrdU for either the first cell cycle or the first and second cell cycles. The cells were then treated with bleomycin, which produces DNA strand breakage; proflavine, which intercalates into DNA; mitomycin C, which produces monoadducts and DNA crosslinks; or aphidicolin, which inhibits DNA polymerase alpha. Chemicals were added before BrdU exposure or during the first, second, or both cell cycles. Only mitomycin C, which induces long-lived lesions, elevated the SCE frequency when cells were treated before BrdU labeling. When bleomycin, proflavine, or mitomycin C was present concurrently with BrdU, the frequency of SCEs was increased independently of the BrdU labeling protocol. Aphidicolin, on the other hand, induced more SCEs when present for the second cell cycle, when DNA replicates on a template DNA strand containing BrdU. We also examined the induction of SCEs in the first cell cycle (twins) and in the second cell cycle (singles) after continuous treatment of cells with BrdU and the test chemicals. Only aphidicolin increased SCE frequency in the second cell cycle. These results indicate that aphidicolin, but not bleomycin, proflavine, or mitomycin C, affects BrdU-substituted DNA and unsubstituted DNA differently. This type of interaction should be taken into consideration when the SCE test is used as an assay system.

Growth rate and sister chromatid exchange (SCE) incidence of bone marrow cells in Acute Myeloblastic Leukemia (AML)

The sister chromatid exchange (SCE) incidence and growth kinetics have been studied by means of an in vitro bromodeoxyuridine (BrdU) chromosome labeling method in the bone marrow cells of 17 acute myeloblastic leukemia (AML) patients with only diploid cells at diagnosis, remission, and relapse of the disease. At diagnosis, the cells tended to exhibit a low SCE frequency as compared to that during remission. An increased SCE frequency was observed after chemotherapy during remission or relapse. At diagnosis and relapse, when leukemic blast cells predominated in the marrow, they were characterized by the predominance of cells that had undergone only one cell cycle after BrdU exposure. In contrast, the marrow cells during remission tended to resemble the control pattern of growth kinetics, with a predominance of cells undergoing second and third cell cycles in the presence of BrdU. These results suggest that the growth rate of leukemic and nonleukemic cells is different, and that chemotherapy can cause an increased SCE frequency in the marrow cells of AML patients irrespective of the state of the disease.