Concentrations Of BrdUrd Research Articles

A new method for the identification of SCE's per cell cycle in BrdUrd — Substituted chromosomes

If cells are allowed to replicate in the presence of different concentrations of BrdUrd throughout three consecutive cell cycles, FPG stained third division chromosomes show three different levels of staining. This fact allows identification of four different types of SCE's, corresponding to the first-, second-and third-cycle exchanges. Finally, the relationship between the degree of BrdUrd-substitution and the frequency of SCE's per cell cycle supports that most of the SCE's detected in BrdUrd-substituted chromosomes are induced by this thymidine analogue incorporated into DNA.

X irradiation and sister chromatid exchange in cultured human lymphocytes.

Sister chromatid exchange (SCE) frequency was not increased in G0 lymphocytes following irradiation up to 400 rads. Lymphocytes irradiation after 42 or 60 hours of culture showed a dose-dependent increases in exchange frequency at 100 and 200 rads. SCE was not increased in cells irradiated in G2 (68.5 hours). Lymphocytes maintained in a 5-Bromodeoxyuridine (BrdUrd) free medium and irradiated 42 hours after culture initiation showed an increase in SCE if BrdUrd was added immediately after irradiation, but no increase was found if there was a 5 hour holding period to the additional of BrdUrd. The effect on induced SCE frequency of heightened radiosensitivity due to increased amounts of BrdUrd was also investigated. When the BrdUrd concentration was increased form 10 micrograms/ml, the percent increase in X-ray-induced SCE was lower at 50 micrograms/ml. In addition, increased BrdUrd concentration only slightly increased the sensitivity of the SCE technique to irradiation doses of 50 rads.

Species variation in BrdUrd-induced sister-chromatid exchanges

Peripheral blood lymphocytes from cattle, pigs, sheep and humans were cultured in the presence of 0.5, 1, 2, 5,10 or 20 μg/ml of BrdUrd. Sister-chromatid exchanges were scored in 25 second-division metaphases from each donor at each level of the chemical. Dose-response curves for all 4 species increased steeply to 2 μg/ml; above this level, SCE numbers increased less rapidly but maintained a linear relationship to increasing BrdUrd concentration. Comparisons of the straight-line portions of the dose-response curves showed human cells to be significantly more sensitive to increasing BrdUrd level than cow or pig cells and different from sheep at the 10% level of confidence.

On the mechanism of 5-bromodeoxyuridine induction of prolactin synthesis in rat pituitary tumor cells.

GH12C1, a clonal strain of rat pituitary tumor cells in culture (GH cells), does not produce detectable amounts of prolactin. 5-Bromodeoxyuridine (BrdUrd), the thymidine analogue, at sublethal concentrations (3-5 microgram/ml) induces prolactin synthesis in these cells. BrdUrd also induces prolactin synthesis in F1BGH12C1 cells, a BrdUrd resistant (BrdUrdr) substrain isolated from GH12C1 cells. The F1BGH12C1 strain is not drug dependent, but its resistance to BrdUrd is a stable phenotype. The significant features of the induction of prolactin synthesis in the BrdUrdr strain are the increased net synthesis of prolactin and the shortening of the lag period of prolactin induction. As BrdUrd concentration in the growth medium is increased, the rise in prolactin synthesis parallels the increased incorporation of BrdUrd into DNA. Prolactin synthesis is first detected when BrdUrd replaces 20-25% of the thymidine in DNA. BrdUrd can replace up to 75-80% of the thymidine within 2 d of treatment. Partial starvation of these cells under specified growth conditions does not affect the general growth pattern of the cells, general protein synthesis, and thymidine uptake, but does affect DNA synthesis. When cells are cultured under conditions in which DNA synthesis is preferentially inhibited, BrdUrd does not induce prolactin synthesis, suggestive of a DNA-mediated mechanism of action for the drug.

Effect of 5-bromodeoxyuridine on heterogeneous nuclear RNA in rat hepatoma cells.

Heterogeneous nuclear RNA HnRNA) was isolated from untreated and 5-bromodeoxyuridine (BrdUrd) treated hepatoma tissue culture (HTC) cells. analysis of this RNA by either electrophoresis on polyacrylamide-agarose gels or centrifugation in sucrose gradients demonstrated that BrdUrd caused a shift in the labeled HnRNA population toward a smaller size distribution. This effect was produced by concentrations of BrdUrd which specifically lower the level of the differentiated enzyme tyrosine aminotransferase, but do not greatly affect cell growth. Differential binding to oligo(dT) cellulose was used to fractionate HnRNA further into classes containing poly(A) (alpha), oligo(A) (beta) or neither category of A-rich sequences (gamma). BrdUrd did not alter the relative rates of uridine incorporation into the three classes. The shift in the labeled HnRNA population due to BrdUrd was observed in all three subclasses of HnRNA.

Bromodeoxyuridine mutagenesis in mammalian cells: mutagenesis is independent of the amount of bromouracil in DNA.

Studies were undertaken to determine how a line of mutant Syrian hamster melanoma cells (HAB-2E) that displays unlimited growth potential when all of the thymine residues in nuclear DNA are replaced by bromouracil (BrUra) could avoid the deleterious effects of bromodeoxyuridine (BrdUrd) mutagenicity. It was found that BrdUrd could be mutagenic to these cells. However, there was a nonlinear relationship between mutagenicity and the amount of BrUra in the DNA of the HAB-2E cells. With these cells, mutagenicity apparently is determined by the concentration of BrdUrd to which the cells are exposed rather than the amount of BrUra in DNA. These results were obtained with both the induction of ouabain resistance and thioguanine resistance as markers for mutagenesis. The dependence of BrdUrd mutagenicity on BrdUrd concentration was also observed for the parental melanoma cells.

Sister chromatid exchanges (SCE) as sensitive indicators of the effects of mutagenic carcinogens

If cells are allowed to replicate in the presence of different concentrations of BrdUrd throughout three consecutive cell cycles, FPG stained third division chromosomes show three different levels of staining. This fact allows identification of four different types of SCE's, corresponding to the first-, second-and third-cycle exchanges. Finally, the relationship between the degree of BrdUrd-substitution and the frequency of SCE's per cell cycle supports that most of the SCE's detected in BrdUrd-substituted chromosomes are induced by this thymidine analogue incorporated into DNA.

Adenosine 3′,5′-Monophosphate in Glial Tumor Cells: ALTERATIONS BY 5-BROMODEOXYURIDINE

Abstract The effect of 5-bromodeoxyuridine (BrdUrd) on the ability of the glial tumor cell line, C-6, to elevate its adenosine 3',5'-monophosphate (cAMP) content in response to norepinephrine has been examined. Increasing concentrations of BrdUrd were found to inhibit the norepinephrine response progressively, with a maximal inhibition of approximately 80%. No effect of BrdUrd was seen on basal cAMP levels. Analysis of individual enzymes demonstrated that BrdUrd increased the cAMP phosphodiesterase activities but had no effect on the adenylate cyclase or its sensitivity to norepinephrine. In addition, BrdUrd caused a change in the shape of the cells. The effect on shape also occurred in confluent cells, whereas the effect on the norepinephrine response required dividing cells, suggesting that incorporation of the BrdUrd into DNA was required. N6, O2'-Dibutyryl adenosine 3',5'-monophosphate also increased the cAMP phosphodiesterase activities of the cells, and caused a similar change in their morphology. Although BrdUrd had no effect on the adenylate cyclase, variations in the culture conditions were found to have a large effect on the enzyme's norepinephrine sensitivity. Following subculturing, there was little elevation of cAMP in response to norepinephrine until after the cells began to enter the stationary growth phase. Phosphodiesterase activity was found to remain constant throughout this period. The results suggest that cell-to-cell contact may regulate certain cellular functions.