Mutagenicity of 5-halodeoxyuridines to diploid human lymphoblasts

The thymidine base analogues 5-chlorodeoxyuridine, 5-bromodeoxyuridine, and 5-iododeoxyuridine were found to be mutagenic in diploid human lymphoblasts. Mutation was measured as the loss of hypoxanthine-guanine phosphoribosyl transferase activity, which is expressed phenotypically as resistance to 6-thioguanine. Concentration dependence of induced mutant fraction exhibited a maximum for all three compounds. It is postulated that at higher concentrations these thymidine analogues inhibit cytidine diphosphate reductase. Slowed DNA synthesis would result in lower analogue incorporation and thus a lower mutant fraction.     

Sister chromatid exchanges induced by light flashes to 5-bromodeoxyuridine- and 5-iododeoxyuridine substituted Chinese hamster chromosomes

Incorporation of 5-bromodeoxyuridine (BUdR) or 5-iododeoxyuridine (IUdR) into the chromosomal DNA of Chinese hamster ovary (CHO) cells during two rounds of replication causes sister chromatids to be differentiated so that they can be discriminated from one another by staining and morphology. Chromatids that contain BUdR or IUdR in both DNA strands stain lighter and are less condensed than their sister chromatids with only unifilar substitution. The halogenated pyrimidine nucleosides also induce sister chromatid exchanges that can be detected without autoradiography. The frequency of these exchanges is markedly increased by exposing the cells to light flashes.     

Induction of alpha- and beta-crystallin synthesis in organ cultures of the adenohypophyseal anlage of chickens as affected by 5-iododeoxyuridine and 5-bromodeoxyuridine

The effects of 5-iododeoxyuridine and 5-bromodeoxyuridine on differentiation of the cells of adenohypophysis rudiment from 3, 4, and 5 day old chick embryos were studied in the in vitro organ culture. On the 7th day of cultivation most explants from 3 and 4 day old embryos formed lentoids and individual cells with the lens phenotype among the adenohypophysis tissue. Alpha-, beta- and delta-crystalline were immunochemically detected in them. When cultivating explants from 5 day old embryos, no lentoids formed. But the immunochemical study of serial sections made it possible to detect in individual explants single alpha-crystalline-containing cells. There is a period in the development of chick adenohypophysis, which lasts five days of incubation and during which the adenohypophysis rudiment retained its capacity for lens differentiation despite the fact that it is already determined in the adenohypophysis direction.     

The degradation of 5-iododeoxyuridine and 5-bromodeoxyuridine by serum from different sources and its consequences for the use of the compounds for incorporation into DNA

Enzymes are present in sera that convert 5-iododeoxyuridine (IdU) to deoxyuridine (dU) and 5-iodouracil (IU). Although in the presence of serum 5-bromodeoxyuridine (BrdU) is not subject to extensive debromination it is converted to 5-bromouracil (BrU) at approx. 50% of the rate for IdU. These conversions are likely brought about by the enzymes thymidylate synthetase and thymidine phosphorylase. In vivo and in culture the dU enters DNA as thymidine 5'-monophosphate (dTMP) via the de novo pathway. Deoxyuridine is often found as a contaminant of [3H]IdU and [3H]BrdU. For these reasons, complications can arise in the interpretation of experimental work using these radioactive compounds. The problems may be overcome by purifying the compounds by high performance liquid chromatography (HPLC) before use together with identification of the DNA components with which the 3H is associated by chromatographic analysis.     

Telomeric chromosome fusion in cells with micronuclei under the combined action of hyperthermia and hypothermia and halogenated analogs of thymidine]

The reaction of cells with micronuclei in respect of the induction of specific dicentric chromosomes with halogenated analogs of thymidine at various temperatures was studied. The positive correlation between the temperature and frequency of dicentrics was shown for all halogenated analogs of thymidine. The minimum frequency of dicentrics was found in the case when used 5-iododeoxyuridine and hypothermia (34 degrees C). The using of 5-bromodeoxyuridine at different temperatures displayed the intermediate results. The maximum level of dicentrics discovered under action of 5-chlorodeoxyuridine and hyperthermia (40 degrees C). In the former case the depression of mitotic chromosome condensation of micronuclei registered, in the latter one the chromosomes with portions of delayed spiralization were not found.     

Effect of thymidine analogs on tyrosinase activity and mRNA accumulation in mouse melanoma cells

The thymidine analog 5-bromodeoxyuridine (BrdU) has been found to inhibit terminal differentiation of a variety of cell types without significantly affecting the growth of these cells. We have compared the effect of BrdU with two other thymidine analogs, 5-iododeoxyuridine and 5-fluorodeoxyuridine, on the growth, tyrosinase activity, and tyrosinase-mRNA accumulation in BL-6 mouse melanoma cells. We show that all three analogs inhibit growth and tyrosinase activity, but only BrdU significantly inhibits the accumulation of tyrosinase mRNA. We consider these results in the light of current understanding of BrdU action.     

Effect of 5-bromodeoxyuridine on incorporation of RNA precursors in sea urchin embryos

Exposure of early sea urchin embryos to 5-bromodeoxyuridine (at concentrations up to 100 μg per ml) severely decreases the uptake of exogenous ³H-uridine into RNA. However, the actual gross rate of DNA or RNA synthesis in these embryos appears not to be affected by the presence of 5-bromodeoxyuridine.     

Epstein-Barr Virus-associated Antigens in Non-producing Clones of Human Lymphoblastoid Cell Lines

NUCLEIC acid hybridization suggests that the Epstein-Barr virus (EBV) genome may be present in human lymphoblastoid cell lines that are free of detectable EBV^1,2. We describe here a plentiful appearance of EBV-associated early antigens (EA) and the viral capsid antigen (VCA) in non-producing Raji and NC-37 cell lines when exposed to 5-bromodeoxyuridine (BUdR) or 5-iododeoxyuridine (IUdR). These antigens were synthesized in all the Raji and NC-37 clones exposed to BUdR or IUdR, strongly suggesting that a complete, but unexpressed, EBV genome exists in the cells of these non-producing lines.     

Phenomenon of delayed disruption of telomeric links between chromosomes in polykaryocytes formed from human-Chinese hamster hybrid cells

The clones MOM-8-1 and MOM-8-3 of human--Chinese hamster cell hybrids were used for induction of the phenomenon of delayed disruption of the telomeric links between chromosomes. Colcemide (0.08 microgram/ml) and 5-bromodeoxyuridine (20 micrograms/ml) were present in cell culture for 30 hours. The dicentrics were observed in tetraploid, hypotetraploid and hypodiploid metaphases. No differences between the clones were found. The hybrid cells were the second object in which the phenomenon under discussion was reproduced.     

Effects of 5-bromodeoxyuridine on the ACTH-dependent mitochondrial biogenesis in cortical cells of fetal rat adrenals in tissue culture

Cortical cells of fetal rat adrenals in tissue culture were treated with 5-bromodeoxyuridine (BrdU) during their proliferative phase and during ACTH stimulation when nuclear DNA synthesis has almost ceased. Pretreatment with 0.5 mug/ml/day of BrdU inhibited the ACTH-induced differentiation of cortical cells as well as the secretion of corticosterone and 18-OH-deoxycorticosterone (18-OHDOC). When nuclear DNA synthesis was suppressed and mitochondrial DNA synthesis was stimulated by ACTH BrdU addition (30 mug/ml/day) permitted normal untrastructural differentiation of cortical cells, except that the development of mitochondrial inner membranes was inhibited. Simultaneously mitochondrial inner membranes was inhibited. Simultaneously mitochondrial 11beta- and 18-hydroxylations were strongly inhibited while cytoplasmic 21-hydroxylation was not affected.     

Anionradicals from 5-halouracil substituted oriented DNA after X-irradiation at low temperatures

Conclusions The model of two initial radiation - induced radical components in oriented fibers of DNA,T ^– andG ⁺ [1], should be amended, for 5-halouracil substituted DNA by the finding that the primary anion is formed on the 5-halouracil base specifically but differentiates, depending on the halogen substituent, into*- and*-forms. The occurrence of the latter appears, from theoretical calculations, to be connected with a C-halogen bond elongation. The DNA matrix apparently allows for this whereas in the corresponding single crystals of 5-bromodeoxyuridine [1] or 5-iododeoxyuridine [4] the crystal packing prevents the elongation thus allowing for*-anion formation only.     

Induction of cytopathogenicity in mammalian cell lines challenged with culturable enteric viruses and its enhancement by 5-iododeoxyuridine

Cultures of 17 established cell lines were tested against 105 enteric virus types for capacity to support viral replication as indicated by cytopathogenic effect production. Enhancement of susceptibility by treatment of the cells with 5-iododeoxyuridine was evaluated in parallel with untreated cells. Cytopathogenic effect was produced in two or more cell lines by every virus tested except six strains of group A coxsackie virus. No cell line was found to be susceptible to these six virus types. In general, treatment with 5-iododeoxyuridine provided a more rapid onset of cytopathogenic effect in susceptible cells and in some instances resulted in refractory cells becoming permissive to viral replication. The use of 5-iododeoxyuridine allowed two human embryonic lines (HEL-299 and L-132), in combination, to be susceptible to all but the six group A coxsackie virus strains.     

Induction of cytopathogenicity in mammalian cell lines challenged with culturable enteric viruses and its enhancement by 5-iododeoxyuridine.

Cultures of 17 established cell lines were tested against 105 enteric virus types for capacity to support viral replication as indicated by cytopathogenic effect production. Enhancement of susceptibility by treatment of the cells with 5-iododeoxyuridine was evaluated in parallel with untreated cells. Cytopathogenic effect was produced in two or more cell lines by every virus tested except six strains of group A coxsackie virus. No cell line was found to be susceptible to these six virus types. In general, treatment with 5-iododeoxyuridine provided a more rapid onset of cytopathogenic effect in susceptible cells and in some instances resulted in refractory cells becoming permissive to viral replication. The use of 5-iododeoxyuridine allowed two human embryonic lines (HEL-299 and L-132), in combination, to be susceptible to all but the six group A coxsackie virus strains.     

Production and characteristics of a Djzungarian hamster cell line (DX-TK-) resistant to 5-bromodeoxyuridine]

New biochemically marked Djungarian hamster cell line (DX-TK-) was established. These cells are resistant to 5-bromodeoxyuridine (25 mkg/ml) and deficient in thymidine kinase activity (TK-). Due to this biochemical defect they have lost the ability to grow in HAT medium. DX-TK- cells are malignant. They grow as tumours after the inoculation to newborn Djungarian hamsters. Tumorigenecity of DX-TK- cells was decreased as compared with the parent TK+ cell line. DX-TK- cell line is a hypodiploid cell culture (26 chromosomes) with 7 chromosome markers easily identified by means of G-band staining. This line is a new model for somatic cell genetic experiments, particularly for somatic cell hybridization.     

Agglutination by concanavalin A of normal chick embryo fibroblasts treated by 5-bromodeoxyuridine (BrdU)

When chick embryo fibroblasts are grown for two days in the presence of low doses (18 μg/ml) of 5-bromodeoxyuridine (BrdU), their agglu-tinability by Concanavalin A is increased to about the same degree as following viral transformation of the cells by Rous sarcoma virus. Simultaneous addition of a large excess, but not of a low dose of thymidine prevents this effect of BrdU. Treatment with BrdU also enhances the agglutinability of fibroblasts infected with a conditional It mutant of Rcus sarcoma virus at the temperature of 41° which restricts morphological transformation.     

Culture media and species-related variations in the requirement of 5-bromodeoxyuridine for differential sister-chromatic staining

Various concentrations of 5-bromodeoxyuridine (BrdU) ranging from 0.01 to 10.0 μg/ml were tried for finding the minimal concentration required for differential staining of sister chromatids in lymphocytes of man, muntjac and cattle grown in three commonly used culture media, namely TC 199, Dulbecco's MEM and Ham's F-10. The lymphocytes grown in TC 199 required the lowest concentration of BrdU whereas it was highest for the lymphocytes grown in F-10. The minimal concentration varied for the 3 species studied, and it was not related to their DNA content. The differing amounts of thymidine and folic acid present in the various culture media seemed to account for the difference in the quantity of BrdU required for eliciting differential staining. Staining may also have depended on the intracellular nucleotide pool and/or on the difference in the substitutable dT sites of the genomes.     

Effects of thymidine analogs on Syrian hamster melanoma cells: phenotypes arising from selection for analog resistance

In order to compare the biological effects of different thymidine (dT) analogs, two unusual cell lines (B-4 and HAB) previously isolated from a Syrian hamster melanoma line by selection with 5-bromodeoxyuridine (BrdU) were analyzed for their response to other analogs. B-4 cells require high concentrations of BrdU for optimal growth, and it was seen that the requirement for BrdU could be satisfied partially by 5-chlorodeoxyuridine (CldU) but not by the other dT analogs tested. HAB cells are able to grow with all the dT residues in nuclear DNA replaced by BrdU, and it was found that they could also grow with essentially all the dT residues in nuclear DNA replaced by CldU but not by other analogs. New cell lines resistant to 100 micrometer concentrations of CldU, 5-iododeoxyuridine (IdU), and 5-hydroxymethyldeoxyuridine (HMdU) were isolated from the melanoma line and tested for cross-resistance to the other dT analogs. A high level of cross-resistance was observed only with BrdU and CldU. The ability of the cell lines resistant to BrdU, CldU, and IdU to incorporate these analogs into nuclear DNA also was determined. BrdU and CldU were incorporated efficiently by all of the lines tested, but the IdU-resistant cells seemed to preferentially exclude IdU.     

Increases in the number of adventitious roots caused by 2-thiouracil and 5-bromodeoxyuridine in Phaseolus mungo cuttings

A cutting of Phaseolus mungoproduced about 4 adventitious rootsat the basal 1 mm region when the basal part of the cuttingwas dipped in water. Rootlets became visible after a 36 hr lagperiod in untreated cuttings. Treatment with 2-thiouracil or5-bromodeoxyuridine increased the number of roots formed onthe cutting and prolonged the lag period. Effects of 2-thiouraciland 5-bromodeoxyuridine were reversed by simultaneously applieduracil and thymidine. The number of roots decreased and thelength of lag period was shortened. The increases in the numberof roots by 2-thiouracil or 5-bromodeoxyuridine was reducedby gibberellic acid, which did not cause a decrease in the numberof roots to be formed on control cuttings. Roots formed at thebasal region seem to suppress further root formation at theupper part of the hypocotyl. Inhibitors used here probably workby blocking the formation of these bottommost roots. (Received April 30, 1971; )     

Increased susceptibility of murine teratocarcinoma cells to Simian virus 40 and polyoma virus following treatment with 5-bromodeoxyuridine.

Cultures of the multipotential stem cell, embryonal carcinoma (EC), of a murine teratocarcinoma were treated with 5-bromodeoxyuridine (BrdU). Within 2-4 days at concentrations of 1-50 mugm/ml of BrdU, there was a marked change in the morphology of cells observed by light and electron microscopy. A comparison of the growth potential showed that for up to four days the BrdU-treated cultures were similar to untreated cultures. When these BrdU-treated cells were infected with Simian virus 40 (SV40) and polyoma virus (Py), there was an increase in susceptibility of the treated cells. The untreated embryonal carcinoma cells were refractory. These results suggest that BrdU modifies the embryonal carcinoma cells to allow infection with two DNA viruses.     

5-Chlorodeoxyuridine, more effectively than 5-bromodeoxyuridine, induces the formation of specific dicentric chromosomes in cells with micronuclei

5-chlorodeoxyuridine (5-ChldU) and 5-bromodeoxyuridine (5-BrdU) were tested in respect of the formation of specific dicentric chromosomes in cells with micronuclei. It is shown that the frequency of dicentrics is higher when 5-ChldU is used. On the ground of results of this study and of the previous data it is concluded that the activity of halogenated deoxyuridines is falling with molecular weight increasing.