Demonstration of spontaneous sister chromatid exchanges in vivo

The frequency of sister chromatid exchanges (SCEs) was examined as a function of bromodeoxyuridine (BUdR) concentration in vivo. Oneyear-old Wistar rats were continuously infused with BUdR through the tail vein for 24 h, sacrificed, and mitotic preparations prepared from femur bone marrow. It was observed that from the minimum concentration of BUdR which permitted accurate scoring (1.9 μg/g wt/h) to a BUdR concentration of 7 μg/g wt/h, SCE frequency remained constant. Above 7 μg BUdR/g wt/h SCE frequency increased, saturating at higher BUdR concentrations. The stability of SCE frequency at low BUdR concentrations is interpreted to indicate the existence of spontaneous SCEs in vivo.     

Effects of 5-bromo-2-deoxyuridine concentration and alpha-naphthoflavone on the association between smoking and the frequency of sister-chromatid exchanges in lymphocytes from maternal and cord blood

The frequency of sister-chromatid exchanges was analyzed in maternal and cord blood lymphocytes obtained at delivery from 23 nonsmokers and 21 smokers. Lymphocytes were cultured under 3 conditions: in the presence of 100 microM 5-bromo-2-deoxyuridine (BUdR), 20 microM BUdR and 20 microM BUdR with 40 microM alpha-naphthoflavone (ANF). Under all assay conditions, frequencies of SCEs were consistently higher for maternal lymphocytes than for cord lymphocytes. There was no association between SCE values for cultures of the same blood specimen with 100 microM BUdR and 20 microM BUdR. When cultured with 100 microM BUdR, maternal lymphocytes from smokers had a mean SCE frequency of 13.5, which was significantly higher than the value of 11.1 observed for nonsmokers (p = 0.001 by the Wilcoxon rank sum test). Maternal smoking had no significant effect on overall frequencies of SCEs in maternal blood cultured with 20 microM BUdR either with or without ANF or when the differential between cells cultured with and without ANF was considered. Use of caffeinated beverages was associated with increased SCE values for maternal lymphocytes cultured with 20 microM BUdR (Tau beta = 0.36, p = 0.02 for the Kendall's Rank Correlation), but no such association was seen with 100 microM BUdR. For cord blood lymphocytes, however, neither smoking nor caffeine use were associated with SCE values obtained by any of the assay conditions used. The findings suggest that results of human monitoring studies using SCEs could differ depending on the concentration of BUdR used in cultures.     

Effects of 5-bromodeoxyuridine on metamorphosis and on cell cycle kinetics of ganglion cells in Drosophila melanogaster

In order to determine suitable experimental conditions for estimating the accurate spontaneous frequency of sister chromatid exchanges (SCEs) in vivo in somatic cells of Drosophila melanogaster, the effects of bromodeoxyuridine (BUdR) on metamorphosis as well as on cell cycle kinetics were examined. The rate of growth of third-instar larvae, fed on BUdR-containing synthetic medium, markedly delayed with increasing concentrations of BUdR, but this toxic effect of BUdR was not observed below 150 μg/ml.Furthermore, the rate of eclosion drastically decreased by the incorporation of BUdR: it was reduced to about one-half of that in the control when the larvae were exposed to 100 (μg/ml. On the other hand, little difference in the rate of pupation was found within the range of 0–800 μg/ml BUdR. These results indicate that the developmental stage from pupa to adult is the most sensitive phase to BUdR.To test the effect of BUdR on cell cycle, metaphase cells were classified as having undergone each replication cycle in the presence of different BUdR concentrations according to the pattern of differential staining of sister chromatids, and the proportion of each replication cycle cells examined. No inhibition of cellular kinetics was observed at BUdR concentrations below 200 μg/ml.On the basis of these results, 100 μg/ml was chosen as suitable BUdR concentration for the analysis of cell cycle kinetics and according to the distribution of replication cycle metaphase cells as a function of time after the initiation of BUdR treatment, the cell cycle duration of the third-instar larval ganglion cells was roughly estimated to be about 7–8 h, at least under our experimental conditions.     

Effects of superoxide dismutase/catalase mimetics on life span and oxidative stress resistance in the housefly,Musca domestica

The purpose of this study was to determine whether superoxide dismutase/catalase mimetics lengthen the life span of the housefly, Musca domestica, as previously demonstrated for the nematode Caenorhabditis elegans. Various concentrations of Eukarion-8 or Eukarion-134 were administered via the drinking water and the effects on the life span of the flies and amounts of protein carbonyls were determined under normoxic and hyperoxic conditions. These SOD/catalase mimetics neither extended the life span of the flies nor attenuated the protein carbonyl content under normoxic conditions and shortened life span under hyperoxic conditions. Thus, the effect of these SOD/catalase mimetics on the life span of animals seem to be species-specific.     

Immunohistochemical Double Staining with Immunogold-Silver and Alkaline Phosphatase to Identify Nuclear Markers of Cellular Proliferation

To facilitate cell kinetics studies of brain tumors labeled with thymidine analogs, we developed a new method to identify nuclei labeled sequentially with bromodeoxyuridine (BUdR) and iododeoxyuridine (IUdR) by double staining with immunogold-silver and alkaline phosphatase. Patients received an intraoperative infusion of BUdR: excised tumor specimens were immediately labeled with IUdR in vitro. fixed with 70% alcohol, embedded in paraffin, and cut into 6 pm sections. The sections were incubated first with BR-3. a monoclonal antibody that recognizes only BUdR, and then with IU-4. a monoclonal antibody that recognizes both BUdR and IUdR: sections were counterstained with hematoxylin to identify unlabeled nuclei. Nuclei labeled only with IUdR stained red, whereas those labeled with BUdR or with both BUdR and IUdR stained black against a red background: unlabeled nuclei stained blue. This method was the most efficient differential staining technique to identify nuclei labeled only with IUdR and those labeled with BUdR among unlabeled nuclei.     

Mutagenic mechanism of 5-bromodeoxyuridine in Chinese hamster cells

Resistance to 6-thioguanine was induced by 5-bromodeoxyuridine (BUdR) in synchronous Chinese hamster cells. The yield of mutant colonies was not proportional to the amount of BUdR incorporated into DNA; thus mutants were not due to mispairing of BUdR with guanine during replication. Few mutants were induced until BUdR concentrations exceeded that of the intracellular thymidine triphosphate pool and mutant yield was depressed by addition of thymidine to the medium. These data suggest that BUdR exerts an allosteric effect on the DNA synthesizing system which renders it more error prone.     

For the reproductive death of a cell, damage to a single chromosome is sufficient

The Chinese hamster cells V-79 were treated with BUdR during one cell cycle; after that the cells were grown in the medium without BUdR and were irradiated by longwave-UV-light at different time. The cell survival after photolysis was compared with the percentage of metaphase plates with different number of chromosomes containing BUdR. It is concluded that for cell inactivation the presence of only one destroyed chromosome (or its part) is enough.     

INHIBITION OF MYOBLAST FUSION AFTER ONE ROUND OF DNA SYNTHESIS IN 5-BROMODEOXYURIDINE

The thymidine analogue 5-bromodeoxyuridine (BUdR) has a differential effect on the synthesis of tissue-specific products and molecules required for growth and division. Proliferating myogenic cells cultured in BUdR fail to fuse and fail to initiate the synthesis of contractile protein filaments. Conversely, BUdR has but a minor effect on cell viability and reproductive integrity. Low concentrations of BUdR result in an enhancement of cell number relative to the controls; higher concentrations are cytotoxic. Suppression of myogenesis is reversible after at least 10 cell generations of growth in the analogue. Cells that do not synthesize DNA, such as postmitotic myoblasts and myotubes, are not affected by BUdR. Incorporation of BUdR for one round of DNA synthesis was accomplished by first incubating myogenic cells, prior to fusion, in 5-fluorodeoxyuridine (FUdR) to block DNA synthesis and collect cells in the presynthetic phase. The cells were then allowed to synthesize either normal DNA or BU-DNA for one S period by circumventing the FUdR block with BUdR or BUdR plus thymidine (TdR). The cultures were continued in FUdR to prevent dilution of the incorporated analogue by further division. After 3 days, the cultures from the FUdR-BUdR series showed the typical BUdR effect; the cells were excessively flattened and few multinucleated myotubes formed. Cells in the control cultures were of normal morphology, and multinucleated myotubes were present. These results were confirmed in another experiment in which BUdR-³H was added to 2-day cultures in which myotubes were forming. Fusion of thymidine-³H-labeled cells begins at 8 hr after the preceding S phase. In contrast, cells which incorporate BUdR-³H for one S period do not fuse with normal myotubes.     

Use of Bromodeoxyuridine For Cell Kinetic Studies In Intact Animals

Abstract. A method is described for the use of BUdR for tracing cell proliferation patterns in the intestinal mucosa of intact mice.
The method has several distinct advantages over existing methods.
Bromodeoxyuridine (BUdR) is a well-established alternative to tritiated thymidine ([³H]TdR) as a tracer for studying DNA replication. However, its use in cytological as opposed to biochemical studies has been largely confined to examination of metaphase spreads, particularly analysis of sister chromatid exchange (Block, 1982). For this, BUdR incorporation into DNA has been demonstrated using the fluorescent dye Hoechst 33258, together with fluorescence microscopy (Latt, 1973), or Giemsa staining (Perry & Wolf, 1974). Recently, introduction of a monoclonal antibody which recognizes BUdR in single-stranded DNA (Gratzner, 1982) has enabled BUdR to be used for studying cell cycle kinetics in a manner exactly analogous to the use of [³H]TdR. This has been reported for whole cells in suspension and in monolayer (Dolbeare et al. , 1983; Dean et al. , 1984; Raza et al. , 1984). BUdR included in tissue culture medium is taken up and incorporated into newly synthesized DNA via the same pyrimidine salvage pathway as [³H]TdR (thymidine kinase). A concentration of as little as 10 μm—well below cytotoxic levels (Cerni, 1984)—is sufficient to give readily detectable labelling by immunocytochemistry with a pulse of less than 15 min. the validity of BUdR labelling for cell kinetic studies has been well established in comparisons with other methods by Dolbeare et al. (1983), Dean et al. (1984), and Raza et al. (1984).
We describe here the use of BUdR together with an immunocytochemical detection system applied to sections of wax-embedded tissues, which provides a convenient method of cell cycle analysis in intact animals.     

Prenatal alcohol exposure shortens life span in rats

In a study of the effects of in utero alcohol exposure on life span in rats, pregnant rats were intubated twice daily with 3.5 gm/kg alcohol on gestational days 11-21 or with an isocaloric sucrose solution. These latter animals were pair-fed and pair-watered to alcohol-treated animals. A third group served as nontreated ad lib-fed controls. At birth, all offspring were removed from their biological mothers, culled to eight per litter, and placed with nontreated surrogate dams. Alcohol-exposed animals died at a significantly younger age than pair-fed and ad lib controls and never attained the same maximum body weights as control animals. For females prenatally exposed to alcohol, life span was shortened by about 20 weeks; in male cohorts, life span was shortened by about 2.5-7 weeks.     

The amplified expression of factors regulating myogenesis in L6 myoblasts

A strategy for increasing the expression of the factors regulating myogenesis was developed based upon the observation that increased amounts of regulatory factors could overcome the inhibition of differentiation produced by 5-bromodeoxyuridine (BUdR). L6 rat myoblasts were subjected to multiple cycles of cloning in progressively increasing concentrations of BUdR. The first clones to differentiate were picked and replated for the next cycle of selection. After 28 cycles in BUdR, cells were isolated that could differentiate in the presence of 8 microM BUdR. Cell hybrids between myoblasts subjected to 21 cycles of selection (BU21 cells) and differentiation-defective myoblasts exhibited a high probability of differentiation, consistent with the hypothesis that BU21 cells were overproducing factor(s) involved in the decision to differentiate. The selection of cells able to differentiate in the presence of BUdR may provide a general approach for increasing the expression of the regulatory molecules controlling terminal differentiation.     

Inhibition of sporulation in Bacillus subtilis by bromodeoxyuridine and the effect on DNA replication

A 5-bromo-2'-deoxyuridine (BUdR)-tolerant derivative of a thymidine (TdR)-requiring strain of Bacillus subtilis was used to examine the effect of BUdR, an analogue of TdR, on sporulation. At a TdR:BUdR ratio which had little effect on growth, sporulation was inhibited if cells were exposed to BUdR during the period of DNA synthesis at the onset of the process. Cells recovered from BUdR inhibition of sporulation if the analogue was removed and DNA replication allowed to continue with TdR alone. BUdR prolonged the period of DNA synthesis during sporulation and experiments with chloramphenicol suggested that this was due in part to unscheduled initiation of new rounds of replication.     

Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.     

The effects of 5-bromodeoxyuridine on fusion of the cranial neural folds in the mouse embryo

The effects of 500 and 300 mg/kg bromodeoxyuridine (BUdR) on the process of fusion of the neural folds were tested after injection into pregnant mice on day 8 of gestation (192 hours postcoitum). Various doses of the natural nucleoside, thymidine (TdR), were also tested. Both doses of BUdR retarded growth to the same extent, but only the larger dose caused neural tube defects in 28.8% of embryos. Treatment with the larger dose also caused extensive cell necrosis to appear in the neuroepithelium of the neural folds between 12 and 15 hours after treatment. No changes were detectable with the light microscope up to this time. Measurement of the cell generation time in treated and control embryos indicated that the BUdR prolonged the cycle by about 2 hours and that the dying cells were in the second DNA synthetic phase following incorporation of the analog. Treatment with the smaller dose of BUdR caused minimal cell necrosis. This was taken as evidence for the importance of cell necrosis in the pathogenesis of BUdR-induced neural tube defects. Treatment with excess TdR did not cause either neural tube defects or cell necrosis, and a dose of TdR equimolar with the large dose of BUdR (400 mg/kg TdR) did not retard growth. Doses of 800 and 1,200 mg/kg TdR retarded growth to the same extent as BUdR. The administration of an equimolar amount of TdR, along with the teratogenic dose of BUdR, prevented the occurrence of cell necrosis and neural tube defects. When treatments were given on day 9 of gestation, 500 mg/kg BUdR caused cell necrosis in the neuroepithelium about 15 hours after treatment but no neural tube defects were produced by day 9 after treatment. It is suggested that in this case cell necrosis occurred too late to interfere with neural fold fusion. It was concluded that the ability of BUdR to cause exencephaly in mouse embryos was due to cell necrosis in the neuroepithelium.     

The UV sensitivity of the bromodeoxyuridine-substituted chromosomes in the Chinese hamster

Chinese hamster cells with chromosomes differently substituted for BUdR (TT-TT, TT-TB, TB-TB, TB-BB, where T is thymidine containing chromatid and B is BUdR substituted chromatid) were exposed to UV-light in phase G2 and chromosome aberrations (mainly chromatid breaks) were analysed. Breaks frequency per chromosome was proportional to BUdR content. No breaks were found in TT-TT chromosomes. The frequency of breaks per TB chromatid was similar with TT-TB and TB-BB chromosomes. In TB-BB chromosomes, however, virtually no breaks occurred in TB chromatids whereas in BB chromatids, their frequency was much higher than was expected.     

Dietary restriction in two rotifer species: the effect of the length of food deprivation on life span and reproduction

According to resource allocation theory, animals face a trade off between the allocation of resources into reproduction and into individual growth/maintenance. This trade off is reinforced when food conditions decline. It is well established in biological research that many animals increase their life span when food is in suboptimal supply for growth and/or reproduction. Such a situation of reduced food availability is called dietary restriction. An increase in life span under dietary restricted conditions is seen as a strategy to tolerate periods of food shortage so that the animals can start reproduction again when food is in greater supply. In this study, the effect of dietary restriction on life span and reproduction in two rotifer species, Cephalodella sp. and Elosa worallii, was investigated using life table experiments. The food concentration under dietary restricted conditions was below the threshold for population growth. It was (1) tested whether the rotifers start reproduction again after food replenishment, and (2) estimated whether the time scale of dietary restricted conditions is relevant for the persistence of a population in the field. Only E. worallii responded to dietary restriction with an increase in life span at the expense of reproduction. After replenishment of food, E. worallii started to reproduce again within 1 day. With an increase in the duration of dietary restricted conditions of up to 15 days, which is longer than the median life span of E. worallii under food saturation, the life span increased and the life time reproduction decreased. These results suggest that in a temporally (or spatially) variable environment, some rotifer populations can persist even during long periods of severe food deprivation.     

Genetics of life span in mice

Thymic involution that occurs earlier in some individuals than others may be the result of complex interactions between genetic factors and the environment. Such interactions may produce defects of thymus-dependent immune regulation associated with susceptibility to developing autoimmune diseases, malignancy, and an increased number of infections associated with aging.The major histocompatibility complex may be important in determining profiles of cause of death and length of life in mice. Genetic influences on life span involve interactions between loci and allelic interactions during life which may change following viral infections or exposure to other environmental factors. We have used different experimental protocols to study the influence of H-2 on life span and found that interactions between genetic regions, are inconsistent, particularly when comparing mice infected or not infected with Sendai virus.Genes important for life span need to be studied against many genetic backgrounds and under differing environmental conditions because of the complexity of the genetics of life span. Several genetic models were used to demonstrate that the MHC is a marker of life span in backcross and intercross male mice of the H-2^d and H-2^b genotypes in B10 congenic mice. Females lived longer than males in backcross and intercross mice, while males lived longer than females in B10 congenics. H-2^d was at a disadvantage for life span in backcross mice of the dilute brown and brown males exposed to Sendai infection, but intercross mice not exposed to Sendai virus of the same genotype were not at a disadvantage. H-2^d mice were not disadvantaged when compared to H-2^b in B10 congenics that had not been exposed to Sendai virus infection but the reverse was true when they were exposed. Overall, all our studies suggest that genetic influences in life span may involve interactions between loci and many allelic interactions in growing animals or humans. These genetic influences on life span may vary after they are exposed to infections or other environmental conditions. This paper emphasizes the need to use several genetic models, especially animals that have been monitored for infections, to study the genetics of life span.     

FLOW CYTOMETRIC CELL CYCLE ANALYSIS USING THE QUENCHING OF 33258 HOECHST FLUORESCENCE BY BROMODEOXYURIDINE INCORPORATION

Cultures of L cells were grown in medium containing 2.0 mg/l bromodeoxyuridine (BUdR) and stained with the fluorescent dye 33258 Hoechst for flow cytometric analysis. During exposure to BUdR, the cells replace thymidine by BUdR in the newly synthesized DNA. The new DNA is not stainable with 33258 Hoechst, which is highly specific for thymidine. The temporal development of the fluorescence distributions after addition of BUdR to the growth medium has been investigated in the flow cytometer, and the data were used to calculate the mean durations of the phases G₁, S and G₂+ M in exponentially growing cultures as well as the cycle transit times in synchronized cultures. The percentage of non-cycling cells was determined in each experiment.