Bromodeoxyuridine amplifies the inhibitory effect of oxygen on cell proliferation

The BrdUrd-Hoechst method was used to analyze the interaction of various oxygen concentrations with BrdUrd substituted DNA with respect to cellular proliferation. At oxygen concentrations above 5%, human diploid fibroblast-like cells and amniotic fluid fibroblast-like cells showed reduced proliferation rates, which resulted from an increase in noncycling cells and from a permanent arrest of cells in the G2 phase of the cell cycle. At 35% oxygen the increase in noncyling cell fraction and the permanent arrest in G2 was strongly dependent upon the concentration of BrdUrd. Incorporation of BrdUrd into DNA, therefore, amplifies the adverse effects of increasing oxygen concentrations upon cell proliferation. The mechanism of this amplification might involve a free radical attack on DNA similar to the radiation sensitizing effect of BrdUrd.     

Testosterone amplifies excitotoxic damage of cultured oligodendrocytes

An overactivation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors has been implicated in the pathophysiology of oligodendrocyte damage in demyelinating disorders of the CNS. We decided to examine the effect of testosterone on excitotoxic death of oligodendrocytes because a gender difference exists in the incidence and disease course of multiple sclerosis. Short-term pure cultures of oligodendrocytes (4 days in vitro) were exposed to a brief pulse with kainate or AMPA + cyclothiazide for the induction of excitotoxicity. Exposure to testosterone enantate was slightly toxic per se and amplified both AMPA and kainate toxicity. Testosterone treatment induced all gene targets of p53, and amplified the induction of these genes induced by kainate. The effect of testosterone was mediated by the activation of androgen receptors and was resistant to the aromatase inhibitors, dl-aminoglutethimide and 4-hydroxyandrost-4-ene-3,17-dione. Testosterone treatment also potentiated the stimulation of 45Ca2+ influx induced by AMPA + cyclothiazide or kainate without changing the expression of the glutamate receptor (GluR) 1, -2/3, and -4 subunits of AMPA receptors or the GluR6/7 subunits of kainate receptors. We conclude that testosterone amplifies excitotoxic damage of oligodendrocytes acting at an early step of the death cascade triggered by AMPA/kainate receptors.     

Bromodeoxyuridine tablet methodology for in vivo studies of DNA synthesis.

5-Bromodeoxyuridine (BrdU) tablets with different physical characteristics are useful in a wide variety of studies requiring detection of DNA replication in vivo. These tablets can effect a high substitution of BrdU in DNA, thereby permitting sister chromatid differentiation in chromosomes stained with 33258 Hoechst alone or in conjunction with Giemsa. Baseline and cyclophosphamide-induced in vivo sister chromatid exchange frequencies in mouse spleen, marrow, and thymus were measured and found to be significantly greater than those in spermatogonia. Sister chromatid exchange analysis was also extended to mouse liver and to Chinese hamster and Armenian hamster marrow cells. Sister chromatid differentiation was observed in Armenian hamster meiotic tissue, and evidence for interhomolog chromatid exchange obtained.     

Indices of free-radical-mediated damage following maximum voluntary eccentric and concentric muscular work

This study monitored plasma and skeletal muscle markers of free-radical-mediated damage following maximum eccentric and concentric exercise, to examine the potential role of free radicals in exercise-induced muscle damage. Fourteen male volunteers performed either (1) a bout of 70 maximum eccentric and a bout of 70 maximum concentric muscle actions of the forearm flexors (the bouts being separated by 4 weeks; n = 8) or (2) a bout of 80 maximum eccentric and a bout of 80 maximum concentric muscle actions of the knee extensors (the bouts being separated by 1 week; n=6). Plasma markers of lipid peroxidation, thiobarbituric acid-reactive substances (TBARS) and diene-conjugated compounds (DCC) were monitored in the arm protocol and skeletal muscle markers of oxidative lipid and protein damage, malondialdehyde (MDA) and protein carbonyl derivatives (PCD) respectively, were monitored in the leg protocol. In both protocols, the contralateral limb was used for the second bout and the order of the bouts was randomised between limbs. Repeated measures ANOVA indicated significant changes from baseline following eccentric arm work on the measures of serum creatine kinase activity (P < 0.05), maximum voluntary torque production (P < 0.01) and relaxed arm angle (P < 0.01). Subjective muscle soreness peaked 2 days after eccentric arm work (P < 0.05, Wilcoxon test). However, there were no changes in the plasma levels of TBARS or DCC following the eccentric or concentric arm exercise. Immediately after concentric leg exercise, skeletal muscle PCD concentrations was significantly higher than that observed immediately after eccentric work (P < 0.05). However, no significant difference between the eccentric and concentric knee extensor bouts was observed on the measure of skeletal muscle MDA concentration. The results of this study offer no support for the involvement of oxygen free radicals in exercise-induced muscle damage.     

Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step

We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([3H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [3H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [3H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.     

Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step

Summary We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([³H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [³H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [³H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.     

Bromodeoxyuridine substitution in mammalian DNA can both stimulate and inhibit restriction cleavage

Total replacement of thymidine by 5-bromodeoxyuridine in mammalian DNA causes a 5-fold stimulation in the rate of DNA cleavage by Mbo I. This is the first report of the stimulation of restriction endonuclease activity by 5-bromodeoxyuridine and is in contrast to the inhibition found with other restriction enzymes. We propose a hypothesis to rationalize these results.     

Singlet oxygen induced DNA damage.

Singlet oxygen generated by photoexcitation and by chemiexcitation selectively reacts with the guanine moiety in nucleosides (kq + kr about 5 x 10(6) M-1s-1) and in DNA. The oxidation products include 8-oxo-7-hydro-deoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Singlet oxygen also causes alkali-labile sites and single-strand breaks in DNA. The biological consequences include a loss of transforming activity as studied with plasmids and bacteriophage DNA, and mutagenicity and genotoxicity. Employing shuttle vectors, it was shown that double-stranded vectors carrying singlet oxygen induced lesions seem to be processed in mammalian cells by DNA repair mechanisms efficient in preserving the biological activity of the plasmid but highly mutagenic in mammalian cells. Biological protection against singlet oxygen is afforded by quenchers, notably carotenoids and tocopherols. Major repair occurs by excision of the oxidized deoxyguanosine moieties by the Fpg protein, preventing mismatch of 8-oxodG with dA, which would generate G:C to T:A transversions.     

Bromodeoxyuridine incorporation into DNA of human leukemia cells is not concentration dependent

Leukemia blasts isolated from bone marrow aspirates of 44 adults with acute leukemia were incubated for 1 h with 0.008-32 microM bromodeoxyuridine (Brd-Urd). After dual labeling with monoclonal anti-BrdUrd antibodies and propidium iodide, the cells were analyzed by flow cytometry. Percent labeled cells and intensity of labeling were similar over concentrations of BrdUrd ranging from 0.8-32 microM--a 40-fold range. Therefore, despite potential interpatient variability in nucleoside pharmacokinetics, commonly used doses of BrdUrd which are intended to achieve steady-state plasma concentrations in the 8.0 microM range can be expected to provide a reliable estimate of the S-phase fraction.     

Engineering a DNA damage response without DNA damage

Recent work has achieved the feat of activating the DNA damage checkpoint in the absence of DNA damage, revealing the importance of protein-chromatin associations for the activation, amplification and maintenance of the DNA damage response.     

DNA damage by peroxynitrite characterized with DNA repair enzymes.

The DNA damage induced by peroxynitrite in isolated bacteriophage PM2 DNA was characterized by means of several repair enzymes with defined substrate specificities. Similar results were obtained with peroxynitrite itself and with 3-morpholinosydnonimine (SIN-1), a compound generating the precursors of peroxynitrite, nitric oxide and superoxide. A high number of base modifications sensitive to Fpg protein which, according to HPLC analysis, were mostly 8-hydroxyguanine residues, and half as many single-strand breaks were observed, while the numbers of oxidized pyrimidines (sensitive to endonuclease III) and of sites of base loss (sensitive to exonuclease III or T4 endonuclease V) were relatively low. This DNA damage profile caused by peroxynitrite is significantly different from that obtained with hydroxyl radicals or with singlet molecular oxygen. The effects of various radical scavengers and other additives (t-butanol, selenomethionine, selenocystine, desferrioxamine) were the same for single-strand breaks and Fpg-sensitive modifications and indicate that a single reactive intermediate but not peroxynitrite itself is responsible for the damage.     

DNA damage and DNA sequence retrieval from ancient tissues.

Gas chromatography/mass spectrometry (GC/MS) was used to determine the amounts of eight oxidative base modifications in DNA extracted from 11 specimens of bones and soft tissues, ranging in age from 40 to >50 000 years. Among the compounds assayed hydantoin derivatives of pyrimidines were quantitatively dominant. From five of the specimens endogenous ancient DNA sequences could be amplified by PCR. The DNA from these specimens contained substantially lower amounts of hydantoins than the six specimens from which no DNA could be amplified. Other types of damage, e.g. oxidation products of purines, did not correlate with the inability to retrieve DNA sequences. Furthermore, all samples with low amounts of damage and from which DNA could be amplified stemmed from regions where low temperatures have prevailed throughout the burial period of the specimens.     

Multiple Initiation of Bacteriophage T4 DNA Replication: Delaying Effect of Bromodeoxyuridine

Effects of bromodeoxyuridine (BUdR) substitutions in phage T4 DNA on the initial stages of DNA replication were investigated. Electron microscope studies of partially replicated, light (thymidine-containing) T4 DNA revealed the presence of multiple loops and forks. These DNA preparations had no BUdR in either parental or newly synthesized DNA, and the observations thus show that multiple initiation of DNA replication is a normal event in T4 development and is not caused by the presence of BUdR. A comparison of early replicative stages of light and heavy (BUdR-containing) DNA in cells mixedly infected with light and heavy T4 phage showed that early DNA synthesis occurs preferentially on the light template. Heavy and light parental DNA became associated with the protein complex of replicative DNA with equal efficiency, and there was no effect of BUdR on the net rate of DNA synthesis after infection. Newly synthesized DNA from heavy templates sedimented more slowly through alkaline sucrose gradients than did newly synthesized DNA from light templates and appeared to represent fewer replicative regions per molecule. These data indicate that BUdR substitutions in the DNA caused a slight delay in initiation but that replication of heavy DNA proceeded normally once initiated.     

DNA damage checkpoint in budding yeast.

Eukaryotic cells have evolved a network of control mechanisms, known as checkpoints, which coordinate cell-cycle progression in response to internal and external cues. The yeast Saccharomyces cerevisiae has been invaluable in dissecting genetically the DNA damage checkpoint pathway. Recent results on posttranslational modifications and protein-protein interactions of some key factors provide new insights into the architecture of checkpoint protein complexes and their order of function.     

Immunochemical detection of glyoxal DNA damage.

The relevance of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases is widely documented. Immunochemical detection of ROS DNA adducts has been developed, however, recognition of glyoxal-DNA adducts has not previously been described. We have generated a polyclonal antibody that has shown increased antibody binding to ROS-modified DNA in comparison to native DNA. In addition, dose-dependent antibody binding to DNA modified with ascorbate alone was shown, with significant inhibition by desferrioxamine, catalase, and ethanol. Minimal inhibition was observed with uric acid, 1,10-phenanthroline and DMSO. However, antibody binding in the presence of EDTA increased 3500-fold. The involvement of hydrogen peroxide and hydroxyl radical in ascorbate-mediated DNA damage is consistent with ascorbate acting as a reducing agent for DNA-bound metal ions. Glyoxal is known to be formed during oxidation of ascorbate. Glyoxylated DNA, that previously had been proposed as a marker of oxidative damage, was recognised in a dose dependent manner using the antibody. We describe the potential use of our anti-ROS DNA antibody, that detects predominantly Fenton-type mediated damage to DNA and report on its specificity for the recognition of glyoxal-DNA adducts.     

Bleomycin-iron complexes and DNA radiation damage.

Radiation in the form of high-energy electrons dose-dependently activates bleomycin-Fe3+ in oxygen-containing DNA solutions. This activation causes a DNA fragmentation and a release of oxidative degradation products from the DNA. During irradiation, bleomycin-chelated Fe3+ is reduced to Fe2+. The kinetics of DNA base propenal-formation (measured by reaction with thiobarbituric acid) and iron-reduction (measured by bathophenanthroline chelation) are similar, with a yield of 1 mol base propenal/6 mol Fe3+ reduced. The activation of bleomycin-Fe3+ by irradiation could be instrumental in the synergistic action of radiotherapy and bleomycin observed on simultaneous administration in vivo.