Detection of free radical-induced DNA damage with bromodeoxyuridine/Hoechst flow cytometry: implications for Bloom's syndrome.

The clinical radiosensitizer bromodeoxyuridine (BrdU) was shown to enhance oxygen free radical-mediated growth inhibition. Cells from Bloom's syndrome, a rare autosomal recessive disorder characterized by pre- and post-natal growth deficits, telangiectatic erythema, recurrent respiratory infections and a high incidence of cancer, exhibit in culture a hypersensitivity to BrdU. We analysed disturbed cell kinetics of Bloom's syndrome fibroblasts and permanent B-cell lines with a novel cell kinetic method: BrdU/Hoechst flow cytometry. Fibroblasts show a pattern similar to that of normal cells exposed to a breakdown product of lipid peroxides, whereas B-cells exhibit the cell kinetic disturbance provoked by elevated oxygen concentrations in normal cells. In both cell types the cell kinetic pattern was dependent upon the BrdU concentration in the culture medium. These data suggest an elevated endogenous generation of oxygen free radicals in Bloom's syndrome cells, which may relate to the elevated incidence of malignancies in these patients.     

Continuous bromodeoxyuridine labeling and bivariate ethidium bromide/Hoechst flow cytometry in cell kinetics

Most techniques of flow cytometric cell cycle analysis are not capable of distinguishing the number of rounds of DNA synthesis that a cell has undergone since the start of an experiment. Continuous labeling with 5-bromodeoxyuridine (BrdUrd) offers such a potential. We illustrate here that the bivariate analysis of non-BrdUrd-quenched ethidium bromide vs. BrdUrd-quenched Hoechst 33258 fluorescence offers a high degree of resolution that enhances the analytical power of the technique, and that this approach can be applied to the analysis of a broad range of human and murine primary cells and established cell lines.     

Mechanisms of free radical-induced damage to DNA

Abstract

Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5′-cyclopurine-2′-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.     

Mechanisms of free radical-induced damage to DNA

Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5'-cyclopurine-2'-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.     

Chemical determination of free radical-induced damage to DNA.

Free radical-induced damage to DNA in vivo can result in deleterious biological consequences such as the initiation and promotion of cancer. Chemical characterization and quantitation of such DNA damage is essential for an understanding of its biological consequences and cellular repair. Methodologies incorporating the technique of gas chromatography/mass spectrometry (GC/MS) have been developed in recent years for measurement of free radical-induced DNA damage. The use of GC/MS with selected-ion monitoring (SIM) facilitates unequivocal identification and quantitation of a large number of products of all four DNA bases produced in DNA by reactions with hydroxyl radical, hydrated electron, and H atom. Hydroxyl radical-induced DNA-protein cross-links in mammalian chromatin, and products of the sugar moiety in DNA are also unequivocally identified and quantitated. The sensitivity and selectivity of the GC/MS-SIM technique enables the measurement of DNA base products even in isolated mammalian chromatin without the necessity of first isolating DNA, and despite the presence of histones. Recent results reviewed in this article demonstrate the usefulness of the GC/MS technique for chemical determination of free radical-induced DNA damage in DNA as well as in mammalian chromatin under a vast variety of conditions of free radical production.     

Inhibition of free radical-induced DNA damage by uric acid

Single-strand DNA breaks were produced in isolated rat liver nuclei incubated with 3 separate oxygen free radical generating systems: xanthine oxidase-acetaldehyde plus Fe(II); hematin-R(H)OOH; Fe(II)-H2O2. Uric acid inhibited the induction of damage in the first two systems only. At concentrations below those found in human plasma, it was particularly effective against strand breaks produced by hematin-cumene hydroperoxide. These results offer additional evidence that uric acid may function as a cellular protective agent against superoxide and hydroperoxyl free radical-induced cytotoxicity toxicity.     

Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry.

Hairless mice were injected intraperitoneally with bromodeoxyuridine (Brd-Urd). Basal cells were isolated from epidermis, fixed in 70% ethanol, and prepared for bivariate BrdUrd/DNA flow cytometric (FCM) analysis. Optimum detection of incorporated BrdUrd in DNA was obtained by combining pepsin digestion and acid denaturation. The cell loss was reduced to a minimum by using phosphate-buffered saline containing Ca2+ and Mg2+ to neutralize the acid. The percentage of cells in S phase and the average uptake of BrdUrd per labelled cell in eight consecutive windows throughout the S phase were measured after pulse labelling at intervals during a 24 h period. Furthermore, the cell cycle progression of a pulse-labelled cohort of cells was followed up to 96 h after BrdUrd injection. In general the results from both experiments were in good agreement with previous data from 3H-thymidine labelling studies. The percentage of cells in S phase was highest at night and lowest in the afternoon, whereas the average uptake of BrdUrd per labelled cell showed only minor circadian variations. There were no indications that BrdUrd significantly perturbed normal epidermal growth kinetics. A cell cycle time of about 36 h was observed for the labelled cohort. Indications of heterogeneity in traverse through G1 phase were found, and the existence of slowly cycling or temporarily resting cells in G2 phase was confirmed. There was, however, no evidence of a significant population of temporarily resting cells in the S phase. Bivariate DNA/keratin FCM analysis revealed a high purity of basal cells in the suspensions and indicated that the synthesis of the differentiation-keratin K10 was turned on only in G1 phase and after the last division.     

Characterization of free radical-induced base damage in DNA at biologically relevant levels

DNA damage induced by oxygen radicals, e.g., hydroxyl radicals generated in living cells either by cellular metabolism or external agents such as ionizing radiations, appears to play an important role in mutagenesis, carcinogenesis, and aging. Elucidation of the chemical nature of such DNA lesions at biologically significant quantities is required for the assessment of their biological consequences and repair. For this purpose, a sensitive method using gas chromatography-mass spectrometry with the selected-ion-monitoring technique (GC-MS/SIM) was developed in the present work. DNA was exposed to hydroxyl radicals and hydrogen atoms produced by ionizing radiation in N2O-saturated aqueous solution. DNA samples were subsequently hydrolyzed with formic acid, trimethylsilylated, and analyzed by GC-MS/SIM. Characteristic ions from previously known mass spectra of DNA base products as their trimethylsilyl derivatives were recorded and the area counts of each ion were integrated. From these acquired data, a partial mass spectrum of each product was generated and then compared with those of authentic materials. This technique permitted the detection and characterization of a large number of free radical-induced based products of DNA, i.e., 5,6-dihydrothymine, 5-hydroxy-5,6-dihydrothymine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-hydroxycytosine, thymine glycol, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine, simultaneously in a single sample after radiation doses from 0.1 to 10 Gy. Detectable amounts of the base products were found to be as low as approximately 10 fmol per injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rate of DNA synthesis determined by flow cytometry using the BrdUrd/Hoechst technique in combination with propidium-iodide staining

Concanavalin A (conA) and phytohemagglutinin (PHA), at relatively high concentrations, induce spreading of human T lymphocytes on adhesive surfaces. After 24–48 h of mitogen stimulation of such lymphocytes in suspension, approx. 50% of the cells had acquired the capacity to develop prominent substrate-attached actin-containing projections with a length of 1–7 μm when subsequently induced to spread on a surface. In addition, cells stimulated with mitogen when in continuous contact with a surface developed similar projections after the same stimulation period. The spreading of lymphocytes was accompanied by a disappearance of the microvilli with a length of 0.2–0.9 μm present in large numbers on activated cells in suspension. Thus, on the basis both of their size as well as on the presence in relation to substrate contact, these microvilli and the substrate-attached projections are separate structures. Acquisition of the capacity to form projections after substrate contact was dependent on protein synthesis during the stimulation period and not detectable until 10–18 h after starting the stimulation. Control experiments indicated that the inhibiting of projection formation by inhibitors of protein synthesis was not due to a toxic effect, since the presence of these inhibitors did not prevent the formation of actin-containing projections in cells that had acquired the capacity to form such projections. T-enriched lymphocytes did not develop substrate-attached projections during continuous adhesion to a surface mediated by the non-mitogenic ligands poly-l-lysine and wheat germ lectin. Nor did cells cultured under these conditions develop prominent projections when subsequently transferred to another substrate and induced to spread in the presence of conA.     

Measurement of DNA damage in mammalian cells using flow cytometry

A technique for the detection of DNA damage induced by radiation insult has been developed. Cells were lysed with a buffer containing 2 M sodium chloride to release the DNA in a supercoiled form, the nucleoid. These were stained with the DNA intercalating dye, ethidium bromide, and exposed to laser light within a flow cytometer. Scattered and fluorescent light was analyzed from the laser/nucleoid interaction following irradiation of viable cells with gamma rays. The addition of ethidium bromide to prepared nucleoids caused a reduction in scattered light due to condensation of the nucleoid. Irradiation of cells prior to nucleoid production and ethidium bromide treatment restricted this condensation and produced a dose-dependent increase in laser scatter. Nucleoids derived from human lymphocytes showed enhanced light scatter from 5 Gy, compared to Chinese hamster ovary (CHO) fibroblasts where doses above 10 Gy were required. Up to 30 Gy CHO nucleoids showed a dose-dependent reduction in the ethidium bromide fluorescence. This technique allows detection of altered light scattering and fluorescent behavior of nucleoids after cellular irradiation; these may be related to structural changes within the nucleus induced by the radiation. The use of flow cytometry compared to other methods allows a rapid analysis of nuclear damage within individual cells.     

Clinical evaluation for the mechanism of cisplatin with bromodeoxyuridine by flow cytometry and clinico-histological study

The tumor cell kinetics before and after intra-arterial injection of cisplatin (CDDP) was examined in a patient with maxillary carcinoma using flow cytometry and propidium iodide and fluorescein isothiocyanate labeled bromodeoxyuridine double staining. In addition, the viability was determined with fluorescein diacetate and compared with the histological picture. The action mechanism which the authors obtained 24 hours after intra-arterial injection of CDDP to the clinical case was considered to be S phase block, especially early S phase block. Markedly decreased viability also makes us consider the cytocidal effect. These findings correlated with the data of the fundamental investigation reported previously. Histological examination after the intra-arterial injection of CDDP revealed remarkable necrosis in the tissue.     

Basic study for measurement of radiation damage of DNA using a flow cytometry

Radiation damage of DNA in HeLa cells was measured according to a method reported by Milner, et al. Cells were suspended in lysis buffer to obtain nucleoid. They were stained with ethidium bromide immediately before the measurement by using a system of flow cytometry. The mean position of channels for forward scatters increased at first and decreased thereafter as the concentration of ethidium bromide increased. The biphasic response disappeared with irradiation given to the cells. When the concentration of ethidium bromide was constant, the mean position of channels for forward scatters increased as the dose of irradiation increased. It might be possible to use the method in predicting the response of a tumor to irradiation in the clinical practice.     

Analysis of free radical damage within single cells using flow cytometry.

The data presented here illustrates the additional information that can be gained on single cell biological effects by using a method of damage estimation based on single cells. The experiments involving primarily free radical damage carried out using H2O2 and the radioprotectors cysteamine and WR 1065, both revealed data that could not have been obtained from a macroscopic study of free radical-DNA chemistry and analysis of reaction products. This serves to emphasise the difficulty in extrapolating both free radical based and other chemical reactions to effects seen in living systems.     

BrdU—Hoechst flow cytometry: A unique tool for quantitative cell cycle analysis

Unlike other techniques, flow cytometric analysis of BrdU-quenched 33258 Hoechst fluorescence may be used to measure cell activation and the G₁, S, and G₂/M compartment distributions in each of three successive cell cycles after growth stimulation of human peripheral blood lymphocytes. Cell cycle kinetic curves can be constructed from the BrdU—Hoechst flow data which allow the simultaneous assessment of growth fraction, lagtime, compartment exit rate, compartment duration, and compartment arrest. Applications of this new versatile technique include the evaluation of drug and growth factor effets, cell aging, and diagnosis in medicine and immunology.     

An improved method for the immunocytochemical detection of bromodeoxyuridine labeled nuclei using flow cytometry

A new staining protocol is described for the immunocytochemical detection of BrdUrd labeled nuclei. Pepsin treatment of ethanol fixed cells or tissue, followed by DNA denaturation at low pH, resulted in increased sensitivity of BrdUrd staining comparable to the thermal denaturation protocol, and decreased background binding. This technique is applicable to cell suspensions, including cultured cells and bone marrow cells. Furthermore, pepsin digestion of ethanol fixed tissue fragments resulted in a high recovery of nuclei in which incorporated BrdUrd could be detected. This possibility, together with the high sensitivity, make this method especially suitable for cell kinetic studies of human solid tumors in vivo.     

Detection of DNA strand breaks in single cells using flow cytometry

A preliminary method is reported of alkaline unwinding of DNA within single cells and quantitation of the single-stranded and double-stranded DNA with the fluorescent probe acridine orange. A suspension of alkali-treated cells is obtained and analysed by flow cytometry. An increase in the amount of single-stranded DNA is taken as an indication of strand breaks. An advantage of this method is that a large number of cells can be individually analysed for DNA strand breaks. A measurement of DNA content is also obtained, making it possible to discriminate between cells in various parts of the cell cycle.     

Multiparameter flow cytometry of bacteria: implications for diagnostics and therapeutics

BACKGROUND: Flow cytometric studies of antibiotic susceptibilities of bacteria have typically measured a single fluorescence parameter, such as membrane potential (indicating viability), or permeability to nucleic acid stains such as propidium (indicating nonviability). Cytometry of bacteria stained simultaneously with a membrane potential dye and a permeability indicator reveals unanticipated complexity. METHODS: Aliquots of cultures of three bacterial species were stained with the potential-sensitive dye hexamethylindocarbocyanine [DiIC1(3)] and the permeability indicator TO-PRO-3, in the presence and absence of a proton ionophore which collapses the potential gradient. They were analyzed using a dual-laser flow cytometer. RESULTS: Cultures grown under suboptimal conditions appear to contain cells that take up TO-PRO-3 while maintaining membrane potential, although some events showing both high DiIC1(3) fluorescence and high TO-PRO-3 fluorescence may represent clumps. CONCLUSIONS: Variations in metabolic patterns between species and within organisms under suboptimal culture conditions or following antibiotic exposure may make it difficult to develop flow cytometric clinical assays for antibiotic susceptibility. However, transient permeabilization of otherwise resistant organisms by sublethal doses of antibiotics may make it possible to treat infections by such organisms with suitably derivatized, otherwise toxic molecules; multiparameter cytometry should be useful in pursuing this approach to therapy.     

Cytochemistry for bromodeoxyuridine/DNA analysis: stoichiometry and sensitivity

This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0.1 M HCl and then heating them to 80 degrees C in a 50% formamide solution. The cells are then immunofluorescently stained using a monoclonal antibody against BrdUrd in single-strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double-strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd-linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.     

Cell kinetics of mouse kidney using bromodeoxyuridine incorporation and flow cytometry: preparation and staining

The kidney is recognized as a dose-limiting tissue by certain radiation treatments. The relationship between the onset of compensatory proliferation in response to irradiation and the expression of functional damage is difficult to study because of the low cell turnover in slowly proliferating tissues. We report on a method to obtain a suitable cell preparation from mouse kidney for study by flow cytometry using the recently developed staining techniques for bromodeoxyuridine incorporation. The labelling index of 0.3% in untreated mouse kidney was easily measured because large numbers of cells could be analysed rapidly. We show that compensatory proliferation after unilateral nephrectomy remains elevated for up to 3 weeks after surgery. Using the BrdU/FCM technique we were able to measure the duration of the S phase in normal and nephrectomized kidneys which we found to be 8.5 hr in both cases. The estimates of potential doubling time were similar to the time scale observed to elapse before functional damage is observed in normal kidneys and those in which damage is precipitated by surgery.     

Cell kinetics of mouse kidney using bromodeoxyuridine incorporation and flow cytometry: preparation and staining

Abstract. The kidney is recognized as a dose-limiting tissue by certain radiation treatments. The relationship between the onset of compensatory proliferation in response to irradiation and the expression of functional damage is difficult to study because of the low cell turnover in slowly proliferating tissues. We report on a method to obtain a suitable cell preparation from mouse kidney for study by flow cytometry using the recently developed staining techniques for bromodeoxyuridine incorporation. The labelling index of 0.3% in untreated mouse kidney was easily measured because large numbers of cells could be analysed rapidly. We show that compensatory proliferation after unilateral nephrectomy remains elevated for up to 3 weeks after surgery. Using the BrdU/FCM technique we were able to measure the duration of the S phase in normal and nephrectomized kidneys which we found to be 8.5 hr in both cases. The estimates of potential doubling time were similar to the time scale observed to elapse before functional damage is observed in normal kidneys and those in which damage is precipitated by surgery.