Multiparametric cell-cycle analysis of peripheral blood-activated lymphocyte subsets using staining based on the TdT method for incorporated BrdUrd.

This study describes a new method for the simultaneous assessment of the distribution of a cell population in the G0/G1, S, and G2/M cell-cycle phases by using multiparameter flow cytometry and single staining based on BrdUrd incorporation. Both the K562 cell line and PHA-stimulated peripheral blood lymphocytes (PBL) were analyzed. Cells were cultured in the presence of BrdUrd for 30 min prior to cell harvesting. Once collected, cells were exposed to ultraviolet light for 5 min and then fixed immediately in 70% ethanol (-20 degrees C) for at least 30 min. Once fixed, the cells were placed for 30 min at 37 degrees C in the presence of terminal deoxynucleotidyl transferase (TdT) and dUTP labeled with digoxigenin; they were then stained with FITC-labeled anti-digoxigenin. Our results show that G0/G1, s, and G2/M cell populations can be clearly discriminated according to FITC fluorescence and light-scatter parameters. In this way, S-phase cells can be identified by their FITC staining. From the cells which were negative for anti-digoxigenin-FITC antibody, two clear populations could be resolved in a forward scatter, side scatter, and fluorescence pulse-width three-dimensional plot; the values obtained for G0/G1 cells were lower than those obtained for G2/M cells in all three parameters. Multiparameter analysis of PBL stained for two surface antigens (CD3 and CD8) and for BrdUrd by direct or indirect TdT method permitted cell-cycle analysis of different subpopulations, including CD3+/CD8+, CD3+/CD8-, CD3-/CD8+, and CD3-/CD8-.

Comparison of Vindelov et al. and bromodeoxyuridine/DNA double-staining flow cytometry methods for analysis of cell cycle distribution in rat thymocytes.

This study compares the cell cycle distribution in rat thymocytes obtained by means of bromodeoxyuridine (BrdUrd) labeling of S-phase cells and the analysis of the S-phase fraction obtained according to the technique of Vindelov et al. (Cytometry 3:332-338, 1983). The proportion of BrdUrd-labeled cells was analyzed in single cell suspensions of adult rat thymocytes after in vivo injection of BrdUrd and the results then compared with those obtained after measuring the cell DNA contents according to the Vindelov et al. method. The percentage of BrdUrd-positive cells was greater than the S-phase fraction obtained using the Vindelov et al. technique. By contrast, no major differences were observed between the percentage of BrdUrd-positive cells and the S-phase fraction obtained after analyzing the DNA histograms of the same data files with the RFIT mathematical model. The elimination of trypsin treatment used in the Vindelov et al. method did not alter the results, whereas the use of DNA denaturation with 2N HCl was shown to increase the percentage of S-phase rat thymocytes (calculated from DNA histograms) independently of whether trypsin treatment was used or not. However, the value of the S-phase fraction was not as great as that obtained after BrdUrd labeling. Thus when comparing BrdUrd-labeling and the Vindelov et al. technique, important differences in the percentage of S-phase adult rat thymocytes were observed. Selective G0/G1 cell loss during washing and centrifugation steps performed after the DNA denaturation used for BrdUrd detection was the main reason for these differences.

Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part III. Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites and in situ hybridization.

This paper is a continuation of parts I (history, methods and cell kinetics) and II (clinical applications and carcinogenesis) published previously (Dolbeare, 1995 Histochem. J. 27, 339, 923). Incorporation of bromodeoxyuridine (BrdUrd) into DNA is used to measure proliferation in normal, diseased and injured tissue and to follow the effect of growth factors. Immunochemical detection of BrdUrd can be used to determine proliferative characteristics of differentiating tissues and to obtain birth dates for actual differentiation events. Studies are also described in which BrdUrd is used to follow the order of DNA replication in specific chromosomes, DNA replication sites in the nucleus and to monitor DNA repair. BrdUrd incorporation has been used as a tool for in situ hybridization experiments.

Comparative flow cytometric analysis of DNA-bound PCNA and DNA content as estimators of S-phase cells in cell cultures.

Flow cytometric estimations of S-phase cells were carried out on cultures from three different cell lines and in frozen aliquots. A PCNA-extraction protocol was applied. Measurements of the S fraction estimated from bivariate PCNA/DNA analysis after detergent extraction of DNA non-bound PCNA were compared with those obtained from total DNA histograms (Vindelöv and Christensen's technique, methanol-fixed whole cells and PCNA-extracted nuclei). No significant differences between methods, or between fresh and frozen specimens, were found in the measurements of the percentage of S-phase cells. Nevertheless, nuclei yield following PCNA extraction was highly variable, ranging from 63% to 10% (mean: 26%). In some cases, the extraction was not complete and samples had to be discarded. Usually, boundaries between S-phase events and G0/G1 or G2/M subpopulations were not clearly defined. Because of these shortcomings, and the fact that is more costly and time consuming, the estimation of the S-phase fraction by means of bivariate DNA-bound PCNA/total DNA flow cytometric studies does not seem to surpass that obtained from standard DNA cell cycle analyses.

Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part II: Oncology, chemotherapy and carcinogenesis.

This paper follows on from Part 1 of my review of bromodeoxyuridine published earlier this year (Dolbeare, 1995).

In vitro bromodeoxyuridine-labelling of single cell suspensions: effects of time and temperature of sample storage.

The present study was aimed to explore how the in vitro BrdUrd-labelling of rat thymocytes might be affected by both the time elapsed between obtaining the sample and the beginning of the labelling (0, 15, 30 or 60 min) and the effect of the temperature of storage (4 degrees C versus room temperature). Single cell suspensions obtained after in vivo labelling with BrdUrd were used as controls. The S phase fraction was calculated by flow cytometry both according to BrdUrd-immunolabelling and DNA content. Immediate incubation with BrdUrd after the sample was obtained resulted in a slight decrease of the proportion of S phase cells analysed either according to DNA content or to BrdUrd-immunolabelling. Regardless of storage-temperature, the S phase fraction decreased in samples kept for 15 min or more before BrdUrd incubation. No BrdUrd-positive cells were detected in samples stored for 60 min at room temperature. This effect was related to temperature since positive cells were found when the samples were kept at 4 degrees C during the same time period. Our results suggest that during in vitro incubation a relative loss of S phase cells exists and that a delay beyond 15 min between obtaining the sample and the in vitro labelling seriously compromises the results of this technique.

Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part I: Historical perspectives, histochemical methods and cell kinetics.

Bromodeoxyuridine (BrdUrd), a thymidine analogue incorporated into DNA, can be quantified by fluorescent or chromophoric quenching of dyes bound to DNA or with antibodies to BrdUrd. These technologies have been used since the 1970s as tools for measuring DNA synthesis in isolated chromosomes and in cells and tissues. This paper is Part I of a three-part comprehensive review of the literature over the last 20 years (to the end of 1993) describing the histochemical methods for measuring BrdUrd in cells and tissues. Fixation, denaturation and staining procedures are compared for quantifying BrdUrd for microscopy and flow cytometry. Non-immunochemical methods related to the quenching of fluorescent DNA stains by BrdUrd are also described. Methods are described for the comparative assay of cell kinetic parameters by tritiated thymidine and bromodeoxyuridine. The multivariate BrdUrd/DNA assay of Ts and Tc, and a comparison of recent methods based on the single biopsy bivariate analysis of Tpot, is presented. Recent developments in the use of double halopyrimidine label to determine kinetic parameters are also reviewed.

Mutagenic activity and heterocyclic amine content of the human diet.

The mutagenic activity and the mass amount of heterocyclic amines responsible for the mutagenic activity have been measured in some cooked foods. Cooked meats are the predominant source of mutagenic activity in the diet with values ranging from 0 to 10,000 revertants per gram reported in the Ames/Salmonella test with strain TA98. Several heterocyclic amines are present and have been quantified using solid-phase extraction followed by HPLC. Frying at higher temperatures and for longer times produces the greatest mutagenic response, and concomitantly, the largest amounts of heterocyclic amines. Most of the mutagenic activity in fried meat samples can be accounted for by MeIQx(2-amino-3,8-dimethylimidazo[4,5-b]quinoxaline), DiMeIQx (2-amino-3,4,8-dimethylimidazo [4,5-f]quinoxaline) and IQ (2-amino-3-methylimidazo [4,5-f]quinoline), although other heterocyclic amines are present and PhIP (2-amino-3-methyl-6-phenylimidazo[4,5-b]pyridine) mutagenic activity becomes significant at higher temperatures. Non-meat products such as baked breads can also form significant mutagenic activity, particularly when overcooked. Commercially prepared hamburgers made from meat substitutes such as tofu, wheat gluten or tempeh and fried at 210 degrees C have up to 10% of the mutagenic activity of a fried beef patty cooked under the same conditions. When detected, amounts of heterocyclic amines in fried beef patties range from a total of 0.35 ng/g for commercial beef hamburgers to 142 ng/g for a beef patty cooked over a barbecue. Dietary intake is expected to have a large range, from less than one microgram per day to over 50 micrograms per day based on current knowledge of known heterocyclic amine chemicals and heterocyclic amine-containing foods.

Effect of microwave pretreatment on heterocyclic aromatic amine mutagens/carcinogens in fried beef patties.

To investigate a method to reduce the amount of mutagenic/carcinogenic heterocyclic aromatic amines formed during frying of ground beef, the mutagenic activity in Salmonella strain TA98 was assessed and the amount of known heterocyclic amines was determined by solid-phase extraction and HPLC. The beef patties received microwave treatment for various times before frying. Microwave pretreatment for 0, 1, 1.5, 2 or 3 min before frying at either 200 degrees C or 250 degrees C for 6 min per side reduced heterocyclic aromatic amine precursors (creatine, creatinine, amino acids, glucose), water, and fat up to 30%, in the patties and resulted in a decrease in mutagenic activity up to 95%. The sum of the four heterocyclic aromatic amines shown to be present--2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4,8-trimethylimidazo-[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)--decreased three- to nine-fold compared with control, non-microwaved beef patties fried under identical conditions.

Mutagenic activity of heterocyclic amines in cooked foods.

Mutagenic heterocyclic amines are generated in foods when they are cooked at temperatures over 150 degrees C. These compounds are present from 0.1 to 50 ppb, depending on the food and cooking conditions. These heterocyclic amines are not only present in cooked red meat, fish, and chicken, but are also present at lower levels in baked and fried foods derived from grain. Mutagenicity of fried beef hamburgers cooked at 230 degrees C is 800 +/- 37 TA98 revertants per gram cooked weight. We measured 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMelQx), and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) formation at this temperature and found 3.0 +/- 2.0, 1.0 +/- 0.18, and 0.06 +/- 0.03 ng/g, respectively. 2-amino-1-methyl-6-phenylimidaz[4,5-b]pyridine (PhIP) was found at a higher concentration of 9.6 ng/g. In our laboratory we have shown these heterocyclic amines are capable of producing both reverse and forward mutations in Salmonella bacteria and forward mutations in Chinese hamster ovary cells (CHO). We have also been able to show a statistically significant increase in mutations in the pancreas of the "mutamouse" following PhIP exposure. The pancreas also shows relatively high DNA binding compared to other organs in the mouse. The number and type of mutations depend on the repair capacity of the cells for both Salmonella and CHO. In Salmonella the mutations are primarily 2-base deletions when the cells lack uvrB repair, but mutations are more complex (larger deletions and insertions) but lower in frequency when repair is functional.(ABSTRACT TRUNCATED AT 250 WORDS)

Validation of a flow cytometric in vitro DNA repair (UDS) assay in rat hepatocytes.

An in vitro flow cytometric (FCM) DNA repair assay has been developed and validated by comparison to conventional autoradiography (ARG). Both assays measure unscheduled DNA synthesis (UDS). Cultures of hepatocytes from young male Sprague-Dawley rats were exposed to a battery of 26 chemicals plus bromodeoxyuridine (BrdUrd) or 3H-thymidine (3H-dT) for 18-20 h before harvest. Selection of test chemicals was based upon both their genotoxicity classifications and carcinogenicity bioassay results in male rats. DNA repair in chemically treated cultures was detected flow cytometrically by measuring the uptake of BrdUrd in non-replicating (G1, G2, mitotic and 4C) cells. Intracellular levels of incorporated BrdUrd were visualized by immunochemical labeling with fluorescein isothiocyanate (FITC), and total cellular DNA content was simultaneously estimated by counterstaining samples with the nucleic acid intercalator, propidium iodide (PI). Information was obtained from 10(4) cells/sample. Since repairing cells incorporate significantly less BrdUrd per unit of time than replicating cells, low intensity BrdUrd-FITC fluorescent signals from repairing cells are readily discriminated from high intensity signals from replicating cells when displayed on linear univariate histograms. Further distinction between repairing and replicating cells was achieved by displaying the DNA contents of all cells on linear bivariate histograms. Thus, repairing cells were resolved without subjecting these cultures to agents which suppress replicative synthesis (e.g., hydroxyurea). Results from these concurrent FCM and ARG investigations include the following: (1) conclusions (DNA repair positive or negative) were in agreement, with one exception, cinnamyl anthranilate, for which cytotoxic doses produced a positive FCM response, but lack of intact hepatocytes in parallel ARG preparations prevented analysis; (2) similar sensitivities for most of the positive chemicals were reported; (3) a high correlation (85%) exists between the reported genotoxicity classification and these DNA repair results in the absence of overt cytotoxicity; (4) a poor correlation exists between these DNA repair results and hepatocarcinogenesis (only 4/11 liver carcinogens tested positive) or overall carcinogenesis in the male rat (only 9/21 carcinogens tested positive). This FCM assay provides a rapid, sensitive, safe and reliable means of identifying agents which induce DNA repair in mammalian cells.

Effect of cooking time and temperature on the heterocyclic amine content of fried beef patties.

The mutagenic heterocyclic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) were measured in ground-beef patties fried at 150, 190 and 230 degrees C for 2-10 min on each side. Heterocyclic amines were purified using solid-phase extraction and analysed by HPLC. Recovery-corrected amounts of each heterocyclic amine were determined by the method of standard addition based on spiked samples with recoveries ranging from 40 to 70%. Mutagenic activity measured by the Ames/Salmonella test was determined for each sample. The amounts of MeIQx, PhIP, DiMeIQx and IQ increased with time and temperature of cooking. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-9H-pyrido[2,3-b]indole (A alpha C) were not detected in any sample. The mutagenic activity response measured for the meat extracts (TA98 revertants) was similar to the mutagenic activity calculated from the mass of heterocyclic amines present. The rate of formation of PhIP in a model system containing creatinine and phenylalanine heated in 80% diethylene glycol was compared with PhIP formation during meat frying. The apparent heats of activation were 6.5 kcal/mol in the model system compared with 6.0 kcal/mol in the fried meat patties. The increase in PhIP and MeIQx formation fitted an exponential function over the range 0 to 11 min and from 150 to 230 degrees C. This report shows clearly that increases in cooking temperature and time can have a profound effect on the amounts of heterocyclic amines generated and subsequently consumed in the diet.

Immunochemical quantitation of bromodeoxyuridine: application to cell-cycle kinetics.

We have described several laboratory procedures for the immunochemical staining of the halopyrimidines, BrdUrd and IdUrd, in cell suspensions for flow cytometry and a method for staining histological sections on slides. Halogenated pyrimidine quantitation allows cell-cycle parameters, including total cell-cycle time, phase durations, and growth fraction to be determined. We have presented some flow cytometric data to demonstrate the use of these methods in determining bivariate BrdUrd/DNA histograms with CHO cells and in kinetic studies with the brown Norway rat myeloid leukemia model.

Statistical confirmation that immunofluorescent detection of DNA repair in human fibroblasts by measurement of bromodeoxyuridine incorporation is stoichiometric and sensitive.

Diploid human fibroblasts (IMR-90 cells), grown to confluency and growth-arrested by serum starvation, were irradiated with a variety of doses of UV light (0.025-40 J/m2) or incubated with broad dose ranges of four direct-acting mutagens [ethyl methanesulfonate (EMS), ICR-170, methyl methanesulfonate (MMS), and 4-nitroquinoline oxide (4-NQO)] and pulsed with a thymidine analog, 5-bromodeoxyuridine (BrdUrd) to detect evidence of DNA repair. These cells and appropriate controls were immunochemically stained and subjected to flow cytometric analysis to quantify BrdUrd incorporation into DNA and simultaneously measure cellular DNA content. Since the maximal quantity of BrdUrd incorporated with repairing cells is profoundly less than the amount incorporated during replicative synthesis and flow cytometric analysis collects information on a cell-to-cell basis, data collection using linear histograms succeeded both in revealing repairing cellular populations and eliminating replicative cells from the analysis. Technical modifications necessary to achieve stoichiometry with UV-irradiated IMR-90 fibroblasts included a 48h repair (and pulse) period, followed by denaturing cellular DNA for 15 min at 90 degrees C. The limit of detection was equal to or below the lowest dose investigated (0.025 J/m2). DNA repair was also detected with cultures incubated with low doses of all chemicals and pulsed with BrdUrd for a 24 h period. The limits of detection were near or below 500 microM EMS, 5 microM MMS, 0.25 microM 4-NQO, and 0.1 microM ICR-170.

Flow cytofluorometric analysis of enzyme reactions based on quenching of fluorescence by the final reaction product: detection of glucose-6-phosphate dehydrogenase deficiency in human erythrocytes.

We developed a method for accurate cytofluorometric analysis of the final reaction product of enzyme reactions in individual cells. Glucose-6-phosphate dehydrogenase (G6PD) activity in human erythrocytes was demonstrated cytochemically, and the amount of final reaction product (formazan) per cell was detected indirectly by quenching of autofluorescence generated by glutaraldehyde fixation. Formazan quenches fluorescence in a dose-dependent manner. The method has been used for detection of G6PD deficiency. Heterozygous and homo(hemi)zygous deficiency could easily be established, even in cases of extreme "Lyonization" where microscopic inspection failed to discriminate between either normal individuals and heterozygously deficient patients or heterozygously and homozygously deficient patients. The principle of quenching of fluorescence by final reaction products of enzymes can be applied to flow cytofluorometric analysis of enzyme activity in individual cells in general.

Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining.

We describe an enzymatic procedure for exposure of single-stranded DNA (ssDNA) containing the halogenated pyrimidines (HdUrd) bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in single cells to antibodies that bind to HrdUrd only in ssDNA. Production of ssDNA was accomplished by digesting the DNA using either restriction endonucleases alone or endonucleases followed by exonuclease III. The enzymatic production of ssDNA was maximal when 0.1 N HCl or 0.1 M citric acid plus Triton X-100 was added to extract nuclear proteins prior to enzymatic denaturation. The restriction endonucleases Bam HI, Dde I, Eco RI, and Hind III produced significant ssDNA when used alone to allow binding of detectable amounts of the anti-HdUrd antibody IU-4 in Chinese hamster ovary cells labeled with 10 microM BrdUrd or 10 microM IdUrd. However, these treatments did not expose sufficient ssDNA to allow binding of IU-1, an anti-HdUrd antibody with lower binding affinity. IU-4 binding was most intense after treatment with Eco RI. Treatment with exonuclease III following endonuclease digestion allowed substantially more IU-4 binding.