To compare the cell cycle effects of different culture conditions on starved BEAS-2B cell lines treated with H₂O₂, the number of proliferating cells (defined as BrdU-positive cells) and apoptotic cells were measured after culturing with growth factors (GF) or in GF-free medium. Quiescent cells starved of GF for 48 h were exposed to H₂O₂ (100–500 μM) for 1 h, after which the cells were cultured in fresh media without GF. After 1 day, cycle analysis indicated that the cells treated with H₂O₂ were similar to untreated controls (Fig. 1A and 1C). However, when quiescent cells were exposed to H₂O₂ followed by culturing in fresh medium containing GF to initiate entry into cycle, a higher percentage of G₁ phase-cells (83%) and a lower percentage of S phase-cells (6%) were found compared to the controls (70% and 16% respectively; Fig. 1B and 1C). H₂O₂ inhibited cell proliferation in a concentration-dependent manner in conjunction with GF (Fig. 1C). As a result, few apoptotic cells (3%) were detected, seen as a "pre-G₁" phase population (Fig. 1B).

It had previously been shown by high-performance liquid chromatography (HPLC) that H₂O₂ induces 8-oxo-dG, a DNA damage marker 16. Exposure of quiescent WI-26VA4 cells, another SV-40 transformed epithelial cell line, to H₂O₂ had no effect on the number of 8-oxo-dG-positive cells (Fig. 2A). In the present study, we used a direct binding fluorescent probe to detect 8-oxo-dG in the DNA, which was quantified by LSC. The rate of DNA damage measured was that induced by a 1 h exposure of BEAS-2B cells to H₂O₂. We also assessed the number of 8-oxo-dG-positive cells by LSC (Fig. 2B). H₂O₂ treatment increased the number of 8-oxo-dG-positive cells increased in a concentration-dependent manner (2.1 versus 19.6%; Fig. 2C).

To determine the effect of H₂O₂ on single-strand DNA breaks in BEAS-2B cells, cells were treated with H₂O₂ (200–1000 μM) and the tail moment (TM) determined. The mean tail moment relative to time of H₂O₂ treatment is shown in Figure 3B. For the first 1h after H₂O₂ treatment, the mean TM showed both a time- and concentration-dependent decrease. However, the mean TM increased at every concentration of H₂O₂ between 60 and 120 min.

Previous studies have suggested that a decline of ΔΨm may be an early event in the process of cell death, and so we determined ΔΨm at various times after H₂O₂ treatment in BEAS-2B cells, using the membrane potential-sensitive probe, JC-1, which forms monomers (green fluorescence) at a low membrane potential and J-aggregates (red fluorescence) at a higher membrane potential 17. The ratio between the red and the green signals is indicative of the ΔΨm. There was a dramatic drop of the red fluorescence in cells exposed to H₂O₂, indicating a loss of ΔΨm. Figure 4A shows a clear increase in the percentage of cells that emitted only green fluorescence after H₂O₂ treatment, representing cells with a depolarized mitochondrial membrane. H₂O₂ caused a concentration-dependent reduction or loss in ΔΨm (Fig. 4B).

An important pathway that enhances NO cytotoxicity is the rapid reaction with superoxide anion radicals (O_2·-) to yield peroxynitrite (ONOO^-), which has a wide range of biologic activities that includes oxidation of biomolecules and protein tyrosine nitration 18. It is widely accepted that peroxynitrite is a key mediator of tissue injury in oxidative stress. To determine whether H₂O₂-induced peroxynitrite formation occurred in BEAS-2B cells, cells were stained with an anti-nitrotyrosine antibody. H₂O₂ (60 min, 500–1000 μM) induced clear perinuclear nitrotyrosine staining (Fig. 5B and 5C). The peroxynitrite donor, SIN-1, induced a predominantly nuclear membrane distribution of nitrotyrosine staining (Fig. 5D).

BEAS-2B cells, which are SV-40 immortalized cells derived from normal human bronchial epithelium 45, were obtained from American Type Culture Collection (Manassas, VA). The cells (passages 32 to 70) were cultured in serum-free keratinocyte basal medium (Sigma; St. Louis, MO), which was supplemented with 10 ng/ml of epidermal growth factor (Gibco BRL; Grand Island, NY) and 30 μg/ml of bovine pituitary extract (Gibco BRL) in the presence of 5% CO₂ at 37°C. The human lung cell line WI-26VA4 was kindly provided by Human Science Research Resource Bank (Osaka, Japan). Cells (passages 162 to 180) were cultured in Dulbecco's modified Eagle's medium (DMEM; IWAKI, Chiba, Japan) supplemented with 10% fetal calf serum, amino acids (L-arginine HCl 252.8 mg/l, L-cystine 48.0 mg/l, L-histidine HCl 83.8 mg/l, L-isoleucine 105.0 mg/l, L-leucine 105.0 mg/l, L-lysine HCl 146.2 mg/l, L-methionine 29.8 mg/l, L-phenylalanine 66.0 mg/l, L-threonine 95.2 mg/l, L-tryptophan 20.4 mg/l, L-tyrosine 72.4 mg/l, and L-valine 93.8 mg/l), mixed vitamins (folic acid 1.0 mg/ml, nicotinamide 1.0 mg/ml, pyridoxal HCl 1.0 mg/ml, riboflavin 0.1 mg/ml, and thiamin HCl 1.0 mg/ml), penicillin (100 U/ml), and streptomycin (100 μg/ml) in the presence of 5% CO₂ at 37°C.

BEAS-2B cells exposed to H₂O₂ were pulse-labeled with 50 μM 5-bromo-2'-deoxyuridine (BrdU) (Sigma Aldrich; Tokyo, Japan) for 30 min one day after exposure, and harvested by trypsinization, pelleted, and gently resuspended in phosphate buffered saline (PBS; OXOID, Hampshire, England). The cells were fixed with ice-cold 70% ethanol, centrifuged and resuspended in 1 ml 2 N HCl containing 0.05% Triton X-100 (v/v). The cells were incubated at room temperature for 30 min followed by centrifugation, rinsed in 0.1 M borate buffer, pH 8.5, and PBS/bovine serum albumin (BSA; Sigma-Aldrich) (1 mg/ml BSA in PBS), after washing with PBS/0.5% Tween 20/1% BSA. Appropriately diluted anti-BrdU antibody (clone BU-1; Amersham, Buckinghamshire, UK) was added to the cell pellet. After incubation for 1 h at room temperature, the cells were rinsed twice in PBS/BSA. For visualization, fluorescein isothiocyanate (FITC)-conjugated Fab2 fragments of rabbit anti-mouse Ig (DAKO, Glostrup, Denmark) antibody were added in a 1:30 dilution. After incubation for 45 min at room temperature, the samples were rinsed twice in PBS/BSA and the cells were resuspended in 0.5 ml cold PBS supplemented with 100 μg/ml RNase (Sigma-Aldrich) and 20 μg/ml propidium iodide (PI; Sigma-Aldrich). The samples were allowed to stand for 15 min on ice in the dark before flow cytometric analysis. For the negative control, the primary antibody was omitted. The stained cells were analyzed by FACScan (Becton-Dickinson, Franklin Lakes, NJ), using the CELL QUEST program.

The extent of cell DNA damage was determined using a commercial kit (Calbiochem-Novabiochem Corporation; Darmstadt, Germany) on WI-26VA4 cells at 50 to 70% confluence on coverslips in a 6-well culture plate. The cells were fixed with fresh 4% paraformaldehyde (Wako Life Science, Osaka, Japan) in PBS and dehydrated in 70% followed by 95% methanol at -20°C. Cells were permeabilized in 99% methanol at -20°C, and incubated for 30 min on ice, rehydrated with 95% and 70% methanol at -20°C. The rehydrated cells were washed with PBS/Tween 20, containing thimerosal. To block non-specific binding sites, a blocking solution was added to each well and incubated for 1 h at 37°C. The blocking solution was removed and the cells washed with PBS/Tween 20 before being incubated with FITC-conjugated substrate for 1 h at 37°C, and washed 5 times with PBS/Tween 20 with 3 min of gentle shaking per wash. Next, the cells were incubated with PI-RNase for 10 min at 37°C, washed 5 times with PBS/Tween 20 with 3 min of gentle shaking per wash, and rinsed once with distilled water. The coverslips were freed, mounting solution was added, and placed on slides to be observed under fluorescent microscopy with FITC filters.

Cells were subsequently analyzed with an LSC (LSC101; Olympus, Tokyo, Japan) as previously described 46, using a krypton/argon laser operating at 488 nm and 568 nm. Dual-channel imaging was performed with two photomultipliers, using a band-pass filter specifically designed to detect FITC fluorescence and a long-pass (590 nm) filter for detecting PI fluorescence. Images were generated using integral computational and display software. To assess mean fluorescence intensity (MFI) of 8-oxo-dG, each nucleus was identified by areas counterstained with PI. We analyzed a 10-mm² scan area, occupied by approximately ~1 × 10⁴ cells.

The alkaline version of the Comet assay was performed according to the method of Singh et al. (1988) with minor modifications 15. Briefly, fully-frosted microscope slides were pre-coated with 1 ml of 0.75% normal melting point agarose (NMA; Promega, Madison, WI) and stored at 4°C. This layer was removed before use and 120 μl of 0.75% NMA was pipetted onto the slides, which were then covered with coverslips. Cell suspensions of BEAS-2B cells (1 × 10⁴ cells/10 μl) were mixed with 70 μl of 1% low melting point agarose (LMA) and pipetted over the first layer of agarose. NMA (80 μl) was used as a final protective layer. After each step, the slides were incubated at 4°C for 10 min to allow the agarose to gel. Slides were placed in cold lysing solution (2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris, pH 10, and 1% sodium N-lauroyl sarcosinate to which 10% dimethyl sulfoxide and 1% Triton X-100 had been added immediately prior to use) for 1 h. After lysis, the slides were placed in electrophoresis buffer (300 mM NaOH and 1 mM Na₂EDTA, pH 13) for 20 min to allow unwinding of the DNA. Electrophoresis was conducted in the same buffer by applying an electric current of 0.8 V/cm (300 mA) for 20 min, using an electrophoresis power supply (Pharmacia LKB, Sweden). Finally, the slides were washed in neutralization buffer (0.4 M Tris, pH 7.5) 3 times for 5 min each, dried and stained with Hoechst 33342 (10 μg/ml).

Image analysis involved a microscope equipped with an LSC. Nuclei were excited with green light and the emitted red spectrum was captured by a 40 × dry objective. Images of 50 randomly selected nuclei were analyzed per duplicate slide (100 nuclei in total). The tail moment (TM) was determined to assess the extent of DNA damage. The TM value was defined as the product of the percentage of DNA in the comet tail and the tail length.

BEAS-2B cells were stained with 1 μg/ml of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine (JC-1; Molecular Probes, Eugene, OR) for 30 min at 37°C, trypsinized, resuspended in medium at a density of approximately 1 × 10⁷/ml, and transferred on ice to the flow cytometer. JC-1 was excited at 488 nm and the monomer signal (green) was analyzed at 525 nm (FL1) on a FACScan (Becton-Dickinson, Mountain-View, CA) flow cytometer. Simultaneously, the aggregate signal (red) was analyzed at 590 nm (FL2). In BEAS-2B cells, the percentage of PI-negative cells (1 × 10⁴) was analyzed and the mean fluorescence was calculated for each sample.

BEAS-2B cells were cultured in 6-well plates on coverslips and were untreated or treated with H₂O₂ alone or with a peroxynitrite generator 3-morpholinosyndonimine (SIN-1), which is the precursor of peroxynitrite, for 1 h. The coverslips were then washed with PBS, prefixed in 2% formalin at room temperature for 10 min, and fixed in methanol/acetone (1:1) at -20°C for 10 min. The coverslips were washed with PBS and then were blocked in PBS containing 20% normal swine serum (Dako), 0.4% Triton X and 0.1% BSA. The fixed cells were stained with a primary antibody against nitrotyrosine (Upstate Biotechnology, Lake Placid, NY) diluted at 1:50 in PBS containing 0.4% Triton X and 0.1% BSA for 1 h. After removing the first antibody by washing, the cells were incubated with biotinylated goat anti-rabbit Ig (Dako) in PBS containing 0.4% Triton X and 0.1% BSA for 45 min. After removing the second antibody, the cells were incubated with FITC-conjugated streptavidin (Dako) in PBS containing 0.4% Triton X and 0.1% BSA for 45 min. Finally, the cells were counter-stained with 20% hematoxylin and the coverslips were mounted on slides with mounting medium (DPX). The stained cells were visualized and photographed using a confocal laser scanning microscope (Leica, Heidelberg, Germany).

Group data are expressed as the mean (± SD). Comparisons between data were tested using the Mann-Whitney U test. A p value < 0.05 was considered significant.