MRN-100 is an iron-based compound derived from bivalent and trivalent ferrates and was prepared in distilled water (DW) with the concentration of Fe²⁺ and Fe³⁺ ions at ~2 × 10^-12 mol/l. MRN-100 is obtained from phytosin as previously described 13. MRN-100 was provided by ACM Co., Ltd., Japan.

Complete medium (CM) consisted of RPMI-1640, supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and 100 μg/ml of streptomycin and penicillin.

C57BL/6 (4–5 weeks old, 20–25 g female mice) were purchased from Harlan Laboratories (Chicago, IL, USA) and from Saitama Experimental Animal Co. Ltd., (Saitama, Japan). The mice were accommodated for 1 week prior to experimentation. Mice were maintained in the animal facility at Charles Drew University of Medicine and Science, Los Angeles, CA, USA and in the Laboratory Animal Center at Jichi Medical University in Japan. Mice were housed 2 per micro-isolator cage and were fed sterilized standard cube pellets and water ad libitum. Animal protocols were in compliance with the Guide for the Care and Use of Laboratory Animals in the USA.

Mice were killed by cervical dislocation. Spleens were removed, teased in CM, and contaminating erythrocytes were lysed with distilled water for 20 seconds. Splenic lymphocytes were centrifuged, and washed three times with HBSS. Cell viability was 95%, as determined by the trypan blue exclusion test. Cells were counted using a hemocytometer and a light microscope, and were re-suspended to a concentration of 10⁷ cells/ml in CM.

Splenic lymphocytes (1 × 10⁶ cells/ml) were cultured in CM and divided into four groups: group 1 – cells were incubated with 25 μm hydrogen peroxide (H₂O₂) for 16 hrs; group 2 – cells were incubated with MRN-100 (100 μl/ml) for 16 hrs; group 3 – cells were incubated with MRN-100 for 2 hrs and were subsequently exposed to H₂O₂ for 14 additional hrs; group 4 – control group, in which cells were incubated with CM alone for 16 hrs.

Splenic lymphocytes were cultured as described above. The percentage of hypodiploid, apoptotic cells was examined by the propidium iodide (PI) technique using flow cytometry. Briefly, splenic cells (1 × 10⁶/ml) were fixed in 70% methanol, re-suspended in the DNA extraction buffer, washed in PBS and incubated with 50 μg/ml of PI for 30 min at room temperature in the dark, and analyzed by FACScan (Becton Dickinson, San Jose, California, USA).

At 16-hrs post-lymphocyte culture, cells from the different groups (H₂O₂, MRN-100, MRN-100 + H₂O₂, and control [CM]) were treated with trypan blue stain (100 μl/ml) for 5 minutes and counted using a light microscope (Nikon, Tokyo, Japan) and hemocytometer. The percent of dead cells was calculated blindly out of a total of 300 cells by the same investigator.

Splenic lymphocytes (1 × 10⁶ cells/ml) were cultured in the presence or absence of H₂O₂ at concentrations of 0–50 μM for 16 hrs. The mouse macrophage cell line, RAW-264.7, was also tested for NO production in the presence or absence of lipopolysaccharide (LPS) as well as H₂O₂ at the above-mentioned concentrations. Production of NO was determined as the amount of nitrite, a stable end-product of NO, in the culture supernatant obtained at 48 hr post stimulation. Nitrite was measured by a colorimetric assay using the Griess reagent (1% sulfanilamide and 0.1% N-1-naphtylethylendiamine dihydrochloride in 2.5% H₃PO₄ solution) 14. The absorbance at 540 nm was measured and the nitrite concentration was quantified (in μM) using sodium nitrite as the standard in each assay.

The effect of H₂O₂ on Ca²⁺ levels was examined in two models: splenic lymphocytes and the mouse macrophage cell line, RAW264.7 cells. Cellular Ca²⁺ flux was determined using Screen Quest™ Fluo-8 NW Calcium Assay Kit (ABD Bioquest Inc, Sunnyvale, California, USA) as follows. Splenic lymphocytes (1 × 10⁷/2 ml CM) were cultured for 2 hrs with either PBS (control) or MRN-100 (10% v/v CM) in a 60 mm culture plate (Corning Inc, Corning, New York, USA). The cells were harvested by centrifugation and resuspended with 1.5 ml of Fluo-8 NW dye-loading solution in the kit. The cell suspension was plated 100 μl per well in a 96-well black well/clear bottom plate (Costar, Cambridge, Massachussetts, USA). In the case of RAW264.7 cells, adherent cells (1 × 10⁵ cells/well) in a 96-well black well/clear bottom plate were cultured with either PBS or MRN-100. At 2 hrs, the supernatant was discarded and 100 μl Fluo-8 NW dye-loading solution was added. The plates were incubated at 37°C for 30 minutes, and then placed at room temperature for another 30 minutes. H₂O₂ solution (25 μl/well) was added to a final concentration of 10, 50 or 250 μM. Fluorescence at Ex = 490/Em = 525 nm was measured by SPECTRAmax M5 (Molecular Devices Corp, Sunnyvale, California, USA).

The protein levels of Bcl-2 and Bax were determined using Western blot analysis. Splenic cells were treated as described above. Cells were harvested, washed with cold PBS [10 mM (pH 7.4)], lysed with ice-cold M-PER mammalian protein extraction reagent (Pierce Biotechnology, Rockford, Illinois, USA) for 30 minutes, and centrifuged at 14,000 g for 20 minutes at 4°C. The supernatant was collected and protein concentration was determined using BCA protein assay (Pierce Biotechnology). Seventy-five μg of cell lysates were subjected to Western blot analysis by 4%–12% SDS polyacrylamide gel electrophoresis. Membranes were probed using 1:500 anti-Bcl-2 or anti-Bax (BD Bioscience, San Diego, California USA) primary antibody. The washed PVDF membranes were then incubated with 1:2000 dilution of monoclonal secondary antibody. Immunoreactive bands were visualized by using ECL detection system (Amersham, Buckinghamshire, UK). To verify equal protein loading and transfer, the blots were stripped and re-probed with β-actin using an anti-actin rabbit polyclonal antibody. Data are represented as mean ± SEM. The ratio of Bcl-2/Bax was examined using densitometry of each band.

Using the Student's t-test, we tested the significance of the difference in the percent of apoptotic splenic cells between treated and control cells. We considered the cutoff of p < 0.05 as significant.

Results in Figure 1 show, as expected, that culture of splenic cells with H₂O₂ alone results in a significant increase in cell apoptosis as compared to control (CM) cells. In contrast, pre-treatment of cells with MRN-100 followed by H₂O₂ treatment results in significantly reduced levels of apoptosis and inhibits the H₂O₂-induced apoptosis to the level of control (CM) cells.

The protective effect of MRN-100 against H₂O₂-induced cell death was further confirmed by trypan blue exclusion tests. Results in Figure 2 show a high level of dead splenic cells post-treatment with H₂O₂. In contrast, pre-treatment of splenic cells with MRN-100 prevents H₂O₂-induced cell death.

Splenic cells were stimulated with H₂O₂ at concentrations of 0–50 μM and nitric oxide (NO) production was examined. Results show that NO production is not detected at any of the concentrations of H₂O₂ tested. Similar results were obtained using the mouse macrophage cell line, RAW-264.7; although this cell line produces NO upon stimulation with LPS, NO was not detected following treatment with H₂O₂ (data not shown).

Intracellular Ca²⁺ was examined in mouse splenic cells and RAW264.7 cells upon stimulation with H₂O₂. Results show that an increase of intracellular Ca²⁺ was scarcely detected in these cells up to 50 μM H₂O₂ (data not shown). A high concentration of H₂O₂ (250 μM), results in little increase of intracellular Ca²⁺, but MRN-100 pre-treatment shows no suppressive effect (data not shown).

The effect of MRN-100 on Bcl-2 and Bax protein levels was determined by Western blot. Bcl-2 is known as an anti-apoptotic protein that has been associated with the inhibition of apoptosis, whereas Bax is a pro-apoptotic protein. Splenic cells were treated for 16 hrs with MRN-100 alone, H₂O₂ alone, MRN-100 + H₂O₂, or control (CM) cells. The effects of H₂O₂ and MRN-100 on Bcl-2 and Bax protein levels in splenic cells are shown in Fig 3A and 3B, respectively. Results show that H₂O₂ causes a decrease in protein level of Bcl-2 and an increase in the protein level of Bax in untreated spleen cells (compare lane 1 with lane 2). In contrast, pre-treatment with MRN-100 partially counteracts the effect of H₂O₂ on both Bcl-2 and Bax protein levels (compare lane 2 with lane 4). Figure 3C depicts the effect of MRN-100 on H₂O₂-induced changes in Bcl-2. The ratio of Bcl-2/Bax was also examined (Figure 3D) by analyzing the densitometry of bands from the Bcl-2 blot (Figure 3A) and Bax blot (Figure 3B). The ratio of Bcl-2 to Bax is reduced significantly in H₂O₂ treated spleen cells. Pre-treatment with MRN-100 partially prevents the H₂O₂-induced decrease in the ratio of Bcl-2/Bax.