RBE4 cells were immortalized using the adenovirus E1A gene without oncogenic transformation. Transfection was carried out using a calcium phosphate precipitation procedure on endothelial microvessel cells. Each line was packaged with trypsin-ethylenediaminetetraacetic acid and RBE4 medium, which was composed of 500 mL of Dulbecco's Modified Eagle's Medium (DMEM; obtained from Gibco, Grand Island, NY), 55 mL of fetal bovine serum (Gibco), 11 mL of HEPES (Sigma, St. Louis, MO, USA), 146 g of L-glutamine, 167 g of geneticin (Sigma) and 55 µL of basic fibroblast growth factor (Biomedical Technologies, Stoughton, MA, USA). Trypsin (4 mL) was added to each 10-cm² dish and the dishes were incubated for 5 minutes. Cells were washed into a 50-mL conical tube and then the dish was washed again with 4 mL of DMEM. The conical tube was then centrifuged at 1,500 rpm for 5 minutes. The supernatant was aspirated and the pellet was resuspended in RBE4 medium based on the desired dilution factor. Cell lines were diluted at concentrations of 1:3 to 1:6 with RBE4 medium.

Certain cell lines were frozen in liquid nitrogen to allow for their use at a later time. Two 10-cm² dishes at 100% confluence were frozen per vial diluted with freeze medium. The freeze medium consisted of 10% dimethyl sulfoxide and 90% DMEM. Cells were trypsinized and centrifuged. Each pellet was resuspended in 500 µL of DMEM and transferred to a vial. Each vial was chilled on ice for 5 minutes, and then another 500 µL of ice-cold freeze media was added to each vial. The vials were then stored at -20℃ for 1 hour prior to storage at -70℃ for 4 to 15 hours, after which vials were put into liquid nitrogen.

Cell lines were passaged on 10-cm² dishes and 3-(4,5-dim-ethylthiazolyl-2)-2,5-diphenyltetrazoliµM bromide (MTT) experiments were conducted on 24-well plates. The plates were coated with fibronectin to allow for the growth of cells. The experiments were comprised of three stages: induction, sensitization, and hydrogen peroxide. The induction phase was initiated at time 0 and continued for 1 hour. The sensitization phase occurred at 16 hours and was followed by the hydrogen peroxide phase, where the cell monolayers received 500 µM of H₂O₂ at 17 hours. Hydrogen peroxide was applied for 1.5 hours before the 10% MTT solution (diluted with DMEM) was placed on the cells for 3 hours. A solubilization buffer was then applied to the cells overnight. The next day, wells were measured in a spectrophotometer at 575 and 675 nm. The average of the differences between the 575 and 675 nm readings from triplicate sets was divided by the control reading. This quotient represented the percentage of cells that survived.

Cells underwent oxidative stress in the peroxide phase due to the addition of 0.5 mL of 500 µM hydrogen peroxide to each well. The induction and sensitization phases represented times during which various known compounds were introduced into the medium to test several hypotheses regarding the antioxidant role of heme oxygenase. The experiments were conducted using different concentrations of zinc deuteroporphyrin IX 2,4 bis glycol (ZnBG, Porphyrin Products Inc., Logan, UT, USA), bilirubin (Porphyrin Products Inc.), cycloheximide (Sigma), desferoxamine (Sigma), and hemin (Sigma).

The MTT cell proliferation kit I (Boehringer Mannheim, Mannheim, Germany) was used to evaluate the viability of the RBE4 cells. The MTT kit was selected to quantify cell viability because it provides a colorimetric assay that can be run expediently on a large nµMber of cell monolayers and does not rely on cell counting. This colorimetric assay is based upon the cleavage of the tetrazoliµM salt, MTT, to purple formazin crystals by dehydrogenase activity in active mitochondria.

Tests were run to determine whether the variables used in the MTT assay experiments affected the cleavage of the tetrazoliµM salt, which could skew the data. Specific concentrations of bilirubin, hemin, ZnBG, cycloheximide, and desferoxamine were placed on cells during the last 10 minutes of the sensitization phase. Nothing was placed on the cells during the induction phase. Subsequently, cells underwent the aforementioned procedure, and it was determined that none of the variables of the experiments affected the cleavage of the salt.

All data are presented as the means±standard errors of the means. One-way analysis of variance was conducted and followed by Dunnett's post hoc test.

To study the role of HO activity in the protective effects observed under conditions of oxidative stress, we employed in vitro models with hemin, which is the HO-1 substrate for induction and sensitization under oxidative stress attributable to hydrogen peroxide (500 µM H₂O₂) (Fig. 1A). Cell death was prevented by treatment with hemin (none vs. hemin [10 µM]; 11.58 vs. 36.32) (Fig. 1B). The results of the cell viability assay suggested that inducible HO-1 played an important role in the survival of RBE4 cells under oxidative stress.

HO appears to exert most of its actions via enzymatic activity and the generation of various enzymatic byproducts that act on multiple pathways. For example, HO can promote: 1) the generation of biliverdin and its subsequent reduction to bilirubin, 2) the release of CO, and 3) the release and safe disposal of iron. These actions modulate various pathways with a high degree of overlap and/or crossover, which may help to ensure the induction of the desired biological effects [13].

To confirm the roles of HO-1, CO, and cyclic guanosine monophosphate (cGMP) in the antioxidant effects of HO-1 induced by hemin, we treated cells with cycloheximide, desferoxamine, and ZnBG, respectively, in the induction phase of experiments for 1 hour (Fig. 2A). After the induction phase, hemin was used in the sensitization phase for 16 hours. Study results revealed that the HO-1 activity induced by hemin was blocked by cycloheximide, an inhibitor of protein biosynthesis. Furthermore, cycloheximide induction completely blocked the survival effect of HO-1. Conversely, ZnBG, which is an inhibitor of heme oxygenase induction, did not block the survival effect of HO-1 following hemin induction (cycloheximide induction vs. ZnBG induction; 16.5 vs. 39.6) (Fig. 2B). Desferoxamine, which binds specifically to iron and removes the relevant effect of iron induction, only partially blocked the survival effect of HO-1. Study results indicated that iron, not CO, was involved in the survival effects attributable to HO-1.

Bilirubin acts as a scavenger against reactive oxygen species via its own oxidation [14,15,16]. Indeed, bilirubin is a powerful antioxidant that can inhibit the oxidation of low-density lipoprotein and other lipids [17], scavenge oxygen radicals [18], and counteract oxidative stress in general [19]. The production of bilirubin could participate in an amplification cycle via its oxidization into biliverdin and then reduction back into bilirubin by biliverdin reductase [20].

To confirm the roles of HO-1, CO, and cGMP in the antioxidant effect of bilirubin, we used cycloheximide, desferoxamine, and ZnBG, respectively, during the induction phase of the experiments for 1 hour. After the induction phase, bilirubin was used in the sensitization phase for 16 hours (Fig. 3A). Cycloheximide induction partially blocked the survival effect of bilirubin, suggesting that HO-1 played an important role in the antioxidant effect of bilirubin. Furthermore, bilirubin did not require CO or cGMP to increase cell viability (Fig. 3B). Study results indicated that HO-1 required heme and iron to produce the observed survival effect. However, bilirubin, as an HO-1 effector for oxidative stress, did not require CO or cGMP.