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(The FASEB Journal. 1999;13:1800-1809.)
© 1999 FASEB

Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron

DENISE M. SUTTNER and PHYLLIS A. DENNERY1

Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94304, USA

1Correspondence: Department of Pediatrics, Stanford University School of Medicine, 750 Welch Rd #315, Palo Alto, CA 94304, USA. E-mail: dennery{at}leland.stanford.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is often postulated that the cytoprotective nature of heme oxygenase (HO-1) explains the inducible nature of this enzyme. However, the mechanisms by which protection occurs are not verified by systematic evaluation of the physiological effects of HO. To explain how induction of HO-1 results in protection against oxygen toxicity, hamster fibroblasts (HA-1) were stably transfected with a tetracycline response plasmid containing the full-length rat HO-1 cDNA construct to allow for regulation of gene expression by varying concentrations of doxycycline (Dox). Transfected cells were exposed to hyperoxia (95% O2/5% CO2) for 24 h and several markers of oxidative injury were measured. With varying concentrations of Dox, HO activity was regulated between 3- and 17-fold. Despite cytoprotection with low (less than fivefold) HO activity, high levels of HO-1 expression (greater than 15-fold) were associated with significant oxygen cytotoxicity. Levels of non-heme reactive iron correlated with cellular injury in hyperoxia whereas lower levels of heme were associated with cytoprotection. Cellular levels of cyclic GMP and bilirubin were not significantly altered by modification of HO activity, precluding a substantial role for activation of guanylate cyclase by carbon monoxide or for accumulation of bile pigments in the physiological consequences of HO-1 overexpression. Inhibition of HO activity or chelation of cellular iron prior to hyperoxic exposure decreased reactive iron levels in the samples and significantly reduced oxygen toxicity. We conclude that there is a beneficial threshold of HO-1 overexpression related to the accumulation of reactive iron released in the degradation of heme. Therefore, despite the ready induction of HO-1 in oxidant stress, accumulation of reactive iron formed makes it unlikely that exaggerated expression of HO-1 is a cytoprotective response.—Suttner, D. M., Dennery, P. A. Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron.


Key Words: heme • hyperoxia • doxycycline • carbon monoxide • bilirubin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IT IS WELL RECOGNIZED that induction of heme oxygenase (HO)2 is a generalized response to oxidative stress (1) . The reason for the induction of HO-1 could be the protective effect against oxidative injury since HO metabolic activity leads to the formation of bile pigments, which have been shown to behave as an antioxidants (2) . Another possible cytoprotective effect of HO-1 induction may be related to the decrease in heme iron and hemoproteins and the increased expression of iron responsive genes such as ferritin and aconitase (3) . The role of other metabolites of HO, namely, carbon monoxide (CO), have not been as well understood in the context of antioxidant defense, although recent reports indicate increased CO in inflammatory lung diseases such as asthma (4) . Several studies have demonstrated that low overexpression of HO-1 (less than a fivefold increase in HO activity) is associated with protection against oxidative stress. For example, a threefold overexpression of HO-1 was associated with protection against heme-mediated damage (5) , and fibroblasts with endogenous 1.8-fold overexpression of HO-1 protein were protected against oxygen toxicity (6) . Furthermore, suppressing HO activity using antisense transfection (7 , 8) or inhibitors (7 , 9) led to worsened oxidative stress. Nonetheless, in some clinically relevant circumstances (10 , 11) and experimental models (12 , 13) , the resultant level of HO activity is well beyond the modest elevation achieved with transfection systems and it is not clear whether HO is beneficial at these higher levels. Exaggerated HO activity may not be beneficial because in addition to the formation of bile pigments and CO, iron is released from the degradation of heme via HO. Iron is well known to mediate reactions leading to the formation of hydroxyl radicals (14) This could result in increased susceptibility to oxidative stress, most likely at the higher range of HO-1 expression. We have shown that despite improved resistance to oxygen toxicity with a 1.8-fold increase in activity, a mere doubling of the level of HO-1 expression in fibroblasts resulted in a loss of cytoprotection (7) . When HO activity was elevated eight- to ninefold in a model of endotoxic shock, significant hypotension was observed (11) . It would therefore be crucial to understand what mediates protection in the HO reaction and to identify circumstances under which HO-1 induction is protective so as to allow for judicious use of HO in therapeutic interventions. To systematically test whether overexpression of HO-1 is beneficial, we established HO-1 transfected cells with low (2–5-fold), moderate (10- to 14-fold), and high (15- to 18-fold) HO activity as compared to untransfected controls. After careful accounting of all HO reaction products, a diametrically opposed, concentration-dependent effect of HO-1 in susceptibility to oxygen toxicity was observed due to reactive iron accumulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell line and culture conditions
Hamster fibroblast (HA-1) cells were used in all experiments. The cells were grown in Eagle's minimum essential medium (EMEM, Sigma, St. Louis, Mo.) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) and maintained at 37°C in 95% air/5% CO2. No exogenous heme was added to the media. The calculated heme content was <= 0.3 µM per flask.

Experimental design
Establishment and maintenance of variable HO-1 cell line
To establish variable HO-1 overexpression, the Tet-Off Gene Expression System (CLONTECH Laboratories, Palo Alto, Calif.) was used. As recommended, 1 x 105 HA-1 cells were grown in 6-well dishes to 60% confluence in a 95% room air, 5% CO2 incubator. The cells were then incubated overnight with 2 µg of the pTet-Off plasmid complexed with 6 µl of LipofectAMINE Reagent (Life Technologies, Inc., Gaithersburg, Md.). Because the pTet-Off plasmid contains a neomycin resistance site, transformed colonies were isolated after 3 wk by growing in the presence of G418 (400 µg/ml, Life Technologies). Transient transfection assays were performed with the luciferase containing plasmid (pTRE-Luc) in order to identify a cell line that had a high level of luciferase induction in the absence of Dox with low background expression in the presence of Dox. This antibiotic is a derivative of tetracycline and is as useful in regulating gene expression with the Tet-Off system. Once identified, this cell line was transfected with 2 µg each of pBI-L containing the full-length rat HO-1 cDNA inserted at the HindIII and EcoRV restriction sites and pTK-Hyg to allow for selection of transformed colonies by growth in hygromycin (Hyg) in a liposome complex. The cells were grown in G418 (400 µg/ml) and Hyg (200 µg/ml; Life Technologies). After 3 wk, transformed colonies were screened using luciferase activity in order to isolate a cell line with high induction of HO-1 in the absence of Dox and low background in the presence of Dox. The cell line was maintained in EMEM with 10% fetal calf serum supplemented with G418 (200 µg/ml) and Hyg (100 µg/ml). Regulation of variable HO-1 expression was accomplished by incubation in media containing 0 to 1 ng/ml of Dox.

Incubation in Dox and hyperoxia exposure
Four days before hyperoxic exposure, transfected cells were plated at 2 x 105 cells per 75 cm2 flask and varying concentrations of Dox were added (1, 0.1, 0.05, 0.01, 0.005, 0.001, and 0 ng/ml) to the maintenance media. The HO-1-expressing cells and HA-1 controls were then exposed to hyperoxia (95% O2/5% CO2) at 37°C in a humidified chamber and harvested for assays after 24 h of exposure.

Incubation with SnMP and DTPA
To verify that the results were due solely to the overexpression of HO-1, high HO-1-expressing cells were incubated with an inhibitor of HO activity, tin mesoporphyrin (SnMP, 10 µM; Porphyrin Products, Logan, Utah). Cell were also incubated with an iron chelator, diethylenetriaminepentaacetic acid (DTPA, 25 µM/ml), to determine whether increased reactive iron associated with HO activity was involved in mediating increased cell damage. After incubation with either SnMP or DTPA for 4 h, cell cultures were exposed to hyperoxia. After 24 h of hyperoxia, cells were harvested for assays.

Verification of gene expression in cultured cells
Since the pBI-L/HO-1 transfected cells expressed the luciferase gene, this was used as a reporter system to assess the level of gene expression with doxycycline. Luciferase activity was monitored in the cells incubated with varying concentrations of doxycycline by incubating culture dishes with 20 µl of luciferin (30 mg/ml) for 20 min and then visualizing the photon emission from each dish on an enhanced charge couple device (CCD) camera as described previously (15) .

Total HO activity
Assays were conducted in subdued lighting. Twenty microliter aliquots of cell sonicates were reacted with 20 µl hemin (150 µM; Sigma) and 20 µl NADPH (4.5 mM; Sigma) in a septum sealed vial at 37°C. Blanks consisted of cell sonicates reacted with hemin only. Vials were purged with CO-free air and allowed to incubate for 15 min. The reaction was stopped with dry ice (-78°C), and CO generation in the vial gas head space was analyzed by gas chromatography (16) . HO activity was derived by subtracting the blank value from the sample value and expressing this quantity as nmol CO/mg protein (16) .

Cell protein content
Sonicates were analyzed for protein content by the method of Bradford (17) and read at absorbance 595 nm.

Antibodies
Polyclonal rabbit anti-rat HO-1 antibodies were raised against a 30 kDa soluble HO-1 protein expressed in Escherichia coli from rat liver cDNA (18) (gift of Angela Wilks, University of California San Francisco, Calif.) by Berkeley Antibodies (Berkeley, Calif.), as described previously (6) . Rabbit polyclonal immunoglobulin G (IgG) anti-p53 corresponding to the full-length p53 of human origin as well as mouse anti-PCNA IgG and goat polyclonal IgG anti-FGF-2 corresponding to amino acids 40–63 of the amino-terminal domain of the FGF-2 precursor (all antibodies from Santa Cruz Biotechnology, Santa Cruz, Calif.) were used. This latter antibody is specific for basic fibroblast growth factor (bFGF) and does not cross react with acidic FGF.

Western analysis For HO-1, p53, PCNA, and bFGF
This technique was used to determine the level of HO-1 expression achieved in the transfected cells and to assess the effect of HO-1 overexpression on markers of cellular proliferation. Aliquots (20 µg protein) of cell sonicates or nuclear proteins (for PCNA detection) were used. Nuclear proteins were derived from cell sonicates collected in a buffer containing 0.5 M sucrose, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 10% glycerol, 1 mM ethylenediaminetetraacetic acid disodium dihydrate (EDTA), and 1 mM phenylmethyl sulfonyl fluoride (PMSF). The sonicates were centrifuged at 4000 x g for 20 min at 4°C and the pellet was resuspended in a high-salt buffer containing 20 mM HEPES (pH 7.9), 25% glycerol, 0.5M KCl, 1.5 mM MgCl2, 0.4 mM EDTA, and 0.5 mM PMSF. After incubation on ice for 10 min, the supernatant was centrifuged at 14,000 x g for 15 min at 4°C. The supernatant was aliquoted and stored at -80°C. Prior to assays, nuclear protein contents were determined as described above (see `Cell protein content').

The samples were electrophoresed on a 12% polyacrylamide gel according to the methods of Laemmli (19) . Proteins from cell sonicates or from nuclear extracts were transferred for 24 h to PVDF membrane (Bio-Rad, Hercules, Calif.) with a Bio-Rad transblot apparatus according to the method of Towbin et al. (20) . Blots were washed briefly in 1x Tris-buffered saline (TBS: 200 mM Tris, 1.5 M NaCl), then incubated overnight at 25°C with rabbit anti-rat HO-1 immunoglobulin G diluted 1:800 in blocking solution [1% non-fat milk + 0.5% bovine serum albumin in 0.05% Tween 20-TBS (T-TBS)]. Blots were washed in T-TBS and incubated for 2 h at 37°C with a 1:5000 dilution of horseradish peroxidase- (HRP) conjugated goat anti-rabbit IgG (Caltag Laboratories, So. San Francisco, Calif.). Antigen–antibody complexes were visualized with the HRP chemiluminescence system according to the manufacturer's instructions (Bio-Rad). Quantification was performed using Molecular Analyst image analysis software (Bio-Rad).

Immunohistochemical detection of HO-1 protein
This method was used to further verify the level of HO-1 overexpression achieved. HO-1 transfected cells, grown to >80% confluence on glass slides for 4 days in Dox (1, 0.5, 0.001, and 0 ng/ml), were washed in phosphate-buffered saline (PBS), then fixed in ice-cold 100% acetone. The cells were permeabilized in 0.3% saponin in PBS and blocked in a PBS solution containing 5% milk, 1% bovine serum albumin, and 0.03% saponin. The slides were incubated with a 1:25 dilution of rabbit anti-rat HO-1 IgG overnight in a humidified chamber. After incubation, the slides were washed twice in PBS with 0.03% saponin and 1% milk, subsequently incubated with a 1:50 dilution of fluorescein isothiocyanate- (FITC) conjugated goat anti-rabbit IgG (Southern Biotechnologies Inc, Birmingham, Ala.) for 2 h at 37°C, and washed twice in PBS. The slides were then mounted in phenylene diamine and viewed with an Axioskop fluorescent microscope (Zeiss, Germany) fitted with a 100W Mercury HBO100W/2 (Zeiss) lamp at excitation 493 nm and photographed with a Nikon camera.

Determination of cell viability
Trypan blue exclusion was used to determine cell viability. Cells were released with 0.05% trypsin-EDTA and 20 µl aliquots were mixed with 20 µl of 0.5% trypan blue. The number of dead (stained) cells was expressed as a ratio of the total (stained and unstained) cells counted.

Determination of lactate dehydrogenase (LDH)
Release of LDH into the medium serves as a marker of altered cellular membrane integrity (21) . Therefore, we assessed whether overexpression of HO-1 altered membrane function in hyperoxia. Media from HO-1 transfected and sham transfected control samples (100 µl) was mixed with 200 µg of NADH in 0.1 M KPO4 buffer and allowed to incubate for 10 min in a multiwell plate. Sodium pyruvate (2.3 µmol) was then added and samples were read at 340 nm at 2 s intervals for 2 min. LDH concentration was calculated automatically from the slope of the absorbance curve. Accuracy was determined with a standard LDH enzyme solution (Enzyme control 2-E, Sigma).

Determination of protein oxidation
To evaluate changes in oxidative injury with HO-1 transfection and hyperoxic exposure, protein oxidation was measured by detecting oxidatively generated carbonyl groups using the OxyBlot Kit (Oncor, Gaithersburg, Md.). The antigen-antibody signal was visualized by chemiluminescence using the HRP chemiluminescence system according to the manufacturer's instructions (Bio-Rad).

Detection of lipid peroxidation
To further corroborate cellular oxidative injury, formation of thiobarbituric acid-reactive substances (TBA-RS) was measured as an estimate of membrane lipid peroxidation using the method of Wright et al. (22) . The concentration of TBA-RS was calculated from the molar extinction coefficient 1.55 x 10-3 M-1 cm-1.

Total glutathione content of cells
A change in cellular glutathione content can be used as a marker of oxidative injury since the synthesis of this tripeptide can be increased in oxidative stress (23) . Total glutathione was measured using the Glutathione Assay Kit (Cayman Chemical, Ann Arbor, Mich.). The samples were deproteinated per the manufacturer's protocol. The samples were read at 405 nm using a microtiter plate at 5 min intervals for 30 min. Total glutathione was determined by comparison with standards and expressed per milligram of protein.

Heme content
To detect alterations in the substrate of the HO reaction with transfection, cellular heme content was determined by quantitation of the hemochrome in a reaction with pyridine in alkali. Cells were washed twice with cold 0.15 M KCl, scraped, and centrifuged. The samples were then resuspended in 0.9% NaCl, mixed with a solution of 25% (v:v) pyridine in 0.075 M NaOH and scanned at absorbance 350–600 nm. The hemochromes have strong Soret bands in the range of 405–440 nm. The absorbance peak corresponding to the Sorret band (414 nm) in the samples was quantitated using a published molar extinction coefficient (24) . Values were expressed as µM heme/mg protein.

Iron content
Since iron is released from the HO reaction and can serve in oxidative reactions, cellular reactive iron was detected using the bleomycin assay. Iron contamination was obviated by using Chelex-100 Resin (Bio-Rad) in all buffers. Fifty microliter aliquots of cell lysates were incubated with a solution, pH 7.4, containing 500 µl DNA [(1 mg/ml; Sigma), 50 µl bleomycin(1.5 U/ml, Sigma), 100 µl MgCl2 (50 mM; Mallinkrodt Baker, Inc., Paris, Ky.), and 100 µl L-ascorbic acid (75 mM; Sigma) in a 37°C shaking water bath for 1 h. The reaction was stopped with 100 µl EDTA (0.1 M; ICN Biochemical, Cleveland, Ohio). The samples were reacted with 500 µl 2-thiobarbituric acid (1% w/v, in 50 mM NaOH; Sigma) and 500 µl hydrochloric acid (25%; Mallinkrodt Baker) in 80°C for 20 min. The absorbance at 532 nm was measured and the iron content is expressed as µM/mg protein (25) .

Determination of cellular bilirubin content
Bile pigments such as bilirubin are formed during the HO reaction. These may alter oxidative injury due to their antioxidant properties. The effect of HO-1 overexpression on cellular bilirubin content was evaluated to document any dose-dependent increase in bilirubin. The moist pellet from 10 to 15 million cells (~20 µl) was mixed with 80–100 µl of 0.1 M di-octamylamine acetate in methanol, sonicated, and centrifuged. Twenty microliters of the supernatant were chromatographed by reversed phase high-performance liquid chromatography (HPLC) as described previously (26) .

Guanosine 3',5'-cyclic monophosphate (cGMP) content
Since CO is released from the HO reaction and this volatile gas has been implicated in the activation of guanylate cyclase (27) , cGMP was measured in the HO-1 transfected cells using the Format A cyclic GMP enzyme immunoassay kit (BIOMOL Research Laboratories, Inc., Plymouth Meeting, Pa.). The samples were read at 405 nm and the cGMP content was determined by comparison to standards per the manufacturer's instructions.

Statistical analysis
For comparison between treatment groups, the Null hypothesis that there was no difference between treatment means was tested by a single-factor analysis of variance for multiple groups or unpaired t test for two groups (Statview 4.02; Abacus Concepts, Berkeley, Calif.). Statistical significance (P<0.05) between groups was determined by means of the Fisher method of multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gene expression in Tet-Off transfected cells
To determine gene expression with doxycycline, luciferase activity was used as a reporter system since the pBI-L/HO-1 gene contained the luciferase gene. The photon emission detected from culture dishes varied with the concentration of doxycycline in the medium in an inversely proportional manner, demonstrating the efficient regulation of gene expression in the HA-1 cells transfected with pBI-L/HO-1 (Fig. 1 A).



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  		Figure 1. A) Representative pseudoimages of light emission in cultured HA-1 cells overexpressing HO-1. Cells were plated in 6-well culture dishes; once 80% confluent, they were incubated with varying concentrations of doxycycline for 24 h The dishes were then each incubated with 20 µl of luciferin (30 mg/ml) for 20 min. Luciferase activity was detected by collecting light emission with an enhanced CCD camera. All images were obtained by collecting photons for the same duration (5 min) to allow for comparison. Lane (-): negative control, empty dish. Lane C: untransfected HA-1 cells. Since these were not transfected, no luciferase activity was observed beyond the level seen in an empty dish. The numbers represent the concentration of doxycycline in the media for each dish. Note the increasing light intensity with decreasing doxycycline concentration. The image shown is representative of 4 experiments per condition. B) Representative slides of immunoreactive HO-1 detection in cultured HA-1 cells overexpressing HO-1. Four slides in each group were incubated with HO-1 antibody and FITC, as described in Materials and Methods. All images were obtained at the same intensity to allow for comparison. Panel (-): negative control: cells incubated with secondary antibody alone. C) HA-1 untransfected controls. The numbers denote the dose (ng/ml) of doxycycline in the medium for a given slide. Note the increasing HO-1 signal in the cytoplasm with decreasing doxycycline concentration.

HO expression in Tet-Off transfected cells
In transfected cells, HO activity was modulated 2.4 ± 0.1 to 17.6 ± 0.5-fold by incubation with 0–1 ng/ml of Dox (Table 1 ). We also verified that Dox did not have any effect on HO-1 expression in the absence of the Tet-Off promoter (data not shown).


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  		Table 1. Effect of HO-1 overexpression on oxygen toxicity in transfected HA-1 cells

Similarly, HO-1 protein was increased 2.8 ± 0.3 to 10.1 ± 0.3-fold with the varying Dox concentration (Table 1) . Immunohistochemical localization of HO-1 revealed intensity of HO-1 protein signal inversely related to the Dox concentration (Fig. 1B ). Based on the consistent levels of HO activity and HO-1 protein observed in repeated experiments (n=6 in each group), HO-1 overexpression was classified as low, moderate, or high based on the degree of HO activity and HO-1 protein achieved. Therefore, for clarification, low was defined as 2–4 to 4.3-fold, moderate was a 10- to 13.5-fold, and high was a 14.9–17.6-fold increase in HO activity.

Cellular injury in HO-1-overexpressing cells
When cells with 2.8- and 10-fold increase in HO activity were exposed to hyperoxia for 48 h, cell viability was significantly increased compared to controls (Table 1) . However, at other low or moderate levels of HO-1 expression, no differences were observed. In contrast, at high levels of HO-1 expression, cell viability was significantly lowered consistent with cytotoxicity at high levels of HO-1 overexpression despite protection at low levels (Table 1) .

As another indicator of oxidative injury, LDH release was also determined. Cellular LDH release was similar to that of control at the low and moderate range of HO-1 expression. However, at the high level of HO-1 expression, LDH release was significantly increased (>2.5-fold) compared to controls (Table 1) . This further documents increased cytotoxicity when HO-1 expression is highest.

Effect of HO-1 overexpression on cell proliferation
The protective effect of HO-1 have been ascribed in some instances to alterations in cellular proliferation (28) . To assess the effect of HO-1 expression on cell proliferation, we determined the level of immunoreactive PCNA in cell nuclei. This protein was elevated when HO-1 was expressed in the low range, but decreased at intermediate levels of HO-1 expression. At high levels of HO-1 expression, increased proliferation was again observed in the viable cells (Fig. 2 ). With the increase in PCNA at high HO-1 expression, immunoreactive bFGF, a fibroblast growth factor and marker of cellular proliferation and transformation (29 30 31) . was also detected (Fig. 2) whereas low and moderate HO-1 expression was not associated with an increased bFGF compared to controls, suggesting that the increased proliferation denoted at the highest levels of HO-1 expression was more likely abnormal. Increases in p53 were detected at high levels of HO-1 expression, further (Fig. 2) suggesting that perturbations in the cell cycle are noted when HO is maximally expressed in the HA-1 cells.



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  		Figure 2. Representative of 4 Western analyses for immunoreactive HO-1, PCNA, bFGF, and p53 protein content in HA-1 cells overexpressing HO-1. Lane C: untransfected HA-1 cells serving as controls. The numbers represent the concentration of doxycycline in the medium for each lane. The level of HO-1, p53, and bFGF from a single membrane is illustrated. The signal for PCNA was obtained from nuclear protein rather than total protein.

Oxidative injury with HO-1 overexpression
As for more specific determinants of oxidative injury, the formation of protein carbonyls was also evaluated. These are formed during oxidative modification of proteins and have been correlated with oxidative damage (32 , 33) . There was a visible decrease in protein carbonyl content at low levels of HO-1 expression, whereas moderate levels of HO-1 overexpression did not demonstrate differences compared to controls. At high levels of HO-1 overexpression, protein carbonyl content was visibly increased compared to controls (Fig. 3 ).



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  		Figure 3. Representative of 4 Western analyses of protein carbonyl detection in HA-1 cells overexpressing HO-1 exposed to hyperoxia. Samples were treated with DNPH, neutralized, then subjected to Western analysis using anti-DNP antibodies. Lane (-): unoxidized control; lane (+): oxidized control; lane C: untransfected HA-1 cells. The numbers denote the concentration of doxycycline in the medium for each lane. Inset: Representative of 4 Western analyses of protein carbonyl detection in HA-1 cells overexpressing HO-1 at high levels exposed to hyperoxia. Control: untransfected HA-1 cells; high: HA-1 cells overexpressing HO-1 at high levels (17-fold increase in activity) exposed to hyperoxia; SnMP: high HO-1-expressing cells incubated with SnMP; DTPA: high expressing cells incubated with DTPA.

Another parameter of oxidative injury is the formation of lipid peroxide byproducts such as TBA-RS. As with the other markers of injury, these were significantly lower in cultured cells with low HO-1 expression, no different in cells with moderate HO-1 expression, and significantly increased in the cells with high HO-1 expression compared to controls (Table 1) .

Total glutathione content was also evaluated since glutathione is an important antioxidant molecule. At the low or moderate levels of HO-1 expression, no differences in glutathione content could be detected when compared to controls. However, at the high levels of HO-1 expression, there was a 2.5-fold increase in total glutathione content compared to controls (Table 1) .

Determination of HO reaction products in HO-1-overexpressing cells
Since CO from HO metabolic activity can modulate guanylate cyclase and thereby alter cGMP (27) , we evaluated cGMP levels in the HO-1-overexpressing cells to determine whether this by-product of the HO-1 reaction could to lead to alterations in cellular toxicity in hyperoxia. No significant differences in cGMP were observed at any levels of HO-1 overexpression.

Bilirubin is another important byproduct of HO that may modulate oxidative damage (34) . This was determined in the high and low HO-1-expressing cells. Neither bilirubin nor bilirubin glucuronides could be detected in extracts of cells by a sensitive HPLC assay.

To evaluate whether the changes in oxidative injury were correlated to the levels of heme or reactive iron within the cells, we evaluated these parameters. Heme content was significantly lower at the low levels of HO-1 expression, but returned to control levels and did not vary significantly at the moderate or high levels of HO-1 expression (Fig. 4 ). As for reactive iron content, this was highest, as expected, at the high levels of HO-1 expression compared to controls (Fig. 5 ).



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  		Figure 4. Heme content in HA-1 cells overexpressing HO-1 exposed to hyperoxia. Heme was detected with spectrophotometry from cell pellets incubated with varying concentrations of doxycycline as shown on the x axis. Values represent the mean ± SE of 5 determinations. *P < 0.05 vs. control (untransfected HA-1 cells).



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  		Figure 5. Reactive iron content in HA-1 cells overexpressing HO-1 exposed to hyperoxia. Iron was determined by the bleomycin assay from cells after incubation with varying concentrations of doxycycline as shown on the x axis. Values represent the mean ± SE of 5 determinations. *P < 0.05 vs. control (untransfected HA-1 cells).

Since induction of HO and consequent iron release is associated with increased expression of ferritin protein (35) , we determined immunoreactive ferritin protein content of cells variably expressing HO-1. Ferritin protein was consistently detected in the low and moderate HO-1-expressing cells but was visibly increased in the high HO-1-overexpressing cells (Fig. 6 ), corroborating the iron-responsive nature of ferritin gene regulation (36) and increased iron release with HO-1 expression. Nonetheless, despite increased ferritin protein with high HO-1 expression, no protection against oxidative injury was found, but significant oxygen toxicity could be detected.



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  		Figure 6. Representative of 3 Western analyses of immunoreactive ferritin protein in HA-1 cells overexpressing HO-1 exposed to hyperoxia. Lane C: untransfected HA-1 cells. The numbers represent the concentration of doxycycline in the medium. Inset: Representative of 3 Western analyses of ferritin protein in HA-1 cells overexpressing HO-1 at high levels exposed to hyperoxia. Control: untransfected HA-1 cells; high: HA-1 cells overexpressing HO-1 at high levels (17-fold increase in activity) exposed to hyperoxia; SnMP: high HO-1-expressing cells incubated with SnMP; DTPA: high expressing cells incubated with DTPA.

To further document that the differences in oxidative stress observed were related to reactive iron content, cells with high levels of HO-1 expression were treated with SnMP, an inhibitor of heme oxygenase, or DTPA, an iron chelator. Total HO activity was reduced by greater than 50% with 10 µM SnMP resulting in a level of HO activity equal in the low to moderate range of HO-1 expression (specifically, a seven- to eightfold increase in HO activity) (Table 2 ). Treatment with DTPA however significantly increased HO activity in the high HO-1-overexpressing cells (Table 2) . Nonetheless, iron levels were significantly lower in the DTPA-treated HO-1 high expressors than in the untreated samples, resulting in iron levels similar to those of untreated low and moderate HO-1-overexpressing cells (Table 2) . Treatment with SnMP resulted in a significant but less dramatic lowering of reactive iron in the high HO-1-overexpressing cells than in untreated high HO-1-expressing samples. The resulting iron levels were comparable to that of the moderate HO-1-expressing cells (Table 2) . Whether achieved with SnMP or DTPA, the lowering of reactive iron content was associated with a significant decrease in injury parameters when compared to the untreated high HO-1-expressing cells, as demonstrated by lowered LDH release (Table 2) and lowered protein oxidation (Fig. 3 , inset). It is important to note that treatment with DTPA or SnMP did not increase the level of ferritin protein expression in the HO-1-overexpressing cells, thereby precluding an increased sequestration of redox active iron mediated by ferritin with the use of these agents (Fig. 6) .


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  		Table 2. Effect of suppression of HO or iron chelation on oxygen toxicity in transfected HA-1 cellsa


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although several reports have demonstrated a protective effect of modest overexpression of HO-1, it is still difficult to explain why the release of ferric iron (Fe2+) from the porphyrin ring of heme during this reaction would result in beneficial effects, since this form of iron is known to catalyze oxidative reactions (14 , 37) . To corroborate this, some reports have suggested a possible duality of effects of HO-1 overexpression in oxidative stress (7 , 38 , 39) . Since it is feasible that catalytically active (reactive) iron would accumulate when HO-1 expression is highest, we used a variable overexpression model, where HO activity varied between 3- and 17-fold and HO-1 protein content was modified 3- to 10-fold. This level of expression was consistent from experiment to experiment.

With hyperoxic exposure, protein oxidation, lipid peroxidation, and glutathione content were significantly increased in the cells expressing greater than 15-fold HO activity, demonstrating a strong association between HO-1 expression and exacerbation of oxidative injury per several reliable parameters. An increase in lung protein carbonyl content was detected in premature infants exposed to high oxygen, which correlated with the incidence of chronic lung disease (40 , 41) . A significant increase in total glutathione is frequently observed early in the process of oxidative injury (42) .

Despite the cytotoxic effect of high HO-1 expression, there was also a consistent cytoprotective effect of HO-1 at low levels of expression. This finding was consistent with many other reports similarly documenting a protective effect of HO-1 when HO activity achieved was in the range of the low overexpressing cells of this current study (5 , 7 , 9 , 28 , 43) , but the deleterious effects of high expression were unmasked with the present study.

Decreased cell proliferation has been noted with HO-1 transfection, and this effect was tied to the protective nature of the HO-1 enzyme (28) . In the present study, moderate levels of HO-1 expression were associated with decreased cell proliferation, but this was reversed at the high levels of HO-1 expression. Furthermore, the increased cellular proliferation at the highest levels of HO-1 expression was associated with possible cellular transformation. Increased expression of bFGF was noted in the high HO-1-expressing cells. This growth factor is specific for fibroblasts, and increased expression has been noted in several malignancies and with exposure to carcinogens (29 30 31) . In the high HO-1 expressors, there was also increased expression of p53, a well-characterized oncogene, further suggesting abnormal cell growth and differentiation (44 , 45) . Taken together, these data suggest that high HO-1 expression perturbs the cell cycle. In clinical settings, abnormal fibroblast proliferation is observed with oxidant stress and may lead to abnormal lung architecture or malignancy (46 47 48) .

We documented a lowered heme content in the low HO-1-overexpressing cells. This corroborates other observations of a 40% reduction of heme with a threefold increase in HO activity mediated by ultraviolet A radiation (49) and a lowered cellular heme iron with a nitric oxide donor that increased HO activity fivefold (3) . Nonetheless, no reports have evaluated the effect of high HO-1 expression on cellular heme content. A paradoxical increase in cellular heme content was observed with moderate and high HO-1 expression compared to low HO-1-overexpressing cells. It is not clear whether this finding is explained by increased iron release from the HO reaction at the higher levels of HO-1 expression and up-regulation of the heme synthetic pathway through iron response elements (50) . This remains to be systematically investigated.

To verify whether the increased oxidative injury was related to changes in the levels of metabolic products of heme oxygenase, we looked at bilirubin, reactive iron content, and cGMP levels. No significant change in cGMP levels was detected with modification of HO expression. Several studies have demonstrated activation of guanylyl cyclase through NO and CO (51 52 53 54) . Nonetheless, some evidence suggests that NO is a better modulator of guanylyl cyclase than is CO (55 , 56) . Our studies would corroborate these observations. We also did not see significant alterations in cellular bilirubin content in the low or high expressors, implying that the generation of bilirubin via the HO reaction is not sufficiently different to explain the cytoprotective effect of low HO-1 expression or that other factors may obviate the protective effects of bilirubin at the high levels of HO-1 expression (57) , at least in this model.

Iron detected with the bleomycin assay is thought to be associated with increased generation of oxygen radicals. In fact, many studies have shown that reactive iron is associated with increased oxidative stress in human disease (58 59 60) . Cells with the highest levels of HO-1 expression had the highest reactive iron content. At the high levels of HO-1 expression, ferritin protein was up-regulated in the presence of iron, but this did not result in increased cytoprotection. In fact, worsened oxygen toxicity was observed, suggesting that ferritin iron could participate in oxidative reactions when released from ferritin in oxidative stress (61 , 62) . Nonetheless, it is not clear whether this ferritin was iron loaded and therefore not useful in iron sequestration.

To further understand whether there was an association between the higher reactive iron content seen at the higher levels of HO-1 expression and the increased oxidative injury, iron levels were modified either by treatment by SnMP or with an iron chelator. As predicted, SnMP reduced heme oxygenase activity from 15-fold to 4- or 5-fold compared to controls. At this range of HO-1 expression, the cultured cells were relatively protected against oxidative injury, as demonstrated by decreases in LDH release and decreases in protein oxidation in hyperoxia compared to untreated high HO-1-expressing controls.

Treatment with DTPA resulted in increased HO activity compared to controls, perhaps suggesting that reactive iron may serve to inhibit HO activity as is the case with other enzymes (63) . Despite the increase in measured HO activity, reactive iron content was reduced to control values with DTPA and there was a significant reduction in oxidative injury, as evidenced by decreased LDH release and protein oxidation. Since both SnMP and DTPA resulted in decreased reactive iron content, the data strongly suggest that released reactive iron from the HO reaction dictates the toxicity of HO-1 at high levels of expression.

In this study, the balance of heme and reactive iron determined the antioxidant effect of HO-1. At low HO-1 expression, low cellular heme and low iron may allow for decreased oxidative injury and up-regulation of important enzymes, whereas excessive accumulation of reactive iron at high HO-1 expression would result in increased oxidative stress, cytotoxicity, and abnormal cellular proliferation.


   ACKNOWLEDGMENTS
 
The authors are grateful to Anthony McDonagh, Ph.D., for performing the bilirubin assays and to Arthur Tatarov and Pavani Kuruma for their expert technical assistance. This work was funded in part by National Institutes of Health grant HL25701 (P.A.D.), the McCormick Funds (D.M.S.), Court Ballanger funds, and Johnson funds from Stanford University.


   FOOTNOTES
 
2 Abbreviations: bFGF, basic fibroblast growth factor; cGMP, guanosine 3',5'-cyclic monophosphate; CO, carbon monoxide; DTPA, diethylenetriaminepentaacetic acid; EDTA, ethylenediaminetetraacetic acid disodium dihydrate; EMEM, Eagle's minimum essential medium; FITC, fluorescein isothiocyanate; HA-1, hamster fibroblast; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HO, heme oxygenase; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; Hyg, hygromycin; Ig, immunoglobulin; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; PMSF, phenylmethyl sulfonyl fluoride; SnMP, tin mesoporphyrin; TBA-RS, thiobarbituric acid-reactive substances; TBS, Tris-buffered saline; T-TBS, Tween-TBS.

Received for publication January 25, 1999. Revised for publication April 15, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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