The role of hydroxyl radical as a messenger in Cr(VI)-induced p53 activation

Suwei Wang1,2, Stephen S. Leonard1,2, Jianping Ye1, Min Ding1, and Xianglin Shi1

1 Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown 26505; and 2 Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, West Virginia 26506


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study investigates whether reactive oxygen species (ROS) are involved in p53 activation, and if they are, which species is responsible for the activation. Our hypothesis is that hydroxyl radical (·OH) functions as a messenger for the activation of this tumor suppressor protein. Human lung epithelial cells (A549) were used to test this hypothesis. Cr(VI) was employed as the source of ROS due to its ability to generate a whole spectrum of ROS inside the cell. Cr(VI) is able to activate p53 by increasing the protein levels and enhancing both the DNA binding activity and transactivation ability of the protein. Increased cellular levels of superoxide radicals (O2-·), hydrogen peroxide (H2O2), and ·OH radicals were detected on the addition of Cr(VI) to the cells. Superoxide dismutase, by enhancing the production of H2O2 from O2-· radicals, increased p53 activity. Catalase, an H2O2 scavenger, eliminated ·OH radical generation and inhibited p53 activation. Sodium formate and aspirin, ·OH radical scavengers, also suppressed p53 activation. Deferoxamine, a metal chelator, inhibited p53 activation by chelating Cr(V) to make it incapable of generating radicals from H2O2. NADPH, which accelerated the one-electron reduction of Cr(VI) to Cr(V) and increased ·OH radical generation, dramatically enhanced p53 activation. Thus ·OH radical generated from Cr(VI) reduction in A549 cells is responsible for Cr(VI)-induced p53 activation.

Cr(VI) carcinogenesis; reactive oxygen species


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE TUMOR SUPPRESSOR PROTEIN p53 plays an important role in protecting cells from tumorgenetic alteration (19, 22). Mutational inactivation of p53 has been found to be involved in various human cancers, which indicates the importance of p53 in human carcinogenesis (42). It has been reported that >50% of human cancers contain mutations in the p53 gene (15, 22). The p53 is activated in response to a variety of stimuli, such as UV, gamma  radiation, hypoxia, nucleotide deprivation, etc. (7, 10, 18, 23). The activation of p53 may cause either cell division cycle arrest or apoptosis (1, 9, 19, 40).

The p53 can be considered as one of the oxidative stress response transcription factors (42). There are several cysteine residues in the central domain of the p53 protein. These residues are crucial for the p53 protein binding to the specific DNA sequence. Redox modulation at a posttranslational level often occurs by reduction or oxidation of the disulfide bond. A reduced state is required for these cysteine residues to ensure that p53 protein would bind to specific consensus DNA and transactivate target genes (12, 27, 29). Among these genes, more than one-half of them are reported to be involved in the metabolism of reactive oxygen species (ROS) (28). ROS are also believed to be involved in the activation of p53 protein induced by UV and ion irradiation (11, 44). Questions remain to be answered concerning which species among ROS plays a key role in the process of p53 activation.

Cr(VI)-containing compounds are considered to be well-established carcinogens (4). They are potential inducers of tumors in experimental animals and active agents in the induction of DNA damage, such as DNA strand breakage (4, 13, 20, 21, 41, 43, 46). Industrial exposure to these compounds is reported to be associated with a higher incidence of human lung cancer (13, 21). Environmental exposure to Cr(VI) could induce lung toxicity in the short term and carcinogenecity over the long term (6). Cr(VI) and Cr(III) are the two stable chromium oxidation states found in nature. Unlike Cr(III), Cr(VI) can enter the cell through the anion transport system (3, 5). After it enters the cell, Cr(VI) is reduced by cellular reductants to its lower oxidation states, Cr(V) and Cr(IV) (31). These reactive chromium intermediates are capable of generating a whole spectrum of ROS (33, 35, 36, 39). ROS generated by these reactions can cause DNA strand breaks, base modification, lipid peroxidation, and nuclear transcription factor NF-kappa B activation (30, 33, 35, 36, 39, 48). In the present studies, Cr(VI) was used as the source of ROS to investigate the role of ROS in p53 activation, and the following questions will be answered: 1) whether Cr(VI) is able to activate p53 protein, and 2) if so, whether ROS play a role in this process and which species is responsible for p53 activation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Sodium dichromate (Na2Cr2O7) and chromium fluoride [Cr(III)] were purchased from Aldrich (Milwaukee, WI). Deferoxamine, 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), superoxide dismutase (SOD), catalase, sodium formate, aspirin, NADPH, and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO). Dihydroethidium (DE) was purchased from Molecular Probes (Eugene, OR). The spin trap, DMPO, was purified by charcoal decolorization and vacuum distillation and was free of electron spin resonance (ESR) detectable impurities.

Cell culture. A human lung epithelial cell line (A549 cells) was obtained from American Type Culture Collection (Rockville, MD). The cells were maintained in F-12K (Kaighn's modification) nutrient mixture medium (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 1,000 U/ml penicillin-streptomycin.

Nuclear extraction. Nuclear extracts were prepared by a modified method of Ye et al. (47). A549 cells suspended in F-12K plus 10% FBS were cultured in 35-mm cell culture plates at 5 × 106 cells/plate for 24 h. The cells were treated with chromium and other agents for 3 h. After washing the cells with 5 ml PBS twice, we added fresh F-12K medium with 10% FBS. The cells were then incubated at 37°C for various times. At the end of the culture period, cells were harvested and treated with 500 µl of lysis buffer [50 mM KCl, 0.5% Nonidet P-40 (NP-40), 25 mM HEPES (pH 7.8), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 100 µM dithiothreitol (DTT)] on ice for 4 min. After centrifugation at 14,000 rpm for 1 min, we saved the supernatant as a cytoplasmic extract. The nuclei were washed once with the same buffer without NP-40. The washed nuclei were suspended in a 100-µl volume of extraction buffer (500 mM KCl, 10% glycerol with the same concentrations of HEPES, PMSF, leupeptin, aprotinin, and DTT as the lysis buffer) and pipetted three times for proper mixing. After centrifugation at 14,000 rpm for 5 min, we harvested the supernatant and stored this nuclear protein extract at -70°C. The concentration was determined using a bicinchoninic acid protein assay reagent (Pierce, Rockford, IL).

Western blot. The nuclear extraction proteins were used for Western blot analysis to determine the p53 protein level. This analysis was carried out as described before (8). Briefly, samples (20 µg protein) denatured with SDS were electrophoretically separated on 10% Tris-glycine gels (Novex, San Diego, CA) and were transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH). The membrane was preblocked in milk buffer [Tris-buffered saline Tween (TBST) containing 5% nonfat milk] for 20 min and exposed for 1 h to 0.8 µg/ml affinity-purified mouse antibody to p53 protein in fresh milk buffer. The membrane was rinsed and incubated with a 1:2,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG for 1 h. The membrane was then washed with TBST, and antibody binding sites were visualized by enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ). All the antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).

Oligonucleotides. Oligonucleotides were synthesized by the phosphoramidite method on a DNA/RNA synthesizer (model 392; Applied Biosystems, Foster City, CA). A p53 binding sequence (5'-AGACATGCCTAGACATTGT-3') was used to synthesize a p53 binding oligonucleotide. The synthesized single-stranded oligonucleotides were deprotected at 50°C overnight, dried in a speed vacuum, and then dissolved in the Tris-EDTA buffer. Complementary strands were denatured at 80°C for 5 min and annealed at room temperature. The double-stranded probe was labeled with [alpha -32P]ATP (Amersham, Arlington Heights, IL) using a T4 kinase (Bethesda Research Laboratories, Gaithersburg, MD).

Electrophoretic mobility shift assay. The DNA-protein binding reaction was conducted in a 24-µl reaction mixture including 1 µg poly[dI-dC] (Sigma), 3 µg nuclear protein extract, 3 µg BSA, 4 × 104 cpm of 32P-labeled oligonucleotide probe (1 µg), 0.1 µg p53 antibody Ab-1 (Oncogene, Cambridge, MA), 3 µl distilled water, and 12 µl of 2× reaction buffer (24% glycerol, 24 mM HEPES, pH 7.9, 8 mM Tris · HCl, pH 7.9, 2 mM EDTA, and 2 mM DTT). This mixture was incubated on ice for 10 min in the absence of the radiolabeled probe, then incubated for 20 min at room temperature in the presence of the radiolabeled probe. After the incubation, the DNA-protein complexes were resolved on a 3.5% acrylamide gel that had been prerun at 210 V for 30 min with 0.5× Tris-boric acid-EDTA buffer. The loaded gel was run at 210 V for 60 min, then dried and placed on Kodak X-OMAT film (Eastman Kodak, Rochester, NY) for autoradiography. The film was developed after overnight exposure at -70°C.

The concentrations given in the figure legends are final concentrations. All experiments were performed at room temperature and under ambient air except those specifically indicated otherwise.

Assay for p53 transcriptional activation. Human lung epithelial A549 cells (3 × 105) suspended in 1 ml of 10% FBS F-12K medium were seeded into each well of a six-well plate. After being incubated at 37°C for 24 h, the cells were transit transfected by 1 µg p53-luciferase reporter plasmid and 1 µg beta -gal plasmid in reduced-serum medium. The cells were incubated overnight and then washed. Fresh 10% FBS F-12K medium was added. Twenty-four hours later, the cells were exposed to various concentrations of Cr(VI) for 3 h. After being washed, the transfected cells were incubated overnight in fresh F-12K medium. The cells were then extracted with 400 µl reporter lysis buffer in 4°C for 2 h. The luciferase activity was measured with 80 µl of cell lysate using a Monolight luminometer (model 3010; Analytical Luminescence Laboratory, Sparks, MD). beta -gal activity was determined by using a method described before (47). The results are expressed as relative p53 activity compared with controls after normalizing by beta -gal activity.

ESR measurements. ESR spin trapping was used to examine free radical generation. The use of this method is necessary because of the reactive nature of the free radicals to be studied. This technique involves an addition-type reaction of a short-lived radical with a diamagnetic compound (spin trap) to form a relatively long-lived free radical product, the so-called spin adduct, which can be studied by conventional ESR. The intensity of the spin adduct signal corresponds to the amount of short-lived radicals trapped, and the hyperfine splittings of the spin adduct are generally characteristic of the original, short-lived trapped radical.

All measurements were conducted with a Varian E9 ESR spectrometer and a flat cell assembly. Hyperfine couplings were measured (to 0.1 Gauss) directly from magnetic field separation with the use of potassium tetraperoxochromate (K3CrO8) and 1,1-diphenyl-2-picrylhydrazyl as reference standards. An EPRDAP 2.0 program (US EPR, Clarksville, MD) was employed for data acquisition and analysis. Reactants were mixed in test tubes to a total final volume of 0.5 ml. The reaction mixture was then transferred to a flat cell for ESR measurement.

Cellular O2-· and H2O2 assay. DE is a superoxide radical (O2-·)-specific dye (2, 26). DE was dissolved in DMSO at 2 mM and kept at -20°C. The cells were plated onto a glass slip in the 24-well plate at 1 × 104/well 16 h before treatment. DE was added to the cell culture together with the various treatments, and the cells were incubated at 37°C for 30 min. The final DMSO concentration in the cell culture was 0.1%. After the cells were stained, they were washed with PBS and fixed with 10% buffered formalin. The slip was mounted on a glass slide and observed with the use of a Saratro 2000 (Molecular Dynamics, Sunnyvale, CA) laser scanning confocal microscope (Optiphot-2; Nikon, Melville, PA) fitted with an argon-ion laser. DCFH-DA at 2 µM was used to monitor the H2O2 level inside the cells according to the methods reported earlier (14, 17, 49).

Oxygen consumption measurements. Oxygen consumption measurements were carried out with a Gilson oxygraph equipped with a Clark electrode (Gilson Medical Electronics, Middleton, WI). These measurements were made from mixtures containing 1.0 × 106/ml cells and various treatments in a total volume of 1.5 ml. The oxygraph was calibrated with media equilibrated with oxygen of known concentrations.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The p53 activation by Cr(VI) is dose and time dependent. Human lung epithelial A549 cells were used to study induction of p53 activation by Cr(VI). The cells were incubated with different concentrations of Cr(VI) for 3 h. After washing the cells, fresh F-12K medium supplemented with 10% FBS was added. The cells were then incubated at 37°C overnight. The p53 protein level and DNA binding activity were analyzed in the nuclear extracts. As shown in Fig. 1A, the p53 protein level increased after exposure to increasing concentrations of Cr(VI). The DNA binding activity of the same protein was also investigated by electrophoretic mobility shift assay. The results are shown in Fig. 1B. From this figure, a similar concentration-dependent pattern was found, i.e., the higher the concentration of Cr(VI), the higher the binding activity of p53 protein.


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Fig. 1.   Dose-dependent p53 activation induced by Cr(VI). The A549 cells were adjusted to a density of 1 × 106/ml and treated for 3 h with different concentrations of Cr(VI). The cells were washed, added to fresh medium, and incubated overnight. The nuclear extraction proteins were used for Western blot and DNA binding activity assay. A: Western blot for p53 protein level. Lane 1, untreated cells; lane 2, cells + 50 µM Cr(VI); lane 3, cells + 100 µM Cr(VI); lane 4, cells + 150 µM Cr(VI); lane 5, cells + 200 µM Cr(VI); lane 6, cells + 300 µM Cr(VI). B: DNA binding assay for p53 protein. Lane 1, untreated cells; lane 2, cells + 50 µM Cr(VI); lane 3, cells + 100 µM Cr(VI); lane 4, cells + 150 µM Cr(VI); lane 5, cells + 200 µM Cr(VI).

Figure 2 shows the effect of time on the p53 protein level and its DNA binding activity. A549 cells were treated with 150 µM Cr(VI) for 3 h. Fresh F-12K medium with 10% FBS was added. The cells were then collected at different times. The same nuclear extractions were used for measuring protein level (Fig. 2A) and DNA binding activity (Fig. 2B). The results indicate that both p53 protein level and DNA binding activity were enhanced in a time-dependent fashion from 0 to 16 h postexposure.


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Fig. 2.   Time-dependent p53 activation induced by Cr(VI). The A549 cells were adjusted to a density of 1 ×106/ml and treated for 3 h with 150 µM Cr(VI). The cells were washed, added to fresh medium, and collected at different times. The nuclear extraction proteins were used for Western blot and DNA binding activity assay. A: Western blot for p53 protein level. Lane 1, 0 h; lane 2, 3 h; lane 3, 4 h after the 3 h of treatment; lane 4, 8 h after the 3 h of treatment; lane 5, overnight after the 3 h of treatment. B: DNA binding assay for p53 protein. The experimental conditions are the same as A.

Figure 3 shows the results of the luciferase assay for p53 activity. Increasing the Cr(VI) concentration up to 100 µM enhanced p53 activation, whereas further increase in Cr(VI) concentration resulted in lower p53 activation, possibly due to Cr(VI)-induced apoptosis.


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Fig. 3.   Luciferase assay for Cr(VI)-induced p53 activation. Human lung epithelial A549 cells (3 × 105) suspended in 1 ml of 10% FBS F-12K (Kaighn's modification) medium were seeded into a 6-well plate. After being incubated at 37°C in a humidified atmosphere of 5% CO2 for 24 h, the cells were transit transfected by p53-luciferase reporter plasmid and beta -gal plasmid in reduced-serum medium. The cells were incubated overnight, then washed, added to fresh 10% FBS F-12K medium, and incubated for 24 h. The cells were exposed to various concentrations of Cr(VI) for 3 h, washed, added to fresh F-12K medium, and incubated overnight. The p53 activity was measured by luciferase activity assay as described in MATERIALS AND METHODS. Results are presented as relative p53 induction compared with the untreated control cells (means and SD of 3 repeated assays). * Indicates a significant increase from control (P < 0.05).

Detection of free radicals. ESR spin trapping was used to detect free radical generation from Cr(VI)-stimulated A549 cells. Fig. 4A shows the spectrum recorded from a mixture containing Cr(VI), A549 cells, and DMPO (a spin-trapping reagent). This spectrum consists of a 1:2:2:1 quartet with splittings of aH = aN = 14.9 G, where aH and aN denote hyperfine splittings of the alpha -hydrogen and the nitroxyl nitrogen, respectively. On the basis of these splitting constants, the 1:2:2:1 quartet was assigned to DMPO/·OH adduct. The arrow at the right side shows a small peak that was assigned to a Cr(V)-NADPH complex as reported earlier (38). Addition of SOD, a O2-· scavenger whose function is to convert O2-· to H2O2, dramatically increased the DMPO/·OH adduct signal (Fig. 4B). Catalase, an H2O2 scavenger, and sodium formate, a scavenger of ·OH radical, decreased the generation of ·OH radical (Fig. 4, C and D). A metal chelator, deferoxamine, suppressed the DMPO/·OH signal (Fig. 4E). NADPH, a cofactor of certain flavoenzymes, such as GSSG-R (glutathione reductase), which catalyzes the conversion of Cr(VI) to Cr(V), enhanced the generation of both Cr(V) and ·OH radical (Fig. 4F). H2O2 dramatically increased the DMPO/·OH signal with reduction of the Cr(V) peak (Fig. 4G).


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Fig. 4.   Generation of free radicals from Cr(VI)-stimulated A549 cells. A: electron spin resonance (ESR) spectrum recorded 10 min after the addition of 1 mM Cr(VI) to 1 × 106 cells/ml and 100 mM 5,5-dimethyl-1-pyrroline 1-oxide in a phosphate-buffered solution (pH 7.4). B: same as A but with 1,000 U/ml superoxide dismutase (SOD) added. C: same as A but with 10,000 U/ml catalase added. D: same as A but with 50 mM sodium formate added. E: same as A but with 2 mM deferoxamine added. F: same as A but with 2 mM NADPH added. G: same as A but with 2 mM H2O2 added.

Specific fluorescent dyes were used to directly visualize free radical generation inside the A549 cells. DE, a specific fluorescent dye for O2-·, and DCFH-DA, a fluorescent dye for H2O2, were used to detect the generation of O2-· and H2O2, respectively. Both O2-· and H2O2 can be visualized in untreated cells (Fig. 5). On stimulation with Cr(VI), O2-· radicals were dramatically enhanced (Fig. 5A). The H2O2 level also increased, although not as significantly as O2-· radicals (Fig. 5B). Addition of catalase decreased the amount of H2O2 inside the cell [Fig. 5B, Cr(VI) + Cat], whereas addition of SOD increased its level [Fig. 5B, Cr(VI) + SOD]. The changes occurred within 30 min. The fluorescent dyes exhibited a red or orange color after being oxidized by ROS.


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Fig. 5.   Generation of O2-· radicals and H2O2 inside the A549 cells. A: fluorescent staining for O2-· radicals. The cells were incubated in the absence (left) or presence (right) of 150 µM Cr(VI) in the presence of 2 µM dihydroethidium for 30 min. B: fluorescent staining of H2O2. The cells were incubated with 5 µM 2',7'-dichlorofluorescein diacetate in the absence (control) or presence of 150 µM Cr(VI), Cr(VI) + 10,000 U/ml catalase, and Cr(VI) + 1,000 U/ml SOD for 30 min. The cells were washed once in PBS and fixed with 10% formalin. The images were captured with a confocal microscope.

Oxygen consumption. As shown before, ·OH radicals were produced via a Cr(V)-mediated Fenton-like reaction [H2O2 + Cr(V) right-arrow Cr(VI) + ·OH + OH-]. The H2O2 was generated by the dismutation of O2-· radicals. O2-· radicals were generated by one-electron reduction of molecular oxygen during the reduction of Cr(VI) by flavoenzymes in the presence of NADPH. A Gilson oxygraph equipped with a Clark electrode was used to measure oxygen consumption in Cr(VI)-stimulated A549 cells. As shown in Fig. 6, addition of Cr(VI) enhanced the oxygen consumption, and NADPH slightly accelerated it. Cr(III) did not have any significant effect.


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Fig. 6.   Oxygen consumption by Cr(VI)-stimulated A549 cells. An incubation mixture contained 1 mM Cr(VI), 1 × 106 cells/ml cells, and various reagents [1 mM NADPH; 1 mM Cr(III)] as indicated. Values are means and SD of 3 repeated experiments. * Indicates a significant increase from the control (P < 0.05).

·OH radical is responsible for Cr(VI)-induced p53 activation. A549 cells were incubated with Cr(VI) in the presence of various reagents for 3 h (Fig. 7). After the cells were washed, the fresh F-12K medium with 10% FBS was added. The cells were then incubated overnight and were harvested for extraction of nuclear proteins. The protein level was analyzed by Western blot. The results are shown in Fig. 7. As shown in this figure, the p53 protein level in the untreated A549 cells (lane 1) was below the detection limits. Cells treated with Cr(VI) showed an elevated p53 protein level (lane 2). Cr(III), because of its inability to enter the cell, had no effect (lane 3). SOD increased the protein level through the generation of H2O2 and ·OH radical (lane 4), whereas catalase, sodium formate, and aspirin scavenged ·OH radical and diminished the protein level (lanes 5-7). Deferoxamine chelated Cr(V) and decreased p53 protein induction (lane 8). NADPH, on the other hand, enhanced the generation of ·OH radical and increased p53 protein induction in A549 cells (lane 9). These results suggest that ·OH radical is responsible for Cr(VI)-induced p53 protein activation.


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Fig. 7.   Effects of reactive oxygen species on Cr(VI)-induced p53 activation. The A549 cells were adjusted to a density of 1 × 106/ml and treated for 3 h with different stimuli. The cells were washed, added to fresh medium, and incubated overnight. The nuclear extraction proteins were used for Western blot. Lane 1, untreated cells; lane 2, cells + 150 µM Cr(VI); lane 3, cells + 150 µM Cr(III); lane 4, cells + 150 µM Cr(VI) + 1,000 U/ml SOD; lane 5, cells + 150 µM Cr(VI) + 10,000 U/ml catalase; lane 6, cells + 150 µM Cr(VI) + 50 mM sodium formate; lane 7, cells + 150 µM Cr(VI) + 2 mM aspirin; lane 8, cells + 150 µM Cr(VI) + 2 mM deferoxamine; lane 9, cells + 150 µM Cr(VI) + 2 mM NADPH.

Results shown above support the generation of ROS in this cellular system. The reaction scheme presented in Fig. 8 may best accommodate the pathways for ·OH radical generation in Cr(VI)-stimulated A549 cells and the resultant p53 activation.


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Fig. 8.   Schematic representation of possible mechanism of ·OH radical generation in Cr(VI)-stimulated A549 cells. GSSG-R, glutathione reductase.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies show that Cr(VI) is able to induce the activation of p53 protein in the epithelial cell line A549. ·OH radicals play a key role in this Cr(VI)-induced p53 activation.

With the use of ESR with a loop-gap resonator, previous studies have demonstrated that reduction of Cr(VI) in live animals generates Cr(V) (24). Flavoenzymes, such as GSSG-R, are likely reductants for Cr(V) generation. With the use of a noncellular chemical system, our earlier studies have shown that reduction of Cr(VI) by GSSG-R in the presence of NADPH generates Cr(V) (31). The results obtained from the present studies show that Cr(VI) can be reduced by the whole cells to Cr(V). During the reduction process, molecular oxygen is reduced to O2-· radical, which generates H2O2 via dismutation. Cr(V) reacts with H2O2 to generate ·OH radicals via a Fenton-like reaction [Cr(V) + H2O2right-arrowCr(VI) + ·OH+ OH-] (Fig. 8).

The ·OH radical is the key species among ROS responsible for p53 activation. The following experimental observations support the above conclusion. 1) Sodium formate, an ·OH radical scavenger, inhibited p53 activation. 2) Deferoxamine, which chelated Cr(V) to make it incapable of generating ·OH radical from H2O2, diminished p53 activation. 3) SOD enhanced the p53 activation instead of attenuating it. This is due to the fact that SOD increased the H2O2 generation by catalyzing O2-· dismutation, as demonstrated by fluorescent staining, and increased the ·OH radical generation through a Fenton-like reaction, as demonstrated by ESR spin trapping. 4) A mixture of Cr(VI) and A549 cells increased molecular oxygen consumption. 5) Catalase, which depleted H2O2 and blocked ·OH radical generation, inhibited the p53 activation.

H2O2 is an endogenous oxidant that is commonly used to investigate the role of ROS in p53 activation. However, inconsistent observations were reported from different laboratories (16, 27). For example, it has been observed that H2O2 can activate p53 protein and its target gene Bax (16). It has also been reported that H2O2 decreased p53 transactivation by weakening its binding to the specific DNA sequence (27). The results obtained from present study show that whereas H2O2 itself is unable to cause p53 activation, it can indirectly activate p53 through its ability to generate ·OH radical on reaction with metal ions. The following experimental observations support this conclusion. 1) Sodium formate, an ·OH radical scavenger, inhibited p53 activation. This ·OH radical scavenger did not react with H2O2. Similar results were obtained with the use of a different ·OH radical scavenger, aspirin. 2) Deferoxamine, which inhibited p53 activation due to chelation of Cr(V) to make this metal ion unable to generate ·OH radical from H2O2, did not react with H2O2. Although H2O2 is not a direct p53 stimulating agent, it functions as a precursor for ·OH radical generation. Thus elimination of H2O2 inhibited p53 activation. This may explain the reported controversy concerning the role of H2O2 in p53 activation.

As for O2-· radical, it is not a direct p53 stimulating agent either. As shown in the present studies, a specific O2-· radical scavenger, SOD, enhanced p53 activation instead of causing inhibition. The enhancement of p53 activation is due to the generation of H2O2 from O2-· radical catalyzed by SOD. O2-· radical is generated via reduction of molecular oxygen as demonstrated by the oxygen consumption assay. Thus the following scheme can best accommodate p53 activation induced by ROS reaction
O<SUB><IT>2</IT></SUB><IT>→</IT>O<SUP>−</SUP><SUB>2</SUB>·  <LIM><OP><ARROW>→</ARROW></OP><UL>SOD</UL></LIM> H<SUB><IT>2</IT></SUB>O<SUB><IT>2</IT></SUB> <LIM><OP><ARROW>→</ARROW></OP><UL>Cr(V)</UL></LIM>  ·OH<IT>→</IT>p<IT>53 </IT>activation
Cr(VI) is a well-established carcinogen. Although the mechanism of its action is still unclear, free radical reactions mediated by this metal ion are believed to play a key role. For example, ·OH radicals generated by reactive chromium intermediates, such as Cr(V) and Cr(IV), can cause NF-kappa B activation, DNA strand breakage, and cell injury. These radicals also function as messengers to induce p53 activation in response to Cr(VI)-induced cellular injury. The process of p53-dependent cell cycle arrest or apoptosis functions to repair or remove the damaged cells. Because of the fundamental importance of cell cycle arrest and apoptosis, which are regulated by p53, alternations of these pathways can enhance cancer development. Thus understanding of p53 activation, especially the role of ROS, is essential for understanding the overall carcinogenic process induced not only by Cr(VI), but also by other chemical carcinogens as well.

It may be noted that many other metal ions, mineral particles, and chemical carcinogens, such as cobalt, nickel, vanadium, asbestos, and silica are reported to be capable of generating ·OH radical (25, 32, 34, 37, 45). It is possible that those agents may have the same function as Cr(VI); i.e., they may cause p53 activation through ·OH radical as a common messenger. Because p53 is involved in various biological processes such as regulation of genes involved in the cell cycle, cell growth arrest after DNA damage, and apoptosis, ·OH radical could be an important messenger in signal transduction pathways involved in carcinogenesis processes.

In conclusion, 1) Cr(VI) is able to induce the p53 activation, 2) ROS is involved in its mechanism of activation, and 3) ·OH radical functions as a messenger in the p53 activation processes.


    FOOTNOTES

Address for reprint requests and other correspondence: X. Shi, Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV 26505 (E-mail: xas0{at}cdc.gov).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 27 December 1999; accepted in final form 27 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Agarwal, ML, Taylor WR, Chernov MV, Chernova OB, and Stark GR. The p53 network. J Biol Chem 273: 1-4, 1998[Free Full Text].

2.   Carter, WO, Narayanan PK, and Robinson JP. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol 55: 253-258, 1994[Abstract].

3.   Connett, PH, and Wetterhahn KE. Metabolism of the carcinogenic chromate by cellular constituents. Struct Bonding 54: 93-124, 1983[ISI].

4.   De Flora, S, Bagnasco M, Serra D, and Zanacchi P. Genotoxicity of chromium compounds: a review. Mutat Res 238: 99-172, 1990[ISI][Medline].

5.   De Flora, S, and Wetterhahn KE. Mechanisms of chromium metabolism and genotoxicity. Life Chem Rep 7: 169-244, 1989.

6.   Freeman, N, and Lioy PJ. Exposure to chromium dust from homes in a chromium surveillance project. Arch Environ Health 52: 213-226, 1997[ISI][Medline].

7.   Fritsche, M, Haessler C, and Brandner G. Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents. Oncogene 8: 307-318, 1993[ISI][Medline].

8.   Ghosh, P, Sica A, Young HA, Ye J, Franco JL, Wiltrout RH, Longo DL, Rice NR, and Komschlies KL. Alterations in NF kappa  B/Rel family proteins in splenic T-cells from tumor-bearing mice and reversal following therapy. Cancer Res 54: 2969-2972, 1994[Abstract].

9.   Gottlieb, TM, and Oren M. p53 in growth control and neoplasia. Biochim Biophys Acta 1287: 77-102, 1996[ISI][Medline].

10.   Graeber, TG, Peterson JF, Tsai M, Monica K, Fornace AJ, Jr, and Giaccia AJ. Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by low-oxygen conditions is independent of p53 status. Mol Cell Biol 14: 6264-6277, 1994[Abstract].

11.   Griffiths, HR, Mistry P, Herbert KE, and Lunec J. Molecular and cellular effects of ultraviolet light-induced genotoxicity. Crit Rev Clin Lab Sci 135: 189-237, 1998.

12.   Hainaut, P, and Milner J. Redox modulation of p53 conformation and sequence-specific DNA binding in vitro. Cancer Res 53: 4469-4473, 1993[Abstract].

13.   Hayes, RB. Review of occupational epidemiology of chromium chemicals and respiratory cancer. Sci Total Environ 71: 331-339, 1988[ISI][Medline].

14.   Hockenbery, DM, Oltvai ZN, Yin XM, Milliman CL, and Lorsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75: 241-251, 1993[ISI][Medline].

15.   Hollstein, M, Rice K, Greenblatt MS, Soussi T, Fuchs R, Sorlie T, Hovig E, Smith-Sorensen B, Montesano R, and Harris CC. Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res 22: 3551-3555, 1994[Abstract].

16.   Jiang, M, Liang H, Liao C, and Lu F. Methyl methanesulfonate and hydrogen peroxide differentially regulate p53 accumulation in hepatoblastoma cells. Toxicol Lett 106: 201-208, 1999[ISI][Medline].

17.   Johnson, TM, Yu ZX, Ferrans VJ, Lowenstein RA, and Finkel T. Reactive oxygen species are downstream mediators of p53-dependent apoptosis. Proc Natl Acad Sci USA 93: 11848-11852, 1996[Abstract/Free Full Text].

18.   Kastan, MB, Onyekwere O, Sidransky D, Vogelstein B, and Craig RW. Participation of p53 protein in the cellular response to DNA damage. Cancer Res 51: 6304-6311, 1991[Abstract].

19.   Ko, JL, and Prives C. p53: puzzle and paradigm. Genes Dev 10: 1054-1072, 1996[ISI][Medline].

20.   Kortenkamp, A, Ozolins Z, Beyersmann D, and O'Brien P. Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione. Mutat Res 216: 19-26, 1989[ISI][Medline].

21.   Langard, S. One hundred years of chromium and cancer: a review of epidemiological evidence and selected case reports. Am J Ind Med 17: 189-215, 1990[ISI][Medline].

22.   Levine, AJ. p53, the cellular gatekeeper for growth and division. Cell 88: 323-331, 1997[ISI][Medline].

23.   Linke, SP, Clarkin KC, DiLeonardo A, Tsou A, and Wahl GM. A reversible, p53-dependent G0/G1 cell cycle arrest induced by ribonucleotide depletion in the absence of detectable DNA damage. Genes Dev 10: 934-947, 1996[Abstract].

24.   Liu, K, Jiang J, Swartz HM, and Shi X. Low frequency ESR detection of chromium(V) formation by one-electron reduction of chromium(VI) in whole live mice. Arch Biochem Biophys 313: 248-252, 1994[ISI][Medline].

25.   Mao, Y, Liu K, Jiang J, and Shi X. Generation of reactive oxygen species by Co(II) from H2O2 in the presence of chelators in relation to DNA damage and 2'-deoxyguanosine hydroxylation. J Toxicol Environ Health 47: 61-75, 1996[ISI][Medline].

26.   Marchetti, P, Castedo M, Susin SA, Zamzami N, Hirsch T, Macho A, Haeffner A, Hirsch F, Geuskens M, and Kroemer G. Mitochondrial permeability transition is a central coordinating event of apoptosis. J Exp Med 184: 1155-1160, 1996[Abstract].

27.   Parks, D, Bolinger R, and Mann K. Redox state regulates binding of p53 to sequence-specific DNA, but not to nonspecific or mismatched DNA. Nucleic Acids Res 25: 1289-1295, 1997[Abstract/Free Full Text].

28.   Polyak, K, Xia Y, Zweier JL, Kinzler KW, and Vogelstein B. A model for p53-induced apoptosis. Nature 389: 300-305, 1997[ISI][Medline].

29.   Rainwater, R, Parks D, Anderson ME, Tegtmeyer P, and Mann K. Role of cysteine residues in regulation of p53 function. Mol Cell Biol 15: 3892-3903, 1995[Abstract].

30.   Shi, X, Castranova V, Halliwell B, and Vallyathan V. Reactive oxygen species and silica-induced carcinogenesis. J Toxicol Environ Health 1: 181-197, 1998[ISI].

31.   Shi, X, Chiu A, Chen CT, Halliwell B, Castranova V, and Vallyathan V. Reduction of chromium(VI) and its relationship to carcinogenesis. J Toxicol Environ Health 2: 87-104, 1999[ISI].

32.   Shi, X, Dalal N, and Kasprzak K. Enhanced generation of hydroxyl radical and sulfur trioxide anion radical from oxidation of sodium sulfite, nickel (II) sulfite, and nickel subsulfide in the presence of nickel (II) complexes. Environ Health Perspect 102, Suppl3: 91-96, 1994[ISI][Medline].

33.   Shi, X, and Dalal NS. Evidence for a Fenton-type mechanism for the generation of hydroxyl radical in the reduction of Cr(VI) in cellular media. Arch Biochem Biophys 281: 90-95, 1990[ISI][Medline].

34.   Shi, X, and Dalal NS. Hydroxyl radical generation in the NADH/microsomal reduction of vanadate. Free Radic Res Commun 17: 369-376, 1992[ISI][Medline].

35.   Shi, X, and Dalal NS. Chromium(V) and hydroxyl radical formation during the glutathione reductase-catalyzed reduction of chromium(VI). Biochem Biophys Res Commun 163: 627-634, 1989[ISI][Medline].

36.   Shi, X, and Dalal NS. On the hydroxyl radical formation in the reaction between hydrogen peroxide and biologically generated chromium(V) species. Arch Biochem Biophys 277: 342-350, 1990[ISI][Medline].

37.   Shi, X, Dalal NS, and Vallyathan V. ESR evidence for the hydroxyl radical formation in aqueous suspension of quartz particles and its possible significance to lipid peroxidation in silicosis. J Toxicol Environ Health 25: 237-245, 1988[ISI][Medline].

38.   Shi, X, Ding M, Wang S, Leonard SS, Zang L, Castranova V, Vallyathan V, Chiu A, Dalal NS, and Liu K. Cr(IV) causes activation of nuclear transcription factor NF-kappa B, DNA strand breaks and dG hydroxylation via free radical reactions. J Inorg Biochem 75: 37-44, 1999[ISI][Medline].

39.   Shi, X, Mao Y, Knapton A, Ding M, Rojanasakul Y, Gannett PM, Dalal NS, and Liu K. Reaction of Cr(VI) with ascorbate and hydrogen peroxide generates hydroxyl radicals and causes DNA damage: role of a Cr(VI)-mediated Fenton-like reaction. Carcinogenesis 15: 2475-2478, 1994[Abstract].

40.   Smith, ML, and Fornace AJ, Jr. p53-mediated protective responses to UV irradiation. Proc Natl Acad Sci USA 94: 12255-12257, 1997[Free Full Text].

41.   Sugiyama, M, Tsuzuki K, and Haramaki N. DNA single-strand breaks and cytotoxicity induced by sodium chromate(VI) in hydrogen peroxide-resistant cell lines. Mutat Res 299: 95-102, 1993[ISI][Medline].

42.   Sun, Y, and Oberley LW. Redox regulation of transcriptional activators. Free Radic Biol Med 21: 335-348, 1996[ISI][Medline].

43.   Tsapakos, MJ, Hampton TH, and Wetterhahn KA. Chromium(VI)-induced DNA lesions and chromium distribution in rat kidney, liver, and lung. Cancer Res 43: 5662-5667, 1983[Abstract].

44.   Vile, GF. Active oxygen species mediate the solar ultraviolet radiation-dependent increase in the tumour suppressor protein p53 in human skin fibroblasts. FEBS Lett 412: 70-74, 1997[ISI][Medline].

45.   Weitzman, SA, and Graceffa P. Asbestos catalyzes hydroxyl and superoxide radical generation from hydrogen peroxide. Arch Biochem Biophys 228: 373-376, 1984[ISI][Medline].

46.   Xu, J, Wise JP, and Patierno SR. DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells. Mutat Res 280: 129-136, 1992[ISI][Medline].

47.   Ye, J, Ghosh P, Cippitelli M, Subleski J, Hardy KJ, Ortaldo JR, and Young HA. Characterization of a silencer regulatory element in the human interferon-gamma promoter. J Biol Chem 269: 25728-25734, 1994[Abstract/Free Full Text].

48.   Ye, J, Zhang X, Young HA, Mao Y, and Shi X. Chromium(VI)-induced nuclear factor-kappa B activation in intact cells via free radical reactions. Carcinogenesis 16: 2401-2405, 1995[Abstract].

49.   Zamzami, N, Marchetti P, Castedo M, Zanin C, Vayssiere JL, Petit PX, and Kroemer G. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med 181: 1661-1672, 1995[Abstract].


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