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Evidence That Nitric Oxide Enhances Cadmium Toxicity by Displacing the Metal from Metallothionein R. Rita Misra,† James F. Hochadel,‡ George T. Smith,† John C. Cook,§ Michael P. Waalkes,*,† and David A. Wink† Inorganic Carcinogenesis and Chemistry Sections, Laboratory of Comparative Carcinogenesis, Division of Cancer Etiology, and BCDP, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, and Tumor Biology Section, Radiation Biology Branch, National Cancer Institute, Bethesda, Maryland 20892 Received June 21, 1995X
Cadmium is carcinogenic in humans and rodents. Although extensive evidence indicates that the toxicity and genotoxicity of Cd is ameliorated by binding to cysteine clusters in metallothionein (MT), the factors governing Cd release at intracellular target sites remain unknown. Nitric oxide is a pollutant gas and an important intercellular messenger in the inflammatory immune response. When growing Chinese hamster ovary cells were treated for 24 h with 0.5, 0.75, or 1.0 mM CdCl2 followed by a 1-h exposure to 1.0, 1.5, or 2.0 mM 1,1diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), an NO-generating sodium salt, NO enhanced Cd-induced inhibition of colony forming ability without affecting Cd-induced cytolethality. In experiments designed to determine whether NO acts by displacing Cd from cellular MT, cells treated with 2.0 mM CdCl2 followed by 1.5 or 3.0 mM DEA/NO exhibited 29 and 38% reductions, respectively, in the amount of Cd bound to MT. When purified rat liver MT was used to further characterize NO-induced release of Cd from MT, dose-related increases in Cd displacement were observed at DEA/NO concentrations between 0.1 and 0.5 mM, and a plateau was reached at 3 mol of Cd displaced/mol of MT at higher DEA/NO concentrations. Compared to cells exposed to Cd or DEA/NO alone, cells treated with Cd followed by DEA/NO also exhibited a transient 2-3-fold decrease in c-myc proto-oncogene expression. Taken together, our results support the hypothesis that NO mediates Cd release from MT in vivo and suggest that intracellular generation of free Cd may induce DNA damage and force cells into a period of growth arrest. Such findings may have particular relevance with regard to the etiology of Cd-induced carcinogenesis in human populations.
Introduction Chronic inhalation of cadmium has been linked to pulmonary carcinogenesis in humans (1). Humans may be exposed to Cd occupationally during smelting and refining of zinc, lead, and copper ores, electroplating, welding, and the manufacture of pigments, plastic stabilizers, and nickel-cadmium batteries (1). Other sources of exposure include cigarette smoke and contaminated food, water, and air. In laboratory rats and mice, Cd appears to be carcinogenic by essentially all routes of exposure thus far tested, and exposures have been associated with tumors of the lung, prostate, testes, and hematopoietic system (2). Although Cd tends to concentrate in the kidney and liver, both organs are typically refractory to the metal’s carcinogenic effects. Tissue specificity appears to be mediated by the presence of large amounts of small, cysteine-rich, metal-storage proteins, metallothioneins (MTs),1 that sequester and thereby inactivate free Cd ions intracellularly (3). Mammalian MTs are approximately * Author to whom correspondence should be directed at NCIFCRDC, Building 538, Room 205E, Frederick, MD 21702. † Inorganic Carcinogenesis and Chemistry Sections. ‡ BCDP. § Tumor Biology Section. X Abstract published in Advance ACS Abstracts, December 15, 1995. 1 Abbreviations: Cd, cadmium; MT, metallothionein; NO, nitric oxide; DEA/NO, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine; MTT, 3-4, (5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEPES, N-(2 hydroxyethyl)piperazine-N′-(2 ethanesulfonic acid).
This article not subject to U.S. Copyright.
6 Kda in size and contain 20 cysteine residues arranged to form two distinct domains which together bind a total of seven divalent metal ions (4). Besides a putative role in metal detoxication, recent data suggest that MT may protect cells against oxidative damage as well (5). At the transcriptional level, metal-induced MT synthesis is regulated by interaction between upstream gene regulatory sequences and a zinc finger containing DNAbinding protein, MTF-1 (6). Cd is an effective inducer of MT synthesis, and under normal circumstances, the metal is rapidly sequestered by MT. While it has become increasingly clear that MT is necessary for zinc homeostasis, the factors governing intracellular release of metal ions from MT remain unknown. Nitric oxide is an important intercellular messenger involved in blood pressure control, platelet aggregation, and the inflammatory immune response (7, 8). This diatomic radical is also a constituent of auto exhaust, cigarette smoke, and gas cooking and heating fumes (9). In the presence of oxygen, NO forms intermediates (NOx) which react efficiently with thiol groups in proteins according to the following scheme (10):
NO + O2 f NOx NOx + RSH f RSNO These reactive intermediates can alter biomolecules such as cytochromes P450 (11), glyceraldehyde-3-phosphate
Published 1996 by the American Chemical Society
NO Enhancement of Cd Toxicity
dehydrogenase (12), Fpg protein (13), and O6-alkylguanine DNA alkyl transferase (14), as well as deaminate bases in DNA (15, 16). In addition, NOx can labilize zinc from metal-thiol loci such as those found in zinc-finger motifs and MT (13, 17). Due to its demonstrated affinity for thiol proteins, we suspected that NO may be particularly effective at displacing Cd from MT. Thus, in the present study, pretreatment of cells with Cd resulting in the formation of Cd-MT complex was followed by exposure to 1,1diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), an NOgenerating sodium salt, and alterations in cell proliferation and cytolethality were monitored. In addition, CdMT binding assays were used to characterize the ability of NO to release Cd ions from MT in vitro and in cultured cells. The results from these assays along with results from analysis of c-myc proto-oncogene expression in similarly treated cells suggest that NO may be an effector molecule responsible for releasing Cd from MT in vivo and that the resultant increase in free intracellular Cd may induce DNA damage and force cells into a period of growth arrest.
Materials and Methods Caution: Cd has been classified by IARC as a Group 1 carcinogen; therefore, all precautions should be taken to prevent exposure. 3-(4,5-Dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) may be mutagenic; inhalation of dust and contact with eyes and skin should be avoided. RNAzol B contains an irritant (guanidinium thiocyanate) and a poison (phenol); inhalation of vapors and contact with eyes, skin, and clothing should be avoided. Materials. Cadmium chloride and MTT were purchased from Sigma Chemical Co. (St. Louis, MO). DEA/NO was a generous gift from Dr. Joseph E. Saavedra (SAIC, NCI-FCRDC, Frederick, MD) and was synthesized as previously described (18, 19). Radioactive 109Cd was purchased from Amersham (Arlington Heights, IL). All other reagents were of the highest purity commercially available. Cell Cultures. CHO-K1 Chinese hamster ovary cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were grown at 37 °C and 5% CO2 in Ham’s F-12 media supplemented with 5% fetal calf serum. Cells were routinely fed every 3 or 4 days and passaged weekly. Assays of Cytotoxicity. Chemically induced inhibition of cell growth was monitored as described previously (20). Cells were seeded at densities of 100, 250, and 500 cells/100-mm dish, and various concentrations of CdCl2 were delivered in complete medium containing 10 mM N-(2 hydroxyethyl) piperazine-N′(2 ethanesulfonic acid) (HEPES) 16 h later. At 24 h after Cd administration, the cultures were treated for 1 h with various amounts of DEA/NO in fresh media containing 50 mM HEPES. The cells were fed once over the course of the 7-day growth period, and colonies were visualized with Giemsa stain. Three individual dishes of each seeding density were assayed per treatment, and mean values were used to estimate cytotoxicity. Cytotoxicity was determined from the percent survival of colonyforming ability (the cloning efficiency of the treated cells divided by the cloning efficiency of the untreated cells, multiplied by 100). Chemically induced cell death was monitored using a slightly modified version of a method described by Hansen et al. (21). This assay measures the conversion of MTT to formazan by dehydrogenase enzymes in the intact mitochondria of living cells. Cells were seeded into 96-well plates, and 16 h later, growing cells were treated with various concentrations of Cd, followed 24 h later by DEA/NO. After exactly 1 h the media in each well was replaced with 100 µL of a 1 mg/mL solution of MTT in 131 mM NaCl, 5.2 mM KC1, 0.9 mM MgSO4, 1 mM CaCl2, 11.1 mM glucose, 10 mM Tris-HCl, pH 7.4, and the plates
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 327 were incubated at 37 °C; 2 h later, 200 µL of lysis solution (20% sodium dodecyl sulfate solubilized in 50% N,N-dimethylformamide, 2.5% glacial acetic acid, and 2.5% 1 N HC1) was added to each well and the plates were allowed to incubate at 37 °C overnight. Optical densities at 570 and 630 nm were measured using a Biotek Model EL340 automated microplate reader (Biotek Inc., Winooski,VT) and 570/630 ratios were used to correct for slight variations in background absorbance between individual wells on the microtiter plates. Twenty-four individual wells were assayed per treatment, and mean values were used to estimate cytotoxicity. Estimation of Intracellular Release of Cd from MT. A modified version of the Cd/heme assay originally described by Onosaka et al. (22) was used to estimate the amount of Cd released by DEA/NO from MT in cells. Briefly, growing CHOK1 cells were treated for 24 h with 2.0 mM CdCl2 (42 mCi of 109Cd/mL) in complete media containing 10 mM HEPES. Immediately following Cd treatment the cells were treated for 1 h with various concentrations of DEA/NO in fresh media containing 50 mM HEPES. Cells from each treatment group (four flasks per DEA/NO dose) were rinsed with phosphate-buffered saline, pooled, resuspended in 500 mL of Tris buffer (10 mM Tris-HCl, pH 7.4), and sonicated on ice. Cytosolic fractions were obtained by centrifugation at 4 °C for 15 min at 10000g. Aliquots (100 mL) of each sample were then mixed with 100 mL of Tris buffer and 50 mL of a 2% bovine hemoglobin solution (Sigma), heated for 2 min at 100 °C, and centrifuged for 3 min at 10000g. Another 50 mL of hemoglobin solution was added to each tube, and the tubes were heated and centrifuged again. In this context, hemoglobin was used to remove all Cd not associated with MT. The amount of radioactivity in a 100-mL aliquot of the final MT-containing supernatant was measured using a Packard 5650 γ counter set for 109Cd. All samples were assayed in duplicate, and mean values were used to calculate the amount of bound Cd per 1 × 106 cells. Calculations involved multiplying the total amount of radioactivity (cpm) remaining in the final Cd/heme supernatant by the specific activity of the 109Cd solution (cpm/mol of CdCl ) and then dividing by the total 2 number of cells. Experiments were performed in duplicate, and averages were used to estimate the percent Cd released from saturated MT. Preparation of Hepatic MT. Partially purified MT was prepared from the pooled livers of two 6-week-old female Fischer F344 rats (Animal Production Area, FCRDC) which received 10 mmol of CdCl2/kg of body weight delivered subcutaneously in saline 24 h prior to MT preparation. The animals were killed with CO2, and hepatic MT was purified as previously described (23). Briefly, livers were homogenized in ice-cold 20 mM TrisHCl, pH 8.6, containing 250 mM sucrose. The cytosolic fraction was obtained by differential centrifugation (15 min at 9000g followed by 45 min at 100000g) and subsequently heated for 10 min at 85 °C. Heat-stable MT protein was selectively purified using increasing concentrations of (40, 60, 80%) acetone with centrifugation for 15 min at 27000g between each step. The final MT pellet, composed almost exclusively (90%) of MT isoforms I and II (23), was stored under argon at -20 °C until used. Determination of Cd Release from MT in Vitro. Aliquots (100 µL) of partially purified rat liver MT (3.6 mg/mL in Tris buffer) were mixed with 100 µL of 109Cd solution (2.0 µg of CdCl2/ mL; 1.0 µCi/mL in Tris buffer) and allowed to incubate for 10 min at room temperature in 1.5-mL polypropylene microcentrifuge tubes. By using the Cd/heme assay originally described by Onosaka et al. (22) and evaluated by Eaton and Toal (24), we determined that, under the conditions used, all seven metalbinding sites in the MT molecule were saturated with Cd. 109Cd-MT mixtures were subsequently diluted in 1.5 mL of Tris buffer, transferred to individual Centricon 3 concentrators (molecular weight cutoff 3000, Amicon, Inc., Beverly, MA), and centrifuged at room temperature for 1.5 h at 7000g. The initial filtrates were discarded, freshly prepared DEA/NO solutions (in 1.5 mL of Tris buffer) were added to the retenates, and the samples were centrifuged again. Filtrates from the second
328 Chem. Res. Toxicol., Vol. 9, No. 1, 1996 centrifugation step were collected, and the total amount of radioactivity was measured using a Packard 5650 γ counter set for 109Cd. All samples were assayed in duplicate, and mean values were used in the following equation to calculate the amount of Cd displaced per molecule of MT:
mol of Cd displaced/mol of MT )
Misra et al.
A
(Xt - Xo) MCd Co MMT
where,Co is the total cpm of 109Cd added to each sample tube, Xo is the cpm in the filtrate of the control sample (no DEA/NO treatment), Xt is the cpm in the filtrate of a DEA/NO-treated sample, Mcd is the moles of Cd added to each sample tube, and MMT is the moles of MT per sample tube (determined using the Cd/heme assay and by assuming 7 mol of Cd/mol of MT). Assay of Proto-Oncogene Expression. Total cellular RNA was extracted using RNAzol B (Cinna/Biotecx, Inc., Friendswood, TX), according to the manufacturer’s instructions. Each sample was prepared from three individually treated flasks of cells that were pooled prior to extraction. RNA samples were denatured in formaldehyde and c-myc expression was analyzed by slot-blot hybridization and by Northern hybridization after RNA fractionation by agarose-formaldehyde gel electrophoresis. Hybridizations were performed on Nytran membranes (Schleicher and Schuell, Keene, NH) using T4 polynucleotide kinase (United States Biochemical, Cleveland, OH) to 32P-end label an oligonucleotide probe specific for rodent c-myc (Oncogene Science, Uniondale, NY). Under these conditions, c-myc mRNA migrates as a single band approximately 2.2 kb in length. This is consistent with the size of rodent c-myc transcripts as reported by Stanton et al. (25). Prehybridization and hybridization were performed according to the manufacturer’s instructions. A human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe (Clontech, Inc., Palo Alto, CA) labeled by random primer extension using the services of Lofstrand Labs, Ltd. (Gaithersburg, MD), was used for standardization, and prehybridization and hybridization of that probe were performed according to the manufacturer’s instructions. Autoradiograms were prepared using Kodak XOMAT film at -80 °C with intensifying screens, and quantification of autoradiogram band intensities was achieved by scanning densitometry using an LKB Ultroscan XL equipped with GelScan XL software. Determination of NOx Production. The rate of NOx production from DEA/NO was estimated by measuring the formation of S-nitrosothiol adducts of GSH as a function of time (14). For this assay GSH (10 mM in Tris buffer containing 50 mM HEPES, pH 7.4) was exposed to DEA/NO (0.5 mM final concentration) at 37 °C and absorbance changes were monitored at 340 nm using a Hewlett-Packard 8451 spectrophotometer equipped with a UV-visible diode array detector.
Results Effect of NO on Cd-Induced Cytotoxicity in CHOK1 Cells. Two well-established assays were used to determine whether NO had any effect on Cd-induced cytotoxicity. In both cases, growing CHO-K1 cells were treated for 24 h with various concentrations of CdCl2, followed by a 1-h exposure to DEA/NO. As indicated in Figure 1A, DEA/NO enhanced Cd-induced inhibition of colony-forming ability in a dose-related manner. Furthermore, such effects were apparent at DEA/NO concentrations that had little effect on cell death as determined by loss of mitochondrial function (Figure 1B). These results suggest that NO may act by triggering intracellular release of Cd from MT and that the liberated Cd is particularly effective at interfering with the normal proliferation of cells. Effect of NO on Intracellular Release of Cd from MT. In experiments designed to determine whether NO
B
Figure 1. (A) Effect of NO on Cd-induced inhibition of cell colony-forming ability. CHO-K1 cells were seeded at 100, 200, and 500 cells per 100-mm plate, exposed to 0, 0.5, 0.75, or 1.0 µM CdCl2 for 24 h followed by 1-h exposure to 0, 1.0, 2.0, or 3.0 mM DEA/NO, and colonies were allowed to grow for 1 week before counting. Three dishes of each seeding density were assayed per treatment, and averages were used to estimate cytotoxicity. The data shown represent results from cultures seeded at 100 cell/plate. Similar results were obtained at the higher seeding densities. (B) Lack of effect of NO on Cd-induced loss of mitochondrial function. Growing CHO-K1 cells were seeded into 96-well plates, treated with 0, 1.0, or 2.0 µM CdCl2 for 24 h followed by 1-h exposure to 0, 1.0, 1.5, or 2.0 mM DEA/ NO, and then assayed for cell death using a modified version of the MTT assay described in the Materials and Methods. Twentyfour wells were assayed per treatment and averages were used to estimate cell death.
was capable of releasing Cd from MT in cells, we determined that CHO-K1 cells which received 2.0 mM Cd but no DEA/NO contained (7.7 ( 1.2) × 10-13 mol of MT-bound Cd/106 cells. In comparison, cultures that received 2.0 mM Cd followed 24 h later by 1.5 or 3.0 mM DEA/NO contained (5.5 ( 1.4) × 10-13 and (4.8 ( 1.0) × 10-13 mol of MT-bound Cd/106 cells, respectively. These values represent 29 and 38% reductions in the amount of MT-bound Cd in NO-treated cells, indicating that NO is indeed capable of releasing Cd in vivo. Effect of NO on Cd Release from Purified MT. In experiments designed to further characterize the kinetics
NO Enhancement of Cd Toxicity
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Rate of NOx Production by DEA/NO. Using an in vitro assay to estimate the rate of NOx production from DEA/NO, we determined that NOx and subsequent Snitrosoglutathione formation occurs extremely rapidly. As illustrated in Figure 4, maximum levels of S-nitrosoglutathione were achieved less than 300 s after DEA/ NO exposure.
Discussion
Figure 2. Effect of NO on the release of Cd from purified MT. MT was purified from rat liver, all seven protein-binding sites were saturated with excess Cd, and Cd-MT complexes were subsequently exposed to 0, 0.1, 0.5, 1.0, 1.5, 2.0, 3.0, or 10 mM DEA/NO. Samples were assayed in duplicate, and the means were used to calculate moles of Cd displaced per mole of MT using the equation described in the Materials and Methods. Error bars represent standard deviation between replicate experiments.
of NO-induced release of Cd from MT in vitro, a sharp dose-related increase in Cd displacement was observed at DEA/NO concentrations between 0.1 and 0.5 mM; a plateau at 3 mol of Cd displaced/mol of MT was reached at higher DEA/NO concentrations (Figure 2). Analysis of MT samples treated with DEA/NO that had been allowed to degrade to diethylamine and nitrite for 16 h at 25 °C prior to protein exposure indicated that NO breakdown products were not responsible for triggering the release of Cd from MT, and analysis of samples that received fresh DEA/NO 20 h prior to centrifugation revealed no evidence of Cd-MT reassociation (data not shown). Effect of NO on Cd-Induced Perturbations in Proto-Oncogene Expression. Slot-blot and Northern hybridization analyses of mRNA from cells treated with Cd plus DEA/NO were performed to further characterize the impact of NO on Cd exposure. In these experiments, perturbations in c-myc expression were used as a sensitive indicator of Cd genotoxicity, and proto-oncogene expression was standardized against levels of GAPDHmRNA, a gene whose expression is known to be refractory to the effects of a wide variety of genotoxic agents. As illustrated in Figure 3A-C, compared to controls, cells that received 1.0 µM CdCl2 for 24 h followed by 1-h exposure to 1.0 mM DEA/NO exhibited 2-3-fold decreases in c-myc expression. In addition, analysis of samples treated with DEA/NO that had been allowed to degrade to diethylamine and nitrite prior to protein exposure indicated that NO breakdown products were not responsible for the decreased c-myc expression (data not shown). In another experiment where the effects of exposure to Cd followed by DEA/NO were monitored over time (0.5, 1, 2, and 4 h after DEA/NO treatment), a boost in c-myc expression (approximately 2-fold) occurred 4 h after DEA/NO administration (Figure 3D). These results lend additional support to the hypothesis that NO enhances Cd toxicity by displacing the metal from MT and that the mechanism underlying Cd toxicity involves DNA damage and subsequent dysregulation of cell proliferation.
In the present study, we demonstrated that NO triggers the release of Cd from MT in vitro and provided evidence that NO is capable of releasing Cd from MT in cells. We have also shown that NO enhances Cd-induced inhibition of cell growth in a dose-related manner at concentrations that have little effect on cytolethality and that sequential exposure to Cd and NO causes a significant, but transient, decrease in c-myc proto-oncogene expression. Taken together these results suggest that NO can mediate the release of Cd from MT in vivo and in turn generate increased concentrations of free intracellular Cd which may induce DNA damage and force cells into a period of growth arrest. The mechanism by which NO mediates the release of Cd from MT appears to involve NOx, a presumed isomer of N2O3 derived from the NO/O2 reaction, (26) which preferentially reacts with thiol moieties (10). As shown by Walker et al. (27), acute exposures to NO under aerobic conditions can lead to a reduction of intracellular GSH levels, presumably through formation of S-nitrosoglutathione. Nitrosation of intracellular GSH by NOx is thought to play a key role in detoxication of this reactive intermediate (10, 27). Further support for thiol residues protecting against NOx toxicity was recently demonstrated using a cell line that overexpresses MT (28). In that study, the authors concluded that thiol groups in MT efficiently scavenge NOx and thereby reduce cytotoxicity. In this context it is quite conceivable that NOx could nitrosate cysteine residues coordinated to Cd, resulting in the formation of S-nitrosothiol adducts and the release of Cd from MT. While such an occurrence would indeed result in the detoxification of NOx, it would simultaneously “activate” Cd, posing a new toxicological threat to cells. The detection of RSNO by Kroncke et al. using Raman techniques (17) along with the release of Cd described in the present study strongly supports such a mechanism. In spite of the fact that each mammalian MT molecule can bind up to seven divalent metal ions (4), results from the present study revealed that NO is capable of displacing only three Cd ions per molecule of MT in vitro (Figure 2). Similar results were also obtained in cells. A possible explanation for this finding could be related to differences in the binding affinities of the two protein domains that comprise MT. Studies on the order of metal binding have shown that the COOH terminal R domain of MT becomes filled with four metal ions before filling of the NH2 terminal β domain begins (29). NO may be capable of releasing Cd from the β domain but may not be capable of interfering with higher affinity metal binding to the R domain. A model illustrating the three-dimensional structure of MT and the mechanisms by which NO might displace Cd from MT is presented (Figure 5). NO concentrations ranging from 10 to 50 µM can be achieved in the presence of 1-2 mM DEA/NO as deter-
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Figure 3. (A, upper left) Northern hybridization after RNA fractionation by agarose-formaldehyde gel electrophoresis using a 32Pend-labeled oligonucleotide probe specific for rodent c-myc. RNA was prepared from cells treated for 24 h with 1.0 µM CdCl2 followed by 1-h exposure to 1.0 mM DEA/NO. Control samples included RNA from untreated cells and cells that received Cd or DEA/NO alone. (B, lower left) Slot-blot analysis of c-myc expression in cells treated for 24 h with 1.0 µM CdCl2 followed by 1-h exposure to 1.0 mM DEA/NO. Control samples included RNA from untreated cells and cells that received Cd or DEA/NO alone. (C, upper right) Results from slot-blot analysis of c-myc expression in cells treated for 24 h with 1 mM CdCl2 followed by 1-h exposure to 1 mM DEA/NO. The data was normalized against GAPDH mRNA. (D, lower right) Results from slot-blot analysis of c-myc expression in cells treated for 24 h with 1.0 mM Cd Cl2followed by exposure to 1.0 mM DEA/NO for varying lengths of time. The data were normalized against GAPDH mRNA.
mined by electrochemical methods (30). However, such high concentrations would be expected only in the proximity of cells containing induced levels of nitric oxide synthase associated with cytokine activation. Nitrosation of thiols and amines occurs by reactive chemical intermediates such as those formed during the NO/O2 reaction. Because of the second-order nature of this reaction and competing pathways of biological consumption (31, 32), low concentrations of NO such as those expected for normal regulatory function do not produce reactive intermediates in vivo. At higher concentrations, such as those expected during the inflammatory immune response, such reactions can occur (32). Originally it was shown that activated macrophages could nitrosate amines in vitro (33). Later, Liu et al. showed that amines became nitrosated in woodchucks infected with hepatitis (34). Further evidence of nitrosation in vivo was demonstrated by Gaston et al. (35); in that report, pulmonary inflammation and exposure to NO gas resulted in the formation of S-nitrosothiol-peptide adducts. Thiol-nitrosated proteins have also been observed in vivo, supporting the notion that RSNO adducts are indeed formed (36). On
the basis of these observations, it is conceivable that production of NO by immune cells or exogenous exposure to NO could result in nitrosation of thiol groups in the MT protein and thereby stimulate the release of toxic metals from MT. The correlation between the amount of DEA/NO required for Cd release and that required to inhibit the activity of DNA repair enzymes is intriguing. Approximately 0.1 mM DEA/NO is required to release 50% of the Cd ultimately released from MT in vitro, and at 0.5 mM DEA/NO, little if any further release of Cd from MT was observed (Figure 2). Similar results were obtained with O6-alkylguanine DNA alkyl transferase and Fpg protein, where exposure to 0.1 mM DEA/NO inhibited 50% of activity in vitro (13, 14). The requirement for higher DEA/NO concentrations in cells may be explained by the cells’ ablility to scavenge NOx via GSH. Although the carcinogenic potential of Cd is widely accepted, the mechanisms underlying Cd genotoxicity remain poorly understood. At present three different hypotheses exist: (1) Cd may interact directly with chromatin to induce strand breaks, cross-links, or conformational changes in DNA; (2) Cd may act indirectly by inhibiting various proteins involved in DNA replica-
NO Enhancement of Cd Toxicity
Figure 4. Time course of S-nitrosoglutathione formation from DEA/NO. Glutathione (10 mM) was combined with DEA/NO (0.5 mM) at 37 °C. Absorbance changes were monitored at 340 nm for 30 min.
Figure 5. Proposed mechanism by which NO displaces Cd from MT. This model illustrates complete saturation at four Cd ions per R domain and three Cd ions per β domain of the MT molecule. Cd release from multiple β domain sites may occur by successive nitrosation of thiol groups or after intramolecular disulfide formation or conformational changes following initial NO-induced Cd displacement.
tion and repair (including a number of proteins containing zinc-finger motifs); or (3) Cd may act by catalyzing cellular redox reactions whose byproducts subsequently produce damage in proteins or DNA. At first glance our results appear to support the position that sublethal concentrations of Cd are particularly effective at inhibiting proteins involved in DNA replication/repair. However, inhibition of cell growth could also be a sensitive indicator of direct damage to the genetic material.
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Others have reported that relatively low doses of Cd cause rapid induction of DNA damage-inducible genes such as gadd153 (37) and c-myc (38, 39) and that exposure to higher concentrations of Cd results in dramatic reductions in c-myc expression (38). An association between altered expression of c-myc, direct DNA damage, and growth arrest is also well established (4043) and is consistent with the present findings. Occupational exposure to Cd occurs mainly in the form of airborne dust and fumes (1). Cigarette smoke represents another important source of Cd exposure, and inhalation of the metal has been linked to chronic obstructive airway disease and pulmonary carcinogenesis (1). In this context, our results indicate that coexposure to NO (e.g., as a consequence of pulmonary inflammation or inhalation of contaminated air or cigarette smoke) could trigger release of Cd ions from MT within lung cells and thereby play an important role in augmenting the toxic and/or carcinogenic effects of Cd in human populations.
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