Effects of Cadmium(II) - American Chemical Society

Feb 14, 2004 - New York College at Buffalo, 1300 Elmwood Avenue, Buffalo, New York 14222. Received November 7, 2003. Cadmium, a widespread ...
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Chem. Res. Toxicol. 2004, 17, 287-293

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Effects of Cadmium(II) on (()-anti-Benzo[a]pyrene-7,8-diol-9,10-epoxide-Induced DNA Damage Response in Human Fibroblasts and DNA Repair: A Possible Mechanism of Cadmium’s Cogenotoxicity Jagat J. Mukherjee, Suresh K. Gupta, Subodh Kumar, and Harish C. Sikka* Environmental Toxicology and Chemistry Laboratory, Great Lakes Center, State University of New York College at Buffalo, 1300 Elmwood Avenue, Buffalo, New York 14222 Received November 7, 2003

Cadmium, a widespread environmental pollutant and a cigarette smoke constituent, enhances the genotoxicity of benzo[a]pyrene (BP). The mechanism(s) underlying the potentiation of BPinduced genotoxicity by Cd2+ is not clearly understood. Our studies of the effects of noncytotoxic concentrations of Cd2+ on the levels of p53 and p21 in (()-anti-benzo[a]pyrene-7,8-diol-9,10epoxide (BPDE)-treated human fibroblasts showed that Cd2+ decreased BPDE-induced p21 levels in a dose-dependent manner whereas p53 accumulation is attenuated only at higher noncytotoxic concentrations of cadmium. These findings suggest that both the activity and the accumulation of p53 in response of BPDE treatment are inhibited by Cd2+ although the possibility of p53-independent p21 transactivation cannot be ruled out. Exposure of synchronized human fibroblast cells to 0.5 µM of BPDE caused 72% of the cells remaining in G1 phase as compared to 52% in the case of untreated cells. Treatment of the cells with CdCl2 prior to exposing them to BPDE caused a decrease in the G1 population (72 to 54%) in a dose-dependent manner. An in vitro repair assay of BPDE-damaged pUC18 plasmid DNA using untreated and cadmium-treated nucleotide excision repair (NER) proficient HeLa extract showed that cadmium impaired the ability of HeLa cell extract to repair BPDE-damaged pUC18 DNA. Our findings indicate that cadmium not only inhibits NER pathway-dependent repair of BPDEdamaged DNA but also impairs p53 and p21 responses and overrides BPDE-induced G1-S cell cycle arrest. The effect of cadmium on these processes may explain, at least partly, the potentiating effect of the metal on the genotoxicity of BP.

Introduction PAHs1 and the heavy metals such as cadmium are widespread environmental pollutants (1-7). They are also among the major constituents of tobacco smoke. Despite the simultaneous occurrence of PAHs and metals in the environment and in tobacco smoke, only limited information is available on the interaction of these chemicals in biological systems. Cadmium, while only weakly mutagenic by itself, produced a synergistic enhancement in the frequency of morphological transformation in mammalian cells in combination with BP, a potent mutagenic/carcinogenic PAH (8). The mechanism underlying the potentiation of BP-induced genotoxicity by cadmium is not known. A likely mechanism by which cadmium exerts its potentiating effect on the genotoicity of BP is that the metal interferes with the BP-induced DNA damage response and that it inhibits the repair of BP-induced DNA damage. To exert its carcinogenic effect, BP is first metabolically activated by the cytochrome P-450-dependent monooxy* To whom correspondence should be addressed. Tel: 716-878-5422. E-mail: [email protected]. 1 Abbreviations: BP, benzo[a]pyrene; BPDE, (()-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide; PAH, polynuclear aromatic hydrocarbon; NER, nucleotide excision repair; [R-32P]dCTP, [R-32P]-2′-deoxycytidine 5′-triphosphate; dG, 2′-deoxyguanosine; DTT, 1,4-dithio-DL-threitol; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid.

genase system preferentially to BPDE, a reactive electrophile that binds to cellular DNA predominantly at the N2 position of dG and is implicated as the ultimate carcinogenic metabolite of BP (9). DNA adduct formation is known to be the initial stage in BP-induced carcinogenesis (10). Once the DNA is damaged by BPDE-DNA adduct formation, a cascade of cellular responses to DNA damage is initiated to protect the cells from the detrimental effect of DNA damage. An important component of cellular response to DNA damage is the induction of p53 protein (11, 12). There is considerable evidence that BP and other PAHs induce p53 in a number of animal or cell systems (13, 14) and that the increase in cellular p53 protein levels in cells exposed to various genotoxic agents is due mainly to an increase in p53 protein stability rather than to an increase in steady state p53 mRNA levels (11, 12). Wildtype p53 gene product plays an important role in growth arrest, DNA repair, and apoptosis (15-18). p53 induced in response to DNA damage may transcriptionally activate various target genes, such as the cyclin-dependent kinase inhibitor p21WAF1, which causes subsequent cell cycle arrest in G1 (19-21). Agents that override the G1-S checkpoint could dramatically enhance genomic instability in response to other DNA-damaging agents.

10.1021/tx034229e CCC: $27.50 © 2004 American Chemical Society Published on Web 02/14/2004

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Physical and chemical damage to DNA in prokaryotes and eukaryotes can be removed by different repair pathways. NER is the major repair system that is capable of removing a remarkably broad spectrum of bulky DNA lesions, with varying degrees of efficiency (22). These include UV-induced photoproducts (cyclobutane pyrimidine dimers and 6-4 products), a variety of bulky chemical adducts formed by mutagenic/carcinogenic chemicals, and both interstrand and intrastrand cross-links. In Escherichia coli and mammalian systems, BP adducts can be repaired by NER (23, 24). p53 can act as a transcriptional activator of repair-related genes such as gadd45 and associate with NER proteins XPB, XPD, and CSB in vitro and in vivo (25, 26) although in some instances the role of p53 in the repair of damaged DNA is less prominent (27, 28). Because NER is an important defense mechanism by which the cells protect themselves from the mutational fixation, inhibition of NER may promote mutagenesis/tumorigenesis. Cadmium is known to interfere with the repair of UV-induced DNA damage (29, 30). Most of the information on the mechanism underlying the inhibition of DNA repair by metals is limited to UV-induced damage. It was observed that (31, 32) cadmium at low, noncytotoxic concentrations interferes with the UV-induced DNA damage recognition step of NER. It is also suggested that cadmium may induce conformational change of wild-type p53 (33). To gain an insight into the molecular mechanism by which cadmium potentiates BP-induced genotoxicity, we examined the effects of the metal on p53 induction and cell cycle arrest in (()-BPDE-treated human fibroblast cell cultures as well as its effect on in vitro repair of (()BPDE-damaged DNA. We have used human fibroblasts that have wild-type p53 and downstream genes. Our results demonstrate that cadmium at noncytotoxic levels impairs p53 induction, inhibits p21 transactivation, overrides G1 cell cycle arrest in response to (()-BPDE, and interferes with in vitro repair of (()-BPDE-damaged DNA.

Materials and Methods Chemicals and Reagents. Modified Eagle’s medium (MEM), fetal bovine serum (FBS), l-glutamine, and trypsin-EDTA were purchased from Invitrogen Life Technologies (CA); acrylamide, bis-acrylamide, TEMED, propidium iodide, and pUC18 plasmid were purchased from Sigma Chemical Co. (MO); JM109 competent cells were purchased from Promega. Rabbit polyclonal antibodies against p53 (FL-393) and p21 (C-19) were obtained from Santa Cruz (CA). [R-32P]dCTP (3000Ci/mmol) was purchased from Amersham Biosciences (NJ). (()-anti-BPDE was purchased from the NCI Chemical Carcinogen Reference Standard Repository. All other chemicals were of analytical grade. Cell Culture, Treatments, and Preparation of Cell Extract. Human fibroblasts in MEM were supplemented with 15% FBS, nonessential amino acids, and pyruvate in an atmosphere of 5% CO2 in air at 37 °C and 85% humidity. The cells were checked on a routine basis for Mycoplasma contamination by the Gibco Mycotect. The cells in mid-log growth were treated with 0.5 µM (()-anti-BPDE (unless mentioned otherwise) dissolved in DMSO (0.05-0.1% of the culture volume) for 90 min in serum-free medium and further incubated in 15% serum for 16 h in the absence of (()-anti-BPDE. For treatment with cadmium, the cells in mid-log growth were treated with different concentrations of cadmium chloride below the cytotoxic level (100 µM) for 4 h prior to 90 min of treatment with (()anti-BPDE. After 16 h, the cells were washed three times with ice cold PBS, scrapped gently, and lysed in 100 µL of a buffer

Mukherjee et al. consisting of 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM EDTA, 10 µg/mL pepstatin, 1 mM phenylmethanesulfonyl fluoride, 5 µg/mL leupeptin, and 100 µg/mL aprotinin (Sigma). The supernatant collected after centrifugation (14 000 rpm for 20 min at 4 °C) was either used immediately or flash-frozen in liquid nitrogen and stored at -86 °C for subsequent analyses of p53 and p21 expressions. Cytotoxicity Measurement. For the cadmium toxicity assay, the cells at the exponential growth phase were treated with different concentrations (0-2 mM) of CdCl2 for 4 h followed by washing with MEM to remove the extracellular cadmium. The cells were further incubated with MEM containing 15% FBS/2 mM glutamine for 24 h and then processed for the determination of viable cells using CellTiter 96R AQueous One solution (Promega) in a 96 well plate reader according to the procedure described by the manufacturer (Promega Technical Bulletin No. 245). Five data points were obtained at each CdCl2 concentration and then averaged. The cytotoxicity of cadmium was also determined by trypan blue staining (0.9% NaCl-0.5% trypan blue) of the cells. The number of viable cells was calculated from the ratio of unstained to the total number of cells. Immunoblotting of p53 and p21. An equal amount of the cell extract (75 µg protein) was separated by 12% SDS-PAGE and electroblotted onto Immobilon-P (Millipore) membrane. After the proteins were transferred, the membrane was blocked in 5% skim milk powder. For p53 detection, the membrane was incubated with rabbit polyclonal antibody p53 (FL-393; Santa Cruz) at a concentration of 0.7 µg antibody/ml solution. Rabbit polyclonal antibody p21 (C-19; Santa Cruz) at a concentration of 0.6 µg antibody/ml solution was used for the detection of p21WAF1 protein. Goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma) was used as a secondary antibody to detect the proteins. The proteins were visualized by enhanced chemiluminescence using Amersham’s ECL Western Blotting Detection Reagents (Amersham Biosciences). Cell Synchronization, Treatment, and Cell Cycle Analysis. The cells were synchronized by holding the confluency for 3 days to bring them to the G0 phase as described for the synchronization of human fibroblast cells (34). The cells were split in a 1:5 ratio and incubated for 18 h to allow the cells to re-enter the G1 phase. The cells were then treated with different noncytotoxic concentrations of cadmium chloride (0-80 µM) for 4 h followed by washing with fresh medium and incubated in the absence or presence of 0.5 µM (()-anti-BPDE in serum-free medium for 90 min. The cells were further incubated for 24 h in 15% serum-containing MEM, harvested by trypsinization, fixed in 70% ethanol, and subjected to cell cycle analysis using the propidium iodide staining method (35). The phases of growing cells were analyzed with a Becton Dickinson FACScan instrument using Winlist (Verity Software House) and Multicycle for Windows (Phoenix Flow Systems) software systems. Preparation of BPDE-Modified DNA. Plasmid pUC18 DNA was propagated in JM109 competent cells and purified by using Qiagen plasmid DNA purification kit (Qiagen Inc., CA)as described by the manufacturer. Damaged pUC18 DNA was obtained according to the previously described procedure (36) with modifications. Briefly, 50 µg of pUC18 DNA was incubated at 37 °C for 3 h in the dark in a 500 µL reaction mixture containing TE buffer (10 mM tris HCl, pH 7.5, 1 mM EDTA), 20% ethanol, and 10 µM (()-anti-BPDE. The modified DNA was purified by repeated extractions with 2 vol of ethyl acetate followed by a single extraction with ethyl ether (37). The (()anti-BPDE-modified DNA was precipitated by adding 0.7 vol of 2-propanol at room temperature followed by centrifugation at 15 000g for 30 min at 4 °C. The precipitated DNA was washed with 70% ethanol, air-dried briefly, and resuspended in 100 µL TE buffer, pH 7.6. pUC18 plasmid DNA without (()-anti-BPDE treatment served as control. Preparation of HeLa Cell Extracts. HeLa cell extracts were prepared according to the procedure described by Manley et al. (38). Briefly, HeLa cells were either untreated or treated

Effect of Cadmium on Cellular Response to BPDE

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Figure 2. Dose-dependent accumulation of p53 protein by (()anti-BPDE in GM03349 fibroblast. Cells were either untreated (lane 1) or treated with 0.1 (lane 2), 0.5 (lane 3), and 1.0 µM (lane 4) of (()-anti-BPDE, respectively, for 90 min, and after 16 h, the levels of p53 were measured by western immunoblot.

Figure 1. Effect of cadmium chloride on the survival of normal human GM03349 fibroblast. The number of viable cells at each concentration of cadmium chloride was monitored colorimetrically at 490 nm in a 96 well plate reader as described in the Materials and Methods. All values were from the mean of five samples. with 5 µM cadmium chloride for 2 h before lysis. The cell suspension was centrifuged at 1900 rpm for 5 min, washed with PBS, and then disrupted in a Dounce homogenizer in hypotonic buffer containing 0.01 M Tris-HCl (pH 7.9), 1 mM EDTA, and 5 mM DTT. After addition of 50 mM Tris-HCl (pH 7.9) containing 10 mM MgCl2, 2 mM DTT, 25% sucrose, and 50% glycerol, the protein was precipitated in the presence of 10% ammonium sulfate and discarded. The proteins in the supernatant were further precipitated with ammonium sulfate (0.33 g/mL), and the pelleted proteins were dissolved in dialysis buffer (25 mM HEPES, pH 7.9, containing 100 mM KCl, 12 mM MgCl2, 0.5 mM EDTA, 2 mM DTT, and 17% glycerol). The extracts were dialyzed against two changes of 50-100 vol each of the dialysis buffer for 8-12 h. The precipitate formed after dialysis was removed by centrifugation at 10 000g for 10 min, and the supernatant was used for repair synthesis. In Vitro DNA Repair Assay. The DNA repair assay was performed according to the procedure described previously (36). Briefly, the assay was carried out in a total volume of 50 µL of reaction mixture containing 45 mM HEPES-KOH buffer (pH 7.8), 7.4 mM MgCl2, 0.9 mM DTT, 0.4 mM EDTA, 200 ng each of damaged pUC18 DNA, 2 mM ATP, 20 µM each dATP, dGTP, and dTTP, 4 µM dCTP, 1 µCi [R-32P]dCTP (3000 Ci/mmol), 40 mM phosphocreatine (disodium salt), 2.5 µg of creatine phosphokinase, 4% glycerol, 100 µg/mL bovine serum albumin, and cell-free HeLa extract (150 µg of protein). The reaction was carried out at 30 °C for 2 h and then stopped by adding EDTA and RNase A to a final concentration of 20 mM and 20 µg/mL, respectively. The reaction mixture was then incubated at 37 °C for 10 min followed by further incubation at 37 °C for 30 min in the presence of 0.5% SDS (final) and 200 µg/mL proteinase K (final). The DNA was then extracted with phenol/chloroform: isoamyl alcohol and precipitated with ethanol. A control reaction was performed simultaneously in the same way using undamaged pUC18 DNA. The repair products were digested with Hind III and separated by electrophoresis on a 1.5% agarose gel containing ethidium bromide. The DNA bands were visualized by UV light, and the repair synthesis was assessed by autoradiography of the dried gel.

Results Cytotoxicity of Cadmium Chloride to Human Fibroblast Cells. The effect of cadmium chloride on the survival of GM03349 fibroblasts is shown in Figure 1. Treatment of cells with cadmium chloride up to 100 µM for 4 h did not cause any significant cell death (88-90% survival) as compared to the untreated cells. Cad-

Figure 3. Effect of cadmium chloride on p53 protein accumulation in (()-anti-BPDE-treated human fibroblast. Cells were pretreated for 4 h with noncytotoxic concentrations of cadmium chloride (20-70 µM, lanes 3-7) followed by treatment with 0.5 µM (()-anti-BPDE (lanes 2-7) for 90 min. Control cells did not get any treatment (lane 1). After 16 h, p53 protein was detected by western analyses as described in the Materials and Methods.

mium(II) at a concentration of 200 µM was significantly toxic to the cells. Trypan Blue Exclusion data also showed a similar effect of cadmium(II) on the survival of fibroblasts (data not shown). Noncytotoxic cadmium chloride concentrations below 100 µM were used in the subsequent experiments. Effect of Cadmium on (()-anti-BPDE-Induced Expression of p53 and p21 Proteins in Human Fibroblasts. BPDE, an ultimate carcinogenic metabolite of BP, causes DNA damage by forming a BPDE-DNA adduct, which elicits a response of p53 induction. To assess the dose-dependent effect of BPDE on the p53 level, the cells were treated with increasing doses of (()anti-BPDE for 90 min. After 16 h, a dose-dependent increase in the induction of p53 protein was observed (Figure 2). Even at very low concentrations of (()-antiBPDE (0.1 µM), a significant increase in the level of p53 protein was observed as compared to the untreated cells. On the contrary, the fibroblasts treated with 1-10 µM concentrations of the parent compound BP showed an extremely low induction of p53 protein even at a 10 µM dose of BP (data not shown). This is probably due to the inability of human fibroblasts to metabolize BP to BPDE (39). Therefore, instead of BP, we used (()-anti-BPDE for treatment of the cells throughout our studies. Figure 3 shows the effect of cadmium on (()-antiBPDE-induced p53 accumulation. Cadmium chloride up to a concentration of 40 µM had practically no effect on the level of p53 accumulation in response to (()-antiBPDE. However, a significant reduction of p53 accumulation was observed when the cells were pretreated with 50 µM or higher cadmium chloride but below the cytotoxic level. More than 60% attenuation of p53 accumulation was observed in the cells treated with 70 µM cadmium chloride (determined by measuring the band intensity using Inotech software). This experiment has been repeated three times, and each time, the same trend of the effect of cadmium chloride on (()-anti-BPDEinduced p53 accumulation was observed.

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Figure 4. Effect of cadmium chloride on p21WAF1 protein accumulation in (()-anti-BPDE-treated human fibroblast. Cells were pretreated for 4 h with noncytotoxic concentrations of cadmium chloride (20-70 µM) followed by treatment with 0.5 µM (()-anti-BPDE for 90 min. After 16 h, p21WAF1 protein was detected by western analyses as described in the Materials and Methods.

One major function of p53 protein induced in response of DNA damage is the transactivation of p21 gene (19). p21 protein inactivates the cyclin-CDK complex resulting in cell cycle arrest (40). Figure 4 shows the effect of cadmium on (()-anti-BPDE-induced p21 response. The p21 level was increased significantly when the fibroblasts were treated with 0.5 µM (()-anti-BPDE. Pretreatment of the cells with varying noncytotoxic concentrations of cadmium chloride (20-70 µM) attenuated p21 protein levels induced in response to (()-anti-BPDE. This experiment was repeated three times, and each time, the same trend was observed. Most interestingly, induction of p21 protein in response to (()-anti-BPDE treatment is significantly reduced even at 20-40 µM cadmium chloride, the concentration range at which there is practically no effect of cadmium on (()-anti-BPDE-induced p53 accumulation (Figure 3). At higher concentrations of cadmium chloride (50-70 µM), both p53 and p21 protein levels are reduced significantly (Figures 3 and 4). The present results suggest that cadmium affects the cellular response to (()-anti-BPDE-induced DNA damage by inhibiting p53 accumulation as well as by interfering with p53 function in relation to transactivation of p21. Effect of Cadmium on Cell Cycle Distribution. Because cadmium attenuated (()-anti-BPDE-induced the p21 level in the human fibroblasts, we were interested in finding out whether the metal has any effect on (()anti-BPDE-induced cell cycle arrest in these cells. To examine this effect, we treated the synchronized cells first with different concentrations of cadmium chloride (2080 µM) for 4 h and then with 0.5 µM (()-anti-BPDE for 2 h. The cells were harvested after 28 h and processed for determining their relative distribution in G1, S, and G2 phases, as described earlier. Figure 5 is the representative of three different experiments performed to study the cell cycle profiles obtained 28 h after the treatment of fibroblasts with (()-anti-BPDE in the absence and presence of different noncytotoxic concentrations of cadmium chloride. Table 1 summarizes the data obtained from Figure 5. Upon treatment with 0.5 µM (()anti-BPDE, the percentage of cells that remained in G1 phase increased from 52 (control) to 72% (Figure 5 and Table 1), indicating arrest in the G1 phase of the cell cycle. The percentage of cells in S phase decreased from 32 to 23%. However, the cell population in G2 phase also decreased from 17 to 5%. Pretreatment of the cells with different noncytoxic concentrations of cadmium chloride showed a gradual abrogation of G1 arrest caused by (()anti-BPDE. At 80 µM Cd2+, the percentage of cells in G1 phase returned almost to the control level accompanied by an increase of cell population in the S phase. These results suggest that (()-anti-BPDE causes G1/S cell cycle arrest in human fibroblasts, and this arrest is overridden in a dose-dependent manner by noncytotoxic concentra-

Figure 5. Cell cycle profile showing the percentage of GM03349 fibroblasts in different phases of cell cycle, 24 h after treatment with (()-anti-BPDE. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. Statistically, this cell cycle profile is the representative of three different experiments, which show the same trend of the effect of cadmium chloride on (()-anti-BPDE-induced G1 arrest. Table 1. Effect of Cadmium on (()-anti-BPDE-Induced G1 Cell Cycle Arrest in GM03349 Fibroblasta,b treatment

% G1

%S

% G2

control (no treatment) BPDE (0.5 µM) CdCl2 (20 µM) + BPDE (0.5 µM) CdCl2 (40 µM) + BPDE (0.5 µM) CdCl2 (60 µM) + BPDE (0.5 µM) CdCl2 (80 µM) + BPDE (0.5 µM)

52 72 66 63 57 54

32 23 20 21 33 27

17 5 14 16 10 19

a Table 1 represents the data from Figure 5, which is the representative profile of three different experiments. b The synchronized human fibroblast cells were treated with different concentrations of cadmium chloride followed by treatment with (()-anti-BPDE, and each cell sample was subjected to cell cycle analysis by flow cytometry as described in the Materials and Methods.

tions of cadmium chloride. The overriding effect of cell cycle arrest by cadmium is observed even at a low concentration of the metal (20 µM). This is in correspondence with the observation that at low cadmium chloride concentration (20 µM), (()-anti-BPDE-induced p21 protein level is also attenuated (Figure 4). Effect of Cadmium on the Repair of DNA Lesions Produced by (()-anti-BPDE. It is well-known that anti-BPDE-induced DNA damage is repaired by NER (23, 24). Because cadmium may inactivate the DNA repair proteins by binding to the protein -SH groups, we were further interested to know whether cadmium has any effect on the repair of (()-anti-BPDE-adducted DNA. Wood et al. (42) developed a human cell-free system for DNA repair, which also faithfully reflects in vivo DNA repair properties. Employing this in vitro system, we examined the effect of cadmium on the NER proficient human HeLa cell extract-mediated repair of (()-antiBPDE-adducted pUC18 plasmid DNA. The rationales for using HeLa cell extract include (i) NER proficiency of HeLa cells and (ii) availability of sufficient quantity of proteins required for repair assay. The preparation of (()anti-BPDE-damaged pUC18 plasmid DNA and the conditions of the repair reaction were described earlier.

Effect of Cadmium on Cellular Response to BPDE

Figure 6. Effect of cadmium chloride on in vitro repair of (()anti-BPDE-damaged pUC18 plasmid DNA. NER was performed using human cell-free extracts (90 µg) prepared from untreated and 5 µM cadmium chloride-treated NER proficient HeLa cells, respectively. pUC18 DNA was damaged with 10 µM (()-antiBPDE and purified by solvent extractions and precipitation with 2-propanol as described in the Materials and Methods. Undamaged pUC18 DNA (lane 1); (()-anti-BPDE-damaged pUC18 DNA (lanes 2-5). (Top) Ethidium bromide stained gel; (bottom) autoradiograph showing incorporation of [R-32P]dCTP as a measure of in vitro repair synthesis.

Following the incorporation of radiolabeled [R-32P]CMP into damaged plasmid DNA, excision repair was measured during repair synthesis catalyzed by HeLa cell extract. After the (()-anti-BPDE-damaged pUC18 plasmid DNA was incubated with 90 µg of human HeLa cell extract, repair synthesis was prominent (Figure 6, lane 2), whereas in control no repair synthesis was observed (lane 1). When the repair reaction was carried out in the presence of the cell extract obtained from HeLa cells treated with 5 µM cadmium chloride, a significant decrease in repair synthesis was observed (lane 3) as compared to that in the presence of untreated HeLa cell extract. When the repair reaction was performed using a mixture of different proportions of untreated and cadmium chloride-treated HeLa cell extracts keeping the total amount of protein constant (90 µg), a gradual increase in the rate of repair synthesis with increasing proportions of untreated HeLa cell extract was observed (lanes 4 and 5). The experiment was performed three times, and the same trend was observed in each time. These results indicate that cadmium interferes with the repair of (()-anti-BPDE-induced DNA damage.

Discussion In this investigation, we examined the effect of cadmium on the cellular responses induced by BPDE in order to obtain an insight into the mechanism by which the metal potentiates the mutagenicity and the cell transformation ability of BP, a potent mutagenic/ carcinogenic PAH. An important component of cellular response to DNA damage by genotoxic agents is the induction of p53 (11, 12). Our results show that BPDE induces p53 protein in human fibroblasts in a dosedependent manner and that cadmium attenuates BPDEinduced p53 accumulation at noncytotoxic concentrations (50-70 µM). Down-regulation of p53 accumulation in response to DNA damage has been observed with tumor promoters (43, 44). Because p53 plays an important role in the protection of cells from the detrimental effects caused by DNA-damaging agents, the attenuation of BPDE-induced p53 accumulation by cadmium may rep-

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resent a mechanism by which the metal potentiates BPinduced genotoxicity. p53 response to genotoxic stress involves activation of its ability to transactivate target genes. The most important of the transactivating genes in terms of cell cycle arrest is p21WAF1/CIP1 (p21), an inhibitor of the cyclinD1/cdk4/6-depenedent phoshorylation of pRB (19, 41). DNA damage-induced p53 accumulation without functionally active p53 protein is detrimental to a cell’s ability to protect itself from genotoxic stress. We observed that 0.5 µM BPDE induces p53 as well as p21 proteins in human fibroblasts and that cadmium attenuates this p21 response at noncytotoxic concentrations (20-70 µM) (Figures 3 and 4). Interestingly, the levels of attenuation of p21 and p53 responses do not follow the similar trend at all concentrations of cadmium tested. Although cadmium at a low concentration (20-40 µM) significantly reduces the p21 protein level, there was no effect of the metal on BPDE-induced p53 accumulation at this concentration range. However, cadmium chloride at concentrations ranging from 50 to 70 µM had an inhibitory effect on both p21 and p53 protein levels. These results suggest that one of the possible mechanisms by which cadmium potentiates the genotoxicity of BP is the attenuation of BPDE-induced p53 response by inhibiting both p53 accumulation (only at higher concentrations below cytotoxic level) and the ability of p53 to transactivate p21, although the possibility of p53-independent p21 transactivation as evident in other instances (20, 21) cannot be ruled out. p53 accumulation in response to DNA damage can happen either by transcriptionl activation of p53 gene by NFkappaB or by increased stability of p53 protein (45-47). Studies of the effect of cadmium on these two aspects of p53 regulation are currently under progress. Recent studies have suggested that the stability and activation of p53 are regulated by posttranslational phosphorylation of p53 protein itself and its negative regulator MDM2 protein by several signaling kinases, e.g., phosphatidylinositol 3-kinase (PI3-K), AKT (PKB), ERK1/2, and PKC, respectively (48-50). Whether cadmium attenuates BPDEinduced p53 response by interfering with the function of the above signal transducing kinases remains to be elucidated. Because the cells treated with (()-BPDE showed increased levels of p21 as compared to untreated cells (Figure 4), it was expected that (()-BPDE treatment would promote growth arrest of the cells. Treatment of the synchronized fibroblasts with 0.5 µM (()-BPDE resulted in growth arrest in the G1 phase of the cell cycle (Figure 5 and Table 1). The finding that cadmium inhibits p21 response in a dose-dependent manner (Figure 4) corresponds to our results that cadmium overrides G1 cell cycle arrest in response to (()-BPDE in a dose-dependent manner. The data suggest that the enhancement of BPinduced genotoxicity by cadmium may be due to the abrogation of p21-dependent cell cycle arrest at G1 phase by the metal. Cellular response of human fibroblasts to BPDEinduced DNA damage results in the induction of cell cycle arrest in G1 phase, which fulfills the requirements for subsequent repair process that may remove the DNA adducts prior to DNA replication. Bulky DNA lesions induced by UV light and mutagenic/carcinogenic chemicals including PAHs and aromatic amines are removed by the NER system (22). Most of the studies reported on the effect of cadmium on NER deal with the UV-induced

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DNA damage; practically no information is available on the effect of the metal on the repair of PAH-induced DNA damage. Asmuss et al. (31) demonstrated that cadmium decreased the binding of XPA (a zinc finger DNA repair protein involved in the recognition of DNA damage) to UV-irradiated DNA in vitro. They suggested that a possible mechanism underlying the inhibition of XPA binding to UV-damaged DNA by cadmium is the displacement of Zn2+ by Cd2+ in the zinc finger structure of XPA. Because cadmium may inactivate the NER proteins and BPDE-DNA adducts are preferentially repaired by NER pathway (23, 24), we have examined the effect of cadmium on the repair of (()-anti-BPDE-adducted pUC18 plasmid DNA in cell-free system using NER proficient HeLa cell extract. Besides NER proficiency, HeLa cells grow fast and a sufficient quantity of cellular proteins needed for the assay of in vitro repair of BPDE-adducted DNA is readily available. Our results demonstrated a decreased ability of cadmium-treated HeLa cell extract to repair (()-BPDE-adducted plasmid DNA in vitro as compared with the untreated HeLa extract. This suggests that cadmium may have a direct or indirect effect on the proteins involved in the repair of BPDE-adducted DNA. There is evidence that (i) HeLa cell extract-mediated in vitro DNA repair faithfully reflects in vivo DNA repair properties (42) and (ii) in vitro NER mimics more closely global genomic repair (GGR) whereas transcriptioncoupled repair (TCR) of active genes plays a major role in vivo (52, 53). On the basis of this information, our present result of the inhibitory effect of cadmium on in vitro DNA repair suggests that cadmium inhibits BPDEadducted DNA repair by interfering with the GGR pathway possibly by direct interaction with repair proteins, but it does not exclude the possibility of interference of cadmium with the TCR pathway in vivo. Because both the TCR and the GGR pathways are involved in the repair of BPDE-dG adduct (54), a detailed in vivo study is needed to decipher the interaction of cadmium with the above two subpathways of NER using XPC and CSA cells deficient in GGR and TCR pathways, respectively. In conclusion, the data from these studies demonstrate that cadmium not only inhibits the repair of BPDEdamaged DNA via the NER pathway but also interferes with p53-related processes involved in DNA repair such as attenuation of p53 and p21 responses and abrogation of cell cycle arrest. The ability of cadmium to interfere with these processes may explain, at least partly, the potentiating effect of the metal on the genotoxicity of BP.

Acknowledgment. We gratefully acknowledge the technical assistance of Alice M. Deltoro and Tonniele M. Naeher. This work was supported by Philip Morris USA, Inc.

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