Induction of Cytotoxicity, Aldehydic DNA Lesions ... - ACS Publications

Jan 19, 2005 - Chia-Hua Lin,† Hong-Tsee Leow,† Shih-Chien Huang,† Jun ... Department of Environmental Engineering, National Chung Hsing Universi...
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Chem. Res. Toxicol. 2005, 18, 257-264

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Induction of Cytotoxicity, Aldehydic DNA Lesions, and Poly(ADP-Ribose) Polymerase-1 Activation by Catechol Derivatives of Pentachlorophenol in Calf Thymus DNA and in Human Breast Cancer Cells Chia-Hua Lin,† Hong-Tsee Leow,† Shih-Chien Huang,† Jun Nakamura,‡ James A. Swenberg,‡ and Po-Hsiung Lin*,† Department of Environmental Engineering, National Chung Hsing University, Taichung 402, Taiwan, and Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27599-7400 Received June 4, 2004

The purpose of this study was to investigate the degree of chlorination of catechol (CAT) derivatives of pentachlorophenol (PCP) on the induction of cytotoxicity and DNA damaging effects in calf thymus DNA (ct-DNA) and in two human breast carcinoma cell lines. Results indicated that with the addition of the transition metal copper(II), increases in the amount of aldehydic DNA lesions (ADL) were detected in ct-DNA exposed to PCP-derived CATs over the corresponding control. The DNA lesions induced by various degrees of chlorination of PCPderived CATs decrease in the rank order CAT = 4-chlorocatechol (4-ClCAT) > 4,5-dichlorocatechol (4,5-Cl2CAT) > 3,4,5-trichlorocatechol (3,4,5-Cl3CAT) > tetrachlorocatechol (Cl4CAT). In contrast, Cl4CAT was the only congeneric form of PCP-derived catechols that induced a significant increase in the number of ADL in human MCF-7 cells, and this only occurred when glutathione was depleted. Pretreatment with copper(I) and iron(II) chelators significantly reduced the formation of ADL in cells exposed to Cl4CAT. The data also indicated that the ADL induced by Cl4CAT in MCF-7 cells contain ∼70% putrescine excisable ADL. This evidence confirmed that the ADL induced by Cl4CAT in MCF-7 cells were derived from oxidative events. In addition, we demonstrated that the depletion of NAD(P)H in human T47D cells exposed to chlorinated CATs decreased in the rank order Cl4CAT . 4-ClCAT = CAT. The depletion of NAD(P)H induced by Cl4CAT in T47D cells was partially blocked by catalase, superoxide dismutase, dimethyl sulfoxide, and copper(I) and iron(II) specific chelators. Additionally, the depletion of NAD(P)H in T47D cells exposed to Cl4CAT (1-10 µM) was completely blocked by three types of poly(ADP-ribose) polymerase-1 inhibitors. This evidence suggests that Cl4CAT induces an imbalance in DNA repair and the subsequent accumulation of DNA strand breaks in human cultured cells. Overall, these findings indicate that dechlorination may decrease the potentials of chlorinated catechols to induce oxidative DNA lesions and cytotoxic effects in living cells.

Introduction Accumulating evidence suggests that chlorinated catechols (CATs)1 are introduced into the environment via discharge from industrial manufacturing processes as well as biodegradation products of chlorinated aromatics (1). Microbial degradation of chlorinated aromatics, such as pentachlorophenol (PCP), may lead to the formation of CAT derivatives (1-8). In mammals, exposure to PCP also gives rise to the formation of CAT derivatives * To whom correspondence should be addressed. Tel: 886/4/ 22840441 ext. 515. Fax: 886/4/22858970. † National Chung Hsing University. ‡ University of North Carolina. 1 Abbreviation: 3-AB, 3-aminobenzamide; 3,4,5-Cl CAT, 3,4,53 trichlorocatechol; 4-ClCAT, 4-chlorocatechol; 4,5-Cl2CAT, 4,5-dichlorocatechol; ADL, aldehydic DNA lesions; AP sites, apurinic/apyrimidinic sites; ASB assay, aldehyde reactive probe slot-blot assay; BA, benzamide; BAT, bathocupronine; PCP, pentachlorophenol; CAT, catechol; CATA, catalase; Cl4CAT, tetrachlorocatechol; DMSO, dimethyl sufoxide; ct-DNA, calf thymus DNA; DPD, 2,2′-dipyridyl; DPH, 2,9-dimethyl1,10-phenanthroline hydrochloride; PARP-1, poly(ADP-ribose) polymerase-1; ROS, reactive oxygen species; SOD, superoxide dismutase.

following metabolic transformation (3, 4, 9). Evidence indicates that liver microsomal cytochrome P450s mediate the conversion of PCP to tetrachlorohydroquinone (Cl4HQ) and tetrachlorocatechol (Cl4CAT), which are oxidized to tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) and tetrachloro-1,2-benzoquinone (Cl4-1,2-BQ), respectively (9-14). Cl4HQ may undergo autooxidation and/or enzymemediated redox cycling cascades and generate reactive oxygen species (ROS) (15). Our previous investigation indicated that Cl4HQ was capable of generating parallel formation of oxidized bases, abasic sites, and DNA strand breaks in purified DNA and in human cultured cells (15, 16). In addition, the para-isomeric form of chlorinated quinonoids is known to induce micronuclei and mutation in mammalian cells (16-19). However, evidence of Cl4CAT-induced genotoxicity in mammalian cells has not been shown. One mutagenicity test conducted in growing cells of Saccharomyces cerevisiae exposed to Cl4CAT yielded negative results (20). However, Cl4-1,2-BQ, the corresponding quinone of Cl4CAT, increased the forma-

10.1021/tx0498511 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/19/2005

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MCF-7 and T47D breast cancer cells exposed to PCPderived CATs.

Materials and Methods

Figure 1. Chemical structure of various CAT derivatives.

tion of 8-hydroxydeoxyguanosine in Chinese hamster V79 cells (18). This event had been suggested to be involved in PCP-induced carcinogenesis (18, 21-23). ROS-mediated DNA damage in mammalian cells is subject to repair primarily by base excision repair enzymes such as 8-hydroxy-2′-deoxyguanosine glycosylse/ lyase, which removes oxidized bases to generate abasic sites (24). If not repaired, abasic sites are promutagenic DNA lesions and are strong blockers of DNA synthesis leading to cell death. The abasic sites resulting from the spontaneous depurination/depyrimidination of the modified bases and the aldehydic base and sugar lesions resulting from the oxidative damage to deoxyribose moieties in the DNA molecules will lead to aldehydic forms of DNA lesions. Information regarding the direct assay of the number of aldehydic DNA lesions (ADL) induced by CAT derivatives of PCP has not been reported. Chlorinated CATs are known to be cytotoxic. Chlorine substitutions in the phenol and CAT molecules are modulating factors of its toxicity (25). Increases in ring chlorination of phenolic chemicals are known to enhance the cytotoxic effects of these chemicals (26). Transition metals may modulate redox reactions of chlorinated quinonoids and subsequent formation of ROS (19, 27). Evidence indicates that chlorinated CATs alone do not induce DNA strand breaks but with the inclusion of Cu(II) and Fe(III) significantly induce DNA strand breaks (25, 28). Dechlorination increases in the extent of DNA strand breaks in PTZ18R plasmid induced by chlorinated CATs and transition metals (25). However, the types and extent of damage induced by chlorinated CATs in human cultured cells have not been fully characterized thus far. The origin of chlorinated CAT-induced DNA damage as well as cell toxicity in mammalian cells remains elusive. To investigate the cell toxicity and DNA damage induced by CAT derivatives of PCP, we undertook the present investigation to study the differences in the induction of cytotoxicity, ADL, and poly(ADP-ribose) polymerase-1 (PARP-1) activation in calf thymus DNA (ct-DNA) and in human breast tumor cells by CAT derivatives of PCP, including CAT, 4-chlorocatechol (4ClCAT), 4,5-dichlorocatechol (4,5-Cl2CAT), 3,4,5-trichlorocatechol (3,4,5-Cl3CAT), and Cl4CAT (structures are as shown in Figure 1). We demonstrate that the dechlorination process potentiates the oxidant-mediated formation of ADL in ct-DNA induced by chlorinated CATs in the presence of Cu(II) and NADP(H) whereas the dechlorination process decreases the parallel induction of cytotoxicity, ADL, and PARP-1 activation in human

Chemicals and Cell Culture. MCF-7 cells and T47D cells, human breast cancer cell lines, were purchased from Culture Collection and Research Center (Hsinchu, Taiwan) and were used in this experiment. MCF-7 cells were grown in a humidified atmosphere containing 5% CO2 in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 5% fetal bovine serum (Sigma), 1% antibiotics (Gibco), and 0.6 µg/mL insulin at 37 °C. T47D cells were grown in a humidified atmosphere containing 5% CO2 in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (Sigma), 1% antibiotics, 0.6 µg/mL insulin, and 1 mM sodium pyruvate (Gibco) and held at 37 °C. CAT, 4-ClCAT, 4,5-Cl2CAT, 3,4,5-Cl3CAT, Cl4CAT, glutathione (GSH), bathocupronic acid, catalase (CATA), superoxide dismutase (SOD), cupric chloride, NAD(P)H, dimethyl sufoxide (DMSO), 3-aminobenzamide (3-AB), benzamide (BA), coumarin, and o-phthalaldehyde were purchased from Sigma Chemical Co. (St. Louis, MO). Kodak XAR-5 film was obtained from Eastman Kodak (Rochester, NY) for autoradiography. Reagents used to measure the ADL assay by the aldehyde reactive probe slot-blot assay (ASB assay) were as described by Nakamura and Swenberg (29). CCK-8 solution was purchased from Dojindo Molecular Technology. All other chemicals were purchased from Sigma, Aldrich, or Fisher unless stated otherwise and used without further purification. Reaction of ct-DNA with CAT Derivatives of PCP. To determine the induction of various types of DNA damage by chlorinated ortho-quinonoids, ct-DNA was incubated with CAT, 4-ClCAT, 4,5-Cl2CAT, 3,4,5-Cl3CAT, and Cl4CAT under physiological conditions. The incubation medium (final volume, 0.5 mL) consisted of 150 mM phosphate-buffered saline (pH 7.4) and ctDNA (250 µg). The ct-DNA used in the analysis of the induction of ADL was pretreated with 100 mM methoxylamine (Sigma) to reduce the background number of ADL (29). To these preparations, chlorinated CATs (dissolved in DMSO) were added and the reaction was carried out at 37 °C. After 2 h of incubation, chilling the mixture in an ice bath terminated the reaction. DNA was isolated by ethanol precipitation as previously described (16). For some experiments, Cu(II) (20-100 µM; prepared from cupric chloride), NAD(P)H (100 µM), CATA (30 U), SOD (40 U), GSH (100 µM), bathocupronine (BAT) (100 µM), and DMSO (6% v/v) were added before the addition of PCPderived CATs to examine the effects of these modulators on the induction of ADL in ct-DNA by chlorinated CATs. Reaction of Human MCF-7 Breast Cancer Cells with CAT Derivatives of PCP. To determine the induction of ADL induced by CAT derivatives of PCP, the MCF-7 cells were seeded in 10 cm dishes and were pretreated with 50 µM N-ethylmaleimide (NEM) for 30 min to reduce the level of GSH. To these preparations, 4-ClCAT and Cl4CAT (1-1000 µM, dissolved in DMSO) were added and the reactions were carried out at 37 °C for 18 h. After incubation, the mixture was chilled in an ice bath to terminate the reaction. DNA was isolated by ethanol precipitation as previously described (16) and was stored under -80 °C before the measurements of the number of ADL by the ASB assay. For some experiments, the copper(I) specific chelator, 2,9dimethyl-1,10-phenanthroline hydrochloride (DPH) (100 µM), and the iron(III) specific chelator, 2,2′-dipyridyl (DPD) (100 µM), were added before the addition of PCP CATs to examine the effects of these modulators on the induction of ADL in MCF-7 cells. Survival of cells evaluated by trypan blue exclusion assay showed the survival rate at 75% or more after exposure (30). Analysis of GSH by o-Phthalaldehyde Assay. The total GSH content was measured using the method as previously described by Siraki et al. (31) with modifications. In brief, MCF-7 cells (seeded in the 96 well black plates) were treated with 50 µM NEM for 30 min and 10 µL of 62.5% trichloroacetic acid was then added to the medium. After 5 min, 40 µL of 1 M

Catechol Derivatives of Pentachlorophenol sodium phosphate (pH 7) was added to each well and the reaction was carried out for 15 min. One hundred microliters of 0.16 M sodium phosphate solution containing 37.5 mM o-phthalaldehyde was added to each well. The fluorescence was recorded (λex) 355 nm excitation and λem ) 430 nm) by a fluorometer after 30 min of reaction at room temperature in the dark and was compared to the values of the control. Analysis of ADL by the ASB Assay. The abasic sites resulting from the spontaneous depurination/depyrimidination of the modified bases and the aldehydic base and sugar lesions resulting from the oxidative damage to deoxyribose moieties in the DNA molecules were measured by an aldehyde reactive probe. ADL were assayed based upon the reaction of the aldehydic group in an apurinic/apyrimidinic site (AP site) with a probe bearing a biotin residue as described by Nakamura et al. (29). Putrescine Cleavage Assays. The putrescine cleavage assays were performed as described by Nakamura et al. (30). DNA, 10 mM EDTA, and 100 mM putrescine were incubated in 10 mM Tris-HCl/KOH at 37 °C for 30 min and immediately analyzed by the ASB assay. Analysis of Cytotoxicity in T47D Cells by the Sulforhodamine B (SRB) Assays. The SRB assay was used in this experiment to determine the total cell number by measuring cellular proteins as previously described by Andersen et al. (32). In brief, 150 µL of Hank’s balanced salt solution was added in the 96 well plates, and then, the solution was discarded. One hundred microliters of the 10% TCA was added, and the plates were incubated in ice for 30 min. The solution was aspirated off, and the plates were rinsed four times with deionized water. The plates were dried at 37 °C. Fifty microliters of the SRB was added to each well, and then, the plates were stained for 30 min. The stain was removed, and the cells were rinsed three times with 100 µL of 1% acetic acid. The plates were dried at 37 °C again. One hundred microliters of Tris solution was added to the plates. The samples were determined by a spectrophotometer and were compared to the values of control. Visible absorbance was recorded in a 96 well plate reader at 492 nm. Analysis of Intracellular NAD(P)H in T47D Cells by a Water Soluble Tetrazolium Salt. Intracellular NAD(P)H was assayed using a commercially available assay as previously described by Nakamura et al. (33) with modifications. A water soluble tetrazolium salt was used to monitor the amount of NAD(P)H through its reduction to a yellow-colored formazan dye and determined periodically by a spectrophotometer. Cells were seeded in 96 well plates (8 × 103 cells/well) and were treated with chlorinated CATs for 24 h at indicated concentrations (1-500 µM). After incubation, the reaction medium was discarded and the wells were washed with PBS. Then, 100 µL of fresh medium and 1/10 volume of CCK-8 solution were added. Cells were further cultured for up to 4 h and were examined periodically (30 min) by a spectrophotometer. Visible absorbance was recorded in a 96 well plate reader at 450 nm with 650 nm as a reference filter. CCK-8 consisted of highly water soluble tetrazolium salt, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], which produces a water soluble formazan dye upon reduction in the presence of an electron carrier, NAD(P)H. Therefore, the amount of intracellular NAD(P)H was determined by the reduction in tetrazolium salts. The decrease in the intracellular NAD(P)H was assessed by comparing the absorbance of a well containing cells treated with chlorinated CAT against the control, which was treated with DMSO only. When necessary, CATA (1000 u/mL), SOD (500 u/mL), CATA (1000 u/mL) plus SOD (500 u/mL), DMSO (1%), DPD (50 µM), DPH (5 µM), and specific PARP-1 inhibitors, including 3-AB (15 mM), BA (5 mM), and coumarin (1.5 mM), were applied 2 h prior to the treatment and kept in the medium during chlorinated CATs exposure until the cells were analyzed. Statistical Analysis. All data are expressed as means ( SD. The significance of differences in the results was evaluated with ANOVA, followed by Dunnett’s multiple comparison tests.

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Figure 2. Formation of ADL in ct-DNA treated with (A) a different degree of chlorination of CATs (100 µM) and Cu(II) (20 µM) and (B) 4-ClCAT (0.01 and 0.1 µM) plus Cu(II) (20 µM) or Fe(III) (20 µM) and NADP(H) (100 µM) at 37 °C for 2 h. Modified DNA was assayed by the ASB assay. Data represent the mean ( SD of three determinations. * and ** indicate statistically significant differences from control [DMSO plus Cu(II) and NADP(H)] or [DMSO plus Cu(II)]; p < 0.05 and p < 0.01.

Results ADL Induced by CAT and Chlorinated CATs in ct-DNA. To determine if CAT and chlorinated CATs induce ADL in ct-DNA, we incubated ct-DNA with various CAT derivatives, including CAT, 4-ClCAT, 4,5Cl2CAT, 3,4,5-Cl3CAT, and Cl4CAT, under physiological conditions. Results indicated that CAT and chlorinated CATs alone did not induce statistically significant increases in the number of ADL (data not shown). In contrast, the addition of transition metals Cu(II) induced appreciable amounts of ADL in ct-DNA treated with 4-ClCAT, 4,5-Cl2CAT, and 3,4,5-Cl3CAT as compared to controls (p < 0.05) (Figure 2A). In the presence of Cu(II) (20 µM), Cl4CAT did not induce statistically significant increases in the number of ADL (p > 0.05). The amounts of ADL induced by chlorinated CATs plus Cu(II) decreased in the order 4-ClCAT > 4,5-Cl2CAT > 3,4,5-Cl3CAT > Cl4CAT. To test whether the inclusion of NAD(P)H further potentiated the DNA damage induced by chlorinated CATs plus transition metals, we incubated ct-DNA with chlorinated CATs in the presence of Cu(II) (20 µM) and NADP(H) (100 µM). Results indicated that in the presence of both Cu(II) and NAD(P)H, the number of ADL in ct-DNA exposed to Cl4CAT (100 µM) increased ∼10-fold over control (Figure 2A). This result suggests that inclusion of NAD(P)H further potentiates the DNA damage induced by chlorinated CATs plus Cu(II). In

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Figure 3. Formation of ADL in ct-DNA treated with 4-ClCAT (1 µM) plus Cu(II) (20 µM), NADP(H) (100 µM), and ROS modulators at 37 °C for 2 h. Data represent the mean ( SD of three determinations. * indicates a statistically significant difference from control [4-ClCAT plus Cu(II)]; p < 0.05.

addition, inclusion of NAD(P)H induced a significant increase in the number of ADL in ct-DNA exposed to CAT and 4-ClCAT over control and the increase in the number of ADL was concentration-dependent (Figure 2B). In contrast, in the presence of both Fe(III) (20 µM) and NAD(P)H (100 µM), 4-ClCAT (0.1 µM) did not induce statistically significant increases in the number of ADL over control (p > 0.05) (Figure 2B). This evidence suggests that CAT derivatives of PCP are capable of inducing DNA damage at nanomolar concentrations. These findings are in good agreement with results reported by Schweigert et al. (25). Effects of ROS Modulators and Transition Metals on the Formation of ADL in ct-DNA Induced by Chlorinated CATs. To determine whether chlorinated CATs mediate the induction of ADL in ct-DNA via the formation of ROS, we incubated ct-DNA with 4-ClCAT (50 µM) plus Cu(II) (20 µM) in the presence of ROS modulators, including SOD (40 U), CATA (30 U), BAT (100 µM), GSH (1 mM), or DMSO (6%). The addition of CATA, BAT, and GSH to the incubates reduced the concentrations of ADL to levels comparable to that of the control [4-ClCAT plus Cu(II)] (p < 0.001) (Figure 3). This finding confirmed the involvement of hydrogen peroxide and Cu(I) in the induction of ADL in ct-DNA by chlorinated CATs (21). ADL Induced by Chlorinated CATs in Human MCF-7 Breast Cancer Cells. To examine the induction of ADL in living cells by chlorinated CATs, human MCF-7 cells were pretreated with 50 µM NEM for 30 min to reduce the level of GSH. ClCAT (100-1000 µM) and Cl4CAT (50-500 µM) were then added to the medium for an additional 18 h. Nuclear DNA was isolated, and the number of ADL was determined by the ASB assay. Results indicated that increases in the number of ADL were detected in cells exposed to Cl4CAT at 500 µM (p < 0.01). ClCAT did not induce significant increases in the number of ADL in NEM-treated MCF-7 cells. The addition of DPD and DPH to the incubates reduced the concentrations of ADL to levels comparable to those of controls (p > 0.05) (Figure 4). This evidence suggests that the dechlorination process reduces the capacity of DNA damaging effects of Cl4CAT in living cells. This result also confirmed that transition metals, Cu(I) and Fe(II), may participate in the induction of ADL by Cl4CAT in MCF-7 cells.

Figure 4. Formation of ADL in MCF-7 cells treated with (A) 4-ClCAT (100-1000 µM) and Cl4CAT (50-500 µM) plus NEM and (B) Cl4CAT (500 µM) plus NEM (50 µM) with the presence of DPH and DPD at 37 °C for 2 h. Data represent the mean ( SD of three determinations. * indicates a statistically significant difference from control (DMSO plus NEM); p < 0.05.

GSH Depletion Induced by NEM in Human MCF-7 Breast Cancer Cells. To confirm the effects of NEM on the level of GSH in living cells, the total GSH content was determined by detection of the fluorescent product of GSH and o-phthalaldehyde. Results indicated that levels of GSH were depleted to approximately 20% of control levels in MCF-7 cells treated with NEM. Putrescine Excisable ADL Induced by Chlorinated CATs. It is believed that ROS are capable of inducing parallel formation of various types of DNA damage (23, 32), including base oxidation, DNA strand breaks, and abasic sites. To further characterize whether chlorinated CATs induce the formation of oxidatantmediated ADL, ct-DNA that had been treated with 4-ClCAT (1 µM) plus Cu(II) (20 µM) and NAD(P)H (100 µM) and nuclear DNA isolated from MCF-7 cells that had been treated with Cl4CAT (500 µM) plus NEM (50 µM) were incubated with putrescine followed by the ASB assay (30). Results indicated that a significant reduction of ADL was detected after putrescine treatment (Figure 5A,B). In the presence of Cu(II) and NAD(P)H, 4-ClCAT produced 67% putrescine excisable ADL in ct-DNA where Cl4CAT produced 70% putrescine excisable ADL in NEMpretreated MCF-7 cells. This evidence suggests that the ADL induced by CAT derivatives of PCP in ct-DNA and in MCF-7 cells are mediated by oxidative events. Cytotoxicity Induced by CAT and Chlorinated CATs in Human T47D Breast Cancer Cells. To determine if CAT and chlorinated CATs induce cytotox-

Catechol Derivatives of Pentachlorophenol

Figure 5. Formation of putrescine excisable ADL (per 106 total nucleotides) in (A) ct-DNA treated with DMSO plus Cu(II) (20 µM) and NADP(H) (100 µM) or 4-ClCAT (1 µM) plus Cu(II) (20 µM) and NADP(H) (100 µM) and in (B) MCF-7 cells exposed to DMSO plus NEM (50 µM) or Cl4CAT (500 µM) plus NEM (50 µM). Modified DNA was treated with putrescine (putre), which cleaved ADL at 3′- or 5′-sites to ADL, followed by the ASB assay. -/- indicates that modified DNA was analyzed for ADL by the ASB assay without pretreatment with putre.

icity in living cells, we incubated human T47D cells with CAT derivatives, including CAT, 4-ClCAT, and Cl4CAT, at concentrations ranging from 1 to 500 µM. A viable cell number was determined by the SRB assay. After 24 h of exposure, Cl4CAT induced concentration-dependent increases in cytotoxic response (∼60% of control, p < 0.01) at concentrations ranging from 250 to 500 µM. Significant cytotoxic effects (∼80% of control, p < 0.05) were observed in CAT- and 4-ClCAT-treated cells at 500 µM (Figure 6A). In addition, we used a commercially available assay, which can monitor the amount of intracellular NAD(P)H levels in T47D cells exposed to CAT, 4-ClCAT, and Cl4CAT (1-500 µM) for 24 h, and monitored the reduction in NAD(P)H after exposure. The intracellular NAD(P)H in T47D cells was significantly reduced by treatment with Cl4CAT in a dose-dependent manner (∼20-90% of control, p < 0.01), but significant reduction was only detected in CAT- and 4-ClCAT-treated cells at concentrations ranging from 250 to 500 µM when compared to control (∼80% of control, p < 0.05) (Figure 6B). These results were in good agreement with what has been observed in the induction of ADL in chlorinated CAT-treated MCF-7 cells where Cl4CAT was more efficient than 4-ClCAT in the induction of ADL in cells. Effects of ROS Modulators and Transition Metals on the Decrease in NAD(P)H Induced by Cl4CAT. To determine the roles of transition metals and ROS in mediating the intracellular NAD(P)H depletion induced by chlorinated CATs in T47D cells, we incubated T47D cells with Cl4CAT (10 µM) in the presence of ROS modulators and metal chelators, including CATA (1000 u/mL), SOD (500 u/mL), CATA (1000 u/mL) plus SOD (500 u/mL), DMSO (1%), DPD (50 µM), and DPH (5 µM).

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Figure 6. (A) Induction of cytotoxicity in T47D cells treated with CAT (1-500 µM), 4-ClCAT (1-500 µM), and Cl4CAT (1500 µM). (B) Induction of intracellular NAD(P)H depletion in T47D cells treated with CAT (1-500 µM), 4-ClCAT (1-500 µM), and Cl4CAT (1-500 µM) under physiological conditions at 37 °C for 24 h. b, CAT; ], 4-ClCAT; and 4, Cl4CAT. Data represent the mean ( SD of three determinations. *, **, and *** indicate statistically significant differences from control (DMSO); p < 0.05, p < 0.01, and p < 0.001.

After 24 h of exposure, SOD, CATA, CATA + SOD, DMSO, DPD, and DPH partially blocked the Cl4CATinduced decreases in the amount of intracellular NAD(P)H in T47D cells (Figure 7A,B). This evidence suggests that superoxide, hydrogen peroxide, hydroxyl radical, and transition metals, including Fe(II) and Cu(I), are involved in Cl4CAT-mediated depletion of NAD(P)H levels in T47D cells. Effects of PARP-1 Inhibitors on the Decrease in NAD(P)H Induced by Cl4CAT. PARP-1 is an enzyme mediating the cellular response to DNA single or double strand breaks and is also involved in early DNA damage recognition and base excision repair (34). Evidence suggests that accumulation of DNA strand breaks activates PARP-1 that catalyzes the formation of polymers of poly (ADP-ribose) and NAD+ depletion (33). To distinguish whether the reduction in NAD(P)H is due to the depletion of NAD+ by PARP-1 activation, T47D cells were cotreated with Cl4CAT and specific PARP inhibitors, i.e., 3-AB, BA, and coumarin, for 24 h and monitored the reduction in NAD(P)H levels. Results indicated that all of the PARP inhibitors completely blocked the Cl4CAT-induced decrease in the amount of intracellular NAD(P)H in T47D cells at concentrations of 1 and 10 µM (p < 0.05) (Figure 8A,B). These results suggested that the decreases in the intracellular levels of NAD(P)H in Cl4CAT-treated T47D cells were primarily due to depletion of NAD+ mediated by PARP-1 activation.

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Figure 7. Induction of intracellular NAD(P)H depletion in T47D cells treated with Cl4CAT (10 µM) plus (A) ROS modulators and (B) transition metals chelates under physiological conditions at 37 °C for 24 h. Data represent the mean ( standard deviation of three determinations. ** and *** indicate statistically significant differences from control (Cl4CAT); p < 0.01 and p < 0.001.

Discussion In this study, we examined the degree of chlorination of CAT derivatives of PCP and the induction of cytotoxicity and DNA damaging effects in ct-DNA and in human MCF-7 and T47D breast cancer cell lines. When ct-DNA was treated with PCP-derived CATs alone, we did not observe a significant increase in the number of ADL (data not shown). In contrast, increases in ADL were detected in ct-DNA exposed to PCP-derived CATs with the inclusion of Cu(II) (Figure 2A). The addition of NAD(P)H further induced increases in the number of ADL in ctDNA treated with PCP-derived CATs plus Cu(II) at a nanomolar concentration (Figure 2A,B). Overall, we concluded that the DNA lesions induced by various degrees of chlorination of PCP-derived CATs decrease in the rank order CAT = 4-ClCAT > 4,5-Cl2CAT > 3,4,5Cl3CAT = Cl4CAT. In addition, results from our analyses support the notion that similar to the para-quinonoid counterparts, PCP-derived CATs may induce the formation of oxidant-mediated ADL in the presence of Cu(II) and NADP(H) in ct-DNA where hydrogen peroxide and Cu(I) may be involved in the process (Figure 3). Evidence suggests that GSH plays a pivotal role in the biological antioxidant defense systems, and the depletion of GSH after stress is considered as a cause of oxidative stress (35). Results from our analyses indicated that GSH completely inhibits the induction of ADL by chlorinated CATs in ct-DNA (Figure 3). It is likely that GSH inhibits

Lin et al.

Figure 8. Induction of intracellular NAD(P)H depletion in T47D cells treated with (A) Cl4CAT (1 µM) and (B) Cl4CAT (10 µM) plus PARP-I inhibitors under physiological conditions at 37 °C for 24 h. Data represent the mean ( standard deviation of three determinations. ** indicates a statistically significant difference from control (DMSO); p < 0.01.

the formation of ADL in ct-DNA induced by 4-ClCAT plus Cu(II) by reacting with H2O2 to generate GSSG and H2O. When we examined the induction of ADL in MCF-7 cells exposed to all four chlorinated CATs alone, we did not observe significant increases in the number of ADL when compared to control (data not shown). However, when we pretreated MCF-7 cells with NEM to deplete cellular GSH, increases in the number of ADL were detected in MCF-7 cells exposed to Cl4CAT but not in ClCAT-treated cells (Figure 4A,B). These results were in good agreement with the finding that acute and membrane toxicity increased with increasing degree of chlorination of CAT derivatives in Escherichia coli (36). This evidence suggests that dechlorination may attenuate the deleterious effect induced by chlorinated CATs in living cells. Evidence suggests that CAT estrogens and tetrachlorohydroquinones can undergo redox cycling in the presence of Cu(II) to generate ROS and induce DNA strand breaks (19, 28, 37-39). Results from our observation confirmed the involvement of hydrogen peroxide and Cu(I) in mediating the oxidative DNA lesions induced by chlorinated CATs in ct-DNA (Figure 3). A similar observation was also detected in MCF-7 cells where Fe(II) and Cu(I) were involved in mediating the oxidative DNA lesions induced by Cl4CAT (Figure 4A,B). As transition metal ions are always present in cells and tissues at levels as high as 100 µM for Cu(II) (39), it is likely that the presence of transition metals and reducing equivalent promotes the redox cycling of chlorinated CATs to gener-

Catechol Derivatives of Pentachlorophenol

ate ROS and initiate oxidative DNA damage in human cultured cells. Our current investigation also demonstrated that ∼70% of the ADL induced by chlorinated CATs in ct-DNA and in MCF-7 cells were excised by putrescine (Figure 5A,B). Because regular abasic sites generated by spontaneous depurination/depyrimidination are not excisable by putrescine (40), the ADL induced by chlorinated CATs in ct-DNA and in MCF-7 cells are likely to be derived from oxidation processes rather than depurination/depyrimidination of labile chlorinated quinone-DNA adducts. The ADL induced by chlorinated CATs may be derived from hydrogen abstraction by ROS on the C5′position of deoxyribose, as proposed in tetrachlorohydroquinone and estrogen CATs (19, 41). Taken together, these results provide evidence that Cl4CAT is capable of inducing similar types of oxidative damage to the DNA backbone in human cultured cells. It has been shown that the intracellular apoptotic signaling mechanisms are very different between human MCF-7 and T47D breast cancer cells. The mechanism of cell death was kinetically different with events occurring earlier in MCF-7 cells than in T47D cells (42-45). In this study, we expanded our investigations to include the T47D cell line to study the relationship between DNA damage and cell toxicity induced by chlorinated and nonchlorinated CATs. When human T47D cells were exposed to CAT, 4-ClCAT, and Cl4CAT for 24 h, a concentration-dependent increase in cytotoxic response was detected in cells treated with Cl4CAT as determined by cell viability and the extent of NAD(P)H depletion (Figure 6A,B). Cl4CAT was more potent than CAT and 4-ClCAT in mediating the cytotoxic effects in T47D cells. This finding is in agreement with the finding in the induction of ADL by chlorinated CATs in MCF-7 cells (Figure 4A). It has been shown that the first acidity constants, octanol-water partitioning coefficients, and cell toxicity of chlorinated CATs were reported to increase with increasing degree of chlorination (46). Chlorinated CATs are more hydrophobic than nonchlorinated CATs, which allow them to diffuse through cellular membranes (31). We speculated that differences in the acidity dissociation constants and lipophilicity contribute to the variation in the induction of cell toxicity and DNA damaging effects between Cl4CAT and ClCAT (47). The addition of SOD, CATA, SOD + CAT, DMSO, DPD, and DPH to the incubate reduced the Cl4CAT-mediated depletion of NADPH in T47D cells (Figure 7A,B). This finding confirmed that the cytotoxic response induced by Cl4CAT in T47D cells was mediated by ROS and transition metals, including Cu(I) and Fe(II). It has been reported that to counteract DNA alkylation, repair-mediated indirect formation of DNA strand breaks would start to accumulate, further leading to NAD(P)H depletion through overactivation of PARP-1 (48). By monitoring the intracellular NAD(P)H level, we can indirectly assess an accumulation of DNA strand breaks (33). Results from our observation confirmed that the intracellular NAD(P)H levels in human T47D cells were significantly reduced by treatment with Cl4CAT in a dosedependent manner at concentrations ranging from 1 to 500 µM (up to 20% of control) but to a lesser extent in 4-ClCAT-treated cells at concentrations above 250 µM (∼80% of control) (Figure 6B). To distinguish whether the reduction in NAD(P)H is due to the depletion of NAD+ by PARP-1 activation, we coexposed T47D cells

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to Cl4CAT and specific PARP-1 inhibitors, i.e., 3-AB, BA, and coumarin. Results clearly demonstrated that the PARP inhibitors completely blocked the Cl4CAT-induced decrease in the amount of intracellular NAD(P)H in T47D cells exposed to Cl4CAT at concentrations 1-10 µM (Figure 8A,B). These results suggest that decreases in the intracellular levels of NAD(P)H in T47D cells exposed to Cl4CAT were primarily due to PARP-1 activation through formation of DNA strand breaks. Again, our investigation indicates that results obtained from ct-DNA were at odds with those in living cells. As dechlorination may not alleviate the potentials of oxidative damage to ct-DNA induced by CAT derivatives of PCP in the presence of Cu(II) and NADP(H), dechlorination may decrease the potentials of oxidant-mediated DNA damage and cytotoxic effects to human cells induced by chlorinated CATs. In summary, we conclude that dechlorination inhibits the parallel induction of cytotoxicity, ADL, and DNA strand breaks in human cultured cells exposed to chlorinated CATs and that similar to its counterpart, Cl4HQ, Cl4CAT may play a critical role in PCP carcinogenesis in rodents via the induction of oxidative DNA lesions in target organs.

Acknowledgment. This work was supported by the National Science Council, Taiwan, through Grants NSC892317-B005-018, NSC90-2320-005-004, NSC91-3112-B005-001, and NSC92-3112-B-005-002 and by the National Institute of Environmental Health Sciences through Grants P42ES05948, F32ES05868, T32ES07126, and P30ES010126.

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