Sulfhydryl Binding and Topoisomerase Inhibition ... - ACS Publications

PCB metabolism generates ROS in vitro and in cells in culture and this leads ..... and serum levels of CB-153 and p,p'-DDE in European and Inuit p...
0 downloads 0 Views 165KB Size
Download PDF
Chem. Res. Toxicol. 2002, 15, 497-505

497

Sulfhydryl Binding and Topoisomerase Inhibition by PCB Metabolites A. Srinivasan,† L. W. Robertson,*,† and G. Ludewig‡ Graduate Center for Toxicology, 306 Health Sciences Research Building, University of Kentucky Medical Center, Lexington, Kentucky 40536-0305, and Department of Nutrition & Food Science, 208 Funkhouser Building, University of Kentucky, Lexington, Kentucky 40506-0054 Received July 31, 2001

Polychlorinated biphenyls (PCBs) are highly persistent contaminants in our environment. Their persistence is due to a general resistance to metabolic attack. Lower halogenated PCBs, however, are metabolized to mono- and dihydroxy compounds, and the latter may be further oxidized to quinones with the formation of reactive oxygen species (ROS). We have shown that PCB metabolism generates ROS in vitro and in cells in culture and this leads to oxidative DNA damage, like DNA strand breaks and 8-oxo-dG formation. In the present study, we have evaluated the reactivity of PCB metabolites with other nucleophiles, like glutathione (GSH), by assessing (1) quantitative GSH binding in vitro, (2) GSH and thiol (sulfhydryl) depletion in HL-60 cells, (3) the associated cytotoxicity, and (4) the inhibition of topoisomerase II activity in vitro. PCB quinones were found to bind GSH in vitro at a ratio of 1:1.5 and to deplete GSH in HL-60 cells as measured by both spectrophotometric and spectrofluorometric methods. By flow cytometry analysis, we confirmed that there was intracellular GSH depletion in HL-60 cells by PCB quinones and this is associated with cytotoxicity. On the other hand, the PCB hydroquinone metabolites did not bind GSH or other thiols within 1 h of exposure. However, by spectral analyses we found that the PCB hydroquinones could be oxidized enzymatically to the quinones, which could then bind GSH. The resulting hydroquinone-glutathione addition product(s) could undergo a second and third cycle of oxidation and GSH addition with the formation of di- and tri-GSH-PCB adducts. The effect of the PCB metabolites was also tested on a sulfhydryl-containing enzyme, topoisomerase II. PCB quinones inhibited topoisomerase II activity while the PCB hydroquinone metabolites did not. Hence, the oxidation of PCB hydroquinone metabolites to quinones in cells followed by the binding of quinones to GSH and to protein sulfhydryl groups and the resulting oxidative stress may be important aspects of the toxicity of these compounds.

Introduction (PCBs)1

Polychlorinated biphenyls were large-scale industrial chemicals from the 1930s to the 1960s that were used in diverse applications as organic diluents, plasticizers, pesticides, cutting oils, flame retardants, dielectrics in transformers, and sealants (1, 2). Commercial PCBs exhibit high stability and inertness that * Correspondence should be addressed to this author at the Graduate Center for Toxicology, 306 HSRB, University of Kentucky Medical Center, Lexington, KY 40536-0305. Phone: (859) 257-3952, Fax: (859) 323-1059, E-mail: [email protected]. † Graduate Center for Toxicology, University of Kentucky Medical Center. ‡ Department of Nutrition & Food Science, University of Kentucky. 1 Abbreviations: 2ClPh-HQ, 2-(2′-chlorophenyl)-1,4-hydroquinone; 3ClPh-HQ, 2-(3′-chlorophenyl)-1,4-hydroquinone; 4ClPh-HQ, 2-(4′chlorophenyl)-1,4-hydroquinone; 3,4ClPh-HQ, 2-(3′,4′-dichlorophenyl)1,4-hydroquinone; 3,5ClPh-HQ, 2-(3′,5′-dichlorophenyl)-1,4-hydroquinone; 3,4,5ClPh-HQ, 2-(3′,4′,5′-chlorophenyl)-1,4-hydroquinone; 2ClPhpQ, 2-(2′-chlorophenyl)-1,4-benzoquinone; 3ClPh-pQ, 2-(3′-chlorophenyl)1,4-benzoquinone; 4ClPh-pQ, 2-(4′-chlorophenyl)-1,4-benzoquinone; 3,4ClPh-pQ, 2-(3′,4′-dichlorophenyl)-1,4-benzoquinone; 3,5ClPh-Q, 2-(3′,5′dichlorophenyl)-1,4-benzoquinone; DMSO, dimethyl sulfoxide; DTNB, dithionitrobenzoic acid; GSH, glutathione; HRP, horseradish peroxidase; KCl, potassium chloride; kDNA, kinetoplast DNA; mBBr, monobromobimane; MgCl2, magnesium chloride; NEM, N-ethylmaleimide; 8-oxo-dG, 8-oxo-deoxyguanosine; PCB, polychlorinated biphenyl; PI, propidium iodide; ROS, reactive oxygen species; TAE, Tris-acetate/ EDTA; TNB, thionitrobenzoic acid.

are dependent on their degree of chlorination (1, 3). The same physical properties for which PCBs are well suited in industry have also contributed to their persistence as environmental contaminants (4). PCBs are highly lipophilic and show a low biodegradability. Hence, they bioaccumulate and biomagnify through the food chain (1). For these reasons, their use in the US in open systems was banned in the late 1970s. However, significant quantities are still in use in older electrical equipment, especially transformers, and can be introduced into the environment through careless disposal and leakage (1, 3). These compounds, therefore, continue to pose a threat to human health. Chronic exposure to PCBs has led to a variety of human health effects including decreased body weight, chloracne, hepatic hypertrophy, and immunosuppression (2). Animal studies have also indicated liver abnormalities including hepatocellular carcinoma (2, 5). In poisoned humans, liver damage, dermal lesions, respiratory disorders, severe ocular signs, neurological symptoms, endocrine damage, immunodeficiency, and reproductive disorders were seen (6). PCBs have also been implicated or related to cancers such as malignant melanoma, breast and lung cancers in exposed populations (7). However, the exact roles of PCBs in the carcinogenic process

10.1021/tx010128+ CCC: $22.00 © 2002 American Chemical Society Published on Web 03/19/2002

498

Chem. Res. Toxicol., Vol. 15, No. 4, 2002

remain unclear. It has been shown that PCBs are good promoters of cancer (2, 8), but the role of PCBs as initiators remain unknown despite some evidence of mutagenic potential (9). Most chemical carcinogens are metabolized by enzymes to the ultimate electrophilic forms, which can react with DNA and other nucleophiles, leading to mutations, and eventually cancer (2). The hydroquinone/quinone metabolites of benzene, o-phenylphenols, polycyclic aromatic hydrocarbons (PAHs), and estrogens have been shown to play an important role in their respective toxicities (10-14). Since PCBs are structurally similar to these compounds, we have hypothesized that the hydroquinone and quinone metabolites of PCBs play a role in PCBmediated toxicity. Our laboratory has shown that lower halogenated PCBs can be metabolized by cytochrome P450s in vitro to o- or p-dihydroxy metabolites (catechols or hydroquinones, respectively) and peroxidases can further metabolize these to quinones (15-18). PCB hydroquinones can be converted to PCB quinones by autoxidation or by peroxidases and/or prostaglandin synthase (15-17, 19). This process is accompanied by reactive oxygen species (ROS) production and also by the probable formation of the semiquinone as an intermediate (17, 19). This ROS production by the PCB metabolites has been shown to cause 8-oxo-dG (8-oxo-deoxyguanosine) formation and DNA strand breaks in vitro (17, 19). PCB metabolites also produce ROS in HL-60 cells (19). PCB quinones react instantaneously with GSH to form 1,4Michael addition products (20), and ROS may be generated during the oxidation of the products (19, 20). These reactions in cells would deplete antioxidants and potentially further increase oxidative stress. Our goal was to investigate the reactivity of these PCB metabolites with sufhydryl groups: how many molecules GSH could be bound per molecule of PCB metabolite, whether PCB metabolites could inhibit vital cellular proteins, and whether sulfhydryl binding and redox cycling that was described in vitro would also occur in cells in culture. We therefore investigated the reactivity of hydroquinone and quinone metabolites of PCBs both in vitro and in cell systems by (1) estimating quantitative GSH binding in vitro with/without enzymatic oxidation, (2) testing GSH and thiol depletion of HL-60 cells in culture, (3) determining the correlation between GSH status and cytotoxicity of these compounds in HL-60 cells, and (4) assessing topoisomerase II activity, an essential sulfhydryl-containing cellular protein, after exposure to the PCB metabolites in vitro. These studies should give us insight into the mechanisms of toxicity of the analyzed PCB metabolites.

Materials and Methods Chemicals. Agarose, glutathione (GSH), EDTA, monobromobimane (mBBr), dimethyl sulfoxide (DMSO), dithionitrobenzoic acid (DTNB), NADPH, Tris, ethidium bromide, glutathione reductase (EC 1.6.4.2), N-ethylmaleimide (NEM), propidium iodide (PI), and horseradish peroxidase (HRP, EC 1.11.1.7) were purchased from Sigma Chemical Co. (St. Louis, MO). Metaphosphoric acid and H2O2 (3%) were from Aldrich Chemical Co. (Milwaukee, MI). Topoisomerase II kits were purchased from TopoGen Inc. (Columbus, OH). RPMI 1640, fetal calf serum (FCS), penicillin, and streptomycin were obtained from Gibco BRL (Grand Island, NY). Test Compounds. The structures and abbreviated nomenclature of the test compounds used in this study are shown in

Srinivasan et al.

Figure 1. Chemical structures and nomenclature of the PCB derivatives used. Figure 1. The detailed chemical synthesis and characterization of these compounds have been described (16, 17, 19, 21). Caution: Synthetic PCB metabolites should be considered potentially toxic and hazardous and should therefore be handled in an appropriate manner. Determination of in Vitro Binding of PCB Metabolites to GSH. (A) Monobromobimane Assay. Monobromobimane (mBBr) is a dye that is fluorescent after binding to thiols such as GSH. For the first quantitative binding studies, 10 µM aliquots of selected quinones and hydroquinones were incubated with 5, 10, 20, 30, and 40 µM concentrations of GSH in 3 mL of 10 mM phosphate buffer for 1 h at 37 °C. Then mBBr was added to a final concentration of 26.6 µM, and the samples were incubated for an additional 30 min. The samples were cooled on ice, and the fluorescence was measured using a RF-5301 PC Shimadzu spectrofluorophotometer at 395 nm excitation wavelength and 470 nm emission wavelength. Using a GSH standard curve, the amount of free GSH in the PCB-GSH mixtures was determined and used to calculate the amount of GSH bound to PCB metabolites. (B) Enzyme Recycling Method. A modified method of Tietze et al. (22) was used to more accurately quantify GSH binding to PCB quinones and hydroquinones. Briefly, a 100 µM sample of each PCB metabolite was incubated for 1 h at 37 °C in an aqueous solution with 50, 100, 200, and 400 µM GSH. The amount of remaining free GSH was determined by adding a 100 µL aliquot of the incubated samples to 800 µL of phosphate/ EDTA buffer (0.2 mM potassium phosphate, 0.01 M EDTA, pH 7.4) containing GSH reductase (0.5 unit/mL) and dithionitrobenzoic acid (DTNB, 0.2 mg/mL). After 2 min incubation at 25 °C, 100 µL of a NADPH solution (5 mg/mL) was added to the samples to start the reaction, and the increase in absorbance at 412 nm (TNB formation) was monitored with a Shimadzu MPS-2000 spectrophotometer for 6 min. Using a GSH standard curve, the amount of free GSH in the PCB-GSH mixtures was determined and used to calculate the amount of GSH bound to PCB metabolites. Data shown are the average binding ratio of three independent experiments. A Pearson correlation analysis was performed to compare the binding ratios obtained by the two methods described above. Spectral Analysis. Changes in the spectra of the test compounds were used to monitor oxidation and reduction reactions. 2-ClPh-HQ (10 µL of a 10 mM stock in DMSO) was

Sulfhydryl Binding by PCB Metabolites added to 1 mL of phosphate buffer (20 mM, pH 7) at 37 °C containing 1 unit of HRP (10 µL of 0.1 unit/µL stock). The HQ was completely oxidized to the quinone by sequential additions of H2O2 (5 µL of 1 mM stock each). The resulting quinone was reduced to the HQ adduct by adding equal amounts of GSH (10 µL of 10 mM stock; 100 µM final concentration). The product of this reaction was again reoxidized with HRP and sequential additions of H2O2 and reduced once more with GSH. This cycle of reactions was continued until the product could no longer be oxidized. The reactions were monitored by recording each spectrum over a range of 275-650 nm with a Shimadzu MPS2000 UV-vis spectrophotometer. Two independent experiments were performed to confirm these results. Cell Culture. The human promyelocytic leukemia cell line HL-60 was obtained from Dr. M. Doukas, University of Kentucky, Lexington, KY. HL-60 cells (passages 60-90) were cultured in RPMI 1640 medium supplemented with 10% FCS, penicillin (0.1 unit/mL), and streptomycin (0.1 µg/mL). Cells were grown in a humidified atmosphere in 5% CO2 and 37 °C. Quantitative Determination of Intracellular Total GSH by an Enzyme Recycling Assay. HL-60 cells (1 × 106/mL, 5 mL per sample) were exposed to different concentrations of 2ClPh-pQ or 2ClPh-pHQ (5, 10, or 40 µM) for different times (1, 3, or 6 h) at 37 °C. Cells were then centrifuged, washed in PBS, resuspended in 500 µL of 1:1 PBS/10% metaphosphoric acid, and stored immediately at -80 °C. For determination of total intracellular GSH, cells were thawed, sonicated, and centrifuged, and an aliquot of the supernatant was analyzed for total GSH by the enzyme recycling method as described above. A GSH standard curve was used to determine the nanomoles of GSH/106 cells per sample. The assay was carried out in triplicate. Statistical significance (ANOVA) was calculated using the General Linear Model (GLM) and a post-hoc Bonferroni test. Determination of Relative Intracellular Free Thiol Groups with mBBr. A total of 3 × 106 HL-60 cells in 3 mL of PBS at 37 °C was exposed for 1 h to 1.25-40 µM test compound. Then mBBr was added to a final concentration of 26.6 µM, and the samples were incubated for an additional 30 min at 37 °C in the dark. The samples were cooled on ice, and fluorescence was measured immediately using a RF-5301 PC Shimadzu spectrofluorophotometer at 395 nm excitation and 470 nm emission wavelengths. Two independent experiments were performed with triplicate samples. Statistical significance was calculated using a SAS program (PROC MIXED), the General Linear Model for ANOVA, and the post-hoc Bonferroni test. Flow Cytometry Analysis of Cytotoxicity and Intracellular Thiol Groups. Cells were divided into two groups, and one group was pretreated for 30 min with 20 µM NEM to deplete intracellular GSH. Both groups were then divided into batches of (1-2) × 106 HL-60 cells/mL of PBS and exposed to different concentrations of 2ClPh-pQ or 2ClPh-HQ at 37 °C for 30 min. Then mBBr was added to a final concentration of 20 µM, and the samples were further incubated for an additional 30 min after which they were analyzed using a Mo Flo (Cytomation, Fort Collins, CO) flow cytometer. For cytotoxicity determination, propidium iodide (PI) was added to a final concentration of 10 µg/mL immediately before flow cytometry analysis. Intracellular fluorescence due to mBBr and PI was measured in about 20 000 cells. At least two independent experiments were performed to confirm the results. Statistical significance was calculated using the General Linear Model for ANOVA and the post-hoc Bonferroni test. Inhibition of Topoisomerase II. Topoisomerase II assays were performed according to the protocol provided by TopoGen Inc. (Columbus, OH). The test compound in 0.5 µL of DMSO was added to reaction buffer (50 mM Tris-HCl, pH 8, 0.5 mM ATP, 0.5 mM dithiothreitol, 10 mM MgCl2, 120 mM KCl, 30 µg of bovine serum albumin/mL) containing 0.2 µg of kDNA, topoisomerase II (4 units, 170 kDa form). Deionized water was added to a final volume of 20 µL. After incubation for 1 h at 37 °C, the reactions were terminated with 5 µL of stop solution

Chem. Res. Toxicol., Vol. 15, No. 4, 2002 499 Table 1. Binding Ratios of PCB Metabolites with Glutathione mol of GSH/ mol of PCB-pQ

2ClPh3ClPh4ClPh3,4ClPh3,5ClPh3,4,5ClPh-

mol of GSH/ mol of PCB-HQ

mBBr assay

enzyme assay

mBBr assay

enzyme assay

1.45 ( 0.08a 1.36 1.64 1.32 1.65 ntb

1.29 ( 0.12 1.33 ( 0.08 1.43 ( 0.11 1.35 1.94 ( 1.11 nt

0.00 ( 0.00 0.00 0.00 0.00 0.00 0.00

0.17 ( 0.04 0.22 0.15 0.46 0.31 0.51

a Where standard deviations are as shown, values are means ( SD with n ) 5. b nt: not tested.

(5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol). The samples were loaded immediately onto 1% agarose gels containing ethidium bromide (0.5 µg/mL). The products were resolved by gel electrophoresis for 1-2 h at 4 V/cm in Tris-acetate buffer (1× TAE, pH 8), which separated the catenated kinetoplast DNA from the decatenated DNA monomers. Following electrophoresis, the gels were destained in water for 30 min, and the DNA was visualized with an UV transilluminator. Pictures were taken with a Sony video graphics printer video camera attached to a Biophotonica gel print 2000I apparatus and used for documentation. Every experiment was repeated at least twice for confirmation of the results.

Results In Vitro Glutathione Binding. To determine nonenzymatic GSH binding, PCB metabolites were incubated for 1 h with 0.5×, 1×, 2×, and 4× molar equivalents of GSH, and the remaining free GSH was determined with two different methods. From those data, the molar binding ratio was calculated (Table 1). The mono- and di-chlorinated p-quinones tested bound GSH at a ratio of about 1:1.5. The standard variation in the enzyme recycling method reflects the variations among three independent experiments. No significant differences were seen among the various PCB-Qs in GSH binding. Also, both assay methods gave similar results with a Pearson correlation factor of 0.712 between the two methods. With mono-, di-, and tri-chlorinated PCB-HQs, no (mBBr assay) or very little (enzyme recycling method) GSH binding was observed. No difference in GSH binding among the different PCB hydroquinones was detected, and the results from the two different assays were comparable. Elucidation of GSH Binding and Product Oxidation through Spectral Changes. PCB quinones and hydroquinones are aromatic compounds with distinct spectral characteristics. We therefore attempted to follow the conjugation reaction between quinones and GSH and the enzymatic oxidation of PCB hydroquinones or their GSH conjugates by spectral analysis. Synthetic 2ClPhHQ has a characteristic absorbance maximum at about 300 nm (Figure 2A, marked with downward arrow). Repeated additions of H2O2 to the HRP-containing solution of 2ClPh-HQ resulted in the disappearance of this peak and a new peak at about 350 nm (Figure 2A, marked with upward arrow). This new spectrum is identical with that of synthetic 2ClPh-pQ, indicating the enzymatic oxidation of the HQ to the corresponding quinone. Addition of equimolar GSH changed the spectrum again. Now a peak at about 325 nm was visible, presumably representing a mono-glutathionyl hydroquinone (unmarked peak). Figure 2B shows that the mono-glutathionyl hydroquinone may be enzymatically

500

Chem. Res. Toxicol., Vol. 15, No. 4, 2002

Srinivasan et al.

Figure 2. Spectra of 2ClPh-HQ after alternate additions of HRP and H2O2 (A, C, E) and GSH (B, D, F).

oxidized, presumably to the mono-glutathionyl quinone, by the stepwise addition of H2O2 (decrease in the peak at 326 nm, increase of the peak at 350 nm). A second addition of equimolar amounts of GSH to the monoglutathionyl quinone caused an immediate change from the quinone-like to a hydroquinone-like spectrum with a new peak at about 325 nm, indicative of the formation of a di-glutathionyl hydroquinone conjugate (Figure 2C). Figure 2D shows that this di-glutathionyl hydroquinone can be oxidized once more to the corresponding diglutathionyl quinone. Again, this di-glutathionyl quinone may be reduced by the addition of GSH, presumably to a tri-glutathionyl hydroquinone (Figure 2E). Figure 2F shows that the tri-glutathionyl hydroquinone did not undergo enzymatic oxidation when H2O2 was added. Depletion of GSH and Other Free Thiols by PCB Metabolites in Cells in Culture. (A) Quantification of Intracellular GSH with the Enzyme Recycling Method. The intracellular total GSH content of untreated HL-60 cells was approximately 2.5 nmol/106 cells, as determined by a modified enzyme recycling method of Tietze et al. (22). Treatment for 1 h with 5-40 µM 2ClPh-HQ slightly reduced the amount of intracellular GSH, but this reduction was not statistically significant (Figure 3A). Exposure to 5 µM 2ClPh-pQ also had no significant effect; however, a 10 µM aliquot of the quinone reduced intracellular total GSH by about 28% to approximately 1.8 nmol/106 cells, which was significantly different from solvent-treated control. Treatment for 1 h with 40 µM 2ClPh-pQ resulted in a nearly complete depletion of intracellular GSH (>90%). Exposure of HL-60 cells to 5 µM 2ClPh-HQ for 1-6 h resulted in a small, nonsignificant reduction of GSH after 1 h and a small, but not significant increase of GSH after 3 and 6 h of treatment (Figure 3B). Exposure of cells for 1-6 h to 5 µM 2ClPhpQ caused a reduction of intracellular GSH at all time points measured, but this reduction was only statistically significant after 6 h of exposure. (B) Determination of Cellular Thiol Depletion by mBBr Spectrofluorometry. Monobromobimane can diffuse into living cells and forms fluorescent adducts after binding to free -SH groups (23). Since GSH is the major thiol present in cells, most, but not all, of the fluorescence may

Figure 3. (A) Intracellular GSH concentration in HL-60 cells treated for 1 h with different concentrations (5, 10, and 40 µM) of 2ClPh-HQ or 2ClPh-pQ, measured with the enzyme recycling method. (*) Significantly different from solvent control (p < 0.05). (B) Intracellular GSH concentration in HL-60 cells treated with 5 µM 2ClPh-HQ or 2ClPh-pQ for 1, 3, and 6 h, measured with the enzyme recycling method. (*) Significantly different from solvent control (p < 0.05).

be due to the binding to intracellular reduced GSH. Total fluorescence of a cell suspension can be determined by spectrofluorometry. To analyze the dose-response relationship between PCB treatment and fluorescence, HL60 cells were treated for 1 h with 1.25-40 µM 2ClPhpQ, after which mBBr was added for an additional 30 min. Figure 4A shows that all concentrations of 2ClPhpQ reduced intracellular free thiol groups. This effect was statistically significant at concentrations of 2.5 µM or more. Free intracellular thiol groups were depleted by more than 75% at concentrations of 10 µM 2ClPh-pQ or more. Figure 4A also shows the results with different concentrations of 2ClPh-HQ. The lower concentrations tested (2.5 and 10 µM) did not significantly deplete GSH from HL-60 cells. At the highest concentration (40 µM), slight but not significant depletion of GSH was seen. The four other mono- and di-chlorinated PCB quinones tested caused a similar decrease in intracellular free thiol groups at the three concentrations (3, 10, 40 µM) tested (Figure 4B). No structure-activity relationship (SAR) could be observed among the different PCB quinones in this assay, because all quinones were about equally as potent as efficacious under the conditions of this assay. Thiol Depletion vs Cytotoxicity of PCB Treatments. (A) Flow Cytometry Analysis of Intracellular Free Thiols and Cell Viability. Thiol depletion may be a cause or a consequence of cell death. To determine the relative

Sulfhydryl Binding by PCB Metabolites

Chem. Res. Toxicol., Vol. 15, No. 4, 2002 501

Figure 4. (A) Intracellular GSH/thiol status in HL-60 cells treated with different concentrations of 2ClPh-pQ or 2ClPh-HQ determined with the monobromobimane assay. (*) Significantly different from solvent control (p < 0.01). (B) Intracellular GSH/ thiol status in HL-60 cells after treatment with different PCB quinones determined with the monobromobimane assay. All treatments caused significant effects (p < 0.01) compared to solvent control. Table 2. Influence of Intracellular GSH Status on HL-60 Viability after Treatment with PCB-HQ or PCB-pQ with/without 20 µM NEM Pretreatment Measured by Flow Cytometry compound

concn (µM)

control 2ClPh-HQ

0 2.5 10 40 2.5 10 40

2ClPh-pQ

PI negative cells (% of total)

% mBBr positive cells (of PI negative)

-

with NEM

-

with NEM

82.9 ( 1.32 80.4 ( 1.73 79.6 ( 0.44 80.8 ( 2.10 78.5 ( 2.11 40.2 ( 5.69a 0.34 ( 0.03a

77.7 ( 3.19 63.9 ( 12.2 71.7 ( 8.76 70.6 ( 2.03 52.2 ( 9.41a 18.6 ( 4.54a 0.27 ( 0.05a

91.4 ( 9.41 91.4 ( 5.95 87.3 ( 7.83 86.7 ( 16.3 85.2 ( 11.4 65.9 ( 11.8 0.41 ( 0.71a

12.2 ( 7.72 5.03 ( 2.55 7.57 ( 4.93 4.67 ( 3.23 6.09 ( 7.09 15.2 ( 8.59 0.00 ( 0.00

a Statistically significant as compared to DMSO control (p < 0.01).

thiol concentration in living and dead cells, the samples were double-stained with mBBr and PI, and the fluorescence in PI-negative and -positive cells was determined. PI can only penetrate into dead cells through the impaired cell membranes. The data in Table 2 show that approximately 80% of cells treated with solvent alone, 2.5, 10, or 40 µM 2ClPh-HQ or 2.5 µM 2ClPh-pQ, were PI-negative (living). However, at higher concentrations of 2ClPh-pQ (10 and 40 µM), only 40% and 0.34% of the cells were PI-negative, respectively. These data indicate that 2ClPh-HQ was not toxic; however, 2ClPh-pQ resulted in about 50% (10 µM) and 100% (40 µM) toxicity during this treatment. Of these PI-negative cells, about

91% were mBBr-positive in the solvent control sample. 2ClPh-HQ caused a slight, dose-dependent, but not statistically significant reduction in the percent of mBBrpositive cells. High concentrations of 2ClPh-pQ caused a significant reduction in the percent of cells that were mBBr-positive and PI-negative. All PI-positive (dead) cells were mBBr-negative (thiol depleted, data not shown). (B) Cytotoxicity of PCB Treatment in GSH-Depleted Cells. N-Ethylmaleimide (NEM) is a known GSH-depleting agent. Table 2 shows the results of an experiment in which cells were pretreated for 30 min with noncytotoxic concentrations of NEM to deplete intracellular GSH, followed by a 60 min treatment with PCB metabolites and determination of GSH and viability. NEM-treatment alone did not reduce viability significantly, but reduced the viable mBBr positive cells to about 12%. Treatment for 60 min with PCB-HQ slightly reduced viability, but this was not statistically significant, whereas PCB-Q significantly reduced viability even at the lowest concentrations tested (2.5 µM). Higher concentrations reduced the number of viable cells to less than 20%. mBBr staining in viable cells of the PCB metabolite treated groups was further reduced to about 5-6% on an average. Inhibition of Topoisomerase II. Topoisomerase II is a nuclear enzyme that induces organized double strand breaks and reattachment during DNA replication and other events. To measure this activity, the Topo II assay uses purified human topoisomerase II and kDNA, a high molecular weight catenated network of mitochondrial DNA rings, isolated from Crithidia fasciculata (24, 25). The Topo II reaction results in a decatenation of the kDNA and the appearance of 2.5 kilobase monomers in either the open circle or the relaxed forms following gel electrophoresis. Using this assay, the PCB quinones and hydroquinones were tested for inhibitory activity on purified human topoisomerase II. The first three lanes and the last lane in Figure 5A show a marker, kDNA in the presence of Topo II and kDNA alone or in the presence of the solvent DMSO (lanes 1, 2, 3, and 11, respectively). High molecular weight kDNA does not migrate in the gel under our conditions, but active Topo II cuts and reunites the rings so that individual relaxed circles and open circles are formed. The solvent DMSO had no effect on the kDNA (lane 11); neither did 100 µM 2ClPh-pQ in the absence of Topo II (lane 10). 2ClPh-pQ at 100, 50, or 25 µM concentrations inhibited Topo II completely, about 50%, or slightly, as can be seen in lanes 4, 5, and 6, respectively. Lower concentrations of 2ClPh-pQ had no visible effect (lanes 7-10). These results indicate a dose-dependent inhibition of topoisomerase II activity by 2ClPh-pQ. Figure 5B shows the effect of GSH on Topo II inhibition by 2ClPh-pQ. At half the molar concentration (50 µM), GSH prevented topoisomerase II inhibition by 100 µM 2ClPh-pQ almost by 50% (lane 3). An equimolar concentration of 100 µM GSH prevented topoisomerase II inhibition by almost 70%. Higher concentrations (200 and 400 µM) of GSH resulted in complete prevention of the inhibitory activity of 2ClPh-pQ on topoisomerase II activity. Figure 5C is similar to Figure 5A, with the exception that the 2ClPh-HQ was added instead of the 2ClPh-pQ. As can be seen in lanes 4-10, this PCB hydroquinone did not inhibit topoisomerase II at any of the concentrations tested (100, 50, 25, 10, 5, 2.5, and 1 µM). For comparison, a marker DNA (lane 1) and kDNA in the

502

Chem. Res. Toxicol., Vol. 15, No. 4, 2002

Figure 5. (A) Topoisomerase II inhibition by 2ClPh-pQ. Lane 1, kDNA linear marker; lane 2, Topo II (4 units) alone; lane 3, kDNA alone; lanes 4-10, topoisomerase II (4 units) with different concentrations of 2ClPh-pQ; lane 11, vehicle control; lane 12, 2ClPh-pQ alone. NM, normal; OC, open circle; REL, relaxed circle. (B) Topoisomerase II inhibition by 100 µM 2ClPhpQ in the absence (lane 2) or presence of 50 µM GSH (3), 100 µM GSH (4), 200 µM GSH (5), or 400 µM GSH (6). Lane 1, Topo II without 2ClPh-pQ (control). NM, normal; OC, open circle; REL, relaxed circle. (C) Topoisomerase II inhibition by 2ClPhpHQ. Lane 1, kDNA linear marker; lane 2, Topo II (4 units) alone; lane 3, kDNA alone; lanes 4-10, topoisomerase II (4 units) with different concentrations of 2ClPh-pHQ; lane 11, vehicle control; and lane 12, 2ClPh-pHQ alone. NM, normal; OC, open circle; REL, relaxed circle.

presence (lane 2) or absence of Topo II (lane 3) were included in the experiment. The solvent DMSO alone did not inhibit Topo II activity (lane 11), nor did 100 µM 2ClPh-HQ in the absence of Topo II induce any strand breaks (lane 12).

Discussion PCBs are metabolized to hydroxylated derivatives (16, 26-31), some of which were shown to persist in human tissues (32). In this study, we analyzed the reactivity of PCB hydroquinones and their oxidation products, the corresponding quinones, with glutathione and cellular proteins in vitro and in cells in culture. We used two different methods, a spectrophotometric assay with mBBr and an enzymatic method, to determine

Srinivasan et al.

quantitatively the amount of spontaneous GSH binding to different mono- and di-chlorinated PCB quinones and mono- to tri-chlorinated PCB hydroquinones. Both methods show that PCB quinones bind GSH in vitro at a ratio of about 1.5 GSH per molecule of PCB quinone under these assay conditions (Table 1). No SAR could be observed, although 3,5ClPh-pQ had the highest binding ratio of all PCB quinones tested in both test systems. If the test compounds would cause an oxidation of GSH to GSSG, the binding ratios in the mBBr assay should be higher than in the enzyme assay. Since this was not the case, GSH oxidation seems to be of minor importance under these assay conditions. The PCB hydroquinones caused only little reduction of unbound GSH (Table 1). Binding ratios between 0 and 0.51 indicate that only limited autoxidation to the quinone occurred under these conditions. Amaro et al. (20) have shown that 4ClPh-pQ binds GSH in vitro instantaneously. After incubating 4ClPhpQ with approximately a 25-fold excess of GSH at pH 9.0 for 5 min, they isolated the major adduct and characterized the chemical structure by NMR and FAB/ MS determination as a monoglutathionyl-hydroquinone adduct, with GSH bound in 3-position on the quinone ring. A second peak, representing a diglutathionylhydroquinone adduct, was also observed. Since in our experiments the binding ratios of the quinones were between 1.29 and 1.94, we have to assume that some of the PCB-glutathione adducts autoxidized and underwent a second addition with GSH. To quantify the maximum possible glutathione addition, we applied several cycles of enzymatic oxidation followed by equimolar GSH addition. With this approach, up to three GSH molecules could be added to 2ClPh-HQ, before further enzymatic oxidation failed (Figure 2). The formation of mono-, di-, and tri-GSH conjugates of 2ClPh-HQ was confirmed by HPLC (data not shown). This means that these PCB metabolites could possibly cause significant GSH depletion in cells containing PCB-HQ oxidizing enzymes. Although GSH is present at high levels in mammalian cells (millimolar concentrations) (33), it can be depleted by electrophilic compounds, either nonenzymatically or by the action of glutathione-S-transferases. Therefore, we tested the GSH status of HL-60 human leukemia cells, which are rich in myeloperoxidase, after exposure to our PCB derivatives. 2ClPh-pQ depleted total GSH, measured with the enzyme recycling method, in HL-60 cells in a dose- and time-dependent manner (Figure 3A,B). With the mBBr method, significant effects were seen with as little as 2.5 µM 2ClPh-pQ after a total of 1.5 h incubation (Figure 4A). All other PCB quinones tested were equally active (Figure 4B). No significant effect was seen with 2ClPh-HQ using either method, enzyme recycling or mBBr (Figures 3A,B and and 4A). We have reported previously that in vitro oxidation of PCB hydroquinones to the corresponding quinone is accompanied by the production of reactive oxygen species (ROS), and we observed oxidative stress in HL-60 cells treated with PCB hydroquinones (19). Intracellular ROS are detoxified by GSH with the formation of oxidized GSH. However, no significant decrease in intracellular thiols after 2ClPhHQ treatment was seen with enzyme recycling (total GSH) or mBBr (free thiols). We also did not observe a significant increase in the levels of oxidized glutathione measured in HL-60 cells by HPLC after exposure to

Sulfhydryl Binding by PCB Metabolites

2ClPh-pQ or 2ClPh-HQ (data not shown). This suggests that oxidized GSH accumulation is not a sensitive marker of exposure in this system. Previous studies in liver cells have shown that severe GSH depletion is accompanied by pathological consequences such as lipid peroxidation and liver necrosis (34). To study the correlation between toxicity and GSH status, we used flow cytometry and double staining with mBBr and PI for GSH and toxicity, respectively. These flow cytometry analyses showed that high concentrations of 2ClPh-pQ (g10 µM) strongly decreased the fraction of living cells and also intracellular free thiols in these living cells (Table 2). Lower concentrations of 2ClPh-pQ and 2ClPh-HQ had no significant effect on either parameter. This suggests a connection between GSH/thiol depletion in HL-60 cells by PCB quinones and cell death. To further analyze this connection, we pretreated cells with NEM, a known GSH depleting agent. Although NEM reduced free intracellular thiols to about 12%, this alone or together with 2ClPh-HQ had no significant effect on cell viability (Table 2). However, GSH depletion increased the toxicity of the PCB quinone. This suggests that GSH protects cells from PCB quinone-induced toxicity. GSH is an ubiquitous intracellular tripeptide involved in many physiological and protective functions such as conjugation of xenobiotics, thus facilitating their excretion, detoxification of metal ions, scavenging and removal of ROS and their reaction products such as lipid peroxides, and maintenance of protein sulfhydryls (35-37). When GSH depletion reaches a certain threshold, lipid peroxidation develops in an abrupt and extensive way (34). GSH depletion of 20-30% of the total levels can impair the cell’s defense against oxidative damage from H2O2 and lead to cell death (38). We had observed an increase in intracellular oxidative stress in cells treated with PCB-HQs or -quinones (19). It remains to be studied whether GSH depletion or redox reactions of PCB metabolites are responsible for this increase in intracellular oxidative stress and its role in 2ClPh-pQ-induced cell death. GSH also maintains cell viability via the maintenance of protein thiols, which are essential for Ca2+ transport and other membrane activities. Loss of protein thiols can lead to a disturbance in the cellular calcium homeostatis and cell death (34). However, NEM depleted intracellular GSH even stronger than 2ClPh-pQ without being cytotoxic. At the same time, NEM increased the toxicity of the 2ClPh-pQ. This suggests that the binding of PCB quinone to other, vital cellular thiols may be an important factor in its toxicity. One important cellular protein is topoisomerase II, a nuclear enzyme that is inhibited by quinone or quinoneforming compounds (24). Hence, the PCB metabolites, particularly the quinones, were considered potential topoisomerase II inhibitors. This could be one mechanism involved in PCB quinone-induced toxicity, since topoisomerase inhibitors are cytotoxic and often used as anticancer agents (39-41). Our results show that indeed 2ClPh-pQ does inhibit topoisomerase II in vitro in a dosedependent manner (Figure 5A), and that this inhibition is preventable by increasing concentrations of GSH (Figure 5B). Two groups reported that 1,4-benzoquinone inhibits topoisomerase II in vitro (42, 43). In this case as well, GSH was protective, probably by competing with the topoisomerase II protein for binding to the quinone (42). Most medicinal topoisomerase II inhibitors, like mitoxantrone, BE 10988, doxorubicin, and etoposide,

Chem. Res. Toxicol., Vol. 15, No. 4, 2002 503

exhibit a quinone or quinone-forming structure (39, 44, 45). Therefore, it is assumed that this essential protein has a free -SH group or nucleophile present in the DNA binding/active site of the enzyme, resulting in inactivation after binding of a quinone to this moiety. The relative potency of topoisomerase II inhibition by 2-ClPh-pQ should be considered cautiously, since the commercial assay kit contains high amounts of dithiothreitol and BSA, both of which may react with and inactivate the quinone. Indeed, Franz and co-workers (42) reported that inhibition of topoisomerase II by 1,4-benzoquinone could be seen at about 50× lower concentrations, if the compound was added directly to the enzyme. This could mean that 2ClPh-pQ might inhibit topoisomerase II already at nanomolar concentrations, with complete inhibition at the low micromolar range. Topoisomerase II is needed to release the tortional tension of the double helix during DNA synthesis, replication, and other events through double strand scission and rejoining. Topoisomerase II inhibitors have been shown to be strong clastogens in mammalian cells (46, 47). Thus, the PCB quinones are potential clastogens. Indeed, chronic exposure to PCBs was reported to cause chromosomal aberrations (48). From our studies, we predict the involvement of PCB quinones in these effects. Correlating with our in vitro GSH binding and cellular GSH depletion results, the PCB hydroquinones did not inhibit topoisomerase II in vitro (Figure 5C), but like hydroxylated benzene metabolites (24) can be expected to be bioactivated by peroxidases. According to O’Brien, quinones can be cytotoxic by two mechanisms: (i) by binding to GSH and cellular macromolecules, causing irreversible changes resulting in cell death, the so-called “covalent binding to proteins mechanism”; and (ii) by the generation of ROS through redox cycling, called the “oxidative stress theory” (49). Toxicity and depletion of cellular GSH by naphthoquinones was shown to be caused by both direct arylation of GSH and redox cycling (50). This seems also to be true for the PCB metabolites we tested. Undoubtedly, PCB quinones do bind to GSH and free protein thiols, and this is involved in their toxicity, since GSH depletion increases vulnerability to PCB quinone cytotoxicity. Thus, the toxicity of PCB quinones, like other quinones, could be due to the interaction with GSH and subsequent disturbance of calcium and cellular energy homeostasis (51), and/or inhibition of many other essential cellular proteins, like topoisomerase II, the example shown here. In addition, PCB quinones can redox-cycle via enzymatic one- and two-electron reduction-oxidation reactions (52), and the PCB hydroquinones and GSH adducts of PCB quinones can be oxidized to the corresponding quinone, both reactions resulting in the formation of ROS (19). Here we have shown that PCB hydroquinones can undergo up to three rounds of oxidation and reduction by GSH addition. Hence, this mechanism may be of major importance for the biological reactivity of the quinones mainly because of their ability to form multiple addition products called quinone-thioethers. In addition, such quinone-thioethers in turn can redox-cycle and generate ROS (53). Previous in vitro studies in our laboratory have shown the formation of PCB quinone thioethers, ROS production, and DNA damage in the form of 8-oxo-dG formation and DNA strand breaks (17, 19, 20). We have also shown increased intracellular ROS in cells in culture treated with PCB hydroquinones and quinones (19). PCB

504

Chem. Res. Toxicol., Vol. 15, No. 4, 2002

quinone toxicity hence can also be due to oxidative stress by redox cycling. Therefore, PCB quinones may be toxic by either or both mechanisms. PCB hydroquinones, on the other hand, are fairly inactive themselves; however, they can be oxidized to a PCB quinone and thereby activated. This activation occurs with the formation of reactive intermediates, the semiquinones, which may be involved in DNA adduct formation (15, 17, 18, 20), and is, of course, accompanied by the generation of ROS both in vitro and in HL-60 cells (19). In vitro, this resulted in the production of oxidative DNA damage like 8-oxo-dG and DNA strand breaks (17, 19). In addition, exposure to ROS results in other forms of DNA damage which include lesions such as modified bases, abasic sites, single and double strand breaks, and DNA-protein cross-links, eventually leading to cytotoxicity (54). Although the hydroquinones were not toxic in our experiments during the 1 h exposure period, PCB hydroquinones should be cytotoxic as well, providing that the cellular environment allows for oxidation reactions of this PCB metabolite. Altogether, the formation of reactive metabolites and/ or intermediates via the oxidation of PCBs to dihydroxy and quinone metabolites may be an important parameter in PCB toxicity. Investigations reveal that hydroxylated metabolites of PCBs are retained in human plasma and in rodent tissue (32, 55, 56). Further, these hydroxylated metabolites can be oxidized to reactive intermediates and further to the PCB quinones. Lin and co-workers reported the formation of quinone-derived protein adducts in the liver and brain of Sprague-Dawley rats treated with 2,2′,5,5′-tetrachlorobiphenyl (57). In this study, we have shown that these PCB metabolites (the PCB quinones in particular) bind to GSH both in vitro and in HL-60 cells, inhibit topoisomerase II in vitro, and cause cytotoxicity. Hence, these metabolites may play an important role in mediating the effects of PCBs through the arylation and inactivation of cellular proteins, by increasing the susceptibility to oxidative damage by depleting radical scavengers such as GSH, and also through the production of ROS by redox cycling reactions. Thus, the persistence of these PCB metabolites in vivo is concerning and might explain the PCB-mediated toxicities in humans and other organisms.

Acknowledgment. This publication was made possible by Grant DAMD 17-96-1-6262 from the DOD, Grant P42 ES 07380 from the NIEHS, and Grant 85-001-13IRG from the American Cancer Society. We thank Dr. Parvaneh Espandiari for technical assistance, Dr. Marta Mendiando and Dr. Daria Pereg for help with the statistical analyses, and Ms. Nilufer Tampal and Dr. Wendy Smith for useful suggestions. Flow cytometry analyses were performed by Jennifer Strange and Greg Bauman, Department of Microbiology/Immunology, University of Kentucky, Lexington, KY. A.S. was supported in part by a training grant from the Superfund Basic Research Program (ES-07380). Contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH, DOD, or ACS.

References (1) Safe, S. H. (1994) Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment. Crit. Rev. Toxicol. 24, 87-149.

Srinivasan et al. (2) Silberhorn, E. M., Glauert, H. P., and Robertson, L. W. (1990) Carcinogenicity of polyhalogenated biphenyls: PCBs and PBBs. Crit. Rev. Toxicol. 20, 440-496. (3) Tanabe, S. (1988) PCB problems in the future: Foresight from curent knowledge. Environ. Pollut. 50, 5-28. (4) Jensen, S. (1966) Report of a new chemical hazard. New Sci. 32, 612. (5) Mayes, B. A., McConnell, E. E., Neal, B. H., Brunner, M. J., Hamilton, S. B., Sullivan, T. M., Peters, A. C., Ryan, M. J., Toft, J. D., Singer, A. W., Brown, J. F., Jr., Menton, R. G., and Moore, J. A. (1998) Comparative carcinogenicity in Sprague-Dawley rats of the polychlorinated biphenyl mixtures Aroclors 1016, 1242, 1254, and 1260. Toxicol. Sci. 41, 62-76. (6) Lang, V. (1992) Polychlorinated biphenyls in the environment. J. Chromatogr. 595, 1-43. (7) Cogliano, V. J. (1998) Assessing the cancer risk from environmental PCBs. Environ. Health Perspect. 106, 317-323. (8) Glauert, H. P., Robertson, L. W., and Silberhorn, E. M. (2001) PCBs and Tumor Promotion. In PCBs: Recent Advances in the Environmental Toxicology and Health Effects (Robertson, L. W., and Hansen, L. G., Eds.) pp 355-371, The University Press of Kentucky, Lexington, KY. (9) Ludewig, G. (2001) Cancer Initiation by PCBs. In PCBs: Recent Advances in the Environmental Toxicology and Health Effects (Robertson, L. W., and Hansen, L. G., Eds.) pp 337-354, The University Press of Kentucky, Lexington, KY. (10) Li, Y., and Trush, M. A. (1993) DNA damage resulting from the oxidation of hydroquinone by copper: role for a Cu(II)/Cu(I) redox cycle and reactive oxygen generation. Carcinogenesis 14, 13031311. (11) Li, Y., and Trush, M. A. (1994) Reactive oxygen-dependent DNA damage resulting from the oxidation of phenolic compounds by a copper-redox cycle mechanism. Cancer Res. 54, 1895s-1898s. (12) Flowers, L., Ohnishi, S. T., and Penning, T. M. (1997) DNA strand scission by polycyclic aromatic hydrocarbon o-quinones: role of reactive oxygen species, Cu(II)/Cu(I) redox cycling, and o-semiquinone anion radicals. Biochemistry 36, 8640-8648. (13) Chen, Y., Shen, L., Zhang, F., Lau, S. S., van Breemen, R. B., Nikolic, D., and Bolton, J. L. (1998) The equine estrogen metabolite 4-hydroxyequilenin causes DNA single-strand breaks and oxidation of DNA bases in vitro. Chem. Res. Toxicol. 11, 11051111. (14) Bolton, J. L., Trush, M. A., Penning, T. M., Dryhurst, G., and Monks, T. J. (2000) Role of quinones in toxicology. Chem. Res. Toxicol. 13, 135-160. (15) McLean, M. R., Robertson, L. W., and Gupta, R. C. (1996) Detection of PCB adducts by the 32P-postlabeling technique. Chem. Res. Toxicol. 9, 165-171. (16) McLean, M. R., Bauer, U., Amaro, A. R., and Robertson, L. W. (1996) Identification of catechol and hydroquinone metabolites of 4-monochlorobiphenyl. Chem. Res. Toxicol. 9, 158-164. (17) Oakley, G. G., Devanaboyina, U., Robertson, L. W., and Gupta, R. C. (1996) Oxidative DNA damage induced by activation of polychlorinated biphenyls (PCBs): implications for PCB-induced oxidative stress in breast cancer. Chem. Res. Toxicol. 9, 12851292. (18) Oakley, G. G., Robertson, L. W., and Gupta, R. C. (1996) Analysis of polychlorinated biphenyl-DNA adducts by 32P-postlabeling. Carcinogenesis 17, 109-114. (19) Srinivasan, A., Lehmler, H.-J., Robertson, L. W., and Ludewig, G. (2001) Production of DNA strand breaks in vitro and reactive oxygen species in vitro and in HL-60 cells by PCB metabolites. Toxicol. Sci. 60, 92-102. (20) Amaro, A. R., Oakley, G. G., Bauer, U., Spielmann, H. P., and Robertson, L. W. (1996) Metabolic activation of PCBs to quinones: reactivity toward nitrogen and sulfur nucleophiles and influence of superoxide dismutase. Chem. Res. Toxicol. 9, 623629. (21) Bauer, U., Amaro, A. R., and Robertson, L. W. (1995) A new strategy for the synthesis of polychlorinated biphenyl metabolites. Chem. Res. Toxicol. 8, 92-95. (22) Tietze, F. (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27, 502-522. (23) Newton, G. L., and Fahey, R. C. (1995) Determination of biothiols by bromobimane labeling and high-performance liquid chromatography. Methods Enzymol. 251, 148-166. (24) Chen, H., and Eastmond, D. A. (1995) Topoisomerase inhibition by phenolic metabolites: a potential mechanism for benzene’s clastogenic effects. Carcinogenesis 16, 2301-2307.

Sulfhydryl Binding by PCB Metabolites (25) Marini, J. C., Miller, K. G., and Englund, P. T. (1980) Decatenation of kinetoplast DNA by topoisomerases. J. Biol. Chem. 255, 4976-4979. (26) Ariyoshi, N., Shigeto, M., Oguri, K., and Yoshimura, H. (1993) Metabolism in vivo of 2,4,6,2′,4′,6′-hexachlorobiphenyl in dog and partial characterization of cytochrome P-450 isozyme responsible for biotransformation of the congener. Fukuoka Igaku Zasshi 84, 181-188. (27) Ariyoshi, N., Yoshimura, H., and Oguri, K. (1993) Identification of in vitro metabolites of 2,4,6,2′,4′,6′-hexachlorobiphenyl from phenobarbital-treated dog liver microsomes. Biol. Pharm. Bull. 16, 852-857. (28) Borlakoglu, J. T., and Haegele, K. D. (1991) Comparative aspects on the bioaccumulation, metabolism and toxicity with PCBs. Comp. Biochem. Physiol. C 100, 327-338. (29) Borlakoglu, J. T., Wilkins, J. P., Quick, M. P., Walker, C. H., and Dils, R. R. (1991) Metabolism of [14C]4-chlorobiphenyl by hepatic microsomes isolated from razorbills, pigeons and rats. Comp. Biochem. Physiol. C 99, 287-291. (30) Goto, M., Sugiura, K., Hattori, M., Miyagawa, T., and Okamura, M. (1974) Metabolism of 2,3-dichlorobiphenyl-14C and 2,4,6trichlorobiphenyl-14C in rat. Chemosphere 5, 227-323. (31) Koga, N., Beppu, M., Ishida, C., and Yoshimura, H. (1989) Further studies on metabolism in vivo of 3,4,3′,4′-tetrachlorobiphenyl in rats: identification of minor metabolites in rat faeces. Xenobiotica 19, 1307-1318. (32) Bergman, A., Klasson-Wehler, E., and Kuroki, H. (1994) Selective retention of hydroxylated PCB metabolites in blood. Environ. Health Perspect. 102, 464-469. (33) Wernerman, J., and Hammarqvist, F. (1999) Modulation of endogenous glutathione availability. Curr. Opin. Clin. Nutr. Metab. Care 2, 487-492. (34) Comporti, M., Maellaro, E., Del Bello, B., and Casini, A. F. (1991) Glutathione depletion: its effects on other antioxidant systems and hepatocellular damage. Xenobiotica 21, 1067-1076. (35) Hanna, P. M., and Mason, R. P. (1992) Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique. Arch. Biochem. Biophys. 295, 205-213. (36) Meister, A. (1985) Methods for the selective modification of glutathione metabolism and study of glutathione transport. Methods Enzymol. 113, 571-585. (37) Spear, N., and Aust, S. D. (1995) Effects of glutathione on Fenton reagent-dependent radical production and DNA oxidation. Arch. Biochem. Biophys. 324, 111-116. (38) Pascoe, G. A., and Reed, D. J. (1989) Cell calcium, vitamin E, and the thiol redox system in cytotoxicity. Free Radical Biol. Med. 6, 209-224. (39) D’Arpa, P., and Liu, L. F. (1989) Topoisomerase-targeting antitumor drugs. Biochim. Biophys. Acta 989, 163-177. (40) Kaufmann, S. H. (1998) Cell death induced by topoisomerasetargeted drugs: more questions than answers. Biochim. Biophys. Acta 1400, 195-211. (41) Larsen, A. K., and Skladanowski, A. (1998) Cellular resistance to topoisomerase-targeted drugs: from drug uptake to cell death. Biochim. Biophys. Acta 1400, 257-274. (42) Frantz, C. E., Chen, H., and Eastmond, D. A. (1996) Inhibition

Chem. Res. Toxicol., Vol. 15, No. 4, 2002 505

(43)

(44) (45)

(46)

(47) (48)

(49) (50)

(51) (52)

(53) (54)

(55)

(56)

(57)

of human topoisomerase II in vitro by bioactive benzene metabolites. Environ. Health Perspect. 104 Suppl. 6, 1319-1323. Hutt, A. M., and Kalf, G. F. (1996) Inhibition of human DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene. Environ. Health Perspect. 104 Suppl. 6, 1265-1269. Burden, D. A., and Osheroff, N. (1998) Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochim. Biophys. Acta 1400, 139-154. Catrycke, M. O., Houssin, R., Henichart, J. P., Pfeiffer, B., Renard, P., Dassonneville, L., and Bailly, C. (1999) Synthesis and cytotoxicity of analogues of the antibiotic BE 10988 inhibitors of DNA topoisomerase II. Bioorg. Med. Chem. Lett. 9, 2025-2030. Anderson, R. D., and Berger, N. A. (1994) International Commission for Protection Against Environmental Mutagens and Carcinogens. Mutagenicity and carcinogenicity of topoisomeraseinteractive agents. Mutat. Res. 309, 109-142. Ferguson, L. R., and Baguley, B. C. (1994) Topoisomerase II enzymes and mutagenicity. Environ. Mol. Mutagen. 24, 245-261. Kalina, I., Sram, R. J., Konecna, H., and Ondrussekova, A. (1991) Cytogenetic analysis of peripheral blood lymphocytes in workers occupationally exposed to polychlorinated biphenyls. Teratog. Carcinog. Mutagen. 11, 77-82. O’Brien, P. J. (1991) Molecular mechanisms of quinone cytotoxicity. Chem.-Biol. Interact. 80, 1-41. Prins, B., Dartee, W. P., Verboom, W., Reinhoudt, D. N., and Koster, A. S. (1994) Quantitative structure activity relationship for the acute cytotoxicity of 13 (bis)aziridinyl-benzoquinones: relation to cellular ATP depletion. Arch. Toxicol. 68, 255-260. Koster, A. S. (1991) Bioreductive activation of quinones: a mixed blessing. Pharm. Weekbl. Sci. 13, 123-126. McLean, M. R., Twaroski, T. P., and Robertson, L. W. (2000) Redox cycling of 2-(x′-mono, -di, -trichlorophenyl)-1,4-benzoquinones, oxidation products of polychlorinated biphenyls. Arch. Biochem. Biophys. 376, 449-455. Monks, T. J., and Lau, S. S. (1997) Biological reactivity of polyphenolic-glutathione conjugates. Chem. Res. Toxicol. 10, 1296-1313. Milne, L., Nicotera, P., Orrenius, S., and Burkitt, M. J. (1993) Effects of glutathione and chelating agents on copper-mediated DNA oxidation: pro-oxidant and antioxidant properties of glutathione. Arch. Biochem. Biophys. 304, 102-109. Letcher, R. J., Klasson-Wehler, E., and Bergman, A. (2000) Methyl Sulfone and Hydroxylated Metabolites of Polychlorinated Biphenyls. In The Handbook of Environmental Chemistry, Vol. 3, Part K, Springer-Verlag, Berlin and Heidelberg. Wehler, E. K., Bergman, A., Brandt, I., Darnerud, P. O., and Wachtmeister, C. A. (1989) 3,3′,4,4′-Tetrachlorobiphenyl. Excretion and tissue retention of hydroxylated metabolites in the mouse. Drug Metab. Dispos. 17, 441-448. Lin, P. H., Sangaiah, R., Ranasinghe, A., Upton, P. B., La, D. K., Gold, A., and Swenberg, J. A. (2000) Formation of quinonoidderived protein adducts in the liver and brain of Sprague-Dawley rats treated with 2,2′,5,5′-tetrachlorobiphenyl. Chem. Res. Toxicol. 13, 710-718.

TX010128+