* Graduate Center for Toxicology, 306 Health Sciences Research Building, University of Kentucky Medical Center, Lexington, Kentucky 405360305; and
Department of Nutrition and Food Science, 208 Funkhouser Building, University of Kentucky, Lexington, Kentucky 405060054
Received June 22, 2000; accepted October 30, 2000
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ABSTRACT |
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Key Words: PCBs; ROS production; DNA strand breaks; quinones; hydroquinones.
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INTRODUCTION |
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Several mixtures of PCBs and a few synthetic congeners have been tested in short-term tests for genotoxicity (reviewed in Silberhorn et al., 1990). The results were mostly negative. This could be due in part to slow rate of metabolism of these compounds under the assay conditions. We and many others have shown that PCBs in general (reviewed in Sipes and Schnellmann, 1987), and lower halogenated PCBs in particular (McLean et al., 1996a), are metabolized by microsomal enzymes to mono- and dihydroxy-metabolites.2 Further metabolism of the dihydroxy-metabolites, in which the hydroxyl groups are ortho (catechols, Cat) or para to each other (hydroquinones, HQ), may result in the formation of semiquinones and quinones, themselves reactive electrophiles. We have also shown that the metabolic activation of PCBs leads to the formation of adducts with nucleotides and DNA in vitro (McLean et al., 1996b
; Oakley et al., 1996a
). Minor adducts may be derived from arene oxide intermediates, whereas several major adducts are more likely to be derived from (semi-) quinone(s). Chemically synthesized PCB quinones are potent electrophiles that react instantaneously with sulfhydryls (N-acetyl cysteine, GSH) and more slowly with N-nucleophiles (Gly, L-Arg, L-His and L-Lys), forming ring addition products (Amaro et al., 1996
). Reaction of PCB-derived semiquinones or quinones with nucleotides and DNA has also been demonstrated, and several DNA adducts have been separated by thin layer chromatography and detected by 32P-postlabeling (Oakley et al., 1996a
). By measuring NADPH oxidation, it was shown that synthesized PCB quinones redox-cycle in the presence of NADPH and rat liver microsomes (McLean et al., 2000
).
The oxidation of dihydroxy-metabolites to quinones may generate superoxide anion radicals and other reactive oxygen species (ROS) via the reduction of molecular oxygen (Fridovich, 1999). ROS are known to cause DNA strand breaks that may play a role in mutagenesis and carcinogenesis (Flowers et al., 1997
). Chronic exposure to PCBs was reported to result in chromosomal aberrations (Kalina et al., 1991
), which are the result of chromosome breakage. Quinones of polycyclic aromatic hydrocarbons and dihydroxylated metabolites of benzene were shown to induce strand breaks in DNA in vitro in the presence of Cu(II), probably through redox reactions and ROS production (Flowers et al., 1997
; Li and Trush, 1993
, 1994
). In vitro incubation of DNA with PCB hydroquinones in the presence of copper-dichloride (Cu II) increased the levels of 8-oxo-deoxyguanosine (8-oxodG) (Oakley et al., 1996b
), probably because of ROS production during oxidation of dihydroxylated PCBs to quinones.
We hypothesize that PCBs are involved in carcinogenicity by several mechanisms, one involving the production of ROS that may cause DNA damage. Although several dihydroxy-PCBs have been shown to produce 8-oxo-dG in vitro, no direct evidence of ROS production by these metabolites has been shown. Moreover, the effect of PCB quinones with respect to ROS production has not been studied, and it remains to be shown whether these PCB metabolites will result in oxidative stress in cells. In this study, we therefore analyzed the potential of various dihydroxy- and quinone-PCBs (mostly hydroquinones and p-quinones) to generate superoxide anion radicals and other ROS both in vitro and in cells in culture and their ability to induce DNA damage in the form of strand breaks in vitro.
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MATERIALS AND METHODS |
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Synthesis and characterization of dihydroxy-PCBs and PCB p-benzoquinones.
The structures and abbreviated nomenclature of the test compounds used in this study are shown in Figure 1. The p-benzoquinones of PCBs were synthesized using the Meerwein arylation as described (Brassard and Ecuyer, 1958
) and shown in Scheme 1
. The PCB hydroquinones were prepared from the corresponding p-benzoquinones by reduction with sodium dithionite (Oakley et al., 1996b
). The detailed synthetic procedure and characterization of the p-benzoquinones and several PCB hydroquinones are given elsewhere (Bauer et al., 1995
; McLean et al., 1996a
; Oakley et al., 1996b
). The PCB catechols were obtained from Suzuki coupling of a suitable dimethoxy benzene boronic acid with a bromochlorobenzene and subsequent deprotection of the dimethoxy-PCB with boron tribromide (BBr3) (Bauer et al., 1995
). Three new compounds (3ClPh-HQ, 3,5ClPh-HQ, and 3,4,5ClPh-HQ) were characterized by 1H and 13C NMR, FT-IR, and GC/MS spectroscopy. The IR spectra were obtained using a Nicolet Magna-IR 560 Spectrometer E.S.P. The 1H and 13C NMR spectra were recorded on a Varian Gemini-200 spectrometer by using acetone-d6 or deuteriated chloroform (CDCl3, Cambridge Isotope Laboratories Inc., Andover, MA) as solvents and internal standards. GC/MS spectra were recorded in the mass spectrometer facility of the University of Kentucky on a Finnigan INCOS 50 and Kratos Concept 1H by using a fused silica capillary column (DB-5MS 15 m x 0.25 mm and OV-1, 25 m, J&W Scientific, Folsom, CA). For GC/MS all dihydroxybiphenyls were silylated with bis(trimethylsilyl)trifluoroacetamide (BSTFA)/pyridine (1:1). All dihydroxybiphenyls showed a purity of > 99 % by GS/MS based on relative peak area after several recrystallizations from chloroform/petroleum ether. The p-benzoquinones were analyzed with a Hewlett Packard 5890A gas chromatograph equipped with an HP-1 (methyl silicone gum) column (Hewlett Packard, Avondale, PA) and showed a purity of > 99 % based on relative peak area. Melting points were determined on an MEL-TEMP apparatus and are uncorrected. All solvents were obtained from commercial sources and used without further purification. The dihydroxybiphenyls were stored at 4°C under an argon atmosphere to minimize oxidation to the corresponding p-benzoquinone. To avoid depletion of any single compound, 2ClPh-HQ was used as standard compound in the NBT assays and 4ClPh-HQ in the strand break assays. For the assays stock solutions of PCB metabolites were prepared in DMSO. Caution: Synthetic PCB metabolites should be considered potentially toxic and hazardous and should therefore be handled in an appropriate manner.
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2-(3',5'-Dichlorophenyl)-1,4-hydroquinone (3,5ClPh-HQ)1: mp = 118°C (white solid from chloroform/petroleum ether at 4°C). 1H-NMR (acetone-d6, 200 MHz) 6.75 (d, J = 8.7 Hz, d, 2.7 Hz, 4-H), 6.84 (d, J = 2.7 Hz, 6-H), 6.86 (d, J = 8.7 Hz, 3-H), 7.39 (t, J = 1.8 Hz, 4'-H), 7.58 (d, J = 1.8 Hz, 2'- and 6'-H), 8.04 (br s, 2- and 5-OH). 13C-NMR (acetone-d6, 50 MHz)
117.07, 117.32, 118.09, 127.07, 130.13, 130.80, 131.01, 132.07, 132.16, 140.51, 148.03, 151.80. IR [cm1]: 3211 (
(OH)), 1588, 1559, 1452, 1409, 1384, 1242, 1192, 1126, 857, 811, 790, 782, 758, 684. MS m/z (relative intensity, %): 45 (21), 73 (100), 93 (10), 275 (20), 348 (10), 398/400 (32/23, C16H24Si2O2Cl2+).
2-(3',4',5'-Trichlorophenyl)-1,4-hydroquinone (3,4,5ClPh-HQ)1: mp = 196197°C (white solid from chloroform/petroleum ether at 4°C). 1H-NMR (acetone-d6, 200 MHz) 6.346.61 (3H, m, H-3/4/6), 7.43 (2H, s, H-2'/6'), 7.78 (2H, br s, OH). 13C-NMR (acetone-d6, 50 MHz)
117.19, 117.68, 118.22, 125.83, 130.58 (2C), 133.99, 140.69, 148.07, 151.86. IR [cm1]: 3248 (
(OH)), 1582, 1540, 1508, 1470, 1429, 1383, 1320, 1305, 1258, 1209, 1192, 1154, 1131, 868, 813, 802, 776, 730, 694, 634, 617. MS m/z (relative intensity, %): 45 (22), 73 (100), 93 (12), 382/384 (17/22), 442/434 (30/33, C16H23Si2O2Cl3+).
Measurement of superoxide production in vitro (NBT assay).
The NBT assay used is a modified version of the assay as described by Epe et al. (1986). Two milliliters of solutions containing 2 mM phosphate buffer (pH 7.0), 5 mM CTAB, and 100 µM NBT alone or with a) Cu(II) (100 µM); b) SOD (600U/ml); c) HRP (2.5U/ml) or LactoPx (0.1U/ml) and H2O2 (10 µM); or d) GSH (50, 100, 200 µM) were prepared and added to both the reference and sample quartz cuvettes. The reaction was started by adding PCB metabolite to a final concentration of 100 µM to the sample cuvette and DMSO to the reference cuvette. The rate of NBT reduction was monitored at 540 nm (37°C) for 10 min with a Shimadzu MPS 2000 UV/VIS spectrophotometer. Statistical analysis was performed using a SAS program (PROC mixed for repeated measures).
Measurement of strand break induction in plasmid DNA.
Supercoiled plasmid DNA (pBS sk, about 3kb size; 1 µg/15 µl) was exposed to the test compound and modifiers for 1 h at 37°C in 2 mM phosphate buffer (total sample volume = 15 µl). Immediately after exposure, the DNA was loaded onto 0.8% agarose gels in 1x Tris acetate/EDTA (TAE) buffer (40 mM Tris acetate, 1 mM EDTA, pH 7.0) and gel electrophoresis was carried out for 2 h at 60 V in a horizontal gel electrophoresis apparatus (Owl Scientific, MA). The gels were stained with ethidium bromide (1 µg/ml water) and the DNA visualized with a UV transilluminator. Pictures were taken with a Sony video graphics printer video camera attached to a Biophotonica gel print 2000i transilluminator and used for documentation. The gel pictures were scanned and total amount of DNA in each lane as well as the relative amounts of plasmid DNA with no strand breaks (supercoiled), single strand breaks (open circle), and double strand breaks (linear form) were determined. The amount of DNA in each form was expressed as percentage of the total DNA in each lane (% of intensity of optical density, IOD).
In order to establish standard assay conditions for our experiments, we performed first strand break experiments with 4ClPh-HQ + Cu(II) (100 µM each) using different buffers and pHs. We observed the same extent of strand break induction in 2 mM phosphate and 4 mM Tris buffer, but reduced strand breaks when 2 mM Na-citrate or a Tris-EDTA buffer was used. Because citrate and EDTA chelate Cu(II) and thus inhibit copper mediated dismutation of ROS, and because Tris is a reactive oxygen scavenger (Tsou et al., 1996), we decided to use 2 mM phosphate buffer for all experiments. We also found that at acidic pHs, less damage was observed than at alkaline or neutral pHs. Therefore, all experiments described here were performed at pH 7.0.
Cell culture and viability test.
The human promyelocytic leukemia cell line HL-60 was obtained from Dr. M. Doukas, University of Kentucky, Lexington, KY. Cells (passages 7090) were cultured in RPMI 1640 medium supplemented with 5% FCS, penicillin (0.1 U/ml) and streptomycin (0.1 µg/ml) and grown in a humidified atmosphere at 5% CO2 and 37°C.
For viability testing, 1 x 106 HL-60 cells in 1 ml PBS (pH 7.4) were exposed to the test compound or solvent alone. After 1 and 3 h at 37°C, aliquots of cells were removed and treated with an equal volume of 10% trypan blue solution. The number of viable cells in each sample was determined by counting unstained cells using a hemocytometer and a light microscope. All determinations were done in triplicate. Data are presented as percent of solvent-treated control. Statistical significance was determined using the ANOVA and post hoc Tukey's test.
Measurement of ROS in HL-60 cells using DCFH-DA.
3 x 105 HL-60 cells in 3 ml PBS (pH 7.4) were treated with various concentrations of 4ClPh-pQ, 4ClPh-HQ, or solvent alone (control) in the presence of 10 µM DCFH-DA. After 1 h or 3 h of incubation at 37°C, fluorescence of the cell suspension was determined using a RF-5301 PC Shimadzu spectrofluorophotometer with an excitation wavelength at 485 nm and emission wavelength at 530 nm. All experiments were carried out in triplicate, and the means and standard deviations (SD) were calculated per data point. Net ROS production was calculated by subtracting the fluorescence of the compounds in PBS without cells from the corresponding incubation with cells in suspension. Statistical significance was determined using the ANOVA and post hoc Tukey's test.
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RESULTS |
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Strand Break Induction
Strand break induction in DNA can be monitored using supercoiled plasmid DNA. When treated with an agent that induces DNA strand breaks, supercoiled DNA is converted to the open-circle form by a single strand break and to linear DNA by a double strand break. Extensive double strand breaks lead to DNA degradation. These effects can be monitored by gel electrophoresis. The migration pattern of a 3-kb plasmid under our gel electrophoresis conditions is: supercoiled > linear > open circle (Fig. 3A).
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DISCUSSION |
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For risk assessment, it is important to have information about structure-activity relationships. We observed more open-circle form after treatment with 4ChPh-HQ than with the two catechols (Fig. 5). This may be explainable by differences in the reduction potentials. Indeed, Amaro and coworkers (Amaro et al., 1996
) reported that in nonaqueous solution the reduction potentials for the pQ/SQ and the SQ/HQ pairs of 4ClPh- and 3,4ClPh-para-metabolites were about 100130 mV lower than the ones for the corresponding 2,3-ortho metabolites, indicating a lower likelihood of oxidation for the catechols. PCB-HQs were also more active than 3',4'-catechols in inducing 8-oxodG formation in vitro (Oakley et al., 1996b
), probably due to a common mechanisms for induction of strand breaks and 8-oxodG.
We did not see an influence of the number or position of chlorines on strand break induction in our gels, possibly due to the generally slow autoxidation rate. However, we observed a structure-activity relationship in the NBT reduction assay: mono-ClPh- > di-ClPh- > triClPh-HQ (Fig. 2). The reported reduction potentials for mono- and dichlorinated metabolites point to a small (about 20 mV), but consistent decrease in the redox potential with the higher chlorination (Amaro et al., 1996, measured in nonaqueous medium), but other factors such as solubility or protonation may also be involved in this effect.
The PCB-quinones were not (NBT) or hardly (strand breaks) active, but were activated by the addition of GSH (Figs. 2C and 6, respectively). Amaro and coworkers have shown that PCB-quinones react instantaneously with GSH by nucelophilic addition and have characterized the resulting hydroquinone thioethers (Amaro et al., 1996
). As the addition of GSH to a quinone can actually lower the reduction potential, as was shown for menadione (reviewed in Monks and Lau, 1997), GSH adducts may repeatedly autoxidize with the formation of superoxide. In combination with the PCB quinones, GSH was clearly activating by promoting the generation of superoxide. The reduction of NBT by PCB hydroquinones was also increased, perhaps by the same quinone-recycling mechanism. Strand break induction by PCB-HQs + Cu(II), however, was strongly reduced in the presence of GSH, possibly by stabilizing copper in the cuprous (+1) form (Milne et al., 1993
), thus preventing redox cycling of copper and OH formation from H2O2 by a Fenton reaction, and/or scavenging of OH (Hanna and Mason, 1992
).
Copper, which was essential for strand break induction, is an essential micronutrient present in all mammalian tissues. It has a preferential accumulation in the nucleus (Agarwal et al., 1989), where it is involved in the formation of higher order chromatin structures due to its role in the attachment of DNA to nuclear scaffolding proteins (Lewis and Laemmli, 1982
). Copper is highly active as a fenton reagent in converting H2O2 to the very reactive OH (Cu+ + H2O2
Cu2+ + OH + OH) (Goldstein et al., 1993
). It may also result in singlet oxygen production (Yamamoto et al., 1993
). Because copper is localized in such close proximity to DNA, it would produce locally high concentrations of these radicals; thus, its role in PCB toxicity is of particular interest. We have shown previously that the presence of copper significantly increased 8-oxodG formation in DNA by various dihydroxy-PCBs (Oakley et al., 1996b
). It was reported that DNA-bound copper ions produce primarily base modifications (Drouin et al., 1996
). In this paper we report that copper is also required for strand break induction by dihydroxy and quinone metabolites of PCBs, an effect that was also seen with other compounds like o-quinones of polycyclic aromatic hydrocarbons (PAHs; Flowers et al., 1997), hydroquinones (Carstens et al., 1990
; Li and Trush, 1993
), estrogen metabolites (Chen et al., 1998
), and polyphenolic compounds (Li and Trush, 1994
). EDTA, which chelates Cu(II) by forming a redox inert complex (Agarwal et al., 1989
; Li and Trush, 1993
), prevented 4ClPh-HQ + Cu(II)induced strand breaks, as did bathocuproine, a Cu(I)-specific chelator. This suggests that it is not Cu(II)-mediated HQ oxidation, but Cu(I)-mediated formation of other ROS from superoxide or its dismutation product H2O2 that is required for strand break induction.
Some scientists reported that the reaction of cuprous ions with hydrogen peroxide generates a Cu(I)-OOH complex that may cause the DNA damage (Murata et al., 1999). Others suggest that this complex is only formed initially and then decomposes into OH, which is the major species derived from the reaction of Cu(I) with H2O2 (Eberhardt et al., 1989
). To determine experimentally the exact nature of the DNA-breaking radical, we included various modifiers in the incubations during the strand break assay. The superoxide scavenger Tiron did not decrease stand break induction, possibly because the concentration tested was not high enough. SOD had no effect, perhaps because superoxide production and not nonenzymatic dismutation of superoxide is the rate-limiting step. Adding H2O2, the product of SOD metabolism of superoxide, to plasmid DNA + Cu(II) resulted in totally destroyed DNA (unpublished observation), indicating that H2O2 or a H2O2-derived oxygen radical is the ultimate clastogen. Two highly reactive oxygen species are OH and singlet oxygen. The OH scavenger Na-benzoate caused only slight protection of the DNA from 4ClPh-HQ + Cu(II)induced strand breaks, and itself produced DNA degradation in this system. The OH scavengers GSH, DMSO, and thiourea decreased strand breaks in a dose-dependent mannerDMSO marginally, GSH and thiourea completely. Hence, OH seems to be a major player in strand break induction by the PCB metabolites. This is in agreement with results from strand break experiments with PAH-quinones, polyphenols, and estrogen metabolites (Carstens et al., 1990
; Flowers et al., 1997
; Li and Trush, 1993
, 1994
). We observed in addition, however, that the singlet oxygen scavengers sodium azide and Tris also reduced strand break generation by 4ClPh-HQ + Cu(II). This radical could be produced by spontaneous dismutation of superoxide (Corey et al., 1987
), or be more likely derived from Cu/peroxide (Yamamoto et al., 1993
). This suggests that these two oxygen species, OH and singlet oxygen, are involved in PCB strand break induction.
The data as discussed above clearly show that PCB metabolites do produce ROS and consequently DNA damage under various conditions in vitro, but what are the effects of these metabolites in cells in culture? Our data provide for the first time direct evidence of intracellular production of ROS by PCB metabolites. The DCFH-DA dye has been used to detect ROS in many types of cells and tissues (LeBel et al., 1992). DCFH-DA is a nonionic and nonpolar substance that can cross the cell membrane and is hydrolyzed by cellular esterases to nonfluorescent DCFH. In the presence of ROS, DCFH is converted rapidly to highly fluorescent DCF (LeBel et al., 1992
). It was reported that oxidation of DCFH occurs almost exclusively in the cytosol (LeBel et al., 1992
). We saw some background production of DCF in PBS, much less, however, than in the presence of cells, indicating that the PCB metabolites do produce a strong net increase in intracellular ROS in HL-60 cells in a dose- and time-dependent manner (Fig. 8
). To confirm that the fluorescence was intracellular and not due to leakage of DCFH or DCF from damaged cells, we examined the cells by confocal fluorescence microscopy. A clear increase in the number of fluorescing cells and intensity of intracellular fluorescence was visible in samples treated with PCB metabolites. In addition, our toxicity tests show that the HL-60 cells were mostly viable at the concentrations tested for ROS production, with the exception of the high concentration of the quinone. HL-60 cells contain considerable amounts of myeloperoxidase (Shen et al., 1996
), which is efficient in PCB-HQ oxidation to the quinone. The PCB quinone was even more active than the PCB-HQ in producing ROS, probably by being activated by conjugation and by depletion of intracellular GSH.
Altogether, our studies demonstrate the production of ROS by PCB metabolites both in vitro and in cells in culture. The activated oxygen species generated may attack cellular DNA and other nucleophiles, resulting in oxidative damage. Indeed, DNA strand breaks were produced in vitro with these metabolites. These mechanisms may play an important role in the different toxic effects of PCBs.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Dihydroxy is used as a general term for all dihydroxylated PCBs. Para metabolites are described by the term hydroquinone (HQ). For ortho metabolites, the term catechol (Cat) is used.
A portion of this work was presented at the PCB Workshop, Recent Advances in the Environmental Toxicology and Health Effects of PCBs, April 912, 2000, Lexington, KY.
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