Aldehydic DNA lesions in calf thymus DNA and HeLa S3 cells produced by bacterial quinone metabolites of fluoranthene and pyrene

Joanna Zielinska-Park, Jun Nakamura1, James A. Swenberg and Michael D. Aitken

Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, NC 27599-7431, USA

1 To whom correspondence should be addressed Email: ynakamur{at}email.unc.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is increasing concern that compounds formed during the chemical or biological transformation of pollutants in the environment may be more detrimental to human and environmental health than the original pollutant. In this study, two bacterial transformation products of polycyclic aromatic hydrocarbons (PAHs), pyrene-4,5-quinone (P45Q) and fluoranthene-2,3-quinone (F23Q), were evaluated for mutagenicity by measuring aldehydic DNA lesions (ADL) in calf thymus DNA and HeLa S3 cells. Both quinones caused oxidative DNA damage in vitro through a copper-mediated redox cycle and subsequent production of reactive oxygen species (ROS). Hydrogen peroxide and copper were essential for causing oxidative DNA damage and glutathione (GSH) prevented DNA damage from F23Q better than from P45Q. In experiments using HeLa cells, F23Q decreased cell viability, but did not produce measurable levels of ADL or base oxidation. To test the hypothesis that DNA damage was being prevented by conjugation of F23Q with GSH, GSH-depleted cells were treated with both quinones. GSH depletion did not increase the toxicity of F23Q or cause it to oxidize DNA. Treatment of HeLa cells with metal chelators did not decrease F23Q toxicity. It is therefore possible that F23Q affected cell viability through a ROS-independent mechanism, either by conjugation with essential cellular proteins or through cellular or mitochondrial membrane damage, which precluded oxidation of DNA. In contrast, P45Q caused both ADL and base oxidation in cells. Neocuproine reduced the amount of ADL caused by P45Q, indicating that copper was still important for the intracellular generation of damaging oxidants. P45Q is a novel metabolite and its effects on DNA have not been investigated previously. This study exemplifies the importance of considering not only primary environmental pollutants, but also their biologically or chemically generated transformation products.

Abbreviations: ADL, aldehydic DNA lesions; AP, apurinic/apyrimidinic; ARP, aldehyde-reactive probe; ASB, ARP–slot blot; BCS, bathocuproine disulfonic acid; CAT, catalase; CT-DNA, calf thymus DNA; DMSO, dimethyl sulfoxide; F23Q, fluoranthene-2,3-quinone; GSH, glutathione; PAH, polycyclic aromatic hydrocarbons; PBS, phosphate-buffered saline; P45Q, pyrene-4,5-quinone; ROS, reactive oxygen species; SOD, superoxide dismutase


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitously distributed pollutants in the environment. Of the 16 priority PAH compounds regulated by the United States Environmental Protection Agency, seven are considered to be probable human carcinogens (1). PAHs must undergo enzymatic transformation before they can adversely interact with DNA in vivo and cause mutations leading to cancer. One of the principal pathways of PAH metabolism in mammalian cells is their transformation by mixed-function oxidases and dehydrogenases into redox-active PAH quinones (2).

PAH quinones also occur in the environment as a result of bacterial and fungal metabolism of PAHs in soil or sediment (35), as combustion products in airborne particulate matter (6,7), or as a result of atmospheric chemical transformation of parent PAHs (8,9). The metabolites pyrene-4,5-quinone (P45Q) (10) and fluoranthene-2,3-quinone (F23Q) (11) (Figure 1) have recently been isolated from laboratory cultures of PAH-degrading soil bacteria as terminal products from the transformation of pyrene and fluoranthene, respectively. Although not considered to be probable human carcinogens (12), the four-ring PAHs fluoranthene and pyrene are among the most predominant PAHs in contaminated soils and in the atmosphere (1214).



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Fig. 1. Structures of F23Q (left) and P45Q (right).

 
Given the known genotoxicity of several PAH quinones (2), quinones formed during photolysis, bioremediation or natural attenuation of PAHs could pose an additional risk to human health. Increases in toxicity and mutagenicity have been observed in PAH-contaminated soil extracts (15) and leachates from PAH-contaminated soil columns (16) after they had undergone bioremediation. Fernández and co- workers (17) identified several PAH quinones, including those derived from fluoranthene and pyrene, in fractionated organic extracts of coastal sediments. The oxy-PAH fraction that contained high concentrations of PAH quinones was found to be strongly mutagenic in Salmonella assays. Fluoranthene-2,3-quinone resulting from the incubation of fluoranthene with rat hepatic microsomal enzymes was found to be strongly mutagenic to Salmonella typhimurium (18). Information about P45Q is sparse in the literature and its mutagenicity and toxicity do not appear to have been investigated.

Several researchers have identified PAH o-quinones as alkylating agents that form stable and depurinating DNA adducts via Michael addition of nucleophilic sites such as Gua N2 and N7 to the quinone ring (19,20). PAH quinones can also enter into redox cycles in the presence of intracellular reducing equivalents, producing reactive oxygen species (ROS) that can damage DNA by causing strand scission and base modification, especially in the presence of copper (2,21). PAH quinones have also been shown to be directly mutagenic and cytotoxic in assays using various S.typhimurium tester strains and rat hepatoma cells (22).

The purpose of this study was to characterize the activity of P45Q and F23Q towards DNA using the aldehyde-reactive probe (ARP)–slot blot technique (ASB) (23) developed for the determination of aldehydic DNA lesions (ADL). The ASB assay has been used previously to quantify endogenous ADL in genomic DNA from mammalian tissues (24), ADL caused by quinonoid metabolites of pentachlorophenol (25,26) and catechol metabolites of estrogen (27). ADL include oxidative lesions resulting from hydrogen abstraction from deoxyribose and apurinic/apyrimidinic (AP) sites formed from the spontaneous release of depurinating adducts or enzymatic excision of oxidatively modified bases (23).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Calf thymus DNA (CT-DNA) was purchased from Sigma (St Louis, MO) and treated with 100 mM methoxyamine (MX) or 360 mM sodium borohydride before being re-suspended in reagent water to reduce the background number of aldehydic lesions. Pyrene-4,5-quinone and fluoranthene-2,3-quinone were synthesized according to published methods (28,29) and their purity was verified by nuclear magnetic resonance spectroscopy. The ARP was purchased from Dojindo Molecular Technologies (Gaithersburg, MD). Catalase (CAT), superoxide dismutase (SOD), bathocuproine disulfonic acid (BCS), glutathione (GSH) neocuproine, 2,2,6,6-tetramethylpiperidinoxyl (TEMPO), NADPH, dimethyl sulfoxide (DMSO) and cupric chloride were purchased from Sigma and were of the highest purity available. D-Mannitol was purchased from ICN Biomedicals (Aurora, OH). Escherichia coli endonuclease III and hOGG1 were kindly provided by Y.W.Kow (Emory University) and T.Arai and S.Nishimura (Banyu Tsukuba Research Institute), respectively.

Aldehydic lesions induced by P45Q and F23Q
Pre-treated CT-DNA (~1 µg/µl) was suspended in phosphate-buffered saline (PBS) and exposed to 10–100 µM P45Q or 1–100 µM F23Q dissolved in DMSO with or without the addition of 100 µM NADPH and 20 µM Cu(II) or Fe(III) (26). As a solvent, DMSO in the samples was present at a concentration of 1%. When used, BCS and free radical scavengers were dissolved in reagent water and added to the samples prior to the addition of the quinones. Triplicate samples were prepared for each experiment and incubated with shaking for 2 h at 37°C after which DNA was precipitated and washed with ethanol before being suspended in reagent water. The ASB assay was performed according to the protocol of Nakamura et al. (23). Briefly, 8 µg of treated DNA was incubated with 1 mM ARP in PBS for 10 min at 37°C, precipitated and washed with ethanol, and re-suspended in Tris–EDTA buffer (pH 7.4). After dilution to 1.25 µg/ml, the DNA was denatured for 10 min at 99°C and immobilized on a nitrocellulose membrane in a slot-blot apparatus. The membrane was dried, blocked with a bovine casein/albumin hybrid mix, and treated with streptavidin-conjugated horseradish peroxidase (BioGenex, San Ramon, CA). The enzymatic activity was enhanced with ECL western blotting detection reagents (Amersham Biosciences, UK) and an exposure was taken on X-ray film. The films were read with a Kodak Imagestation 440 and analyzed with Kodak ID Image Analysis Software. The number of aldehydic lesions per million nucleotides was determined based on a standard curve.

Depurination of heat-labile base adducts
CT-DNA exposed to 100 µM P45Q or F23Q in the presence or absence of NADPH was treated with 100 mM MX at 37°C for 2 h to eliminate the existing ADL. The DNA was then precipitated and washed with ethanol to remove residual MX, re-suspended in PBS and incubated at 70°C for 2 h. The resulting increase in aldehydic sites was attributed to the depurination/depyrimidation of unstable quinone base adducts, as determined by the ASB assay.

Assessment of ADL in HeLa cells
HeLa S3 cells obtained from the Lineberger Comprehensive Cancer Center (University of North Carolina Chapel Hill School of Medicine) were maintained at 37°C and 5% CO2 in Dulbecco's Modified Eagle Medium/nutrient mixture F12 with 5% fetal bovine serum and 100 µg/ml each penicillin and streptomycin (Invitrogen Life Technologies, Carlsbad, CA). The cells were plated at ~0.5 x 105 cells/ml of fresh F12 medium and cultured for 24 h before being incubated with varying concentrations of P45Q and F23Q in DMSO at 37°C for 4 h. The concentration of DMSO in cell cultures was <1% (v/v) and three plates were used for each set of exposure conditions. The cells were then washed three times with cold PBS, harvested by scraping and frozen at –80°C (30). Metal chelators, when used, were dissolved in reagent water and added to the plates 1.5 h prior to the addition of the quinones. DNA was extracted using a PureGene DNA extraction kit (Gentra Systems, Minneapolis, MN), re-suspended in reagent water containing 1 mM TEMPO to protect from further oxidative damage, and stored at –80°C. ASB assays were performed as described previously for CT-DNA. Heat-labile bases were also determined using the same methods as for CT-DNA.

Endonuclease III and hOGG1 assays
To determine the extent of base oxidation produced by P45Q and F23Q, extracted DNA from quinone-exposed HeLa cells was allowed to react with 340 mM NaBH4 for 15 min on ice to eliminate aldehydic lesions before being incubated with EndoIII or hOgg1 to enzymatically excise oxidized bases. Eight micrograms of DNA and 0.1 µg EndoIII were incubated at 37°C for 30 min in 10 mM Tris–HCl buffer containing 1 mM EDTA and 100 mM NaCl prior to reaction with ARP (24). Similarly, 8 µg of DNA, 140 ng hOgg1 and 3 µg of bovine serum albumin were incubated in Tris–HCl/EDTA/NaCl (31,32). The resulting AP sites from both treatments were quantified using the ASB assay.

Cytotoxicity
The toxicity of P45Q and F23Q to HeLa cells was evaluated using Trypan Blue exclusion after incubation with the compounds for 4 h. All incubations were performed in triplicate. Cells were counted directly after exposure or were first washed, supplemented with fresh F12 medium and counted 24 h later to evaluate recovery. After removal of the incubation medium, cells were washed with cold PBS three times and detached from the culture dish using trypsin (Invitrogen Life Technologies). Ten microliters of cell suspension was diluted 1:1 with Trypan Blue and counted on a hematocytometer slide.

GSH-depleted HeLa cells
HeLa cells were treated with 1 mM buthionine sulfoximine (BSO) for 24 h to inhibit GSH synthesis. The resulting levels of GSH in treated and non-treated cells were determined using a Total Glutathione Quantification Kit (Dojindo Molecular Technologies, Gaithersburg, MD). GSH concentrations were 106 ± 12 µM for the control cells and 7 ± 0.6 µM for the BSO-treated cells. GSH-depleted and control cells were exposed to quinones as described and the resulting ADL and cytotoxicity was evaluated as described above.

Statistical analysis
All data presented are the results of at least three separate experiments. The significance of the effects was tested using ANOVA, followed by either Dunnett's or Tukey's Multiple Comparison test.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
ADL in CT-DNA treated with quinones
Both P45Q and F23Q produced ADL in CT-DNA in the presence of 20 µM Cu(II) and 100 µM NADPH. Approximately 10 times more damage was casued by F23Q than by P45Q at equimolar concentrations (Figure 2), indicating that it had greater redox cycling capability. At concentrations above 50 µM the number of ADL remained constant, and the reaction appeared to be limited by the concentration of metal, dissolved oxygen and/or reducing equivalent. Neither compound was able to produce an increase in ADL without NADPH (data not shown). P45Q did not produce ADL in the absence of copper or in the presence of Fe(III), while F23Q produced only a small amount of ADL in the presence of NADPH alone and with NADPH and Fe(III) (Figure 3). Thermal hydrolysis at 70°C and subsequent analysis of resulting ADL did not demonstrate the formation of depurinating adducts from any of the treatments (data not shown).



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Fig. 2. Dose–response curves for ADL in CT-DNA caused by F23Q and P45Q in the presence of 20 µM Cu(II) and 100 µM NADPH. Error bars represent the standard deviation of the results from three individual samples.

 


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Fig. 3. ADL produced by 100 µM P45Q or F23Q in the absence of Cu(II) and in the presence of 100 µM NADPH or 100 µM NADPH with 20 µM Fe(III). Error bars represent the standard deviation of three separate experiments. The symbol # indicates P < 0.001 relative to control.

 
Inhibition of ADL formation by BCS and free radical scavengers
There is ample evidence that PAH quinones can enter into redox cycles with Cu(II) to produce superoxide ion, hydrogen peroxide, Cu(I) and ultimately hydroxyl radicals that lead to oxidative DNA damage (21,33). ADL formation from P45Q and F23Q was substantially reduced by CAT and BCS (Figure 4), indicating that both hydrogen peroxide and Cu(I) were involved in the production of ADL. CAT that had been de-activated by boiling did not decrease ADL from either compound, indicating that the reduction by active CAT was a result of hydrogen peroxide dismutation (data not shown). The hydroxyl radical scavengers mannitol and DMSO were not effective in reducing ADL from P45Q or F23Q. GSH reduced ADL in a concentration-dependent manner, and had a greater effect on samples treated with F23Q. SOD did not significantly reduce ADL from either quinone.



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Fig. 4. Effects of CAT, free radical scavengers and bathocuproine, a Cu(I)-specific chelator, on ADL formation by P45Q and F23Q. Values represent the percentage of ADL induced by P45Q (50 µM) and F23Q (5 µM) in the presence of scavenging agents relative to samples with no scavengers. Average values and standard deviations were determined based on three separate observations. The symbol * indicates P < 0.001; + indicates P < 0.005; # indicates P < 0.05 relative to 100%.

 
ADL and base oxidation in HeLa cells
In contrast to its effect on DNA in vitro, F23Q did not induce oxidative DNA damage in intact cells at doses ranging from 0.1 to 100 µM during 4 h of exposure (data not shown). P45Q, which was not as efficient as F23Q at causing ADL in CT-DNA, produced significant DNA damage in cells at a concentration of 10 µM (Figure 5). Thermal hydrolysis yielded no evidence of heat-labile DNA adducts, similar to the results obtained when using CT-DNA (data not shown). Treatment of cells with the membrane-permeable copper(I)-specific chelator neocuproine 90 min prior to quinone exposure reduced the number of ADL caused by P45Q by ~40% (Figure 5). Pyrimidine oxidation measured as AP sites (ADL) formed after treatment of exposed cell DNA with EndoIII was elevated in cells treated with 10 µM P45Q while cells treated with 1 µM F23Q had oxidized base levels similar to that of the DMSO-treated controls (Figure 6). Apurinic sites from the removal of oxidized purine bases by hOGG1 were below the detection limit in control and F23Q-exposed cells, while cells treated with P45Q had increased levels of ADL from hOGG1 treatment. GSH depletion did not increase ADL levels in P45Q-treated cells, and did not cause any measurable DNA damage in F23Q treated cells (data not shown).



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Fig. 5. Effect of 100 µM neocuproine on direct ADL in HeLa cells produced by a 4-h exposure to a solvent control or 10 µM P45Q. Error bars represent the standard deviations of three separate observations. The symbol # indicates P < 0.005 relative to DMSO control; {dagger} indicates P < 0.005 relative to P45Q without neocuproine.

 


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Fig. 6. Direct ADL in HeLa cells and ADL resulting from enzymatic excision of oxidized bases with EndoIII and hOGG1 after 4 h of exposure to quinones in DMSO. ADL from hOGG1 excisable sites were below the detection limit for both the solvent control and F23Q. Error bars represent the standard deviation of measurements from three individual plates. The symbol # indicates P < 0.0001 relative to control; {dagger} indicates P < 0.001 relative to control.

 
Cytotoxicity of quinones to HeLa cells
At concentrations of 10–100 µM, F23Q considerably reduced the viability of HeLa cells after a 4-h incubation period as measured by Trypan Blue exclusion, while P45Q appeared to have no effect on short-term viability (Figure 7). The cytotoxicity of F23Q was not reduced by the addition of 100 µM neocuproine, the Fe(II)-specific chelator dipyridyl or BCS (data not shown). When cells were incubated in fresh medium for 24 h after exposure to the quinones, the amount of viable cells in F23Q-treated cultures remained unchanged, and neocuproine had no significant effects on cell recovery. In contrast, P45Q-treated cells were completely killed after being incubated in fresh medium for 24 h (Figure 7). Cells treated with P45Q were suspended in the culture medium and were therefore not treated with trypsin. Staining of a 10 µl aliquot of cells floating in culture medium with Trypan Blue indicated a 100% reduction in viability even at P45Q concentrations as low as 5 µM. Pre-incubation with neocuproine (100 and 200 µM) had no effect on the long-term viability of P45Q treated cells (data not shown). GSH depletion did not increase the toxicity of F23Q or P45Q, and also did not cause a decrease in viability in control (DMSO) treated cells (data not shown).



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Fig. 7. Dose–response curves for cytotoxicity of F23Q and P45Q to HeLa cells after 4 h of incubation with the quinones (closed symbols) and a 24-h recovery period in clean growth medium (open symbols). Cytotoxicity was measured by Trypan Blue exclusion and error bars represent the standard deviation of three separate culture dishes.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many redox-active compounds have been shown to damage DNA through the production of ROS in the presence of metals (3437). In the case of quinones, redox cycling via reduction by NADPH to either the semiquinone or hydroquinone initiates a redox cascade, which produces superoxide anion and hydrogen peroxide (H2O2). In the presence of copper, H2O2 can decompose to yield hydroxyl radical, a potent oxidant of DNA (21,3842). Aldehydic lesions were produced in CT-DNA as a result of incubation with NADPH, CuCl2 and both PAH-quinones. F23Q was a more powerful producer of ADL in CT-DNA by approximately an order of magnitude, indicating that it is a much more efficient redox cycler than P45Q.

Adducts of PAH o-quinones with DNA (19,20) and other cellular constituents, most notably cysteine residues on proteins (43) and GSH (44), have been identified primarily as 1,4-Michael addition products. Neither quinone used in this study can react to give 1,4-Michael addition products, which may account for the absence of depurinating adducts. It is also possible that F23Q and P45Q formed stable conjugates with DNA, or that the conditions used for hydrolysis of covalent adduct–DNA bonds did not cause additional AP sites.

GSH reduced ADL caused by F23Q to a greater extent than those caused by P45Q. Since dose–response experiments suggest that F23Q produced larger amounts of reactive oxygen species than P45Q, it is possible that another mechanism of detoxification by GSH in addition to ROS scavenging and copper complexation is important for these quinones. Conjugation of PAH quinones with GSH via Michael addition has been observed (44), but it has also been noted that this reaction does not abolish redox cycling, and may in fact enhance the toxic activity of redox-active quinones (2,45). Furthermore, as noted above, Michael addition can most probably be ruled out, based on the structures of these quinones. Another potential detoxification mechanism for GSH may be through nucleophilic addition of sulfur to a ketone carbonyl. Such additions have been observed with naphthoquinones in experimental systems (46,47) and would disrupt redox cycling. No attempts were made in this study to identify GSH–quinone adducts or to quantify the rates of complex formation.

In additional scavenger experiments, the largest reductions in ADL were achieved with CAT and BCS, confirming the involvement of the Cu(I)/Cu(II) redox cycle and possibly a Fenton-type reaction between Cu(I) and H2O2 to produce the highly-reactive hydroxyl radical (OH). Mannitol and DMSO however were not effective at alleviating DNA damage in these systems, indicating that free hydroxyl radical was not the ultimate oxidizing agent. Although DNA damage stemming from the interaction of redox-active organic compounds or hydrogen peroxide with metals has been extensively reviewed in the literature (34,48,49), the exact nature of the ultimate DNA-damaging species is still a matter of debate. Flowers et al. (21) studied DNA damage from the o-quinones of naphthalene and benzo[a]pyrene in the presence of NADPH and Cu(II) and concluded that OH was responsible for the majority of DNA damage based on a significant reduction in strand scission and malondialdehyde release when mannitol and other OH scavengers were added to the samples. However, since iron is also effective at producing OH from H2O2 (4850), it would be expected that ADL would be produced to a similar extent in experiments where Fe(III) was substituted for Cu(II), which was not observed in the present study or by Flowers et al. (21).

It has also been proposed that singlet oxygen resulting from the metal-catalyzed Haber–Weiss reaction between H2O2 and superoxide (37,39,51) as well as the conjugate acid of superoxide (the hydroperoxyl or perhydroxyl radical) (52) are the principal DNA damaging species in these types of systems. However, since SOD had no effect on ADL resulting from incubation of DNA with either P45Q or F23Q, it is unlikely that 1O2 or HOO were important ROS in these experiments.

Copper–hydroperoxo complexes such as Cu(I)–OOH resulting from the incubation of DNA with NADH, H2O2 and copper (53), or catechol and copper (54), have also been proposed as probable candidates for causing oxidative DNA damage. Copper forms stable complexes with DNA, that can react with H2O2 to form DNA–Cu(I)–OOH complexes that are believed to release OH radicals in very close proximity to DNA. As a result of the high reactivity of OH, the probability of reaction with proximate DNA bases or sugars is high (53,54), and may explain why hydroxyl radical scavengers in the bulk solution did not have an effect on the extent of oxidative damage. Iron, however, has also been shown to cause oxidative damage to DNA from reaction with hydrogen peroxide (30,55), indicating that the ADL observed in these experiments may be a result of more complex interactions. Since the metal-catalyzed reduction of oxygen to hydrogen peroxide proceeds via successive one-electron transfers to form superoxide as an intermediate, it would be reasonable to expect SOD to have an effect on the rate of redox cycling and the resulting DNA damage. Since no inhibition was observed, it is possible that H2O2 formation is the result of a concerted transfer of two electrons to oxygen within an inner-sphere complex between the (hydro/semi)quinone, copper and molecular oxygen (56). When complexed in this way, superoxide may be too transient, or too tightly sequestered to be accessible to scavengers. More work is needed to fully investigate this phenomenon.

In contrast to the results obtained using extracted CT-DNA, F23Q was not able to induce ADL in HeLa S3 cells at any of the concentrations tested (0.3–100 µM). P45Q, which was ~10 times weaker as an ADL inducer in vitro, caused extensive damage to cellular DNA. The membrane-permeable Cu(I) chelator neocuproine reduced ADL from P45Q, indicating that copper is still important in the intracellular reaction and that ADL could be due to quinone–copper redox cycling. Li and co-workers (39) demonstrated an increase in 7,8- dihydro-8-oxo-guanine (8-OHdG) levels in DNA from cultured rat hepatocytes exposed to the quinone metabolite of butylated hydroxyanisole, and found that oxidized base levels were reduced by membrane-permeable copper chelators. In the present study, HeLa cells treated with P45Q had elevated levels of oxidized purine lesions as measured by hOGG1. Oxidized pyrimidine lesions, as detected by treatment with the DNA glycosylase E.coli EndoIII were also observed in cells treated with P45Q and were present in similar amounts as direct ADL. EndoIII has broad substrate specificity and excises ring- saturated, ring-opened and ring-fragmented pyrimidines (57,58), including thymine glycols and 4,6-diamino-5- formamido-pyrimidine (fapyAde) (59). The hOGG1 protein catalyzes the excision of damaged purine bases, including the most common oxidized base, 8-OHdG resulting from OH attack on the C8 position of guanine (60,61). Both proteins excise DNA bases through hydrolysis of the N-glycosylic bond between the target base and deoxyribose, leaving behind an AP site (57,58). HeLa cells exposed to P45Q had elevated levels of AP sites after treatment with both enzymes, indicating that P45Q was able to redox-cycle intracellularly and promote base oxidation. AP sites are non-coding lesions in DNA and can lead to mutation and apoptosis if their incidence exceeds the capacity of DNA repair proteins (62).

Although no DNA damage was detected in HeLa cells treated with F23Q, the compound did reduce the viability of HeLa cells in a concentration-dependent manner, and this effect was unchanged after 24 h. In contrast, cells treated with P45Q were completely viable after 4 h of exposure, but were completely killed after 24 h even at low concentrations of P45Q (Figure 7). Neocuproine offered no protection from the long-term toxicity of P45Q despite its ability to reduce ADL formation. However, since neocuproine was only able to reduce, and not completely abolish ADL from P45Q, cell death may be attributed to accumulation of residual DNA damage and apoptosis. Neocuproine and the iron-specific chelator DPD also had no effect on the decrease in cell viability caused by F23Q, indicating that it was potentially not a result of iron or copper-dependent redox-cycling (data not shown).

Several studies focusing on the effects of arylating and redox-cycling o-quinones on platelets (63) and colon carcinoma cells (64) concluded that arylation of sulfhydryl groups and the resulting alteration in intracellular protein thiol and Ca2+ homeostasis is the principal mechanism of quinone toxicity. The fact that no oxidative lesions were observed in cells treated with F23Q suggests that the quinone was either detoxified before it was able to adversely interact with DNA or was localized away from the nucleus. It is therefore possible that F23Q became covalently bound to other cellular constituents before it was able to react with DNA. McAmis et al. (65) found that menadione (2-methyl-1,4-napthoquinone) was toxic to endothelial cells, producing endothelial membrane failure by modifying intracellular protein and non-protein thiols and not by oxidant production. Depletion of intracellular GSH however did not enhance the toxicity of F23Q in the present study. Zhu et al. (66) showed that a decrease in cellular ATP and changes in mitochondrial structure preceded a decrease in the survival of stromal cells treated with benzo[a]pyrene quinone. These researchers also found that GSH depletion did not potentiate quinone toxicity and speculated that disruption of the mitochondrial respiratory chain did not depend on reduced oxygen species. Quinones can depolarize mitochondrial membrane potential by arylation or redox cycling, resulting in ATP depletion and cellular toxicity (67,68). Cytoskeletal, cellular and sub-cellular membrane proteins can also be altered by quinones, which can lead to permeability changes and toxicity (65,67). Polycyclic aromatic hydrocarbon quinones found in diesel exhaust have also been shown to preferentially react with dithiol compounds, resulting in modifications of their thiol groups, and ultimate cytotoxicity, while at the same time displaying minimal reactivity with monothiol compounds, such as GSH (69). A compound introduced into culture liquid must first cross the cellular membrane and diffuse through the cytoplasmic space before it can interact with nuclear DNA. It is therefore reasonable to assume that F23Q was localized and stabilized, either through covalent interaction or intercalation with cellular membrane constituents, mitochondria, or cytoskeletal proteins, which precluded its interaction with cellular DNA.

It has been established that K-region dihydrodiols such as pyrene-4,5-dihydrodiol are not substrates for the dihydrodiol dehydrogenase enzymes that ultimately convert them to quinones during mammalian metabolism of PAHs (70,71). Therefore, it is not likely that K-region PAH quinones are formed in vivo. This may be a reason for the apparent lack of carcinogenicity from the parent compound, pyrene, and indicates that P45Q from environmental sources could pose an additional risk to human health. The importance of potentially toxic and persistent daughter products of environmental pollutants is only beginning to be explored. There is increasing concern over the fact that risk assessment studies fail to account for these environmentally relevant transformation products, mainly because there are insufficient data available as to their characteristics, formation kinetics and toxicity (72,73). This study contributes to the latter need by evaluating the possible cytotoxicity and mutagenicity of products that are formed as a result of chemical or biological reactions in the environment, and may have the potential to pose a greater risk to human health than the parent compounds.


    Acknowledgments
 
The authors thank Ramiah Sangaiah and the UNC Superfund Basic Research Program Chemistry Core for synthesis and purification of the quinones. We also gratefully acknowledge Po-Hsiung Lin and Betsy Purvis for aid with experimental set-up and enzyme assays, respectively, and Avram Gold for helpful discussion and critical reading of the manuscript. This work was supported in part by grants from the National Institutes of Health (P42 ES05948, P30 CA16086 and P30 ES10126).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 27, 2003; revised April 14, 2004; accepted April 20, 2004.