Induction of direct adducts, apurinic/apyrimidinic sites and oxidized bases in nuclear DNA of human HeLa S3 tumor cells by tetrachlorohydroquinone

Po-Hsiung Lin1,, Jun Nakamura, Shuji Yamaguchi, David K. La, Patricia B. Upton and James A. Swenberg,2

Department of Environmental Sciences and Engineering, The University of North Carolina, Chapel Hill, NC 27599-7400, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA damage induced by tetrachlorohydroquinone (Cl4HQ), the quinonoid metabolite of pentachlorophenol (PCP), was investigated in human HeLa S3 tumor cells. Formation of one major and two minor DNA adducts in cells treated with Cl4HQ (50–300 µM) was detected by 32P-post-labeling assay and the adducts accumulated over the course of the experiment (0.5–2 h), with total adduct levels estimated to be 3–6 per 108 nucleotides. These adducts did not correspond to those derived from calf thymus DNA treated with tetrachloro-1,4-benzoquinone. Results from the apurinic/apyrimidinic (AP) sites assay indicated that the number of AP sites was 2-fold greater in cells exposed to Cl4HQ (300 µM) than the corresponding control. Further characterization of the AP sites confirmed that Cl4HQ induced predominantly (75%) putrescine-excisable AP sites in HeLa S3 cells. In parallel, the concentration of 8-hydroxy-2'-deoxyguanosine (8-HO-dG) in cells treated with Cl4HQ for 0.5 and 2 h was increased 2- and 5-fold, respectively, compared with the control. The extent of oxidative DNA damage induced by Cl4HQ was approximately two orders of magnitude greater than those of direct DNA adducts. Overall, it appears that reactive oxygen species mediate the parallel formation of AP sites and 8-HO-dG in HeLa S3 cells following treatment with Cl4HQ and that the contribution of depurination/depyrimidination of direct DNA adducts is relatively insignificant compared with the formation of oxidized AP sites. We conclude that putrescine-excisable AP sites represent a major type of ROS-mediated oxidative DNA damage in cellular DNA induced by Cl4HQ and may play a role in PCP-induced clastogenicity in mammalian cells.

Abbreviations: AP, apurinic/apyrimidinic; DMEM, Dulbecco's modified Eagle's medium; ESA, electrochemical array detector; BHT, butylated hydroxytolvene; HPLC-ECD, HPLC/electrochemical detection; 8-HO-dG, 8-hydroxy-2'-deoxyguanosine; PCP, pentachlorophenol; PEI, polyethyleneimine; ROS, reactive oxygen species; RAL, relative adduct levels; Cl4HQ, tetrachlorohydroquinone.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tetrachlorohydroquinone (Cl4HQ), one of the reactive quinonoid metabolites of pentachlorophenol (PCP), is mutagenic and clastogenic in mammalian cells (18). Evidence indicates that Cl4HQ induces mutations at the hprt locus, micronuclei in Chinese hamster cells (1,2) and DNA single-strand breaks in Chinese hamster ovary cells and human fibroblasts (35). Cl4HQ may also have contributed to the chromosomal damage induced by PCP in mammalian cells (810).

Tetrachlorohydroquinone and its semiquinone and quinone counterparts can undergo redox cycling to generate reactive oxygen species (ROS) as well as alkylate proteins and genomic DNA (8,1113). Cl4HQ-derived ROS induces multiple types of DNA lesions in calf thymus DNA in the presence of Cu(II) (8,14), including the parallel formation of an oxidized base, DNA fragmentation and apurinic/apyrimidinic (AP) sites. The AP sites induced by Cl4HQ/Cu(II) in calf thymus DNA were characterized as predominantly putrescine-excisable AP sites, which is similar to those of the endogenous AP sites present in mammalian tissues and of calf thymus DNA treated with H2O2 (15). ROS are the main sources for the formation of AP sites in calf thymus DNA induced by PCP-derived quinonoids, whereas depurination/depyrimidination of direct DNA adducts of PCP quinonoids, such as tetrachloro-1,4-benzoquinone, is relatively insignificant (14). This raises the question as to whether similar types of DNA damage can be produced by Cl4HQ in mammalian cells and if such damage could have contributed to Cl4HQ-induced clastogenicity in mammalian cells. A diagram of the chemical structures of PCP, Cl4HQ and the respective benzoquinone are depicted in Figure 1Go.



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Fig. 1. Diagram of the chemical structures of pentachlorophenol, tetrachlorohydroquinone and tetrachloro-1,4-benzoquinone.

 
ROS-induced DNA damage in mammalian cells is subject to repair primarily by base-excision repair enzymes, such as 8-OH-dG glycosylase/lyase, which removes oxidized bases to generate AP sites (16). An additional repair enzyme, AP endonuclease, excises 3'-nicked AP sites to generate one nucleotide gaps. This process is followed by repair synthesis by DNA polymerase ß and completed by ligase. If not repaired, AP sites can result in mutations, as well as chromosome aberrations (17).

To investigate the types of DNA damage induced by Cl4HQ in mammalian cells, we undertook the present investigation to study the formation of direct adducts and oxidative damage in nuclear DNA of human HeLa S3 tumor cells exposed to Cl4HQ. We examined the formation of AP sites and 8-HO-dG, as well as direct DNA adducts by the 32P-post-labeling method in Cl4HQ-treated and control HeLa S3 cells. We tested the hypothesis that putrescine-excisable AP sites are a major type of ROS-mediated oxidative DNA damage in cellular DNA induced by Cl4HQ.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Cl4HQ (99%) and Cl4BQ (98%) were from Aldrich (Milwaukee, WI). Micrococcal nuclease, nuclease P1, potato apyrase (type VII) and spleen phosphodiesterase (type I) were purchased from Sigma (St Louis, MO). Whatman chromatography paper was from Fisher Scientific (Pittsburgh, PA). T4 polynucleotide kinase was purchased from Amersham (Arlington Heights, IL). Polyethyleneimine (PEI)-cellulose thin layers were purchased from Alltech (Deerfield, IL). [{gamma}-32P]ATP (sp. act. > 7000 Ci/mmol) was from ICN Pharmaceutical (Irvine, CA). Kodak XAR-5 film was obtained from Eastman Kodak (Rochester, NY) for autoradiography. Reagents used for the AP site assay were as described in Nakamura et al. (18). Trypsin, fetal bovine calf serum (FBS), trypan blue stain and Dulbecco's modified Eagle's medium (DMEM)/F12 were purchased from Life Technologies (Gaithersburg, MD). All other chemicals were purchased from Sigma, Aldrich and Fisher unless stated otherwise, and used without further purification.

Reaction of human HeLa S3 tumor cells with Cl4HQ
To measure the induction of DNA damage in nuclear DNA by PCP-derived quinonoid metabolites, 40 ml HeLa S3 cells (5x105 cell/ml) were suspended in (DMEM)/F12 (1:1) supplemented with 10% FBS at 37°C for 4 h until cells were seeded in a tissue culture flask (162 cm2). The medium was decanted and the cells were maintained in (DMEM)/F12 (1:1) supplemented with 0.5% FBS at 37°C. Cells were treated with Cl4HQ (prepared in acetone; final concentration 0, 50 and 300 µM) by adding stock solutions directly to the medium and were incubated at 37°C for 0.5 or 2 h. Following treatment, the medium was decanted and the seeded HeLa cells were washed twice with PBS, digested with 0.25% trypsin for 5 min at 37°C. Immediately following the digestion, the cells were pelleted (200 g at 4°C) and stored under –80°C until further processing. Cytotoxicity evaluated by trypan blue dye exclusion showed high survival rates (85% or more) after exposure.

DNA isolation
Nuclear DNA of HeLa S3 cells was isolated by phenol–chloroform extraction as described in Nakamura and Swenberg (15), with modifications. Cells were added to 4 ml ice-cold lysis buffer (Applied Biosystems, Foster City, CA), proteinase K (Applied Biosystems) and 20 µl BHT (20 mg/ml in acetone) and mixed for 12–16 h at 4°C. The cell lysates were extracted manually twice with 4 ml 70% phenol/chloroform/water (Applied Biosystems) containing 20 µl 20 mg/ml BHT and once with 4 ml Sevag mix containing 20 µl 20 mg/ml BHT on ice. DNA was precipitated from the aqueous layer with the addition of 1/10 vol 4 M NaCl and 2.5 vol ice-cold 100% ethanol. It was then washed with 5 ml 70% ethanol (ice cold, –20°C). The DNA pellet was resuspended in 2 ml PBS containing RNase A, RNase T1 and 5 µl 20 mg/ml BHT and was mixed at 4°C for 1–16 h to make a homogenous DNA solution. Following the incubation at 37°C for 30 min, an additional 2 ml deionized H2O was added, and DNA was precipitated with the addition of 2.5 vol ice-cold 100% ethanol, and washed with 70% ice-cold ethanol. The DNA pellets were resuspended in deionized water for 16 h by mixing at 4°C. DNA was quantified by spectrophotometry, assuming a A260 of 1 when the DNA concentration was 50 µg/ml.

DNA adduct analysis by 32P-post-labeling assay
Covalent modification of DNA was analyzed by 32P-post-labeling after enrichment by nuclease P1 digestion, as described in Reddy and Randerath (19). DNA (5–10 µg) was digested with micrococcal nuclease and spleen phosphodiesterase, and the adducts were enriched by nuclease P1. The adducts were labeled with [{gamma}-32P]ATP (~100 µCi, sp. act. >7000 Ci/mmol) and T4 polynucleotide kinase as described by Gupta (20). Aliquots were spotted onto a polyethyleneimine cellulose sheet (12x20 cm) with a 15 cm wick (Whatman 1 chromatography paper). The plates were first developed in 1.0 M sodium phosphate (pH 5.8) (D1) overnight. The adducts were separated by development in the opposite direction as D1 with 3.6 M lithium formate and 8.5 M urea (pH 3.5). This was followed by development in 0.6 M lithium chloride and 0.5 M Tris–HCl, and 8.5 M urea (pH 8.0) perpendicular to the previous development onto a 2 cm wick (Whatman 3 mm chromatography paper). A final development with 1.7 M sodium phosphate (pH 6.0) onto a 2 cm wick (Whatman 1 mm chromatography paper) was performed in the same direction to reduce the background radioactivity. Adducts were visualized by autoradiography. Quantitation was performed by counting the radioactivity for each adduct and total nucleotides to determine the relative adduct levels (RAL) as described by Reddy and Randerath (19).

Apurinic/apyrimidinic sites analyzed by ASB assay
Apurinic/apyrimidinic sites were assayed based upon the reaction of the aldehyde group in an AP site with a probe bearing a biotin residue as described by Nakamura et al. (18). The AP site cleavage assay was performed as described by Nakamura and Swenberg (15).

Analysis of 8-HO-dG by HPLC-ECD
Quantitation of 8-HO-dG was based on a HPLC/electrochemical detection method (HPLC-ECD). DNA (100 µg) was hydrolyzed enzymatically to deoxyribonucleosides using deoxyribonuclease I, spleen phosphodiesterase, snake venom phosphodiesterase and alkaline phosphatase. The digest was separated by reversed phase HPLC, and 8-HO-dG quantitated using an electrochemical array detector (ESA, Chelmsford, MA), as described in Lin et al. (7).

Statistical analysis
All data are expressed as mean ± SD. The significance of differences in the results was evaluated with ANOVA, followed by Dunnett's multiple comparison test.


    Results
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 Results
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DNA adduct analysis by 32P-post-labeling assay
To determine the formation of direct DNA adducts, DNA isolated from HeLa S3 cells treated with Cl4HQ was analyzed by 32P-post-labeling assay. One major and two minor adducts (adducts 1, 2 and 3) were detected in cells treated with 300 µM Cl4HQ for 0.5 (Figure 2BGo) and 2 h (Figure 2CGo). The number of DNA adducts was estimated to be 3–6 adducts per 108 total nucleotides (Table IGo). The relative percentages of the adducts were estimated as follows: 1, 47%; 2, 26% and 3, 27%. The adduct levels measured in cells treated with 300 µM Cl4HQ for 2 h were 1.8-fold greater than those for the 0.5 h treatment, suggesting adduct accumulation. Relatively small amounts of direct adducts were detected in cells treated with 50 µM Cl4HQ for 2 h and the levels of individual adducts were estimated to be ~1–5 adducts per 109 nucleotides (autoradiographs not shown) which was close to the limit of detection (1 adduct per 109 nucleotides). Further investigation confirmed that these adducts did not correspond to those produced in calf thymus DNA treated with Cl4BQ (Figure 2DGo).



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Fig. 2. 32P-post-labeling maps of adducts of tetrachlorohydroquinone (Cl4HQ) with HeLa S3 cells treated with Cl4HQ: (A) control only; (B) 300 µM Cl4HQ for 0.5 h; (C) 300 µM Cl4HQ for 2 h; (D) DNA adducts in calf thymus DNA treated with tetrachlorohydroquinone Cl4BQ (1 mM) at 37°C for 2 h. The autoradiograph exposure time was 72 h at – 80°C. Chromatography conditions are as described in Materials and methods.

 

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Table I. Formation of DNA adducts (per 108 total nucleotides) in nuclear DNA of HeLa S3 cells treated with Cl4HQ (300 µM) for 0.5–2 h at physiological condition
 
Detection and characterization of AP sites
To investigate whether Cl4HQ induces increases in AP sites in intact cells, human HeLa S3 tumor cells were incubated with Cl4HQ (0–300 µM) for 0.5–2 h under physiological conditions. Nuclear DNA was isolated and analyzed by ASB assay for AP sites. Results from the measurement of AP sites indicated that an increased number of AP sites was detected in cells treated with 300 µM Cl4HQ for 0.5 h (13.4 ± 1.34 versus 10.2 ± 1.49 AP sites per 106 nucleotides) (P < 0.05) and 2 h (15.7 ± 3.38 versus 7.86 ± 1.26 AP sites per 106 nucleotides) (P < 0.01) (Figure 3Go). Increases in the number of AP sites in cells treated with Cl4HQ at 50 µM for 0.5–2 h were not statistically significantly increased over the corresponding control. To test the hypothesis that AP sites induced by Cl4HQ in HeLa cells are excisable by putrescine, DNA was incubated with putrescine and immediately followed by the ASB assay. Results indicated that Cl4HQ induced predominantly putrescine-excisable AP sites (75%) in HeLa S3 cells (Figure 4Go).



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Fig. 3. Formation of AP sites in nuclear DNA of human HeLa S3 tumor cells treated with 0–300 µM Cl4HQ under physiological conditions at 37°C for 0.5–2 h. Data represent the mean ± SD of three to five determinations. Treatments marked with asterisks are statistically significantly different from control: *P < 0.05; **P < 0.01.

 


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Fig. 4. Formation of putrescine-excisable AP sites in nuclear DNA of human HeLa S3 tumor cells treated with 0–300 µM Cl4HQ under physiological condition at 37°C for 0.5–2 h. Data represent the mean ± SD of three to five determinations. DNA isolated from HeLa S3 cells treated with Cl4HQ and vehicle control were reacted with putrescine (Putre) and without putrescine (-/-) followed by ASB assay for AP sites. The number of putrescine-excisable AP sites was calculated from the original number of AP sites () minus the number of AP sites left after putrescine treatment (Putre).

 
Analysis of the formation of 8-HO-dG
To determine whether Cl4HQ induces the formation of 8-HO-dG in HeLa S3 cells in parallel to the induction of AP sites, nuclear DNA derived from the control and treated HeLa S3 cells was assayed for the presence of 8-HO-dG by HPLC-ECD method as described above. Results indicated that the concentration of 8-OH-dG increased 2-fold in cells treated with 300 µM Cl4HQ for 0.5 h over the corresponding control (0.43 ± 0.09 versus 0.21 ± 0.03 8-HO-dG per 105 dG) (Figure 5Go). The concentration of 8-OH-dG increased 5-fold in cells treated with 300 µM Cl4HQ compared with the control 2 h after treatment (0.69 ± 0.13 versus 0.14 ± 0.03 8-HO-dG per 105 dG) (P < 0.001). No increase in 8-HO-dG was detected in cells treated with 50 µM Cl4HQ.



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Fig. 5. Formation of 8-HO-dG in nuclear DNA of human HeLa S3 tumor cells treated with 0–300 µM Cl4HQ under physiological conditions at 37°C for 0.5–2 h. Data represent the mean ± SD of three to five determinations. Treatments marked with asterisks are statistically significantly different from control: *P < 0.05; **P < 0.01.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our previous investigation provided clear evidence of parallel formation of 8-HO-dG, DNA fragmentation, and AP sites in calf thymus DNA treated with Cl4HQ in the presence of the metal ion, Cu(II) (14). We demonstrated that, unlike estrogen and PAH-derived quinones, the AP sites induced by PCP quinonoids in calf thymus DNA were primarily mediated by ROS, whereas the contribution derived from depurination/depyrimidination of direct DNA adducts was relatively insignificant for the formation of AP sites (21,22). Our objective here was to extend our work in PCP quinonoid-induced DNA damage in calf thymus DNA to mammalian cells, where DNA damage is subject to the repair process.

It is increasingly evident that various types of oxidative DNA damage can be derived from ROS that interact with DNA. In an effort to understand the genetic consequences of oxidative damage in intact cells induced by PCP quinone and hydroquinone, we analyzed the relationship between the formation of direct adducts and oxidative damage in the nuclear DNA of mammalian cells following treatment with Cl4HQ. Results of our analyses suggest that there is a trend for concentration- and time-dependent formation of direct adducts in nuclear DNA of HeLa S3 cells treated with Cl4HQ. We observed that Cl4HQ-derived DNA adducts accumulate over time and that these DNA adducts did not co-elute with those generated by Cl4BQ in calf thymus DNA. Adduct levels were estimated to be ~10–20% of that observed in calf thymus DNA treated with Cl4HQ (14).

In parallel, an increased number of AP sites and 8-HO-dG was detected in cells treated with Cl4HQ over the corresponding control. Levels of AP sites and 8-HO-dG were estimated to be approximately two orders of magnitude greater than those of direct DNA adducts. However, we do not exclude the possibility that the relative levels of these distinct DNA lesions could be underestimated since the 32P-post-labeling method may not fully recover the direct adducts induced by Cl4HQ. Further investigation indicated that the AP sites induced by Cl4HQ in HeLa S3 cells were predominantly (75%) excised by putrescine. This result is comparable with those observed in calf thymus DNA treated with Cl4HQ plus Cu(II), Cl4BQ plus Cu(II) and NADPH (14), and is in good agreement with those observed in HeLa S3 cells treated with hydrogen peroxide (15). The predominant formation of putrescine-excisable AP sites along with the persistent presence of direct DNA adducts suggest that ROS are the main source for the induction of AP sites by Cl4HQ in HeLa cells. Altogether, these results point to the formation of putrescine-excisable AP sites as one of the major types of oxidative DNA damage in intact cells induced by Cl4HQ.

The ratio of the net increases of 8-HO-dG to AP sites (per nucleotide) in HeLa S3 cells induced by Cl4HQ is estimated to be 1:6, which is 4-fold more than that estimated in calf thymus DNA treated with Cl4HQ plus Cu(II) (ratio ~1:1.6) (14). We recently demonstrated that the Fenton reaction predominantly induced 8-HO-dG in calf thymus DNA compared with AP sites and oxidized pyrimidine bases (23). In contrast, 8-HO-dG was less than the number of AP sites in HeLa cells exposed to H2O2. In addition, while AP sites were persistent in HeLa cells during a 6 h repair period, 8-HO-dG was significantly repaired (~83%) within 6 h. Based on these results, we believe that repair enzymes, 8-HO-dG glycosylase/lyase (mOGG1), that cleave 8-HO-dG to form 3' AP sites, is responsible for the difference observed between calf thymus DNA treated with Cl4HQ plus Cu(II) and cells exposed to Cl4HQ (24,25). Taken together, we theorize that quinonoid-derived ROS mediate the formation of putrescine-excisable AP sites, which may explain, in part, PCP-induced clastogenicity in mammalian cells.


    Notes
 
1 Present address: Department of Environmental Engineering, National Chung-Hsing University, Taichung, Taiwan Back

2 To whom correspondence should be addressed Back


    Acknowledgments
 
This work was supported by the National Institute of Environmental Health Sciences through grants P42ES05948, F32ES05868 and T32ES07126.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 18, 2000; revised November 2, 2000; accepted December 20, 2000.