Analysis of DNA adducts in rats exposed to pentachlorophenol

Po-Hsiung Lin1,2, David K. La1,3, Patricia B. Upton1 and James A. Swenberg1,4

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Pentachlorophenol (PCP) is a widely used biocide that has been reported to be hepatocarcinogenic in mice. Its effects in rats are equivocal, but the liver clearly is not a target organ for carcinogenesis. The carcinogenic effects of PCP in mice may relate to reactive oxygen species generated during metabolism. PCP is known to increase the hydroxyl radical-derived DNA lesion, 8-oxodeoxyguanosine (ohdG), in the liver of exposed mice. To investigate whether the generation of oxidative DNA damage and direct DNA adducts may explain the species difference in carcinogenicity, we have analyzed ohdG in hepatic DNA from PCP-exposed rats. Rats were exposed acutely to PCP for 1 or 5 days. Tissues also were obtained from a 27 week interim sacrifice of the 2 year National Toxicology Program carcinogenesis bioassay. We used HPLC with electrochemical array detection for ohdG analysis. Single or 5 day exposure to PCP (up to 120 or 60 mg/kg/day, respectively) did not increase ohdG. Dietary exposure to 1000 p.p.m. PCP (equivalent to 60 mg/kg/day) for 27 weeks induced a 2-fold increase in ohdG (1.8 versus 0.91x10–6 in controls). In parallel, formation of direct DNA adducts was analyzed by 32P-post-labeling following nuclease P1 adduct enrichment. We detected two major DNA adducts with relative adduct labeling of 0.78x107 adducts per total nucleotides. One of these adducts was found to co-migrate with the adduct induced by the metabolite, tetrachloro-1,4-benzoquinone. We observed differences in DNA adduct formation between acute and chronic studies, with acute studies not inducing any detectable amount of DNA adducts. These results indicated that chronic, but not acute exposure to PCP increased ohdG and direct adducts in hepatic DNA. As the same exposure conditions that enhanced ohdG did not produce liver cancer in rats, the generation of reactive oxygen species, oxidative DNA damage and direct DNA adducts is not sufficient for the induction of hepatocarcinogenesis by PCP in the rat.

Abbreviations: ohdG, 8-oxodeoxyguanosine; PCP, pentachlorophenol


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Pentachlorophenol (PCP) is a wide-spectrum biocide that has been used as a wood preservative, herbicide and defoliant (1). Its extensive use and long persistence have resulted in significant environmental contamination and potential exposure to the general population. The carcinogenic effects of PCP have been evaluated in several chronic animal bioassays. PCP was reported to be carcinogenic in B6C3F1 mice, inducing hepatocellular adenomas and carcinomas, hemangiosarcomas and adrenal pheochromocytomas (2). Its effects in rats were equivocal. PCP did not induce tumors in Sprague–Dawley rats (3). A recent NTP bioassay reported increased incidences of mesotheliomas (tunica vaginalis) and nasal squamous cell carcinomas, but these effects were observed only when the maximum tolerated dose was exceeded (4). The liver is a major site for PCP metabolism, but is not a target organ for carcinogenesis in the rat.

The mechanism underlying PCP carcinogenesis in the mouse is unknown, but may relate to reactive oxygen species generated during PCP metabolism. PCP is metabolized to tetrachlorohydroquinone and tetrachlorocatechol, which can be oxidized further to tetrachloro-1,4-benzoquinone and tetrachloro-1,2-benzoquinone via the corresponding semiquinones (5,6). Redox cycling associated with the oxidation of tetrachlorohydroquinone and/or the reduction of tetrachloro-1,4-benzoquinone to semiquinones generates oxygen radicals. This cascade has been reported to increase the concentration of the hydroxyl radical-derived DNA lesion, such as 8-oxodeoxyguanosine (ohdG), in the livers of PCP and tetrachlorohydroquinone-treated mice (7–11). In addition, the formation of direct DNA adducts by the quinonoid metabolite, tetrachloro-1,4-benzoquinone has been reported in the liver of B6C3F1 mice treated with multiple doses of PCP (at 15 mg/kg body wt once a day for 7 days) (12).

We are unaware of any published studies that have reported oxidative DNA damage and direct DNA adducts in the rat. To investigate whether the generation of reactive oxygen species and oxidative DNA damage, as well as direct DNA adducts, may explain the species difference in carcinogenicity, we have analyzed ohdG and direct DNA adducts in hepatic DNA from PCP-exposed rats. Fischer-344 rats were exposed acutely to PCP for 1 or 5 days and for 27 weeks in an interim sacrifice of the 2 year National Toxicology Program (NTP) bioassay.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Male Fischer-344 rats, ~200 g, were purchased from Charles River Laboratories (Raleigh, NC) and acclimated prior to treatment. PCP was dissolved in corn oil, and administered to rats by gavage in a volume of 5 ml/kg. Rats (n = 3–4 per group) were administered 30, 60 or 120 mg PCP/kg for 1 day and killed 4 h following exposure. Additional rats (n = 3–4 per group) were exposed to 30 or 60 mg/kg/day for 5 days. We isolated DNA from the liver by phenol and chloroform extractions and ethanol precipitation. Artifactual generation of ohdG was minimized during sample work-up by the addition of radical scavengers (e.g. butylated hydroxytoluene). We also obtained tissues from a 27 week interim sacrifice of the 2 year NTP carcinogenesis bioassay, in which Fischer-344 rats were fed 1000 p.p.m. PCP daily. DNA was isolated from the liver of 10 exposed and 10 control rats.

Quantification of ohdG was based on HPLC/electrochemical detection approach, which was modified from a method described previously by Richter et al. (13). DNA 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 ohdG quantified using an electrochemical array detector (ESA, Chelmsford, MA). Electrochemical oxidation was monitored at 300, 375, 450, 525, 600, 700, 800 and 900 mV. The electrochemical array system, with multiple electrodes, has significantly improved the sensitivity and specificity of detection. In a serial array of electrodes at increasing potential, compounds reacted and measured at one potential are effectively eliminated from detection by subsequent electrodes in the array. By this process, compounds that co-elute are clearly differentiated. Moreover, the electrochemical oxidation pattern at different potentials provides a fingerprint that facilitates qualitative characterization of samples. The concentration of ohdG was normalized to the amount of deoxyguanosine present in tissues, as determined by HPLC/electrochemical detection.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We detected ohdG and dG in DNA digests using HPLC/electrochemical detection, with maximum oxidation occurring at an applied potential of 375 mV (electrochemical oxidation monitored at 300, 375, 450, 525, 600, 700, 800 and 900 mV). The linear range of the standard calibration curves for ohdG and dG were 5–500 fmol (r2 > 0.990) and 0.1–5.2 nmol (r2 > 0.999), respectively. The limit of detection was defined as 1 ohdG/107 dG when 100 mg of DNA was assayed. Values of the ohdG and dG were within the linear range of the standard curves. Concentrations of ohdG were 2-fold greater in hepatic DNA of chronically exposed rats (1.8 ± 0.65x 10–6) relative to controls (0.91 ± 0.42x10–6) (Figure 1CGo). This difference was statistically significant when compared using Student's t-test (P = 0.01). We did not observe an increase in ohdG in hepatic DNA following a single exposure or the 5 day exposure to PCP (Figure 1A and BGo).



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Fig. 1. Comparison of ohdG in liver DNA of rats exposed to PCP. Fischer-344 rats were exposed to PCP (A) by single administration, (B) for 5 days and (C) 27 weeks. Levels of ohdG were detected by HPLC/electrochemical array analysis. Electrochemical oxidation was monitored at potentials of 300, 375, 450, 525, 600, 700, 800 and 900 mV. Quantitation of ohdG was based upon the applied potential of 375 mV. Treatment marked with different symbols (*) are statistically significantly different from control (P < 0.05).

 
Several investigations have suggested that PCP-induced mouse hepatocarcinogenesis involves the generation of DNA reactive oxygen species (8,10,11,14–16). In this study, we investigated the effects of PCP exposure on steady state concentrations of the oxidative DNA lesion, ohdG, in Fischer-344 rats. Chronic exposure to PCP (1000 p.p.m.) induced 2-fold greater concentrations of ohdG in the hepatic DNA of exposed rats relative to controls. The increase in ohdG in the rat liver was comparable with that reported previously for B6C3F1 mice. Umemura et al. (11) reported a 2.4–2.8-fold increase in ohdG in hepatic DNA of mice exposed up to 0.12% PCP in the diet for 2–4 weeks. Based on food consumption, the amount of PCP ingested by the mice was 120 mg/kg/day compared with 60 mg/kg/day in the Fischer-344 rats exposed to 1000 p.p.m.

We did not observe an increase in ohdG in rats following a single or the 5 day exposures to PCP (Figure 1A and BGo), whereas Sai-Kato et al. (10) reported increased ohdG in mice exposed similarly. This difference may result from quantitative differences in PCP metabolism. PCP exposure has been shown to produce a 4-fold greater nuclear dose of total quinone metabolites in mouse liver relative to the rat (17). It is not known whether differences in ohdG following short-term exposure to PCP are important in the observed species differences in carcinogenicity. It is interesting to note that while acute exposure to PCP increased ohdG in mouse liver DNA, such increases were not observed in non-target organs (10).

The generation of reactive oxygen species and oxidative DNA damage from PCP exposure does provide a plausible explanation for the increased incidence of mesotheliomas in rats. Previous research with mineral fibers strongly suggests that free radicals are involved in the development of mesotheliomas (18,19). The oxidant chemical, potassium bromate, also causes increased incidences of mesotheliomas (20). Mesothelial cells have relatively low amounts of antioxidants and are susceptible to free radical toxicity. In the chronic bioassay of PCP, rats in the 600 p.p.m. exposure group received a greater cumulative dose than the 1000 p.p.m. group, as the former was dosed for 2 years compared with 1 year for the 1000 p.p.m. group (4). Yet, mesotheliomas were observed only in the latter group, indicating a possible threshold. Oxidative DNA damage commonly exhibits a threshold, where effects are not pronounced until cellular defences are significantly depleted.

Events in addition to the formation of oxidative DNA lesions also may be important in PCP carcinogenesis. There are species differences in PCP toxicity, with greater hepatotoxicity reported in mice than in rats. PCP induces hepatocellular karyomegaly, cytomegaly and degeneration in mice, whereas only mild hepatotoxicity has been observed in exposed rats (2,21). Umemura et al. (11) reported that PCP exposure induced a sustained increase in cell proliferation in the liver of B6C3F1 mice. While the effects of PCP on cell proliferation have not been investigated in the rat, the species difference in toxicity suggests a possible species difference in cell proliferation.

Covalent modification of DNA by endogenous and exogenous electrophiles is generally considered to be important in carcinogenesis. In addition to ohdG, PCP induces other DNA lesions. Randerath et al. (22) reported that wood preserving waste extracts, which include PCP, increased concentrations of bulky oxidative DNA lesions. The quinone and semiquinone metabolites of PCP also are strongly electrophilic and capable of binding to macromolecules (5,23). The covalent modification of DNA was analyzed by 32P-post-labeling after enrichment of assay sensitivity by nuclease P1 digestion as described (24). Using the 32P-post-labeling procedure, we observed adduct formation in liver DNA of Fischer-344 rats chronically exposed to 1000 p.p.m. PCP (Figure 2BGo). Values of relative adduct levels of these two adducts were estimated to be 0.78 ± 0.04 adducts/107 total nucleotides. Comparison of DNA adducts 1 and 2 produced in rats treated with PCP and in calf thymus DNA by Cl4-1,4-BQ (Figure 2CGo) suggests that adduct 1 is derived from Cl4-1,4-BQ. Such an induction of adducts in the liver by chronic administration of PCP to rats was not detected in rats treated with single (0–120 mg/kg body wt) and multiple doses (0–60 mg/kg body wt) of PCP by gavage (data not shown). Formation of adducts in kidney DNA was also observed in these PCP-treated rats (15). The chemical structure of the adduct, while unknown, appears to be the same as a DNA adduct induced by the PCP metabolite, tetrachloro-1,4-benzoquinone based on chromatographic migration. This adduct was present at a concentration 10-fold lower than the amount of ohdG present in the DNA. In addition, we noticed that there was evidence of parallel formation of DNA adducts and ohdG in the liver of rats chronically exposed to PCP, whereas single and multiple doses of PCP to rats did not induce these DNA lesions. The tetrachloro-1,4-benzoquinone-derived DNA adduct also was detected in mouse liver DNA following exposure to PCP (15 mg PCP/kg body wt/day for 7 days) at 8 adducts/107 nucleotides which is significantly greater compared with the rat (12). This evidence is in good agreement with the finding that a greater nuclear dose of total PCP-derived quinone metabolites in mouse liver relative to the rat (17).



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Fig. 2. Autoradiographs from 32P-post-labeling analysis of adducts in liver nuclear DNA of male Fischer 344 rats fed (A) normal diet, (B) PCP (1000 p.p.m.) for 27 weeks and (C) reaction of tetrachloro-1,4-benzoquinone (5 mM) with calf thymus DNA. DNA (5–10 µg) was digested with micrococcal nuclease, spleen phosphodiesterase, and enriched by nuclease P1. Adducts were labeled with 32P-ATP (~100 mCi, sp. act. > 7000 Ci/mmol) and T4 polynucleotide kinase phosphorylation. Aliquots were spotted onto polyethylene imine cellulose sheet (12x20 cm) with a 15 cm wick (Whatman 17 Chromatography paper). The plates were first developed in 1.0 M sodium phosphate (pH 5.7) (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 is followed by development in 0.6 M lithium chloride and 0.5 M Tris–HCl, and 8.5 M urea (pH 8.0) (D4) with a 2 cm of wick perpendicular to D3. A final development with 1.7 M sodium phosphate (pH 5.8) in the same direction of D4 is to reduce the background radioactivity. Screen-intensified autoradiography was for 20 h at –80°C. Quantification of adducts were estimated by relative adduct levels and were performed by counting the radioactivity for each adduct versus the respective total nucleotides by scintillation counter.

 
It is known that DNA modification by PCP can be mediated by endogenous and exogenous electrophiles (Figure 3Go), but their relative importance in PCP carcinogenesis is unclear. Our finding of increased ohdG in rat liver suggests that it is not sufficient to induce hepatic carcinogenesis. However, ohdG is but one of many DNA lesions that derive from reactive oxygen species. Recently, PCP has been reported to induce apurinic/apyrimidinic sites in DNA by an oxygen radical mechanism that involves cleavage of the deoxyribose, as well as by glycosylase cleavage of oxidized bases in DNA (25,26). Additional investigations of these secondary DNA lesions will be important to improve the understanding of PCP carcinogenesis.



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Fig. 3. Proposed pathway for the generation of reactive oxygen species during PCP metabolism. PCP is metabolized to quinols [i.e. tetrachlorocatechol (Cl4CAT) and tetrachlorohydroquinone (Cl4HQ)], which undergo auto-oxidation and/or enzyme-mediated oxidation to the corresponding semiquinones [tetrachloro-1,2-benzosemiquinones (Cl4-1,2-SQ) and tetrachloro-1,2-benzosemiquinones (Cl4-1,4-SQ)] and quinones [i.e. tetrachloro-1,2-benzoquinone (Cl4-1,2-BQ) and tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ)]. Subsequent reduction of quinones to semiquinones initiates redox cycling cascades and generates reactive oxygen species (i.e. H2O2), which induce oxidative DNA damage. Additionally, PCP quinones and semiquinones (not shown) are capable of reacting with genomic DNA to form direct DNA adducts.

 
The greater amounts of both oxidative and direct DNA damage, together with increased hepatotoxicity and cell proliferation, may provide the critical events necessary for hepatic carcinogenesis in the mouse. In contrast, the decreased amount of DNA damage and the lack of hepatotoxicity and cell proliferation in the rat do not result in such critical changes. The data suggest that a high degree of uncertainty will be associated with extrapolation of high dose cancer risks based on the mouse data to lower human exposures.


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

3 Alza Corporation, 1900 and Charleston Road, Mountain View, CA 94043, USA Back

4 To whom correspondence should be addressed Email: janes_swenberg{at}unc.edu Back


    Acknowledgments
 
The authors are grateful to the National Toxicology Program for providing the liver tissue of rats in a chronic bioassay and Dr Jun Nakamura's comments on the manuscript. This research was supported in part by grants from the National Institute of Environmental Health Sciences (P42ES05948, P30ES10126, F32ES05868 and T32ES07126) and the National Cancer Institute (P30CA16086 and CA 83369).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

  1. IARC (1979) IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans. Pentachlorophenol, Vol. 20. IARC Scientific Publications, Lyon.
  2. National Toxicology Program (NTP) (1989) Toxicology and Carcinogenesis Studies of Two Pentachlorophenol Technical-Grade Mixtures (CAS No. 87-86-5) in B6C3F1 Mice (Feed Studies). Technical Report No. 349. NTP, Research Triangle Park, NC.
  3. Schwetz,B.A., Quast,I.F., Keeler,P.A., Humiston,C.G. and Kociba,R.J. (1978) Results of two-year toxicity and reproduction studies on pentachlorophenol in rats. In Rao,K.R. (ed.) Pentachlorophenol: Chemistry, Pharmacology and Environmental Toxicology. Plenum Press, New York, pp. 301–309.
  4. National Toxicology Program (NTP) (1999) Toxicology and Carcinogenesis Studies of Pentachlorophenol (CAS No. 87-86-5) in F344/N Rats (Feed Studies). Technical Report No. 483. NTP, Research Triangle Park, NC.
  5. van Ommen,B., Adang,A., Muller,F. and van Bladeren,P.J. (1986) The microsomal metabolism of pentachlorophenol and its covalent binding to protein and DNA. Chem. Biol. Interact., 60, 1–11.[ISI][Medline]
  6. Lin,P.H., Nakamura,J. and Swenberg,J.A. (1999) Quantitative analysis of DNA adducts and abasic sites induced by pentachlorophenol-derived quinone and hydroquinone in calf-thymus DNA. Toxicologist, 48, 232–233.
  7. Carstens,C.P., Blum,J.K. and Witte,I. (1990) The role of hydroxyl radicals in tetra-chlorohydroquinone induced DNA strand break formation in PM2 DNA and human fibroblasts. Chem. Biol. Interact., 74, 305–314.[ISI][Medline]
  8. Dahlhaus,M., Almstadt,E. and Appel,K.E. (1994) The pentachlorophenol metabolite tetrachloro-p-hydroquinone induces the formation of 8-hydroxy-2-deoxyguanosine in liver DNA of male B6C3F1 mice. Toxicol. Lett., 74, 265–274.[ISI][Medline]
  9. Naito,S., Ono,Y., Somiya,I., Inoue,S., Ito,K., Yamamoto,K. and Kawanishi,S. (1994) Role of active oxygen species in DNA damage by pentachlorophenol metabolites. Mutat. Res., 310, 79–88.[ISI][Medline]
  10. Sai-Kato,K., Umemura,T., Takagi,A., Hasegawa,R., Tanimura,A. and Kurokawa,Y. (1995) Pentachlorophenol-induced oxidative DNA damage in mouse liver and protective effect of antioxidants. Food Chem. Toxicol., 33, 877–882.[ISI][Medline]
  11. Umemura,T., Sai-Kato,K., Takagi,A., Hasegawa,R. and Kurokawa,Y. (1996) Oxidative DNA damage and cell proliferation in the livers of B6C3F1 mice exposed to pentachlorophenol in their diet. Fund. Appl. Toxicol., 30, 285–289.[ISI][Medline]
  12. Bodell,W.J. and Pathak,D.N. (1998) Detection of DNA adducts in B6C3F1 mice treated with pentachlorophenol. Proc. Am. Assoc. Cancer Res., 39, 332.
  13. Richter,C., Park,J.W. and Ames,B.N. (1988) Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl Acad. Sci., 85, 6465–7467.[Abstract]
  14. Dahlhaus,M., Almstadt,E., Henschke,P., Luttgert,S. and Appel,K.E. (1995) Induction of 8-hydroxy-2-deoxyguanosine and single-strand breaks in DNA of V79 cells by tetrachloro-p-hydroquinone. Mutat. Res., 329, 29–36.[ISI][Medline]
  15. La,D.K., Lin,P.H. and Swenberg,J.A. (1998) Analysis of DNA adducts in rats chronically exposed to pentachlorophenol. Proc. Am. Assoc. Cancer Res., 39, 330.
  16. Dalhaus,M., Almstadt,E., Henschke,P., Luttgert,S. and Appel,K.E. (1996) Oxidative DNA lesions in V79 cells mediated by pentachlorophenol metabolites. Arch. Toxicol., 70, 457–460.[ISI][Medline]
  17. Lin,P.H., Waidyanatha,S., Pollack,G.M. and Rappaport,S.M. (1997) Dosimetry of chlorinated quinone metabolites of pentachlorophenol in the livers of rats and mice based upon measurement of protein adducts. Toxicol. Appl. Pharmacol., 145, 399–408.[ISI][Medline]
  18. Adachi,S., Yoshida,S., Kawamura,K., Takahashi,M., Uchida,H., Odagiri,Y. and Takemoto,K. (1994) Inductions of oxidative DNA damage and mesothelioma by crocidolite, with special reference to the presence of iron inside and outside of asbestos fiber. Carcinogenesis, 15, 753–758.[Abstract]
  19. Murata-Kamiya,N., Tsutsui,T., Fujino,A., Kasai,H. and Kaji,H. (1997) Determination of carcinogenic potential of mineral fibers by 8-hydroxydeoxyguanosine as a marker of oxidative DNA damage in mammalian cells. Int. Arch. Occup. Environ. Health, 70, 321–326.[ISI][Medline]
  20. Kurokawa,Y., Takayama,S., Konishi,Y., Hiasa,Y., Asahina,S., Takahasi,M., Maekawa,A. and Hayashi,Y. (1986) Long-term in vivo carcinogenicity tests of potassium bromate, sodium hypochlorite and sodium chlorite conducted in Japan. Environ. Health Perspect., 69, 221–235.[ISI][Medline]
  21. Kimbrough,R.D. and Linder,R.E. (1978) The effect of technical and purified pentachlorophenol on the rat liver. Toxicol. Appl. Pharmacol., 46, 151–162.[ISI][Medline]
  22. Randerath,K., Zhou,G.D., Randerath,E., Safe,S.H. and Donnelly,K.C. (1997) Comparative 32P-postlabeling analysis of exogenous and endogenous DNA adducts in mouse skin exposed to a wood-preserving waste extract, a complex mixture of polycyclic and polychlorinated chemicals. Environ. Mol. Mutagen., 29, 372–378.[ISI][Medline]
  23. Witte,I., Juhl,U. and Butte,W. (1985) DNA-damaging properties and cytotoxicity in human fibroblasts of tetrachlorohydroquinone, a pentachlorophenol metabolite. Mutat. Res., 145, 71–75.[ISI][Medline]
  24. Reddy,M.V. and Randerath,K. (1986) Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis, 7, 1543–1551.[Abstract]
  25. Lin,P.H., Nakamura,J., Yamaguchi,S., La,D.K., Upton,P.B. and Swenberg,J.A. (2001) Oxidative damage and direct adducts in calf thymus DNA induced by tetrachlorohydroquinone and tetrachloro-1,4-benzoquinone. Carcinogenesis, 22, 627–634.[Abstract/Free Full Text]
  26. Lin,P.H., Nakamura,J., Yamaguchi,S., La,D.K., Upton,P.B. and Swenberg,J.A. (2001) Induction of direct adducts, apurinic/apyrimidinic sites and oxidized bases in nuclear DNA of human HeLa S3 tumor cells by tetrachlorohydroquinone. Carcinogenesis, 22, 635–639.[Abstract/Free Full Text]
Received July 13, 2001; revised November 21, 2001; accepted November 27, 2001.