Induction of Hepatic 8-Oxo-deoxyguanosine Adducts by 2,3,7,8

Chem. Res. Toxicol. 2001, 14, 849-855

849

Induction of Hepatic 8-Oxo-deoxyguanosine Adducts by 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Sprague-Dawley Rats Is Female-Specific and Estrogen-Dependent Michael E. Wyde,†,‡ Victoria A. Wong,§ Amy H. Kim,†,‡ George W. Lucier,‡ and Nigel J. Walker*,‡ National Institute of Environmental Health Sciences, Environmental Toxicology Program, Research Triangle Park, North Carolina 27709, Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27709, and CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709 Received December 29, 2000

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a hepatocarcinogen that induces sex-specific hepatic neoplastic alterations in female, but not male, rats. It has been hypothesized that TCDD-induced alterations in estrogen metabolism lead to increased generation of reactive oxygen species. The resulting oxidative damage to DNA may contribute to TCDD-induced tumor promotion and hepatocarcinogenesis. This hypothesis is supported by previous observations of increased 8-oxo-deoxyguanosine (8-oxo-dG) adduct formation in the livers of intact, but not ovariectomized (OVX), rats following chronic exposure to TCDD. The aim of the current study was to more clearly define the roles of hormonal regulation, gender, dose-response, and exposure duration in TCDD induction of 8-oxo-dG adducts. Diethylnitrosamine (DEN)-initiated male and female (both intact and OVX) rats were exposed to TCDD in the presence or absence of 17 β-estradiol. Following 30 weeks of exposure, hepatic 8-oxo-dG adduct levels were significantly higher in TCDD-treated intact female rats, and TCDD-treated OVX female rats receiving supplemental 17 β-estradiol, when compared to respective corn oil vehicle controls. In DEN-initiated female rats exposed to a range of TCDD concentrations for 30 weeks, TCDD induced 8-oxo-dG adduct levels in a dose-dependent manner. However, 8-oxo-dG adduct levels were not altered in TCDD-treated male or OVX female rats following 30 weeks of exposure. In noninitiated female rats, the level of 8-oxo-dG adducts 4 days following a single dose of TCDD was not significantly different than in control rats. Additionally, 8-oxo-dG adduct formation was not affected by exposure to TCDD for 20 weeks in intact female rats. These data suggest that the induction of 8-oxo-dG adduct levels by TCDD is likely a response to chronic oxidative imbalance. These studies provide strong evidence that the induction of 8-oxo-dG by TCDD occurs via a chronic, sex-specific, estrogen-dependent mechanism.

Introduction 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a multisite rodent carcinogen in both sexes (1-3). In chronic 2-year bioassays, TCDD is a potent hepatocarcinogen in female, but not male, rats (1, 3). Although TCDD is not directly genotoxic (4-6), it has been hypothesized that TCDD may indirectly result in genotoxicity via an oxidative pathway. TCDD induces the cytochrome P450 isozymes CYP1 (7) which metabolize estradiol to catechol estrogens (8, 9). The induction of estradiol metabolism by TCDD-inducible cytochromes P450 may result in an oxidative imbalance that leads to DNA damage (10-12). Exposure to TCDD specifically induces 2- and 4-hydroxylase activity for 17 β-estradiol in rat liver (13, 14). The catechol estrogen, 4-hydroxyestradiol, is as equipotent a carcinogen as 17 β-estradiol in the Syrian hamster model of estrogen-induced carcinogenesis (15). In CD-1 mice, * To whom correspondence should be addressed. Phone: (919) 5414893. Fax: (919) 558-7053. E-mail: [email protected]. † University of North Carolina. ‡ National Institute of Environmental Health Sciences. § CIIT Centers for Health Research.

exposure to either 2- or 4-hydroxyestradiol during neonatal development resulted in the development of uterine adenocarcinomas (16). Oxidative damage resulting from redox cycling between catechol estrogen semiquinones and quinones is believed to be a major factor in carcinogenesis induced by estrogen and catechol estrogens (1719) and may be involved in the mechanism of TCDDinduced hepatocarcinogenesis. The interaction between reactive oxygen species and DNA leads to base modifications (20). If this oxidative damage to DNA is not repaired, modified bases may give rise to mutations during DNA replication. Since many of the agents that induce oxidative DNA damage are also complete carcinogens or tumor promoters, these cellular events are believed to contribute to multiple stages of the carcinogenic process (21-23). Of the wide variety of potential products of oxidative damage (24), the analysis of 8-oxo-deoxyguanosine (8-oxo-dG)1 adducts has com1 Abbreviations: 8-oxo-dG, 8-oxo-deoxyguanosine; dG, 2′-deoxyguanosine; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; OVX, ovariectomized; DEN, diethylnitrosamine; HPLC, high-performance liquid chromatography; EDTA, disodium ethylenediamine tetraacetate.

10.1021/tx000266j CCC: $20.00 © 2001 American Chemical Society Published on Web 05/25/2001

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monly been used as a potential surrogate marker for oxidative DNA damage (20, 25, 26). The induction of 8-oxo-dG adducts is associated with exposure to certain carcinogens (27-29) and can induce G to T transversions in DNA (30-32) if left unrepaired. The hypothesis of indirect oxidative DNA damage induced by TCDD is supported by observations that 8-oxo-dG adduct levels are higher in the liver of intact rats chronically exposed to TCDD compared to OVX rats (10). Additionally, exposure to 17 β-estradiol or 4-hydroxyestradiol increased 8-oxo-dG formation in the Syrian hamster model (33). Park et al. demonstrated the leakage of oxygen radicals from TCDD-induced CYP1A1 during presumed futile metabolism of TCDD, which is a poor substrate for CYP1A1 (34). Leakage of oxygen radicals from the catalytic site of cytochrome P4501A1 or potentially other TCDD-inducible cytochromes P450 such as CYP1A2 or CYP1B1 may contribute to the induction of oxidative DNA damage by TCDD in female rats. The aim of the current study was to more clearly define the roles of hormonal regulation, gender, dose-response, and exposure duration in TCDD-induced formation of 8-oxo-dG adducts in rat liver. To investigate the formation of 8-oxo-dG adducts by TCDD, diethylnitrosamine (DEN)-initiated female Sprague-Dawley rats received multiple doses of TCDD ranging from 0 to 125 ng/kg/day for 30 weeks and uninitiated rats 4 days following a single dose of TCDD. To test the hypothesis that estrogen enhances the induction of 8-oxo-dG adducts, DEN-initiated and OVX female rats were treated with TCDD for 20 or 30 weeks and males for 30 weeks in the presence and absence of estrogen administered continuously by subcutaneous implanted pellets (35).

Materials and Methods Animals. Estrogen-dependency of adduct formation was investigated in a previously described supplemental estrogen model (35). In addition, the dependency of adduct formation on gender was investigated in male Sprague-Dawley rats obtained from Charles River (Raleigh, NC). Male and female SpragueDawley rats (Charles River, Raleigh, NC) were housed three to a cage under conditions of controlled temperature (70 ( 0.5 °F), humidity (50 ( 5%), and lighting (12 h light/12 h dark), and received food and water ad libitum. Female rats were ovariectomized or sham-operated at 8 weeks of age. All animals were initiated with a single intraperitoneal (i.p.) injection of 175 mg of DEN/kg at 10 weeks of age. One week after initiation, males and OVX females were implanted with 90-day release pellets containing 0 mg (placebo) or 0.18 mg of 17 β-estradiol/pellet (Innovative Research). Intact female rats received placebo pellets only. New pellets were implanted after 90 days. Starting 1 week after pellet implantation, animals were treated once per week with a gavage dose of 700 ng of TCDD/kg or corn oil vehicle for 20 or 30 weeks. (This dose of TCDD is equivalent to an average daily dose of 100 ng of TCDD/kg/day). Each treatment group of males and females contained eight and nine animals, respectively. The dose-response relationship of TCDD-induced 8-oxo-dG adduct formation was analyzed in liver tissue obtained from a previous study (36). In this study, 8-week-old female SpragueDawley rats (Charles River, Raleigh, NC) were acclimated for 2 weeks. At 10 weeks of age, animals were initiated with single i.p. injections of 175 mg of DEN/kg of body weight. Starting 2 weeks later, animals were treated biweekly with gavage doses ranged from 49 to 1750 ng of TCDD/kg of body weight or corn oil vehicle for 30 weeks. These doses of TCDD are equivalent to average daily doses of 3.5, 10.7, 35.7, and 125 ng of TCDD/kg/

Wyde et al. day. Liver samples were stored at -70 °C. Each treatment group contained 8-10 animals. The effect of acute exposure to TCDD on the formation of 8-oxo-dG adducts was determined in an additional experimental protocol. Eight-week-old female Sprague-Dawley rats (Taconic, Germantown, NY) were acclimatized for 2 weeks. At 10 weeks of age, noninitiated animals received single gavage doses of 0, 1000, or 3000 ng of TCDD/kg of body weight or corn oil vehicle. As predicted by the model of Kohn et al. (37), doses of 1000 and 3000 ng of TCDD/kg should result in hepatic concentrations of TCDD 4 days after exposure of 7 and 23 ppb wet weight, respectively. These concentrations are approximately equivalent to those reported in chronically exposed rats (35, 38). Each treatment group contained five animals. All animals were killed by asphyxiation with CO2, and livers were removed, weighed, and frozen in liquid nitrogen. DNA Isolation. DNA was isolated by the chaotropic sodium iodide-based method of Wang et al. (39) adapted by Nakae et al. (40). WB DNA extractor kits were obtained from Wako Chemicals (Richmond, VA). Frozen rat liver tissue (100 mg) was homogenized on ice in 10 mM tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 7.5) containing 1% Triton X-100, 320 mM saccharose, and 5 mM MgCl2 (kit lysis solution) at 4 °C. Nuclei were isolated by centrifugation, washed in lysis solution, and resuspended in 10 mM Tris-HCl buffer (pH 8.0) containing 1% sodium dodecyl sulfate and 5 mM disodium ethylenediamine tetraacetate (kit enzyme reaction solution) at 4 °C. Residual RNA was eliminated with 95 units/mL RNase T1 and 0.19 mg/ mL RNase A (final concentration) at 50 °C. After 10 min, proteinase K (kit proteinase solution) at a final concentration of 739 µg/mL was added and incubated for an additional 60 min at 50 °C. After incubation, 7.6 M sodium iodide and 20 mM disodium ethylenediamine tetraacetate (EDTA) in 40 mM TrisHCl buffer (pH 8.0) (kit sodium iodide solution) was added. DNA was precipitated with an equal volume of isopropyl alcohol. Precipitated DNA was washed sequentially with each of two alcohol-based washing solutions (kit washing solution A and washing solution B) and vacuum-dried for 2 min. DNA was dissolved in 0.5 mL of 10 mM Tris buffer with 1 mM EDTA (pH 7.4) overnight at 4 °C. HPLC Analysis of 8-Oxo-dG Adducts. DNA was analyzed as described by Kasai et al. (41) and Sausen et al. (42). Dissolved DNA (0.5 mL) was hydrolyzed with 10 units of nuclease P1 (1 units/µL stock solution prepared in 20 mM sodium acetate buffer) (Sigma, St. Louis, MO) and 2 mM ZnCl2 (final concentration) for 60 min at 37 °C. The pH was alkaline-adjusted with 100 mM Tris and 25 mM MgCl2 (pH 8.0); 2 units of alkaline phosphatase (Type VII-S, 1 unit/5 µL) (Sigma, St. Louis, MO) was added and DNA was incubated for 60 min at 37 °C. Nucleosides were filtered through Ultrafree C3TK (Millipore, Bedford, MA) to remove high molecular weight contaminants. Digested DNA was analyzed by HPLC on a system comprised of an ESA model 580 pump, an SSI pulse dampener, a HewlettPackard 1050 autosampling injector, an ABI 785A UV detector, and an ESA Coulochem II electrochemical detector equipped with a high sensitivity analytical cell (model 5011). 8-Oxo-dG and 2’-deoxyguanosine (dG) (Sigma, St. Louis, MO) were measured from the same injection by electrochemical detection [parameters: guard cell, 500 mV; E1, 150 mV; E2, 350 mV) and UV absorbance (254 nm) detection]. 8-Oxo-dG levels in hepatic genomic DNA were reported as molecules of 8-oxo-dG/106 molecules of dG. Baseline measurements in control animals were routinely in the range of 1-5 adducts/106 dG. Statistics. Data were log transformed since nontransformed data were not normally distributed as tested by Levene’s test. Significant differences were determined by analysis of variance (ANOVA) and pairwise comparisons by Fisher’s least significant difference on log transformed data (P < 0.05). Dose-response relationships were tested by Wald’s test (P < 0.01). Multiple comparisons between the treatment groups and corn oil control group were made in dose-response study by Dunnet’s test (P < 0.01).

TCDD-Induced 8-Oxo-dG Adducts in Sprague-Dawley Rats

Chem. Res. Toxicol., Vol. 14, No. 7, 2001 851 Table 1. 8-Oxo-dG Adduct Levels in DEN-Initiated Intact Rats Following 30 Weeks of Treatment 8-oxo-dG adducts/106 dG

Figure 1. TCDD-induced 8-oxo-dG adduct formation in intact, OVX, and 17 β-estradiol-supplemented OVX rats. DEN-initiated female Sprague-Dawley rats were treated with weekly gavage doses of 700 ng of TCDD/kg (equivalent to an average daily dose of 100 ng of TCDD/kg) and placebo or 0.18 mg/pellet 17 β-estradiol. 8-Oxo-dG adducts were quantitated by HPLC in nuclear DNA from livers of rats exposed to TCDD or corn oil vehicle for (A) 20 weeks of promotion or (B) 30 weeks of promotion. Differences between groups were determined by ANOVA and pairwise comparisons by Fisher’s least significant difference on log transformed data. (*) Significantly higher than 17 β-estradiol-supplemented OVX controls (P < 0.01). (**) Significantly higher than intact controls (P < 0.01).

Results 8-Oxo-dG adduct levels in hepatic DNA were quantitated in DEN-initiated intact and OVX female SpragueDawley rats treated weekly with 700 ng of TCDD/kg (an average daily dose of 100 ng/kg/day) for 20 and 30 weeks of treatment. Following 20 weeks of treatment, mean background levels of 8-oxo-dG adducts in intact and OVX rats were 3.11 ( 1.46 and 2.64 ( 1.43 molecules of 8-oxodG/106 molecules of unadducted 2’-deoxyguanosine (dG), respectively. In intact rats, 8-oxo-dG adduct levels were higher in TCDD-treated rats, but not significantly different, than corn oil controls (Figure 1A). No differences were observed between TCDD-treated and control OVX rats. The contribution of 17 β-estradiol to 8-oxo-dG adduct formation was analyzed in OVX rats in the presence and absence of TCDD. Although estradiol supplementation in OVX rats resulted in supra-physiological serum estradiol levels, terminal serum concentrations and estrogenic responses were equivalent to physiological conditions in intact rats (35). Background adduct formation in 17 β-estradiol-supplemented OVX rats was not significantly different from that in nonsupplemented rats. In TCDD-treated OVX rats, however, 8-oxo-dG formation was significantly higher in the presence of 17 β-estradiol than in the absence of 17 β-estradiol. Following 30 weeks of treatment, background levels of 8-oxo-dG adducts were similar to levels following 20 weeks of treatment. Mean values in intact and OVX rats were 2.56 ( 1.29 and 2.98 ( 1.13 molecules of 8-oxo-dG/

rat

control

TCDD

1 2 3 4 5 6 7 8 9

1.77 1.64 2.37 3.27 4.23 1.95 4.86 1.01 1.98

81.67 11.45 26.56 56.97 72.98 4.92 54.25 81.08 34.09

106 molecules of dG, respectively. In intact rats, 8-oxodG adduct formation was significantly higher in TCDDtreated than in control rats (Figure 1B). Although there was a large range of individual 8-oxo-dG levels, mean adduct formation was 18-fold higher in TCDD-treated rats than in control rats (Table 1). In OVX rats, no differences were observed in 8-oxo-dG adduct formation between TCDD-treated and control rats. However, in 17 β-estradiol-supplemented OVX rats, 8-oxo-dG formation was significantly higher in TCDD-treated rats than in control rats. The number of 8-oxo-dG adducts in TCDDtreated 17 β-estradiol-supplemented OVX rats was not significantly different from TCDD-treated intact rats. No differences were observed in adduct formation between 17 β-estradiol-supplemented OVX rats and nonsupplemented rats in the absence of TCDD. The effects of TCDD exposure on 8-oxo-dG adduct formation in hepatic DNA were quantitated in DENinitiated male Sprague-Dawley rats to assess sex differences in response. Exposure to TCDD for 30 weeks in male rats receiving placebo and 17 β-estradiol pellets resulted in mean hepatic TCDD concentrations of 12 595 and 12 927 ppt wet weight, respectively. These concentrations in males are consistent with hepatic concentrations of TCDD in female rats exposed to similar doses of TCDD for a similar duration of exposure (35). Mean background concentrations of TCDD in corn oil control rats was 34 and 68 ppt wet weight in placebo and 17 β-estradiol-treated rats, respectively. Following 30 weeks of treatment, the formation of 8-oxo-dG in males was lower but not significantly different than in female rats. The mean background values of 8-oxo-dG adducts in untreated male rats were 1.66 ( 1.39 compared to 2.56 ( 1.29 molecules of 8-oxo-dG/106 molecules of dG in untreated female rats. In male rats, no significant differences were observed between TCDDtreated and control rats (Figure 2) (P ) 0.175). Similarly, no significant differences were observed between TCDDtreated and control male rats in the presence of 17 β-estradiol (P ) 0.095). As observed in female rats, no differences were observed in 8-oxo-dG adduct formation between 17 β-estradiol-treated and placebo-control male rats and rats not exposed to TCDD. To examine the dose-response relationship of TCDD exposure, 8-oxo-dG adduct formation in hepatic DNA was quantitated in DEN-initiated female Sprague-Dawley rats exposed to a range of TCDD doses for 30 weeks (Table 2). 8-Oxo-dG adduct formation was significantly higher in rats receiving biweekly gavage doses of 500 ng of TCDD/kg (equivalent to 35.7 ng/kg/day) and 1750 ng of TCDD/kg (equivalent to 125 ng of TCDD/kg/day) than in corn oil control rats (P < 0.01). A nonlinear dose-

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Figure 2. Effects of TCDD exposure on 8-oxo-dG adduct formation in male rats following 30 weeks of treatment. DENinitiated male Sprague-Dawley rats received subcutaneous pellets containing 0.18 mg of 17 β-estradiol/pellet or placebo and were treated with weekly gavage doses of 700 ng of TCDD/kg (equivalent to an average daily dose of 100 ng of TCDD/kg). 8-Oxo-dG adducts were quantitated by HPLC in nuclear DNA from livers of rats. Differences between groups were evaluated by ANOVA, which indicated no significant differences (P ) 0.6). Table 2. Dose-Dependent Induction of 8-Oxo-dG Adducts per 106 dG in DEN-Initiated Female Sprague Dawley Rats Exposed to TCDD for 30 Weeksa dose (ng of TCDD/ kg/day)

group size

mean of log10 transformed 8-oxo-dG levels (5%, 95% confidence intervals)a

0 3.5 10.7 35.7 125.0

10 9 8 10 9

1.25 (0.67, 2.35) 2.40 (1.48, 3.90) 2.06 (1.11, 3.85) 14.60 (1.23, 173.56)b 15.83 (1.89, 132.85)b,c

a Values expressed as adducts per 106 dG, confidence intervals calculated using mean ( 2 standard deviations of the log transformed data. b Significantly different than corn oil control as determined by Dunnet’s test (P < 0.01). c Significant nonlinear dose-response trend determined by Wald’s test (P < 0.01).

response relationship was observed following exposure to TCDD (P < 0.01). The effect of single-dose, acute exposure to TCDD on 8-oxo-dG formation in hepatic DNA was also quantitated in noninitiated female Sprague-Dawley rats. According to the model of Kohn et al. (37), a single-dose exposure to 1000 or 3000 ng of TCDD/kg body weight is predicted to result in a liver burden 4 days after exposure of approximately 7 and 23 ppb, respectively. These predicted values for tissue burden are comparable to previously observed liver burdens of 13-17 ppb wet weight in the livers of female rats chronically exposed to average daily doses of 100 ng/kg (35). No overall significant differences in 8-oxo-dG adducts were observed between control and treated rats (Figure 3) (P ) 0.601 by ANOVA). The mean number of background adducts was 1.64 ( 0.67 in control rats. In TCDD-treated rats, the number of 8-oxo-dG adducts in hepatic DNA was 1.43 ( 0.28 and 2.17 ( 1.64/106 molecules of dG in rats receiving 1000 and 3000 ng/kg TCDD, respectively.

Discussion In this study, we examined the effect of TCDD on induction of 8-oxo-dG levels in male and female SpragueDawley rat liver. These studies clearly demonstrate that the induction of hepatic 8-oxo-dG adducts by TCDD is female-specific, ovarian hormone- and estrogen dependent and observed only following chronic, but not acute, exposure.

Wyde et al.

Figure 3. Effects of single-dose exposure to TCDD on 8-oxodG adduct formation in female rats. Female Sprague-Dawley rats were treated with a single dose of 1000 or 3000 ng of TCDD/ kg or corn oil vehicle. 8-oxo-dG adducts were quantitated by HPLC in hepatic nuclear DNA. Differences between groups were evaluated by ANOVA.

The induction of 8-oxo-dG adducts was observed in female rats exposed to TCDD for 30 weeks, but not following acute exposure or chronic exposure for 20 weeks. The hepatic TCDD concentration in rats exposed to TCDD for 20 weeks was similar to those exposed for 30 weeks (35). Similarly, expected hepatic TCDD concentrations 4 days following single dose exposures of TCDD were expected within equivalent range of the rats treated with TCDD for 20 or 30 weeks. Additionally, the levels of induction of cytochrome P4501A1 mRNA expression are not significantly different in livers of rats following acute and chronic exposure to TCDD (43, 44). These data suggest that the induction of 8-oxo-dG adducts by TCDD is a chronic effect and not solely dependent upon TCDD liver burden or levels of P450 enzyme induction. Previously observed effects in male B6CF1 mice are consistent with the contribution of the duration of exposure to increased oxidative damage. Slezak et al. (45) demonstrated that increases in nonspecific oxidation as measured by cytochrome C reduction and decreased antioxidant capacity required significantly higher hepatic concentrations of TCDD in acutely exposed than subchronically exposed male B6CF1 mice. Exposure to TCDD for 30 weeks induced 8-oxo-dG adduct formation in intact, but not in OVX, rats. These results are consistent with previously results in female Sprague-Dawley rats exposed to TCDD for 30 weeks (10). Supplementation of 17 β-estradiol to OVX rats enhanced the formation of 8-oxo-dG adducts in TCDDtreated rats. The induction of 8-oxo-dG adduct formation by TCDD in female rats was not observed in similarly exposed male rats. These data indicate that TCDDinduction of 8-oxo-dG adduct formation is sex dependent. These data are consistent with the sex-specific hepatocarcinogenic effects of TCDD in chronic two-year bioassays (1, 3). In addition, 8-oxo-dG adduct levels were not significantly higher in TCDD-treated male rats compared to controls even in the presence of 17 β-estradiol exposure. Potentially, gender differences in estrogen metabolism and relative differences in formation and clearance of specific catechol estrogen metabolites may play a role in this TCDD-estrogen interaction. For example, in the Syrian hamster kidney model of estrogen carcinogenesis, single exposures to 4-hydroxyestradiol but not to 2-hydroxyestradiol significantly induced 8-oxo-dG adduct formation (46). In 17 β-estradiol-supplemented OVX rats, TCDD induced levels of 8-oxo-dG formation following 30 weeks of


exposure were equivalent to those in intact rats. These data support the hypothesis that the induction of 8-oxodG adducts in rat liver by TCDD is estrogen-dependent. Following 20 weeks of treatment, however, the levels of 8-oxo-dG adducts were significantly higher in 17 β-estradiol-supplemented TCDD-treated rats than in 17 β-estradiol-supplemented corn oil control rats. No significant effects were observed in intact rats between control and TCDD-treated rats following 20 weeks of exposure. Serum estradiol concentrations in 17 β-estradiol-supplemented rats were continuous at levels at or above peak physiological levels (35) achieved naturally during the estrous cycle. Persistently high, noncycling levels of estrogen may result in an increase in the formation of 8-oxo-dG adducts via multiple potential sources: (i) increased formation of redox cycling catechol estrogens, (ii) futile cycling from cytochrome P450 during the metabolism of excess 17 β-estradiol, or (iii) futile cycling from cytochrome P450 during the attempted metabolism of the poorly metabolized TCDD. Additionally, binding of catechol estrogens to glutathione may result in glutathione depletion and cytotoxicity-induced macrophage activation and DNA oxidation (47). If the hypothesis of estrogen-mediated increases in oxidative damage is correct, increased 8-oxo-dG adduct formation would be expected in the presence of excess 17 β-estradiol. In the Syrian golden hamster liver, metabolism of 17 β-estradiol is concentration dependent, with a higher percentage of catechol estrogens formed at higher concentrations (48). If these effects also occur in the Sprague-Dawley rat, periods of increased catechol estrogen formation there may exist in the estrous cycle. Periods of increased oxidative damage may be a subsequent result of peak estrogen levels during the estrus cycle in female rats or the supra-physiological levels achieved in this model. 8-Oxo-dG adduct formation has commonly been used as a marker for oxidative DNA damage since these adducts result in biologically significant mutations and are detectable in the femtomolar range by HPLC analysis with electrochemical detection (49, 50). However, there is considerable controversy over the significance of 8-oxodG adducts as a marker of overall oxidative DNA damage since it represents approximately only a small fraction of the total potential oxidative DNA damage products (51). In addition, there is concern over its measurement since analytical variation in the measurement of 8-oxodG has been observed between different laboratories using similar methodologies (52). Studies comparing different methods of DNA isolation show that tissue handling is a potential source of artifactual oxidation of unmodified bases to form 8-oxo-dG adducts (53, 54). In an effort to minimize artifactual oxidation, we used the chaotrophic sodium iodide method. This method results in lower and less variable values of 8-oxo-dG adducts compared with other methods typically employed for DNA isolation (54, 55). This suggests that isolation of DNA by this method induces less artifactual oxidation and consequently may be one of the better methods currently available for the isolation of DNA for subsequent 8-oxo dG analysis (54). Additionally, we used HPLC with coulometric detection which is one of the recommended methodologies used for detection and measurement of 8-oxo-dG adducts (52). Despite the caveats over measurement of 8-oxo-dG, the magnitude of the observed effects of TCDD on 8-oxo-dG

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are high, suggesting that they are unlikely to be solely due to analytical variation. The observed effects of TCDD on 8-oxo-dG levels may be a consequence of (1) an effect of TCDD on 8-oxo-dG that is representative of a general effect on oxidative DNA damage as whole in vivo, (2) a specific effect of TCDD on 8-oxo-dG alone that is not representative of an effect on general oxidative DNA damage in vivo, (3) a treatment related effect on the capacity of the samples to undergo auto-oxidation during processing ex vivo. Nonetheless, the observed chronic dose dependence, female specificity, ovarian hormone and estrogen dependence of TCDD effects on 8-oxo-dG adducts clearly suggests a chemical specific effect and are consistent with observed effect of TCDD on tumor promotion and carcinogenicity in rat liver (3, 56, 57). In DNA, oxidative imbalance increases base modifications, strand breaks, abasic sites, and DNA-protein cross-links (20). Although alterations in the oxidative balance also lead to the damage of other macromolecules, damage to DNA potentially gives rise to mutations, which may genetically alter cells and contribute to the carcinogenic process. The measurement of 8-oxo-dG adducts depends on the extent of cellular DNA repair processes and the loss of adducts by incorporation of a G to T transversion at the adduct site during DNA replication. Oxidative imbalance is a two-sided process involving an increase in oxidative damage, a decrease in oxidative repair and defense mechanisms, or both. Cellular antioxidant mechanisms such as superoxide dismutase detoxify reactive oxygen species that cause oxidative damage and prevent 8-oxo-dG adduct formation. Additionally, endogenous and xenobiotic-induced 8-oxo-dG adducts are removed by repair enzymes. Studies in Escherichia coli demonstrate that 8-oxo-dG adducts are removed by base excision repair (58). The mutation rate of 8-oxo-dG adducts is estimated to be 0.5-4.8% in vitro (30-32, 59). Mitogenesis and mutagenesis are associated processes in carcinogenesis. Since DNA lesions that escape repair may give rise to a mutation during DNA synthesis, increased rates of cell replication may lead to increased mutations. If both the rate of DNA adduct formation and cell replication are increased, the effect on mutagenesis is multiplicative (60). Exposure to TCDD for 30 weeks in intact female rats increases the rate of cell proliferation (38, 56). TCDD also induces cell proliferation in 17 β-estradiol-supplemented OVX rats (57). However, no differences were observed between corn oil control and TCDD-treated OVX rats. The effects of TCDD on 8-oxodG adduct formation in intact, OVX, and 17 β-estradiolsupplemented OVX rats were induced in a fashion similar to cell replication. These data suggest that the probability of a mutagenic event may be greater in TCDD-treated intact and 17 β-estradiol-supplemented rats than in OVX rats. The potential increases in mutations in a highly proliferative environment may contribute to increased damage to initiated cells. TCDD-induced oxidative damage may contribute to progression of lesions to tumors. Increased initiation of normal cells by mutations arising from oxidative damage may contribute to increases in the number of a subset of altered hepatocellular foci per cubic centimeter by TCDD in intact and 17 β-estradiolsupplemented rats, but not in OVX rats. These data likely reflect increases in both mutation probability and cell proliferation. This is the first set of experimental in vivo data suggesting that TCDD is indirectly genotoxic via an estrogen-dependent mechanism.

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Acknowledgment. The authors would like to thank Louise Harris, Larry Judd, James Clark, John Seely, Bill Ross, Amy Kim, Scott Masten, Diane Spencer, Page Myers, Jean Grassman, Frank Ye, and Heather Vahdat for technical assistance. Additionally, the authors would like to thank Michael L. Cunningham and Russ Cattley for providing their expertise in the area of oxidative DNA damage. This work supported by a collaborative agreement with Byron Butterworth at CIIT. Thanks to Bennett Van Houten and Retha Newbold at NIEHS and Stephen Ploch, Michael DeLorme, and Barbara Kuyper at CIIT for critical review of this manuscript.

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Induction of Hepatic 8-Oxo-deoxyguanosine Adducts by 2,3,7,8

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