Promotion of Altered Hepatic Foci by 2,3,7,8-Tetrachlorodibenzo-p-dioxin and 17β-estradiol in Male Sprague-Dawley Rats

Abstract

The determination of differences in hormonal regulation of tumor promotion-related response to 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) between males and females may identify factors contributing to the female-specific hepatocarcinogenicity of TCDD in rats. In the current study, diethylnitrosamine-initiated male Sprague-Dawley rats were exposed to TCDD or corn oil vehicle in the presence and absence of 17β-estradiol (E2), and cell proliferation and development of preneoplastic altered hepatic foci (AHF) were determined. After 30 weeks of exposure, γ-glutamyltranspeptidase (GGT)-positive AHF and the number of placental glutathione-s-transferase (PGST)-positive AHF were significantly higher in TCDD-treated rats than in control rats. Both the number and volume fraction of GGT-positive AHF were significantly lower in rats cotreated with E2 regardless of TCDD exposure compared with corresponding non-E2-treated groups and were unaffected by TCDD. In contrast, the number of PGST-positive AHF was significantly higher in E2-treated rats in the absence of TCDD treatment. In addition, whereas E2 had no effect on the volume fraction of PGST-positive foci, the levels in rats cotreated with both E2 and TCDD were significantly higher than in controls. No differences were observed in cell proliferation between TCDD-treated and control rats, although cell proliferation was lower in rats exposed to E2 compared with placebo controls. The weaker potency of tumor promotion and lack of induction of cell replication and DNA damage in male rats likely explain the female-specific hepatocarcinogenicity of TCDD in chronic bioassays.

Article

The ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a multisite rodent carcinogen in both sexes (Kociba et al., 1978; NTP, 1982). TCDD is considered a nongenotoxic carcinogen because it fails to exhibit genotoxicity in in vivo and in vitro assays (Kociba, 1984; Poland and Glover, 1979; Turteltaub et al., 1990; Wassom et al., 1977). In a two-stage initiation-promotion model for hepatocarcinogenesis, TCDD is a potent promoter in rodent skin, liver, and lung (Beebe et al., 1995; Pitot et al., 1980; Poland et al., 1982). In tumor promotion studies, TCDD promotes the formation of liver tumors (Pitot et al., 1980; Walker et al., 2000) and various phenotypes of preneoplastic enzyme-altered hepatocellular foci (AHF) (Flodström and Ahlborg, 1992; Pitot et al., 1980; Waern et al., 1991) in initiated rats at a similar exposure level reported in the chronic 2-year bioassay by Kociba et al.(1978). Hepatic tumor development after chronic exposure to TCDD is specific to female rats (Kociba et al., 1978; NTP, 1982). Although the early Ah receptor-mediated molecular events are well characterized (Schmidt and Bradfield, 1996; Sutter and Greenlee, 1992; Swanson and Bradfield, 1993), it is not currently known how TCDD, the most potent ligand for the Ah-receptor, causes cancer in rats.

The induction of preneoplastic AHF by TCDD in chronic tumor promotion studies is well characterized. Chronic exposure of female Sprague-Dawley rats to more than 100 ng/kg/day of TCDD significantly induces AHF expressing the placental form of glutathione-s-transferase (PGST) (Bager et al., 1997; Flodström and Ahlborg, 1992; Maronpot et al., 1993; Tritscher et al., 1995; van der Plas et al., 1999; Walker et al., 2000) and AHF expressing γ-glutamyltranspeptidase (GGT) (Flodström and Ahlborg, 1989; Flodström et al., 1991; Graham et al., 1988; Hemming et al., 1995; Waern et al., 1991; Wyde et al., 2001a). To elucidate biological and physiological contributing factors to the mechanism of tumor promotion, studies have investigated the hormonal regulation of tumor promotion by TCDD. Ovarian hormones have been shown to contribute to tumor promotion by TCDD in female Sprague-Dawley rats (Lucier et al., 1991; Wyde et al., 2001a). Ovariectomized (OVX) rats had significantly diminished development of GGT-positive AHF induced by TCDD compared with sham-operated rats (Lucier et al., 1991). Additionally, the induction of GGT-positive AHF by TCDD in intact female rats is not observed in OVX female rats (Wyde et al., 2001a).

Oxidative DNA damage and increased cell proliferation may be critical factors that contribute to the process of tumor promotion (Ames and Gold, 1990, 1991; Butterworth et al., 1992). Left unrepaired, lesions that form as a result of oxidative DNA damage may be fixed into the genome during subsequent cell replication. Resulting mutations in genes involved in growth and differentiation may, therefore, result in an increase in the population of initiated hepatocytes and altered growth properties of initiated hepatocytes undergoing clonal expansion.

The induction of cell proliferation and 8-oxo-deoxyguanosine (8-oxo-dG) oxidative DNA adducts depends on ovarian hormones (Lucier et al., 1991; Tritscher et al., 1996; Wyde et al., 2001a,b). TCDD-induced cell proliferation after 30 weeks of exposure is observed in intact, but not OVX, rats (Lucier et al., 1991; Wyde et al., 2001a). Similarly, 8-oxo-dG adducts are significantly induced by TCDD in intact rats but not OVX female rats (Tritscher et al., 1996; Wyde et al., 2001b). Additionally, TCDD-induced adduct formation is observed in female rats but not male rats (Wyde et al., 2001b). These data provide further evidence of ovarian hormone modulation of 8-oxo-dG adduct formation. Through increased cell proliferation in combination with increased DNA damage, TCDD may indirectly induce mutation frequency via an ovarian hormone-dependent mechanism.

The endogenous estrogen 17β-estradiol (E2) may contribute to the ovarian hormone-modulated mechanism of TCDD-induced tumor promotion in female rats. It has been shown that continuous treatment of OVX female rats with estradiol can compensate for the inhibitory effect of ovariectomy on TCDD-induced tumor promotion (Wyde et al., 2001b). Although E2 itself does not induce AHF formation or cell proliferation in OVX Sprague-Dawley rats, it does enhance TCDD-induced increases in GGT-positive AHF and cell proliferation (Wyde et al., 2001a). No significant induction of GGT-positive AHF is observed in OVX rats without E2 exposure. In OVX rats receiving supplemental E2, TCDD significantly induces hepatocyte proliferation (Wyde et al., 2001a). Additionally, 8-oxo-dG adduct formation is induced by TCDD in OVX female rats receiving supplemental E2 (Wyde et al., 2001b). These data support the hypothesis that E2 enhances tumor promotion by TCDD. The hormonal regulation of AHF, cell proliferation, and oxidative DNA damage is consistent with the sex and ovarian hormone influence on induction of hepatic tumor incidence by TCDD.

TCDD induces the expression and activity of cytochrome P-450 isozymes that metabolize E2 to catechol estrogens in Sprague-Dawley rats (Drahushuk et al., 1996; Graham et al., 1988; Hakansson et al., 1994; Vanden Heuvel et al., 1994). Catechol estrogens are rodent carcinogens (Liehr et al., 1986; Newbold and Liehr, 2000) that may contribute to estrogen-induced carcinogenesis through reactive oxygen intermediates (Li et al., 1995; Liehr, 1994; Liehr and Roy, 1990; Yager and Liehr, 1996). TCDD-induced tumor promotion may involve catechol estrogen-derived reactive oxygen formation or leakage of oxygen radicals from the active site of TCDD-inducible cytochrome P-450, including P-4501A1 (Park et al., 1996). Just as E2 or 4-hydroxyestradiol increases oxidative DNA damage in the Syrian hamster model (Han and Liehr, 1994), TCDD induces oxidative DNA damage in female Sprague-Dawley rat liver. Additionally, estrogen-modulated cell proliferation may, in combination with oxidative damage, contribute to the mechanism of tumor promotion by TCDD. In all likelihood, TCDD acts through multiple mechanisms, both estrogen modulated and estrogen independent, to induce hepatocarcinogenesis and liver tumor promotion.

Because TCDD is a potent liver hepatocarcinogen in female but not male rats, most studies in the past 2 decades have further investigated the promotional effects of TCDD on liver tumor promotion in female rats. These studies have mainly characterized temporal and dose-related effects of TCDD on the formation of AHF in female rats (Dragan et al., 1992; Maronpot et al., 1993; Pitot et al., 1980; Teeguarden et al., 1999; Walker et al., 2000). Despite extensive studies, little is known about the mechanism of induction of liver tumor by TCDD in the female rat. Advances have been made in the understanding of the role of ovarian hormones and E2 in TCDD-induced tumor promotion (Wyde et al., 2001b). However, further investigations are required to determine the contribution of these factors and identify key events in the mechanism of tumor promotion and hepatocarcinogenesis by TCDD. Although comparisons between control and TCDD-treated rats have effectively identified TCDD-induced responses, the identification of responses in female rats not occurring in male rats may indicate critical contributing factors or key events in the mechanism of tumor promotion by TCDD in Sprague-Dawley rats.

The aim of the current study was to investigate tumor promotion by TCDD in male rats to elucidate key factors and critical events in the mechanism of TCDD in female rats. To determine differences in tumor promotion between male and female rats, diethylnitrosamine (DEN)-initiated male rats were treated with TCDD for 30 weeks. TCDD-induced alterations in GGT- and PGST-positive AHF formation and development as markers of tumor promotion were analyzed and compared with results from similarly treated female rats (Wyde et al., 2001a). Additionally, the effects of TCDD on the incorporation of 5`-bromodeoxyuridine (BrdU) into the DNA of replicating hepatocytes were determined in male rats and compared with results from similarly treated female rats (Wyde et al., 2001a). Because tumor development is specific to female Sprague-Dawley rats, it was expected that exposure to TCDD in male rats would not result in a significant increase in preneoplastic AHF or an induction of hepatocyte proliferation. However, because E2 potentiates TCDD-induced increases in GGT-positive AHF and cell proliferation in female rats, it may be expected to act similarly in male rats modulating TCDD-induced development of AHF lesions and cell proliferation. To test this hypothesis, male rats were treated with TCDD in the presence and absence of E2 administered by subcutaneously implanted pellets.

MATERIALS AND METHODS

Animals.

Male Sprague-Dawley rats (Charles River, Raleigh, NC) were treated as previously described (Wyde et al., 2000). Rats were housed 2 per 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. Rats were divided into 4 groups of 8 rats each. All rats were initiated with 175 mg DEN/kg (administered intraperitoneally) at 10 weeks of age. One week after initiation, 2 groups were implanted with 90-day release pellets containing mg (placebo) and 2 groups were implanted with pellets containing 0.18 mg of E2/pellet (Innovative Research). New pellets were implanted after 90 days. Starting 1 week later, 1 group of placebo-treated rats and 1 group of E2-treated rats were treated once per week with an oral gavage dose of 700 ng TCDD/kg for 30 weeks. This dose of TCDD is equivalent to a daily average dose of 100 ng TCDD/kg/day, comparable to exposure levels that induced liver tumors in female rats in the chronic bioassay described by Kociba et al.(1978). The remaining placebo and E2 groups were treated weekly by oral gavage with corn oil vehicle for 30 weeks. Osmotic pumps (Alzet model 2ML1; 10 μl/h delivery rate; Alzet Corp., Palo Alto, CA) containing 30 mg/ml 5-bromo-2`-deoxyuridine in saline were implanted subcutaneously 7 days before necropsy to allow for evaluation of hepatocyte proliferation. Rats were killed by asphyxiation with carbon dioxide, and liver tissues were removed, weighed, sectioned, and frozen in liquid nitrogen. Liver slices were also fixed in 4% paraformaldehyde or ethanol and embedded in paraffin. Serial liver sections (5 μm thick) were cut and placed on microscope slides.

AHF formation.

PGST immunohistochemistry, GGT enzyme histochemistry, and stereological analysis were performed on paraformaldehyde- and ethanol-fixed liver sections, respectively, as previously described (Wyde et al., 2001a). The promotion index was calculated using the following formula:Formulawhere Vf is the volume fraction of treated rats, Vc is the volume fraction in control rats, and mmol per week is the mean amount of promoter administered to each rat during the period of promotion. The calculation for the promotion index allows for comparisons of potency of tumor promotion activity between sexes, species, and strains from different studies.

Cell replication.

The incorporation of BrdU into DNA of replicating hepatocytes was determined by immunohistochemistry according to the methods of Goldsworthy et al.(1991). Positively stained nuclei were scored in at least 1000 hepatocytes determined to be histopathologically nonfocal as assessed by a trained pathologist in hematoxylin and eosin counterstained tissue. The BrdU labeling index is expressed as the percentage of all nuclei counted that were positively labeled with BrdU.

Statistics.

Because of the presence of a large number of zero values in the GGT AHF data, these data sets were analyzed by nonparametric statistical methods. Significant between-group differences were first determined by one-way Kruskal-Wallis tests (p < 0.05). Because these were significant, pairwise group comparisons were subsequently made by pairwise rank-order-based Mann-Whitney U-tests (p < 0.05). Untransformed PGST-positive AHF and BrdU LI data exhibited homogeneity of variance when tested by Bartlett’s tests. Log10 transformed CYP data exhibited homogeneity of variance when tested by Bartlett’s tests. Significant differences between all groups were determined by one-way ANOVA. Because the ANOVA tests indicated significant between-group effects, pairwise group comparisons were then tested using Fisher’s least significant difference test (p < 0.05).

Real-time and quantitative RT-PCR.

Quantitation of CYP1A1 messenger RNA levels was determined using real-time fluorescence detection reverse-transcriptase polymerase chain reaction (RT-PCR) (Walker, 2001). Total RNA was isolated from frozen liver samples using an acid-guanidinium-phenol-chloroform technique (Tri-reagent) (Sigma, St. Louis, MO). Real-time RT-PCR reactions were prepared using a Perkin-Elmer Syber Green RT-PCR kit according the manufacturer’s recommendations with the modification of the use of a CYP1A1-specific reverse-transcription primer (5`cca atc act gtg). Thermal cycling and real-time detection of the Syber Green-associated fluorescence was performed using an Applied Biosystems 7700 Sequence Detection system with default temperature parameters. PCR primers were designed using Primer Express; forward, 5`-tcaaagagcactacaggacatttg; reverse, 5`gggttggttaccaggtacatgag. Quantitation of CYP1A1 was determined by interpolation of the sample-specific cycle-threshold value against a standard curve prepared on the same plate using a 10-fold serial dilution of a quantitated liver total RNA sample. The expression level of CYP1A1 in the quantitated liver total RNA sample was determined using a competitive RT-PCR titration assay as previously described (Walker et al., 1999).

RESULTS

Formation and Development of GGT-Positive AHF

In male rats treated weekly with 700 ng TCDD/kg (a daily average dose of 100 ng/kg/day) for 30 weeks, there were no significant differences in the number of GGT-positive AHF/cm3 compared with control male rats (Fig. 1). Additionally, there were no significant differences in the number of GGT-positive AHF/cm3 between TCDD-treated and control rats in the presence of E2. However, in rats exposed to E2, the number of GGT-positive AHF/cm3 was significantly lower than in male rats not receiving E2. The median number of GGT-positive AHF/cm3 was 59.7 and in placebo-control and E2-treated male rats, respectively. The suppression of GGT-positive AHF/cm3 by E2 was also observed in rats exposed to TCDD. In TCDD-treated rats, exposure to E2 resulted in 3.5-fold lower median numbers of GGT-positive AHF/cm3 than in TCDD-treated rats not exposed to E2.

The percentage of the liver occupied by GGT-positive AHF (volume fraction) in TCDD-treated rats was significantly higher than in control rats (Fig. 2). A 2-fold induction in GGT-positive volume fraction by TCDD was observed with median values of 0.012% and 0.024% in control and TCDD-treated rats, respectively. The GGT-positive volume fraction was significantly lower in male rats receiving E2 than in placebo-control rats. In rats exposed to TCDD, the volume fraction of GGT-positive AHF was significantly lower in E2-treated rats than in control rats. These results are similar to the effects of E2 on the number of GGT-positive AHF/cm3 (see Fig. 1) in both control and TCDD-treated rats.

Formation and Development of PGST-Positive AHF

PGST-positive AHF have been demonstrated to represent approximately 97% of all AHF in Sprague-Dawley rats (Teeguarden et al., 1999) and greater than 65% of all AHF in Fischer F344 rats (Hendrich et al., 1987). Therefore, alterations in the development of PGST-positive AHF by TCDD and E2 were analyzed in addition to GGT-positive AHF in male Sprague-Dawley rats.

In contrast to the observations of the suppressive effects of E2 on GGT positive AHF, the number of PGST-positive AHF/cm3 in TCDD-treated male rats was significantly higher than in control male rats (Fig. 3). The mean number of PGST-positive AHF was more than 2-fold higher in TCDD-treated rats than in control rats. In rats treated with E2, no significant difference was observed between TCDD-treated and control rats. However, the number of PGST-positive AHF/cm3 was significantly higher in rats treated with E2 only compared with placebo-controls.

No significant differences were observed in the volume fraction of PGST-positive AHF between TCDD-treated or E2-treated rats compared with placebo-control rats (Fig. 4). However, in rats exposed to both E2 and TCDD, the volume fraction of PGST-positive AHF was significantly higher than in rats receiving E2 alone. The mean volume fraction was 0.8% in E2-treated rats and 2.2% in male rats cotreated with E2 and TCDD.

Alterations in Cell Replication

The effect of TCDD and coexposure to TCDD and E2 on cell proliferation, as determined by the uptake of BrdU into the DNA of replicating hepatocytes, was determined. No significant differences were observed in BrdU labeling index between TCDD-treated and control rats; however, exposure to E2 suppressed the BrdU labeling index more than 5-fold compared with rats receiving placebo pellets (Fig. 5). Although the BrdU labeling index in TCDD-treated rats was equivalent between groups receiving E2 or placebo pellets, the BrdU labeling index was significantly higher in rats cotreated with both TCDD and E2 compared with those receiving only E2. These data may reflect a suppression of cell proliferation by E2 treatment that is inhibited by TCDD in coexposed rats.

Relative Potency of Promotion by TCDD

Relative potency of promoters based on differences in induction of the development of AHF has previously been reported (Dragan and Pitot, 1992; Pitot et al., 1987). These calculations for the promotion index allow for comparisons of potency of tumor promotion activity among sexes, species, and strains. Promotion index is described by the following formula in which Vf is the volume fraction of treated rats, Vc is the volume fraction in control rats, and millimoles per week is the mean amount of promoter administered to each rat during the period of promotion:FormulaTable 1 shows promotion indices for GGT-positive AHF from the current study in male rats and a prior study in female rats and values previously reported in the literature. The promotion index calculated from the prior study in similarly treated female rats (Wyde et al., 2001a) for TCDD was 2.6 × 107 in intact female rats after 30 weeks of exposure (see Table 1). With the exception of 1 study (Hemming et al., 1995), these results are consistent with previously calculated potency of TCDD in female Sprague-Dawley rats ranging from 1.5 × 107 to 2.8 × 107 (Hemming et al., 1993; Pitot et al., 1980; Waern et al., 1991). In male rats, the promotion index was 2.76 × 106, an order of magnitude lower than in female rats. In E2-treated male rats, the promotion index of TCDD was 7.7 × 104. In female rats, it has been demonstrated that ovarian hormones contribute to TCDD-induced tumor promotion (Lucier et al., 1991; Wyde et al., 2001a) and E2 may supplement for the inhibitory effect of ovariectomy (Wyde et al., 2001a). The promotional index in OVX rats in the prior study (Wyde et al., 2001a) was 7.5 × 105. The calculated promotion index for TCDD in E2-supplemented OVX rats (Wyde et al., 2001a) was 8.42 × 106, which is 10-fold greater than in OVX rats. The value of the promotion index, based on GGT-positive AHF, is consistent with the inhibitory effect of ovariectomy and enhancing effect of E2 on the potency of tumor promotion by TCDD.

Expression of CYP1A1

To determine whether the differences in the AHF promotion were due to differences in the level of responsiveness of male and female rats to TCDD, the expression of CYP1A1 was measured by quantitative RT-PCR (Table 2). In control rats, the expression of CYP1A1 was significantly higher in males compared with females. In male and female TCDD-treated rats, CYP1A1 was significantly higher compared with their respective controls regardless of gender or hormonal status. In addition, there was no significant difference between male and females in the level of CYP1A1 expression in similarly treated TCDD-treated groups. Of note, the TCDD-induced expression of CYP1A1 was significantly higher in OVX versus intact female rats. These data indicate that although the fold induction of CYP1A1 was higher in females, the level to which the male and female rats were induced by TCDD was equivalent. These data are consistent with prior observations that tissue levels of TCDD in male and female rats after similar chronic exposures are equivalent (Wyde et al., 2001b). Given the similarity in the levels of TCDD-induced expression of CYP1A1, it is unlikely that the observed differences in AHF between TCDD-treated groups are due to differences in the level of activation of the Ah receptor.

DISCUSSION

Because TCDD is not a hepatocarcinogen in male rats, hepatic tumor promotion studies of TCDD have focused on female rats. These studies demonstrate dose- and duration-dependent induction of multiple phenotypes of preneoplastic AHF by TCDD in female Sprague-Dawley rats. However, little has been determined regarding the mechanism by which TCDD induces these lesions and, subsequently, liver tumors. In the current study, the development of preneoplastic AHF lesions and alterations in cell proliferation were determined as markers and contributors to tumor promotion by TCDD in male Sprague-Dawley rats. TCDD significantly induced the volume fraction of GGT-positive AHF. The volume fraction of AHF is the most applicable in determining the extent and efficacy of tumor promotion (Pitot et al., 1989). However, although the median volume fraction of GGT-positive AHF was induced 2-fold over controls in male rats, in a prior study in female Sprague-Dawley rats (Wyde et al., 2001a), TCDD induced a 14-fold increase in GGT-positive volume fraction. Additional studies in females have consistently demonstrated a 6- to 12-fold induction of GGT-positive AHF volume fraction after 27 to 30 weeks of exposure to TCDD depending on the initiation-promotion protocol (Flodström and Ahlborg, 1989; Graham et al., 1988; Lucier et al., 1991). Using the GGT-positive AHF data, the promotion index, a measure of potency of tumor promotion, was an order of magnitude higher in female rats than male rats. Taken together, these data indicate that, based on induction of GGT-positive AHF, TCDD is a more potent promoter in female than in male Sprague-Dawley rats.

TCDD exposure did not significantly induce the number of GGT-positive AHF/cm3 in male Sprague-Dawley rats. This quantitative measure of AHF tends to reflect the potency and efficacy of initiation (Pitot et al., 1989). In similarly treated female rats, TCDD resulted in an elevated number of GGT-positive AHF/cm3 (p = 0.085) (Wyde et al., 2001a). Although not statistically significant, an elevated number of GGT-positive AHF/cm3 is consistent with previous observations of significant increases in GGT-positive AHF/cm3 by TCDD (Flodström and Ahlborg, 1989, 1992; Waern et al., 1991). Because comparable doses of DEN were used as the initiating agent in both studies, differences in the number of AHF/cm3 may reflect differences in contribution of TCDD to continued initiation of hepatocytes throughout the duration of exposure. These data are consistent with TCDD-induced oxidative DNA damage in female, but not male, Sprague Dawley rats (Wyde et al., 2000). Additionally, in the current study, TCDD did not alter hepatocyte replication in male rats. In female Sprague-Dawley rats, TCDD-induced cell proliferation (Wyde et al., 2001a), combined with oxidative DNA damage, may contribute to increased mutation frequency. Subsequent mutations in altered or initiated hepatocytes may contribute to the progression of preneoplastic lesions to hepatocellular carcinomas in female rats. This hypothesis is consistent with the sex-specific induction of liver tumors. These data suggest that the induction of cell proliferation and oxidative DNA damage may be critical events in the mechanism of hepatocarcinogenesis by TCDD.

The development of AHF has been demonstrated to reflect clonal expansion of a single initiated hepatocyte (Rabes et al., 1982; Scherer and Hoffmann, 1971; Tsuji et al., 1988; Weinberg et al., 1987). Therefore, the induction of the number of PGST-positive AHF/cm3 by TCDD suggests additional conversion of normal hepatocytes to PGST-positive hepatocytes. These results are consistent with the induction of the number of PGST-positive AHF/cm3 by TCDD in female rats (Wyde et al., 2001a). Additionally, E2 exposure alone induced the number of PGST-positive AHF/cm3 but not the volume fraction in male rats. These data suggest that more, but smaller, individual AHF may contribute to the same percentage of PGST-positive liver tissue. However, because neither TCDD nor E2 induce oxidative 8-oxo-dG DNA adducts in male Sprague-Dawley rats (Wyde et al., 2001a), subsequent mutations to normal hepatocytes must be derived via an alternative mechanism. No effect on PGST-positive volume fraction was observed after exposure to TCDD. These data indicate that the growth of PGST-positive AHF was not induced by TCDD. In contrast, TCDD induced the volume fraction of PGST-positive AHF in rats coexposed to TCDD and E2. Because the numbers of PGST-positive AHF/cm3 were similar between E2-treated and cotreated rats, these data suggest that E2 potentiates TCDD-induced growth of PGST-positive AHF. Although these data are consistent with TCDD induction of PGST-positive AHF in female rats regardless of hormonal status, they suggest a contribution of E2 to TCDD-induced promotion of the PGST-positive phenotype of AHF in male rats.

Although the effects of TCDD and E2 on the development of PGST-positive AHF are not entirely consistent between male and female rats, previous studies suggest that PGST-positive AHF may not the most appropriate marker for assessing the effects of TCDD exposure on promotion. The GGT-positive AHF are more responsive to hormonal regulation of tumor promotion by TCDD and more appropriately reflect TCDD-induced molecular and pathological alterations in female rats (Wyde et al., 2001a). The influence of ovarian hormones and E2 on TCDD-induced GGT-positive AHF is more consistent with TCDD-induced effects on cell proliferation (Lucier et al., 1991; Wyde et al., 2001a), oxidative DNA damage (Tritscher et al., 1996; Wyde et al., 2001b), and sex-specific tumor induction (Kociba et al., 1978; NTP, 1982) than alterations in PGST-positive AHF. Additionally, the relative potency of tumor promotion by TCDD in intact and OVX rats as determined by PGST-positive AHF are equivalent. The promotional index for TCDD based on PGST-positive AHF is 4.56 × 106 in intact rats and 3.25 × 106 in OVX rats. These data suggest that alterations of GGT-positive AHF may be more biologically relevant in assessing tumor promotional effects of TCDD (Wyde et al., 2001a). Therefore, inhibition of AHF development observed in the GGT-positive phenotype may be more accurate in evaluating the effect of E2 on tumor promotion by TCDD in male rats than the enhancement observed in the PGST-positive phenotype.

Exposure to E2 significantly reduced hepatocyte replication and the development of GGT-positive AHF in male rats regardless of exposure to TCDD. These results are consistent with the hormonal responsiveness of GGT-positive AHF in female Sprague-Dawley rats (Wyde et al., 2001a). An increase in the development of GGT-positive AHF is reflected in the removal of ovarian hormones by ovariectomy. Supplementation with E2 in OVX rats reduces GGT-positive AHF development regardless of TCDD exposure. Although the absolute values for GGT-positive development are lower in E2-supplemented OVX rats than in intact or nonsupplemented rats, exposure to E2 in OVX female rats may contribute to the effect of TCDD on hepatic tumor promotion. Lower absolute values for GGT-positive AHF parameters may reflect an inhibitory effect of supraphysiological levels of E2 (Wyde et al., 2000) on GGT-positive AHF. Lower values were also observed in the current study in E2-treated male rats. However, in male rats, GGT-positive AHF were not induced by TCDD in the presence of E2 exposure. This is reflected in differences in the promotion index of 2.7 × 106 in rats receiving placebo pellets and 7.7 × 104 in rats treated with E2 pellets. These data suggest that potentiation of TCDD-induced tumor promotion by E2 is specific to female rats.

This study indicates that TCDD is capable of promoting the development of preneoplastic foci in male Sprague-Dawley rats. However, continuous exposure to TCDD in male rats does not induce liver tumor incidence. Differences in response to TCDD in hepatocyte proliferation, oxidative DNA damage, and preneoplastic AHF development may account for sex differences in tumor response between male and female rats. TCDD-induced cell proliferation and oxidative DNA damage in female Sprague-Dawley rats (Wyde et al., 2001a,b) were not observed in male Sprague-Dawley rats after 30 weeks of exposure. Increased rates of mutation may result in highly proliferative hepatocytes that exhibit higher levels of DNA damage (Ames et al., 1993). However, increased oxidative DNA damage and increased rates of cell proliferation are not observed in female Sprague-Dawley rats after 20 weeks of exposure (Wyde et al., 2001a,b). These data suggest that the induction of cell proliferation and oxidative DNA damage may contribute to the progression of TCDD-induced preneoplastic lesions to tumors in female rats but not male rats. Additionally, differences in the potency of promotion by TCDD in males and females may contribute to the sex specificity of tumor development. Although the number of preneoplastic AHF is suggested to plateau after extended periods of promotion, the growth of preneoplastic lesions is believed to continue until the development of hepatocellular carcinomas (Hendrich et al., 1986; Pitot et al., 1989). Induction of AHF volume fraction in female rats is substantially greater than in male rats. These data provide a mechanistic foundation for differences in hepatocarcinogenesis of TCDD between male and female Sprague-Dawley rats.

Acknowledgments

The authors gratefully thank Louise Harris, Larry Judd, James Clark, John Seely, Bill Ross, Amy Kim, Diane Spencer, Page Myers, Jean Grassman, and Heather Vahdat for technical assistance and Nancy Ress and Dori Germolec for critical review of this work.

Footnotes

REFERENCES