Hepatocarcinogenesis in Female Sprague-Dawley Rats following Discontinuous Treatment with 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Nigel J. Walker1, Angelika M. Tritscher2, Robert C. Sills, George W. Lucier and Christopher J. Portier

Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Received August 5, 1999; accepted December 1, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we investigated the time course of promotion of tumors and putatively preneoplastic altered hepatic foci in the livers of diethylnitrosamine (DEN)-initiated female Sprague-Dawley rats. These rats had been treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) under different dosing regimens, but we used the same administered biweekly dose of 1.75 µg/kg of body weight. Animals were treated continuously for up to 60 weeks, or continuously for 30 weeks, followed by cessation of treatment for up to 30 weeks. In addition, TCDD treatment in these groups was begun either 2 or 18 weeks after initiation with DEN. Liver tumors were only observed in animals after 60 weeks on the study and were increased by continuous TCDD treatment, relative to controls. The incidence of hepatocellular adenoma and carcinoma combined, in animals treated with TCDD for 30 weeks followed by no TCDD treatment for 30 weeks (17%), was lower than in animals receiving either TCDD (79%) or vehicle control (corn oil) alone (55%) for 60 weeks. The lower liver-tumor incidence after cessation of TCDD treatment paralleled time-dependent decreases in the volume fraction occupied by placental glutathione S-transferase-positive altered hepatic foci and the number of foci per unit volume, but not the mean focus volume that exhibited a time-dependent increase after cessation of TCDD treatment. Cessation of TCDD treatment led to reductions in liver TCDD levels, and these changes were reflected in a cessation of reduced body weight because of TCDD treatment. These data indicate that liver-tumor promotion by TCDD in female rats is dependent upon continuous exposure to TCDD, and that alterations in patterns of TCDD exposure can have significant effects on tumor incidence not reflected by standard measures of dioxin exposure.

Key Words: TCDD; dioxin; carcinogenesis; altered hepatocellular foci; liver tumors; placental glutathione S-transferase; half-life.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a multi-site, multi-species carcinogen in multiple rodent models (Huff et al., 1994Go). The incidence of liver tumors in TCDD-treated female rats has often been used by regulatory agencies to help in establishing appropriate guidelines for human exposure to dioxin-like compounds that have a common mechanism of action through the aryl hydrocarbon receptor (AHR). Humans are exposed to lower levels of dioxins than those that induce liver tumors in rodent studies of carcinogenesis (DeVito et al., 1995Go). Consequently, studies aimed at understanding the dose response, time course, and mechanism of hepatocarcinogenesis induced by TCDD in rats are necessary to provide a scientific basis for the extrapolation of rat tumor data to humans.

While TCDD is not a direct-acting genotoxic agent, several studies have shown that it acts as a potent liver tumor promoter (Graham et al., 1988Go; Maronpot et al., 1993Go; Pitot et al., 1980Go). In a chronic 2-stage initiation/promotion study, significant increases in cell proliferation and enzyme-altered hepatic foci are observed in the livers of female Sprague-Dawley rats treated biweekly with 1.75 µg TCDD/kg, to approximate a daily dose of 125 ng TCDD/kg/day for 30 weeks (Maronpot et al., 1993Go). Despite the fact that activation of gene transcription via the AHR is well-understood (Schmidt and Bradfield, 1996Go), the development of liver tumors and preneoplastic altered hepatic foci (AHF) as a result of TCDD exposure is not. Effects of TCDD on tumor promotion may be through multiple mechanisms, including activation of endogenous estrogens (Graham et al., 1988Go), oxidative stress (Tritscher et al., 1996Go), increased cell proliferation (Walker et al., 1998Go) and reductions in inducible apoptosis (Stinchcombe et al., 1995Go).

Mathematical 2-stage models of carcinogenesis have been used to describe the effects of tumor promoters on the development of AHF and therefore can be used to test hypotheses regarding the specific action of tumor promoters. Mathematical models describing the effect of TCDD on development of AHF in the rat have been described, although current data sets are inadequate to effectively determine which model is most appropriate (Conolly and Andersen, 1997Go; Moolgavkar et al., 1996Go; Portier et al., 1996Go). Mathematical modeling requires adequate data sets, especially regarding the dose response and time course of tumor promotion. The study by Maronpot and co-workers is the most well-characterized dose-response study on the effect of TCDD on liver tumor promotion (Maronpot et al., 1993Go). While the reversibility of tumor promotion by TCDD has been previously described (Dragan et al., 1992Go; Tritscher et al., 1995Go) limitations in the respective designs for these studies have hindered their use in carcinogenesis modeling of TCDD.

The ability to distinguish between different mathematical models relies on the availability of tumor-promotion data that adequately describe both the dose response and the time course of development foci. One key issue in carcinogenesis modeling is that of distinguishing the effect of promoters on the initiated "stem cell" versus effects of preneoplastic foci later in development. It has been suggested that studies that separate the initiation phase from the promotion phase by a period of no-treatment may be suitable for such discriminatory analyses (Portier et al., 1993Go). The study presented in this manuscript was designed for direct application to the continued development of mathematical models describing tumor promotion by TCDD. It describes the time course and reversibility of AHF development and liver tumor promotion by TCDD within the framework of a 60-week initiation/promotion model. In addition, the effect of a recovery period following initiation on promotion of hepatocarcinogenesis in the female Sprague-Dawley rat was also tested. Data from this present study describing the effects of TCDD on hepatocyte-cell proliferation, liver weights, TCDD tissue dosimetry and CYP1A1 enzyme induction have been published previously (Walker et al., 1998Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal handling and treatment.
All procedures and experiments with animals in this study were approved by the Animal Care and Use Committee at the NIEHS. Female Sprague-Dawley rats were randomly divided into 21 groups containing 5–15 animals per group. Animals were housed 3/cage under conditions of controlled temperature (70 ± 0.5°F), humidity (50 ± 5%), and lighting (12-h light/dark cycle), and received food and water ad libitum. Animals were initiated with 175 mg diethylnitrosamine/kg body weight at 10 weeks of age (D), or received saline alone (S). Stock TCDD solutions were prepared by Radian Corporation (Morrisville, NC) in corn oil and concentrations measured by GC/MS analysis. TCDD treatment was by oral gavage, biweekly with 1.75 µg/kg, to give a daily averaged dose of 125 ng TCDD/kg/day. Control animals received corn oil alone. One week prior to necropsy, osmotic pumps (Alzet model 2ML1; 10 µl/h delivery rate; Alzet Corp., Palo Alto, CA) containing 30 mg/ml 5-bromo-2'-deoxyuridine (BrdU) in distilled water, were implanted subcutaneously. Animals were killed by asphyxiation with CO2, one week after the last treatment, with either corn oil or TCDD.

The treatment groups used in this study are shown in Figure 1Go and as previously described (Walker et al., 1998Go). The study consisted of 4 basic exposure groups:

(I) Continuous group. Beginning 2 weeks after initiation, animals were dosed biweekly with TCDD for up to 60 weeks. Necropsies were performed 15, 31, and 61 weeks after the start of treatment. Control animals received corn oil alone (C) and necropsies were done at 15, 31, 47 (Group C30–16), and 61 weeks.
(II) Withdrawal group. After 30 weeks of TCDD treatment, it was stopped, and the animals subsequently received corn oil alone. Necropsies were done at 17 weeks (Group 30–16) and 31 weeks (Group 30–30) after the last TCDD treatment.
(III) Waiting group. First TCDD treatment was 18 weeks after initiation. Necropsies were done one week after the last dosing, at 31 weeks (Group 0–16–14) and 47 weeks (0–16–30) of TCDD treatment.
(IV) Waiting + withdrawal. For some animals in the waiting group, TCDD treatment was stopped after 30 weeks, and the animals were subsequently dosed with corn oil (Group 0–16–30–16). Necropsies were done 17 weeks after the last treatment with TCDD.



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FIG. 1. Experimental design used in this study (Walker et al., 1998Go). Female Sprague-Dawley rats were initiated with either 175-mg diethylnitrosamine/kg body weight (D) or received saline (S) at 10 weeks-of-age. Starting 2 or 18 weeks after initiation, animals were treated biweekly with TCDD (T) at a daily average dose of 125 ng TCDD/kg/day for up to 61 weeks (solid bars) or received corn oil (C) alone (solid lines).

 
Histologic and biochemical procedures.
Representative sections of liver were fixed overnight in freshly prepared 4% paraformaldehyde and embedded in paraffin. Other sections of the liver were removed, minced, and aliquots frozen immediately in liquid nitrogen. Serum was obtained from blood withdrawn by cardiac puncture and used for analysis of serum markers of liver toxicity and hormone analyses. Altered hepatic foci (AHF) expressing the placental form of glutathione S-transferase (PGST) were detected immunohistochemically. The number of AHF and area of each AHF were measured by computer-assisted analysis of a digitized image of stained liver sections. Mathematical 3-D stereological transformation of the 2-dimensional, trans-sectional PGST data was carried out according to the calculations as outlined by the works of Campbell and Pugh (Campbell et al., 1982Go; Pugh et al., 1983Go). AHF measurements were calculated as percentage of liver occupied by AHF (volume fraction), and AHF/cm3. The ratio of these 2 measures was calculated to give an estimation of the "mean focus volume" (Campbell et al., 1982Go). Pathological evaluations were carried out by Experimental Pathology Laboratories Inc., Research Triangle Park, NC. Measurement of levels of TCDD in the frozen liver tissue taken at necropsy was performed by Triangle Laboratories Inc. (Durham, NC) by GC/MS as described. TCDD levels are shown in the text as ppt relative to tissue wet weight and represent the actual level of TCDD in the tissue taken at necropsy, which for all groups was one week after the last dosing with either TCDD or corn oil.

TCDD half-life determinations.
Due to the complexity of the design, half-lives were determined using a modified bolus dosing model with first-order kinetics. The basic model is of the form C(t) = C0e–at + B, where C(t) is the concentration in the tissue under study at time t, C0 is the concentration at time 0, B is the steady-state background concentration in the tissue (determined from the untreated animals), e is Euler's constant (2.718 ...) and a is a parameter or group of parameters to be estimated from the data. In all analyses, bolus doses every 2 weeks were modeled by the addition of C0 units of concentration at the time of dosing. To test for statistical significance of several design factors (e.g., age, delay of exposure) and other factors (e.g., body and liver weights) on the value of the half-lives, a was treated as a function of the independent factors. For the largest model considered, a was of the form

(1)
where a0, a1, a2, ...are parameters estimated from the data, Iage is an indicator for young versus older animals (Iage = 0 if age < 35 weeks, Iage = 1 if age > 35 weeks), Idelay is an indicator variable for whether the start of TCDD exposure was delayed (Idelay = 0 if the start of TCDD exposure was 2 weeks post-initiation, Idelay = 1 if start of TCDD exposure was 18 weeks post initiation), Iconc is an indicator variable for high versus low tissue concentrations (Iconc = 0 if tissue concentration was below some critical concentration, (t), and 1 if above this value; different values of t were tried over a broad range (this is discussed in the findings). Iinitiation was an indicator of whether the animals were initiated (Iinitiation = 1) or not initiated (Iinitiation = 0), bw is body weight evaluated every 2 weeks, and lw is liver weight evaluated at the time of animal sacrifice.

Non-linear, least-squares methods were used to estimate the model parameters and statistical tests (based upon Wald-type statistics) were used to determine if the estimated parameters (a1, a2 ...a6) were significantly different from zero. When a parameter could include 0, this indicated that the associated factor did not significantly alter the half-life. These parameters were sequentially removed from Equation 1, leaving only those factors that significantly affected half-life in the rodents. Once a formula for a (Equation 1) was finalized, half-life was calculated using the routine formula T1/2 = ln(2)/a, where ln represents the natural logarithm.

Statistical analyses.
Homogeneity of variance was initially tested by Bartlett's test. Because data was not normally distributed, non-parametric methods were used to assess overall group differences (Kruskal-Wallis tests) and pairwise comparisons (Mann-Whitney U tests). Due to the non-normal distribution of the altered hepatic foci data, data is represented as the median and inter-quartile range from the 25th to the 75th percentiles. For comparisons between TCDD-treated and control groups, C30 served as the control group for T0–16–14; C30–16 for Group T0–16–30; and C60 for Group T30–30 and T0–16–30–16 (Fig. 1Go). While statistical analyses of all pairwise comparisons are not shown, in most cases 2 groups were significantly different at the p < 0.05 level if there was no overlap in the inter-quartile ranges. For the tumor data, Chi-square tests were carried out to see if there were any overall significant differences among groups. If differences were detected, then pairwise comparisons were made by 2-sided Fisher's exact tests. Overall differences in tumors per animal were assessed by Kruskal-Wallis tests, with pairwise comparisons carried out by Mann-Whitney U tests.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Liver Tumor Incidence
There were no observable tumors in non-initiated animals receiving either 125 ng TCDD/kg/day or corn oil vehicle, for 60 weeks (data not shown). By comparison, there was a high incidence of liver tumors (adenomas and carcinomas combined) in animals that were initiated with a single dose of 175 mg DEN/kg and treated with either 125 ng TCDD/kg/day (T60) or corn oil (C60) for 60 weeks (79 and 55% respectively) (Table 1Go). The incidence of tumors, the numbers of animals with multiple tumors, and mean tumors per animal were all higher in the animals treated with TCDD for 60 weeks (T60) compared to corn oil controls (C60). While these data are consistent with induction of hepatic tumors by TCDD (Kociba et al., 1978Go; NTP, 1982Go), in this experimental initiation/promotion model, these differences were not statistically significant.


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TABLE 1 Liver Tumor Formation in DEN-Initiated Animals after Discontinuous Treatment with TCDD
 
We examined the effect of discontinuous exposure on hepatocarcinogenesis induced by TCDD, in animals exposed to TCDD for only 30 weeks, starting either 2 weeks (T30) or 18 weeks (T0–16–30) following initiation with DEN (Table 1Go). In the DEN-initiated group that received TCDD for 30 weeks, followed by corn oil for 30 weeks (T30–30), the incidence of tumors (either adenoma alone or adenoma/carcinoma combined) and the number of animals with multiple tumors was significantly lower than in the group that received TCDD continuously for 60 weeks (T60) (Table 1Go). Similarly, in the DEN-initiated group that received TCDD for 30 weeks, starting 18 weeks after initiation, followed by corn oil for 16 weeks (T0–16–30–16), the number of animals with multiple liver tumors was significantly lower than in the group that received TCDD continuously for 60 weeks (T60) (Table 1Go). There were no significant differences between this group (T0–16–30–16) and DEN-initiated animals that received corn oil alone for 60 weeks (C60).

PGST-Positive Altered Hepatocellular Foci Formation
The expression of the placental form of glutathione S-transferase (PGST) in hepatocytes is believed to be a biomarker of carcinogenesis in rodent liver (Hendrich and Pitot, 1987Go). The development of putatively preneoplastic PGST positive altered hepatocellular foci (PGST +ve AHF) was analyzed in DEN-initiated animals following biweekly treatment with 1750 ng TCDD/kg (an approximate daily dose of 125 ng TCDD/kg/day) using different exposure regimens (Table 2Go). Initiation with a single dose of 175 mg DEN/kg resulted in a significant increase in all measurements of PGST +ve AHF (number of AHF/cm3, AHF volume fraction, and mean focus volume) in animals receiving corn oil alone, compared with non-initiated animals (data not shown). For clarity only the data on DEN-initiated animals is presented.


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TABLE 2 Time Course of Development of PGST-Positive Altered Hepatic Foci in DEN-Initiated TCDD-Treated Rats
 
There was a time-dependent increase in all AHF measures in control animals and in animals treated biweekly with 1750 ng TCDD/kg (Fig. 2Go). Continuous TCDD treatment resulted in increases in all AHF measures after both 14 weeks (T14) and 30 weeks (T30) (Table 2Go). Mean focus volume was the only measure not significantly increased after treatment for 14 weeks (T14). The volume fraction in animals treated with TCDD for 60 weeks (T60) was significantly elevated over controls (C60). However, very-high volume fractions (>30%) and extensive positive staining for PGST were observed in the livers of animals treated with TCDD for 60 weeks (T60). This was likely due to both the presence of tumors in these animals and to the merging of individual AHF, thus precluding a valid evaluation of individual PGST +ve AHF in this group. Therefore, the number of PGST +ve AHF/cm3 and mean focus volume in this group (T60) were not determined.



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FIG. 2. Graphic representation of the time course of tumor promotion in TCDD-treated rats. The x-axis represents the number of weeks after start of treatment until necropsy. Lines represent interpolations between individual group values within each of the exposure groups as shown in Figure 1Go (for mean values, error bars indicate ± standard deviation; for median values error bars indicate range from the 25th to the 75th percentile). (A) Mean body weights measured at biweekly intervals for groups C60 (continuous controls), T60 (continuous TCDD), T30–30 (withdrawal) and T0–16–30–16 (waiting/waiting + withdrawal). (B) Mean liver TCDD levels (ppt wet weight); (C) median PGST +ve AHF/cm3; (D) median PGST +ve AHF liver volume fraction (for purposes of clarity, data from the T60 group has been omitted in this figure). (E) median PGST +ve "mean focus volume."

 
The effect of withdrawal of TCDD treatment was also examined in DEN-initiated animals. The volume fraction and mean focus volume, 30 weeks after withdrawal of treatment (T30–30), was not significantly different from age-matched corn oil controls (C60). Furthermore, the AHF/cm3, after 30 weeks of withdrawal (T30–30), was significantly lower than in age-matched animals that received corn oil alone(C60) (p < 0.05, Mann-Whitney U-test).

The effect of TCDD on PGST +ve AHF was examined in animals that began treatment 18 weeks after initiation rather than only 2 weeks after initiation (waiting groups). Significant increases in volume fraction and AHF/cm3 were observed after both 14 (T0–16–14) and 30 weeks (T0–16–30) of TCDD treatment relative to age matched controls (C30 and C30–16, respectively). There was a significantly higher volume fraction and AHF/cm3 in animals treated with TCDD for 14 weeks after the 16 week DEN-initiation recovery period (T0–16–14) compared with animals treated with TCDD for 14 weeks after a 2-week DEN-initiation recovery period (T14). In contrast there was no difference in any AHF measurement in animals treated with TCDD for 30 weeks, either 2 (T30) or 18 weeks (T0–16–30) after initiation. The volume fraction and AHF/cm3 in the waiting + withdrawal group (T0–16–30–16) were significantly lower compared with animals exposed to TCDD for 30 weeks (T0–16–30). Volume fraction and mean focus volume in this group (T0–16–30–16) were not significantly different from age-matched controls (C60), but AHF/cm3 was significantly lower than age-matched controls (C60).

Alterations in liver TCDD with different dosing regimens were reflected in the time-dependent differences in observed mean body weights (Fig. 2Go). The time course of reduction in AHF/cm3 paralleled the reduction in TCDD present in the liver at terminal necropsy (Fig. 2Go). However, the time courses for changes in mean focus volume and volume fraction were not reflective of changes in the liver concentration of TCDD.

An interesting observation made was that there was an apparent heterogeneity in staining for PGST in the AHF in animals in the withdrawal groups. This was characterized by an apparent variability in the intensity of staining within an individual focus (data not shown). The variability was also observed in separate slides indicating that this was not an artifact of staining within a given slide. The intensity of staining in many AHF in the withdrawal groups appeared to be faint in comparison with AHF in the continuously-treated groups that were stained at the same time. In addition, intense staining for PGST in AHF in animals treated with either corn oil or TCDD, continuously, was homogeneous within individual foci. While this may have been indicative of a heterogeneity in phenotype of PGST +ve AHF in the withdrawal groups, all AHF were considered the same in the AHF measurements, regardless of staining intensity.

TCDD Tissue Concentration and Elimination Half-life
The mean liver concentrations of TCDD from this study have previously been reported (Walker et al., 1998Go). Using these data, the half-life for TCDD in the liver was estimated using the methods outlined in Materials and Methods. The design was developed in such a way as to allow us to test for changes in the half-life of TCDD due to age, initiation, high vs. low concentration, delayed first exposure, body weight, and liver weight. For liver, the magnitude of the concentration and delay of exposure both had a significant impact on the half-life. The half-life for TCDD in the liver was determined to be bi-phasic (p < 0.01), with the half-life decreasing when the concentration of TCDD rose above 1000 ppt wet weight (this value was not estimated but chosen from 3 possibilities, 500, 1000, and 2000 ppt, because it yielded the most significant change and best fit the data). In addition, it was observed that animals that were given their first treatment with TCDD after a waiting period of 18 weeks following initiation by DEN (rather than the 2 weeks) had a significantly longer half-life (p = 0.04). This yielded 4 different half-life estimates for the liver (with or without waiting period, above or below 1000 ppt concentration). For animals with only a 2-week waiting period following initiation, when the tissue concentration was in the range of 0 to 1000 ppt, the half-life was estimated to be 33.1 days (95% CI; 25.2 to 43.5 days). When the concentration increased above 1000 ppt, the half-life in these animals dropped to just 16.7 days (95% CI; 9.1 to 36.4 days). For animals with an 18-week waiting period following initiation, when the tissue concentration was in the range of 0 to 1000 ppt, the half-life was estimated to be 45.3 days (95% CI; 25.3 to 93.0 days). When the concentration increased above 1000 ppt, the half-life in these animals dropped to just 19.2 days (95% CI; 9.1 to 66.0 days).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
While extensive research has resulted in a thorough understanding of the activation of the Ah receptor and subsequent alteration of biological responses, our understanding of the mechanism of hepatocarcinogenesis induced by TCDD is still limited. Mathematical models of carcinogenesis have been applied to data from tumor promotion studies and other chronic rodents studies, in order to provide insights into the underlying mechanism involved in tumor promotion by TCDD (Conolly and Andersen, 1997Go; Moolgavkar et al., 1996Go; Portier et al., 1996Go). While these models differ in their description of the development of altered hepatic foci in TCDD-treated rat liver, current data sets are inadequate for the determination of the most appropriate model. The study described in this paper was designed to generate a suitable data set that will be used for the mathematical modeling of the time course of development of altered hepatic foci during tumor promotion by TCDD.

Studies using a simple 1-cell/2-stage model of cancer, suggest that TCDD may be classified as an "activator" of carcinogenesis, in that it causes an increase in the transition rate of normal cells into the initiated cell (preneoplastic) population (Moolgavkar et al., 1996Go; Portier et al., 1996Go). Several hypotheses have been suggested to account for this activation, including formation of potentially genotoxic catechol estrogens or other endogenous/dietary pro-genotoxicants, by TCDD-inducible cytochromes P450. Other studies however indicate that a 2-cell model may also be used to describe tumor promotion by TCDD, in the absence of any effect of TCDD on this mutation rate (Conolly and Andersen, 1997Go). Two-cell models are based on the hypothesis for tumor promotion, whereby different promoters may promote out a subset of initiated cells to produce AHF that exhibit a programmed phenotype different from those that would be promoted by other promoters. For example, TCDD exposure may result in the development of both "TCDD-responsive," and "TCDD-non-responsive" AHF. The number and size of PGST positive AHF in the TCDD-treated liver would be reflective of the both the magnitude and duration of exposure to TCDD.

With regard to the interpretation of the AHF data, it is important to note that within the limited number of animals used in this study there was a high degree of inter-animal variability in the induction of AHF by TCDD. This may have been due to differences in the degree of DNA damage caused by DEN, differences in the compensatory proliferative response following the necrosis induced by DEN treatment, or due to differences in the response to tumor promotion by TCDD. Within this context, large inter-animal differences in hepatocyte cell replication, induced by TCDD, were also observed in this study (Walker et al., 1998Go). The reversibility of AHF after withdrawal of TCDD treatment is consistent with previous observations indicating that after withdrawal there is a reduction in the number of foci but an increase in the size of the subset of foci (Dragan et al., 1992Go; Tritscher et al., 1995Go). The reversibility of promotion is likely due to reduction in TCDD levels within the liver after cessation of treatment, and to subsequent changes in growth-signaling pathways that are known to be altered by exposure. It is important to note that 16 weeks after withdrawal of treatment, TCDD levels in the liver are still significantly elevated over control animals (Walker et al., 1998Go), but lower than that necessary for the induction of tumor promotion by TCDD (Maronpot et al., 1993Go). Currently, the most appropriate measure of "dose" for evaluating the relationship between TCDD exposure and AHF development is not known.

The observation of increasing mean focal volume and volume fraction after cessation of treatment suggests that some AHF remain, and that at some point in their development the growth of these AHF may become TCDD-independent. Alternatively, within the context of a single-cell model, the reduction of TCDD in the liver after cessation of treatment may simply result in the altered growth rate of the AHF population, during which some of the AHF lose their enzyme-altered phenotype. The variability in staining for PGST within the AHF after withdrawal of TCDD is consistent with the notion that the phenotype of a focus may change as the promotional environment changes (Kopp-Schneider et al., 1998Go).

Significant reductions in AHF/cm3 were observed after only 16 weeks of withdrawal in the waiting group (T0–16–30–16) compared with 30 weeks in the group exposed to TCDD only 2 weeks after initiation (T30–30). However, there was no significant difference in the number of AHF/cm3 in the livers between these two groups. In addition, while the AHF/cm3 in these withdrawal groups was significantly lower than controls, there was no difference in the tumor incidence. The observation of a high tumor incidence in the control animals, using this experimental model, suggests that a necrogenic dose of DEN induced the formation of truly preneoplastic lesions that are capable of progressing to tumors in the absence of an exogenous promotional stimulus. The absence of an effect of 30 weeks of TCDD treatment on the tumor incidences in the withdrawal groups indicates that a subset of the AHF may be not only preneoplastic but are likely also to be insensitive to altered growth signaling by TCDD. It is not clear at this point why the number of AHF/cm3 in the T30–30 and T0–16–30–16 groups is lower than that observed in controls (C60). It may be a result of a TCDD-induced loss of a subset of AHF after cessation of treatment, either via a physical loss of cells or by loss of the PGST phenotype. Alternatively, promotion by TCDD may inhibit the growth of a subset of AHF that would normally develop into observable clones in the absence of an exogenous promoter. Subsequent cessation of treatment, and reduction in growth promotion of AHF that are responsive to TCDD, would lead to a loss of a large proportion of AHF (as seen in the T30–16 and T30–30 groups) thereby leaving fewer observable AHF than in controls. The observation that volume fractions in the T30–30 and T0–16–30–16 groups were not significantly different from the C60 group also raises the possibility that coalescence of some AHF may have occurred, resulting in the scoring of multiple AHF as a single AHF, and hence leading to a lower AHF/cm3 and higher predicted mean focal volumes, as was observed.

At 60 weeks, the volume fraction was very high. Individual AHF analyses could not be determined in this group, since there were large areas stained positive for PGST. This indicates that at some point there was likely some coalescence of individual foci. Consequently a characteristic of AHF promotion by TCDD is to a situation where there are fewer AHF but a higher volume fraction and hence a larger mean focus volume. In the T30-16 withdrawal group, the volume fraction was higher than after 30 weeks of continuous treatment, and therefore its is likely that some of the reduction in foci number may have been due to focal coalescence in addition to focal regression.

This study extends previous studies to show that cessation of TCDD treatment and subsequent reductions in liver TCDD levels and foci number lead to a lower tumor-incidence rate compared with animals continuously exposed to TCDD. Within the context of tumor promotion, if its assumed that a PGST +ve AHF represents a preneoplastic lesion, then clearly not all AHF progress to tumors, and a high mean focus volume AHF does not equate with a higher tumor incidence. This is in part due to the fact that mean focus volume, does not adequately represent the true size distribution of the measured AHF but is an estimate of how the volume fraction and AHF/cm3 are related.

It will be important to determine if there are TCDD-dependent and TCDD-independent foci phenotypes and if certain phenotypes of foci have a greater probability to progress to tumors. This may help to explain why alteration of the promotional environment may result in an alteration in tumor incidence observed. These rodent studies are relevant to the human situation in that, in human studies, TCDD exposure varies in magnitude, duration, and over different portions of an individuals lifetime. Such observations suggest that one of the reasons why the epidemiological data on increased cancer incidence in human cohorts exposed to TCDD are not strong, is that the most suitable measure of exposure and/or dose for dioxin-induced carcinogenesis is not known.


    ACKNOWLEDGMENTS
 
The authors would like to thank Joe Haseman for statistical analyses, Peter Mann (Experimental Pathology Laboratories) for pathological evaluations, Louise Harris for dosing the animals, Willie Purdie and Larry Judd for assistance with animal procedures, George Clark, John Seely (Pathco), Charles Sewall, and Brian Miller and Ralph Wilson, for help in carrying out this study, and Angelique VanBirgelen and Scott Masten for critical review of this manuscript.


    NOTES
 
1 To whom correspondence should be addressed at the National Institute of Environmental Health Sciences, Building 101, Room D452, 111 Alexander Drive, P.O. Box 12233, Research Triangle Park, NC 27709. Fax: (919) 558-7053. E-mail: walker3{at}niehs.nih.gov. Back

2 Present address: Nestle Research Center, P.O. Box 44, CH-1000 Lausanne 26, Switzerland. Back

Portions of this paper were presented at the 17th Symposium on Chlorinated Dioxins and Related Compounds (Dioxin '97), Indianapolis, Indiana, August 25th–29th, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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