* Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden;
Chemical Industry Institute of Toxicology, Six Davis Drive, Research Triangle Park, North Carolina 27709; and
AstraZeneca, R&D Södertälje, Safety Assessment, S-151 85, Södertälje, Sweden
Received December 22, 1999; accepted May 9, 2000
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ABSTRACT |
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Key Words: polychlorinated biphenyl (PCB); 2,4,5,3',4'-pentachlorobiphenyl; tumor promotion; cell proliferation; quantitative foci growth model; altered hepatic foci.
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INTRODUCTION |
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The carcinogenic properties of the chlorinated aromatic hydrocarbons are one of the critical effects of these substances (Ahlborg et al., 1992; IARC, 1997) and in some risk assessments of TCDD the recommended low risk exposure levels have been based on carcinogenicity (U. S. Environmental Protection Agency [U.S. EPA], 1985; Nordic Council, 1988
). Mixtures of PCBs are carcinogenic in rat liver (reviewed by Ahlborg et al., 1992). Most data suggest a nongenotoxic mode of action and individual PCBs and mixtures of PCBs can promote liver tumorigenesis in rats and mice following initiation with various genotoxic agents (reviewed by Silberhorn et al., 1990).
The liver tumor promoting effect of the mono-orthosubstituted 2,4,5,3',4'-pentachlorobiphenyl (PCB118) has been studied in a 2-stage initiation-promotion protocol (Haag-Grönlund et al., 1997). Preneoplastic hepatic foci positive for the placental form of glutathione S-transferase (GST-P) were analyzed after various doses of PCB administered for 20 or 52 weeks. The results showed that PCB118 enhances foci growth in rat liver, indicating a tumor-promoting activity. The occurrence of foci increased with both time and dose, but further conclusions regarding the growth kinetics of the PCB-induced foci could not be drawn without a more sophisticated analysis.
Biologically based, quantitative foci growth models have been proposed as an additional tool for the analysis of foci growth (Conolly and Kimbell, 1994; Dewanji et al., 1989
). These models can be used to distinguish between the initiating and promoting modes of action and to estimate birth, death, and mutation rates (Fig. 1
). Research utilizing such models can provide important insights in cancer mechanisms by demonstrating which mechanistic assumptions are consistent with the dose-response data. The first quantitative models of focus growth (Conolly and Kimbell, 1994
; Dewanji et al., 1989
) assumed only one homogenous population of initiated cells (Fig. 1A
). Conolly and Andersen (1997) proposed a two-cell model leading to the formation of 2 types of initiated cells (Fig. 1B
). In the present study, the biologically based quantitative model proposed by Conolly and Andersen (1997) and Conolly and Kimbell (1994) was used for analyses of the hepatic foci growth caused by weekly administration of PCB118 for 20 or 52 weeks (Haag-Grönlund et al., 1997
). In addition, hepatocyte proliferation in foci as well as in surrounding nonfocal liver tissue was quantitated experimentally and the data were used to guide parameter estimation. The aim of this study was to get a better understanding of the kinetics and potential mechanisms involved in hepatic foci growth induced by PCB treatment in DEN-initiated animals.
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MATERIALS AND METHODS |
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Proliferation of hepatocytes was analyzed by incorporation of the thymidine analogue bromodeoxyuridine (BrdU) in the DNA of dividing cells and subsequent immunohistochemical staining of the labeled cells. BrdU tablets (Innovation Research of America, Toledo, Ohio) of 100 mg each were surgically implanted subcutaneously in 5 animals per dose group one week before the animals were killed. Serial sections (4 µm) of the right lateral lobe of formalin-fixed, paraffin-embedded liver were stained for GST-P and BrdU respectively. The GST-P staining was performed as described by Haag-Grönlund et al. (1997). For the BrdU staining, the sections were deparaffinized in xylene and ethanol. Endogenous peroxidase activity was blocked with 1% fresh H2O2 in methanol for 10 min. Four consecutive microwave treatments for 4 min in 10 mM citrate buffer pH 6.0 were required for retrieval of the BrdU antigen. The sections were then incubated with 10% goat serum for 10 min followed by a 1-h incubation with the primary BrdU antibody (Becton-Dickinson, Sweden) diluted 1:50. Following 1 h of incubation with the secondary antibody, which was a peroxidase conjugated goat-antimouse IgG (Dako, Denmark) diluted 1:100, the BrdU antigen was localized by final incubation with the chromogen diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich, Sweden). The sections were counterstained with Meyer's hematoxylin and mounted in a glycerol-based mounting medium. All chemicals were obtained commercially in appropriate grades of purity.
BrdU-labeled nuclei, which were brown in contrast to the blue nonlabeled nuclei, were recorded in focal and nonfocal hepatocytes of animals treated with 0, 40, 640, and 10,000 µg/kg bw/week for 20 and 52 weeks, using a Leitz diaplan microscope (400x magnification). Photographs of GST-Pstained serial sections were used as guides for the localization of the focal areas on the BrdU-stained sections. At least 1000 nonfocal hepatocytes and 1000 focal hepatocytes were counted from each animal. The labeling index (LI) was calculated as the number of BrdU-labeled nuclei divided by the total number of counted nuclei, expressed as a percentage. The division rate, (day1), was calculated as described by Moolgavkar and Luebeck (1992):
![]() | (1) |
Differences in proliferation between treatment groups were statistically analyzed with the Kruskal-Wallis one-way test and with nonparametric multiple comparisons (Siegel and Castellan, 1988). Differences between focal and nonfocal tissue were analyzed with the Mann-Whitney test. Correlation was analyzed with the Pearson Correlation Test. The statistical analyses were carried out with the SPSS for Windows statistical package (SPSS, Inc. Chicago, IL) and the significance level chosen for all analyses was p < 0.05.
Simulation model.
The simulation model used for the analysis of the foci data was a modification of the model proposed initially by Conolly and Kimbell (1994) and in a refined form by Conolly and Andersen (1997) (Fig. 1). The model postulates that normal cells may either undergo cell division, giving rise to 2 normal daughter cells, or die (or differentiate), and thereby leave the pool of susceptible cells, or they may divide into one normal cell and one mutated cell. The mutated cell may in turn divide, die (or differentiate), or undergo a second mutation resulting in one mutated daughter cell and one malignant cell. The second mutation step has not been considered here, since tumor data were not available for modeling. The model of Conolly and Kimbell (1994) assumed only one homogenous pool of initiated cells (Fig. 1A
), while the model of Conolly and Andersen (1997) was extended to describe 2 independent types of initiated cells (A and B) (Fig. 1B
). In the present study, we tried to model the focus data with both the one-cell and two-cell models.
The one-cell model was not able to provide accurate descriptions of the data despite the use of extensive manual adjustment of parameter values and formal optimization (ACSLopt, Pharsight, Mountain View, CA). A promotional mechanism is most consistent with the current understanding of the mechanism of PCB carcinogenesis (Ahlborg et al., 1992; Silberhorn et al., 1990
). Therefore, we did not attempt to fit a version of the one-cell model that includes an effect of PCB on the mutation of normal cells, as was previously done for DEN/TCDD initiation-promotion experiments by Moolgavkar et al. (1996) and Portier et al. (1996). The following is a brief description of the quantitative aspects of the simulation model. The reader interested in a more complete formal description of the model should refer to Conolly and Andersen (1997) and Conolly and Kimbell (1994).
The liver is treated as consisting of 1.69 x 108 hepatocytes/cm3 (Weibel et al., 1969) and this cell density is used for both normal and mutated cells. Both normal hepatocytes and hepatocytes in foci behave independently of each other. These simplifying assumptions are used because no data are available to support modeling at a greater level of detail. Growth kinetics of hepatocytes are described by division rates (
) and death rates (ß) (rate expressed as day1):
![]() | (2) |
![]() | (3) |
where Nm is the number of normal hepatocytes mutating during the time interval t and µ is the mutation parameter, i.e., probability of mutation per cell division. A random deviate about Nm, denoting the number of mutations during
t, is drawn from a Poisson distribution using the function POIDEV (Press et al., 1989
). Inputs to POIDEV are the mean of the Poisson distribution (Nm) and a pseudorandom number between 0 and 1 generated with the algorithm UNIFL (Bratley et al., 1987
).
Once a mutated cell is created, it may either divide or die, with probabilities per time step calculated from the division and death rates for altered cells according to Conolly and Kimbell (1994). Division of these single altered cells, derived directly from normal cells, may give rise to clones of altered cells. The simulation program keeps track of each clone over time, allowing average clone size (cells/clone) and number (clones/cm3) to be described. Volume fraction is calculated directly from average clone size and number of clones/cm3.
The behavior of each cell in a clone is calculated stochastically for each time step as long as the clone is smaller than 1000 cells. When clones become sufficiently large, greater than 1000 cells in size, their growth kinetics become effectively deterministic. At this point, the program reverts to a deterministic calculation of growth for each clone to save computer time. If clone size falls below 1000 cells, the calculation switches back to the stochastic mode. A number of simulations were run showing that growth of individual clones is effectively deterministic at 1000 cells by varying the cutoff point at which the calculation switches between stochastic and deterministic modes.
Clones smaller than 24 cells in 3 dimensions, approximately corresponding to a 2-dimensional radius of 35 mm (Arias et al., 1988; Marsman, personal communication; Weibel et al., 1969
), are considered to be undetectable, since this was the cut-off limit applied when the foci were analyzed (Haag-Grönlund et al., 1997
).
Partial hepatectomy is described as an instantaneous decrease in liver weight. The remaining liver starts to regenerate the missing tissue 24 h later, and the liver is fully regenerated in 7 days (Higgins and Anderson, 1931; Tatematsu et al., 1988
) after partial hepatectomy. Regeneration is achieved by increasing the division rate (
) of hepatocytes.
The effect of DEN is modeled as a transiently sustained increase in the probability of mutation per cell division while the liver is undergoing regenerative cellular replication. This increased probability of mutation lasts until the liver is fully regenerated, at which point the probability returns to its basal level. This part of the model is intended to approximate the actual sequence of events relating to regenerative proliferation and mutation after partial hepatectomy and DEN treatment.
Parameter values.
Since no data on the number of mutated cells after initiation were available, this parameter was estimated from the number of foci/cm3. Thus, the highest average number of detectable foci in any of the treatment groups in Haag-Grönlund et al. (1997), 24,000 per cm3, which was obtained in DEN-initiated (30 mg/kg, 24 h after partial hepatectomy) animals subsequently treated with the positive-control compound 3,4,5,3',4'-pentachlorobiphenyl (PCB126) for 20 weeks, was used as an approximation of the number of mutated cells available for a promoting action. This number includes all foci with an equivalent radius of > 25 µm and is therefore higher than the number reported in Haag-Grönlund et al. (1997), which was based on an equivalent radius of > 35 µm. The 2 mutation parameters of the two-cell model were constrained so that the total number of mutated cells after DEN treatment was about 24,000/cm3. The actual number of mutated cells due to DEN varies from run to run of the model, as this is a stochastic parameter (Equation 3). The strategy for partitioning the (approximately) 24,000 mutated cells/cm3 between A and B cells was similar to that used by Conolly and Andersen (1997). Briefly, the control foci are assumed to consist mostly of A cells, and the high-dose foci mostly of B cells. Sufficient DEN-related mutations of normal hepatocytes to the A genotype are required to account for the amount of A cells in DEN control animals. Having fixed the mutation parameter for A cells in this manner, the mutation parameter for B cells is adjusted to provide enough mutations to account for the amount of B cells in DEN-initiated, high dose PCB-treated animals. The low and intermediate dose data consist of mixtures of A and B cells with the proportion of B cells increasing with increasing dose.
During the regenerative proliferation, the division rate for all hepatocytes was calculated according to Conolly and Andersen (1997). In addition, the division rate of mutated A cells was assumed to increase by a factor of 43.5, compared to normal hepatocytes, during the regenerative proliferation and for 2 weeks thereafter. This assumption was necessary in order to obtain the measured volume fraction in the controls, particularly after 20 weeks of exposure. No data were available for a direct estimate of ß. The basal death rate for both A and B cells was set to a value close to zero (1.0 x 104/day), to reach the number and volume fraction of foci at the end of the study. This value of ß is approximately equal to the birth rate of normal hepatocytes in adult rats, where there is no net growth of the liver (Conolly and Kimbell, 1994).
When the liver had recovered from partial hepatectomy, the mutation rates were set to 0 and the measured occurrence of foci was modeled by estimation of the suitable division and death rates. While there is some ongoing mutation of normal hepatocytes in the absence of DEN-related effects, it is reasonable to assume that this is quantitatively insignificant compared to the mutation rate induced by DEN. Visual fitting of the model to the foci data was the primary method used to estimate the birth and death rates. Formal parameter estimation using the ACSLopt software was used on a limited basis, primarily to evaluate the ability of the one-cell model to fit the data. More extensive use of formal optimization was not possible as the two-cell model is parameter-rich, and sufficient data are not currently available to support a statistically rigorous approach to parameter estimation. When formal methods were not used for parameter estimation, a criterion of biological plausibility was applied, i.e., the model was fit to the data using parameter values that were biologically plausible. The resultant model can thus be classified as biologically plausible but not statistically optimal. The experimentally derived cell proliferation data were used to guide the estimation of the division rates in foci. The parameters were first established for the DEN-initiated control group and were then adjusted to fit the data in the PCB-exposed groups. For each dose level, the adjustments were confined to 5 time points during the course of the study: (1) the day of partial hepatectomy, (2) the day before the first PCB administration, (3) the day of the first PCB administration, (4) 20 weeks after the first PCB administration, and (5) 52 weeks after the first PCB administration. Division and death rates in the time interval between 2 of these time points were linearly interpolated between division and death rates at the 2 time points.
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RESULTS |
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Estimation of division rates during PCB treatment was constrained by the labeling index data. Although it was not possible to use division rates calculated directly from the labeling index data, the same overall pattern of dose-dependent response seen with the labeling index data (Table 1, Fig. 2
) was maintained for the estimated division rates. This constraint required that the B cell population had to undergo a rapid increase in proliferation at the start of PCB dosing. The estimated division rate of B cells was 0.0035 day1 in all dose groups before PCB administration. The rate transiently increased to 0.0090 day1 in the 3 lowest dose groups and to 0.01, 0.025, and 0.07 day1, respectively, in the 3 highest dose groups, as an immediate response to PCB treatment. At 20 weeks of administration, the estimated division rates for B cells declined to lower values. However, with the exception of the lowest dose group, these rates were still higher when compared to the values before PCB administration. Importantly, the increase of proliferation at the start of PCB dosing, predicted by the model, would not have been detected in the experiment, since no animals were killed at that point in time. Relative to the B cells, the division rates of A cells was found to be small and fairly invariable throughout the study, with ranges of only 0.00720.0074 and 0.00450.0074 at 20 weeks and 52 weeks, respectively (Table 3
). Moreover, no PCB-related burst of proliferation of A cells was required to model the foci data.
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The model predicted that the number of foci/cm3 would plateau at the 10,000 µg/kg/week dose level (Fig. 3), because under these experimental conditions, all the initiated cells are predicted to have either become extinct (lost all cells due to the stochastic birth-death process) or have grown large enough to become visible. Since no foci remain invisible, no further increase in the number of foci/cm3 is possible.
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DISCUSSION |
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The shape of the dose-response curve for hepatocyte proliferation in focal and nonfocal tissue correlated well with foci growth, which was further supported by correlation analysis of individual animal data. Regenerative cell proliferation induced by cytotoxicity may act as a stimulus for preferential growth of pre-cancerous and cancerous cells (Butterworth et al., 1992). Although alternative mechanisms cannot be excluded, it is reasonable to assume that the mechanisms of foci growth in the higher dose groups after 52 weeks involved regenerative proliferation, as indicated by the high nonfocal LI and the observed liver histopathology, e.g., hyperplasia and focal hemorrhagic necrosis (Haag-Grönlund et al., 1997
). After 20 weeks of treatment with the highest dose of PCB, neither proliferation nor volume fraction was significantly different from the corresponding DEN controls, and furthermore, no correlation was found between these variables. Apparently, a longer treatment period is necessary to experimentally detect differences in proliferation and foci growth between DEN-initiated controls and DEN + PCB118-dosed animals.
A decreased cell death in foci cells compared to surrounding tissue is another mechanism that may give foci cells a growth advantage over nonfocal cells. The balance between cell proliferation and cell death has a profound impact on the net growth of initiated, preneoplastic and neoplastic cell populations (Goldsworthy et al., 1996; Schulte-Hermann et al., 1995
). Although quantitative studies of cell death were not performed in this study, the outcome of the modeling suggests that PCB118 affects the death rate in the 3 high-dose groups and induces a selective increase in the death rate of A cells at 20 and 52 weeks. The histopathological observation of necrosis and regenerative growth (Haag-Grönlund et al., 1997
), and the increase in proliferation of both focal and nonfocal tissue in the highest dose group at 52 weeks, suggest that the increase in cell death is at least partially attributed to a cytotoxic mode of action involving necrotic cell death. However, apoptogenic mechanisms cannot be excluded, and future work needs to be focused on this important issue.
Moolgavkar et al. (1996) and Portier et al. (1996) have modeled hepatic foci growth caused by TCDD, a substance with structural and toxicological properties similar to the coplanar PCBs, assuming that TCDD increases the mutation rate of normal cells. However, TCDD fails to show mutagenic action in a variety of assays (IARC, 1997; Shu et al., 1987) and in a recent paper by Conolly and Andersen (1997), a two-cell model was proposed, which could model the same TCDD data set without an assumption of mutagenicity by TCDD. For the present simulation, the mutation rate was assumed to be unaffected by the PCB exposure. The potential genotoxic activities of PCBs have been analyzed in several test systems and current data indicate that PCBs have little, if any in vivo genotoxic potential (reviewed by Ahlborg et al., 1992). Short-term exposure to PCBs during liver-cell proliferation have not shown initiating action in a 2-stage initiation-promotion protocol (Hayes et al., 1985
). These results are further supported by the lack of a significant increase in volume fraction and in number of foci/cm3 in noninitiated animals after long-term treatment with PCB118 (Haag-Grönlund et al., 1997
). Thus, available data indicate a nongenotoxic mode of action of PCBs, in support of the zero mutation rate assumed in the models after PCB treatment.
The experimental data on PCB-promoted hepatic foci could be adequately simulated if the model assumes 2 initiated populations of hepatocytes, but no adequate simulation was possible with only one homogenous cell population of initiated hepatocytes. Experimental and theoretical support for a model of foci growth based on 2 or more cell types has been reported in the literature. The existence of different types of foci cells is demonstrated by the great heterogeneity in phenotypes of foci, which can be classified into subgroups based on, e.g., morphology or enzyme expression (Bannasch and Zerban, 1992). Various subpopulations of initiated cells have been postulated after treatment with DEN as well as with other initiating agents (Dragan et al., 1993
; Jirtle et al., 1994
; Kaufmann et al., 1992
). Jirtle et al. (1994) showed that in a subset (15%) of preneoplastic liver lesions, the level of the mitoinhibitory transforming growth factor-ß1 (TGF-ß1) was lower than in other preneoplastic lesions and in surrounding normal tissue after exposure to the tumor promoter phenobarbital. The majority of liver carcinomas were either partially or uniformly negative for TGF-ß1. A relative decrease in the ability of initiated hepatocytes to express TGF-ß1 could give these cells a selective growth advantage over the surrounding normal hepatocytes during phenobarbital exposure. Another finding in support of a growth model with more than one cell population is that administration of phenobarbital after DEN initiation resulted in a different distribution of hepatic foci phenotypes compared with initiated animals who did not receive this promoting agent (Dragan et al., 1993
). Furthermore, at least 3 types of DNA adducts are formed after administration of DEN, all of which may induce dissimilar types of mutations and thus result in initiated cells of distinct phenotypes (Dragan et al., 1994
).
An accurate description of the volume fraction in the DEN control group required introduction into the model of a parameter describing a selective growth advantage for initiated cells, relative to normal cells, during and 2 weeks after the period of regenerative proliferation. Introduction of this parameter postulates that initiated cells have a growth advantage over normal hepatocytes after partial hepatectomy and DEN treatment, resulting in selective clonal growth of these cells before PCB administration. DEN is a potent, complete carcinogen and single doses of 50 mg/kg have been reported to cause tumors after partial hepatectomy in rats (Scherer and Emmelot, 1975). Clusters of GST-P foci have been demonstrated 14 days after partial hepatectomy and DEN treatment (Dragan et al., 1994
). Examination of the experimental data used for the present study revealed that the number and volume fraction of foci in initiated control animals were substantially higher than in PCB-treated animals without initiation (Haag-Grönlund et al., 1997
). Furthermore, the volume fraction of foci, but not the number of foci, is higher at the 52 week time point compared to the 20 week time point in the initiated control animals (Fig. 3
). These facts show that the initiation treatment (PH + 30 mg/kg of DEN) without subsequent promotion treatment stimulates growth of foci.
Another assumption that was introduced in the present model was an initial dose-dependent, transient increase of proliferation of B cells immediately after the first PCB administration (Table 1). This assumption was necessary to increase the number of foci and percentage of liver occupied by foci to the observed levels after PCB administration, without reaching proliferation rates unrealistically higher than the values experimentally measured at 20 and 52 weeks. In order to quickly reach effective target organ doses, the first dose of PCB was 5 times higher than the subsequent doses. Since high doses of tumor promoters may cause high cell proliferation, the loading dose could explain such a transient increase. However, considering the slow metabolism and elimination of PCBs, the loading dose probably does not result in higher tissue doses in the liver at early time points, relative to later time points. Also with doses that are maintained over time, an increase in cell proliferation of normal and preneoplastic liver cells during the first period of treatment often returns to lower levels during continuous treatment (Butterworth et al., 1992
; Schulte-Hermann et al., 1990
). Such an early increase in nonfocal liver-cell proliferation, reaching a peak a few days after the first administration and returning back to control levels within some weeks, despite continued administration, has been observed for some rodent tumor promoters such as phenobarbital and ethinyl estradiol (Peraino et al., 1975
; Yager et al., 1986
). In addition, there are numbers of studies reporting peaks in proliferation within 1 to 2 days after single administrations of tumor promoters (Butterworth et al., 1992
). Furthermore, single doses of a number of liver mitogens, known or assumed to promote liver tumor development, have been shown to produce high proliferating activity in foci cells (LI up to 50%), whereas proliferation in normal liver cells increased slightly to moderately (Schulte-Hermann et al., 1981
). Equivocal findings have been reported in the literature regarding proliferation after single or short-term exposure to TCDD (Büsser and Lutz, 1987
; Conaway and Matsumura, 1975
; Fox et al., 1993
). Buchmann et al. (1994) demonstrated a sustained increase of liver cell proliferation in foci during 17 weeks of continuous TCDD exposure. In a study by Wölfe et al. (1988) hepatocyte proliferation was increased one week after exposure of rats to 3,4,3',4'-tetrachlorobiphenyl (PCB 77). In the present study, it was not possible to experimentally confirm the simulated proliferation peak, since no animals were killed until 20 weeks after the first administration of PCB. It is worth noting that the negative selection hypothesis for tumor promotion (Andersen et al., 1995
; Jirtle et al., 1991
, 1994
; Solt and Farber, 1976
) predicts that an initial burst of cellular proliferation due to promotional activity is overcome by growth regulatory factors subsequently released by the affected tissue. Experimentally observed promotion occurs when cells acquire mutations that allow them to divide in spite of the presence of the growth regulatory factors. This hypothesis is consistent with the transient burst of cellular proliferation found in the present analysis to be consistent with the observed foci data.
A closer investigation of the dose response curve for PCB118 after 20 weeks of treatment revealed a slight nonsignificant drop in number of foci/cm3 and volume fraction in the dose group given 40 µg/kg bw/week (Fig. 3). The number of foci/cm3 was 6.4 x 103, 5.0 x 103 and 6.0 x 103 and the volume fraction 2.5%, 1.6%, and 2.2% in the groups treated with 10, 40, and 160 µg/kg bw/week, respectively (Haag-Grönlund et al., 1997
). Interestingly, a parallel nonsignificant drop in focal cell proliferation was observed in this dose group (Fig. 2
), supporting the biological relevance of the slight drop in foci development observed at 40 µg/kg bw/week. The same phenomenon, generally referred to as hormesis, has been observed for hepatic effects of TCDD (Kociba et al., 1978
; Maronpot et al., 1993
; Pitot et al., 1987
) as well as for toxic effects of other groups of chemical compounds (Calabrese and Baldwin, 1997
; Stebbing, 1997
), and several mechanistic explanations have been proposed for such J or U shaped dose-response curves (Andersen and Barton, 1998
; Stebbing, 1997
; Teeguarden et al., 1998
). Since this drop was small compared to the high variability in foci data, and not statistically verified, no assumption about a J shape was included in the simulation model. However, assumption of at least 2 cell populations, as proposed in this study, would have been required for simulation of such a drop in foci growth (Conolly and Andersen, 1997
).
As was noted in Materials and Methods, the available data are not sufficient to support formal statistical estimation of parameter values for the two-cell model. Instead, the more relaxed constraint that parameter values should be biologically plausible was applied. While a good description of the foci data was obtained using this approach (Fig. 3), we cannot use statistical arguments to guarantee that a different set of parameter values would not also give a good fit to the data. The parameterization of the model can thus be considered to represent a biologically plausible form of the model, but cannot presently be statistically confirmed.
In summary, in the present study, a biologically based mathematical model and experimental data on hepatocyte proliferation were used to further explore the growth characteristics of PCB-promoted foci growth. The primary goals of this analysis were to determine if the two-cell model could provide good fits to the foci data while using biologically reasonable parameter values, and to better understand the kinetics and potential mechanisms involved in hepatic foci growth induced by PCB treatment in DEN-initiated animals. The foci data were adequately simulated by the two-cell model, suggesting that mutations are not pivotal to explaining the experimental foci growth by PCB118. A strong correlation was found between foci growth and cell proliferation in foci and nonfocal tissue. An increase in cell death of A cells, was also predicted by the model for high doses, after long-term exposure. These data in parallel with the histopathology (Haag-Grönlund et al., 1997) and proliferation data suggest that regenerative proliferation is involved in the development of PCB118-induced foci. Moreover, the results of the analysis suggest that at least 2 separate populations of initiated cells may be involved in the foci growth caused by PCB118 after DEN initiation, and that, under the experimental conditions of this study, initiated cells have a growth advantage compared to noninitiated cells, before PCB administration. Furthermore, at higher PCB doses, a rapid increase in proliferation of initiated cells after the first PCB treatment, followed by a decrease in spite of continuous administration, was suggested by the model. This behavior, initially after PCB treatment, is consistent with the negative selection hypothesis for tumor promotion (Andersen et al., 1995
; Jirtle et al., 1991
; Solt and Farber, 1976
).
This study has raised a number of new questions regarding the growth characteristics of PCB-induced foci and has suggested some new, hitherto unknown, hepatocyte growth events after PCB exposure that could affect the final outcome of the experiment. An interesting continuation of the investigation of PCB-induced foci growth would be experiments designed to describe the hepatocyte growth shortly after the start of PCB exposure. Measurement of cell density (cells/cm3) as a function of dose and time would also be of interest, as variability in this parameter could affect the values of other parameters estimated by fitting the model to the data. Results from such studies would also help in the validation of the simulation model. For the purpose of model validation, an examination of the kinetics of focus growth after DEN treatment and partial hepatectomy, but before the first PCB treatment, would also be of interest.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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