Toxicological Sciences 55, 44-51 (2000)
Copyright © 2000 by the Society of Toxicology
Carcinogenicity |
Dose Response of Early Effects Related to Tumor Promotion of 2-Acetylaminofluorene
Institut für Toxikologie der Universität Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany
Received October 19, 1999; accepted January 6, 2000
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The genotoxic effects of 2-acetylaminofluorene (AAF) alone cannot explain the formation of rat liver tumors. It has been proposed that mitochondrial effects are associated with its tumor-promoting properties. These mitochondrial effects are thought to trigger apoptosis and regenerative proliferation, which alters the liver lobule in a cirrhosis-like manner. A situation is generated which favors the growth of initiated cells. To test this sequence of events, the dose dependence of early effects with time was studied. Male Wistar rats received 50, 100, 200, 400, or 800 ppm AAF in the diet and the following endpoints were analyzed at 2, 4, 8, and 16 weeks of feeding: apoptotic cell death, cell proliferation, GST-P-positive foci (placental form of glutathione S-transferase), and morphological alterations. Hydrolyzable hemoglobin adducts were used as a dosimeter for metabolic activation after 2 weeks of feeding. The hemoglobin adduct levels increased linearly with dose. With the conventional carcinogenic concentration of 200-ppm AAF in the diet, the number of apoptoses increased first, predominantly in the periportal area (2 weeks). Cell proliferation followed with some delay (4 weeks). This reflects regenerative tissue response and not the growth of initiated cells, because the number of enzyme-altered foci was still extremely low at that time. Foci developed only later when the morphology had changed. With 50 ppm AAF in the diet, a no-effect level had not been reached for any of the endpoints, but foci developed much more gradually than with higher doses. Unexpectedly, the proliferative response stabilized at 8 weeks with a labeling index of 12–17, with all AAF concentrations. The observed sequence of events supports the hypothesis. It is concluded that (1) The proliferation of initiated cells—defined as promotable cells—is largely determined by the cellular environment, such as morphologically altered liver. (2) The morphological alterations in rat liver result from imperfect regeneration from toxic effects. (3) Imperfect regeneration results from limitations in the possibilities to adapt to chemical stress. (4) If these limits are overwhelmed and morphology has changed to a certain extent, preneoplastic foci develop; this occurs much faster, at least, than without these changes.
Key Words: 2-acetylaminofluorene; toxicity; tumor promotion; rat liver; tissue specificity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-Acetylaminofluorene (AAF) is one of the key model substances in chemical carcinogenesis. An important contribution regarding dose response came from an extraordinarily large chronic feeding study—the so-called ED01 study (Staffa and Mehlmann, 1979
)—in which 7 different dietary concentrations of AAF were given to BALB/c mice. Tumors developed in liver and urinary bladder. Whereas tumor incidence in liver increased in a linear manner with dose, the increase in incidence in the bladder was nonlinear. The tumor rate in bladder rose from spontaneous 0.3% to only 2% at 75 ppm of AAF in the diet, but increased dramatically with higher doses, i.e., 17 and 75% at doses of 100 and 150 ppm, respectively. Bladder hyperplasia preceded tumor formation and it was concluded that cell proliferation caused or accompanied the increase in tumor formation (Cohen and Ellwein, 1990). In a parallel DNA adduct study (Poirier et al., 1991), it was shown that DNA adducts as a dosimeter for the biologically active dose in the target tissue increased in a linear manner with dose in both tissues. The adduct levels were even 2–4-fold higher in bladder than in liver and it was concluded that genotoxic effects were necessary but not sufficient to explain the tissue-specific tumor formation. The question of what actually causes toxicity or cell proliferation remains to be answered.
A similar situation exists in rat liver. AAF is a carcinogen in this tissue but genotoxic effects are not sufficient to explain the tissue-specific effects (Neumann et al., 1994). Acute and chronic toxic effects were proposed to be responsible for a cirrhosis like transformation of liver as a prerequisite for the expansion of initiated foci (Neumann et al., 1994, 1997). During these studies, reactions of AAF metabolites in mitochondria were detected that may provide a biochemical basis of AAF-toxicity. 2-Nitrosofluorene causes redox-cycling by draining electrons from the mitochondrial respiratory chain, which interferes with oxidative phosphorylation and potently induces the mitochondrial permeability transition pore in vitro (Klöhn and Neumann, 1997; Klöhn et al., 1998). As these mitochondrial effects may trigger apoptosis, a hypothesis of the sequence of events was brought forward. The homeostatic equilibria in biological systems can be disturbed in many ways. At low, nontoxic doses, tissues react with adaptive responses (Neumann et al., 1992). If these defense mechanisms are overwhelmed and cells are stressed too much, they are eliminated by apoptosis. Regenerative proliferation takes place. In the case of rat liver, oval-cell proliferation is supposed to provide the tissue with new hepatocytes, but substitution is not perfect and bile duct-like, fiber-producing cells are formed. The architecture of the liver lobule is disturbed. This generates a condition that favors the expansion of initiated cells or existing small foci.
We describe now part of the results of an extended experiment designed to test this hypothesis. If the hypothesis is correct, foci are expected to grow less than proportionate with dose, or not at all with nontoxic doses. Studying the dose dependence of early effects should allow demonstration of the sequence of events, and whether the early effects of the process depend linearly or nonlinearly on dose. Moreover, the existence of a threshold dose for the tumor-promoting effects, if they exist, should become visible. To this end, male Wistar rats were treated for up to 16 weeks with 5 different AAF concentrations in the diet: a putative nontoxic concentration (50 ppm), a concentration below the conventional tumor-producing level (100 ppm), the usual carcinogenic concentration (200 ppm), a concentration above that (400 ppm), and a putative toxic dose (800 ppm). The following parameters were measured after 2, 4, 8, and 16 weeks of feeding: apoptotic cell death, cell proliferation, and GST-P-positive foci. After 2 weeks, hemoglobin adducts served as a dosimeter to control for the dose dependence of metabolic activation. Other endpoints will be described elsewhere.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and Treatment
Male Wistar rats (180–240 g, HsdBrt:WH) were obtained from Harlan Winkelmann (Borchen, Germany). They were fed standard diet (Altromin 1324, Alrogge, Lage/Lippe, Germany) or standard diet containing different concentrations of AAF (Sigma, Deisenhofen, Germany, purity 95–97%).
The supplemented diet was prepared by the feed company and the content was controlled in this laboratory. Four animals for each dose were sacrificed after 2, 4, 8, and 16 weeks on the diet (a total of 92 animals).
At the time of sacrifice, livers were perfused under ether anesthesia. Two adjacent slices of liver, 2 mm thick, were cut from the right posterior and right anterior lobes. One of the slices of each lobe was fixed in 10% buffered formalin (pH 7.4) for routine paraffin-embedding. The other one was embedded in OCT tissue tek, snap frozen in liquid nitrogen, and stored at –80°C until use.
Measurement of Endpoints
Apoptoses were determined by in situ nick end labeling according to the procedure of Gavrieli et al. (1992), with slight modifications using the in situ cell death detection kit (AP-kit, Boehringer, Mannheim, Germany). Sections preincubated for 10 min with 100 U/mL DNase I (RNase-free, Boehringer Mannheim) served as positive controls. An apoptotic labeling index (apoptotic cells/100 cells) was determined by counting the numbers of labeled cells in 10 high-power fields per liver and calculating the average. Data obtained from 4 animals by 2 independent observers were used for the calculations. The results were confirmed by eosin fluorescence (data not shown).
Cell proliferation was determined by PCNA immunostaining as described previously (Hadjiolov and Bitsch, 1997). The antigen retrieval was done with 10 mM citrate buffer, pH 6, and sections were incubated with a mouse monoclonal antibody (PC 10, Dako Corp., Denmark) in dilutions from 1:50 to 1:80. A peroxidase conjugated to a goat anti-mouse IgG (Dianova, Hamburg, Germany) was used as secondary antibody in a dilution of 1:300. The brown to black reaction product that correlates with the different phases of the cell cycle was considered to indicate positive staining (Foley et al., 1993). The PCNA proliferation index (proliferating cells/100 cells) was defined as average count of all cells in G1, S, G2, and M-phase in 10 high-power fields. Labeled hepatocytes and proliferating oval cells, observed in the periportal area at 2 and 4 weeks, were counted together.
GST-P-positive foci were determined immunohistochemically (Sato et al., 1984) with a rabbit polyclonal antibody (Biotrin Int., Dublin, Ireland), diluted 1:200 in 2% phosphate-buffered saline, pH 7.4. A goat anti-rabbit IgG antibody (Dianova, Hamburg, FRG; 1:300) served as secondary antibody. Sections incubated with rat-immune serum instead of primary antibody served as negative control. Positive immunostaining was quantified by counting labeled foci and assessing areas in 10 high-power fields using x40 objective and x10 ocular lenses.
Hemoglobin Adducts
The formation of hemoglobin adducts of AAF is discussed and adduct analysis has been performed essentially as described in Zwirner-Baier and Neumann (1998) with minor modifications. To hydrolyze the hemoglobin adducts, 5 ml 1 N NaOH and 500 µl SDS (0.5%), as well as 100 ng 4-aminobiphenyl as recovery standard, were added to the protein (50 mg) and the samples were sonicated for 10 min and stirred for another 50 min at room temperature. After centrifugation, the supernatant was processed by solid phase extraction. All samples were processed in duplicate. Recoveries were 87 to 101% as determined by spiking hemoglobin from non-exposed animals with 1, 5, 10, and 100 ng 2-aminofluorene and 4-aminobiphenyl.
The extracts were analyzed by HPLC/UV 287 nm using LiChrospher 60 RP-select B, 5 µm, 250 x 4 mm, and as eluent, sodium phosphate buffer (0.02 M, pH 5.4) with acetonitril (5%) and methanol (50%); flow rate 0.8 ml/min. Calibration curves were established by measuring standard solutions of 2-aminofluorene, 4-aminobiphenyl (Sigma-Aldrich, Deisenhofen, Germany) and AAF (1:1:2.5) at 6 different concentrations. A 2-level calibration was made every working day. Adduct levels are expressed as nmol/g hemoglobin.
Statistical Analyses
Means were compared using the Student`s t-test (Microsoft Excel, Office 98). Statistical differences are noted in the legends of Figures 1, 3–5.
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0.05) and 800 ppm (p
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Toxic effects are reflected very sensitively by any increase in body weight. The doses were chosen in this experiment such that the lowest dose should be nontoxic, and the weights of animals fed 50 ppm AAF in the diet should have developed undistinguishable from controls. This was not quite the case (Fig. 1
). But, with the exception of the group fed with 400 ppm AAF, which started with somewhat higher body weights, the growth curves reflect nicely the effects of increasing concentrations. Feeding with 800 ppm AAF had to be stopped after 4 weeks because the animals showed severe signs of toxicity. They recovered quickly thereafter but were killed at week 8 of the experiment. These results show the narrow range (1:16) of doses between almost nontoxic and almost lethal.
To interpret the dose-response relationship, it is important to demonstrate that AAF is metabolically activated proportionately with dose within the administered dose range. We used the hydrolyzable adduct of AAF resulting from the reaction of 2-nitrosofluorene with the SH-group of cysteine in hemoglobin as a dosimeter for the biologically active dose at 2 weeks of feeding (Zwirner-Baier and Neumann, 1998). The hemoglobin adduct levels increased linearly with dose (Fig. 2). From the consumption of feed, the cumulative AAF dose was calculated as 0.29, 0.48, 0.87, 1.68, and 2.79 mmol/kg, respectively, for the AAF concentrations 50, 100, 200, 400, and 800 ppm.
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Time Course of Changes in Different End Points
The apoptotic index in liver as determined by in-situ DNA nick end labeling (TUNEL) was significantly and dose-dependently elevated over the control values after only 2 weeks, with all AAF concentrations (Fig. 3
). At this early stage, the 2 lowest concentrations produced notably fewer apoptoses than the 3 highest doses. Surprisingly, a comparable level of apoptoses was observed with all AAF concentrations at 4 and 8 weeks, which only increased further at 16 weeks, when necroses are also seen and liver morphology is altered. At the 2 early time points, apoptotic cells were localized predominantly in the periportal area. As preneoplastic foci grow, apoptoses are observed within foci, but these occurred also throughout the liver lobule.
Cell proliferation changed with time quite differently (Fig. 4). The 3 lowest doses had no effect at 2 weeks, proliferation increased significantly at that time only in livers of animals fed 400 ppm in the diet. The proliferation index increased gradually with time in the 50-ppm group. At 4 weeks, proliferation had increased rapidly with the higher concentrations, but stabilized at these levels at 8 weeks, such that it was very similar with all concentration groups at that time.
GST-P-positive foci as indicator for preneoplastic lesions increased notably later (Fig. 5). Up to 4 weeks, only occasional small foci were seen. Even at 8 weeks, foci numbers remained rather low in feeding groups 50 and 100 ppm. At 16 weeks, the number of foci had increased significantly with all concentrations except the lowest one: in the 50-ppm group, foci developed notably slower.
Changes in Morphology
Some enlarged and clear cell hepatocytes could be seen after feeding the animals for 2 weeks only with the 2 highest AAF concentrations. A few oval cells appeared in the periportal area.
At four weeks, liver morphology was already visibly altered. Clear cell hepatocytes and foci, acidophilic cells, as well as cells of the mixed type appeared. Many more oval cells proliferated in the periportal area and, depending on AAF concentration, individual cell necrosis and shrinkage were seen more often. In 2 of 4 animals receiving the 800-ppm diet, dystrophic and necrotic areas occurred widespread at 4 weeks. Due to these signs of toxicity, feeding was not continued in this group. Moreover, signs of chronic persistent hepatitis were noted in 2 of 4 animals of the 400-ppm group at 8 weeks, which may explain the low number of foci at that time point (Fig. 5).
At eight weeks, morphologic appearance was dominated by altered hepatocytes (clear cells, acidophilic cells, and cells of the mixed type) in all treatment groups. Oval cells and bile duct-like cells proliferated in the periportal area. Strands of connective tissue extended from one portal area to the next one, particularly in the 200- and 400-ppm groups.
At 16 weeks of AAF feeding, the histologic findings were even more impressive, again particularly in the 200- and 400-ppm groups. Marked oval cell and bile duct-like cell proliferation was observed in the periportal area. Strands of connective tissue often invaded the liver lobules giving the tissue a "cirrhosis-like" appearance. More details of histologic observations will be described elsewhere (Hadjiolov et al., in preparation).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This extended experiment was designed to demonstrate the development with time of very early effects in liver tumor production by AAF, to test the hypothesis that acute and chronic toxicity plays a major role in this process. The results clearly support this hypothesis. The experiments were also intended to show how these early events depend on the administered dose and to look for possible nonlinear effects or threshold phenomena. In this regard, the results are not as clear as expected.
The sequence of events can be assessed best if the time courses of the 3 different end points (cf. Figs. 3–5) are combined. Figure 6 shows a schematic representation. In animals fed the typical carcinogenic AAF concentration of 200 ppm, the number of apoptoses increases first, which indicates intolerable toxic stress to a significant number of cells, predominantly in the periportal area as mentioned above. With notable delay, increased cell proliferation can be seen. This proliferation essentially does not reflect the growth of initiated cells whose numbers are still extremely low at 4 weeks as measured by GST-P-positive cells and foci; rather it indicates the regenerative tissue response. This concept is clearly supported by the morphological observations. As also has been described previously (Hadjiolov et al., 1995), the enzyme-altered foci develop only later, after morphology has been altered.
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Essentially the same time pattern emerged in the next lower dose group fed 100-ppm AAF (Fig. 6
). The lowest dose (50 ppm) was not entirely nontoxic, as indicated by the smaller increase in body weight (Fig. 1). Accordingly, the increase of apoptoses indicates cellular stress, which must have been rather limited, since the proliferative response is comparatively low. Enzyme-altered foci develop much more gradually than with the higher doses (Fig. 6). Nevertheless, a no-effect level has not been reached for any of the endpoints. Most surprising, however, is how the different endpoints depend on dose. Whereas toxicokinetics, including metabolic activation, appear to be independent of dose, as indicated by the linear increase of hemoglobin adducts as a dosimeter, the liver tissue does not respond linearly with dose. With a 5-fold increase to 2–3 apoptoses/100 hepatocytes, a kind of maximum is reached with all doses. Higher levels are present after only 16 weeks, when about 4–5 apoptoses/100 cells are observed with all doses. This may be explained partly by our observation that 2-nitrosofluorene inhibits dose-dependent opening of the permeability pore in mitochondria, which is a key control device in apoptosis. Under chronic AAF exposure, the pore is regulated towards low-open probability, and inhibition is maximal only with 400 ppm AAF in ex vivo mitochondria (Bitsch et al. 1999, Klöhn et al., in preparation). We interpret this inhibition of apoptosis as an adaptive response, which may prevent excess cell elimination.
A first wave of dose-dependent proliferation is observed at 4 weeks (Fig. 4). Then, at eight weeks the rate of proliferation of cells appears to stabilize at a moderate level of 12 to 17 labeled cells/100 cells, with all doses. This unexpected observation could mean that the proliferative response reflects in some way the limited dose-independent cell elimination. A proliferative index above 20 is reached only with 400 ppm AAF at 16 weeks, at the time when histology shows that the tissue is already severely altered.
The number of GST-P-positive foci begins to increase notably at 8 weeks. At this time point, the effects depend on the administered dose, except with 400 ppm AAF (Fig. 5). As mentioned above, in this group 2 out of 4 animals had developed signs of chronic persistent hepatitis with many necroses, such that the observable number of foci was exceptionally low. At 16 weeks, the number of foci had increased to 200–300 foci/cm2, which occupy 20-30% of the area. The difference between the 3 higher doses, however, unexpectedly is not significant. The results confirm our notion that morphological changes precede the development of preneoplastic lesions but raise questions about the dose dependence of this process. It seems that the development of foci reflects the growth stimuli from the surrounding tissue rather than the potential for autonomous growth of the initiated cells. Nevertheless, we would have expected that the number of promotable lesions would increase with dose, which should have become apparent by 16 weeks of feeding.
With 50 ppm, a no-effect level had not been reached for any of the endpoints. However, the effects were clearly less pronounced and the preneoplastic foci developed considerably less rapidly than with the higher concentrations. The morphological alterations of the tissue were almost negligible.
Hepatotoxic effects have often been considered to be involved in AAF rat liver-tumor formation. Usually, more complicated tumor models were applied using additional tumor initiators or partial hepatectomy (Peraino et al., 1971; Tatematsu et al., 1983; Tiwawech et al., 1991). It is therefore difficult to compare the results of those studies with ours. The study that comes closest is by Williams et al. (1998). Cumulative total exposures were 0.126, 0.42, and 1.26 mmol AAF/kg body weight, administered for 12 weeks by daily intragastric instillation to male Fischer rats. This treatment was followed by feeding 500-ppm phenobarbital for another 24 weeks. The 2 lower exposures did not increase cell proliferation, and it was concluded that tumor initiation and carcinogenicity are restricted to exposures that produce toxicity and compensatory cell proliferation. Below a threshold, in this case below 0.5 mmol AAF/kg body weight, DNA lesions are considered by these authors not to be fixed as mutations in significant excess to background lesions. These authors selected doses even lower than 50 ppm, because they had shown in previous studies that 50 ppm AAF in the diet induced a low frequency of GGT-positive foci in liver by 12 weeks in F344 rats, and some tumors (more than anticipated) after 76 weeks of feeding (Williams et al., 1991). Moreover, they had found that a cumulative dose of 2.0 mmol/kg administered by gavage for 8 weeks (corresponding to 200 ppm in the diet) was toxic, whereas 0.5 mmol/kg was not (Umemura et al., 1993).
With 50 ppm, we reached a cumulative dose of 0.43-mmol AAF/kg body weight within 4 weeks, and 0.48 mmol/kg with 100 ppm within 2 weeks. In both situations we see some, however small, increase of the proliferation index, but clearly an enhanced number of apoptotic cells, which indicates that cells affected by toxic effects are eliminated. Since the AAF exposures do not overlap sufficiently and the endpoints of toxicity are not the same, the 2 studies cannot be compared exactly, but may be used to discuss the interpretation of a threshold. Williams et al. (1998) base their argument on the notion that sufficient DNA damage must be generated to fix mutations in excess to background DNA lesions, which then produce an observable increase in initiated, i.e., promotable cells. This does not exclude linear DNA damage at low doses. Following a different concept, we wanted to demonstrate a non-linear dose dependence of the development of preneoplastic lesions based on non-genotoxic effects. Among the many cellular targets, mitochondria are particularly sensitive as a target for cytotoxic effects of 2-nitrosofluorene (Klöhn et al., 1998) and we find it attractive to determine their role in the process. The metabolite of AAF potently induces the mitochondrial permeability transition. Increased resistance of mitochondria to permeability transition may alter the threshold for apoptosis as a means of adaptation to chemical stress, and we proposed that the regulation of the permeability transition pore is altered by chronic AAF feeding (Klöhn et al., 1998). New results with regard to this issue will be discussed in more detail elsewhere (Klöhn et al., in preparation).
Although the exposures were possibly too high to see a threshold, our results do not exclude its existence, since toxicity was low and as a consequence, preneoplastic foci developed, notably delayed with feeding dosage of 50-ppm AAF. However, it is interesting to note that on the one hand apoptoses are already increased with this low exposure and on the other hand an intermediate level of up to 3 apoptoses/100 cells is not exceeded by feeding higher AAF doses over 8 weeks. We assume that the stress of mitochondria correlates with dose and cells are eliminated. But at the same time the mechanism which triggers apoptosis is inhibited dose dependently with increasing concentrations up to feedings of 400 ppm AAF, such that the number of apoptoses is kept within the observed limits.
The role of the selection of drug-resistant hepatocytes and remodeling of nodules (Tatematsu et al., 1983) can also be considered in the light of the present results. These authors say that nodules do not disappear by regression or replacement from the surrounding liver. Remodeling is considered a form of physiological adaptation to exposure to xenobiotics. This implies that most of the foci and nodules are characterized by the reversible expression of an adaptation program. Nevertheless, this adaptation should not be restricted to focal cells. In agreement with earlier results, the whole liver responds with numerous changes in enzyme activities, among others, not only related to the metabolism of xenobiotics but also to that of carbohydrates (Neumann et al., 1994). We show now that consequences of stress are observed at rather low exposures, which obviously are not restricted to resistant foci, because they do not exist at the early time points.
It is concluded that (1) The proliferation of initiated cells (defined as promotable cells) is largely determined by the cellular environment, such as morphologically altered liver. (2) The morphological alterations in rat liver result from imperfect regeneration from toxic effects. (3) Imperfect regeneration results from limitations in the possibilities to adapt to chemical stress. (4) If these limits are overwhelmed and morphology has changed to a certain extent, preneoplastic foci develop—at least much faster than without these changes.
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ACKNOWLEDGMENTS |
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The expert technical assistance of Elisabeth Rüb-Spiegel and Elisabeth Stein is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 172).
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NOTES |
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1 To whom correspondence should be addressed. Fax: 49 931 2013988. E-mail: neumann{at}toxi.uni-wuerzburg.de.
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