1 Department of Medical Biophysics,
2 Department of Laboratory Medicine and Pathobiology and
3 Department of Nutritional Sciences, University of Toronto, Toronto M5S 3E2, Canada
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Abstract |
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Abbreviations: 2-AAF, 2-acetylaminofluorene; AI, apoptotic index; BrdU, 5'-bromodeoxyuridine; Cop, Copenhagen; DEN, diethylnitrosamine; GST 7-7, glutathione S-transferase 7-7; HGF, hepatocyte growth factor; LI, labeling index; PBS, phosphate-buffered saline; PH, 2/3 partial hepatectomy; RH, resistant hepatocyte.
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Introduction |
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In the RH model of hepatocarcinogenesis, the initiating agent results in the formation of single cells and small foci of putative initiated hepatocytes (2,3). Phenotypic markers for these lesions include altered expression of a number of enzymes that can be detected histochemically, such as the placental form of glutathione S-transferase (GST 7-7) (4,5). The lesions are then promoted with three doses of 2-acetylaminofluorene (2-AAF) followed by a partial hepatectomy (PH) and then a fourth dose of 2-AAF (6). 2-AAF is mito-inhibitory to normal hepatocytes but does not inhibit a subpopulation of the GST 7-7-positive hepatocytes. The PH acts as a mitotic stimulus that results in the rapid growth of this subpopulation into larger GST 7-7-positive lesions, while the surrounding liver remains quiescent. Some lesions eventually develop into grossly visible nodules and, over months, tumors arise (6).
Two other phenomena that occur following the promotional stimulus of the RH protocol are remodeling (or redifferentiation) of lesions and the proliferation and migration of oval cells. Hepatocytes within remodeling lesions lose expression of GST 7-7 and other enzymes common to preneoplastic liver lesions and also revert to a more normal liver architecture (7,8). Remodeling occurs in most lesions over the months following promotion. Lesions that remain are one site within which hepatocellular carcinoma can develop. Proliferation and migration of oval cells occurs in rat liver following treatment with a variety of chemical compounds (9). Oval cells are thought by some to be bipotential cells that can differentiate into either bile duct cells or hepatocytes (1012). Oval cells have been proposed to play a role in this model of hepatocarcinogenesis since their proliferation and migration may be an attempt by the liver to regenerate under conditions where hepatocytes cannot divide, such as occurs during the RH protocol (13,14).
In addition to establishing the time course of resistance in Cop rats, in the present study we have also explored potential mechanisms of resistance. The proliferation of hepatocytes within lesions and in surrounding normal liver as well as apoptosis in lesions of both strains were examined. A lower proliferation rate and/or higher apoptotic rate in lesions of Cop rats could reduce their overall growth rate. We also determined whether Cop rats have a greater proportion of lesions undergoing remodeling and/or a diminished oval cell response compared with susceptible F344 rats.
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Materials and methods |
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Animals
Cop and F344 rats, purchased from Harlan SpragueDawley (Indianapolis, IN), were allowed to acclimatize for 1 week prior to the start of experiments at which time they were 78 weeks old. Food (Harlan Teklad, 6% fat, Madison, WI) and acidified water (pH 2.8) were provided ad libitum and a 12 h lightdark cycle was maintained automatically.
Animal treatments
Twenty-five Cop and 25 F344 rats were administered a single i.p. dose of 200 mg/kg DEN dissolved in 0.9% NaCl solution. All rats were then treated using a modified RH protocol (6). Briefly, 18 days after DEN, three daily gavages of 20 mg/kg 2-AAF in DMSO and corn oil (1:29, vol:vol) were given followed by a PH. A fourth 20 mg/kg dose of 2-AAF was given the day after PH. Groups of five rats from each strain were killed on days 2 and 4 post-PH, six rats from each strain on day 7, and four Cop and three F344 rats on day 14 post PH. Representative liver samples were fixed in 5% acetic acid in methanol. Using this regime we found that a number of rats of both strains became moribund around day 11 post-PH such that there were too few rats to make comparisons at our intended 21 day time point. Therefore, to obtain data at 21 days, a group of five Cop and eight F344 rats were treated as above except the fourth dose of 2-AAF was given 4 days after PH and was lowered to 5 mg/kg. This is a commonly used regime that has a promoting effect equivalent to the protocol that we used for the rats killed on days 214 (6). The broken line connecting points in Figures 2, 3, 4 and 7 indicates this change in protocol.
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Immunohistochemical staining
After fixing overnight, tissues were embedded in paraffin wax and 2 µm sections were prepared on microscope slides. Double immunohistochemical staining was carried out for GST 7-7 using X-Gal as a chromogen, and for BrdU using DAB as a chromogen, as described by Stinchcombe et al. (15). Briefly, following deparaffinization and rehydration, sections were blocked with 10% normal swine serum for 10 min, then rabbit anti-GST 7-7 antibody was applied (1:2000) overnight at 4°C. The following were then applied to the sections at room temperature, washing with PBS between steps: biotinylated swine anti-rabbit antibody at 1:500 for 2 h, streptavidin-ß-galactosidase complex at 1:200 for 2 h and X-Gal substrate for 1 h. All dilutions were made in 1% normal swine serum in PBS.
Sections were then dehydrated and placed in 1% hydrogen peroxide in methanol for 15 min to block endogenous peroxidase activity, rehydrated and incubated in 4 N HCl for 15 min followed by rinsing in water and then PBS. BrdU staining was carried out by blocking with 1% bovine serum albumin in PBS, applying primary antibody (1:200) at 4°C overnight, rinsing in PBS, and then applying secondary antibody conjugated to peroxidase (1:200) for 2 h at room temperature. Sections were washed again and DAB was used as a chromogen. Dilutions for BrdU staining were made in 0.1% bovine serum albumin in PBS. Sections were finally counterstained with hematoxylin and eosin following the method described by Stinchcombe et al. (15).
Analysis of stained sections
The areas occupied by GST 7-7-positive lesions in liver sections were measured using a morphometric analysis machine (Bioquant IV; Zeiss, Wetzler, Germany) and converted to the percent of liver volume occupied by lesions and the number of lesions per liver using the method of Enzmann et al. (16). Only lesions with radii >35 µm in diameter were included in the analysis (17,18). Lesions undergoing remodeling often had indistinct borders but their areas were measured in the same manner as non-remodeling lesions, tracing the outermost edge of the lesion.
Random microscope fields from all lobes were scored for BrdU-positive hepatocytes within GST 7-7-positive lesions. The labeling index (LI) in each rat was calculated as the percent of hepatocytes within lesions that were BrdU-positive after at least 1000 hepatocytes were counted per rat. The same procedure was used to obtain an LI for surrounding GST 7-7-negative hepatocytes.
The mean apoptotic indices (AI) within lesions of each group of rats were expressed as the percent of apoptotic cells after at least 2000 hepatocytes within lesions had been counted per rat. Lesions in F344 rats on days 14 and 21 post-PH were very large, so at least 5000 hepatocytes within lesions were examined in these livers. Apoptotic cells within lesions were identified using the property of eosin fluorescence of apoptotic bodies (15) in random microscope fields, and their identity was confirmed using normal transmitted light.
Lesions undergoing remodeling or redifferentiation were identified by their patchy GST 7-7 staining and indistinct borders (7). The extent of remodeling in each rat was expressed as the percent of all lesions in a section undergoing remodeling. Oval cells were identified by their characteristic ovoid nucleus and scant cytoplasm (9).
Since individual lesions may have differences in LI or AI that could influence their growth rate, we measured these parameters as well as the extent of remodeling in 20 randomly selected individual lesions in each rat.
Statistical tests
Results were analyzed using a 2-way ANOVA, and a Tukey post-test was used to compare one group of animals to another. P-values <0.05 were considered significant.
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Results |
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Percent liver volume occupied by GST 7-7-positive lesions
Following immunohistochemical analysis for GST 7-7-positive lesions, no difference in the percent of liver volume occupied by lesions was seen between strains until day 14, when a clear difference between the Cop and F344 rats became evident (Figure 3). This difference was even greater on day 21.
BrdU labeling index
To estimate cell proliferation rates, BrdU was injected into rats 2 h before killing. The labeling indices of hepatocytes within GST 7-7-positive lesions and in the surrounding normal parenchyma are shown in Figure 4. There were no differences in hepatocyte labeling indices between rat strains, either within lesions or surrounding normal liver, at any time point during the experiment. The intestinal epithelial cells of the duodenum used as a positive control had a high LI in all of the rats (data not shown).
Apoptotic index
Apoptotic cells within lesions were identified using eosin fluorescence of apoptotic bodies followed by confirmation using transmitted light. The apoptotic indices were not significantly different between the strains at any time point (Figure 5).
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The percent of lesions undergoing remodeling was not significantly different between the strains on days 4 and 7 post-PH. On days 14 and 21, however, liver sections from Cop rats appeared to contain many more remodeling lesions than F344 rats (Figure 6F and H versus E and G). When this was quantified it became apparent that remodeling in Cop rats was significantly more extensive than in F344s (Figure 7
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Oval cell response
Figure 6 shows the pattern of oval cell response from day 4 to day 21 post-PH. On day 4 post-PH, in both strains, there is a small amount of oval cell proliferation with little or no migration of these cells (Figure 6A and B
). On days 7 and 14, however, F344 livers had extensive infiltration of oval cells from the portal areas into the liver parenchyma (Figure 6C and E
), whereas in Cop livers this response remained localized to the portal areas (Figure 6D and F
). On day 21, much of the liver volume was occupied by nodules in F344 livers, and the surrounding parenchyma had fewer oval and duct-like cells than on day 14 (Figure 6G
). In Cop livers the oval cell response appeared to reach a maximum around day 7 and declined thereafter to levels similar to day 4.
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Discussion |
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The number of lesions per liver was the same in Cop and F344 rats on days 2 and 4 post-PH, but on days 7, 14 and 21, Cop rats had more lesions per liver than F344 rats (Figure 2). Cop rats may indeed have more lesions per liver at these time points, but it is possible that the stereological method we used may have produced incorrect estimates of this parameter. In particular, on days 14 and 21 post-PH, F344 livers contained large lesions that were coalescing, such that several lesions may have been counted as one. This would lead to an underestimation of the number of individual lesions present in F344 rats. Regardless of the possible errors in the estimates of lesions per liver, Cop rats are clearly not resistant to the initiation of lesions and may actually be more susceptible to initiation than F344 rats.
The percent of the liver volume occupied by GST 7-7-positive lesions was the same in both strains up to day 7 post-PH (Figure 3). On day 14, however, the two strains diverged such that Cop rats showed no significant increase in percent volume occupied by lesions compared with day 7, while F344 rats had ~8-fold more liver occupied by lesions on day 14 than day 7. By day 21 post-PH, the percent of liver volume occupied by lesions in Cop rats had not changed compared with day 14, whereas F344 liver lesions continued to grow, approximately doubling the percentage of liver volume the lesions occupied on day 14.
The overall rate of lesion growth during promotion is influenced by the rate of cell division and the rate of cell loss (19,20). During the RH protocol, GST 7-7-positive hepatocytes can be lost either through cell death or through loss of GST 7-7 expression (remodeling) (21). We investigated these factors in both strains by measuring the BrdU LI of lesions and surrounding liver, the AI in lesions and the extent of lesion remodeling.
In order for liver lesions to grow rapidly during the RH protocol, 2-AAF must have a mito-inhibitory effect on normal hepatocytes. The PH then provides a potent proliferative stimulus to which only the `resistant' hepatocytes that make up the lesions respond (22,23). Previously, we have shown that the degree of 2-AAF-induced mito-inhibition of normal hepatocytes following a PH is the same in Cop and F344 rats (1). The current study corroborates this finding by showing that surrounding normal hepatocytes have the same LI in both strains at all time points examined (Figure 4). It is also evident from Figure 4
that GST 7-7-positive hepatocytes are resistant to 2-AAF mito-inhibition to the same extent in both strains. These `resistant' hepatocytes had the same high LI in both strains even on day 2 post-PH and maintained this growth advantage over surrounding hepatocytes to the same extent in both strains at least until day 7. By day 14 post-PH, there was no growth advantage for GST 7-7-positive hepatocytes in either strain.
Another factor influencing lesion growth is the rate of cell death. Some promoting agents, such as phenobarbital (24,25) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (15) are thought to act, at least in part, by inhibiting apoptosis. Dietary restriction and S-adenosyl-L-methionine are both potent inhibitors of carcinogenesis and at least some of their anti-carcinogenic effects appear to be mediated by an increase in apoptosis within liver lesions (21,26). Although we used the same methods as the above studies to quantify apoptotic cells, we were unable to show a higher AI in Cop compared with F344 lesions.
There are several shortcomings of the methods used to quantify apoptosis in the liver. The popular TUNEL method has been shown to label cells undergoing necrosis as well as apoptosis and also does not detect apoptotic bodies that lack chromatin (27). The technique we have used that detects apoptotic bodies by eosin fluorescence, takes advantage of the eosinophilia of apoptotic bodies in the liver and is thought to increase the sensitivity and speed of detection (15). It is also relatively easy to check that a fluorescent structure represents an apoptotic body by switching to normal transmitted light to confirm its identity. Detection of apoptotic cells by any method, however, has a limited window of time in which these cells are visible. Using the gold standard of morphology, apoptotic cells are only visible for ~3h in the liver before they are completely degraded by other cells (25). Given these limitations, it is possible that a small difference in AI between lesions in Cop and F344 rats may be undetectable. A small difference maintained over a long period of time, however, could potentially result in a significant difference in the growth rate of lesions in the two strains.
Another factor that might explain the difference in lesion growth between the strains is the proportion of lesions that are resistant to 2-AAF mito-inhibition. Such resistant lesions have previously been defined as having a higher LI than the highest LI in the surrounding liver (28). Using this criterion, we found no differences between the strains in the percent of lesions that were resistant on days 4, 7 or 14 post-PH (data not shown).
Remodeling or redifferentiation has been characterized in the RH model of hepatocarcinogenesis (7). Typically, 95% of hepatic lesions induced by the RH protocol eventually undergo remodeling (8). Our results show that a significantly higher percent of lesions in Cop rats undergo remodeling early after PH compared with F344s, the difference becoming significant between 7 and 14 days post-PH (Figure 7
). This precocious remodeling could account for the small volume of liver occupied by GST 7-7-positive hepatocytes in the Cop rats following the RH protocol, even though the proliferative and apoptotic indices in the lesions of the two strains were indistinguishable. Brown Norway rats that are also known to be resistant to hepatocarcinogenesis, likewise also have a greater proportion of their liver lesions remodeling compared with F344 rats following an RH protocol (18). Cop and Brown Norway rats may, therefore, share a common mechanism of resistance.
Other groups have found a lower LI and a higher AI in remodeling compared with non-remodeling lesions (19,21). There were, however, no such differences in our animals. A possible explanation is that we examined lesions very early after the PH in the RH protocol when the promotional stimulus was still present, while the other studies examined lesions many weeks after PH. Furthermore, in our study, almost all of the remodeling lesions were in Cop rats and the process by which these lesions remodel may be very different from that in F344 rats.
2-AAF administration followed by PH in rats is known to stimulate proliferation and migration of small duct-like epithelial cells, called oval cells (9), that originate in portal areas and express GST 7-7 (29). On day 7 post-PH in F344 rats, oval cells can be found invading far into the parenchyma and on day 14 surround GST 7-7-positive lesions (Figure 6C and E). In contrast, although we showed the presence of oval cells in the portal regions of Cop livers, these cells failed to migrate and remained localized to the periportal areas (Figure 6D and F
). The process of oval cell proliferation and migration is associated with the production of several growth factors including hepatocyte growth factor (HGF), transforming growth factor
, acidic fibroblast growth factor and stem cell factor (30,31), and the production of urokinase-type plasminogen activator (32,33) which can cleave pro-HGF to produce active HGF (34,35). Although the role of oval cells in hepatocarcinogenesis is currently being debated (13,14,36,37), local production of growth factors associated with the oval cell response could contribute to the growth of preneoplastic lesions. Thus, the weak oval cell response that we observed in Cop rats may play a role in their resistance. This is the first report, to our knowledge, where 2-AAF/PH treatment resulted in mito-inhibition without an extensive oval cell response. Cop rats appear to be unique in this respect and thus provide a model to study the potential independence of these events in the RH protocol.
In summary, we have shown that Cop rats form similar numbers of GST 7-7-positive lesions as F344 rats following DEN initiation. However, Cop rats are resistant to the promotion of growth of these lesions using a modified RH protocol. The proliferative rates within lesions were indistinguishable in Cop and F344 rats, as were the proliferative rates in the surrounding normal hepatocytes. The apoptotic indices within lesions were also not different between strains. Two characteristics of Cop rats that were different from F344s were precocious remodeling of liver lesions and a diminished oval cell response. These differences can potentially account for the resistance of Cop rats to the growth of GST 7-7-positive hepatic lesions.
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Acknowledgments |
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Notes |
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References |
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