Japanese Journal of Clinical Oncology Advance Access published online on August 16, 2008
Japanese Journal of Clinical Oncology, doi:10.1093/jjco/hyn080
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© The Author (2008). Published by Oxford University Press. All rights reserved
Bile Duct-bound Growth of Precursor Cells of Preneoplastic Foci Inducible in the Initiation Stage of Rat Chemical Hepatocarcinogenesis by 2-Acetylaminofluorene
1 Department of Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori
2 Department of Signal Transduction, Research Institute of Microbial Disease, Osaka University, Osaka
3 Department of Neuropharmacology, Hokkaido University Graduate School of Medicine, Sapporo
4 Department of Pathologic Analysis, Aomori
5 Department of Pharmaceutical Science, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori, Japan
For reprints and all correspondence: Kimihiko Satoh, Department of Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, Hon-Cho 66-1, Hirosaki 036-8564, Japan. E-mail: kisatoh{at}cc.hirosaki-u.ac.jp
Received April 7, 2008; accepted July 22, 2008
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Abstract |
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Background: We previously detected precursor cell populations of preneoplastic foci, GST-P+/GGT– and GST-P+/GGT+ minifoci, in rat liver in the initiation stage of chemical hepatocarcinogenesis, where GST-P and GGT represent glutathione S-transferase P-form and
-glutamyltranspeptidase, respectively. Methods: Sprague–Dawley male rats were fed a basal diet containing 2-acetylaminofluorene (0.02%) over 16 weeks. Precursor cells were detected by our sensitive staining method for GGT activity and immunocytochemical staining for GST-P.
Results: GST-P+/GGT– single cells were overproduced maximally in the animal liver after the 6 weeks followed by a gradual growth of GST-P+/GGT– and GST-P+/GGT+ minifoci, which were bound to bile ducts and ductules. GGT was expressed within GST-P+ minifoci gradually with time forming GGT+ lane-like structures. The bile duct binding and lane-like structure formation were prominent especially when minifoci-bearing rats were subjected to two-thirds partial hepatectomy.
Conclusions: A variety of precursor minifoci were noted to be selectively bound to bile ducts and ductules in rat liver, which may be of physiologic significance in excretion of carcinogens during initiation.
Key Words: hepatocarcinogenesis • tumor marker enzyme • -glutamyltranspeptidase • glutathione S-transferase • 2-acetylaminofluorene
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INTRODUCTION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFundingReferences |
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The molecular and cellular mechanisms of cancer initiation require elucidation in the basic cancer research and clinical oncology (1–3). One of the principal difficulties in solving this fundamental problem is the lack of appropriate approaches for detecting the initial carcinogenic changes involved in normal cells in vivo. However, preneoplastic and neoplastic marker enzymes such as glutathione S-transferase P-form (GST-P, EC 2.5.1.1 [EC] 8) and
-glutamyltranspeptidase (GGT, EC 2.3.2.2) may be of use in addressing to this problem (4–7). We have previously identified two precursor cells of preneoplastic foci, GST-P+/GGT– and GST-P+/GGT+ phenotypic minifoci, in rat liver in the chemical hepatocarcinogenesis protocol according to Solt and Farber, using sensitive staining method for GGT activity in combination with an immunocytochemical staining method for GST-P (8). GGT expression was noted to be confined to GST-P+ precursor minifoci that are induced in the periportal area (zone 1) of the liver. In addition, GGT expression levels differed from one minifocus to another, and GST-P+/GGT+ phenotypic minifoci and foci were much more proliferative than GST-P+/GGT– phenotypic ones. Given the paucity of data on precursor cell populations and the complexity of the Solt–Farber protocol (9), we report here the results from our investigation on a precursor cell induction process by 2-acetylaminofluorene (AAF).
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MATERIALS AND METHODS |
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Chemicals
L-Glutamic acid
-(4-methoxy-2-naphthylamide) (GMNA) was purchased from Sigma–Aldrich (Tokyo, Japan). Affinity-purified anti-GST-P antibody (rabbit) was obtained from Medical and Biologic Laboratories (Nagoya, Japan).
Animals and Treatments
Sprague–Dawley male rats (5-week old) were purchased from Clea (Tokyo, Japan) and maintained in the Institute for Animal Experiments of Hirosaki University. All animal experiments were conducted according to the guidelines for animal experimentation of Hirosaki University. A total of 62 rats were fed a basal diet containing AAF (0.02%), and groups of 4–6 animals were killed after 2, 4, 5, 6, 7, 8, 9, 10, 12 and 16 weeks. Rat liver lobes were excised, cut into 3–4 mm thick slices and fixed with cold acetone. The fixative was replaced once after 2 or 3 days.
Measurement of Precursor Cell Populations
Liver slices were sectioned into 25 µm thick sections using a microslicer (Vibratome 1500 sectioning system; Vibratome Product, NY, USA) in PBS. GGT-activity staining was performed with GMNA as substrate (10), and immunocytochemical staining for GST-P was performed as described previously (7). Microphotographs were taken with either a microscope (Axioskop, Carl Zeiss, Oberkochen, Germany) equipped with a digital camera or a digital microscope (Coolscope, Nikon, Tokyo, Japan), in which an LED (light emitting diode) was employed as the light source. It was necessary with Axioskop, but not with the Coolscope, to take photographs within 10–20 s of exposure in strong lighting to avoid drying and deformation of specimens. Despite this disadvantage, the Axioskop microscope was superior to the Coolscope in resolution. Measurement of cell composition and size classification were performed using NIH image software version 1.62 as previously reported (8).
Statistical Analysis
Statistical analysis was performed using the Student's t-test. Data are expressed as mean ± SD where indicated.
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RESULTS |
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Macroscopic Induction Processes of Precursor Cells
Precursor cell populations of preneoplastic foci induced in rat liver were examined quantitatively and qualitatively according to the protocol shown schematically in Fig. 1A. Animals of 6 weeks of age (0-week feeding) were fed a basal diet containing 0.02% AAF for 16 weeks. A number of GST-P+/GGT– and GST-P+/GGT+ minifoci were macroscopically detectable
6 weeks after administration of AAF, and these rapidly and progressively increased in size and density up to 16 weeks (Fig. 1B). When some animals were subjected to two-thirds partial hepatectomy (PH) at Week 6, large foci were rapidly induced 7 days later as compared with a non-operated control fed the indicated diet for 7 weeks (Fig. 1B, 6W & PH3d, 6W & PH7d and 7W). GST-P+ single cells and minifoci were hardly detectable in the liver of control animals fed a basal diet for 16 weeks. Although GGT+ and GST-P+ minifoci and foci appeared to correspond well with each other macroscopically, there were significant microscopic differences. As described in the following sections, a number of precursor cells were microscopically detectable 2–6 weeks after the administration of AAF. The induction process was thus tentatively divided into latent and logarithmic growth phases before and after 6 weeks, respectively.
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Size Distribution of GST-P+ and GGT+ Cells During the Induction Process
The size distribution of GST-P+ and GGT+ cell populations induced in rat livers have revealed that the induction processes of GST-P+ single cells and minifoci were fairly different. A number of GST-P+(/GGT–) single cells were detectable 2–3 weeks after administration of AAF and reached a maximum of 396 ± 168 cells/cm2 in liver after 6 weeks (n = 5), followed by a rapid decrease from 8 to 16 weeks (Fig. 2A). Single cells were selectively induced in the periportal area of zone 1 at an average rate of 64–85% of a total of 3540 cells when examined from 2 to 8 weeks (Table 1). GST-P+ minifoci were much smaller than the single cells in number, but gradually increased with time. According to the stereological method of Campbell et al. (12), the number of GST-P+ single cells and the total number of GST-P+ minifoci other than the single cells induced after 6 weeks were approximated to be (1.86 ± 0.79) x 105/cm3 and (9.8 ± 6.1) x 103/cm3, respectively. Thus, the number of single cells was
20-fold greater than the number of whole minifoci at 6 weeks after exposure to AAF (*, P < 0.005). The number of minifoci composed of 11–50 cells induced after 8 weeks (**, 110 ± 55/cm2) appeared to be higher than that induced after 10 and 12 weeks, but was not statistically significant. This may be due to the fact that smaller minifoci were obscured by larger foci and nodules predominated with time. In contrast, the induction process of GGT+ cell populations was fairly different from that of GST-P+ cell populations (Fig. 2B). First, GGT+ single hepatocytes were practically undetectable during the latent and logarithmic growth phases in the animal livers examined. Second, during our examination, the numbers of GGT+(/GST-P+) minifoci composed of 2–10 or 11–50 cells were much smaller than those of corresponding GST-P+ single cells up to 6 weeks. Third, the number of GGT+ minifoci and foci increased rapidly after 8 weeks with continued AAF administration and became comparable to corresponding GST-P+ cells after 12–16 weeks. The number of GGT+ minifoci composed of 11–50 cells induced after 10 weeks (
) appeared to be greater than those induced after 12 and 16 weeks, but not significant. Although GGT activity within precursor minifoci was generally very low and comparable with the activity in bile duct and ductular cells non-specifically induced in the animal liver at the latent phase, the enzymatic activity expressed within minifoci and foci was much higher than that observed in non-specifically induced bile epithelial cells during the logarithmic growth phase. Neither GST-P+ single cells nor GST-P+ minifoci were detectable in practice in the liver of control animals fed a basal diet alone for up to 16 weeks as reported previously (11).
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Bile Duct-Bound Growth of Minifoci Forming Lane-like Structures
Various types of precursor minifoci were microscopically detected in the animal liver during the latent phase of 4–6 weeks (Fig. 3). GST-P+ minifoci were noted to be induced in the periportal area of liver lobules (Fig. 3A, GST-P stain,
, low magnification). GST-P+ single cells (Fig. 3B, GST-P stain, , high magnification) were negative for GGT (Fig. 3B, GGT stain), whereas bile ducts and ductules (BD) were non-specifically positive under the conditions examined. Minifocus 
'/ is typical of the GST-P+/GGT– phenotype, which was observed to be composed of 55 GST-P+ cells (Fig. 3C). Minifocus β'/β is typical of the GST-P+/GGT+ minifoci, in which GGT is partially expressed [32% = (28/88 cells) x 100] and is adjacent to the bile duct (Fig. 3D, arrowhead). Direct binding is, therefore, the most prominent with the minifocus β'/β. GGT+ lane-like structures were noted to be formed within minifoci '/ and 
'/ that grew alongside the periphery of liver lobules (Fig. 3E and F). GGT was almost fully expressed in minifoci '/ and '/ whose relative areas were 87.5 and 104%, respectively. Relatively fine and coarse networks were seen in the mirror images of GGT staining of the two minifoci. The minifocus '/-bound bile duct was noted to be full of brownish bile juice, which is indicative of enhanced biliary excretion of carcinogens when animals were fed AAF. In the mirror images of the three minifoci (Fig. 3G), tangled lane-like networks were observed in minifocus 
'/ in the direction of bile ducts, and a lane-like structure is seen within a minifocus 
'/. The intercellular networks formed within minifoci '/ and '/ could thus be ‘nascent’ networks that give rise to mature ones such as in minifoci '/ and '/.
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Effect of PH on the Growth and Structure of Minifoci
When rats were subjected to two-thirds PH at 6 weeks, marked changes were observed in both minifoci and foci. Three days post-operation, GST-P and GGT expression was greatly enhanced and the membrane domains of GGT+ foci
'(/) and β'(/β) were heavily positive for GGT and appeared as striped bands (Fig. 4, 6W & PH3d1 and 3d2), both of which were bound to GGT+ bile ducts and ductules at multiple sites as indicated by arrows (). Morphologic changes were much more prominent 7 days later. Tree-like structures were seen in GGT+ focus '(/), whose relative area was 68% GST-P+ focus and through which the large bile duct was deeply invaginated in the focus (Fig. 4, 6W & PH7d1). Finely branched structures were seen in a large focus '(/) (Fig. 4, 6W & PH7d2). Distinct networks were, however, rather unclear in foci '(/) and ’(/), which were induced in the liver of non-operated control animals fed AAF alone for 7 weeks (Fig. 4, 7W1 and 7W2). Overall GGT expression was much lower in the control animals compared with operated ones. Ordered structures were less obvious when animals were administered CCl4 through gavage or fed a sublethal dose of AAF (0.04%) or phenobarbital (0.05%) in their basal diet (data not shown). PH was found to be the most effective at inducing the formation of finely ordered structures within minifoci from among the carcinogenic stimuli tested.
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DISCUSSION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFundingReferences |
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Inasmuch as precursor cell populations of preneoplastic foci are readily detectable in rat liver aside from minor technical difficulties, it is of interest to gain insight into the molecular and cellular mechanisms of initiation of rat chemical hepatocarcinogenesis. Rats were, therefore, fed a basal diet containing AAF (0.02%) alone over 16 weeks in the present study, as it is a complete carcinogen (13–15). As compared with the Solt–Farber carcinogenesis protocol (8), the induction process of precursor cells was generally very slow allowing us to observe them in more detail. Quantitative and qualitative analyses have revealed that GST-P+/GGT– single hepatocytes were overproduced prior to the induction of precursor minifoci. It was noteworthy that minifoci were selectively bound to bile ducts forming lane-like structures intra-minifocally.
Overproduction of GST-P+/GGT– Single Cells in Rat Liver
As many as 186 000 ± 7900 GST-P+/GGT– single hepatocytes per gram of liver were estimated to be induced prior to the induction of minifoci after 6 weeks of AAF exposure. This value is one order of magnitude greater than 9150 ± 4190 cells/g of liver induced by N,N'-diethylnitrosamine (11), or 13 500 cells/g of liver induced by N-ethyl-N'-niroso-morpholine (16). Despite the differences in the degree and the time of maximal induction according to the carcinogens, the rapid rise-and-fall of GST-P+/GGT– single cells is suggestive of their common short half-life times of 1–2 weeks. Thus, single cells appear to be much weaker and more susceptible to cell death than normal hepatocytes whose half-life times are known to be 100–200 days. The drastic rise-and-fall of single cells are therefore implicating that almost all cells are destined for death except for a few induced in the periportal area of the animal liver. However, it is at present difficult to discriminate which single cells are proliferating and which are necrotic.
Bile Duct-Bound Growth of Minifoci Forming Lane-Like Structures
In contrast to the overproduction of single cells, minifoci were increased gradually with time accompanied by the expression of GGT extra- and intra-minifocally. Various types of minifoci were detectable under the microscope especially 4–6 weeks after AAF administration (Fig. 3). Most of the minifoci were selectively bound to bile ducts in accord with our previous observation in the carcinogenesis protocol according to Solt and Farber (8). GGT was expressed gradually and heterogeneously within GST-P+ minifoci at levels ranging from no (GST-P+/GGT– minifocus) to partial or full (GST-P+/GGT+ minifocus) expression forming lane-like structures therein. Extra- and intra-structural changes were better exemplified in minifoci and foci when animals were subjected to two-thirds hepatectomy (Fig. 4). Three days post-operation, foci were noted to be bound to bile ducts and ductules at multiple sites. GGT+ tree-like structures or branched lane-like structures were developed within foci 7 days later. After PH, carcinogen-containing blood would flow at least 3-fold faster than before in the residual right liver lobe of the animal. Responding to the drastic increase in carcinogenic stimuli, GGT and GST-P expression was greatly enhanced to form finely branched lane-like structures within minifoci and foci. In some large foci, bile ducts were integrated or invaginated deeply. Thus, a variety of minifoci and foci were observed to be tightly bound to bile ducts and ductules. As is well known, bile is a major route of excretion for drugs and carcinogens, and bile ducts proliferate excessively in response to the administration of these compounds (17). Irving et al. (18) demonstrated that a large fraction of AAF was excreted in bile through glucuronic acid conjugation. In addition, Fardel et al. (19) have shown that the ATP-binding cassette transporter family, P-glycoprotein, multidrug resistance-associated protein 2 and bile salt export pump (BSEP) were upregulated by AAF in rat liver. It may, therefore, be of prime importance for minifoci and foci to bind to bile ducts and ductules to excrete toxic compounds efficiently.
As a matter of course, the cellular origin(s) of preneoplastic and neoplastic cells remains unclear as yet. Farber and Cameron (1) and Farber (20) mentioned briefly that carcinomas appeared to develop within hyperplastic liver nodules, which are surrounded by bile ducts occasionally. Since nodules are very large, it is uncertain whether they selectively bind to bile ducts and ductules or they are simply compressed within the complicated histology of preneoplastic and neoplastic tissues. In contrast, most minifoci were noted to be directly bound to bile ducts and ductules, forming lane-like structures therein when examined by our sensitive method. Compression could rationally be ruled out with the small cell populations, and histological changes involved in the early stages were much simpler than those involved in the later stages of chemical carcinogenesis. These structural features are therefore indicative that minifoci and foci are advantageous for the efficient excretion of carcinogens. Rat hepatic preneoplastic foci and nodules are known to be ‘resistant cells’ against carcinogens including AAF, a strong mito-inhibitor to hepatocytes (21,22). The resistance mechanism has been regarded to be due, in principle, to the activation of phase II detoxification enzymes (20–23). Thus, it is of interest to note that minifoci and foci have additional advantages in their structure/morphology for excretion of carcinogens.
Enzymatic Roles of GGT and GST-P During Initiation
The physiological roles of the preneoplastic and neoplastic marker enzymes GST-P and GGT are of particular importance during initiation. As noted previously in detail, GST-P is a measure of carcinogens accumulated intracellularly, as it could be a binding protein for endogenous and exogenous carcinogens (24). In other words, single cells and minifoci are those that suffer from the accumulation of toxic compounds intracellularly up to lethal levels. On the other hand, GGT is a measure of the excretory potency of carcinogens for minifoci and foci. In fact, GGT is weakly expressed in the epithelial cells of bile ducts and ductules in the normal liver and is induced by many drugs and carcinogens as a result of bile duct proliferation. GGT is thus localized to the excretory pathways of drugs and carcinogens (25,26). As GGT accounts for the selective binding of minifoci and foci to bile ducts and ductules together with the development of fine and ordered networks therein, it may be of prime importance for survival and growth of premalignant cells to excrete carcinogenic compounds as soon as possible.
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Funding |
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This work was supported by grants from Nippon Boehringer-Ingelheim Co. Ltd (Osaka); Hirosaki University Fund for Promotion of International Scientific Research (Hirosaki, Japan) and The Foundation for Promotion of Cancer Research in Japan.
Conflict of interest statement
None declared.
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References |
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