Fumonisin B1-induced hepatocellular and cholangiocellular tumors in male Fischer 344 rats: potentiating effects of 2-acetylaminofluorene on oval cell proliferation and neoplastic development in a discontinued feeding study
Eric R. Lemmer1,5,
Carina J. Vessey2,
Wentzel C. A. Gelderblom3,6,
Enid G. Shephard1,
Dirk J. Van Schalkwyk4,
Rochelle A. Van Wijk2,
Walter F. O. Marasas3,
Ralph E. Kirsch1 and
Pauline de la M. Hall2
1 MRC/UCT Liver Research Center and 2 Department of Anatomical Pathology, University of Cape Town, Observatory, Cape Town, South Africa, 3 Program on Mycotoxins and Experimental Carcinogenesis (PROMEC), Medical Research Council, Tygerberg, South Africa and 4 Business Informatics, Cape Technicon, Cape Town, South Africa
5 Present address: Laboratory of Experimental Carcinogenesis, National Cancer Institute, Building 37, Room 4146A, 37 Convent Drive, Bethesda, MD 20892, USA
6 To whom correspondence should be addressed Email: wentzel.gelderblom{at}mrc.ca.za
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Abstract
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Fumonisin B1 (FB1) is a naturally occurring mycotoxin produced by Fusarium verticillioides. Dietary exposure to FB1 has been linked to human cancer in certain parts of the world, and treatment with FB1 causes oval cell proliferation and liver tumors in rats. To study the potential role of oval (liver progenitor) cells in the cellular pathogenesis of FB1-induced liver tumors, we gave male F344 rats prolonged treatment with FB1 for 25 weeks, followed by return to control diet until 50 weeks (stop study). The time course of FB1-induced liver lesions was followed by examination of serial liver biopsies at set time intervals and post-mortem liver tissue at the end of the study. The effects of different FB1 treatment regimens (5 versus 25 weeks), as well as the modulating effect of 2-acetylaminofluorene (2-AAF), on the kinetics of oval cell proliferation and development of liver tumors were compared. Prolonged treatment with FB1 in normal diet caused persistent oval cell proliferation and generation of both hepatic adenomas and cholangiofibromas (CFs). These liver lesions occurred in the setting of chronic toxic hepatitis and liver fibrosis/cirrhosis, similar to that seen in human hepatocarcinogenesis. Some adenomas and CFs were dysplastic, and one post-mortem liver contained a hepatocellular carcinoma. OV-6+ oval cells were noted in close relation to proliferative neoplastic liver lesions, and some of these lesions expressed OV-6, suggesting that all these cell types were derived from a common progenitor cell. 2-AAF enhanced the size of FB1-induced glutathione S-transferase pi+ hepatocellular lesions and the incidence of CFs in post-mortem liver specimens, but this was not statistically significant. In conclusion, this study supports the involvement of dietary FB1 in liver carcinogenesis in male F344 rats. Oval cells may be the source of both the hepatocellular and cholangiocellular tumors induced by prolonged treatment with FB1. 2-AAF appears to have an enhancing effect on FB1-induced liver tumors, presumably due to its potent inhibitory effects on hepatocyte regeneration.
Abbreviations: 2-AAF, 2-acetylaminofluorene; CF, cholangiofibroma; FAH, foci of altered hepatocytes; FB1, fumonisin B1; GSTP, glutathione S-transferase pi; HA, hepatocellular adenoma; HCC, hepatocellular carcinoma; H&E, hematoxylineosin; LTFB, long-term fumonisin B1 treatment; LTFB/AAF, long-term fumonisin B1 treatment plus 2-AAF; STFB, short-term fumonisin B1 treatment; STFB/AAF, short-term fumonisin B1 treatment plus 2-AAF
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Introduction
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Fumonisin B1 (FB1) is a food-borne mycotoxin produced by the fungus Fusarium verticillioides (previously Fusarium moniliforme) (1) that occurs worldwide and causes a variety of naturally occurring toxicoses in animals, including fatal illnesses in horses (2) and pigs (3). Human dietary consumption of Fusarium-contaminated corn products has been linked epidemiologically to increased rates of esophageal cancer (4,5), and perhaps hepatocellular carcinoma (HCC) (6), in regions of the world in which corn is the staple grain, such as South Africa and China. FB1 is hepatotoxic and hepatocarcinogenic in rats. Short-term feeding with FB1 causes severe toxic hepatitis (1), while continued FB1 administration leads to a chronic toxic hepatitis and fibrosis, which progresses to cirrhosis, and sometimes terminates in HCC or cholangiocarcinoma (7). FB1 appears to be a unique carcinogen that causes regenerative hyperplasia of hepatocytes and promotion of tumors despite striking pro-apoptotic effects (8,9). The molecular mechanisms underlying FB1-induced hepatocarcinogenesis are not known, but recent data implicate the activation of specific apoptotic and oncogenic pathways, including the TGF-ß pathway (10) and the Akt/cyclinD1 pathway (11).
The cellular origin of HCCs induced by FB1 is unclear, and most studies have focused on the role of the mature hepatocyte (9). However, we recently showed that short-term treatment of rats with FB1 resulted in early proliferation of oval cells, which coincided with the appearance of foci of altered hepatocytes (FAH) (10). Microscopically, oval cells are small cells with elongated nuclei and scanty cytoplasm (12), which appear to arise from cells in the terminal bile ductules (canals of Hering) or from periductular cells (13). Proliferating oval cells have been noted to appear early in several experimental carcinogenesis protocols, and these cells may represent the progeny of a liver stem cell (14). Oval cells are bipotential and can differentiate into either hepatocytes or bile duct epithelial cells, and they have been shown to have clonogenic potential in vivo and in vitro. Oval cells are not normally involved in liver regeneration, due to the enormous replicative capacity of hepatocytes, but are induced to proliferate under conditions of severe liver injury when mature hepatocytes are overwhelmed or prevented from proliferating (14). There is now good evidence that bipotential ductular progenitor cells (oval cells) may give rise to HCCs under certain experimental conditions (15).
In order to study the potential role of oval cell proliferation in the generation of FB1-induced proliferative and neoplastic liver lesions, male F344 rats were treated with FB1 for 25 weeks, followed by return to control diet (stop study). Different FB1-containing treatment regimens (5 versus 25 weeks) were compared, and the modulating effect of 2-acetylaminofluorene (2-AAF) on FB1-induced oval cell proliferation and tumorigenesis was also assessed. The sequential development of hepatic histopathologic lesions in the different treatment groups was monitored by examination of serial liver biopsies as well as post-mortem liver tissue.
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Materials and methods
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Chemicals
FB1 was purified from corn cultures of F.verticillioides strain MRC 826 according to a method described previously (16). The purity as compared with an analytical standard by high-performance liquid chromatography was in the order of 9295% (24). The monomethylester derivatives of FB1, which are artifacts of the purification procedure, constituted the remainder of the FB1 preparation. 2-AAF stock powder was purchased from Sigma-Aldrich (St Louis, MO).
Animals and diets
The study was approved by the Animal Ethics and Research Committee of the Faculty of Health Sciences, University of Cape Town and the experiments were conducted in accordance with the laws and regulations controlling experiments on live animals in South Africa. Fifty-four male Fischer 344 rats weighing between 150 and 200 g were used for the experiments. The animals were caged individually in a controlled environment at 2324°C and 50% humidity with a 12 h artificial light cycle. Food and water were available ad libitum, and rats were weighed weekly. All the animals received American Institute of Nutrition (AIN)-76 diet (17) with the following modifications: the corn starch was replaced with glucose/sucrose/corn starch (1:1:1) while sunflower oil was used instead of corn oil as a fat source. Corn products were excluded from the control diet in order to prevent any possibility of contamination by F.verticillioides.
Treatments
The FB1-containing treatments were prepared as described previously (18). The 2-AAF containing treatments were prepared by mixing 2-AAF stock powder into AIN-76 diet to give a concentration of 0.02% (w/w), a dose known to cause effective mitoinhibition of hepatocytes (19,20).
Experimental
The different FB1-containing treatment regimens are shown in Figure 1, and the details of the individual treatment groups (in order of decreasing intensities) are as follows: group I rats (long-term FB1 plus 2-AAF, LTFB/AAF, n = 12) initially received FB1 250 mg/kg diet for 5 weeks, a dose shown previously to induce oval cell proliferation and appearance of FAH (10). The animals then received a 2-week course of 2-AAF 0.02% in the diet (weeks 57), and were then continued on FB1 100 mg/kg until 25 weeks. The maintenance dose of FB1 was reduced from the initial dose in order to avoid fulminant hepatotoxicity while maintaining a pro-carcinogenic effect (21); group II rats (long-term FB1, LTFB, n = 12) initially received FB1 250 mg/kg diet for 5 weeks, and were then continued on FB1 100 mg/kg until 25 weeks; group III rats (short-term FB1 plus 2-AAF, STFB/AAF, n = 12) received FB1 250 mg/kg diet for 5 weeks only, followed by 2-AAF 0.02% from weeks 57; group IV rats (short-term FB1, STFB, n = 12) received FB1 250 mg/kg diet for 5 weeks only; and group V rats (controls, n = 6) received control (modified AIN-76) diet. A treatment group of rats given only a 2-week course of 2-AAF was not included in this study, but this regimen is reported to produce little cellular change (22). Following treatment with the different active (FB1-containing) treatment regimens in groups IIV, all animals were returned to control diet until week 50, in order to assess the reversibility of FB1-induced proliferative and neoplastic liver lesions.

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Fig. 1. Schema of FB1-containing treatment regimens (see text for details of individual treatment groups). Dashed arrow indicate open liver biopsies and solid arrow indicate post-mortem liver tissue.
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Serial liver biopsies
The sequential development of histopathologic liver lesions was followed by histologic examination of liver tissue obtained by repeated open wedge liver biopsies (Figure 1), which were performed under ether anesthesia (to reduce the potential for hepatotoxicity) according to the method described by Cmielewski et al. (23). Biopsies were performed via a small (23 cm) midline incision, and bleeding from the biopsy site was controlled by diathermy. A different lobe was biopsied each time, commencing with the posteriorly situated left lobe, and rotating to the more accessible median lobe for repeat biopsy. Biopsies were performed at weeks 5 and 7, and then 4-weekly. At each session, two rats from each of the active treatment groups and one control animal were biopsied. Rats receiving biopsies were rotated at each session until all the animals in the different groups had undergone a liver biopsy, thereafter repeat biopsies were performed in the same order. As a result of the high surgical mortality rate (see Results), serial liver biopsies were discontinued at 39 weeks.
Post-mortem liver examinations
Post-mortem liver tissue was obtained upon the death of surviving rats at 50 weeks (Figure 1), or following deaths at any time during the course of the study period. The livers were weighed and sectioned. Macroscopic tumor nodules near the peritoneal surface or on the cut surface were recorded. Slices from each of the four liver lobes were processed for histological examination (see below).
Light microscopy and immunohistochemistry
Four to five millimeter-thick slices of liver were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 4 µm for staining with hematoxylineosin (H&E) for routine light microscopy and Sirius red stain for collagen (10). Immunohistochemical staining for desmin (stellate cells), OV-6 (oval cells) and glutathione S-transferase pi (GSTP) was performed as described previously (10). Diagnoses of proliferative and neoplastic liver lesions were made according to the criteria of Bannasch and Zerban (24). Oval cells were identified by characteristic light microscopic features (12), and confirmed by staining with OV-6 monoclonal antibody.
Scoring of hepatic histopathological lesions
In order to enable semi-quantitative assessments and comparisons between treatment groups, numerical scoring systems were devised for hepatic fibrosis, oval cell proliferation and area (%) occupied by GSTP+ lesions (Table I). In this study, only GSTP+ lesions >1 mm were included in the measurements. The liver pathologist examined coded liver sections, and was unaware of the treatment given in each case.
Statistical analysis
For comparison of liver biopsy histopathological parameters (scores) amongst the different treatment groups, statistical analysis was performed on the unweighted means of the treatment-time combinations of the groups, using the general linear model of analysis of variance (ANOVA). Histopathological data from post-mortem livers for the different treatment groups were compared using two-way ANOVA. The level of statistical significance was set at P < 0.05.
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Results
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Surgical mortality
The 54 rats underwent 84 open liver biopsies at 10 time points during the study period. All 54 rats had one liver biopsy, and 30 rats underwent a second biopsy. The numbers of biopsies performed in the different groups were as follows: group I (LTFB/AAF), 19; group II (LTFB), 18; group III (STFB/AAF), 19; group IV (STFB), 17; and group V (controls), 11. The surgical mortality rates by treatment group were as follows: group I, 5.0% (1/20); group II, 19.0% (4/21); group III, 10.5% (2/19); group IV, 23.3% (5/19); and group V, 11.1% (1/9). Most deaths appeared to be related to general anesthesia and liver surgery in animals with severe chronic liver toxicity, and the single operative death in the control group was an unexpected event related to anesthesia overdose. Because of the high surgical mortality rate encountered, serial liver biopsies were not extended beyond week 39 of the study.
Hepatic histopathological lesions caused by treatment of male F344 rats with FB1-containing feeding regimens
Group I rats (LTFB/AAF). The early hepatic histopathological lesions induced by treatment with FB1 (at 5 weeks) were similar to those described previously (10), although the degree of toxic liver injury and oval cell proliferation appeared to be less severe. Proliferation of hepatic stellate cells was maximal at weeks 57 (during treatment with 2-AAF), and desmin-positive stellate cells and portal fibroblasts were seen to radiate out from portal tracts into the surrounding hepatic parenchyma (data not shown). By week 7, there was established hepatic fibrosis, resulting in portalportal linkage and architectural distortion. Thereafter, liver fibrosis progressed more gradually, eventuating in septal fibrosis or (less commonly) cirrhosis (Figure 2A) during the study period. Many of the larger nodules in cirrhotic livers were GSTP+, indicating that these nodules were in fact hepatic adenomas (HAs) (Figure 2A, inset). Proliferation of OV-6+ oval cells and ductules was maximal at 7 weeks and declined thereafter, despite continued treatment with FB1. The size of HAs appeared to enlarge progressively, attaining maximal size after completion of FB1 treatment (
27 weeks). However, several HAs persisted and even enlarged from weeks 2550, following discontinuation of FB1. Histologically, basophilic and clear cell types predominated in the HAs (Figure 2B). Some HA hepatocytes showed features of nuclear atypia (Figure 2B). Proliferating oval cells and ductules were seen within several GSTP+ (adenomatous) lesions. In addition, several HAs contained curious cystic lesions lined by cuboidal cells. These lining cells had the morphology of hepatocytes, but they expressed OV-6 antigen (Figure 2C). The intimate spatial relationships and common phenotypic markers of these different epithelial cell types suggested that they might have all originated from proliferating oval cells and ductules. Most livers contained typical cholangiofibromas (CFs) (often multiple) by the end of the study, characterized by proliferating bile ducts embedded in dense connective tissue (Figure 3A), presumably laid down by proliferating stellate cells (Figure 3A, inset). Some CFs showed cytologic features of dysplasia (Figure 3B), but no unequivocal cholangiocarcinomas were seen. In post-mortem livers, some GSTP+ lesions were noted to be located directly adjacent to CFs (Figure 3C), and proliferating oval cells were intertwined with both of these lesions (Figure 3C). Furthermore, some HAs contained hepatocytes that appeared to form ductular structures, and these cells showed expression of OV-6 in a peripheral pattern (Figure 3D). Multilocular cystic cholangiomas were seen in some livers, and the lining epithelial cells of these biliary lesions also expressed OV-6 (Figure 3D). Taken together, these close spatial and immunophenotypic relationships raised the possibility that proliferating oval and ductular cells might be involved in the generation of both the hepatocellular and cholangiocellular lesions seen in these animals.

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Fig. 2. Hepatic histopathological changes in F344 rats treated with FB1-containing regimens. (A) Sirius red stain of a liver biopsy from group I rat (LTFB/AAF) at week 23 showing loss of the normal acinar architecture due to the presence of regenerative nodules of hepatocytes, which are partially or completely surrounded by bands of fibrous tissue, indicating established cirrhosis. Sirius red, 40x. (Inset) Low power view of liver biopsy from another group I rat at week 23 showing cirrhotic liver with regenerative nodules of different sizes. Several of the larger nodules are composed of GSTP+ hepatocytes, indicating altered enzyme phenotype and suggesting that these are HAs. GSTP; 40x. (B) Post-mortem liver from group I (LTFB/AAF) rat at week 50 death showing a HA with features of dysplasia. The HA contains both basophilic and clear hepatocytes, and these cells show evidence of cytological atypia. A mitotic figure (arrow) is seen in a basophilic hepatocyte. H&E, 200x. (C) OV-6 immunostaining of liver from a group I (LTFB/AAF) rat at week 50 death showing large cystic lesions that are lined by cuboidal cells, and which have the morphology of hepatocytes but express OV-6 antigen (arrows). These cystic lesions are located within a HA, and some of the surrounding hepatocytes also express OV-6 (arrowheads). OV-6, 200x. (D) Liver biopsy specimen from group II rat (LTFB) at week 19 showing cells with the morphology of oval cells (arrowheads) which are proliferating inside a lesion composed of GSTP+ hepatocytes. The oval cells do not stain with GSTP, which aids their identification within the GSTP+ lesion. GSTP, 200x. (E) Post-mortem liver from a group II rat (LTFB) at week 50 death showing an unequivocal HCC. The malignant tumor is moderately differentiated and has a predominantly trabecular pattern, but in areas the formation of pseudoglands can be seen (arrows). H&E, 200x. (F) Liver biopsy from group III animal (STFB/AAF) at week 7 showing extensive proliferation of OV-6-positive oval cells and ductules, which are radiating out from a portal tract (PT) as ribbons and cords into the hepatic parenchyma. OV-6, 200x.
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Fig. 3. Hepatic histopathological changes in F344 rats treated with FB1-containing regimens. (A) Post-mortem liver from a group I rat (LTFB/AAF) at week 50 death showing a CF with dense fibrosis surrounding proliferative bile duct lesions. Strands of fibrotic tissue extend into the surrounding liver, which shows disturbance of the normal acinar architecture and early regenerative nodules. Sirius red, 100x. (Inset) Staining of the same liver specimen with desmin showed the presence of proliferating vessels and hepatic stellate cells within the fibrous tissue surrounding the bile duct lesions in a CF. Desmin, 100x. (B) Liver biopsy specimen from a group I rat (LTFB/AAF) at week 15 showing a CF containing irregular tapered bile ducts, which show features of cytological atypia, e.g. crowding of cells, nuclear hyperchromasia, and a mitotic figure (arrow). H&E, 400x. (C) Post-mortem liver from group I rat (LTFB/AAF) at week 50 death showing a HA containing of GSTP+ hepatocytes (upper right), and which is directly adjacent to proliferative bile duct lesions of a CF. Furthermore, cells with the morphology of oval cells and ductules are proliferating in close relationship to the GSTP+ hepatocytes (arrowheads) and proliferative bile duct lesions. Of note is that the proliferating oval cells, ductules, and bile duct lesions do not stain with GSTP. A mitotic figure (arrow) is seen within one of the GSTP+ hepatocytes. GSTP, 200x. (D) OV-6 immunostaining of a post-mortem liver from a group I rat (LTFB/AAF) at week 50 death showing HA-containing hepatocytes that are expressing OV-6 antigen in a peripheral (membranous) pattern. Many of the OV-6-positive hepatocytes within the nodule appear to be forming ductules (arrowheads). A multilobular cholangioma (*) is seen adjacent to the adenoma, and the flattened epithelial lining cells also express OV-6. OV-6, 200x.
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Group II rats (LTFB). The histopathological changes were similar to those described for group I animals (LTFB/AAF) above. Once again, proliferating oval cells were noted within and around GSTP+ (Figure 2D) lesions, suggesting a common cell of origin. One post-mortem liver contained a trabecular HCC (Figure 2E), and this was the only animal in the study that had developed an unequivocal HCC.
Group III rats (STFB/AAF). 2-AAF appeared to enhance FB1-induced proliferation of OV-6+ oval cells and ductules seen at 7 weeks (Figure 2F), but oval cell proliferation declined rapidly following discontinuation of treatment. HAs were small and no HCCs were found. However, 2-AAF appeared to have a potentiating effect on the incidence and number of CFs induced by short-term treatment with FB1.
Group IV rats (STFB). Minimal long-term effects, e.g. fibrosis, HAs and CFs were noted in livers from animals that received short-term treatment with FB1 only.
Group V (controls). Livers from control rats all showed normal histology, apart from occasional single cell GSTP+ foci by the end of the study.
Kinetics of oval cell proliferation and development of liver tumors in rats from the different FB1-containing treatment groups
Liver biopsy data. As shown in Figure 4A, mean oval cell scores (treatment-time combinations) were significantly increased in all active (FB1-containing) treatment groups compared with controls. There was no clear increase in oval cell score by either 2-AAF [group I (LTFB/AAF) versus II (LTFB) and III (STFB/AAF) versus IV (STFB)] or prolonged FB1 treatment [group I (LTFB/AAF) versus III (STFB/AAF) and II (LTFB) versus IV (STFB)]. However, the combination of 2-AAF and prolonged FB1 (LTFB/AAF) resulted in a significant increase in mean oval cell score when compared with brief FB1 treatment (STFB) alone (Figure 4A), suggesting an enhancing effect. Despite marked variability at individual time points, there appeared to be a decline in oval cell scores over time in all active treatment groups (data not shown). Biopsy GSTP scores were significantly increased in all treatment groups compared with controls, but there were no differences between the different active treatment groups (Figure 4B). Unlike the sequential biopsy oval cell scores, GSTP scores appeared to remain stable or increase over time in all active treatment groups (data not shown). Scores for fibrosis were increased in all active treatment groups compared with controls, but there were no differences between treatment groups, and no significant increases over time (data not shown).

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Fig. 4. Histopathological scores for liver lesions caused by different FB1-containing treatment regimens (liver biopsy data). The combined (treatment-time) mean histological score for each treatment group is shown. (A) Oval cell proliferation. (B) GSTP+ lesions. The numbers of animals per group are indicated in brackets and vertical bars represent the standard error (SE). *P < 0.01, active treatment groups (groups IIV) versus controls; P(0.05), group I (LTFB/AAF) versus IV (STFB). See Table I for numerical histologic scoring systems and Figure 1 for details of treatment protocols.
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Post-mortem liver data. Scores for oval cell proliferation were increased in all active (FB1-containing) treatment groups compared with controls, despite return to control diet after completion of the FB1-containing regimens (Table II). Similar to the biopsy findings (see above), there is evidence of enhancement of oval cell scores by either 2-AAF or prolonged FB1 treatment alone, but in combination (LTFB/AAF) the two treatments had an augmented effect compared with brief FB1 treatment (STFB) alone. The scores for GSTP+ lesions were increased in all active treatment groups compared with controls. GSTP+ scores were increased by 15% for group I (LTFB/AAF) versus group II (LTFB) and by 50% for group III (STFB/AAF) versus group IV (STFB) (data not shown), but these differences were not statistically significant. However, the ranges of GSTP+ lesion sizes (percentage liver area occupied) that were chosen for the different scoring levels were quite broad (Table I), and could hide marked differences in sizes of lesions. Examination of the raw data for percentage area occupied by GSTP+ lesions showed that there was clear evidence of considerable enhancement of GSTP+ lesions caused by both 2-AAF (group I versus II and III versus IV) and prolonged FB1 treatment (group I versus III and II versus IV) (Table II), which was of borderline statistical significance. Furthermore, the effects of 2-AAF and prolonged FB1 treatment on GSTP+ lesions appeared to be augmented, as evidenced by a significant increase in percentage area of GSTP+ lesions in group I (LTFB/AAF) compared with group IV (STFB). Scores for fibrosis were increased in all treatment groups compared with controls, but there were no significant differences in fibrosis scores (or the development of cirrhosis) between the different active treatment groups. Interestingly, there appeared to be an enhancing effect of 2-AAF (but not prolonged FB1) on the development of CFs, as evidenced by a non-significant increase in the number of post-mortem livers that contained CF in groups I (LTFB/AAF) and III (STFB/AAF) compared with groups II (LTFB) and IV (STFB), respectively. Only one post-mortem liver was found to contain unequivocal HCC, and this liver came from a group II (LTFB) animal.
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Table II. Comparisons of hepatic histopathological lesions caused by different FB1-containing treatment regimens (post-mortem liver data)a
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Discussion
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The present study shows that prolonged treatment with FB1 in normal diet causes liver tumors (both hepatocellular and cholangiocellular) in rats. Male F344 rats given FB1 (250 mg/kg diet for 5 weeks followed by 100 mg/kg diet until 25 weeks) developed GSTP+ hepatic foci (FAH) and adenomas (HA), and these lesions persisted following discontinuation of FB1, indicating autonomous (neoplastic) growth. The FB1-induced hepatocellular tumors developed in a setting of chronic (toxic) hepatitis and marked hepatic fibrosis, sometimes progressing to cirrhosis. These findings are reminiscent of human HCC, which typically develops in the setting of chronic necroinflammatory liver disease and cirrhosis (25). The FB1-fed rat may thus be a useful model to study the histopathogenesis of tumor development in human HCC. Prolonged FB1 treatment also resulted in the appearance of CFs, and some hepatocellular and cholangiocellular tumors showed cytological features of dysplasia. However, only one post-mortem liver (from a rat that received prolonged FB1 without 2-AAF) showed a trabecular HCC, and no unequivocal cholangiocarcinomas were found. It is likely that a longer period of FB1 treatment is required to cause HCC and cholangiocarcinomas in these animals. In this regard, Gelderblom et al. (7) showed previously that treatment of BD IX rats with FB1 (50 mg/kg diet) for 26 months resulted in the development of HCCs (in 60% of rats) and cholangiocarcinomas. However, the basal diet used in the study was deficient in multiple vitamins and lipotropes, and such dietary deficiencies could modulate FB1-induced hepatocarcinogenesis (26). More recently, Howard et al. (27) found that male F344/N rats fed diets containing FB1 (up to 150 mg/kg diet) for 2 years developed renal tubular neoplasms but no liver tumors, and there was no evidence of carcinogenic activity of FB1 in females. The reason for the lack of hepatocarcinogenic effects of FB1 in this long-term feeding study is unclear.
To date, in vivo studies on cellular carcinogenesis by FB1 have focused on the role of initiated hepatocytes and the foci-nodule-carcinoma sequence (9), according to the resistant hepatocyte model of Solt and Farber (28,29). Here we provide evidence of a potential role for oval cells in FB1-induced hepatocarcinogenesis. Prolonged treatment of rats with FB1 caused both hepatocellular and cholangiocellular tumors (frequently in the same animal). This propensity of FB1 to cause both hepatocellular and bile duct tumors is shared by only a few other experimental carcinogens, e.g. 2-AAF (30) and furan (31), and suggests that both the hepatocelluar and cholangiocellular tumors induced by FB1 may originate from a common bipotential progenitor cell capable of differentiating along both the hepatocytic and biliary lineages. OV-6 is a mouse monoclonal antibody directed at cytokeratin 14/19 (32), which normally stains oval cells and also may stain some bile duct cells, adenomatous hepatocytes (within HAs) and HCCs in rat liver (33,34). We noted an intimate spatial relationship between proliferating OV-6+ oval cells and ductules, GSTP+ hepatocellular lesions (foci and adenomas) and bile duct lesions (CFs). Furthermore, several cell types besides oval cells and ductular cells were found to express OV-6 antigen, including some adenomatous hepatocytes and also cells within HAs that resembled hepatocytes, but which lined curious cystic structures or appeared to form ductules. While these observations do not provide direct proof that oval cells are the source of the hepatocellular and cholangiocellular tumors in the FB1-fed rat, they add to the growing weight of evidence from other carcinogenesis models of such a concept (15,22).
From the biopsy and post-mortem liver data, it appears that 2-AAF and prolonged FB1 treatment have transient effects only on oval cell proliferation, but they exert sustained and enhancing effects on the growth of GSTP+ hepatocellular lesions. Although not specifically monitored in this study, it is unlikely that the liver biopsy procedures themselves resulted in any significant enhancing effects on the development of liver lesions. The enhancing effects of 2-AAF and FB1 on GSTP+ lesions were particularly evident in post-mortem liver specimens, especially when using the raw data for percentage area occupied by GSTP+ lesions rather than the GSTP+ score. Examination of whole liver specimens avoids the potential problem of sampling error, which is inherent in liver biopsy specimens, and is thus the most accurate method for assessing focal liver lesions in the rat. Although there appeared to be discordance in the kinetics of oval cell proliferation and GSTP+ lesion growth during the course of the study, it is quite possible that some oval cells may have differentiated into adenomatous hepatocytes at an early time point. These findings thus do not militate against the possibility of an oval cell origin for the hepatocellular tumors caused by FB1. Interestingly, 2-AAF (but not prolonged FB1 treatment) had a marked enhancing effect on the incidence of CFs in post-mortem livers. Although not specifically measured in this study, 2-AAF is known for marked inhibition of proliferation of normal hepatocytes (19,20), and this property was thought to underlie its potent promoting effects on FB1-induced GSTP+ lesions and CFs, possibly through resultant activation of a facultative stem cell compartment (oval cells) in the liver.
In conclusion, this study supports the involvement of dietary FB1 in liver carcinogenesis in male F344 rats. Oval cells may be the source of both the hepatocellular and cholangiocellular tumors induced by prolonged treatment with FB1. 2-AAF appears to have an enhancing effect on FB1-induced liver tumors, presumably due to its potent inhibitory effects on hepatocyte regeneration.
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Acknowledgments
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The authors wish to thank Mrs Heather McCleod, Mr Willem Ryneveldt and Mr Gert Engelbrecht for expert technical assistance; Mrs Petra Snijman for the purification of FB1 and Ms Charon Lambrechts for humane care of the animals. We are grateful to Dr Peter Cruse for valuable discussions and to Dr Stewart Sell for the OV-6 antibody.
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References
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Received September 11, 2003;
revised February 6, 2004;
accepted February 19, 2004.