Histopathology and gene expression changes in rat liver during feeding of fumonisin B1, a carcinogenic mycotoxin produced by Fusarium moniliforme

Eric R. Lemmer1,5, Pauline de la Motte Hall2,3, Nobuhiko Omori5, Masako Omori5, Enid G. Shephard1,6, Wentzel C.A. Gelderblom4, J. Peter Cruse2, Rochelle A. Barnard2, Walter F.O. Marasas4, Ralph E. Kirsch1 and Snorri S. Thorgeirsson5

1 MRC/UCT Liver Research Centre and
2 Departments of Anatomical Pathology, University of Cape Town, Observatory, Cape 7925, South Africa,
3 Flinders University of South Australia, Bedford Park 5042, South Australia,
4 MRC Programme on Mycotoxins and Experimental Carcinogenesis, Tygerberg 7505, South Africa and
5 Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Fumonisin B1 (FB1) is a carcinogenic mycotoxin produced by the fungus Fusarium moniliforme in corn. Feeding of FB1 to rats causes acute liver injury, chronic liver injury progressing to cirrhosis, and sometimes terminates in hepatocellular carcinoma or cholangiocarcinoma. This study describes the histolopathology and changes in gene expression in the rat liver during short-term feeding of FB1. Male Fischer rats were fed either FB1 250 mg/kg or control diet, and were killed weekly for 5 weeks. FB1 caused a predominantly zone 3 `toxic' liver injury, with hepatocyte death due to necrosis and apoptosis. Hepatocyte injury and death were mirrored by hepatic stellate cell proliferation and marked fibrosis, with progressive disturbance of architecture and formation of regenerative nodules. Despite ongoing hepatocyte mitotic activity, oval cell proliferation was noted from week 2, glutathione S-transferase {pi}-positive hepatic foci and nodules developed and, at later time points, oval cells were noted inside some of the `atypical' nodules. Northern blot (mRNA) analysis of liver specimens from weeks 3 to 5 showed a progressive increase in gene expression for {alpha}-fetoprotein, hepatocyte growth factor, transforming growth factor alpha (TGF-{alpha}) and especially TGF-ß1 and c-myc. Immunostaining with LC(130) antibody demonstrated a progressive increase in expression of mature TGF-ß1 protein by hepatocytes over the 5 week feeding period. The overexpression of TGF-ß1 may be causally related to the prominent apoptosis and fibrosis seen with FB1-induced liver injury. Increased expression of c-myc may be involved in the cancer promoting effects of FB1.

Abbreviations: AAF, acetylaminofluorene; AFP, {alpha}-fetoprotein; AIN, American Institute of Nutrition; FB1, fumonisin B1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST{pi}, {pi} class glutathione S-transferase; H & E, haematoxylin and eosin; HCC, hepatocellular carcinoma; HGF, hepatocyte growth factor; PH, partial hepatectomy; TGF-{alpha}/ß, transforming growth factor alpha/beta.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
The fumonisin B mycotoxins are natural contaminants of corn infected with the fungus Fusarium moniliforme (1). Ingestion of fumonisin B1 (FB1), the major fumonisin produced by the fungus, causes a variety of toxicoses in animals, including equine leukoencephalomalacia (2) and porcine pulmonary edema (3). Human dietary consumption of Fusarium-contaminated corn products has been epidemiologically linked to increased rates of esophageal cancer in regions of the world in which corn is the staple grain, such as South Africa (4) and China (5). In rats, feeding with FB1 causes acute and chronic liver toxicity, bile duct proliferation (`hyperplasia'), fibrosis progressing to cirrhosis, cholangiofibrosis and, often, hepatocellular carcinoma (HCC) and/or cholangiocarcinoma (1,6,7). These carcinogenic mycotoxins are non-genotoxic, and appear to act mainly as promoters (and possibly weak initiators) of tumors (6,8,9).

In addition to initiated hepatocytes, another group of cells, the `oval cells', proliferate in the liver during experimental hepatocarcinogenesis. This non-specific term was introduced by Farber in 1956 (10), although these cells had been described previously (11). Oval cells have elongated nuclei, scanty cytoplasm and appear very early in the development of several experimental hepatic cancer protocols. Oval cells may either arise from the biliary epithelium (12,13), or else be derived from pluripotential hepatic stem cells (1416). Oval cells were shown to have the capacity to differentiate to both biliary duct cells and hepatocytes (1719). Some authors claim that oval cells play no role in hepatocarcinogenesis (20,21), while others have reported that oval cells can give rise to preneoplastic foci and nodules that can progress to cancer (14,15).

The present study describes the histopathology and changes in gene expression in the rat liver during short-term feeding of FB1.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Chemicals
FB1 was purified from corn cultures of F.moniliforme strain MRC 826 as described previously (22). The purity as compared with an analytical standard by high performance liquid chromatography (23) was in the order of 92–95%. The monomethylester derivatives of FB1, which are artifacts of the purification procedure (22), constituted the remainder of the FB1 preparation.

Animals and diet
Animal care was according to the criteria outlined in the guide for the care and use of laboratory animals, prepared by the National Academy of Sciences (24). Thirty male Fischer 344 rats weighing between 150 and 200 g were used in all the experiments. The animals were caged individually in a controlled environment at 23–24°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 the AIN-76 diet (25) 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.moniliforme. The cellulose was donated by Sappi Saicor, Umkomaas, Natal, South Africa.

Treatments
The FB1-containing diet (250 mg FB1/kg diet) was prepared as follows: FB1 stock sample dissolved in methanol (50 ml) was evaporated onto a subsample (200 g) of the diet, and dried in a fume hood at room temperature for 12 h. Subsequently, the subsample was thoroughly mixed into the diet (6 kg) to obtain the desired concentration of FB1. The control diet was treated in a similar way using only an equal volume of methanol. Each diet was prepared in 6 g quantities at a time and stored under nitrogen at 4°C until used.

Experimental
The 30 animals were randomly allocated into a group of 20 (treatment group) and a group of 10 (control group), and fed for up to 5 weeks with 250 mg FB1/kg or control AIN diet, respectively. Four rats from the treatment group and two rats from the control group were killed weekly till the end of 5 weeks. The livers were harvested, and slices of liver were fixed in 10% neutral buffered formalin for light microscopy and immunohistochemistry. The remaining liver was snap frozen in liquid nitrogen and stored at –70°C for mRNA analysis. Liver specimens from day 9 post partial hepatectomy combined with acetylaminofluorene (AAF–PH regimen) from another study (26) were included to serve as positive controls for oval cell proliferation. Maximal oval cell proliferation is known to occur at this time point post AAF–PH (27).

Light microscopy
For routine light microscopy, slices of liver 4–5 mm in thickness were immersion fixed in 10% neutral buffered formalin for 24 h before processing, embedding in paraffin wax and sectioning at 4 µm. Stains included haematoxylin and eosin (H & E), sirius red for collagen, and Gordon and Sweet's method for reticulin. Coded sections were examined for evidence of hepatocyte injury, apoptotic bodies, fatty change, mitoses, architectural distortion, fibrosis, regenerative nodules and oval cell proliferation. Cells undergoing apoptosis were detected in situ by TUNEL specific labeling of nuclear DNA strand breaks, as described by Gavrieli et al. (28).

Immunohistochemistry
Staining with Desmin D33 (Dako, Copenhagen, Denmark) for hepatic stellate (Ito) cells, and with rabbit polyclonal glutathione S-transferase {pi} (GST{pi}; Novacastra, Newcastle-Upon-Tyne, UK) for enzyme-altered hepatic foci and preneoplastic nodules was performed on paraffin sections. After sequential layering with biotinylated rabbit anti-mouse or swine anti-rabbit (Dako) 1:250 dilution as link antibodies, peroxidase conjugated Streptavidin (Dako) 1:500 was applied for 30 min at room temperature. The OV-6 mouse monoclonal antibody, which stains both oval cells and bile duct cells, was a generous gift from Professor Stewart Sell (Albany, NY). Acetone fixed cryostat sections were brought to room temperature and stained by means of a standard two-stage indirect peroxidase conjugated technique (Dako P161). Antibody to mature TGF-ß1 protein was a generous gift from Dr K.Flanders (National Cancer Institiute, Bethesda, MD). Immunohistochemical staining for transforming growth factor beta-1 (TGF-ß1) protein was performed on 5 µm deparaffinized sections with an indirect immunoperoxidase antiserum detection protocol (Elite kit; Vector Laboratories). Mature TGF-ß1 protein was detected by the rabbit polyclonal LC(130) antibody, as described previously (29).

Probes
Antisense riboprobes labeled with [32P]CTP were utilized for each of the following. A 429 bp piece of the 5' end rat {alpha}-fetoprotein (AFP) cDNA subcloned into pGEM-4Z (kindly provided by Dr Thomas D.Sargent, National Institute of Child Health and Human Development, Bethesda, MD) was linearized by PstI and transcribed by SP6 RNA polymerase (30). A 600 bp cDNA fragment encoding the 3' end of rat hepatocyte growth factor (HGF) subcloned into the pBluescript SK vector (kindly provided by Dr Brian Carr, University of Pittsburgh School of Medicine, Pittsburgh, PA) was linearized by HindIII and transcribed by T3 RNA polymerase. A 335 bp fragment of rat TGF-{alpha} cDNA was obtained by RT–PCR and cloned as described previously (31). EcoRV and SP6 RNA polymerase were used for its linearization and in vitro transcription. A 985 bp fragment of rat TGF-ß1 cDNA cloned in pBluescript II KS+ vector (kindly provided by Dr Su Wen Qian, National Cancer Institute, Bethesda, MD) was linearized by XhoI and transcribed by T3 RNA polymerase. Mouse cDNA for c-myc subcloned into pGEM4 was linearized by EcoRI and transcribed by T7 RNA polymerase. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe labeled with [32P]dCTP by the random primer extension method was used as an internal control (32).

RNA isolation and northern blot analysis
RNA was extracted from rat liver with guanidium thiocyanate followed by centrifugation in cesium chloride solution. Poly(A)+ RNA was selected by oligo(dT)–cellulose chromatography. Ten micrograms of poly(A) RNAs per lane were electophoresed on 0.8% agarose gels containing 2.22 mol/l formaldehyde and were later transferred to nylon filters. After UV cross-linking, the filters were hybridized with riboprobes at 60°C and at 42°C with cDNA probes. Blots were washed twice each with 1x standard sodium citrate/0.1% sodium dodecyl sulfate (SSC/SDS) at room temperature, 0.1x SSC/SDS at room temperature, and 0.1x SSC/SDS at 60°C. Autoradiography was performed on Kodak X-OMAT AR film (Rochester, NY) at –70°C using an intensifying screen.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Light microscopy and immunohistochemistry
Staining with H & E showed that FB1 250 mg/kg diet caused a toxic injury in livers of all treated animals. At week 1, necroinflammatory lesions were confined to zone 3 of the liver acinus. Hepatocyte death was due to both necrosis and apoptosis, and numerous apoptotic bodies and a mild infiltrate of mononuclear cells were seen (Figure 1AGo). Loss of hepatocytes, due to fragmentation of the apoptotic cells, was accompanied by collapse of the reticulin framework. Scattered mitoses and mild to moderate macrovesicular fatty change in hepatocytes were also present. By week 2, oval cell proliferation was just discernible in the portal tracts (Figure 1BGo) and apoptosis was apparent in all zones of the liver together with mild and variable fatty change. Apoptosis continued to be seen in all zones of the liver at weeks 3, 4, and 5, and was confirmed by the TUNEL method (Figure 1CGo). Frequent hepatocyte mitoses were also seen in all zones at each time point. By week 5, marked oval cell proliferation, seen as single small epithelial cells, cords of cells and small ductules, was seen in all portal tracts with extension into the adjacent hepatic parenchyma (Figure 1DGo). By week 4, there was considerable distortion of the architecture due to the presence of bands of fibrous tissue linking terminal hepatic venules to mildly fibrotic portal tracts, and early regenerative nodule formation. The architectural distortion progressed and by week 5 the features were those of a developing cirrhosis (Figure 1E and FGo). Numerous desmin positive hepatic stellate cells were seen in the injured zone 3 regions at week 1 (Figure 2AGo). At all subsequent time points, stellate cells were seen in the portal tracts and were also scattered through zones 1, 2 and 3. However, hepatic stellate cell proliferation appeared to be maximal at 3 weeks, whereafter the overall numbers appeared to decrease, but with a predominance in the portal tracts and zone 1 (Figure 2BGo).



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Fig. 1. Liver pathology in the fumonisin-fed rat. (A) Liver at week 1 showing a terminal hepatic venule (center right), numerous apoptotic bodies (arrows), focal hepatocyte drop-out and a sparse infiltrate of mononuclear cells in zone 3. H & E, objective x20. (B) Liver at week 2 showing mild oval cell proliferation seen as a cord of small epithelial cells and a small ductule (center top) in a portal tract and a sparse infiltrate of mononuclear cells. H & E, objective x10. (C) Liver at week 3 showing a terminal hepatic venule (center) and numerous apoptotic hepatocytes scattered throughout the parenchyma. TUNEL method, objective x10. (D) Liver at week 5 showing moderately fibrotic portal tracts which are linked by bands of fibrous tissue, and numerous small, proliferating ductules (arrows). (E) Liver at week 5 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. Gordon and Sweets stain for reticulin, objective x4. (F) Liver at week 5 showing the features of an early/developing cirrhosis. Sirius red, objective x10.

 


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Fig. 2. Immunohistochemical studies of livers from FB1-fed rats. (A) Liver at week 1 showing numerous desmin positive hepatic stellate cells which are located in the zone 3 region of the liver (arrows) with a similar distribution to the liver injury shown in Figure 1Go. P-A-P, objective x20. (B) Liver at week 3 showing numerous desmin positive hepatic stellate cells which are located in the portal tracts and zone 1 regions (arrows). Numerous proliferating oval cells are also seen in the portal tracts, and the hepatocytes show mild fatty change. Objective x10. (C) Liver at week 2 showing small numbers of OV-6 positive single cells and small ductules in the portal tracts. The OV-6 positive cells correspond to the small epithelial cells and ductules of the type seen in Figure 1BGo. Objective x10. (D) Liver at week 5 showing numerous proliferating oval cells, which are seen as single cells and small ductules that are OV-6 positive, located in the portal tracts and adjacent liver parenchyma. The OV-6 positive cells correspond to the small ductules seen in Figure 1DGo. Objective x4. (E) Liver at week 5 showing an atypical nodule composed of GST{pi}-positive hepatocytes which is located next to a portal tract (left). Proliferating oval cells, which are GST{pi} negative, are seen in the portal tract and within the nodule (arrow). Objective x20. (F) Higher magnification of the liver in Figure 2EGo showing oval cells (arrow) with a group of GST{pi}-positive hepatocytes. Objective x40.

 
Positive staining with OV-6 confirmed that the small epithelial cells, seen as single cells, cords and ductules (Figure 1BGo) were oval cells (Figure 2CGo). Maximal proliferation of oval cells was seen during weeks 3–5 (Figure 2DGo). Initially, the oval cells stained with GST{pi}, but at 4 weeks following treatment with FB1, many oval cells did not express GST{pi} (Figure 2EGo).

After 1 week, single GST{pi}-positive hepatocytes were scattered throughout the acini, and by week 2 there were GST{pi}-positive enzyme-altered hepatic foci, varying from one to three per section of liver. The GST{pi}-positive hepatic foci increased in number and size from weeks 3 to 5. Some foci were close to the terminal hepatic venules while others abutted on the portal tracts. By weeks 4 and 5 some rats had developed large atypical nodules in the liver, which were readily seen in the H & E sections and confirmed by GST{pi} staining. Of note was the close relationship of some hepatic foci and nodules to portal tracts that contained the proliferating oval cells, and the presence of cells with morphological features of oval cells inside several of these atypical nodules (Figure 2FGo).

Expression of AFP, HGF and TGF-{alpha}
Transcripts for AFP were not detected in normal liver. Feeding with FB1 250 mg/kg resulted in a progressive increase in the expression of the 2.1 kb AFP transcripts from weeks 3 to 5. The peak level of AFP expression was, however, much less than that seen at day 9 post AAF–PH (Figure 3Go).



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Fig. 3. Analysis of expression of poly(A)+ RNA (10 µg per lane) for AFP, HGF and TGF-{alpha} by northern blotting during weeks 3–5 of FB1 feeding. Timed rat liver specimens from day 9 post AAF–PH served as positive controls.

 
Transcripts for HGF were also not detected in normal liver. Feeding with FB1 250 mg/kg resulted in a moderate but sustained increase in expression of HGF from weeks 3 to 5. HGF expression in FB1-fed animals was much less than that seen at day 9 post AAF–PH (Figure 3Go).

There was no expression of TGF-{alpha} mRNA in normal liver. Short-term feeding with FB1 250 mg/kg resulted in a moderate and fluctuating increase in expression of TGF-{alpha} to levels that were similar to that seen at day 9 post AAF–PH (Figure 3Go).

Expression of TGF-ß1 and c-myc
Some expression of TGF-ß1 mRNA was detected in normal liver. Feeding with FB1 250 mg/kg resulted in a marked and progressive increase in expression of TGF-ß1 from weeks 3 to 5. The maximum level of expression of TGF-ß1 in FB1-fed rats greatly exceeded that seen at day 9 post AAF–PH (Figure 4Go).



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Fig. 4. Analysis of expression of poly(A)+ RNA (10 µg per lane) for TGF-ß1 and c-myc by northern blotting during weeks 3–5 of FB1 feeding. Timed rat liver specimens from day 9 post AAF–PH served as positive controls. GAPDH poly(A)+ RNA was used as loading control.

 
Transcripts for c-myc were detectable at a low level in normal liver. Feeding with FB1 250 mg/kg resulted in a marked increase in expression of c-myc, with maximum levels being expressed at week 4. This level of expression of c-myc was similar to that seen at day 9 post AAF–PH (Figure 4Go).

Immunostaining for TGF-ß1 protein
Staining with LC(130) antibody demonstrated a progressive increase in expression of mature TGF-ß1 protein in the rat liver over the 5 week FB1 feeding period (Figure 5B–DGo). Expression of TGF-ß1 protein by hepatocytes was initially focal (Figure 5BGo), but with continued feeding the protein was abundantly expressed in most hepatocytes (Figure 5C and DGo). TGF-ß1 protein was not detected in zone 3 hepatocytes, which showed evidence of frequent mitoses and apoptosis. Oval cells, stellate cells and Kuppfer cells were consistently negative for TGF-ß1 protein.



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Fig. 5. Mature TGF-ß1 protein in timed liver specimens from FB1-fed rats, detected by staining with LC(130) antibody. (A) Control, with omission of the primary antibody. (B) Liver at 1 week, showing patchy expression of TGF-ß1 by hepatocytes. (C) Liver at 2 weeks, showing expression of TGF-ß1 by most hepatocytes in zones 1 and 2. (D) Liver at 3 weeks, showing extensive expression of TGF-ß1, but persistent lack of staining by zone 3 hepatocytes. DAB, objective x10. (Straight arrows indicate central veins; curved arrows indicate portal tracts.)

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
FB1 has been shown to be hepatotoxic (1) and hepatocarcinogenic (6) in rats. The principal pathological change in rats treated with FB1 in the diet (1000 mg/kg) in short-term toxicity tests (21–33 days) is progressive toxic hepatitis characterized by hepatocellular necrosis, bile duct proliferation (`hyperplasia') and fibrosis (1). During a chronic feeding study over a period of 26 months with FB1 50 mg/kg, animals developed a chronic toxic hepatitis that progressed to cirrhosis and cholangiofibrosis, and which terminated in hepatocellular carcinoma and cholangiocarcinoma, respectively (6). In a preliminary study, Voss et al. (7) reported that FB1 was hepatotoxic in rats fed a diet containing 150 mg/kg for 4 weeks and also nephrotoxic at 15–50 mg/kg. Scattered single cell necrosis, bile duct proliferation and an increase in mitotic cells were described. The scattered single cell necrosis was subsequently confirmed as apoptosis (33).

In this short-term feeding study, FB1 250 mg/kg caused toxic liver injury, initially with apoptosis in zone 3, followed by apoptosis and mitoses in all zones of the liver, progressive hepatic fibrosis and regenerative nodule formation and development of enzyme-altered hepatic foci and atypical nodules. The bile duct proliferation (hyperplasia) previously described by Gelderblom et al. (1) was confirmed in this study as proliferation of OV-6 positive oval cells, radiating from portal tracts into the adjacent liver parenchyma. Oval cell proliferation occurred despite clear evidence of continued hepatocyte regeneration at all time points. Proliferation of adult hepatocytes has been noted in other models for oval cell activation, including galactosamine (34) and dipin (35). FB1 has been reported to cause a dose-dependent inhibition of PH-induced incorporation of [3H]thymidine in hepatocytes (36), and bile ductular hyperplasia has been noted at FB1 dosages of 50 mg/kg diet (unpublished data). FB1-induced oval cell proliferation does not appear to be an `all-or-nothing' phenomenon, which occurs only when hepatocyte regeneration is absent. Thus, complete mitoinhibition of hepatocytes does not appear to be an absolute requirement for the activation of the progenitor cell compartment, and other factors (e.g. functional impairment of hepatocytes) may conceivably also play a role.

Immunohistochemical staining demonstrated marked proliferation of desmin positive hepatic stellate cells from week 2, which appeared to mirror the distribution of the hepatocyte injury as well as the development of hepatic fibrosis. Hepatic stellate cells are perisinusoidal non-parenchymal cells which in normal liver are non-proliferative and are the main storage site for vitamin A (37). Following liver injury of any kind, stellate cells undergo `activation' and transformation with loss of intracellular retinoid, enhanced production of extracellular matrix proteins, increased contractility and secretion of a variety of growth factors and cytokines which act in an autocrine and paracrine fashion on cells in the liver (27). Hepatic stellate cells thus play a major role in hepatic fibrogenesis (37).

There is currently no information on changes in hepatic gene expression during feeding of FB1. Feeding with FB1 250 mg/kg resulted in increased expression of HGF, TGF-{alpha} and AFP transcripts at weeks 3, 4 and 5, which coincided with desmin positive stellate cell and OV-6 positive oval cell proliferation. AFP expression was used as a marker for oval cell proliferation (38). HGF and TGF-{alpha} are important growth factors in the liver, and are involved in both normal liver regeneration (39,40) and activation/proliferation of the oval cell compartment (16,41). Expression of transcripts for HGF and TGF-{alpha} during feeding of FB1 was less than that seen at day 9 post AAF–PH (maximal oval cell proliferation). The moderately increased but sustained expression of genes for liver growth factors during feeding with FB1 presumably reflects ongoing hepatotoxicity. This contrasts with the AAF–PH model, in which massive but transient liver injury and growth factor expression occurs.

FB1 feeding resulted in marked overexpression of TGF-ß1 in rat liver. Contrary to what was anticipated, TGF-ß1 protein was expressed almost exclusively by hepatocytes, while stellate cells and oval cells were persistently negative. Immunostaining demonstrated a progressive increase in the numbers of hepatocytes expressing mature TGF-ß1 protein during FB1 feeding, and by 3 weeks most hepatocytes in the liver lobule were stained by LC(130) antibody. However, zone 3 hepatocytes remained persistently negative for TGF-ß1 protein, and many of these hepatocytes were undergoing mitosis or apoptosis. The markedly increased levels of TGF-ß1 expression by hepatocytes may be responsible for the marked apoptosis and fibrosis seen in the FB1-fed rat.

The prominent pro-apoptotic effects of FB1 in rat liver are intriguing, and contrast with the in vitro effects of this mycotoxin on chemotherapy-mediated tumor cell destruction (42). FB1 has been found to have specific, potent activity as an inhibitor of sphingolipid biosynthesis by blocking the conversion of sphinganine to ceramide (4345). Ceramide is a recently identified lipid second messenger that is believed to be one of the immediate signals for cell death generated in tumor cells treated with the chemotherapeutic agent, daunorubicin (46). The production of ceramide and the ensuing onset of apoptosis in murine leukemia cells cultured in the presence of daunorubicin is prevented by pretreatment with FB1 (42). The molecular mechanisms of action of FB1 are thus complex, and it appears that this fungal toxin may act as either an inhibitor or promoter of apoptosis, depending on the experimental situation (47).

At later time points in this study, we noted the close relationship of some `premalignant' hepatic foci and nodules to portal tracts that contained proliferating oval cells and ductules, and the presence of oval cells and ductules inside several of these nodules. It is possible that some of these oval cells were in fact adjacent to (`swirling around') the premalignant nodules, depending on the plane of section of the liver specimens. These cells showed typical morphological features of oval cells, but did not express GST{pi}, thus aiding their recognition within the GST{pi}-positive nodules.

Feeding of FB1 also resulted in overexpression of c-myc, an important nuclear oncogene. The myc oncoproteins act as sequence-specific transcription factors that regulate a variety of genes important in normal cellular growth and differentiation processes (48,49). Deregulation of c-myc expression is frequently observed in experimentally induced HCC in rodents, as well as in primary human tumors (5053). Transgenic mice chronically overexpressing c-myc develop liver tumors, and this process is accelerated by concomitant overexpression of TGF-{alpha} (54). Mechanisms of oncogenesis in c-myc/TGF-{alpha} double transgenic mice appear to involve disruption of the Rb/E2F pathway and deregulation of cell cycle control (55). Both transgenes contribute to induction of cyclin D1 expression and resultant inactivation of Rb, and c-myc may directly induce E2F (55). Short-term feeding of FB1 has recently been shown to cause overexpression of cyclin D1 (56), and it would be important in the future to study the co-expression of c-myc, cyclin D1 and E2F in rat liver during FB1-induced carcinogenesis.


    Conclusion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Short-term feeding with FB1 causes a severe toxic liver injury characterized by hepatocyte death due to apoptosis and necrosis, hepatic stellate cell proliferation, fibrosis, oval cell proliferation and the appearance of premalignant hepatic foci and nodules. Oval cells closely related to foci and nodules appeared to be undergoing phenotypic changes, and long-term FB1 feeding studies are required to determine the ultimate fate of these oval cells. The markedly increased expression of TGF-ß1 by hepatocytes may be causally related to the prominent apoptosis and fibrosis seen in FB1-induced liver injury, and overexpression of c-myc may be involved in the cancer promoting effects of FB1. The FB1-fed rat is an attractive model for the study of liver injury, apoptosis, fibrosis, oval cell proliferation and hepatocarcinogenesis.


    Acknowledgments
 
The authors wish to thank Ms Tanya Hoang, Mrs Heather McCleod and Mrs Marietjie Levey for expert technical assistance, and Mrs Petra Bakkes for the purification of FB1. We are grateful to Dr Valentina Factor for help with TGF-ß immunohistochemical staining. E.R.L. was the recipient of a combined travel award from the South African Gastroenterology Society (SAGES) and Byk Gulden Pharmaceuticals.


    Notes
 
6 To whom correspondence should be addressed Email: enid{at}liver.uct.ac.za Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 

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Received April 27, 1998; revised December 24, 1998; accepted January 25, 1999.