Ethanol interactions with a choline-deficient, ethionine-supplemented feeding regime potentiate pre-neoplastic cellular alterations in rat liver

Emma J. Croager1, Patrick G.J. Smith and George C.T. Yeoh

Western Australian Institute for Medical Research, Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, The University of Western Australia, Crawley 6009, Australia


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To examine the effect of ethanol on hepatocarcinogenesis induced by a choline-deficient, ethionine-supplemented (CDE) diet, rats were fed either an ethanol-supplemented diet or ethanol-free, isocaloric diet for 2 months, followed by a CDE diet or control diet for up to 8 months. Changes to cellular composition and pattern of gene expression in the liver were determined at 0 and 3 days, and 1, 2 and 3 weeks after commencing the CDE diet, using histological/immunochemical techniques and northern analysis. Oval cells in the liver were identified morphologically and by expression of pi-glutathione S-transferase ({pi}-GST), alpha-fetoprotein (AFP) and the embryonic isoform of pyruvate kinase (M2-PK). Oval cell numbers and changes in the pattern of gene expression induced by the CDE diet were accelerated by pre-treatment with ethanol. At all stages, the proportion of oval cells in the test group exceeded that in controls. After 1 week, oval cells had spread sufficiently from the periportal region to be observed pericentrally in test animals and by 3 weeks, extensive formation of ductal structures was apparent, which were absent in controls. Additionally, M2-PK and AFP mRNA were detected earlier, and in greater abundance in animals pre-treated with ethanol. After 8 months of CDE treatment, one or two small hepatic foci (<10 hepatocytes), strongly positive for {pi}-GST, were detected in the liver of ethanol-pre-treated animals. These foci were absent in CDE-treated animals; however, animals pre-treated with ethanol followed by chronic CDE treatment showed increased size (>40 hepatocytes) and numbers of foci, correlating with the extent of liver damage and varying from 5 to 50% of the liver section. Our data suggest that ethanol pre-treatment potentiates the short-term effects of the CDE diet by enhancing oval cell proliferation, while chronic CDE administration enhances the appearance of pre-malignant hepatic foci that are observed with ethanol pre-treatment alone.

Abbreviations: AFP, alpha-fetoprotein; CDE, choline-deficient, ethionine-supplemented; CLD, control liquid diet; ELD, ethanol liquid diet; {pi}-GST, pi-isoform of glutathione S-transferase; HCC, hepatocellular carcinoma; M2-PK, embryonic isoform of pyruvate kinase.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is widely accepted that excess alcohol consumption plays a role in tumourigenesis (18), although some reports question a direct carcinogenic role of ethanol, suggesting instead that it acts as a co-carcinogen (611). In the case of hepatocarcinogenesis, ethanol may affect hepatocytes directly, causing cellular injury (8,12), or indirectly by altering drug metabolizing pathways (4,7,12,13) and hence acts in the classical role as a promoter.

There exists a substantial amount of epidemiological data indicating that alcohol consumption increases the risk of developing liver cancer (2,47,1417). Ethanol can also interact with other hepatotoxic agents, such as the hepatitis B and C viruses, further enhancing liver carcinogenesis (3,5,12,13). These findings suggest an ancillary role for ethanol, rather than that of a primary carcinogenic agent. This notion has been substantiated in some experimental models of hepatocarcinogenesis (1,5,18).

Rat models for studying the molecular and cellular events associated with carcinogenesis include partial hepatectomy plus diethylnitrosamine (1,19), administration of 3-dimethyl-4-dimethylaminoazobenzene (9), feeding a choline-deficient, ethionine-supplemented (CDE) diet (2024), the Solt-Farber regime (25,26) or the choline-deficient, N-2-fluorenylacetamide-supplemented diet (27). In most, though not all of these experimental models and during chemically induced chronic liver damage, oval cells originating from the portal triads are seen to proliferate throughout the liver lobule prior to tumour formation (28). As a consequence, the induction of oval cells in the liver in some models of hepatocarcinogenesis is interpreted as a pre-neoplastic condition.

It has been suggested that oval cells may represent a liver stem cell compartment (29,30), or are themselves the progeny of stem cells that are bipotential. This evidence suggests that, under appropriate conditions, oval cells proliferate and give rise to hepatocytes (30,31), duct-like structures (22), or a mixture of hepatocellular (HCC) and cholangiocellular carcinomas (HCCs) (22,23).

Oval cells induced by the CDE feeding regime have been characterized by establishing their pattern of expression of alpha-fetoprotein (AFP); the embryonic (M2-PK) and adult forms of pyruvate kinase; alpha, mu and pi class glutathione S-transferase (GST); albumin; transferrin; and tyrosine aminotransferase (21,22). Chronic ethanol exposure is known to induce oval cell proliferation in rat liver (21) and oval cells have been identified in livers of patients with chronic alcoholic liver disease (32). As such, the purpose of the present study was to determine if ethanol not only induces a pre-neoplastic change in the liver, represented by oval cell proliferation, but can also potentiate the effect of short-term and chronic exposure to hepatocarcinogens.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Males (180–200 g) of the Wistar strain of the albino rat Rattus norvegicus were used for all experiments. The animal study protocols were conducted according to guidelines set by the National Health and Medical Research Council of Australia.

Ethanol diet
Animals were fed a totally liquid diet according to the all-purpose liquid diet regime of Lieber–DeCarli (33). Thirty-two animals received the ethanol liquid diet (ELD) containing 5% (w/v) ethanol, and the same number of animals received the corresponding liquid diet (CLD) where the ethanol component was replaced by additional carbohydrate. Both groups were pair-fed for 2 months, after which time blood samples were taken from six animals in each group and analysed for alcohol content (PathCentre, Australia).

CDE diet
Following 2 months of ELD or CLD treatment, 26 animals from each group were administered a CDE diet comprising choline-deficient chow (Teklad, USA) and 0.15% (w/v) DL-ethionine dissolved in the drinking water. Based on their consumption of liquid, this was equivalent to giving rats a 0.07% (w/v) ethionine-supplemented diet in solid form. Four animals from each group were killed at 0 and 3 days, and 1, 2 and 3 weeks and six animals were killed 8 months after commencement of the CDE diet. The remaining six animals from the ELD and CLD groups were placed on a normal laboratory diet and killed after 8 months.

Elutriation of liver cells
The animals were anaesthetized and perfused by the portal vein with perfusion buffer (34) for 10 min. Livers were collagenase digested according to the method of Seglen (34). Liver cells were separated according to size and density by centrifugal elutriation as described by Yaswen et al. (24). Four fractions were collected at flow rates of 18, 24, 40 and 50 ml/min and a fifth fraction was obtained by shutting down the rotor (from 2500 r.p.m.) while the flow rate was maintained at 50 ml/min. Where oval cells were present, the vast majority were collected in fraction three, with low numbers (10%) in fraction four. Routine cytochemistry and immunocytochemical staining confirmed fraction three was free of adult hepatocytes. Cell fractions were fixed by mixing with an equal volume of 2x fixative to give a final concentration of 4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.45). Fractions were attached to poly-L-lysine coated glass slides by cytocentrifugation.

Immunostaining techniques
Following perfusion of the liver with perfusion buffer (34), and prior to digestion with collagenase, a small piece of liver (~0.5 cm3) was removed, immerse-fixed in Carnoy’s solution for 1.5 h and embedded in paraffin wax. For light microscopy, 6 µm sections were cut and attached to poly- L-lysine coated slides. Liver sections and cytocentrifuged cell preparations were stained with hematoxylin and eosin (H&E) to ascertain morphological changes. Immunohistochemistry was performed at room temperature using the indirect immunoperoxidase method of Clement et al. (35). Endogenous peroxidase activity was eliminated by treatment with 2.5% aqueous periodic acid for 5 min, followed by 0.02% sodium borohydride for 2 min. Samples were blocked for 1 h in 10% fetal calf serum/0.2% saponin in PBS then incubated for 1 h with M2-PK (diluted 1:500, a gift from Dr T.Noguchi, Japan), {pi}-GST (diluted 1:200, Biotrin, Ireland), AFP (diluted 1:400, prepared in-house), albumin (diluted 1:200, prepared in-house) or non-immune serum (diluted 1:100, negative control). Following three washes with 0.2% saponin-PBS, samples were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (diluted 1:100, Sanofi, France), for 1 h. Peroxidase activity was detected using liquid DAB (Dako, Germany) and samples were mounted in Kaiser’s glycerol gelatine (Merck, Germany). For each sample, 20 fields were scored using a 40x objective and the number of oval cells as a fraction of hepatocytes was determined by scoring M2-PK-positive oval cells relative to albumin-positive hepatocytes.

Pathological assessment of liver damage
Livers from animals used in the long-term study were removed, sliced (4–5 mm thickness), immerse-fixed in Carnoy’s solution for 1.5 h, embedded in paraffin wax and sectioned at 5 µm. Stains included H&E, modified trichrome and Gomori’s method for reticulin. Immunostaining for {pi}-GST was carried out as described above. Individual sections were examined independently, and without prior knowledge of feeding regime, for evidence of fibrosis, fatty change, architectural distortion and regenerative nodules. Hepatic fibrosis was quantified using a modified Knodell Index (36) applying the following scores: 0, absence of fibrosis; 1, fibrosis expansion of some portal areas, with or without short fibrous septa; 2, fibrosis expansion of most portal areas, with or without short fibrous septa; 3, fibrosis expansion of most portal areas, with portal–portal bridging; 4, fibrous expansion with marked portal–portal and portal–central bridging; 5, marked bridging with occasional nodules. Results are expressed as mean ± SE and statistically analysed using Student’s t-test. Results were considered significant when P < 0.05. Multiple samples of liver from each animal were used for evaluations.

Northern analysis
Following perfusion, and prior to digestion with collagenase, a sample of liver was removed, snap-frozen and stored in liquid nitrogen for RNA isolation (37). For northern blots, either 5 or 15 µg of RNA from each sample was electrophoresed according to the method of Lehrach et al. (38) and transferred to a GenescreenPlus membrane (Dupont, USA) using a vacuum blotter (Hoefer Scientific Instruments, USA). 18S ribosomal RNA, M2-PK and AFP mRNAs were detected by hybridization to specific cDNA probes 32P-labelled by nick translation using the Nick Translation Reagent Kit (Amersham International, UK). Pre-hybridization and hybridization were performed at 42°C in 50% formamide, 5x SSC, 0.1% SDS, 5x Denhardt’s solution and 250 µg/ml salmon sperm DNA. After overnight pre-hybridization, 106 c.p.m. of labelled probe per ml of pre-hybridization buffer was added to the hybridization bottle. Following hybridization for 24 h, membranes were washed twice at room temperature with 2x SSPE, once at 65°C with 2x SSPE in 2% SDS, then twice more with 0.1x SSPE before autoradiography with X-ray film (Fujifilm, Japan). Films were exposed for 3–5 days at –70°C in contact with Fuji G-12 intensifying screens. Autoradiographs were processed and scanned with a document scanner in transmission mode. The intensity of the autoradiographic signal of the digitized image was quantified using NIH Image software (NIH, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Blood alcohol levels
Rats consumed an average of –1.5 ml of either the control or ethanol-supplemented Lieber–DeCarli liquid diet per 100 g body wt/day for 2 months. For the ethanol-treated group, this represented an average consumption of 1.58 g ethanol/100 g body wt/day, and resulted in serum alcohol levels of 0.076 ± 0.018% (mean ± SE, n=6) as judged by blood samples taken prior to death. Alcohol was not detected in the blood of the control-fed group.

Pathological assessment of liver damage
H&E staining of sections from 0 and 3 day CDE-treated control liver (data not shown) reveal normal liver morphology. There is no evidence of small basophilic cells in regions surrounding the portal triads, the site of initial oval cell proliferation. Oval-like cells are first observed around some portal triads after 1 week of CDE treatment (Figure 1aGo, arrow). After 2 weeks of CDE treatment, oval-like cells are present in the majority of portal triads, with increased numbers present in each triad (Figure 1bGo). However, extensive areas of normal parenchyma still exist in central regions.



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Fig. 1. Morphology of liver from rats fed either control liquid diet (CLD) for 2 months followed by CDE diet for up to 2 weeks (a and b) or ELD for 2 months followed by CDE diet for up to 2 weeks (c and d). (a) CLD + 1 week CDE; first evidence of oval-like cells (arrow) extending from some portal triads. (b) CLD + 2 weeks CDE; more extensive spreading of oval cells around the portal triads (arrow) with some migrating centrally. (c) ELD + 1 week CDE; extensive oval cell proliferation periportally (arrow), and some have formed into ducts. (d) ELD + 2 weeks CDE; extensive periportal oval cell proliferation, some with small basophilic nuclei seen centrally (small arrow). Some clusters of inflammatory cells with larger more lightly staining nuclei (large arrow) are also present. Morphology was assessed by H&E staining. Magnification bar represents 20 µm.

 
In contrast, small basophilic cells are already visible in approximately one-third of portal triads in liver sections from ethanol-treated animals prior to CDE treatment (0 day CDE, data not shown). After 3 days of exposure to the CDE diet, the number of portal triads containing oval cells has increased. Additionally, the number of oval cells per triad is more abundant, as large foci of oval cells are observed periportally with occasional extension of these cells centrally (data not shown). By 1 week of CDE feeding, ductal structures are present (Figure 1cGo) and after 2 weeks, the architecture of the liver is grossly disrupted with extensive periportal oval cell proliferation extending as tracts infiltrating the central zone (Figure 1dGo).

Livers from animals fed CLD followed by standard laboratory chow were histologically normal after 8 months, with evidence of mild pericentral macrovesicular steatosis in two animals. There was no evidence of fibrosis in these animals (Table IGo). Two months of ethanol feeding prior to 8 months of normal diet resulted in increased collagen deposition around portal veins (Figure 2aGo, arrow) in all animals, with excess portal collagen deposition and increased ductular proliferation (Figure 2bGo, arrow) in four out of five animals. Two animals displayed very mild pericentral macrovesicular steatosis. These animals showed evidence of fibrosis; however, the difference between the ELD plus control and CLD plus control groups was not statistically significant (Table IGo). Moderate to severe mixed-vesicular steatosis and a slight increase in collagen deposition was apparent in the livers of all animals (4/4) fed the CDE diet alone. All livers displayed architectural distortion and the appearance of fibrosis (Table IGo).


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Table I. Evaluation of liver fibrosis
 


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Fig. 2. Histochemical and immunohistochemical analysis of liver from rats fed ELD for 2 months followed by CDE diet or normal chow for 8 months. Liver sections were stained with trichrome to assess fibrogenesis (a, b, e) or H&E to assess tumourigenesis (c, d, f). ELD + 8 month normal chow; increased central vein collagen deposition (a) and increased ductular proliferation (b). ELD + CDE; livers showed marked histological changes ranging from minimal disruption of architecture (c), to extensive fibrosis (d, e) and tumour formation (f). A small {pi}-GST-positive enzyme-altered hepatic focus present in the liver of an animal fed ELD + 8 month normal chow (g). The hepatocellular adenoma from an ELD + CDE animal stains strongly for {pi}-GST (h). Magnification bar represents 100 µm.

 
Moderate to severe mixed-vesicular steatosis was present in livers from all animals (6/6) fed the ELD plus CDE diet. In addition, marked histological changes were apparent in all livers (6/6), ranging from minimal disruption of liver architecture and a slight increase (2/6) in collagen deposition (Figure 2cGo), to extensive collagen deposition (4/6, Figure 2d–eGo) with nodule formation (1/6, Figure 2fGo). Macroscopic nodules were observed on the surface of the liver lobule, varying in size from 1 to 5 mm in diameter. The largest nodule was composed of cells closely resembling normal hepatocytes, with little variation in size or shape and containing small, uniform nuclei (Figure 2fGo, arrow). These cells were separated by trabeculae two to three cells thick, with inconspicuous sinusoids, and were completely encapsulated. The nodule was histologically characterized as a hepatocellular adenoma. Architectural distortion and the appearance of fibrosis was apparent in all livers; however, the difference between the CLD plus CDE and ELD plus CDE groups was not statistically significant (Table IGo).

For each experimental group the pattern of {pi}-GST staining was distinctly different. Only cells lining portal tracts were {pi}-GST-positive in animals fed CLD plus standard laboratory chow. In contrast, in all animals fed ethanol prior to normal chow, one or two small {pi}-GST-positive enzyme-altered hepatic foci were present per liver section (Figure 2gGo, arrow). These foci comprise no more than 10 hepatocytes and strongly express {pi}-GST. In animals fed the CDE diet alone, low levels of {pi}-GST staining were observed in hepatocytes throughout the entire liver section, with staining slightly increased in central regions for some animals (2/4). However, there was no evidence of strongly staining {pi}-GST-positive enzyme-altered hepatic foci in any of these livers. Most notably, livers of all animals fed the ELD plus CDE diet (6/6) contained large {pi}-GST-positive enzyme-altered hepatic foci, comprising of at least 40 hepatocytes strongly expressing {pi}-GST. In addition, hepatocytes with nuclear {pi}-GST staining were scattered throughout the acini. The number of {pi}-GST-positive enzyme-altered hepatic foci per section correlated with the extent of liver damage (as assessed by scoring of hepatic fibrosis) and varied from 5 to 50% of the liver section, with the hepatocellular adenoma staining strongly for {pi}-GST (Figure 2hGo, arrow).

Pattern of gene expression
Immunostained liver sections from control and ethanol-treated animals confirmed the histological findings with respect to the abundance of oval cells in the respective groups. Prior to CDE treatment, small cells with ovoid nuclei expressing M2-PK (Figure 3aGo), AFP or {pi}-GST (data not shown) are not observed, either periportally or centrally in control animals. A similar result is obtained after 3 days on the CDE diet (data not shown). A small number of oval cells staining positively for M2-PK are first identified after 1 week of CDE treatment (Figure 3bGo, arrow). These cells are weakly positive for AFP and {pi}-GST (data not shown). After 2 weeks, the proportion of oval cells increases markedly (Figure 3cGo, arrow). Examination of an oval cell fraction purified by centrifugal elutriation shows the majority expressed {pi}-GST (~95%), while ~25% expressed AFP. Samples from 3 week CDE-treated liver show small tracts of M2-PK-positive oval cells located periportally, with only a small proportion of these cells located in central regions (data not shown). Nearly all (>95%) of these cells expressed {pi}-GST and ~25% expressed AFP (data not shown).



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Fig. 3. Immunohistochemical localization of M2-PK-positive cells in liver of rats fed either CLD for 2 months followed by CDE diet for up to 2 weeks (ac) or ELD for 2 months followed by CDE diet for up to 2 weeks (df). (a) CLD + 0 day CDE; no evidence of M2-PK-positive oval cells. (b) CLD + 1 week CDE; a few M2-PK-positive oval cells (arrow) around some portal regions. (c) CLD + 2 week CDE; periportal, as well as centrally located oval cells (arrow). (d) ELD + 0 day CDE; some M2-PK-positive oval cells (arrow) are present around most portal triads. (e) ELD + 1 week CDE; M2-PK-positive oval cells are evident throughout the lobule, many (arrow) are positioned between portal triads. (f) ELD + 2 week CDE; shows extensive oval cells scattered throughout the liver lobule; and in many instances, tracts emanate from portal areas (arrow). Magnification bar represents 20 µm.

 
In contrast, small numbers of M2-PK-positive oval cells are already present in ethanol-treated animals prior to CDE treatment (0 day CDE, Figure 3dGo, arrow). All these periportal cells strongly express {pi}-GST and ~15% weakly express AFP. Larger groups of M2-PK, {pi}-GST and AFP-positive oval cells are present 3 days after treatment with CDE (data not shown). By 1 week, foci of oval-like cells identified in H&E stained sections strongly express M2-PK (Figure 3eGo, arrow) and {pi}-GST (data not shown). Immunostaining of the elutriated oval cell fraction shows that 33% of these cells express AFP (data not shown). After 2 weeks of CDE treatment, extensive populations of M2-PK-positive (Figure 3fGo) and {pi}-GST-positive (Figure 4aGo) oval cells are observed in livers of ethanol-treated animals and the percentage of AFP-positive oval cells (Figure 4bGo) increased to 50%. By 3 weeks of CDE treatment, many AFP-positive cells are present (data not shown) and examination of elutriated cells established that 62% of the oval cell population stain positively. Ductular M2-PK staining was not observed in the liver of control or ethanol-treated rats prior to CDE administration; however, after CDE-diet administration M2-PK staining was observed in bile duct epithelia.



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Fig. 4. Immunohistochemical localization of {pi}-GST- and AFP-positive cells in liver of rats fed ELD for 2 months followed by a CDE diet for 2 weeks. There is extensive proliferation of {pi}-GST-positive cells (a) around the portal triad (arrow) with some individual positive cells located more centrally. Insert shows a duct where nearly all cells stain positively in the nucleus. AFP stains cells surrounding the portal triad (b); not the bile duct cells, but oval cells (arrow), which proliferate in response to the CDE diet. About 60% of {pi}-GST-positive cells are AFP positive. Magnification bar represents 20 µm.

 
By scoring M2-PK-positive oval cells relative to albumin-positive cells it is apparent that oval cells appear earlier and in significantly greater numbers in animals which received ethanol prior to CDE treatment (Figure 5Go).



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Fig. 5. Oval cell numbers in the liver of rats fed either CLD for 2 months followed by CDE diet for up to 3 weeks or ELD for 2 months followed by CDE diet for up to 3 weeks. Oval cell numbers are expressed as M2-PK-positive cells (with oval cell morphology) per 100 hepatocytes (identified by albumin staining and with hepatocyte morphology). The data presented are the mean ± SE of percentages for 20 fields for each time point from at least three animals.

 
Northern analysis
Northern analysis shows low levels of M2-PK and AFP mRNA expression in control liver at all stages following the CDE diet (Figure 6aGo). Levels of both genes, which are strongly expressed in oval cells, are significantly elevated in the livers of ethanol-treated animals 1, 2 and 3 weeks after they are placed on the CDE diet (Figure 6a and bGo). In every instance, the level of AFP (Figure 6aGo) and M2-PK (Figure 6a and bGo) is higher in ethanol-treated animals than control animals.



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Fig. 6. (a) Northern blot of RNA prepared from 15 day gestation fetal rat liver (positive control for M2-PK and AFP probe); adult liver; and liver of rats fed either CLD or ELD for 2 months then placed on a CDE diet for up to 3 weeks. RNA loading was checked by hybridization against an 18S ribosomal RNA probe (15 µg loaded in all lanes except lane 1 which contained 5 µg). The membrane was sequentially probed for M2-PK, then AFP. (b) Histogram showing northern blot signals quantified by densitometry and normalized against the 18S ribosomal RNA signal. Two groups of animals were analyzed with similar results and representative data for one experiment is presented.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In a previous study, we reported the appearance of oval cells in the liver of rats chronically exposed to ethanol (21). These cells are identical in terms of morphology and pattern of gene expression to those derived from rat liver after short-term exposure to a CDE diet. It has been well documented that oval cell induction is one of the earliest pre-neoplastic changes observed in some models of experimental hepatocarcinogenesis (29,3942). Therefore, the purpose of the present study was to determine if ethanol not only induces a pre-neoplastic change in the liver, but can also potentiate the effect of short-term and chronic exposure to hepatocarcinogens.

This study illustrates that administration of ethanol prior to CDE treatment enhances the number of periportal oval cells in rat liver. These oval cells were detected and quantified by morphology and the cellular markers M2-PK, {pi}-GST and AFP. Prior to CDE exposure there is already a difference between the control and ethanol-fed groups, an observation compatible with previous findings, which reported that chronic ethanol exposure induces oval cell proliferation (21). Oval cell proliferation occurs in instances where hepatocyte proliferation is inhibited and it is well documented that ethanol inhibits both in vivo and in vitro hepatocyte proliferation (4349).

Following CDE-treatment, the ethanol-fed group showed pronounced acceleration of the cellular alterations commonly associated with the early stages of experimental hepatocarcinogenesis. At all time points, these samples displayed greater numbers of oval cells than the control group. Additionally, the pattern of the oval cell proliferation differed between the two groups. Oval cells were detected in control samples specifically in periportal regions after 1 and 2 weeks of CDE treatment; only after 3 weeks were any detected centrally. In contrast, prior exposure to ethanol resulted in the premature appearance of periportal and centrally located oval cells after 1 week of CDE treatment, and ductal structures after only 2 weeks of CDE treatment. Ductal hyperplasia was not observed at any stage of this study in control rat liver. In addition, ductular M2-PK staining was not observed in the liver of control or ethanol-treated rats prior to CDE administration. This appears contrary to previous observations, which demonstrated very weak M2-PK staining in bile duct cells of 5-week-old rat liver (50). This difference may be attributed to the age of the rats used in this study, which are 2 months older due to ethanol pre-treatment.

Tarsetti et al. (51) report that the severity of most histological abnormalities caused by the CDE diet, particularly oval cell proliferation and cholangiofibrosis, are directly related to the administered dose of ethionine. In contrast, the incidence of HCC is not linearly related to the amount of ethionine used in CDE feeding regimes (51). After 6–12 months, ethionine increased the incidence of hepatocellular nodules and HCC when present at a concentration of 0.05%. However, 0.1% ethionine failed to induce pre-malignant and malignant parenchymal lesions (51). In the present study, after long-term exposure to a CDE diet equivalent to 0.07% (w/v) ethionine, the incidence of liver tumourigenesis is lower than would be predicted from the previous observations of Tarsetti et al. (51). Additionally, the fraction of AFP-positive oval cells observed in the test animals is greater in the short-term study than in the control group and lends further support for the view that ethanol potentiates the effect of carcinogens. Yet, the yield of AFP-positive cells in control animals (25%) is lower in this study, after 3 weeks of CDE treatment, than in a previous study (38%) conducted in our laboratory (21).

We attribute the observed differences between this and previous studies to the age at which the rats were introduced to the CDE diet. In the present study, rats are 2 months older, due to the ethanol/CLD treatment prior to CDE diet administration. Further, we note that increased numbers of immature fetal or oval-like cells reside in the liver of young rats (unpublished data) that could potentially function as a facultative population under the influence of carcinogens. A decline in the number of these cells with development may explain the reduced efficacy of the CDE diet observed in this study.

Nevertheless, the histological alterations observed in ethanol-pre-treated rats are maintained during chronic CDE diet administration, as illustrated by expansion of the ethanol-induced {pi}-GST-positive enzyme-altered hepatic foci. It has been reported that a strong relationship exists between the appearance of {pi}-GST-positive liver foci in medium term assays and liver cancer incidences in the long-term (52,53). Therefore, {pi}-GST-positive foci are considered pre-neoplastic lesions and have been employed as biomarkers in liver carcinogenicity assays (54). The presence of {pi}-GST-positive foci in both ethanol pre-treated experimental groups suggests that ethanol-induced cellular alterations are responsible for their appearance. These alterations are enhanced by chronic CDE treatment, as expansion of the {pi}-GST-positive foci, in size and numbers per section, correlates with the severity of liver damage. Thus, our data suggest that ethanol pre-treatment potentiates short-term effects of the CDE diet by enhancing oval cell proliferation, while chronic CDE administration enhances cellular changes initially induced by ethanol pre-treatment. The numbers and size of {pi}-GST-positive foci developing in the liver under medium-term bioassay conditions have been shown to correlate with eventual incidence of hepatocarcinomas (51). Therefore, it is probable that the {pi}-GST-positive foci observed in our model of ethanol pre-treatment-CDE diet administration would ultimately result in tumour formation. In this study, it is difficult to establish the cellular origin of the observed hepatocellular adenoma. However, as the adenoma expresses {pi}-GST, it is more likely to have originated from a pre-neoplastic {pi}-GST-positive hepatic focus.

Takada et al. (1) and Porta et al. (18) have proposed that ethanol has a promoter-like effect on hepatocarcinogenesis. Ethanol is seen as an agent capable of modifying pathways by which the carcinogen is normally metabolized. This is the classical view of how a promoter or co-carcinogen acts. It has also been reported that ethanol feeding leads to hypomethylation of DNA in colonic mucosal cells, which could increase their susceptibility to carcinogenesis (55). A similar alteration in liver cells could also enhance the susceptibility of oval cells to cancer. This study suggests that an additional consequence of ethanol activity should be considered; this is its ability to alter the cellular composition of the liver, as illustrated by expansion of the ethanol-induced {pi}-GST-positive enzyme-altered hepatic foci.

Our results indicate that ethanol enhances the effectiveness of the CDE diet in the short-term, inducing oval cell proliferation. It is possible that this enhancement involves TNF signalling as TNF has been implicated in both alcohol-induced liver injury and the CDE-mediated oval cell response (56,57). Ethanol consumption also increases permeability of the small intestine, resulting in Kupffer cell activation and induction of TNF{alpha} expression (58). Therefore, ethanol treatment may enhance the activation of Kupffer cells, which release TNF{alpha}, thus augmenting the oval cell proliferation induced by the CDE diet.

Our data indicate that ethanol has the potential to prime the liver to be more responsive to a carcinogenic diet, a situation that may be relevant in humans. Thus, cellular changes in the composition of the liver may ultimately provide an explanation for the association between increasing oval cell number and severity of liver disease observed in chronic alcoholics (32). Additionally, as animal models of liver cancer, which show increased oval cell numbers, can lead to cholangiocellular and hepatocellular carcinoma (22,23), this study has implications for the development of both forms of liver cancer in alcoholic liver disease.


    Notes
 
1 To whom correspondence should be addressed Email: ecroager{at}cyllene.uwa.edu.au Back


    Acknowledgments
 
The authors wish to thank Dr Ross Glancy for assistance with interpreting liver pathology and Dr Elizabeth Quail for critical discussion of this manuscript. This study was supported by grants from the National Health and Medical Research Council of Australia and the Cancer Foundation of Western Australia. Dr Emma Croager is supported by a fellowship from the Healy Medical Research Foundation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 6, 2002; revised June 6, 2002; accepted June 6, 2002.





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