Pyruvate kinase isoenzyme shift from L-type to M2-type is a late event in hepatocarcinogenesis induced in rats by a choline-deficient/DL- ethionine-supplemented diet

Rats received a choline-deficient diet containing 0.1% (w/w) DL- ethionine (CDE) for 4, 10, 14 or 22 weeks. A separate group was treated for 4 weeks with CDE and then received a normal diet for 4 weeks. The L and M2 isoenzymes of pyruvate kinase were immunocytochemically demonstrated in liver sections. L-PK expression was strongly reduced in the hepatocytes after 4 weeks of treatment and remained low until the end of the study. Withdrawal of CDE after 4 weeks followed by 4 weeks normal diet resulted in a nearly full recovery of L-PK expression as compared to untreated controls. At later stages (10-22 weeks of CDE- treatment) many pseudolobules, preneoplastic foci of altered hepatocytes (FAH) such as combined clear/acidophilic cell foci (CCF/ACF) and mixed/basophilic cell foci (MCF/BCF), and hepatocellular adenomas (HCA) were observed. Pseudolobules showed a slight reduction in L-PK-expression, and were negative for M2-PK. In all clear cell components of CCF/ACF excessively storing glycogen, L-PK-expression was increased compared to both the surrounding parenchyma and hepatocytes of controls. In acidophilic cell components with less pronounced glycogen storage L-PK expression was similar to that of pseudolobules showing a slightly reduced content of this enzyme protein. M2-PK was invariably negative in CCF/ACF. In most MCF glycogen-storing subpopulations expressed L-PK, whereas in all glycogen-poor basophilic populations L-PK protein was strongly reduced. M2-PK was not expressed in most of these MCF. However, in rare MCF the reduction in L-PK expression was combined with a significant expression of M2-PK. In HCA M2-PK underwent a further increase, although to a variable degree, while L-PK remained strongly reduced. Our results show that an isoenzyme shift from L-PK to M2-PK takes place at a late stage of the hepatocarcinogenic process, and that those MCF with a low L-PK expression and a reexpression of M2-PK most probably represent the direct precursor lesions of hepatocellular neoplasms.      

Expression of alpha, mu and pi class glutathione S-transferases in oval and ductal cells in liver of rats placed on a choline-deficient, ethionine-supplemented diet.

Expression of the alpha, mu and pi class glutathione S-transferases (GSTs) in hepatocytes, oval cells and ductal cells derived from the livers of rats placed on a choline-deficient, ethionine-supplemented (CDE) diet for 5 weeks was investigated. An overall decrease in the expression of alpha and mu class GSTs and an over-expression of pi class GST was observed in the liver after CDE treatment as indicated by Northern blotting analysis. Massive disruption of the liver with oval cell infiltration in the sinusoids throughout the lobule occurred after 5 weeks CDE treatment. 'Duct-like' structures consisting of oval-like cells (ductal cells) with rounder nuclei and more cytoplasm than oval cells within the sinusoids were also apparent. Immunocytochemical analysis revealed that the altered expression of GST in the whole liver is attributed to a differential expression of alpha, mu and pi class GSTs in the different cell types in the liver, including hepatocytes, oval cells around the portal region and among the sinusoids, and oval-like cells (ductal cells) in the 'duct-like' structures. In vitro studies using purified oval-ductal cells and hepatocyte populations confirmed the differential expression of GSTs in the varying cell populations in situ. The expression of the alpha and mu class GSTs in hepatocytes does not appear to be altered by the CDE diet. Heterogeneity in distribution of pi class GST was observed in the hepatocyte population, some hepatocytes were stained strongly while no staining was observed in others. Oval and ductal cells represent two distinct populations displaying different expression of GSTs. Pi class GST was detected in the majority of oval and ductal cells. Alpha class GST was detected in < 5% of the oval cell population and was found in > 50% of the ductal cell population. In contrast, mu class GST was absent in ductal cells and was present in 24% of oval cells around the portal region. This supports the view that ductal cells are not of bile ductal origin since mu GST is present in normal bile duct epithelial cells. Furthermore the change in expression of GSTs in the liver after CDE treatment is attributed to the large increase in oval and ductal cell populations.     

In situ hybridization studies on expression of albumin and alpha-fetoprotein during the early stage of neoplastic transformation in rat liver

The expressions of albumin and alpha-fetoprotein (AFP) genes were studied in early preneoplastic liver lesions produced by the Solt-Farber protocol using "in situ" hybridization with single stranded RNA probes. In normal rat liver, albumin was expressed at a lower level in the centrilobular than in the periportal areas of the liver acinus, whereas the bile duct epithelium did not show any expression. Five weeks after initiation with diethylnitrosamine, islands of hepatocytes were present which showed heterogeneous expression of albumin and were surrounded by cells comprised of albumin negative hepatocytes and oval cells. gamma-Glutamyltranspeptidase positive foci of enzyme altered cells were located in albumin positive areas. Albumin expression gradually decreased in permanent nodules but increased in the hepatocytes outside the nodules during the first five months after initiation with diethylnitrosamine. Remodeling nodules, which were partly gamma-glutamyltranspeptidase and albumin positive, were also present. However, no consistent correlation was found between gamma-glutamyltranspeptidase positive and albumin negative areas during the first 5 months after initiation. Occasionally, cells showing an elevated expression of albumin were found in permanent nodules. These cells were located in the vicinity of oval type cells, which also showed a weak expression of albumin. AFP was expressed at high level in oval cells 5 weeks after the initiation. However, oval cells observed at later time points, either around the neoplastic nodules or inside the nodules showed only low expression of AFP. Hepatocytes in the enzyme-altered foci and in neoplastic nodules were always negative for AFP. The presence of strongly albumin positive cells inside the neoplastic nodules in close proximity to oval type cells suggests that these cells may be derived from primitive "stem-cell"-like oval cells.     

In vivo differentiation of rat liver oval cells into hepatocytes

The Solt-Farber protocol, in the absence of an initiating agent, was used to examine the precursor-product relationship between oval cells and hepatocytes in rat liver. The animals were administered 2-acetylaminofluorene (AAF) by gavage for 2 wk combined with partial hepatectomy 1 wk after administering AAF Two dose levels of AAF were used: 9- and 21-mg total dose for animals in Groups I and II, respectively. [3H]Thymidine was administered i.p. to one-half of the animals at Day 6 post-partial hepatectomy. Animals were sacrificed 7, 9, 11, and 13 days after surgery. Only oval cells became labeled on Day 7 in both groups. On Day 9 both labeled oval cells and labeled basophilic hepatocytes were present in Group I, whereas in Group II only oval cells remained labeled. On Days 11 and 13 both oval cells and basophilic hepatocytes were labeled in both groups. The total amount of radioactivity in Group II livers remained the same on Day 9 when only labeled oval cells were present and on Days 11 and 13 when both labeled oval cells and labeled basophilic hepatocytes were present. The calculated half-life for basophilic hepatocytes was about 50 h. The differentiation of oval cells into basophilic hepatocytes was delayed in Group II as compared to Group I, and the higher dose of AAF also induced the formation of both intestinal metaplasia and bile duct formation. In situ hybridization with an alpha-fetoprotein probe showed a strong expression in groups of typical oval cells and in cells arranged in duct-like structures. In addition a transient expression of AFP was also observed in the areas of basophilic hepatocytes 9 to 11 days after partial hepatectomy. Administration of AAF decreased the level of albumin mRNA in preexisting hepatocytes and caused a significant decrease of serum albumin. In contrast, oval cells showed a strong albumin expression, and basophilic hepatocytes formed islands of albumin-expressing cells. Oval cells and the foci of early basophilic hepatocytes lacked glucose-6-phosphatase activity. At Day 13 significant numbers of basophilic hepatocytes were positive for glucose-6-phosphatase. Oval cells were strongly gamma-glutamyltranspeptidase positive, whereas the foci of basophilic hepatocytes were negative for gamma-glutamyltranspeptidase. Only occasionally were transiently gamma-glutamyltranspeptidase-positive hepatocytes observed in basophilic foci. In summary our data indicate that oval cells can differentiate to hepatocytes and may have an important physiological function as a source of major serum proteins when hepatocytes are unable to synthesize these proteins.     

Carcinohistogenesis and expression of alpha fetoprotein in experimental hepatocarcinoma

In1973,WhenAnthony,etal.inUganda[']werestudyinghumanhepatocirrhosisandhepatocarcinomatheyfirstreportedthatatypicalhyperplasia(AH)ofhepatocyteswasprecancerosisofhepatocellularcarcinoma(HCC).Roncalli,etal.[2]dividedAHintolargehepatocyticandsmallhepatocyticAH,andtheyconsideredthelatterwascloselyrelatedtoformingHCC.In1988,Xu,etal.[3]pointedoutthatHCCoccursfromproliferativenodulesofbasophilicandacidophilichepatocytes.Indeed,carcinohistogenesis,inparticularthechangeofalpha-fetoprotein(AFP)i…     

Presence of alpha-fetoprotein-positive cells in hepatocellular foci and microcarcinomas induced by single injections of diethylnitrosamine in infant mice

Single injections of diethylnitrosamine (5 and 50 micrograms/g body weight) in male C57BL/6J X C3HeB/FeJ F1 mice when they were 15 days old resulted in the induction of RNA-rich hepatocellular foci and nodules that contained alpha-fetoprotein (AFP)-positive hepatocytes after 20 and 28 weeks. The focal lesions were composed of 1- to 2-cell-thick plates of hepatocytes or closely packed clusters of cells, but they did not show the histological patterns that are diagnostic of trabecular hepatocellular carcinoma. AFP-positive hepatocytes were found in almost one-fourth (14 of 60) of the foci and nodules in a serially sectioned block of liver from a mouse given one injection of 50 micrograms/g body weight diethylnitrosamine and killed at 28 weeks. In general, the presence or absence of AFP-positive cells correlated with the size of the foci and nodules. All six nodules with diameters greater than 1.5 mm contained AFP-positive cells, while all 12 foci smaller than 0.24 mm in diameter were negative for AFP. However, among the 42 foci that were intermediate in size, there were 8 AFP-positive foci, the sizes of which appeared rather randomly distributed among the negative foci. Reactive changes in hepatocytes could be ruled out as a cause of the induction of AFP because the foci first appeared many weeks after the administration of diethylnitrosamine in these mice. Since bile ductules or oval cells, which occasionally appeared in these foci, were lacking entirely in AFP and since ductules are absent from the early-appearing and smallest foci, we believe that in this model the AFP-positive foci arise only from hepatocytes. The presence of AFP in the focal lesions and in tumor thrombi that extended from them into hepatic vein branches supports the hypothesis that some foci undergo progression to invasive microcarcinomas and that these in turn are precursors of late-appearing (after 1 year) metastasizing trabecular hepatocellular carcinomas.     

Persistence of the cholangiocellular and hepatocellular lesions observed in rats fed a choline-deficient/DL-ethionine-supplemented diet.

Male outbred Sprague-Dawley rats were fed a choline-deficient diet containing 0.10% DL-ethionine (CDE) for 4, 6, 10, 14 or 22 weeks followed by a standard diet for up to 59 weeks. Liver sections were histochemically analyzed for the following parameters: basophilia, glycogen content and the activities of glycogen synthase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6PASE), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerin-3-phosphate dehydrogenase (G3PDH), 'malic enzyme' (MDH), alkaline phosphatase (ALKPASE) and gamma-glutamyltranspeptidase (GGT). The stop experiments revealed that many of the oval cells proliferating during the first 4-6 weeks may undergo necrotic changes and disappear with time, whereas cholangiofibroses appearing in animals fed CDE for at least 10 weeks are persistent lesions. The sequence of lesions seen in this study, leading from persistent oval cells through cholangiofibroses to cholangiofibromas, strongly suggests that the oval cells are the precursor cells of cholangiocellular tumors. The proliferating oval cells and the hepatic foci consisting of clear and acidophilic or mixed cell populations were always spatially separated and no transitions between oval and parenchymal cells were observed. These results argue against a precursor-product relationship between oval and parenchymal cells. Both proliferating and persistent oval cells, cholangiofibroses and cholangiofibromas showed a strong staining for G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT; low PHO, SYN and G6PASE activities were also detected in these lesions. Persistent glycogen-storage foci, which developed in all rats fed CDE for 4-14 weeks followed by a normal lab chow for over a year, had increased PHO, G6PDH, MDH, ALKPASE and GGT activities, while SYN, GAPDH and G3PDH activities remained unaltered and G6PASE activity decreased. Mixed cell foci appearing in animals fed CDE for 22 weeks followed by a normal lab chow for 59 weeks had strongly increased G6PDH, GAPDH, G3PDH, MDH, ALKPASE and GGT activities as well as decreased G6PASE activity. These results indicate that the characteristic metabolic pattern of preneoplastic hepatic foci is independent of the further administration of the carcinogenic diet. The shift from glycogen metabolism to glycolysis and the pentose phosphate pathway occurring during the later stages of CDE-induced hepatocarcinogenesis is an autogenous process apparently directing the disturbed carbohydrate metabolism towards alternative metabolic pathways. A similar metabolic shift also seems to take place during cholangiocarcinogenesis.     

Increased yield in GST-P-positive liver pre-neoplastic foci induced by dena or enu in rats pre-treated with estradiol or tamoxifen

We investigated the mechanisms by which partial hepatec-tomy (PH) increases the ability of chemical hepatocarcinogens to induce pre-neoplastic liver foci. Comparison of the effects of pre-treatment with PH, estradiol (E2) or tamoxifen (TAM) on the yield in glutathione-S-transferase(GST-P)-positive pre-neoplastic foci in rat liver induced by subsequent treatment with ethylnitrosourea (ENU) or diethylnitrosamine (DENA) showed that pre-treatment with E2 increased the yield in foci induced by subsequent treatment with ENU or DENA, as compared with that in animals not pre-treated, the increase being of similar magnitude with either carcinogen. Compared with that of PH, the effect of the hormone was much more pronounced than would be expected from the relative mito-genic effect of the hormonal and surgical pre-treatments if the mitotic rate were the cause. On the other hand, the average volume of pre-neoplastic liver lesions in rats treated with ENU or DENA was 2.5 to 5.0 times higher than in rats not pre-treated whenever PH was included in the pre-treatment, whereas it was not affected by any other pre-treatment.     

Bone marrow-derived cells fuse with hepatic oval cells but are not involved in hepatic tumorigenesis in the choline-deficient ethionine-supplemented diet rat model

Bone marrow cells (BMCs) have been reported to behave as tissue-specific stem cells in some organs and to participate in tumorigenesis. However, the roles of BMCs in hepatic regeneration and carcinogenesis are still unknown. A choline-deficient, ethionine-supplemented (CDE) diet leads to the appearance of oval cells, a type of hepatic progenitor cell, and activates their replication. Furthermore, this type of diet induces preneoplastic nodules and hepatocellular carcinomas (HCCs) derived from oval cell progenitors. The aims of this study were to determine whether oval cells are derived from BMCs and whether preneoplastic nodules or HCCs originate from BMCs in the CDE diet rat model. To clarify the origin of constituent cells in the liver, we transplanted BMCs from green fluorescent protein (GFP) transgenic female rats into male Lewis rats, which were then exposed to a CDE diet to induce hepatocarcinogenesis. Some oval cells showed both donor-derived GFP expression and the recipient-specific Y chromosome, indicating that donor BMCs fused with recipient oval cells. Several preneoplastic nodules (precancerous lesions) identified by their glutathione S-transferase placental (GSTp) positivity were induced by CDE treatment. However, these preneoplastic GSTp-positive nodules were not GFP positive. In conclusion, this study has produced two major findings. First, BMCs fuse with some oval cells. Second, BMC-fused oval cells and BMCs might not have malignant potential in the CDE-treated rat model.     

Differential expression of alpha-fetoprotein and gamma-glutamyltranspeptidase in chemical and spontaneous hepatocarcinogenesis.

The expression of two markers of fetal liver, alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase (GGT), was studied in chemical and spontaneous hepatocarcinogenesis in mice. Serum AFP concentration increased within 3 weeks 3 weeks from the commencement of feeding of o-aminoazotuluene. This early elevation subsided about 3 months after the beginning of the administration of the carcinogen. A new, sustained elevation of the serum AFP level followed at 5 to 6 months accompanied by the appearance of liver tumors. In immunofluorescence, some small oval cells and scattered adult-type hepatocytes contained AFP during the early stage of chemical carcinogenesis. During the later phase, AFP was detected in a few of the nodular areas, in solitary hepatocytes, and in groups of carcinoma cells. GGT activity in the liver increased within 1 week after the carcinogen regimen was started, preceding the early increase of AFP production. At the final stage, the chemically induced hepatomas contained about 80 times more GGT than did normal liver. In histochemical staining, proliferating oval cells and small areas of hepatocytes stained for GGT during the early weeks, and later most nodules, small areas of nonnodular parenchyma, and carcinomas contained GGT. During spontaneous carcinogenesis in male C3HeB/FeJ mice, premalignant lesions, accompanied by a slight increase of serum AFP, precede the appearance of liver tumors. No cells staining for AFP were detected during this early stage. Once overt liver cancers had developed, AFP was readily detectable in the tumors and was localized to some but not all carcinoma cells. The corresponding serum AFP levels were highly elevated. In contrast to the high levels of GGT found during chemical carcinogenesis, no elevation of GGT was found in livers at various stages of spontaneous carcinogenesis, including cancers in eight individual mice. These results indicate that the production of AFP and GGT is not turned on as a single "genetic package," and that these two markers differ in their behaviour in liver carcinogenesis.     

Differential cytokeratin and alpha-fetoprotein expression in morphologically distinct epithelial cells emerging at the early stage of rat hepatocarcinogenesis

The various liver cell populations emerging during the transitory reappearance of alpha-fetoprotein (AFP) in serum of 3'-methyl-4-dimethylaminoazobenzene-ingesting rats were analyzed in situ and in vitro on isolated cell preparations in terms of their cytokeratin and AFP expression using single and double indirect immunofluorescence microscopy. A polyclonal guinea pig antibody raised against cow hoof prekeratin, which recognized a Mr 52,000 cytokeratin, was found to react with bile ductular epithelial cells and oval cells but not with hepatocytes. A monoclonal antibody against a Mr 55,000 cytokeratin reacted not only with bile ductular and oval cells but also with hepatocytes. In contrast, a polyclonal antibody against porcine eye lens vimentin reacted with sinusoidal cells and stroma cells. To assess further the heterogeneity of the emerging cell populations, liver cells were isolated after 4 weeks of treatment and fractionated according to cell size and ploidy level into 4 fractions (I to IV) by velocity sedimentation at 1 X g. A cell-type analysis using AFP and albumin as functional markers revealed the presence of AFP-producing cells in Fraction IV at a mean velocity equivalent to that of newborn diploid rat hepatocytes, whereas most of the albumin-producing cells were distributed in Fractions I to III at velocities similar to those of adult tetraploid rat hepatocytes. A similar analysis based on the differential expression of Mr 52,000 and Mr 55,000 cytokeratins and vimentin in bile ductular and other diploid epithelial cells, hepatocytes, and mesenchymal cells showed that large cells in Fractions I to III were tetraploid hepatocytes, whereas viable cells present in Fraction IV were diploid epithelial cells and mesenchymal cells in proportion of 62 and 38%, respectively. These cell populations could be resolved further by changing the sedimentation time. A subsequent examination of the Mr 55,000 cytokeratin-containing diploid epithelial cells in Fraction IV using double immunofluorescence microscopy resolved three cell populations with respect to Mr 52,000 cytokeratin and AFP expression, namely, two cell populations expressing either protein marker and a third one containing both markers. These results suggest a ductular origin of oval cells and a possible relation to immature hepatocytes.     

Effect of ethanol on dimethylnitrosamine activation and DNA synthesis in rat liver

Male Wistar rats were treated for 2 weeks with 20% ethanol inthe drinking water. Twenty four hours after the terminationof treatment animals were injected with different doses of [¹⁴C]dimethylnitrosamineand killed 4 h thereafter. The amounts of 7-methylguanine andO⁶-methylguanine present in liver DNA were determined. Therewas no significant difference in the levels of either DNA alkylationproduct between untreated controls and animals pre-treated withethanol. The O⁶-methylguanine/7-methylguanine ratio was alsounchanged. Specific radioactivity levels in the cellular proteinof [¹⁴C]dimethylnitrosamine injected animals were slightly loweredafter ethanol pre-treatment. In contrast, incorporation of labelledguanine into liver DNA was greatly enhanced at all DMN dosesin ethanol pre-treated animals indicating an increase in DNAsynthesis. This enhancement of DNA synthesis was confirmed atdifferent ethanol doses, given either in the drinking wateror by stomach tube, by the increase in specific radioactivityof liver DNA and the increase in the number of labelled nucleifollowing [³H]thymidine pulse-labelling.     

Autoradiography of "oval cells" appearing rapidly in the livers of rats fed N-2-fluorenylacetamide in a choline devoid diet

Autoradiographic analysis of liver sections from rats fed thehepatocarcinogen N-2-fluorenylacetamide (FAA) in a choline devoid(CD) diet suggests that proliferating small "oval" cells arisefrom a few small portally-situated cells, and spread rapidlyacross the entire liver lobule. Small cells with detectablegrains are first located where liver plates meet the portalareas. This cell type gradually increases in number over a 10–12day period, then proliferates rapidly. After 28 days, microscopicnodules consisting of heavily labeled large eosinophilic cellsappear, whereas residual hepatocytes are not labeled. Combinedimmunofluorescent and autoradiographic labeling studies revealthat many of the small cells contain AFP; approximately halfof the a-fetoprotein-containing cells are labeled with [³H]thymidine(dT). Feeding CD-FAA diets to rats with hepatocytes prelabeledwith [³H]dT after 70% hepa-tectomy 7 weeks earlier providesdata which suggest that small "oval" cells do not arise fromprelabeled hepatocytes but, instead, infiltrate the prelabeledhepatocytes during the diet induced proliferative phase. Weconclude that "oval" cells arise from a small number of portalcells, not from hepatocytes. Exact identification of the ovalcell precursor is not possible, but it could be a "stem" cell.Although hepatocyte-like properties are found in small cells(e.g., albumin staining), there is no evidence that they differentiateinto normally functioning hepatocytes.     

Elevation of Serum Alpha-fetoprotein and Proliferation of Oval Cells in the Livers of LEC Rats

Alpha-fetoprotein (AFP) in the sera of 35 LEC (Long-Evans with a cinnamon-like coat color) rats between 7 and 25 weeks of age was evaluated by enzyme-linked immunosorbent assay (ELISA). Elevation of serum AFP and proliferation of oval cells in the liver were observed in most LEC rats, which suffered from acute hepatitis. On the other hand, the serum AFP level was within the normal range before the onset of hepatitis. Immunohistochemical staining for AFP revealed that some of the proliferating oval cells produced AFP, Morphotnetric analysis of AFP-positive cells and ELISA for serum AFP demonstrated that there was a statistically significant correlation between the number of AFP-positive cells in the liver and the concentration of AFP in the serum. Histological examination revealed the transition and differentiation of the oval cells to small hepatocytes. These results suggested that the phenomena which occurred in LEC rats suffering from acute hepatitis were similar to those that occurred during the early stage of azo dye hepatocarcinogenesis, although the extent of the oval cell proliferation and the elevation of serum AFP in LEC rats were not as great as those in rats treated with azo dye. This is the first report on a rat strain with proliferation of AFP-producing oval cells during its natural history.     

Inhibition of adult liver progenitor (oval) cell growth and viability by an agonist of the peroxisome proliferator activated receptor (PPAR) family member gamma, but not alpha or delta

Multifaceted evidence links the development of liver tumours to the activation and proliferation of adult liver progenitor (oval) cells during the early stages of chronic liver injury. The aim of this study was to examine the role of the peroxisome proliferator activated receptors (PPARs): PPARalpha, delta and gamma, in mediating the behaviour of liver progenitor cells during pre-neoplastic disease and to investigate their potential as therapeutic targets for the treatment of chronic liver injury. We observed increased liver expression of PPARalpha and gamma in concert with expanding oval cell numbers during the first 21 days following commencement of the choline deficient, ethionine supplemented (CDE) dietary model of carcinogenic liver injury in mice. Both primary and immortalized liver progenitor cells were found to express PPARalpha, delta and gamma, but not gamma2, the alternate splice form of PPARgamma. WY14643 (PPARalpha agonist), GW501516 (PPARdelta agonist) and ciglitazone (PPARgamma agonist) were tested for their ability to modulate the behaviour of p53-immortalized liver (PIL) progenitor cell lines in vitro. Both PPARdelta and gamma agonists induced dose-dependent growth inhibition and apoptosis of PIL cells. In contrast, the PPARalpha agonist had no effect on PIL cell growth. None of the drugs affected the maturation of PIL cells along either the hepatocytic or biliary lineages, as judged by their patterns of hepatic gene expression prior to and following treatment. Administration of the PPARgamma agonist ciglitazone to mice fed with the CDE diet for 14 days resulted in a significantly diminished oval cell response and decreased fibrosis compared with those receiving placebo. In contrast, GW501516 did not affect oval cell numbers or liver fibrosis, but inhibited CDE-induced hepatic steatosis. In summary, PPARgamma agonists reduce oval cell proliferation and fibrosis during chronic liver injury and may be useful in the prevention of hepatocellular carcinoma.     

Elevation of serum alpha-fetoprotein and proliferation of oval cells in the livers of LEC rats

Alpha-fetoprotein (AFP) in the sera of 35 LEC (Long-Evans with a cinnamon-like coat color) rats between 7 and 25 weeks of age was evaluated by enzyme-linked immunosorbent assay (ELISA). Elevation of serum AFP and proliferation of oval cells in the liver were observed in most LEC rats, which suffered from acute hepatitis. On the other hand, the serum AFP level was within the normal range before the onset of hepatitis. Immunohistochemical staining for AFP revealed that some of the proliferating oval cells produced AFP. Morphometric analysis of AFP-positive cells and ELISA for serum AFP demonstrated that there was a statistically significant correlation between the number of AFP-positive cells in the liver and the concentration of AFP in the serum. Histological examination revealed the transition and differentiation of the oval cells to small hepatocytes. These results suggested that the phenomena which occurred in LEC rats suffering from acute hepatitis were similar to those that occurred during the early stage of azo dye hepatocarcinogenesis, although the extent of the oval cell proliferation and the elevation of serum AFP in LEC rats were not as great as those in rats treated with azo dye. This is the first report on a rat strain with proliferation of AFP-producing oval cells during its natural history.     

Modulation of cytotoxic and genotoxic effects of 2-acetylaminofluorene in rat and hamster hepatocytes by 3-methylcholanthrene pre-treatment

It is well known that 2-acetylaminofluorene (AAF)-induced livercancer is reduced by simultaneous administration of 3-methylcholanthrene(MC) in the rat, but not in the hamster. The present reportexamines the effects of MC pre-treatment on the metabolism andtoxkity of AAF in monolayer cultures of hepatocytes. Hepatocytesisolated from pre-treated animals of both species metabolizedAAF and 2-aminofluorene (AF) to metabolites mutagenic to Salmonellatyphimurium more efficiently than hepatocytes from control animals.MC-pre-treated rat hepatocytes showed increased responses toAAF-and AF-induced unscheduled DNA synthesis, while MC-pre-treatedhamster hepatocytes were less responsive than the untreatedhepatocytes. Increased cytotoxic effects of AAF were observedin MC-pre-treated rat hepatocytes, whereas AAF was not cytotoxicin hamster hepatocytes from either pre-treated or control animals.MC pre-treatment caused increased rates of formation of C-hydroxylated,N-hydroxylated, water-soluble and covalently macromolecularbound AAF metabolites in both species. No significant effectof MC pre-treatment was seen on the formation of AF from AAF.A large decrease in the ratio between covalently macromolecularbound (activated) metabolites and the sum of C-hydroxylatedand water-soluble (detoxified) AAF metabolites, was seen afterMC pre-treatment of rat hepatocytes, whereas no or only a minordecrease was observed in hamster hepatocytes. This ratio correlatedmuch better with the in vivo carcinogen-icity data than theother parameters such as mutagenicity, DNA repair or covalentmacromolecular binding. Thus, the hypothesis that A AF-inducedliver cancer depends less on the rate at which AAF is activated,but more on the relative proportion of the dose which is activated,is supported by the present data.